肝癌患者单个核细胞线粒体DNA拷贝数与抗氧化能力的变化

目的:探讨肝癌患者单个核细胞(PBMC)线粒体DNA(mt DNA)拷贝数的变化及其与机体抗氧化能力的关系。方法:用Ficoll Hypaque离心法分离外周血单个核细胞(PBMC),采用实时荧光定量PCR反应,检测线粒体NADH脱氢酶亚基1(ND1)基因的拷贝数,并以核基因组的β-actin作为内参基因,比较25例肝癌患者与30例健康人外周血单个核细胞中mt DNA拷贝数的差异。流式细胞术检测单个核细胞活性氧(ROS)含量的变化。生化检测法检测血浆中总抗氧化能力(T-AOC)的变化。结果:肝癌组外周血单个核细胞ND1基因拷贝数是健康对照组的73%,表明肝癌患者外周血单个核细胞mt DNA拷贝数明显下降。肝癌组单个核细胞活性氧含量的平均荧光强度为(417.82±110.62),健康对照组为(301.82±75.54),肝癌组单个核细胞活性氧含量显著高于健康对照组(P0.01)。肝癌组血浆总抗氧化能力(单位/毫升血浆)吸光度为(1.30±0.85),健康对照组为(3.20±1.62),肝癌组血浆总抗氧化能力显著低于健康对照组(P0.01)。结论:肝癌患者的外周血单个核细胞线粒体DNA拷贝数减少可能与机体抗氧化水平下降有关。     

荧光定量PCR精确检测人胚胎干细胞线粒体DNA拷贝数方法的建立

建立一种精确定量人胚胎干细胞线粒体DNA拷贝数的方法。构建包含线粒体DNANDl和核单拷贝基β-globin基因序列的重组质粒作为标准品;收集无饲养层培养体系下人胚胎干细胞DNA样本,结合2个单独的Taqman探针实时荧光定量PCR对待测样本中线粒体NDl和核β-globin基因分别进行定量,从而对人胚胎干细胞线粒体DNA的含量进行了精确定量。结果提示,人胚胎干细胞线粒体DNA的平均拷贝数／细胞为1321±228。研究表明,该技术可对人胚胎干细胞线粒体DNA拷贝数进行准确的测定,为研究培养条件对人胚胎干细胞线粒体DNA拷贝数的影响及优化体外培养条件奠定了基础。     

基于数字PCR的不同品种鸭组织中线粒体与核DNA拷贝数差异研究

肉和肉制品是人类生活的重要营养来源,但近年来肉制品中发生的掺假使假事件屡见不鲜,使得肉品的质量安全问题已经成为全世界关注的热点话题。以核酸为目标的动物源鉴定是当前普遍使用的方法。在核酸检测中,常用线粒体基因或核基因作为靶标,缺乏统一标准。以绍兴鸭和北京鸭等不同品种及生鲜组织(鸭血、鸭胸肉、鸭肝、鸭皮、鸭心和鸭腿肉)为实验材料,提取DNA后利用微滴式数字PCR开展线粒体和核DNA拷贝数的比较研究,以两者拷贝数及其比值的变异系数为判定依据。结果显示,核DNA的拷贝数在不同品种鸭组织间相对稳定,且变异系数小于线粒体DNA,表明核DNA是开展鸭肉制品掺假定量检测的最适DNA来源。鸭腿肉中线粒体/核DNA拷贝数比值的变异系数最小,表明线粒体DNA作为靶基因的鸭肉掺假比例定量检测时,鸭腿肉来源的肉制品是最佳选择。     

线粒体DNA数目不同的人角质形成细胞模型的建立

该研究采用溴化乙锭(ethidium bromide,Et Br)对人永生化角质形成细胞(HaCaT)进行诱导,采用RT-PCR法、MTT法和健那绿染色技术联合鉴定Et Br对HaCaT细胞线粒体DNA(mitochondrial DNA,mt DNA)拷贝数的影响,建立不同mt DNA拷贝数目的HaCaT细胞模型。结果显示,HaCaT细胞经100 ng/m L和50 ng/m L Et Br分别处理10 d后,细胞形态随着处理时间的延长发生改变,由规则铺路石形状逐渐变圆,某些细胞体积变小,成团生长,细胞膜边缘不光滑,且随着mt DNA数目的减少,细胞增殖速率明显下降。RT-PCR分析结果显示,与对照组相比,50 ng/m L和100 ng/m L Et Br处理10 d的HaCaT细胞中,mt DNA的拷贝数分别减少了52.9%和97.6%[以线粒体DNA脱氢酶1(mitochondrial NADH dehydrogenase 1 gene,ND1)与甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)的比值来表示mt DNA的相对拷贝数变化情况]。健那绿染色显示,对照组HaCaT细胞中可见分散存在的蓝绿色线粒体颗粒,100 ng/m L Et Br处理10 d后的HaCaT细胞中,显微镜下蓝绿色线粒体颗粒明显减少。综上所述,该研究证明了HaCaT细胞可通过Et Br诱导被成功培养成ρ–细胞,且Et Br浓度与HaCaT细胞mt DNA拷贝数的变化在一定的范围内存在着剂量–效应关系。     

首发未服药精神分裂症患者线粒体DNA拷贝数及其与血脂指标的相关性

精神分裂症是一种重度的精神疾病,伴发严重的能量代谢失衡。其中,脂代谢的异常近年来受到越来越多的关注。多项研究表明,线粒体在精神分裂症患者代谢异常的过程中发挥重要的作用。线粒体DNA拷贝数是线粒体含量及完整性的重要指标,直接参与线粒体的多项重要功能。本研究旨在通过考察首发精神分裂症患者线粒体DNA拷贝数的变化特征及其与相关代谢指标之间的相关性,为阐明精神分裂症代谢异常的机制提供支持。本研究共计纳入82例首发且未服用抗精神病药物的精神分裂症患者和77例健康对照,采用荧光定量PCR技术进行线粒体DNA拷贝数的检测,并进行了临床信息和血脂指标的采集。结果表明,精神分裂症患者组的线粒体DNA拷贝数显著低于健康对照组(P=0.000572,FC=-1.22)。血脂指标中,患者组的HDL-c显著低于健康对照组(P=0.001,FC=-1.12),LDL-c(P=0.009,FC=1.09)、CHOL/HDL-c(P=0.000019,FC=1.17)、TG/HDL-c(P=0.000656,FC=1.31)和LDL-c/HDL-c(P=0.000004,FC=1.30)均显著高于健康对照组。相关性分析表示,在健康对照组中,mtDNA拷贝数与TG显著负相关(r=-0.232,P=0.0499),与TG/HDL-c(r=-0.235,P=0.052)呈负相关,但不显著;在精神分裂症患者组,mtDNA拷贝数与各血脂指标的关系都不显著。本研究为精神分裂症患者的线粒体功能障碍、脂代谢异常提供了证据,提示了线粒体在精神分裂症患者并发心血管代谢疾病高风险中可能的重要作用。     

线粒体DNA单体型M8a对转线粒体细胞线粒体能量代谢的影响

为了观察线粒体DNA(mitochondrial DNA,mt DNA)单体型M8a对细胞线粒体能量代谢的影响,该研究利用聚乙二醇(polyethylene glycol,PEG)介导血小板–细胞融合技术,构建mt DNA单体型M8a和G2a转线粒体细胞(transmitochondrial cytoplasmic hybrid cell,cybrid)模型。后续运用Real-time PCR检测细胞mt DNA拷贝数和RNA转录水平,多功能酶标仪检测细胞活性氧类(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,MMP),线粒体呼吸测定仪检测细胞内源性氧呼吸(oxygen consumption,OC)情况。结果表明,与G2a细胞相比,M8a细胞mt DNA拷贝数和线粒体轻链(L链)转录水平明显降低(P0.01),细胞基础耗氧量、ATP合成耗氧量、最大氧呼吸能力和MMP显著下降(P0.05),细胞ROS水平显著上升(P0.05)。mt DNA单体型M8a细胞的线粒体呼吸功能受损,可能增加了该单体型人群罹患2型糖尿病的风险。     

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。     

mtDNA拷贝数的生物学意义及其调控

线粒体是细胞能量和自由基代谢中心,并在细胞凋亡、钙调控、细胞周期和信号转导中发挥重要作用,维持线粒体功能正常对于细胞正常行使职能意义重大。线粒体的功能与线粒体DNA（mitochondrial DNA,mtDNA）的数量和质量紧密相关,mtDNA的数量即mtDNA拷贝数又受到mtDNA质量的影响,因此mtDNA拷贝数可作为线粒体功能的重要表征。mtDNA拷贝数变异引起线粒体功能紊乱,进而导致疾病发生。本文综述了mtDNA拷贝数变异与神经退行性疾病、心血管疾病、肿瘤等疾病的发生发展和个体衰老之间的关系,以及mtDNA复制转录相关因子、氧化应激、细胞自噬等因素介导mtDNA拷贝数变异的调控机制。以期为进一步深入探究mtDNA拷贝数调控的分子机制,以及未来治疗神经退行性疾病、肿瘤及延缓衰老等提供一定的理论基础。     

mtDNA单倍型D4和B4a细胞线粒体功能及其影响衰老发生的机制研究

该文初步分析了两个与衰老发生抑制及促进的线粒体DNA(mitochondrial DNA,mtDNA)单倍型D4和B4a细胞的线粒体功能情况,并对其影响衰老的可能机制进行了探究。通过细胞融合构建两个核背景一致但mtDNA单倍型分别是D4和B4a的胞质杂合细胞。应用RT-PCR方法检测细胞mt DNA拷贝数以及线粒体基因的mRNA水平,氧电极法和非变性梯度聚丙烯酰胺凝胶电泳(blue native polyacrylamide gelelectrophoresis,BN-PAGE)技术检测细胞线粒体氧化呼吸能力及线粒体复合体水平,TMRM染料和DCFH-DA染料法检测细胞线粒体膜电位和活性氧(reactive oxygen species,ROS)生成水平。结果显示,D4单倍型细胞线粒体氧呼吸能力、膜电位水平以及线粒体基因(mt-ND1、mt-7S RNA)转录水平均明显高于B4a单倍型细胞,而ROS生成水平明显降低,mt DNA拷贝数则并无明显差异。该结果表明,在D4单倍型细胞中,通过提高某些线粒体基因的表达,使得线粒体呼吸复合体表达得到提升,继而提高线粒体氧化呼吸功能,降低细胞氧化损伤,从而延缓衰老的发生。     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Apoptotic Regulation of Caspase Activity

Intracellular cysteine aspartate-specific proteases (caspases) play both signaling and effector roles in realizing the program of cell death. Caspases function as proteolytic cascades unique for each cell type and signal triggering apoptosis. All parts of the proteolytic cascades are duplicated and controlled by feedback signals. Amplification cycles between pairs of caspases (the third and the sixth, the ninth and the third, the twelfth and the sixth, and others) help multiply the initial apoptotic signal. The presence of physiological inhibitors of apoptosis that directly interact with caspases creates a multilevel regulatory network of apoptosis in cell. The caspase proteolytic cascades are also regulated by sphingolipid secondary messengers, among them ceramide, sphingosine, and their phosphates. Moreover, an association of the caspase signaling with ubiquitin-dependent proteolysis is shown in cells. In particular, the use of extracellular activators and inhibitors of caspases allows irreversible activation of apoptosis in tumor cells or the prevention of apoptosis in cortical neurons under neurodegenerative diseases.     

Analysis of twin of eyeless regulation during early embryogenesis in Drosophila melanogaster

The Drosophila Pax6 homolog twin of eyeless (toy) is so far the first zygotically expressed gene involved in eye morphogenesis in Drosophila. The study of its expression during embryogenesis is therefore informative of the initial events of eye development in Drosophila. We have analyzed how the initial expression domain of toy at cellular blastoderm is regulated. We show that the three maternal patterning systems active in the cephalic region (the anterior, terminal and dorsal-ventral systems) cooperate with zygotically activated gap genes to shape the initial expression domain of toy. Whereas Bicoid, Dorsal and Torso signaling synergistically act as activators, Hunchback, Knirps and Decapentaplegic act as repressors.     

Host plants of someRhinanthoideae (Scrophulariaceae) of eastern North America

Twenty three species in 11 genera were examined in the field to determine hosts. OnlyStriga asiatica andSeymeria cassioides have a narrow host range being restricted to grasses and pines, respectively. These are the only species which cause pronounced and sometimes serious host damage. The other species attach to a great diversity of hosts.     

New species of Peruvian Orchidaceae III

Sixteen new species are proposed in the generaAckermania, Dressleria, Epidendrum, Maxillaria, Oncidium, Rodriguezia, Sigmatostalix, andTrigonidium. All new species are illustrated.Maxillaria vittariifolia L. O. Williams is newly recorded for Peru. A key is provided forTrigonidium of Peru.Trigonidium loretoense Schltr. andT. peruvianum Schltr. are lectotypified.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Interaction of Synthetic Analogues of Natural Chalcones and Flavones with Guanidine

The reaction of chalcones and flavones with guanidine in ethanol results in 2-(2-aminopyrimidinyl-4)-phenols.