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Sample records for antimycin

  1. The regulation and biosynthesis of antimycins

    Directory of Open Access Journals (Sweden)

    Ryan F. Seipke

    2013-11-01

    Full Text Available Antimycins (>40 members were discovered nearly 65 years ago but the discovery of the gene cluster encoding antimycin biosynthesis in 2011 has facilitated rapid progress in understanding the unusual biosynthetic pathway. Antimycin A is widely used as a piscicide in the catfish farming industry and also has potent killing activity against insects, nematodes and fungi. The mode of action of antimycins is to inhibit cytochrome c reductase in the electron transport chain and halt respiration. However, more recently, antimycin A has attracted attention as a potent and selective inhibitor of the mitochondrial anti-apoptotic proteins Bcl-2 and Bcl-xL. Remarkably, this inhibition is independent of the main mode of action of antimycins such that an artificial derivative named 2-methoxyantimycin A inhibits Bcl-xL but does not inhibit respiration. The Bcl-2/Bcl-xL family of proteins are over-produced in cancer cells that are resistant to apoptosis-inducing chemotherapy agents, so antimycins have great potential as anticancer drugs used in combination with existing chemotherapeutics. Here we review what is known about antimycins, the regulation of the ant gene cluster and the unusual biosynthetic pathway.

  2. Laboratory studies on antimycin A as a fish toxicant

    Science.gov (United States)

    Berger, Bernard L.; Lennon, Robert E.; Hogan, James W.

    1969-01-01

    Liquid and sand formulations of antimycin A were tested in laboratory waters of various temperature, hardness, pH, and turbidity against 31 species of fresh-water fish of various sizes and life stages. Each formulation of toxicant was lethal under all water conditions to fish eggs, fry, fingerlings, and adult fish. Trouts are the most sensitive and catfishes the least sensitive. Of the 31 species, 24 succumb to 5 p.p.b. or less of the toxicant; only certain catfishes survive 25 p.p.b, The order of toxicity to various species of fish suggests that antimycin has possibilities for selective or partial control of certain unwanted fish. Although toxic to fish under ice, antimycin is more active in warm water than in cold. It is slightly more active in soft water than in hard; it is more active and persists far longer in water at pH 5 to 8 than at pH 9 or 10. It is active on fish in either clear and turbid waters, and it can be detoxified by potassium permanganate, The results contributed to registration of antimycin A in Fintrol-5 formulation as a fish toxicant.

  3. Regulation of antimycin biosynthesis by the orphan ECF RNA polymerase sigma factor σAntA

    Directory of Open Access Journals (Sweden)

    Ryan F. Seipke

    2014-02-01

    Full Text Available Antimycins are an extended family of depsipeptides that are made by filamentous actinomycete bacteria and were first isolated more than 60 years ago. Recently, antimycins have attracted renewed interest because of their activities against the anti-apoptotic machineries inside human cells which could make them promising anti-cancer compounds. The biosynthetic pathway for antimycins was recently characterised but very little is known about the organisation and regulation of the antimycin (ant gene cluster. Here we report that the ant gene cluster in Streptomyces albus is organized into four transcriptional units; the antBA, antCDE, antGF and antHIJKLMNO operons. Unusually for secondary metabolite clusters, the antG and antH promoters are regulated by an extracytoplasmic function (ECF RNA polymerase sigma factor named σAntA which represents a new sub-family of ECF σ factors that is only found in antimycin producing strains. We show that σAntA controls production of the unusual precursor 3-aminosalicylate which is absolutely required for the production of antimycins. σAntA is highly conserved in antimycin producing strains and the −10 and −35 elements at the σAntA regulated antG and antH promoters are also highly conserved suggesting a common mechanism of regulation. We also demonstrate that altering the C-terminal Ala-Ala residues found in all σAntA proteins to Asp-Asp increases expression of the antFG and antGHIJKLMNO operons and we speculate that this Ala-Ala motif may be a signal for the protease ClpXP.

  4. Two Antimycin A Analogues from Marine-Derived Actinomycete Streptomyces lusitanus

    Directory of Open Access Journals (Sweden)

    Peiyuan Qian

    2012-03-01

    Full Text Available Two new antimycin A analogues, antimycin B1 and B2 (1–2, were isolated from a spent broth of a marine-derived bacterium, Streptomyces lusitanus. The structures of 1 and 2 were established on the basis of spectroscopic analyses and chemical methods. The isolated compounds were tested for their anti-bacterial potency. Compound 1 was found to be inactive against the bacteria Bacillus subtilis, Staphyloccocus aureus, and Loktanella hongkongensis. Compound 2 showed antibacterial activities against S. aureus and L. hongkongensis with MIC values of 32.0 and 8.0 μg/mL, respectively.

  5. Effect of rainbow trout size on response to rotenone and antimycin.

    Science.gov (United States)

    Brown, Peter J.; Johnson, Heather; Zale, Alexander V.

    2011-01-01

    The piscicides rotenone and antimycin are commonly used to eradicate unwanted fish populations. However, the relationships (if present) between their toxicities and fish sizes are unknown and could be especially important when bioassay fish are used to detect piscicide presence and effectiveness. Size-mediated toxicity could lead to either excessive or inadequate piscicide applications if bioassay fish are larger or smaller than the fish being eradicated. The relationships between time to death and weight of rainbow trout Oncorhynchus mykiss (0.7–574.0 g) at an antimycin concentration of 7.5 μg/L and a rotenone concentration of 12.5 μg/L were determined. Antimycin took significantly longer than rotenone to kill rainbow trout at concentrations typically used in eradication projects. Significant positive relationships existed between fish size and time to death for rotenone and antimycin exposures and were probably caused by size-mediated differences in metabolic rate; however, these relationships accounted for less than 21% of the variation in time to death. Smaller fish appeared to be affected by the chemicals more quickly, but their deaths did not consistently occur before the deaths of larger fish.

  6. Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

    Science.gov (United States)

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  7. A Preclinical Evaluation of Antimycin A as a Potential Antilung Cancer Stem Cell Agent

    Directory of Open Access Journals (Sweden)

    Chi-Tai Yeh

    2013-01-01

    Full Text Available Drug resistance and tumor recurrence are major obstacles in treating lung cancer patients. Accumulating evidence considers lung cancer stem cells (CSCs as the major contributor to these clinical challenges. Agents that can target lung CSCs could potentially provide a more effective treatment than traditional chemotherapy. Here, we utilized the side-population (SP method to isolate lung CSCs from A549 and PC-9 cell lines. Subsequently, a high throughput platform, connectivity maps (CMAPs, was used to identify potential anti-CSC agents. An antibiotic, antimycin A (AMA, was identified as a top candidate. SP A549 cells exhibited an elevated stemness profile, including Nanog, β-catenin, Sox2, and CD133, and increased self-renewal ability. AMA treatment was found to suppress β-catenin signaling components and tumor sphere formation. Furthermore, AMA treatment decreased the proliferation of gefitinib-resistant PC-9/GR cells and percentage of SP population. AMA demonstrated synergistic suppression of PC-9/GR cell viability when combined with gefitinib. Finally, AMA treatment suppressed tumorigenesis in mice inoculated with A549 SP cells. Collectively, we have identified AMA using CMAP as a novel antilung CSC agent, which acts to downregulate β-catenin signaling. The combination of AMA and targeted therapeutic agents could be considered for overcoming drug resistance and relapse in lung cancer patients.

  8. Binding of the Respiratory Chain Inhibitor Antimycin to theMitochondrial bc1 Complex: A New Crystal Structure Reveals an AlteredIntramolecular Hydrogen-Bonding Pattern

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Cobessi, David; Tung, Eric Y.; Berry, Edward A.

    2005-05-10

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex.Structure-activity-relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28Angstrom resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cyt b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alpha-A helix.

  9. Binding of the respiratory chain inhibitor antimycin to the mitochondrial bc1 complex: a new crystal structure reveals an altered intramolecular hydrogen-bonding pattern.

    Science.gov (United States)

    Huang, Li-Shar; Cobessi, David; Tung, Eric Y; Berry, Edward A

    2005-08-19

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Two previous X-ray structures of antimycin bound to vertebrate bc1 complex gave conflicting results. A new structure reported here of the bovine mitochondrial bc1 complex at 2.28 A resolution with antimycin bound, allows us for the first time to reliably describe the binding of antimycin and shows that the intramolecular hydrogen bond described in solution and in the small-molecule structure is replaced by one involving the NH rather than carbonyl O of the amide linkage, with rotation of the amide group relative to the aromatic ring. The phenolic OH and formylamino N form H-bonds with conserved Asp228 of cytochrome b, and the formylamino O H-bonds via a water molecule to Lys227. A strong density, the right size and shape for a diatomic molecule is found between the other side of the dilactone ring and the alphaA helix.

  10. BINDING OF THE RESPIRATORY CHAIN INHIBITOR ANTIMYCIN TO THE MITOCHONDRIAL bc1 COMPLEX: A NEW CRYSTAL STRUCTURE REVEALS AN ALTERED INTRAMOLECULAR HYDROGEN-BONDING PATTERN.

    OpenAIRE

    Huang, Li-shar; Cobessi, David; Tung, Eric Y.; Berry, Edward A.

    2005-01-01

    Antimycin A (antimycin), one of the first known and most potent inhibitors of the mitochondrial respiratory chain, binds to the quinone reduction site of the cytochrome bc1 complex. Structure-activity-relationship studies have shown that the N-formylamino-salicyl-amide group is responsible for most of the binding specificity, and suggested that a low pKa for the phenolic OH group and an intramolecular H-bond between that OH and the carbonyl O of the salicylamide linkage are important. Tw...

  11. Open reading frame ssr2016 is required for antimycin A-sensitive photosystem I-driven cyclic electron flow in the cyanobacterium Synechocystis sp. PCC 6803

    NARCIS (Netherlands)

    Yeremenko, Nataliya; Jeanjean, Robert; Prommeenate, Peerada; Krasikov, Vladimir; Nixon, Peter J.; Vermaas, Wim F. J.; Havaux, Michel; Matthijs, Hans C. P.

    2005-01-01

    Open reading frame ssr2016 encodes a protein with substantial sequence similarities to PGR5 identified as a component of the antimycin A-sensitive ferredoxin:plastoquinone reductase (FQR) in PSI cyclic photophosphorylation in Arabidopsis thaliana. We studied cyclic electron flow in Synechocystis sp.

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

    Science.gov (United States)

    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.

  13. Anti-apoptotic effects of pan-caspase inhibitor (Z-VAD), SOD or catalase on antimycin A-induced HeLa cell death.

    Science.gov (United States)

    Han, Yong Hwan; Kim, Suhn Hee; Kim, Sung Zoo; Park, Woo Hyun

    2009-01-01

    Antimycin A (AMA) is an inhibitor of the electron transport chain in mitochondria. In this study, we investigated the anti-apoptotic effects of pan-caspase inhibitor (Z-VAD), superoxide dismutase (SOD) or catalase on AMA-induced HeLa cell death in relation to the cell cycle. Treatment with Z-VAD, SOD or catalase rescued some HeLa cells from AMA-induced apoptosis, but did not prevent the growth inhibition of HeLa cells by AMA. DNA flow cytometric analysis indicated that treatment with AMA significantly induced an S-phase arrest of the cell cycle at 72 h. Interestingly, Z-VAD, SOD and catalase intensified S-phase arrest in AMA-treated cells. In conclusion, treatment with Z-VAD, SOD or catalase decreased apoptotic levels in AMA-treated cells, which was associated with the enhancement of the S-phase arrest of the cell cycle in these cells.

  14. 31P NMR saturation-transfer measurements in Saccharomyces cerevisiae: characterization of phosphate exchange reactions by iodoacetate and antimycin A inhibition

    International Nuclear Information System (INIS)

    Campbell-Burk, S.L.; Jones, K.A.; Shulman, R.G.

    1987-01-01

    31 P nuclear magnetic resonance (NMR) saturation-transfer (ST) techniques have been used to measure steady-state flows through phosphate-adenosine 5'-triphosphate (ATP) exchange reactions in glucose-grown derepressed yeast. The results have revealed that the reactions catalyzed by glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK) and by the mitochondrial ATPase contribute to the observed ST. Contributions from these reactions were evaluated by performing ST studies under various metabolic conditions in the presence and absence of either iodoacetate, a specific inhibitor of GAPDH, or the respiratory chain inhibitor antimycin A. Intracellular phosphate (P/sub i/) longitudinal relaxation times were determined by performing inversion recovery experiments during steady-state ATP/sub λ/ saturation and were used in combination with ST data to determine P/sub i/ consumption rates. 13 C NMR and O 2 electrode measurements were also conducted to monitor changes in rates of glucose consumption and O 2 consumption, respectively, under the various metabolic conditions examined. The results suggest that GAPDH/PGK-catalyzed P/sub i/-ATP exchange is responsible for antimycin-resistant saturation transfer observed in anaerobic and aerobic glucose-fed yeast. Kinetics through GAPDH/PGK were found to depend on metabolic conditions. The coupled system appears to operate in a unidirectional manner during anaerobic glucose metabolism and bidirectionally when the cells are respiring on exogenously supplied ethanol. Additionally, mitochondrial ATPase activity appears to be responsible for the transfer observed in iodoacetate-treated aerobic cells supplied with either glucose or ethanol, with synthesis of ATP occurring unidirectionally

  15. Cellular Quantitative Structure–Activity Relationship (Cell-QSAR): Conceptual Dissection of Receptor Binding and Intracellular Disposition in Antifilarial Activities of Selwood Antimycins

    Science.gov (United States)

    2012-01-01

    We present the cellular quantitative structure–activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug’s lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition. PMID:22468611

  16. Cellular quantitative structure-activity relationship (Cell-QSAR): conceptual dissection of receptor binding and intracellular disposition in antifilarial activities of Selwood antimycins.

    Science.gov (United States)

    Natesan, Senthil; Wang, Tiansheng; Lukacova, Viera; Bartus, Vladimir; Khandelwal, Akash; Subramaniam, Rajesh; Balaz, Stefan

    2012-04-26

    We present the cellular quantitative structure-activity relationship (cell-QSAR) concept that adapts ligand-based and receptor-based 3D-QSAR methods for use with cell-level activities. The unknown intracellular drug disposition is accounted for by the disposition function (DF), a model-based, nonlinear function of a drug's lipophilicity, acidity, and other properties. We conceptually combined the DF with our multispecies, multimode version of the frequently used ligand-based comparative molecular field analysis (CoMFA) method, forming a single correlation function for fitting the cell-level activities. The resulting cell-QSAR model was applied to the Selwood data on filaricidal activities of antimycin analogues. Their molecules are flexible, ionize under physiologic conditions, form different intramolecular H-bonds for neutral and ionized species, and cross several membranes to reach unknown receptors. The calibrated cell-QSAR model is significantly more predictive than other models lacking the disposition part and provides valuable structure optimization clues by factorizing the cell-level activity of each compound into the contributions of the receptor binding and disposition.

  17. Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b

    DEFF Research Database (Denmark)

    Grimmelikhuijzen, C J; Marres, C A; Slater, Conor

    1975-01-01

    1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reo...

  18. Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels.

    NARCIS (Netherlands)

    Graaf, A.O. de; Meijerink, J.P.P.; Heuvel, L.P.W.J. van den; Abreu, R.A. de; Witte, T.J.M. de; Jansen, J.H.; Smeitink, J.A.M.

    2002-01-01

    Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production

  19. Acetate oxidation by bloodstream forms of Trypanosoma cruzi.

    Science.gov (United States)

    Docampo, R; Cruz, F S; Leon, W; Schmunis, G A

    1979-05-01

    Bloodstream forms of Trypanosoma cruzi had a substantial increase in respiration in the presence of acetate. Oxidation of acetate took place via the tricarboxylic acid cycle and involved an antimycin A-sensitive respiratory pathway. Oxygen uptake in the presence of acetate was a sensitive to antimycin A inhibition as was CO2 production. There was a 6--7% residual O2 uptake which was not inhibited by high antimycin concentrations. Human anti-T. cruzi sera had no effect on oxygen uptake.

  20. Respiration in the yeast and mycelial phases of Histoplasma capsulatum.

    Science.gov (United States)

    Maresca, B; Lambowitz, A M; Kobayashi, G S; Medoff, G

    1979-05-01

    Respiration in the yeast and mycelial phases of Histoplasma capsulatum proceeds via a cytochrome system and an alternate oxidase, both present constitutively. The mycelial cytochrome system is distinguished by an additional partial shunt around the antimycin-sensitive site.

  1. Differential reduction of reactive oxygen species by human ...

    Indian Academy of Sciences (India)

    In this study, for the first time, we investigated the differences in the reactive oxygen species (ROS) reductionabilities of tissue-specific MSCs to mitigate cellular damage in oxidative stress. Hepatic Stellate cells (LX-2) and cardiomyocyteswere treated with Antimycin A (AMA) to induce oxidative stress and tissue specific ...

  2. Differential reduction of reactive oxygen species by human tissue ...

    Indian Academy of Sciences (India)

    Swati Paliwal

    2017-06-24

    Jun 24, 2017 ... Keywords. Oxidative stress; reactive oxygen species; tissue-specific mesenchymal stem cells. Abbreviations: AD, adipose; AMA, Antimycin A; AU, arbitrary units; BM, bone marrow; DP, dental pulp; MFI, mean fluorescence intensity; MSC, mesenchymal stem cell; ROS, reactive oxygen species. 1. Introduction.

  3. Metabolomic Profiling and Genomic Study of a Marine Sponge-Associated Streptomyces sp.

    Directory of Open Access Journals (Sweden)

    Christina Viegelmann

    2014-06-01

    Full Text Available Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8 isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1, 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2, and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3 that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.

  4. Inhibition of oxidative phosphorylation for enhancing citric acid production by Aspergillus niger.

    Science.gov (United States)

    Wang, Lu; Zhang, Jianhua; Cao, Zhanglei; Wang, Yajun; Gao, Qiang; Zhang, Jian; Wang, Depei

    2015-01-16

    The spore germination rate and growth characteristics were compared between the citric acid high-yield strain Aspergillus niger CGMCC 5751 and A. niger ATCC 1015 in media containing antimycin A or DNP. We inferred that differences in citric acid yield might be due to differences in energy metabolism between these strains. To explore the impact of energy metabolism on citric acid production, the changes in intracellular ATP, NADH and NADH/NAD+ were measured at various fermentation stages. In addition, the effects of antimycin A or DNP on energy metabolism and citric acid production was investigated by CGMCC 5751. By comparing the spore germination rate and the extent of growth on PDA plates containing antimycin A or DNP, CGMCC 5751 was shown to be more sensitive to antimycin A than ATCC 1015. The substrate-level phosphorylation of CGMCC 5751 was greater than that of ATCC 1015 on PDA plates with DNP. DNP at tested concentrations had no apparent effect on the growth of CGMCC 5751. There were no apparent effects on the mycelial morphology, the growth of mycelial pellets or the dry cell mass when 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP was added to medium at the 24-h time point. The concentrations of intracellular ATP, NADH and NADH/NAD+ of CGMCC 5751 were notably lower than those of ATCC 1015 at several fermentation stages. Moreover, at 96 h of fermentation, the citric acid production of CGMCC 5751 reached up to 151.67 g L(-1) and 135.78 g L(-1) by adding 0.2 mg L(-1) antimycin A or 0.1 mg L(-1) DNP, respectively, at the 24-h time point of fermentation. Thus, the citric acid production of CGMCC 5751 was increased by 19.89% and 7.32%, respectively. The concentrations of intracellular ATP, NADH and NADH/NAD+ of the citric acid high-yield strain CGMCC 5751 were notably lower than those of ATCC 1015. The excessive ATP has a strong inhibitory effect on citric acid accumulation by A. niger. Increasing NADH oxidation and appropriately reducing the concentration of

  5. Characterization of the respiratory chain of Helicobacter pylori

    DEFF Research Database (Denmark)

    Chen, M; Andersen, L P; Zhai, L

    1999-01-01

    The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH......-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c...... reductase was inhibited by antimycin, implying the presence of a classical pathway from complex II to complex III in this bacterium. The presence of NADH-fumarate reductase (FRD) was demonstrated in H. pylori and fumarate could reduce H2O2 production from NADH, indicating fumarate to be an endogenous...

  6. Oxidative DNA damage causes mitochondrial genomic instability in Saccharomyces cerevisiae.

    Science.gov (United States)

    Doudican, Nicole A; Song, Binwei; Shadel, Gerald S; Doetsch, Paul W

    2005-06-01

    Mitochondria contain their own genome, the integrity of which is required for normal cellular energy metabolism. Reactive oxygen species (ROS) produced by normal mitochondrial respiration can damage cellular macromolecules, including mitochondrial DNA (mtDNA), and have been implicated in degenerative diseases, cancer, and aging. We developed strategies to elevate mitochondrial oxidative stress by exposure to antimycin and H(2)O(2) or utilizing mutants lacking mitochondrial superoxide dismutase (sod2Delta). Experiments were conducted with strains compromised in mitochondrial base excision repair (ntg1Delta) and oxidative damage resistance (pif1Delta) in order to delineate the relationship between these pathways. We observed enhanced ROS production, resulting in a direct increase in oxidative mtDNA damage and mutagenesis. Repair-deficient mutants exposed to oxidative stress conditions exhibited profound genomic instability. Elimination of Ntg1p and Pif1p resulted in a synergistic corruption of respiratory competency upon exposure to antimycin and H(2)O(2). Mitochondrial genomic integrity was substantially compromised in ntg1Delta pif1Delta sod2Delta strains, since these cells exhibit a total loss of mtDNA. A stable respiration-defective strain, possessing a normal complement of mtDNA damage resistance pathways, exhibited a complete loss of mtDNA upon exposure to antimycin and H(2)O(2). This loss was preventable by Sod2p overexpression. These results provide direct evidence that oxidative mtDNA damage can be a major contributor to mitochondrial genomic instability and demonstrate cooperation of Ntg1p and Pif1p to resist the introduction of lesions into the mitochondrial genome.

  7. Characterization of the respiratory chain of Helicobacter pylori

    DEFF Research Database (Denmark)

    Chen, M; Andersen, L P; Zhai, L

    1999-01-01

    The respiratory chain of Helicobacter pylori has been investigated. The total insensitivity of activities of NADH dehydrogenase to rotenone and of NADH-cytochrome c reductase to antimycin is indicative of the absence of the classical complex I of the electron transfer chain in this bacterium. NADPH......-dependent respiration was significantly stronger than NADH-dependent respiration, indicating that this is a major respiratory electron donor in H. pylori. Fumarate and malonate exhibited a concentration-dependent inhibitory effect on the activity of succinate dehydrogenase. The activity of succinate-cytochrome c...

  8. Dependence of anaphylactic histamine release from rat mast cells on cellular energy metabolism

    DEFF Research Database (Denmark)

    Johansen, Torben

    1981-01-01

    The relation between anaphylactic histamine release and the adenosine triphosphate (ATP) content of the mast cells was studied. The cells were incubated with glycolytic (2-deoxyglucose) and respiratory inhibitors (antimycin A and oligomycin) in order to decrease the ATP content of the cells prior...... pretreated with 2-deoxyglucose. The release of histamine from these cells was reduced when respiratory inhibitors were added to the cell suspension 5 to 20 sec after exposure of the cells to antigen. This may indicate that the secretory process requires energy, and it seems necessary that energy should...... be produced as the release of histamine takes place....

  9. The critical glucose concentration for respiration-independent proliferation of fission yeast, Schizosaccharomyces pombe.

    Science.gov (United States)

    Takeda, Kojiro; Starzynski, Caroline; Mori, Ayaka; Yanagida, Mitsuhiro

    2015-05-01

    Glucose is the fundamental energy source for life; thus cells need to respond appropriately to changes in available glucose concentration. We investigated the relationship between media glucose concentration and respiration-dependency of proliferation, using Schizosaccharomyces pombe. In media containing ≥ 0.2% glucose, neither antimycin A, an inhibitor of Complex III, nor gene deletions of essential electron transfer chain components, impaired cell division, while these factors completely inhibited cell division in media containing ≤ 0.1% glucose. These results indicate the existence of a threshold in glucose concentration that governs respiration-dependency of S. pombe proliferation. Copyright © 2015. Published by Elsevier B.V.

  10. A High-Throughput Flow Cytometry Screen Identifies Molecules That Inhibit Hantavirus Cell Entry.

    Science.gov (United States)

    Buranda, Tione; Gineste, Catherine; Wu, Yang; Bondu, Virginie; Perez, Dominique; Lake, Kaylin R; Edwards, Bruce S; Sklar, Larry A

    2018-04-01

    Hantaviruses cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS), which infects more than 200,000 people worldwide. Sin Nombre virus (SNV) and Andes virus (ANDV) cause the most severe form of HCPS, with case fatality ratios of 30%-40%. There are no specific therapies or vaccines for SNV. Using high-throughput flow cytometry, we screened the Prestwick Chemical Library for small-molecule inhibitors of the binding interaction between UV-inactivated and fluorescently labeled SNV R18 particles, and decay-accelerating factor (DAF) expressed on Tanoue B cells. Eight confirmed hit compounds from the primary screen were investigated further in secondary screens that included infection inhibition, cytotoxicity, and probe interference. Antimycin emerged as a bona fide hit compound that inhibited cellular infection of the major HCPS (SNV)- and HCPS (Hantaan)-causing viruses. Confirming our assay's ability to detect active compounds, orthogonal testing of the hit compound showed that antimycin binds directly to the virus particle and blocks recapitulation of physiologic integrin activation caused by SNV binding to the integrin PSI domain.

  11. Alternative respiration and fumaric acid production of Rhizopus oryzae.

    Science.gov (United States)

    Gu, Shuai; Xu, Qing; Huang, He; Li, Shuang

    2014-06-01

    Under the conditions of fumaric acid fermentation, Rhizopus oryzae ME-F14 possessed at least two respiratory systems. The respiration of mycelia was partially inhibited by the cytochrome respiration inhibitor antimycin A or the alternative respiration inhibitor salicylhydroxamic acid and was completely inhibited in the presence of both antimycin A and salicylhydroxamic acid. During fumaric acid fermentation process, the activity of alternative respiration had a great correlation with fumaric acid productivity; both of them reached peak at the same time. The alternative oxidase gene, which encoded the mitochondrial alternative oxidase responsible for alternative respiration in R. oryzae ME-F14, was cloned and characterized in Escherichia coli. The activity of alternative respiration, the alternative oxidase gene transcription level, as well as the fumaric acid titer were measured under different carbon sources and different carbon-nitrogen ratios. The activity of alternative respiration was found to be comparable to the transcription level of the alternative oxidase gene and the fumaric acid titer. These results indicated that the activity of the alternative oxidase was regulated at the transcription stage under the conditions tested for R. oryzae ME-F14.

  12. Cyanide-insensitive respiration in Acanthamoeba castellanii. Changes in sensitivity of whole cell respiration during exponential growth

    Energy Technology Data Exchange (ETDEWEB)

    Edwards, S.W.; Lloyd, D.

    1977-01-01

    Respiration of Acanthamoeba castellanii shows varying sensitivity to cyanide during exponential growth in a medium containing proteose peptone, glucose and yeast extract. After 20 h growth, respiration was stimulated up to 40% by I mM-cyanide; sensitivity to cyanide then gradually increased until 90% inhibition of respiration was attained in late exponential phase cultures. Salicyl hydroxamic acid alone never stimulated or inhibited respiration by more than 20% but, when added together with cyanide, inhibition was always 70 to 100% from 3 h onward. Sensitivity to antimycin A was similar, but not identical to that shown to cyanide; when antimycin A was added together with salicyl hydroxamic acid, the inhibition was greater. Increased sensitivities to arsenite and malonate were also observed in late-exponential phase cultures. These changes in sensitivities were not associated with alterations in the growth medium since similar changes in sensitivity to inhibitors were observed during growth in conditioned medium. A rotenone-sensitive site is associated with cyanide-stimulated respiration and the results suggest that A. castellanii possesses a branched electron transport system.

  13. Substance P receptor desensitization requires receptor activation but not phospholipase C

    International Nuclear Information System (INIS)

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

    1988-01-01

    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 [ 3 H]inositol 1,4,5-trisphosphate (IP 3 ), and persistent desensitization of the substance P response. In cells treated with antimycin in medium containing glucose, ATP was decreased to ∼20% of control values, IP 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 ∼5% of control values, and both IP 3 formation and desensitization were blocked. A series of substance P-related peptides increased the formation of [ 3 H]IP 3 and induced desensitization of the substance P response with a similar rank order of potencies. The substance P antagonist, [D-Pro 2 , D-Try 7,9 ]-substance P, inhibited substance P-induced IP 3 formation and desensitization but did not induce desensitization. These results suggest that the desensitization of substance P-induced IP 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

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

    Directory of Open Access Journals (Sweden)

    Daniel P Lindsay

    2015-03-01

    Full Text Available Excessive mitochondrial reactive oxygen species (ROS emission is a critical component in the etiolo-gy 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 complex 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 rote-none, 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. These results indicate that ROS production by complex I and by III is differently affected by buffer pH and Ca2+ loading with mPTP opening. The study sug-gests 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.

  15. Ethacrynic acid inhibition of histamine release from rat mast cells: effect on cellular ATP levels and thiol groups

    DEFF Research Database (Denmark)

    Johansen, Torben

    1983-01-01

    The experiments concerned the effect of ethacrynic acid (0.5 mM) on the adenosine triphosphate (ATP) content of rat mast cells and the effect on histamine release induced by the ionophore A23187 (10 microM). Ethacrynic acid decreased the ATP level of the cells in presence of antimycin A and glucose....... Furthermore, the inhibition of histamine release occurred mainly through interaction with thiol groups, although inhibition of cellular energy production may have been an additional mechanism....... as well as in presence of 2-deoxyglucose. A23187-induced histamine release was inhibited by ethacrynic acid, and this inhibition was completely reversed by dithiothreitol. These observations may indicate that ethacrynic acid inhibits glycolytic and respiratory energy production in rat mast cells...

  16. Energy metabolism in rat mast cells in relation to histamine secretion

    DEFF Research Database (Denmark)

    Johansen, T

    1987-01-01

    of histamine was induced by the antigen-antibody reaction, the polymeric amine compound 48/80, and the divalent ionophore A23187. 2. In presence of low concentrations of metabolic inhibitors (oligomycin or antimycin A) a linear relation between the secretion of histamine induced by all three liberators...... and the cellular ATP content at the time of cell activation was demonstrated. This may indicate a direct link between ATP and the secretory mechanism. 3. The possibility of an increased utilization of ATP during histamine secretion was explored in mast cells exposed to metabolic inhibitors. Incubation of mast...... cells with 2-deoxyglucose (2-DG) decreased the ATP content of the cells, and a long-lasting and stable level of mast cell ATP was observed. This is explained by a small decrease in the rate of ATP-synthesis by 2-DG. In 2-DG-treated cells secretion of histamine in response to compound 48...

  17. Utilization of adenosine triphosphate in rat mast cells during histamine release induced by the ionophore A23187

    DEFF Research Database (Denmark)

    Johansen, Torben

    1979-01-01

    The role of endogenous adenosine triphosphate (ATP) in histamine release from rat mast cells induced by the ionophore A23187 in vitro has been studied. 2 The amount of histamine released by calcium from rat mast cells primed with the ionophore A23187 was dependent on the ATP content of the mast...... cells. 3 In aerobic experiments a drastic reduction in mast cell ATP content was found during the time when histamine release induced by A23187 takes place. 4 Anaerobic experiments were performed with metabolic inhibitors (antimycin A, oligomycin, and carbonyl cyanide p...... The observations are consistent with the view that energy requiring processes are involved in ionophore-induced histamine release from rat mast cells although part of the ATP reduction in the aerobic experiments may be due to an uncoupling effect of calcium on the oxidative phosphorylation....

  18. Evaluation of whole antioxidant defenses of human mononuclear cells by a new in vitro biological test: lack of correlation between erythrocyte and mononuclear cell resistance to oxidative stress.

    Science.gov (United States)

    Caspar-Bauguil, Sylvie; Maestre, Nicolas; Segafredo, Céline; Galinier, Anne; Garcia, Jésus; Prost, Michel; Périquet, Brigitte; Pénicaud, Luc; Salvayre, Robert; Casteilla, Louis

    2009-04-01

    This work aims to evaluate the resistance of mononuclear cells to oxidative stress using a "KRL" test, formerly utilized to evaluate the resistance of erythrocyte to free radicals. The "KRL" test evaluates the resistance to lysis of cells treated by free radicals generated under standardized conditions. We defined new analytical parameters (level of radical production, time course, number of cells) to obtain an accurate assay determining the resistance to oxidative stress of mononuclear cells, in comparison to that of erythrocytes. This test allows the evaluation of change in the redox state of mononuclear cells (improved by an antioxidant mix or deteriorated by antimycin A-induced mitochondrial radical overproduction). Interestingly, our data show that the sensitivity of mononuclear cells to oxidative stress is not correlated with the susceptibility of erythrocytes to oxidative stress. The quantification of the susceptibility of mononuclear cells to oxidative stress gives additional information (in addition to erythrocyte resistance) and could be helpful for patients with chronic inflammation.

  19. Expression of Arabidopsis gdh2 gene depends on activity of alternative electron transfer pathway in mitochondria

    Directory of Open Access Journals (Sweden)

    Konstantinov Yu. M.

    2012-09-01

    Full Text Available Aim. We studied the expression level of gdh2 gene, encoding subunit of mitochondrial glutamate dehydrogenase, in Arabidopsis suspension culture cells with genetically modified level of alternative oxidase AOX1a. Methods. Polymerase chain reaction, Northern-blotting. Results. The treatment with main electron transfer pathway inhibitor antimycin A led to an increase in gdh2 transcript level in the wild-type cells and the cells with decreased level of alternative oxidase, but not in the cells with elevated level of alternative oxidase. Conclusions. It is known that an increase in alternative oxidase level in the plant cell results in more oxidized state of ubiquinone pool in respiratory chain. Therefore, the obtained results support the earlier proposed model according to which the expression level of gdh2 gene depends on the redox state of ubiquinone pool.

  20. Identification of a New Isoindole-2-yl Scaffold as a Qo and Qi Dual Inhibitor of Cytochrome bc1Complex: Virtual Screening, Synthesis, and Biochemical Assay.

    Science.gov (United States)

    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.

  1. The effect of metabolic inhibitors on the development of respiration in anaerobically grown yeast

    Science.gov (United States)

    Bartley, W.; Tustanoff, E. R.

    1966-01-01

    1. Iodoacetate and fluoride did not prevent the development of respiration in aerobically grown yeast. 2. The effect of dinitrophenol suggested that phosphorylation developed simultaneously with respiration in anaerobically grown yeast, but the effect of oligomycin suggested that the phosphorylation and oxidation were not tightly coupled. 3. Inhibitors of electron transport showed that both the respiratory peak and the subsequent respiration were cyanide-sensitive, but the peak respiration was insensitive to antimycin. 4. Of the inhibitors of protein or RNA synthesis tested, only p-fluorophenylalanine inhibited the development of respiration. The results are not consistent with a new synthesis of mitochondria. 5. 2-Phenylethanol inhibited the development of respiration in anaerobically grown yeast and also yeast growth. Other inhibitors of DNA synthesis had no effect on the development of respiration. 6. The relevance of the results to mitochondrial morphogenesis is discussed. PMID:4290405

  2. The influence of ascorbic acid on the oxygen consumption and the heat production by the cells of wheat seedling roots with their mitochondrial electron transport chain inhibited at complexes I and III

    International Nuclear Information System (INIS)

    Gordon, L.K.; Rakhmatullina, D.F.; Ogorodnikova, T.I.; Alyabyev, A.J.; Minibayeva, F.V.; Loseva, N.L.; Mityashina, S.Y.

    2007-01-01

    The influence of exogenous ascorbic acid (AsA) on oxidative phosphorylation was studied using wheat seedling roots. Treatment of them with AsA stimulated the rates of oxygen consumption and the heat production and caused a decrease of the respiratory coefficient. The increase in respiration was prevented by inhibitors of ascorbate oxidase, diethyldithiocarbamate (DEDTC), and of cytochrome oxidase, cyanide (KCN). Exogenous AsA sharply stimulated the rate of oxygen consumption of roots when complexes I and III of the mitochondrial electron transport chain were inhibited by rotenone and antimycin A, respectively, while the rates of heat production did not change significantly. It is concluded that AsA is a potent energy substrate, which can be used in conditions of failing I and III complexes in the mitochondrial electron transport chain

  3. Increased Na+/K(+)-pump activity and adenosine triphosphate utilization after compound 48/80-induced histamine secretion from rat mast cells

    DEFF Research Database (Denmark)

    Johansen, Torben; Praetorius, Birger Hans

    1994-01-01

    The Na+/K(+)-pump activity and the utilization of adenosine triphosphate (ATP) were studied in rat peritoneal mast cells after histamine secretion induced by compound 48/80. We measured the ouabain-sensitive K(+)-uptake by a radioactive technique (86Rb+). The ATP content and the glycolytic ATP......-production were measured by the bioluminescence technique (firefly lantern) and by measurement of the lactate production under anaerobic conditions (antimycin A, oligomycin), respectively. There was an increased requirement for ATP after the secretory response associated with an increased activity of the Na+/K(+)-pump....... The anaerobic, but not the aerobic, pathway for ATP-synthesis was able to respond to the increased ATP-requirement. The ATP-requirement of the Na+/K(+)-pump was only partly satisfied when ATP was supplied from either the glycolytic or the oxidative pathway. This may indicate that the availability of ATP...

  4. Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra.

    Science.gov (United States)

    Sergeant, Gerard P; Bradley, Eamonn; Thornbury, Keith D; McHale, Noel G; Hollywood, Mark A

    2008-10-01

    Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca(2+) waves. The purpose of this study was to investigate if this activity was influenced by Ca(2+) uptake into mitochondria. Spontaneous Ca(2+) waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 microm) and antimycin A (5 microm) abolished Ca(2+) waves and increased basal Ca(2+) levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 microm) and CCCP (1 microm). Spontaneous Ca(2+) waves were not inhibited by the ATP synthase inhibitor oligomycin (1 microm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at -60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 microm) also inhibited STICS. Stimulation of Ca(2+) uptake into mitochondria using the plant flavonoid kaempferol (10 microm) induced a series of propagating Ca(2+) waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca(2+), but was not significantly affected by the IP(3) receptor blocker 2-APB (100 microm). These data suggest that spontaneous Ca(2+) waves in urethral ICC are regulated by buffering of cytoplasmic Ca(2+) by mitochondria.

  5. On the location of the H+-extruding steps in site 2 of the mitochondrial electron transport chain.

    Science.gov (United States)

    Alexandre, A; Galiazzo, F; Lehninger, A L

    1980-11-25

    The location of the H+-translocating reactions within energy-conserving Site 2 of the mitochondrial electron transport chain was evaluated from two sets of data. In the first, the H+/2e- ejection ratios and Ca2+/2e- uptake ratios were compared for electron flow from succinate dehydrogenase, whose active site is on the matrix side of the inner membrane and from glycerol phosphate dehydrogenase, whose active site is on the cytosolic side. In intact rat liver mitochondria both substrates yielded H+/2e- ejection ratios close to 4.0 and Ca2+/2e- uptake ratios close to 1.0 during antimycin-sensitive reduction of ferricyanide. With rat liver mitoplasts and ferricytochrome c as electron acceptor, both substrates again gave the same stoichiometric ratios. The second approach involved determination of the sidedness of H+ formation during electron flow from succinate to ferricyanide via bypass of the antimycin block of the cytochrome b.c1 complex provided by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), under conditions in which the TMPD-TMPD+ couple does not act as a membrane-penetrating protonophore. Electron flow in this system was inhibited by 2-then-oyltrifluoroacetone, indicating that TMPD probably accepts electrons from ubiquinol. The 2 H+ formed in this system were not delivered into the matrix but appeared directly in the medium in the absence of a protonophore. To accommodate the available evidence on Site 2 substrates, it is concluded that the substrate hydrogens are first transferred to ubiquinone, 2 H+ per 2e then appear in the medium by protolytic dehydrogenation of a species of ubiquinol or ubiquinol-protein having the appropriate sidedness (designated Site 2A), and the other 2 H+ are translocated from the matrix to the medium on passage of 2e- through the cytochrome b x c1 complex (designated Site 2B).

  6. Role of mitochondria in modulation of spontaneous Ca2+ waves in freshly dispersed interstitial cells of Cajal from the rabbit urethra

    Science.gov (United States)

    Sergeant, Gerard P; Bradley, Eamonn; Thornbury, Keith D; McHale, Noel G; Hollywood, Mark A

    2008-01-01

    Interstitial cells of Cajal (ICC) isolated from the rabbit urethra exhibit pacemaker activity that results from spontaneous Ca2+ waves. The purpose of this study was to investigate if this activity was influenced by Ca2+ uptake into mitochondria. Spontaneous Ca2+ waves were recorded using a Nipkow spinning disk confocal microscope and spontaneous transient inward currents (STICs) were recorded using the whole-cell patch clamp technique. Disruption of the mitochondrial membrane potential with the electron transport chain inhibitors rotenone (10 μm) and antimycin A (5 μm) abolished Ca2+ waves and increased basal Ca2+ levels. Similar results were achieved when mitochondria membrane potential was collapsed using the protonophores FCCP (0.2 μm) and CCCP (1 μm). Spontaneous Ca2+ waves were not inhibited by the ATP synthase inhibitor oligomycin (1 μm), suggesting that these effects were not attributable to an effect on ATP levels. STICs recorded under voltage clamp at −60 mV were also inhibited by CCCP and antimycin A. Dialysis of cells with the mitochondrial uniporter inhibitor RU360 (10 μm) also inhibited STICS. Stimulation of Ca2+ uptake into mitochondria using the plant flavonoid kaempferol (10 μm) induced a series of propagating Ca2+ waves. The kaempferol-induced activity was inhibited by application of caffeine (10 mm) or removal of extracellular Ca2+, but was not significantly affected by the IP3 receptor blocker 2-APB (100 μm). These data suggest that spontaneous Ca2+ waves in urethral ICC are regulated by buffering of cytoplasmic Ca2+ by mitochondria. PMID:18703577

  7. A single Sfp-type phosphopantetheinyl transferase plays a major role in the biosynthesis of PKS and NRPS derived metabolites in Streptomyces ambofaciens ATCC23877.

    Directory of Open Access Journals (Sweden)

    Robert Bunet

    Full Text Available 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.

  8. A single Sfp-type phosphopantetheinyl transferase plays a major role in the biosynthesis of PKS and NRPS derived metabolites in Streptomyces ambofaciens ATCC23877.

    Science.gov (United States)

    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.

  9. Anticancer Drug 2-Methoxyestradiol Protects against Renal Ischemia/Reperfusion Injury by Reducing Inflammatory Cytokines Expression

    Directory of Open Access Journals (Sweden)

    Ying-Yin Chen

    2014-01-01

    Full Text Available Background. Ischemia/reperfusion (I/R injury is a major cause of acute renal failure and allograft dysfunction in kidney transplantation. ROS/inflammatory cytokines are involved in I/R injury. 2-Methoxyestradiol (2ME2, an endogenous metabolite of estradiol, inhibits inflammatory cytokine expression and is an antiangiogenic and antitumor agent. We investigated the inhibitory effect of 2ME2 on renal I/R injury and possible molecular actions. Methods. BALB/c mice were intraperitoneally injected with 2ME2 (10 or 20 mg/kg or vehicle 12 h before and immediately after renal I/R experiments. The kidney weight, renal function, tubular damages, and apoptotic response were examined 24 h after I/R injury. The expression of mRNA of interleukin-1β, tumor necrosis factor- (TNF α, caspase-3, hypoxia inducible factor- (HIF 1α, and proapoptotic Bcl-2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3 in kidney tissue was determined using RT-PCR, while the expression of nuclear factor κB (NF-κB, BCL-2, and BCL-xL, activated caspase-9, and HIF-1α was determined using immunoblotting. In vitro, we determined the effect of 2ME2 on reactive oxygen species (ROS production and cell viability in antimycin-A-treated renal mesangial (RMC and tubular (NRK52E cells. Results. Serum creatinine and blood urea nitrogen were significantly higher in mice with renal I/R injury than in sham control and in I/R+2ME2-treated mice. Survival in I/R+2ME2-treated mice was higher than in I/R mice. Histological examination showed that 2ME2 attenuated tubular damage in I/R mice, which was associated with lower expression TNF-α, IL-1β, caspase-9, HIF-1α, and BNIP3 mRNA in kidney tissue. Western blotting showed that 2ME2 treatment substantially decreased the expression of activated caspase-9, NF-κB, and HIF-1α but increased the antiapoptotic proteins BCL-2 and BCL-xL in kidney of I/R injury. In vitro, 2MR2 decreased ROS production and increased cell viability in antimycin

  10. Molecular Characterization of Endoplasmic Reticulum Oxidoreductin 1 from Bombyx mori

    Directory of Open Access Journals (Sweden)

    Minchul Seo

    2015-11-01

    Full Text Available We isolated a complementary DNA (cDNA clone encoding endoplasmic reticulum oxidoreductin 1 (bERO1, a specific oxidant of protein disulfide isomerase (PDI from Bombyx mori. This protein has a putative open reading frame (ORF of 489 amino acids and a predicted size of 57.4 kDa. Although bERO1 protein shares less than 57% amino acid sequence homology with other reported ERO1s, it contains two conserved redox active motifs, a Cys-X-X-X-X-Cys motif of N-terminal and Cys-X-X-Cys-X-X-Cys motif of C-terminal. Both motifs are typically present in ERO1 protein family members. The bEro1 mRNA expression was highest in posterior silk gland on the sixth day of the 5th instar larvae. Expression of bEro1 mRNA also markedly increased during endoplasmic reticulum (ER stress induced by stimulation with antimycin, calcium ionophore A23187, dithiothreitol, H2O2, monencin, and tunicamycin. In addition, expression levels of bEro1 exactly coincided with that of bPdi. This is the first result suggesting that bERO1 plays an essential role in ER quality control through the combined activities of bERO1 and bPDI as a catalyst of protein folding in the ER and sustaining cellular redox homeostasis.

  11. The enhancement of cyclic electron flow around photosystem I improves the recovery of severely desiccated Porphyra yezoensis (Bangiales, Rhodophyta).

    Science.gov (United States)

    Gao, Shan; Wang, Guangce

    2012-07-01

    Porphyra yezoensis, a representative species of intertidal macro-algae, is able to withstand periodic desiccation at low tide but is submerged in seawater at high tide. In this study, changes in photosynthetic electron flow in P. yezoensis during desiccation and re-hydration were investigated. The results suggested that the cyclic electron flow around photosystem I (PSI) increased significantly during desiccation, continued to operate at times of severe desiccation, and showed greater tolerance to desiccation than the electron flow around PSII. In addition, PSI activity in desiccated blades recovered faster than PSII activity during re-hydration. Even though linear electron flow was suppressed by DCMU [3-(3',4'-dichlorophenyl)-1,1-dimethylurea], cyclic electron flow could still be restored. This process was insensitive to antimycin A and could be suppressed by dibromothymoquinone (DBMIB). The prolonged dark treatment of blades reduced the speed in which the cyclic electron flow around PSI recovered, suggesting that stromal reductants, including NAD(P)H, played an important role in the donation of electrons to PSI and were the main cause of the rapid recovery of cyclic electron flow in desiccated blades during re-hydration. These results suggested that cyclic electron flow in P. yezoensis played a significant physiological role during desiccation and re-hydration and may be one of the most important factors allowing P. yezoensis blades to adapt to intertidal environments.

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

    Science.gov (United States)

    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.

  13. Diabetes increases susceptibility of primary cultures of rat proximal tubular cells to chemically induced injury

    International Nuclear Information System (INIS)

    Zhong Qing; Terlecky, Stanley R.; Lash, Lawrence H.

    2009-01-01

    Diabetic nephropathy is characterized by increased oxidative stress and mitochondrial dysfunction. In the present study, we prepared primary cultures of proximal tubular (PT) cells from diabetic rats 30 days after an ip injection of streptozotocin and compared their susceptibility to oxidants (tert-butyl hydroperoxide, methyl vinyl ketone) and a mitochondrial toxicant (antimycin A) with that of PT cells isolated from age-matched control rats, to test the hypothesis that PT cells from diabetic rats exhibit more cellular and mitochondrial injury than those from control rats when exposed to these toxicants. PT cells from diabetic rats exhibited higher basal levels of reactive oxygen species (ROS) and higher mitochondrial membrane potential, demonstrating that the PT cells maintain the diabetic phenotype in primary culture. Incubation with either the oxidants or mitochondrial toxicant resulted in greater necrotic and apoptotic cell death, greater evidence of morphological damage, greater increases in ROS, and greater decreases in mitochondrial membrane potential in PT cells from diabetic rats than in those from control rats. Pretreatment with either the antioxidant N-acetyl-L-cysteine or a catalase mimetic provided equivalent protection of PT cells from both diabetic and control rats. Despite the greater susceptibility to oxidative and mitochondrial injury, both cytoplasmic and mitochondrial glutathione concentrations were markedly higher in PT cells from diabetic rats, suggesting an upregulation of antioxidant processes in diabetic kidney. These results support the hypothesis that primary cultures of PT cells from diabetic rats are a valid model in which to study renal cellular function in the diabetic state.

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

    Science.gov (United States)

    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 oxidation rate of C18:1 was greater in mitochondria than peroxisomes, while the oxidation of C22:1 was higher in peroxisomes than mitochondria ( p 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.

  15. Piscicides and invertebrates: after 70 years, does anyone really know?

    Science.gov (United States)

    Vinson, M.R.; Dinger, E.C.; Vinson, D.K.

    2010-01-01

    The piscicides rotenone and antimycin have been used for more than 70 years to manage fish populations by eliminating undesirable fish species. The effects of piscicides on aquatic invertebrate assemblages are considered negligible by some and significant by others. This difference of opinion has created contentious situations and delayed native fish restoration projects. We review the scientific evidence and report that short-term ( 1 year) impacts are largely unknown. Recovery of invertebrate assemblages following treatments ranged from a few months for abundances of common taxa to several years for rarer taxa. Variation in reported effects was primarily due to natural variation among species and habitats and a lack of adequate pre- and post-treatment sampling which prevents determining the true impacts to invertebrate assemblages. The factors most likely to influence impacts and recovery of aquatic invertebrate assemblages following piscicide treatments are: (1) concentration, duration, and breadth of the piscicide treatment; (2) invertebrate morphology and life history characteristics, including surface area to volume ratios, type of respiration organs, generation time, and propensity to disperse; (3) refugia presence; and (4) distance from colonization sources.

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

    Science.gov (United States)

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

    2013-02-07

    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.

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

    Directory of Open Access Journals (Sweden)

    Yonghui He

    2017-12-01

    Full Text Available 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 [1-14C]-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.

  18. The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells.

    Science.gov (United States)

    Murphy, T. M.; Auh, C. K.

    1996-01-01

    Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells. PMID:12226208

  19. The relationship between energy metabolism and the action of inhibitors of histamine release

    DEFF Research Database (Denmark)

    Garland, L G; Johansen, Torben

    1977-01-01

    1 Dextran-induced release of histamine from rat mast cells was inhibited equally in complete and glucose-free Tyrode solution by doxantrazole (0.03-3 micronmol/l), theophylline (0.1-3 mmol/l) and dicumarol (0.01-10 micronmol/litre). 2 Doxantrazole (3 micronmol/l), theophylline (3 mmol/l) and dicu......1 Dextran-induced release of histamine from rat mast cells was inhibited equally in complete and glucose-free Tyrode solution by doxantrazole (0.03-3 micronmol/l), theophylline (0.1-3 mmol/l) and dicumarol (0.01-10 micronmol/litre). 2 Doxantrazole (3 micronmol/l), theophylline (3 mmol...... of histamine release from mast cells incubated in glucose-free solution than in complete Tyrode solution (dose-ratio = 20). Like antimycin A (L MICRONMOL/L), PAPAVERINE (3 MICRONMOL/L) CAUSED A DEPLETION OF MAST CELL ATP that was greater in the absence (85%) than in the presence (25%) of extracellular glucose....... 4 These results suggest that dicumarol, like doxantrazole and theophylline, inhibits histamine release without affecting mast cell energy metabolism. In contrast, papaverine probably inhibits release by depleting ATP that is required for exocytosis. 5 Inhibition of histamine release by dibutyryl...

  20. Mitochondrial Malfunctioning, Proteasome Arrest and Apoptosis in Cancer Cells by Focused Intracellular Generation of Oxygen Radicals

    Directory of Open Access Journals (Sweden)

    Ilaria Postiglione

    2015-08-01

    Full Text Available Photofrin/photodynamic therapy (PDT at sub-lethal doses induced a transient stall in proteasome activity in surviving A549 (p53+/+ and H1299 (p53−/− cells as indicated by the time-dependent decline/recovery of chymotrypsin-like activity. Indeed, within 3 h of incubation, Photofrin invaded the cytoplasm and localized preferentially within the mitochondria. Its light activation determined a decrease in mitochondrial membrane potential and a reversible arrest in proteasomal activity. A similar result is obtained by treating cells with Antimycin and Rotenone, indicating, as a common denominator of this effect, the ATP decrease. Both inhibitors, however, were more toxic to cells as the recovery of proteasomal activity was incomplete. We evaluated whether combining PDT (which is a treatment for killing tumor cells, per se, and inducing proteasome arrest in the surviving ones with Bortezomib doses capable of sustaining the stall would protract the arrest with sufficient time to induce apoptosis in remaining cells. The evaluation of the mitochondrial membrane depolarization, residual proteasome and mitochondrial enzymatic activities, colony-forming capabilities, and changes in protein expression profiles in A549 and H1299 cells under a combined therapeutic regimen gave results consistent with our hypothesis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1982-10-01

    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:B8 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.

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

    International Nuclear Information System (INIS)

    Kondo-Nakamura, Mihoko; Shintani-Ishida, Kaori; Uemura, Koichi; Yoshida, Ken-ichi

    2010-01-01

    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 2 - generation, which was inhibited by a mitochondrial complex III inhibitor (antimycin A) but not by a NADH oxidase inhibitor (apocynin). The CO-enhanced Akt phosphorylation was suppressed by an O 2 - scavenger (Tiron), catalase or a superoxide dismutase (SOD) inhibitor (DETC). These results suggest that CO pretreatment induces mitochondrial generation of O 2 - , which is then converted by SOD to H 2 O 2 , and subsequent Akt activation by H 2 O 2 attenuates apoptosis in ischemia-reperfusion.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kondo-Nakamura, Mihoko [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Shintani-Ishida, Kaori, E-mail: kaori@m.u-tokyo.ac.jp [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan); Uemura, Koichi; Yoshida, Ken-ichi [Department of Forensic Medicine, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan)

    2010-03-12

    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) 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.

  4. The role of mitochondria for the regulation of cardiac alternans

    Directory of Open Access Journals (Sweden)

    Stela M Florea

    2010-11-01

    Full Text Available Electromechanical and Ca alternans is a beat-to-beat alternation of action potential duration, contraction strength and Ca transient amplitude observed in cardiac myocytes at regular stimulation frequency. Ca alternans is a multifactorial process that is causally linked to cardiac arrhythmias. At the cellular level, conditions that increase fractional release from the sarcoplasmic reticulum or reduce diastolic Ca sequestration favor the occurrence of alternans. Mitochondria play a significant role in cardiac excitation-contraction coupling and Ca signaling by providing the energy for contraction and ATP-dependent processes and possibly by serving as Ca buffering organelles. Here we tested the hypothesis that impairment of mitochondrial function generates conditions that favor the occurrence of Ca alternans. Alternans were elicited by electrical pacing (>1 Hz in single cat atrial myocytes and intracellular Ca ([Ca]i was measured with the fluorescent Ca indicator Indo-1. The degree of alternans was quantified as the alternans ratio (AR=1-S/L, where S/L is the ratio of the small to the large amplitude of a pair of alternating Ca transients. Dissipation of mitochondrial membrane potential (with FCCP as well as inhibition of mitochondrial F1/F0-ATP synthase (oligomycin, electrontransport chain (rotenone, antimycin, CN-, Ca-dependent dehydrogenases and mitochondrial Ca uptake or extrusion, all enhanced AR and lowered the threshold for the occurrence of Ca alternans. The data indicate that impairment of mitochondrial function adversely affects cardiac Ca cycling leading to proarrhythmic Ca alternans.

  5. Hyphal formation of Candida albicans is controlled by electron transfer system

    International Nuclear Information System (INIS)

    Watanabe, Toshihiko; Ogasawara, Ayako; Mikami, Takeshi; Matsumoto, Tatsuji

    2006-01-01

    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 growth 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

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

    Science.gov (United States)

    Karpinska, Barbara; Alomrani, Sarah Owdah; Foyer, Christine H

    2017-09-26

    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'. © 2017 The Authors.

  7. Ascorbate Biosynthesis in Mitochondria Is Linked to the Electron Transport Chain between Complexes III and IV1

    Science.gov (United States)

    Bartoli, Carlos G.; Pastori, Gabriela M.; Foyer, Christine H.

    2000-01-01

    Ascorbic acid is synthesized from galactono-γ-lactone (GL) in plant tissues. An improved extraction procedure involving ammonium sulfate precipitation of membrane proteins from crude leaf homogenates yielded a simple, quick method for determining tissue activities of galactono-γ-lactone dehydrogenase (GLDH). Total foliar ascorbate and GLDH activity decreased with leaf age. Subcellular fractionation experiments using marker enzymes demonstrated that 80% of the total GLDH activity was located on the inner mitochondrial membrane, and 20% in the microsomal fraction. Specific antibody raised against potato (Solanum tuberosum L.) tuber GLDH recognized a 56-kD polypeptide in extracts from the mitochondrial membranes but failed to detect the equivalent polypeptide in microsomes. We demonstrate that isolated intact mitochondria synthesize ascorbate in the presence of GL. GL stimulated mitochondrial electron transport rates. The respiration inhibitor antimycin A stimulated ascorbate biosynthesis, while cyanide inhibited both respiration and ascorbate production. GL-dependent oxygen uptake was observed in isolated intact mitochondria. This evidence suggests that GLDH delivers electrons to the mitochondrial electron transport chain between complexes III and IV. PMID:10806250

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

    Science.gov (United States)

    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.

  9. Metabolic profiles show specific mitochondrial toxicities in vitro in myotube cells

    International Nuclear Information System (INIS)

    Xu Qiuwei; Vu, Heather; Liu Liping; Wang, Ting-Chuan; Schaefer, William H.

    2011-01-01

    Mitochondrial toxicity has been a serious concern, not only in preclinical drug development but also in clinical trials. In mitochondria, there are several distinct metabolic processes including fatty acid β-oxidation, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation (OXPHOS), and each process contains discrete but often intimately linked steps. Interruption in any one of those steps can cause mitochondrial dysfunction. Detection of inhibition to OXPHOS can be complicated in vivo because intermediate endogenous metabolites can be recycled in situ or circulated systemically for metabolism in other organs or tissues. Commonly used assays for evaluating mitochondrial function are often applied to ex vivo or in vitro samples; they include various enzymatic or protein assays, as well as functional assays such as measurement of oxygen consumption rate, membrane potential, or acidification rates. Metabolomics provides quantitative profiles of overall metabolic changes that can aid in the unraveling of explicit biochemical details of mitochondrial inhibition while providing a holistic view and heuristic understanding of cellular bioenergetics. In this paper, we showed the application of quantitative NMR metabolomics to in vitro myotube cells treated with mitochondrial toxicants, rotenone and antimycin A. The close coupling of the TCA cycle to the electron transfer chain (ETC) in OXPHOS enables specific diagnoses of inhibition to ETC complexes by discrete biochemical changes in the TCA cycle.

  10. Putative Structural and Functional Coupling of the Mitochondrial BKCa Channel to the Respiratory Chain.

    Directory of Open Access Journals (Sweden)

    Piotr Bednarczyk

    Full Text Available Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels regulate the mitochondrial membrane potential, the matrix volume and respiration. The activation of these channels is cytoprotective. In our study, the single-channel activity of a large-conductance Ca(2+-regulated potassium channel (mitoBKCa channel was measured by patch-clamping mitoplasts isolated from the human astrocytoma (glioblastoma U-87 MG cell line. A potassium-selective current was recorded with a mean conductance of 290 pS in symmetrical 150 mM KCl solution. The channel was activated by Ca(2+ at micromolar concentrations and by the potassium channel opener NS1619. The channel was inhibited by paxilline and iberiotoxin, known inhibitors of BKCa channels. Western blot analysis, immuno-gold electron microscopy, high-resolution immunofluorescence assays and polymerase chain reaction demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membrane of the human astrocytoma cells. We showed that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate, decrease the activity of the channel at positive voltages. This effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The putative interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated using blue native electrophoresis. Our findings indicate possible structural and functional coupling of the mitoBKCa channel with the mitochondrial respiratory chain in human astrocytoma U-87 MG cells.

  11. Mitochondrial respiratory pathways inhibition in Rhizopus oryzae potentiates activity of posaconazole and itraconazole via apoptosis.

    Directory of Open Access Journals (Sweden)

    Fazal Shirazi

    Full Text Available The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA and benzohydroxamate (BHAM, inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ and itraconazole (ICZ against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS, phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents.

  12. Mitochondrial Swelling Induced by Glutathione

    Science.gov (United States)

    Lehninger, Albert L.; Schneider, Marion

    1959-01-01

    Reduced glutathione, in concentrations approximating those occurring in intact rat liver, causes swelling of rat liver mitochondria in vitro which is different in kinetics and extent from that yielded by L-thyroxine. The effect is also given by cysteine, which is more active, and reduced coenzyme A, but not by L-ascorbate, cystine, or oxidized glutathione. The optimum pH is 6.5, whereas thyroxine-induced swelling is optimal at pH 7.5. The GSH-induced swelling is not inhibited by DNP or dicumarol, nor by high concentrations of sucrose, serum albumin, or polyvinylpyrrolidone, in contrast to thyroxine-induced swelling. ATP inhibits the GSH swelling, but ADP and AMP are ineffective. Mn-+ is a very potent inhibitor, but Mg++ is ineffective. Ethylenediaminetetraacetate is also an effective inhibitor of GSH-induced swelling. The respiratory inhibitors amytal and antimycin A do not inhibit the swelling action of GSH, but cyanide does; these findings are consistent with the view that the oxidation-reduction state of the respiratory chain between cytochrome c and oxygen is a determinant of GSH-induced swelling. Reversal of GSH-induced swelling by osmotic means or by ATP in KCl media could not be observed. Large losses of nucleotides and protein occur during the swelling by GSH, suggesting that the action is irreversible. The characteristically drastic swelling action of GSH could be prevented if L-thyroxine was also present in the medium. PMID:13630941

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

    Science.gov (United States)

    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.

  14. Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

    International Nuclear Information System (INIS)

    Chapman, K.D.; Trelease, R.N.

    1991-01-01

    Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) activities, which equilibrated with the main peak of Anti-mycin A-insensitive NADH: cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes in 0.2 millimolar MgCl 2 followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with 14 C-CCP choline (or 14 C-CDPethanolamine) resulted in synthesis and incorporation of 14 C-phosphatidylcholine (PC) (or 14 C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg 2+ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. under these conditions, newly synthesized and incorporated 14 C-PC (or PE) was recovered in these membranes. These results indicate that Er in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis

  15. Generation of adenosine tri-phosphate in Leishmania donovani amastigote forms.

    Science.gov (United States)

    Mondal, Subhasish; Roy, Jay Jyoti; Bera, Tanmoy

    2014-03-01

    Leishmania, the causative agent of various forms of leishmaniasis, is the significant cause of morbidity and mortality. Regarding energy metabolism, which is an essential factor for the survival, parasites adapt to the environment under low oxygen tension in the host using metabolic systems which are very different from that of the host mammals. We carried out the study of susceptibilities to different inhibitors of mitochondrial electron transport chain and studies on substrate level phosphorylation in wild-type L. donovani. The amastigote forms of L. donovani are independent on oxidative phosphorylation for ATP production. Indeed, its cell growth was not inhibited by excess oligomycin and dicyclohexylcarbodiimide, which are the most specific inhibitors of the mitochondrial Fo/F1-ATP synthase. In contrast, mitochondrial complex I inhibitor rotenone and complex III inhibitor antimycin A inhibited amastigote cell growth, suggesting the role of complex I and complex III in cell survival. Complex II appeared to have no role in cell survival. To further investigate the site of ATP production, we studied the substrate level phosphorylation, which was involved in the synthesis of ATP. Succinate-pyruvate couple showed the highest substrate level phosphorylation in amastigotes whereas NADH-fumarate and NADH-pyruvate couples failed to produce ATP. In contrast, NADPH-fumarate showed the highest rate of ATP formation in promastigotes. Therefore, we can conclude that substrate level phosphorylation is essential for the survival of amastigote forms of Leishmania donovani.

  16. Investigations on the mechanism of oxygen-dependent plant processes: ethylene biosynthesis and cyanide-resistant respiration

    International Nuclear Information System (INIS)

    Stegink, S.J.

    1985-01-01

    Two oxygen-dependent plant processes were investigated. A cell-free preparation from pea (Pisum sativum L., cv. Alaska) was used to study ethylene biosynthesis from 1-aminocyclopropane-1-carboxylic acid. Mitochondrial cyanide-resistant respiration was investigated in studies with 14 C-butyl gallate and other respiratory effectors. Ethylene biosynthesis was not due to a specific enzyme, or oxygen radicals. Rather, hydrogen peroxide, generated at low levels, coupled with endogenous manganese produced ethylene. 14 C-butyl gallate bound specifically to mitochondria from cyanide-sensitive and -resistant higher plants and Neurospora crassa mitochondria. The amount of gallate bound was similar for all higher plant mitochondria. Rat liver mitochondria bound very little 14 C-butyl gallate. Plant mitochondria in which cyanide-resistance was induced bound as much 14 C-butyl gallate as before induction. However mitochondria from recently harvested white potato tubers did not bind the gallate. The observations suggest that an engaging factor couples with a gallate binding site in the mitochondrial membrane. With skunk cabbage spadix mitochondria the I 5 0 for antimycin A inhibition of oxygen uptake was decreased by salicylhydroxamic acid pretreatment; this was also true for reverse order additions. No shift was observed with mung bean hypocotyl or Jerusalem artichoke tuber mitochondria

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

    Science.gov (United States)

    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.

  18. Effect of Hexavalent Chromium on Electron Leakage of Respiratory Chain in Mitochondria Isolated from Rat Liver

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    Ying Xie

    2013-03-01

    Full Text Available Background/Aims: 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. Methods: 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. Results: 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. Conclusion: 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.

  19. Bovine mitochondrial oxygen consumption effects on oxymyoglobin in the presence of lactate as a substrate for respiration.

    Science.gov (United States)

    Ramanathan, R; Mancini, R A; Joseph, P; Suman, S P

    2013-04-01

    Research suggests that NADH formed from lactate addition can increase mitochondrial oxygen consumption. However, limited research has assessed the subsequent effects of lactate-mediated mitochondrial oxygen consumption on oxymyoglobin. Therefore, our objective was to assess the effects of bovine mitochondrial oxygen consumption on oxymyoglobin in the presence of lactate, LDH, and NAD. Isolated beef cardiac and skeletal muscle mitochondria (n=5) were reacted with lactate (40 mM), LDH (100 units), and NAD (0.02 mM) to initiate oxygen consumption. Myoglobin redox state was measured to assess the effects of oxygen consumption on oxymyoglobin. The addition of lactate-LDH-NAD to mitochondria increased (p<0.05) both oxygen consumption and conversion of oxymyoglobin to deoxymyoglobin compared with control mitochondria without substrates. The addition of antimycin A to mitochondria decreased oxygen consumption and deoxymyoglobin formation (p<0.05). The results suggest that increased oxygen consumption can influence myoglobin redox state and this effect might be partially responsible for the darkening effect in lactate enhanced beef. Copyright © 2012 Elsevier Ltd. All rights reserved.

  20. Effect of long-term cyanide exposure on cyanide-sensitive respiration and phosphate metabolism in the fungus Phycomyces blakesleeanus

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    Stanić Marina

    2014-01-01

    Full Text Available The effects of long-term exposure (5 h of Phycomyces blakesleeanus mycelium to 5 mM KCN on respiration and phosphate metabolites were tested. Exposure to cyanide, antimycin A and azide lead to a decrease in the activity of cyanide-sensitive respiration (CSR, and the ratio of core polyphosphates (PPc and inorganic phosphates (Pi, which is a good indicator of the metabolic state of a cell. After 5 h of incubation, the activity of CSR returned to control values. For this, the recovery of cytochrome c oxidase (COX was required. In addition, the PPc/Pi ratio started to recover shortly after initiation of COX recovery, but never reached control values. This led us to conclude that the regulation of polyphosphate (PPn levels in the cell is tightly coupled to respiratory chain functioning. In addition, acutely applied cyanide caused two different responses, observed by 31P NMR spectroscopy, that were probably mediated through the mechanism of glycolytic oscillations, triggered by the effect of cyanide on mitochondria. [Projekat Ministarstva nauke Republike Srbije, br. 173040

  1. The localization of glycollate-pathway enzymes in Euglena.

    Science.gov (United States)

    Collins, N; Merrett, M J

    1975-01-01

    Isolation of organelles from broken-cell suspensions of phototrophically grown Euglena gracilis Klebs was achieved by isopycnic centrifugation on sucrose gradients. 2. Equilibrium densities of 1.23g/cm3 for peroxisome-like particles, 1.22g/cm3 for mitochondria and 1.17g/cm3 for chloroplasts were recorded. 3. The enzymes glycollate dehydrogenase, glutamate-glyoxylate aminotransferase, serineglyoxylate aminotransferase, aspartate-alpha-oxoglutarate aminotransferase, hydroxy pyruvate reductase and malate dehydrogenase were present in peroxisome-like particles. 4. Unlike higher plants glycollate dehydrogenase and glutamate-glyoxylate aminotransferase were present in the mitochondria of Euglena. 5. Rates of glycollate and D-lactate oxidation were additive in the mitochondria, and, although glycollate dehydrogenase was inhibited by cyanide, D-lactate dehydrogenase activity was unaffected. 6. Glycollate oxidation was linked to O2 uptake in mitochondria but not in peroxisome-like particles. This glycollate-dependent O2 uptake was inhibited by antimycin A or cyanide. 7. The physiological significance of glycollate metabolism in Euglena mitochondria is discussed, with special reference to its role in photorespiration in algae. PMID:1156408

  2. Synergism of Antifungal Activity between Mitochondrial Respiration Inhibitors and Kojic Acid

    Directory of Open Access Journals (Sweden)

    Ronald P. Haff

    2013-01-01

    Full Text Available Co-application of certain types of compounds to conventional antimicrobial drugs can enhance the efficacy of the drugs through a process termed chemosensitization. We show that kojic acid (KA, a natural pyrone, is a potent chemosensitizing agent of complex III inhibitors disrupting the mitochondrial respiratory chain in fungi. Addition of KA greatly lowered the minimum inhibitory concentrations of complex III inhibitors tested against certain filamentous fungi. Efficacy of KA synergism in decreasing order was pyraclostrobin > kresoxim-methyl > antimycin A. KA was also found to be a chemosensitizer of cells to hydrogen peroxide (H2O2, tested as a mimic of reactive oxygen species involved in host defense during infection, against several human fungal pathogens and Penicillium strains infecting crops. In comparison, KA-mediated chemosensitization to complex III inhibitors/H2O2 was undetectable in other types of fungi, including Aspergillus flavus, A. parasiticus, and P. griseofulvum, among others. Of note, KA was found to function as an antioxidant, but not as an antifungal chemosensitizer in yeasts. In summary, KA could serve as an antifungal chemosensitizer to complex III inhibitors or H2O2 against selected human pathogens or Penicillium species. KA-mediated chemosensitization to H2O2 seemed specific for filamentous fungi. Thus, results indicate strain- and/or drug-specificity exist during KA chemosensitization.

  3. Inhibitory Effects of Respiration Inhibitors on Aflatoxin Production

    Directory of Open Access Journals (Sweden)

    Shohei Sakuda

    2014-03-01

    Full Text Available 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.

  4. Growth of Streptococcus faecalis var. zymogenes on glycerol: the effect of aerobic and anaerobic growth in the presence and absence of haematin on enzyme synthesis.

    Science.gov (United States)

    Pugh, S Y; Knowles, C J

    1982-05-01

    Streptococcus faecalis var. zymogenes was grown aerobically and anaerobically in the presence and absence of haematin, with glycerol as the carbon and energy source. Aerobic growth was stimulated by the inclusion of haematin in the medium but fumarate had no effect on growth. The bacterium was unable to grow anaerobically on glycerol unless fumarate was present; haematin had no effect on growth. NADH oxidase activity, which catalysed the oxidation of NADH + H+ to form H2O rather than H2O2, was found in the soluble fraction and was induced by aerobic growth but partially repressed when haematin was present in the medium. In contrast, a particulate NADH oxidase, which was sensitive to inhibition by antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, was induced by aerobic growth in the presence of haematin. NADH peroxidase was massively induced by aerobic growth, whereas more lactate dehydrogenase activity was found in anaerobically grown bacteria. Catalase was formed only during aerobic growth in the presence of haematin.

  5. Unsaturated fatty acids-dependent linkage between respiration and fermentation revealed by deletion of hypoxic regulatory KlMGA2 gene in the facultative anaerobe-respiratory yeast Kluyveromyces lactis.

    Science.gov (United States)

    Ottaviano, Daniela; Montanari, Arianna; De Angelis, Lorenzo; Santomartino, Rosa; Visca, Andrea; Brambilla, Luca; Rinaldi, Teresa; Bello, Cristiano; Reverberi, Massimo; Bianchi, Michele M

    2015-08-01

    In the yeast Kluyveromyces lactis, the inactivation of structural or regulatory glycolytic and fermentative genes generates obligate respiratory mutants which can be characterized by sensitivity to the mitochondrial drug antimycin A on glucose medium (Rag(-) phenotype). Rag(-) mutations can occasionally be generated by the inactivation of genes not evidently related to glycolysis or fermentation. One such gene is the hypoxic regulatory gene KlMGA2. In this work, we report a study of the many defects, in addition to the Rag(-) phenotype, generated by KlMGA2 deletion. We analyzed the fermentative and respiratory metabolism, mitochondrial functioning and morphology in the Klmga2Δ strain. We also examined alterations in the regulation of the expression of lipid biosynthetic genes, in particular fatty acids, ergosterol and cardiolipin, under hypoxic and cold stress and the phenotypic suppression by unsaturated fatty acids of the deleted strain. Results indicate that, despite the fact that the deleted mutant strain had a typical glycolytic/fermentative phenotype and KlMGA2 is a hypoxic regulatory gene, the deletion of this gene generated defects linked to mitochondrial functions suggesting new roles of this protein in the general regulation and cellular fitness of K. lactis. Supplementation of unsaturated fatty acids suppressed or modified these defects suggesting that KlMga2 modulates membrane functioning or membrane-associated functions, both cytoplasmic and mitochondrial. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Deletion of mitochondrial ATPase inhibitor in the yeast Saccharomyces cerevisiae decreased cellular and mitochondrial ATP levels under non-nutritional conditions and induced a respiration-deficient cell-type.

    Science.gov (United States)

    Lu, Y M; Miyazawa, K; Yamaguchi, K; Nowaki, K; Iwatsuki, H; Wakamatsu, Y; Ichikawa, N; Hashimoto, T

    2001-12-01

    T(1), a mutant yeast lacking three regulatory proteins of F(1)F(o)ATPase, namely ATPase inhibitor, 9K protein and 15K protein, grew on non-fermentable carbon source at the same rate as normal cells but was less viable when incubated in water. During the incubation, the cellular ATP content decreased rapidly in the T(1) cells but not in normal cells, and respiration-deficient cells appeared among the T(1) cells. The same mutation was also induced in D26 cells lacking only the ATPase inhibitor. Overexpression of the ATPase inhibitor in YC63 cells, which were derived from the D26 strain harboring an expression vector containing the gene of the ATPase inhibitor, prevented the decrease of cellular ATP level and the mutation. Isolated T(1) mitochondria exhibited ATP hydrolysis for maintenance of membrane potential when antimycin A was added to the mitochondrial suspension, while normal and YC63 mitochondria continued to show low hydrolytic activity and low membrane potential. Thus, it is likely that deletion of the ATPase inhibitor induces ATPase activity of F(1)F(o)ATPase to create a dispensable membrane potential under the non-nutritional conditions and that this depletes mitochondrial and cellular ATP. The depletion of mitochondrial ATP in turn leads to occurrence of aberrant DNA in mitochondria.

  7. Evaluation of the electron transport chain inhibition and uncoupling of mitochondrial bioelectrocatalysis with antibiotics and nitro-based compounds

    International Nuclear Information System (INIS)

    Arechederra, Marguerite N.; Fischer, Caitlin N.; Wetzel, David J.; Minteer, Shelley D.

    2010-01-01

    Mitochondrial bioelectrocatalysis can be useful for sensing applications due to the unique metabolic pathways than can be selectively inhibited and uncoupled in mitochondria. This paper details the comparison of different inhibitors and nitro-containing explosive uncouplers in a mitochondria-catalyzed biofuel cell for self-powered explosive sensing. Previous research has reported inhibition of pyruvate oxidation at a mitochondria-modified electrode followed by nitroaromatic uncoupling of current and power. We have previously used oligomycin as the antibiotic and nitrobenzene as the uncoupler of the membrane in the mitochondria-catalyzed biofuel cell, but no comprehensive comparison of various mitochondria inhibitors or explosives has been performed. Results are discussed here for inhibitors targeting complex I, complex III, ATP synthases, adenine nucleotide transport and monocarboxylic acid transport. Reactivation with nitrobenzene was possible in the presence of these inhibitors: oligomycin, 3,3'-diindolylmethane, atractyloside, rotenone, α-cyano-4-hydroxy cinnamic acid and antimycin A. All eleven explosives studied, including: 2,4,6-trinitrotoluene (TNT) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX), caused uncoupling of the mitochondria function and could be detected by the biosensor.

  8. Studies on the mechanism of synthesis of ethyl acetate in Kluyveromyces marxianus DSM 5422.

    Science.gov (United States)

    Löser, Christian; Urit, Thanet; Keil, Peter; Bley, Thomas

    2015-02-01

    Kluyveromyces marxianus converts whey-borne sugar into ethyl acetate, an environmentally friendly solvent with many applications. K. marxianus DSM 5422 presumably synthesizes ethyl acetate from acetyl-SCoA. Iron limitation as a trigger for this synthesis is explained by a diminished aconitase and succinate dehydrogenase activity (both enzymes depend on iron) causing diversion of acetyl-SCoA from the tricarboxic acid cycle to ester synthesis. Copper limitation as another trigger for ester synthesis in this yeast refers to involvement of the electron transport chain (all ETC complexes depend on iron and complex IV requires copper). This hypothesis was checked by using several ETC inhibitors. Malonate was ineffective but carboxin partially inhibited complex II and initiated ester synthesis. Antimycin A and cyanide as complexes III and IV inhibitors initiated ester synthesis only at moderate levels while higher concentrations disrupted all respiration and caused ethanol formation. A restricted supply of oxygen (the terminal electron acceptor) also initiated some ester synthesis but primarily forced ethanol production. A switch from aerobic to anaerobic conditions nearly stopped ester synthesis and induced ethanol formation. Iron-limited ester formation was compared with anaerobic ethanol production; the ester yield was lower than the ethanol yield but a higher market price, a reduced number of process stages, a faster process, and decreased expenses for product recovery by stripping favor biotechnological ester production.

  9. Mitochondrial Respiratory Pathways Inhibition in Rhizopus oryzae Potentiates Activity of Posaconazole and Itraconazole via Apoptosis

    Science.gov (United States)

    Shirazi, Fazal; Kontoyiannis, Dimitrios P.

    2013-01-01

    The incidence of mucormycosis has increased drastically in immunocompromised patients. Also the array of targets whose inhibition results in Mucorales death is limited. Recently, researchers identified mitochondria as important regulators of detoxification and virulence mechanisms in fungi. In this context, targeting the mitochondrial respiratory chain may provide a new platform for antifungal development. We hypothesized that targeting respiratory pathways potentiates triazoles activity via apoptosis. We found that simultaneous administration of antimycin A (AA) and benzohydroxamate (BHAM), inhibitors of classical and alternative mitochondrial pathways respectively, resulted in potent activity of posaconazole (PCZ) and itraconazole (ICZ) against Rhizopus oryzae. We observed cellular changes characteristic of apoptosis in R. oryzae cells treated with PCZ or ICZ in combination with AA and BHAM. The fungicidal activity of this combination against R. oryzae was correlated with intracellular reactive oxygen species accumulation (ROS), phosphatidylserine externalization, mitochondrial membrane depolarization, and increased caspase like activity. DNA fragmentation and condensation assays also revealed apoptosis of R. oryzae cells. These apoptotic features were prevented by the addition of the ROS scavenger N-acetyl-cysteine. Taken together, these findings suggest that the use of PCZ or ICZ in combination with AA and BHAM makes R. oryzae exquisitely sensitive to treatment with triazoles via apoptosis. This strategy may serve as a new model for the development of improved or novel antifungal agents. PMID:23696824

  10. Effects of chitosan and oligochitosan on development and mitochondrial function of Rhizopus stolonifer.

    Science.gov (United States)

    Robles-Martínez, Leobarda; Guerra-Sánchez, María Guadalupe; Hernández-Lauzardo, Ana Niurka; Pardo, Juan Pablo; Velázquez-del Valle, Miguel Gerardo

    2014-07-01

    The antifungal activities of chitosan and oligochitosan have been used to control postharvest decay of the fruits. The effect of chitosan and oligochitosan on mycelium growth, spore germination, and mitochondrial function of Rhizopus stolonifer was evaluated in order to establish a connection between fungus development and the main organelle in charge to provide energy to the cell. The mycelium growth of R. stolonifer was significantly reduced on minimum media amended with chitosan or oligochitosan. The highest antifungal indexes were obtained on media containing chitosan or oligochitosan at 2.0 mg ml(-1). Microscopic observation showed that chitosan and oligochitosan affected the spore germination and hyphae morphology. Both polymers increased oxygen consumption of R. stolonifer. Respiratory activity was restored with NADH in permeabilized treated and untreated cells, and was inhibited with rotenone and flavones. Complex III and IV were inhibited by antimycin A and cyanide, respectively, in treated and untreated cells. Chitosan and oligochitosan increased NADH dehydrogenase activity in isolated mitochondria. However, there were not changes in the cytochrome c oxidase and ATPase activities by effect of these polymers. These results suggest that both chitosan and oligochitosan affect the development of R. stolonifer and might be implicated in the mitochondrial dysfunction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Peroxynitrite formation in nitric oxide-exposed submitochondrial particles: detection, oxidative damage and catalytic removal by Mn-porphyrins.

    Science.gov (United States)

    Valez, Valeria; Cassina, Adriana; Batinic-Haberle, Ines; Kalyanaraman, Balaraman; Ferrer-Sueta, Gerardo; Radi, Rafael

    2013-01-01

    Peroxynitrite (ONOO(-)) formation in mitochondria may be favored due to the constant supply of superoxide radical (O(2)(∙-)) by the electron transport chain plus the facile diffusion of nitric oxide ((∙)NO) to this organelle. Herein, a model system of submitochondrial particles (SMP) in the presence of succinate plus the respiratory inhibitor antimycin A (to increase O(2)(∙-) rates) and the (∙)NO-donor NOC-7 was studied to directly establish and quantitate peroxynitrite by a multiplicity of methods including chemiluminescence, fluorescence and immunochemical analysis. While all the tested probes revealed peroxynitrite at near stoichiometric levels with respect to its precursor radicals, coumarin boronic acid (a probe that directly reacts with peroxynitrite) had the more straightforward oxidation profile from O(2)(∙-)-forming SMP as a function of the (∙)NO flux. Interestingly, immunospintrapping studies verified protein radical generation in SMP by peroxynitrite. Substrate-supplemented SMP also reduced Mn(III)porphyrins (MnP) to Mn(II)P under physiologically-relevant oxygen levels (3-30 μM); then, Mn(II)P were capable to reduce peroxynitrite and protect SMP from the inhibition of complex I-dependent oxygen consumption and protein radical formation and nitration of membranes. The data directly support the formation of peroxynitrite in mitochondria and demonstrate that MnP can undergo a catalytic redox cycle to neutralize peroxynitrite-dependent mitochondrial oxidative damage. Copyright © 2012 Elsevier Inc. All rights reserved.

  12. Biochemical factors affecting the quantum efficiency of hydrogen production by membranes of green photosynthetic bacteria

    Energy Technology Data Exchange (ETDEWEB)

    Bernstein, J.D.; Olson, J.M.

    1981-01-01

    Photohydrogen production, 200-700 ..mu..mol H/sub 2/ h/sup -1/ (mg bacteriochlorophyll a)/sup -1/ has been obtained in a system containing unit membrane vesicles (Complex I) from the green photosynthetic bacterium Chlorobium limicola f. thiosulfatophilum, ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine, dithioerythritol, an oxygen scavenging mixture, either methyl viologen (MV) or clostridial ferredoxin (CPS Fd) as electron carrier, and either CPS hydrogenase or platinum asbestos as catalyst. All components are necessary for maximum activity, and spinach Fd cannot be substituted for CPS Fd. Higher rates of photohydrogen production are obtained using MV or CPS Fd with hydrogenase than with MV and Pt asbestos. The highest quantum efficiencies (7-10% at 0.2-0.9 mW absorbed light and over 20% at lower light) were obtained with CPS Fd, hydrogenase and non-saturating 812 nm light. With saturating white light, however, rates of photohydrogen production varied relatively little among the various combinations of electron carrier and catalyst tested. The reaction rate is unaffected by 0.03% Triton X-100, and is insensitive to treatment with antimycin a or m-chloro-carbonyl cyanide phenylhydrazone.This indicates that neither electron flow through an endogenous cyclic chain, nor maintenance of a proton gradient are involved in this process.

  13. Lacidipine Attenuates Apoptosis via a Caspase-3 Dependent Pathway in Human Kidney Cells

    Directory of Open Access Journals (Sweden)

    Aiqi Zhang

    2013-10-01

    Full Text Available Background: Acute kidney injury (AKI is common in hospitalised patients and has a poor prognosis. Therefore, new therapeutic strategies are anticipated. Lacidipine, a novel third-generation dihydropyridine calcium channel blocker, has been demonstrated effective for hypertension. However, its potential effect on renal injury remains unknown. In the present study, an in vitro model of renal ischemia reperfusion (I/R injury was used to investigate the protective effect and underlying mechanisms of lacidipine on human kidney cell (HKC apoptosis. Methods: HKCs were subjected to adenosine triphosphate (ATP depletion and recovery (0.01 µM AA, depletion for 2 h and recovery for 30 min, with or without lacidipine (1 µM and 10 µM, 24 h, then cell viability and apoptosis were determined using the cell counting kit-8 (CCK-8 assay and Annexin V flow cytometry. The expression of Bcl-2, Bax, and cytochrome c (cyt c was examined by western blot. Results: Antimycin A (AA was found to induce apoptosis of HKCs. The proportion of early apoptosis and activity of caspase-3 peaked at 30 min after ATP depletion and recovery and were attenuated by lacidipine. The expression of cyt c and Bax was decreased, while that of Bcl-2 was increased significantly in lacidipine treated group. Conclusion: We conclude that lacidipine protects HKCs against apoptosis induced by ATP depletion and recovery by regulating the caspase-3 pathway.

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

    Science.gov (United States)

    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.

  15. The glucose sensor-like protein Hxs1 is a high-affinity glucose transporter and required for virulence in Cryptococcus neoformans.

    Directory of Open Access Journals (Sweden)

    Tong-Bao Liu

    Full Text Available Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2 in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.

  16. The Glucose Sensor-Like Protein Hxs1 Is a High-Affinity Glucose Transporter and Required for Virulence in Cryptococcus neoformans

    Science.gov (United States)

    Baker, Gregory M.; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence. PMID:23691177

  17. The glucose sensor-like protein Hxs1 is a high-affinity glucose transporter and required for virulence in Cryptococcus neoformans.

    Science.gov (United States)

    Liu, Tong-Bao; Wang, Yina; Baker, Gregory M; Fahmy, Hany; Jiang, Linghuo; Xue, Chaoyang

    2013-01-01

    Cryptococcus is a major fungal pathogen that frequently causes systemic infection in patients with compromised immunity. Glucose, an important signal molecule and the preferred carbon source for Cryptococcus, plays a critical role in fungal development and virulence. Cryptococcus contains more than 50 genes sharing high sequence homology with hexose transporters in Saccharomyces cerevisiae. However, there is no report on their function in glucose sensing or transport. In this study, we investigated two hexose transporter-like proteins (Hxs1 and Hxs2) in Cryptococcus that share the highest sequence identity with the glucose sensors Snf3 and Rgt2 in S. cerevisiae. The expression of HXS1 is repressed by high glucose, while the HXS2 expression is not regulated by glucose. Functional studies showed that Hxs1 is required for fungal resistance to oxidative stress and fungal virulence. The hxs1Δ mutant exhibited a significant reduction in glucose uptake activity, indicating that Hxs1 is required for glucose uptake. Heterologous expression of Cryptococcus HXS1 rendered the S. cerevisiae mutant lacking all 20 hexose transporters a high glucose uptake activity, demonstrating that Hxs1 functions as a glucose transporter. Heterologous expression of HXS1 in the snf3Δ rgt2Δ double mutant did not complement its growth in YPD medium containing the respiration inhibitor antimycin A, suggesting that Hxs1 may not function as a glucose sensor. Taken together, our results demonstrate that Hxs1 is a high-affinity glucose transporter and required for fungal virulence.

  18. Iron speciation in human cancer cells by K-edge total reflection X-ray fluorescence-X-ray absorption near edge structure analysis

    Energy Technology Data Exchange (ETDEWEB)

    Polgari, Zs. [Eoetvoes Lorand University, Institute of Chemistry, Department of Analytical Chemistry, Laboratory of Environmental Chemistry and Bioanalytics, P.O. Box 32, H-1518, Budapest (Hungary); Meirer, F. [Institute of Atomic and Subatomic Physics, Vienna University of Technology, Vienna (Austria); MiNALab, CMM-irst, Fondazione Bruno Kessler, Povo, Trento (Italy); Sasamori, S.; Ingerle, D. [Institute of Atomic and Subatomic Physics, Vienna University of Technology, Vienna (Austria); Pepponi, G. [MiNALab, CMM-irst, Fondazione Bruno Kessler, Povo, Trento (Italy); Streli, C. [Institute of Atomic and Subatomic Physics, Vienna University of Technology, Vienna (Austria); Rickers, K. [Hamburger Synchrotronstrahlungslabor at DESY, Hamburg (Germany); Reti, A.; Budai, B. [Department of Clinical Research, National Institute of Oncology, Budapest (Hungary); Szoboszlai, N. [Eoetvoes Lorand University, Institute of Chemistry, Department of Analytical Chemistry, Laboratory of Environmental Chemistry and Bioanalytics, P.O. Box 32, H-1518, Budapest (Hungary); Zaray, G., E-mail: zaray@ludens.elte.hu [Eoetvoes Lorand University, Institute of Chemistry, Department of Analytical Chemistry, Laboratory of Environmental Chemistry and Bioanalytics, P.O. Box 32, H-1518, Budapest (Hungary)

    2011-03-15

    X-ray absorption near edge structure (XANES) analysis in combination with synchrotron radiation induced total reflection X-ray fluorescence (SR-TXRF) acquisition was used to determine the oxidation state of Fe in human cancer cells and simultaneously their elemental composition by applying a simple sample preparation procedure consisting of pipetting the cell suspension onto the quartz reflectors. XANES spectra of several inorganic and organic iron compounds were recorded and compared to that of different cell lines. The XANES spectra of cells, independently from the phase of cell growth and cell type were very similar to that of ferritin, the main Fe store within the cell. The spectra obtained after CoCl{sub 2} or NiCl{sub 2} treatment, which could mimic a hypoxic state of cells, did not differ noticeably from that of the ferritin standard. After 5-fluorouracil administration, which could also induce an oxidative-stress in cells, the absorption edge position was shifted toward higher energies representing a higher oxidation state of Fe. Intense treatment with antimycin A, which inhibits electron transfer in the respiratory chain, resulted in minor changes in the spectrum, resembling rather the N-donor Fe-{alpha},{alpha}'-dipyridyl complex at the oxidation energy of Fe(III), than ferritin. The incorporation of Co and Ni in the cells was followed by SR-TXRF measurements.

  19. Role of Mitochondrial Electron Transport Chain Dysfunction in Cr(VI-Induced Cytotoxicity in L-02 Hepatocytes

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    Fang Xiao

    2014-04-01

    Full Text Available Background: Hexavalent chromium [Cr(VI] and its compounds (e.g. chromates, which are extensively used in numerous industrial processes including leather tanning and steel manufacturing, are considered as priority pollutants. There is growing evidence supporting that Cr(VI could be a human carcinogen that induces primary liver cancer after oral exposure, and this sheds light on the importance of the investigation of Cr(VI-induced hepatotoxicity. Although it is known that mitochondria are major targets for heavy metals, the mechanisms of electron transfer chain (ETC dysfunction involved in Cr(VI-induced cytotoxicity are unclear. Methods: In the present study, by using mitochondrial respiratory chain complex (MRCC I inhibitor rotenone (ROT and its substrates glutamate/malate (Glu/Mal, MRCC III inhibitor antimycin A (AA and its substrate coenzyme Q (CoQ, and the antioxidant Vitamin C (Vit C, we aimed to elucidate the role of mitochondrial ETC dysfunction in Cr(VI-induced cytotoxicity. Results: We found that Cr(VI targeted and inhibited MRCC I and III to induce ETC dysfunction, which played an important role in Cr(VI-induced cytotoxicity. Conclusion: Our current data provides novel important insight into the mechanisms of mitochondrial ETC dysfunction in Cr(VI-induced cytotoxicity in the hepatocytes, and we will be poised to develop new methods in the prevention and treatment of liver diseases involving mitochondrial ETC dysfunction for the occupational exposure population.

  20. A broad distribution of the alternative oxidase in microsporidian parasites.

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    Bryony A P Williams

    2010-02-01

    Full Text Available Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of iron-sulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX, a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1 as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosomes.

  1. Endocytosis of heat-denatured albumin by cultured rat Kupffer cells

    International Nuclear Information System (INIS)

    Brouwer, A.; Knook, D.L.

    1982-01-01

    Purified Kupffer cells were obtained by centrifugal elutriation of sinusoidal cells isolated by pronase treatment of the rat liver. The endocytosis of radioactively labeled heat-aggregated colloidal albumin (CA 125 I) was investigated in maintenance cultures of the purified Kupffer cells. The endocytic capacity of the cells was studied during 4 days of culture. Maximum uptake was observed after 24 hr of culture, with a gradual decline during the following days. When the uptake was measured after incubation with increasing concentrations of CA 125 I, a saturation effect was observed. This finding and the observed high rate of uptake are strong indications that receptor sites on the cell membrane are involved in the mechanism of endocytosis. The uptake of CA 125 I by Kupffer cells was inhibited by the metabolic inhibitors fluoride and antimycin A, indicating that endocytosis of CA 125 I is dependent on energy derived from both glycolysis and mitochondrial respiration. The mechanism of internalization may also require the action of microfilaments as well as intact microtubules, since both cytochalasin B and colchicine inhibited the uptake of CA 125 I. The intracellular degradation of CA 125 I by Kupffer cells was strongly inhibited by chloroquine but not by colchicine. The degradation of ingested CA 125 I occurred within the Kupffer cell lysosomes

  2. GENE TRANSFER IN TOBACCO MITOCHONDRIA IN VITRO AND IN VIVO

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    Katyshev A.I.

    2012-08-01

    -cob* genetic construct with integrative properties. It contains the selective gene and the gene of interest under control of the 5'-regulatory regions of Arabidopsis orf262 gene and the tobacco cob gene. We used modified variant of the tobacco apocytochrome b gene as a gene for selection with the nucleotide substitution G128T (G43V which results in antimycin A resistance. The maize sod3.1 gene was used as a gene of interest. The construct was delivered into tobacco callus cells and leaf disks by biolistic method. The callus lines demonstrating the high growth rates in the presence of antimycin A in comparison with the non-transformed control lines were selected. PCR analysis of transformed callus lines revealed the presence of heterologous maize sod3.1 sequence and the integration of the construct elements in tobacco mitochondrial genome.

  3. Quinol-cytochrome c Oxidoreductase and Cytochrome c4 Mediate Electron Transfer during Selenate Respiration in Thauera selenatis*

    Science.gov (United States)

    Lowe, Elisabeth C.; Bydder, Sarah; Hartshorne, Robert S.; Tape, Hannah L. U.; Dridge, Elizabeth J.; Debieux, Charles M.; Paszkiewicz, Konrad; Singleton, Ian; Lewis, Richard J.; Santini, Joanne M.; Richardson, David J.; Butler, Clive S.

    2010-01-01

    Selenate reductase (SER) from Thauera selenatis is a periplasmic enzyme that has been classified as a type II molybdoenzyme. The enzyme comprises three subunits SerABC, where SerC is an unusual b-heme cytochrome. In the present work the spectropotentiometric characterization of the SerC component and the identification of redox partners to SER are reported. The mid-point redox potential of the b-heme was determined by optical titration (Em + 234 ± 10 mV). A profile of periplasmic c-type cytochromes expressed in T. selenatis under selenate respiring conditions was undertaken. Two c-type cytochromes were purified (∼24 and ∼6 kDa), and the 24-kDa protein (cytc-Ts4) was shown to donate electrons to SerABC in vitro. Protein sequence of cytc-Ts4 was obtained by N-terminal sequencing and liquid chromatography-tandem mass spectrometry analysis, and based upon sequence similarities, was assigned as a member of cytochrome c4 family. Redox potentiometry, combined with UV-visible spectroscopy, showed that cytc-Ts4 is a diheme cytochrome with a redox potential of +282 ± 10 mV, and both hemes are predicted to have His-Met ligation. To identify the membrane-bound electron donors to cytc-Ts4, growth of T. selenatis in the presence of respiratory inhibitors was monitored. The specific quinol-cytochrome c oxidoreductase (QCR) inhibitors myxothiazol and antimycin A partially inhibited selenate respiration, demonstrating that some electron flux is via the QCR. Electron transfer via a QCR and a diheme cytochrome c4 is a novel route for a member of the DMSO reductase family of molybdoenzymes. PMID:20388716

  4. NQO1-dependent redox cycling of idebenone: effects on cellular redox potential and energy levels.

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    Roman H Haefeli

    Full Text Available Short-chain quinones are described as potent antioxidants and in the case of idebenone have already been under clinical investigation for the treatment of neuromuscular disorders. Due to their analogy to coenzyme Q10 (CoQ10, a long-chain quinone, they are widely regarded as a substitute for CoQ10. However, apart from their antioxidant function, this provides no clear rationale for their use in disorders with normal CoQ10 levels. Using recombinant NAD(PH:quinone oxidoreductase (NQO enzymes, we observed that contrary to CoQ10 short-chain quinones such as idebenone are good substrates for both NQO1 and NQO2. Furthermore, the reduction of short-chain quinones by NQOs enabled an antimycin A-sensitive transfer of electrons from cytosolic NAD(PH to the mitochondrial respiratory chain in both human hepatoma cells (HepG2 and freshly isolated mouse hepatocytes. Consistent with the substrate selectivity of NQOs, both idebenone and CoQ1, but not CoQ10, partially restored cellular ATP levels under conditions of impaired complex I function. The observed cytosolic-mitochondrial shuttling of idebenone and CoQ1 was also associated with reduced lactate production by cybrid cells from mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS patients. Thus, the observed activities separate the effectiveness of short-chain quinones from the related long-chain CoQ10 and provide the rationale for the use of short-chain quinones such as idebenone for the treatment of mitochondrial disorders.

  5. 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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  7. Mitochondrial function is involved in regulation of cholesterol efflux to apolipoprotein (apoA-I from murine RAW 264.7 macrophages

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    Allen Anne Marie

    2012-12-01

    Full Text Available Abstract Background Mitochondrial DNA damage, increased production of reactive oxygen species and progressive respiratory chain dysfunction, together with increased deposition of cholesterol and cholesteryl esters, are hallmarks of atherosclerosis. This study investigated the role of mitochondrial function in regulation of macrophage cholesterol efflux to apolipoprotein A-I, by the addition of established pharmacological modulators of mitochondrial function. Methods Murine RAW 264.7 macrophages were treated with a range of concentrations of resveratrol, antimycin, dinitrophenol, nigericin and oligomycin, and changes in viability, cytotoxicity, membrane potential and ATP, compared with efflux of [3H]cholesterol to apolipoprotein (apo A-I. The effect of oligomycin treatment on expression of genes implicated in macrophage cholesterol homeostasis were determined by quantitative polymerase chain reaction, and immunoblotting, relative to the housekeeping enzyme, Gapdh, and combined with studies of this molecule on cholesterol esterification, de novo lipid biosynthesis, and induction of apoptosis. Significant differences were determined using analysis of variance, and Dunnett’s or Bonferroni post t-tests, as appropriate. Results The positive control, resveratrol (24 h, significantly enhanced cholesterol efflux to apoA-I at concentrations ≥30 μM. By contrast, cholesterol efflux to apoA-I was significantly inhibited by nigericin (45%; ppAbca1 mRNA. Oligomycin treatment did not affect cholesterol biosynthesis, but significantly inhibited cholesterol esterification following exposure to acetylated LDL, and induced apoptosis at ≥30 μM. Finally, oligomycin induced the expression of genes implicated in both cholesterol efflux (Abca1, Abcg4, Stard1 and cholesterol biosynthesis (Hmgr, Mvk, Scap, Srebf2, indicating profound dysregulation of cholesterol homeostasis. Conclusions Acute loss of mitochondrial function, and in particular Δψm, reduces

  8. 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.

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    Marcus Wallgren

    Full Text Available 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.

  9. Mitochondrial Complexes I and II Are More Susceptible to Autophagy Deficiency in Mouse β-Cells

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    Min Joo Kim

    2015-03-01

    Full Text Available BackgroundDamaged mitochondria are removed by autophagy. Therefore, impairment of autophagy induces the accumulation of damaged mitochondria and mitochondrial dysfunction in most mammalian cells. Here, we investigated mitochondrial function and the expression of mitochondrial complexes in autophagy-related 7 (Atg7-deficient β-cells.MethodsTo evaluate the effect of autophagy deficiency on mitochondrial function in pancreatic β-cells, we isolated islets from Atg7F/F:RIP-Cre+ mice and wild-type littermates. Oxygen consumption rate and intracellular adenosine 5'-triphosphate (ATP content were measured. The expression of mitochondrial complex genes in Atg7-deficient islets and in β-TC6 cells transfected with siAtg7 was measured by quantitative real-time polymerase chain reaction.ResultsBaseline oxygen consumption rate of Atg7-deficient islets was significantly lower than that of control islets (P<0.05. Intracellular ATP content of Atg7-deficient islets during glucose stimulation was also significantly lower than that of control islets (P<0.05. By Oxygraph-2k analysis, mitochondrial respiration in Atg7-deficient islets was significantly decreased overall, although state 3 respiration and responses to antimycin A were unaffected. The mRNA levels of mitochondrial complexes I, II, III, and V in Atg7-deficient islets were significantly lower than in control islets (P<0.05. Down-regulation of Atg7 in β-TC6 cells also reduced the expression of complexes I and II, with marginal significance (P<0.1.ConclusionImpairment of autophagy in pancreatic β-cells suppressed the expression of some mitochondrial respiratory complexes, and may contribute to mitochondrial dysfunction. Among the complexes, I and II seem to be most vulnerable to autophagy deficiency.

  10. Modulation of intracellular calcium waves and triggered activities by mitochondrial ca flux in mouse cardiomyocytes.

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    Zhenghang Zhao

    Full Text Available Recent studies have suggested that mitochondria may play important roles in the Ca(2+ homeostasis of cardiac myocytes. However, it is still unclear if mitochondrial Ca(2+ flux can regulate the generation of Ca(2+ waves (CaWs and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca(2+ (Cai (2+ was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR Ca(2+ release and CaWs were induced in the presence of high (4 mM external Ca(2+ (Cao (2+. The protonophore carbonyl cyanide p-(trifluoromethoxyphenylhydrazone (FCCP reversibly raised basal Cai (2+ levels even after depletion of SR Ca(2+ in the absence of Cao (2+ , suggesting Ca(2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψ m and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψ m or Ru360 (a mitochondrial Ca(2+ uniporter inhibitor, but not by oligomycin (an ATP synthase inhibitor or iodoacetic acid (a glycolytic inhibitor, excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca(2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca(2+ release and uptake exquisitely control the local Ca(2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis.

  11. Xylose uptake by the ruminal bacterium Selenomonas ruminantium.

    Science.gov (United States)

    Williams, D K; Martin, S A

    1990-01-01

    Selenomonas ruminantium HD4 does not use the phosphoenolpyruvate phosphotransferase system to transport xylose (S. A. Martin and J. B. Russell, J. Gen. Microbiol. 134:819-827, 1988). Xylose uptake by whole cells of S. ruminantium HD4 was inducible. Uptake was unaffected by monensin or lasalocid, while oxygen, o-phenanthroline, and HgCl2 were potent inhibitors. Menadione, antimycin A, and KCN had little effect on uptake, and acriflavine inhibited uptake by 23%. Sodium fluoride decreased xylose uptake by 10%, while N,N'-dicyclohexylcarbodiimide decreased uptake by 31%. Sodium arsenate was a strong inhibitor (83%), and these results suggest the involvement of a high-energy phosphate compound and possibly a binding protein in xylose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and SF6847 inhibited xylose uptake by 88, 82, and 43%, respectively. The cations Na+ and K+ did not stimulate xylose uptake. The kinetics of xylose uptake were nonlinear, and it appeared that more than one uptake mechanism may be involved or that two proteins (i.e., a binding protein and permease protein) with different affinities for xylose were present. Excess (10 mM) glucose, sucrose, or maltose decreased xylose uptake less than 40%. Uptake was unaffected at extracellular pH values between 6.0 and 8.0, while pH values of 5.0 and 4.0 decreased uptake 28 and 24%, respectively. The phenolic monomers p-coumaric acid and vanillin inhibited growth on xylose and xylose uptake more than ferulic acid did. The predominant end products resulting from the fermentation of xylose were lactate (7.5 mM), acetate (4.4 mM), and propionate (5.1 nM), and the Yxylose was 24.1 g/mol. PMID:2383009

  12. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

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    Jakob D Wikstrom

    Full Text Available The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets.The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets.The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  13. The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation.

    Science.gov (United States)

    Beavis, A D; Lehninger, A L

    1986-07-15

    Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.

  14. Molecular and functional characterization of two pyruvate decarboxylase genes, PDC1 and PDC5, in the thermotolerant yeast Kluyveromyces marxianus.

    Science.gov (United States)

    Choo, Jin Ho; Han, Changpyo; Lee, Dong Wook; Sim, Gyu Hun; Moon, Hye Yun; Kim, Jae-Young; Song, Ji-Yoon; Kang, Hyun Ah

    2018-04-01

    Pyruvate decarboxylase (Pdc) is a cytosolic enzyme located at the branch point between fermentative and respiratory sugar catabolism. Here, we identified and functionally characterized KmPDC1 and KmPDC5 encoding two homologs of Pdc in the thermotolerant yeast Kluyveromyces marxianus KCTC 17555. Despite the conservation of important Pdc domains, a few amino acid sequences essential for enzymatic activity are not conserved in KmPdc5p. Deletion of KmPDC1 alone eliminated most of Pdc activity, but the growth of the Kmpdc1Δ strain on glucose was comparable to that of the wild type (WT) strain under aerobic conditions. In contrast to the WT, Kmpdc1Δ could not grow on glucose under oxygen-limited conditions. The KmPDC5 deletion did not generate any apparent change in Pdc activity or growth patterns under several tested conditions. Whereas the expression of KmPDC1 was enhanced by glucose, the basic expression levels of KmPDC5 were very low, without a detectable difference between glucose and nonfermentable carbon sources. Moreover, KmPDC5 overexpression was unable to complement the growth defect of Kmpdc1Δ in the presence of antimycin A, and the purified recombinant KmPdc5p was inactive in Pdc activity assay, supporting the notion that KmPdc5p may lack Pdc enzymatic activity. Notably, compared to the WT, Kmpdc1Δ single and Kmpdc1Δpdc5Δ double mutants produced significantly less glycerol, acetate, and ethanol while accumulating pyruvate. Altogether, our data indicate that a single deletion of KmPDC1 is sufficient in Crabtree-negative K. marxianus strains to generate a starting host strain for engineering of production of high-value biomaterials derived from pyruvate without byproduct formation.

  15. The metabolism of malate by cultured rat brain astrocytes

    Energy Technology Data Exchange (ETDEWEB)

    McKenna, M.C.; Tildon, J.T.; Couto, R.; Stevenson, J.H.; Caprio, F.J. (Department of Pediatrics, University of Maryland School of Medicine, Baltimore (USA))

    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 biphasic 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.

  16. Effects of Ca2+, Mg2+, and depolarizing agents, on the 32Pi-labeling and degradation of phosphatidylinositols in rat brain synaptosomes.

    Science.gov (United States)

    Marinetti, G V; Morris, T W; Leaky, P

    1993-03-01

    In isolated synaptosomes from rat brain, 100 microM antimycin A and 10 microM oxamic acid inhibit the 32Pi-labeling of phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylinositol-4-phosphate (PIP) by 90% and 95-99% respectively. 10 mM sodium fluoride inhibits the labeling by 50-60% and 10 mM A23187 inhibits the labeling by 63-70%. Phospholipase A2 inhibits the labeling of PIP2 and PIP by 93-94% and stimulates their degradation by 84-92%. Depolarization of synaptosomes with 75 mM K+ or 100 microM veratrine decreases the labeling of PIP2 and PIP by 66-74%. The decreased labeling results in large part from the Ca(2+)-dependent degradation of 32P-labeled PIP2 and PIP as shown by pulse-chase experiments in which PIP2 and PIP were prelabeled with 32Pi. Depolarization of synaptosomes results in the stimulation of 45Ca2+ uptake with the concomitant hydrolysis of PIP and PIP2. Addition of 1 mM Ca2+ accounts for 25% of the enhanced degradation whereas depolarization with 75 mM K+ accounts for 75% of the enhanced degradation of PIP2 and PIP. Depolarization with 100 mM veratrine results in a 223% increase in inositol trisphosphate as evidenced by stimulation of 45Ca2+ uptake. EGTA (10 mM) and Mg2+ (5-10 mM) inhibit the degradation of PIP and PIP2 and counteract the action of 1 mM Ca2+. Our data demonstrate that 45Ca2+, Mg2+, and membrane depolarization play an important role in the turnover of membrane phosphatidylinositols.

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

    Science.gov (United States)

    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.

  18. Kinetics of Molybdenum Reduction to Molybdenum Blue by Bacillus sp. Strain A.rzi

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    A. R. Othman

    2013-01-01

    Full Text Available 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 pmax, Ks, Sm, 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.

  19. Similar Transition States Mediate the Q-cycle and Superoxide Production by the Cytochrome bc1 Complex

    International Nuclear Information System (INIS)

    Forquer, Isaac P.; Covian, Raul; Bowman, Michael K.; Trumpower, Bernard; Kramer, David M.

    2006-01-01

    The cytochrome bc complexes found in mitochondria, chloroplasts and many bacteria catalyze a critical reaction in their respective electron transport chains. The quinol oxidase (Qo) site in this complex oxidizes a hydroquinone (quinol), reducing two one-electron carriers, a low-potential cytochrome b heme and a ''Rieske'' iron-sulfur cluster. The overall electron transfer reactions are coupled to transmembrane translocation of protons via a ''Q-cycle'' mechanism, which generates proton motive force for ATP synthesis. Since semiquinone intermediates of quinol oxidation are generally highly reactive, one of the key questions in this field is: how does the Qo site oxidize quinol without the production of deleterious side reactions including superoxide production? We attempt to test three possible general models to account for this behavior: (1) The Qo site semiquinone (or quinol:imidazolate complex) is unstable and thus occurs at a very low steady-state concentration, limiting O2 reduction; (2) the Qo site semiquinone is highly stabilized making it unreactive towards oxygen; and (3) the Qo site catalyzes a quantum mechanically-coupled two-electron/two proton transfer without a semiquinone intermediate. Enthalpies of activation were found to be almost identical between the uninhibited Q-cycle and superoxide production in the presence of Antimycin A in wild type. This behavior was also preserved in a series of mutants with altered driving forces for quinol oxidation. Overall, the data supports models where the rate-limiting step for both Q-cycle and superoxide production are essentially identical, consistent with model 1 but requiring modifications to models 2 and 3

  20. Endoplasmic Reticulum Stress-Induced Autophagy Provides Cytoprotection from Chemical Hypoxia and Oxidant Injury and Ameliorates Renal Ischemia-Reperfusion Injury.

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    Bhavya B Chandrika

    Full Text Available We examined whether endoplasmic reticulum (ER stress-induced autophagy provides cytoprotection from renal tubular epithelial cell injury due to oxidants and chemical hypoxia in vitro, as well as from ischemia-reperfusion (IR injury in vivo. We demonstrate that the ER stress inducer tunicamycin triggers an unfolded protein response, upregulates ER chaperone Grp78, and activates the autophagy pathway in renal tubular epithelial cells in culture. Inhibition of ER stress-induced autophagy accelerated caspase-3 activation and cell death suggesting a pro-survival role of ER stress-induced autophagy. Compared to wild-type cells, autophagy-deficient MEFs subjected to ER stress had enhanced caspase-3 activation and cell death, a finding that further supports the cytoprotective role of ER stress-induced autophagy. Induction of autophagy by ER stress markedly afforded cytoprotection from oxidants H2O2 and tert-Butyl hydroperoxide and from chemical hypoxia induced by antimycin A. In contrast, inhibition of ER stress-induced autophagy or autophagy-deficient cells markedly enhanced cell death in response to oxidant injury and chemical hypoxia. In mouse kidney, similarly to renal epithelial cells in culture, tunicamycin triggered ER stress, markedly upregulated Grp78, and activated autophagy without impairing the autophagic flux. In addition, ER stress-induced autophagy markedly ameliorated renal IR injury as evident from significant improvement in renal function and histology. Inhibition of autophagy by chloroquine markedly increased renal IR injury. These studies highlight beneficial impact of ER stress-induced autophagy in renal ischemia-reperfusion injury both in vitro and in vivo.

  1. Sustained Submicromolar H2O2 Levels Induce Hepcidin via Signal Transducer and Activator of Transcription 3 (STAT3)*

    Science.gov (United States)

    Millonig, Gunda; Ganzleben, Ingo; Peccerella, Teresa; Casanovas, Guillem; Brodziak-Jarosz, Lidia; Breitkopf-Heinlein, Katja; Dick, Tobias P.; Seitz, Helmut-Karl; Muckenthaler, Martina U.; Mueller, Sebastian

    2012-01-01

    The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H2O2 (0.3–6 μm), concentrations that are comparable with levels of H2O2 released by inflammatory cells. In contrast, bolus treatment of H2O2 has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 μm. H2O2 treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H2O2-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H2O2 responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H2O2 released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H2O2. Thus, similar to cytokines, H2O2 provides an important regulatory link between inflammation and iron metabolism. PMID:22932892

  2. Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle.

    Science.gov (United States)

    Taddeo, E P; Laker, R C; Breen, D S; Akhtar, Y N; Kenwood, B M; Liao, J A; Zhang, M; Fazakerley, D J; Tomsig, J L; Harris, T E; Keller, S R; Chow, J D; Lynch, K R; Chokki, M; Molkentin, J D; Turner, N; James, D E; Yan, Z; Hoehn, K L

    2014-04-01

    Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.

  3. Mitochondrial Superoxide Generation Enhances P2X7R-mediated Loss of Cell Surface CD62L on Naïve Human CD4+ T Lymphocytes1

    Science.gov (United States)

    Foster, John G.; Carter, Edward; Kilty, Iain; MacKenzie, Amanda B.; Ward, Stephen G.

    2013-01-01

    Migration of naïve CD4+ T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule CD62L, critical for this process, is highly expressed on naïve CD4+ T lymphocytes and is down-regulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naïve CD4+ T lymphocytes and show functional channel activity in whole cell patch clamp recordings. CD62L down-regulation occurs rapidly in response to extracellular ATP, a process which is blocked by selective antagonists of P2X7R. This loss of surface CD62L expression was not associated with externalization of phosphatidyl serine. While investigating the mechanisms for this process we revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. Enhanced superoxide generation in the mitochondria of Rotenone and Antimycin A treated cells was observed and may contribute to the enhanced sensitivity of P2X7R to ATP. P2X7R dependent exposure of phosphatidylserine was also revealed by pre-incubation with mitochondrial un-couplers prior to ATP treatment. This may present a novel mechanism whereby P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. The clearance of apoptotic cells may therefore be enhanced by this mechanism which requires functional P2X7R expression. PMID:23319734

  4. Mitochondrial superoxide generation enhances P2X7R-mediated loss of cell surface CD62L on naive human CD4+ T lymphocytes.

    Science.gov (United States)

    Foster, John G; Carter, Edward; Kilty, Iain; MacKenzie, Amanda B; Ward, Stephen G

    2013-02-15

    Migration of naive CD4(+) T lymphocytes into lymphoid tissue is essential for their activation and subsequent roles in adaptive immunity. The adhesion molecule L-selectin (CD62L), critical for this process, is highly expressed on naive CD4(+) T lymphocytes and is downregulated upon T lymphocyte activation. We demonstrate protein expression of P2X7R on naive CD4(+) T lymphocytes and show functional channel activity in whole-cell patch clamp recordings. CD62L downregulation occurs rapidly in response to extracellular ATP, a process that is blocked by selective antagonists of P2X7R. This loss of surface CD62L expression was not associated with externalization of phosphatidylserine. While investigating the mechanisms for this process, we revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R-dependent CD62L downregulation by increasing ATP potency. Enhanced superoxide generation in the mitochondria of rotenone- and antimycin A-treated cells was observed and may contribute to the enhanced sensitivity of P2X7R to ATP. P2X7R-dependent exposure of phosphatidylserine was also revealed by preincubation with mitochondrial uncouplers prior to ATP treatment. This may present a novel mechanism whereby P2X7R-dependent phosphatidylserine exposure occurs only when cells have enhanced mitochondrial reactive oxygen species generation. The clearance of apoptotic cells may therefore be enhanced by this mechanism which requires functional P2X7R expression.

  5. Reactive oxygen species in unstimulated hemocytes of the pacific oyster Crassostrea gigas: a mitochondrial involvement.

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    Ludovic Donaghy

    Full Text Available The Pacific oyster Crassostrea gigas is a sessile bivalve mollusc whose homeostasis relies, at least partially, upon cells circulating in hemolymph and referred to as hemocytes. Oyster's hemocytes have been reported to produce reactive oxygen species (ROS, even in absence of stimulation. Although ROS production in bivalve molluscs is mostly studied for its defence involvement, ROS may also be involved in cellular and tissue homeostasis. ROS sources have not yet been described in oyster hemocytes. The objective of the present work was to characterize the ROS sources in unstimulated hemocytes. We studied the effects of chemical inhibitors on the ROS production and the mitochondrial membrane potential (Δψ(m of hemocytes. First, this work confirmed the specificity of JC-10 probe to measure Δψ(m in oyster hemocytes, without being affected by ΔpH, as reported in mammalian cells. Second, results show that ROS production in unstimulated hemocytes does not originate from cytoplasmic NADPH-oxidase, nitric oxide synthase or myeloperoxidase, but from mitochondria. In contrast to mammalian cells, incubation of hemocytes with rotenone (complex I inhibitor had no effect on ROS production. Incubation with antimycin A (complex III inhibitor resulted in a dose-dependent ROS production decrease while an over-production is usually reported in vertebrates. In hemocytes of C. gigas, the production of ROS seems similarly dependent on both Δψ(m and ΔpH. These findings point out differences between mammalian models and bivalve cells, which warrant further investigation about the fine characterization of the electron transfer chain and the respective involvement of mitochondrial complexes in ROS production in hemocytes of bivalve molluscs.

  6. Cytotoxic mechanisms of Zn2+ and Cd2+ involve Na+/H+ exchanger (NHE) activation by ROS

    International Nuclear Information System (INIS)

    Koutsogiannaki, Sophia; Evangelinos, Nikolaos; Koliakos, George; Kaloyianni, Martha

    2006-01-01

    The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in ·O 2 - production, with Cd to be more potent (216 ± 15%) than Zn (150 ± 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na + /H + exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on ·O 2 - production was mediated via the interaction of metal ions with α 1 - and β-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi (ΔpHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi (ΔpHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O 2 - production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-γ receptors. In addition, differences between the two metals concerning NHE activation, O 2 - production and interaction with adrenergic receptors were observed

  7. Physiological Levels of Nitric Oxide Diminish Mitochondrial Superoxide. Potential Role of Mitochondrial Dinitrosyl Iron Complexes and Nitrosothiols

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    Sergey I. Dikalov

    2017-11-01

    Full Text Available Mitochondria are the major source of superoxide radicals and superoxide overproduction contributes to cardiovascular diseases and metabolic disorders. Endothelial dysfunction and diminished nitric oxide levels are early steps in the development of these pathological conditions. It is known that physiological production of nitric oxide reduces oxidative stress and inflammation, however, the precise mechanism of “antioxidant” effect of nitric oxide is not clear. In this work we tested the hypothesis that physiological levels of nitric oxide diminish mitochondrial superoxide production without inhibition of mitochondrial respiration. In order to test this hypothesis we analyzed effect of low physiological fluxes of nitric oxide (20 nM/min on superoxide and hydrogen peroxide production by ESR spin probes and Amplex Red in isolated rat brain mitochondria. Indeed, low levels of nitric oxide substantially attenuated both basal and antimycin A-stimulated production of reactive oxygen species in the presence of succinate or glutamate/malate as mitochondrial substrates. Furthermore, slow releasing NO donor DPTA-NONOate (100 μM did not change oxygen consumption in State 4 and State 3. However, the NO-donor strongly inhibited oxygen consumption in the presence of uncoupling agent CCCP, which is likely associated with inhibition of the over-reduced complex IV in uncoupled mitochondria. We have examined accumulation of dinitrosyl iron complexes and nitrosothiols in mitochondria treated with fast-releasing NO donor MAHMA NONOate (10 μM for 30 min until complete release of NO. Following treatment with NO donor, mitochondria were frozen for direct detection of dinitrosyl iron complexes using Electron Spin Resonance (ESR while accumulation of nitrosothiols was measured by ferrous-N-Methyl-D-glucamine dithiocarbamate complex, Fe(MGD2, in lysed mitochondria. Treatment of mitochondria with NO-donor gave rise to ESR signal of dinitrosyl iron complexes while ESR

  8. Effect of artificial and natural phospholipid membranes on rate of sperm whale oxymyoglobin autooxidation.

    Science.gov (United States)

    Postnikova, G B; Shekhovtsova, E A

    2013-03-01

    We were the first to show that MbO2 deoxygenation in the cell occurs only upon interaction of myoglobin with mitochondrial membrane, which must be accompanied by changes in the heme cavity conformation of the protein and its affinity for the ligand. Under aerobic conditions, some changes in the equilibrium O2 dissociation constant (Kdis) can be detected by changes of the rate of MbO2 autooxidation, i.e. spontaneous turning it into metMb (kox), as far as a direct correlation between Kdis and kox is experimentally shown. In this work, we studied the effect on MbO2 autooxidation rate of phospholipid liposomes from neutral soybean phosphatidylcholine (lecithin) and from negatively charged 1-palmitoyl-2-oleylphosphatidylglycerol (POPG) at various phospholipid/MbO2 ratios from 25 : 1 to 100 : 1, and also the effect of rat liver mitochondria at concentration of 1 and 2 mg/ml mitochondrial protein (at 22 and 37°C). In all cases, kox was found to increase due to interaction of the protein with phospholipid membranes. The effect of negatively charged liposomes from POPG on kox is significantly greater than that of neutral lecithin liposomes. At the POPG/MbO2 molar ratio of 25 : 1, MbO2 autooxidation rate is almost 25-fold increased compared to the control, whereas in the presence of 50-fold molar excess of lecithin, kox is only ~10 times higher (10 mM buffer, pH 7.2, 22°C). With the same phospholipid/MbO2 ratio of 100 : 1, kox is 7 times higher for the POPG than for lecithin liposomes. In the presence of mitochondria inhibited by antimycin A, kox grows proportionally to their concentration (about 10-fold per 1 mg/ml of mitochondrial protein), and practically does not change after adding superoxide dismutase in the reaction mixture. The kox value decreases markedly at high ionic strength, thus suggesting an important role of coulombic electrostatics in the myoglobin-mitochondrial interaction. The increase in the autooxidation rate of MbO2 (and hence its Kdis) due to the

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

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    Dionizio Ramos-Filho

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  11. [Organ transplantation. One of the biochemical reasons for the race against the clock].

    Science.gov (United States)

    Zénatti, M; Emeric-Blanchouin, N; Bitker, M O; Barrou, B; Lugagne, P M; Thallier, M; Aupetit-Faisant, B

    1991-11-27

    The authors have studied the subcellular functioning of human adrenal glands removed from subjects in a stage IV coma. The present study has a two-fold interest: on the one hand, it offers biochemical information on a key element in the intermediate metabolism (namely, the mitochondrial energetic metabolism, occurring in a fragile tissue which, under a state of shock, is primarily affected; the results obtained on such type of tissue may therefore be inferred to other organs); on the other hand, it allows a wider approach of the adrenal biochemical mechanisms during a stage IV coma. The mitochondrial fraction was obtained by differential centrifugation carried out immediately after organ removal. The steroid synthesis, studied using radioactive precursors, turned out to be similar to that found in other mammals. Respiratory characteristics, determined by polarography with a Clark oxygen electrode, at 37 degrees C, were satisfactory: respiratory intensity was 77.25 +/- 12.16 nanomoles O2/min/mg mitochondrial protein in the presence of succinate 15 mM and respiratory control was 1.93 +/- 0.15 in the presence of ADP 37 microM. The respiratory chain functioned in a classical manner: rotenone 25 microM did not inhibit respiration in the presence of succinate 15 mM, while it did with L-malate 15 mM. In the presence of succinate 15 mM, the respiratory intensity was inhibited at 87.4 percent and 76.7 percent by KCN 0.01 microM and antimycin A 0.09 microM respectively; with DNP 85 microM, it was multiplied by 5. However, the value of the P/O ratio was low (0.24 +/- 0.04). Under the present conditions, this may highlight the difficulty to synthetize ATP whenever neither the functioning nor the regulation of the multi-enzymatic complex accounting for oxygen consumption are affected. This result clearly confirms that the shortest possible delay between organ removal and transplantation is crucial, as the renewal of cell structures requires energy. These fundamental research

  12. Hearts deficient in both Mfn1 and Mfn2 are protected against acute myocardial infarction.

    Science.gov (United States)

    Hall, A R; Burke, N; Dongworth, R K; Kalkhoran, S B; Dyson, A; Vicencio, J M; Dorn, G W; Yellon, D M; Hausenloy, D J

    2016-05-26

    Mitochondria alter their shape by undergoing cycles of fusion and fission. Changes in mitochondrial morphology impact on the cellular response to stress, and their interactions with other organelles such as the sarcoplasmic reticulum (SR). Inhibiting mitochondrial fission can protect the heart against acute ischemia/reperfusion (I/R) injury. However, the role of the mitochondrial fusion proteins, Mfn1 and Mfn2, in the response of the adult heart to acute I/R injury is not clear, and is investigated in this study. To determine the effect of combined Mfn1/Mfn2 ablation on the susceptibility to acute myocardial I/R injury, cardiac-specific ablation of both Mfn1 and Mfn2 (DKO) was initiated in mice aged 4-6 weeks, leading to knockout of both these proteins in 8-10-week-old animals. This resulted in fragmented mitochondria (electron microscopy), decreased mitochondrial respiratory function (respirometry), and impaired myocardial contractile function (echocardiography). In DKO mice subjected to in vivo regional myocardial ischemia (30 min) followed by 24 h reperfusion, myocardial infarct size (IS, expressed as a % of the area-at-risk) was reduced by 46% compared with wild-type (WT) hearts. In addition, mitochondria from DKO animals had decreased MPTP opening susceptibility (assessed by Ca(2+)-induced mitochondrial swelling), compared with WT hearts. Mfn2 is a key mediator of mitochondrial/SR tethering, and accordingly, the loss of Mfn2 in DKO hearts reduced the number of interactions measured between these organelles (quantified by proximal ligation assay), attenuated mitochondrial calcium overload (Rhod2 confocal microscopy), and decreased reactive oxygen species production (DCF confocal microscopy) in response to acute I/R injury. No differences in isolated mitochondrial ROS emissions (Amplex Red) were detected in response to Ca(2+) and Antimycin A, further implicating disruption of mitochondria/SR tethering as the protective mechanism. In summary, despite apparent

  13. 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).

    Science.gov (United States)

    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

  14. 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.

    Science.gov (United States)

    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

  15. The electron transport system of the anaerobic Propionibacterium shermanii: cytochrome and inhibitor studies.

    Science.gov (United States)

    Schwartz, A C; Sporkenbach, J

    1975-03-10

    1. Electron transport particles obtained from cell-free extracts of Propionibacterium shermanii by centrifugation at 105000 times g for 3 hrs oxidized NADH, D,L-lactate, L-glycerol-3-phosphate and succinate with oxygen and, except for succinate, with fumarate, too. 2. Spectral investigation of the electron transport particles revealed the presence of cytochromes b, d and o, and traces of cytochrome alpha1 and a c-type cytochrome. Cytochrome b was reduced by succinate to about 50%, and by NADH, lactate or glycerol-3-phosphate to 80--90%. 3. The inhibitory effects of amytal and rotenone on NADH oxidation, but not on the oxidation of the other substrates, indicated the presence of the NADH dehydrogenase complex, or "site I region", in the electron transport system of P. shermanii. 4. NQNO inhibited substrate oxidations by oxygen and fumarate, as well as equilibration of the flavoproteins of the substrate dehydrogenases by way of menaquinone. The inhibition occurred at low concentrations of the inhibitor and reached 80--100%, depending on the substrate tested. The site of inhibition of the respiratory activity was located between menaquinone and cytochrome b. In addition, inhibition of flavoprotein equilibration suggested that NQNO acted upon the electron transfer directed from menaquinol towards the acceptor to be reduced, either cytochrome b or the flavoproteins, which would include fumarate reductase. 5. In NQNO-inhibited particles, cytochrome b was not oxidized by oxygen-free fumarate, but readily oxidized by oxygen. It was concluded from this and the above evidence that the branching-point of the electron transport chain towards fumarate reductase was located at the menaquinone in P. shermanii. It was further concluded that all cytochromes were situated in the oxygen-linked branch of the chain, which formed a dead end of the system under anaerobic conditions. 6. Antimycin A inhibited only oxygen-linked reactions of the particles to about 50% at high concentrations

  16. Kalispel Non-Native Fish Suppression Project 2007 Annual Report.

    Energy Technology Data Exchange (ETDEWEB)

    Wingert, Michele; Andersen, Todd [Kalispel Natural Resource Department

    2008-11-18

    Non-native salmonids are impacting native salmonid populations throughout the Pend Oreille Subbasin. Competition, hybridization, and predation by non-native fish have been identified as primary factors in the decline of some native bull trout (Salvelinus confluentus) and westslope cutthroat trout (Oncorhynchus clarki lewisi) populations. In 2007, the Kalispel Natural Resource Department (KNRD) initiated the Kalispel Nonnative Fish Suppression Project. The goal of this project is to implement actions to suppress or eradicate non-native fish in areas where native populations are declining or have been extirpated. These projects have previously been identified as critical to recovering native bull trout and westslope cutthroat trout (WCT). Lower Graham Creek was invaded by non-native rainbow (Oncorhynchus mykiss) and brook trout (Salvelinus fontinalis) after a small dam failed in 1991. By 2003, no genetically pure WCT remained in the lower 700 m of Graham Creek. Further invasion upstream is currently precluded by a relatively short section of steep, cascade-pool stepped channel section that will likely be breached in the near future. In 2008, a fish management structure (barrier) was constructed at the mouth of Graham Creek to preclude further invasion of non-native fish into Graham Creek. The construction of the barrier was preceded by intensive electrofishing in the lower 700 m to remove and relocate all captured fish. Westslope cutthroat trout have recently been extirpated in Cee Cee Ah Creek due to displacement by brook trout. We propose treating Cee Cee Ah Creek with a piscicide to eradicate brook trout. Once eradication is complete, cutthroat trout will be translocated from nearby watersheds. In 2004, the Washington Department of Fish and Wildlife (WDFW) proposed an antimycin treatment within the subbasin; the project encountered significant public opposition and was eventually abandoned. However, over the course of planning this 2004 project, little public

  17. Hypoxic vasoconstriction of partial muscular intra-acinar pulmonary arteries in murine precision cut lung slices

    Directory of Open Access Journals (Sweden)

    Goldenberg Anna

    2006-06-01

    Full Text Available Abstract Background Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV which serves to match lung perfusion to ventilation. The underlying mechanisms are not fully resolved yet. The major vascular segment contributing to HPV, the intra-acinar artery, is mostly located in that part of the lung that cannot be selectively reached by the presently available techniques, e.g. hemodynamic studies of isolated perfused lungs, recordings from dissected proximal arterial segments or analysis of subpleural vessels. The aim of the present study was to establish a model which allows the investigation of HPV and its underlying mechanisms in small intra-acinar arteries. Methods Intra-acinar arteries of the mouse lung were studied in 200 μm thick precision-cut lung slices (PCLS. The organisation of the muscle coat of these vessels was characterized by α-smooth muscle actin immunohistochemistry. Basic features of intra-acinar HPV were characterized, and then the impact of reactive oxygen species (ROS scavengers, inhibitors of the respiratory chain and Krebs cycle metabolites was analysed. Results Intra-acinar arteries are equipped with a discontinuous spiral of α-smooth muscle actin-immunoreactive cells. They exhibit a monophasic HPV (medium gassed with 1% O2 that started to fade after 40 min and was lost after 80 min. This HPV, but not vasoconstriction induced by the thromboxane analogue U46619, was effectively blocked by nitro blue tetrazolium and diphenyleniodonium, indicating the involvement of ROS and flavoproteins. Inhibition of mitochondrial complexes II (3-nitropropionic acid, thenoyltrifluoroacetone and III (antimycin A specifically interfered with HPV, whereas blockade of complex IV (sodium azide unspecifically inhibited both HPV and U46619-induced constriction. Succinate blocked HPV whereas fumarate had minor effects on vasoconstriction. Conclusion This study establishes the first model for investigation of basic characteristics of HPV

  18. 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

    Directory of Open Access Journals (Sweden)

    Zoll J

    2015-07-01

    Full Text Available Joffrey Zoll,1 Jamal Bouitbir,1 Pascal Sirvent,2 Alexis Klein,3 Antoine Charton,1,4 Liliana Jimenez,3 François R Péronnet,5 Bernard Geny,1 Ruddy Richard61Physiology Department, Faculty of Medicine and EA3072, Université de Strasbourg, Strasbourg, 2Clermont Université, Université Blaise Pascal, EA 3533, Laboratoire des Adaptations Métaboliques à l’Exercice en Conditions Physiologiques et Pathologiques, Clermont-Ferrand, 3Danone Research, Centre Daniel Carasso, Palaiseau, 4Department of Anesthesia and Critical Care and EA3072, Hôpital de Hautepierre, Université de Strasbourg, France; 5Kinesiology Department, Université de Montréal, Montréal, QC, Canada; 6Department of Sport Medicine and Functional Explorations and INRA UMR 1019, Faculty of Medicine, Université d’Auvergne, Clermont-Ferrand, FranceBackground: 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

  19. Metformin as a prevention and treatment for preeclampsia: effects on soluble fms-like tyrosine kinase 1 and soluble endoglin secretion and endothelial dysfunction.

    Science.gov (United States)

    Brownfoot, Fiona C; Hastie, Roxanne; Hannan, Natalie J; Cannon, Ping; Tuohey, Laura; Parry, Laura J; Senadheera, Sevvandi; Illanes, Sebastian E; Kaitu'u-Lino, Tu'uhevaha J; Tong, Stephen

    2016-03-01

    explant-conditioned media (to whole vessels). Finally, we examined the effects of metformin on angiogenesis on maternal omental vessel explants. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary endothelial cells, villous cytotrophoblast cells, and preterm preeclamptic placental villous explants. The reduction in soluble fms-like tyrosine kinase 1 and soluble endoglin secretion was rescued by coadministration of succinate, which suggests that the effects of metformin on soluble fms-like tyrosine kinase 1 and soluble endoglin were likely to be regulated at the level of the mitochondria. In addition, the mitochondrial electron transport chain inhibitors rotenone and antimycin reduced soluble fms-like tyrosine kinase 1 secretion, which further suggests that soluble fms-like tyrosine kinase 1 secretion is regulated through the mitochondria. Mitochondrial electron transport chain activity in preterm preeclamptic placentas was increased compared with gestation-matched control subjects. Metformin improved features of endothelial dysfunction relevant to preeclampsia. It reduced endothelial cell messenger RNA expression of vascular cell adhesion molecule 1 that was induced by tumor necrosis factor-α (vascular cell adhesion molecule 1 is an inflammatory adhesion molecule up-regulated with endothelial dysfunction and is increased in preeclampsia). Placental conditioned media impaired bradykinin-induced vasodilation; this effect was reversed by metformin. Metformin also improved whole blood vessel angiogenesis impaired by fms-like tyrosine kinase 1. Metformin reduced soluble fms-like tyrosine kinase 1 and soluble endoglin secretion from primary human tissues, possibly by inhibiting the mitochondrial electron transport chain. The activity of the mitochondrial electron transport chain was increased in preterm preeclamptic placenta. Metformin reduced endothelial dysfunction, enhanced vasodilation in omental arteries, and induced