Pretreatment mitochondrial priming correlates with clinical response to cytotoxic chemotherapy

Cytotoxic chemotherapy targets elements common to all nucleated human cells, such as DNA and microtubules, yet it selectively kills tumor cells. Here we show that clinical response to these drugs correlates with, and may be partially governed by, the pretreatment proximity of tumor cell mitochondria to the apoptotic threshold, a property called mitochondrial priming. We used BH3 profiling to measure priming in tumor cells from patients with multiple myeloma, acute myelogenous and lymphoblastic leukemia, and ovarian cancer. This assay measures mitochondrial response to peptides derived from proapoptotic BH3 domains of proteins critical for death signaling to mitochondria. Patients with highly primed cancers exhibited superior clinical response to chemotherapy. In contrast, chemoresistant cancers and normal tissues were poorly primed. Manipulation of mitochondrial priming might enhance the efficacy of cytotoxic agents.     

mtDNA A3243G 点突变小鼠模型的建立及其致病机制探讨

    利用显微注射线粒体技术建立转人线粒体小鼠模型,研究外源突变mtDNA在不同组织的分布及遗传规律,探讨mtDNA A3243G点突变对线粒体功能的影响.从健康成人及2型糖尿病患者(携带mtDNA 3243A-G突变)血液标本中分离有活性的线粒体,将其显微注射至小鼠受精卵,胚胎移植,产出仔鼠后利用分子生物学方法检测人mtDNA及mtDNA A3243G点突变.获得嵌合体小鼠后,对其空腹血糖和全血乳酸进行测定,并使用荧光法和比色法分析A3243G点突变小鼠重要脏器组织细胞活性氧生成量(ROS)、线粒体复合酶Ⅰ和Ⅳ活力及线粒体ATP合成活力的变化.研究结果显示:在1只雌性(转健康人线粒体)和2只雄性小鼠(转患者线粒体)中检测到人mtDNA,其中2只雄性小鼠携带mtDNA 3243A-G突变;将嵌和体雌鼠与野生型C57BL/6J 雄鼠交配后,在1只后代仔鼠中检测到人mtDNA;人mtDNA仅在嵌合小鼠的部分组织中表达.在含有mtDNA A3243G突变的组织中发现,线粒体复合酶Ⅰ、Ⅳ活力降低,ATP合成速率下降,ROS水平升高,说明A3243G点突变能损伤线粒体正常功能从而导致疾病的发生.综上所述,本研究利用显微注射法成功建立了嵌和小鼠,引入了致病性的点突变,为线粒体疾病的研究提供了良好的思路.     

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier. Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria, resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP production.     

ER tubules mark sites of mitochondrial division

Mitochondrial structure and distribution are regulated by division and fusion events. Mitochondrial division is regulated by Dnm1/Drp1, a dynamin-related protein that forms helices around mitochondria to mediate fission. Little is known about what determines sites of mitochondrial fission within the mitochondrial network. The endoplasmic reticulum (ER) and mitochondria exhibit tightly coupled dynamics and have extensive contacts. We tested whether ER plays a role in mitochondrial division. We found that mitochondrial division occurred at positions where ER tubules contacted mitochondria and mediated constriction before Drp1 recruitment. Thus, ER tubules may play an active role in defining the position of mitochondrial division sites.     

归芪多糖延缓细胞衰老的分子作用机制

以SIRT1和线粒体为切入点，采用分子生物学技术以及多种线粒体分析技术，深入探讨SIRT1和线粒体在衰老细胞中的作用，揭示归芪多糖延缓细胞衰老的分子作用机制。结果表明，AAP显著降低细胞的衰老程度并提高细胞活力，而Ex527阻断AAP的作用。同时，发现AAP增强细胞内SIRT1和CyclinD1的表达，降低p53的表达水平，在Ex527组中未观察到类似的逆转作用。线粒体分析结果显示，AAP可显著降低细胞内的活性氧水平，降低线粒体膜电位，减轻线粒体肿胀程度和增加线粒体内ATP含量，而Ex527的预处理消除这些作用。基于上述结果，推测AAP可能通过信号通路p53/p16和CyclinD/CDK4来改善线粒体功能，从而达到延缓衰老的作用，且这些作用与SIRT1密切相关。     

基于线粒体能量代谢途径的金针菇采后纳米包装保鲜机制

【目的】从线粒体水平上揭示不同包装对金针菇能量代谢的影响,为进一步揭示纳米包装保鲜机制提供新思路。【方法】以金针菇为材料,对比不连续密度梯度离心法（DDGCM）、酵母线粒体提取试剂盒方法（YMEKM）和改进普利莱法（IPKM）3种提取方法对金针菇子实体与菌丝体中线粒体功能活性的影响,确定最佳提取方法。通过测定不同包装及不同贮藏期金针菇线粒体中ATP代谢系统物质含量（ATP、ADP、AMP及能荷）和线粒体主要复合体活性,揭示不同包装金针菇采后能量代谢规律。【结果】通过标志性污染物酶和线粒体呼吸速率指标测定,3种方法以IPKM提取的线粒体内乙醇脱氢酶活性及胞液内细胞色素C氧化酶活性最低,呼吸速率最高,证明IPKM所提的线粒体结构完整性最好;IPKM提取的线粒体超氧化物歧化酶活性最高,达到17.82 U·mg^-1 pro,比DDGCM-1和YMEKM分别高7.31%和25.59%。结合透射电镜和健那绿染色结果可知,IPKM提取的线粒体结构相对完整,活性线粒体数量最多。在金针菇冷藏期间,纳米包装金针菇的ATP水平、能荷含量以及线粒体主要复合体活性都显著高于普通PE包装。纳米包装材料通过维持高水平的ATP含量,延缓能荷值的下降,促进了线粒体的氧化磷酸化过程。纳米包装组在第21天的ATP含量仍然比普通PE包装组高113.83 μg·g^-1FW,从而避免金针菇线粒体复合体I、Ⅲ活性降低,减缓线粒体复合体Ⅳ的活性降低。普通PE包装金针菇的线粒体复合体Ⅳ活性在第9天达到第一个峰值,而纳米包装金针菇线粒体复合体IV活性在第15天达到第一个峰值（5.14 U·mg^-1 pro）,显著高于普通PE包装（1.12 U·mg^-1 pro）,相差达到78.23%。证明纳米包装能有效控制线粒体呼吸链中的电子传递不受阻碍,保证细胞能量供应,从而更好地保持金针菇的能量状态。【结论】改进普利莱法对线粒体损伤最小,并维持线粒体活性,适合金针菇采后线粒体能量代谢研究。同时,纳米包装通过维持较高水平的ATP含量与线粒体复合体I、Ⅲ活性并延缓能荷与线粒体复合体Ⅳ活性的下降,进而保持金针菇的能量状态并延缓衰老,延长贮藏期。     

4种合成活性肽对小鼠生长性能和免疫机能的影响

本试验选用4种合成生物活性肽(肽1、肽2、肽3、肽4分别由15、7、9、7个氨基酸构成,采用固相合成柱手工合成的直链肽)对小鼠进行灌喂饲养试验,旨在探讨其对小鼠的生长性能、免疫机能和器官发育的影响.选取清洁级健康小鼠90只,随机分为9组,每组10只,试验组分别连续灌喂10 μg/kg体重和50 μg/kg体重剂量的4种活性肽,空白对照组灌喂等量蒸馏水.试验期32 d.结果表明:(1)灌喂活性肽15 d和32 d,小鼠体重、平均日耗料量各试验组之间及与对照组间差异均不显著(P>0.05).(2)小鼠灌喂活性肽15 d,50 μg/kg体重肽3能显著提高小鼠血清IgG和IFN-γ含量(P<0.05).(3)小鼠灌喂活性肽15 d,50 μg/kg体重肽1组小鼠肝脏系数显著高于10 μg/kg体重肽1组(P<0.05),其余各组之间无显著差异(P>0.05);心脏、脾脏和肾脏系数各组之间差异均不显著(P>0.05).小鼠灌喂活性肽32 d时,50 μg/kg体重活性肽2、肽3、肽4对小鼠心脏、肝脏和肾脏系数有显著提高作用.综上可见,4种人工合成生物活性肽对小鼠生长性能未见明显影响,对免疫机能和脏器系数有一定的促进作用.     

Structure and expression of a complementary DNA for the nuclear coded precursor of human mitochondrial ornithine transcarbamylase

Most mitochondrial proteins are encoded in the nucleus and are translated on free cytoplasmic ribosomes as larger precursors containing amino-terminal "leader" sequences, which are removed after the precursors are taken up by mitochondria. We have deduced the complete primary structure of the precursor of a human mitochondrial matrix enzyme, ornithine transcarbamylase (OTC), from the nucleotide sequence of cloned complementary DNA. The amino-terminal leader peptide of OTC is 32 amino acids in length and contains four arginines but no acidic residues. Cleavage of the leader peptide from the "mature" protein occurs between glutamine and asparagine residues. The sequence of mature human OTC resembles that of the subunits of both OTC and aspartate transcarbamylase from Escherichia coli. The biological activity of the cloned OTC complementary DNA was tested by joining it with SV40 (an animal virus) regulatory elements and transfecting cultured HeLa cells, which do not normally express OTC. Both the precursor and mature forms of the OTC subunit were identified; in stable transformants, enzymatic activity was also detected.     

Hydrolyzing Condition and Immunocompetence of Sheep Bone Protein Enzymatic Lysates

Utilizing collagen of sheep bone as material to get immunocompetent peptide, enzymatic hydrolysis conditions were optimized using quadratic regression general rotation design. The effect of temperature （T）, time （t）, enzyme/substrate （E/S） ratio, and substrate concentration （S） on the amount of tricarboxylix acid cycle （TCA） soluble peptides were investigated. The content of soluble peptide in the acquisition was measured by Folin-hydrozybebzebe method, and the correlation between soluble peptide content and immunocompetence was analyzed by SAS software. The best enzymatic hydrolysis condition was gotten from Design Expert 7.1.2 software. The optimal condition under which immunocompetent peptides could be prepared was 1 576 U g^-1 （E/S）, 64.05℃ （T）, 0.271 kg L^-1 （S）, and 7.22 h （t）. The correlation coefficient between TCA soluble peptides and the immuneocompetence was 0.045 〈r0.05 = 0.355, which indicated that they had no significant correlation. The result showed that the soluble peptide contained immunocompetent peptides which content was independent of immunocompetence in the hydrolasates.     

家蚕细胞色素C基因的克隆及其蛋白在家蚕凋亡细胞中的释放

 【目的】细胞色素C是线粒体凋亡路径上的关键因子,通过家蚕细胞色素C基因的克隆和其蛋白在家蚕凋亡细胞中的释放研究,为家蚕细胞凋亡的研究奠定基础。【方法】利用生物信息学和分子生物学方法克隆家蚕细胞色素C基因,通过紫外线照射诱导家蚕细胞凋亡,应用流式细胞仪和Western-blotting检测家蚕凋亡细胞的线粒体膜电位变化和细胞色素C的释放。【结果】在家蚕中克隆了一个含有细胞色素C保守结构域的同源基因,该基因cDNA长570 bp,有3个外显子,开放阅读框（open reading frame,ORF）长324 bp,编码108个氨基酸,存在参与细胞凋亡时与下游凋亡蛋白酶激活因子（Apaf－1）相互作用的保守关键位点;紫外线诱导家蚕胚胎细胞BmE-SWU1凋亡后12 h,细胞线粒体跨膜电位明显下降,同时检测到了细胞色素C由线粒体向细胞质释放。【结论】在家蚕细胞凋亡中可能存在细胞色素C由线粒体释放的路径。     

Ca2+诱导损伤线粒体超微结构的原子力显微镜研究

采用Ca²⁺急性诱导构建线粒体损伤模型,分别通过透射电子显微镜和原子力显微镜观测线粒体内部结构和表面形貌的变化,用以探究线粒体损伤过程中结构变化的规律性。结果显示,线粒体在Ca²⁺诱导下的损伤过程依照内部结构的变化规律可分为前后2个阶段：前期以嵴有序结构的破坏为特征,线粒体内部特异性结构逐步被破坏,无明显肿胀趋势;后期以线粒体嵴间隙膨胀、嵴囊泡化为特征,线粒体迅速膨胀并向球形变化,线粒体的最终裂解应存在临界条件,决定于线粒体外膜的张力特性。     

Mitochondrial dynamics caused by QoIs and SDHIs fungicides depended on FgDnm1 in Fusarium graminearum

Fusarium head blight (FHB) caused by Fusarium graminearum is a devastating fungal disease on small grain cereal crops, because it reduces yield and quality and causes the mycotoxin contamination to the grain. Dynamins and dynamin-related proteins (DRPs) are large GTPase superfamily members, which are typically involved in the budding and division of vesicles in eukaryotic cells, but their roles in Fusarium spp. remain unexplored. Here, we found that FgDnm1, a DRP and homolog to Dnm1 in Saccharomyces cerevisiae, contributes to the normal fungal growth, sexual reproduction and sensitivity to fungicides. In addition, we found FgDnm1 co-localizes with mitochondria and is involved in toxisome formation and deoxynivalenol (DON) production. Several quinone outside inhibitors (QoIs) and succinate dehydrogenase inhibitors (SDHIs) cause fragmentated morphology of mitochondria. Importantly, the deletion of FgDnm1 displays filamentous mitochondria and blocks the mitochondrial fragmentation induced by QoIs and SDHIs. Taken together, our studies uncover the effect of mitochondrial dynamics in fungal normal growth and how such events link to fungicides sensitivity and toxisome formation. Thus, we concluded that altered mitochondrial morphology induced by QoIs and SDHIs depends on FgDnm1.     

水稻线粒体nad1基因超表达载体的构建和遗传转化

[目的]克隆线粒体相关基因nad1,获得转nad1的转基因水稻植株。[方法]采用TRIzol法提取水稻幼苗总RNA,以反转录的cD-NA为模板,扩增得到nad1;将nad1接到线粒体信号肽Rf1b的5’（Rf1b5’）,装载到pCAMBIA1305.1双元表达载体,采用农杆菌介导的愈伤组织侵染法进行遗传转化。[结果]克隆的目的基因nad1大小为978bp,成功构建了携带线粒体信号肽的nad1植物表达载体,并获得了转nad1基因的阳性植株。[结论]为探讨水稻中过表达nad1对水稻生长的影响奠定了基础。     

Development of a Synthetic Peptide ELISA Assay for the Detection of Foot-and-Mouth Disease Virus Nonstructural Protein Antibodies

In order to develop an ELISA assay with synthetic peptides for the detection of antibody to the nonstructural proteins of foot-and-mouth disease virus, specific peptides were synthesized by a solid-phase method according to FMDV NSPs B-cell epitopes, and were conjugated to carrier protein BSA. An ELISA system was developed to detect FMDV NSPs antibody with the conjugated proteins as the coating antigen. The optimal coating concentration of the antigen was determined as 2.5 μg mL-1. The comparative study of this assay with UBI NSP ELISA kit and national commercial 3ABC ELISA kit in the detection of 199 serum samples showed that they were very coincident, and the identity rates were 96.48 and 97.48%, respectively. The development of ELISA using the synthetic peptides as coating antigen is specific, reproducible, stable, and easy, and can be used to differentiate FMDV infected pigs from immunized pigs.     

Hypoxic coordinate regulation of mitochondrial enzymes in mammalian cells

The effect of hypoxic exposure on various mitochondrial enzymes and on cell mitochondrial genomic content was studied in two types of mammalian cells. Hypoxia depressed the activity of six enzymes to the same degree. The kinetics of depression and of recovery during reexposure to normoxia were statistically similar for three marker enzymes. Despite the global and symmetrical decrease in enzyme activities, mitochondrial DNA remained constant. This suggests either symmetrical loss of mitochondrial enzymes from all mitochondria or complete loss of enzymes from a subpopulation of mitochondria with retention of an intact mitochondrial genome.     

超声协助提取维药芹菜籽蛋白多肽类物质的研究

采用单因素和正交试验,以磷酸盐缓冲溶液提取并用硫酸铵沉淀芹菜籽中的多肽类成分,且以不同条件下硫酸铵沉淀（80％）的多肽量为考察指标,来确定活性多肽的超声波提取最佳条件。结果表明,芹菜籽中多肽的最佳提取条件为：磷酸盐缓;中溶液pH9．0,超声提取30min,液固比5．0：1（ml：g）,提取的多肽具有较好的清除ABTS自由基的能力。关键词芹菜籽;活性多肽;超声波提取;最佳条件;抗氧化活性     

Mitochondrial autonomy: incorporation of monosaccharides into glycoprotein by isolated mitochondria

Isolated intact mitochondria selectively incorporate monosaccharides from nucleotide diphosphate monosaccharides into protein. Fucose, mannose, glucose, and galactose were incorporated by the mitochondria into glycoprotein; xylose was not. Structural integrity of the mitochondria was not necessary for the incorporation of monosaccharide into glycoprotein; mitochondria broken by homogenization also incorporated monosaccharide. The monosaccharides incorporated into glycoprotein were localized in the inner mitochondrial membranes, the same membranes which contain the protein into which leucine is incorporated by the isolated mitochondria.     

Method for Isolating Mitochondrial DNA from Etiolated Tissue of Cabbage

Isolation of high-quality mitochondrial DNA（mtDNA） is an important premise for researching molecular mechanisms in cytoplasmic male sterility of cabbage（Brassica oleracea L.var.capitata）. An efficient protocol for separation and purification of mitochondria and extraction of mitochondrial DNA（mtDNA） from etiolated tissues of cabbage was developed. We took a method combined mannitol density gradient with differential centrifugation, selected appropriate rotational speed, extended DNase I treating time and changed mitochondria cracking condition. The results showed that the extracted mitochondria in this protocol had complete structure, appeared to ellipsoid and had not been contaminated with other impurities under the Jannus Green B staining. The isolated mitochondrial DNA had high purity and yield through detecting the optical density, nuclear specific primer PCR and agarose gel electrophoresis. The results indicated that mitochondrial DNA extracted by this protocol had high quality and enabled to be used in futher genetic studies.     

Regulated cycling of mitochondrial Hsp70 at the protein import channel

Hsp70 of the mitochondrial matrix (mtHsp70) provides a critical driving force for the import of proteins into mitochondria. Tim44, a peripheral inner-membrane protein, tethers it to the import channel. Here, regulated interactions were found to maximize occupancy of the active, adenosine 5'-triphosphate (ATP)-bound mtHsp70 at the channel through its intrinsic high affinity for Tim44, as well as through release of adenosine diphosphate (ADP)-bound mtHsp70 from Tim44 by the cofactor Mge1. A model peptide substrate rapidly released mtHsp70 from Tim44, even in the absence of ATP hydrolysis. In vivo, the analogous interaction of translocating polypeptide would release mtHsp70 from the channel. Consistent with the ratchet model of translocation, subsequent hydrolysis of ATP would trap the polypeptide, driving import by preventing its movement back toward the cytosol.