Reactive oxygen species, mitochondria, apoptosis and aging

In this paper, we shall review various antioxygen defense systems of the cell paying particular attention to those that prevent superoxide formation rather than scavenge already formed superoxide and its products. The role of uncoupled, decoupled and non-coupled respiration, mitochondrial pore, mitochondrion-linked apoptosis will be considered. Mitochondrial theory of aging will be regarded in context of reactive oxygen species-induced damage of mitochondrial DNA. (Mol Cell Biochem 174: 305–319, 1997)     

Mitochondrial Disease: A Historical,Biochemical, and London Perspective

Roland Luft is credited with describing the first truly mitochondrial disorder in the late 1950s and early 1960s. Cases such as his have proven to be exceptionally rare. Some years later, methods of mitochondrial analysis--enzymatic, polarographic, and spectroscopic, which had been developed primarily by groups in Philadelphia--were applied to the study of mitochondria isolated from skeletal muscle biopsies of patients thought to have defects of oxidative phosphorylation. In the vanguard of these investigations were groups in New York and London. John Clark led the latter group. Application of biochemical studies, more recently supplemented by molecular mtDNA and nuclear DNA studies, have revealed that mitochondrial disorders are among the most common of all metabolic disorders.     

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

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

Mitochondrial behaviour,morphology, and animal performance

We have a limited understanding of the proximate mechanisms that are responsible for the development of variation in animal performance and life‐history strategies. Provided that components of an organism's successful life history – for example, mate competition, gestation, lactation, etc. – are energetically demanding, increased energy production within mitochondria is likely the foundation from which organisms are able to perform these tasks. Mitochondrial behaviour (positioning within the cell and communication between mitochondria) and morphology affect variation in energy production at the molecular, cellular, and organismal levels. Therefore, adaptations in mitochondrial behaviour and morphology that favour efficient energy production likely influence variation in animal performance. Previous work has linked greater proportions of inter‐mitochondrial junctions and density of the inner mitochondrial membrane, among other traits, with increased energetic demand. Future research should focus on how inter‐mitochondrial junctions and morphology of the inner mitochondrial membrane, in particular, influence animal performance in accordance with mitochondrial density, fission, and fusion.     

Silver Carp, Hypophthalmichthys molitrix, in the Poyang Lake belong to the Ganjiang River Population Rather than the Changjiang River Population

The Poyang Lake is the largest lake and the main nursery area in the middle basin of the Changjiang (Yangtze) River. We compared molecular genetic markers of silver carp among populations of the Changjiang River, the Ganjiang River and the Poyang Lake using the ND5/6 region of mtDNA. Analysis of restriction fragment length polymorphisms (RFLPs) of this region revealed distinct variation between the Ganjiang River and the Changjiang River populations. The Poyang Lake is linked with the Ganjiang River and the Changjiang River. Shared RFLP fragments between the Ganjiang River population and the Poyang Lake population are as high as 61.4%. The value is 47.74% between the populations of the Changjiang River and that of the Poyang Lake. Frequencies of bands peculiar to the Ganjiang River population are the same as in the Poyang Lake population. We conclude that the Poyang Lake silver carp population consists mainly of the Ganjiang River population. The water level of the Poyang Lake outlet, which is higher than that of the Changjiang River in the silver carp spawning season, supports this conclusion.     

Visualizing,quantifying, and manipulating mitochondrial DNA in vivo

Mitochondrial DNA (mtDNA) encodes proteins and RNAs that support the functions of mitochondria and thereby numerous physiological processes. Mutations of mtDNA can cause mitochondrial diseases and are implicated in aging. The mtDNA within cells is organized into nucleoids within the mitochondrial matrix, but how mtDNA nucleoids are formed and regulated within cells remains incompletely resolved. Visualization of mtDNA within cells is a powerful means by which mechanistic insight can be gained. Manipulation of the amount and sequence of mtDNA within cells is important experimentally and for developing therapeutic interventions to treat mitochondrial disease. This review details recent developments and opportunities for improvements in the experimental tools and techniques that can be used to visualize, quantify, and manipulate the properties of mtDNA within cells.     

综述: 细胞自噬和线粒体质量控制与健康衰老

拥有健康的晚年是每一个人的祈盼,这也是目前应对即将到来的社会老龄化危机而需要解决的重要课题.实现健康衰老需要对人类衰老发生的机制有深入的了解,比如在此过程中扮演着重要角色的线粒体的研究.线粒体是细胞能量和自由基代谢中心,也是细胞凋亡调控中心,并在信号转导和基因表达调控中发挥重要作用.线粒体一旦受损,一方面能量代谢发生紊乱,另一方面产生大量自由基,影响细胞的正常生长,并导致细胞甚至机体的衰老.正常情况下,细胞通过自噬溶酶体机制选择性清除受损伤和不需要的线粒体,这是线粒体质量控制的重要机制.研究发现,线粒体质量控制异常可能在衰老发生过程中起关键作用.限食及增强运动能有效促进线粒体质量控制,改善线粒体功能并延缓衰老.     

Respiratory supercomplexes: structure,function and assembly

The mitochondrial respiratory chain consists of 5 enzyme complexes that are responsible for ATP generation. The paradigm of the electron transport chain as discrete enzymes diffused in the inner mitochondrial membrane has been replaced by the solid state supercomplex model wherein the respiratory complexes associate with each other to form supramolecular complexes. Defects in these supercomplexes, which have been shown to be functionally active and required for forming stable respiratory complexes, have been associated with many genetic and neurodegenerative disorders demonstrating their biomedical significance. In this review, we will summarize the functional and structural significance of supercomplexes and provide a comprehensive review of their assembly and the assembly factors currently known to play a role in this process.     

Mitochondrial respiration in heart, liver, and kidney of copper-deficient rats

Morphological observations in some tissues indicate that dietary copper deficiency results in structural damage to mitochondria. The purpose of this study was to determine whether mitochondrial function is impaired as well. Male, weanling Sprague-Dawley rats were fed diets deficient or sufficient in copper for 4 weeks. Mitochondria were isolated from heart, liver, kidney cortex, and kidney medulla. P/O ratio, state 3 and state 4 respiration rates (oxygen consumed in the presence and absence of ADP, respectively), and acceptor control index (ratio of state 3:state 4) were determined using succinate or pyruvate/malate as substrate. State 3 respiration rate in mitochondria from copper-deficient hearts and livers was lower than in mitochondria from copper-sufficient hearts. Copper deficiency reduced the state 4 respiration rate only in cardiac mitochondria. Neither respiration rate was affected by copper deficiency in mitochondria from kidney medulla or cortex. P/O ratio was not significantly affected by copper deficiency in any tissue examined. Acceptor control index was reduced only in liver mitochondria. The observed decreases in respiration rates are consistent with decreased cytochrome c oxidase activity, shown by others to occur in mitochondria isolated from hearts and livers of copper-deficient rats.     

Regulation of mitochondrial contact sites in neonatal,juvenile and diabetic hearts

Mitochondrial contact sites (MiCS) are dynamic structures involved in high capacity transport of energy from mitochondria into the cytosole. Previous studies revealed that in normal conditions the actual number of MiCS is in correlation with the energy requirements of the heart, particularly with those for its contractile work. Although the detailed mechanisms of signalling between the processes of energy utilisation and MiCS formation in the heart are not yet elucidated, it is known that intracellular Ca²⁺ transients are intimately involved in this crosstalk. The present study is devoted to investigation of Ca²⁺-linked MiCS formation in healthy adult hearts and in hearts with modified Ca²⁺-handling such as in developing, in juvenile and diabetic myocardium. Experiments were performed on hearts of healthy rats on the 22nd embryonal day, 1st, 4th, 7th and 14th postnatal days as well as on adult hearts. Diabetic hearts were investigated on the 8th day after streptozotocin injection (45 mg.kg^–1 i.v.) to adult rats. Intracellular Ca²⁺ movements were affected by modulation of Ca²⁺ concentration in perfusion solution (1.6 or 2.2 mmol.l^–1) in isolated, Langendorff-perfused hearts, by calcium paradox (CaP) or by replacing of Ca²⁺ by Cd²⁺ ions. Elevation of extracellular Ca²⁺ was reflected by 30.1, 10.4 and 24.1% increase in intracellular free Ca²⁺ concentration in healthy adult, diabetic and 14-day old hearts respectively. In developing hearts the amount of MiCS was culminating on the 4th postnatal day. In adult hearts, elevated calcium in the perfusion solution, CaP as well as diabetes led to a significant increase in the amounts of MiCS formed (58.1, 77.2 and 86.5% respectively; p < 0.05). Diabetic and 14-day old hearts naturally exhibited amounts of MiCS comparable to those obtained by Ca²⁺-stimulation of MiCS formation in adult healthy hearts. In contrast to healthy controls, perfusion of diabetic and 14-day old hearts with elevated Ca²⁺ as well as induction of CaP exerted little influence on MiCS formation (4.4 and 8.2% for elevated Ca²⁺; 2.9 and 10.7% for CaP; p > 0.05). A replacement of Ca²⁺ by Cd²⁺ ions lowered the amount of MiCS in healthy adult and diabetic hearts (61 and 52.2%; p < 0.05). In conclusion, during development, the formation of MiCS may be influenced by both, permanent stimulation by Ca²⁺-signalling and the availability of mCPK. In healthy adult hearts the amount of MiCS is modulated by intracellular Ca²⁺ transients in response to changes in extracellular Ca²⁺ concentration. In diabetic hearts the modulation of MiCS formation is naturally attenuated, apparently as a consequence of persisting alterations in Ca²⁺-handling.     

Crystal structure and functional analysis of MiD49, a receptor for the mitochondrial fission protein Drp1

Mitochondrial fission requires recruitment of dynamin‐related protein 1 (Drp1) to the mitochondrial surface, where assembly leads to activation of its GTP‐dependent scission function. MiD49 and MiD51 are two receptors on the mitochondrial outer membrane that can recruit Drp1 to facilitate mitochondrial fission. Structural studies indicated that MiD51 has a variant nucleotidyl transferase fold that binds an ADP co‐factor essential for activation of Drp1 function. MiD49 shares sequence homology with MiD51 and regulates Drp1 function. However, it is unknown if MiD49 binds an analogous co‐factor. Because MiD49 does not readily crystallize, we used structural predictions and biochemical screening to identify a surface entropy reduction mutant that facilitated crystallization. Using molecular replacement, we determined the atomic structure of MiD49 to 2.4 Å. Like MiD51, MiD49 contains a nucleotidyl transferase domain; however, the electron density provides no evidence for a small‐molecule ligand. Structural changes in the putative nucleotide‐binding pocket make MiD49 incompatible with an extended ligand like ADP, and critical nucleotide‐binding residues found in MiD51 are not conserved. MiD49 contains a surface loop that physically interacts with Drp1 and is necessary for Drp1 recruitment to the mitochondrial surface. Our results suggest a structural basis for the differential regulation of MiD51‐ versus MiD49‐mediated fission.     

Phylogeography of the Italian wall lizard, Podarcis sicula, as revealed by mitochondrial DNA sequences

In a phylogeographical survey of the Italian wall lizard, Podarcis sicula, DNA sequence variation along an 887-bp segment of the cytochrome b gene was examined in 96 specimens from 86 localities covering the distribution range of the species. In addition, parts of the 12S rRNA and 16S rRNA genes from 12 selected specimens as representatives of more divergent cytochrome b haploclades were sequenced (together about 950 bp). Six phylogeographical main groups were found, three representing samples of the nominate subspecies Podarcis sicula sicula and closely related subspecies and the other three comprising Podarcis sicula campestris as well as all subspecies described from northern and eastern Adriatic islands. In southern Italy a population group with morphological characters of P. s. sicula but with the mitochondrial DNA features of P. s. campestris was detected indicating a probably recent hybridization zone. The present distribution patterns were interpreted as the consequence of natural events like retreats to glacial refuges and postglacial area expansions, but also as the results of multiple introductions by man.     

贵州侗族、仡佬族、土家族和彝族人群线粒体DNA多态性研究

从母系遗传的角度揭示世居贵州的侗族、仡佬族、土家族和彝族人群的的遗传结构和遗传分化关系,并对各民族的族源和迁徙进行初步的探讨。采用高变区序列分析与编码区PCR-RFLP分析相结合的方法对4个群体108例样本进行mtDNA多态性分析,共鉴定了37种(亚)单倍群,单倍群分布频率及主成分分析显示:侗族含有高比例的南方优势单倍群,表现出典型的南方群体特征;彝族兼有高比例的南北方优势单倍群,提示它同时具有南北方群体的一些母系遗传特征;彝族和仡佬族聚在一起,可能是由于历史上两个民族的先民曾发生过广泛的基因交流。     

Mitochondrial DNA deletion-dependent podocyte injuries in Mito-miceΔ, a murine model of mitochondrial disease

Focal segmental glomerulosclerosis (FSGS) is a major renal complication of human mitochondrial disease. However, its pathogenesis has not been fully explained. In this study, we focused on the glomerular injury of mito-miceΔ and investigated the pathogenesis of their renal involvement. We analyzed biochemical data and histology in mito-miceΔ. The proteinuria began to show in some mito-miceΔ with around 80% of mitochondrial DNA deletion, then proteinuria developed dependent with higher mitochondrial DNA deletion, more than 90% deletion. Mito-miceΔ with proteinuria histologically revealed FSGS. Immunohistochemistry demonstrated extensive distal tubular casts due to abundant glomerular proteinuria. Additionally, the loss of podocyte-related protein and podocyte’s number were found. Therefore, the podocyte injuries and its depletion had a temporal relationship with the development of proteinuria. This study suggested mitochondrial DNA deletion-dependent podocyte injuries as the pathogenesis of renal involvement in mito-miceΔ. The podocytes are the main target of mitochondrial dysfunction originated from the accumulation of mitochondrial DNA abnormality in the kidney.     

Mitochondrial DNA as a genetic marker for brown trout, Salmo trutta L., populations

Mitochondrial DNA was examined in natural and hatchery-reared stocks of brown trout, using different methods of restriction analysis. The methods included the development of a brown trout mt DNA hybridization probe through cloning part of the brown trout mitochondrial genome. In addition, fragments were analysed by ethidium bromide staining and end-labelling. The relative merits of each of these methods in assessing levels of genetic relatedness between the natural and hatchery-reared brown trout stocks were evaluated. In addition, the study revealed a diagnostic mtDNA restriction pattern which could be used as a genetic marker for the discrimination of these two groups of brown trout.     

A Comment on the Siberian, Acipenser baerii, and Russian, Acipenser gueldenstaedtii, Sturgeons

Contrary to the opinion of Kynard et al. (2002), the Siberian sturgeon, Acipenser baerii, does not belong to the Ponto-Caspian species. It inhabits Siberian rivers and Lake Baikal. Acipenser baerii is a typical potamodromous species and the comparison of the behavior of its embryos and larvae with those of the anadromous Russian sturgeon, Acipenser gueldenstaedtii, should be done with understanding that these species have different life histories. The statement by Kynard et al. (2002) that larvae of the Russian sturgeon do not migrate contradicts results of previous studies.     

Embryo quality,and not chromosome nondiploidy,affects mitochondrial DNA content in mouse blastocysts

It has been shown recently that there is premature mitochondria biosynthesis in blastocysts from older women whose egg or embryo quality is poor and that aneuploid blastocysts also have a high number of mitochondrial DNA (mtDNA) copies. Whether nondiploidy/aneuploidy or reduced egg or embryo quality causes premature mitochondrial biosynthesis is not known. This study constructed haploid, diploid, triploid, and tetraploid blastocysts by parthenogenetic activation, intracytoplasmic sperm injection with one or two sperm heads, blastomere electrofusion, respectively, and generated reduced cytoplasm quality embryos from diabetic mouse and in vitro fertilization of aged oocytes, and examined whether nondiploidy or reduced cytoplasm quality causes premature mitochondrial biosynthesis. MtDNA numbers of each blastocyst from different models were tested by absolute quantitative real-time polymerase chain reaction. It was found that mtDNA content in preimplantation embryos was not associated with their chromosome ploidy, while mtDNA copy numbers in embryos with suboptimal quality were increased. Therefore, it might be the reduced cytoplasmic quality, and not chromosome nondiploidy, that causes premature mitochondria biosynthesis in blastocysts.