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Sample records for disrupts mitochondrial membrane

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

    PubMed

    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.

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

    PubMed Central

    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

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

    PubMed Central

    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. PMID:26091109

  4. Cyclosporin A does not protect the disruption of the inner mitochondrial membrane potential induced by potassium ionophores in intact K562 cells.

    PubMed

    Marques-Santos, Luis F; Coqueiro, Vivian M; Rumjanek, Vivian M

    2006-03-01

    Mitochondrial dysfunction has been widely associated with programmed cell death. Studies of intact cells are important for the understanding of the process of cell death and its relation to mitochondrial physiology. Using cytofluorometric approaches we studied the mitochondrial behavior in an erythroleukemic cell line. The effects of protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), potassium exchanger (nigericin), potassium ionophore (valinomycin), Na+K+-ATPase inhibitor (ouabain) and mitochondrial permeability transition pore inhibitor (cyclosporin A) were evaluated. Cyclosporin A (CSA) was very effective in attenuating the disruption of inner mitochondrial membrane potential induced by CCCP. However, CSA failed to protect the loss of inner mitochondrial membrane potential induced by potassium intracellular flux manipulation. Our findings suggest that mitochondrial cyclophilin is not involved in the cell events mediated by deregulation of potassium flux, underlining the need for further studies in intact tumor cells for a better understanding of the involvement of mitochondria physiology in cell death events.

  5. Antifungal Action of Methylene Blue Involves Mitochondrial Dysfunction and Disruption of Redox and Membrane Homeostasis in C. albicans

    PubMed Central

    Ansari, Moiz A.; Fatima, Zeeshan; Hameed, Saif

    2016-01-01

    Candida albicans is known to cause infections ranging from superficial and systemic in immunocompromised person. In this study, we explored that the antifungal action of Methylene blue (MB) is mediated through mitochondrial dysfunction and disruption of redox and membrane homeostasis against C. albicans. We demonstrated that MB displayed its antifungal potential against C. albicans and two clinical isolates tested. We also showed that MB is effective against two non- albicans species as well. Notably, the antifungal effect of MB seems to be independent of the major drug efflux pumps transporter activity. We explored that MB treated Candida cells were sensitive on non-fermentable carbon source leading us to propose that MB inhibits mitochondria. This sensitive phenotype was reinforced with the fact that sensitivity of Candida cells to MB could be rescued upon the supplementation of ascorbic acid, an antioxidant. This clearly suggests that disturbances in redox status are linked with MB action. We further demonstrated that Candida cells were susceptible to membrane perturbing agent viz. SDS which was additionally confirmed by transmission electron micrographs showing disruption of membrane integrity. Moreover, the ergosterol levels were significantly decreased by 66% suggesting lipid compositional changes due to MB. Furthermore, we could demonstrate that MB inhibits the yeast to hyphal transition in C. albicans which is one of the major virulence attribute in most of the hyphal inducing conditions. Taken together, the data generated from present study clearly establishes MB as promising antifungal agent that could be efficiently employed in strategies to treat Candida infections. PMID:27006725

  6. A mechanism of virulence: virulent Mycobacterium tuberculosis strain H37Rv, but not attenuated H37Ra, causes significant mitochondrial inner membrane disruption in macrophages leading to necrosis.

    PubMed

    Chen, Minjian; Gan, Huixian; Remold, Heinz G

    2006-03-15

    Infection of human monocyte-derived macrophages with Mycobacterium tuberculosis at low multiplicities of infection leads 48-72 h after the infection to cell death with the characteristics of apoptosis or necrosis. Predominant induction of one or the other cell death modality depends on differences in mitochondrial membrane perturbation induced by attenuated and virulent strains. Infection of macrophages with the attenuated H37Ra or the virulent H37Rv causes mitochondrial outer membrane permeabilization characterized by cytochrome c release from the mitochondrial intermembrane space and apoptosis. Mitochondrial outer membrane permeabilization is transient, peaks 6 h after infection, and requires Ca(2+) flux and B cell chronic lymphocytic leukemia/lymphoma 2-associated protein X translocation into mitochondria. In contrast, only the virulent H37Rv induces significant mitochondrial transmembrane potential (Deltapsi(m)) loss caused by mitochondrial permeability transition. Dissipation of Deltapsi(m) also peaks at 6 h after infection, is transient, is inhibited by the classical mitochondrial permeability transition inhibitor cyclosporine A, has a requirement for mitochondrial Ca(2+) loading, and is independent of B cell chronic lymphocytic leukemia/lymphoma translocation into the mitochondria. Transient dissipation of Deltapsi(m) 6 h after infection is essential for the induction of macrophage necrosis by Mtb, a mechanism that allows further dissemination of the pathogen and development of the disease.

  7. PUMA-mediated mitochondrial apoptotic disruption by hypoxic postconditioning.

    PubMed

    Li, YuZhen; Guo, Qi; Liu, XiuHua; Wang, Chen; Song, DanDan

    2015-08-01

    Postconditioning can reduce ischemia-reperfusion (I/R)-induced cardiomyocyte apoptosis by targeting mitochondria. p53 upregulated modulator of apoptosis (PUMA) is involved in lethal I/R injury. Here, we hypothesized that postconditioning might inhibit mitochondrial pathway-mediated cardiomyocyte apoptosis by controlling PUMA expression. The cultured neonatal rat cardiomyocytes underwent 3 h of hypoxia and 3 h of reoxygenation. Postconditioning consisted of three cycles of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Hypoxic postconditioning reduced the levels of PUMA mRNA and protein. Concomitantly, the loss of mitochondrial membrane potential, cytochrome c release and caspase-3 activation were decreased significantly by postconditioning. Overexpression of PUMA increased greatly not only the number of apoptotic cardiomyocytes, but also the collapse of mitochondrial membrane potential, cytochrome c release and caspase-3 activation under postconditioning condition. The data suggest that reduction of PUMA expression mediates the endogenous cardioprotective mechanisms of postconditioning by disrupting mitochondrial apoptotic pathway.

  8. Anticancer and apoptotic activities of oleanolic acid are mediated through cell cycle arrest and disruption of mitochondrial membrane potential in HepG2 human hepatocellular carcinoma cells

    PubMed Central

    ZHU, YUE-YONG; HUANG, HONG-YAN; WU, YIN-LIAN

    2015-01-01

    Hepatocellular carcinoma (HCC) is an aggressive form of cancer, with high rates of morbidity and mortality, a poor prognosis and limited therapeutic options. The objective of the present study was to demonstrate the anticancer activity of oleanolic acid in HepG2 human HCC cells. Cell viability was evaluated using an MTT assay, following administration of various doses of oleanolic acid. The effect of oleanolic acid on cell cycle phase distribution and mitochondrial membrane potential was evaluated using flow cytometry with propidium iodide and rhodamine-123 DNA-binding cationic fluorescent dyes. Fluorescence microscopy was employed to detect morphological changes in HepG2 cells following oleanolic acid treatment. The results revealed that oleanolic acid induced a dose-dependent, as well as time-dependent inhibition in the growth of HepG2 cancer cells. Following acridine orange and ethidium bromide staining, treatment with various doses (0, 5, 25 and 50 µM) of oleanolic acid induced typical morphological changes associated with apoptosis, including cell shrinkage, membrane blebbing, nuclear condensation and apoptotic body formation. Cell cycle analysis revealed that oleanolic acid induced cell cycle arrest in HepG2 cells at the sub-G1 (apoptotic) phase of the cell cycle, in a dose-dependent manner. Staining with Annexin V-fluorescein isothiocyanate and propidium iodide revealed that apoptosis occurred early in these cells. Oleanolic acid treatment also resulted in fragmentation of nuclear DNA in a dose-dependent manner, producing the typical features of DNA laddering on an agarose gel. The results also demonstrated that oleanolic acid treatment resulted in a potent loss of mitochondrial membrane potential, which also occurred in a dose-dependent manner. Therefore, oleanolic acid may be used as a therapeutic agent in the treatment of human HCC. PMID:26151733

  9. Mitochondrial membrane permeabilization with nanosecond electric pulses.

    PubMed

    Vernier, P Thomas

    2011-01-01

    Ultra-short, high-field electric pulses permeabilize plasma and intracellular membranes. We report here nanosecond pulse-induced permeabilization of mitochondrial membranes in living cells. Using four independent methods based on fluorescent dyes--JC-1, rhodamine 123, tetramethyl rhodamine ethyl ester, and cobalt-quenched calcein--we show that as few as five, 4 ns, 10 MV/m pulses delivered at 1 kHz cause an increase of the inner mitochondrial membrane permeability and an associated loss of mitochondrial membrane potential. The most likely interpretation of these results is a pulse-induced permeabilization of the inner mitochondrial membrane.

  10. Plasma membrane disruption: repair, prevention, adaptation

    NASA Technical Reports Server (NTRS)

    McNeil, Paul L.; Steinhardt, Richard A.

    2003-01-01

    Many metazoan cells inhabit mechanically stressful environments and, consequently, their plasma membranes are frequently disrupted. Survival requires that the cell rapidly repair or reseal the disruption. Rapid resealing is an active and complex structural modification that employs endomembrane as its primary building block, and cytoskeletal and membrane fusion proteins as its catalysts. Endomembrane is delivered to the damaged plasma membrane through exocytosis, a ubiquitous Ca2+-triggered response to disruption. Tissue and cell level architecture prevent disruptions from occurring, either by shielding cells from damaging levels of force, or, when this is not possible, by promoting safe force transmission through the plasma membrane via protein-based cables and linkages. Prevention of disruption also can be a dynamic cell or tissue level adaptation triggered when a damaging level of mechanical stress is imposed. Disease results from failure of either the preventive or resealing mechanisms.

  11. Enhanced induction of cell cycle arrest and apoptosis via the mitochondrial membrane potential disruption in human U87 malignant glioma cells by aloe emodin.

    PubMed

    Ismail, Samhani; Haris, Khalilah; Abdul Ghani, Abdul Rahman Izaini; Abdullah, Jafri Malin; Johan, Muhammad Farid; Mohamed Yusoff, Abdul Aziz

    2013-09-01

    Aloe emodin, one of the active compounds found in Aloe vera leaves, plays an important role in the regulation of cell growth and death. It has been reported to promote the anti-cancer effects in various cancer cells by inducing apoptosis. However, the mechanism of inducing apoptosis by this agent is poorly understood in glioma cells. This research is to investigate the apoptosis and cell cycle arrest inducing by aloe emodin on U87 human malignant glioma cells. Aloe emodin showed a time- and dose-dependent inhibition of U87 cells proliferation and decreased the percentage of viable U87 cells via the induction of apoptosis. Characteristic morphological changes, such as the formation of apoptotic bodies, were observed with confocal microscope by Annexin V-FITC/PI staining, supporting our viability study and flow cytometry analysis results. Our data also demonstrated that aloe emodin arrested the cell cycle in the S phase and promoted the loss of mitochondrial membrane potential in U87 cells that indicated the early event of the mitochondria-induced apoptotic pathway.

  12. 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.

    PubMed

    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.

  13. Effect of short-term exposure to diesel exhaust particles and carboxylic acids on mitochondrial membrane disruption in airway epithelial cells

    EPA Science Inventory

    Rationale: Diesel exhaust has been shown to induce adverse pulmonary health effects; however, the underlying mechanisms for these effects are still unclear. Previous studies have imlplicated mitochondrial dysfunction in the toxicity of diesel exhaust particles (DEP). DEP contain...

  14. DISRUPTION OF YEAST MEMBRANES BY METHYLPHENIDATE.

    DTIC Science & Technology

    Methylphenidate blocked sorbose uptake and loss by yeast spheroplasts and, at higher concentrations, disrupted spheroplasts. At high concentrations methylphenidate also ruptured the membranes of whole yeast cells; sorbose and 280 nm-absorbing materials were lost from the cells, and methylene blue stained them. Intracellular structures were extensively affected as shown by electron micrographs, and evidently were more sensitive to methylphenidate than the external membrane. N-ethylmaleimide and Ca(++) enhanced the rupture of external membranes by methylphenidate. (Author)

  15. An analysis of membrane fusion between mitochondrial double membranes and MITO-Porter, mitochondrial fusogenic vesicles.

    PubMed

    Yamada, Yuma; Fukuda, Yutaka; Harashima, Hideyoshi

    2015-09-01

    To achieve mitochondrial gene therapy, therapeutic molecules need to be transported through the outer and inner membranes of mitochondria into the innermost space (mitochondrial matrix), which contains the mtDNA pool. We previously reported on the construction of a MITO-Porter with a high fusogenic activity for the mitochondrial outer membrane for delivering molecules to the mitochondria of human cells. Here, we report on an investigation of a fusogenic lipid composition for the inner membrane, and an analysis of the fusogenic compositions for the outer and inner membranes. A significant relationship was found between fusion activity and the mitochondrial delivery of nucleic acids.

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

    PubMed

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

    PubMed

    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, <0.9). For the first time, we have demonstrated that mitochondrial 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.

  18. Novel channels of the inner mitochondrial membrane.

    PubMed

    Zoratti, Mario; De Marchi, Umberto; Gulbins, Erich; Szabò, Ildikò

    2009-05-01

    Along with a large number of carriers, exchangers and "pumps", the inner mitochondrial membrane contains ion-conducting channels which endow it with controlled permeability to small ions. Some have been shown to be the mitochondrial counterpart of channels present also in other cellular membranes. The manuscript summarizes the current state of knowledge on the major inner mitochondrial membrane channels, properties, identity and proposed functions. Considerable attention is currently being devoted to two K(+)-selective channels, mtK(ATP) and mtBK(Ca). Their activation in "preconditioning" is considered by many to underlie the protection of myocytes and other cells against subsequent ischemic damage. We have recently shown that in apoptotic lymphocytes inner membrane mtK(V)1.3 interacts with the pro-apoptotic protein Bax after the latter has inserted into the outer mitochondrial membrane. Whether the just-discovered mtIK(Ca) has similar cellular role(s) remains to be seen. The Ca(2+) "uniporter" has been characterized electrophysiologically, but still awaits a molecular identity. Chloride-selective channels are represented by the 107 pS channel, the first mitochondrial channel to be observed by patch-clamp, and by a approximately 400 pS pore we have recently been able to fully characterize in the inner membrane of mitochondria isolated from a colon tumour cell line. This we propose to represent a component of the Permeability Transition Pore. The available data exclude the previous tentative identification with porin, and indicate that it coincides instead with the still molecularly unidentified "maxi" chloride channel.

  19. Monitoring mitochondrial membranes permeability in live neurons and mitochondrial swelling through electron microscopy analysis.

    PubMed

    Arrázola, Macarena S; Inestrosa, Nibaldo C

    2015-01-01

    Maintenance of mitochondrial membrane integrity is essential for mitochondrial function and neuronal viability. Apoptotic stimulus or calcium overload leads to mitochondrial permeability transition pore (mPTP ) opening and induces mitochondrial swelling, a common feature of mitochondrial membrane permeabilization. The first phenomenon can be evaluated in cells loaded with the dye calcein -AM quenched by cobalt, and mitochondrial swelling can be detected by electron microscopy through the analysis of mitochondrial membrane integrity. Here, we describe a live cell imaging assay to detect mitochondrial permeability transition and the development of a detailed analysis of morphological and ultrastructural changes that mitochondria undergo during this process.

  20. Fullerenol cytotoxicity in kidney cells is associated with cytoskeleton disruption, autophagic vacuole accumulation, and mitochondrial dysfunction

    SciTech Connect

    Johnson-Lyles, Denise N.; Peifley, Kimberly; Lockett, Stephen; Neun, Barry W.; Hansen, Matthew; Clogston, Jeffrey; Stern, Stephan T.; McNeil, Scott E.

    2010-11-01

    Water soluble fullerenes, such as the hydroxylated fullerene, fullerenol (C{sub 60}OH{sub x}), are currently under development for diagnostic and therapeutic biomedical applications in the field of nanotechnology. These molecules have been shown to undergo urinary clearance, yet there is limited data available on their renal biocompatibility. Here we examine the biological responses of renal proximal tubule cells (LLC-PK1) exposed to fullerenol. Fullerenol was found to be cytotoxic in the millimolar range, with viability assessed by the sulforhodamine B and trypan blue assays. Fullerenol-induced cell death was associated with cytoskeleton disruption and autophagic vacuole accumulation. Interaction with the autophagy pathway was evaluated in vitro by Lysotracker Red dye uptake, LC3-II marker expression and TEM. Fullerenol treatment also resulted in coincident loss of cellular mitochondrial membrane potential and ATP depletion, as measured by the Mitotracker Red dye and the luciferin-luciferase assays, respectively. Fullerenol-induced ATP depletion and loss of mitochondrial potential were partially ameliorated by co-treatment with the autophagy inhibitor, 3-methyladenine. In vitro fullerenol treatment did not result in appreciable oxidative stress, as measured by lipid peroxide and glutathione content. Based on these data, it is hypothesized that cytoskeleton disruption may be an initiating event in fullerenol cytotoxicity, leading to subsequent autophagy dysfunction and loss of mitochondrial capacity. As nanoparticle-induced cytoskeleton disruption, autophagic vacuole accumulation and mitochondrial dysfunction are commonly reported in the literature, the proposed mechanism may be relevant for a variety of nanomaterials.

  1. Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca(2+) and ROS in Candida albicans.

    PubMed

    Tian, Jun; Lu, Zhaoqun; Wang, Yanzhen; Zhang, Man; Wang, Xueyan; Tang, Xudong; Peng, Xue; Zeng, Hong

    2017-02-14

    The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca(2+)) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca(2+) and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca(2+) and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.

  2. Aging leads to elevation of O-GlcNAcylation and disruption of mitochondrial homeostasis in retina.

    PubMed

    Zhao, Lin; Feng, Zhihui; Zou, Xuan; Cao, Ke; Xu, Jie; Liu, Jiankang

    2014-01-01

    Retina is particularly susceptible to aging as oxidative damage accumulates within retina, leading to age-related retinal dysfunction or even visual loss. However, the underlying mechanisms still remain obscure and effective therapeutic strategy is urgently in need. Here, we quested for the answer particularly focusing on mitochondrial homeostasis and O-GlcNAcylation in rat retina. By comparing expression of electron transfer chain complexes and key factors in mitochondrial biogenesis and dynamics in retinas of aged and young Sprague-Dawley rats, we found that mitochondrial Complex I, II, IV and V were increased in aged retina with decreased mtTFA and Mfn2. Also, we noticed that p38 and JNK of MAPK signaling were substantially more activated in aged retina, suggesting stress induction. In addition, we found that pan-O-GlcNAcylation was remarkably stronger with lower OGA expression in aged retina. To further elucidate the roles of Mfn2 and O-GlcNAcylation, we employed ARPE-19 cells and found that ATP production, oxygen consumption, and mitochondrial membrane potential were reduced and ROS level was increased by Mfn2 knockdown, while treating with PUGNAc or UDP-GlcNAc heightened oxygen consumption and reduced ROS. Our results suggest disrupted mitochondrial homeostasis may increase oxidative stress; yet enhanced O-GlcNAcylation might defend against oxidative stress and promote mitochondrial respiration in aged retina.

  3. Membrane disruption mechanism of antimicrobial peptides

    NASA Astrophysics Data System (ADS)

    Lee, Ka Yee

    2012-04-01

    Largely distributed among living organisms, antimicrobial peptides are a class of small (<100 residues) host defense peptides that induce selective membrane lytic activity against microbial pathogens. The permeabilizing behavior of these diverse peptides has been commonly attributed to the formation of pores, and such pore formation has been categorized as barrel-stave, toroidal, or carpet-like. With the continuing discovery of new peptide species, many are uncharacterized and the exact mechanism is unknown. Through the use of atomic force microscopy, the disruption of supported lipid bilayer patches by protegrin-1 is concentration-dependent. The intercalation of antimicrobial peptide into the bilayer results in structures beyond that of pore formation, but with the formation of worm-like micelles at high peptide concentration. Our results suggest that antimicrobial peptide acts to lower the interfacial energy of the bilayer in a way similar to detergents. Antimicrobial peptides with structural differences, magainin-1 and aurein 1.1, exhibit a mechanistic commonality.

  4. Mitochondrial glutathione peroxidase 4 disruption causes male infertility.

    PubMed

    Schneider, Manuela; Förster, Heidi; Boersma, Auke; Seiler, Alexander; Wehnes, Helga; Sinowatz, Fred; Neumüller, Christine; Deutsch, Manuel J; Walch, Axel; Hrabé de Angelis, Martin; Wurst, Wolfgang; Ursini, Fulvio; Roveri, Antonella; Maleszewski, Marek; Maiorino, Matilde; Conrad, Marcus

    2009-09-01

    Selenium is linked to male fertility. Glutathione peroxidase 4 (GPx4), first described as an antioxidant enzyme, is the predominant selenoenzyme in testis and has been suspected of being vital for spermatogenesis. Cytosolic, mitochondrial, and nuclear isoforms are all encoded by the same gene. While disruption of entire GPx4 causes early embryonic lethality in mice, inactivation of nuclear GPx4 does not impair embryonic development or fertility. Here, we show that deletion of mitochondrial GPx4 (mGPx4) allows both normal embryogenesis and postnatal development, but causes male infertility. Infertility was associated with impaired sperm quality and severe structural abnormalities in the midpiece of spermatozoa. Knockout sperm display higher protein thiol content and recapitulate features typical of severe selenodeficiency. Interestingly, male infertility induced by mGPx4 depletion could be bypassed by intracytoplasmic sperm injection. We also show for the first time that mGPx4 is the prevailing GPx4 product in male germ cells and that mGPx4 disruption has no effect on proliferation or apoptosis of germinal or somatic tissue. Our study finally establishes that mitochondrial GPx4 confers the vital role of selenium in mammalian male fertility and identifies cytosolic GPx4 as the only GPx4 isoform being essential for embryonic development and apoptosis regulation.

  5. Sustained Early Disruption of Mitochondrial Function Contributes to Arsenic-Induced Prostate Tumorigenesis.

    PubMed

    Singh, B; Kulawiec, M; Owens, K M; Singh, A; Singh, K K

    2016-10-01

    Arsenic is a well-known human carcinogen that affects millions of people worldwide, but the underlying mechanisms of carcinogenesis are unclear. Several epidemiological studies have suggested increased prostate cancer incidence and mortality due to exposure to arsenic. Due to lack of an animal model of arsenic-induced carcinogenesis, we used a prostate epithelial cell culture model to identify a role for mitochondria in arsenic-induced prostate cancer. Mitochondrial morphology and membrane potential was impacted within a few hours of arsenic exposure of non-neoplastic prostate epithelial cells. Chronic arsenic treatment induced mutations in mitochondrial genes and altered mitochondrial functions. Human non-neoplastic prostate epithelial cells continuously cultured for seven months in the presence of 5 µM arsenite showed tumorigenic properties in vitro and induced tumors in SCID mice, which indicated transformation of these cells. Protein and mRNA expression of subunits of mtOXPHOS complex I were decreased in arsenic-transformed cells. Alterations in complex I, a main site for reactive oxygen species (ROS) production as well as increased expression of ROS-producing NOX4 in arsenic-transformed cells suggested a role of oxidative stress in tumorigenic transformation of prostate epithelial cells. Whole genome cGH array analyses of arsenic-transformed prostate cells identified extensive genomic instability. Our study revealed mitochondrial dysfunction induced oxidative stress and decreased expression of p53 in arsenic-transformed cells as an underlying mechanism of the mitochondrial and nuclear genomic instability. These studies suggest that early changes in mitochondrial functions are sustained during prolong arsenic exposure. Overall, our study provides evidence that arsenic disruption of mitochondrial function is an early and key step in tumorigenic transformation of prostate epithelial cells.

  6. The outer mitochondrial membrane in higher plants.

    PubMed

    Duncan, Owen; van der Merwe, Margaretha J; Daley, Daniel O; Whelan, James

    2013-04-01

    The acquisition and integration of intracellular organelles, such as mitochondria and plastids, were important steps in the emergence of complex multicellular life. Although the outer membranes of these organelles have lost many of the functions of their free-living bacterial ancestor, others were acquired during organellogenesis. To date, the biological roles of these proteins have not been systematically characterized. In this review, we discuss the evolutionary origins and functions of outer membrane mitochondrial (OMM) proteins in Arabidopsis thaliana. Our analysis, using phylogenetic inference, indicates that several OMM proteins either acquired novel functional roles or were recruited from other subcellular localizations during evolution in Arabidopsis. These observations suggest the existence of novel communication routes and functions between organelles within plant cells.

  7. Amyloid Aggregation and Membrane Disruption by Amyloid Proteins

    NASA Astrophysics Data System (ADS)

    Ramamoorthy, Ayyalusamy

    2013-03-01

    Amyloidogenesis has been the focus of intense basic and clinical research, as an increasing number of amyloidogenic proteins have been linked to common and incurable degenerative diseases including Alzheimer's, type II diabetes, and Parkinson's. Recent studies suggest that the cell toxicity is mainly due to intermediates generated during the assembly process of amyloid fibers, which have been proposed to attack cells in a variety of ways. Disruption of cell membranes is believed to be one of the key components of amyloid toxicity. However, the mechanism by which this occurs is not fully understood. Our research in this area is focused on the investigation of the early events in the aggregation and membrane disruption of amyloid proteins, Islet amyloid polypeptide protein (IAPP, also known as amylin) and amyloid-beta peptide, on the molecular level. Structural insights into the mechanisms of membrane disruption by these amyloid proteins and the role of membrane components on the membrane disruption will be presented.

  8. A male sterility-associated mitochondrial protein in wild beets causes pollen disruption in transgenic plants.

    PubMed

    Yamamoto, Masayuki P; Shinada, Hiroshi; Onodera, Yasuyuki; Komaki, Chihiro; Mikami, Tetsuo; Kubo, Tomohiko

    2008-06-01

    In higher plants, male reproductive (pollen) development is known to be disrupted in a class of mitochondrial mutants termed cytoplasmic male sterility (CMS) mutants. Despite the increase in knowledge regarding CMS-encoding genes and their expression, definitive evidence that CMS-associated proteins actually cause pollen disruption is not yet available in most cases. Here we compare the translation products of mitochondria between the normal fertile cytoplasm and the male-sterile I-12CMS(3) cytoplasm derived from wild beets. The results show a unique 12 kDa polypeptide that is present in the I-12CMS(3) mitochondria but is not detectable among the translation products of normal mitochondria. We also found that a mitochondrial open reading frame (named orf129) was uniquely transcribed in I-12CMS(3) and is large enough to encode the novel 12 kDa polypeptide. Antibodies against a GST-ORF129 fusion protein were raised to establish that this 12 kDa polypeptide is the product of orf129. ORF129 was shown to accumulate in flower mitochondria as well as in root and leaf mitochondria. As for the CMS-associated protein (PCF protein) in petunia, ORF129 is primarily present in the matrix and is loosely associated with the inner mitochondrial membrane. The orf129 sequence was fused to a mitochondrial targeting pre-sequence, placed under the control of the Arabidopsis apetala3 promoter, and introduced into the tobacco nuclear genome. Transgenic expression of ORF129 resulted in male sterility, which provides clear supporting evidence that ORF129 is responsible for the male-sterile phenotype in sugar beet with wild beet cytoplasm.

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

    PubMed

    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.

  10. Isolation of mitochondria by gentle cell membrane disruption, and their subsequent characterization.

    PubMed

    Shibata, Takahiro; Yamashita, Saki; Hirusaki, Kotoe; Katoh, Kaoru; Ohta, Yoshihiro

    2015-08-07

    Mitochondria play a key role in several physiological processes as in integrating signals in the cell. However, understanding of the mechanism by which mitochondria sense and respond to signals has been limited due to the lack of an appropriate model system. In this study, we developed a method to isolate and characterize mitochondria without cell homogenization. By gently pipetting cells treated with streptolysin-O, a pore-forming membrane protein, we disrupted the cell membrane and were able to isolate both elongated and spherical mitochondria. Fluorescence imaging combined with super resolution microscopy showed that both the outer and inner membranes of the elongated mitochondria isolated using the newly developed method were intact. In addition, a FRET-based ATP sensor expressed in the mitochondrial matrix demonstrated that ATP generation by FoF1-ATPase in the isolated elongated mitochondria was as high as that in intracellular mitochondria. On the other hand, some of the spherical mitochondria isolated with this method had the outer membrane that no longer encapsulated the inner membrane. In addition, all mitochondria isolated using conventional procedures involving homogenization were spherical, many of them had damaged membranes, and low levels of ATP generation. Our results suggest that elongated mitochondria isolated from cells through gentle cell membrane disruption using a pore-forming protein tend to be more similar to intracellular mitochondria, having an intact membrane system and higher activity than spherical mitochondria.

  11. ChChd3, an Inner Mitochondrial Membrane Protein, Is Essential for Maintaining Crista Integrity and Mitochondrial Function*

    PubMed Central

    Darshi, Manjula; Mendiola, Vincent L.; Mackey, Mason R.; Murphy, Anne N.; Koller, Antonius; Perkins, Guy A.; Ellisman, Mark H.; Taylor, Susan S.

    2011-01-01

    The mitochondrial inner membrane (IM) serves as the site for ATP production by hosting the oxidative phosphorylation complex machinery most notably on the crista membranes. Disruption of the crista structure has been implicated in a variety of cardiovascular and neurodegenerative diseases. Here, we characterize ChChd3, a previously identified PKA substrate of unknown function (Schauble, S., King, C. C., Darshi, M., Koller, A., Shah, K., and Taylor, S. S. (2007) J. Biol. Chem. 282, 14952–14959), and show that it is essential for maintaining crista integrity and mitochondrial function. In the mitochondria, ChChd3 is a peripheral protein of the IM facing the intermembrane space. RNAi knockdown of ChChd3 in HeLa cells resulted in fragmented mitochondria, reduced OPA1 protein levels and impaired fusion, and clustering of the mitochondria around the nucleus along with reduced growth rate. Both the oxygen consumption and glycolytic rates were severely restricted. Ultrastructural analysis of these cells revealed aberrant mitochondrial IM structures with fragmented and tubular cristae or loss of cristae, and reduced crista membrane. Additionally, the crista junction opening diameter was reduced to 50% suggesting remodeling of cristae in the absence of ChChd3. Analysis of the ChChd3-binding proteins revealed that ChChd3 interacts with the IM proteins mitofilin and OPA1, which regulate crista morphology, and the outer membrane protein Sam50, which regulates import and assembly of β-barrel proteins on the outer membrane. Knockdown of ChChd3 led to almost complete loss of both mitofilin and Sam50 proteins and alterations in several mitochondrial proteins, suggesting that ChChd3 is a scaffolding protein that stabilizes protein complexes involved in maintaining crista architecture and protein import and is thus essential for maintaining mitochondrial structure and function. PMID:21081504

  12. MINOS is plus: a Mitofilin complex for mitochondrial membrane contacts.

    PubMed

    Herrmann, Johannes M

    2011-10-18

    Cristae junctions mark the boundaries of respiratory compartments in the inner mitochondrial membrane. In this issue of Developmental Cell, von der Malsburg et al. (2011) identify a complex, MINOS, that organizes cristae junctions. Mitofilin/Fcj1, the central component of the MINOS complex, also connects the inner membrane to outer membrane protein import machinery.

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

    PubMed

    Sauvanet, Cécile; Duvezin-Caubet, Stéphane; Salin, Bénédicte; David, Claudine; Massoni-Laporte, Aurélie; di Rago, Jean-Paul; Rojo, Manuel

    2012-01-01

    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 the network of

  14. Mitochondrial membrane permeabilization and cell death during myocardial infarction: roles of calcium and reactive oxygen species

    PubMed Central

    Webster, Keith A

    2013-01-01

    Excess generation of reactive oxygen species (ROS) and cytosolic calcium accumulation play major roles in the initiation of programmed cell death during acute myocardial infarction. Cell death may include necrosis, apoptosis and autophagy, and combinations thereof. During ischemia, calcium handling between the sarcoplasmic reticulum and myofilament is disrupted and calcium is diverted to the mitochondria causing swelling. Reperfusion, while essential for survival, reactivates energy transduction and contractility and causes the release of ROS and additional ionic imbalance. During acute ischemia–reperfusion, the principal death pathways are programmed necrosis and apoptosis through the intrinsic pathway, initiated by the opening of the mitochondrial permeability transition pore and outer mitochondrial membrane permeabilization, respectively. Despite intense investigation, the mechanisms of action and modes of regulation of mitochondrial membrane permeabilization are incompletely understood. Extrinsic apoptosis, necroptosis and autophagy may also contribute to ischemia–reperfusion injury. In this review, the roles of dysregulated calcium and ROS and the contributions of Bcl-2 proteins, as well as mitochondrial morphology in promoting mitochondrial membrane permeability change and the ensuing cell death during myocardial infarction are discussed. PMID:23176689

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

    PubMed

    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+.

  16. The development of mitochondrial membrane affinity chromatography columns for the study of mitochondrial transmembrane proteins

    PubMed Central

    Habicht, K-L.; Singh, N.S.; Indig, F.E.; Wainer, I.W.; Moaddel, R.; Shimmo, R.

    2015-01-01

    Mitochondrial membrane fragments from U-87 MG (U87MG) and HEK-293 cells were successfully immobilized on to Immobilized Artificial Membrane (IAM) chromatographic support and surface of activated open tubular (OT) silica capillary resulting in mitochondrial membrane affinity chromatography (MMAC) columns. Translocator protein (TSPO), located in mitochondrial outer membrane as well as sulfonylurea and mitochondrial permeability transition pore (mPTP) receptors, localized to the inner membrane, were characterized. Frontal displacement experiments with multiple concentrations of dipyridamole (DIPY) and PK-11195 were run on MMAC-(U87MG) column and the binding affinities (Kd) determined were 1.08 ± 1.49 and 0.0086 ± 0.0006 μM respectively, which was consistent with previously reported values. Further, binding affinities (Ki) for DIPY binding site were determined for TSPO ligands, PK-11195, mesoporphyrin IX, protoporphyrin IX and rotenone. Additionally, the relative ranking of these TSPO ligands based on single displacement studies using DIPY as marker on MMAC-(U87MG) was consistent with the obtained Ki values. The immobilization of mitochondrial membrane fragments was also confirmed by confocal microscopy. PMID:26049098

  17. Accumulation of Mitochondrial DNA Mutations Disrupts Cardiac Progenitor Cell Function and Reduces Survival.

    PubMed

    Orogo, Amabel M; Gonzalez, Eileen R; Kubli, Dieter A; Baptista, Igor L; Ong, Sang-Bing; Prolla, Tomas A; Sussman, Mark A; Murphy, Anne N; Gustafsson, Åsa B

    2015-09-04

    Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

  18. Deleterious variants in TRAK1 disrupt mitochondrial movement and cause fatal encephalopathy.

    PubMed

    Barel, Ortal; Christine V Malicdan, May; Ben-Zeev, Bruria; Kandel, Judith; Pri-Chen, Hadass; Stephen, Joshi; Castro, Inês G; Metz, Jeremy; Atawa, Osama; Moshkovitz, Sharon; Ganelin, Eti; Barshack, Iris; Polak-Charcon, Sylvie; Nass, Dvora; Marek-Yagel, Dina; Amariglio, Ninette; Shalva, Nechama; Vilboux, Thierry; Ferreira, Carlos; Pode-Shakked, Ben; Heimer, Gali; Hoffmann, Chen; Yardeni, Tal; Nissenkorn, Andreea; Avivi, Camila; Eyal, Eran; Kol, Nitzan; Glick Saar, Efrat; Wallace, Douglas C; Gahl, William A; Rechavi, Gideon; Schrader, Michael; Eckmann, David M; Anikster, Yair

    2017-03-01

    Cellular distribution and dynamics of mitochondria are regulated by several motor proteins and a microtubule network. In neurons, mitochondrial trafficking is crucial because of high energy needs and calcium ion buffering along axons to synapses during neurotransmission. The trafficking kinesin proteins (TRAKs) are well characterized for their role in lysosomal and mitochondrial trafficking in cells, especially neurons. Using whole exome sequencing, we identified homozygous truncating variants in TRAK1 (NM_001042646:c.287-2A > C), in six lethal encephalopathic patients from three unrelated families. The pathogenic variant results in aberrant splicing and significantly reduced gene expression at the RNA and protein levels. In comparison with normal cells, TRAK1-deficient fibroblasts showed irregular mitochondrial distribution, altered mitochondrial motility, reduced mitochondrial membrane potential, and diminished mitochondrial respiration. This study confirms the role of TRAK1 in mitochondrial dynamics and constitutes the first report of this gene in association with a severe neurodevelopmental disorder.

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

    PubMed

    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.

  20. Simple Strategy for Taming Membrane-Disrupting Antibiotics

    PubMed Central

    2016-01-01

    A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity. PMID:27801580

  1. Tau accumulation impairs mitophagy via increasing mitochondrial membrane potential and reducing mitochondrial Parkin

    PubMed Central

    Wang, Zhi-hao; Luo, Yu; Zhang, Xiangnan; Liu, Xiu-Ping; Feng, Qiong; Wang, Qun; Yue, Zhenyu; Chen, Zhong; Ye, Keqiang; Wang, Jian-Zhi; Liu, Gong-Ping

    2016-01-01

    Intracellular accumulation of wild type tau is a hallmark of sporadic Alzheimer's disease (AD). However, the molecular mechanisms underlying tau toxicity is not fully understood. Here, we detected mitophagy deficits evidenced by the increased levels of mitophagy markers, including COX IV, TOMM20, and the ratio of mtDNA to genomic DNA indexed as mt-Atp6/Rpl13, in the AD brains and in the human wild type full-length tau (htau) transgenic mice. More interestingly, the mitophagy deficit was only shown in the AD patients who had an increased total tau level. Further studies demonstrated that overexpression of htau induced mitophagy deficits in HEK293 cells, the primary hippocampal neurons and in the brains of C57 mice. Upon overexpression of htau, the mitochondrial membrane potential was increased and the levels of PTEN-induced kinase 1 (PINK1) and Parkin decreased in the mitochondrial fraction, while upregulation of Parkin attenuated the htau-induced mitophagy deficits. Finally, we detected a dose-dependent allocation of tau proteins into the mitochondrial outer membrane fraction along with its cytoplasmic accumulation. These data suggest that intracellular accumulation of htau induces mitophagy deficits by direct inserting into the mitochondrial membrane and thus increasing the membrane potential, which impairs the mitochondrial residence of PINK1/Parkin. Our findings reveal a novel mechanism underlying the htau-induced neuronal toxicities in AD and other tauopathies. PMID:26943044

  2. Proteome analysis of mitochondrial outer membrane from Neurospora crassa

    SciTech Connect

    Schmitt, Simone; Prokisch, Holger; Schlunk, Tilman; Camp, David G.; Ahting, Uwe; Waizenegger, Thomas; Scharfe, Curt M.; Meitinger, Thomas; Imhof, Axel; Neupert, Walter; Oefner, Peter J.; Rapaport, Doron

    2006-01-01

    The mitochondrial outer membrane mediates numerous interactions between the metabolic and genetic systems of mitochondria and the rest of the eukaryotic cell. We performed a proteomic study to discover novel functions of components of the mitochondrial outer membrane. Proteins of highly pure outer membrane vesicles (OMV) from Neurospora crassa were identified by a combination of liquid chromatography tandem mass spectrometry of tryptic peptide digests and gel electrophoresis of solubilized OMV proteins, followed by their identification using MALDI-MS peptide fingerprinting. Among the 30 proteins found in at least three of four separate analyses were 23 proteins with known functions in the outer membrane. These included components of the import machinery (the TOM and TOB complexes), a pore-forming component (Porin), and proteins that control fusion and fission of the organelle. In addition, proteins playing a role in various biosynthetic pathways, whose intracellular location had not been established previously, could be localized to the mitochondrial outer membrane. Thus, the proteome of the outer membrane can help in identifying new mitochondria-related functions.

  3. Interaction of tau protein with model lipid membranes induces tau structural compaction and membrane disruption

    PubMed Central

    Jones, Emmalee M.; Dubey, Manish; Camp, Phillip J.; Vernon, Briana C.; Biernat, Jacek; Mandelkow, Eckhard; Majewski, Jaroslaw; Chi, Eva Y.

    2012-01-01

    The misfolding and aggregation of the intrinsically disordered, microtubule-associated tau protein into neurofibrillary tangles is implicated in the pathogenesis of Alzheimer's disease. However, the mechanisms of tau aggregation and toxicity remain unknown. Recent work has shown that lipid membrane can induce tau aggregation and that membrane permeabilization may serve as a pathway by which protein aggregates exert toxicity, suggesting that the plasma membrane may play dual roles in tau pathology. This prompted our investigation to assess tau's propensity to interact with membranes and to elucidate the mutually disruptive structural perturbations the interactions induce in both tau and the membrane. We show that although highly charged and soluble, the full-length tau (hTau40) is also highly surface active, selectively inserts into anionic DMPG lipid monolayers and induces membrane morphological changes. To resolve molecular-scale structural details of hTau40 associated with lipid membranes, X-ray and neutron scattering techniques are utilized. X-ray reflectivity indicates hTau40's presence underneath a DMPG monolayer and penetration into the lipid headgroups and tailgroups, whereas grazing incidence X-ray diffraction shows that hTau40 insertion disrupts lipid packing. Moreover, both air/water and DMPG lipid membrane interfaces induce the disordered hTau40 to partially adopt a more compact conformation with density similar to that of a folded protein. Neutron reflectivity shows that tau completely disrupts supported DMPG bilayers while leaving the neutral DPPC bilayer intact. Our results show that hTau40's strong interaction with anionic lipids induces tau structural compaction and membrane disruption, suggesting possible membrane-based mechanisms of tau aggregation and toxicity in neurodegenerative diseases. PMID:22401494

  4. Membrane Curvature-sensing and Curvature-inducing Activity of Islet Amyloid Polypeptide and Its Implications for Membrane Disruption*♦

    PubMed Central

    Kegulian, Natalie C.; Sankhagowit, Shalene; Apostolidou, Melania; Jayasinghe, Sajith A.; Malmstadt, Noah; Butler, Peter C.; Langen, Ralf

    2015-01-01

    Islet amyloid polypeptide (IAPP) is a 37-amino acid amyloid protein intimately associated with pancreatic islet β-cell dysfunction and death in type II diabetes. In this study, we combine spectroscopic methods and microscopy to investigate α-helical IAPP-membrane interactions. Using light scattering and fluorescence microscopy, we observe that larger vesicles become smaller upon treatment with human or rat IAPP. Electron microscopy shows the formation of various highly curved structures such as tubules or smaller vesicles in a membrane-remodeling process, and spectrofluorometric detection of vesicle leakage shows disruption of membrane integrity. This effect is stronger for human IAPP than for the less toxic rat IAPP. From CD spectra in the presence of different-sized vesicles, we also uncover the membrane curvature-sensing ability of IAPP and find that it transitions from inducing to sensing membrane curvature when lipid negative charge is decreased. Our in vivo EM images of immunogold-labeled rat IAPP and human IAPP show both forms to localize to mitochondrial cristae, which contain not only locally curved membranes but also phosphatidylethanolamine and cardiolipin, lipids with high spontaneous negative curvature. Disruption of membrane integrity by induction of membrane curvature could apply more broadly to other amyloid proteins and be responsible for membrane damage observed in other amyloid diseases as well. PMID:26283787

  5. Topological Transitions in Mitochondrial Membranes controlled by Apoptotic Proteins

    NASA Astrophysics Data System (ADS)

    Hwee Lai, Ghee; Sanders, Lori K.; Mishra, Abhijit; Schmidt, Nathan W.; Wong, Gerard C. L.; Ivashyna, Olena; Schlesinger, Paul H.

    2010-03-01

    The Bcl-2 family comprises pro-apoptotic proteins, capable of permeabilizing the mitochondrial membrane, and anti-apoptotic members interacting in an antagonistic fashion to regulate programmed cell death (apoptosis). They offer potential therapeutic targets to re-engage cellular suicide in tumor cells but the extensive network of implicated protein-protein interactions has impeded full understanding of the decision pathway. We show, using synchrotron x-ray diffraction, that pro-apoptotic proteins interact with mitochondrial-like model membranes to generate saddle-splay (negative Gaussian) curvature topologically required for pore formation, while anti-apoptotic proteins can deactivate curvature generation by molecules drastically different from Bcl-2 family members and offer evidence for membrane-curvature mediated interactions general enough to affect very disparate systems.

  6. Bcl-xL-mediated antioxidant function abrogates the disruption of mitochondrial dynamics induced by LRRK2 inhibition.

    PubMed

    Saez-Atienzar, Sara; Bonet-Ponce, Luis; da Casa, Carmen; Perez-Dolz, Laura; Blesa, Jose R; Nava, Eduardo; Galindo, Maria F; Jordan, Joaquín

    2016-01-01

    We have used the human neuroblastoma cell line SH-SY5Y overexpressing Bcl-xL (SH-SY5Y/Bcl-xL) to clarify the effects of this mitochondrial protein on the control of mitochondrial dynamics and the autophagic processes which occur after the inhibition of leucine-rich repeat kinase 2 (LRRK2) with GSK2578215A. In wild type (SH-SY5Y/Neo) cells, GSK2578215A (1nM) caused a disruption of mitochondrial morphology and an imbalance in intracellular reactive oxygen species (ROS) as indicated by an increase in dichlorofluorescein fluorescence and 4-hydroxynonenal. However, SH-SY5Y/Bcl-xL cells under GSK2578215A treatment, unlike the wild type, preserved a high mitochondrial membrane potential and did not exhibit apoptotical chromatins. In contrast to wild type cells, in SH-SY5Y/Bcl-xL cells, GSK2578215A did not induce mitochondrial translocation of neither dynamin related protein-1 nor the proapoptotic protein, Bax. In SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, mitochondrial fragmentation elicited by GSK2578215A precedes an autophagic response. Furthermore, the overexpression of Bcl-xL protein restores the autophagic flux pathway disrupted by this inhibitor. SH-SY5Y/Neo, but not SH-SY5Y/Bcl-xL cells, responded to LRRK2 inhibition by an increase in the levels of acetylated tubulin, indicating that this was abrogated by Bcl-xL overexpression. This hyperacetylation of tubulin took place earlier than any of the above-mentioned events suggesting that it is involved in the autophagic flux interruption. Pre-treatment with tempol prevented the GSK2578215A-induced mitochondrial fragmentation, autophagy and the rise in acetylated tubulin in SH-SY5Y/Neo cells. Thus, these data support the notion that ROS act as a second messenger connexion between LRRK2 inhibition and these deleterious responses, which are markedly alleviated by the Bcl-xL-mediated ROS generation blockade.

  7. Two-Step Mechanism of Membrane Disruption by Aβ through Membrane Fragmentation and Pore Formation

    PubMed Central

    Sciacca, Michele F.M.; Kotler, Samuel A.; Brender, Jeffrey R.; Chen, Jennifer; Lee, Dong-kuk; Ramamoorthy, Ayyalusamy

    2012-01-01

    Disruption of cell membranes by Aβ is believed to be one of the key components of Aβ toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aβ occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aβ1–40, defects form on the membrane that share many of the properties of Aβ channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aβ1–40 is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aβ and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer’s disease. PMID:22947931

  8. Atmospheric-pressure guided streamers for liposomal membrane disruption

    SciTech Connect

    Svarnas, P.; Aleiferis, Sp.; Matrali, S. H.; Gazeli, K.; Clement, F.; Antimisiaris, S. G.

    2012-12-24

    The potential to use liposomes (LIPs) as a cellular model in order to study interactions of cold atmospheric-pressure plasma with cells is herein investigated. Cold atmospheric-pressure plasma is formed by a dielectric-barrier discharge reactor. Large multilamellar vesicle liposomes, consisted of phosphatidylcholine and cholesterol, are prepared by the thin film hydration technique, to encapsulate a small hydrophilic dye, i.e., calcein. The plasma-induced release of calcein from liposomes is then used as a measure of liposome membrane integrity and, consequently, interaction between the cold atmospheric plasma and lipid bilayers. Physical mechanisms leading to membrane disruption are suggested, based on the plasma characterization including gas temperature calculation.

  9. Altered Skeletal Muscle Mitochondrial Proteome As the Basis of Disruption of Mitochondrial Function in Diabetic Mice

    PubMed Central

    Zabielski, Piotr; Lanza, Ian R.; Gopala, Srinivas; Holtz Heppelmann, Carrie J.; Bergen, H. Robert; Dasari, Surendra

    2016-01-01

    Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress. PMID:26718503

  10. Urinary mitochondrial DNA is a biomarker of mitochondrial disruption and renal dysfunction in acute kidney injury

    PubMed Central

    Whitaker, Ryan M.; Stallons, L. Jay; Kneff, Joshua E.; Alge, Joseph L.; Harmon, Jennifer L.; Rahn, Jennifer J.; Arthur, John M.; Beeson, Craig C.; Chan, Sherine L.; Schnellmann, Rick G.

    2015-01-01

    Recent studies show the importance of mitochondrial dysfunction in the initiation and progression of acute kidney injury (AKI). However, no biomarkers exist linking renal injury to mitochondrial function and integrity. To this end, we evaluated urinary mitochondrial DNA (UmtDNA) as a biomarker of renal injury and function in humans with AKI following cardiac surgery. mtDNA was isolated from the urine of patients following cardiac surgery and quantified by qPCR. Patients were stratified into no AKI, stable AKI and progressive AKI groups based on Acute Kidney Injury Network (AKIN) staging. UmtDNA was elevated in progressive AKI patients, and was associated with progression of patients with AKI at collection to higher AKIN stages. To evaluate the relationship of UmtDNA to measures of renal mitochondrial integrity in AKI, mice were subjected to sham surgery or varying degrees of ischemia followed by 24 hours of reperfusion. UmtDNA increased in mice after 10-15 minutes of ischemia and positively correlated with ischemia time. Furthermore, UmtDNA was predictive of AKI in the mouse model. Finally, UmtDNA levels were negatively correlated with renal cortical mtDNA and mitochondrial gene expression. These translational studies demonstrate that UmtDNA is associated with recovery from AKI following cardiac surgery by serving as an indicator of mitochondrial integrity. Thus, UmtDNA may serve as valuable biomarker for the development of mitochondrial targeted therapies in AKI. PMID:26287315

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

    NASA Astrophysics Data System (ADS)

    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.

  12. The CT20 peptide causes detachment and death of metastatic breast cancer cells by promoting mitochondrial aggregation and cytoskeletal disruption.

    PubMed

    Lee, M W; Bassiouni, R; Sparrow, N A; Iketani, A; Boohaker, R J; Moskowitz, C; Vishnubhotla, P; Khaled, A S; Oyer, J; Copik, A; Fernandez-Valle, C; Perez, J M; Khaled, A R

    2014-05-22

    Metastasis accounts for most deaths from breast cancer, driving the need for new therapeutics that can impede disease progression. Rationally designed peptides that take advantage of cancer-specific differences in cellular physiology are an emerging technology that offer promise as a treatment for metastatic breast cancer. We developed CT20p, a hydrophobic peptide based on the C terminus of Bax that exhibits similarities with antimicrobial peptides, and previously reported that CT20p has unique cytotoxic actions independent of full-length Bax. In this study, we identified the intracellular actions of CT20p which precede cancer cell-specific detachment and death. Previously, we found that CT20p migrated in the heavy membrane fractions of cancer cell lysates. Here, using MDA-MB-231 breast cancer cells, we demonstrated that CT20p localizes to the mitochondria, leading to fusion-like aggregation and mitochondrial membrane hyperpolarization. As a result, the distribution and movement of mitochondria in CT20p-treated MDA-MB-231 cells was markedly impaired, particularly in cell protrusions. In contrast, CT20p did not associate with the mitochondria of normal breast epithelial MCF-10A cells, causing little change in the mitochondrial membrane potential, morphology or localization. In MDA-MB-231 cells, CT20p triggered cell detachment that was preceded by decreased levels of α5β1 integrins and reduced F-actin polymerization. Using folate-targeted nanoparticles to encapsulate and deliver CT20p to murine tumors, we achieved significant tumor regression within days of peptide treatment. These results suggest that CT20p has application in the treatment of metastatic disease as a cancer-specific therapeutic peptide that perturbs mitochondrial morphology and movement ultimately culminating in disruption of the actin cytoskeleton, cell detachment, and loss of cell viability.

  13. Contribution of liver mitochondrial membrane-bound glutathione transferase to mitochondrial permeability transition pores

    SciTech Connect

    Hossain, Quazi Sohel; Ulziikhishig, Enkhbaatar; Lee, Kang Kwang; Yamamoto, Hideyuki; Aniya, Yoko

    2009-02-15

    We recently reported that the glutathione transferase in rat liver mitochondrial membranes (mtMGST1) is activated by S-glutathionylation and the activated mtMGST1 contributes to the mitochondrial permeability transition (MPT) pore and cytochrome c release from mitochondria [Lee, K.K., Shimoji, M., Quazi, S.H., Sunakawa, H., Aniya, Y., 2008. Novel function of glutathione transferase in rat liver mitochondrial membrane: role for cytochrome c release from mitochondria. Toxcol. Appl. Pharmacol. 232, 109-118]. In the present study we investigated the effect of reactive oxygen species (ROS), generator gallic acid (GA) and GST inhibitors on mtMGST1 and the MPT. When rat liver mitochondria were incubated with GA, mtMGST1 activity was increased to about 3 fold and the increase was inhibited with antioxidant enzymes and singlet oxygen quenchers including 1,4-diazabicyclo [2,2,2] octane (DABCO). GA-mediated mtMGST1 activation was prevented by GST inhibitors such as tannic acid, hematin, and cibacron blue and also by cyclosporin A (CsA). In addition, GA induced the mitochondrial swelling which was also inhibited by GST inhibitors, but not by MPT inhibitors CsA, ADP, and bongkrekic acid. GA also released cytochrome c from the mitochondria which was inhibited completely by DABCO, moderately by GST inhibitors, and somewhat by CsA. Ca{sup 2+}-mediated mitochondrial swelling and cytochrome c release were inhibited by MPT inhibitors but not by GST inhibitors. When the outer mitochondrial membrane was isolated after treatment of mitochondria with GA, mtMGST1 activity was markedly increased and oligomer/aggregate of mtMGST1 was observed. These results indicate that mtMGST1 in the outer mitochondrial membrane is activated by GA through thiol oxidation leading to protein oligomerization/aggregation, which may contribute to the formation of ROS-mediated, CsA-insensitive MPT pore, suggesting a novel mechanism for regulation of the MPT by mtMGST1.

  14. Mitochondrial respiratory chain inhibitors modulate the metal-induced inner mitochondrial membrane permeabilization.

    PubMed

    Belyaeva, Elena A

    2010-01-01

    To elucidate the molecular mechanisms of the protective action of stigmatellin (an inhibitor of complex III of mitochondrial electron transport chain, mtETC) against the heavy metal-induced cytotoxicity, we tested its effectiveness against mitochondrial membrane permeabilization produced by heavy metal ions Cd²(+), Hg²(+), Cu²(+) and Zn²(+), as well as by Ca²(+) (in the presence of P(i)) or Se (in form of Na₂SeO₃) using isolated rat liver mitochondria. It was shown that stigmatellin modulated mitochondrial swelling produced by these metals/metalloids in the isotonic sucrose medium in the presence of ascorbate plus tetramethyl-p-phenylenediamine (complex IV substrates added for energization of the mitochondria). It was found that stigmatellin and other mtETC inhibitors enhanced the mitochondrial swelling induced by selenite. However, in the same medium, all the mtETC inhibitors tested as well as cyclosporin A and bongkrekic acid did not significantly affect Cu²(+)-induced swelling. In contrast, the high-amplitude swelling produced by Cd²(+), Hg²(+), Zn²(+), or Ca²(+) plus P(i) was significantly depressed by these inhibitors. Significant differences in the action of these metals/metalloids on the redox status of pyridine nucleotides, transmembrane potential and mitochondrial respiration were also observed. In the light of these results as well as the data from the recent literature, our hypothesis on a possible involvement of the respiratory supercomplex, formed by complex I (P-site) and complex III (S-site) in the mitochondrial permeabilization mediated by the mitochondrial transition pore, is updated.

  15. Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes

    PubMed Central

    1981-01-01

    Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane

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

    PubMed Central

    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

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

    PubMed

    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 Ca(2+) transients which are further transduced by Ca(2+) sensor proteins into a transcriptional and metabolic response. Most of the research on Ca(2+) signaling in plants has been focused on the transport mechanisms for Ca(2+) across the plasma- and the vacuolar membranes as well as on the components involved in decoding of cytoplasmic Ca(2+) signals, but how intracellular organelles such as mitochondria are involved in the process of Ca(2+) signaling is just emerging. The combination of the molecular players and the elicitors of Ca(2+) signaling in mitochondria together with newly generated detection systems for measuring organellar Ca(2+) 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 Ca(2+) across the outer and inner mitochondrial membranes in plants. We briefly discuss the relevance of the mitochondrial Ca(2+) homeostasis for ensuring optimal bioenergetic performance of this organelle.

  18. Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.

    PubMed

    Kurup, C K; Sanadi, D R

    1977-02-01

    3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.

  19. Disruption of mitochondrial homeostasis in organic acidurias: insights from human and animal studies.

    PubMed

    Wajner, Moacir; Goodman, Stephen I

    2011-02-01

    Organic acidurias or organic acidemias constitute a group of inherited disorders caused by deficient activity of specific enzymes of amino acids, carbohydrates or lipids catabolism, leading to large accumulation and excretion of one or more carboxylic (organic) acids. Affected patients usually present neurologic symptoms and abnormalities, sometimes accompanied by cardiac and skeletal muscle alterations, whose pathogenesis is poorly known. However, in recent years growing evidence has emerged indicating that mitochondrial dysfunction is directly or indirectly involved in the pathology of various organic acidemias. Mitochondrial impairment in some of these diseases are generally due to mutations in nuclear genes of the tricarboxylic acid cycle or oxidative phosphorylation, while in others it seems to result from toxic influences of the endogenous organic acids to the mitochondrion. In this minireview, we will briefly summarize the present knowledge obtained from human and animal studies showing that disruption of mitochondrial homeostasis may represent a relevant pathomechanism of tissue damage in selective organic acidemias. The discussion will focus on mitochondrial alterations found in patients affected by organic acidemias and by the deleterious effects of the accumulating organic acids on mitochondrial pathways that are crucial for ATP formation and transfer. The elucidation of the mechanisms of toxicity of these acidic compounds offers new perspectives for potential novel adjuvant therapeutic strategies in selected disorders of this group.

  20. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli*

    PubMed Central

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H.; Pessi, Gabriella; Eberl, Leo; Robinson, John A.

    2016-01-01

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. PMID:26627837

  1. Role of the mitochondrial contact site and cristae organizing system in membrane architecture and dynamics.

    PubMed

    Rampelt, Heike; Zerbes, Ralf M; van der Laan, Martin; Pfanner, Nikolaus

    2017-04-01

    The elaborate membrane architecture of mitochondria is a prerequisite for efficient respiration and ATP generation. The cristae membranes, invaginations of the inner mitochondrial membrane, represent a specialized compartment that harbors the complexes of the respiratory chain and the F1Fo-ATP synthase. Crista junctions form narrow openings that connect the cristae membranes to the inner boundary membrane. The mitochondrial contact site and cristae organizing system (MICOS) is located at crista junctions where it stabilizes membrane curvature and forms contact sites between the mitochondrial inner and outer membranes. MICOS is a large machinery, consisting of two dynamic subcomplexes that are anchored in the inner membrane and expose domains to the intermembrane space. The functions of MICOS in mitochondrial membrane architecture and biogenesis are influenced by numerous interaction partners and the phospholipid environment.

  2. Sulfite disrupts brain mitochondrial energy homeostasis and induces mitochondrial permeability transition pore opening via thiol group modification.

    PubMed

    Grings, Mateus; Moura, Alana P; Amaral, Alexandre U; Parmeggiani, Belisa; Gasparotto, Juciano; Moreira, José C F; Gelain, Daniel P; Wyse, Angela T S; Wajner, Moacir; Leipnitz, Guilhian

    2014-09-01

    Sulfite oxidase (SO) deficiency is biochemically characterized by the accumulation of sulfite, thiosulfate and S-sulfocysteine in tissues and biological fluids of the affected patients. The main clinical symptoms include severe neurological dysfunction and brain abnormalities, whose pathophysiology is still unknown. The present study investigated the in vitro effects of sulfite and thiosulfate on mitochondrial homeostasis in rat brain mitochondria. It was verified that sulfite per se, but not thiosulfate, decreased state 3, CCCP-stimulated state and respiratory control ratio in mitochondria respiring with glutamate plus malate. In line with this, we found that sulfite inhibited the activities of glutamate and malate (MDH) dehydrogenases. In addition, sulfite decreased the activity of a commercial solution of MDH, that was prevented by antioxidants and dithiothreitol. Sulfite also induced mitochondrial swelling and reduced mitochondrial membrane potential, Ca(2+) retention capacity, NAD(P)H pool and cytochrome c immunocontent when Ca(2+) was present in the medium. These alterations were prevented by ruthenium red, cyclosporine A (CsA) and ADP, supporting the involvement of mitochondrial permeability transition (MPT) in these effects. We further observed that N-ethylmaleimide prevented the sulfite-elicited swelling and that sulfite decreased free thiol group content in brain mitochondria. These findings indicate that sulfite acts directly on MPT pore containing thiol groups. Finally, we verified that sulfite reduced cell viability in cerebral cortex slices and that this effect was prevented by CsA. Therefore, it may be presumed that disturbance of mitochondrial energy homeostasis and MPT induced by sulfite could be involved in the neuronal damage characteristic of SO deficiency.

  3. The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance.

    PubMed

    Shepard, K A; Yaffe, M P

    1999-02-22

    The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.

  4. Geldanamycin disrupts platelet-membrane structure, leading to membrane permeabilization and inhibition of platelet aggregation.

    PubMed Central

    Suttitanamongkol, S; Gear, A R; Polanowska-Grabowska, R

    2000-01-01

    Geldanamycin (GA), a benzoquinoid ansamycin antibiotic, has been used as a tyrosine kinase inhibitor and an anti-tumour agent and is known to bind to heat-shock protein 90. In the present study on human platelets we have found that GA inhibited platelet aggregation induced by ADP, thrombin and the thrombin-receptor-activating peptide and caused platelet plasma-membrane damage, detected by leakage of adenine nucleotides as well as serotonin. Scanning electron microscopy (SEM) revealed that platelet exposure to GA led to the formation of holes or fenestrations in the platelet plasma membrane, confirming GA's ability to initiate membrane damage. In addition, GA itself caused both the dephosphorylation and phosphorylation of proteins in resting platelets and prevented agonist-induced phosphorylation of pleckstrin, the 20-kDa myosin light chain and other proteins. Another ansamycin, herbimycin A, also inhibited platelet aggregation, but caused minimal membrane permeabilization, as detected by (3)H release from platelets labelled previously with [(3)H]adenine, and much less membrane damage, revealed by SEM. Overall, GA is able to disrupt membrane structure and inhibit platelet aggregation, an ability which may be linked to alterations in the activity of protein kinases and phosphatases. PMID:10620508

  5. Controls and constrains of the membrane disrupting action of Aurein 1.2

    NASA Astrophysics Data System (ADS)

    Shahmiri, Mahdi; Enciso, Marta; Mechler, Adam

    2015-11-01

    Aurein 1.2 is a 13 residue antimicrobial peptide secreted by the Australian tree frog Litoria Aurea. It is a surface-acting membrane disrupting peptide that permeabilizes bacterial membranes via the carpet mechanism; the molecular details of this process are mostly unknown. Here the mechanism of action of Aurein 1.2 was investigated with an emphasis on the role of membrane charge and C-terminal amidation of the peptide. Using quartz crystal microbalance (QCM) fingerprinting it was found that the membrane charge correlates with membrane affinity of the peptide, however the binding and the membrane disrupting processes are not charge driven; increased membrane charge reduces the membrane disrupting activity. Coarse grain simulations revealed that phenylalanine residues act as membrane anchors. Accordingly Aurein 1.2 has the ability to bind to any membrane. Furthermore, bundling precludes membrane disruption in case of wild type peptides, while non C-terminal amidated peptides form random aggregates leading to detachment from the membrane. Hence C-terminal amidation is crucial for Aurein 1.2 action. Our results suggest that Aurein 1.2 acts via aggregation driven membrane penetration. The concomitant change in the tension of the outer leaflet imposes a spontaneous curvature on the membrane, leading to disintegration.

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

    EPA Science Inventory

    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...

  7. Mode of action of pyrazinamide: disruption of Mycobacterium tuberculosis membrane transport and energetics by pyrazinoic acid.

    PubMed

    Zhang, Ying; Wade, Mary Margaret; Scorpio, Angelo; Zhang, Hao; Sun, Zhonghe

    2003-11-01

    Pyrazinamide is an important sterilizing drug that shortens tuberculosis (TB) therapy. However, the mechanism of action of pyrazinamide is poorly understood because of its unusual properties. Here we show that pyrazinoic acid, the active moiety of pyrazinamide, disrupted membrane energetics and inhibited membrane transport function in Mycobacterium tuberculosis. The preferential activity of pyrazinamide against old non-replicating bacilli correlated with their low membrane potential and the disruption of membrane potential by pyrazinoic acid and acid pH. Inhibitors of membrane energetics increased the antituberculous activity of pyrazinamide. These findings shed new light on the mode of action of pyrazinamide and may help in the design of new drugs that shorten therapy.

  8. Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein.

    PubMed Central

    Roucou, Xavier; Montessuit, Sylvie; Antonsson, Bruno; Martinou, Jean-Claude

    2002-01-01

    In response to various apoptotic stimuli, Bax, a pro-apoptotic member of the Bcl-2 family, is oligomerized and permeabilizes the mitochondrial outer membrane to apoptogenic factors, including cytochrome c. Bax oligomerization can also be induced by incubating isolated mitochondria containing endogenous Bax with recombinant tBid (caspase-8-cleaved Bid) in vitro. The mechanism by which Bax oligomerizes under these conditions is still unknown. To address this question, recombinant human full-length Bax was purified as a monomeric protein. Bax failed to oligomerize spontaneously in isolated mitochondria or in liposomes composed of either cardiolipin or lipids extracted from mitochondria. However, in the presence of tBid, the protein formed large complexes in mitochondrial membranes and induced the release of cytochrome c. tBid also induced Bax oligomerization in isolated mitochondrial outer membranes, but not in other membranes, such as plasma membranes or microsomes. Moreover, tBid-induced Bax oligomerization was inhibited when mitochondria were pretreated with protease K. The presence of the voltage-dependent anion channel was not required either for Bax oligomerization or for Bax-induced cytochrome c release. Finally, Bax oligomerization was reconstituted in proteoliposomes made from mitochondrial membrane proteins. These findings imply that tBid is necessary but not sufficient for Bax oligomerization; a mitochondrial protein is also required. PMID:12193163

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

    PubMed

    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.

  10. Ceramide forms channels in mitochondrial outer membranes at physiologically relevant concentrations

    PubMed Central

    Siskind, Leah J.; Kolesnick, Richard N.; Colombini, Marco

    2007-01-01

    Recent evidence suggests that the ability of ceramides to induce apoptosis is due to a direct action on mitochondria. Mitochondria are known to contain enzymes responsible for ceramide synthesis and hydrolysis and mitochondrial ceramide levels have been shown to be elevated prior to the mitochondrial phase of apoptosis. Ceramides have been reported to induce the release of intermembrane space proteins from mitochondria, which has been linked to their ability to form large channels in membranes. The aim of this study was to determine if the membrane concentration of ceramide required for the formation of protein permeable channels is within the range that is present in mitochondria during the induction phase of apoptosis. Only a very small percentage of the ceramide actually inserts into the mitochondrial membranes. The permeability of the mitochondrial outer membrane correlates directly with the level of ceramide in the membrane. Importantly, the concentration of ceramide at which significant channel formation occurs is consistent with the level of mitochondrial ceramide that occurs during the induction phase of apoptosis (4 pmol ceramide/nanomole phospholipid). Similar results were obtained with short- and long-chain ceramide. Ceramide channel formation is specific to mitochondrial membranes in that no channel formation occurs in the plasma membranes of erythrocytes even at concentrations 20 times higher than those required for channel formation in mitochondrial outer membranes. Thus, ceramide channels are good candidates for the pathway by which proapoptotic proteins are released from mitochondria during the induction phase of apoptosis. PMID:16713754

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

    PubMed

    Jensen, Kristian H R; Rekling, Jens C

    2010-10-01

    Mitochondrial dysfunction is a hallmark of several diseases and may also result from drugs with unwanted side effects on mitochondrial biochemistry. The mitochondrial membrane potential is a good indicator of mitochondrial function. Here, the authors have developed a no-wash mitochondrial membrane 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, which is a suspected mitochondrial toxicant. CCCP and DNP have short-term depolarizing effects, and thioridazine has long-term hyperpolarizing effects on the mitochondrial membrane potential of Chinese hamster ovary (CHO) cells. The assay also detected changes of the mitochondrial membrane potential in CHO cells exposed to cobalt (mimicking hypoxia) and in PC12 cells exposed to amyloid β, demonstrating that the assay can be used in cellular models of hypoxia and Alzheimer's disease. The assay needs no washing steps, has a Z' value >0.5, can be used on standard fluorometers, has good post liquid-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. Photodynamic action of chlorin e6 on thymocyte plasmatic and mitochondrial membrane potentials

    NASA Astrophysics Data System (ADS)

    Gyulkhandanyan, Grigor V.

    2005-08-01

    Transmembrane potentials appear to be cell state sensitive characteristics and can give information about cell damage initial stage. Photodynamic action of the photosensitizer chlorin e6 on plasmatic and mitochondrial membrane potentials of the rat thymus lymphocytes was studied using voltage-sensitive dye rhodamine 6G. It has been revealed that mitochondrial membrane potential is more sensitive characteristic of membrane disfunction than plasmatic one at the cell photodamage.

  13. Sodium/proton antiporters in the mitochondrial inner membrane.

    PubMed

    Garlid, K D

    1988-01-01

    The two mitochondrial Na+/H+ antiporters differ in several important respects, and the most physiologically significant of these may be their differences in regulation. The Mg2+-dependent Na+/H+ antiporter controls mitochondrial volume in a dangerous, high-K+ environment. To play this vital role, this porter must always lie poised far from K+/H+ equilibrium; i.e., it must be under dynamic regulation, as proposed in the Mg2+ carrier-brake hypothesis (7). Being regulated, it is not necessary for this antiporter to be cation-selective, since all electroneutral cation movements will be followed by redistributions of anions and water. On the other hand, there is no indication at present that the Mg2+-independent Na+/H+ antiporter is regulated. This transporter is therefore required to exhibit high discrimination against K+ in order to prevent the collapse of matrix volume dueto uncontrolled loss of K+ salts and water (4). Do the properties of the mitochondrial Na+/H+ antiporters help us in any way to understand the plasmalemmal Na+/H+ antiporters? I believe they do, if we allow that there are a limited number of ways in which nature constructs such porters. The difference in cation selectivities very likely reflects a fundamental structural difference between the two mitochondrial antiporters, and this difference appears to be mirrored in two types of plasmalemmal Na+/H+ antiporters. Thus, the Mg2+-independent Na+/H+ antiporter resembles the renal tubular Na+/H+ antiporter in its discrimination against K+ and its competitive inhibition by Li+. On the other hand, the Mg2+-dependent Na+/H+ antiporter resembles a cardiac sarcolemmal Na+/H+ antiporter which transports all alkali cations, including Na+ and K+, and which is inhibited by DCCD and amphiphilic amines (S. Kakar, A. Askari and K. Garlid, in preparation). The existence of the latter class of antiporter in plasmalemma may seem unlikely at first glance, since it would tend to catalyze Na+/K+ exchange and dissipate

  14. Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties[S

    PubMed Central

    Sousa, Tânia; Castro, Rui E.; Pinto, Sandra N.; Coutinho, Ana; Lucas, Susana D.; Moreira, Rui; Rodrigues, Cecília M. P.; Prieto, Manuel; Fernandes, Fábio

    2015-01-01

    Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure. PMID:26351365

  15. Deoxycholic acid modulates cell death signaling through changes in mitochondrial membrane properties.

    PubMed

    Sousa, Tânia; Castro, Rui E; Pinto, Sandra N; Coutinho, Ana; Lucas, Susana D; Moreira, Rui; Rodrigues, Cecília M P; Prieto, Manuel; Fernandes, Fábio

    2015-11-01

    Cytotoxic bile acids, such as deoxycholic acid (DCA), are responsible for hepatocyte cell death during intrahepatic cholestasis. The mechanisms responsible for this effect are unclear, and recent studies conflict, pointing to either a modulation of plasma membrane structure or mitochondrial-mediated toxicity through perturbation of mitochondrial outer membrane (MOM) properties. We conducted a comprehensive comparative study of the impact of cytotoxic and cytoprotective bile acids on the membrane structure of different cellular compartments. We show that DCA increases the plasma membrane fluidity of hepatocytes to a minor extent, and that this effect is not correlated with the incidence of apoptosis. Additionally, plasma membrane fluidity recovers to normal values over time suggesting the presence of cellular compensatory mechanisms for this perturbation. Colocalization experiments in living cells confirmed the presence of bile acids within mitochondrial membranes. Experiments with active isolated mitochondria revealed that physiologically active concentrations of DCA change MOM order in a concentration- and time-dependent manner, and that these changes preceded the mitochondrial permeability transition. Importantly, these effects are not observed on liposomes mimicking MOM lipid composition, suggesting that DCA apoptotic activity depends on features of mitochondrial membranes that are absent in protein-free mimetic liposomes, such as the double-membrane structure, lipid asymmetry, or mitochondrial protein environment. In contrast, the mechanism of action of cytoprotective bile acids is likely not associated with changes in cellular membrane structure.

  16. Mutation of C20orf7 Disrupts Complex I Assembly and Causes Lethal Neonatal Mitochondrial Disease

    PubMed Central

    Sugiana, Canny; Pagliarini, David J.; McKenzie, Matthew; Kirby, Denise M.; Salemi, Renato; Abu-Amero, Khaled K.; Dahl, Hans-Henrik M.; Hutchison, Wendy M.; Vascotto, Katherine A.; Smith, Stacey M.; Newbold, Robert F.; Christodoulou, John; Calvo, Sarah; Mootha, Vamsi K.; Ryan, Michael T.; Thorburn, David R.

    2008-01-01

    Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease. PMID:18940309

  17. The intra-operative radius joystick test to diagnose complete disruption of the interosseous membrane.

    PubMed

    Soubeyrand, M; Ciais, G; Wassermann, V; Kalouche, I; Biau, D; Dumontier, C; Gagey, O

    2011-10-01

    Disruption of the interosseous membrane is easily missed in patients with Essex-Lopresti syndrome. None of the imaging techniques available for diagnosing disruption of the interosseous membrane are completely dependable. We undertook an investigation to identify whether a simple intra-operative test could be used to diagnose disruption of the interosseous membrane during surgery for fracture of the radial head and to see if the test was reproducible. We studied 20 cadaveric forearms after excision of the radial head, ten with and ten without disruption of the interosseous membrane. On each forearm, we performed the radius joystick test: moderate lateral traction was applied to the radial neck with the forearm in maximal pronation, to look for lateral displacement of the proximal radius indicating that the interosseous membrane had been disrupted. Each of six surgeons (three junior and three senior) performed the test on two consecutive days. Intra-observer agreement was 77% (95% confidence interval (CI) 67 to 85) and interobserver agreement was 97% (95% CI 92 to 100). Sensitivity was 100% (95% CI 97 to 100), specificity 88% (95% CI 81 to 93), positive predictive value 90% (95% CI 83 to 94), and negative predictive value 100%). This cadaveric study suggests that the radius joystick test may be useful for detecting disruption of the interosseous membrane in patients undergoing open surgery for fracture of the radial head and is reproducible. A confirmatory study in vivo is now required.

  18. Destabilization of the Outer and Inner Mitochondrial Membranes by Core and Linker Histones

    PubMed Central

    Cascone, Annunziata; Bruelle, Celine; Lindholm, Dan; Bernardi, Paolo; Eriksson, Ove

    2012-01-01

    Background Extensive DNA damage leads to apoptosis. Histones play a central role in DNA damage sensing and may mediate signals of genotoxic damage to cytosolic effectors including mitochondria. Methodology/Principal Findings We have investigated the effects of histones on mitochondrial function and membrane integrity. We demonstrate that both linker histone H1 and core histones H2A, H2B, H3, and H4 bind strongly to isolated mitochondria. All histones caused a rapid and massive release of the pro-apoptotic intermembrane space proteins cytochrome c and Smac/Diablo, indicating that they permeabilize the outer mitochondrial membrane. In addition, linker histone H1, but not core histones, permeabilized the inner membrane with a collapse of the membrane potential, release of pyridine nucleotides, and mitochondrial fragmentation. Conclusions We conclude that histones destabilize the mitochondrial membranes, a mechanism that may convey genotoxic signals to mitochondria and promote apoptosis following DNA damage. PMID:22523586

  19. Human Mitochondrial DNA-Protein Complexes Attach to a Cholesterol-Rich Membrane Structure

    PubMed Central

    Gerhold, Joachim M.; Cansiz-Arda, Şirin; Lõhmus, Madis; Engberg, Oskar; Reyes, Aurelio; van Rennes, Helga; Sanz, Alberto; Holt, Ian J.; Cooper, Helen M.; Spelbrink, Johannes N.

    2015-01-01

    The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol. PMID:26478270

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

    PubMed

    Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi

    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.

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

    SciTech Connect

    Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi

    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.

  2. Selective sorting and destruction of mitochondrial membrane proteins in aged yeast

    PubMed Central

    Hughes, Adam L; Hughes, Casey E; Henderson, Kiersten A; Yazvenko, Nina; Gottschling, Daniel E

    2016-01-01

    Mitochondrial dysfunction is a hallmark of aging, and underlies the development of many diseases. Cells maintain mitochondrial homeostasis through a number of pathways that remodel the mitochondrial proteome or alter mitochondrial content during times of stress or metabolic adaptation. Here, using yeast as a model system, we identify a new mitochondrial degradation system that remodels the mitochondrial proteome of aged cells. Unlike many common mitochondrial degradation pathways, this system selectively removes a subset of membrane proteins from the mitochondrial inner and outer membranes, while leaving the remainder of the organelle intact. Selective removal of preexisting proteins is achieved by sorting into a mitochondrial-derived compartment, or MDC, followed by release through mitochondrial fission and elimination by autophagy. Formation of MDCs requires the import receptors Tom70/71, and failure to form these structures exacerbates preexisting mitochondrial dysfunction, suggesting that the MDC pathway provides protection to mitochondria in times of stress. DOI: http://dx.doi.org/10.7554/eLife.13943.001 PMID:27097106

  3. Oligopolyphenylenevinylene-Conjugated Oligoelectrolyte Membrane Insertion Molecules Selectively Disrupt Cell Envelopes of Gram-Positive Bacteria

    PubMed Central

    Poh, Wee Han; Chu, Justin Jang Hann; Loo, Joachim Say Chye; Bazan, Guillermo C.; Hancock, Lynn E.

    2015-01-01

    The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone. PMID:25576607

  4. Oligopolyphenylenevinylene-conjugated oligoelectrolyte membrane insertion molecules selectively disrupt cell envelopes of Gram-positive bacteria.

    PubMed

    Hinks, Jamie; Poh, Wee Han; Chu, Justin Jang Hann; Loo, Joachim Say Chye; Bazan, Guillermo C; Hancock, Lynn E; Wuertz, Stefan

    2015-03-01

    The modification of microbial membranes to achieve biotechnological strain improvement with exogenous small molecules, such as oligopolyphenylenevinylene-conjugated oligoelectrolyte (OPV-COE) membrane insertion molecules (MIMs), is an emerging biotechnological field. Little is known about the interactions of OPV-COEs with their target, the bacterial envelope. We studied the toxicity of three previously reported OPV-COEs with a selection of Gram-negative and Gram-positive organisms and demonstrated that Gram-positive bacteria are more sensitive to OPV-COEs than Gram-negative bacteria. Transmission electron microscopy demonstrated that these MIMs disrupt microbial membranes and that this occurred to a much greater degree in Gram-positive organisms. We used a number of mutants to probe the nature of MIM interactions with the microbial envelope but were unable to align the membrane perturbation effects of these compounds to previously reported membrane disruption mechanisms of, for example, cationic antimicrobial peptides. Instead, the data support the notion that OPV-COEs disrupt microbial membranes through a suspected interaction with diphosphatidylglycerol (DPG), a major component of Gram-positive membranes. The integrity of model membranes containing elevated amounts of DPG was disrupted to a greater extent by MIMs than those prepared from Escherichia coli total lipid extracts alone.

  5. The mitochondrial outer membrane protein hFis1 regulates mitochondrial morphology and fission through self-interaction

    SciTech Connect

    Serasinghe, Madhavika N.; Yoon, Yisang

    2008-11-15

    Mitochondrial fission in mammals is mediated by at least two proteins, DLP1/Drp1 and hFis1. DLP1 mediates the scission of mitochondrial membranes through GTP hydrolysis, and hFis1 is a putative DLP1 receptor anchored at the mitochondrial outer membrane by a C-terminal single transmembrane domain. The cytosolic domain of hFis1 contains six {alpha}-helices ({alpha}1-{alpha}6) out of which {alpha}2-{alpha}5 form two tetratricopeptide repeat (TPR) folds. In this study, by using chimeric constructs, we demonstrated that the cytosolic domain contains the necessary information for hFis1 function during mitochondrial fission. By using transient expression of different mutant forms of the hFis1 protein, we found that hFis1 self-interaction plays an important role in mitochondrial fission. Our results show that deletion of the {alpha}1 helix greatly increased the formation of dimeric and oligomeric forms of hFis1, indicating that {alpha}1 helix functions as a negative regulator of the hFis1 self-interaction. Further mutational approaches revealed that a tyrosine residue in the {alpha}5 helix and the linker between {alpha}3 and {alpha}4 helices participate in hFis1 oligomerization. Mutations causing oligomerization defect greatly reduced the ability to induce not only mitochondrial fragmentation by full-length hFis1 but also the formation of swollen ball-shaped mitochondria caused by {alpha}1-deleted hFis1. Our data suggest that oligomerization of hFis1 in the mitochondrial outer membrane plays a role in mitochondrial fission, potentially through participating in fission factor recruitment.

  6. Yeast mitochondrial fission proteins induce antagonistic Gaussian membrane curvatures to regulate apoptosis

    NASA Astrophysics Data System (ADS)

    Lee, Michelle; Hwee Lai, Ghee; Schmidt, Nathan; Xian, Wujing; Wong, Gerard C. L.

    2013-03-01

    Mitochondria form a dynamic and interconnected network, which disintegrates during apoptosis to generate numerous smaller mitochondrial fragments. This process is at present not well understood. Yeast mitochondrial fission machinery proteins, Dnm1 and Fis1, are believed to regulate programmed cell death in yeast. Yeast Dnm1 has been previously shown to promote mitochondrial fragmentation and degradation characteristic of apoptotic cells, while yeast Fis1 inhibits cell death by limiting the mitochondrial fission induced by Dnm1 [Fannjiang et al, Genes & Dev. 2004. 18: 2785-2797]. To better understand the mechanisms of these antagonistic fission proteins, we use synchrotron small angle x-ray scattering (SAXS) to investigate their interaction with model cell membranes. The relationship between each protein, Dnm1 and Fis1, and protein-induced changes in membrane curvature and topology is examined. Through the comparison of the membrane rearrangement and phase behavior induced by each protein, we will discuss their respective roles in the regulation of mitochondrial fission.

  7. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>500) known and predicted TA proteins in Arabidopsis for those annotated, based on Gene Ontology, to possess mitochondrial...

  8. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy

    PubMed Central

    1981-01-01

    Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans- membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria- associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level. PMID:6783667

  9. Phytosphingosine kills Candida albicans by disrupting its cell membrane.

    PubMed

    Veerman, Enno C I; Valentijn-Benz, Marianne; van't Hof, Wim; Nazmi, Kamran; van Marle, Jan; Amerongen, Arie V Nieuw

    2010-01-01

    The mechanism of action of phytosphingosine (PHS), a member of the sphingosine family which has candidacidal activity when added externally, was investigated. Previously, it has been reported that the fungicidal activity of PHS is based on the induction of caspase-independent apoptosis. In contrast, we found that addition of PHS causes a direct permeabilization of the plasma membrane of yeast, highlighted by the influx of the membrane probe propidium iodide, and the efflux of small molecules (i.e., adenine nucleotides) as well as large cellular constituents such as proteins. Freeze-fracture electron microscopy revealed that PHS treatment causes severe damage of the plasma membrane of the cell, which seems to have lost its integrity completely. We also found that PHS reverts the azide-induced insensitivity to histatin 5 (Hst5) of Candida albicans. In a previous study, we had found that the decreased sensitivity to Hst5 of energy-depleted cells is due to rigidification of the plasma membrane, which could be reverted by the membrane fluidizer benzyl alcohol. In line with the increased membrane permeabilization and ultrastructural damage, this reversal of the azide-induced insensitivity by PHS also points to a direct interaction between PHS and the cytoplasmic membrane of C. albicans.

  10. Mitochondrial membrane potential: a novel biomarker of oxidative environmental stress.

    PubMed Central

    Vayssier-Taussat, Muriel; Kreps, Sarah E; Adrie, Christophe; Dall'Ava, Josette; Christiani, David; Polla, Barbara S

    2002-01-01

    Epidemiologic analyses, traditionally based on long-term cohort or case-control studies, provide retrospective causal associations between exposure to a particular environmental stressor and an exposure-related disease end point. Recent research initiatives have propelled a shift toward exploring molecular epidemiology and molecular biological markers (biomarkers) as a means of providing more immediate, quantitative risk assessment of potentially deleterious environmental exposures. We compared, in normal human monocytes isolated from the blood of healthy donors, variations in Hsp70 expression and mitochondrial membrane potential (delta psi m) in response to exposure to either tobacco smoke or gamma-irradiation, two models for environmentally mediated oxidant exposure. On the basis of its mechanistic specificity for oxidants and little baseline variation in cells from distinct individuals, we propose that delta psi m represents a selective in vitro and in vivo biomarker for oxidant exposure. delta psi m may be used to gauge risks associated with oxidant-mediated air pollution and radiation. PMID:11882482

  11. Reoxygenation-induced mitochondrial damage is caused by the Ca2+-dependent mitochondrial inner membrane permeability transition.

    PubMed

    Tanaka, T; Hakoda, S; Takeyama, N

    1998-07-01

    Anoxia/reoxygenation injury of isolated rat liver mitochondria was investigated. During anoxia of up to 60 min, the membrane potential was largely preserved and mitochondrial swelling was not observed. Reoxygenation of anoxic mitochondria rapidly caused swelling, cyclosporin A-sensitive Ca2+ efflux, [14C]sucrose trapping, and loss of the membrane potential along with increased generation of reactive oxygen intermediates (ROI). Although pretreatment with catalase and superoxide dismutase completely abolished reoxygenation-induced generation of ROI, mitochondrial damage was not prevented, as indicated by swelling, loss of the membrane potential, a decrease of the ATP content, and cyclosporin A-sensitive Ca2+ efflux. However, addition of the immunosuppressant cyclosporin A or addition of ADP completely prevented the mitochondrial damage induced by reoxygenation. The same protective effect was noted when Ca2+ cycling was prevented, either by chelating Ca2+ with EGTA or by inhibiting Ca2+ reuptake with ruthenium red. These findings indicate that mitochondrial anoxia/reoxygenation injury is caused by the cyclosporin A-sensitive and Ca2+-dependent membrane permeability transition. In contrast, reoxygenation injury does not appear to be triggered by the enhanced production of ROI.

  12. Biochemical and molecular characterization of mitochondrial membrane-bound arginase in Heteropneustes fossilis.

    PubMed

    Mishra, Suman; Mishra, Rajnikant

    2016-05-01

    The two predominant forms of arginase, cytosolic Arginase-I and mitochondrial Arginase-II, catalyze hydrolysis of arginine into ornithine and urea. Based on presence of arginase activity in extracts using potassium chloride (KCl), mitochondrial membrane-bound arginase has also been suggested. However, the activity of arginase in fractions obtained after KCl-treatment may be either due to leakage of mitochondrial arginase or release of adhered cytosolic arginase to cell organelles having altered net charge. Therefore, it has been intended to analyse impact of KCl on ultra-structural properties of mitochondria, and biochemical analysis of mitochondrial membrane-bound proteins and arginase of Heteropneustes fossilis. Liver of H. fossilis was used for isolating mitochondria for analysis of ultrastructural properties, preparing cytosolic, mitochondrial, and mitochondrial-membrane bound extracts after treatment of KCl. Extracts were analysed for arginase activity assay, protein profiling through SDS-PAGE and MALDI MS/MS. The KCl-mediated modulation in polypeptides and arginase were also evaluated by PANTHER, MitoProt and IPSORT servers. The effects of KCl on ultra-structural integrity of mitochondria, activity of arginase, modulation on mitochondrial proteins and enzymes including arginase were observed. The 48 kDa polypeptide of mitochondrial fraction, that showed KCl-dependent alteration matched with Myb binding protein and 30 kDa bands resembles to arginase after MALDI MS/MS analysis. Results indicate KCl-dependent ultrastructural changes in mitochondria and release of mitochondrial arginase. The proposed membrane bound mitochondrial arginase could be mitochondrial arginase-II or altered form of cytosolic arginase-I contributing to KCl-induced arginase activity in H. fossilis.

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

    PubMed

    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.

  14. The role of the mitochondrial ribosome in human disease: searching for mutations in 12S mitochondrial rRNA with high disruptive potential

    PubMed Central

    Smith, Paul M.; Elson, Joanna L.; Greaves, Laura C.; Wortmann, Saskia B.; Rodenburg, Richard J.T.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

    2014-01-01

    Mutations of mitochondrial DNA are linked to many human diseases. Despite the identification of a large number of variants in the mitochondrially encoded rRNA (mt-rRNA) genes, the evidence supporting their pathogenicity is, at best, circumstantial. Establishing the pathogenicity of these variations is of major diagnostic importance. Here, we aim to estimate the disruptive effect of mt-rRNA variations on the function of the mitochondrial ribosome. In the absence of direct biochemical methods to study the effect of mt-rRNA variations, we relied on the universal conservation of the rRNA fold to infer their disruptive potential. Our method, named heterologous inferential analysis or HIA, combines conservational information with functional and structural data obtained from heterologous ribosomal sources. Thus, HIA's predictive power is superior to the traditional reliance on simple conservation indexes. By using HIA, we have been able to evaluate the disruptive potential for a subset of uncharacterized 12S mt-rRNA variations. Our analysis revealed the existence of variations in the rRNA component of the human mitoribosome with different degrees of disruptive power. In cases where sufficient information regarding the genetic and pathological manifestation of the mitochondrial phenotype is available, HIA data can be used to predict the pathogenicity of mt-rRNA mutations. In other cases, HIA analysis will allow the prioritization of variants for additional investigation. Eventually, HIA-inspired analysis of potentially pathogenic mt-rRNA variations, in the context of a scoring system specifically designed for these variants, could lead to a powerful diagnostic tool. PMID:24092330

  15. Increased androgen levels in rats impair glucose-stimulated insulin secretion through disruption of pancreatic beta cell mitochondrial function.

    PubMed

    Wang, Hongdong; Wang, Xiaping; Zhu, Yunxia; Chen, Fang; Sun, Yujie; Han, Xiao

    2015-11-01

    Although insulin resistance is recognized to contribute to the reproductive and metabolic phenotypes of polycystic ovary syndrome (PCOS), pancreatic beta cell dysfunction plays an essential role in the progression from PCOS to the development of type 2 diabetes. However, the role of insulin secretory abnormalities in PCOS has received little attention. In addition, the precise changes in beta cells and the underlying mechanisms remain unclear. In this study, we therefore attempted to elucidate potential mechanisms involved in beta cell alterations in a rat model of PCOS. Glucose-induced insulin secretion was measured in islets isolated from DHT-treated and control rats. Oxygen consumption rate (OCR), ATP production, and mitochondrial copy number were assayed to evaluate mitochondrial function. Glucose-stimulated insulin secretion is significantly decreased in islets from DHT-treated rats. On the other hand, significant reductions are observed in the expression levels of several key genes involved in mitochondrial biogenesis and in mitochondrial OCR and ATP production in DHT-treated rat islets. Meanwhile, we found that androgens can directly impair beta cell function by inducing mitochondrial dysfunction in vitro in an androgen receptor dependent manner. For the first time, our study demonstrates that increased androgens in female rats can impair glucose-stimulated insulin secretion partly through disruption of pancreatic beta cell mitochondrial function. This work has significance for hyperandrogenic women with PCOS: excess activation of the androgen receptor by androgens may provoke beta cell dysfunction via mitochondrial dysfunction.

  16. Permeabilized myocardial fibers as model to detect mitochondrial dysfunction during sepsis and melatonin effects without disruption of mitochondrial network.

    PubMed

    Doerrier, Carolina; García, José A; Volt, Huayqui; Díaz-Casado, María E; Luna-Sánchez, Marta; Fernández-Gil, Beatriz; Escames, Germaine; López, Luis C; Acuña-Castroviejo, Darío

    2016-03-01

    Analysis of mitochondrial function is crucial to understand their involvement in a given disease. High-resolution respirometry of permeabilized myocardial fibers in septic mice allows the evaluation of the bioenergetic system, maintaining mitochondrial ultrastructure and intracellular interactions, which are critical for an adequate functionality. OXPHOS and electron transport system (ETS) capacities were assessed using different substrate combinations. Our findings show a severe septic-dependent impairment in OXPHOS and ETS capacities with mitochondrial uncoupling at early and late phases of sepsis. Moreover, sepsis triggers complex III (CIII)-linked alterations in supercomplexes structure, and loss of mitochondrial density. In these conditions, melatonin administration to septic mice prevented sepsis-dependent mitochondrial injury in mitochondrial respiration. Likewise, melatonin improved cytochrome b content and ameliorated the assembly of CIII in supercomplexes. These results support the use of permeabilized fibers to identify properly the respiratory deficits and specific melatonin effects in sepsis.

  17. Phenotypes of gene disruptants in relation to a putative mitochondrial malate-citrate shuttle protein in citric acid-producing Aspergillus niger.

    PubMed

    Kirimura, Kohtaro; Kobayashi, Keiichi; Ueda, Yuka; Hattori, Takasumi

    2016-09-01

    The mitochondrial citrate transport protein (CTP) functions as a malate-citrate shuttle catalyzing the exchange of citrate plus a proton for malate between mitochondria and cytosol across the inner mitochondrial membrane in higher eukaryotic organisms. In this study, for functional analysis, we cloned the gene encoding putative CTP (ctpA) of citric acid-producing Aspergillus niger WU-2223L. The gene ctpA encodes a polypeptide consisting 296 amino acids conserved active residues required for citrate transport function. Only in early-log phase, the ctpA disruptant DCTPA-1 showed growth delay, and the amount of citric acid produced by strain DCTPA-1 was smaller than that by parental strain WU-2223L. These results indicate that the CTPA affects growth and thereby citric acid metabolism of A. niger changes, especially in early-log phase, but not citric acid-producing period. This is the first report showing that disruption of ctpA causes changes of phenotypes in relation to citric acid production in A. niger.

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

    PubMed

    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.

  19. Increased mitochondrial ROS formation by acetaminophen in human hepatic cells is associated with gene expression changes suggesting disruption of the mitochondrial electron transport chain.

    PubMed

    Jiang, Jian; Briedé, Jacob J; Jennen, Danyel G J; Van Summeren, Anke; Saritas-Brauers, Karen; Schaart, Gert; Kleinjans, Jos C S; de Kok, Theo M C M

    2015-04-16

    Acetaminophen (APAP) overdosage results in hepatotoxicity, but the underlying molecular mechanisms are still not completely understood. In the current study, we focused on mitochondrial-specific oxidative liver injury induced by APAP exposure. Owning to genetic polymorphisms in the CYP2E1 gene or varying inducibility by xenobiotics, the CYP2E1 mRNA level and protein activity vary extensively among individuals. As CYP2E1 is a known ROS generating enzyme, we chose HepG2 to minimize CYP2E1-induced ROS formation, which will help us better understand the APAP induced mitochondrial-specific hepatotoxicity in a subpopulation with low CYP2E1 activity. HepG2 cells were exposed to a low and toxic dose (0.5 and 10mM) of APAP and analyzed at four time points for genome-wide gene expression. Mitochondria were isolated and electron spin resonance spectroscopy was performed to measure the formation of mitochondrial ROS. The yield of ATP was measured to confirm the impact of the toxic dose of APAP on cellular energy production. Our results indicate that 10mM APAP significantly influences the expression of mitochondrial protein-encoding genes in association with an increase in mitochondrial ROS formation. Additionally, 10mM APAP affects the expression of genes encoding the subunits of electron transport chain (ETC) complexes, which may alter normal mitochondrial functions by disrupting the assembly, stability, and structural integrity of ETC complexes, leading to a measurable depletion of ATP, and cell death. The expression of mitochondrium-specific antioxidant enzyme, SOD2, is reduced which may limit the ROS scavenging ability and cause imbalance of the mitochondrial ROS homeostasis. Overall, transcriptome analysis reveals the molecular processes involved in the observed APAP-induced increase of mitochondrial ROS formation and the associated APAP-induced oxidative stress.

  20. Preliminary crystallographic studies of yeast mitochondrial peripheral membrane protein Tim44p

    SciTech Connect

    Josyula, Ratnakar; Jin, Zhongmin; McCombs, Deborah; DeLucas, Lawrence; Sha, Bingdong

    2006-02-01

    Tim44p is an essential mitochondrial peripheral membrane protein. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p has been crystallized. Protein translocations across mitochondrial membranes play critical roles in mitochondrion biogenesis. Protein transport from the cell cytosol to the mitochondrial matrix is carried out by the translocase of the outer membrane (TOM) complex and the translocase of the inner membrane (TIM) complexes. Tim44p is an essential mitochondrial peripheral membrane protein and a major component of the TIM23 translocon. To investigate the mechanism by which Tim44p functions in the TIM23 translocon to deliver the mitochondrial protein precursors, the yeast Tim44p was crystallized. The crystals diffract to 3.2 Å using a synchrotron X-ray source and belong to space group P6{sub 3}22, with unit-cell parameters a = 124.25, c = 77.83 Å. There is one Tim44p molecule in one asymmetric unit, which corresponds to a solvent content of approximately 43%. Structure determination by MAD methods is under way.

  1. Superresolution Imaging Identifies That Conventional Trafficking Pathways Are Not Essential for Endoplasmic Reticulum to Outer Mitochondrial Membrane Protein Transport.

    PubMed

    Salka, Kyle; Bhuvanendran, Shivaprasad; Wilson, Kassandra; Bozidis, Petros; Mehta, Mansi; Rainey, Kristin; Sesaki, Hiromi; Patterson, George H; Jaiswal, Jyoti K; Colberg-Poley, Anamaris M

    2017-12-01

    Most nuclear-encoded mitochondrial proteins traffic from the cytosol to mitochondria. Some of these proteins localize at mitochondria-associated membranes (MAM), where mitochondria are closely apposed with the endoplasmic reticulum (ER). We have previously shown that the human cytomegalovirus signal-anchored protein known as viral mitochondria-localized inhibitor of apoptosis (vMIA) traffics from the ER to mitochondria and clusters at the outer mitochondrial membrane (OMM). Here, we have examined the host pathways by which vMIA traffics from the ER to mitochondria and clusters at the OMM. By disruption of phosphofurin acidic cluster sorting protein 2 (PACS-2), mitofusins (Mfn1/2), and dynamin related protein 1 (Drp1), we find these conventional pathways for ER to the mitochondria trafficking are dispensable for vMIA trafficking to OMM. Instead, mutations in vMIA that change its hydrophobicity alter its trafficking to mitochondria. Superresolution imaging showed that PACS-2- and Mfn-mediated membrane apposition or hydrophobic interactions alter vMIA's ability to organize in nanoscale clusters at the OMM. This shows that signal-anchored MAM proteins can make use of hydrophobic interactions independently of conventional ER-mitochondria pathways to traffic from the ER to mitochondria. Further, vMIA hydrophobic interactions and ER-mitochondria contacts facilitate proper organization of vMIA on the OMM.

  2. Interaction of the Intermembrane Space Domain of Tim23 Protein with Mitochondrial Membranes*

    PubMed Central

    Bajaj, Rakhi; Munari, Francesca; Becker, Stefan; Zweckstetter, Markus

    2014-01-01

    Tim23 mediates protein translocation into mitochondria. Although inserted into the inner membrane, the dynamic association of its intermembrane space (IMS) domain with the outer membrane promotes protein import. However, little is known about the molecular basis of this interaction. Here, we demonstrate that the IMS domain of Tim23 tightly associates with both inner and outer mitochondrial membrane-like membranes through a hydrophobic anchor at its N terminus. The structure of membrane-bound Tim23IMS is highly dynamic, allowing recognition of both the incoming presequence and other translocase components at the translocation contact. Cardiolipin enhances Tim23 membrane attachment, suggesting that cardiolipin can influence preprotein import. PMID:25349212

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

    PubMed

    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.

  4. The Role of Cationic Group Structure in Membrane Binding and Disruption by Amphiphilic Copolymers

    PubMed Central

    Palermo, Edmund F.; Lee, Dong-Kuk; Ramamoorthy, Ayyalusamy; Kuroda, Kenichi

    2010-01-01

    Cationic, amphiphilic polymers are currently being used as antimicrobial agents which disrupt biomembranes, although their mechanism(s) remain poorly understood. Herein, membrane association and disruption by amphiphilic polymers bearing primary, tertiary, or quaternary ammonium salt groups reveals the role of cationic group structure in the polymer-membrane interaction. The dissociation constants of polymers to liposomes of POPC were obtained by a fluorometric assay, exploiting the environmental sensitivity of dansyl moieties in the polymer end groups. Dye leakage from liposomes and solid-state NMR provided further insights into the polymer-induced membrane disruption. Interestingly, the polymers with primary amine groups induced reorganization of the bilayer structure to align lipid headgroups perpendicular to the membrane. The results showed that polymers bearing primary amines exceed the tertiary and quaternary ammonium counterparts in membrane binding and disrupting abilities. This is likely due to enhanced complexation of primary amines to the phosphate groups in the lipids, through a combination of hydrogen bonding and electrostatic interactions. PMID:21171655

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

    PubMed

    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.

  6. Membrane raft disruption promotes axonogenesis in n2a neuroblastoma cells.

    PubMed

    Petro, Kimberly A; Schengrund, Cara-Lynne

    2009-01-01

    Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

  7. Palmitoleic acid induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

    PubMed

    Oyanagi, Eri; Uchida, Masataka; Miyakawa, Takeshi; Miyachi, Motohiko; Yamaguchi, Hidetaka; Nagami, Kuniatsu; Utsumi, Kozo; Yano, Hiromi

    Although palmitoleic acid (C16:1) is associated with arrhythmias, and increases in an age-dependent matter, the effects of L-carnitine, which is essential for the transport of long-chain fatty acids into the mitochondria, are unclear. It has been postulated that L-carnitine may attenuate palmitate (C16:0)-induced mitochondrial dysfunction and the apoptosis of cardiomyocytes. The aim of this study was to elucidate the activity of L-carnitine in the prevention of the palmitoleic acid-induced mitochondrial membrane permeability transition and cytochrome c release using isolated cardiac mitochondria from rats. Palmitoleoyl-CoA-induced mitochondrial respiration was not accelerated by L-carnitine treatment, and this respiration was slightly inhibited by oligomycin, which is an inhibitor of ATP synthase. Despite pretreatment with L-carnitine, the mitochondrial membrane potential decreased and mitochondrial swelling was induced by palmitoleoyl-CoA. In the presence of a combination of L-carnitine and tiron, a free radical scavenger, there was attenuated mitochondrial swelling and cytochrome c release following palmitoleoyl-CoA treatment. We concluded that palmitoleic acid, but not palmitate, induces the cardiac mitochondrial membrane permeability transition despite the presence of L-carnitine.

  8. The fusogenic lipid phosphatidic acid promotes the biogenesis of mitochondrial outer membrane protein Ugo1

    PubMed Central

    Keller, Michael; Taskin, Asli A.; Horvath, Susanne E.; Guan, Xue Li; Prinz, Claudia; Opalińska, Magdalena; Zorzin, Carina; van der Laan, Martin; Wenk, Markus R.; Schubert, Rolf; Wiedemann, Nils; Holzer, Martin

    2015-01-01

    Import and assembly of mitochondrial proteins depend on a complex interplay of proteinaceous translocation machineries. The role of lipids in this process has been studied only marginally and so far no direct role for a specific lipid in mitochondrial protein biogenesis has been shown. Here we analyzed a potential role of phosphatidic acid (PA) in biogenesis of mitochondrial proteins in Saccharomyces cerevisiae. In vivo remodeling of the mitochondrial lipid composition by lithocholic acid treatment or by ablation of the lipid transport protein Ups1, both leading to an increase of mitochondrial PA levels, specifically stimulated the biogenesis of the outer membrane protein Ugo1, a component of the mitochondrial fusion machinery. We reconstituted the import and assembly pathway of Ugo1 in protein-free liposomes, mimicking the outer membrane phospholipid composition, and found a direct dependency of Ugo1 biogenesis on PA. Thus, PA represents the first lipid that is directly involved in the biogenesis pathway of a mitochondrial membrane protein. PMID:26347140

  9. Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

    SciTech Connect

    Montaigne, David; Marechal, Xavier; Baccouch, Riadh; Modine, Thomas; Preau, Sebastien; Zannis, Konstantinos; Marchetti, Philippe; Lancel, Steve; Neviere, Remi

    2010-05-01

    The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 muM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt{sub max} of 105 +- 8 mN/s in control hearts vs. 49 +- 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 +- 0.2 in control hearts vs. 2.2 +- 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 +- 1 muM cytochrome c/min/mg in control hearts vs. 14 +- 3 muM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

  10. Loss of Prohibitin Membrane Scaffolds Impairs Mitochondrial Architecture and Leads to Tau Hyperphosphorylation and Neurodegeneration

    PubMed Central

    Merkwirth, Carsten; Morbin, Michela; Brönneke, Hella S.; Jordan, Sabine D.; Rugarli, Elena I.; Langer, Thomas

    2012-01-01

    Fusion and fission of mitochondria maintain the functional integrity of mitochondria and protect against neurodegeneration, but how mitochondrial dysfunctions trigger neuronal loss remains ill-defined. Prohibitins form large ring complexes in the inner membrane that are composed of PHB1 and PHB2 subunits and are thought to function as membrane scaffolds. In Caenorhabditis elegans, prohibitin genes affect aging by moderating fat metabolism and energy production. Knockdown experiments in mammalian cells link the function of prohibitins to membrane fusion, as they were found to stabilize the dynamin-like GTPase OPA1 (optic atrophy 1), which mediates mitochondrial inner membrane fusion and cristae morphogenesis. Mutations in OPA1 are associated with dominant optic atrophy characterized by the progressive loss of retinal ganglion cells, highlighting the importance of OPA1 function in neurons. Here, we show that neuron-specific inactivation of Phb2 in the mouse forebrain causes extensive neurodegeneration associated with behavioral impairments and cognitive deficiencies. We observe early onset tau hyperphosphorylation and filament formation in the hippocampus, demonstrating a direct link between mitochondrial defects and tau pathology. Loss of PHB2 impairs the stability of OPA1, affects mitochondrial ultrastructure, and induces the perinuclear clustering of mitochondria in hippocampal neurons. A destabilization of the mitochondrial genome and respiratory deficiencies manifest in aged neurons only, while the appearance of mitochondrial morphology defects correlates with tau hyperphosphorylation in the absence of PHB2. These results establish an essential role of prohibitin complexes for neuronal survival in vivo and demonstrate that OPA1 stability, mitochondrial fusion, and the maintenance of the mitochondrial genome in neurons depend on these scaffolding proteins. Moreover, our findings establish prohibitin-deficient mice as a novel genetic model for tau pathologies

  11. The presequence pathway is involved in protein sorting to the mitochondrial outer membrane

    PubMed Central

    Wenz, Lena-Sophie; Opaliński, Łukasz; Schuler, Max-Hinderk; Ellenrieder, Lars; Ieva, Raffaele; Böttinger, Lena; Qiu, Jian; van der Laan, Martin; Wiedemann, Nils; Guiard, Bernard; Pfanner, Nikolaus; Becker, Thomas

    2014-01-01

    The mitochondrial outer membrane contains integral α-helical and β-barrel proteins that are imported from the cytosol. The machineries importing β-barrel proteins have been identified, however, different views exist on the import of α-helical proteins. It has been reported that the biogenesis of Om45, the most abundant signal-anchored protein, does not depend on proteinaceous components, but involves direct insertion into the outer membrane. We show that import of Om45 occurs via the translocase of the outer membrane and the presequence translocase of the inner membrane. Assembly of Om45 in the outer membrane involves the MIM machinery. Om45 thus follows a new mitochondrial biogenesis pathway that uses elements of the presequence import pathway to direct a protein to the outer membrane. PMID:24781695

  12. Characterization of the mitochondrial inner membrane protein translocator Tim17 from Trypanosoma brucei

    PubMed Central

    Singha, Ujjal K.; Peprah, Emmanuel; Williams, Shuntae; Walker, Robert; Saha, Lipi; Chaudhuri, Minu

    2010-01-01

    Mitochondrial protein translocation machinery in the kinetoplastid parasites, like Trypanosoma brucei, has been characterized poorly. In T. brucei genome data base, one homolog for a protein translocator of mitochondrial inner membrane (Tim) has been found, which is closely related to Tim17 from other species. The T. brucei Tim17 (TbTim17) has a molecular mass 16.2 kDa and it possesses four characteristic transmembrane domains. The protein is localized in the mitochondrial inner membrane. The level of TbTim17 protein is 6–7 fold higher in the procyclic form that has a fully active mitochondrion, than in the mammalian bloodstream form of T. brucei, where many of the mitochondrial activities are suppressed. Knockdown of TbTim17 expression by RNAi caused a cessation of cell growth in the procyclic form and reduced growth rate in the bloodstream form. Depletion of TbTim17 decreased mitochondrial membrane potential more in the procyclic than bloodstream form. However, TbTim17 knockdown reduced the expression level of several nuclear encoded mitochondrial proteins in both the forms. Furthermore, import of presequence containing nuclear encoded mitochondrial proteins was significantly reduced in TbTim17 depleted mitochondria of the procyclic as well as the bloodstream form, confirming that TbTim17 is critical for mitochondrial protein import in both developmental forms. Together, these show that TbTim17 is the translocator of nuclear encoded mitochondrial proteins and its expression is regulated according to mitochondrial activities in T. brucei. PMID:18325611

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  15. SV40 late protein VP4 forms toroidal pores to disrupt membranes for viral release

    PubMed Central

    Raghava, Smita; Giorda, Kristina M.; Romano, Fabian B.; Heuck, Alejandro P.; Hebert, Daniel N.

    2014-01-01

    Nonenveloped viruses are generally released from the cell by the timely lysis of host cell membranes. SV40 has been used as a model virus for the study of the lytic nonenveloped virus life cycle. The expression of SV40 VP4 at later times during infection is concomitant with cell lysis. To investigate the role of VP4 in viral release and its mechanism of action, VP4 was expressed and purified from bacteria as a fusion protein for use in membrane disruption assays. Purified VP4 perforated membranes as demonstrated by the release of fluorescent markers encapsulated within large unilamellar vesicles or liposomes. Dynamic light scattering results found that VP4 treatment did not cause membrane lysis or change the size of the liposomes. Liposomes encapsulated with bodipy-labeled streptavidin were used to show that VP4 formed stable pores in membranes. These VP4 pores had an inner diameter of between 1 and 5 nm. Asymmetrical liposomes containing pyrene-labeled lipids in the outer monolayer were employed to monitor transbilayer lipid diffusion. Consistent with VP4 forming toroidal pore structures in membranes, VP4 induced transbilayer lipid diffusion or lipid flip-flop. Altogether, these studies support a central role for VP4 acting as a viroporin in the disruption of cellular membranes to trigger SV40 viral release by forming toroidal pores that unite the outer and inner leaflets of membrane bilayers. PMID:23651212

  16. Ca2+ acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by phenylarsine oxide.

    PubMed

    Kowaltowski, A J; Castilho, R F

    1997-12-15

    Mitochondrial permeability transition (MPT) induced by the thiol cross-linker phenylarsine oxide (PhAsO) in Ca(2+)-depleted mitochondria incubated in the presence of ruthenium red, an inhibitor of the Ca2+ uniporter, is stimulated by the addition of extramitochondrial Ca2+. The presence of extramitochondrial Ca2+ stimulates the reaction of mitochondrial membrane protein thiol groups with PhAsO. Both Ca(2+)-induced increase in mitochondrial membrane permeabilization and protein thiol group reaction with PhAsO are dependent on time (5-10 min to be complete) and the concentration of Ca2+ (1-25 microM). Mitochondrial permeabilization induced by PhAsO (15 microM) and extramitochondrial Ca2+ is inhibited by ADP, cyclosporin A, dibucaine and Mg2+, while mitochondrial permeabilization induced by high concentrations of PhAsO (60 microM) in the absence of Ca2+ is inhibited only by ADP and cyclosporin A. These results suggest that dibucaine and Mg2+ can inhibit mitochondrial permeabilization by antagonizing the effect of Ca2+ on the mitochondrial membrane. Once mitochondrial permeabilization induced by 15 microM PhAsO and extramitochondrial Ca2+ has already occurred, the addition of the Ca2+ chelator EGTA restores mitochondrial membrane potential (MPT pore closure), suggesting that the presence of Ca2+ is essential for the maintenance of the permeability of the mitochondrial membrane to protons (MPT pore opening). In conclusion, the results presented indicate that low Ca2+ concentrations acting at the external side of the inner mitochondrial membrane can stimulate mitochondrial permeability transition induced by PhAsO, due to increased accessibility of protein thiol groups to the reaction with PhAsO and increased probability of MPT pore opening.

  17. 5-HTR3 and 5-HTR4 located on the mitochondrial membrane and functionally regulated mitochondrial functions

    PubMed Central

    Wang, Qingyi; Zhang, Huiyuan; Xu, Hao; Guo, Dongqing; Shi, Hui; Li, Yuan; Zhang, Weiwei; Gu, Yuchun

    2016-01-01

    5-HT has been reported to possess significant effects on cardiac activities, but activation of 5-HTR on the cell membrane failed to illustrate the controversial cardiac reaction. Because 5-HT constantly comes across the cell membrane via 5-HT transporter (5-HTT) into the cytoplasm, whether 5-HTR is functional present on the cellular organelles is unknown. Here we show 5-HTR3 and 5-HTR4 were located in cardiac mitochondria, and regulated mitochondrial activities and cellular functions. Knock down 5-HTR3 and 5-HTR4 in neonatal cardiomyocytes resulted in significant increase of cell damage in response to hypoxia, and also led to alternation in heart beating. Activation of 5-HTR4 attenuated mitochondrial Ca2+ uptake under the both normoxic and hypoxic conditions, whereas 5-HTR3 augmented Ca2+ uptake only under hypoxia. 5-HTR3 and 5-HTR4 exerted the opposite effects on the mitochondrial respiration: 5-HTR3 increased RCR (respiration control ratio), but 5-HTR4 reduced RCR. Moreover, activation of 5-HTR3 and 5-HTR4 both significantly inhibited the opening of mPTP. Our results provided the first evidence that 5-HTR as a GPCR and an ion channel, functionally expressed in mitochondria and participated in the mitochondria function and regulation to maintain homeostasis of mitochondrial [Ca2+], ROS, and ATP generation efficiency in cardiomyocytes in response to stress and O2 tension. PMID:27874067

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

    PubMed

    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 < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for 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.

  19. Mechanism of action of cytotoxic cyclotides: cycloviolacin O2 disrupts lipid membranes.

    PubMed

    Svangård, Erika; Burman, Robert; Gunasekera, Sunithi; Lövborg, Henrik; Gullbo, Joachim; Göransson, Ulf

    2007-04-01

    In recent years, the cyclotides have emerged as the largest family of naturally cyclized proteins. Cyclotides display potent cytotoxic activity that varies with the structure of the proteins, and combined with their unique structure, they represent novel cytotoxic agents. However, their mechanism of action is yet unknown. In this work we show that disruption of cell membranes plays a crucial role in the cytotoxic effect of the cyclotide cycloviolacin O2 (1), which has been isolated from Viola odorata. Cell viability and morphology studies on the human lymphoma cell line U-937 GTB showed that cells exposed to 1 displayed disintegrated cell membranes within 5 min. Functional studies on calcein-loaded HeLa cells and on liposomes showed rapid concentration-dependent release of their respective internal contents. The present results show that cyclotides have specific membrane-disrupting activity.

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

    PubMed

    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.

  1. [HOMOCYSTEINE-INDUCED MEMBRANE CURRENTS, CALCIUM RESPONSES AND CHANGES OF MITOCHONDRIAL POTENTIAL IN RAT CORTICAL NEURONS].

    PubMed

    Abushik, P A; Karelina, T V; Sibarov, D A; Stepanenko, J D; Giniatullin, R; Antonov, S M

    2015-01-01

    Homocysteine, a sulfur-containing amino acid, exhibits neurotoxic effects and is involved in the pathogenesis of several major neurodegenerative disorders. In contrast to well studied excitoxicity of glutamate, the mechanism of homocysteine neurotoxicity is not clearly understood. By using whole-cell patch-clamp, calcium imaging (fluo-3) and measurements of mitochondrial membrane potential (rhodamine 123) we studied transmembrane currents, calcium signals and changes in mitochondrial membrane potential induced by homocysteine versus responses induced by NMDA and glutamate in cultured rat cortical neurons. L-homocysteine (50 µM) induced inward currents that could be completely blocked by the selective antagonist of NMDA receptors - AP-5. In contrast to NMDA-induced currents, homocysteine-induced currents had a smaller steady-state amplitude. Comparison of calcium responses to homocysteine, NMDA or glutamate demonstrated that in all cortical neurons homocysteine elicited short, oscillatory-type calcium responses, whereas NMDA or glutamate induced sustained increase of intracellular calcium. Analysis of mitochondrial changes demonstrated that in contrast to NMDA homocysteine did not cause a drop of mitochondrial membrane potential at the early stages of action. However, after its long-term action, as in the case of NMDA and glutamate, the changes in mitochondrial membrane potential were comparable with the full drop of respiratory chain induced by protonophore FCCP. Our data suggest that in cultured rat cortical neuron homocysteine at the first stages of action induces neurotoxic effects through activation of NMDA-type ionotropic glutamate receptors with strong calcium influx through the channels of these receptors. The long-term action of homocysteine may lead to mitochondrial disfuction and appears as a drop of mitochondrial membrane potential.

  2. Bacillus thuringiensis Cyt2Aa2 toxin disrupts cell membranes by forming large protein aggregates

    PubMed Central

    Tharad, Sudarat; Toca-Herrera, José L.; Promdonkoy, Boonhiang; Krittanai, Chartchai

    2016-01-01

    Bacillus thuringiensis (Bt) Cyt2Aa2 showed toxicity against Dipteran insect larvae and in vitro lysis activity on several cells. It has potential applications in the biological control of insect larvae. Although pore-forming and/or detergent-like mechanisms were proposed, the mechanism underlying cytolytic activity remains unclear. Analysis of the haemolytic activity of Cyt2Aa2 with osmotic stabilizers revealed partial toxin inhibition, suggesting a distinctive mechanism from the putative pore formation model. Membrane permeability was studied using fluorescent dye entrapped in large unilamellar vesicles (LUVs) at various protein/lipid molar ratios. Binding of Cyt2Aa2 monomer to the lipid membrane did not disturb membrane integrity until the critical protein/lipid molar ratio was reached, when Cyt2Aa2 complexes and cytolytic activity were detected. The complexes are large aggregates that appeared as a ladder when separated by agarose gel electrophoresis. Interaction of Cyt2Aa2 with Aedes albopictus cells was investigated by confocal microscopy and total internal reflection fluorescent microscopy (TIRF). The results showed that Cyt2Aa2 binds on the cell membrane at an early stage without cell membrane disruption. Protein aggregation on the cell membrane was detected later which coincided with cell swelling. Cyt2Aa2 aggregations on supported lipid bilayers (SLBs) were visualized by AFM. The AFM topographic images revealed Cyt2Aa2 aggregates on the lipid bilayer at low protein concentration and subsequently disrupts the lipid bilayer by forming a lesion as the protein concentration increased. These results supported the mechanism whereby Cyt2Aa2 binds and aggregates on the lipid membrane leading to the formation of non-specific hole and disruption of the cell membrane. PMID:27612497

  3. Detergent disruption of bacterial inner membranes and recovery of protein translocation activity

    SciTech Connect

    Cunningham, K.; Wickner, W.T. )

    1989-11-01

    Isolation of the integral membrane components of protein translocation requires methods for fractionation and functional reconstitution. The authors treated inner-membrane vesicles of Escherichia coli with mixtures of octyl {beta}-D-glucoside, phospholipids, and an integral membrane carrier protein under conditions that extract most of the membrane proteins into micellar solution. Upon dialysis, proteoliposomes were reconstituted that supported translocation of radiochemically pure ({sup 35}S)pro-OmpA (the precursor of outer membrane protein A). Translocation into these proteoliposomes required ATP hydrolysis and membrane proteins, indicating that the reaction is that of the inner membrane. The suspension of membranes in detergent was separated into supernatant and pellet fractions by ultracentrifugation. After reconstitution, translocation activity was observed in both fractions, but processing by leader peptidase of translocated pro-OmpA to OmpA was not detectable in the reconstituted pellet fraction. Processing activity was restored by addition of pure leader peptidase as long as this enzyme was added before detergent removal, indicating that the translocation activity is not associated with detergent-resistant membrane vesicles. These results show that protein translocation activity can be recovered from detergent-disrupted membrane vesicles, providing a first step towards the goal of isolating the solubilized components.

  4. [Development of the MITO-porter, a nano device for mitochondrial drug delivery via membrane fusion].

    PubMed

    Yamada, Yuma

    2014-01-01

    Many human diseases have been reported to be associated with mitochondrial dysfunction. Therefore, mitochondrial therapy would be expected to be useful and productive in the treatment of various diseases. To achieve such an innovative therapy, it will be necessary to deliver therapeutic agents into mitochondria. However, only a limited number of methods are available for accomplishing this. We previously developed the MITO-Porter, a liposome-based carrier that permits macromolecular cargos to be transported into mitochondria via membrane fusion. Intracellular observations using the green fluorescence protein as a model macromolecule confirmed the mitochondrial delivery of a macromolecule by the MITO-Porter. Moreover, when we attempted the mitochondrial delivery of bongkrekic acid (BKA), an antiapoptosis agent, the MITO-Porter enhanced the antiapoptosis effect compared with naked BKA. To construct a device with enhanced performance, the MITO-Porter was coated with cell membrane-fusogenic outer envelopes to produce the dual function (DF)-MITO-Porter. Intracellular observations indicated that the DF-MITO-Porter was more effective in delivering exogenous macromolecules into mitochondria than the conventional MITO-Porter. Furthermore, when biomacromolecules were delivered using the DF-MITO-Porter to estimate the mitochondrial gene targeting of the carrier, the results confirmed that the MITO-Porter system has the potential for use in therapies aimed at mitochondrial DNA. This paper sumarizes our findings on mitochondrial drug delivery systems that are directed toward mitochondrial medicine development and mitochondrial gene therapy. It is expected that the MITO-Porter system will open new research areas in mitochondrial drug delivery systems and have a significant impact on the medical and life sciences.

  5. Applying Fluorescence Correlation Spectroscopy to Investigate Peptide-Induced Membrane Disruption.

    PubMed

    Kristensen, Kasper; Henriksen, Jonas R; Andresen, Thomas L

    2017-01-01

    There is considerable interest in understanding the interactions of antimicrobial peptides with phospholipid membranes. Fluorescence correlation spectroscopy (FCS) is a powerful experimental technique that can be used to gain insight into these interactions. Specifically, FCS can be used to quantify leakage of fluorescent molecules of different sizes from large unilamellar lipid vesicles, thereby providing a tool for estimating the size of peptide-induced membrane disruptions. If fluorescently labeled lipids are incorporated into the membranes of the vesicles, FCS can also be used to obtain information about whether leakage occurs due to localized membrane perturbations or global membrane destabilization. Here, we outline a detailed step-by-step protocol on how to optimally implement an FCS-based leakage assay. To make the protocol easily accessible to other researchers, it has been supplemented with a number of practical tips and tricks.

  6. Protective action of methylglyoxal bis (guanylhydrazone) on the mitochondrial membrane.

    PubMed

    Toninello, A; Siliprandi, D; Castagnini, P; Novello, M C; Siliprandi, N

    1988-09-15

    At low concentrations (0.5-1.0 mM) methylglyoxal bis (guanylhydrazone) (MGBG) exhibited a clearcut protection of rat liver mitochondria against the deenergizing action of either Ca2+, or oxidizing agents (butylhydroperoxide and oxaloacetate). Such a protection resulted from the prevention of transmembrane potential decay, discharge of accumulated Ca2+, release of mitochondrial Mg2+, adenine nucleotides and pyridine nucleotides and mitochondrial swelling. At high concentrations (5-10 mM) MGBG induced functional alterations of mitochondria (decrease of transmembrane potential, lower capability to accumulate and to retain Ca2+) which can be reversed by resuspension of mitochondria in a MGBG free medium. These reversible mitochondrial alterations by high MGBG concentrations are interpreted as a consequence of an aggregation and coprecipitation of suspended mitochondria.

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

    PubMed Central

    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. PMID:26752045

  8. Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane.

    PubMed

    Zeczycki, Tonya N; Whelan, Jarrett; Hayden, William Tyler; Brown, David A; Shaikh, Saame Raza

    2014-07-18

    It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia-reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.

  9. KCl-Dependent Release of Mitochondrial Membrane-Bound Arginase Appears to Be a Novel Variant of Arginase-II

    PubMed Central

    Suman, Mishra; Rajnikant, Mishra

    2016-01-01

    Arginase regulates arginine metabolism, ornithine-urea cycle, and immunological surveillance. Arginase-I is predominant in cytosol, and arginase-II is localised in the mitochondria. A mitochondrial membrane-bound arginase has also been proposed to be adsorbed with outer membrane of mitochondria which gets released by 150 mM potassium chloride (KCl). It is presumed that inclusion of 150 mM KCl in the homogenization medium would not only facilitate release of arginase bound with outer membrane of mitochondria but also affect functional anatomy of mitochondria, mitochondrial enzymes, and proteins. Therefore, it has been intended to characterize KCl-dependent release of mitochondrial membrane-bound arginase from liver of mice. Results provide advancement in the area of arginase biology and suggest that fraction of mitochondrial membrane-bound arginase contains mitochondrial arginase-II and a variant of arginase-II. PMID:27293971

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

    PubMed

    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.

  11. Polypeptide and phospholipid composition of the membrane of rat liver peroxisomes: comparison with endoplasmic reticulum and mitochondrial membranes

    PubMed Central

    1982-01-01

    Membranes were isolated from highly purified peroxisomes, mitochondria, and rough and smooth microsomes of rat liver by the one-step Na2CO3 procedure described in the accompanying paper (1982, J. Cell Biol. 93:97-102). The polypeptide compositions of these membranes were determined by SDS PAGE and found to be greatly dissimilar. The peroxisomal membrane contains 12% of the peroxisomal protein and consists of three major polypeptides (21,700, 67,700 and 69,700 daltons) as well as some minor polypeptides. The major peroxisomal membrane proteins as well as most of the minor ones are absent from the endoplasmic reticulum (ER). Conversely, most ER proteins are absent from peroxisomes. By electron microscopy, purified peroxisomal membranes are approximately 6.8 nm thick and have a typical trilaminar appearance. The phospholipid/protein ratio of peroxisomal membranes is approximately 200 nmol/mg; the principal phospholipids are phosphatidyl choline and phosphatidyl ethanolamine as in ER and mitochondrial membranes. In contrast to the mitochondria, peroxisomal membranes contain no cardiolipin. All the membranes investigated contain a polypeptide band with a molecular mass of approximately 15,000 daltons. Whether this represents an exceptional common membrane protein or a coincidence is unknown. The implications of these results for the biogenesis of peroxisomes are discussed. PMID:7068748

  12. Mitochondrial Sirtuin Network Reveals Dynamic SIRT3-Dependent Deacetylation in Response to Membrane Depolarization.

    PubMed

    Yang, Wen; Nagasawa, Koji; Münch, Christian; Xu, Yingjie; Satterstrom, Kyle; Jeong, Seungmin; Hayes, Sebastian D; Jedrychowski, Mark P; Vyas, F Sejal; Zaganjor, Elma; Guarani, Virginia; Ringel, Alison E; Gygi, Steven P; Harper, J Wade; Haigis, Marcia C

    2016-11-03

    Mitochondrial sirtuins, SIRT3-5, are NAD(+)-dependent deacylases and ADP-ribosyltransferases that are critical for stress responses. However, a comprehensive understanding of sirtuin targets, regulation of sirtuin activity, and the relationships between sirtuins remains a key challenge in mitochondrial physiology. Here, we employ systematic interaction proteomics to elucidate the mitochondrial sirtuin protein interaction landscape. This work reveals sirtuin interactions with numerous functional modules within mitochondria, identifies candidate sirtuin substrates, and uncovers a fundamental role for sequestration of SIRT3 by ATP synthase in mitochondrial homeostasis. In healthy mitochondria, a pool of SIRT3 binds ATP synthase, but upon matrix pH reduction with concomitant loss of mitochondrial membrane potential, SIRT3 dissociates. This release correlates with rapid deacetylation of matrix proteins, and SIRT3 is required for recovery of membrane potential. In vitro reconstitution experiments, as well as analysis of CRISPR/Cas9-engineered cells, indicate that pH-dependent SIRT3 release requires H135 in the ATP5O subunit of ATP synthase. Our SIRT3-5 interaction network provides a framework for discovering novel biological functions regulated by mitochondrial sirtuins.

  13. A Rapid and Quantitative Flow Cytometry Method for the Analysis of Membrane Disruptive Antimicrobial Activity

    PubMed Central

    O’Brien-Simpson, Neil M.; Pantarat, Namfon; Attard, Troy J.; Walsh, Katrina A.; Reynolds, Eric C.

    2016-01-01

    We describe a microbial flow cytometry method that quantifies within 3 hours antimicrobial peptide (AMP) activity, termed Minimum Membrane Disruptive Concentration (MDC). Increasing peptide concentration positively correlates with the extent of bacterial membrane disruption and the calculated MDC is equivalent to its MBC. The activity of AMPs representing three different membranolytic modes of action could be determined for a range of Gram positive and negative bacteria, including the ESKAPE pathogens, E. coli and MRSA. By using the MDC50 concentration of the parent AMP, the method provides high-throughput, quantitative screening of AMP analogues. A unique feature of the MDC assay is that it directly measures peptide/bacteria interactions and lysed cell numbers rather than bacteria survival as with MIC and MBC assays. With the threat of multi-drug resistant bacteria, this high-throughput MDC assay has the potential to aid in the development of novel antimicrobials that target bacteria with improved efficacy. PMID:26986223

  14. Membrane Incorporation, Channel Formation, and Disruption of Calcium Homeostasis by Alzheimer's β-Amyloid Protein

    PubMed Central

    Kawahara, Masahiro; Ohtsuka, Isao; Yokoyama, Shoko; Kato-Negishi, Midori; Sadakane, Yutaka

    2011-01-01

    Oligomerization, conformational changes, and the consequent neurodegeneration of Alzheimer's β-amyloid protein (AβP) play crucial roles in the pathogenesis of Alzheimer's disease (AD). Mounting evidence suggests that oligomeric AβPs cause the disruption of calcium homeostasis, eventually leading to neuronal death. We have demonstrated that oligomeric AβPs directly incorporate into neuronal membranes, form cation-sensitive ion channels (“amyloid channels”), and cause the disruption of calcium homeostasis via the amyloid channels. Other disease-related amyloidogenic proteins, such as prion protein in prion diseases or α-synuclein in dementia with Lewy bodies, exhibit similarities in the incorporation into membranes and the formation of calcium-permeable channels. Here, based on our experimental results and those of numerous other studies, we review the current understanding of the direct binding of AβP into membrane surfaces and the formation of calcium-permeable channels. The implication of composition of membrane lipids and the possible development of new drugs by influencing membrane properties and attenuating amyloid channels for the treatment and prevention of AD is also discussed. PMID:21547225

  15. Human metapneumovirus glycoprotein G disrupts mitochondrial signaling in airway epithelial cells.

    PubMed

    Bao, Xiaoyong; Kolli, Deepthi; Ren, Junping; Liu, Tianshuang; Garofalo, Roberto P; Casola, Antonella

    2013-01-01

    Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.

  16. DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in C. elegans

    PubMed Central

    Lane, Rebecca K.; Borror, Megan B.; Bokov, Alex F.; Link, Christopher D.; Rea, Shane L.

    2016-01-01

    Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension. PMID:27420916

  17. DLK-1, SEK-3 and PMK-3 Are Required for the Life Extension Induced by Mitochondrial Bioenergetic Disruption in C. elegans.

    PubMed

    Munkácsy, Erin; Khan, Maruf H; Lane, Rebecca K; Borror, Megan B; Park, Jae H; Bokov, Alex F; Fisher, Alfred L; Link, Christopher D; Rea, Shane L

    2016-07-01

    Mitochondrial dysfunction underlies numerous age-related pathologies. In an effort to uncover how the detrimental effects of mitochondrial dysfunction might be alleviated, we examined how the nematode C. elegans not only adapts to disruption of the mitochondrial electron transport chain, but in many instances responds with extended lifespan. Studies have shown various retrograde responses are activated in these animals, including the well-studied ATFS-1-dependent mitochondrial unfolded protein response (UPRmt). Such processes fall under the greater rubric of cellular surveillance mechanisms. Here we identify a novel p38 signaling cascade that is required to extend life when the mitochondrial electron transport chain is disrupted in worms, and which is blocked by disruption of the Mitochondrial-associated Degradation (MAD) pathway. This novel cascade is defined by DLK-1 (MAP3K), SEK-3 (MAP2K), PMK-3 (MAPK) and the reporter gene Ptbb-6::GFP. Inhibition of known mitochondrial retrograde responses does not alter induction of Ptbb-6::GFP, instead induction of this reporter often occurs in counterpoint to activation of SKN-1, which we show is under the control of ATFS-1. In those mitochondrial bioenergetic mutants which activate Ptbb-6::GFP, we find that dlk-1, sek-3 and pmk-3 are all required for their life extension.

  18. Distinct Pathways Mediate the Sorting of Tail-anchored Mitochondrial Outer Membrane Proteins

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Little is known about the biogenesis of tail-anchored (TA) proteins localized to the mitochondrial outer membrane in plant cells. To address this issue, we screened all of the (>600) known and predicted TA proteins in Arabidopsis thaliana for those annotated, based on Gene Ontology, to possess mitoc...

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

    PubMed

    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.

  20. Synchronism in mitochondrial ROS flashes, membrane depolarization and calcium sparks in human carcinoma cells.

    PubMed

    Kuznetsov, Andrey V; Javadov, Sabzali; Saks, Valdur; Margreiter, Raimund; Grimm, Michael

    2017-03-06

    Mitochondria are major producers of reactive oxygen species (ROS) in many cells including cancer cells. However, complex interrelationships between mitochondrial ROS (mitoROS), mitochondrial membrane potential (ΔΨm) and Ca(2+) are not completely understood. Using human carcinoma cells, we further highlight biphasic ROS dynamics: - gradual mitoROS increase followed by mitoROS flash. Also, we demonstrate heterogeneity in rates of mitoROS generation and flash initiation time. Comparing mitochondrial and near-extra-mitochondrial signals, we show that mechanisms of mitoROS flashes in single mitochondria, linked to mitochondrial permeability transition pore opening (ΔΨm collapse) and calcium sparks, may involve flash triggering by certain levels of external ROS released from the same mitochondria. In addition, mitochondria-mitochondria interactions can produce wave propagations of mitoROS flashes and ΔΨm collapses in cancer cells similar to phenomena of ROS-induced ROS release (RIRR). Our data suggest that in cancer cells RIRR, activation of mitoROS flashes and mitochondrial depolarization may involve participation of extramitochondrial-ROS produced either by individual mitochondria and/or by neighboring mitochondria. This could represent general mechanisms in ROS-ROS signaling with suggested role in both mitochondrial and cellular physiology and signaling.

  1. Tuning the activity of mitochondria-penetrating peptides for delivery or disruption.

    PubMed

    Horton, Kristin L; Pereira, Mark P; Stewart, Kelly M; Fonseca, Sonali B; Kelley, Shana O

    2012-02-13

    Mitochondrially targeted agents have the capacity to be both vehicles for the delivery of bioactive agents and mitochondrial disrupters and show promise for the treatment of various diseases. Engineering these agents to specifically accumulate or disrupt the mitochondrion is challenging, as there is a fine line between characteristics of the molecules that accomplish each task. Here, we assess the physicochemical properties governing mitochondrial matrix accumulation or membrane disruption caused by mitochondria-penetrating peptides. Increases in peptide length and hydrophobicity were uncovered as the dominant factors in deriving membrane disruptive activity. Shorter, less hydrophobic peptides did not disrupt the mitochondrial membrane, but rather accumulated in the mitochondrial matrix without interfering with cellular activity. These shorter peptides, however, can trigger cytochrome c release through activation of the permeability transition pore complex (PTPC), but only at very high concentrations. This study illustrates that the activity of a mitochondria-localizing agent can be controlled through alterations in peptide hydrophobicity and dosing concentrations.

  2. Simulations of Membrane-Disrupting Peptides II: AMP Piscidin 1 Favors Surface Defects over Pores.

    PubMed

    Perrin, B Scott; Fu, Riqiang; Cotten, Myriam L; Pastor, Richard W

    2016-09-20

    Antimicrobial peptides (AMPs) that disrupt bacterial membranes are promising therapeutics against the growing number of antibiotic-resistant bacteria. The mechanism of membrane disruption by the AMP piscidin 1 was examined with multimicrosecond all-atom molecular dynamics simulations and solid-state NMR spectroscopy. The primary simulation was initialized with 20 peptides in four barrel-stave pores in a fully hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol bilayer. The four pores relaxed to toroidal by 200 ns, only one porelike structure containing two transmembrane helices remained at 26 μs, and none of the 18 peptides released to the surface reinserted to form pores. The simulation was repeated at 413 K with an applied electric field and all peptides were surface-bound by 200 ns. Trajectories of surface-bound piscidin with and without applied fields at 313 and 413 K and totaling 6 μs show transient distortions of the bilayer/water interface (consistent with (31)P NMR), but no insertion to transmembrane or pore states. (15)N chemical shifts confirm a fully surface-bound conformation. Taken together, the simulation and experimental results imply that transient defects rather than stable pores are responsible for membrane disruption by piscidin 1, and likely other AMPs.

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

    PubMed

    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.

  4. A nuclear restorer-of-fertility mutation disrupts accumulation of mitochondrial ATP synthase subunit alpha in developing pollen of S male-sterile maize.

    PubMed Central

    Wen, Lanying; Ruesch, Kimberly L; Ortega, Victor M; Kamps, Terry L; Gabay-Laughnan, Susan; Chase, Christine D

    2003-01-01

    Mitochondrial biogenesis and function depend upon the interaction of mitochondrial and nuclear genomes. Forward genetic analysis of mitochondrial function presents a challenge in organisms that are obligated to respire. In the S-cytoplasmic male sterility (CMS-S) system of maize, expression of mitochondrial open reading frames (orf355-orf77) conditions collapse of developing haploid pollen. Nuclear restorer-of-fertility mutations that circumvent pollen collapse are often homozygous lethal. These spontaneous mutations potentially result from disruption of nuclear genes required for mitochondrial gene expression, in contrast to homozygous-viable restorer-of-fertility alleles that function to block or compensate for the expression of mitochondrial CMS genes. Consistent with this hypothesis, the homozygous-lethal restoring allele historically designated RfIII was shown to be recessive in diploid pollen produced by tetraploid CMS-S plants. Accordingly, the symbol for this allele has been changed to restorer-of-fertility lethal 1 (rfl1). In haploid rfl1 pollen, orf355-orf77 transcripts and mitochondrial transcripts encoding the alpha-subunit of the ATP synthase (ATPA) were decreased in abundance. Haploid rfl1 pollen failed to accumulate wild-type levels of ATPA protein, indicating that functional requirements for mitochondrial protein accumulation are relaxed in maize pollen. The CMS-S system and rfl mutations therefore allow for the selection of nuclear mutations disrupting mitochondrial biogenesis in a multicellular eukaryote. PMID:14573487

  5. Bioavailability of endocrine disrupting chemicals (EDCs): Liposome-water partitioning and lipid membrane permeation

    NASA Astrophysics Data System (ADS)

    Kwon, Jung-Hwan

    The bioavailability of endocrine disrupting chemicals (EDCs) is a function of a number of parameters including the ability of the chemical to partition into organic tissue and reach receptor sites within an organism. In this dissertation, equilibrium partition coefficients between water and lipid membrane vesicles and artificial lipid membrane permeability were investigated for evaluating bioavailability of aqueous pollutants. Structurally diverse endocrine disrupting chemicals were chosen as model compounds for partitioning experiments and simple hydrophobic organic chemicals were used for the evaluation of a parallel artificial membrane device developed to mimic bioconcentration rates in fish. Hydrophobic interactions represented by octanol/water partition coefficients (KOWs) were not appropriate for estimating lipid membrane/water partition coefficients (Klipws) for the selected EDCs having a relatively large molar liquid volume (MLV) and containing polar functional groups. Correlations that include MLV and polar surface area (PSA) reduce the predicted value of log K lipw, suggesting that lipid membranes are less favorable than 1-octanol for a hydrophobic solute because of the changes in membrane fluidity and the amount of cholesterol in the lipid bilayers. These results suggested that KOW alone has limited potential for estimating K lipw, and MLV or PSA may be used as additional descriptors for developing quantitative structure-activity relationships (QSARs). The poor correlations between KOW and Klipw observed in this research may be due to the highly organized structure of lipid bilayers. Measured thermodynamic constants demonstrated that the entropy contribution becomes more dominant for more organized liposomes having saturated lipid tails. This implies that entropy-driven partitioning process makes Klipw different from KOW especially for more saturated lipid bilayer membranes. In the parallel artificial membrane system developed, a membrane filter

  6. Caspase cleavage of cytochrome c1 disrupts mitochondrial function and enhances cytochrome c release.

    PubMed

    Zhu, Yushan; Li, Min; Wang, Xiaohui; Jin, Haijing; Liu, Shusen; Xu, Jianxin; Chen, Quan

    2012-01-01

    Mitochondrial catastrophe can be the cause or consequence of apoptosis and is associated with a number of pathophysiological conditions. The exact relationship between mitochondrial catastrophe and caspase activation is not completely understood. Here we addressed the underlying mechanism, explaining how activated caspase could feedback to attack mitochondria to amplify further cytochrome c (cyto.c) release. We discovered that cytochrome c1 (cyto.c1) in the bc1 complex of the mitochondrial respiration chain was a novel substrate of caspase 3 (casp.3). We found that cyto.c1 was cleaved at the site of D106, which is critical for binding with cyto.c, following apoptotic stresses or targeted expression of casp.3 into the mitochondrial intermembrane space. We demonstrated that this cleavage was closely linked with further cyto.c release and mitochondrial catastrophe. These mitochondrial events could be effectively blocked by expressing non-cleavable cyto.c1 (D106A) or by caspase inhibitor z-VAD-fmk. Our results demonstrate that the cleavage of cyto.c1 represents a critical step for the feedback amplification of cyto.c release by caspases and subsequent mitochondrial catastrophe.

  7. Mitochondrial metabolic states and membrane potential modulate mtNOS activity.

    PubMed

    Valdez, Laura B; Zaobornyj, Tamara; Boveris, Alberto

    2006-03-01

    The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.

  8. Definition of the mitochondrial proteome by measurement of molecular masses of membrane proteins.

    PubMed

    Carroll, Joe; Fearnley, Ian M; Walker, John E

    2006-10-31

    The covalent structure of a protein is incompletely defined by its gene sequence, and mass spectrometric analysis of the intact protein is needed to detect the presence of any posttranslational modifications. Because most membrane proteins are purified in detergents that are incompatible with mass spectrometric ionization techniques, this essential measurement has not been made on many hydrophobic proteins, and so proteomic data are incomplete. We have extracted membrane proteins from bovine mitochondria and detergent-purified NADH:ubiquinone oxidoreductase (complex I) with organic solvents, fractionated the mixtures by hydrophilic interaction chromatography, and measured the molecular masses of the intact membrane proteins, including those of six subunits of complex I that are encoded in mitochondrial DNA. These measurements resolve long-standing uncertainties about the interpretation of the mitochondrial genome, and they contribute significantly to the definition of the covalent composition of complex I.

  9. Profiling of the Tox21 Chemical Collection for Mitochondrial Function to Identify Compounds that Acutely Decrease Mitochondrial Membrane Potential

    PubMed Central

    Attene-Ramos, Matias S.; Huang, Ruili; Michael, Sam; Witt, Kristine L.; Richard, Ann; Tice, Raymond R.; Simeonov, Anton; Austin, Christopher P.

    2014-01-01

    Background: Mitochondrial dysfunction has been implicated in the pathogenesis of a variety of disorders including cancer, diabetes, and neurodegenerative and cardiovascular diseases. Understanding whether different environmental chemicals and druglike molecules impact mitochondrial function represents an initial step in predicting exposure-related toxicity and defining a possible role for such compounds in the onset of various diseases. Objectives: We sought to identify individual chemicals and general structural features associated with changes in mitochondrial membrane potential (MMP). Methods: We used a multiplexed [two end points in one screen; MMP and adenosine triphosphate (ATP) content] quantitative high throughput screening (qHTS) approach combined with informatics tools to screen the Tox21 library of 10,000 compounds (~ 8,300 unique chemicals) at 15 concentrations each in triplicate to identify chemicals and structural features that are associated with changes in MMP in HepG2 cells. Results: Approximately 11% of the compounds (913 unique compounds) decreased MMP after 1 hr of treatment without affecting cell viability (ATP content). In addition, 309 compounds decreased MMP over a concentration range that also produced measurable cytotoxicity [half maximal inhibitory concentration (IC50) in MMP assay/IC50 in viability assay ≤ 3; p < 0.05]. More than 11% of the structural clusters that constitute the Tox21 library (76 of 651 clusters) were significantly enriched for compounds that decreased the MMP. Conclusions: Our multiplexed qHTS approach allowed us to generate a robust and reliable data set to evaluate the ability of thousands of drugs and environmental compounds to decrease MMP. The use of structure-based clustering analysis allowed us to identify molecular features that are likely responsible for the observed activity. Citation: Attene-Ramos MS, Huang R, Michael S, Witt KL, Richard A, Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox

  10. Making heads or tails of mitochondrial membranes in longevity and aging: a role for comparative studies

    PubMed Central

    2014-01-01

    Mitochondria play vital roles in metabolic energy transduction, intermediate molecule metabolism, metal ion homeostasis, programmed cell death and regulation of the production of reactive oxygen species. As a result of their broad range of functions, mitochondria have been strongly implicated in aging and longevity. Numerous studies show that aging and decreased lifespan are also associated with high reactive oxygen species production by mitochondria, increased mitochondrial DNA and protein damage, and with changes in the fatty acid composition of mitochondrial membranes. It is possible that the extent of fatty acid unsaturation of the mitochondrial membrane determines susceptibility to lipid oxidative damage and downstream protein and genome toxicity, thereby acting as a determinant of aging and lifespan. Reviewing the vast number of comparative studies on mitochondrial membrane composition, metabolism and lifespan reveals some evidence that lipid unsaturation ratios may correlate with lifespan. However, we caution against simply relating these two traits. They may be correlative but have no functional relation. We discuss an important methodology for body mass and phylogenetic correction in comparative studies. PMID:24588808

  11. Arabidopsis chloroplast lipid transport protein TGD2 disrupts membranes and is part of a large complex.

    PubMed

    Roston, Rebecca; Gao, Jinpeng; Xu, Changcheng; Benning, Christoph

    2011-06-01

    In most plants the assembly of the photosynthetic thylakoid membrane requires lipid precursors synthesized at the endoplasmic reticulum (ER). Thus, the transport of lipids from the ER to the chloroplast is essential for biogenesis of the thylakoids. TGD2 is one of four proteins in Arabidopsis required for lipid import into the chloroplast, and was found to bind phosphatidic acid in vitro. However, the significance of phosphatidic acid binding for the function of TGD2 in vivo and TGD2 interaction with membranes remained unclear. Developing three functional assays probing how TGD2 affects lipid bilayers in vitro, we show that it perturbs membranes to the point of fusion, causes liposome leakage and redistributes lipids in the bilayer. By identifying and characterizing five new mutant alleles, we demonstrate that these functions are impaired in specific mutants with lipid phenotypes in vivo. At the structural level, we show that TGD2 is part of a protein complex larger than 500 kDa, the formation of which is disrupted in two mutant alleles, indicative of the biological relevance of this TGD2-containing complex. Based on the data presented, we propose that TGD2, as part of a larger complex, forms a lipid transport conduit between the inner and outer chloroplast envelope membranes, with its N terminus anchored in the inner membrane and its C terminus binding phosphatidic acid in the outer membrane.

  12. Disruption of Renal Tubular Mitochondrial Quality Control by Myo-Inositol Oxygenase in Diabetic Kidney Disease

    PubMed Central

    Zhan, Ming; Usman, Irtaza M.; Sun, Lin

    2015-01-01

    Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2–Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD. PMID:25270067

  13. Loss of Drp1 function alters OPA1 processing and changes mitochondrial membrane organization

    SciTech Connect

    Moepert, Kristin; Hajek, Petr; Frank, Stephan; Chen, Christiane; Kaufmann, Joerg; Santel, Ansgar

    2009-08-01

    RNAi mediated loss of Drp1 function changes mitochondrial morphology in cultured HeLa and HUVEC cells by shifting the balance of mitochondrial fission and fusion towards unopposed fusion. Over time, inhibition of Drp1 expression results in the formation of a highly branched mitochondrial network along with 'bulge'-like structures. These changes in mitochondrial morphology are accompanied by a reduction in levels of Mitofusin 1 (Mfn1) and 2 (Mfn2) and a modified proteolytic processing of OPA1 isoforms, resulting in the inhibition of cell proliferation. In addition, our data imply that bulge formation is driven by Mfn1 action along with particular proteolytic short-OPA1 (s-OPA1) variants: Loss of Mfn2 in the absence of Drp1 results in an increase of Mfn1 levels along with processed s-OPA1-isoforms, thereby enhancing continuous 'fusion' and bulge formation. Moreover, bulge formation might reflect s-OPA1 mitochondrial membrane remodeling activity, resulting in the compartmentalization of cytochrome c deposits. The proteins Yme1L and PHB2 appeared not associated with the observed enhanced OPA1 proteolysis upon RNAi of Drp1, suggesting the existence of other OPA1 processing controlling proteins. Taken together, Drp1 appears to affect the activity of the mitochondrial fusion machinery by unbalancing the protein levels of mitofusins and OPA1.

  14. Radiation inactivation method provides evidence that membrane-bound mitochondrial creatine kinase is an oligomer

    SciTech Connect

    Quemeneur, E.; Eichenberger, D.; Goldschmidt, D.; Vial, C.; Beauregard, G.; Potier, M.

    1988-06-30

    Lyophilized suspensions of rabbit heart mitochondria have been irradiated with varying doses of gamma rays. Mitochondrial creatine kinase activity was inactivated exponentially with a radiation inactivation size of 352 or 377 kDa depending upon the initial medium. These values are in good agreement with the molecular mass previously deduced from by permeation experiments: 357 kDa. This is the first direct evidence showing that the native form of mitochondrial creatine kinase is associated to the inner membrane as an oligomer, very likely an octamer.

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

    PubMed Central

    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

  16. Function of the mitochondrial outer membrane as a diffusion barrier in health and diseases.

    PubMed

    Gellerich, F N; Trumbeckaite, S; Opalka, J R; Seppet, E; Rasmussen, H N; Neuhoff, C; Zierz, S

    2000-02-01

    The mitochondrial outer membrane separates the intermembrane space from the cytosol. The whole exchange of metabolites, cations and information between mitochondria and the cell occurs through the outer membrane. Experimental evidence is reviewed supporting the hypothesis of dynamic ADP compartmentation within the intermembrane space. The outer membrane creates a diffusion barrier for small molecules (adenine nucleotides, creatine phosphate, creatine etc.) causing rate-dependent concentration gradients as a prerequisite for the action of ADP shuttles via creatine kinases or adenylate kinases. If the outer membrane becomes leaky, cytochrome c and apoptosis-inducing factor can be released, leading to apoptosis, and as a bioenergetic consequence the cytosolic phosphorylation potential decreases. Leaky outer membranes can be detected in saponin-skinned fibres with spectrophotometric and oxygraphic methods. This is of special interest in respect to acute impairment of mitochondria during ischaemia/reperfusion.

  17. Superresolution Fluorescence Imaging of Mitochondrial Nucleoids Reveals Their Spatial Range, Limits, and Membrane Interaction ▿ †

    PubMed Central

    Brown, Timothy A.; Tkachuk, Ariana N.; Shtengel, Gleb; Kopek, Benjamin G.; Bogenhagen, Daniel F.; Hess, Harald F.; Clayton, David A.

    2011-01-01

    A fundamental objective in molecular biology is to understand how DNA is organized in concert with various proteins, RNA, and biological membranes. Mitochondria maintain and express their own DNA (mtDNA), which is arranged within structures called nucleoids. Their functions, dimensions, composition, and precise locations relative to other mitochondrial structures are poorly defined. Superresolution fluorescence microscopy techniques that exceed the previous limits of imaging within the small and highly compartmentalized mitochondria have been recently developed. We have improved and employed both two- and three-dimensional applications of photoactivated localization microscopy (PALM and iPALM, respectively) to visualize the core dimensions and relative locations of mitochondrial nucleoids at an unprecedented resolution. PALM reveals that nucleoids differ greatly in size and shape. Three-dimensional volumetric analysis indicates that, on average, the mtDNA within ellipsoidal nucleoids is extraordinarily condensed. Two-color PALM shows that the freely diffusible mitochondrial matrix protein is largely excluded from the nucleoid. In contrast, nucleoids are closely associated with the inner membrane and often appear to be wrapped around cristae or crista-like inner membrane invaginations. Determinations revealing high packing density, separation from the matrix, and tight association with the inner membrane underscore the role of mechanisms that regulate access to mtDNA and that remain largely unknown. PMID:22006021

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

    PubMed

    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.

  19. Disruption of the mitochondrial thioredoxin system as a cell death mechanism of cationic triphenylmethanes.

    PubMed

    Zhang, Xu; Zheng, Yujuan; Fried, Levi E; Du, Yatao; Montano, Sergio J; Sohn, Allie; Lefkove, Benjamin; Holmgren, Lars; Arbiser, Jack L; Holmgren, Arne; Lu, Jun

    2011-04-01

    Alterations in mitochondrial structure and function are a hallmark of cancer cells compared to normal cells and thus targeting mitochondria has emerged as an novel approach to cancer therapy. The mitochondrial thioredoxin 2 (Trx2) system is critical for cell viability, but its role in cancer biology is not well understood. Recently some cationic triphenylmethanes such as brilliant green (BG) and gentian violet were shown to have antitumor and antiangiogenic activity with unknown mechanisms. Here we demonstrate that BG killed cells at nanomolar concentrations and targeted mitochondrial Trx2, which was oxidized and degraded. HeLa cells were more sensitive to BG than fibroblasts. In HeLa cells, Trx2 down-regulation by siRNA resulted in increased sensitivity to BG, whereas for fibroblasts, the same treatments had no effect. BG was observed to accumulate in mitochondria and cause a rapid and dramatic decrease in mitochondrial Trx2 protein. With a redox Western blot method, we found that treatment with BG caused oxidation of both Trx1 and Trx2, followed by release of cytochrome c and apoptosis-inducing factor from the mitochondria into the cytosol. Moreover, this treatment resulted in an elevation of the mRNA level of Lon protease, a protein quality control enzyme in the mitochondrial matrix, suggesting that the oxidized Trx2 may be degraded by Lon protease.

  20. Disrupted mitochondrial function in the Opa3L122P mouse model for Costeff Syndrome impairs skeletal integrity

    PubMed Central

    Navein, Alice E.; Cooke, Esther J.; Davies, Jennifer R.; Smith, Terence G.; Wells, Lois H. M.; Ohazama, Atsushi; Healy, Christopher; Sharpe, Paul T.; Evans, Sam L.; Evans, Bronwen A. J.; Votruba, Marcela; Wells, Timothy

    2016-01-01

    Mitochondrial dysfunction connects metabolic disturbance with numerous pathologies, but the significance of mitochondrial activity in bone remains unclear. We have, therefore, characterized the skeletal phenotype in the Opa3L122P mouse model for Costeff syndrome, in which a missense mutation of the mitochondrial membrane protein, Opa3, impairs mitochondrial activity resulting in visual and metabolic dysfunction. Although widely expressed in the developing normal mouse head, Opa3 expression was restricted after E14.5 to the retina, brain, teeth and mandibular bone. Opa3 was also expressed in adult tibiae, including at the trabecular surfaces and in cortical osteocytes, epiphyseal chondrocytes, marrow adipocytes and mesenchymal stem cell rosettes. Opa3L122P mice displayed craniofacial abnormalities, including undergrowth of the lower mandible, accompanied in some individuals by cranial asymmetry and incisor malocclusion. Opa3L122P mice showed an 8-fold elevation in tibial marrow adiposity, due largely to increased adipogenesis. In addition, femoral length and cortical diameter and wall thickness were reduced, the weakening of the calcified tissue and the geometric component of strength reducing overall cortical strength in Opa3L122P mice by 65%. In lumbar vertebrae reduced vertebral body area and wall thickness were accompanied by a proportionate reduction in marrow adiposity. Although the total biomechanical strength of lumbar vertebrae was reduced by 35%, the strength of the calcified tissue (σmax) was proportionate to a 38% increase in trabecular number. Thus, mitochondrial function is important for the development and maintenance of skeletal integrity, impaired bone growth and strength, particularly in limb bones, representing a significant new feature of the Costeff syndrome phenotype. PMID:27106103

  1. Structural and functional alterations in mitochondrial membrane in picrotoxin-induced epileptic rat brain.

    PubMed

    Acharya, Munjal M; Katyare, Surendra S

    2005-03-01

    Mitochondrial function is a key determinant of both excitability and viability of neurons. Present studies were carried out to decipher cerebral mitochondrial oxidative energy metabolism and membrane function in the chronic condition of generalized seizures induced by picrotoxin (PTX) in rats. PTX-induced convulsions resulted in decreased respiration rates (14-41%) with glutamate, pyruvate + malate, and succinate as substrate. The ADP phosphorylation rates were drastically reduced by 44-65%. An opposite trend was observed with ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine [corrected] (TMPD) as substrate. In general, uncoupling of the mitochondrial electron transport was observed after PTX treatment. Malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities were decreased by 20-80%; also, there was significant reduction in cytochrome b content after PTX treatment, while the F(o)F(1) ATPase (complex V) activity increased in basal and 2,4-dinitrophenol (DNP)-stimulated condition, indicating increased membrane fragility. The substrate kinetics analysis had shown that K(m) and V(max) of the higher affinity kinetic component of ATPase increased significantly by 1.2- to 1.4-fold in epileptic condition. Temperature kinetic analysis revealed 1.2-fold increase in energies of activation with decreased transition temperature. The total phospholipid (TPL) and cholesterol (CHL) contents decreased significantly with lowering of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylserine (PS), while lysophospholipid (lyso), sphingomyelin (SPM), and phosphatidylcholine components were found to be elevated. Brain mitochondrial membrane was somewhat more fluidized in epileptic animals. Possible consequences of mitochondrial respiratory chain (MRC) dysfunction are discussed. In conclusion, impairment of MRC function along with structural alterations suggests novel pathophysiological mechanisms important for

  2. Blood-Brain Barrier Disruption and Neurovascular Unit Dysfunction in Diabetic Mice: Protection with the Mitochondrial Carbonic Anhydrase Inhibitor Topiramate.

    PubMed

    Salameh, Therese S; Shah, Gul N; Price, Tulin O; Hayden, Melvin R; Banks, William A

    2016-12-01

    All forms of diabetes mellitus are characterized by chronic hyperglycemia, resulting in the development of a number of microvascular and macrovascular pathologies. Diabetes is also associated with changes in brain microvasculature, leading to dysfunction and ultimately disruption of the blood-brain barrier (BBB). These changes are correlated with a decline in cognitive function. In diabetes, BBB damage is associated with increased oxidative stress and reactive oxygen species. This occurs because of the increased oxidative metabolism of glucose caused by hyperglycemia. Decreasing the production of bicarbonate with the use of a mitochondrial carbonic anhydrase inhibitor (mCAi) limits oxidative metabolism and the production of reactive oxygen species. In this study, we have demonstrated that 1) streptozotocin-induced diabetes resulted in BBB disruption, 2) ultrastructural studies showed a breakdown of the BBB and changes to the neurovascular unit (NVU), including a loss of brain pericytes and retraction of astrocytes, the two cell types that maintain the BBB, and 3) treatment with topiramate, a mCAi, attenuated the effects of diabetes on BBB disruption and ultrastructural changes in the neurovascular unit.

  3. MitoQ regulates autophagy by inducing a pseudo-mitochondrial membrane potential.

    PubMed

    Sun, Chao; Liu, Xiongxiong; Di, Cuixia; Wang, Zhenhua; Mi, Xiangquan; Liu, Yang; Zhao, Qiuyue; Mao, Aihong; Chen, Weiqiang; Gan, Lu; Zhang, Hong

    2017-04-03

    During the process of oxidative phosphorylation, protons are pumped into the mitochondrial intermembrane space to establish a mitochondrial membrane potential (MMP). The electrochemical gradient generated allows protons to return to the matrix through the ATP synthase complex and generates ATP in the process. MitoQ is a lipophilic cationic drug that is adsorbed to the inner mitochondrial membrane; however, the cationic moiety of MitoQ remains in the intermembrane space. We found that the positive charges in MitoQ inhibited the activity of respiratory chain complexes I, III, and IV, reduced proton production, and decreased oxygen consumption. Therefore, a pseudo-MMP (PMMP) was formed via maintenance of exogenous positive charges. Proton backflow was severely impaired, leading to a decrease in ATP production and an increase in AMP production. Excess AMP activates AMP kinase, which inhibits the MTOR (mechanistic target of rapamycin) pathway and induces macroautophagy/autophagy. Therefore, we conclude that MitoQ increases PMMP via proton displacement with exogenous positive charges. In addition, PMMP triggered autophagy in hepatocellular carcinoma HepG2 cells via modification of mitochondrial bioenergetics pathways.

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

    PubMed

    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.

  5. The anti-cancer agent guttiferone-A permeabilizes mitochondrial membrane: Ensuing energetic and oxidative stress implications

    SciTech Connect

    Pardo-Andreu, Gilberto L.; Tudella, Valeria G.

    2011-06-15

    Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with cytotoxic action in vitro and anti-tumor action in rodent models. We addressed a potential involvement of mitochondria in GA toxicity (1-25 {mu}M) toward cancer cells by employing both hepatic carcinoma (HepG2) cells and succinate-energized mitochondria, isolated from rat liver. In HepG2 cells GA decreased viability, dissipated mitochondrial membrane potential, depleted ATP and increased reactive oxygen species (ROS) levels. In isolated rat-liver mitochondria GA promoted membrane fluidity increase, cyclosporine A/EGTA-insensitive membrane permeabilization, uncoupling (membrane potential dissipation/state 4 respiration rate increase), Ca{sup 2+} efflux, ATP depletion, NAD(P)H depletion/oxidation and ROS levels increase. All effects in cells, except mitochondrial membrane potential dissipation, as well as NADPH depletion/oxidation and permeabilization in isolated mitochondria, were partly prevented by the a NAD(P)H regenerating substrate isocitrate. The results suggest the following sequence of events: 1) GA interaction with mitochondrial membrane promoting its permeabilization; 2) mitochondrial membrane potential dissipation; 3) NAD(P)H oxidation/depletion due to inability of membrane potential-sensitive NADP{sup +} transhydrogenase of sustaining its reduced state; 4) ROS accumulation inside mitochondria and cells; 5) additional mitochondrial membrane permeabilization due to ROS; and 6) ATP depletion. These GA actions are potentially implicated in the well-documented anti-cancer property of GA/structure related compounds. - Graphical abstract: Guttiferone-A permeabilizes mitochondrial membrane and induces cancer cell death Display Omitted Highlights: > We addressed the involvement of mitochondria in guttiferone (GA) toxicity toward cancer cells. > GA promoted membrane permeabilization, membrane potential dissipation, NAD(P)H depletion, ROS accumulation and ATP depletion. > These actions

  6. Apricot Melanoidins Prevent Oxidative Endothelial Cell Death by Counteracting Mitochondrial Oxidation and Membrane Depolarization

    PubMed Central

    Giordo, Roberta; Emanueli, Costanza; Sanguinetti, Anna Maria; Piscopo, Amalia; Poiana, Marco; Capobianco, Giampiero; Piga, Antonio; Pintus, Gianfranco

    2012-01-01

    The cardiovascular benefits associated with diets rich in fruit and vegetables are thought to be due to phytochemicals contained in fresh plant material. However, whether processed plant foods provide the same benefits as unprocessed ones is an open question. Melanoidins from heat-processed apricots were isolated and their presence confirmed by colorimetric analysis and browning index. Oxidative injury of endothelial cells (ECs) is the key step for the onset and progression of cardiovascular diseases (CVD), therefore the potential protective effect of apricot melanoidins on hydrogen peroxide-induced oxidative mitochondrial damage and cell death was explored in human ECs. The redox state of cytoplasmic and mitochondrial compartments was detected by using the redox-sensitive, fluorescent protein (roGFP), while the mitochondrial membrane potential (MMP) was assessed with the fluorescent dye, JC-1. ECs exposure to hydrogen peroxide, dose-dependently induced mitochondrial and cytoplasmic oxidation. Additionally detected hydrogen peroxide-induced phenomena were MMP dissipation and ECs death. Pretreatment of ECs with apricot melanoidins, significantly counteracted and ultimately abolished hydrogen peroxide-induced intracellular oxidation, mitochondrial depolarization and cell death. In this regard, our current results clearly indicate that melanoidins derived from heat-processed apricots, protect human ECs against oxidative stress. PMID:23144984

  7. Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane.

    PubMed

    Kushnareva, Y E; Sokolove, P M

    2000-04-15

    Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5

  8. Large Plasma Membrane Disruptions Are Rapidly Resealed by Ca2+-dependent Vesicle–Vesicle Fusion Events

    PubMed Central

    Terasaki, Mark; Miyake, Katsuya; McNeil, Paul L.

    1997-01-01

    A microneedle puncture of the fibroblast or sea urchin egg surface rapidly evokes a localized exocytotic reaction that may be required for the rapid resealing that follows this breach in plasma membrane integrity (Steinhardt, R.A,. G. Bi, and J.M. Alderton. 1994. Science (Wash. DC). 263:390–393). How this exocytotic reaction facilitates the resealing process is unknown. We found that starfish oocytes and sea urchin eggs rapidly reseal much larger disruptions than those produced with a microneedle. When an ∼40 by 10 μm surface patch was torn off, entry of fluorescein stachyose (FS; 1,000 mol wt) or fluorescein dextran (FDx; 10,000 mol wt) from extracellular sea water (SW) was not detected by confocal microscopy. Moreover, only a brief (∼5–10 s) rise in cytosolic Ca2+ was detected at the wound site. Several lines of evidence indicate that intracellular membranes are the primary source of the membrane recruited for this massive resealing event. When we injected FS-containing SW deep into the cells, a vesicle formed immediately, entrapping within its confines most of the FS. DiI staining and EM confirmed that the barrier delimiting injected SW was a membrane bilayer. The threshold for vesicle formation was ∼3 mM Ca2+ (SW is ∼10 mM Ca2+). The capacity of intracellular membranes for sealing off SW was further demonstrated by extruding egg cytoplasm from a micropipet into SW. A boundary immediately formed around such cytoplasm, entrapping FDx or FS dissolved in it. This entrapment did not occur in Ca2+-free SW (CFSW). When egg cytoplasm stratified by centrifugation was exposed to SW, only the yolk platelet–rich domain formed a membrane, suggesting that the yolk platelet is a critical element in this response and that the ER is not required. We propose that plasma membrane disruption evokes Ca2+ regulated vesicle–vesicle (including endocytic compartments but possibly excluding ER) fusion reactions. The function in resealing of this cytoplasmic fusion

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

    PubMed

    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

  10. Toxicity and Loss of Mitochondrial Membrane Potential Induced by Alkyl Gallates in Trypanosoma cruzi

    PubMed Central

    Andréo, Rogério; Regasini, Luís Octávio; Petrônio, Maicon Segalla; Chiari-Andréo, Bruna Galdorfini; Tansini, Aline; Silva, Dulce Helena Siqueira; Cicarelli, Regina Maria Barretto

    2015-01-01

    American trypanosomiasis or Chagas disease is a debilitating disease representing an important social problem that affects, approximately, 10 million people in the world. The main aggravating factor of this situation is the lack of an effective drug to treat the different stages of this disease. In this context, the search for trypanocidal substances isolated from plants, synthetic or semi synthetic molecules, is an important strategy. Here, the trypanocidal potential of gallates was assayed in epimastigotes forms of T. cruzi and also, the interference of these substances on the mitochondrial membrane potential of the parasites was assessed, allowing the study of the mechanism of action of the gallates in the T. cruzi organisms. Regarding the preliminary structure-activity relationships, the side chain length of gallates plays crucial role for activity. Nonyl, decyl, undecyl, and dodecyl gallates showed potent antitrypanosomal effect (IC50 from 1.46 to 2.90 μM) in contrast with benznidazole (IC50 = 34.0 μM). Heptyl gallate showed a strong synergistic activity with benznidazole, reducing by 105-fold the IC50 of benznidazole. Loss of mitochondrial membrane potential induced by these esters was revealed. Tetradecyl gallate induced a loss of 53% of the mitochondrial membrane potential, at IC50 value. PMID:27347554

  11. Two distinct membrane potential-dependent steps drive mitochondrial matrix protein translocation.

    PubMed

    Schendzielorz, Alexander Benjamin; Schulz, Christian; Lytovchenko, Oleksandr; Clancy, Anne; Guiard, Bernard; Ieva, Raffaele; van der Laan, Martin; Rehling, Peter

    2017-01-02

    Two driving forces energize precursor translocation across the inner mitochondrial membrane. Although the membrane potential (Δψ) is considered to drive translocation of positively charged presequences through the TIM23 complex (presequence translocase), the activity of the Hsp70-powered import motor is crucial for the translocation of the mature protein portion into the matrix. In this study, we show that mitochondrial matrix proteins display surprisingly different dependencies on the Δψ. However, a precursor's hypersensitivity to a reduction of the Δψ is not linked to the respective presequence, but rather to the mature portion of the polypeptide chain. The presequence translocase constituent Pam17 is specifically recruited by the receptor Tim50 to promote the transport of hypersensitive precursors into the matrix. Our analyses show that two distinct Δψ-driven translocation steps energize precursor passage across the inner mitochondrial membrane. The Δψ- and Pam17-dependent import step identified in this study is positioned between the two known energy-dependent steps: Δψ-driven presequence translocation and adenosine triphosphate-driven import motor activity.

  12. The mechanisms of fatty acid-induced proton permeability of the inner mitochondrial membrane.

    PubMed

    Wojtczak, L; Wieckowski, M R

    1999-10-01

    Nonesterified long-chain fatty acids have long been known as uncouplers of oxidative phosphorylation. They are efficient protonophores in the inner mitochondrial membrane but not so in artificial phospholipid membranes. In the un-ionized form, they undergo a rapid spontaneous transbilayer movement (flip-flop). However, the transbilayer passage of the dissociated (anionic) form is hindered by the negatively charged hydrophilic carboxylic group. In the inner mitochondrial membrane, the transfer of fatty acid anions is mediated by the adenine nucleotide translocase, the dicarboxylate carrier, and the glutamate/aspartate carrier. As a result, the passage of protons and electric charges is a concerted effect of the spontaneous flip-flop of the undissociated (protonated) form in one direction and carrier-facilitated transfer of the ionized (deprotonated) form in the other direction. In addition, fatty acids also promote opening of the mitochondrial permeability transition pore, presumably due to their interaction with one of its constituents, the adenine nucleotide translocase, thus forming an additional route for dissipation of the proton gradient. Structural prerequisites for these proton-conducting mechanisms are (1) a weakly ionized carboxylic group and (2) a hydrocarbon chain of appropriate length without substituents limiting its mobility and hydrophobicity.

  13. Mitochondrial Outer Membrane Proteins Assist Bid in Bax-mediated Lipidic Pore Formation

    PubMed Central

    Schafer, Blanca; Quispe, Joel; Choudhary, Vineet; Chipuk, Jerry E.; Ajero, Teddy G.; Du, Han; Schneiter, Roger

    2009-01-01

    Mitochondrial outer membrane permeabilization (MOMP) is a critical step in apoptosis and is regulated by Bcl-2 family proteins. In vitro systems using cardiolipin-containing liposomes have demonstrated the key features of MOMP induced by Bax and cleaved Bid; however, the nature of the “pores” and how they are formed remain obscure. We found that mitochondrial outer membranes contained very little cardiolipin, far less than that required for liposome permeabilization, despite their responsiveness to Bcl-2 family proteins. Strikingly, the incorporation of isolated mitochondrial outer membrane (MOM) proteins into liposomes lacking cardiolipin conferred responsiveness to cleaved Bid and Bax. Cardiolipin dependence was observed only when permeabilization was induced with cleaved Bid but not with Bid or Bim BH3 peptide or oligomerized Bax. Therefore, we conclude that MOM proteins specifically assist cleaved Bid in Bax-mediated permeabilization. Cryoelectron microscopy of cardiolipin-liposomes revealed that cleaved Bid and Bax produced large round holes with diameters of 25–100 nm, suggestive of lipidic pores. In sum, we propose that activated Bax induces lipidic pore formation and that MOM proteins assist cleaved Bid in this process in the absence of cardiolipin. PMID:19244344

  14. Fluctuations of the proton-electromotive force across the inner mitochondrial membrane

    NASA Astrophysics Data System (ADS)

    Procopio, Joaquim; Fornés, José A.

    1997-05-01

    The intermembrane mitochondrial space (IMMS) is delimited by the inner and outer mitochondrial membranes and defines a region of molecular dimension where fluctuations of the number of free protons and of transmembrane voltage can give rise to fluctuations in the proton-electromotive force EPMF across the inner mitochondrial membrane (IMM). We have applied the fluctuation-dissipation theorem to an electrical equivalent circuit consisting of a resistor Rm in parallel with a capacitor Cm representing the passive electrical properties of the IMM, in series with another capacitor Cb representing the proton-buffering power of the IMMS fluid. An access resistance Ra was defined as a link between the capacitor Cb and the membrane. Average EPMF fluctuations across the IMM were calculated for different assumptions concerning the intermembrane space dimensions. The calculated average EPMF fluctuations were in the vicinity of 100 mV for relaxation times in the few-microseconds range. The corresponding fluctuational protonic free energy is about 10 kJ/mole, which is comparable to the binding energy for protons in different transporters. This suggests that fluctuations in EPMF can be of relevance in the universe of forces influencing the molecular machinery embedded in the IMM.

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  18. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization.

    PubMed Central

    Rodrigues, C. M.; Solá, S.; Silva, R.; Brites, D.

    2000-01-01

    BACKGROUND: The pathogenesis of bilirubin encephalopathy and Alzheimer's disease appears to result from accumulation of unconjugated bilirubin (UCB) and amyloid-beta (Abeta) peptide, respectively, which may cause apoptosis. Permeabilization of the mitochondrial membrane, with release of intermembrane proteins, has been strongly implicated in cell death. Inhibition of the mitochondrial permeability is one pathway by which ursodeoxycholate (UDC) and tauroursodeoxycholate (TUDC) protect against apoptosis in hepatic and nonhepatic cells. In this study, we further characterize UCB- and Abeta-induced cytotoxicty in isolated neural cells, and investigate membrane perturbation during incubation of isolated mitochondria with both agents. In addition, we evaluate whether the anti-apoptotic drugs UDC and TUDC prevent any changes from occurring. MATERIALS AND METHODS: Primary rat neuron and astrocyte cultures were incubated with UCB or Abeta peptide, either alone or in the presence of UDC. Apoptosis was assessed by DNA fragmentation and nuclear morphological changes. Isolated mitochondria were treated with each toxic, either alone or in combination with UDC, TUDC, or cyclosporine A. Mitochondrial swelling was measured spectrophotometrically and cytochrome c protein levels determined by Western blot. RESULTS: Incubation of neural cells with both UCB and Abeta induced apoptosis (p < 0.01). Coincubation with UDC reduced apoptosis by > 50% (p < 0.05). Both toxins caused membrane permeabilization in isolated mitochondria (p < 0.001); whereas, pretreatment with UDC was protective (p < 0.05). TUDC was even more effective at preventing matrix swelling mediated by Abeta (p < 0.01). UDC and TUDC markedly reduced cytochrome c release associated with mitochondrial permeabilization induced by UCB and Abeta, respectively (p < 0.05). Moreover, cyclosporine A significantly inhibited mitochondrial swelling and cytochrome c efflux mediated by UCB (p < 0.05). CONCLUSION: UCB and Abeta peptide

  19. High fat diet-induced modifications in membrane lipid and mitochondrial-membrane protein signatures precede the development of hepatic insulin resistance in mice

    PubMed Central

    Kahle, M.; Schäfer, A.; Seelig, A.; Schultheiß, J.; Wu, M.; Aichler, M.; Leonhardt, J.; Rathkolb, B.; Rozman, J.; Sarioglu, H.; Hauck, S.M.; Ueffing, M.; Wolf, E.; Kastenmueller, G.; Adamski, J.; Walch, A.; Hrabé de Angelis, M.; Neschen, S.

    2014-01-01

    Objective Excess lipid intake has been implicated in the pathophysiology of hepatosteatosis and hepatic insulin resistance. Lipids constitute approximately 50% of the cell membrane mass, define membrane properties, and create microenvironments for membrane-proteins. In this study we aimed to resolve temporal alterations in membrane metabolite and protein signatures during high-fat diet (HF)-mediated development of hepatic insulin resistance. Methods We induced hepatosteatosis by feeding C3HeB/FeJ male mice an HF enriched with long-chain polyunsaturated C18:2n6 fatty acids for 7, 14, or 21 days. Longitudinal changes in hepatic insulin sensitivity were assessed via the euglycemic-hyperinsulinemic clamp, in membrane lipids via t-metabolomics- and membrane proteins via quantitative proteomics-analyses, and in hepatocyte morphology via electron microscopy. Data were compared to those of age- and litter-matched controls maintained on a low-fat diet. Results Excess long-chain polyunsaturated C18:2n6 intake for 7 days did not compromise hepatic insulin sensitivity, however, induced hepatosteatosis and modified major membrane lipid constituent signatures in liver, e.g. increased total unsaturated, long-chain fatty acid-containing acyl-carnitine or membrane-associated diacylglycerol moieties and decreased total short-chain acyl-carnitines, glycerophosphocholines, lysophosphatidylcholines, or sphingolipids. Hepatic insulin sensitivity tended to decrease within 14 days HF-exposure. Overt hepatic insulin resistance developed until day 21 of HF-intervention and was accompanied by morphological mitochondrial abnormalities and indications for oxidative stress in liver. HF-feeding progressively decreased the abundance of protein-components of all mitochondrial respiratory chain complexes, inner and outer mitochondrial membrane substrate transporters independent from the hepatocellular mitochondrial volume in liver. Conclusions We assume HF-induced modifications in membrane lipid

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

    PubMed Central

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-01-01

    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. PMID:26137589

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

    PubMed

    Buelli, Simona; Perico, Luca; Galbusera, Miriam; Abbate, Mauro; Morigi, Marina; Novelli, Rubina; Gagliardini, Elena; Tentori, Chiara; Rottoli, Daniela; Sabadini, Ettore; Saito, Takao; Kawano, Mitsuhiro; Saeki, Takako; Zoja, Carlamaria; Remuzzi, Giuseppe; Benigni, Ariela

    2015-05-01

    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.

  2. Mitochondrial membrane protein thiol reactivity with N-ethylmaleimide or mersalyl is modified by Ca2+: correlation with mitochondrial permeability transition.

    PubMed

    Kowaltowski, A J; Vercesi, A E; Castilho, R F

    1997-02-15

    The content of mitochondrial membrane protein thiol groups accessible to react with the monofunctional thiol reagents mersalyl or N-ethylmaleimide (NEM) was determined using Ellman's reagent. Deenergized mitochondria incubated in the presence of Ca2+ (0-500 microM) undergo a very significant decrease in the content of membrane protein thiols accessible to NEM, and an increase in the content of thiols accessible to mersalyl. This process is time-dependent and inhibited by Mg2+, ruthenium red and ADP, but not by cyclosporin A. This suggests that Ca2+ binding to the inner mitochondrial membrane promotes extensive alterations in the conformation of membrane proteins that result in location changes of thiol groups. The relationship between these alterations and mitochondrial membrane permeability transition was studied through the effect of NEM and mersalyl on mitochondrial swelling induced by Ca2+ plus t-butyl hydroperoxide (t-bOOH) or Ca2+ plus the thiol cross-linkers 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or phenylarsine oxide (PhAsO). We observed that the hydrophobic thiol reagent NEM inhibits the effects of t-bOOH, DIDS and PhAsO, while the hydrophilic thiol reagent mersalyl inhibits only the effect of DIDS. Permeability transition in all the situations studied is accompanied by a significant decrease in the total membrane protein thiol content. In addition, mitochondrial membrane permeabilization induced by PhAsO is inhibited by EGTA, but not by ruthenium red. This result suggests that PhAsO leads to permeability transition through a mechanism independent of intramitochondrial Ca2(+)-induced alterations of thiol group reactivity, but dependent on Ca2+ binding to an extramitochondrial site. This site is sensitive to extramitochondrial Ca2+ concentrations in range of 1-50 microM.

  3. Studies of membrane topology of mitochondrial cholesterol hydroxylases CYPs 27A1 and 11A1

    PubMed Central

    Mast, Natalia; Liao, Wei-Li; Turko, Illarion V.

    2010-01-01

    Mitochondrial cytochrome P450 enzymes (CYP or P450, EC 1.14.13.15) play an important role in metabolism of cholesterol. CYP27A1 hydroxylates cholesterol at position 27 and, thus, initiates cholesterol removal from many extrahepatic tissues. CYP11A1 is a steroidogenic P450 that converts cholesterol to pregnenolone, the first step in the biosynthesis of all steroid hormones. We utilized a new approach to study membrane topology of CYPs 27A1 and 11A1. This approach involves heterologous expression of membrane-bound P450 in E. coli, isolation of the P450-containing E. coli membranes, treatment of the membranes with protease, removal of the digested soluble portion and extraction of the membrane-associated peptides, which are then identified by mass spectrometry. By using this approach, we found four membrane-interacting peptides in CYP27A1, and two peptides in CYP11A1. Peptides in CYP27A1 represent a contiguous portion of the polypeptide chain (residues 210-251) corresponding to the putative F-G loop and adjacent portions of the F and G helices. Peptides in CYP11A1 are from the putative F-G loop (residues 218-225) and the C-terminal portion of the G helix (residues 238-250). This data is consistent with those obtained previously by us and others and provide new information about membrane topology of CYPs 27A1 and 11A1. PMID:18791760

  4. Fluorescent probe environment and the structural and charge changes in energy coupling of mitochondrial membranes.

    PubMed

    Chance, B

    1970-10-01

    The use of fluorescent probes to give continuous readouts of the structural states of mitochondrial membranes during energy coupling seems a logical extension of their use in the study of protein structural changes. A clear correlation of the probes' fluorescence characteristics with the acquisition of energy coupling can be demonstrated in fragmented and natural membrane using 1-anilinonaphthalene-8-sulfonate (ANS) and ethidium bromide respectively. The present contribution attempts to bring together contemporary viewpoints of this and other laboratories and the recent experimental data and give some detailed information on probe environment and on the structural or charge changes occurring upon energization. The energy-dependent region of the membrane is located at an aqueous interface between an outer layer of proteins (presumably cytochromes) and the membrane permeability barrier; the aromatic portion of ANS appears to be located in the lipid phase and the sulfonic acid group in the aqueous phase. The aqueous phase is probably a structured water region near paramagnetic membrane components such as cytochrome. Membrane energization arising from altered redox potential changes of cytochromes (b(T)) is communicated to the water structure through altered structural states of the hemoproteins, causing a decreased volume of the structured water region and increased interaction with the paramagnetic components in the energized state. Attendant alterations of protonic equilibria of membrane components induce both local and transmembrane changes in charge distribution, with consequent movements of ions, including the probe molecules themselves.

  5. Mammea E/BB, an isoprenylated dihydroxycoumarin protonophore that potently uncouples mitochondrial electron transport, disrupts hypoxic signaling in tumor cells.

    PubMed

    Du, Lin; Mahdi, Fakhri; Jekabsons, Mika B; Nagle, Dale G; Zhou, Yu-Dong

    2010-11-29

    The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC(50) values of 0.96 and 0.89 μM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 μM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlie their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines.

  6. Mammea E/BB, An Isoprenylated Dihydroxycoumarin Protonophore that Potently Uncouples Mitochondrial Electron Transport Disrupts Hypoxic Signaling in Tumor Cells

    PubMed Central

    Du, Lin; Mahdi, Fakhri; Jekabsons, Mika B.; Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    The mammea-type coumarin mammea E/BB (1) was found to inhibit both hypoxia-induced and iron chelator-induced hypoxia-inducible factor-1 (HIF-1) activation in human breast tumor T47D cells with IC50 values of 0.96 and 0.89 µM, respectively. Compound 1 suppressed the hypoxic induction of secreted VEGF protein (T47D cells) and inhibited cell viability/proliferation in four human tumor cell lines. Compound 1 (at 5 and 20 µM) inhibited human breast tumor MDA-MB-231 cell migration. While the mechanisms that underlay their biological activities have remained unknown, prenylated mammea coumarins have been shown to be cytotoxic to human tumor cells, suppress tumor growth in animal models, and display a wide variety of antimicrobial effects. Mechanistic studies revealed that 1 appears to exert an assemblage of cellular effects by functioning as an anionic protonophore that potently uncouples mitochondrial electron transport and disrupts mitochondrial signaling in human tumor cell lines. PMID:20929261

  7. Mitochondrial membrane potential is reduced in copper-deficient C2C12 cells in the absence of apoptosis.

    PubMed

    Chen, Xiulian; Medeiros, Denis M; Jennings, Dianne

    2005-07-01

    Mitochondrial membrane potential is reduced in copper-deficient rat hearts, but it is uncertain if this will lead to the onset of apoptosis. To determine if copper deficiency per se leads to apoptosis, C2C12 cells were made copper deficient by treatment with the copper chelator tetraethylenepentamine (TEPA). In TEPA-treated cells, the activity of Cu, Zn-superoxide dismutase and cytochrome-c oxidase decreased dramatically. The protein levels of nuclear-encoded subunits of the cytochromie-c oxidase decreased, but the mitochondrial-encoded subunits remained unchanged. Decreased mitochondrial membrane potential was indicated in TEPA-treated cells, but further investigation of the potential induction of apoptosis by measuring caspase-3 activity, protein concentrations of Bcl-2 and Bax, and DNA fragmentation suggested that apoptosis is not induced in TEPA-treated C2C12 cells. Cells with decreased mitochondrial membrane potential were not destined to apoptosis as a result of copper deficiency.

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

    PubMed

    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.

  9. High-fat Diet Accelerates Intestinal Tumorigenesis Through Disrupting Intestinal Cell Membrane Integrity

    PubMed Central

    Park, Mi-Young; Kim, Min Young; Seo, Young Rok; Kim, Jong-Sang; Sung, Mi-Kyung

    2016-01-01

    Background: Excess energy supply induces chronic low-grade inflammation in association with oxidative stress in various tissues including intestinal epithelium. The objective of this study was to investigate the effect of high-fat diet (HFD) on intestinal cell membrane integrity and intestinal tumorigenesis in ApcMin/+ mice. Methods: Mice were fed with either normal diet (ND) or HFD for 12 weeks. The number of intestinal tumors were counted and biomarkers of endotoxemia, oxidative stress, and inflammation were determined. Changes in intestinal integrity was measured by fluorescein isothiocyanate (FITC)-dextran penetration and membrane gap junction protein expression. Results: HFD group had significantly higher number of tumors compared to ND group (P < 0.05). Blood total antioxidant capacity was lower in HFD group, while colonic 8-hydroxy-2′-deoxyguanosine level, a marker of oxidative damage, was higher in HFD group compared to that of ND group (P < 0.05). The penetration of FITC-dextran was substantially increased in HFD group (P < 0.05) while the expressions of membrane gap junction proteins including zonula occludens-1, claudin-1, and occludin were lower in HFD group (P < 0.05) compared to those in ND group. Serum concentration of lipopolysaccharide (LPS) receptor (CD14) and colonic toll-like receptor 4 (a LPS receptor) mRNA expression were significantly higher in HFD group than in ND group (P < 0.05), suggesting that significant endotoxemia may occur in HFD group due to the increased membrane permeability. Serum interleukin-6 concentration and myeloperoxidase activity were also higher in HFD group compared to those of ND group (P < 0.05). Conclusions: HFD increases oxidative stress disrupting intestinal gap junction proteins, thereby accelerating membrane permeability endotoxemia, inflammation, and intestinal tumorigenesis. PMID:27390738

  10. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

    PubMed Central

    Westphal, Dana; Dewson, Grant; Menard, Marie; Frederick, Paul; Iyer, Sweta; Bartolo, Ray; Gibson, Leonie; Czabotar, Peter E.; Smith, Brian J.; Adams, Jerry M.; Kluck, Ruth M.

    2014-01-01

    The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores. PMID:25228770

  11. Cristae remodeling causes acidification detected by integrated graphene sensor during mitochondrial outer membrane permeabilization

    PubMed Central

    Pham, Ted D.; Pham, Phi Q.; Li, Jinfeng; Letai, Anthony G.; Wallace, Douglas C.; Burke, Peter J.

    2016-01-01

    The intrinsic apoptotic pathway and the resultant mitochondrial outer membrane permeabilization (MOMP) via BAK and BAX oligomerization, cytochrome c (cytc) release, and caspase activation are well studied, but their effect on cytosolic pH is poorly understood. Using isolated mitochondria, we show that MOMP results in acidification of the surrounding medium. BAK conformational changes associated with MOMP activate the OMA1 protease to cleave OPA1 resulting in remodeling of the cristae and release of the highly concentrated protons within the cristae invaginations. This was revealed by utilizing a nanomaterial graphene as an optically clear and ultrasensitive pH sensor that can measure ionic changes induced by tethered mitochondria. With this platform, we have found that activation of mitochondrial apoptosis is accompanied by a gradual drop in extra-mitochondrial pH and a decline in membrane potential, both of which can be rescued by adding exogenous cytc. These findings have importance for potential pharmacological manipulation of apoptosis, in the treatment of cancer. PMID:27786282

  12. Mechanics of membrane bulging during cell-wall disruption in Gram-negative bacteria

    NASA Astrophysics Data System (ADS)

    Daly, Kristopher E.; Huang, Kerwyn Casey; Wingreen, Ned S.; Mukhopadhyay, Ranjan

    2011-04-01

    The bacterial cell wall is a network of sugar strands crosslinked by peptides that serve as the primary structure for bearing osmotic stress. Despite its importance in cellular survival, the robustness of the cell wall to network defects has been relatively unexplored. Treatment of the Gram-negative bacterium Escherichia coli with the antibiotic vancomycin, which disrupts the crosslinking of new material during growth, leads to the development of pronounced bulges and eventually of cell lysis. Here, we model the mechanics of the bulging of the cytoplasmic membrane through pores in the cell wall. We find that the membrane undergoes a transition between a nearly flat state and a spherical bulge at a critical pore radius of ~20 nm. This critical pore size is large compared to the typical distance between neighboring peptides and glycan strands, and hence pore size acts as a constraint on network integrity. We also discuss the general implications of our model to membrane deformations in eukaryotic blebbing and vesiculation in red blood cells.

  13. Comparative Analysis of the Bacterial Membrane Disruption Effect of Two Natural Plant Antimicrobial Peptides

    PubMed Central

    Farkas, Attila; Maróti, Gergely; Kereszt, Attila; Kondorosi, Éva

    2017-01-01

    In the Medicago truncatula genome about 700 genes code for nodule-specific cysteine-rich (NCR) small peptides that are expressed in the symbiotic organ, the root nodule, where they control terminal differentiation of the endosymbiotic rhizobium bacteria to nitrogen-fixing bacteroids. Cationic NCR peptides were predicted to have antimicrobial activities. Here antibacterial activities of NCR247, NCR335, polymyxin B (PMB), and streptomycin were investigated and compared on two foodborne pathogens Salmonella enterica and Listeria monocytogenes as representatives of Gram-negative and Gram-positive bacteria. The integrity of the bacterial membrane was seriously compromised by these NCR peptides. Different localization was observed for NCR247 and NCR335 in the treated bacteria, the peptides mostly accumulated in the cytosol in S. enterica while they remained in the bacterial membrane in L. monocytogenes. Scanning electron microscopy revealed distinct membrane morphology of the peptide-treated bacteria. Complete cell disruption was induced by PMB and NCR335 in S. enterica while NCR247 treatment resulted in extensive budding observed on the cell surface of Salmonella. PMB had no effect on L. monocytogenes while NCR335 and NCR247 provoked morphological changes on this bacterium, the whole Listeria cell content was released in response to NCR335 treatment. PMID:28167938

  14. Mdm31 and Mdm32 are inner membrane proteins required for maintenance of mitochondrial shape and stability of mitochondrial DNA nucleoids in yeast.

    PubMed

    Dimmer, Kai Stefan; Jakobs, Stefan; Vogel, Frank; Altmann, Katrin; Westermann, Benedikt

    2005-01-03

    The MDM31 and MDM32 genes are required for normal distribution and morphology of mitochondria in the yeast Saccharomyces cerevisiae. They encode two related proteins located in distinct protein complexes in the mitochondrial inner membrane. Cells lacking Mdm31 and Mdm32 harbor giant spherical mitochondria with highly aberrant internal structure. Mitochondrial DNA (mtDNA) is instable in the mutants, mtDNA nucleoids are disorganized, and their association with Mmm1-containing complexes in the outer membrane is abolished. Mutant mitochondria are largely immotile, resulting in a mitochondrial inheritance defect. Deletion of either one of the MDM31 and MDM32 genes is synthetically lethal with deletion of either one of the MMM1, MMM2, MDM10, and MDM12 genes, which encode outer membrane proteins involved in mitochondrial morphogenesis and mtDNA inheritance. We propose that Mdm31 and Mdm32 cooperate with Mmm1, Mmm2, Mdm10, and Mdm12 in maintenance of mitochondrial morphology and mtDNA.

  15. Evaluation of mitochondrial function and membrane integrity by dual fluorescent staining for assessment of sperm status in rats.

    PubMed

    Kato, Masashi; Makino, Sachiko; Kimura, Hitoshi; Ota, Takao; Furuhashi, Tadakazu; Nagamura, Yoichi

    2002-02-01

    Dual fluorescent staining (DFS) with calcein acetoxy methyl ester (CAM), which labels the cellular esterase activity that is a major component of energy metabolism in cellular mitochondria, and with ethidium homodimer-1 (EthD-1) was used to evaluate mitochondrial function and membrane integrity in rat spermatozoa. The spermatozoa stained by DFS could be classified into three different populations microscopically when excited at 490 nm after 60 min incubation. 1) Spermatozoa, which were stained with CAM alone and had maintained either mitochondrial function or membrane integrity, were identified as live during incubation. 2) Spermatozoa, which were stained with EthD-1 alone and had lost either mitochondrial function or membrane integrity, were identified as already dead at the beginning of incubation. 3) Spermatozoa, which were stained with both CAM and EthD-1 and had maintained mitochondrial function with membrane breached, were identified as having died during incubation. Two toxicological tests, an in vitro triton X-100 experiment and an in vivo nitrobenzene experiment, were done. All spermatozoa were immobilized and lost either mitochondrial function or membrane integrity by 1.0% triton X-100 treatment. Almost no motile sperm were found at 0.1% in the triton X-100 group and in the groups treated with 60 and 40 mg/kg/day of nitrobenzene, and these spermatozoa maintained their mitochondrial function but had their membrane breached. In conclusion, the DFS procedure, which uses CAM and EthD-1, can clearly and visually identify the population of viable and dead spermatozoa simultaneously by fluorescence microscopy in rats. This is a useful technique to characterize sperm status, which is determined by the mitochondrial function assessed by CAM and membrane integrity evaluated by EthD-1.

  16. Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies

    PubMed Central

    Nameta, Masaaki; Saijo, Yoko; Ohmoto, Yasukazu; Katsuragi, Kiyonori; Yamamoto, Keiko; Yamamoto, Tadashi; Ishibashi, Kenichi; Sasaki, Sei

    2016-01-01

    Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs. PMID:27681727

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

    PubMed

    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.

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

    PubMed

    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.

  19. The cytotoxic effects of VE-3N, a novel 1,4-dihydropyridine derivative, involve the mitochondrial bioenergetic disruption via uncoupling mechanisms.

    PubMed

    Marín-Prida, Javier; Pardo Andreu, Gilberto L; Rossignoli, Camila Pederiva; Durruthy, Michael González; Rodríguez, Estael Ochoa; Reyes, Yamila Verdecia; Acosta, Roberto Fernández; Uyemura, Sergio A; Alberici, Luciane C

    2017-03-29

    Several 1,4-dihydropyridine derivatives overcome the multidrug resistance in tumors, but their intrinsic cytotoxic mechanisms remain unclear. Here we addressed if mitochondria are involved in the cytotoxicity of the novel 1,4-dihydropyridine derivative VE-3N [ethyl 6-chloro-5-formyl-2-methyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylate] towards cancer cells by employing hepatic carcinoma (HepG2) cells and isolated rat liver mitochondria. In HepG2 cells, VE-3N induced mitochondrial membrane potential dissipation, ATP depletion, annexin V/propidium iodide double labeling, and Hoechst staining; events indicating apoptosis induction. In isolated rat liver mitochondria, VE-3N promoted mitochondrial uncoupling by exerting protonophoric actions and by increasing membrane fluidity. Mitochondrial uncoupling was evidenced by an increase in resting respiration, dissipation of mitochondrial membrane potential, inhibition of Ca(2+) uptake, stimulation of Ca(2+) release, decrease in ATP synthesis, and swelling of valinomycin-treated organelles in hyposmotic potassium acetate media. Furthermore, uncoupling concentrations of VE-3N in the presence of Ca(2+) plus ruthenium red induced the mitochondrial permeability transition process. These results indicate that mitochondrial uncoupling is potentially involved in the VE-3N cytotoxic actions towards HepG2 cells. Considering that hepatocellular carcinoma is the most common form of liver cancer, our findings may open a new avenue for the development of VE-3N-based cancer therapies, and help to unravel the cytotoxic mechanisms of 1,4-dihydropyridines towards cancer cells.

  20. The force exerted by the membrane potential during protein import into the mitochondrial matrix

    NASA Technical Reports Server (NTRS)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2004-01-01

    The force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated on the basis of continuum electrostatics. The force is found to vary from 3.0 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 6.5 to 12 A, its measured range. In the present model, the decrease in force with increasing pore width arises from the shielding effect of water. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a purely membranous pore without water gives a force of 3.2 pN per unit charge, which should represent an upper limit. When applied to mitochondrial import experiments on the protein barnase, these results imply that forces between 11 +/- 2 pN and 13.5 +/- 2.5 pN catalyze the unfolding of barnase in those experiments. A comparison of these results with unfolding forces measured using atomic force microscopy is made.

  1. Stimulation of mitochondrial proton conductance by hydroxynonenal requires a high membrane potential.

    PubMed

    Parker, Nadeene; Vidal-Puig, Antonio; Brand, Martin D

    2008-04-01

    Mild uncoupling of oxidative phosphorylation, caused by a leak of protons back into the matrix, limits mitochondrial production of ROS (reactive oxygen species). This proton leak can be induced by the lipid peroxidation products of ROS, such as HNE (4-hydroxynonenal). HNE activates uncoupling proteins (UCP1, UCP2 and UCP3) and ANT (adenine nucleotide translocase), thereby providing a negative feedback loop. The mechanism of activation and the conditions necessary to induce uncoupling by HNE are unclear. We have found that activation of proton leak by HNE in rat and mouse skeletal muscle mitochondria is dependent on incubation with respiratory substrate. In the presence of HNE, mitochondria energized with succinate became progressively more leaky to protons over time compared with mitochondria in the absence of either HNE or succinate. Energized mitochondria must attain a high membrane potential to allow HNE to activate uncoupling: a drop of 10-20 mV from the resting value is sufficient to blunt induction of proton leak by HNE. Uncoupling occurs through UCP3 (11%), ANT (64%) and other pathways (25%). Our findings have shown that exogenous HNE only activates uncoupling at high membrane potential. These results suggest that both endogenous HNE production and high membrane potential are required before mild uncoupling will be triggered to attenuate mitochondrial ROS production.

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

    EPA Science Inventory

    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...

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

    PubMed

    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.

  4. Copper effects on key metabolic enzymes and mitochondrial membrane potential in gills of the estuarine crab Neohelice granulata at different salinities.

    PubMed

    Lauer, Mariana Machado; de Oliveira, Camila Bento; Yano, Natalia Lie Inocencio; Bianchini, Adalto

    2012-11-01

    The estuarine crab Neohelice granulata was exposed (96 h) to a sublethal copper concentration under two different physiological conditions (hyperosmoregulating crabs: 2 ppt salinity, 1 mg Cu/L; isosmotic crabs: 30 ppt salinity, 5 mg Cu/L). After exposure, gills (anterior and posterior) were dissected and activities of enzymes involved in glycolysis (hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase), Krebs cycle (citrate synthase), and mitochondrial electron transport chain (cytochrome c oxidase) were analyzed. Membrane potential of mitochondria isolated from anterior and posterior gill cells was also evaluated. In anterior gills of crabs acclimated to 2 ppt salinity, copper exposure inhibited hexokinase, phosphofructokinase, pyruvate kinase, and citrate synthase activity, increased lactate dehydrogenase activity, and reduced the mitochondrial membrane potential. In posterior gills, copper inhibited hexokinase and pyruvate kinase activity, and increased citrate synthase activity. In anterior gills of crabs acclimated to 30 ppt salinity, copper exposure inhibited phosphofructokinase and citrate synthase activity, and increased hexokinase activity. In posterior gills, copper inhibited phosphofructokinase and pyruvate kinase activity, and increased hexokinase and lactate dehydrogenase activity. Copper did not affect cytochrome c oxidase activity in either anterior or posterior gills of crabs acclimated to 2 and 30 ppt salinity. These findings indicate that exposure to a sublethal copper concentration affects the activity of enzymes involved in glycolysis and Krebs cycle, especially in anterior (respiratory) gills of hyperosmoregulating crabs. Changes observed indicate a switch from aerobic to anaerobic metabolism, characterizing a situation of functional hypoxia. In this case, reduced mitochondrial membrane potential would suggest a decrease in ATP production. Although gills of isosmotic crabs were also affected by copper exposure, changes

  5. β2-Microglobulin Amyloid Fibril-Induced Membrane Disruption Is Enhanced by Endosomal Lipids and Acidic pH

    PubMed Central

    Goodchild, Sophia C.; Sheynis, Tania; Thompson, Rebecca; Tipping, Kevin W.; Xue, Wei-Feng; Ranson, Neil A.; Beales, Paul A.; Hewitt, Eric W.; Radford, Sheena E.

    2014-01-01

    Although the molecular mechanisms underlying the pathology of amyloidoses are not well understood, the interaction between amyloid proteins and cell membranes is thought to play a role in several amyloid diseases. Amyloid fibrils of β2-microglobulin (β2m), associated with dialysis-related amyloidosis (DRA), have been shown to cause disruption of anionic lipid bilayers in vitro. However, the effect of lipid composition and the chemical environment in which β2m-lipid interactions occur have not been investigated previously. Here we examine membrane damage resulting from the interaction of β2m monomers and fibrils with lipid bilayers. Using dye release, tryptophan fluorescence quenching and fluorescence confocal microscopy assays we investigate the effect of anionic lipid composition and pH on the susceptibility of liposomes to fibril-induced membrane damage. We show that β2m fibril-induced membrane disruption is modulated by anionic lipid composition and is enhanced by acidic pH. Most strikingly, the greatest degree of membrane disruption is observed for liposomes containing bis(monoacylglycero)phosphate (BMP) at acidic pH, conditions likely to reflect those encountered in the endocytic pathway. The results suggest that the interaction between β2m fibrils and membranes of endosomal origin may play a role in the molecular mechanism of β2m amyloid-associated osteoarticular tissue destruction in DRA. PMID:25100247

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

    PubMed

    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.

  7. Anti-Candida activity of geraniol involves disruption of cell membrane integrity and function.

    PubMed

    Sharma, Y; Khan, L A; Manzoor, N

    2016-09-01

    Candidiasis is a major problem in immunocompromised patients. Candida, an opportunistic fungal pathogen, is a major health concern today as conventional drugs are highly toxic with undesirable side effects. Their fungistatic nature is responsible for drug resistance in continuously evolving strains. Geraniol, an acyclic monoterpene alcohol, is a component of several plant essential oils. In the present study, an attempt has been made to understand the antifungal activity of geraniol at the cell membrane level in three Candida species. With an MIC of 30-130μg/mL, this natural compound was fungicidal at concentrations 2×MIC. There was complete suppression of fungal growth at MIC values (growth curves) and encouragingly geraniol is non-toxic even at the concentrations approaching 5×MIC (hemolysis assay). Exposed cells showed altered morphology, wherein the cells appeared either broken or shrivelled up (SEM studies). Significant reduction was seen in ergosterol levels at sub-MIC and glucose-induced H(+) efflux at concentrations>MIC values. Our results suggest that geraniol disrupts cell membrane integrity by interfering with ergosterol biosynthesis and inhibiting the very crucial PM-ATPase. It may hence be used in the management and treatment of both superficial and invasive candidiasis but further studies are required to elaborate its mode of action.

  8. Removal of typical endocrine disrupting chemicals by membrane bioreactor: in comparison with sequencing batch reactor.

    PubMed

    Zhou, Yingjun; Huang, Xia; Zhou, Haidong; Chen, Jianhua; Xue, Wenchao

    2011-01-01

    The removal of endocrine disrupting chemicals (EDCs) by a laboratory-scale membrane bioreactor (MBR) fed with synthetic sewage was evaluated and moreover, compared with that by a sequencing batch reactor (SBR) operated under same conditions in parallel. Eight kinds of typical EDCs, including 17β-estradiol (E2), estrone (E1), estriol (E3), 17α-ethynilestradiol (EE2), 4-octylphenol (4-OP), 4-nonylphenol (4-NP), bisphenol A (BPA) and nonylphenol ethoxylates (NPnEO), were spiked into the feed. Their concentrations in influent, effluent and supernatant were determined by gas chromatography-mass spectrometry method. The overall estrogenecity was evaluated as 17β-estradiol equivalent quantity (EEQ), determined via yeast estrogen screen (YES) assay. E2, E3, BPA and 4-OP were well removed by both MBR and SBR, with removal rates more than 95% and no significant differences between the two reactors. However, with regard to the other four EDCs, of which the removal rates were lower, MBR performed better. Comparison between supernatant and effluent of the two reactors indicated that membrane separation of sludge and effluent, compared with sedimentation, can relatively improve elimination of target EDCs and total estrogenecity. By applying different solids retention times (SRTs) (5, 10, 20 and 40 d) to the MBR, 10 and 5 d were found to be the lower critical SRTs for efficient target EDCs and EEQ removal, respectively.

  9. Mitochondrial outer-membrane E3 ligase MUL1 ubiquitinates ULK1 and regulates selenite-induced mitophagy

    PubMed Central

    Li, Jie; Qi, Wei; Chen, Guo; Feng, Du; Liu, Jinhua; Ma, Biao; Zhou, Changqian; Mu, Chenglong; Zhang, Weilin; Chen, Quan; Zhu, Yushan

    2015-01-01

    Mitochondria serve as membrane sources and signaling platforms for regulating autophagy. Accumulating evidence has also shown that damaged mitochondria are removed through both selective mitophagy and general autophagy in response to mitochondrial and oxidative stresses. Protein ubiquitination through mitochondrial E3 ligases plays an integrative role in mitochondrial outer membrane protein degradation, mitochondrial dynamics, and mitophagy. Here we showed that MUL1, a mitochondria-localized E3 ligase, regulates selenite-induced mitophagy in an ATG5 and ULK1-dependent manner. ULK1 partially translocated to mitochondria after selenite treatment and interacted with MUL1. We also demonstrated that ULK1 is a novel substrate of MUL1. These results suggest the association of mitochondria with autophagy regulation and provide a new mechanism for the beneficial effects of selenium as a chemopreventive agent. PMID:26018823

  10. [Peroxidation of lipids in mitochondrial membranes, induced by enzymatic deamination of biogenic amines].

    PubMed

    Kagan, V E; Smirnov, A V; Savov, V M; Gorkin, V Z

    1984-01-01

    In presence of ferrous cations and ascorbate lipid peroxidation in mitochondrial membranes has been induced by incubation of fragments of the membrane devoid of catalase activity with amines which are substrates of monoamine oxidases of the B type (2-phenyl ethylamine, benzylamine) or transformed monoamine oxidases of type A (cadaverine). In the samples containing both cadaverine and benzylamine the highest stimulation of lipid peroxidation was noted. To the contrary, a substrate of the monoamine oxidases of the type A (serotonin) caused under the same conditions an antioxidative effect. The following conditions are obligatory to induce lipid peroxidation in mitochondria by incubation with amines: I. absence of catalase activity in the biomembranes; 2. presence of physiological concentrations of Fe2+. Physiological concentrations of ascorbate or alterations of pH in the samples caused additional stimulation of the lipid peroxidation.

  11. Lipid peroxidation in mitochondrial membranes induced by enzymatic deamination of biogenic amines.

    PubMed

    Kagan, V E; Smirnov, A V; Savov, V M; Prilipko, L L; Gorkin, V Z

    1983-01-01

    In the presence of Fe2+ and ascorbate lipid peroxidation in mitochondrial membranes is induced by incubation of membrane fragments devoid of catalase activity with amines which are the substrates of monoamine oxidases of the type B (2-phenylethylamine, benzylamine) or transformed monoamine oxidases of the type A (cadaverine). The highest stimulation of lipid peroxidation is observed in the samples containing both cadaverine and benzylamine. On the contrary, the substrate of the monoamine oxidases of the type A, serotonin, causes an antioxidative effect under these conditions. The necessary prerequisites for lipid peroxidation induction in mitochondria during their incubation with amines are i) the absence of catalase activity in the biomembranes and, ii) the presence of physiological concentrations of Fe2+. Physiological concentrations of ascorbate or pH shifts cause additional stimulation of lipid peroxidation.

  12. A new method for evaluating stallion sperm viability and mitochondrial membrane potential in fixed semen samples.

    PubMed

    Peña, F J; Ball, B A; Squires, E L

    2016-12-29

    Multiparametric assessment of stallion sperm quality using flow cytometry can be a useful adjunct in semen evaluation; however, the availability of flow cytometers in veterinary practice is limited. The ability to preserve and transport sperm samples for later flow cytometric analysis using fixable probes would potentially facilitate this process. In the current study, we validated the combination of live/dead Zombie Green(®) (a fixable dye used to assess live and dead sperm) and MitoTracker Deep Red(®) (used to assess mitochondrial membrane potential). The assay was validated against classic, non-fixable, membrane assays (SYBR-14/PI). Our results demonstrated the feasibility of the assay. In conclusion, stained and fixed semen samples stored for 72 h obtained equivalent results to the exam on the same day; this new protocol shall facilitate the wider use of flow cytometry in stallion andrology in the future. © 2016 International Clinical Cytometry Society.

  13. Dimethyl Sulfoxide Damages Mitochondrial Integrity and Membrane Potential in Cultured Astrocytes

    PubMed Central

    Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

    2014-01-01

    Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO. PMID:25238609

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

    PubMed

    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.

  15. Dimethyl sulfoxide damages mitochondrial integrity and membrane potential in cultured astrocytes.

    PubMed

    Yuan, Chan; Gao, Junying; Guo, Jichao; Bai, Lei; Marshall, Charles; Cai, Zhiyou; Wang, Linmei; Xiao, Ming

    2014-01-01

    Dimethyl sulfoxide (DMSO) is a polar organic solvent that is used to dissolve neuroprotective or neurotoxic agents in neuroscience research. However, DMSO itself also has pharmacological and pathological effects on the nervous system. Astrocytes play a central role in maintaining brain homeostasis, but the effect and mechanism of DMSO on astrocytes has not been studied. The present study showed that exposure of astrocyte cultures to 1% DMSO for 24 h did not significantly affect cell survival, but decreased cell viability and glial glutamate transporter expression, and caused mitochondrial swelling, membrane potential impairment and reactive oxygen species production, and subsequent cytochrome c release and caspase-3 activation. DMSO at concentrations of 5% significantly inhibited cell variability and promoted apoptosis of astrocytes, accompanied with more severe mitochondrial damage. These results suggest that mitochondrial impairment is a primary event in DMSO-induced astrocyte toxicity. The potential cytotoxic effects on astrocytes need to be carefully considered during investigating neuroprotective or neurotoxic effects of hydrophobic agents dissolved by DMSO.

  16. Eukaryote-wide sequence analysis of mitochondrial β-barrel outer membrane proteins

    PubMed Central

    2011-01-01

    Background The outer membranes of mitochondria are thought to be homologous to the outer membranes of Gram negative bacteria, which contain 100's of distinct families of β-barrel membrane proteins (BOMPs) often forming channels for transport of nutrients or drugs. However, only four families of mitochondrial BOMPs (MBOMPs) have been confirmed to date. Although estimates as high as 100 have been made in the past, the number of yet undiscovered MBOMPs is an open question. Fortunately, the recent discovery of a membrane integration signal (the β-signal) for MBOMPs gave us an opportunity to look for undiscovered MBOMPs. Results We present the results of a comprehensive survey of eukaryotic protein sequences intended to identify new MBOMPs. Our search employs recent results on β-signals as well as structural information and a novel BOMP predictor trained on both bacterial and mitochondrial BOMPs. Our principal finding is circumstantial evidence suggesting that few MBOMPs remain to be discovered, if one assumes that, like known MBOMPs, novel MBOMPs will be monomeric and β-signal dependent. In addition to this, our analysis of MBOMP homologs reveals some exceptions to the current model of the β-signal, but confirms its consistent presence in the C-terminal region of MBOMP proteins. We also report a β-signal independent search for MBOMPs against the yeast and Arabidopsis proteomes. We find no good candidates MBOMPs in yeast but the Arabidopsis results are less conclusive. Conclusions Our results suggest there are no remaining MBOMPs left to discover in yeast; and if one assumes all MBOMPs are β-signal dependent, few MBOMP families remain undiscovered in any sequenced organism. PMID:21272379

  17. Kalkitoxin Inhibits Angiogenesis, Disrupts Cellular Hypoxic Signaling, and Blocks Mitochondrial Electron Transport in Tumor Cells

    PubMed Central

    Morgan, J. Brian; Liu, Yang; Coothankandaswamy, Veena; Mahdi, Fakhri; Jekabsons, Mika B.; Gerwick, William H.; Valeriote, Frederick A.; Zhou, Yu-Dong; Nagle, Dale G.

    2015-01-01

    The biologically active lipopeptide kalkitoxin was previously isolated from the marine cyanobacterium Moorea producens (Lyngbya majuscula). Kalkitoxin exhibited N-methyl-d-aspartate (NMDA)-mediated neurotoxicity and acted as an inhibitory ligand for voltage-sensitive sodium channels in cultured rat cerebellar granule neurons. Subsequent studies revealed that kalkitoxin generated a delayed form of colon tumor cell cytotoxicity in 7-day clonogenic cell survival assays. Cell line- and exposure time-dependent cytostatic/cytotoxic effects were previously observed with mitochondria-targeted inhibitors of hypoxia-inducible factor-1 (HIF-1). The transcription factor HIF-1 functions as a key regulator of oxygen homeostasis. Therefore, we investigated the ability of kalkitoxin to inhibit hypoxic signaling in human tumor cell lines. Kalkitoxin potently and selectively inhibited hypoxia-induced activation of HIF-1 in T47D breast tumor cells (IC50 5.6 nM). Mechanistic studies revealed that kalkitoxin inhibits HIF-1 activation by suppressing mitochondrial oxygen consumption at electron transport chain (ETC) complex I (NADH-ubiquinone oxidoreductase). Further studies indicate that kalkitoxin targets tumor angiogenesis by blocking the induction of angiogenic factors (i.e., VEGF) in tumor cells. PMID:25803180

  18. Dietary Tocotrienol/γ-Cyclodextrin Complex Increases Mitochondrial Membrane Potential and ATP Concentrations in the Brains of Aged Mice

    PubMed Central

    Schloesser, Anke; Esatbeyoglu, Tuba; Piegholdt, Stefanie; Dose, Janina; Ikuta, Naoko; Okamoto, Hinako; Ishida, Yoshiyuki; Terao, Keiji; Matsugo, Seiichi; Rimbach, Gerald

    2015-01-01

    Brain aging is accompanied by a decrease in mitochondrial function. In vitro studies suggest that tocotrienols, including γ- and δ-tocotrienol (T3), may exhibit neuroprotective properties. However, little is known about the effect of dietary T3 on mitochondrial function in vivo. In this study, we monitored the effect of a dietary T3/γ-cyclodextrin complex (T3CD) on mitochondrial membrane potential and ATP levels in the brain of 21-month-old mice. Mice were fed either a control diet or a diet enriched with T3CD providing 100 mg T3 per kg diet for 6 months. Dietary T3CD significantly increased mitochondrial membrane potential and ATP levels compared to those of controls. The increase in MMP and ATP due to dietary T3CD was accompanied by an increase in the protein levels of the mitochondrial transcription factor A (TFAM). Furthermore, dietary T3CD slightly increased the mRNA levels of superoxide dismutase, γ-glutamyl cysteinyl synthetase, and heme oxygenase 1 in the brain. Overall, the present data suggest that T3CD increases TFAM, mitochondrial membrane potential, and ATP synthesis in the brains of aged mice. PMID:26301044

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

    SciTech Connect

    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.

  20. Yeast Vps13 promotes mitochondrial function and is localized at membrane contact sites

    PubMed Central

    Park, Jae-Sook; Thorsness, Mary K.; Policastro, Robert; McGoldrick, Luke L.; Hollingsworth, Nancy M.; Thorsness, Peter E.; Neiman, Aaron M.

    2016-01-01

    The Vps13 protein family is highly conserved in eukaryotic cells. Mutations in human VPS13 genes result in a variety of diseases, such as chorea acanthocytosis (ChAc), but the cellular functions of Vps13 proteins are not well defined. In yeast, there is a single VPS13 orthologue, which is required for at least two different processes: protein sorting to the vacuole and sporulation. This study demonstrates that VPS13 is also important for mitochondrial integrity. In addition to preventing transfer of DNA from the mitochondrion to the nucleus, VPS13 suppresses mitophagy and functions in parallel with the endoplasmic reticulum–mitochondrion encounter structure (ERMES). In different growth conditions, Vps13 localizes to endosome–mitochondrion contacts and to the nuclear–vacuole junctions, indicating that Vps13 may function at membrane contact sites. The ability of VPS13 to compensate for the absence of ERMES correlates with its intracellular distribution. We propose that Vps13 is present at multiple membrane contact sites and that separation-of-function mutants are due to loss of Vps13 at specific junctions. Introduction of VPS13A mutations identified in ChAc patients at cognate sites in yeast VPS13 are specifically defective in compensating for the lack of ERMES, suggesting that mitochondrial dysfunction might be the basis for ChAc. PMID:27280386

  1. The Force Exerted by the Membrane Potential During Protein Import into the Mitochondrial Matrix

    NASA Technical Reports Server (NTRS)

    Shariff, Karim; Ghosal, Sandip; Matouschek, Andreas

    2002-01-01

    The electrostatic force exerted on a targeting sequence by the electrical potential across the inner mitochondrial membrane is calculated and found to vary from 1.4 pN to 2.2 pN (per unit elementary charge) as the radius of the inner membrane pore (assumed aqueous) is varied from 12 to 6.5 Angstroms, its measured range. Since the pore is not very much wider than the distance between water molecules, the full shielding effect of water may not be present; the extreme case of a nonaqueous pore gives a force of 3.1 pN per unit charge, which represents an upper limit. When applied to mitochondrial import experiments on the protein harness, these results imply that a force of 11 plus or minus 4 pN is sufficient to catalyze the unfolding of harness during import. Comparison of these results with unfolding forces measured using atomic force microscopy suggests that the two are not inconsistent.

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

    PubMed

    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.

  3. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor.

    PubMed

    Clinton, Ryan W; Francy, Christopher A; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.

  4. Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor*

    PubMed Central

    Clinton, Ryan W.; Francy, Christopher A.; Ramachandran, Rajesh; Qi, Xin; Mears, Jason A.

    2016-01-01

    Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction. PMID:26578514

  5. Critical roles of Rho-associated kinase in membrane blebbing and mitochondrial pathway of apoptosis caused by 1-butanol.

    PubMed

    Noritake, Kanako; Aki, Toshihiko; Funakoshi, Takeshi; Unuma, Kana; Nara, Akina; Kato, Chizuru; Uemura, Koichi

    2012-09-01

    Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome c, and the activation of caspase 9 and 3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.

  6. Decreased Levels of Proapoptotic Factors and Increased Key Regulators of Mitochondrial Biogenesis Constitute New Potential Beneficial Features of Long-lived Growth Hormone Receptor Gene–Disrupted Mice

    PubMed Central

    2013-01-01

    Decreased somatotrophic signaling is among the most important mechanisms associated with extended longevity. Mice homozygous for the targeted disruption of the growth hormone (GH) receptor gene (GH receptor knockout; GHRKO) are obese and dwarf, are characterized by a reduced weight and body size, undetectable levels of GH receptor, high concentration of serum GH, and greatly reduced plasma levels of insulin and insulin-like growth factor-I, and are remarkably long lived. Recent results suggest new features of GHRKO mice that may positively affect longevity—decreased levels of proapoptotic factors and increased levels of key regulators of mitochondrial biogenesis. The alterations in levels of the proapoptotic factors and key regulators of mitochondrial biogenesis were not further improved by two other potential life-extending interventions—calorie restriction and visceral fat removal. This may attribute the primary role to GH resistance in the regulation of apoptosis and mitochondrial biogenesis in GHRKO mice in terms of increased life span. PMID:23197187

  7. A novel antimicrobial peptide derived from fish goose type lysozyme disrupts the membrane of Salmonella enterica.

    PubMed

    Kumaresan, Venkatesh; Bhatt, Prasanth; Ganesh, Munuswamy-Ramanujam; Harikrishnan, Ramasamy; Arasu, MariadhasValan; Al-Dhabi, Naif Abdullah; Pasupuleti, Mukesh; Marimuthu, Kasi; Arockiaraj, Jesu

    2015-12-01

    In aquaculture, accumulation of antibiotics resulted in development of resistance among bacterial pathogens. Consequently, it became mandatory to find alternative to synthetic antibiotics. Antimicrobial peptides (AMPs) which are described as evolutionary ancient weapons have been considered as promising alternates in recent years. In this study, a novel antimicrobial peptide had been derived from goose type lysozyme (LyzG) which was identified from the cDNA library of freshwater fish Channa striatus (Cs). The identified lysozyme cDNA contains 585 nucleotides which encodes a protein of 194 amino acids. CsLyzG was closely related to Siniperca chuatsi with 92.8% homology. The depicted protein sequence contained a GEWL domain with conserved GLMQ motif, 7 active residues and 2 catalytic residues. Gene expression analysis revealed that CsLyzG was distributed in major immune organs with highest expression in head kidney. Results of temporal expression analysis after bacterial (Aeromonas hydrophila) and fungal (Aphanomyces invadans) challenges indicated a stimulant-dependent expression pattern of CsLyzG. Two antimicrobial peptides IK12 and TS10 were identified from CsLyzG and synthesized. Antibiogram showed that IK12 was active against Salmonella enterica, a major multi-drug resistant (MDR) bacterial pathogen which produces beta lactamase. The IK12 induced loss of cell viability in the bacterial pathogen. Flow cytometry assay revealed that IK12 disrupt the membrane of S. enterica which is confirmed by scanning electron microscope (SEM) analysis that reveals blebs around the bacterial cell membrane. Conclusively, CsLyzG is a potential innate immune component and the identified antimicrobial peptide has great caliber to be used as an ecofriendly antibacterial substance in aquaculture.

  8. Mevalonolactone disrupts mitochondrial functions and induces permeability transition pore opening in rat brain mitochondria: Implications for the pathogenesis of mevalonic aciduria.

    PubMed

    Cecatto, Cristiane; Amaral, Alexandre Umpierrez; da Silva, Janaína Camacho; Wajner, Alessandro; Godoy, Kálita Dos Santos; Ribeiro, Rafael Teixeira; Gonçalves, Aline de Mello; Vargas, Carmen Regla; Wajner, Moacir

    2017-03-09

    Mevalonic aciduria (MVA) is caused by severe deficiency of mevalonic kinase activity leading to tissue accumulation and high urinary excretion of mevalonic acid (MA) and mevalonolactone (ML). Patients usually present severe neurologic symptoms whose pathophysiology is poorly known. Here, we tested the hypothesis that the major accumulating metabolites are toxic by investigating the in vitro effects of MA and ML on important mitochondrial functions in rat brain and liver mitochondria. ML, but not MA, markedly decreased mitochondrial membrane potential (ΔΨm), NAD(P)H content and the capacity to retain Ca(2+) in the brain, besides inducing mitochondrial swelling. These biochemical alterations were totally prevented by the classical inhibitors of mitochondrial permeability transition (MPT) cyclosporine A and ADP, as well as by ruthenium red in Ca(2+)-loaded mitochondria, indicating the involvement of MPT and an important role for mitochondrial Ca(2+) in these effects. ML also induced lipid peroxidation and markedly inhibited aconitase activity, an enzyme that is highly susceptible to free radical attack, in brain mitochondrial fractions, indicating that lipid and protein oxidative damage may underlie some of ML-induced deleterious effects including MTP induction. In contrast, ML and MA did not compromise oxidative phosphorylation in the brain and all mitochondrial functions evaluated in the liver, evidencing a selective toxicity of ML towards the central nervous system. Our present study provides for the first time evidence that ML impairs essential brain mitochondrial functions with the involvement of MPT pore opening. It is therefore presumed that disturbance of brain mitochondrial homeostasis possibly contributes to the neurologic symptoms in MVA.

  9. Supplementation of T3 Recovers Hypothyroid Rat Liver Cells from Oxidatively Damaged Inner Mitochondrial Membrane Leading to Apoptosis

    PubMed Central

    Mukherjee, Sutapa; Samanta, Luna; Roy, Anita; Bhanja, Shravani; Chainy, Gagan B. N.

    2014-01-01

    Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation. PMID:24987693

  10. Supplementation of T3 recovers hypothyroid rat liver cells from oxidatively damaged inner mitochondrial membrane leading to apoptosis.

    PubMed

    Mukherjee, Sutapa; Samanta, Luna; Roy, Anita; Bhanja, Shravani; Chainy, Gagan B N

    2014-01-01

    Hypothyroidism is a growing medical concern. There are conflicting reports regarding the mechanism of oxidative stress in hypothyroidism. Mitochondrial oxidative stress is pivotal to thyroid dysfunction. The present study aimed to delineate the effects of hepatic inner mitochondrial membrane dysfunction as a consequence of 6-n-propyl-2-thiouracil-induced hypothyroidism in rats. Increased oxidative stress predominance in the submitochondrial particles (SMP) and altered antioxidant defenses in the mitochondrial matrix fraction correlated with hepatocyte apoptosis. In order to check whether the effects caused by hypothyroidism are reversed by T3, the above parameters were evaluated in a subset of T3-treated hypothyroid rats. Complex I activity was inhibited in hypothyroid SMP, whereas T3 supplementation upregulated electron transport chain complexes. Higher mitochondrial H2O2 levels in hypothyroidism due to reduced matrix GPx activity culminated in severe oxidative damage to membrane lipids. SMP and matrix proteins were stabilised in hypothyroidism but exhibited increased carbonylation after T3 administration. Glutathione content was higher in both. Hepatocyte apoptosis was evident in hypothyroid liver sections; T3 administration, on the other hand, exerted antiapoptotic and proproliferative effects. Hence, thyroid hormone level critically regulates functional integrity of hepatic mitochondria; hypothyroidism injures mitochondrial membrane lipids leading to hepatocyte apoptosis, which is substantially recovered upon T3 supplementation.

  11. Direct Membrane Association Drives Mitochondrial Fission by the Parkinson Disease-associated Protein α-Synuclein*♦

    PubMed Central

    Nakamura, Ken; Nemani, Venu M.; Azarbal, Farnaz; Skibinski, Gaia; Levy, Jon M.; Egami, Kiyoshi; Munishkina, Larissa; Zhang, Jue; Gardner, Brooke; Wakabayashi, Junko; Sesaki, Hiromi; Cheng, Yifan; Finkbeiner, Steven; Nussbaum, Robert L.; Masliah, Eliezer; Edwards, Robert H.

    2011-01-01

    The protein α-synuclein has a central role in Parkinson disease, but the mechanism by which it contributes to neural degeneration remains unknown. We now show that the expression of α-synuclein in mammalian cells, including neurons in vitro and in vivo, causes the fragmentation of mitochondria. The effect is specific for synuclein, with more fragmentation by α- than β- or γ-isoforms, and it is not accompanied by changes in the morphology of other organelles or in mitochondrial membrane potential. However, mitochondrial fragmentation is eventually followed by a decline in respiration and neuronal death. The fragmentation does not require the mitochondrial fission protein Drp1 and involves a direct interaction of synuclein with mitochondrial membranes. In vitro, synuclein fragments artificial membranes containing the mitochondrial lipid cardiolipin, and this effect is specific for the small oligomeric forms of synuclein. α-Synuclein thus exerts a primary and direct effect on the morphology of an organelle long implicated in the pathogenesis of Parkinson disease. PMID:21489994

  12. [Change in the lipid composition of the inner mitochondrial membranes in rat organs during adaptation to heat].

    PubMed

    Zubareva, E V; Seferova, R I; Denisova, N A

    1991-01-01

    Under conditions of adaptation to heating lipid composition in mitochondrial membranes of rat inner tissues was altered as follows: an increase in relative concentration of plasmalogenous forms of phospholipids (kidney, heart) and in content of saturated fatty acids (liver tissue), a decrease in the index of fatty acids unsaturation and in the ratio of fatty acids omega-3/omega-6. The alterations observed enabled the membranes to keep sufficient amount of liquidity essential for functional activity of mitochondria in heating.

  13. Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes*

    PubMed Central

    Schlame, Michael

    2008-01-01

    In this article, the formation of prokaryotic and eukaryotic cardiolipin is reviewed in light of its biological function. I begin with a detailed account of the structure of cardiolipin, its stereochemistry, and the resulting physical properties, and I present structural analogs of cardiolipin that occur in some organisms. Then I continue to discuss i) the de novo formation of cardiolipin, ii) its acyl remodeling, iii) the assembly of cardiolipin into biological membranes, and iv) the degradation of cardiolipin, which may be involved in apoptosis and mitochondrial fusion. Thus, this article covers the entire metabolic cycle of this unique phospholipid. It is shown that mitochondria produce cardiolipin species with a high degree of structural uniformity and molecular symmetry, among which there is often a dominant form with four identical acyl chains. The subsequent assembly of cardiolipin into functional membranes is largely unknown, but the analysis of crystal structures of membrane proteins has revealed a first glimpse into the underlying principles of cardiolipin-protein interactions. Disturbances of cardiolipin metabolism are crucial in the pathophysiology of human Barth syndrome and perhaps also play a role in diabetes and ischemic heart disease. PMID:18077827

  14. High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation.

    PubMed

    Azzu, Vian; Parker, Nadeene; Brand, Martin D

    2008-07-15

    Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.

  15. Real-time imaging of laser-induced membrane disruption of a living cell observed with multifocus coherent anti-Stokes Raman scattering microscopy.

    PubMed

    Minamikawa, Takeo; Niioka, Hirohiko; Araki, Tsutomu; Hashimoto, Mamoru

    2011-02-01

    We demonstrate the real-time imaging of laser-induced disruption of the cellular membrane in a living HeLa cell and its cellular response with a multifocus coherent anti-Stokes Raman scattering (CARS) microscope. A near-infrared pulsed laser beam tightly focused on the cellular membrane of a living cell induces ablation at the focal point causing a local disruption of the cellular membrane. After the membrane disruption a dark spot decreasing CARS intensity of 2840 cm(-1) Raman shift at the disrupted site appears. This dark spot immediately disappears and a strong CARS signal is observed around the disrupted site. This increase of the CARS signal might be caused by resealing of the disrupted site via aggregation of the patch lipid vesicles in the cytoplasm. The accumulation of lipids around the disrupted site is also confirmed with three-dimensional CARS images of a cell before and after membrane disruption. The temporal behavior of the CARS signal at the disrupted site is observed to detect the fusion dynamics of patch vesicles.

  16. Cardiac mitochondrial membrane stability after deep hypothermia using a xenon clathrate cryostasis protocol - an electron microscopy study.

    PubMed

    Sheleg, Sergey; Hixon, Hugh; Cohen, Bruce; Lowry, David; Nedzved, Mikhail

    2008-01-01

    We investigated a new cryopreservation method using xenon, a clathrate-forming gas, under medium pressure (100psi). The objective of the study was to determine whether this cryostasis protocol could protect cardiac mitochondria at cryogenic temperatures (below 100 degrees Celsius).We analyzed transmission electron microscopy images to obtain information about changes in mitochondrial morphology induced by cryopreservation of the hearts. Our data showed absence of mitochondrial swelling, rupture of inner and outer membranes, and leakage of mitochondrial matrix into the cytoplasm after applying this cryostasis protocol. The electron microscopy results provided the first evidence that a cryostasis protocol using xenon as a clathrate-forming gas under pressure may have protective effects on intracellular membranes. This cryostasis technology may find applications in developing new approaches for long-term cryopreservation protocols.

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

    PubMed

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

    2016-10-20

    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.

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

    PubMed

    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.

  19. Cytochrome c oxidase is regulated by modulations in protein expression and mitochondrial membrane phospholipid composition in estivating African lungfish.

    PubMed

    Frick, N T; Bystriansky, J S; Ip, Y K; Chew, S F; Ballantyne, J S

    2010-03-01

    We examined some of the potential mechanisms lungfish (Protopterus dolloi) use to regulate cytochrome c oxidase (CCO), during metabolic depression. CCO activity was reduced by 67% in isolated liver mitochondria of estivating fish. This was likely accomplished, in part, by the 46% reduction in CCO subunit I protein expression in the liver. No change in the mRNA expression levels of CCO subunits I, II, III, and IV were found in the liver, suggesting CCO is under translational regulation; however, in the kidney, messenger limitation may be a factor as the expression of subunits I and II were depressed ( approximately 10-fold) during estivation, suggesting tissue-specific mechanisms of regulation. CCO is influenced by mitochondrial membrane phospholipids, particularly cardiolipin (CL). In P. dolloi, the phospholipid composition of the liver mitochondrial membrane changed during estivation, with a approximately 2.3-fold reduction in the amount of CL. Significant positive correlations were found between CCO activity and the amount of CL and phosphatidylethanolamine within the mitochondrial membrane. It appears CCO activity is regulated through multiple mechanisms in P. dolloi, and individual subunits of CCO are regulated independently, and in a tissue-specific manner. It is proposed that altering the amount of CL within the mitochondrial membrane may be a means of regulating CCO activity during metabolical depression in the African lungfish, P. dolloi.

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

    PubMed

    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 (P<0.05) in pore opening. The difference in pore opening was found to be statistically significant (P<0.05) when the artesunate and ferrous 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.

  1. Cerium oxide nanoparticles prevent apoptosis in primary cortical culture by stabilizing mitochondrial membrane potential.

    PubMed

    Arya, A; Sethy, N K; Das, M; Singh, S K; Das, A; Ujjain, S K; Sharma, R K; Sharma, M; Bhargava, K

    2014-07-01

    Cerium oxide nanoparticles (CNPs) of spherical shape have unique antioxidant capacity primarily due to alternating + 3 and + 4 oxidation states and crystal defects. Several studies revealed the protective efficacies of CNPs in cells and tissues against the oxidative damage. However, its effect on mitochondrial functioning, downstream effectors of radical burst and apoptosis remains unknown. In this study, we investigated whether CNPs treatment could protect the primary cortical cells from loss of mitochondrial membrane potential (Δψm) and Δψm-dependent cell death. CNPs with spherical morphology and size range 7-10 nm were synthesized and utilized at a concentration of 25 nM on primary neuronal culture challenged with 50 μM of hydrogen peroxide (H2O2). We showed that optimal dose of CNPs minimized ROS content of the cells and also curbed related surge in cellular calcium flux. Importantly, CNPs treatment prevented apoptotic loss of cell viability. Reduction in the apoptosis could be successfully attributed to the maintenance of Δψm and restoration of major redox equivalents NADH/NAD(+) ratio and cellular ATP. These findings, therefore, suggest possible route of CNPs protective efficacies in primary cortical culture.

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

    SciTech Connect

    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.

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

    PubMed

    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.

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

    PubMed Central

    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. PMID:28280332

  5. Multiple Lines of Evidence Localize Signaling, Morphology, and Lipid Biosynthesis Machinery to the Mitochondrial Outer Membrane of Arabidopsis[W][OA

    PubMed Central

    Duncan, Owen; Taylor, Nicolas L.; Carrie, Chris; Eubel, Holger; Kubiszewski-Jakubiak, Szymon; Zhang, Botao; Narsai, Reena; Millar, A. Harvey; Whelan, James

    2011-01-01

    The composition of the mitochondrial outer membrane is notoriously difficult to deduce by orthology to other organisms, and biochemical enrichments are inevitably contaminated with the closely associated inner mitochondrial membrane and endoplasmic reticulum. In order to identify novel proteins of the outer mitochondrial membrane in Arabidopsis (Arabidopsis thaliana), we integrated a quantitative mass spectrometry analysis of highly enriched and prefractionated samples with a number of confirmatory biochemical and cell biology approaches. This approach identified 42 proteins, 27 of which were novel, more than doubling the number of confirmed outer membrane proteins in plant mitochondria and suggesting novel functions for the plant outer mitochondrial membrane. The novel components identified included proteins that affected mitochondrial morphology and/or segregation, a protein that suggests the presence of bacterial type lipid A in the outer membrane, highly stress-inducible proteins, as well as proteins necessary for embryo development and several of unknown function. Additionally, proteins previously inferred via orthology to be present in other compartments, such as an NADH:cytochrome B5 reductase required for hydroxyl fatty acid accumulation in developing seeds, were shown to be located in the outer membrane. These results also revealed novel proteins, which may have evolved to fulfill plant-specific requirements of the mitochondrial outer membrane, and provide a basis for the future functional characterization of these proteins in the context of mitochondrial intracellular interaction. PMID:21896887

  6. Prediction of ultrasound-mediated disruption of cell membranes using machine learning techniques and statistical analysis of acoustic spectra.

    PubMed

    Lee, Eva K; Gallagher, Richard J; Campbell, Ann Melissa; Prausnitz, Mark R

    2004-01-01

    Although biological effects of ultrasound must be avoided for safe diagnostic applications, ultrasound's ability to disrupt cell membranes has attracted interest as a method to facilitate drug and gene delivery. This paper seeks to develop "prediction rules" for predicting the degree of cell membrane disruption based on specified ultrasound parameters and measured acoustic signals. Three techniques for generating prediction rules (regression analysis, classification trees and discriminant analysis) are applied to data obtained from a sequence of experiments on bovine red blood cells. For each experiment, the data consist of four ultrasound parameters, acoustic measurements at 400 frequencies, and a measure of cell membrane disruption. To avoid over-training, various combinations of the 404 predictor variables are used when applying the rule generation methods. The results indicate that the variable combination consisting of ultrasound exposure time and acoustic signals measured at the driving frequency and its higher harmonics yields the best rule for all three rule generation methods. The methods used for deriving the prediction rules are broadly applicable, and could be used to develop prediciton rules in other scenarios involving different cell types or tissues. These rules and the methods used to derive them could be used for real-time feedback about ultrasound's biological effects.

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

    PubMed

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

    2008-12-01

    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 Apaf1(fog/fog) (fog/fog) mice for initiator caspase-9 activation by cellular stresses. When the mitochondrial inner membrane potential (Deltapsim) was disrupted by staurosporine, +/+ cells but not fog/fog cells activated caspase-9 to cause apoptosis, indicating the lack of apoptosome (apoptosis protease activating factor 1 (Apaf-1)/cytochrome c/(d)ATP/procaspase-9) function in fog/fog cells. However, when a marginal ( approximately 20%) decrease in Deltapsim was caused by hydrogen peroxide (0.1 mM), peroxynitritedonor 3-morpholinosydnonimine (0.1 mM) and UV-C irradiation (20 J/m(2)), both +/+ and fog/fog cells triggered procaspase-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 genotypes. Cellular ATP concentration significantly decreased under the hypoactive Deltapsim 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.

  8. Targeted gene disruption identifies three PPR-DYW proteins involved in RNA editing for five editing sites of the moss mitochondrial transcripts.

    PubMed

    Ohtani, Shotaro; Ichinose, Mizuho; Tasaki, Eiji; Aoki, Yoshiaki; Komura, Yoshihiro; Sugita, Mamoru

    2010-11-01

    In plant organelles, RNA editing frequently occurs in many transcripts, but little is known about its molecular mechanism. Eleven RNA editing sites are present in the moss Physcomitrella patens mitochondria. Recently PpPPR_71, one member of 10 DYW-subclass pentatricopeptide repeat (PPR-DYW) proteins, has been identified as a site-specific recognition factor for RNA editing in the mitochondrial transcript. In this study, we disrupted three genes encoding a PPR-DYW protein-PpPPR_56, PpPPR_77, and PpPPR_91-to investigate whether they are involved in RNA editing. Transient expression of an N-terminal amino acid sequence fused to the green fluorescent protein (GFP) suggests that the three PPR-DYW proteins are targeted to mitochondria. Disruption of each gene by homologous recombination revealed that PpPPR_56 was involved in RNA editing at the nad3 and nad4 sites, PpPPR_77 at the cox2 and cox3 sites, and PpPPR_91 at the nad5-2 site in the mitochondrial transcripts. The nucleotide sequences surrounding the two editing sites targeted by a single PPR-DYW protein share 42 to 56% of their identities. Thus, moss PPR-DYW proteins seem to be site-specific factors for RNA editing in mitochondrial transcripts.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  12. Malaria parasite CelTOS targets the inner leaflet of cell membranes for pore-dependent disruption

    PubMed Central

    Jimah, John R; Salinas, Nichole D; Sala-Rabanal, Monica; Jones, Nathaniel G; Sibley, L David; Nichols, Colin G; Schlesinger, Paul H; Tolia, Niraj H

    2016-01-01

    Apicomplexan parasites contain a conserved protein CelTOS that, in malaria parasites, is essential for traversal of cells within the mammalian host and arthropod vector. However, the molecular role of CelTOS is unknown because it lacks sequence similarity to proteins of known function. Here, we determined the crystal structure of CelTOS and discovered CelTOS resembles proteins that bind to and disrupt membranes. In contrast to known membrane disruptors, CelTOS has a distinct architecture, specifically binds phosphatidic acid commonly present within the inner leaflet of plasma membranes, and potently disrupts liposomes composed of phosphatidic acid by forming pores. Microinjection of CelTOS into cells resulted in observable membrane damage. Therefore, CelTOS is unique as it achieves nearly universal inner leaflet cellular activity to enable the exit of parasites from cells during traversal. By providing novel molecular insight into cell traversal by apicomplexan parasites, our work facilitates the design of therapeutics against global pathogens. DOI: http://dx.doi.org/10.7554/eLife.20621.001 PMID:27906127

  13. Brevibacillin, a cationic lipopeptide that binds to lipoteichoic acid and subsequently disrupts cytoplasmic membrane of Staphylococcus aureus.

    PubMed

    Yang, Xu; Huang, En; Yousef, Ahmed E

    2017-01-01

    Brevibacillin is a newly-discovered antimicrobial lipopeptide produced by Brevibacillus laterosporus OSY-I1. It is active against Gram-positive bacteria, including antibiotic resistant strains. This research was initiated to investigate the mechanism of action of brevibacillin against an indicator strain, Staphylococcus aureus ATCC 6538. Results of the study proved that brevibacillin binds to lipoteichoic acid (LTA) on cell wall before interacting with cell membrane. Additionally, brevibacillin disrupts S. aureus cytoplasmic membrane by increasing its permeability, depolarization and potassium leakage. Therefore, cytoplasmic membrane serves as a major target for brevibacillin. Despite the presence of multiple sites on S. aureus cell envelope, scanning electron microscope observation didn't reveal evidence of cell lysis or any morphological defects in cells treated with brevibacillin. Based on the results of this study, we propose that the electrostatic interaction between the cationic brevibacillin and the anionic LTA helped the accumulation of the antimicrobial agent at cell surface; this was followed by translocation of the lipopeptide to the cytoplasmic membrane and disrupting its vital functions.

  14. Dependence of mitochondrial coenzyme A uptake on the membrane electrical gradient

    SciTech Connect

    Tahiliani, A.G. )

    1989-11-05

    Coenzyme A (CoA) transport was studied in isolated rat heart mitochondria. Uptake of CoA was assayed by determining (3H)CoA associated with mitochondria under various conditions. Various oxidizable substrates including alpha-ketoglutarate, succinate, or malate stimulated CoA uptake. The membrane proton (delta pH) and electrical (delta psi) gradients, which dissipated with time in the absence of substrate, were maintained at their initial levels throughout the incubation in the presence of substrate. Addition of phosphate caused a concentration-dependent decrease of both delta pH and CoA uptake. Nigericin also dissipated the proton gradient and prevented CoA uptake. Valinomycin also prevented CoA uptake into mitochondria. Although the proton gradient was unaffected, the electrical gradient was completely abolished in the presence of valinomycin. Addition of 5 mM phosphate 10 min after the start of incubation prevented further uptake of CoA into mitochondria. A rapid dissipation of the proton gradient upon addition of phosphate was observed. Addition of nigericin or valinomycin 10 min after the start of incubation also resulted in no further uptake of CoA into with mitochondria; valinomycin caused an apparent efflux of CoA from mitochondria. Uptake was found to be sensitive to external pH displaying a pH optimum at pHext 8.0. Although nigericin significantly inhibited CoA uptake over the pHext range of 6.75-8, maximal transport was observed around pHext 8.0-8.25. Valinomycin, on the other hand, abolished transport over the entire pH range. The results suggest that mitochondrial CoA transport is determined by the membrane electrical gradient. The apparent dependence of CoA uptake on an intact membrane pH gradient is probably the result of modulation of CoA transport by matrix pH.

  15. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    PubMed

    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.

  16. Direct regulation of complex I by mitochondrial MEF2D is disrupted in a mouse model of Parkinson disease and in human patients

    PubMed Central

    She, Hua; Yang, Qian; Shepherd, Kennie; Smith, Yoland; Miller, Gary; Testa, Claudia; Mao, Zixu

    2011-01-01

    The transcription factors in the myocyte enhancer factor 2 (MEF2) family play important roles in cell survival by regulating nuclear gene expression. Here, we report that MEF2D is present in rodent neuronal mitochondria, where it can regulate the expression of a gene encoded within mitochondrial DNA (mtDNA). Immunocytochemical, immunoelectron microscopic, and biochemical analyses of rodent neuronal cells showed that a portion of MEF2D was targeted to mitochondria via an N-terminal motif and the chaperone protein mitochondrial heat shock protein 70 (mtHsp70). MEF2D bound to a MEF2 consensus site in the region of the mtDNA that contained the gene NADH dehydrogenase 6 (ND6), which encodes an essential component of the complex I enzyme of the oxidative phosphorylation system; MEF2D binding induced ND6 transcription. Blocking MEF2D function specifically in mitochondria decreased complex I activity, increased cellular H2O2 level, reduced ATP production, and sensitized neurons to stress-induced death. Toxins known to affect complex I preferentially disrupted MEF2D function in a mouse model of Parkinson disease (PD). In addition, mitochondrial MEF2D and ND6 levels were decreased in postmortem brain samples of patients with PD compared with age-matched controls. Thus, direct regulation of complex I by mitochondrial MEF2D underlies its neuroprotective effects, and dysregulation of this pathway may contribute to PD. PMID:21393861

  17. Thermotropic lateral translational motion of intramembrane particles in the inner mitochondrial membrane and its inhibition by artificial peripheral proteins

    PubMed Central

    1977-01-01

    Freeze fracturing and deep etching have been used to study thermotropic lateral translational motion of intramembrane particles and membrane surface anionic groups in the inner mitochondrial membrane. When the inner membrane is equilibrated at low temperature, the fracture faces of both halves of the membrane reveal a lateral separation between intramembrane particles and particle free, large smooth patches. Such separation is completely reversed through free lateral translational diffusion by reversing the temperature. The low temperature induced, particle-free, smooth membrane patches appear to represent regions of protein-excluding, ordered bilayer lipid which form during thermotropic liquid crystalline to gel state phase transitions. When polycationic ferritin is electrostatically bound to anionic groups exposed at the membrane surface at concentrations which inhibit the activities of cytochrome c oxidase and succinate permease, the bound ferritin migrates with intramembrane particles during the thermotropic lateral separation between the membrane particles and smooth patches. When bound polycationic ferritin is cross-bridged with native ferritin, an artificial peripheral protein lattice forms in association with the surface anionic groups and diminishes the thermotropic lateral translational motion of intramembrane particles in the membrane. These results reveal that the anionic groups of metabolically active integral proteins which are known to be exposed at the surface of the inner mitochondrial membrane migrate with intramembrane particles in the plane of the membrane under conditions which induce lipid-protein lateral separations. In addition, cross-bridging of the anionic groups through an artificial peripheral protein lattice appears to diminish such induced lipid protein lateral separations. PMID:833199

  18. A perforin-like protein mediates disruption of the erythrocyte membrane during egress of Plasmodium berghei male gametocytes.

    PubMed

    Deligianni, Elena; Morgan, Rhiannon N; Bertuccini, Lucia; Wirth, Christine C; Silmon de Monerri, Natalie C; Spanos, Lefteris; Blackman, Michael J; Louis, Christos; Pradel, Gabriele; Siden-Kiamos, Inga

    2013-08-01

    Successful gametogenesis of the malaria parasite depends on egress of the gametocytes from the erythrocytes within which they developed. Egress entails rupture of both the parasitophorous vacuole membrane and the erythrocyte plasma membrane, and precedes the formation of the motile flagellated male gametes in a process called exflagellation. We show here that egress of the male gametocyte depends on the function of a perforin-like protein, PPLP2. A mutant of Plasmodium berghei lacking PPLP2 displayed abnormal exflagellation; instead of each male gametocyte forming eight flagellated gametes, it produced gametocytes with only one, shared thicker flagellum. Using immunofluorescence and transmission electron microscopy analysis, and phenotype rescue with saponin or a pore-forming toxin, we conclude that rupture of the erythrocyte membrane is blocked in the mutant. The parasitophorous vacuole membrane, on the other hand, is ruptured normally. Some mutant parasites are still able to develop in the mosquito, possibly because the vigorous motility of the flagellated gametes eventually leads to escape from the persisting erythrocyte membrane. This is the first example of a perforin-like protein in Plasmodium parasites having a role in egress from the host cell and the first parasite protein shown to be specifically required for erythrocyte membrane disruption during egress.

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

    PubMed

    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).

  20. Characterization of rat TOM70 as a receptor of the preprotein translocase of the mitochondrial outer membrane.

    PubMed

    Suzuki, Hiroyuki; Maeda, Maki; Mihara, Katsuyoshi

    2002-05-01

    We cloned a approximately 70 kDa rat mitochondrial outer membrane protein (OM70) with a sequence identity of 28.1% and 20.1% with N. crassa and S. cerevisiae Tom70, respectively. Even with this low sequence identity, however, the proteins share a remarkable structural similarity: they have 7-10 tetratricopeptide repeat motifs and are anchored to the outer membrane through the N-terminal transmembrane domain with the bulk portion located in the cytosol. Antibodies against OM70 inhibited import of preproteins, such as the ADP/ATP carrier and rTOM40, that use internal targeting signals but not the import of cleavable presequence-containing preproteins. Blue native gel electrophoresis and immunoprecipitation of digitoninsolubilized mitochondrial outer membranes revealed that OM70 was loosely associated with the approximately 400 kDa translocase complex of the mitochondrial outer membrane, which contains rTOM22 and rTOM40. A yeast two-hybrid system demonstrated that OM70 interacted with rTOM20 and rTOM22 through the cytoplasmic domains. Thus, OM70 is a functional homologue of fungal Tom70 and functions as a receptor of the preprotein import machinery of the rat mitochondrial outer membrane. Furthermore, the N-terminal 66 residue region of OM70, which comprises a hydrophilic 41 residue N-terminal domain, a 22 residue transmembrane domain and three arginine residues, is sufficient to act as a mitochondria-targeting signal, and the arginine cluster is crucial for this function.

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

    PubMed

    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.

  2. [Changes in polarization of myometrial cells plasma and internal mitochondrial membranes under calixarenes action as inhibitors of plasma membrane Na+, K+-ATPase].

    PubMed

    Danylovych, H V; Danylovych, Iu V; Kolomiiets', O V; Kosterin, S O; Rodik, R V; Cherenok, S O; Kal'chenko, V I; Chunikhin, O Iu; Horchev, V F; Karakhim, S O

    2012-01-01

    The influence of supramolecular macrocyclic compounds--calix[4]arenes C-97, C-99, C-107, which are ouabainomymetic high affinity inhibitors of Na+, K(+)-ATPase, on the polarization level of plasmic and mitochondrial membranes of rat uterine smooth muscle cells was investigated. The influence of these compounds on the myocytes characteristic size was studied. By using a confocal microscopy and specific for mitochondrial MitoTracker Orange CM-H2TMRos dye it was proved that the potential-sensitive fluorescent probe DiOC6(3) interacts with mitochondria. Artificial potential collapse of plasmic membrane in this case was modeled by myocytes preincubation with ouabain (1 mM). Further experiments performed using the method of flow cytometry with DiOC6(3) have shown that the compounds C-97, C-99 and C-107 at concentration 50-100 nM caused depolarization of the plasma membrane (at the level of 30% relative to control values) in conditions of artificial collapse of mitochondrial potential by myocytes preincubation in the presence of 5 mM of sodium azide. Under artificial sarcolemma depolarization by ouabain, calixarenes C-97, C-99 and C-107 at 100 nM concentrations caused a transient increase of mitochondrial membrane potential, that is 40% of the control level and lasted about 5 minutes. Calixarenes C-99 and C-107 caused a significant increase in fluorescence of myocytes in these conditions, which was confirmed by confocal microscopy too. It was proved by photon correlation spectroscopy method that the C-99 and C-107 caused an increase of characteristic size of myocytes.

  3. Glutaredoxin 2 catalyzes the reversible oxidation and glutathionylation of mitochondrial membrane thiol proteins: implications for mitochondrial redox regulation and antioxidant DEFENSE.

    PubMed

    Beer, Samantha M; Taylor, Ellen R; Brown, Stephanie E; Dahm, Christina C; Costa, Nikola J; Runswick, Michael J; Murphy, Michael P

    2004-11-12

    The redox poise of the mitochondrial glutathione pool is central in the response of mitochondria to oxidative damage and redox signaling, but the mechanisms are uncertain. One possibility is that the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) and the consequent change in the GSH/GSSG ratio causes protein thiols to change their redox state, enabling protein function to respond reversibly to redox signals and oxidative damage. However, little is known about the interplay between the mitochondrial glutathione pool and protein thiols. Therefore we investigated how physiological GSH/GSSG ratios affected the redox state of mitochondrial membrane protein thiols. Exposure to oxidized GSH/GSSG ratios led to the reversible oxidation of reactive protein thiols by thiol-disulfide exchange, the extent of which was dependent on the GSH/GSSG ratio. There was an initial rapid phase of protein thiol oxidation, followed by gradual oxidation over 30 min. A large number of mitochondrial proteins contain reactive thiols and most of these formed intraprotein disulfides upon oxidation by GSSG; however, a small number formed persistent mixed disulfides with glutathione. Both protein disulfide formation and glutathionylation were catalyzed by the mitochondrial thiol transferase glutaredoxin 2 (Grx2), as were protein deglutathionylation and the reduction of protein disulfides by GSH. Complex I was the most prominent protein that was persistently glutathionylated by GSSG in the presence of Grx2. Maintenance of complex I with an oxidized GSH/GSSG ratio led to a dramatic loss of activity, suggesting that oxidation of the mitochondrial glutathione pool may contribute to the selective complex I inactivation seen in Parkinson's disease. Most significantly, Grx2 catalyzed reversible protein glutathionylation/deglutathionylation over a wide range of GSH/GSSG ratios, from the reduced levels accessible under redox signaling to oxidized ratios only found under severe oxidative

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

    PubMed

    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 < 0.05) higher percentages of viable spermatozoa with high content of peroxides and of superoxides than PFE. In contrast, and despite cryopreservation of stallion spermatozoa increasing DNA fragmentation and disrupting disulphide bonds in 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.

  5. Tespa1 is a novel component of mitochondria-associated endoplasmic reticulum membranes and affects mitochondrial calcium flux.

    PubMed

    Matsuzaki, Hiroshi; Fujimoto, Takahiro; Tanaka, Masatoshi; Shirasawa, Senji

    2013-04-12

    Regulation of intracellular Ca(2+) concentration is critical in numerous biological processes. Inositol 1,4,5-trisphosphate receptor (IP3R) functions as the Ca(2+) release channel on endoplasmic reticulum (ER) membranes. Much attention has been dedicated to mitochondrial Ca(2+) uptake via mitochondria-associated ER membranes (MAM) which is involved in intracellular Ca(2+) homeostasis; however, the molecular mechanisms that link the MAM to mitochondria still remain elusive. We previously reported that Tespa1 (thymocyte-expressed, positive selection-associated gene 1) expressed in lymphocytes physically interacts with IP3R. In this study, we first performed double-immunocytochemical staining of Tespa1 with a mitochondrial marker or an ER marker on an acute T lymphoblastic leukemia cell line, Jurkat cells, by using anti-ATP synthase or anti-calnexin antibody, respectively, and demonstrated that Tespa1 was localized very close to mitochondria and the Tespa1 localization was overlapped with restricted portion of ER. Next, we examined the effects of Tespa1 on the T cell receptor (TCR) stimulation-induced Ca(2+) flux by using Ca(2+) imaging in Jurkat cells. Reduction of Tespa1 protein by Tespa1-specific siRNA diminished TCR stimulation-induced Ca(2+) flux into both mitochondria and cytoplasm through the analyses of the mitochondrial Ca(2+) indicator (Rhod-2) and the cytoplasmic Ca(2+) indicator (Fluo-4), respectively. Furthermore, co-immunoprecipitation assay in HEK293 cells revealed that exogenous Tespa1 protein physically interacted with a MAM-associated protein, GRP75 (glucose-regulated protein 75), but not with an outer mitochondrial membrane protein, VDAC1 (voltage-dependent anion channel 1). All these results suggested that Tespa1 will participate in the molecular link between IP3R-mediated Ca(2+) release and mitochondrial Ca(2+) uptake in the MAM compartment.

  6. The effects of the ergosteroid 7-oxo-dehydroepiandrosterone on mitochondrial membrane potential: possible relationship to thermogenesis.

    PubMed

    Bobyleva, V; Bellei, M; Kneer, N; Lardy, H

    1997-05-01

    Administered 3 beta-hydroxyandrost-5-ene-7,17-dione (7-oxo-DHEA) is more effective than 3 beta-hydroxyandrost-5-en-7-one (DHEA) as an inducer of liver mitochondrial sn-glycerol-3-phosphate dehydrogenase and cytosolic malic enzyme in rats. Like DHEA, the 7-oxo metabolite enhances liver catalase, fatty acylCoA oxidase, cytosolic sn-glycerol-3-phosphate dehydrogenase, mitochondrial substrate oxidation rate, and the reconstructed sn-glycerol 3-phosphate shuttle. The mitochondrial adenine nucleotide carrier is diminished by thyroidectomy and is restored to normal activity by administering 7-oxo-DHEA. The relationship between respiratory rate and proton motive force across the mitochondrial membrane was measured in the nonphosphorylating state. When treated with increasing concentrations of respiratory inhibitors liver mitochondria from rats treated with 7-oxo-DHEA or thyroid hormones show a more rapid decline of membrane potential than do normal liver mitochondria. Thus 7-oxo-DHEA induces an increased proton leak or slip as has been reported for the thyroid hormone by M.D. Brand [(1990) Biochem. Biophys. Acta 1018, 128-133]. This process may contribute to the enhanced thermogenesis caused by ergosteroids as well as by thyroid hormones.

  7. The three domains of the mitochondrial outer membrane protein Mim1 have discrete functions in assembly of the TOM complex.

    PubMed

    Lueder, Franziska; Lithgow, Trevor

    2009-05-06

    The assembly of mitochondrial outer membrane proteins is an essential process, mediated by the SAM complex and a set of additional protein modules. We show that one of these, Mim1, is anchored in the outer membrane with its N-terminus exposed to the cytosol and its C-terminus in the mitochondrial intermembrane space. Using an in vitro assay to measure the multi-step pathway for assembly of Tom40 into the TOM complex, we find that an "early reaction" mediated by the SAM complex is regulated by the N-terminal domain of Mim1. In addition, a "late reaction" catalysed by the Sam37 subunit of the SAM complex is also influenced by Mim1. Thus, Mim1 participates at multiple stages in the assembly of the TOM complex.

  8. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein.

    PubMed

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-12-29

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins.

  9. Atomistic MD simulations reveal the protective role of cholesterol in dimeric beta-amyloid induced disruptions in neuronal membrane mimics

    NASA Astrophysics Data System (ADS)

    Qiu, Liming; Buie, Creighton; Cheng, Sara; Chou, George; Vaughn, Mark; Cheng, K.

    2011-10-01

    Interactions of oligomeric beta-amyloid peptides with neuronal membranes have been linked to the pathogenesis of Alzheimer's disease (AD). The molecular details of the interactions of different lipid components, particularly cholesterol (CHOL), of the membranes with the peptides are not clear. Using an atomistic MD simulations approach, the water permeability barrier, structural geometry and order parameters of binary phosphatidylcholine (PC) and PC/CHOL lipid bilayers were examined from various 200 ns-simulation replicates. Our results suggest that the longer length dimer (2 x 42 residues) perturbs the membrane more than the shorter one (2 x 40 residues). In addition, we discovered a significant protective role of cholesterol in protein-induced disruptions of the membranes. The use of a new Monte-Carlo method in characterizing the structures of the conformal annular lipids in close proximity with the proteins will be introduced. We propose that the neurotoxicity of beta-amyloid peptide may be associated with the nanodomain or raft-like structures of the neuronal membranes in-vivo in the development of AD.

  10. Rejection of pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs) by low pressure reverse osmosis membranes.

    PubMed

    Ozaki, H; Ikejima, N; Shimizu, Y; Fukami, K; Taniguchi, S; Takanami, R; Giri, R R; Matsui, S

    2008-01-01

    This paper aims to elucidate retention characteristics of some pharmaceuticals and personal care products (PPCPs), and endocrine disrupting chemicals (EDCs), by two polyamide low pressure reverse osmosis (LPRO) membranes. Feed solution pH did not have an influence on rejections of undissociated solutes, which was most likely governed by adsorption, size exclusion and diffusion simultaneously. Size exclusion was presumably dominant, especially with tight membranes (UTC-70U). Rejections of the solutes with low dipole moment (<1.0 debye) decreased with increasing octanol-water partition coefficient (K(ow)). The solutes with large K(ow) values were most likely adsorbed on membrane and subsequently passed through it resulting in larger diffusion coefficient (D(p)). The rejections decreased with increasing D(p) values irrespective of their dipole moments. Rejections of solutes with comparatively larger dipole moments might be dominated by diffusion and/or convection rather than their hydrophobicity. However, rejections of solutes with hydroxyl and carboxyl functional groups by UTC-60 increased with solution pH. More than 80% rejections were obtained for degree of dissociation (alpha)>0.5. Electrostatic repulsion played a key role for rejection of dissociated solutes, especially by loose LPRO membranes. Therefore, assessing the dissociation degree at desired pH values can be a key step to obtain an insight of rejection mechanisms by polyamide membranes.

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

    SciTech Connect

    Lizard, G.; Fournel, S.; Genestier, L.; Dhedin, N.

    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.

  12. Opposing effects on mitochondrial membrane potential by malonate and levamisole, whose effect on cell-mediated mineralization is antagonistic.

    PubMed

    Klein, B Y; Gal, I; Libergal, M; Ben-Bassat, H

    1996-01-01

    The act of chondrocyte preparation for primary, enchondral, mineralization is associated with a decline in mitochondrial respiration toward the end of the proliferative zone and the hypertrophic zone in the growth plate. Dexamethasone (Dex)-stimulated cultures of rat marrow stroma constitute a differentiation model simulating, in its energy metabolism, chondrocyte mineralization. In this model, early inhibition of succinate dehydrogenase (SDH) enriches the culture with mineralizing cells, whereas levamisole inhibits mineralization. Dex also increases mitochondrial membrane potential in stromal cells, especially on days 7-8 of stimulation. In the present study, suicide inhibition of SDH, by nitropropionic acid (NPA), in Dex-stimulated cells showed a dose-dependent increase in day 21 mineralization; the maximal effect was induced on days 2-4 of stimulation. Mineralization under 2-day-long exposure to NPA showed a similar trend to the previously studied effect of continuous exposure to malonate applied between days 3-11. Unlike malonate, the effect of NPA required its presence in the cultures for only 2 days and resulted in higher mineralization than that seen under 8 days of malonate. NPA delineated a period, days 2/4 to 7/9, in which inhibition of succinate oxidation is necessary to augment mineralization. During this period, NPA also exhibited OPC selection capacity. Early application of levamisole, under conditions previously shown to decrease day 21 mineralization, maintained mitochondrial membrane potential at the beginning of Dex stimulation but decreased or had little effect on it during days 5-10. By contrast, malonate previously found to increase day 21 mineralization decreased the membrane potential at the beginning of Dex stimulation but increased it later on day 7, or during days 5-10. These results indicate that during osteoprogenitor differentiation, before the mineralization stage, a surge in mitochondrial inner membrane potential during late matrix

  13. Protective effect of silymarin on viability, motility and mitochondrial membrane potential of ram sperm treated with sodium arsenite

    PubMed Central

    Eskandari, Farzaneh; Momeni, Hamid Reza

    2016-01-01

    Background: Sodium arsenite can impair male reproductive function by inducing oxidative stress. Silymarin is known as a potent antioxidant. Objective: This study was performed to investigate if silymarin can prevent the adverse effect of sodium arsenite on ram sperm viability, motility and mitochondrial membrane potential. Materials and Methods: Epidydimal spermatozoa obtained from ram were divided into five groups: 1) Spermatozoa at 0 hr, 2) spermatozoa at 180 min (control), 3) spermatozoa treated with sodium arsenite (10 μM) for 180 min, 4) spermatozoa treated with silymarin (20 μM) + sodium arsenite (10 μM) for 180 min and 5) spermatozoa treated with silymarin (20 μM) for 180 min. MTT assay and Rhodamine 123 staining were used to assess sperm viability and mitochondrial membrane potential respectively. Sperm motility was performed according to World Health Organization (WHO) guidelines. Results: Viability (p<0.01), nonprogressive motility (p<0.001) and intact mitochondrial membrane potential (p<0.001) of the spermatozoa were significantly decreased in sodium arsenite treated group compared to control group. In silymarin + sodium arsenite group, silymarin could significantly reverse the adverse effect of sodium arsenite on these sperm parameters compared to sodium arsenite group (p<0.001). In addition, the application of silymarin alone for 180 minutes could significantly increase progressively motile sperm (p<0.001) and decrease non motile sperm (p<0.01) compared to the control. Conclusion: Silymarin could compensate the adverse effect of sodium arsenite on viability, nonprogressive motility and mitochondrial membrane potential of ram sperm. PMID:27525323

  14. The AAA+ ATPase ATAD3A Controls Mitochondrial Dynamics at the Interface of the Inner and Outer Membranes

    PubMed Central

    Gilquin, Benoît; Taillebourg, Emmanuel; Cherradi, Nadia; Hubstenberger, Arnaud; Gay, Olivia; Merle, Nicolas; Assard, Nicole; Fauvarque, Marie-Odile; Tomohiro, Shiho; Kuge, Osamu; Baudier, Jacques

    2010-01-01

    Dynamic interactions between components of the outer (OM) and inner (IM) membranes control a number of critical mitochondrial functions such as channeling of metabolites and coordinated fission and fusion. We identify here the mitochondrial AAA+ ATPase protein ATAD3A specific to multicellular eukaryotes as a participant in these interactions. The N-terminal domain interacts with the OM. A central transmembrane segment (TMS) anchors the protein in the IM and positions the C-terminal AAA+ ATPase domain in the matrix. Invalidation studies in Drosophila and in a human steroidogenic cell line showed that ATAD3A is required for normal cell growth and cholesterol channeling at contact sites. Using dominant-negative mutants, including a defective ATP-binding mutant and a truncated 50-amino-acid N-terminus mutant, we showed that ATAD3A regulates dynamic interactions between the mitochondrial OM and IM sensed by the cell fission machinery. The capacity of ATAD3A to impact essential mitochondrial functions and organization suggests that it possesses unique properties in regulating mitochondrial dynamics and cellular functions in multicellular organisms. PMID:20154147

  15. Bcl-x(L) blocks a mitochondrial inner membrane channel and prevents Ca2+ overload-mediated cell death.

    PubMed

    Tornero, Daniel; Posadas, Inmaculada; Ceña, Valentín

    2011-01-01

    Apoptosis is an active process that plays a key role in many physiological and pathological conditions. One of the most important organelles involved in apoptosis regulation is the mitochondrion. An increase in intracellular Ca(2+) is a general mechanism of toxicity in neurons which occurs in response to different noxious stimuli like excitotoxicity and ischemia producing apoptotic and necrotic cell death through mitochondria-dependent mechanisms. The Bcl-2 family of proteins modulate the release of pro-apoptotic factors from the mitochondrial intermembrane space during cell death induction by different stimuli. In this work, we have studied, using single-cell imaging and patch-clamp single channel recording, the mitochondrial mechanisms involved in the neuroprotective effect of Bcl-x(L) on Ca(2+) overload-mediated cell death in human neuroblastoma SH-SY5Y cells. We have found that Bcl-x(L) neuroprotective actions take place at mitochondria where this antiapoptotic protein delays both mitochondrial potential collapse and opening of the permeability transition pore by preventing Ca(2+)-mediated mitochondrial multiple conductance channel opening. Bcl-x(L) neuroprotective actions were antagonized by the Bcl-x(L) inhibitor ABT-737 and potentiated by the Ca(2+) chelator BAPTA-AM. As a consequence, this would prevent free radical production, mitochondrial membrane permeabilization, release from mitochondria of pro-apoptotic molecules, caspase activation and cellular death.

  16. Mechanisms for the transport of alpha,omega-dicarboxylates through the mitochondrial inner membrane.

    PubMed

    Liu, G; Hinch, B; Beavis, A D

    1996-10-11

    alpha,omega-Dicarboxylates have antibacterial properties, have been used in the treatment of hyperpigmentary disorders, are active against various melanoma cell lines, and can also undergo beta-oxidation. Little, however, is known about their transport. In this paper, we examine the mitochondrial transport of alpha, omega-dicarboxylates ranging from oxalate (DC2) to sebacate (DC10). DC2-DC10 are transported by the inner membrane anion channel (IMAC). DC6-DC10 are also transported by an electroneutral mechanism that appears to reflect transport of the acid through the lipid bilayer. At 37 degrees C and pH 7.0, DC10 is transported very rapidly at 3 micromol/min.mg, and respiring mitochondria swell in the K+ salts of these acids. This transport mechanism is probably the major pathway by which the longer dicarboxylates enter cells, bacteria, and mitochondria. We also demonstrate that DC5-DC10 can also be transported by an electroneutral mechanism mediated by tributyltin, a potent inhibitor of IMAC. The mechanism appears to involve electroneutral exchange of a TBT-dicarboxylate-H complex for TBT-OH. Finally, we present evidence that of all the dicarboxylates tested only DC2-DC4 can be transported by the classical dicarboxylate carrier.

  17. Mitochondrial Membrane Potential Identifies Cells with Enhanced Stemness for Cellular Therapy.

    PubMed

    Sukumar, Madhusudhanan; Liu, Jie; Mehta, Gautam U; Patel, Shashank J; Roychoudhuri, Rahul; Crompton, Joseph G; Klebanoff, Christopher A; Ji, Yun; Li, Peng; Yu, Zhiya; Whitehill, Greg D; Clever, David; Eil, Robert L; Palmer, Douglas C; Mitra, Suman; Rao, Mahadev; Keyvanfar, Keyvan; Schrump, David S; Wang, Ena; Marincola, Francesco M; Gattinoni, Luca; Leonard, Warren J; Muranski, Pawel; Finkel, Toren; Restifo, Nicholas P

    2016-01-12

    Long-term survival and antitumor immunity of adoptively transferred CD8(+) T cells is dependent on their metabolic fitness, but approaches to isolate therapeutic T cells based on metabolic features are not well established. Here we utilized a lipophilic cationic dye tetramethylrhodamine methyl ester (TMRM) to identify and isolate metabolically robust T cells based on their mitochondrial membrane potential (ΔΨm). Comprehensive metabolomic and gene expression profiling demonstrated global features of improved metabolic fitness in low-ΔΨm-sorted CD8(+) T cells. Transfer of these low-ΔΨm T cells was associated with superior long-term in vivo persistence and an enhanced capacity to eradicate established tumors compared with high-ΔΨm cells. Use of ΔΨm-based sorting to enrich for cells with superior metabolic features was observed in CD8(+), CD4(+) T cell subsets, and long-term hematopoietic stem cells. This metabolism-based approach to cell selection may be broadly applicable to therapies involving the transfer of HSC or lymphocytes for the treatment of viral-associated illnesses and cancer.

  18. Clearing the outer mitochondrial membrane from harmful proteins via lipid droplets

    PubMed Central

    Bischof, Johannes; Salzmann, Manuel; Streubel, Maria Karolin; Hasek, Jiri; Geltinger, Florian; Duschl, Jutta; Bresgen, Nikolaus; Briza, Peter; Haskova, Danusa; Lejskova, Renata; Sopjani, Mentor; Richter, Klaus; Rinnerthaler, Mark

    2017-01-01

    In recent years it turned out that there is not only extensive communication between the nucleus and mitochondria but also between mitochondria and lipid droplets (LDs) as well. We were able to demonstrate that a number of proteins shuttle between LDs and mitochondria and it depends on the metabolic state of the cell on which organelle these proteins are predominantly localized. Responsible for the localization of the particular proteins is a protein domain consisting of two α-helices, which we termed V-domain according to the predicted structure. So far we have detected this domain in the following proteins: mammalian BAX, BCL-XL, TCTP and yeast Mmi1p and Erg6p. According to our experiments there are two functions of this domain: (1) shuttling of proteins to mitochondria in times of stress and apoptosis; (2) clearing the outer mitochondrial membrane from pro- as well as anti-apoptotic proteins by moving them to LDs after the stress ceases. In this way the LDs are used by the cell to modulate stress response. PMID:28386457

  19. Capillarisin Exhibits Anticancer Effects by Inducing Apoptosis, Cell Cycle Arrest and Mitochondrial Membrane Potential Loss in Osteosarcoma Cancer Cells (HOS).

    PubMed

    Chen, N-J; Hao, F-Y; Liu, H; Zhao, H; Li, J-M

    2015-08-01

    The aim of the present study was to assess the anticancer activity of capillarisin against human osteosarcoma (HOS) cancer cells in vitro. Cell viability after capillarisin drug treatment and evaluated by MTT assay. The extent of cell death induced by capillarisin was estimated by using lactate dehydrogenase (LDH) assay. The effect of capillarisin on cell cycle phase distribution and mitochondrial membrane potential (ΛΨm) was demonstrated via flow cytometry using propidium iodide (PI) and rhodamine-123 (Rh-123) DNA-binding fluorescent dyes respectively. Fluorescence microscopy was employed to examine the morphological changes in osteosarcoma cancer cells and presence of apoptotic bodies following capillarisin treatment. The results of this study revealed that capillarisin induced dose-dependent growth inhibition of these cancer cells after 12-h of incubation. Further, capillarisin induced significant release of LDH from these cell cultures and this LDH release was much more noticeable at higher concentrations of capillarisin. Hoechst 33258 staining revealed characteristic morphological features of apoptosis triggered by capillarisin treatment. Cell cycle analysis revealed that capillarisin induced dose-dependent G0/G1-phase cell cycle arrest. Capillarisin also trigerred a progressive and dose-dependent reduction in the mitochondrial membrane potential. In conclusion, capillarisin inhibits cancer cell growth of osteosarcoma cells by inducing apoptosis accompanied with G0/G1-phase cell cycle arrest and loss in mitochondrial membrane potential.

  20. The Bradyrhizobium japonicum cycM gene encodes a membrane-anchored homolog of mitochondrial cytochrome c.

    PubMed Central

    Bott, M; Ritz, D; Hennecke, H

    1991-01-01

    Mitochondrial cytochrome c is a water-soluble protein in the intermembrane space which catalyzes electron transfer from the cytochrome bc1 complex to the terminal oxidase cytochrome aa3. In Bradyrhizobium japonicum, a gene (cycM) which apparently encodes a membrane-anchored homolog of mitochondrial cytochrome c was discovered. The apoprotein deduced from the nucleotide sequence of the cycM gene consists of 184 amino acids with a calculated Mr of 19,098 and an isoelectric point of 8.35. At the N-terminal end (positions 9 to 31), there was a strongly hydrophobic domain which, by forming a transmembrane helix, could serve first as a transport signal and then as a membrane anchor. The rest of the protein was hydrophilic and, starting at position 72, shared about 50% sequence identity with mitochondrial cytochrome c. The heme-binding-site motif Cys-Gly-Ala-Cys-His was located at positions 84 to 88. A B. japonicum cycM insertion mutant (COX122) exhibited an oxidase-negative phenotype and apparently lacked cytochrome aa3 in addition to the CycM protein. The wild-type phenotype with respect to all characteristics tested was restored by providing the cycM gene in trans. The data supported the conclusion that the assembly of cytochrome aa3 depended on the prior incorporation of the CycM protein in the cytoplasmic membrane. Images FIG. 3 PMID:1657867

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

    NASA Astrophysics Data System (ADS)

    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

  2. Relation between cell death progression, reactive oxygen species production and mitochondrial membrane potential in fermenting Saccharomyces cerevisiae cells under heat-shock conditions.

    PubMed

    Pyatrikas, Darya V; Fedoseeva, Irina V; Varakina, Nina N; Rusaleva, Tatyana M; Stepanov, Alexei V; Fedyaeva, Anna V; Borovskii, Gennadii B; Rikhvanov, Eugene G

    2015-06-01

    Moderate heat shock increased reactive oxygen species (ROS) production that led to cell death in glucose-grown Saccharomyces cerevisiae cells. Conditions that disturb mitochondrial functions such as treatment by uncouplers and petite mutation were shown to inhibit ROS production and protects cell from thermal death. Hence, mitochondria are responsible for ROS production and play an active role in cell death. An increase in ROS production was accompanied by hyperpolarization of inner mitochondrial membrane. All agents suppressing hyperpolarization also suppressed heat-induced ROS production. It was supposed that generation of ROS under moderate heat shock in glucose-grown S. cerevisiae cells is driven by the mitochondrial membrane potential.

  3. The Mitochondrial Complex V–Associated Large-Conductance Inner Membrane Current Is Regulated by Cyclosporine and Dexpramipexole

    PubMed Central

    Alavian, Kambiz N.; Dworetzky, Steven I.; Bonanni, Laura; Zhang, Ping; Sacchetti, Silvio; Li, Hongmei; Signore, Armando P.; Smith, Peter J. S.; Gribkoff, Valentin K.

    2015-01-01

    Inefficiency of oxidative phosphorylation can result from futile leak conductance through the inner mitochondrial membrane. Stress or injury may exacerbate this leak conductance, putting cells, and particularly neurons, at risk of dysfunction and even death when energy demand exceeds cellular energy production. Using a novel method, we have recently described an ion conductance consistent with mitochondrial permeability transition pore (mPTP) within the c-subunit of the ATP synthase. Excitotoxicity, reactive oxygen species–producing stimuli, or elevated mitochondrial matrix calcium opens the channel, which is inhibited by cyclosporine A and ATP/ADP. Here we show that ATP and the neuroprotective drug dexpramipexole (DEX) inhibited an ion conductance consistent with this c-subunit channel (mPTP) in brain-derived submitochondrial vesicles (SMVs) enriched for F1FO ATP synthase (complex V). Treatment of SMVs with urea denatured extramembrane components of complex V, eliminated DEX- but not ATP-mediated current inhibition, and reduced binding of [14C]DEX. Direct effects of DEX on the synthesis and hydrolysis of ATP by complex V suggest that interaction of the compound with its target results in functional conformational changes in the enzyme complex. [14C]DEX bound specifically to purified recombinant b and oligomycin sensitivity–conferring protein subunits of the mitochondrial F1FO ATP synthase. Previous data indicate that DEX increased the efficiency of energy production in cells, including neurons. Taken together, these studies suggest that modulation of a complex V–associated inner mitochondrial membrane current is metabolically important and may represent an avenue for the development of new therapeutics for neurodegenerative disorders. PMID:25332381

  4. Coupling membranes as energy-transmitting cables. I. Filamentous mitochondria in fibroblasts and mitochondrial clusters in cardiomyocytes.

    PubMed

    Amchenkova, A A; Bakeeva, L E; Chentsov, Y S; Skulachev, V P; Zorov, D B

    1988-08-01

    An hypothesis considering mitochondria as intracellular power-transmitting protonic cables was tested in human fibroblasts where mitochondria are thin and long and in rat cardiomyocytes where they show cluster organization. Mitochondria in the cell were specifically stained with fluorescent-penetrating cation ethylrhodamine, which electrophoretically accumulates in the mitochondrial matrix. A 40-micron-long mitochondrial filament of fibroblast was illuminated by a very narrow (less than or equal to 0.5 micron) laser beam to induce local damage of the mitochondrial membranes. Such a treatment was found to induce quenching of the ethylrhodamine fluorescence in the entire filament. According to the electron microscope examination, the laser-treated filament retained its continuity after the laser illumination. Other mitochondrial filaments (some of which were localized at a distance less than 10 micron from the laser-treated one) remained fluorescent. In a cell where mitochondrial filaments seemed to be united in a network, laser illumination of one filament resulted in fluorescence quenching in the whole network, whereas fluorescence of small mitochondria not connected with the network was unaffected. The illumination of cardiomyocyte was found to result in the fluorescence quenching not only in a laser-illuminated mitochondrion but also in a large cluster of organelles composed of many mitochondria. Electron microscopy showed that all the mitochondria in the cluster change from the orthodox to the condensed state. It was also found that mitochondria in the cluster are connected to one another with specific junctions. If a mitochondrion did not form junctions with a quenched cluster, its fluorescence was not decreased even when this mitochondrion was localized close to an illuminated one. The size of the mitochondrial cluster may be as long as 50 micron. The cluster is formed by branched chains of contacting mitochondria, which may be defined as Streptio

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

    PubMed Central

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

    2015-01-01

    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 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. PMID:26647379

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

    PubMed

    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.

  7. Delivery of bioactive molecules to the mitochondrial genome using a membrane-fusing, liposome-based carrier, DF-MITO-Porter.

    PubMed

    Yamada, Yuma; Harashima, Hideyoshi

    2012-02-01

    Mitochondrial dysfunction has been implicated in a variety of human diseases. It is now well accepted that mutations and defects in the mitochondrial genome form the basis of these diseases. Therefore, mitochondrial gene therapy and diagnosis would be expected to have great medical benefits. To achieve such a strategy, it will be necessary to deliver therapeutic agents into mitochondria in living cells. We report here on an approach to accomplish this via the use of a Dual Function (DF)-MITO-Porter, aimed at the mitochondrial genome, so-called mitochondrial DNA (mtDNA). The DF-MITO-Porter, a nano carrier for mitochondrial delivery, has the ability to penetrate the endosomal and mitochondrial membranes via step-wise membrane fusion. We first constructed a DF-MITO-Porter encapsulating DNase I protein as a bioactive cargo. It was expected that mtDNA would be digested, when the DNase I was delivered to the mitochondria. We observed the intracellular trafficking of the carriers, and then measured mitochondrial activity and mtDNA-levels after the delivery of DNase I by the DF-MITO-Porter. The findings confirm that the DF-MITO-Porter effectively delivered the DNase I into the mitochondria, and provides a demonstration of its potential use in therapies that are selective for the mitochondrial genome.

  8. Stability of Mitochondrial Membrane Proteins in Terrestrial Vertebrates Predicts Aerobic Capacity and Longevity

    PubMed Central

    Kitazoe, Yasuhiro; Kishino, Hirohisa; Hasegawa, Masami; Matsui, Atsushi; Lane, Nick; Tanaka, Masashi

    2011-01-01

    The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5–10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs. PMID:21824868

  9. The effect of aminoguanidine on sperm motility and mitochondrial membrane potential in varicocelized rats

    PubMed Central

    Alizadeh, Rafieh; Navid, Shadan; Abbasi, Niloofar; Yari, Abazar; Mazaheri, Zohreh; Daneshi, Erfan; Agarwal, Ashok; Abbasi, Mehdi

    2016-01-01

    Objective(s): Increased levels of nitric oxide (NO) in the testicular veins of people suffering from varicocele have already been reported. However, the role of NO-synthase (NOS) isozymes and their inhibitors have not been extensively studied. We aimed to evaluate the inhibitory effects of aminoguanidine (AG), on sperm motility, vitality, and mitochondrial membrane potential (MMP) in varicocelized rats. Materials and Methods: Twenty fore male Wister rats were divided into control, sham, varicocele, and treatment groups. Varicocele and treatment groups underwent partial ligation of left renal vein. Rats in the sham group underwent the same procedures as the varicocele group with the exception of vein ligation. 10 weeks after varicocele induction, sperm parameters were evaluated in all groups. The treatment group received 50 mg/kg AG injection daily for 10 weeks after which they were sacrificed prior to assessment of the parameters. Sperm viability and MMP were assessed by flow cytometry using propidium iodide (PI) and rhodamine 123 (Rh123), respectively. Results: The results of this study show a decrease in sperm viability, motility and MMP in the varicocele group compared with the other groups. After AG injection, we observed that all the parameters were significantly enhanced in the treatment group compared with the other groups. Rh123 staining revealed a positive relation between MMP and sperm motility, whereas PI staining showed a positive relation between sperm motility and viability. Conclusion: The findings of our study show that AG improves sperm motility and MMP, and thus, might be useful in the management of varicocele-related infertility. PMID:28096959

  10. Depletion of zebrafish Tcap leads to muscular dystrophy via disrupting sarcomere-membrane interaction, not sarcomere assembly.

    PubMed

    Zhang, Ruilin; Yang, Jingchun; Zhu, Jin; Xu, Xiaolei

    2009-11-01

    Tcap/telethonin encodes a Z-disc protein that plays important roles in sarcomere assembly, sarcomere-membrane interaction and stretch sensing. It remains unclear why mutations in Tcap lead to limb-girdle muscular dystrophy 2G (LGMD2G) in human patients. Here, we cloned tcap in zebrafish and conducted genetic studies. We show that tcap is functionally conserved, as the Tcap protein appears in the sarcomeric Z-disc and reduction of Tcap resulted in muscular dystrophy-like phenotypes including deformed muscle structure and impaired swimming ability. However, the observations that Tcap integrates into the sarcomere at a stage after the Z-disc becomes periodic, and that the sarcomere remains intact in tcap morphants, suggest that defective sarcomere assembly does not contribute to this particular type of muscular dystrophy. Instead, a defective interaction between the sarcomere and plasma membrane was detected, which was further underscored by the disrupted development of the T-tubule system. Pertinent to a potential function in stretch sensor signaling, zebrafish tcap exhibits a variable expression pattern during somitogenesis. The variable expression is inducible by stretch force, and the expression level of Tcap is negatively regulated by integrin-link kinase (ILK), a protein kinase that is involved in stretch sensing signaling. Together, our genetic studies of tcap in zebrafish suggested that pathogenesis in LGMD2G is due to a disruption of sarcomere-T-tubular interaction, but not of sarcomere assembly per se. In addition, our data prompted a novel hypothesis that predicts that the transcription level of Tcap can be regulated by the stretch force to ensure proper sarcomere-membrane interaction in striated muscles.

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

    PubMed

    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.

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

    PubMed Central

    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

  13. Impact of operating conditions on the removal of endocrine disrupting chemicals by membrane photocatalytic reactor.

    PubMed

    López Fernández, Raquel; Coleman, Heather M; Le-Clech, Pierre

    2014-08-01

    This study focuses on the performance of a submerged membrane photocatalytic reactor for the removal of 17beta-oestradiol (E2) in the presence of humic acid (HA). In addition to the impact of operating parameters, such as membrane pore size, ultraviolet (UV) intensity and hydraulic retention time (HRT), the influence of long-term operation was also assessed by advanced characterization of the fouling layer formed on the membrane. The tighter (0.04 microm) hollow fibre polyvinylydene fluoride (PVDF) membrane was found to exhibit not only higher HA removal than the (0.2 microm) module (85% and 75%, respectively), but also greater transmembrane pressure (TMP) values and higher irreversible fouling. Long-term operation conditions have been simulated by conducting an ageing catalyst process and demonstrated a decrease in performance obtained with time. The artificially aged TiO2 resulted in higher TMP values and lower HA removals (about 10-20% decrease) compared with the non-aged catalyst. For E2 removal in the presence of HA, the passive adsorption of the oestrogen onto the organic matter was found to be significant (40% of the E2 adsorbed after I h), demonstrating the importance of the nature of the water matrix for this type of treatment process. An increase in the UV light intensity was observed to favour the E2 elimination, leading to more than 90% removal when using 64 W combined with PVDF membrane and an HRT of 3 h.

  14. Interactive HIV-1 Tat and morphine-induced synaptodendritic injury is triggered through focal disruptions in Na⁺ influx, mitochondrial instability, and Ca²⁺ overload.

    PubMed

    Fitting, Sylvia; Knapp, Pamela E; Zou, Shiping; Marks, William D; Bowers, M Scott; Akbarali, Hamid I; Hauser, Kurt F

    2014-09-17

    Synaptodendritic injury is thought to underlie HIV-associated neurocognitive disorders and contributes to exaggerated inflammation and cognitive impairment seen in opioid abusers with HIV-1. To examine events triggering combined transactivator of transcription (Tat)- and morphine-induced synaptodendritic injury systematically, striatal neuron imaging studies were conducted in vitro. These studies demonstrated nearly identical pathologic increases in dendritic varicosities as seen in Tat transgenic mice in vivo. Tat caused significant focal increases in intracellular sodium ([Na(+)]i) and calcium ([Ca(2+)]i) in dendrites that were accompanied by the emergence of dendritic varicosities. These effects were largely, but not entirely, attenuated by the NMDA and AMPA receptor antagonists MK-801 and CNQX, respectively. Concurrent morphine treatment accelerated Tat-induced focal varicosities, which were accompanied by localized increases in [Ca(2+)]i and exaggerated instability in mitochondrial inner membrane potential. Importantly, morphine's effects were prevented by the μ-opioid receptor antagonist CTAP and were not observed in neurons cultured from μ-opioid receptor knock-out mice. Combined Tat- and morphine-induced initial losses in ion homeostasis and increases in [Ca(2+)]i were attenuated by the ryanodine receptor inhibitor ryanodine, as well as pyruvate. In summary, Tat induced increases in [Na(+)]i, mitochondrial instability, excessive Ca(2+) influx through glutamatergic receptors, and swelling along dendrites. Morphine, acting via μ-opioid receptors, exacerbates these excitotoxic Tat effects at the same subcellular locations by mobilizing additional [Ca(2+)]i and by further disrupting [Ca(2+)]i homeostasis. We hypothesize that the spatiotemporal relationship of μ-opioid and aberrant AMPA/NMDA glutamate receptor signaling is critical in defining the location and degree to which opiates exacerbate the synaptodendritic injury commonly observed in neuroAIDS.

  15. Disruption of the membrane-bound alcohol dehydrogenase-encoding gene improved glycerol use and dihydroxyacetone productivity in Gluconobacter oxydans.

    PubMed

    Habe, Hiroshi; Fukuoka, Tokuma; Morita, Tomotake; Kitamoto, Dai; Yakushi, Toshiharu; Matsushita, Kazunobu; Sakaki, Keiji

    2010-01-01

    Dihydroxyacetone (DHA) production from glycerol by Gluconobacter oxydans is an industrial form of fermentation, but some problems exist related to microbial DHA production. For example, glycerol inhibits DHA production and affects its biological activity. G. oxydans produces both DHA and glyceric acid (GA) from glycerol simultaneously, and membrane-bound glycerol dehydrogenase and membrane-bound alcohol dehydrogenases are involved in the two reactions, respectively. We discovered that the G. oxydans mutant DeltaadhA, in which the membrane-bound alcohol dehydrogenase-encoding gene (adhA) was disrupted, significantly improved its ability to grow in a higher concentration of glycerol and to produce DHA compared to a wild-type strain. DeltaadhA grew on 220 g/l of initial glycerol and produced 125 g/l of DHA during a 3-d incubation, whereas the wild-type did not. Resting DeltaadhA cells converted 230 g/l of glycerol aqueous solution to 139.7 g/l of DHA during a 3-d incubation. The inhibitory effect of glycerate sodium salt on DeltaadhA was investigated. An increase in the glycerate concentration at the beginning of growth resulted in decreases in both growth and DHA production.

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

    PubMed Central

    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.

    2016-01-01

    Summary 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. PMID:26712463

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

    PubMed

    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

  18. Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body.

    PubMed Central

    Duchen, M R; Biscoe, T J

    1992-01-01

    1. In the accompanying paper (Duchen & Biscoe, 1992) we have described graded changes in autofluorescence derived from mitochondrial NAD(P)H in type I cells of the carotid body in response to changes of PO2 over a physiologically significant range. These observations suggest that mitochondrial function in these cells is unusually sensitive to oxygen and could play a role in oxygen sensing. We have now explored further the relationships between hypoxia, mitochondrial membrane potential (delta psi m) and [Ca2+]i. 2. The fluorescence of Rhodamine 123 (Rh 123) accumulated within mitochondria is quenched by delta psi m. Mitochondrial depolarization thus increases the fluorescence signal. Blockade of electron transport (CN-, anoxia, rotenone) and uncoupling agents (e.g. carbonyl cyanide p-trifluoromethoxy-phenylhydrazone; FCCP) increased fluorescence by up to 80-120%, while fluorescence was reduced by blockade of the F0 proton channel of the mitochondrial ATP synthase complex (oligomycin). 3. delta psi m depolarized rapidly with anoxia, and was usually completely dissipated within 1-2 min. The depolarization of delta psi m with anoxia (or CN-) and repolarization on reoxygenation both followed a time course well characterized as the sum of two exponential processes. Oligomycin (0.2-2 micrograms/ml) hyperpolarized delta psi m and abolished the slower components of both the depolarization with anoxia and of the subsequent repolarization. These data (i) illustrate the role of the F1-F0 ATP synthetase in slowing the rate of dissipation of delta psi m on cessation of electron transport, (ii) confirm blockade of the ATP synthetase by oligomycin at these concentrations, and (iii) indicate significant accumulation of intramitochondrial ADP during 1-2 min of anoxia. 4. Depolarization of delta psi m was graded with graded changes in PO2 below about 60 mmHg. The stimulus-response curves thus constructed strongly resemble those for [Ca2+]i and NAD(P)H with PO2. The change in delta

  19. PrP106-126 peptide disrupts lipid membranes: Influence of C-terminal amidation

    SciTech Connect

    Zheng Wenfu; Wang Lijun; Hong Yuankai; Sha Yinlin

    2009-02-06

    PrP106-126 is located within the important domain concerning membrane related conformational conversion of human Prion protein (from cellular isoform PrP{sup C} to scrapie isoform PrP{sup Sc}). Recent advances reveal that the pathological and physicochemical properties of PrP106-126 peptide are very sensitive to its N-terminal amidation, however, the detailed mechanism remains unclear. In this work, we studied the interactions of the PrP106-126 isoforms (PrP106-126{sub CONH2} and PrP106-126{sub COOH}) with the neutral lipid bilayers by atomic force microscopy, surface plasmon resonance and fluorescence spectroscopy. The membrane structures were disturbed by the two isoforms in a similarly stepwise process. The distinct morphological changes of the membrane were characterized by formation of semi-penetrated defects and sigmoidal growth of flat high-rise domains on the supported lipid bilayers. However, PrP106-126{sub COOH} displayed a higher peptide-lipid binding affinity than PrP106-126{sub CONH2} ({approx}2.9 times) and facilitated the peptide-lipid interactions by shortening the lag time. These results indicate that the C-terminal amidation may influence the pathological actions of PrP106-126 by lowering the interaction potentials with lipid membranes.

  20. Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth

    PubMed Central

    Sthanam, Lakshmi Kavitha; Srivastava, Rohit; Basu, Bhakti; Dutta, Suryendu; Sen, Shamik; Modi, Deepak

    2016-01-01

    Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death. PMID:27583406

  1. A dual antibacterial mechanism involved in membrane disruption and DNA binding of 2R,3R-dihydromyricetin from pine needles of Cedrus deodara against Staphylococcus aureus.

    PubMed

    Wu, Yanping; Bai, Jinrong; Zhong, Kai; Huang, Yina; Gao, Hong

    2017-03-01

    The antibacterial activity and mechanism of 2R,3R-dihydromyricetin (DMY) against Staphylococcus aureus were investigated. The minimum inhibitory concentration of DMY against S. aureus was 0.125mg/ml, and the growth inhibitory assay also revealed that DMY showed a potent antibacterial activity against S. aureus. Massive nucleotide leakage and flow cytometric analysis demonstrated that DMY disrupted the membrane integrity of S. aureus. Morphological changes and membrane hyperpolarization of S. aureus cells treated with DMY further suggested that DMY destroyed cell membrane. Meanwhile, DMY probably interacted with membrane lipids and proteins, causing a significant reduction in membrane fluidity and changes in conformation of membrane protein. Moreover, DMY could interact with S. aureus DNA through the groove binding mode. Overall, the results suggested that DMY could be applied as a candidate for the development of new food preservatives as it achieved bactericidal activity by damaging cell membrane and binding to intracellular DNA.

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

    PubMed

    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.

  3. Phellinus linteus polysaccharide extracts increase the mitochondrial membrane potential and cause apoptotic death of THP-1 monocytes

    PubMed Central

    2013-01-01

    Background The differentiation resp. death of human monocytic THP-1 cells induced by polysaccharide extracts of the medicinal mushrooms Phellinus linteus, Agaricus bisporus and Agaricus brasiliensis have been studied. This study aims to identify leads for the causal effects of these mushroom components on cell differentiation and death. Methods THP-1 cells were treated with different polysaccharide extracts of mushrooms and controls. Morphological effects were observed by light microscopy. Flow cytometry was applied to follow the cell differentiation by cell cycle shifts after staining with propidium iodide, changes of mitochondrial membrane potential after incubation with JC-1, and occurrence of intracellular reactive oxygen species after incubation with hydroethidine. Principal component analysis of the data was performed to evaluate the cellular effects of the different treatments. Results P. linteus polysaccharide extracts induced dose-dependent apoptosis of THP-1 cells within 24 h, while A. bisporus and A. brasiliensis polysaccharide extracts caused differentiation into macrophages. A pure P. linteus polysaccharide had no effect. Apoptosis was inhibited by preincubating THP-1 cells with human serum. The principal component analysis revealed that P. linteus, A. bisporus and A. brasiliensis polysaccharide extracts increased reactive oxygen species production. Both A. bisporus and A. brasiliensis polysaccharide extracts decreased the mitochondrial membrane potential, while this was increased by P. linteus polysaccharide extracts. Conclusions P. linteus polysaccharide extracts caused apoptosis of THP-1 monocytes while A. bisporus and A. brasiliensis polysaccharide extracts caused these cells to differentiate into macrophages. The protective effects of human serum suggested that P. linteus polysaccharide extract induced apoptosis by extrinsic pathway, i.e. by binding to the TRAIL receptor. The mitochondrial membrane potential together with reactive oxygen species

  4. The presence of highly disruptive 16S rRNA mutations in clinical samples indicates a wider role for mutations of the mitochondrial ribosome in human disease

    PubMed Central

    Elson, Joanna L.; Smith, Paul M.; Greaves, Laura C.; Lightowlers, Robert N.; Chrzanowska-Lightowlers, Zofia M.A.; Taylor, Robert W.; Vila-Sanjurjo, Antón

    2015-01-01

    Mitochondrial DNA mutations are well recognized as an important cause of disease, with over two hundred variants in the protein encoding and mt-tRNA genes associated with human disorders. In contrast, the two genes encoding the mitochondrial rRNAs (mt-rRNAs) have been studied in far less detail. This is because establishing the pathogenicity of mt-rRNA mutations is a major diagnostic challenge. Only two disease causing mutations have been identified at these loci, both mapping to the small subunit (SSU). On the large subunit (LSU), however, the evidence for the presence of pathogenic LSU mt-rRNA changes is particularly sparse. We have previously expanded the list of deleterious SSU mt-rRNA mutations by identifying highly disruptive base changes capable of blocking the activity of the mitoribosomal SSU. To do this, we used a new methodology named heterologous inferential analysis (HIA). The recent arrival of near-atomic-resolution structures of the human mitoribosomal LSU, has enhanced the power of our approach by permitting the analysis of the corresponding sites of mutation within their natural structural context. Here, we have used these tools to determine whether LSU mt-rRNA mutations found in the context of human disease and/or ageing could disrupt the function of the mitoribosomal LSU. Our results clearly show that, much like the for SSU mt-rRNA, LSU mt-rRNAs mutations capable of compromising the function of the mitoribosomal LSU are indeed present in clinical samples. Thus, our work constitutes an important contribution to an emerging view of the mitoribosome as an important element in human health. PMID:26349026

  5. Mitochondrial dysfunction and organophosphorus compounds

    SciTech Connect

    Karami-Mohajeri, Somayyeh; Abdollahi, Mohammad

    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.

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

    PubMed

    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.

  7. Mitochondrial dynamics in the mouse liver infected by Schistosoma mansoni.

    PubMed

    Chen, Tina Tu-Wen; Wu, Lawrence Shih Hsin; Hsu, Paul Wei-Che; Pang, Cheng-Yoong; Lee, Kin-Mu; Cheng, Po-Ching; Peng, Shih-Yi

    2015-08-01

    Mitochondrial dynamics is crucial for regulation of cell homeostasis. Schistosoma mansoni is one of the most common parasites known to cause liver disease. Mice infected by S. mansoni show acute symptoms of schistosomiasis after 8 weeks. Hence, in this study, we attempted to assess the direct effects of S. mansoni infection on mice liver, and to explore the expression of mitochondrial morphology, dynamics, and function. Our recent findings show that S. mansoni infection changes mitochondrial morphology and affects mitochondrial functions, which attenuates mitochondrial membrane potential and ATP generation. S. mansoni-infected mice increases mitochondrial numbers by upregulating of genes involved in mitochondrial biogenesis, including peroxisome proliferator-activated receptor c co-activator 1α (PGC1α) and mitochondrial transcription factor A (Tfam). This may promote mitochondria generation for accelerating the recovery of mitochondrial functions. Moreover, S. mansoni would disrupt mitochondrial dynamics including induced mitochondrial fission and promoted mitochondrial fragmentation in mice liver. More importantly, S. mansoni further stimulated upregulation both extrinsic and intrinsic apoptosis pathway in infected mice liver. The intrinsic pathway was triggered by cytochrome c release. Additionally, NFκB (nuclear factor-kappa B, p65) could play a protective role to inhibit apoptosis through reducing active caspase-3 expression. Therefore, our results confirmed the liver damage mechanism of experimental schistosomiasis in mice model.

  8. The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

    PubMed Central

    Harsman, Anke; Oeljeklaus, Silke; Wenger, Christoph; Huot, Jonathan L.; Warscheid, Bettina; Schneider, André

    2016-01-01

    Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins. PMID:27991487

  9. Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis.

    PubMed

    Brenzan, M A; Santos, A O; Nakamura, C V; Filho, B P Dias; Ueda-Nakamura, T; Young, M C M; Côrrea, A G; Júnior, J Alvim; Morgado-Díaz, J A; Cortez, D A G

    2012-02-15

    We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD₅₀ of 0.9 μM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.

  10. Topogenesis of Mammalian Oxa1, a Component of the Mitochondrial Inner Membrane Protein Export Machinery*S⃞

    PubMed Central

    Sato, Takashi; Mihara, Katsuyoshi

    2009-01-01

    Oxa1 is a mitochondrial inner membrane protein with a predicted five-transmembrane segment (TM1∼5) topology in which the N terminus and a hydrophilic loop, L2, are exposed to the intermembrane space and the C-terminal region and two loops, L1 and L3, are exposed to the matrix. Oxa1 mediates the insertion of mitochondrial DNA-encoded subunits of respiratory complexes and several nuclear DNA-encoded proteins into the inner membrane from the matrix. Compared with yeast Oxa1, little is known about the import and function of mammalian Oxa1. Here, we investigated the topogenesis of Oxa1 in HeLa cells using systematic deletion or mutation constructs and found that (i) the N-terminal 64-residue segment formed a presequence, and its deletion directed the mature protein to the endoplasmic reticulum, indicating that the presequence arrests cotranslational activation of the potential endoplasmic reticulum-targeting signal within mature Oxa1, (ii) systematic deletion of Oxa1 TM segments revealed that the presence of all five TMs is essential for efficient membrane integration, (iii) the species-conserved hexapeptide (GLPWWG) located near the N terminus of TM1 was essential for export of the N-terminal segment and L2 into the intermembrane space from the matrix, i.e. for correct topogenesis of Oxa1, and (iv) GLPWWG placed near the N terminus of TM2 or TM3 in the reporter construct also supported its membrane integration in the Nout-Cin orientation. Together, these results demonstrated that topogenesis of Oxa1 is a cooperative event of all five TMs, and GLPWWG followed immediately by TM1 is essential for correct Oxa1 topogenesis. PMID:19349278

  11. Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40.

    PubMed

    Kuszak, Adam J; Jacobs, Daniel; Gurnev, Philip A; Shiota, Takuya; Louis, John M; Lithgow, Trevor; Bezrukov, Sergey M; Rostovtseva, Tatiana K; Buchanan, Susan K

    2015-10-23

    Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function.

  12. Evidence of Distinct Channel Conformations and Substrate Binding Affinities for the Mitochondrial Outer Membrane Protein Translocase Pore Tom40*

    PubMed Central

    Kuszak, Adam J.; Jacobs, Daniel; Gurnev, Philip A.; Shiota, Takuya; Louis, John M.; Lithgow, Trevor; Bezrukov, Sergey M.; Rostovtseva, Tatiana K.; Buchanan, Susan K.

    2015-01-01

    Nearly all mitochondrial proteins are coded by the nuclear genome and must be transported into mitochondria by the translocase of the outer membrane complex. Tom40 is the central subunit of the translocase complex and forms a pore in the mitochondrial outer membrane. To date, the mechanism it utilizes for protein transport remains unclear. Tom40 is predicted to comprise a membrane-spanning β-barrel domain with conserved α-helical domains at both the N and C termini. To investigate Tom40 function, including the role of the N- and C-terminal domains, recombinant forms of the Tom40 protein from the yeast Candida glabrata, and truncated constructs lacking the N- and/or C-terminal domains, were functionally characterized in planar lipid membranes. Our results demonstrate that each of these Tom40 constructs exhibits at least four distinct conductive levels and that full-length and truncated Tom40 constructs specifically interact with a presequence peptide in a concentration- and voltage-dependent manner. Therefore, neither the first 51 amino acids of the N terminus nor the last 13 amino acids of the C terminus are required for Tom40 channel formation or for the interaction with a presequence peptide. Unexpectedly, substrate binding affinity was dependent upon the Tom40 state corresponding to a particular conductive level. A model where two Tom40 pores act in concert as a dimeric protein complex best accounts for the observed biochemical and electrophysiological data. These results provide the first evidence for structurally distinct Tom40 conformations playing a role in substrate recognition and therefore in transport function. PMID:26336107

  13. Mutations in Lama1 disrupt retinal vascular development and inner limiting membrane formation.

    PubMed

    Edwards, Malia M; Mammadova-Bach, Elmina; Alpy, Fabien; Klein, Annick; Hicks, Wanda L; Roux, Michel; Simon-Assmann, Patricia; Smith, Richard S; Orend, Gertraud; Wu, Jiang; Peachey, Neal S; Naggert, Jürgen K; Lefebvre, Olivier; Nishina, Patsy M

    2010-03-05

    The Neuromutagenesis Facility at the Jackson Laboratory generated a mouse model of retinal vasculopathy, nmf223, which is characterized clinically by vitreal fibroplasia and vessel tortuosity. nmf223 homozygotes also have reduced electroretinogram responses, which are coupled histologically with a thinning of the inner nuclear layer. The nmf223 locus was mapped to chromosome 17, and a missense mutation was identified in Lama1 that leads to the substitution of cysteine for a tyrosine at amino acid 265 of laminin alpha1, a basement membrane protein. Despite normal localization of laminin alpha1 and other components of the inner limiting membrane, a reduced integrity of this structure was suggested by ectopic cells and blood vessels within the vitreous. Immunohistochemical characterization of nmf223 homozygous retinas demonstrated the abnormal migration of retinal astrocytes into the vitreous along with the persistence of hyaloid vasculature. The Y265C mutation significantly reduced laminin N-terminal domain (LN) interactions in a bacterial two-hybrid system. Therefore, this mutation could affect interactions between laminin alpha1 and other laminin chains. To expand upon these findings, a Lama1 null mutant, Lama1(tm1.1Olf), was generated that exhibits a similar but more severe retinal phenotype than that seen in nmf223 homozygotes. The increased severity of the Lama1 null mutant phenotype is probably due to the complete loss of the inner limiting membrane in these mice. This first report of viable Lama1 mouse mutants emphasizes the importance of this gene in retinal development. The data presented herein suggest that hypomorphic mutations in human LAMA1 could lead to retinal disease.

  14. Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane.

    PubMed

    Alakomi, H L; Skyttä, E; Saarela, M; Mattila-Sandholm, T; Latva-Kala, K; Helander, I M

    2000-05-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl(2). Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances.

  15. Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane

    PubMed Central

    Alakomi, H.-L.; Skyttä, E.; Saarela, M.; Mattila-Sandholm, T.; Latva-Kala, K.; Helander, I. M.

    2000-01-01

    The effect of lactic acid on the outer membrane permeability of Escherichia coli O157:H7, Pseudomonas aeruginosa, and Salmonella enterica serovar Typhimurium was studied utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis. For control purposes, similar assays were performed with EDTA (a permeabilizer acting by chelation) and with hydrochloric acid, the latter at pH values corresponding to those yielded by lactic acid, and also in the presence of KCN. Already 5 mM (pH 4.0) lactic acid caused prominent permeabilization in each species, the effect in the fluorescence assay being stronger than that of EDTA or HCl. Similar results were obtained in the presence of KCN, except for P. aeruginosa, for which an increase in the effect of HCl was observed in the presence of KCN. The permeabilization by lactic and hydrochloric acid was partly abolished by MgCl2. Lactic acid sensitized E. coli and serovar Typhimurium to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than did HCl, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was effectively sensitized to lysozyme by lactic acid and by HCl. Considerable proportions of lipopolysaccharide were liberated from serovar Typhimurium by these acids; analysis of liberated material by electrophoresis and by fatty acid analysis showed that lactic acid was more active than EDTA or HCl in liberating lipopolysaccharide from the outer membrane. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the pH, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances. PMID:10788373

  16. The Function of the Mitochondrial Calcium Uniporter in Neurodegenerative Disorders

    PubMed Central

    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

  17. Erastin Disrupts Mitochondrial Permeability Transition Pore (mPTP) and Induces Apoptotic Death of Colorectal Cancer Cells

    PubMed Central

    Huo, Haizhong; Zhou, Zhiyuan; Qin, Jian; Liu, Wenyong; Wang, Bing; Gu, Yan

    2016-01-01

    We here evaluated the potential anti-colorectal cancer activity by erastin, a voltage-dependent anion channel (VDAC)-binding compound. Our in vitro studies showed that erastin exerted potent cytotoxic effects against multiple human colorectal cancer cell lines, possibly via inducing oxidative stress and caspase-9 dependent cell apoptosis. Further, mitochondrial permeability transition pore (mPTP) opening was observed in erastin-treated cancer cells, which was evidenced by VDAC-1 and cyclophilin-D (Cyp-D) association, mitochondrial depolarization, and cytochrome C release. Caspase inhibitors, the ROS scavenger MnTBAP, and mPTP blockers (sanglifehrin A, cyclosporin A and bongkrekic acid), as well as shRNA-mediated knockdown of VDAC-1, all significantly attenuated erastin-induced cytotoxicity and apoptosis in colorectal cancer cells. On the other hand, over-expression of VDAC-1 augmented erastin-induced ROS production, mPTP opening, and colorectal cancer cell apoptosis. In vivo studies showed that intraperitoneal injection of erastin at well-tolerated doses dramatically inhibited HT-29 xenograft growth in severe combined immunodeficient (SCID) mice. Together, these results demonstrate that erastin is cytotoxic and pro-apoptotic to colorectal cancer cells. Erastin may be further investigated as a novel anti-colorectal cancer agent. PMID:27171435

  18. A novel transferrin/TfR2-mediated mitochondrial iron transport system is disrupted in Parkinson's disease

    PubMed Central

    Mastroberardino, Pier Giorgio; Hoffman, Eric K.; Horowitz, Maxx P.; Betarbet, Ranjita; Taylor, Georgia; Cheng, Dongmei; Na, Hye Mee; Gutekunst, Claire-Anne; Gearing, Marla; Trojanowski, John Q.; Anderson, Marjorie; Chu, Charleen T.; Peng, Junmin; Greenamyre, J. Timothy

    2009-01-01

    More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinson's disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell – mitochondria and the respiratory complexes – has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention. PMID:19250966

  19. CoQ deficiency causes disruption of mitochondrial sulfide oxidation, a new pathomechanism associated with this syndrome.

    PubMed

    Luna-Sánchez, Marta; Hidalgo-Gutiérrez, Agustín; Hildebrandt, Tatjana M; Chaves-Serrano, Julio; Barriocanal-Casado, Eliana; Santos-Fandila, Ángela; Romero, Miguel; Sayed, Ramy Ka; Duarte, Juan; Prokisch, Holger; Schuelke, Markus; Distelmaier, Felix; Escames, Germaine; Acuña-Castroviejo, Darío; López, Luis C

    2017-01-01

    Coenzyme Q (CoQ) is a key component of the mitochondrial respiratory chain, but it also has several other functions in the cellular metabolism. One of them is to function as an electron carrier in the reaction catalyzed by sulfide:quinone oxidoreductase (SQR), which catalyzes the first reaction in the hydrogen sulfide oxidation pathway. Therefore, SQR may be affected by CoQ deficiency. Using human skin fibroblasts and two mouse models with primary CoQ deficiency, we demonstrate that severe CoQ deficiency causes a reduction in SQR levels and activity, which leads to an alteration of mitochondrial sulfide metabolism. In cerebrum of Coq9(R239X) mice, the deficit in SQR induces an increase in thiosulfate sulfurtransferase and sulfite oxidase, as well as modifications in the levels of thiols. As a result, biosynthetic pathways of glutamate, serotonin, and catecholamines were altered in the cerebrum, and the blood pressure was reduced. Therefore, this study reveals the reduction in SQR activity as one of the pathomechanisms associated with CoQ deficiency syndrome.

  20. Genomic and Proteomic Profiling Reveals Reduced Mitochondrial Function and Disruption of the Neuromuscular Junction Driving Rat Sarcopenia

    PubMed Central

    Ibebunjo, Chikwendu; Chick, Joel M.; Kendall, Tracee; Eash, John K.; Li, Christine; Zhang, Yunyu; Vickers, Chad; Wu, Zhidan; Clarke, Brian A.; Shi, Jun; Cruz, Joseph; Fournier, Brigitte; Brachat, Sophie; Gutzwiller, Sabine; Ma, QiCheng; Markovits, Judit; Broome, Michelle; Steinkrauss, Michelle; Skuba, Elizabeth; Galarneau, Jean-Rene; Gygi, Steven P.

    2013-01-01

    Molecular mechanisms underlying sarcopenia, the age-related loss of skeletal muscle mass and function, remain unclear. To identify molecular changes that correlated best with sarcopenia and might contribute to its pathogenesis, we determined global gene expression profiles in muscles of rats aged 6, 12, 18, 21, 24, and 27 months. These rats exhibit sarcopenia beginning at 21 months. Correlation of the gene expression versus muscle mass or age changes, and functional annotation analysis identified gene signatures of sarcopenia distinct from gene signatures of aging. Specifically, mitochondrial energy metabolism (e.g., tricarboxylic acid cycle and oxidative phosphorylation) pathway genes were the most downregulated and most significantly correlated with sarcopenia. Also, perturbed were genes/pathways associated with neuromuscular junction patency (providing molecular evidence of sarcopenia-related functional denervation and neuromuscular junction remodeling), protein degradation, and inflammation. Proteomic analysis of samples at 6, 18, and 27 months confirmed the depletion of mitochondrial energy metabolism proteins and neuromuscular junction proteins. Together, these findings suggest that therapeutic approaches that simultaneously stimulate mitochondrogenesis and reduce muscle proteolysis and inflammation have potential for treating sarcopenia. PMID:23109432

  1. The acylphloroglucinols hyperforin and myrtucommulone A cause mitochondrial dysfunctions in leukemic cells by direct interference with mitochondria.

    PubMed

    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.

  2. Protein translocation channel of mitochondrial inner membrane and matrix-exposed import motor communicate via two-domain coupling protein

    PubMed Central

    Banerjee, Rupa; Gladkova, Christina; Mapa, Koyeli; Witte, Gregor; Mokranjac, Dejana

    2015-01-01

    The majority of mitochondrial proteins are targeted to mitochondria by N-terminal presequences and use the TIM23 complex for their translocation across the mitochondrial inner membrane. During import, translocation through the channel in the inner membrane is coupled to the ATP-dependent action of an Hsp70-based import motor at the matrix face. How these two processes are coordinated remained unclear. We show here that the two domain structure of Tim44 plays a central role in this process. The N-terminal domain of Tim44 interacts with the components of the import motor, whereas its C-terminal domain interacts with the translocation channel and is in contact with translocating proteins. Our data suggest that the translocation channel and the import motor of the TIM23 complex communicate through rearrangements of the two domains of Tim44 that are stimulated by translocating proteins. DOI: http://dx.doi.org/10.7554/eLife.11897.001 PMID:26714107

  3. Mitochondrial motility in axons: membranous organelles may interact with the force generating system through multiple surface binding sites.

    PubMed

    Martz, D; Lasek, R J; Brady, S T; Allen, R D

    1984-01-01

    In living tissue, membrane-bound organelles, including mitochondria, move along parallel cytoplasmic pathways. Motion is directed and tends to be confined to a single path. Deviations from this single path motion are rare. When present, however, they tend to occur at points of intersection of cytoskeletal linear elements (LE). Such intersections are relatively uncommon in intact axons and extruded axoplasm. However, we have found that such intersections can be produced in extruded preparations by shear forces directed tangential to the axoplasmic surface. We have studied the detailed behavior of mitochondria in extruded squid axoplasm. Special attention was directed to the relationship between mitochondrial shape changes and orientation of cytoskeletal LE. The most striking of these changes in shape is branching. In this process, the mitochondrion transiently assumes a triradial (three-ended) shape. This appearance may be maintained for seconds to minutes before the normal cylindrical shape is resumed by absorption of either the newly formed end or, more commonly, one of the original ends. The frequency of branching appears to be dependent on the degree of cytoskeletal organization. It becomes more common as the number of apparent intersections between cytoskeletal LE increases. Further, the formation of new ends seems to occur along paths defined by cytoskeletal elements. These observations suggest that the mitochondrial membrane is multivalent. That is, it contains multiple sites capable of interacting with the axonal force generation apparatus. Furthermore, LE in the cytoskeleton may indicate the paths along which these interactions are permissible.

  4. VCP cooperates with UBXD1 to degrade mitochondrial outer membrane protein MCL1 in model of Huntington's disease.

    PubMed

    Guo, Xing; Qi, Xin

    2017-02-01

    Proteasome-dependent turnover of mitochondrial outer membrane (OMM)-associated proteins is one of the mechanisms for maintaining proper mitochondrial quality and function. However, the underlying pathways and their implications in human disease are poorly understood. Huntington's disease (HD) is a fatal, inherited neurodegenerative disorder caused by expanded CAG repeats in the N terminal of the huntingtin gene (mutant Huntingtin, mtHtt). In this study, we show an extensive degradation of the OMM protein MCL1 (Myeloid cell leukemia sequence 1) in both HD mouse striatal cells and HD patient fibroblasts. The decrease in MCL1 level is associated with mitochondrial and cellular damage. Valosin-containing-protein (VCP) is an AAA-ATPase central to protein turnover via the ubiquitin proteasome system (UPS). We found that VCP translocates to mitochondria and promotes MCL1 degradation in HD cell cultures. Either down-regulation of VCP by RNA interference or inhibition of VCP by a dominant negative mutant abolishes MCL1 degradation in HD cell cultures. We further show that UBX-domain containing protein 1 (UBXD1), a known co-factor of VCP assisting in the recognition of substrates for protein degradation, selectively binds to MCL1 and interacts with VCP to mediate MCL1 extraction from the mitochondria. These results indicate that the OMM protein MCL1 is degraded by the VCP-UBXD1 complex and that the process is promoted by the presence of mtHtt. Therefore, our finding provides a new insight into the mechanism of mitochondrial dysfunction in HD.

  5. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling.

    PubMed

    Silver, Kristopher; Littlejohn, Alaina; Thomas, Laurel; Marsh, Elizabeth; Lillich, James D

    2015-12-15

    Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

  6. Inhibition of Kv channel expression by NSAIDs depolarizes membrane potential and inhibits cell migration by disrupting calpain signaling

    PubMed Central

    Silver, Kristopher; Littlejohn, Alaina; Thomas, Laurel; Marsh, Elizabeth; Lillich, James D.

    2015-01-01

    Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40 mM K+, or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity. PMID:26549367

  7. Plasma membrane fatty acid-binding protein and mitochondrial glutamic-oxaloacetic transaminase of rat liver are related

    SciTech Connect

    Berk, P.D.; Potter, B.J.; Sorrentino, D.; Zhou, S.L.; Isola, L.M.; Stump, D.; Kiang, C.L.; Thung, S. ); Wada, H.; Horio, Y. )

    1990-05-01

    The hepatic plasma membrane fatty acid-binding protein (h-FABP{sub PM}) and the mitochondrial isoenzyme of glutamic-oxaloacetic transaminase (mGOT) of rat liver have similar amino acid compositions and identical amino acid sequences for residues 3-24. Both proteins migrate with an apparent molecular mass of 43 kDa on SDS/polyacrylamide gel electrophoresis, have a similar pattern of basic charge isomers on isoelectric focusing, are eluted similarly from four different high-performance liquid chromatographic columns, have absorption maxima at 435 nm under acid conditions and 354 nm at pH 8.3, and bind oleate. Sinusoidally enriched liver plasma membranes and purified h-FABP{sub PM} have GOT enzymatic activity. Monospecific rabbit antiserum against h-FABP{sub PM} reacts on Western blotting with mGOT, and vice versa. Antisera against both proteins produce plasma membrane immunofluorescence in rat hepatocytes and selectively inhibit the hepatocellular uptake of ({sup 3}H)oleate but not that of ({sup 35}S)sulfobromophthalein or ({sup 14}C)taurocholate. The inhibition of oleate uptake produced by anti-h-FABP{sub PM} can be eliminated by preincubation of the antiserum with mGOT; similarly, the plasma membrane immunofluorescence produced by either antiserum can be eliminated by preincubation with the other antigen. These data suggest that h-FABP{sub PM} and mGOT are closely related.

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

    PubMed

    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] (P<0.05). The Mn(2+) concentrations in the semen were positively correlated with the incidence of sperm with IMe, DA, and LM and negatively correlated with number of sperm with DMe, IA, and LM. Therefore, dietary Mn(2+) supplementation for Nelore bulls 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.

  9. Intramolecular Disulfide Bond of Tim22 Protein Maintains Integrity of the TIM22 Complex in the Mitochondrial Inner Membrane*

    PubMed Central

    Okamoto, Hiroaki; Miyagawa, Akiko; Shiota, Takuya; Tamura, Yasushi; Endo, Toshiya

    2014-01-01

    Mitochondrial proteins require protein machineries called translocators in the outer and inner membranes for import into and sorting to their destination submitochondrial compartments. Among them, the TIM22 complex mediates insertion of polytopic membrane proteins into the inner membrane, and Tim22 constitutes its central insertion channel. Here we report that the conserved Cys residues of Tim22 form an intramolecular disulfide bond. By comparison of Tim22 Cys → Ser mutants with wild-type Tim22, we show that the disulfide bond of Tim22 stabilizes Tim22 especially at elevated temperature through interactions with Tim18, which are also important for the stability of the TIM22 complex. We also show that lack of the disulfide bond in Tim22 impairs the assembly of TIM22 pathway substrate proteins into the inner membrane especially when the TIM22 complex handles excess amounts of substrate proteins. Our findings provide a new insight into the mechanism of the maintenance of the structural and functional integrity of the TIM22 complex. PMID:24385427

  10. Disruption of the phagosomal membrane and egress of Legionella pneumophila into the cytoplasm during the last stages of intracellular infection of macrophages and Acanthamoeba polyphaga.

    PubMed

    Molmeret, Maëlle; Bitar, Dina M; Han, Lihui; Kwaik, Yousef Abu

    2004-07-01

    Although the early stages of intracellular infection by Legionella pneumophila are well established at the ultrastructural level, a detailed ultrastructural analysis of late stages of intracellular replication has never been done. Here we show that the membrane of the L. pneumophila-containing phagosome (LCP) is intact for up to 8 h postinfection of macrophages and Acanthamoeba polyphaga. At 12 h, 71 and 74% of the LCPs are disrupted within macrophages and A. polyphaga, respectively, while the plasma membrane remains intact. At 18 and 24 h postinfection, cytoplasmic elements such as mitochondria, lysosomes, vesicles, and amorphous material are dispersed among the bacteria and these bacteria are considered cytoplasmic. At 18 h, 77% of infected macrophages and 32% of infected A. polyphaga amoebae harbor cytoplasmic bacteria. At 24 h, 99 and 78% of infected macrophages and amoebae, respectively, contain cytoplasmic bacteria. On the basis of lysosomal acid phosphatase staining of infected macrophages and A. polyphaga, the lysosomal enzyme is present among the bacteria when host vesicles are dispersed among bacteria. Our data indicate that bacterial replication proceeds despite physical disruption of the phagosomal membrane. We also show that an lspG mutant that is defective in the type II secretion system and therefore does not secrete the hydrolytic enzymes metalloprotease, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A is as efficient as the wild-type strain in disruption of the LCP. Therefore, L. pneumophila disrupts the phagosomal membrane and becomes cytoplasmic at the last stages of infection in both macrophages and A. polyphaga. Lysosomal elements, mitochondria, cytoplasmic vesicles, and amorphous material are all dispersed among the bacteria, after phagosomal disruption, within both human macrophages and A. polyphaga. The disruption of the LCP is independent of the hydrolytic enzymes exported by the type II secretion

  11. Clostridium difficile Toxins Disrupt Epithelial Barrier Function by Altering Membrane Microdomain Localization of Tight Junction Proteins

    PubMed Central

    Nusrat, A.; von Eichel-Streiber, C.; Turner, J. R.; Verkade, P.; Madara, J. L.; Parkos, C. A.

    2001-01-01

    The anaerobic bacterium Clostridium difficile is the etiologic agent of pseudomembranous colitis. C. difficile toxins TcdA and TcdB are UDP-glucosyltransferases that monoglucosylate and thereby inactivate the Rho family of GTPases (W. P. Ciesla, Jr., and D. A. Bobak, J. Biol. Chem. 273:16021–16026, 1998). We utilized purified reference toxins of C. difficile, TcdA-10463 (TcdA) and TcdB-10463 (TcdB), and a model intestinal epithelial cell line to characterize their influence on tight-junction (TJ) organization and hence to analyze the mechanisms by which they contribute to the enhanced paracellular permeability and disease pathophysiology of pseudomembranous colitis. The increase in paracellular permeability induced by TcdA and TcdB was associated with disorganization of apical and basal F-actin. F-actin restructuring was paralleled by dissociation of occludin, ZO-1, and ZO-2 from the lateral TJ membrane without influencing the subjacent adherens junction protein, E-cadherin. In addition, we observed decreased association of actin with the TJ cytoplasmic plaque protein ZO-1. Differential detergent extraction and fractionation in sucrose density gradients revealed TcdB-induced redistribution of occludin and ZO-1 from detergent-insoluble fractions constituting “raft-like” membrane microdomains, suggesting an important role of Rho proteins in maintaining the association of TJ proteins with such microdomains. These toxin-mediated effects on actin and TJ structure provide a mechanism for early events in the pathophysiology of pseudomembranous colitis. PMID:11179295

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

    PubMed

    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

  13. Mitochondrial Dysfunction, Disruption of F-Actin Polymerization, and Transcriptomic Alterations in Zebrafish Larvae Exposed to Trichloroethylene.

    PubMed

    Wirbisky, Sara E; Damayanti, Nur P; Mahapatra, Cecon T; Sepúlveda, Maria S; Irudayaraj, Joseph; Freeman, Jennifer L

    2016-02-15

    Trichloroethylene (TCE) is primarily used as an industrial degreasing agent and has been in use since the 1940s. TCE is released into the soil, surface, and groundwater. From an environmental and regulatory standpoint, more than half of Superfund hazardous waste sites on the National Priority List are contaminated with TCE. Occupational exposure to TCE occurs primarily via inhalation, while environmental TCE exposure also occurs through ingestion of contaminated drinking water. Current literature links TCE exposure to various adverse health effects including cardiovascular toxicity. Current studies aiming to address developmental cardiovascular toxicity utilized rodent and avian models, with the majority of studies using relatively higher parts per million (mg/L) doses. In this study, to further investigate developmental cardiotoxicity of TCE, zebrafish embryos were treated with 0, 10, 100, or 500 parts per billion (ppb; μg/L) TCE during embryogenesis and/or through early larval stages. After the appropriate exposure period, angiogenesis, F-actin, and mitochondrial function were assessed. A significant dose-response decrease in angiogenesis, F-actin, and mitochondrial function was observed. To further complement this data, a transcriptomic profile of zebrafish larvae was completed to identify gene alterations associated with the 10 ppb TCE exposure. Results from the transcriptomic data revealed that embryonic TCE exposure caused significant changes in genes associated with cardiovascular disease, cancer, and organismal injury and abnormalities with a number of targets in the FAK signaling pathway. Overall, results from our study support TCE as a developmental cardiovascular toxicant, provide molecular targets and pathways for investigation in future studies, and indicate a need for continued priority for environmental regulation.

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

    PubMed

    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. Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

    PubMed

    Sileikyte, Justina; Petronilli, Valeria; Zulian, Alessandra; Dabbeni-Sala, Federica; Tognon, Giuseppe; Nikolov, Peter; Bernardi, Paolo; Ricchelli, Fernanda

    2011-01-14

    We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

  16. High frequency application of nanosecond pulsed electric fields alters cellular membrane disruption and fluorescent dye uptake

    NASA Astrophysics Data System (ADS)

    Steelman, Zachary A.; Tolstykh, Gleb P.; Beier, Hope T.; Ibey, Bennett L.

    2016-03-01

    Cells exposed to nanosecond-pulsed electric fields (nsPEF) exhibit a wide variety of nonspecific effects, including blebbing, swelling, intracellular calcium bursts, apoptotic and necrotic cell death, formation of nanopores, and depletion of phosphatidylinositol 4,5-biphosphate (PIP2) to induce activation of the inositol trisphosphate/diacylglycerol pathway. While several studies have taken place in which multiple pulses were delivered to cells, the effect of pulse repetition rate (PRR) is not well understood. To better understand the effects of PRR, a laser scanning confocal microscope was used to observe CHO-K1 cells exposed to ten 600ns, 200V pulses at varying repetition rates (5Hz up to 500KHz) in the presence of either FM 1-43, YO-PRO-1, or Propidium Iodide (PI) fluorescent dyes, probes frequently used to indicate nanoporation or permeabilization of the plasma membrane. Dye uptake was monitored for 30 seconds after pulse application at a rate of 1 image/second. In addition, a single long pulse of equivalent energy (200V, 6 μs duration) was applied to test the hypothesis that very fast PRR will approximate the biological effects of a single long pulse of equal energy. Upon examination of the data, we found strong variation in the relationship between PRR and uptake in each of the three dyes. In particular, PI uptake showed little frequency dependence, FM 1-43 showed a strong inverse relationship between frequency and internal cell fluorescence, and YO-PRO-1 exhibited a "threshold" point of around 50 KHz, after which the inverse trend observed in FM 1-43 was seen to reverse itself. Further, a very high PRR of 500 KHz only approximated the biological effects of a single 6 μs pulse in cells stained with YO-PRO-1, suggesting that uptake of different dyes may proceed by different physical mechanisms.

  17. Demonstration of an intramitochondrial invertase activity and the corresponding sugar transporters of the inner mitochondrial membrane in Jerusalem artichoke (Helianthus tuberosus L.) tubers.

    PubMed

    Szarka, András; Horemans, Nele; Passarella, Salvatore; Tarcsay, Akos; Orsi, Ferenc; Salgó, András; Bánhegyi, Gábor

    2008-10-01

    Genetic evidences indicate that alkaline/neutral invertases are present in plant cell organelles, and they might have a novel physiological function in mitochondria. The present study demonstrates an invertase activity in the mitochondrial matrix of Helianthus tuberosus tubers. The pH optimum, the kinetic parameters and the inhibitor profile of the invertase activity indicated that it belongs to the neutral invertases. In accordance with this topology, transport activities responsible for the mediation of influx/efflux of substrate/products were studied in the inner mitochondrial membrane. The transport of sucrose, glucose and fructose was shown to be bidirectional, saturable and independent of the mitochondrial respiration and membrane potential. Sucrose transport was insensitive to the inhibitors of the proton-sucrose symporters. The different kinetic parameters and inhibitors as well as the absence of cross-inhibition suggest that sucrose, glucose and fructose transport are mediated by separate transporters in the inner mitochondrial membrane. The mitochondrial invertase system composed by an enzyme activity in the matrix and the corresponding sugar transporters might have a role in both osmoregulation and intermediary metabolism.

  18. Effect of actin cytoskeleton disruption on electric pulse-induced apoptosis and electroporation in tumour cells.

    PubMed

    Xiao, Deyou; Tang, Liling; Zeng, Chao; Wang, Jianfei; Luo, Xiao; Yao, Chenguo; Sun, Caixin

    2011-02-01

    Electric pulses are known to affect the outer membrane and intracellular structures of tumour cells. By applying electrical pulses of 450 ns duration with electric field intensity of 8 kV/cm to HepG2 cells for 30 s, electric pulse-induced changes in the integrity of the plasma membrane, apoptosis, viability and mitochondrial transmembrane potential were investigated. Results demonstrated that electric pulses induced cell apoptosis and necrosis accompanied with the decrease of mitochondrial transmembrane potential and the formation of pores in the membrane. The role of cytoskeleton in cellular response to electric pulses was investigated. We found that the apoptotic and necrosis percentages of cells in response to electric pulses decreased after cytoskeletal disruption. The electroporation of cell was not affected by cytoskeletal disruption. The results suggest that the disruption of actin skeleton is positive in protecting cells from killing by electric pulses, and the skeleton is not involved in the electroporation directly.

  19. A potential role of X-linked inhibitor of apoptosis protein in mitochondrial membrane permeabilization and its implication in cancer therapy

    PubMed Central

    Bhat, Tariq A.; O'Malley, Jordan; Kumar, Sandeep; Chandra, Dhyan

    2015-01-01

    X-chromosome-linked inhibitor of apoptosis protein (XIAP) has an important regulatory role in programmed cell death by inhibiting the caspase cascade. Activation of XIAP-dependent signaling culminates into regulation of multiple cellular processes including apoptosis, innate immunity, epithelial-to-mesenchymal transition, cell migration, invasion, metastasis and differentiation. Although XIAP localizes to the cytosolic compartment, XIAP-mediated cellular signaling encompasses mitochondrial and post-mitochondrial levels. Recent findings demonstrate that XIAP also localizes to mitochondria and regulates mitochondria functions. XIAP acts upstream of mitochondrial cytochrome c release and modulates caspase-dependent apoptosis. The new function of XIAP has potential to enhance mitochondrial membrane permeabilization and other cellular functions controlling cytochrome c release. These findings could exploit the overexpression of XIAP in human tumors for therapeutic benefits. PMID:26232549

  20. Evaluation of a photocatalytic reactor membrane pilot system for the removal of pharmaceuticals and endocrine disrupting compounds from water.

    PubMed

    Benotti, Mark J; Stanford, Benjamin D; Wert, Eric C; Snyder, Shane A

    2009-04-01

    A photocatalytic reactor membrane pilot system, employing UV/TiO(2) photocatalysis, was evaluated for its ability to remove thirty-two pharmaceuticals, endocrine disrupting compounds, and estrogenic activity from water. Concentrations of all compounds decreased following treatment, and removal followed pseudo-first-order kinetics as a function of the amount of treatment. Twenty-nine of the targeted compounds in addition to total estrogenic activity were greater than 70% removed while only three compounds were less than 50% removed following the highest level of treatment (4.24 kW h/m(3)). No estrogenically active transformation products were formed during treatment. Additionally, the unit was operated in photolytic mode (UV only) and photolytic plus H(2)O(2) mode (UV/H(2)O(2)) to determine the relative amount of energy required. Based on the electrical energy per order (EEO), the unit achieved the greatest efficiency when operated in photolytic plus H(2)O(2) mode for the conditions tested.

  1. Novel function of glutathione transferase in rat liver mitochondrial membrane: Role for cytochrome c release from mitochondria

    SciTech Connect

    Lee, Kang Kwang; Shimoji, Manami; Hossain, Quazi Sohel; Sunakawa, Hajime; Aniya, Yoko

    2008-10-01

    Microsomal glutathione transferase (MGST1) is activated by oxidative stress. Although MGST1 is found in mitochondrial membranes (mtMGST1), there is no information about the oxidative activation of mtMGST1. In the present study, we aimed to determine whether mtMGST1 also undergoes activation and about its function. When rats were treated with galactosamine/lipopolysaccharide (GalN/LPS), mtMGST1 activity was significantly increased, and the increased activity was reduced by the disulfide reducing agent dithiothreitol. In mitochondria from GalN/LPS-treated rats, disulfide-linked mtMGST1 dimer and mixed protein glutathione disulfides (glutathionylation) were detected. In addition, cytochrome c release from mitochondria isolated from GalN/LPS-treated rats was observed, and the release was inhibited by anti-MGST1 antibodies. Incubation of mitochondria from control rats with diamide and diamide plus GSH in vitro resulted in dimer- and mixed disulfide bond-mediated activation of mtMGST1, respectively. The activation of mtMGST1 by diamide plus GSH caused cytochrome c release from the mitochondria, and the release was prevented by treatment with anti-MGST1 antibodies. In addition, diamide plus GSH treatment caused mitochondrial swelling accompanied by cytochrome c release, which was inhibited by cyclosporin A (CsA) and bongkrekic acid (BKA), inhibitors of the mitochondrial permeability transition (MPT) pore. Furthermore, mtMGST1 activity was also inhibited by CsA and BKA. These results indicate that mtMGST1 is activated through mixed disulfide bond formation that contributes to cytochrome c release from mitochondria through the MPT pore.

  2. Anaerobic respiration sustains mitochondrial membrane potential in a prolyl hydroxylase pathway-activated cancer cell line in a hypoxic microenvironment.

    PubMed

    Takahashi, Eiji; Sato, Michihiko

    2014-02-15

    To elucidate how tumor cells produce energy in oxygen-depleted microenvironments, we studied the possibility of mitochondrial electron transport without oxygen. We produced well-controlled oxygen gradients (ΔO2) in monolayer-cultured cells. We then visualized oxygen levels and mitochondrial membrane potential (ΔΦm) in individual cells by using the red shift of green fluorescent protein (GFP) fluorescence and a cationic fluorescent dye, respectively. In this two-dimensional tissue model, ΔΦm was abolished in cells >500 μm from the oxygen source [the anoxic front (AF)], indicating limitations in diffusional oxygen delivery. This result perfectly matched GFP-determined ΔO2. In cells pretreated with dimethyloxaloylglycine (DMOG), a prolyl hydroxylase domain-containing protein (PHD) inhibitor, the AF was expanded to 1,500-2,000 μm from the source. In these cells, tissue ΔO2 was substantially decreased, indicating that PHD pathway activation suppressed mitochondrial respiration. The expansion of the AF and the reduction of ΔO2 were much more prominent in a cancer cell line (Hep3B) than in the equivalent fibroblast-like cell line (COS-7). Hence, the results indicate that PHD pathway-activated cells can sustain ΔΦm, despite significantly decreased electron flux to complex IV. Complex II inhibition abolished the effect of DMOG in expanding the AF, although tissue ΔO2 remained shallow. Separate experiments demonstrated that complex II plays a substantial role in sustaining ΔΦm in DMOG-pretreated Hep3B cells with complex III inhibition. From these results, we conclude that PHD pathway activation can sustain ΔΦm in an otherwise anoxic microenvironment by decreasing tissue ΔO2 while activating oxygen-independent electron transport in mitochondria.

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

    PubMed

    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.

  4. Downregulation of HSP60 disrupts mitochondrial proteostasis to promote tumorigenesis and progression in clear cell renal cell carcinoma

    PubMed Central

    Tang, Haiping; Chen, Yuling; Liu, Xiaohui; Wang, Shiyu; Lv, Yang; Wu, Di; Wang, Qingtao; Luo, Minkui; Deng, Haiteng

    2016-01-01

    In the present study, we demonstrate that HSP60 is unequivocally downregulated in clear cell renal cell carcinoma (ccRCC) tissues compared to pericarcinous tissues. Overexpression of HSP60 in ccRCC cancer cells suppresses cell growth. HSP60 knockdown increases cell growth and proliferation in both cell culture and nude mice xenografts, and drives cells to undergo epithelial to mesenchymal transition (EMT). Our results propose that HSP60 silencing disrupts the integrity of the respiratory complex I and triggers the excessive ROS production, which promotes tumor progression in the following aspects: (1) ROS activates the AMPK pathway that promotes acquisition of the Warburg phenotype in HSP60-KN cells; (2) ROS generated by HSP60 knockdown or by rotenone inhibition drives cells to undergo EMT; and (3) the high level of ROS may also fragment the Fe-S clusters that up regulates ADHFe1 expression and the 2-hydroxygluterate (2-HG) production leading to changes in DNA methylation. These results suggest that the high level of ROS is needed for tumorigenesis and progression in tumors with the low HSP60 expression and HSP60 is a potential diagnostic biomarker as well as a therapeutic target in ccRCC. PMID:27246978

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

    PubMed

    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.

  6. IκΒα inhibits apoptosis at the outer mitochondrial membrane independently of NF-κB retention

    PubMed Central

    Pazarentzos, Evangelos; Mahul-Mellier, Anne-Laure; Datler, Christoph; Chaisaklert, Wanwisa; Hwang, Ming-Shih; Kroon, Jan; Qize, Ding; Osborne, Foy; Al-Rubaish, Abdullah; Al-Ali, Amein; Mazarakis, Nicholas D; Aboagye, Eric O; Grimm, Stefan

    2014-01-01

    IκBα resides in the cytosol where it retains the inducible transcription factor NF-κB. We show that IκBα also localises to the outer mitochondrial membrane (OMM) to inhibit apoptosis. This effect is especially pronounced in tumour cells with constitutively active NF-κB that accumulate high amounts of mitochondrial IκBα as a NF-κB target gene. 3T3 IκBα−/− cells also become protected from apoptosis when IκBα is specifically reconstituted at the OMM. Using various IκBα mutants, we demonstrate that apoptosis inhibition and NF-κB inhibition can be functionally and structurally separated. At mitochondria, IκBα stabilises the complex of VDAC1 and hexokinase II (HKII), thereby preventing Bax recruitment to VDAC1 and the release of cytochrome c for apoptosis induction. When IκBα is reduced in tumour cells with constitutively active NF-κB, they show an enhanced response to anticancer treatment in an in vivo xenograft tumour model. Our results reveal the unexpected activity of IκBα in guarding the integrity of the OMM against apoptosis induction and open possibilities for more specific interference in tumours with deregulated NF-κB. PMID:25361605

  7. SB203580 enhances the RV-induced loss of mitochondrial membrane potential and apoptosis in A549 cells

    NASA Astrophysics Data System (ADS)

    Li, Hai-yang; Zhuang, Cai-ping; Wang, Xiao-ping; Chen, Tong-sheng

    2012-03-01

    Resveratrol (RV), a naturally occurring phytoalexin, is known to possess a wide spectrum of chemopreventive and chemotherapeutic effects in various stages of human tumors. p38, a member of the mitogen-activated protein kinase (MAPK) superfamily, is always activated by some extracellular stimulus to regulate many cellular signal transduction pathways, such as apoptosis, proliferation, and inflammation and so on. In this report, we assessed the effect of SB203580, a specific inhibitor of p38 MAPK signaling pathway, on the RV-induced apoptosis in human lung adenocarcinoma (A549) cells. CCK-8 assay showed that pretreatment with SB203580 significantly enhanced the cytotoxicity of RV, which was further verified by analyzing the phosphatidylserine externalization using flow cytometry. In order to further confirm whether SB203580 accelerated apoptosis via the intrinsic apoptosis pathway, we analyzed the dysfunction of mitochondrial membrane potential (Δψm) of cells stained with rhodamine 123 by using flow cytometry after treatment with RV in the absence and presence of SB203580. Our data for the first time reported that p38 inhibitor SB203580 enhanced the RV-induced apoptosis via a mitochondrial pathway.

  8. Effect of staining and sorting on boar sperm membrane integrity, mitochondrial activity and in vitro blastocyst development.

    PubMed

    Spinaci, M; De Ambrogi, M; Volpe, S; Galeati, G; Tamanini, C; Seren, E

    2005-07-01

    The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.

  9. Benzo[a]pyrene-induced nitric oxide production acts as a survival signal targeting mitochondrial membrane potential.

    PubMed

    Hardonnière, Kévin; Huc, Laurence; Podechard, Normand; Fernier, Morgane; Tekpli, Xavier; Gallais, Isabelle; Sergent, Odile; Lagadic-Gossmann, Dominique

    2015-10-01

    Benzo[a]pyrene (B[a]P), the prototype molecule of polycyclic aromatic hydrocarbons, exhibits genotoxic and carcinogenic effects, which has led the International Agency for Research on Cancer to recognize it as a human carcinogen. Besides the well-known apoptotic signals triggered by B[a]P, survival signals have also been suggested to occur, both signals likely involved in cancer promotion. Our previous work showed that B[a]P induced an hyperpolarization of mitochondrial membrane potential (ΔΨm) in rat hepatic epithelial F258 cells. Elevated ΔΨm plays a role in tumor development and progression, and nitric oxide (NO) has been suggested to be responsible for increases in ΔΨm. The present study therefore aimed at evaluating the impact of B[a]P on NO level in F258 cells, and at testing the putative role for NO as a survival signal, notably in link with ΔΨm. Our data demonstrated that B[a]P exposure resulted in an NO production which was dependent upon the activation of the inducible NO synthase. This enzyme activation involved AhR and possibly p53 activation. Preventing NO production not only increased B[a]P-induced cell death but also blocked mitochondrial hyperpolarization. This therefore points to a role for NO as a survival signal upon B[a]P exposure, possibly targeting ΔΨm.

  10. Membrane-potential-dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain.

    PubMed

    Murphy, M P; Brand, M D

    1988-05-02

    The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (delta psi). The technique used was a variation of the steady-state method of Al-Shawi and Brand [(1981) Biochem. J. 200, 539-546]. We show that q+/O was about 2.7 at high values of delta psi (170 mV). As delta psi was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0. A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in delta psi after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as delta psi is varied.

  11. Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis

    PubMed Central

    Fossale, Elisa; Wolf, Pavlina; Espinola, Janice A; Lubicz-Nawrocka, Tanya; Teed, Allison M; Gao, Hanlin; Rigamonti, Dorotea; Cattaneo, Elena; MacDonald, Marcy E; Cotman, Susan L

    2004-01-01

    Background JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. Results To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. Conclusions These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. PMID:15588329

  12. Biomolecular Interaction Assays Identified Dual Inhibitors of Glutaminase and Glutamate Dehydrogenase That Disrupt Mitochondrial Function and Prevent Growth of Cancer Cells.

    PubMed

    Zhu, Min; Fang, Jinzhang; Zhang, Jingjing; Zhang, Zheng; Xie, Jianhui; Yu, Yan; Ruan, Jennifer Jin; Chen, Zhao; Hou, Wei; Yang, Gensheng; Su, Weike; Ruan, Benfang Helen

    2017-02-07

    Glutaminase (KGA/isoenzyme GAC) is an emerging and important drug target for cancer. Traditional methods for assaying glutaminase activity are coupled with several other enzymes. Such coupled assays do not permit the direct and stringent characterization of specific glutaminase inhibitors. Ebselen was identified as a potent 9 nM KGA inhibitor in the KGA/glutamate oxidase (GO)/horse radish peroxidase (HRP) coupled assay but showed very weak activity in inhibiting the growth of glutamine-dependent cancer cells. For rigorous characterization, we developed a direct kinetic binding assay for KGA using bio-layer interferometry (BLI) as the detection method; Ebselen was identified as a GDH inhibitor but not a KGA inhibitor. Furthermore, we designed and synthesized several benzo[d][1,2]selenazol-3(2H)-one dimers which were subjected to SAR analysis by several glutaminolysis specific biochemical and cell based assays. Novel glutamate dehydrogenase (GDH) or dual KGA/GDH inhibitors were discovered from the synthetic compounds; the dual inhibitors completely disrupt mitochondrial function and demonstrate potent anticancer activity with a minimum level of toxicity.

  13. Inhibition of free radical scavenging enzymes affects mitochondrial membrane permeability transition during growth and aging of yeast cells.

    PubMed

    Deryabina, Yulia; Isakova, Elena; Sekova, Varvara; Antipov, Alexey; Saris, Nils-Erik L

    2014-12-01

    In this study, we investigated the change in the antioxidant enzymes activity, cell respiration, reactive oxygen species (ROS), and impairment of membrane mitochondria permeability in the Endomyces magnusii yeasts during culture growth and aging. We showed that the transition into stationary phase is the key tool to understanding interaction of these processes. This growth stage is distinguished by two-fold increase in ROS production and respiration rate as compared to those in the logarithmic phase. It results in induction of alternative oxidase (AO) in the stationary phase, decline of the main antioxidant enzymes activities, ROS-production, and mitochondria membrane permeability. Significant increase in the share of mitochondrial isoform of superoxide dismutase (SOD2) occurred in the stationary phase from 51.8% (24 h of cultivation) to 68.6% (48 h of cultivation). Upon blocking the essential ROS-scavenging enzymes, SODs and catalases (CATs) some heterogeneity of cell population was observed: 80-90% of cells displayed evident signs of early apoptosis (such as disorientation of mitochondria cristae, mitochondrial fragmentation and deformation of nuclear chromatine). However, 10-20% of the population were definitely healthy. It allowed to draw the conclusion that a complete system of cell antioxidant protection underlies normal mitochondria functioning while the E. magnusii yeasts grow and age. Moreover, this system provides unimpaired cell physiology under oxidative stress during culture aging in the stationary phase. Failures in mitochondria functions due to inhibition of ROS-scavenging enzymes of CATs and SODs could lead to damage of the cells and some signs of early apoptosis.

  14. Real-Time Observation of Antimicrobial Polycation Effects on Escherichia coli: Adapting the Carpet Model for Membrane Disruption to Quaternary Copolyoxetanes.

    PubMed

    Wang, Congzhou; Zolotarskaya, Olga Y; Nair, Sithara S; Ehrhardt, Christopher J; Ohman, Dennis E; Wynne, Kenneth J; Yadavalli, Vamsi K

    2016-03-29

    Real-time atomic force microscopy (AFM) was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 μg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 μg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 μg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water-soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

  15. Reconstitution of Proapoptotic BAK Function in Liposomes Reveals a Dual Role for Mitochondrial Lipids in the BAK-driven Membrane Permeabilization Process*

    PubMed Central

    Landeta, Olatz; Landajuela, Ane; Gil, David; Taneva, Stefka; DiPrimo, Carmelo; Sot, Begoña; Valle, Mikel; Frolov, Vadim A.; Basañez, Gorka

    2011-01-01

    BAK is a key effector of mitochondrial outer membrane permeabilization (MOMP) whose molecular mechanism of action remains to be fully dissected in intact cells, mainly due to the inherent complexity of the intracellular apoptotic machinery. Here we show that the core features of the BAK-driven MOMP pathway can be reproduced in a highly simplified in vitro system consisting of recombinant human BAK lacking the carboxyl-terminal 21 residues (BAKΔC) and tBID in combination with liposomes bearing an appropriate lipid environment. Using this minimalist reconstituted system we established that tBID suffices to trigger BAKΔC membrane insertion, oligomerization, and pore formation. Furthermore, we demonstrate that tBID-activated BAKΔC permeabilizes the membrane by forming structurally dynamic pores rather than a large proteinaceous channel of fixed size. We also identified two distinct roles played by mitochondrial lipids along the molecular pathway of BAKΔC-induced membrane permeabilization. First, using several independent approaches, we showed that cardiolipin directly interacts with BAKΔC, leading to a localized structural rearrangement in the protein that “primes” BAKΔC for interaction with tBID. Second, we provide evidence that selected curvature-inducing lipids present in mitochondrial membranes specifically modulate the energetic expenditure required to create the BAKΔC pore. Collectively, our results support the notion that BAK functions as a direct effector of MOMP akin to BAX and also adds significantly to the growing evidence indicating that mitochondrial membrane lipids are actively implicated in BCL-2 protein family function. PMID:21196599

  16. Calcium-dependent nonspecific permeability of the inner mitochondrial membrane is not induced in mitochondria of the yeast Endomyces magnusii.

    PubMed

    Deryabina, Y I; Isakova, E P; Shurubor, E I; Zvyagilskaya, R A

    2004-09-01

    Mitochondria of the yeast Endomyces magnusii were examined for the presence of a Ca2+- and phosphate-induced permeability of the inner mitochondrial membrane (pore). For this purpose, coupled mitochondria were incubated under conditions known to induce the permeability transition pore in animal mitochondria, i.e., in the presence of high concentrations of Ca2+ and P(i), prooxidants (t-butylhydroperoxide), oxaloacetate, atractyloside (an inhibitor of ADP/ATP translocator), SH-reagents, by depletion of adenine nucleotide pools, and deenergization of the mitochondria. Large amplitude swelling, collapse of the membrane potential, and efflux of the accumulated Ca2+ were used as parameters for demonstrating pore induction. E. magnusii mitochondria were highly resistant to the above-mentioned substances. Deenergization of mitochondria or depletion of adenine nucleotide pools have no effect on low-amplitude swelling or the other parameters. Cyclosporin A, a specific inhibitor of the nonspecific permeability transition in animal mitochondria, did not affect the parameters measured. It is thus evident that E. magnusii mitochondria lack a functional Ca2+-dependent pore, or possess a pore differently regulated as compared to that of mammalian mitochondria.

  17. Tamoxifen inhibits mitochondrial oxidative stress damage induced by copper orthophenanthroline.

    PubMed

    Buelna-Chontal, Mabel; Hernández-Esquivel, Luz; Correa, Francisco; Díaz-Ruiz, Jorge Luis; Chávez, Edmundo

    2016-12-01

    In this work, we studied the effect of tamoxifen and cyclosporin A on mitochondrial permeability transition caused by addition of the thiol-oxidizing pair Cu(2+) -orthophenanthroline. The findings indicate that tamoxifen and cyclosporin A circumvent the oxidative membrane damage manifested by matrix Ca(2+) release, mitochondrial swelling, and transmembrane electrical gradient collapse. Furthermore, it was found that tamoxifen and cyclosporin A prevent the generation of TBARs promoted by Cu(2+) -orthophenanthroline, as well as the inactivation of the mitochondrial enzyme aconitase and disruption of mDNA. Electrophoretic analysis was unable to demonstrate a cross-linking reaction between membrane proteins. Yet, it was found that Cu(2+) -orthophenanthroline induced the generation of reactive oxygen species. It is thus plausible that membrane leakiness is due to an oxidative stress injury.

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

    PubMed Central

    Mühling, Tobias; Duda, Johanna; Weishaupt, Jochen H.; Ludolph, Albert C.; Liss, Birgit

    2014-01-01

    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, the endstage superoxide dismutase SOD1G93A transgenic mouse, 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 Na+/Ca2+ exchanger NCX1 were also about 2-fold higher in hMNs from SOD1G93A mice. Thus, pharmacological stimulation of Ca2+ transporters in highly vulnerable hMNs might offer a neuroprotective strategy for ALS. PMID:25452714

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

    SciTech Connect

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Characterization of a novel β-barrel protein (AtOM47) from the mitochondrial outer membrane of Arabidopsis thaliana

    PubMed Central

    Li, Lu; Kubiszewski-Jakubiak, Szymon; Radomiljac, Jordan; Wang, Yan; Law, Simon R.; Keech, Olivier; Narsai, Reena; Berkowitz, Oliver; Duncan, Owen; Murcha, Monika W.; Whelan, James

    2016-01-01

    In plant cells, mitochondria are major providers of energy and building blocks for growth and development as well as abiotic and biotic stress responses. They are encircled by two lipid membranes containing proteins that control mitochondrial function through the import of macromolecules and metabolites. Characterization of a novel β-barrel protein, OUTER MEMBRANE PROTEIN 47 (OM47), unique to the green lineage and related to the voltage-dependent anion channel (VDAC) protein family, showed that OM47 can complement a VDAC mutant in yeast. Mutation of OM47 in Arabidopsis thaliana by T-DNA insertion had no effect on the import of proteins, such as the β-barrel proteins translocase of the outer membrane 40 (TOM40) or sorting and assembly machinery 50 (SAM50), into mitochondria. Molecular and physiological analyses revealed a delay in chlorophyll breakdown, higher levels of starch, and a delay in the induction of senescence marker genes in the mutant lines. While there was a reduction of >90% in OM47 protein in mitochondria isolated from 3-week-old om47 mutants, in mitochondria isolated from 8-week-old plants OM47 levels were similar to that of the wild type. This recovery was achieved by an up-regulation of OM47 transcript abundance in the mutants. Combined, these results highlight a role in leaf senescence for this plant-specific β-barrel protein, probably mediating the recovery and recycling of chloroplast breakdown products by transporting metabolic intermediates into and out of mitochondria. PMID:27811077

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

    SciTech Connect

    Gourley, Paul Lee; Copeland, Robert Guild; McDonald, Anthony Eugene; Hendricks, Judy K.; Naviaux, Robert K.

    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.

  3. Disruption of the mitochondrial alternative oxidase (AOX) and uncoupling protein (UCP) alters rates of foliar nitrate and carbon assimilation in Arabidopsis thaliana.

    PubMed

    Gandin, Anthony; Denysyuk, Mykhaylo; Cousins, Asaph B

    2014-07-01

    Under high light, the rates of photosynthetic CO2 assimilation can be influenced by reductant consumed by both foliar nitrate assimilation and mitochondrial alternative electron transport (mAET). Additionally, nitrate assimilation is dependent on reductant and carbon skeletons generated from both the chloroplast and mitochondria. However, it remains unclear how nitrate assimilation and mAET coordinate and contribute to photosynthesis. Here, hydroponically grown Arabidopsis thaliana T-DNA insertional mutants for alternative oxidase (AOX1A) and uncoupling protein (UCP1) fed either NO3 (-) or NH4 (+) were used to determine (i) the response of NO3 (-) uptake and assimilation to the disruption of mAET, and (ii) the interaction of N source (NO3 (-) versus NH4 (+)) and mAET on photosynthetic CO2 assimilation and electron transport. The results showed that foliar NO3 (-) assimilation was enhanced in both aox1a and ucp1 compared with the wild-type, suggesting that foliar NO3 (-) assimilation is probably driven by a decreased capacity of mAET and an increase in reductant within the cytosol. Wild-type plants had also higher rates of net CO2 assimilation (A net) and quantum yield of PSII (ϕPSII) under NO3 (-) feeding compared with NH4 (+) feeding. Additionally, under NO3 (-) feeding, A net and ϕPSII were decreased in aox1a and ucp1 compared with the wild type; however, under NH4 (+) they were not significantly different between genotypes. This indicates that NO3 (-) assimilation and mAET are both important to maintain optimal rates of photosynthesis, probably in regulating reductant accumulation and over-reduction of the chloroplastic electron transport chain. These results highlight the importance of mAET in partitioning energy between foliar nitrogen and carbon assimilation.

  4. Disruption of the mitochondrial alternative oxidase (AOX) and uncoupling protein (UCP) alters rates of foliar nitrate and carbon assimilation in Arabidopsis thaliana

    PubMed Central

    Gandin, Anthony; Denysyuk, Mykhaylo; Cousins, Asaph B.

    2014-01-01

    Under high light, the rates of photosynthetic CO2 assimilation can be influenced by reductant consumed by both foliar nitrate assimilation and mitochondrial alternative electron transport (mAET). Additionally, nitrate assimilation is dependent on reductant and carbon skeletons generated from both the chloroplast and mitochondria. However, it remains unclear how nitrate assimilation and mAET coordinate and contribute to photosynthesis. Here, hydroponically grown Arabidopsis thaliana T-DNA insertional mutants for alternative oxidase (AOX1A) and uncoupling protein (UCP1) fed either NO3 – or NH4 + were used to determine (i) the response of NO3 – uptake and assimilation to the disruption of mAET, and (ii) the interaction of N source (NO3 – versus NH4 +) and mAET on photosynthetic CO2 assimilation and electron transport. The results showed that foliar NO3 – assimilation was enhanced in both aox1a and ucp1 compared with the wild-type, suggesting that foliar NO3 – assimilation is probably driven by a decreased capacity of mAET and an increase in reductant within the cytosol. Wild-type plants had also higher rates of net CO2 assimilation (A net) and quantum yield of PSII (ϕPSII) under NO3 – feeding compared with NH4 + feeding. Additionally, under NO3 – feeding, A net and ϕPSII were decreased in aox1a and ucp1 compared with the wild type; however, under NH4 + they were not significantly different between genotypes. This indicates that NO3 – assimilation and mAET are both important to maintain optimal rates of photosynthesis, probably in regulating reductant accumulation and over-reduction of the chloroplastic electron transport chain. These results highlight the importance of mAET in partitioning energy between foliar nitrogen and carbon assimilation. PMID:24799562

  5. Lipid-Loving ANTs: Molecular Simulations of Cardiolipin Interactions and the Organization of the Adenine Nucleotide Translocase in Model Mitochondrial Membranes

    PubMed Central

    2016-01-01

    The exchange of ADP and ATP across the inner mitochondrial membrane is a fundamental cellular process. This exchange is facilitated by the adenine nucleotide translocase, the structure and function of which are critically dependent on the signature phospholipid of mitochondria, cardiolipin (CL). Here we employ multiscale molecular dynamics simulations to investigate CL interactions within a membrane environment. Using simulations at both coarse-grained and atomistic resolutions, we identify three CL binding sites on the translocase, in agreement with those seen in crystal structures and inferred from nuclear magnetic resonance measurements. Characterization of the free energy landscape for lateral lipid interaction via potential of mean force calculations demonstrates the strength of interaction compared to those of binding sites on other mitochondrial membrane proteins, as well as their selectivity for CL over other phospholipids. Extending the analysis to other members of the family, yeast Aac2p and mouse uncoupling protein 2, suggests a degree of conservation. Simulation of large patches of a model mitochondrial membrane containing multiple copies of the translocase shows that CL interactions persist in the presence of protein–protein interactions and suggests CL may mediate interactions between translocases. This study provides a key example of how computational microscopy may be used to shed light on regulatory lipid–protein interactions. PMID:27786441

  6. Disrupted-in-schizophrenia 1 (DISC1) plays essential roles in mitochondria in collaboration with Mitofilin

    PubMed Central

    Park, Young-Un; Jeong, Jaehoon; Lee, Haeryun; Mun, Ji Young; Kim, Joung-Hun; Lee, Jong Seo; Nguyen, Minh Dang; Han, Sung Sik; Suh, Pann-Ghill; Park, Sang Ki

    2010-01-01

    Disrupted-in-schizophrenia 1 (DISC1) has emerged as a schizophrenia-susceptibility gene affecting various neuronal functions. In this study, we characterized Mitofilin, a mitochondrial inner membrane protein, as a mediator of the mitochondrial function of DISC1. A fraction of DISC1 was localized to the inside of mitochondria and directly interacts with Mitofilin. A reduction in DISC1 function induced mitochondrial dysfunction, evidenced by decreased mitochondrial NADH dehydrogenase activities, reduced cellular ATP contents, and perturbed mitochondrial Ca2+ dynamics. In addition, deficiencies in DISC1 and Mitofilin induced a reduction in mitochondrial monoamine oxidase-A activity. The mitochondrial dysfunctions evoked by the deficiency of DISC1 were partially phenocopied by an overexpression of truncated DISC1 that is associated with schizophrenia in human. DISC1 deficiencies induced the ubiquitination of Mitofilin, suggesting that DISC1 is critical for the stability of Mitofilin. Finally, the mitochondrial dysfunction induced by DISC1 deficiency was partially reversed by coexpression of Mitofilin, confirming a functional link between DISC1 and Mitofilin for the normal mitochondrial function. According to these results, we propose that DISC1 plays essential roles for mitochondrial function in collaboration with a mitochondrial interacting partner, Mitofilin. PMID:20880836

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

    PubMed

    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.

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

    PubMed Central

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

    2017-01-01

    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. PMID:28007883

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

    PubMed Central

    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

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

    PubMed

    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 < 0.001; n = 125) and the number of spermatozoa with DNA fragmentation (rs  = 0.43, p = 0.032; n = 25) was found. However, only a trend of positive correlation between ROS and the number of spermatozoa with TUNEL-detected DNA fragmentation was observed. Moreover, this latter was not correlated with loss of 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.

  11. 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.

    PubMed

    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 < 0.05) in flutamide-treated 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 < 0.05). No further decrease in the membrane integrity was found when the effect of anti-androgen lasted for 24 h. On the other hand, a decrease in sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p < 0.05). Characterization of 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.

  12. TIMMDC1/C3orf1 functions as a membrane-embedded mitochondrial complex I assembly factor through association with the MCIA complex.

    PubMed

    Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L; Gygi, Steven P; Harper, J Wade

    2014-03-01

    Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology.

  13. TIMMDC1/C3orf1 Functions as a Membrane-Embedded Mitochondrial Complex I Assembly Factor through Association with the MCIA Complex

    PubMed Central

    Guarani, Virginia; Paulo, Joao; Zhai, Bo; Huttlin, Edward L.; Gygi, Steven P.

    2014-01-01

    Complex I (CI) of the electron transport chain, a large membrane-embedded NADH dehydrogenase, couples electron transfer to the release of protons into the mitochondrial inner membrane space to promote ATP production through ATP synthase. In addition to being a central conduit for ATP production, CI activity has been linked to neurodegenerative disorders, including Parkinson's disease. CI is built in a stepwise fashion through the actions of several assembly factors. We employed interaction proteomics to interrogate the molecular associations of 15 core subunits and assembly factors previously linked to human CI deficiency, resulting in a network of 101 proteins and 335 interactions (edges). TIMMDC1, a predicted 4-pass membrane protein, reciprocally associated with multiple members of the MCIA CI assembly factor complex and core CI subunits and was localized in the mitochondrial inner membrane, and its depletion resulted in reduced CI activity and cellular respiration. Quantitative proteomics demonstrated a role for TIMMDC1 in assembly of membrane-embedded and soluble arms of the complex. This study defines a new membrane-embedded CI assembly factor and provides a resource for further analysis of CI biology. PMID:24344204

  14. Evidence that StAR and MLN64 act on the outer mitochondrial membrane as molten globules.

    PubMed

    Bose, H S; Baldwin, M A; Miller, W L

    2000-11-01

    StAR increases the flow of cholesterol from the outer to inner mitochondrial membrane (OMM to IMM), but its mechanism of action remains unclear. MLN64 is a 445 amino acid protein of unknown function that has four N-terminal transmembrane domains and whose C-terminal domain from 218-445 is 37% identical to StAR. N-62 StAR is as active as wild-type StAR, and N-234 MLN64 has 1/3 to 1/2 of StAR's activity. N-62 StAR lacks a mitochondrial leader and is confined to the cytoplasm, indicating that it acts on the OMM. Bacterially expressed N-62 StAR and N-218 MLN64 are active with isolated MA-10 cell mitochondria, indicating the proteins were properly folded. Far-UV CD spectroscopy, unfolding in urea, and fluorescence spectroscopy indicate that StAR undergoes a pH-dependent transition to a molten globule (retained secondary structure, partially lost tertiary structure) and stabilizes in mildly acid conditions. Far-UV CD data indicate that MLN64 undergoes a much less pronounced transition. Western blotting shows that normal human placenta has abundant N-terminally-cleaved 30 kDa MLN64. Partial proteolysis followed by mass spectrometry shows that the C-termini of StAR and MLN64 are sensitive to proteolysis, indicating looser folding. Our model of StAR action is that the protease-resistant domain unfolds slowly during normal mitochondrial entry, keeping StAR in contact with the OMM longer, increasing activity. The transition to the molten globule may be related to interaction with the OMM. These data are consistent with the recent crystallographic structure of N-216 MLN64 in which MLN64 binds cholesterol one molecule at a time, but are not consistent with the suggestion that StAR/MLN64 must reside in the intramembraneous space to transfer cholesterol form the OMM to the IMM.

  15. Cryopreservation of bull semen shipped overnight and its effect on post-thaw sperm motility, plasma membrane integrity, mitochondrial membrane potential and normal acrosomes.

    PubMed

    Anzar, M; Kroetsch, T; Boswall, L

    2011-06-01

    In the Canadian Animal Genetic Resource Program, bull semen is donated in frozen or fresh (diluted) states. This study was designed to assess the cryopreservation of diluted bull semen shipped at 4°C overnight, and to determine the post-thaw quality of shipped semen using different straw volumes and freezing rates. Semen was collected from four breeding bulls (three ejaculates per bull). Semen was diluted in Tris-citric acid-egg yolk-glycerol (TEYG) extender, cooled to 4°C and frozen as per routine (control semen). After cooling to 4°C, a part of semen was removed and shipped overnight to the research laboratory via express courier (shipped semen). Semen was packaged in 0.25 or 0.5 ml straws and frozen in a programmable freezer using three freezing rates, i.e., -10, -25 or -40°C/min. Control semen was also shipped to the research laboratory. Post-thaw sperm motility characteristics were assessed using CASA, and post-thaw sperm plasma membrane, mitochondrial membrane potential and normal acrosomes were assessed using flow cytometry. Post-thaw sperm quality was greater in shipped semen as compared to control (P<0.001). The shipped semen packaged in 0.25 ml straws had better post-thaw sperm quality than in 0.5 ml straws (P<0.001). Freezing rate had no effect on post-thaw sperm quality. In conclusion, bull semen can be shipped overnight for subsequent cryopreservation and gene banking. Overnight shipping of semen was found advantageous for bull semen cryopreservation. Semen packaging in 0.25 ml straws yielded better post-thaw quality than 0.5 ml straws.

  16. The plasma membrane Na+/Ca2+ exchange inhibitor KB-R7943 is also a potent inhibitor of the mitochondrial Ca2+ uniporter

    PubMed Central

    Santo-Domingo, J; Vay, L; Hernández-SanMiguel, E; Lobatón, C D; Moreno, A; Montero, M; Alvarez, J

    2007-01-01

    Background and purpose: The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes. Experimental approach. [Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells. Key results: KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU. Conclusions and Implications: Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future. PMID:17471180

  17. Influence of mitochondrial membrane potential of spermatozoa on in vitro fertilisation outcome.

    PubMed

    Marchetti, P; Ballot, C; Jouy, N; Thomas, P; Marchetti, C

    2012-04-01

    To determine whether the outcome of in vitro fertilisation (IVF) is influenced by the percentage of spermatozoa with functional mitochondria, a total of 91 random couples undergoing IVF were included. Mitochondrial function was determined by flow cytometry and expressed as percentage of spermatozoa. Conventional sperm parameters were studied by light microscopy. Reproductive outcome parameters were fertilisation rate, embryo quality and clinical pregnancy. It was found that the fertilisation rate was correlated with the percentage of spermatozoa (r = 0.24, P = 0.01) as well as with the percentage of highly motile spermatozoa. However, we did not find any relationship between the percentage of spermatozoa and embryo quality. Nevertheless, no patient who exhibited less than 64% of spermatozoa achieved pregnancy. It is concluded that determination of Δψ(m) provides accurate information to guide physicians to identify male patients for whom IVF will be unlikely to result in pregnancy. Therefore, we suggest that the percentage of spermatozoa may contribute to identify the most appropriate treatment for an individual patient.

  18. A study of properties and abundance of the components of liver carnitine palmitoyltransferases in mitochondrial inner and outer membranes. Effects of hypothyroidism, fasting and a ketotic diabetic state.

    PubMed

    Ghadiminejad, I; Saggerson, E D

    1991-08-01

    1. Liver mitochondrial outer and inner membranes were isolated from normal, 48 h-fasted, streptozotocin-diabetic and hypothyroid rats. 2. Relative to membrane protein, fasting and diabetes substantially increased the activity of carnitine palmitoyltransferase (CPT) in outer membranes. Inner-membrane CPT specific activity was only slightly altered, being increased in diabetes and decreased in hypothyroidism. Abundance of an inner-membrane Mr-68,000 polypeptide that cross-reacted with an anti-CPT serum was significantly increased in diabetes and hypothyroidism. Relative to inner-membrane CPT activity, this cross-reactivity was increased by 37% in diabetes and by 400% in hypothyroidism, suggesting modification of the intrinsic activity of the CPT in these states. 3. CPT in outer membranes was inhibitable by malonyl-CoA, whereas inner-membrane CPT was insensitive to malonyl-CoA. Fasting and diabetes increased the IC50 (concentration of malonyl-CoA causing 50% inhibition) for outer-membrane CPT, whereas the IC50 was decreased in hypothyroidism. 4. Binding of [14C]malonyl-CoA was observed with both outer and inner membranes and was fitted to two-site models in each case. Fasting, diabetes and hypothyroidism changed the KD for binding at the higher-affinity site in outer membranes in a manner that correlated closely with changes in IC50 for inhibition of outer-membrane CPT by malonyl-CoA. Fasting and diabetes increased the abundance of this outer-membrane high-affinity malonyl-CoA-binding site, whereas hypothyroidism decreased its abundance.

  19. Regulation of the mitochondrial permeability transition pore by the outer membrane does not involve the peripheral benzodiazepine receptor (Translocator Protein of 18 kDa (TSPO)).

    PubMed

    Šileikytė, Justina; Blachly-Dyson, Elizabeth; Sewell, Randall; Carpi, Andrea; Menabò, Roberta; Di Lisa, Fabio; Ricchelli, Fernanda; Bernardi, Paolo; Forte, Michael

    2014-05-16

    Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.

  20. In vitro cytotoxicity of Mokko lactone in human leukemia HL-60 cells: induction of apoptotic cell death by mitochondrial membrane potential collapse.

    PubMed

    Yun, Y G; Oh, H; Oh, G S; Pae, H O; Choi, B M; Kwon, J W; Kwon, T O; Jang, S I; Chung, Hun-Taeg

    2004-08-01

    We studied the effect of mokko lactone (ML) isolated from the roots of Saussurea lappa (Compositae), a plant that is used for medicinal purposes in Korea, on the induction of apoptosis in human leukemia HL-60 cells. ML was cytotoxic to HL-60 cells, and this cytotoxic effect of ML appears to be attributable to its induction of apoptotic cell death, as ML induced nuclear morphologic changes and internucleosomal DNA fragmentation and increased the proportion of Annexin V-positive cells and the activity of caspase-3. Further studies revealed that the induction of apoptosis by ML was associated with the loss of mitochondrial membrane potential. Collectively, our results suggest that apoptosis induced by ML in HL-60 cells was executed by a collapse of mitochondrial membrane potential followed by the activation of caspase-3. This is the first report on the mechanism of apoptosis-inducing effect of ML.

  1. Ferric nitrilotriacetate (Fe-NTA)-induced reactive oxidative species protects human hepatic stellate cells from apoptosis by regulating Bcl-2 family proteins and mitochondrial membrane potential

    PubMed Central

    Liu, Mei; Li, Shu-Jie; Xin, Yong-Ning; Ji, Shu-Sheng; Xie, Rui-Jin; Xuan, Shi-Ying

    2015-01-01

    Reactive oxidative species (ROS)-induced apoptosis of human hepatic stellate (HSC) is one of the treatments for liver fibrosis. However, how ROS (reactive oxygen species) affect HSC apoptosis and liver fibrosis is still unknown. In our study, ROS in human HSC cell line LX-2 was induced by ferric nitrilotriacetate (Fe-NTA) and assessed by superoxide dismutase (SOD) activity and methane dicarboxylic aldehyde (MDA) level. We found that in LX2 cells Fe-NTA induced notable ROS, which played a protective role in HSCs cells apoptosis by inhibiting Caspase-3 activation. Fe-NTA-induced ROS increased mRNA and protein level of anti-apoptosis Bcl-2 and decreased mRNA protein level of pro-apoptosis gene Bax, As a result, maintaining mitochondrial membrane potential of HSCs. Fe-NTA-induced ROS play a protective role in human HSCs by regulating Bcl-2 family proteins and mitochondrial membrane potential. PMID:26770403

  2. β2 and γ3 laminins are critical cortical basement membrane components: ablation of Lamb2 and Lamc3 genes disrupts cortical lamination and produces dysplasia.

    PubMed

    Radner, Stephanie; Banos, Charles; Bachay, Galina; Li, Yong N; Hunter, Dale D; Brunken, William J; Yee, Kathleen T

    2013-03-01

    Cortical development is dependent on the timely production and migration of neurons from neurogenic sites to their mature positions. Mutations in several receptors for extracellular matrix (ECM) molecules and their downstream signaling cascades produce dysplasia in brain. Although mutation of a critical binding site in the gene that encodes the ECM molecule laminin γ1 (Lamc1) disrupts cortical lamination, the ECM ligand(s) for many ECM receptors have not been demonstrated directly in the cortex. Several isoforms of the heterotrimeric laminins, all containing the β2 and γ3 chain, have been isolated from the brain, suggesting they are important for CNS function. Here, we report that mice homozygous null for the laminin β2 and γ3 chains exhibit cortical laminar disorganization. Mice lacking both of these laminin chains exhibit hallmarks of human cobblestone lissencephaly (type II, nonclassical): they demonstrate severe laminar disruption; midline fusion; perturbation of Cajal-Retzius cell distribution; altered radial glial cell morphology; and ectopic germinal zones. Surprisingly, heterozygous mice also exhibit laminar disruption of cortical neurons, albeit with lesser severity. In compound null mice, the pial basement membrane is fractured, and the distribution of a key laminin receptor, dystroglycan, is altered. These data suggest that β2 and γ3-containing laminins play an important dose-dependent role in development of the cortical pial basement membrane, which serves as an attachment site for Cajal-Retzius and radial glial cells, thereby guiding neural development.

  3. Quantitative measurement of mitochondrial membrane potential in cultured cells: calcium-induced de- and hyperpolarization of neuronal mitochondria

    PubMed Central

    Gerencser, Akos A; Chinopoulos, Christos; Birket, Matthew J; Jastroch, Martin; Vitelli, Cathy; Nicholls, David G; Brand, Martin D

    2012-01-01

    Mitochondrial membrane potential (ΔΨM) is a central intermediate in oxidative energy metabolism. Although ΔΨM is routinely measured qualitatively or semi-quantitatively using fluorescent probes, its quantitative assay in intact cells has been limited mostly to slow, bulk-scale radioisotope distribution methods. Here we derive and verify a biophysical model of fluorescent potentiometric probe compartmentation and dynamics using a bis-oxonol-type indicator of plasma membrane potential (ΔΨP) and the ΔΨM probe tetramethylrhodamine methyl ester (TMRM) using fluorescence imaging and voltage clamp. Using this model we introduce a purely fluorescence-based quantitative assay to measure absolute values of ΔΨM in millivolts as they vary in time in individual cells in monolayer culture. The ΔΨP-dependent distribution of the probes is modelled by Eyring rate theory. Solutions of the model are used to deconvolute ΔΨP and ΔΨM in time from the probe fluorescence intensities, taking into account their slow, ΔΨP-dependent redistribution and Nernstian behaviour. The calibration accounts for matrix:cell volume ratio, high- and low-affinity binding, activity coefficients, background fluorescence and optical dilution, allowing comparisons of potentials in cells or cell types differing in these properties. In cultured rat cortical neurons, ΔΨM is −139 mV at rest, and is regulated between −108 mV and −158 mV by concerted increases in ATP demand and Ca2+-dependent metabolic activation. Sensitivity analysis showed that the standard error of the mean in the absolute calibrated values of resting ΔΨM including all biological and systematic measurement errors introduced by the calibration parameters is less than 11 mV. Between samples treated in different ways, the typical equivalent error is ∼5 mV. PMID:22495585

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

    PubMed

    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.

  5. Repetitive Transient Depolarizations of the Inner Mitochondrial Membrane Induced by Proton Pumping

    PubMed Central

    Hattori, Tomohiro; Watanabe, Koichi; Uechi, Yukiko; Yoshioka, Hisashi; Ohta, Yoshihiro

    2005-01-01

    Single mitochondria show the spontaneous fluctuations of ΔΨm. In this study, to examine the mechanism of the fluctuations, we observed ΔΨm in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K+ from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca2+, CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of ΔΨm are discussed in relation to the matrix pH and the permeability transitions. PMID:15653749

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

    SciTech Connect

    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.

  7. Mulberry leaf phenolics ameliorate hyperglycemia-induced oxidative stress and stabilize mitochondrial membrane potential in HepG2 cells.

    PubMed

    Zou, Yu-Xiao; Shen, Wei-Zhi; Liao, Sen-Tai; Liu, Fan; Zheng, Shan-Qing; Blumberg, Jeffrey B; Chen, C-Y Oliver

    2014-12-01

    To investigate the effect of phenolics in mulberry leaves (mulberry leaf phenolics; MLP) on hyperglycemia-induced oxidative stress and mitochondrial membrane potential (ΔΨm) in HepG2 cells; we treated HepG2 with glucose [5.5 (N-Glc) or 50 mmol/L (Hi-Glc)] with or without MLP at 10 or 100 µmol/L gallic acid equivalents and assessed level of reactive oxidant species (ROS), ΔΨm, malondialdehyde (MDA) and nuclear factor-kappaB (NF-κB) activation. Hi-Glc-induced oxidative damage was demonstrated by a series of increase in superoxides (560%, 0.5 h), MDA (400%, 24 h), NF-κB activation (474%, 4 h) and a wild fluctuation of ΔΨm relative to the control cells (p ≤ 0.05). MLP treatments ameliorate Hi-Glc-induced negative effects by a 40% reduction in ROS production, 34-44% reduction in MDA production, over 35% inhibition of NF-κB activation, as well as exert protective effect on HepG2 cells from change in ΔΨm. Our data show that MLP in vitro can protect hepatoctyes from hyperglycemia-induced oxidative damages.

  8. Sodium Fluoride Induces Apoptosis in H9c2 Cardiomyocytes by Altering Mitochondrial Membrane Potential and Intracellular ROS Level.

    PubMed

    Yan, Xiaoyan; Yang, Xia; Hao, Xianhui; Ren, Qiurong; Gao, Jiping; Wang, Yu; Chang, Na; Qiu, Yulan; Song, Guohua

    2015-08-01

    Chronic excessive fluoride intake is known to be toxic, and effects of long-term fluorosis on different organ systems have been examined. However, there are few studies about the effects of fluorosis on cardiovascular systems. Here, we studied the fluoride-induced apoptosis in H9c2 cells and determined the underlying molecular mechanisms including the cell viability, intracellular reactive oxygen species (ROS) level, the changes of mitochondrial membrane potential (ΔΨm), and the cell apoptosis. Sodium fluoride (NaF) at concentrations of 0, 2, 4, 8, and 16 mg/L was administered to cultured H9c2 cells for up to 48 h. After the treatment, H9c2 cells were collected and the associated parameters were measured by flow cytometry. Our study found that fluoride not only inhibited H9c2 cell proliferation but also induced cell apoptosis. With the increment of NaF concentration, the apoptotic rates and ROS generation were increased, while the ΔΨm was decreased. In summary, these data suggested that NaF-induced H9c2 cell apoptosis is mediated by direct increased intracellular ROS and downregulated ΔΨm.

  9. Effect of protection and repair of injury of mitochondrial membrane-phospholipid on prognosis in patients with dilated cardiomyopathy.

    PubMed

    Ma, A; Zhang, W; Liu, Z

    1996-01-01

    We have already proved that the mitochondrial membrane-phospholipid (MMP) injury changes of peripheral lymphocytes in patients with heart failure can be used as an injury indicator of myocardia, and are related to the long-term prognosis. In the present study, MMP localization of the peripheral lymphocytes was performed by modified Demer's tricomplex flocculation method, and we compared the changes, after classification, between the pre-treatment and the 12-week post-treatment, of coenzyme Q10 (Co.Q10) and captopril in 61 hospitalized patients with dilated cardiomyopathy (DCM). They were followed up for 16.1 +/- 7.8 months (mean). The results showed that compared with the placebo, Co.Q10 and captopril could significantly protect against and repair MMP injury and improve the heart function of patients with DCM after 12 weeks, and the 2-year survival rate rose significantly by 72.7% for Co.Q10, and 64.0% for captopril, vs 24.7% for placebo. As for Longrank test, X2 equals 4.660 and 6.318, respectively, with both p < 0.05. The aforementioned results indicate that MMP injury of peripheral lymphocytes can predict the prognosis of the patients with DCM, thus the protection and repairment of MMP injury can improve the life-quality and prolong the life-span of the patients.

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

    SciTech Connect

    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.

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

    PubMed

    Liu, Zhizhen; Xie, Jun; Luo, Tian'e; Zhang, Tao; Zhao, Xia; Zhao, Hong; Li, Peizhen

    2012-02-25

    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.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Mitochondrial genetics

    PubMed Central

    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 conducted using PubMed (http://www.ncbi.nlm.nih.gov/pubmed/). In addition, this review makes use of a growing number of publically available databases including MITOMAP, a human mitochondrial genome database (www.mitomap.org), the Human DNA polymerase Gamma Mutation Database (http://tools.niehs.nih.gov/polg/) and PhyloTree.org (www.phylotree.org), a repository of global mtDNA variation. Areas of agreement The disruption in cellular energy, resulting from defects in mtDNA or defects in the nuclear-encoded genes responsible for mitochondrial maintenance, manifests in a growing number of human diseases. Areas of controversy The exact mechanisms which govern the inheritance of mtDNA are hotly debated. Growing points Although still in the early stages, the development of in vitro genetic manipulation could see an end to the inheritance of the most severe mtDNA disease. PMID:23704099

  14. Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein.

    PubMed Central

    Nemoto, Y; De Camilli, P

    1999-01-01

    Synaptojanin 1 is an inositol 5'-phosphatase highly enriched in nerve terminals with a putative role in recycling of synaptic vesicles. We have previously described synaptojanin 2, which is more broadly expressed as multiple alternatively spliced forms. Here we have identified and characterized a novel mitochondrial outer membrane protein, OMP25, with a single PDZ domain that specifically binds to a unique motif in the C-terminus of synaptojanin 2A. This motif is encoded by the exon sequence specific to synaptojanin 2A. OMP25 mRNA is widely expressed in rat tissues. OMP25 is localized to the mitochondrial outer membrane via the C-terminal transmembrane region, with the PDZ domain facing the cytoplasm. Overexpression of OMP25 results in perinuclear clustering of mitochondria in transfected cells. This effect is mimicked by enforced expression of synaptojanin 2A on the mitochondrial outer membrane, but not by the synaptojanin 2A mutants lacking the inositol 5'-phosphatase domain. Our findings provide evidence that OMP25 mediates recruitment of synaptojanin 2A to mitochondria and that modulation of inositol phospholipids by synaptojanin 2A may play a role in maintenance of the intracellular distribution of mitochondria. PMID:10357812

  15. Membrane-disruptive properties of the bioinsecticide Jaburetox-2Ec: implications to the mechanism of the action of insecticidal peptides derived from ureases.

    PubMed

    Barros, Pedro R; Stassen, Hubert; Freitas, Mônica S; Carlini, Célia R; Nascimento, Marco A C; Follmer, Cristian

    2009-12-01

    Jaburetox-2Ec, a recombinant peptide derived from an urease isoform (JBURE-II), displays high insecticidal activity against important pests such as Spodoptera frugiperda and Dysdercus peruvianus. Although the molecular mechanism of action of ureases-derived peptides remains unclear, previous ab initio data suggest the presence of structural motifs in Jaburetox-2Ec with characteristics similar to those found in a class of pore-forming peptides. Here, we investigated the molecular aspects of the interaction between Jaburetox-2Ec and large unilamellar vesicles. Jaburetox-2Ec displays membrane-disruptive ability on acidic lipid bilayers and this effect is greatly influenced by peptide aggregation. Corroborating with this finding, molecular modeling studies revealed that Jaburetox-2Ec might adopt a well-defined beta-hairpin conformation similar to those found in antimicrobial peptides with membrane disruption properties. In addition, molecular dynamics simulations suggest that the protein is able to anchor at a polar/non-polar interface. In the light of these findings, for the first time it was possible to point out some evidence that the peptide Jaburetox-2Ec interacting with lipid vesicles promotes membrane permeabilization.

  16. Three-dimensional organization of the endoplasmic reticulum membrane around the mitochondrial constriction site in mammalian cells revealed by using focused-ion beam tomography.

    PubMed

    Ohta, Keisuke; Okayama, Satoko; Togo, Akinobu; Nakamura, Kei-Ichiro

    2014-11-01

    The endoplasmic reticulum (ER) and mitochondria associate at multiple contact sites to form specific domains known as mitochondria-ER associated membranes (MAMs) that play a role in the regulation of various cellular processes such as Ca2+ transfer, autophagy, and inflammation. Recently, it has been suggested that MAMs are also involved in mitochondrial dynamics, especially fission events. Cytological analysis showed that ER tubules were frequently located close to each other in mitochondrial fission sites that accumulate fission-related proteins. Three-dimensional (3D) imaging of ER-mitochondrial contacts in yeast mitochondria by using cryo-electron tomography also showed that ER tubules were attached near the constriction site, which is considered to be a fission site1). MAMs have been suggested to play a role in the initiation of mitochondrial fission, although the molecular relationships between MAMs and the mitochondrial fission process have not been established. Although an ER-mitochondrial membrane association has also been observed at the fission site in mammalian mitochondria, the detailed organization of MAMs around mammalian mitochondria remains to be established. To visualize the 3D distribution of the ER-mitochondrial contacts around the mitochondria, especially around the constriction site in mammalian cells, we attempted 3D structural analysis of the mammalian cytoplasm using high-resolution focused ion-beam scanning electron microscopy (FIB-SEM) tomography, and observed the distribution pattern of ER contacts around the mammalian mitochondrial constriction site.Rat hepatocytes and HeLa cells were used. Liver tissue was obtained from male rats (Wistar, 6W) fixed by transcardial perfusion of 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) under deep anesthesia. HeLa cells were fixed with the same fixative. The specimens were then stained en bloc to enhance membrane contrast and embedded in epoxy resin2). The surface of

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

    PubMed

    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; P<0.001; n=50) between the area of relaxed chromatin in the nucleus and the degree of nucleotide labelling. While the chromatin of some spermatozoa increased more than eight times its normal size, not all sperm nuclei with relaxed chromatin showed evidence of nucleotide incorporation. Preferential staining associated with sperm DNA fragmentation (SDF) was typically located in the peri-acrosomal and peripheral regions of the sperm head and at the base of the spermatozoa where it appear to be 'hot spots' of DNA damage following cryopreservation. Results of the comparative effects of anisotonic media and cryopreservation on the integrity of koala spermatozoa revealed that injury induced by exposure to osmotic flux, essentially imitated the results found following cryopreservation. Plasma 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; P<0.05; n=5); however, no correlation was found when the percentage of sperm with relaxed chromatin was compared. While a cryo-induced osmotic

  18. Stabilization of c-myc G-Quadruplex DNA, inhibition of telomerase activity, disruption of mitochondrial functions and tumor cell apoptosis by platinum(II) complex with 9-amino-oxoisoaporphine.

    PubMed

    Qin, Jiao-Lan; Qin, Qi-Pin; Wei, Zu-Zhuang; Yu, Yan-Cheng; Meng, Ting; Wu, Chen-Xuan; Liang, Yue-Lan; Liang, Hong; Chen, Zhen-Feng

    2016-11-29

    [Pd(L)(DMSO)Cl2] (1) and [Pt(L)(DMSO)Cl2] (2) with 9-amino-oxoisoaporphine (L), were synthesized and characterized. 1 and 2 are more selectively cytotoxic to Hep-G2 cells versus normal liver cells (HL-7702). Various experiments showed that 2 acted as telomerase inhibitors targeting G4-DNA and triggered cell apoptosis by interacting with c-myc G4-DNA. Furthermore, 2 significantly induced cell cycle arrest at both G2/M and S phase, which leading to the down-regulation of cdc25 A, cyclin D, cyclin B, cyclin A and CDK2 and the up-regulation of p53, p27, p21,chk1 and chk2. In addition, 2 also caused mitochondrial dysfunction. Taken together, we found that 2 exerted its cytotoxic activity mainly via inhibiting telomerase by interaction with c-myc G4-DNA and disruption of mitochondrial function.

  19. Mitochondrial targeted peptides for cancer therapy.

    PubMed

    Farsinejad, Sadaf; Gheisary, Zohre; Ebrahimi Samani, Sanaz; Alizadeh, Ali Mohammad

    2015-08-01

    Mitochondria are a key pharmacological target in all cancer cells, since the structure and function of this organelle is different between healthy and malignant cells. Oxidative damage, disruption of mitochondrial ATP synthesis, calcium dyshomeostasis, mtDNA damage, and induction of the mitochondrial outer membrane permeabilization (MOMP) lead to the mitochondrial dysfunctionality and increase the probability of the programmed cell death or apoptosis. A variety of the signaling pathways have been developed to promote cell death including overexpression of pro-apoptotic members of Bcl-2 family, overloaded calcium, and elevated reactive oxygen species (ROS) play a key role in the promoting mitochondrial cytochrome c release through MOMP and eventually leads to cell death. There are a wide range of the therapeutic-based peptide drugs, known mitochondrial targeted peptides (MTPs), which specifically target mitochondrial pathways into death. They have prominent advantages such as low toxicity, high specificity, and easy to synthesis. Some of these therapeutic peptides have shown to increased the clinical activity alone or in combination with other agents. In this review, we will outline the biological properties of MTPs for cancer therapy. Understanding the molecular mechanisms and signaling pathways controlling cell death by MTPs can be critical for the development of the therapeutic strategies for cancer patients that would be valuable for researchers in both fields of molecular and clinical oncology.

  20. Specific requirements of nonbilayer phospholipids in mitochondrial respiratory chain function and formation

    PubMed Central

    Baker, Charli D.; Basu Ball, Writoban; Pryce, Erin N.; Gohil, Vishal M.

    2016-01-01

    Mitochondrial membrane phospholipid composition affects mitochondrial function by influencing the assembly of the mitochondrial respiratory chain (MRC) complexes into supercomplexes. For example, the loss of cardiolipin (CL), a signature non–bilayer-forming phospholipid of mitochondria, results in disruption of MRC supercomplexes. However, the functions of the most abundant mitochondrial phospholipids, bilayer-forming phosphatidylcholine (PC) and non–bilayer-forming phosphatidylethanolamine (PE), are not clearly defined. Using yeast mutants of PE and PC biosynthetic pathways, we show a specific requirement for mitochondrial PE in MRC complex III and IV activities but not for their formation, whereas loss of PC does not affect MRC function or formation. Unlike CL, mitochondrial PE or PC is not required for MRC supercomplex formation, emphasizing the specific requirement of CL in supercomplex assembly. Of interest, PE biosynthesized in the endoplasmic reticulum (ER) can functionally substitute for the lack of mitochondrial PE biosynthesis, suggesting the existence of PE transport pathway from ER to mitochondria. To understand the mechanism of PE transport, we disrupted ER–mitochondrial contact sites formed by the ERMES complex and found that, although not essential for PE transport, ERMES facilitates the efficient rescue of mitochondrial PE deficiency. Our work highlights specific roles of non–bilayer-forming phospholipids in MRC function and formation. PMID:27226479

  1. Processing of beta-dystroglycan by matrix metalloproteinase disrupts the link between the extracellular matrix and cell membrane via the dystroglycan complex.

    PubMed

    Yamada, H; Saito, F; Fukuta-Ohi, H; Zhong, D; Hase, A; Arai, K; Okuyama, A; Maekawa, R; Shimizu, T; Matsumura, K

    2001-07-15

    The dystroglycan complex is a membrane-spanning complex composed of two subunits, alpha- and beta-dystroglycan. alpha-dystroglycan is a cell surface peripheral membrane protein which binds to the extracellular matrix (ECM), whereas beta-dystroglycan is an integral membrane protein which anchors alpha-dystroglycan to the cell membrane. The dystroglycan complex provides a tight link between the ECM and cell membrane. Dysfunction of the dystroglycan complex has commonly been implicated in the molecular pathogenesis of severe forms of hereditary neuromuscular diseases, including Duchenne muscular dystrophy, Fukuyama-type congenital muscular dystrophy and sarcoglycanopathy (LGMD2C, -D, -E and -F). To begin to clarify the pathway by which the dysfunction of the dystroglycan complex could lead to muscle cell degeneration, we investigated the proteolytic processing of the dystroglycan complex in this study. We demonstrate that (i) a 30 kDa fragment of beta-dystroglycan is expressed in peripheral nerve, kidney, lung and smooth muscle, but not skeletal muscle, cardiac muscle or brain, and (ii) this fragment is the product of proteolytic processing of the extracellular domain of beta-dystroglycan by the membrane-associated matrix metalloproteinase (MMP) activity. Importantly, furthermore, we demonstrate that this processing disintegrates the dystroglycan complex. Our results indicate that the processing of beta-dystroglycan by MMP causes the disruption of the link between the ECM and cell membrane via the dystroglycan complex, which could have profound effects on cell viability. Based on these and previously reported findings, we propose a hypothesis that this processing may play a crucial role in the molecular pathogenesis of sarcoglycanopathy.

  2. The inhibition by bromothymol blue of anion translocation across the mitochondrial membrane.

    PubMed

    Aleksandrowicz, Z; Sweirczyński, J

    1975-02-28

    1. In rat liver mitochondria bromothymol blue inhibited the exchange of [14C]succinate for succinate, malonate, L-malate and inorganic phosphate; the [14C]citrate/citrate and [14C]citrate/malate exchange reactions and the phosphate/hydroxyl exchange were also inhibited by this dye. The inhibition of the rate of succinate, citrate and phosphate uptake by bromothymol blue is found to be competitive. 2. The degree of inhibition by bromothymol blue of the ]14C]succinate/malonate exchange reaction was pH dependent. It has been shown that the inhibition increased linearly while the pH was increased from 6.0 to 8.2. However, the binding rate of bromothymol blue to the mitochondria decreased with the rising pH of the medium. It is concluded that the binding of acidic bromothymol blue was not essential for the inhibitory effect. 3. Other sulfonephthalein derivatives also inhibited [14C]succinate/malonate exchange reaction. At pH 7.2 the relative order of the strength of the inhibitory action of the sulfonephthalein compounds tested was: thymol blue greater than bronocresol green greater than bromothymol blue greater than phenol red greater than bromocresol purple. The results do not indicate any correlation between the pK values of pH values of pH indicators and their extents of inhibition. 4. It is suggested that the negatively charged bromothymol blue interacts with the positively charged centers of the anion carrier systems causing inhibition of membrane permeability for anions.

  3. Triiodothyronine Facilitates Weaning From Extracorporeal Membrane Oxygenation by Improved Mitochondrial Substrate Utilization

    PubMed Central

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

    2014-01-01

    Background 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. We tested the hypothesis that although ECMO partially ameliorates metabolic abnormalities induced by ischemia‐reperfusion, these abnormalities persist or recur with weaning. We also determined if thyroid hormone supplementation (triiodothyronine) during ECMO improves oxidative metabolism and cardiac function. Methods and Results Neonatal piglets underwent transient coronary ischemia to induce cardiac injury then were separated into 4 groups based on loading status. Piglets without coronary ischemia served as controls. We infused into the left coronary artery [2‐13C]pyruvate and [13C6, 15N]l‐leucine to evaluate oxidative metabolism by gas chromatography‐mass spectroscopy and nuclear magnetic resonance methods. ECMO improved survival, increased oxidative substrate contribution through pyruvate dehydrogenase, reduced succinate and fumarate accumulation, and ameliorated ATP depletion induced by ischemia. The functional and metabolic benefit of ECMO was lost with weaning, yet triiodothyronine supplementation during ECMO restored function, increased relative pyruvate dehydrogenase flux, reduced succinate and fumarate, and preserved ATP stores. Conclusions Although ECMO provides metabolic rest by decreasing energy demand, metabolic impairments persist, and are exacerbated with weaning. Treating ECMO‐induced thyroid depression with triiodothyronine improves substrate flux, myocardial oxidative capacity and cardiac contractile function. This translational model suggests that metabolic targeting can improve weaning. PMID:24650924

  4. In vitro effects of triterpenic acids from olive leaf extracts on the mitochondrial membrane potential of promastigote stage of Leishmania spp.

    PubMed

    Sifaoui, Ines; López-Arencibia, Atteneri; Martín-Navarro, Carmen Maria; Ticona, Juan Carlos; Reyes-Batlle, María; Mejri, Mondher; Jiménez, Antonio Ignacio; Lopez-Bazzocchi, Isabel; Valladares, Basilio; Lorenzo-Morales, Jacob; Abderabba, Manef; Piñero, José E

    2014-10-15

    Protozoan diseases, such as leishmaniasis, are a cause of considerable morbidity throughout the world, affecting millions every year. In this study, two triterpenic acids (maslinic and oleanolic acids) were isolated from Tunisian olive leaf extracts and their in vitro activity against the promastigotes stage of Leishmania (L.) infantum and Leishmania (L.) amazonensis was investigated. Maslinic acid showed the highest activity with an IC50 of 9.32 ± 1.654 and 12.460 ± 1.25 μg/ml against L. infantum and L. amazonensis, respectively. The mechanism of action of these drugs was investigated by detecting changes in the phosphatidylserine (PS) exposure, the plasma membrane permeability, the mitochondrial membrane potential and the ATP level production in the treated parasites. By using the fluorescent probe SYTOX® Green, both triterpenic acids showed that they produce a time-dependent plasma membrane permeabilization in the treated Leishmania species. In addition, spectrofluorimeteric data revealed the surface exposure of PS in promastigotes. Both molecules reduced the mitochondrial membrane potential and decreased the ATP levels to 15% in parasites treated with IC90 for 24h. We conclude that the triterpenic acids tested in this study, show potential as future therapeutic alternative against leishmaniasis. Further studies are needed to confirm this.

  5. The Phosphatidylcholine Transfer Protein Stard7 is Required for Mitochondrial and Epithelial Cell Homeostasis.

    PubMed

    Yang, Li; Na, Cheng-Lun; Luo, Shiyu; Wu, David; Hogan, Simon; Huang, Taosheng; Weaver, Timothy E

    2017-04-12

    Mitochondria synthesize select phospholipids but lack the machinery for synthesis of the most abundant mitochondrial phospholipid, phosphatidylcholine (PC). Although the phospholipid transfer protein Stard7 promotes uptake of PC by mitochondria, the importance of this pathway for mitochondrial and cellular homeostasis represents a significant knowledge gap. Haploinsufficiency for Stard7 is associated with significant exacerbation of allergic airway disease in mice, including an increase in epithelial barrier permeability. To test the hypothesis that Stard7 deficiency leads to altered barrier structure/function downstream of mitochondrial dysfunction, Stard7 expression was knocked down in a bronchiolar epithelial cell line (BEAS-2B) and specifically deleted in lung epithelial cells of mice (Stard7(epi∆/∆)). Stard7 deficiency was associated with altered mitochondrial size and membrane organization both in vitro and in vivo. Altered mitochondrial structure was accompanied by disruption of mitochondrial homeostasis, including decreased aerobic respiration, increased oxidant stress, and mitochondrial DNA damage that, in turn, was linked to altered barrier integrity and function. Both mitochondrial and barrier defects were largely corrected by targeting Stard7 to mitochondria or treating epithelial cells with a mitochondrial-targeted antioxidant. These studies suggest that Stard7-mediated transfer of PC is crucial for mitochondrial homeostasis and that mitochondrial dysfunction contributes to altered barrier permeability in Stard7-deficient mice.

  6. Design and membrane-disruption mechanism of charge-enriched AMPs exhibiting cell selectivity, high-salt resistance, and anti-biofilm properties.

    PubMed

    Han, Hyo Mi; Gopal, Ramamourthy; Park, Yoonkyung

    2016-02-01

    Cationic antimicrobial peptides (AMPs) are essential components of the innate immune system, offering protection against invading pathogenic bacteria. In nature, AMPs serve as antibiotics with broad-spectrum antimicrobial and anti-biofilm properties. However, low effective stability in high-salt environments and physiological instability in biological membranes limit the applicability of naturally occurring AMPs as novel therapeutics. We therefore designed short synthetic cationic peptides by substituting key residues in myxinidin, an AMP derived from the epidermal mucus of hagfish, with lysine (Lys, K), arginine (Arg, R), and tryptophan (Trp, W). The resultant myxinidin analogs exhibited strong antimicrobial activity against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains, even under high-salt conditions. Moreover, these peptides showed high binding affinity for both lipopolysaccharides and lipoteichoic acids and inhibited biofilm formation by most bacteria, but did not cause significant lysis of human red blood cells and were not cytotoxic to normal human keratinocytes. Circular dichroism analysis revealed that myxinidin and its analogs assumed α-helical or β-sheet structures within artificial liposomes and bacterial membranes. In addition, bacterial killing and membrane permeation experiments demonstrated that the myxinidin analogs permeated through bacterial membranes, leading to cytoplasmic disruption and cell death. Taken together, these findings suggest myxinidin analogs may be promising candidate antibiotic agents for therapeutic application against antibiotic-resistant bacteria.

  7. Effects of eprosartan on mitochondrial membrane potential and H2O2 levels in leucocytes in hypertension.

    PubMed

    Labiós, M; Martínez, M; Gabriel, F; Guiral, V; Ruiz-Aja, S; Beltrán, B; Muñoz, A

    2008-07-01

    We investigated whether circulating leucocytes from hypertensive patients exhibit more spontaneous, stimulated hydrogen peroxide (H2O2) production and greater mitochondrial membrane potential (Deltapsi) than those from normotensive individuals. We also investigated the effects of oral treatment with the angiotensin II (AT II) type 1 receptor blocker eprosartan (600 mg day(-1)) on these markers of oxidative stress. In 25 hypertensive patients and 28 healthy volunteers, spontaneous H2O2 formation was measured by flow cytometry after preincubation of buffy coat-leucocytes from fresh peripheral venous blood at 37 degrees C with 2',7' dichlorofluorescein. Stimulation of H2O2 formation by circulating leucocytes was elicited by the addition of tert-butylhydroperoxide (tBHP). Deltapsi was determined by flow cytometry after the addition of tetramethylrhodamine methyl ester (TMRM). Compared with healthy individuals, lymphocytes from hypertensive patients exhibited higher Deltapsi (12.28+/-3.20 vs 16.25+/-2.88 arbitrary fluorescence units (AFU), respectively; P<0.001) and greater spontaneous H2O2 production (4.75+/-5.15 vs 8.98+/-9.97 AFU, respectively; P<0.05). tBHP stimulation was associated with higher H2O2 levels in circulating leucocytes in patients with uncorrected hypertension than in normotensive individuals. H2O2 overproduction was corrected by eprosartan treatment. These results suggest that oxidative stress could be important in the pathogenesis of hypertension. Furthermore, measurement of leucocyte oxidant activities may be useful for the evaluation of oxidative stress, which may be reduced with the use of antihypertensive drugs. Our results demonstrate that treatment of hypertension with eprosartan normalizes blood pressure and corrects oxidative disturbances, suggesting that leucocytes could be a target for this drug.

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

    PubMed Central

    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. PMID:27404273

  9. Progesterone attenuates Aβ(25-35)-induced neuronal toxicity via JNK inactivation and progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway.

    PubMed

    Qin, Yabin; Chen, Zesha; Han, Xiaolei; Wu, Honghai; Yu, Yang; Wu, Jie; Liu, Sha; Hou, Yanning

    2015-11-01

    Progesterone, which acts as a neurosteroid in nervous system, has been shown to have neuroprotective effects in different experiments in vitro and in vivo. Our previous study demonstrates that progesterone exerts neuroprotections in Alzheimer's disease-like rats. Present study attempted to evaluate the protective effects of progesterone on Aβ-treated neurons and potential mechanisms involved in neuroprotection. Results showed that treatment with progesterone protected primary cultured rat cortical neurons against Aβ(25-35)-induced apoptosis. Furthermore, we observed that progesterone alleviated mitochondrial dysfunction by rescuing mitochondrial membrane potential under Aβ challenge. Moreover, progesterone could also attenuate Bax/Bcl-2 proteins ratio upregulation and inhibit the activation of caspase-3 in Aβ-treated neurons. These indicate that progesterone attenuates Aβ(25-35)-induced neuronal toxicity by inhibiting mitochondria-associated apoptotic pathway. Both classic progesterone receptors (classic PR) and progesterone receptor membrane component 1 (PGRMC1), a special progesterone membrane receptor, are broadly expressed throughout the brain. The protective effect of progesterone was partially abolished by PGRMC1 inhibitor AG205 rather than classic PR antagonist RU486 in this study. Additionally, progesterone protected neurons by inhibiting Aβ-induced activation of JNK, which was an upstream signaling component in Aβ-induced mitochondria-associated apoptotic pathway. But this process was independent of PGRMC1. Taken together, these results suggest that progesterone exerts a protective effect against Aβ(25-35)-induced insults at least in part by two complementary pathways: (1) progesterone receptor membrane component 1-dependent inhibition of mitochondrial apoptotic pathway, and (2) blocking Aβ-induced JNK activation. The present study provides new insights into the mechanism by which progesterone brings neuroprotection. This article is part of a

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

    PubMed

    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.

  11. Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology.

    PubMed

    Wang, Yajie; Wu, Fengyi; Pan, Haining; Zheng, Wenzhong; Feng, Chi; Wang, Yunfu; Deng, Zixin; Wang, Lianrong; Luo, Jie; Chen, Shi

    2016-02-29

    Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) deposition in the brain. Aβ plaques are produced through sequential β/γ cleavage of amyloid precursor protein (APP), of which there are three main APP isoforms: APP695, APP751 and APP770. KPI-APPs (APP751 and APP770) are known to be elevated in AD, but the reason remains unclear. Transcription activator-like (TAL) effector nucleases (TALENs) induce mutations with high efficiency at specific genomic loci, and it is thus possible to knock out specific regions using TALENs. In this study, we designed and expressed TALENs specific for the C-terminus of APP in HeLa cells, in which KPI-APPs are predominantly expressed. The KPI-APP mutants lack a 12-aa region that encompasses a 5-aa trans-membrane (TM) region and 7-aa juxta-membrane (JM) region. The mutated KPI-APPs exhibited decreased mitochondrial localization. In addition, mitochondrial morphology was altered, resulting in an increase in spherical mitochondria in the mutant cells through the disruption of the balance between fission and fusion. Mitochondrial dysfunction, including decreased ATP levels, disrupted mitochondrial membrane potential, increased ROS generation and impaired mitochondrial dehydrogenase activity, was also found. These results suggest that specific regions of KPI-APPs are important for mitochondrial localization and function.

  12. High-molecular-weight polyphenols from oolong tea and black tea: purification, some properties, and role in increasing mitochondrial membrane potential.

    PubMed

    Fujihara, Takashi; Nakagawa-Izumi, Akiko; Ozawa, Tetsuo; Numata, Osamu

    2007-03-01

    High-molecular-weight polyphenols from oolong and black teas increased mitochondrial membrane potential, as measured by a method using ciliated protozoan Tetrahymena and rhodamine 123. These polyphenols, referred to as mitochondrial activation factors (MAFs), were purified from oolong and black teas by solvent extraction and Toyopearl column chromatography. The number-average molecular weights of the MAFs were 9,000 to 18,000, and the weight-average molecular weights were 15,000 to 25,000. The MAFs increased the mitochondrial membrane potential more than catechins did. Treatment of the MAFs with tannase indicated that they contained galloyl residues. When the MAFs were hydrolyzed with HCl-n-BuOH, cyanidin and delphinidin were detected. The partial structure of the MAFs was analyzed by pyrolysis-gas chromatography-mass spectrometry, and nine compounds were identified. These results suggest that MAFs are heterogeneous polymers of flavan-3-ols and flavan-3-ol gallates with intermonomeric linkages of B-ring to B-ring and C-ring to A-ring.

  13. Direct modulation of the outer mitochondrial membrane channel, voltage-dependent anion channel 1 (VDAC1) by cannabidiol: a novel mechanism for cannabinoid-induced cell death

    PubMed Central

    Rimmerman, N; Ben-Hail, D; Porat, Z; Juknat, A; Kozela, E; Daniels, M P; Connelly, P S; Leishman, E; Bradshaw, H B; Shoshan-Barmatz, V; Vogel, Z

    2013-01-01

    Cannabidiol (CBD) is a non-psychoactive plant cannabinoid that inhibits cell proliferation and induces cell death of cancer cells and activated immune cells. It is not an agonist of the classical CB1/CB2 cannabinoid receptors and the mechanism by which it functions is unknown. Here, we studied the effects of CBD on various mitochondrial functions in BV-2 microglial cells. Our findings indicate that CBD treatment leads to a biphasic increase in intracellular calcium levels and to changes in mitochondrial function and morphology leading to cell death. Density gradient fractionation analysis by mass spectrometry and western blotting showed colocalization of CBD with protein markers of mitochondria. Single-channel recordings of the outer-mitochondrial membrane protein, the voltage-dependent anion channel 1 (VDAC1) functioning in cell energy, metabolic homeostasis and apoptosis revealed that CBD markedly decreases channel conductance. Finally, using microscale thermophoresis, we showed a direct interaction between purified fluorescently labeled VDAC1 and CBD. Thus, VDAC1 seems to serve as a novel mitochondrial target for CBD. The inhibition of VDAC1 by CBD may be responsible for the immunosuppressive and anticancer effects of CBD. PMID:24309936

  14. Juglone disrupts root plasma membrane H+-ATPase activity and impairs water uptake, root respiration, and growth in soybean (Glycine max) and corn (Zea mays).

    PubMed

    Hejl, Angela M; Koster, Karen L

    2004-02-01

    Juglone is phytotoxic, but the mechanisms of growth inhibition have not been fully explained. Previous studies have proposed that disruption of electron transport functions in mitochondria and chloroplasts contribute to observed growth reduction in species exposed to juglone. In studies reported here, corn and soybean seedlings grown in nutrient solution amended with 10, 50, or 100 microM juglone showed significant decreases in root and shoot dry weights and lengths with increasing concentrations. However, no significant differences in leaf chlorophyll fluorescence or CO2-dependent leaf oxygen evolution were observed, even in seedlings that were visibly affected. Disruption of root oxygen uptake was positively correlated with increasing concentrations of juglone, suggesting that juglone may reach mitochondria in root cells. Water uptake and acid efflux also decreased for corn and soybean seedlings treated with juglone, suggesting that juglone may affect metabolism of root cells by disrupting root plasma membrane function. Therefore, the effect of juglone on H+-ATPase activity in corn and soybean root microsomes was tested. Juglone treatments from 10 to 1000 microM significantly reduced H+-ATPase activity compared to controls. This inhibition of H+-ATPase activity and observed reduction of water uptake offers a logical explanation for previously documented phytotoxicity of juglone. Impairment of this enzyme's activity could affect plant growth in a number of ways because proton-pumping in root cells drives essential plant processes such as solute uptake and, hence, water uptake.

  15. Dose-Dependent Thresholds of 10-ns Electric Pulse Induced Plasma Membrane Disruption and Cytotoxicity in Multiple Cell Lines

    DTIC Science & Technology

    2011-01-01

    staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat...staining positive) for propidium (Pr) iodide uptake, and phosphatidylserine (PS) externalization for several commonly studied cell lines (HeLa, Jurkat...membrane, we chose to monitor the externalization of phosphatidylserine (PS) molecules on the outer leaflet of the plasma membrane by utilizing FITC

  16. Lipid, detergent, and Coomassie Blue G-250 affect the migration of small membrane proteins in blue native gels: mitochondrial carriers migrate as monomers not dimers.

    PubMed

    Crichton, Paul G; Harding, Marilyn; Ruprecht, Jonathan J; Lee, Yang; Kunji, Edmund R S

    2013-07-26

    Blue native gel electrophoresis is a popular method for the determination of the oligomeric state of membrane proteins. Studies using this technique have reported that mitochondrial carriers are dimeric (composed of two ∼32-kDa monomers) and, in some cases, can form physiologically relevant associations with other proteins. Here, we have scrutinized the behavior of the yeast mitochondrial ADP/ATP carrier AAC3 in blue native gels. We find that the apparent mass of AAC3 varies in a detergent- and lipid-dependent manner (from ∼60 to ∼130 kDa) that is not related to changes in the oligomeric state of the protein, but reflects differences in the associated detergent-lipid micelle and Coomassie Blue G-250 used in this technique. Higher oligomeric state species are only observed under less favorable solubilization conditions, consistent with aggregation of the protein. Calibration with an artificial covalent AAC3 dimer indicates that the mass observed for solubilized AAC3 and other mitochondrial carriers corresponds to a monomer. Size exclusion chromatography of purified AAC3 in dodecyl maltoside under blue native gel-like conditions shows that the mass of the monomer is ∼120 kDa, but appears smaller on gels (∼60 kDa) due to the unusually high amount of bound negatively charged dye, which increases the electrophoretic mobility of the protein-detergent-dye micelle complex. Our results show that bound lipid, detergent, and Coomassie stain alter the behavior of mitochondrial carriers on gels, which is likely to be true for other small membrane proteins where the associated lipid-detergent micelle is large when compared with the mass of the protein.

  17. Overexpression of ErbB2 renders breast cancer cells susceptible to 3-BrPA through the increased dissociation of hexokinase II from mitochondrial outer membrane

    PubMed Central

    GAO, SUJIE; CHEN, XUEBO; JIN, HONGYONG; REN, SHENGNAN; LIU, ZHUO; FANG, XUEDONG; ZHANG, GUIZHEN

    2016-01-01

    ErbB2 is known to upregulate glycolysis in breast cancer, however, the precise mechanisms remain unclear. In the present study, ErbB2 upregulated Hexokinase II (HK II) activity by increasing the binding of HK II to the mitochondrial outer membrane. Dysregulated glucose metabolism in high ErbB2-expressing breast cancer cells induces susceptibility to glucose starvation and glycolysis inhibition. Additionally, HK II has a tendency to dissociate from the mitochondria outer membrane in ErbB2-overexpressing cells following treatment with the HK II inhibitor, 3-BrPA. Furthermore, 3-BrPA treatment results in decreased mitochondria membrane potential and release of cytochrome c into cytoplasm in ErbB2-overexpressing cells, leading to activation of the mitochondrial apoptotic signaling pathway. In summary, the results demonstrate a novel mechanism for ErbB2-activated glycolysis and reveal that 3-BrPA is effective in reducing ErbB2-positive breast cancer cell viability by targeting HK II in vitro and in vivo. PMID:26893781

  18. Mitochondrial-bacterial hybrids of BamA/Tob55 suggest variable requirements for the membrane integration of β-barrel proteins

    PubMed Central

    Pfitzner, Anna-Katharina; Steblau, Nadja; Ulrich, Thomas; Oberhettinger, Philipp; Autenrieth, Ingo B.; Schütz, Monika; Rapaport, Doron

    2016-01-01

    β-Barrel proteins are found in the outer membrane (OM) of Gram-negative bacteria, chloroplasts and mitochondria. The assembly of these proteins into the corresponding OM is facilitated by a dedicated protein complex that contains a central conserved β-barrel protein termed BamA in bacteria and Tob55/Sam50 in mitochondria. BamA and Tob55 consist of a membrane-integral C-terminal domain that forms a β-barrel pore and a soluble N-terminal portion comprised of one (in Tob55) or five (in BamA) polypeptide transport-associated (POTRA) domains. Currently the functional significance of this difference and whether the homology between BamA and Tob55 can allow them to replace each other are unclear. To address these issues we constructed hybrid Tob55/BamA proteins with differently configured N-terminal POTRA domains. We observed that constructs harboring a heterologous C-terminal domain could not functionally replace the bacterial BamA or the mitochondrial Tob55 demonstrating species-specific requirements. Interestingly, the various hybrid proteins in combination with the bacterial chaperones Skp or SurA supported to a variable extent the assembly of bacterial β-barrel proteins into the mitochondrial OM. Collectively, our findings suggest that the membrane assembly of various β-barrel proteins depends to a different extent on POTRA domains and periplasmic chaperones. PMID:27982054

  19. The rescue of microtubule-dependent traffic recovers mitochondrial function in Parkinson's disease.

    PubMed

    Esteves, A R; Gozes, I; Cardoso, S M

    2014-01-01

    In Parkinson's disease mitochondrial dysfunction can lead to a deficient ATP supply to microtubule protein motors leading to mitochondrial axonal transport disruption. Compromised axonal transport will then lead to a disorganized distribution of mitochondria and other organelles in the cell, as well as, the accumulation of aggregated proteins like alpha-synuclein. Moreover, axonal transport disruption can trigger synaptic accumulation of autophagosomes packed with damaged mitochondria and protein aggregates promoting synaptic failure. We previously observed that neuronal-like cells with an inherent mitochondrial impairment derived from PD patients contain a disorganized microtubule network, as well as, alpha-synuclein oligomer accumulation. In this work we provide new evidence that an agent that promotes microtubule network assembly, NAP (davunetide), improves microtubule-dependent traffic, restores the autophagic flux and potentiates autophagosome-lysosome fusion leading to autophagic vacuole clearance in Parkinson's disease cells. Moreover, NAP is capable of efficiently reducing alpha-synuclein oligomer content and its sequestration by the mitochondria. Most interestingly, NAP decreases mitochondrial ubiquitination levels, as well as, increases mitochondrial membrane potential indicating a rescue in mitochondrial function. Overall, we demonstrate that by improving microtubule-mediated traffic, we can avoid mitochondrial-induced damage and thus recover cell homeostasis. These results prove that NAP may be a promising therapeutic lead candidate for neurodegenerative diseases that involve axonal transport failure and mitochondrial impairment as hallmarks, like Parkinson's disease and related disorders.

  20. Overexpression of human SOD1 in VDAC1-less yeast restores mitochondrial functionality modulating beta-barrel outer membrane protein genes.

    PubMed

    Magrì, Andrea; Di Rosa, Maria Carmela; Tomasello, Marianna Flora; Guarino, Francesca; Reina, Simona; Messina, Angela; De Pinto, Vito

    2016-06-01

    Cu/Zn Superoxide Dismutase (SOD1), the most important antioxidant defense against ROS in eukaryotic cells, localizes in cytosol and intermembrane space of mitochondria (IMS). Several evidences show a SOD1 intersection with both fermentative and respiratory metabolism. The Voltage Dependent Anion Channel (VDAC) is the main pore-forming protein in the mitochondrial outer membrane (MOM), and is considered the gatekeeper of mitochondrial metabolism. Saccharomyces cerevisiae lacking VDAC1 (Δpor1) is a very convenient model system, since it shows an impaired growth rate on non-fermentable carbon source. Transformation of Δpor1 yeast with human SOD1 completely restores the cell growth deficit in non-fermentative conditions and re-establishes the physiological levels of ROS, as well as the mitochondrial membrane potential. No similar result was found upon yeast SOD1 overexpression. A previous report highlighted the action of SOD1 as a transcription factor. Quantitative Real-Time PCR showed that β-barrel outer-membrane encoding-genes por2, tom40, sam50 are induced by hSOD1, but the same effect was not obtained in Δpor1Δpor2 yeast, indicating a crucial function for yVDAC2. Since the lack of VDAC1 in yeast can be considered a stress factor for the cell, hSOD1 could relieve it stimulating the expression of genes bringing to the recovery of the MOM function. Our results suggest a direct influence of SOD1 on VDAC.

  1. Estrogen amelioration of Aβ-induced defects in mitochondria is mediated by mitochondrial signaling pathway involving ERβ, AKAP and Drp1.

    PubMed

    Sarkar, Saumyendra; Jun, Sujung; Simpkins, James W

    2015-08-07

    Perturbations in dynamic properties of mitochondria including fission, fusion, and movement lead to disruption of energy supply to synapses contributing to neuropathology and cognitive dysfunction in Alzheimer׳s disease (AD). The molecular mechanisms underlying these defects are still unclear. Previously, we have shown that ERβ is localized in the mitochondria and ERβ knock down disrupts mitochondrial functions. Because a selective ERβ modulator (DPN) can activate PKA, and localized PKA signaling in the mitochondrial membrane regulates mitochondrial structure and functions, we reasoned that ERβ signaling in the mitochondrial membrane rescues many of the mitochondrial defects caused by soluble Aβ oligomer. We now report that DPN treatment in primary hippocampal neurons attenuates soluble Aβ-oligomer induced dendritic mitochondrial fission and reduced mobility. Additionally, Aβ treatment reduced the respiratory reserve capacity of hippocampal neuron and inhibited phosphorylation of Drp1 at its PKA site, which induces excessive mitochondrial fission, and DPN treatment ameliorates these inhibitions. Finally, we discovered a direct interaction of ERβ with a mitochondrial resident protein AKAP1, which induces the PKA-mediated local signaling pathway involved in increased oxidative phosphorylation and inhibition of mitochondrial fission. Taken together, our findings highlight the possibility that ERβ signaling pathway may be a useful mitochondria-directed therapeutic target for AD.

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

    SciTech Connect

    Fa, Svetlana; Pogrmic-Majkic, Kristina; Samardzija, Dragana; Hrubik, Jelena; Glisic, Branka; Kovacevic, Radmila; Andric, Nebojsa

    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

  3. Haploinsufficiency of the 22q11.2-microdeletion gene Mrpl40 disrupts short-term synaptic plasticity and working memory through dysregulation of mitochondrial calcium

    PubMed Central

    Devaraju, Prakash; Yu, Jing; Eddins, Donnie; Mellado-Lagarde, Marcia M.; Earls, Laurie R.; Westmoreland, Joby J.; Quarato, Giovanni; Green, Douglas R.; Zakharenko, Stanislav S.

    2016-01-01

    Hemizygous deletion of a 1.5- to 3-megabase region on chromosome 22 causes 22q11.2 deletion syndrome (22q11DS), which constitutes one of the strongest genetic risks for schizophrenia. Mouse models of 22q11DS have abnormal short-term synaptic plasticity (STP) that contributes to working memory deficiencies similar to those in schizophrenia. We screened mutant mice carrying hemizygous deletions of 22q11DS genes and identified haploinsufficiency of Mrpl40 (mitochondrial large ribosomal subunit protein 40) as a contributor to abnormal STP. Two-photon imaging of the genetically encoded fluorescent calcium indicator GCaMP6, expressed in presynaptic cytosol or mitochondria, showed that Mrpl40 haploinsufficiency deregulates STP via impaired calcium extrusion from the mitochondrial matrix through the mitochondrial permeability transition pore. This led to abnormally high cytosolic calcium transients in presynaptic terminals and deficient working memory but did not affect long-term spatial memory. Thus, we propose that mitochondrial calcium deregulation is a novel pathogenic mechanism of cognitive deficiencies in schizophrenia. PMID:27184122

  4. Bound simian virus 40 translocates to caveolin-enriched membrane domains, and its entry is inhibited by drugs that selectively disrupt caveolae.

    PubMed

    Anderson, H A; Chen, Y; Norkin, L C

    1996-11-01

    Simian virus 40 (SV40) entry leading to infection occurred only after the virus was at the cell surface for 1.5 to 2 h. SV40 infectious entry was not sensitive to cytosol acidification, a treatment that blocks endocytosis via clathrin-coated vesicles. Instead, SV40 infectious entry was blocked by treating cells with the phorbol ester PMA or nystatin, which selectively disrupts caveolae. In control experiments, transferrin internalization was sensitive to cytosol acidification but was not sensitive to PMA or nystatin. Also, absorbed transferrin entered cells within minutes. Finally, bound SV40 translocated to caveolin-enriched membrane complexes isolated by a Triton X-100 insolubility protocol. Treatment with nystatin did not impair SV40 binding but did block the partitioning of virus into the caveolin-enriched complexes.