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Sample records for abt-737-induced mitochondrial membrane

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

    Science.gov (United States)

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

    2016-01-01

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

  2. ABT737 enhances cholangiocarcinoma sensitivity to cisplatin through regulation of mitochondrial dynamics

    International Nuclear Information System (INIS)

    Fan, Zhongqi; Yu, Huimei; Cui, Ni; Kong, Xianggui; Liu, Xiaomin; Chang, Yulei; Wu, Yao; Sun, Liankun; Wang, Guangyi

    2015-01-01

    Cholangiocarcinoma responses weakly to cisplatin. Mitochondrial dynamics participate in the response to various stresses, and mainly involve mitophagy and mitochondrial fusion and fission. Bcl-2 family proteins play critical roles in orchestrating mitochondrial dynamics, and are involved in the resistance to cisplatin. Here we reported that ABT737, combined with cisplatin, can promote cholangiocarcinoma cells to undergo apoptosis. We found that the combined treatment decreased the Mcl-1 pro-survival form and increased Bak. Cells undergoing cisplatin treatment showed hyperfused mitochondria, whereas fragmentation was dominant in the mitochondria of cells exposed to the combined treatment, with higher Fis1 levels, decreased Mfn2 and OPA1 levels, increased ratio of Drp1 60 kD to 80 kD form, and more Drp1 located on mitochondria. More p62 aggregates were observed in cells with fragmented mitochondria, and they gradually translocated to mitochondria. Mitophagy was induced by the combined treatment. Knockdown p62 decreased the Drp1 ratio, increased Tom20, and increased cell viability. Our data indicated that mitochondrial dynamics play an important role in the response of cholangiocarcinoma to cisplatin. ABT737 might enhance cholangiocarcinoma sensitivity to cisplatin through regulation of mitochondrial dynamics and the balance within Bcl-2 family proteins. Furthermore, p62 seems to be critical in the regulation of mitochondrial dynamics. - Highlights: • Cholangiocarcinoma may adapt to cisplatin through mitochondrial fusion. • ABT737 sensitizes cholangiocarcinoma to cisplatin by promoting fission and mitophagy. • p62 might participate in the regulation of mitochondrial fission and mitophagy

  3. The combination of BH3-mimetic ABT-737 with the alkylating agent temozolomide induces strong synergistic killing of melanoma cells independent of p53.

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    Steven N Reuland

    Full Text Available Metastatic melanoma has poor prognosis and is refractory to most conventional chemotherapies. The alkylating agent temozolomide (TMZ is commonly used in treating melanoma but has a disappointing response rate. Agents that can act cooperatively with TMZ and improve its efficacy are thus highly sought after. The BH3 mimetic ABT-737, which can induce apoptosis by targeting pro-survival Bcl-2 family members, has been found to enhance the efficacy of many conventional chemotherapeutic agents in multiple cancers. We found that combining TMZ and ABT-737 induced strong synergistic apoptosis in multiple human melanoma cell lines. When the drugs were used in combination in a mouse xenograft model, they drastically reduced tumor growth at concentrations where each individual drug had no significant effect. We found that TMZ treatment elevated p53 levels, and that the pro-apoptotic protein Noxa was elevated in TMZ/ABT-737 treated cells. Experiments with shRNA demonstrated that the synergistic effect of TMZ and ABT-737 was largely dependent on Noxa. Experiments with nutlin-3, a p53 inducer, demonstrated that p53 induction was sufficient for synergistic cell death with ABT-737 in a Noxa-dependent fashion. However, p53 was not necessary for TMZ/ABT-737 synergy as demonstrated by a p53-null line, indicating that TMZ and ABT-737 together induce Noxa in a p53-independent fashion. These results demonstrate that targeting anti-apoptotic Bcl-2 members is a promising method for treating metastatic melanoma, and that clinical trials with TMZ and Bcl-2 inhibitors are warranted.

  4. Inhibition of p38 MAPK enhances ABT-737-induced cell death in melanoma cell lines: novel regulation of PUMA.

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    Keuling, Angela M; Andrew, Susan E; Tron, Victor A

    2010-06-01

    The mitogen-activated protein kinase (MAPK) pathway is constitutively activated in the majority of melanomas, promoting cell survival, proliferation and migration. In addition, anti-apoptotic Bcl-2 family proteins Mcl-1, Bcl-xL and Bcl-2 are frequently overexpressed, contributing to melanoma's well-documented chemoresistance. Recently, it was reported that the combination of MAPK pathway inhibition by specific MEK inhibitors and Bcl-2 family inhibition by BH3-mimetic ABT-737 synergistically induces apoptotic cell death in melanoma cell lines. Here we provide the first evidence that inhibition of another key MAPK, p38, synergistically induces apoptosis in melanoma cells in combination with ABT-737. We also provide novel mechanistic data demonstrating that inhibition of p38 increases expression of pro-apoptotic Bcl-2 protein PUMA. Furthermore, we demonstrate that PUMA can be cleaved by a caspase-dependent mechanism during apoptosis and identify what appears to be the PUMA cleavage product. Thus, our findings suggest that the combination of ABT-737 and inhibition of p38 is a promising, new treatment strategy that acts through a novel PUMA-dependent mechanism.

  5. Cafestol overcomes ABT-737 resistance in Mcl-1-overexpressed renal carcinoma Caki cells through downregulation of Mcl-1 expression and upregulation of Bim expression.

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    Woo, S M; Min, K-J; Seo, B R; Nam, J-O; Choi, K S; Yoo, Y H; Kwon, T K

    2014-11-06

    Although ABT-737, a small-molecule Bcl-2/Bcl-xL inhibitor, has recently emerged as a novel cancer therapeutic agent, ABT-737-induced apoptosis is often blocked in several types of cancer cells with elevated expression of Mcl-1. Cafestol, one of the major compounds in coffee beans, has been reported to have anti-carcinogenic activity and tumor cell growth-inhibitory activity, and we examined whether cafestol could overcome resistance against ABT-737 in Mcl-1-overexpressed human renal carcinoma Caki cells. ABT-737 alone had no effect on apoptosis, but cafestol markedly enhanced ABT-737-mediated apoptosis in Mcl-1-overexpressed Caki cells, human glioma U251MG cells, and human breast carcinoma MDA-MB231 cells. By contrast, co-treatment with ABT-737 and cafestol did not induce apoptosis in normal human skin fibroblast. Furthermore, combined treatment with cafestol and ABT-737 markedly reduced tumor growth compared with either drug alone in xenograft models. We found that cafestol inhibited Mcl-1 protein expression, which is important for ABT-737 resistance, through promotion of protein degradation. Moreover, cafestol increased Bim expression, and siRNA-mediated suppression of Bim expression reduced the apoptosis induced by cafestol plus ABT-737. Taken together, cafestol may be effectively used to enhance ABT-737 sensitivity in cancer therapy via downregulation of Mcl-1 expression and upregulation of Bim expression.

  6. Role of p38 MAPK in enhanced human cancer cells killing by the combination of aspirin and ABT-737

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    Zhang, Chong; Shi, Jing; Mao, Shi-ying; Xu, Ya-si; Zhang, Dan; Feng, Lin-yi; Zhang, Bo; Yan, You-you; Wang, Si-cong; Pan, Jian-ping; Yang, You-ping; Lin, Neng-ming

    2015-01-01

    Regular use of aspirin after diagnosis is associated with longer survival among patients with mutated-PIK3CA colorectal cancer, but not among patients with wild-type PIK3CA cancer. In this study, we showed that clinically achievable concentrations of aspirin and ABT-737 in combination could induce a synergistic growth arrest in several human PIK3CA wild-type cancer cells. In addition, our results also demonstrated that long-term combination treatment with aspirin and ABT-737 could synergistically induce apoptosis both in A549 and H1299 cells. In the meanwhile, short-term aspirin plus ABT-737 combination treatment induced a greater autophagic response than did either drug alone and the combination-induced autophagy switched from a cytoprotective signal to a death-promoting signal. Furthermore, we showed that p38 acted as a switch between two different types of cell death (autophagy and apoptosis) induced by aspirin plus ABT-737. Moreover, the increased anti-cancer efficacy of aspirin combined with ABT-737 was further validated in a human lung cancer A549 xenograft model. We hope that this synergy may contribute to failure of aspirin cancer therapy and ultimately lead to efficacious regimens for cancer therapy. PMID:25388762

  7. Knockdown of BAG3 sensitizes bladder cancer cells to treatment with the BH3 mimetic ABT-737.

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    Mani, Jens; Antonietti, Patrick; Rakel, Stefanie; Blaheta, Roman; Bartsch, Georg; Haferkamp, Axel; Kögel, Donat

    2016-02-01

    BAG3 is overexpressed in several malignancies and mediates a non-canonical, selective form of (macro)autophagy. By stabilizing pro-survival Bcl-2 proteins in complex with HSP70, BAG3 can also exert an apoptosis-antagonizing function. ABT-737 is a high affinity Bcl-2 inhibitor that fails to target Mcl-1. This failure may confer resistance in various cancers. Urothelial cancer cells were treated with the BH3 mimetics ABT-737 and (-)-gossypol, a pan-Bcl-2 inhibitor which inhibits also Mcl-1. To clarify the importance of the core autophagy regulator ATG5 and BAG3 in ABT-737 treatment, cell lines carrying a stable lentiviral knockdown of ATG5 and BAG3 were created. The synergistic effect of ABT-737 and pharmaceutical inhibition of BAG3 with the HSF1 inhibitor KRIBB11 or sorafenib was also evaluated. Total cell death and apoptosis were quantified by FACS analysis of propidium iodide, annexin. Target protein analysis was conducted by Western blotting. Knockdown of BAG3 significantly downregulated Mcl-1 protein levels and sensitized urothelial cancer cells to apoptotic cell death induced by ABT-737, while inhibition of bulk autophagy through depletion of ATG5 had no discernible effect on cell death. Similar to knockdown of BAG3, pharmacological targeting of the BAG3/Mcl-1 pathway with KRIBB11 was capable to sensitize both cell lines to treatment with ABT-737. Our results show that BAG3, but not bulk autophagy has a major role in the response of bladder cancer cells to BH3 mimetics. They also suggest that BAG3 is a suitable target for combined therapies aimed at synergistically inducing apoptosis in bladder cancer.

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

    DEFF Research Database (Denmark)

    Yin, Shutao; Dong, Yinhui; Li, Jinghua

    2012-01-01

    ABT-737, a novel small molecule inhibitor of Bcl-2 family proteins, holds great promise to complement current cancer therapies. However many types of solid cancer cells are resistant to ABT-737. One practical approach to improve its therapeutic efficacy is to combine with the agents that can...

  9. Synergistic anticancer effects of the 9.2.27PE immunotoxin and ABT-737 in melanoma.

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    Karianne Risberg

    Full Text Available In cancer, combinations of drugs targeting different cellular functions is well accepted to improve tumor control. We studied the effects of a Pseudomonas exotoxin A (PE-based immunotoxin, the 9.2.27PE, and the BH-3 mimetic compound ABT-737 in a panel of melanoma cell lines. The drug combination resulted in synergistic cytotoxicity, and the cell death observed was associated with apoptosis, as activation of caspase-3, inactivation of Poly (ADP-ribose polymerase (PARP and increased DNA fragmentation could be prevented by pre-treatment with caspase and cathepsin inhibitors. We further show that ABT-737 caused endoplasmic reticulum (ER stress with increased GRP78 and phosphorylated eIF2α protein levels. Moreover, treatment with ABT-737 increased the intracellular calcium levels, an effect which was enhanced by 9.2.27PE, which as a single entity drug had minimal effect on calcium release from the ER. In addition, silencing of Mcl-1 by short hairpin RNA (shRNA enhanced the intracellular calcium levels and cytotoxicity caused by ABT-737. Notably, the combination of 9.2.27PE and ABT-737 caused growth delay in a human melanoma xenograft mice model, supporting further investigations of this particular drug combination.

  10. Cooperation of IRAK1/4 inhibitor and ABT-737 in nanoparticles for synergistic therapy of T cell acute lymphoblastic leukemia

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    Wu X

    2017-10-01

    Full Text Available Xiaoyan Wu,1 Lin Wang,1 Yining Qiu,1 Bingyu Zhang,1 Zhenhua Hu,2 Runming Jin1 1Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 2Department of Pharmacy, Shanghai Jiao Tong University, Shanghai, China Abstract: T cell acute lymphoblastic leukemia (T-ALL is caused by clonal expansion of variant T cell progenitors and is considered as a high risk leukemia. Contemporary single chemotherapy has a limited effect due to dynamic and versatile properties of T-ALL. Here IRAK1/4 inhibitor and ABT-737 were co-encapsulated into polyethylene glycol modified poly (lactic-co-glycolic acid nanoparticles (IRAK/ABT-NP to enhance synergistic therapy of T-ALL. The formulation was optimized to achieve high drug loading using Box-Behnken design and response surface methodology. The optimal parameter comprised 2.98% polymer in acetonitrile, a ratio of oil phase to water phase of 1:8.33, and 2.12% emulsifier concentration. High drug loading and uniform spherical shape was achieved. In vitro release study showed sustained release of IRAK1/4 inhibitor for 72 hours as well as sustained release of ABT-737 for more than 120 hours. Uptake efficiency of IRAK/ABT-NP and induced apoptotic T-ALL fraction by IRAK/ABT-NP were much higher than the IRAK1/4 and ABT-737 combined solution. IC50 of IRAK/ABT-NP was two-fold lower than free drug combination in Jurkat cells. Additionally, we conducted in vivo experiments in which IRAK/ABT-NP exhibited greater cytotoxicity toward T-ALL cells, the capacity to significantly restore white blood cell number in peripheral blood, and improved survival time of T-ALL mouse model compared to the IRAK1/4 and ABT-737 combined solution. Keywords: T cell acute lymphoblastic leukemia, IRAK1/4 inhibitor, ABT-737, Box-Behnken design and response surface methodology, PEG-PLGA

  11. Chemotherapeutic drugs sensitize human renal cell carcinoma cells to ABT-737 by a mechanism involving the Noxa-dependent inactivation of Mcl-1 or A1

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    Zantl Niko

    2010-06-01

    Full Text Available Abstract Background Human renal cell carcinoma (RCC is very resistant to chemotherapy. ABT-737 is a novel inhibitor of anti-apoptotic proteins of the Bcl-2 family that has shown promise in various preclinical tumour models. Results We here report a strong over-additive pro-apoptotic effect of ABT-737 and etoposide, vinblastine or paclitaxel but not 5-fluorouracil in cell lines from human RCC. ABT-737 showed very little activity as a single agent but killed RCC cells potently when anti-apoptotic Mcl-1 or, unexpectedly, A1 was targeted by RNAi. This potent augmentation required endogenous Noxa protein since RNAi directed against Noxa but not against Bim or Puma reduced apoptosis induction by the combination of ABT-737 and etoposide or vinblastine. At the level of mitochondria, etoposide-treatment had a similar sensitizing activity and allowed for ABT-737-induced release of cytochrome c. Conclusions Chemotherapeutic drugs can overcome protection afforded by Mcl-1 and A1 through endogenous Noxa protein in RCC cells, and the combination of such drugs with ABT-737 may be a promising strategy in RCC. Strikingly, A1 emerged in RCC cell lines as a protein of similar importance as the well-established Mcl-1 in protection against apoptosis in these cells.

  12. The stress protein BAG3 stabilizes Mcl-1 protein and promotes survival of cancer cells and resistance to antagonist ABT-737.

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    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D; Marnett, Lawrence J

    2013-03-08

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery.

  13. The Stress Protein BAG3 Stabilizes Mcl-1 Protein and Promotes Survival of Cancer Cells and Resistance to Antagonist ABT-737*

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    Boiani, Mariana; Daniel, Cristina; Liu, Xueyuan; Hogarty, Michael D.; Marnett, Lawrence J.

    2013-01-01

    Members of the Bcl-2 family of proteins are important inhibitors of apoptosis in human cancer and are targets for novel anticancer agents such as the Bcl-2 antagonists, ABT-263 (Navitoclax), and its analog ABT-737. Unlike Bcl-2, Mcl-1 is not antagonized by ABT-263 or ABT-737 and is considered to be a major factor in resistance. Also, Mcl-1 exhibits differential regulation when compared with other Bcl-2 family members and is a target for anticancer drug discovery. Here, we demonstrate that BAG3, an Hsp70 co-chaperone, protects Mcl-1 from proteasomal degradation, thereby promoting its antiapoptotic activity. Using neuroblastoma cell lines, with a defined Bcl-2 family dependence, we found that BAG3 expression correlated with Mcl-1 dependence and ABT-737 resistance. RNA silencing of BAG3 led to a marked reduction in Mcl-1 protein levels and overcame ABT-737 resistance in Mcl-1-dependent cells. In ABT-737-resistant cells, Mcl-1 co-immunoprecipitated with BAG3, and loss of Mcl-1 after BAG3 silencing was prevented by proteasome inhibition. BAG3 and Mcl-1 were co-expressed in a panel of diverse cancer cell lines resistant to ABT-737. Silencing BAG3 reduced Mcl-1 protein levels and overcame ABT-737 resistance in several of the cell lines, including triple-negative breast cancer (MDA-MB231) and androgen receptor-negative prostate cancer (PC3) cells. These studies identify BAG3-mediated Mcl-1 stabilization as a potential target for cancer drug discovery. PMID:23341456

  14. Exploiting the synergy between carboplatin and ABT-737 in the treatment of ovarian carcinomas.

    KAUST Repository

    Jain, Harsh Vardhan

    2014-01-06

    Platinum drug-resistance in ovarian cancers mediated by anti-apoptotic proteins such as Bcl-xL is a major factor contributing to the chemotherapeutic resistance of recurrent disease. Consequently, concurrent inhibition of Bcl-xL in combination with chemotherapy may improve treatment outcomes for patients. Here, we develop a mathematical model to investigate the potential of combination therapy with ABT-737, a small molecule inhibitor of Bcl-xL, and carboplatin, a platinum-based drug, on a simulated tumor xenograft. The model is calibrated against in vivo experimental data, wherein xenografts established in mice were treated with ABT-737 and/or carboplatin on a fixed periodic schedule. The validated model is used to predict the minimum drug load that will achieve a predetermined level of tumor growth inhibition, thereby maximizing the synergy between the two drugs. Our simulations suggest that the infusion-duration of each carboplatin dose is a critical parameter, with an 8-hour infusion of carboplatin given weekly combined with a daily bolus dose of ABT-737 predicted to minimize residual disease. The potential of combination therapy to prevent or delay the onset of carboplatin-resistance is also investigated. When resistance is acquired as a result of aberrant DNA-damage repair in cells treated with carboplatin, drug delivery schedules that induce tumor remission with even low doses of combination therapy can be identified. Intrinsic resistance due to pre-existing cohorts of resistant cells precludes tumor regression, but dosing strategies that extend disease-free survival periods can still be identified. These results highlight the potential of our model to accelerate the development of novel therapeutics such as BH3 mimetics.

  15. The BH3 mimetic ABT-737 targets selective Bcl-2 proteins and efficiently induces apoptosis via Bak/Bax if Mcl-1 is neutralized

    OpenAIRE

    van Delft, Mark F.; Wei, Andrew H.; Mason, Kylie D.; Vandenberg, Cassandra J.; Chen, Lin; Czabotar, Peter E.; Willis, Simon N.; Scott, Clare L.; Day, Catherine L.; Cory, Suzanne; Adams, Jerry M.; Roberts, Andrew W.; Huang, David C.S.

    2006-01-01

    Since apoptosis is impaired in malignant cells overexpressing pro-survival Bcl-2 proteins, drugs mimicking their natural antagonists, BH3-only proteins, might overcome chemoresistance. Of seven putative BH3 mimetics tested, only ABT-737 triggered Bax/Bak-mediated apoptosis. Despite its high affinity for Bcl-2, Bcl-xL and Bcl-w, many cell types proved refractory to ABT-737. We show that this resistance reflects its inability to target another pro-survival relative, Mcl-1. Down-regulation of Mc...

  16. A microtubule inhibitor, ABT-751, induces autophagy and delays apoptosis in Huh-7 cells

    Energy Technology Data Exchange (ETDEWEB)

    Wei, Ren-Jie [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Lin, Su-Shuan [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Wu, Wen-Ren [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Chen, Lih-Ren [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Division of Physiology, Livestock Research Institute, Council of Agriculture, Taiwan (China); Li, Chien-Feng [Department of Biotechnology, Southern Taiwan University of Science and Technology, Tainan, Taiwan (China); Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan (China); National Institute of Cancer Research, National Health Research Institute, Tainan, Taiwan (China); Institute of Clinical Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chen, Han-De; Chou, Chien-Ting; Chen, Ya-Chun [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Liang, Shih-Shin [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan (China); Chien, Shang-Tao [Department of Pathology, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan (China); Shiue, Yow-Ling, E-mail: ylshiue@mail.nsysu.edu.tw [Institute of Biomedical Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan (China); Doctoral Degree Program in Marine Biotechnology, National Sun Yat-sen University, Kaohsiung, Taiwan (China)

    2016-11-15

    The objective was to investigate the upstream mechanisms of apoptosis which were triggered by a novel anti-microtubule drug, ABT-751, in hepatocellular carcinoma-derived Huh-7 cells. Effects of ABT-751 were evaluated by immunocytochemistry, flow cytometric, alkaline comet, soft agar, immunoblotting, CytoID, green fluorescent protein-microtubule associated protein 1 light chain 3 beta detection, plasmid transfection, nuclear/cytosol fractionation, coimmunoprecipitation, quantitative reverse transcription-polymerase chain reaction, small-hairpin RNA interference and mitochondria/cytosol fractionation assays. Results showed that ABT-751 caused dysregulation of microtubule, collapse of mitochondrial membrane potential, generation of reactive oxygen species (ROS), DNA damage, G{sub 2}/M cell cycle arrest, inhibition of anchorage-independent cell growth and apoptosis in Huh-7 cells. ABT-751 also induced early autophagy via upregulation of nuclear TP53 and downregulation of the AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin (MTOR) pathway. Through modulation of the expression levels of DNA damage checkpoint proteins and G{sub 2}/M cell cycle regulators, ABT-751 induced G{sub 2}/M cell cycle arrest. Subsequently, ABT-751 triggered apoptosis with marked downregulation of B-cell CLL/lymphoma 2, upregulation of mitochondrial BCL2 antagonist/killer 1 and BCL2 like 11 protein levels, and cleavages of caspase 8 (CASP8), CASP9, CASP3 and DNA fragmentation factor subunit alpha proteins. Suppression of ROS significantly decreased ABT-751-induced autophagic and apoptotic cells. Pharmacological inhibition of autophagy significantly increased the percentages of ABT-751-induced apoptotic cells. The autophagy induced by ABT-751 plays a protective role to postpone apoptosis by exerting adaptive responses following microtubule damage, ROS and/or impaired mitochondria. - Highlights: • An anti-microtubule agent, ABT-751, induces autophagy and apoptosis in Huh-7 cells.

  17. Bcl-xL stimulates Bax relocation to mitochondria and primes cells to ABT-737.

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    Renault, Thibaud T; Teijido, Oscar; Missire, Florent; Ganesan, Yogesh Tengarai; Velours, Gisèle; Arokium, Hubert; Beaumatin, Florian; Llanos, Raul; Athané, Axel; Camougrand, Nadine; Priault, Muriel; Antonsson, Bruno; Dejean, Laurent M; Manon, Stéphen

    2015-07-01

    Bax cytosol-to-mitochondria translocation is a central event of the intrinsic pathway of apoptosis. Bcl-xL is an important regulator of this event and was recently shown to promote the retrotranslocation of mitochondrial Bax to the cytosol. The present study identifies a new aspect of the regulation of Bax localization by Bcl-xL: in addition to its role in Bax inhibition and retrotranslocation, we found that, like with Bcl-2, an increase of Bcl-xL expression levels led to an increase of Bax mitochondrial content. This finding was substantiated both in pro-lymphocytic FL5.12 cells and a yeast reporting system. Bcl-xL-dependent increase of mitochondrial Bax is counterbalanced by retrotranslocation, as we observed that Bcl-xLΔC, which is unable to promote Bax retrotranslocation, was more efficient than the full-length protein in stimulating Bax relocation to mitochondria. Interestingly, cells overexpressing Bcl-xL were more sensitive to apoptosis upon treatment with the BH3-mimetic ABT-737, suggesting that despite its role in Bax inhibition, Bcl-xL also primes mitochondria to permeabilization and cytochrome c release. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

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    Keuling, Angela M; Felton, Kathleen E A; Parker, Arabesque A M; Akbari, Majid; Andrew, Susan E; Tron, Victor A

    2009-08-17

    Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10) activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

  19. RNA silencing of Mcl-1 enhances ABT-737-mediated apoptosis in melanoma: role for a caspase-8-dependent pathway.

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    Angela M Keuling

    Full Text Available BACKGROUND: Malignant melanoma is resistant to almost all conventional forms of chemotherapy. Recent evidence suggests that anti-apoptotic proteins of the Bcl-2 family are overexpressed in melanoma and may contribute to melanoma's striking resistance to apoptosis. ABT-737, a small-molecule inhibitor of Bcl-2, Bcl-xl and Bcl-w, has demonstrated efficacy in several forms of leukemia, lymphoma as well as solid tumors. However, overexpression of Mcl-1, a frequent observance in melanoma, is known to confer ABT-737 resistance. METHODOLOGY/PRINCIPAL FINDINGS: Here we report that knockdown of Mcl-1 greatly reduces cell viability in combination with ABT-737 in six different melanoma cell lines. We demonstrate that the cytotoxic effect of this combination treatment is due to apoptotic cell death involving not only caspase-9 activation but also activation of caspase-8, caspase-10 and Bid, which are normally associated with the extrinsic pathway of apoptosis. Caspase-8 (and caspase-10 activation is abrogated by inhibition of caspase-9 but not by inhibitors of the death receptor pathways. Furthermore, while caspase-8/-10 activity is required for the full induction of cell death with treatment, the death receptor pathways are not. Finally, we demonstrate that basal levels of caspase-8 and Bid correlate with treatment sensitivity. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that the combination of ABT-737 and Mcl-1 knockdown represents a promising, new treatment strategy for malignant melanoma. We also report a death receptor-independent role for extrinsic pathway proteins in treatment response and suggest that caspase-8 and Bid may represent potential markers of treatment sensitivity.

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

    Science.gov (United States)

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

    2016-02-01

    Does induction of mitochondrial outer membrane permeabilization (MOMP) in vitro affect specific functional parameters of human spermatozoa? Our findings show that MOMP induction increases intracellular reactive oxygen species (ROS) and decreases mean sperm velocity but does not alter DNA integrity. MOMP in somatic cells is related to a variety of apoptotic traits, such as alteration of mitochondrial membrane potential (ΔΨm), and increase in ROS production and DNA fragmentation. Although the presence of these apoptotic features has been reported in spermatozoa, to date the effects of MOMP on sperm function and DNA integrity have not been analysed. The study included spermatozoa from fertile donors. Motile sperm were obtained using the swim-up method. The highly motile sperm were collected and diluted with human tubal fluid to a final cell concentration of 5 × 10(6) ml(-1). To induce MOMP, selected sperm were treated at 37°C for 4 h with a mimetic of a Bcl-2 pro-apoptotic protein, ABT-737. MOMP was evaluated by relocating of cytochrome c. In addition, the effect of ABT-737 on mitochondrial inner membrane permeabilization was assessed using the calcein-AM/cobalt chloride method. In turn, ΔΨm was evaluated with JC-1 staining, intracellular ROS production with dihydroethidium, sperm motility was analysed by computer-assisted sperm analysis and DNA fragmentation by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL) assay. Measurements were performed by flow cytometry. MOMP was associated with ΔΨm dissipation (P < 0.05), increased ROS production (P < 0.05) and decreased mean sperm velocity (P < 0.05), but it was not associated with DNA fragmentation. MOMP did not induce a large increase in ROS, which could explain the negligible effect of MOMP on sperm DNA fragmentation under our experimental conditions. The study was carried out in vitro using highly motile sperm, selected by swim-up, from healthy donors. The results obtained in this

  1. In vivo efficacy of the Bcl-2 antagonist ABT-737 against aggressive Myc-driven lymphomas

    OpenAIRE

    Mason, Kylie D.; Vandenberg, Cassandra J.; Scott, Clare L.; Wei, Andrew H.; Cory, Suzanne; Huang, David C. S.; Roberts, Andrew W.

    2008-01-01

    Deregulated Myc expression drives many human cancers, including Burkitt's lymphoma and a highly aggressive subset of diffuse large cell lymphomas. Myc-driven tumors often display resistance to chemotherapeutics because of acquisition of mutations that impair the apoptosis pathway regulated by the Bcl-2 protein family. Given the need to identify new therapies for such lymphomas, we have evaluated the efficacy of ABT-737, a small molecule that mimics the action of the BH3-only proteins, natural...

  2. Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis.

    Science.gov (United States)

    Renault, Thibaud T; Floros, Konstantinos V; Elkholi, Rana; Corrigan, Kelly-Ann; Kushnareva, Yulia; Wieder, Shira Y; Lindtner, Claudia; Serasinghe, Madhavika N; Asciolla, James J; Buettner, Christoph; Newmeyer, Donald D; Chipuk, Jerry E

    2015-01-08

    Proapoptotic BCL-2 proteins converge upon the outer mitochondrial membrane (OMM) to promote mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Here we investigated the mechanistic relationship between mitochondrial shape and MOMP and provide evidence that BAX requires a distinct mitochondrial size to induce MOMP. We utilized the terminal unfolded protein response pathway to systematically define proapoptotic BCL-2 protein composition after stress and then directly interrogated their requirement for a productive mitochondrial size. Complementary biochemical, cellular, in vivo, and ex vivo studies reveal that Mfn1, a GTPase involved in mitochondrial fusion, establishes a mitochondrial size that is permissive for proapoptotic BCL-2 family function. Cells with hyperfragmented mitochondria, along with size-restricted OMM model systems, fail to support BAX-dependent membrane association and permeabilization due to an inability to stabilize BAXα9·membrane interactions. This work identifies a mechanistic contribution of mitochondrial size in dictating BAX activation, MOMP, and apoptosis. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. Salvianolic Acid-A Induces Apoptosis, Mitochondrial Membrane ...

    African Journals Online (AJOL)

    using Hoechst 33258 staining. The effect of the compound on mitochondrial membrane potential loss ... Fluorescence microscopy demonstrated that salvianolic acid-A induced dose- dependent ..... aggregation and anticancer properties. It has.

  4. PEGylated anticancer-carbon nanotubes complex targeting mitochondria of lung cancer cells

    Science.gov (United States)

    Kim, Sang-Woo; Lee, Yeon Kyung; Lee, Jong Yeon; Hong, Jeong Hee; Khang, Dongwoo

    2017-11-01

    Although activating apoptosis in cancer cells by targeting the mitochondria is an effective strategy for cancer therapy, insufficient targeting of the mitochondria in cancer cells restricts the availability in clinical treatment. Here, we report on a polyethylene glycol-coated carbon nanotube (CNT)-ABT737 nanodrug that improves the mitochondrial targeting of lung cancer cells. The polyethylene glycol-coated CNT-ABT737 nanodrug internalized into the early endosomes via macropinocytosis and clathrin-mediated endocytosis in advance of early endosomal escape and delivered into the mitochondria. Cytosol release of the nanodrug led to apoptosis of lung cancer cells by abruption of the mitochondrial membrane potential, inducing Bcl-2-mediated apoptosis and generating intracellular reactive oxygen species. As such, this study provides an effective strategy for increasing the anti-lung cancer efficacy by increasing mitochondria accumulation rate of cytosol released anticancer nanodrugs.

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

    International Nuclear Information System (INIS)

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

    2010-01-01

    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 μM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt 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 μM cytochrome c/min/mg in control hearts vs. 14 ± 3 μM 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.

  6. Induced mitochondrial membrane potential for modeling solitonic conduction of electrotonic signals.

    Directory of Open Access Journals (Sweden)

    R R Poznanski

    Full Text Available A cable model that includes polarization-induced capacitive current is derived for modeling the solitonic conduction of electrotonic potentials in neuronal branchlets with microstructure containing endoplasmic membranes. A solution of the nonlinear cable equation modified for fissured intracellular medium with a source term representing charge 'soakage' is used to show how intracellular capacitive effects of bound electrical charges within mitochondrial membranes can influence electrotonic signals expressed as solitary waves. The elastic collision resulting from a head-on collision of two solitary waves results in localized and non-dispersing electrical solitons created by the nonlinearity of the source term. It has been shown that solitons in neurons with mitochondrial membrane and quasi-electrostatic interactions of charges held by the microstructure (i.e., charge 'soakage' have a slower velocity of propagation compared with solitons in neurons with microstructure, but without endoplasmic membranes. When the equilibrium potential is a small deviation from rest, the nonohmic conductance acts as a leaky channel and the solitons are small compared when the equilibrium potential is large and the outer mitochondrial membrane acts as an amplifier, boosting the amplitude of the endogenously generated solitons. These findings demonstrate a functional role of quasi-electrostatic interactions of bound electrical charges held by microstructure for sustaining solitons with robust self-regulation in their amplitude through changes in the mitochondrial membrane equilibrium potential. The implication of our results indicate that a phenomenological description of ionic current can be successfully modeled with displacement current in Maxwell's equations as a conduction process involving quasi-electrostatic interactions without the inclusion of diffusive current. This is the first study in which solitonic conduction of electrotonic potentials are generated by

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

    Science.gov (United States)

    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

    2015-01-01

    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. 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. 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. We assume HF-induced modifications in membrane lipid- and protein-signatures prior to and

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

    Directory of Open Access Journals (Sweden)

    Elisa Balboa

    2017-08-01

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

  9. Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

    Science.gov (United States)

    Harrison, Luke R.E.; Micha, Dimitra; Brandenburg, Martin; Simpson, Kathryn L.; Morrow, Christopher J.; Denneny, Olive; Hodgkinson, Cassandra; Yunus, Zaira; Dempsey, Clare; Roberts, Darren; Blackhall, Fiona; Makin, Guy; Dive, Caroline

    2011-01-01

    Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic induction of apoptosis was mediated through downregulation of myeloid cell leukemia sequence 1 (Mcl-1), a Bcl-2 family protein that serves as a biomarker for ABT-737 resistance. Downregulation of Mcl-1 in hypoxia was independent of hypoxia-inducible factor 1 (HIF-1) activity and was consistent with decreased global protein translation. In addition, ABT-737 induced apoptosis deep within tumor spheroids, consistent with an optimal hypoxic oxygen tension being necessary to promote ABT-737induced cell death. Tumor xenografts in ABT-737–treated mice also displayed significantly more apoptotic cells within hypoxic regions relative to normoxic regions. Synergies between ABT-737 and other cytotoxic drugs were maintained in hypoxia, suggesting that this drug may be useful in combination with chemotherapeutic agents. Taken together, these findings suggest that Mcl-1–sparing BH-3 mimetics may induce apoptosis in hypoxic tumor cells that are resistant to other chemotherapeutic agents and may have a role in combinatorial chemotherapeutic regimens for treatment of solid tumors. PMID:21393866

  10. Cisplatin impairs rat liver mitochondrial functions by inducing changes on membrane ion permeability: Prevention by thiol group protecting agents

    International Nuclear Information System (INIS)

    Custodio, Jose B.A.; Cardoso, Carla M.P.; Santos, Maria S.; Almeida, Leonor M.; Vicente, Joaquim A.F.; Fernandes, Maria A.S.

    2009-01-01

    Cisplatin (CisPt) is the most important platinum anticancer drug widely used in the treatment of head, neck, ovarian and testicular cancers. However, the mechanisms by which CisPt induces cytotoxicity, namely hepatotoxicity, are not completely understood. The goal of this study was to investigate the influence of CisPt on rat liver mitochondrial functions (Ca 2+ -induced mitochondrial permeability transition (MPT), mitochondrial bioenergetics, and mitochondrial oxidative stress) to better understand the mechanism underlying its hepatotoxicity. The effect of thiol group protecting agents and some antioxidants against CisPt-induced mitochondrial damage was also investigated. Treatment of rat liver mitochondria with CisPt (20 nmol/mg protein) induced Ca 2+ -dependent mitochondrial swelling, depolarization of membrane potential (ΔΨ), Ca 2+ release, and NAD(P)H fluorescence intensity decay. These effects were prevented by cyclosporine A (CyA), a potent and specific inhibitor of the MPT. In the concentration range of up to 40 nmol/mg protein, CisPt slightly inhibited state 3 and stimulated state 2 and state 4 respiration rates using succinate as respiratory substrate. The respiratory indexes, respiratory control ratio (RCR) and ADP/O ratios, the ΔΨ, and the ADP phosphorylation rate were also depressed. CisPt induced mitochondrial inner membrane permeabilization to protons (proton leak) but did not induce significant changes on mitochondrial H 2 O 2 generation. All the effects induced by CisPt on rat liver mitochondria were prevented by thiol group protecting agents namely, glutathione (GSH), dithiothreitol (DTT), N-acetyl-L-cysteine (NAC) and cysteine (CYS), whereas superoxide-dismutase (SOD), catalase (CAT) and ascorbate (ASC) were without effect. In conclusion, the anticancer drug CisPt: (1) increases the sensitivity of mitochondria to Ca 2+ -induced MPT; (2) interferes with mitochondrial bioenergetics by increasing mitochondrial inner membrane permeabilization to

  11. Soluble products of Escherichia coli induce mitochondrial dysfunction-related sperm membrane lipid peroxidation which is prevented by lactobacilli.

    Directory of Open Access Journals (Sweden)

    Arcangelo Barbonetti

    Full Text Available Unidentified soluble factors secreted by E. coli, a frequently isolated microorganism in genitourinary infections, have been reported to inhibit mitochondrial membrane potential (ΔΨm, motility and vitality of human spermatozoa. Here we explore the mechanisms involved in the adverse impact of E. coli on sperm motility, focusing mainly on sperm mitochondrial function and possible membrane damage induced by mitochondrial-generated reactive oxygen species (ROS. Furthermore, as lactobacilli, which dominate the vaginal ecosystem of healthy women, have been shown to exert anti-oxidant protective effects on spermatozoa, we also evaluated whether soluble products from these microorganisms could protect spermatozoa against the effects of E. coli. We assessed motility (by computer-aided semen analysis, ΔΨm (with JC-1 dye by flow cytometry, mitochondrial ROS generation (with MitoSOX red dye by flow cytometry and membrane lipid-peroxidation (with the fluorophore BODIPY C11 by flow cytometry of sperm suspensions exposed to E. coli in the presence and in the absence of a combination of 3 selected strains of lactobacilli (L. brevis, L. salivarius, L. plantarum. A Transwell system was used to avoid direct contact between spermatozoa and microorganisms. Soluble products of E. coli induced ΔΨm loss, mitochondrial generation of ROS and membrane lipid-peroxidation, resulting in motility loss. Soluble factors of lactobacilli prevented membrane lipid-peroxidation of E. coli-exposed spermatozoa, thus preserving their motility. In conclusion, sperm motility loss by soluble products of E. coli reflects a mitochondrial dysfunction-related membrane lipid-peroxidation. Lactobacilli could protect spermatozoa in the presence of vaginal disorders, by preventing ROS-induced membrane damage.

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

    Directory of Open Access Journals (Sweden)

    Ramzi J Khairallah

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

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

    Directory of Open Access Journals (Sweden)

    Seong-Woon Yu

    2009-10-01

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

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

    LENUS (Irish Health Repository)

    Gupta, Sanjeev

    2010-01-01

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

  15. Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN

    Directory of Open Access Journals (Sweden)

    Annalisa Canta

    2015-06-01

    Full Text Available The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN. This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy.

  16. Mitochondrial Dysfunction in Chemotherapy-Induced Peripheral Neuropathy (CIPN)

    Science.gov (United States)

    Canta, Annalisa; Pozzi, Eleonora; Carozzi, Valentina Alda

    2015-01-01

    The mitochondrial dysfunction has a critical role in several disorders including chemotherapy-induced peripheral neuropathies (CIPN). This is due to a related dysregulation of pathways involving calcium signalling, reactive oxygen species and apoptosis. Vincristine is able to affect calcium movement through the Dorsal Root Ganglia (DRG) neuronal mitochondrial membrane, altering its homeostasis and leading to abnormal neuronal excitability. Paclitaxel induces the opening of the mitochondrial permeability transition pore in axons followed by mitochondrial membrane potential loss, increased reactive oxygen species generation, ATP level reduction, calcium release and mitochondrial swelling. Cisplatin and oxaliplatin form adducts with mitochondrial DNA producing inhibition of replication, disruption of transcription and morphological abnormalities within mitochondria in DRG neurons, leading to a gradual energy failure. Bortezomib is able to modify mitochondrial calcium homeostasis and mitochondrial respiratory chain. Moreover, the expression of a certain number of genes, including those controlling mitochondrial functions, was altered in patients with bortezomib-induced peripheral neuropathy. PMID:29056658

  17. TCA Cycle and Mitochondrial Membrane Potential Are Necessary for Diverse Biological Functions.

    Science.gov (United States)

    Martínez-Reyes, Inmaculada; Diebold, Lauren P; Kong, Hyewon; Schieber, Michael; Huang, He; Hensley, Christopher T; Mehta, Manan M; Wang, Tianyuan; Santos, Janine H; Woychik, Richard; Dufour, Eric; Spelbrink, Johannes N; Weinberg, Samuel E; Zhao, Yingming; DeBerardinis, Ralph J; Chandel, Navdeep S

    2016-01-21

    Mitochondrial metabolism is necessary for the maintenance of oxidative TCA cycle function and mitochondrial membrane potential. Previous attempts to decipher whether mitochondria are necessary for biological outcomes have been hampered by genetic and pharmacologic methods that simultaneously disrupt multiple functions linked to mitochondrial metabolism. Here, we report that inducible depletion of mitochondrial DNA (ρ(ο) cells) diminished respiration, oxidative TCA cycle function, and the mitochondrial membrane potential, resulting in diminished cell proliferation, hypoxic activation of HIF-1, and specific histone acetylation marks. Genetic reconstitution only of the oxidative TCA cycle function specifically in these inducible ρ(ο) cells restored metabolites, resulting in re-establishment of histone acetylation. In contrast, genetic reconstitution of the mitochondrial membrane potential restored ROS, which were necessary for hypoxic activation of HIF-1 and cell proliferation. These results indicate that distinct mitochondrial functions associated with respiration are necessary for cell proliferation, epigenetics, and HIF-1 activation. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. Bcl-xL regulates mitochondrial energetics by stabilizing the inner membrane potential.

    Science.gov (United States)

    Chen, Ying-Bei; Aon, Miguel A; Hsu, Yi-Te; Soane, Lucian; Teng, Xinchen; McCaffery, J Michael; Cheng, Wen-Chih; Qi, Bing; Li, Hongmei; Alavian, Kambiz N; Dayhoff-Brannigan, Margaret; Zou, Shifa; Pineda, Fernando J; O'Rourke, Brian; Ko, Young H; Pedersen, Peter L; Kaczmarek, Leonard K; Jonas, Elizabeth A; Hardwick, J Marie

    2011-10-17

    Mammalian Bcl-x(L) protein localizes to the outer mitochondrial membrane, where it inhibits apoptosis by binding Bax and inhibiting Bax-induced outer membrane permeabilization. Contrary to expectation, we found by electron microscopy and biochemical approaches that endogenous Bcl-x(L) also localized to inner mitochondrial cristae. Two-photon microscopy of cultured neurons revealed large fluctuations in inner mitochondrial membrane potential when Bcl-x(L) was genetically deleted or pharmacologically inhibited, indicating increased total ion flux into and out of mitochondria. Computational, biochemical, and genetic evidence indicated that Bcl-x(L) reduces futile ion flux across the inner mitochondrial membrane to prevent a wasteful drain on cellular resources, thereby preventing an energetic crisis during stress. Given that F(1)F(O)-ATP synthase directly affects mitochondrial membrane potential and having identified the mitochondrial ATP synthase β subunit in a screen for Bcl-x(L)-binding partners, we tested and found that Bcl-x(L) failed to protect β subunit-deficient yeast. Thus, by bolstering mitochondrial energetic capacity, Bcl-x(L) may contribute importantly to cell survival independently of other Bcl-2 family proteins.

  19. Formation and Regulation of Mitochondrial Membranes

    Directory of Open Access Journals (Sweden)

    Laila Cigana Schenkel

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sergio A. Rosales-Corral

    2012-01-01

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

  1. Pharmacological and protein profiling suggest venetoclax (ABT-199) as optimal partner with ibrutinib in chronic lymphocytic leukemia

    Science.gov (United States)

    Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A.; Wierda, William G.; Keating, Michael J.; Balakrishnan, Kumudha; Gandhi, Varsha

    2015-01-01

    Purpose Bruton’s tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Experimental design Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199) and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. Results The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted highly cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family anti-apoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Conclusions Our biological and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. PMID:25829398

  2. Pharmacological and Protein Profiling Suggests Venetoclax (ABT-199) as Optimal Partner with Ibrutinib in Chronic Lymphocytic Leukemia.

    Science.gov (United States)

    Cervantes-Gomez, Fabiola; Lamothe, Betty; Woyach, Jennifer A; Wierda, William G; Keating, Michael J; Balakrishnan, Kumudha; Gandhi, Varsha

    2015-08-15

    Bruton's tyrosine kinase (BTK) is a critical enzyme in the B-cell receptor pathway and is inhibited by ibrutinib due to covalent binding to the kinase domain. Though ibrutinib results in impressive clinical activity in chronic lymphocytic leukemia (CLL), most patients achieve only partial remission due to residual disease. We performed a pharmacologic profiling of residual circulating CLL cells from patients receiving ibrutinib to identify optimal agents that could induce cell death of these lymphocytes. Ex vivo serial samples of CLL cells from patients on ibrutinib were obtained prior and after (weeks 2, 4, and 12) the start of treatment. These cells were incubated with PI3K inhibitors (idelalisib or IPI-145), bendamustine, additional ibrutinib, or BCL-2 antagonists (ABT-737 or ABT-199), and cell death was measured. In vitro investigations complemented ex vivo studies. Immunoblots for BTK signaling pathway and antiapoptotic proteins were performed. The BCL-2 antagonists, especially ABT-199, induced high cell death during ex vivo incubations. In concert with the ex vivo data, in vitro combinations also resulted in high cytotoxicity. Serial samples of CLL cells obtained before and 2, 4, 12, or 36 weeks after the start of ibrutinib showed inhibition of BTK activity and sensitivity to ABTs. Among the three BCL-2 family antiapoptotic proteins that are overexpressed in CLL, levels of MCL-1 and BCL-XL were decreased after ibrutinib while ABT-199 selectively antagonizes BCL-2. Our biologic and molecular results suggest that ibrutinib and ABT-199 combination should be tested clinically against CLL. ©2015 American Association for Cancer Research.

  3. The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis*

    Science.gov (United States)

    Barnwal, Bhaskar; Karlberg, Helen; Mirazimi, Ali; Tan, Yee-Joo

    2016-01-01

    Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells. PMID:26574543

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

    International Nuclear Information System (INIS)

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

    2009-01-01

    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 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

  5. Mitofilin complexes: conserved organizers of mitochondrial membrane architecture.

    Science.gov (United States)

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

    2012-11-01

    Mitofilin proteins are crucial organizers of mitochondrial architecture. They are located in the inner mitochondrial membrane and interact with several protein complexes of the outer membrane, thereby generating contact sites between the two membrane systems of mitochondria. Within the inner membrane, mitofilins are part of hetero-oligomeric protein complexes that have been termed the mitochondrial inner membrane organizing system (MINOS). MINOS integrity is required for the maintenance of the characteristic morphology of the inner mitochondrial membrane, with an inner boundary region closely apposed to the outer membrane and cristae membranes, which form large tubular invaginations that protrude into the mitochondrial matrix and harbor the enzyme complexes of the oxidative phosphorylation machinery. MINOS deficiency comes along with a loss of crista junction structures and the detachment of cristae from the inner boundary membrane. MINOS has been conserved in evolution from unicellular eukaryotes to humans, where alterations of MINOS subunits are associated with multiple pathological conditions.

  6. A mitochondrially targeted compound delays aging in yeast through a mechanism linking mitochondrial membrane lipid metabolism to mitochondrial redox biology

    Directory of Open Access Journals (Sweden)

    Michelle T. Burstein

    2014-01-01

    Full Text Available A recent study revealed a mechanism of delaying aging in yeast by a natural compound which specifically impacts mitochondrial redox processes. In this mechanism, exogenously added lithocholic bile acid enters yeast cells, accumulates mainly in the inner mitochondrial membrane, and elicits an age-related remodeling of phospholipid synthesis and movement within both mitochondrial membranes. Such remodeling of mitochondrial phospholipid dynamics progresses with the chronological age of a yeast cell and ultimately causes significant changes in mitochondrial membrane lipidome. These changes in the composition of membrane phospholipids alter mitochondrial abundance and morphology, thereby triggering changes in the age-related chronology of such longevity-defining redox processes as mitochondrial respiration, the maintenance of mitochondrial membrane potential, the preservation of cellular homeostasis of mitochondrially produced reactive oxygen species, and the coupling of electron transport to ATP synthesis.

  7. Mitofilin complexes : conserved organizers of mitochondrial membrane architecture

    NARCIS (Netherlands)

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

    2012-01-01

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

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

    International Nuclear Information System (INIS)

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

    2012-01-01

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

  9. KB-R7943, a plasma membrane Na(+)/Ca(2+) exchanger inhibitor, blocks opening of the mitochondrial permeability transition pore.

    Science.gov (United States)

    Wiczer, Brian M; Marcu, Raluca; Hawkins, Brian J

    2014-01-31

    The isothiourea derivative, KB-R7943, inhibits the reverse-mode of the plasma membrane sodium/calcium exchanger and protects against ischemia/reperfusion injury. The mechanism through which KB-R7943 confers protection, however, remains controversial. Recently, KB-R7943 has been shown to inhibit mitochondrial calcium uptake and matrix overload, which may contribute to its protective effects. While using KB-R7943 for this purpose, we find here no evidence that KB-R7943 directly blocks mitochondrial calcium uptake. Rather, we find that KB-R7943 inhibits opening of the mitochondrial permeability transition pore in permeabilized cells and isolated liver mitochondria. Furthermore, we find that this observation correlates with protection against calcium ionophore-induced mitochondrial membrane potential depolarization and cell death, without detrimental effects to basal mitochondrial membrane potential or complex I-dependent mitochondrial respiration. Our data reveal another mechanism through which KB-R7943 may protect against calcium-induced injury, as well as a novel means to inhibit the mitochondrial permeability transition pore. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Common effects of lithium and valproate on mitochondrial functions: protection against methamphetamine-induced mitochondrial damage.

    Science.gov (United States)

    Bachmann, Rosilla F; Wang, Yun; Yuan, Peixiong; Zhou, Rulun; Li, Xiaoxia; Alesci, Salvatore; Du, Jing; Manji, Husseini K

    2009-07-01

    Accumulating evidence suggests that mitochondrial dysfunction plays a critical role in the progression of a variety of neurodegenerative and psychiatric disorders. Thus, enhancing mitochondrial function could potentially help ameliorate the impairments of neural plasticity and cellular resilience associated with a variety of neuropsychiatric disorders. A series of studies was undertaken to investigate the effects of mood stabilizers on mitochondrial function, and against mitochondrially mediated neurotoxicity. We found that long-term treatment with lithium and valproate (VPA) enhanced cell respiration rate. Furthermore, chronic treatment with lithium or VPA enhanced mitochondrial function as determined by mitochondrial membrane potential, and mitochondrial oxidation in SH-SY5Y cells. In-vivo studies showed that long-term treatment with lithium or VPA protected against methamphetamine (Meth)-induced toxicity at the mitochondrial level. Furthermore, these agents prevented the Meth-induced reduction of mitochondrial cytochrome c, the mitochondrial anti-apoptotic Bcl-2/Bax ratio, and mitochondrial cytochrome oxidase (COX) activity. Oligoarray analysis demonstrated that the gene expression of several proteins related to the apoptotic pathway and mitochondrial functions were altered by Meth, and these changes were attenuated by treatment with lithium or VPA. One of the genes, Bcl-2, is a common target for lithium and VPA. Knock-down of Bcl-2 with specific Bcl-2 siRNA reduced the lithium- and VPA-induced increases in mitochondrial oxidation. These findings illustrate that lithium and VPA enhance mitochondrial function and protect against mitochondrially mediated toxicity. These agents may have potential clinical utility in the treatment of other diseases associated with impaired mitochondrial function, such as neurodegenerative diseases and schizophrenia.

  11. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A.

    Science.gov (United States)

    Siu, Woen Ping; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan; Boelsterli, Urs A

    2008-03-15

    Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (>500 microM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 microM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca2+-Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury.

  12. Bax-mediated mitochondrial outer membrane permeabilization (MOMP), distinct from the mitochondrial permeability transition, is a key mechanism in diclofenac-induced hepatocyte injury: Multiple protective roles of cyclosporin A

    International Nuclear Information System (INIS)

    Siu, W.P.; Pun, Pamela Boon Li; Latchoumycandane, Calivarathan; Boelsterli, Urs A.

    2008-01-01

    Diclofenac, a widely used nonsteroidal anti-inflammatory drug, has been associated with rare but severe cases of clinical hepatotoxicity. Diclofenac causes concentration-dependent cell death in human hepatocytes (after 24-48 h) by mitochondrial permeabilization via poorly defined mechanisms. To explore whether the cyclophilin D (CyD)-dependent mitochondrial permeability transition (mPT) and/or the mitochondrial outer membrane permeabilization (MOMP) was primarily involved in mediating cell death, we exposed immortalized human hepatocytes (HC-04) to apoptogenic concentrations of diclofenac (> 500 μM) in the presence or absence of inhibitors of upstream mediators. The CyD inhibitor, cyclosporin A (CsA, 2 μM) fully inhibited diclofenac-induced cell injury, suggesting that mPT was involved. However, CyD gene silencing using siRNA left the cells susceptible to diclofenac toxicity, and CsA still protected the CyD-negative cells from lethal injury. Diclofenac induced early (9 h) activation of Bax and Bak and caused mitochondrial translocation of Bax, indicating that MOMP was involved in cell death. Inhibition of Bax protein expression by using siRNA significantly protected HC-04 from diclofenac-induced cell injury. Diclofenac also induced early Bid activation (tBid formation, 6 h), which is an upstream mechanism that initiates Bax activation and mitochondrial translocation. Bid activation was sensitive to the Ca 2+ chelator, BAPTA. In conclusion, we found that Bax/Bak-mediated MOMP is a key mechanism of diclofenac-induced lethal cell injury in human hepatocytes, and that CsA can prevent MOMP through inhibition of Bax activation. These data support our concept that the Ca 2+ -Bid-Bax-MOMP axis is a critical pathway in diclofenac (metabolite)-induced hepatocyte injury

  13. Mitochondrial fusion through membrane automata.

    Science.gov (United States)

    Giannakis, Konstantinos; Andronikos, Theodore

    2015-01-01

    Studies have shown that malfunctions in mitochondrial processes can be blamed for diseases. However, the mechanism behind these operations is yet not sufficiently clear. In this work we present a novel approach to describe a biomolecular model for mitochondrial fusion using notions from the membrane computing. We use a case study defined in BioAmbient calculus and we show how to translate it in terms of a P automata variant. We combine brane calculi with (mem)brane automata to produce a new scheme capable of describing simple, realistic models. We propose the further use of similar methods and the test of other biomolecular models with the same behaviour.

  14. Mulberry Fruit Extract Affords Protection against Ethyl Carbamate-Induced Cytotoxicity and Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-01-01

    Full Text Available Ethyl carbamate (EC is a food and environmental toxicant and is a cause of concern for human exposure. Several studies indicated that EC-induced toxicity was associated with oxidative stress. Mulberry fruits are reported to have a wide range of bioactive compounds and pharmacological activities. The present study was therefore aimed to investigate the protective property of mulberry fruit extract (MFE on EC-induced cytotoxicity and oxidative stress. Chemical composition analysis showed that total phenolic content and total flavonoid content in MFE were 502.43 ± 5.10 and 219.12 ± 4.45 mg QE/100 g FW. Cyanidin-3-O-glucoside and cyanidin-3-O-rutinoside were the major anthocyanins in MFE. In vitro antioxidant studies (DPPH, ABTS, and FRAP assays jointly exhibited the potent antioxidant capacity of MFE. Further study indicated that MFE protected human liver HepG2 cells from EC-induced cytotoxicity by scavenging overproduced cellular ROS. EC treatment promoted intracellular glutathione (GSH depletion and caused mitochondrial membrane potential (MMP collapse, as well as mitochondrial membrane lipid peroxidation, whereas MFE pretreatment significantly inhibited GSH depletion and restored the mitochondrial membrane function. Overall, our study suggested that polyphenolic-rich MFE could afford a potent protection against EC-induced cytotoxicity and oxidative stress.

  15. BH3-mimetics- and cisplatin-induced cell death proceeds through different pathways depending on the availability of death-related cellular components.

    Directory of Open Access Journals (Sweden)

    Vicente Andreu-Fernández

    Full Text Available BACKGROUND: Owing to their important function in regulating cell death, pharmacological inhibition of Bcl-2 proteins by dubbed BH3-mimetics is a promising strategy for apoptosis induction or sensitization to chemotherapy. However, the role of Apaf-1, the main protein constituent of the apoptosome, in the process has yet not been analyzed. Furthermore as new chemotherapeutics develop, the possible chemotherapy-induced toxicity to rapidly dividing normal cells, especially sensitive differentiated cells, has to be considered. Such undesirable effects would probably be ameliorated by selectively and locally inhibiting apoptosis in defined sensitive cells. METHODOLOGY AND PRINCIPAL FINDINGS: Mouse embryonic fibroblasts (MEFS from Apaf-1 knock out mouse (MEFS KO Apaf-1 and Bax/Bak double KO (MEFS KO Bax/Bak, MEFS from wild-type mouse (MEFS wt and human cervix adenocarcinoma (HeLa cells were used to comparatively investigate the signaling cell death-induced pathways of BH3-mimetics, like ABT737 and GX15-070, with DNA damage-inducing agent cisplatin (cis-diammineplatinum(II dichloride, CDDP. The study was performed in the absence or presence of apoptosis inhibitors namely, caspase inhibitors or apoptosome inhibitors. BH3-mimetic ABT737 required of Apaf-1 to exert its apoptosis-inducing effect. In contrast, BH3-mimetic GX15-070 and DNA damage-inducing CDDP induced cell death in the absence of both Bax/Bak and Apaf-1. GX15-070 induced autophagy-based cell death in all the cell lines analyzed. MEFS wt cells were protected from the cytotoxic effects of ABT737 and CDDP by chemical inhibition of the apoptosome through QM31, but not by using general caspase inhibitors. CONCLUSIONS: BH3-mimetic ABT737 not only requires Bax/Bak to exert its apoptosis-inducing effect, but also Apaf-1, while GX15-070 and CDDP induce different modalities of cell death in the absence of Bax/Bak or Apaf-1. Inclusion of specific Apaf-1 inhibitors in topical and well

  16. ABT-263 induces G1/G0-phase arrest, apoptosis and autophagy in human esophageal cancer cells in vitro.

    Science.gov (United States)

    Lin, Qing-Huan; Que, Fu-Chang; Gu, Chun-Ping; Zhong, De-Sheng; Zhou, Dan; Kong, Yi; Yu, Le; Liu, Shu-Wen

    2017-12-01

    Both the anti- and pro-apoptotic members of the Bcl-2 family are regulated by a conserved Bcl-2 homology (BH3) domain. ABT-263 (Navitoclax), a novel BH3 mimetic and orally bioavailable Bcl-2 family inhibitor with high affinity for Bcl-xL, Bcl-2 and Bcl-w has entered clinical trials for cancer treatment. But the anticancer mechanisms of ABT-263 have not been fully elucidated. In this study we investigated the effects of ABT-263 on human esophageal cancer cells in vitro and to explore its anticancer mechanisms. Treatment with ABT-263 dose-dependently suppressed the viability of 3 human esophageal cancer cells with IC 50 values of 10.7±1.4, 7.1±1.5 and 8.2±1.6 μmol/L, in EC109, HKESC-2 and CaES-17 cells, respectively. ABT-263 (5-20 μmol/L) dose-dependently induced G 1 /G 0 -phase arrest in the 3 cancer cell lines and induced apoptosis evidenced by increased the Annexin V-positive cell population and elevated levels of cleaved caspase 3, cleaved caspase 9 and PARP. We further demonstrated that ABT-263 treatment markedly increased the expression of p21 Waf1/Cip1 and decreased the expression of cyclin D1 and phospho-Rb (retinoblastoma tumor suppressor protein) (Ser780) proteins that contributed to the G 1 /G 0 -phase arrest. Knockdown of p21 Waf1/Cip1 attenuated ABT-263-induced G 1 /G 0 -phase arrest. Moreover, ABT-263 treatment enhanced pro-survival autophagy, shown as the increased LC3-II levels and decreased p62 levels, which counteracted its anticancer activity. Our results suggest that ABT-263 exerts cytostatic and cytotoxic effects on human esophageal cancer cells in vitro and enhances pro-survival autophagy, which counteracts its anticancer activity.

  17. Calcium and mitochondrial metabolism in ceramide-induced cardiomyocyte death.

    Science.gov (United States)

    Parra, Valentina; Moraga, Francisco; Kuzmicic, Jovan; López-Crisosto, Camila; Troncoso, Rodrigo; Torrealba, Natalia; Criollo, Alfredo; Díaz-Elizondo, Jessica; Rothermel, Beverly A; Quest, Andrew F G; Lavandero, Sergio

    2013-08-01

    Ceramides are important intermediates in the biosynthesis and degradation of sphingolipids that regulate numerous cellular processes, including cell cycle progression, cell growth, differentiation and death. In cardiomyocytes, ceramides induce apoptosis by decreasing mitochondrial membrane potential and promoting cytochrome-c release. Ca(2+) overload is a common feature of all types of cell death. The aim of this study was to determine the effect of ceramides on cytoplasmic Ca(2+) levels, mitochondrial function and cardiomyocyte death. Our data show that C2-ceramide induces apoptosis and necrosis in cultured cardiomyocytes by a mechanism involving increased Ca(2+) influx, mitochondrial network fragmentation and loss of the mitochondrial Ca(2+) buffer capacity. These biochemical events increase cytosolic Ca(2+) levels and trigger cardiomyocyte death via the activation of calpains. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

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

  19. Ebselen induces mitochondrial permeability transition because of its interaction with adenine nucleotide translocase.

    Science.gov (United States)

    Pavón, Natalia; Correa, Francisco; Buelna-Chontal, Mabel; Hernández-Esquivel, Luz; Chávez, Edmundo

    2015-10-15

    Mitochondrial permeability transition is a process established through massive Ca(2+) load in addition to an inducer reagent. Ebselen (Ebs), an antioxidant seleno compound, has been introduced as a reagent which inhibits mitochondrial dysfunction induced by permeability transition. Paradoxically enough, it has been shown that Ebs may also be able to induce the opening of the mitochondrial non-selective pores. This study was performed with the purpose of establishing the membrane system involved in Ebs-induced pore opening. Permeability transition was appraised by analyzing the following: i) matrix Ca(2+) release, and mitochondrial swelling, ii) efflux of cytochrome c, and iii) the inhibition of superoxide dismutase. All of these adverse reactions were inhibited by N-ethylmaleimide and cyclosporin A. At concentrations from 5 to 20 μM, we found that Ebs induces non-specific membrane permeability. Remarkably, Ebs blocks the binding of the fluorescent reagent eosin-5-maleimide to the thiol groups of the adenine nucleotide translocase. Based on the above, it is tempting to hypothesize that Ebs induces pore opening through its binding to the ADP/ATP carrier. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. In vitro and in vivo activation of mitochondrial membrane permeability transition pore using triiodothyronine.

    Science.gov (United States)

    Endlicher, R; Drahota, Z; Červinková, Z

    2016-06-20

    Using a novel method for evaluating mitochondrial swelling (Drahota et al. 2012a) we studied the effect of calcium (Ca(2+)), phosphate (P(i)), and triiodothyronine (T(3)) on the opening of mitochondrial membrane permeability transition pore and how they interact in the activation of swelling process. We found that 0.1 mM P(i), 50 microM Ca(2+) and 25 microM T(3) when added separately increase the swelling rate to about 10 % of maximal values when all three factors are applied simultaneously. Our findings document that under experimental conditions in which Ca(2+) and P(i) are used as activating factors, the addition of T(3) doubled the rate of swelling. T(3) has also an activating effect on mitochondrial membrane potential. The T(3) activating effect was also found after in vivo application of T(3). Our data thus demonstrate that T(3) has an important role in opening the mitochondrial membrane permeability pore and activates the function of the two key physiological swelling inducers, calcium and phosphate ions.

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

    Science.gov (United States)

    Roundhill, Elizabeth; Turnbull, Doug; Burchill, Susan

    2016-05-01

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

  2. Protective effects of physical exercise on MDMA-induced cognitive and mitochondrial impairment.

    Science.gov (United States)

    Taghizadeh, Ghorban; Pourahmad, Jalal; Mehdizadeh, Hajar; Foroumadi, Alireza; Torkaman-Boutorabi, Anahita; Hassani, Shokoufeh; Naserzadeh, Parvaneh; Shariatmadari, Reyhaneh; Gholami, Mahdi; Rouini, Mohammad Reza; Sharifzadeh, Mohammad

    2016-10-01

    Debate continues about the effect of 3, 4-methylenedioxymethamphetamine (MDMA) on cognitive and mitochondrial function through the CNS. It has been shown that physical exercise has an important protective effect on cellular damage and death. Therefore, we investigated the effect of physical exercise on MDMA-induced impairments of spatial learning and memory as well as MDMA effects on brain mitochondrial function in rats. Male wistar rats underwent short-term (2 weeks) or long-term (4 weeks) treadmill exercise. After completion of exercise duration, acquisition and retention of spatial memory were evaluated by Morris water maze (MWM) test. Rats were intraperitoneally (I.P) injected with MDMA (5, 10, and 15mg/kg) 30min before the first training trial in 4 training days of MWM. Different parameters of brain mitochondrial function were measured including the level of ROS production, mitochondrial membrane potential (MMP), mitochondrial swelling, mitochondrial outermembrane damage, the amount of cytochrome c release from the mitochondria, and ADP/ATP ratio. MDMA damaged the spatial learning and memory in a dose-dependent manner. Brain mitochondria isolated from the rats treated with MDMA showed significant increase in ROS formation, collapse of MMP, mitochondrial swelling, and outer membrane damage, cytochrome c release from the mitochondria, and finally increased ADP/ATP ratio. This study also found that physical exercise significantly decreased the MDMA-induced impairments of spatial learning and memory and also mitochondrial dysfunction. The results indicated that MDMA-induced neurotoxicity leads to brain mitochondrial dysfunction and subsequent oxidative stress is followed by cognitive impairments. However, physical exercise could reduce these deleterious effects of MDMA through protective effects on brain mitochondrial function. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Effects of the histamine H₃ receptor antagonist ABT-239 on cognition and nicotine-induced memory enhancement in mice.

    Science.gov (United States)

    Kruk, Marta; Miszkiel, Joanna; McCreary, Andrew C; Przegaliński, Edmund; Filip, Małgorzata; Biała, Grażyna

    2012-01-01

    The strong correlation between central histaminergic and cholinergic pathways on cognitive processes has been reported extensively. However, the role of histamine H(3) receptor mechanisms interacting with nicotinic mechanisms has not previously been extensively investigated. The current study was conducted to determine the interactions of nicotinic and histamine H(3) receptor systems with regard to learning and memory function using a modified elevated plus-maze test in mice. In this test, the latency for mice to move from the open arm to the enclosed arm (i.e., transfer latency) was used as an index of memory. We tested whether ABT-239 (4-(2-{2-[(2R)-2-methylpyrrolidinyl]ethyl}-benzofuran-5-yl), an H(3) receptor antagonist/inverse agonist, had influence on two different stages of memory, i.e., memory acquisition and consolidation (administered prior to or immediately after the first trial, respectively) and whether ABT-239 influenced nicotine-induced memory enhancement. Our results revealed that the acute administration of nicotine (0.035 and 0.175 mg/kg), but not of ABT-239 (0.1-3 mg/kg) reduced transfer latency in the acquisition and consolidation phases. In combination studies, concomitant administration of either ABT-239 (1 and 3 mg/kg) and nicotine (0.035 mg/kg), or ABT-239 (0.1 mg/kg) and nicotine (0.0175 mg/kg) further increased nicotine-induced improvement in both memory acquisition and consolidation. The present data confirm an important role for H(3) receptors in regulating nicotine-induced mnemonic effects since inhibition of H(3) receptors augmented nicotine-induced memory enhancement in mice.

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

    Directory of Open Access Journals (Sweden)

    Divya Padmaraj

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    ,3'-diethyloxacarbocyanine iodide. The responses of glycolytic and membrane potential oscillations to a number of inhibitors of glycolysis, mitochondrial electron flow, and mitochondrial and plasma membrane H(+)-ATPase were investigated. Furthermore, the glycolytic flux was determined as the rate of production of ethanol....../ATP antiporter and the mitochondrial F(0)F(1)-ATPase. The results further suggest that ATP hydrolysis, through the action of the mitochondrial F(0)F(1)-ATPase and plasma membrane H(+)-ATPase, are important in regulating these oscillations. We conclude that it is glycolysis that drives the oscillations...

  6. Modulation of myometrium mitochondrial membrane potential by calmodulin antagonists

    Directory of Open Access Journals (Sweden)

    S. G. Shlykov

    2014-02-01

    Full Text Available Influence of calmodulin antagonists on mitochondrial membrane potential was investigated using­ a flow cytometry method, confocal microscopy and fluorescent potential-sensitive probes TMRM and MTG. Influence of different concentrations of calmodulin antagonists on mitochondrial membrane potential was studied using flow cytometry method and a fraction of myometrium mitochondria of unpregnant rats. It was shown that 1-10 µМ calmidazolium gradually reduced mitochondria membrane potential. At the same time 10-100 µМ trifluope­razine influenced as follows: 10 µМ – increased polarization, while 100 µМ – caused almost complete depolarization of mitochondrial membranes. In experiments which were conducted with the use of confocal microscopy method and myometrium cells it was shown, that MTG addition to the incubation medium­ led to the appearance of fluorescence signal in a green range. Addition of the second probe (ТМRM resulted in the appearance of fluorescent signal in a red range. Mitochondrial membrane depolarization by 1µМ СССР or 10 mМ NaN3 was accompanied by the decline of “red” fluo­rescence intensity, “green” fluorescence was kept. The 10-15 minute incubation of myometrium cells in the presen­ce 10 µМ calmidazolium or 100 µМ trifluoperazine was accompanied by almost complete decrease of the TMRM fluorescent signal. Thus, with the use of potential-sensitive fluorescent probes TMRM and MTG it was shown, that calmodulin antagonists modulate mitochondrial membrane potential of myometrium cells.

  7. Influenza virus PB1-F2 protein induces cell death through mitochondrial ANT3 and VDAC1.

    Directory of Open Access Journals (Sweden)

    Dmitriy Zamarin

    2005-09-01

    Full Text Available The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.

  8. The Involvement of Mitochondrial Membrane Potential in Cross-Resistance Between Radiation and Docetaxel

    Energy Technology Data Exchange (ETDEWEB)

    Kuwahara, Yoshikazu [Department of Radiation Biology and Medicine, Faculty of Medicine, Tohoku Medical and Pharmaceutical University, Sendai (Japan); Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Roudkenar, Mehryar Habibi; Suzuki, Masatoshi; Urushihara, Yusuke; Fukumoto, Motoi [Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Saito, Yohei [Department of Radiopharmacy, Tohoku Medical and Pharmaceutical University, Sendai (Japan); Fukumoto, Manabu, E-mail: manabu.fukumoto.a8@tohoku.ac.jp [Department of Pathology, Institute of Development, Aging and Cancer, Tohoku University, Sendai (Japan); Department of Molecular Pathology, Tokyo Medical University, Tokyo (Japan)

    2016-11-01

    Purpose: To understand the molecular mechanisms underlying cancer cell radioresistance, clinically relevant radioresistant (CRR) cells that continue to proliferate during exposure to 2 Gy/day X-rays for more than 30 days were established. A modified high-density survival assay for anticancer drug screening revealed that CRR cells were resistant to an antimicrotubule agent, docetaxel (DTX). The involvement of reactive oxygen species (ROS) from mitochondria (mtROS) in the cross-resistance to X-rays and DTX was studied. Methods and Materials: Sensitivity to anticancer agents was determined by a modified high-density cell survival or water-soluble tetrazolium salt assay. DTX-induced mtROS generation was determined by MitoSOX red staining. JC-1 staining was used to visualize mitochondrial membrane potential. DTX-induced DNA double-strand breaks were determined by γ-H2AX staining. To obtain mitochondrial DNA-lacking (ρ{sup 0}) cells, the cells were cultured for 3 to 4 weeks in medium containing ethidium bromide. Results: Treatment with DTX increased mtROS in parental cells but not in CRR cells. DTX induced DNA double-strand breaks in parental cells. The mitochondrial membrane potential of CRR cells was lower in CRR cells than in parental cells. Depletion of mtDNA induced DTX resistance in parental cells. Treatment with dimethyl sulfoxide also induced DTX resistance in parental cells. Conclusions: The mitochondrial dysfunction observed in CRR cells contributes to X-ray and DTX cross-resistance. The activation of oxidative phosphorylation in CRR cells may represent an effective approach to overcome radioresistant cancers. In general, the overexpression of β-tubulin or multidrug efflux pumps is thought to be involved in DTX resistance. In the present study, we discovered another DTX resistant mechanism by investigating CRR cells.

  9. Tributyltin induces mitochondrial fission through Mfn1 degradation in human induced pluripotent stem cells.

    Science.gov (United States)

    Yamada, Shigeru; Asanagi, Miki; Hirata, Naoya; Itagaki, Hiroshi; Sekino, Yuko; Kanda, Yasunari

    2016-08-01

    Organotin compounds, such as tributyltin (TBT), are well-known endocrine disruptors. TBT is also known to cause various forms of cytotoxicity, including neurotoxicity and immunotoxicity. However, TBT toxicity has not been identified in normal stem cells. In the present study, we examined the effects of TBT on cell growth in human induced pluripotent stem cells (iPSCs). We found that exposure to nanomolar concentrations of TBT decreased intracellular ATP levels and inhibited cell viability in iPSCs. Because TBT suppressed energy production, which is a critical function of the mitochondria, we further assessed the effects of TBT on mitochondrial dynamics. Staining with MitoTracker revealed that nanomolar concentrations of TBT induced mitochondrial fragmentation. TBT also reduced the expression of mitochondrial fusion protein mitofusin 1 (Mfn1), and this effect was abolished by knockdown of the E3 ubiquitin ligase membrane-associated RING-CH 5 (MARCH5), suggesting that nanomolar concentrations of TBT could induce mitochondrial dysfunction via MARCH5-mediated Mfn1 degradation in iPSCs. Thus, mitochondrial function in normal stem cells could be used to assess cytotoxicity associated with metal exposure. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Mitochondrial mutagenesis induced by tumor-specific radiation bystander effects.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The radiation bystander effect is a cellular process whereby cells not directly exposed to radiation display cellular alterations similar to directly irradiated cells. Cellular targets including mitochondria have been postulated to play a significant role in this process. In this study, we utilized the Random Mutation Capture assay to quantify the levels of random mutations and deletions in the mitochondrial genome of bystander cells. A significant increase in the frequency of random mitochondrial mutations was found at 24 h in bystander cells exposed to conditioned media from irradiated tumor explants (p = 0.018). CG:TA mutations were the most abundant lesion induced. A transient increase in the frequency of random mitochondrial deletions was also detected in bystander cells exposed to conditioned media from tumor but not normal tissue at 24 h (p = 0.028). The increase in both point mutations and deletions was transient and not detected at 72 h. To further investigate mitochondrial dysfunction, mitochondrial membrane potential and reactive oxygen species were assessed in these bystander cells. There was a significant reduction in mitochondrial membrane potential and this was positively associated with the frequency of random point mutation and deletions in bystander cells treated with conditioned media from tumor tissue (r = 0.71, p = 0.02). This study has shown that mitochondrial genome alterations are an acute consequence of the radiation bystander effect secondary to mitochondrial dysfunction and suggests that this cannot be solely attributable to changes in ROS levels alone.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1972-05-01

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

  12. Ionizing radiation induces mitochondrial reactive oxygen species production accompanied by upregulation of mitochondrial electron transport chain function and mitochondrial content under control of the cell cycle checkpoint.

    Science.gov (United States)

    Yamamori, Tohru; Yasui, Hironobu; Yamazumi, Masayuki; Wada, Yusuke; Nakamura, Yoshinari; Nakamura, Hideo; Inanami, Osamu

    2012-07-15

    Whereas ionizing radiation (Ir) instantaneously causes the formation of water radiolysis products that contain some reactive oxygen species (ROS), ROS are also suggested to be released from biological sources in irradiated cells. It is now becoming clear that these ROS generated secondarily after Ir have a variety of biological roles. Although mitochondria are assumed to be responsible for this Ir-induced ROS production, it remains to be elucidated how Ir triggers it. Therefore, we conducted this study to decipher the mechanism of Ir-induced mitochondrial ROS production. In human lung carcinoma A549 cells, Ir (10 Gy of X-rays) induced a time-dependent increase in the mitochondrial ROS level. Ir also increased mitochondrial membrane potential, mitochondrial respiration, and mitochondrial ATP production, suggesting upregulation of the mitochondrial electron transport chain (ETC) function after Ir. Although we found that Ir slightly enhanced mitochondrial ETC complex II activity, the complex II inhibitor 3-nitropropionic acid failed to reduce Ir-induced mitochondrial ROS production. Meanwhile, we observed that the mitochondrial mass and mitochondrial DNA level were upregulated after Ir, indicating that Ir increased the mitochondrial content of the cell. Because irradiated cells are known to undergo cell cycle arrest under control of the checkpoint mechanisms, we examined the relationships between cell cycle and mitochondrial content and cellular oxidative stress level. We found that the cells in the G2/M phase had a higher mitochondrial content and cellular oxidative stress level than cells in the G1 or S phase, regardless of whether the cells were irradiated. We also found that Ir-induced accumulation of the cells in the G2/M phase led to an increase in cells with a high mitochondrial content and cellular oxidative stress level. This suggested that Ir upregulated mitochondrial ETC function and mitochondrial content, resulting in mitochondrial ROS production, and that

  13. Alcohol dehydrogenase accentuates ethanol-induced myocardial dysfunction and mitochondrial damage in mice: role of mitochondrial death pathway.

    Directory of Open Access Journals (Sweden)

    Rui Guo

    2010-01-01

    Full Text Available Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH.ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p. for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways were examined.Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O(2 (*-. Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-alpha, Fas receptor, Fas L and cytosolic AIF.Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.

  14. Alcohol dehydrogenase accentuates ethanol-induced myocardial dysfunction and mitochondrial damage in mice: role of mitochondrial death pathway.

    Science.gov (United States)

    Guo, Rui; Ren, Jun

    2010-01-18

    Binge drinking and alcohol toxicity are often associated with myocardial dysfunction possibly due to accumulation of the ethanol metabolite acetaldehyde although the underlying mechanism is unknown. This study was designed to examine the impact of accelerated ethanol metabolism on myocardial contractility, mitochondrial function and apoptosis using a murine model of cardiac-specific overexpression of alcohol dehydrogenase (ADH). ADH and wild-type FVB mice were acutely challenged with ethanol (3 g/kg/d, i.p.) for 3 days. Myocardial contractility, mitochondrial damage and apoptosis (death receptor and mitochondrial pathways) were examined. Ethanol led to reduced cardiac contractility, enlarged cardiomyocyte, mitochondrial damage and apoptosis, the effects of which were exaggerated by ADH transgene. In particular, ADH exacerbated mitochondrial dysfunction manifested as decreased mitochondrial membrane potential and accumulation of mitochondrial O(2) (*-). Myocardium from ethanol-treated mice displayed enhanced Bax, Caspase-3 and decreased Bcl-2 expression, the effect of which with the exception of Caspase-3 was augmented by ADH. ADH accentuated ethanol-induced increase in the mitochondrial death domain components pro-caspase-9 and cytochrome C in the cytoplasm. Neither ethanol nor ADH affected the expression of ANP, total pro-caspase-9, cytosolic and total pro-caspase-8, TNF-alpha, Fas receptor, Fas L and cytosolic AIF. Taken together, these data suggest that enhanced acetaldehyde production through ADH overexpression following acute ethanol exposure exacerbated ethanol-induced myocardial contractile dysfunction, cardiomyocyte enlargement, mitochondrial damage and apoptosis, indicating a pivotal role of ADH in ethanol-induced cardiac dysfunction possibly through mitochondrial death pathway of apoptosis.

  15. Metabolic activation of hepatotoxic drug (benzbromarone) induced mitochondrial membrane permeability transition

    Energy Technology Data Exchange (ETDEWEB)

    Shirakawa, Maho; Sekine, Shuichi; Tanaka, Ayaka [The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba (Japan); Horie, Toshiharu [Faculty of Pharmaceutical Sciences, Teikyo Heisei University, Tokyo (Japan); Ito, Kousei, E-mail: itokousei@chiba-u.jp [The Laboratory of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba (Japan)

    2015-10-01

    The risk of drug-induced liver injury (DILI) is of great concern to the pharmaceutical industry. It is well-known that metabolic activation of drugs to form toxic metabolites (TMs) is strongly associated with DILI onset. Drug-induced mitochondrial dysfunction is also strongly associated with increased risk of DILI. However, it is difficult to determine the target of TMs associated with exacerbation of DILI because of difficulties in identifying and purifying TMs. In this study, we propose a sequential in vitro assay system to assess TM formation and their ability to induce mitochondrial permeability transition (MPT) in a one-pot process. In this assay system, freshly-isolated rat liver mitochondria were incubated with reaction solutions of 44 test drugs preincubated with liver microsomes in the presence or absence of NADPH; then, NADPH-dependent MPT pore opening was assessed as mitochondrial swelling. In this assay system, several hepatotoxic drugs, including benzbromarone (BBR), significantly induced MPT in a NADPH-dependent manner. We investigated the rationality of using BBR as a model drug, since it showed the most prominent MPT in our assay system. Both the production of a candidate toxic metabolite of BBR (1′,6-(OH){sub 2} BBR) and NADPH-dependent MPT were inhibited by several cytochrome P450 (CYP) inhibitors (clotrimazole and SKF-525A, 100 μM). In summary, this assay system can be used to evaluate comprehensive metabolite-dependent MPT without identification or purification of metabolites. - Highlights: • We constructed a sequential assay system for toxic metabolite induced MPT in one pot. • 14 drugs (e.g. benzbromarone (BBR)) induced toxic metabolite dependent MPT. • Both the production of toxic metabolite and MPT could be inhibited by CYP inhibitors. • This system could evaluate the comprehensive MPT without purification of metabolites.

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

    Science.gov (United States)

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

    2013-01-01

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

  17. Apricot melanoidins prevent oxidative endothelial cell death by counteracting mitochondrial oxidation and membrane depolarization.

    Directory of Open Access Journals (Sweden)

    Annalisa Cossu

    Full Text Available 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.

  18. Air pollution induces enhanced mitochondrial oxidative stress in cystic fibrosis airway epithelium.

    Science.gov (United States)

    Kamdar, O; Le, Wei; Zhang, J; Ghio, A J; Rosen, G D; Upadhyay, D

    2008-10-29

    We studied the effects of airborne particulate matters (PM) on cystic fibrosis (CF) epithelium. We noted that PM enhanced human CF bronchial epithelial apoptosis, activated caspase-9 and PARP-1; and reduced mitochondrial membrane potential. Mitochondrial inhibitors (4,4-diisothiocyanatostilbene-2,2'disulfonic acid, rotenone and thenoyltrifluoroacetone) blocked PM-induced generation of reactive oxygen species and apoptosis. PM upregulated pro-apoptotic Bad, Bax, p53 and p21; and enhanced mitochondrial localization of Bax. The anti-apoptotic Bcl-2, Bcl-xl, Mcl-1 and Xiap remained unchanged; however, overexpression of Bcl-xl blocked PM-induced apoptosis. Accordingly, we provide the evidence that PM enhances oxidative stress and mitochondrial signaling mediated apoptosis via the modulation of Bcl family proteins in CF.

  19. [The effects of electromagnetic pulse on fluidity and lipid peroxidation of mitochondrial membrane].

    Science.gov (United States)

    Wang, Changzhen; Cong, Jianbo; Xian, Hong; Cao, Xiaozhe; Sun, Cunpu; Wu, Ke

    2002-08-01

    To study the effects of intense electromagnetic pulse(EMP) on the biological effects of mitochondrial membrane. Rat liver mitochondrial suspension was exposed to EMP at 60 kV/m level. The changes of membrane lipid fluidity and membrane protein mobility were detected by ESR and spin label technique. Malondialdehyde(MDA) was detected by spectrophotometer. The mobility of membrane protein decreased significantly(P < 0.05). Correlation time (tau c) of control group was (0.501 +/- 0.077) x 10(-9)s, and tau c of EMP group was (0.594 +/- 0.049) x 10(-9)s, indicating that the mobility of protein was restricted. The fluidity of mitochondrial membrane increased significantly(P < 0.05) at the same time. Order parameter(S) of mitochondrial membrane lipid in control group was 0.63 +/- 0.01, while S of EMP group was 0.61 +/- 0.01(P < 0.05). MDA decreased significantly. The mobility and lipid peroxidation of mitochondrial membrane may be disturbed after EMP exposure.

  20. B cell lymphoma-2 (BCL-2) homology domain 3 (BH3) mimetics demonstrate differential activities dependent upon the functional repertoire of pro- and anti-apoptotic BCL-2 family proteins.

    Science.gov (United States)

    Renault, Thibaud T; Elkholi, Rana; Bharti, Archana; Chipuk, Jerry E

    2014-09-19

    The B cell lymphoma-2 (BCL-2) family is the key mediator of cellular sensitivity to apoptosis during pharmacological interventions for numerous human pathologies, including cancer. There is tremendous interest to understand how the proapoptotic BCL-2 effector members (e.g. BCL-2-associated X protein, BAX) cooperate with the BCL-2 homology domain only (BH3-only) subclass (e.g. BCL-2 interacting mediator of death, BIM; BCL-2 interacting-domain death agonist, BID) to induce mitochondrial outer membrane permeabilization (MOMP) and apoptosis and whether these mechanisms may be pharmacologically exploited to enhance the killing of cancer cells. Indeed, small molecule inhibitors of the anti-apoptotic BCL-2 family members have been designed rationally. However, the success of these "BH3 mimetics" in the clinic has been limited, likely due to an incomplete understanding of how these drugs function in the presence of multiple BCL-2 family members. To increase our mechanistic understanding of how BH3 mimetics cooperate with multiple BCL-2 family members in vitro, we directly compared the activity of several BH3-mimetic compounds (i.e. ABT-263, ABT-737, GX15-070, HA14.1, TW-37) in biochemically defined large unilamellar vesicle model systems that faithfully recapitulate BAX-dependent mitochondrial outer membrane permeabilization. Our investigations revealed that the presence of BAX, BID, and BIM differentially regulated the ability of BH3 mimetics to derepress proapoptotic molecules from anti-apoptotic proteins. Using mitochondria loaded with fluorescent BH3 peptides and cells treated with inducers of cell death, these differences were supported. Together, these data suggest that although the presence of anti-apoptotic BCL-2 proteins primarily dictates cellular sensitivity to BH3 mimetics, additional specificity is conferred by proapoptotic BCL-2 proteins. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Targeted Modification of Mitochondrial ROS Production Converts High Glucose-Induced Cytotoxicity to Cytoprotection: Effects on Anesthetic Preconditioning.

    Science.gov (United States)

    Sedlic, Filip; Muravyeva, Maria Y; Sepac, Ana; Sedlic, Marija; Williams, Anna Marie; Yang, Meiying; Bai, Xiaowen; Bosnjak, Zeljko J

    2017-01-01

    Contradictory reports on the effects of diabetes and hyperglycemia on myocardial infarction range from cytotoxicity to cytoprotection. The study was designed to investigate acute effects of high glucose-driven changes in mitochondrial metabolism and osmolarity on adaptive mechanisms and resistance to oxidative stress of isolated rat cardiomyocytes. We examined the effects of high glucose on several parameters of mitochondrial bioenergetics, including changes in oxygen consumption, mitochondrial membrane potential, and NAD(P)H fluorometry. Effects of high glucose on the endogenous cytoprotective mechanisms elicited by anesthetic preconditioning (APC) and the mediators of cell injury were also tested. These experiments included real-time measurements of reactive oxygen species (ROS) production and mitochondrial permeability transition pore (mPTP) opening in single cells by laser scanning fluorescence confocal microscopy, and cell survival assay. High glucose rapidly enhanced mitochondrial energy metabolism, observed by increase in NAD(P)H fluorescence intensity, oxygen consumption, and mitochondrial membrane potential. This substantially elevated production of ROS, accelerated opening of the mPTP, and decreased survival of cells exposed to oxidative stress. Abrogation of high glucose-induced mitochondrial hyperpolarization with 2,4 dinitrophenol (DNP) significantly, but not completely, attenuated ROS production to a level similar to hyperosmotic mannitol control. DNP treatment reversed high glucose-induced cytotoxicity to cytoprotection. Hyperosmotic mannitol treatment also induced cytoprotection. High glucose abrogated APC-induced mitochondrial depolarization, delay in mPTP opening and cytoprotection. In conclusion, high glucose-induced mitochondrial hyperpolarization abolishes APC and augments cell injury. Attenuation of high glucose-induced ROS production by eliminating mitochondrial hyperpolarization protects cardiomyocytes. J. Cell. Physiol. 232: 216-224, 2017

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

    Energy Technology Data Exchange (ETDEWEB)

    Josyula, Ratnakar [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Jin, Zhongmin [SER-CAT, APS, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439 (United States); McCombs, Deborah; DeLucas, Lawrence [Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States); Sha, Bingdong, E-mail: bdsha@uab.edu [Department of Cell Biology, Center for Biophysical Sciences and Engineering, University of Alabama at Birmingham (United States)

    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.

  3. Ethanol induced hepatic mitochondrial dysfunction is attenuated by all trans retinoic acid supplementation.

    Science.gov (United States)

    Nair, Saritha S; Prathibha, P; Rejitha, S; Indira, M

    2015-08-15

    Alcoholics have reduced vitamin A levels in serum since vitamin A and ethanol share the same metabolic pathway. Vitamin A supplementation has an additive effect on ethanol induced toxicity. Hence in this study, we assessed the impact of supplementation of all trans retinoic acid (ATRA), an active metabolite of vitamin A on ethanol induced disruptive alterations in liver mitochondria. Male Sprague Dawley rats were grouped as follows: I: Control; II: Ethanol (4 g/kg b.wt./day); III: ATRA (100 μg/kg b.wt./day); and IV: Ethanol (4 g/kg b.wt./day)+ATRA (100 μg/kg b.wt./day). Duration of the experiment was 90 days, after which the animals were sacrificed for the study. The key enzymes of energy metabolism, reactive oxygen species, mitochondrial membrane potential and hepatic mRNA expressions of Bax, Bcl-2, c-fos and c-jun were assessed. Ethanol administration increased the reactive oxygen species generation in mitochondria. It also decreased the activities of the enzymes of citric acid cycle and oxidative phosphorylation. ATP content and mitochondrial membrane potential were decreased and cytosolic cytochrome c was increased consequently enhancing apoptosis. All these alterations were altered significantly on ATRA supplementation along with ethanol. These results were reinforced by our histopathological studies. ATRA supplementation to ethanol fed rats, led to reduction in oxidative stress, decreased calcium overload in the matrix and increased mitochondrial membrane potential, which might have altered the mitochondrial energy metabolism and elevated ATP production thereby reducing the apoptotic alterations. Hence ATRA supplementation seemed to be an effective intervention against alcohol induced mitochondrial dysfunction. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Enhanced oxidative stress and aberrant mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells during methamphetamine induced apoptosis

    International Nuclear Information System (INIS)

    Wu, C.-W.; Ping, Y.-H.; Yen, J.-C.; Chang, C.-Y.; Wang, S.-F.; Yeh, C.-L.; Chi, C.-W.; Lee, H.-C.

    2007-01-01

    Methamphetamine (METH) is an abused drug that may cause psychiatric and neurotoxic damage, including degeneration of monoaminergic terminals and apoptosis of non-monoaminergic cells in Brain. The cellular and molecular mechanisms underlying these METH-induced neurotoxic effects remain to be clarified. In this study, we performed a time course assessment to investigate the effects of METH on intracellular oxidative stress and mitochondrial alterations in a human dopaminergic neuroblastoma SH-SY5Y cell line. We characterized that METH induces a temporal sequence of several cellular events including, firstly, a decrease in mitochondrial membrane potential within 1 h of the METH treatment, secondly, an extensive decline in mitochondrial membrane potential and increase in the level of reactive oxygen species (ROS) after 8 h of the treatment, thirdly, an increase in mitochondrial mass after the drug treatment for 24 h, and finally, a decrease in mtDNA copy number and mitochondrial proteins per mitochondrion as well as the occurrence of apoptosis after 48 h of the treatment. Importantly, vitamin E attenuated the METH-induced increases in intracellular ROS level and mitochondrial mass, and prevented METH-induced cell death. Our observations suggest that enhanced oxidative stress and aberrant mitochondrial biogenesis may play critical roles in METH-induced neurotoxic effects

  5. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function.

    Directory of Open Access Journals (Sweden)

    Sun Woo Sophie Kang

    Full Text Available Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK. When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury.

  6. AMPK Activation Prevents and Reverses Drug-Induced Mitochondrial and Hepatocyte Injury by Promoting Mitochondrial Fusion and Function

    Science.gov (United States)

    Taniane, Caitlin; Farrell, Geoffrey; Arias, Irwin M.; Lippincott-Schwartz, Jennifer; Fu, Dong

    2016-01-01

    Mitochondrial damage is the major factor underlying drug-induced liver disease but whether conditions that thwart mitochondrial injury can prevent or reverse drug-induced liver damage is unclear. A key molecule regulating mitochondria quality control is AMP activated kinase (AMPK). When activated, AMPK causes mitochondria to elongate/fuse and proliferate, with mitochondria now producing more ATP and less reactive oxygen species. Autophagy is also triggered, a process capable of removing damaged/defective mitochondria. To explore whether AMPK activation could potentially prevent or reverse the effects of drug-induced mitochondrial and hepatocellular damage, we added an AMPK activator to collagen sandwich cultures of rat and human hepatocytes exposed to the hepatotoxic drugs, acetaminophen or diclofenac. In the absence of AMPK activation, the drugs caused hepatocytes to lose polarized morphology and have significantly decreased ATP levels and viability. At the subcellular level, mitochondria underwent fragmentation and had decreased membrane potential due to decreased expression of the mitochondrial fusion proteins Mfn1, 2 and/or Opa1. Adding AICAR, a specific AMPK activator, at the time of drug exposure prevented and reversed these effects. The mitochondria became highly fused and ATP production increased, and hepatocytes maintained polarized morphology. In exploring the mechanism responsible for this preventive and reversal effect, we found that AMPK activation prevented drug-mediated decreases in Mfn1, 2 and Opa1. AMPK activation also stimulated autophagy/mitophagy, most significantly in acetaminophen-treated cells. These results suggest that activation of AMPK prevents/reverses drug-induced mitochondrial and hepatocellular damage through regulation of mitochondrial fusion and autophagy, making it a potentially valuable approach for treatment of drug-induced liver injury. PMID:27792760

  7. A cyclopalladated complex interacts with mitochondrial membrane thiol-groups and induces the apoptotic intrinsic pathway in murine and cisplatin-resistant human tumor cells

    International Nuclear Information System (INIS)

    Serrano, Fabiana A; Machado, Joel Jr; Santos, Edson L; Pesquero, João B; Martins, Rafael M; Travassos, Luiz R; Caires, Antonio CF; Rodrigues, Elaine G; Matsuo, Alisson L; Monteforte, Priscila T; Bechara, Alexandre; Smaili, Soraya S; Santana, Débora P; Rodrigues, Tiago; Pereira, Felipe V; Silva, Luis S

    2011-01-01

    Systemic therapy for cancer metastatic lesions is difficult and generally renders a poor clinical response. Structural analogs of cisplatin, the most widely used synthetic metal complexes, show toxic side-effects and tumor cell resistance. Recently, palladium complexes with increased stability are being investigated to circumvent these limitations, and a biphosphinic cyclopalladated complex {Pd 2 [S (-) C 2 , N-dmpa] 2 (μ-dppe)Cl 2 } named C7a efficiently controls the subcutaneous development of B16F10-Nex2 murine melanoma in syngeneic mice. Presently, we investigated the melanoma cell killing mechanism induced by C7a, and extended preclinical studies. B16F10-Nex2 cells were treated in vitro with C7a in the presence/absence of DTT, and several parameters related to apoptosis induction were evaluated. Preclinical studies were performed, and mice were endovenously inoculated with B16F10-Nex2 cells, intraperitoneally treated with C7a, and lung metastatic nodules were counted. The cytotoxic effects and the respiratory metabolism were also determined in human tumor cell lines treated in vitro with C7a. Cyclopalladated complex interacts with thiol groups on the mitochondrial membrane proteins, causes dissipation of the mitochondrial membrane potential, and induces Bax translocation from the cytosol to mitochondria, colocalizing with a mitochondrial tracker. C7a also induced an increase in cytosolic calcium concentration, mainly from intracellular compartments, and a significant decrease in the ATP levels. Activation of effector caspases, chromatin condensation and DNA degradation, suggested that C7a activates the apoptotic intrinsic pathway in murine melanoma cells. In the preclinical studies, the C7a complex protected against murine metastatic melanoma and induced death in several human tumor cell lineages in vitro, including cisplatin-resistant ones. The mitochondria-dependent cell death was also induced by C7a in human tumor cells. The cyclopalladated C7a complex is

  8. An Unusual Prohibitin Regulates Malaria Parasite Mitochondrial Membrane Potential

    Directory of Open Access Journals (Sweden)

    Joachim Michael Matz

    2018-04-01

    Full Text Available Summary: Proteins of the stomatin/prohibitin/flotillin/HfIK/C (SPFH family are membrane-anchored and perform diverse cellular functions in different organelles. Here, we investigate the SPFH proteins of the murine malaria model parasite Plasmodium berghei, the conserved prohibitin 1, prohibitin 2, and stomatin-like protein and an unusual prohibitin-like protein (PHBL. The SPFH proteins localize to the parasite mitochondrion. While the conserved family members could not be deleted from the Plasmodium genome, PHBL was successfully ablated, resulting in impaired parasite fitness and attenuated virulence in the mammalian host. Strikingly, PHBL-deficient parasites fail to colonize the Anopheles vector because of complete arrest during ookinete development in vivo. We show that this arrest correlates with depolarization of the mitochondrial membrane potential (ΔΨmt. Our results underline the importance of SPFH proteins in the regulation of core mitochondrial functions and suggest that fine-tuning of ΔΨmt in malarial parasites is critical for colonization of the definitive host. : Matz et al. present an experimental genetics study of an unusual prohibitin-like protein in the malaria parasite and find that it regulates mitochondrial membrane polarity. Ablation of this protein causes almost complete mitochondrial depolarization in the mosquito vector, which, in turn, leads to a block in malaria parasite transmission. Keywords: Plasmodium berghei, malaria, SPFH, prohibitin, stomatin-like protein, mitochondrion, membrane potential, ookinete, transmission

  9. Reconstitution of proapoptotic BAK function in liposomes reveals a dual role for mitochondrial lipids in the BAK-driven membrane permeabilization process.

    Science.gov (United States)

    Landeta, Olatz; Landajuela, Ane; Gil, David; Taneva, Stefka; Di Primo, Carmelo; Sot, Begoña; Valle, Mikel; Frolov, Vadim A; Basañez, Gorka

    2011-03-11

    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.

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

    DEFF Research Database (Denmark)

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

    and endoplasmic reticulum membrane are known to closely interact with the cell membrane, we hypothesize that narcotics can be further partitioned into these organelle membranes where they can disrupt essential membrane-bound processes. The electron transport chain (ETC) is an example of a crucial mitochondrial...

  11. Apoptosis of leukemia K562 and Molt-4 cells induced by emamectin benzoate involving mitochondrial membrane potential loss and intracellular Ca2+ modulation.

    Science.gov (United States)

    Yun, Xinming; Rao, Wenbing; Xiao, Ciying; Huang, Qingchun

    2017-06-01

    Leukemia threatens millions of people's health and lives, and the pesticide-induced leukemia has been increasingly concerned because of the etiologic exposure. In this paper, cytotoxic effect of emamectin benzoate (EMB), an excellent natural-product insecticide, was evaluated through monitoring cell viability, cell apoptosis, mitochondrial membrane potential and intracellular Ca 2+ concentration ([Ca 2+ ] i ) in leukemia K562 and Molt-4 cells. Following the exposure to EMB, cell viability was decreased and positive apoptosis of K562 and Molt-4 cells was increased in a concentration- and time- dependent fashion. In the treatment of 10μM EMB, apoptotic cells accounted for 93.0% to K562 cells and 98.9% to Molt-4 cells based on the control, meanwhile, 63.47% of K562 cells and 81.15% of Molt-4 cells exhibited late apoptotic and necrotic features with damaged cytoplasmic membrane. 48h exposure to 10μM EMB increased significantly the great number of cells with mitochondrial membrane potential (MMP) loss, and the elevation of [Ca 2+ ] i level was peaked and persisted within 70s in K562 cells whilst 50s in Molt-4 cells. Moreover, a stronger cytotoxicity of EMB was further observed than that of imatinib. The results authenticate the efficacious effect of EMB as a potential anti-leukemia agent and an inconsistency with regard to insecticide-induced leukemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Lipophilic triphenylphosphonium cations inhibit mitochondrial electron transport chain and induce mitochondrial proton leak.

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    Jan Trnka

    Full Text Available The lipophilic positively charged moiety of triphenylphosphonium (TPP+ has been used to target a range of biologically active compounds including antioxidants, spin-traps and other probes into mitochondria. The moiety itself, while often considered biologically inert, appears to influence mitochondrial metabolism.We used the Seahorse XF flux analyzer to measure the effect of a range of alkylTPP+ on cellular respiration and further analyzed their effect on mitochondrial membrane potential and the activity of respiratory complexes. We found that the ability of alkylTPP+ to inhibit the respiratory chain and decrease the mitochondrial membrane potential increases with the length of the alkyl chain suggesting that hydrophobicity is an important determinant of toxicity.More hydrophobic TPP+ derivatives can be expected to have a negative impact on mitochondrial membrane potential and respiratory chain activity in addition to the effect of the biologically active moiety attached to them. Using shorter linker chains or adding hydrophilic functional groups may provide a means to decrease this negative effect.

  13. Mutations in valosin-containing protein (VCP) decrease ADP/ATP translocation across the mitochondrial membrane and impair energy metabolism in human neurons.

    Science.gov (United States)

    Ludtmann, Marthe H R; Arber, Charles; Bartolome, Fernando; de Vicente, Macarena; Preza, Elisavet; Carro, Eva; Houlden, Henry; Gandhi, Sonia; Wray, Selina; Abramov, Andrey Y

    2017-05-26

    Mutations in the gene encoding valosin-containing protein (VCP) lead to multisystem proteinopathies including frontotemporal dementia. We have previously shown that patient-derived VCP mutant fibroblasts exhibit lower mitochondrial membrane potential, uncoupled respiration, and reduced ATP levels. This study addresses the underlying basis for mitochondrial uncoupling using VCP knockdown neuroblastoma cell lines, induced pluripotent stem cells (iPSCs), and iPSC-derived cortical neurons from patients with pathogenic mutations in VCP Using fluorescent live cell imaging and respiration analysis we demonstrate a VCP mutation/knockdown-induced dysregulation in the adenine nucleotide translocase, which results in a slower rate of ADP or ATP translocation across the mitochondrial membranes. This deregulation can explain the mitochondrial uncoupling and lower ATP levels in VCP mutation-bearing neurons via reduced ADP availability for ATP synthesis. This study provides evidence for a role of adenine nucleotide translocase in the mechanism underlying altered mitochondrial function in VCP-related degeneration, and this new insight may inform efforts to better understand and manage neurodegenerative disease and other proteinopathies. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model.

    Science.gov (United States)

    Lemasson, Benjamin; Wang, Hanxiao; Galbán, Stefanie; Li, Yinghua; Zhu, Yuan; Heist, Kevin A; Tsein, Christina; Chenevert, Thomas L; Rehemtulla, Alnawaz; Galbán, Craig J; Holland, Eric C; Ross, Brian D

    2016-02-01

    Despite the use of ionizing radiation (IR) and temozolomide (TMZ), outcome for glioblastoma (GBM) patients remains dismal. Poly (ADP-ribose) polymerase (PARP) is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Evaluation of Concurrent Radiation, Temozolomide and ABT-888 Treatment Followed by Maintenance Therapy with Temozolomide and ABT-888 in a Genetically Engineered Glioblastoma Mouse Model

    Directory of Open Access Journals (Sweden)

    Benjamin Lemasson

    2016-02-01

    Full Text Available Despite the use of ionizing radiation (IR and temozolomide (TMZ, outcome for glioblastoma (GBM patients remains dismal. Poly (ADP-ribose polymerase (PARP is important in repair pathways for IR-induced DNA damage and TMZ-induced alkylation at N7-methylguanine and N3-methyldenine. However, optimized protocols for administration of PARP inhibitors have not been delineated. In this study, the PARP inhibitor ABT-888 was evaluated in combination with and compared to current standard-of-care in a genetically engineered mouse GBM model. Results demonstrated that concomitant TMZ/IR/ABT-888 with adjuvant TMZ/ABT-888 was more effective in inducing apoptosis and reducing proliferation with significant tumor growth delay and improved overall survival over concomitant TMZ/IR with adjuvant TMZ. Diffusion-weighted MRI, an early translatable response biomarker detected changes in tumors reflecting response at 1 day post TMZ/IR/ABT-888 treatment. This study provides strong scientific rationale for the development of an optimized dosing regimen for a PARP inhibitor with TMZ/IR for upfront treatment of GBM.

  16. Changes in mitochondrial dynamics during ceramide-induced cardiomyocyte early apoptosis.

    Science.gov (United States)

    Parra, Valentina; Eisner, Veronica; Chiong, Mario; Criollo, Alfredo; Moraga, Francisco; Garcia, Alejandra; Härtel, Steffen; Jaimovich, Enrique; Zorzano, Antonio; Hidalgo, Cecilia; Lavandero, Sergio

    2008-01-15

    In cells, mitochondria are organized as a network of interconnected organelles that fluctuate between fission and fusion events (mitochondrial dynamics). This process is associated with cell death. We investigated whether activation of apoptosis with ceramides affects mitochondrial dynamics and promotes mitochondrial fission in cardiomyocytes. Neonatal rat cardiomyocytes were incubated with C(2)-ceramide or the inactive analog dihydro-C(2)-ceramide for up to 6 h. Three-dimensional images of cells loaded with mitotracker green were obtained by confocal microscopy. Dynamin-related protein-1 (Drp-1) and mitochondrial fission protein 1 (Fis1) distribution and levels were studied by immunofluorescence and western blot. Mitochondrial membrane potential (DeltaPsi(m)) and cytochrome c (cyt c) distribution were used as indexes of early activation of apoptosis. Cell viability and DNA fragmentation were determined by propidium iodide staining/flow cytometry, whereas cytotoxicity was evaluated by lactic dehydrogenase activity. To decrease the levels of the mitochondrial fusion protein mitofusin 2, we used an antisense adenovirus (AsMfn2). C(2)-ceramide, but not dihydro-C(2)-ceramide, promoted rapid fragmentation of the mitochondrial network in a concentration- and time-dependent manner. C(2)-ceramide also increased mitochondrial Drp-1 and Fis1 content, Drp-1 colocalization with Fis1, and caused early activation of apoptosis. AsMfn2 accentuated the decrease in DeltaPsi(m) and cyt c redistribution induced by C(2)-ceramide. Doxorubicin, which induces cardiomyopathy and apoptosis through ceramide generation, also stimulated mitochondrial fragmentation. Ceramides stimulate mitochondrial fission and this event is associated with early activation of cardiomyocyte apoptosis.

  17. Rebamipide suppresses diclofenac-induced intestinal permeability via mitochondrial protection in mice.

    Science.gov (United States)

    Diao, Lei; Mei, Qiao; Xu, Jian-Ming; Liu, Xiao-Chang; Hu, Jing; Jin, Juan; Yao, Qiang; Chen, Mo-Li

    2012-03-14

    To investigate the protective effect and mechanism of rebamipide on small intestinal permeability induced by diclofenac in mice. Diclofenac (2.5 mg/kg) was administered once daily for 3 d orally. A control group received the vehicle by gavage. Rebamipide (100 mg/kg, 200 mg/kg, 400 mg/kg) was administered intragastrically once a day for 3 d 4 h after diclofenac administration. Intestinal permeability was evaluated by Evans blue and the FITC-dextran method. The ultrastructure of the mucosal barrier was evaluated by transmission electron microscopy (TEM). Mitochondrial function including mitochondrial swelling, mitochondrial membrane potential, mitochondrial nicotinamide adenine dinucleotide-reduced (NADH) levels, succinate dehydrogenase (SDH) and ATPase activities were measured. Small intestinal mucosa was collected for assessment of malondialdehyde (MDA) content and myeloperoxidase (MPO) activity. Compared with the control group, intestinal permeability was significantly increased in the diclofenac group, which was accompanied by broken tight junctions, and significant increases in MDA content and MPO activity. Rebamipide significantly reduced intestinal permeability, improved inter-cellular tight junctions, and was associated with decreases in intestinal MDA content and MPO activity. At the mitochondrial level, rebamipide increased SDH and ATPase activities, NADH level and decreased mitochondrial swelling. Increased intestinal permeability induced by diclofenac can be attenuated by rebamipide, which partially contributed to the protection of mitochondrial function.

  18. Nicotine induces resistance to chemotherapy by modulating mitochondrial signaling in lung cancer.

    Science.gov (United States)

    Zhang, Jingmei; Kamdar, Opal; Le, Wei; Rosen, Glenn D; Upadhyay, Daya

    2009-02-01

    Continued smoking causes tumor progression and resistance to therapy in lung cancer. Carcinogens possess the ability to block apoptosis, and thus may induce development of cancers and resistance to therapy. Tobacco carcinogens have been studied widely; however, little is known about the agents that inhibit apoptosis, such as nicotine. We determine whether mitochondrial signaling mediates antiapoptotic effects of nicotine in lung cancer. A549 cells were exposed to nicotine (1 muM) followed by cisplatin (35 muM) plus etoposide (20 muM) for 24 hours. We found that nicotine prevented chemotherapy-induced apoptosis, improved cell survival, and caused modest increases in DNA synthesis. Inhibition of mitogen-activated protein kinase (MAPK) and Akt prevented the antiapoptotic effects of nicotine and decreased chemotherapy-induced apoptosis. Small interfering RNA MAPK kinase-1 blocked antiapoptotic effects of nicotine, whereas small interfering RNA MAPK kinase-2 blocked chemotherapy-induced apoptosis. Nicotine prevented chemotherapy-induced reduction in mitochondrial membrane potential and caspase-9 activation. Antiapoptotic effects of nicotine were blocked by mitochondrial anion channel inhibitor, 4,4'diisothiocyanatostilbene-2,2'disulfonic acid. Chemotherapy enhanced translocation of proapoptotic Bax to the mitochondria, whereas nicotine blocked these effects. Nicotine up-regulated Akt-mediated antiapoptotic X-linked inhibitor of apoptosis protein and phosphorylated proapoptotic Bcl2-antagonist of cell death. The A549-rho0 cells, which lack mitochondrial DNA, demonstrated partial resistance to chemotherapy-induced apoptosis, but blocked the antiapoptotic effects of nicotine. Accordingly, we provide evidence that nicotine modulates mitochondrial signaling and inhibits chemotherapy-induced apoptosis in lung cancer. The mitochondrial regulation of nicotine imposes an important mechanism that can critically impair the treatment of lung cancer, because many cancer

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

    Science.gov (United States)

    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

  20. Polychlorinated Biphenyls Induce Mitochondrial Dysfunction in SH-SY5Y Neuroblastoma Cells.

    Directory of Open Access Journals (Sweden)

    Stefania Cocco

    Full Text Available Chronic exposure to polychlorinated biphenyls (PCBs, ubiquitous environmental contaminants, can adversely affect the development and function of the nervous system. Here we evaluated the effect of PCB exposure on mitochondrial function using the PCB mixture Aroclor-1254 (A1254 in SH-SY5Y neuroblastoma cells. A 6-hour exposure to A1254 (5 μg/ml reduced cellular ATP production by 45%±7, and mitochondrial membrane potential, detected by TMRE, by 49%±7. Consistently, A1254 significantly decreased oxidative phosphorylation and aerobic glycolysis measured by extracellular flux analyzer. Furthermore, the activity of mitochondrial protein complexes I, II, and IV, but not V (ATPase, measured by BN-PAGE technique, was significantly reduced after 6-hour exposure to A1254. The addition of pyruvic acid during exposure to A1254 significantly prevent A1254-induced cell injury, restoring resting mitochondrial membrane potential, ATP levels, oxidative phosphorylation and aerobic glycolysis. Furthermore, pyruvic acid significantly preserved the activity of mitochondrial complexes I, II and IV and increased basal activity of complex V. Collectively, the present results indicate that the neurotoxicity of A1254 depends on the impairment of oxidative phosphorylation, aerobic glycolysis, and mitochondrial complexes I, II, and IV activity and it was counteracted by pyruvic acid.

  1. Water-Soluble Coenzyme Q10 Inhibits Nuclear Translocation of Apoptosis Inducing Factor and Cell Death Caused by Mitochondrial Complex I Inhibition

    Directory of Open Access Journals (Sweden)

    Haining Li

    2014-07-01

    Full Text Available The objectives of the study were to explore the mechanism of rotenone-induced cell damage and to examine the protective effects of water-soluble Coenzyme Q10 (CoQ10 on the toxic effects of rotenone. Murine hippocampal HT22 cells were cultured with mitochondrial complex I inhibitor rotenone. Water-soluble CoQ10 was added to the culture media 3 h prior to the rotenone incubation. Cell viability was determined by alamar blue, reactive oxygen species (ROS production by dihydroethidine (DHE and mitochondrial membrane potential by tetramethyl rhodamine methyl ester (TMRM. Cytochrome c, caspase-9 and apoptosis-inducing factor (AIF were measured using Western blotting after 24 h rotenone incubation. Rotenone caused more than 50% of cell death, increased ROS production, AIF nuclear translocation and reduction in mitochondrial membrane potential, but failed to cause mitochondrial cytochrome c release and caspase-9 activation. Pretreatment with water-soluble CoQ10 enhanced cell viability, decreased ROS production, maintained mitochondrial membrane potential and prevented AIF nuclear translocation. The results suggest that rotenone activates a mitochondria-initiated, caspase-independent cell death pathway. Water-soluble CoQ10 reduces ROS accumulation, prevents the fall of mitochondrial membrane potential, and inhibits AIF translocation and subsequent cell death.

  2. Role of mitochondrial permeability transition in human renal tubular epithelial cell death induced by aristolochic acid

    International Nuclear Information System (INIS)

    Qi Xinming; Cai Yan; Gong Likun; Liu Linlin; Chen Fangping; Xiao Ying; Wu Xiongfei; Li Yan; Xue Xiang; Ren Jin

    2007-01-01

    Aristolochic acid (AA), a natural nephrotoxin and carcinogen, can induce a progressive tubulointerstitial nephropathy. However, the mechanism by which AA causes renal injury remains largely unknown. Here we reported that the mitochondrial permeability transition (MPT) plays an important role in the renal injury induced by aristolochic acid I (AAI). We found that in the presence of Ca 2+ , AAI caused mitochondrial swelling, leakage of Ca 2+ , membrane depolarization, and release of cytochrome c in isolated kidney mitochondria. These alterations were suppressed by cyclosporin A (CsA), an agent known to inhibit MPT. Culture of HK-2 cell, a human renal tubular epithelial cell line for 24 h with AAI caused a decrease in cellular ATP, mitochondrial membrane depolarization, cytochrome c release, and increase of caspase 3 activity. These toxic effects of AAI were attenuated by CsA and bongkrekic acid (BA), another specific MPT inhibitor. Furthermore, AAI greatly inhibited the activity of mitochondrial adenine nucleotide translocator (ANT) in isolated mitochondria. We suggested that ANT may mediate, at least in part, the AAI-induced MPT. Taken together, these results suggested that MPT plays a critical role in the pathogenesis of HK-2 cell injury induced by AAI and implied that MPT might contribute to human nephrotoxicity of aristolochic acid

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

    Science.gov (United States)

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

    2013-02-01

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

  4. Resveratrol induces mitochondrial biogenesis in endothelial cells.

    Science.gov (United States)

    Csiszar, Anna; Labinskyy, Nazar; Pinto, John T; Ballabh, Praveen; Zhang, Hanrui; Losonczy, Gyorgy; Pearson, Kevin; de Cabo, Rafael; Pacher, Pal; Zhang, Cuihua; Ungvari, Zoltan

    2009-07-01

    Pathways that regulate mitochondrial biogenesis are potential therapeutic targets for the amelioration of endothelial dysfunction and vascular disease. Resveratrol was shown to impact mitochondrial function in skeletal muscle and the liver, but its role in mitochondrial biogenesis in endothelial cells remains poorly defined. The present study determined whether resveratrol induces mitochondrial biogenesis in cultured human coronary arterial endothelial cells (CAECs). In CAECs resveratrol increased mitochondrial mass and mitochondrial DNA content, upregulated protein expression of electron transport chain constituents, and induced mitochondrial biogenesis factors (proliferator-activated receptor-coactivator-1alpha, nuclear respiratory factor-1, mitochondrial transcription factor A). Sirtuin 1 (SIRT1) was induced, and endothelial nitric oxide (NO) synthase (eNOS) was upregulated in a SIRT1-dependent manner. Knockdown of SIRT1 (small interfering RNA) or inhibition of NO synthesis prevented resveratrol-induced mitochondrial biogenesis. In aortas of type 2 diabetic (db/db) mice impaired mitochondrial biogenesis was normalized by chronic resveratrol treatment, showing the in vivo relevance of our findings. Resveratrol increases mitochondrial content in endothelial cells via activating SIRT1. We propose that SIRT1, via a pathway that involves the upregulation of eNOS, induces mitochondrial biogenesis. Resveratrol induced mitochondrial biogenesis in the aortas of type 2 diabetic mice, suggesting the potential for new treatment approaches targeting endothelial mitochondria in metabolic diseases.

  5. Protective effects of myricetin on acute hypoxia-induced exercise intolerance and mitochondrial impairments in rats.

    Directory of Open Access Journals (Sweden)

    Dan Zou

    Full Text Available Exercise tolerance is impaired in hypoxia. The aim of this study was to evaluate the effects of myricetin, a dietary flavonoid compound widely found in fruits and vegetables, on acute hypoxia-induced exercise intolerance in vivo and in vitro.Male rats were administered myricetin or vehicle for 7 days and subsequently spent 24 hours at a barometric pressure equivalent to 5000 m. Exercise capacity was then assessed through the run-to-fatigue procedure, and mitochondrial morphology in skeletal muscle cells was observed by transmission electron microscopy (TEM. The enzymatic activities of electron transfer complexes were analyzed using an enzyme-linked immuno-sorbent assay (ELISA. mtDNA was quantified by real-time-PCR. Mitochondrial membrane potential was measured by JC-1 staining. Protein expression was detected through western blotting, immunohistochemistry, and immunofluorescence.Myricetin supplementation significantly prevented the decline of run-to-fatigue time of rats in hypoxia, and attenuated acute hypoxia-induced mitochondrial impairment in skeletal muscle cells in vivo and in vitro by maintaining mitochondrial structure, mtDNA content, mitochondrial membrane potential, and activities of the respiratory chain complexes. Further studies showed that myricetin maintained mitochondrial biogenesis in skeletal muscle cells under hypoxic conditions by up-regulating the expressions of mitochondrial biogenesis-related regulators, in addition, AMP-activated protein kinase(AMPK plays a crucial role in this process.Myricetin may have important applications for improving physical performance under hypoxic environment, which may be attributed to the protective effect against mitochondrial impairment by maintaining mitochondrial biogenesis.

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

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Soy lecithin interferes with mitochondrial function in frozen-thawed ram spermatozoa.

    Science.gov (United States)

    Del Valle, I; Gómez-Durán, A; Holt, W V; Muiño-Blanco, T; Cebrián-Pérez, J A

    2012-01-01

    Egg yolk and milk are the 2 major membrane cryoprotectants commonly used in freezing media for the long-term preservation of semen (alone or in combination with others). However, in recent years, there have been increasing arguments against the use of egg yolk or milk because of the risk of introducing diseases through the use of cryopreserved semen. In this study, we analyzed the protective effect of lecithin as an alternative to egg yolk for the cryopreservation of ram semen, using a range of functional markers for sperm viability, motility, apoptosis, and mitochondrial functionality analyses (mitochondrial inner membrane surface [MIMS], mitochondrial inner membrane potential [MIMP], and cell membrane potential) as methods of assessment in samples diluted in 3 different media: Tris-citrate-glucose as control and 2 media supplemented with soy lecithin or egg yolk. The results showed that lecithin was able to effectively protect certain sperm quality characteristics against freezing-induced damage. However, lecithin induced loss of mitochondrial membrane potential or mitochondrial loss that was not reflected by modifications in sperm motility in fresh semen. MIMS and MIMP values decreased in thawed lecithin-treated samples, concomitant with a lower (P lecithin may have affected the inner mitochondrial membrane in frozenthawed spermatozoa and confirmed that sublethal damages that seriously affect sperm functionality, not detected by classic sperm quality analyses, can be evidenced by changes in the inner mitochondrial membrane surface. These findings strengthen the relationship between mitochondrial membrane potential and motility and show that the mitochondrial alterations induced by the cryopreservation process could be specific targets for the improvement of semen cryopreservation protocols.

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

    Directory of Open Access Journals (Sweden)

    Sutapa Mukherjee

    2014-01-01

    Full Text Available 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.

  9. Metabolic remodeling precedes mitochondrial outer membrane permeabilization in human glioma xenograft cells.

    Science.gov (United States)

    Ponnala, Shivani; Chetty, Chandramu; Veeravalli, Krishna Kumar; Dinh, Dzung H; Klopfenstein, Jeffrey D; Rao, Jasti S

    2012-02-01

    Glioma cancer cells adapt to changing microenvironment and shift from mitochondrial oxidative phosphorylation to aerobic glycolysis for their metabolic needs irrespective of oxygen availability. In the present study, we show that silencing MMP-9 in combination with uPAR/cathepsin B switch the glycolytic metabolism of glioma cells to oxidative phosphorylation (OXPHOS) and generate reactive oxygen species (ROS) to predispose glioma cells to mitochondrial outer membrane permeabilization. shRNA for MMP-9 and uPAR (pMU) as well as shRNA for MMP-9 and cathepsin B (pMC) activated complexes of mitochondria involved in OXPHOS and inhibited glycolytic hexokinase expression. The decreased interaction of hexokinase 2 with mitochondria in the treated cells indicated the inhibition of glycolysis activation. Overexpression of Akt reversed the pMU- and pMC-mediated OXPHOS to glycolysis switch. The OXPHOS un-coupler oligomycin A altered the expression levels of the Bcl-2 family of proteins; treatment with pMU or pMC reversed this effect and induced mitochondrial outer membrane permeabilization. In addition, our results show changes in mitochondrial pore transition to release cytochrome c due to changes in the VDAC-Bcl-XL and BAX-BAK interaction with pMU and pMC treatments. Taken together, our results suggest that pMU and pMC treatments switch glioma cells from the glycolytic to the OXPHOS pathway through an inhibitory effect on Akt, ROS induction and an increase of cytosolic cytochrome c accumulation. These results demonstrate the potential of pMU and pMC as therapeutic candidates for the treatment of glioma.

  10. Pan-Bcl-2 inhibitor obatoclax delays cell cycle progression and blocks migration of colorectal cancer cells.

    Directory of Open Access Journals (Sweden)

    Bruno Christian Koehler

    Full Text Available Despite the fact that new treatment regimes have improved overall survival of patients challenged by colorectal cancer (CRC, prognosis in the metastatic situation is still restricted. The Bcl-2 family of proteins has been identified as promising anti cancer drug target. Even though small molecules targeting Bcl-2 proteins are in clinical trials, little is known regarding their effects on CRC. The aim of this study was to preclinically investigate the value of ABT-737 and Obatoclax as anticancer drugs for CRC treatment. The effects of the BH3-mimetics ABT-737 and Obatoclax on CRC cells were assessed using viability and apoptosis assays. Wound healing migration and boyden chamber invasion assays were applied. 3-dimensional cell cultures were used for long term assessment of invasion and proliferation. Clinically relevant concentrations of pan-Bcl-2 inhibitor Obatoclax did not induce cell death. In contrast, the BH3-mimetic ABT-737 induced apoptosis in a dose dependent manner. Obatoclax caused a cell line specific slowdown of CRC cell growth. Furthermore, Obatoclax, but not ABT-737, recovered E-Cadherin expression and led to impaired migration and invasion of CRC cells. The proliferative capacity and invasiveness of CRC cells was strikingly inhibited by low dose Obatoclax in long term 3-dimensional cell cultures. Obatoclax, but not ABT-737, caused a G1-phase arrest accompanied by a downregulation of Cyclin D1 and upregulation of p27 and p21. Overexpression of Mcl-1, Bcl-xL or Bcl-2 reversed the inhibitory effect of Obatoclax on migration but failed to restore the proliferative capacity of Obatoclax-treated CRC cells. The data presented indicate broad and multifaceted antitumor effects of the pan-Bcl-2 inhibitor Obatoclax on CRC cells. In contrast to ABT-737, Obatoclax inhibited migration, invasion and proliferation in sublethal doses. In summary, this study recommends pan-Bcl-2 inhibition as a promising approach for clinical trials in CRC.

  11. Characterization of the Respiration-Induced Yeast Mitochondrial Permeability Transition Pore

    OpenAIRE

    Bradshaw, Patrick C.; Pfeiffer, Douglas R.

    2013-01-01

    When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable throug...

  12. Sodium valproate induces mitochondrial respiration dysfunction in HepG2 in vitro cell model.

    Science.gov (United States)

    Komulainen, Tuomas; Lodge, Tiffany; Hinttala, Reetta; Bolszak, Maija; Pietilä, Mika; Koivunen, Peppi; Hakkola, Jukka; Poulton, Joanna; Morten, Karl J; Uusimaa, Johanna

    2015-05-04

    Sodium valproate (VPA) is a potentially hepatotoxic antiepileptic drug. Risk of VPA-induced hepatotoxicity is increased in patients with mitochondrial diseases and especially in patients with POLG1 gene mutations. We used a HepG2 cell in vitro model to investigate the effect of VPA on mitochondrial activity. Cells were incubated in glucose medium and mitochondrial respiration-inducing medium supplemented with galactose and pyruvate. VPA treatments were carried out at concentrations of 0-2.0mM for 24-72 h. In both media, VPA caused decrease in oxygen consumption rates and mitochondrial membrane potential. VPA exposure led to depleted ATP levels in HepG2 cells incubated in galactose medium suggesting dysfunction in mitochondrial ATP production. In addition, VPA exposure for 72 h increased levels of mitochondrial reactive oxygen species (ROS), but adversely decreased protein levels of mitochondrial superoxide dismutase SOD2, suggesting oxidative stress caused by impaired elimination of mitochondrial ROS and a novel pathomechanism related to VPA toxicity. Increased cell death and decrease in cell number was detected under both metabolic conditions. However, immunoblotting did not show any changes in the protein levels of the catalytic subunit A of mitochondrial DNA polymerase γ, the mitochondrial respiratory chain complexes I, II and IV, ATP synthase, E3 subunit dihydrolipoyl dehydrogenase of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and glutathione peroxidase. Our results show that VPA inhibits mitochondrial respiration and leads to mitochondrial dysfunction, oxidative stress and increased cell death, thus suggesting an essential role of mitochondria in VPA-induced hepatotoxicity. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    Huang, Wenmin; Li, Dunhai; Liu, Yongding

    2014-09-01

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

  14. [Effect of 3-bromopyruvate on mitochondrial membrane potential and apoptosis of human breast carcinoma SK-BR-3 cells].

    Science.gov (United States)

    Zhang, Yuanyuan; Liu, Zhe; Zhang, Qianwen; Chao, Zhenhua; Zhang, Pei; Xia, Fei; Jiang, Chenchen; Liu, Hao; Jiang, Zhiwen

    2013-09-01

    To study the effect of glycolysis inhibitor 3-bromopyruvate (3-BrPA) in inducing apoptosis of human breast carcinoma cells SK-BR-3 and the possible mechanism. MTT assay was used to detect the growth inhibition induced by 3-BrPA in breast cancer cells SK-BR-3. The apoptotic cells were detected by flow cytometry with propidium iodide (PI). ATP levels in the cells were detected by ATP assay kit, and DHE fluorescent probe technique was used to determine superoxide anion levels; the mitochondrial membrane potential was assessed using JC-1 staining assay. MTT assay showed that the proliferation of SK-BR-3 cells was inhibited by 3-BrPA in a time- and concentration-dependent manner. Exposure to 80, 160, and 320 µmol·L(-1) 3-BrPA for 24 h resulted in cell apoptosis rates of 6.7%, 22.3%, and 79.6%, respectively, and the intracellular ATP levels of SK-BR-3 cells treated with 80, 160, 320 µmol·L(-1) 3-BrPA for 5 h were 87.7%, 60.6%, and 23.7% of the control levels. 3-BrPA at 160 µmol·L(-1) increased reactive oxygen levels and lowered mitochondrial membrane potential of SK-BR-3 cells. 3-BrPA can inhibit cell proliferation, reduce the mitochondrial membrane potential and induce apoptosis in SK-BR-3 cells, the mechanism of which may involve a reduced ATP level by inhibiting glycolysis and increasing the reactive oxygen level in the cells.

  15. Lowered iPLA2γ activity causes increased mitochondrial lipid peroxidation and mitochondrial dysfunction in a rotenone-induced model of Parkinson's disease.

    Science.gov (United States)

    Chao, Honglu; Liu, Yinlong; Fu, Xian; Xu, Xiupeng; Bao, Zhongyuan; Lin, Chao; Li, Zheng; Liu, Yan; Wang, Xiaoming; You, Yongping; Liu, Ning; Ji, Jing

    2018-02-01

    iPLA 2 γ, calcium-independent phospholipase A 2 γ, discerningly hydrolyses glycerophospholipids to liberate free fatty acids. iPLA 2 γ-deficiency has been associated with abnormal mitochondrial function. More importantly, the iPLA 2 family is causative proteins in mitochondrial neurodegenerative disorders such as parkinsonian disorders. However, the mechanisms by which iPLA 2 γ affects Parkinson's disease (PD) remain unknown. Mitochondrion stress has a key part in rotenone-induced dopaminergic neuronal degeneration. The present evaluation revealed that lowered iPLA 2 γ function provokes the parkinsonian phenotype and leads to the reduction of dopamine and its metabolites, lowered survival, locomotor deficiencies, and organismal hypersensitivity to rotenone-induced oxidative stress. In addition, lowered iPLA 2 γ function escalated the amount of mitochondrial irregularities, including mitochondrial reactive oxygen species (ROS) regeneration, reduced ATP synthesis, reduced glutathione levels, and abnormal mitochondrial morphology. Further, lowered iPLA 2 γ function was tightly linked with strengthened lipid peroxidation and mitochondrial membrane flaws following rotenone treatment, which can cause cytochrome c release and eventually apoptosis. These results confirmed the important role of iPLA 2 γ, whereby decreasing iPLA 2 γ activity aggravates mitochondrial degeneration to induce neurodegenerative disorders in a rotenone rat model of Parkinson's disease. These findings may be useful in the design of rational approaches for the prevention and treatment of PD-associated symptoms. Copyright © 2017 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    2014-01-24

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

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

    Science.gov (United States)

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

    2014-01-01

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

  18. BID is cleaved by caspase-8 within a native complex on the mitochondrial membrane

    NARCIS (Netherlands)

    Schug, Z. T.; Gonzalvez, F.; Houtkooper, R. H.; Vaz, F. M.; Gottlieb, E.

    2011-01-01

    Caspase-8 stably inserts into the mitochondrial outer membrane during extrinsic apoptosis. Inhibition of caspase-8 enrichment on the mitochondria impairs caspase-8 activation and prevents apoptosis. However, the function of active caspase-8 on the mitochondrial membrane remains unknown. In this

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

    DEFF Research Database (Denmark)

    Andersen, Ann Zahle; Poulsen, Allan K.; Brasen, Jens Christian

    2007-01-01

    We employed the fluorescent cyanine dye DiOC(2)(3) to measure membrane potential in semi-anaerobic yeast cells under conditions where glycolysis was oscillating. Oscillations in glycolysis were studied by means of the naturally abundant nicotinamide adenine dinucleotide (NADH). We found...... studies showed that glycolytic oscillations perturb the mitochondrial membrane potential and that the mitochondria do not have any controlling effect on the dynamics of glycolysis under these conditions. Depolarization of the mitochondrial membrane by addition of FCCP quenched mitochondrial membrane...... potential oscillations and delocalized DiOC(2)(3), while glycolysis continued to oscillate unaffected....

  20. Ketamine Causes Mitochondrial Dysfunction in Human Induced Pluripotent Stem Cell-Derived Neurons

    Science.gov (United States)

    Ito, Hiroyuki; Uchida, Tokujiro; Makita, Koshi

    2015-01-01

    Purpose Ketamine toxicity has been demonstrated in nonhuman mammalian neurons. To study the toxic effect of ketamine on human neurons, an experimental model of cultured neurons from human induced pluripotent stem cells (iPSCs) was examined, and the mechanism of its toxicity was investigated. Methods Human iPSC-derived dopaminergic neurons were treated with 0, 20, 100 or 500 μM ketamine for 6 and 24 h. Ketamine toxicity was evaluated by quantification of caspase 3/7 activity, reactive oxygen species (ROS) production, mitochondrial membrane potential, ATP concentration, neurotransmitter reuptake activity and NADH/NAD+ ratio. Mitochondrial morphological change was analyzed by transmission electron microscopy and confocal microscopy. Results Twenty-four-hour exposure of iPSC-derived neurons to 500 μM ketamine resulted in a 40% increase in caspase 3/7 activity (P ketamine (100 μM) decreased the ATP level (22%, P ketamine concentration, which suggests that mitochondrial dysfunction preceded ROS generation and caspase activation. Conclusions We established an in vitro model for assessing the neurotoxicity of ketamine in iPSC-derived neurons. The present data indicate that the initial mitochondrial dysfunction and autophagy may be related to its inhibitory effect on the mitochondrial electron transport system, which underlies ketamine-induced neural toxicity. Higher ketamine concentration can induce ROS generation and apoptosis in human neurons. PMID:26020236

  1. Hypoxia induces mitochondrial mutagenesis and dysfunction in inflammatory arthritis.

    LENUS (Irish Health Repository)

    Biniecka, Monika

    2012-02-01

    OBJECTIVE: To assess the levels and spectrum of mitochondrial DNA (mtDNA) point mutations in synovial tissue from patients with inflammatory arthritis in relation to in vivo hypoxia and oxidative stress levels. METHODS: Random Mutation Capture assay was used to quantitatively evaluate alterations of the synovial mitochondrial genome. In vivo tissue oxygen levels (tPO(2)) were measured at arthroscopy using a Licox probe. Synovial expression of lipid peroxidation (4-hydroxynonenal [4-HNE]) and mitochondrial cytochrome c oxidase subunit II (CytcO II) deficiency were assessed by immunohistochemistry. In vitro levels of mtDNA point mutations, reactive oxygen species (ROS), mitochondrial membrane potential, and markers of oxidative DNA damage (8-oxo-7,8-dihydro-2\\'-deoxyguanine [8-oxodG]) and lipid peroxidation (4-HNE) were determined in human synoviocytes under normoxia and hypoxia (1%) in the presence or absence of superoxide dismutase (SOD) or N-acetylcysteine (NAC) or a hydroxylase inhibitor (dimethyloxalylglycine [DMOG]). Patients were categorized according to their in vivo tPO(2) level (<20 mm Hg or >20 mm Hg), and mtDNA point mutations, immunochemistry features, and stress markers were compared between groups. RESULTS: The median tPO(2) level in synovial tissue indicated significant hypoxia (25.47 mm Hg). Higher frequency of mtDNA mutations was associated with reduced in vivo oxygen tension (P = 0.05) and with higher synovial 4-HNE cytoplasmic expression (P = 0.04). Synovial expression of CytcO II correlated with in vivo tPO(2) levels (P = 0.03), and levels were lower in patients with tPO(2) <20 mm Hg (P < 0.05). In vitro levels of mtDNA mutations, ROS, mitochondrial membrane potential, 8-oxo-dG, and 4-HNE were higher in synoviocytes exposed to 1% hypoxia (P < 0.05); all of these increased levels were rescued by SOD and DMOG and, with the exception of ROS, by NAC. CONCLUSION: These findings demonstrate that hypoxia-induced mitochondrial dysfunction drives

  2. UCP2 muscle gene transfer modifies mitochondrial membrane potential.

    Science.gov (United States)

    Marti, A; Larrarte, E; Novo, F J; Garcia, M; Martinez, J A

    2001-01-01

    The aim of this work was to evaluate the effect of uncoupling protein 2 (UCP2) muscle gene transfer on mitochondrial activity. Five week-old male Wistar rats received an intramuscular injection of plasmid pXU1 containing UCP2 cDNA in the right tibialis anterior muscles. Left tibialis anterior muscles were injected with vehicle as control. Ten days after DNA injection, tibialis anterior muscles were dissected and muscle mitochondria isolated and analyzed. There were two mitochondrial populations in the muscle after UCP2 gene transfer, one of low fluorescence and complexity and the other, showing high fluorescence and complexity. UCP2 gene transfer resulted in a 3.6 fold increase in muscle UCP2 protein levels compared to control muscles assessed by Western blotting. Furthermore, a significant reduction in mitochondria membrane potential assessed by spectrofluorometry and flow cytometry was observed. The mitochondria membrane potential reduction might account for a decrease in fluorescence of the low fluorescence mitochondrial subpopulation. It has been demonstrated that UCP2 muscle gene transfer in vivo is associated with a lower mitochondria membrane potential. Our results suggest the potential involvement of UCP2 in uncoupling respiration. International Journal of Obesity (2001) 25, 68-74

  3. Mitochondrial shaping cuts.

    Science.gov (United States)

    Escobar-Henriques, Mafalda; Langer, Thomas

    2006-01-01

    A broad range of cellular processes are regulated by proteolytic events. Proteolysis has now also been established to control mitochondrial morphology which results from the balanced action of fusion and fission. Two out of three known core components of the mitochondrial fusion machinery are under proteolytic control. The GTPase Fzo1 in the outer membrane of mitochondria is degraded along two independent proteolytic pathways. One controls mitochondrial fusion in vegetatively growing cells, the other one acts upon mating factor-induced cell cycle arrest. Fusion also depends on proteolytic processing of the GTPase Mgm1 by the rhomboid protease Pcp1 in the inner membrane of mitochondria. Functional links of AAA proteases or other proteolytic components to mitochondrial dynamics are just emerging. This review summarises the current understanding of regulatory roles of proteolytic processes for mitochondrial plasticity.

  4. Radiation and chemotherapy bystander effects induce early genomic instability events: telomere shortening and bridge formation coupled with mitochondrial dysfunction.

    LENUS (Irish Health Repository)

    Gorman, Sheeona

    2012-02-01

    The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapy-treated explant tissues. Bystander cells exposed to media from 2Gy, 5Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0Gy) at 24h. There was no significant difference between 2Gy and 5Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p=0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p=0.02) and mitochondrial membrane potential increased (p=0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial

  5. The mitochondrial outer membrane protein MDI promotes local protein synthesis and mtDNA replication.

    Science.gov (United States)

    Zhang, Yi; Chen, Yong; Gucek, Marjan; Xu, Hong

    2016-05-17

    Early embryonic development features rapid nuclear DNA replication cycles, but lacks mtDNA replication. To meet the high-energy demands of embryogenesis, mature oocytes are furnished with vast amounts of mitochondria and mtDNA However, the cellular machinery driving massive mtDNA replication in ovaries remains unknown. Here, we describe a Drosophila AKAP protein, MDI that recruits a translation stimulator, La-related protein (Larp), to the mitochondrial outer membrane in ovaries. The MDI-Larp complex promotes the synthesis of a subset of nuclear-encoded mitochondrial proteins by cytosolic ribosomes on the mitochondrial surface. MDI-Larp's targets include mtDNA replication factors, mitochondrial ribosomal proteins, and electron-transport chain subunits. Lack of MDI abolishes mtDNA replication in ovaries, which leads to mtDNA deficiency in mature eggs. Targeting Larp to the mitochondrial outer membrane independently of MDI restores local protein synthesis and rescues the phenotypes of mdi mutant flies. Our work suggests that a selective translational boost by the MDI-Larp complex on the outer mitochondrial membrane might be essential for mtDNA replication and mitochondrial biogenesis during oogenesis. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  6. Nitric oxide partitioning into mitochondrial membranes and the control of respiration at cytochrome c oxidase

    Science.gov (United States)

    Shiva, Sruti; Brookes, Paul S.; Patel, Rakesh P.; Anderson, Peter G.; Darley-Usmar, Victor M.

    2001-06-01

    An emerging and important site of action for nitric oxide (NO) within cells is the mitochondrial inner membrane, where NO binds to and inhibits members of the electron transport chain, complex III and cytochrome c oxidase. Although it is known that inhibition of cytochrome c oxidase by NO is competitive with O2, the mechanisms that underlie this phenomenon remain unclear, and the impact of both NO and O2 partitioning into biological membranes has not been considered. These properties are particularly interesting because physiological O2 tensions can vary widely, with NO having a greater inhibitory effect at low O2 tensions (mitochondrial membranes in the absence of substrate, in a nonsaturable process that is O2 dependent. This consumption modulates inhibition of cytochrome c oxidase by NO and is enhanced by the addition of exogenous membranes. From these data, it is evident that the partition of NO into mitochondrial membranes has a major impact on the ability of NO to control mitochondrial respiration. The implications of this conclusion are discussed in the context of mitochondrial lipid:protein ratios and the importance of NO as a regulator of respiration in pathophysiology.

  7. Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H+-dependent mitochondrial pathway

    International Nuclear Information System (INIS)

    Pelin, Marco; Ponti, Cristina; Sosa, Silvio; Gibellini, Davide; Florio, Chiara; Tubaro, Aurelia

    2013-01-01

    In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na + influx due to the transformation of Na + /K + ATPase in a cationic channel. Recently, we have demonstrated that Na + overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na + intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O 2 − production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na + -mediated H + -imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O 2 − production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O 2 − production induced by PLTX-mediated ionic imbalance. Indeed, the H + intracellular overload that follows PLTX-induced intracellular Na + accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O 2 − production by reversing mitochondrial electron transport. Highlights: ► PLTX induces superoxide (O 2 − ) production by reversing mitochondrial transport chain. ► The mechanism of O 2 − production is dependent on PLTX-induced ionic imbalance. ► The results led to the

  8. Qing Dai attenuates nonsteroidal anti-inflammatory drug-induced mitochondrial reactive oxygen species in gastrointestinal epithelial cells.

    Science.gov (United States)

    Saito, Rie; Tamura, Masato; Matsui, Hirofumi; Nagano, Yumiko; Suzuki, Hideo; Kaneko, Tsuyoshi; Mizokami, Yuji; Hyodo, Ichinosuke

    2015-01-01

    Treatments with nonsteroidal anti-inflammatory drugs (NSAIDs) have increased the number of patients with gastrointestinal complications. Qing Dai has been traditionally used in Chinese herbal medicine for various inflammatory diseases such as ulcerative colitis. We previously reported that Qing Dai suppressed inflammations by scavenging reactive oxygen species (ROS) in ulcerative colitis patients. Thus, Qing Dai can attenuate the production of ROS, which play an important role in NSAID-induced gastrointestinal injuries. In this study, we aimed to elucidate whether Qing Dai decreased mitochondrial ROS production in NSAID-treated gastrointestinal cells by examining cellular injury, mitochondrial membrane potentials, and ROS production with specific fluorescent indicators. We also performed electron paramagnetic resonance measurement in isolated mitochondria with a spin-trapping reagent (CYPMPO or DMPO). Treatments with indomethacin and aspirin induced cellular injury and mitochondrial impairment in the gastrointestinal cells. Under these conditions, mitochondrial alterations were observed on electron microscopy. Qing Dai prevented these complications by suppressing ROS production in gastrointestinal cells. These results indicate that Qing Dai attenuated the ROS production from the NSAID-induced mitochondrial alteration in the gastrointestinal epithelial cells. Qing Dai treatment may be considered effective for the prevention NSAID-induced gastrointestinal injury.

  9. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    Science.gov (United States)

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. A biophysical approach to menadione membrane interactions: relevance for menadione-induced mitochondria dysfunction and related deleterious/therapeutic effects.

    Science.gov (United States)

    Monteiro, João P; Martins, André F; Nunes, Cláudia; Morais, Catarina M; Lúcio, Marlene; Reis, Salette; Pinheiro, Teresa J T; Geraldes, Carlos F G C; Oliveira, Paulo J; Jurado, Amália S

    2013-08-01

    Menadione (MEN), a polycyclic aromatic ketone, was shown to promote cell injury by imposing massive oxidative stress and has been proposed as a promising chemotherapeutic agent for the treatment of cancer diseases. The mechanisms underlying MEN-induced mitochondrial dysfunction and cell death are not yet fully understood. In this work, a systematic study was performed to unveil the effects of MEN on membrane lipid organization, using models mimicking mitochondrial membranes and native mitochondrial membranes. MEN was found to readily incorporate in membrane systems composed of a single phospholipid (phosphatidylcholine) or the lipids dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine and tetraoleoylcardiolipin at 1:1:1 molar ratio, as well as in mitochondrial membranes. Increased permeability in both membrane models, monitored by calcein release, seemed to correlate with the extent of MEN incorporation into membranes. MEN perturbed the physical properties of vesicles composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine plus tetraoleoylcardiolipin (at 7:3 molar ratio), as reflected by the downshift of the lipid phase transition temperature and the emergence of a new transition peak in the mixed lipid system, detected by DSC. (31)P NMR studies revealed that MEN favored the formation of non-lamellar structures. Also, quenching studies with the fluorescent probes DPH and TMA-DPH showed that MEN distributed across the bilayer thickness in both model and native mitochondrial membranes. MEN's ability to promote alterations of membrane lipid organization was related with its reported mitochondrial toxicity and promotion of apoptosis, predictably involved in its anti-carcinogenic activity. Copyright © 2013 Elsevier B.V. All rights reserved.

  11. Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

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    Carolina Paola García

    Full Text Available Human embryonic stem cells (hESCs are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs and hESCs-derived neuroprogenitors (NP. We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help

  12. Mitochondrial ceramide-rich macrodomains functionalize Bax upon irradiation.

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    Hyunmi Lee

    Full Text Available Evidence indicates that Bax functions as a "lipidic" pore to regulate mitochondrial outer membrane permeabilization (MOMP, the apoptosis commitment step, through unknown membrane elements. Here we show mitochondrial ceramide elevation facilitates MOMP-mediated cytochrome c release in HeLa cells by generating a previously-unrecognized mitochondrial ceramide-rich macrodomain (MCRM, which we visualize and isolate, into which Bax integrates.MCRMs, virtually non-existent in resting cells, form upon irradiation coupled to ceramide synthase-mediated ceramide elevation, optimizing Bax insertion/oligomerization and MOMP. MCRMs are detected by confocal microscopy in intact HeLa cells and isolated biophysically as a light membrane fraction from HeLa cell lysates. Inhibiting ceramide generation using a well-defined natural ceramide synthase inhibitor, Fumonisin B1, prevented radiation-induced Bax insertion, oligomerization and MOMP. MCRM deconstruction using purified mouse hepatic mitochondria revealed ceramide alone is non-apoptogenic. Rather Bax integrates into MCRMs, oligomerizing therein, conferring 1-2 log enhanced cytochrome c release. Consistent with this mechanism, MCRM Bax isolates as high molecular weight "pore-forming" oligomers, while non-MCRM membrane contains exclusively MOMP-incompatible monomeric Bax.Our recent studies in the C. elegans germline indicate that mitochondrial ceramide generation is obligate for radiation-induced apoptosis, although a mechanism for ceramide action was not delineated. Here we demonstrate that ceramide, generated in the mitochondrial outer membrane of mammalian cells upon irradiation, forms a platform into which Bax inserts, oligomerizes and functionalizes as a pore. We posit conceptualization of ceramide as a membrane-based stress calibrator, driving membrane macrodomain organization, which in mitochondria regulates intensity of Bax-induced MOMP, and is pharmacologically tractable in vitro and in vivo.

  13. The seleno-organic compound ebselen impairs mitochondrial physiology and induces cell death in AR42J cells.

    Science.gov (United States)

    Santofimia-Castaño, Patricia; Garcia-Sanchez, Lourdes; Ruy, Deborah Clea; Fernandez-Bermejo, Miguel; Salido, Gines M; Gonzalez, Antonio

    2014-09-17

    Ebselen is a seleno-organic compound that causes cell death in several cancer cell types. The mechanisms underlying its deleterious effects have not been fully elucidated. In this study, the effects of ebselen (1 μM-40 μM) on AR42J tumor cells have been examined. Cell viability was studied using AlamarBlue(®) test. Cell cycle phase determination was carried out by flow cytometry. Changes in intracellular free Ca(2+) concentration were followed by fluorimetry analysis of fura-2-loaded cells. Distribution of mitochondria, mitochondrial Ca(2+) concentration and mitochondrial membrane potential were monitored by confocal microscopy of cells loaded with Mitotracker Green™ FM, rhod-2 or TMRM respectively. Caspase-3 activity was calculated following the luorogenic substrate ACDEVD-AMC signal with a spectrofluorimeter. Results show that cell viability decreased in the presence of ebselen. An increase in the number of cells in the S-phase of the cell cycle was observed. Ebselen induced a concentration-dependent mobilization of Ca(2+) from agonist- and thapsigargin-sensitive Ca(2+) pools. Ebselen induced also a transient increase in mitochondrial Ca(2+) concentration, a progressive decrease of the mitochondrial membrane potential and a disruption of the mitochondrial network. Finally, a concentration-dependent increase in caspase-3 activity was detected. We conclude that ebselen exerts deleterious actions on the cells that involve the impairment of mitochondrial physiology and the activation of caspase-3-mediated apoptotic pathway. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  14. Protection against oxidant-induced apoptosis by mitochondrial thioredoxin in SH-SY5Y neuroblastoma cells

    International Nuclear Information System (INIS)

    Chen Yan; Yu Min; Jones, Dean P.; Greenamyre, J. Timothy; Cai Jiyang

    2006-01-01

    Mitochondrial oxidative stress plays important roles in aging and age-related degenerative disorders. The newly identified mitochondrial thioredoxin (mtTrx; Trx2) is a key component of the mitochondrial antioxidant system which is responsible for the clearance of reactive intermediates and repairs proteins with oxidative damage. Here, we show that in cultured SH-SY5Y human neuroblastoma 1cells, overexpression of mtTrx inhibited apoptosis and loss of mitochondrial membrane potential induced by a chemical oxidant, tert-butylhydroperoxide (tBH). The effects of calcium ionophore (Br-A23187) were not affected by mtTrx, suggesting the protection was specific against oxidative injury. The mitochondrial glutathione pool was oxidized by tBH, and this oxidation was not inhibited by increased mtTrx. Consequently, the antioxidant function of mtTrx is not redundant, but rather in addition, to that of GSH. Mutations of Cys90 and Cys93 to serines rendered mtTrx ineffective in protection against tBH-induced cytoxicity. These data indicate that mtTrx controls the mitochondrial redox status independently of GSH and is a key component of the defensive mechanism against oxidative stress in cultured neuronal cells

  15. Tempol, a superoxide dismutase mimetic agent, ameliorates cisplatin-induced nephrotoxicity through alleviation of mitochondrial dysfunction in mice.

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    Lamiaa A Ahmed

    Full Text Available Mitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice.Nephrotoxicity was assessed 72 h after a single i.p. injection of cisplatin (25 mg/kg with or without oral administration of tempol (100 mg/kg/day. Serum creatinine and urea as well as glucosuria and proteinuria were evaluated. Both kidneys were isolated for estimation of oxidative stress markers, adenosine triphosphate (ATP content and caspase-3 activity. Moreover, mitochondrial oxidative phosphorylation capacity, complexes I-IV activities and mitochondrial nitric oxide synthase (mNOS protein expression were measured along with histological examinations of renal tubular damage and mitochondrial ultrastructural changes. Tempol was effective against cisplatin-induced elevation of serum creatinine and urea as well as glucosuria and proteinuria. Moreover, pretreatment with tempol notably inhibited cisplatin-induced oxidative stress and disruption of mitochondrial function by restoring mitochondrial oxidative phosphorylation, complexes I and III activities, mNOS protein expression and ATP content. Tempol also provided significant protection against apoptosis, tubular damage and mitochondrial ultrastructural changes. Interestingly, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma.This study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction.

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

    International Nuclear Information System (INIS)

    Yasuzaki, Yukari; Yamada, Yuma; Harashima, Hideyoshi

    2010-01-01

    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.

  17. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between Beclin 1 and Bcl-2/Bcl-X(L).

    Science.gov (United States)

    Maiuri, Maria Chiara; Criollo, Alfredo; Tasdemir, Ezgi; Vicencio, José Miguel; Tajeddine, Nicolas; Hickman, John A; Geneste, Olivier; Kroemer, Guido

    2007-01-01

    Beclin 1 has recently been identified as novel BH3-only protein, meaning that it carries one Bcl-2-homology-3 (BH3) domain. As other BH3-only proteins, Beclin 1 interacts with anti-apoptotic multidomain proteins of the Bcl-2 family (in particular Bcl-2 and its homologue Bcl-X(L)) by virtue of its BH3 domain, an amphipathic alpha-helix that binds to the hydrophobic cleft of Bcl-2/Bcl-X(L). The BH3 domains of other BH3-only proteins such as Bad, as well as BH3-mimetic compounds such as ABT737, competitively disrupt the inhibitory interaction between Beclin 1 and Bcl-2/Bcl-X(L). This causes autophagy of mitochondria (mitophagy) but not of the endoplasmic reticulum (reticulophagy). Only ER-targeted (not mitochondrion-targeted) Bcl-2/Bcl-X(L) can inhibit autophagy induced by Beclin 1, and only Beclin 1-Bcl-2/Bcl-X(L) complexes present in the ER (but not those present on heavy membrane fractions enriched in mitochondria) are disrupted by ABT737. These findings suggest that the Beclin 1-Bcl-2/Bcl-X(L) complexes that normally inhibit autophagy are specifically located in the ER and point to an organelle-specific regulation of autophagy. Furthermore, these data suggest a spatial organization of autophagy and apoptosis control in which BH3-only proteins exert two independent functions. On the one hand, they can induce apoptosis, by (directly or indirectly) activating the mitochondrion-permeabilizing function of pro-apoptotic multidomain proteins from the Bcl-2 family. On the other hand, they can activate autophagy by liberating Beclin 1 from its inhibition by Bcl-2/Bcl-X(L) at the level of the endoplasmic reticulum.

  18. Mesenchymal Stem Cells Protect Nucleus Pulposus Cells from Compression-Induced Apoptosis by Inhibiting the Mitochondrial Pathway

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    Sheng Chen

    2017-01-01

    Full Text Available Objective. Excessive apoptosis of nucleus pulposus cells (NPCs induced by various stresses, including compression, contributes to the development of intervertebral disc degeneration (IVDD. Mesenchymal stem cells (MSCs can benefit the regeneration of NPCs and delay IVDD, but the underlying molecular mechanism is poorly understood. This study aimed to evaluate the antiapoptosis effects of bone marrow-derived MSC (BMSC on rat NPCs exposed to compression and investigate whether the mitochondrial pathway was involved. Methods. BMSCs and NPCs were cocultured in the compression apparatus at 1.0 MPa for 36 h. Cell viability, apoptosis, mitochondrial function, and the expression of apoptosis-related proteins were evaluated. Results. The results showed that coculturing with BMSCs increased the cell viability and reduced apoptosis of NPCs exposed to compression. Meanwhile, BMSCs could relieve the compression-induced mitochondrial damage of NPCs by decreasing reactive oxygen species level and maintaining mitochondrial membrane potential as well as mitochondrial integrity. Furthermore, coculturing with BMSCs suppressed the activated caspase-3 and activated caspase-9, decreased the expressions of cytosolic cytochrome c and Bax, and increased the expression of Bcl-2. Conclusions. Our results suggest that BMSCs can protect against compression-induced apoptosis of NPCs by inhibiting the mitochondrial pathway and thus enhance our understanding on the MSC-based therapy for IVDD.

  19. A nontoxic, photostable and high signal-to-noise ratio mitochondrial probe with mitochondrial membrane potential and viscosity detectivity

    Science.gov (United States)

    Chen, Yanan; Qi, Jianguo; Huang, Jing; Zhou, Xiaomin; Niu, Linqiang; Yan, Zhijie; Wang, Jianhong

    2018-01-01

    Herein, we reported a yellow emission probe 1-methyl-4-(6-morpholino-1, 3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl) pyridin-1-ium iodide which could specifically stain mitochondria in living immortalized and normal cells. In comparison to the common mitochondria tracker (Mitotracker Deep Red, MTDR), this probe was nontoxic, photostable and ultrahigh signal-to-noise ratio, which could real-time monitor mitochondria for a long time. Moreover, this probe also showed high sensitivity towards mitochondrial membrane potential and intramitochondrial viscosity change. Consequently, this probe was used for imaging mitochondria, detecting changes in mitochondrial membrane potential and intramitochondrial viscosity in physiological and pathological processes.

  20. Mitochondrial cardiolipin/phospholipid trafficking: the role of membrane contact site complexes and lipid transfer proteins.

    Science.gov (United States)

    Schlattner, Uwe; Tokarska-Schlattner, Malgorzata; Rousseau, Denis; Boissan, Mathieu; Mannella, Carmen; Epand, Richard; Lacombe, Marie-Lise

    2014-04-01

    Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

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

    Science.gov (United States)

    Botta, Amy; Laher, Ismail; Beam, Julianne; Decoffe, Daniella; Brown, Kirsty; Halder, Swagata; Devlin, Angela; Gibson, Deanna L; Ghosh, Sanjoy

    2013-01-01

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

  2. Tempol, a Superoxide Dismutase Mimetic Agent, Ameliorates Cisplatin-Induced Nephrotoxicity through Alleviation of Mitochondrial Dysfunction in Mice

    Science.gov (United States)

    Ahmed, Lamiaa A.; Shehata, Nagwa I.; Abdelkader, Noha F.; Khattab, Mahmoud M.

    2014-01-01

    Background Mitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice. Methods and Findings Nephrotoxicity was assessed 72 h after a single i.p. injection of cisplatin (25 mg/kg) with or without oral administration of tempol (100 mg/kg/day). Serum creatinine and urea as well as glucosuria and proteinuria were evaluated. Both kidneys were isolated for estimation of oxidative stress markers, adenosine triphosphate (ATP) content and caspase-3 activity. Moreover, mitochondrial oxidative phosphorylation capacity, complexes I–IV activities and mitochondrial nitric oxide synthase (mNOS) protein expression were measured along with histological examinations of renal tubular damage and mitochondrial ultrastructural changes. Tempol was effective against cisplatin-induced elevation of serum creatinine and urea as well as glucosuria and proteinuria. Moreover, pretreatment with tempol notably inhibited cisplatin-induced oxidative stress and disruption of mitochondrial function by restoring mitochondrial oxidative phosphorylation, complexes I and III activities, mNOS protein expression and ATP content. Tempol also provided significant protection against apoptosis, tubular damage and mitochondrial ultrastructural changes. Interestingly, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma. Conclusion This study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction

  3. Rapeseed oil-rich diet alters in vitro menadione and nimesulide hepatic mitochondrial toxicity.

    Science.gov (United States)

    Monteiro, João P; Silva, Ana M; Jurado, Amália S; Oliveira, Paulo J

    2013-10-01

    Diet-induced changes in the lipid composition of mitochondrial membranes have been shown to influence physiological processes. However, the modulation effect of diet on mitochondrially-active drugs has not yet received the deserved attention. Our hypothesis is that modulation of membrane dynamics by diet impacts drug-effects on liver mitochondrial functioning. In a previous work, we have shown that a diet rich in rapeseed oil altered mitochondrial membrane composition and bioenergetics in Wistar rats. In the present work, we investigated the influence of the modified diet on hepatic mitochondrial activity of two drugs, menadione and nimesulide, and FCCP, a classic protonophore, was used for comparison. The results showed that the effects of menadione and nimesulide were less severe on liver mitochondria for rats fed the modified diet than on rats fed the control diet. A specific effect on complex I seemed to be involved in drug-induced mitochondria dysfunction. Liver mitochondria from the modified diet group were more susceptible to nimesulide effects on MPT induction. The present work demonstrates that diet manipulation aimed at modifying mitochondrial membrane properties alters the toxicity of mitochondria active agents. This work highlights that diet may potentiate mitochondrial pharmacologic effects or increase drug-induced liabilities. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. Elastocapillary Instability in Mitochondrial Fission

    Science.gov (United States)

    Gonzalez-Rodriguez, David; Sart, Sébastien; Babataheri, Avin; Tareste, David; Barakat, Abdul I.; Clanet, Christophe; Husson, Julien

    2015-08-01

    Mitochondria are dynamic cell organelles that constantly undergo fission and fusion events. These dynamical processes, which tightly regulate mitochondrial morphology, are essential for cell physiology. Here we propose an elastocapillary mechanical instability as a mechanism for mitochondrial fission. We experimentally induce mitochondrial fission by rupturing the cell's plasma membrane. We present a stability analysis that successfully explains the observed fission wavelength and the role of mitochondrial morphology in the occurrence of fission events. Our results show that the laws of fluid mechanics can describe mitochondrial morphology and dynamics.

  5. Impact of Storage and Purification on Mitochondrial Membrane Potential of Boar Spermatozoa

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    Aristotelis G. Lymberopoulos

    2013-05-01

    Full Text Available This study aimed to evaluate the effect of semen purification and storage on sperm mitochondrial membrane potential (ΔΨm. Gel-free whole ejaculates were collected from five proven fertile Large White boars aged two to three years. Aliquots of fresh semen were split, diluted in one step with commercial extenders and incubated at 37oC for 5-10 minutes. Semen was cooled to 18oC and packaged in 15-ml sterile propylene tubes. After 4-10 hours post-semen collection, stored semen was purified by colloidal centrifugation. After 48 hours post-semen collection, stored semen was incubated at 37oC and evaluated after 45 minutes for motility, velocity and sperm ΔΨm. Samples were stained with 2.99 μM JC-1 and 2.32 μM EH-1 and assessed by Fluorescence microscopy. After centrifugation a significant improvement of motility (P<0.035, and velocity (P<0.012 was noticed. The percentage of spermatozoa with intact plasma membrane and high/low mitochondrial membrane potential was statistical higher after centrifugation and storage at 18°C for 48 hours. In conclusion, colloidal purification of boar semen can improve sperm quality and  mitochondrial membrane potential.

  6. Biological rational for sequential targeting of Bruton tyrosine kinase and Bcl-2 to overcome CD40-induced ABT-199 resistance in mantle cell lymphoma.

    Science.gov (United States)

    Chiron, David; Dousset, Christelle; Brosseau, Carole; Touzeau, Cyrille; Maïga, Sophie; Moreau, Philippe; Pellat-Deceunynck, Catherine; Le Gouill, Steven; Amiot, Martine

    2015-04-20

    The aggressive biological behavior of mantle cell lymphoma (MCL) and its short response to current treatment highlight a great need for better rational therapy. Herein, we investigate the ability of ABT-199, the Bcl-2-selective BH3 mimetic, to kill MCL cells. Among MCL cell lines tested (n = 8), only three were sensitive (LD50 < 200 nM). In contrast, all primary MCL samples tested (n = 11) were highly sensitive to ABT-199 (LD50 < 10 nM). Mcl-1 and Bcl-xL both confer resistance to ABT-199-specific killing and BCL2/(BCLXL+MCL1) mRNA ratio is a strong predictor of sensitivity. By mimicking the microenvironment through CD40 stimulation, we show that ABT-199 sensitivity is impaired through activation of NF-kB pathway and Bcl-x(L) up-regulation. We further demonstrate that resistance is rapidly lost when MCL cells detach from CD40L-expressing fibroblasts. It has been reported that ibrutinib induces lymphocytosis in vivo holding off malignant cells from their protective microenvironment. We show here for two patients undergoing ibrutinib therapy that mobilized MCL cells are highly sensitive to ABT-199. These results provide evidence that in situ ABT-199 resistance can be overcome when MCL cells escape from the lymph nodes. Altogether, our data support the clinical application of ABT-199 therapy both as a single agent and in sequential combination with BTK inhibitors.

  7. Mitochondrial Dysfunction Causes Oxidative Stress and Tapetal Apoptosis in Chemical Hybridization Reagent-Induced Male Sterility in Wheat

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    Shuping Wang

    2018-01-01

    Full Text Available Male sterility in plants has been strongly linked to mitochondrial dysfunction. Chemical hybridization agent (CHA-induced male sterility is an important tool in crop heterosis. Therefore, it is important to better understand the relationship between mitochondria and CHA-induced male sterility in wheat. This study reports on the impairment of mitochondrial function duo to CHA-SQ-1, which occurs by decreasing cytochrome oxidase and adenosine triphosphate synthase protein levels and theirs activities, respiratory rate, and in turn results in the inhibition of the mitochondrial electron transport chain (ETC, excessive production of reactive oxygen species (ROS and disruption of the alternative oxidase pathway. Subsequently, excessive ROS combined with MnSOD defects results in damage to the mitochondrial membrane, followed by ROS release into the cytoplasm. The microspores underwent severe oxidative stress during pollen development. Furthermore, chronic oxidative stress, together with the overexpression of type II metacaspase, triggered premature tapetal apoptosis, which resulted in pollen abortion. Accordingly, we propose a metabolic pathway for mitochondrial-mediated male sterility in wheat, which provides information on the molecular events underlying CHA-SQ-1-induced abortion of anthers and may serve as an additional guide to the practical application of hybrid breeding.

  8. Biguanide-induced mitochondrial dysfunction yields increased lactate production and cytotoxicity of aerobically-poised HepG2 cells and human hepatocytes in vitro

    International Nuclear Information System (INIS)

    Dykens, James A.; Jamieson, Joseph; Marroquin, Lisa; Nadanaciva, Sashi; Billis, Puja A.; Will, Yvonne

    2008-01-01

    As a class, the biguanides induce lactic acidosis, a hallmark of mitochondrial impairment. To assess potential mitochondrial impairment, we evaluated the effects of metformin, buformin and phenformin on: 1) viability of HepG2 cells grown in galactose, 2) respiration by isolated mitochondria, 3) metabolic poise of HepG2 and primary human hepatocytes, 4) activities of immunocaptured respiratory complexes, and 5) mitochondrial membrane potential and redox status in primary human hepatocytes. Phenformin was the most cytotoxic of the three with buformin showing moderate toxicity, and metformin toxicity only at mM concentrations. Importantly, HepG2 cells grown in galactose are markedly more susceptible to biguanide toxicity compared to cells grown in glucose, indicating mitochondrial toxicity as a primary mode of action. The same rank order of potency was observed for isolated mitochondrial respiration where preincubation (40 min) exacerbated respiratory impairment, and was required to reveal inhibition by metformin, suggesting intramitochondrial bio-accumulation. Metabolic profiling of intact cells corroborated respiratory inhibition, but also revealed compensatory increases in lactate production from accelerated glycolysis. High (mM) concentrations of the drugs were needed to inhibit immunocaptured respiratory complexes, supporting the contention that bioaccumulation is involved. The same rank order was found when monitoring mitochondrial membrane potential, ROS production, and glutathione levels in primary human hepatocytes. In toto, these data indicate that biguanide-induced lactic acidosis can be attributed to acceleration of glycolysis in response to mitochondrial impairment. Indeed, the desired clinical outcome, viz., decreased blood glucose, could be due to increased glucose uptake and glycolytic flux in response to drug-induced mitochondrial dysfunction

  9. The protective effect of lipid emulsion in preventing bupivacaine-induced mitochondrial injury and apoptosis of H9C2 cardiomyocytes.

    Science.gov (United States)

    Chen, Zhe; Jin, Zhousheng; Xia, Yun; Zhao, Shishi; Xu, Xuzhong; Papadimos, Thomas J; Wang, Quanguang

    2017-11-01

    Lipid emulsion (LE) has been shown to be effective in the resuscitation of bupivacaine-induced cardiac arrest, but the precise mechanism of this action has not been fully elucidated. Pursuant to this lack of information on the mechanism in which LE protects the myocardium during bupivacaine-induced toxicity, we explored mitochondrial function and cell apoptosis. H9C2 cardiomyocytes were used in study. Cells were randomly divided in different groups and were cultivated 6 h, 12 h, and 24 h. The mitochondria were extracted and mitochondrial ATP content was measured, as was mitochondrial membrane potential, the concentration of calcium ion (Ca2+), and the activity of Ca2+-ATP enzyme (Ca2+-ATPase). Cells from groups Bup1000, LE group, and Bup1000LE were collected to determine cell viability, cell apoptosis, and electron microscopy scanning of mitochondrial ultrastructure (after 24 h). We found that LE can reverse the inhibition of the mitochondrial function induced by bupivacaine, regulate the concentration of calcium ion in mitochondria, resulting in the protection of myocardial cells from toxicity induced by bupivacaine.

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

    Science.gov (United States)

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

    2016-04-01

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

  11. Mitochondrial phospholipase A2 activated by reactive oxygen species in heart mitochondria induces mild uncoupling

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; Jabůrek, Martin; Zelenka, Jaroslav; Ježek, Petr

    2010-01-01

    Roč. 59, č. 5 (2010), s. 737-747 ISSN 0862-8408 R&D Projects: GA ČR(CZ) GA303/07/0105; GA MŠk ME09018; GA AV ČR(CZ) KJB500110902 Institutional research plan: CEZ:AV0Z50110509 Keywords : Heart mitochondrial phospholipase A2 * Fatty Acids * Adenine nucleotide translocase Subject RIV: ED - Physiology Impact factor: 1.646, year: 2010

  12. Ethanol Influences on Bax Associations with Mitochondrial Membrane Proteins in Neonatal Rat Cerebellum

    Science.gov (United States)

    Heaton, Marieta Barrow; Siler-Marsiglio, Kendra; Paiva, Michael; Kotler, Alexandra; Rogozinski, Jonathan; Kubovec, Stacey; Coursen, Mary; Madorsky, Vladimir

    2012-01-01

    These studies investigated interactions taking place at the mitochondrial membrane in neonatal rat cerebellum following ethanol exposure, and focused on interactions between pro-apoptotic Bax and proteins of the permeability transition pore (PTP), voltage-dependent anion channel (VDAC), and adenine nucleotide translocator (ANT), of the outer and inner mitochondrial membranes, respectively. Cultured cerebellar granule cells were used to assess the role of these interactions in ethanol neurotoxicity. Analyses were made at the age of maximal cerebellar ethanol vulnerability (P4), compared to the later age of relative resistance (P7), to determine whether differential ethanol sensitivity was mirrored by differences in these molecular interactions. We found that following ethanol exposure, Bax pro-apoptotic associations with both VDAC and ANT were increased, particularly at the age of greater ethanol sensitivity, and these interactions were sustained at this age for at least two hours post-exposure. Since Bax:VDAC interactions disrupt protective VDAC interactions with mitochondrial hexokinase (HXK), we also assessed VDAC:HXK associations following ethanol treatment, and found such interactions were altered by ethanol treatment, but only at two-hours post-exposure, and only in the P4, ethanol-sensitive cerebellum. Ethanol neurotoxicity in cultured neuronal preparations was abolished by pharmacological inhibition of both VDAC and ANT interactions with Bax, but not by a Bax channel blocker. Therefore, we conclude that at this age, within the constraints of our experimental model, a primary mode of Bax-induced initiation of the apoptosis cascade following ethanol insult involves interactions with proteins of the PTP complex, and not channel formation independent of PTP constituents. PMID:22767450

  13. Technology evaluation: ABT-510, Abbott.

    NARCIS (Netherlands)

    Westphal, J.R.

    2004-01-01

    ABT-510 is a small peptide thrombospondin-1 mimetic angiogenesis inhibitor under development by Abbott Laboratories for the potential treatment of solid tumors. ABT-510 is undergoing phase II clinical trials.

  14. GTP-binding-defective ARL4D alters mitochondrial morphology and membrane potential.

    Directory of Open Access Journals (Sweden)

    Chun-Chun Li

    Full Text Available ARL4D, ARL4A, and ARL4C are closely related members of the ADP-ribosylation factor/ARF-like protein (ARF/ARL family of GTPases. All three ARL4 proteins contain nuclear localization signals (NLSs at their C-termini and are primarily found at the plasma membrane, but they are also present in the nucleus and cytoplasm. ARF function and localization depends on their controlled binding and hydrolysis of GTP. Here we show that GTP-binding-defective ARL4D is targeted to the mitochondria, where it affects mitochondrial morphology and function. We found that a portion of endogenous ARL4D and the GTP-binding-defective ARL4D mutant ARL4D(T35N reside in the mitochondria. The N-terminal myristoylation of ARL4D(T35N was required for its localization to mitochondria. The localization of ARL4D(T35N to the mitochondria reduced the mitochondrial membrane potential (ΔΨm and caused mitochondrial fragmentation. Furthermore, the C-terminal NLS region of ARL4D(T35N was required for its effect on the mitochondria. This study is the first to demonstrate that the dysfunctional GTP-binding-defective ARL4D is targeted to mitochondria, where it subsequently alters mitochondrial morphology and membrane potential.

  15. Role of polyhydroxybutyrate in mitochondrial calcium uptake

    Science.gov (United States)

    Smithen, Matthew; Elustondo, Pia A.; Winkfein, Robert; Zakharian, Eleonora; Abramov, Andrey Y.; Pavlov, Evgeny

    2013-01-01

    Polyhydroxybutyrate (PHB) is a biological polymer which belongs to the class of polyesters and is ubiquitously present in all living organisms. Mammalian mitochondrial membranes contain PHB consisting of up to 120 hydroxybutyrate residues. Roles played by PHB in mammalian mitochondria remain obscure. It was previously demonstrated that PHB of the size similar to one found in mitochondria mediates calcium transport in lipid bilayer membranes. We hypothesized that the presence of PHB in mitochondrial membrane might play a significant role in mitochondrial calcium transport. To test this, we investigated how the induction of PHB hydrolysis affects mitochondrial calcium transport. Mitochondrial PHB was altered enzymatically by targeted expression of bacterial PHB hydrolyzing enzyme (PhaZ7) in mitochondria of mammalian cultured cells. The expression of PhaZ7 induced changes in mitochondrial metabolism resulting in decreased mitochondrial membrane potential in HepG2 but not in U87 and HeLa cells. Furthermore, it significantly inhibited mitochondrial calcium uptake in intact HepG2, U87 and HeLa cells stimulated by the ATP or by the application of increased concentrations of calcium to the digitonin permeabilized cells. Calcium uptake in PhaZ7 expressing cells was restored by mimicking calcium uniporter properties with natural electrogenic calcium ionophore - ferutinin. We propose that PHB is a previously unrecognized important component of the mitochondrial calcium uptake system. PMID:23702223

  16. Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H{sup +}-dependent mitochondrial pathway

    Energy Technology Data Exchange (ETDEWEB)

    Pelin, Marco, E-mail: marco.pelin@phd.units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Ponti, Cristina, E-mail: cponti@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Sosa, Silvio, E-mail: silvio.sosa@econ.units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Gibellini, Davide, E-mail: davide.gibellini@unibo.it [Department of Haematology and Oncological Sciences, University of Bologna, Via Massarenti 9, 40138 Bologna (Italy); Florio, Chiara, E-mail: florioc@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy); Tubaro, Aurelia, E-mail: tubaro@units.it [Department of Life Science, University of Trieste, Via L. Giorgieri 7/9, 34127 Trieste (Italy)

    2013-01-01

    In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na{sup +} influx due to the transformation of Na{sup +}/K{sup +} ATPase in a cationic channel. Recently, we have demonstrated that Na{sup +} overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na{sup +} intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O{sub 2}{sup −} production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na{sup +}-mediated H{sup +}-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O{sub 2}{sup −} production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O{sub 2}{sup −} production induced by PLTX-mediated ionic imbalance. Indeed, the H{sup +} intracellular overload that follows PLTX-induced intracellular Na{sup +} accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O{sub 2}{sup −} production by reversing mitochondrial electron transport. Highlights: ► PLTX induces superoxide (O{sub 2}{sup −}) production by reversing mitochondrial transport chain. ► The mechanism of

  17. Anti-apoptotic BFL-1 is the major effector in activation-induced human mast cell survival.

    Directory of Open Access Journals (Sweden)

    Maria Ekoff

    Full Text Available Mast cells are best known for their role in allergic reactions, where aggregation of FcεRI leads to the release of mast cell mediators causing allergic symptoms. The activation also induces a survival program in the cells, i.e., activation-induced mast cell survival. The aim of the present study was to investigate how the activation-induced survival is mediated. Cord blood-derived mast cells and the mast cell line LAD-2 were activated through FcεRI crosslinking, with or without addition of chemicals that inhibit the activity or expression of selected Bcl-2 family members (ABT-737; roscovitine. Cell viability was assessed using staining and flow cytometry. The expression and function of Bcl-2 family members BFL-1 and MCL-1 were investigated using real-time quantitative PCR and siRNA treatment. The mast cell expression of Bfl-1 was investigated in skin biopsies. FcεRI crosslinking promotes activation-induced survival of human mast cells and this is associated with an upregulation of the anti-apoptotic Bcl-2 family member Bfl-1. ABT-737 alone or in combination with roscovitine decreases viability of human mast cells although activation-induced survival is sustained, indicating a minor role for Bcl-X(L, Bcl-2, Bcl-w and Mcl-1. Reducing BFL-1 but not MCL-1 levels by siRNA inhibited activation-induced mast cell survival. We also demonstrate that mast cell expression of Bfl-1 is elevated in birch-pollen-provocated skin and in lesions of atopic dermatitis and psoriasis patients. Taken together, our results highlight Bfl-1 as a major effector in activation-induced human mast cell survival.

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

    Science.gov (United States)

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

    2015-01-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor–Couette–Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis. PMID:25742953

  19. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    International Nuclear Information System (INIS)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-01-01

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1

  20. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zheng, Ruimao, E-mail: rmzheng@pku.edu.cn [Department of Anatomy, Histology and Embryology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China); Zhu, Shigong, E-mail: sgzhu@bjmu.edu.cn [Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University Health Science Center, Beijing (China)

    2014-07-18

    Highlights: • 14,15-EET inhibits OGD-induced apoptosis in cortical neurons. • Mitochondrial biogenesis of cortical neurons is promoted by 14,15-EET. • 14,15-EET preserves mitochondrial function of cortical neurons under OGD. • CREB mediates effect of 14,15-EET on mitochondrial biogenesis and function. - Abstract: 14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen–glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1.

  1. BIM-Mediated Membrane Insertion of the BAK Pore Domain Is an Essential Requirement for Apoptosis

    Directory of Open Access Journals (Sweden)

    Kathrin Weber

    2013-10-01

    Full Text Available BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models.

  2. Reperfusion promotes mitochondrial dysfunction following focal cerebral ischemia in rats.

    Directory of Open Access Journals (Sweden)

    Jun Li

    Full Text Available BACKGROUND AND PURPOSE: Mitochondrial dysfunction has been implicated in the cell death observed after cerebral ischemia, and several mechanisms for this dysfunction have been proposed. Reperfusion after transient cerebral ischemia may cause continued and even more severe damage to the brain. Many lines of evidence have shown that mitochondria suffer severe damage in response to ischemic injury. The purpose of this study was to observe the features of mitochondrial dysfunction in isolated mitochondria during the reperfusion period following focal cerebral ischemia. METHODS: Male Wistar rats were subjected to focal cerebral ischemia. Mitochondria were isolated using Percoll density gradient centrifugation. The isolated mitochondria were fixed for electron microscopic examination; calcium-induced mitochondrial swelling was quantified using spectrophotometry. Cyclophilin D was detected by Western blotting. Fluorescent probes were used to selectively stain mitochondria to measure their membrane potential and to measure reactive oxidative species production using flow cytometric analysis. RESULTS: Signs of damage were observed in the mitochondrial morphology after exposure to reperfusion. The mitochondrial swelling induced by Ca(2+ increased gradually with the increasing calcium concentration, and this tendency was exacerbated as the reperfusion time was extended. Cyclophilin D protein expression peaked after 24 hours of reperfusion. The mitochondrial membrane potential was decreased significantly during the reperfusion period, with the greatest decrease observed after 24 hours of reperfusion. The surge in mitochondrial reactive oxidative species occurred after 2 hours of reperfusion and was maintained at a high level during the reperfusion period. CONCLUSIONS: Reperfusion following focal cerebral ischemia induced significant mitochondrial morphological damage and Ca(2+-induced mitochondrial swelling. The mechanism of this swelling may be mediated by

  3. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by beta-amyloid peptide.

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, W E

    2010-05-01

    beta-Amyloid peptide (Abeta) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Abeta-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Abeta and on neurite outgrowth in PC12 cells were investigated. Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Abeta(1-42). Similar protective effects against Abeta(1-42) were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Abeta load was markedly diminished in the brain of those animals after treatment with piracetam. Abeta production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Abeta-induced mitochondrial dysfunction and Abeta-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Abeta on brain function.

  4. Inhibition of Bcl-2 potentiates AZD-2014-induced anti-head and neck squamous cell carcinoma cell activity

    International Nuclear Information System (INIS)

    Li, Yi; Cui, Jiang-Tao

    2016-01-01

    Mammalian target of rapamycin (mTOR) is a therapeutic target for head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the activity of AZD-2014, a potent mTOR complex 1/2 (mTORC1/2) dual inhibitor, against HNSCC cells. We showed that AZD-2014 blocked mTORC1/2 activation in established and primary human HNSCC cells, where it was anti-proliferative and pro-apoptotic. Yet, AZD-2014 was non-cytotoxic to the human oral epithelial cells with low basal mTORC1/2 activation. In an effect to identify possible AZD-2014 resistance factors, we showed that the anti-apoptosis protein Bcl-2 was upregulated in AZD-2014-resistant SQ20B HNSCC cells. Inhibition of Bcl-2 by ABT-737 (a known Bcl-2 inhibitor) or Bcl-2 shRNA dramatically potentiated AZD-2014 lethality against HNSCC cells. On the other hand, exogenous overexpression of Bcl-2 largely attenuated AZD-2014’s activity against HNSCC cells. For the in vivo studies, we showed that oral gavage of AZD-2014 suppressed SQ20B xenograft growth in severe combined immunodeficient (SCID) mice. It also significantly improved mice survival. Importantly, AZD-2014’s anti-HNSCC activity in vivo was potentiated with co-administration of ABT-737. The preclinical results of this study suggest that AZD-2014 could be further tested as a valuable anti-HNSCC agent, either alone or in combination with Bcl-2 inhibitors. - Highlights: • AZD-2014 blocks mTORC1/2 activation in HNSCC cells. • AZD-2014 suppresses HNSCC cell proliferation. • AZD-2014 activates caspase-3 and apoptosis in HNSCC cells. • Bcl-2 is the key resistance factor of AZD-2014 in HNSCC cells. • ABT-737 sensitizes AZD-2014-induced anti-HNSCC activity in vivo.

  5. Inhibition of Bcl-2 potentiates AZD-2014-induced anti-head and neck squamous cell carcinoma cell activity

    Energy Technology Data Exchange (ETDEWEB)

    Li, Yi; Cui, Jiang-Tao, E-mail: cuijingtaopaper@126.com

    2016-09-02

    Mammalian target of rapamycin (mTOR) is a therapeutic target for head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the activity of AZD-2014, a potent mTOR complex 1/2 (mTORC1/2) dual inhibitor, against HNSCC cells. We showed that AZD-2014 blocked mTORC1/2 activation in established and primary human HNSCC cells, where it was anti-proliferative and pro-apoptotic. Yet, AZD-2014 was non-cytotoxic to the human oral epithelial cells with low basal mTORC1/2 activation. In an effect to identify possible AZD-2014 resistance factors, we showed that the anti-apoptosis protein Bcl-2 was upregulated in AZD-2014-resistant SQ20B HNSCC cells. Inhibition of Bcl-2 by ABT-737 (a known Bcl-2 inhibitor) or Bcl-2 shRNA dramatically potentiated AZD-2014 lethality against HNSCC cells. On the other hand, exogenous overexpression of Bcl-2 largely attenuated AZD-2014’s activity against HNSCC cells. For the in vivo studies, we showed that oral gavage of AZD-2014 suppressed SQ20B xenograft growth in severe combined immunodeficient (SCID) mice. It also significantly improved mice survival. Importantly, AZD-2014’s anti-HNSCC activity in vivo was potentiated with co-administration of ABT-737. The preclinical results of this study suggest that AZD-2014 could be further tested as a valuable anti-HNSCC agent, either alone or in combination with Bcl-2 inhibitors. - Highlights: • AZD-2014 blocks mTORC1/2 activation in HNSCC cells. • AZD-2014 suppresses HNSCC cell proliferation. • AZD-2014 activates caspase-3 and apoptosis in HNSCC cells. • Bcl-2 is the key resistance factor of AZD-2014 in HNSCC cells. • ABT-737 sensitizes AZD-2014-induced anti-HNSCC activity in vivo.

  6. TanshinoneIIA and cryptotanshinone protect against hypoxia-induced mitochondrial apoptosis in H9c2 cells.

    Directory of Open Access Journals (Sweden)

    Hyou-Ju Jin

    Full Text Available Mitochondrial apoptosis pathway is an important target of cardioprotective signalling. Tanshinones, a group of major bioactive compounds isolated from Salvia miltiorrhiza, have been reported with actions against inflammation, oxidative stress, and myocardial ischemia reperfusion injury. However, the actions of these compounds on the chronic hypoxia-related mitochondrial apoptosis pathway have not been investigated. In this study, we examined the effects and molecular mechanisms of two major tanshonones, tanshinone IIA (TIIA and cryptotanshinone (CT on hypoxia induced apoptosis in H9c2 cells. Cultured H9c2 cells were treated with TIIA and CT (0.3 and 3 μΜ 2 hr before and during an 8 hr hypoxic period. Chronic hypoxia caused a significant increase in hypoxia inducible factor 1α expression and the cell late apoptosis rate, which was accompanied with an increase in caspase 3 activity, cytochrome c release, mitochondria membrane potential and expression of pro-apoptosis proteins (Bax and Bak. TIIA and CT (0.3 and 3 μΜ, in concentrations without affecting the cell viability, significantly inhibited the late apoptosis and the changes of caspase 3 activity, cytochrome c release, and mitochondria membrane potential induced by chronic hypoxia. These compounds also suppressed the overexpression of Bax and reduced the ratio of Bax/Bcl-2. The results indicate that TIIA and CT protect against chronic hypoxia induced cell apoptosis by regulating the mitochondrial apoptosis signaling pathway, involving inhibitions of mitochondria hyperpolarization, cytochrome c release and caspase 3 activity, and balancing anti- and pro-apoptotic proteins in Bcl-2 family proteins.

  7. Benzaldehyde thiosemicarbazone derived from limonene complexed with copper induced mitochondrial dysfunction in Leishmania amazonensis.

    Directory of Open Access Journals (Sweden)

    Elizandra Aparecida Britta

    Full Text Available BACKGROUND: Leishmaniasis is a major health problem that affects more than 12 million people. Treatment presents several problems, including high toxicity and many adverse effects, leading to the discontinuation of treatment and emergence of resistant strains. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the in vitro antileishmanial activity of benzaldehyde thiosemicarbazone derived from limonene complexed with copper, termed BenzCo, against Leishmania amazonensis. BenzCo inhibited the growth of the promastigote and axenic amastigote forms, with IC(50 concentrations of 3.8 and 9.5 µM, respectively, with 72 h of incubation. Intracellular amastigotes were inhibited by the compound, with an IC(50 of 10.7 µM. BenzCo altered the shape, size, and ultrastructure of the parasites. Mitochondrial membrane depolarization was observed in protozoa treated with BenzCo but caused no alterations in the plasma membrane. Additionally, BenzCo induced lipoperoxidation and the production of mitochondrial superoxide anion radicals in promastigotes and axenic amastigotes of Leishmania amazonensis. CONCLUSION/SIGNIFICANCE: Our studies indicated that the antileishmania activity of BenzCo might be associated with mitochondrial dysfunction and oxidative damage, leading to parasite death.

  8. ABT-773 (Abbott Laboratories).

    Science.gov (United States)

    Lawrence, L E

    2001-06-01

    ABT-773 is a macrolide antibacterial agent under development by Abbott Laboratories and Taisho Pharmaceutical Co Ltd for the potential treatment of bacterial infection [266579]. As of February 2001, ABT-773 had entered phase III trials in the US [398274]. Japanese phase II trials were expected to commence in June 2000 and a phase II trial is being designed for respiratory infections, with Abbott expecting filing in March 2002 [360455]. The bioavailability of ABT-773 in humans is unaffected by food [383228] and in a phase I, randomized, double-blind trial in healthy males only mild adverse effects, usually affecting the gastrointestinal system, were observed [383208]. Under an agreement, Abbott and Taisho are conducting joint research to discover new compounds; Abbott will have worldwide marketing, manufacturing and supply rights (except in Japan), and Taisho will receive royalties on Abbott's sales in consideration of granted rights. In Japan, the companies will co-market any resulting compounds [266579]. ABT-773 demonstrated good activity in vitro and in vivo against Streptococcus pneumoniae and Staphylococcus aureus [383229], [383231], and was highly potent even against macrolide-resistant [382149], [382150] and invasive [383782] S pneumoniae.

  9. Elevated hydrostatic pressures induce apoptosis and oxidative stress through mitochondrial membrane depolarization in PC12 neuronal cells: A cell culture model of glaucoma.

    Science.gov (United States)

    Tök, Levent; Nazıroğlu, Mustafa; Uğuz, Abdülhadi Cihangir; Tök, Ozlem

    2014-10-01

    Despite the importance of oxidative stress and apoptosis through mitochondrial depolarization in neurodegenerative diseases, their roles in etiology of glaucoma are poorly understood. We aimed to investigate whether oxidative stress and apoptosis formation are altered in rat pheochromocytoma-derived cell line-12 (PC12) neuronal cell cultures exposed to elevated different hydrostatic pressures as a cell culture model of glaucoma. Cultured PC12 cells were subjected to 0, 15 and 70 mmHg hydrostatic pressure for 1 and 24 h. Then, the following values were analyzed: (a) cell viability; (b) lipid peroxidation and intracellular reactive oxygen species production; (c) mitochondrial membrane depolarization; (d) cell apoptosis; (e) caspase-3 and caspase-9 activities; (f) reduced glutathione (GSH) and glutathione peroxidase (GSH-Px). The hydrostatic pressures (15 and 70 mmHg) increased oxidative cell damage through a decrease of GSH and GSH-Px values, and increasing mitochondrial membrane potential. Additionally, 70 mmHg hydrostatic pressure for 24 h indicated highest apoptotic effects, as demonstrated by plate reader analyses of apoptosis, caspase-3 and -9 values. The present data indicated oxidative stress, apoptosis and mitochondrial changes in PC12 cell line during different hydrostatic pressure as a cell culture model of glaucoma. This findings support the view that mitochondrial oxidative injury contributes early to glaucomatous optic neuropathy.

  10. Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

    International Nuclear Information System (INIS)

    Watanabe, Tomoyuki; Saotome, Masao; Nobuhara, Mamoru; Sakamoto, Atsushi; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi; Funaki, Makoto; Hayashi, Hideharu

    2014-01-01

    Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ m ) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H 2 O 2 ), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ m depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H 2 O 2 -induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ m depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin-resistance. • Inhibition of DRP or ROS

  11. Roles of mitochondrial fragmentation and reactive oxygen species in mitochondrial dysfunction and myocardial insulin resistance

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Tomoyuki [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Saotome, Masao, E-mail: msaotome@hama-med.ac.jp [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Nobuhara, Mamoru; Sakamoto, Atsushi; Urushida, Tsuyoshi; Katoh, Hideki; Satoh, Hiroshi [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan); Funaki, Makoto [Clinical Research Center for Diabetes, Tokushima University Hospital, 2-50-1 Kuramoto-cho, Tokushima 770-8503 (Japan); Hayashi, Hideharu [Internal Medicine III, Hamamatsu University School of Medicine, 1-20-1 Handayama, Higashi-ku, Hamamatsu 431-3192 (Japan)

    2014-05-01

    Purpose: Evidence suggests an association between aberrant mitochondrial dynamics and cardiac diseases. Because myocardial metabolic deficiency caused by insulin resistance plays a crucial role in heart disease, we investigated the role of dynamin-related protein-1 (DRP1; a mitochondrial fission protein) in the pathogenesis of myocardial insulin resistance. Methods and Results: DRP1-expressing H9c2 myocytes, which had fragmented mitochondria with mitochondrial membrane potential (ΔΨ{sub m}) depolarization, exhibited attenuated insulin signaling and 2-deoxy-D-glucose (2-DG) uptake, indicating insulin resistance. Treatment of the DRP1-expressing myocytes with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (TMPyP) significantly improved insulin resistance and mitochondrial dysfunction. When myocytes were exposed to hydrogen peroxide (H{sub 2}O{sub 2}), they increased DRP1 expression and mitochondrial fragmentation, resulting in ΔΨ{sub m} depolarization and insulin resistance. When DRP1 was suppressed by siRNA, H{sub 2}O{sub 2}-induced mitochondrial dysfunction and insulin resistance were restored. Our results suggest that a mutual enhancement between DRP1 and reactive oxygen species could induce mitochondrial dysfunction and myocardial insulin resistance. In palmitate-induced insulin-resistant myocytes, neither DRP1-suppression nor TMPyP restored the ΔΨ{sub m} depolarization and impaired 2-DG uptake, however they improved insulin signaling. Conclusions: A mutual enhancement between DRP1 and ROS could promote mitochondrial dysfunction and inhibition of insulin signal transduction. However, other mechanisms, including lipid metabolite-induced mitochondrial dysfunction, may be involved in palmitate-induced insulin resistance. - Highlights: • DRP1 promotes mitochondrial fragmentation and insulin-resistance. • A mutual enhancement between DRP1 and ROS ipromotes insulin-resistance. • Palmitate increases DRP1 expression and induces insulin

  12. Hypoxic human cancer cells are sensitized to BH-3 mimetic–induced apoptosis via downregulation of the Bcl-2 protein Mcl-1

    OpenAIRE

    Harrison, Luke R.E.; Micha, Dimitra; Brandenburg, Martin; Simpson, Kathryn L.; Morrow, Christopher J.; Denneny, Olive; Hodgkinson, Cassandra; Yunus, Zaira; Dempsey, Clare; Roberts, Darren; Blackhall, Fiona; Makin, Guy; Dive, Caroline

    2011-01-01

    Solid tumors contain hypoxic regions in which cancer cells are often resistant to chemotherapy-induced apoptotic cell death. Therapeutic strategies that specifically target hypoxic cells and promote apoptosis are particularly appealing, as few normal tissues experience hypoxia. We have found that the compound ABT-737, a Bcl-2 homology domain 3 (BH-3) mimetic, promotes apoptotic cell death in human colorectal carcinoma and small cell lung cancer cell lines exposed to hypoxia. This hypoxic indu...

  13. Application of ABTS radical cation for selective on-line detection of radical scavengers in HPLC eluates

    NARCIS (Netherlands)

    Koleva, [No Value; Niederlander, HAG; van Beek, TA

    2001-01-01

    The radical cation 2,2 ' -azinobis-(3 -ethylbenzothiazoline-6-sulfonate), (ABTS(.+)) was utilized in an on-line HPLC method for the detection of radical scavengers in complex matrixes. The HPLC-separated analytes react postcolumn with the preformed ABTS(.+), and the induced bleaching is detected as

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

    Science.gov (United States)

    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 Tice RR, Simeonov A, Austin CP, Xia M. 2015. Profiling of the Tox21 chemical collection for mitochondrial function to identify compounds that acutely decrease mitochondrial membrane potential. Environ Health Perspect 123:49–56; http://dx.doi.org/10.1289/ehp.1408642 PMID:25302578

  15. Piracetam improves mitochondrial dysfunction following oxidative stress

    Science.gov (United States)

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

    2005-01-01

    Mitochondrial dysfunction including decrease of mitochondrial membrane potential and reduced ATP production represents a common final pathway of many conditions associated with oxidative stress, for example, hypoxia, hypoglycemia, and aging. Since the cognition-improving effects of the standard nootropic piracetam are usually more pronounced under such pathological conditions and young healthy animals usually benefit little by piracetam, the effect of piracetam on mitochondrial dysfunction following oxidative stress was investigated using PC12 cells and dissociated brain cells of animals treated with piracetam. Piracetam treatment at concentrations between 100 and 1000 μM improved mitochondrial membrane potential and ATP production of PC12 cells following oxidative stress induced by sodium nitroprusside (SNP) and serum deprivation. Under conditions of mild serum deprivation, piracetam (500 μM) induced a nearly complete recovery of mitochondrial membrane potential and ATP levels. Piracetam also reduced caspase 9 activity after SNP treatment. Piracetam treatment (100–500 mg kg−1 daily) of mice was also associated with improved mitochondrial function in dissociated brain cells. Significant improvement was mainly seen in aged animals and only less in young animals. Moreover, the same treatment reduced antioxidant enzyme activities (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in aged mouse brain only, which are elevated as an adaptive response to the increased oxidative stress with aging. In conclusion, therapeutically relevant in vitro and in vivo concentrations of piracetam are able to improve mitochondrial dysfunction associated with oxidative stress and/or aging. Mitochondrial stabilization and protection might be an important mechanism to explain many of piracetam's beneficial effects in elderly patients. PMID:16284628

  16. Tempo enhances heat-induced apoptosis by mitochondrial targeting of Bax

    International Nuclear Information System (INIS)

    Zhao, Q.-L.; Fujiwara, Y.; Kondo, T.

    2003-01-01

    A stable membrane-permeable nitroxide, Tempo, exerts an SOD-like antioxidant activity against ROS. Reportedly, Tempo inhibits ROS-induced thymocyte apoptosis, while 10 mM Tempo activates JNK1 to induce apoptosis in breast cancer cells. We have observed that nontoxic 5 mM Tempo enhances suboptimal hyperthermia (44 deg C/10 min)-induced apoptosis in U937 cells. Here we report the enhancing mechanism, focusing on activation and targeting of Bax to mitochondria and cytochrome c release. Methods: U937 cells were treated with either Tempo (5 mM, 37 deg C/10 min), heating (44 deg C/10 min), or Tempo-plus-heating, washed and incubated for various times up to 6 h, until assessing apoptosis, mitochondrial potential (ΔΨ>), and amount of superoxide by flow cytometry using Annexin V-FITC/PI, TMRM, and dihydroethidium, respectively. Bax, Bcl-2 and Bcl-XL, and cytochrome c were detected by western blotting, activated Bax was by immunoprecipitation, and ATP was by a luciferase assay. Bax targeting to and cytochrome c release from mitochorndria were also detected immunocytochemically under fluorescent microscopy. Results and Discussion: Treatment of U937 cells with 5 mM Tempo for 10 min at 37 deg C or suboptimal heating (44 deg C/ 10 min) alone did not induce apoptosis. The combined treatment with 5 mM Tempo and 44 deg C for 10 min dramatically induced ∼90% apoptosis in 6 h, as did the 44 deg C/30 min heating. During the enhanced apoptosis, cytochrome c release progressed. Although signals of Bcl-2, Bcl-XL and Bax in cell lysates were not altered, Bax was specifically activated and translocated to mitochondria after the combined treatment. Further, loss of ΔΨ>and decreases in superoxide and ATP progressed after the combined treatment, suggesting that the treatment may disturb mitochondrial electron transport. Thus, Tempo sensitizes the heat-induced apoptosis through (1) targeting of Bax to mitochondria and releasing cytochrome c, and (2) mitochondrial dysfunction

  17. Zinc and calcium alter the relationship between mitochondrial respiration, ROS and membrane potential in rainbow trout (Oncorhynchus mykiss) liver mitochondria.

    Science.gov (United States)

    Sharaf, Mahmoud S; Stevens, Don; Kamunde, Collins

    2017-08-01

    At excess levels, zinc (Zn) disrupts mitochondrial functional integrity and induces oxidative stress in aquatic organisms. Although much is known about the modulation of Zn toxicity by calcium (Ca) in fish, their interactions at the mitochondrial level have scarcely been investigated. Here we assessed the individual and combined effects of Zn and Ca on the relationship between mitochondrial respiration, ROS and membrane potential (ΔΨ mt ) in rainbow trout liver mitochondria. We tested if cation uptake through the mitochondrial calcium uniporter (MCU) is a prerequisite for Zn- and/or Ca-induced alteration of mitochondrial function. Furthermore, using our recently developed real-time multi-parametric method, we investigated the changes in respiration, ΔΨ mt , and reactive oxygen species (ROS, as hydrogen peroxide (H 2 O 2 )) release associated with Ca-induced mitochondrial depolarization imposed by transient and permanent openings of the mitochondrial permeability transition pore (mPTP). We found that independent of the MCU, Zn precipitated an immediate depolarization of the ΔΨ mt that was associated with relatively slow enhancement of H 2 O 2 release, inhibition of respiration and reversal of the positive correlation between ROS and ΔΨ mt . In contrast, an equitoxic dose of Ca caused transient depolarization, and stimulation of both respiration and H 2 O 2 release, effects that were completely abolished when the MCU was blocked. Contrary to our expectation that mitochondrial transition ROS Spike (mTRS) would be sensitive to both Zn and Ca, only Ca suppressed it. Moreover, Zn and Ca in combination immediately depolarized the ΔΨ mt , and caused transient and sustained stimulation of respiration and H 2 O 2 release, respectively. Lastly, we uncovered and characterized an mPTP-independent Ca-induced depolarization spike that was associated with exposure to moderately elevated levels of Ca. Importantly, we showed the stimulation of ROS release associated with

  18. Involvment of cytosolic and mitochondrial GSK-3beta in mitochondrial dysfunction and neuronal cell death of MPTP/MPP-treated neurons.

    Directory of Open Access Journals (Sweden)

    Agnès Petit-Paitel

    Full Text Available Aberrant mitochondrial function appears to play a central role in dopaminergic neuronal loss in Parkinson's disease (PD. 1-methyl-4-phenylpyridinium iodide (MPP(+, the active metabolite of N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP, is a selective inhibitor of mitochondrial complex I and is widely used in rodent and cell models to elicit neurochemical alterations associated with PD. Recent findings suggest that Glycogen Synthase Kinase-3beta (GSK-3beta, a critical activator of neuronal apoptosis, is involved in the dopaminergic cell death. In this study, the role of GSK-3beta in modulating MPP(+-induced mitochondrial dysfunction and neuronal death was examined in vivo, and in two neuronal cell models namely primary cultured and immortalized neurons. In both cell models, MPTP/MPP(+ treatment caused cell death associated with time- and concentration-dependent activation of GSK-3beta, evidenced by the increased level of the active form of the kinase, i.e. GSK-3beta phosphorylated at tyrosine 216 residue. Using immunocytochemistry and subcellular fractionation techniques, we showed that GSK-3beta partially localized within mitochondria in both neuronal cell models. Moreover, MPP(+ treatment induced a significant decrease of the specific phospho-Tyr216-GSK-3beta labeling in mitochondria concomitantly with an increase into the cytosol. Using two distinct fluorescent probes, we showed that MPP(+ induced cell death through the depolarization of mitochondrial membrane potential. Inhibition of GSK-3beta activity using well-characterized inhibitors, LiCl and kenpaullone, and RNA interference, prevented MPP(+-induced cell death by blocking mitochondrial membrane potential changes and subsequent caspase-9 and -3 activation. These results indicate that GSK-3beta is a critical mediator of MPTP/MPP(+-induced neurotoxicity through its ability to regulate mitochondrial functions. Inhibition of GSK-3beta activity might provide protection against

  19. Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress

    Energy Technology Data Exchange (ETDEWEB)

    Muhsain, Siti Nur Fadzilah, E-mail: sitinurfadzilah077@ppinang.uitm.edu.my [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Faculty of Pharmacy, University Teknologi Mara (Malaysia); Lang, Matti A., E-mail: m.lang@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

    2015-01-01

    The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200 mg pyrazole/kg/day for 3 days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. - Highlights: • Pyrazole induces oxidative stress in the mouse liver. • Pyrazole-induced oxidative stress induces mitochondrial targeting of key bilirubin regulatory enzymes, HMOX1

  20. Mitochondrial targeting of bilirubin regulatory enzymes: An adaptive response to oxidative stress

    International Nuclear Information System (INIS)

    Muhsain, Siti Nur Fadzilah; Lang, Matti A.; Abu-Bakar, A'edah

    2015-01-01

    The intracellular level of bilirubin (BR), an endogenous antioxidant that is cytotoxic at high concentrations, is tightly controlled within the optimal therapeutic range. We have recently described a concerted intracellular BR regulation by two microsomal enzymes: heme oxygenase 1 (HMOX1), essential for BR production and cytochrome P450 2A5 (CYP2A5), a BR oxidase. Herein, we describe targeting of these enzymes to hepatic mitochondria during oxidative stress. The kinetics of microsomal and mitochondrial BR oxidation were compared. Treatment of DBA/2J mice with 200 mg pyrazole/kg/day for 3 days increased hepatic intracellular protein carbonyl content and induced nucleo-translocation of Nrf2. HMOX1 and CYP2A5 proteins and activities were elevated in microsomes and mitoplasts but not the UGT1A1, a catalyst of BR glucuronidation. A CYP2A5 antibody inhibited 75% of microsomal BR oxidation. The inhibition was absent in control mitoplasts but elevated to 50% after treatment. An adrenodoxin reductase antibody did not inhibit microsomal BR oxidation but inhibited 50% of mitochondrial BR oxidation. Ascorbic acid inhibited 5% and 22% of the reaction in control and treated microsomes, respectively. In control mitoplasts the inhibition was 100%, which was reduced to 50% after treatment. Bilirubin affinity to mitochondrial and microsomal CYP2A5 enzyme is equally high. Lastly, the treatment neither released cytochrome c into cytoplasm nor dissipated membrane potential, indicating the absence of mitochondrial membrane damage. Collectively, the observations suggest that BR regulatory enzymes are recruited to mitochondria during oxidative stress and BR oxidation by mitochondrial CYP2A5 is supported by mitochondrial mono-oxygenase system. The induced recruitment potentially confers membrane protection. - Highlights: • Pyrazole induces oxidative stress in the mouse liver. • Pyrazole-induced oxidative stress induces mitochondrial targeting of key bilirubin regulatory enzymes, HMOX1

  1. 47 CFR 74.737 - Antenna location.

    Science.gov (United States)

    2010-10-01

    ... 47 Telecommunication 4 2010-10-01 2010-10-01 false Antenna location. 74.737 Section 74.737 Telecommunication FEDERAL COMMUNICATIONS COMMISSION (CONTINUED) BROADCAST RADIO SERVICES EXPERIMENTAL RADIO... Booster Stations § 74.737 Antenna location. (a) An applicant for a new low power TV, TV translator, or TV...

  2. Mitochondrial Reactive Oxygen Species (ROS) and ROS-Induced ROS Release

    Science.gov (United States)

    Zorov, Dmitry B.; Juhaszova, Magdalena; Sollott, Steven J.

    2014-01-01

    Byproducts of normal mitochondrial metabolism and homeostasis include the buildup of potentially damaging levels of reactive oxygen species (ROS), Ca2+, etc., which must be normalized. Evidence suggests that brief mitochondrial permeability transition pore (mPTP) openings play an important physiological role maintaining healthy mitochondria homeostasis. Adaptive and maladaptive responses to redox stress may involve mitochondrial channels such as mPTP and inner membrane anion channel (IMAC). Their activation causes intra- and intermitochondrial redox-environment changes leading to ROS release. This regenerative cycle of mitochondrial ROS formation and release was named ROS-induced ROS release (RIRR). Brief, reversible mPTP opening-associated ROS release apparently constitutes an adaptive housekeeping function by the timely release from mitochondria of accumulated potentially toxic levels of ROS (and Ca2+). At higher ROS levels, longer mPTP openings may release a ROS burst leading to destruction of mitochondria, and if propagated from mitochondrion to mitochondrion, of the cell itself. The destructive function of RIRR may serve a physiological role by removal of unwanted cells or damaged mitochondria, or cause the pathological elimination of vital and essential mitochondria and cells. The adaptive release of sufficient ROS into the vicinity of mitochondria may also activate local pools of redox-sensitive enzymes involved in protective signaling pathways that limit ischemic damage to mitochondria and cells in that area. Maladaptive mPTP- or IMAC-related RIRR may also be playing a role in aging. Because the mechanism of mitochondrial RIRR highlights the central role of mitochondria-formed ROS, we discuss all of the known ROS-producing sites (shown in vitro) and their relevance to the mitochondrial ROS production in vivo. PMID:24987008

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

    Science.gov (United States)

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

    2006-01-01

    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. PMID:17060615

  4. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2017-05-01

    Full Text Available Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz. 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH, apoptosis analysis by annexin V and propidium iodide (PI double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM and transmission electron microscope (TEM, mitochondrial transmembrane potential (ΔΨm assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly (p < 0.05 release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 (p < 0.01 and casepase-3 (p < 0.05 levels, significantly (p < 0.01 increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of

  5. Nocardia cyriacigeogica from Bovine Mastitis Induced In vitro Apoptosis of Bovine Mammary Epithelial Cells via Activation of Mitochondrial-Caspase Pathway.

    Science.gov (United States)

    Chen, Wei; Liu, Yongxia; Zhang, Limei; Gu, Xiaolong; Liu, Gang; Shahid, Muhammad; Gao, Jian; Ali, Tariq; Han, Bo

    2017-01-01

    Nocardia is one of the causing agents of bovine mastitis and increasing prevalence of nocardial mastitis in shape of serious outbreaks has been reported from many countries. However, the mechanisms by which this pathogen damages the bovine mammary epithelial cells (bMECs) is not yet studied. Therefore, this study was designed with the aim to evaluate the apoptotic effects elicited by Nocardia and to investigate the pathway by which the Nocardia induce apoptosis in bMECs. Clinical Nocardia cyriacigeorgica strain from bovine mastitis was used to infect the bMECs for different time intervals, viz . 1, 3, 6, 12, and 18 h, and then the induced effects on bMECs were studied using adhesion and invasion assays, release of lactate dehydrogenase (LDH), apoptosis analysis by annexin V and propidium iodide (PI) double staining, morphological, and ultrastructural observations under scanning electron microscope (SEM) and transmission electron microscope (TEM), mitochondrial transmembrane potential (ΔΨm) assay using flow cytometry, and the protein quantification of mitochondrial cytochrome c and caspase-9 and caspase-3 by western blotting. The results of this study showed that N. cyriacigeorgica possessed the abilities of adhesion and invasion to bMECs. N. cyriacigeorgica was found to collapse mitochondrial transmembrane potential, significantly ( p < 0.05) release mitochondrial cytochrome c and ultimately induce cell apoptosis. Additionally, it promoted casepase-9 ( p < 0.01) and casepase-3 ( p < 0.05) levels, significantly ( p < 0.01) increased the release of LDH and promoted DNA fragmentation which further confirmed the apoptosis. Furthermore, N. cyriacigeorgica induced apoptosis/necrosis manifested specific ultrastructure features under TEM, such as swollen endoplasmic reticulum, cristae degeneration, and swelling of mitochondria, vesicle formation on the cell surface, rupturing of cell membrane and nuclear membrane, clumping, fragmentation, and margination of chromatin

  6. The Acetyl Group Buffering Action of Carnitine Acetyltransferase Offsets Macronutrient-Induced Lysine Acetylation of Mitochondrial Proteins

    Directory of Open Access Journals (Sweden)

    Michael N. Davies

    2016-01-01

    Full Text Available Lysine acetylation (AcK, a posttranslational modification wherein a two-carbon acetyl group binds covalently to a lysine residue, occurs prominently on mitochondrial proteins and has been linked to metabolic dysfunction. An emergent theory suggests mitochondrial AcK occurs via mass action rather than targeted catalysis. To test this hypothesis, we performed mass spectrometry-based acetylproteomic analyses of quadriceps muscles from mice with skeletal muscle-specific deficiency of carnitine acetyltransferase (CrAT, an enzyme that buffers the mitochondrial acetyl-CoA pool by converting short-chain acyl-CoAs to their membrane permeant acylcarnitine counterparts. CrAT deficiency increased tissue acetyl-CoA levels and susceptibility to diet-induced AcK of broad-ranging mitochondrial proteins, coincident with diminished whole body glucose control. Sub-compartment acetylproteome analyses of muscles from obese mice and humans showed remarkable overrepresentation of mitochondrial matrix proteins. These findings reveal roles for CrAT and L-carnitine in modulating the muscle acetylproteome and provide strong experimental evidence favoring the nonenzymatic carbon pressure model of mitochondrial AcK.

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Benjamin Clémençon

    2012-02-01

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

  9. S-52, a novel nootropic compound, protects against β-amyloid induced neuronal injury by attenuating mitochondrial dysfunction.

    Science.gov (United States)

    Gao, Xin; Zheng, Chun Yan; Qin, Guo Wei; Tang, Xi Can; Zhang, Hai Yan

    2012-10-01

    Accumulating evidence suggests that β-amyloid (Aβ)-induced oxidative DNA damage and mitochondrial dysfunction may initiate and contribute to the progression of Alzheimer's disease (AD). This study evaluated the neuroprotective effects of S-52, a novel nootropic compound, on Aβ-induced mitochondrial failure. In an established paradigm of moderate cellular injury induced by Aβ, S-52 was observed to attenuate the toxicity of Aβ to energy metabolism, mitochondrial membrane structure, and key enzymes in the electron transport chain and tricarboxylic acid cycle. In addition, S-52 also effectively inhibited reactive oxygen species accumulation dose dependently not only in Aβ-harmed cells but also in unharmed, normal cells. The role of S-52 as a scavenger of free radicals is involved in the antioxidative effect of this compound. The beneficial effects on mitochondria and oxidative stress extend the neuroprotective effects of S-52. The present study provides crucial information for better understanding the beneficial profiles of this compound and discovering novel potential drug candidates for AD therapy. Copyright © 2012 Wiley Periodicals, Inc.

  10. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells

    Directory of Open Access Journals (Sweden)

    Gábor J. Szebeni

    2017-10-01

    Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  11. Achiral Mannich-Base Curcumin Analogs Induce Unfolded Protein Response and Mitochondrial Membrane Depolarization in PANC-1 Cells.

    Science.gov (United States)

    Szebeni, Gábor J; Balázs, Árpád; Madarász, Ildikó; Pócz, Gábor; Ayaydin, Ferhan; Kanizsai, Iván; Fajka-Boja, Roberta; Alföldi, Róbert; Hackler, László; Puskás, László G

    2017-10-07

    Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G₀/G₁ cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5 , ATF4, XBP1 , and DDIT3 . The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.

  12. [Relationship between mitochondrial DNA copy number, membrane potential of human embryo and embryo morphology].

    Science.gov (United States)

    Zhao, H; Teng, X M; Li, Y F

    2017-11-25

    Objective: To explore the relationship between the embryo with the different morphological types in the third day and its mitochondrial copy number, the membrane potential. Methods: Totally 117 embryos with poor development after normal fertilization and were not suitable transferred in the fresh cycle and 106 frozen embryos that were discarded voluntarily by infertility patients with in vitro fertilization-embryo transfer after successful pregnancy were selected. According to evaluation of international standard in embryos, all cleavage stage embryos were divided into class Ⅰ frozen embryo group ( n= 64), class Ⅱ frozen embryo group ( n= 42) and class Ⅲ fresh embryonic group (not transplanted embryos; n= 117). Real-time PCR and confocal microscopy methods were used to detect mitochondrial DNA (mtDNA) copy number and the mitochondrial membrane potential of a single embryo. The differences between embryo quality and mtDNA copy number and membrane potential of each group were compared. Results: The copy number of mtDNA and the mitochondrial membrane potential in class Ⅲ fresh embryonic group [(1.7±1.0)×10(5) copy/μl, 1.56±0.32] were significantly lower than those in class Ⅰ frozen embryo group [(3.4±1.7)×10(5) copy/μl, 2.66±0.21] and class Ⅱ frozen embryo group [(2.6±1.2)×10(5) copy/μl, 1.80±0.32; all Pembryo group were significantly higher than those in classⅡ frozen embryo group (both Pembryos of the better quality embryo are higher.

  13. Oxidative stress induces mitochondrial fragmentation in frataxin-deficient cells

    Energy Technology Data Exchange (ETDEWEB)

    Lefevre, Sophie [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); ED515 UPMC, 4 place Jussieu 75005 Paris (France); Sliwa, Dominika [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); Rustin, Pierre [Inserm, U676, Physiopathology and Therapy of Mitochondrial Disease Laboratory, 75019 Paris (France); Universite Paris-Diderot, Faculte de Medecine Denis Diderot, IFR02 Paris (France); Camadro, Jean-Michel [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France); Santos, Renata, E-mail: santos.renata@ijm.univ-paris-diderot.fr [Mitochondria, Metals and Oxidative Stress Laboratory, Institut Jacques Monod, CNRS-Universite Paris-Diderot, Sorbonne Paris Cite, 15 rue Helene Brion, 75205 Paris cedex 13 (France)

    2012-02-10

    Highlights: Black-Right-Pointing-Pointer Yeast frataxin-deficiency leads to increased proportion of fragmented mitochondria. Black-Right-Pointing-Pointer Oxidative stress induces complete mitochondrial fragmentation in {Delta}yfh1 cells. Black-Right-Pointing-Pointer Oxidative stress increases mitochondrial fragmentation in patient fibroblasts. Black-Right-Pointing-Pointer Inhibition of mitochondrial fission in {Delta}yfh1 induces oxidative stress resistance. -- Abstract: Friedreich ataxia (FA) is the most common recessive neurodegenerative disease. It is caused by deficiency in mitochondrial frataxin, which participates in iron-sulfur cluster assembly. Yeast cells lacking frataxin ({Delta}yfh1 mutant) showed an increased proportion of fragmented mitochondria compared to wild-type. In addition, oxidative stress induced complete fragmentation of mitochondria in {Delta}yfh1 cells. Genetically controlled inhibition of mitochondrial fission in these cells led to increased resistance to oxidative stress. Here we present evidence that in yeast frataxin-deficiency interferes with mitochondrial dynamics, which might therefore be relevant for the pathophysiology of FA.

  14. p53 represses autophagy in a cell cycle-dependent fashion.

    Science.gov (United States)

    Tasdemir, Ezgi; Maiuri, Maria Chiara; Orhon, Idil; Kepp, Oliver; Morselli, Eugenia; Criollo, Alfredo; Kroemer, Guido

    2008-10-01

    Autophagy is one of the principal mechanisms of cellular defense against nutrient depletion and damage to cytoplasmic organelles. When p53 is inhibited by a pharmacological antagonist (cyclic pifithrin-alpha), depleted by a specific small interfering RNA (siRNA) or deleted by homologous recombination, multiple signs of autophagy are induced. Here, we show by epistatic analysis that p53 inhibition results in a maximum level of autophagy that cannot be further enhanced by a variety of different autophagy inducers including lithium, tunicamycin-induced stress of the endoplasmic reticulum (ER) or inhibition of Bcl-2 and Bcl-X(L) with the BH3 mimetic ABT737. Chemical inducers of autophagy (including rapamycin, lithium, tunicamycin and ABT737) induced rapid depletion of the p53 protein. The absence or the inhibition of p53 caused autophagy mostly in the G(1) phase, less so in the S phase and spares the G(2)/M phase of the cell cycle. The possible pathophysiological implications of these findings are discussed.

  15. 14,15-EET promotes mitochondrial biogenesis and protects cortical neurons against oxygen/glucose deprivation-induced apoptosis.

    Science.gov (United States)

    Wang, Lai; Chen, Man; Yuan, Lin; Xiang, Yuting; Zheng, Ruimao; Zhu, Shigong

    2014-07-18

    14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen-glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1. Copyright © 2014 Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Anke Schloesser

    2015-01-01

    Full Text Available 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.

  17. ER-mediated stress induces mitochondrial-dependent caspases activation in NT2 neuron-like cells.

    Science.gov (United States)

    Arduino, Daniela M; Esteves, A Raquel; Domingues, A Filipa; Pereira, Claudia M F; Cardoso, Sandra M; Oliveira, Catarina R

    2009-11-30

    Recent studies have revealed that endoplasmic reticulum (ER) disturbance is involved in the pathophysiology of neurodegenerative disorders, contributing to the activation of the ER stress-mediated apoptotic pathway. Therefore, we investigated here the molecular mechanisms underlying the ER-mitochondria axis, focusing on calcium as a potential mediator of cell death signals. Using NT2 cells treated with brefeldin A or tunicamycin, we observed that ER stress induces changes in the mitochondrial function, impairing mitochondrial membrane potential and distressing mitochondrial respiratory chain complex Moreover, stress stimuli at ER level evoked calcium fluxes between ER and mitochondria. Under these conditions, ER stress activated the unfolded protein response by an overexpression of GRP78, and also caspase-4 and-2, both involved upstream of caspase-9. Our findings show that ER and mitochondria interconnection plays a prominent role in the induction of neuronal cell death under particular stress circumstances.

  18. Aqueous Fraction of Nephelium ramboutan-ake Rind Induces Mitochondrial-Mediated Apoptosis in HT-29 Human Colorectal Adenocarcinoma Cells

    Directory of Open Access Journals (Sweden)

    Muhamad Noor Alfarizal Kamarudin

    2012-05-01

    Full Text Available The aim of this study was to investigate the cytotoxic and apoptotic effects of Nephelium ramboutan-ake (pulasan rind in selected human cancer cell lines. The crude ethanol extract and fractions (ethyl acetate and aqueous of N. ramboutan-ake inhibited the growth of HT-29, HCT-116, MDA-MB-231, Ca Ski cells according to MTT assays. The N. ramboutan-ake aqueous fraction (NRAF was found to exert the greatest cytotoxic effect against HT-29 in a dose-dependent manner. Evidence of apoptotic cell death was revealed by features such as chromatin condensation, nuclear fragmentation and apoptotic body formation. The result from a TUNEL assay strongly suggested that NRAF brings about DNA fragmentation in HT-29 cells. Phosphatidylserine (PS externalization on the outer leaflet of plasma membranes was detected with annexin V-FITC/PI binding, confirming the early stage of apoptosis. The mitochondrial permeability transition is an important step in the induction of cellular apoptosis, and the results clearly suggested that NRAF led to collapse of mitochondrial transmembrane potential in HT-29 cells. This attenuation of mitochondrial membrane potential (Δψm was accompanied by increased production of ROS and depletion of GSH, an increase of Bax protein expression, and induced-activation of caspase-3/7 and caspase-9. These combined results suggest that NRAF induces mitochondrial-mediated apoptosis.

  19. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Chunhua [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China); Ma, Xa; Shi, Shangshi [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Zhao, Jianya; Nie, Xiaoke [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Han, Jingling; Xiao, Jing; Wang, Xiaoke [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiang, Shengyang [Department of Nutrition and Food Hygiene, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China); Jiang, Junkang, E-mail: Jiang_junkang@163.com [Department of Occupational Medicine and Environmental Toxicology, School of Public Health, Nantong University, Nantong 226019 Jiangsu (China); Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong 226019 Jiangsu (China)

    2014-12-15

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H{sub 2}O{sub 2} production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death. - Highlights: • p53 is

  20. Pivotal roles of p53 transcription-dependent and -independent pathways in manganese-induced mitochondrial dysfunction and neuronal apoptosis

    International Nuclear Information System (INIS)

    Wan, Chunhua; Ma, Xa; Shi, Shangshi; Zhao, Jianya; Nie, Xiaoke; Han, Jingling; Xiao, Jing; Wang, Xiaoke; Jiang, Shengyang; Jiang, Junkang

    2014-01-01

    Chronic exposure to excessive manganese (Mn) has been known to lead to neuronal loss and a clinical syndrome resembling idiopathic Parkinson's disease (IPD). p53 plays an integral role in the development of various human diseases, including neurodegenerative disorders. However, the role of p53 in Mn-induced neuronal apoptosis and neurological deficits remains obscure. In the present study, we showed that p53 was critically involved in Mn-induced neuronal apoptosis in rat striatum through both transcription-dependent and -independent mechanisms. Western blot and immunohistochemistrical analyses revealed that p53 was remarkably upregulated in the striatum of rats following Mn exposure. Coincidentally, increased level of cleaved PARP, a hallmark of apoptosis, was observed. Furthermore, using nerve growth factor (NGF)-differentiated PC12 cells as a neuronal cell model, we showed that Mn exposure decreased cell viability and induced apparent apoptosis. Importantly, p53 was progressively upregulated, and accumulated in both the nucleus and the cytoplasm. The cytoplasmic p53 had a remarkable distribution in mitochondria, suggesting an involvement of p53 mitochondrial translocation in Mn-induced neuronal apoptosis. In addition, Mn-induced impairment of mitochondrial membrane potential (ΔΨm) could be partially rescued by pretreatment with inhibitors of p53 transcriptional activity and p53 mitochondrial translocation, Pifithrin-α (PFT-α) and Pifithrin-μ (PFT-μ), respectively. Moreover, blockage of p53 activities with PFT-α and PFT-μ significantly attenuated Mn-induced reactive oxidative stress (ROS) generation and mitochondrial H 2 O 2 production. Finally, we observed that pretreatment with PFT-α and PFT-μ ameliorated Mn-induced apoptosis in PC12 cells. Collectively, these findings implicate that p53 transcription-dependent and -independent pathways may play crucial roles in the regulation of Mn-induced neuronal death. - Highlights: • p53 is robustly

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

    Science.gov (United States)

    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.

  2. Cr(VI) induces mitochondrial-mediated and caspase-dependent apoptosis through reactive oxygen species-mediated p53 activation in JB6 Cl41 cells

    International Nuclear Information System (INIS)

    Son, Young-Ok; Hitron, J. Andrew; Wang Xin; Chang Qingshan; Pan Jingju; Zhang Zhuo; Liu Jiankang; Wang Shuxia; Lee, Jeong-Chae; Shi Xianglin

    2010-01-01

    Cr(VI) compounds are known to cause serious toxic and carcinogenic effects. Cr(VI) exposure can lead to a severe damage to the skin, but the mechanisms involved in the Cr(VI)-mediated toxicity in the skin are unclear. The present study examined whether Cr(VI) induces cell death by apoptosis or necrosis using mouse skin epidermal cell line, JB6 Cl41 cells. We also investigated the cellular mechanisms of Cr(VI)-induced cell death. This study showed that Cr(VI) induced apoptotic cell death in a dose-dependent manner, as demonstrated by the appearance of cell shrinkage, the migration of cells into the sub-G1 phase, the increase of Annexin V positively stained cells, and the formation of nuclear DNA ladders. Cr(VI) treatment resulted in the increases of mitochondrial membrane depolarization and caspases activation. Electron spin resonance (ESR) and fluorescence analysis revealed that Cr(VI) increased intracellular levels of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide anion radical in dose-dependent manner. Blockage of p53 by si-RNA transfection suppressed mitochondrial changes of Bcl-2 family composition, mitochondrial membrane depolarization, caspase activation and PARP cleavage, leading to the inhibition of Cr(VI)-induced apoptosis. Further, catalase treatment prevented p53 phosphorylation stimulated by Cr(VI) with the concomitant inhibition of caspase activation. These results suggest that Cr(VI) induced a mitochondrial-mediated and caspase-dependent apoptosis in skin epidermal cells through activation of p53, which are mainly mediated by reactive oxidants generated by the chemical.

  3. L-carnitine protects against nickel-induced neurotoxicity by maintaining mitochondrial function in Neuro-2a cells

    International Nuclear Information System (INIS)

    He Mindi; Xu Shangcheng; Lu Yonghui; Li Li; Zhong Min; Zhang Yanwen; Wang Yuan; Li Min; Yang Ju; Zhang Guangbin; Yu Zhengping; Zhou Zhou

    2011-01-01

    Mitochondrial dysfunction is thought to be a part of the mechanism underlying nickel-induced neurotoxicity. L-carnitine (LC), a quaternary ammonium compound biosynthesized from the amino acids lysine and methionine in all mammalian species, manifests its neuroprotective effects by improving mitochondrial energetics and function. The purpose of this study was to investigate whether LC could efficiently protect against nickel-induced neurotoxicity. Here, we exposed a mouse neuroblastoma cell line (Neuro-2a) to different concentrations of nickel chloride (NiCl 2 ) (0.25, 0.5, 1, and 2 mM) for 24 h, or to 0.5 mM and 1 mM NiCl 2 for various periods (0, 3, 6, 12, or 24 h). We found that nickel significantly increased the cell viability loss and lactate dehydrogenase (LDH) release in Neuro-2a cells. In addition, nickel exposure significantly elevated reactive oxygen species (ROS) and malondialdehyde (MDA) levels, disrupted the mitochondrial membrane potential (ΔΨ m ), reduced adenosine-5'-triphosphate (ATP) concentrations and decreased mitochondrial DNA (mtDNA) copy numbers and mtRNA transcript levels. However, all of the cytotoxicities and mitochondrial dysfunctions that were triggered by nickel were efficiently attenuated by pretreatment with LC. These protective effects of LC may be attributable to its role in maintaining mitochondrial function in nickel-treated cells. Our results suggest that LC may have great pharmacological potential in protecting against the adverse effects of nickel in the nervous system.

  4. Proteasomal Dysfunction Induced By Diclofenac Engenders Apoptosis Through Mitochondrial Pathway.

    Science.gov (United States)

    Amanullah, Ayeman; Upadhyay, Arun; Chhangani, Deepak; Joshi, Vibhuti; Mishra, Ribhav; Yamanaka, Koji; Mishra, Amit

    2017-05-01

    Diclofenac is the most commonly used phenylacetic acid derivative non-steroidal anti-inflammatory drug (NSAID) that demonstrates significant analgesic, antipyretic, and anti-inflammatory effects. Several epidemiological studies have demonstrated anti-proliferative activity of NSAIDs and examined their apoptotic induction effects in different cancer cell lines. However, the precise molecular mechanisms by which these pharmacological agents induce apoptosis and exert anti-carcinogenic properties are not well known. Here, we have observed that diclofenac treatment induces proteasome malfunction and promotes accumulation of different critical proteasome substrates, including few pro-apoptotic proteins in cells. Exposure of diclofenac consequently elevates aggregation of various ubiquitylated misfolded proteins. Finally, we have shown that diclofenac treatment promotes apoptosis in cells, which could be because of mitochondrial membrane depolarization and cytochrome c release into cytosol. This study suggests possible beneficial insights of NSAIDs-induced apoptosis that may improve our existing knowledge in anti-proliferative interspecific strategies development. J. Cell. Biochem. 118: 1014-1027, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Characterization of the respiration-induced yeast mitochondrial permeability transition pore.

    Science.gov (United States)

    Bradshaw, Patrick C; Pfeiffer, Douglas R

    2013-12-01

    When isolated mitochondria from the yeast Saccharomyces cerevisiae oxidize respiratory substrates in the absence of phosphate and ADP, the yeast mitochondrial unselective channel, also called the yeast permeability transition pore (yPTP), opens in the inner membrane, dissipating the electrochemical gradient. ATP also induces yPTP opening. yPTP opening allows mannitol transport into isolated mitochondria of laboratory yeast strains, but mannitol is not readily permeable through the yPTP in an industrial yeast strain, Yeast Foam. The presence of oligomycin, an inhibitor of ATP synthase, allowed for respiration-induced mannitol permeability in mitochondria from this strain. Potassium (K+) had varied effects on the respiration-induced yPTP, depending on the concentration of the respiratory substrate added. At low respiratory substrate concentrations K+ inhibited respiration-induced yPTP opening, while at high substrate concentrations this effect diminished. However, at the high respiratory substrate concentrations, the presence of K+ partially prevented phosphate inhibition of yPTP opening. Phosphate was found to inhibit respiration-induced yPTP opening by binding a site on the matrix space side of the inner membrane in addition to its known inhibitory effect of donating protons to the matrix space to prevent the pH change necessary for yPTP opening. The respiration-induced yPTP was also inhibited by NAD, Mg2+, NH4 + or the oxyanion vanadate polymerized to decavanadate. The results demonstrate similar effectors of the respiration-induced yPTP as those previously described for the ATP-induced yPTP and reconcile previous strain-dependent differences in yPTP solute selectivity. Copyright © 2013 John Wiley & Sons, Ltd.

  6. PGC-1α-Dependent Mitochondrial Adaptation Is Necessary to Sustain IL-2-Induced Activities in Human NK Cells.

    Science.gov (United States)

    Miranda, Dante; Jara, Claudia; Ibañez, Jorge; Ahumada, Viviana; Acuña-Castillo, Claudio; Martin, Adrian; Córdova, Alexandra; Montoya, Margarita

    2016-01-01

    Human Natural Killer (NK) cells are a specialized heterogeneous subpopulation of lymphocytes involved in antitumor defense reactions. NK cell effector functions are critically dependent on cytokines and metabolic activity. Among various cytokines modulating NK cell function, interleukin-2 (IL-2) can induce a more potent cytotoxic activity defined as lymphokine activated killer activity (LAK). Our aim was to determine if IL-2 induces changes at the mitochondrial level in NK cells to support the bioenergetic demand for performing this enhanced cytotoxic activity more efficiently. Purified human NK cells were cultured with high IL-2 concentrations to develop LAK activity, which was assessed by the ability of NK cells to lyse NK-resistant Daudi cells. Here we show that, after 72 h of culture of purified human NK cells with enough IL-2 to induce LAK activity, both the mitochondrial mass and the mitochondrial membrane potential increased in a PGC-1α-dependent manner. In addition, oligomycin, an inhibitor of ATP synthase, inhibited IL-2-induced LAK activity at 48 and 72 h of culture. Moreover, the secretion of IFN-γ from NK cells with LAK activity was also partially dependent on PGC-1α expression. These results indicate that PGC-1α plays a crucial role in regulating mitochondrial function involved in the maintenance of LAK activity in human NK cells stimulated with IL-2.

  7. Effects of Insecticides on the Fluidity of Mitochondrial Membranes of the Diamondback Moth, Plutella xylostella, Resistant and Susceptible to Avermectin

    Science.gov (United States)

    Hu, J.; Liang, P.; Shi, X.; Gao, X.

    2008-01-01

    The effects of various insecticides on the fluidity of mitochondrial membranes and cross-resistance were investigated in the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae) using strains that were both resistant and susceptible to avermectin. The resistant strain of P. xylostella, AV-R, developed 1078-fold resistance to avermetins with a high level of cross-resistance to the analogs of avermectins, ivermectin and emamectin benzoate. It had more than 1000 times greater resistance when compared with the avermectin-susceptible strain, XH-S. Mitochondrial membrane fluidity was measured by detecting fluorescence polarization using DPH (1,6-Diphenyl -1,3,5-hexatriene) as the fluorescence probe. Abamectin, emamectin benzoate, ivermectin, cypermethrin and fenvalerate decreased the fluidity of mitochondrial membranes in the XH-S strain at 25°C. However, fipronil and acephate did not change the fluidity of mitochondrial membrane when the concentration of these insecticides was 1×10-4 mol/L. Membrane fluidity increased as the temperature increased. The thermotropic effect on the polarization value of DPH increased as the insecticide concentration was increased. There was a significant difference of mitochondrial membrane fluidity between both XH-S and AV-R when temperature was less than 25°C and no difference was observed when the temperature was more than 25°C. The low-dose abamectin (0.11 mg/L) in vivo treatment caused a significant change of membrane fluidity in the XH-S strain and no change in the AV-R strain. However, a high-dose abamectin (11.86 mg/L) resulted in 100% mortality of the XH-S strain. In vivo treatment may cause a significant change of membrane fluidity in the AV-R strain PMID:20345311

  8. Hypoxia-induced decrease of UCP3 gene expression in rat heart parallels metabolic gene switching but fails to affect mitochondrial respiratory coupling.

    Science.gov (United States)

    Essop, M Faadiel; Razeghi, Peter; McLeod, Chris; Young, Martin E; Taegtmeyer, Heinrich; Sack, Michael N

    2004-02-06

    Mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) are postulated to contribute to antioxidant defense, nutrient partitioning, and energy efficiency in the heart. To distinguish isotype function in response to metabolic stress we measured cardiac mitochondrial function and cardiac UCP gene expression following chronic hypobaric hypoxia. Isolated mitochondrial O(2) consumption and ATP synthesis rate were reduced but respiratory coupling was unchanged compared to normoxic groups. Concurrently, left ventricular UCP3 mRNA levels were significantly decreased with hypoxia (pheart as opposed to uncoupling of mitochondria. Moreover, the divergent hypoxia-induced regulation of UCP2 and UCP3 supports distinct mitochondrial regulatory functions of these inner mitochondrial membrane proteins in the heart in response to metabolic stress.

  9. Fluoride induces apoptosis in H9c2 cardiomyocytes via the mitochondrial pathway.

    Science.gov (United States)

    Yan, Xiaoyan; Wang, Lu; Yang, Xia; Qiu, Yulan; Tian, Xiaolin; Lv, Yi; Tian, Fengjie; Song, Guohua; Wang, Tong

    2017-09-01

    Numerous studies have shown that chronic excessive fluoride intake can adversely affect different organ systems. In particular, the cardiovascular system is susceptible to disruption by a high concentration of fluoride. The objectives of this study were to explore the mechanism of apoptosis by detecting the toxic effects of different concentrations of sodium fluoride (NaF) in H9c2 cells exposed for up to 96 h. NaF not only inhibited H9c2 cell proliferation but also induced apoptosis and morphological damage. With increasing NaF concentrations, early apoptosis of H9c2 cells was increased while the mitochondrial membrane potential was decreased. Compared with the control group, the mRNA levels of caspase-3, caspase-9, and cytochrome c all increased with increasing concentrations of NaF. In summary, these data suggest that apoptosis is involved in NaF-induced H9c2 cell toxicity and that activation of the mitochondrial pathway may occur. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Fipronil induces apoptosis through caspase-dependent mitochondrial pathways in Drosophila S2 cells.

    Science.gov (United States)

    Zhang, Baoyan; Xu, Zhiping; Zhang, Yixi; Shao, Xusheng; Xu, Xiaoyong; Cheng, Jiaogao; Li, Zhong

    2015-03-01

    Fipronil is the first phenylpyrazole insecticide widely used in controlling pests, including pyrethroid, organophosphate and carbamate insecticides. It is generally accepted that fipronil elicits neurotoxicity via interactions with GABA and glutamate receptors, although alternative mechanisms have recently been proposed. This study evaluates the genotoxicity of fipronil and its likely mode of action in Drosophila S2 cells, as an in vitro model. Fipronil administrated the concentration- and time-dependent S2 cell proliferation. Intracellular biochemical assays showed that fipronil-induced S2 cell apoptosis coincided with a decrease in the mitochondrial membrane potential and an increase reactive oxygen species generation, a significant decrease of Bcl-2 and DIAP1, and a marked augmentation of Cyt c and caspase-3. Because caspase-3 is the major executioner caspase downstream of caspase-9 in Drosophila, enzyme activity assays were used to determine the activities of caspase-3 and caspase-9. Our results indicated that fipronil effectively induced apoptosis in Drosophila S2 cells through caspase-dependent mitochondrial pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Resveratrol-Sensitized UVA Induced Apoptosis in Human Keratinocytes through Mitochondrial Oxidative Stress and Pore Opening

    Science.gov (United States)

    Boyer, Jean Z; Jandova, Jana; Janda, Jaroslav; Vleugels, Frank R; Elliott, David; Sligh, James E

    2012-01-01

    Resveratrol (3, 5, 4′-trihydroxy- trans- stilbene), a polyphenol compound, is derived from natural products such as the skin of red grapes, blueberries and cranberries. Resveratrol not only exhibits antioxidant, cardioprotection, and anti-aging properties, but can also inhibit cancer cell growth and induce apoptosis. It has been shown that resveratrol inhibits the activation of Nf-kB and subsequently down regulates the expression of Nf-kB regulated genes such as interleukin-2 and Bcl-2, leading to cell cycle arrest and increased apoptosis in multiple myeloma cells. In the skin, resveratrol has been reported to sensitize keratinocytes to UVA induced apoptosis. However, the effect of resveratrol on opening of the mitochondrial permeability transition pore has not been previously examined. Our data show that UVA (14J/cm2) along with resveratrol causes massive oxidative stress in mitochondria. As a consequence of oxidative stress, the mitochondrial membrane potential decreases which results in opening of the mitochondrial pores ultimately leading to apoptosis in human keratinocytes. These results may have clinical implications for development of future chemotherapeutic treatment for tumors of the skin. PMID:22673012

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    in genomic maintenance and transcriptome regulation. By immunocytochemistry, mitochondrial fractionation, and Western blotting, we demonstrate that CSB localizes to mitochondria in different types of cells, with increased mitochondrial distribution following menadione-induced oxidative stress. Moreover, our...

  13. Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Choe, Tae-Boo [Department of Microbiological Engineering, Kon-Kuk University, Gwangjin-gu, Seoul (Korea, Republic of); Hong, Seok-Il [Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Yi, Jae-Youn [Laboratory of Modulation of Radiobiological Responses, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Hwang, Sang-Gu [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of); Lee, Hyun-Gyu [Department of Microbiology and Immunology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Lee, Yun-Han, E-mail: yhlee87@yuhs.ac [Department of Radiation Oncology, College of Medicine, Yonsei University, 250 Seongsan-no, Seodaemun-gu, Seoul (Korea, Republic of); Park, In-Chul, E-mail: parkic@kcch.re.kr [Division of Radiation Cancer Research, Korea Institute of Radiological and Medical Sciences, 215-4 Gongneung-dong, Nowon-gu, Seoul (Korea, Republic of)

    2014-07-11

    Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

  14. Mitochondrial dysfunction in lyssavirus-induced apoptosis.

    Science.gov (United States)

    Gholami, Alireza; Kassis, Raïd; Real, Eléonore; Delmas, Olivier; Guadagnini, Stéphanie; Larrous, Florence; Obach, Dorothée; Prevost, Marie-Christine; Jacob, Yves; Bourhy, Hervé

    2008-05-01

    Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis.

  15. Promotion of mitochondrial membrane complex assembly by a proteolytically inactive yeast Lon

    NARCIS (Netherlands)

    Rep, M; van Dijl, J M; Suda, K; Schatz, G; Grivell, L A; Suzuki, C K

    1996-01-01

    Afg3p and Rca1p are adenosine triphosphate (ATP)-dependent metalloproteases in yeast mitochondria. Cells lacking both proteins exhibit defects in respiration-dependent growth, degradation of mitochondrially synthesized proteins, and assembly of inner-membrane complexes. Defects in growth and protein

  16. DJ-1 KNOCK-DOWN IMPAIRS ASTROCYTE MITOCHONDRIAL FUNCTION

    Science.gov (United States)

    LARSEN, N. J.; AMBROSI, G.; MULLETT, S. J.; BERMAN, S. B.; HINKLE, D. A.

    2012-01-01

    Mitochondrial dysfunction has long been implicated in the pathogenesis of Parkinson’s disease (PD). PD brain tissues show evidence for mitochondrial respiratory chain Complex I deficiency. Pharmacological inhibitors of Complex I, such as rotenone, cause experimental parkinsonism. The cytoprotective protein DJ-1, whose deletion is sufficient to cause genetic PD, is also known to have mitochondria-stabilizing properties. We have previously shown that DJ-1 is over-expressed in PD astrocytes, and that DJ-1 deficiency impairs the capacity of astrocytes to protect co-cultured neurons against rotenone. Since DJ-1 modulated, astrocyte-mediated neuroprotection against rotenone may depend upon proper astrocytic mitochondrial functioning, we hypothesized that DJ-1 deficiency would impair astrocyte mitochondrial motility, fission/fusion dynamics, membrane potential maintenance, and respiration, both at baseline and as an enhancement of rotenone-induced mitochondrial dysfunction. In astrocyte-enriched cultures, we observed that DJ-1 knock-down reduced mitochondrial motility primarily in the cellular processes of both untreated and rotenone treated cells. In these same cultures, DJ-1 knock-down did not appreciably affect mitochondrial fission, fusion, or respiration, but did enhance rotenone-induced reductions in the mitochondrial membrane potential. In neuron–astrocyte co-cultures, astrocytic DJ-1 knock-down reduced astrocyte process mitochondrial motility in untreated cells, but this effect was not maintained in the presence of rotenone. In the same co-cultures, astrocytic DJ-1 knock-down significantly reduced mitochondrial fusion in the astrocyte cell bodies, but not the processes, under the same conditions of rotenone treatment in which DJ-1 deficiency is known to impair astrocyte-mediated neuroprotection. Our studies therefore demonstrated the following new findings: (i) DJ-1 deficiency can impair astrocyte mitochondrial physiology at multiple levels, (ii) astrocyte

  17. 36 CFR 905.737-102 - Enforcement proceedings.

    Science.gov (United States)

    2010-07-01

    ... or 5 CFR part 737 after a final decision by: (1) Prohibiting the individual from making, on behalf of... CORPORATION STANDARDS OF CONDUCT Conduct and Responsibilities of Former Employees-Enforcement § 905.737-102... there is reasonable cause to believe that a former employee has violated 18 U.S.C. 207 or 5 CFR part 737...

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

    Directory of Open Access Journals (Sweden)

    Michelle Barbi de Moura

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

  19. Mitochondrial respiration is sensitive to cytoarchitectural breakdown.

    Science.gov (United States)

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

    2016-11-07

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

  20. PGC-1{alpha} is required for AICAR induced expression of GLUT4 and mitochondrial proteins in mouse skeletal muscle

    DEFF Research Database (Denmark)

    Leick, Lotte; Fentz, Joachim; Biensø, Rasmus S

    2010-01-01

    We tested the hypothesis that repeated activation of AMPK induces mitochondrial and glucose membrane transporter gene/protein expression via a peroxisome proliferator activated receptor Upsilon co-activator (PGC)-1alpha dependent mechanism. Whole body PGC-1alpha knockout (KO) and littermate wild...... GLUT4, cytochrome c oxidase (COX)I and cytochrome (cyt) c protein expression ~10-40% relative to saline in white muscles of the WT mice, but not of the PGC-1alpha KO mice. In line, GLUT4 and cyt c mRNA content increased 30-60% 4h after a single AICAR injection relative to saline only in WT mice. One...... and PGC-1alpha KO mice. In conclusion, we here provide genetic evidence for a major role of PGC-1alpha in AMPK mediated regulation of mitochondrial and glucose membrane transport protein expression in skeletal muscle....

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

    Directory of Open Access Journals (Sweden)

    Daniel O Frank

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

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

    Science.gov (United States)

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

    2015-01-01

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

  3. Mitochondrial nucleoid interacting proteins support mitochondrial protein synthesis.

    Science.gov (United States)

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

    2012-07-01

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

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

    Science.gov (United States)

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

    2011-03-01

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

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

    Czech Academy of Sciences Publication Activity Database

    Bischof, J.; Salzmann, M.; Streubel, M.K.; Hašek, Jiří; Geltinger, F.; Duschl, J.; Bresgen, N.; Briza, P.; Hašková, Danuša; Lejsková, Renata; Sopjani, M.; Richter, K.; Rinnerthaler, M.

    2017-01-01

    Roč. 3, March 20 (2017), č. článku 17016. E-ISSN 2058-7716 R&D Projects: GA ČR(CZ) GA16-05497S; GA MŠk(CZ) 7AMB16AT006 Institutional support: RVO:61388971 Keywords : mitochondrial membrane * harmful protein s * lipid droplets Subject RIV: EE - Microbiology, Virology OBOR OECD: Microbiology

  6. MBA1 encodes a mitochondrial membrane-associated protein required for biogenesis of the respiratory chain.

    Science.gov (United States)

    Rep, M; Grivell, L A

    1996-06-17

    The yeast MBA 1 gene (Multi-copy Bypass of AFG3) is one of three genes whose overexpression suppresses afg3-null and rca1-null mutations. Bypass of AFG3 and RCA1, whose products are essential for assembly of mitochondrial inner membrane enzyme complexes, suggests a related role for MBA1. The predicted translation product is a 30 kDa hydrophilic protein with a putative mitochondrial targeting sequence and no homology to any sequence in protein or EST databases. Gene disruption leads to a partial respiratory growth defect, which is more pronounced at temperatures above 30 degrees C. Concomitantly, amounts of cytochromes b and aa3 are reduced. A C-terminal c-myc-tagged MBA1 gene product is functional and is found associated with the mitochondrial inner membrane, from which it can he extracted by carbonate, but not by high salt. These observations give further support to a role of MBA1 in assembly of the respiratory chain.

  7. Hypoxic stress induces, but cannot sustain trophoblast stem cell differentiation to labyrinthine placenta due to mitochondrial insufficiency

    Directory of Open Access Journals (Sweden)

    Yufen Xie

    2014-11-01

    Full Text Available Dysfunctional stem cell differentiation into placental lineages is associated with gestational diseases. Of the differentiated lineages available to trophoblast stem cells (TSC, elevated O2 and mitochondrial function are necessary to placental lineages at the maternal–placental surface and important in the etiology of preeclampsia. TSC lineage imbalance leads to embryonic failure during uterine implantation. Stress at implantation exacerbates stem cell depletion by decreasing proliferation and increasing differentiation. In an implantation site O2 is normally ~2%. In culture, exposure to 2% O2 and fibroblast growth factor 4 (FGF4 enabled the highest mouse TSC multipotency and proliferation. In contrast, hypoxic stress (0.5% O2 initiated the most TSC differentiation after 24 h despite exposure to FGF4. However, hypoxic stress supported differentiation poorly after 4–7 days, despite FGF4 removal. At all tested O2 levels, FGF4 maintained Warburg metabolism; mitochondrial inactivity and aerobic glycolysis. However, hypoxic stress suppressed mitochondrial membrane potential and maintained low mitochondrial cytochrome c oxidase (oxidative phosphorylation/OxPhos, and high pyruvate kinase M2 (glycolysis despite FGF4 removal. Inhibiting OxPhos inhibited optimum differentiation at 20% O2. Moreover, adding differentiation-inducing hyperosmolar stress failed to induce differentiation during hypoxia. Thus, differentiation depended on OxPhos at 20% O2; hypoxic and hyperosmolar stresses did not induce differentiation at 0.5% O2. Hypoxia-limited differentiation and mitochondrial inhibition and activation suggest that differentiation into two lineages of the labyrinthine placenta requires O2 > 0.5–2% and mitochondrial function. Stress-activated protein kinase increases an early lineage and suppresses later lineages in proportion to the deviation from optimal O2 for multipotency, thus it is the first enzyme reported to prioritize differentiation.

  8. Curcumin prevents cisplatin-induced renal alterations in mitochondrial bioenergetics and dynamic.

    Science.gov (United States)

    Ortega-Domínguez, Bibiana; Aparicio-Trejo, Omar Emiliano; García-Arroyo, Fernando E; León-Contreras, Juan Carlos; Tapia, Edilia; Molina-Jijón, Eduardo; Hernández-Pando, Rogelio; Sánchez-Lozada, Laura Gabriela; Barrera-Oviedo, Diana; Pedraza-Chaverri, José

    2017-09-01

    Cisplatin is widely used as chemotherapeutic agent for treatment of diverse types of cancer, however, acute kidney injury (AKI) is an important side effect of this treatment. Diverse mechanisms have been involved in cisplatin-induced AKI, such as oxidative stress, apoptosis and mitochondrial damage. On the other hand, curcumin is a polyphenol extracted from the rhizome of Curcuma longa L. Previous studies have shown that curcumin protects against the cisplatin-induced AKI; however, it is unknown whether curcumin can reduce alterations in mitochondrial bioenergetics and dynamic in this model. It was found that curcumin prevents cisplatin-induced: (a) AKI and (b) alterations in the following mitochondrial parameters: bioenergetics, ultrastructure, hydrogen peroxide production and dynamic. In fact, curcumin prevented the increase of mitochondrial fission 1 protein (FIS1), the decrease of optic atrophy 1 protein (OPA1) and the decrease of NAD + -dependent deacetylase sirtuin-3 (SIRT3), a mitochondrial dynamic regulator as well as the increase in the mitophagy associated proteins parkin and phosphatase and tensin homologue (PTEN)-induced putative kinase protein 1 (PINK1). In conclusion, the protective effect of curcumin in cisplatin-induced AKI was associated with the prevention of the alterations in mitochondrial bioenergetics, ultrastructure, redox balance, dynamic, and SIRT3 levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Carvedilol-mediated antioxidant protection against doxorubicin-induced cardiac mitochondrial toxicity

    International Nuclear Information System (INIS)

    Oliveira, Paulo J.; Bjork, James A.; Santos, Maria S.; Leino, Richard L.; Froberg, M. Kent; Moreno, Antonio J.; Wallace, Kendall B.

    2004-01-01

    The cardiotoxicity associated with doxorubicin (DOX) therapy limits the total cumulative dose and therapeutic success of active anticancer chemotherapy. Cardiac mitochondria are implicated as primary targets for DOX toxicity, which is believed to be mediated by the generation of highly reactive free radical species of oxygen from complex I of the mitochondrial electron transport chain. The objective of this study was to determine if the protection demonstrated by carvedilol (CV), a β-adrenergic receptor antagonist with strong antioxidant properties, against DOX-induced mitochondrial-mediated cardiomyopathy [Toxicol. Appl. Pharmacol. 185 (2002) 218] is attributable to its antioxidant properties or its β-adrenergic receptor antagonism. Our results confirm that DOX induces oxidative stress, mitochondrial dysfunction, and histopathological lesions in the cardiac tissue, all of which are inhibited by carvedilol. In contrast, atenolol (AT), a β-adrenergic receptor antagonist lacking antioxidant properties, preserved phosphate energy charge but failed to protect against any of the indexes of DOX-induced oxidative mitochondrial toxicity. We therefore conclude that the cardioprotective effects of carvedilol against DOX-induced mitochondrial cardiotoxicity are due to its inherent antioxidant activity and not to its β-adrenergic receptor antagonism

  10. TSA protects H9c2 cells against thapsigargin-induced apoptosis related to endoplasmic reticulum stress-mediated mitochondrial injury.

    Science.gov (United States)

    Li, Zhiping; Liu, Yan; Dai, Xinlun; Zhou, Qiangqiang; Liu, Xueli; Li, Zeyu; Chen, Xia

    2017-05-01

    Endoplasmic reticulum stress (ERS) activates an adaptive unfolded protein response (UPR) that facilitates cellular repair, however, under prolonged ER stress, the UPR can ultimately trigger apoptosis thereby terminating damaged cells. Recently, TSA has shown protective effects on ERS and its mechanisms related to ER pathway has been previously characterized. However, whether TSA exerts its protective role via metabolic events remain largely undefined. Objectives : To explore the possible involvement of the metabolic changes during ERS and to better understand how TSA influence mitochondrial function to facilitate cellular adaptation. Results : TSA is an inhibitor of histone deacetylase which could significantly inhibit H9c2 cell apoptosis induced by Thapsigargin (TG). It also intervene the decrease of mitochondrial membrane potential. By immunofluorescence staining, we have shown that GRP78 was concentrated in the perinuclear region and co-localized with ER. However, treatments with TG and TSA could let it overlap with the mitochondrial marker MitoTracker. Cellular fractionation also confirmed the location of GRP78 in mitochondrion. TSA decreases ERS-induced cell apoptosis and mitochondrial injury may related to enhance the location of GRP78 in mitochondrion.

  11. Protective Effect of Edaravone Against Aβ25-35-Induced Mitochondrial Oxidative Damage in SH-SY5Y Cells.

    Science.gov (United States)

    Zhang, G-L; Zhang, L; Guo, Y-Y; Ma, Z-L; Wang, H-Y; Li, T; Liu, J; Du, Y; Yao, L; Li, T-T; Du, J-M

    2017-05-20

    Amyloid-β (Aβ)-induced oxidative stress plays an important role in the pathogenesis of Alzheimer's disease (AD). Recent studies show that Aβ accumulation may lead to mitochondrial oxidative damage. In the present study, we investigated the protective effect of edaravone on mitochondrial damage in SH-SY5Y cells treated with Aβ25-35. SH-SY5Y cells were pre-treated with 20, 40 or 80 μM edaravone before treatment with 25 μM Aβ25-35. After 24h cell culture, cellular apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), ATP levels and mitochondrial morphology were evaluated. SH-SY5Y cells exposed to Aβ25-35 had high levels of apoptosis and ROS; loss of ΔΨm, decreased ATP levels and presence of mitochondrial swelling. However, these effects were significantly inhibited by edaravone pre-treatment. These results indicate that edaravone prevents mitochondria oxidative damage caused by Aβ in SH-SY5Y cells, which suggests that it may have potential clinical application in AD therapy.

  12. The metabolic enhancer piracetam ameliorates the impairment of mitochondrial function and neurite outgrowth induced by ß-amyloid peptide

    Science.gov (United States)

    Kurz, C; Ungerer, I; Lipka, U; Kirr, S; Schütt, T; Eckert, A; Leuner, K; Müller, WE

    2010-01-01

    Background and purpose: β-Amyloid peptide (Aβ) is implicated in the pathogenesis of Alzheimer's disease by initiating a cascade of events from mitochondrial dysfunction to neuronal death. The metabolic enhancer piracetam has been shown to improve mitochondrial dysfunction following brain aging and experimentally induced oxidative stress. Experimental approach: We used cell lines (PC12 and HEK cells) and murine dissociated brain cells. The protective effects of piracetam in vitro and ex vivo on Aβ-induced impairment of mitochondrial function (as mitochondrial membrane potential and ATP production), on secretion of soluble Aβ and on neurite outgrowth in PC12 cells were investigated. Key results: Piracetam improves mitochondrial function of PC12 cells and acutely dissociated brain cells from young NMRI mice following exposure to extracellular Aβ1-42. Similar protective effects against Aβ1-42 were observed in dissociated brain cells from aged NMRI mice, or mice transgenic for mutant human amyloid precursor protein (APP) treated with piracetam for 14 days. Soluble Aβ load was markedly diminished in the brain of those animals after treatment with piracetam. Aβ production by HEK cells stably transfected with mutant human APP was elevated by oxidative stress and this was reduced by piracetam. Impairment of neuritogenesis is an important consequence of Aβ-induced mitochondrial dysfunction and Aβ-induced reduction of neurite growth in PC12 cells was substantially improved by piracetam. Conclusion and implications: Our findings strongly support the concept of improving mitochondrial function as an approach to ameliorate the detrimental effects of Aβ on brain function. This article is commented on by Moncada, pp. 217–219 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00706.x and to view related papers by Pravdic et al. and Puerta et al. visit http://dx.doi.org/10.1111/j.1476-5381.2010.00698.x and http://dx.doi.org/10.1111/j

  13. Mitochondrial dysfunction is responsible for fatty acid synthase inhibition-induced apoptosis in breast cancer cells by PdpaMn.

    Science.gov (United States)

    Wang, Qiang; Du, Xia; Zhou, Bingjie; Li, Jing; Lu, Wenlong; Chen, Qiuyun; Gao, Jing

    2017-12-01

    Targeting cellular metabolism is becoming a hallmark to overcome drug resistance in breast cancer treatment. Activation of fatty acid synthase (FASN) has been shown to promote breast cancer cell growth. However, there is no concrete report underlying the mechanism associated with mitochondrial dysfunction in relation to fatty acid synthase inhibition-induced apoptosis in breast cancer cells. The current study is aimed at exploring the effect of the novel manganese (Mn) complex, labeled as PdpaMn, on lipid metabolism and mitochondrial function in breast cancer cells. Herein, we observed that PdpaMn displayed strong cytotoxicity on breast cancer cell lines and selectively targeted the tumor without affecting the normal organs or cells in vivo. We also observed that PdpaMn could bind to TE domain of FASN and decrease the activity and the level of expression of FASN, which is an indication that FASN could serve as a target of PdpaMn. In addition, we demonstrated that PdpaMn increased intrinsic apoptosis in breast cancer cells relayed by a suppressed the level of expression of FASN, followed by the release of mitochondrial cytochrome c and the activation of caspases-9. Instigated by the above observations, we hypothesized that PdpaMn-induced apoptosis events are dependent on mitochondrial dysfunction. Indeed, we found that mitochondrial membrane potential (MMP) collapse, mitochondrial oxygen consumption reduction and adenosine triphosphate (ATP) release were deeply repressed. Furthermore, our results showed that PdpaMn significantly increased the reactive oxygen species (ROS) production, and the protection conferred by the free radical scavenger N-acetyl-cysteine (NAC) indicates that PdpaMn-induced apoptosis through an oxidative stress-associated mechanism. More so, the above results have demonstrated that mitochondrial dysfunction participated in FASN inhibition-induce apoptosis in breast cancer cells by PdpaMn. Therefore, PdpaMn may be considered as a good candidate

  14. Using Förster-Resonance Energy Transfer to Measure Protein Interactions Between Bcl-2 Family Proteins on Mitochondrial Membranes.

    Science.gov (United States)

    Pogmore, Justin P; Pemberton, James M; Chi, Xiaoke; Andrews, David W

    2016-01-01

    The Bcl-2 family of proteins regulates the process of mitochondrial outer membrane permeabilization, causing the release of cytochrome c and committing a cell to apoptosis. The majority of the functional interactions between these proteins occur at, on, or within the mitochondrial outer membrane, complicating structural studies of the proteins and complexes. As a result most in vitro studies of these protein-protein interactions use truncated proteins and/or detergents which can cause artificial interactions. Herein, we describe a detergent-free, fluorescence-based, in vitro technique to study binding between full-length recombinant Bcl-2 family proteins, particularly cleaved BID (cBID) and BCL-XL, on the membranes of purified mitochondria.

  15. N-(1-Pyrenyl Maleimide Induces Bak Oligomerization and Mitochondrial Dysfunction in Jurkat Cells

    Directory of Open Access Journals (Sweden)

    Pei-Rong Huang

    2015-01-01

    Full Text Available N-(1-pyrenyl maleimide (NPM is a fluorescent reagent that is frequently used as a derivatization agent for the detection of thio-containing compounds. NPM has been shown to display a great differential cytotoxicity against hematopoietic cancer cells. In this study, the molecular mechanism by which NPM induces apoptosis was examined. Here, we show that treatment of Jurkat cells with NPM leads to Bak oligomerization, loss of mitochondrial membrane potential (Δψm, and release of cytochrome C from mitochondria to cytosol. Induction of Bak oligomerization appears to play a critical role in NPM-induced apoptosis, as downregulation of Bak by shRNA significantly prevented NPM-induced apoptosis. Inhibition of caspase 8 by Z-IETD-FMK and/or depletion of Bid did not affect NPM-induced oligomerization of Bak. Taken together, these results suggest that NPM-induced apoptosis is mediated through a pathway that is independent of caspase-8 activation.

  16. Influence of Glucose Deprivation on Membrane Potentials of Plasma Membranes, Mitochondria and Synaptic Vesicles in Rat Brain Synaptosomes.

    Science.gov (United States)

    Hrynevich, Sviatlana V; Pekun, Tatyana G; Waseem, Tatyana V; Fedorovich, Sergei V

    2015-06-01

    Hypoglycemia can cause neuronal cell death similar to that of glutamate-induced cell death. In the present paper, we investigated the effect of glucose removal from incubation medium on changes of mitochondrial and plasma membrane potentials in rat brain synaptosomes using the fluorescent dyes DiSC3(5) and JC-1. We also monitored pH gradients in synaptic vesicles and their recycling by the fluorescent dye acridine orange. Glucose deprivation was found to cause an inhibition of K(+)-induced Ca(2+)-dependent exocytosis and a shift of mitochondrial and plasma membrane potentials to more positive values. The sensitivity of these parameters to the energy deficit caused by the removal of glucose showed the following order: mitochondrial membrane potential > plasma membrane potential > pH gradient in synaptic vesicles. The latter was almost unaffected by deprivation compared with the control. The pH-dependent dye acridine orange was used to investigate synaptic vesicle recycling. However, the compound's fluorescence was shown to be enhanced also by the mixture of mitochondrial toxins rotenone (10 µM) and oligomycin (5 µg/mL). This means that acridine orange can presumably be partially distributed in the intermembrane space of mitochondria. Glucose removal from the incubation medium resulted in a 3.7-fold raise of acridine orange response to rotenone + oligomycin suggesting a dramatic increase in the mitochondrial pH gradient. Our results suggest that the biophysical characteristics of neuronal presynaptic endings do not favor excessive non-controlled neurotransmitter release in case of hypoglycemia. The inhibition of exocytosis and the increase of the mitochondrial pH gradient, while preserving the vesicular pH gradient, are proposed as compensatory mechanisms.

  17. Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

    Energy Technology Data Exchange (ETDEWEB)

    Seo, Kyuhwa; Seo, Suho; Han, Jae Yun; Ki, Sung Hwan; Shin, Sang Mi, E-mail: smshin@chosun.ac.kr

    2014-10-15

    Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2

  18. Resveratrol attenuates methylglyoxal-induced mitochondrial dysfunction and apoptosis by Sestrin2 induction

    International Nuclear Information System (INIS)

    Seo, Kyuhwa; Seo, Suho; Han, Jae Yun; Ki, Sung Hwan; Shin, Sang Mi

    2014-01-01

    Methylglyoxal is found in high levels in the blood and other tissues of diabetic patients and exerts deleterious effects on cells and tissues. Previously, we reported that resveratrol, a polyphenol in grapes, induced the expression of Sestrin2 (SESN2), a novel antioxidant protein, and inhibited hepatic lipogenesis. This study investigated whether resveratrol protects cells from the methylglyoxal-induced toxicity via SESN2 induction. Methylglyoxal significantly induced cell death in HepG2 cells. However, cells pretreated with resveratrol were rescued from methylglyoxal-induced apoptosis. Resveratrol attenuated glutathione (GSH) depletion and ROS production promoted by methylglyoxal. Moreover, mitochondrial damage was observed by methylglyoxal treatment, but resveratrol restored mitochondrial function, as evidenced by the observed lack of mitochondrial permeability transition and increased ADP/ATP ratio. Resveratrol treatment inhibited SESN2 depletion elicited by methylglyoxal. SESN2 overexpression repressed methylglyoxal-induced mitochondrial dysfunction and apoptosis. Likewise, rotenone-induced cytotoxicity was not observed in SESN2 overexpressed cells. Furthermore, siRNA knockdown of SESN2 reduced the ability of resveratrol to prevent methylglyoxal-induced mitochondrial permeability transition. In addition, when mice were exposed to methylglyoxal after infection of Ad-SESN2, the plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and GSH depletion by methylglyoxal in liver was reduced in Ad-SESN2 infected mice. Our results demonstrated that resveratrol is capable of protecting cells from methylglyoxal-induced mitochondrial dysfunction and oxidative stress via SESN2 induction. - Highlights: • Resveratrol decreased methylglyoxal-induced apoptosis. • Resveratrol attenuated GSH depletion and ROS production promoted by methylglyoxal. • Resveratrol restored the mitochondrial function by Sestrin2 induction. • Induction of Sestrin2

  19. ABT-773: pharmacokinetics and interactions with ranitidine and sucralfate.

    Science.gov (United States)

    Pletz, M W; Preechachatchaval, V; Bulitta, J; Allewelt, M; Burkhardt, O; Lode, H

    2003-03-01

    We assessed the pharmacokinetics and interaction of ABT-773 in 12 volunteers receiving ABT-773 alone or concomitantly with ranitidine or sucralfate. Data for 150 mg of ABT-773 were as follows: the maximum concentration of the drug in plasma (C(max)) was 318 ng/ml, its half-life was 5.66 h, and its area under the plasma concentration-time curve from 0 h to infinity (AUC(0- infinity )) was 1,662 ng. h/ml. Coadministration of ranitidine, reduced the C(max) (-25.7%) and AUC(0- infinity ) (-15.8%) significantly. Sucralfate had no impact on the bioavailability of ABT-773.

  20. Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells

    International Nuclear Information System (INIS)

    Arriba, G. de; Perez de Hornedo, J.; Ramirez Rubio, S.; Calvino Fernandez, M.; Benito Martinez, S.; Maiques Camarero, M.; Parra Cid, T.

    2009-01-01

    Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis. CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.

  1. Drp1-Dependent Mitochondrial Autophagy Plays a Protective Role Against Pressure Overload-Induced Mitochondrial Dysfunction and Heart Failure.

    Science.gov (United States)

    Shirakabe, Akihiro; Zhai, Peiyong; Ikeda, Yoshiyuki; Saito, Toshiro; Maejima, Yasuhiro; Hsu, Chiao-Po; Nomura, Masatoshi; Egashira, Kensuke; Levine, Beth; Sadoshima, Junichi

    2016-03-29

    Mitochondrial autophagy is an important mediator of mitochondrial quality control in cardiomyocytes. The occurrence of mitochondrial autophagy and its significance during cardiac hypertrophy are not well understood. Mice were subjected to transverse aortic constriction (TAC) and observed at multiple time points up to 30 days. Cardiac hypertrophy developed after 5 days, the ejection fraction was reduced after 14 days, and heart failure was observed 30 days after TAC. General autophagy was upregulated between 1 and 12 hours after TAC but was downregulated below physiological levels 5 days after TAC. Mitochondrial autophagy, evaluated by electron microscopy, mitochondrial content, and Keima with mitochondrial localization signal, was transiently activated at ≈3 to 7 days post-TAC, coinciding with mitochondrial translocation of Drp1. However, it was downregulated thereafter, followed by mitochondrial dysfunction. Haploinsufficiency of Drp1 abolished mitochondrial autophagy and exacerbated the development of both mitochondrial dysfunction and heart failure after TAC. Injection of Tat-Beclin 1, a potent inducer of autophagy, but not control peptide, on day 7 after TAC, partially rescued mitochondrial autophagy and attenuated mitochondrial dysfunction and heart failure induced by overload. Haploinsufficiency of either drp1 or beclin 1 prevented the rescue by Tat-Beclin 1, suggesting that its effect is mediated in part through autophagy, including mitochondrial autophagy. Mitochondrial autophagy is transiently activated and then downregulated in the mouse heart in response to pressure overload. Downregulation of mitochondrial autophagy plays an important role in mediating the development of mitochondrial dysfunction and heart failure, whereas restoration of mitochondrial autophagy attenuates dysfunction in the heart during pressure overload. © 2016 American Heart Association, Inc.

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  3. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    International Nuclear Information System (INIS)

    Liu, Cong; Sekine, Shuichi; Ito, Kousei

    2016-01-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

  4. Assessment of mitochondrial dysfunction-related, drug-induced hepatotoxicity in primary rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Cong; Sekine, Shuichi, E-mail: ssekine@faculty.chiba-u.jp; Ito, Kousei

    2016-07-01

    Evidence that mitochondrial dysfunction plays a central role in drug-induced liver injury is rapidly accumulating. In contrast to physiological conditions, in which almost all adenosine triphosphate (ATP) in hepatocytes is generated in mitochondria via aerobic respiration, the high glucose content and limited oxygen supply of conventional culture systems force primary hepatocytes to generate most ATP via cytosolic glycolysis. Thus, such anaerobically poised cells are resistant to xenobiotics that impair mitochondrial function, and are not suitable to identify drugs with mitochondrial liabilities. In this study, primary rat hepatocytes were cultured in galactose-based medium, instead of the conventional glucose-based medium, and in hyperoxia to improve the reliance of energy generation on aerobic respiration. Activation of mitochondria was verified by diminished cellular lactate release and increased oxygen consumption. These conditions improved sensitivity to the mitochondrial complex I inhibitor rotenone. Since oxidative stress is also a general cause of mitochondrial impairment, cells were exposed to test compounds in the presence of transferrin to increase the generation of reactive oxygen species via increased uptake of iron. Finally, 14 compounds with reported mitochondrial liabilities were tested to validate this new drug-induced mitochondrial toxicity assay. Overall, the culture of primary rat hepatocytes in galactose, hyperoxia and transferrin is a useful model for the identification of mitochondrial dysfunction-related drug-induced hepatotoxicity. - Highlights: • Drug-induced mitochondrial toxicity was evaluated using primary rat hepatocytes. • Galactose and hyperoxia could activate OXPHOS in primary rat hepatocytes. • Cells with enhanced OXPHOS exhibit improved sensitivity to mitochondrial toxins. • Transferrin potentiate mitochondrial toxicity via increased ROS production.

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

    Directory of Open Access Journals (Sweden)

    Fujita Naoya

    2011-01-01

    Full Text Available Abstract 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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-07-15

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

  7. Mitochondrial Dysfunction in Lyssavirus-Induced Apoptosis▿ †

    Science.gov (United States)

    Gholami, Alireza; Kassis, Raïd; Real, Eléonore; Delmas, Olivier; Guadagnini, Stéphanie; Larrous, Florence; Obach, Dorothée; Prevost, Marie-Christine; Jacob, Yves; Bourhy, Hervé

    2008-01-01

    Lyssaviruses are highly neurotropic viruses associated with neuronal apoptosis. Previous observations have indicated that the matrix proteins (M) of some lyssaviruses induce strong neuronal apoptosis. However, the molecular mechanism(s) involved in this phenomenon is still unknown. We show that for Mokola virus (MOK), a lyssavirus of low pathogenicity, the M (M-MOK) targets mitochondria, disrupts the mitochondrial morphology, and induces apoptosis. Our analysis of truncated M-MOK mutants suggests that the information required for efficient mitochondrial targeting and dysfunction, as well as caspase-9 activation and apoptosis, is held between residues 46 and 110 of M-MOK. We used a yeast two-hybrid approach, a coimmunoprecipitation assay, and confocal microscopy to demonstrate that M-MOK physically associates with the subunit I of the cytochrome c (cyt-c) oxidase (CcO) of the mitochondrial respiratory chain; this is in contrast to the M of the highly pathogenic Thailand lyssavirus (M-THA). M-MOK expression induces a significant decrease in CcO activity, which is not the case with M-THA. M-MOK mutations (K77R and N81E) resulting in a similar sequence to M-THA at positions 77 and 81 annul cyt-c release and apoptosis and restore CcO activity. As expected, the reverse mutations, R77K and E81N, introduced in M-THA induce a phenotype similar to that due to M-MOK. These features indicate a novel mechanism for energy depletion during lyssavirus-induced apoptosis. PMID:18321977

  8. Legionella pneumophila secretes a mitochondrial carrier protein during infection.

    Directory of Open Access Journals (Sweden)

    Pavel Dolezal

    2012-01-01

    Full Text Available The Mitochondrial Carrier Family (MCF is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionellanucleotide carrier Protein (LncP, encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.

  9. Cerebral energy metabolism during induced mitochondrial dysfunction

    DEFF Research Database (Denmark)

    Nielsen, T H; Bindslev, TT; Pedersen, S M

    2013-01-01

    In patients with traumatic brain injury as well as stroke, impaired cerebral oxidative energy metabolism may be an important factor contributing to the ultimate degree of tissue damage. We hypothesize that mitochondrial dysfunction can be diagnosed bedside by comparing the simultaneous changes...... in brain tissue oxygen tension (PbtO(2)) and cerebral cytoplasmatic redox state. The study describes cerebral energy metabolism during mitochondrial dysfunction induced by sevoflurane in piglets....

  10. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

    International Nuclear Information System (INIS)

    Yan, Jidong; Xu, Jing; Fei, Yao; Jiang, Congshan; Zhu, Wenhua; Han, Yan; Lu, Shemin

    2016-01-01

    Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of

  11. TrxR2 deficiencies promote chondrogenic differentiation and induce apoptosis of chondrocytes through mitochondrial reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Jidong [Department of Human Anatomy, Histology and Embryology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Xu, Jing [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Fei, Yao [College of Life Sciences, Northwest University, Xi’an, Shaanxi Province 710069 (China); Jiang, Congshan; Zhu, Wenhua; Han, Yan [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Lu, Shemin, E-mail: lushemin@xjtu.edu.cn [Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, Xi’an, Shaanxi 710061 (China); Key Laboratory of Environment and Genes Related to Diseases, Xi’an Jiaotong University, Ministry of Education of China (China)

    2016-05-15

    Thioredoxin reductase 2 (TrxR2) is a selenium (Se) containing protein. Se deficiency is associated with an endemic osteoarthropathy characterized by impaired cartilage formation. It is unclear whether TrxR2 have roles in cartilage function. We examined the effects of TrxR2 on chondrogenic ATDC5 cells through shRNA-mediated gene silencing of TrxR2. We demonstrated TrxR2 deficiencies could enhance chondrogenic differentiation and apoptosis of ATDC5 cells. TrxR2 deficiencies increased accumulation of cartilage glycosaminoglycans (GAGs) and mineralization. TrxR2 deficiencies also stimulated expression of extracellular (ECM) gene including Collagen II and Aggrecan. The enhanced chondrogenic properties were further confirmed by activation of Akt signaling which are required for chondrogenesis. In addition, TrxR2 deficiencies promoted chondrocyte proliferation through acceleration of cell cycle progression by increase in both S and G2/M phase cell distribution accompanied with induction of parathyroid hormone-related protein (PTHrP). Moreover, TrxR2 deficiencies induced chondrocyte death via apoptosis and increased cell sensitivity to exogenous oxidative stress. Furthermore, TrxR2 deficiencies induced emission of mitochondrial reactive oxygen species (ROS) without alteration of mitochondrial membrane potential and intracellular ATP content. Finally, treatment of TrxR2 deficiency cells with N-acetylcysteine (NAC) inhibited mitochondrial ROS production and chondrocyte apoptosis. NAC also prevented chondrogenic differentiation of TrxR2 deficiency cells by suppression of ECM gene expression, GAGs accumulation and mineralization, as well as attenuation of Akt signaling. Thus, TrxR2-mediated mitochondrial integrity is indispensable for chondrogenic differentiation of ATDC5 cells. TrxR2 deficiency-induced impaired proliferation and death of chondrocytes may be the pathological mechanism of the osteoarthropathy due to Se deficiency. Notably, this study also uncover the roles of

  12. Humanin rescues cultured rat cortical neurons from NMDA-induced toxicity through the alleviation of mitochondrial dysfunction

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    Cui A

    2017-04-01

    Full Text Available Ai-Ling Cui,1 Ying-Hua Zhang,2 Jian-Zhong Li,3 Tianbin Song,4 Xue-Min Liu,1 Hui Wang,2 Ce Zhang,5 Guo-Lin Ma,6 Hui Zhang,7 Kefeng Li8 1Anatomy Department, Changzhi Medical College, Changzhi, Shanxi, 2Key Laboratory of Tissue Regeneration of Henan Province, Xinxiang Medical University, Xinxiang, Henan, 3Clinical Laboratory of Heji Hospital Affiliated to Changzhi Medical College, Changzhi, Shanxi, 4Department of Nuclear Medicine, Xuanwu Hospital, Capital Medical University, Beijing, 5Department of Physiology, Shanxi Medical University, Taiyuan, Shanxi, 6Department of Radiology, China-Japan Friendship Hospital, Beijing, 7Department of Radiology, First Clinical Medical College, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China; 8School of Medicine, University of California – San Diego, San Diego, CA, USA Abstract: N-methyl-D-aspartate (NDMA receptor-mediated excitotoxicity has been implicated in a variety of pathological situations such as Alzheimer’s disease (AD and Parkinson’s disease. However, no effective treatments for the same have been developed so far. Humanin (HN is a 24-amino acid peptide originally cloned from the brain of patients with AD and it prevents stress-induced cell death in many cells/tissues. In our previous study, HN was found to effectively rescue rat cortical neurons. It is still not clear whether HN protects the neurons through the attenuation of mitochondrial dysfunction. In this study, excitatory toxicity was induced by NMDA, which binds the NMDA receptor in primarily cultured rat cortical neurons. We found that NMDA (100 µmol/L dramatically induced the decrease of cell viability and caused mitochondrial dysfunction. Pretreatment of the neurons with HN (1 µmol/L led to significant increases of mitochondrial succinate dehydrogenase (SDH activity and membrane potential. In addition, HN pretreatment significantly reduced the excessive production of both reactive oxygen species (ROS and nitric

  13. Ginsenoside Rb1 Protects Neonatal Rat Cardiomyocytes from Hypoxia/Ischemia Induced Apoptosis and Inhibits Activation of the Mitochondrial Apoptotic Pathway

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    Xu Yan

    2014-01-01

    Full Text Available Aim. To investigate the effect of Ginsenoside Rb1 (GS-Rb1 on hypoxia/ischemia (H/I injury in cardiomyocytes in vitro and the mitochondrial apoptotic pathway mediated mechanism. Methods. Neonatal rat cardiomyocytes (NRCMs for the H/I groups were kept in DMEM without glucose and serum, and were placed into a hypoxic jar for 24 h. GS-Rb1 at concentrations from 2.5 to 40 µM was given during hypoxic period for 24 h. NRCMs injury was determined by MTT and lactate dehydrogenase (LDH leakage assay. Cell apoptosis, ROS accumulation, and mitochondrial membrane potential (MMP were assessed by flow cytometry. Cytosolic translocation of mitochondrial cytochrome c and Bcl-2 family proteins were determined by Western blot. Caspase-3 and caspase-9 activities were determined by the assay kit. Results. GS-Rb1 significantly reduced cell death and LDH leakage induced by H/I. It also reduced H/I induced NRCMs apoptosis induced by H/I, in accordance with a minimal reactive oxygen species (ROS burst. Moreover, GS-Rb1 markedly decreased the translocation of cytochrome c from the mitochondria to the cytosol, increased the Bcl-2/ Bax ratio, and preserved mitochondrial transmembrane potential (ΔΨm. Its administration also inhibited activities of caspase-9 and caspase-3. Conclusion. Administration of GS-Rb1 during H/I in vitro is involved in cardioprotection by inhibiting apoptosis, which may be due to inhibition of the mitochondrial apoptotic pathway.

  14. Acute inhibition of selected membrane-proximal mouse T cell receptor signaling by mitochondrial antagonists.

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    Kwangmi Kim

    2009-11-01

    Full Text Available T cells absorb nanometric membrane vesicles, prepared from plasma membrane of antigen presenting cells, via dual receptor/ligand interactions of T cell receptor (TCR with cognate peptide/major histocompatibility complex (MHC plus lymphocyte function-associated antigen 1 (LFA-1 with intercellular adhesion molecule 1. TCR-mediated signaling for LFA-1 activation is also required for the vesicle absorption. Exploiting those findings, we had established a high throughput screening (HTS platform and screened a library for isolation of small molecules inhibiting the vesicle absorption. Follow-up studies confirmed that treatments (1 hour with various mitochondrial antagonists, including a class of anti-diabetic drugs (i.e., Metformin and Phenformin, resulted in ubiquitous inhibition of the vesicle absorption without compromising viability of T cells. Further studies revealed that the mitochondrial drug treatments caused impairment of specific membrane-proximal TCR signaling event(s. Thus, activation of Akt and PLC-gamma1 and entry of extracellular Ca(2+ following TCR stimulation were attenuated while polymerization of monomeric actins upon TCR triggering progressed normally after the treatments. Dynamic F-actin rearrangement concurring with the vesicle absorption was also found to be impaired by the drug treatments, implying that the inhibition by the drug treatments of downstream signaling events (and the vesicle absorption could result from lack of directional relocation of signaling and cell surface molecules. We also assessed the potential application of mitochondrial antagonists as immune modulators by probing effects of the long-term drug treatments (24 hours on viability of resting primary T cells and cell cycle progression of antigen-stimulated T cells. This study unveils a novel regulatory mechanism for T cell immunity in response to environmental factors having effects on mitochondrial function.

  15. Regorafenib impairs mitochondrial functions, activates AMP-activated protein kinase, induces autophagy, and causes rat hepatocyte necrosis.

    Science.gov (United States)

    Weng, Zuquan; Luo, Yong; Yang, Xi; Greenhaw, James J; Li, Haibo; Xie, Liming; Mattes, William B; Shi, Qiang

    2015-01-02

    The tyrosine kinase inhibitor regorafenib was approved by regulatory agencies for cancer treatment, albeit with strong warnings of severe hepatotoxicity included in the product label. The basis of this toxicity is unknown; one possible mechanism, that of mitochondrial damage, was tested. In isolated rat liver mitochondria, regorafenib directly uncoupled oxidative phosphorylation (OXPHOS) and promoted calcium overload-induced swelling, which were respectively prevented by the recoupler 6-ketocholestanol (KC) and the mitochondrial permeability transition (MPT) pore blocker cyclosporine A (CsA). In primary hepatocytes, regorafenib uncoupled OXPHOS, disrupted mitochondrial inner membrane potential (MMP), and decreased cellular ATP at 1h, and triggered MPT at 3h, which was followed by necrosis but not apoptosis at 7h and 24h, all of which were abrogated by KC. The combination of the glycolysis enhancer fructose plus the mitochondrial ATPase synthase inhibitor oligomycin A abolished regorafenib induced necrosis at 7h. This effect was not seen at 24h nor with the fructose or oligomycin A separately. CsA in combination with trifluoperazine, both MPT blockers, showed similar effects. Two compensatory mechanisms, activation of AMP-activated protein kinase (AMPK) to ameliorate ATP shortage and induction of autophagy to remove dysfunctional mitochondria, were found to be mobilized. Hepatocyte necrosis was enhanced either by the AMPK inhibitor Compound C or the autophagy inhibitor chloroquine, while autophagy inducer rapamycin was strongly cytoprotective. Remarkably, all toxic effects were observed at clinically-relevant concentrations of 2.5-15μM. These data suggest that uncoupling of OXPHOS and the resulting ATP shortage and MPT induction are the key mechanisms for regorafenib induced hepatocyte injury, and AMPK activation and autophagy induction serve as pro-survival pathways against such toxicity. Published by Elsevier Ireland Ltd.

  16. Adhesion Regulating Molecule 1 Mediates HAP40 Overexpression-Induced Mitochondrial Defects

    Science.gov (United States)

    Huang, Zih-Ning; Chung, Her Min; Fang, Su-Chiung; Her, Lu-Shiun

    2017-01-01

    Striatal neuron death in Huntington's disease is associated with abnormal mitochondrial dynamics and functions. However, the mechanisms for this mitochondrial dysregulation remain elusive. Increased accumulation of Huntingtin-associated protein 40 (HAP40) has been shown to be associated with Huntington's disease. However, the link between increased HAP40 and Huntington's disease remains largely unknown. Here we show that HAP40 overexpression causes mitochondrial dysfunction and reduces cell viability in the immortalized mouse striatal neurons. HAP40-associated mitochondrial dysfunction is associated with reduction of adhesion regulating molecule 1 (ADRM1) protein. Consistently, depletion of ADRM1 by shRNAs impaired mitochondrial functions and increased mitochondrial fragmentation in mouse striatal cells. Moreover, reducing ADRM1 levels enhanced activity of fission factor dynamin-related GTPase protein 1 (Drp1) via increased phosphorylation at serine 616 of Drp1 (Drp1Ser616). Restoring ADRM1 protein levels was able to reduce HAP40-induced ROS levels and mitochondrial fragmentation and improved mitochondrial functions and cell viability. Moreover, reducing Drp1 activity by Drp1 inhibitor, Mdivi-1, ameliorates both HAP40 overexpression- and ADRM1 depletion-induced mitochondrial dysfunction. Taken together, our studies suggest that HAP40-mediated reduction of ADRM1 alters the mitochondrial fission activity and results in mitochondrial fragmentation and mitochondrial dysfunction. PMID:29209146

  17. BID links ferroptosis to mitochondrial cell death pathways

    Directory of Open Access Journals (Sweden)

    Sandra Neitemeier

    2017-08-01

    Full Text Available Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the Xc- system or inhibition of glutathione peroxidase 4 (Gpx4 to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation.In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by Xc- inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Keywords: Ferroptosis, BID, Mitochondria, CRISPR, Oxytosis, Neuronal death

  18. Manganese induces mitochondrial dynamics impairment and apoptotic cell death: a study in human Gli36 cells.

    Science.gov (United States)

    Alaimo, Agustina; Gorojod, Roxana M; Miglietta, Esteban A; Villarreal, Alejandro; Ramos, Alberto J; Kotler, Mónica L

    2013-10-25

    Manganese (Mn) is an essential trace element due to its participation in many physiological processes. However, overexposure to this metal leads to a neurological disorder known as Manganism whose clinical manifestations and molecular mechanisms resemble Parkinson's disease. Several lines of evidence implicate astrocytes as an early target of Mn neurotoxicity being the mitochondria the most affected organelles. The aim of this study was to investigate the possible mitochondrial dynamics alterations in Mn-exposed human astrocytes. Therefore, we employed Gli36 cells which express the astrocytic markers GFAP and S100B. We demonstrated that Mn triggers the mitochondrial apoptotic pathway revealed by increased Bax/Bcl-2 ratio, by the loss of mitochondrial membrane potential and by caspase-9 activation. This apoptotic program may be in turn responsible of caspase-3/7 activation, PARP-1 cleavage, chromatin condensation and fragmentation. In addition, we determined that Mn induces deregulation in mitochondria-shaping proteins (Opa-1, Mfn-2 and Drp-1) expression levels in parallel with the disruption of the mitochondrial network toward to an exacerbated fragmentation. Since mitochondrial dynamics is altered in several neurodegenerative diseases, these proteins could become future targets to be considered in Manganism treatment. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  19. Melatonin-induced increase of lipid droplets accumulation and in vitro maturation in porcine oocytes is mediated by mitochondrial quiescence.

    Science.gov (United States)

    He, Bin; Yin, Chao; Gong, Yabin; Liu, Jie; Guo, Huiduo; Zhao, Ruqian

    2018-01-01

    Melatonin, the major pineal secretory product, has a significant impact on the female reproductive system. Recently, the beneficial effects of melatonin on mammalian oocyte maturation and embryonic development have drawn increased attention. However, the exact underlying mechanisms remain to be fully elucidated. This study demonstrates that supplementing melatonin to in vitro maturation (IVM) medium enhances IVM rate, lipid droplets (LDs) accumulation as well as triglyceride content in porcine oocytes. Decrease of mitochondrial membrane potential, mitochondrial respiratory chain complex IV activity as well as mitochondrial reactive oxygen species (mROS) content indicated that melatonin induced a decrease of mitochondrial activity. The copy number of mitochondrial DNA (mtDNA) which encodes essential subunits of oxidative phosphorylation (OXPHOS), was not affected by melatonin. However, the expression of mtDNA-encoded genes was significantly down-regulated after melatonin treatment. The DNA methyltransferase DNMT1, which regulates methylation and expression of mtDNA, was increased and translocated into the mitochondria in melatonin-treated oocytes. The inhibitory effect of melatonin on the expression of mtDNA was significantly prevented by simultaneous addition of DNMT1 inhibitor, which suggests that melatonin regulates the transcription of mtDNA through up-regulation of DNMT1 and mtDNA methylation. Increase of triglyceride contents after inhibition of OXPHOS indicated that mitochondrial quiescence is crucial for LDs accumulation in oocytes. Taken together, our results suggest that melatonin-induced reduction in mROS production and increase in IVM, and LDs accumulation in porcine oocytes is mediated by mitochondrial quiescence. © 2017 Wiley Periodicals, Inc.

  20. Radiation-induced damage of membranes

    International Nuclear Information System (INIS)

    Yonei, Shuji

    1977-01-01

    An outline of membranous structure was stated, and radiation-induced damage of membranes were surveyed. By irradiation, permeability of membranes, especially passive transportation mechanism, was damaged, and glycoprotein in the surface layers of cells and the surface layer structures were changed. The intramembranous damage was induced by decrease of electrophoresis of nuclear mambranes and a quantitative change of cytochrome P450 of microsomal membranes of the liver, and peroxidation of membranous lipid and SH substitute damage of membranous protein were mentioned as the mechanism of membranous damage. Recovery of membranous damage depends on radiation dose and temperature, and membranous damage participates largely in proliferation death. (tsunoda, M.)

  1. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Aibin; Liu, Jingyi [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Institute of Cardiovascular Disease, General Hospital of Beijing Command, PLA, Beijing (China); Liu, Peilin; Jia, Min; Wang, Han [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China); Tao, Ling, E-mail: lingtao2006@gmail.com [Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an (China)

    2014-04-18

    Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H{sub 2}O{sub 2} led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H{sub 2}O{sub 2} and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the

  2. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome

    International Nuclear Information System (INIS)

    Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

    2014-01-01

    Highlights: • Metabolic syndrome exacerbated MI/R induced injury accompanied by decreased Nur77. • ROS led to Nur77 translocation in metabolic syndrome. • Inhibiting relocation of Nur77 to mitochondria reduced ROS-induced cardiomyocyte injury in metabolic syndrome. - Abstract: Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H 2 O 2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H 2 O 2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process

  3. Non-bilayer structures in mitochondrial membranes regulate ATP synthase activity.

    Science.gov (United States)

    Gasanov, Sardar E; Kim, Aleksandr A; Yaguzhinsky, Lev S; Dagda, Ruben K

    2018-02-01

    Cardiolipin (CL) is an anionic phospholipid at the inner mitochondrial membrane (IMM) that facilitates the formation of transient non-bilayer (non-lamellar) structures to maintain mitochondrial integrity. CL modulates mitochondrial functions including ATP synthesis. However, the biophysical mechanisms by which CL generates non-lamellar structures and the extent to which these structures contribute to ATP synthesis remain unknown. We hypothesized that CL and ATP synthase facilitate the formation of non-bilayer structures at the IMM to stimulate ATP synthesis. By using 1 H NMR and 31 P NMR techniques, we observed that increasing the temperature (8°C to 37°C), lowering the pH (3.0), or incubating intact mitochondria with CTII - an IMM-targeted toxin that increases the formation of immobilized non-bilayer structures - elevated the formation of non-bilayer structures to stimulate ATP synthesis. The F 0 sector of the ATP synthase complex can facilitate the formation of non-bilayer structures as incubating model membranes enriched with IMM-specific phospholipids with exogenous DCCD-binding protein of the F 0 sector (DCCD-BPF) elevated the formation of immobilized non-bilayer structures to a similar manner as CTII. Native PAGE assays revealed that CL, but not other anionic phospholipids, specifically binds to DCCD-BPF to promote the formation of stable lipid-protein complexes. Mechanistically, molecular docking studies identified two lipid binding sites for CL in DCCD-BPF. We propose a new model of ATP synthase regulation in which CL mediates the formation of non-bilayer structures that serve to cluster protons and ATP synthase complexes as a mechanism to enhance proton translocation to the F 0 sector, and thereby increase ATP synthesis. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Single-cell analysis of dihydroartemisinin-induced apoptosis through reactive oxygen species-mediated caspase-8 activation and mitochondrial pathway in ASTC-a-1 cells using fluorescence imaging techniques

    Science.gov (United States)

    Lu, Ying-Ying; Chen, Tong-Sheng; Wang, Xiao-Ping; Li, Li

    2010-07-01

    Dihydroartemisinin (DHA), a front-line antimalarial herbal compound, has been shown to possess promising anticancer activity with low toxicity. We have previously reported that DHA induced caspase-3-dependent apoptosis in human lung adenocarcinoma cells. However, the cellular target and molecular mechanism of DHA-induced apoptosis is still poorly defined. We use confocal fluorescence microscopy imaging, fluorescence resonance energy transfer, and fluorescence recovery after photobleaching techniques to explore the roles of DHA-elicited reactive oxygen species (ROS) in the DHA-induced Bcl-2 family proteins activation, mitochondrial dysfunction, caspase cascade, and cell death. Cell Counting Kit-8 assay and flow cytometry analysis showed that DHA induced ROS-mediated apoptosis. Confocal imaging analysis in a single living cell and Western blot assay showed that DHA triggered ROS-dependent Bax translocation, mitochondrial membrane depolarization, alteration of mitochondrial morphology, cytochrome c release, caspase-9, caspase-8, and caspase-3 activation, indicating the coexistence of ROS-mediated mitochondrial and death receptor pathway. Collectively, our findings demonstrate for the first time that DHA induces cell apoptosis by triggering ROS-mediated caspase-8/Bid activation and the mitochondrial pathway, which provides some novel insights into the application of DHA as a potential anticancer drug and a new therapeutic strategy by targeting ROS signaling in lung adenocarcinoma therapy in the future.

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

    Science.gov (United States)

    Haupt, Sara; Malik, Zvi; Ehrenberg, Benjamin

    2014-01-01

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

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

    International Nuclear Information System (INIS)

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

    1988-01-01

    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

  7. ACS6, a Hydrogen sulfide-donating derivative of sildenafil, inhibits homocysteine-induced apoptosis by preservation of mitochondrial function

    Directory of Open Access Journals (Sweden)

    Tang Xiao-Qing

    2011-08-01

    Full Text Available Abstract Background The hydrogen sulfide-releasing sildenafil, ACS6, has been demonstrated to inhibit superoxide formation through donating hydrogen sulfide (H2S. We have found that H2S antagonizes homocysteine-induced oxidative stress and neurotoxicity. The aim of the present study is to explore the protection of ACS6 against homocysteine-triggered cytotoxicity and apoptosis and the molecular mechanisms underlying in PC12 cells. Methods Cell viability was determined by Cell Counting Kit-8 assay. Cell apoptosis was observed using the chromatin dye Hoechst 33258 and analyzed by Flow Cytometry after propidium iodide staining. Mitochondrial membrane potential was monitored using the fluorescent dye Rh123. Intracellular reactive oxygen species were determined by oxidative conversion of cell permeable 2',7'-dichlorfluorescein-diacetate to fluorescent 2',7'-dichlorfluorescein. The expression of cleaved caspase-3 and bcl-2 and the accumulation of cytosolic cytochrome c were analyzed by Western blot. Results We show that ACS6 protects PC12 cells against cytotoxicity and apoptosis induced by homocysteine and blocks homocysteine-triggered cytochrome c release and caspase-3 activation. ACS6 treatment results in not only prevention of homocysteine-caused mitochondrial membrane potential (Δψ loss and reactive oxygen species (ROS overproduction but also reversal of Bcl-2 down-expression. Conclusions These results indicate that ACS6 protects PC12 cells against homocysteine-induced cytotoxicity and apoptosis by preservation of mitochondrial function though inhibiting both loss of Δψ and accumulation of ROS as well as modulating the expression of Bcl-2. Our study provides evidence both for a neuroprotective effect of ACS6 and for further evaluation of ACS6 as novel neuroprotectants for Alzheimer's disease associated with homocysteine.

  8. Hydrogen sulfide protects HUVECs against hydrogen peroxide induced mitochondrial dysfunction and oxidative stress.

    Directory of Open Access Journals (Sweden)

    Ya-Dan Wen

    Full Text Available BACKGROUND: Hydrogen sulfide (H₂S has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer's disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂ induced endothelial cells damage. METHODOLOGY AND PRINCIPAL FINDINGS: H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE, diphenyl-l-pyrenylphosphine (DPPP and malonaldehyde (MDA levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG, a selective inhibitor of cystathionine γ-lyase (CSE, abolished the protective effects of H₂S donors. INNOVATION: This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences. SIGNIFICANCE: H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.

  9. Rat liver mitochondrial damage under acute or chronic carbon tetrachloride-induced intoxication: Protection by melatonin and cranberry flavonoids

    International Nuclear Information System (INIS)

    Cheshchevik, V.T.; Lapshina, E.A.; Dremza, I.K.; Zabrodskaya, S.V.; Reiter, R.J.; Prokopchik, N.I.; Zavodnik, I.B.

    2012-01-01

    In current societies, the risk of toxic liver damage has markedly increased. The aim of the present work was to carry out further research into the mechanism(s) of liver mitochondrial damage induced by acute (0.8 g/kg body weight, single injection) or chronic (1.6 g/ kg body weight, 30 days, biweekly injections) carbon tetrachloride – induced intoxication and to evaluate the hepatoprotective potential of the antioxidant, melatonin, as well as succinate and cranberry flavonoids in rats. Acute intoxication resulted in considerable impairment of mitochondrial respiratory parameters in the liver. The activity of mitochondrial succinate dehydrogenase (complex II) decreased (by 25%, p 4 displayed obvious irreversible impairments. Long-term melatonin administration (10 mg/kg, 30 days, daily) to chronically intoxicated rats diminished the toxic effects of CCl 4 , reducing elevated plasma activities of alanine aminotransferase and aspartate aminotransferase and bilirubin concentration, prevented accumulation of membrane lipid peroxidation products in rat liver and resulted in apparent preservation of the mitochondrial ultrastructure. The treatment of the animals by the complex of melatonin (10 mg/kg) plus succinate (50 mg/kg) plus cranberry flavonoids (7 mg/kg) was even more effective in prevention of toxic liver injury and liver mitochondria damage. Highlights: ► After 30-day chronic CCl 4 intoxication mitochondria displayed considerable changes. ► The functional parameters of mitochondria were similar to the control values. ► Melatonin + succinate + flavonoids prevented mitochondrial ultrastructure damage. ► The above complex enhanced regenerative processes in the liver.

  10. CoQ10 Deficiency May Indicate Mitochondrial Dysfunction in Cr(VI Toxicity

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    Xiali Zhong

    2017-04-01

    Full Text Available To investigate the toxic mechanism of hexavalent chromium Cr(VI and search for an antidote for Cr(VI-induced cytotoxicity, a study of mitochondrial dysfunction induced by Cr(VI and cell survival by recovering mitochondrial function was performed. In the present study, we found that the gene expression of electron transfer flavoprotein dehydrogenase (ETFDH was strongly downregulated by Cr(VI exposure. The levels of coenzyme 10 (CoQ10 and mitochondrial biogenesis presented by mitochondrial mass and mitochondrial DNA copy number were also significantly reduced after Cr(VI exposure. The subsequent, Cr(VI-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species (ROS accumulation, caspase-3 and caspase-9 activation, decreased superoxide dismutase (SOD and ATP production, increased methane dicarboxylic aldehyde (MDA content, mitochondrial membrane depolarization and mitochondrial permeability transition pore (MPTP opening, increased Ca2+ levels, Cyt c release, decreased Bcl-2 expression, and significantly elevated Bax expression. The Cr(VI-induced deleterious changes were attenuated by pretreatment with CoQ10 in L-02 hepatocytes. These data suggest that Cr(VI induces CoQ10 deficiency in L-02 hepatocytes, indicating that this deficiency may be a biomarker of mitochondrial dysfunction in Cr(VI poisoning and that exogenous administration of CoQ10 may restore mitochondrial function and protect the liver from Cr(VI exposure.

  11. Mitochondrial uncoupling proteins in unicellular eukaryotes.

    Science.gov (United States)

    Jarmuszkiewicz, Wieslawa; Woyda-Ploszczyca, Andrzej; Antos-Krzeminska, Nina; Sluse, Francis E

    2010-01-01

    Uncoupling proteins (UCPs) are members of the mitochondrial anion carrier protein family that are present in the mitochondrial inner membrane and mediate free fatty acid (FFA)-activated, purine nucleotide (PN)-inhibited proton conductance. Since 1999, the presence of UCPs has been demonstrated in some non-photosynthesising unicellular eukaryotes, including amoeboid and parasite protists, as well as in non-fermentative yeast and filamentous fungi. In the mitochondria of these organisms, UCP activity is revealed upon FFA-induced, PN-inhibited stimulation of resting respiration and a decrease in membrane potential, which are accompanied by a decrease in membranous ubiquinone (Q) reduction level. UCPs in unicellular eukaryotes are able to divert energy from oxidative phosphorylation and thus compete for a proton electrochemical gradient with ATP synthase. Our recent work indicates that membranous Q is a metabolic sensor that might utilise its redox state to release the PN inhibition of UCP-mediated mitochondrial uncoupling under conditions of phosphorylation and resting respiration. The action of reduced Q (QH2) could allow higher or complete activation of UCP. As this regulatory feature was demonstrated for microorganism UCPs (A. castellanii UCP), plant and mammalian UCP1 analogues, and UCP1 in brown adipose tissue, the process could involve all UCPs. Here, we discuss the functional connection and physiological role of UCP and alternative oxidase, two main energy-dissipating systems in the plant-type mitochondrial respiratory chain of unicellular eukaryotes, including the control of cellular energy balance as well as preventive action against the production of reactive oxygen species. Copyright © 2009 Elsevier B.V. All rights reserved.

  12. Mitochondrial vulnerability and increased susceptibility to nutrient-induced cytotoxicity in fibroblasts from leigh syndrome French canadian patients.

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    Yan Burelle

    Full Text Available Mutations in LRPPRC are responsible for the French Canadian variant of Leigh Syndrome (LSFC, a severe disorder characterized biochemically by a tissue-specific deficiency of cytochrome c oxidase (COX and clinically by the occurrence of severe and deadly acidotic crises. Factors that precipitate these crises remain unclear. To better understand the physiopathology and identify potential treatments, we performed a comprehensive analysis of mitochondrial function in LSFC and control fibroblasts. Furthermore, we have used this cell-based model to screen for conditions that promote premature cell death in LSFC cells and test the protective effect of ten interventions targeting well-defined aspects of mitochondrial function. We show that, despite maintaining normal ATP levels, LSFC fibroblasts present several mitochondrial functional abnormalities under normal baseline conditions, which likely impair their capacity to respond to stress. This includes mitochondrial network fragmentation, impaired oxidative phosphorylation capacity, lower membrane potential, increased sensitivity to Ca2+-induced permeability transition, but no changes in reactive oxygen species production. We also show that LSFC fibroblasts display enhanced susceptibility to cell death when exposed to palmitate, an effect that is potentiated by high lactate, while high glucose or acidosis alone or in combination were neutral. Furthermore, we demonstrate that compounds that are known to promote flux through the electron transport chain independent of phosphorylation (methylene blue, dinitrophenol, or modulate fatty acid (L-carnitine or Krebs cycle metabolism (propionate are protective, while antioxidants (idebenone, N-acetyl cysteine, resveratrol exacerbate palmitate plus lactate-induced cell death. Collectively, beyond highlighting multiple alterations in mitochondrial function and increased susceptibility to nutrient-induced cytotoxicity in LSFC fibroblasts, these results raise

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

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    Susannah K Rogers

    2014-02-01

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

  14. Modulation of mitochondrial morphology by bioenergetics defects in primary human fibroblasts

    DEFF Research Database (Denmark)

    Guillery, O.; Malka, F.; Frachon, P.

    2008-01-01

    induced partial but significant mitochondrial fragmentation, whereas dissipation of mitochondrial membrane potential (D Psi m) provoked complete fragmentation, and glycolysis inhibition had no effect. Oxidative phosphorylation defective fibroblasts had essentially normal filamentous mitochondria under...... basal conditions, although when challenged some of them presented with mild alteration of fission or fusion efficacy. Severely defective cells disclosed complete mitochondrial fragmentation under glycolysis inhibition. In conclusion, mitochondrial morphology is modulated by D Psi m but loosely linked...... to mitochondrial oxidative phosphorylation. Its alteration by glycolysis, inhibition points to a severe oxidative phosphorylation defect. (C) 2008 Elsevier B.V. All rights reserved Udgivelsesdato: 2008/4...

  15. Inhibition of NAPDH Oxidase 2 (NOX2 Prevents Oxidative Stress and Mitochondrial Abnormalities Caused by Saturated Fat in Cardiomyocytes.

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    Leroy C Joseph

    Full Text Available Obesity and high saturated fat intake increase the risk of heart failure and arrhythmias. The molecular mechanisms are poorly understood. We hypothesized that physiologic levels of saturated fat could increase mitochondrial reactive oxygen species (ROS in cardiomyocytes, leading to abnormalities of calcium homeostasis and mitochondrial function. We investigated the effect of saturated fat on mitochondrial function and calcium homeostasis in isolated ventricular myocytes. The saturated fatty acid palmitate causes a decrease in mitochondrial respiration in cardiomyocytes. Palmitate, but not the monounsaturated fatty acid oleate, causes an increase in both total cellular ROS and mitochondrial ROS. Palmitate depolarizes the mitochondrial inner membrane and causes mitochondrial calcium overload by increasing sarcoplasmic reticulum calcium leak. Inhibitors of PKC or NOX2 prevent mitochondrial dysfunction and the increase in ROS, demonstrating that PKC-NOX2 activation is also required for amplification of palmitate induced-ROS. Cardiomyocytes from mice with genetic deletion of NOX2 do not have palmitate-induced ROS or mitochondrial dysfunction. We conclude that palmitate induces mitochondrial ROS that is amplified by NOX2, causing greater mitochondrial ROS generation and partial depolarization of the mitochondrial inner membrane. The abnormal sarcoplasmic reticulum calcium leak caused by palmitate could promote arrhythmia and heart failure. NOX2 inhibition is a potential therapy for heart disease caused by diabetes or obesity.

  16. Loss of mitochondrial transmembrane potential and caspase-9 activation during apoptosis induced by the novel styryl-lactone goniothalamin in HL-60 leukemia cells.

    Science.gov (United States)

    Inayat-Hussain, S H; Annuar, B O; Din, L B; Ali, A M; Ross, D

    2003-08-01

    Styryl-lactones such as goniothalamin represent a new class of compounds with potential anti-cancer properties. In this study, we investigated the mechanisms of goniothalamin (GTN), a plant styryl-lactone induced apoptosis in human promyelocytic leukemia HL-60 cells. This plant extract resulted in apoptosis in HL-60 cells as assessed by the externalisation of phosphatidylserine. Using the mitochondrial membrane dye (DIOC(6)) in conjunction with flow cytometry, we found that GTN treated HL-60 cells demonstrated a loss of mitochondrial transmembrane potential (Deltapsi(m)). Further immunoblotting on these cells showed activation of initiator caspase-9 and the executioner caspases-3 and -7. Pretreatment with the pharmacological caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD.FMK) abrogated apoptosis as assessed by all of the apoptotic features in this study. In summary, our results demonstrate that goniothalamin-induced apoptosis occurs via the mitochondrial pathway in a caspase dependent manner.

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

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    Yajin Liao

    2017-02-01

    Full Text Available The mitochondrial calcium uniporter (MCU—a calcium uniporter on the inner membrane of mitochondria—controls the mitochondrial calcium uptake in normal and abnormal situations. Mitochondrial calcium is essential for the production of adenosine triphosphate (ATP; however, excessive calcium will induce mitochondrial dysfunction. Calcium homeostasis disruption and mitochondrial dysfunction is observed in many neurodegenerative disorders. However, the role and regulatory mechanism of the MCU in the development of these diseases are obscure. In this review, we summarize the role of the MCU in controlling oxidative stress-elevated mitochondrial calcium and its function in neurodegenerative disorders. Inhibition of the MCU signaling pathway might be a new target for the treatment of neurodegenerative disorders.

  18. Radioprotection by Rubia cordifolia: studies on mitochondrial membranes

    International Nuclear Information System (INIS)

    Kamat, J.P.; Mishra, K.P.

    2005-01-01

    Oxidative stress is inevitable to living cells and induces several adverse effects in human ill health. Ionizing radiations are the major sources of ROS. Our present study was aimed to examine the radioprotective role of one popular ayurvedic herbal drug, Rubia cordifolia (Rubiaceae) (Rc) against cellular damage. The results showed that aqueous extract of Rc ( ∼50 μg/ml) significantly prevented rat liver mitochondrial damage induced by ionizing radiation. Single strand breaks induced in plasmid pBR322 DNA following ionizing radiations was effectively prevented by Rc extract. Based on several biochemical results as well as radical scavenging ability of the extract, it can be suggested that extract of Rc may have possible therapeutic applications in the prevention of radiation-induced cellular damage. (author)

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

    DEFF Research Database (Denmark)

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

    2009-01-01

    , the exceptional nature of cardiolipins is characterized by their small charged head group connected to typically four hydrocarbon chains. In this work, we present atomic-scale molecular dynamics simulations of the inner mitochondrial membrane modeled as a mixture of cardiolipins (CLs), phosphatidylcholines (PCs...

  20. Mitochondrial rejuvenation after induced pluripotency.

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    Steven T Suhr

    2010-11-01

    Full Text Available As stem cells of the early embryo mature and differentiate into all tissues, the mitochondrial complement undergoes dramatic functional improvement. Mitochondrial activity is low to minimize generation of DNA-damaging reactive oxygen species during pre-implantation development and increases following implantation and differentiation to meet higher metabolic demands. It has recently been reported that when the stem cell type known as induced pluripotent stem cells (IPSCs are re-differentiated for several weeks in vitro, the mitochondrial complement progressively re-acquires properties approximating input fibroblasts, suggesting that despite the observation that IPSC conversion "resets" some parameters of cellular aging such as telomere length, it may have little impact on other age-affected cellular systems such as mitochondria in IPSC-derived cells.We have examined the properties of mitochondria in two fibroblast lines, corresponding IPSCs, and fibroblasts re-derived from IPSCs using biochemical methods and electron microscopy, and found a dramatic improvement in the quality and function of the mitochondrial complement of the re-derived fibroblasts compared to input fibroblasts. This observation likely stems from two aspects of our experimental design: 1 that the input cell lines used were of advanced cellular age and contained an inefficient mitochondrial complement, and 2 the re-derived fibroblasts were produced using an extensive differentiation regimen that may more closely mimic the degree of growth and maturation found in a developing mammal.These results - coupled with earlier data from our laboratory - suggest that IPSC conversion not only resets the "biological clock", but can also rejuvenate the energetic capacity of derived cells.

  1. Protosappanin B protects PC12 cells against oxygen-glucose deprivation-induced neuronal death by maintaining mitochondrial homeostasis via induction of ubiquitin-dependent p53 protein degradation.

    Science.gov (United States)

    Zeng, Ke-Wu; Liao, Li-Xi; Zhao, Ming-Bo; Song, Fang-Jiao; Yu, Qian; Jiang, Yong; Tu, Peng-Fei

    2015-03-15

    Protosappanin B (PTB) is a bioactive dibenzoxocin derivative isolated from Caesalpinia sappan L. Here, we investigated the neuroprotective effects and the potential mechanisms of PTB on oxygen-glucose deprivation (OGD)-injured PC12 cells. Results showed that PTB significantly increased cell viability, inhibited cell apoptosis and up-regulated the expression of growth-associated protein 43 (a marker of neural outgrowth). Moreover, our study revealed that PTB effectively maintained mitochondrial homeostasis by up-regulation of mitochondrial membrane potential (MMP), inhibition of cytochrome c release from mitochondria and inactivation of mitochondrial caspase-9/3 apoptosis pathway. Further study showed that PTB significantly promoted cytoplasmic component degradation of p53 protein, a key negative regulator for mitochondrial function, resulting in a release of Bcl-2 from p53-Bcl-2 complex and an enhancing translocation of Bcl-2 to mitochondrial outer membrane. Finally, we found the degradation of p53 protein was induced by PTB via activation of a MDM2-dependent ubiquitination process. Taken together, our findings provided a new viewpoint of neuronal protection strategy for anoxia and ischemic injury with natural small molecular dibenzoxocin derivative by activating ubiquitin-dependent p53 protein degradation as well as increasing mitochondrial function. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. BID links ferroptosis to mitochondrial cell death pathways.

    Science.gov (United States)

    Neitemeier, Sandra; Jelinek, Anja; Laino, Vincenzo; Hoffmann, Lena; Eisenbach, Ina; Eying, Roman; Ganjam, Goutham K; Dolga, Amalia M; Oppermann, Sina; Culmsee, Carsten

    2017-08-01

    Ferroptosis has been defined as an oxidative and iron-dependent pathway of regulated cell death that is distinct from caspase-dependent apoptosis and established pathways of death receptor-mediated regulated necrosis. While emerging evidence linked features of ferroptosis induced e.g. by erastin-mediated inhibition of the X c - system or inhibition of glutathione peroxidase 4 (Gpx4) to an increasing number of oxidative cell death paradigms in cancer cells, neurons or kidney cells, the biochemical pathways of oxidative cell death remained largely unclear. In particular, the role of mitochondrial damage in paradigms of ferroptosis needs further investigation. In the present study, we find that erastin-induced ferroptosis in neuronal cells was accompanied by BID transactivation to mitochondria, loss of mitochondrial membrane potential, enhanced mitochondrial fragmentation and reduced ATP levels. These hallmarks of mitochondrial demise are also established features of oxytosis, a paradigm of cell death induced by X c - inhibition by millimolar concentrations of glutamate. Bid knockout using CRISPR/Cas9 approaches preserved mitochondrial integrity and function, and mediated neuroprotective effects against both, ferroptosis and oxytosis. Furthermore, the BID-inhibitor BI-6c9 inhibited erastin-induced ferroptosis, and, in turn, the ferroptosis inhibitors ferrostatin-1 and liproxstatin-1 prevented mitochondrial dysfunction and cell death in the paradigm of oxytosis. These findings show that mitochondrial transactivation of BID links ferroptosis to mitochondrial damage as the final execution step in this paradigm of oxidative cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  3. Lysosomal membrane permeabilization: Carbon nanohorn-induced reactive oxygen species generation and toxicity by this neglected mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Mei, E-mail: happy_deercn@163.com [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Zhang, Minfang; Tahara, Yoshio; Chechetka, Svetlana; Miyako, Eijiro [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Iijima, Sumio [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Faculty of Science and Technology, Meijo University, 1-501 Shiogamaguchi, Tenpaku, Nagoya 468-8502 (Japan); Yudasaka, Masako, E-mail: m-yudasaka@aist.go.jp [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan)

    2014-10-01

    Understanding the molecular mechanisms responsible for the cytotoxic effects of carbon nanomaterials is important for their future biomedical applications. Carbon nanotubular materials induce the generation of reactive oxygen species (ROS), which causes cell death; however, the exact details of this process are still unclear. Here, we identify a mechanism of ROS generation that is involved in the apoptosis of RAW264.7 macrophages caused by excess uptake of carbon nanohorns (CNHs), a typical type of carbon nanotubule. CNH accumulated in the lysosomes, where they induced lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteases, such as cathepsins, which in turn caused mitochondrial dysfunction and triggered the generation of ROS in the mitochondria. The nicotinamide adenine dinucleotide phosphate oxidase was not directly involved in CNH-related ROS production, and the ROS generation cannot be regulated by mitochondrial electron transport chain. ROS fed back to amplify the mitochondrial dysfunction, leading to the subsequent activation of caspases and cell apoptosis. Carbon nanotubules commonly accumulate in the lysosomes after internalization in cells; however, lysosomal dysfunction has not attracted much attention in toxicity studies of these materials. These results suggest that LMP, a neglected mechanism, may be the primary reason for carbon nanotubule toxicity. - Highlights: • We clarify an apoptotic mechanism of RAW264.7 cells caused by carbon nanohorns. • In the meantime, the mechanism of CNH-induced ROS generation is identified. • LMP is the initial factor of CNH-induced ROS generation and cell death. • Cathepsins work as mediators that connect LMP and mitochondrial dysfunction.

  4. Mitochondrial nucleoid clusters protect newly synthesized mtDNA during Doxorubicin- and Ethidium Bromide-induced mitochondrial stress

    Energy Technology Data Exchange (ETDEWEB)

    Alán, Lukáš, E-mail: lukas.alan@fgu.cas.cz; Špaček, Tomáš; Pajuelo Reguera, David; Jabůrek, Martin; Ježek, Petr

    2016-07-01

    Mitochondrial DNA (mtDNA) is compacted in ribonucleoprotein complexes called nucleoids, which can divide or move within the mitochondrial network. Mitochondrial nucleoids are able to aggregate into clusters upon reaction with intercalators such as the mtDNA depletion agent Ethidium Bromide (EB) or anticancer drug Doxorobicin (DXR). However, the exact mechanism of nucleoid clusters formation remains unknown. Resolving these processes may help to elucidate the mechanisms of DXR-induced cardiotoxicity. Therefore, we addressed the role of two key nucleoid proteins; mitochondrial transcription factor A (TFAM) and mitochondrial single-stranded binding protein (mtSSB); in the formation of mitochondrial nucleoid clusters during the action of intercalators. We found that both intercalators cause numerous aberrations due to perturbing their native status. By blocking mtDNA replication, both agents also prevented mtDNA association with TFAM, consequently causing nucleoid aggregation into large nucleoid clusters enriched with TFAM, co-existing with the normal nucleoid population. In the later stages of intercalation (> 48 h), TFAM levels were reduced to 25%. In contrast, mtSSB was released from mtDNA and freely distributed within the mitochondrial network. Nucleoid clusters mostly contained nucleoids with newly replicated mtDNA, however the nucleoid population which was not in replication mode remained outside the clusters. Moreover, the nucleoid clusters were enriched with p53, an anti-oncogenic gatekeeper. We suggest that mitochondrial nucleoid clustering is a mechanism for protecting nucleoids with newly replicated DNA against intercalators mediating genotoxic stress. These results provide new insight into the common mitochondrial response to mtDNA stress and can be implied also on DXR-induced mitochondrial cytotoxicity. - Highlights: • The mechanism for mitochondrial nucleoid clustering is proposed. • DNA intercalators (Doxorubicin or Ethidium Bromide) prevent TFAM

  5. Influenza virus induces apoptosis via BAD-mediated mitochondrial dysregulation.

    Science.gov (United States)

    Tran, Anh T; Cortens, John P; Du, Qiujiang; Wilkins, John A; Coombs, Kevin M

    2013-01-01

    Influenza virus infection results in host cell death and major tissue damage. Specific components of the apoptotic pathway, a signaling cascade that ultimately leads to cell death, are implicated in promoting influenza virus replication. BAD is a cell death regulator that constitutes a critical control point in the intrinsic apoptosis pathway, which occurs through the dysregulation of mitochondrial outer membrane permeabilization and the subsequent activation of downstream apoptogenic factors. Here we report a novel proviral role for the proapoptotic protein BAD in influenza virus replication. We show that influenza virus-induced cytopathology and cell death are considerably inhibited in BAD knockdown cells and that both virus replication and viral protein production are dramatically reduced, which suggests that virus-induced apoptosis is BAD dependent. Our data showed that influenza viruses induced phosphorylation of BAD at residues S112 and S136 in a temporal manner. Viral infection also induced BAD cleavage, late in the viral life cycle, to a truncated form that is reportedly a more potent inducer of apoptosis. We further demonstrate that knockdown of BAD resulted in reduced cytochrome c release and suppression of the intrinsic apoptotic pathway during influenza virus replication, as seen by an inhibition of caspases-3, caspase-7, and procyclic acidic repetitive protein (PARP) cleavage. Our data indicate that influenza viruses carefully modulate the activation of the apoptotic pathway that is dependent on the regulatory function of BAD and that failure of apoptosis activation resulted in unproductive viral replication.

  6. ABTS-Modified Silica Nanoparticles as Laccase Mediators for Decolorization of Indigo Carmine Dye

    Directory of Open Access Journals (Sweden)

    Youxun Liu

    2015-01-01

    Full Text Available Efficient reuse and regeneration of spent mediators are highly desired for many of the laccases’ biotechnology applications. This investigation demonstrates that a redox mediator 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS covalently attached to silica nanoparticles (SNPs effectively mediated dye decolorization catalyzed by laccase. Characteristics of ABTS-modified silica nanoparticles (ABTS-SNPs were researched by scanning electron microscopy and Fourier-transformed infrared spectroscopy. When ABTS and ABTS-SNPs were used as laccase mediators, the decolorization yields of 96 and 95% were, respectively, obtained for indigo carmine dye. The results suggest that ABTS immobilized on SNPs can be used as laccase mediators as they retain almost the same efficiency as the free ABTS. The oxidized ABTS-SNPs were regenerated by their reduction reaction with ascorbic acid. Decolorization efficiency of regenerated ABTS-SNPs and their initial forms were found to be almost equivalent. Six reuse cycles for spent ABTS-SNPs were run for the treatment of indigo carmine, providing decolorization yields of 96–77%. Compared with free mediator, the immobilized mediators have the advantage of being easily recovered, regenerated, and reused making the whole process environmentally friendly.

  7. Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration

    Directory of Open Access Journals (Sweden)

    Jung Hyun Park

    2017-01-01

    Full Text Available Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP+-dependent isocitrate dehydrogenase (idh2 regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of idh2 in acrolein-induced lung injury using idh2 short hairpin RNA- (shRNA- transfected Lewis lung carcinoma (LLC cells and idh2-deficient (idh2−/− mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In addition, acrolein-induced toxicity in idh2 shRNA-transfected LLC cells and in idh2 knockout mice was ameliorated by the antioxidant, N-acetylcysteine, through attenuation of oxidative stress resulting from idh2 deficiency. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2−/− mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

  8. Idh2 Deficiency Exacerbates Acrolein-Induced Lung Injury through Mitochondrial Redox Environment Deterioration.

    Science.gov (United States)

    Park, Jung Hyun; Ku, Hyeong Jun; Lee, Jin Hyup; Park, Jeen-Woo

    2017-01-01

    Acrolein is known to be involved in acute lung injury and other pulmonary diseases. A number of studies have suggested that acrolein-induced toxic effects are associated with depletion of antioxidants, such as reduced glutathione and protein thiols, and production of reactive oxygen species. Mitochondrial NADP + -dependent isocitrate dehydrogenase ( idh2 ) regulates mitochondrial redox balance and reduces oxidative stress-induced cell injury via generation of NADPH. Therefore, we evaluated the role of idh2 in acrolein-induced lung injury using idh2 short hairpin RNA- (shRNA-) transfected Lewis lung carcinoma (LLC) cells and idh2 -deficient ( idh2 -/- ) mice. Downregulation of idh2 expression increased susceptibility to acrolein via induction of apoptotic cell death due to elevated mitochondrial oxidative stress. Idh2 deficiency also promoted acrolein-induced lung injury in idh2 knockout mice through the disruption of mitochondrial redox status. In addition, acrolein-induced toxicity in idh2 shRNA-transfected LLC cells and in idh2 knockout mice was ameliorated by the antioxidant, N-acetylcysteine, through attenuation of oxidative stress resulting from idh2 deficiency. In conclusion, idh2 deficiency leads to mitochondrial redox environment deterioration, which causes acrolein-mediated apoptosis of LLC cells and acrolein-induced lung injury in idh2 -/- mice. The present study supports the central role of idh2 deficiency in inducing oxidative stress resulting from acrolein-induced disruption of mitochondrial redox status in the lung.

  9. The sensitivity of diffuse large B-cell lymphoma cell lines to histone deacetylase inhibitor-induced apoptosis is modulated by BCL-2 family protein activity.

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    Ryan C Thompson

    Full Text Available BACKGROUND: Diffuse large B-cell lymphoma (DLBCL is a genetically heterogeneous disease and this variation can often be used to explain the response of individual patients to chemotherapy. One cancer therapeutic approach currently in clinical trials uses histone deacetylase inhibitors (HDACi's as monotherapy or in combination with other agents. METHODOLOGY/PRINCIPAL FINDINGS: We have used a variety of cell-based and molecular/biochemical assays to show that two pan-HDAC inhibitors, trichostatin A and vorinostat, induce apoptosis in seven of eight human DLBCL cell lines. Consistent with previous reports implicating the BCL-2 family in regulating HDACi-induced apoptosis, ectopic over-expression of anti-apoptotic proteins BCL-2 and BCL-XL or pro-apoptotic protein BIM in these cell lines conferred further resistance or sensitivity, respectively, to HDACi treatment. Additionally, BCL-2 family antgonist ABT-737 increased the sensitivity of several DLBCL cell lines to vorinostat-induced apoptosis, including one cell line (SUDHL6 that is resistant to vorinostat alone. Moreover, two variants of the HDACi-sensitive SUDHL4 cell line that have decreased sensitivity to vorinostat showed up-regulation of BCL-2 family anti-apoptotic proteins such as BCL-XL and MCL-1, as well as decreased sensitivity to ABT-737. These results suggest that the regulation and overall balance of anti- to pro-apoptotic BCL-2 family protein expression is important in defining the sensitivity of DLBCL to HDACi-induced apoptosis. However, the sensitivity of DLBCL cell lines to HDACi treatment does not correlate with expression of any individual BCL-2 family member. CONCLUSIONS/SIGNIFICANCE: These studies indicate that the sensitivity of DLBCL to treatment with HDACi's is dependent on the complex regulation of BCL-2 family members and that BCL-2 antagonists may enhance the response of a subset of DLBCL patients to HDACi treatment.

  10. Effect of remifentanil on mitochondrial oxygen consumption of cultured human hepatocytes.

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    Siamak Djafarzadeh

    Full Text Available During sepsis, liver dysfunction is common, and failure of mitochondria to effectively couple oxygen consumption with energy production has been described. In addition to sepsis, pharmacological agents used to treat septic patients may contribute to mitochondrial dysfunction. This study addressed the hypothesis that remifentanil interacts with hepatic mitochondrial oxygen consumption. The human hepatoma cell line HepG2 and their isolated mitochondria were exposed to remifentanil, with or without further exposure to tumor necrosis factor-α (TNF-α. Mitochondrial oxygen consumption was measured by high-resolution respirometry, Caspase-3 protein levels by Western blotting, and cytokine levels by ELISA. Inhibitory κBα (IκBα phosphorylation, measurement of the cellular ATP content and mitochondrial membrane potential in intact cells were analysed using commercial ELISA kits. Maximal cellular respiration increased after one hour of incubation with remifentanil, and phosphorylation of IκBα occurred, denoting stimulation of nuclear factor κB (NF-κB. The effect on cellular respiration was not present at 2, 4, 8 or 16 hours of incubation. Remifentanil increased the isolated mitochondrial respiratory control ratio of complex-I-dependent respiration without interfering with maximal respiration. Preincubation with the opioid receptor antagonist naloxone prevented a remifentanil-induced increase in cellular respiration. Remifentanil at 10× higher concentrations than therapeutic reduced mitochondrial membrane potential and ATP content without uncoupling oxygen consumption and basal respiration levels. TNF-α exposure reduced respiration of complex-I, -II and -IV, an effect which was prevented by prior remifentanil incubation. Furthermore, prior remifentanil incubation prevented TNF-α-induced IL-6 release of HepG2 cells, and attenuated fragmentation of pro-caspase-3 into cleaved active caspase 3 (an early marker of apoptosis. Our data suggest that

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

    DEFF Research Database (Denmark)

    Fedosov, Sergey; Belousova, Lubov; Plesner, Igor

    1993-01-01

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

  12. Lycium barbarum Polysaccharides Protect Rat Corneal Epithelial Cells against Ultraviolet B-Induced Apoptosis by Attenuating the Mitochondrial Pathway and Inhibiting JNK Phosphorylation

    Directory of Open Access Journals (Sweden)

    Shaobo Du

    2017-01-01

    Full Text Available Lycium barbarum polysaccharides (LBPs have been shown to play a key role in protecting the eyes by reducing the apoptosis induced by certain types of damage. However, it is not known whether LBPs can protect damaged corneal cells from apoptosis. Moreover, no reports have focused on the role of LBPs in guarding against ultraviolet B- (UVB- induced apoptosis. The present study aimed to investigate the protective effect and underlying mechanism of LBPs against UVB-induced apoptosis in rat corneal epithelial (RCE cells. The results showed that LBPs significantly prevented the loss of cell viability and inhibited cell apoptosis induced by UVB in RCE cells. LBPs also inhibited UVB-induced loss of mitochondrial membrane potential, downregulation of Bcl-2, and upregulation of Bax and caspase-3. Finally, LBPs attenuated the phosphorylation of c-Jun NH2-terminal kinase (JNK triggered by UVB. In summary, LBPs protect RCE cells against UVB-induced damage and apoptosis, and the underlying mechanism involves the attenuation of the mitochondrial apoptosis pathway and the inhibition of JNK phosphorylation.

  13. Mitochondrial Reactive Oxygen Species in Lipotoxic Hearts Induce Post-Translational Modifications of AKAP121, DRP1, and OPA1 That Promote Mitochondrial Fission.

    Science.gov (United States)

    Tsushima, Kensuke; Bugger, Heiko; Wende, Adam R; Soto, Jamie; Jenson, Gregory A; Tor, Austin R; McGlauflin, Rose; Kenny, Helena C; Zhang, Yuan; Souvenir, Rhonda; Hu, Xiao X; Sloan, Crystal L; Pereira, Renata O; Lira, Vitor A; Spitzer, Kenneth W; Sharp, Terry L; Shoghi, Kooresh I; Sparagna, Genevieve C; Rog-Zielinska, Eva A; Kohl, Peter; Khalimonchuk, Oleh; Schaffer, Jean E; Abel, E Dale

    2018-01-05

    Cardiac lipotoxicity, characterized by increased uptake, oxidation, and accumulation of lipid intermediates, contributes to cardiac dysfunction in obesity and diabetes mellitus. However, mechanisms linking lipid overload and mitochondrial dysfunction are incompletely understood. To elucidate the mechanisms for mitochondrial adaptations to lipid overload in postnatal hearts in vivo. Using a transgenic mouse model of cardiac lipotoxicity overexpressing ACSL1 (long-chain acyl-CoA synthetase 1) in cardiomyocytes, we show that modestly increased myocardial fatty acid uptake leads to mitochondrial structural remodeling with significant reduction in minimum diameter. This is associated with increased palmitoyl-carnitine oxidation and increased reactive oxygen species (ROS) generation in isolated mitochondria. Mitochondrial morphological changes and elevated ROS generation are also observed in palmitate-treated neonatal rat ventricular cardiomyocytes. Palmitate exposure to neonatal rat ventricular cardiomyocytes initially activates mitochondrial respiration, coupled with increased mitochondrial polarization and ATP synthesis. However, long-term exposure to palmitate (>8 hours) enhances ROS generation, which is accompanied by loss of the mitochondrial reticulum and a pattern suggesting increased mitochondrial fission. Mechanistically, lipid-induced changes in mitochondrial redox status increased mitochondrial fission by increased ubiquitination of AKAP121 (A-kinase anchor protein 121) leading to reduced phosphorylation of DRP1 (dynamin-related protein 1) at Ser637 and altered proteolytic processing of OPA1 (optic atrophy 1). Scavenging mitochondrial ROS restored mitochondrial morphology in vivo and in vitro. Our results reveal a molecular mechanism by which lipid overload-induced mitochondrial ROS generation causes mitochondrial dysfunction by inducing post-translational modifications of mitochondrial proteins that regulate mitochondrial dynamics. These findings provide a

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

    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.

  16. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

    International Nuclear Information System (INIS)

    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián; Simon, Felipe; Riedel, Claudia; Ferrick, David; Elorza, Alvaro A.

    2013-01-01

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive

  17. Copper deficiency alters cell bioenergetics and induces mitochondrial fusion through up-regulation of MFN2 and OPA1 in erythropoietic cells

    Energy Technology Data Exchange (ETDEWEB)

    Bustos, Rodrigo I.; Jensen, Erik L.; Ruiz, Lina M.; Rivera, Salvador; Ruiz, Sebastián [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Simon, Felipe; Riedel, Claudia [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile); Ferrick, David [Seahorse Bioscience, Billerica, MA (United States); Elorza, Alvaro A., E-mail: aelorza@unab.cl [Center for Biomedical Research, Faculty of Biological Sciences and Faculty of Medicine, Universidad Andres Bello, Santiago (Chile); Millennium Institute of Immunology and Immunotherapy, Santiago (Chile)

    2013-08-02

    Highlights: •In copper deficiency, cell proliferation is not affected. In turn, cell differentiation is impaired. •Enlarged mitochondria are due to up-regulation of MNF2 and OPA1. •Mitochondria turn off respiratory chain and ROS production. •Energy metabolism switch from mitochondria to glycolysis. -- Abstract: Copper is essential in cell physiology, participating in numerous enzyme reactions. In mitochondria, copper is a cofactor for respiratory complex IV, the cytochrome c oxidase. Low copper content is associated with anemia and the appearance of enlarged mitochondria in erythropoietic cells. These findings suggest a connection between copper metabolism and bioenergetics, mitochondrial dynamics and erythropoiesis, which has not been explored so far. Here, we describe that bathocuproine disulfonate-induced copper deficiency does not alter erythropoietic cell proliferation nor induce apoptosis. However it does impair erythroid differentiation, which is associated with a metabolic switch between the two main energy-generating pathways. That is, from mitochondrial function to glycolysis. Switching off mitochondria implies a reduction in oxygen consumption and ROS generation along with an increase in mitochondrial membrane potential. Mitochondrial fusion proteins MFN2 and OPA1 were up-regulated along with the ability of mitochondria to fuse. Morphometric analysis of mitochondria did not show changes in total mitochondrial biomass but rather bigger mitochondria because of increased fusion. Similar results were also obtained with human CD34+, which were induced to differentiate into red blood cells. In all, we have shown that adequate copper levels are important for maintaining proper mitochondrial function and for erythroid differentiation where the energy metabolic switch plus the up-regulation of fusion proteins define an adaptive response to copper deprivation to keep cells alive.

  18. Cudraflavone C Induces Apoptosis of A375.S2 Melanoma Cells through Mitochondrial ROS Production and MAPK Activation.

    Science.gov (United States)

    Lee, Chiang-Wen; Yen, Feng-Lin; Ko, Horng-Huey; Li, Shu-Yu; Chiang, Yao-Chang; Lee, Ming-Hsueh; Tsai, Ming-Horng; Hsu, Lee-Fen

    2017-07-13

    Melanoma is the most malignant form of skin cancer and is associated with a very poor prognosis. The aim of this study was to evaluate the apoptotic effects of cudraflavone C on A375.S2 melanoma cells and to determine the underlying mechanisms involved in apoptosis. Cell viability was determined using the MTT and real-time cytotoxicity assays. Flow cytometric evaluation of apoptosis was performed after staining the cells with Annexin V-FITC and propidium iodide. The mitochondrial membrane potential was evaluated using the JC-1 assay. Cellular ROS production was measured using the CellROX assay, while mitochondrial ROS production was evaluated using the MitoSOX assay. It was observed that cudraflavone C inhibited growth in A375.S2 melanoma cells, and promoted apoptosis via the mitochondrial pathway mediated by increased mitochondrial ROS production. In addition, cudraflavone C induced phosphorylation of MAPKs (p38, ERK, and JNK) and up-regulated the expression of apoptotic proteins (Puma, Bax, Bad, Bid, Apaf-1, cytochrome C, caspase-9, and caspase-3/7) in A375.S2 cells. Pretreatment of A375.S2 cells with MitoTEMPOL (a mitochondria-targeted antioxidant) attenuated the phosphorylation of MAPKs, expression of apoptotic proteins, and the overall progression of apoptosis. In summary, cudraflavone C induced apoptosis in A375.S2 melanoma cells by increasing mitochondrial ROS production; thus, activating p38, ERK, and JNK; and increasing the expression of apoptotic proteins. Therefore, cudraflavone C may be regarded as a potential form of treatment for malignant melanoma.

  19. Mitochondrial localization of the low level p53 protein in proliferative cells

    Energy Technology Data Exchange (ETDEWEB)

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Oliver, Lisa [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Rincheval, Vincent; Renaud, Flore [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vallette, Francois M. [INSERM U601, Universite de Nantes, Faculte de Medecine, Nantes Cedex (France); Mignotte, Bernard [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France); Vayssiere, Jean-Luc, E-mail: jean-luc.vayssiere@uvsq.fr [Laboratoire de Genetique et Biologie Cellulaire - CNRS UMR 8159, Universite de Versailles Saint-Quentin-en-Yvelines, Versailles, France and Laboratoire de Genetique Moleculaire et Physiologique, Ecole Pratique des Hautes Etudes, Versailles (France)

    2009-10-02

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  20. Mitochondrial localization of the low level p53 protein in proliferative cells

    International Nuclear Information System (INIS)

    Ferecatu, Ioana; Bergeaud, Marie; Rodriguez-Enfedaque, Aida; Le Floch, Nathalie; Oliver, Lisa; Rincheval, Vincent; Renaud, Flore; Vallette, Francois M.; Mignotte, Bernard; Vayssiere, Jean-Luc

    2009-01-01

    p53 protein plays a central role in suppressing tumorigenesis by inducing cell cycle arrest or apoptosis through transcription-dependent and -independent mechanisms. Emerging publications suggest that following stress, a fraction of p53 translocates to mitochondria to induce cytochrome c release and apoptosis. However, the localization of p53 under unstressed conditions remains largely unexplored. Here we show that p53 is localized at mitochondria in absence of apoptotic stimuli, when cells are proliferating, localization observed in various cell types (rodent and human). This is also supported by acellular assays in which p53 bind strongly to mitochondria isolated from rat liver. Furthermore, the mitochondria subfractionation study and the alkaline treatment of the mitochondrial p53 revealed that the majority of mitochondrial p53 is present in the membranous compartments. Finally, we identified VDAC, a protein of the mitochondrial outer-membrane, as a putative partner of p53 in unstressed/proliferative cells.

  1. Lycopene Prevents Amyloid [Beta]-Induced Mitochondrial Oxidative Stress and Dysfunctions in Cultured Rat Cortical Neurons.

    Science.gov (United States)

    Qu, Mingyue; Jiang, Zheng; Liao, Yuanxiang; Song, Zhenyao; Nan, Xinzhong

    2016-06-01

    Brains affected by Alzheimer's disease (AD) show a large spectrum of mitochondrial alterations at both morphological and genetic level. The causal link between β-amyloid (Aβ) and mitochondrial dysfunction has been established in cellular models of AD. We observed previously that lycopene, a member of the carotenoid family of phytochemicals, could counteract neuronal apoptosis and cell damage induced by Aβ and other neurotoxic substances, and that this neuroprotective action somehow involved the mitochondria. The present study aims to investigate the effects of lycopene on mitochondria in cultured rat cortical neurons exposed to Aβ. It was found that lycopene attenuated Aβ-induced oxidative stress, as evidenced by the decreased intracellular reactive oxygen species generation and mitochondria-derived superoxide production. Additionally, lycopene ameliorated Aβ-induced mitochondrial morphological alteration, opening of the mitochondrial permeability transition pores and the consequent cytochrome c release. Lycopene also improved mitochondrial complex activities and restored ATP levels in Aβ-treated neuron. Furthermore, lycopene prevented mitochondrial DNA damages and improved the protein level of mitochondrial transcription factor A in mitochondria. Those results indicate that lycopene protects mitochondria against Aβ-induced damages, at least in part by inhibiting mitochondrial oxidative stress and improving mitochondrial function. These beneficial effects of lycopene may account for its protection against Aβ-induced neurotoxicity.

  2. Anthelmintic drug ivermectin inhibits angiogenesis, growth and survival of glioblastoma through inducing mitochondrial dysfunction and oxidative stress

    International Nuclear Information System (INIS)

    Liu, Yingying; Fang, Shanshan; Sun, Qiushi; Liu, Bo

    2016-01-01

    Glioblastoma is one of the most vascular brain tumour and highly resistant to current therapy. Targeting both glioblastoma cells and angiogenesis may present an effective therapeutic strategy for glioblastoma. In our work, we show that an anthelmintic drug, ivermectin, is active against glioblastoma cells in vitro and in vivo, and also targets angiogenesis. Ivermectin significantly inhibits growth and anchorage-independent colony formation in U87 and T98G glioblastoma cells. It induces apoptosis in these cells through a caspase-dependent manner. Ivermectin significantly suppresses the growth of two independent glioblastoma xenograft mouse models. In addition, ivermectin effectively targets angiogenesis through inhibiting capillary network formation, proliferation and survival in human brain microvascular endothelial cell (HBMEC). Mechanistically, ivermectin decreases mitochondrial respiration, membrane potential, ATP levels and increases mitochondrial superoxide in U87, T98G and HBMEC cells exposed to ivermectin. The inhibitory effects of ivermectin are significantly reversed in mitochondria-deficient cells or cells treated with antioxidants, further confirming that ivermectin acts through mitochondrial respiration inhibition and induction of oxidative stress. Importantly, we show that ivermectin suppresses phosphorylation of Akt, mTOR and ribosomal S6 in glioblastoma and HBMEC cells, suggesting its inhibitory role in deactivating Akt/mTOR pathway. Altogether, our work demonstrates that ivermectin is a useful addition to the treatment armamentarium for glioblastoma. Our work also highlights the therapeutic value of targeting mitochondrial metabolism in glioblastoma. - Highlights: • Ivermectin is effective in glioblastoma cells in vitro and in vivo. • Ivermectin inhibits angiogenesis. • Ivermectin induces mitochondrial dysfunction and oxidative stress. • Ivermectin deactivates Akt/mTOR signaling pathway.

  3. Protective effects of l-carnitine and piracetam against mitochondrial permeability transition and PC3 cell necrosis induced by simvastatin.

    Science.gov (United States)

    Costa, Rute A P; Fernandes, Mariana P; de Souza-Pinto, Nadja C; Vercesi, Aníbal E

    2013-02-15

    Mitochondrial oxidative stress followed by membrane permeability transition (MPT) has been considered as a possible mechanism for statins cytotoxicity. Statins use has been associated with reduced risk of cancer incidence, especially prostate cancer. Here we investigated the pathways leading to simvastatin-induced prostate cancer cell death as well as the mechanisms of cell death protection by l-carnitine or piracetam. These compounds are known to prevent and/or protect against cell death mediated by oxidative mitochondrial damage induced by a variety of conditions, either in vivo or in vitro. The results provide evidence that simvastatin induced MPT and cell necrosis were sensitive to either l-carnitine or piracetam in a dose-dependent fashion and mediated by additive mechanisms. When combined, l-carnitine and piracetam acted at concentrations significantly lower than they act individually. These results shed new light into both the cytotoxic mechanisms of statins and the mechanisms underlying the protection against MPT and cell death by the compounds l-carnitine and piracetam. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. Mitochondrial events responsible for morphine's cardioprotection against ischemia/reperfusion injury

    International Nuclear Information System (INIS)

    He, Haiyan; Huh, Jin; Wang, Huihua; Kang, Yi; Lou, Jianshi; Xu, Zhelong

    2016-01-01

    Morphine may induce cardioprotection by targeting mitochondria, but little is known about the exact mitochondrial events that mediate morphine's protection. We aimed to address the role of the mitochondrial Src tyrosine kinase in morphine's protection. Isolated rat hearts were subjected to 30 min ischemia and 2 h of reperfusion. Morphine was given before the onset of ischemia. Infarct size and troponin I release were measured to evaluate cardiac injury. Oxidative stress was evaluated by measuring mitochondrial protein carbonylation and mitochondrial ROS generation. HL-1 cells were subjected to simulated ischemia/reperfusion and LDH release and mitochondrial membrane potential (ΔΨm) were measured. Morphine reduced infarct size as well as cardiac troponin I release which were aborted by the selective Src tyrosine kinase inhibitors PP2 and Src-I1. Morphine also attenuated LDH release and prevented a loss of ΔΨm at reperfusion in a Src tyrosine kinase dependent manner in HL-1 cells. However, morphine failed to reduce LDH release in HL-1 cells transfected with Src siRNA. Morphine increased mitochondrial Src phosphorylation at reperfusion and this was abrogated by PP2. Morphine attenuated mitochondrial protein carbonylation and mitochondrial superoxide generation at reperfusion through Src tyrosine kinase. The inhibitory effect of morphine on the mitochondrial complex I activity was reversed by PP2. These data suggest that morphine induces cardioprotection by preventing mitochondrial oxidative stress through mitochondrial Src tyrosine kinase. Inhibition of mitochondrial complex I at reperfusion by Src tyrosine kinase may account for the prevention of mitochondrial oxidative stress by morphine. - Highlights: • Morphine induced mito-Src phosphorylation and reduced infarct size in rat hearts. • Morphine failed to reduce I/R-induced LDH release in Src-silencing HL-1 cells. • Morphine prevented mitochondria damage caused by I/R through Src. • Morphine reduced

  5. Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

    Science.gov (United States)

    Wang, Wei-jia; Wang, Yuan; Chen, Hang-zi; Xing, Yong-zhen; Li, Feng-wei; Zhang, Qian; Zhou, Bo; Zhang, Hong-kui; Zhang, Jie; Bian, Xue-li; Li, Li; Liu, Yuan; Zhao, Bi-xing; Chen, Yan; Wu, Rong; Li, An-zhong; Yao, Lu-ming; Chen, Ping; Zhang, Yi; Tian, Xu-yang; Beermann, Friedrich; Wu, Mian; Han, Jiahuai; Huang, Pei-qiang; Lin, Tianwei; Wu, Qiao

    2014-02-01

    Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

  6. Mitochondrial-targeted aryl hydrocarbon receptor and the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin on cellular respiration and the mitochondrial proteome

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, Hye Jin [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States); Dornbos, Peter [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Steidemann, Michelle [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI 48824 (United States); Dunivin, Taylor K. [Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 (United States); Rizzo, Mike [Institute for Integrative Toxicology, Michigan State University, East Lansing, MI 48824-1319 (United States); Cell and Molecular Biology Graduate Program, Michigan State University, East Lansing, MI 48824 (United States); LaPres, John J., E-mail: lapres@msu.edu [Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824 (United States); Center for Mitochondrial Science and Medicine, Michigan State University, East Lansing, MI 48824 (United States)

    2016-08-01

    The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor within the Per-Arnt-Sim (PAS) domain superfamily. Exposure to the most potent AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), is associated with various pathological effects including metabolic syndrome. While research over the last several years has demonstrated a role for oxidative stress and metabolic dysfunction in AHR-dependent TCDD-induced toxicity, the role of the mitochondria in this process has not been fully explored. Our previous research suggested that a portion of the cellular pool of AHR could be found in the mitochondria (mitoAHR). Using a protease protection assay with digitonin extraction, we have now shown that this mitoAHR is localized to the inter-membrane space (IMS) of the organelle. TCDD exposure induced a degradation of mitoAHR similar to that of cytosolic AHR. Furthermore, siRNA-mediated knockdown revealed that translocase of outer-mitochondrial membrane 20 (TOMM20) was involved in the import of AHR into the mitochondria. In addition, TCDD altered cellular respiration in an AHR-dependent manner to maintain respiratory efficiency as measured by oxygen consumption rate (OCR). Stable isotope labeling by amino acids in cell culture (SILAC) identified a battery of proteins within the mitochondrial proteome influenced by TCDD in an AHR-dependent manner. Among these, 17 proteins with fold changes ≥ 2 are associated with various metabolic pathways, suggesting a role of mitochondrial retrograde signaling in TCDD-mediated pathologies. Collectively, these studies suggest that mitoAHR is localized to the IMS and AHR-dependent TCDD-induced toxicity, including metabolic dysfunction, wasting syndrome, and hepatic steatosis, involves mitochondrial dysfunction. - Highlights: • The mitoAHR is localized in the mitochondrial intermembrane space. • TOMM20 participates in mitoAHR translocation. • AHR contributes to the maintenance of respiratory control ratio following

  7. Molecular insights into the m-AAA protease-mediated dislocation of transmembrane helices in the mitochondrial inner membrane.

    Science.gov (United States)

    Lee, Seoeun; Lee, Hunsang; Yoo, Suji; Kim, Hyun

    2017-12-08

    Protein complexes involved in respiration, ATP synthesis, and protein import reside in the mitochondrial inner membrane; thus, proper regulation of these proteins is essential for cell viability. The m -AAA protease, a conserved hetero-hexameric AAA (ATPase associated with diverse cellular activities) protease, composed of the Yta10 and Yta12 proteins, regulates mitochondrial proteostasis by mediating protein maturation and degradation. It also recognizes and mediates the dislocation of membrane-embedded substrates, including foreign transmembrane (TM) segments, but the molecular mechanism involved in these processes remains elusive. This study investigated the role of the TM domains in the m -AAA protease by systematic replacement of one TM domain at a time in yeast. Our data indicated that replacement of the Yta10 TM2 domain abolishes membrane dislocation for only a subset of substrates, whereas replacement of the Yta12 TM2 domain impairs membrane dislocation for all tested substrates, suggesting different roles of the TM domains in each m -AAA protease subunit. Furthermore, m -AAA protease-mediated membrane dislocation was impaired in the presence of a large downstream hydrophilic moiety in a membrane substrate. This finding suggested that the m -AAA protease cannot dislocate large hydrophilic domains across the membrane, indicating that the membrane dislocation probably occurs in a lipid environment. In summary, this study highlights previously underappreciated biological roles of TM domains of the m -AAA proteases in mediating the recognition and dislocation of membrane-embedded substrates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Thallium induces hydrogen peroxide generation by impairing mitochondrial function

    International Nuclear Information System (INIS)

    Hanzel, Cecilia E.; Verstraeten, Sandra V.

    2006-01-01

    Thallium (Tl) is highly toxic through yet poorly understood mechanisms. In this study, we comparatively investigated the effects of thallic (Tl(III)) cations on mitochondrial functionality and oxidative stress promotion, and results were compared to those obtained for thallous (Tl(I)) cation. PC12 cells were incubated between 1 and 72 h in the presence of a single dose of Tl(I) or Tl(III) (10-250 μM). A metal concentration- and time-dependent decrease in cell viability was observed evaluated by both MTT reduction and calcein fluorescence. After 24 h in culture, Tl(I) and Tl(III) significantly decreased mitochondrial membrane potential evaluated as the incorporation of rhodamine 123. Along the incubation period assessed, both Tl(I) and Tl(III) (50 and 100 μM) significantly increased mitochondrial H 2 O 2 steady-state levels, being the magnitude of the effect: Tl(III) > Tl(I). Glutathione content, measured by reaction with monochlorobimane, was significantly reduced in Tl-treated cells. Finally, higher oxidant species content in cells cytoplasm was found, which positively correlated with mitochondrial H 2 O 2 content. Together, these results indicate that both ionic species of Tl enhance cells reactive oxygen species production, decreasing mitochondrial functionality. These effects could partially be responsible for the loss of cell viability, and account for the metabolic alterations found in Tl intoxication

  9. Mitochondrial calcium uniporter silencing potentiates caspase-independent cell death in MDA-MB-231 breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Curry, Merril C.; Peters, Amelia A. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Kenny, Paraic A. [Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461 (United States); Roberts-Thomson, Sarah J. [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia); Monteith, Gregory R., E-mail: gregm@uq.edu.au [School of Pharmacy, The University of Queensland, Brisbane, Queensland 4072 (Australia)

    2013-05-10

    Highlights: •Some clinical breast cancers are associated with MCU overexpression. •MCU silencing did not alter cell death initiated with the Bcl-2 inhibitor ABT-263. •MCU silencing potentiated caspase-independent cell death initiated by ionomycin. •MCU silencing promoted ionomycin-mediated cell death without changes in bulk Ca{sup 2+}. -- Abstract: The mitochondrial calcium uniporter (MCU) transports free ionic Ca{sup 2+} into the mitochondrial matrix. We assessed MCU expression in clinical breast cancer samples using microarray analysis and the consequences of MCU silencing in a breast cancer cell line. Our results indicate that estrogen receptor negative and basal-like breast cancers are characterized by elevated levels of MCU. Silencing of MCU expression in the basal-like MDA-MB-231 breast cancer cell line produced no change in proliferation or cell viability. However, distinct consequences of MCU silencing were seen on cell death pathways. Caspase-dependent cell death initiated by the Bcl-2 inhibitor ABT-263 was not altered by MCU silencing; whereas caspase-independent cell death induced by the calcium ionophore ionomycin was potentiated by MCU silencing. Measurement of cytosolic Ca{sup 2+} levels showed that the promotion of ionomycin-induced cell death by MCU silencing occurs independently of changes in bulk cytosolic Ca{sup 2+} levels. This study demonstrates that MCU overexpression is a feature of some breast cancers and that MCU overexpression may offer a survival advantage against some cell death pathways. MCU inhibitors may be a strategy to increase the effectiveness of therapies that act through the induction of caspase-independent cell death pathways in estrogen receptor negative and basal-like breast cancers.

  10. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction.

    Science.gov (United States)

    Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

    2013-12-15

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. © 2013.

  11. PFOS prenatal exposure induce mitochondrial injury and gene expression change in hearts of weaned SD rats

    International Nuclear Information System (INIS)

    Xia, Wei; Wan, Yanjian; Li, Yuan-yuan; Zeng, Huaicai; Lv, Ziquan; Li, Gengqi; Wei, Zhengzheng; Xu, Shun-qing

    2011-01-01

    Xenobiotics exposure in early life may have adverse effects on animals' development through mitochondrial injury or dysfunction. The current study demonstrated the possibility of cardiac mitochondrial injury in prenatal PFOS-exposed weaned rat heart. Pregnant Sprague-Dawley (SD) rats were exposed to perfluorooctane sulfonate (PFOS) at doses of 0.1, 0.6 and 2.0 mg/kg/d and 0.05% Tween 80 as control by gavage from gestation days 2-21. The dams were allowed to give nature delivery and then heart tissues from weaned (postnatal day 21) offspring rats were analyzed for mitochondrial injury through ultrastructure observation by electron microscope, global gene expression profile by microarray, as well as related mRNA and proteins expression levels by quantitative PCR and western blot. Ultrastructural analysis revealed significant vacuolization and inner membrane injury occurred at the mitochondria of heart tissues from 2.0 mg/kg/d dosage group. Meanwhile, the global gene expression profile showed significant difference in level of some mRNA expression associated with mitochondrial function at 2.0 mg/kg/d dosage group, compared to the control. Furthermore, dose-response trends for the expression of selected genes were analyzed by quantitative PCR and western blot analysis. The selected genes were mainly focused on those encoding for proteins involved in energy production, control of ion levels, and maintenance of heart function. The down-regulation of mitochondrial ATP synthetase (ATP5E, ATP5I and ATP5O) implicated a decrease in energy supply. This was accompanied by down-regulation of gene transcripts involved in energy consumption such as ion transporting ATPase (ATP1A3 and ATP2B2) and inner membrane protein synthesis (SLC25A3, SLC25A4, SLC25A10, SLC25A29). The up-regulation of gene transcripts encoding for uncoupling proteins (UCP1 and UCP3), epidermal growth factor receptor (EGFR) and connective tissue growth factor (CTGF), was probably a protective process to maintain

  12. Laminarin Induces Apoptosis of Human Colon Cancer LOVO Cells through a Mitochondrial Pathway

    Directory of Open Access Journals (Sweden)

    He Zhang

    2012-08-01

    Full Text Available Many scientific studies have shown that laminarin has anti-tumor effects, but the anti-tumor mechanism was unclear. The purpose of this study was to investigate the effect of laminarin on the induction of apoptosis in human colon cancer LOVO cells and the molecular mechanism involved. LOVO cells were treated with different concentrations of laminarin at different times. Morphology observations were performed to determine the effects of laminarin on apoptosis of LOVO cells. Flow cytometry (FCM was used to detect the level of intracellular reactive oxygen species (ROS and pH. Laser scanning confocal microscope (LSCM was used to analyze intracellular calcium ion concentration, mitochondrion permeability transition pore (MPTP and mitochondrial membrane potential (MMP. Western blotd were performed to analyze the expressions of Cyt-C, Caspase-9 and -3. The results showed the apoptosis morphology, which showed cell protuberance, concentrated cytoplasm and apoptotic bodies, was obvious after 72 h treatment. Laminarin treatment for 24 h increased the intracellular level of ROS and Ca2+; decreased pH value; activated intracellular MPTP and decreased MMP in dose-dependent manners. It also induced the release of Cyt-C and the activation of Caspase-9 and -3. In conclusion, laminarin induces LOVO cell apoptosis through a mitochondrial pathway, suggesting that it could be a potent agent for cancer prevention and treatment.

  13. Cisplatin Induces a Mitochondrial-ROS Response That Contributes to Cytotoxicity Depending on Mitochondrial Redox Status and Bioenergetic Functions

    Science.gov (United States)

    Marullo, Rossella; Werner, Erica; Degtyareva, Natalya; Moore, Bryn; Altavilla, Giuseppe; Ramalingam, Suresh S.; Doetsch, Paul W.

    2013-01-01

    Cisplatin is one of the most effective and widely used anticancer agents for the treatment of several types of tumors. The cytotoxic effect of cisplatin is thought to be mediated primarily by the generation of nuclear DNA adducts, which, if not repaired, cause cell death as a consequence of DNA replication and transcription blockage. However, the ability of cisplatin to induce nuclear DNA (nDNA) damage per se is not sufficient to explain its high degree of effectiveness nor the toxic effects exerted on normal, post-mitotic tissues. Oxidative damage has been observed in vivo following exposure to cisplatin in several tissues, suggesting a role for oxidative stress in the pathogenesis of cisplatin-induced dose-limiting toxicities. However, the mechanism of cisplatin-induced generation of ROS and their contribution to cisplatin cytotoxicity in normal and cancer cells is still poorly understood. By employing a panel of normal and cancer cell lines and the budding yeast Saccharomyces cerevisiae as model system, we show that exposure to cisplatin induces a mitochondrial-dependent ROS response that significantly enhances the cytotoxic effect caused by nDNA damage. ROS generation is independent of the amount of cisplatin-induced nDNA damage and occurs in mitochondria as a consequence of protein synthesis impairment. The contribution of cisplatin-induced mitochondrial dysfunction in determining its cytotoxic effect varies among cells and depends on mitochondrial redox status, mitochondrial DNA integrity and bioenergetic function. Thus, by manipulating these cellular parameters, we were able to enhance cisplatin cytotoxicity in cancer cells. This study provides a new mechanistic insight into cisplatin-induced cell killing and may lead to the design of novel therapeutic strategies to improve anticancer drug efficacy. PMID:24260552

  14. Mitochondrial mislocalization underlies Abeta42-induced neuronal dysfunction in a Drosophila model of Alzheimer's disease.

    Directory of Open Access Journals (Sweden)

    Kanae Iijima-Ando

    2009-12-01

    Full Text Available The amyloid-beta 42 (Abeta42 is thought to play a central role in the pathogenesis of Alzheimer's disease (AD. However, the molecular mechanisms by which Abeta42 induces neuronal dysfunction and degeneration remain elusive. Mitochondrial dysfunctions are implicated in AD brains. Whether mitochondrial dysfunctions are merely a consequence of AD pathology, or are early seminal events in AD pathogenesis remains to be determined. Here, we show that Abeta42 induces mitochondrial mislocalization, which contributes to Abeta42-induced neuronal dysfunction in a transgenic Drosophila model. In the Abeta42 fly brain, mitochondria were reduced in axons and dendrites, and accumulated in the somata without severe mitochondrial damage or neurodegeneration. In contrast, organization of microtubule or global axonal transport was not significantly altered at this stage. Abeta42-induced behavioral defects were exacerbated by genetic reductions in mitochondrial transport, and were modulated by cAMP levels and PKA activity. Levels of putative PKA substrate phosphoproteins were reduced in the Abeta42 fly brains. Importantly, perturbations in mitochondrial transport in neurons were sufficient to disrupt PKA signaling and induce late-onset behavioral deficits, suggesting a mechanism whereby mitochondrial mislocalization contributes to Abeta42-induced neuronal dysfunction. These results demonstrate that mislocalization of mitochondria underlies the pathogenic effects of Abeta42 in vivo.

  15. Targeting the unmet medical need: the Abbott Laboratories oncology approach.

    Science.gov (United States)

    Carlson, Dawn M; Steinberg, Joyce L; Gordon, Gary

    2005-09-01

    While significant advances in the treatment of cancer occured during the last half of the twentieth century, parallel decreases in overall cancer death rates were not observed. Cancer therapy remains an area of significant unmet medical need. Abbott's oncology research programs are focused on pioneering trageted, less toxic therapies, aimed at different aspects of tumor growth and development. Oncology drugs in development at Abbott target several mechanisms of cancer progression by interfering with multiple processes necessary for tumor growth: recruitment of a blood supply, cell proliferation, and the development of metastases. They include a selective endothelin A-receptor antagonist (atrasentan/Xinlay), 3 angiogenesis inhibitors (ABT 510, a thrombospondin mimetic: ABT-869, a multitargeted receptor tyrosine kinase inhibitor; and ABT 828, recombinant human plasminogen kringle 5), a cell proliferation inhibitor (ABT-751, an antimitotic agent), an apoptosis inducer (ABT 737, a Bcl-2 family inhibitor), and a poly(ADP-ribose)polymerase inhibitor.

  16. Celastrol targets mitochondrial respiratory chain complex I to induce reactive oxygen species-dependent cytotoxicity in tumor cells

    Directory of Open Access Journals (Sweden)

    Xu Yuanji

    2011-05-01

    Full Text Available Abstract Background Celastrol is an active ingredient of the traditional Chinese medicinal plant Tripterygium Wilfordii, which exhibits significant antitumor activity in different cancer models in vitro and in vivo; however, the lack of information on the target and mechanism of action of this compound have impeded its clinical application. In this study, we sought to determine the mode of action of celastrol by focusing on the processes that mediate its anticancer activity. Methods The downregulation of heat shock protein 90 (HSP90 client proteins, phosphorylation of c-Jun NH2-terminal kinase (JNK, and cleavage of PARP, caspase 9 and caspase 3 were detected by western blotting. The accumulation of reactive oxygen species (ROS was analyzed by flow cytometry and fluorescence microscopy. Cell cycle progression, mitochondrial membrane potential (MMP and apoptosis were determined by flow cytometry. Absorption spectroscopy was used to determine the activity of mitochondrial respiratory chain (MRC complexes. Results Celastrol induced ROS accumulation, G2-M phase blockage, apoptosis and necrosis in H1299 and HepG2 cells in a dose-dependent manner. N-acetylcysteine (NAC, an antioxidative agent, inhibited celastrol-induced ROS accumulation and cytotoxicity. JNK phosphorylation induced by celastrol was suppressed by NAC and JNK inhibitor SP600125 (SP. Moreover, SP significantly inhibited celastrol-induced loss of MMP, cleavage of PARP, caspase 9 and caspase 3, mitochondrial translocation of Bad, cytoplasmic release of cytochrome c, and cell death. However, SP did not inhibit celastrol-induced ROS accumulation. Celastrol downregulated HSP90 client proteins but did not disrupt the interaction between HSP90 and cdc37. NAC completely inhibited celastrol-induced decrease of HSP90 client proteins, catalase and thioredoxin. The activity of MRC complex I was completely inhibited in H1299 cells treated with 6 μM celastrol in the absence and presence of NAC

  17. Ginsenoside Re protects against phencyclidine-induced behavioral changes and mitochondrial dysfunction via interactive modulation of glutathione peroxidase-1 and NADPH oxidase in the dorsolateral cortex of mice.

    Science.gov (United States)

    Tran, The-Vinh; Shin, Eun-Joo; Dang, Duy-Khanh; Ko, Sung Kwon; Jeong, Ji Hoon; Nah, Seung-Yeol; Jang, Choon-Gon; Lee, Yu Jeung; Toriumi, Kazuya; Nabeshima, Toshitaka; Kim, Hyoung-Chun

    2017-12-01

    We investigated whether ginsenoside Re (Re) modulates phencyclidine (PCP)-induced sociability deficits and recognition memory impairments to extend our recent finding. We examined the role of GPx-1 gene in the pharmacological activity of Re against mitochondrial dysfunction induced by PCP in the dorsolateral cortex of mice. Since mitochondrial oxidative stress activates NADPH oxidase (PHOX), we applied PHOX inhibitor apocynin for evaluating interactive modulation between GPx-1 and PHOX against PCP neurotoxicity. Sociability deficits and recognition memory impairments induced by PCP were more pronounced in GPx-1 knockout (KO) than in wild type (WT) mice. PCP-induced mitochondrial oxidative stress, mitochondrial dysfunction, and membrane translocation of p47phox were more evident in GPx-1 KO than in WT. Re treatment significantly attenuated PCP-induced neurotoxic changes. Re also significantly attenuated PCP-induced sociability deficits and recognition memory impairments. The attenuation by Re was comparable to that by apocynin. The attenuation was more obvious in GPx-1 KO than in WT. Importantly, apocynin did not show any additional positive effects on the neuroprotective activity of Re, indicating that PHOX is a molecular target for therapeutic activity of Re. Our results suggest that Re requires interactive modulation between GPx activity and PHOX (p47phox) to exhibit neuroprotective potentials against PCP insult. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    Moepert, Kristin [Silence Therapeutics AG, 13125 Berlin (Germany); Hajek, Petr [Division of Biology, California Institute of Technology, Pasadena, CA 91125 (United States); Frank, Stephan [Department of Neuropathology, Institute of Pathology, University Hospital Basel, CH-4031 Basel (Switzerland); Chen, Christiane [Department of Pediatric Hematology and Oncology, University Children' s Hospital Muenster, 48149 Muenster (Germany); Kaufmann, Joerg [Silence Therapeutics AG, 13125 Berlin (Germany); Santel, Ansgar, E-mail: a.santel@silence-therapeutics.com [Silence Therapeutics AG, 13125 Berlin (Germany)

    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.

  19. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chun-Ru [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Liao, Wei-Siang [Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Wu, Ya-Hui [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Murugan, Kaliyappan [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chen, Chinpiao, E-mail: chinpiao@mail.ndhu.edu.tw [Department of Chemistry, National Dong Hwa University, Hualien 974, Taiwan (China); Chao, Jui-I, E-mail: jichao@faculty.nctu.edu.tw [Department of Biological Science and Technology, National Chiao Tung University, Hsinchu 30068, Taiwan (China); Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu 30068, Taiwan (China)

    2013-12-15

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy.

  20. CR108, a novel vitamin K3 derivative induces apoptosis and breast tumor inhibition by reactive oxygen species and mitochondrial dysfunction

    International Nuclear Information System (INIS)

    Yang, Chun-Ru; Liao, Wei-Siang; Wu, Ya-Hui; Murugan, Kaliyappan; Chen, Chinpiao; Chao, Jui-I

    2013-01-01

    Vitamin K3 derivatives have been shown to exert anticancer activities. Here we show a novel vitamin K3 derivative (S)-2-(2-hydroxy-3-methylbutylthio)naphthalene-1,4-dione, which is named as CR108 that induces apoptosis and tumor inhibition through reactive oxygen species (ROS) and mitochondrial dysfunction in human breast cancer. CR108 is more effective on the breast cancer cell death than other vitamin K3 derivatives. Moreover, CR108 induced apoptosis in both the non-HER-2-overexpressed MCF-7 and HER-2-overexpressed BT-474 breast cancer cells. CR108 caused the loss of mitochondrial membrane potential, cytochrome c released from mitochondria to cytosol, and cleaved PARP proteins for apoptosis induction. CR108 markedly increased ROS levels in breast cancer cells. N-acetylcysteine (NAC), a general ROS scavenger, completely blocked the CR108-induced ROS levels, mitochondrial dysfunction and apoptosis. Interestingly, CR108 increased the phosphorylation of p38 MAP kinase but conversely inhibited the survivin protein expression. NAC treatment prevented the activation of p38 MAP kinase and rescued the survivin protein levels. SB202190, a specific p38 MAP kinase inhibitor, recovered the survivin protein levels and attenuated the cytotoxicity of CR108-treated cells. Furthermore, CR108 inhibited the xenografted human breast tumor growth in nude mice. Together, we demonstrate that CR108 is a novel vitamin K3 derivative that induces apoptosis and tumor inhibition by ROS production and mitochondrial dysfunction and associates with the phosphorylation of p38 MAP kinase and the inhibition of survivin in the human breast cancer. - Highlights: • CR108 is more effective on the cell death than other vitamin K3 derivatives. • CR108 induces apoptosis and tumor inhibition by ROS and mitochondrial dysfunction. • CR108 induces apoptosis by p38 kinase activation and survivin inhibition. • CR108 is a potent vitamin K3 analog that can develop for breast cancer therapy

  1. Cyanidin-3-glucoside inhibits glutamate-induced Zn2+ signaling and neuronal cell death in cultured rat hippocampal neurons by inhibiting Ca2+-induced mitochondrial depolarization and formation of reactive oxygen species.

    Science.gov (United States)

    Yang, Ji Seon; Perveen, Shazia; Ha, Tae Joung; Kim, Seong Yun; Yoon, Shin Hee

    2015-05-05

    Cyanidin-3-glucoside (C3G), a member of the anthocyanin family, is a potent natural antioxidant. However, effects of C3G on glutamate-induced [Zn(2+)]i increase and neuronal cell death remain unknown. We studied the effects of C3G on glutamate-induced [Zn(2+)]i increase and cell death in cultured rat hippocampal neurons from embryonic day 17 maternal Sprague-Dawley rats using digital imaging methods for Zn(2+), Ca(2+), reactive oxygen species (ROS), mitochondrial membrane potential and a MTT assay for cell survival. Treatment with glutamate (100 µM) for 7 min induces reproducible [Zn(2+)]i increase at 35 min interval in cultured rat hippocampal neurons. The intracellular Zn(2+)-chelator TPEN markedly blocked glutamate-induced [Zn(2+)]i increase, but the extracellular Zn(2+) chelator CaEDTA did not affect glutamate-induced [Zn(2+)]i increase. C3G inhibited the glutamate-induced [Zn(2+)]i response in a concentration-dependent manner (IC50 of 14.1 ± 1.1 µg/ml). C3G also significantly inhibited glutamate-induced [Ca(2+)]i increase. Two antioxidants such as Trolox and DTT significantly inhibited the glutamate-induced [Zn(2+)]i response, but they did not affect the [Ca(2+)]i responses. C3G blocked glutamate-induced formation of ROS. Trolox and DTT also inhibited the formation of ROS. C3G significantly inhibited glutamate-induced mitochondrial depolarization. However, TPEN, Trolox and DTT did not affect the mitochondrial depolarization. C3G, Trolox and DTT attenuated glutamate-induced neuronal cell death in cultured rat hippocampal neurons, respectively. Taken together, all these results suggest that cyanidin-3-glucoside inhibits glutamate-induced [Zn(2+)]i increase through a release of Zn(2+) from intracellular sources in cultured rat hippocampal neurons by inhibiting Ca(2+)-induced mitochondrial depolarization and formation of ROS, which is involved in neuroprotection against glutamate-induced cell death. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Reactive oxygen species mediates homocysteine-induced mitochondrial biogenesis in human endothelial cells: Modulation by antioxidants

    International Nuclear Information System (INIS)

    Perez-de-Arce, Karen; Foncea, Rocio; Leighton, Federico

    2005-01-01

    It has been proposed that homocysteine (Hcy)-induces endothelial dysfunction and atherosclerosis by generation of reactive oxygen species (ROS). A previous report has shown that Hcy promotes mitochondrial damage. Considering that oxidative stress can affect mitochondrial biogenesis, we hypothesized that Hcy-induced ROS in endothelial cells may lead to increased mitochondrial biogenesis. We found that Hcy-induced ROS (1.85-fold), leading to a NF-κB activation and increase the formation of 3-nitrotyrosine. Furthermore, expression of the mitochondrial biogenesis factors, nuclear respiratory factor-1 and mitochondrial transcription factor A, was significantly elevated in Hcy-treated cells. These changes were accompanied by increase in mitochondrial mass and higher mRNA and protein expression of the subunit III of cytochrome c oxidase. These effects were significantly prevented by pretreatment with the antioxidants, catechin and trolox. Taken together, our results suggest that ROS is an important mediator of mitochondrial biogenesis induced by Hcy, and that modulation of oxidative stress by antioxidants may protect against the adverse vascular effects of Hcy

  3. PREVENTION OF PHOSPHATE - INDUCED MITOCHONDRIAL SWELLING

    Science.gov (United States)

    Kroll, Arnold J.; Kuwabara, Toichiro

    1962-01-01

    The prevention of phosphate-induced mitochondrial swelling in the whole retina of the rabbit was studied with the electron microscope. It was found that a mixture of ATP, Mg++, and bovine serum albumin protected the mitochondria in vitro. This finding confirmed the results obtained spectrophotometrically with isolated rat liver mitochondria by Lehninger. PMID:13927020

  4. Transcranial low-level laser therapy improves brain mitochondrial function and cognitive impairment in D-galactose-induced aging mice.

    Science.gov (United States)

    Salehpour, Farzad; Ahmadian, Nahid; Rasta, Seyed Hossein; Farhoudi, Mehdi; Karimi, Pouran; Sadigh-Eteghad, Saeed

    2017-10-01

    Mitochondrial function plays a key role in the aging-related cognitive impairment, and photoneuromodulation of mitochondria by transcranial low-level laser therapy (LLLT) may contribute to its improvement. This study focused on the transcranial LLLT effects on the D-galactose (DG)-induced mitochondrial dysfunction, apoptosis, and cognitive impairment in mice. For this purpose, red and near-infrared (NIR) laser wavelengths (660 and 810 nm) at 2 different fluencies (4 and 8 J/cm 2 ) at 10-Hz pulsed wave mode were administrated transcranially 3 d/wk in DG-received (500 mg/kg/subcutaneous) mice model of aging for 6 weeks. Spatial and episodic-like memories were assessed by the Barnes maze and What-Where-Which (WWWhich) tasks. Brain tissues were analyzed for mitochondrial function including active mitochondria, adenosine triphosphate, and reactive oxygen species levels, as well as membrane potential and cytochrome c oxidase activity. Apoptosis-related biomarkers, namely, Bax, Bcl-2, and caspase-3 were evaluated by Western blotting method. Laser treatments at wavelengths of 660 and 810 nm at 8 J/cm 2 attenuated DG-impaired spatial and episodic-like memories. Also, results showed an obvious improvement in the mitochondrial function aspects and modulatory effects on apoptotic markers in aged mice. However, same wavelengths at the fluency of 4 J/cm 2 had poor effect on the behavioral and molecular indexes in aging model. This data indicates that transcranial LLLT at both of red and NIR wavelengths at the fluency of 8 J/cm 2 has a potential to ameliorate aging-induced mitochondrial dysfunction, apoptosis, and cognitive impairment. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Discovery and molecular characterization of a Bcl-2-regulated cell death pathway in schistosomes.

    Science.gov (United States)

    Lee, Erinna F; Clarke, Oliver B; Evangelista, Marco; Feng, Zhiping; Speed, Terence P; Tchoubrieva, Elissaveta B; Strasser, Andreas; Kalinna, Bernd H; Colman, Peter M; Fairlie, W Douglas

    2011-04-26

    Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2-regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2-like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with "BH3 mimetic" drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment.

  6. Discovery and molecular characterization of a Bcl-2–regulated cell death pathway in schistosomes

    Science.gov (United States)

    Lee, Erinna F.; Clarke, Oliver B.; Evangelista, Marco; Feng, Zhiping; Speed, Terence P.; Tchoubrieva, Elissaveta B.; Strasser, Andreas; Kalinna, Bernd H.; Colman, Peter M.; Fairlie, W. Douglas

    2011-01-01

    Schistosomiasis is an infectious disease caused by parasites of the phylum platyhelminthe. Here, we describe the identification and characterization of a Bcl-2–regulated apoptosis pathway in Schistosoma japonicum and S. mansoni. Genomic, biochemical, and cell-based mechanistic studies provide evidence for a tripartite pathway, similar to that in humans including BH3-only proteins that are inhibited by prosurvival Bcl-2–like molecules, and Bax/Bak-like proteins that facilitate mitochondrial outer-membrane permeabilization. Because Bcl-2 proteins have been successfully targeted with “BH3 mimetic” drugs, particularly in the treatment of cancer, we investigated whether schistosome apoptosis pathways could provide targets for future antischistosomal drug discovery efforts. Accordingly, we showed that a schistosome prosurvival protein, sjA, binds ABT-737, a well-characterized BH3 mimetic. A crystal structure of sjA bound to a BH3 peptide provides direct evidence for the feasibility of developing BH3 mimetics to target Bcl-2 prosurvival proteins in schistosomes, suggesting an alternative application for this class of drugs beyond cancer treatment. PMID:21444803

  7. Mitochondrial translocation of Nur77 induced by ROS contributed to cardiomyocyte apoptosis in metabolic syndrome.

    Science.gov (United States)

    Xu, Aibin; Liu, Jingyi; Liu, Peilin; Jia, Min; Wang, Han; Tao, Ling

    2014-04-18

    Metabolic syndrome is a major risk factor for cardiovascular diseases, and increased cardiomyocyte apoptosis which contributes to cardiac dysfunction after myocardial ischemia/reperfusion (MI/R) injury. Nur77, a nuclear orphan receptor, is involved in such various cellular events as apoptosis, proliferation, and glucose and lipid metabolism in several cell types. Apoptosis is positively correlated with mitochondrial translocation of Nur77 in the cancer cells. However, the roles of Nur77 on cardiac myocytes in patients with metabolic syndrome remain unclear. The objective of this study was to determine whether Nur77 may contribute to cardiac apoptosis in patients with metabolic syndrome after I/R injury, and, if so, to identify the underlying molecular mechanisms responsible. We used leptin-deficient (ob/ob) mice to make metabolic syndrome models. In this report, we observed that, accompanied by the substantial decline in apoptosis inducer Nur77, MI/R induced cardiac dysfunction was manifested as cardiomyopathy and increased ROS. Using the neonatal rat cardiac myocytes cultured in a high-glucose and high-fat medium, we found that excessive H2O2 led to the significant alteration in mitochondrial membrane potential and translocation of Nur77 from the nucleus to the mitochondria. However, inhibition of the relocation of Nur77 to mitochondria via Cyclosporin A reversed the changes in membrane potential mediated by H2O2 and reduced myocardial cell injury. Therefore, these data provide a potential underlying mechanism for cardiac dysfunction in metabolic syndrome and the suppression of Nur77 translocation may provide an effective approach to reduce cardiac injury in the process. Copyright © 2014 Elsevier Inc. All rights reserved.

  8. Autophagy capacity and sub-mitochondrial heterogeneity shape Bnip3-induced mitophagy regulation of apoptosis.

    Science.gov (United States)

    Choe, Sehyo Charley; Hamacher-Brady, Anne; Brady, Nathan Ryan

    2015-08-08

    Mitochondria are key regulators of apoptosis. In response to stress, BH3-only proteins activate pro-apoptotic Bcl2 family proteins Bax and Bak, which induce mitochondrial outer membrane permeabilization (MOMP). While the large-scale mitochondrial release of pro-apoptotic proteins activates caspase-dependent cell death, a limited release results in sub-lethal caspase activation which promotes tumorigenesis. Mitochondrial autophagy (mitophagy) targets dysfunctional mitochondria for degradation by lysosomes, and undergoes extensive crosstalk with apoptosis signaling, but its influence on apoptosis remains undetermined. The BH3-only protein Bnip3 integrates apoptosis and mitophagy signaling at different signaling domains. Bnip3 inhibits pro-survival Bcl2 members via its BH3 domain and activates mitophagy through its LC3 Interacting Region (LIR), which is responsible for binding to autophagosomes. Previously, we have shown that Bnip3-activated mitophagy prior to apoptosis induction can reduce mitochondrial activation of caspases, suggesting that a reduction to mitochondrial levels may be pro-survival. An outstanding question is whether organelle dynamics and/or recently discovered subcellular variations of protein levels responsible for both MOMP sensitivity and crosstalk between apoptosis and mitophagy can influence the cellular apoptosis decision event. To that end, here we undertook a systems biology analysis of mitophagy-apoptosis crosstalk at the level of cellular mitochondrial populations. Based on experimental findings, we developed a multi-scale, hybrid model with an individually adaptive mitochondrial population, whose actions are determined by protein levels, embedded in an agent-based model (ABM) for simulating subcellular dynamics and local feedback via reactive oxygen species signaling. Our model, supported by experimental evidence, identified an emergent regulatory structure within canonical apoptosis signaling. We show that the extent of mitophagy is

  9. Nucleo-mitochondrial interaction of yeast in response to cadmium sulfide quantum dot exposure

    International Nuclear Information System (INIS)

    Pasquali, Francesco; Agrimonti, Caterina; Pagano, Luca; Zappettini, Andrea; Villani, Marco; Marmiroli, Marta; White, Jason C.; Marmiroli, Nelson

    2017-01-01

    Highlights: • CdS QDs induce oxidative stress in yeast. • CdS QDs disrupt mitochondrial membrane potentials and morphology. • CdS QDs do not affect mtDNA content. • CdS QDs modify the expression of genes involved in mitochondrial organization and function. • Deletion of some of these genes induces either tolerant or sensitive phenotypes to CdS QDs. - Abstract: Cell sensitivity to quantum dots (QDs) has been attributed to a cascade triggered by oxidative stress leading to apoptosis. The role and function of mitochondria in animal cells are well understood but little information is available on the complex genetic networks that regulate nucleo-mitochondrial interaction. The effect of CdS QD exposure in yeast Saccharomyces cerevisiae was assessed under conditions of limited lethality (<10%), using cell physiological and morphological endpoints. Whole-genomic array analysis and the screening of a deletion mutant library were also carried out. The results showed that QDs: increased the level of reactive oxygen species (ROS) and decreased the level of reduced vs oxidized glutathione (GSH/GSSG); reduced oxygen consumption and the abundance of respiratory cytochromes; disrupted mitochondrial membrane potentials and affected mitochondrial morphology. Exposure affected the capacity of cells to grow on galactose, which requires nucleo-mitochondrial involvement. However, QDs exposure did not materially induce respiratory deficient (RD) mutants but only RD phenocopies. All of these cellular changes were correlated with several key nuclear genes, including TOM5 and FKS1, involved in the maintenance of mitochondrial organization and function. The consequences of these cellular effects are discussed in terms of dysregulation of cell function in response to these “pathological mitochondria”.

  10. Nucleo-mitochondrial interaction of yeast in response to cadmium sulfide quantum dot exposure

    Energy Technology Data Exchange (ETDEWEB)

    Pasquali, Francesco; Agrimonti, Caterina [Department of Life Sciences, University of Parma, Parma (Italy); Pagano, Luca [Department of Life Sciences, University of Parma, Parma (Italy); Stockbridge school of Agriculture, University of Massachusetts, Amherst, MA (United States); The Connecticut Agricultural Experiment Station, New Haven, CT (United States); Zappettini, Andrea; Villani, Marco [IMEM-CNR - Istituto dei Materiali per l' Elettronica ed il Magnetismo, Parma (Italy); Marmiroli, Marta [Department of Life Sciences, University of Parma, Parma (Italy); White, Jason C. [The Connecticut Agricultural Experiment Station, New Haven, CT (United States); Marmiroli, Nelson, E-mail: nelson.marmiroli@unipr.it [Department of Life Sciences, University of Parma, Parma (Italy); CINSA - Consorzio Interuniversitario Nazionale per le Scienze Ambientali, University of Parma, Parma (Italy)

    2017-02-15

    Highlights: • CdS QDs induce oxidative stress in yeast. • CdS QDs disrupt mitochondrial membrane potentials and morphology. • CdS QDs do not affect mtDNA content. • CdS QDs modify the expression of genes involved in mitochondrial organization and function. • Deletion of some of these genes induces either tolerant or sensitive phenotypes to CdS QDs. - Abstract: Cell sensitivity to quantum dots (QDs) has been attributed to a cascade triggered by oxidative stress leading to apoptosis. The role and function of mitochondria in animal cells are well understood but little information is available on the complex genetic networks that regulate nucleo-mitochondrial interaction. The effect of CdS QD exposure in yeast Saccharomyces cerevisiae was assessed under conditions of limited lethality (<10%), using cell physiological and morphological endpoints. Whole-genomic array analysis and the screening of a deletion mutant library were also carried out. The results showed that QDs: increased the level of reactive oxygen species (ROS) and decreased the level of reduced vs oxidized glutathione (GSH/GSSG); reduced oxygen consumption and the abundance of respiratory cytochromes; disrupted mitochondrial membrane potentials and affected mitochondrial morphology. Exposure affected the capacity of cells to grow on galactose, which requires nucleo-mitochondrial involvement. However, QDs exposure did not materially induce respiratory deficient (RD) mutants but only RD phenocopies. All of these cellular changes were correlated with several key nuclear genes, including TOM5 and FKS1, involved in the maintenance of mitochondrial organization and function. The consequences of these cellular effects are discussed in terms of dysregulation of cell function in response to these “pathological mitochondria”.

  11. Bisphenol A induces oxidative stress and mitochondrial dysfunction in lymphoblasts from children with autism and unaffected siblings.

    Science.gov (United States)

    Kaur, Kulbir; Chauhan, Ved; Gu, Feng; Chauhan, Abha

    2014-11-01

    Autism is a behaviorally defined neurodevelopmental disorder. Although there is no single identifiable cause for autism, roles for genetic and environmental factors have been implicated in autism. Extensive evidence suggests increased oxidative stress and mitochondrial dysfunction in autism. In this study, we examined whether bisphenol A (BPA) is an environmental risk factor for autism by studying its effects on oxidative stress and mitochondrial function in the lymphoblasts. When lymphoblastoid cells from autistic subjects and age-matched unaffected sibling controls were exposed to BPA, there was an increase in the generation of reactive oxygen species (ROS) and a decrease in mitochondrial membrane potential in both groups. A further subdivision of the control group into two subgroups-unaffected nontwin siblings and twin siblings-showed significantly higher ROS levels without any exposure to BPA in the unaffected twin siblings compared to the unaffected nontwin siblings. ROS levels were also significantly higher in the autism vs the unaffected nontwin siblings group. The effect of BPA on three important mtDNA genes-NADH dehydrogenase 1, NADH dehydrogenase 4, and cytochrome b-was analyzed to observe any changes in the mitochondria after BPA exposure. BPA induced a significant increase in the mtDNA copy number in the lymphoblasts from the unaffected siblings group and in the unaffected twin siblings group vs the unaffected nontwin siblings. In all three genes, the mtDNA increase was seen in 70% of the subjects. These results suggest that BPA exposure results in increased oxidative stress and mitochondrial dysfunction in the autistic subjects as well as the age-matched sibling control subjects, particularly unaffected twin siblings. Therefore, BPA may act as an environmental risk factor for autism in genetically susceptible children by inducing oxidative stress and mitochondrial dysfunction. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Mitochondrial Membrane Permeability Inhibitors in Acute Myocardial Infarction

    Directory of Open Access Journals (Sweden)

    Cory Trankle, MD

    2016-10-01

    Full Text Available Despite therapeutic advances, acute myocardial infarction (AMI remains a leading cause of morbidity and mortality worldwide. One potential limitation of the current treatment paradigm is the lack of effective therapies to optimize reperfusion after ischemia and prevent reperfusion-mediated injury. Experimental studies indicate that this process accounts for up to 50% of the final infarct size, lending it importance as a potential target for cardioprotection. However, multiple therapeutic approaches have shown potential in pre-clinical and early phase trials but a paucity of clear clinical benefit when expanded to larger studies. Here we explore this history of trials and errors of the studies of cyclosporine A and other mitochondrial membrane permeability inhibitors, agents that appeared to have a promising pre-clinical record yet provided disappointing results in phase III clinical trials.

  13. A reaction-diffusion model of ROS-induced ROS release in a mitochondrial network.

    Directory of Open Access Journals (Sweden)

    Lufang Zhou

    2010-01-01

    Full Text Available Loss of mitochondrial function is a fundamental determinant of cell injury and death. In heart cells under metabolic stress, we have previously described how the abrupt collapse or oscillation of the mitochondrial energy state is synchronized across the mitochondrial network by local interactions dependent upon reactive oxygen species (ROS. Here, we develop a mathematical model of ROS-induced ROS release (RIRR based on reaction-diffusion (RD-RIRR in one- and two-dimensional mitochondrial networks. The nodes of the RD-RIRR network are comprised of models of individual mitochondria that include a mechanism of ROS-dependent oscillation based on the interplay between ROS production, transport, and scavenging; and incorporating the tricarboxylic acid (TCA cycle, oxidative phosphorylation, and Ca(2+ handling. Local mitochondrial interaction is mediated by superoxide (O2.- diffusion and the O2.(--dependent activation of an inner membrane anion channel (IMAC. In a 2D network composed of 500 mitochondria, model simulations reveal DeltaPsi(m depolarization waves similar to those observed when isolated guinea pig cardiomyocytes are subjected to a localized laser-flash or antioxidant depletion. The sensitivity of the propagation rate of the depolarization wave to O(2.- diffusion, production, and scavenging in the reaction-diffusion model is similar to that observed experimentally. In addition, we present novel experimental evidence, obtained in permeabilized cardiomyocytes, confirming that DeltaPsi(m depolarization is mediated specifically by O2.-. The present work demonstrates that the observed emergent macroscopic properties of the mitochondrial network can be reproduced in a reaction-diffusion model of RIRR. Moreover, the findings have uncovered a novel aspect of the synchronization mechanism, which is that clusters of mitochondria that are oscillating can entrain mitochondria that would otherwise display stable dynamics. The work identifies the

  14. The mitochondrial inner membrane AAA metalloprotease family in metazoans.

    Science.gov (United States)

    Juhola, M K; Shah, Z H; Grivell, L A; Jacobs, H T

    2000-09-15

    Three metalloproteases belonging to the AAA superfamily (Yme1p, Afg3p and Rca1p) are involved in protein turnover and respiratory chain complex assembly in the yeast inner mitochondrial membrane. Analysis of the completed genome sequences of Caenorhabditis elegans and Drosophila melanogaster indicates that this gene family typically comprises 3-4 members in metazoans. Phylogenetic analysis reveals three main branches represented, respectively, by Saccharomyces cerevisiae YME1, human SPG7 (paraplegin) and S. cerevisiae AFG3 and RCA1. mt-AAA metalloproteases are weak candidates for several previously studied Drosophila mutants. A full elucidation of the cellular and physiological roles of mt-AAA metalloproteases in metazoans will require the creation of targeted mutations.

  15. Standardized extracts of Bacopa monniera protect against MPP+- and paraquat-induced toxicity by modulating mitochondrial activities, proteasomal functions, and redox pathways.

    Science.gov (United States)

    Singh, Manjeet; Murthy, Ven; Ramassamy, Charles

    2012-01-01

    Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases and affects millions of people worldwide. Strong evidence supports the role of free radicals, oxidative stress, mitochondrial, and proteasomal dysfunctions underlying neuronal death in PD. Environmental factors, especially pesticides, represent one of the primary classes of neurotoxic agents associated with PD, and several epidemiological studies have identified the exposure of the herbicide paraquat (PQ) as a potential risk factor for the onset of PD. The objective of our study was to investigate the neuroprotective effects of the standardized extracts of Bacopa monniera (BM) against PQ-induced and 1-methyl-4-phenyl-pyridinium iodide (MPP(+))-induced toxicities and to elucidate the mechanisms underlying this protection. Our results show that a pretreatment with the BM extract from 50 μg/ml protected the dopaminergic SK-N-SH cell line against MPP(+)- and PQ-induced toxicities in various cell survival assays. We demonstrate that BM pretreatment prevented the depletion of glutathione (GSH) besides preserving the mitochondrial membrane potential and maintaining the mitochondrial complex I activity. BM pretreatment from 10.0 μg/ml also prevented the generation of intracellular reactive oxygen species and decreased the mitochondrial superoxide level. BM treatment activated the nuclear factor erythroid 2-related factor 2 pathway by modulating the expression of Keap1, thereby upregulating the endogenous GSH synthesis. The effect of BM on the phosphorylation of Akt further strengthens its role in the promotion of cell survival. By preserving the cellular redox homeostasis and mitochondrial activities and by promoting cell survival pathways, BM extract may have therapeutic uses in various age-related neurodegenerative diseases such as PD.

  16. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation

    Directory of Open Access Journals (Sweden)

    Fang Chen

    2016-04-01

    Full Text Available Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O, has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu and Cat D inhibitor (pepA inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05 in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  17. Rat liver mitochondrial damage under acute or chronic carbon tetrachloride-induced intoxication: Protection by melatonin and cranberry flavonoids

    Energy Technology Data Exchange (ETDEWEB)

    Cheshchevik, V.T. [Institute for Pharmacology and Biochemistry, National Academy of Sciences of Belarus, Len. Kom. Blvd. - 50, 230017 Grodno (Belarus); Department of Biochemistry, Yanka Kupala Grodno State University, Len. Kom. Blvd. - 50, 230017 Grodno (Belarus); Lapshina, E.A.; Dremza, I.K.; Zabrodskaya, S.V. [Institute for Pharmacology and Biochemistry, National Academy of Sciences of Belarus, Len. Kom. Blvd. - 50, 230017 Grodno (Belarus); Reiter, R.J. [Department of Cellular and Structural Biology, University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78229–3900 (United States); Prokopchik, N.I. [Grodno State Medical University, Gorkogo - 80, 230015 Grodno (Belarus); Zavodnik, I.B., E-mail: zavodnik_il@mail.ru [Institute for Pharmacology and Biochemistry, National Academy of Sciences of Belarus, Len. Kom. Blvd. - 50, 230017 Grodno (Belarus); Department of Biochemistry, Yanka Kupala Grodno State University, Len. Kom. Blvd. - 50, 230017 Grodno (Belarus)

    2012-06-15

    In current societies, the risk of toxic liver damage has markedly increased. The aim of the present work was to carry out further research into the mechanism(s) of liver mitochondrial damage induced by acute (0.8 g/kg body weight, single injection) or chronic (1.6 g/ kg body weight, 30 days, biweekly injections) carbon tetrachloride – induced intoxication and to evaluate the hepatoprotective potential of the antioxidant, melatonin, as well as succinate and cranberry flavonoids in rats. Acute intoxication resulted in considerable impairment of mitochondrial respiratory parameters in the liver. The activity of mitochondrial succinate dehydrogenase (complex II) decreased (by 25%, p < 0.05). Short-term melatonin treatment (10 mg/kg, three times) of rats did not reduce the degree of toxic mitochondrial dysfunction but decreased the enhanced NO production. After 30-day chronic intoxication, no significant change in the respiratory activity of liver mitochondria was observed, despite marked changes in the redox-balance of mitochondria. The activities of the mitochondrial enzymes, succinate dehydrogenase and glutathione peroxidase, as well as that of cytoplasmic catalase in liver cells were inhibited significantly. Mitochondria isolated from the livers of the rats chronically treated with CCl{sub 4} displayed obvious irreversible impairments. Long-term melatonin administration (10 mg/kg, 30 days, daily) to chronically intoxicated rats diminished the toxic effects of CCl{sub 4}, reducing elevated plasma activities of alanine aminotransferase and aspartate aminotransferase and bilirubin concentration, prevented accumulation of membrane lipid peroxidation products in rat liver and resulted in apparent preservation of the mitochondrial ultrastructure. The treatment of the animals by the complex of melatonin (10 mg/kg) plus succinate (50 mg/kg) plus cranberry flavonoids (7 mg/kg) was even more effective in prevention of toxic liver injury and liver mitochondria damage

  18. Changes in mitochondrial function by lipid peroxidation and their inhibition by biscoclaurin alkaloid

    International Nuclear Information System (INIS)

    Aono, K.; Shiraishi, N.; Arita, T.; Inouye, B.; Nakazawa, T.; Utsumi, K.

    1981-01-01

    During in vitro investigation of changes in mitochondrial function accompanying lipid peroxidation, it was found that cepharanthine, a biscoclaurin alkaloid, protects against such change. Results obtained were as follows: (1) Fe2+ induces lipid peroxidation of isolated mitochondria, resulting in diminished oxidative phosphorylation. (2) This diminishment largely depends on deterioration of ion compartmentation of the membrane and an increase in latent ATPase activity. (3) The Fe2+-induced deterioration in ion compartmentation is inhibited by cepharanthine. (4) Cepharanthine inhibits the mitochondrial lipid peroxidation induced by Fe2+. (5) Cepharanthine inhibits the lipid peroxidation of soybean lecithin liposomes by 60Co-irradiation

  19. Oxidative Stress Induced Mitochondrial Failure and Vascular Hypoperfusion as a Key Initiator for the Development of Alzheimer Disease

    Directory of Open Access Journals (Sweden)

    Valentin Bragin

    2010-01-01

    Full Text Available Mitochondrial dysfunction may be a principal underlying event in aging, including age-associated brain degeneration. Mitochondria provide energy for basic metabolic processes. Their decay with age impairs cellular metabolism and leads to a decline of cellular function. Alzheimer disease (AD and cerebrovascular accidents (CVAs are two leading causes of age-related dementia. Increasing evidence strongly supports the theory that oxidative stress, largely due to reactive oxygen species (ROS, induces mitochondrial damage, which arises from chronic hypoperfusion and is primarily responsible for the pathogenesis that underlies both disease processes. Mitochondrial membrane potential, respiratory control ratios and cellular oxygen consumption decline with age and correlate with increased oxidant production. The sustained hypoperfusion and oxidative stress in brain tissues can stimulate the expression of nitric oxide synthases (NOSs and brain endothelium probably increase the accumulation of oxidative stress products, which therefore contributes to blood brain barrier (BBB breakdown and brain parenchymal cell damage. Determining the mechanisms behind these imbalances may provide crucial information in the development of new, more effective therapies for stroke and AD patients in the near future.

  20. Obesity-induced down-regulation of the mitochondrial translocator protein (TSPO) impairs placental steroid production.

    Science.gov (United States)

    Lassance, Luciana; Haghiac, Maricela; Minium, Judi; Catalano, Patrick; Hauguel-de Mouzon, Sylvie

    2015-01-01

    Low concentrations of estradiol and progesterone are hallmarks of adverse pregnancy outcomes as is maternal obesity. During pregnancy, placental cholesterol is the sole source of sex steroids. Cholesterol trafficking is the limiting step in sex steroid biosynthesis and is mainly mediated by the translocator protein (TSPO), present in the mitochondrial outer membrane. The objective of the study was to investigate the effects of maternal obesity in placental sex steroid biosynthesis and TSPO regulation. One hundred forty-four obese (body mass index 30-35 kg/m(2)) and 90 lean (body mass index 19-25 kg/m(2)) pregnant women (OP and LP, respectively) recruited at scheduled term cesarean delivery. Placenta and maternal blood were collected. This study was conducted at MetroHealth Medical Center (Cleveland, Ohio). Maternal metabolic components (fasting glucose, insulin, leptin, estradiol, progesterone, and total cholesterol) and placental weight were measured. Placenta (mitochondria and membranes separated) and cord blood cholesterol values were verified. The expression and regulation of TSPO and mitochondrial function were analyzed. Plasma estradiol and progesterone concentrations were significantly lower (P < .04) in OP as compared with LP women. Maternal and cord plasma cholesterol were not different between groups. Placental citrate synthase activity and mitochondrial DNA, markers of mitochondrial density, were unchanged, but the mitochondrial cholesterol concentrations were 40% lower in the placenta of OP. TSPO gene and protein expressions were decreased 2-fold in the placenta of OP. In vitro trophoblast activation of the innate immune pathways with lipopolysaccharide and long-chain saturated fatty acids reduced TSPO expression by 2- to 3-fold (P < .05). These data indicate that obesity in pregnancy impairs mitochondrial steroidogenic function through the negative regulation of mitochondrial TSPO.

  1. Mitochondrial dysfunction and organophosphorus compounds

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-07-01

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

  2. Mitochondrial dysfunction and organophosphorus compounds

    International Nuclear Information System (INIS)

    Karami-Mohajeri, Somayyeh; Abdollahi, Mohammad

    2013-01-01

    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

  3. 31 CFR 15.737-19 - Administrative Law Judge.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Administrative Law Judge. 15.737-19... CONFLICT OF INTEREST Administrative Enforcement Proceedings § 15.737-19 Administrative Law Judge. (a) Appointment. An Administrative Law Judge appointed as provided by 5 U.S.C. 3105 (1966), shall conduct...

  4. Importance of mitochondrial calcium uniporter in high glucose-induced endothelial cell dysfunction.

    Science.gov (United States)

    Chen, Wei; Yang, Jie; Chen, Shuhua; Xiang, Hong; Liu, Hengdao; Lin, Dan; Zhao, Shaoli; Peng, Hui; Chen, Pan; Chen, Alex F; Lu, Hongwei

    2017-11-01

    Mitochondrial Ca 2+ overload is implicated in hyperglycaemia-induced endothelial cell dysfunction, but the key molecular events responsible remain unclear. We examined the involvement of mitochondrial calcium uniporter, which mediates mitochondrial Ca 2+ uptake, in endothelial cell dysfunction resulting from high-glucose treatment. Human umbilical vein endothelial cells were exposed to various glucose concentrations and to high glucose (30 mM) following mitochondrial calcium uniporter inhibition or activation with ruthenium red and spermine, respectively. Subsequently, mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA and protein expression was measured by real-time polymerase chain reaction and western blotting. Ca 2+ concentrations were analysed by laser confocal microscopy, and cytoplasmic and mitochondrial oxidative stress was detected using 2',7'-dichlorofluorescein diacetate and MitoSOX Red, respectively. Apoptosis was assessed by annexin V-fluorescein isothiocyanate/propidium iodide staining, and a wound-healing assay was performed using an in vitro model. High glucose markedly upregulated mitochondrial calcium uniporter and mitochondrial calcium uniporter regulator 1 messenger RNA expression, as well as protein production, in a dose- and time-dependent manner with a maximum effect demonstrated at 72 h and 30 mM glucose concentration. Moreover, high-glucose treatment significantly raised both mitochondrial and cytoplasmic Ca 2+ and reactive oxygen species levels, increased apoptosis and compromised wound healing (all p calcium uniporter, respectively. Mitochondrial calcium uniporter plays an important role in hyperglycaemia-induced endothelial cell dysfunction and may constitute a therapeutic target to reduce vascular complications in diabetes.

  5. Diclofenac induces proteasome and mitochondrial dysfunction in murine cardiomyocytes and hearts.

    Science.gov (United States)

    Ghosh, Rajeshwary; Goswami, Sumanta K; Feitoza, Luis Felipe B B; Hammock, Bruce; Gomes, Aldrin V

    2016-11-15

    One of the most common nonsteroidal anti-inflammatory drugs (NSAIDs) used worldwide, diclofenac (DIC), has been linked to increased risk of cardiovascular disease (CVD). The molecular mechanism(s) by which DIC causes CVD is unknown. Proteasome activities were studied in hearts, livers, and kidneys from male Swiss Webster mice treated with either 100mg/kg DIC for 18h (acute treatment) or 10mg/kg DIC for 28days (chronic treatment). Cultured H9c2 cells and neonatal cardiomyocytes were also treated with different concentrations of DIC and proteasome function, cell death and ROS generation studied. Isolated mouse heart mitochondria were utilized to determine the effect of DIC on various electron transport chain complex activities. DIC significantly inhibited the chymotrypsin-like proteasome activity in rat cardiac H9c2 cells, murine neonatal cardiomyocytes, and mouse hearts, but did not affect proteasome subunit expression levels. Proteasome activity was also affected in liver and kidney tissues from DIC treated animals. The levels of polyubiquitinated proteins increased in hearts from DIC treated mice. Importantly, the levels of oxidized proteins increased while the β5i immunoproteasome activity decreased in hearts from DIC treated mice. DIC increased ROS production and cell death in H9c2 cells and neonatal cardiomyocytes while the cardioprotective NSAID, aspirin, had no effect on ROS levels or cell viability. DIC inhibited mitochondrial Complex III, a major source of ROS, and impaired mitochondrial membrane potential suggesting that mitochondria are the major sites of ROS generation. These results suggest that DIC induces cardiotoxicity by a ROS dependent mechanism involving mitochondrial and proteasome dysfunction. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Polyglutamine toxicity in yeast induces metabolic alterations and mitochondrial defects

    KAUST Repository

    Papsdorf, Katharina

    2015-09-03

    Background Protein aggregation and its pathological effects are the major cause of several neurodegenerative diseases. In Huntington’s disease an elongated stretch of polyglutamines within the protein Huntingtin leads to increased aggregation propensity. This induces cellular defects, culminating in neuronal loss, but the connection between aggregation and toxicity remains to be established. Results To uncover cellular pathways relevant for intoxication we used genome-wide analyses in a yeast model system and identify fourteen genes that, if deleted, result in higher polyglutamine toxicity. Several of these genes, like UGO1, ATP15 and NFU1 encode mitochondrial proteins, implying that a challenged mitochondrial system may become dysfunctional during polyglutamine intoxication. We further employed microarrays to decipher the transcriptional response upon polyglutamine intoxication, which exposes an upregulation of genes involved in sulfur and iron metabolism and mitochondrial Fe-S cluster formation. Indeed, we find that in vivo iron concentrations are misbalanced and observe a reduction in the activity of the prominent Fe-S cluster containing protein aconitase. Like in other yeast strains with impaired mitochondria, non-fermentative growth is impossible after intoxication with the polyglutamine protein. NMR-based metabolic analyses reveal that mitochondrial metabolism is reduced, leading to accumulation of metabolic intermediates in polyglutamine-intoxicated cells. Conclusion These data show that damages to the mitochondrial system occur in polyglutamine intoxicated yeast cells and suggest an intricate connection between polyglutamine-induced toxicity, mitochondrial functionality and iron homeostasis in this model system.

  7. In vitro pharmacological characterization of a novel selective alpha7 neuronal nicotinic acetylcholine receptor agonist ABT-107.

    Science.gov (United States)

    Malysz, John; Anderson, David J; Grønlien, Jens H; Ji, Jianguo; Bunnelle, William H; Håkerud, Monika; Thorin-Hagene, Kirten; Ween, Hilde; Helfrich, Rosalind; Hu, Min; Gubbins, Earl; Gopalakrishnan, Sujatha; Puttfarcken, Pamela S; Briggs, Clark A; Li, Jinhe; Meyer, Michael D; Dyhring, Tino; Ahring, Philip K; Nielsen, Elsebet Ø; Peters, Dan; Timmermann, Daniel B; Gopalakrishnan, Murali

    2010-09-01

    cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity alpha7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

  8. Induction of necrosis and apoptosis to KB cancer cells by sanguinarine is associated with reactive oxygen species production and mitochondrial membrane depolarization

    International Nuclear Information System (INIS)

    Chang, M.-C.; Chan, C.-P.; Wang, Y.-J.; Lee, P.-H.; Chen, L.-I; Tsai, Y.-L.; Lin, B.-R.; Wang, Y.-L.; Jeng, J.-H.

    2007-01-01

    Sanguinarine is a benzopheanthridine alkaloid present in the root of Sanguinaria canadensis L. and Chellidonium majus L. In this study, sanguinarine (2 and 3 μM) exhibited cytotoxicity to KB cancer cells by decreasing MTT reduction to 83% and 52% of control after 24-h of exposure. Sanguinarine also inhibited the colony forming capacity (> 52-58%) and growth of KB cancer cells at concentrations higher than 0.5-1 μM. Short-term exposure to sanguinarine (> 0.5 μM) effectively suppressed the adhesion of KB cells to collagen and fibronectin (FN). Sanguinarine (2 and 3 μM) induced evident apoptosis as indicated by an increase in sub-G0/G1 populations, which was detected after 6-h of exposure. Only a slight increase in cells arresting in S-phase and G2/M was noted. Induction of KB cell apoptosis and necrosis by sanguinarine (2 and 3 μM) was further confirmed by Annexin V-PI dual staining flow cytometry and the presence of DNA fragmentation. The cytotoxicity by sanguinarine was accompanied by an increase in production of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential as indicated by single cell flow cytometric analysis of DCF and rhodamine fluorescence. NAC (1 and 3 mM) and catalase (2000 U/ml) prevented the sanguinarine-induced ROS production and cytotoxicity, whereas dimethylthiourea (DMT) showed no marked preventive effect. These results suggest that sanguinarine has anticarcinogenic properties with induction of ROS production and mitochondrial membrane depolarization, which mediate cancer cell death

  9. Role of Lactobacillus plantarum MTCC1325 in membrane-bound transport ATPases system in Alzheimer’s disease-induced rat brain

    Directory of Open Access Journals (Sweden)

    Nimgampalle Mallikarjuna

    2016-12-01

    Results: Chronic injection of D-Galactose caused lipid peroxidation, oxidative stress, and mitochondrial dysfunction leading to the damage of neurons in the brain, finally bringing a significant decrease (-20% in the brain total membrane bound ATPases over the controls. Contrary to this, treatment of AD-induced rats with L. plantarum MTCC1325 reverted all the constituents of ATPase enzymes to near normal levels within 30 days. Conclusion: Lactobacillus plantarum MTCC1325 exerted a beneficial action on the entire ATPases system in AD-induced rat brain by delaying neurodegeneration.

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

    Directory of Open Access Journals (Sweden)

    Tobias eMühling

    2014-11-01

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

  11. SMG-1 kinase attenuates mitochondrial ROS production but not cell respiration deficits during hyperoxia.

    Science.gov (United States)

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

    Supplemental oxygen (hyperoxia) used to treat individuals in respiratory distress causes cell injury by enhancing the production of toxic reactive oxygen species (ROS) and inhibiting mitochondrial respiration. The suppressor of morphogenesis of genitalia (SMG-1) kinase is activated during hyperoxia and promotes cell survival by phosphorylating the tumor suppressor p53 on serine 15. Here, we investigate whether SMG-1 and p53 blunt this vicious cycle of progressive ROS production and decline in mitochondrial respiration seen during hyperoxia. Human lung adenocarcinoma A549 and H1299 or colon carcinoma HCT116 cells were depleted of SMG-1, UPF-1, or p53 using RNA interference, and then exposed to room air (21% oxygen) or hyperoxia (95% oxygen). Immunoblotting was used to evaluate protein expression; a Seahorse Bioanalyzer was used to assess cellular respiration; and flow cytometry was used to evaluate fluorescence intensity of cells stained with mitochondrial or redox sensitive dyes. Hyperoxia increased mitochondrial and cytoplasmic ROS and suppressed mitochondrial respiration without changing mitochondrial mass or membrane potential. Depletion of SMG-1 or its cofactor, UPF1, significantly enhanced hyperoxia-induced mitochondrial but not cytosolic ROS abundance. They did not affect mitochondrial mass, membrane potential, or hyperoxia-induced deficits in mitochondrial respiration. Genetic depletion of p53 in A549 cells and ablation of the p53 gene in H1299 or HCT116 cells revealed that SMG-1 influences mitochondrial ROS through activation of p53. Our findings show that hyperoxia does not promote a vicious cycle of progressive mitochondrial ROS and dysfunction because SMG-1-p53 signaling attenuates production of mitochondrial ROS without preserving respiration. This suggests antioxidant therapies that blunt ROS production during hyperoxia may not suffice to restore cellular respiration.

  12. The production of reactive oxygen species and the mitochondrial membrane potential are modulated during onion oil-induced cell cycle arrest and apoptosis in A549 cells.

    Science.gov (United States)

    Wu, Xin-jiang; Stahl, Thorsten; Hu, Ying; Kassie, Fekadu; Mersch-Sundermann, Volker

    2006-03-01

    Protective effects of Allium vegetables against cancers have been shown extensively in experimental animals and epidemiologic studies. We investigated cell proliferation and the induction of apoptosis by onion oil extracted from Allium cepa, a widely consumed Allium vegetable, in human lung cancer A549 cells. GC/MS analysis suggested that propyl sulfides but not allyl sulfides are major sulfur-containing constituents of onion oil. Onion oil at 12.5 mg/L significantly induced apoptosis (13% increase of apoptotic cells) as indicated by sub-G1 DNA content. It also caused cell cycle arrest at the G2/M phase; 25 mg/L onion oil increased the percentage of G2/M cells almost 6-fold compared with the dimethyl sulfoxide control. The action of onion oil may occur via a reactive oxygen species-dependent pathway because cell cycle arrest and apoptosis were blocked by the antioxidants N-acetylcysteine and exogenous glutathione. Marked collapse of the mitochondrial membrane potential suggested that dysfunction of the mitochondria may be involved in the oxidative burst and apoptosis induced by onion oil. Expression of phospho-cdc2 and phospho-cyclin B1 were downregulated by onion oil, perhaps accounting for the G2/M arrest. Overall, these results suggest that onion oil may exert chemopreventive action by inducing cell cycle arrest and apoptosis in tumor cells.

  13. Mitochondrial Reactive Oxygen Species Mediate Cardiac Structural, Functional, and Mitochondrial Consequences of Diet-Induced Metabolic Heart Disease.

    Science.gov (United States)

    Sverdlov, Aaron L; Elezaby, Aly; Qin, Fuzhong; Behring, Jessica B; Luptak, Ivan; Calamaras, Timothy D; Siwik, Deborah A; Miller, Edward J; Liesa, Marc; Shirihai, Orian S; Pimentel, David R; Cohen, Richard A; Bachschmid, Markus M; Colucci, Wilson S

    2016-01-11

    Mitochondrial reactive oxygen species (ROS) are associated with metabolic heart disease (MHD). However, the mechanism by which ROS cause MHD is unknown. We tested the hypothesis that mitochondrial ROS are a key mediator of MHD. Mice fed a high-fat high-sucrose (HFHS) diet develop MHD with cardiac diastolic and mitochondrial dysfunction that is associated with oxidative posttranslational modifications of cardiac mitochondrial proteins. Transgenic mice that express catalase in mitochondria and wild-type mice were fed an HFHS or control diet for 4 months. Cardiac mitochondria from HFHS-fed wild-type mice had a 3-fold greater rate of H2O2 production (P=0.001 versus control diet fed), a 30% decrease in complex II substrate-driven oxygen consumption (P=0.006), 21% to 23% decreases in complex I and II substrate-driven ATP synthesis (P=0.01), and a 62% decrease in complex II activity (P=0.002). In transgenic mice that express catalase in mitochondria, all HFHS diet-induced mitochondrial abnormalities were ameliorated, as were left ventricular hypertrophy and diastolic dysfunction. In HFHS-fed wild-type mice complex II substrate-driven ATP synthesis and activity were restored ex vivo by dithiothreitol (5 mmol/L), suggesting a role for reversible cysteine oxidative posttranslational modifications. In vitro site-directed mutation of complex II subunit B Cys100 or Cys103 to redox-insensitive serines prevented complex II dysfunction induced by ROS or high glucose/high palmitate in the medium. Mitochondrial ROS are pathogenic in MHD and contribute to mitochondrial dysfunction, at least in part, by causing oxidative posttranslational modifications of complex I and II proteins including reversible oxidative posttranslational modifications of complex II subunit B Cys100 and Cys103. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

  14. Ursolic Acid-enriched herba cynomorii extract induces mitochondrial uncoupling and glutathione redox cycling through mitochondrial reactive oxygen species generation: protection against menadione cytotoxicity in h9c2 cells.

    Science.gov (United States)

    Chen, Jihang; Wong, Hoi Shan; Ko, Kam Ming

    2014-01-27

    Herba Cynomorii (Cynomorium songaricum Rupr., Cynomoriaceae) is one of the most commonly used 'Yang-invigorating' tonic herbs in Traditional Chinese Medicine (TCM). An earlier study in our laboratory has demonstrated that HCY2, an ursolic acid-enriched fraction derived from Herba Cynomorii, increased mitochondrial ATP generation capacity (ATP-GC) and induced mitochondrial uncoupling as well as a cellular glutathione response, thereby protecting against oxidant injury in H9c2 cells. In this study, we demonstrated that pre-incubation of H9c2 cells with HCY2 increased mitochondrial reactive oxygen species (ROS) generation in these cells, which is likely an event secondary to the stimulation of the mitochondrial electron transport chain. The suppression of mitochondrial ROS by the antioxidant dimethylthiourea abrogated the HCY2-induced enhancement of mitochondrial uncoupling and glutathione reductase (GR)-mediated glutathione redox cycling, and also protected against menadione-induced cytotoxicity. Studies using specific inhibitors of uncoupling protein and GR suggested that the HCY2-induced mitochondrial uncoupling and glutathione redox cycling play a determining role in the cytoprotection against menadione-induced oxidant injury in H9c2 cells. Experimental evidence obtained thus far supports the causal role of HCY2-induced mitochondrial ROS production in eliciting mitochondrial uncoupling and glutathione antioxidant responses, which offer cytoprotection against oxidant injury in H9c2 cells.

  15. Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing.

    Science.gov (United States)

    Sommer, Natascha; Hüttemann, Maik; Pak, Oleg; Scheibe, Susan; Knoepp, Fenja; Sinkler, Christopher; Malczyk, Monika; Gierhardt, Mareike; Esfandiary, Azadeh; Kraut, Simone; Jonas, Felix; Veith, Christine; Aras, Siddhesh; Sydykov, Akylbek; Alebrahimdehkordi, Nasim; Giehl, Klaudia; Hecker, Matthias; Brandes, Ralf P; Seeger, Werner; Grimminger, Friedrich; Ghofrani, Hossein A; Schermuly, Ralph T; Grossman, Lawrence I; Weissmann, Norbert

    2017-08-04

    Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Isolated ventilated and perfused lungs from Cox4i2 -/- mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2 -/- mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild-type PASMCs was absent in Cox4i2 -/- PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2 -/- PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2 -/- PASMCs compared with wild-type PASMCs, which could be normalized by the application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion

  16. Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

    Energy Technology Data Exchange (ETDEWEB)

    Shekhar, Tanmay M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Green, Maja M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Department of Anatomy & Neuroscience, The University of Melbourne, Parkville 3010 (Australia); Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M. [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia); Hawkins, Christine J., E-mail: c.hawkins@latrobe.edu.au [Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Bundoora 3083 (Australia)

    2015-07-15

    Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

  17. Inhibition of Bcl-2 or IAP proteins does not provoke mutations in surviving cells

    International Nuclear Information System (INIS)

    Shekhar, Tanmay M.; Green, Maja M.; Rayner, David M.; Miles, Mark A.; Cutts, Suzanne M.; Hawkins, Christine J.

    2015-01-01

    Graphical abstract: - Highlights: • Mutagenicities of anti-cancer drugs were tested using HPRT, γH2AX and comet assays. • TRAIL, doxorubicin and etoposide were more mutagenic than BH3- or Smac-mimetics. • Physiologically achievable levels of the BH3-mimetic ABT-737 were not mutagenic. • High concentrations of ABT-737 provoked mutations via an off-target mechanism. • Even very high concentrations of IAP antagonists were not mutagenic. - Abstract: Chemotherapy and radiotherapy can cause permanent damage to the genomes of surviving cells, provoking severe side effects such as second malignancies in some cancer survivors. Drugs that mimic the activity of death ligands, or antagonise pro-survival proteins of the Bcl-2 or IAP families have yielded encouraging results in animal experiments and early phase clinical trials. Because these agents directly engage apoptosis pathways, rather than damaging DNA to indirectly provoke tumour cell death, we reasoned that they may offer another important advantage over conventional therapies: minimisation or elimination of side effects such as second cancers that result from mutation of surviving normal cells. Disappointingly, however, we previously found that concentrations of death receptor agonists like TRAIL that would be present in vivo in clinical settings provoked DNA damage in surviving cells. In this study, we used cell line model systems to investigate the mutagenic capacity of drugs from two other classes of direct apoptosis-inducing agents: the BH3-mimetic ABT-737 and the IAP antagonists LCL161 and AT-406. Encouragingly, our data suggest that IAP antagonists possess negligible genotoxic activity. Doses of ABT-737 that were required to damage DNA stimulated Bax/Bak-independent signalling and exceeded concentrations detected in the plasma of animals treated with this drug. These findings provide hope that cancer patients treated by BH3-mimetics or IAP antagonists may avoid mutation-related illnesses that afflict

  18. Lost region in amyloid precursor protein (APP) through TALEN-mediated genome editing alters mitochondrial morphology.

    Science.gov (United States)

    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.

  19. 31 CFR 15.737-12 - Contents of complaint.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Contents of complaint. 15.737-12 Section 15.737-12 Money and Finance: Treasury Office of the Secretary of the Treasury POST EMPLOYMENT... charges against him/her so that the respondent is able to prepare a defense. (b) Demand for answer. In the...

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

    Directory of Open Access Journals (Sweden)

    Andrej Korenić

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

  1. Characterization of canine mitochondrial protein expression in natural and induced forms of idiopathic dilated cardiomyopathy.

    Science.gov (United States)

    Lopes, Rosana; Solter, Philip F; Sisson, D David; Oyama, Mark A; Prosek, Robert

    2006-06-01

    To map canine mitochondrial proteins and identify qualitative and quantitative differences in heart mitochondrial protein expression between healthy dogs and dogs with naturally occurring and induced dilated cardiomyopathy (DCM). Left ventricle samples were obtained from 7 healthy dogs, 7 Doberman Pinschers with naturally occurring DCM, and 7 dogs with induced DCM. Fresh and frozen mitochondrial fractions were isolated from the left ventricular free wall and analyzed by 2-dimensional electrophoresis. Protein spots that increased or decreased in density by >or= 2-fold between groups were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometry. Within narrow pH gradients of control canine heart mitochondrial samples, a total of 1,528 protein spots were revealed. Forty subunits of heart mitochondrial proteins that differ significantly from control tissues were altered in tissue specimens from dogs with naturally occurring and induced forms of DCM. The most affected heart mitochondrial proteins in both groups were those of oxidative phosphorylation (55%). Upregulation of manganese superoxide dismutase was suggestive of heart oxidative injury in tissue specimens from dogs with both forms of DCM. Evidence of apoptosis was associated with overexpression of the heart mitochondrial voltage-dependent anion channel-2 protein and endonuclease G in tissue specimens from dogs with induced DCM. Alterations of heart mitochondrial proteins related to oxidative phosphorylation dysfunction were more prevalent in tissue specimens from dogs with induced or naturally occurring DCM, compared with those of control dogs.

  2. The voltage-dependent anion selective channel 1 (VDAC1 topography in the mitochondrial outer membrane as detected in intact cell.

    Directory of Open Access Journals (Sweden)

    Marianna F Tomasello

    Full Text Available Voltage-Dependent Anion selective Channel maintains the permeability of the outer mitochondrial membrane and is relevant in bioenergetic metabolism and apoptosis. The structure of the protein was shown to be a β-barrel formed by 19 strands. The topology or sideness of the pore has been predicted with various approaches but a general consensus was never reached. This is an important issue since VDAC is considered receptor of Hexokinase and Bcl-2. We fused at VDAC1 C-terminus two tags separated by a caspase cleavage site. Activation in cellulo of caspases was used to eventually separate the two reporters. This experiment did not require the isolation of mitochondria and limited the possibility of outer membrane rupture due to similar procedures. Our results show that the C-terminus end of VDAC faces the mitochondrial inter-membrane space.

  3. STING-IRF3 Triggers Endothelial Inflammation in Response to Free Fatty Acid-Induced Mitochondrial Damage in Diet-Induced Obesity

    Science.gov (United States)

    Mao, Yun; Luo, Wei; Zhang, Lin; Wu, Weiwei; Yuan, Liangshuai; Xu, Hao; Song, Juhee; Fujiwara, Keigi; Abe, Jun-ichi; LeMaire, Scott A.; Wang, Xing Li; Shen, Ying. H.

    2017-01-01

    Objective Metabolic stress in obesity induces endothelial inflammation and activation, which initiates adipose tissue inflammation, insulin resistance, and cardiovascular diseases. However, the mechanisms underlying endothelial inflammation induction are not completely understood. Stimulator of interferon genes (STING) is an important molecule in immunity and inflammation. In the present study, we sought to determine the role of STING in palmitic acid (PA)-induced endothelial activation/inflammation. Approach and Results In cultured endothelial cells, PA treatment activated STING, as indicated by its perinuclear translocation and binding to interferon regulatory factor 3 (IRF3), leading to IRF3 phosphorylation and nuclear translocation. The activated IRF3 bound to the promoter of intercellular adhesion molecule 1 (ICAM-1) and induced ICAM-1 expression and monocyte–endothelial cell adhesion. When analyzing the upstream signaling, we found that PA activated STING by inducing mitochondrial damage. PA treatment caused mitochondrial damage and leakage of mitochondrial DNA (mtDNA) into the cytosol. Through the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS), the mitochondrial damage and leaked cytosolic mtDNA activated the STING-IRF3 pathway and increased ICAM-1 expression. In mice with diet-induced obesity, the STING-IRF3 pathway was activated in adipose tissue. However, STING deficiency (Stinggt/gt) partially prevented diet-induced adipose tissue inflammation, obesity, insulin resistance, and glucose intolerance. Conclusions The mitochondrial damage-cGAS-STING-IRF3 pathway is critically involved in metabolic stress-induced endothelial inflammation. STING may be a potential therapeutic target for preventing cardiovascular diseases and insulin resistance in obese individuals. PMID:28302626

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

    Directory of Open Access Journals (Sweden)

    Carlos Logullo

    2011-06-01

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

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

    Science.gov (United States)

    Campos, Eldo; Façanha, Arnoldo R.; Costa, Evenilton P.; Fraga, Amanda; Moraes, Jorge; da Silva Vaz, Itabajara; Masuda, Aoi; Logullo, Carlos

    2011-01-01

    The physiological roles of polyphosphates (polyP) recently found in arthropod mitochondria remain obscure. Here, the relationship between the mitochondrial membrane exopolyphosphatase (PPX) and the energy metabolism of hard tick Rhipicephalus microplus embryos are investigated. Mitochondrial respiration was activated by adenosine diphosphate using polyP as the only source of inorganic phosphate (Pi) and this activation was much greater using polyP3 than polyP15. After mitochondrial subfractionation, most of the PPX activity was recovered in the membrane fraction and its kinetic analysis revealed that the affinity for polyP3 was 10 times stronger than that for polyP15. Membrane PPX activity was also increased in the presence of the respiratory substrate pyruvic acid and after addition of the protonophore carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Furthermore, these stimulatory effects disappeared upon addition of the cytochrome oxidase inhibitor potassium cyanide and the activity was completely inhibited by 20 μg/mL heparin. The activity was either increased or decreased by 50% upon addition of dithiothreitol or hydrogen peroxide, respectively, suggesting redox regulation. These results indicate a PPX activity that is regulated during mitochondrial respiration and that plays a role in adenosine-5′-triphosphate synthesis in hard tick embryos. PMID:21747692

  6. ABTS assay of phenol oxidase activity in soil.

    Science.gov (United States)

    Floch, Carine; Alarcon-Gutiérrez, Enrique; Criquet, Stéven

    2007-12-01

    Phenol oxidases (PO) are involved in degradation of many recalcitrant aromatic compounds and may be sensitive to some pollutants. Hence, their activities may be a useful indicator for evaluating soil quality and health. To this end, the aim of this study was to develop a simple method to assay PO activity directly in bulk samples by spectrophotometric test using 2,2'-azinobis-(-3 ethylbenzothiazoline-6-sulfononic acid) diammonium salt (ABTS) as the substrate. Three Mediterranean soils were used as models. For each soil, we studied the kinetic parameters and the effects of certain factors (i.e. amount of soil, pH, temperature, incubation time and substrate concentration) in order to determine the optimum conditions for the ABTS assay. Results showed that PO attain their optimum activities when incubating 0.1 g of soil at 30 degrees C for 5 min with 10 ml of a Modified Universal Buffer (MUB) at pH 2 and 200 microl of a 0.1 M ABTS solution.

  7. Molecular basis for mitochondrial signaling

    CERN Document Server

    2017-01-01

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

  8. The cyclophilin D/Drp1 axis regulates mitochondrial fission contributing to oxidative stress-induced mitochondrial dysfunctions in SH-SY5Y cells

    International Nuclear Information System (INIS)

    Xiao, Anqi; Gan, Xueqi; Chen, Ruiqi; Ren, Yanming; Yu, Haiyang; You, Chao

    2017-01-01

    Oxidative stress plays a central role in the pathogenesis of various neurodegenerative diseases. Increasing evidences have demonstrated that structural abnormalities in mitochondria are involved in oxidative stress related nerve cell damage. And Drp1 plays a critical role in mitochondrial dynamic imbalance insulted by oxidative stress-derived mitochondria. However, the status of mitochondrial fusion and fission pathway and its relationship with mitochondrial properties such as mitochondrial membrane permeability transition pore (mPTP) have not been fully elucidated. Here, we demonstrated for the first time the role of Cyclophilin D (CypD), a crucial component for mPTP formation, in the regulation of mitochondrial dynamics in oxidative stress treated nerve cell. We observed that CypD-mediated phosphorylation of Drp1 and subsequently augmented Drp1 recruitment to mitochondria and shifts mitochondrial dynamics toward excessive fission, which contributes to the mitochondrial structural and functional dysfunctions in oxidative stress-treated nerve cells. CypD depletion or over expression accompanies mitochondrial dynamics/functions recovery or aggravation separately. We also demonstrated first time the link between the CypD to mitochondrial dynamics. Our data offer new insights into the mechanism of mitochondrial dynamics which contribute to the mitochondrial dysfunctions, specifically the role of CypD in Drp1-mediated mitochondrial fission. The protective effect of CsA, or other molecules affecting the function of CypD hold promise as a potential novel therapeutic strategy for governing oxidative stress pathology via mitochondrial pathways. - Highlights: • Demonstrated first time the link between the mPTP to mitochondrial dynamics. • The role of Cyclophilin D in the regulation of Drp1-mediated mitochondrial fission. • CsA as a potential target for governing oxidative stress related neuropathology.

  9. Fast-twitch glycolytic skeletal muscle is predisposed to age-induced impairments in mitochondrial function

    DEFF Research Database (Denmark)

    Jacobs, Robert A; Díaz, Víctor; Soldini, Lavinia

    2013-01-01

    The etiology of mammalian senescence is suggested to involve the progressive impairment of mitochondrial function; however, direct observations of age-induced alterations in actual respiratory chain function are lacking. Accordingly, we assessed mitochondrial function via high-resolution respirom......The etiology of mammalian senescence is suggested to involve the progressive impairment of mitochondrial function; however, direct observations of age-induced alterations in actual respiratory chain function are lacking. Accordingly, we assessed mitochondrial function via high......-resolution respirometry and mitochondrial protein expression in soleus, quadricep, and lateral gastrocnemius skeletal muscles, which represent type 1 slow-twitch oxidative muscle (soleus) and type 2 fast-twitch glycolytic muscle (quadricep and gastrocnemius), respectively, in young (10-12 weeks) and mature (74-76 weeks......) mice. Electron transport through mitochondrial complexes I and III increases with age in quadricep and gastrocnemius, which is not observed in soleus. Mitochondrial coupling efficiency during respiration through complex I also deteriorates with age in gastrocnemius and shows a tendency (p = .085...

  10. Glucose-stimulated insulin secretion of insulinoma INS-1E cells is associated with elevation of both respiration and mitochondrial membrane potential

    Czech Academy of Sciences Publication Activity Database

    Špaček, Tomáš; Šantorová, Jitka; Zacharovová, K.; Berková, Z.; Hlavatá, Lydie; Saudek, F.; Ježek, Petr

    2008-01-01

    Roč. 40, č. 8 (2008), s. 1522-1535 ISSN 1357-2725 R&D Projects: GA MZd(CZ) NR7917 Institutional research plan: CEZ:AV0Z50110509 Keywords : in situ mitochondrial membrane potential * in situ mitochondrial respiration * glucose-stimulated insulin secretion Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 4.178, year: 2008

  11. Phenolic extract from oleaster (Olea europaea var. Sylvestris) leaves reduces colon cancer growth and induces caspase-dependent apoptosis in colon cancer cells via the mitochondrial apoptotic pathway.

    Science.gov (United States)

    Zeriouh, Wafa; Nani, Abdelhafid; Belarbi, Meriem; Dumont, Adélie; de Rosny, Charlotte; Aboura, Ikram; Ghanemi, Fatima Zahra; Murtaza, Babar; Patoli, Danish; Thomas, Charles; Apetoh, Lionel; Rébé, Cédric; Delmas, Dominique; Khan, Naim Akhtar; Ghiringhelli, François; Rialland, Mickael; Hichami, Aziz

    2017-01-01

    Dietary polyphenols, derived from natural products, have received a great interest for their chemopreventive properties against cancer. In this study, we investigated the effects of phenolic extract of the oleaster leaves (PEOL) on tumor growth in mouse model and on cell death in colon cancer cell lines. We assessed the effect of oleaster leaf infusion on HCT116 (human colon cancer cell line) xenograft growth in athymic nude mice. We observed that oleaster leaf polyphenol-rich infusion limited HCT116 tumor growth in vivo. Investigations of PEOL on two human CRC cell lines showed that PEOL induced apoptosis in HCT116 and HCT8 cells. We demonstrated an activation of caspase-3, -7 and -9 by PEOL and that pre-treatment with the pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), prevented PEOL-induced cell death. We observed an involvement of the mitochondrial pathway in PEOL-induced apoptosis evidenced by reactive oxygen species (ROS) production, a decrease of mitochondrial membrane potential, and cytochrome c release. Increase in intracellular Ca2+ concentration induced by PEOL represents the early event involved in mitochondrial dysfunction, ROS-induced endoplasmic reticulum (ER) stress and apoptosis induced by PEOL, as ruthenium red, an inhibitor of mitochondrial calcium uptake inhibited apoptotic effect of PEOL, BAPTA/AM inhibited PEOL-induced ROS generation and finally, N-acetyl-L-cysteine reversed ER stress and apoptotic effect of PEOL. These results demonstrate that polyphenols from oleaster leaves might have a strong potential as chemopreventive agent in colorectal cancer.

  12. Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants.

    Science.gov (United States)

    Echtay, Karim S; Murphy, Michael P; Smith, Robin A J; Talbot, Darren A; Brand, Martin D

    2002-12-06

    Superoxide activates nucleotide-sensitive mitochondrial proton transport through the uncoupling proteins UCP1, UCP2, and UCP3 (Echtay, K. S., et al. (2002) Nature 415, 1482-1486). Two possible mechanisms were proposed: direct activation of the UCP proton transport mechanism by superoxide or its products and a cycle of hydroperoxyl radical entry coupled to UCP-catalyzed superoxide anion export. Here we provide evidence for the first mechanism and show that superoxide activates UCP2 in rat kidney mitochondria from the matrix side of the mitochondrial inner membrane: (i) Exogenous superoxide inhibited matrix aconitase, showing that external superoxide entered the matrix. (ii) Superoxide-induced uncoupling was abolished by low concentrations of the mitochondrially targeted antioxidants 10-(6'-ubiquinonyl)decyltriphenylphosphonium (mitoQ) or 2-[2-(triphenylphosphonio)ethyl]-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol bromide (mitoVit E), which are ubiquinone (Q) or tocopherol derivatives targeted to the matrix by covalent attachment to triphenylphosphonium cation. However, superoxide-induced uncoupling was not affected by similar concentrations of the nontargeted antioxidants Q(o), Q(1), decylubiquinone, vitamin E, or 6-hydroxy-2,5,7,8-tetramethylchroman 2-carboxylic acid (TROLOX) or of the mitochondrially targeted but redox-inactive analogs decyltriphenylphosphonium or 4-chlorobutyltriphenylphosphonium. Thus matrix superoxide appears to be necessary for activation of UCP2 by exogenous superoxide. (iii) When the reduced to oxidized ratio of mitoQ accumulated by mitochondria was increased by inhibiting cytochrome oxidase, it induced nucleotide-sensitive uncoupling that was not inhibited by external superoxide dismutase. Under these conditions quinols are known to produce superoxide, and because mitoQ is localized within the mitochondrial matrix this suggests that production of superoxide in the matrix was sufficient to activate UCP2. Furthermore, the superoxide

  13. ALDH2 restores exhaustive exercise-induced mitochondrial dysfunction in skeletal muscle

    International Nuclear Information System (INIS)

    Zhang, Qiuping; Zheng, Jianheng; Qiu, Jun; Wu, Xiahong; Xu, Yangshuo; Shen, Weili; Sun, Mengwei

    2017-01-01

    Background: Mitochondrial aldehyde dehydrogenase 2 (ALDH2) is highly expressed in heart and skeletal muscles, and is the major enzyme that metabolizes acetaldehyde and toxic aldehydes. The cardioprotective effects of ALDH2 during cardiac ischemia/reperfusion injury have been recognized. However, less is known about the function of ALDH2 in skeletal muscle. This study was designed to evaluate the effect of ALDH2 on exhaustive exercise-induced skeletal muscle injury. Methods: We created transgenic mice expressing ALDH2 in skeletal muscles. Male wild-type C57/BL6 (WT) and ALDH2 transgenic mice (ALDH2-Tg), 8-weeks old, were challenged with exhaustive exercise for 1 week to induce skeletal muscle injury. Animals were sacrificed 24 h post-exercise and muscle tissue was excised. Results: ALDH2-Tg mice displayed significantly increased treadmill exercise capacity compared to WT mice. Exhaustive exercise caused an increase in mRNA levels of the muscle atrophy markers, Atrogin-1 and MuRF1, and reduced mitochondrial biogenesis and fusion in WT skeletal muscles; these effects were attenuated in ALDH2-Tg mice. Exhaustive exercise also enhanced mitochondrial autophagy pathway activity, including increased conversion of LC3-I to LC3-II and greater expression of Beclin1 and Bnip3; the effects of which were mitigated by ALDH2 overexpression. In addition, ALDH2-Tg reversed the increase of an oxidative stress biomarker (4-hydroxynonenal) and decreased levels of mitochondrial antioxidant proteins, including manganese superoxide dismutase and NAD(P)H:quinone oxidoreductase 1, in skeletal muscle induced by exhaustive exercise. Conclusion: ALDH2 may reverse skeletal muscle mitochondrial dysfunction due to exhaustive exercise by regulating mitochondria dynamic remodeling and enhancing the quality of mitochondria. - Highlights: • Skeletal muscle ALDH2 expression and activity declines during exhaustive exercise. • ALDH2 overexpression enhances physical performance and restores muscle

  14. Linezolid-induced lactic acidosis: the thin line between bacterial and mitochondrial ribosomes.

    Science.gov (United States)

    Santini, Alessandro; Ronchi, Dario; Garbellini, Manuela; Piga, Daniela; Protti, Alessandro

    2017-07-01

    Linezolid inhibits bacterial growth by targeting bacterial ribosomes and by interfering with bacterial protein synthesis. Lactic acidosis is a rare, but potentially lethal, side effect of linezolid. Areas covered: The pathogenesis of linezolid-induced lactic acidosis is reviewed with special emphasis on aspects relevant to the recognition, prevention and treatment of the syndrome. Expert opinion: Linezolid-induced lactic acidosis reflects the untoward interaction between the drug and mitochondrial ribosomes. The inhibition of mitochondrial protein synthesis diminishes the respiratory chain enzyme content and thus limits aerobic energy production. As a result, anaerobic glycolysis and lactate generation accelerate independently from tissue hypoxia. In the absence of any confirmatory test, linezolid-induced lactic acidosis should be suspected only after exclusion of other, more common, causes of lactic acidosis such as hypoxemia, anemia or low cardiac output. Normal-to-high whole-body oxygen delivery, high venous oxygen saturation and lack of response to interventions that effectively increase tissue oxygen provision all suggest a primary defect in oxygen use at the mitochondrial level. During prolonged therapy with linezolid, blood drug and lactate levels should be regularly monitored. The current standard-of-care treatment of linezolid-induced lactic acidosis consists of drug withdrawal to reverse mitochondrial intoxication and intercurrent life support.

  15. Effect of Mucuna pruriens (Linn.) on mitochondrial dysfunction and DNA damage in epididymal sperm of streptozotocin induced diabetic rat.

    Science.gov (United States)

    Suresh, Sekar; Prithiviraj, Elumalai; Lakshmi, Nagella Venkata; Ganesh, Mohanraj Karthik; Ganesh, Lakshmanan; Prakash, Seppan

    2013-01-09

    Mucuna pruriens Linn. (M. pruriens) is a leguminous plant that has been recognized as an herbal medicine for improving fertility and related disorders in the Indian traditional system of medicine, however without proper scientific validations. To study the effect of ethanolic seed extract of M. pruriens on mitochondrial dysfunction and the DNA damage in hyperglycemic rat epididymal spermatozoa. Male Wistar albino rats were divided as control (Sham), diabetes induced [streptozotocin 60 mg/kg of body weight (b.w.) in 0.1M citrate buffer] (STZ), diabetic rats administered with 200mg/kg b.w. of extract (STZ+MP) and normal rats administered with 200mg/kg b.w. of extract (Sham+MP). M. pruriens was administered (gavage) once daily for a period of 60 days. On 60th day animals were sacrificed by cervical dislocation sperm were collected from epididymis and subjected various analysis like antioxidants, ROS, lipid peroxidation (LPO), DNA damage, chromosomal integrity and mitochondrial membrane potential (MMP). Significant reduction in the sperm count, motility, viability and significant increase in the number of abnormal sperm in STZ compared to sham was noticed. STZ rat sperm showed significant increase in LPO and DNA damage. Both the enzymic and non-enzymic were decreased; MMP and the mitochondrial functions were severely affected in STZ group. The diabetic rats supplemented with M. pruriens showed a remarkable recovery in antioxidant levels and reduced LPO with well preserved sperm DNA. MMP and mitochondrial function test were also preserved in STZ+MP rat sperm. The present study has clearly demonstrated the potency of M. pruriens to reduce the diabetic induced sperm damage induced by oxidative stress (OS). These observations are encouraging to perform similar studies in human. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  16. Spermine selectively inhibits high-conductance, but not low-conductance calcium-induced permeability transition pore.

    Science.gov (United States)

    Elustondo, Pia A; Negoda, Alexander; Kane, Constance L; Kane, Daniel A; Pavlov, Evgeny V

    2015-02-01

    The permeability transition pore (PTP) is a large channel of the mitochondrial inner membrane, the opening of which is the central event in many types of stress-induced cell death. PTP opening is induced by elevated concentrations of mitochondrial calcium. It has been demonstrated that spermine and other polyamines can delay calcium-induced swelling of isolated mitochondria, suggesting their role as inhibitors of the mitochondrial PTP. Here we further investigated the mechanism by which spermine inhibits the calcium-induced, cyclosporine A (CSA) -sensitive PTP by using three indicators: 1) calcium release from the mitochondria detected with calcium green, 2) mitochondrial membrane depolarization using TMRM, and 3) mitochondrial swelling by measuring light absorbance. We found that despite calcium release and membrane depolarization, indicative of PTP activation, mitochondria underwent only partial swelling in the presence of spermine. This was in striking contrast to the high-amplitude swelling detected in control mitochondria and in mitochondria treated with the PTP inhibitor CSA. We conclude that spermine selectively prevents opening of the high-conductance state, while allowing activation of the lower conductance state of the PTP. We propose that the existence of lower conductance, stress-induced PTP might play an important physiological role, as it is expected to allow the release of toxic levels of calcium, while keeping important molecules (e.g., NAD) within the mitochondrial matrix. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Nicotinamide induces mitochondrial-mediated apoptosis through oxidative stress in human cervical cancer HeLa cells.

    Science.gov (United States)

    Feng, Yi; Wang, Yonghua; Jiang, Chengrui; Fang, Zishui; Zhang, Zhiqiang; Lin, Xiaoying; Sun, Liwei; Jiang, Weiying

    2017-07-15

    Nicotinamide participates in energy metabolism and influences cellular redox status and modulates multiple pathways related with both cellular survival and death. Recent studies have shown that it induced proliferation inhibition and apoptosis in many cancer cells. However, little is known about the effects of nicotinamide on human cervical cancer cells. We aimed to evaluate the effects of the indicated concentrations nicotinamide on cell proliferation, apoptosis and redox-related parameters in HeLa cells and investigated the apoptotic mechanism. After the treatment of the indicated concentrations nicotinamide, HeLa cell proliferation was evaluated by the CCK-8 assay and the production of ROS (reactive oxygen species) was measured using 2',7'-Dichlorofluorescin diacetate. The apoptotic effect was confirmed by observing the cellular and nuclear morphologies with fluorescence microscope and apoptotic rate of HeLa cell apoptosis was measured by flow cytometry using Annexin-V method. Moreover, we examined the mitochondrial membrane potential by JC-1 method and measured the expression of apoptosis related genes using qRT-PCR and immunoblotting. Nicotinamide restrained the HeLa cell proliferation and significantly increased the accumulation of ROS and depletion of GSH at relatively high concentrations. Furthermore, nicotinamide promoted HeLa cell apoptosis via the intrinsic mitochondrial apoptotic pathway. Our study revealed that nicotinamide induced the apoptosis through oxidative stress and intrinsic mitochondrial apoptotic pathways in HeLa cell. The results emerge that nicotinamide may be an inexpensive, safe and promising therapeutic agent or a neoadjuvant chemotherapy for cervical cancer patients, as well useful to find new drugs for cervical cancer therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Current-Induced Membrane Discharge

    DEFF Research Database (Denmark)

    Andersen, Mathias Bækbo; van Soestbergen, M.; Mani, A.

    2012-01-01

    . Salt depletion leads to a large electric field resulting in a local pH shift within the membrane with the effect that the membrane discharges and loses its ion selectivity. Since salt co-ions, H+ ions, and OH- ions contribute to OLC, CIMD interferes with electrodialysis (salt counterion removal...... neglects chemical effects and remains to be quantitatively tested. Here, we show that charge regulation and water self-ionization can lead to OLC by "current-induced membrane discharge'' (CIMD), even in the absence of fluid flow, in ion-exchange membranes much thicker than the local Debye screening length...

  19. Training Enhances Immune Cells Mitochondrial Biosynthesis, Fission, Fusion, and Their Antioxidant Capabilities Synergistically with Dietary Docosahexaenoic Supplementation

    Directory of Open Access Journals (Sweden)

    Carla Busquets-Cortés

    2016-01-01

    Full Text Available Exercise training induces adaptations in mitochondrial metabolism, dynamics, and oxidative protection. Omega-3 fatty acids change membrane lipid composition and modulate mitochondrial function. The aim was to investigate the effect of 8-week training and docosahexaenoic acid (DHA supplementation (1.14 g/day on the mitochondria dynamics and antioxidant status in peripheral blood mononuclear cells (PBMCs from sportsmen. Subjects were assigned to an intervention (N=9 or placebo groups (N=7 in a randomized double-blind trial. Nutritional intervention significantly increased the DHA content in erythrocyte membranes from the experimental group. No significant differences were reported in terms of circulating PBMCs, Mn-superoxide dismutase protein levels, and their capability to produce reactive oxygen species. The proteins related to mitochondrial dynamics were, in general, increased after an 8-week training and this increase was enhanced by DHA supplementation. The content in mitofusins Mtf-1 and Mtf-2, optic atrophy protein-1 (Opa-1, and mitochondrial transcription factor A (Tfam were significantly higher in the DHA-supplemented group after intervention. Cytochrome c oxidase (COX-IV activity and uncoupling proteins UCP-2 and UCP-3 protein levels were increased after training, with higher UCP-3 levels in the supplemented group. In conclusion, training induced mitochondrial adaptations which may contribute to improved mitochondrial function. This mitochondrial response was modulated by DHA supplementation.

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

    Science.gov (United States)

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

    2016-02-09

    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. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Elena A. Matveeva

    2015-10-01

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

  2. Myostatin induces mitochondrial metabolic alteration and typical apoptosis in cancer cells

    Science.gov (United States)

    Liu, Y; Cheng, H; Zhou, Y; Zhu, Y; Bian, R; Chen, Y; Li, C; Ma, Q; Zheng, Q; Zhang, Y; Jin, H; Wang, X; Chen, Q; Zhu, D

    2013-01-01

    Myostatin, a member of the transforming growth factor-β superfamily, regulates the glucose metabolism of muscle cells, while dysregulated myostatin activity is associated with a number of metabolic disorders, including muscle cachexia, obesity and type II diabetes. We observed that myostatin induced significant mitochondrial metabolic alterations and prolonged exposure of myostatin induced mitochondria-dependent apoptosis in cancer cells addicted to glycolysis. To address the underlying mechanism, we found that the protein levels of Hexokinase II (HKII) and voltage-dependent anion channel 1 (VDAC1), two key regulators of glucose metabolisms as well as metabolic stress-induced apoptosis, were negatively correlated. In particular, VDAC1 was dramatically upregulated in cells that are sensitive to myostatin treatment whereas HKII was downregulated and dissociated from mitochondria. Myostatin promoted the translocation of Bax from cytosol to mitochondria, and knockdown of VDAC1 inhibited myostatin-induced Bax translocation and apoptosis. These apoptotic changes can be partially rescued by repletion of ATP, or by ectopic expression of HKII, suggesting that perturbation of mitochondrial metabolism is causally linked with subsequent apoptosis. Our findings reveal novel function of myostatin in regulating mitochondrial metabolism and apoptosis in cancer cells. PMID:23412387

  3. Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells.

    Directory of Open Access Journals (Sweden)

    Arwa S Kathiria

    Full Text Available Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn's disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα, both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB, which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A(1 or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells

  4. Mitochondrial Protein Synthesis, Import, and Assembly

    Science.gov (United States)

    Fox, Thomas D.

    2012-01-01

    The mitochondrion is arguably the most complex organelle in the budding yeast cell cytoplasm. It is essential for viability as well as respiratory growth. Its innermost aqueous compartment, the matrix, is bounded by the highly structured inner membrane, which in turn is bounded by the intermembrane space and the outer membrane. Approximately 1000 proteins are present in these organelles, of which eight major constituents are coded and synthesized in the matrix. The import of mitochondrial proteins synthesized in the cytoplasm, and their direction to the correct soluble compartments, correct membranes, and correct membrane surfaces/topologies, involves multiple pathways and macromolecular machines. The targeting of some, but not all, cytoplasmically synthesized mitochondrial proteins begins with translation of messenger RNAs localized to the organelle. Most proteins then pass through the translocase of the outer membrane to the intermembrane space, where divergent pathways sort them to the outer membrane, inner membrane, and matrix or trap them in the intermembrane space. Roughly 25% of mitochondrial proteins participate in maintenance or expression of the organellar genome at the inner surface of the inner membrane, providing 7 membrane proteins whose synthesis nucleates the assembly of three respiratory complexes. PMID:23212899

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

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

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

  6. 31 CFR 15.737-28 - Notice of disciplinary action.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 1 2010-07-01 2010-07-01 false Notice of disciplinary action. 15.737... period of suspension. (b) The Director shall take other appropriate disciplinary action as may be... EMPLOYMENT CONFLICT OF INTEREST Administrative Enforcement Proceedings § 15.737-28 Notice of disciplinary...

  7. Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

    Directory of Open Access Journals (Sweden)

    Aditya Sen

    2015-06-01

    Full Text Available Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1 and Parkin (Park, either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu, a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

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

    Science.gov (United States)

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

    2017-02-01

    Proteins localized to mitochondria by a carboxyl-terminal tail anchor (TA) play roles in apoptosis, mitochondrial dynamics, and mitochondrial protein import. To reveal characteristics of TAs that may be important for mitochondrial targeting, we focused our attention upon the TA of the Saccharomyces cerevisiae Fis1 protein. Specifically, we generated a library of Fis1p TA variants fused to the Gal4 transcription factor, then, using next-generation sequencing, revealed which Fis1p TA mutations inhibited membrane insertion and allowed Gal4p activity in the nucleus. Prompted by our global analysis, we subsequently analyzed the ability of individual Fis1p TA mutants to localize to mitochondria. Our findings suggest that the membrane-associated domain of the Fis1p TA may be bipartite in nature, and we encountered evidence that the positively charged patch at the carboxyl terminus of Fis1p is required for both membrane insertion and organelle specificity. Furthermore, lengthening or shortening of the Fis1p TA by up to three amino acids did not inhibit mitochondrial targeting, arguing against a model in which TA length directs insertion of TAs to distinct organelles. Most importantly, positively charged residues were more acceptable at several positions within the membrane-associated domain of the Fis1p TA than negatively charged residues. These findings, emerging from the first high-resolution analysis of an organelle targeting sequence by deep mutational scanning, provide strong, in vivo evidence that lysine and arginine can "snorkel," or become stably incorporated within a lipid bilayer by placing terminal charges of their side chains at the membrane interface. Copyright © 2017 by the Genetics Society of America.

  9. Evaluation of the water disinfection by-product dichloroacetonitrile-induced biochemical, oxidative, histopathological, and mitochondrial functional alterations: Subacute oral toxicity in rats.

    Science.gov (United States)

    Dong, Ying; Li, Fang; Shen, Haijun; Lu, Rongzhu; Yin, Siqi; Yang, Qi; Li, Zhuangfa; Wang, Suhua

    2018-03-01

    Dichloroacetonitrile (DCAN), an emerging nitrogenous disinfection by-product, is more genotoxic and cytotoxic than the currently regulated carbonaceous disinfection by-products such as haloacetic acids. Few mechanistic studies have been conducted on the hepatic and renal toxicities of DCAN. This study examined the clinical biochemical, hematological, histopathological, oxidative, and mitochondrial functional alterations to evaluate the systematic toxicity after subacute oral exposure of 11 or 44 mg/kg/day in rats for 28 days. Body and spleen weights were lower, and organ-to-body weight ratios of the liver and kidney were higher in rats administered 44-mg/kg DCAN than in controls. The activities of serum alanine aminotransferase and alkaline phosphatase, and concentrations of blood serum urea nitrogen and retinol-binding protein were increased in rats administered 44-mg/kg DCAN compared with those of controls, thereby indicating hepatic and renal damage in this group. This was confirmed by histopathological alterations, including hepatic sinus dilation, extensive hemorrhage, vacuolar degeneration in the liver and glomerulus hemorrhage, and renal tubular swelling, in DCAN-exposed rats. Exposure to 44-mg/kg DCAN induced hepatic oxidative damage shown by the significant increase in malonaldehyde levels, a poisonous product of lipid peroxidation. Exposure to 44-mg/kg DCAN significantly increased hepatic glutathione content and mitochondrial bioenergy as noted by the elevation of mitochondrial membrane potential and cytochrome c oxidase activity, which might be attributed to compensatory pathophysiologic responses to DCAN-induced hepatic mitochondrial damage.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

    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

  12. Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

    International Nuclear Information System (INIS)

    Sappal, Ravinder; MacDonald, Nicole; Fast, Mark; Stevens, Don; Kibenge, Fred; Siah, Ahmed; Kamunde, Collins

    2014-01-01

    Highlights: • Interacting effects of Cu and temperature were investigated in rainbow trout liver mitochondria. • Mitochondrial functional indices are highly sensitive to temperature change. • High and low temperatures sensitize mitochondria to adverse effects of Cu. • Cu induces a highly temperature-sensitive mitochondrial permeability transition pore. • Cu-imposed mitochondrial membrane potential dissipation is mediated by reactive oxygen species. - Abstract: Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q 10 effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control, Cu

  13. Interactions of copper and thermal stress on mitochondrial bioenergetics in rainbow trout, Oncorhynchus mykiss

    Energy Technology Data Exchange (ETDEWEB)

    Sappal, Ravinder [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); MacDonald, Nicole [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Fast, Mark [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Stevens, Don [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Kibenge, Fred [Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada); Siah, Ahmed [British Columbia Centre for Aquatic Health Sciences, 871A Island Highway, Campbell River, BC V9W 2C2 (Canada); Kamunde, Collins, E-mail: ckamunde@upei.ca [Department of Biomedical Sciences, Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE C1A 4P3 (Canada)

    2014-12-15

    Highlights: • Interacting effects of Cu and temperature were investigated in rainbow trout liver mitochondria. • Mitochondrial functional indices are highly sensitive to temperature change. • High and low temperatures sensitize mitochondria to adverse effects of Cu. • Cu induces a highly temperature-sensitive mitochondrial permeability transition pore. • Cu-imposed mitochondrial membrane potential dissipation is mediated by reactive oxygen species. - Abstract: Thermal stress may influence how organisms respond to concurrent or subsequent chemical, physical and biotic stressors. To unveil the potential mechanisms via which thermal stress modulates metals-induced bioenergetic disturbances, the interacting effects of temperature and copper (Cu) were investigated in vitro. Mitochondria isolated from rainbow trout livers were exposed to a range of Cu concentrations at three temperatures (5, 15 and 25 °C) with measurement of mitochondrial complex I (mtCI)-driven respiratory flux indices and uncoupler-stimulated respiration. Additional studies assessed effects of temperature and Cu on mtCI enzyme activity, induction of mitochondrial permeability transition pore (MPTP), swelling kinetics and mitochondrial membrane potential (MMP). Maximal and basal respiration rates, as well as the proton leak, increased with temperature with the Q{sub 10} effects being higher at lower temperatures. The effect of Cu depended on the mitochondrial functional state in that the maximal respiration was monotonically inhibited by Cu exposure while low and high Cu concentrations stimulated and inhibited the basal respiration/proton leak, respectively. Importantly, temperature exacerbated the effects of Cu by lowering the concentration of the metal required for toxicity and causing loss of thermal dependence of mitochondrial respiration. Mitochondrial complex I activity was inhibited by Cu but was not affected by incubation temperature. Compared with the calcium (Ca) positive control

  14. Impact of Storage and Purification on Mitochondrial Membrane Potential of Boar Spermatozoa

    OpenAIRE

    Aristotelis G. Lymberopoulos; TAREK KHALIFA

    2013-01-01

    This study aimed to evaluate the effect of semen purification and storage on sperm mitochondrial membrane potential (ΔΨm). Gel-free whole ejaculates were collected from five proven fertile Large White boars aged two to three years. Aliquots of fresh semen were split, diluted in one step with commercial extenders and incubated at 37oC for 5-10 minutes. Semen was cooled to 18oC and packaged in 15-ml sterile propylene tubes. After 4-10 hours post-semen collection, stored semen was purified by co...

  15. Chemotherapeutic Drugs and Mitochondrial Dysfunction: Focus on Doxorubicin, Trastuzumab, and Sunitinib

    Directory of Open Access Journals (Sweden)

    Stefania Gorini

    2018-01-01

    Full Text Available Many cancer therapies produce toxic side effects whose molecular mechanisms await full elucidation. The most feared and studied side effect of chemotherapeutic drugs is cardiotoxicity. Also, skeletal muscle physiology impairment has been recorded after many chemotherapeutical treatments. However, only doxorubicin has been extensively studied for its side effects on skeletal muscle. Chemotherapeutic-induced adverse side effects are, in many cases, mediated by mitochondrial damage. In particular, trastuzumab and sunitinib toxicity is mainly associated with mitochondria impairment and is mostly reversible. Vice versa, doxorubicin-induced toxicity not only includes mitochondria damage but can also lead to a more robust and extensive cell injury which is often irreversible and lethal. Drugs interfering with mitochondrial functionality determine the depletion of ATP reservoirs and lead to subsequent reversible contractile dysfunction. Mitochondrial damage includes the impairment of the respiratory chain and the loss of mitochondrial membrane potential with subsequent disruption of cellular energetic. In a context of increased stress, AMPK has a key role in maintaining energy homeostasis, and inhibition of the AMPK pathway is one of the proposed mechanisms possibly mediating mitochondrial toxicity due to chemotherapeutics. Therapies targeting and protecting cell metabolism and energy management might be useful tools in protecting muscular tissues against the toxicity induced by chemotherapeutic drugs.

  16. Is cell aging caused by respiration-dependent injury to the mitochondrial genome

    Science.gov (United States)

    Fleming, J. E.; Yengoyan, L. S.; Miquel, J.; Cottrell, S. F.; Economos, A. C.

    1982-01-01

    Though intrinsic mitochondrial aging has been considered before as a possible cause of cellular senescence, the mechanisms of such mitochondrial aging have remained obscure. In this article, the hypothesis of free-radical-induced inhibition of mitochondrial replenishment in fixed postmitotic cells is expanded. It is maintained that the respiration-dependent production of superoxide and hydroxyl radicals may not be fully counteracted, leading to a continuous production of lipoperoxides and malonaldehyde in actively respiring mitochondria. These compounds, in turn, can easily react with the mitochondrial DNA which is in close spatial relationship with the inner mitochondrial membrane, producing an injury that the mitochondria may be unable to counteract because of their apparent lack of adequate repair mechanisms. Mitochondrial division may thus be inhibited leading to age-related reduction of mitochondrial numbers, a deficit in energy production with a concomitant decrease in protein synthesis, deterioration of physiological performance, and, therefore, of organismic performance.

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

    Science.gov (United States)

    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.

  18. Estrogen-related receptor α is essential for maintaining mitochondrial integrity in cisplatin-induced acute kidney injury.

    Science.gov (United States)

    Tsushida, Keigo; Tanabe, Katsuyuki; Masuda, Kana; Tanimura, Satoshi; Miyake, Hiromasa; Arata, Yuka; Sugiyama, Hitoshi; Wada, Jun

    2018-04-15

    Acute kidney injury (AKI) has been associated with not only higher in-hospital mortality but also the subsequent development of chronic kidney disease (CKD). Recent evidence has suggested the involvement of mitochondrial dysfunction and impaired dynamics in the pathogenesis of AKI. Estrogen-related receptor α (ERRα) is an orphan nuclear receptor that acts as a transcription factor to regulate the transcription of genes required for mitochondrial biogenesis and oxidative phosphorylation. In the present study, we examined the effects of ERRα deficiency on the progression of AKI induced by cisplatin. Male C57BL/6 J wild-type and ERRα -/- mice received a single intraperitoneal injection of 20 mg/kg cisplatin. Seventy-two hours after the injection, kidney function and morphology were evaluated. ERRα expression was observed in renal tubules, and cisplatin inhibited its translocation into nuclei. ERRα deficiency exacerbated cisplatin-induced renal dysfunction and tubular injury, as well as oxidative stress and apoptosis. ERRα -/- mice kidneys revealed lower mitochondrial DNA content and swollen mitochondria with reduced cristae. In addition, these mice had lower expression of the mitochondrial fusion protein mitofusin-2. The cisplatin-induced decrease in mitochondrial DNA and altered mitochondrial structure were more severe in ERRα -/- mice. In cultured mouse proximal tubular epithelial cells, the ERRα inverse agonist XCT-790 significantly inhibited mitofusin-2 expression and induced mitochondrial fragmentation. Taken together, our findings suggest the involvement of ERRα in the progression of cisplatin-induced AKI probably through impaired mitochondrial dynamics. Copyright © 2018 Elsevier Inc. All rights reserved.

  19. Comparative study of the in vitro activity of a new fluoroquinolone, ABT-492.

    Science.gov (United States)

    Harnett, S J; Fraise, A P; Andrews, J M; Jevons, G; Brenwald, N P; Wise, R

    2004-05-01

    The in vitro activity of a new fluoroquinolone, ABT-492, was determined. MICs were compared with those of two beta-lactams, telithromycin, ciprofloxacin and four later generation fluoroquinolones. The effects of human serum and of inoculum concentration were also investigated. MIC data indicate that ABT-492 has potent activity against Gram-positive organisms with enhanced anti-staphylococcal activity compared with earlier fluoroquinolones, in addition to activity against beta-haemolytic streptococci, pneumococci including penicillin- and fluoroquinolone-resistant strains and vancomycin-susceptible and -resistant Enterococcus faecalis but not Enterococcus faecium. ABT-492 was the most active agent tested against Haemophilus influenzae, Moraxella catarrhalis, Neisseria meningitidis, fluoroquinolone-susceptible Neisseria gonorrhoeae and anaerobes. Good activity was observed for ABT-492 amongst the Enterobacteriaceae and anaerobes tested, but ciprofloxacin showed superior activity for species of Proteus, Morganella and Providencia, as well as for