Coordinate regulation of cytochrome and alternative pathway respiration in tobacco.

PubMed

Vanlerberghe, G C; McIntosh, L

1992-12-01

In suspension cells of NT1 tobacco (Nicotiana tabacum L. cv bright yellow), inhibition of the cytochrome pathway of respiration with antimycin A induced a large increase in the capacity of the alternative pathway over a period of approximately 12 h, as confirmed in both whole cells and isolated mitochondria. The increase in alternative pathway capacity required de novo RNA and protein synthesis and correlated closely with the increase of a 35-kD alternative oxidase protein. When the cytochrome pathway of intact cells was inhibited by antimycin A, respiration proceeded exclusively through the alternative pathway, reached rates significantly higher than before antimycin A addition, and was not stimulated by p-trifluoromethoxycarbonylcyanide (FCCP). When inhibition of the cytochrome pathway was relieved, alternative pathway capacity and the level of the 35-kD alternative oxidase protein declined. Respiration rate also declined and could once again be stimulated by FCCP. These observations show that the capacities of the mitochondrial electron transport pathways can be regulated in a coordinate fashion.

A Single Streptomyces Symbiont Makes Multiple Antifungals to Support the Fungus Farming Ant Acromyrmex octospinosus

PubMed Central

Seipke, Ryan F.; Barke, Jörg; Brearley, Charles; Hill, Lionel; Yu, Douglas W.; Goss, Rebecca J. M.; Hutchings, Matthew I.

2011-01-01

Attine ants are dependent on a cultivated fungus for food and use antibiotics produced by symbiotic Actinobacteria as weedkillers in their fungus gardens. Actinobacterial species belonging to the genera Pseudonocardia, Streptomyces and Amycolatopsis have been isolated from attine ant nests and shown to confer protection against a range of microfungal weeds. In previous work on the higher attine Acromyrmex octospinosus we isolated a Streptomyces strain that produces candicidin, consistent with another report that attine ants use Streptomyces-produced candicidin in their fungiculture. Here we report the genome analysis of this Streptomyces strain and identify multiple antibiotic biosynthetic pathways. We demonstrate, using gene disruptions and mass spectrometry, that this single strain has the capacity to make candicidin and multiple antimycin compounds. Although antimycins have been known for >60 years we report the sequence of the biosynthetic gene cluster for the first time. Crucially, disrupting the candicidin and antimycin gene clusters in the same strain had no effect on bioactivity against a co-evolved nest pathogen called Escovopsis that has been identified in ∼30% of attine ant nests. Since the Streptomyces strain has strong bioactivity against Escovopsis we conclude that it must make additional antifungal(s) to inhibit Escovopsis. However, candicidin and antimycins likely offer protection against other microfungal weeds that infect the attine fungal gardens. Thus, we propose that the selection of this biosynthetically prolific strain from the natural environment provides A. octospinosus with broad spectrum activity against Escovopsis and other microfungal weeds. PMID:21857911

Endophytic Streptomyces sp. AC35, a producer of bioactive isoflavone aglycones and antimycins.

PubMed

Ondrejíčková, P; Šturdíková, M; Hushegyi, A; Švajdlenka, E; Markošová, K; Čertík, M

2016-09-01

In this research, a microbial endophytic strain obtained from the rhizosphere of the conifer Taxus baccata and designated as Streptomyces sp. AC35 (FJ001754.1 Streptomyces, GenBank) was investigated. High 16S rDNA gene sequence similarity suggests that this strain is closely related to S. odorifer. The major fatty acid profile of intracellular lipids was also carried out to further identify this strain. Atomic force microscopy and scanning acoustic microscopy were used to image our strain. Its major excreted substances were extracted, evaluated for antimicrobial activity, purified, and identified by ultraviolet-visible spectroscopy (UV-vis), liquid chromatography-mass spectrometry (LC-MS/MS) and nuclear magnetic resonance as the bioactive isoflavone aglycones-daidzein, glycitein and genistein. Batch cultivation, performed under different pH conditions, revealed enhanced production of antimycin components when the pH was stable at 7.0. Antimycins were detected by HPLC and identified by UV-vis and LC-MS/MS combined with the multiple reaction monitoring. Our results demonstrate that Streptomyces sp. AC35 might be used as a potential source of effective, pharmaceutically active compounds.

Discovery of potent broad spectrum antivirals derived from marine actinobacteria.

PubMed

Raveh, Avi; Delekta, Phillip C; Dobry, Craig J; Peng, Weiping; Schultz, Pamela J; Blakely, Pennelope K; Tai, Andrew W; Matainaho, Teatulohi; Irani, David N; Sherman, David H; Miller, David J

2013-01-01

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds.

Pharmacological Modulation of the Mitochondrial Electron Transport Chain in Paclitaxel-Induced Painful Peripheral Neuropathy.

PubMed

Griffiths, Lisa A; Flatters, Sarah J L

2015-10-01

Paclitaxel is an effective first-line chemotherapeutic with the major dose-limiting side effect of painful neuropathy. Mitochondrial dysfunction and oxidative stress have been implicated in paclitaxel-induced painful neuropathy. Here we show the effects of pharmacological modulation of mitochondrial sites that produce reactive oxygen species using systemic rotenone (complex I inhibitor) or antimycin A (complex III inhibitor) on the maintenance and development of paclitaxel-induced mechanical hypersensitivity in adult male Sprague Dawley rats. The maximally tolerated dose (5 mg/kg) of rotenone inhibited established paclitaxel-induced mechanical hypersensitivity. However, some of these inhibitory effects coincided with decreased motor coordination; 3 mg/kg rotenone also significantly attenuated established paclitaxel-induced mechanical hypersensitivity without any motor impairment. The maximally tolerated dose (.6 mg/kg) of antimycin A reversed established paclitaxel-induced mechanical hypersensitivity without any motor impairment. Seven daily doses of systemic rotenone or antimycin A were given either after paclitaxel administration or before and during paclitaxel administration. Rotenone had no significant effect on the development of paclitaxel-induced mechanical hypersensitivity. However, antimycin A significantly inhibited the development of paclitaxel-induced mechanical hypersensitivity when given before and during paclitaxel administration but had no effect when given after paclitaxel administration. These studies provide further evidence of paclitaxel-evoked mitochondrial dysfunction in vivo, suggesting that complex III activity is instrumental in paclitaxel-induced pain. This study provides further in vivo evidence that mitochondrial dysfunction is a key contributor to the development and maintenance of chemotherapy-induced painful neuropathy. This work also indicates that selective modulation of the electron transport chain can induce antinociceptive effects in a preclinical model of paclitaxel-induced pain. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Discovery of Potent Broad Spectrum Antivirals Derived from Marine Actinobacteria

PubMed Central

Raveh, Avi; Delekta, Phillip C.; Dobry, Craig J.; Peng, Weiping; Schultz, Pamela J.; Blakely, Pennelope K.; Tai, Andrew W.; Matainaho, Teatulohi; Irani, David N.; Sherman, David H.; Miller, David J.

2013-01-01

Natural products provide a vast array of chemical structures to explore in the discovery of new medicines. Although secondary metabolites produced by microbes have been developed to treat a variety of diseases, including bacterial and fungal infections, to date there has been limited investigation of natural products with antiviral activity. In this report, we used a phenotypic cell-based replicon assay coupled with an iterative biochemical fractionation process to identify, purify, and characterize antiviral compounds produced by marine microbes. We isolated a compound from Streptomyces kaviengensis, a novel actinomycetes isolated from marine sediments obtained off the coast of New Ireland, Papua New Guinea, which we identified as antimycin A1a. This compound displays potent activity against western equine encephalitis virus in cultured cells with half-maximal inhibitory concentrations of less than 4 nM and a selectivity index of greater than 550. Our efforts also revealed that several antimycin A analogues display antiviral activity, and mechanism of action studies confirmed that these Streptomyces-derived secondary metabolites function by inhibiting the cellular mitochondrial electron transport chain, thereby suppressing de novo pyrimidine synthesis. Furthermore, we found that antimycin A functions as a broad spectrum agent with activity against a wide range of RNA viruses in cultured cells, including members of the Togaviridae, Flaviviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Finally, we demonstrate that antimycin A reduces central nervous system viral titers, improves clinical disease severity, and enhances survival in mice given a lethal challenge with western equine encephalitis virus. Our results provide conclusive validation for using natural product resources derived from marine microbes as source material for antiviral drug discovery, and they indicate that host mitochondrial electron transport is a viable target for the continued development of broadly active antiviral compounds. PMID:24349254

Liquid-chromatography thermospray mass-spectrometric study of n-acylamino dilactones and 4-butyrolactones derived from Antimycin-A

USGS Publications Warehouse

Abidi, S.L.; Ha, S.C.; Rosen, R.T.

1990-01-01

Reversed-phase high-performance liquid chromatography—thermospray mass spectrometric (HPLC—MS) characteristics of four sets of lactonic complexes (one 4-butyrolactones and three dilactone complexes) derived from antimycin A were investigated. Three types of 8-hydroxy analogues were also included in the study. Pairs of a–b structures isomeric at the 8-acyloxy ester side-chains were best separated with a high-efficiency octadecylsilica column prior to analysis by HPLC—MS. Mass spectra of the a–b pairs each with identical molecular weights exhibited virtually indistinguishable fragmentation patterns, although their relative intensities were not superimposable. In some cases, HPLC—MS of the title compounds yielded mass chromatograms showing the minor components more easily recognizable than the HPLC—UV counter parts because of the apparent higher ionization efficiency of the minor isomers and increased resolution of subcomponents in the MS system. Under the mobile phase conditions employed, analyte ionization occurred with variable degrees of gas phase ammonolysis depending upon the ammonia concentration of the buffer. Potential applicability of the on-line HPLC—MS technique for the characterization of components in mixtures of antimycin analogues and isomers is demonstrated.

Determination of Antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation

USGS Publications Warehouse

Bernardy, Jeffry A.; Hubert, Terrance D.; Ogorek, Jacob M.; Schmidt, Larry J.

2013-01-01

An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples ≥500 ng/L and 4.8 to 17% RSD with samples ≤100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.

Determination of antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation.

PubMed

Bernardy, Jeffry A; Hubert, Terrance D; Ogorek, Jacob M; Schmidt, Larry J

2013-01-01

An LC/MS method was developed and validated for the quantitative determination and confirmation of antimycin-A (ANT-A) in water from lakes or streams. Three different water sample volumes (25, 50, and 250 mL) were evaluated. ANT-A was stabilized in the field by immediately extracting it from water into anhydrous acetone using SPE. The stabilized concentrated samples were then transported to a laboratory and analyzed by LC/MS using negative electrospray ionization. The method was determined to have adequate accuracy (78 to 113% recovery), precision (0.77 to 7.5% RSD with samples > or = 500 ng/L and 4.8 to 17% RSD with samples < or = 100 ng/L), linearity, and robustness over an LOQ range from 8 to 51 600 ng/L.

Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes.

PubMed

Shao, Zuo-Hui; Becker, Lance B; Vanden Hoek, Terry L; Schumacker, Paul T; Li, Chang-Qing; Zhao, Danhong; Wojcik, Kim; Anderson, Travis; Qin, Yimin; Dey, Lucy; Yuan, Chun-Su

2003-06-01

This study sought to test whether grape seed proanthocyanidin extract (GSPE) attenuates exogenous and endogenous oxidant stress induced in chick cardiomyocytes and whether this cytoprotection is mediated by PKC activation, mito K(ATP) channel opening, NO production, oxidant scavenging, or iron chelating effects. Cells were exposed to hydrogen peroxide (H(2)O(2)) (exogenous oxidant stress, 0.5mM) or antimycin A (endogenous oxidant stress, 100 micro M) for 2h following pretreatment with GSPE at various concentrations for 2h. Cells were also pretreated with GSPE or with inhibitors of PKC (chelerytherine), mito K(ATP) channel (5-hydroxydecanoate), nitric oxide synthase (nitro-L-arginine methyl ester) for 2h. Oxidant stress was measured by 2',7'-dichlorofluorescin diacetate and cell viability was assessed using propidium iodide. Free radical scavenging and iron chelating ability was tested in vitro. GSPE dose-dependently attenuated oxidant formation and significantly improved cell survival and contractile function. However, inhibitors of PKC, mito K(ATP) channel or NO synthase failed to abolish the protective action of GSPE during H(2)O(2) or antimycin A exposure. In vitro studies suggested that GSPE scavenges H(2)O(2), hydroxyl radical and superoxide, and may chelate iron. These results indicate that GSPE confers cardioprotection against exogenous H(2)O(2)- or antimycin A-induced oxidant injury. Its effect does not require PKC, mito K(ATP) channel, or NO synthase, presumably because it acts by reactive oxygen species scavenging and iron chelating directly.

[Oxidative stress promotes hepatocyte apoptosis mediated by glycogen synthase kinase 3β].

PubMed

Zhang, Xiangying; Guo, Yuanyuan; Zhang, Li; Wen, Tao; Piao, Zhengfu; Shi, Hongbo; Chen, Dexi; Duan, Zhongping; Ren, Feng

2015-01-01

To analyze the role of glycogen synthase kinase 3β (GSK3β) in hepatocyte apoptosis induced by oxidative stress. Human HL-7702 hepatoma cells were induced by H₂O₂/antimycin A to establish oxidative stress-induced cell apoptosis models. SB216763, a specific inhibitor of GSK3β, was given to the cells two hours before H₂O₂/antimycin A induction. Cell survival was observed using calcein acetoxymethyl ester/propidium iodide (PI) double staining, and cell apoptosis was detected using annexin V-FITC/PI staining combined with flow cytometry. In the meanwhile, the cell culture supernatant was subjected to lactate dehydrogenase (LDH) assay to evaluate the extent of cell death. The expressions of p-GSK3β, GSK3β, caspase-3, cleaved caspase-3, c-Jun N-terminal kinase (JNK) and cytochrome C (CytC) proteins were examined using Western blotting. Oxidative stress triggered by H₂O₂/antimycin A promoted GSK3β activity; inhibition of GSK3β activity by SB216763 relieved oxidative stress and reduced cell apoptosis induced by oxidative stress. Compared with the model groups, SB216763 intervened group showed that the cell apoptosis rate and the level of LDH were reduced significantly, and that the expressions of cleaved caspase-3, JNK, CytC proteins decreased. GSK3β is an important signaling molecule in the apoptosis pathway induced by oxidative stress. The inhibition on GSK3β may alleviate the oxidative stress-induced hepatocyte apoptosis.

Environmental conditions affecting the efficiency and efficacy of piscicides for use in nonnative fish eradication

USGS Publications Warehouse

Brown, Peter James

2010-01-01

Conservation of native fish is a pressing issue for fisheries managers. Conservation efforts often require eliminating threats posed by nonnative fish by eradicating them with piscicides. The piscicides rotenone and antimycin are used for eradication but their application is often inefficient or ineffective. My goal was to increase the efficiency and efficacy of nonnative fish eradication using piscicides. I identified environmental conditions affecting piscicide application, researched methods to overcome these problems, and provided tools that piscicide applicators can use to make piscicide application more efficient and effective. Rotenone and antimycin were exposed to varying levels of sunlight, turbulence, and dissolved organic matter (DOM) to determine the effect these environmental conditions have on piscicides. Bioassay fish were used to determine the toxicity of the piscicides. Sunlight and turbulence affected rotenone and antimycin but DOM did not. Increasing the concentration of chemical can increase the resistance to the effects of these environmental conditions; however, the effects of these conditions are considerable in natural settings. Observations of bioassay fish in stream applications of rotenone were used to develop a statistical model to predict the persistence of the piscicide. The model can be used to predict rotenone persistence in small montane streams and to estimate where rotenone concentrations need to be fortified. I measured the mixing rate of a chemical plume in different channel morphologies and at center or edge applications. Center application had a significantly shorter mixing distance than edge application, but mixing distance was not different among meandering, straight, and riffle/pool morphologies. Application of my findings will increase the efficiency and efficacy of native fish conservation using piscicides.

Measurements of bovine sperm velocities under true anaerobic and aerobic conditions.

PubMed

Krzyzosiak, J; Molan, P; Vishwanath, R

1999-04-30

Velocities of bovine spermatozoa in a medium containing glucose were similar under true anaerobic and aerobic conditions. Spermatozoa were not able to sustain motility under anaerobic conditions when glycolysis was inhibited, but regained motility when re-aerated. This demonstrates that immobilisation was due to lack of oxygen and that conditions under which motility was analysed were truly anaerobic. Sperm motility parameters were not significantly different in the presence and absence of 4 microM antimycin A and 4 microM rotenone when glucose was present in the medium. After each incubation, functionality of sperm mitochondria was assayed by washing sperm into the medium which supported respiration but not glycolysis, and motility was visually assessed. All sperm samples were highly motile in this medium indicating that their mitochondria were functional. When glycolysis was inhibited, antimycin and rotenone abolished sperm motility immediately after addition. Bovine sperm can maintain similar levels of motility aerobically and anaerobically if a glycolysable substrate is available. Available data on bovine sperm energetics support this view.

Effect of the oral administration homeopathic Arnica montana on mitochondrial oxidative stress.

PubMed

de Camargo, Ronaldo Antônio; da Costa, Ellen Dias; Catisti, Rosana

2013-01-01

To analyze the effect of homeopathic Arnica on mitochondrial oxidative stress induced by Ca(2+) plus inorganic phosphate and/or Fe(2+)-citrate-mediated lipid peroxidation through changes in oxygen consumption rates. Mitochondria were isolated by differential centrifugation from the livers of adult male Wistar rats which had been treated with Arnica montana 6cH, 12cH, 30cH or succussed 30% ethanol (control) for 21 days. In the presence of antimycin-A, electron transport chain inhibitor, as evidenced by antimycin-A insensitive O(2) consumption, Arnica inhibited lipid peroxidation of mitochondrial membranes. In oxidative stress conditions, in the presence of Ca(2+) and inorganic phosphate, animals receiving Arnica 30cH had a significant decrease in mitochondrial O(2) consumption compared to control animals. When administrated orally, Arnica 30cH protects against hepatic mitochondrial membrane permeabilization induced by Ca(2+) and/or Fe(2+)-citrate-mediated lipid peroxidation and fragmentation of proteins due to the attack by reactive oxygen species. Copyright © 2012 The Faculty of Homeopathy. Published by Elsevier Ltd. All rights reserved.

[Respiration of wheat root cells under simultaneous inhibition of parts I and III of the electron transport chain of mitochondria by rotenone and antimycine A].

PubMed

Rakhmatullina, D F; Gordon, L Kh; Ogorodnikova, T I

2005-01-01

Respiration of excised roots of 5 day old wheat seedlings with blocked mitochondrial oxidation under simultaneous action of rotenone and antimycine A was studied. A reduced rate of oxygen uptake was observed within the first hour of root treatment inhibitors. However, after a 5 h exposure there was an increase in oxygen uptake, which was prevented by KCN but amplified by malate and ascorbate. The application of inhibitors caused a considerable increase in the respiratory coefficient (RC) up to 2.1, that suggests a significant CO2 release, when the initial sites of mitochondrial electron transport chain were inhibited. RC did not raise, when ascorbate was added in the presence of inhibitors. We assume that inhibition of mitochondrial oxidation at I and III sites of electron transport chain facilitates switching on the alternative paths of reductant translocation to oxygen. Participation of ATPases and redox system of plasma membrane in the response reactions of respiration directed to the restoration of ion, particularly, proton homeostasis in conditions of inhibited mitochondrial oxidation is discussed.

Anti-Candida Properties of Urauchimycins from Actinobacteria Associated with Trachymyrmex Ants

PubMed Central

Mendes, Thais D.; Borges, Warley S.; Solomon, Scott E.; Vieira, Paulo C.; Duarte, Marta C. T.; Pagnocca, Fernando C.

2013-01-01

After decades of intensive searching for antimicrobial compounds derived from actinobacteria, the frequency of isolation of new molecules has decreased. To cope with this concern, studies have focused on the exploitation of actinobacteria from unexplored environments and actinobacteria symbionts of plants and animals. In this study, twenty-four actinobacteria strains isolated from workers of Trachymyrmex ants were evaluated for antifungal activity towards a variety of Candida species. Results revealed that seven strains inhibited the tested Candida species. Streptomyces sp. TD025 presented potent and broad spectrum of inhibition of Candida and was selected for the isolation of bioactive molecules. From liquid shake culture of this bacterium, we isolated the rare antimycin urauchimycins A and B. For the first time, these molecules were evaluated for antifungal activity against medically important Candida species. Both antimycins showed antifungal activity, especially urauchimycin B. This compound inhibited the growth of all Candida species tested, with minimum inhibitory concentration values equivalent to the antifungal nystatin. Our results concur with the predictions that the attine ant-microbe symbiosis may be a source of bioactive metabolites for biotechnology and medical applications. PMID:23586060

The Vacuolar-Type H+-ATPase in Ovine Rumen Epithelium is Regulated by Metabolic Signals

PubMed Central

Kuzinski, Judith; Zitnan, Rudolf; Warnke-Gurgel, Christina; Schweigel, Monika

2010-01-01

In this study, the effect of metabolic inhibition (MI) by glucose substitution with 2-deoxyglucose (2-DOG) and/or application of antimycin A on ovine rumen epithelial cells (REC) vacuolar-type H+-ATPase (vH+-ATPase) activity was investigated. Using fluorescent spectroscopy, basal pHi of REC was measured to be 7.3 ± 0.1 in HCO3−-free, glucose-containing NaCl medium. MI induced a strong pHi reduction (−0.44 ± 0.04 pH units) with a more pronounced effect of 2-DOG compared to antimycin A (−0.30 ± 0.03 versus −0.21 ± 0.03 pH units). Treatment with foliomycin, a specific vH+-ATPase inhibitor, decreased REC pHi by 0.21 ± 0.05 pH units. After MI induction, this effect was nearly abolished (−0.03 ± 0.02 pH units). In addition, membrane-associated localization of vH+-ATPase B subunit disappeared. Metabolic control of vH+-ATPase involving regulation of its assembly state by elements of the glycolytic pathway could provide a means to adapt REC ATP consumption according to energy availability. PMID:20069127

Genome Sequence of Streptomyces wadayamensis Strain A23, an Endophytic Actinobacterium from Citrus reticulata

PubMed Central

Tormet Gonzalez, Gabriela D.; Samborsky, Markyian; Marcon, Joelma; Araujo, Welington L.; de Azevedo, João Lucio

2014-01-01

The actinobacterium Streptomyces wadayamensis A23 is an endophyte of Citrus reticulata that produces the antimycin and mannopeptimycin antibiotics, among others. The strain has the capability to inhibit Xylella fastidiosa growth. The draft genome of S. wadayamensis A23 has ~7.0 Mb and 6,006 protein-coding sequences, with a 73.5% G+C content. PMID:24994795

Superoxide produced in the matrix of mitochondria enhances methylmercury toxicity in human neuroblastoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mailloux, Ryan J.; Yumvihoze, Emmanuel; Chan, Hing Man, E-mail: laurie.chan@uottawa.ca

2015-12-15

The mechanism of intracellular metabolism of methylmercury (MeHg) is not fully known. It has been shown that superoxide (O{sub 2}·{sup −}), the proximal reactive oxygen species (ROS) generated by mitochondria, is responsible for MeHg demethylation. Here, we investigated the impact of different mitochondrial respiratory inhibitors, namely rotenone and antimycin A, on the O{sub 2}·{sup −} mediated degradation of MeHg in human neuroblastoma cells SH-K-SN. We also utilized paraquat (PQ) which generates O{sub 2}·{sup −} in the mitochondrial matrix. We found that the cleavage of the carbon-metal bond in MeHg was highly dependent on the topology of O{sub 2}·{sup −} productionmore » by mitochondria. Both rotenone and PQ, which increase O{sub 2}·{sup −} in the mitochondrial matrix at a dose-dependent manner, enhanced the conversion of MeHg to inorganic mercury (iHg). Surprisingly, antimycin A, which prompts emission of O{sub 2}·{sup −} into the intermembrane space, did not have the same effect even though antimycin A induced a dose dependent increase in O{sub 2}·{sup −} emission. Rotenone and PQ also enhanced the toxicity of sub-toxic doses (0.1 μM) MeHg which correlated with the accumulation of iHg in mitochondria and depletion of mitochondrial protein thiols. Taken together, our results demonstrate that MeHg degradation is mediated by mitochondrial O{sub 2}·{sup −}, specifically within the matrix of mitochondria when O{sub 2}·{sup −} is in adequate supply. Our results also show that O{sub 2}·{sup −} amplifies MeHg toxicity specifically through its conversion to iHg and subsequent interaction with protein cysteine thiols (R-SH). The implications of our findings in mercury neurotoxicity are discussed herein. - Highlights: • Superoxide produced in the matrix of mitochondria degrades MeHg. • Superoxide produced in intermembrane space does not degrade MeHg. • Matrix-generated superoxide enhances Hg toxicity by converting MeHg to iHg.« less

Localization of superoxide anion production to mitochondrial electron transport chain in 3-NPA-treated cells

PubMed Central

Bacsi, Attila; Woodberry, Mitchell; Widger, William; Papaconstantinou, John; Mitra, Sankar; Peterson, Johnny W.; Boldogh, Istvan

2011-01-01

3-Nitropropionic acid (3-NPA), an inhibitor of succinate dehydrogenase (SDH) at complex II of the mitochondrial electron transport chain induces cellular energy deficit and oxidative stress-related neurotoxicity. In the present study, we identified the site of reactive oxygen species production in mitochondria. 3-NPA increased O2•− generation in mitochondria respiring on the complex I substrates pyruvate + malate, an effect fully inhibited by rotenone. Antimycin A increased O2•− production in the presence of complex I and/or II substrates. Addition of 3-NPA markedly increased antimycin A-induced O2•− production by mitochondria incubated with complex I substrates, but 3-NPA inhibited O2•− formation driven with the complex II substrate succinate. At 0.6 μM, myxothiazol inhibits complex III, but only partially decreases complex I activity, and allowed 3-NPA-induced O2•− formation; however, at 40 μM myxothiazol (which completely inhibits both complexes I and III) eliminated O2•− production from mitochondria respiring via complex I substrates. These results indicate that in the presence of 3-NPA, mitochondria generate O2•− from a site between the ubiquinol pool and the 3-NPA block in the respiratory complex II. PMID:17011837

Differential effects of buffer pH on Ca2+-induced ROS emission with inhibited mitochondrial complexes I and III

PubMed Central

Lindsay, Daniel P.; Camara, Amadou K. S.; Stowe, David F.; Lubbe, Ryan; Aldakkak, Mohammed

2015-01-01

Excessive mitochondrial reactive oxygen species (ROS) emission is a critical component in the etiology of ischemic injury. Complex I and complex III of the electron transport chain are considered the primary sources of ROS emission during cardiac ischemia and reperfusion (IR) injury. Several factors modulate ischemic ROS emission, such as an increase in extra-matrix Ca2+, a decrease in extra-matrix pH, and a change in substrate utilization. Here we examined the combined effects of these factors on ROS emission from respiratory complexes I and III under conditions of simulated IR injury. Guinea pig heart mitochondria were suspended in experimental buffer at a given pH and incubated with or without CaCl2. Mitochondria were then treated with either pyruvate, a complex I substrate, followed by rotenone, a complex I inhibitor, or succinate, a complex II substrate, followed by antimycin A, a complex III inhibitor. H2O2 release rate and matrix volume were compared with and without adding CaCl2 and at pH 7.15, 6.9, or 6.5 with pyruvate + rotenone or succinate + antimycin A to simulate conditions that may occur during in vivo cardiac IR injury. We found a large increase in H2O2 release with high [CaCl2] and pyruvate + rotenone at pH 6.9, but not at pHs 7.15 or 6.5. Large increases in H2O2 release rate also occurred at each pH with high [CaCl2] and succinate + antimycin A, with the highest levels observed at pH 7.15. The increases in H2O2 release were associated with significant mitochondrial swelling, and both H2O2 release and swelling were abolished by cyclosporine A, a desensitizer of the mitochondrial permeability transition pore (mPTP). These results indicate that ROS production by complex I and by complex III is differently affected by buffer pH and Ca2+ loading with mPTP opening. The study suggests that changes in the levels of cytosolic Ca2+ and pH during IR alter the relative amounts of ROS produced at mitochondrial respiratory complex I and complex III. PMID:25805998

Redox signaling in the growth and development of colonial hydroids.

PubMed

Blackstone, Neil W

2003-02-01

Redox signaling provides a quick and efficient mechanism for clonal or colonial organisms to adapt their growth and development to aspects of the environment, e.g. the food supply. A 'signature' of mitochondrial redox signaling, particularly as mediated by reactive oxygen species (ROS), can be elucidated by experimental manipulation of the electron transport chain. The major sites of ROS formation are found at NADH dehydrogenase of complex I and at the interface between coenzyme Q and complex III. Inhibitors of complex III should thus upregulate ROS from both sites; inhibitors of complex I should upregulate ROS from the first but not the second site, while uncouplers of oxidative phosphorylation should downregulate ROS from both sites. To investigate the possibility of such redox signaling, perturbations of colony growth and development were carried out using the hydroid Podocoryna carnea. Oxygen uptake of colonies was measured to determine comparable physiological doses of antimycin A(1) (an inhibitor of complex III), rotenone (an inhibitor of complex I) and carbonyl cyanide m-chlorophenylhydrazone (CCCP; an uncoupler of oxidative phosphorylation). Using these doses, clear effects on colony growth and development were obtained. Treatment with antimycin A(1) results in 'runner-like' colony growth, with widely spaced polyps and stolon branches, while treatment with CCCP results in 'sheet-like' growth, with closely spaced polyps and stolon branches. Parallel results have been obtained previously with azide, an inhibitor of complex IV, and dinitrophenol, another uncoupler of oxidative phosphorylation. Perhaps surprisingly, rotenone produced effects on colony development similar to those of CCCP. Assays of peroxides using 2',7'-dichlorofluorescin diacetate and fluorescent microscopy suggest a moderate difference in ROS formation between the antimycin and rotenone treatments. The second site of ROS formation (the interface between coenzyme Q and complex III) may thus predominate in the signaling that regulates colony development. The fat-rich, brine shrimp diet of these hydroids may be relevant in this context. Acyl CoA dehydrogenase, which catalyzes the first step in the mitochondrial beta-oxidation of fatty acids, carries electrons to coenzyme Q, thus bypassing complex I. These results support a role for redox signaling, mediated by ROS, in colony development. Nevertheless, other redox sensors between complexes I and III may yet be found.

Effectiveness of piscicides for controlling round gobies (Neogobius melanostomus)

USGS Publications Warehouse

Schreier, Theresa M.; Dawson, V.K.; Larson, W.

2008-01-01

Round gobies (Neogobius melanostomus) were introduced to the Great Lakes presumably as a result of ballast water releases from seagoing freighters returning from European water bodies. These unwelcome fish have become established in the Great Lakes region and are expanding their range to suitable portions of other interior drainage basins including the Mississippi River traversing the central United States and the Trent-Severn waterway spanning south-central Ontario. If the invasion continues, use of chemical toxicants as a control measure may be necessary. Toxicity tests of the currently registered piscicides antimycin, rotenone, 3-trifluoromethyl-4-nitrophenol (TFM), and Bayluscide?? were conducted with three fish species native to the Great Lakes and round gobies collected from the Illinois Waterway. Tests indicated that round gobies are sensitive to all of the piscicides, however, the level of sensitivity is similar to that of the native fish species tested. Therefore, currently registered piscicides have limited potential to selectively remove round gobies. Bottom-release formulations of Bayluscide?? and antimycin were also evaluated as control agents for the normally bottom-dwelling round goby. Avoidance behavior tests demonstrated that the round goby did not react to the presence of either chemical. Therefore, the bottom-release formulations may have some application for the selective removal of round gobies, and may be one of the few tools presently available to fishery managers to help limit the range expansion of this invasive fish.

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement.

PubMed

Pallotta, Maria Luigia; Valenti, Daniela; Iacovino, Michelina; Passarella, Salvatore

2004-02-15

In this work we looked at whether and how mitochondria isolated from Saccharomyces cerevisiae (SCM) oxidize d-lactate. We found that: (1). externally added d-lactate causes oxygen uptake by SCM with P/O ratio equal to 1.5; in the presence of antimycin A (AA), P/O ratio was 1.8, differently in the presence of the non-penetrant alpha-cyanocinnamate (alpha-CCN-) no P/O ratio could be measured. Consistently, mitochondrial electrical membrane potential (deltapsi) generation was found, due to externally added d-lactate in the presence of antimycin A, but not of alpha-CCN-. (2). SCM oxidize d-lactate in two different manners: (i). via inner membrane d-lactate dehydrogenase which leads to d-lactate oxidation without driving deltapsi generation and ATP synthesis and (ii). via the matrix d-lactate dehydrogenase, which drives deltapsi generation and ATP synthesis by using taken up d-lactate. (3). Pyruvate newly synthesised in the mitochondrial matrix is exported via the novel d-lactate/pyruvate antiporter. d-Lactate/pyruvate antiport proved to regulate the rate of pyruvate efflux in vitro. (4). The existence of the d-lactate/H+ symporter is also proposed as shown by mitochondrial swelling. The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.

Substance P receptor desensitization requires receptor activation but not phospholipase C

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sugiya, Hiroshi; Putney, J.W. Jr.

1988-08-01

Previous studies have shown that exposure of parotid acinar cells to substance P at 37{degree}C results in activation of phospholipase C, formation of ({sup 3}H)inositol 1,4,5-trisphosphate (IP{sub 3}), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to {approximately}20% of control values, IP{sub 3} formation was completely inhibited, but desensitization was unaffected. When cells were treated with antimycin in the absence of glucose, cellular ATP was decreased to {approximately}5% of control values, and both IP{sub 3} formation and desensitization were blocked. A series of substance P-related peptides increased themore » formation of ({sup 3}H)IP{sub 3} and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, (D-Pro{sup 2}, D-Try{sup 7,9})-substance P, inhibited substance P-induced IP{sub 3} formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP{sub 3} formation requires agonist activation of a P-type substance P receptor, and that one or more cellular ATP-dependent processes are required for this reaction. However, activation of phospholipase C and the generation of inositol phosphates does not seem to be a prerequisite for desensitization.« less

Antimycin A inhibits cytochrome b559-mediated cyclic electron flow within photosystem II.

PubMed

Takagi, Daisuke; Ifuku, Kentaro; Nishimura, Taishi; Miyake, Chikahiro

2018-05-22

The light reactions of photosynthesis are known to comprise both linear and cyclic electron flow in order to convert light energy into chemical energy in the form of NADPH and ATP. Antimycin A (AA) has been proposed as an inhibitor of ferredoxin-dependent cyclic electron flow around photosystem I (CEF-PSI) in photosynthesis research. However, its precise inhibitory mechanism and target site had not been elucidated yet. Here we show that AA inhibits the cyclic (alternative) electron flow via cytochrome b 559 (Cyt b 559 ) within photosystem II (CEF-PSII). When AA was applied to thylakoid membranes isolated from spinach leaves, the high potential form of Cyt b 559 , which was reduced in the dark, was transformed into the lower potential forms and readily oxidized by molecular oxygen. In the absence of AA, the reduced Cyt b 559 was oxidized by P680 + upon light illumination and re-reduced in the dark, mainly by the electron from the Q B site on the acceptor side of PSII. In contrast, AA suppressed the oxidation of Cyt b 559 and induced its reduction under the illumination. This inhibition of Cyt b 559 oxidation by AA enhanced photoinhibition of PSII. Based on the above results, we propose caution regarding the use of AA for evaluating CEF-PSI per se and concurrently propose that AA provides for new insights into, and interpretations of, the physiological importance of Cyt b 559 , rather than that of CEF-PSI in photosynthetic organisms.

Assessing the efficacy of corn-based bait containing antimycin-a to control common carp populations using laboratory and pond experiments

USGS Publications Warehouse

Poole, Joshua R.; Sauey, Blake W.; Amberg, Jon J.; Bajer, Przemyslaw G.

2018-01-01

Strategic use of oral toxicants could allow for practical and sustainable control schemes for the invasive common carp (Cyprinus carpio, or ‘carp’) if a toxicant selectively targeted carp and not native species. In this study, we incorporated antimycin-a (ANT-A), a known fish toxicant, into a corn-based bait and conducted a series of experiments to determine its toxicity, leaching rate, and species-specificity. Our results showed that ANT-A was lethal to carp at doses ≥ 4 mg/kg and that the amount of ANT-A that leached out of the bait in 72 h was not lethal to carp or bluegill (Lepomis macrochirus). Species-specificity trials were conducted in 227 L tanks, in which carp were stocked with three native species representing families that occur sympatrically with carp in our study region: the fathead minnow (Pimephales promelas), yellow perch (Perca flavescens) and bluegill. These trials showed high mortality of carp (46%) and fathead minnows (76%) but no significant mortality of perch or bluegill. Finally, a pond study, which used the same species composition except for fathead minnows, resulted in 37% morality among adult carp and no mortality among perch or bluegill. Our results suggest that corn-based bait that contains ANT-A could be used to selectively control carp in ecosystems dominated by percids or centrarchids, such as lakes across the Great Plains ecoregion of North America, where carp are especially problematic.

Characteristics of alpha-glycerophosphate-evoked H2O2 generation in brain mitochondria.

PubMed

Tretter, Laszlo; Takacs, Katalin; Hegedus, Vera; Adam-Vizi, Vera

2007-02-01

Characteristics of reactive oxygen species (ROS) production in isolated guinea-pig brain mitochondria respiring on alpha-glycerophosphate (alpha-GP) were investigated and compared with those supported by succinate. Mitochondria established a membrane potential (DeltaPsi(m)) and released H(2)O(2) in parallel with an increase in NAD(P)H fluorescence in the presence of alpha-GP (5-40 mm). H(2)O(2) formation and the increase in NAD(P)H level were inhibited by rotenone, ADP or FCCP, respectively, being consistent with a reverse electron transfer (RET). The residual H(2)O(2) formation in the presence of FCCP was stimulated by myxothiazol in mitochondria supported by alpha-GP, but not by succinate. ROS under these conditions are most likely to be derived from alpha-GP-dehydrogenase. In addition, huge ROS formation could be provoked by antimycin in alpha-GP-supported mitochondria, which was prevented by myxothiazol, pointing to the generation of ROS at the quinol-oxidizing center (Q(o)) site of complex III. FCCP further stimulated the production of ROS to the highest rate that we observed in this study. We suggest that the metabolism of alpha-GP leads to ROS generation primarily by complex I in RET, and in addition a significant ROS formation could be ascribed to alpha-GP-dehydrogenase in mammalian brain mitochondria. ROS generation by alpha-GP at complex III is evident only when this complex is inhibited by antimycin.

A Single Sfp-Type Phosphopantetheinyl Transferase Plays a Major Role in the Biosynthesis of PKS and NRPS Derived Metabolites in Streptomyces ambofaciens ATCC23877

PubMed Central

Bunet, Robert; Riclea, Ramona; Laureti, Luisa; Hôtel, Laurence; Paris, Cédric; Girardet, Jean-Michel; Spiteller, Dieter; Dickschat, Jeroen S.; Leblond, Pierre; Aigle, Bertrand

2014-01-01

The phosphopantetheinyl transferases (PPTases) are responsible for the activation of the carrier protein domains of the polyketide synthases (PKS), non ribosomal peptide synthases (NRPS) and fatty acid synthases (FAS). The analysis of the Streptomyces ambofaciens ATCC23877 genome has revealed the presence of four putative PPTase encoding genes. One of these genes appears to be essential and is likely involved in fatty acid biosynthesis. Two other PPTase genes, samT0172 (alpN) and samL0372, are located within a type II PKS gene cluster responsible for the kinamycin production and an hybrid NRPS-PKS cluster involved in antimycin production, respectively, and their products were shown to be specifically involved in the biosynthesis of these secondary metabolites. Surprisingly, the fourth PPTase gene, which is not located within a secondary metabolite gene cluster, appears to play a pleiotropic role. Its product is likely involved in the activation of the acyl- and peptidyl-carrier protein domains within all the other PKS and NRPS complexes encoded by S. ambofaciens. Indeed, the deletion of this gene affects the production of the spiramycin and stambomycin macrolide antibiotics and of the grey spore pigment, all three being PKS-derived metabolites, as well as the production of the nonribosomally produced compounds, the hydroxamate siderophore coelichelin and the pyrrolamide antibiotic congocidine. In addition, this PPTase seems to act in concert with the product of samL0372 to activate the ACP and/or PCP domains of the antimycin biosynthesis cluster which is also responsible for the production of volatile lactones. PMID:24498152

A Membrane-Bound NAC Transcription Factor, ANAC017, Mediates Mitochondrial Retrograde Signaling in Arabidopsis[W][OPEN

PubMed Central

Ng, Sophia; Ivanova, Aneta; Duncan, Owen; Law, Simon R.; Van Aken, Olivier; De Clercq, Inge; Wang, Yan; Carrie, Chris; Xu, Lin; Kmiec, Beata; Walker, Hayden; Van Breusegem, Frank; Whelan, James; Giraud, Estelle

2013-01-01

Plants require daily coordinated regulation of energy metabolism for optimal growth and survival and therefore need to integrate cellular responses with both mitochondrial and plastid retrograde signaling. Using a forward genetic screen to characterize regulators of alternative oxidase1a (rao) mutants, we identified RAO2/Arabidopsis NAC domain-containing protein17 (ANAC017) as a direct positive regulator of AOX1a. RAO2/ANAC017 is targeted to connections and junctions in the endoplasmic reticulum (ER) and F-actin via a C-terminal transmembrane (TM) domain. A consensus rhomboid protease cleavage site is present in ANAC017 just prior to the predicted TM domain. Furthermore, addition of the rhomboid protease inhibitor N-p-Tosyl-l-Phe chloromethyl abolishes the induction of AOX1a upon antimycin A treatment. Simultaneous fluorescent tagging of ANAC017 with N-terminal red fluorescent protein (RFP) and C-terminal green fluorescent protein (GFP) revealed that the N-terminal RFP domain migrated into the nucleus, while the C-terminal GFP tag remained in the ER. Genome-wide analysis of the transcriptional network regulated by RAO2/ANAC017 under stress treatment revealed that RAO2/ANAC017 function was necessary for >85% of the changes observed as a primary response to cytosolic hydrogen peroxide (H2O2), but only ∼33% of transcriptional changes observed in response to antimycin A treatment. Plants with mutated rao2/anac017 were more stress sensitive, whereas a gain-of-function mutation resulted in plants that had lower cellular levels of H2O2 under untreated conditions. PMID:24045017

A Functional Antagonistic Relationship between Auxin and Mitochondrial Retrograde Signaling Regulates Alternative Oxidase1a Expression in Arabidopsis1[W][OPEN

PubMed Central

Ivanova, Aneta; Law, Simon R.; Narsai, Reena; Duncan, Owen; Lee, Jae-Hoon; Zhang, Botao; Van Aken, Olivier; Radomiljac, Jordan D.; van der Merwe, Margaretha; Yi, KeKe; Whelan, James

2014-01-01

The perception and integration of stress stimuli with that of mitochondrion function are important during periods of perturbed cellular homeostasis. In a continuous effort to delineate these mitochondrial/stress-interacting networks, forward genetic screens using the mitochondrial stress response marker alternative oxidase 1a (AOX1a) provide a useful molecular tool to identify and characterize regulators of mitochondrial stress signaling (referred to as regulators of alternative oxidase 1a [RAOs] components). In this study, we reveal that mutations in genes coding for proteins associated with auxin transport and distribution resulted in a greater induction of AOX1a in terms of magnitude and longevity. Three independent mutants for polarized auxin transport, rao3/big, rao4/pin-formed1, and rao5/multidrug-resistance1/abcb19, as well as the Myb transcription factor rao6/asymmetric leaves1 (that displays altered auxin patterns) were identified and resulted in an acute sensitivity toward mitochondrial dysfunction. Induction of the AOX1a reporter system could be inhibited by the application of auxin analogs or reciprocally potentiated by blocking auxin transport. Promoter activation studies with AOX1a::GUS and DR5::GUS lines further confirmed a clear antagonistic relationship between the spatial distribution of mitochondrial stress and auxin response kinetics, respectively. Genome-wide transcriptome analyses revealed that mitochondrial stress stimuli, such as antimycin A, caused a transient suppression of auxin signaling and conversely, that auxin treatment repressed a part of the response to antimycin A treatment, including AOX1a induction. We conclude that mitochondrial stress signaling and auxin signaling are reciprocally regulated, balancing growth and stress response(s). PMID:24820025

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

PubMed Central

2011-01-01

Background Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1. Results Antimycin A (an alternative respiratory pathway inducer and also an inducer of resistance to viruses) and SA triggered resistance to tobacco mosaic virus (TMV). Resistance to TMV induced by antimycin A, but not by SA, was inhibited in Aox transgenic plants while SA-induced resistance to this virus appeared to be stronger in Aox-E transgenic plants. These effects, which were limited to directly inoculated leaves, were not affected by the presence or absence of a transgene constitutively expressing a functional RNA-dependent RNA polymerase (MtRDR1). Unexpectedly, Aox-transgenic plants infected with potato virus X (PVX) showed markedly increased susceptibility to systemic disease induction and virus accumulation in inoculated and systemically infected leaves. SA-induced resistance to PVX was compromised in Aox-transgenic plants but plants expressing AOX-E exhibited enhanced SA-induced resistance to this virus. Conclusions We conclude that AOX-regulated mechanisms not only play a role in SA-induced resistance but also make an important contribution to basal resistance against certain viruses such as PVX. PMID:21356081

The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes.

PubMed

Milner, P I; Wilkins, R J; Gibson, J S

2007-07-01

To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.

Segregation and transmission of mitochondrial markers in fusion products of the asporogenous yeast Torulopsis glabrata.

PubMed

Sriprakash, K S; Batum, C

1981-09-01

Using a protoplast fusion technique we have been able to locate to the mitochondrial genome of the asporogenous yeast Torulopsis glabrata mutations conferring resistance to oligomycin, antimycin and diuron. When two strains differing in the size of their mtDNAs were fused the mitochondrial markers from the parent with the larger mtDNA (71-91) were transmitted predominantly among the fusion products. Both genetical and physical evidence support the occurrence of recombination in T. glabrata mitochondrial genome. Segregation of the mitochondrial genome appears to take place before the separation of the first bud from the fusion product.

Uncovering production of specialized metabolites by Streptomyces argillaceus: Activation of cryptic biosynthesis gene clusters using nutritional and genetic approaches.

PubMed

Becerril, Adriana; Álvarez, Susana; Braña, Alfredo F; Rico, Sergio; Díaz, Margarita; Santamaría, Ramón I; Salas, José A; Méndez, Carmen

2018-01-01

Sequencing of Streptomyces genomes has revealed they harbor a high number of biosynthesis gene cluster (BGC), which uncovered their enormous potentiality to encode specialized metabolites. However, these metabolites are not usually produced under standard laboratory conditions. In this manuscript we report the activation of BGCs for antimycins, carotenoids, germicidins and desferrioxamine compounds in Streptomyces argillaceus, and the identification of the encoded compounds. This was achieved by following different strategies, including changing the growth conditions, heterologous expression of the cluster and inactivating the adpAa or overexpressing the abrC3 global regulatory genes. In addition, three new carotenoid compounds have been identified.

Overexpression of Endogenous Anti-Oxidants with Selenium Supplementation Protects Trophoblast Cells from Reactive Oxygen Species-Induced Apoptosis in a Bcl-2-Dependent Manner.

PubMed

Khera, Alisha; Vanderlelie, Jessica J; Holland, Olivia; Perkins, Anthony V

2017-06-01

The human placenta provides life support for the developing foetus, and a healthy placenta is a prerequisite to a healthy start to life. Placental tissue is subject to oxidative stress which can lead to pathological conditions of pregnancy such as preeclampsia, preterm labour and intrauterine growth restriction. Up-regulation of endogenous anti-oxidants may alleviate placental oxidative stress and provide a therapy for these complications of pregnancy. In this study, selenium supplementation, as inorganic sodium selenite (NaSel) or organic selenomethionine (SeMet), was used to increase the protein production and cellular activity of the important redox active proteins glutathione peroxidase (GPx) and thioredoxin reductase (Thx-Red). Placental trophoblast cell lines, BeWo, JEG-3 and Swan-71, were cultured in various concentrations of NaSel or SeMet for 24 h and cell extracts prepared for western blots and enzyme assays. Rotenone and antimycin were used to stimulate mitochondrial reactive oxygen species (ROS) production and induce apoptosis. Trophoblast cells supplemented with 100 nM NaSel and 500 nM SeMet exhibited significantly enhanced expression and activity of both GPx and Thx-Red. Antimycin and rotenone were found to generate ROS when measured by 2',7'-dichlorofluorescein diacetate (DCFDA) assay, and selenium supplementation was shown to reduce ROS production in a dose-dependent manner. Rotenone, 100 μM treatment for 4 h, caused trophoblast cell apoptosis as evidenced by increased Annexin V binding and decreased expression of Bcl-2. In both assays of apoptosis, selenium supplementation was able to prevent apoptosis, preserve Bcl-2 expression and protect trophoblast cells from mitochondrial oxidative stress. This data suggests that selenoproteins such as GPx and Thx-Red have an important role in protecting trophoblast cells from mitochondrial oxidative stress and that selenium supplementation may be important in treating some placental pathologies.

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility

PubMed Central

Plaza Davila, María; Martin Muñoz, Patricia; Tapia, Jose A.; Ortega Ferrusola, Cristina; Balao da Silva C, Carolina; Peña, Fernando J.

2015-01-01

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37°C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence. PMID:26407142

Redox modulation of thimet oligopeptidase activity by hydrogen peroxide.

PubMed

Icimoto, Marcelo Y; Ferreira, Juliana C; Yokomizo, César H; Bim, Larissa V; Marem, Alyne; Gilio, Joyce M; Oliveira, Vitor; Nantes, Iseli L

2017-07-01

Thimet oligopeptidase (EC 3.4.24.15, TOP) is a cytosolic mammalian zinc protease that can process a diversity of bioactive peptides. TOP has been pointed out as one of the main postproteasomal enzymes that process peptide antigens in the MHC class I presentation route. In the present study, we describe a fine regulation of TOP activity by hydrogen peroxide (H 2 O 2 ). Cells from a human embryonic kidney cell line (HEK293) underwent an ischemia/reoxygenation-like condition known to increase H 2 O 2 production. Immediately after reoxygenation, HEK293 cells exhibited a 32% increase in TOP activity, but no TOP activity was observed 2 h after reoxygenation. In another model, recombinant rat TOP (rTOP) was challenged by H 2 O 2 produced by rat liver mitoplasts (RLMt) alone, and in combination with antimycin A, succinate, and antimycin A plus succinate. In these conditions, rTOP activity increased 17, 30, 32 and 38%, respectively. Determination of H 2 O 2 concentration generated in reoxygenated cells and mitoplasts suggested a possible modulation of rTOP activity dependent on the concentration of H 2 O 2 . The measure of pure rTOP activity as a function of H 2 O 2 concentration corroborated this hypothesis. The data fitted to an asymmetrical bell-shaped curve in which the optimal activating H 2 O 2 concentration was 1.2 nM, and the maximal inhibition (75% about the control) was 1 μm. Contrary to the oxidation produced by aging associated with enzyme oligomerization and inhibition, H 2 O 2 oxidation produced sulfenic acid and maintained rTOP in the monomeric form. Consistent with the involvement of rTOP in a signaling redox cascade, the H 2 O 2 -oxidized rTOP reacted with dimeric thioredoxin-1 (TRx-1) and remained covalently bound to one subunit of TRx-1.

Inhibition of Mitochondrial Complex I Leads to Decreased Motility and Membrane Integrity Related to Increased Hydrogen Peroxide and Reduced ATP Production, while the Inhibition of Glycolysis Has Less Impact on Sperm Motility.

PubMed

Plaza Davila, María; Martin Muñoz, Patricia; Tapia, Jose A; Ortega Ferrusola, Cristina; Balao da Silva C, Carolina; Peña, Fernando J

2015-01-01

Mitochondria have been proposed as the major source of reactive oxygen species in somatic cells and human spermatozoa. However, no data regarding the role of mitochondrial ROS production in stallion spermatozoa are available. To shed light on the role of the mitochondrial electron transport chain in the origin of oxidative stress in stallion spermatozoa, specific inhibitors of complex I (rotenone) and III (antimycin-A) were used. Ejaculates from seven Andalusian stallions were collected and incubated in BWW media at 37 °C in the presence of rotenone, antimycin-A or control vehicle. Incubation in the presence of these inhibitors reduced sperm motility and velocity (CASA analysis) (p<0.01), but the effect was more evident in the presence of rotenone (a complex I inhibitor). These inhibitors also decreased ATP content. The inhibition of complexes I and III decreased the production of reactive oxygen species (p<0.01) as assessed by flow cytometry after staining with CellRox deep red. This observation suggests that the CellRox probe mainly identifies superoxide and that superoxide production may reflect intense mitochondrial activity rather than oxidative stress. The inhibition of complex I resulted in increased hydrogen peroxide production (p<0.01). The inhibition of glycolysis resulted in reduced sperm velocities (p<0.01) without an effect on the percentage of total motile sperm. Weak and moderate (but statistically significant) positive correlations were observed between sperm motility, velocity and membrane integrity and the production of reactive oxygen species. These results indicate that stallion sperm rely heavily on oxidative phosphorylation (OXPHOS) for the production of ATP for motility but also require glycolysis to maintain high velocities. These data also indicate that increased hydrogen peroxide originating in the mitochondria is a mechanism involved in stallion sperm senescence.

Effect of simvastatin on vascular tone in porcine coronary artery: Potential role of the mitochondria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Almukhtar, H.; Garle, M.J.; Smith, P.A.

2016-08-15

Statins induce acute vasorelaxation which may contribute to the overall benefits of statins in the treatment of cardiovascular disease. The mechanism underlying this relaxation is unknown. As statins have been shown to alter mitochondrial function, in this study we investigated the role of mitochondria in the relaxation to simvastatin. Relaxation of porcine coronary artery segments by statins was measured using isolated tissue baths. Mitochondrial activity was determined by measuring changes in rhodamine 123 fluorescence. Changes in intracellular calcium levels were determined in freshly isolated smooth muscle cells with Fluo-4 using standard epifluorescent imaging techniques. Simvastatin, but not pravastatin, produced amore » slow relaxation of the coronary artery, which was independent of the endothelium. The relaxation was attenuated by the mitochondrial complex I inhibitor rotenone (10 μM) and the complex III inhibitor myxothiazol (10 μM), or a combination of the two. The complex III inhibitor antimycin A (10 μM) produced a similar time-dependent relaxation of the porcine coronary artery, which was attenuated by rotenone. Changes in rhodamine 123 fluorescence showed that simvastatin (10 μM) depolarized the membrane potential of mitochondria in both isolated mitochondria and intact blood vessels. Simvastatin and antimycin A both inhibited calcium-induced contractions in isolated blood vessels and calcium influx in smooth muscle cells and this inhibition was prevented by rotenone. In conclusion, simvastatin produces an endothelium-independent relaxation of the porcine coronary artery which is dependent, in part, upon effects on the mitochondria. The effects on the mitochondria may lead to a reduction in calcium influx and hence relaxation of the blood vessel. - Highlights: • Simvastatin produces a relaxation of the porcine coronary artery. • This relaxation is inhibited by mitochondrial complex inhibitors. • Simvastatin alters mitochondrial membrane potential in intact blood vessels. • Simvastatin inhibits calcium influx in smooth muscle cells, prevented by a mitochondrial inhibitor. • Simvastatin-induced relaxations are dependent upon mitochondrial activity.« less

Mangifera indica L. extract (Vimang) inhibits Fe2+-citrate-induced lipoperoxidation in isolated rat liver mitochondria.

PubMed

Pardo Andreu, Gilberto; Delgado, René; Velho, Jesus; Inada, Natalia M; Curti, Carlos; Vercesi, Anibal E

2005-05-01

The extract of Mangifera indica L. (Vimang) is able to prevent iron mediated mitochondrial damage by means of oxidation of reduced transition metals required for the production of superoxide and hydroxyl radicals and direct free radical scavenging activity. In this study we report for the first time the iron-complexing ability of Vimang as a primary mechanism for protection of rat liver mitochondria against Fe2+ -citrate-induced lipoperoxidation. Thiobarbituric acid reactive substances (TBARS) and antimycin A-insensitive oxygen consumption were used as quantitative measures of lipoperoxidation. Vimang at 10 microM mangiferin concentration equivalent induced near-full protection against 50 microM Fe2+ -citrate-induced mitochondrial swelling and loss of mitochondrial transmembrane potential (DeltaPsi). The IC50 value for Vimang protection against Fe2+ -citrate-induced mitochondrial TBARS formation (7.89+/-1.19 microM) was around 10 times lower than that for tert-butylhydroperoxide mitochondrial induction of TBARS formation. The extract also inhibited the iron citrate induction of mitochondrial antimycin A-insensitive oxygen consumption, stimulated oxygen consumption due to Fe2+ autoxidation and prevented Fe3+ ascorbate reduction. The extracted polyphenolic compound, mainly mangiferin, could form a complex with Fe2+, accelerating Fe2+ oxidation and the formation of more stable Fe3+ -polyphenol complexes, unable to participate in Fenton-type reactions and lipoperoxidation propagation phase. The strong DPPH radical scavenging activity with an apparent IC50 of 2.45+/-0.08 microM suggests that besides its iron-complexing capacity, Vimang could also protect mitochondria from Fe2+ -citrate lipoperoxidation through direct free radical scavenging ability, mainly lipoperoxyl and alcoxyl radicals, acting as both a chain-breaking and iron-complexing antioxidant. These results are of pharmacological relevance since Vimang could be a potential candidate for antioxidant therapy in diseases related to abnormal intracellular iron distribution or iron overload.

Effects of toxicants on community metabolism in pools

USGS Publications Warehouse

Whitworth, Walter R.; Lane, Thomas H.

1969-01-01

Estimates of community metabolism of simulated natural environments were dcrivcd by diel oxygen techniques over a period of nine months. During this time, toxicants were added to some of the pools. "Natural" environmental factors and toxicants that did not affect the communities (0.02 mg/liter p,p' DDT; 0.1 mg/liter antimycin A; and 9.2 mg/liter KMnO,I) usually resulted in simultaneous changes, up or down, in both community photosynthesis and respiration. Concentrations (mg/liter) of 0.9 and 2.6 formalin, 10.0 nigrosine black, 10.0 and 2.0 malachite green, 0.1 Diquat, and 0,.5 and 5.0 CUSO.~ usually tlcprcsscd community oxygen production and increased community respiration for periods of 1 to 20 weeks.

Cross Talk among Calcium, Hydrogen Peroxide, and Nitric Oxide and Activation of Gene Expression Involving Calmodulins and Calcium-Dependent Protein Kinases in Ulva compressa Exposed to Copper Excess1[C][W][OA

PubMed Central

González, Alberto; Cabrera, M. de los Ángeles; Henríquez, M. Josefa; Contreras, Rodrigo A.; Morales, Bernardo; Moenne, Alejandra

2012-01-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H2O2) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H2O2, ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H2O2 increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H2O2 accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H2O2. In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H2O2, and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases. PMID:22234999

Cross talk among calcium, hydrogen peroxide, and nitric oxide and activation of gene expression involving calmodulins and calcium-dependent protein kinases in Ulva compressa exposed to copper excess.

PubMed

González, Alberto; Cabrera, M de Los Ángeles; Henríquez, M Josefa; Contreras, Rodrigo A; Morales, Bernardo; Moenne, Alejandra

2012-03-01

To analyze the copper-induced cross talk among calcium, nitric oxide (NO), and hydrogen peroxide (H(2)O(2)) and the calcium-dependent activation of gene expression, the marine alga Ulva compressa was treated with the inhibitors of calcium channels, ned-19, ryanodine, and xestospongin C, of chloroplasts and mitochondrial electron transport chains, 3-(3,4-dichlorophenyl)-1,1-dimethylurea and antimycin A, of pyruvate dehydrogenase, moniliformin, of calmodulins, N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide, and of calcium-dependent protein kinases, staurosporine, as well as with the scavengers of NO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, and of H(2)O(2), ascorbate, and exposed to a sublethal concentration of copper (10 μm) for 24 h. The level of NO increased at 2 and 12 h. The first peak was inhibited by ned-19 and 3-(2,3-dichlorophenyl)-1,1-dimethylurea and the second peak by ned-19 and antimycin A, indicating that NO synthesis is dependent on calcium release and occurs in organelles. The level of H(2)O(2) increased at 2, 3, and 12 h and was inhibited by ned-19, ryanodine, xestospongin C, and moniliformin, indicating that H(2)O(2) accumulation is dependent on calcium release and Krebs cycle activity. In addition, pyruvate dehydrogenase, 2-oxoxglutarate dehydrogenase, and isocitrate dehydrogenase activities of the Krebs cycle increased at 2, 3, 12, and/or 14 h, and these increases were inhibited in vitro by EGTA, a calcium chelating agent. Calcium release at 2, 3, and 12 h was inhibited by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide and ascorbate, indicating activation by NO and H(2)O(2). In addition, the level of antioxidant protein gene transcripts decreased with N-(6-aminohexyl)-5-chloro-1-naphtalene sulfonamide and staurosporine. Thus, there is a copper-induced cross talk among calcium, H(2)O(2), and NO and a calcium-dependent activation of gene expression involving calmodulins and calcium-dependent protein kinases.

Assaying Mitochondrial Respiration as an Indicator of Cellular Metabolism and Fitness.

PubMed

Smolina, Natalia; Bruton, Joseph; Kostareva, Anna; Sejersen, Thomas

2017-01-01

Mitochondrial respiration is the most important generator of cellular energy under most circumstances. It is a process of energy conversion of substrates into ATP. The Seahorse equipment allows measuring oxygen consumption rate (OCR) in living cells and estimates key parameters of mitochondrial respiration in real-time mode. Through use of mitochondrial inhibitors, four key mitochondrial respiration parameters can be measured: basal, ATP production-linked, maximal, and proton leak-linked OCR. This approach requires application of mitochondrial inhibitors-oligomycin to block ATP synthase, FCCP-to make the inner mitochondrial membrane permeable for protons and allow maximum electron flux through the electron transport chain, and rotenone and antimycin A-to inhibit complexes I and III, respectively. This chapter describes the protocol of OCR assessment in the culture of primary myotubes obtained upon satellite cell fusion.

Early cysteine-dependent inactivation of 26S proteasomes does not involve particle disassembly.

PubMed

Hugo, Martín; Korovila, Ioanna; Köhler, Markus; García-García, Carlos; Cabrera-García, J Daniel; Marina, Anabel; Martínez-Ruiz, Antonio; Grune, Tilman

2018-06-01

Under oxidative stress 26S proteasomes suffer reversible disassembly into its 20S and 19S subunits, a process mediated by HSP70. This inhibits the degradation of polyubiquitinated proteins by the 26S proteasome and allows the degradation of oxidized proteins by a free 20S proteasome. Low fluxes of antimycin A-stimulated ROS production caused dimerization of mitochondrial peroxiredoxin 3 and cytosolic peroxiredoxin 2, but not peroxiredoxin overoxidation and overall oxidation of cellular protein thiols. This moderate redox imbalance was sufficient to inhibit the ATP stimulation of 26S proteasome activity. This process was dependent on reversible cysteine oxidation. Moreover, our results show that this early inhibition of ATP stimulation occurs previous to particle disassembly, indicating an intermediate step during the redox regulation of the 26S proteasome with special relevance under redox signaling rather than oxidative stress conditions. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Genetic Evidence for the Action of Oxathiin and Thiazole Derivatives on the Succinic Dehydrogenase System of Ustilago maydis Mitochondria

PubMed Central

Georgopoulos, S. G.; Alexandri, E.; Chrysayi, M.

1972-01-01

The inhibitory effect of fungitoxic derivatives of 1,4-oxathiin on substrate oxidation by the basidiomycete Ustilago maydis is diminished by a single-gene mutation (oxr). The difference between mutant and wild type is approximately the same on the basis of inhibition of either growth and operation of the tricarboxylic acid cycle in intact cells or succinate-driven reduction of ferricyanide by mitochondrial preparations. The mutation affects the behavior of the succinic dehydrogenase system of mitochondria not only in the presence but also in the absence of the toxicant, from which it is concluded that some component of the system itself has been modified. The malonate and the antimycin A sensitivity of the oxr mutant is similar to that of the wild type but cross-resistance to thiazole derivatives is easily demonstrated. Images PMID:5030620

Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase)

PubMed Central

Miyadera, Hiroko; Shiomi, Kazuro; Ui, Hideaki; Yamaguchi, Yuichi; Masuma, Rokuro; Tomoda, Hiroshi; Miyoshi, Hideto; Osanai, Arihiro; Kita, Kiyoshi; Ōmura, Satoshi

2003-01-01

Enzymes in the mitochondrial respiratory chain are involved in various physiological events in addition to their essential role in the production of ATP by oxidative phosphorylation. The use of specific and potent inhibitors of complex I (NADH-ubiquinone reductase) and complex III (ubiquinol-cytochrome c reductase), such as rotenone and antimycin, respectively, has allowed determination of the role of these enzymes in physiological processes. However, unlike complexes I, III, and IV (cytochrome c oxidase), there are few potent and specific inhibitors of complex II (succinate-ubiquinone reductase) that have been described. In this article, we report that atpenins potently and specifically inhibit the succinate-ubiquinone reductase activity of mitochondrial complex II. Therefore, atpenins may be useful tools for clarifying the biochemical and structural properties of complex II, as well as for determining its physiological roles in mammalian tissues. PMID:12515859

Identification of a New Isoindole-2-yl Scaffold as a Qo and Qi Dual Inhibitor of Cytochrome bc 1 Complex: Virtual Screening, Synthesis, and Biochemical Assay.

PubMed

Azizian, Homa; Bagherzadeh, Kowsar; Shahbazi, Sophia; Sharifi, Niusha; Amanlou, Massoud

2017-09-18

Respiratory chain ubiquinol-cytochrome (cyt) c oxidoreductase (cyt bc 1 or complex III) has been demonstrated as a promising target for numerous antibiotics and fungicide applications. In this study, a virtual screening of NCI diversity database was carried out in order to find novel Qo/Qi cyt bc 1 complex inhibitors. Structure-based virtual screening and molecular docking methodology were employed to further screen compounds with inhibition activity against cyt bc 1 complex after extensive reliability validation protocol with cross-docking method and identification of the best score functions. Subsequently, the application of rational filtering procedure over the target database resulted in the elucidation of a novel class of cyt bc 1 complex potent inhibitors with comparable binding energies and biological activities to those of the standard inhibitor, antimycin.

Mechanism of Siglec-8-induced human eosinophil apoptosis: role of caspases and mitochondrial injury.

PubMed

Nutku, Esra; Hudson, Sherry A; Bochner, Bruce S

2005-10-28

Sialic acid binding immunoglobulin like lectin (Siglec)-8 crosslinking with specific antibodies causes human eosinophil apoptosis. Mechanisms by which Siglec-8 crosslinking induces apoptosis are not known. Peripheral blood eosinophils were examined for caspase, mitochondria and reactive oxygen species (ROS) involvement after incubating the cells with anti-Siglec-8 crosslinking Abs or control Abs, in the presence or absence of selective inhibitors. Siglec-8 crosslinking induced rapid cleavage of caspase-3, caspase-8, and caspase-9 in eosinophils. Selective caspase-8 and/or caspase-9 inhibitors inhibited this apoptosis. Siglec-8 crosslinking on eosinophils increased dissipation of mitochondrial membrane potential upstream of caspase activation. Rotenone and antimycin, inhibitors of mitochondrial respiratory chain components, completely inhibited apoptosis. Additional experiments with an inhibitor of ROS, diphenyleneiodonium, demonstrated that ROS was also essential for Siglec-8-mediated apoptosis and preceded Siglec-8-mediated mitochondrial dissipation. These experiments show that Siglec-8-induced apoptosis occurs through the sequential production of ROS, followed by induction of mitochondrial injury and caspase cleavage.

Appearance of an Alternate Pathway Cyanide-resistant during Germination of Seeds of Cicer arietinum

PubMed Central

Burguillo, Placido De La Fuente; Nicolás, Gregorio

1977-01-01

The combined action of the inhibitors antimycin A and cyanide with benzohydroxamic acid indicates the presence of a cyanide-resistant pathway of respiration in chick pea (Cicer arietinum L.) seeds. The appearance of this pathway takes place during germination. During the first 12 hours of germination, the respiration is predominantly cyanide-sensitive, showing after this time a shift to an “alternate” respiration which is sensitive to benzohydroxamic acid, reaching the maximal cyanide resistance between 72 and 96 hours of germination. The appearance of the alternate pathway is initiated by high O2 concentrations and depends on cytoplasmic protein synthesis, since its appearance is inhibited by cycloheximide but not by chloramphenicol. Actinomycin D has no effect on the appearance of the alternate pathway. Our results indicate, in agreement with other authors, that the branching point is located between the flavoproteins and cytochromes b, probably at the level of ubiquinone, but the possibility of more than one branching point of the electron flow is also considered. PMID:16660130

Reduction of molybdate to molybdenum blue by Klebsiella sp. strain hkeem.

PubMed

Lim, H K; Syed, M A; Shukor, M Y

2012-06-01

A novel molybdate-reducing bacterium, tentatively identified as Klebsiella sp. strain hkeem and based on partial 16s rDNA gene sequencing and phylogenetic analysis, has been isolated. Strain hkeem produced 3 times more molybdenum blue than Serratia sp. strain Dr.Y8; the most potent Mo-reducing bacterium isolated to date. Molybdate was optimally reduced to molybdenum blue using 4.5 mM phosphate, 80 mM molybdate and using 1% (w/v) fructose as a carbon source. Molybdate reduction was optimum at 30 °C and at pH 7.3. The molybdenum blue produced from cellular reduction exhibited absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of electron transport system such as antimycin A, rotenone, sodium azide, and potassium cyanide did not inhibit the molybdenum-reducing enzyme. Mercury, silver, and copper at 1 ppm inhibited molybdenum blue formation in whole cells of strain hkeem. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Removal of toxic chemicals from water with activated carbon

USGS Publications Warehouse

Dawson, V.K.; Marking, L.L.; Bills, T.D.

1976-01-01

Activated carbon was effective in removing fish toxicants and anesthetics from water solutions. Its capacity to adsorb 3-trifluoromethyl-4-nitrophenol (TFM), antimycin, NoxfishA? (5% rotenone), Dibrorms, juglone, MSa??222, and benzocaine ranged from 0.1 to 64 mg per gram of carbon. The adsorptive capacity (end point considered as a significant discharge) of activated carbon for removal of TFM was determined at column depths of 15, 30, and 60 cm; temperatures of 7, 12, 17, and 22 C; pH's of 6.5, 7.5, 8.5, and 9.5; and flow rates of 50, 78, 100, 200, and 940 ml/min. Adsorptive capacity increased when the contact time was increased by reducing the flow rate or increasing the column depth. The adsorptive capacity was not significantly influenced by temperature but was substantially higher at pH 6.5 than at the other pH's tested. A practical and efficient filter for purifying chemically treated water was developed.

Aminobenzoates as building blocks for natural product assembly lines.

PubMed

Walsh, Christopher T; Haynes, Stuart W; Ames, Brian D

2012-01-01

The ortho-, meta-, and para- regioisomers of aminobenzoate are building blocks for a wide range of microbial natural products. Both the ortho-isomer (anthranilate) and PABA derive from the central shikimate pathway metabolite chorismate while the meta-isomer is not available by that route and starts from UDP-3-aminoglucose. PABA is largely funnelled into folate biosynthesis while anthranilate is the scaffold for biosynthetic elaboration into many natural heterocycles, most notably with its role in indole formation for tryptophan biosynthesis. Anthranilate is also converted to benzodiazepinones, fumiquinazolines, quinoxalines, phenoxazines, benzoxazolinates, quinolones, and phenazines, often with redox enzyme participation. The 5-hydroxy form of 3-aminobenzaote is the starter unit for ansa-bridged rifamycins, ansamitocins, and geldanamycins, whereas regioisomers 2-hydroxy, 4-hydroxy and 2,4-dihydroxy-3-aminobenzoate are key components of antimycin, grixazone, and platencin and platensimycin biosynthesis, respectively. The enzymatic mechanisms for generation of the aminobenzoate regioisomers and their subsequent utilization for diverse heterocycle and macrocycle construction are examined.

Reverse electron transport effects on NADH formation and metmyoglobin reduction.

PubMed

Belskie, K M; Van Buiten, C B; Ramanathan, R; Mancini, R A

2015-07-01

The objective was to determine if NADH generated via reverse electron flow in beef mitochondria can be used for electron transport-mediated reduction and metmyoglobin reductase pathways. Beef mitochondria were isolated from bovine hearts (n=5) and reacted with combinations of succinate, NAD, and mitochondrial inhibitors to measure oxygen consumption and NADH formation. Mitochondria and metmyoglobin were reacted with succinate, NAD, and mitochondrial inhibitors to measure electron transport-mediated metmyoglobin reduction and metmyoglobin reductase activity. Addition of succinate and NAD increased oxygen consumption, NADH formation, electron transport-mediated metmyoglobin reduction, and reductase activity (p<0.05). Addition of antimycin A prevented electron flow beyond complex III, therefore, decreasing oxygen consumption and electron transport-mediated metmyoglobin reduction. Addition of rotenone prevented reverse electron flow, increased oxygen consumption, increased electron transport-mediated metmyoglobin reduction, and decreased NADH formation. Succinate and NAD can generate NADH in bovine tissue postmortem via reverse electron flow and this NADH can be used by both electron transport-mediated and metmyoglobin reductase pathways. Copyright © 2015 Elsevier Ltd. All rights reserved.

Maltotriose fermentation by Saccharomyces cerevisiae.

PubMed

Zastrow, C R; Hollatz, C; de Araujo, P S; Stambuk, B U

2001-07-01

Maltotriose, the second most abundant sugar of brewer's wort, is not fermented but is respired by several industrial yeast strains. We have isolated a strain capable of growing on a medium containing maltotriose and the respiratory inhibitor, antimycin A. This strain produced equivalent amounts of ethanol from 20 g l(-1) glucose, maltose, or maltotriose. We performed a detailed analysis of the rates of active transport and intracellular hydrolysis of maltotriose by this strain, and by a strain that does not ferment this sugar. The kinetics of sugar hydrolysis by both strains was similar, and our results also indicated that yeast cells do not synthesize a maltotriose-specific alpha-glucosidase. However, when considering active sugar transport, a different pattern was observed. The maltotriose-fermenting strain showed the same rate of active maltose or maltotriose transport, while the strain that could not ferment maltotriose showed a lower rate of maltotriose transport when compared with the rates of active maltose transport. Thus, our results revealed that transport across the plasma membrane, and not intracellular hydrolysis, is the rate-limiting step for the fermentation of maltotriose by these Saccharomyces cerevisiae cells.

Synchronization of calcium waves by mitochondrial substrates in Xenopus laevis oocytes

NASA Astrophysics Data System (ADS)

Jouaville, Laurence S.; Ichas, François; Holmuhamedov, Ekhson L.; Camacho, Patricia; Lechleiter, James D.

1995-10-01

INXenopus oocytes, as well as other cells, inositol-l,4,5-tris-phosphate (Ins(l,4,5)P3)-induced Ca2+ release1-4 is an excitable process that generates propagating Ca2+ waves5-7 that annihilate upon collision8-12. The fundamental property responsible for excitability13 appears to be the Ca2+ dependency of the Ins(l,4,5)P3 receptor9. Here we report that Ins(l,4,5)P3-induced Ca2+ wave activity is strengthened by oxidizable substrates that energize mitochondria, increasing Ca2+ wave amplitude, velocity and interwave period. The effects of pyruvate/malate are blocked by ruthenium red at the Ca2+ uniporter, by rotenone at complex I, and by antimycin A at complex III, and are subsequently rescued at complex IV by ascorbate tetramethylphenylenediamine (TMPD)14. Our data reveal that potential-driven mitochondrial Ca2+ uptake is a major factor in the regulation of Ins(l,4,5)P3-induced Ca2+ release and clearly demonstrate a physiological role of mitochondria in intracellular Ca2+ signalling.

Calmodulin stimulation of calcium transport in carrot microsomal vesicles. [Daucus carota

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pierce, W.S.; Sze, H.

1987-04-01

ATP-dependent /sup 45/Ca/sup 2 +/ uptake into microsomal vesicles isolated from cultured carrot cells (Daucus carota Danvers) was stimulated 2-3 fold by 5 ug/ml calmodulin (CaM). Microsomal vesicles separated with a linear sucrose gradient showed two peaks with CaM-stimulated Ca/sup 2 +/ uptake activities. One peak (at 1.12 g/cc) comigrated with the activity of the antimycin A-insensitive NADH-dependent cytochrome c reductase. This transport activity was enhanced 10-20 fold by 10 mM oxalate and appeared to be associates with vesicles derived primarily from the ER. The other peak of CaM-stimulated Ca/sup 2 +/ uptake (at 1.17 g/cc) was not affected bymore » oxalate. These vesicles are probably derived from the plasma membrane. Preliminary experiments with the low-density vesicles (ER) vesicles, indicate that inositol-1,4,5-trisphosphate caused a transient reduction in intravesicular Ca/sup 2 +/. These results are consistent with the ER being an important site of intracellular Ca/sup 2 +/ regulation.« less

Mitochondrial electron transport chain is involved in microcystin-RR induced tobacco BY-2 cells apoptosis.

PubMed

Huang, Wenmin; Li, Dunhai; Liu, Yongding

2014-09-01

Microcystin-RR (MC-RR) has been suggested to induce apoptosis in tobacco BY-2 cells through mitochondrial dysfunction including the loss of mitochondrial membrane potential (ΔΨm). To further elucidate the mechanisms involved in MC-RR induced apoptosis in tobacco BY-2 cells, we have investigated the role of mitochondrial electron transport chain (ETC) as a potential source for reactive oxygen species (ROS). Tobacco BY-2 cells after exposure to MC-RR (60mg/L) displayed apoptotic changes in association with an increased production of ROS and loss of ΔΨm. All of these adverse effects were significantly attenuated by ETC inhibitors including Rotenone (2μmol/L, complex I inhibitor) and antimycin A (0.01μmol/L, complex III inhibitor), but not by thenoyltrifluoroacetone (5μmol/L, complex II inhibitor). These results suggest that mitochondrial ETC plays a key role in mediating MC-RR induced apoptosis in tobacco BY-2 cells through an increased mitochondrial production of ROS. Copyright © 2014. Published by Elsevier B.V.

Mode of action of the qcr9 and cat3 mutations in restoring the ability of Saccharomyces cerevisiae tps1 mutants to grow on glucose.

PubMed

Blázquez, M A; Gancedo, C

1995-12-20

Mutations in the TPS1 gene, which encodes trehalose-6-P synthase, cause a glucose-negative phenotype in Saccharomyces cerevisiae. Antimycin A or disruption of the QCR9 gene, which encodes one subunit of the cytochrome bc1 complex, restore the ability to grow in glucose-containing media. Under these conditions the cell excreted a large amount of glycerol, corresponding to about 20% of the glucose taken up. Suppression appears to be achieved by diversion of accumulated glycolytic intermediates to the production of glycerol, thereby providing NAD+ and phosphate for the glyceraldehyde-3-P dehydrogenase reaction. Analysis of the mutation sci1-1, which also suppresses the glucose-negative phenotype of tps1 mutants, showed that glucose transport was decreased in sci1-1 mutants. The gene SCI1 was cloned and its nucleotide sequence revealed it to be identical to CAT3/SNF4. The suppression mediated by sci1-1 is attributable to a decrease in glycolytic flux.

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes

PubMed Central

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D.

2010-01-01

The relationship between ethanol induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. When hepatocytes were exposed to ethanol (50 mM, 24 hr) in the presence of N-acetyl cystein (ROS reducer) or dietary antioxidants (quercetin, resveratrol), or NADPH oxidase inhibitor apocynin, ethanol induced increases in ROS and H3AcK9, both were significantly reduced. On the other hand, l-buthionine-sulfoximine (ROS inducer) and inhibitor of mitochondrial complex I (rotenone) and III (antimycin) increased ethanol induced H3AcK9 (p<0.01). Oxidative stress also affected ethanol induced alcohol dehydrogenase 1 (ADH1) mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol induced histone H3 acetylation in hepatocytes. PMID:20705415

Evidence for the role of oxidative stress in the acetylation of histone H3 by ethanol in rat hepatocytes.

PubMed

Choudhury, Mahua; Park, Pil-Hoon; Jackson, Daniel; Shukla, Shivendra D

2010-09-01

The relationship between ethanol-induced oxidative stress and acetylation of histone H3 at lysine 9 (H3AcK9) remains unknown and was therefore investigated in primary cultures of rat hepatocytes. Cells were treated with ethanol, and a select group of pharmacological agents and the status of H3AcK9 and reactive oxygen species (ROS) were monitored. Pretreatment of hepatocytes with N-acetyl cystein (ROS reducer), or dietary antioxidants (quercetin, reserveratrol), or NADPH (reduced nicotinamide adenine dinucleotide phosphate) oxidase inhibitor apocynin, significantly reduced ethanol (50 mM, 24 h) induced increases in ROS and H3AcK9. In contrast, l-buthionine sulfoximine (ROS inducer) and inhibitor of mitochondrial complexes I (rotenone) and III (antimycin) increased ethanol-induced H3AcK9 (P<.01). Oxidative stress also affected ethanol-induced alcohol dehydrogenase 1 mRNA expression. These results demonstrate for the first time that oxidative stress is involved in the ethanol-induced histone H3 acetylation in hepatocytes. Copyright © 2010 Elsevier Inc. All rights reserved.

History of fish toxicants in the United States

USGS Publications Warehouse

Cumming, K.B.

1975-01-01

Many bodies of water have been overrun with fish species that are undesirable for the well-being of endemic aquatic biota and are of little or no interest to anglers. This situation has resulted from the transplanting of fish, creation of new waters, increasing fishing pressure and changing water quality. Although as many as 30 toxicants have been tried, only about 30 major piscicides have been used in rehabilitation programs to remove the unwanted species. Explicit knowledge of the mode of action of toxicants in the environment and improvements in fish farming, have permitted fishery managers to reduce the amount of control chemical that is applied to obtain a desired result. The number and volume of waters being reclaimed have increased, but this trend may change soon because environmental laws involving the use of registered pesticides are becoming more restrictive. Registration research has established the safety of some fish toxicants but has eliminated others. Only four piscicides are presently registered (or nonfood use application: antimycin and rotenone as general fish toxicants and 3-trifluoromethyl-4-nitrophenol (TFM) and Bayluscide as selective lampricides.

Photooxidation of the cytochrome b-559 in the presence of various substituted 2-anilinothiophenes and of some other compounds, in Chlamydomonas reinhardtii.

PubMed

Maroc, J; Garnier, J

1979-11-08

Five substituted 2-anilinothiophenes and two substituted carbonylcyanide-phenylhydrazones were comparatively studied with respect to their capacities for inducing photooxidation of the cytochrome b-559 in chloroplast fragments and in whole cells of Chlamydomonas reinhardtii (wild type and P-700-lacking mutant Fl 5). In addition, some other compounds: antimycin A, picric acid, tetraphenylboron and NH4Cl were also tested. Cytochrome b-559 photooxidations were clearly observed in the presence of 2-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene (ANT 2p), 2-(3,4,5-trichloro)anilino-3,5-dinitrothiophene (ANT 2s), 2-(4-chloro)anilino-3,5-dinitrothiophene and, with greater amplitudes, in the presence of carbonylcyanide-p-trifluoromethoxyphenylhydrazone and carbonylcyanide-m-chlorophenylhydrazone, both in whole cells and in chloroplast fragments. Picric acid, antimycin A and tetraphenylboron were also able to induce cytochrome b-559 photooxidation in chloroplast fragments, but not in whole cells. In the wild type, the highest photoinduced redox changes were 1.1 (carbonylcyanide-p-trifluoromethoxyphenylhydrazone, carbonylcyanide-m-chlorophenyl-hydrazone) and 0.6 (ANT 2p, ANT 2s) mumol of oxidized cytochrome b-559/1 mmol of chlorophyll, corresponding to 40% and 23% of the redox changes which could be induced chemically. All these cytochrome b-559 photooxidations, the greater part of which was inhibited by 3-(3,4-dichloropheny)-1,1-dimethylurea and occurred in the mutant Fl 5, appeared to be mainly Photosystem II-dependent reactions. But 3-(3,4-dichlorophenyl)-1,1-dimethylurea-insensitive Photosystem I-dependent photooxidations of cytochrome b-559 occurred also in the wild type. On the other hand, 2-(4-dimethylamine)-anilino-3,5-dinitrothiophene, 2-N-methyl-(3-chloro-4-trifluoromethyl)anilino-3,5-dinitrothiophene and NH4Cl did not induce any cytochrome b-559 photooxidation. These results were discussed taking in consideration the nature of the molecular substitutions of the various tested substances and their respective acceleration of the deactivation reactions of the water-splitting enzyme system Y of photosynthesis capacities which had been defined elsewhere by Renger (Renger, G. (1972) Biochim. Biophys. Acta 256, 428-439) for spinach chloroplasts. Like the acceleration of the deactivation reactions of the water-splitting enzyme system Y effect, the capacity for inducing the cytochrome b-559 photooxidation appeared dependent on the acidity of the NH group and on the number of halogenous substituents in the aromatic ring of the molecule. The greatest action towards cytochrome b-559 photooxidation was obtained with the most active acceleration of the deactivation reactions of the water-splitting enzyme system Y agents: carbonylcyanide-p-trifluoromethoxyphenylhydrazone, ANT 2p and ANT 2s.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Babu, Dinesh, E-mail: dinesh.babu@ugent.be; Leclercq, Georges; Goossens, Vera

2015-10-15

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H{sub 2}O{sub 2}-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrialmore » superoxide anion (O{sub 2}·{sup −}) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψ{sub m}) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H{sub 2}O{sub 2}-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O{sub 2}·{sup −} production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O{sub 2}·{sup −} levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol. - Highlights: • In MODE-K IECs, TNF-α/CHX induces correlating ROS, mitochondrial O{sub 2}·{sup −} and cell death. • CORM-A1 does not influence basal intracellular ROS and mitochondrial O{sub 2}·{sup −} levels. • Resveratrol increases basal intracellular ROS but decreases mitochondrial O{sub 2}·{sup −} levels. • CORM-A1 acts solely on NOX-derived ROS to protect from cell death by TNF-α/CHX. • Cytoprotection by resveratrol is predominantly due to reduction of mitochondrial O{sub 2}·{sup −}.« less

Effect of Magnolol on the Function of Osteoblastic MC3T3-E1 Cells

PubMed Central

Kwak, Eun Jung; Lee, Young Soon; Choi, Eun Mi

2012-01-01

Objectives. In the present study, the ability of magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, to stimulate osteoblast function and inhibit the release of bone-resorbing mediators was investigated in osteoblastic MC3T3-E1 cells. Methods. Osteoblast function was measured by cell growth, alkaline phosphatase activity, collagen synthesis, and mineralization. Glutathione content was also measured in the cells. Bone-resorbing cytokines, receptor activator of nuclear factor-κB ligand (RANKL), TNF-α, and IL-6 were measured with an enzyme immunoassay system. Results. Magnolol caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, mineralization, and glutathione content in the cells (P < 0.05). Skeletal turnover is orchestrated by a complex network of regulatory factors. Among cytokines, RANKL, TNF-α, and IL-6 were found to be key osteoclastogenetic molecules produced by osteoblasts. Magnolol significantly (P < 0.05) decreased the production of osteoclast differentiation inducing factors such as RANKL, TNF-α, and IL-6 in the presence of antimycin A, which inhibits mitochondrial electron transport and has been used as an ROS generator. Conclusion. Magnolol might be a candidate as an agent for the prevention of bone disorders such as osteoporosis. PMID:22474400

Identification of a mitochondrial alcohol dehydrogenase in Schizosaccharomyces pombe: new insights into energy metabolism

PubMed Central

Crichton, Paul G.; Affourtit, Charles; Moore, Anthony L.

2006-01-01

In the present study we have shown that mitochondria isolated from Schizosaccharomyces pombe exhibit antimycin A-sensitive oxygen uptake activity that is exclusively dependent on ethanol and is inhibited by trifluoroethanol, a potent inhibitor of ADH (alcohol dehydrogenase). Ethanol-dependent respiratory activity has, to our knowledge, not been reported in S. pombe mitochondria to date, which is surprising as it has been concluded previously that only one ADH gene, encoding a cytosolic enzyme, occurs in this yeast. Spectrophotometric enzyme assays reveal that ADH activity in isolated mitochondria is increased ∼16-fold by Triton X-100, which demonstrates that the enzyme is located in the matrix. Using genetic knockouts, we show conclusively that the novel mitochondrial ADH is encoded by adh4 and, as such, is unrelated to ADH isoenzymes found in mitochondria of other yeasts. By performing a modular-kinetic analysis of mitochondrial electron transfer, we furthermore show how ethanol-dependent respiratory activity (which involves oxidation of matrix-located NADH) compares with that observed when succinate or externally added NADH are used as substrates. This analysis reveals distinct kinetic differences between substrates which fully explain the lack of respiratory control generally observed during ethanol oxidation in yeast mitochondria. PMID:16999687

Hyphal formation of Candida albicans is controlled by electron transfer system

DOE Office of Scientific and Technical Information (OSTI.GOV)

Watanabe, Toshihiko; Ogasawara, Ayako; Mikami, Takeshi

2006-09-15

Most Candida albicans cells cultured in RPMI1640 medium at 37 deg. C grow in hyphal form in aerobic conditions, but they grow in yeast form in anaerobic conditions. The hyphal growth of C. albicans was inhibited in glucose-deficient conditions. Malonic acid, an inhibitor of succinate dehydrogenase, enhanced the yeast proliferation of C. albicans, indicating that the hyphal-formation signal was derived from the glycolysis system and the signal was transmitted to the electron transfer system via the citric acid cycle. Thenoyl trifluoro acetone (TTFA), an inhibitor of the signal transmission between complex II and Co Q, significantly inhibited the hyphal growthmore » of C. albicans. Antimycin, KCN, and oligomycin, inhibitors of complex III, IV, and V, respectively, did not inhibit the hyphal growth of C. albicans. The production of mRNAs for the hyphal formation signal was completely inhibited in anaerobic conditions. These results indicate that the electron transfer system functions upstream of the RAS1 signal pathway and activates the expression of the hyphal formation signal. Since the electron transfer system is inactivated in anaerobic conditions, C. albicans grew in yeast form in this condition.« less

Effect of mitochondrial dysfunction and oxidative stress on endogenous levels of coenzyme Q(10) in human cells.

PubMed

Yen, Hsiu-Chuan; Chen, Feng-Yuan; Chen, Shih-Wei; Huang, Yu-Hsiang; Chen, Yun-Ru; Chen, Chih-Wei

2011-01-01

Little is known about the regulation of endogenous CoQ(10) levels in response to mitochondrial dysfunction or oxidative stress although exogenous CoQ(10) has been extensively used in humans. In this study, we first demonstrated that acute treatment of antimycin A, an inhibitor of mitochondrial complex III, and the absence of mitochondrial DNA suppressed CoQ(10) levels in human 143B cells. Because these two conditions also enhanced formation of reactive oxygen species (ROS), we further investigated whether oxidative stress or mitochondrial dysfunction primarily contributed to the decrease of CoQ(10) levels. Results showed that H(2)O(2) augmented CoQ(10) levels, but carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a chemical uncoupler, decreased CoQ(10) levels in 143B cells. However, H(2)O(2) and FCCP both increased mRNA levels of multiple COQ genes for biosynthesis of CoQ(10) . Our findings suggest that ROS induced CoQ(10) biosynthesis, whereas mitochondrial energy deficiency caused secondary suppression of CoQ(10) levels possibly due to impaired import of COQ proteins into mitochondria. Copyright © 2011 Wiley Periodicals, Inc.

Brief exposure to carbon monoxide preconditions cardiomyogenic cells against apoptosis in ischemia-reperfusion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kondo-Nakamura, Mihoko; Shintani-Ishida, Kaori, E-mail: kaori@m.u-tokyo.ac.jp; Uemura, Koichi

We examined whether and how pretreatment with carbon monoxide (CO) prevents apoptosis of cardioblastic H9c2 cells in ischemia-reperfusion. Reperfusion (6 h) following brief ischemia (10 min) induced cytochrome c release, activation of caspase-9 and caspase-3, and apoptotic nuclear condensation. Brief CO pretreatment (10 min) or a caspase-9 inhibitor (Z-LEHD-FMK) attenuated these apoptotic changes. Ischemia-reperfusion increased phosphorylation of Akt at Ser472/473/474, and this was enhanced by CO pretreatment. A specific Akt inhibitor (API-2) blunted the anti-apoptotic effects of CO in reperfusion. In normoxic cells, CO enhanced O{sub 2}{sup -} generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A)more » but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O{sub 2}{sup -} scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O{sub 2}{sup -}, which is then converted by SOD to H{sub 2}O{sub 2}, and subsequent Akt activation by H{sub 2}O{sub 2} attenuates apoptosis in ischemia-reperfusion.« less

Oxidative stress in duckweed (Lemna minor L.) induced by glyphosate: Is the mitochondrial electron transport chain a target of this herbicide?

PubMed

Gomes, Marcelo Pedrosa; Juneau, Philippe

2016-11-01

We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H 2 O 2 ) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l -1 . Inhibition of mitochondrial ETC Complex I by rotenone reduced H 2 O 2 concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H 2 O 2 formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis.

PubMed

Wilson, A J; Bhattacharjee, J K

1986-12-01

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.

Respiratory chain components involved in the glycerophosphate dehydrogenase-dependent ROS production by brown adipose tissue mitochondria.

PubMed

Vrbacký, Marek; Drahota, Zdenek; Mrácek, Tomás; Vojtísková, Alena; Jesina, Pavel; Stopka, Pavel; Houstek, Josef

2007-07-01

Involvement of mammalian mitochondrial glycerophosphate dehydrogenase (mGPDH, EC 1.1.99.5) in reactive oxygen species (ROS) generation was studied in brown adipose tissue mitochondria by different spectroscopic techniques. Spectrofluorometry using ROS-sensitive probes CM-H2DCFDA and Amplex Red was used to determine the glycerophosphate- or succinate-dependent ROS production in mitochondria supplemented with respiratory chain inhibitors antimycin A and myxothiazol. In case of glycerophosphate oxidation, most of the ROS originated directly from mGPDH and coenzyme Q while complex III was a typical site of ROS production in succinate oxidation. Glycerophosphate-dependent ROS production monitored by KCN-insensitive oxygen consumption was highly activated by one-electron acceptor ferricyanide, whereas succinate-dependent ROS production was unaffected. In addition, superoxide anion radical was detected as a mGPDH-related primary ROS species by fluorescent probe dihydroethidium, as well as by electron paramagnetic resonance (EPR) spectroscopy with DMPO spin trap. Altogether, the data obtained demonstrate pronounced differences in the mechanism of ROS production originating from oxidation of glycerophosphate and succinate indicating that electron transfer from mGPDH to coenzyme Q is highly prone to electron leak and superoxide generation.

Mitochondria mediate tumor necrosis factor-alpha/NF-kappaB signaling in skeletal muscle myotubes

NASA Technical Reports Server (NTRS)

Li, Y. P.; Atkins, C. M.; Sweatt, J. D.; Reid, M. B.; Hamilton, S. L. (Principal Investigator)

1999-01-01

Tumor necrosis factor-alpha (TNF-alpha) is implicated in muscle atrophy and weakness associated with a variety of chronic diseases. Recently, we reported that TNF-alpha directly induces muscle protein degradation in differentiated skeletal muscle myotubes, where it rapidly activates nuclear factor kappaB (NF-kappaB). We also have found that protein loss induced by TNF-alpha is NF-kappaB dependent. In the present study, we analyzed the signaling pathway by which TNF-alpha activates NF-kappaB in myotubes differentiated from C2C12 and rat primary myoblasts. We found that activation of NF-kappaB by TNF-alpha was blocked by rotenone or amytal, inhibitors of complex I of the mitochondrial respiratory chain. On the other hand, antimycin A, an inhibitor of complex III, enhanced TNF-alpha activation of NK-kappaB. These results suggest a key role of mitochondria-derived reactive oxygen species (ROS) in mediating NF-kappaB activation in muscle. In addition, we found that TNF-alpha stimulated protein kinase C (PKC) activity. However, other signal transduction mediators including ceramide, Ca2+, phospholipase A2 (PLA2), and nitric oxide (NO) do not appear to be involved in the activation of NF-kappaB.

Fungal mitochondrial oxygen consumption induces the growth of strict anaerobic bacteria.

PubMed

Lambooij, Joost M; Hoogenkamp, Michel A; Brandt, Bernd W; Janus, Marleen M; Krom, Bastiaan P

2017-12-01

Fungi are commonly encountered as part of a healthy oral ecosystem. Candida albicans is the most often observed and investigated fungal species in the oral cavity. The role of fungi in the oral ecosystem has remained enigmatic for decades. Recently, it was shown that C. albicans, in vitro, influences the bacterial composition of young oral biofilms, indicating it possibly plays a role in increasing diversity in the oral ecosystem. C. albicans favored growth of strictly anaerobic species under aerobic culture conditions. In the present study, the role of mitochondrial respiration, as mechanism by which C. albicans modifies its environment, was investigated. Using oxygen sensors, a rapid depletion of dissolved oxygen (dO 2 ) was observed. This decrease was not C. albicans specific as several non-albicans Candida species showed similar oxygen consumption. Heat inactivation as well as addition of the specific mitochondrial respiration inhibitor Antimycin A inhibited depletion of dO 2 . Using 16S rDNA sequencing, it is shown that mitochondrial activity, more than physical presence of C. albicans is responsible for inducing growth of strictly anaerobic oral bacteria in aerobic growth conditions. The described mechanism of dO 2 depletion may be a general mechanism by which fungi modulate their direct environment. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Mitochondrial origin of extracelullar transferred electrons in yeast-based biofuel cells.

PubMed

Hubenova, Yolina; Mitov, Mario

2015-12-01

The influence of mitochondrial electron transport chain inhibitors on the electricity outputs of Candida melibiosica yeast-based biofuel cell was investigated. The addition of 30 μM rotenone or antimycin A to the yeast suspension results in a decrease in the current generation, corresponding to 25.7±1.3%, respectively 38.8±1.9% reduction in the electric charge passed through the bioelectrochemical system. The latter percentage coincides with the share of aerobic respiration in the yeast catabolic processes, determined by the decrease of the ethanol production during cultivation in the presence of oxygen compared with that obtained under strict anaerobic conditions. It was established that the presence of both inhibitors leads to almost complete mitochondrial dysfunction, expressed by inactivation of cytochrome c oxidase and NADH:ubiquinone oxidoreductase as well as reduced electrochemical activity of isolated yeast mitochondria. It was also found that methylene blue partially neutralized the rotenone poisoning, probably serving as alternative intracellular electron shuttle for by-passing the complex I blockage. Based on the obtained results, we suppose that electrons generated through the aerobic respiration processes in the mitochondria participate in the extracellular electron transfer from the yeast cells to the biofuel cell anode, which contributes to higher current outputs at aerobic conditions. Copyright © 2014 Elsevier B.V. All rights reserved.

[Phenolic antioxidant TS-13 regulating ARE-dependent genes induces tumor cell death by mitochondria-dependent pathway].

PubMed

Martinovich, G G; Martinovich, I V; Zenkov, N K; Men'shikova, E B; Kandalintseva, N V; Cherenkevich, S N

2015-01-01

Effects of water-soluble phenolic antioxidant sodium 3-(3'-tret-butyl-4'-hydroxyphenyl)-propyl thiosulfonate (TS-13), potassium 3,5-dimethyl-4-hydroxybenzyl thioetanoate (BEP-11-K) and potassium 3-(3',5'-ditretbutyl-4'-hydroxyphenyl)-propionate (potassium phenosan) on tumor cells proliferative activity and the role of redox-dependent and calcium-dependent signaling mechanisms in realization of tumor cell response to the antioxidant action were studied. Potassium phenosan and BEP-11-K were found to stimulate proliferation and ARE-inducing phenolic antioxidant TS-13 was found to inhibit tumor cell growth in culture. The tumor cell growth rate depended on the rate of intracellular reactive oxygen species production and was decreased by apocynin (a NADPH-oxidase inhibitor) and antimycin A (an ubiquinol-cytochrome c oxidoreductase inhibitor). TS-13 action on tumor cells was accompanied by a transient increase in intracellular reactive oxygen species production and the intracellular calcium concentration, whereas cell incubation with potassium phenosan and BEP-11-K did not influence the reactive oxygen species level and intracellular calcium ions. Cyclosporine A blocked the inhibitory effect of TS-13. Thus, it can be reasonably speculated that phenolic antioxidant TS-13 starts mitochondria-dependent apoptosis in tumor cells by the opening of permeability transition pores.

Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

NASA Astrophysics Data System (ADS)

Jahn, Karolina; Buschmann, Volker; Hille, Carsten

2015-09-01

In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

Sex as a response to oxidative stress: a twofold increase in cellular reactive oxygen species activates sex genes.

PubMed

Nedelcu, Aurora M; Marcu, Oana; Michod, Richard E

2004-08-07

Organisms are constantly subjected to factors that can alter the cellular redox balance and result in the formation of a series of highly reactive molecules known as reactive oxygen species (ROS). As ROS can be damaging to biological structures, cells evolved a series of mechanisms (e.g. cell-cycle arrest, programmed cell death) to respond to high levels of ROS (i.e. oxidative stress). Recently, we presented evidence that in a facultatively sexual lineage--the multicellular green alga Volvox carteri--sex is an additional response to increased levels of stress, and probably ROS and DNA damage. Here we show that, in V. carteri, (i) sex is triggered by an approximately twofold increase in the level of cellular ROS (induced either by the natural sex-inducing stress, namely heat, or by blocking the mitochondrial electron transport chain with antimycin A), and (ii) ROS are responsible for the activation of sex genes. As most types of stress result in the overproduction of ROS, we believe that our findings will prove to extend to other facultatively sexual lineages, which could be indicative of the ancestral role of sex as an adaptive response to stress and ROS-induced DNA damage. Copyright 2004 The Royal Society

Attenuation of noise-induced hearing loss using methylene blue

PubMed Central

Park, J-S; Jou, I; Park, S M

2014-01-01

The overproduction of reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been known to contribute to the pathogenesis of noise-induced hearing loss. In this study, we discovered that in BALB/c mice pretreatment with methylene blue (MB) for 4 consecutive days significantly protected against cochlear injury by intense broad-band noise for 3 h. It decreased both compound threshold shift and permanent threshold shift and, further, reduced outer hair cell death in the cochlea. MB also reduced ROS and RNS formation after noise exposure. Furthermore, it protected against rotenone- and antimycin A-induced cell death and also reversed ATP generation in the in vitro UB-OC1 cell system. Likewise, MB effectively attenuated the noise-induced impairment of complex IV activity in the cochlea. In addition, it increased the neurotrophin-3 (NT-3) level, which could affect the synaptic connections between hair cells and spiral ganglion neurons in the noise-exposed cochlea, and also promoted the conservation of both efferent and afferent nerve terminals on the outer and inner hair cells. These findings suggest that the amelioration of impaired mitochondrial electron transport and the potentiation of NT-3 expression by treatment with MB have a significant therapeutic value in preventing ROS-mediated sensorineural hearing loss. PMID:24763057

Binding of /sup 125/I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

DOE Office of Scientific and Technical Information (OSTI.GOV)

Meyers, K.M.; Boehme, M.; Inbar, O.

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using /sup 125/I-labeled E coli O127:B8more » endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.« less

Inhibitory Effects of Respiration Inhibitors on Aflatoxin Production

PubMed Central

Sakuda, Shohei; Prabowo, Diyan Febri; Takagi, Keiko; Shiomi, Kazuro; Mori, Mihoko; Ōmura, Satoshi; Nagasawa, Hiromichi

2014-01-01

Aflatoxin production inhibitors, which do not inhibit the growth of aflatoxigenic fungi, may be used to control aflatoxin without incurring a rapid spread of resistant strains. A respiration inhibitor that inhibits aflatoxin production was identified during a screening process for natural, aflatoxin-production inhibitors. This prompted us to evaluate respiration inhibitors as potential aflatoxin control agents. The inhibitory activities of four natural inhibitors, seven synthetic miticides, and nine synthetic fungicides were evaluated on aflatoxin production in Aspergillus parasiticus. All of the natural inhibitors (rotenone, siccanin, aptenin A5, and antimycin A) inhibited fungal aflatoxin production with IC50 values around 10 µM. Among the synthetic miticides, pyridaben, fluacrypyrim, and tolfenpyrad exhibited strong inhibitory activities with IC50 values less than 0.2 µM, whereas cyflumetofen did not show significant inhibitory activity. Of the synthetic fungicides, boscalid, pyribencarb, azoxystrobin, pyraclostrobin, and kresoxim-methyl demonstrated strong inhibitory activities, with IC50 values less than 0.5 µM. Fungal growth was not significantly affected by any of the inhibitors tested at concentrations used. There was no correlation observed between the targets of respiration inhibitors (complexes I, II, and III) and their IC50 values for aflatoxin-production inhibitory activity. This study suggests that respiration inhibitors, including commonly used pesticides, are useful for aflatoxin control. PMID:24674936

Effect of hexavalent chromium on electron leakage of respiratory chain in mitochondria isolated from rat liver.

PubMed

Xie, Ying; Zhong, Caigao; Zeng, Ming; Guan, Lan; Luo, Lei

2013-01-01

In the present study, we explored reactive axygen species (ROS) production in mitochondria, the mechanism of hexavalent chromium (Cr(VI)) hepatotoxicity, and the role of protection by GSH. Intact mitochondria were isolated from rat liver tissues and mitochondrial basal respiratory rates of NADH and FADH2 respiratory chains were determined. Mitochondria were treated with Cr(VI), GSH and several complex inhibitors. Mitochondria energized by glutamate/malate were separately or jointly treated with Rotenone (Rot), diphenyleneiodonium (DPI) and antimycinA (Ant), while mitochondria energized by succinate were separately or jointly treated with Rot, DPI ' thenoyltrifluoroacetone (TTFA) and Ant. Cr(VI) concentration-dependently induced ROS production in the NADH and FADH2 respiratory chain in liver mitochondria. Basal respiratory rate of the mitochondrial FADH2 respiratory chain was significantly higher than that of NADH respiratory chain. Hepatic mitochondrial electron leakage induced by Cr(VI) from NADH respiratory chain were mainly from ubiquinone binding sites of complex I and complex III. Treatment with 50µM Cr(VI) enhances forward movement of electrons through FADH2 respiratory chain and leaking through the ubiquinone binding site of complex III. Moreover, the protective effect of GSH on liver mitochondria electron leakage is through removing excess H2O2 and reducing total ROS. Copyright © 2013 S. Karger AG, Basel.

The occurrence and control of nitric oxide generation by the plant mitochondrial electron transport chain.

PubMed

Alber, Nicole A; Sivanesan, Hampavi; Vanlerberghe, Greg C

2017-07-01

The plant mitochondrial electron transport chain (ETC) is bifurcated such that electrons from ubiquinol are passed to oxygen via the usual cytochrome path or through alternative oxidase (AOX). We previously showed that knockdown of AOX in transgenic tobacco increased leaf concentrations of nitric oxide (NO), implying that an activity capable of generating NO had been effected. Here, we identify the potential source of this NO. Treatment of leaves with antimycin A (AA, Q i -site inhibitor of Complex III) increased NO amount more than treatment with myxothiazol (Myxo, Q o -site inhibitor) despite both being equally effective at inhibiting respiration. Comparison of nitrate-grown wild-type with AOX knockdown and overexpression plants showed a negative correlation between AOX amount and NO amount following AA. Further, Myxo fully negated the ability of AA to increase NO amount. With ammonium-grown plants, neither AA nor Myxo strongly increased NO amount in any plant line. When these leaves were supplied with nitrite alongside the AA or Myxo, then the inhibitor effects across lines mirrored that of nitrate-grown plants. Hence the ETC, likely the Q-cycle of Complex III generates NO from nitrite, and AOX reduces this activity by acting as a non-energy-conserving electron sink upstream of Complex III. © 2016 John Wiley & Sons Ltd.

Malate-aspartate shuttle and exogenous NADH/cytochrome c electron transport pathway as two independent cytosolic reducing equivalent transfer systems.

PubMed

Abbrescia, Daniela Isabel; La Piana, Gianluigi; Lofrumento, Nicola Elio

2012-02-15

In mammalian cells aerobic oxidation of glucose requires reducing equivalents produced in glycolytic phase to be channelled into the phosphorylating respiratory chain for the reduction of molecular oxygen. Data never presented before show that the oxidation rate of exogenous NADH supported by the malate-aspartate shuttle system (reconstituted in vitro with isolated liver mitochondria) is comparable to the rate obtained on activation of the cytosolic NADH/cytochrome c electron transport pathway. The activities of these two reducing equivalent transport systems are independent of each other and additive. NADH oxidation induced by the malate-aspartate shuttle is inhibited by aminooxyacetate and by rotenone and/or antimycin A, two inhibitors of the respiratory chain, while the NADH/cytochrome c system remains insensitive to all of them. The two systems may simultaneously or mutually operate in the transfer of reducing equivalents from the cytosol to inside the mitochondria. In previous reports we suggested that the NADH/cytochrome c system is expected to be functioning in apoptotic cells characterized by the presence of cytochrome c in the cytosol. As additional new finding the activity of reconstituted shuttle system is linked to the amount of α-ketoglutarate generated inside the mitochondria by glutamate dehydrogenase rather than by aspartate aminotransferase. Copyright © 2011 Elsevier Inc. All rights reserved.

Dermal fibroblasts from long-lived Ames dwarf mice maintain their in vivo resistance to mitochondrial generated reactive oxygen species (ROS)

PubMed Central

Hsieh, Ching-Chyuan; Papaconstantinou, John

2009-01-01

Activation of p38 MAPK by ROS involves dissociation of an inactive, reduced thioredoxin-ASK1 complex [(SH)2Trx-ASK1]. Release of ASK1 activates its kinase activity thus stimulating the p38 MAPK pathway. The level of p38 MAPK activity is, therefore, regulated by the balance of free vs. bound ASK1. Longevity of Ames dwarf mice is attributed to their resistance to oxidative stress. The levels of (SH)2 Trx-ASK1 are more abundant in young and old dwarf mice compared to their age-matched controls suggesting that the levels of this complex may play a role in their resistance to oxidative stress. In these studies we demonstrate that dermal fibroblasts from these long-lived mice exhibit (a) higher levels of (SH)2Trx-ASK1 that correlate with their resistance to ROS generated by inhibitors of electron transport chain complexes CI (rotenone), CII (3-nitropropionic acid), CIII, (antimycin A), and H2O2-mediated activation of p38 MAPK, and (b) maintain their in vivo resistance to ROS generated by 3NPA. We propose that elevated levels of (SH)2Trx-ASK1 play a role in conferring resistance to mitochondrial generated oxidative stress and decreased endogenous ROS which are characteristics of longevity determination. PMID:20157567

Mechanism of triclosan toxicity: Mitochondrial dysfunction including complex II inhibition, superoxide release and uncoupling of oxidative phosphorylation.

PubMed

Teplova, Vera V; Belosludtsev, Konstantin N; Kruglov, Alexey G

2017-06-05

Triclosan (5-chloro-2'-(2,4-dichlorophenoxy)phenol), a widely used antibacterial agent, exerts adverse effects on the organism of mammals. Recent research reviled that triclosan at low micromolar concentrations causes mitochondrial dysfunction in many cell types, but the mechanisms of its effect are not fully understood. Here we show that exposure to triclosan disrupted membrane potential, prevented the calcium uptake-driven high-amplitude mitochondrial swelling, stimulated the respiration in the presence of complex I substrates, and suppressed the ADP-stimulated respiration in the presence of complex II substrate, succinate. Triclosan directly inhibited complex II activity. Similar to the complex II inhibitor thenoyltrifluoroacetone, triclosan induced the oxidation of the cytochromes b566 and b562 and caused the release of mitochondrial superoxide. Opposite to thenoyltrifluoroacetone, triclosan increased superoxide release synergistically with myxothiazol but not with antimycin A, indicating different topology of superoxide-producing sites. We concluded that triclosan is unique by its capability of acting as both a protonophore and an unusual complex II inhibitor, which interferes with the mitochondrial respiration by blocking the electron transfer between ubiquinone at the Q d -binding site and heme b. Our data can provide an insight into the mechanisms of the carcinogenic effect of triclosan in the liver and other tissues. Copyright © 2017 Elsevier B.V. All rights reserved.

Effects of catecholamines on rat myocardial metabolism. II. Influence of catecholamines on 32p-incorporation into rat myocardial adenylic nucleotides and their turn-over.

PubMed

Merouze, P; Gaudemer, Y; Gautheron, D

1975-01-01

1. The influence of catecholamines (adrenaline and noradrenaline) on 32Pi incorporation into intracellular phosphate and adenylic nucleotides has been studied on rat myocardium slices; consequently, the turn-over of nucleotides could be determined and compared under the influence of these two hormones. 2. In order to specify the site of action of these catecholamines, several inhibitors and activators of energetic metabolism were included in the incubation medium: 3'5'-AMP, caffein, ouabain, oligomycin, rotenone + antimycin. 3. Both catecholamines favour Pi exchanges between intra and extracellular spaces; ATP turn-over is greatly increased, while ADP turn-over is slightly decreased, and 32P-incorporation into ADP is increased. 4. 3'5'-AMP and caffein are without effect on Pi penetration; however, caffein increases catecholamine effects on this penetration. ATP turn-over is slightly increased by 3'5'-AMP or caffein. 5. Ouabain decreases ATP turn-over but does not prevent the adrenaline induced acceleration. Inhibitors of oxidative phosphorylation and electron transport decrease ATP-turn-over severely; this inhibition is not released by catecholamines. 6. It is concluded that the catecholamine effects observed are dependent on the oxidative phosphorylations process. The increase of Pi exchange by catecholamines may be related to the increase of extracellular space and cation translocations we observed with the hormones.

Mitochondrial Respiratory Chain Inhibitors Involved in ROS Production Induced by Acute High Concentrations of Iodide and the Effects of SOD as a Protective Factor

PubMed Central

Wang, Lingyan; Duan, Qi; Wang, Tingting; Ahmed, Mohamed; Zhang, Na; Li, Yongmei; Li, Lanying; Yao, Xiaomei

2015-01-01

A major source of reactive oxygen species (ROS) generation is the mitochondria. By using flow cytometry of the mitochondrial fluorescent probe, MitoSOX Red, western blot of mitochondrial ROS scavenger Peroxiredoxin (Prx) 3 and fluorescence immunostaining, ELISA of cleaved caspases 3 and 9, and TUNEL staining, we demonstrated that exposure to 100 μM KI for 2 hours significantly increased mitochondrial superoxide production and Prx 3 protein expression with increased expressions of cleaved caspases 3 and 9. Besides, we indicated that superoxide dismutase (SOD) at 1000 unit/mL attenuated the increase in mitochondrial superoxide production, Prx 3 protein expression, and lactate dehydrogenase (LDH) release and improved the relative cell viability at 100 μM KI exposure. However, SOD inhibitor diethyldithiocarbamic acid (DETC) (2 mM), Rotenone (0.5 μM), a mitochondrial complex I inhibitor, and Antimycin A (10 μM), a complex III inhibitor, caused an increase in mitochondrial superoxide production, Prx 3 protein expression, and LDH release and decreased the relative cell viability. We conclude that the inhibitors of mitochondrial respiratory chain complex I or III may be involved in oxidative stress caused by elevated concentrations of iodide, and SOD demonstrates its protective effect on the Fischer rat thyroid cell line (FRTL) cells. PMID:26294939

Association between ROS production, swelling and the respirasome integrity in cardiac mitochondria.

PubMed

Jang, Sehwan; Javadov, Sabzali

2017-09-15

Although mitochondrial Ca 2+ overload and ROS production play a critical role in mitochondria-mediated cell death, a cause-effect relationship between them remains elusive. This study elucidated the crosstalk between mitochondrial swelling, ROS production, and electron transfer chain (ETC) supercomplexes in rat heart mitochondria in response to Ca 2+ and tert-butyl hydroperoxide (TBH), a lipid-soluble organic peroxide. Results showed that ROS production induced by TBH was significantly increased in the presence of Ca 2+ in a dose-dependent manner. TBH markedly inhibited the state 3 respiration rate with no effect on the mitochondrial swelling. Ca 2+ exerted a slight effect on mitochondrial respiration that was greatly aggravated by TBH. Analysis of supercomplexes revealed a minor difference in the presence of TBH and/or Ca 2+ . However, incubation of mitochondria in the presence of high Ca 2+ (1 mM) or inhibitors of ETC complexes (rotenone and antimycin A) induced disintegration of the main supercomplex, respirasome. Thus, PTP-dependent swelling of mitochondria solely depends on Ca 2+ but not ROS. TBH has no effect on the respirasome while Ca 2+ induces disintegration of the supercomplex only at a high concentration. Intactness of individual ETC complexes I and III is important for maintenance of the structural integrity of the respirasome. Copyright © 2017 Elsevier Inc. All rights reserved.

Uncovering the formation and selection of benzylmalonyl-CoA from the biosynthesis of splenocin and enterocin reveals a versatile way to introduce amino acids into polyketide carbon scaffolds.

PubMed

Chang, Chenchen; Huang, Rong; Yan, Yan; Ma, Hongmin; Dai, Zheng; Zhang, Benying; Deng, Zixin; Liu, Wen; Qu, Xudong

2015-04-01

Selective modification of carbon scaffolds via biosynthetic engineering is important for polyketide structural diversification. Yet, this scope is currently restricted to simple aliphatic groups due to (1) limited variety of CoA-linked extender units, which lack aromatic structures and chemical reactivity, and (2) narrow acyltransferase (AT) specificity, which is limited to aliphatic CoA-linked extender units. In this report, we uncovered and characterized the first aromatic CoA-linked extender unit benzylmalonyl-CoA from the biosynthetic pathways of splenocin and enterocin in Streptomyces sp. CNQ431. Its synthesis employs a deamination/reductive carboxylation strategy to convert phenylalanine into benzylmalonyl-CoA, providing a link between amino acid and CoA-linked extender unit synthesis. By characterization of its selection, we further validated that AT domains of splenocin, and antimycin polyketide synthases are able to select this extender unit to introduce the phenyl group into their dilactone scaffolds. The biosynthetic machinery involved in the formation of this extender unit is highly versatile and can be potentially tailored for tyrosine, histidine and aspartic acid. The disclosed aromatic extender unit, amino acid-oriented synthetic pathway, and aromatic-selective AT domains provides a systematic breakthrough toward current knowledge of polyketide extender unit formation and selection, and also opens a route for further engineering of polyketide carbon scaffolds using amino acids.

Activation of cyclic electron flow by hydrogen peroxide in vivo

DOE PAGES

Strand, Deserah D.; Livingston, Aaron K.; Satoh-Cruz, Mio; ...

2015-04-13

Cyclic electron flow (CEF) around photosystem I is thought to balance the ATP/NADPH energy budget of photosynthesis, requiring that its rate be finely regulated. The mechanisms of this regulation are not well understood. We observed that mutants that exhibited constitutively high rates of CEF also showed elevated production of H 2O 2. We thus tested the hypothesis that CEF can be activated by H 2O 2 in vivo. CEF was strongly increased by H 2O 2 both by infiltration or in situ production by chloroplast-localized glycolate oxidase, implying that H 2O 2 can activate CEF either directly by redox modulationmore » of key enzymes, or indirectly by affecting other photosynthetic processes. CEF appeared with a half time of about 20 min after exposure to H 2O 2, suggesting activation of previously expressed CEF-related machinery. H 2O 2-dependent CEF was not sensitive to antimycin A or loss of PGR5, indicating that increased CEF probably does not involve the PGR5-PGRL1 associated pathway. In contrast, the rise in CEF was not observed in a mutant deficient in the chloroplast NADPH:PQ reductase (NDH), supporting the involvement of this complex in CEF activated by H 2O 2. In conclusion, we propose that H 2O 2 is a missing link between environmental stress, metabolism, and redox regulation of CEF in higher plants.« less

Occurrence of the malate-aspartate shuttle in various tumor types.

PubMed

Greenhouse, W V; Lehninger, A L

1976-04-01

The activity of the malate-aspartate shuttle for the reoxidation of cytoplasmic reduced nicotinamide adenine dinucleotide (NADH) by mitochondria was assessed in six lines of rodent ascites tumor cells (two strains of Ehrlich ascites carcinoma, Krebs II carcinoma, Novikoff hepatoma, AS-30D hepatoma, and L1210 mouse leukemia). All the tumor cells examined showed mitochondrial reoxidation of cytoplasmic NADH, as evidenced by the accumulation of pyruvate when the cells were incubated aerobically with L-lactate. Reoxidation of cytoplasmic NADH thus generated was completely inhibited by the transaminase inhibitor aminooxyacetate. The involvement of the respiratory chain in the reoxidation of cytoplasmic NADH was demonstrated by the action of cyanide, rotenone, and antimycin A, which strongly inhibited the formation of pyruvate from added L-lactate. Compounds that inhibit the carrier-mediated entry of malate into mitochondria, such as butylmalonate, benzenetricarboxylate, and iodobenzylmalonate, also inhibited the accumulation of pyruvate from added L-lactate by the tumor cells. The maximal rate of the malate-aspartate shuttle was established by addtion of arsenite to inhibit the mitochondrial oxidation of the pyruvate formed from added lactate. The capacity of the various tumor lines for the reoxidation of cytoplasmic NADH via the malate-aspartate shuttle approaches 20% of the total respiratory rate of the cells and thus appears to be sufficient to account for the mitochondrial reoxidation of that fraction of glycolytic NADH not reoxidized by pyruvate and lactate dehydrognenase in the cytoplasm.

Kinetics of molybdenum reduction to molybdenum blue by Bacillus sp. strain A.rzi.

PubMed

Othman, A R; Bakar, N A; Halmi, M I E; Johari, W L W; Ahmad, S A; Jirangon, H; Syed, M A; Shukor, M Y

2013-01-01

Molybdenum is very toxic to agricultural animals. Mo-reducing bacterium can be used to immobilize soluble molybdenum to insoluble forms, reducing its toxicity in the process. In this work the isolation of a novel molybdate-reducing Gram positive bacterium tentatively identified as Bacillus sp. strain A.rzi from a metal-contaminated soil is reported. The cellular reduction of molybdate to molybdenum blue occurred optimally at 4 mM phosphate, using 1% (w/v) glucose, 50 mM molybdate, between 28 and 30 °C and at pH 7.3. The spectrum of the Mo-blue product showed a maximum peak at 865 nm and a shoulder at 700 nm. Inhibitors of bacterial electron transport system (ETS) such as rotenone, sodium azide, antimycin A, and potassium cyanide could not inhibit the molybdenum-reducing activity. At 0.1 mM, mercury, copper, cadmium, arsenic, lead, chromium, cobalt, and zinc showed strong inhibition on molybdate reduction by crude enzyme. The best model that fitted the experimental data well was Luong followed by Haldane and Monod. The calculated value for Luong's constants p max, K(s), S(m), and n was 5.88 μmole Mo-blue hr(-1), 70.36 mM, 108.22 mM, and 0.74, respectively. The characteristics of this bacterium make it an ideal tool for bioremediation of molybdenum pollution.

Ex vivo hyperpolarized MR spectroscopy on isolated renal tubular cells: A novel technique for cell energy phenotyping.

PubMed

Juul, Troels; Palm, Fredrik; Nielsen, Per Mose; Bertelsen, Lotte Bonde; Laustsen, Christoffer

2017-08-01

It has been demonstrated that hyperpolarized 13 C MR is a useful tool to study cultured cells. However, cells in culture can alter phenotype, which raises concerns regarding the in vivo significance of such findings. Here we investigate if metabolic phenotyping using hyperpolarized 13 C MR is suitable for cells isolated from kidney tissue, without prior cell culture. Isolation of tubular cells from freshly excised kidney tissue and treatment with either ouabain or antimycin A was investigated with hyperpolarized MR spectroscopy on a 9.4 Tesla preclinical imaging system. Isolation of tubular cells from less than 2 g of kidney tissue generally resulted in more than 10 million live tubular cells. This amount of cells was enough to yield robust signals from the conversion of 13 C-pyruvate to lactate, bicarbonate and alanine, demonstrating that metabolic flux by means of both anaerobic and aerobic pathways can be quantified using this technique. Ex vivo metabolic phenotyping using hyperpolarized 13 C MR in a preclinical system is a useful technique to study energy metabolism in freshly isolated renal tubular cells. This technique has the potential to advance our understanding of both normal cell physiology as well as pathological processes contributing to kidney disease. Magn Reson Med 78:457-461, 2017. © 2016 International Society for Magnetic Resonance in Medicine. © 2016 International Society for Magnetic Resonance in Medicine.

A new inhibitor of the CoQ-dependent redox reactions in mitochondria and chromatophores.

PubMed

Kolesova, G M; Belyakova, M M; Mamedov, M D; Yaguzhinsky, L S

2000-05-01

The effects of 3,4-dimethoxyphenyl-1-amylketone (DPK) on the CoQ-dependent stages of the electron transport systems in mitochondria and Rhodobacter sphaeroides chromatophores were studied. The two systems contain the complete Q-cycle. The sensitivities of the Q-cycles of two electron transport systems to antimycin, myxothiazole, and other inhibitors are virtually indistinguishable from one another, but these systems have different CoQ reduction processes. The dependence of the inhibition extent of the mitochondrial succinate oxidase on the DPK concentration was studied. The effective concentration of DPK is 0.5-2.5 mM. The presence of the point of inflection in the titration curve indicates that there are two mechanisms of inhibition. The effects of DPK on the extent of reduction of cytochromes b and c1 + c in mitochondria as well as on the electrogenic stages of the Q-cycle in chromatophores were examined. The experiments showed that DPK prevents three CoQ-dependent reactions related to the Q-cycle: electron transport between succinate dehydrogenase and the Q-cycle in mitochondria and functioning of the Z (o) and C (i) sites of the Q-cycle in chromatophores. DPK does not affect the electrogenic reaction associated with protonation of the secondary quinone acceptor QB in the reaction center of chromatophores. The mitochondrial NADH-dehydrogenase is inhibited by DPK at lower but comparable concentrations (C50 = 0.2 mM).

Characterization of oxidative phosphorylation in the colorless chlorophyte Polytomella sp. Its mitochondrial respiratory chain lacks a plant-like alternative oxidase.

PubMed

Reyes-Prieto, Adrián; El-Hafidi, Mohammed; Moreno-Sánchez, Rafael; González-Halphen, Diego

2002-07-01

The presence of an alternative oxidase (AOX) in Polytomella sp., a colorless relative of Chlamydomonas reinhardtii, was explored. Oxygen uptake in Polytomella sp. mitochondria was inhibited by KCN (94%) or antimycin (96%), and the remaining cyanide-resistant respiration was not blocked by the AOX inhibitors salicylhydroxamic acid (SHAM) or n-propylgallate. No stimulation of an AOX activity was found upon addition of either pyruvate, alpha-ketoglutarate, or AMP, or by treatment with DTT. An antibody raised against C. reinhardtii AOX did not recognized any polypeptide band of Polytomella sp. mitochondria in Western blots. Also, PCR experiments and Southern blot analysis failed to identify an Aox gene in this colorless alga. Finally, KCN exposure of cell cultures failed to stimulate an AOX activity. Nevertheless, KCN exposure of Polytomella sp. cells induced diminished mitochondrial respiration (20%) and apparent changes in cytochrome c oxidase affinity towards cyanide. KCN-adapted cells exhibited a significant increase of a-type cytochromes, suggesting accumulation of inactive forms of cytochrome c oxidase. Another effect of KCN exposure was the reduction of the protein/fatty acid ratio of mitochondrial membranes, which may affect the observed respiratory activity. We conclude that Polytomella lacks a plant-like AOX, and that its corresponding gene was probably lost during the divergence of this colorless genus from its close photosynthetic relatives.

The dopamine-D2-receptor agonist ropinirole dose-dependently blocks the Ca2+-triggered permeability transition of mitochondria.

PubMed

Parvez, Suhel; Winkler-Stuck, Kirstin; Hertel, Silvia; Schönfeld, Peter; Siemen, Detlef

2010-01-01

Ropinirole, an agonist of the post-synaptic dopamine D2-receptor, exerts neuroprotective activity. The mechanism is still under discussion. Assuming that this neuroprotection might be associated with inhibition of the apoptotic cascade underlying cell death, we examined a possible effect of ropinirole on the permeability transition pore (mtPTP) in the mitochondrial inner membrane. Using isolated rat liver mitochondria, the effect of ropinirole was studied on Ca2+-triggered large amplitude swelling, membrane depolarization and cytochrome c release. In addition, the effect of ropinirole on oxidation of added, membrane-impermeable NADH was investigated. The results revealed doubtlessly, that ropinirole can inhibit permeability transition. In patch-clamp experiments on mitoplasts, we show directly that ropinirole interacts with the mtPTP. Thus, ropinirole reversibly inhibits the opening of mtPTP with an IC50 of 3.4 microM and a Hill coefficient of 1.3. In both systems (i.e. energized mitochondria and mitoplasts) the inhibitory effect on permeability transition was attenuated by increasing concentrations of inorganic phosphate. In addition, we showed with antimycin A-treated mitochondria that ropinirole failed to suppress respiratory chain-linked reactive oxygen species release. In conclusion, our data suggest that the neuroprotective activity of ropinirole is due to the blockade of the Ca2+-triggered permeability transition. Copyright © 2010 Elsevier B.V. All rights reserved.

Release of mitochondrial glutathione and calcium by a cyclosporin A-sensitive mechanism occurs without large amplitude swelling.

PubMed

Savage, M K; Reed, D J

1994-11-15

Treatment of isolated mitochondria with calcium and inorganic phosphate induces inner membrane permeability that is thought to be mediated through a non-selective, calcium-dependent pore. The inner membrane permeability results in the rapid efflux of small matrix solutes such as glutathione and calcium, loss of coupled functions, and large amplitude swelling. We have identified conditions of permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing calcium and inorganic phosphate abolished large-amplitude swelling of mitochondria but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial glutathione and calcium. The release of both glutathione and calcium was inhibited by the addition of cyclosporin A, a potent inhibitor of permeability transition. Transmission electron microscopy analysis, combined with the glutathione and calcium release data, indicate that permeability transition can be observed in the absence of large-amplitude swelling. Permeability transition occurring both with and without large-amplitude swelling was accompanied by a collapse of the membrane potential. We conclude that cyclosporin A-sensitive permeability transition can occur without obvious morphological changes such as large-amplitude swelling. Monitoring the cyclosporin A-sensitive release of concentrated endogenous matrix solutes, such as GSH, may be a sensitive and useful indicator of permeability transition.

Effects of mitochondrial poisons on glutathione redox potential and carotid body chemoreceptor activity.

PubMed

Gomez-Niño, A; Agapito, M T; Obeso, A; Gonzalez, C

2009-01-01

Low oxygen sensing in chemoreceptor cells involves the inhibition of specific plasma membrane K(+) channels, suggesting that mitochondria-derived reactive oxygen species (ROS) link hypoxia to K(+) channel inhibition, subsequent cell depolarization and activation of neurotransmitter release. We have used several mitochondrial poisons, alone and in combination with the antioxidant N-acetylcysteine (NAC), and quantify their capacity to alter GSH/GSSG levels and glutathione redox potential (E(GSH)) in rat diaphragm. Selected concentrations of mitochondrial poisons with or without NAC were tested for their capacity to activate neurotransmitter release in chemoreceptor cells and to alter ATP levels in intact rat carotid body (CB). We found that rotenone (1 microM), antimycin A (0.2 microg/ml) and sodium azide (5mM) decreased E(GSH); NAC restored E(GSH) to control values. At those concentrations mitochondrial poisons activated neurotransmitter release from CB chemoreceptor cells and decreased CB ATP levels, NAC being ineffective to modify these responses. Additional experiments with 3-nitroprionate (5mM), lower concentrations of rotenone and dinitrophenol revealed variable relationships between E(GSH) and chemoreceptor cell neurotransmitter release responses and ATP levels. These findings indicate a lack of correlation between mitochondrial-generated modifications of E(GSH) and chemoreceptor cells activity. This lack of correlation renders unlikely that alteration of mitochondrial production of ROS is the physiological pathway chemoreceptor cells use to signal hypoxia.

The components of an α-glycerophosphate cycle and their relation to oxidative metabolism in the lens

PubMed Central

Griffiths, M. H.

1966-01-01

1. The concentration of ATP in a lens brei is maintained when the brei is incubated in oxygen with α-glycerophosphate. Lack of α-glycerophosphate or incubation in nitrogen causes the concentration to decrease. α-Glycerophosphate has some effect under anaerobic conditions but this is not sufficient to account for the maintenance in oxygen. 2. Manometric experiments show that α-glycerophosphate enhances the respiration of lens preparations. This respiration can be further increased by the addition of ADP and is abolished by cyanide and antimycin. The inference from these experiments is that a mitochondrial system able to oxidize α-glycerophosphate is present, i.e. the particulate half of the α-glycerophosphate cycle. 3. More than the calculated proportion of NADH is used when limiting amounts of dihydroxyacetone phosphate are added to lens tissue in spectrophotometric experiments. Dihydroxyacetone phosphate is therefore regenerated and an α-glycerophosphate cycle is operative. 4. A preparation of a particulate α-glycerophosphate dehydrogenase that takes up oxygen with methylene blue as electron acceptor is described. 5. Methods for obtaining mitochondria from lens are compared, and a useful extraction medium is defined. 6. Mitochondria with activities of the same order of magnitude as those obtained from liver, with α-glycerophosphate and glutamate as substrates, are prepared from epithelium detached from the capsule; some respiratory control is observed. PMID:4290550

Mobilization of Ca2+ by Cyclic ADP-Ribose from the Endoplasmic Reticulum of Cauliflower Florets1

PubMed Central

Navazio, Lorella; Mariani, Paola; Sanders, Dale

2001-01-01

The NAD+ metabolite cADP-Rib (cADPR) elevates cytosolic free Ca2+ in plants and thereby plays a central role in signal transduction pathways evoked by the drought and stress hormone abscisic acid. cADPR is known to mobilize Ca2+ from the large vacuole of mature cells. To determine whether additional sites for cADPR-gated Ca2+ release reside in plant cells, microsomes from cauliflower (Brassica oleracea) inflorescences were subfractionated on sucrose density gradients, and the distribution of cADPR-elicited Ca2+ release was monitored. cADPR-gated Ca2+ release was detected in the heavy-density fractions associated with rough endoplasmic reticulum (ER). cADPR-dependent Ca2+ release co-migrated with two ER markers, calnexin and antimycin A-insensitive NADH-cytochrome c reductase activity. To investigate the possibility that contaminating plasma membrane in the ER-rich fractions was responsible for the observed release, plasma membrane vesicles were purified by aqueous two-phase partitioning, everted with Brij-58, and loaded with Ca2+: These vesicles failed to respond to cADPR. Ca2+ release evoked by cADPR at the ER was fully inhibited by ruthenium red and 8-NH2-cADPR, a specific antagonist of cADPR-gated Ca2+ release in animal cells. The presence of a Ca2+ release pathway activated by cADPR at higher plant ER reinforces the notion that, alongside the vacuole, the ER participates in Ca2+ signaling. PMID:11299392

Ubiquinol-binding site in the alternative oxidase: mutagenesis reveals features important for substrate binding and inhibition.

PubMed

Albury, Mary S; Elliott, Catherine; Moore, Anthony L

2010-12-01

The alternative oxidase (AOX) is a non-protonmotive ubiquinol oxidase that is found in all plants, some fungi, green algae, bacteria and pathogenic protozoa. The lack of AOX in the mammalian host renders this protein an important potential therapeutic target in the treatment of pathogenic protozoan infections. Bioinformatic searches revealed that, within a putative ubiquinol-binding crevice in AOX, Gln242, Asn247, Tyr253, Ser256, His261 and Arg262 were highly conserved. To confirm that these amino-acid residues are important for ubiquinol-binding and hence activity substitution mutations were generated and characterised. Assessment of AOX activity in isolated Schizosaccharomyces pombe mitochondria revealed that mutation of either Gln242, Ser256, His261 and Arg262 resulted in >90% inhibition of antimycin A-insensitive respiration suggesting that hydroxyl, guanidino, imidazole groups, polar and charged residues in addition to the size of the amino-acid chain are important for ubiquinone-binding. Substitution of Asn247 with glutamine or Tyr253 with phenylalanine had little effect upon the respiratory rate indicating that these residues are not critical for AOX activity. However replacement of Tyr253 by alanine resulted in a 72% loss of activity suggesting that the benzoquinone group and not hydroxyl group is important for quinol binding. These results provide important new insights into the ubiquinol-binding site of the alternative oxidase, the identity of which maybe important for future rational drug design. Copyright © 2010 Elsevier B.V. All rights reserved.

Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

PubMed

Charlton, R R; Wenner, C E

1978-03-15

1. The interaction of intact Ehrlich ascites-tumour cells with Ca2+ at 37 degrees C consists of Ca2+ uptake followed by efflux from the cells. Under optimum conditions, two or three cycles of uptake and efflux are observed in the first 15 min after Ca2+ addition. 2. The respiratory substrates malate, succinate and ascorbate plus p-phenylenediamine support Ca2+ uptake. Ca2+ uptake at 37 degrees C is sensitive to the respiratory inhibitors rotenone and antimycin A when appropriate substrates are present. Ca2+ uptake and retention are inhibited by the uncoupler S-13. 3. Increasing extracellular Pi (12 to 30 mM) stimulates uncoupler-sensitive Ca2+ uptake, which reaches a maximum extent of 15 nmol/mg of protein when supported by succinate respiration. Ca2+ efflux is partially inhibited at 30 mM-Pi. 4. Optimum Ca2+ uptake occurs in the presence of succinate and Pi, suggesting that availability of substrate and Pi are rate-limiting. K. Ca2+ uptake occurs at 4 degrees C and is sensitive to uncouplers and oligomycin. Ca2+ efflux at this temperature is minimal. These data are consistent with a model in which passive diffusion of Ca2+ through the plasma membrane is followed by active uptake by the mitochondria. Ca2+ uptake is supported by substrates entering respiration at all three energy-coupling sites. Ca2+ efflux appears to be an active process with a high temperature coefficient.

Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release.

PubMed

Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen Hm

2017-06-01

The detection of intracellular molecular oxygen (O 2 ) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6-pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O 2 -independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O 2 . Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O 2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at 'physiological' tissue O 2 levels (5% O 2 ). Multiplexing also allowed us to monitor intracellular O 2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O 2 despite a decrease in mitochondrial membrane potential.

Single-cell time-lapse imaging of intracellular O2 in response to metabolic inhibition and mitochondrial cytochrome-c release

PubMed Central

Düssmann, Heiko; Perez-Alvarez, Sergio; Anilkumar, Ujval; Papkovsky, Dmitri B; Prehn, Jochen HM

2017-01-01

The detection of intracellular molecular oxygen (O2) levels is important for understanding cell physiology, cell death, and drug effects, and has recently been improved with the development of oxygen-sensitive probes that are compatible with live cell time-lapse microscopy. We here provide a protocol for the use of the nanoparticle probe MitoImage-MM2 to monitor intracellular oxygen levels by confocal microscopy under baseline conditions, in response to mitochondrial toxins, and following mitochondrial cytochrome-c release. We demonstrate that the MitoImage-MM2 probe, which embeds Pt(II)-5,10,15,20-tetrakis-(2,3,4,5,6–pentafluorophenyl)-porphyrin as oxygen sensor and poly(9,9-dioctylfluorene) as an O2-independent component, enables quantitative, ratiometric time-lapse imaging of intracellular O2. Multiplexing with tetra-methyl-rhodamine-methyl ester in HeLa cervical cancer cells showed significant increases in intracellular O2 accompanied by strong mitochondrial depolarization when respiratory chain complexes III or IV were inhibited by Antimycin A or sodium azide, respectively, and when cells were maintained at ‘physiological’ tissue O2 levels (5% O2). Multiplexing also allowed us to monitor intracellular O2 during the apoptotic signaling process of mitochondrial outer membrane permeabilization in HeLa expressing cytochrome-c-eGFP, and demonstrated that mitochondria post cytochrome-c release are able to retain their capacity to respire at physiological O2 despite a decrease in mitochondrial membrane potential. PMID:28569778

Isolating antifungals from fungus-growing ant symbionts using a genome-guided chemistry approach.

PubMed

Seipke, Ryan F; Grüschow, Sabine; Goss, Rebecca J M; Hutchings, Matthew I

2012-01-01

We describe methods used to isolate and identify antifungal compounds from actinomycete strains associated with the leaf-cutter ant Acromyrmex octospinosus. These ants use antibiotics produced by symbiotic actinomycete bacteria to protect themselves and their fungal cultivar against bacterial and fungal infections. The fungal cultivar serves as the sole food source for the ant colony, which can number up to tens of thousands of individuals. We describe how we isolate bacteria from leaf-cutter ants collected in Trinidad and analyze the antifungal compounds made by two of these strains (Pseudonocardia and Streptomyces spp.), using a combination of genome analysis, mutagenesis, and chemical isolation. These methods should be generalizable to a wide variety of insect-symbiont situations. Although more time consuming than traditional activity-guided fractionation methods, this approach provides a powerful technique for unlocking the complete biosynthetic potential of individual strains and for avoiding the problems of rediscovery of known compounds. We describe the discovery of a novel nystatin compound, named nystatin P1, and identification of the biosynthetic pathway for antimycins, compounds that were first described more than 60 years ago. We also report that disruption of two known antifungal pathways in a single Streptomyces strain has revealed a third, and likely novel, antifungal plus four more pathways with unknown products. This validates our approach, which clearly has the potential to identify numerous new compounds, even from well-characterized actinomycete strains. Copyright © 2012 Elsevier Inc. All rights reserved.

Inhibitor-induced oxidation of the nucleus and cytosol in Arabidopsis thaliana: implications for organelle to nucleus retrograde signalling

PubMed Central

Karpinska, Barbara; Alomrani, Sarah Owdah

2017-01-01

Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction–oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1, WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression. This article is part of the themed issue ‘Enhancing photosynthesis in crop plants: targets for improvement’. PMID:28808105

Mitochondrial impacts of insecticidal formate esters in insecticide-resistant and insecticide-susceptible Drosophila melanogaster.

PubMed

Song, Cheol; Scharf, Michael E

2009-06-01

Previous research on insecticidal formate esters in flies and mosquitoes has documented toxicity profiles, metabolism characteristics and neurological impacts. The research presented here investigated mitochondrial impacts of insecticidal formate esters and their hydrolyzed metabolite formic acid in the model dipteran insect Drosophila melanogaster Meig. These studies compared two Drosophila strains: an insecticide-susceptible strain (Canton-S) and a strain resistant by cytochrome P450 overexpression (Hikone-R). In initial studies investigating inhibition of mitochondrial cytochrome c oxidase, two proven insecticidal materials (hydramethylnon and sodium cyanide) caused significant inhibition. However, for insecticidal formate esters and formic acid, no significant inhibition was identified in either fly strain. Mitochondrial impacts of formate esters were then investigated further by tracking toxicant-induced cytochrome c release from mitochondria into the cytoplasm, a biomarker of apoptosis and neurological dysfunction. Formic acid and three positive control treatments (rotenone, antimycin A and sodium cyanide) induced cytochrome c release, verifying that formic acid is capable of causing mitochondrial disruption. However, when comparing formate ester hydrolysis and cytochrome c release between Drosophila strains, formic acid liberation was only weakly correlated with cytochrome c release in the susceptible Canton-S strain (r(2) = 0.70). The resistant Hikone-R strain showed no correlation (r(2) < 0.0001) between formate ester hydrolysis and cytochrome c release. The findings of this study provide confirmation of mitochondrial impacts by insecticidal formate esters and suggest links between mitochondrial disruption, respiratory inhibition, apoptosis and formate-ester-induced neurotoxicity.

Activation of PPARα by Oral Clofibrate Increases Renal Fatty Acid Oxidation in Developing Pigs.

PubMed

He, Yonghui; Khan, Imad; Bai, Xiumei; Odle, Jack; Xi, Lin

2017-12-08

The objective of this study was to evaluate the effects of peroxisome proliferator-activated receptor α (PPARα) activation by clofibrate on both mitochondrial and peroxisomal fatty acid oxidation in the developing kidney. Ten newborn pigs from 5 litters were randomly assigned to two groups and fed either 5 mL of a control vehicle (2% Tween 80) or a vehicle containing clofibrate (75 mg/kg body weight, treatment). The pigs received oral gavage daily for three days. In vitro fatty acid oxidation was then measured in kidneys with and without mitochondria inhibitors (antimycin A and rotenone) using [1- 14 C]-labeled oleic acid (C18:1) and erucic acid (C22:1) as substrates. Clofibrate significantly stimulated C18:1 and C22:1 oxidation in mitochondria ( p < 0.001) but not in peroxisomes. In addition, the oxidation rate of C18:1 was greater in mitochondria than peroxisomes, while the oxidation of C22:1 was higher in peroxisomes than mitochondria ( p < 0.001). Consistent with the increase in fatty acid oxidation, the mRNA abundance and enzyme activity of carnitine palmitoyltransferase I (CPT I) in mitochondria were increased. Although mRNA of mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (mHMGCS) was increased, the β-hydroxybutyrate concentration measured in kidneys did not increase in pigs treated with clofibrate. These findings indicate that PPARα activation stimulates renal fatty acid oxidation but not ketogenesis.

Are mitochondria a permanent source of reactive oxygen species?

PubMed

Staniek, K; Nohl, H

2000-11-20

The observation that in isolated mitochondria electrons may leak out of the respiratory chain to form superoxide radicals (O(2)(radical-)) has prompted the assumption that O(2)(radical-) formation is a compulsory by-product of respiration. Since mitochondrial O(2)(radical-) formation under homeostatic conditions could not be demonstrated in situ so far, conclusions drawn from isolated mitochondria must be considered with precaution. The present study reveals a link between electron deviation from the respiratory chain to oxygen and the coupling state in the presence of antimycin A. Another important factor is the analytical system applied for the detection of activated oxygen species. Due to the presence of superoxide dismutase in mitochondria, O(2)(radical-) release cannot be realistically determined in intact mitochondria. We therefore followed the release of the stable dismutation product H(2)O(2) by comparing most frequently used H(2)O(2) detection methods. The possible interaction of the detection systems with the respiratory chain was avoided by a recently developed method, which was compared with conventional methods. Irrespective of the methods applied, the substrates used for respiration and the state of respiration established, intact mitochondria could not be made to release H(2)O(2) from dismutating O(2)(radical-). Although regular mitochondrial respiration is unlikely to supply single electrons for O(2)(radical-) formation our study does not exclude the possibility of the respiratory chain becoming a radical source under certain conditions.

Spathaspora passalidarum selected for resistance to AFEX hydrolysate shows decreased cell yield.

PubMed

Su, Yi-Kai; Willis, Laura B; Rehmann, Lars; Smith, David; Jeffries, Thomas W

2018-06-21

This study employed cell recycling, batch adaptation, cell mating and high throughput screening to select adapted Spathaspora passalidarum strains with improved fermentative ability.The most promising candidate, YK208-E11 (E11) showed a 3-fold increase in specific fermentation rate compared to the parental strain and an ethanol yield greater than 0.45 g/g substrate while co-utilizing cellobiose, glucose, and xylose. Further characterization showed that strain E11 also makes 40% less biomass compared to the parental strain when cultivated in rich media under aerobic conditions. A tetrazolium agar overlay assay in the presence of respiration inhibitors, including rotenone, antimycin A, KCN, and salicylhydroxamic acid elucidated the nature of the mutational events. Results indicated that E11 has a deficiency in its respiration system that could contribute to its low cell yield. Strain E11 was subjected to whole genome sequencing and a ∼11 kb deletion was identified; the open reading frames absent in strain E11 code for proteins with predicted functions in respiration, cell division and the actin cytoskeleton, and may contribute to the observed physiology of the adapted strain. Results of the tetrazolium overlay also suggest that cultivation on xylose affects the respiration capacity in the wild-type strain, which could account for its faster fermentation of xylose as compared to glucose. These results support our previous finding that S. passalidarum has highly unusual physiological responses to xylose under oxygen limitation.

Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction.

PubMed

Resseguie, Emily A; Staversky, Rhonda J; Brookes, Paul S; O'Reilly, Michael A

2015-08-01

High levels of oxygen (hyperoxia) are often used to treat individuals with respiratory distress, yet prolonged hyperoxia causes mitochondrial dysfunction and excessive reactive oxygen species (ROS) that can damage molecules such as DNA. Ataxia telangiectasia mutated (ATM) kinase is activated by nuclear DNA double strand breaks and delays hyperoxia-induced cell death through downstream targets p53 and p21. Evidence for its role in regulating mitochondrial function is emerging, yet it has not been determined if mitochondrial dysfunction or ROS activates ATM. Because ATM maintains mitochondrial homeostasis, we hypothesized that hyperoxia induces both mitochondrial dysfunction and ROS that activate ATM. In A549 lung epithelial cells, hyperoxia decreased mitochondrial respiratory reserve capacity at 12h and basal respiration by 48 h. ROS were significantly increased at 24h, yet mitochondrial DNA double strand breaks were not detected. ATM was not required for activating p53 when mitochondrial respiration was inhibited by chronic exposure to antimycin A. Also, ATM was not further activated by mitochondrial ROS, which were enhanced by depleting manganese superoxide dismutase (SOD2). In contrast, ATM dampened the accumulation of mitochondrial ROS during exposure to hyperoxia. Our findings suggest that hyperoxia-induced mitochondrial dysfunction and ROS do not activate ATM. ATM more likely carries out its canonical response to nuclear DNA damage and may function to attenuate mitochondrial ROS that contribute to oxygen toxicity. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Mitochondrial Respiratory Chain Dysfunction in Dorsal Root Ganglia of Streptozotocin-Induced Diabetic Rats and Its Correction by Insulin Treatment

PubMed Central

Chowdhury, Subir K. Roy; Zherebitskaya, Elena; Smith, Darrell R.; Akude, Eli; Chattopadhyay, Sharmila; Jolivalt, Corinne G.; Calcutt, Nigel A.; Fernyhough, Paul

2010-01-01

OBJECTIVE Impairments in mitochondrial physiology may play a role in diabetic sensory neuropathy. We tested the hypothesis that mitochondrial dysfunction in sensory neurons is due to abnormal mitochondrial respiratory function. RESEARCH DESIGN AND METHODS Rates of oxygen consumption were measured in mitochondria from dorsal root ganglia (DRG) of 12- to- 22-week streptozotocin (STZ)-induced diabetic rats, diabetic rats treated with insulin, and age-matched controls. Activities and expression of components of mitochondrial complexes and reactive oxygen species (ROS) were analyzed. RESULTS Rates of coupled respiration with pyruvate + malate (P + M) and with ascorbate + TMPD (Asc + TMPD) in DRG were unchanged after 12 weeks of diabetes. By 22 weeks of diabetes, respiration with P + M was significantly decreased by 31–44% and with Asc + TMPD by 29–39% compared with control. Attenuated mitochondrial respiratory activity of STZ-diabetic rats was significantly improved by insulin that did not correct other indices of diabetes. Activities of mitochondrial complexes I and IV and the Krebs cycle enzyme, citrate synthase, were decreased in mitochondria from DRG of 22-week STZ-diabetic rats compared with control. ROS levels in perikarya of DRG neurons were not altered by diabetes, but ROS generation from mitochondria treated with antimycin A was diminished compared with control. Reduced mitochondrial respiratory function was associated with downregulation of expression of mitochondrial proteins. CONCLUSIONS Mitochondrial dysfunction in sensory neurons from type 1 diabetic rats is associated with impaired rates of respiratory activity and occurs without a significant rise in perikaryal ROS. PMID:20103706

The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria.

PubMed

Macchioni, Lara; Davidescu, Magdalena; Roberti, Rita; Corazzi, Lanfranco

2014-10-01

Tumor cells favor abnormal energy production via aerobic glycolysis and show resistance to apoptosis, suggesting the involvement of mitochondrial dysfunction. The differences between normal and cancer cells in their energy metabolism provide a biochemical basis for developing new therapeutic strategies. The energy blocker 3-bromopyruvate (3BP) can eradicate liver cancer in animals without associated toxicity, and is a potent anticancer towards glioblastoma cells. Since mitochondria are 3BP targets, in this work the effects of 3BP on the bioenergetics of normal rat brain mitochondria were investigated in vitro, in comparison with the anticancer agent lonidamine (LND). Whereas LND impaired oxygen consumption dependent on any complex of the respiratory chain, 3BP was inhibitory to malate/pyruvate and succinate (Complexes I and II), but preserved respiration from glycerol-3-phosphate and ascorbate (Complex IV). Accordingly, although electron flow along the respiratory chain and ATP levels were decreased by 3BP in malate/pyruvate- and succinate-fed mitochondria, they were not significantly influenced from glycerol-3-phosphate- or ascorbate-fed mitochondria. LND produced a decrease in electron flow from all substrates tested. No ROS were produced from any substrate, with the exception of 3BP-induced H(2)O(2) release from succinate, which suggests an antimycin-like action of 3BP as an inhibitor of Complex III. We can conclude that 3BP does not abolish completely respiration and ATP synthesis in brain mitochondria, and has a limited effect on ROS production, confirming that this drug may have limited harmful effects on normal cells.

Mitochondrion-Derived Reactive Oxygen Species Lead to Enhanced Amyloid Beta Formation

PubMed Central

Schütt, Tanja; Kurz, Christopher; Eckert, Schamim H.; Schiller, Carola; Occhipinti, Angelo; Mai, Sören; Jendrach, Marina; Eckert, Gunter P.; Kruse, Shane E.; Palmiter, Richard D.; Brandt, Ulrich; Dröse, Stephan; Wittig, Ilka; Willem, Michael; Haass, Christian; Reichert, Andreas S.; Müller, Walter E.

2012-01-01

Abstract Aims: Intracellular amyloid beta (Aβ) oligomers and extracellular Aβ plaques are key players in the progression of sporadic Alzheimer's disease (AD). Still, the molecular signals triggering Aβ production are largely unclear. We asked whether mitochondrion-derived reactive oxygen species (ROS) are sufficient to increase Aβ generation and thereby initiate a vicious cycle further impairing mitochondrial function. Results: Complex I and III dysfunction was induced in a cell model using the respiratory inhibitors rotenone and antimycin, resulting in mitochondrial dysfunction and enhanced ROS levels. Both treatments lead to elevated levels of Aβ. Presence of an antioxidant rescued mitochondrial function and reduced formation of Aβ, demonstrating that the observed effects depended on ROS. Conversely, cells overproducing Aβ showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. Again, the capability of these cells to generate Aβ was partly reduced by an antioxidant, indicating that Aβ formation was also ROS dependent. Moreover, mice with a genetic defect in complex I, or AD mice treated with a complex I inhibitor, showed enhanced Aβ levels in vivo. Innovation: We show for the first time that mitochondrion-derived ROS are sufficient to trigger Aβ production in vitro and in vivo. Conclusion: Several lines of evidence show that mitochondrion-derived ROS result in enhanced amyloidogenic amyloid precursor protein processing, and that Aβ itself leads to mitochondrial dysfunction and increased ROS levels. We propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic AD. Antioxid. Redox Signal. 16, 1421–1433. PMID:22229260

Putting together a plasma membrane NADH oxidase: a tale of three laboratories.

PubMed

Löw, Hans; Crane, Frederick L; Morré, D James

2012-11-01

The observation that high cellular concentrations of NADH were associated with low adenylate cyclase activity led to a search for the mechanism of the effect. Since cyclase is in the plasma membrane, we considered the membrane might have a site for NADH action, and that NADH might be oxidized at that site. A test for NADH oxidase showed very low activity, which could be increased by adding growth factors. The plasma membrane oxidase was not inhibited by inhibitors of mitochondrial NADH oxidase such as cyanide, rotenone or antimycin. Stimulation of the plasma membrane oxidase by iso-proterenol or triiodothyronine was different from lack of stimulation in endoplasmic reticulum. After 25 years of research, three components of a trans membrane NADH oxidase have been discovered. Flavoprotein NADH coenzyme Q reductases (NADH cytochrome b reductase) on the inside, coenzyme Q in the middle, and a coenzyme Q oxidase on the outside as a terminal oxidase. The external oxidase segment is a copper protein with unique properties in timekeeping, protein disulfide isomerase and endogenous NADH oxidase activity, which affords a mechanism for control of cell growth by the overall NADH oxidase and the remarkable inhibition of oxidase activity and growth of cancer cells by a wide range of anti-tumor drugs. A second trans plasma membrane electron transport system has been found in voltage dependent anion channel (VDAC), which has NADH ferricyanide reductase activity. This activity must be considered in relation to ferricyanide stimulation of growth and increased VDAC antibodies in patients with autism. Copyright © 2012 Elsevier Ltd. All rights reserved.

Respiration, oxidative phosphorylation, and uncoupling protein in Candida albicans.

PubMed

Cavalheiro, R A; Fortes, F; Borecký, J; Faustinoni, V C; Schreiber, A Z; Vercesi, A E

2004-10-01

The respiration, membrane potential (Deltapsi), and oxidative phosphorylation of mitochondria in situ were determined in spheroplasts obtained from Candida albicans control strain ATCC 90028 by lyticase treatment. Mitochondria in situ were able to phosphorylate externally added ADP (200 microM) in the presence of 0.05% BSA. Mitochondria in situ generated and sustained stable mitochondrial Deltapsi respiring on 5 mM NAD-linked substrates, 5 mM succinate, or 100 microM N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride plus 1 mM ascorbate. Rotenone (4 microM) inhibited respiration by 30% and 2 micro M antimycin A or myxothiazole and 1 mM cyanide inhibited it by 85%. Cyanide-insensitive respiration was partially blocked by 2 mM benzohydroxamic acid, suggesting the presence of an alternative oxidase. Candida albicans mitochondria in situ presented a carboxyatractyloside-insensitive increase of Deltapsi induced by 5 mM ATP and 0.5% BSA, and Deltapsi decrease induced by 10 microM linoleic acid, both suggesting the existence of an uncoupling protein. The presence of this protein was subsequently confirmed by immunodetection and respiration experiments with isolated mitochondria. In conclusion, Candida albicans ATCC 90028 possesses an alternative electron transfer chain and alternative oxidase, both absent in animal cells. These pathways can be exceptional targets for the design of new chemotherapeutic agents. Blockage of these respiratory pathways together with inhibition of the uncoupling protein (another potential target for drug design) could lead to increased production of reactive oxygen species, dysfunction of Candida mitochondria, and possibly to oxidative cell death.

Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.

PubMed

Moreno-Sánchez, R; Bravo, C; Westerhoff, H V

1999-09-01

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.

Decreased NAA in gray matter is correlated with decreased availability of acetate in white matter in postmortem multiple sclerosis cortex.

PubMed

Li, S; Clements, R; Sulak, M; Gregory, R; Freeman, E; McDonough, J

2013-11-01

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) which leads to progressive neurological disability. Our previous studies have demonstrated mitochondrial involvement in MS cortical pathology and others have documented decreased levels of the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in the MS brain. While NAA is synthesized in neurons, it is broken down in oligodendrocytes into aspartate and acetate. The resulting acetate is incorporated into myelin lipids, linking neuronal mitochondrial function to oligodendrocyte-mediated elaboration of myelin lipids in the CNS. In the present study we show that treating human SH-SY5Y neuroblastoma cells with the electron transport chain inhibitor antimycin A decreased levels of NAA as measured by HPLC. To better understand the significance of the relationship between mitochondrial function and levels of NAA and its breakdown product acetate on MS pathology we then quantitated the levels of NAA and acetate in MS and control postmortem tissue blocks. Regardless of lesion status, we observed that levels of NAA were decreased 25 and 32 % in gray matter from parietal and motor cortex in MS, respectively, compared to controls. Acetate levels in adjacent white matter mirrored these decreases as evidenced by the 36 and 45 % reduction in acetate obtained from parietal and motor cortices. These data suggest a novel mechanism whereby mitochondrial dysfunction and reduced NAA levels in neurons may result in compromised myelination by oligodendrocytes due to decreased availability of acetate necessary for the synthesis of myelin lipids.

Decreased NAA in Gray Matter is Correlated with Decreased Availability of Acetate in White Matter in Postmortem Multiple Sclerosis Cortex

PubMed Central

Li, S.; Clements, R.; Sulak, M.; Gregory, R.; Freeman, E.; McDonough, J.

2013-01-01

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system (CNS) which leads to progressive neurological disability. Our previous studies have demonstrated mitochondrial involvement in MS cortical pathology and others have documented decreased levels of the neuronal mitochondrial metabolite N-acetyl aspartate (NAA) in the MS brain. While NAA is synthesized in neurons, it is broken down in oligodendrocytes into aspartate and acetate. The resulting acetate is incorporated into myelin lipids, linking neuronal mitochondrial function to oligodendrocyte-mediated elaboration of myelin lipids in the CNS. In the present study we show that treating human SH-SY5Y neuroblastoma cells with the electron transport chain inhibitor antimycin A decreased levels of NAA as measured by HPLC. To better understand the significance of the relationship between mitochondrial function and levels of NAA and its breakdown product acetate on MS pathology we then quantitated the levels of NAA and acetate in MS and control postmortem tissue blocks. Regardless of lesion status, we observed that levels of NAA were decreased 25 and 32 % in gray matter from parietal and motor cortex in MS, respectively, compared to controls. Acetate levels in adjacent white matter mirrored these decreases as evidenced by the 36 and 45 % reduction in acetate obtained from parietal and motor cortices. These data suggest a novel mechanism whereby mitochondrial dysfunction and reduced NAA levels in neurons may result in compromised myelination by oligodendrocytes due to decreased availability of acetate necessary for the synthesis of myelin lipids. PMID:24078261

D-Lactate transport and metabolism in rat liver mitochondria.

PubMed

de Bari, Lidia; Atlante, Anna; Guaragnella, Nicoletta; Principato, Giovanni; Passarella, Salvatore

2002-07-15

In the present study we investigated whether isolated rat liver mitochondria can take up and metabolize D-lactate. We found the following: (1) externally added D-lactate causes oxygen uptake by mitochondria [P/O ratio (the ratio of mol of ATP synthesized to mol of oxygen atoms reduced to water during oxidative phosphorylation)=2] and membrane potential (Delta(psi)) generation in processes that are rotenone-insensitive, but inhibited by antimycin A and cyanide, and proton release from coupled mitochondria inhibited by alpha-cyanocinnamate, but not by phenylsuccinate; (2) the activity of the putative flavoprotein (D-lactate dehydrogenase) was detected in inside-out submitochondrial particles, but not in mitochondria and mitoplasts, as it is localized in the matrix phase of the mitochondrial inner membrane; (3) three novel separate translocators exist to mediate D-lactate traffic across the mitochondrial inner membrane: the D-lactate/H(+) symporter, which was investigated by measuring fluorimetrically the rate of endogenous flavin reduction, the D-lactate/oxoacid antiporter (which mediates both the D-lactate/pyruvate and D-lactate/oxaloacetate exchanges) and D-lactate/malate antiporter studied by monitoring photometrically the appearance of the D-lactate counteranions outside mitochondria. The D-lactate translocators, in the light of their different inhibition profiles separate from the monocarboxylate carrier, were found to differ from each other in the V(max) values and in the inhibition and pH profiles and were shown to regulate mitochondrial D-lactate metabolism in vitro. The D-lactate translocators and the D-lactate dehydrogenase could account for the removal of the toxic methylglyoxal from cytosol, as well as for D-lactate-dependent gluconeogenesis.

Influence of metabolic inhibitors on mitochondrial permeability transition and glutathione status.

PubMed

Reed, D J; Savage, M K

1995-05-24

Treatment of isolated mitochondria with Ca2+ and inorganic phosphate (Pi) induces an inner membrane permeability that appears to be mediated through a cyclosporin A (CsA)-inhibitable Ca(2+)-dependent pore. Isolated mitochondria during inner membrane permeability undergo rapid efflux of matrix solutes such as glutathione as GSH and Ca2+, loss of coupled functions, and large amplitude swelling. Permeability transition without large amplitude swelling, a parameter often used to assess inner membrane permeability, has been observed. The addition of either oligomycin, antimycin, or sulfide to incubation buffer containing Ca2+ and Pi abolished large amplitude swelling of mitochondria. The GSH status during a Ca(2+)- and Pi-dependent mechanism of mitochondrial GSH release in isolated mitochondria was influenced significantly by metabolic inhibitors of the respiratory chain but did not prevent inner membrane permeability as demonstrated by the release of mitochondrial GSH and Ca2+. The release of GSH was inhibited by the addition of CsA, a potent inhibitor of permeability transition. Under these conditions we did not find GSSG; however, rapid oxidation of pyridine nucleotides and depletion of ATP and ADP with conversion to AMP occurred. The addition of CsA, prevented the oxidation of pyridine nucleotides and depletion of ATP and ADP. Since NADH and NADPH were extensively oxidized, protection against oxidative stress is reflected in maintenance of GSH and not observable lipid peroxidation. Evidence from transmission electron microscopy analysis, combined with the GSH release data, indicate that permeability transition can be observed in the absence of large amplitude swelling.

Rapid mitochondrial adjustments in response to short-term hypoxia and re-oxygenation in the Pacific oyster, Crassostrea gigas.

PubMed

Sussarellu, Rossana; Dudognon, Tony; Fabioux, Caroline; Soudant, Philippe; Moraga, Dario; Kraffe, Edouard

2013-05-01

As oxygen concentrations in marine coastal habitats can fluctuate rapidly and drastically, sessile marine organisms such as the oyster Crassostrea gigas can experience marked and rapid oxygen variations. In this study, we investigated the responses of oyster gill mitochondria to short-term hypoxia (3 and 12 h, at 1.7 mg O2 l(-1)) and subsequent re-oxygenation. Mitochondrial respiratory rates (states 3 and 4 stimulated by glutamate) and phosphorylation efficiency [respiratory control ratio (RCR) and the relationship between ADP and oxygen consumption (ADP/O)] were measured. Cytochrome c oxidase (CCO) activity and cytochrome concentrations (a, b, c1 and c) were measured to investigate the rearrangements of respiratory chain subunits. The potential implication of an alternative oxidase (AOX) was investigated using an inhibitor of the respiratory chain (antimycin A) and through gene expression analysis in gills and digestive gland. Results indicate a downregulation of mitochondrial capacity, with 60% inhibition of respiratory rates after 12 h of hypoxia. RCR remained stable, while ADP/O increased after 12 h of hypoxia and 1 h of re-oxygenation, suggesting increased phosphorylation efficiency. CCO showed a fast and remarkable increase of its catalytic activity only after 3 h of hypoxia. AOX mRNA levels showed similar patterns in gills and digestive gland, and were upregulated after 12 and 24 h of hypoxia and during re-oxygenation. Results suggest a set of controls regulating mitochondrial functions in response to oxygen fluctuations, and demonstrate the fast and extreme plasticity of oyster mitochondria in response to oxygen variations.

Low-Level Light Therapy Potentiates NPe6-mediated Photodynamic Therapy in a Human Osteosarcoma Cell Line via Increased ATP

PubMed Central

Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R.

2015-01-01

Background Low-Level Light Therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). Methods We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-L-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5 J/cm2 of 810 nm near infrared radiation (NIR) followed by addition of 10 μM NPe6 and after 2 h incubation by 1.5 J/cm2 of 652 nm red light for PDT. Results PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Conclusions Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. PMID:25462575

The End of the Line: Can Ferredoxin and Ferredoxin NADP(H) Oxidoreductase Determine the Fate of Photosynthetic Electrons?

PubMed Central

Goss, Tatjana; Hanke, Guy

2014-01-01

At the end of the linear photosynthetic electron transfer (PET) chain, the small soluble protein ferredoxin (Fd) transfers electrons to Fd:NADP(H) oxidoreductase (FNR), which can then reduce NADP+ to support C assimilation. In addition to this linear electron flow (LEF), Fd is also thought to mediate electron flow back to the membrane complexes by different cyclic electron flow (CEF) pathways: either antimycin A sensitive, NAD(P)H complex dependent, or through FNR located at the cytochrome b6f complex. Both Fd and FNR are present in higher plant genomes as multiple gene copies, and it is now known that specific Fd iso-proteins can promote CEF. In addition, FNR iso-proteins vary in their ability to dynamically interact with thylakoid membrane complexes, and it has been suggested that this may also play a role in CEF. We will highlight work on the different Fd-isoproteins and FNR-membrane association found in the bundle sheath (BSC) and mesophyll (MC) cell chloroplasts of the C4 plant maize. These two cell types perform predominantly CEF and LEF, and the properties and activities of Fd and FNR in the BSC and MC are therefore specialized for CEF and LEF respectively. A diversity of Fd isoproteins and dynamic FNR location has also been recorded in C3 plants, algae and cyanobacteria. This indicates that the principles learned from the extreme electron transport situations in the BSC and MC of maize might be usefully applied to understanding the dynamic transition between these states in other systems. PMID:24678667

Disrupting mitochondrial Ca2+ homeostasis causes tumor-selective TRAIL sensitization through mitochondrial network abnormalities.

PubMed

Ohshima, Yohei; Takata, Natsuhiko; Suzuki-Karasaki, Miki; Yoshida, Yukihiro; Tokuhashi, Yasuaki; Suzuki-Karasaki, Yoshihiro

2017-10-01

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has emerged as a promising anticancer agent with high tumor-selective cytotoxicity. The congenital and acquired resistance of some cancer types including malignant melanoma and osteosarcoma impede the current TRAIL therapy of these cancers. Since fine tuning of the intracellular Ca2+ level is essential for cell function and survival, Ca2+ dynamics could be a promising target for cancer treatment. Recently, we demonstrated that mitochondrial Ca2+ removal increased TRAIL efficacy toward malignant melanoma and osteosarcoma cells. Here we report that mitochondrial Ca2+ overload leads to tumor-selective sensitization to TRAIL cytotoxicity. Treatment with the mitochondrial Na+/Ca2+ exchanger inhibitor CGP-37157 and oxidative phosphorylation inhibitor antimycin A and FCCP resulted in a rapid and persistent mitochondrial Ca2+ rise. These agents also increased TRAIL sensitivity in a tumor-selective manner with a switching from apoptosis to a nonapoptotic cell death. Moreover, we found that mitochondrial Ca2+ overload led to increased mitochondrial fragmentation, while mitochondrial Ca2+ removal resulted in mitochondrial hyperfusion. Regardless of their reciprocal actions on the mitochondrial dynamics, both interventions commonly exacerbated TRAIL-induced mitochondrial network abnormalities. These results expand our previous study and suggest that an appropriate level of mitochondrial Ca2+ is essential for maintaining the mitochondrial dynamics and the survival of these cells. Thus, disturbing mitochondrial Ca2+ homeostasis may serve as a promising approach to overcome the TRAIL resistance of these cancers with minimally compromising the tumor-selectivity.

Different molecular mechanisms involved in spontaneous and oxidative stress-induced mitochondrial fragmentation in tripeptidyl peptidase-1 (TPP-1)-deficient fibroblasts

PubMed Central

Van Beersel, Guillaume; Tihon, Eliane; Demine, Stéphane; Hamer, Isabelle; Jadot, Michel; Arnould, Thierry

2012-01-01

NCLs (neuronal ceroid lipofuscinoses) form a group of eight inherited autosomal recessive diseases characterized by the intralysosomal accumulation of autofluorescent pigments, called ceroids. Recent data suggest that the pathogenesis of NCL is associated with the appearance of fragmented mitochondria with altered functions. However, even if an impairement in the autophagic pathway has often been evoked, the molecular mechanisms leading to mitochondrial fragmentation in response to a lysosomal dysfunction are still poorly understood. In this study, we show that fibroblasts that are deficient for the TPP-1 (tripeptidyl peptidase-1), a lysosomal hydrolase encoded by the gene mutated in the LINCL (late infantile NCL, CLN2 form) also exhibit a fragmented mitochondrial network. This morphological alteration is accompanied by an increase in the expression of the protein BNIP3 (Bcl2/adenovirus E1B 19 kDa interacting protein 3) as well as a decrease in the abundance of mitofusins 1 and 2, two proteins involved in mitochondrial fusion. Using RNAi (RNA interference) and quantitative analysis of the mitochondrial morphology, we show that the inhibition of BNIP3 expression does not result in an increase in the reticulation of the mitochondrial population in LINCL cells. However, this protein seems to play a key role in cell response to mitochondrial oxidative stress as it sensitizes mitochondria to antimycin A-induced fragmentation. To our knowledge, our results bring the first evidence of a mechanism that links TPP-1 deficiency and oxidative stress-induced changes in mitochondrial morphology. PMID:23249249

Regulation of glycolysis in Kluyveromyces lactis: role of KlGCR1 and KlGCR2 in glucose uptake and catabolism.

PubMed

Neil, H; Lemaire, M; Wésolowski-Louvel, M

2004-03-01

In Kluyveromyces lactis, the casein kinase I (Rag8p) regulates the transcription of glycolytic genes and the expression of the low-affinity glucose transporter gene RAG1. This control involves the transcription factor Sck1p, a homologue of Sgc1p of Saccharomyces cerevisiae. SGC1 is known to interact genetically with ScGCR1 and ScGCR2, which code for regulators of glycolytic gene expression. Therefore, we studied the role of KlGCR1 and KlGCR2 genes in K. lactis. The Klgcr1 null mutant could not grow on glucose when respiration was blocked by antimycin A (Rag(- )phenotype). In contrast, the Klgcr2 null mutant could grow under the same conditions, although at a reduced rate. In both mutants, the transcription of glycolytic genes was affected, while that of ribosomal protein genes was not modified. Furthermore, the transcription of the glucose permease genes was also found to be affected in the two mutants, although dissimilarly. While RAG1 transcription decreased at high glucose concentrations, the expression of the high-affinity glucose permease gene HGT1 was unexpectedly impaired under gluconeogenic conditions, in the absence of glucose. Gel mobility shift assays performed with purified maltose-binding protein-KlGcr1p showed that KlGcr1p could interact directly with the promoters of the glycolytic genes, but not with the promoters of the glucose permease genes. Thus, the control exerted by KlGcr1p and KlGcr2p upon glucose transporter genes is probably indirect.

Evaluation of functioning of mitochondrial electron transport chain with NADH and FAD autofluorescence

PubMed

Danylovych, H V

2016-01-01

We prove the feasibility of evaluation of mitochondrial electron transport chain function in isolated mitochondria of smooth muscle cells of rats from uterus using fluorescence of NADH and FAD coenzymes. We found the inversely directed changes in FAD and NADH fluorescence intensity under normal functioning of mitochondrial electron transport chain. The targeted effect of inhibitors of complex I, III and IV changed fluorescence of adenine nucleotides. Rotenone (5 μM) induced rapid increase in NADH fluorescence due to inhibition of complex I, without changing in dynamics of FAD fluorescence increase. Antimycin A, a complex III inhibitor, in concentration of 1 μg/ml caused sharp increase in NADH fluorescence and moderate increase in FAD fluorescence in comparison to control. NaN3 (5 mM), a complex IV inhibitor, and CCCP (10 μM), a protonophore, caused decrease in NADH and FAD fluorescence. Moreover, all the inhibitors caused mitochondria swelling. NO donors, e.g. 0.1 mM sodium nitroprusside and sodium nitrite similarly to the effects of sodium azide. Energy-dependent Ca2+ accumulation in mitochondrial matrix (in presence of oxidation substrates and Mg-ATP2- complex) is associated with pronounced drop in NADH and FAD fluorescence followed by increased fluorescence of adenine nucleotides, which may be primarily due to Ca2+- dependent activation of dehydrogenases of citric acid cycle. Therefore, the fluorescent signal of FAD and NADH indicates changes in oxidation state of these nucleotides in isolated mitochondria, which may be used to assay the potential of effectors of electron transport chain.

Genetic and Functional Investigation of Zn2Cys6 Transcription Factors RSE2 and RSE3 in Podospora anserina

PubMed Central

Bovier, Elodie; Sellem, Carole H.; Humbert, Adeline

2014-01-01

In Podospora anserina, the two zinc cluster proteins RSE2 and RSE3 are essential for the expression of the gene encoding the alternative oxidase (aox) when the mitochondrial electron transport chain is impaired. In parallel, they activated the expression of gluconeogenic genes encoding phosphoenolpyruvate carboxykinase (pck) and fructose-1,6-biphosphatase (fbp). Orthologues of these transcription factors are present in a wide range of filamentous fungi, and no other role than the regulation of these three genes has been evidenced so far. In order to better understand the function and the organization of RSE2 and RSE3, we conducted a saturated genetic screen based on the constitutive expression of the aox gene. We identified 10 independent mutations in 9 positions in rse2 and 11 mutations in 5 positions in rse3. Deletions were generated at some of these positions and the effects analyzed. This analysis suggests the presence of central regulatory domains and a C-terminal activation domain in both proteins. Microarray analysis revealed 598 genes that were differentially expressed in the strains containing gain- or loss-of-function mutations in rse2 or rse3. It showed that in addition to aox, fbp, and pck, RSE2 and RSE3 regulate the expression of genes encoding the alternative NADH dehydrogenase, a Zn2Cys6 transcription factor, a flavohemoglobin, and various hydrolases. As a complement to expression data, a metabolome profiling approach revealed that both an rse2 gain-of-function mutation and growth on antimycin result in similar metabolic alterations in amino acids, fatty acids, and α-ketoglutarate pools. PMID:24186951

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection

PubMed Central

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

Background It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Materials and methods Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate–malate or N, N, N′, N′-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)–ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate–malate as substrate. Results As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate–malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD–ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. Conclusion In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process. PMID:26203225

Apparent Km of mitochondria for oxygen computed from Vmax measured in permeabilized muscle fibers is lower in water enriched in oxygen by electrolysis than injection.

PubMed

Zoll, Joffrey; Bouitbir, Jamal; Sirvent, Pascal; Klein, Alexis; Charton, Antoine; Jimenez, Liliana; Péronnet, François R; Geny, Bernard; Richard, Ruddy

2015-01-01

It has been suggested that oxygen (O2) diffusion could be favored in water enriched in O2 by a new electrolytic process because of O2 trapping in water superstructures (clathrates), which could reduce the local pressure/content relationships for O2 and facilitate O2 diffusion along PO2 gradients. Mitochondrial respiration was compared in situ in saponin-skinned fibers isolated from the soleus muscles of Wistar rats, in solution enriched in O2 by injection or the electrolytic process 1) at an O2 concentration decreasing from 240 µmol/L to 10 µmol/L (132 mmHg to 5 mmHg), with glutamate-malate or N, N, N', N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (with antimycin A) as substrates; and 2) at increasing adenosine diphosphate (ADP) concentration with glutamate-malate as substrate. As expected, maximal respiration decreased with O2 concentration and, when compared to glutamate-malate, the apparent Km O2 of mitochondria for O2 was significantly lower with TMPD-ascorbate with both waters. However, when compared to the water enriched in O2 by injection, the Km O2 was significantly lower with both electron donors in water enriched in O2 by electrolysis. This was not associated with any increase in the sensitivity of mitochondria to ADP; no significant difference was observed for the Km ADP between the two waters. In this experiment, a higher affinity of the mitochondria for O2 was observed in water enriched in O2 by electrolysis than by injection. This observation is consistent with the hypothesis that O2 diffusion can be facilitated in water enriched in O2 by the electrolytic process.

Age-dependent reductions in mitochondrial respiration are exacerbated by calcium in the female rat heart.

PubMed

Hunter, J Craig; Machikas, Alexandra M; Korzick, Donna H

2012-06-01

Cardiovascular disease mortality increases rapidly after menopause by poorly defined mechanisms. Because mitochondrial function and Ca(2+) sensitivity are important regulators of cell death after myocardial ischemia, we sought to determine whether aging and/or estrogen deficiency (ovariectomy) increased mitochondrial Ca(2+) sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6 months; n = 26) and aged (24 months; n = 25), intact or ovariectomized female rats using the substrates α-ketoglutarate/malate (complex I); succinate/rotenone (complex II); ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/antimycin (complex IV). State 2 and 3 respiration was initiated by sequential addition of mitochondria and adenosine diphosphate. Ca(2+) sensitivity was assessed by Ca(2+)-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered intraperitoneally 45 minutes before euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for complexes I and II by 12% and 8%, respectively, independent of ovary status (P < 0.05). Of interest, Ca(2+) induced a greater decrease (18%-30%; P < 0.05) in complex I state 3 respiration in aged and ovariectomized animals, and mitochondrial swelling occurred twice as quickly in aged (vs adult) female rats (P < 0.05). Pretreatment with PPT increased RCI by 8% and 7% at complexes I and II, respectively (P < 0.05) but surprisingly increased Ca(2+) sensitivity. Age-dependent decreases in RCI and sensitization to Ca(2+) may explain in part the age-associated reductions in female ischemic tolerance; however, protection afforded by ER agonism involves more complex mechanisms. Copyright © 2012 Elsevier HS Journals, Inc. All rights reserved.

Age-Dependent Reductions in Mitochondrial Respiration are Exacerbated by Calcium in the Female Rat Heart

PubMed Central

Hunter, J. Craig; Machikas, Alexandra M.; Korzick, Donna H.

2012-01-01

Cardiovascular disease mortality increases rapidly following menopause by poorly defined mechanisms. Since mitochondrial function and Ca2+ sensitivity are important regulators of cell death following myocardial ischemia, we sought to determine if aging and/or estrogen deficiency (ovx) increased mitochondrial Ca2+ sensitivity. Mitochondrial respiration was measured in ventricular mitochondria isolated from adult (6mo; n=26) and aged (24mo; n=25), intact or ovariectomized female rats using the substrates: α-ketoglutarate/malate (Complex I); succinate/rotenone (Complex II); ascorbate/TMPD/Antimycin (Complex IV). State 2 and State 3 respiration was initiated by sequential addition of mitochondria and ADP. Ca2+ sensitivity was assessed by Ca2+-induced swelling of de-energized mitochondria and reduction in state 3 respiration. Propylpyrazole triol (PPT) was administered i.p. 45 min prior to euthanasia to assess mitochondrial protective effects through estrogen receptor (ER) α activation. Aging decreased the respiratory control index (RCI; state 3/state 2) for Complexes I and II by 12% and 8%, respectively, independent of ovary status (p<0.05). Of interest, Ca2+ induced a greater decrease (18–30%; p<0.05) in Complex I state 3 respiration in aged and ovx animals, and mitochondrial swelling occurred twice as quickly in aged (vs. adult) female rats (p<0.05). Pretreatment with PPT increased RCI by 8% and 7% at Complexes I and II, respectively (p<0.05) but surprisingly increased Ca2+ sensitivity. Age-dependent decreases in RCI and sensitization to Ca2+ may explain in part the age-associated reductions in female ischemic tolerance; however protection afforded by ER agonism involves more complex mechanisms. PMID:22555015

Coenzyme Q10 instilled as eye drops on the cornea reaches the retina and protects retinal layers from apoptosis in a mouse model of kainate-induced retinal damage.

PubMed

Lulli, Matteo; Witort, Ewa; Papucci, Laura; Torre, Eugenio; Schipani, Christian; Bergamini, Christian; Dal Monte, Massimo; Capaccioli, Sergio

2012-12-17

To evaluate if coenzyme Q10 (CoQ10) can protect retinal ganglion cells (RGCs) from apoptosis and, when instilled as eye drops on the cornea, if it can reach the retina and exert its antiapoptotic activity in this area in a mouse model of kainate (KA)-induced retinal damage. Rat primary or cultured RGCs were subjected to glutamate (50 μM) or chemical hypoxia (Antimycin A, 200 μM) or serum withdrawal (FBS, 0.5%) in the presence or absence of CoQ10 (10 μM). Cell viability was evaluated by light microscopy and fluorescence-activated cell sorting analyses. Apoptosis was evaluated by caspase 3/7 activity and mitochondrion depolarization tetramethylrhodamine ethyl ester analysis. CoQ10 transfer to the retina following its instillation as eye drops on the cornea was quantified by HPLC. Retinal protection by CoQ10 (10 μM) eye drops instilled on the cornea was then evaluated in a mouse model of KA-induced excitotoxic retinal cell apoptosis by cleaved caspase 3 immunohistofluorescence, caspase 3/7 activity assays, and quantification of inhibition of RGC loss. CoQ10 significantly increased viable cells by preventing RGC apoptosis. Furthermore, when topically applied as eye drops to the cornea, it reached the retina, thus substantially increasing local CoQ10 concentration and protecting retinal layers from apoptosis. The ability of CoQ10 eye drops to protect retinal cells from apoptosis in the mouse model of KA-induced retinal damage suggests that topical CoQ10 may be evaluated in designing therapies for treating apoptosis-driven retinopathies.

Low-level light therapy potentiates NPe6-mediated photodynamic therapy in a human osteosarcoma cell line via increased ATP.

PubMed

Tsai, Shang-Ru; Yin, Rui; Huang, Ying-Ying; Sheu, Bor-Ching; Lee, Si-Chen; Hamblin, Michael R

2015-03-01

Low-level light therapy (LLLT) is used to stimulate healing, reduce pain and inflammation, and preserve tissue from dying. LLLT has been shown to protect cells in culture from dying after various cytotoxic insults, and LLLT is known to increase the cellular ATP content. Previous studies have demonstrated that maintaining a sufficiently high ATP level is necessary for the efficient induction and execution of apoptosis steps after photodynamic therapy (PDT). We asked whether LLLT would protect cells from cytotoxicity due to PDT, or conversely whether LLLT would enhance the efficacy of PDT mediated by mono-l-aspartyl chlorin(e6) (NPe6). Increased ATP could lead to enhanced cell uptake of NPe6 by the energy dependent process of endocytosis, and also to more efficient apoptosis. In this study, human osteosarcoma cell line MG-63 was subjected to 1.5J/cm(2) of 810nm near infrared radiation (NIR) followed by addition of 10μM NPe6 and after 2h incubation by 1.5J/cm(2) of 652nm red light for PDT. PDT combined with LLLT led to higher cell death and increased intracellular reactive oxygen species compared to PDT alone. The uptake of NPe6 was moderately increased by LLLT, and cellular ATP was increased. The mitochondrial respiratory chain inhibitor antimycin A abrogated the LLLT-induced increase in cytotoxicity. Taken together, these results demonstrate that LLLT potentiates NPe6-mediated PDT via increased ATP synthesis and is a potentially promising strategy that could be applied in clinical PDT. Copyright © 2014 Elsevier B.V. All rights reserved.

Oxygen sensitivity of mitochondrial function in rat arterial chemoreceptor cells

PubMed Central

Buckler, Keith J; Turner, Philip J

2013-01-01

The mechanism of oxygen sensing in arterial chemoreceptors is unknown but has often been linked to mitochondrial function. A common criticism of this hypothesis is that mitochondrial function is insensitive to physiological levels of hypoxia. Here we investigate the effects of hypoxia (down to 0.5% O2) on mitochondrial function in neonatal rat type-1 cells. The oxygen sensitivity of mitochondrial [NADH] was assessed by monitoring autofluorescence and increased in hypoxia with a P50 of 15 mm Hg (1 mm Hg = 133.3 Pa) in normal Tyrode or 46 mm Hg in Ca2+-free Tyrode. Hypoxia also depolarised mitochondrial membrane potential (ψm, measured using rhodamine 123) with a P50 of 3.1, 3.3 and 2.8 mm Hg in normal Tyrode, Ca2+-free Tyrode and Tyrode containing the Ca2+ channel antagonist Ni2+, respectively. In the presence of oligomycin and low carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP; 75 nm) ψm is maintained by electron transport working against an artificial proton leak. Under these conditions hypoxia depolarised ψm/inhibited electron transport with a P50 of 5.4 mm Hg. The effects of hypoxia upon cytochrome oxidase activity were investigated using rotenone, myxothiazol, antimycin A, oligomycin, ascorbate and the electron donor tetramethyl-p-phenylenediamine. Under these conditions ψm is maintained by complex IV activity alone. Hypoxia inhibited cytochrome oxidase activity (depolarised ψm) with a P50 of 2.6 mm Hg. In contrast hypoxia had little or no effect upon NADH (P50= 0.3 mm Hg), electron transport or cytochrome oxidase activity in sympathetic neurons. In summary, type-1 cell mitochondria display extraordinary oxygen sensitivity commensurate with a role in oxygen sensing. The reasons for this highly unusual behaviour are as yet unexplained. PMID:23671162

Imaging of oxygen gradients in giant umbrella cells: an ex vivo PLIM study.

PubMed

Zhdanov, A V; Golubeva, A V; Okkelman, I A; Cryan, J F; Papkovsky, D B

2015-10-01

O2 plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O2 levels are well documented; however, the physiological significance of O2 microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O2 probe Pt-Glc and confocal fluorescence microscopy, we visualized O2 distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O2 gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5-15 μM O2) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O2 gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O2 distribution potentially can be used for the modeling of intracellular O2-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O2 gradients using confocal imaging can shed light on the molecular mechanisms regulating O2-dependent (patho)physiological processes in the bladder and other tissues. Copyright © 2015 the American Physiological Society.

Cytotoxicity and cellular response mechanisms of water-soluble platinum(II) complexes of lidocaine and phenylcyanamide derivatives.

PubMed

Tabrizi, Leila; Chiniforoshan, Hossein

2017-02-01

Three new platinum(II) complexes of lidocaine and phenylcyanamide derivative ligands of formula K[Pt(3,5-(NO 2 ) 2 pcyd) 2 (LC)], 1, K[Pt(3,5-(CF 3 ) 2 pcyd) 2 (LC)], 2, K[Pt(3,5-Cl 2 pcyd) 2 (LC)], 3 (LC: lidocaine, 3,5-(NO 2 ) 2 pcyd: 3,5-dinitro phenylcyanamide, 3,5-(CF 3 ) 2 pcyd: 3,5-bis(trifluoromethyl) phenylcyanamide, 3,5-Cl 2 pcyd: 3,5-dichloro phenylcyanamide) have been synthesized and fully characterized. Cellular uptake, DNA platination and cytotoxicity against a panel of human tumor cell lines were evaluated. The complexes 1-3 revealed a significant in vitro antiproliferative activity against human ovarian carcinoma (A2780), colorectal adenocarcinoma (HT29), breast (MCF-7), liver hepatocellular carcinoma (HepG-2) and lung adenocarcinoma (A549) cancer cell lines. All the complexes are more active than cisplatin and follow the trend 1 > 2 > 3. Mechanistic studies showed that the trend in cytotoxicity of the Pt(II) complexes is mainly consistent with their ability to accumulate into cancer cells and to increase intracellular basal reactive oxygen species levels, which consequently results in the loss of mitochondrial membrane potential and apoptosis induction. The complex 1 caused to approximately 80-fold higher DNA platination level with respect to cisplatin. The complexes 1-3 can considerably stimulate the production of hydrogen peroxide in a time-dependent manner. Also, the complexes 1-3 induced an increase in reactive oxygen species (ROS) production that was superior to that induced by antimycin. The complex 1 had the most effect on ROS production in comparison with other complexes.

Pyruvate metabolism in castor-bean mitochondria.

PubMed Central

Brailsford, M A; Thompson, A G; Kaderbhai, N; Beechey, R B

1986-01-01

We report the isolation of mitochondria from the endosperm of castor beans (Ricinus communis). These mitochondria oxidized succinate, external NADH, malate and pyruvate with respiratory-control and ADP/O ratios consistent with those found previously with mitochondria from other plant sources. The mitochondria exhibited considerable sensitivity to the electron-transport-chain inhibitors antimycin A and cyanide when oxidizing succinate and external NADH. Pyruvate-dependent O2 uptake was relatively insensitive to these inhibitors, although the residual O2 uptake could be inhibited by salicylhydroxamic acid. We conclude that a cyanide-insensitive alternative terminal oxidase is functional in these mitochondria. However, electrons from the succinate dehydrogenase or external NADH dehydrogenase seem to have no access to this pathway. There is little interconnection between the salicylhydroxamic acid-sensitive and cyanide-sensitive pathways of electron transport. alpha-Cyanocinnamate and its analogues, compound UK5099 [alpha-cyano-beta-(1-phenylindol-3-yl)acrylate] and alpha-cyano-4-hydroxycinnamate, were all found to be potent non-competitive inhibitors of pyruvate oxidation in castor-bean mitochondria. The accumulation of pyruvate by castor-bean mitochondria was determined by using a silicone-oil-centrifugation technique. The accumulation was shown to observe Michaelis-Menten kinetics, with a Km for pyruvate of 0.10 mM and a Vmax. of 0.95 nmol/min per mg of mitochondrial protein. However, the observed rates of pyruvate accumulation were insufficient to account for the pyruvate oxidation rates found in the oxygen-electrode studies. We were able to demonstrate that this is due to the immediate export of the accumulated radiolabel in the form of malate and citrate. Compound UK5099 inhibited the accumulation of [2-14C]pyruvate by castor-bean mitochondria at concentrations similar to those required to inhibit pyruvate oxidation. PMID:3814077

Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells.

PubMed

Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M; Eguchi, Satoru; Brown, Michael D; Park, Joon-Young

2015-08-01

The concept of enhancing structural integrity of mitochondria has emerged as a novel therapeutic option for cardiovascular disease. Flow-induced increase in laminar shear stress is a potent physiological stimulant associated with exercise, which exerts atheroprotective effects in the vasculature. However, the effect of laminar shear stress on mitochondrial remodeling within the vascular endothelium and its related functional consequences remain largely unknown. Using in vitro and in vivo complementary studies, here, we report that aerobic exercise alleviates the release of endothelial microparticles in prehypertensive individuals and that these salutary effects are, in part, mediated by shear stress-induced mitochondrial biogenesis. Circulating levels of total (CD31(+)/CD42a(-)) and activated (CD62E(+)) microparticles released by endothelial cells were significantly decreased (∼40% for both) after a 6-mo supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced the release of endothelial microparticles, which was accompanied by an increase in mitochondrial biogenesis through a sirtuin 1 (SIRT1)-dependent mechanism. Resveratrol, a SIRT1 activator, treatment showed similar effects. SIRT1 knockdown using small-interfering RNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or peroxisome proliferator-activated receptor-γ coactivator-1α small-interfering RNA significantly increased the number of total, and activated, released endothelial microparticles, and shear stress restored these back to basal levels. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. Moreover, prolonged laminar shear stress, which is systemically elevated during aerobic exercise in the vessel wall, mitigates endothelial dysfunction by promoting mitochondrial biogenesis. Copyright © 2015 the American Physiological Society.

Ethanol stimulates ROS generation by mitochondria through Ca2+ mobilization and increases GFAP content in rat hippocampal astrocytes.

PubMed

González, Antonio; Pariente, José A; Salido, Ginés M

2007-10-31

We have employed rat hippocampal astrocytes in culture to investigate the effect of ethanol on reactive oxygen species (ROS) production as well as its effect on [Ca2+]c and GFAP expression. Cells were loaded with the fluorescent probes fura-2 and H2DCFDA for the determination of changes in [Ca2+]c and ROS production respectively, employing spectrofluorimetry. GFAP content was determined by immunocytochemistry and confocal scanning microscopy. Our results show ROS production in response to 50 mM ethanol, that was reduced in Ca2+-free medium (containing 0.5 mM EGTA) and in the presence of the intracellular Ca2+ chelator BAPTA (10 microM). The effect of ethanol on ROS production was significantly reduced in the presence of the alcohol dehydrogenase inhibitor 4-methylpyrazole (1 mM), and the antioxidants resveratrol (100 microM) or catalase (300 U/ml). Preincubation of astrocytes in the presence of 10 microM antimycin plus 10 microM oligomycin to inhibit mitochondria completely blocked ethanol-evoked ROS production. In addition, ethanol led to a sustained increase in [Ca2+]c that reached a constant level over the prestimulation values. Finally, incubation of astrocytes in the presence of ethanol increased the content of GFAP that was significantly reduced in the absence of extracellular Ca2+ and by resveratrol and catalase pretreatment. The data obtained in the present study suggest that astrocytes are able to metabolize ethanol, which induces two effects on intracellular homeostasis: an immediate response (Ca2+ release and ROS generation) and later changes involving GFAP expression. Both effects may underline various signaling pathways which are important for cell proliferation, differentiation and function.

Characterization of carbohydrate metabolism and demonstration of glycosomes in a Phytomonas sp. isolated from Euphorbia characias.

PubMed

Sanchez-Moreno, M; Lasztity, D; Coppens, I; Opperdoes, F R

1992-09-01

Phytomonas sp. isolated from Euphorbia characias was adapted to SDM-79 medium. Cells isolated in the early stationary phase of growth were analyzed for their capacity to utilize plant carbohydrates for their energy requirements. The cellulose-degrading enzymes amylase, amylomaltase, invertase, carboxymethylcellulase, and the pectin-degrading enzymes polygalacturonase and oligo-D-galactosiduronate lyase were present in Phytomonas sp. and were all, except for amylomaltase, excreted into the external medium. Glucose, fructose and mannose served as the major energy substrates. Catabolism of carbohydrates occurred mainly via aerobic glycolysis according to the Embden-Meyerhof pathway, of which all the enzymes were detected. Likewise, the end-products of glycolysis, acetate and pyruvate, glycerol, succinate and ethanol were detected in the culture medium, as were the enzymes responsible for their production. Mitochondria were incapable of oxidizing succinate, 2-oxoglutarate, pyruvate, malate and proline, but had a high capacity to oxidize glycerol 3-phosphate. This oxidation was completely inhibited by salicylhydroxamic acid. No cytochromes could be detected either in intact mitochondria or in sub-mitochondrial particles. Mitochondrial respiration was not inhibited by antimycin, azide or cyanide. The glycolytic enzymes, from hexokinase to phosphoglycerate kinase, and the enzymes glycerol kinase, glycerol-3-phosphate dehydrogenase, phosphoenolpyruvate carboxykinase, malate dehydrogenase and adenylate kinase, were all associated with glycosomes that had a buoyant density of about 1.24 g cm-1 in sucrose. Cytochemical staining revealed the presence of catalase in these organelles. The cytosolic enzyme pyruvate kinase was activated by fructose 2,6-bisphosphate, typical of all other pyruvate kinases from Kinetoplastida. The energy metabolism of the plant parasite Phytomonas sp. isolated from E. characias resembled that of the bloodstream form of the mammalian parasite Trypanosoma brucei.

Mitochondrial-derived hydrogen peroxide inhibits relaxation of bovine coronary arterial smooth muscle to hypoxia through stimulation of ERK MAP kinase.

PubMed

Gao, Qun; Zhao, Xiangmin; Ahmad, Mansoor; Wolin, Michael S

2009-12-01

Mitochondrial reactive oxygen species (ROS) are potentially important in vascular oxygen-sensing mechanisms because hypoxia appears to be a stimulus for mitochondrial ROS generation; however, scavenging of endogenous ROS does not alter relaxation of endothelium-denuded bovine coronary arteries (BCA) to hypoxia. The purpose of this study was to investigate the influence of increasing mitochondrial ROS on the relaxation of BCA to hypoxia. Increasing mitochondrial superoxide with inhibitors of electron transport (10 microM rotenone and antimycin) and by opening mitochondrial ATP-dependent K+ channels with 100 microM diazoxide were observed in this study to attenuate relaxation of BCA precontracted with 30 mM KCl to hypoxia by 68-76% and 38%, respectively. This effect of rotenone is not prevented by inhibiting NADPH oxidase (Nox) activation or scavenging superoxide with Peg-SOD; however, it is reversed 85% and 26% by increasing the consumption of intracellular peroxide by 0.1 mM ebselen and 32.5 U/ml Peg-catalase. Because inhibition of extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase (10 microM PD-98059), but not src kinase or rho kinase, also reverses the effects of rotenone by 69%, the peroxide-elicited force-enhancing effects of ERK appear to be attenuating the response to hypoxia. Rotenone increased the phosphorylation of ERK (by 163%). Activation of ERK in BCA with 0.1 mM peroxide or endogenous peroxide generated by stimulating Nox2 with a stretch treatment or contraction with 100 nM U-46619 also attenuated relaxation to hypoxia. Thus coronary arterial relaxation to hypoxia may be attenuated by pathophysiological conditions associated with increased peroxide generation by mitochondria or other sources that stimulate ERK.

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

PubMed

Bai, Y; Hájek, P; Chomyn, A; Chan, E; Seo, B B; Matsuno-Yagi, A; Yagi, T; Attardi, G

2001-10-19

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

An in vitro hepatic zonation model with a continuous oxygen gradient in a microdevice.

PubMed

Sato, Asako; Kadokura, Kanae; Uchida, Hideyuki; Tsukada, Kosuke

2014-10-31

In a hepatic lobule, different sets of metabolic enzymes are expressed in the periportal (PP) and pericentral (PC) regions, forming a functional zonation, and the oxygen gradient is considered a determinant of zone formation. It is desirable to reproduce lobular microenvironment in vitro, but incubation of primary hepatocytes in conventional culture dishes has been limited at fixed oxygen concentrations due to technical difficulties. We designed a cell culture microdevice with an oxygen gradient to reproduce the hepatic microenvironment in vitro. The oxygen gradient during cell culture was monitored using a laser-assisted phosphorescence quenching method, and the cellular oxygen consumption rate could be estimated from changes in the gradient. Culture medium was continuously exchanged through microchannels installed in the device to maintain the oxygen gradient for a long term without transient hyper-oxygenation. The oxygen consumption rates of hepatocytes at 70.0mmHg and 31.4mmHg of partial oxygen pressure, which correspond to PP and PC regions in the microdevice, were 3.67×10(-10) and 3.15×10(-10)mol/s/10(6) cells, respectively. Antimycin A changed the oxygen gradient profile, indicating that cellular respiration can be estimated during cell culture. RT-PCR analysis of hepatocytes cultured under the oxygen gradient showed that mRNA expression of PEPCK and GK significantly increased in culture areas corresponding to PP and PC regions, respectively. These results indicate that the developed microdevice can reproduce the hepatic lobular microenvironment. The oxygen gradient in the microdevice can be closely controlled by changing the sizes of gas channels and the ambient oxygen concentration around the device; therefore, it could be expected to mimic the oxygen gradient of various organs, and it may be applicable to other pathological models. Copyright © 2014 Elsevier Inc. All rights reserved.

Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals.

PubMed

Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K; Waagepetersen, Helle S

2017-01-01

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.

CORM-A1 prevents blood-brain barrier dysfunction caused by ionotropic glutamate receptor-mediated endothelial oxidative stress and apoptosis.

PubMed

Basuroy, Shyamali; Leffler, Charles W; Parfenova, Helena

2013-06-01

In cerebral microvascular endothelial cells (CMVEC) of newborn pigs, glutamate at excitotoxic concentrations (mM) causes apoptosis mediated by reactive oxygen species (ROS). Carbon monoxide (CO) produced by CMVEC or delivered by a CO-releasing molecule, CORM-A1, has antioxidant properties. We tested the hypothesis that CORM-A1 prevents cerebrovascular endothelial barrier dysfunction caused by glutamate excitotoxicity. First, we identified the glutamate receptors (GluRs) and enzymatic sources of ROS involved in the mechanism of endothelial apoptosis. In glutamate-exposed CMVEC, ROS formation and apoptosis were blocked by rotenone, 2-thenoyltrifluoroacetone (TTFA), and antimycin, indicating that mitochondrial complexes I, II, and III are the major sources of oxidative stress. Agonists of ionotropic GluRs (iGluRs) N-methyl-D-aspartate (NMDA), cis-ACPD, AMPA, and kainate increased ROS production and apoptosis, whereas iGluR antagonists exhibited antiapoptotic properties, suggesting that iGluRs mediate glutamate-induced endothelial apoptosis. The functional consequences of endothelial injury were tested in the model of blood-brain barrier (BBB) composed of CMVEC monolayer on semipermeable membranes. Glutamate and iGluR agonists reduced transendothelial electrical resistance and increased endothelial paracellular permeability to 3-kDa dextran. CORM-A1 exhibited potent antioxidant and antiapoptotic properties in CMVEC and completely prevented BBB dysfunction caused by glutamate and iGluR agonists. Overall, the endothelial component of the BBB is a cellular target for excitotoxic glutamate that, via a mechanism involving a iGluR-mediated activation of mitochondrial ROS production and apoptosis, leads to BBB opening that may be prevented by the antioxidant and antiapoptotic actions of CORMs. Antioxidant CORMs therapy may help preserve BBB functional integrity in neonatal cerebrovascular disease.

Further characterization of [3H]gamma-aminobutyric acid release from isolated neuronal growth cones: role of intracellular Ca2+ stores.

PubMed

Lockerbie, R O; Gordon-Weeks, P R

1986-04-01

We have recently shown that growth cones isolated from neonatal rat forebrain possess uptake and release mechanisms for the neurotransmitter gamma-aminobutyric acid. About half of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones is dependent on extracellular Ca2+. The remaining component of the [3H]gamma-aminobutyric acid release is unaffected by removal of extracellular Ca2+ and is resistant to blockade by the voltage-sensitive Ca2+-channel blocker methoxyverapamil. In the present series of experiments we have used caffeine to assess the possible role of intracellular stores of Ca2+ in supporting that component of the K+-induced release of [3H]gamma-aminobutyric acid from isolated growth cones that is independent of extracellular Ca2+. We have chosen caffeine because of its well established effect of releasing Ca2+ from smooth endoplasmic reticulum in muscle. We found that caffeine can release [3H]gamma-aminobutyric acid from isolated growth cones. This effect persists in Ca2+-free medium, in the presence of methoxyverapamil and in the absence of Na+. Furthermore, isobutylmethylxanthine could not substitute for caffeine suggesting that the caffeine effect is not due to phosphodiesterase inhibition and the subsequent rise in intracellular cyclic nucleotides. A combination of the mitochondrial poisons, Antimycin A and sodium azide had no effect on the release of [3H]gamma-aminobutyric acid induced either by caffeine or by high K+. We conclude that caffeine causes the release of Ca2+ from a non-mitochondrial store within the growth cone and that this Ca2+ store supports that component of the K+-induced release of [3H]gamma-aminobutyric acid that is independent of extracellular Ca2+.

Posttranslational Regulation of Human DNA Polymerase ι.

PubMed

McIntyre, Justyna; McLenigan, Mary P; Frank, Ekaterina G; Dai, Xiaoxia; Yang, Wei; Wang, Yinsheng; Woodgate, Roger

2015-11-06

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys(11)- and Lys(48)-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys(11) and Lys(48) rather than oxidative damage per se. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

The absorption of protons with specific amino acids and carbohydrates by yeast

PubMed Central

Seaston, A.; Inkson, C.; Eddy, A. A.

1973-01-01

1. Proton uptake in the presence of various amino acids was studied in washed yeast suspensions containing deoxyglucose and antimycin to inhibit energy metabolism. A series of mutant strains of Saccharomyces cerevisiae with defective amino acid permeases was used. The fast absorption of glycine, l-citrulline and l-methionine through the general amino acid permease was associated with the uptake of about 2 extra equivalents of protons per mol of amino acid absorbed, whereas the slower absorption of l-methionine, l-proline and, possibly, l-arginine through their specific permeases was associated with about 1 proton equivalent. l-Canavanine and l-lysine were also absorbed with 1–2 equivalents of protons. 2. A strain of Saccharomyces carlsbergensis behaved similarly with these amino acids. 3. Preparations of the latter yeast grown with maltose subsequently absorbed it with 2–3 equivalents of protons. The accelerated rate of proton uptake increased up to a maximum value with the maltose concentration (Km=1.6mm). The uptake of protons was also faster in the presence of α-methylglucoside and sucrose, but not in the presence of glucose, galactose or 2-deoxyglucose. All of these compounds except the last could cause acid formation. The uptake of protons induced by maltose, α-methylglucoside and sucrose was not observed when the yeast was grown with glucose, although acid was then formed both from sucrose and glucose. 4. A strain of Saccharomyces fragilis that both fermented and formed acid from lactose absorbed extra protons in the presence of lactose. 5. The observations show that protons were co-substrates in the systems transporting the amino acids and certain of the carbohydrates. PMID:4587071

Obesity-exposed oocytes accumulate and transmit damaged mitochondria due to an inability to activate mitophagy.

PubMed

Boudoures, Anna L; Saben, Jessica; Drury, Andrea; Scheaffer, Suzanne; Modi, Zeel; Zhang, Wendy; Moley, Kelle H

2017-06-01

Mitochondria are the most prominent organelle in the oocyte. Somatic cells maintain a healthy population of mitochondria by degrading damaged mitochondria via mitophagy, a specialized autophagy pathway. However, evidence from previous work investigating the more general macroautophagy pathway in oocytes suggests that mitophagy may not be active in the oocyte. This would leave the vast numbers of mitochondria - poised to be inherited by the offspring - vulnerable to damage. Here we test the hypothesis that inactive mitophagy in the oocyte underlies maternal transmission of dysfunctional mitochondria. To determine whether oocytes can complete mitophagy, we used either CCCP or AntimycinA to depolarize mitochondria and trigger mitophagy. After depolarization, we did not detect co-localization of mitochondria with autophagosomes and mitochondrial DNA copy number remained unchanged, indicating the non-functional mitochondrial population was not removed. To investigate the impact of an absence of mitophagy in oocytes with damaged mitochondria on offspring mitochondrial function, we utilized in vitro fertilization of high fat high sugar (HF/HS)-exposed oocytes, which have lower mitochondrial membrane potential and damaged mitochondria. Here, we demonstrate that blastocysts generated from HF/HS oocytes have decreased mitochondrial membrane potential, lower metabolites involved in ATP generation, and accumulation of PINK1, a mitophagy marker protein. This mitochondrial phenotype in the blastocyst mirrors the phenotype we show in HF/HS exposed oocytes. Taken together, these data suggest that the mechanisms governing oocyte mitophagy are fundamentally distinct from those governing somatic cell mitophagy and that the absence of mitophagy in the setting of HF/HS exposure contributes to the oocyte-to-blastocyst transmission of dysfunctional mitochondria. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Critical role of mitochondrial ROS is dependent on their site of production on the electron transport chain in ischemic heart.

PubMed

Madungwe, Ngonidzashe B; Zilberstein, Netanel F; Feng, Yansheng; Bopassa, Jean C

2016-01-01

Reactive oxygen species (ROS) generation has been implicated in many pathologies including ischemia/reperfusion (I/R) injury. This led to multiple studies on antioxidant therapies to treat cardiovascular diseases but paradoxically, results have so far been mixed as ROS production can be beneficial as a signaling mechanism and in cardiac protection via preconditioning interventions. We investigated whether the differential impact of increased ROS in injury as well as in protection could be explained by their site of production on the mitochondrial electron transport chain. Using amplex red to measure ROS production, we found that mitochondria isolated from hearts after I/R produced more ROS than non-ischemic when complex I substrate (glutamate/malate) was used. Interestingly, the substrates of complex II (succinate) and ubiquinone (sn-glycerol 3-phosphate, G3P) produced less ROS in mitochondria from I/R hearts compared to normal healthy hearts. The inhibitors of complex I (rotenone) and complex III (antimycin A) increased ROS production when glutamate/malate and G3P were used; in contrast, they reduced ROS production when the complex II substrate was used. Mitochondrial calcium retention capacity required to induce mitochondrial permeability transition pore (mPTP) opening was measured using calcium green fluorescence and was found to be higher when mitochondria were treated with G3P and succinate compared to glutamate/malate. Furthermore, Langendorff hearts treated with glutamate/malate exhibited reduced cardiac functional recovery and increased myocardial infarct size compared to hearts treated with G3P. Thus, ROS production by the stimulated respiratory chain complexes I and III has opposite roles: cardio-deleterious when produced in complex I and cardio-protective when produced in complex III. The mechanism of these ROS involves the inhibition of the mPTP opening, a key event in cell death following ischemia/reperfusion injury.

Calcium transport in tonoplast and endoplasmic reticulum vesicles isolated from cultured carrot cells. [Daucus carota Danvers

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bush, D.R.; Sze, H.

1986-02-01

Two active calcium (Ca/sup 2 +/) transport systems have been identified and partially characterized in membrane vesicles isolated from cultured carrot cells (Daucus carota Danvers). Both transport systems required MgATP for activity and were enhanced by 10 millimolar oxalate. Ca/sup 2 +/ transport in membrane vesicles derived from isolated vacuoles equilibrated at 1.10 grams per cubic centimeter and comigrated with Cl/sup -/-stimulated, NO/sub 3//sup -/-inhibited ATPase activity on sucrose density gradients. Ca/sup 2 +/ transport in this system was insensitive to vanadate, but was inhibited by nitrate, carbonyl cyanide-m-chlorophenylhydrazone (CCCP), N,N'-dicyclohexylcarbodiimide (DCCD), and 4,4-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS). The K/sub m/more » for MgATP and Ca/sup 2 +/ were 0.1 mM and 21 micromolar, respectively. The predominant Ca/sup 2 +/ transport system detectable in microsomal membrane preparations equilibrated at a density of 1.13 grams per cubic centimeter and comigrated with the endoplasmic reticulum (ER) marker, antimycin A-insensitive NADH-dependent cytochrome c reductase. Ca/sup 2 +/ transport activity and the ER marker also shifted in parallel in ER shifting experiments. This transport system was inhibited by vanadate (I/sub 50/ = 12 micromolar) and was insensitive to nitrate, CCCP, DCCD, and DIDS. Transport exhibited cooperative MgATP dependent kinetics. Ca/sup 2 +/ dependent kinetics were complex with an apparent K/sub m/ ranging from 0.7 to 2 micromolar. We conclude that the vacuolar-derived system is a Ca/sup 2 +//H/sup +/ antiport located on the tonoplast and that the microsomal transport system is a Ca,Mg-ATPase enriched on the ER. These two Ca/sup 2 +/ transport systems are proposed to restore and maintain cytoplasmic Ca/sup 2 +/ homeostasis under changing cellular and environmental conditions.« less

Ischemia/Reperfusion Model Impairs Endocannabinoid Signaling and Na+/K+ ATPase Expression and Activity in Kidney Proximal Tubule Cells.

PubMed

Sampaio, Luzia S; Iannotti, Fabio A; Veneziani, Luciana; Borelli-Tôrres, Rosa T; De Maio, Fabrizia; Piscitelli, Fabiana; Reis, Ricardo A M; Di Marzo, Vincenzo; Einicker-Lamas, Marcelo

2018-06-08

LLC-PK1 cells, an immortalized epithelial cell line derived from pig renal proximal tubules, express all the major players of the endocannabinoid system (ECS) such as CB1, CB2 and TRPV1 receptor, as well as the main enzymes involved in the biosynthesis and degradation of the major endocannabinoids named 2-arachidonoylglycerol, 2-AG and anandamide, AEA. Here we investigated whether the damages caused by ischemic insult either in vitro using LLC-PK1 cells exposed to antimycin A (an inductor of ATP-depletion) or in vivo using Wistar rats in a classic renal ischemia and reperfusion (IR) protocol, lead to changes in AEA and 2-AG levels, as well as altered expression of genes from the main enzymes involved in the regulation of the ECS. Our data show that the mRNA levels of CB1 receptor gene were downregulated, while the transcript levels of monoacylglycerol lipase (MAGL), the main 2-AG degradative enzyme, are upregulated in LLC-PK1 cells after IR model. Accordingly, IR was accompanied by a significant reduction in the levels of 2-AG and AEA, as well as of the two endocannabinoid related molecules, oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) in LLC-PK1 cells. In kidney cortex homogenates, the AEA levels were selectively significantly decreased. In addition, we found that both the in vitro and in vivo model of IR caused a reduction in the expression and activity of the Na + /K + ATPase. These changes were reversed by the CB1/CB2 agonist WIN55,212, in a CB1-receptor dependent manner on LLC-PK1 IR model. In conclusion, the ECS and Na + /K + ATPase are down-regulated following IR model in LLC-PK1 cells and rat kidney. We suggest that CB1 agonists might represent a potential strategy to reverse the consequences of IR injury in kidney tissues. Copyright © 2018 Elsevier Inc. All rights reserved.

Mitochondrial Dysfunction Leads to Deconjugation of Quercetin Glucuronides in Inflammatory Macrophages

PubMed Central

Miki, Satomi; Shiba, Yuko; Minekawa, Shoko; Nishikawa, Tomomi; Mukai, Rie; Terao, Junji; Kawai, Yoshichika

2013-01-01

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that β-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of β-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the β-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular β-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites. PMID:24260490

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions

PubMed Central

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja

2018-01-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/106 cells after cold storage, 5 ± 3 nmol/106 cells after rewarming vs. control 29 ± 6 nmol/106 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec−1 per 106 cells after rewarming vs. control 232 ± 83 pmol sec−1 per 106 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/106 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production—as elicited by an inhibitor of the respiratory chain, antimycin A—can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions. PMID:29390882

Bioprospecting Deep-Sea Actinobacteria for Novel Anti-infective Natural Products

PubMed Central

Xu, Dongbo; Han, Linna; Li, Chunhui; Cao, Qi; Zhu, Duolong; Barrett, Nolan H.; Harmody, Dedra; Chen, Jing; Zhu, Haining; McCarthy, Peter J.; Sun, Xingmin; Wang, Guojun

2018-01-01

The global prevalence of drug resistance has created an urgent need for the discovery of novel anti-infective drugs. The major source of antibiotics in current clinical practice is terrestrial actinobacteria; the less-exploited deep-sea actinobacteria may serve as an unprecedented source of novel natural products. In this study, we evaluated 50 actinobacteria strains derived from diverse deep water sponges and environmental niches for their anti-microbial activities against a panel of pathogens including Candida albicans, Clostridium difficile, Staphylococcus aureus, and methicillin-resistant S. aureus (MRSA), and Pseudomonas aeruginosa. More than half of the tested strains (27) were identified as active in at least one assay. The rare earth salt lanthanum chloride (LaCl3) was shown to be as an effective elicitor. Among the 27 strains, the anti-microbial activity of 15 were induced or enhanced by the addition of LaCl3. This part of study focused on one strain R818, in which potent antifungal activity was induced by the addition of LaCl3. We found that the LaCl3-activated metabolites in R818 are likely antimycin-type compounds. One of them, compound 1, has been purified. Spectroscopic analyses including HR-MS and 1D NMR indicated that this compound is urauchimycin D. The antifungal activity of compound 1 was confirmed with a minimal inhibitory concentration (MIC) of 25 μg/mL; the purified compound also showed a moderate activity against C. difficile. Additional notable strains are: strain N217 which showed both antifungal and antibacterial (including P. aeruginosa) activities and strain M864 which showed potent activity against C. difficile with an MIC value (0.125 μg/mL) lower than those of vancomycin and metronidazole. Our preliminary studies show that deep-sea actinobacteria is a promising source of anti-infective natural products. PMID:29760684

Evidence of ROS generation by mitochondria in cells with impaired electron transport chain and mitochondrial DNA damage.

PubMed

Indo, Hiroko P; Davidson, Mercy; Yen, Hsiu-Chuan; Suenaga, Shigeaki; Tomita, Kazuo; Nishii, Takeshi; Higuchi, Masahiro; Koga, Yasutoshi; Ozawa, Toshihiko; Majima, Hideyuki J

2007-01-01

Mitochondrial damage is a well known cause of mitochondria-related diseases. A major mechanism underlying the development of mitochondria-related diseases is thought to be an increase in intracellular oxidative stress produced by impairment of the mitochondrial electron transport chain (ETC). However, clear evidence of intracellular free radical generation has not been clearly provided for mitochondrial DNA (mtDNA)-damaged cells. In this study, using the novel fluorescence dye, 2-[6-(4'-hydroxy)phenoxy-3H-xanthen-3-on-9-yl]benzoic acid (HPF), which was designed to detect hydroxyl radicals (*OH), intracellular free radical formation was examined in 143B cells (parental cells), 143B-rho(0) cells (mtDNA-lacking cells), 87 wt (cybrid), and cybrids of 4977-bp mtDNA deletion (common deletion) cells containing the deletion with 0%, 5%, 50% and >99% frequency (HeLacot, BH5, BH50 and BH3.12, respectively), using a laser confocal microscope detection method. ETC inhibitors (rotenone, 3-nitropropionic acid, thenoyltrifluoroacetone, antimycin A and sodium cyanide) were also tested to determine whether inhibitor treatment increased intracellular reactive oxygen species (ROS) generation. A significant increase in ROS for 143B-rho(0) cells was observed compared with 143B cells. However, for the 87 wt cybrid, no increase was observed. An increase was also observed in the mtDNA-deleted cells BH50 and BH3.12. The ETC inhibitors increased intracellular ROS in both 143B and 143B-rho(0) cells. Furthermore, in every fluorescence image, the fluorescence dye appeared localized around the nuclei. To clarify the localization, we double-stained cells with the dye and MitoTracker Red. The resulting fluorescence was consistently located in mitochondria. Furthermore, manganese superoxide dismutase (MnSOD) cDNA-transfected cells had decreased ROS. These results suggest that more ROS are generated from mitochondria in ETC-inhibited and mtDNA-damaged cells, which have impaired ETC.

The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7).

PubMed

Charizanis, C; Juhnke, H; Krems, B; Entian, K D

1999-10-01

In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation groupfap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F, which is characterized by a 10(4)-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.

Reconstitution of the anti-apoptotic Bcl-2 protein into lipid membranes and biophysical evidence for its detergent-driven association with the pro-apoptotic Bax protein.

PubMed

Wallgren, Marcus; Lidman, Martin; Pedersen, Anders; Brännström, Kristoffer; Karlsson, B Göran; Gröbner, Gerhard

2013-01-01

The anti-apoptotic B-cell CLL/lymphoma-2 (Bcl-2) protein and its counterpart, the pro-apoptotic Bcl-2-associated X protein (Bax), are key players in the regulation of the mitochondrial pathway of apoptosis. However, how they interact at the mitochondrial outer membrane (MOM) and there determine whether the cell will live or be sentenced to death remains unknown. Competing models have been presented that describe how Bcl-2 inhibits the cell-killing activity of Bax, which is common in treatment-resistant tumors where Bcl-2 is overexpressed. Some studies suggest that Bcl-2 binds directly to and sequesters Bax, while others suggest an indirect process whereby Bcl-2 blocks BH3-only proteins and prevents them from activating Bax. Here we present the results of a biophysical study in which we investigated the putative interaction of solubilized full-length human Bcl-2 with Bax and the scope for incorporating the former into a native-like lipid environment. Far-UV circular dichroism (CD) spectroscopy was used to detect direct Bcl-2-Bax-interactions in the presence of polyoxyethylene-(23)-lauryl-ether (Brij-35) detergent at a level below its critical micelle concentration (CMC). Additional surface plasmon resonance (SPR) measurements confirmed this observation and revealed a high affinity between the Bax and Bcl-2 proteins. Upon formation of this protein-protein complex, Bax also prevented the binding of antimycin A2 (a known inhibitory ligand of Bcl-2) to the Bcl-2 protein, as fluorescence spectroscopy experiments showed. In addition, Bcl-2 was able to form mixed micelles with Triton X-100 solubilized neutral phospholipids in the presence of high concentrations of Brij-35 (above its CMC). Following detergent removal, the integral membrane protein was found to have been fully reconstituted into a native-like membrane environment, as confirmed by ultracentrifugation and subsequent SDS-PAGE experiments.

Mitochondrial Impairment as a Key Factor for the Lack of Attachment after Cold Storage of Hepatocyte Suspensions.

PubMed

Pless-Petig, Gesine; Walter, Björn; Bienholz, Anja; Rauen, Ursula

2017-12-01

Isolated primary hepatocytes, which are widely used for pharmacological and clinical purposes, usually undergo certain periods of cold storage in suspension during processing. While adherent hepatocytes were shown previously to suffer iron-dependent cell death during cold (4 °C) storage and early rewarming, we previously found little iron-dependent hepatocyte death in suspension but severely decreased attachment ability unless iron chelators were added. Here, we focus on the role of mitochondrial impairment in this nonattachment of hepatocyte suspensions. Rat hepatocyte suspensions were stored in a chloride-poor, glycine-containing cold storage solution with and without iron chelators at 4 °C. After 1 wk of cold storage in the basic cold storage solution, cell viability in suspension was unchanged, while cell attachment was decreased by >80%. In the stored cells, a loss of mitochondrial membrane potential (MMP), a decrease in adenosine triphosphate (ATP) content (2 ± 2 nmol/10 6 cells after cold storage, 5 ± 3 nmol/10 6 cells after rewarming vs. control 29 ± 6 nmol/10 6 cells), and a decrease in oxygen consumption (101 ± 59 pmol sec -1 per 10 6 cells after rewarming vs. control 232 ± 83 pmol sec -1 per 10 6 cells) were observed. Addition of iron chelators to the cold storage solution increased cell attachment to 53% ± 20% and protected against loss of MMP, and cells were able to partially regenerate ATP during rewarming (15 ± 10 nmol/10 6 cells). Increased attachment could also be achieved by addition of the inhibitor combination of mitochondrial permeability transition, trifluoperazine + fructose. Attached hepatocytes displayed normal MMP and mitochondrial morphology. Additional experiments with freshly isolated hepatocytes confirmed that impaired energy production-as elicited by an inhibitor of the respiratory chain, antimycin A-can decrease cell attachment without decreasing viability. Taken together, these results suggest that mitochondrial impairment with subsequent energy deficiency is a key factor for the lack of attachment of cold-stored hepatocyte suspensions.

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines : a mechanism for induction of apoptosis via the 18 kDa mitochondrial translocator protein.

PubMed

Veenman, Leo; Alten, Julia; Linnemannstöns, Karen; Shandalov, Yulia; Zeno, Sivan; Lakomek, Max; Gavish, Moshe; Kugler, Wilfried

2010-07-01

Erucylphosphohomocholine (ErPC3, Erufosine) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F(O)F(1)-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F(O) and F(1) subunits of the mitochondrial F(O)F(1)-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Deltapsi(m)). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Deltapsi(m), normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F(O) subunit of the mitochondrial F(O)F(1)-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Deltapsi(m) as well as ROS generation by ErPC3 and TSPO.

Low level tumor necrosis factor-alpha protects cardiomyocytes against high level tumor necrosis factor-alpha: brief insight into a beneficial paradox.

PubMed

Cacciapaglia, Fabio; Salvatorelli, Emanuela; Minotti, Giorgio; Afeltra, Antonella; Menna, Pierantonio

2014-12-01

Whether tumor necrosis factor-alpha (TNFα) caused beneficial or detrimental cardiovascular effects remains poorly defined. Anti-TNFα agents improved cardiac end points in chronic rheumatic diseases characterized by progressive deterioration of cardiac function. In contrast, anti-TNFα agents did not always improve but actually worsened cardiac function in non-rheumatic patients with heart failure (HF), in spite of that HF usually accompanies with high circulating levels of TNFα. To shed light on these mixed findings, we characterized the effects of TNFα in H9c2 cardiomyocytes. Cells were incubated for 24 h with increasing concentrations of TNFα, hydrogen peroxide, aminotriazole, or etoposide. Posttreatment cell viability was assessed by antimycin A-inhibitable reduction of 3-(4,dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, and the IC50 value of each test compound was defined. H9c2 cells were also preconditioned with a low non-toxic concentration of TNFα and then re-challenged with increasing concentrations of TNFα and other stressor agents. In re-challenge experiments, all of the IC50 values increased significantly, with the IC50 value of TNFα increasing approximately 16-fold. TNFα preconditioning increased cardiomyocytes shedding of the external portion of transmembrane type 1 and type 2 TNFα receptors [(soluble TNFα receptors (sTNFR)]. Levels of survival-oriented soluble TNFR2 (sTNFR2) always exceeded those of death-oriented sTNFR1. When exposed to TNFα at its IC50 value, preconditioned cardiomyocytes showed an increased release of sTNFR2 but not sTNFR1. These results denoted that preconditioning by "low TNFα" helped cardiomyocyte to withstand toxicity from "high TNFα" or other agents. These results also suggested that beneficial or detrimental effects of anti-TNFα agents might well depend on whether these agents spared or intercepted discrete amounts of TNFα that preconditioned cardiomyocytes and made them more resistant to high concentrations of TNFα.

The metabolism of malate by cultured rat brain astrocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

McKenna, M.C.; Tildon, J.T.; Couto, R.

1990-12-01

Since malate is known to play an important role in a variety of functions in the brain including energy metabolism, the transfer of reducing equivalents and possibly metabolic trafficking between different cell types; a series of biochemical determinations were initiated to evaluate the rate of 14CO2 production from L-(U-14C)malate in rat brain astrocytes. The 14CO2 production from labeled malate was almost totally suppressed by the metabolic inhibitors rotenone and antimycin A suggesting that most of malate metabolism was coupled to the electron transport system. A double reciprocal plot of the 14CO2 production from the metabolism of labeled malate revealed biphasicmore » kinetics with two apparent Km and Vmax values suggesting the presence of more than one mechanism of malate metabolism in these cells. Subsequent experiments were carried out using 0.01 mM and 0.5 mM malate to determine whether the addition of effectors would differentially alter the metabolism of high and low concentrations of malate. Effectors studied included compounds which could be endogenous regulators of malate metabolism and metabolic inhibitors which would provide information regarding the mechanisms regulating malate metabolism. Both lactate and aspartate decreased 14CO2 production from malate equally. However, a number of effectors were identified which selectively altered the metabolism of 0.01 mM malate including aminooxyacetate, furosemide, N-acetylaspartate, oxaloacetate, pyruvate and glucose, but had little or no effect on the metabolism of 0.5 mM malate. In addition, alpha-ketoglutarate and succinate decreased 14CO2 production from 0.01 mM malate much more than from 0.5 mM malate. In contrast, a number of effectors altered the metabolism of 0.5 mM malate more than 0.01 mM. These included methionine sulfoximine, glutamate, malonate, alpha-cyano-4-hydroxycinnamate and ouabain.« less

Accelerated recovery of mitochondrial membrane potential by GSK-3β inactivation affords cardiomyocytes protection from oxidant-induced necrosis.

PubMed

Sunaga, Daisuke; Tanno, Masaya; Kuno, Atsushi; Ishikawa, Satoko; Ogasawara, Makoto; Yano, Toshiyuki; Miki, Takayuki; Miura, Tetsuji

2014-01-01

Loss of mitochondrial membrane potential (ΔΨm) is known to be closely linked to cell death by various insults. However, whether acceleration of the ΔΨm recovery process prevents cell necrosis remains unclear. Here we examined the hypothesis that facilitated recovery of ΔΨm contributes to cytoprotection afforded by activation of the mitochondrial ATP-sensitive K+ (mKATP) channel or inactivation of glycogen synthase kinase-3β (GSK-3β). ΔΨm of H9c2 cells was determined by tetramethylrhodamine ethyl ester (TMRE) before or after 1-h exposure to antimycin A (AA), an inducer of reactive oxygen species (ROS) production at complex III. Opening of the mitochondrial permeability transition pore (mPTP) was determined by mitochondrial loading of calcein. AA reduced ΔΨm to 15 ± 1% of the baseline and induced calcein leak from mitochondria. ΔΨm was recovered to 51 ± 3% of the baseline and calcein-loadable mitochondria was 6 ± 1% of the control at 1 h after washout of AA. mKATP channel openers improved the ΔΨm recovery and mitochondrial calcein to 73 ± 2% and 30 ± 7%, respectively, without change in ΔΨm during AA treatment. Activation of the mKATP channel induced inhibitory phosphorylation of GSK-3β and suppressed ROS production, LDH release and apoptosis after AA washout. Knockdown of GSK-3β and pharmacological inhibition of GSK-3β mimicked the effects of mKATP channel activation. ROS scavengers administered at the time of AA removal also improved recovery of ΔΨm. These results indicate that inactivation of GSK-3β directly or indirectly by mKATP channel activation facilitates recovery of ΔΨm by suppressing ROS production and mPTP opening, leading to cytoprotection from oxidant stress-induced cell death.

Rotenone Induction of Hydrogen Peroxide Inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E Pathways, Leading to Neuronal Apoptosis

PubMed Central

Zhou, Qian; Liu, Chunxiao; Liu, Wen; Zhang, Hai; Zhang, Ruijie; Liu, Jia; Zhang, Jinfei; Xu, Chong; Liu, Lei; Huang, Shile; Chen, Long

2015-01-01

Rotenone, a common pesticide and inhibitor of mitochondrial complex I, induces loss of dopaminergic neurons and consequential aspects of Parkinson’s disease (PD). However, the exact mechanism of rotenone neurotoxicity is not fully elucidated. Here, we show that rotenone induced reactive oxygen species (ROS), leading to apoptotic cell death in PC12 cells and primary neurons. Pretreatment with catalase (CAT), a hydrogen peroxide-scavenging enzyme, attenuated rotenone-induced ROS and neuronal apoptosis, implying hydrogen peroxide (H2O2) involved, which was further verified by imaging intracellular H2O2 using a peroxide-selective probe H2DCFDA. Using thenoyltrifluoroacetone (TTFA), antimycin A, or Mito-TEMPO, we further demonstrated rotenone-induced mitochondrial H2O2-dependent neuronal apoptosis. Rotenone dramatically inhibited mTOR-mediated phosphorylation of S6K1 and 4E-BP1, which was also attenuated by CAT in the neuronal cells. Of interest, ectopic expression of wild-type mTOR or constitutively active S6K1, or downregulation of 4E-BP1 partially prevented rotenone-induced H2O2 and cell apoptosis. Furthermore, we noticed that rotenone-induced H2O2 was linked to the activation of caspase-3 pathway. This was evidenced by the finding that pretreatment with CAT partially blocked rotenone-induced cleavages of caspase-3 and poly (ADP-ribose) polymerase. Of note, zVAD-fmk, a pan caspase inhibitor, only partially prevented rotenone-induced apoptosis in PC12 cells and primary neurons. Expression of mTOR-wt, S6K1-ca, or silencing 4E-BP1 potentiated zVAD-fmk protection against rotenone-induced apoptosis in the cells. The results indicate that rotenone induction of H2O2 inhibits mTOR-mediated S6K1 and 4E-BP1/eIF4E pathways, resulting in caspase-dependent and -independent apoptosis in neuronal cells. Our findings suggest that rotenone-induced neuronal loss in PD may be prevented by activating mTOR signaling and/or administering antioxidants. PMID:25304210

Visualizing changes in electron distribution in coupled chains of cytochrome bc(1) by modifying barrier for electron transfer between the FeS cluster and heme c(1).

PubMed

Cieluch, Ewelina; Pietryga, Krzysztof; Sarewicz, Marcin; Osyczka, Artur

2010-02-01

Cytochrome c(1) of Rhodobacter (Rba.) species provides a series of mutants which change barriers for electron transfer through the cofactor chains of cytochrome bc(1) by modifying heme c(1) redox midpoint potential. Analysis of post-flash electron distribution in such systems can provide useful information about the contribution of individual reactions to the overall electron flow. In Rba. capsulatus, the non-functional low-potential forms of cytochrome c(1) which are devoid of the disulfide bond naturally present in this protein revert spontaneously by introducing a second-site suppression (mutation A181T) that brings the potential of heme c(1) back to the functionally high levels, yet maintains it some 100 mV lower from the native value. Here we report that the disulfide and the mutation A181T can coexist in one protein but the mutation exerts a dominant effect on the redox properties of heme c(1) and the potential remains at the same lower value as in the disulfide-free form. This establishes effective means to modify a barrier for electron transfer between the FeS cluster and heme c(1) without breaking disulfide. A comparison of the flash-induced electron transfers in native and mutated cytochrome bc(1) revealed significant differences in the post-flash equilibrium distribution of electrons only when the connection of the chains with the quinone pool was interrupted at the level of either of the catalytic sites by the use of specific inhibitors, antimycin or myxothiazol. In the non-inhibited system no such differences were observed. We explain the results using a kinetic model in which a shift in the equilibrium of one reaction influences the equilibrium of all remaining reactions in the cofactor chains. It follows a rather simple description in which the direction of electron flow through the coupled chains of cytochrome bc(1) exclusively depends on the rates of all reversible partial reactions, including the Q/QH2 exchange rate to/from the catalytic sites. 2009 Elsevier B.V. All rights reserved.

Antioxidant mechanism of mitochondria-targeted plastoquinone SkQ1 is suppressed in aglycemic HepG2 cells dependent on oxidative phosphorylation.

PubMed

Ježek, Jan; Engstová, Hana; Ježek, Petr

2017-09-01

Previously suggested antioxidant mechanisms for mitochondria-targeted plastoquinone SkQ1 included: i) ion-pairing of cationic SkQ1 + with free fatty acid anions resulting in uncoupling; ii) SkQ1H 2 ability to interact with lipoperoxyl radical; iii) interference with electron flow at the inner ubiquinone (Q) binding site of Complex III (Q i ), involving the reduction of SkQ1 to SkQ1H 2 by ubiquinol. We elucidated SkQ1 antioxidant properties by confocal fluorescence semi-quantification of mitochondrial superoxide (J m ) and cytosolic H 2 O 2 (J c ) release rates in HepG2 cells. Only in glycolytic cells, SkQ1 prevented the rotenone-induced enhancement of J m and J c but not basal releases without rotenone. The effect ceased in glutaminolytic aglycemic cells, in which the redox parameter NAD(P)H/FAD increased after rotenone in contrast to its decrease in glycolytic cells. Autofluorescence decay indicated decreased NADPH/NADH ratios with rotenone in both metabolic modes. SkQ1 did not increase cell respiration and diminished J m established high by antimycin or myxothiazol but not by stigmatellin. The revealed SkQ1 antioxidant modes reflect its reduction to SkQ1H 2 at Complex I I Q or Complex III Q i site. Both reductions diminish electron diversions to oxygen thus attenuating superoxide formation. Resulting SkQ1H 2 oxidizes back to SkQ1at the second (flavin) Complex I site, previously indicated for MitoQ 10 . Regeneration proceeds only at lower NAD(P)H/FAD in glycolytic cells. In contrast, cyclic SkQ1 reduction/SkQ1H 2 oxidation does not substantiate antioxidant activity in intact cells in the absence of oxidative stress (neither pro-oxidant activity, representing a great advantage). A targeted delivery to oxidative-stressed tissues is suggested for the effective antioxidant therapy based on SkQ1. Copyright © 2017 Elsevier B.V. All rights reserved.

Site of mitochondrial reactive oxygen species production in skeletal muscle of chronic obstructive pulmonary disease and its relationship with exercise oxidative stress.

PubMed

Puente-Maestu, Luis; Tejedor, Alberto; Lázaro, Alberto; de Miguel, Javier; Alvarez-Sala, Luis; González-Aragoneses, Federico; Simón, Carlos; Agustí, Alvar

2012-09-01

Exercise triggers skeletal muscle oxidative stress in patients with chronic obstructive pulmonary disease (COPD). The objective of this research was to study the specific sites of reactive oxygen species (ROS) production in mitochondria isolated from skeletal muscle of patients with COPD and its relationship with local oxidative stress induced by exercise. Vastus lateralis biopsies were obtained in 16 patients with COPD (66 ± 10 yr; FEV(1), 54 ± 12% ref) and in 14 control subjects with normal lung function who required surgery because of lung cancer (65 ± 7 yr; FEV(1), 91 ± 14% ref) at rest and after exercise. In these biopsies we isolated mitochondria and mitochondrial membrane fragments and determined in vitro mitochondrial oxygen consumption (Mit$$\\stackrel{.}{\\hbox{ V }}$$o(2)) and ROS production before and after inhibition of complex I (rotenone), complex II (stigmatellin), and complex III (antimycin-A). We related the in vitro ROS production during state 3 respiration), which mostly corresponds to the mitochondria respiratory state during exercise, with skeletal muscle oxidative stress after exercise, as measured by thiobarbituric acid reactive substances.State 3 Mit$$\\stackrel{.}{\\hbox{ V }}$$o(2) was similar in patients with COPD and control subjects (191 ± 27 versus 229 ± 46 nmol/min/mg; P = 0.058), whereas H(2)O(2) production was higher in the former (147 ± 39 versus 51 ± 8 pmol/mg/h; P < 0.001). The addition of complexI, II, and III inhibitors identify complex III as the main site of H(2)O(2) release by mitochondria in patients with COPD and in control subjects. The mitochondrial production of H(2)O(2) in state 3 respiration was related (r = 0.69; P < 0.001) to postexercise muscle thiobarbituric acid reactive substance levels. Our results show that complex III is the main site of the enhanced mitochondrial H(2)O(2) production that occurs in skeletal muscle of patients with COPD, and the latter appears to contribute to muscle oxidative damage.

The origin of the post-tetanic hyperpolarization of mammalian motor nerve terminals

PubMed Central

Gage, P. W.; Hubbard, J. I.

1966-01-01

1. Motor nerve terminals in magnesium-poisoned rat hemidiaphragm-phrenic nerve preparations in vitro were stimulated with short depolarizing pulses of approximately threshold strength and the evoked antidromic responses recorded from the phrenic nerve. The percentage of these 1/sec or 0·5/sec stimuli to which there was no antidromic response was used as a quantitative measure of the terminal excitability. After standard tetanic stimulation (1000 impulses at 100/sec) the excitability of the terminals was depressed for an average duration of 60-70 sec, during most of which time no antidromic responses to stimuli of pretetanic intensity were recorded. There was no significant interaction between stimuli to the terminals at rates of 1 or 0·5/sec. 2. Potassium-free solutions at first increased, then decreased, the post-tetanic depression of excitability. Raising [K]o threefold (15 mM) abolished the post-tetanic depression and often converted it to an exaltation of excitability. 3. Polarizing currents were applied to the terminals with a second electrode. Depolarizing currents increased, while hyperpolarizing currents decreased, the post-tetanic depression of excitability. 4. In solutions with 70% of the normal NaCl content replaced by sucrose, the post-tetanic depression of excitability was reversibly prolonged. 5. In the presence of 7·7 × 10-6 M digoxin or 0·42 mM ouabain there was a small reversible reduction of post-tetanic excitability. 6. After exposure to solutions containing no glucose or to solutions containing 3-5 mM sodium azide the excitability of the terminals was not altered by the tetanus. After washing with the control solution, post-tetanic depression of excitability returned. Antimycin-A (1·8 × 10-6 M) had little or no effect upon post-tetanic excitability. 7. It was concluded that the post-tetanic depression of excitability reflected hyperpolarization of the terminals and that this hyperpolarization was caused by a shift of the membrane potential towards the potassium equilibrium potential because of an increase in potassium permeability. ImagesFig. 1 PMID:5921834

High Intensity Interval Training (HIIT) Induces Specific Changes in Respiration and Electron Leakage in the Mitochondria of Different Rat Skeletal Muscles

PubMed Central

de-Souza-Ferreira, Eduardo; Guerra Martinez, Camila; Kurtenbach, Eleonora; Casimiro-Lopes, Gustavo; Galina, Antonio

2015-01-01

High intensity interval training (HIIT) is characterized by vigorous exercise with short rest intervals. Hydrogen peroxide (H2O2) plays a key role in muscle adaptation. This study aimed to evaluate whether HIIT promotes similar H2O2 formation via O2 consumption (electron leakage) in three skeletal muscles with different twitch characteristics. Rats were assigned to two groups: sedentary (n=10) and HIIT (n=10, swimming training). We collected the tibialis anterior (TA-fast), gastrocnemius (GAST-fast/slow) and soleus (SOL-slow) muscles. The fibers were analyzed for mitochondrial respiration, H2O2 production and citrate synthase (CS) activity. A multi-substrate (glycerol phosphate (G3P), pyruvate, malate, glutamate and succinate) approach was used to analyze the mitochondria in permeabilized fibers. Compared to the control group, oxygen flow coupled to ATP synthesis, complex I and complex II was higher in the TA of the HIIT group by 1.5-, 3.0- and 2.7-fold, respectively. In contrast, oxygen consumed by mitochondrial glycerol phosphate dehydrogenase (mGPdH) was 30% lower. Surprisingly, the oxygen flow coupled to ATP synthesis was 42% lower after HIIT in the SOL. Moreover, oxygen flow coupled to ATP synthesis and complex II was higher by 1.4- and 2.7-fold in the GAST of the HIIT group. After HIIT, CS activity increased 1.3-fold in the TA, and H2O2 production was 1.3-fold higher in the TA at sites containing mGPdH. No significant differences in H2O2 production were detected in the SOL. Surprisingly, HIIT increased H2O2 production in the GAST via complex II, phosphorylation, oligomycin and antimycin by 1.6-, 1.8-, 2.2-, and 2.2-fold, respectively. Electron leakage was 3.3-fold higher in the TA with G3P and 1.8-fold higher in the GAST with multiple substrates. Unexpectedly, the HIIT protocol induced different respiration and electron leakage responses in different types of muscle. PMID:26121248

High Intensity Interval Training (HIIT) Induces Specific Changes in Respiration and Electron Leakage in the Mitochondria of Different Rat Skeletal Muscles.

PubMed

Ramos-Filho, Dionizio; Chicaybam, Gustavo; de-Souza-Ferreira, Eduardo; Guerra Martinez, Camila; Kurtenbach, Eleonora; Casimiro-Lopes, Gustavo; Galina, Antonio

2015-01-01

High intensity interval training (HIIT) is characterized by vigorous exercise with short rest intervals. Hydrogen peroxide (H2O2) plays a key role in muscle adaptation. This study aimed to evaluate whether HIIT promotes similar H2O2 formation via O2 consumption (electron leakage) in three skeletal muscles with different twitch characteristics. Rats were assigned to two groups: sedentary (n=10) and HIIT (n=10, swimming training). We collected the tibialis anterior (TA-fast), gastrocnemius (GAST-fast/slow) and soleus (SOL-slow) muscles. The fibers were analyzed for mitochondrial respiration, H2O2 production and citrate synthase (CS) activity. A multi-substrate (glycerol phosphate (G3P), pyruvate, malate, glutamate and succinate) approach was used to analyze the mitochondria in permeabilized fibers. Compared to the control group, oxygen flow coupled to ATP synthesis, complex I and complex II was higher in the TA of the HIIT group by 1.5-, 3.0- and 2.7-fold, respectively. In contrast, oxygen consumed by mitochondrial glycerol phosphate dehydrogenase (mGPdH) was 30% lower. Surprisingly, the oxygen flow coupled to ATP synthesis was 42% lower after HIIT in the SOL. Moreover, oxygen flow coupled to ATP synthesis and complex II was higher by 1.4- and 2.7-fold in the GAST of the HIIT group. After HIIT, CS activity increased 1.3-fold in the TA, and H2O2 production was 1.3-fold higher in the TA at sites containing mGPdH. No significant differences in H2O2 production were detected in the SOL. Surprisingly, HIIT increased H2O2 production in the GAST via complex II, phosphorylation, oligomycin and antimycin by 1.6-, 1.8-, 2.2-, and 2.2-fold, respectively. Electron leakage was 3.3-fold higher in the TA with G3P and 1.8-fold higher in the GAST with multiple substrates. Unexpectedly, the HIIT protocol induced different respiration and electron leakage responses in different types of muscle.

NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK-2 cells.

PubMed

Dong, Wei; Li, Zhilian; Chen, Yuanhan; Zhang, Li; Ye, Zhiming; Liang, Huaban; Li, Ruizhao; Xu, Lixia; Zhang, Bin; Liu, Shuangxin; Wang, Weidong; Li, Chunling; Luo, Jialun; Shi, Wei; Liang, Xinling

2017-09-01

The aim of the present study was to investigate the protective effect of the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) against necroptosis in renal tubular epithelial cells. A necroptosis model of HK-2 cells was established using tumor necrosis factor-α, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone and antimycin A (collectively termed TZA), as in our previous research. The necroptosis inhibitor, necrostatin-1 (Nec-1) or the NADPH oxidase inhibitor, DPI were administered to the necroptosis model. Production of reactive oxygen species (ROS) was detected by dichlorodihydrofluorescein diacetate in the different groups, and the manner of cell death was identified by flow cytometry. Western blot analysis was used to determine the levels of phosphorylation of receptor-interacting protein kinase 3 (RIP-3) and mixed lineage kinase domain-like (MLKL), which are essential to necroptosis. The results revealed that TZA increased the percentages of propidium iodide-positive HK-2 cells from 1.22±0.69 to 8.98±0.73% (P<0.001), and augmented the phosphorylation of RIP-3 and MLKL. ROS levels were increased in the TZA group compared with the control group (27.74±1.60×10 4 vs. 18.51±1.10×10 4 , respectively; P<0.001), and could be inhibited by Nec-1 (TZA + Nec-1 group, 22.90±2.22×10 4 vs. TZA group, 27.74±1.60×10 4 ; P=0.01). DPI decreased ROS production (TZA + DPI group, 22.13±1.86×10 4 vs. TZA group, 27.74±1.60×10 4 ; P<0.001) and also reduced the proportions of necrosis in the necroptosis model (TZA + DPI group, 4.40±1.51% vs. TZA group, 8.98±0.73%; P<0.001). Phosphorylated RIP-3 and MLKL were also decreased by DPI treatment. The results indicate that ROS production increases in HK-2 cells undergoing necroptosis, and that the NADPH oxidase inhibitor, DPI may protect HK-2 cells from necroptosis via inhibition of ROS production.

Association of cultured myotubes and fasting plasma metabolite profiles with mitochondrial dysfunction in type 2 diabetes subjects.

PubMed

Abu Bakar, Mohamad Hafizi; Sarmidi, Mohamad Roji

2017-08-22

Accumulating evidence implicates mitochondrial dysfunction-induced insulin resistance in skeletal muscle as the root cause for the greatest hallmarks of type 2 diabetes (T2D). However, the identification of specific metabolite-based markers linked to mitochondrial dysfunction in T2D has not been adequately addressed. Therefore, we sought to identify the markers-based metabolomics for mitochondrial dysfunction associated with T2D. First, a cellular disease model was established using human myotubes treated with antimycin A, an oxidative phosphorylation inhibitor. Non-targeted metabolomic profiling of intracellular-defined metabolites on the cultured myotubes with mitochondrial dysfunction was then determined. Further, a targeted MS-based metabolic profiling of fasting blood plasma from normal (n = 32) and T2D (n = 37) subjects in a cross-sectional study was verified. Multinomial logical regression analyses for defining the top 5% of the metabolites within a 95% group were employed to determine the differentiating metabolites. The myotubes with mitochondrial dysfunction exhibited insulin resistance, oxidative stress and inflammation with impaired insulin signalling activities. Four metabolic pathways were found to be strongly associated with mitochondrial dysfunction in the cultured myotubes. Metabolites derived from these pathways were validated in an independent pilot investigation of the fasting blood plasma of healthy and diseased subjects. Targeted metabolic analysis of the fasting blood plasma with specific baseline adjustment revealed 245 significant features based on orthogonal partial least square discriminant analysis (PLS-DA) with a p-value < 0.05. Among these features, 20 significant metabolites comprised primarily of branched chain and aromatic amino acids, glutamine, aminobutyric acid, hydroxyisobutyric acid, pyroglutamic acid, acylcarnitine species (acetylcarnitine, propionylcarnitine, dodecenoylcarnitine, tetradecenoylcarnitine hexadecadienoylcarnitine and oleylcarnitine), free fatty acids (palmitate, arachidonate, stearate and linoleate) and sphingomyelin (d18:2/16:0) were identified as predictive markers for mitochondrial dysfunction in T2D subjects. The current study illustrates how cellular metabolites provide potential signatures associated with the biochemical changes in the dysregulated body metabolism of diseased subjects. Our finding yields additional insights into the identification of robust biomarkers for T2D associated with mitochondrial dysfunction in cultured myotubes.

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana

PubMed Central

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-01-01

Background and Aims The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Methods Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. Key Results In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. Conclusions These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III. PMID:26292995

Importance of the alternative oxidase (AOX) pathway in regulating cellular redox and ROS homeostasis to optimize photosynthesis during restriction of the cytochrome oxidase pathway in Arabidopsis thaliana.

PubMed

Vishwakarma, Abhaypratap; Tetali, Sarada Devi; Selinski, Jennifer; Scheibe, Renate; Padmasree, Kollipara

2015-09-01

The importance of the alternative oxidase (AOX) pathway, particularly AOX1A, in optimizing photosynthesis during de-etiolation, under elevated CO2, low temperature, high light or combined light and drought stress is well documented. In the present study, the role of AOX1A in optimizing photosynthesis was investigated when electron transport through the cytochrome c oxidase (COX) pathway was restricted at complex III. Leaf discs of wild-type (WT) and aox1a knock-out mutants of Arabidopsis thaliana were treated with antimycin A (AA) under growth-light conditions. To identify the impact of AOX1A deficiency in optimizing photosynthesis, respiratory O2 uptake and photosynthesis-related parameters were measured along with changes in redox couples, reactive oxygen species (ROS), lipid peroxidation and expression levels of genes related to respiration, the malate valve and the antioxidative system. In the absence of AA, aox1a knock-out mutants did not show any difference in physiological, biochemical or molecular parameters compared with WT. However, after AA treatment, aox1a plants showed a significant reduction in both respiratory O2 uptake and NaHCO3-dependent O2 evolution. Chlorophyll fluorescence and P700 studies revealed that in contrast to WT, aox1a knock-out plants were incapable of maintaining electron flow in the chloroplastic electron transport chain, and thereby inefficient heat dissipation (low non-photochemical quenching) was observed. Furthermore, aox1a mutants exhibited significant disturbances in cellular redox couples of NAD(P)H and ascorbate (Asc) and consequently accumulation of ROS and malondialdehyde (MDA) content. By contrast, WT plants showed a significant increase in transcript levels of CSD1, CAT1, sAPX, COX15 and AOX1A in contrast to aox1a mutants. These results suggest that AOX1A plays a significant role in sustaining the chloroplastic redox state and energization to optimize photosynthesis by regulating cellular redox homeostasis and ROS generation when electron transport through the COX pathway is disturbed at complex III. © The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genetic localization of diuron- and mucidin-resistant mutants relative to a group of loci of the mitochondrial DNA controlling coenzyme QH2-cytochrome c reductase in Saccharomyces cerevisiae.

PubMed

Colson, A M; Slonimski, P P

1979-01-02

Diuron-resistance, DIU (Colson et al., 1977), antimycin-resistance, ANA (Michaelis, 1976; Burger et al., 1976), funiculosin-resistance, FUN (Pratje and Michaelis, 1977; Burger et al., 1977) and mucidin-resistance, MUC (Subik et al., 1977) are each coded by a pair of genetic loci on the mit DNA of S. cerevisiae. In the present paper, these respiratiory-competent, drug-resistant loci are localized relative to respiratory-deficient BOX mutants deficient in coenzyme QH2-cytochrome c reductase (Kotylak and Slonimski, 1976, 1977) using deletion and recombination mapping. Three drug-resistant loci possessing distinct mutated allelic forms are distinguished. DIU1 is allelic or closely linked to ANA2, FUN1 and BOX1; DIU2 is allelic or closely linked to ANA1, MUC1 and BOX4/5; MUC2 is allelic to BOX6. The high recombinant frequencies observed between the three loci (13% on the average for 33 various combinations analyzed) suggest the existence of either three genes coding for three distinct polypeptides or of a single gene coding for a single polypeptide but subdivided into three easily separable segments. The resistance of the respiratory-chain observed in vitro in the drug-resistant mutants and the allelism relationships between respiratory-competent, drug-resistant loci and coQH2-cyt c reductase deficient, BOX, loci strongly suggest that each of the three drug-resistant loci codes for a structural gene-product which is essential for the normal coQH2-cyt c reductase activity and is obviously a good candidate for a gene product of the drug-resistant loci mapped in this paper. Polypeptide length modifications of cytochrome b were observed in mutants deficient in the coQH2-cyt c red and localized at the BOX1, BOX4 and BOX6 genetic loci (Claisse et al., 1977, 1978) which are precisely the loci allelic to drug resistant mutants as shown in the present work. Taken together these two sets of data provide a strong evidence in favor of the idea that there exist three non contiguous segments of the mitochondrial DNA sequence which code for a single polypeptide sequence of cytochrome b. In each segment mutations which modify the polypeptide sequence can occur leading to the loss (BOX mutants) or to a modification (drug resistant mutants) of the enzyme activity.

Studies on the synthesis and processing of the asparagine-linked carbohydrate units of glycoproteins.

PubMed

Spiro, R G; Spiro, M J

1982-12-24

It has become apparent in recent years from the work of a number of laboratories that the N-glycosylation of both membrane and secretory glycoproteins is effected by the transfer en bloc to nascent polypeptides of a glucose-containing oligosaccharide (Glc3Man9GlcNAc2) from a dolichyl pyrophosphoryl carrier; this is followed by a series of modifying reactions to yield the mature polymannose and complex asparagine-linked carbohydrate units. The enzymic steps involved in the assembly of the precursor oligosaccharide, its transfer to protein and its subsequent processing represent potential sites for the regulation of glycoprotein synthesis. Studies performed in our laboratory have dealt primarily with thyroid slices and particulate enzymes with special regard to the role of glucose in these events. Thyroglobulin, the major secretory glycoprotein of this tissue, has well defined complex and polymannose saccharide units, and indeed the most complete form of the latter (Man9GlcNAc2) has the same structure as the lipid-linked oligosaccharide without the glucose. Our studies indicate that effective N-glycosylation requires a complete glucose chain (Glc3) and that the glucose sequence is assembled from dolichol-P-glucose in a stepwise manner through the concerted action of at least two transferases in a fashion complementary to the subsequent excision of this sugar by glucosidases. Pulse-chase studies indicate that, after the transfer to protein, the removal of all three glucose residues as well as of the first mannose takes place in the endoplasmic reticulum and three additional mannoses are excised in the Golgi complex, because in the presence of an inhibitor of intracellular transport, carbonyl cyanide m-chlorophenylhydrazone (CCCP), there is a pronounced accumulation of protein-linked Man8GlcNAc2. Studies with metabolic inhibitors (CCCP, antimycin, N2) indicate that, under conditions of energy depletion, glucosylation of oligosaccharide-lipid is selectively impaired, resulting in an accumulation, as measured chemically or metabolically, of high-mannose-containing (Man9GlcNAc2 and Man8GlcNAc2) lipid-linked saccharides. Further evidence that the glucosylation reaction is very sensitive to the metabolic state is suggested by the observation that tissues not rapidly frozen after removal from the animal show a similar depletion of the glucose-containing oligosaccharide lipids. Another important aspect for the regulation of N-glycosylation of proteins is the availability of dolichyl phosphate for the formation of the lipid-linked mono- and oligosaccharides. Our studies with puromycin suggest that there is a limited supply of the lipid carrier, because in the presence of this inhibitor there is no accumulation of any of the oligosaccharide-lipid species.

Control of electron transfer in the cytochrome system of mitochondria by pH, transmembrane pH gradient and electrical potential. The cytochromes b-c segment.

PubMed

Papa, S; Lorusso, M; Izzo, G; Capuano, F

1981-02-15

1. A study is presented of the effects of pH, transmembrane pH gradient and electrical potential on oxidoreductions of b and c cytochromes in ox heart mitochondria and 'inside-out' submitochondrial particles. 2. Kinetic analysis shows that, in mitochondria at neutral pH, there is a restraint on the aerobic oxidation of cytochrome b566 with respect to cytochrome b562. Valinomycin plus K+ accelerates cytochrome b566 oxidation and retards net oxidation of cytochrome b562. At alkaline pH the rate of cytochrome b566 oxidation approaches that of cytochrome b562 and the effects of valinomycin on b cytochromes are impaired. 3. At slightly acidic pH, oxygenation of antimycin-supplemented mitochondria causes rapid reduction of cytochrome b566 and small delayed reduction of cytochrome b562. Valinomycin or a pH increase in the medium promote reduction of cytochrome b562 and decrease net reduction of cytochrome b566. 4. Addition of valinomycin to mitochondria and submitochondrial particles in the respiring steady state causes, at pH values around neutrality, preferential oxidation of cytochrome b566 with respect to cytochrome b562. The differential effect of valinomycin on oxidation of cytochromes b566 and b562 is enhanced by substitution of 1H2O of the medium with 2H2O and tends to disappear as the pH of the medium is raised to alkaline values. 5. Nigericin addition in the aerobic steady state causes, both in mitochondria and submitochondrial particles, preferential oxidation of cytochrome b562 with respect to cytochrome b566. This is accompanied by c cytochrome oxidation in mitochondria but c cytochrome reduction in submitochondrial particles. 6. In mitochondria as well as in submitochondrial particles, the aerobic transmembrane potential (delta psi) does not change by raising the pH of the external medium from neutrality to alkalinity. The transmembrane pH gradient (delta pH) on the other hand, decrease slightly. 7. The results presented provide evidence that the delta psi component of the aerobic delta microH+ (the sum of the proton chemical and electrical activities) exerts a pH-dependent constraint on forward electron flow from cytochrome b566 to cytochrome b562. This effect is explained as a consequence of anisotropic location of cytochromes b566 and b562 in the membrane and the pH-dependence of the redox function of these cytochromes. Transmembrane delta pH, on the other hand, exerts control on electron flow from cytochrome b562 to c cytochromes.

The origin of the initial heat associated with a single impulse in mammalian non-myelinated nerve fibres

PubMed Central

Howarth, J. V.; Keynes, R. D.; Ritchie, J. M.

1968-01-01

1. A study has been made of the temperature changes associated with the passage of a single impulse in rabbit desheated vagus nerves. 2. The initial changes consist of an evolution of positive heat followed by a reabsorption of most of it; i.e. there is a phase of positive and a phase of negative heat production. 3. The size of the positive heat, its time of onset, and the time of onset of the negative heat have been measured by an analogue method of analysis. In addition, these parameters, together with the size of the negative heat and the duration of both phases of initial heat, have been studied with the aid of a computer, and also by conventional heat block analysis. 4. At about 5° C the measured positive heat is 7·2 μcal/g. impulse. It starts as soon as the compound action potential reaches the thermopile and lasts for about 107 msec. 5. This positive heat decreases with increasing temperature, the ratio of heat at 4° C to that at 14° C being 1·86. 6. The measured negative heat at about 5° C is 4·9 μcal/g. impulse. It starts 102 msec after the onset of positive heat, and lasts for about 240 msec. 7. When the sodium of Locke solution is replaced by lithium the positive heat is reduced by 19%, but the negative heat is increased by 22%. 8. In potassium-free solutions the positive heat is hardly affected (increase of 5%), but the negative heat is more than doubled. As a result the nerve may become briefly colder than its initial temperature by about 2 μ° C. 9. The effect of sodium-deficient solutions on the positive heat is somewhat variable, but the negative heat is consistently diminished. 10. Replacement of the chloride of Locke solution by sulphate or nitrate has little effect on the positive heat. The negative heat is reduced in size by 26% and in duration by 22%. 11. Replacement of most of the sodium of Locke solution by barium reduces or abolishes the negative heat, and increases the measured size of the positive heat nearly threefold. 12. Veratrine (10-5 g/ml.) produces a nearly tenfold increase in the net positive heat. 13. Ouabain (1 mM) and antimycin A (1 μg/ml.) applied for 30-60 min have little effect on the initial heat production. 14. Over the temperature range 5-15° C, and for the ionic solution changes described above, there is close agreement in timing between the positive heat and the rising phase of the action potential, and between the negative heat and the falling phase. 15. Because of the inevitable temporal dispersion of the action potential over the face of the thermopile, the observed temperature changes are smaller than those which occur at a single point in the nerve close to a stimulating electrode. The corrected value of the positive heat at 5° C is 24·5 μcal/g. impulse, while that of the negative heat is 22·2 μcal/g. impulse. 16. The heats of mixing of the ions in solution that interchange during the action potential are much too small to account for the observed initial heats, but an exchange of ions associated with fixed charges might make a significant contribution to the heats. 17. The condenser theory, according to which the positive heat represents the dissipation of electrical energy stored in the membrane capacity, while the negative heat results from the recharging of the capacity, appears unable to account for more than half of the observed temperature changes. 18. It seems probable that the greater part of the initial heat results from changes in the entropy of the nerve membrane when it is depolarized and repolarized. PMID:5636997