Sample records for antimycin

  1. The regulation and biosynthesis of antimycins

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    Ryan F. Seipke


    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. Comparative kinetics of Qi site inhibitors of cytochrome bc1 complex: picomolar antimycin and micromolar cyazofamid. (United States)

    Li, Hui; Zhu, Xiao-Lei; Yang, Wen-Chao; Yang, Guang-Fu


    Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase (SCR) with Ki  = 0.033 ± 0.00027 nm and non-competitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of SCR with Ki  = 12.90 ± 0.91 μm and a non-competitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed an H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow tight-binding inhibitor, whereas cyazofamid is a classic inhibitor.

  3. All-atom molecular dynamics simulations reveal significant differences in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc1 complexes. (United States)

    Kokhan, Oleksandr; Shinkarev, Vladimir P


    Antimycin A is the most frequently used specific and powerful inhibitor of the mitochondrial respiratory chain. We used all-atom molecular dynamics (MD) simulations to study the dynamic aspects of the interaction of antimycin A with the Q(i) site of the bacterial and bovine bc(1) complexes embedded in a membrane. The MD simulations revealed considerable conformational flexibility of antimycin and significant mobility of antimycin, as a whole, inside the Q(i) pocket. We conclude that many of the differences in antimycin binding observed in high-resolution x-ray structures may have a dynamic origin and result from fluctuations of protein and antimycin between multiple conformational states of similar energy separated by low activation barriers, as well as from the mobility of antimycin within the Q(i) pocket. The MD simulations also revealed a significant difference in interaction between antimycin and conserved amino acid residues in bovine and bacterial bc(1) complexes. The strong hydrogen bond between antimycin and conserved Asp-228 (bovine numeration) was observed to be frequently broken in the bacterial bc(1) complex and only rarely in the bovine bc(1) complex. In addition, the distances between antimycin and conserved His-201 and Lys-227 were consistently larger in the bacterial bc(1) complex. The observed differences could be responsible for a weaker interaction of antimycin with the bacterial bc(1) complex.

  4. U.S.D.A. Forest Service environmental analysis report on the use of antimycin to rehabilitate lakes and streams (United States)

    US Fish and Wildlife Service, Department of the Interior — Report identifies characteristics of use of antimycin to destroy stickleback, chub, suckers and other competitors to salmon and dolly varden.

  5. Inhibition of aldehyde dehydrogenase 2 activity enhances antimycin-induced rat cardiomyocytes apoptosis through activation of MAPK signaling pathway. (United States)

    Zhang, Peng; Xu, Danling; Wang, Shijun; Fu, Han; Wang, Keqiang; Zou, Yunzeng; Sun, Aijun; Ge, Junbo


    Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial-specific enzyme, has been proved to be involved in oxidative stress-induced cell apoptosis, while little is known in cardiomyocytes. This study was aimed at investigating the role of ALDH2 in antimycin A-induced cardiomyocytes apoptosis by suppressing ALDH2 activity with a specific ALDH2 inhibitor Daidzin. Antimycin A (40μg/ml) was used to induce neonatal cardiomyocytes apoptosis. Daidzin (60μM) effectively inhibited ALDH2 activity by 50% without own effect on cell apoptosis, and significantly enhanced antimycin A-induced cardiomyocytes apoptosis from 33.5±4.4 to 56.5±6.4% (Hochest method, pdaidzin treated cardiomyocytes compared to the cells treated with antimycin A alone. These findings indicated that modifying mitochondrial ALDH2 activity/expression might be a potential therapeutic option on reducing oxidative insults induced cardiomyocytes apoptosis.

  6. Analysis of antimycin A by reversed-phase liquid chromatography/nuclear magnetic-resonance spectrometry (United States)

    Ha, Steven T.K.; Wilkins, Charles L.; Abidi, Sharon L.


    A mixture of closely related streptomyces fermentation products, antimycin A, Is separated, and the components are identified by using reversed-phase high-performance liquid chromatography with directly linked 400-MHz proton nuclear magnetic resonance detection. Analyses of mixtures of three amino acids, alanine, glycine, and valine, are used to determine optimal measurement conditions. Sensitivity increases of as much as a factor of 3 are achieved, at the expense of some loss in chromatographic resolution, by use of an 80-μL NMR cell, Instead of a smaller 14-μL cell. Analysis of the antimycin A mixture, using the optimal analytical high performance liquid chromatography/nuclear magnetic resonance conditions, reveals it to consist of at least 10 closely related components.

  7. High-performance liquid chromatographic separation of subcomponents of antimycin A. (United States)

    Abidi, S L


    Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, A1a, A1b, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins A1, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpreted based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

  8. Reversed-phase thin-layer chromatography of homologs of Antimycin-A and related derivatives (United States)

    Abidi, Sharon L.


    Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

  9. High-performance liquid-chromatographic separation of subcomponents of antimycin-A (United States)

    Abidi, S.L.


    Using a reversed-phase high-performance liquid chromatographic (HPLC) technique, a mixture of antimycins A was separated into eight hitherto unreported subcomponents, Ala, Alb, A2a, A2b, A3a, A3b, A4a, and A4b. Although a base-line resolution of the known four major antimycins Al, A2, A3, and A4 was readily achieved with mobile phases containing acetate buffers, the separation of the new antibiotic subcomponents was highly sensitive to variation in mobile phase conditions. The type and composition of organic modifiers, the nature of buffer salts, and the concentration of added electrolytes had profound effects on capacity factors, separation factors, and peak resolution values. Of the numerous chromatographic systems examined, a mobile phase consisting of methanol-water (70:30) and 0.005 M tetrabutylammonium phosphate at pH 3.0 yielded the most satisfactory results for the separation of the subcomponents. Reversed-phase gradient HPLC separation of the dansylated or methylated antibiotic compounds produced superior chromatographic characteristics and the presence of added electrolytes was not a critical factor for achieving separation. Differences in the chromatographic outcome between homologous and structural isomers were interpretated based on a differential solvophobic interaction rationale. Preparative reversed-phase HPLC under optimal conditions enabled isolation of pure samples of the methylated antimycin subcomponents for use in structural studies.

  10. Spermine binding to liver mitochondria deenergized by ruthenium red plus either FCCP or antimycin A. (United States)

    Dalla Via, L; Di Noto, V; Toninello, A


    Thermodynamic analysis of spermine binding to mitochondria treated with ruthenium red and deenergized with either FCCP or antimycin A confirms the presence of two polyamine binding sites, S1 and S2, both with monocoordination, as previously observed in energized mitochondria [Dalla Via et al., Biochim. Biophys. Acta 1284 (1996) 247-252]. Both sites undergo a marked change in binding capacity and binding affinity upon mitochondrial deenergization. This change is most likely responsible for the incomplete or delayed spermine-mediated inhibition of the permeability transition induced in deenergized mitochondria.

  11. Bypasses of the antimycin a block of mitochondrial electron transport in relation to ubisemiquinone function. (United States)

    Alexandre, A; Lehninger, A L


    Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.

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

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    Chi-Tai Yeh


    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.

  13. Mitochondrial morphology and dynamics in Triticum aestivum roots in response to rotenone and antimycin A. (United States)

    Rakhmatullina, Daniya; Ponomareva, Anastasiya; Gazizova, Natalia; Minibayeva, Farida


    Mitochondria are dynamic organelles, capable of fusion and fission as a part of cellular responses to various signals, such as the shifts in the redox status of a cell. The mitochondrial electron transport chain (ETC.) is involved in the generation of reactive oxygen species (ROS), with complexes I and III contributing the most to this process. Disruptions of ETC. can lead to increased ROS generation. Here, we demonstrate the appearance of giant mitochondria in wheat roots in response to simultaneous application of the respiratory inhibitors rotenone (complex I of mitochondrial ETC.) and antimycin A (complex III of mitochondrial ETC.). The existence of such megamitochondria was temporary, and following longer treatment with inhibitors mitochondria resumed their conventional size and oval shape. Changes in mitochondrial morphology were accompanied with a decrease in mitochondrial potential and an unexpected increase in oxygen consumption. Changes in mitochondrial morphology and activity may result from the fusion and fission of mitochondria induced by the disruption of mitochondrial ETC. Results from experiments with the inhibitor of mitochondrial fission Mdivi-1 suggest that the retarded fission may facilitate plant mitochondria to appear in a fused shape. The processes of mitochondrial fusion and fission are involved in the regulation of the efficacy of the functions of the respiratory chain complexes and ROS metabolism during stresses. The changes in morphology of mitochondria, along with the changes in their functional activity, can be a part of the strategy of the plant adaptation to stresses.

  14. Celastrol Protects against Antimycin A-Induced Insulin Resistance in Human Skeletal Muscle Cells

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    Mohamad Hafizi Abu Bakar


    Full Text Available Mitochondrial dysfunction and inflammation are widely accepted as key hallmarks of obesity-induced skeletal muscle insulin resistance. The aim of the present study was to evaluate the functional roles of an anti-inflammatory compound, celastrol, in mitochondrial dysfunction and insulin resistance induced by antimycin A (AMA in human skeletal muscle cells. We found that celastrol treatment improved insulin-stimulated glucose uptake activity of AMA-treated cells, apparently via PI3K/Akt pathways, with significant enhancement of mitochondrial activities. Furthermore, celastrol prevented increased levels of cellular oxidative damage where the production of several pro-inflammatory cytokines in cultures cells was greatly reduced. Celastrol significantly increased protein phosphorylation of insulin signaling cascades with amplified expression of AMPK protein and attenuated NF-κB and PKC θ activation in human skeletal muscle treated with AMA. The improvement of insulin signaling pathways by celastrol was also accompanied by augmented GLUT4 protein expression. Taken together, these results suggest that celastrol may be advocated for use as a potential therapeutic molecule to protect against mitochondrial dysfunction-induced insulin resistance in human skeletal muscle cells.

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

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


    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.

  16. Determination of antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation. (United States)

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


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

  17. Determination of Antimycin-A in water by liquid chromatographic/mass spectrometry: single-laboratory validation (United States)

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


    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.

  18. Mechanisms involved in the inhibition of glycolysis by cyanide and antimycin A in Candida albicans and its reversal by hydrogen peroxide. A common feature in Candida species. (United States)

    Peña, Antonio; Sánchez, Norma Silvia; González-López, Omar; Calahorra, Martha


    In Candida albicans, cyanide and antimycin A inhibited K(+) transport, not only with ethanol-O2 as the substrate, but also with glucose. The reason for this was that they inhibited not only respiration, but also fermentation, decreasing ATP production. Measurements of oxygen levels in cell suspensions allowed identification of the electron pathways involved. NADH fluorescence levels increased in the presence of the inhibitors, indirectly indicating lower levels of NAD(+) and so pointing to glyceraldehyde-3-phosphate dehydrogenase as the limiting step responsible for the inhibition of glycolysis, which was confirmed by the levels of glycolytic intermediaries. The cyanide effect could be reversed by hydrogen peroxide, mainly due to an activity by which H2O2 can be reduced by electrons flowing from NADH through a pathway that can be inhibited by antimycin A, and appears to be a cytochrome c peroxidase. Therefore, the inhibition of glycolysis by the respiratory inhibitors seems to be due to the decreased availability of NAD(+), resulting in a decreased activity of glyceraldehyde-3-phosphate dehydrogenase. Compartmentalization of pyridine nucleotides in favor of the mitochondria can contribute to explaining the low fermentation capacity of C. albicans. Similar results were obtained with three C. albicans strains, Candida dubliniensis and, to a lower degree, Candida parapsilosis.

  19. Extramitochondrial release of hydrogen peroxide from insect and mouse liver mitochondria using the respiratory inhibitors phosphine, myxothiazol, and antimycin and spectral analysis of inhibited cytochromes. (United States)

    Bolter, C J; Chefurka, W


    The fumigant insecticide phosphine (PH3) is known to inhibit cytochrome c oxidase in vitro. Inhibition of the respiratory chain at this site has been shown to stimulate the generation of superoxide radicals (O2-), which dismutate to form hydrogen peroxide (H2O2). This study was performed in order to investigate the production of H2O2 by mitochondria isolated from granary weevil (Sitophilus granarius) and mouse liver on exposure to PH3. Other respiratory inhibitors, antimycin, myxothiazol, and rotenone were used with insect mitochondria. Hydrogen peroxide was measured spectrophotometrically using yeast cytochrome c peroxidase as an indicator. Insect and mouse liver mitochondria, utilizing endogenous substrate, both produced H2O2 after inhibition by PH3. Insect organelles released threefold more H2O2 than did mouse organelles, when exposed to PH3. Production of H2O2 by PH3-treated insect mitochondria was increased significantly on addition of the substrate alpha-glycerophosphate. Succinate did not enhance H2O2 production, however, indicating that the H2O2 did not result from the autoxidation of ubiquinone. NAD(+)-linked substrates, malate and pyruvate also had no effect on H2O2 production, suggesting that NADH-dehydrogenase was not the source of H2O2. Data obtained using antimycin and myxothiazol, both of which stimulated the release of H2O2 from insect mitochondria, lead to the conclusion that glycerophosphate dehydrogenase is a source of H2O2. The effect of combining PH3, antimycin, and myxothiazol on cytochrome spectra in insect mitochondria was also recorded. It was observed that PH3 reduces cytochrome c oxidase but none of the other cytochromes in the electron transport chain. There was no movement of electrons to cytochrome b when insect mitochondria are inhibited with PH3. The spectral data show that the inhibitors interact with the respiratory chain in a way that would allow the production of H2O2 from the sites proposed previously.

  20. Physiological Concentration of Exogenous Lactate Reduces Antimycin A Triggered Oxidative Stress in Intestinal Epithelial Cell Line IPEC-1 and IPEC-J2 In Vitro (United States)

    Kahlert, Stefan; Junnikkala, Sami; Renner, Lydia; Hynönen, Ulla; Hartig, Roland; Nossol, Constanze; Barta-Böszörményi, Anikó; Dänicke, Sven; Souffrant, Wolfgang-Bernhard; Palva, Airi; Rothkötter, Hermann-Josef; Kluess, Jeannette


    Weaning triggers an adaptation of the gut function including luminal lactate generation by lactobacilli, depending on gastrointestinal site. We hypothesized that both lactobacilli and lactate influence porcine intestinal epithelial cells. In vivo experiments showed that concentration of lactate was significantly higher in gastric, duodenal and jejunal chyme of suckling piglets compared to their weaned counterparts. In an in vitro study we investigated the impact of physiological lactate concentration as derived from the in vivo study on the porcine intestinal epithelial cells IPEC-1 and IPEC-J2. We detected direct adherence of lactobacilli on the apical epithelial surface and a modulated F-actin structure. Application of lactobacilli culture supernatant alone or lactate (25 mM) at low pH (pH 4) changed the F-actin structure in a similar manner. Treatment of IPEC cultures with lactate at near neutral pH resulted in a significantly reduced superoxide-generation in Antimycin A-challenged cells. This protective effect was nearly completely reversed by inhibition of cellular lactate uptake via monocarboxylate transporter. Lactate treatment enhanced NADH autofluorescence ratio (Fcytosol/Fnucleus) in non-challenged cells, indicating an increased availability of reduced nucleotides, but did not change the overall ATP content of the cells. Lactobacilli-derived physiological lactate concentration in intestine is relevant for alleviation of redox stress in intestinal epithelial cells. PMID:27054581

  1. Induction of the AOX1D Isoform of Alternative Oxidase in A. thaliana T-DNA Insertion Lines Lacking Isoform AOX1A Is Insufficient to Optimize Photosynthesis when Treated with Antimycin A

    Institute of Scientific and Technical Information of China (English)

    Inga Strodtkotter; Kollipara Padmasree; Challabathula Dinakar; Birgit Speth; Pamela S. Niazi; Joanna Wojtera; Ingo Voss; Phuc Thi Do; Adriano Nunes-Nesi; Alisdair R. Fernie; Vera Linke; Agepati S. Raghavendra; Renate Scheibe


    Plant respiration is characterized by two pathways for electron transfer to O2, namely the cytochrome path-way (CP) that is linked to ATP production, and the alternative pathway (AP), where electrons from ubiquinol are directly transferred to O2 via an alternative oxidase (AOX) without concomitant ATP production. This latter pathway is well suited to dispose of excess electrons in the light, leading to optimized photosynthetic performance. We have characterized T-DNA-insertion mutant lines of Arabidopsis thaliana that do not express the major isoform, AOXIA. In standard growth conditions, these plants did not show any phenotype, but restriction of electron flow through CP by antimycin A, which induces AOX1A expression in the wild-type, led to an increased expression of AOX1D in leaves of the aox1a-knockout mutant. Despite the increased presence of the AOX1D isoform in the mutant, antimycin A caused inhibition of photosyn-thesis, increased ROS, and ultimately resulted in amplified membrane leakage and necrosis when compared to the wild-type, which was only marginally affected by the inhibitor. It thus appears that AOX1D was unable to fully compensate for the loss of AOX1A when electron flow via the CP is restricted. A combination of inhibition studies, coupled to metabolite profiling and targeted expression analysis of the P-protein of glycine decarboxylase complex (GDC), suggests that the aox1a mutants attempt to increase their capacity for photorespiration. However, given their deficiency, it is intriguing that increase in expression neither of AOX1D nor of GDC could fully compensate for the lack of AOX1A to optimize pho-tosynthesis when treated with antimycin A. We suggest that the aox1a mutants can further be used to substantiate the current models concerning the influence of mitochondrial redox on photosynthetic performance and gene expression.

  2. Increased sensitivity of photosynthesis to antimycin A induced by inactivation of the chloroplast ndhB gene. Evidence for a participation of the NADH-dehydrogenase complex to cyclic electron flow around photosystem I. (United States)

    Joët, T; Cournac, L; Horvath, E M; Medgyesy, P; Peltier, G


    Tobacco (Nicotiana tabacum var Petit Havana) ndhB-inactivated mutants (ndhB-) obtained by plastid transformation (E.M. Horvath, S.O. Peter, T. Joët, D. Rumeau, L. Cournac, G.V. Horvath, T.A. Kavanagh, C. Schäfer, G. Peltier, P. MedgyesyHorvath [2000] Plant Physiol 123: 1337-1350) were used to study the role of the NADH-dehydrogenase complex (NDH) during photosynthesis and particularly the involvement of this complex in cyclic electron flow around photosystem I (PSI). Photosynthetic activity was determined on leaf discs by measuring CO2 exchange and chlorophyll fluorescence quenchings during a dark-to-light transition. In the absence of treatment, both non-photochemical and photochemical fluorescence quenchings were similar in ndhB- and wild type (WT). When leaf discs were treated with 5 microM antimycin A, an inhibitor of cyclic electron flow around PSI, both quenchings were strongly affected. At steady state, maximum photosynthetic electron transport activity was inhibited by 20% in WT and by 50% in ndhB-. Under non-photorespiratory conditions (2% O2, 2,500 microL x L(-1) CO2), antimycin A had no effect on photosynthetic activity of WT, whereas a 30% inhibition was observed both on quantum yield of photosynthesis assayed by chlorophyll fluorescence and on CO2 assimilation in ndhB-. The effect of antimycin A on ndhB- could not be mimicked by myxothiazol, an inhibitor of the mitochondrial cytochrome bc1 complex, therefore showing that it is not related to an inhibition of the mitochondrial electron transport chain but rather to an inhibition of cyclic electron flow around PSI. We conclude to the existence of two different pathways of cyclic electron flow operating around PSI in higher plant chloroplasts. One of these pathways, sensitive to antimycin A, probably involves ferredoxin plastoquinone reductase, whereas the other involves the NDH complex. The absence of visible phenotype in ndhB- plants under normal conditions is explained by the complement of these two

  3. 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


    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...


    Institute of Scientific and Technical Information of China (English)

    江昇平; 田晓清; 廖丽; 杨桥; 陆亚男; 马丽艳; 陈波; 樊成奇


    极地微生物代谢产生芳香族硝基化合物、肽类、萜类等多种骨架类型的化学成分.为从北极放线菌中发现结构新颖、活性显著的小分子化合物,本研究采用Sephadex LH-20凝胶和半制备HPLC等柱层析分离纯化化合物手段,结合LC-MS、1H-和13C-NMR等波谱学数据和文献对比进行结构解析,从链霉菌Streptomycessp.604F提取物中分离鉴定出抗霉素Alb(1)、抗霉素A4a(2)、抗霉素A2a(3)、抗霉素A3a(4)、抗霉素Ala(5)以及伏尔加霉素(6)和8-脱氧伏尔加霉素(7)这7个化合物.化合物1-7是首次从北极海洋沉积物来源的微生物中发现,而且是首次从同一菌株中发现抗霉素A和伏尔加霉素这两类化合物.

  5. Estimation of the rate of energy production of rat mast cells in vitro

    DEFF Research Database (Denmark)

    Johansen, Torben


    Rat mast cells were treated with glycolytic and respiratory inhibitors. The rate of adenosine triphosphate depletion of cells incubated with both types of inhibitors and the rate of lactate produced in presence of antimycin A and glucose were used to estimate the rate of oxidative and glycolytic...

  6. A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus.

    Directory of Open Access Journals (Sweden)

    Ryan F Seipke

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

  7. A single Streptomyces symbiont makes multiple antifungals to support the fungus farming ant Acromyrmex octospinosus. (United States)

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


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

  8. 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


    -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...

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


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


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

  10. Characterization of the respiratory chain of Helicobacter pylori

    DEFF Research Database (Denmark)

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


    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...

  11. [Bioactivity of endophytic actinomycetes from medicinal plants and secondary metabolites from strain D62]. (United States)

    Liu, Ning; Zhang, Hui; Zheng, Wen; Huang, Ying; Wang, Hai-Bin


    It is believed that genetic recombination of the endophytes with the hosts that occurred in evolutionary time could result in some endophytes producing certain phytochemical originally characteristic of the host. Based on this widely accepted hypothesis, there have been increasing research efforts focused on screening for novel natural products from endophytes. In this study, antimicrobial and antitumor activities of 165 actinomycetes isolated from medicinal plants collected from Xishuangbanna were tested by agar diffusion method and WST-8 assay respectively. The results showed that over 42% of the isolates exhibited antagonism against pathogenic strains, and 54.5% displayed excellent inhibition against mouse melanoma cell line B16 or/and human alveolar epithelial cell line A549. These results are superior to those of soil actinomycetes, indicating tremendous potential of endophytic of actinomycetes for exploration. Six compounds that had both antimicrobial and antitumor activities were separated and purified from isolate Streptomyces sp. D62 by resin adsorption, silica-gel column and sephadex chromatography, etc. On the basis of spectral analyses, they were identified as antimycin A4a (1), antimycin A7a (2), antimycin A2a (3), antimycin A1a (4), 10-hydroxy-10-methyl-dodec-2-en-1,4-olide (5) and 6-(2-(4-aminophenyl)-2-oxoethyl)-3,5-dimethyl-tetrahydropyran-2-one(6), with the last one defined as a novel compound. Based on all these results, it is convinced that endophytic actinomycetes are a promising resource for bioactive natural product discovery.

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

    DEFF Research Database (Denmark)

    Juul, Troels; Palm, Fredrik; Nielsen, Per Mose;


    ) C MR is suitable for cells isolated from kidney tissue, without prior cell culture. METHODS: 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...

  13. Chemo-sensitization of fungal pathogens to antimicrobial agents using benzaldehyde analogs (United States)

    Activity of conventional antifungal agents, fludioxonil, strobilurin and antimycinA, which target the oxidative and osmotic stress response systems, was elevated by co-application of certain analogs of benzaldehyde. Fungal tolerance to 2,3-dihydroxybenzaldehyde or 2,3-dihydroxybenzoic acid was foun...

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

    Directory of Open Access Journals (Sweden)

    Christina Viegelmann


    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.

  15. Regulation of cyclic photophosphorylation during ferredoxin-mediated electron transport. Effect of DCMU and the NADPH/NADP/sup +/ ratio

    Energy Technology Data Exchange (ETDEWEB)

    Hosler, J.P.; Yocum, C.F.


    Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O/sub 2/ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP/sup +/ ratio is varied, as is the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.

  16. Decreased Pasteur effect in platelets of aged individuals. (United States)

    D'Aurelio, M; Merlo Pich, M; Catani, L; Sgarbi, G L; Bovina, C; Formiggini, G; Parenti Castelli, G; Baum, H; Tura, S; Lenaz, G


    We have investigated the mitochondrial energy state in human platelets of young (19-30 years old) and aged individuals (65-87 years old) exploiting the Pasteur effect, i.e. stimulation of lactate production by incubation of the purified platelets with the mitochondrial respiratory chain inhibitor, antimycin A. This assay allows the determination of mitochondrial function with respect to glycolysis, and the ratio of mitochondrial adenosine triphosphate (ATP) to glycolytic ATP. A significant increase of basal, non-stimulated lactate production and decrease of the stimulation by antimycin A were observed in the older individuals, suggesting that the impairment of oxidative phosphorylation detectable in post-mitotic tissues of aged individuals can be observed also in easily collectable blood cells.

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


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


    Background 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 s...

  18. Bioactivity of endophytic actinomycetes from medicinal plants and secondary metabolites from strain D62%药用植物内生放线菌的生物活性及菌株D62的代谢产物分析

    Institute of Scientific and Technical Information of China (English)

    刘宁; 张辉; 郑文; 黄英; 王海彬; 雒文军


    利用琼脂移块法及WST-8法分别对分离自西双版纳药用植物的165株内生放线菌进行了抗菌、抗肿瘤活性测定.结果显示,超过42%的菌株对病原菌表现出拮抗活性,且对病原真菌的总体拮抗活性明显强于土壤放线菌;78%的菌株表现出抗肿瘤活性,且大部分菌株(54.5%)具有强抗肿瘤活性.选取其中对真菌及肿瘤细胞均有高抑制活性的菌株D62,并对其次生代谢产物进行了进一步的研究,共分离得6个化合物,分别是Antimycin A4a(1),Antimycin A7a(2)、Antimycin A2a(3)、Antimycin A1a(4)、10-hydroxy-10-methyl-dodec-2-en-1,4-olide(5)及6-(2-(4-aminophenyl)-2-oxoethyl)-3,5-dimethyl-tetrahydropyran-2-one(6),其中化合物6为新化合物.以上结果表明药用植物内生放线菌作为一类新的微生物资源具有很好的开发潜力.

  19. Influence of growth regulators and respiration inhibitors on dark transformation of phytochrome in coleoptiles of oat seedlings

    Directory of Open Access Journals (Sweden)

    Jan Kopcewicz


    Full Text Available Irradiation with red light leads to the formation of an unstable, undergoing gradual destruction, physiologically active PFR form of phytochrome in the coleoptiles of oat seedlings. Growth substances: IAA, GA3, kinetin, ABA, ethrel as well acetylcholine do not influence the nature and rate of phytochrome dark transformation. Inhibitors of energy-producing processes such as KCN, 2,4-DNP, DCCD and antimycin A inhibit the process of dark destruction of the PFR form of phytochrome.

  20. Ubisemiquinone is the electron donor for superoxide formation by complex III of heart mitochondria. (United States)

    Turrens, J F; Alexandre, A; Lehninger, A L


    Much evidence indicates that superoxide is generated from O2 in a cyanide-sensitive reaction involving a reduced component of complex III of the mitochondrial respiratory chain, particularly when antimycin A is present. Although it is generally believed that ubisemiquinone is the electron donor to O2, little experimental evidence supporting this view has been reported. Experiments with succinate as electron donor in the presence of antimycin A in intact rat heart mitochondria, which contain much superoxide dismutase but little catalase, showed that myxothiazol, which inhibits reduction of the Rieske iron-sulfur center, prevented formation of hydrogen peroxide, determined spectrophotometrically as the H2O2-peroxidase complex. Similarly, depletion of the mitochondria of their cytochrome c also inhibited formation of H2O2, which was restored by addition of cytochrome c. These observations indicate that factors preventing the formation of ubisemiquinone also prevent H2O2 formation. They also exclude ubiquinol, which remains reduced under these conditions, as the reductant of O2. Since cytochrome b also remains fully reduced when myxothiazol is added to succinate- and antimycin A-supplemented mitochondria, reduced cytochrome b may also be excluded as the reductant of O2. These observations, which are consistent with the Q-cycle reactions, by exclusion of other possibilities leave ubisemiquinone as the only reduced electron carrier in complex III capable of reducing O2 to O2-.

  1. Strong Pasteur effect in rabbit corneal endothelium preserves fluid transport under anaerobic conditions. (United States)

    Riley, M V; Winkler, B S


    1. The hydration and transparency of the mammalian cornea are maintained by a metabolically dependent fluid transport system located in the endothelial cell layer. The purpose of the study was to determine whether this pump activity is dependent upon aerobic or anaerobic metabolism. 2. The ability of the cornea, superfused in vitro with a bicarbonate-Ringer solution containing glucose and adenosine, to maintain normal hydration (thickness) when respiration is inhibited has been examined in intact and de-epithelialized preparations and correlated with glycolytic activity and cellular concentrations of ATP. 3. In respiring intact and de-epithelialized corneas thickness was maintained for periods up to 5 h during superfusion with the control Ringer solution. 4. KCN (10(-3) M) or antimycin A (10(-6) M) caused the intact cornea to swell at 15 +/- 3 microns h-1, whereas the de-epithelialized tissue maintained normal thickness under these conditions. This swelling of the intact tissue appears to be due to the osmotic effect of increased epithelial lactate production under anaerobic conditions. 5. Pre-swollen de-epithelialized corneas deturgesced fully to original thickness at a rate of 43 +/- 7 microns h-1 under aerobic conditions, but with KCN or antimycin they deturgesced at only 65% of that rate and generally failed to return to their original thickness. 6. Ouabain (10(-4) M) caused de-epithelialized corneas to swell in the presence of KCN or antimycin, as it did under aerobic conditions, showing that maintenance of hydration or deturgescence are pump-dependent processes under both conditions. 7. Lactate production was markedly stimulated by KCN or antimycin in intact and de-epithelialized preparations, but not in the stroma alone. The magnitude of the Pasteur effect was calculated to be 5-fold in the endothelium and 2.5-fold in the epithelium. Ouabain inhibited anaerobic lactate production in the endothelium by 50%. 8. ATP content of the epithelium following 5 h

  2. Low-dose methotrexate enhances cycling of highly anaplastic cancer cells (United States)

    Marzi, Ilaria; Olivotto, Massimo


    ABSTRACT We previously showed that cellular RedOx state governs the G1-S transition of AH130 hepatoma, a tumor spontaneously reprogrammed to the embryonic stem cell stage. This transition is impaired when the mithocondrial electron transport system is blocked by specific inhibitors (antimycin A) or the respiratory chain is saturated by adding to the cells high concentrations of pyruvate. The antimycin A or pyruvate block is removed by the addition of adequate concentrations of folate (F). This suggests that the G1-S transition of AH130 cells depends on a respiration-linked step of DNA synthesis related to folate metabolism. In the study reported here, we characterized the effects of methotrexate (MTX), an inhibitor of dihydofolate-reductase, on the G1-S transition of hepatoma cells, in the absence or the presence of exogenously added F, dihydrofolate (FH2) or tetrahydrofolate (FH4). MTX, at 1 μM or higher concentrations, inhibited G1-S transition. This inhibition was completely removed by exogenous folates. Surprisingly, 10 nM MTX stimulated G1-S transition. The addition of F, but not FH2 or FH4, significantly increased this effect. Furthermore, 10 nM MTX removed the block of the G1-S transition operated by antimycin A or pyruvate, an effect which was enhanced in the presence of F. Finally, the stimulatory effect of 10 nM MTX was inhibited in the presence of serine. Our findings indicated that, under certain conditions, MTX may stimulate, rather than inhibiting, the cycling of cancer cells exhibiting a stem cell-like phenotype, such as AH130 cells. This may impact the therapeutic use of MTX and of folates as supportive care. PMID:27841718

  3. Modification of the spectral properties of cytochrome b in mutants of Saccharomyces cerevisiae resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Mapping at two distinct genetic loci of the split mitochondrial gene of cytochrome b. (United States)

    Briquet, M; Goffeau, A


    The effects of five inhibitors of the cytochrome bc1 complex: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), 2-n-heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuron-resistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b. 1. The oxidation of NADH and of cytochrome b-561 exhibits a similar resistance to diuron and HpHOQnO in Diu 1 and Diu 2 mutants. 2. No extra reduction of cytochrome b-561 and cytochrome b-565 is observed in the presence of diuron and HpHOQnO. 3. Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b-561 induced by concentrations of HpHOQno 2 -- 3-times higher than those required in the parental strains. 4. The spectral and respiratory effects of antimycin A, funiculosin and mucidin and generally similar in the diuron-resistant mutants and in their parental strains. However a cross-resistance between diuron and antimycin A is indicated in one Diu 2 mutant. 5. From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in the central region of the apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b-565 and b-561 as well as the flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b-561 is only slightly affected by the diu-1 and diu-2 mutations.

  4. Evaluation of cellular energetics by the pasteur effect in intact cardiomyoblasts and isolated perfused hearts. (United States)

    Muscari, Claudio; Gamberini, Chiara; Bonafe', Francesca; Giordano, Emanuele; Bianchi, Cristina; Lenaz, Giorgio; Caldarera, Claudio Marcello


    This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (delta-lactate = stimulated [lactate] -basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 micromol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control delta-lactate was 20.9 +/- 2.31 (S.E.M.) microg/mL and markedly decreased after reperfusion (7.66 +/- 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when delta-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.

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

    DEFF Research Database (Denmark)

    Johansen, Torben


    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....

  6. 40 CFR Appendix A to Part 355 - The List of Extremely Hazardous Substances and Their Threshold Planning Quantities (United States)


    ... Pentafluoride 500 500 1397-94-0 Antimycin A b 1,000 1,000/10,000 86-88-4 ANTU 100 500/10,000 1303-28-2 Arsenic... 500 814-49-3 Diethyl Chlorophosphate d 500 500 71-63-6 Digitoxin b 100 100/10,000 2238-07-5 Diglycidyl... Acetate 500 500/10,000 80-63-7 Methyl 2-Chloroacrylate 500 500 74-83-9 Methyl Bromide f 1,000 1,000...

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

    DEFF Research Database (Denmark)

    Johansen, Torben


    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...... 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...

  8. Terpenoid Metabolism in Plastids 1 (United States)

    Camara, Bilal; Bardat, Françoise; Dogbo, Odette; Brangeon, Judy; Monéger, René


    A technique for the isolation and the purification of Capsicum annum L. var. Yolo Wonder chromoplasts is described. The degree of purity of the isolated chromoplasts is greatly improved by the absence of MgCl2 in the extraction medium and in the gradient purification system, as shown by electron micrographs and the near absence of antimycin-insensitive NADH:cytochrome c reductase activity and succinate:cytochrome c reductase activity. Furthermore, phosphatidylserine was absent and the phosphatidylethanolamine content was reduced by a factor of 5 in these preparations, which were active in the synthesis of galactolipids, prenylquinones, and carotenoids. Images Fig. 1 PMID:16663194

  9. Anti-Candida properties of urauchimycins from actinobacteria associated with trachymyrmex ants. (United States)

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


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

  10. Mitochondrial lipid peroxidation by cumene hydroperoxide and its prevention by succinate. (United States)

    Bindoli, A; Cavallini, L; Jocelyn, P


    Rat liver mitochondria form lipid hydroperoxides when they are incubated aerobically with cumene hydroperoxide. The rate of reaction is dependent on the initial concentration of the latter and involves the consumption of oxygen. Gradient-separated and cytochrome c-depleted mitochondria, mitoplasts and submitochondrial fractions also undergo this peroxidation. Mitochondrial lipid peroxidation by cumene hydroperoxide is strongly inhibited by SKF52A (an inhibitor of cytochrome P-450), by antioxidants and to a lesser extent by the enzymes superoxide dismutase and catalase. Conversely, rotenone and N-ethylmaleimide stimulate the reaction. Succinate protects against the lipid peroxidation and in some mitochondrial fractions the associated oxygen uptake is also inhibited. This protection by succinate is prevented by malonate but not by N-ethylmaleimide or antimycin. Lipid hydroperoxides present in previously peroxidised mitochondria are partly lost on reincubation with succinate and this reaction is also unaffected by N-ethylmaleimide but inhibited by both malonate and antimycin. The results suggest that reduction of mitochondrial ubiquinone may prevent the generation of lipid hydroperoxides but that their subsequent removal may require reduction at or beyond cytochrome b.

  11. 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.


    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.

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

    Directory of Open Access Journals (Sweden)

    Thais D. Mendes


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

  13. Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity. (United States)

    de Visser, R; Blacquière, T


    The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K(i) of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path.

  14. Electron transport chain inhibitors induce microglia activation through enhancing mitochondrial reactive oxygen species production. (United States)

    Ye, Junli; Jiang, Zhongxin; Chen, Xuehong; Liu, Mengyang; Li, Jing; Liu, Na


    Reactive oxygen species (ROS) are believed to be mediators of excessive microglial activation, yet the resources and mechanism are not fully understood. Here we stimulated murine microglial BV-2 cells and primary microglial cells with different inhibitors of electron transport chain (ETC), rotenone, thenoyltrifluoroacetone (TTFA), antimycin A, and NaN3 to induce mitochondrial ROS production and we observed the role of mitochondrial ROS in microglial activation. Our results showed that ETC inhibitors resulted in significant changes in cell viability, microglial morphology, cell cycle arrest and mitochondrial ROS production in a dose-dependent manner in both primary cultural microglia and BV-2 cell lines. Moreover, ETC inhibitors, especially rotenone and antimycin A stimulated secretion of interleukin 1β (IL-1β), interleukin 6 (IL-6), interleukin 12 (IL-12) and tumor necrosis factor α (TNF-α) by microglia with marked activation of mitogen-activated proteinkinases (MAPKs) and nuclear factor κB (NF-κB), which could be blocked by specific inhibitors of MAPK and NF-κB and mitochondrial antioxidants, Mito-TEMPO. Taken together, our results demonstrated that inhibition of mitochondrial respiratory chain in microglia led to production of mitochondrial ROS and therefore may activate MAPK/NF-кB dependent inflammatory cytokines release in microglia, which indicated that mitochondrial-derived ROS were contributed to microglial activation.

  15. The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes. (United States)

    Anari, M R; Khan, S; O'Brien, P J


    Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.

  16. Alternative respiration and fumaric acid production of Rhizopus oryzae. (United States)

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


    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.

  17. Effects of the thyroid hormone derivatives 3-iodothyronamine and thyronamine on rat liver oxidative capacity (United States)

    Venditti, P.; Napolitano, G.; Di Stefano, L.; Chiellini, G.; Zucchi, R.; Scanlan, T.S.; Di Meo, S.


    Thyronamines T0AM and T1AM are naturally occurring decarboxylated thyroid hormone derivatives. Their in vivo administration induces effects opposite to those induced by thyroid hormone, including lowering of body temperature. Since the mitochondrial energy-transduction apparatus is known to be a potential target of thyroid hormone and its derivatives, we investigated the in vitro effects of T0AM and T1AM on the rates of O2 consumption and H2O2 release by rat liver mitochondria. Hypothyroid animals were used because of the low levels of endogenous thyronamines. We found that both compounds are able to reduce mitochondrial O2 consumption and increase H2O2 release. The observed changes could be explained by a partial block, operated by thyronamines, at a site located near the site of action of antimycin A. This hypothesis was confirmed by the observation that thyronamines reduced the activity of Complex III where the site of antimycin action is located. Because thyronamines exerted their effects at concentrations comparable to those found in hepatic tissue, it is conceivable that they can affect in vivo mitochondrial O2 consumption and H2O2 production acting as modulators of thyroid hormone action. PMID:21664427

  18. Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus. (United States)

    Higa, Ataru; Mori, Yuko; Kitamura, Yoshie


    Hyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions. In order to examine which elements of the mitochondrial electron transport chain might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity. Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H

  19. 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


    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.

  20. Utilization of adenosine triphosphate in rat mast cells during and after secretion of histamine in response to compound 48/80

    DEFF Research Database (Denmark)

    Johansen, Torben


    The adenosine triphosphate (ATP) content of rat mast cells and their lactate production were measured during and after secretion of histamine induced by compound 48/80. Antimycin A and oligomycin were used to block oxidative ATP synthesis, and 2-deoxyglucose (2-DG) was used to block glycolytic ATP...... synthesis. Histamine secretion was completed after 10 sec. exposure of the cells to compound 48/80. During that time period there was an increased ATP-utilization of 0.15 pmol/10(3) cells. After completion of the secretory process there seemed to be an enhanced utilization of ATP of 0.40 pmol/10(3) cells....../min., which may be associated with recovery of the cells....

  1. Anomalous cell wall synthesis in Mucor mucedo (L.) Fres. induced by some fungicides and other compounds related to the problem of dimorphism. (United States)

    Lyr, H; Casperson, G


    An anomalous cell wall thickening in Mucor mucedo is induced already within 60-120 min by some fungicides (etridiazol, chloroneb, pentachloronitrobenzene, dicloran, drazoxolon, biphenyl) as well as with a N2-atmosphere or high concentrations of glucose, but not with 2,4-dinitrophenol, chlorinated phenols, dichlofluanid and antimycin A. This effect seems to be identical to the change from the mycelial (M-) to the yeast (Y-) form in dimorphic fungi, which can be achieved by culture conditions as well as by addition of chemicals. The cause seems to be a specific, complex change in the metabolic state. A scheme of regulation is presented which explains most of the experimental results described till now.

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

    DEFF Research Database (Denmark)

    Johansen, Torben


    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......-trifluorometroxyphenylnydrazone), which are known to block the energy-dependent calcium uptake by isolated mitochondria. The mast cell ATP content was reduced during A23187-induced histamine release under anaerobic conditions in the presence of glucose. This indicates an increased utilization of ATP during the release process. 5...

  3. Insulin resistance and the mitochondrial link. Lessons from cultured human myotubes

    DEFF Research Database (Denmark)

    Gaster, Michael


    In order to better understand the impact of reduced mitochondrial function for the development of insulin resistance and cellular metabolism, human myotubes were established from lean, obese, and T2D subjects and exposed to mitochondrial inhibitors, either affecting the electron transport chain...... lipid uptake. The metabolic phenotype during respiratory uncoupling resembled the above picture, except for an increase in glucose and palmitate oxidation. Antimycin A and oligomycin treatment induced insulin resistance at the level of glucose and palmitate uptake in all three study groups while......, at the level of glycogen synthesis, insulin resistance was only seen in lean myotubes. Primary insulin resistance in diabetic myotubes was significantly worsened at the level of glucose and lipid uptake. The present study is the first convincing data linking functional mitochondrial impairment per se...

  4. Screening for microbial metabolites affecting phenotype of Caenorhabditis elegans. (United States)

    Yamamuro, Daisuke; Uchida, Ryuji; Takahashi, Yoko; Masuma, Rokuro; Tomoda, Hiroshi


    Microbial samples, including our library of known microbial compounds (ca. 300) and microbial culture broths (ca. 9000), were screened for small molecules affecting the phenotype of Caenorhabditis elegans. As a result, seven known compounds were found to induce phenotypic abnormality of C. elegans. Staurosporine exhibited morphological defects in the vulva and tail of C. elegans, avermectin B1a exhibited hatching inhibition of starting eggs on day 1 at 25-100 µM and growth inhibition at 0.01-12.5 µM, siccanin and antimycin A inhibited the growth of C. elegans, and fluorouracil inhibited hatching of eggs newly spawned by adult C. elegans. Toromycin induced morphological defects in the intestine. 5-(4-Methoxyphenyl)-oxazole, isolated as a fungal metabolite for the first time, inhibited the hatching of eggs newly spawned by adult C. elegans.

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

    DEFF Research Database (Denmark)

    Hohnholt, Michaela C; Andersen, Vibe H; Bak, Lasse K;


    and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone...... and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity....... Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes...

  6. Characterization of cereulide synthetase, a toxin-producing macromolecular machine.

    Directory of Open Access Journals (Sweden)

    Diego A Alonzo

    Full Text Available Cereulide synthetase is a two-protein nonribosomal peptide synthetase system that produces a potent emetic toxin in virulent strains of Bacillus cereus. The toxin cereulide is a depsipeptide, as it consists of alternating aminoacyl and hydroxyacyl residues. The hydroxyacyl residues are derived from keto acid substrates, which cereulide synthetase selects and stereospecifically reduces with imbedded ketoreductase domains before incorporating them into the growing depsipeptide chain. We present an in vitro biochemical characterization of cereulide synthetase. We investigate the kinetics and side chain specificity of α-keto acid selection, evaluate the requirement of an MbtH-like protein for adenylation domain activity, assay the effectiveness of vinylsulfonamide inhibitors on ester-adding modules, perform NADPH turnover experiments and evaluate in vitro depsipeptide biosynthesis. This work also provides biochemical insight into depsipeptide-synthesizing nonribosomal peptide synthetases responsible for other bioactive molecules such as valinomycin, antimycin and kutzneride.

  7. D-chiro-inositol affects accumulation of raffinose family oligosaccharides in developing embryos of Pisum sativum. (United States)

    Lahuta, Lesław B; Dzik, Tomasz


    Developing garden pea embryos are able to take up exogenously applied cyclitols: myo-inositol, which naturally occurs in pea, and two cyclitols absent in pea plants: d-chiro-inositol and d-pinitol. The competition in the uptake of cyclitols by pea embryo, insensitivity to glucose and sucrose, and susceptibility to inhibitor(s) of H(+)-symporters (e.g. CCCP and antimycin A) suggest that a common cyclitol transporter is involved. Both d-chiro-inositol and d-pinitol can be translocated through the pea plant to developing embryos. During seed maturation drying, they are used for synthesis of mainly mono-galactosides, such as fagopyritol B1 and galactosyl pinitol A. Accumulation of d-chiro-inositol (and formation of fagopyritols), but not d-pinitol, strongly reduces accumulation of verbascose, the main raffinose oligosaccharide in pea seeds. The reasons for the observed changes are discussed.

  8. Activation of AMP-activated protein kinase regulates hippocampal neuronal pH by recruiting Na(+)/H(+) exchanger NHE5 to the cell surface. (United States)

    Jinadasa, Tushare; Szabó, Elöd Z; Numat, Masayuki; Orlowski, John


    Strict regulation of intra- and extracellular pH is an important determinant of nervous system function as many voltage-, ligand-, and H(+)-gated cationic channels are exquisitely sensitive to transient fluctuations in pH elicited by neural activity and pathophysiologic events such as hypoxia-ischemia and seizures. Multiple Na(+)/H(+) exchangers (NHEs) are implicated in maintenance of neural pH homeostasis. However, aside from the ubiquitous NHE1 isoform, their relative contributions are poorly understood. NHE5 is of particular interest as it is preferentially expressed in brain relative to other tissues. In hippocampal neurons, NHE5 regulates steady-state cytoplasmic pH, but intriguingly the bulk of the transporter is stored in intracellular vesicles. Here, we show that NHE5 is a direct target for phosphorylation by the AMP-activated protein kinase (AMPK), a key sensor and regulator of cellular energy homeostasis in response to metabolic stresses. In NHE5-transfected non-neuronal cells, activation of AMPK by the AMP mimetic AICAR or by antimycin A, which blocks aerobic respiration and causes acidification, increased cell surface accumulation and activity of NHE5, and elevated intracellular pH. These effects were effectively blocked by the AMPK antagonist compound C, the NHE inhibitor HOE694, and mutation of a predicted AMPK recognition motif in the NHE5 C terminus. This regulatory pathway was also functional in primary hippocampal neurons, where AMPK activation of NHE5 protected the cells from sustained antimycin A-induced acidification. These data reveal a unique role for AMPK and NHE5 in regulating the pH homeostasis of hippocampal neurons during metabolic stress.

  9. On the location of the H+-extruding steps in site 2 of the mitochondrial electron transport chain. (United States)

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


    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).

  10. Ascorbic acid is a key participant during the interactions between chloroplasts and mitochondria to optimize photosynthesis and protect against photoinhibition

    Indian Academy of Sciences (India)

    Saikrishna Talla; Khateef Riazunnisa; Lolla Padmavathi; Pidakala Rajsheel; Agepati S Raghavendra


    The possible role of L-ascorbate (AsA) as a biochemical signal during the interactions between photosynthesis and respiration was examined in leaf discs of Arabidopsis thaliana. AsA content was either decreased as in AsA-deficient vtc1 mutants or increased by treatment with L-galactono-1, 4-lactone (L-GalL, a precursor of AsA; EC In mutants, photosynthesis was extremely sensitive to both antimycin A (inhibitor of the cytochrome oxidase pathway [COX pathway]) and salicylhydroxamic acid (SHAM, inhibitor of the alternative pathway [AOX pathway]), particularly at high light conditions. Mitochondrial inhibitors lowered the ratio of reduced AsA to total AsA, at high light, indicating oxidative stress in leaf discs. Elevation of AsA by L-GalL decreased the sensitivity of photosynthesis at high light to antimycin A or SHAM, sustained photosynthesis at supraoptimal light and relieved the extent of photoinhibition. High ratios of reduced AsA to total AsA in L-GalL-treated leaf discs suggests that L-GalL lowers oxidative stress. The protection by L-GalL of photosynthesis against the mitochondrial inhibitors and photoinhibition was quite pronounced in vtc1 mutants. Our results suggest that the levels and redox state of AsA modify the pattern of modulation of photosynthesis by mitochondrial metabolism. The extent of the AOX pathway as a percentage of the total respiration in Arabidopsis mesophyll protoplasts was much higher in vtc1 than in wild type. We suggest that the role of AsA becomes pronounced at high light and/or when the AOX pathway is inhibited. While acknowledging the importance of the COX pathway, we hypothesize that AsA and the AOX pathway may complement each other to protect photosynthesis against photoinhibition.

  11. Sites and mechanisms responsible for the low rate of free radical production of heart mitochondria in the long-lived pigeon. (United States)

    Herrero, A; Barja, G


    Basal (substrate alone) and maximum rates of H2O2 production, oxygen consumption and free radical leak in the respiratory chain were higher in heart mitochondria of the short-lived rat (4 years) than in the long-lived pigeon (35 years). This suggests that the low free radical production of pigeon heart mitochondria is due in part to both a low electron flow and a low percent leak of electrons out of sequence in the respiratory chain. Thenoyltrifluoroacetone did not increase H2O2 production with succinate either in rats or pigeons. Mitochondrial H2O2 production was higher with pyruvate/malate than with succinate in both animal species. Rotenone and antimycin A increased H2O2 production with pyruvate/malate to the maximum levels observed in each species. Addition of myxothiazol to antimycin A-treated mitochondria supplemented with pyruvate/malate decreased H2O2 production in both species. All the combinations of inhibitors added with pyruvate/malate resulted in higher rates of H2O2 production in rats than in pigeons. When succinate instead of pyruvate/malate was used as substrate, rotenone and thenoyltrifluoroacetone decreased mitochondrial H2O2 production in the rat and did not change it in the pigeon. The results indicate that Complexes I and III are the main H2O2 generators of heart mitochondria in rats and pigeons and that both Complexes are responsible for the low H2O2 production of the bird. p-Chloromercuribenzoate and ethoxyformic anhydride strongly inhibited the H2O2 production induced by rotenone with pyruvate/malate in both species. This suggests that the free radical generator of Complex I is located after the ferricyanide reduction site, between the ethoxyformic and the rotenone-sensitive sites.

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

    Directory of Open Access Journals (Sweden)

    Ying-Yin Chen


    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

  13. Respiratory Contribution of the Alternate Path during Various Stages of Ripening in Avocado and Banana Fruits. (United States)

    Theologis, A; Laties, G G


    The respiration of fresh slices of preclimacteric avocado (Persea americana Mill. var. Hass) and banana (Musa cavendishii var. Valery) fruits is stimulated by cyanide and antimycin. The respiration is sensitive to m-chlorobenzhydroxamic acid in the presence of cyanide but much less so in the presence of antimycin. In the absence of cyanide the contribution of the cyanide-resistant pathway to the coupled preclimacteric respiration is zero. In uncoupled slices, by contrast, the alternate path is engaged and utilized fully in avocado, and extensively in banana. Midclimacteric and peak climacteric slices are also cyanide-resistant and, in the presence of cyanide, sensitive to m-chlorobenzhydroxamic acid. In the absence of uncoupler there is no contribution by the alternate path in either tissue. In uncoupled midclimacteric avocado slices the alternate path is fully engaged. Midclimacteric banana slices, however, do not respond to uncouplers, and the alternate path is not engaged. Avocado and banana slices at the climacteric peak neither respond to uncouplers nor utilize the alternate path in the presence or absence of uncoupler.The maximal capacities of the cytochrome and alternate paths, V(cyt) and V(alt), respectively, have been estimated in slices from preclimacteric and climacteric avocado fruit and found to remain unchanged. The total respiratory capacity in preclimacteric and climacteric slices exceeds the respiratory rise which attends fruit ripening. In banana V(alt) decreases slightly with ripening.The aging of thin preclimacteric avocado slices in moist air results in ripening with an accompanying climacteric rise. In this case the alternate path is fully engaged at the climacteric peak, and the respiration represents the total potential respiratory capacity present in preclimacteric tissue. The respiratory climacteric in intact avocado and banana fruits is cytochrome path-mediated, whereas the respiratory climacteric of ripened thin avocado slices comprises

  14. Low-level laser therapy activates NF-kB via generation of reactive oxygen species in mouse embryonic fibroblasts.

    Directory of Open Access Journals (Sweden)

    Aaron C-H Chen

    Full Text Available BACKGROUND: Despite over forty years of investigation on low-level light therapy (LLLT, the fundamental mechanisms underlying photobiomodulation at a cellular level remain unclear. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we isolated murine embryonic fibroblasts (MEF from transgenic NF-kB luciferase reporter mice and studied their response to 810 nm laser radiation. Significant activation of NF-kB was observed at fluences higher than 0.003 J/cm(2 and was confirmed by Western blot analysis. NF-kB was activated earlier (1 hour by LLLT compared to conventional lipopolysaccharide treatment. We also observed that LLLT induced intracellular reactive oxygen species (ROS production similar to mitochondrial inhibitors, such as antimycin A, rotenone and paraquat. Furthermore, we observed similar NF-kB activation with these mitochondrial inhibitors. These results, together with inhibition of laser induced NF-kB activation by antioxidants, suggests that ROS play an important role in the laser induced NF-kB signaling pathways. However, LLLT, unlike mitochondrial inhibitors, induced increased cellular ATP levels, which indicates that LLLT also upregulates mitochondrial respiration. CONCLUSION: We conclude that LLLT not only enhances mitochondrial respiration, but also activates the redox-sensitive NFkB signaling via generation of ROS. Expression of anti-apoptosis and pro-survival genes responsive to NFkB could explain many clinical effects of LLLT.

  15. In vitro reactive oxygen species production by mitochondria from the rabbitfish Siganus fuscessens livers and the effects of Irgarol-1051. (United States)

    Liang, Bo; Wang, Li; He, Tangtian; Liu, Wenhua; Li, Qi; Li, Mingfeng


    In this study, the mitochondria from the livers of Siganus fuscessens were exposed to the Irgarol-1051with or without respiratory chain inhibitors using succinate or malate as the substrate, and the effects on mitochondrial ROS production were tested. The mitochondrial ROS production was significantly enhanced by antimycin A with an increase of more than three folds but not by rotenone and NaN3, and this may suggest complex III is the major ROS-producing site. Irgarol-1051 treatments gave a somewhat contradictory result: this chemical can inhibit the mitochondrial ROS production but the inhibition decreased with the increase of doses. These contradictory data about Irgarol-1051 may be explained by the balance between the effects of inhibition through the opening of small-size pores and stimulation through blocking electron transfer, but the mechanism laid behind needs more evidence to support. As Irgarol-1051 was continuously used in antifouling and its bio-concentration factor is up to 160 in fish, the toxic effect of Irgarol-1051 on aquatic animals should be paid more attention to.

  16. Microscopic energy transfer spectroscopy to determine mitochondrial malfunction in human myotubes (United States)

    Gschwend, Michael H.; Strauss, Wolfgang S. L.; Brinkmeier, H.; Ruedel, R.; Steiner, Rudolf W.; Schneckenburger, Herbert


    A microscopic equipment is reported for examination of cellular autofluorescence and determination of energy transfer in vitro, which is proposed to be an appropriate tool to investigate mitochondrial malfunction. The method includes fluorescence microscopy combined with time-gated (nanosecond) fluorescence emission spectroscopy and is presently used to study mitochondrial metabolism of human myotube primary cultures Enzyme complexes of the respiratory chain, located at the inner mitochondrial membrane, were inhibited by various drugs, and fluorescence of the mitochondrial coenzyme nicotinamide adenine dinucleotide (NADH) as well as of the mitochondrial marker rhodamine 123 (R123) was examined. After inhibition of enzyme complex I (NADH-coenzyme Q reductase) by rotenone or enzyme complex III (coenzyme QH2-cytochrome c reductase) by antimycin a similar or increased NADH fluorescence was observed. In addition, energy transfer from excited states of NADH (energy donor) to R123 (energy acceptor) was deduced from a decrease of NADH fluorescence after coincubation with these inhibitors and R123. Application of microscopic energy transfer spectroscopy for diagnosis of congenital mitochondrial deficiencies is currently in preparation.

  17. Mitochondrial modulation of store-operated Ca(2+) entry in model cells of Alzheimer's disease. (United States)

    Ma, Tuo; Gong, Kai; Yan, Yufang; Song, Bo; Zhang, Xiufang; Gong, Yandao


    Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer's disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca(2+) signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca(2+), which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer's disease.

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

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


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

  19. Reduction of molybdate to molybdenum blue by Klebsiella sp. strain hkeem. (United States)

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


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

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

    DEFF Research Database (Denmark)

    Garland, L G; Johansen, Torben


    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...

  1. Reduction of molybdate to molybdenum blue by Enterobacter sp. strain Dr.Y13. (United States)

    Shukor, M Y; Rahman, M F; Shamaan, N A; Syed, M A


    Extensive use of metals in various industrial applications has caused substantial environmental pollution. Molybdenum-reducing bacteria isolated from soils can be used to remove molybdenum from contaminated environments. In this work we have isolated a local bacterium with the capability to reduce soluble molybdate to the insoluble molybdenum blue. We studied several factors that would optimize molybdate reduction. Electron donor sources such as glucose, sucrose, lactose, maltose and fructose (in decreasing efficiency) supported molybdate reduction after 24 h of incubation with optimum glucose concentration for molybdate reduction at 1.5% (w/v). The optimum pH, phosphate and molybdate concentrations, and temperature for molybdate reduction were pH 6.5, 5.0, 25 to 50 mM and 37 degrees C, respectively. The Mo-blue produced by cellular reduction exhibited a unique absorption spectrum with a maximum peak at 865 nm and a shoulder at 700 nm. Metal ions such as chromium, cadmium, copper, silver and mercury caused approximately 73, 71, 81, 77 and 78% inhibition of the molybdenum-reducing activity, respectively. All of the respiratory inhibitors tested namely rotenone, azide, cyanide and antimycin A did not show any inhibition to the molybdenum-reducing activity suggesting components of the electron transport system are not responsible for the reducing activity. The isolate was tentatively identified as Enterobacter sp. strain Dr.Y13 based on carbon utilization profiles using Biolog GN plates and partial 16S rDNA molecular phylogeny.

  2. Molybdate Reduction to Molybdenum Blue by an Antarctic Bacterium

    Directory of Open Access Journals (Sweden)

    S. A. Ahmad


    Full Text Available A molybdenum-reducing bacterium from Antarctica has been isolated. The bacterium converts sodium molybdate or Mo6+ to molybdenum blue (Mo-blue. Electron donors such as glucose, sucrose, fructose, and lactose supported molybdate reduction. Ammonium sulphate was the best nitrogen source for molybdate reduction. Optimal conditions for molybdate reduction were between 30 and 50 mM molybdate, between 15 and 20°C, and initial pH between 6.5 and 7.5. The Mo-blue produced had a unique absorption spectrum with a peak maximum at 865 nm and a shoulder at 710 nm. Respiratory inhibitors such as antimycin A, sodium azide, potassium cyanide, and rotenone failed to inhibit the reducing activity. The Mo-reducing enzyme was partially purified using ion exchange and gel filtration chromatography. The partially purified enzyme showed optimal pH and temperature for activity at 6.0 and 20°C, respectively. Metal ions such as cadmium, chromium, copper, silver, lead, and mercury caused more than 95% inhibition of the molybdenum-reducing activity at 0.1 mM. The isolate was tentatively identified as Pseudomonas sp. strain DRY1 based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system. The characteristics of this strain would make it very useful in bioremediation works in the polar and temperate countries.

  3. Molybdate reduction to molybdenum blue by an Antarctic bacterium. (United States)

    Ahmad, S A; Shukor, M Y; Shamaan, N A; Mac Cormack, W P; Syed, M A


    A molybdenum-reducing bacterium from Antarctica has been isolated. The bacterium converts sodium molybdate or Mo⁶⁺ to molybdenum blue (Mo-blue). Electron donors such as glucose, sucrose, fructose, and lactose supported molybdate reduction. Ammonium sulphate was the best nitrogen source for molybdate reduction. Optimal conditions for molybdate reduction were between 30 and 50 mM molybdate, between 15 and 20°C, and initial pH between 6.5 and 7.5. The Mo-blue produced had a unique absorption spectrum with a peak maximum at 865 nm and a shoulder at 710 nm. Respiratory inhibitors such as antimycin A, sodium azide, potassium cyanide, and rotenone failed to inhibit the reducing activity. The Mo-reducing enzyme was partially purified using ion exchange and gel filtration chromatography. The partially purified enzyme showed optimal pH and temperature for activity at 6.0 and 20°C, respectively. Metal ions such as cadmium, chromium, copper, silver, lead, and mercury caused more than 95% inhibition of the molybdenum-reducing activity at 0.1 mM. The isolate was tentatively identified as Pseudomonas sp. strain DRY1 based on partial 16s rDNA molecular phylogenetic assessment and the Biolog microbial identification system. The characteristics of this strain would make it very useful in bioremediation works in the polar and temperate countries.

  4. Enzymic analysis of NADPH metabolism in beta-lactam-producing Penicillium chrysogenum: presence of a mitochondrial NADPH dehydrogenase. (United States)

    Harris, Diana M; Diderich, Jasper A; van der Krogt, Zita A; Luttik, Marijke A H; Raamsdonk, Léonie M; Bovenberg, Roel A L; van Gulik, Walter M; van Dijken, Johannes P; Pronk, Jack T


    Based on assumed reaction network structures, NADPH availability has been proposed to be a key constraint in beta-lactam production by Penicillium chrysogenum. In this study, NADPH metabolism was investigated in glucose-limited chemostat cultures of an industrial P. chrysogenum strain. Enzyme assays confirmed the NADP(+)-specificity of the dehydrogenases of the pentose-phosphate pathway and the presence of NADP(+)-dependent isocitrate dehydrogenase. Pyruvate decarboxylase/NADP(+)-linked acetaldehyde dehydrogenase and NADP(+)-linked glyceraldehyde-3-phosphate dehydrogenase were not detected. Although the NADPH requirement of penicillin-G-producing chemostat cultures was calculated to be 1.4-1.6-fold higher than that of non-producing cultures, in vitro measured activities of the major NADPH-providing enzymes were the same. Isolated mitochondria showed high rates of antimycin A-sensitive respiration of NADPH, thus indicating the presence of a mitochondrial NADPH dehydrogenase that oxidises cytosolic NADPH. The presence of this enzyme in P. chrysogenum might have important implications for stoichiometric modelling of central carbon metabolism and beta-lactam production and may provide an interesting target for metabolic engineering.

  5. Efflux of radioactive nucleotides from mouse pancreatic islets prelabelled with 2-/sup 3/H-adenosine

    Energy Technology Data Exchange (ETDEWEB)

    Welsh, M.


    Cultured mouse pancreatic islets were prelabelled with 2-/sup 3/H-adenosine in order to monitor the efflux pattern of radioactivity and insulin. The outflow of radioactivity decreased continuously when the islets were perifused with glucose (1.67 mmol/l). When raising the glucose concentration to 16.7 mmol/l, there was a prompt inhibition of the radioactive efflux concomitant with an increased rate of insulin release. These effects were reversed when the high glucose challenge was withdrawn. Similar radioactive efflux patterns were obtained after addition of ..cap alpha..-ketoisocaproic acid, leucine or pyruvate to the perifusion medium, and also when the islets were challenged with high glucose concentrations in the absence of calcium. Both antimycin A and glipizide stimulated the efflux of radioactivity, although only the addition of glipizide was accompanied by a stimulation of the insulin release. Nucleotides constituted approximately 90% of the total effluent radioactivity. Decrease in the radioactive AMP and ADP efflux due to high glucose was furthermore found to be the cause of the observed inhibition of the total radioactive efflux. The changes in radioactive efflux induced by glucose probably reflect changes in the intracellular concentrations of AMP and ADP. It is concluded that no simple correlation exists between radioactive efflux and insulin release and that changes in the intracellular concentrations of nucleotides may be an early event in the stimulus-secretion coupling of glucose-induced insulin release.

  6. Hyperoxia activates ATM independent from mitochondrial ROS and dysfunction. (United States)

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


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

  7. 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.

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

    Directory of Open Access Journals (Sweden)

    Bryony A P Williams


    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.

  9. Biochemical effects of the flavanol-rich lychee fruit extract on the melanin biosynthesis and reactive oxygen species. (United States)

    Hagiwara, Kazuya; Okura, Masae; Sumikawa, Yasuyuki; Hida, Tokimasa; Kuno, Atsushi; Horio, Yoshiyuki; Yamashita, Toshiharu


    An ingredient of fruit polyphenol, resveratrol, is known to have an inhibitory effect on melanogenesis. In order to examine the functional differences between resveratrol and other fruit polyphenols, we compared biochemical effects of a resveratrol-free polyphenol, flavanol-rich lychee fruit extract (FRLFE), with other phenolic compounds including resveratrol. FRLFE as well as hydroquinone and resveratrol suppressed growth of B16F1 melanoma cells more significantly than rhododendrol or arbutin. Resveratrol suppressed mushroom tyrosinase at the lowest concentration (23.0 μmol/L) among the compounds tested. FRLFE and hydroquinone suppressed tyrosinase at almost the same concentration (half maximal inhibitory concentration [IC50 ], 83.5 and 94.6 μmol/L, respectively), which was higher than rhododendrol, ascorbic acid and arbutin (IC50 , 245, 345 and 421 μmol/L, respectively). Western blot analysis revealed that although resveratrol decreased expressions of tyrosinase and tyrosinase-related protein 1, FRLFE did not affect their expressions. Both FRLFE and resveratrol suppressed antimycin A-mediated reactive oxygen species (ROS) production in melanocytic cells. Resveratrol-mediated ROS suppression was inhibited by nicotinamide, a SIRT1 inhibitor. However, FRLFE-mediated suppression was not affected by nicotinamide. Moreover, FRLFE directly decreased superoxide in vitro, as detected by superoxide dismutase-like scavenging activity assay. These results suggest that FRLFE can protect melanocytes from cytotoxicity caused by an excess amount of melanin and ROS in a different manner from resveratrol.

  10. Energy-dependent intracellular translocation of proparathormone. (United States)

    Chu, L L; MacGregor, R R; Cohn, D V


    We previously suggested that after synthesis, proparathormone is transferred from rough endoplasmic reticulum to the Golgi region where its conversion to parathormone occurs. We have attempted to define more closely this transfer process. In the first type of study, bovine parathyroid slices were incubated with [3H]leucine for 10 min and then radioisotope labeling was restricted by addition of a large excess of nonradioactive leucine. Under these conditions, more than 90% of the initially labeled proparathormone was converted to parathormone in 40 min. Lowered temperature in the chase period markedly inhibited the conversion. Several chemical agents were employed individually in the chase period to examine their effect on the conversion process. Antimycin A, dinitrophenol, oligomycin, and anaerobiosis (N2) inhibited the conversion, whereas sodium flouride and cycloheximide had no effect. In the second type of study, parathyroid slices were incubated with [3H]leucine for the entire incubation period. Lowered temperature and inhibitors of energy metabolism and microtubular function all lengthened the interval (lag) between the initial synthesis of [3H]parathormone. Cycloheximide, Tris, and chloroquine decreased the rates of protein synthesis and conversion, respectively, but none had any effect on the lag. We interpret the lag to represent the time of transit for proparathormone from rough endoplasmic reticulum to the Golgi region. We conclude that this transfer process is independent of the synthesis of the prohormone and its conversion to the hormone. Moreover, this translocation requires metabolic energy and appears to be mediated by microtubules.

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

    Directory of Open Access Journals (Sweden)

    Ilaria Postiglione


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Xu Qiuwei, E-mail:; Vu, Heather; Liu Liping; Wang, Ting-Chuan; Schaefer, William H. [Merck Research Laboratories (United States)


    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 {beta}-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.

  13. Existence of aa3-type ubiquinol oxidase as a terminal oxidase in sulfite oxidation of Acidithiobacillus thiooxidans. (United States)

    Sugio, Tsuyoshi; Hisazumi, Tomohiro; Kanao, Tadayoshi; Kamimura, Kazuo; Takeuchi, Fumiaki; Negishi, Atsunori


    It was found that Acidithiobacillus thiooxidans has sulfite:ubiquinone oxidoreductase and ubiquinol oxidase activities in the cells. Ubiquinol oxidase was purified from plasma membranes of strain NB1-3 in a nearly homogeneous state. A purified enzyme showed absorption peaks at 419 and 595 nm in the oxidized form and at 442 and 605 nm in the reduced form. Pyridine ferrohaemochrome prepared from the enzyme showed an alpha-peak characteristic of haem a at 587 nm, indicating that the enzyme contains haem a as a component. The CO difference spectrum of ubiquinol oxidase showed two peaks at 428 nm and 595 nm, and a trough at 446 nm, suggesting the existence of an aa(3)-type cytochrome in the enzyme. Ubiquinol oxidase was composed of three subunits with apparent molecular masses of 57 kDa, 34 kDa, and 23 kDa. The optimum pH and temperature for ubiquinol oxidation were pH 6.0 and 30 degrees C. The activity was completely inhibited by sodium cyanide at 1.0 mM. In contrast, the activity was inhibited weakly by antimycin A(1) and myxothiazol, which are inhibitors of mitochondrial bc(1) complex. Quinone analog 2-heptyl-4-hydoroxyquinoline N-oxide (HOQNO) strongly inhibited ubiquinol oxidase activity. Nickel and tungstate (0.1 mM), which are used as a bacteriostatic agent for A. thiooxidans-dependent concrete corrosion, inhibited ubiquinol oxidase activity 100 and 70% respectively.

  14. Essentiality of respiratory activity for pentose utilization in thermotolerant yeast Kluyveromyces marxianus DMKU 3-1042. (United States)

    Lertwattanasakul, Noppon; Suprayogi; Murata, Masayuki; Rodrussamee, Nadchanok; Limtong, Savitree; Kosaka, Tomoyuki; Yamada, Mamoru


    By random integrative mutagenesis with a kanMX4 cassette in Kluyveromyces marxianus DMKU 3-1042, we obtained three mutants of COX15, ATP25 and CYC3 encoding a cytochrome oxidase assembly factor (singleton), a transcription factor required for assembly of the Atp9p subunit of mitochondrial ATP synthase and cytochrome c heme lyase, respectively, as mutants lacking growth capability on xylose and/or arabinose. They exhibited incapability of growth on non-fermentable carbon sources, such as acetate or glycerol, and thermosensitiveness. Their biomass formation in glucose medium was reduced, but ethanol yields were increased with a high ethanol level in the medium, compared to those of the parental strain. Experiments with respiratory inhibitors showed that cox15 and cyc3, but not atp25, were able to grow in glucose medium containing antimycin A and that the atp25 mutant was KCN-resistant. Activities of NADH and ubiquinol oxidases in membrane fractions of each mutant became a half of that of the parent and negligible, respectively, and their remaining NADH oxidase activities were found to be resistant to KCN. Absolute absorption spectral analysis revealed that the peak corresponding to a + a 3 was very small in atp25 and negligible in cox15 and cyc3. These findings suggest that the K. marxianus strain possesses an alternative KCN-resistant oxidase that is located between primary dehydrogenases and the ubiquinone pool and that the respiratory activity is essential for utilization of pentoses.

  15. Studies on the mechanism of synthesis of ethyl acetate in Kluyveromyces marxianus DSM 5422. (United States)

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


    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.

  16. Molecular cloning of alcohol dehydrogenase genes of the yeast Pichia stipitis and identification of the fermentative ADH. (United States)

    Passoth, V; Schäfer, B; Liebel, B; Weierstall, T; Klinner, U


    Two Pichia stipitis ADH genes (PsADH1 and PsADH2) were isolated by complementation of a Saccharomyces cerevisiae Adh(-)-mutant. The genes enabled the transformants to grow in the presence of antimycin A on glucose, to use ethanol as sole carbon source and made them sensitive to allylalcohol. The sequences of the genes showed similarities of 70-77% to sequences of ADH genes of Candida albicans, Kluyveromyces lactis, K. marxianus, and S. cerevisiae and about 60% homology to those of Schizosaccharomyces pombe and Aspergillus flavus. Southern hybridization experiments suggested that P. stipitis has only these two ADH genes. Both genes are located on the largest chromosome of P. stipitis. PsADH2 encodes for the ADH activity that is responsible for ethanol formation at oxygen limitation. The gene is regulated at the transcriptional level. Moreover, also in cells grown on ethanol, only PsADH2 transcript was found. PsADH1 transcript was detected under aerobic conditions on fermentable carbon sources.

  17. Assay of mitochondrial functions by resazurin in vitro

    Institute of Scientific and Technical Information of China (English)

    Hai-xia ZHANG; Guan-hua DU; Jun-tian ZHANG


    AIM: To study the mechanism of resazurin as indicator of mitochondrial function and to develop a rapid and sensitive assay for measuring metabolic activity of isolated mitochondria from rat liver in vitro. METHODS: The screening was carried out on 96-well microtitre plates by monitoring fluorescence intensity of resazurin reduced by mitochondria. Experimental conditions were optimized and influences of several inhibitors on mitochondrial function were observed. RESULTS: Fluorescence intensity increased in a linear manner when the mitochondrial protein concentration from 5 to 50 μg protein per well was incubated with resazurin (5 μmol/L) during 230 min period at 37 ℃. Edetic acid could promote the reduction of resazurin in mitochondria. The fluorescence intensity decreased greatly after pretreatment with NaN3, antimycin A, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP),and oligomycin compared with the control. However, the typical complex I inhibitor, rotenone enhanced the fluorescence intensity without mitochondria. CONCLUSION: Using resazurin to determine mitochondrial function is sensitive, inexpensive and could be easily automated for high throughput screening.

  18. Mitochondrial swelling impairs the transport of organelles in cerebellar granule neurons. (United States)

    Kaasik, Allen; Safiulina, Dzhamilja; Choubey, Vinay; Kuum, Malle; Zharkovsky, Alexander; Veksler, Vladimir


    Organelle transport in neuronal processes is central to the organization, developmental fate, and functions of neurons. Organelles must be transported through the slender, highly branched neuronal processes, making the axonal transport vulnerable to any perturbation. However, some intracellular structures like mitochondria are able to considerably modify their volume. We therefore hypothesized that swollen mitochondria could impair the traffic of other organelles in neurite shafts. To test this hypothesis, we have investigated the effects of mitochondrial swellers on the organelle traffic. Our data demonstrate that treatment of neurons with potassium ionophore valinomycin led to the fast time-dependent inhibition of organelle movement in cerebellar granule neurons. Similar inhibition was observed in neurons treated with the inhibitors of the mitochondrial respiratory chain, sodium azide and antimycin, which also induced swelling. No decrease in the motility of organelles was observed in cultures treated with inhibitors of ATP production or transport, oligomycin or bongkrekic acid, suggesting that inhibition of the ATP-generating activity itself without swelling does not affect the motility of organelles. The effect of swellers on the traffic was more important in thin processes, thus indicating the role of steric hindrance of swollen mitochondria. We propose that the size and morphology of the transported cargo is also relevant for seamless axonal transport and speculate that mitochondrial swelling could be one of the reasons for impaired organelle transport in neuronal processes.

  19. Ceramide synthesis from free fatty acids in rat brain: function of NADPH and substrate specificity

    Energy Technology Data Exchange (ETDEWEB)

    Singh, I.


    At the subcellular level, the synthesis of ceramide from free lignoceric acid and sphingosine in brain required reconstituted enzyme system (particulate fraction, heat-stable and heat-labile factors) and pyridine nucleotide (NADPH). The mitochondrial electron transfer inhibitors (KCN and antimycin A), energy uncouplers (oligomycin and 2,4-dinitrophenol), and carboxyatractyloside, which prevents the transport of ATP and ADP through the mitochondrial wall, inhibit the synthesis of ceramide in the presence of NADPH but have very little effect in the presence of ATP. Similar to the synthesis of ceramide, the synthesis of ATP from NADPH and NADH by the particulate fraction also required cytoplasmic factors (heat-stable and heat-labile factors). Moreover, ATP, but not its analog (AMP-CH2-P-O-P), can replace NADPH, thus suggesting that the function of the pyridine nucleotide is to provide ATP for the synthesis of ceramide. The cytoplasmic factors were not required for the synthesis of ceramide in the presence of ATP. The maximum velocity for synthesis of ceramide from free fatty acids of different chain lengths (C16-C26) was bimodal, with maxima around stearic acid (C18) and behenic acid (C22). The relative rate of synthesis of ceramide parallels the relative distribution of these fatty acids in brain cerebrosides and sulfatides.

  20. 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: [Eoetvoes Lorand University, Institute of Chemistry, Department of Analytical Chemistry, Laboratory of Environmental Chemistry and Bioanalytics, P.O. Box 32, H-1518, Budapest (Hungary)


    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Arechederra, Marguerite N.; Fischer, Caitlin N.; Wetzel, David J. [Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO (United States); Minteer, Shelley D., E-mail: minteers@slu.ed [Department of Chemistry, Saint Louis University, 3501 Laclede Ave., St. Louis, MO (United States)


    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, {alpha}-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.

  2. [Generation of superoxide radicals by the mitochondrial respiratory chain of isolated cardiomyocytes]. (United States)

    Kashkarov, K P; Vasil'eva, E V; Ruuge, E K


    Generation of superoxide radicals by the mitochondrial respiratory chain of cardiomyocites isolated from rat heart and treated with saponin was studied. The rate of O2- production was measured by electron paramagnetic resonance (EPR) spectroscopy using hydroxylamine TEMPONE-H as spin trap. A device has been constructed which provided permanent stirring of cardiomyocyte samples directly in the cavity and prevented cell aggregation. When substrates and antimycin A and/or rotenone are added, the radical production rate increased and reached its maximum in the presence of the both inhibitors. Superoxide dismutase as well as KCN suppressed the radical production, thus being suggestive of the generation of superoxide radicals in the bc1 complex, while the mechanism of O2- production is the same as was suggested for isolated mitochondria. The ratio between rates of O2- generation by isolated cardiomyocytes under various experimental conditions is in a good accord with corresponding parameter of isolated mitochondria. However, in the case of cardiomyocytes the absolute values of the O2- production rate are approximately twice as high as those in isolated mitochondria, presumably due to the partial damage of the mitochondrial respiratory chain during the isolation procedure.

  3. Fe(III) shifts the mitochondria permeability transition-eliciting capacity of mangiferin to protection of organelle. (United States)

    Pardo-Andreu, Gilberto L; Cavalheiro, Renata A; Dorta, Daniel J; Naal, Zeki; Delgado, René; Vercesi, Aníbal E; Curti, Carlos


    Mangiferin acts as a strong antioxidant on mitochondria. However, when in the presence of Ca(2+), mangiferin elicits mitochondrial permeability transition (MPT), as evidenced by cyclosporin A-sensitive mitochondrial swelling. We now provide evidence, by means of electrochemical and UV-visible spectroscopical analysis, that Fe(III) coordinates with mangiferin. The resulting mangiferin-Fe(III) complex does not elicit MPT and prevents MPT by scavenging reactive oxygen species. Indeed, the complex protects mitochondrial membrane protein thiols and glutathione from oxidation. Fe(III) also significantly increases the ability of mangiferin to scavenge the 2,2-diphenyl-1-picrylhydrazyl radical, as well as to display antioxidant activity toward antimycin A-induced H(2)O(2) production and t-butyl hydroperoxide-promoted membrane lipid peroxidation in mitochondria. We postulate that coordination with Fe(III) constitutes a potential protective mechanism toward the prooxidant action of mangiferin and other catechol-containing antioxidants regarding MPT induction. Potential therapeutic relevance of this finding for conditions of pathological iron overload is discussed.

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

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    Stanić Marina


    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

  5. Cells lacking Rieske iron-sulfur protein have a reactive oxygen species-associated decrease in respiratory complexes I and IV. (United States)

    Diaz, Francisca; Enríquez, José Antonio; Moraes, Carlos T


    Mitochondrial respiratory complexes of the electron transport chain (CI, CIII, and CIV) can be assembled into larger structures forming supercomplexes. We analyzed the assembly/stability of respiratory complexes in mouse lung fibroblasts lacking the Rieske iron-sulfur protein (RISP knockout [KO]cells), one of the catalytic subunits of CIII. In the absence of RISP, most of the remaining CIII subunits were able to assemble into a large precomplex that lacked enzymatic activity. CI, CIV, and supercomplexes were decreased in the RISP-deficient cells. Reintroduction of RISP into KO cells restored CIII activity and increased the levels of active CI, CIV, and supercomplexes. We found that hypoxia (1% O(2)) resulted in increased levels of CI, CIV, and supercomplex assembly in RISP KO cells. In addition, treatment of control cells with different oxidative phosphorylation (OXPHOS) inhibitors showed that compounds known to generate reactive oxygen species (ROS) (e.g., antimycin A and oligomycin) had a negative impact on CI and supercomplex levels. Accordingly, a superoxide dismutase (SOD) mimetic compound and SOD2 overexpression provided a partial increase in supercomplex levels in the RISP KO cells. Our data suggest that the stability of CI, CIV, and supercomplexes is regulated by ROS in the context of defective oxidative phosphorylation.

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

    Gomes, Marcelo Pedrosa; Juneau, Philippe


    We investigated the physiological responses of Lemna minor plants exposed to glyphosate. The deleterious effects of this herbicide on photosynthesis, respiration, and pigment concentrations were related to glyphosate-induced oxidative stress through hydrogen peroxide (H2O2) accumulation. By using photosynthetic and respiratory electron transport chain (ETC) inhibitors we located the primary site of reactive oxygen species (ROS) production in plants exposed to 500 mg glyphosate l(-1). Inhibition of mitochondrial ETC Complex I by rotenone reduced H2O2 concentrations in glyphosate-treated plants. Complex III activity was very sensitive to glyphosate which appears to act much like antimycin A (an inhibitor of mitochondrial ETC Complex III) by shunting electrons from semiquinone to oxygen, with resulting ROS formation. Confocal evaluations for ROS localization showed that ROS are initially produced outside of the chloroplasts upon initial glyphosate exposure. Our results indicate that in addition to interfering with the shikimate pathway, glyphosate can induce oxidative stress in plants through H2O2 formation by targeting the mitochondrial ETC, which would explain its observed effects on non-target organisms.

  7. Expression of the alternative oxidase mitigates beta-amyloid production and toxicity in model systems. (United States)

    El-Khoury, Riyad; Kaulio, Eveliina; Lassila, Katariina A; Crowther, Damian C; Jacobs, Howard T; Rustin, Pierre


    Mitochondrial dysfunction has been widely associated with the pathology of Alzheimer's disease, but there is no consensus on whether it is a cause or consequence of disease, nor on the precise mechanism(s). We addressed these issues by testing the effects of expressing the alternative oxidase AOX from Ciona intestinalis, in different models of AD pathology. AOX can restore respiratory electron flow when the cytochrome segment of the mitochondrial respiratory chain is inhibited, supporting ATP synthesis, maintaining cellular redox homeostasis and mitigating excess superoxide production at respiratory complexes I and III. In human HEK293-derived cells, AOX expression decreased the production of beta-amyloid peptide resulting from antimycin inhibition of respiratory complex III. Because hydrogen peroxide was neither a direct product nor substrate of AOX, the ability of AOX to mimic antioxidants in this assay must be indirect. In addition, AOX expression was able to partially alleviate the short lifespan of Drosophila models neuronally expressing human beta-amyloid peptides, whilst abrogating the induction of markers of oxidative stress. Our findings support the idea of respiratory chain dysfunction and excess ROS production as both an early step and as a pathologically meaningful target in Alzheimer's disease pathogenesis, supporting the concept of a mitochondrial vicious cycle underlying the disease.

  8. Occurrence of the malate-aspartate shuttle in various tumor types. (United States)

    Greenhouse, W V; Lehninger, A L


    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.

  9. Protective effects of rilmenidine and AGN 192403 on oxidative cytotoxicity and mitochondrial inhibitor-induced cytotoxicity in astrocytes. (United States)

    Choi, Dong-Hee; Kim, Dong-Hoon; Park, Yun-Gyu; Chun, Boe-Gwun; Choi, Sang-Hyun


    Oxidative stress and mitochondrial dysfunction are important aspects of pathogenesis, particularly in the brain, which is highly dependent on oxygen, and the protection of astrocytes is essential for neuroprotection. In this context, imidazoline drugs have been reported to be neuroprotective. Our recent study showed that imidazoline drugs, including guanabenz, inhibit the naphthazarin-induced oxidative cytotoxicity associated with lysosomal destabilization. We now report on a study into the protective effects of rilmenidine and AGN 192403, which have affinity for imidazoline-1 receptors, on the cytotoxicity induced by naphthazarin and inhibitors of mitochondrial respiration in astrocytes. Cytotoxicity was measured grossly by LDH release and by measuring changes in lysosomal membrane stability and features of mitochondrial membrane permeabilization. Naphthazarin-induced cytotoxicity was evidenced by the ordered development of lysosomal acridine orange relocation, decrease in mitochondrial potential, cytochrome c release, and caspase-9 activation, and was inhibited by guanabenz, rilmenidine, and AGN 192403. Antimycin A and rotenone induced mitochondrial dysfunction primarily, and their cytotoxicities were inhibited only by AGN 192403. Rilmenidine and guanabenz may have a lysosomal stabilizing effect, which underlies their protective effects. AGN 192403 might affect the mitochondrial cell death cascades, and had a novel protective effect on the cytotoxicity associated with mitochondrial dysfunction.

  10. Identification of interacting partners of Human Mpv17-like protein with a mitigating effect of mitochondrial dysfunction through mtDNA damage. (United States)

    Iida, Reiko; Ueki, Misuzu; Yasuda, Toshihiro


    Human Mpv17-like protein (M-LPH) has been suggested to participate in mitochondrial function. In this study, we investigated the proteins that interact with M-LPH, and identified four: H2A histone family, member X (H2AX), ribosomal protein S14 (RPS14), ribosomal protein S3 (RPS3) and B-cell receptor-associated protein 31 (Bap31). Immunofluorescence and subcellular fractionation studies revealed that M-LPH is localized predominantly in the nucleus, to some extent in a subset of mitochondria, and marginally in the cytosol. Mitochondrial M-LPH appeared as punctate foci, and these were co-localized with a subset of mitochondrial transcription factor A (TFAM) and mtDNA, indicating that M-LPH is localized in or in close proximity to mitochondrial nucleoids. RNAi-mediated knockdown of M-LPH resulted in an increase of mtDNA damage and reduced the expression of mtDNA-encoded genes. A ROS inducer, antimycin A, caused an increase in both the number and size of the mitochondrial M-LPH foci, and these foci were co-localized with two enzymes, DNA polymerase γ (POLG) and DNA ligase III (LIG3), both involved in mtDNA repair. Furthermore, knockdown of M-LPH hampered mitochondrial localization of these enzymes. Taken together, these observations suggest that M-LPH is involved in the maintenance of mtDNA and protects cells from mitochondrial dysfunction.

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

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    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. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells. (United States)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten


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

  13. Effects of chitosan and oligochitosan on development and mitochondrial function of Rhizopus stolonifer. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Ronald P. Haff


    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.

  15. Anoxic depolarization of hippocampal astrocytes: possible modulation by P2X7 receptors. (United States)

    Leichsenring, Anna; Riedel, Thomas; Qin, Ying; Rubini, Patrizia; Illes, Peter


    Current responses from CA1 neurons and stratum oriens astrocytes were recorded from hippocampal brain slices by means of the whole-cell patch-clamp technique. Anoxic depolarization (AD) was induced by an oxygen/glucose-deprived (OGD) medium also containing sodium iodoacetate and antimycin, in order to block glycolysis and oxidative phosphorylation, respectively. Anoxic depolarization has been reported to be due to the sudden increase of the extracellular K(+) concentration and the accompanying explosive rise in glutamate concentration. We asked ourselves whether the release of ATP activating P2X7 receptors is also involved in the AD. Although, the AD was evoked in absolute synchrony in neurons and astrocytes, and the NMDA receptor antagonistic AP-5 depressed these responses, neither the non-selective P2 receptor antagonist PPADS, nor the highly selective P2X7 receptor antagonist A438079 interfered with the AD or its delay time in neurons/astrocytes after inducing chemical hypoxia. However, A438079, but not PPADS increased in astrocytes the slow inward current observed in a hypoxic medium. It is concluded that ATP co-released with glutamate by hypoxic stimulation has only a minor function in the present brain slice system.

  16. 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: [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)


    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.

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

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    Lingyan Wang


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

  18. Antioxidant properties of Neu2000 on mitochondrial free radicals and oxidative damage. (United States)

    Visavadiya, Nishant P; McEwen, Melanie L; Pandya, Jignesh D; Sullivan, Patrick G; Gwag, Byoung Joo; Springer, Joe E


    Neu2000 [2-hydroxy-5-(2,3,5,6-tetrafluoro-4 trifluoromethylbenzylamino) benzoic acid] is a dual-acting neuroprotective agent that functions both as a noncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist and a free radical scavenger. In the present study, we investigated the scavenging activity of Neu2000 on various classes of reactive oxygen species and reactive nitrogen species (ROS/RNS) as well as its efficacy for reducing free radicals and oxidative stress/damage induced in spinal cord mitochondrial preparations. Neu2000 exerted scavenging activity against superoxide, nitric oxide, and hydroxyl radicals, and efficiently scavenged peroxynitrite. In the mitochondrial studies, Neu2000 markedly inhibited ROS/RNS and hydrogen peroxide levels following antimycin treatment. In addition, Neu2000 effectively scavenged hydroxyl radicals generated by iron(III)-ascorbate, reduced protein carbonyl formation mediated by hydroxyl radicals and peroxynitrite, and prevented glutathione oxidation caused by tert-butyl hydroperoxide in isolated mitochondria. Interestingly, incubation of isolated mitochondria with Neu2000 followed by centrifugation and removal of the supernatant also resulted in a concentration-dependent decrease in lipid peroxidation. This observation suggests that Neu2000 enters mitochondria to target free radicals or indirectly affects mitochondrial function in a manner that promotes antioxidant activity. The results of the present study demonstrate that Neu2000 possesses potent in vitro antioxidant activity due, most likely, to its active phenoxy group.


    Directory of Open Access Journals (Sweden)

    Katyshev A.I.


    -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.

  20. 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.

  1. Inhibition of the mitochondrial respiratory chain function abrogates quartz induced DNA damage in lung epithelial cells

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    Li Hui [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Haberzettl, Petra [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Albrecht, Catrin [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Hoehr, Doris [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Knaapen, Ad M. [Department of Health Risk Analysis and Toxicology, Nutrition and Toxicology Research Institute Maastricht (NUTRIM), University of Maastricht (Netherlands); Borm, Paul J.A. [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany); Hogeschool Zuyd Heerlen (Netherlands); Schins, Roel P.F. [Institut fuer umweltmedizinische Forschung (IUF) at the Heinrich-Heine-University, Auf' m Hennekamp 50, D-40225 Duesseldorf (Germany)]. E-mail:


    Respirable quartz dust has been classified as a human carcinogen by the International Agency for Research on Cancer. The aim of our study was to investigate the mechanisms of DNA damage by DQ12 quartz in RLE-6TN rat lung epithelial type II cells (RLE). Transmission electron microscopy and flow-cytometry analysis showed a rapid particle uptake (30 min to 4 h) of quartz by the RLE cells, but particles were not found within the cell nuclei. This suggests that DNA strand breakage and induction of 8-hydroxydeoxyguanosine - as also observed in these cells during these treatment intervals - did not result from direct physical interactions between particles and DNA, or from short-lived particle surface-derived reactive oxygen species. DNA damage by quartz was significantly reduced in the presence of the mitochondrial inhibitors rotenone and antimycin-A. In the absence of quartz, these inhibitors did not affect DNA damage, but they reduced cellular oxygen consumption. No signs of apoptosis were observed by quartz. Flow-cytometry analysis indicated that the reduced DNA damage by rotenone was not due to a possible mitochondria-mediated reduction of particle uptake by the RLE cells. Further proof of concept for the role of mitochondria was shown by the failure of quartz to elicit DNA damage in mitochondria-depleted 143B (rho-0) osteosarcoma cells, at concentrations where it elicited DNA damage in the parental 143B cell line. In conclusion, our data show that respirable quartz particles can elicit oxidative DNA damage in vitro without entering the nuclei of type II cells, which are considered to be important target cells in quartz carcinogenesis. Furthermore, our observations indicate that such indirect DNA damage involves the mitochondrial electron transport chain function, by an as-yet-to-be elucidated mechanism.

  2. Alternative oxidase: distribution, induction, properties, structure, regulation, and functions. (United States)

    Rogov, A G; Sukhanova, E I; Uralskaya, L A; Aliverdieva, D A; Zvyagilskaya, R A


    The respiratory chain in the majority of organisms with aerobic type metabolism features the concomitant existence of the phosphorylating cytochrome pathway and the cyanide- and antimycin A-insensitive oxidative route comprising a so-called alternative oxidase (AOX) as a terminal oxidase. In this review, the history of AOX discovery is described. Considerable evidence is presented that AOX occurs widely in organisms at various levels of organization and is not confined to the plant kingdom. This enzyme has not been found only in Archaea, mammals, some yeasts and protists. Bioinformatics research revealed the sequences characteristic of AOX in representatives of various taxonomic groups. Based on multiple alignments of these sequences, a phylogenetic tree was constructed to infer their possible evolution. The ways of AOX activation, as well as regulatory interactions between AOX and the main respiratory chain are described. Data are summarized concerning the properties of AOX and the AOX-encoding genes whose expression is either constitutive or induced by various factors. Information is presented on the structure of AOX, its active center, and the ubiquinone-binding site. The principal functions of AOX are analyzed, including the cases of cell survival, optimization of respiratory metabolism, protection against excess of reactive oxygen species, and adaptation to variable nutrition sources and to biotic and abiotic stress factors. It is emphasized that different AOX functions complement each other in many instances and are not mutually exclusive. Examples are given to demonstrate that AOX is an important tool to overcome the adverse aftereffects of restricted activity of the main respiratory chain in cells and whole animals. This is the first comprehensive review on alternative oxidases of various organisms ranging from yeasts and protists to vascular plants.

  3. Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation. (United States)

    Hsu, Hung-Chih; Chang, Wen-Ming; Wu, Jin-Yi; Huang, Chin-Chin; Lu, Fung-Jou; Chuang, Yi-Wen; Chang, Pey-Jium; Chen, Kai-Hua; Hong, Chang-Zern; Yeh, Rang-Hui; Liu, Tsan-Zon; Chen, Ching-Hsein


    Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.

  4. Ultrastructure and function of mitochondria in gametocytic stage of Plasmodium falciparum

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    Krungkrai J.


    Full Text Available Morphological properties of the mitochondrial organelles in the asexual and sexual gametocytic stages of Plasmodium falciparum have been analyzed and found to be markedly different. From in vitro cultures of both stages in human erythrocytes, it has been demonstrated that the asexual stages contained a defined double-membrane organelle having a few tubular-like cristae. The numbers of mitochondria in the gametocytes were found to be ~ 6 organelles per parasite, and they showed a greater density of the cristae than that of the asexual stage parasite. The organelles of the gametocytes were successfully purified by differential centrifugation following Percoll density gradient separation with the results of ~ 7 % yields and ~ 5 folds. The gametocytic organelles contained much more activities of mitochondrial electron transporting enzymes (i.e., cytochrome c reductase, cytochrome c oxidase than the asexual stage organelles. Mitochondrial function as measured by oxygen consumption were found to be different between these two stages organelles. Their rates of oxygen consumption were relatively low, as compared to those of human leukocyte and mouse liver mitochondria. In contrast to the coupled mammalian mitochondria, the gametocytic organelles were in the uncoupling state between oxidation and phosphorylation reactions during their respiration. However, they were sensitive to inhibitors of the electron transport system, e.g., antimycin A, cyanide. Our results suggest that the mitochondria of the gametocytic stages are metabolically active and still underdeveloped, although their inner membranes are extensively folded. The biochemical significance of the unique structure of the mitochondria in these developing stages in host erythrocytes remains to be elucidated.

  5. Cytotoxic mechanisms of Zn{sup 2+} and Cd{sup 2+} involve Na{sup +}/H{sup +} exchanger (NHE) activation by ROS

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    Koutsogiannaki, Sophia [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Evangelinos, Nikolaos [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Koliakos, George [Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, P.O. Box 17034, 54124 Thessaloniki (Greece); Kaloyianni, Martha [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)]. E-mail:


    The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in {center_dot}O{sub 2} {sup -} 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{sup +}/H{sup +} exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on {center_dot}O{sub 2} {sup -} production was mediated via the interaction of metal ions with {alpha}{sub 1}- and {beta}-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 ({delta}pHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi ({delta}pHi -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{sub 2} {sup -} 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-{gamma} receptors. In addition, differences between the two metals concerning NHE activation, O{sub 2} {sup -} production and interaction with adrenergic receptors were observed.

  6. Induction of mitochondrial alternative oxidase in response to a cell signal pathway down-regulating the cytochrome pathway prevents programmed cell death. (United States)

    Vanlerberghe, Greg C; Robson, Christine A; Yip, Justine Y H


    Treatment of tobacco (Nicotiana tabacum L. cv Petit Havana SR1) cells with cysteine (Cys) triggers a signal pathway culminating in a large loss of mitochondrial cytochrome (cyt) pathway capacity. This down-regulation of the cyt path likely requires events outside the mitochondrion and is effectively blocked by cantharidin or endothall, indicating that protein dephosphorylation is one critical process involved. Generation of reactive oxygen species, cytosolic protein synthesis, and Ca(2+) flux from organelles also appear to be involved. Accompanying the loss of cyt path is a large induction of alternative oxidase (AOX) protein and capacity. Induction of AOX allows the cells to maintain high rates of respiration, indicating that the lesion triggered by Cys is in the cyt path downstream of ubiquinone. Consistent with this, transgenic (AS8) cells unable to induce AOX (due to the presence of an antisense transgene) lose all respiratory capacity upon Cys treatment. This initiates in AS8 a programmed cell death pathway, as evidenced by the accumulation of oligonucleosomal fragments of DNA as the culture dies. Alternatively, wild-type cells remain viable and eventually recover their cyt path. Induction of AOX in response to a chemical inhibition of the cyt path (by antimycin A) is also dependent upon protein dephosphorylation and the generation of reactive oxygen species. Common events required for both down-regulation of the cyt path and induction of AOX may represent a mechanism to coordinate the biogenesis of these two electron transport paths. Such coordinate regulation may be necessary, not only to satisfy metabolic demands, but also to modulate the initiation of a programmed cell death pathway responsive to mitochondrial respiratory status.

  7. Ischemic proximal tubular injury primes mice to endotoxin-induced TNF-alpha generation and systemic release. (United States)

    Zager, R A; Johnson, Ali C M; Hanson, Sherry Y; Lund, Steve


    Endotoxemia (LPS) can exacerbate ischemic tubular injury and acute renal failure (ARF). The present study tested the following hypothesis: that acute ischemic damage sensitizes the kidney to LPS-mediated TNF-alpha generation, a process that can worsen inflammation and cytotoxicity. CD-1 mice underwent 15 min of unilateral renal ischemia. LPS (10 mg/kg iv), or its vehicle, was injected either 45 min before, or 18 h after, the ischemic event. TNF-alpha responses were gauged 2 h post-LPS injection by measuring plasma/renal cortical TNF-alpha and renal cortical TNF-alpha mRNA. Values were contrasted to those obtained in sham-operated mice or in contralateral, nonischemic kidneys. TNF-alpha generation by isolated mouse proximal tubules (PTs), and by cultured proximal tubule (HK-2) cells, in response to hypoxia-reoxygenation (H/R), oxidant stress, antimycin A (AA), or LPS was also assessed. Ischemia-reperfusion (I/R), by itself, did not raise plasma or renal cortical TNF-alpha or its mRNA. However, this same ischemic insult dramatically sensitized mice to LPS-mediated TNF-alpha increases in both plasma and kidney (approximately 2-fold). During late reperfusion, increased TNF-alpha mRNA levels also resulted. PTs generated TNF-alpha in response to injury. Neither AA nor LPS alone induced an HK-2 cell TNF-alpha response. However, when present together, AA+LPS induced approximately two- to fivefold increases in TNF-alpha/TNF-alpha mRNA. We conclude that modest I/R injury, and in vitro HK-2 cell mitochondrial inhibition (AA), can dramatically sensitize the kidney/PTs to LPS-mediated TNF-alpha generation and increases in TNF-alpha mRNA. That ischemia can "prime" tubules to LPS response(s) could have potentially important implications for sepsis syndrome, concomitant renal ischemia, and for the induction of ARF.

  8. The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation. (United States)

    Beavis, A D; Lehninger, A L


    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.

  9. 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.

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

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    A. R. Othman


    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.

  11. A possible role of oxidative stress in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells. (United States)

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Kedzior, Jakub; Słomińska, Ewa; Lipiński, Marcin; Wakabayashi, Takashi


    Apoptosis is induced not only during morphogenesis and embryogenesis but also under various pathological conditions, especially related to oxidative stress. Apoptotic cells are phagocytized by neighboring cells while necrotic cells cause local and general reactions sometimes lethal to our bodies. Data have been accumulated to demonstrate that the switch of the cell death mode from apoptosis to necrosis does occur. However, detailed mechanisms involved in the switch mechanism remain unsolved although decreases in the intracellular level of ATP and a burst in the cellular level of reactive oxygen species (ROS) have been proposed. Recently, we have shown that the population of apoptotic cells reaches maximum in human osteosarcoma 143B cells treated for 6h with menadione (MEN) while necrotic cells become predominant at 9h of the treatment. In the present study we have attempted to clarify the role of cellular ATP in the switch mechanism using rho(0) cells derived from human osteosarcoma rho+ cells. Results are summarized as follows: (1) Apoptotic and necrotic changes in rho(0) cells are much faster than rho+ cells after the treatment with MEN. (2) Cellular level of ATP in rho(0) cells remains essentially in the same level before and after the MEN-treatment while intracellular levels of superoxide continuously increase after the MEN-treatment. (3) rho+ cells treated with MEN in the presence of antimycin A plus oligomycin show similar changes to those of MEN-treated rho(0) cells. (4) MEN-induced increases in the cellular level of superoxide are distinctly suppressed by inhibitors of NADPH oxidase. These results suggest that the intracellular level of superoxide may be a key factor directly related to the switch mechanism from apoptosis to necrosis, and that decreases in cellular level of ATP accelerate both apoptotic and necrotic changes of the cells.

  12. Role of mitochondria in the switch mechanism of the cell death mode from apoptosis to necrosis--studies on rho0 cells. (United States)

    Wochna, Agnieszka; Niemczyk, Edyta; Kurono, Chieko; Masaoka, Makoto; Majczak, Anna; Kedzior, Jakub; Slominska, Ewa; Lipinski, Marcin; Wakabayashi, Takashi


    Detailed mechanisms of the switch of the cell death mode from apoptosis to necrosis remain to be solved, although the intracellular level of ATP and that of free radicals have been postulated to be the major factors involved in the mechanisms. In the present study menadione (MEN)-induced cell injury processes were studied using rho0 cells derived from human osteosarcoma 143B cells and parental rho+ cells co-treated with inhibitors of electron transfer chain of mitochondria or oligomycin, an inhibitor of ATP synthesis. Treatment of rho+ cells with 100 microM MEN induced apoptosis, which reached the maximum at 6 h, and was followed by an abrupt decrease thereafter, while necrotic cells (NC) increased continuously when they were judged by Annexin V and PI double staining. On the other hand, MEN induced apoptotic and necrotic changes much faster in rho0 cells compared to rho+ cells. The frequency to find apoptotic cells (AP) in the former cells was distinctly smaller than that to find NC judged by Annexin V and PI double staining. Electron microscopically, a major population of rho0 cells treated with MEN for 6 h consisted of intermediate cells, and a small number of AP co-existed. At 9 h of the treatment intermediate cells were exclusively seen, and AP were hardly detected. When parental rho+ cells were treated with MEN in the presence of oligomycin or oligomycin plus antimycin A both apoptotic and necrotic changes of the cells were distinctly accelerated. The intracellular level of superoxide in rho0 cells continuously increased after the MEN treatment, whereas that of ATP remained distinctly low before and after the MEN treatment compared to that in rho+ cells. These data suggest that the intracellular level of superoxide may be a key factor controlling the switch from apoptosis to necrosis.

  13. Evidence for the role of cyclic electron flow in photoprotection for oxygen-evolving complex. (United States)

    Huang, Wei; Yang, Ying-Jie; Hu, Hong; Zhang, Shi-Bao; Cao, Kun-Fang


    Cyclic electron flow (CEF) alleviates PSII photo-inhibition under high light by at least two different mechanisms: one is liked to thermal energy dissipation (qE) and the other one is independent of qE. However, the latter mechanism is unclear. Because the photodamage to PSII primarily occurred at the oxygen-evolving complex (OEC), and the stability of OEC is dependent on proton gradient across thylakoid membrane (ΔpH), we hypothesize that the CEF-dependent generation of ΔpH can alleviate photodamage to OEC. To test this hypothesis, we determined the effects of antimycin A (AA), methyl viologen (MV), chloramphenicol (CM), nigericin (Nig) on PSII activity and the stability of OEC for leaves of a light-demanding tropical tree species Erythrophleum guineense by the analysis of OKJIP chlorophyll a fluorescence transient. After high light treatment, the stronger decrease in Fv/Fm in the AA-, CM-, MV-, and Nig-treated samples was accompanied with larger photo damage of OEC. The AA-treated samples significantly showed lower CEF activity than the H2O-treated samples. Although the AA-treated leaves significantly showed stronger PSII photo-inhibition and photo-damage of OEC compared to the H2O-treated leaves, the value of non-photochemical quenching did not differ between them. Therefore, CEF activity was partly inhibited in the AA-treated samples, and the stronger PSII photo-inhibition in the AA-treated leaves was independent of qE. Taking together, we propose a hypothesis that CEF-dependent generation of ΔpH under high light plays an important role in photoprotection for the OEC activity.

  14. Elucidation of Photoprotective Mechanisms of PSI Against Fluctuating Light photoinhibition. (United States)

    Kono, Masaru; Terashima, Ichiro


    It has been claimed that the cyclic electron flow around PSI (CEF-PSI) plays an important role in protection of PSI against fluctuating light photoinhibition. However, the photoprotective mechanism of PSI is not fully elucidated. Here, we examined the mechanism, using two CEF-PSI mutants of Arabidopsis thaliana, and antimycin A, an inhibitor of the PGR5 (proton gradient regulation 5)-mediated CEF-PSI. Dark-adapted leaves in these plants were illuminated in fluctuating light alternating between high light at 1,200 µmol m(-2) s(-1) and low light at 30 µmol m(-2) s(-1) every 2 min, and PSI and PSII parameters were simultaneously measured for 160 min with 830 nm absorption and Chl fluorescence, respectively. When CEF-PSI, especially PGR5-mediated CEF-PSI, did not operate, the acceptor-side limitation of PSI, Y(NA), increased stepwise, leading to marked PSI photoinhibition. The deficiency of CFE-PSI decreased not only the electron transport rate through PSI but also the donor-side limitation of PSI, Y(ND), in high light phases. These results showed that the large Y(ND), observed only when CEF-PSI operated, contributed to suppression of PSI photoinhibition. Taken together with our previous report that high Y(NA) was alleviated by the enhancement of CEF-PSI, a model for the protective mechanisms of PSI is proposed. In this model, both alleviation of Y(NA) and acceleration of Y(ND) are indispensable, and for realization of such a situation, regulation of the electron flows, especially the PGR5-mediated CEF-PSI, plays a key role. It is important for effective protection to regulate the balance of Y(ND) and Y(NA) through CEF-PSI.

  15. The mitochondrial PPR protein LOVASTATIN INSENSITIVE 1 plays regulatory roles in cytosolic and plastidial isoprenoid biosynthesis through RNA editing. (United States)

    Tang, Jianwei; Kobayashi, Keiko; Suzuki, Masashi; Matsumoto, Shogo; Muranaka, Toshiya


    Unlike animals, plants synthesize isoprenoids via two pathways, the cytosolic mevalonate (MVA) pathway and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway. Little information is known about the mechanisms that regulate these complex biosynthetic networks over multiple organelles. To understand such regulatory mechanisms of the biosynthesis of isoprenoids in plants, we previously characterized the Arabidopsis mutant, lovastatin insensitive 1 (loi1), which is resistant to lovastatin and clomazone, specific inhibitors of the MVA and MEP pathways, respectively. LOI1 encodes a pentatricopeptide repeat (PPR) protein localized in mitochondria that is thought to have RNA binding ability and function in post-transcriptional regulation of mitochondrial gene expression. LOI1 belongs to the DYW subclass of PPR proteins, which is hypothesized to be correlated with RNA editing. As a result of analysis of RNA editing of mitochondrial genes in loi1, a defect in RNA editing of three genes, nad4, ccb203 and cox3, was identified in loi1. These genes are related to the respiratory chain. Wild type (WT) treated with some respiration inhibitors mimicked the loi1 phenotype. Interestingly, HMG-CoA reductase activity of WT treated with lovastatin combined with antimycin A, an inhibitor of complex III in the respiratory chain, was higher than that of WT treated with only lovastatin, despite the lack of alteration of transcript or protein levels of HMGR. These results suggest that HMGR enzyme activity is regulated through the respiratory cytochrome pathway. Although various mechanisms exist for isoprenoid biosynthesis, our studies demonstrate the novel possibility that mitochondrial respiration plays potentially regulatory roles in isoprenoid biosynthesis.

  16. 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.

  17. 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


    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

  18. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning. (United States)

    Leonard, Anthony P; Cameron, Robert B; Speiser, Jaime L; Wolf, Bethany J; Peterson, Yuri K; Schnellmann, Rick G; Beeson, Craig C; Rohrer, Bärbel


    Understanding the processes of mitochondrial dynamics (fission, fusion, biogenesis, and mitophagy) has been hampered by the lack of automated, deterministic methods to measure mitochondrial morphology from microscopic images. A method to quantify mitochondrial morphology and function is presented here using a commercially available automated high-content wide-field fluorescent microscopy platform and R programming-language-based semi-automated data analysis to achieve high throughput morphological categorization (puncta, rod, network, and large & round) and quantification of mitochondrial membrane potential. In conjunction with cellular respirometry to measure mitochondrial respiratory capacity, this method detected that increasing concentrations of toxicants known to directly or indirectly affect mitochondria (t-butyl hydroperoxide [TBHP], rotenone, antimycin A, oligomycin, ouabain, and carbonyl cyanide-p-trifluoromethoxyphenylhydrazone [FCCP]), decreased mitochondrial networked areas in cultured 661w cells to 0.60-0.80 at concentrations that inhibited respiratory capacity to 0.20-0.70 (fold change compared to vehicle). Concomitantly, mitochondrial swelling was increased from 1.4- to 2.3-fold of vehicle as indicated by changes in large & round areas in response to TBHP, oligomycin, or ouabain. Finally, the automated identification of mitochondrial location enabled accurate quantification of mitochondrial membrane potential by measuring intramitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence intensity. Administration of FCCP depolarized and administration of oligomycin hyperpolarized mitochondria, as evidenced by changes in intramitochondrial TMRM fluorescence intensities to 0.33- or 5.25-fold of vehicle control values, respectively. In summary, this high-content imaging method accurately quantified mitochondrial morphology and membrane potential in hundreds of thousands of cells on a per-cell basis, with sufficient throughput for pharmacological

  19. 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.

  20. Differential production of superoxide by neuronal mitochondria

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    Levin Leonard A


    Full Text Available Abstract Background Mitochondrial DNA (mtDNA mutations, which are present in all mitochondria-containing cells, paradoxically cause tissue-specific disease. For example, Leber's hereditary optic neuropathy (LHON results from one of three point mutations mtDNA coding for complex I components, but is only manifested in retinal ganglion cells (RGCs, a central neuron contained within the retina. Given that RGCs use superoxide for intracellular signaling after axotomy, and that LHON mutations increase superoxide levels in non-RGC transmitochondrial cybrids, we hypothesized that RGCs regulate superoxide levels differently than other neuronal cells. To study this, we compared superoxide production and mitochondrial electron transport chain (METC components in isolated RGC mitochondria to mitochondria isolated from cerebral cortex and neuroblastoma SK-N-AS cells. Results In the presence of the complex I substrate glutamate/malate or the complex II substrate succinate, the rate of superoxide production in RGC-5 cells was significantly lower than cerebral or neuroblastoma cells. Cerebral but not RGC-5 or neuroblastoma cells increased superoxide production in response to the complex I inhibitor rotenone, while neuroblastoma but not cerebral or RGC-5 cells dramatically decreased superoxide production in response to the complex III inhibitor antimycin A. Immunoblotting and real-time quantitative PCR of METC components demonstrated different patterns of expression among the three different sources of neuronal mitochondria. Conclusion RGC-5 mitochondria produce superoxide at significantly lower rates than cerebral and neuroblastoma mitochondria, most likely as a result of differential expression of complex I components. Diversity in METC component expression and function could explain tissue specificity in diseases associated with inherited mtDNA abnormalities.

  1. Modulation of mitochondrial bioenergetics in a skeletal muscle cell line model of mitochondrial toxicity

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    William Dott


    Full Text Available Mitochondrial toxicity is increasingly being implicated as a contributing factor to many xenobiotic-induced organ toxicities, including skeletal muscle toxicity. This has necessitated the need for predictive in vitro models that are able to sensitively detect mitochondrial toxicity of chemical entities early in the research and development process. One such cell model involves substituting galactose for glucose in the culture media. Since cells cultured in galactose are unable to generate sufficient ATP from glycolysis they are forced to rely on mitochondrial oxidative phosphorylation for ATP generation and consequently are more sensitive to mitochondrial perturbation than cells grown in glucose. The aim of this study was to characterise cellular growth, bioenergetics and mitochondrial toxicity of the L6 rat skeletal muscle cell line cultured in either high glucose or galactose media. L6 myoblasts proliferated more slowly when cultured in galactose media, although they maintained similar levels of ATP. Galactose cultured L6 cells were significantly more sensitive to classical mitochondrial toxicants than glucose-cultured cells, confirming the cells had adapted to galactose media. Analysis of bioenergetic function with the XF Seahorse extracellular flux analyser demonstrated that oxygen consumption rate (OCR was significantly increased whereas extracellular acidification rate (ECAR, a measure of glycolysis, was decreased in cells grown in galactose. Mitochondria operated closer to state 3 respiration and had a lower mitochondrial membrane potential and basal mitochondrial O2·– level compared to cells in the glucose model. An antimycin A (AA dose response revealed that there was no difference in the sensitivity of OCR to AA inhibition between glucose and galactose cells. Importantly, cells in glucose were able to up-regulate glycolysis, while galactose cells were not. These results confirm that L6 cells are able to adapt to growth in a

  2. The energy blockers 3-bromopyruvate and lonidamine: effects on bioenergetics of brain mitochondria. (United States)

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


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

  3. 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 BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSIONS/SIGNIFICANCE: 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.

  4. Recombinant Bcl-xL attenuates vascular hyperpermeability in a rat model of hemorrhagic shock (United States)

    Tharakan, B; McNeal, SI; Hunter, FA; Sawant, DA; Smythe, WR; Childs, EW


    Following hemorrhagic shock (HS), vascular hyperpermeability, that is, the leakage of fluid, nutrients and proteins into the extravascular space occurs primarily due to the disruption of the endothelial cell–cell adherens junctional complex. Studies from our laboratory demonstrate that activation of the mitochondria-mediated ‘intrinsic’ apoptotic signaling cascade has a significant role in modulating HS-induced hyperpermeability. Here we report the novel use of recombinant Bcl-xL, an anti-apoptotic protein, to control HS-induced vascular hyperpermeability. Our results corroborate involvement of vascular hyperpermeability and apoptotic signaling. HS (the mean arterial pressure (MAP) was reduced to 40 mm Hg for 60 min followed by resuscitation to 90 mm Hg for 60 min) in rats resulted in vascular hyperpermeability as determined by intravital microscopy. Treatment of Bcl-xL (2.5 µg/ml of rat blood in non-lipid cationic polymer, i.v.) before, during and even after HS attenuated or reversed HS-induced vascular hyperpermeability significantly (P<0.05). Conversely, treatment using Bcl-xL inhibitors, 2-methoxy antimycin (2-OMeAA) and ABT 737, significantly increased vascular hyperpermeability compared with sham (P<0.05). Bcl-xL treatment also decreased the amount of fluid volume required to maintain a MAP of 90 mm Hg during resuscitation (P<0.05). HS resulted in an increased mitochondrial reactive oxygen species formation, reduction of ΔΨm, mitochondrial release of cytochrome c and significant activation of caspase-3 (P<0.05). All of these effects were significantly inhibited by Bcl-xL pre-treatment (P<0.05). Our results show that recombinant Bcl-xL is effective against HS-induced vascular hyperpermeability that appears to be mediated through the preservation of ΔΨm and subsequent prevention of caspase-3 activation. PMID:27042339

  5. Computational modeling analysis of mitochondrial superoxide production under varying substrate conditions and upon inhibition of different segments of the electron transport chain. (United States)

    Markevich, Nikolai I; Hoek, Jan B


    A computational mechanistic model of superoxide (O2•-) formation in the mitochondrial electron transport chain (ETC) was developed to facilitate the quantitative analysis of factors controlling mitochondrial O2•- production and assist in the interpretation of experimental studies. The model takes into account all individual electron transfer reactions in Complexes I and III. The model accounts for multiple, often seemingly contradictory observations on the effects of ΔΨ and ΔpH, and for the effects of multiple substrate and inhibitor conditions, including differential effects of Complex III inhibitors antimycin A, myxothiazol and stigmatellin. Simulation results confirm that, in addition to O2•- formation in Complex III and at the flavin site of Complex I, the quinone binding site of Complex I is an additional superoxide generating site that accounts for experimental observations on O2•- production during reverse electron transfer. However, our simulation results predict that, when cytochrome c oxidase is inhibited during oxidation of succinate, ROS production at this site is eliminated and almost all superoxide in Complex I is generated by reduced FMN, even when the redox pressure for reverse electron transfer from succinate is strong. In addition, the model indicates that conflicting literature data on the kinetics of electron transfer in Complex III involving the iron-sulfur protein-cytochrome bL complex can be resolved in favor of a dissociation of the protein only after electron transfer to cytochrome bH. The model predictions can be helpful in understanding factors driving mitochondrial superoxide formation in intact cells and tissues.

  6. Glucose, Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals. (United States)

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


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

  7. The metabolism of malate by cultured rat brain astrocytes

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    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))


    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.

  8. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

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    Shilpi Khare


    Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

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

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    Goldenberg Anna


    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

  10. Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes. (United States)

    Fulks, R M; Stadtman, E R


    When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged by the incorporation of [14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incorporation of [14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogenously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe2+ (or Ni2+) together with ATP (or other nucleoside di- and triphosphates. After 40-60 h dialysis Fe3+ together with NADH (but

  11. Non-endothelial endothelin counteracts hypoxic vasodilation in porcine large coronary arteries

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    Fröbert Ole


    glycol catalase (300 u/ml inhibited H2O2 vasodilation, but failed to affect vasodilation to O2 lowering. Neither did PEG-SOD (polyethylene glycol superoxide dismutase(70 u/ml affect vasodilation to O2 lowering. The mitochondrial inhibitors rotenone (1 μM and antimycin A (1 μM both inhibited hypoxic vasodilatation. Conclusion The present results in porcine coronary arteries suggest NO contributes to hypoxic vasodilation, probably through K channel opening, which is reversed by addition of ET-1 and enhanced by endothelin receptor antagonism. These latter findings suggest that endothelin receptor activation counteracts hypoxic vasodilation.

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

    Colson, A M; Slonimski, P P


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

  13. Mitochondrial dysfunction leads to deconjugation of quercetin glucuronides in inflammatory macrophages.

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    Akari Ishisaka

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

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

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


    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

  15. Evaluation of methylmercury biotransformation using rat liver slices

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    Yasutake, A. [Biochemistry Section, National Inst. for Minamata Disease, Minamata, Kumamoto (Japan); Hirayama, K. [Kumamoto University College of Medical Science, Kuhonji (Japan)


    To examine the demethylation reaction of methylmercury (MeHg) in rat liver, slices prepared from MeHg-treated rats were incubated in L-15 medium under 95% O{sub 2}/5% CO{sub 2} atmosphere. During the incubation, the amount of inorganic Hg in the slices markedly increased in a time-dependent manner, although the concentration of total Hg remained unchanged. Since the C-Hg bond in MeHg was demonstrated to be cleaved by the action of some reactive oxygen species, the effects on MeHg demethylation of several reagents that could modify reactive oxygen production were examined in the present system. Methylviologen was found to be an effective enhancer of the demethylation reaction with only a minor effect on lipid peroxidation. On the other hand, ferrous ion added to the medium showed no effect on demethylation in the presence or absence of methylviologen, although lipid peroxide levels were increased significantly by ferrous ion. Similarly, deferoxamine mesylate, which effectively suppressed the increase in lipid peroxide levels, also had no effect on demethylation. Furthermore, hydroxy radical scavengers, such as mannitol and dimethylsulfoxide, had no effect on inorganic Hg production. Rotenone, an inhibitor of complex I in the mitochondrial electron transport system, increased levels of both inorganic Hg and lipid peroxide. However, other inhibitors, such as antimycin A, myxothiazole and NaCN, significantly suppressed the demethylation reaction. Cell fractionation of the MeHg-treated rat liver revealed that the ratio of inorganic Hg to total Hg was highest in the mitochondrial fraction. Furthermore, superoxide anion could degrade MeHg in an organic solvent but not in water. These results suggested that the demethylation of MeHg by the liver slice would proceed with the aid of superoxide anion produced in the electron transfer system at the hydrophobic mitochondrial inner membrane. Furthermore, the involvement of hydroxy radicals, which have been demonstrated to be

  16. 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.

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

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


    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.

  18. Inhibition of membrane lipid peroxidation by a radical scavenging mechanism: a novel function for hydroxyl-containing ionophores. (United States)

    Grijalba, M T; Andrade, P B; Meinicke, A R; Castilho, R F; Vercesi, A E; Schreier, S


    In the present study we show that K+/H+ hydroxyl-containing ionophores lasalocid-A (LAS) and nigericin (NIG) in the nanomolar concentration range, inhibit Fe2+-citrate and 2,2'-azobis(2-amidinopropane) dihydrochloride (ABAP)-induced lipid peroxidation in intact rat liver mitochondria and in egg phosphatidylcholine (PC) liposomes containing negatively charged lipids--dicetyl phosphate (DCP) or cardiolipin (CL)--and KCl as the osmotic support. In addition, monensin (MON), a hydroxyl-containing ionophore with higher affinity for Na+ than for K+, promotes a similar effect when NaCl is the osmotic support. The protective effect of the ionophores is not observed when the osmolyte is sucrose. Lipid peroxidation was evidenced by mitochondrial swelling, antimycin A-insensitive O2 consumption, formation of thiobarbituric acid-reactive substances (TBARS), conjugated dienes, and electron paramagnetic resonance (EPR) spectra of an incorporated lipid spin probe. A time-dependent decay of spin label EPR signal is observed as a consequence of lipid peroxidation induced by both inductor systems in liposomes. Nitroxide destruction is inhibited by butylated hydroxytoluene, a known antioxidant, and by the hydroxyl-containing ionophores. In contrast, valinomycin (VAL), which does not possess alcoholic groups, does not display this protective effect. Effective order parameters (Seff), determined from the spectra of an incorporated spin label are larger in the presence of salt and display a small increase upon addition of the ionophores, as a result of the increase of counter ion concentration at the negatively charged bilayer surface. This condition leads to increased formation of the ion-ionophore complex, the membrane binding (uncharged) species. The membrane-incorporated complex is the active species in the lipid peroxidation inhibiting process. Studies in aqueous solution (in the absence of membranes) showed that NIG and LAS, but not VAL, decrease the Fe2+-citrate-induced production

  19. 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

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    Zoll J


    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


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    Subota I.Yu.


    Full Text Available We studied an impact of the widely spread intra-cellular signals Ca2+ and сAMP on activity of the protein phosphorylation in maize mitochondria. The use of the isolated mitochondria is a convenient model system for investigation of the different physiological processes, for example for simulation of the different stress conditions. The treatment of maize mitochondria with high concentration of calcium ions which mimics the initial stage of apoptosis led to an increase of the phosphorylation level of some proteins and to an additional phosphorylation of the 59 and 66 kDa proteins. The treatment of the mitoplasts, i.e., the mitochondria devoid of the outer membrane with calcium ions insignificantly induced the activity of protein phosphorylation. It is assumed that the outer membrane is essential for Ca2+ signal transduction to plant mitochondria. We also identified a 94 kDa protein involved in phosphorylation of the mitochondrial proteins. This protein might be a single-subunit protein kinase or one of the subunits of the protein kinase complex. Antimycin A and KCN which are the inhibitors of mitochondria respiration increased the phosphorylation activity of the mitochondrial polypeptides. The effect of this inhibitors was similar both in in organello system and at the level of the whole plant. It should be noticed that at the level of the whole plant the effect of KCN on activity of the mitochondrial protein phosphorylation was more essential. Some considerable differences were found both at the level of protein phosphorylation and in electrophoresis patterns representing the intact mitochondria, the mitoplasts and the outer membrane fraction. The activity of protein phosphorylation in mitoplasts and the outer membrane fraction was extremely high compared to the phosphorylation activity of the mitochondrial proteins. This could be explained by the higher level of “substrate phosphoprotein phosphatase” in the outer membrane of mitochondria

  1. Modulation of the Pasteur effect in retinal cells: implications for understanding compensatory metabolic mechanisms. (United States)

    Winkler, Barry S; Sauer, Michael W; Starnes, Catherine A


    The purpose of the present experiments was to enhance understanding of the factors that are critical for the survival of retinal cells exposed to mitochondrial inhibition. Confluent cultures of Müller cells (rMC-1) and human retinal pigment epithelial cells (hRPE) were incubated in Dulbecco's minimal essential medium in the presence and absence of 1x10(-5)M Antimycin A, an inhibitor of mitochondrial electron transport. To modulate the rates of aerobic and anaerobic glycolysis, cells were incubated in media containing varying concentrations of glucose and 1-100 micro M of iodoacetic acid (IAA), an inhibitor of glyceraldehdye-3-phosphate dehydrogenase (G3PDH). Measurements were made of G3PDH, lactic acid production, and cellular ATP levels, along with an examination of cellular morphology, the latter providing an index of cellular viability. Control rMC-1 and hRPE produced lactate aerobically, respectively, at 0.48 and 1.50 micro molhr(-1)/10(6) cells. Anaerobically, lactate production increased 2-fold in rMC-1 and 3-fold in hRPE. Anaerobic ATP levels in both types of cells were maintained at control levels over 8hr. Experimental conditions were sought that would modulate only the capacity of rMC-1 and hRPE to increase glycolysis following mitochondrial inhibition, i.e. alter their Pasteur effect. We used low concentrations of IAA to partially inhibit G3PDH. Incubation of rMC-1 with IAA for 6hr caused a graded inhibition of G3PDH: 70% inhibition with 1 micro M, 90% with 5 micro M, 97% with 10 micro M, and 100% with 100 micro M. While the aerobic and anaerobic rates of lactic acid production were not altered by 1 micro M IAA, both were suppressed completely by 100 micro M IAA. However, incubation of rMC-1 with 5 micro M IAA caused a decrease of 30% in the rate of anaerobic lactic acid production but no change in the rate of aerobic glycolysis. Moreover, with 5 micro M IAA, rMC-1 incubated aerobically maintained ATP levels, but anaerobic ATP content decreased to a low