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1.
In the present work, we show that after induction of mitochondrial damage by oxidative stress, in the presence of calcium, matrix DNA content decreased to 42+/-6%. Mitochondrial damage was analyzed by measuring aconitase activity, a marker enzyme of mitochondrial oxidative stress. The genes were identified by amplifying them through the polymerase chain reaction (PCR), using specific primers for each mitochondrial gene (MTCO1, MTCO2, MTCO3, MTND3, MTND5, MTATP6, MTATP8, and MTCYB). The results show that after oxidative stress, the amount of MTCO1, MTND3, and MTCYB genes in the mitochondria approximately decreased by 46, 22, and 54%, respectively. This effect was inhibited in the presence of cyclosporin A. These genes were found outside the mitochondria after permeability transition was induced. Mitochondrial integrity was evaluated by observing the activity of adenylate kinase and malate dehydrogenase. 相似文献
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Mitochondrial DNA Repair Pathways 总被引:5,自引:0,他引:5
It has long been held that there is no DNA repair in mitochondria. Early observations suggestedthat the reason for the observed accumulation of DNA damage in mitochondrial DNA is thatDNA lesions are not removed. This is in contrast to the very efficient repair that is seen inthe nuclear DNA. Mitochondrial DNA does not code for any DNA repair proteins, but it hasbeen observed that a number of repair factors can be found in mitochondrial extracts. Mostof these participate in the base excision DNA repair pathway which is responsible for theremoval of simple lesions in DNA. Recent work has shown that there is efficient base excisionrepair in mammalian mitochondria and there are also indications of the presence of morecomplex repair processes. Thus, an active field of mitochondrial DNA repair is emerging. Anunderstanding of the DNA repair processes in mammalian mitochondria is an important currentchallenge and it is likely to lead to clarification of the etiology of the common mutations anddeletions that are found in mitochondria, and which are thought to cause various humandisorders and to play a role in the aging phenotype. 相似文献
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Seok-Jo Kim Paul Cheresh David Williams Yuan Cheng Karen Ridge Paul T. Schumacker Sigmund Weitzman Vilhelm A. Bohr David W. Kamp 《The Journal of biological chemistry》2014,289(9):6165-6176
Mitochondria-targeted human 8-oxoguanine DNA glycosylase (mt-hOgg1) and aconitase-2 (Aco-2) each reduce oxidant-induced alveolar epithelial cell (AEC) apoptosis, but it is unclear whether protection occurs by preventing AEC mitochondrial DNA (mtDNA) damage. Using quantitative PCR-based measurements of mitochondrial and nuclear DNA damage, mtDNA damage was preferentially noted in AEC after exposure to oxidative stress (e.g. amosite asbestos (5–25 μg/cm2) or H2O2 (100–250 μm)) for 24 h. Overexpression of wild-type mt-hOgg1 or mt-long α/β 317–323 hOgg1 mutant incapable of DNA repair (mt-hOgg1-Mut) each blocked A549 cell oxidant-induced mtDNA damage, mitochondrial p53 translocation, and intrinsic apoptosis as assessed by DNA fragmentation and cleaved caspase-9. In contrast, compared with controls, knockdown of Ogg1 (using Ogg1 shRNA in A549 cells or primary alveolar type 2 cells from ogg1−/− mice) augmented mtDNA lesions and intrinsic apoptosis at base line, and these effects were increased further after exposure to oxidative stress. Notably, overexpression of Aco-2 reduced oxidant-induced mtDNA lesions, mitochondrial p53 translocation, and apoptosis, whereas siRNA for Aco-2 (siAco-2) enhanced mtDNA damage, mitochondrial p53 translocation, and apoptosis. Finally, siAco-2 attenuated the protective effects of mt-hOgg1-Mut but not wild-type mt-hOgg1 against oxidant-induced mtDNA damage and apoptosis. Collectively, these data demonstrate a novel role for mt-hOgg1 and Aco-2 in preserving AEC mtDNA integrity, thereby preventing oxidant-induced mitochondrial dysfunction, p53 mitochondrial translocation, and intrinsic apoptosis. Furthermore, mt-hOgg1 chaperoning of Aco-2 in preventing oxidant-mediated mtDNA damage and apoptosis may afford an innovative target for the molecular events underlying oxidant-induced toxicity. 相似文献
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Mitochondrial DNA disorders are an important cause of neurological disease, yet despite our awareness of the importance of these conditions, relatively little is known about the neuropathology of these disorders and even less about the mechanisms involved in neuronal dysfunction and death. In this review we detail important features from neuropathological studies available and highlight deficiencies that are currently limiting our understanding of mitochondrial DNA disease. We also discuss possible future approaches that might resolve some of these outstanding issues. Further study of these disorders is critical because mitochondria play a central role in neuronal survival and it is likely that an understanding of the mechanisms involved in neuronal dysfunction and cell death in mitochondrial DNA disease may have implications for other neurodegenerative diseases. 相似文献
5.
线粒体是存在于绝大多数真核细胞内的一种基本的重要的细胞器,其具有相对独立的遗传系统。线粒体基因在真核生物具有高保守性,线粒体DNA(mtDNA)已被广泛应用于发病机理、临床诊断、遗传变异、生物进化等多方面的研究。1981年,Anderson用氯化铯密度梯度分离得到线粒体DNA(mtDNA),进行了全序列分析。此后,mtDNA的研究日益得到重视。已有的mtDNA提取方法概括起来可分为密度梯度离心法、酶消化法、柱层析法、氯化铯超速离心法、碱变性法和改进高盐沉淀法等,通过对以上方法的比较,发现改进高盐沉淀法具有简便、经济、易重复等优点。 相似文献
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Petr Jeek Lydie Plecit-Hlavat 《The international journal of biochemistry & cell biology》2009,41(10):1790-1804
A single mitochondrial network in the cell undergoes constant fission and fusion primarily depending on the local GTP gradients and the mitochondrial energetics. Here we overview the main properties and regulation of pro-fusion and pro-fission mitodynamins, i.e. dynamins-related GTPases responsible for mitochondrial shape-forming, such as pro-fusion mitofusins MFN1, MFN2, and the inner membrane-residing long OPA1 isoforms, and pro-fission mitodynamins FIS1, MFF, and DRP1 multimers required for scission. Notably, the OPA1 cleavage into non-functional short isoforms at a diminished ATP level (collapsed membrane potential) and the DRP1 recruitment upon phosphorylation by various kinases are overviewed. Possible responses of mitodynamins to the oxidative stress, hypoxia, and concomitant mtDNA mutations are also discussed. We hypothesize that the increased GTP formation within the Krebs cycle followed by the GTP export via the ADP/ATP carrier shift the balance between fission and fusion towards fusion by activating the GTPase domain of OPA1 located in the peripheral intermembrane space (PIMS). Since the protein milieu of PIMS is kept at the prevailing oxidized redox potential by the TOM, MIA40 and ALR/Erv1 import-redox trapping system, redox regulations shift the protein environment of PIMS to a more reduced state due to the higher substrate load and increased respiration. A higher cytochrome c turnover rate may prevent electron transfer from ALR/Erv1 to cytochrome c. Nevertheless, the putative links between the mitodynamin responses, mitochondrial morphology and the changes in the mitochondrial bioenergetics, superoxide production, and hypoxia are yet to be elucidated, including the precise basis for signaling by the mitochondrion-derived vesicles. 相似文献
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Peizhong MaoPatience Gallagher Samira NedungadiMaria Manczak Ulziibat P. shirendebSteven G. Kohama Betsy FergusonByung S. Park P. Hemachandra Reddy 《生物化学与生物物理学报:疾病的分子基础》2012,1822(2):111-119
The purpose of this study was to determine the relationship between mitochondrial DNA (mtDNA) deletions, mtDNA content and aging in rhesus monkeys. Using 2 sets of specific primers, we amplified an 8 kb mtDNA fragment covering a common 5.7 kb deletion and the entire 16.5 kb mitochondrial genome in the brain and buffy-coats of young and aged monkeys. We studied a total of 66 DNA samples: 39 were prepared from a buffy-coat and 27 were prepared from occipital cortex tissues. The mtDNA data were assessed using a permutation test to identify differences in mtDNA, in the different monkey groups. Using real-time RT-PCR strategy, we also assessed both mtDNA and nuclear DNA levels for young, aged and male and female monkeys. We found a 5.7 kb mtDNA deletion in 81.8% (54 of 66) of the total tested samples. In the young group of buffy-coat DNA, we found 5.7 kb deletions in 7 of 17 (41%), and in the aged group, we found 5.7 kb deletions in 12 of 22 (54%), suggesting that the prevalence of mtDNA deletions is related to age. We found decreased mRNA levels of mtDNA in aged monkeys relative to young monkeys. The increases in mtDNA deletions and mtDNA levels in aged rhesus monkeys suggest that damaged DNA accumulates as rhesus monkeys age and these altered mtDNA changes may have physiological relevance to compensate decreased mitochondrial function. 相似文献
11.
鼠毛及脑线粒体DNA片段缺失与增龄的关系 总被引:10,自引:0,他引:10
以聚合酶链反应(PCR)技术检测不同年龄Balb/c小鼠脑细胞线粒体DNA片段缺失与增龄的关系.发现老年鼠脑细胞线粒体3867bp片段缺失率为50%;而断奶鼠与青年鼠均无此缺失片段出现;用鼠毛为材料进行无损伤检测亦获类似的结果.有人认为线粒体DNA片段缺失率可作为生物衰老的一种生物学标志 相似文献
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Mitochondrial dynamics in the regulation of neuronal cell death 总被引:1,自引:0,他引:1
Cheung EC McBride HM Slack RS 《Apoptosis : an international journal on programmed cell death》2007,12(5):979-992
Mitochondria undergo continuous fission and fusion events in physiological situations. Fragmentation of mitochondria during
cell death has been shown to play a key role in cell death progression, including release of the mitochondrial apoptotic proteins.
Ultrastructural changes in mitochondria, such as cristae remodeling, is also involved in cell death initiation. Here, we emphasize
the important role of mitochondrial fission/fusion machinery in neuronal cell death. Unlike many other cell types such as
immortalized cell lines, neurons are distinct morphologically and functionally. We will discuss how this uniqueness presents
special challenges in the cellular response to neurotoxic stresses, and how this affects the mitochondrial dynamics in the
regulation of cell death in neurons. 相似文献
14.
Oxidative stress can induce mitochondrial dysfunction, mitochondrial DNA (mtDNA) depletion, and neurodegeneration, although
the underlying mechanisms are poorly understood. The major mitochondrial antioxidant system that protects cells consists of
manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx) and glutathione (GSH). To investigate the putative adaptive
changes in antioxidant enzyme protein expression and targeting to mitochondria as mtDNA depletion occurs, we progressively
depleted U87 astrocytoma cells of mtDNA by chronic treatment with ethidium bromide (EB, 50 ng/ml). Cellular MnSOD protein
expression was markedly increased in a time-related manner while that of GPx showed time-related decreases. The mtDNA depletion
also altered targeting or subcellular distribution of GPx, suggesting the importance of intact mtDNA in mitochondrial genome-nuclear
genome signaling/communication. Cellular NADP+-ICDH activity also showed marked, time-related increases while their GSH content decreased. Thus, our findings suggest that
interventions to elevate MnSOD, GPx, NADP+-ICDH, and GSH levels may protect brain cells from oxidative stress. 相似文献
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P. Chetrit C. Mathieu F. Vedel G. Pelletier C. Primard 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》1985,69(4):361-366
Summary The mitochondrial genomes of five rapeseed somatic hybrid plants, which combine in a first experimentBrassica napus chloroplasts and a cytoplasmic male sterility trait coming fromRaphanus sativus, and in a second experiment chloroplasts of a triazine resistantB. compestris and a cytoplasmic male sterility trait fromR. sativus, were analyzed by restriction endonucleases. Restriction fragment patterns indicate that these genomes differ from each other and from both parents. The presence of new bands in the somatic hybrid mitochondrial DNA restriction patterns is evidence of mitochondrial recombination in somatic hybrid cells. In both parental and somatic hybrid plants large quantitative variations in a mitochondrial plasmid-like DNA have been observed. Our results suggest that the cytoplasmic support for male sterility is located in the chromosomal mitochondrial DNA instead of the plasmid-like DNA. 相似文献
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《Cell calcium》2018
Ca2+-activated K+ channels (KCa) are expressed at the plasma membrane and in cellular organelles. Expression of all KCa channel subtypes (BK, IK and SK) has been detected at the inner mitochondrial membrane of several cell types. Primary functions of these mitochondrial KCa channels include the regulation of mitochondrial ROS production, maintenance of the mitochondrial membrane potential and preservation of mitochondrial calcium homeostasis. These channels are therefore thought to contribute to cellular protection against oxidative stress through mitochondrial mechanisms of preconditioning. In this review, we summarize the current knowledge on mitochondrial KCa channels, and their role in mitochondrial function in relation to cell death and survival pathways. More specifically, we systematically discuss studies on the role of these mitochondrial KCa channels in pharmacological preconditioning, and according protective effects on ischemic insults to the brain and the heart. 相似文献
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Excessive nuclear or mitochondrial DNA damage can lead to mitochondrial dysfunction, decreased energy production, and increased generation of reactive oxygen species (ROS). Although numerous cell signaling pathways are activated when cells are injured, the ataxia telangiectasia mutant (ATM) protein has emerged as a major regulator of the response to both mitochondrial dysfunction and nuclear DNA double-strand breaks (DSBs). Because mitochondrial dysfunction is often a response to excessive DNA damage, it has been difficult to determine whether nuclear and/or mitochondrial DNA DSBs activate ATM independent of mitochondrial dysfunction. In this study, mitochondrial and nuclear DNA DSBs were generated in the A549 human lung adenocarcinoma cell line by infecting with retroviruses expressing the restriction endonuclease PstI fused to a mitochondrial targeting sequence (MTS) or nuclear localization sequence (NLS) and a hemagglutinin antigen epitope tag (HA). Expression of MTS-PstI-HA or NLS-PstI-HA activated the DNA damage response defined by phosphorylation of ATM, the tumor suppressor protein p53 (TP53), KRAB-associated protein (KAP)-1, and structural maintenance of chromosomes (SMC)-1. Phosphorylated ATM and SMC1 were detected in nuclear fractions, whereas phosphorylated TP53 and KAP1 were detected in both mitochondrial and nuclear fractions. PstI also enhanced expression of the cyclin-dependent kinase inhibitor p21 and inhibited cell growth. This response to DNA damage occurred in the absence of detectable mitochondrial dysfunction and excess production of ROS. These findings reveal that DNA DSBs are sufficient to activate ATM independent of mitochondrial dysfunction and suggest that the activated form of ATM and some of its substrates are restricted to the nuclear compartment, regardless of the site of DNA damage. 相似文献
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Paik-Seong Lim Yi-Shing Ma Yueh-Mei Cheng Henry Chai Cheng-Feng Lee Tzu-Ling Chen Prof. Yau-Huei Wei 《Journal of biomedical science》2002,9(6):549-560
Abundant evidence has been gathered to suggest that mitochondrial DNA (mtDNA) sustains many more mutations and greater oxidative damage than does nuclear DNA in human tissues. Uremic patients are subject to a state of enhanced oxidative stress due to excess production of oxidants and a defective antioxidant defense system. This study was conducted to investigate mtDNA mutations and oxidative damage in skeletal muscle of patients with chronic uremia. Results showed that large-scale deletions between nucleotide position (np) 7,900 and 16,300 of mtDNA occurred at a high frequency in muscle of uremic patients. Among them, the 4,977-bp deletion (mtDNA4977) was the most frequent and most abundant large-scale mtDNA deletion in uremic skeletal muscle. The proportion of mtDNA4977 was found to correlate positively with the level of 8-hydroxy 2-deoxyguanosine (8-OHdG) in the total DNA of skeletal muscle (r=0.62, p<0.05). Using long-range PCR and DNA sequencing, we identified and characterized multiple deletions of mtDNA in skeletal muscle of 16 of 19 uremic patients examined. The 8,041-bp deletion, which occurred between np 8035 and 16,075, was flanked by a 5-bp direct repeat of 5-CCCAT-3. Some of the deletions were found in more than 1 patient. On the other hand, we found that the mean 8-OHdG/105 dG ratio in the total cellular DNA of muscle of uremic patients was significantly higher than that of the controls (182.7 ± 63.6 vs. 50.9 ± 21.5, p=0.05). In addition, the mean 8-OHdG/105 dG ratio in muscle mtDNA of uremic patients was significantly higher than that in nuclear DNA (344.0 ± 56.9 vs. 146.3 ± 95.8, p=0.001). Moreover, we found that the average content of lipid peroxides in mitochondrial membranes of skeletal muscle of uremic patients was significantly higher than that of age-matched healthy subjects (23.76 ± 6.06 vs. 7.67 ± 0.95 nmol/mg protein; p<0.05). The average content of protein carbonyls in the mitochondrial membranes prepared from uremic skeletal muscles was significantly higher than that in normal controls (24.90 ± 4.00 vs. 14.48 ± 1.13 nmol/mg protein; p<0.05). Taken together, these findings suggest that chronic uremia leads to mtDNA mutations together with enhanced oxidative damage to DNA, lipids, and proteins of mitochondria in skeletal muscle, which may contribute to the impairment of mitochondrial bioenergetic function and to skeletal myopathy commonly seen in uremic patients. 相似文献
20.
The mechanism by which we age has sparked a huge number of theories, and is an area of intense debate. As the elderly population rises, the importance of elucidating these mechanisms is becoming more apparent as age is the single biggest risk factor for a number of diseases such as cancer, diabetes and neurodegenerative disease. Mitochondrial DNA (MtDNA) mutations have been shown to accumulate in cells and tissues during the ageing process; however the question as to whether these mutations have a causal role in the ageing process remains an area of uncertainty. Here we review the current literature, and discuss the evidence for and against a causal role of mtDNA mutations in ageing and in the pathogenesis of age-related disease. 相似文献