线粒体呼吸功能与精子活力、核DNA损伤的相关性分析

为探讨线粒体呼吸功能与精子活力、核DNA损伤程度之间的相关性,按WHO标准收集34例不同活力的精液标本,采用蔗糖差速离心法或密度梯度离心法提取精子线粒体,通过铂氧电极-溶氧仪测定线粒体呼吸耗氧率并计算状态III呼吸、状态IV呼吸、呼吸控制率（RCR）、磷氧比（P／0）及氧化磷酸化效率（0PR）;应用精子染色质扩散（sperm chromatin dispersion,SCD）实验检测精子DNA损伤情况。结果表明：不同活力精子线粒体状态Ⅲ呼吸耗氧量之间具有显著差异俨〈0．01）;弱精子症组RcR和OPR与正常对照组比较,分别降低了17．03％（P〈0．05）和40．74％（P〈0。01）;精子DNA损伤程度与精子活力、状态III呼吸及OPR均呈极显著负相关（r值分别是-0．812、-0．788和-0．696）。以上结果提示：精子线粒体呼吸耗氧和氧化磷酸化功能与精子活力之间存在着密切的联系;精子DNA（包括mtDNA）损伤可能影响精子的正常功能。     

人类多种疾病中mtDNA拷贝数的变化

线粒体是细胞能量和自由基代谢中心,在细胞生命活动中发挥着重要作用,而mt DNA拷贝数是决定线粒体功能的重要因素。由于mt DNA缺乏组蛋白的保护和损伤修复系统,又暴露在氧化磷酸化产生的高氧环境中,易受损引起拷贝数变化,从而影响线粒体的功能。大量研究表明,多种疾病的发生发展与mt DNA拷贝数变化密切相关。该文综述了癌症、神经退行性疾病、心脑血管疾病、精神疾病等各种疾病中mt DNA拷贝数的变化情况,以期从中发现疾病相关mt DNA拷贝数的变化规律和调控机制。     

人线粒体RNA加工及调控

线粒体是细胞内氧化磷酸化（oxidative phosphorylation,OXPHOS）和合成三磷酸腺苷（adenosine triphosphate,ATP）的细胞器，是细胞能量代谢的“动力工厂”。线粒体几乎存在于所有真核生物中，参与细胞凋亡、钙稳态以及先天免疫反应的调节等过程，对细胞行使正常的生理功能至关重要。线粒体是半自主细胞器，拥有自身的基因组DNA，编码37个基因，包括2个rRNA基因、13个m RNA基因和22个tRNA基因。线粒体的基因表达需要经过复杂的转录和转录后加工过程，包括多顺反子RNA的切割、RNA的修饰以及RNA的末端加工等过程。异常的线粒体RNA加工会导致线粒体RNA表达谱发生变化、线粒体翻译紊乱、线粒体功能失常等，从而造成多种线粒体相关疾病。本文综述了线粒体DNA的转录、RNA转录后加工以及影响RNA加工的因素方面的最新研究进展。     

线粒体呼吸链膜蛋白复合体的结构

线粒体作为真核细胞的重要“能量工厂”,是细胞进行呼吸作用的场所,呼吸作用包括柠檬酸循环和氧化磷酸化两个过程,其中氧化磷酸化过程的电子传递链（又称线粒体呼吸链）位于线粒体内膜上,由四个相对分子质量很大的跨膜蛋白复合体（Ⅰ、Ⅱ、Ⅲ、和Ⅳ）、介于Ⅰ／Ⅱ与Ⅲ之间的泛醌以及介于Ⅲ与Ⅳ之间的细胞色素c共同组成。线粒体呼吸链的功能是进行生物氧化,并与称之为复合物V的ATP合成酶（磷酸化过程）相偶联,共同完成氧化磷酸化过程,并生产能量分子ATP。线粒体呼吸链的结构生物学研究对于彻底了解电子传递和能量转化的机理是至关重要的,本文分别论述线粒体呼吸链复合体Ⅰ、Ⅱ、Ⅲ和Ⅳ的结构,并跟踪线粒体呼吸链超复合体的结构研究进展。     

线粒体病的基因治疗

线粒体病是一组因线粒体DNA缺失或突变而致氧化磷酸化及能量供应异常的疾病,目前该类疾病的治疗主要是支持治疗。然而由于线粒体结构和功能的特殊性,该疗效并不确切。因此,线粒体病的基因治疗显得越来越迫切和重要。目前,基因治疗的策略包括降低突变型mtDNA／野生型mtDNA的比例、错位表达、输入其他同源性基因以及利用限制性内切酶修复突变型mtDNA等。     

肿瘤能量代谢：糖酵解还是氧化磷酸化？

正常细胞代谢活动所需要的能量主要由线粒体氧化磷酸化产生的ATP提供。与正常细胞不同,肿瘤细胞糖酵解增强,氧化磷酸化功能降低。长期以来,肿瘤细胞的有氧糖酵解被认为是由于线粒体出现不可逆的损伤。最近有不少研究结果对这一观点提出质疑,认为多数肿瘤的线粒体氧化磷酸化功能是完好的,肿瘤有氧糖酵解的改变被认为是其他多种因素（例如癌基因、肿瘤抑制基因、低氧微环境、mtDNA突变等）综合作用的结果。     

锌转运体Zip2对心肌缺血再灌注小鼠心肌线粒体呼吸的调控作用

本研究旨在探讨锌转运体Zip2 (SLC39A2)在心肌缺血再灌注(ischemia/reperfusion, I/R)过程中对线粒体呼吸的调控作用及其机制。通过冠状动脉左前降支结扎建立小鼠在体心肌I/R损伤模型,用电感耦合离子发射光谱仪(inductively coupled plasma-optical emission spectrometer, ICP-OES)测量心肌组织的锌含量,用高分辨呼吸测定系统(Oxygraph-2K)检测小鼠心肌线粒体呼吸功能和氧化磷酸化水平,采用Western blot技术检测小鼠心肌组织STAT3和ERK的磷酸化水平。结果显示:(1)与假手术组相比,野生型小鼠I/R心肌组织的锌含量明显降低,Zip2基因敲除小鼠I/R心肌组织的锌含量进一步降低;(2)与野生对照组相比,Zip2基因敲除组小鼠心肌线粒体呼吸控制率(respiratory control ratio, RCR)和氧化磷酸化水平降低,I/R后上述指标进一步降低;(3)与野生对照组相比,I/R后Zip2基因敲除组小鼠心肌组织STAT3 (Ser~(727))和ERK的蛋白磷酸化水平均明显降低;(4)与空载体感染组相比,I/R后STAT3感染组心肌线粒体呼吸功能明显提高,而STAT3负突变体感染组心肌线粒体呼吸功能则降低。STAT3过表达可逆转Zip2基因敲除对线粒体呼吸的抑制作用。以上结果提示,心肌I/R时Zip2通过STAT3来调控线粒体呼吸,其机制可能与STAT3 (Ser~(727))的磷酸化有关,这可能是Zip2保护心肌的分子机制之一。     

运动训练对小鼠心肌线粒体能量转换功能增龄性改变的影响

目的和方法：以C57BL／6J雄性小鼠跑转笼为运动方式,研究以5月龄开始进行为期8个月或15个月的运动训练对小鼠心肌线粒体能量转换功能的影响。结果：以α－酮戊二酸为底物时,线粒体RCR、ADP／O均呈现随年龄的增加而下降,尤其以衰老晚期（小鼠20月龄）下降明显。结论：衰老过程中氧化磷酸化偶联程度降低,能量产出减少,长期运动训练的小鼠心肌线粒体出现适应性变化,表现为线粒体功能随增龄而下降的程度减小。     

线粒体与多潜能干细胞功能

线粒体是细胞内重要的细胞器,主要功能是通过氧化磷酸化为细胞生命活动提供能量。近年来,研究表明,在多潜能干细胞（Pluripotent stem cells, PSCs）中线粒体表现出独有的特征,即在多能性状态下,PSCs主要依靠糖酵解提供能量,其分化期间线粒体氧化磷酸化代谢能力逐渐增强。相反,体细胞重编程为多潜能干细胞期间,线粒体氧化磷酸化向糖酵解途径的转变是其成功重编程必需的代谢过程。另外,线粒体通过生物合成和形态结构的动态重塑维持了PSCs多能性、诱导分化及诱导多能干细胞（Induced pluripotent stem cells, iPSCs）的重编程。因此,本文综述了PSCs线粒体形态结构及其在调控PSCs多能性、合成代谢、氧化还原状态的平衡、分化及重新编程中的作用,为深入了解线粒体调控PSCs功能的作用提供理论基础。     

Attempts to understand the mechanisms of mitochondrial diseases: The reverse genetics of mouse models for mitochondrial disease

BackgroundMitochondrial disease is a general term for a disease caused by a decline in mitochondrial function. The pathology of this disease is extremely diverse and complex, and the mechanism of its pathogenesis is still unknown. Using mouse models that develop the disease via the same processes as in humans is the easiest path to understanding the underlying mechanism. However, creating a mouse model is extremely difficult due to the lack of technologies that enable editing of mitochondrial DNA (mtDNA).Scope of reviewThis paper outlines the complex pathogenesis of mitochondrial disease, and the difficulties in producing relevant mouse models. Then, the paper provides a detailed discussion on several mice created with mutations in mtDNA. The paper also introduces the pathology of mouse models with mutations including knockouts of nuclear genes that directly affect mitochondrial function.Major conclusionsSeveral mice with mtDNA mutations and those with nuclear DNA mutations have been established. Although these models help elucidate the pathological mechanism of mitochondrial disease, they lack sufficient diversity to enable a complete understanding. Considering the variety of factors that affect the cause and mechanism of mitochondrial disease, it is necessary to account for this background diversity in mouse models as well.General significanceMouse models are indispensable for understanding the pathological mechanism of mitochondrial disease, as well as for searching new treatments. There is a need for the creation and examination of mouse models with more diverse mutations and altered nuclear backgrounds and breeding environments.     

Production of transmitochondrial mice

With the advancement of various gene transfer technologies, the establishment of mitochondria transfer as a viable technique to genetically engineer mouse models paradoxically lagged behind other genetic technologies. The lack of demonstrable recombination in mtDNA necessitates different approaches to conventional transgenesis-based techniques. Initially, heteroplasmic mice were created to explore disease pathogenesis and mitochondrial dynamics in an in vivo system. Ultimately, transmitochondrial mouse models will be used to explore the role of the mitochondrial genome in human disease processes and in the development of novel human gene therapies. Here, we describe methodology to produce transmitochondrial mice (both homoplasmic and heteroplasmic models) harboring foreign mitochondrial genomes, using both embryo microinjection and embryonic stem (ES) cell-based approaches. Specific modeling and the procedures for mitochondrial transfer will be of considerable importance toward our understanding of discrete mitochondrial mutations, as well as lead to the development of novel strategies and therapies for human diseases influenced by mitochondrial DNA mutations.     

Mitochondrial DNA mutations cause resistance to opening of the permeability transition pore

The age-related accumulation of mitochondrial DNA mutations has the potential to impair organ function and contribute to disease. In support of this hypothesis, accelerated mitochondrial mutagenesis is pathogenic in the mouse heart, and there is an increase in myocyte apoptosis. The current study sought to identify functional alterations in cell death signaling via mitochondria. Of particular interest is the mitochondrial permeability transition pore, opening of which can initiate cell death, while pore inhibition is protective. Here, we show that mitochondria from transgenic mice that develop mitochondrial DNA mutations have a marked inhibition of calcium-induced pore opening. Temporally, inhibited pore opening coincides with disease. Pore inhibition also correlates with an increase in Bcl-2 protein integrated into the mitochondrial membrane. We hypothesized that pore inhibition was mediated by mitochondrial Bcl-2. To test this hypothesis, we treated isolated mitochondria with Bcl-2 antagonistic peptides (derived from the BH3 domain of Bax or Bid). These peptides released the inhibition to pore opening. The data are consistent with a Bcl-2-mediated inhibition of pore opening. Thus, mitochondrial DNA mutations induce an adaptive-protective response in the heart that inhibits opening of the mitochondrial permeability pore.     

Mechanism of mitochondrial DNA replication in mouse L-cells: kinetics of synthesis and turnover of the initiation sequence

Pulse-chase radioactive labeling experiments using thymidine kinase-plus mouse LA9 cells have shown that the 7 S mitochondrial DNA initiation sequence of mitochondrial DNA is synthesized and turned over at a faster rate than previously determined. These pulse-chase labeling experiments have also determined that the replication time of mouse LA9 cell mitochondrial DNA is one hour. The halflife of pulse-labeled 7 S mitochondrial DNA initiation sequences is approximately 70 minutes. This turnover is so rapid that at least 95% of the mitochondrial DNA initiation sequences synthesized are lost to turnover without acting as primers for expansion synthesis of the mitochondrial DNA heavy strand. The mechanism of 7 S mitochondrial DNA turnover does not lead to significant accumulation of free 7 S mitochondrial DNA single-strands within mitochondria. Resynthesis of the 7 S mitochondrial DNA initiation sequence is sufficiently rapid that the majority of mitochondrial DNA molecules are maintained as displacement loop molecules. Approximately 20% of all nucleotides polymerized into mitochondrial DNA are incorporated into the 7 S initiation sequences. The size of newly synthesized 7 S mitochondrial DNA strands varies from about 500 to 620 nucleotides. Several size classes are resolved by polyacrylamide/urea gel electrophoresis and each class has approximately the same turnover rate.Mouse LD cells maintain their mitochondrial DNA genomes as unicircular, head-to-tail dimers. Since a significant fraction of these unicircular dimers contain only one displacement loop, the size of the initiation sequence in such molecules should be twice as long if synthesis of the strand is limited by the free energy of superhelix formation. An identical array of size classes of 7 S strands is obtained from this cell line as compared to mouse LA9 cells. This indicates that the extent of 7 S mitochondrial DNA synthesis is most likely determined by a nucleotide sequence specific event.     

Proteins associated with mitochondrial DNA protect it against the action of X-rays and hydrogen peroxide

It has been suggested in a number of investigations that the high vulnerability of mitochondrial DNA to reactive oxygen species and other damaging agents is due to the absence in mitochondria of histones complexed with DNA. In the present study it was shown that DNA-binding proteins of mitochondrial nucleoids were able to shield mitochondrial DNA from X-ray radiation and hydrogen peroxide, as nuclear histones did. Mitochondria, mitochondrial nucleoid proteins, and histones were isolated from mouse liver cells. The degree of damage to or protection of mitochondrial DNA was assessed from the yield of its PCR amplification product. The in vitro experiments demonstrated that mouse mitochondrial DNA, when in complex with mitochondrial nucleoids or nuclear histones, was damaged much less by radiation and/or hydrogen peroxide than in the absence of these proteins and histones. No significant difference between mitochondrial nucleoid proteins and nuclear histones was revealed in their efficiency to protect mitochondrial DNA from the damaging effect of radiation and hydrogen peroxide. It is likely that the nucleoid proteins in the mitochondria shield mitochondrial DNA against the attack of reactive oxygen species, thus significantly decreasing the level of the oxidative damage to mitochondrial DNA.     

Isolation of two DNA polymerases from a mitochondrial membrane-DNA complex of mouse cell cultures

A membrane-DNA complex isolated from the mitochondria of thymidine kinase deficient mouse cells could be shown to contain in addition to mitochondrial DNA two different DNA polymerases: (i) Mitochondrial DNA polymerase 1 exhibiting characteristics of the DNA polymerase described for HeLa cell mitochondria and (ii) mitochondrial DNA polymerase 2 showing properties comparable to those of the DNA polymerase isolated from mouse liver mitochondria.     

A method for mutagenesis of mouse mtDNA and a resource of mouse mtDNA mutations for modeling human pathological conditions

Mutations in human mitochondrial DNA (mtDNA) can cause mitochondrial disease and have been associated with neurodegenerative disorders, cancer, diabetes and aging. Yet our progress toward delineating the precise contributions of mtDNA mutations to these conditions is impeded by the limited availability of faithful transmitochondrial animal models. Here, we report a method for the isolation of mutations in mouse mtDNA and its implementation for the generation of a collection of over 150 cell lines suitable for the production of transmitochondrial mice. This method is based on the limited mutagenesis of mtDNA by proofreading-deficient DNA-polymerase γ followed by segregation of the resulting highly heteroplasmic mtDNA population by means of intracellular cloning. Among generated cell lines, we identify nine which carry mutations affecting the same amino acid or nucleotide positions as in human disease, including a mutation in the ND4 gene responsible for 70% of Leber Hereditary Optic Neuropathies (LHON). Similar to their human counterparts, cybrids carrying the homoplasmic mouse LHON mutation demonstrated reduced respiration, reduced ATP content and elevated production of mitochondrial reactive oxygen species (ROS). The generated resource of mouse mtDNA mutants will be useful both in modeling human mitochondrial disease and in understanding the mechanisms of ROS production mediated by mutations in mtDNA.     

Mitochondrial DNA in the mouse preimplantation embryo

Total DNA was extracted from mouse embryos that were collected from CD-1 random-bred females on Day 1 of pregnancy and cultured for up to 4 days in vitro, or from the reproductive tracts of pregnant females on Days 1, 3, 4 and 5 of pregnancy. Southern blot analyses with a cloned mouse mitochondrial DNA probe were performed to determine the relative levels of mitochondrial DNA in the zygote, morula, blastocyst and early egg cylinder stage embryos. The results indicated that the total amount of mitochondrial DNA does not change during development of the mouse embryo up to the egg cylinder stage and is not altered during in-vitro culture of the fertilized one-cell embryo to the blastocyst stage.     

Structure,function and evolution of the animal mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is essential for animal mitochondrial DNA (mtDNA) maintenance. Deleterious mutations in the gene that encodes it cause mitochondrial dysfunction manifested in developmental delays, defects and arrest, limited life span, and a number of human pathogenic phenotypes that are recapitulated in animals across taxa. In fact, the replicative mtDNA helicase was discovered with the identification of human disease mutations in its nuclear gene, and based upon its deduced amino acid sequence homology with bacteriophage T7 gene 4 protein (T7 gp4), a bi-functional primase-helicase. Since that time, numerous investigations of its structure, mechanism, and physiological relevance have been reported, and human disease alleles have been modeled in the human, mouse, and Drosophila systems. Here, we review this literature and draw evolutionary comparisons that serve to shed light on its divergent features.