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Sample records for abt-737-induced mitochondrial membrane

  1. Biguanides sensitize leukemia cells to ABT-737-induced apoptosis by inhibiting mitochondrial electron transport

    Science.gov (United States)

    Velez, Juliana; Pan, Rongqing; Lee, Jason T.C.; Enciso, Leonardo; Suarez, Marta; Duque, Jorge Eduardo; Jaramillo, Daniel; Lopez, Catalina; Morales, Ludis; Bornmann, William; Konopleva, Marina; Krystal, Gerald; Andreeff, Michael; Samudio, Ismael

    2016-01-01

    Metformin displays antileukemic effects partly due to activation of AMPK and subsequent inhibition of mTOR signaling. Nevertheless, Metformin also inhibits mitochondrial electron transport at complex I in an AMPK-independent manner, Here we report that Metformin and rotenone inhibit mitochondrial electron transport and increase triglyceride levels in leukemia cell lines, suggesting impairment of fatty acid oxidation (FAO). We also report that, like other FAO inhibitors, both agents and the related biguanide, Phenformin, increase sensitivity to apoptosis induction by the bcl-2 inhibitor ABT-737 supporting the notion that electron transport antagonizes activation of the intrinsic apoptosis pathway in leukemia cells. Both biguanides and rotenone induce superoxide generation in leukemia cells, indicating that oxidative damage may sensitize toABT-737 induced apoptosis. In addition, we demonstrate that Metformin sensitizes leukemia cells to the oligomerization of Bak, suggesting that the observed synergy with ABT-737 is mediated, at least in part, by enhanced outer mitochondrial membrane permeabilization. Notably, Phenformin was at least 10-fold more potent than Metformin in abrogating electron transport and increasing sensitivity to ABT-737, suggesting that this agent may be better suited for targeting hematological malignancies. Taken together, our results suggest that inhibition of mitochondrial metabolism by Metformin or Phenformin is associated with increased leukemia cell susceptibility to induction of intrinsic apoptosis, and provide a rationale for clinical studies exploring the efficacy of combining biguanides with the orally bioavailable derivative of ABT-737, Venetoclax. PMID:27283492

  2. BAX and BAK1 are dispensable for ABT-737-induced dissociation of the BCL2-BECN1 complex and autophagy.

    Science.gov (United States)

    Pedro, Jose Manuel Bravo-San; Wei, Yongjie; Sica, Valentina; Maiuri, Maria Chiara; Zou, Zhongju; Kroemer, Guido; Levine, Beth

    2015-01-01

    Disruption of the complex of BECN1 with BCL2 or BCL2L1/BCL-XL is an essential switch that turns on cellular autophagy in response to environmental stress or treatment with BH3 peptidomimetics. Recently, it has been proposed that BCL2 and BCL2L1/BCL-XL may inhibit autophagy indirectly through a mechanism dependent on the proapoptotic BCL2 family members, BAX and BAK1. Here we report that the BH3 mimetic, ABT-737, induces autophagy in parallel with disruption of BCL2-BECN1 binding in 2 different apoptosis-deficient cell types lacking BAX and BAK1, namely in mouse embryonic fibroblasts cells and in human colon cancer HCT116 cells. We conclude that the BH3 mimetic ABT-737 induces autophagy through a BAX and BAK1-independent mechanism that likely involves disruption of BECN1 binding to antiapoptotic BCL2 family members.

  3. Formation and Regulation of Mitochondrial Membranes

    Directory of Open Access Journals (Sweden)

    Laila Cigana Schenkel

    2014-01-01

    Full Text Available Mitochondrial membrane phospholipids are essential for the mitochondrial architecture, the activity of respiratory proteins, and the transport of proteins into the mitochondria. The accumulation of phospholipids within mitochondria depends on a coordinate synthesis, degradation, and trafficking of phospholipids between the endoplasmic reticulum (ER and mitochondria as well as intramitochondrial lipid trafficking. Several studies highlight the contribution of dietary fatty acids to the remodeling of phospholipids and mitochondrial membrane homeostasis. Understanding the role of phospholipids in the mitochondrial membrane and their metabolism will shed light on the molecular mechanisms involved in the regulation of mitochondrial function and in the mitochondrial-related diseases.

  4. Mitofilin complexes : conserved organizers of mitochondrial membrane architecture

    NARCIS (Netherlands)

    Zerbes, Ralf M.; van der Klei, Ida J.; Veenhuis, Marten; Pfanner, Nikolaus; van der Laan, Martin; Bohnert, Maria

    2012-01-01

    Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membran

  5. Methylseleninic acid potentiates multiple types of cancer cells to ABT-737-induced apoptosis by targeting Mcl-1 and Bad

    DEFF Research Database (Denmark)

    Yin, Shutao; Dong, Yinhui; Li, Jinghua

    2012-01-01

    ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can over...... found that expression of Bax was required for the efficient execution of synergistic sensitization. Our findings, for the first time, provide a strong mechanistic rationale for developing MSeA as a novel sensitizing agent of ABT-737....

  6. Effect of tricyclic drugs on mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Eto,Kohei

    1985-08-01

    Full Text Available The effects of tricyclic drugs (clomipramine, imipramine, chlorpromazine and promethazine on isolated liver mitochondria of rats were examined. All the drugs tested accelerated state 4 respiration. Their stimulative potency at concentrations below 100 microM was in the order of chlorpromazine greater than clomipramine greater than imipramine, promethazine. On state 3 respiration, the chlorine containing drugs had an inhibitive effect at high concentrations, while the other drugs seemed to have a slightly stimulative effect. These drugs stimulated latent ATPase activity of mitochondria. Clomipramine and chlorpromazine inhibited 2, 4-dinitrophenol-stimulated ATPase activity in a dose-dependent fashion. Imipramine also inhibited 2, 4-dinitrophenol-stimulated ATPase activity at high concentrations. Promethazine, however, had almost no effect. All the drugs induced potassium release from mitochondrial vesicles, and their potency was in the order of clomipramine greater than chlorpromazine greater than imipramine greater than promethazine. These results suggest that clomipramine, imipramine, chlorpromazine and promethazine cause impediments in both mitochondrial respiration and ion compartmentation, and that the chlorine containing drugs are more toxic than others on the functions of the mitochondrial membrane.

  7. Appoptosin interacts with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

    Science.gov (United States)

    Zhang, Cuilin; Shi, Zhun; Zhang, Lingzhi; Zhou, Zehua; Zheng, Xiaoyuan; Liu, Guiying; Bu, Guojun; Fraser, Paul E; Xu, Huaxi; Zhang, Yun-Wu

    2016-03-01

    Mitochondrial morphology is regulated by fusion and fission machinery. Impaired mitochondria dynamics cause various diseases, including Alzheimer's disease. Appoptosin (encoded by SLC25A38) is a mitochondrial carrier protein that is located in the mitochondrial inner membrane. Appoptosin overexpression causes overproduction of reactive oxygen species (ROS) and caspase-dependent apoptosis, whereas appoptosin downregulation abolishes β-amyloid-induced mitochondrial fragmentation and neuronal death during Alzheimer's disease. Herein, we found that overexpression of appoptosin resulted in mitochondrial fragmentation in a manner independent of its carrier function, ROS production or caspase activation. Although appoptosin did not affect levels of mitochondrial outer-membrane fusion (MFN1 and MFN2), inner-membrane fusion (OPA1) and fission [DRP1 (also known as DNM1L) and FIS1] proteins, appoptosin interacted with MFN1 and MFN2, as well as with the mitochondrial ubiquitin ligase MITOL (also known as MARCH5) but not OPA1, FIS1 or DRP1. Appoptosin overexpression impaired the interaction between MFN1 and MFN2, and mitochondrial fusion. By contrast, co-expression of MFN1, MITOL and a dominant-negative form of DRP1, DRP1(K38A), partially rescued appoptosin-induced mitochondrial fragmentation and apoptosis, whereas co-expression of FIS1 aggravated appoptosin-induced apoptosis. Together, our results demonstrate that appoptosin can interact with mitochondrial outer-membrane fusion proteins and regulates mitochondrial morphology.

  8. Tamoxifen inhibits mitochondrial membrane damage caused by disulfiram.

    Science.gov (United States)

    Pavón, Natalia; Buelna-Chontal, Mabel; Correa, Francisco; Yoval-Sánchez, Belem; Belmont, Javier; Hernández-Esquivel, Luz; Rodríguez-Zavala, José S; Chávez, Edmundo

    2017-10-01

    In this work, we studied the protective effects of tamoxifen (TAM) on disulfiram (Dis)-induced mitochondrial membrane insult. The results indicate that TAM circumvents the inner membrane leakiness manifested as Ca(2+) release, mitochondrial swelling, and collapse of the transmembrane electric gradient. Furthermore, it was found that TAM prevents inactivation of the mitochondrial enzyme aconitase and detachment of cytochrome c from the inner membrane. Interestingly, TAM also inhibited Dis-promoted generation of hydrogen peroxide. Given that TAM is an antioxidant molecule, it is plausible that its protection may be due to the inhibition of Dis-induced oxidative stress.

  9. Mitochondrial DNA mutations provoke dominant inhibition of mitochondrial inner membrane fusion.

    Directory of Open Access Journals (Sweden)

    Cécile Sauvanet

    Full Text Available Mitochondria are highly dynamic organelles that continuously move, fuse and divide. Mitochondrial dynamics modulate overall mitochondrial morphology and are essential for the proper function, maintenance and transmission of mitochondria and mitochondrial DNA (mtDNA. We have investigated mitochondrial fusion in yeast cells with severe defects in oxidative phosphorylation (OXPHOS due to removal or various specific mutations of mtDNA. We find that, under fermentative conditions, OXPHOS deficient cells maintain normal levels of cellular ATP and ADP but display a reduced mitochondrial inner membrane potential. We demonstrate that, despite metabolic compensation by glycolysis, OXPHOS defects are associated to a selective inhibition of inner but not outer membrane fusion. Fusion inhibition was dominant and hampered the fusion of mutant mitochondria with wild-type mitochondria. Inhibition of inner membrane fusion was not systematically associated to changes of mitochondrial distribution and morphology, nor to changes in the isoform pattern of Mgm1, the major fusion factor of the inner membrane. However, inhibition of inner membrane fusion correlated with specific alterations of mitochondrial ultrastructure, notably with the presence of aligned and unfused inner membranes that are connected to two mitochondrial boundaries. The fusion inhibition observed upon deletion of OXPHOS related genes or upon removal of the entire mtDNA was similar to that observed upon introduction of point mutations in the mitochondrial ATP6 gene that are associated to neurogenic ataxia and retinitis pigmentosa (NARP or to maternally inherited Leigh Syndrome (MILS in humans. Our findings indicate that the consequences of mtDNA mutations may not be limited to OXPHOS defects but may also include alterations in mitochondrial fusion. Our results further imply that, in healthy cells, the dominant inhibition of fusion could mediate the exclusion of OXPHOS-deficient mitochondria from

  10. Role of MINOS in Mitochondrial Membrane Architecture : Cristae Morphology and Outer Membrane Interactions Differentially Depend on Mitofilin Domains

    NARCIS (Netherlands)

    Zerbes, Ralf M.; Bohnert, Maria; Stroud, David A.; von der Malsburg, Karina; Kram, Anita; Oeljeklaus, Silke; Warscheid, Bettina; Becker, Thomas; Wiedemann, Nils; Veenhuis, Marten; van der Klei, Ida J.; Pfanner, Nikolaus; van der Laan, Martin

    2012-01-01

    The mitochondrial inner membrane contains a large protein complex crucial for membrane architecture, the mitochondrial inner membrane organizing system (MINOS). MINOS is required for keeping cristae membranes attached to the inner boundary membrane via crista junctions and interacts with protein com

  11. Mechanisms of ER Stress-Mediated Mitochondrial Membrane Permeabilization.

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

    During apoptosis, the process of mitochondrial outer membrane permeabilization (MOMP) represents a point-of-no-return as it commits the cell to death. Here we have assessed the role of caspases, Bcl-2 family members and the mitochondrial permeability transition pore on ER stress-induced MOMP and subsequent cell death. Induction of ER stress leads to upregulation of several genes such as Grp78, Edem1, Erp72, Atf4, Wars, Herp, p58ipk, and ERdj4 and leads to caspase activation, release of mitochondrial intermembrane proteins and dissipation of mitochondrial transmembrane potential (DeltaPsim). Mouse embryonic fibroblasts (MEFs) from caspase-9, -2 and, -3 knock-out mice were resistant to ER stress-induced apoptosis which correlated with decreased processing of pro-caspase-3 and -9. Furthermore, pretreatment of cells with caspase inhibitors (Boc-D.fmk and DEVD.fmk) attenuated ER stress-induced loss of DeltaPsim. However, only deficiency of caspase-9 and -2 could prevent ER stress-mediated loss of DeltaPsim. Bcl-2 overexpression or pretreatment of cells with the cell permeable BH4 domain (BH4-Tat) or the mitochondrial permeability transition pore inhibitors, bongkrekic acid or cyclosporine A, attenuated the ER stress-induced loss of DeltaPsim. These data suggest a role for caspase-9 and -2, Bcl-2 family members and the mitochondrial permeability transition pore in loss of mitochondrial membrane potential during ER stress-induced apoptosis.

  12. Mitochondrial outer membrane proteome of Trypanosoma brucei reveals novel factors required to maintain mitochondrial morphology.

    Science.gov (United States)

    Niemann, Moritz; Wiese, Sebastian; Mani, Jan; Chanfon, Astrid; Jackson, Christopher; Meisinger, Chris; Warscheid, Bettina; Schneider, André

    2013-02-01

    Trypanosoma brucei is a unicellular parasite that causes devastating diseases in humans and animals. It diverged from most other eukaryotes very early in evolution and, as a consequence, has an unusual mitochondrial biology. Moreover, mitochondrial functions and morphology are highly regulated throughout the life cycle of the parasite. The outer mitochondrial membrane defines the boundary of the organelle. Its properties are therefore key for understanding how the cytosol and mitochondria communicate and how the organelle is integrated into the metabolism of the whole cell. We have purified the mitochondrial outer membrane of T. brucei and characterized its proteome using label-free quantitative mass spectrometry for protein abundance profiling in combination with statistical analysis. Our results show that the trypanosomal outer membrane proteome consists of 82 proteins, two-thirds of which have never been associated with mitochondria before. 40 proteins share homology with proteins of known functions. The function of 42 proteins, 33 of which are specific to trypanosomatids, remains unknown. 11 proteins are essential for the disease-causing bloodstream form of T. brucei and therefore may be exploited as novel drug targets. A comparison with the outer membrane proteome of yeast defines a set of 17 common proteins that are likely present in the mitochondrial outer membrane of all eukaryotes. Known factors involved in the regulation of mitochondrial morphology are virtually absent in T. brucei. Interestingly, RNAi-mediated ablation of three outer membrane proteins of unknown function resulted in a collapse of the network-like mitochondrion of procyclic cells and for the first time identified factors that control mitochondrial shape in T. brucei.

  13. Bcl-2 apoptosis proteins, mitochondrial membrane curvature, and cancer

    Science.gov (United States)

    Hwee Lai, Ghee; Schmidt, Nathan; Sanders, Lori; Mishra, Abhijit; Wong, Gerard; Ivashyna, Olena; Christenson, Eric; Schlesinger, Paul; Akabori, Kiyotaka; Santangelo, Christian

    2012-02-01

    Critical interactions between Bcl-2 family proteins permeabilize the outer mitochondrial membrane, a common decision point early in the intrinsic apoptotic pathway that irreversibly commits the cell to death. However, a unified picture integrating the essential non-passive role of lipid membranes with the contested dynamics of Bcl-2 regulation remains unresolved. Correlating results between synchrotron x-ray diffraction and microscopy in cell-free assays, we report activation of pro-apoptotic Bax induces strong pure negative Gaussian membrane curvature topologically necessary for pore formation and membrane remodeling events. Strikingly, Bcl-xL suppresses not only Bax-induced pore formation, but also membrane remodeling by disparate systems including cell penetrating, antimicrobial or viral fusion peptides, and bacterial toxin, none of which have BH3 allosteric domains to mediate direct binding. We propose a parallel mode of Bcl-2 pore regulation in which Bax and Bcl-xL induce antagonistic and mutually interacting Gaussian membrane curvatures. The universal nature of curvature-mediated interactions allows synergy with direct binding mechanisms, and potentially accounts for the Bcl-2 family modulation of mitochondrial fission/fusion dynamics.

  14. Role of cardiolipins in the inner mitochondrial membrane: insight gained through atom-scale simulations

    DEFF Research Database (Denmark)

    Róg, Tomasz; Martinez-Seara, Hector; Munck, Nana

    2009-01-01

    Mitochondrial membranes are unique in many ways. Unlike other cellular membranes, they are comprised of two membranes instead of just one, and cardiolipins, one of the abundant lipid species in mitochondrial membranes, are not found in significant amounts elsewhere in the cell. Among other aspect...

  15. Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity

    DEFF Research Database (Denmark)

    Larsen, Anna Karina; Malinska, Dominika; Koszela-Piotrowska, Izabela

    2012-01-01

    The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time......-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 µg/mL, however, lower PEI levels still exert...... some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 µg/mL PEI) followed by a slower...

  16. Detergent-resistant membrane subfractions containing proteins of plasma membrane, mitochondrial, and internal membrane origins.

    Science.gov (United States)

    Mellgren, Ronald L

    2008-04-24

    HEK293 cell detergent-resistant membranes (DRMs) isolated by the standard homogenization protocol employing a Teflon pestle homogenizer yielded a prominent opaque band at approximately 16% sucrose upon density gradient ultracentrifugation. In contrast, cell disruption using a ground glass tissue homogenizer generated three distinct DRM populations migrating at approximately 10%, 14%, and 20% sucrose, named DRM subfractions A, B, and C, respectively. Separation of the DRM subfractions by mechanical disruption suggested that they are physically associated within the cellular environment, but can be dissociated by shear forces generated during vigorous homogenization. All three DRM subfractions possessed cholesterol and ganglioside GM1, but differed in protein composition. Subfraction A was enriched in flotillin-1 and contained little caveolin-1. In contrast, subfractions B and C were enriched in caveolin-1. Subfraction C contained several mitochondrial membrane proteins, including mitofilin and porins. Only subfraction B appeared to contain significant amounts of plasma membrane-associated proteins, as revealed by cell surface labeling studies. A similar distribution of DRM subfractions, as assessed by separation of flotillin-1 and caveolin-1 immunoreactivities, was observed in CHO cells, in 3T3-L1 adipocytes, and in HEK293 cells lysed in detergent-free carbonate. Teflon pestle homogenization of HEK293 cells in the presence of the actin-disrupting agent latrunculin B generated DRM subfractions A-C. The microtubule-disrupting agent vinblastine did not facilitate DRM subfraction separation, and DRMs prepared from fibroblasts of vimentin-null mice were present as a single major band on sucrose gradients, unless pre-treated with latrunculin B. These results suggest that the DRM subfractions are interconnected by the actin cytoskeleton, and not by microtubes or vimentin intermediate filaments. The subfractions described may prove useful in studying discrete protein

  17. Mitochondrial DNA damage associated with lipid peroxidation of the mitochondrial membrane induced by Fe2+-citrate

    OpenAIRE

    2006-01-01

    Iron imbalance/accumulation has been implicated in oxidative injury associated with many degenerative diseases such as hereditary hemochromatosis, beta-thalassemia, and Friedreich's ataxia. Mitochondria are particularly sensitive to iron-induced oxidative stress - high loads of iron cause extensive lipid peroxidation and membrane permeabilization in isolated mitochondria. Here we detected and characterized mitochondrial DNA damage in isolated rat liver mitochondria exposed to a Fe2+-citrate c...

  18. Calcium Flux across Plant Mitochondrial Membranes: Possible Molecular Players

    Science.gov (United States)

    Carraretto, Luca; Checchetto, Vanessa; De Bortoli, Sara; Formentin, Elide; Costa, Alex; Szabó, Ildikó; Teardo, Enrico

    2016-01-01

    Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca2+ transients which are further transduced by Ca2+ sensor proteins into a transcriptional and metabolic response. Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter (MCU), LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore (PTP), that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle. PMID:27065186

  19. Calcium flux across plant mitochondrial membranes: possible molecular players

    Directory of Open Access Journals (Sweden)

    Luca eCarraretto

    2016-03-01

    Full Text Available Plants, being sessile organisms, have evolved the ability to integrate external stimuli into metabolic and developmental signals. A wide variety of signals, including abiotic, biotic, and developmental stimuli, were observed to evoke specific spatio-temporal Ca2+ transients which are further transduced by Ca2+ sensor proteins into a transcriptional and metabolic response. Most of the research on Ca2+ signaling in plants has been focused on the transport mechanisms for Ca2+ across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca2+ signals, but how intracellular organelles such as mitochondria are involved in the process of Ca2+ signaling is just emerging. The combination of the molecular players and the elicitors of Ca2+ signaling in mitochondria together with newly generated detection systems for measuring organellar Ca2+ concentrations in plants has started to provide fruitful grounds for further discoveries. In the present review we give an updated overview of the currently identified/hypothesized pathways, such as voltage-dependent anion channels, homologs of the mammalian mitochondrial uniporter, LETM1, a plant glutamate receptor family member, adenine nucleotide/phosphate carriers and the permeability transition pore, that may contribute to the transport of Ca2+ across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca2+ homeostasis for ensuring optimal bioenergetic performance of this organelle.

  20. Phylogenetic Analysis of Mitochondrial Outer Membrane β-Barrel Channels

    Science.gov (United States)

    Wojtkowska, Małgorzata; Jąkalski, Marcin; Pieńkowska, Joanna R.; Stobienia, Olgierd; Karachitos, Andonis; Przytycka, Teresa M.; Weiner, January; Kmita, Hanna; Makałowski, Wojciech

    2012-01-01

    Transport of molecules across mitochondrial outer membrane is pivotal for a proper function of mitochondria. The transport pathways across the membrane are formed by ion channels that participate in metabolite exchange between mitochondria and cytoplasm (voltage-dependent anion-selective channel, VDAC) as well as in import of proteins encoded by nuclear genes (Tom40 and Sam50/Tob55). VDAC, Tom40, and Sam50/Tob55 are present in all eukaryotic organisms, encoded in the nuclear genome, and have β-barrel topology. We have compiled data sets of these protein sequences and studied their phylogenetic relationships with a special focus on the position of Amoebozoa. Additionally, we identified these protein-coding genes in Acanthamoeba castellanii and Dictyostelium discoideum to complement our data set and verify the phylogenetic position of these model organisms. Our analysis show that mitochondrial β-barrel channels from Archaeplastida (plants) and Opisthokonta (animals and fungi) experienced many duplication events that resulted in multiple paralogous isoforms and form well-defined monophyletic clades that match the current model of eukaryotic evolution. However, in representatives of Amoebozoa, Chromalveolata, and Excavata (former Protista), they do not form clearly distinguishable clades, although they locate basally to the plant and algae branches. In most cases, they do not posses paralogs and their sequences appear to have evolved quickly or degenerated. Consequently, the obtained phylogenies of mitochondrial outer membrane β-channels do not entirely reflect the recent eukaryotic classification system involving the six supergroups: Chromalveolata, Excavata, Archaeplastida, Rhizaria, Amoebozoa, and Opisthokonta. PMID:22155732

  1. Chemical synthesis of yeast mitochondrial ATP synthase membranous subunit 8.

    Science.gov (United States)

    Goetz, M; Schmitter, J M; Geoffre, S; Dufourc, E J

    1999-06-01

    Chemical synthesis of highly hydrophobic peptides and proteins remains a challenging problem. Strong interchain associations within the peptide-resin matrix have to be overcome. A synthetic strategy for solid phase peptide synthesis is proposed, mainly based on prolonged coupling time using aprotic polar solvent mixtures. A tailored chromatographic purification was required to obtain a sample sufficiently pure for structural analysis. In this work, the total chemical synthesis of the membrane-embedded yeast mitochondrial ATP synthase subunit 8 is described. The quality of the synthetic protein was checked by electrospray mass spectrometry, its tendency to adopt alpha-helical secondary structure is evidenced by circular dichroism spectroscopy.

  2. Use of carbonate extraction in analyzing moderately hydrophobic transmembrane proteins in the mitochondrial inner membrane.

    Science.gov (United States)

    Kim, Hayoung; Botelho, Salomé Calado; Park, Kwangjin; Kim, Hyun

    2015-12-01

    Resistance to sodium carbonate extraction is regarded as a canonical way to distinguish integral membrane proteins (MPs) from other membrane-associated proteins. However, it has been observed that carbonate extraction releases some mitochondrial integral MPs. Here, by analyzing both artificially designed and native mitochondrial inner MPs containing transmembrane domains (TMDs) of different hydrophobicities, we show that carbonate treatment can release moderately hydrophobic TMDs from the mitochondrial inner membrane. These results suggest that resistance and sensitivity to carbonate extraction may be interpreted with caution when analyzing the nature of mitochondrial inner MPs.

  3. Computer-assisted live cell analysis of mitochondrial membrane potential, morphology and calcium handling.

    NARCIS (Netherlands)

    Koopman, W.J.H.; Distelmaier, F.; Esseling, J.J.; Smeitink, J.A.M.; Willems, P.H.G.M.

    2008-01-01

    Mitochondria are crucial for many aspects of cellular homeostasis and a sufficiently negative membrane potential (Deltapsi) across the mitochondrial inner membrane (MIM) is required to sustain most mitochondrial functions including ATP generation, MIM fusion, and calcium uptake and release. Here, we

  4. Mitochondrial membrane potential: a trait involved in organelle inheritance?

    Science.gov (United States)

    Milani, Liliana

    2015-10-01

    Which mitochondria are inherited across generations? Are transmitted mitochondria functionally silenced to preserve the integrity of their genetic information, or rather are those mitochondria with the highest levels of function (as indicated by membrane potential Δψm) preferentially transmitted? Based on observations of the unusual system of doubly uniparental inheritance of mitochondria and of the common strictly maternal inheritance mode, I formulate a general hypothesis to explain which mitochondria reach the primordial germ cells (PGCs), and how this happens. Several studies indicate that mitochondrial movements are driven by microtubules and that mitochondria with high Δψm are preferentially transported. This can be applied also to the mitochondria that eventually populate embryonic PGCs, so I propose that Δψm may be a trait that allows for the preferential transmission of the most active (and healthy) mitochondria. The topics discussed here are fundamental in cell biology and genetics but remain controversial and a subject of heated debate; I propose an explanation for how a Δψm-dependent mechanism can cause the observed differences in mitochondrial transmission.

  5. Regulation of glycolytic oscillations by mitochondrial and plasma membrane H+-ATPases

    DEFF Research Database (Denmark)

    Olsen, Lars Folke; Andersen, Ann Zahle; Lunding, Anita

    2009-01-01

    We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3,3'-diethylo......We investigated the coupling between glycolytic and mitochondrial membrane potential oscillations in Saccharomyces cerevisiae under semianaerobic conditions. Glycolysis was measured as NADH autofluorescence, and mitochondrial membrane potential was measured using the fluorescent dye 3......,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol...... in a number of different situations (changing pH or the presence and absence of inhibitors). Finally, the intracellular pH was determined and shown to oscillate. The results support earlier work suggesting that the coupling between glycolysis and mitochondrial membrane potential is mediated by the ADP...

  6. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study.

    Science.gov (United States)

    Picard, Martin; White, Kathryn; Turnbull, Douglass M

    2013-01-15

    Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology.

  7. p53's mitochondrial translocation and MOMP action is independent of Puma and Bax and severely disrupts mitochondrial membrane integrity

    Institute of Scientific and Technical Information of China (English)

    Sonja Wolff; Susan Erster; Gustavo Palacios; Ute M Moll

    2008-01-01

    p53's apoptotic program consists of transcription-dependent and transcription-independent pathways. In the latter, physical interactions between mitochondrial p53 and anti-and pro-apoptotic members of the Bcl2 family of mitochondrial permeability regulators are central. Using isogenic cell systems with defined deficiencies, we characterize in detail how mitochondrial p53 contributes to mitochondrial permeabilization, to what extent its action depends on other key Bcl2 family members and define its release activity. We show that mitochondrial p53 is highly efficient in inducing the release of soluble and insoluble apoptogenic factors by severely disrupting outer and inner mitochondrial membrane integrity. This action is associated with wild-type p53-induced oligomerization of Bax, Bak and VDAC and the formation of a stress-induced endogenous complex between p53 and cyclophilin D, normally located at the inner membrane. Tumor-derived p53 mutants are deficient in activating the Bax/Bak lipid pore. These actions are independent of Puma and Bax. Importantly, the latter distinguishes the mitochondrial from the cytosolic p53 death pathway.

  8. Effect of narcotics on membrane-bound mitochondrial processes in fish

    DEFF Research Database (Denmark)

    Vergauwen, Lucia; Nørgaard Schmidt, Stine; Michiels, Ellen

    observed decreasing growth, heart rate and motility with increasing exposure concentration of all narcotics, consistent with the general assumption of reduced cardiorespiratory function. At the cellular level, the cell membrane is expected to be the first target of narcotics. Since the mitochondrial...... and endoplasmic reticulum membrane are known to closely interact with the cell membrane, we hypothesize that narcotics can be further partitioned into these organelle membranes where they can disrupt essential membrane-bound processes. The electron transport chain (ETC) is an example of a crucial mitochondrial...

  9. Dual Role of Mitofilin in Mitochondrial Membrane Organization and Protein Biogenesis

    NARCIS (Netherlands)

    von der Malsburg, Karina; Mueller, Judith M.; Bohnert, Maria; Oeljeklaus, Silke; Kwiatkowska, Paulina; Becker, Thomas; Loniewska-Lwowska, Adrianna; Wiese, Sebastian; Rao, Sanjana; Milenkovic, Dusanka; Hutu, Dana P.; Zerbes, Ralf M.; Schulze-Specking, Agnes; Meyer, Helmut E.; Martinou, Jean-Claude; Rospert, Sabine; Rehling, Peter; Meisinger, Chris; Veenhuis, Marten; Warscheid, Bettina; van der Klei, Ida J.; Pfanner, Nikolaus; Chacinska, Agnieszka; van der Laan, Martin; Müller, Judith M.

    2011-01-01

    The mitochondrial inner membrane consists of two domains, inner boundary membrane and cristae membrane that are connected by crista junctions. Mitofilin/Fcj1 was reported to be involved in formation of crista junctions, however, different views exist on its function and possible partner proteins. We

  10. Mitochondrial matrix delivery using MITO-Porter, a liposome-based carrier that specifies fusion with mitochondrial membranes

    Energy Technology Data Exchange (ETDEWEB)

    Yasuzaki, Yukari; Yamada, Yuma [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan); Harashima, Hideyoshi, E-mail: harasima@pharm.hokudai.ac.jp [Laboratory for Molecular Design of Pharmaceutics, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812 (Japan)

    2010-06-25

    Mitochondria are the principal producers of energy in cells of higher organisms. It was recently reported that mutations and defects in mitochondrial DNA (mtDNA) are associated with various mitochondrial diseases including a variety of neurodegenerative and neuromuscular diseases. Therefore, an effective mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve this, therapeutic agents need to be delivered into the innermost mitochondrial space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the development of MITO-Porter, a liposome-based carrier that introduces macromolecular cargos into mitochondria via membrane fusion. In this study, we provide a demonstration of mitochondrial matrix delivery and the visualization of mitochondrial genes (mtDNA) in living cells using the MITO-Porter. We first prepared MITO-Porter containing encapsulated propidium iodide (PI), a fluorescent dye used to stain nucleic acids to detect mtDNA. We then confirmed the emission of red-fluorescence from PI by conjugation with mtDNA, when the carriers were incubated in the presence of isolated rat liver mitochondria. Finally, intracellular observation by confocal laser scanning microscopy clearly verified that the MITO-Porter delivered PI to the mitochondrial matrix.

  11. Toward high-content screening of mitochondrial morphology and membrane potential in living cells

    NARCIS (Netherlands)

    Iannetti, E.F.; Willems, P.H.G.M.; Pellegrini, M.; Beyrath, J.D.; Smeitink, J.; Blanchet, L.M.; Koopman, W.J.H.

    2015-01-01

    Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These "mitochondrial dynamics" are bi-directionally linked to mitochondrial and cell functional state in space and time

  12. Life cell quantification of mitochondrial membrane potential at the single organelle level.

    NARCIS (Netherlands)

    Distelmaier, F.; Koopman, W.J.; Testa, E.R.; Jong, AS de; Swarts, H.G.P.; Mayatepek, E.; Smeitink, J.A.M.; Willems, P.H.G.M.

    2008-01-01

    Mitochondrial membrane potential (Deltapsi) is key to mitochondrial function and cellular survival. Here, we aimed to develop an automated protocol allowing sensitive quantification of Deltapsi in living cells at the level of individual mitochondria. Human skin fibroblasts were stained with the fluo

  13. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

    Overexpression of plasma membrane multidrug resistance-associated protein 1 (MRP-1) in Ewing's sarcoma (ES) predicts poor outcome. MRP-1 is also expressed in mitochondria, and we have examined the submitochondrial localization of MRP-1 and investigated the mechanism of MRP-1 transport and role of this organelle in the response to doxorubicin. The mitochondrial localization of MRP-1 was examined in ES cell lines by differential centrifugation and membrane solubilization by digitonin. Whether MRP-1 is chaperoned by heat shock proteins (HSPs) was investigated by immunoprecipitation, immunofluorescence microscopy, and HSP knockout using small hairpin RNA and inhibitors (apoptozole, 17-AAG, and NVPAUY). The effect of disrupting mitochondrial MRP-1-dependent efflux activity on the cytotoxic effect of doxorubicin was investigated by counting viable cell number. Mitochondrial MRP-1 is glycosylated and localized to the outer mitochondrial membrane, where it is coexpressed with HSP90. MRP-1 binds to both HSP90 and HSP70, although only inhibition of HSP90β decreases expression of MRP-1 in the mitochondria. Disruption of mitochondrial MRP-1-dependent efflux significantly increases the cytotoxic effect of doxorubicin (combination index, MRP-1 is expressed in the outer mitochondrial membrane and is a client protein of HSP90β, where it may play a role in the doxorubicin-induced resistance of ES.-Roundhill, E., Turnbull, D., Burchill, S. Localization of MRP-1 to the outer mitochondrial membrane by the chaperone protein HSP90β.

  14. Degradation of transplanted rat liver mitochondrial-outer-membrane proteins in hepatoma cells.

    OpenAIRE

    Russell, S.M.; Mayer, R J

    1983-01-01

    Reductively [3H]methylated 3H mitochondrial-outer-membrane vesicles from rat liver and vesicles where monoamine oxidase has been derivatized irreversibly by [3H]-pargyline have been deliberately miscompartmentalized by heterologous transplantation into hepatoma (HTC) cells by poly(ethylene glycol)-mediated vesicle-cell fusion. Fluorescein-conjugated mitochondrial-outer-membrane vesicles have also been used to show that transplanted material is patched, capped and internalized. Reductively met...

  15. Mitochondrial membrane studies using impedance spectroscopy with parallel pH monitoring.

    Directory of Open Access Journals (Sweden)

    Divya Padmaraj

    Full Text Available A biological microelectromechanical system (BioMEMS device was designed to study complementary mitochondrial parameters important in mitochondrial dysfunction studies. Mitochondrial dysfunction has been linked to many diseases, including diabetes, obesity, heart failure and aging, as these organelles play a critical role in energy generation, cell signaling and apoptosis. The synthesis of ATP is driven by the electrical potential across the inner mitochondrial membrane and by the pH difference due to proton flux across it. We have developed a tool to study the ionic activity of the mitochondria in parallel with dielectric measurements (impedance spectroscopy to gain a better understanding of the properties of the mitochondrial membrane. This BioMEMS chip includes: 1 electrodes for impedance studies of mitochondria designed as two- and four-probe structures for optimized operation over a wide frequency range and 2 ion-sensitive field effect transistors for proton studies of the electron transport chain and for possible monitoring other ions such as sodium, potassium and calcium. We have used uncouplers to depolarize the mitochondrial membrane and disrupt the ionic balance. Dielectric spectroscopy responded with a corresponding increase in impedance values pointing at changes in mitochondrial membrane potential. An electrical model was used to describe mitochondrial sample's complex impedance frequency dependencies and the contribution of the membrane to overall impedance changes. The results prove that dielectric spectroscopy can be used as a tool for membrane potential studies. It can be concluded that studies of the electrochemical parameters associated with mitochondrial bioenergetics may render significant information on various abnormalities attributable to these organelles.

  16. Toward high-content screening of mitochondrial morphology and membrane potential in living cells.

    Science.gov (United States)

    Iannetti, Eligio F; Willems, Peter H G M; Pellegrini, Mina; Beyrath, Julien; Smeitink, Jan A M; Blanchet, Lionel; Koopman, Werner J H

    2015-06-01

    Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These "mitochondrial dynamics" are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial "health" and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations. This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

  17. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide.

    Science.gov (United States)

    Perry, Seth W; Norman, John P; Barbieri, Justin; Brown, Edward B; Gelbard, Harris A

    2011-02-01

    Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.

  18. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    Science.gov (United States)

    Korenić, Andrej; Boltze, Johannes; Deten, Alexander; Peters, Myriam; Andjus, Pavle; Radenović, Lidija

    2014-01-01

    Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m)) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m), visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m) during reperfusion, whereas GD caused a robust Δψ(m) negativation. In case no Δψ(m) negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m) hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψ(m) during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

  19. Astrocytic mitochondrial membrane hyperpolarization following extended oxygen and glucose deprivation.

    Directory of Open Access Journals (Sweden)

    Andrej Korenić

    Full Text Available Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψ(m in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD, OGD and combinations of both conditions varying in duration and sequence. Changes in Δψ(m, visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψ(m during reperfusion, whereas GD caused a robust Δψ(m negativation. In case no Δψ(m negativation was observed after OGD, subsequent chemical oxygen deprivation (OD induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψ(m hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen and their hyperpolarizing effect on Δψ(m during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

  20. Metaxin deficiency alters mitochondrial membrane permeability and leads to resistance to TNF-induced cell killing.

    Science.gov (United States)

    Ono, Koh; Wang, Xiaofei; Kim, Sung Ouk; Armstrong, Lucas C; Bornstein, Paul; Han, Jiahuai

    2010-02-01

    Metaxin, a mitochondrial outer membrane protein, is critical for TNF-induced cell death in L929 cells. Its deficiency, caused by retroviral insertion-mediated mutagenesis, renders L929 cells resistance to TNF killing. In this study, we further characterized metaxin deficiency-caused TNF resistance in parallel with Bcl-X(L) overexpression-mediated death resistance. We did not find obvious change in mitochondria membrane potential in metaxin-deficient (Met(mut)) and Bcl-X(L)-overexpressing cells, but we did find an increase in the release rate of the mitochondrial membrane potential probe rhodamine 123 (Rh123) that was preloaded into mitochondria. In addition, overexpression of a function-interfering mutant of metaxin (MetaΔTM/C) or Bcl-X(L) in MCF-7.3.28 cells also resulted in an acquired resistance to TNF killing and a faster rate of Rh123 release, indicating a close correlation between TNF resistance and higher rates of the dye release from the mitochondria. The release of Rh123 can be controlled by the mitochondrial membrane permeability transition (PT) pore, as targeting an inner membrane component of the PT pore by cyclosporin A (CsA) inhibited Rh123 release. However, metaxin deficiency and Bcl-X(L) overexpression apparently affect Rh123 release from a site(s) different from that of CsA, as CsA can overcome their effect. Though both metaxin and Bcl-X(L) appear to function on the outer mitochondrial membrane, they do not interact with each other. They may use different mechanisms to increase the permeability of Rh123, since previous studies have suggested that metaxin may influence certain outer membrane porins while Bcl-X(L) may form pores on the outer membrane. The alteration of the mitochondrial outer membrane properties by metaxin deficiency and Bcl-X(L) overexpression, as indicated by a quicker Rh123 release, may be helpful in maintaining mitochondrial integrity.

  1. Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption.

    Science.gov (United States)

    Soares, S S; Gutiérrez-Merino, C; Aureliano, M

    2007-05-01

    Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 microM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 microM, whereas no effect was observed up to 100 microM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 M decavanadate and 10 microM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 microM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither F(O)F(1)-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.

  2. Integrity of the plasma membrane, the acrosomal membrane, and the mitochondrial membrane potential of sperm in Nelore bulls from puberty to sexual maturity

    Directory of Open Access Journals (Sweden)

    L.S.L.S. Reis

    2016-06-01

    Full Text Available ABSTRACT This study evaluated the plasma membrane integrity, acrosomal membrane integrity, and mitochondrial membrane potential of Nelore bull sperm from early puberty to early sexual maturity and their associations with sperm motility and vigor, the mass motility of the spermatozoa (wave motion, scrotal circumference, and testosterone. Sixty Nelore bulls aged 18 to 19 months were divided into four lots (n=15 bulls/lot and evaluated over 280 days. Semen samples, collected every 56 days by electroejaculation, were evaluated soon after collection for motility, vigor and wave motion under an optical microscope. Sperm membrane integrity, acrosomal integrity, and mitochondrial activity were evaluated under a fluorescent microscope using probe association (FITC-PSA, PI, JC-1, H342. The sperm were classified into eight integrity categories depending on whether they exhibited intact or damaged membranes, an intact or damaged acrosomal membrane, and high or low mitochondrial potential. The results show that bulls have a low amount of sperm with intact membranes at puberty, and the sperm show low motility, vigor, and wave motion; however, in bulls at early sexual maturity, the integrity of the sperm membrane increased significantly. The rate of sperm membrane damage was negatively correlated with motility, vigor, wave motion, and testosterone in the bulls, and a positive correlation existed between sperm plasma membrane integrity and scrotal circumference. The integrity of the acrosomal membrane was not influenced by puberty. During puberty and into early sexual maturity, bulls show low sperm mitochondrial potential, but when bulls reached sexual maturity, high membrane integrity with high mitochondrial potential was evident.

  3. Interaction of ADP, atractyloside, and gummiferin on the ADP translocase of the inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Vignais, P.V.; Vignais, P.M.; Defaye, G.; Lauquin, G.; Doussiere, J.; Chabert, J.; Brandolin, G.

    1972-05-01

    From international conference on mechanism in bioenergetica; Bari, Italy (1 May 1972). Two specific inhibitors of the adenine nucleotide translocation, gummiferin (GUM), identified to 4-carboxyatractyloside and atractyloside (ATR), were labeled with /sup 35/S and their binding properties to whole mitochondria and inner mitochondrial membrane vesicles used to monitor changes of membrane conformation induced by ADP. (auth)

  4. Lipid unsaturation per se does not explain the physical state of mitochondrial membranes in Mytilus galloprovincialis.

    Science.gov (United States)

    Fiorini, Rosamaria; Pagliarani, Alessandra; Nesci, Salvatore; Trombetti, Fabiana; Pirini, Maurizio; Fabbri, Micaela; Ventrella, Vittoria

    2016-01-01

    Through a multiple approach, the present study on the mitochondrial membranes from mussel gills and swine heart combines some biochemical information on fatty acid composition, sterol pattern, and temperature dependence of the F1FO-ATPase activity (EC 3.6.3.14.) with fluorescence data on mitochondrial membranes and on liposomes obtained from lipid extracts of mitochondria. The physical state of mussel gills and swine heart was investigated by Laurdan steady state fluorescence. Quite surprisingly, the similar temperature dependence of the F1FO complex, illustrated as Arrhenius plot which in both mitochondria exhibits the same discontinuity at approximately 21°C and overlapping activation energies above and below the discontinuity, is apparently compatible with a different composition and physical state of mitochondrial membranes. Accordingly, mussel membranes contain highly unsaturated fatty acids, abundant sterols, including phytosterols, while mammalian membranes only contain cholesterol and in prevalence shorter and less unsaturated fatty acids, leading to a lower membrane unsaturation with respect to mussel mitochondria. As suggested by fluorescence data, the likely formation of peculiar microdomains interacting with the membrane-bound enzyme complex in mussel mitochondria could produce an environment which somehow approaches the physical state of mammalian mitochondrial membranes. Thus, as an adaptive strategy, the interaction between sterols, highly unsaturated phospholipids and proteins in mussel gill mitochondria could allow the F1FO-ATPase activity to maintain the same activation energy as the mammalian enzyme.

  5. Cytosolic BNIP3 Dimer Interacts with Mitochondrial BAX Forming Heterodimers in the Mitochondrial Outer Membrane under Basal Conditions.

    Science.gov (United States)

    Hendgen-Cotta, Ulrike B; Esfeld, Sonja; Rudi, Katharina; Miinalainen, Ilkka; Klare, Johann P; Rassaf, Tienush

    2017-03-23

    The primary function of mitochondria is energy production, a task of particular importance especially for cells with a high energy demand like cardiomyocytes. The B-cell lymphoma (BCL-2) family member BCL-2 adenovirus E1B 19 kDa-interacting protein 3 (BNIP3) is linked to mitochondrial targeting after homodimerization, where it functions in inner membrane depolarization and permeabilization of the mitochondrial outer membrane (MOM) mediating cell death. We investigated the basal distribution of cardiac BNIP3 in vivo and its physical interaction with the pro-death protein BCL2 associated X, apoptosis regulator (BAX) and with mitochondria using immunoblot analysis, co-immunoprecipitation, and continuous wave and pulsed electron paramagnetic resonance spectroscopy techniques. We found that BNIP3 is present as a dimer in the cytosol and in the outer membrane of cardiac mitochondria under basal conditions. It forms disulfide-bridged, but mainly non-covalent dimers in the cytosol. Heterodimers with BAX are formed exclusively in the MOM. Furthermore, our results suggest that BNIP3 interacts with the MOM directly via mitochondrial BAX. However, the physical interactions with BAX and the MOM did not affect the membrane potential and cell viability. These findings suggest that another stimulus other than the mere existence of the BNIP3/BAX dimer in the MOM is required to promote BNIP3 cell-death activity; this could be a potential disturbance of the BNIP3 distribution homeostasis, namely in the direction of the mitochondria.

  6. Air bubble contact with endothelial cells causes a calcium-independent loss in mitochondrial membrane potential.

    Directory of Open Access Journals (Sweden)

    Peter Sobolewski

    Full Text Available OBJECTIVE: Gas microembolism remains a serious risk associated with surgical procedures and decompression. Despite this, the signaling consequences of air bubbles in the vasculature are poorly understood and there is a lack of pharmacological therapies available. Here, we investigate the mitochondrial consequences of air bubble contact with endothelial cells. METHODS AND RESULTS: Human umbilical vein endothelial cells were loaded with an intracellular calcium indicator (Fluo-4 and either a mitochondrial calcium indicator (X-Rhod-1 or mitochondrial membrane potential indicator (TMRM. Contact with 50-150 µm air bubbles induced concurrent rises in intracellular and mitochondrial calcium, followed by a loss of mitochondrial membrane potential. Pre-treating cells with 1 µmol/L ruthenium red, a TRPV family calcium channel blocker, did not protect cells from the mitochondrial depolarization, despite blocking the intracellular calcium response. Mitigating the interactions between the air-liquid interface and the endothelial surface layer with 5% BSA or 0.1% Pluronic F-127 prevented the loss of mitochondrial membrane potential. Finally, inhibiting protein kinase C-α (PKCα, with 5 µmol/L Gö6976, protected cells from mitochondrial depolarization, but did not affect the intracellular calcium response. CONCLUSIONS: Our results indicate that air bubble contact with endothelial cells activates a novel, calcium-independent, PKCα-dependent signaling pathway, which results in mitochondrial depolarization. As a result, mitochondrial dysfunction is likely to be a key contributor to the pathophysiology of gas embolism injury. Further, this connection between the endothelial surface layer and endothelial mitochondria may also play an important role in vascular homeostasis and disease.

  7. Biophysical significance of the inner mitochondrial membrane structure on the electrochemical potential of mitochondria

    Science.gov (United States)

    Song, Dong Hoon; Park, Jonghyun; Maurer, Laura L.; Lu, Wei; Philbert, Martin A.; Sastry, Ann Marie

    2013-12-01

    The available literature supports the hypothesis that the morphology of the inner mitochondrial membrane is regulated by different energy states, that the three-dimensional morphology of cristae is dynamic, and that both are related to biochemical function. Examination of the correlation between the inner mitochondrial membrane (IMM) structure and mitochondrial energetic function is critical to an understanding of the links between mesoscale morphology and function in progressive mitochondrial dysfunction such as aging, neurodegeneration, and disease. To investigate this relationship, we develop a model to examine the effects of three-dimensional IMM morphology on the electrochemical potential of mitochondria. The two-dimensional axisymmetric finite element method is used to simulate mitochondrial electric potential and proton concentration distribution. This simulation model demonstrates that the proton motive force (Δp) produced on the membranes of cristae can be higher than that on the inner boundary membrane. The model also shows that high proton concentration in cristae can be induced by the morphology-dependent electric potential gradient along the outer side of the IMM. Furthermore, simulation results show that a high Δp is induced by the large surface-to-volume ratio of an individual crista, whereas a high capacity for ATP synthesis can primarily be achieved by increasing the surface area of an individual crista. The mathematical model presented here provides compelling support for the idea that morphology at the mesoscale is a significant driver of mitochondrial function.

  8. Translocation of chicken heart apocytochrome c and its mutants (C17S, H18D) across mitochondrial membrane

    Institute of Scientific and Technical Information of China (English)

    朱勇; 韩学海; 杨福愉

    1999-01-01

    Cytochrome c is a component of mitochondrial respiratory chain, located at the outer side of mitochondrial inner membrane. Its precursor, apocytochrome c, is encoded by a nuclear gene, synthesized on cytoplasmic ribosomes, and posttranslationally imported into mitochondria, but apocytochrome c is unique in the translocation compared with most mitochondrial proteins. It does not carry a cleavable amino terminal targeting sequence; no proteinous receptor on the mitochondrial outer membrane is identified for its import and its translocation does not compete with other preproteins for translocation machinery in the outer membrane. Besides, neither ATP nor membrane potential is required for its translocation across mitochonctria.

  9. The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

    Science.gov (United States)

    Macchi, Marc; El Fissi, Najla; Tufi, Roberta; Bentobji, Mélanie; Liévens, Jean-Charles; Martins, L Miguel; Royet, Julien; Rival, Thomas

    2013-02-01

    Cristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape.

  10. Mitochondrial uncouplers act synergistically with the fumigant phosphine to disrupt mitochondrial membrane potential and cause cell death.

    Science.gov (United States)

    Valmas, Nicholas; Zuryn, Steven; Ebert, Paul R

    2008-10-30

    Phosphine is the most widely used fumigant for the protection of stored commodities against insect pests, especially food products such as grain. However, pest insects are developing resistance to phosphine and thereby threatening its future use. As phosphine inhibits cytochrome c oxidase (complex IV) of the mitochondrial respiratory chain and reduces the strength of the mitochondrial membrane potential (DeltaPsi(m)), we reasoned that mitochondrial uncouplers should act synergistically with phosphine. The mitochondrial uncouplers FCCP and PCP caused complete mortality in populations of both wild-type and phosphine-resistant lines of Caenorhabditis elegans simultaneously exposed to uncoupler and phosphine at concentrations that were individually nonlethal. Strong synergism was also observed with a third uncoupler DNP. We have also tested an alternative complex IV inhibitor, azide, with FCCP and found that this also caused a synergistic enhancement of toxicity in C. elegans. To investigate potential causes of the synergism, we measured DeltaPsi(m), ATP content, and oxidative damage (lipid hydroperoxides) in nematodes subjected to phosphine-FCCP treatment and found that neither an observed 50% depletion in ATP nor oxidative stress accounted for the synergistic effect. Instead, a synergistic reduction in DeltaPsi(m) was observed upon phosphine-FCCP co-treatment suggesting that this is directly responsible for the subsequent mortality. These results support the hypothesis that phosphine-induced mortality results from the in vivo disruption of normal mitochondrial activity. Furthermore, we have identified a novel pathway that can be targeted to overcome genetic resistance to phosphine.

  11. Knockdown of cytosolic glutaredoxin 1 leads to loss of mitochondrial membrane potential: implication in neurodegenerative diseases.

    Directory of Open Access Journals (Sweden)

    Uzma Saeed

    Full Text Available Mitochondrial dysfunction including that caused by oxidative stress has been implicated in the pathogenesis of neurodegenerative diseases. Glutaredoxin 1 (Grx1, a cytosolic thiol disulfide oxido-reductase, reduces glutathionylated proteins to protein thiols and helps maintain redox status of proteins during oxidative stress. Grx1 downregulation aggravates mitochondrial dysfunction in animal models of neurodegenerative diseases, such as Parkinson's and motor neuron disease. We examined the mechanism underlying the regulation of mitochondrial function by Grx1. Downregulation of Grx1 by shRNA results in loss of mitochondrial membrane potential (MMP, which is prevented by the thiol antioxidant, alpha-lipoic acid, or by cyclosporine A, an inhibitor of mitochondrial permeability transition. The thiol groups of voltage dependent anion channel (VDAC, an outer membrane protein in mitochondria but not adenosine nucleotide translocase (ANT, an inner membrane protein, are oxidized when Grx1 is downregulated. We then examined the effect of beta-N-oxalyl amino-L-alanine (L-BOAA, an excitatory amino acid implicated in neurolathyrism (a type of motor neuron disease, that causes mitochondrial dysfunction. Exposure of cells to L-BOAA resulted in loss of MMP, which was prevented by overexpression of Grx1. Grx1 expression is regulated by estrogen in the CNS and treatment of SH-SY5Y cells with estrogen upregulated Grx1 and protected from L-BOAA mediated MMP loss. Our studies demonstrate that Grx1, a cytosolic oxido-reductase, helps maintain mitochondrial integrity and prevents MMP loss caused by oxidative insult. Further, downregulation of Grx1 leads to mitochondrial dysfunction through oxidative modification of the outer membrane protein, VDAC, providing support for the critical role of Grx1 in maintenance of MMP.

  12. Mitochondrial membrane potential probes and the proton gradient: a practical usage guide

    OpenAIRE

    Seth W Perry; Norman, John P.; Barbieri, Justin; Brown, Edward B.; Gelbard, Harris A.

    2011-01-01

    Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations...

  13. Bax assembles into large ring-like structures remodeling the mitochondrial outer membrane in apoptosis.

    Science.gov (United States)

    Große, Lena; Wurm, Christian A; Brüser, Christian; Neumann, Daniel; Jans, Daniel C; Jakobs, Stefan

    2016-02-15

    The Bcl-2 family proteins Bax and Bak are essential for the execution of many apoptotic programs. During apoptosis, Bax translocates to the mitochondria and mediates the permeabilization of the outer membrane, thereby facilitating the release of pro-apoptotic proteins. Yet the mechanistic details of the Bax-induced membrane permeabilization have so far remained elusive. Here, we demonstrate that activated Bax molecules, besides forming large and compact clusters, also assemble, potentially with other proteins including Bak, into ring-like structures in the mitochondrial outer membrane. STED nanoscopy indicates that the area enclosed by a Bax ring is devoid of mitochondrial outer membrane proteins such as Tom20, Tom22, and Sam50. This strongly supports the view that the Bax rings surround an opening required for mitochondrial outer membrane permeabilization (MOMP). Even though these Bax assemblies may be necessary for MOMP, we demonstrate that at least in Drp1 knockdown cells, these assemblies are not sufficient for full cytochrome c release. Together, our super-resolution data provide direct evidence in support of large Bax-delineated pores in the mitochondrial outer membrane as being crucial for Bax-mediated MOMP in cells. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  14. Protective role of mitochondrial K-ATP channel and mitochondrial membrane transport pore in rat kidney ischemic postconditioning

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-liang; ZHAO Yan-li; LIU Xiao-ming; CHEN Jing; ZHANG Dong

    2011-01-01

    Background Previous studies suggested that mechanical intervention during early reperfusion, or ischemia postconditioning (Ipo), could protect kidneys against renal ischemia reperfusion injury (RIRI). However, the mechanisms responsible for this protection remain unclear. This study therefore investigated the protection afforded by Ipo in rat kidneys in vivo, and the roles of mitochondrial KATP channels (mitOKATP) and mitochondrial permeability transition pores (MPTPs), by inhibiting mitOKATP with 5-hydroxydecanoate (5-HD), and by directly detecting open MPTPs using calcein-AM and CoCl2.Methods Thirty-five male Sprague-Dawley rats were randomly assigned to sham-operation (S), ischemia-reperfusion (I/R),Ipo, ischemia reperfusion with 5-HD (I/R+5-HD), or Ipo with 5-HD (Ipo +5-HD) groups. Rats in each group were sacrificed after 6 hours of reperfusion by heart exsanguination or cervical dislocation under anesthesia. RIRI was assessed by determination of creatinine and blood urea nitrogen (BUN), and by examination of histologic sections. The roles of mitoKATP and MPTP were investigated by analyzing fluorescence intensities of mitochondria, mitochondrial membrane potential,intracellular reactive oxygen species (ROS) and intracellular calcium, using appropriate fluorescent markers. The relationship between apoptosis and RIRI was assessed by determining the apoptotic index (Al) of kidney tubular epithelial cells.Results The RIRI model was shown to be successful. Significantly higher levels of creatinine and BUN, and abnormal pathology of histologic sections, were observed in group I/R, compared with group S. 5-HD eliminated the renoprotective effects of Ipo. Mitochondrial and mitochondrial membrane potential fluorescence intensities increased, and intracellular calcium, ROS fluorescence intensities and AI decreased in group Ipo, compared with group I/R. However, mitochondrial and mitochondrial membrane potential fluorescence intensities decreased, and intracellular

  15. Mitochondrial DNA damage associated with lipid peroxidation of the mitochondrial membrane induced by Fe2+-citrate

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    Andréa M. Almeida

    2006-09-01

    Full Text Available Iron imbalance/accumulation has been implicated in oxidative injury associated with many degenerative diseases such as hereditary hemochromatosis, beta-thalassemia, and Friedreich's ataxia. Mitochondria are particularly sensitive to iron-induced oxidative stress - high loads of iron cause extensive lipid peroxidation and membrane permeabilization in isolated mitochondria. Here we detected and characterized mitochondrial DNA damage in isolated rat liver mitochondria exposed to a Fe2+-citrate complex, a small molecular weight complex. Intense DNA fragmentation was induced after the incubation of mitochondria with the iron complex. The detection of 3' phosphoglycolate ends at the mtDNA strand breaks by a 32P-postlabeling assay, suggested the involvement of hydroxyl radical in the DNA fragmentation induced by Fe2+-citrate. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine also suggested that Fe2+-citrate-induced oxidative stress causes mitochondrial DNA damage. In conclusion, our results show that iron-mediated lipid peroxidation was associated with intense mtDNA damage derived from the direct attack of reactive oxygen species.Desequilíbrio/acúmulo de ferro tem sido implicado em injúria oxidativa associada a diversas doenças degenerativas tais como, hemocromatose hereditária, beta-talassemia e ataxia de Friedreich. As mitocôndrias são particularmente sensíveis a estresse oxidativo induzido por ferro - um carregamento alto de ferro em mitocôndrias isoladas pode causar uma extensiva peroxidação lipídica e a permeabilização de membrana. Nesse estudo, nós detectamos e caracterizamos danos do DNA mitocondrial em mitocôndrias isoladas de fígado de rato, expostas ao complexo Fe2+-citrato, um dos complexos de baixo peso molecular. A intensa fragmentação do DNA foi induzida após a incubação das mitocôndrias com o complexo de ferro. A detecção de finais 3' de fosfoglicolato nas quebras de fitas de DNA mitocondrial pelo ensaio 32

  16. Interactions of mitochondrial presequence peptides with the mitochondrial outer membrane preprotein translocase TOM.

    Science.gov (United States)

    Romero-Ruiz, Mercedes; Mahendran, Kozhinjampara R; Eckert, Reiner; Winterhalter, Mathias; Nussberger, Stephan

    2010-08-04

    TOM protein-conducting channels serve as the main entry sites into mitochondria for virtually all mitochondrial proteins. When incorporated into lipid bilayers, they form large, relatively nonspecific ion channels that are blocked by peptides derived from mitochondrial precursor proteins. Using single-channel electrical recordings, we analyzed the interactions of mitochondrial presequence peptides with single TOM pores. The largest conductance state of the translocon represents the likely protein-conducting conformation of the channel. The frequency (but not the duration) of the polypeptide-induced blockage is strongly modulated by the substrate concentration. Structural differences between substrates are reflected in characteristic blockage frequencies and duration of blockage. To our knowledge, this study provides first quantitative data regarding the kinetics of polypeptide interaction with the mitochondrial TOM machinery. 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  17. The presence of phosphate-binding protein in inner mitochondrial membrane

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    Hatase,Osamu

    1976-06-01

    Full Text Available Phosphate-binding protein(s was found in the inner mitochondrial membrane of calf heart by Sephadex G-200 and G-25 gel filtration. The binding activity was inhibited by N-ethylmaleimide and competed by a large amount of cold phosphate. The amount of phosphate bound to the fraction was 29 nmoles per mg of protein. Affinity chromatography with phosphate-bound Sepharose 4B confirmed the presence of phosphate-binding protein(s in the active fraction of mitochondrial membrane fractionated by gel filtration.

  18. Mitochondrial glutathione transferases involving a new function for membrane permeability transition pore regulation.

    Science.gov (United States)

    Aniya, Yoko; Imaizumi, Naoki

    2011-05-01

    The mitochondria in mammalian cells are a predominant resource of reactive oxygen species (ROS), which are produced during respiration-coupled oxidative metabolism or various chemical stresses. End-products from membrane-lipid peroxidation caused by ROS are highly toxic, thereby their elimination/scavenging are protective of mitochondria and cells against oxidative damages. In mitochondria, soluble (kappa, alpha, mu, pi, zeta) and membrane-bound glutathione transferases (GSTs) (MGST1) are distributed. Mitochondrial GSTs display both glutathione transferase and peroxidase activities that detoxify such harmful products through glutathione (GSH) conjugation or GSH-mediated peroxide reduction. Some GST isoenzymes are induced by oxidative stress, an adaptation mechanism for the protection of cells from oxidative stress. Membrane-bound MGST1 is activated through the thiol modification in oxidative conditions. Protective action of MGST1 against oxidative stress has been confirmed using MCF7 cells highly expressed of MGST1. In recent years, mitochondria have been recognized as a regulator of cell death via both apoptosis and necrosis, where oxidative stress-induced alteration of the membrane permeability is an important step. Recent studies have shown that MGST1 in the inner mitochondrial membrane could interact with the mitochondrial permeability transition (MPT) regulator proteins, such as adenine nucleotide translocator (ANT) and/or cyclophilin D, and could contribute to oxidant-induced MPT pores. Interaction of GST alpha with ANT has also been shown. In this review, functions of the mitochondrial GSTs, including a new role for mitochondria-mediated cell death, are described.

  19. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

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    Akihiro Yamashita

    Full Text Available Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs, while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles. Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN. The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence.

  20. Formation of Mitochondrial Outer Membrane Derived Protrusions and Vesicles in Arabidopsis thaliana.

    Science.gov (United States)

    Yamashita, Akihiro; Fujimoto, Masaru; Katayama, Kenta; Yamaoka, Shohei; Tsutsumi, Nobuhiro; Arimura, Shin-Ichi

    2016-01-01

    Mitochondria are dynamic organelles that have inner and outer membranes. In plants, the inner membrane has been well studied but relatively little is known about the outer membrane. Here we report that Arabidopsis cells have mitochondrial outer membrane-derived structures, some of which protrude from the main body of mitochondria (mitochondrial outer-membrane protrusions; MOPs), while others form vesicle-like structures without a matrix marker. The latter vesicle-like structures are similar to some mammalian MDVs (mitochondrial-derived vesicles). Live imaging demonstrated that a plant MDV budded off from the tip of a MOP. MDVs were also observed in the drp3a drp3b double mutant, indicating that they could be formed without the mitochondrial fission factors DRP3A and DRP3B. Double staining studies showed that the MDVs were not peroxisomes, endosomes, Golgi apparatus or trans-Golgi network (TGN). The numbers of MDVs and MOPs increased in senescent leaves and after dark treatment. Together, these results suggest that MDVs and MOPs are related to leaf senescence.

  1. Variations in mitochondrial membrane potential correlate with malic acid production by natural isolates of Saccharomyces cerevisiae sake strains.

    Science.gov (United States)

    Oba, Takahiro; Kusumoto, Kenichi; Kichise, Yuki; Izumoto, Eiji; Nakayama, Shunichi; Tashiro, Kosuke; Kuhara, Satoru; Kitagaki, Hiroshi

    2014-08-01

    Research on the relationship between mitochondrial membrane potential and fermentation profile is being intensely pursued because of the potential for developing advanced fermentation technologies. In the present study, we isolated naturally occurring strains of yeast from sake mash that produce high levels of malic acid and demonstrate that variations in mitochondrial membrane potential correlate with malic acid production. To define the underlying biochemical mechanism, we determined the activities of enzymes required for malic acid synthesis and found that pyruvate carboxylase and malate dehydrogenase activities in strains that produce high levels of malic acid were elevated compared with the standard sake strain K901. These results inspired us to hypothesize that decreased mitochondrial membrane potential was responsible for increased malic acid synthesis, and we present data supporting this hypothesis. Thus, the mitochondrial membrane potential of high malic acid producers was lower compared with standard strains. We conclude that mitochondrial membrane potential correlates with malic acid production.

  2. Yeast Mitochondrial Interactosome Model: Metabolon Membrane Proteins Complex Involved in the Channeling of ADP/ATP

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

    Full Text Available The existence of a mitochondrial interactosome (MI has been currently well established in mammalian cells but the exact composition of this super-complex is not precisely known, and its organization seems to be different from that in yeast. One major difference is the absence of mitochondrial creatine kinase (MtCK in yeast, unlike that described in the organization model of MI, especially in cardiac, skeletal muscle and brain cells. The aim of this review is to provide a detailed description of different partner proteins involved in the synergistic ADP/ATP transport across the mitochondrial membranes in the yeast Saccharomyces cerevisiae and to propose a new mitochondrial interactosome model. The ADP/ATP (Aacp and inorganic phosphate (PiC carriers as well as the VDAC (or mitochondrial porin catalyze the import and export of ADP, ATP and Pi across the mitochondrial membranes. Aacp and PiC, which appear to be associated with the ATP synthase, consist of two nanomotors (F0, F1 under specific conditions and form ATP synthasome. Identification and characterization of such a complex were described for the first time by Pedersen and co-workers in 2003.

  3. Mitochondrial membrane potential changes in osteoblasts treated with parathyroid hormone and estradiol.

    Science.gov (United States)

    Troyan, M B; Gilman, V R; Gay, C V

    1997-06-15

    This study assessed mitochondrial membrane potential changes in cultured osteoblasts treated with hormones known to regulate osteoblasts. A fluorescent carbocyanine dye, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine++ + iodide, also called JC-1, was used as a probe. JC-1 emits photons at 585 nm (orange-red) when the membrane potential in mitochondria is highly negative, but when the potential becomes reduced emission occurs at 527 nm (green). Osteoblasts were rinsed in serum-free medium for 5 min, then loaded with 1 x 10(-6) M JC-1 for 10 min. The distribution and intensity of JC-1 fluorescence were evaluated with a laser-scanning confocal microscope system. Hormone treatments included parathyroid hormone (PTH; 10(-8) M), 17beta-estradiol (10(-8) M), and thyroxine (T4; 10(-8) M). The potassium ionophore valinomycin (10(-6) M) was used as a control since it is known to disrupt the electrochemical gradient of mitochondria without interfering with the pH gradient. Valinomycin caused a profound, rapid increase (22.5% above untreated values) in the green/red ratio, which indicated a lowering of the mitochondrial membrane potential in all samples evaluated. PTH caused a less pronounced, but significant (7-14%), reduction in membrane potential in all cells examined. PTH is known to affect osteoblasts in a number of ways and is inhibitory to mitochondrial respiration; the results confirm this effect. For estradiol, half of the cells responded at a significant level, with a membrane potential reduction of 6 to 13% being recorded; the other half did not respond. Thyroxine did not alter mitochondrial membrane potential. Responses were detectable within 20 s for valinomycin, but occurred at a slower rate, over 200 to 300 s, following PTH and estradiol treatment. Responses to PTH and estradiol could be due to mitochondrial uptake of cytosolic Ca2+.

  4. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease.

  5. Assessment of mitochondrial membrane potential using an on-chip microelectrode in a microfluidic device.

    Science.gov (United States)

    Lim, Tae-Sun; Dávila, Antonio; Wallace, Douglas C; Burke, Peter

    2010-07-07

    The mitochondrial membrane potential is used to generate and regulate energy in living systems, driving the conversion of ADP to ATP, regulating ion homeostasis, and controlling apoptosis, all central to human health and disease. Therefore, there is a need for tools to study its regulation in a controlled environment for potential clinical and scientific applications. For this aim, an on-chip tetraphenylphosphonium (TPP(+)) selective microelectrode sensor was constructed in a microfluidic environment. The concentration of isolated mitochondria (Heb7A) used in a membrane potential measurement was 0.3 ng microL(-1), four orders of magnitude smaller than the concentration used in conventional assays (3 microg microL(-1)). In addition, the volume of the chamber (85 microL) is 2 orders of magnitude smaller than traditional experiments. As a demonstration, changes in the membrane potential are clearly measured in response to a barrage of well-known substrates and inhibitors of the electron transport chain. This general approach, which to date has not been demonstrated for study of mitochondrial function and bio-energetics in generally, can be instrumental in advancing the field of mitochondrial research and clinical applications by allowing high throughput studies of the regulation, dynamics, and statistical properties of the mitochondrial membrane potential in response to inhibitors and inducers of apoptosis in a controlled (microfluidic) chemical environment.

  6. Deacylation on the matrix side of the mitochondrial inner membrane regulates cardiolipin remodeling.

    Science.gov (United States)

    Baile, Matthew G; Whited, Kevin; Claypool, Steven M

    2013-06-01

    The mitochondrial-specific lipid cardiolipin (CL) is required for numerous processes therein. After its synthesis on the matrix-facing leaflet of the inner membrane (IM), CL undergoes acyl chain remodeling to achieve its final form. In yeast, this process is completed by the transacylase tafazzin, which associates with intermembrane space (IMS)-facing membrane leaflets. Mutations in TAZ1 result in the X-linked cardiomyopathy Barth syndrome. Amazingly, despite this clear pathophysiological association, the physiological importance of CL remodeling is unresolved. In this paper, we show that the lipase initiating CL remodeling, Cld1p, is associated with the matrix-facing leaflet of the mitochondrial IM. Thus monolysocardiolipin generated by Cld1p must be transported to IMS-facing membrane leaflets to gain access to tafazzin, identifying a previously unknown step required for CL remodeling. Additionally, we show that Cld1p is the major site of regulation in CL remodeling; and that, like CL biosynthesis, CL remodeling is augmented in growth conditions requiring mitochondrially produced energy. However, unlike CL biosynthesis, dissipation of the mitochondrial membrane potential stimulates CL remodeling, identifying a novel feedback mechanism linking CL remodeling to oxidative phosphorylation capacity.

  7. Outer membrane VDAC1 controls permeability transition of the inner mitochondrial membrane in cellulo during stress-induced apoptosis

    Institute of Scientific and Technical Information of China (English)

    Flora Tomasello; Angela Messina; Lydia Lartigue; Laura Schembri; Chantal Medina; Simona Reina; Didier Thorava; Marc Crouzet; Francois Ichas; Vito De Pinto; Francesca De Giorgi

    2009-01-01

    Voltage-dependent anion channel (VDAC)l is the main channel of the mitochondrial outer membrane (MOM) and it has been proposed to be part of the permeability transition pore (PTP), a putative multiprotein complex candidate agent of the mitochondrial permeability transition (MPT). Working at the single live cell level, we found that over-expression of VDAC1 triggers MPT at the mitochondrial inner membrane (MIM). Conversely, silencing VDAC1 ex-pression results in the inhibition of MPT caused by selenite-induced oxidative stress. This MOM-M1M crosstalk was modulated by Cyclosporin A and mitochondrial Cyclophilin D, but not by Bcl-2 and Bcl-XL, indicative of PTP opera-tion. VDAC1-dependent MPT engages a positive feedback loop involving reactive oxygen species and p38-MAPK, and secondarily triggers a canonical apoptotic response including Bax activation, cytochrome c release and caspase 3 activation. Our data thus support a model of the PTP complex involving VDAC1 at the MOM, and indicate that VDAC1-dependent MPT is an upstream mechanism playing a causal role in oxidative stress-induced apoptosis.

  8. Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization.

    Science.gov (United States)

    Lam, M; Oleinick, N L; Nieminen, A L

    2001-12-14

    Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.

  9. Plasma membrane electron transport in Saccharomyces cerevisiae depends on the presence of mitochondrial respiratory subunits.

    Science.gov (United States)

    Herst, Patries M; Perrone, Gabriel G; Dawes, Ian W; Bircham, Peter W; Berridge, Michael V

    2008-09-01

    Most investigations into plasma membrane electron transport (PMET) in Saccharomyces cerevisiae have focused on the inducible ferric reductase responsible for iron uptake under iron/copper-limiting conditions. In this paper, we describe a PMET system, distinct from ferric reductase, which reduces the cell-impermeable water-soluble tetrazolium dye, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulphophenyl)-2H-tetrazolium monosodium salt (WST-1), under normal iron/copper conditions. WST-1/1-methoxy-phenazine methosulphate reduction was unaffected by anoxia and relatively insensitive to diphenyleneiodonium. Dye reduction was increased when intracellular NADH levels were high, which, in S. cerevisiae, required deletion of numerous genes associated with NADH recycling. Genome-wide screening of all viable nuclear gene-deletion mutants of S. cerevisiae revealed that, although mitochondrial electron transport per se was not required, the presence of several nuclear and mitochondrially encoded subunits of respiratory complexes III and IV was mandatory for PMET. This suggests some form of interaction between components of mitochondrial and plasma membrane electron transport. In support of this, mitochondrial tubular networks in S. cerevisiae were shown to be located in close proximity to the plasma membrane using confocal microscopy.

  10. Alterations in Lipid Levels of Mitochondrial Membranes Induced by Amyloid-ß: A Protective Role of Melatonin

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    Sergio A. Rosales-Corral

    2012-01-01

    Full Text Available Alzheimer pathogenesis involves mitochondrial dysfunction, which is closely related to amyloid-ß (Aß generation, abnormal tau phosphorylation, oxidative stress, and apoptosis. Alterations in membranal components, including cholesterol and fatty acids, their characteristics, disposition, and distribution along the membranes, have been studied as evidence of cell membrane alterations in AD brain. The majority of these studies have been focused on the cytoplasmic membrane; meanwhile the mitochondrial membranes have been less explored. In this work, we studied lipids and mitochondrial membranes in vivo, following intracerebral injection of fibrillar amyloid-ß (Aß. The purpose was to determine how Aß may be responsible for beginning of a vicious cycle where oxidative stress and alterations in cholesterol, lipids and fatty acids, feed back on each other to cause mitochondrial dysfunction. We observed changes in mitochondrial membrane lipids, and fatty acids, following intracerebral injection of fibrillar Aß in aged Wistar rats. Melatonin, a well-known antioxidant and neuroimmunomodulator indoleamine, reversed some of these alterations and protected mitochondrial membranes from obvious damage. Additionally, melatonin increased the levels of linolenic and n-3 eicosapentaenoic acid, in the same site where amyloid ß was injected, favoring an endogenous anti-inflammatory pathway.

  11. Alterations in Lipid Levels of Mitochondrial Membranes Induced by Amyloid-β: A Protective Role of Melatonin

    Science.gov (United States)

    Rosales-Corral, Sergio A.; Lopez-Armas, Gabriela; Cruz-Ramos, Jose; Melnikov, Valery G.; Tan, Dun-Xian; Manchester, Lucien C.; Munoz, Ruben; Reiter, Russel J.

    2012-01-01

    Alzheimer pathogenesis involves mitochondrial dysfunction, which is closely related to amyloid-β (Aβ) generation, abnormal tau phosphorylation, oxidative stress, and apoptosis. Alterations in membranal components, including cholesterol and fatty acids, their characteristics, disposition, and distribution along the membranes, have been studied as evidence of cell membrane alterations in AD brain. The majority of these studies have been focused on the cytoplasmic membrane; meanwhile the mitochondrial membranes have been less explored. In this work, we studied lipids and mitochondrial membranes in vivo, following intracerebral injection of fibrillar amyloid-β (Aβ). The purpose was to determine how Aβ may be responsible for beginning of a vicious cycle where oxidative stress and alterations in cholesterol, lipids and fatty acids, feed back on each other to cause mitochondrial dysfunction. We observed changes in mitochondrial membrane lipids, and fatty acids, following intracerebral injection of fibrillar Aβ in aged Wistar rats. Melatonin, a well-known antioxidant and neuroimmunomodulator indoleamine, reversed some of these alterations and protected mitochondrial membranes from obvious damage. Additionally, melatonin increased the levels of linolenic and n-3 eicosapentaenoic acid, in the same site where amyloid β was injected, favoring an endogenous anti-inflammatory pathway. PMID:22666620

  12. Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

    Science.gov (United States)

    Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

    2013-01-01

    Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

  13. Polyhydroxybutyrate targets mammalian mitochondria and increases permeability of plasmalemmal and mitochondrial membranes.

    Directory of Open Access Journals (Sweden)

    Pia A Elustondo

    Full Text Available Poly(3-hydroxybutyrate (PHB is a polyester of 3-hydroxybutyric acid (HB that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB. We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle.

  14. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

    OpenAIRE

    2016-01-01

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and saf...

  15. Outer mitochondrial membrane localization of apoptosis-inducing factor: mechanistic implications for release

    Directory of Open Access Journals (Sweden)

    Seong‑Woon Yu

    2009-11-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  16. Outer Mitochondrial Membrane Localization of Apoptosis-Inducing Factor: Mechanistic Implications for Release

    Directory of Open Access Journals (Sweden)

    Seong-Woon Yu

    2009-10-01

    Full Text Available Poly(ADP-ribose polymerase-1-dependent cell death (known as parthanatos plays a pivotal role in many clinically important events including ischaemia/reperfusion injury and glutamate excitotoxicity. A recent study by us has shown that uncleaved AIF (apoptosis-inducing factor, but not calpain-hydrolysed truncated-AIF, was rapidly released from the mitochondria during parthanatos, implicating a second pool of AIF that might be present in brain mitochondria contributing to the rapid release. In the present study, a novel AIF pool is revealed in brain mitochondria by multiple biochemical analyses. Approx. 30% of AIF loosely associates with the outer mitochondrial membrane on the cytosolic side, in addition to its main localization in the mitochondrial intermembrane space attached to the inner membrane. Immunogold electron microscopic analysis of mouse brain further supports AIF association with the outer, as well as the inner, mitochondrial membrane in vivo. In line with these observations, approx. 20% of uncleaved AIF rapidly translocates to the nucleus and functionally causes neuronal death upon NMDA (N-methyl-d-aspartate treatment. In the present study we show for the first time a second pool of AIF in brain mitochondria and demonstrate that this pool does not require cleavage and that it contributes to the rapid release of AIF. Moreover, these results suggest that this outer mitochondrial pool of AIF is sufficient to cause cell death during parthanatos. Interfering with the release of this outer mitochondrial pool of AIF during cell injury paradigms that use parthanatos hold particular promise for novel therapies to treat neurological disorders.

  17. Mitochondrial respiration and membrane potential after low-flow ischemia are not affected by ischemic preconditioning.

    Science.gov (United States)

    Boengler, Kerstin; Gres, Petra; Dodoni, Giuliano; Konietzka, Ina; Di Lisa, Fabio; Heusch, Gerd; Schulz, Rainer

    2007-11-01

    Mitochondrial function following prolonged ischemia and subsequent reperfusion is better preserved by ischemic preconditioning (IP). In the present study, we analyzed whether or not IP has an impact on mitochondrial function at the end of a sustained ischemic period. Göttinger minipigs were subjected to 90-min low-flow ischemia without (n=5) and with (n=5) a preconditioning cycle of 10-min ischemia and 15-min reperfusion. Mitochondria were isolated from the ischemic or preconditioned anterior wall (AW) and the control posterior wall (PW) at the end of ischemia. Basal mitochondrial respiration was not different between AW and PW. The ADP-stimulated (state 3) respiration in AW mitochondria compared to PW mitochondria was equally decreased in non-preconditioned and preconditioned pigs. The uncoupled respiration as well as the membrane potential (rhodamine 123 fluorescence) were not significantly different between groups. However, the recovery of the membrane potential (Delta rhodamine 123 fluorescence/s) after the addition of ADP was delayed in mitochondria obtained from AW compared to PW, both in non-preconditioned and in preconditioned pig hearts. Neither the amount of marker proteins for complexes of the electron transport chain nor the level of reactive oxygen species were affected by ischemia without or with IP. State 3 respiration and recovery of membrane potential were impaired in pig mitochondria after 90 min of low-flow ischemia. IP did not improve mitochondrial function during ischemia. Therefore, the preservation of mitochondrial function by IP may occur during reperfusion rather than during the sustained ischemic period.

  18. Mitochondrial OXA Translocase Plays a Major Role in Biogenesis of Inner-Membrane Proteins.

    Science.gov (United States)

    Stiller, Sebastian B; Höpker, Jan; Oeljeklaus, Silke; Schütze, Conny; Schrempp, Sandra G; Vent-Schmidt, Jens; Horvath, Susanne E; Frazier, Ann E; Gebert, Natalia; van der Laan, Martin; Bohnert, Maria; Warscheid, Bettina; Pfanner, Nikolaus; Wiedemann, Nils

    2016-05-10

    The mitochondrial inner membrane harbors three protein translocases. Presequence translocase and carrier translocase are essential for importing nuclear-encoded proteins. The oxidase assembly (OXA) translocase is required for exporting mitochondrial-encoded proteins; however, different views exist about its relevance for nuclear-encoded proteins. We report that OXA plays a dual role in the biogenesis of nuclear-encoded mitochondrial proteins. First, a systematic analysis of OXA-deficient mitochondria led to an unexpected expansion of the spectrum of OXA substrates imported via the presequence pathway. Second, biogenesis of numerous metabolite carriers depends on OXA, although they are not imported by the presequence pathway. We show that OXA is crucial for the biogenesis of the Tim18-Sdh3 module of the carrier translocase. The export translocase OXA is thus required for the import of metabolite carriers by promoting assembly of the carrier translocase. We conclude that OXA is of central importance for the biogenesis of the mitochondrial inner membrane.

  19. Evidence for several cysteine transport mechanisms in the mitochondrial membranes of Arabidopsis thaliana.

    Science.gov (United States)

    Lee, Chun Pong; Wirtz, Markus; Hell, Rüdiger

    2014-01-01

    Cysteine is essential for many mitochondrial processes in plants, including translation, iron-sulfur cluster biogenesis and cyanide detoxification. Its biosynthesis is carried out by serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL) which can be found in the cytosol, plastids and mitochondria. Mutants lacking one compartment-specific OAS-TL isoform show viable phenotypes, leading to the hypothesis that the organellar membranes are permeable to substrates and products of the cysteine biosynthetic pathway. In this report, we show that exogenouslly supplied [(35)S]cysteine accumulates in the mitochondrial fraction and is taken up into isolated mitochondria for in organello protein synthesis. Analysis of cysteine uptake by isolated mitochondria and mitoplasts indicates that cysteine is transported by multiple facilitated mechanisms that operate in a concentration gradient-dependent manner. In addition, cysteine uptake is dependent mainly on the ΔpH across the inner membrane. The rates of mitochondrial cysteine transport can be mildly altered by specific metabolites in the cyanide detoxification-linked sulfide oxidation, but not by most substrates and products of the cysteine biosynthetic pathway. Based on these results, we propose that the transport of cysteine plays a pivotal role in regulating cellular cysteine biosynthesis as well as modulating the availability of sulfur for mitochondrial metabolism.

  20. Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

    NARCIS (Netherlands)

    van der Toorn, Marco; Kauffman, Henk F.; van der Deen, Margaretha; Slebos, Dirk-Jan; Koeter, Gerard H.; Gans, Rijk O. B.; Bakker, Stephan J. L.

    2007-01-01

    Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (Delta Psi(m)) and the production of reactive oxygen species. Fluorescent probes were used to assess Delt

  1. A model of mitochondrial creatine kinase binding to membranes: adsorption constants, essential amino acids and the effect of ionic strength

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Belousova, Lubov; Plesner, Igor

    1993-01-01

    The quantitative aspects of mitochondrial creatinekinase (mitCK) binding to mitochondrial membranes were investigated. A simple adsorption and binding model was used for data fitting, taking into account the influence of protein concentration, pH, ionic strength and substrate concentration on the...

  2. Cyclosporin A-induced oxidative stress is not the consequence of an increase in mitochondrial membrane potential

    NARCIS (Netherlands)

    van der Toorn, Marco; Kauffman, Henk F.; van der Deen, Margaretha; Slebos, Dirk-Jan; Koeter, Gerard H.; Gans, Rijk O. B.; Bakker, Stephan J. L.

    2007-01-01

    Cyclosporin A induces closure of the mitochondrial permeability transition pore. We aimed to investigate whether this closure results in concomitant increases in mitochondrial membrane potential (Delta Psi(m)) and the production of reactive oxygen species. Fluorescent probes were used to assess Delt

  3. Mitochondrial calcium ion and membrane potential transients follow the pattern of epileptiform discharges in hippocampal slice cultures.

    Science.gov (United States)

    Kovács, Richard; Kardos, Julianna; Heinemann, Uwe; Kann, Oliver

    2005-04-27

    Emerging evidence suggests that mitochondrial dysfunction contributes to the pathophysiology of epilepsy. Recurrent mitochondrial Ca2+ ion load during seizures might act on mitochondrial membrane potential (DeltaPsim) and proton motive force. By using electrophysiology and confocal laser-scanning microscopy, we investigated the effects of epileptiform activity, as induced by low-Mg2+ ion perfusion in hippocampal slice cultures, on changes in DeltaPsim and in mitochondrial Ca2+ ion concentration ([Ca2+]m). The mitochondrial compartment was identified by monitoring DeltaPsim in the soma and dendrites of patched CA3 pyramidal cells using the mitochondria-specific voltage-sensitive dye rhodamine-123 (Rh-123). Interictal activity was accompanied by localized mitochondrial depolarization that was restricted to a few mitochondria in small dendrites. In contrast, robust Rh-123 release into the cytosol was observed during seizure-like events (SLEs), indicating simultaneous depolarization of mitochondria. This was critically dependent on Ca2+ ion uptake and extrusion, because inhibition of the mitochondrial Ca2+ ion uniporter by Ru360 and the mitochondrial Na+/Ca2+ ion exchanger by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one but not the inhibitor of mitochondrial permeability transition pore, cyclosporin A, decreased the SLE-associated mitochondrial depolarization. The Ca2+ ion dependence of simultaneous mitochondrial depolarization suggested enhanced Ca2+ ion cycling across mitochondrial membranes during epileptiform activity. Indeed, [Ca2+]m fluctuated during interictal activity in single dendrites, and these fluctuations spread over the entire mitochondrial compartment during SLEs, as revealed using mitochondria-specific dyes (rhod-2 and rhod-ff) and spatial frequency-based image analysis. These findings strengthen the hypothesis that epileptic activity results in Ca2+ ion-dependent changes in mitochondrial function that might contribute to the

  4. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA depletion.

    Science.gov (United States)

    Spinazzola, Antonella; Viscomi, Carlo; Fernandez-Vizarra, Erika; Carrara, Franco; D'Adamo, Pio; Calvo, Sarah; Marsano, René Massimiliano; Donnini, Claudia; Weiher, Hans; Strisciuglio, Pietro; Parini, Rossella; Sarzi, Emmanuelle; Chan, Alicia; DiMauro, Salvatore; Rötig, Agnes; Gasparini, Paolo; Ferrero, Iliana; Mootha, Vamsi K; Tiranti, Valeria; Zeviani, Massimo

    2006-05-01

    The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.

  5. A mitochondrial-focused genetic interaction map reveals a scaffold-like complex required for inner membrane organization in mitochondria.

    Science.gov (United States)

    Hoppins, Suzanne; Collins, Sean R; Cassidy-Stone, Ann; Hummel, Eric; Devay, Rachel M; Lackner, Laura L; Westermann, Benedikt; Schuldiner, Maya; Weissman, Jonathan S; Nunnari, Jodi

    2011-10-17

    To broadly explore mitochondrial structure and function as well as the communication of mitochondria with other cellular pathways, we constructed a quantitative, high-density genetic interaction map (the MITO-MAP) in Saccharomyces cerevisiae. The MITO-MAP provides a comprehensive view of mitochondrial function including insights into the activity of uncharacterized mitochondrial proteins and the functional connection between mitochondria and the ER. The MITO-MAP also reveals a large inner membrane-associated complex, which we term MitOS for mitochondrial organizing structure, comprised of Fcj1/Mitofilin, a conserved inner membrane protein, and five additional components. MitOS physically and functionally interacts with both outer and inner membrane components and localizes to extended structures that wrap around the inner membrane. We show that MitOS acts in concert with ATP synthase dimers to organize the inner membrane and promote normal mitochondrial morphology. We propose that MitOS acts as a conserved mitochondrial skeletal structure that differentiates regions of the inner membrane to establish the normal internal architecture of mitochondria.

  6. Mitochondrial-dependent Autoimmunity in Membranous Nephropathy of IgG4-related Disease

    Directory of Open Access Journals (Sweden)

    Simona Buelli

    2015-05-01

    Full Text Available The pathophysiology of glomerular lesions of membranous nephropathy (MN, including seldom-reported IgG4-related disease, is still elusive. Unlike in idiopathic MN where IgG4 prevails, in this patient IgG3 was predominant in glomerular deposits in the absence of circulating anti-phospholipase A2 receptor antibodies, suggesting a distinct pathologic process. Here we documented that IgG4 retrieved from the serum of our propositus reacted against carbonic anhydrase II (CAII at the podocyte surface. In patient's biopsy, glomerular CAII staining increased and co-localized with subepithelial IgG4 deposits along the capillary walls. Patient's IgG4 caused a drop in cell pH followed by mitochondrial dysfunction, excessive ROS production and cytoskeletal reorganization in cultured podocytes. These events promoted mitochondrial superoxide-dismutase-2 (SOD2 externalization on the plasma membrane, becoming recognizable by complement-binding IgG3 anti-SOD2. Among patients with IgG4-related disease only sera of those with IgG4 anti-CAII antibodies caused low intracellular pH and mitochondrial alterations underlying SOD2 externalization. Circulating IgG4 anti-CAII can cause podocyte injury through processes of intracellular acidification, mitochondrial oxidative stress and neoantigen induction in patients with IgG4 related disease. The onset of MN in a subset of patients could be due to IgG4 antibodies recognizing CAII with consequent exposure of mitochondrial neoantigen in the context of multifactorial pathogenesis of disease.

  7. Investigating membrane and mitochondrial cryobiological responses of HUVEC using interrupted cooling protocols.

    Science.gov (United States)

    Reardon, Anthony J F; Elliott, Janet A W; McGann, Locksley E

    2015-10-01

    The success of cryopreservation protocols is largely based on membrane integrity assessments after thawing, since membrane integrity can be considered to give an upper limit in assessment of cell viability and the plasma membrane is considered to be a primary site of cryoinjury. However, the exposure of cells to conditions associated with low temperatures can induce injury to cellular structure and function that may not be readily identified by membrane integrity alone. Interrupted cooling protocols (including interrupted slow cooling without a hold time (graded freezing), and interrupted rapid cooling with a hold time (two-step freezing)), can yield important information about cryoinjury by separating the damage that occurs upon cooling to (and possibly holding at) a critical intermediate temperature range from the damage that occurs upon plunging to the storage temperature (liquid nitrogen). In this study, we used interrupted cooling protocols in the absence of cryoprotectant to investigate the progression of damage to human umbilical vein endothelial cells (HUVEC), comparing an assessment of membrane integrity with a mitochondrial polarization assay. Additionally, the membrane integrity response of HUVEC to interrupted cooling was investigated as a function of cooling rate (for interrupted slow cooling) and hold time (for interrupted rapid cooling). A key finding of this work was that under slow cooling conditions which resulted in a large number of membrane intact cells immediately post thaw, mitochondria are predominantly in a non-functional depolarized state. This study, the first to look directly at mitochondrial polarization throughout interrupted cooling profiles and a detailed study of HUVEC response, highlights the complexity of the progression of cell damage, as the pattern and extent of cell injury throughout the preservation process differs by injury site.

  8. Ethanol reduces mitochondrial membrane integrity and thereby impacts carbon metabolism of Saccharomyces cerevisiae.

    Science.gov (United States)

    Yang, Kyung-Mi; Lee, Na-Rae; Woo, Ji-Min; Choi, Wonja; Zimmermann, Martin; Blank, Lars M; Park, Jin-Byung

    2012-09-01

    Saccharomyces cerevisiae is an excellent ethanol producer, but is rather sensitive to high concentration of ethanol. Here, influences of ethanol on cellular membrane integrity and carbon metabolism of S. cerevisiae were investigated to rationalize mechanism involved in ethanol toxicity. Addition of 5% (v/v) ethanol did neither significantly change the permeability of the cytoplasmic membrane of the reference strain S. cerevisiae BY4741 nor of the ethanol-tolerant strain iETS3. However, the addition of ethanol resulted in a marked decrease in the mitochondrial membrane potential and in increased concentrations of intracellular reactive oxygen species (ROS). The carbon flux was redistributed under these conditions from mainly ethanol production to the TCA cycle. This redistribution was possibly a result of increased energy demand for cell maintenance that increased from about zero to 20-40 mmol ATP (g(CDW)  h)(-1) . This increase in maintenance energy might be explained by the ethanol-induced reduction of the proton motive force and the required removal of ROS. Thus, the stability of the mitochondrial membrane and subsequently the capacity to keep ROS levels low could be important factors to improve tolerance of S. cerevisiae against ethanol. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

  9. Heat shock induces production of reactive oxygen species and increases inner mitochondrial membrane potential in winter wheat cells.

    Science.gov (United States)

    Fedyaeva, A V; Stepanov, A V; Lyubushkina, I V; Pobezhimova, T P; Rikhvanov, E G

    2014-11-01

    Heat shock leads to oxidative stress. Excessive ROS (reactive oxygen species) accumulation could be responsible for expression of genes of heat-shock proteins or for cell death. It is known that in isolated mammalian mitochondria high protonic potential on the inner membrane actuates the production of ROS. Changes in viability, ROS content, and mitochondrial membrane potential value have been studied in winter wheat (Triticum aestivum L.) cultured cells under heat treatment. Elevation of temperature to 37-50°C was found to induce elevated ROS generation and increased mitochondrial membrane potential, but it did not affect viability immediately after treatment. More severe heat exposure (55-60°C) was not accompanied by mitochondrial potential elevation and increased ROS production, but it led to instant cell death. A positive correlation between mitochondrial potential and ROS production was observed. Depolarization of the mitochondrial membrane by the protonophore CCCP inhibited ROS generation under the heating conditions. These data suggest that temperature elevation leads to mitochondrial membrane hyperpolarization in winter wheat cultured cells, which in turn causes the increased ROS production.

  10. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax.

    Science.gov (United States)

    Lidman, Martin; Pokorná, Šárka; Dingeldein, Artur P G; Sparrman, Tobias; Wallgren, Marcus; Šachl, Radek; Hof, Martin; Gröbner, Gerhard

    2016-06-01

    Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Toxins in Botanical Dietary Supplements: Blue Cohosh Components Disrupt Cellular Respiration and Mitochondrial Membrane Potential

    Science.gov (United States)

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B.; Khan, Ikhlas A.; Nagle, Dale G.; Zhou, Yu-Dong

    2014-01-01

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA “Black Box” warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3) exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage. PMID:24328138

  12. Vimentin is involved in regulation of mitochondrial motility and membrane potential by Rac1

    Directory of Open Access Journals (Sweden)

    Elena A. Matveeva

    2015-10-01

    Full Text Available In this study we show that binding of mitochondria to vimentin intermediate filaments (VIF is regulated by GTPase Rac1. The activation of Rac1 leads to a redoubling of mitochondrial motility in murine fibroblasts. Using double-mutants Rac1(G12V, F37L and Rac1(G12V, Y40H that are capable to activate different effectors of Rac1, we show that mitochondrial movements are regulated through PAK1 kinase. The involvement of PAK1 kinase is also confirmed by the fact that expression of its auto inhibitory domain (PID blocks the effect of activated Rac1 on mitochondrial motility. The observed effect of Rac1 and PAK1 kinase on mitochondria depends on phosphorylation of the Ser-55 of vimentin. Besides the effect on motility Rac1 activation also decreases the mitochondrial membrane potential (MMP which is detected by ∼20% drop of the fluorescence intensity of mitochondria stained with the potential sensitive dye TMRM. One of important consequences of the discovered regulation of MMP by Rac1 and PAK1 is a spatial differentiation of mitochondria in polarized fibroblasts: at the front of the cell they are less energized (by ∼25% than at the rear part.

  13. Toxins in botanical dietary supplements: blue cohosh components disrupt cellular respiration and mitochondrial membrane potential.

    Science.gov (United States)

    Datta, Sandipan; Mahdi, Fakhri; Ali, Zulfiqar; Jekabsons, Mika B; Khan, Ikhlas A; Nagle, Dale G; Zhou, Yu-Dong

    2014-01-24

    Certain botanical dietary supplements have been associated with idiosyncratic organ-specific toxicity. Similar toxicological events, caused by drug-induced mitochondrial dysfunction, have forced the withdrawal or U.S. FDA "black box" warnings of major pharmaceuticals. To assess the potential mitochondrial liability of botanical dietary supplements, extracts from 352 authenticated plant samples used in traditional Chinese, Ayurvedic, and Western herbal medicine were evaluated for the ability to disrupt cellular respiration. Blue cohosh (Caulophyllum thalictroides) methanol extract exhibited mitochondriotoxic activity. Used by some U.S. midwives to help induce labor, blue cohosh has been associated with perinatal stroke, acute myocardial infarction, congestive heart failure, multiple organ injury, and neonatal shock. The potential link between mitochondrial disruption and idiosyncratic herbal intoxication prompted further examination. The C. thalictroides methanol extract and three saponins, cauloside A (1), saponin PE (2), and cauloside C (3), exhibited concentration- and time-dependent mitochondriotoxic activities. Upon treatment, cell respiration rate rapidly increased and then dramatically decreased within minutes. Mechanistic studies revealed that C. thalictroides constituents impair mitochondrial function by disrupting membrane integrity. These studies provide a potential etiological link between this mitochondria-sensitive form of cytotoxicity and idiosyncratic organ damage.

  14. Vanadate induces necrotic death in neonatal rat cardiomyocytes through mitochondrial membrane depolarization.

    Science.gov (United States)

    Soares, Sandra Sofia; Henao, Fernando; Aureliano, Manuel; Gutiérrez-Merino, Carlos

    2008-03-01

    Besides the well-known inotropic effects of vanadium in cardiac muscle, previous studies have shown that vanadate can stimulate cell growth or induce cell death. In this work, we studied the toxicity to neonatal rat ventricular myocytes (cardiomyocytes) of two vanadate solutions containing different oligovanadates distribution, decavanadate (containing decameric vanadate, V 10) and metavanadate (containing monomeric vanadate and also di-, tetra-, and pentavanadate). Incubation for 24 h with decavanadate or metavanadate induced necrotic cell death of cardiomyocytes, without significant caspase-3 activation. Only 10 microM total vanadium of either decavanadate (1 microM V 10) or metavanadate (10 microM total vanadium) was needed to produce 50% loss of cell viability after 24 h (assessed with MTT and propidium iodide assays). Atomic absorption spectroscopy showed that vanadium accumulation in cardiomyocytes after 24 h was the same when incubation was done with decavanadate or metavanadate. A decrease of 75% of the rate of mitochondrial superoxide anion generation, monitored with dihydroethidium, and a sustained rise of cytosolic calcium (monitored with Fura-2-loaded cardiomyocytes) was observed after 24 h of incubation of cardiomyocytes with decavanadate or metavanadate concentrations close to those inducing 50% loss of cell viability produced. In addition, mitochondrial membrane depolarization within cardiomyocytes, monitored with tetramethylrhodamine ethyl esther or with 3,3',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide, were observed after only 6 h of incubation with decavanadate or metavanadate. The concentration needed for 50% mitochondrial depolarization was 6.5 +/- 1 microM total vanadium for both decavanadate (0.65 microM V 10) and metavanadate. In conclusion, mitochondrial membrane depolarization was an early event in decavanadate- and monovanadate-induced necrotic cell death of cardiomyocytes.

  15. Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

    Science.gov (United States)

    Anand, Sanjeev K; Gaba, Amit; Singh, Jaswant; Tikoo, Suresh K

    2014-02-01

    Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

  16. Profiling of the Tox21 Chemical Collection for Mitochondrial Function: I. Compounds that Decrease Mitochondrial Membrane Potential

    Science.gov (United States)

    Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding how different environmental chemicals and drug-like molecules impact mitochondrial function rep...

  17. High-Content Imaging Assays for Identifying Compounds that Generate Superoxide and Impair Mitochondrial Membrane Potential in Adherent Eukaryotic Cells.

    Science.gov (United States)

    Billis, Puja; Will, Yvonne; Nadanaciva, Sashi

    2014-02-19

    Reactive oxygen species (ROS) are constantly produced in cells as a result of aerobic metabolism. When there is an excessive production of ROS and the cell's antioxidant defenses are overwhelmed, oxidative stress occurs. The superoxide anion is a type of ROS that is produced primarily in mitochondria but is also generated in other regions of the cell including peroxisomes, endoplasmic reticulum, plasma membrane, and cytosol. Here, a high-content imaging assay using the dye dihydroethidium is described for identifying compounds that generate superoxide in eukaryotic cells. A high-content imaging assay using the fluorescent dye tetramethylrhodamine methyl ester is also described to identify compounds that impair mitochondrial membrane potential in eukaryotic cells. The purpose of performing both assays is to identify compounds that (1) generate superoxide at lower concentrations than they impair mitochondrial membrane potential, (2) impair mitochondrial membrane potential at lower concentrations than they generate superoxide, (3) generate superoxide and impair mitochondrial function at similar concentrations, and (4) do not generate superoxide or impair mitochondrial membrane potential during the duration of the assays.

  18. Lipidomics reveals mitochondrial membrane remodeling associated with acute thermoregulation in a rodent with a wide thermoneutral zone.

    Science.gov (United States)

    Pan, Qian; Li, Min; Shi, Yao-Long; Liu, Huwei; Speakman, John R; Wang, De-Hua

    2014-07-01

    Mongolian gerbils (Meriones unguiculatus) have high physiological flexibility in response to acute temperature changes, and have the widest thermoneutral zone (TNZ, 26.5-38.9 °C) reported among small mammals. At the upper critical temperature (T(uc), 38.9 °C), body temperatures of gerbils were significantly increased (39-41 °C) while metabolic rates were maintained at the basal level. In contrast, below the lower critical temperature (T(lc), 26.5 °C), metabolism was elevated and body temperature stable. Rapid changes in mitochondrial membrane lipidome were hypothesized to play an important role during acute thermoregulation of gerbils. Taking advantage of a recent lipidomic technique, we examined changes in the membrane phospholipids environment and free fatty acids (FFA) production in mitochondria between 38 and 27 °C (in the TNZ), and between 27 and 16 °C (below the TNZ). At 38 °C, acute heat stress elicited distinct remodeling in mitochondrial membrane lipidome which related to a potential decrease in mitochondrial respiration and membrane fluidity compared to 27 °C. At 16 °C, a sharply decreased unsaturation index and increased chain lengths were detected in mitochondrial FFA production both in muscle and brown adipose tissue. Our results suggest that mitochondrial membrane lipid remodeling may stabilize membrane function and activity of respiration related membrane protein to maintain a stable metabolic rate at T(uc), and improve heat production by decomposing less fluid fatty acid conjugates of membrane lipids under acute cold exposure. These data therefore imply an important role of membrane remodeling during acute thermoregulation in a non-hibernating endotherm.

  19. Ocean acidification impacts on sperm mitochondrial membrane potential bring sperm swimming behaviour near its tipping point.

    Science.gov (United States)

    Schlegel, Peter; Binet, Monique T; Havenhand, Jonathan N; Doyle, Christopher J; Williamson, Jane E

    2015-04-01

    Broadcast spawning marine invertebrates are susceptible to environmental stressors such as climate change, as their reproduction depends on the successful meeting and fertilization of gametes in the water column. Under near-future scenarios of ocean acidification, the swimming behaviour of marine invertebrate sperm is altered. We tested whether this was due to changes in sperm mitochondrial activity by investigating the effects of ocean acidification on sperm metabolism and swimming behaviour in the sea urchin Centrostephanus rodgersii. We used a fluorescent molecular probe (JC-1) and flow cytometry to visualize mitochondrial activity (measured as change in mitochondrial membrane potential, MMP). Sperm MMP was significantly reduced in ΔpH -0.3 (35% reduction) and ΔpH -0.5 (48% reduction) treatments, whereas sperm swimming behaviour was less sensitive with only slight changes (up to 11% decrease) observed overall. There was significant inter-individual variability in responses of sperm swimming behaviour and MMP to acidified seawater. We suggest it is likely that sperm exposed to these changes in pH are close to their tipping point in terms of physiological tolerance to acidity. Importantly, substantial inter-individual variation in responses of sperm swimming to ocean acidification may increase the scope for selection of resilient phenotypes, which, if heritable, could provide a basis for adaptation to future ocean acidification. © 2015. Published by The Company of Biologists Ltd.

  20. Anomalous diffusion induced by cristae geometry in the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Valerii M Sukhorukov

    Full Text Available Diffusion of inner membrane proteins is a prerequisite for correct functionality of mitochondria. The complicated structure of tubular, vesicular or flat cristae and their small connections to the inner boundary membrane impose constraints on the mobility of proteins making their diffusion a very complicated process. Therefore we investigate the molecular transport along the main mitochondrial axis using highly accurate computational methods. Diffusion is modeled on a curvilinear surface reproducing the shape of mitochondrial inner membrane (IM. Monte Carlo simulations are carried out for topologies resembling both tubular and lamellar cristae, for a range of physiologically viable crista sizes and densities. Geometrical confinement induces up to several-fold reduction in apparent mobility. IM surface curvature per se generates transient anomalous diffusion (TAD, while finite and stable values of projected diffusion coefficients are recovered in a quasi-normal regime for short- and long-time limits. In both these cases, a simple area-scaling law is found sufficient to explain limiting diffusion coefficients for permeable cristae junctions, while asymmetric reduction of the junction permeability leads to strong but predictable variations in molecular motion rate. A geometry-based model is given as an illustration for the time-dependence of diffusivity when IM has tubular topology. Implications for experimental observations of diffusion along mitochondria using methods of optical microscopy are drawn out: a non-homogenous power law is proposed as a suitable approach to TAD. The data demonstrate that if not taken into account appropriately, geometrical effects lead to significant misinterpretation of molecular mobility measurements in cellular curvilinear membranes.

  1. Improved mitochondrial function with diet-induced increase in either docosahexaenoic acid or arachidonic acid in membrane phospholipids.

    Directory of Open Access Journals (Sweden)

    Ramzi J Khairallah

    Full Text Available Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP. We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA docosahexaenoic acid (DHA; 22:6n3 and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6 in mitochondrial membranes is associated with a greater Ca(2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6. Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca(2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca(2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.

  2. Lipid, membrane, and mitochondrial characteristics of Ustilago maydis following exposure to ergosterol biosynthesis inhibitors

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    Waterfield, W.F. III

    1986-01-01

    Pencoazole at 0.5 ..mu..g/ml inhibited ergosterol biosynthesis in U. maydis. Polar lipids of sporidia grown with 0.5 ..mu..g/ml penconazole for 7.5 or 22 hr or 1.0 ..mu..g/ml fenarimol for 7.5 hr contained more 18:2 than 18:1 fatty acids. There was usually more 18:1 than 18:2 fatty acids in polar lipids of untreated sporidia but this ratio was influenced by culture cell density. The high 18:2 to 18:1 ratio in the polar lipids from penconazole grown cells was unaffected by cell density. There was an increase in free fatty acids and these were enriched with 18:2 members in cells grown with 0.5 ..mu..g/ml penconazole for 22 hr. Unsaturation of triglycerides fatty acids did not differ appreciably from that of untreated sporidia. Untreated WT U. maydis protoplasts lysed more slowly in 0.3 M sorbitol than those prepared from WT sporidia grown for 16 hr with 1.0 ..mu..g/ml penconazole or 2.0 ..mu..g/ml fenarimol or from untreated erg-40 sporidia. Protoplasts were more permeable to crystal violet than were those from untreated WT sporidia. Mitochondria from untreated WT sporidia oxidizing pyruvate plus malate or succinate yielded higher ADP/O rations than mitochondria from erg-40 or penconazole grown WT sporidia. The mitochondrial ATPase of control cells had a Km of 0.8 mM ATP whereas the mitochondrial ATPase of penconazole grown WT and erg-40 had a Km value of 3.7 and 3.2 mM ATP, respectively. When the mitochondrial catalytic subunit of the ATPase from these mitochondria were solubilized, the Km did not differ. These studies suggest that changes in sterols and membrane fatty acids resulting from treatments with EBI fungicides cause increased membrane fluidity which affects membrane stability, permeability and activity of the mitochondrial ATPase.

  3. Bacterial origin of a mitochondrial outer membrane protein translocase: new perspectives from comparative single channel electrophysiology.

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    Harsman, Anke; Niemann, Moritz; Pusnik, Mascha; Schmidt, Oliver; Burmann, Björn M; Hiller, Sebastian; Meisinger, Chris; Schneider, André; Wagner, Richard

    2012-09-07

    Mitochondria are of bacterial ancestry and have to import most of their proteins from the cytosol. This process is mediated by Tom40, an essential protein that forms the protein-translocating pore in the outer mitochondrial membrane. Tom40 is conserved in virtually all eukaryotes, but its evolutionary origin is unclear because bacterial orthologues have not been identified so far. Recently, it was shown that the parasitic protozoon Trypanosoma brucei lacks a conventional Tom40 and instead employs the archaic translocase of the outer mitochondrial membrane (ATOM), a protein that shows similarities to both eukaryotic Tom40 and bacterial protein translocases of the Omp85 family. Here we present electrophysiological single channel data showing that ATOM forms a hydrophilic pore of large conductance and high open probability. Moreover, ATOM channels exhibit a preference for the passage of cationic molecules consistent with the idea that it may translocate unfolded proteins targeted by positively charged N-terminal presequences. This is further supported by the fact that the addition of a presequence peptide induces transient pore closure. An in-depth comparison of these single channel properties with those of other protein translocases reveals that ATOM closely resembles bacterial-type protein export channels rather than eukaryotic Tom40. Our results support the idea that ATOM represents an evolutionary intermediate between a bacterial Omp85-like protein export machinery and the conventional Tom40 that is found in mitochondria of other eukaryotes.

  4. Assessment of mitochondrial membrane potential in proximal tubules after hypoxia-reoxygenation.

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    Feldkamp, Thorsten; Kribben, Andreas; Weinberg, Joel M

    2005-06-01

    Proximal tubules develop a severe energetic deficit during hypoxia-reoxygenation (H/R) that previous studies using fluorescent potentiometric probes have suggested is characterized by sustained, partial mitochondrial deenergization. To validate the primary occurrence of mitochondrial deenergization in the process, optimize approaches for estimating changes in mitochondrial membrane potential (DeltaPsim) in the system, and clarify the mechanisms for the defect, we further investigated the behavior of 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazocarbocyanine iodide (JC-1) in these cells and introduce a more dynamic and quantitative approach employing safranin O for use with the tubule system. Although use of JC-1 can be complicated by decreases in the plasma membrane potential that limit cellular uptake of JC-1 and such behavior was demonstrated in ouabain-treated tubules, changes in DeltaPsim entirely accounted for the decreases in the formation of red fluorescent JC-1 aggregates and in the ratio of red/green fluorescence observed after H/R. The red JC-1 aggregates did not readily dissociate when tubules were deenergized after JC-1 uptake, making it unsuitable for dynamic studies of energization. Safranin O uptake by digitonin-permeabilized tubules required very small numbers of tubules, permitted measurements of DeltaPsim for relatively prolonged periods after the end of the experimental maneuvers, was rapidly reversible during deenergization, and allowed for direct assessment of both substrate-dependent, electron transport-mediated DeltaPsim, and ATP hydrolysis-supported DeltaPsim. Both types of energization measured using safranin O in tubules permeabilized after H/R were impaired, but combining substrates and ATP substantially restored DeltaPsim.

  5. Permethrin may disrupt testosterone biosynthesis via mitochondrial membrane damage of Leydig cells in adult male mouse.

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    Zhang, Shu-Yun; Ito, Yuki; Yamanoshita, Osamu; Yanagiba, Yukie; Kobayashi, Miya; Taya, Kazuyoshi; Li, ChunMei; Okamura, Ai; Miyata, Maiko; Ueyama, Jun; Lee, Chul-Ho; Kamijima, Michihiro; Nakajima, Tamie

    2007-08-01

    Permethrin, a popular synthetic pyrethroid insecticide used to control noxious insects in agriculture, forestry, households, horticulture, and public health throughout the world, poses risks of environmental exposure. Here we evaluate the reproductive toxicity of cis-permethrin in adult male ICR mice that were orally administered cis-permethrin (0, 35, or 70 mg/kg d) for 6 wk. Caudal epididymal sperm count and sperm motility in the treated groups were statistically reduced in a dose-dependent manner. Testicular testosterone production and plasma testosterone concentration were significantly and dose-dependently decreased with an increase in LH, and a significant regression was observed between testosterone levels and cis-permethrin residues in individual mice testes after exposure. However, no significant changes were observed in body weight, reproductive organ absolute and relative weights, sperm morphology, and plasma FSH concentration after cis-permethrin treatment. Moreover, cis-permethrin exposure significantly diminished the testicular mitochondrial mRNA expression levels of peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) and enzyme and protein expression levels of StAR and P450scc. At the electron microscopic level, mitochondrial membrane damage was found in Leydig cells of the exposed mouse testis. Our results suggest that the insecticide permethrin may cause mitochondrial membrane impairment in Leydig cells and disrupt testosterone biosynthesis by diminishing the delivery of cholesterol into the mitochondria and decreasing the conversion of cholesterol to pregnenolone in the cells, thus reducing subsequent testosterone production.

  6. Heterogeneity in mitochondrial morphology and membrane potential is independent of the nuclear division cycle in multinucleate fungal cells.

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    Gerstenberger, John P; Occhipinti, Patricia; Gladfelter, Amy S

    2012-03-01

    In the multinucleate filamentous fungus Ashbya gossypii, nuclei divide asynchronously in a common cytoplasm. We hypothesize that the division cycle machinery has a limited zone of influence in the cytoplasm to promote nuclear autonomy. Mitochondria in cultured mammalian cells undergo cell cycle-specific changes in morphology and membrane potential and therefore can serve as a reporter of the cell cycle state of the cytoplasm. To evaluate if the cell cycle state of nuclei in A. gossypii can influence the adjacent cytoplasm, we tested whether local mitochondrial morphology and membrane potential in A. gossypii are associated with the division state of a nearby nucleus. We found that mitochondria exhibit substantial heterogeneity in both morphology and membrane potential within a single multinucleated cell. Notably, differences in mitochondrial morphology or potential are not associated with a specific nuclear division state. Heterokaryon mutants with a mixture of nuclei with deletions of and wild type for the mitochondrial fusion/fission genes DNM1 and FZO1 exhibit altered mitochondrial morphology and severe growth and sporulation defects. This dominant effect suggests that the gene products may be required locally near their expression site rather than diffusing widely in the cell. Our results demonstrate that mitochondrial dynamics are essential in these large syncytial cells, yet morphology and membrane potential are independent of nuclear cycle state.

  7. Changes of proton transportation across the inner mitochondrial membrane and H+-ATPase in endotoxic shock rats

    Institute of Scientific and Technical Information of China (English)

    LU Song-min 陆松敏; SONG Shuang-ming 宋双明; LIU Jian-cang 刘建仓; YANG He-ming 杨鹤鸣; LI Ping 李萍; WANG Zheng-guo 王正国

    2003-01-01

    Objective: To investigate the changes of proton transportation across the inner mitochondrial membrane (IMM) and H+-ATPase of hepatocytes in endotoxic shock rats.Methods: Endotoxin from E.Coil of 5.0 mg/kg or saline of 1 ml/kg was injected into the femoral vein.The rats were sacrificed pre-injection and 1, 3, 5, 8 hours after injection, and plasma and liver tissue samples were collected respectively.The liver tissue samples were used for preparation of mitochondria and submitochondrial particles (SMPs).The proton-translocation of SMPs and H+-ATPase, phospholipase A2 (PLA2) activities and malondialdehyde (MDA) content, membrane fluidities of different level of mitochondria membrane and plasma MDA content were assayed.Results: (1) Five hours after E.Coli.O111B4 injection, the maximum fluorescence quenching ACMA after adding ATP, nicotinamide adenin dinucleoacid hydrogen (NADH), and the succinate were significantly decreased (P<0.05).The time of maximum fluorescent quenching and the half time of fluorescent quenching were significantly prolonged (P<0.01), especially when NADH was used as a substrate.(2) The mitochondrial H+-ATPase activity was significantly increased at early stage of endotoxic shock (P<0.05), and significantly decreased at late stage of endotoxic shock (P<0.01).(3) The mitochondrial membrane bound PLA2 activity, plasmal and mitochondrial MDA content were significantly increased and succinate dehydrogenase (SDH) activity of mitochondria decreased markedly in endotoxic shock rats (P<0.05).(4) The mitochondrial membrane fluidity of different lipid regions was decreased, especially in the head of phospholipid.Conclusions: Proton transportation across IMM and mitochondrial H+-ATPase activity are significantly decreased in endotoxic shock.

  8. Role of Pterocarpus santalinus against mitochondrial dysfunction and membrane lipid changes induced by ulcerogens in rat gastric mucosa.

    Science.gov (United States)

    Narayan, Shoba; Devi, R S; Devi, C S Shyamala

    2007-11-20

    Free radicals produced by ulcerogenic agents affect the TCA cycle enzymes located in the outer membrane of the mitochondria. Upon induction with ulcerogens, peroxidation of membrane lipids bring about alterations in the mitochondrial enzyme activity. This indicates an increase in the permeability levels of the mitochondrial membrane. The ability of PSE to scavenge the reactive oxygen species results in restoration of activities of TCA cycle enzymes. NSAIDs interfere with the mitochondrial beta-oxidation of fatty acids in vitro and in vivo, resulting in uncoupling of mitochondrial oxidative phosphorylation process. This usually results in diminished cellular ATP production. The recovery of gastric mucosal barrier function through maintenance of energy metabolism results in maintenance of ATP levels, as observed in this study upon treatment with PSE. Membrane integrity altered by peroxidation is known to have a modified fatty acid composition, a disruption of permeability, a decrease in electrical resistance, and increase in flip-flopping between monolayers and inactivated cross-linked proteins. The severe depletion of arachidonic acid in ulcer induced groups was prevented upon treatment with PSE. The acid inhibitory property of the herbal extract enables the maintenance of GL activity upon treatment with PSE. The ability to prevent membrane peroxidation has been traced to the presence of active constituents in the PSE. In essence, PSE has been found to prevent mitochondrial dysfunction, provide mitochondrial cell integrity, through the maintenance of lipid bilayer by its ability to provide a hydrophobic character to the gastric mucosa, further indicating its ability to reverse the action of NSAIDs and mast cell degranulators in gastric mucosa.

  9. Glutaminolysis and glycolysis regulation by troglitazone in breast cancer cells: Relationship to mitochondrial membrane potential.

    Science.gov (United States)

    Friday, Ellen; Oliver, Robert; Welbourne, Tomas; Turturro, Francesco

    2011-02-01

    We studied the roles of glycolysis and glutaminolysis following an acute reduction in mitochondrial membrane potential (Ψ(m)) induced by the thiazolidinedione troglitazone (TRO) and compared the responses with CCCP-induced depolarization in breast cancer derived MCF-7 and MDA-MB-231 cells as well as in the MCF-10A normal breast cell line. TRO and CCCP both acutely reduced Ψ(m) but after 24 h TRO-treated cells had restored Ψ(m) associated with both increased glycolysis and glutaminolysis. In contrast, CCCP-treated cells exhibited only a partial restoration of Ψ(m) associated with increased glycolysis but decreased glutaminolysis. TRO-induced glutaminolysis was coupled to increased ammonium (GDH flux) and decreased alanine production (ALT flux) in all three cell lines. Both cancer cell lines exhibited a higher spontaneous GDH/ALT flux than the normal breast cell line associated with a reduced keto-acid pool. TRO's effect on GDH/ALT fluxes and mitochondrial keto-acid pool homeostasis was additive with glucose withdrawal suggesting limited intramitochondrial pyruvate availability. The TRO-induced acceleration in GDH flux supplies substrate for Complex I contributing to the restoration of Ψ(m) as well as Krebs cycle intermediates for biosynthesis. Inhibiting mitochondrial proton ATPase with oligomycin or nullifying the proton gradient with CCCP prevented both the TRO-induced recovery of Ψ(m) and accelerated GDH flux but restored ALT flux consonant with important roles for proton pumping in regulating GDH flux and Ψ(m) recovery. Blocking enhanced GDH flux reduced DNA synthesis consistent with glutaminolysis via GDH playing an important biosynthetic role in tumorigenesis. © 2010 Wiley-Liss, Inc.

  10. MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells.

    Science.gov (United States)

    Vowinckel, Jakob; Hartl, Johannes; Butler, Richard; Ralser, Markus

    2015-09-01

    Mitochondria assemble into flexible networks. Here we present a simple method for the simultaneous quantification of mitochondrial membrane potential and network morphology that is based on computational co-localisation analysis of differentially imported fluorescent marker proteins. Established in, but not restricted to, Saccharomyces cerevisiae, MitoLoc reproducibly measures changes in membrane potential induced by the uncoupling agent CCCP, by oxidative stress, in respiratory deficient cells, and in ∆fzo1, ∆ref2, and ∆dnm1 mutants that possess fission and fusion defects. In combination with super-resolution images, MitoLoc uses 3D reconstruction to calculate six geometrical classifiers which differentiate network morphologies in ∆fzo1, ∆ref2, and ∆dnm1 mutants, under oxidative stress and in cells lacking mtDNA, even when the network is fragmented to a similar extent. We find that mitochondrial fission and a decline in membrane potential do regularly, but not necessarily, co-occur. MitoLoc hence simplifies the measurement of mitochondrial membrane potential in parallel to detect morphological changes in mitochondrial networks. Marker plasmid open-source software as well as the mathematical procedures are made openly available.

  11. Naja naja oxiana Cobra Venom Cytotoxins CTI and CTII Disrupt Mitochondrial Membrane Integrity: Implications for Basic Three-Fingered Cytotoxins.

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    Gasanov, Sardar E; Shrivastava, Indira H; Israilov, Firuz S; Kim, Aleksandr A; Rylova, Kamila A; Zhang, Boris; Dagda, Ruben K

    2015-01-01

    Cobra venom cytotoxins are basic three-fingered, amphipathic, non-enzymatic proteins that constitute a major fraction of cobra venom. While cytotoxins cause mitochondrial dysfunction in different cell types, the mechanisms by which cytotoxins bind to mitochondria remain unknown. We analyzed the abilities of CTI and CTII, S-type and P-type cytotoxins from Naja naja oxiana respectively, to associate with isolated mitochondrial fractions or with model membranes that simulate the mitochondrial lipid environment by using a myriad of biophysical techniques. Phosphorus-31 nuclear magnetic resonance (31P-NMR) spectroscopy data suggest that both cytotoxins bind to isolated mitochondrial fractions and promote the formation of aberrant non-bilayer structures. We then hypothesized that CTI and CTII bind to cardiolipin (CL) to disrupt mitochondrial membranes. Collectively, 31P-NMR, electron paramagnetic resonance (EPR), proton NMR (1H-NMR), deuterium NMR (2H-NMR) spectroscopy, differential scanning calorimetry, and erythrosine phosphorescence assays suggest that CTI and CTII bind to CL to generate non-bilayer structures and promote the permeabilization, dehydration and fusion of large unilamellar phosphatidylcholine (PC) liposomes enriched with CL. On the other hand, CTII but not CTI caused biophysical alterations of large unilamellar PC liposomes enriched with phosphatidylserine (PS). Mechanistically, single molecule docking simulations identified putative CL, PS and PC binding sites in CTI and CTII. While the predicted binding sites for PS and PC share a high number of interactive amino acid residues in CTI and CTII, the CL biding sites in CTII and CTI are more divergent as it contains additional interactive amino acid residues. Overall, our data suggest that cytotoxins physically associate with mitochondrial membranes by binding to CL to disrupt mitochondrial structural integrity.

  12. The inner mitochondrial membrane has aquaporin-8 water channels and is highly permeable to water.

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    Calamita, Giuseppe; Ferri, Domenico; Gena, Patrizia; Liquori, Giuseppa E; Cavalier, Annie; Thomas, Daniel; Svelto, Maria

    2005-04-29

    Mitochondria are remarkably plastic organelles constantly changing their shape to fulfil their various functional activities. Although the osmotic movement of water into and out of the mitochondrion is central for its morphology and activity, the molecular mechanisms and the pathways for water transport across the inner mitochondrial membrane (IMM), the main barrier for molecules moving into and out of the organelle, are completely unknown. Here, we show the presence of a member of the aquaporin family of water channels, AQP8, and demonstrate the strikingly high water permeability (Pf) characterizing the rat liver IMM. Immunoblotting, electron microscopy, and biophysical studies show that the largest mitochondria feature the highest AQP8 expression and IMM Pf. AQP8 was also found in the mitochondria of other organs, whereas no other known aquaporins were seen. The osmotic water transport of liver IMM was partially inhibited by the aquaporin blocker Hg2+, while the related activation energy remained low, suggesting the presence of a Hg2+-insensitive facilitated pathway in addition to AQP8. It is suggested that AQP8-mediated water transport may be particularly important for rapid expansions of mitochondrial volume such as those occurring during active oxidative phosphorylation and those following apoptotic signals.

  13. Antioxidant activity of capsaicin on radiation-induced oxidation of murine hepatic mitochondrial membrane preparation

    Directory of Open Access Journals (Sweden)

    Gangabhagirathi R

    2015-06-01

    Full Text Available Ramachandran Gangabhagirathi,1 Ravi Joshi,2 1Bioorganic Division, 2Radiation and Photochemistry Division, Bhabha Atomic Research Center, Trombay, Mumbai, India Abstract: Capsaicin is the major capsaicinoid in chili peppers and is widely used as a spice. It is also used for topical applications in cases of peripheral neuropathy. The present study deals with its role in modulation of gamma radiation-induced damages of the biochemical constituents of rat liver mitochondrial membrane (RLM preparation. The extent of lipid hydroperoxide formation, depletion in protein thiols, and formation of protein carbonyls have been biochemically assessed in the presence of varying concentrations of capsaicin in RLM. Decrease in the activities of the important antioxidant enzyme superoxide dismutase, which is involved in the scavenging of free radicals, and the mitochondrial marker enzyme succinate dehydrogenase have been also looked into. Capsaicin has been found to efficiently inhibit radiation-induced biochemical alterations, namely lipid peroxidation and protein oxidation. It also significantly prevented radiation-induced loss in the activity of antioxidant enzyme and the important endogenous antioxidant glutathione. The study suggests that capsaicin can act as an antioxidant and radioprotector in physiological systems. Keywords: capsaicin, gamma radiation, radioprotection, lipid peroxidation, protein oxidation, enzyme activity

  14. Effect of oral coadministration of artesunate with ferrous sulfate on rat liver mitochondrial membrane permeability transition.

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    Fafowora, Mosebolatan V; Atanu, Francis; Sanya, Olayinka; Olorunsogo, Olufunso O; Erukainure, Ochuko L

    2011-07-01

    The recent resurgence of interest in the study of mitochondria has been fuelled in large part by the recognition that genetic and/or metabolic alterations in this organelle are causative or contributing factors in a variety of human diseases including cancer. This study hypothesizes that co-administration of artesunate and ferrous sulfate could induce apoptosis which can be targeted on cancerous cells in such a manner, thus providing a novel, viable and perhaps inexpensive way of dealing with the cancer scourge. Artesunate and Ferrous sulfate were co-administered to rats at various doses for seven days. At the end of the treatment, the rats were fasted overnight and sacrificed by cervical dislocation. Low ionic strength mitochondria were isolated from hepatic cells of the rats and assayed for protein content; changes in the absorbance of the liver mitochondria; and mitochondrial swelling. Co-administration of artesunate and ferrous sulfate resulted in a significant increase (Pferrous iron-treated groups were compared with the artesunate only treated group. Results from this study show that co-administration of artesunate and ferrous sulfate can cause an opening in the mitochondrial membrane transition pore. A combined dose of ferrous sulfate and artesunate may prove to be a more potent therapy for targeting cancerous cells.

  15. Peripheral-type benzodiazepine receptor: a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands

    Energy Technology Data Exchange (ETDEWEB)

    Snyder, S.H.; Verma, A.; Trifiletti, R.R.

    1987-10-01

    The peripheral-type benzodiazepine receptor is a site identified by its nanomolar affinity for (/sup 3/H)diazepam, similar to the affinity of diazepam for the central-type benzodiazepine receptor in the brain. The peripheral type benzodiazepine receptor occurs in many peripheral tissues but has discrete localizations as indicated by autoradiographic studies showing uniquely high densities of the receptors in the adrenal cortex and in Leydig cells of the testes. Subcellular localization studies reveal a selective association of the receptors with the outer membrane of mitochondria. Photoaffinity labeling of the mitochondrial receptor with (/sup 3/H)flunitrazepam reveals two discrete labeled protein bands of 30 and 35 kDa, respectively. The 35-kDa band appears to be identical with the voltage-dependent anion channel protein porin. Fractionation of numerous peripheral tissues reveals a single principal endogenous ligand for the receptor, consisting of porphyrins, which display nanomolar affinity. Interactions of porphyrins with the mitochondrial receptor may clarify its physiological role and account for many pharmacological actions of benzodiazepines.

  16. Inorganic nanoparticles kill Toxoplasma gondii via changes in redox status and mitochondrial membrane potential

    Science.gov (United States)

    Adeyemi, Oluyomi Stephen; Murata, Yuho; Sugi, Tatsuki; Kato, Kentaro

    2017-01-01

    This study evaluated the anti-Toxoplasma gondii potential of gold, silver, and platinum nanoparticles (NPs). Inorganic NPs (0.01–1,000 µg/mL) were screened for antiparasitic activity. The NPs caused >90% inhibition of T. gondii growth with EC50 values of ≤7, ≤1, and ≤100 µg/mL for gold, silver, and platinum NPs, respectively. The NPs showed no host cell cytotoxicity at the effective anti-T. gondii concentrations; the estimated selectivity index revealed a ≥20-fold activity toward the parasite versus the host cell. The anti-T. gondii activity of the NPs, which may be linked to redox signaling, affected the parasite mitochondrial membrane potential and parasite invasion, replication, recovery, and infectivity potential. Our results demonstrated the antiparasitic potential of NPs. The findings support the further exploration of NPs as a possible source of alternative and effective anti-T. gondii agents.

  17. Biogenesis of cytochrome oxidase-sophisticated assembly lines in the mitochondrial inner membrane.

    Science.gov (United States)

    Herrmann, Johannes M; Funes, Soledad

    2005-07-18

    Biogenesis of the cytochrome oxidase complex in the mitochondrial inner membrane depends on the concerted action of a variety of proteins. Recent studies shed light on this biological assembly process revealing an astonishingly complex procedure by which the different subunits of the enzymes are put together and the required cofactors are supplied. In this review we present a hypothetical model for the assembly process of cytochrome oxidase based on the current knowledge of the functions of specific assembly factors. According to this model the two largest subunits of the complex are first equipped with their respective cofactors on independent assembly lines. Prior to their assembly with the residual subunits that complete the whole complex, these two subcomplexes remain bound to substrate-specific chaperones. We propose that these chaperones, Mss51 for subunit 1 and Cox20 for subunit 2, control the coordinate assembly process to prevent potentially harmful redox reactions of unassembled or misassembled subunits.

  18. Modification of the biosynthesis and composition of polyglycerophosphatides in outer and inner mitochondrial membranes by cytidine liponucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Stuhne-Sekalec, L.; Stanacev, N.Z. (Univ. of Toronto (Canada))

    1989-01-01

    The biosynthesis of (3H)polyglycerophosphatides ((3H)phosphatidylglycerophosphate and (3H)phosphatidylglycerol) in mitochondrial and submitochondrial (outer and inner) membranes isolated from guinea pig liver was examined. Experimental results have established that the amount of biosynthesized (3H)polyglycerophosphatides and the relative amounts of biosynthesized (3H)phosphatidylglycerol and (3H)phosphatidylglycerolphosphate can be influenced by varying the composition of fatty acids in CDP-diglycerides and by altering the incubation time of the mixture containing CDP-diglycerides (obligatory precursor), sn-(2-3H)glycerol-3-phosphate and mitochondria or submitochondrial membranes. The changes thus obtained in respect to the amount and composition of biosynthesized (3H)polyglycerophosphatides are different in mitochondria and submitochondrial membranes. The highest amount of biosynthesized (3H)polyglycerophosphatides was obtained with CDP-didecanoin and inner mitochondrial membranes. The greatest accumulation of (3H)phosphatidylglycerol with CDP-didecanoin was obtained in mitochondria and outer mitochondrial membranes, while in inner mitochondrial membranes the amounts of (3H)phosphatidylglycerol and (3H)phosphatidylglycerolphosphate accumulated were approximately the same. In general, prolongation of the incubation time decreased the relative amounts of (3H)phosphatidylglycerolphosphate and increased the amount of accumulated (3H)phosphatidylglycerol, but the absolute amounts of these (3H)polyglycerophosphatides were more dependent on fatty acid composition of CDP-diglycerides tested. The following cytidine liponucleotides were tested: CDP-didecanoin, CDP-dipalmitin, CDP-diolein, and CDP-diglycerides containing saturated and unsaturated fatty acids similar to those in egg yolk lecithin.

  19. Redox-active nanoceria depolarize mitochondrial membrane of human colon cancer cells

    Science.gov (United States)

    Jana, Saikat Kumar; Banerjee, Priyanka; Das, Soumen; Seal, Sudipta; Chaudhury, Koel

    2014-06-01

    Nanotherapeutics is emerging as a promising option to the various limitations and side effects associated with conventional chemotherapy. The present study investigates the cytotoxic effect of redox-active cerium oxide nanoparticles (nanoceria) on human colorectal adenocarcinoma-derived cell line (HCT 15). Exposure of these cells to nanoceria for 24 h with concentration ranging between 10 and 100 μM resulted in a significant reduction of cell viability in a dose-dependent manner. Further, at a concentration of 10 µM, nanoceria exhibited time-dependent cytotoxic effect when exposed to the cells for 24, 48, and 72 h. Upon treatment of the cells with nanoceria, reactive oxygen species (ROS) and lipid peroxidation which are indicators of oxidative stress and cytotoxicity increased significantly, in a dose-dependent manner. Nanoceria was also found to depolarize the mitochondrial membrane, thereby collapsing the membrane potential and leading to initiation of apoptosis. Scanning electron microscopic study of nanoceria-treated HCT 15 cells showed morphological changes and loss of filopodia and lamellipodia, indicating arrest of metastatic spread. Summarizing, when cultured HCT 15 cells are exposed to nanoceria, a dose-dependent cytotoxic effect mediated by ROS generation is observed.

  20. Interaction of carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) with lipid membrane systems: a biophysical approach with relevance to mitochondrial uncoupling.

    Science.gov (United States)

    Monteiro, João P; Martins, André F; Lúcio, Marlene; Reis, Salette; Geraldes, Carlos F G C; Oliveira, Paulo J; Jurado, Amália S

    2011-06-01

    FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone), a classical uncoupler of mitochondrial oxidative phosphorylation, is used in this study as a model to clarify how interactions of uncouplers with membrane lipid bilayers may influence membrane biophysics and their protonophoric activity itself. In order to disclose putative effects that may be important when considering using uncouplers for pharmacological purposes, an extensive characterization of FCCP membrane lipid interactions using accurate biophysical approaches and simple model lipid systems was carried out. Differential scanning calorimetry studies showed that FCCP molecules disturb lipid bilayers and favor lateral phase separation in mixed lipid systems. (31)P NMR assays indicated that FCCP alters the curvature elastic properties of membrane models containing non-bilayer lipids, favoring lamellar/H(II) transition, probably by alleviation of hydrocarbon-packing constraints in the inverted hexagonal phase. Taking advantage of FCCP quenching effects on the fluorescent probes DPH (1,6-diphenyl-1,3,5-hexatriene) and DPH-PA (3-(p-(6-phenyl)-1,3,5-hexatrienyl)phenylpropionic acid), it is demonstrated that FCCP distributes across the bilayer thickness in both a single and a ternary lipid system mimicking the inner mitochondrial membrane. This behavior is consistent with the ability of the compound to migrate through the thickness of the inner mitochondrial membrane, an event required for its protonophoric activity. Finally, the study of the membrane fluidity in different lipid systems, as reported by the rotational correlation time (θ) of DPH or DPH-PA, showed that the extension at which FCCP disturbs membrane properties associated with the dynamics and the order of lipid molecules depends on the lipid composition of the model lipid system assayed.

  1. BH3-only proteins are tail-anchored in the outer mitochondrial membrane and can initiate the activation of Bax.

    Science.gov (United States)

    Wilfling, F; Weber, A; Potthoff, S; Vögtle, F-N; Meisinger, C; Paschen, S A; Häcker, G

    2012-08-01

    During mitochondrial apoptosis, pro-apoptotic BH3-only proteins cause the translocation of cytosolic Bcl-2-associated X protein (Bax) to the outer mitochondrial membrane (OMM) where it is activated to release cytochrome c from the mitochondrial intermembrane space, but the mechanism is under dispute. We show that most BH3-only proteins are mitochondrial proteins that are imported into the OMM via a C-terminal tail-anchor domain in isolated yeast mitochondria, independently of binding to anti-apoptotic Bcl-2 proteins. This C-terminal domain acted as a classical mitochondrial targeting signal and was sufficient to direct green fluorescent protein to mitochondria in human cells. When expressed in mouse fibroblasts, these BH3-only proteins localised to mitochondria and were inserted in the OMM. The BH3-only proteins Bcl-2-interacting mediator of cell death (Bim), tBid and p53-upregulated modulator of apoptosis sensitised isolated mitochondria from Bax/Bcl-2 homologous antagonist/killer-deficient fibroblasts to cytochrome c-release by recombinant, extramitochondrial Bax. For Bim, this activity is shown to require the C-terminal-targeting signal and to be independent of binding capacity to and presence of anti-apoptotic Bcl-2 proteins. Bim further enhanced Bax-dependent killing in yeast. A model is proposed where OMM-tail-anchored BH3-only proteins permit passive 'recruitment' and catalysis-like activation of extra-mitochondrial Bax. The recognition of C-terminal membrane-insertion of BH3-only proteins will permit the development of a more detailed concept of the initiation of mitochondrial apoptosis.

  2. Mitochondrial Ca(2+) uniporter (MCU)-dependent and MCU-independent Ca(2+) channels coexist in the inner mitochondrial membrane.

    Science.gov (United States)

    Bondarenko, Alexander I; Jean-Quartier, Claire; Parichatikanond, Warisara; Alam, Muhammad Rizwan; Waldeck-Weiermair, Markus; Malli, Roland; Graier, Wolfgang F

    2014-07-01

    A protein referred to as CCDC109A and then renamed to mitochondrial calcium uniporter (MCU) has recently been shown to accomplish mitochondrial Ca(2+) uptake in different cell types. In this study, we investigated whole-mitoplast inward cation currents and single Ca(2+) channel activities in mitoplasts prepared from stable MCU knockdown HeLa cells using the patch-clamp technique. In whole-mitoplast configuration, diminution of MCU considerably reduced inward Ca(2+) and Na(+) currents. This was accompanied by a decrease in occurrence of single channel activity of the intermediate conductance mitochondrial Ca(2+) current (i-MCC). However, ablation of MCU yielded a compensatory 2.3-fold elevation in the occurrence of the extra large conductance mitochondrial Ca(2+) current (xl-MCC), while the occurrence of bursting currents (b-MCC) remained unaltered. These data reveal i-MCC as MCU-dependent current while xl-MCC and b-MCC seem to be rather MCU-independent, thus, pointing to the engagement of at least two molecularly distinct mitochondrial Ca(2+) channels.

  3. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    Science.gov (United States)

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  4. Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

    Science.gov (United States)

    Dispersyn, G; Nuydens, R; Connors, R; Borgers, M; Geerts, H

    1999-08-05

    This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).

  5. Bax Activation Initiates the Assembly of a Multimeric Catalyst that Facilitates Bax Pore Formation in Mitochondrial Outer Membranes

    Science.gov (United States)

    Kushnareva, Yulia; Andreyev, Alexander Y.; Kuwana, Tomomi; Newmeyer, Donald D.

    2012-01-01

    Bax/Bak-mediated mitochondrial outer membrane permeabilization (MOMP) is essential for “intrinsic” apoptotic cell death. Published studies used synthetic liposomes to reveal an intrinsic pore-forming activity of Bax, but it is unclear how other mitochondrial outer membrane (MOM) proteins might facilitate this function. We carefully analyzed the kinetics of Bax-mediated pore formation in isolated MOMs, with some unexpected results. Native MOMs were more sensitive than liposomes to added Bax, and MOMs displayed a lag phase not observed with liposomes. Heat-labile MOM proteins were required for this enhanced response. A two-tiered mathematical model closely fit the kinetic data: first, Bax activation promotes the assembly of a multimeric complex, which then catalyzes the second reaction, Bax-dependent pore formation. Bax insertion occurred immediately upon Bax addition, prior to the end of the lag phase. Permeabilization kinetics were affected in a reciprocal manner by [cBid] and [Bax], confirming the “hit-and-run” hypothesis of cBid-induced direct Bax activation. Surprisingly, MOMP rate constants were linearly related to [Bax], implying that Bax acts non-cooperatively. Thus, the oligomeric catalyst is distinct from Bax. Moreover, contrary to common assumption, pore formation kinetics depend on Bax monomers, not oligomers. Catalyst formation exhibited a sharp transition in activation energy at ∼28°C, suggesting a role for membrane lipid packing. Furthermore, catalyst formation was strongly inhibited by chemical antagonists of the yeast mitochondrial fission protein, Dnm1. However, the mammalian ortholog, Drp1, was undetectable in mitochondrial outer membranes. Moreover, ATP and GTP were dispensable for MOMP. Thus, the data argue that oligomerization of a catalyst protein, distinct from Bax and Drp1, facilitates MOMP, possibly through a membrane-remodeling event. PMID:23049480

  6. Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection.

    Science.gov (United States)

    Mukherjee, Madhuchhanda; Basu Ball, Writoban; Das, Pijush K

    2014-10-01

    Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3β to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent transcriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.

  7. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; Sorensen, Martin M; Hvitby, Christina Poulsen;

    2010-01-01

    Cockayne syndrome (CS) is a human premature aging disorder associated with severe developmental deficiencies and neurodegeneration, and phenotypically it resembles some mitochondrial DNA (mtDNA) diseases. Most patients belong to complementation group B, and the CS group B (CSB) protein plays a role...

  8. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N. [Hospital Edouard Herriot, Lyon (France)] [and others

    1995-11-01

    Programmed cell death or apoptosis is characterized by typical morphological alterations. By transmission electron microscopy, apoptotic cells are identified by condensation of the chromatin in tight apposition to the nuclear envelope, alteration of the nuclear envelope and fragmentation of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display and early desintegration of cytoplasmic membrane and swelling of mitochondria. In this study we assessed by flow cytometry the sequential alterations of forward angle light scatter, 90{degrees} light scatter, and fluorescence associated with fluorescein diacetate, rhodamine 123, and propidium iodide in two human B cell lines undergoing apoptosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by the topoisomerase II inhibitor VP-16. The kinetics of these modifications were compared to those of cells undergoing necrosis induced by sodium azide. At the same time intervals, cells were examined by transmission electron microscopy and by UV microscopy after staining with Hoechst 33342. We report that sequential changes in light scatters and fluorescein diacetate are similar in cells undergoing apoptosis or necrosis, whereas apoptosis is characterized by a slightly delayed decrease of mitochondrial activity as assessed by rhodamine 123 staining. Surprisingly, a part of cells undergoing apoptosis displayed an early uptake of propidium iodide followed by a condensation and then a fragmentation of their nuclei. It is concluded that uptake of propidium iodide is a very early marker of cell death which does not discriminate between necrosis and apoptosis. Along with biochemical criteria, nuclear morphology revealed by staining with Hoechst 33342 would seem to be of the most simple and most discriminative assay of apoptosis. 33 refs., 5 figs., 1 tab.

  9. Mitochondria-associated endoplasmic reticulum membranes allow adaptation of mitochondrial metabolism to glucose availability in the liver.

    Science.gov (United States)

    Theurey, Pierre; Tubbs, Emily; Vial, Guillaume; Jacquemetton, Julien; Bendridi, Nadia; Chauvin, Marie-Agnès; Alam, Muhammad Rizwan; Le Romancer, Muriel; Vidal, Hubert; Rieusset, Jennifer

    2016-04-01

    Mitochondria-associated endoplasmic reticulum membranes (MAM) play a key role in mitochondrial dynamics and function and in hepatic insulin action. Whereas mitochondria are important regulators of energy metabolism, the nutritional regulation of MAM in the liver and its role in the adaptation of mitochondria physiology to nutrient availability are unknown. In this study, we found that the fasted to postprandial transition reduced the number of endoplasmic reticulum-mitochondria contact points in mouse liver. Screening of potential hormonal/metabolic signals revealed glucose as the main nutritional regulator of hepatic MAM integrity both in vitro and in vivo Glucose reduced organelle interactions through the pentose phosphate-protein phosphatase 2A (PP-PP2A) pathway, induced mitochondria fission, and impaired respiration. Blocking MAM reduction counteracted glucose-induced mitochondrial alterations. Furthermore, disruption of MAM integrity mimicked effects of glucose on mitochondria dynamics and function. This glucose-sensing system is deficient in the liver of insulin-resistant ob/ob and cyclophilin D-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission, and altered mitochondrial respiration. These data indicate that MAM contribute to the hepatic glucose-sensing system, allowing regulation of mitochondria dynamics and function during nutritional transition. Chronic disruption of MAM may participate in hepatic mitochondrial dysfunction associated with insulin resistance. © The Author (2016). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

  10. Translocation of connexin 43 to the inner mitochondrial membrane of cardiomyocytes through the heat shock protein 90-dependent TOM pathway and its importance for cardioprotection.

    Science.gov (United States)

    Rodriguez-Sinovas, Antonio; Boengler, Kerstin; Cabestrero, Alberto; Gres, Petra; Morente, Miriam; Ruiz-Meana, Marisol; Konietzka, Ina; Miró, Elisabet; Totzeck, Andreas; Heusch, Gerd; Schulz, Rainer; Garcia-Dorado, David

    2006-07-07

    We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.

  11. Effect of Qingkailing injection on rat embryonic neuronal apoptosis and mitochondrial membrane potential

    Institute of Scientific and Technical Information of China (English)

    He Pang; Lingqun Zhu; Shuoren Wang; Fuing Niu; Wei Cui

    2006-01-01

    BACKGROUND:The decrease of mitochondrial membrane potential(MMP)is an irreversible marker of neuronal apoptosis during ischemla/reperfusion(I/R)injury of brain tissue.Qingkaiing injection is proved to have protective effect on neuronal ischemic injury.Whether inhibiting the decrease of MMP can inhibit apoptosis when I/R injury of brain tissue occurs is unclear.OBJECTIVE:To observe the effect of Qingkaiing injection on rat embryonic hippocampal neuronal apoptosis,MMP and mitochondroal activity after hypoxia/hypoglycamia and reoxygenation,and make a comparison of therapeutic effect on I/R injury between Oingkaiing injection and nimodipine.DESIGN:Observation and controlled trial.SETTING:Peropheral Vascular Center,Dongzhimen Hospital, Beijing University of Chinese Medicine;the Key Laboratory of Chinese Internal Medicine of Ministry of Education and Beijing Key Laboratory.Dongzhimen Hospital,Beijing University of Chinese Medicine.MATERIALS:Eight Wistar rats at embryonic 18 days,provided by Breeding Farm of Experimental Animals,Chinese Academy of Medical Sciences(Permission No.SCXK-11-00-0006) were employed in this trial.Qingkaiing injection (Pharmaceutical Factory of Beijing University of Chinese Medicine,Batch No.213710A,10 Ml each,baicalin 50 g and total nitrogen 25 mg included)and nimodipine(ICN company,USA)were also used.METHODS:This experiment was carried out in the Key Laboratory of Chinese Internal Medicine of Ministry of Education,Dongzhimen Hospital,Beijing University of Chinese Medicine and Beijing Key Laboratory from January 2003 to December 2005.①The pregnant rats were anesthetized and fetal rats were isolated for culturong fetal rat hippocampal neurons.The neurons cultured for 10 days were used for expedment.The neurons were divided into 5 groups:model group,control group,nimodipine group.Qingkailing high-dose group and Oingkailing low-dose group.Hypoxia/hypoglycemia and reoxygenation models served as model group,and they were used to simulate reperfusion

  12. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

    Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

  13. Relationship between ATPase activity and conjugated polyamines in mitochondrial membrane from wheat seedling roots under osmotic stress

    Institute of Scientific and Technical Information of China (English)

    LIU Huai-pan; LIU Jun; ZHANG Yan-yan; LIU You-liang

    2004-01-01

    The effects of osmotic stress on the ATPase activity, the contents of -SH group and conjugated polyamines in mitochondrial membrane from wheat seedling [Triticum aestivum L. cv. Yumai No.18(drought-tolerant) and cv. Yumai No.9(drought-sensitive)] roots were investigated. The results showed that ATPase activity and -SH group content decreased with polyethylene glycol(PEG) 6000(-0.55 MPa) treatment for 7 d, in concert with the decrease of the ratio of noncovalently conjugated spermidine(NCC-Spd)/noncovalently conjugated putrescine(NCC-Put) and increase of the covalently conjugated putrescine(CC-Put). Osmotic stress injury to Yangmai No.9 seedlings was alleviated greatly with 1 mmol/L exogenous spermidined(Spd), in concert with marked increases of the ratio of NCC-Spd/NCC-Put, -SH group contents and ATPase activity in mitochondrial membrane. Under osmotic stress, the concomitant treatment of Yumai No.18 seedlings with methylglyoxyl bis(guanylhydrazone) (MGBG), an inhibitor of S-adenosyl methionine decarboxylase(SAMDC), and phenanthrolin (o-Phen), an inhibitor of transglutaminase(TGase), caused a significant decrease of the ratio of NCC-Spd / NCC-Put, CC-Put contents, respectively, in concert with the marked decreases of ATPase activity, -SH group content and its tolerance to osmotic stress. All the results above suggested that osmotic stress tolerance of wheat seedlings was associated with the ATPase activity, the contents of -SH group, NCC-Spd and CC-Put in mitochondrial membrane.

  14. Differential effects of insecticides on mitochondrial membrane lfuidity and ATPase activity between the wolf spider and the rice stem borer

    Institute of Scientific and Technical Information of China (English)

    LI Hai-ping; CHANG Jing; FENG Tao; GAO Xi-wu

    2015-01-01

    Differential effects of methamidophos and three pyrethroids on ATPase activity and membrane lfuidity of mitochondria were investigated between the wolf spider (Pirata subpiraticus(Boes. et Str.)) and the rice stem borer (Chilo suppressalis (Walker)). Based on a comparison of LD50values, the toxicities of the tested insecticides were higher to the wolf spider than to the rice stem borer. Cyhalothrin at 1×10–4 mmol L–1 caused inhibition of the mitochondrial Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities, and it’s inhibitions on Na+-K+-ATPase and Ca2+-Mg2+-ATPase activities were signiifcantly higher in the wolf spider (44 and 28%) than in the rice stem borer (19 and 11%). Methamidophos at 1×10–4 mmol L–1 decreased Ca2+-Mg2+-ATPase activity by 16 and 27% in the wolf spider and the rice stem borer, respectively, but no signiifcant effect on the speciifc activity of Na+-K+-ATPase was observed. The DPH (1,6-diphenyl-1,3,5-hexatriene) lfuorescence polarization values of mitochondrial membranes were not signiifcantly affected by methamidophos in either species. However, cyhalothrin and alpha-cyperme-thrin induced the values of DPH polarization of mitochondrial membrane increasing with the concentration of cyhalothrin and alpha-cypermethrin from 20 to 100 µmol L–1 in the rice stem borer and the wolf spider. Effect of ethofenprox on lfuidity of the wolf spider and the rice stem borer was contrary. These results suggest that both inhibition of membrane ATPase and changes of membrane lfuidity could be appended to the action mechanisms of pyrethroid insecticides.

  15. Extraction of yeast mitochondrial membrane proteins by solubilization and detergent/polymer aqueous two-phase partitioning.

    Science.gov (United States)

    Everberg, Henrik; Gustavsson, Niklas; Tjerneld, Folke

    2009-01-01

    Identification and characterization of membrane proteins is of increasing importance in modern proteomic studies. It is of central interest to have access to methods that combine efficient solubilization with enrichment of proteins and intact protein complexes. Separation methods have been developed based on nondenaturing detergent extraction of yeast mitochondrial membrane proteins followed by enrichment of hydrophobic proteins in aqueous two-phase system. Combining the zwitterionic detergent Zwittergent 3-10 and the nonionic detergent Triton X-114 results in a complementary solubilization of proteins, which is similar to that of the anionic detergent sodium dodecyl sulfate (SDS) but with the important advantage of being nondenaturing. Detergent/polymer two-phase system partitioning offers removal of soluble proteins, which can be further improved by manipulation of the driving forces governing protein distribution between the phases. Integral and peripheral membrane protein subunits from intact membrane protein complexes partition to the detergent phase while soluble proteins are found in the polymer phase. A protocol is presented which combines nondenaturing solubilization of membrane proteins with extraction in detergent/polymer two-phase system for application in proteomic studies as a mild and efficient method for enrichment of membrane proteins and membrane protein complexes.

  16. Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria.

    Science.gov (United States)

    Chowdhury, Subir Roy; Djordjevic, Jelena; Albensi, Benedict C; Fernyhough, Paul

    2015-12-08

    Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30-35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer's disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.

  17. GATA-4 protects against hypoxia-induced cardiomyocyte injury: effects on mitochondrial membrane potential.

    Science.gov (United States)

    Li, Hong-Xia; Zhou, Ya-Feng; Zhao, Xin; Jiang, Bin; Yang, Xiang-Jun

    2014-08-01

    Our previous studies have suggested that GATA-4 increases the differentiation of bone-marrow-derived mesenchymal stem cells (MSCs) into cardiac phenotypes. This study further investigated whether GATA-4 enhances MSC-mediated cardioprotection following hypoxia. MSCs were harvested from rat bone marrow and transduced with GATA-4 (MSC(GATA-4)). To mimic ischemic injury, cultured cardiomyocytes (CMs) isolated from neonatal rat ventricles were exposed to hypoxia or were pretreated with concentrated conditioned medium (CdM) from MSC(GATA-4) or transduced control MSC (MSC(Null)) for 16 h before exposure to hypoxic culture conditions (low glucose and low oxygen). Myocyte damage was estimated by annexin-V-PE and TUNEL technique and by lactate dehydrogenase (LDH) release. Cell survival was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium (MTT) uptake. Mitochondrial membrane potential was determined using confocal microscopy. ELISA studies indicated that insulin-like growth factor 1 (IGF-1) and vascular endothelial growth factor (VEGF) were significantly increased in MSC(GATA-4) compared with MSC(Null). Hypoxia-induced apoptosis/cell death was significantly reduced when CMs were co-cultured with MSC(GATA-4) in a dual-chamber system. Cell protection mediated by MSC(GATA-4) was mimicked by treating CMs with CdM from MSC(GATA-4) and abrogated with IGF-1- and VEGF-neutralizing antibodies. MSC(GATA-4) protects CMs under hypoxic conditions. The release of IGF-1 and VEGF from MSC(GATA-4) is likely to be responsible for protection of CMs.

  18. Novel ligands that target the mitochondrial membrane protein mitoNEET

    Science.gov (United States)

    Bieganski, Robert M.; Yarmush, Martin L.

    2012-01-01

    Ligands of the thiazolidinedione (TZD) class of compounds, pioglitazone (Actos™) and rosiglitazone (Avandia™) are currently approved for treatment of type 2 diabetes and are known to bind to the PPAR-γ nuclear receptor subtype. Recent evidence suggesting PPAR-γ independent action of the TZDs led to the discovery of a novel integral outer mitochondrial membrane protein, mitoNEET. In spite of the several reported X-ray crystal structures of the unbound form of mitoNEET, the location and nature of the mitoNEET ligand binding sites (LBS) remain unknown. In this study, a molecular blind docking (BD) method was used to discover potential mitoNEET LBS and novel ligands, utilizing the program AutoDock Vina (v 1.0.2). Validation of BD was performed on the PPAR-γ receptor (PDB ID: 1ZGY) with the test compound rosiglitazone, demonstrating that the binding conformation of rosiglitazone determined by AutoDock Vina matches well with that of the cocrystallized ligand (root mean square deviation of the heavy atoms 1.45 Å). The locations and a general ligand binding interaction model for the LBS were determined, leading to the discovery of novel mitoNEET ligands. An in vitro fluorescence binding assay utilizing purified recombinant mitoNEET protein was used to determine the binding affinity of a predicted mitoNEET ligand, and the data obtained is in good agreement with AutoDock Vina results. The discovery of potential mitoNEET ligand binding sites and novel ligands, opens up the possibility for detailed structural studies of mitoNEET–ligand complexes, as well as rational design of novel ligands specifically targeted for mitoNEET. PMID:21531159

  19. Multi-membrane-bound structures of Apicomplexa: II. the ovoid mitochondrial cytoplasmic (OMC) complex of Toxoplasma gondii tachyzoites.

    Science.gov (United States)

    Köhler, Sabine

    2006-03-01

    Apicomplexa including the causative agents of toxoplasmosis and malaria reportedly possess one or few tubular-shaped mitochondria that permeate, more or less branched, throughout these unicellular parasites. Electron micrographs generated herein from serial-sectioned Toxoplasma gondii tachyzoites demonstrated, however, a greater diversity regarding both the shape of the cultured parasite's single mitochondrion and its sub-structural organization. Moreover, a unique subcellular construction was detected that basically comprised a pouch-shaped subdivision of the tachyzoite mitochondrion plus a fraction of parasitic cytoplasm enclosed therein. This composite assembling, termed ovoid mitochondrial cytoplasmic (OMC) complex, characteristically displayed a highly reduced matrix lumen of its mitochondrial border construction, which furthermore often failed to possess any cristae or contained tightly pleated cristae, thus creating a pouch-shaped multi-laminar wall of four or more membranous layers, respectively. Given this architecture, cross-sectioned OMC complexes of T. gondii tachyzoites frequently mimicked in size and shape the parasites' plastid-like organelle (apicoplast). Moreover, like the apicoplast, the OMC complex was often found adjacent to the tachyzoite's single Golgi complex and constantly located in close proximity to the outer membrane of the parasite's nuclear envelope. The T. gondii OMC complex differed, however, from the apicoplast in its exact fine structural organization and a stage-restricted presence that was apparently linked to mitochondrial growth and/or division. Any special function(s) possibly performed by the T. gondii OMC complex remains, nevertheless, to be elucidated.

  20. Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model

    DEFF Research Database (Denmark)

    Cheniour, Mouhedine; Brewer, Jonathan R.; Bagatolli, Luis

    2017-01-01

    Background Mitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds...

  1. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-01-01

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI: http://dx.doi.org/10.7554/eLife.00772.001 PMID:23991283

  2. Bax and Bak function as the outer membrane component of the mitochondrial permeability pore in regulating necrotic cell death in mice.

    Science.gov (United States)

    Karch, Jason; Kwong, Jennifer Q; Burr, Adam R; Sargent, Michelle A; Elrod, John W; Peixoto, Pablo M; Martinez-Caballero, Sonia; Osinska, Hanna; Cheng, Emily H-Y; Robbins, Jeffrey; Kinnally, Kathleen W; Molkentin, Jeffery D

    2013-08-27

    A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death. DOI:http://dx.doi.org/10.7554/eLife.00772.001.

  3. Kinetics of plasma membrane and mitochondrial alterations in cells undergoing apoptosis

    National Research Council Canada - National Science Library

    Lizard, G; Fournel, S; Genestier, L; Dhedin, N; Chaput, C; Flacher, M; Mutin, M; Panaye, G; Revillard, J P

    1995-01-01

    ... of the nucleus, whereas integrity of the plasma membrane and organelles is preserved. Conversely cells undergoing necrosis display an early desintegration of cytoplasmic membrane and swelling of mitochondria...

  4. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.

    Science.gov (United States)

    Logan, Angela; Pell, Victoria R; Shaffer, Karl J; Evans, Cameron; Stanley, Nathan J; Robb, Ellen L; Prime, Tracy A; Chouchani, Edward T; Cochemé, Helena M; Fearnley, Ian M; Vidoni, Sara; James, Andrew M; Porteous, Carolyn M; Partridge, Linda; Krieg, Thomas; Smith, Robin A J; Murphy, Michael P

    2016-02-01

    The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo.

  5. Quantitative analysis of mitochondrial morphology and membrane potential in living cells using high-content imaging, machine learning, and morphological binning.

    Science.gov (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

    2015-02-01

    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

  6. Comparative kinetics of damage to the plasma and mitochondrial membranes by intra-cellularly synthesized and externally-provided photosensitizers using multi-color FACS.

    Science.gov (United States)

    Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

    2014-01-01

    Photodynamic therapy (PDT) of cancer involves inflicting lethal damage to the cells of malignant tumors, primarily by singlet oxygen that is generated following light-absorption in a photosensitizer molecule. Dysfunction of cells is manifested in many ways, including peroxidation of cellular components, membrane rupture, depolarization of electric potentials, termination of mitochondrial activity, onset of apoptosis and necrosis and eventually cell lysis. These events do not necessarily occur in linear fashion and different types of damage to cell components occur, most probably, in parallel. In this report we measured the relative rates of damage to two cellular membranes: the plasma membrane and the mitochondrial membrane. We employed photosensitizers of diverse hydrophobicities and used different incubation procedures, which lead to their different intra-cellular localizations. We monitored the damage that was inflicted on these membranes, by employing optical probes of membrane integrity, in a multi-color FACS experiment. The potentiometric indicator JC-1 monitored the electric cross-membrane potential of the mitochondria and the fluorometric indicator Draq7 monitored the rupture of the plasma membrane. We show that the electric depolarization of the mitochondrial membrane and the damage to the enveloping plasma membrane proceed with different kinetics that reflect the molecular character and intracellular location of the sensitizer: PpIX that is synthesized in the cells from ALA causes rapid mitochondrial damage and very slow damage to the plasma membrane, while externally added PpIX has an opposite effect. The hydrophilic sensitizer HypS4 can be taken up by the cells by different incubation conditions, and these affect its intracellular location, and as a consequence either the plasma membrane or the mitochondria is damaged first. A similar correlation was found for additional extracellularly-provided photosensitizers HP and PpIX.

  7. Mitochondrial membrane potential is a suitable candidate for assessing pollution toxicity in fish

    Energy Technology Data Exchange (ETDEWEB)

    Padmini, Ekambaram, E-mail: dstpadmini@rediffmail.com; Usha Rani, Munuswamy, E-mail: musharani.2007@rediffmail.com

    2011-09-01

    Fish inhabiting polluted estuaries are highly exposed to severe stress characterized by an oxidant-antioxidant imbalance. The aim of the study was to explore the use of stress parameters such as adenosine triphosphate/adenosine diphosphate (ATP/ADP) ratio, mitochondrial membrane potential ({Delta}{psi}m) and total protein expression patterns as biomarkers against oxidant exposures in hepatocytes of Mugil cephalus living in either a contaminated (Test; Ennore) or uncontaminated (Control; Kovalam) estuary. Earlier, the pollutant stress impact was determined through light and electron microscopy studies. The ATP/ADP ratio was measured using high performance liquid chromatography; {Delta}{psi}m by fluorescent probe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl benzimidazolcarbocyanine iodide (JC-1) dye and total protein expression patterns by protein profiling. The preponderance of stress impact was confirmed through microscopy studies that featured cytological alterations, disturbances in the surface morphology and in the cell organelles at the ultrastructural levels. Hepatocytes of test fish demonstrated a decrease in ATP and an increase in ADP and thereby alteration in ATP/ADP ratio (p < 0.05; 20.75%). A significant disturbance (p < 0.05; 26.57%) in {Delta}{psi}m with a ratio of J-aggregates/JC-1 monomer of 1 was observed for test fish hepatocytes compared to control group with a J-aggregates/JC-1 monomer ratio of 1.5. Quantitative assessment of protein expression levels also revealed enhanced induction of both low and high molecular weight proteins in test fish hepatocytes. The findings highlight the use of these parameters as the highly sensitive biomarkers in response to contaminant exposure compared to the routinely used antioxidant and oxidant stress parameters in biomonitoring programs. Among the measured parameters, the determination of {Delta}{psi}m may be suggested as a novel candidate as a biomarker because of its greater specificity

  8. Correlation between fluidising effects on phospholipid membranes and mitochondrial respiration of propofol and p-nitrosophenol homologues.

    Science.gov (United States)

    Momo, Federico; Fabris, Sabrina; Wisniewska, Anna; Fiore, Cristina; Bindoli, Alberto; Scutari, Guido; Stevanato, Roberto

    2003-03-25

    Nitrosopropofol (2-6-diisopropyl-4-nitrosophenol) has dramatic consequences for respiration, ATP synthesis and the transmembrane potential of isolated rat liver mitochondria at concentrations at which propofol (2-6-diisopropylphenol) does not cause any apparent effects. These results correlate well with the observation that nitrosopropofol is also a stronger perturbing agent of phospholipid membranes. In this paper we verify the possible biological activity of different phenols and nitrosophenols on mitochondrial respiration. We then discuss their interactions with phospholipid liposomes, studied with differential scanning calorimetry, spin labelling techniques and UV-Vis spectrophotometry, in order to obtain information on drug distribution and the modifications they impose on lipid bilayer. The results of the experiments performed on mitochondria and model membranes prove an interesting correlation between the effects of the molecules on both systems.

  9. Inner-membrane proteins PMI/TMEM11 regulate mitochondrial morphogenesis independently of the DRP1/MFN fission/fusion pathways.

    Science.gov (United States)

    Rival, Thomas; Macchi, Marc; Arnauné-Pelloquin, Laetitia; Poidevin, Mickael; Maillet, Frédéric; Richard, Fabrice; Fatmi, Ahmed; Belenguer, Pascale; Royet, Julien

    2011-03-01

    Mitochondria are highly dynamic organelles that can change in number and morphology during cell cycle, development or in response to extracellular stimuli. These morphological dynamics are controlled by a tight balance between two antagonistic pathways that promote fusion and fission. Genetic approaches have identified a cohort of conserved proteins that form the core of mitochondrial remodelling machineries. Mitofusins (MFNs) and OPA1 proteins are dynamin-related GTPases that are required for outer- and inner-mitochondrial membrane fusion respectively whereas dynamin-related protein 1 (DRP1) is the master regulator of mitochondrial fission. We demonstrate here that the Drosophila PMI gene and its human orthologue TMEM11 encode mitochondrial inner-membrane proteins that regulate mitochondrial morphogenesis. PMI-mutant cells contain a highly condensed mitochondrial network, suggesting that PMI has either a pro-fission or an anti-fusion function. Surprisingly, however, epistatic experiments indicate that PMI shapes the mitochondria through a mechanism that is independent of drp1 and mfn. This shows that mitochondrial networks can be shaped in higher eukaryotes by at least two separate pathways: one PMI-dependent and one DRP1/MFN-dependent.

  10. Effect of manganese supplementation on the membrane integrity and the mitochondrial potential of the sperm of grazing Nelore bulls.

    Science.gov (United States)

    Reis, L S L S; Ramos, A A; Camargos, A S; Oba, E

    2014-11-10

    The effect of dietary manganese (Mn(2+)) supplementation on the reproductive performance of Nelore bulls was evaluated by assessment of sperm membrane integrity. Sixty Nelore bulls (Bos taurus indicus) aged 18-20 mo were randomly divided into four groups (n=15) receiving dietary Mn(2+) supplementation at 540, 1300, 3800 and 6300mg/kg (treatments TC, T1300, T3800 and T6300, respectively). The diets were changed for the groups every 70d. Semen samples were obtained 15 and 56d after the diet change, which corresponded to the period of adjustment to the new diet and the time required for a complete spermatogenesis cycle, respectively. Sperm integrity was assessed by detection of: intact (IMe) or damaged (DMe) membranes, intact (IA) or damaged (DA) acrosomes, and high (HM) or low (LM) mitochondrial membrane potentials. Only bulls from the TC treatment showed a significant increase in the production of intact sperm [IMe/IA/LM] and decrease in the production of sperm with damaged acrosome [IMe/DA/LM] or completely damaged sperm [DMe/DA/LM] (Pbulls must be limited to 540mg of Mn(2+)/kg given that higher doses are detrimental to the integrity of the plasma and acrosomal sperm membranes.

  11. Development of a no-wash assay for mitochondrial membrane potential using the styryl dye DASPEI

    DEFF Research Database (Denmark)

    Reveles Jensen, Kristian; Rekling, Jens C

    2010-01-01

    potential assay using 2-(4-(dimethylamino)styryl)-N-ethylpyridinium iodide (DASPEI), a rarely used mitochondrial potentiometric probe, in a 96-well format using a fluorescent plate reader. The assay was validated using 2 protonophores (CCCP, DNP), which are known uncouplers, and the neuroleptic thioridazine......-handling stability, and thus is suitable for large-scale screening efforts. In summary, the DASPEI assay is simple and rapid and may be of use in toxicological testing, drug target discovery, and mechanistic models of diseases involving mitochondrial dysfunction....

  12. A Splice-Isoform of Vesicle-associated Membrane Protein-1 (VAMP-1) Contains a Mitochondrial Targeting Signal

    Science.gov (United States)

    Isenmann, Sandra; Khew-Goodall, Yeesim; Gamble, Jennifer; Vadas, Mathew; Wattenberg, Binks W.

    1998-01-01

    Screening of a library derived from primary human endothelial cells revealed a novel human isoform of vesicle-associated membrane protein-1 (VAMP-1), a protein involved in the targeting and/or fusion of transport vesicles to their target membrane. We have termed this novel isoform VAMP-1B and designated the previously described isoform VAMP-1A. VAMP-1B appears to be an alternatively spliced form of VAMP-1. A similar rat splice variant of VAMP-1 (also termed VAMP-1B) has recently been reported. Five different cultured cell lines, from different lineages, all contained VAMP-1B but little or no detectable VAMP-1A mRNA, as assessed by PCR. In contrast, brain mRNA contained VAMP-1A but no VAMP-1B. The VAMP-1B sequence encodes a protein identical to VAMP-1A except for the carboxy-terminal five amino acids. VAMP-1 is anchored in the vesicle membrane by a carboxy-terminal hydrophobic sequence. In VAMP-1A the hydrophobic anchor is followed by a single threonine, which is the carboxy-terminal amino acid. In VAMP-1B the predicted hydrophobic membrane anchor is shortened by four amino acids, and the hydrophobic sequence is immediately followed by three charged amino acids, arginine-arginine-aspartic acid. Transfection of human endothelial cells with epitope-tagged VAMP-1B demonstrated that VAMP-1B was targeted to mitochondria whereas VAMP-1A was localized to the plasma membrane and endosome-like structures. Analysis of C-terminal mutations of VAMP-1B demonstrated that mitochondrial targeting depends both on the addition of positive charge at the C terminus and a shortened hydrophobic membrane anchor. These data suggest that mitochondria may be integrated, at least at a mechanistic level, to the vesicular trafficking pathways that govern protein movement between other organelles of the cell. PMID:9658161

  13. Palmitoylation of the immunity related GTPase, Irgm1: impact on membrane localization and ability to promote mitochondrial fission.

    Directory of Open Access Journals (Sweden)

    Stanley C Henry

    Full Text Available The Immunity-Related GTPases (IRG are a family of large GTPases that mediate innate immune responses. Irgm1 is particularly critical for immunity to bacteria and protozoa, and for inflammatory homeostasis in the intestine. Although precise functions for Irgm1 have not been identified, prior studies have suggested roles in autophagy/mitophagy, phagosome remodeling, cell motility, and regulating the activity of other IRG proteins. These functions ostensibly hinge on the ability of Irgm1 to localize to intracellular membranes, such as those of the Golgi apparatus and mitochondria. Previously, it has been shown that an amphipathic helix, the αK helix, in the C-terminal portion of the protein partially mediates membrane binding. However, in absence of αK, there is still substantial binding of Irgm1 to cellular membranes, suggesting the presence of other membrane binding motifs. In the current work, an additional membrane localization motif was found in the form of palmitoylation at a cluster of cysteines near the αK. An Irgm1 mutant possessing alanine to cysteine substitutions at these amino acids demonstrated little residual palmitoylation, yet it displayed only a small decrease in localization to the Golgi and mitochondria. In contrast, a mutant containing the palmitoylation mutations in combination with mutations disrupting the amphipathic character of the αK displayed a complete loss of apparent localization to the Golgi and mitochondria, as well as an overall loss of association with cellular membranes in general. Additionally, Irgm1 was found to promote mitochondrial fission, and this function was undermined in Irgm1 mutants lacking the palmitoylation domain, and to a greater extent in those lacking the αK, or the αK and palmitoylation domains combined. Our data suggest that palmitoylation together with the αK helix firmly anchor Irgm1 in the Golgi and mitochondria, thus facilitating function of the protein.

  14. FAT/CD36 is located on the outer mitochondrial membrane, upstream of long-chain acyl-CoA synthetase, and regulates palmitate oxidation.

    Science.gov (United States)

    Smith, Brennan K; Jain, Swati S; Rimbaud, Stéphanie; Dam, Aaron; Quadrilatero, Joe; Ventura-Clapier, Renée; Bonen, Arend; Holloway, Graham P

    2011-07-01

    FAT/CD36 (fatty acid translocase/Cluster of Differentiation 36), a plasma membrane fatty-acid transport protein, has been found on mitochondrial membranes; however, it remains unclear where FAT/CD36 resides on this organelle or its functional role within mitochondria. In the present study, we demonstrate, using several different approaches, that in skeletal muscle FAT/CD36 resides on the OMM (outer mitochondrial membrane). To determine the functional role of mitochondrial FAT/CD36 in this tissue, we determined oxygen consumption rates in permeabilized muscle fibres in WT (wild-type) and FAT/CD36-KO (knockout) mice using a variety of substrates. Despite comparable muscle mitochondrial content, as assessed by unaltered mtDNA (mitochondrial DNA), citrate synthase, β-hydroxyacyl-CoA dehydrogenase, cytochrome c oxidase complex IV and respiratory capacities [maximal OXPHOS (oxidative phosphorylation) respiration] in WT and KO mice, palmitate-supported respiration was 34% lower in KO animals. In contrast, palmitoyl-CoA-supported respiration was unchanged. These results indicate that FAT/CD36 is key for palmitate-supported respiration. Therefore we propose a working model of mitochondrial fatty-acid transport, in which FAT/CD36 is positioned on the OMM, upstream of long-chain acyl-CoA synthetase, thereby contributing to the regulation of mitochondrial fatty-acid transport. We further support this model by providing evidence that FAT/CD36 is not located in mitochondrial contact sites, and therefore does not directly interact with carnitine palmitoyltransferase-I as original proposed.

  15. Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells.

    Science.gov (United States)

    Dwivedi, Sourabh; Saquib, Quaiser; Al-Khedhairy, Abdulaziz A; Musarrat, Javed

    2012-12-01

    Butachlor is a systemic herbicide widely applied on rice, tea, wheat, beans and other crops; however, it concurrently exerts toxic effects on beneficial organisms like earthworms, aquatic invertebrates and other non-target animals including humans. Owing to the associated risk to humans, this chloroacetanilide class of herbicide was investigated with the aim to assess its potential for the (i) interaction with DNA, (ii) mitochondria membrane damage and DNA strand breaks and (iii) cell cycle arrest and necrosis in butachlor treated human peripheral blood mononuclear (PBMN) cells. Fluorescence quenching data revealed the binding constant (Ka=1.2×10(4)M(-1)) and binding capacity (n=1.02) of butachlor with ctDNA. The oxidative potential of butachlor was ascertained based on its capacity of inducing reactive oxygen species (ROS) and substantial amounts of promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts in DNA. Also, the discernible butachlor dose-dependent reduction in fluorescence intensity of a cationic dye rhodamine (Rh-123) and increased fluorescence intensity of 2',7'-dichlorodihydro fluorescein diacetate (DCFH-DA) in treated cells signifies decreased mitochondrial membrane potential (ΔΨm) due to intracellular ROS generation. The comet data revealed significantly greater Olive tail moment (OTM) values in butachlor treated PBMN cells vs untreated and DMSO controls. Treatment of cultured PBMN cells for 24h resulted in significantly increased number of binucleated micronucleated (BNMN) cells with a dose dependent reduction in the nuclear division index (NDI). The flow cytometry analysis of annexin V(-)/7-AAD(+) stained cells demonstrated substantial reduction in live population due to complete loss of cell membrane integrity. Overall the data suggested the formation of butachlor-DNA complex, as an initiating event in butachlor-induced DNA damage. The results elucidated the oxidative role of butachlor in intracellular ROS production, and

  16. Molecular Chaperone Hsp70/Hsp90 Prepares the Mitochondrial Outer Membrane Translocon Receptor Tom71 for Preprotein Loading

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jingzhi; Qian, Xinguo; Hu, Junbin; Sha, Bingdong; (UAB)

    2010-11-03

    The preproteins targeted to the mitochondria are transported through the translocase of the outer membrane complex. Tom70/Tom71 is a major surface receptor of the translocase of the outer membrane complex for mitochondrial preproteins. The preproteins are escorted to Tom70/Tom71 by molecular chaperones Hsp70 and Hsp90. Here we present the high resolution crystal structures of Tom71 and the protein complexes between Tom71 and the Hsp70/Hsp90 C terminus. The crystal structures indicate that Tom70/Tom71 may exhibit two distinct states. In the closed state, the N-terminal domain of Tom70/Tom71 partially blocks the preprotein-binding pocket. In the open state, the N-terminal domain moves away, and the preprotein-binding pocket is fully exposed. The complex formation between the C-terminal EEVD motif of Hsp70/Hsp90 and Tom71 could lock Tom71 in the open state where the preprotein-binding pocket of Tom71 is ready to receive preproteins. The interactions between Hsp70/Hsp90 and Tom71 N-terminal domain generate conformational changes that may increase the volume of the preprotein-binding pocket. The complex formation of Hsp70/Hsp90 and Tom71 also generates significant domain rearrangement within Tom71, which may position the preprotein-binding pocket closer to Hsp70/Hsp90 to facilitate the preprotein transfer from the molecular chaperone to Tom71. Therefore, molecular chaperone Hsp70/Hsp90 may function to prepare the mitochondrial outer membrane receptor Tom71 for preprotein loading.

  17. Assessing the efficacy of vesicle fusion with planar membrane arrays using a mitochondrial porin as reporter

    DEFF Research Database (Denmark)

    Pszon-Bartosz, Kamila Justyna; Hansen, Jesper S.; Stibius, Karin B.

    2011-01-01

    reconstitution in biomimetic membrane arrays may be quantified using the developed FomA assay. Specifically, we show that FomA vesicles are inherently fusigenic. Optimal FomA incorporation is obtained with a proteoliposome lipid-to-protein molar ratio (LPR)=50 more than 105 FomA proteins could be incorporated......Reconstitution of functionally active membrane protein into artificially made lipid bilayers is a challenge that must be overcome to create a membrane-based biomimetic sensor and separation device. In this study we address the efficacy of proteoliposome fusion with planar membrane arrays. We...... establish a protein incorporation efficacy assay using the major non-specific porin of Fusobacterium nucleatum (FomA) as reporter. We use electrical conductance measurements and fluorescence microscopy to characterize proteoliposome fusion with an array of planar membranes. We show that protein...

  18. Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

    Science.gov (United States)

    Lydka, M; Piasecka, M; Gaczarzewicz, D; Koziorowski, M; Bilinska, B

    2012-08-01

    Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

  19. Improved glycaemic control decreases inner mitochondrial membrane leak in type 2 diabetes

    DEFF Research Database (Denmark)

    Rabøl, R; Højberg, P M V; Almdal, T;

    2009-01-01

    AIM: Several mechanisms have been targeted as culprits of weight gain during antihyperglycaemic treatment in type 2 diabetes (T2DM). These include reductions in glucosuria, increased food intake from fear of hypoglycaemia, the anabolic effect of insulin, decreased metabolic rate and increased...... efficiency in fuel usage. The purpose of the study was to test the hypothesis that mitochondrial efficiency increases as a result of insulin treatment in patients with type 2 diabetes. METHODS: We included ten patients with T2DM (eight males) on oral antidiabetic treatment, median age: 51.5 years (range: 39......-67) and body mass index (BMI): 30.1 +/- 1.2 kg/m2 (mean +/- s.e.). Muscle biopsies from m. vastus lateralis and m. deltoideus were obtained before and after seven weeks of intensive insulin treatment, and mitochondrial respiration was measured using high-resolution respirometry. State 3 respiration...

  20. Continuous renal replacement therapy (CRRT) attenuates myocardial inflammation and mitochondrial injury induced by venovenous extracorporeal membrane oxygenation (VV ECMO) in a healthy piglet model.

    Science.gov (United States)

    Shen, Juanhong; Yu, Wenkui; Chen, Qiyi; Shi, Jialiang; Hu, Yimin; Zhang, Juanjuan; Gao, Tao; Xi, Fengchan; He, Changsheng; Gong, Jianfeng; Li, Ning; Li, Jieshou

    2013-10-01

    In this study, we investigated the myocardial inflammation and mitochondrial function during venovenous extracorporeal membrane oxygenation (VV ECMO) and further evaluated the effects of continuous renal replacement therapy (CRRT) on them. Eighteen piglets were assigned to the control group, ECMO group, and ECMO+CRRT group. Myocardial inflammation was assessed by the activity of myeloperoxidase (MPO), myocardial concentrations, and mRNA expression of TNF-α, IL-1β, and IL-6; mitochondrial function was assessed by activities of mitochondrial complexes I-V. VV ECMO elicited a general activation of serum and myocardial inflammation and significantly decreased the activities of mitochondrial complexes I and IV. After being combined with CRRT, serum and myocardial concentrations of IL-1β and IL-6, myocardial mRNA expression of IL-6, and the activity of MPO were decreased significantly; the activities of mitochondrial complexes were increased. We conclude that myocardial inflammation was activated during ECMO therapy, inducing mitochondrial injury; moreover, CRRT reduced myocardial inflammation and partially ameliorated mitochondrial function.

  1. Megaconial muscular dystrophy caused by mitochondrial membrane homeostasis defect, new insights from skeletal and heart muscle analyses.

    Science.gov (United States)

    Vanlander, Arnaud V; Muiño Mosquera, Laura; Panzer, Joseph; Deconinck, Tine; Smet, Joél; Seneca, Sara; Van Dorpe, Jo; Ferdinande, Liesbeth; Ceuterick-de Groote, Chantal; De Jonghe, Peter; Van Coster, Rudy; Baets, Jonathan

    2016-03-01

    Megaconial congenital muscular dystrophy is a disease caused by pathogenic mutations in the gene encoding choline kinase beta (CHKB). Microscopically, the disease is hallmarked by the presence of enlarged mitochondria at the periphery of skeletal muscle fibres leaving the centre devoid of mitochondria. Clinical characteristics are delayed motor development, intellectual disability and dilated cardiomyopathy in half of reported cases. This study describes a patient presenting with the cardinal clinical features, in whom a homozygous nonsense mutation (c.248_249insT; p.Arg84Profs*209) was identified in CHKB and who was treated by heart transplantation. Microscopic evaluation of skeletal and heart muscles typically showed enlarged mitochondria. Spectrophotometric evaluation in both tissues revealed a mild decrease of all OXPHOS complexes. Using BN-PAGE analysis followed by activity staining subcomplexes of complex V were detected in both tissues, indicating incomplete complex V assembly. Mitochondrial DNA content was not depleted in analysed tissues. This is the first report describing the microscopic and biochemical abnormalities in the heart from an affected patient. A likely hypothesis is that the biochemical findings are caused by an abnormal lipid profile in the inner mitochondrial membrane resulting from a defective choline kinase B activity.

  2. Chloroquine-induced glioma cells death is associated with mitochondrial membrane potential loss, but not oxidative stress.

    Science.gov (United States)

    Vessoni, Alexandre Teixeira; Quinet, Annabel; de Andrade-Lima, Leonardo Carmo; Martins, Davi Jardim; Garcia, Camila Carrião Machado; Rocha, Clarissa Ribeiro Reily; Vieira, Debora Braga; Menck, Carlos Frederico Martins

    2016-01-01

    Chloroquine (CQ), a quinolone derivative widely used to treat and prevent malaria, has been shown to exert a potent adjuvant effect when combined with conventional glioblastoma therapy. Despite inducing lysosome destabilization and activating p53 in human glioma cells, the mechanisms underlying cell death induced by this drug are poorly understood. Here, we analyzed in a time- and dose-dependent manner, the effects of CQ upon mitochondria integrity, autophagy regulation and redox processes in four human glioma cell lines that differ in their resistance to this drug. NAC-containing media protected cells against CQ-induced loss of mitochondrial membrane potential (MMP), autophagic vacuoles (LC3II) accumulation and loss of cell viability induced by CQ. However, we noticed that part of this protection was due to media acidification in NAC preparations, alerting for problems in experimental procedures using NAC. The results indicate that although CQ induces accumulation of LC3II, mitochondria, and oxidative stress, neither of these events is clearly correlated to cell death induced by this drug. The only event elicited in all cell lines at equitoxic doses of CQ was the loss of MMP, indicating that mitochondrial stability is important for cells resistance to this drug. Finally, the data indicate that higher steady-state MMP values can predict cell resistance to CQ treatment. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Intracellular magnesium content changes during mitochondria-mediated apoptosis: in depth study of early events on mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    Lucia Merolle

    2014-01-01

    Full Text Available A recent study showed the antitumor activity of a new indole-derivative – MM-67 – inducing mitochondria-mediated apoptosis and a decrease of intracellular magnesium (Mg concentration in HT29 colon cancer cells. Aim of this work was to assess cellular Mg levels throughout MM-67-induced apoptosis from the early to the final stage of the process and to evaluate the correlation with mitochondrial membrane potential (ΔΨm variations. All analysis were performed by flow cytometry: ΔΨm was assessed by using mitochondrial potential sensitive dye DiOC6, while free and total intracellular cation concentrations were assessed by using the commercial probe MagFluo4-AM (Kd=4.7 mM, and the new synthesized DCHQ5 (Kd=8.3 mM, respectively. Our results evidenced that the MM67 induced apoptosis is characterized by a direct correlation between ΔΨ and free intracellular Mg content variations.

  4. Involvement of seminal leukocytes, reactive oxygen species, and sperm mitochondrial membrane potential in the DNA damage of the human spermatozoa.

    Science.gov (United States)

    Lobascio, A M; De Felici, M; Anibaldi, M; Greco, P; Minasi, M G; Greco, E

    2015-03-01

    Measurement of reactive oxygen species (ROS) producing leukocytes in semen has been a standard component of the semen analysis, but its true significance remains still unknown. In this study, we have correlated the number of seminal leukocytes to various semen parameters. We found a negative correlation between the leukocyte number and sperm concentration (rs  = -0.22; p = 0.01) and motility (rs  = -0.20; p = 0.02). In contrast, a positive correlation between the number of leukocytes and both seminal ROS (rs  = 0.70, p sperm mitochondrial membrane potential (MMP) (10% vs 35%, rs  = 0.25, p = 0.08; n = 50). Overall these results indicate that the presence of high number of leukocytes in the ejaculate negatively affects key semen parameters, as sperm concentration and motility, associated with infertility conditions. Moreover, they suggest that leukocytes are the major source of the seminal ROS and cause of sperm DNA fragmentation. However, the absence of a clear correlation between ROS and sperm DNA fragmentation, and spermatozoa with damaged DNA and MMP loss, suggest that ROS produced by leukocytes might be not the only cause of DNA damage in spermatozoa and that intrinsic mitochondrial-dependent apoptotic pathways might not have a major impact on sperm DNA fragmentation.

  5. Regulation of brain mitochondrial H2O2 production by membrane potential and NAD(P)H redox state.

    Science.gov (United States)

    Starkov, Anatoly A; Fiskum, Gary

    2003-09-01

    Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH-linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H2O2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (DeltaPsi) and NAD(P)H redox state. DeltaPsi was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H2O2 production was closely related to DeltaPsi and the level of NAD(P)H reduction at high values of DeltaPsi, 30% of the maximal rate of H2O2 formation was still observed in the presence of uncoupler (p-trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of DeltaPsi and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH-dependent substrates is regulated by DeltaPsi and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.

  6. Synthesis, Molecular Structure, DNA/Protein Binding, Cytotoxicity, Apoptosis, Reactive Oxygen Species, and Mitochondrial Membrane Potential of Dibenzoxanthenes Derivatives.

    Science.gov (United States)

    Yang, Hui-Hui; Han, Bing-Jie; Li, Wei; Liu, Yun-Jun; Wang, Xiu-Zhen

    2015-12-01

    Two dibenzoxanthene isomers 3 and 4 were synthesized and characterized. The crystal structures of the two compounds were solved by single-crystal X-ray diffraction. Binding of two compounds with calf thymus DNA (CT DNA) and BSA (bovine serum albumin) has been thoroughly investigated by UV-Vis and fluorescence spectroscopy. The DNA-binding constants were determined to be 2.51 (± 0.09) × 10(3) for compound 3 and 4.55 (± 0.10) × 10(3) for compound 4. Two compounds can cleave pBR322 DNA upon irradiation. Significant nuclear damages of BEL-7402 cells were observed with compound treatment in a comet assay. The cytotoxicity in vitro was investigated by MTT method. These compounds have been found to induce nuclear condensation and fragmentation in BEL-7402 cells. The two compounds can enhance intracellular reactive oxygen species and decrease the mitochondrial membrane potential. The compounds activated caspase-3 and caspase-7, down-regulated the expression levels of anti-apoptotic protein Bcl-2, and up-regulated the expression levels of pro-apoptotic protein Bax. These compounds induce apoptosis of BEL-7402 cells through an ROS-mediated mitochondrial dysfunction pathway.

  7. Mitochondrial membrane potential in human neutrophils is maintained by complex III activity in the absence of supercomplex organisation.

    Directory of Open Access Journals (Sweden)

    Bram J van Raam

    Full Text Available BACKGROUND: Neutrophils depend mainly on glycolysis for their energy provision. Their mitochondria maintain a membrane potential (Deltapsi(m, which is usually generated by the respiratory chain complexes. We investigated the source of Deltapsi(m in neutrophils, as compared to peripheral blood mononuclear leukocytes and HL-60 cells, and whether neutrophils can still utilise this Deltapsi(m for the generation of ATP. METHODS AND PRINCIPAL FINDINGS: Individual activity of the oxidative phosphorylation complexes was significantly reduced in neutrophils, except for complex II and V, but Deltapsi(m was still decreased by inhibition of complex III, confirming the role of the respiratory chain in maintaining Deltapsi(m. Complex V did not maintain Deltapsi(m by consumption of ATP, as has previously been suggested for eosinophils. We show that complex III in neutrophil mitochondria can receive electrons from glycolysis via the glycerol-3-phosphate shuttle. Furthermore, respiratory supercomplexes, which contribute to efficient coupling of the respiratory chain to ATP synthesis, were lacking in neutrophil mitochondria. When HL-60 cells were differentiated to neutrophil-like cells, they lost mitochondrial supercomplex organisation while gaining increased aerobic glycolysis, just like neutrophils. CONCLUSIONS: We show that neutrophils can maintain Deltapsi(m via the glycerol-3-phosphate shuttle, whereby their mitochondria play an important role in the regulation of aerobic glycolysis, rather than producing energy themselves. This peculiar mitochondrial phenotype is acquired during differentiation from myeloid precursors.

  8. Isolation and characterization of a Ca/sup 2 +/ carrier candidate from calf heart inner mitochondrial membrane

    Energy Technology Data Exchange (ETDEWEB)

    Jeng, A.Y.

    1979-01-01

    A protein was isolated from calf heart inner mitochondrial membrane with the aid of an electron paramagnetic resonance assay based on the relative binding properties of Ca/sup 2 +/, Mn/sup 2 +/, and Mg/sup 2 +/ to the protein. Partial delipidation of the protein was performed by using either the organic solvent extraction procedure or the silicic acid column chromatography. Control experiments indicated that the Ca/sup 2 +/ transport properties of the isolated protein were not due to the contaminating phospholipids. A complete delipidation procedure was developd by using Sephadex LH-20 column chromatography. Further characterization of the physical and chemical properties of the delipidated protein showed that delipidated protein becomes more hydrophobic in the presence of Ca/sup 2 +/ and alkaline pH in the organic solvent extraction experiments. Two possible models of calciphorin-mediated Ca/sup 2 +/ transport in mitochondria are proposed. (PCS)

  9. Effects of transmembrane potential and pH gradient on the cytochrome c-promoted fusion of mitochondrial mimetic membranes.

    Science.gov (United States)

    Kawai, Cintia; Pessoto, Felipe S; Graves, Catharine V; Carmona-Ribeiro, Ana Maria; Nantes, Iseli L

    2013-08-01

    The present study investigated the effects of ΔΨ and ΔpH (pH gradient) on the interaction of cytochrome c with a mitochondrial mimetic membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and cardiolipin (CL) leading to vesicle fusion. ΔpH generated by lowered bulk pH (pH(out)) of PCPECL liposomes, with an internal pH (pH(in)) of 8.0, favored vesicle fusion with a titration sigmoidal profile (pK(a) ~ 6.9). Conversely, ΔpH generated by enhanced pH(in) of PCPECL at a pH(out) of 6.0 favored the fusion of vesicles with a linear profile. We did not observe a significant amount of liposome fusion when ΔpH was generated by lowered pH(in) at a pH(out) of 8.0. At bulk acidic pH, ΔΨ generated by Na⁺ gradient also favored cyt c-promoted vesicle fusion. At acidic and alkaline pH(out), the presence of ΔpH and ΔΨ did not affect cytochrome c binding affinity measured by pyrene quenching. Therefore, cytochrome c-mediated PC/PE/CL vesicle fusion is dependent of ionization of the protein site L (acidic pH) and the presence of transmembrane potential. The effect of transmembrane potential is probably related to the generation of defects on the lipid bilayer. These results are consistent with previous reports showing that cytochrome c release prior to the dissipation of the ΔΨ(M) blocks inner mitochondrial membrane fusion during apoptosis.

  10. Interventional effect of phycocyanin on mitochondrial membrane potential and activity of PC12 cells after hypoxia/reoxygenation

    Institute of Scientific and Technical Information of China (English)

    Nan Jiang; Yunliang Guo; Hongbing Chen

    2006-01-01

    BACKGROUND: Phycocyanin can relieve decrease of mitochondrial membrane potential through reducing production of active oxygen so as to protect neurons after hypoxia/reoxygenation.OBJECTIVE: To observe the effect of phycocyanin on activity of PC12 cells and mitochondrial membrane potential after hypoxia/reoxygenation.DESIGN: Randomized controlled study.SETTING: Cerebrovascular Disease Institute of Affiliated Hospital, Medical College of Qingdao University.MATERIALS: The experiment was carried out at the Key Laboratory of Prevention and Cure for cerebropathia in Shandong Province from October to December 2005. PC12 cells, rat chromaffin tumor cells,were provided by Storage Center of Wuhan University; phycocyanin was provided by Ocean Institute of Academia Sinica; Thiazoyl blue tetrazolium bromide (MTT) and rhodamine 123 were purchased from Sigma Company, USA; RPMI-1640 medium, fetal bovine serum and equine serum were purchased from Gibco Company, USA.METHODS: ① Culture of PC12 cells: PC12 cells were put into RPMI-1640 medium which contained 100 g/L heat inactivation equine serum and 0.05 volume fraction of fetal bovine serum and incubated in CO2 incubator at 37 ℃. Number of cells was regulated to 4 × 105 L-1, and cells were inoculated at 96-well culture plate.The final volume was 100 μL. ② Model establishing and grouping: Cultured PC12 cells were randomly divided into three groups: phycocyanin group, model control group and non-hypoxia group. At 24 hours before hypoxia, culture solution in phycocyanin group was added with phycocyanin so as to make sure the final concentration of 3 g/L, but cells in model control group did not add with phycocyanin. Cells in non-hypoxia group were also randomly divided into adding phycocyanin group (the final concentration of 3 g/L) and non-adding phycocyanin group. Cells in model control group and phycocyanin group were cultured with hypoxia for 1 hour and reoxygenation for 1, 2 and 3 hours; meanwhile, cells in non

  11. Fusion of the endoplasmic reticulum and mitochondrial outer membrane in rats brown adipose tissue: activation of thermogenesis by Ca2+.

    Directory of Open Access Journals (Sweden)

    Leopoldo de Meis

    Full Text Available Brown adipose tissue (BAT mitochondria thermogenesis is regulated by uncoupling protein 1 (UCP 1, GDP and fatty acids. In this report, we observed fusion of the endoplasmic reticulum (ER membrane with the mitochondrial outer membrane of rats BAT. Ca(2+-ATPase (SERCA 1 was identified by immunoelectron microscopy in both ER and mitochondria. This finding led us to test the Ca(2+ effect in BAT mitochondria thermogenesis. We found that Ca(2+ increased the rate of respiration and heat production measured with a microcalorimeter both in coupled and uncoupled mitochondria, but had no effect on the rate of ATP synthesis. The Ca(2+ concentration needed for half-maximal activation varied between 0.08 and 0.11 microM. The activation of respiration was less pronounced than that of heat production. Heat production and ATP synthesis were inhibited by rotenone and KCN. Liver mitochondria have no UCP1 and during respiration synthesize a large amount of ATP, produce little heat, GDP had no effect on mitochondria coupling, Ca(2+ strongly inhibited ATP synthesis and had little or no effect on the small amount of heat released. These finding indicate that Ca(2+ activation of thermogenesis may be a specific feature of BAT mitochondria not found in other mitochondria such as liver.

  12. Osmotic stress and cryoinjury of koala sperm: an integrative study of the plasma membrane, chromatin stability and mitochondrial function.

    Science.gov (United States)

    Johnston, S D; Satake, N; Zee, Y; López-Fernández, C; Holt, W V; Gosálvez, J

    2012-06-01

    This study investigated whether cryopreservation-induced injury to koala spermatozoa could be explained using an experimental model that mimics the structural and physiological effects of osmotic flux. DNA labelling after in situ nick translation of thawed cryopreserved spermatozoa revealed a positive correlation (r=0.573; PPlasma membrane integrity, chromatin relaxation and SDF appeared particularly susceptible to extreme hypotonic environments. Mitochondrial membrane potential (MMP), while susceptible to extreme hypo- and hypertonic environments, showed an ability to rebound from hypertonic stress when returned to isotonic conditions. Koala spermatozoa exposed to 64 mOsm/kg media showed an equivalent, or more severe, degree of structural and physiological injury to that of frozen-thawed spermatozoa, supporting the hypothesis that cryoinjury is principally associated with a hypo-osmotic effect. A direct comparison of SDF of thawed cryopreserved spermatozoa and those exposed to a 64 mOsm/kg excursion showed a significant correlation (r=0.878; Pmembrane) and oxidative stress that first leads to the reduction of MMP and the relaxation of chromatin, which is then ultimately followed by an increase in DNA fragmentation.

  13. Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex

    Directory of Open Access Journals (Sweden)

    Gidda Satinder K

    2008-09-01

    Full Text Available Abstract Background Carnation Italian ringspot virus (CIRV is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36, are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined. Results Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM, but not with the sorting and assembly machinery (SAM. Conclusion Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer

  14. A Mitochondrial Membrane Exopolyphosphatase Is Modulated by, and Plays a Role in, the Energy Metabolism of Hard Tick Rhipicephalus (Boophilus microplus Embryos

    Directory of Open Access Journals (Sweden)

    Carlos Logullo

    2011-06-01

    Full Text Available The physiological roles of polyphosphates (polyP recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 µg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5’-triphosphate synthesis in hard tick embryos.

  15. Elevated mRNA-levels of distinct mitochondrial and plasma membrane Ca2+ transporters in individual hypoglossal motor neurons of endstage SOD1 transgenic mice.

    Directory of Open Access Journals (Sweden)

    Tobias eMühling

    2014-11-01

    Full Text Available Disturbances in Ca2+ homeostasis and mitochondrial dysfunction have emerged as major pathogenic features in familial and sporadic forms of Amyotrophic Lateral Sclerosis (ALS, a fatal degenerative motor neuron disease. However, the distinct molecular ALS-pathology remains unclear. Recently, an activity-dependent Ca2+ homeostasis deficit, selectively in highly vulnerable cholinergic motor neurons in the hypoglossal nucleus (hMNs from a common ALS mouse model, endstage superoxide dismutase SOD1G93A transgenic mice, was described. This functional deficit was defined by a reduced hMN mitochondrial Ca2+ uptake capacity and elevated Ca2+ extrusion across the plasma membrane. To address the underlying molecular mechanisms, here we quantified mRNA-levels of respective potential mitochondrial and plasma membrane Ca2+ transporters in individual, choline-acetyltransferase (ChAT positive hMNs from wildtype (WT and endstage SOD1G93A mice, by combining UV laser microdissection with RT-qPCR techniques, and specific data normalization. As ChAT cDNA levels as well as cDNA and genomic DNA levels of the mitochondrially encoded NADH dehydrogenase ND1 were not different between hMNs from WT and endstage SOD1G93A mice, these genes were used to normalize hMN-specific mRNA-levels of plasma membrane and mitochondrial Ca2+ transporters, respectively. We detected about 2-fold higher levels of the mitochondrial Ca2+ transporters MCU/MICU1, Letm1 and UCP2 in remaining hMNs from endstage SOD1G93A mice. These higher expression-levels of mitochondrial Ca2+ transporters in individual hMNs were not associated with a respective increase in number of mitochondrial genomes, as evident from hMN specific ND1 DNA quantification. Normalized mRNA-levels for the plasma membrane Na2+/Ca2+exchanger NCX1 was also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a novel neuroprotective strategy for ALS.

  16. Triiodothyronine facilitates weaning from extracorporeal membrane oxygenation by improved mitochondrial substrate utilization

    Energy Technology Data Exchange (ETDEWEB)

    Files, Matthew D.; Kajimoto, Masaki; Priddy, Colleen M.; Ledee, Dolena R.; Xu, Chun; Des Rosiers, Christine; Isern, Nancy G.; Portman, Michael A.

    2014-03-20

    Extracorporeal membrane oxygenation (ECMO) provides a bridge to recovery after myocardial injury in infants and children, yet morbidity and mortality remain high. Weaning from the circuit requires adequate cardiac contractile function, which can be impaired by metabolic disturbances induced either by ischemia-reperfusion and / or by ECMO.

  17. On-line measurements of oscillating mitochondrial membrane potential in glucose-fermenting Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Andersen, Ann Zahle; Poulsen, Allan K; Olsen, Lars Folke

    2007-01-01

    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found that the ...

  18. New proteomic approaches to study the organization of yeast mitochondrial membranes

    NARCIS (Netherlands)

    Gubbens, J.

    2008-01-01

    Despite their importance, membrane proteins have traditionally been underrepresented in proteomics studies due to their incompatibility with common methods used in this field. Therefore, new methods have to be developed for studying this class of proteins. In this thesis, new approaches are describe

  19. Influence of low-power laser radiation on the activity of some membraneous and mitochondrial enzymes of hepatocytes in rats

    Science.gov (United States)

    Cieslar, Grzegorz; Adamek, Mariusz; Sieron, Aleksander; Kaminski, Marcin

    1995-01-01

    It was observed in some experiments that visible laser radiation activates the enzymatic function of mitochondria, while infrared laser radiation affects the enzymatic activity of cellular membranes. The aim of the study was to estimate the activity of some membranous as well as mitochondrial enzymes of hepatocytes in rats irradiated with infrared laser. Experimental material consisted of 38 Wistar rats divided into 2 groups -- a studied group exposed to infrared laser radiation and a control group, in which no irradiation was made. A semiconductive infrared laser (wavelength -- 904 nm, mean power -- 8.9 mW) was used. The clean-shaven skin of the right infracostal region of animals was irradiated 5 minutes daily for 15 consecutive days. After finishing the experiment in the preparations from obtained segments of the left liver lobe, the enzymatic activity of succinate dehydrogenase (SDH, EC 1.3.99.1), lactic dehydrogenase (LDH, EC 1.1.1.27), Mg2+ dependent ATP-ase (ATP-ase Mg2+, EC 3.1.3.2.) and acid phosphatase (AcP, EC 3.6.1.8.) was estimated with the use of histochemical methods. In the case of SDH and LDH the increase of enzymatic activity was observed in all 3 zones of liver cluster, especially in male rats. In the case of ATP-ase Mg2+ and AcP the increase of enzymatic activity in biliary canaliculi of hepatocytes in all zones of the liver cluster was observed. On the basis of the obtained results it was proved that infrared laser radiation activates significantly the enzymatic activity of most of the analyzed enzymes, which means that it affects not only properties of biological membranes but also activates the oxidoreductive processes of organism, as it has been observed for visible laser radiation. On the basis of the spectrum of energetic levels in macromolecules (Jablonski's diagram) the mechanisms of availed results are discussed both for enzymes possessing and not possessing chromatophores.

  20. Quantum squeezed light for probing mitochondrial membranes and study of neuroprotectants.

    Energy Technology Data Exchange (ETDEWEB)

    Gourley, Paul Lee; Copeland, Robert Guild; McDonald, Anthony Eugene; Hendricks, Judy K.; Naviaux, Robert K. (University of California, San Diego, CA)

    2005-01-01

    We report a new nanolaser technique for measuring characteristics of human mitochondria. Because mitochondria are so small, it has been difficult to study large populations using standard light microscope or flow cytometry techniques. We recently discovered a nano-optical transduction method for high-speed analysis of submicron organelles that is well suited to mitochondrial studies. This ultrasensitive detection technique uses nano-squeezing of light into photon modes imposed by the ultrasmall organelle dimensions in a semiconductor biocavity laser. In this paper, we use the method to study the lasing spectra of normal and diseased mitochondria. We find that the diseased mitochondria exhibit larger physical diameter and standard deviation. This morphological differences are also revealed in the lasing spectra. The diseased specimens have a larger spectral linewidth than the normal, and have more variability in their statistical distributions.

  1. Apaf-1-deficient fog mouse cell apoptosis involves hypopolarization of the mitochondrial inner membrane,ATP depletion and citrate accumulation

    Institute of Scientific and Technical Information of China (English)

    Iyoko Katoh; Shingo Sato; Nahoko Fukunishi; Hiroki Yoshida; Takasuke Imai; Shun-ichi Kurata

    2008-01-01

    To explore how the intrinsic apoptosis pathway is controlled in the spontaneous fog (forebrain overgrowth) mutant mice with an Apaf1 splicing deficiency,we examined spleen and bone marrow cells from Apaf1+/+(+/+) and Apaf1fog/fog (fog/fog) mice for initiator caspase-9 activation by cellular stresses.When the mitochondrial inner membrane potential (△Ψm) was disrupted by staurosporine,+/+ cells but not fog/fog cells activated caspase-9 to cause apoptosis,indicating the lack of apoptosomc (apoptosis protease activating factor 1 (Apaf-1)/cytochrome c/(d)ATP/procaspase-9) function in fog/fog cells.However,when a marginal (~20%) decrease in △Ψm was caused by hydrogen peroxide (0.1 mM),peroxynitrite donor 3-morpholinosydnonimine (0.1 mM) and UV-C irradiation (20 J/m2),both +/+ and fog/fog cells triggeredprocaspase-9 auto-processing and its downstream cascade activation.Supporting our previous results,procaspase-9 pre-existing in the mitochondria induced its auto-processing before the cytosolic caspase activation regardless of the geuotypes.Cellular ATP concentration significantly decreased under the hypoactive AΨm condition.Furthermore,we detected accumulation of citrate,a kosmotrope known to facilitate procaspase-9 dimerization,probably due to a feedback control of the Krebs cycle by the electron transfer system.Thus,mitochondrial in situ caspase-9 activation may be caused by the major metabolic reactions in response to physiological stresses,which may represent a mode of Apaf-1-independent apoptosis hypothesized from recent genetic studies.

  2. Positive Feedback Amplifies the Response of Mitochondrial Membrane Potential to Glucose Concentration in Clonal Pancreatic Beta Cells.

    Science.gov (United States)

    Gerencser, Akos A; Mookerjee, Shona A; Jastroch, Martin; Brand, Martin D

    2017-05-01

    Analysis of the cellular mechanisms of metabolic disorders, including type 2 diabetes mellitus, is complicated by the large number of reactions and interactions in metabolic networks. Metabolic control analysis with appropriate modularization is a powerful method for simplifying and analyzing these networks. To analyze control of cellular energy metabolism in adherent cell cultures of the INS-1 832/13 pancreatic β-cell model we adapted our microscopy assay of absolute mitochondrial membrane potential (ΔψM) to a fluorescence microplate reader format, and applied it in conjunction with cell respirometry. In these cells the sensitive response of ΔψM to extracellular glucose concentration drives glucose-stimulated insulin secretion. Using metabolic control analysis we identified the control properties that generate this sensitive response. Force-flux relationships between ΔψM and respiration were used to calculate kinetic responses to ΔψM of processes both upstream (glucose oxidation) and downstream (proton leak and ATP turnover) of ΔψM. The analysis revealed that glucose-evoked ΔψM hyperpolarization is amplified by increased glucose oxidation activity caused by factors downstream of ΔψM. At high glucose, the hyperpolarized ΔψM is stabilized almost completely by the action of glucose oxidation, whereas proton leak also contributes to the homeostatic control of ΔψM at low glucose. These findings suggest a strong positive feedback loop in the regulation of β-cell energetics, and a possible regulatory role of proton leak in the fasting state. Analysis of islet bioenergetics from published cases of type 2 diabetes suggests that disruption of this feedback can explain the damaged bioenergetic response of β-cells to glucose. This article is part of a Special Issue entitled: Oxidative Stress and Mitochondrial Quality in Diabetes/Obesity and Critical Illness Spectrum of Diseases - edited by P. Hemachandra Reddy. Copyright © 2016 Elsevier B.V. All rights

  3. Simultaneous Single Neuron Recording of O2 Consumption, [Ca2+]i and Mitochondrial Membrane Potential in Glutamate Toxicity

    Science.gov (United States)

    Gleichmann, Marc; Collis, Leon P.; Smith, Peter J.S.; Mattson, Mark P.

    2009-01-01

    To order the cellular processes in glutamate toxicity, we simultaneously recorded O2 consumption, cytosolic Ca2+ concentration ([Ca2+]i) and mitochondrial membrane potential (mΔψ) in single cortical neurons. O2 consumption was measured using an amperometric self-referencing platinum electrode adjacent to neurons in which [Ca2+]i and mΔψ were monitored with Fluo-4 and TMRE+, respectively using a spinning disk laser confocal microscope. Excitotoxic doses of glutamate caused an elevation of [Ca2+]i followed seconds afterwards by an increase in O2 consumption which reached a maximum level within 1 to 5 min. A modest increase in mΔψ occurred during this time period, and then, shortly before maximal O2 consumption was reached, the mΔψ, as indicated by TMRE+ fluorescence, dissipated. Maximal O2 consumption lasted up to 5 min and then declined together with mΔψ and ATP levels, while [Ca2+]i further increased. mΔψ and [Ca2+]i returned to baseline levels when neurons were treated with an N-methyl-D-aspartate receptor antagonist shortly after the [Ca2+]i increased. Our unprecedented spatial and time resolution revealed that this sequence of events is identical in all neurons, albeit with considerable variability in magnitude and kinetics of changes in O2 consumption, [Ca2+]i and mΔψ. The data obtained using this new method are consistent with a model where Ca2+ influx causes ATP depletion, despite maximal mitochondrial respiration, minutes after glutamate receptor activation. PMID:19226367

  4. Evidence for Amino Acid Snorkeling from a High-Resolution, In Vivo Analysis of Fis1 Tail-Anchor Insertion at the Mitochondrial Outer Membrane.

    Science.gov (United States)

    Keskin, Abdurrahman; Akdoğan, Emel; Dunn, Cory D

    2017-02-01

    Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can "snorkel," or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface.

  5. Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes.

    Science.gov (United States)

    Gan, Zhuohui; Audi, Said H; Bongard, Robert D; Gauthier, Kathryn M; Merker, Marilyn P

    2011-05-01

    Our goal was to quantify mitochondrial and plasma potential (Δψ(m) and Δψ(p)) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([R(e)]) were measured over time. R123 [R(e)] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [R(e)] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K(+) concentration ([K(+)]), used to manipulate contributions of membrane potentials, attenuated decreases in [R(e)], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [R(e)]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [R(e)] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψ(m) (-130 ± 7 and -133 ± 4 mV), Δψ(p) (-36 ± 4 and -49 ± 4 mV), and a Pgp activity parameter (K(Pgp), 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψ(m) (-124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψ(m) and Δψ(p.) Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O(2) for 48 h) cells have equivalent resting Δψ(m), hyperoxic cell Δψ(m) was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction.

  6. Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes

    Science.gov (United States)

    Gan, Zhuohui; Audi, Said H.; Bongard, Robert D.; Gauthier, Kathryn M.

    2011-01-01

    Our goal was to quantify mitochondrial and plasma potential (Δψm and Δψp) based on the disposition of rhodamine 123 (R123) or tetramethylrhodamine ethyl ester (TMRE) in the medium surrounding pulmonary endothelial cells. Dyes were added to the medium, and their concentrations in extracellular medium ([Re]) were measured over time. R123 [Re] fell from 10 nM to 6.6 ± 0.1 (SE) nM over 120 min. TMRE [Re] fell from 20 nM to a steady state of 4.9 ± 0.4 nM after ∼30 min. Protonophore or high K+ concentration ([K+]), used to manipulate contributions of membrane potentials, attenuated decreases in [Re], and P-glycoprotein (Pgp) inhibition had the opposite effect, demonstrating the qualitative impact of these processes on [Re]. A kinetic model incorporating a modified Goldman-Hodgkin-Katz model was fit to [Re] vs. time data for R123 and TMRE, respectively, under various conditions to obtain (means ± 95% confidence intervals) Δψm (−130 ± 7 and −133 ± 4 mV), Δψp (−36 ± 4 and −49 ± 4 mV), and a Pgp activity parameter (KPgp, 25 ± 5 and 51 ± 11 μl/min). The higher membrane permeability of TMRE also allowed application of steady-state analysis to obtain Δψm (−124 ± 6 mV). The consistency of kinetic parameter values obtained from R123 and TMRE data demonstrates the utility of this experimental and theoretical approach for quantifying intact cell Δψm and Δψp. Finally, steady-state analysis revealed that although room air- and hyperoxia-exposed (95% O2 for 48 h) cells have equivalent resting Δψm, hyperoxic cell Δψm was more sensitive to depolarization with protonophore, consistent with previous observations of pulmonary endothelial hyperoxia-induced mitochondrial dysfunction. PMID:21239539

  7. Measurement of the Absolute Magnitude and Time Courses of Mitochondrial Membrane Potential in Primary and Clonal Pancreatic Beta-Cells.

    Science.gov (United States)

    Gerencser, Akos A; Mookerjee, Shona A; Jastroch, Martin; Brand, Martin D

    2016-01-01

    The aim of this study was to simplify, improve and validate quantitative measurement of the mitochondrial membrane potential (ΔψM) in pancreatic β-cells. This built on our previously introduced calculation of the absolute magnitude of ΔψM in intact cells, using time-lapse imaging of the non-quench mode fluorescence of tetramethylrhodamine methyl ester and a bis-oxonol plasma membrane potential (ΔψP) indicator. ΔψM is a central mediator of glucose-stimulated insulin secretion in pancreatic β-cells. ΔψM is at the crossroads of cellular energy production and demand, therefore precise assay of its magnitude is a valuable tool to study how these processes interplay in insulin secretion. Dispersed islet cell cultures allowed cell type-specific, single-cell observations of cell-to-cell heterogeneity of ΔψM and ΔψP. Glucose addition caused hyperpolarization of ΔψM and depolarization of ΔψP. The hyperpolarization was a monophasic step increase, even in cells where the ΔψP depolarization was biphasic. The biphasic response of ΔψP was associated with a larger hyperpolarization of ΔψM than the monophasic response. Analysis of the relationships between ΔψP and ΔψM revealed that primary dispersed β-cells responded to glucose heterogeneously, driven by variable activation of energy metabolism. Sensitivity analysis of the calibration was consistent with β-cells having substantial cell-to-cell variations in amounts of mitochondria, and this was predicted not to impair the accuracy of determinations of relative changes in ΔψM and ΔψP. Finally, we demonstrate a significant problem with using an alternative ΔψM probe, rhodamine 123. In glucose-stimulated and oligomycin-inhibited β-cells the principles of the rhodamine 123 assay were breached, resulting in misleading conclusions.

  8. Crystallization of Mitochondrial Respiratory Complex II fromChicken Heart: A Membrane-Protein Complex Diffracting to 2.0Angstrom

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Li-shar; Borders, Toni M.; Shen, John T.; Wang, Chung-Jen; Berry, Edward A.

    2004-12-17

    Procedure is presented for preparation of diffraction-quality crystals of a vertebrate mitochondrial respiratory Complex II. The crystals have the potential to diffract to at least 2.0 Angstrom with optimization of post-crystal-growth treatment and cryoprotection. This should allow determination of the structure of this important and medically relevant membrane protein complex at near-atomic resolution and provide great detail of the mode of binding of substrates and inhibitors at the two substrate-binding sites.

  9. La surexpression de Sirt3 dans le muscle squelettique modifie la composition en lipides des membranes des mitochondries et leur activité

    OpenAIRE

    Chabi, Beatrice; Fouret, Gilles; Lecomte, Jérôme; Pessemesse, Laurence; Cortade, Fabienne; Coudray , Charles; Casas, Francois; Wrutniak-Cabello, Chantal; Feuillet-Coudray, Christine

    2016-01-01

    Introduction et but de l’étude : Des travaux récents indiquent que la fonctionnalité de la mitochondrie peut être influencée par la composition en lipides des membranes mitochondriales, en particulier le cardiolipide, un phospholipide exclusivement présent dans la membrane mitochondriale. La teneur en cardiolipide impacte la fluidité membranaire, l’organisation et le fonctionnement des complexes de la chaîne respiratoire, et la production mitochondriale d’espèces oxygénées réactives. Sirt3...

  10. Low intensity ultrasound induces apoptosis via MPT channel on mitochondrial membrane: Target for regulating cancer therapy or not?

    Science.gov (United States)

    Feng, Yi; Wan, Mingxi

    2017-03-01

    To discuss how the mitochondrion is involved in low intensity ultrasound induced apoptosis, HepG2 cells were irradiated by low intensity focused ultrasound (ISPTA = 3W/cm2, 1 min) and then cultured from 3-12 h post irradiation in the study. The morphological alteration was examined by light and fluorescent microscopy respectively. Cell viability and apoptosis were examined by trypan blue staining and flow cytometry with double staining of FITC-labelled Annexin-V/PI. Key proteins responded to irradiation were screened out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and shotgun proteomic methods with Agilent 1100 HPLC-Chip-MS technology. Representative apoptotic morphological characteristics and increased percentage of apoptotic cells were achieved. Six important proteins (4 up-regulated and 2 down-regulated) were selected and analyzed. It revealed low intensity focused ultrasound could induce apoptosis in HepG2 cells and the US-induced apoptosis was mitochondria-dependent and caspases-dependent. Moreover, mitochondrial membrane permeability transition (MPT) is related to ultrasound induced apoptosis, but VDAC may be not the main MPT channel. Understanding it could help to assist the cancer therapy by regulating the MPT as the target.

  11. Abrin P2 suppresses proliferation and induces apoptosis of colon cancer cells via mitochondrial membrane depolarization and caspase activation.

    Science.gov (United States)

    Yu, Ying; Yang, Runmei; Zhao, Xiuyun; Qin, Dandan; Liu, Zhaoyang; Liu, Fang; Song, Xin; Li, Liqin; Feng, Renqing; Gao, Nannan

    2016-05-01

    To explore the cytotoxic mechanism of abrin P2 on human colon cancer HCT-8 cells, abrin P2 was isolated from the seed of Abrus precatorius L. It was found that abrin P2 exhibited cytotoxicity toward 12 different human cancer cell lines. Our results demonstrated that abrin P2 suppressed the proliferation of human colon cancer cells (HCT-8 cells) and induced cell cycle arrest at the S and G2/M phases. The mechanism by which abrin P2 inhibited cell proliferation was via the down-regulation of cyclin B1, proliferating cell nuclear antigen and Ki67, as well as the up-regulation of P21. In addition, abrin P2 induced a dose- and time-dependent increase in the rate of HCT-8 cell apoptosis. Treatment with both Z-VAD-FMK, a broad-spectrum caspase inhibitor, and abrin P2 demonstrated that abrin P2 induced HCT-8 cell apoptosis via the activation of caspases. Together, our results revealed that abrin P2-induced apoptosis in HCT-8 cells was associated with the activation of caspases-3/-8/-9, the reduction in the Bcl-2/Bax ratio, the loss of mitochondrial membrane potential, and the increase in cytochrome c release. We further showed that abrin P2 administration effectively suppressed the growth of colon cancer xenografts in nude mice. This is the first report that abrin P2 effectively inhibits colon cancer cell growth in vivo and in vitro by suppressing proliferation and inducing apoptosis.

  12. An epidemiologic study of mitochondrial membrane transporter protein gene polymorphism and risk factors for neural tube defects in Shanxi, China

    Institute of Scientific and Technical Information of China (English)

    Zhizhen Liu; Jun Xie; Tiane Luo; Tao Zhang; Xia Zhao; Hong Zhao; Peizhen Li

    2012-01-01

    The present study involved a questionnaire survey of 156 mothers that gave birth to children with neural tube defects or had a history of pregnancy resulting in children with neural tube defects (case group) and 156 control mothers with concurrent healthy children (control group) as well as detection of mitochondrial membrane transporter protein gene [uncoupling protein 2 (UCP2)] polymorphism. The maternal UCP2 3' untranslated region (UTR) D/D genotype and D allele frequency were significantly higher in the case group compared with the control group (odds ratio (OR) 3.233; 95% confidence interval (CI) 1.103-9.476; P = 0.040; OR: 3.484; 95% CI: for neural tube defects 2.109-5.753; P < 0.001). Univariate and multivariate logistic regression analysis of risk factors for neural tube defects showed that a maternal UCP2 3' UTR D/D genotype was negatively interacted with the mothers'consumption of frequent fresh fruit and vegetables (S = 0.007), positively interacted with the mothers'frequency of germinated potato consumption (S = 2.15) and positively interacted with the mothers' body mass index (S = 3.50). These findings suggest that maternal UCP2 3' UTR gene polymorphism, pregnancy time, consumption of germinated potatoes and body mass index are associated with an increased risk for neural tube defects in children from mothers living in Shanxi province, China. Moreover, there is an apparent gene-environment interaction involved in the development of neural tube defects in offspring.

  13. Caspase-8 Activation Precedes Alterations of Mitochondrial Membrane Potential during Monocyte Apoptosis Induced by Phagocytosis and Killing of Staphylococcus aureus

    Science.gov (United States)

    Węglarczyk, Kazimierz; Baran, Jarosław; Zembala, Marek; Pryjma, Juliusz

    2004-01-01

    Human peripheral blood monocytes become apoptotic following phagocytosis and killing of Staphylococcus aureus. Although this type of monocyte apoptosis is known to be initiated by Fas-Fas ligand (FasL) interactions, the downstream signaling pathway has not been determined. In this work the involvement of mitochondria and the kinetics of caspase-8 and caspase-3 activation after phagocytosis of S. aureus were studied. Caspase-8 activity was measured in cell lysates by using the fluorogenic substrate Ac-IETD-AFC. Active caspase-3 levels and mitochondrial membrane potential (Δψm) were measured in whole cells by flow cytometry using monoclonal antibodies reacting with activated caspase-3 and chloromethyl-X-rosamine, respectively. The results show that caspase-8 was activated shortly after phagocytosis of bacteria. Caspase-8 activation was followed by progressive disruption of Δψm, which is associated with the production of reactive oxygen intermediates. The irreversible caspase-8 inhibitor zIETD-FMK prevented the disruption of Δψm and the release of cytochrome c from S. aureus-exposed monocytes. Caspase-3 activation occurred following disruption of Δψm. These results strongly suggest that apoptosis of monocytes that have phagocytosed and killed S. aureus is driven by the Fas-FasL-initiated pathway, which is typical for type II cells. PMID:15102767

  14. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  15. Rapid detection of an ABT-737-sensitive primed for death state in cells using microplate-based respirometry.

    Directory of Open Access Journals (Sweden)

    Pascaline Clerc

    Full Text Available Cells that exhibit an absolute dependence on the anti-apoptotic BCL-2 protein for survival are termed "primed for death" and are killed by the BCL-2 antagonist ABT-737. Many cancers exhibit a primed phenotype, including some that are resistant to conventional chemotherapy due to high BCL-2 expression. We show here that 1 stable BCL-2 overexpression alone can induce a primed for death state and 2 that an ABT-737-induced loss of functional cytochrome c from the electron transport chain causes a reduction in maximal respiration that is readily detectable by microplate-based respirometry. Stable BCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis. Mitochondria within permeabilized BCL-2 overexpressing cells were selectively vulnerable to ABT-737-induced cytochrome c release compared to those from control-transfected cells, consistent with a primed state. ABT-737 treatment caused a dose-dependent impairment of maximal O(2 consumption in MCF10A BCL-2 overexpressing cells but not in control-transfected cells or in immortalized mouse embryonic fibroblasts lacking both BAX and BAK. This impairment was rescued by delivering exogenous cytochrome c to mitochondria via saponin-mediated plasma membrane permeabilization. An ABT-737-induced reduction in maximal O(2 consumption was also detectable in SP53, JeKo-1, and WEHI-231 B-cell lymphoma cell lines, with sensitivity correlating with BCL-2:MCL-1 ratio and with susceptibility (SP53 and JeKo-1 or resistance (WEHI-231 to ABT-737-induced apoptosis. Multiplexing respirometry assays to ELISA-based determination of cytochrome c redistribution confirmed that respiratory inhibition was associated with cytochrome c release. In summary, cell-based respiration assays were able to rapidly identify a primed for death state in cells with either artificially overexpressed or high endogenous BCL-2. Rapid detection of a primed for death state in

  16. Cell membrane penetration and mitochondrial targeting by platinum-decorated ceria nanoparticles

    Science.gov (United States)

    Torrano, Adriano A.; Herrmann, Rudolf; Strobel, Claudia; Rennhak, Markus; Engelke, Hanna; Reller, Armin; Hilger, Ingrid; Wixforth, Achim; Bräuchle, Christoph

    2016-07-01

    In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and focus on their fast uptake and association with mitochondria, the cell's powerhouse. Using live-cell imaging and electron microscopy we clearly show that 46 nm platinum-decorated ceria nanoparticles can rapidly penetrate cell membranes and reach the cytosol. Moreover, if suitably targeted, these particles are able to selectively attach to mitochondria. These results are complemented by cytotoxicity assays, thus providing insights into the biological effects of these particles on cells. Interestingly, no permanent membrane disruption or any other significant adverse effects on cells were observed. The unusual uptake behavior observed for 46 nm nanoparticles was not observed for equivalent but larger 143 nm and 285 nm platinum-decorated particles. Our results demonstrate a remarkable particle size effect in which particles smaller than ~50-100 nm escape the usual endocytic pathway and translocate directly into the cytosol, while particles larger than ~150 nm are internalized by conventional endocytosis. Since the small particles are able to bypass endocytosis they could be explored as drug and gene delivery vehicles. Platinum-decorated nanoparticles are therefore highly interesting in the fields of nanotoxicology and nanomedicine.In this work we investigate the interaction between endothelial cells and nanoparticles emitted by catalytic converters. Although catalyst-derived particles are recognized as growing burden added to environmental pollution, very little is known about their health impact. We use platinum-decorated ceria nanoparticles as model compounds for the actual emitted particles and

  17. A transient increase in lipid peroxidation primes preadipocytes for delayed mitochondrial inner membrane permeabilization and ATP depletion during prolonged exposure to fatty acids.

    Science.gov (United States)

    Rogers, Carlyle; Davis, Barbara; Neufer, P Darrell; Murphy, Michael P; Anderson, Ethan J; Robidoux, Jacques

    2014-02-01

    Preadipocytes are periodically subjected to fatty acid (FA) concentrations that are potentially cytotoxic. We tested the hypothesis that prolonged exposure of preadipocytes of human origin to a physiologically relevant mix of FAs leads to mitochondrial inner membrane (MIM) permeabilization and ultimately to mitochondrial crisis. We found that exposure of preadipocytes to FAs led to progressive cyclosporin A-sensitive MIM permeabilization, which in turn caused a reduction in MIM potential, oxygen consumption, and ATP synthetic capacity and, ultimately, death. Additionally, we showed that FAs induce a transient increase in intramitochondrial reactive oxygen species (ROS) and lipid peroxide production, lasting roughly 30 and 120min for the ROS and lipid peroxides, respectively. MIM permeabilization and its deleterious consequences including mitochondrial crisis and cell death were prevented by treating the cells with the mitochondrial FA uptake inhibitor etomoxir, the mitochondrion-selective superoxide and lipid peroxide antioxidants MitoTempo and MitoQ, or the lipid peroxide and reactive carbonyl scavenger l-carnosine. FAs also promoted a delayed oxidative stress phase. However, the beneficial effects of etomoxir, MitoTempo, and l-carnosine were lost by delaying the treatment by 2h, suggesting that the initial phase was sufficient to prime the cells for the delayed MIM permeabilization and mitochondrial crisis. It also suggested that the second ROS production phase is a consequence of this loss in mitochondrial health. Altogether, our data suggest that approaches designed to diminish intramitochondrial ROS or lipid peroxide accumulation, as well as MIM permeabilization, are valid mechanism-based therapeutic avenues to prevent the loss in preadipocyte metabolic fitness associated with prolonged exposure to elevated FA levels. © 2013 Elsevier Inc. All rights reserved.

  18. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating Serine 65

    Science.gov (United States)

    Kondapalli, Chandana; Kazlauskaite, Agne; Zhang, Ning; Woodroof, Helen I.; Campbell, David G.; Gourlay, Robert; Burchell, Lynn; Walden, Helen; Macartney, Thomas J.; Deak, Maria; Knebel, Axel; Alessi, Dario R.; Muqit, Miratul M. K.

    2012-01-01

    Summary Missense mutations in PTEN-induced kinase 1 (PINK1) cause autosomal-recessive inherited Parkinson's disease (PD). We have exploited our recent discovery that recombinant insect PINK1 is catalytically active to test whether PINK1 directly phosphorylates 15 proteins encoded by PD-associated genes as well as proteins reported to bind PINK1. We have discovered that insect PINK1 efficiently phosphorylates only one of these proteins, namely the E3 ligase Parkin. We have mapped the phosphorylation site to a highly conserved residue within the Ubl domain of Parkin at Ser65. We show that human PINK1 is specifically activated by mitochondrial membrane potential (Δψm) depolarization, enabling it to phosphorylate Parkin at Ser65. We further show that phosphorylation of Parkin at Ser65 leads to marked activation of its E3 ligase activity that is prevented by mutation of Ser65 or inactivation of PINK1. We provide evidence that once activated, PINK1 autophosphorylates at several residues, including Thr257, which is accompanied by an electrophoretic mobility band-shift. These results provide the first evidence that PINK1 is activated following Δψm depolarization and suggest that PINK1 directly phosphorylates and activates Parkin. Our findings indicate that monitoring phosphorylation of Parkin at Ser65 and/or PINK1 at Thr257 represent the first biomarkers for examining activity of the PINK1-Parkin signalling pathway in vivo. Our findings also suggest that small molecule activators of Parkin that mimic the effect of PINK1 phosphorylation may confer therapeutic benefit for PD. PMID:22724072

  19. Sperm DNA fragmentation and mitochondrial membrane potential combined are better for predicting natural conception than standard sperm parameters.

    Science.gov (United States)

    Malić Vončina, Slađana; Golob, Barbara; Ihan, Alojz; Kopitar, Andreja Nataša; Kolbezen, Mojca; Zorn, Branko

    2016-03-01

    To evaluate whether DNA fragmentation and/or mitochondrial membrane potential (MMP) predict natural conception better than standard sperm parameters. Prospective cross-sectional study. University medical center. Eighty-five infertile and 51 fertile men. Assessment of sperm DNA fragmentation, MMP, and standard semen parameters over a 6- to 12-month observation period. Comparison between the results of DNA fragmentation, MMP, and standard sperm parameters alone or combined and achievement of natural conception. Twenty-six of the 85 (31%) men from infertile couples conceived naturally. The median values of DNA fragmentation and MMP in the men who conceived within the observation period were similar to those in the fertile controls. Optimal threshold values of DNA fragmentation and MMP were 25% as determined by receiver operating characteristic analysis (area under the curve [AUC], 0.70; 95% confidence interval (CI) 0.58-0.82) and 62.5% (AUC, 0.68, 95% CI 0.56-0.80), respectively. The men in the infertile group with values of DNA fragmentation ≤25% and with MMP values ≥62.5% had significantly higher odds for conception (odds ratio [OR], 5.22; 95% CI 1.82-14.93] and OR, 4.67; 95% CI 1.74-12.5, respectively). Normal semen analysis alone had no predictive value for natural conception (OR, 1.84; 95% CI 0.67-5.07]). Both sperm function tests combined had significant odds for natural conception (OR, 8.24; 95% CI 2.91-23.33]), with a probability of 0.607 (60.7%) for both normal values and 0.158 (15.8%) for abnormal values. Sperm DNA fragmentation and MMP combined may be superior to standard semen parameters for the prediction of natural conception. Copyright © 2016. Published by Elsevier Inc.

  20. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM.

    Directory of Open Access Journals (Sweden)

    Daniel O Frank

    Full Text Available The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM. In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20 by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  1. The pro-apoptotic BH3-only protein Bim interacts with components of the translocase of the outer mitochondrial membrane (TOM).

    Science.gov (United States)

    Frank, Daniel O; Dengjel, Jörn; Wilfling, Florian; Kozjak-Pavlovic, Vera; Häcker, Georg; Weber, Arnim

    2015-01-01

    The pro-apoptotic Bcl-2-family protein Bim belongs to the BH3-only proteins known as initiators of apoptosis. Recent data show that Bim is constitutively inserted in the outer mitochondrial membrane via a C-terminal transmembrane anchor from where it can activate the effector of cytochrome c-release, Bax. To identify regulators of Bim-activity, we conducted a search for proteins interacting with Bim at mitochondria. We found an interaction of Bim with Tom70, Tom20 and more weakly with Tom40, all components of the Translocase of the Outer Membrane (TOM). In vitro import assays performed on tryptically digested yeast mitochondria showed reduced Bim insertion into the outer mitochondrial membrane (OMM) indicating that protein receptors may be involved in the import process. However, RNAi against components of TOM (Tom40, Tom70, Tom22 or Tom20) by siRNA, individually or in combination, did not consistently change the amount of Bim on HeLa mitochondria, either at steady state or upon de novo-induction. In support of this, the individual or combined knock-downs of TOM receptors also failed to alter the susceptibility of HeLa cells to Bim-induced apoptosis. In isolated yeast mitochondria, lack of Tom70 or the TOM-components Tom20 or Tom22 alone did not affect the import of Bim into the outer mitochondrial membrane. In yeast, expression of Bim can sensitize the cells to Bax-dependent killing. This sensitization was unaffected by the absence of Tom70 or by an experimental reduction in Tom40. Although thus the physiological role of the Bim-TOM-interaction remains unclear, TOM complex components do not seem to be essential for Bim insertion into the OMM. Nevertheless, this association should be noted and considered when the regulation of Bim in other cells and situations is investigated.

  2. Study on the mitochondrial activity and membrane potential after exposing later stage oocytes of two gorgonian corals (Junceella juncea and Junceella fragilis) to cryoprotectants.

    Science.gov (United States)

    Tsai, S; Spikings, E; Huang, I-C; Lin, C

    2011-01-01

    Coral reefs provide a valuable habitat for many economically valuable fish and invertebrates. However, they are in serious jeopardy, threatened by increasing over-exploitation, pollution, habitat destruction, disease and global climate change. Here, we examined the effect of cryoprotectant exposure on mitochondrial activity and membrane potential in coral oocytes in order to find suitable cryoprotectants towards their successful cryopreservation. According to the No Observed Effect Concentrations (NOECs), methanol was found to be the least toxic cryoprotectant whilst DMSO was the most toxic cryoprotectant. The results also demonstrated that there were no significant differences (p > 0.05) in ATP concentrations between Junceella juncea and Junceella fragilis after exposure to all concentrations of all cryoprotectants for 30 min. Using confocal microscopy, JC-1 (5,50,6,60-tetrachloro-1,10,3,30-tetraethyl-imidacarbocyanine iodide) staining indicated that the mitochondrial membrane potential of Junceella fragilis oocytes reduced after 1 M and 2 M methanol treatment and a loss of the mitochondrial distribution pattern and poor green fluorescence after 3M methanol treatment. Therefore, even oocytes that show no adverse effect of cryoprotectants on survival might suffer some more subtle impacts. The results obtained from this study will provide a basis for development of protocols to cryopreserve the oocytes of gorgonian corals.

  3. Oral administration of fumonisin B1 and T-2 individually and in combination affects hepatic total and mitochondrial membrane lipid profile of rabbits.

    Science.gov (United States)

    Szabó, A; Szabó-Fodor, J; Fébel, H; Mézes, M; Bajzik, G; Kovács, M

    2016-09-01

    Weaned rabbits were fed diets contaminated with 2 mg/kg diet T-2 toxin alone, or 10 mg/kg diet fumonisin B1 (FB1) alone, and both toxins in combination (2 + 10 mg/kg, respectively) compared to a toxin-free control diet. Samplings were performed after 4 weeks (blood and liver). Bodyweight of T-2-fed group was lower after 4 weeks; the liver weight was increased dramatically (threefold of control). Liver total phospholipids (PLs) provided slight alterations in the fatty acid (FA) composition; all three toxin-treated groups showed a decrease in palmitoleic acid (C16:1 n7) proportion. In the liver mitochondrial PL FA composition, margaric acid (C17:0) proportion decreased in the separated toxin treatments compared to the combined setting. Oleic acid (C18:1 n9) proportion was increased and arachidonic acid (C20:4 n6) was decreased in the FB1-treated group, while docosapentaenoic acid (C22:5 n3) was decreased in the separated treatments. The total monounsaturation was significantly higher in the FB1 group's mitochondrial PL FA profile. After 4 weeks, all toxin treatments decreased the blood plasma reduced glutathione and glutathione peroxidase activity, and FB1 increased the plasma sphinganine/sphingosine ratio. Both mycotoxins seem to cross the hepatocellular and the hepatic mitochondrial membrane, without drastic membrane disruption, as assessed from the PL FA composition, but inducing detectable lipid peroxidation.

  4. Products of the Parkinson's disease-related glyoxalase DJ-1, D-lactate and glycolate, support mitochondrial membrane potential and neuronal survival

    Directory of Open Access Journals (Sweden)

    Yusuke Toyoda

    2014-07-01

    Full Text Available Parkinson's disease is associated with mitochondrial decline in dopaminergic neurons of the substantia nigra. One of the genes linked with the onset of Parkinson's disease, DJ-1/PARK7, belongs to a novel glyoxalase family and influences mitochondrial activity. It has been assumed that glyoxalases fulfill this task by detoxifying aggressive aldehyde by-products of metabolism. Here we show that supplying either D-lactate or glycolate, products of DJ-1, rescues the requirement for the enzyme in maintenance of mitochondrial potential. We further show that glycolic acid and D-lactic acid can elevate lowered mitochondrial membrane potential caused by silencing PINK-1, another Parkinson's related gene, as well as by paraquat, an environmental toxin known to be linked with Parkinson's disease. We propose that DJ-1 and consequently its products are components of a novel pathway that stabilizes mitochondria during cellular stress. We go on to show that survival of cultured mesencephalic dopaminergic neurons, defective in Parkinson's disease, is enhanced by glycolate and D-lactate. Because glycolic and D-lactic acids occur naturally, they are therefore a potential therapeutic route for treatment or prevention of Parkinson's disease.

  5. The cell-free integration of a polytopic mitochondrial membrane protein into liposomes occurs cotranslationally and in a lipid-dependent manner.

    Directory of Open Access Journals (Sweden)

    Ashley R Long

    Full Text Available The ADP/ATP Carrier (AAC is the most abundant transporter of the mitochondrial inner membrane. The central role that this transporter plays in cellular energy production highlights the importance of understanding its structure, function, and the basis of its pathologies. As a means of preparing proteoliposomes for the study of membrane proteins, several groups have explored the use of cell-free translation systems to facilitate membrane protein integration directly into preformed unilamellar vesicles without the use of surfactants. Using AAC as a model, we report for the first time the detergent-free reconstitution of a mitochondrial inner membrane protein into liposomes using a wheat germ-based in vitro translation system. Using a host of independent approaches, we demonstrate the efficient integration of AAC into vesicles with an inner membrane-mimetic lipid composition and, more importantly, that the integrated AAC is functionally active in transport. By adding liposomes at different stages of the translation reaction, we show that this direct integration is obligatorily cotranslational, and by synthesizing stable ribosome-bound nascent chain intermediates, we show that the nascent AAC polypeptide interacts with lipid vesicles while ribosome-bound. Finally, we show that the presence of the phospholipid cardiolipin in the liposomes specifically enhances AAC translation rate as well as the efficiency of vesicle association and integration. In light of these results, the possible mechanisms of liposome-assisted membrane protein integration during cell-free translation are discussed with respect to the mode of integration and the role of specific lipids.

  6. Formation of palmitic acid/Ca2+ complexes in the mitochondrial membrane: a possible role in the cyclosporin-insensitive permeability transition.

    Science.gov (United States)

    Mironova, Galina D; Gritsenko, Elena; Gateau-Roesch, Odile; Levrat, Christiane; Agafonov, Alexey; Belosludtsev, Konstantin; Prigent, Annie France; Muntean, Danina; Dubois, Madeleine; Ovize, Michel

    2004-04-01

    A possible role of palmitic acid/Ca2+ (PA/Ca2+) complexes in the cyclosporin-insensitive permeability transition in mitochondria has been studied. It has been shown that in the presence of Ca2+, PA induces a swelling of mitochondria, which is not inhibited by cyclosporin A. The swelling is accompanied by a drop in membrane potential, which cannot be explained only by a work of the Ca2+ uniporter. With time, the potential is restored. Evidence has been obtained indicating that the specific content of mitochondrial lipids would favor the PA/Ca2+ -induced permeabilization of the membrane. In experiments with liposomes, the PA/Ca2+ -induced membrane permeabilization was larger for liposomes formed from the mitochondrial lipids, as compared to the azolectin liposomes. Additionally, it has been found that in mitochondria of the TNF (tumor necrosis factor)-sensitive cells (WEHI-164 line), the content of PA is larger than in mitochondria of the TNF-insensitive cells (C6 line), with this difference being mainly provided by PA incorporated in phosphatidylethanolamine and especially, cardiolipin. The PA/Ca2+ -dependent mechanism of permeability transition in mitochondria might be related to some pathologies, e.g. myocardial ischemia. The heaviness of myocardial infarction of ischemic patients has been demonstrated to correlate directly with the content of PA in the human blood serum.

  7. 氟康唑对热带念珠菌活性氧和%Effect of fluconazole on reactive oxygen species and mitochondrial membrane potential of Candida tropicalis

    Institute of Scientific and Technical Information of China (English)

    邱莲女; 周永列; 胡庆丰; 朱永泽; 郭伟; 吕火烊

    2011-01-01

    To explore fluconazole's effect mechanism, we investigated the changes of viability rate, reactive oxygen species (ROS), mitochondrial membrane potential (△Ψm) and cell cycle of Candida tropicalis after treatment with fluconazole. The minimum inhibitory concentration (MIC) of the clinical isolates Candida tropicalis to fluconazole were tested by NCCLS M27-A microdilution method. After treatment wth different concentration of fluconazole, viability rate, the intracellular accumulation of ROS, the loss of mitochondrial membrane potential △Ψm and cell cycle of Candida tropicalis were detected with flow cytometry, respectively. After treatment with fluconazole, there were no significant variation among viability rate, ROS, mitochondrial membrane potential △Ψm and cell cycle in fluconazole-resistant strains, but a decrease of mitochondrial membrane potential △Ψm and viability rate,an increase of ROS accumulation were detected in a time-dose-dependent manner in fluconazole-susceptibile strains. A majority of Candida tropicalis were arrested in G2/M phase and apoptosis peak was seen. Free radicals scavenger glutathione inhibited ROS production, prevented G2/M arrest and decreased apoptosis in fluconazole-susceptibile strains. According to it, fluconazole maybe induce intracellular accumulation of ROS and decrease of mitochondrial membrane potential △Ψm, which could result in apoptosis of Candida tropicalis.%为了探讨氟康唑作用机制,观察它对热带念珠菌作用后存活率、活性氧(Reactive oxygenspecies,ROS)、线粒体膜电位(Mitochondrial membrane potential,,△Ψm)和细胞周期的变化.参照NCCLS M27-A 方案的微量稀释法测定氟康唑对热带念珠菌的最低抑菌浓度(MIC); 热带念珠菌与不同浓度氟康唑共同培养后用流式细胞术(Flow cytometry,FCM)分析热带念珠菌存活率、ROS、线粒体膜电位△Ψm 和细胞周期的变化.结果表明,氟康唑作用后,热带念珠菌氟康唑耐药株的

  8. Levetiracetam differentially alters CD95 expression of neuronal cells and the mitochondrial membrane potential of immune and neuronal cells in vitro

    Directory of Open Access Journals (Sweden)

    Susannah K Rogers

    2014-02-01

    Full Text Available Epilepsy is a neurological seizure disorder that affects over 100 million people worldwide. Levetiracetam, either alone, as monotherapy, or as adjunctive treatment, is widely used to control certain types of seizures. Despite its increasing popularity as a relatively safe and effective anti-convulsive treatment option, its mechanism(s of action are poorly understood. Studies have suggested neuronal, glial, and immune mechanisms of action. Understanding the precise mechanisms of action of Levetiracetam would be extremely beneficial in helping to understand the processes involved in seizure generation and epilepsy. Moreover, a full understanding of these mechanisms would help to create more efficacious treatments while minimizing side effects. The current study examined the effects of Levetiracetam on the mitochondrial membrane potential of neuronal and non-neuronal cells, in vitro, in order to determine if Levetiracetam influences metabolic processes in these cell types. In addition, this study sought to address possible immune-mediated mechanisms by determining if Levetiracetam alters the expression of immune receptor-ligand pairs. The results show that Levetiracetam induces expression of CD95 and CD178 on NGF-treated C17.2 neuronal cells. The results also show that Levetiracetam increases mitochondrial membrane potential on C17.2 neuronal cells in the presence of nerve growth factor. In contrast, Levetiracetam decreases the mitochondrial membrane potential of splenocytes and this effect was dependent on intact invariant chain, thus implicating immune cell interactions. These results suggest that both neuronal and non-neuronal anti-epileptic activities of Levetiracetam involve control over energy metabolism, more specifically, mΔΨ. Future studies are needed to further investigate this potential mechanism of action.

  9. 3T3 fibroblasts transfected with a cDNA for mitochondrial aspartate aminotransferase express plasma membrane fatty acid-binding protein and saturable fatty acid uptake.

    OpenAIRE

    1995-01-01

    To explore the relationship between mitochondrial aspartate aminotransferase (mAspAT; EC 2.6.1.1) and plasma membrane fatty acid-binding protein (FABPpm) and their role in cellular fatty acid uptake, 3T3 fibroblasts were cotransfected with plasmid pMAAT2, containing a full-length mAspAT cDNA downstream of a Zn(2+)-inducible metallothionein promoter, and pFR400, which conveys methotrexate resistance. Transfectants were selected in methotrexate, cloned, and exposed to increasing methotrexate co...

  10. HBCDD-induced sustained reduction in mitochondrial membrane potential, ATP and steroidogenesis in peripubertal rat Leydig cells

    Energy Technology Data Exchange (ETDEWEB)

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa, E-mail: nebojsa.andric@dbe.uns.ac.rs

    2015-01-01

    Hexabromocyclododecane (HBCDD), a brominated flame retardant added to various consumer products, is a ubiquitous environmental contaminant. We have previously shown that 6-hour exposure to HBCDD disturbs basal and human chorionic gonadotropin (hCG)-induced steroidogenesis in rat Leydig cells. Reduction in mitochondrial membrane potential (ΔΨm) and cAMP production was also observed. Here, we further expanded research on the effect of HBCDD on Leydig cells by using a prolonged exposure scenario. Cells were incubated in the presence of HBCDD during 24 h and then treated with HBCDD + hCG for additional 2 h. Results showed that HBCDD caused a sustained reduction in ATP level after 24 h of exposure, which persisted after additional 2-hour treatment with HBCDD + hCG. cAMP and androgen accumulations measured after 2 h of HBCDD + hCG treatment were also inhibited. Real-time PCR analysis showed significant inhibition in the expression of genes for steroidogenic enzymes, luteinizing hormone receptor, regulatory and transport proteins, and several transcription factors under both treatment conditions. Western blot analysis revealed a decreased level of 30 kDa steroidogenic acute regulatory protein (StAR) after HBCDD + hCG treatment. In addition, HBCDD decreased the conversion of 22-OH cholesterol to pregnenolone and androstenedione to testosterone, indicating loss of the activity of cytochrome P450C11A1 (CYP11A1) and 17β-hydroxysteroid dehydrogenase (HSD17β). Cell survival was not affected, as confirmed by cytotoxicity and trypan blue tests or DNA fragmentation analysis. In summary, our data showed that HBCDD inhibits ATP supply, most likely through a decrease in ΔΨm, and targets multiple sites in the steroidogenic pathway in Leydig cells. - Highlights: • HBCDD causes a sustained reduction in ΔΨm and ATP level in Leydig cells. • Prolonged HBCDD exposure decreases hCG-supported steroidogenesis in Leydig cells. • HBCDD targets StAR, HSD17β and CYP11A1 in Leydig

  11. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  12. Mitochondrial phospholipids: role in mitochondrial function.

    Science.gov (United States)

    Mejia, Edgard M; Hatch, Grant M

    2016-04-01

    Mitochondria are essential components of eukaryotic cells and are involved in a diverse set of cellular processes that include ATP production, cellular signalling, apoptosis and cell growth. These organelles are thought to have originated from a symbiotic relationship between prokaryotic cells in an effort to provide a bioenergetic jump and thus, the greater complexity observed in eukaryotes (Lane and Martin 2010). Mitochondrial processes are required not only for the maintenance of cellular homeostasis, but also allow cell to cell and tissue to tissue communication (Nunnari and Suomalainen 2012). Mitochondrial phospholipids are important components of this system. Phospholipids make up the characteristic outer and inner membranes that give mitochondria their shape. In addition, these membranes house sterols, sphingolipids and a wide variety of proteins. It is the phospholipids that also give rise to other characteristic mitochondrial structures such as cristae (formed from the invaginations of the inner mitochondrial membrane), the matrix (area within cristae) and the intermembrane space (IMS) which separates the outer mitochondrial membrane (OMM) and inner mitochondrial membrane (IMM). Phospholipids are the building blocks that make up these structures. However, the phospholipid composition of the OMM and IMM is unique in each membrane. Mitochondria are able to synthesize some of the phospholipids it requires, but the majority of cellular lipid biosynthesis takes place in the endoplasmic reticulum (ER) in conjunction with the Golgi apparatus (Fagone and Jackowski 2009). In this review, we will focus on the role that mitochondrial phospholipids play in specific cellular functions and discuss their biosynthesis, metabolism and transport as well as the differences between the OMM and IMM phospholipid composition. Finally, we will focus on the human diseases that result from disturbances to mitochondrial phospholipids and the current research being performed to help

  13. Kaempferol ameliorates aflatoxin B1 (AFB1) induced hepatocellular carcinoma through modifying metabolizing enzymes, membrane bound ATPases and mitochondrial TCA cycle enzymes

    Institute of Scientific and Technical Information of China (English)

    Kulanthaivel Langeswaran; Rajendran Revathy; Subbaraj Gowtham Kumar; Shanmugam Vijayaprakash

    2012-01-01

    Objective: The present study was aimed to scrutinize the anticancer consequence of kaempferol against aflatoxin B1 induced hepatocarcinogenesis. Epidemiological studies of the incidence of liver cancer in the population, where dietary aflatoxin exposure is high, have provided much circumstantial evidence for the development of aflatoxin B1 induced primary liver cancer in humans. Methods:In the present investigation, aflatoxin B1 (2 mg/kg body weight i.p) was used as a hepatocarcinogen to induce hepatocellular carcinoma in experimental animals. Results: In the present analysis, on treatment with bioflavonoid kaempferol (100 mg/kg body weight p.o) the nucleic acids levels were brought back to normal and also the altered levels of biological enzymes such as membrane bound ATPase, carbohydrate metabolizing enzymes and mitochondrial TCA cycle enzymes levels (P<0.01).Conclusions:Membrane bound ATPase, carbohydrate metabolizing enzymes and mitochondrial TCA cycle enzymes were modulated by kaempferol evaluated on aflatoxin B1 induced primary liver carcinogenesis.

  14. Conformation-dependent accessibility of Cys-136 and Cys-155 of the mitochondrial rat carnitine/acylcarnitine carrier to membrane-impermeable SH reagents.

    Science.gov (United States)

    Giangregorio, Nicola; Tonazzi, Annamaria; Indiveri, Cesare; Palmieri, Ferdinando

    2007-11-01

    During substrate translocation mitochondrial carriers cycle between the cytoplasmic-state (c-state) with substrate-binding site open to the intermembrane space and matrix-state (m-state) with the binding site open to the mitochondrial matrix. Here, the accessibility of Cys-58, Cys-136 and Cys-155 of the rat mitochondrial carnitine/acylcarnitine carrier (CAC) to membrane-impermeable SH reagents was examined as a function of the conformational state. Reconstituted mutant CACs containing the combinations Cys-58/Cys-136, Cys-58/Cys-155, and Cys-136/Cys-155 transport carnitine with a ping-pong mechanism like the wild-type, since increasing substrate concentrations on one side of the membrane decreased the apparent affinity for the substrate on the other side. In view of this mechanism, the effect of SH reagents on the transport activity of mutant CACs was tested by varying the substrate concentration inside or outside the proteoliposomes, keeping the substrate concentration on the opposite side constant. The reagents MTSES, MTSEA and fluorescein-5-maleimide did not affect the carnitine/carnitine exchange activity of the mutant carrier with only Cys-58 in contrast to mutant carriers with Cys-58/Cys-136, Cys-58/Cys-155 or Cys-136/Cys-155. In the latter, the inhibitory effect of the reagents was more pronounced when the intraliposomal carnitine concentration was increased, favouring the m-state of the carrier, whereas the effect was less when the concentration of carnitine was increased in the external compartment of the proteoliposomes, favouring the c-state. Moreover, the mutant carrier proteins with Cys-136/Cys-155, Cys-58/Cys-136 or Cys-58/Cys-155 were more fluorescent when extracted from fluorescein-5-maleimide-treated proteoliposomes containing 15 mM internal carnitine as compared to 2.5 mM. These results are discussed in terms of conformational changes of the carrier occurring during substrate translocation.

  15. Short term exercise induces PGC-1α, ameliorates inflammation and increases mitochondrial membrane proteins but fails to increase respiratory enzymes in aging diabetic hearts.

    Directory of Open Access Journals (Sweden)

    Amy Botta

    Full Text Available PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα and systemic (circulating chemokines and cytokines inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

  16. Cryotolerance of stallion spermatozoa is related to ROS production and mitochondrial membrane potential rather than to the integrity of sperm nucleus.

    Science.gov (United States)

    Yeste, M; Estrada, E; Rocha, L G; Marín, H; Rodríguez-Gil, J E; Miró, J

    2015-03-01

    Although cryopreservation of stallion spermatozoa allows long-term preservation of spermatozoa from particular stallions and facilitates international trade, it is understood to inflict damages on sperm cells that may finally reduce their fertilizing ability. In addition, individual differences are known to exist in the sperm ability to withstand freeze-thawing protocols. To date, these differences have mainly been reported on the basis of sperm motility and membrane integrity. For this reason, the present work sought to determine differences between good (good freezability ejaculates: GFE) and poor (poor freezability ejaculates: PFE) freezability stallion ejaculates in other sperm parameters, including peroxide and superoxide levels, potential of mitochondrial membrane and nuclear integrity. With this purpose, a total of 24 stallion ejaculates were cryopreserved and classified into two groups (GFE vs. PFE), depending on their sperm membrane integrity and motility after freeze-thawing. From the total of 24 ejaculates, 13 were classified as GFE and the other 11 were classified as PFE. Apart from differences in sperm membrane permeability and lipid disorder after freeze-thawing, GFE presented significantly (p sperm head proteins, no significant differences between GFE and PFE were seen. We can thus conclude that good and poor freezability stallion ejaculates differ in their reactive oxygen species levels after cryopreservation, but not in the damage extent on sperm nucleus.

  17. Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and associated changes in the Ca(2+)-induced mitochondrial permeability transition and mitochondrial membrane potential in AML12 hepatocytes.

    Science.gov (United States)

    Chiu, Po Yee; Luk, Ka Fai; Leung, Hoi Yan; Ng, Ka Ming; Ko, Kam Ming

    2009-11-01

    The effects of the schisandrin B stereoisomers, (+/-)gamma-schisandrin [(+/-)gamma-Sch] and (-)schisandrin B [(-)Sch B], on hypoxia/reoxygenation-induced apoptosis were investigated in AML12 hepatocytes. Changes in cellular reduced glutathione (GSH) levels, Ca(2+)-induced mitochondrial permeability transitions (MPTs) and mitochondrial membrane potentials (Deltapsi(m) values) were also examined in (+/-)gamma-Sch- and (-)Sch B-treated cells, without or with hypoxia/reoxygenation challenge. The (+/-)gamma-Sch/(-)Sch B pretreatments (2.5-5.0 microm) protected against hypoxia/reoxygenation-induced apoptosis in AML12 cells in a concentration-dependent manner, with the (-)Sch B effect being more potent. Drug antiapoptotic effects were further evidenced by suppression of hypoxia/reoxygenation-induced mitochondrial cytochrome c release and subsequent cleavage of caspase 3 and poly-ADP-ribose polymerase by (-)Sch B pretreatment. Whereas hypoxia/reoxygenation challenge increased the extent of Ca(2+)-induced MPT pore opening, and Deltapsi(m), in AML12 hepatocytes, cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B pretreatment against hypoxia/reoxygenation-induced apoptosis was associated with a decreased sensitivity to Ca(2+)-induced MPT and an increased Deltapsi(m) in both unchallenged and challenged cells, compared with the drug-free control. The results indicate that (+/-)gamma-Sch/(-)Sch B pretreatment protected against hypoxia/reoxygenation-induced apoptosis in AML12 hepatocytes and that the cytoprotection afforded by (+/-)gamma-Sch/(-)Sch B may at least in part be mediated by a decrease in sensitivity to Ca(2+)-induced MPT, which may in turn result from enhancement of cellular GSH levels by drug pretreatments.

  18. Pharmacologic Effects on Mitochondrial Function

    Science.gov (United States)

    Cohen, Bruce H.

    2010-01-01

    The vast majority of energy necessary for cellular function is produced in mitochondria. Free-radical production and apoptosis are other critical mitochondrial functions. The complex structure, electrochemical properties of the inner mitochondrial membrane (IMM), and genetic control from both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) are…

  19. The antioxidant Trolox restores mitochondrial membrane potential and Ca2+ -stimulated ATP production in human complex I deficiency.

    NARCIS (Netherlands)

    Distelmaier, F.; Visch, H.J.; Smeitink, J.A.M.; Mayatepek, E.; Koopman, W.J.H.; Willems, P.H.G.M.

    2009-01-01

    Malfunction of mitochondrial complex I caused by nuclear gene mutations causes early-onset neurodegenerative diseases. Previous work using cultured fibroblasts of complex-I-deficient patients revealed elevated levels of reactive oxygen species (ROS) and reductions in both total Ca(2+) content of the

  20. Mitochondrial outer membrane permeabilization increases reactive oxygen species production and decreases mean sperm velocity but is not associated with DNA fragmentation in human sperm.

    Science.gov (United States)

    Treulen, F; Uribe, P; Boguen, R; Villegas, J V

    2016-02-01

    Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this study reveal that the alterations of sperm functions caused by MOMP are sufficiently relevant to justify its future study

  1. Membrane-embedded C-terminal segment of rat mitochondrial TOM40 constitutes protein-conducting pore with enriched beta-structure.

    Science.gov (United States)

    Suzuki, Hiroyuki; Kadowaki, Tomoko; Maeda, Maki; Sasaki, Hiroyuki; Nabekura, Junichi; Sakaguchi, Masao; Mihara, Katsuyoshi

    2004-11-26

    TOM40 is the central component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). We purified recombinant rat TOM40 (rTOM40), which was refolded in Brij35 after solubilization from inclusion bodies by guanidine HCl. rTOM40 (i) consisted of a 63% beta-sheet structure and (ii) bound a matrix-targeted preprotein with high affinity and partially translocated it into the rTOM40 pore. This partial translocation was inhibited by stabilization of the mature domain of the precursor. (iii) rTOM40 bound preprotein initially through ionic interactions, followed by salt-resistant non-ionic interactions, and (iv) exhibited presequence-sensitive, cation-specific channel activity in reconstituted liposomes. Based on the domain structure of rTOM40 deduced by protease treatment, we purified the elastase-resistant and membrane-embedded C-terminal segment (rTOM40(DeltaN165)) as a recombinant protein with 62% beta-structure that exhibited properties comparable with those of full-size rTOM40. We concluded that the membrane-embedded C-terminal half of rTOM40 constitutes the preprotein recognition domain with an enriched beta-structure, which forms the preprotein conducting pore containing a salt-sensitive cis-binding site and a salt-resistant trans-binding site.

  2. A short C-terminal tail prevents mis-targeting of hydrophobic mitochondrial membrane proteins to the ER.

    Science.gov (United States)

    Reithinger, Johannes H; Yim, Chewon; Park, Kwangjin; Björkholm, Patrik; von Heijne, Gunnar; Kim, Hyun

    2013-11-01

    Sdh3/Shh3, a subunit of mitochondrial succinate dehydrogenase, contains transmembrane domains with a hydrophobicity comparable to that of endoplasmic reticulum (ER) proteins. Here, we show that a C-terminal reporter fusion to Sdh3/Shh3 results in partial mis-targeting of the protein to the ER. This mis-targeting is mediated by the signal recognition particle (SRP) and depends on the length of the C-terminal tail. These results imply that if nuclear-encoded mitochondrial proteins contain strongly hydrophobic transmembrane domains and a long C-terminal tail, they have the potential to be recognized by SRP and mis-targeted to the ER. Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  3. Ruthenium(II) complexes: DNA-binding, cytotoxicity, apoptosis, cellular localization, cell cycle arrest, reactive oxygen species, mitochondrial membrane potential and western blot analysis.

    Science.gov (United States)

    Li, Wei; Jiang, Guang-Bin; Yao, Jun-Hua; Wang, Xiu-Zhen; Wang, Ji; Han, Bing-Jie; Xie, Yang-Yin; Lin, Gan-Jian; Huang, Hong-Liang; Liu, Yun-Jun

    2014-11-01

    The aim of our study was to investigate DNA-binding and cytotoxic activity of the four new Ru(II) polypyridyl complexes [Ru(dmb)₂(HMHPIP)](ClO₄)₂ (1), [Ru(bpy)₂(HMHPIP)](ClO₄)₂ (2), [Ru(phen)₂(HMHPIP)](ClO₄)₂ (3) and [Ru(dmp)₂(HMHPIP)](ClO₄)₂ (4). The complexes interact with DNA through intercalative mode and show relatively high cytotoxic activity against A549 cells, no cytotoxicity toward MG-63 cells. Complexes 1-4 can enhance the levels of ROS in A549 cells and induce the decrease of the mitochondrial membrane potential. These complexes inhibit the cell growth in A549 cells at G0/G1 or S phase. Complex 3 activated caspase 7, and down-regulated the expression of the anti-apoptotic protein Bcl-2. Complexes 1-4 induce apoptosis in A549 cells through ROS-mediated mitochondrial dysfunction pathway. Crown Copyright © 2014. Published by Elsevier B.V. All rights reserved.

  4. Low-level laser (light) therapy increases mitochondrial membrane potential and ATP synthesis in C2C12 myotubes with a peak response at 3-6 hours

    Science.gov (United States)

    Ferraresi, Cleber; Kaippert, Beatriz; Avci, Pinar; Huang, Ying-Ying; de Sousa, Marcelo Victor Pires; Bagnato, Vanderlei Salvador; Parizotto, Nivaldo Antonio; Hamblin, Michael R

    2014-01-01

    Low level laser (light) therapy has been used before exercise to increase muscle performance in both experimental animals and in humans. However uncertainty exists concerning the optimum time to apply the light before exercise. The mechanism of action is thought to be stimulation of mitochondrial respiration in muscles, and to increase adenosine triphosphate (ATP) needed to perform exercise. The goal of this study was to investigate the time course of the increases in mitochondrial membrane potential (MMP) and ATP in myotubes formed from C2C12 mouse muscle cells and exposed to light-emitting diode therapy (LEDT). LEDT employed a cluster of LEDs with 20 red (630 ± 10 nm, 25 mW) and 20 near-infrared (850 ± 10 nm, 50 mW) delivering 28 mW/cm2 for 90 sec (2.5 J/cm2) with analysis at 5 min, 3 h, 6 h and 24 h post-LEDT. LEDT-6h had the highest MMP, followed by LEDT-3h, LEDT-24h, LEDT-5min and Control with significant differences. The same order (6h>3h>24h>5min>Control) was found for ATP with significant differences. A good correlation was found (r=0.89) between MMP and ATP. These data suggest an optimum time window of 3-6 h for LEDT stimulate muscle cells. PMID:25443662

  5. Effects of the leaf decoction of Momordica charantia (bitter melon) on Mitochondrial Membrane Permeability Transition Pore (MMPTP) and fertility in normal male albino rats.

    Science.gov (United States)

    Odewusi, A F; Oyeyemi, M O; Olayemi, F O; Emikpe, B; Ehigie, L O; Adisa, R A; Olorunsogo, O O

    2010-12-01

    Momordica charantia (M. charantia), a medicinal plant of the family, Cucurbitaceae, is used in treating an array of ailments including diabetes, heamorrhoids, fevers and various cancers. Programmed cell death may be modulated by an intrinsic pathway involving the release of cytochrome C when the mitochondrial membrane permeability transition (MMPTP) pore is opened. Opening of MMPT pore was assayed using the method of Lapidus and Sokolove. The results obtained revealed that there was a dose-dependent and significant increase in the opening of the MMPT pore in rats orally administered the decoction with maximum induction (11-fold increase) at 55mg/100g body weight (bw), although the extent of opening of the pore was reduced at 65mg/100g bw (9-fold increase). An assessment of the blood parameters of animals orally exposed to the decoction showed significant decrease (pfertility in individuals who rely on oral administration of the decoction in treating various ailments.

  6. A Novel Deletion Mutation of Exon 2 of the C19orf12 Gene in an Omani Family with Mitochondrial Membrane Protein-Associated Neurodegeneration (MPAN)

    Science.gov (United States)

    Al Macki, Nabil; Al Rashdi, Ismail

    2017-01-01

    Mutations in the C19orf12 gene are known to cause mitochondrial membrane protein-associated neurodegeneration (MPAN), which is a neurodegeneration with brain iron accumulation (NBIA) type 4 disorder. To the best of our knowledge, this is the first report of a genetically confirmed case of MPAN from Oman. A novel homozygous deletion of exon 2 of the C19orf12 gene was confirmed on the proband, a seven-year-old girl, who presented with gait instability. Brain magnetic resonance imaging showed iron deposition on the basal ganglia. This report highlights the importance of genetic testing of such a clinically and genetically heterogeneous condition among a population with a high consanguinity rate. To overcome the diagnostic difficulty, implementation of a cost-effective approach to perform cascade screening of carriers at risk is needed as well as programs to address risky consanguineous marriages. PMID:28042406

  7. Adverse Effect of H2O2 Change on Morphology, Mitochondrial Membrane Permeability and Antioxidant Enzyme in Root of Dianthus Chinensis L. under Salt Stress

    Directory of Open Access Journals (Sweden)

    Xue-qin He

    2013-04-01

    Full Text Available Dianthus Chinensis L. is a salt-tolerant ornamental plant. Root is the first and critical part of plant to encounter soli salinity. In order to elucidate H2O2 impact on root morphology and mitochondrial permeability transition as well as activities of antioxidant enzymes in root ofDianthus Chinensis L., we treated seedling with H2O2 and DMTU under NaCl. The results revealed that change of H2O2 level under NaCl would negatively influence the root growth, as well as lower the value of mitochodrial membrane absorbance at 540 nm and the ratio of Cyt c/a. Meanwhile, SOD and POD under NaCl plus H2O2 and NaCl plus DMTU were far lower than those under NaCl alone.

  8. Radiosensitization by fullerene-C60 dissolved in squalene on human malignant melanoma through lipid peroxidation and enhanced mitochondrial membrane potential

    Science.gov (United States)

    Kato, Shinya; Kimura, Masatsugu; Miwa, Nobuhiko

    2014-04-01

    We examined fullerene-C60 dissolved in squalene (C60/Sqe) for the ability to potentiate the radiosensitization under X-ray irradiation on human malignant melanoma HMV-II cells, which were treated with C60/Sqe and thereafter irradiated with X-ray. The cell proliferation for C60/Sqe was inhibited more markedly than for Sqe alone. Meanwhile, cell proliferation was almost unaltered for C60/squalane (Sqa) or Sqa, a hydrogenated form of Sqe, as compared to no-additive control. Thus radiosensitization of C60/Sqe is attributed to peroxidation of unsaturated bonds of squalene by X-ray-excited C60 in contrast to squalane. The fluorescence images of HMV-II cells stained with Rhodamine123, an indicator for mitochondrial membrane potential, were monitored for 6 h after X-ray irradiation. C60/Sqe obviously exhibited more augmented fluorescence intensity on perinuclear region of HMV-II cells than Sqe alone. TBARS assay showed that the lipid peroxidation level as malondialdehyde-equivalent increased by combination of C60/Sqe and X-ray dose-dependently on X-ray doses. C60/Sqe exhibited lipid peroxidation more markedly by 1.2-fold than Sqe alone. Thus the level of lipid peroxidation of squalene was sufficiently higher in C60/Sqe than in Sqe in the absence of C60 under X-ray irradiation, suggesting the combination of C60/Sqe and X-ray irradiation induced radiosensitization on HMV-II cells by peroxidation of absorbed Sqe in mitochondrial membrane via oxidative stress mediated by fullerene-C60.

  9. Mucuna pruriens and its major constituent L-DOPA recover spermatogenic loss by combating ROS, loss of mitochondrial membrane potential and apoptosis.

    Directory of Open Access Journals (Sweden)

    Akhand Pratap Singh

    Full Text Available BACKGROUND: The Ayurvedic medicinal system claims Mucuna pruriens (MP to possess pro-male fertility, aphrodisiac and adaptogenic properties. Some scientific evidence also supports its pro-male fertility properties; however, the mechanism of its action is not yet clear. The present study aimed at demonstrating spermatogenic restorative efficacy of MP and its major constituent L-DOPA (LD, and finding the possible mechanism of action thereof in a rat model. METHODOLOGY/FINDINGS: Ethinyl estradiol (EE was administered at a rate of 3 mg/kg body weight (BW/day for a period of 14 days to generate a rat model with compromised spermatogenesis. MP and LD were administered in two separate groups of these animals starting 15(th day for a period of 56 days, and the results were compared with an auto-recovery (AR group. Sperm count and motility, testis histo-architecture, level of reactive oxygen species (ROS, mitochondrial membrane potential (MMP, apoptosis, peripheral hormone levels and testicular germ cell populations were analysed, in all experimental groups. We observed efficient and quick recovery of spermatogenesis in MP and LD groups in comparison to the auto-recovery group. The treatment regulated ROS level, apoptosis, and mitochondrial membrane potential (MMP, recovered the hypothalamic-pituitary-gonadal axis and the number of testicular germ cells, ultimately leading to increased sperm count and motility. CONCLUSION/SIGNIFICANCE: M. pruriens efficiently recovers the spermatogenic loss induced due to EE administration. The recovery is mediated by reduction in ROS level, restoration of MMP, regulation of apoptosis and eventual increase in the number of germ cells and regulation of apoptosis. The present study simplified the complexity of mechanism involved and provided meaningful insights into MP/LD mediated correction of spermatogenic impairment caused by estrogens exposure. This is the first study demonstrating that L-DOPA largely accounts for pro

  10. After Embedding in Membranes Antiapoptotic Bcl-XL Protein Binds Both Bcl-2 Homology Region 3 and Helix 1 of Proapoptotic Bax Protein to Inhibit Apoptotic Mitochondrial Permeabilization*

    Science.gov (United States)

    Ding, Jingzhen; Mooers, Blaine H. M.; Zhang, Zhi; Kale, Justin; Falcone, Domina; McNichol, Jamie; Huang, Bo; Zhang, Xuejun C.; Xing, Chengguo; Andrews, David W.; Lin, Jialing

    2014-01-01

    Bcl-XL binds to Bax, inhibiting Bax oligomerization required for mitochondrial outer membrane permeabilization (MOMP) during apoptosis. How Bcl-XL binds to Bax in the membrane is not known. Here, we investigated the structural organization of Bcl-XL·Bax complexes formed in the MOM, including the binding interface and membrane topology, using site-specific cross-linking, compartment-specific labeling, and computational modeling. We found that one heterodimer interface is formed by a specific interaction between the Bcl-2 homology 1–3 (BH1–3) groove of Bcl-XL and the BH3 helix of Bax, as defined previously by the crystal structure of a truncated Bcl-XL protein and a Bax BH3 peptide (Protein Data Bank entry 3PL7). We also discovered a novel interface in the heterodimer formed by equivalent interactions between the helix 1 regions of Bcl-XL and Bax when their helical axes are oriented either in parallel or antiparallel. The two interfaces are located on the cytosolic side of the MOM, whereas helix 9 of Bcl-XL is embedded in the membrane together with helices 5, 6, and 9 of Bax. Formation of the helix 1·helix 1 interface partially depends on the formation of the groove·BH3 interface because point mutations in the latter interface and the addition of ABT-737, a groove-binding BH3 mimetic, blocked the formation of both interfaces. The mutations and ABT-737 also prevented Bcl-XL from inhibiting Bax oligomerization and subsequent MOMP, suggesting that the structural organization in which interactions at both interfaces contribute to the overall stability and functionality of the complex represents antiapoptotic Bcl-XL·Bax complexes in the MOM. PMID:24616095

  11. Functional Diversity of Human Mitochondrial J-proteins Is Independent of Their Association with the Inner Membrane Presequence Translocase.

    Science.gov (United States)

    Sinha, Devanjan; Srivastava, Shubhi; D'Silva, Patrick

    2016-08-12

    Mitochondrial J-proteins play a critical role in governing Hsp70 activity and, hence, are essential for organellar protein translocation and folding. In contrast to yeast, which has a single J-protein Pam18, humans involve two J-proteins, DnaJC15 and DnaJC19, associated with contrasting cellular phenotype, to transport proteins into the mitochondria. Mutation in DnaJC19 results in dilated cardiomyopathy and ataxia syndrome, whereas expression of DnaJC15 regulates the response of cancer cells to chemotherapy. In the present study we have comparatively assessed the biochemical properties of the J-protein paralogs in relation to their association with the import channel. Both DnaJC15 and DnaJC19 formed two distinct subcomplexes with Magmas at the import channel. Knockdown analysis suggested an essential role for Magmas and DnaJC19 in organellar protein translocation and mitochondria biogenesis, whereas DnaJC15 had dispensable supportive function. The J-proteins were found to have equal affinity for Magmas and could stimulate mitochondrial Hsp70 ATPase activity by equivalent levels. Interestingly, we observed that DnaJC15 exhibits bifunctional properties. At the translocation channel, it involves conserved interactions and mechanism to translocate the precursors into mitochondria. In addition to protein transport, DnaJC15 also showed a dual role in yeast where its expression elicited enhanced sensitivity of cells to cisplatin that required the presence of a functional J-domain. The amount of DnaJC15 expressed in the cell was directly proportional to the sensitivity of cells. Our analysis indicates that the differential cellular phenotype displayed by human mitochondrial J-proteins is independent of their activity and association with Magmas at the translocation channel.

  12. Mitochondrial cholesterol: mechanisms of import and effects on mitochondrial function.

    Science.gov (United States)

    Martin, Laura A; Kennedy, Barry E; Karten, Barbara

    2016-04-01

    Mitochondria require cholesterol for biogenesis and membrane maintenance, and for the synthesis of steroids, oxysterols and hepatic bile acids. Multiple pathways mediate the transport of cholesterol from different subcellular pools to mitochondria. In steroidogenic cells, the steroidogenic acute regulatory protein (StAR) interacts with a mitochondrial protein complex to mediate cholesterol delivery to the inner mitochondrial membrane for conversion to pregnenolone. In non-steroidogenic cells, several members of a protein family defined by the presence of a StAR-related lipid transfer (START) domain play key roles in the delivery of cholesterol to mitochondrial membranes. Subdomains of the endoplasmic reticulum (ER), termed mitochondria-associated ER membranes (MAM), form membrane contact sites with mitochondria and may contribute to the transport of ER cholesterol to mitochondria, either independently or in conjunction with lipid-transfer proteins. Model systems of mitochondria enriched with cholesterol in vitro and mitochondria isolated from cells with (patho)physiological mitochondrial cholesterol accumulation clearly demonstrate that mitochondrial cholesterol levels affect mitochondrial function. Increased mitochondrial cholesterol levels have been observed in several diseases, including cancer, ischemia, steatohepatitis and neurodegenerative diseases, and influence disease pathology. Hence, a deeper understanding of the mechanisms maintaining mitochondrial cholesterol homeostasis may reveal additional targets for therapeutic intervention. Here we give a brief overview of mitochondrial cholesterol import in steroidogenic cells, and then focus on cholesterol trafficking pathways that deliver cholesterol to mitochondrial membranes in non-steroidogenic cells. We also briefly discuss the consequences of increased mitochondrial cholesterol levels on mitochondrial function and their potential role in disease pathology.

  13. Inhibition of N-Methyl-D-aspartate-induced Retinal Neuronal Death by Polyarginine Peptides Is Linked to the Attenuation of Stress-induced Hyperpolarization of the Inner Mitochondrial Membrane Potential.

    Science.gov (United States)

    Marshall, John; Wong, Kwoon Y; Rupasinghe, Chamila N; Tiwari, Rakesh; Zhao, Xiwu; Berberoglu, Eren D; Sinkler, Christopher; Liu, Jenney; Lee, Icksoo; Parang, Keykavous; Spaller, Mark R; Hüttemann, Maik; Goebel, Dennis J

    2015-09-01

    It is widely accepted that overactivation of NMDA receptors, resulting in calcium overload and consequent mitochondrial dysfunction in retinal ganglion neurons, plays a significant role in promoting neurodegenerative disorders such as glaucoma. Calcium has been shown to initiate a transient hyperpolarization of the mitochondrial membrane potential triggering a burst of reactive oxygen species leading to apoptosis. Strategies that enhance cell survival signaling pathways aimed at preventing this adverse hyperpolarization of the mitochondrial membrane potential may provide a novel therapeutic intervention in retinal disease. In the retina, brain-derived neurotrophic factor has been shown to be neuroprotective, and our group previously reported a PSD-95/PDZ-binding cyclic peptide (CN2097) that augments brain-derived neurotrophic factor-induced pro-survival signaling. Here, we examined the neuroprotective properties of CN2097 using an established retinal in vivo NMDA toxicity model. CN2097 completely attenuated NMDA-induced caspase 3-dependent and -independent cell death and PARP-1 activation pathways, blocked necrosis, and fully prevented the loss of long term ganglion cell viability. Although neuroprotection was partially dependent upon CN2097 binding to the PDZ domain of PSD-95, our results show that the polyarginine-rich transport moiety C-R(7), linked to the PDZ-PSD-95-binding cyclic peptide, was sufficient to mediate short and long term protection via a mitochondrial targeting mechanism. C-R(7) localized to mitochondria and was found to reduce mitochondrial respiration, mitochondrial membrane hyperpolarization, and the generation of reactive oxygen species, promoting survival of retinal neurons.

  14. Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry.

    Science.gov (United States)

    Krumschnabel, Gerhard; Eigentler, Andrea; Fasching, Mario; Gnaiger, Erich

    2014-01-01

    The mitochondrial transmembrane potential (Δψmt or mtMP) is directly influenced by oxidative phosphorylation (OXPHOS). The exact nature of the interactions between respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. Here, we describe a combination of high-resolution respirometry and fluorometry based on the OROBOROS Oxygraph-2k and the widely applied mtMP indicator safranin. The analysis of OXPHOS in mouse brain homogenates revealed that, at commonly applied concentrations, safranin inhibits Complex I-driven OXPHOS capacity, primarily targeting the phosphorylation system, but has no effects on LEAK respiration. Conversely, Complex II-driven OXPHOS capacity was inhibited by safranin concentrations normally used for mtMP monitoring. The mtMP was higher in the LEAK state without adenylates than at identical LEAK respiration after ADP stimulation and Complex V inhibition with oligomycin. The maximal electron transfer system (ETS) capacity was reached in uncoupler titrations before the mtMP fully collapsed, whereas respiration was inhibited at increasing uncoupler concentrations, resulting in the progressive reduction of mtMP. In a pharmacologically induced state of Complex II dysfunction, mtMP was rather insensitive to the inhibition of OXPHOS to 50% of its normal capacity, but robustly responded to inhibitors when respiration was limited by substrate depletion. The optimal concentration of uncoupler supporting maximal ETS capacity varied as a function of pharmacological intervention. Taken together, the combined measurement of respiration and mtMP greatly enhances the informative potential of OXPHOS studies. The respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore employed to assess mtMP in any respiratory state, tissue type, and pathophysiological condition. The methodological issues analyzed herein are relevant for the

  15. Flux control exerted by mitochondrial outer membrane carnitine palmitoyltransferase over beta-oxidation, ketogenesis and tricarboxylic acid cycle activity in hepatocytes isolated from rats in different metabolic states.

    Science.gov (United States)

    Drynan, L; Quant, P A; Zammit, V A

    1996-08-01

    The Flux Control Coefficients of mitochondrial outer membrane carnitine palmitoyltransferase (CPT I) with respect to the overall rates of beta-oxidation, ketogenesis and tricarboxylic acid cycle activity were measured in hepatocytes isolated from rats in different metabolic states (fed, 24 h-starved, starved-refed and starved/insulin-treated). These conditions were chosen because there is controversy as to whether, when significant control ceases to be exerted by CPT I over the rate of fatty oxidation [Moir and Zammit (1994) Trends Biochem. Sci. 19, 313-317], this is transferred to one or more steps proximal to acylcarnitine synthesis (e.g. decreased delivery of fatty acids to the liver) or to the reaction catalysed by mitochondrial 3-hydroxy-3-methyl-glutaryl-CoA synthase [Hegardt (1995) Biochem. Soc. Trans. 23, 486-490]. Therefore isolated hepatocytes were used in the present study to exclude the involvement of changes in the rate of delivery of non-esterified fatty acids (NEFA) to the liver, such as occur in vivo, and to ascertain whether, under conditions of constant supply of NEFA, CPT I retains control over the relevant fluxes of fatty acid oxidation to ketones and carbon dioxide, or whether control is transferred to another (intrahepatocytic) site. The results clearly show that the Flux Control Coefficients of CPT I with respect to overall beta-oxidation and ketogenesis are very high under all conditions investigated, indicating that control is not lost to another intrahepatic site during the metabolic transitions studied. The control of CPT I over tricarboxylic acid cycle activity was always very low. The significance of these findings for the integration of fatty acid and carbohydrate metabolism in the liver is discussed.

  16. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

    He, J; Cooper, H M; Reyes, A; Di Re, M; Sembongi, H; Litwin, T R; Gao, J; Neuman, K C; Fearnley, I M; Spinazzola, A; Walker, J E; Holt, I J

    2012-07-01

    Mitochondrial ribosomes and translation factors co-purify with mitochondrial nucleoids of human cells, based on affinity protein purification of tagged mitochondrial DNA binding proteins. Among the most frequently identified proteins were ATAD3 and prohibitin, which have been identified previously as nucleoid components, using a variety of methods. Both proteins are demonstrated to be required for mitochondrial protein synthesis in human cultured cells, and the major binding partner of ATAD3 is the mitochondrial ribosome. Altered ATAD3 expression also perturbs mtDNA maintenance and replication. These findings suggest an intimate association between nucleoids and the machinery of protein synthesis in mitochondria. ATAD3 and prohibitin are tightly associated with the mitochondrial membranes and so we propose that they support nucleic acid complexes at the inner membrane of the mitochondrion.

  17. A Metabotropic-Like Flux-Independent NMDA Receptor Regulates Ca2+ Exit from Endoplasmic Reticulum and Mitochondrial Membrane Potential in Cultured Astrocytes

    Science.gov (United States)

    Montes de Oca Balderas, Pavel; Aguilera, Penélope

    2015-01-01

    Astrocytes were long thought to be only structural cells in the CNS; however, their functional properties support their role in information processing and cognition. The ionotropic glutamate N-methyl D-aspartate (NMDA) receptor (NMDAR) is critical for CNS functions, but its expression and function in astrocytes is still a matter of research and debate. Here, we report immunofluorescence (IF) labeling in rat cultured cortical astrocytes (rCCA) of all NMDAR subunits, with phenotypes suggesting their intracellular transport, and their mRNA were detected by qRT-PCR. IF and Western Blot revealed GluN1 full-length synthesis, subunit critical for NMDAR assembly and transport, and its plasma membrane localization. Functionally, we found an iCa2+ rise after NMDA treatment in Fluo-4-AM labeled rCCA, an effect blocked by the NMDAR competitive inhibitors D(-)-2-amino-5-phosphonopentanoic acid (APV) and Kynurenic acid (KYNA) and dependent upon GluN1 expression as evidenced by siRNA knock down. Surprisingly, the iCa2+ rise was not blocked by MK-801, an NMDAR channel blocker, or by extracellular Ca2+ depletion, indicating flux-independent NMDAR function. In contrast, the IP3 receptor (IP3R) inhibitor XestosponginC did block this response, whereas a Ryanodine Receptor inhibitor did so only partially. Furthermore, tyrosine kinase inhibition with genistein enhanced the NMDA elicited iCa2+ rise to levels comparable to those reached by the gliotransmitter ATP, but with different population dynamics. Finally, NMDA depleted the rCCA mitochondrial membrane potential (mΔψ) measured with JC-1. Our results demonstrate that rCCA express NMDAR subunits which assemble into functional receptors that mediate a metabotropic-like, non-canonical, flux-independent iCa2+ increase. PMID:25954808

  18. Molecular basis for mitochondrial signaling

    CERN Document Server

    2017-01-01

    This book covers recent advances in the study of structure, function, and regulation of metabolite, protein and ion translocating channels, and transporters in mitochondria. A wide array of cutting-edge methods are covered, ranging from electrophysiology and cell biology to bioinformatics, as well as structural, systems, and computational biology. At last, the molecular identity of two important channels in the mitochondrial inner membrane, the mitochondrial calcium uniporter and the mitochondrial permeability transition pore have been established. After years of work on the physiology and structure of VDAC channels in the mitochondrial outer membrane, there have been multiple discoveries on VDAC permeation and regulation by cytosolic proteins. Recent breakthroughs in structural studies of the mitochondrial cholesterol translocator reveal a set of novel unexpected features and provide essential clues for defining therapeutic strategies. Molecular Basis for Mitochondrial Signaling covers these and many more re...

  19. The dose-dependent effect of zearalenone on mitochondrial metabolism, plasma membrane permeabilization and cell cycle in human prostate cancer cell lines.

    Science.gov (United States)

    Kowalska, Karolina; Habrowska-Górczyńska, Dominika Ewa; Domińska, Kamila; Piastowska-Ciesielska, Agnieszka Wanda

    2017-08-01

    Zearalenone (ZEA) is a nonsteroidal mycotoxin produced by several fungi of the genus Fusarium spp. It is known to play various roles in the regulation of the prostate cancer cell cycle, including carcinogenesis. The present study evaluates the influence of ZEA on the mitochondrial metabolism, plasma membrane permeabilization and cell cycle of prostate cancer cells. At concentrations of 100 nM and 0.3 nM, ZEA caused a decrease in the oxidative activity of mitochondria, as well as increases in LDH release, apoptosis induction and the number of cells in the G0/G1 phase. The opposite effect was observed for lower concentrations (0.1 nM and 0.001 nM). These in vitro studies indicate that ZEA might have pro- and antiproliferative properties in prostate cancer cells, at concentrations 0.1 nM, 0.001 nM and 0.3 nM, 100 nM, respectively. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Kinetic activity, membrane mitochondrial potential, lipid peroxidation, intracellular pH and calcium of frozen/thawed bovine spermatozoa treated with metabolic enhancers.

    Science.gov (United States)

    Boni, R; Gallo, A; Cecchini, S

    2017-01-01

    Owing to the progressive decline of sperm motility during storage there is a need to find substances capable of enhancing sperm energy metabolism and motility and/or preserving it from oxidative damage. The aim of this study was to evaluate in frozen/thawed bovine spermatozoa the effect of several compounds, such as myo-inositol, pentoxifylline, penicillamine + hypotaurine + epinephrine mixture (PHE), caffeine and coenzyme Q10+ zinc + d-aspartate mixture (CZA), on either kinetic or metabolic parameters. Sperm kinetics was evaluated by Sperm Class Analyser whereas specific fluorochromes were used to evaluated mitochondrial membrane potential (MMP), intracellular pH, intracellular calcium concentration and lipid peroxidation. Lipid peroxidation was also evaluated by TBARS analysis. Treatments significantly affected total and progressive motility with different dynamics in relation to the incubation time. After the first hour of incubation, CZA treatment produced the best performance in total and progressive sperm motility as well as in curvilinear velocity, average path velocity and amplitude of head displacement, whereas pentoxifylline stimulated the highest straight-line velocity. MMP showed higher values (p kinetic and metabolic parameters. These findings provide new comparative information on the effects of putative metabolic enhancers on kinetics and metabolic activities of bovine spermatozoa. In this study, a rapid methodological approach for evaluating sperm quality is proposed. © 2016 American Society of Andrology and European Academy of Andrology.

  1. THC (Δ9-Tetrahydrocannabinol) Exerts Neuroprotective Effect in Glutamate-affected Murine Primary Mesencephalic Cultures Through Restoring Mitochondrial Membrane Potential and Anti-apoptosis Involving CB1 Receptor-dependent Mechanism.

    Science.gov (United States)

    Nguyen, Chi Huu; Krewenka, Christopher; Radad, Khaled; Kranner, Barbara; Huber, Alexandra; Duvigneau, Johanna Catharina; Miller, Ingrid; Moldzio, Rudolf

    2016-12-01

    Aging-related neurodegenerative diseases, such as Parkinson's disease (PD) or related disorders, are an increasing societal and economic burden worldwide. Δ9-Tetrahydrocannabinol (THC) is discussed as a neuroprotective agent in several in vitro and in vivo models of brain injury. However, the mechanisms by which THC exhibits neuroprotective properties are not completely understood. In the present study, we investigated neuroprotective mechanisms of THC in glutamate-induced neurotoxicity in primary murine mesencephalic cultures, as a culture model for PD. Glutamate was administered for 48 h with or without concomitant THC treatment. Immunocytochemistry staining and resazurin assay were used to evaluate cell viability. Furthermore, superoxide levels, caspase-3 activity, and mitochondrial membrane potential were determined to explore the mode of action of this compound. THC protected dopaminergic neurons and other cell types of primary dissociated cultures from glutamate-induced neurotoxicity. Moreover, THC significantly counteracted the glutamate-induced mitochondrial membrane depolarization and apoptosis. SR141716A, a CB1 receptor antagonist, concentration-dependently blocked the protective effect of THC in primary mesencephalic cultures. In conclusion, THC exerts anti-apoptotic and restores mitochondrial membrane potential via a mechanism dependent on CB1 receptor. It strengthens the fact that THC has a benefit on degenerative cellular processes occurring, among others, in PD and other neurodegenerative diseases by slowing down the progression of neuronal cell death. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Mitochondrial Dynamics in Mitochondrial Diseases

    Directory of Open Access Journals (Sweden)

    Juan M. Suárez-Rivero

    2016-12-01

    Full Text Available Mitochondria are very versatile organelles in continuous fusion and fission processes in response to various cellular signals. Mitochondrial dynamics, including mitochondrial fission/fusion, movements and turnover, are essential for the mitochondrial network quality control. Alterations in mitochondrial dynamics can cause neuropathies such as Charcot-Marie-Tooth disease in which mitochondrial fusion and transport are impaired, or dominant optic atrophy which is caused by a reduced mitochondrial fusion. On the other hand, mitochondrial dysfunction in primary mitochondrial diseases promotes reactive oxygen species production that impairs its own function and dynamics, causing a continuous vicious cycle that aggravates the pathological phenotype. Mitochondrial dynamics provides a new way to understand the pathophysiology of mitochondrial disorders and other diseases related to mitochondria dysfunction such as diabetes, heart failure, or Hungtinton’s disease. The knowledge about mitochondrial dynamics also offers new therapeutics targets in mitochondrial diseases.

  3. Mitochondrial biosensors.

    Science.gov (United States)

    De Michele, Roberto; Carimi, Francesco; Frommer, Wolf B

    2014-03-01

    Biosensors offer an innovative tool for measuring the dynamics of a wide range of metabolites in living organisms. Biosensors are genetically encoded, and thus can be specifically targeted to specific compartments of organelles by fusion to proteins or targeting sequences. Mitochondria are central to eukaryotic cell metabolism and present a complex structure with multiple compartments. Over the past decade, genetically encoded sensors for molecules involved in energy production, reactive oxygen species and secondary messengers have helped to unravel key aspects of mitochondrial physiology. To date, sensors for ATP, NADH, pH, hydrogen peroxide, superoxide anion, redox state, cAMP, calcium and zinc have been used in the matrix, intermembrane space and in the outer membrane region of mitochondria of animal and plant cells. This review summarizes the different types of sensors employed in mitochondria and their main limits and advantages, and it provides an outlook for the future application of biosensor technology in studying mitochondrial biology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. Grape seed extract induces apoptotic death of human prostate carcinoma DU145 cells via caspases activation accompanied by dissipation of mitochondrial membrane potential and cytochrome c release.

    Science.gov (United States)

    Agarwal, Chapla; Singh, Rana P; Agarwal, Rajesh

    2002-11-01

    Grape seed extract (GSE), rich in the bioflavonoids commonly known as procyanidins, is one of the most commonly consumed dietary supplements in the United States because of its several health benefits. Epidemiological studies show that many prostate cancer (PCA) patients use herbal extracts as dietary supplements in addition to their prescription drugs. Accordingly, in recent years, we have focused our attention on assessing the efficacy of GSE against PCA. Our studies showed that GSE inhibits growth and induces apoptotic death of human PCA cells in culture and in nude mice. Here, we performed detailed studies to define the molecular mechanism of GSE-induced apoptosis in advanced human PCA DU145 cells. GSE treatment of cells at various doses (50-200 micro g/ml) for 12-72 h resulted in a moderate to strong apoptotic death in a dose- and time-dependent manner. In the studies assessing the apoptotic-signaling pathway induced by GSE, we observed an increase in cleaved fragments of caspases 3, 7 and 9 as well as PARP in GSE-treated cells after 48 and 72 h of treatment. Pre-treatment of cells with general caspases inhibitor, z-Val-Ala-Asp(OMe)-FMK or caspase 3-like proteases inhibitor [z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-FMK], almost completely (approximately 90%) inhibited the GSE-induced apoptotic cell death. In a later case, GSE-induced caspase-3 activity was completely inhibited. Selective caspase 9 inhibitor [z-Leu-Glu(OMe)-His-Asp(OMe)-FMK] showed only partial inhibition of GSE-induced apoptosis whereas GSE-induced protease activity of caspase 9 was completely inhibited. Upstream of caspase cascade, GSE showed disappearance of mitochondrial membrane potential and an increase in cytochrome c release in cytosol. Together, these results suggest that GSE possibly causes mitochondrial damage leading to cytochrome c release in cytosol and activation of caspases resulting in PARP cleavage and execution of apoptotic death of human PCA DU145 cells. Furthermore, GSE

  5. BH3 domains of BH3-only proteins differentially regulate Bax-mediated mitochondrial membrane permeabilization both directly and indirectly.

    Science.gov (United States)

    Kuwana, Tomomi; Bouchier-Hayes, Lisa; Chipuk, Jerry E; Bonzon, Christine; Sullivan, Barbara A; Green, Douglas R; Newmeyer, Donald D

    2005-02-18

    Using a Bax-dependent membrane-permeabilization assay, we show that peptides corresponding to the BH3 domains of Bcl-2 family "BH3-only" proteins have dual functions. Several BH3 peptides relieved the inhibition of Bax caused by the antiapoptotic Bcl-x(L) and/or Mcl-1 proteins, some displaying a specificity for either Bcl-x(L) or Mcl-1. Besides having this derepression function, the Bid and Bim peptides activated Bax directly and were the only BH3 peptides tested that could potently induce cytochrome c release from mitochondria in cultured cells. Furthermore, Bax activator molecules (cleaved Bid protein and the Bim BH3 peptide) synergistically induced cytochrome c release when introduced into cells along with derepressor BH3 peptides. These observations support a unified model of BH3 domain function, encompassing both positive and negative regulation of other Bcl-2 family members. In this model, the simple inhibition of antiapoptotic functions is insufficient to induce apoptosis unless a direct activator of Bax or Bak is present.

  6. Mitochondrial small conductance SK2 channels prevent glutamate-induced oxytosis and mitochondrial dysfunction.

    Science.gov (United States)

    Dolga, Amalia M; Netter, Michael F; Perocchi, Fabiana; Doti, Nunzianna; Meissner, Lilja; Tobaben, Svenja; Grohm, Julia; Zischka, Hans; Plesnila, Nikolaus; Decher, Niels; Culmsee, Carsten

    2013-04-12

    Small conductance calcium-activated potassium (SK2/K(Ca)2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K(+) currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction.

  7. Mitochondrial Small Conductance SK2 Channels Prevent Glutamate-induced Oxytosis and Mitochondrial Dysfunction*

    Science.gov (United States)

    Dolga, Amalia M.; Netter, Michael F.; Perocchi, Fabiana; Doti, Nunzianna; Meissner, Lilja; Tobaben, Svenja; Grohm, Julia; Zischka, Hans; Plesnila, Nikolaus; Decher, Niels; Culmsee, Carsten

    2013-01-01

    Small conductance calcium-activated potassium (SK2/KCa2.2) channels are known to be located in the neuronal plasma membrane where they provide feedback control of NMDA receptor activity. Here, we provide evidence that SK2 channels are also located in the inner mitochondrial membrane of neuronal mitochondria. Patch clamp recordings in isolated mitoplasts suggest insertion into the inner mitochondrial membrane with the C and N termini facing the intermembrane space. Activation of SK channels increased mitochondrial K+ currents, whereas channel inhibition attenuated these currents. In a model of glutamate toxicity, activation of SK2 channels attenuated the loss of the mitochondrial transmembrane potential, blocked mitochondrial fission, prevented the release of proapoptotic mitochondrial proteins, and reduced cell death. Neuroprotection was blocked by specific SK2 inhibitory peptides and siRNA targeting SK2 channels. Activation of mitochondrial SK2 channels may therefore represent promising targets for neuroprotective strategies in conditions of mitochondrial dysfunction. PMID:23430260

  8. Recombinant Mitochondrial Transcription Factor A with N-terminal Mitochondrial Transduction Domain Increases Respiration and Mitochondrial Gene Expression

    OpenAIRE

    Iyer, Shilpa; Thomas, Ravindar R.; Portell, Francisco R.; Dunham, Lisa D.; Quigley, Caitlin K.; Bennett, James P

    2009-01-01

    We developed a scalable procedure to produce human mitochondrial transcription factor A (TFAM) modified with an N-terminal protein transduction domain (PTD) and mitochondrial localization signal (MLS) that allow it to cross membranes and enter mitochondria through its “mitochondrial transduction domain” (MTD=PTD+MLS). Alexa488-labeled MTD-TFAM rapidly entered the mitochondrial compartment of cybrid cells carrying the G11778A LHON mutation. MTD-TFAM reversibly increased respiration and levels ...

  9. Binding of FUNDC1 with Inositol 1,4,5-Trisphosphate Receptor in Mitochondria-Associated Endoplasmic Reticulum (ER) Membranes Maintains Mitochondrial Dynamics and Function in Hearts In Vivo.

    Science.gov (United States)

    Wu, Shengnan; Lu, Qiulun; Wang, Qilong; Ding, Ye; Ma, Zejun; Mao, Xiaoxiang; Huang, Kai; Xie, Zhonglin; Zou, Ming-Hui

    2017-09-23

    Background -FUN14 domain containing 1 (FUNDC1) is a highly conserved outer mitochondrial membrane protein. The aim of this study is to examine if FUNDC1 modulates the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), mitochondrial morphology, and function in cardiomyocytes and in intact hearts. Methods -The impacts of FUNDC1 on MAMs formation and cardiac functions were studied in mouse neonatal cardiomyocytes, in mice with cardiomyocyte-specific Fundc1 gene knockout (Fundc1(f/Y)/Cre(αMyHC+/-) ), and in the cardiac tissues of the patients with heart failure. Results -In mouse neonatal cardiomyocytes and intact hearts, FUNDC1 was localized in MAMs by binding to ER-resided inositol 1,4,5-trisphosphate type 2 receptor (IP3R2). Fundc1 ablation disrupted MAMs, reduced the levels of IP3R2 and Ca(2+) in both mitochondria and cytosol whereas overexpression of Fundc1 increased the levels of IP3R2 and Ca(2+) in both mitochondria and cytosol. Consistently, Fundc1 ablation increased Ca(2+) levels in ER whereas Fundc1 overexpression lowered ER Ca(2+) levels. Further, Fundc1 ablation in cardiomyocytes elongated mitochondria, and compromised mitochondrial functions. Mechanistically, we found that Fundc1 ablation-induced reduction of intracellular Ca(2+) levels suppressed mitochondrial fission 1 protein (Fis1) expression and mitochondrial fission by reducing the binding of the cAMP response element binding protein (CREB) in the Fis1 promoter. Fundc1(f/Y)/Cre(αMyHC+/-) mice but not their littermate control mice (Fundc1(wt/Y)/Cre(αMyHC+/-)) exhibited cardiac dysfunction. The ligation of the left ventricle artery of Fundc1(f/Y)/Cre(αMyHC+/-) mice caused more severe cardiac dysfunction than those in sham-treated Fundc1(f/Y)/Cre(αMyHC+/-) mice. Finally, we found that the FUNDC1/MAMs/CREB/Fis1 signaling axis was significantly suppressed in the patients with heart failure. Conclusions -We conclude that FUNDC1 binds to IP3R2 to modulate ER Ca(2+) release into

  10. Removal of spermatozoa with externalized phosphatidylserine from sperm preparation in human assisted medical procreation: effects on viability, motility and mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    de Agostini Ariane

    2009-01-01

    Full Text Available Abstract Background Externalization of phosphatidylserine (EPS occurs in apoptotic-like spermatozoa and could be used to remove them from sperm preparations to enhance sperm quality for assisted medical procreation. We first characterized EPS in sperms from infertile patients in terms of frequency of EPS spermatozoa as well as localization of phosphatidylserine (PS on spermatozoa. Subsequently, we determined the impact of depleting EPS spermatozoa on sperm quality. Methods EPS were visualized by fluorescently-labeled annexin V binding assay. Double staining with annexin V and Hoechst differentiates apoptotic from necrotic spermatozoa. We used magnetic-activated cell sorting using annexin V-conjugated microbeads (MACS-ANMB technique to remove EPS spermatozoa from sperm prepared by density gradient centrifugation (DGC. The impact of this technique on sperm quality was evaluated by measuring progressive motility, viability, and the integrity of the mitochondrial membrane potential (MMP by Rhodamine 123. Results Mean percentages of EPS spermatozoa were 14% in DGC sperm. Four subpopulations of spermatozoa were identified: 70% alive, 3% early apoptotic, 16% necrotic and 11% late apoptotic or necrotic. PS were localized on head and/or midpiece or on the whole spermatozoa. MACS efficiently eliminates EPS spermatozoa. MACS combined with DGC allows a mean reduction of 70% in EPS and of 60% in MMP-disrupted spermatozoa with a mean increase of 50% in sperm survival at 24 h. Conclusion Human ejaculates contain EPS spermatozoa which can mostly be eliminated by DGC plus MACS resulting in improved sperm long term viability, motility and MMP integrity. EPS may be used as an indicator of sperm quality and removal of EPS spermatozoa may enhance fertility potential in assisted medical procreation.

  11. Sclareol, a plant diterpene, exhibits potent antiproliferative effects via the induction of apoptosis and mitochondrial membrane potential loss in osteosarcoma cancer cells.

    Science.gov (United States)

    Wang, Lin; He, Hong-Sheng; Yu, Hua-Long; Zeng, Yun; Han, Heng; He, Ning; Liu, Zhi-Gang; Wang, Zhi-Yong; Xu, Shou-Jia; Xiong, Min

    2015-06-01

    The objective of the current study was to evaluate the antiproliferative activity of sclareol against MG63 osteosarcoma cells. A 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide assay was used to evaluate the cell viability of cells following treatment with sclareol. The extent of cell death induced by sclareol was evaluated using a lactate dehydrogenase (LDH) assay. The effect of sclareol on cell cycle progression and mitochondrial membrane potential (ΛΨm) was evaluated with flow cytometry using the DNA‑binding fluorescent dyes propidium iodide and rhodamine‑123, respectively. Fluorescence microscopy was used to detect the morphological changes in the MG63 osteosarcoma cancer cells and the appearance of apoptotic bodies following sclareol treatment. The results revealed that sclareol induced dose‑ and time‑dependent growth inhibition of MG63 cancer cells with an IC50 value of 65.2 µM following a 12‑h incubation. Furthermore, sclareol induced a significant increase in the release of LDH from MG63 cell cultures, which was much more pronounced at higher doses. Fluorescence microscopy revealed that sclareol induced characteristic morphological features of apoptosis and the appearance of apoptotic bodies. Flow cytometry revealed that sclareol induced G1‑phase cell cycle arrest, which showed significant dose‑dependence. Additionally, sclareol induced a progressive and dose‑dependent reduction in the ΛΨm. In summary, sclareol inhibits the growth of osteosarcoma cancer cells via the induction of apoptosis, which is accompanied by G1‑phase cell cycle arrest and loss of ΛΨm.

  12. Mitochondrial ATP is required for the maintenance of membrane integrity in stallion spermatozoa, whereas motility requires both glycolysis and oxidative phosphorylation

    NARCIS (Netherlands)

    Davila, M Plaza; Muñoz, P Martin; Bolaños, J M Gallardo; Stout, T A E; Gadella, B M; Tapia, J A; da Silva, C Balao; Ferrusola, C Ortega; Peña, F J

    2016-01-01

    To investigate the hypothesis that oxidative phosphorylation is a major source of ATP to fuel stallion sperm motility, oxidative phosphorylation was suppressed using the mitochondrial uncouplers CCCP and 2,4,-dinitrophenol (DNP) and by inhibiting mitochondrial respiration at complex IV using sodium

  13. Isolation and quantification of major chlorogenic acids in three major instant coffee brands and their potential effects on H2O2-induced mitochondrial membrane depolarization and apoptosis in PC-12 cells.

    Science.gov (United States)

    Park, Jae B

    2013-11-01

    Coffee is a most consumed drink worldwide, with potential health effects on several chronic diseases including neuronal degenerative diseases. Chlorogenic acids (CHAs) are phenolic compounds found in coffee and they are reported to have strong antioxidant and anti-inflammatory activities. However, the amounts of CHAs often vary in coffee drinks and their potential effects on ROS-induced neuronal cell death still require more investigation. Therefore, in this paper, major CHAs were isolated from three major instant coffee brands, confirmed and quantified using HPLC and NMR spectroscopic methods. Then, their antioxidant activities and protective effects on H2O2-induced apoptosis in PC-12 cells were investigated using radical scavenging, mitochondrial membrane potential and caspase assays. In the coffee samples, three major CHAs (3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, 5-O-caffeoylquinic acid) and some minor CHAs (3-O-feruloylquinic acid, 4-O-feruloylquinic acid, 5-O-feruloylquinic acid, 3,5-O-dicaffeoylquinic acid, 3,4-O-dicaffeoylquinic acid, and 4,5-O-dicaffeoylquinic acid) were detected. The three major CHAs were further isolated and their chemical structures were confirmed using NMR spectroscopic techniques. Also, the amounts of the three major CHAs were individually quantified using a HPLC method. At the concentration of 10 μM, all three major CHAs quenched DPPH and/or xanthine oxidase-generated radical species by 21-51% (P < 0.014). They also inhibited H2O2-induced mitochondrial membrane depolarization and caspase-9 activation by 27% (P < 0.034) and 50% (P < 0.05), respectively. This study suggests that the major CHAs found in coffee are likely to be potent antioxidant compounds able to quench radical species as well as inhibit H2O2-induced apoptosis via suppressing mitochondrial membrane depolarization and caspase-9 activation in the cells.

  14. Mitochondrial Myopathies

    Science.gov (United States)

    ... which stimulates normal beating of the heart. Cardiac muscle damage also may occur. People with mitochondrial disorders may need to have regular examina- tions by a cardiologist. Other potential health issues Some people with mitochondrial disease experience ...

  15. Mitochondrial haplogroups

    DEFF Research Database (Denmark)

    Benn, Marianne; Schwartz, Marianne; Nordestgaard, Børge G

    2008-01-01

    Rare mutations in the mitochondrial genome may cause disease. Mitochondrial haplogroups defined by common polymorphisms have been associated with risk of disease and longevity. We tested the hypothesis that common haplogroups predict risk of ischemic cardiovascular disease, morbidity from other...

  16. Mitochondrial genetics

    OpenAIRE

    Chinnery, Patrick Francis; Hudson, Gavin

    2013-01-01

    Introduction In the last 10 years the field of mitochondrial genetics has widened, shifting the focus from rare sporadic, metabolic disease to the effects of mitochondrial DNA (mtDNA) variation in a growing spectrum of human disease. The aim of this review is to guide the reader through some key concepts regarding mitochondria before introducing both classic and emerging mitochondrial disorders. Sources of data In this article, a review of the current mitochondrial genetics literature was con...

  17. OXPHOS-Dependent Cells Identify Environmental Disruptors of Mitochondrial Function

    Science.gov (United States)

    Mitochondrial dysfunction is associated with numerous chronic diseases including metabolic syndrome. Environmental chemicals can impair mitochondrial function through numerous mechanisms such as membrane disruption, complex inhibition and electron transport chain uncoupling. Curr...

  18. Grape proanthocyanidins induce apoptosis by loss of mitochondrial membrane potential of human non-small cell lung cancer cells in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Tripti Singh

    Full Text Available Lung cancer remains the leading cause of cancer-related deaths worldwide, and non-small cell lung cancer (NSCLC represents approximately 80% of total lung cancer cases. The use of non-toxic dietary phytochemicals can be considered as a chemotherapeutic strategy for the management of the NSCLC. Here, we report that grape seed proanthocyanidins (GSPs induce apoptosis of NSCLC cells, A549 and H1299, in vitro which is mediated through increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose polymerase (PARP. Pre-treatment of A549 and H1299 cells with the caspase-3 inhibitor (z-DEVD-fmk significantly blocked the GSPs-induced apoptosis of these cells confirmed that GSPs-induced apoptosis is mediated through activation of caspases-3. Treatments of A549 and H1299 cells with GSPs resulted in an increase in G1 arrest. G0/G1 phase of the cell cycle is known to be controlled by cyclin dependent kinases (Cdk, cyclin-dependent kinase inhibitors (Cdki and cyclins. Our western blot analyses showed that GSPs-induced G1 cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27, and a simultaneous decrease in the levels of Cdk2, Cdk4, Cdk6 and cyclins. Further, administration of 50, 100 or 200 mg GSPs/kg body weight of mice by oral gavage (5 d/week markedly inhibited the growth of s.c. A549 and H1299 lung tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death, increased expression of Bax, reduced expression of anti-apoptotic proteins and activation of caspase-3 in tumor xenograft cells. Based on the data obtained in animal study, human equivalent dose of GSPs was calculated, which seems affordable and attainable. Together, these results suggest that GSPs may represent a potential therapeutic agent for the non

  19. Comparisons of flux control exerted by mitochondrial outer-membrane carnitine palmitoyltransferase over ketogenesis in hepatocytes and mitochondria isolated from suckling or adult rats.

    Science.gov (United States)

    New, K J; Krauss, S; Elliott, K R; Quant, P A

    1999-02-01

    The primary aim of this paper was to calculate and report flux control coefficients for mitochondrial outer-membrane carnitine palmitoyltransferase (CPT I) over hepatic ketogenesis because its role in controlling this pathway during the neonatal period is of academic importance and immediate clinical relevance. Using hepatocytes isolated from suckling rats as our model system, we measured CPT I activity and carbon flux from palmitate to ketone bodies and to CO2 in the absence and presence of a range of concentrations of etomoxir. (This is converted in situ to etomoxir-CoA which is a specific inhibitor of the enzyme.) From these data we calculated the individual flux control coefficients for CPT I over ketogenesis, CO2 production and total carbon flux (0.51 +/- 0.03; -1.30 +/- 0.26; 0.55 +/- 0.07, respectively) and compared them with equivalent coefficients calculated by similar analyses [Drynan, L., Quant, P.A. & Zammit, V.A. (1996) Biochem. J. 317, 791-795] in hepatocytes isolated from adult rats (0.85 +/- 0.20; 0.23 +/- 0.06; 1.06 +/- 0.29). CPT I exerts significantly less control over ketogenesis in hepatocytes isolated from suckling rats than those from adult rats. In the suckling systems the flux control coefficients for CPT I over ketogenesis specifically and over total carbon flux (< 0.6) are not consistent with the enzyme being rate-limiting. Broadly similar results were obtained and conclusions drawn by reanalysis of previous data {from experiments in mitochondria isolated from suckling or adult rats [Krauss, S., Lascelles, C.V., Zammit, V.A. & Quant, P.A. (1996) Biochem. J. 319, 427-433]} using a different approach of control analysis, although it is not strictly valid to compare flux control coefficients from different systems. Our overall conclusion is that flux control coefficients for CPT I over oxidative fluxes from palmitate (or palmitoyl-CoA) differ markedly according to (a) the metabolic state, (b) the stage of development, (c) the specific

  20. Role of oxidative stress, mitochondrial membrane potential, and calcium homeostasis in human lymphocyte death induced by nickel carbonate hydroxide in vitro

    Energy Technology Data Exchange (ETDEWEB)

    M' Bemba-Meka, Prosper [Faculty of Medicine, Universite de Montreal, QC (Canada); University of Louisville, Department of Pharmacology and Toxicology, Center for Genetics and Molecular Medicine, Louisville, KY (United States); Lemieux, Nicole [Universite de Montreal, Department of Pathology and Cellular Biology, Main Station, P.O. Box 6128, Montreal, QC (Canada); Chakrabarti, Saroj K. [Faculty of Medicine, Universite de Montreal, QC (Canada)

    2006-07-15

    When isolated human lymphocytes were treated in vitro with various concentrations of soluble form of nickel carbonate hydroxide (NiCH) (0-1 mM), at 37 C for 4 h, both concentration- and time-dependent effects of NiCH on lymphocyte death were observed. Increased generation of hydrogen peroxide (H{sub 2}O{sub 2}), superoxide anion (O{sub 2} {sup -}), depletion of both no protein (NP-) and protein (P-) sulfhydryl (SH) contents and lipid peroxidation (LPO) were induced by NiCH. Pretreatment of lymphocytes with either catalase (H{sub 2}O{sub 2} scavenger), or deferoxamine (DFO) (iron chelator), or excess glutathione (GSH) (an antioxidant) not only significantly reduced the NiCH-induced generation of H{sub 2}O{sub 2} and LPO, but also increased the NP-SH and P-SH contents initially reduced by NiCH. NiCH-induced generation of excess O{sub 2} {sup -} but not excess LPO was significantly reduced by pretreatment with superoxide dismutase (SOD). NiCH-induced lymphocyte death was significantly prevented by pre-treatment with either catalase, or dimethylthiourea/mannitol (hydroxyl radical scavengers), or DFO, or excess GSH/N-acetylcysteine. NiCH-induced lymphocyte death was also significantly prevented by pretreatment with excess SOD. Thus, various types of oxidative stresses play an important role in NiCH-induced lymphocyte death. Cotreatment with cyclosporin A, a specific inhibitor of alteration in mitochondrial membrane potential ({delta}{psi}{sub m}), not only inhibited NiCH-induced alteration in {delta}{psi}{sub m}, but also significantly prevented Ni-compound-induced lymphocyte death. Furthermore, NiCH-induced destabilization of cellular calcium homeostasis. As such, NiCH-induced lymphocyte death was significantly prevented by modulating intracellular calcium fluxes such as Ca{sup 2+} channel blockers and intracellular Ca{sup 2+} antagonist. Thus, the mechanism of NiCH (soluble form)-induced activation of lymphocyte death signalling pathways involves not only the excess

  1. Inherited mitochondrial optic neuropathies

    Science.gov (United States)

    Yu-Wai-Man, P; Griffiths, P G; Hudson, G; Chinnery, P F

    2009-01-01

    Leber hereditary optic neuropathy (LHON) and autosomal dominant optic atrophy (DOA) are the two most common inherited optic neuropathies and they result in significant visual morbidity among young adults. Both disorders are the result of mitochondrial dysfunction: LHON from primary mitochondrial DNA (mtDNA) mutations affecting the respiratory chain complexes; and the majority of DOA families have mutations in the OPA1 gene, which codes for an inner mitochondrial membrane protein critical for mtDNA maintenance and oxidative phosphorylation. Additional genetic and environmental factors modulate the penetrance of LHON, and the same is likely to be the case for DOA which has a markedly variable clinical phenotype. The selective vulnerability of retinal ganglion cells (RGCs) is a key pathological feature and understanding the fundamental mechanisms that underlie RGC loss in these disorders is a prerequisite for the development of effective therapeutic strategies which are currently limited. PMID:19001017

  2. Mitochondrial vasculopathy

    Institute of Scientific and Technical Information of China (English)

    Josef Finsterer; Sinda Zarrouk-Mahjoub

    2016-01-01

    Mitochondrial disorders(MIDs)are usually multisystem disorders(mitochondrial multiorgan disorder syndrome)either on from onset or starting at a point during the disease course.Most frequently affected tissues are those with a high oxygen demand such as the central nervous system,the muscle,endocrine glands,or the myocardium.Recently,it has been shown that rarely alsothe arteries may be affected(mitochondrial arteriopathy).This review focuses on the type,diagnosis,and treat-ment of mitochondrial vasculopathy in MID patients.A literature search using appropriate search terms was carried out.Mitochondrial vasculopathy manifests as either microangiopathy or macroangiopathy.Clinical manifestations of mitochondrial microangiopathy include leukoencephalopathy,migraine-like headache,stroke-like episodes,or peripheral retinopathy.Mitochondrial macroangiopathy manifests as atherosclerosis,ectasia of arteries,aneurysm formation,dissection,or spontan-eous rupture of arteries.The diagnosis relies on the documentation and confirmation of the mitochondrial metabolic defect or the genetic cause after exclusion of non-MID causes.Treatment is not at variance compared to treatment of vasculopathy due to non-MID causes.Mitochondrial vasculopathy exists and manifests as micro-or macroangiopathy.Diagnosing mitochondrial vasculopathy is crucial since appropriate treatment may prevent from severe complications.

  3. Effects of nicorandil on cardiac plasma membrane and cardiac mitochondrial membrane potential of guinea-pig%尼可地尔对豚鼠心肌细胞膜及线粒体膜电位的影响

    Institute of Scientific and Technical Information of China (English)

    冯力; 刘伊丽; 刘杰; 金春华

    2001-01-01

    研究KATP通道开放剂尼可地尔(Nic)对豚鼠心肌细胞膜和线粒体膜电位的影响.用激光共聚焦显微镜和特异性荧光探针,观察不同剂量的Nic及KATP通道阻滞剂格列本脲(Gli)引起急性分离的豚鼠心肌细胞膜电位,线粒体膜电位荧光值的变化.Nic1mmol.L-1引起细胞膜电位在1min内迅速超极化〔膜电位荧光值减少(75±12)%〕,Gli3μmol.L-1可阻断其变化;0.1和1mmol.L-1Nic可使线粒体膜电位去极化和膜电位荧光值在1,2,5min分别增加(12±3)%和(32±8)%,(25±6)%和(39±9)%,(34±6)%和(45±12)%;3μmol.L-1Gli可抑制其变化.结果说明低浓度Nic只引起线粒体膜电位去极化,高浓度Nic还可使细胞膜电位发生超极化,引起KATP通道开放.%With digital imaging techniques of advanced laser confocalmicroscope, effects of KATP channel opener nicorandil(Nic) on cardiac plasma membrane(CPM) and cardiac mitochondrial membrane(CMM) potential of guinea-pig were studied. It was found that Nic 1 mmol.L-1 caused the potential of CPM more negative (hyperpolarization), fluorescence intensity(FI) decreased by (75±12)% of baseline within 1 min, but no effect at 0.1 mmol.L-1. CMM was depolarized by 0.1 mmol.L-1 Nic〔FI increased by (12±3)%, (25±6)%, (34±6)% of baseline within 1, 2, 5 min〕, and by 1 mmol.L-1 Nic〔FI remarkably increased by (32±8)%, (39±9)%, (45±12)% of baseline〕. KATP channel blocker glibenclamide 3 μmol.L-1 itself caused no effect on potential of CPM and CMM, but blocked the above effect on potential of CPM and CMM induced by Nic. The results suggest that KATP channel of CMM is activated by low dose of Nic, and the high dose of Nic activate both KATP channels of CPM and CMM.

  4. The mitochondrial contact site complex, a determinant of mitochondrial architecture

    Science.gov (United States)

    Harner, Max; Körner, Christian; Walther, Dirk; Mokranjac, Dejana; Kaesmacher, Johannes; Welsch, Ulrich; Griffith, Janice; Mann, Matthias; Reggiori, Fulvio; Neupert, Walter

    2011-01-01

    Mitochondria are organelles with a complex architecture. They are bounded by an envelope consisting of the outer membrane and the inner boundary membrane (IBM). Narrow crista junctions (CJs) link the IBM to the cristae. OMs and IBMs are firmly connected by contact sites (CS). The molecular nature of the CS remained unknown. Using quantitative high-resolution mass spectrometry we identified a novel complex, the mitochondrial contact site (MICOS) complex, formed by a set of mitochondrial membrane proteins that is essential for the formation of CS. MICOS is preferentially located at the CJs. Upon loss of one of the MICOS subunits, CJs disappear completely or are impaired, showing that CJs require the presence of CS to form a superstructure that links the IBM to the cristae. Loss of MICOS subunits results in loss of respiratory competence and altered inheritance of mitochondrial DNA. PMID:22009199

  5. Association of active caspase 8 with the mitochondrial membrane during apoptosis: potential roles in cleaving BAP31 and caspase 3 and mediating mitochondrion-endoplasmic reticulum cross talk in etoposide-induced cell death.

    Science.gov (United States)

    Chandra, Dhyan; Choy, Grace; Deng, Xiaodi; Bhatia, Bobby; Daniel, Peter; Tang, Dean G

    2004-08-01

    It was recently demonstrated that during apoptosis, active caspase 9 and caspase 3 rapidly accumulate in the mitochondrion-enriched membrane fraction (D. Chandra and D. G. Tang, J. Biol. Chem.278:17408-17420, 2003). We now show that active caspase 8 also becomes associated with the membranes in apoptosis caused by multiple stimuli. In MDA-MB231 breast cancer cells treated with etoposide (VP16), active caspase 8 is detected only in the membrane fraction, which contains both mitochondria and endoplasmic reticulum (ER), as revealed by fractionation studies. Immunofluorescence microscopy, however, shows that procaspase 8 and active caspase 8 predominantly colocalize with the mitochondria. Biochemical analysis demonstrates that both procaspase 8 and active caspase 8 are localized mainly on the outer mitochondrial membrane (OMM) as integral proteins. Functional analyses with dominant-negative mutants, small interfering RNAs, peptide inhibitors, and Fas-associated death domain (FADD)- and caspase 8-deficient Jurkat T cells establish that the mitochondrion-localized active caspase 8 results mainly from the FADD-dependent and tumor necrosis factor receptor-associated death domain-dependent mechanisms and that caspase 8 activation plays a causal role in VP16-induced caspase 3 activation and cell death. Finally, we present evidence that the OMM-localized active caspase 8 can activate cytosolic caspase 3 and ER-localized BAP31. Cleavage of BAP31 leads to the generation of ER- localized, proapoptotic BAP20, which may mediate mitochondrion-ER cross talk through a Ca(2+)-dependent mechanism.

  6. Hsp90 inhibition decreases mitochondrial protein turnover.

    Directory of Open Access Journals (Sweden)

    Daciana H Margineantu

    Full Text Available BACKGROUND: Cells treated with hsp90 inhibitors exhibit pleiotropic changes, including an expansion of the mitochondrial compartment, accompanied by mitochondrial fragmentation and condensed mitochondrial morphology, with ultimate compromise of mitochondrial integrity and apoptosis. FINDINGS: We identified several mitochondrial oxidative phosphorylation complex subunits, including several encoded by mtDNA, that are upregulated by hsp90 inhibitors, without corresponding changes in mRNA abundance. Post-transcriptional accumulation of mitochondrial proteins observed with hsp90 inhibitors is also seen in cells treated with proteasome inhibitors. Detailed studies of the OSCP subunit of mitochondrial F1F0-ATPase revealed the presence of mono- and polyubiquitinated OSCP in mitochondrial fractions. We demonstrate that processed OSCP undergoes retrotranslocation to a trypsin-sensitive form associated with the outer mitochondrial membrane. Inhibition of proteasome or hsp90 function results in accumulation of both correctly targeted and retrotranslocated mitochondrial OSCP. CONCLUSIONS: Cytosolic turnover of mitochondrial proteins demonstrates a novel connection between mitochondrial and cytosolic compartments through the ubiquitin-proteasome system. Analogous to defective protein folding in the endoplasmic reticulum, a mitochondrial unfolded protein response may play a role in the apoptotic effects of hsp90 and proteasome inhibitors.

  7. Piracetam improves mitochondrial dysfunction following oxidative stress

    OpenAIRE

    2005-01-01

    Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging.Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction fol...

  8. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    Science.gov (United States)

    Kandel, Judith; Angelin, Alessia A; Wallace, Douglas C; Eckmann, David M

    2016-11-07

    An abundance of research suggests that cellular mitochondrial and cytoskeletal disruption are related, but few studies have directly investigated causative connections between the two. We previously demonstrated that inhibiting microtubule and microfilament polymerization affects mitochondrial motility on the whole-cell level in fibroblasts. Since mitochondrial motility can be indicative of mitochondrial function, we now further characterize the effects of these cytoskeletal inhibitors on mitochondrial potential, morphology and respiration. We found that although they did not reduce mitochondrial inner membrane potential, cytoskeletal toxins induced significant decreases in basal mitochondrial respiration. In some cases, basal respiration was only affected after cells were pretreated with the calcium ionophore A23187 in order to stress mitochondrial function. In most cases, mitochondrial morphology remained unaffected, but extreme microfilament depolymerization or combined intermediate doses of microtubule and microfilament toxins resulted in decreased mitochondrial lengths. Interestingly, these two particular exposures did not affect mitochondrial respiration in cells not sensitized with A23187, indicating an interplay between mitochondrial morphology and respiration. In all cases, inducing maximal respiration diminished differences between control and experimental groups, suggesting that reduced basal respiration originates as a largely elective rather than pathological symptom of cytoskeletal impairment. However, viability experiments suggest that even this type of respiration decrease may be associated with cell death.

  9. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    Directory of Open Access Journals (Sweden)

    Elisa Balboa

    2017-08-01

    Full Text Available MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

  10. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content.

    Science.gov (United States)

    Balboa, Elisa; Castro, Juan; Pinochet, María-José; Cancino, Gonzalo I; Matías, Nuria; José Sáez, Pablo; Martínez, Alexis; Álvarez, Alejandra R; Garcia-Ruiz, Carmen; Fernandez-Checa, José C; Zanlungo, Silvana

    2017-08-01

    MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. A forskolin derivative, FSK88, induces apoptosis in human gastric cancer BGC823 cells through caspase activation involving regulation of Bcl-2 family gene expression, dissipation of mitochondrial membrane potential and cytochrome c release.

    Science.gov (United States)

    Li, Zhonghai; Wang, Jingze

    2006-11-01

    FSK88, a forskolin derivative, was extracted and purified from cultured tropical plant roots, Coleus forskohlii. Our previous studies have demonstrated that FSK88 can inhibit HL-60 cell proliferation and induce the differentiation of HL-60 cells to monocyte macrophages. In this study, we showed that FSK88 can induce apoptotic death of human gastric cancer BGC823 cells in a dose- and time-dependent manner. Results showed that FSK88-induced apoptosis was accompanied by the mitochondrial release of cytochrome c and activation of caspase-3 in BGC823 cells. Furthermore, treatment with caspase-3 inhibitor (z-DEVD-fmk) was capable of preventing the FSK88-induced caspase-3 activity and apoptosis. FSK88-induced apoptosis in human gastric cancer BGC823 cells was also accompanied by the up-regulation of Bax, Bad and down-regulation of Bcl-2. Theses results clearly demonstrated that the induction of apoptosis by FSK88 involved multiple cellular and molecular pathways and strongly suggest that pro- and anti-apoptotic Bcl-2 family genes, mitochondrial membrane potential (Deltapsi(m)), cytochrome c, and caspase-3, participate in the FSK88-induced apoptotic process in human gastric cancer BGC823 cells.

  12. ERp57 modulates mitochondrial calcium uptake through the MCU.

    Science.gov (United States)

    He, Jingquan; Shi, Weikang; Guo, Yu; Chai, Zhen

    2014-06-01

    ERp57 participates in the regulation of calcium homeostasis. Although ERp57 modulates calcium flux across the plasma membrane and the endoplasmic reticulum membrane, its functions on mitochondria are largely unknown. Here, we found that ERp57 can regulate the expression of the mitochondrial calcium uniporter (MCU) and modulate mitochondrial calcium uptake. In ERp57-silenced HeLa cells, MCU was downregulated, and the mitochondrial calcium uptake was inhibited, consistent with the effect of MCU knockdown. When MCU was re-expressed in the ERp57 knockdown cells, mitochondrial calcium uptake was restored. Thus, ERp57 is a potent regulator of mitochondrial calcium homeostasis.

  13. Mitochondrial disorders.

    Science.gov (United States)

    Zeviani, M; Tiranti, V; Piantadosi, C

    1998-01-01

    Mitochondrial respiration, the most efficient metabolic pathway devoted to energy production, is at the crosspoint of 2 quite different genetic systems, the nuclear genome and the mitochondrial genome (mitochondrial DNA, mtDNA). The latter encodes a few essential components of the mitochondrial respiratory chain and has unique molecular and genetic properties that account for some of the peculiar features of mitochondrial disorders. However, the perpetuation, propagation, and expression of mtDNA, the majority of the subunits of the respiratory complexes, as well as a number of genes involved in their assembly and turnover, are contained in the nuclear genome. Although mitochondrial disorders have been known for more than 30 years, a major breakthrough in their understanding has come much later, with the discovery of an impressive, ever-increasing number of mutations of mitochondrial DNA. Partial deletions or duplications of mtDNA, or maternally inherited point mutations, have been associated with well-defined clinical syndromes. However, phenotypes transmitted as mendelian traits have also been identified. These include clinical entities defined on the basis of specific biochemical defects, and also a few autosomal dominant or recessive syndromes associated with multiple deletions or tissue-specific depletion of mtDNA. Given the complexity of mitochondrial genetics and biochemistry, the clinical manifestations of mitochondrial disorders are extremely heterogenous. They range from lesions of single tissues or structures, such as the optic nerve in Leber hereditary optic neuropathy or the cochlea in maternally inherited nonsyndromic deafness, to more widespread lesions including myopathies, encephalomyopathies, cardiopathies, or complex multisystem syndromes. The recent advances in genetic studies provide both diagnostic tools and new pathogenetic insights in this rapidly expanding area of human pathology.

  14. Melatonin: A Mitochondrial Targeting Molecule Involving Mitochondrial Protection and Dynamics

    Directory of Open Access Journals (Sweden)

    Dun-Xian Tan

    2016-12-01

    Full Text Available Melatonin has been speculated to be mainly synthesized by mitochondria. This speculation is supported by the recent discovery that aralkylamine N-acetyltransferase/serotonin N-acetyltransferase (AANAT/SNAT is localized in mitochondria of oocytes and the isolated mitochondria generate melatonin. We have also speculated that melatonin is a mitochondria-targeted antioxidant. It accumulates in mitochondria with high concentration against a concentration gradient. This is probably achieved by an active transportation via mitochondrial melatonin transporter(s. Melatonin protects mitochondria by scavenging reactive oxygen species (ROS, inhibiting the mitochondrial permeability transition pore (MPTP, and activating uncoupling proteins (UCPs. Thus, melatonin maintains the optimal mitochondrial membrane potential and preserves mitochondrial functions. In addition, mitochondrial biogenesis and dynamics is also regulated by melatonin. In most cases, melatonin reduces mitochondrial fission and elevates their fusion. Mitochondrial dynamics exhibit an oscillatory pattern which matches the melatonin circadian secretory rhythm in pinealeocytes and probably in other cells. Recently, melatonin has been found to promote mitophagy and improve homeostasis of mitochondria.

  15. Melatonin mitigates mitochondrial malfunction.

    Science.gov (United States)

    León, Josefa; Acuña-Castroviejo, Darío; Escames, Germane; Tan, Dun-Xian; Reiter, Russel J

    2005-01-01

    Melatonin, or N-acetyl-5-methoxytryptamine, is a compound derived from tryptophan that is found in all organisms from unicells to vertebrates. This indoleamine may act as a protective agent in disease conditions such as Parkinson's, Alzheimer's, aging, sepsis and other disorders including ischemia/reperfusion. In addition, melatonin has been proposed as a drug for the treatment of cancer. These disorders have in common a dysfunction of the apoptotic program. Thus, while defects which reduce apoptotic processes can exaggerate cancer, neurodegenerative disorders and ischemic conditions are made worse by enhanced apoptosis. The mechanism by which melatonin controls cell death is not entirely known. Recently, mitochondria, which are implicated in the intrinsic pathway of apoptosis, have been identified as a target for melatonin actions. It is known that melatonin scavenges oxygen and nitrogen-based reactants generated in mitochondria. This limits the loss of the intramitochondrial glutathione and lowers mitochondrial protein damage, improving electron transport chain (ETC) activity and reducing mtDNA damage. Melatonin also increases the activity of the complex I and complex IV of the ETC, thereby improving mitochondrial respiration and increasing ATP synthesis under normal and stressful conditions. These effects reflect the ability of melatonin to reduce the harmful reduction in the mitochondrial membrane potential that may trigger mitochondrial transition pore (MTP) opening and the apoptotic cascade. In addition, a reported direct action of melatonin in the control of currents through the MTP opens a new perspective in the understanding of the regulation of apoptotic cell death by the indoleamine.

  16. Mitochondrial medicine

    National Research Council Canada - National Science Library

    Bandyopadhyay, S K; Dutt, Anita

    2010-01-01

    .... With the coming of age for mitochondrial medicine, it is now appropriate that physicians keep themselves well-acquainted with the recent developments in this expanding field of biomedical research.

  17. Mitochondrial Cristae: Where Beauty Meets Functionality.

    Science.gov (United States)

    Cogliati, Sara; Enriquez, Jose A; Scorrano, Luca

    2016-03-01

    Mitochondrial cristae are dynamic bioenergetic compartments whose shape changes under different physiological conditions. Recent discoveries have unveiled the relation between cristae shape and oxidative phosphorylation (OXPHOS) function, suggesting that membrane morphology modulates the organization and function of the OXPHOS system, with a direct impact on cellular metabolism. As a corollary, cristae-shaping proteins have emerged as potential modulators of mitochondrial bioenergetics, a concept confirmed by genetic experiments in mouse models of respiratory chain deficiency. Here, we review our knowledge of mitochondrial ultrastructural organization and how it impacts mitochondrial metabolism.

  18. Aquaporin-8-facilitated mitochondrial ammonia transport.

    Science.gov (United States)

    Soria, Leandro R; Fanelli, Elena; Altamura, Nicola; Svelto, Maria; Marinelli, Raúl A; Calamita, Giuseppe

    2010-03-05

    Aquaporin-8 (AQP8) is a membrane channel permeable to water and ammonia. As AQP8 is expressed in the inner mitochondrial membrane of several mammalian tissues, we studied the effect of the AQP8 expression on the mitochondrial transport of ammonia. Recombinant rat AQP8 was expressed in the yeast Saccharomyces cerevisiae. The presence of AQP8 in the inner membrane of yeast mitochondria was demonstrated by subcellular fractionation and immunoblotting analysis. The ammonia transport was determined in isolated mitochondria by stopped flow light scattering using formamide as ammonia analog. We found that the presence of AQP8 increased by threefold mitochondrial formamide transport. AQP8-facilitated mitochondrial formamide transport in rat native tissue was confirmed in liver (a mitochondrial AQP8-expressing tissue) vs. brain (a mitochondrial AQP8 non-expressing tissue). Comparative studies indicated that the AQP8-mediated mitochondrial movement of formamide was markedly higher than that of water. Together, our data suggest that ammonia diffusional transport is a major function for mitochondrial AQP8. 2010 Elsevier Inc. All rights reserved.

  19. Oxidative stress, mitochondrial damage and neurodegenerative diseases****

    Institute of Scientific and Technical Information of China (English)

    Chunyan Guo; Li Sun; Xueping Chen; Danshen Zhang

    2013-01-01

    Oxidative stress and mitochondrial damage have been implicated in the pathogenesis of several neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Oxidative stress is characterized by the overproduction of reactive oxygen species, which can induce mitochondrial DNA mutations, damage the mitochondrial respiratory chain, alter membrane permeability, and influence Ca2+ homeostasis and mitochondrial defense systems. Al these changes are implicated in the development of these neurodegenerative diseases, mediating or amplifying neuronal dysfunction and triggering neurodegeneration. This paper summarizes the contribution of oxidative stress and mitochondrial damage to the onset of neurodegenerative eases and discusses strategies to modify mitochondrial dysfunction that may be attractive thera-peutic interventions for the treatment of various neurodegenerative diseases.

  20. Constriction of the mitochondrial inner compartment is a priming event for mitochondrial division

    Science.gov (United States)

    Cho, Bongki; Cho, Hyo Min; Jo, Youhwa; Kim, Hee Dae; Song, Myungjae; Moon, Cheil; Kim, Hyongbum; Kim, Kyungjin; Sesaki, Hiromi; Rhyu, Im Joo; Kim, Hyun; Sun, Woong

    2017-01-01

    Mitochondrial division is critical for the maintenance and regulation of mitochondrial function, quality and distribution. This process is controlled by cytosolic actin-based constriction machinery and dynamin-related protein 1 (Drp1) on mitochondrial outer membrane (OMM). Although mitochondrial physiology, including oxidative phosphorylation, is also important for efficient mitochondrial division, morphological alterations of the mitochondrial inner-membrane (IMM) have not been clearly elucidated. Here we report spontaneous and repetitive constriction of mitochondrial inner compartment (CoMIC) associated with subsequent division in neurons. Although CoMIC is potentiated by inhibition of Drp1 and occurs at the potential division spots contacting the endoplasmic reticulum, it appears on IMM independently of OMM. Intra-mitochondrial influx of Ca2+ induces and potentiates CoMIC, and leads to K+-mediated mitochondrial bulging and depolarization. Synergistically, optic atrophy 1 (Opa1) also regulates CoMIC via controlling Mic60-mediated OMM–IMM tethering. Therefore, we propose that CoMIC is a priming event for efficient mitochondrial division. PMID:28598422

  1. Experimental study on mitochondrial membrane potential and ultrastructure injuries of kidney in septic rats%脓毒症大鼠肾脏线粒体膜电位变化和超微结构损伤的研究

    Institute of Scientific and Technical Information of China (English)

    陈飞燕; 曾其毅; 赵明奇; 杨文敏; 连广琬

    2008-01-01

    Objective To study the mitochondrial membrane potential and ultrastructure injuries of kidney in septic rats. Methods Thirty SD rats were randomly divided into three groups: Saline control group, 6 h LPS group and 24 h LPS group. The rats in LPS groups received 10 mg/kg lipopolysaccharide by intraperitonesl injection. The levels of serum Cr and BUN were detected. The swelling (FSC/SSC) and the membrane potential (FL2/FL1) of isolated renal mitochondrion were tested by flow cytometry. The morphologic change of mitochondria in kidney was observed by electronic microscopy. Results Compared with control group, serum Cr and BUN increased in septic rats. Compared with control group, swelling of the mirochondrion significantly increased in 24 h LPS group ( Control: 0.55 ± 0.10; 6 h LPS: 0.58 ± 0.10; 24 h LPS: 0.66 ± 0.12, P<0.05). Renal mitochondrial membrane potential significantly decreased in 6 h LPS group and 24 h LPS group (Control:0.77 ± 0.26; 6 h LPS: 0.32 ± 0.19;24 h LPS: 0.30 ± 0.17, P < 0.01). The mitochondrial membrane potential in kidney were inversely correlated with serum Cr level( r = -0.510, P = 0.004). The injuries of mitochondrial ultrastructure increased in 24 h LPS group. Conclusion The injury of the renal mitochondrial membrane potential has occurred in early stage of sepsis before obvious ultrastructure damage of the renal mitochondrion in septic rats.%目的 探讨脓毒症时大鼠肾脏线粒体的损伤情况.方法 采用腹腔注射内毒素(LPS)建立脓毒症大鼠模型.30只SD大鼠随机分为对照组、6 h脓毒症组和24 h脓毒症组.分别检测各组大鼠血清肌酐(Cr)、尿素氮(BUN)水平;电镜观察肾脏线粒体形态学变化;运用流式细胞术检测分离肾脏线粒体的肿胀程度和线粒体膜电位,用前向角与侧向角的平均荧光强度比值(FSC/SSC)反映线粒体肿胀程度,用二通道和一通道平均荧光强度比值(FL2/FL1)确定线粒体膜电位.结果 脓毒症大鼠的血清Cr、BUN

  2. Genetics Home Reference: MPV17-related hepatocerebral mitochondrial DNA depletion syndrome

    Science.gov (United States)

    ... on PubMed Central Spinazzola A, Santer R, Akman OH, Tsiakas K, Schaefer H, Ding X, Karadimas CL, ... Zeviani M. MPV17 encodes an inner mitochondrial membrane protein and is mutated in infantile hepatic mitochondrial DNA ...

  3. Sphingolipids and mitochondrial apoptosis.

    Science.gov (United States)

    Patwardhan, Gauri A; Beverly, Levi J; Siskind, Leah J

    2016-04-01

    The sphingolipid family of lipids modulate several cellular processes, including proliferation, cell cycle regulation, inflammatory signaling pathways, and cell death. Several members of the sphingolipid pathway have opposing functions and thus imbalances in sphingolipid metabolism result in deregulated cellular processes, which cause or contribute to diseases and disorders in humans. A key cellular process regulated by sphingolipids is apoptosis, or programmed cell death. Sphingolipids play an important role in both extrinsic and intrinsic apoptotic pathways depending on the stimuli, cell type and cellular response to the stress. During mitochondrial-mediated apoptosis, multiple pathways converge on mitochondria and induce mitochondrial outer membrane permeabilization (MOMP). MOMP results in the release of intermembrane space proteins such as cytochrome c and Apaf1 into the cytosol where they activate the caspases and DNases that execute cell death. The precise molecular components of the pore(s) responsible for MOMP are unknown, but sphingolipids are thought to play a role. Here, we review evidence for a role of sphingolipids in the induction of mitochondrial-mediated apoptosis with a focus on potential underlying molecular mechanisms by which altered sphingolipid metabolism indirectly or directly induce MOMP. Data available on these mechanisms is reviewed, and the focus and limitations of previous and current studies are discussed to present important unanswered questions and potential future directions.

  4. Mitochondrial ABC transporters.

    Science.gov (United States)

    Lill, R; Kispal, G

    2001-01-01

    In contrast to bacteria, mitochondria contain only a few ATP binding cassette (ABC) transporters in their inner membrane. The known mitochondrial ABC proteins fall into two major classes that, in the yeast Saccharomyces cerevisiae, are represented by the half-transporter Atm1p and the two closely homologous proteins Mdl1p and Mdl2p. In humans two Atm1p orthologues (ABC7 and MTABC3) and two proteins homologous to Mdll/2p have been localized to mitochondria. The Atm1p-like proteins perform an important function in mitochondrial iron homeostasis and in the maturation of Fe/S proteins in the cytosol. Mutations in ABC7 are causative of hereditary X-linked sideroblastic anemia and cerebellar ataxia (XLSA/A). MTABC3 may be a candidate gene for the lethal neonatal syndrome. The function of the mitochondrial Mdl1/2p-like proteins is not clear at present with the notable exception of murine ABC-me that may transport intermediates of heme biosynthesis from the matrix to the cytosol in erythroid tissues.

  5. Mitochondrial Fusion Proteins and Human Diseases

    Directory of Open Access Journals (Sweden)

    Michela Ranieri

    2013-01-01

    Full Text Available Mitochondria are highly dynamic, complex organelles that continuously alter their shape, ranging between two opposite processes, fission and fusion, in response to several stimuli and the metabolic demands of the cell. Alterations in mitochondrial dynamics due to mutations in proteins involved in the fusion-fission machinery represent an important pathogenic mechanism of human diseases. The most relevant proteins involved in the mitochondrial fusion process are three GTPase dynamin-like proteins: mitofusin 1 (MFN1 and 2 (MFN2, located in the outer mitochondrial membrane, and optic atrophy protein 1 (OPA1, in the inner membrane. An expanding number of degenerative disorders are associated with mutations in the genes encoding MFN2 and OPA1, including Charcot-Marie-Tooth disease type 2A and autosomal dominant optic atrophy. While these disorders can still be considered rare, defective mitochondrial dynamics seem to play a significant role in the molecular and cellular pathogenesis of more common neurodegenerative diseases, for example, Alzheimer’s and Parkinson’s diseases. This review provides an overview of the basic molecular mechanisms involved in mitochondrial fusion and focuses on the alteration in mitochondrial DNA amount resulting from impairment of mitochondrial dynamics. We also review the literature describing the main disorders associated with the disruption of mitochondrial fusion.

  6. Mitochondrial Myopathy

    Science.gov (United States)

    ... diseases are caused by CoQ10 deficiency, and CoQ10 supplementation is clearly beneficial in these cases. It might provide some relief from other mitochondrial diseases. Creatine, L-carnitine, and CoQ10 supplements often are combined into a “ ...

  7. Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

    Full Text Available SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

  8. Mitochondrial dysfunction and organophosphorus compounds

    Energy Technology Data Exchange (ETDEWEB)

    Karami-Mohajeri, Somayyeh [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Kerman University of Medical Sciences, Kerman (Iran, Islamic Republic of); Abdollahi, Mohammad, E-mail: Mohammad.Abdollahi@UToronto.Ca [Department of Toxicology and Pharmacology, Faculty of Pharmacy, and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2013-07-01

    Organophosphorous (OPs) pesticides are the most widely used pesticides in the agriculture and home. However, many acute or chronic poisoning reports about OPs have been published in the recent years. Mitochondria as a site of cellular oxygen consumption and energy production can be a target for OPs poisoning as a non-cholinergic mechanism of toxicity of OPs. In the present review, we have reviewed and criticized all the evidences about the mitochondrial dysfunctions as a mechanism of toxicity of OPs. For this purpose, all biochemical, molecular, and morphological data were retrieved from various studies. Some toxicities of OPs are arisen from dysfunction of mitochondrial oxidative phosphorylation through alteration of complexes I, II, III, IV and V activities and disruption of mitochondrial membrane. Reductions of adenosine triphosphate (ATP) synthesis or induction of its hydrolysis can impair the cellular energy. The OPs disrupt cellular and mitochondrial antioxidant defense, reactive oxygen species generation, and calcium uptake and promote oxidative and genotoxic damage triggering cell death via cytochrome C released from mitochondria and consequent activation of caspases. The mitochondrial dysfunction induced by OPs can be restored by use of antioxidants such as vitamin E and C, alpha-tocopherol, electron donors, and through increasing the cytosolic ATP level. However, to elucidate many aspect of mitochondrial toxicity of Ops, further studies should be performed. - Highlights: • As a non-cholinergic mechanism of toxicity, mitochondria is a target for OPs. • OPs affect action of complexes I, II, III, IV and V in the mitochondria. • OPs reduce mitochondrial ATP. • OPs promote oxidative and genotoxic damage via release of cytochrome C from mitochondria. • OP-induced mitochondrial dysfunction can be restored by increasing the cytosolic ATP.

  9. Cardiolipin and mitochondrial cristae organization.

    Science.gov (United States)

    Ikon, Nikita; Ryan, Robert O

    2017-03-20

    A fundamental question in cell biology, under investigation for over six decades, is the structural organization of mitochondrial cristae. Long known to harbor electron transport chain proteins, crista membrane integrity is key to establishment of the proton gradient that drives oxidative phosphorylation. Visualization of cristae morphology by electron microscopy/tomography has provided evidence that cristae are tube-like extensions of the mitochondrial inner membrane (IM) that project into the matrix space. Reconciling ultrastructural data with the lipid composition of the IM provides support for a continuously curved cylindrical bilayer capped by a dome-shaped tip. Strain imposed by the degree of curvature is relieved by an asymmetric distribution of phospholipids in monolayer leaflets that comprise cristae membranes. The signature mitochondrial lipid, cardiolipin (~18% of IM phospholipid mass), and phosphatidylethanolamine (34%) segregate to the negatively curved monolayer leaflet facing the crista lumen while the opposing, positively curved, matrix-facing monolayer leaflet contains predominantly phosphatidylcholine. Associated with cristae are numerous proteins that function in distinctive ways to establish and/or maintain their lipid repertoire and structural integrity. By combining unique lipid components with a set of protein modulators, crista membranes adopt and maintain their characteristic morphological and functional properties. Once established, cristae ultrastructure has a direct impact on oxidative phosphorylation, apoptosis, fusion/fission as well as diseases of compromised energy metabolism.

  10. The lectin BJcuL induces apoptosis through TRAIL expression, caspase cascade activation and mitochondrial membrane permeability in a human colon adenocarcinoma cell line.

    Science.gov (United States)

    Damasio, Danusa de Castro; Nolte, Stefanie; Polak, Leonardo Puchetti; Brandt, Anna Paula; Bonan, Natália Borges; Zischler, Luciana; Stuelp-Campelo, Patrícia M; Cadena, Silvia Maria S C; Noronha, Lúcia de; Elífio-Esposito, Selene L; Moreno-Amaral, Andréa Novais

    2014-11-01

    It has been demonstrated that the cytotoxic effect of BJcuL, the lectin isolated from Bothrops jararacussu venom, on human gastric carcinoma is accompanied by the inhibition of extracellular matrix adhesion, cytoskeleton disassembly and apoptosis induction. The present study aimed to evaluate the apoptosis mechanisms triggered by the BJcuL interaction with specific glycans on the surface of HT29 human colon adenocarcinoma cells. The results demonstrated that BJcuL interacts with glycoligands targets on the cell, which were inhibited in the presence of d-galactose. It shows a dose-dependently cytotoxic effect that is inhibited in the presence of d-galactose. A dose-dependent cell aggregation decrease was also observed for the HT29 cells. Analysis of cell proliferation inhibition was assessed by anti-PCNA and demonstrated that lectin diminishes PCNA expression when compared with untreated cells. Differences in apoptotic marker expression estimated by immunohistochemistry revealed that the lectin promotes an increase in TRAIL expression, leading to an increase in the expression of FADD, caspase-8 and Bax. Besides the increased expression of apoptosis-related proteins, our results revealed that the lectin promotes a mitochondrial respiration decrease and a 75% increase in the amount of cytochrome c released. Together these results suggest that the cytotoxicity of BJcuL can sensitize pro-apoptotic proteins in the cytoplasm and mitochondria, leading to the apoptotic cascade.

  11. Quantitative affinity chromatographic studies of mitochondrial cytochrome c binding to bacterial photosynthetic reaction center, reconstituted in liposome membranes and immobilized by detergent dialysis and avidin--biotin binding.

    Science.gov (United States)

    Yang, Q; Liu, X Y; Hara, M; Lundahl, P; Miyake, J

    2000-04-10

    In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 micromol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20-0.57 microM. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.

  12. Membrane integrity, mitochondrial activity, ATP content, and motility of the European catfish (Silurus glanis) testicular spermatozoa after freezing with different cryoprotectants.

    Science.gov (United States)

    Ogier de Baulny, B; Labbé, C; Maisse, G

    1999-09-01

    The extent of cellular damage was investigated after freeze-thawing of the European catfish testicular sperm with various cryoprotectants. The best protection was given by dimethylacetamide (10 and 15%) in a sucrose solution. Under these conditions, the percentage of cells with an intact membrane was high (90%), and the protection of the activity of the mitochondria was medium (47%). It was shown that the addition of dimethylacetamide largely increased the ATP content of the spermatozoa. It is suggested that this phenomenon is a decisive factor for the freezing resistance of European catfish testicular spermatozoa in the presence of dimethylacetamide (60% motility after thawing versus 90% before freezing).

  13. MARCH5 inactivation supports mitochondrial function during neurodegenerative stress

    Directory of Open Access Journals (Sweden)

    Lei eFang

    2013-10-01

    Full Text Available Neuronal cell death is accompanied by mitochondrial dysfunction with mitochondrial maintenance critical to neuronal survival. The mitochondrial ubiquitin ligase MARCH5 has dual roles in the upkeep of mitochondrial function. MARCH5 is involved in targeted degradation of proteins harmful to mitochondria and impacts mitochondrial morphology upstream of the fission protein Drp1. In a neuronal cell model, dominant-negative MARCH5 prevents mitochondrial fragmentation during neurodegenerative stress induced by the neuron-specific reactive oxygen generator 6 hydroxydopamine, the complex I inhibitor rotenone or Alzheimer’s-releated Aβ peptide. In addition, preservation of mitochondrial function in terms of membrane potential and lower reactive oxygen generation was observed following inactivation of MARCH5. Our findings connect MARCH5 to neuronal stress responses and further emphasize the link between mitochondrial dynamics and function.

  14. Normal skin and hypertrophic scar fibroblasts differentially regulate collagen and fibronectin expression as well as mitochondrial membrane potential in response to basic fibroblast growth factor

    Directory of Open Access Journals (Sweden)

    Rui Song

    2011-05-01

    Full Text Available Basic fibroblast growth factor (bFGF regulates skin wound healing; however, the underlying mechanism remains to be defined. In the present study, we determined the effects of bFGF on the regulation of cell growth as well as collagen and fibronectin expression in fibroblasts from normal human skin and from hypertrophic scars. We then explored the involvement of mitochondria in mediating bFGF-inducedeffects on the fibroblasts. We isolated and cultivated normal and hypertrophic scar fibroblasts from tissue biopsies of patients who underwent plastic surgery for repairing hypertrophic scars. The fibroblasts were then treated with different concentrations of bFGF (ranging from 0.1 to 1000 ng/mL. The growth of hypertrophic scar fibroblasts became slower with selective inhibition of type I collagen production after exposure to bFGF. However, type III collagen expression was affected in both normal and hypertrophic scar fibroblasts. Moreover, fibronectin expression in the normal fibroblasts was up-regulated after bFGF treatment. bFGF (1000 ng/mL also induced mitochondrial depolarization in hypertrophic scar fibroblasts (P < 0.01. The cellular ATP level decreased in hypertrophic scar fibroblasts (P < 0.05, while it increased in the normal fibroblasts following treatment with bFGF (P < 0.01. These data suggest that bFGF has differential effects and mechanisms on fibroblasts of the normal skin and hypertrophic scars, indicating that bFGF may play a role in the early phase of skin wound healing and post-burn scar formation.

  15. Fisetin inhibits growth, induces G₂ /M arrest and apoptosis of human epidermoid carcinoma A431 cells: role of mitochondrial membrane potential disruption and consequent caspases activation.

    Science.gov (United States)

    Pal, Harish C; Sharma, Samriti; Elmets, Craig A; Athar, Mohammad; Afaq, Farrukh

    2013-07-01

    Non-melanoma skin cancers (NMSCs), one of the most common neoplasms, cause serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and antiproliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fisetin (5-80 μm) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G₂ /M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2; Bcl-xL and Mcl-1); (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad); (iii) disruption of mitochondrial potential; (iv) release of cytochrome c and Smac/DIABLO from mitochondria; (v) activation of caspases; and (vi) cleavage of Poly(ADP-ribose) polymerase (PARP) protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  16. Fisetin inhibits growth, induces G2/M arrest and apoptosis of human epidermoid carcinoma A431 cells: Role of mitochondrial membrane potential disruption and consequent caspases activation

    Science.gov (United States)

    Pal, Harish C.; Sharma, Samriti; Elmets, Craig A.; Athar, Mohammad; Afaq, Farrukh

    2013-01-01

    Non-melanoma skin cancers (NMSCs) one of the most common neoplasms causes serious morbidity and mortality. Therefore, identification of non-toxic phytochemicals for prevention/treatment of NMSCs is highly desirable. Fisetin (3,3′,4′,7-tetrahydroxyflavone), a dietary flavonoid, present in fruits and vegetables possesses anti-oxidant and anti-proliferative properties. The aim of this study was to investigate the chemotherapeutic potential of fisetin in cultured human epidermoid carcinoma A431 cells. Treatment of A431 cells with fistein (5-80 μM) resulted in a significant decrease in cell viability in a dose- and time-dependent manner. Employing clonogenic assay, we found that fisetin treatment significantly reduced colony formation in A431 cells. Fisetin treatment of A431 cells resulted in G2/M arrest and induction of apoptosis. Furthermore, treatment of A431 cells with fisetin resulted in (i) decreased expression of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (ii) increased expression of pro-apoptotic proteins (Bax, Bak and Bad), (iii) disruption of mitochondrial potential, (iv) release of cytchrome c and Smac/DIABLO from mitochondria, (v) activation of caspases, and (vi) cleavage of PARP protein. Pretreatment of A431 cells with the pan-caspase inhibitor (Z-VAD-FMK) blocked fisetin-induced cleavage of caspases and PARP. Taken together, these data provide evidence that fisetin possesses chemotherapeutic potential against human epidermoid carcinoma A431 cells. Overall, these results suggest that fisetin could be developed as a novel therapeutic agent for the management of NMSCs. PMID:23800058

  17. Mitochondrial Machineries for Protein Import and Assembly.

    Science.gov (United States)

    Wiedemann, Nils; Pfanner, Nikolaus

    2017-03-15

    Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics. Expected final online publication date for the Annual Review of Biochemistry Volume 86 is June 20, 2017. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

  18. Typha capensis (Rohrb.)N.E.Br. (bulrush) extract scavenges free radicals, inhibits collagenase activity and affects human sperm motility and mitochondrial membrane potential in vitro: a pilot study.

    Science.gov (United States)

    Henkel, R; Fransman, W; Hipler, U-C; Wiegand, C; Schreiber, G; Menkveld, R; Weitz, F; Fisher, D

    2012-05-01

    The biodiversity in South Africa provides more than 30,000 higher plants, of which more than 3000 are used by traditional healers to treat diseases. Typha capensis (bulrush) is one of the medicinal plants used in South Africa to treat male fertility problems. Considering that South African traditional healers have been recognised by Law and the health benefits of T. capensis have not been scientifically investigated yet, this study aimed at investigating the in vitro effects of aqueous extracts from this plant on male reproductive functions. Both leaves and rhizomes of T. capensis were dried, infused with distilled water and freeze-dried. Motile sperm from 50 men were isolated by swim-up and incubated with 1 μg ml(-1) aqueous extract of Typha rhizome for 1 h at 37 °C. Vitality, motility, sperm production of reactive oxygen species and mitochondrial membrane potential were analysed in the test sample, a control and in the pellet from the swim-up. Results showed that the rhizome extract had significant (P < 0.0001) negative effects on all parameters. The extracts from the leaves and rhizomes revealed dose-dependent inhibitory activity for collagenase and free radical formation. No inhibitory activity for elastase was found. The inhibitory activity for collagenase might indicate possible anti-cancer effects. © 2011 Blackwell Verlag GmbH.

  19. COX assembly factor ccdc56 regulates mitochondrial morphology by affecting mitochondrial recruitment of Drp1.

    Science.gov (United States)

    Ban-Ishihara, Reiko; Tomohiro-Takamiya, Shiho; Tani, Motohiro; Baudier, Jacques; Ishihara, Naotada; Kuge, Osamu

    2015-10-07

    Mitochondria are dynamic organelles that alter their morphology in response to cellular signaling and differentiation through balanced fusion and fission. In this study, we found that the mitochondrial inner membrane ATPase ATAD3A interacted with ccdc56/MITRAC12/COA3, a subunit of the cytochrome oxidase (COX)-assembly complex. Overproduction of ccdc56 in HeLa cells resulted in fragmented mitochondrial morphology, while mitochondria were highly elongated in ccdc56-repressed cells by the defective recruitment of the fission factor Drp1. We also found that mild and chronic inhibition of COX led to mitochondrial elongation, as seen in ccdc56-repressed cells. These results indicate that ccdc56 positively regulates mitochondrial fission via regulation of COX activity and the mitochondrial recruitment of Drp1, and thus, suggest a novel relationship between COX assembly and mitochondrial morphology.

  20. Dynamic organization of the mitochondrial protein import machinery.

    Science.gov (United States)

    Straub, Sebastian P; Stiller, Sebastian B; Wiedemann, Nils; Pfanner, Nikolaus

    2016-11-01

    Mitochondria contain elaborate machineries for the import of precursor proteins from the cytosol. The translocase of the outer mitochondrial membrane (TOM) performs the initial import of precursor proteins and transfers the precursors to downstream translocases, including the presequence translocase and the carrier translocase of the inner membrane, the mitochondrial import and assembly machinery of the intermembrane space, and the sorting and assembly machinery of the outer membrane. Although the protein translocases can function as separate entities in vitro, recent studies revealed a close and dynamic cooperation of the protein import machineries to facilitate efficient transfer of precursor proteins in vivo. In addition, protein translocases were found to transiently interact with distinct machineries that function in the respiratory chain or in the maintenance of mitochondrial membrane architecture. Mitochondrial protein import is embedded in a regulatory network that ensures protein biogenesis, membrane dynamics, bioenergetic activity and quality control.

  1. L-Galactono-1,4-lactone dehydrogenase is an assembly factor of the membrane arm of mitochondrial complex I in Arabidopsis.

    Science.gov (United States)

    Schimmeyer, Joram; Bock, Ralph; Meyer, Etienne H

    2016-01-01

    L-Galactono-1,4-lactone dehydrogenase (GLDH) catalyses the last enzymatic step of the ascorbate biosynthetic pathway in plants. GLDH is localised to mitochondria and several reports have shown that GLDH is associated with complex I of the respiratory chain. In a gldh knock-out mutant, complex I is not detectable, suggesting that GLDH is essential for complex I assembly or stability. GLDH has not been identified as a genuine complex I subunit, instead, it is present in a smaller, lowly abundant version of complex I called complex I*. In addition, GLDH activity has also been detected in smaller protein complexes within mitochondria membranes. Here, we investigated the role of GLDH during complex I assembly. We identified GLDH in complexes co-localising with some complex I assembly intermediates. Using a mutant that accumulates complex I assembly intermediates, we confirmed that GLDH is associated with the complex I assembly intermediates of 400 and 450 kDa. In addition, we detected accumulation of the 200 kDa complex I assembly intermediate in the gldh mutant. Taken together, our data suggest that GLDH is an assembly factor of the membrane arm of complex I. This function appears to be independent of the role of GLDH in ascorbate synthesis, as evidenced by the ascorbate-deficient mutant vtc2-1 accumulating wild-type levels of complex I. Therefore, we propose that GLDH is a dual-function protein that has a second, non-enzymatic function in complex I assembly as a plant-specific assembly factor. We propose an updated model for complex I assembly that includes complex I* as an assembly intermediate.

  2. Mitochondrial Extrusion through the cytoplasmic vacuoles during cell death.

    Science.gov (United States)

    Nakajima, Akihito; Kurihara, Hidetake; Yagita, Hideo; Okumura, Ko; Nakano, Hiroyasu

    2008-08-29

    Under various conditions, noxious stimuli damage mitochondria, resulting in mitochondrial fragmentation; however, the mechanisms by which fragmented mitochondria are eliminated from the cells remain largely unknown. Here we show that cytoplasmic vacuoles originating from the plasma membrane engulfed fragmented mitochondria and subsequently extruded them into the extracellular spaces in undergoing acute tumor necrosis factor alpha-induced cell death in a caspase-dependent fashion. Notably, upon fusion of the membrane encapsulating mitochondria to the plasma membrane, naked mitochondria were released into the extracellular spaces in an exocytotic manner. Mitochondrial extrusion was specific to tumor necrosis factor alpha-induced cell death, because a genotoxic stress-inducing agent such as cisplatin did not elicit mitochondrial extrusion. Moreover, intact actin and tubulin cytoskeletons were required for mitochondrial extrusion as well as membrane blebbing. Furthermore, fragmented mitochondria were engulfed by cytoplasmic vacuoles and extruded from hepatocytes of mice injected with anti-Fas antibody, suggesting that mitochondrial extrusion can be observed in vivo under pathological conditions. Mitochondria are eliminated during erythrocyte maturation under physiological conditions, and anti-mitochondrial antibody is detected in some autoimmune diseases. Thus, elucidating the mechanism underlying mitochondrial extrusion will open a novel avenue leading to better understanding of various diseases caused by mitochondrial malfunction as well as mitochondrial biology.

  3. Intermittent hypoxia protects cerebral mitochondrial function from calcium overload.

    Science.gov (United States)

    Chen, Jian; Liao, Weigong; Gao, Wenxiang; Huang, Jian; Gao, Yuqi

    2013-12-01

    Hypoxia leads to Ca(2+) overload and results in mitochondrial uncoupling, decreased ATP synthesis, and neuronal death. Inhibition of mitochondrial Ca(2+) overload protects mitochondrial function after hypoxia. The present study was aimed to investigate the effect of intermittent hypoxia on mitochondrial function and mitochondrial tolerance to Ca(2+) overload. Wistar rats were divided into control and intermittent hypoxia (IH) groups. The IH group was subject to hypoxia for 4 h daily in a hypobaric cabin (5,000 m) for 7 days. Brain mitochondria were isolated on day 7 following hypoxia. The baseline mitochondrial functions, such as ST3, ST4, and respiratory control ratio (RCR = ST3/ST4), were measured using a Clark-type oxygen electrode. Mitochondrial adenine nucleotide concentrations were measured by HPLC. Mitochondrial membrane potential was determined by measuring rhodamine 123 (Rh-123) fluorescence in the absence and presence of high Ca(2+) concentration (0.1 M), which simulates Ca(2+) overload. Our results revealed that IH did not affect mitochondrial respiratory functions, but led to a reduction in AMP and an increase in ADP concentrations in mitochondria. Both control and IH groups demonstrated decreased mitochondrial membrane potential in the presence of high Ca(2+) (0.1 M), while the IH group showed a relative higher mitochondrial membrane potential. These results indicated that the neuroprotective effect of intermittent hypoxia was resulted partly from preserving mitochondrial membrane potential, and increasing mitochondrial tolerance to high calcium levels. The increased ADP and decreased AMP in mitochondria following intermittent hypoxia may be a mechanism underlying this protection.

  4. Melatonin protects against common deletion of mitochondrial DNA-augmented mitochondrial oxidative stress and apoptosis.

    Science.gov (United States)

    Jou, Mei-Jie; Peng, Tsung-I; Yu, Pai-Zu; Jou, Shuo-Bin; Reiter, Russel J; Chen, Jin-Yi; Wu, Hong-Yueh; Chen, Chih-Chun; Hsu, Lee-Fen

    2007-11-01

    Defected mitochondrial respiratory chain (RC), in addition to causing a severe ATP deficiency, often augments reactive oxygen species (ROS) generation in mitochondria (mROS) which enhances pathological conditions and diseases. Previously, we demonstrated a potent endogenously RC defect-augmented mROS associated dose-dependently with a commonly seen large-scale deletion of 4977 base pairs of mitochondrial DNA (mtDNA), i.e. the common deletion (CD). As current treatments for CD-associated diseases are rather supplementary and ineffective, we investigated whether melatonin, a potential mitochondrial protector, provides beneficial protection for CD-augmented mitochondrial oxidative stress and apoptosis particularly upon the induction of a secondary oxidative stress. Detailed mechanistic investigations were performed by using laser scanning dual fluorescence imaging microscopy to provide precise spatial and temporal resolution of mitochondrial events at single cell level. We demonstrate, for the first time, that melatonin significantly prevents CD-augmented mROS formation under basal conditions as well as at early time-points upon secondary oxidative stress induced by H2O2 exposure. Thus, melatonin prevents mROS-mediated depolarization of mitochondrial membrane potential (DeltaPsim) and subsequent opening of the mitochondrial permeability transition pore (MPTP) and cytochrome c release. Moreover, melatonin prevents depletion of cardiolipin which appears to be crucial for postponing later MPTP opening, disruption of the mitochondrial membrane and apoptosis. Finally, the protection provided by melatonin is superior to those caused by the suppression of mitochondrial Ca2+ regulators including the mitochondrial Na+-Ca2) exchanger, the MPTP, and the mitochondrial Ca2+ uniporter and by antioxidants including vitamin E and mitochondria-targeted coenzyme Q, MitoQ. As RC defect-augmented endogenous mitochondrial oxidative stress is centrally involved in a variety of pathological

  5. What Is Mitochondrial DNA?

    Science.gov (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  6. Mitochondrial endonuclease G mediates breakdown of paternal mitochondria upon fertilization.

    Science.gov (United States)

    Zhou, Qinghua; Li, Haimin; Li, Hanzeng; Nakagawa, Akihisa; Lin, Jason L J; Lee, Eui-Seung; Harry, Brian L; Skeen-Gaar, Riley Robert; Suehiro, Yuji; William, Donna; Mitani, Shohei; Yuan, Hanna S; Kang, Byung-Ho; Xue, Ding

    2016-07-22

    Mitochondria are inherited maternally in most animals, but the mechanisms of selective paternal mitochondrial elimination (PME) are unknown. While examining fertilization in Caenorhabditis elegans, we observed that paternal mitochondria rapidly lose their inner membrane integrity. CPS-6, a mitochondrial endonuclease G, serves as a paternal mitochondrial factor that is critical for PME. We found that CPS-6 relocates from the intermembrane space of paternal mitochondria to the matrix after fertilization to degrade mitochondrial DNA. It acts with maternal autophagy and proteasome machineries to promote PME. Loss of cps-6 delays breakdown of mitochondrial inner membranes, autophagosome enclosure of paternal mitochondria, and PME. Delayed removal of paternal mitochondria causes increased embryonic lethality, demonstrating that PME is important for normal animal development. Thus, CPS-6 functions as a paternal mitochondrial degradation factor during animal development.

  7. Parkin suppresses Drp1-independent mitochondrial division.

    Science.gov (United States)

    Roy, Madhuparna; Itoh, Kie; Iijima, Miho; Sesaki, Hiromi

    2016-07-01

    The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson's disease-associated protein-parkin, which biochemically and genetically interacts with Drp1-in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division.

  8. Piracetam improves mitochondrial dysfunction following oxidative stress.

    Science.gov (United States)

    Keil, Uta; Scherping, Isabel; Hauptmann, Susanne; Schuessel, Katin; Eckert, Anne; Müller, Walter E

    2006-01-01

    1.--Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging. 2.--Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction following oxidative stress was investigated using PC12 cells and dissociated brain cells of animals treated with piracetam. 3.--Piracetam treatment at concentrations between 100 and 1000 microM improved mitochondrial membrane potential and ATP production of PC12 cells following oxidative stress induced by sodium nitroprusside (SNP) and serum deprivation. Under conditions of mild serum deprivation, piracetam (500 microM) induced a nearly complete recovery of mitochondrial membrane potential and ATP levels. Piracetam also reduced caspase 9 activity after SNP treatment. 4.--Piracetam treatment (100-500 mg kg(-1) daily) of mice was also associated with improved mitochondrial function in dissociated brain cells. Significant improvement was mainly seen in aged animals and only less in young animals. Moreover, the same treatment reduced antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in aged mouse brain only, which are elevated as an adaptive response to the increased oxidative stress with aging. 5.--In conclusion, therapeutically relevant in vitro and in vivo concentrations of piracetam are able to improve mitochondrial dysfunction associated with oxidative stress and/or aging. Mitochondrial stabilization and protection might be an important mechanism to explain many of piracetam's beneficial effects in elderly patients.

  9. Mitochondrial DNA repair and association with aging--an update

    DEFF Research Database (Denmark)

    Diaz, Ricardo Gredilla; Bohr, Vilhelm A; Stevnsner, Tinna V.

    2010-01-01

    Mitochondrial DNA is constantly exposed to oxidative injury. Due to its location close to the main site of reactive oxygen species, the inner mitochondrial membrane, mtDNA is more susceptible than nuclear DNA to oxidative damage. The accumulation of DNA damage is thought to play a critical role...

  10. Grape seed proanthocyanidins promote apoptosis in human epidermoid carcinoma A431 cells through alterations in Cdki-Cdk-cyclin cascade, and caspase-3 activation via loss of mitochondrial membrane potential.

    Science.gov (United States)

    Meeran, Syed M; Katiyar, Santosh K

    2007-05-01

    Dietary grape seed proanthocyanidins (GSPs) prevent photocarcinogenesis in mice. Here, we report that in vitro treatment of human epidermoid carcinoma A431 cells with GSPs inhibited cellular proliferation (13-89%) and induced cell death (1-48%) in a dose (5-100 mug/ml)- and time (24, 48 and 72 h)-dependent manner. GSP-induced inhibition of cell proliferation was associated with an increase in G1-phase arrest at 24 h, which was mediated through the inhibition of cyclin-dependent kinases (Cdk) Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and simultaneous increase in protein expression of cyclin-dependent kinase inhibitors (Cdki), Cip1/p21 and Kip1/p27, and enhanced binding of Cdki-Cdk. The treatment of A431 cells with GSPs (20-80 mug/ml) resulted in a dose-dependent increase in apoptotic cell death (26-58%), which was associated with an increased protein expression of proapoptotic Bax, decreased expression of antiapoptotic Bcl-2 and Bcl-xl, loss of mitochondrial membrane potential, and cleavage of caspase-9, caspase-3 and PARP. Pretreatment with the pan-caspase inhibitor (z-VAD-fmk) blocked the GSP-induced apoptosis in A431 cells suggesting that GSP-induced apoptosis is associated primarily with the caspase-3-dependent pathway. Together, our study suggests that GSPs possess chemotherapeutic potential against human epidermoid carcinoma cells in vitro, further in vivo mechanistic studies are required to verify the chemotherapeutic effect of GSPs in skin cancers.

  11. Berberine inhibits growth, induces G1 arrest and apoptosis in human epidermoid carcinoma A431 cells by regulating Cdki-Cdk-cyclin cascade, disruption of mitochondrial membrane potential and cleavage of caspase 3 and PARP.

    Science.gov (United States)

    Mantena, Sudheer K; Sharma, Som D; Katiyar, Santosh K

    2006-10-01

    Chemotherapeutic approach using non-toxic botanicals may be one of the strategies for the management of the skin cancers. Here we report that in vitro treatment of human epidermoid carcinoma A431 cells with berberine, a naturally occurring isoquinoline alkaloid, decreased cell viability (3-77%, P berberine-induced G(1) cell cycle arrest was mediated through the increased expression of Cdki proteins (Cip1/p21 and Kip1/p27), a simultaneous decrease in Cdk2, Cdk4, Cdk6 and cyclins D1, D2 and E and enhanced binding of Cdki-Cdk. In additional studies, treatment of A431 cells with berberine (15-75 microM) for 72 h resulted in a significant dose-dependent increase in apoptosis (31-60%, P berberine-treated control (11.7%), which was associated with an increased expression of pro-apoptotic protein Bax, decreased expression of anti-apoptotic proteins Bcl-2 and Bcl-xl, disruption of mitochondrial membrane potential, and activation of caspases 9, 3 and poly (ADP-ribose) polymerase. Pretreatment of A431 cells with the pan-caspase inhibitor (z-VAD-fmk) significantly blocked the berberine-induced apoptosis in A431 cells confirmed that berberine-induced apoptosis is mediated through activation of caspase 3-dependent pathway. Together, this study for the first time identified berberine as a chemotherapeutic agent against human epidermoid carcinoma A431 cells in vitro, further in vivo studies are required to determine whether berberine could be an effective chemotherapeutic agent for the management of non-melanoma skin cancers.

  12. Nuclear responses to depletion of mitochondrial DNA in human cells.

    Science.gov (United States)

    Li, K; Neufer, P D; Williams, R S

    1995-11-01

    The derivation of human cell lines devoid of mitochondrial (mt) DNA (rho 0) provides an opportunity to study nuclear responses to a chronic impairment of mitochondrial oxidative phosphorylation. Expression of several nuclear genes is induced in human rho 0 cells, including those encoding integral proteins of the mitochondrial inner membrane, intermediate filaments, and ribosomes. In contrast to conditions in which mitochondrial respiration is altered acutely, expression of heat shock proteins and immediate early genes is not induced. Mitochondria from rho 0 cells maintain a transmembrane electrochemical potential and are distributed within the cytoplasm of these cells in a manner indistinguishable from that of wild-type cells. We conclude that a chronic deficiency of mitochondrial oxidative phosphorylation produced by elimination of mtDNA is associated with a different pattern of gene induction than that provoked by other acute or subacute conditions that impair mitochondrial respiration or create energy demands in excess of mitochondrial respiratory capacity.

  13. Mitochondrial fusion, fission, and mitochondrial toxicity.

    Science.gov (United States)

    Meyer, Joel N; Leuthner, Tess C; Luz, Anthony L

    2017-08-05

    Mitochondrial dynamics are regulated by two sets of opposed processes: mitochondrial fusion and fission, and mitochondrial biogenesis and degradation (including mitophagy), as well as processes such as intracellular transport. These processes maintain mitochondrial homeostasis, regulate mitochondrial form, volume and function, and are increasingly understood to be critical components of the cellular stress response. Mitochondrial dynamics vary based on developmental stage and age, cell type, environmental factors, and genetic background. Indeed, many mitochondrial homeostasis genes are human disease genes. Emerging evidence indicates that deficiencies in these genes often sensitize to environmental exposures, yet can also be protective under certain circumstances. Inhibition of mitochondrial dynamics also affects elimination of irreparable mitochondrial DNA (mtDNA) damage and transmission of mtDNA mutations. We briefly review the basic biology of mitodynamic processes with a focus on mitochondrial fusion and fission, discuss what is known and unknown regarding how these processes respond to chemical and other stressors, and review the literature on interactions between mitochondrial toxicity and genetic variation in mitochondrial fusion and fission genes. Finally, we suggest areas for future research, including elucidating the full range of mitodynamic responses from low to high-level exposures, and from acute to chronic exposures; detailed examination of the physiological consequences of mitodynamic alterations in different cell types; mechanism-based testing of mitotoxicant interactions with interindividual variability in mitodynamics processes; and incorporating other environmental variables that affect mitochondria, such as diet and exercise. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. 碘过量对大鼠甲状腺细胞线粒体超氧化物生成和膜电位的影响%Effects of iodine excess on mitochondrial superoxide production and mitochondrial membrane potential in rat thyroid cell line cells

    Institute of Scientific and Technical Information of China (English)

    李敏; 姚小梅; 陈祖培; 李兰英

    2010-01-01

    Objective To investigate the effects of iodine excess on mitochondrial superoxide production and mitoehondrial membrane potential(△ψ)changes in Fisher rat thyroid cell line(FRTL)cells.Methods FRTL cells were treated with 10-4mol/L potassium iodine(KI),10 U/L thyrotropin(TSH),10-4 mol/L KI+10 U/L TSH respectively for 24 h.Effects on cell proliferation were assayed by methyl thiazolyl tetrazolium(MTT)colorimetric method.Changes of mitochondrial superoxide production and △ψ were measured by live cell imaging and spectrofluorometer using MitoSOX and rhodamine 123(rh123)respectively.Results Absorbance(A)in the KI group (0.794±0.144)showed a significant decline compared to the control group(1.000 ±0.183,P0.05),but the former was marked higher than the KI group(P 0.05). Conclusion Iodine excess (10-4 mol/L KI) may lead to peroxide damage on the mitochondria of FRTL cells, and cell proliferation is inhibited. Combining treatment with 10 U/L TSH may attenuate mitochondrial peroxide damage and inhibition of cell proliferation caused by iodine excess.%目的 探讨碘过量对Fisher大鼠甲状腺细胞(FRTL)线粒体超氧化物生成和膜电位(△ψ)的影响.方法 FRTL细胞分别以10-4mol/L碘化钾(KI)、10 U/L促甲状腺素(TSH)、10-4mol/L KI+10 U/L TSH 处理24 h,利用甲基噻唑基四唑(MTT)比色法检测FRTL细胞增殖情况,利用MitoSOX探针通过活细胞影像法检测线粒体超氧化物生成,利用罗丹明123(rh123)通过荧光分光光度计检测△ψ的变化.结果 细胞增殖情况,KI组(0.794±0.144)明显低于对照组(1.000±0.183,P0.05),但明显高于KI组(P0.05).结论 碘过量(10-4mol/L KI)能造成FRTL细胞线粒体过氧化损伤,抑制细胞增殖,10 U/L TSH能够促进FRTL细胞增殖,减轻碘过量对FRTL细胞线粒体的过氧化损伤.

  15. Mitochondrial Cristae Shape Determines Respiratory Chain Supercomplexes Assembly and Respiratory Efficiency

    OpenAIRE

    Cogliati, Sara; Frezza, Christian; Soriano, Maria Eugenia; Varanita, Tatiana; Quintana-Cabrera, Ruben; Corrado, Mauro; Cipolat, Sara; Costa, Veronica; Casarin, Alberto; Gomes, Ligia C.; Perales-Clemente, Ester; Salviati, Leonardo; Fernandez-Silva, Patricio; Enriquez, Jose A.; Scorrano, Luca

    2013-01-01

    Summary Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity o...

  16. Effects of genistein and folic acid on neuronal membrane and mitochondrial membrane damaged by β-amyloid peptides 31-35%染料木黄酮和叶酸对β淀粉样肽31-35诱导的细胞及线粒体膜损伤的拮抗作用

    Institute of Scientific and Technical Information of China (English)

    余焕玲; 张晓红; 肖荣; 李丽; 向丽; 封锦芳; 苑林宏; 麻微微

    2010-01-01

    Objective To observe the neuro-protective effects of genistein (Gen) and folic acid (FA) on neurons membrane and mitochondrial membrane damaged by β-amyloid peptides 31-35 (Aβ31-35).Methods The primary cultured rat cerebral cortical neurons were randomly divided into DMEM(control) ,Aβ31-35 (25 μmol/L) ,Gen( Gen 27 μg/ml) ,FA( FA 40 μg/ml) and Gen + FA( Gen 27 μg/ml + FA 40 μg/ml).Gen and/or FA were added two hours before Aβ31-35 addition.After twenty four hours, MTT assay was performed to measure the viability of cultured neurons.Fluorescence polarization was performed to observe the neuron cell membrane fluidity.The mitochondrial membrane potential(MMP) was determined to investigate the alteration of mitochondrial structure and function of neurons by laser scanning confocal microscope and a flow cytometer was used to investigate the activation of mitochondrial permeability transition pore (MPTP).Each experiment was repeated three times.Results Compared with group Aβ31-35 (0.845 ± 0.050, F = 4.931, P < 0.05 =, the absorbance was significantly higher in group Gen (0.982 ± 0.110, t=3.523,P<0.01=,FA (0.947 ±0.061,t=2.745,P<0.01= and Gen+ FA (0.996 ± 0.090, t = 3.966, P < 0.01 =.The viscosity of cell neuron membrane in group Gen ( 1.75 ± 0.28,t=2.085,P<0.05=,FA (1.66±0.37,t=2.357,P<0.05= andGen + FA (1.50±0.20,t=3.784,P < 0.05 = was significantly lower than that in group Aβ31-35 (2.11 ± 0.44, F = 5.529, P < 0.01 =, which indicated the cell membrane fluidity was significantly higher in group Gen and/or FA than that in group Aβ31-35.MMP was significantly decreased by Aβ31-35 (3.364 ± 1.140, t= 3.949, P< 0.01 = when comparing to control group (6.383 ± 1.683) ,while it was significantly increased by Gen (5.286 ± 1.792,t=2.406,P<0.05=,FA (5.884±2.022,t=2.887,P<0.01= and Gen + FA (6.120 ±2.124,t=3.304,P < 0.01 = when comparing to group Aβ31-35 ( F = 7.585, P < 0.01 =.MPTP was activated by Aβ31

  17. Mitochondrial DNA Alterations and Reduced Mitochondrial Function in Aging

    OpenAIRE

    Hebert, Sadie L.; Lanza, Ian R.; Nair, K. Sreekumaran

    2010-01-01

    Oxidative damage to mitochondrial DNA increases with aging. This damage has the potential to affect mitochondrial DNA replication and transcription which could alter the abundance or functionality of mitochondrial proteins. This review describes mitochondrial DNA alterations and changes in mitochondrial function that occur with aging. Age-related alterations in mitochondrial DNA as a possible contributor to the reduction in mitochondrial function are discussed.

  18. Protein carbonylation and adipocyte mitochondrial function.

    Science.gov (United States)

    Curtis, Jessica M; Hahn, Wendy S; Stone, Matthew D; Inda, Jacob J; Droullard, David J; Kuzmicic, Jovan P; Donoghue, Margaret A; Long, Eric K; Armien, Anibal G; Lavandero, Sergio; Arriaga, Edgar; Griffin, Timothy J; Bernlohr, David A

    2012-09-21

    Carbonylation is the covalent, non-reversible modification of the side chains of cysteine, histidine, and lysine residues by lipid peroxidation end products such as 4-hydroxy- and 4-oxononenal. In adipose tissue the effects of such modifications are associated with increased oxidative stress and metabolic dysregulation centered on mitochondrial energy metabolism. To address the role of protein carbonylation in the pathogenesis of mitochondrial dysfunction, quantitative proteomics was employed to identify specific targets of carbonylation in GSTA4-silenced or overexpressing 3T3-L1 adipocytes. GSTA4-silenced adipocytes displayed elevated carbonylation of several key mitochondrial proteins including the phosphate carrier protein, NADH dehydrogenase 1α subcomplexes 2 and 3, translocase of inner mitochondrial membrane 50, and valyl-tRNA synthetase. Elevated protein carbonylation is accompanied by diminished complex I activity, impaired respiration, increased superoxide production, and a reduction in membrane potential without changes in mitochondrial number, area, or density. Silencing of the phosphate carrier or NADH dehydrogenase 1α subcomplexes 2 or 3 in 3T3-L1 cells results in decreased basal and maximal respiration. These results suggest that protein carbonylation plays a major instigating role in cytokine-dependent mitochondrial dysfunction and may be linked to the development of insulin resistance in the adipocyte.

  19. Trichothecene Mycotoxins Inhibit Mitochondrial Translation—Implication for the Mechanism of Toxicity

    Directory of Open Access Journals (Sweden)

    Susan McCormick

    2011-12-01

    Full Text Available Fusarium head blight (FHB reduces crop yield and results in contamination of grains with trichothecene mycotoxins. We previously showed that mitochondria play a critical role in the toxicity of a type B trichothecene. Here, we investigated the direct effects of type A and type B trichothecenes on mitochondrial translation and membrane integrity in Saccharomyces cerevisiae. Sensitivity to trichothecenes increased when functional mitochondria were required for growth, and trichothecenes inhibited mitochondrial translation at concentrations, which did not inhibit total translation. In organello translation in isolated mitochondria was inhibited by type A and B trichothecenes, demonstrating that these toxins have a direct effect on mitochondrial translation. In intact yeast cells trichothecenes showed dose-dependent inhibition of mitochondrial membrane potential and reactive oxygen species, but only at doses higher than those affecting mitochondrial translation. These results demonstrate that inhibition of mitochondrial translation is a primary target of trichothecenes and is not secondary to the disruption of mitochondrial membranes.

  20. Differential retrotranslocation of mitochondrial Bax and Bak

    Science.gov (United States)

    Todt, Franziska; Cakir, Zeynep; Reichenbach, Frank; Emschermann, Frederic; Lauterwasser, Joachim; Kaiser, Andrea; Ichim, Gabriel; Tait, Stephen WG; Frank, Stephan; Langer, Harald F; Edlich, Frank

    2015-01-01

    The Bcl-2 proteins Bax and Bak can permeabilize the outer mitochondrial membrane and commit cells to apoptosis. Pro-survival Bcl-2 proteins control Bax by constant retrotranslocation into the cytosol of healthy cells. The stabilization of cytosolic Bax raises the question whether the functionally redundant but largely mitochondrial Bak shares this level of regulation. Here we report that Bak is retrotranslocated from the mitochondria by pro-survival Bcl-2 proteins. Bak is present in the cytosol of human cells and tissues, but low shuttling rates cause predominant mitochondrial Bak localization. Interchanging the membrane anchors of Bax and Bak reverses their subcellular localization compared to the wild-type proteins. Strikingly, the reduction of Bax shuttling to the level of Bak retrotranslocation results in full Bax toxicity even in absence of apoptosis induction. Thus, fast Bax retrotranslocation is required to protect cells from commitment to programmed death. PMID:25378477

  1. Aspirin increases mitochondrial fatty acid oxidation.

    Science.gov (United States)

    Uppala, Radha; Dudiak, Brianne; Beck, Megan E; Bharathi, Sivakama S; Zhang, Yuxun; Stolz, Donna B; Goetzman, Eric S

    2017-01-08

    The metabolic effects of salicylates are poorly understood. This study investigated the effects of aspirin on fatty acid oxidation. Aspirin increased mitochondrial long-chain fatty acid oxidation, but inhibited peroxisomal fatty acid oxidation, in two different cell lines. Aspirin increased mitochondrial protein acetylation and was found to be a stronger acetylating agent in vitro than acetyl-CoA. However, aspirin-induced acetylation did not alter the activity of fatty acid oxidation proteins, and knocking out the mitochondrial deacetylase SIRT3 did not affect the induction of long-chain fatty acid oxidation by aspirin. Aspirin did not change oxidation of medium-chain fatty acids, which can freely traverse the mitochondrial membrane. Together, these data indicate that aspirin does not directly alter mitochondrial matrix fatty acid oxidation enzymes, but most likely exerts its effects at the level of long-chain fatty acid transport into mitochondria. The drive on mitochondrial fatty acid oxidation may be a compensatory response to altered mitochondrial morphology and inhibited electron transport chain function, both of which were observed after 24 h incubation of cells with aspirin. These studies provide insight into the pathophysiology of Reye Syndrome, which is known to be triggered by aspirin ingestion in patients with fatty acid oxidation disorders. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Melatonin in Mitochondrial Dysfunction and Related Disorders

    Directory of Open Access Journals (Sweden)

    Venkatramanujam Srinivasan

    2011-01-01

    Full Text Available Mitochondrial dysfunction is considered one of the major causative factors in the aging process, ischemia/reperfusion (I/R, septic shock, and neurodegenerative disorders like Parkinson's disease (PD, Alzheimer's disease (AD, and Huntington's disease (HD. Increased free radical generation, enhanced mitochondrial inducible nitric oxide (NO synthase activity, enhanced NO production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pore all have been suggested as factors responsible for impaired mitochondrial function. Melatonin, the major hormone of the pineal gland, also acts as an antioxidant and as a regulator of mitochondrial bioenergetic function. Both in vitro and in vivo, melatonin was effective for preventing oxidative stress/nitrosative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. In addition, melatonin is known to retard aging and to inhibit the lethal effects of septic shock or I/R lesions by maintaining respiratory complex activities, electron transport chain, and ATP production in mitochondria. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other antioxidants. Melatonin has thus emerged as a major potential therapeutic tool for treating neurodegenerative disorders such as PD or AD, and for preventing the lethal effects of septic shock or I/R.

  3. Ethambutol-induced optic neuropathy linked to OPA1 mutation and mitochondrial toxicity.

    Science.gov (United States)

    Guillet, Virginie; Chevrollier, Arnaud; Cassereau, Julien; Letournel, Franck; Gueguen, Naïg; Richard, Laurence; Desquiret, Valérie; Verny, Christophe; Procaccio, Vincent; Amati-Bonneau, Patrizia; Reynier, Pascal; Bonneau, Dominique

    2010-03-01

    Ethambutol (EMB), widely used in the treatment of tuberculosis, has been reported to cause Leber's hereditary optic neuropathy in patients carrying mitochondrial DNA mutations. We study the effect of EMB on mitochondrial metabolism in fibroblasts from controls and from a man carrying an OPA1 mutation, in whom the drug induced the development of autosomal dominant optic atrophy (ADOA). EMB produced a mitochondrial coupling defect together with a 25% reduction in complex IV activity. EMB induced the formation of vacuoles associated with decreased mitochondrial membrane potential and increased fragmentation of the mitochondrial network. Mitochondrial genetic variations may therefore be predisposing factors in EMB-induced ocular injury.

  4. Protective role of melatonin in mitochondrial dysfunction and related disorders.

    Science.gov (United States)

    Paradies, Giuseppe; Paradies, Valeria; Ruggiero, Francesca M; Petrosillo, Giuseppe

    2015-06-01

    Mitochondria are the powerhouse of the eukaryotic cell through their use of oxidative phosphorylation to generate ATP. Mitochondrial dysfunction is considered an important contributing factor in a variety of physiopathological situations such as aging, heart ischemia/reperfusion injury, diabetes and several neurodegenerative and cardiovascular diseases, as well as in cell death. Increased formation of reactive oxygen species, altered respiratory chain complexes activity and opening of the mitochondrial permeability transition pore have been suggested as possible factors responsible for impaired mitochondrial function. Therefore, preventing mitochondrial dysfunction could be an effective therapeutic strategy against cellular degenerative processes. Cardiolipin is a unique phospholipid located at the level of inner mitochondrial membrane where it plays an important role in mitochondrial bioenergetics, as well as in cell death. Cardiolipin abnormalities have been associated with mitochondrial dysfunction in a variety of pathological conditions and aging. Melatonin, the major secretory product of the pineal gland, is a well-known antioxidant agent and thus an effective protector of mitochondrial bioenergetic function. Melatonin was reported to prevent mitochondrial dysfunction from oxidative damage by preserving cardiolipin integrity, and this may explain, at least in part, the beneficial effect of this compound in mitochondrial physiopathology. In this article, mechanisms through which melatonin exerts its protective role in mitochondrial dysfunction and related disorders are reviewed.

  5. Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm

    OpenAIRE

    Chai, Ran‐Ran; Chen, Guo‐Wu; Shi, Hui‐Juan; O, Wai‐Sum; Martin‐DeLeon, Patricia A.; Chen, Hong

    2016-01-01

    Abstract Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoas...

  6. Betaine is a positive regulator of mitochondrial respiration

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Icksoo, E-mail: icksoolee@dankook.ac.kr

    2015-01-09

    Highlights: • Betaine enhances cytochrome c oxidase activity and mitochondrial respiration. • Betaine increases mitochondrial membrane potential and cellular energy levels. • Betaine’s anti-tumorigenic effect might be due to a reversal of the Warburg effect. - Abstract: Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

  7. A Biophysical Model of the Mitochondrial ATP-Mg/Pi Carrier

    OpenAIRE

    2012-01-01

    Mitochondrial adenine nucleotide (AdN) content is regulated through the Ca2+-activated, electroneutral ATP-Mg/Pi carrier (APC). The APC is a protein in the mitochondrial carrier super family that localizes to the inner mitochondrial membrane (IMM). It is known to modulate a number of processes that depend on mitochondrial AdN content, such as gluconeogenesis, protein synthesis, and citrulline synthesis. Despite this critical role, a kinetic model of the underlying mechanism has not been devel...

  8. A mechanistic view of mitochondrial death decision pores

    Directory of Open Access Journals (Sweden)

    J.E. Belizário

    2007-08-01

    Full Text Available Mitochondria increase their outer and inner membrane permeability to solutes, protons and metabolites in response to a variety of extrinsic and intrinsic signaling events. The maintenance of cellular and intraorganelle ionic homeostasis, particularly for Ca2+, can determine cell survival or death. Mitochondrial death decision is centered on two processes: inner membrane permeabilization, such as that promoted by the mitochondrial permeability transition pore, formed across inner membranes when Ca2+ reaches a critical threshold, and mitochondrial outer membrane permeabilization, in which the pro-apoptotic proteins BID, BAX, and BAK play active roles. Membrane permeabilization leads to the release of apoptogenic proteins: cytochrome c, apoptosis-inducing factor, Smac/Diablo, HtrA2/Omi, and endonuclease G. Cytochrome c initiates the proteolytic activation of caspases, which in turn cleave hundreds of proteins to produce the morphological and biochemical changes of apoptosis. Voltage-dependent anion channel, cyclophilin D, adenine nucleotide translocase, and the pro-apoptotic proteins BID, BAX, and BAK may be part of the molecular composition of membrane pores leading to mitochondrial permeabilization, but this remains a central question to be resolved. Other transporting pores and channels, including the ceramide channel, the mitochondrial apoptosis-induced channel, as well as a non-specific outer membrane rupture may also be potential release pathways for these apoptogenic factors. In this review, we discuss the mechanistic models by which reactive oxygen species and caspases, via structural and conformational changes of membrane lipids and proteins, promote conditions for inner/outer membrane permeabilization, which may be followed by either opening of pores or a rupture of the outer mitochondrial membrane.

  9. Loss of mitochondrial exo/endonuclease EXOG affects mitochondrial respiration and induces ROS-mediated cardiomyocyte hypertrophy.

    Science.gov (United States)

    Tigchelaar, Wardit; Yu, Hongjuan; de Jong, Anne Margreet; van Gilst, Wiek H; van der Harst, Pim; Westenbrink, B Daan; de Boer, Rudolf A; Silljé, Herman H W

    2015-01-15

    Recently, a locus at the mitochondrial exo/endonuclease EXOG gene, which has been implicated in mitochondrial DNA repair, was associated with cardiac function. The function of EXOG in cardiomyocytes is still elusive. Here we investigated the role of EXOG in mitochondrial function and hypertrophy in cardiomyocytes. Depletion of EXOG in primary neonatal rat ventricular cardiomyocytes (NRVCs) induced a marked increase in cardiomyocyte hypertrophy. Depletion of EXOG, however, did not result in loss of mitochondrial DNA integrity. Although EXOG depletion did not induce fetal gene expression and common hypertrophy pathways were not activated, a clear increase in ribosomal S6 phosphorylation was observed, which readily explains increased protein synthesis. With the use of a Seahorse flux analyzer, it was shown that the mitochondrial oxidative consumption rate (OCR) was increased 2.4-fold in EXOG-depleted NRVCs. Moreover, ATP-linked OCR was 5.2-fold higher. This increase was not explained by mitochondrial biogenesis or alterations in mitochondrial membrane potential. Western blotting confirmed normal levels of the oxidative phosphorylation (OXPHOS) complexes. The increased OCR was accompanied by a 5.4-fold increase in mitochondrial ROS levels. These increased ROS levels could be normalized with specific mitochondrial ROS scavengers (MitoTEMPO, mnSOD). Remarkably, scavenging of excess ROS strongly attenuated the hypertrophic response. In conclusion, loss of EXOG affects normal mitochondrial function resulting in increased mitochondrial respiration, excess ROS production, and cardiomyocyte hypertrophy. Copyright © 2015 the American Physiological Society.

  10. Alterations of the mitochondrial proteome caused by the absence of mitochondrial DNA: A proteomic view

    Science.gov (United States)

    Chevallet, Mireille; Lescuyer, Pierre; Diemer, Hélène; van Dorsselaer, Alain; Leize-Wagner, Emmanuelle; Rabilloud, Thierry

    2006-01-01

    The proper functioning of mitochondria requires that both the mitochondrial and the nuclear genome are functional. To investigate the importance of the mitochondrial genome, which encodes only 13 subunits of the respiratory complexes, the mitochondrial rRNAs and a few tRNAs, we performed a comparative study on the 143B cell line and on its Rho-0 counterpart, i.e. devoid of mitochondrial DNA. Quantitative differences were found, of course in the respiratory complexes subunits, but also in the mitochondrial translation apparatus, mainly mitochondrial ribosomal proteins, and in the ion and protein import system, i.e. including membrane proteins. Various mitochondrial metabolic processes were also altered, especially electron transfer proteins and some dehydrogenases, but quite often on a few proteins for each pathway. This study also showed variations in some hypothetical or poorly characterized proteins, suggesting a mitochondrial localization for these proteins. Examples include a stomatin-like protein and a protein sharing homologies with bacterial proteins implicated in tyrosine catabolism. Proteins involved in apoptosis control are also found modulated in Rho-0 mitochondria. PMID:16548050

  11. Characteristics of mitochondrial calpains.

    Science.gov (United States)

    Ozaki, Taku; Tomita, Hiroshi; Tamai, Makoto; Ishiguro, Sei-Ichi

    2007-09-01

    Calpains are considered to be cytoplasmic enzymes, although several studies have shown that calpain-like protease activities also exist in mitochondria. We partially purified mitochondrial calpain from swine liver mitochondria and characterized. Only one type of mitochondrial calpain was detected by the column chromatographies. The mitochondrial calpain was stained with anti-mu-calpain and calpain small subunit antibodies. The susceptibility of mitochondrial calpain to calpain inhibitors and the optimum pH differ from those of cytosolic mu- and m-calpains. The Ca(2+)-dependency of mitochondrial calpain was similar to that of cytosolic mu-calpain. Therefore, we named the protease mitochondrial mu-like calpain. In zymogram analysis, two types of caseinolytic enzymes existed in mitochondria and showed different mobilities from cytosolic mu- and m-calpains. The upper major band was stained with anti-mu-calpain and calpain small subunit antibodies (mitochondrial calpain I, mitochondrial mu-like calpain). The lower band was stained only with anti-calpain small subunit antibody (mitochondrial calpain II, unknown mitochondrial calpain). Calpastatin was not detected in mitochondrial compartments. The mitochondrial calpain processed apoptosis-inducing factor (AIF) to truncated AIF (tAIF), releasing tAIF into the intermembrane space. These results indicate that mitochondrial calpain, which differs from mu- and m-calpains, seems to be a ubiquitous calpain and may play a role in mitochondrial apoptotic signalling.

  12. Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Tomoyuki [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Saotome, Masao, E-mail: msaotome@hama-med.ac.jp [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Nobuhara, Mamoru; Sakamoto, Atsushi; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Funaki, Makoto [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Hayashi, Hideharu [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan)

    2014-05-01

    Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ{sub m}) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H{sub 2}O{sub 2}), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ{sub m} depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H{sub 2}O{sub 2}-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ{sub m} depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin

  13. Almacenamiento en frío de espermatozoides de trucha arcoiris (Oncorhynchus mykiss: Efectos en la motilidad, superóxido intracelular, integridad de la membrana plasmática y potencial de membrana mitocondrial Cold storage of sperm of rainbow trout (oncorhynchus mykiss: Effect on motility, intracellular superoxide, plasma membrane integrity and mitochondrial membrane potential

    Directory of Open Access Journals (Sweden)

    O Berríos

    2010-01-01

    Full Text Available A diferencia de lo que ocurre en mamíferos, en teleósteos la mayoría de los estudios que evalúan la calidad del semen almacenado están orientados a la exposición de los espermatozoides a algunas especies reactivas de oxígeno (ROS, a la incorporación de antioxidantes en la dieta o a la aplicación de éstos en el plasma seminal. No se encuentran trabajos disponibles que traten la presencia del radical superóxido (O2._, ni la función que éste cumple al interior del espermatozoide cuando se encuentran almacenados. En la presente investigación se evaluó el efecto del almacenamiento en el O2._, motilidad, integridad de la membrana plasmática y potencial de membrana mitocondrial (ΔΨMit en espermatozoides de trucha arcoiris (oncorhynchus mykiss. Para ello se extrajo el semen, el cual fue almacenado durante 12 días a 4 ºC. Cada 4 días se evaluó motilidad, ΔΨMit, integridad de la membrana plasmática y se detectó O2._ intracelular en los espermatozoides. Se encontró un 82,59% de células con tinción positiva para O2._ el día de extracción de la muestra, mientras que la motilidad, ΔΨMit y la integridad de la membrana plasmática, solo mostraron deterioro después del octavo día de almacenamiento. Únicamente el ΔΨMit se correlaciona negativamente con O2._ a partir del octavo día de almacenamiento (r = -0,56 P In teleostei, as opposed to what happens in mammals, most of the studies that evaluate the quality storages of semen are oriented toward the exposure of spermatozoa to some reactive oxygen species (ROS, the utilization of antioxidants in the diet, or the incorporation of these in seminal plasma. There is no available the literature covering the presence of superoxide ions (O2._, or the function of these on the interior of the spermatozoa that have been stored. In this study, we evaluated the effect of storage on intracellular O2._, motility, plasmatic membrane integrity, and mitochondrial membrane potential (

  14. Drug-induced mitochondrial neuropathy in children: a conceptual framework for critical windows of development.

    Science.gov (United States)

    Wallace, Kendall B

    2014-09-01

    Mitochondrial disease arises from genetic or nongenetic events that interfere either directly or indirectly with the bioenergetic function of the mitochondrion and manifest clinically in some form of metabolic disorder. In primary mitochondrial disease, the critical molecular target is one or more of the individual subunits of the respiratory complexes or their assembly and incorporation into the inner mitochondrial membrane, whereas with secondary mitochondrial disease the bioenergetic deficits are secondary to effects on targets other than the electron transport chain and oxidative phosphorylation. Primary genetic events include mutations to or altered expression of proteins targeted to the mitochondrial compartment, whether they are encoded by the nuclear or mitochondrial genome. In this review, we emphasize the occurrence of nongenetic mitochondrial disease resulting from therapeutic drug administration, review the broad scope of drugs implicated in affecting specific primary mitochondrial targets, and describe evidence demonstrating critical windows of risk for the developing neonate to drug-induced mitochondrial disease and neuropathy.

  15. Metabolism. AMP-activated protein kinase mediates mitochondrial fission in response to energy stress.

    Science.gov (United States)

    Toyama, Erin Quan; Herzig, Sébastien; Courchet, Julien; Lewis, Tommy L; Losón, Oliver C; Hellberg, Kristina; Young, Nathan P; Chen, Hsiuchen; Polleux, Franck; Chan, David C; Shaw, Reuben J

    2016-01-15

    Mitochondria undergo fragmentation in response to electron transport chain (ETC) poisons and mitochondrial DNA-linked disease mutations, yet how these stimuli mechanistically connect to the mitochondrial fission and fusion machinery is poorly understood. We found that the energy-sensing adenosine monophosphate (AMP)-activated protein kinase (AMPK) is genetically required for cells to undergo rapid mitochondrial fragmentation after treatment with ETC inhibitors. Moreover, direct pharmacological activation of AMPK was sufficient to rapidly promote mitochondrial fragmentation even in the absence of mitochondrial stress. A screen for substrates of AMPK identified mitochondrial fission factor (MFF), a mitochondrial outer-membrane receptor for DRP1, the cytoplasmic guanosine triphosphatase that catalyzes mitochondrial fission. Nonphosphorylatable and phosphomimetic alleles of the AMPK sites in MFF revealed that it is a key effector of AMPK-mediated mitochondrial fission.

  16. Parkin suppresses Drp1-independent mitochondrial division

    Energy Technology Data Exchange (ETDEWEB)

    Roy, Madhuparna, E-mail: mroy17@jhmi.edu; Itoh, Kie, E-mail: kito5@jhmi.edu; Iijima, Miho, E-mail: miijima@jhmi.edu; Sesaki, Hiromi, E-mail: hsesaki@jhmi.edu

    2016-07-01

    The cycle of mitochondrial division and fusion disconnect and reconnect individual mitochondria in cells to remodel this energy-producing organelle. Although dynamin-related protein 1 (Drp1) plays a major role in mitochondrial division in cells, a reduced level of mitochondrial division still persists even in the absence of Drp1. It is unknown how much Drp1-mediated mitochondrial division accounts for the connectivity of mitochondria. The role of a Parkinson’s disease-associated protein—parkin, which biochemically and genetically interacts with Drp1—in mitochondrial connectivity also remains poorly understood. Here, we quantified the number and connectivity of mitochondria using mitochondria-targeted photoactivatable GFP in cells. We show that the loss of Drp1 increases the connectivity of mitochondria by 15-fold in mouse embryonic fibroblasts (MEFs). While a single loss of parkin does not affect the connectivity of mitochondria, the connectivity of mitochondria significantly decreased compared with a single loss of Drp1 when parkin was lost in the absence of Drp1. Furthermore, the loss of parkin decreased the frequency of depolarization of the mitochondrial inner membrane that is caused by increased mitochondrial connectivity in Drp1-knockout MEFs. Therefore, our data suggest that parkin negatively regulates Drp1-indendent mitochondrial division. -- Highlights: •A Drp1-mediated mechanism accounts for ∼95% of mitochondrial division. •Parkin controls the connectivity of mitochondria via a mechanism that is independent of Drp1. •In the absence of Drp1, connected mitochondria transiently depolarize. •The transient depolarization is independent of calcium signaling and uncoupling protein 2.

  17. Altered Mitochondrial Dynamics and TBI Pathophysiology.

    Science.gov (United States)

    Fischer, Tara D; Hylin, Michael J; Zhao, Jing; Moore, Anthony N; Waxham, M Neal; Dash, Pramod K

    2016-01-01

    Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS), and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI) reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1), which translocates to the mitochondrial outer membrane (MOM) to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 h post-injury, followed by a significant decrease in length at 72 h. Post-TBI administration of Mitochondrial division inhibitor-1 (Mdivi-1), a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the

  18. Retro-translocation of mitochondrial intermembrane space proteins

    Science.gov (United States)

    Bragoszewski, Piotr; Wasilewski, Michal; Sakowska, Paulina; Gornicka, Agnieszka; Böttinger, Lena; Qiu, Jian; Wiedemann, Nils; Chacinska, Agnieszka

    2015-01-01

    The content of mitochondrial proteome is maintained through two highly dynamic processes, the influx of newly synthesized proteins from the cytosol and the protein degradation. Mitochondrial proteins are targeted to the intermembrane space by the mitochondrial intermembrane space assembly pathway that couples their import and oxidative folding. The folding trap was proposed to be a driving mechanism for the mitochondrial accumulation of these proteins. Whether the reverse movement of unfolded proteins to the cytosol occurs across the intact outer membrane is unknown. We found that reduced, conformationally destabilized proteins are released from mitochondria in a size-limited manner. We identified the general import pore protein Tom40 as an escape gate. We propose that the mitochondrial proteome is not only regulated by the import and degradation of proteins but also by their retro-translocation to the external cytosolic location. Thus, protein release is a mechanism that contributes to the mitochondrial proteome surveillance. PMID:26056291

  19. The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

    Directory of Open Access Journals (Sweden)

    Yajin Liao

    2017-02-01

    Full Text Available The mitochondrial calcium uniporter (MCU—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP; however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.

  20. The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

    Science.gov (United States)

    Liao, Yajin; Dong, Yuan; Cheng, Jinbo

    2017-01-01

    The mitochondrial calcium uniporter (MCU)—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP); however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders. PMID:28208618

  1. Control mechanisms in mitochondrial oxidative phosphorylation

    Institute of Scientific and Technical Information of China (English)

    Jana Hroudová; Zdeněk Fi(s)ar

    2013-01-01

    Distribution and activity of mitochondria are key factors in neuronal development, synaptic plasticity and axogenesis. The majority of energy sources, necessary for cellular functions, originate from oxidative phosphorylation located in the inner mitochondrial membrane. The adenosine-5'- triphosphate production is regulated by many control mechanism–firstly by oxygen, substrate level, adenosine-5'-diphosphate level, mitochondrial membrane potential, and rate of coupling and proton leak. Recently, these mechanisms have been implemented by "second control mechanisms," such as reversible phosphorylation of the tricarboxylic acid cycle enzymes and electron transport chain complexes, allosteric inhibition of cytochrome c oxidase, thyroid hormones, effects of fatty acids and uncoupling proteins. Impaired function of mitochondria is implicated in many diseases ranging from mitochondrial myopathies to bipolar disorder and schizophrenia. Mitochondrial dysfunctions are usually related to the ability of mitochondria to generate adenosine-5'-triphosphate in response to energy demands. Large amounts of reactive oxygen species are released by defective mitochondria, similarly, decline of antioxidative enzyme activities (e.g. in the elderly) enhances reactive oxygen species production. We reviewed data concerning neuroplasticity, physiology, and control of mitochondrial oxidative phosphorylation and reactive oxygen species production.

  2. Mitochondrial biogenesis and turnover.

    Science.gov (United States)

    Diaz, Francisca; Moraes, Carlos T

    2008-07-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last 20 years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium-dependent protein kinases that in turn activate transcription factors and coactivators such as PGC-1alpha that regulates the expression of genes coding for mitochondrial components. In addition, mitochondrial biogenesis involves the balance of mitochondrial fission-fusion. Mitochondrial malfunction or defects in any of the many pathways involved in mitochondrial biogenesis can lead to degenerative diseases and possibly play an important part in aging.

  3. Upregulation of Mitochondrial Content in Cytochrome c Oxidase Deficient Fibroblasts.

    Science.gov (United States)

    Kogot-Levin, Aviram; Saada, Ann; Leibowitz, Gil; Soiferman, Devorah; Douiev, Liza; Raz, Itamar; Weksler-Zangen, Sarah

    2016-01-01

    Cytochrome-c-oxidase (COX) deficiency is a frequent cause of mitochondrial disease and is associated with a wide spectrum of clinical phenotypes. We studied mitochondrial function and biogenesis in fibroblasts derived from the Cohen (CDs) rat, an animal model of COX deficiency. COX activity in CDs-fibroblasts was 50% reduced compared to control rat fibroblasts (P<0.01). ROS-production in CDs fibroblasts increased, along with marked mitochondrial fragmentation and decreased mitochondrial membrane-potential, indicating mitochondrial dysfunction. Surprisingly, cellular ATP content, oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) were unchanged. To clarify the discrepancy between mitochondrial dysfunction and ATP production, we studied mitochondrial biogenesis and turnover. The content of mitochondria was higher in CDs-fibroblasts. Consistently, AMPK activity and the expression of NRF1-target genes, NRF2 and PGC1-α that mediate mitochondrial biogenesis were increased (P<0.01 vs control fibroblast). In CDs-fibrobalsts, the number of autophagosomes (LC3+ puncta) containing mitochondria in CDs fibroblasts was similar to that in control fibroblasts, suggesting that mitophagy was intact. Altogether, our findings demonstrate that mitochondrial dysfunction and oxidative stress are associated with an increase in mitochondrial biogenesis, resulting in preservation of ATP generation.

  4. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  5. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson’s Disease Patients

    Directory of Open Access Journals (Sweden)

    William Haylett

    2016-01-01

    Full Text Available Mutations in the parkin gene are the most common cause of early-onset Parkinson’s disease (PD. Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093, while exhibiting more fragmented mitochondrial networks (p=0.0304. Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p=0.0001. These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.

  6. Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients

    Science.gov (United States)

    Swart, Chrisna; van der Westhuizen, Francois; van Dyk, Hayley; van der Merwe, Lize; van der Merwe, Celia; Loos, Ben; Carr, Jonathan; Kinnear, Craig; Bardien, Soraya

    2016-01-01

    Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation. PMID:27034887

  7. Effects of MPTP, MPP+ and paraquat on mitochondrial potential and oxidative stress

    OpenAIRE

    Lambert, Ce; Bondy, SC

    1989-01-01

    The effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), 1-methyl-4-phenylpyridinium (MPP+) and 1,1-dimethyl-4,4-bipyridinium (paraquat) upon the electrical potential across the plasma and mitochondrial membranes within synaptosomes has been investigated. MPTP selectively depressed plasma membrane potential while MPP+ specifically reduced mitochondrial potential. The structurally similar compound paraquat had no effect on either membrane potential. Enhancement of the lipid peroxidat...

  8. Reperfusion promotes mitochondrial dysfunction following focal cerebral ischemia in rats.

    Directory of Open Access Journals (Sweden)

    Jun Li

    Full Text Available BACKGROUND AND PURPOSE: Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia. METHODS: Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis. RESULTS: Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca(2+ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period. CONCLUSIONS: Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca(2+-induced mitochondrial swelling. The mechanism of this swelling may be mediated by

  9. Postnatal changes in heart mitochondrial calcium and energy metabolism.

    Science.gov (United States)

    Wolf, W J; Rex, K A; Geshi, E; Sordahl, L A

    1991-07-01

    The newborn mammalian heart has less functional capacity compared with the adult, yet newborn myocardial mitochondrial respiratory activity is the same or exceeds that of adult. This study was aimed at determining the temporal changes in newborn rabbit heart mitochondrial energy-linked Ca2+ transport during early postnatal development. At birth, substrate-supported Ca2+ uptake is twice that of adult and declines toward adult rates during the first 14 days. Both NADH- and succinate-linked respiration are equivalent to adult values at birth, increase transiently during the first 7 days, and then decline toward adult. Newborn heart mitochondrial preparations exhibit the same membrane potential (delta psi) values during Ca2+ uptake and have comparable rates of Na(+)-induced Ca2+ efflux as adult. Creatine kinase (CK) activity is very low in 1- to 7-day-old newborn mitochondria and increases rapidly toward adult values after 10 days of age. The decreasing rates of Ca2+ uptake do not appear to be related to respiratory activity, membrane potential, or increased cycling of Ca2+ but rather to a direct effect on the mitochondrial Ca2+ uniporter. Preliminary studies indicate changes in mitochondrial membrane phospholipids during early development that may be related to the increasing CK activity and decreasing Ca2+ uptake and respiration. We postulate that mitochondrial membrane lipid changes in early postnatal development may be the causative factor underlying these changes in functional activity.

  10. Assessing mitochondrial dysfunction in cells.

    Science.gov (United States)

    Brand, Martin D; Nicholls, David G

    2011-04-15

    Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review.

  11. Nitration of Hsp90 on Tyrosine 33 Regulates Mitochondrial Metabolism.

    Science.gov (United States)

    Franco, Maria C; Ricart, Karina C; Gonzalez, Analía S; Dennys, Cassandra N; Nelson, Pascal A; Janes, Michael S; Mehl, Ryan A; Landar, Aimee; Estévez, Alvaro G

    2015-07-31

    Peroxynitrite production and tyrosine nitration are present in several pathological conditions, including neurodegeneration, stroke, aging, and cancer. Nitration of the pro-survival chaperone heat shock protein 90 (Hsp90) in position 33 and 56 induces motor neuron death through a toxic gain-of-function. Here we show that nitrated Hsp90 regulates mitochondrial metabolism independently of the induction of cell death. In PC12 cells, a small fraction of nitrated Hsp90 was located on the mitochondrial outer membrane and down-regulated mitochondrial membrane potential, oxygen consumption, and ATP production. Neither endogenous Hsp90 present in the homogenate nor unmodified and fully active recombinant Hsp90 was able to compete with the nitrated protein for the binding to mitochondria. Moreover, endogenous or recombinant Hsp90 did not prevent the decrease in mitochondrial activity but supported nitrated Hsp90 mitochondrial gain-of-function. Nitrotyrosine in position 33, but not in any of the other four tyrosine residues prone to nitration in Hsp90, was sufficient to down-regulate mitochondrial activity. Thus, in addition to induction of cell death, nitrated Hsp90 can also regulate mitochondrial metabolism, suggesting that depending on the cell type, distinct Hsp90 nitration states regulate different aspects of cellular metabolism. This regulation of mitochondrial homeostasis by nitrated Hsp90 could be of particular relevance in cancer cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Mitochondrial mutagenesis induced by tumor-specific radiation bystander effects.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The radiation bystander effect is a cellular process whereby cells not directly exposed to radiation display cellular alterations similar to directly irradiated cells. Cellular targets including mitochondria have been postulated to play a significant role in this process. In this study, we utilized the Random Mutation Capture assay to quantify the levels of random mutations and deletions in the mitochondrial genome of bystander cells. A significant increase in the frequency of random mitochondrial mutations was found at 24 h in bystander cells exposed to conditioned media from irradiated tumor explants (p = 0.018). CG:TA mutations were the most abundant lesion induced. A transient increase in the frequency of random mitochondrial deletions was also detected in bystander cells exposed to conditioned media from tumor but not normal tissue at 24 h (p = 0.028). The increase in both point mutations and deletions was transient and not detected at 72 h. To further investigate mitochondrial dysfunction, mitochondrial membrane potential and reactive oxygen species were assessed in these bystander cells. There was a significant reduction in mitochondrial membrane potential and this was positively associated with the frequency of random point mutation and deletions in bystander cells treated with conditioned media from tumor tissue (r = 0.71, p = 0.02). This study has shown that mitochondrial genome alterations are an acute consequence of the radiation bystander effect secondary to mitochondrial dysfunction and suggests that this cannot be solely attributable to changes in ROS levels alone.

  13. The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

    Science.gov (United States)

    Wiechmann, Katja; Müller, Hans; Fischer, Dagmar; Jauch, Johann; Werz, Oliver

    2015-11-01

    The acylphloroglucinols hyperforin (Hypf) and myrtucommulone A (MC A) induce death of cancer cells by triggering the intrinsic/mitochondrial pathway of apoptosis, accompanied by a loss of the mitochondrial membrane potential and release of cytochrome c. However, the upstream targets and mechanisms leading to these mitochondrial events in cancer cells remain elusive. Here we show that Hypf and MC A directly act on mitochondria derived from human leukemic HL-60 cells and thus, disrupt mitochondrial functions. In isolated mitochondria, Hypf and MC A efficiently impaired mitochondrial viability (EC50 = 0.2 and 0.9 µM, respectively), caused loss of the mitochondrial membrane potential (at 0.03 and 0.1 µM, respectively), and suppressed mitochondrial ATP synthesis (IC50 = 0.2 and 0.5 µM, respectively). Consequently, the compounds activated the adenosine monophosphate-activated protein kinase (AMPK) in HL-60 cells, a cellular energy sensor involved in apoptosis of cancer cells. Side by side comparison with the protonophore CCCP and the ATP synthase inhibitor oligomycin suggest that Hypf and MC A act as protonophores that primarily dissipate the mitochondrial membrane potential by direct interaction with the mitochondrial membrane. Together, Hypf and MC A abolish the mitochondrial proton motive force that on one hand impairs mitochondrial viability and on the other cause activation of AMPK due to lowered ATP levels which may further facilitate the intrinsic mitochondrial pathway of apoptosis.

  14. Ionizing radiation accelerates Drp1-dependent mitochondrial fission, which involves delayed mitochondrial reactive oxygen species production in normal human fibroblast-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Kobashigawa, Shinko, E-mail: kobashin@nagasaki-u.ac.jp [Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan); Suzuki, Keiji; Yamashita, Shunichi [Atomic Bomb Disease Institute, Course of Life Sciences and Radiation Research, Nagasaki University Graduate School of Biomedical Sciences, 1-12-4 Sakamoto, Nagasaki 852-8523 (Japan)

    2011-11-04

    Highlights: Black-Right-Pointing-Pointer We report first time that ionizing radiation induces mitochondrial dynamic changes. Black-Right-Pointing-Pointer Radiation-induced mitochondrial fission was caused by Drp1 localization. Black-Right-Pointing-Pointer We found that radiation causes delayed ROS from mitochondria. Black-Right-Pointing-Pointer Down regulation of Drp1 rescued mitochondrial dysfunction after radiation exposure. -- Abstract: Ionizing radiation is known to increase intracellular level of reactive oxygen species (ROS) through mitochondrial dysfunction. Although it has been as a basis of radiation-induced genetic instability, the mechanism involving mitochondrial dysfunction remains unclear. Here we studied the dynamics of mitochondrial structure in normal human fibroblast like cells exposed to ionizing radiation. Delayed mitochondrial O{sub 2}{sup {center_dot}-} production was peaked 3 days after irradiation, which was coupled with accelerated mitochondrial fission. We found that radiation exposure accumulated dynamin-related protein 1 (Drp1) to mitochondria. Knocking down of Drp1 expression prevented radiation induced acceleration of mitochondrial fission. Furthermore, knockdown of Drp1 significantly suppressed delayed production of mitochondrial O{sub 2}{sup {center_dot}-}. Since the loss of mitochondrial membrane potential, which was induced by radiation was prevented in cells knocking down of Drp1 expression, indicating that the excessive mitochondrial fission was involved in delayed mitochondrial dysfunction after irradiation.

  15. Looking Beyond Structure: Membrane Phospholipids of Skeletal Muscle Mitochondria.

    Science.gov (United States)

    Heden, Timothy D; Neufer, P Darrell; Funai, Katsuhiko

    2016-08-01

    Skeletal muscle mitochondria are highly dynamic and are capable of tremendous expansion to meet cellular energetic demands. Such proliferation in mitochondrial mass requires a synchronized supply of enzymes and structural phospholipids. While transcriptional regulation of mitochondrial enzymes has been extensively studied, there is limited information on how mitochondrial membrane lipids are generated in skeletal muscle. Herein we describe how each class of phospholipids that constitute mitochondrial membranes are synthesized and/or imported, and summarize genetic evidence indicating that membrane phospholipid composition represents a significant modulator of skeletal muscle mitochondrial respiratory function. We also discuss how skeletal muscle mitochondrial phospholipids may mediate the effect of diet and exercise on oxidative metabolism. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Mitochondrial Band-7 family proteins: scaffolds for respiratory chain assembly?

    OpenAIRE

    2014-01-01

    The band-7 protein family comprises a diverse set of membrane-bound proteins characterized by the presence of a conserved domain. The exact function of this band-7 domain remains elusive, but examples from animal and bacterial stomatin-type proteins demonstrate binding to lipids and the ability to assemble into membrane-bound oligomers that form putative scaffolds. Some members, such as prohibitins (PHB) and human stomatin-like protein 2 (HsSLP2), localize to the mitochondrial inner membrane ...

  17. Altered Mitochondrial Dynamics and TBI Pathophysiology

    Directory of Open Access Journals (Sweden)

    Tara Diane Fischer

    2016-03-01

    Full Text Available Mitochondrial function is intimately linked to cellular survival, growth, and death. Mitochondria not only generate ATP from oxidative phosphorylation, but also mediate intracellular calcium buffering, generation of reactive oxygen species (ROS, and apoptosis. Electron leakage from the electron transport chain, especially from damaged or depolarized mitochondria, can generate excess free radicals that damage cellular proteins, DNA, and lipids. Furthermore, mitochondrial damage releases pro-apoptotic factors to initiate cell death. Previous studies have reported that traumatic brain injury (TBI reduces mitochondrial respiration, enhances production of ROS, and triggers apoptotic cell death, suggesting a prominent role of mitochondria in TBI pathophysiology. Mitochondria maintain cellular energy homeostasis and health via balanced processes of fusion and fission, continuously dividing and fusing to form an interconnected network throughout the cell. An imbalance of these processes, particularly an excess of fission, can be detrimental to mitochondrial function, causing decreased respiration, ROS production, and apoptosis. Mitochondrial fission is regulated by the cytosolic GTPase, dynamin-related protein 1 (Drp1, which translocates to the mitochondrial outer membrane to initiate fission. Aberrant Drp1 activity has been linked to excessive mitochondrial fission and neurodegeneration. Measurement of Drp1 levels in purified hippocampal mitochondria showed an increase in TBI animals as compared to sham controls. Analysis of cryo-electron micrographs of these mitochondria also showed that TBI caused an initial increase in the length of hippocampal mitochondria at 24 hours post-injury, followed by a significant decrease in length at 72 hours. Post-TBI administration of Mdivi-1, a pharmacological inhibitor of Drp1, prevented this decrease in mitochondria length. Mdivi-1 treatment also reduced the loss of newborn neurons in the hippocampus and improved

  18. Strokes in mitochondrial diseases

    Directory of Open Access Journals (Sweden)

    N V Pizova

    2012-01-01

    Full Text Available It is suggested that mitochondrial diseases might be identified in 22—33% of cryptogenic stroke cases in young subjects. The incidence of mitochondrial disorders in patients with stroke is unknown; it is 0.8 to 7.2% according to the data of some authors. The paper gives data on the prevalence, pathogenesis, and clinical manifestations of mitochondrial diseases, such as mitochondrial encephalopathy, lactic acidosis, and stroke-like syndrome (MELAS and insulin-like episodes; myoclonic epilepsy and ragged-red fibers (MERRF syndrome, and Kearns-Sayre syndrome (sporadic multisystem mitochondrial pathology.

  19. Noninvasive probes of mitochondrial molecular motors

    Science.gov (United States)

    Nawarathna, Dharmakeerthna; Claycomb, James

    2005-03-01

    We report on a noninvasive method of probing mitochondrial molecular motors using nonlinear dielectric spectroscopy. It has been found previously that enzymes in the plasma membrane, particularly H+ ATPase, result in a strong low frequency (less than 100 Hz) nonlinear harmonic response. In this study, we find evidence that molecular motors located in the inner membranes of mitochondria cause the generation of harmonics at relatively high frequencies (1 - 30 kHz). In particular, we find that potassium cyanide (KCN), a respiratory inhibitor that binds to cytochrome c oxidase and thus prevents transport of protons across the mitochondrial inner membrane, suppresses the harmonic response. We observe this behavior in yeast (S. cerevisiae), a eucaryote that typically contains about 300 mitochondria, and B. indicas, a procaryote believed to be related to the ancient ancestor of mitochondria. Our current modeling efforts are focusing on a Brownian ratchet model of the F0 unit of ATP synthase, a remarkable molecular turbine driven by the proton gradient across the mitochondrial inner membrane.

  20. 信息动态%Evaluation of Mitochondrial Damage of lsletβCells by Mitochondrial Permeability Transition Pore

    Institute of Scientific and Technical Information of China (English)

    2011-01-01

    Objective To evaluate the mitochondrial damage of islet β cells under glucolipotoxicity by investigating the mitochondrial permeability transition pore (mPTP). Methods Pancreatic β cell lines INS-1 cells were treated with 0. 4 mmol/L palmitic acid and different concentrations of glucose (5.6 mmol/L or 25 mmol/L). The mitochondrial membrane potential, mPTP and reactive oxygen species (ROS) were measured by flow cytometry and fluorescence staining technique to assess the mitochon drial damage. Cell proliferation was measured by 5-bromodeoxyuridine incorporation and cell apoptosis was detected by Annexin V method. Results Compared with the low glucose concentration, the high glucose concentration resulted in decreased mPTP activity (P<0.05), increased mitochondrial membrane potential (P<0.05) and increased cell proliferation rate (P<0.05). There was no significant change in ROS generation. When cells were exposed to high glucose concentration and palmitic acid, both mPTP activity and mitochonhdrial membrane potential reduced (P<0.05), with increased cell apoptosis rate (P <0.05) and increased ROS generation. Conclusion The high glucose concentration decreases mPTP and increases mitochondrial membrane potential, suggesting that cells may remain in an unstable high metabolic state. Evaluation of mPTP may contribute to a more comprehensive understanding of mitochondrial dysfunction under glucotoxictiy.

  1. Ca2+ signals regulate mitochondrial metabolism by stimulating CREB-mediated expression of the mitochondrial Ca2+ uniporter gene MCU.

    Science.gov (United States)

    Shanmughapriya, Santhanam; Rajan, Sudarsan; Hoffman, Nicholas E; Zhang, Xueqian; Guo, Shuchi; Kolesar, Jill E; Hines, Kevin J; Ragheb, Jonathan; Jog, Neelakshi R; Caricchio, Roberto; Baba, Yoshihiro; Zhou, Yandong; Kaufman, Brett A; Cheung, Joseph Y; Kurosaki, Tomohiro; Gill, Donald L; Madesh, Muniswamy

    2015-03-03

    Cytosolic Ca2+ signals, generated through the coordinated translocation of Ca2+ across the plasma membrane (PM) and endoplasmic reticulum (ER) membrane, mediate diverse cellular responses. Mitochondrial Ca2+ is important for mitochondrial function, and when cytosolic Ca2+ concentration becomes too high, mitochondria function as cellular Ca2+ sinks. By measuring mitochondrial Ca2+ currents, we found that mitochondrial Ca2+ uptake was reduced in chicken DT40 B lymphocytes lacking either the ER-localized inositol trisphosphate receptor (IP3R), which releases Ca2+ from the ER, or Orai1 or STIM1, components of the PM-localized Ca2+ -permeable channel complex that mediates store-operated calcium entry (SOCE) in response to depletion of ER Ca2+ stores. The abundance of MCU, the pore-forming subunit of the mitochondrial Ca2+ uniporter, was reduced in cells deficient in IP3R, STIM1, or Orai1. Chromatin immunoprecipitation and promoter reporter analyses revealed that the Ca2+ -regulated transcription factor CREB (cyclic adenosine monophosphate response element-binding protein) directly bound the MCU promoter and stimulated expression. Lymphocytes deficient in IP3R, STIM1, or Orai1 exhibited altered mitochondrial metabolism, indicating that Ca2+ released from the ER and SOCE-mediated signals modulates mitochondrial function. Thus, our results showed that a transcriptional regulatory circuit involving Ca2+ -dependent activation of CREB controls the Ca2+ uptake capability of mitochondria and hence regulates mitochondrial metabolism.

  2. Get1p and Get2p are required for maintenance of mitochondrial morphology and normal cardiolipin levels.

    Science.gov (United States)

    Joshi, Amit S; Fei, Naomi; Greenberg, Miriam L

    2016-05-01

    Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes. CL deficiency leads to defects in mitochondrial function. Using a targeted synthetic lethality screen to identify defects that exacerbate CL deficiency, we determined that deletion of mitochondrial morphology genes in cells lacking CL leads to severe growth defects. We show that ER membrane proteins Get1p and Get2p are required for maintaining normal levels of CL. We propose that these proteins regulate the level of CL by maintaining wild type-like tubular mitochondrial morphology. The genetic interactions observed in this study identify novel physiological modifiers that are required for maintenance of CL levels and mitochondrial morphology.

  3. Disorders of phospholipid metabolism: an emerging class of mitochondrial disease due to defects in nuclear genes

    Directory of Open Access Journals (Sweden)

    Ya-Wen eLu

    2015-02-01

    Full Text Available The human nuclear and mitochondrial genomes co-exist within each cell. While the mitochondrial genome encodes for a limited number of proteins, transfer RNAs, and ribosomal RNAs, the vast majority of mitochondrial proteins are encoded in the nuclear genome. Of the multitude of mitochondrial disorders known to date, only a fifth are maternally inherited. The recent characterization of the mitochondrial proteome therefore serves as an important step towards delineating the nosology of a large spectrum of phenotypically heterogeneous diseases. Following the identification of the first nuclear gene defect to underlie a mitochondrial disorder, a plenitude of genetic variants that provoke mitochondrial pathophysiology have been molecularly elucidated and classified into six categories that impact: 1 oxidative phosphorylation (subunits and assembly factors; 2 mitochondrial DNA maintenance and expression; 3 mitochondrial protein import and assembly; 4 mitochondrial quality control (chaperones and proteases; 5 iron-sulfur cluster homeostasis; and 6 mitochondrial dynamics (fission and fusion. Here, we propose that an additional class of genetic variant be included in the classification schema to acknowledge the role of genetic defects in phospholipid biosynthesis, remodeling, and metabolism in mitochondrial pathophysiology. This seventh class includes a small but notable group of nuclear-encoded proteins whose dysfunction impacts normal mitochondrial phospholipid metabolism. The resulting human disorders present with a diverse array of pathologic consequences that reflect the variety of functions that phospholipids have in mitochondria and highlight the important role of proper membrane homeostasis in mitochondrial biology.

  4. The plant mitochondrial proteome

    DEFF Research Database (Denmark)

    Millar, A.H.; Heazlewood, J.L.; Kristensen, B.K.

    2005-01-01

    The plant mitochondrial proteome might contain as many as 2000-3000 different gene products, each of which might undergo post-translational modification. Recent studies using analytical methods, such as one-, two- and three-dimensional gel electrophoresis and one- and two-dimensional liquid...... chromatography linked on-line with tandem mass spectrometry, have identified >400 mitochondrial proteins, including subunits of mitochondrial respiratory complexes, supercomplexes, phosphorylated proteins and oxidized proteins. The results also highlight a range of new mitochondrial proteins, new mitochondrial...... functions and possible new mechanisms for regulating mitochondrial metabolism. More than 70 identified proteins in Arabidopsis mitochondrial samples lack similarity to any protein of known function. In some cases, unknown proteins were found to form part of protein complexes, which allows a functional...

  5. Multi-parametric analysis and modeling of relationships between mitochondrial morphology and apoptosis.

    Science.gov (United States)

    Reis, Yara; Bernardo-Faura, Marti; Richter, Daniela; Wolf, Thomas; Brors, Benedikt; Hamacher-Brady, Anne; Eils, Roland; Brady, Nathan R

    2012-01-01

    Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis.

  6. Mitochondrial fusion and ERK activity regulate steroidogenic acute regulatory protein localization in mitochondria.

    Science.gov (United States)

    Duarte, Alejandra; Castillo, Ana Fernanda; Podestá, Ernesto J; Poderoso, Cecilia

    2014-01-01

    The rate-limiting step in the biosynthesis of steroid hormones, known as the transfer of cholesterol from the outer to the inner mitochondrial membrane, is facilitated by StAR, the Steroidogenic Acute Regulatory protein. We have described that mitochondrial ERK1/2 phosphorylates StAR and that mitochondrial fusion, through the up-regulation of a fusion protein Mitofusin 2, is essential during steroidogenesis. Here, we demonstrate that mitochondrial StAR together with mitochondrial active ERK and PKA are necessary for maximal steroid production. Phosphorylation of StAR by ERK is required for the maintenance of this protein in mitochondria, observed by means of over-expression of a StAR variant lacking the ERK phosphorylation residue. Mitochondrial fusion regulates StAR levels in mitochondria after hormone stimulation. In this study, Mitofusin 2 knockdown and mitochondrial fusion inhibition in MA-10 Leydig cells diminished StAR mRNA levels and concomitantly mitochondrial StAR protein. Together our results unveil the requirement of mitochondrial fusion in the regulation of the localization and mRNA abundance of StAR. We here establish the relevance of mitochondrial phosphorylation events in the correct localization of this key protein to exert its action in specialized cells. These discoveries highlight the importance of mitochondrial fusion and ERK phosphorylation in cholesterol transport by means of directing StAR to the outer mitochondrial membrane to achieve a large number of steroid molecules per unit of StAR.

  7. Multi-parametric analysis and modeling of relationships between mitochondrial morphology and apoptosis.

    Directory of Open Access Journals (Sweden)

    Yara Reis

    Full Text Available Mitochondria exist as a network of interconnected organelles undergoing constant fission and fusion. Current approaches to study mitochondrial morphology are limited by low data sampling coupled with manual identification and classification of complex morphological phenotypes. Here we propose an integrated mechanistic and data-driven modeling approach to analyze heterogeneous, quantified datasets and infer relations between mitochondrial morphology and apoptotic events. We initially performed high-content, multi-parametric measurements of mitochondrial morphological, apoptotic, and energetic states by high-resolution imaging of human breast carcinoma MCF-7 cells. Subsequently, decision tree-based analysis was used to automatically classify networked, fragmented, and swollen mitochondrial subpopulations, at the single-cell level and within cell populations. Our results revealed subtle but significant differences in morphology class distributions in response to various apoptotic stimuli. Furthermore, key mitochondrial functional parameters including mitochondrial membrane potential and Bax activation, were measured under matched conditions. Data-driven fuzzy logic modeling was used to explore the non-linear relationships between mitochondrial morphology and apoptotic signaling, combining morphological and functional data as a single model. Modeling results are in accordance with previous studies, where Bax regulates mitochondrial fragmentation, and mitochondrial morphology influences mitochondrial membrane potential. In summary, we established and validated a platform for mitochondrial morphological and functional analysis that can be readily extended with additional datasets. We further discuss the benefits of a flexible systematic approach for elucidating specific and general relationships between mitochondrial morphology and apoptosis.

  8. A role of taurine in mitochondrial function

    DEFF Research Database (Denmark)

    Hansen, Svend Høime; Andersen, Mogens Larsen; Cornett, Claus

    2010-01-01

    The mitochondrial pH gradient across the inner-membrane is stabilised by buffering of the matrix. A low-molecular mass buffer compound has to be localised in the matrix to maintain its alkaline pH value. Taurine is found ubiquitously in animal cells with concentrations in the millimolar range...... and its pKa value is determined to 9.0 (25 degrees C) and 8.6 (37 degrees C), respectively. Localisation of such a low-molecular buffer in the mitochondrial matrix, transforms the matrix into a biochemical reaction chamber for the important matrix-localised enzyme systems. Three acyl-CoA dehydrogenase...... enzymes, which are pivotal for beta-oxidation of fatty acids, are demonstrated to have optimal activity in a taurine buffer. By application of the model presented, taurine depletion caused by hyperglycemia could provide a link between mitochondrial dysfunction and diabetes....

  9. Optic atrophy 1-dependent mitochondrial remodeling controls steroidogenesis in trophoblasts.

    Science.gov (United States)

    Wasilewski, Michał; Semenzato, Martina; Rafelski, Susanne M; Robbins, Jennifer; Bakardjiev, Anna I; Scorrano, Luca

    2012-07-10

    During human pregnancy, placental trophoblasts differentiate and syncytialize into syncytiotrophoblasts that sustain progesterone production [1]. This process is accompanied by mitochondrial fragmentation and cristae remodeling [2], two facets of mitochondrial apoptosis, whose molecular mechanisms and functional consequences on steroidogenesis are unclear. Here we show that the mitochondria-shaping protein Optic atrophy 1 (Opa1) controls efficiency of steroidogenesis. During syncytialization of trophoblast BeWo cells, levels of the profission mitochondria-shaping protein Drp1 increase, and those of Opa1 and mitofusin (Mfn) decrease, leading to mitochondrial fragmentation and cristae remodeling. Manipulation of the levels of Opa1 reveal an inverse relationship with the efficiency of steroidogenesis in trophoblasts and in mouse embryonic fibroblasts where the mitochondrial steroidogenetic pathway has been engineered. In an in vitro assay, accumulation of cholesterol is facilitated in the inner membrane of isolated mitochondria lacking Opa1. Thus, Opa1-dependent inner membrane remodeling controls efficiency of steroidogenesis.

  10. Involvement of the mitochondrial compartment in human NCL fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Pezzini, Francesco; Gismondi, Floriana [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Tessa, Alessandra [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Tonin, Paola [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy); Carrozzo, Rosalba [IRCCS Bambino Gesu Hospital-Molecular Medicine Unit, Roma (Italy); Mole, Sara E. [MRC Laboratory for Molecular Cell Biology, Molecular Medicines Unit, UCL Institute of Child Health and Department of Genetics, Evolution and Environment, University College London (United Kingdom); Santorelli, Filippo M. [IRCCS Fondazione Stella Maris-Molecular Medicine Unit, Pisa (Italy); Simonati, Alessandro, E-mail: alessandro.simonati@univr.it [Department of Neurological, Psychological, Morphological and Motor Sciences, Divisions of Neurology (Child Neurology) and Neuropathology, University of Verona Medical School, Verona (Italy)

    2011-12-09

    Highlights: Black-Right-Pointing-Pointer Mitochondrial reticulum fragmentation occurs in human CLN1 and CLN6 fibroblasts. Black-Right-Pointing-Pointer Likewise mitochondrial shift-to periphery and decreased mitochondrial density are seen. Black-Right-Pointing-Pointer Enhanced caspase-mediated apoptosis occurs following STS treatment in CLN1 fibroblasts. -- Abstract: Neuronal ceroid lipofuscinosis (NCL) are a group of progressive neurodegenerative disorders of childhood, characterized by the endo-lysosomal storage of autofluorescent material. Impaired mitochondrial function is often associated with neurodegeneration, possibly related to the apoptotic cascade. In this study we investigated the possible effects of lysosomal accumulation on the mitochondrial compartment in the fibroblasts of two NCL forms, CLN1 and CLN6. Fragmented mitochondrial reticulum was observed in all cells by using the intravital fluorescent marker Mitotracker, mainly in the perinuclear region. This was also associated with intense signal from the lysosomal markers Lysotracker and LAMP2. Likewise, mitochondria appeared to be reduced in number and shifted to the cell periphery by electron microscopy; moreover the mitochondrial markers VDCA and COX IV were reduced following quantitative Western blot analysis. Whilst there was no evidence of increased cell death under basal condition, we observed a significant increase in apoptotic nuclei following Staurosporine treatment in CLN1 cells only. In conclusion, the mitochondrial compartment is affected in NCL fibroblasts invitro, and CLN1 cells seem to be more vulnerable to the negative effects of stressed mitochondrial membrane than CLN6 cells.

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

    Directory of Open Access Journals (Sweden)

    William Dott

    2014-01-01

    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

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

    Science.gov (United States)

    Dott, William; Mistry, Pratibha; Wright, Jayne; Cain, Kelvin; Herbert, Karl E

    2014-01-01

    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 galactose media model

  13. Connexin 43 impacts on mitochondrial potassium uptake

    Directory of Open Access Journals (Sweden)

    Kerstin eBoengler

    2013-06-01

    Full Text Available In cardiomyocytes, connexin 43 (Cx43 forms gap junctions and unopposed hemichannels at the plasma membrane, but the protein is also present at the inner membrane of subsarcolemmal mitochondria. Both inhibition and genetic ablation of Cx43 reduce ADP-stimulated complex 1 respiration. Since mitochondrial potassium influx impacts on oxygen consumption, we investigated whether or not inhibition or ablation of mitochondrial Cx43 alters mitochondrial potassium uptake.Subsarcolemmal mitochondria were isolated from rat left ventricular (LV myocardium and loaded with the potassium-sensitive dye PBFI. Intramitochondrial potassium was replaced by TEA (tetraethylammonium. Mitochondria were incubated under control conditions or treated with 250 µM Gap19, a peptide that specifically inhibits Cx43-dependent hemichannels at plasma membranes. Subsequently, 140 mM KCl was added and the slope of the increase in PBFI fluorescence over time was calculated. The slope of the PBFI fluorescence of the control mitochondria was set to 100%. In the presence of Gap19, the mitochondrial potassium influx was reduced from 100±11.6 % in control mitochondria to 65.5±10.7 % (n=6, p<0.05. In addition to the pharmacological inhibition of Cx43, potassium influx was studied in mitochondria isolated from conditional Cx43 knockout mice. Here, the ablation of Cx43 was achieved by the injection of 4-hydroxytamoxifen (Cx43Cre-ER(T/fl + 4-OHT. The mitochondria of the Cx43Cre-ER(T/fl + 4-OHT mice contained 3±1% Cx43 (n=6 of that in control mitochondria (100±11%, n=8, p<0.05. The ablation of Cx43 (n=5 reduced the velocity of the potassium influx from 100±11.2 % in control mitochondria (n=9 to 66.6±5.5 % (p<0.05.Taken together, our data indicate that both pharmacological inhibition and genetic ablation of Cx43 reduce mitochondrial potassium influx.

  14. Improved mitochondrial function in brain aging and Alzheimer disease - the new mechanism of action of the old metabolic enhancer piracetam

    OpenAIRE

    2010-01-01

    Piracetam, the prototype of the so-called nootropic drugs’ is used since many years in different countries to treat cognitive impairment in aging and dementia. Findings that piracetam enhances fluidity of brain mitochondrial membranes led to the hypothesis that piracetam might improve mitochondrial function, e.g., might enhance ATP synthesis. This assumption has recently been supported by a number of observations showing enhanced mitochondrial membrane potential, enhanced ATP production, and ...

  15. Mitochondrial helicases and mitochondrial genome maintenance

    DEFF Research Database (Denmark)

    Aamann, Maria Diget; de Souza-Pinto, Nadja C; Kulikowicz, Tomasz

    2010-01-01

    Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases...... have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus....

  16. Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans

    Science.gov (United States)

    Wang, Yang; Zhang, Yi; Chen, Lianwan; Liang, Qian; Yin, Xiao-Ming; Miao, Long; Kang, Byung-Ho; Xue, Ding

    2016-01-01

    In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME. PMID:27581092

  17. Kinetics and specificity of paternal mitochondrial elimination in Caenorhabditis elegans.

    Science.gov (United States)

    Wang, Yang; Zhang, Yi; Chen, Lianwan; Liang, Qian; Yin, Xiao-Ming; Miao, Long; Kang, Byung-Ho; Xue, Ding

    2016-09-01

    In most eukaryotes, mitochondria are inherited maternally. The autophagy process is critical for paternal mitochondrial elimination (PME) in Caenorhabditis elegans, but how paternal mitochondria, but not maternal mitochondria, are selectively targeted for degradation is poorly understood. Here we report that mitochondrial dynamics have a profound effect on PME. A defect in fission of paternal mitochondria delays PME, whereas a defect in fusion of paternal mitochondria accelerates PME. Surprisingly, a defect in maternal mitochondrial fusion delays PME, which is reversed by a fission defect in maternal mitochondria or by increasing maternal mitochondrial membrane potential using oligomycin. Electron microscopy and tomography analyses reveal that a proportion of maternal mitochondria are compromised when they fail to fuse normally, leading to their competition for the autophagy machinery with damaged paternal mitochondria and delayed PME. Our study indicates that mitochondrial dynamics play a critical role in regulating both the kinetics and the specificity of PME.

  18. Impaired expression of the mitochondrial calcium uniporter suppresses mast cell degranulation.

    Science.gov (United States)

    Furuno, Tadahide; Shinkai, Narumi; Inoh, Yoshikazu; Nakanishi, Mamoru

    2015-12-01

    Calcium ion (Ca(2+)) uptake into the mitochondrial matrix influences ATP production, Ca(2+) homeostasis, and apoptosis regulation. Ca(2+) uptake across the ion-impermeable inner mitochondrial membrane is mediated by the mitochondrial Ca(2+) uniporter (MCU) complex. The MCU complex forms a pore structure composed of several proteins. MCU is a Ca(2+)-selective channel in the inner-mitochondrial membrane that allows electrophoretic Ca(2+) entry into the matrix. Mitochondrial Ca(2+) uptake 1 (MICU1) functions as a Ca(2+)-sensing regulator of the MCU complex. Previously, by microscopic analysis at the single-cell level, we found that during mast cell activation, mitochondria capture cytosolic Ca(2+) in two steps. Consequently, mitochondrial Ca(2+) uptake likely plays a role in cellular function through cytosolic Ca(2+) buffering. Here, we investigate the role of MCU and MICU1 in mitochondrial Ca(2+) uptake and mast cell degranulation using MCU- and MICU1-knockdown (KD) mast cells. Whereas MCU- and MICU1-KD mast cells show normal proliferation rates and mitochondrial membrane potential, they exhibit slow and reduced cytosolic and mitochondrial Ca(2+) elevation after antigen stimulation. Moreover, β-hexosaminidase release induced by antigen was significantly suppressed in MCU-KD cells but not MICU1-KD cells. This suggests that both MCU and MICU1 are involved in mitochondrial Ca(2+) uptake in mast cells, while MCU plays a role in mast cell degranulation.

  19. Increased intrinsic mitochondrial function in humans with mitochondrial haplogroup H

    DEFF Research Database (Denmark)

    Larsen, Steen; Díez-Sánchez, Carmen; Rabøl, Rasmus

    2014-01-01

    It has been suggested that human mitochondrial variants influence maximal oxygen uptake (VO2max). Whether mitochondrial respiratory capacity per mitochondrion (intrinsic activity) in human skeletal muscle is affected by differences in mitochondrial variants is not known. We recruited 54 males...... and determined their mitochondrial haplogroup, mitochondrial oxidative phosphorylation capacity (OXPHOS), mitochondrial content (citrate synthase (CS)) and VO2max. Intrinsic mitochondrial function is calculated as mitochondrial OXPHOS capacity divided by mitochondrial content (CS). Haplogroup H showed a 30......% higher intrinsic mitochondrial function compared with the other haplo group U. There was no relationship between haplogroups and VO2max. In skeletal muscle from men with mitochondrial haplogroup H, an increased intrinsic mitochondrial function is present....

  20. Effects of the Czech Propolis on Sperm Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Miroslava Cedikova

    2014-01-01

    Full Text Available Propolis is a natural product that honeybees collect from various plants. It is known for its beneficial pharmacological effects. The aim of our study was to evaluate the impact of propolis on human sperm motility, mitochondrial respiratory activity, and membrane potential. Semen samples from 10 normozoospermic donors were processed according to the World Health Organization criteria. Propolis effects on the sperm motility and mitochondrial activity parameters were tested in the fresh ejaculate and purified spermatozoa. Propolis preserved progressive motility of spermatozoa in the native semen samples. Oxygen consumption determined in purified permeabilized spermatozoa by high-resolution respirometry in the presence of adenosine diphosphate and substrates of complex I and complex II (state OXPHOSI+II was significantly increased in the propolis-treated samples. Propolis also increased uncoupled respiration in the presence of rotenone (state ETSII and complex IV activity, but it did not influence state LEAK induced by oligomycin. Mitochondrial membrane potential was not affected by propolis. This study demonstrates that propolis maintains sperm motility in the native ejaculates and increases activities of mitochondrial respiratory complexes II and IV without affecting mitochondrial membrane potential. The data suggest that propolis improves the total mitochondrial respiratory efficiency in the human spermatozoa in vitro thereby having potential to improve sperm motility.

  1. Mitochondrial function in neuronal cells depends on p97/VCP/Cdc48-mediated quality control

    Directory of Open Access Journals (Sweden)

    Lei eFang

    2015-02-01

    Full Text Available Maintaining mitochondrial function is essential for neuronal survival and offers protection against neurodegeneration. Ubiquitin-mediated, proteasome-dependent protein degradation in the form of outer mitochondrial membrane associated degradation (OMMAD was shown to play roles in maintenance of mitochondria on the level of proteostasis, but also mitophagy and cell death. Recently, the AAA-ATPase p97/VCP/Cdc48 was recognized as part of OMMAD acting as retrotranslocase of ubiquitinated mitochondrial proteins for proteasomal degradation. Thus, p97 likely plays a major role in mitochondrial maintenance. Support for this notion comes from mitochondrial dysfunction associated with amyotrophic lateral sclerosis and hereditary inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia (IBMPFD caused by p97 mutation. Using SH-SY5Y cells stably expressing p97 or dominant-negative p97QQ treated with mitochondrial toxins rotenone, 6-OHDA, or Aβ-peptide as model for neuronal cells suffering from mitochondrial dysfunction, we found mitochondrial fragmentation under normal and stress conditions was significantly increased upon inactivation of p97. Furthermore, inactivation of p97 resulted in loss of mitochondrial membrane potential and increased production of reactive oxygen species (ROS. Under additional stress conditions, loss of mitochondrial membrane potential and increased ROS production was even more pronounced. Loss of mitochondrial fidelity upon inactivation of p97 was likely due to disturbed maintenance of mitochondrial proteostasis as the employed treatments neither induced mitophagy nor cell death. This was supported by the accumulation of oxidatively-damaged proteins on mitochondria in response to p97 inactivation. Dysfunction of p97 under normal and stress conditions in neuron-like cells severely impacts mitochondrial function, thus supporting for the first time a role for p97 as a major component of mitochondrial

  2. The mitochondrial calcium uniporter (MCU): molecular identity and physiological roles.

    Science.gov (United States)

    Patron, Maria; Raffaello, Anna; Granatiero, Veronica; Tosatto, Anna; Merli, Giulia; De Stefani, Diego; Wright, Lauren; Pallafacchina, Giorgia; Terrin, Anna; Mammucari, Cristina; Rizzuto, Rosario

    2013-04-12

    The direct measurement of mitochondrial [Ca(2+)] with highly specific probes demonstrated that major swings in organellar [Ca(2+)] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca(2+) uniporter, the channel allowing rapid Ca(2+) accumulation across the inner mitochondrial membrane.

  3. Deceleration of fusion-fission cycles improves mitochondrial quality control during aging.

    Directory of Open Access Journals (Sweden)

    Marc Thilo Figge

    Full Text Available Mitochondrial dynamics and mitophagy play a key role in ensuring mitochondrial quality control. Impairment thereof was proposed to be causative to neurodegenerative diseases, diabetes, and cancer. Accumulation of mitochondrial dysfunction was further linked to aging. Here we applied a probabilistic modeling approach integrating our current knowledge on mitochondrial biology allowing us to simulate mitochondrial function and quality control during aging in silico. We demonstrate that cycles of fusion and fission and mitophagy indeed are essential for ensuring a high average quality of mitochondria, even under conditions in which random molecular damage is present. Prompted by earlier observations that mitochondrial fission itself can cause a partial drop in mitochondrial membrane potential, we tested the consequences of mitochondrial dynamics being harmful on its own. Next to directly impairing mitochondrial function, pre-existing molecular damage may be propagated and enhanced across the mitochondrial population by content mixing. In this situation, such an infection-like phenomenon impairs mitochondrial quality control progressively. However, when imposing an age-dependent deceleration of cycles of fusion and fission, we observe a delay in the loss of average quality of mitochondria. This provides a rational why fusion and fission rates are reduced during aging and why loss of a mitochondrial fission factor can extend life span in fungi. We propose the 'mitochondrial infectious damage adaptation' (MIDA model according to which a deceleration of fusion-fission cycles reflects a systemic adaptation increasing life span.

  4. Stimulation of glutamate receptors in cultured hippocampal neurons causes Ca2+-dependent mitochondrial contraction.

    Science.gov (United States)

    Brustovetsky, Tatiana; Li, Viacheslav; Brustovetsky, Nickolay

    2009-07-01

    Cultured hippocampal neurons expressing mitochondrially-targeted enhanced yellow fluorescent protein (mito-eYFP) were used to quantitatively examine mitochondrial remodelling in response to excitotoxic glutamate. Mitochondrial morphology was evaluated using laser spinning-disk confocal microscopy followed by calibrated image processing and 3D image rendering. Glutamate triggered an increase in cytosolic Ca(2+) and mitochondrial depolarization accompanied by Ca(2+)-dependent morphological transformation of neuronal mitochondria from "thread-like" to rounded structures. The quantitative analysis of the mitochondrial remodelling revealed that exposure to glutamate resulted in a decrease in mitochondrial volume and surface area concurrent with an increase in sphericity of the organelles. NIM811, an inhibitor of the mitochondrial permeability transition, attenuated the glutamate-induced sustained increase in cytosolic Ca(2+) and suppressed mitochondrial remodelling in the majority of affected neurons, but it did not rescue mitochondrial membrane potential. Shortening, fragmentation, and formation of circular mitochondria with decreased volume and surface area accompanied mitochondrial depolarization with FCCP. However, FCCP-induced morphological alterations appeared to be distinctly different from mitochondrial remodelling caused by glutamate. Moreover, FCCP prevented glutamate-induced mitochondrial remodelling suggesting an important role of Ca(2+) influx into mitochondria in the morphological alterations. Consistent with this, in saponin-permeabilized neurons, Ca(2+) caused mitochondrial remodelling which could be prevented by Ru(360).

  5. MAVS maintains mitochondrial homeostasis via autophagy

    Science.gov (United States)

    Sun, Xiaofeng; Sun, Liwei; Zhao, Yuanyuan; Li, Ying; Lin, Wei; Chen, Dahua; Sun, Qinmiao

    2016-01-01

    Mitochondrial antiviral signalling protein (MAVS) acts as a critical adaptor protein to transduce antiviral signalling by physically interacting with activated RIG-I and MDA5 receptors. MAVS executes its functions at the outer membrane of mitochondria to regulate downstream antiviral signalling, indicating that the mitochondria provides a functional platform for innate antiviral signalling transduction. However, little is known about whether and how MAVS-mediated antiviral signalling contributes to mitochondrial homeostasis. Here we show that the activation of MAVS is sufficient to induce autophagic signalling, which may mediate the turnover of the damaged mitochondria. Importantly, we find MAVS directly interacts with LC3 through its LC3-binding motif ‘YxxI’, suggesting that MAVS might act as an autophagy receptor to mediate mitochondrial turnover upon excessive activation of RLR signalling. Furthermore, we provide evidence that both MAVS self-aggregation and its interaction with TRAF2/6 proteins are important for MAVS-mediated mitochondrial turnover. Collectively, our findings suggest that MAVS acts as a potential receptor for mitochondria-associated autophagic signalling to maintain mitochondrial homeostasis. PMID:27551434

  6. Characteristics of Mitochondrial Transformation into Human Cells.

    Science.gov (United States)

    Kesner, E E; Saada-Reich, A; Lorberboum-Galski, H

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process.

  7. Characteristics of Mitochondrial Transformation into Human Cells

    Science.gov (United States)

    Kesner, E. E.; Saada-Reich, A.; Lorberboum-Galski, H.

    2016-01-01

    Mitochondria can be incorporated into mammalian cells by simple co-incubation of isolated mitochondria with cells, without the need of transfection reagents or any other type of intervention. This phenomenon was termed mitochondrial transformation, and although it was discovered in 1982, currently little is known regarding its mechanism(s). Here we demonstrate that mitochondria can be transformed into recipient cells very quickly, and co-localize with endogenous mitochondria. The isolated mitochondria interact directly with cells, which engulf the mitochondria with cellular extensions in a way, which may suggest the involvement of macropinocytosis or macropinocytosis-like mechanisms in mitochondrial transformation. Indeed, macropinocytosis inhibitors but not clathrin-mediated endocytosis inhibition-treatments, blocks mitochondria transformation. The integrity of the mitochondrial outer membrane and its proteins is essential for the transformation of the mitochondria into cells; cells can distinguish mitochondria from similar particles and transform only intact mitochondria. Mitochondrial transformation is blocked in the presence of the heparan sulfate molecules pentosan polysulfate and heparin, which indicate crucial involvement of cellular heparan sulfate proteoglycans in the mitochondrial transformation process. PMID:27184109

  8. Mitochondrial modulation of phosphine toxicity and resistance in Caenorhabditis elegans.

    Science.gov (United States)

    Zuryn, Steven; Kuang, Jujiao; Ebert, Paul

    2008-03-01

    Phosphine is a fumigant used to protect stored commodities from infestation by pest insects, though high-level phosphine resistance in many insect species threatens the continued use of the fumigant. The mechanisms of toxicity and resistance are not clearly understood. In this study, the model organism, Caenorhabditis elegans, was employed to investigate the effects of phosphine on its proposed in vivo target, the mitochondrion. We found that phosphine rapidly perturbs mitochondrial morphology, inhibits oxidative respiration by 70%, and causes a severe drop in mitochondrial membrane potential (DeltaPsim) within 5 h of exposure. We then examined the phosphine-resistant strain of nematode, pre-33, to determine whether resistance was associated with any changes to mitochondrial physiology. Oxygen consumption was reduced by 70% in these mutant animals, which also had more mitochondrial genome copies than wild-type animals, a common response to reduced metabolic capacity. The mutant also had an unexpected increase in the basal DeltaPsim, which protected individuals from collapse of the membrane potential following phosphine treatment. We tested whether directly manipulating mitochondrial function could influence sensitivity toward phosphine and found that suppression of mitochondrial respiratory chain genes caused up to 10-fold increase in phosphine resistance. The current study confirms that phosphine targets the mitochondria and also indicates that direct alteration of mitochondrial function may be related to phosphine resistance.

  9. The small GTPase Arf1 modulates mitochondrial morphology and function.

    Science.gov (United States)

    Ackema, Karin B; Hench, Jürgen; Böckler, Stefan; Wang, Shyi Chyi; Sauder, Ursula; Mergentaler, Heidi; Westermann, Benedikt; Bard, Frédéric; Frank, Stephan; Spang, Anne

    2014-11-18

    The small GTPase Arf1 plays critical roles in membrane traffic by initiating the recruitment of coat proteins and by modulating the activity of lipid-modifying enzymes. Here, we report an unexpected but evolutionarily conserved role for Arf1 and the ArfGEF GBF1 at mitochondria. Loss of function of ARF-1 or GBF-1 impaired mitochondrial morphology and activity in Caenorhabditis elegans. Similarly, mitochondrial defects were observed in mammalian and yeast cells. In Saccharomyces cerevisiae, aberrant clusters of the mitofusin Fzo1 accumulated in arf1-11 mutants and were resolved by overexpression of Cdc48, an AAA-ATPase involved in ER and mitochondria-associated degradation processes. Yeast Arf1 co-fractionated with ER and mitochondrial membranes and interacted genetically with the contact site component Gem1. Furthermore, similar mitochondrial abnormalities resulted from knockdown of either GBF-1 or contact site components in worms, suggesting that the role of Arf1 in mitochondrial functioning is linked to ER-mitochondrial contacts. Thus, Arf1 is involved in mitochondrial homeostasis and dynamics, independent of its role in vesicular traffic.

  10. Mitochondrial Biogenesis and Turnover

    OpenAIRE

    Diaz, Francisca; Moraes, Carlos T.

    2008-01-01

    Mitochondrial biogenesis is a complex process involving the coordinated expression of mitochondrial and nuclear genes, the import of the products of the latter into the organelle and turnover. The mechanisms associated with these events have been intensively studied in the last twenty years and our understanding of their details is much improved. Mitochondrial biogenesis requires the participation of calcium signaling that activates a series of calcium dependent protein kinases that in turn a...

  11. Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

    Science.gov (United States)

    Del Valle, I; Gómez-Durán, A; Holt, W V; Muiño-Blanco, T; Cebrián-Pérez, J A

    2012-01-01

    Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.

  12. Progress in mitochondrial epigenetics.

    Science.gov (United States)

    Manev, Hari; Dzitoyeva, Svetlana

    2013-08-01

    Mitochondria, intracellular organelles with their own genome, have been shown capable of interacting with epigenetic mechanisms in at least four different ways. First, epigenetic mechanisms that regulate the expression of nuclear genome influence mitochondria by modulating the expression of nuclear-encoded mitochondrial genes. Second, a cell-specific mitochondrial DNA content (copy number) and mitochondrial activity determine the methylation pattern of nuclear genes. Third, mitochondrial DNA variants influence the nuclear gene expression patterns and the nuclear DNA (ncDNA) methylation levels. Fourth and most recent line of evidence indicates that mitochondrial DNA similar to ncDNA also is subject to epigenetic modifications, particularly by the 5-methylcytosine and 5-hydroxymethylcytosine marks. The latter interaction of mitochondria with epigenetics has been termed 'mitochondrial epigenetics'. Here we summarize recent developments in this particular area of epigenetic research. Furthermore, we propose the term 'mitoepigenetics' to include all four above-noted types of interactions between mitochondria and epigenetics, and we suggest a more restricted usage of the term 'mitochondrial epigenetics' for molecular events dealing solely with the intra-mitochondrial epigenetics and the modifications of mitochondrial genome.

  13. [Mitochondrial and oocyte development].

    Science.gov (United States)

    Deng, Wei-Ping; Ren, Zhao-Rui

    2007-12-01

    Oocyte development and maturation is a complicated process. The nuclear maturation and cytoplasmic maturation must synchronize which can ensure normal oocyte fertilization and following development. Mitochondrial is the most important cellular organell in cytoplasm, and the variation of its distribution during oocyte maturation, the capacity of OXPHOS generating ATP as well as the content or copy number or transcription level of mitochondrial DNA play an important role in oocyte development and maturation. Therefore, the studies on the variation of mitochondrial distribution, function and mitochondrial DNA could enhance our understanding of the physiology of reproduction and provide new insight to solve the difficulties of assisted reproduction as well as cloning embryo technology.

  14. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  15. Keshan disease and mitochondrial cardiomyopathy

    Institute of Scientific and Technical Information of China (English)

    YANG Fuyu

    2006-01-01

    Keshan disease (KD) is a potentially fatal form of cardiomyopathy (disease of the heart muscle) endemic in certain areas of China. From 1984 to 1986, a national comprehensive scientific investigation on KD in Chuxiong region of Yunnan Province in the southwest China was conducted. The investigation team was composed of epidemiologists, clinic doctors, pathologists, biochemists, biophysicists and specialists in ecological environment. Results of pathological, biochemical and biophysical as well as clinical studies showed: an obvious increase of enlarged and swollen mitochondria with distended crista membranes in myocardium from patients with KD; significant reductions in the activity of oxidative phosphorylation (succinate dehydrogenase, cytochrome oxidase, succinate oxidase, H+-ATPase) of affected mitochondria; decrease in CoQ, cardiolipin, Se and GSHPx activity, while obvious increase in the Ca2+ content. So, it was suggested that mitochondria are the predominant target of the pathogenic factors of KD. Before Chuxiong KD survey only a few cases of mitochondrial cardiomyopathy were studied. During the multidisciplinary scientific investigation on KD in Chuxiong a large amount of samples from KD cases and the positive controls were examined. On the basis of the results obtained it was suggested that KD might be classified as a "Mitochondrial Cardiomyopathy" endemic in China. This is one of the achievements in the three years' survey in Chuxiong and is valuable not only to the deeper understanding of pathogenic mechanism of KD but also to the study of mitochondrial cardiomyopathy in general.Keshan disease is not a genetic disease, but is closely related to the malnutrition (especially microelement Se deficiency). KD occurs along a low Se belt, and Se supplementation has been effective in prevention of such disease. The incidence of KD has sharply decreased along with the steady raise of living standard and realization of preventive measures. At present, patients of

  16. Mitochondrial Cristae Shape Determines Respiratory Chain Supercomplexes Assembly and Respiratory Efficiency

    Science.gov (United States)

    Cogliati, Sara; Frezza, Christian; Soriano, Maria Eugenia; Varanita, Tatiana; Quintana-Cabrera, Ruben; Corrado, Mauro; Cipolat, Sara; Costa, Veronica; Casarin, Alberto; Gomes, Ligia C.; Perales-Clemente, Ester; Salviati, Leonardo; Fernandez-Silva, Patricio; Enriquez, Jose A.; Scorrano, Luca

    2013-01-01

    Summary Respiratory chain complexes assemble into functional quaternary structures called supercomplexes (RCS) within the folds of the inner mitochondrial membrane, or cristae. Here, we investigate the relationship between respiratory function and mitochondrial ultrastructure and provide evidence that cristae shape determines the assembly and stability of RCS and hence mitochondrial respiratory efficiency. Genetic and apoptotic manipulations of cristae structure affect assembly and activity of RCS in vitro and in vivo, independently of changes to mitochondrial protein synthesis or apoptotic outer mitochondrial membrane permeabilization. We demonstrate that, accordingly, the efficiency of mitochondria-dependent cell growth depends on cristae shape. Thus, RCS assembly emerges as a link between membrane morphology and function. PMID:24055366

  17. Mitochondrial gene therapy augments mitochondrial physiology in a Parkinson's disease cell model.

    Science.gov (United States)

    Keeney, Paula M; Quigley, Caitlin K; Dunham, Lisa D; Papageorge, Christina M; Iyer, Shilpa; Thomas, Ravindar R; Schwarz, Kathleen M; Trimmer, Patricia A; Khan, Shaharyar M; Portell, Francisco R; Bergquist, Kristen E; Bennett, James P

    2009-08-01

    Neurodegeneration in Parkinson's disease (PD) affects mainly dopaminergic neurons in the substantia nigra, where age-related, increasing percentages of cells lose detectable respiratory activity associated with depletion of intact mitochondrial DNA (mtDNA). Replenishment of mtDNA might improve neuronal bioenergetic function and prevent further cell death. We developed a technology ("ProtoFection") that uses recombinant human mitochondrial transcription factor A (TFAM) engineered with an N-terminal protein transduction domain (PTD) followed by the SOD2 mitochondrial localization signal (MLS) to deliver mtDNA cargo to the mitochondria of living cells. MTD-TFAM (MTD = PTD + MLS = "mitochondrial transduction domain") binds mtDNA and rapidly transports it across plasma membranes to mitochondria. For therapeutic proof-of-principle we tested ProtoFection technology in Parkinson's disease cybrid cells, using mtDNA generated from commercially available human genomic DNA (gDNA; Roche). Nine to 11 weeks after single exposures to MTD-TFAM + mtDNA complex, PD cybrid cells with impaired respiration and reduced mtDNA genes increased their mtDNA gene copy numbers up to 24-fold, mtDNA-derived RNAs up to 35-fold, TFAM and ETC proteins, cell respiration, and mitochondrial movement velocities. Cybrid cells with no or minimal basal mitochondrial impairments showed reduced or no responses to treatment, suggesting the possibility of therapeutic selectivity. Exposure of PD but not control cybrid cells to MTD-TFAM protein alone or MTD-TFAM + mtDNA complex increased expression of PGC-1alpha, suggesting activation of mitochondrial biogenesis. ProtoFection technology for mitochondrial gene therapy holds promise for improving bioenergetic function in impaired PD neurons and needs additional development to define its pharmacodynamics and delineate its molecular mechanisms. It also is unclear whether single-donor gDNA for generating mtDNA would be a preferred therapeutic compared with the pooled

  18. Neurodegenerative and Fatiguing Illnesses, Infections and Mitochondrial Dysfunction: Use of Natural Supplements to Improve Mitochondrial Function

    Directory of Open Access Journals (Sweden)

    Garth L. Nicolson

    2014-01-01

    Full Text Available Background: Many chronic diseases and illnesses are associated with one or more chronic infections, dysfunction of mitochondria and reduced production of ATP. This results in fatigue and other symptoms that occur in most if not all chronic conditions and diseases. Methods: This is a review of the published literature on chronic infections in neurodegenerative diseases and fatiguing illnesses that are also typified by mitochondrial dysfunction. This contribution also reviews the use of natural supplements to enhance mitochondrial function and reduce the effects of chronic infections to improve overall function in various chronic illnesses. Results: Mitochondrial function can be enhanced by the use of various natural supplements, notably Lipid Replacement Therapy (LRT using glyerolphospholipids and other mitochondrial supplements. In various chronic illnesses that are characterized by the presence of chronic infections, such as intracellular bacteria (Mycoplasma, Borrelia, Chlamydia and other infections and viruses, LRT has proven useful in multiple clinical trials. For example, in clinical studies on chronic fatigue syndrome, fibromyalgia syndrome and other chronic fatiguing illnesses where a large majority of patients have chronic infections, LRT significantly reduced fatigue by 35-43% in different clinical trials and increased mitochondrial function. In clinical trials on patients with multiple intracellular bacterial infections and intractable fatigue LRT plus other mitochondrial supplements significantly decreased fatigue and improved mood and cognition. Conclusions: LRT formulations designed to improve mitochondrial function appear to be useful as non-toxic dietary supplements for reducing fatigue and restoring mitochondrial and other cellular membrane functions in patients with chronic illnesses and multiple chronic infections.

  19. Mitochondrial function provides instructive signals for activation-induced B-cell fates.

    OpenAIRE

    Jang, Kyoung-Jin; Mano, Hiroto; Aoki, Koji; Hayashi, Tatsunari; Muto, Akihiko; Nambu, Yukiko; Takahashi, Katsu; Itoh, Katsuhiko; Taketani, Shigeru; Stephen L Nutt; Igarashi, Kazuhiko; Shimizu, Akira; Sugai, Manabu

    2015-01-01

    During immune reactions, functionally distinct B-cell subsets are generated by stochastic processes, including class-switch recombination (CSR) and plasma cell differentiation (PCD). In this study, we show a strong association between individual B-cell fates and mitochondrial functions. CSR occurs specifically in activated B cells with increased mitochondrial mass and membrane potential, which augment mitochondrial reactive oxygen species (mROS), whereas PCD occurs in cells with decreased mit...

  20. Characteristics and function of cardiac mitochondrial nitric oxide synthase.

    Science.gov (United States)

    Dedkova, Elena N; Blatter, Lothar A

    2009-02-15

    We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H(2)DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca(2+) uptake by exposure to different cytoplasmic Ca(2+) concentrations ([Ca(2+)](i) = 1, 2 and 5 microm) resulted in a dose-dependent increase of NO production by mitochondria when L-arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca(2+) uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of L-arginine, mitochondrial NO production during stimulation of Ca(2+) uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit L-arginine availability resulted in 50% inhibition of Ca(2+)-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-N(5)-(1-iminoethyl)ornithine (L-NIO) or iNOS inhibitor N-(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca(2+)-induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of L-arginine, mitochondrial Ca(2+) uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and Mn

  1. Mitochondrial aquaporin-8 knockdown in human hepatoma HepG2 cells causes ROS-induced mitochondrial depolarization and loss of viability

    Energy Technology Data Exchange (ETDEWEB)

    Marchissio, Maria Julia; Francés, Daniel Eleazar Antonio; Carnovale, Cristina Ester; Marinelli, Raúl Alberto, E-mail: rmarinel@unr.edu.ar

    2012-10-15

    Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H{sub 2}O{sub 2} across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H{sub 2}O{sub 2} release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72 h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p < 0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H{sub 2}O{sub 2} release, assessed by Amplex Red, was reduced by about 45% (p < 0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+ 120%, p < 0.05) and loss of mitochondrial membrane potential (− 80%, p < 0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H{sub 2}O{sub 2} release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death. -- Highlights: ► Aquaporin-8 is expressed in mitochondria of human hepatoma HepG2 cells. ► Aquaporin-8 knockdown impairs mitochondrial H{sub 2}O{sub 2} release and increases ROS. ► Aquaporin

  2. MICU1 motifs define mitochondrial calcium uniporter binding and activity.

    Science.gov (United States)

    Hoffman, Nicholas E; Chandramoorthy, Harish C; Shamugapriya, Santhanam; Zhang, Xueqian; Rajan, Sudarsan; Mallilankaraman, Karthik; Gandhirajan, Rajesh Kumar; Vagnozzi, Ronald J; Ferrer, Lucas M; Sreekrishnanilayam, Krishnalatha; Natarajaseenivasan, Kalimuthusamy; Vallem, Sandhya; Force, Thomas; Choi, Eric T; Cheung, Joseph Y; Madesh, Muniswamy

    2013-12-26

    Resting mitochondrial matrix Ca(2+) is maintained through a mitochondrial calcium uptake 1 (MICU1)-established threshold inhibition of mitochondrial calcium uniporter (MCU) activity. It is not known how MICU1 interacts with MCU to establish this Ca(2+) threshold for mitochondrial Ca(2+) uptake and MCU activity. Here, we show that MICU1 localizes to the mitochondrial matrix side of the inner mitochondrial membrane and MICU1/MCU binding is determined by a MICU1 N-terminal polybasic domain and two interacting coiled-coil domains of MCU. Further investigation reveals that MICU1 forms homo-oligomers, and this oligomerization is independent of the polybasic region. However, the polybasic region confers MICU1 oligomeric binding to MCU and controls mitochondrial Ca(2+) current (IMCU). Moreover, MICU1 EF hands regulate MCU channel activity, but do not determine MCU binding. Loss of MICU1 promotes MCU activation leading to oxidative burden and a halt to cell migration. These studies establish a molecular mechanism for MICU1 control of MCU-mediated mitochondrial Ca(2+) accumulation, and dysregulation of this mechanism probably enhances vascular dysfunction.

  3. Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

    Directory of Open Access Journals (Sweden)

    Hyunmi Lee

    Full Text Available Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP, the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM, which we visualize and isolate, into which Bax integrates.MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

  4. Defects of mitochondrial DNA replication.

    Science.gov (United States)

    Copeland, William C

    2014-09-01

    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease.

  5. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats

    Science.gov (United States)

    Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R.; Cortés-Rojo, Christian

    2015-01-01

    Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨm), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress. PMID:26180820

  6. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats.

    Science.gov (United States)

    Ortiz-Avila, Omar; Esquivel-Martínez, Mauricio; Olmos-Orizaba, Berenice Eridani; Saavedra-Molina, Alfredo; Rodriguez-Orozco, Alain R; Cortés-Rojo, Christian

    2015-01-01

    Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats). Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential (ΔΨ m ), besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  7. Structural Basis for Recruitment of Mitochondrial Fission Complexes By Fis1

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Y.; Chan, D.C.

    2009-06-04

    Mitochondrial fission controls mitochondrial shape and physiology, including mitochondrial remodeling in apoptosis. During assembly of the yeast mitochondrial fission complex, the outer membrane protein Fis1 recruits the dynamin-related GTPase Dnm1 to mitochondria. Fis1 contains a tetratricopeptide repeat (TPR) domain and interacts with Dnm1 via the molecular adaptors Mdv1 and Caf4. By using crystallographic analysis of adaptor-Fis1 complexes, we show that these adaptors use two helices to bind to both the concave and convex surfaces of the Fis1 TPR domain. Fis1 therefore contains two interaction interfaces, a binding mode that, to our knowledge, has not been observed previously for TPR domains. Genetic and biochemical studies indicate that both binding interfaces are important for binding of Mdv1 and Caf4 to Fis1 and for mitochondrial fission activity in vivo. Our results reveal how Fis1 recruits the mitochondrial fission complex and will facilitate efforts to manipulate mitochondrial fission.

  8. Repeated Administration of Mercury Intensifies Brain Damage in Multiple Sclerosis through Mitochondrial Dysfunction

    Science.gov (United States)

    Kahrizi, Farzad; Salimi, Ahmad; Noorbakhsh, Farshid; Faizi, Mehrdad; Mehri, Freshteh; Naserzadeh, Parvaneh; Naderi, Nima; Pourahmad, Jalal

    2016-01-01

    In this study we investigated the additive effect of mercury on the brain mitochondrial dysfunction in experimental autoimmune encephalomyelitis (EAE) model. Experimental animals (female C57BL/6 mice) are divided into four groups (n = 8); control, Hg, EAE, EAE with Hg. EAE model of MS induced by injecting myelin oligodendrocyte glycoprotein (MOG). Neurobehavioral alterations are recorded and then mice were sacrificed at day 28 and brain mitochondria were isolated and mitochondrial toxicity parameters including mitochondrial swelling, reactive oxygen species (ROS) formation, collapse of mitochondrial membrane potential (MMP) and cytochrome c release were measured. Our results showed that repeated treatment of mercury following induction of EAE in mice significantly increased the neurobehavioral scores, as well as mitochondrial toxicity through ROS formation, mitochondrial swelling, collapse of MMP and cytochrome c release. Our findings proved that repeated exposure with mercury accelerates progression of MS through mitochondrial damage related to oxidative stress and finally apoptosis.

  9. Avocado Oil Improves Mitochondrial Function and Decreases Oxidative Stress in Brain of Diabetic Rats

    Directory of Open Access Journals (Sweden)

    Omar Ortiz-Avila

    2015-01-01

    Full Text Available Diabetic encephalopathy is a diabetic complication related to the metabolic alterations featuring diabetes. Diabetes is characterized by increased lipid peroxidation, altered glutathione redox status, exacerbated levels of ROS, and mitochondrial dysfunction. Although the pathophysiology of diabetic encephalopathy remains to be clarified, oxidative stress and mitochondrial dysfunction play a crucial role in the pathogenesis of chronic diabetic complications. Taking this into consideration, the aim of this work was to evaluate the effects of 90-day avocado oil intake in brain mitochondrial function and oxidative status in streptozotocin-induced diabetic rats (STZ rats. Avocado oil improves brain mitochondrial function in diabetic rats preventing impairment of mitochondrial respiration and mitochondrial membrane potential ΔΨm, besides increasing complex III activity. Avocado oil also decreased ROS levels and lipid peroxidation and improved the GSH/GSSG ratio as well. These results demonstrate that avocado oil supplementation prevents brain mitochondrial dysfunction induced by diabetes in association with decreased oxidative stress.

  10. Wnt Signaling Prevents the Aβ Oligomer-Induced Mitochondrial Permeability Transition Pore Opening Preserving Mitochondrial Structure in Hippocampal Neurons

    Science.gov (United States)

    Arrázola, Macarena S.; Ramos-Fernández, Eva; Cisternas, Pedro; Ordenes, Daniela; Inestrosa, Nibaldo C.

    2017-01-01

    Alzheimer’s disease (AD) is a neurodegenerative disorder mainly known for synaptic impairment and neuronal cell loss, affecting memory processes. Beside these damages, mitochondria have been implicated in the pathogenesis of AD through the induction of the mitochondrial permeability transition pore (mPTP). The mPTP is a non-selective pore that is formed under apoptotic conditions, disturbing mitochondrial structure and thus, neuronal viability. In AD, Aβ oligomers (Aβos) favor the opening of the pore, activating mitochondria-dependent neuronal cell death cascades. The Wnt signaling activated through the ligand Wnt3a has been described as a neuroprotective signaling pathway against amyloid-β (Aβ) peptide toxicity in AD. However, the mechanisms by which Wnt signaling prevents Aβos-induced neuronal cell death are unclear. We proposed here to study whether Wnt signaling protects neurons earlier than the late damages in the progression of the disease, through the preservation of the mitochondrial structure by the mPTP inhibition. To study specific events related to mitochondrial permeabilization we performed live-cell imaging from primary rat hippocampal neurons, and electron microscopy to analyze the mitochondrial morphology and structure. We report here that Wnt3a prevents an Aβos-induced cascade of mitochondrial events that leads to neuronal cell death. This cascade involves (a) mPTP opening, (b) mitochondrial swelling, (c) mitochondrial membrane potential loss and (d) cytochrome c release, thus leading to neuronal cell death. Furthermore, our results suggest that the activation of the Wnt signaling prevents mPTP opening by two possible mechanisms, which involve the inhibition of mitochondrial GSK-3β and/or the modulation of mitochondrial hexokinase II levels and activity. This study suggests a possible new approach for the treatment of AD from a mitochondrial perspective, and will also open new lines of study in the field of Wnt signaling in neuroprotection

  11. Mitochondrial dynamics and inherited peripheral nerve diseases.

    Science.gov (United States)

    Pareyson, Davide; Saveri, Paola; Sagnelli, Anna; Piscosquito, Giuseppe

    2015-06-02

    Peripheral nerves have peculiar energetic requirements because of considerable length of axons and therefore correct mitochondria functioning and distribution along nerves is fundamental. Mitochondrial dynamics refers to the continuous change in size, shape, and position of mitochondria within cells. Abnormalities of mitochondrial dynamics produced by mutations in proteins involved in mitochondrial fusion (mitofusin-2, MFN2), fission (ganglioside-induced differentiation-associated protein-1, GDAP1), and mitochondrial axonal transport usually present with a Charcot-Marie-Tooth disease (CMT) phenotype. MFN2 mutations cause CMT type 2A by altering mitochondrial fusion and trafficking along the axonal microtubule system. CMT2A is an axonal autosomal dominant CMT type which in most cases is characterized by early onset and rather severe course. GDAP1 mutations also alter fission, fusion and transport of mitochondria and are associated either with recessive demyelinating (CMT4A) and axonal CMT (AR-CMT2K) and, less commonly, with dominant, milder, axonal CMT (CMT2K). OPA1 (Optic Atrophy-1) is involved in fusion of mitochondrial inner membrane, and its heterozygous mutations lead to early-onset and progressive dominant optic atrophy which may be complicated by other neurological symptoms including peripheral neuropathy. Mutations in several proteins fundamental for the axonal transport or forming the axonal cytoskeleton result in peripheral neuropathy, i.e., CMT, distal hereditary motor neuropathy (dHMN) or hereditary sensory and autonomic neuropathy (HSAN), as well as in hereditary spastic paraplegia. Indeed, mitochondrial transport involves directly or indirectly components of the kinesin superfamily (KIF5A, KIF1A, KIF1B), responsible of anterograde transport, and of the dynein complex and related proteins (DYNC1H1, dynactin, dynamin-2), implicated in retrograde flow. Microtubules, neurofilaments, and chaperones such as heat shock proteins (HSPs) also have a fundamental

  12. Mitochondrial alterations in PINK1 deficient cells are influenced by calcineurin-dependent dephosphorylation of dynamin-related protein 1.

    Directory of Open Access Journals (Sweden)

    Anna Sandebring

    Full Text Available PTEN-induced novel kinase 1 (PINK1 mutations are associated with autosomal recessive parkinsonism. Previous studies have shown that PINK1 influences both mitochondrial function and morphology although it is not clearly established which of these are primary events and which are secondary. Here, we describe a novel mechanism linking mitochondrial dysfunction and alterations in mitochondrial morphology related to PINK1. Cell lines were generated by stably transducing human dopaminergic M17 cells with lentiviral constructs that increased or knocked down PINK1. As in previous studies, PINK1 deficient cells have lower mitochondrial membrane potential and are more sensitive to the toxic effects of mitochondrial complex I inhibitors. We also show that wild-type PINK1, but not recessive mutant or kinase dead versions, protects against rotenone-induced mitochondrial fragmentation whereas PINK1 deficient cells show lower mitochondrial connectivity. Expression of dynamin-related protein 1 (Drp1 exaggerates PINK1 deficiency phenotypes and Drp1 RNAi rescues them. We also show that Drp1 is dephosphorylated in PINK1 deficient cells due to activation of the calcium-dependent phosphatase calcineurin. Accordingly, the calcineurin inhibitor FK506 blocks both Drp1 dephosphorylation and loss of mitochondrial integrity in PINK1 deficient cells but does not fully rescue mitochondrial membrane potential. We propose that alterations in mitochondrial connectivity in this system are secondary to functional effects on mitochondrial membrane potential.

  13. Effect of Anthocyanin Extract of Mulberry Fruit on the Apoptosis and the Mitochondrial Membrane Potential of Breast Cancer Cells%桑葚花色苷提取物对乳腺癌细胞凋亡及线粒体膜电位的影响

    Institute of Scientific and Technical Information of China (English)

    常徽; 王湛; 袁丽佳; 付钰洁; 糜漫天

    2012-01-01

    目的:观察桑葚花色苷提取物对人乳腺癌细胞株MDA-MB-453、MDA-MB-231和MCF-7细胞凋亡及线粒体膜电位的影响.方法:利用超声辅助乙醇萃取法提取桑葚花色苷,pH示差法测定提取物花色苷总含量,以50、100和150 mg/mL桑葚花色苷提取物作用三种乳腺癌细胞MDA-MB-231、MDA-MB-453和MCF-7 24h,采用Annexin V/PI双染流式细胞分析法检测细胞凋亡水平变化,JC-1探针染色激光共聚焦扫描显微镜观察MDA-MB-453细胞线粒体膜电位水平变化.结果:凋亡分析结果表明,桑葚花色苷提取物作用后三种乳腺癌细胞凋亡率均升高,显示出促凋亡效应,且具有剂量-效应关系,100和150 mg/mL组凋亡率显著升高(P<0.05).激光共聚焦扫描显微镜检测结果显示,桑葚花色苷提取物作用24h,可使MDA-MB-453细胞线粒体膜电位显著下降,表现为红色/绿色荧光的比值显著降低(P<0.05).结论:桑葚花色苷提取物可显著降低乳腺癌细胞线粒体膜电位,并促发细胞凋亡.%Objective: To study the effect of anthocyanin extract of mulberry fruit on the apoptosis and the mitochondrial membrane potential of breast cancer cells MDA-MB-453, MDA-MB-231 and MCF-7. Methods: Preparation of anthocyanin-rich extract from mulberry fruits was carried out by ultrasonic extraction with acidified-ethanol. The breast cancer cells MDA-MB-453, MDA-MB-231 and MCF-7 were reated with 50, 100 or 150 mg/mL of the anthocyanin-rich extract for 24h; the cells apoptosis were analyzed by Annexin V/PI dyeing and flow cytometric assay. To characterize the upstream factors involved in the intrinsic apoptosis pathway, the mitochondrial permeability of MDA-MB-453 cells were measured by JC-1 staining and the laser confocal scanning microscopy. Results: The apoptosis analysis results indicated that the anthocyanin-rich mulberry fruit extract treated for 24 h, the apoptosis rate of these three breast cells in 100 and 150 mg/mL treated groups markedly

  14. Betaine is a positive regulator of mitochondrial respiration.

    Science.gov (United States)

    Lee, Icksoo

    2015-01-09

    Betaine protects cells from environmental stress and serves as a methyl donor in several biochemical pathways. It reduces cardiovascular disease risk and protects liver cells from alcoholic liver damage and nonalcoholic steatohepatitis. Its pretreatment can rescue cells exposed to toxins such as rotenone, chloroform, and LiCl. Furthermore, it has been suggested that betaine can suppress cancer cell growth in vivo and in vitro. Mitochondrial electron transport chain (ETC) complexes generate the mitochondrial membrane potential, which is essential to produce cellular energy, ATP. Reduced mitochondrial respiration and energy status have been found in many human pathological conditions including aging, cancer, and neurodegenerative disease. In this study we investigated whether betaine directly targets mitochondria. We show that betaine treatment leads to an upregulation of mitochondrial respiration and cytochrome c oxidase activity in H2.35 cells, the proposed rate limiting enzyme of ETC in vivo. Following treatment, the mitochondrial membrane potential was increased and cellular energy levels were elevated. We propose that the anti-proliferative effects of betaine on cancer cells might be due to enhanced mitochondrial function contributing to a reversal of the Warburg effect.

  15. A tale of two mitochondrial channels, MAC and PTP, in apoptosis.

    Science.gov (United States)

    Kinnally, Kathleen W; Antonsson, Bruno

    2007-05-01

    The crucial step in the intrinsic, or mitochondrial, apoptotic pathway is permeabilization of the mitochondrial outer membrane. Permeabilization triggers release of apoptogenic factors, such as cytochrome c, from the mitochondrial intermembrane space into the cytosol where these factors ensure propagation of the apoptotic cascade and execution of cell death. However, the mechanism(s) underlying permeabilization of the outer membrane remain controversial. Two mechanisms, involving opening of two different mitochondrial channels, have been proposed to be responsible for the permeabilization; the permeability transition pore (PTP) in the inner membrane and the mitochondrial apoptosis-induced channel (MAC) in the outer membrane. Opening of PTP would lead to matrix swelling, subsequent rupture of the outer membrane, and an unspecific release of intermembrane proteins into the cytosol. However, many believe PTP opening is a consequence of apoptosis and this channel is thought to principally play a role in necrosis, not apoptosis. Activation of MAC is exquisitely regulated by Bcl-2 family proteins, which are the sentinels of apoptosis. MAC provides specific pores in the outer membrane for the passage of intermembrane proteins, in particular cytochrome c, to the cytosol. The electrophysiological characteristics of MAC are very similar to Bax channels and depletion of Bax significantly diminishes MAC activity, suggesting that Bax is an essential constituent of MAC in some systems. The characteristics of various mitochondrial channels and Bax are compared. The involvement of MAC and PTP activities in apoptosis of disease and their pharmacology are discussed.

  16. Reciprocal Degradation of YME1L and OMA1 Adapts Mitochondrial Proteolytic Activity during Stress

    Directory of Open Access Journals (Sweden)

    T. Kelly Rainbolt

    2016-03-01

    Full Text Available The mitochondrial inner membrane proteases YME1L and OMA1 are critical regulators of essential mitochondrial functions, including inner membrane proteostasis maintenance and mitochondrial dynamics. Here, we show that YME1L and OMA1 are reciprocally degraded in response to distinct types of cellular stress. OMA1 is degraded through a YME1L-dependent mechanism in response to toxic insults that depolarize the mitochondrial membrane. Alternatively, insults that depolarize mitochondria and deplete cellular ATP stabilize active OMA1 and promote YME1L degradation. We show that the differential degradation of YME1L and OMA1 alters their proteolytic processing of the dynamin-like GTPase OPA1, a critical regulator of mitochondrial inner membrane morphology, which influences the recovery of tubular mitochondria following membrane-depolarization-induced fragmentation. Our results reveal the differential stress-induced degradation of YME1L and OMA1 as a mechanism for sensitively adapting mitochondrial inner membrane protease activity and function in response to distinct types of cellular insults.

  17. Inhibition of NAPDH Oxidase 2 (NOX2 Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

    Directory of Open Access Journals (Sweden)

    Leroy C Joseph

    Full Text Available Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

  18. Labeling the plasma membrane with TMA-DPH.

    Science.gov (United States)

    Chazotte, Brad

    2011-05-01

    INTRODUCTION TMA-DPH (trimethylamine-diphenylhexatriene) is a fluorescent membrane probe that has classically been used to label the outer leaflet of a membrane bilayer, to label the outer leaflet of the plasma membrane in cells, and to report on membrane dynamics using the techniques of fluorescence polarization and/or fluorescence lifetime. This probe has also been used to follow exocytosis and endocytosis of labeled plasma membranes. The interaction of the aqueous environment with mitochondrial inner membrane dynamics has also been studied following the fluorescence polarization and the lifetime of TMA-DPH. This protocol describes the use of TMA-DPH to label the plasma membrane.

  19. The mitochondrial free radical theory of aging.

    Science.gov (United States)

    Barja, Gustavo

    2014-01-01

    The mitochondrial free radical theory of aging is reviewed. Only two parameters currently correlate with species longevity in the right sense: the mitochondrial rate of reactive oxygen species (mitROS) production and the degree of fatty acid unsaturation of tissue membranes. Both are low in long-lived animals. In addition, the best-known manipulation that extends longevity, dietary restriction, also decreases the rate of mitROS production and oxidative damage to mtDNA. The same occurs during protein restriction as well as during methionine restriction. These two manipulations also increase maximum longevity in rodents. The decrease in mitROS generation and oxidative stress that takes place in caloric restriction seems to be due to restriction of a single dietary substance: methionine. The information available supports a mitochondrial free radical theory of aging focused on low generation of endogenous damage and low sensitivity of membranes to oxidation in long-lived animals. © 2014 Elsevier Inc. All rights reserved.

  20. Mitochondrial benzodiazepine receptors regulate steroid biosynthesis

    Energy Technology Data Exchange (ETDEWEB)

    Mukhin, A.G.; Papadopoulos, V.; Costa, E.; Krueger, K.E. (Georgetown Univ. School of Medicine, Washington, DC (USA))

    1989-12-01

    Recent observations on the steroid synthetic capability within the brain open the possibility that benzodiazepines may influence steroid synthesis in nervous tissue through interactions with peripheral-type benzodiazepine recognition sites, which are highly expressed in steroidogenic cells and associated with the outer mitochondrial membrane. To examine this possibility nine molecules that exhibit a greater than 10,000-fold difference in their affinities for peripheral-type benzodiazepine binding sites were tested for their effects on a well-established steroidogenic model system, the Y-1 mouse adrenal tumor cell line. 4{prime}-Chlorodiazepam, PK 11195, and PK 14067 stimulated steroid production by 2-fold in Y-1 cells, whereas diazepam, flunitrazepam, zolpidem, and PK 14068 displayed a lower (1.2- to 1.5-fold) maximal stimulation. In contrast, clonazepam and flumazenil did not stimulate steroid synthesis. The potencies of these compounds to inhibit {sup 3}H-labeled PK 11195 binding to peripheral-type benzodiazepine recognition sites correlated with their potencies to stimulate steroid production. Similar findings were observed in bovine and rat adrenocortical cell preparations. These results suggest that ligands of the peripheral-type benzodiazepine recognition site acting on this mitochondrial receptor can enhance steroid production. This action may contribute specificity to the pharmacological profile of drugs preferentially acting on the benzodiazepine recognition site associated with the outer membrane of certain mitochondrial populations.

  1. Mitochondrial biogenesis: pharmacological approaches.

    Science.gov (United States)

    Valero, Teresa

    2014-01-01

    Organelle biogenesis is concomitant to organelle inheritance during cell division. It is necessary that organelles double their size and divide to give rise to two identical daughter cells. Mitochondrial biogenesis occurs by growth and division of pre-existing organelles and is temporally coordinated with cell cycle events [1]. However, mitochondrial biogenesis is not only produced in association with cell division. It can be produced in response to an oxidative stimulus, to an increase in the energy requirements of the cells, to exercise training, to electrical stimulation, to hormones, during development, in certain mitochondrial diseases, etc. [2]. Mitochondrial biogenesis is therefore defined as the process via which cells increase their individual mitochondrial mass [3]. Recent discoveries have raised attention to mitochondrial biogenesis as a potential target to treat diseases which up to date do not have an efficient cure. Mitochondria, as the major ROS producer and the major antioxidant producer exert a crucial role within the cell mediating processes such as apoptosis, detoxification, Ca2+ buffering, etc. This pivotal role makes mitochondria a potential target to treat a great variety of diseases. Mitochondrial biogenesis can be pharmacologically manipulated. This issue tries to cover a number of approaches to treat several diseases through triggering mitochondrial biogenesis. It contains recent discoveries in this novel field, focusing on advanced mitochondrial therapies to chronic and degenerative diseases, mitochondrial diseases, lifespan extension, mitohormesis, intracellular signaling, new pharmacological targets and natural therapies. It contributes to the field by covering and gathering the scarcely reported pharmacological approaches in the novel and promising field of mitochondrial biogenesis. There are several diseases that have a mitochondrial origin such as chronic progressive external ophthalmoplegia (CPEO) and the Kearns- Sayre syndrome (KSS

  2. Mitochondrial Ca2+ uptake in skeletal muscle health and disease

    CERN Document Server

    Zhou, Jingsong; Yi, Jianxun

    2016-01-01

    Muscle uses Ca2+ as a messenger to control contraction and relies on ATP to maintain the intracellular Ca2+ homeostasis. Mitochondria are the major sub-cellular organelle of ATP production. With a negative inner membrane potential, mitochondria take up Ca2+ from their surroundings, a process called mitochondrial Ca2+ uptake. Under physiological conditions, Ca2+ uptake into mitochondria promotes ATP production. Excessive uptake causes mitochondrial Ca2+ overload, which activates downstream adverse responses leading to cell dysfunction. Moreover, mitochondrial Ca2+ uptake could shape spatio-temporal patterns of intracellular Ca2+ signaling. Malfunction of mitochondrial Ca2+ uptake is implicated in muscle degeneration. Unlike non-excitable cells, mitochondria in muscle cells experience dramatic changes of intracellular Ca2+ levels. Besides the sudden elevation of Ca2+ level induced by action potentials, Ca2+ transients in muscle cells can be as short as a few milliseconds during a single twitch or as long as min...

  3. CoMIC, the hidden dynamics of mitochondrial inner compartments.

    Science.gov (United States)

    Cho, Bongki; Sun, Woong

    2017-08-14

    Mitochondria have evolutionarily, functionally and structurally distinct outer- (OMM) and inner-membranes (IMM). Thus, mitochondrial morphology is controlled by independent but coordinated activity of fission and fusion of the OMM and IMM. Constriction and division of the OMM are mediated by endocytosis-like machineries, which include dynamin-related protein 1 with additional cytosolic vesicle scissoring machineries such as actin filament and Dynamin 2. However, structural alteration of the IMM during mitochondrial division has been poorly understood. Recently, we found that the IMM and the inner compartments undergo transient and reversible constriction prior to the OMM division, which we termed CoMIC, Constriction of Mitochondrial Inner Compartment. In this short review, we further discuss the evolutionary perspective and the regulatory mechanism of CoMIC during mitochondrial division.

  4. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    Science.gov (United States)

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

  5. Mitochondrial dysfunction induced by different concentrations of gadolinium ion.

    Science.gov (United States)

    Zhao, Jie; Zhou, Zhi-Qiang; Jin, Jian-Cheng; Yuan, Lian; He, Huan; Jiang, Feng-Lei; Yang, Xiao-Gang; Dai, Jie; Liu, Yi

    2014-04-01

    Gadolinium-based compounds are the most widely used paramagnetic contrast agents in magnetic resonance imaging on the world. But the tricationic gadolinium ion (Gd(3+)) could induce cell apoptosis probably because of its effects on mitochondria. Until now, the mechanism about how Gd(3+) interacts with mitochondria is not well elucidated. In this work, mitochondrial swelling, collapsed transmembrane potential and decreased membrane fluidity were observed to be important factors for mitochondrial permeability transition pore (mtPTP) opening induced by Gd(3+). The protection effect of CsA (Cyclosporin A) could confirm high concentration of Gd(3+) (500 μM) would trigger mtPTP opening. Moreover, mitochondrial outer membrane breakdown and volume expansion observed clearly by transmission electron microscopy and the release of Cyt c (Cytochrome c) could explain the mtPTP opening from another aspect. In addition, MBM(+) (monobromobimane(+)) and DTT (dithiothreitol) could protect thiol (-SH) groups from oxidation so that the toxicity of Gd(3+) might be resulted from the chelation of -SH of membrane proteins by free Gd(3+). Gd(3+) could inhibit the initiation of mitochondrial membrane lipid peroxidation, so it might interact with anionic lipids too. These findings will highly contribute to the safe applications of Gd-based agents.

  6. The causes and functions of mitochondrial proton leak.

    Science.gov (United States)

    Brand, M D; Chien, L F; Ainscow, E K; Rolfe, D F; Porter, R K

    1994-08-30

    The non-linear relationship between respiration rate and protonmotive force in isolated mitochondria is explained entirely by delta p-dependent changes in the proton conductance of the mitochondrial inner membrane and is not caused by redox slip in the proton pumps. Mitochondrial proton leak occurs in intact cells and tissues: the futile cycle of proton pumping and proton leak accounts for 26% +/- 7% of the total oxygen consumption rate or 33% +/- 7% of the mitochondrial respiration rate of isolated hepatocytes (mean +/- S.D. for 43 rats); 52% of the oxygen consumption rate of resting perfused muscle and up to 38% of the basal metabolic rate of a rat, suggesting that heat production may be an important function in the proton leak in homeotherms. Together with non-mitochondrial oxygen consumption, it lowers the effective P/O ratio in cells from maximum possible values of 2.33 (palmitate oxidation) or 2.58 (glucose oxidation) to as low as 1.1 in liver or 0.8 in muscle. The effective P/O ratio increases in response to ATP demand; the ability to allow rapid switching of flux from leak to ATP turnover may be an even more important function of the leak reaction than heat production. The mitochondrial proton conductance in isolated mitochondria and in hepatocytes is greatly modulated by thyroid hormones, by phylogeny and by body mass. Usually the reactions of ATP turnover change in parallel so that the coupling ratio is not greatly affected. Changes in proton leak in tissues are brought about in the short term by changes in mitochondrial protonmotive force and in the longer term by changes in the surface area and proton permeability of the mitochondrial inner membrane. Permeability changes are probably caused by changes in the fatty acid composition of the membrane phospholipids.

  7. Membrane reactor. Membrane reactor

    Energy Technology Data Exchange (ETDEWEB)

    Shindo, Y.; Wakabayashi, K. (National Chemical Laboratory for Industry, Tsukuba (Japan))

    1990-08-05

    Many reaction examples were introduced of membrane reactor, to be on the point of forming a new region in the field of chemical technology. It is a reactor to exhibit excellent function, by its being installed with membrane therein, and is generally classified into catalyst function type and reaction promotion type. What firstly belongs to the former is stabilized zirconia, where oxygen, supplied to the cathodic side of membrane with voltage, impressed thereon, becomes O {sup 2 {minus}} to be diffused through the membrane and supplied, as variously activated oxygenous species, on the anodic side. Examples with many advantages can be given such as methane coupling, propylene oxidation, methanating reaction of carbon dioxide, etc. Apart, palladium film and naphion film also belong to the former. While examples of the latter comprise, among others, decomposition of hydrogen sulfide by porous glass film and dehydrogenation of cyclohexane or palladium alloy film, which are expected to be developed and materialized in the industry. 33 refs., 8 figs.

  8. Alcohol dehydrogenase accentuates ethanol-induced myocardial dysfunction and mitochondrial damage in mice: role of mitochondrial death pathway.

    Science.gov (United States)

    Guo, Rui; Ren, Jun

    2010-01-18

    Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined. Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O(2) (*-). Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-alpha, Fas receptor, Fas L and cytosolic AIF. Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.

  9. United Mitochondrial Disease Foundation

    Science.gov (United States)

    ... to Mitochondrial Disease FAQ's MitoFirst Handbook More Information Mito 101 Symposium Archives Get Connected Find an Event Adult Advisory Council Team Ask The Mito Doc Grand Rounds Kids & Teens Medical Child Abuse ...

  10. Experimental treatments for mitochondrial dysfunction in sepsis: A narrative review

    Directory of Open Access Journals (Sweden)

    Guilang Zheng

    2015-01-01

    Full Text Available Sepsis is a systemic inflammatory response to infection. Sepsis, which can lead to severe sepsis, septic shock, and multiple organ dysfunction syndrome, is an important cause of mortality. Pathogenesis is extremely complex. In recent years, cell hypoxia caused by mitochondrial dysfunction has become a hot research field. Sepsis damages the structure and function of mitochondria, conversely, mitochondrial dysfunction aggravated sepsis. The treatment of sepsis lacks effective specific drugs. The aim of this paper is to undertake a narrative review of the current experimental treatment for mitochondrial dysfunction in sepsis. The search was conducted in PubMed databases and Web of Science databases from 1950 to January 2014. A total of 1,090 references were retrieved by the search, of which 121 researches met all the inclusion criteria were included. Articles on the relationship between sepsis and mitochondria, and drugs used for mitochondrial dysfunction in sepsis were reviewed retrospectively. The drugs were divided into four categories: (1 Drug related to mitochondrial matrix and respiratory chain, (2 drugs of mitochondrial antioxidant and free radical scavengers, (3 drugs related to mitochondrial membrane stability, (4 hormone therapy for septic mitochondria. In animal experiments, many drugs show good results. However, clinical research lacks. In future studies, the urgent need is to develop promising drugs in clinical trials.

  11. Crosstalk between circadian rhythmicity, mitochondrial dynamics and macrophage bactericidal activity

    Science.gov (United States)

    Oliva-Ramírez, Jacqueline; Moreno-Altamirano, María Maximina B; Pineda-Olvera, Benjamín; Cauich-Sánchez, Patricia; Sánchez-García, F Javier

    2014-01-01

    Biological functions show rhythmic fluctuations with 24-hr periodicity regulated by circadian proteins encoded by the so-called ‘clock’ genes. The absence or deregulation of circadian proteins in mice leads to metabolic disorders and in vitro models have shown that the synthesis of pro-inflammatory cytokines by macrophages follows a circadian rhythm so showing a link between circadian rhythmicity, metabolism and immunity. Recent evidence reveals that mitochondrial shape, position and size, collectively referred to as mitochondrial dynamics, are related to both cell metabolism and immune function. However, studies addressing the simultaneous crosstalk between circadian rhythm, mitochondrial dynamics and cell immune function are scarce. Here, by using an in vitro model of synchronized murine peritoneal macrophages, we present evidence that the mitochondrial dynamics and the mitochondrial membrane potential (Δψm) follow a circadian rhythmic pattern. In addition, it is shown that the fusion of mitochondria along with high Δψm, indicative of high mitochondrial activity, precede the highest phagocytic and bactericidal activity of macrophages on Salmonella typhimurium. Taken together, our results suggest a timely coordination between circadian rhythmicity, mitochondrial dynamics, and the bactericidal capacity of macrophages. PMID:24903615

  12. Advances in Human Mitochondrial Diseases Molecular Genetic Analysis of Pathogenic mtDNA Mutations.

    Science.gov (United States)

    Davidson, E; King, M P

    1997-01-01

    The mitochondrial diseases are a heterogeneous group of disorders that have been defined by specific morphological alterations in muscle and by deficits of the mitochondrial respiratory chain. The morphological hallmarks of these diseases include ragged-red fibers (an extensive proliferation of mitochondria in muscle fibers) and abnormal paracrystalline inclusions and membrane structures in mitochondria. The identification of pathogenic mutations in mitochondrial DNA (mtDNA) has resulted in a genetic classification of mitochondrial diseases. Investigations are being conducted to understand the molecular basis for the biochemical and morphological alterations of mitochondria associated with mtDNA mutations. © 1997, Elsevier Science Inc. (Trends Cardiovasc Med 1997;7:16-24).

  13. Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using yeast models of OXPHOS deficiencies.

    Science.gov (United States)

    Fontanesi, Flavia; Diaz, Francisca; Barrientos, Antoni

    2009-10-01

    The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. Several approaches for the assessment of the OXPHOS system enzymes have been developed. Based on the methods described elsewhere, this unit describes the creation and study of yeast models of mitochondrial OXPHOS deficiencies.

  14. [Mitochondrial diseases and stroke].

    Science.gov (United States)

    Irimia, P; Oliveros-Cid, A; Martínez-Vila, E

    1998-04-01

    We review the mitochondrial diseases in which cerebrovascular changes are seen, such as the MERRF syndrome (myoclonic epilepsy and ragged red fibers) or the Kearns-Sayre syndrome (progressive external ophthalmoplegia, retinitis pigmentaria, cerebellar disorders and disorders of cardiac conduction), focusing on the syndrome involving mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS). We consider the different clinical aspects, diagnostic methods, pathophysiological mechanisms of the cerebrovascular involvement as well as therapeutic approaches.

  15. Mitochondrial protection by resveratrol.

    Science.gov (United States)

    Ungvari, Zoltan; Sonntag, William E; de Cabo, Rafael; Baur, Joseph A; Csiszar, Anna

    2011-07-01

    Mitochondrial dysfunction and oxidative stress are thought to play important roles in mammalian aging. Resveratrol is a plant-derived polyphenol that exerts diverse antiaging activities, mimicking some of the molecular and functional effects of dietary restriction. This review focuses on the molecular mechanisms underlying the mitochondrial protective effects of resveratrol, which could be exploited for the prevention or amelioration of age-related diseases in the elderly.

  16. The effect of ethidium bromide and chloramphenicol on mitochondrial biogenesis in primary human fibroblasts.

    Science.gov (United States)

    Kao, Li-Pin; Ovchinnikov, Dmitry; Wolvetang, Ernst

    2012-05-15

    The expression of mitochondrial components is controlled by an intricate interplay between nuclear transcription factors and retrograde signaling from mitochondria. The role of mitochondrial DNA (mtDNA) and mtDNA-encoded proteins in mitochondrial biogenesis is, however, poorly understood and thus far has mainly been studied in transformed cell lines. We treated primary human fibroblasts with ethidium bromide (EtBr) or chloramphenicol for six weeks to inhibit mtDNA replication or mitochondrial protein synthesis, respectively, and investigated how the cells recovered from these insults two weeks after removal of the drugs. Although cellular growth and mitochondrial gene expression were severely impaired after both inhibitor treatments we observed marked differences in mitochondrial structure,membrane potential, glycolysis, gene expression, and redox status between fibroblasts treated with EtBr and chloramphenicol. Following removal of the drugs we further detected clear differences in expression of both mtDNA-encoded genes and nuclear transcription factors that control mitochondrial biogenesis, suggesting that the cells possess different compensatory mechanisms to recover from drug-induced mitochondrial dysfunction. Our data reveal new aspects of the interplay between mitochondrial retrograde signaling and the expression of nuclear regulators of mitochondrial biogenesis, a process with direct relevance to mitochondrial diseases and chloramphenicol toxicity in humans.

  17. Mitochondrial bioenergetics and redox state are unaltered in Trypanosoma cruzi isolates with compromised mitochondrial complex I subunit genes.

    Science.gov (United States)

    Carranza, Julio César; Kowaltowski, Alicia J; Mendonça, Marco Aurélio G; de Oliveira, Thays C; Gadelha, Fernanda R; Zingales, Bianca

    2009-06-01

    In trypanosomatids the involvement of mitochondrial complex I in NADH oxidation has long been debated. Here, we took advantage of natural Trypanosoma cruzi mutants which present conspicuous deletions in ND4, ND5 and ND7 genes coding for complex I subunits to further investigate its functionality. Mitochondrial bioenergetics of wild type and complex I mutants showed no significant differences in oxygen consumption or respiratory control ratios in the presence of NADH-linked substrates or FADH(2)-generating succinate. No correlation could be established between mitochondrial membrane potentials and ND deletions. Since release of reactive oxygen species occurs at complex I, we measured mitochondrial H(2)O(2) formation induced by different substrates. Significant differences not associated to ND deletions were observed among the parasite isolates, demonstrating that these mutations are not important for the control of oxidant production. Our data support the notion that complex I has a limited function in T. cruzi.

  18. Time representation of mitochondrial morphology and function after acute spinal cord injury

    Institute of Scientific and Technical Information of China (English)

    Zhi-qiang Jia; Gang Li; Zhen-yu Zhang; Hao-tian Li; Ji-quan Wang; Zhong-kai Fan; Gang Lv

    2016-01-01

    Changes in mitochondrial morphology and function play an important role in secondary damage after acute spinal cord injury. We re-corded the time representation of mitochondrial morphology and function in rats with acute spinal cord injury. Results showed that mitochondria had an irregular shape, and increased in size. Mitochondrial cristae were disordered and mitochondrial membrane rupture was visible at 2–24 hours after injury. Fusion protein mitofusin 1 expression gradually increased, peaked at 8 hours after injury, and then decreased to its lowest level at 24 hours. Expression of dynamin-related protein 1, amitochondrial ifssion protein, showed the opposite kinetics. At 2–24 hours after acute spinal cord injury, malondialdehyde content, cytochrome c levels and caspase-3 expression were in-creased, but glutathione content, adenosine triphosphate content, Na+-K+-ATPase activity and mitochondrial membrane potential were gradually reduced. Furthermore, mitochondrial morphology altered during the acute stage of spinal cord injury. Fusion was important within the ifrst 8 hours, but ifssion played a key role at 24 hours. Oxidative stress was inhibited, biological productivity was diminished, and mitochondrial membrane potential and permeability were reduced in the acute stage of injury. In summary, mitochondrial apoptosis is activated when the time of spinal cord injury is prolonged.

  19. Regulation of mitochondrial function by voltage dependent anion channels in ethanol metabolism and the Warburg effect.

    Science.gov (United States)

    Lemasters, John J; Holmuhamedov, Ekhson L; Czerny, Christoph; Zhong, Zhi; Maldonado, Eduardo N

    2012-06-01

    Voltage dependent anion channels (VDAC) are highly conserved proteins that are responsible for permeability of the mitochondrial outer membrane to hydrophilic metabolites like ATP, ADP and respiratory substrates. Although previously assumed to remain open, VDAC closure is emerging as an important mechanism for regulation of global mitochondrial metabolism in apoptotic cells and also in cells that are not dying. During hepatic ethanol oxidation to acetaldehyde, VDAC closure suppresses exchange of mitochondrial metabolites, resulting in inhibition of ureagenesis. In vivo, VDAC closure after ethanol occurs coordinately with mitochondrial uncoupling. Since acetaldehyde passes through membranes independently of channels and transporters, VDAC closure and uncoupling together foster selective and more rapid oxidative metabolism of toxic acetaldehyde to nontoxic acetate by mitochondrial aldehyde dehydrogenase. In single reconstituted VDAC, tubulin decreases VDAC conductance, and in HepG2 hepatoma cells, free tubulin negatively modulates mitochondrial membrane potential, an effect enhanced by protein kinase A. Tubulin-dependent closure of VDAC in cancer cells contributes to suppression of mitochondrial metabolism and may underlie the Warburg phenomenon of aerobic glycolysis. This article is part of a Special Issue entitled: VDAC structure, function, and regulation of mitochondrial metabolism.

  20. Low-dose ionizing radiation induces mitochondrial fusion and increases expression of mitochondrial complexes I and III in hippocampal neurons.

    Science.gov (United States)

    Chien, Ling; Chen, Wun-Ke; Liu, Szu-Ting; Chang, Chuang-Rung; Kao, Mou-Chieh; Chen, Kuan-Wei; Chiu, Shih-Che; Hsu, Ming-Ling; Hsiang, I-Chou; Chen, Yu-Jen; Chen, Linyi

    2015-10-13

    High energy ionizing radiation can cause DNA damage and cell death. During clinical radiation therapy, the radiation dose could range from 15 to 60 Gy depending on targets. While 2 Gy radiation has been shown to cause cancer cell death, studies also suggest a protective potential by low dose radiation. In this study, we examined the effect of 0.2-2 Gy radiation on hippocampal neurons. Low dose 0.2 Gy radiation treatment increased the levels of MTT. Since hippocampal neurons are post-mitotic, this result reveals a possibility that 0.2 Gy irradiation may increase mitochondrial activity to cope with stimuli. Maintaining neural plasticity is an energy-demanding process that requires high efficient mitochondrial function. We thus hypothesized that low dose radiation may regulate mitochondrial dynamics and function to ensure survival of neurons. Our results showed that five days after 0.2 Gy irradiation, no obvious changes on neuronal survival, neuronal synapses, membrane potential of mitochondria, reactive oxygen species levels, and mitochondrial DNA copy numbers. Interestingly, 0.2 Gy irradiation promoted the mitochondria fusion, resulting in part from the increased level of a mitochondrial fusion protein, Mfn2, and inhibition of Drp1 fission protein trafficking to the mitochondria. Accompanying with the increased mitochondrial fusion, the expressions of complexes I and III of the electron transport chain were also increased. These findings suggest that, hippocampal neurons undergo increased mitochondrial fusion to modulate cellular activity as an adaptive mechanism in response to low dose radiation.

  1. Progress in surface and membrane science

    CERN Document Server

    Cadenhead, D A; Rosenberg, M D

    1974-01-01

    Progress in Surface and Membrane Science, Volume 8 covers the developments in the study of surface and membrane science. The book discusses the applications of statistical mechanics to physical adsorption; the impact of electron spectroscopy and cognate techniques on the study of solid surfaces; and the ellipsometric studies of thin films. The text also describes the interfacial photochemistry of bilayer lipid membranes; cell junctions and their development; and the composition and function of the inner mitochondrial membrane. The role of the cell surface in contact inhibition of cell division

  2. Biobased Membrane

    NARCIS (Netherlands)

    Koenders, E.A.B.; Zlopasa, J.; Picken, S.J.

    2015-01-01

    The present invention is in the field of a composition for forming a bio-compatible membrane applicable to building material, such as concrete, cement, etc., to a meth od of applying said composition for forming a bio-compatible membrane, a biocompatible membrane, use of said membrane for various pu

  3. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  4. Peripheral neuropathy in mitochondrial disorders.

    Science.gov (United States)

    Pareyson, Davide; Piscosquito, Giuseppe; Moroni, Isabella; Salsano, Ettore; Zeviani, Massimo

    2013-10-01

    Why is peripheral neuropathy common but mild in many mitochondrial disorders, and why is it, in some cases, the predominant or only manifestation? Although this question remains largely unanswered, recent advances in cellular and molecular biology have begun to clarify the importance of mitochondrial functioning and distribution in the peripheral nerve. Mutations in proteins involved in mitochondrial dynamics (ie, fusion and fission) frequently result in a Charcot-Marie-Tooth phenotype. Peripheral neuropathies with different phenotypic presentations occur in mitochondrial diseases associated with abnormalities in mitochondrial DNA replication and maintenance, or associated with defects in mitochondrial respiratory chain complex V. Our knowledge of mitochondrial disorders is rapidly growing as new nuclear genes are identified and new phenotypes described. Early diagnosis of mitochondrial disorders, essential to provide appropriate genetic counselling, has become crucial in a few treatable conditions. Recognising and diagnosing an underlying mitochondrial defect in patients presenting with peripheral neuropathy is therefore of paramount importance.

  5. Ocular manifestations of mitochondrial disease

    Directory of Open Access Journals (Sweden)

    S. D. Mathebula

    2012-12-01

    Full Text Available Mitochondrial disease caused by mutations in mitochondrial DNA is recognized as one of the most common causes of inherited neurological disease. Neuro-ophthalmic manifestations are a common feature of mitochondrial disease.  Optic atrophy causing central visual loss is the dominant feature of mitochondrial DNA diseases. Nystagmus is also encountered in mitochondrial disease.Although optometrists are not involved with the management of mitochondrial disease, they are likely to see more patients with this disease. Oph-thalmic examination forms part of the clinical assessment of mitochondrial disease. Mitochondrial disease should be suspected in any patient with unexplained optic neuropathy, ophthalmoplegia, pigmentary retinopathy or retrochiasmal visual loss. Despite considerable advances in the under-standing of mitochondrial genetics and the patho-genesis of mtDNA diseases, no effective treatment options are currently available for patients withmitochondrial dysfunction. (S Afr Optom 201271(1 46-50

  6. Phosphodiesterase-3 inhibitor (cilostazol) attenuates oxidative stress-induced mitochondrial dysfunction in the heart

    Institute of Scientific and Technical Information of China (English)

    Siriporn C.Chattipakorn; Savitree Thummasorn; Jantira Sanit; Nipon Chattipakorn

    2014-01-01

    Background Cilostazol is a type 3 phosphodiesterase inhibitor which has been previously demonstrated to prevent the occurrence of tachyarrhythmia and improve defibrillation efficacy. However, the mechanism for this beneficial effect is still unclear. Since cardiac mito-chondria have been shown to play a crucial role in fatal cardiac arrhythmias and that oxidative stress is one of the main contributors to arr-hythmia generation, we tested the effects of cilostazol on cardiac mitochondria under severe oxidative stress. Methods Mitochondria were isolated from rat hearts and treated with H2O2 to induce oxidative stress. Cilostazol, at various concentrations, was used to study its protective effects. Pharmacological interventions, including a mitochondrial permeability transition pore (mPTP) blocker, cyclosporine A (CsA), and an inner membrane anion channel (IMAC) blocker, 4’-chlorodiazepam (CDP), were used to investigate the mechanistic role of cilostazol on cardiac mitochondria. Cardiac mitochondrial reactive oxygen species (ROS) production, mitochondrial membrane potential change and mi-tochondrial swelling were determined as indicators of cardiac mitochondrial function. Results Cilostazol preserved cardiac mitochondrial function when exposed to oxida