动物线粒体基因组全序列测定的研究策略

动物线粒体基因组全序列测定是近年来生物学研究中的一个热点。科学技术的发展,对动物线粒体基因组全序列测定的研究策略也产生了深远的影响。在此对几种在动物线粒体基因组全序列测定中常用的几种策略进行了综述,并对其优缺点进行了比较分析。     

Reactive oxygen species are crucial for hydroxychavicol toxicity toward KB epithelial cells

Betel quid (BQ) chewing shows a strong correlation to the incidence of oral submucous fibrosis (OSF), leukoplakia and oral cancer. BQ contains mainly areca nut, lime, Piper betle leaf (PBL) and the inflorescence of P. betle (IPB). Hydroxychavicol (4-allyl-catechol, HC), as a major phenolic compound in PBL and IPB, is shown to induce oxidative stress, glutathione (GSH) depletion and cell cycle deregulation. Using bivariate BrdU/PI flow cytometry, KB cells in DNA synthesis (S phase) are shown to be sensitive to the toxic effect of HC and show cell cycle arrest and apoptosis following exposure to 0.1 and 0.3 mM HC. HC-induced apoptosis and cell cycle arrest are associated with mitochondrial membrane potential (m) depolarization as revealed by a decrease in rhodamine fluorescence. N-acetyl-L-cysteine (1 mM), superoxide dismutase (100 U/ml) and catalase (1000 U/ml) were effective in prevention of HC-induced GSH depletion (as indicated by chloromethylfluorescein fluorescence), reactive oxygen species (ROS) production (by dichlorofluorescein fluorescence), cell cycle arrest and apoptosis. However, dimethylthiourea (2 mM), neocuproine (1 mM), 1,10-phenanthroline (200 M) and desferrioxamine (0.5 mM) showed little effect on HC-induced cell changes. HC elevated the cellular and mitochondrial GSH levels at moderate concentrations (0.05–0.1 mM), whereas at a concentration of 0.3 mM, inhibitory effects were noted. These results indicate that HC consumption may be associated with BQ-chewing-related oral mucosal diseases via GSH depletion, ROS production, mitochondrial dysfunction, cell cycle disturbance and the induction of apoptosis. These events are related to the production of superoxide radicals and hydrogen peroxide.Received 9 July 2003; received after revision 28 September 2003; accepted 24 October 2003     

Effect of Salvianolic Acid B on Mitochondrial Function of CerebralIschemia in Mice

The effects of salvianolic acid B (SalB) on the mitochondrial membrane potential (MMP), calcium, and apoptosis of neurons with cerebral ischemia in mice were investigated using an acute cerebral ischemia model established by ligating the bilateral common carotid arteries in mice. The MMP, the intracellular calcium concentration, and the apoptosis rate of cortical neurons were measured at 6 min, 12 min, 18 min, 24 min, and 30 min after cerebral ischemia by a flow cytometer. The experiments show that SalB increases the MMP and reduces the intracellular calcium and the apoptosis rate at different stages of the cerebral ischemia in mice. The results show that the protective mechanism of SalB on cerebral ischemia enhances the MMP and maintains intracellular calcium homeostasis.     

DBP对大鼠睾丸支持细胞内Ca2+、线粒体膜电位及Caspase活性的影响

目的通过测定DBP对大鼠睾丸支持细胞内Ca2+、线粒体膜电位及Caspase活性的影响,探讨DBP是否通过影响线粒体膜电位而引起支持细胞凋亡.方法建立支持细胞原代培养体系,将细胞分为0.1%DMSO(对照组)、1 mg/L(低剂量组)、10 mg/L(中剂量组)、100 mg/L(高剂量组),分别处理细胞24 h;荧光分光光度计测定支持细胞内Ca2+的浓度,流式细胞仪检测线粒体膜电位,酶标仪测定Caspase-9和Caspase-3的活性.结果与对照组相比,高剂量组中Ca2+浓度明显升高,差异具有统计学意义(P0.05),而低、中剂量组与对照组比较无统计学意义(P0.05).DBP导致的线粒体膜电位升高在中、高剂量组中具有统计学意义(P0.05),随着DBP浓度的增加,Caspase-9与Caspase-3的活性升高,差异均具有统计学意义(P0.05).结论 DBP能显著降低支持细胞线粒体膜电位,改变线粒体膜的通透性,进一步升高Caspase-9及Caspase-3的活性,并最终导致睾丸支持细胞凋亡.     

几种鹤类线粒体DNA的差异和亲缘关系分析

采用聚合酶链式反应,从丹顶鹤(G.joponensis♂)、白枕鹤(G.vipio♀)、灰鹤(G.grus)及丹顶鹤♂和白枕鹤♀的4个杂交子代中分别扩增出线粒体DNA细胞色素b基因,并测定出细胞色素b碱基序列,它们之间的序列差异在0.1%～7.2%之间.序列分析表明几种鹤类细胞色素b基因序列的碱基变化主要是转换和颠换,转换的比率大于颠换;绘制几种鹤类细胞色素b之间的亲缘关系进化树表明丹顶鹤和灰鹤的进化距离更近,4个杂交子代与母本白枕鹤的亲缘关系更近.     

厦门海域真刺唇角水蚤mtCOI序列分析

采用PCR扩增、克隆和序列测定等技术对厦门海域真刺唇角水蚤(Labidocera euchaetaGiesbrecht)线粒体细胞色素氧化酶I亚基基因(mitochondrial COI,mtCOI)进行了初步研究,得到3个雌性个体的mtCOI基因片段序列,序列的长度为709 bp,其A,T,C,G平均含量分别为23%、37%、15%和25%.三条核苷酸序列经比较后,得到其遗传相似性(i-dentity)为99.29%,共有15个差异位点,核苷酸差异均为颠换(transversion)类型.比较了厦门海域真刺唇角水蚤与GenBank数据库中的角水蚤科(Pontellidae)6个种的mtDNA COI基因片段序列(索引号AY145428、AY145429、AB206443、AB206444、AB206445、AB206446),其同源性为87.34%.文中讨论了上述7种桡足类的系统发生关系.     

Flying insects: model systems in exercise physiology

Insect flight is the most energy-demanding exercise known. It requires very effective coupling of adenosine triphosphate (ATP) hydrolysis and regeneration in the working flight muscles.³¹P nuclear magnetic resonance (NMR) spectroscopy of locust flight muscle in vivo has shown that flight causes only a small decrease in the content of ATP, whereas the free concentrations of inorganic phosphate (P _i ), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were estimated to increase by about 3-, 5- and 27-fold, respectively. These metabolites are potent activators of glycogen phosphorylase and phosphofructokinase (PFK). Activation of glycolysis by AMP and P _i is reinforced synergistically by fructose 2,6-bisphosphate (F2,6P₂), a very potent activator of PFK. During prolonged flight locusts gradually change from using carbohydrate to lipids as their main fuel. This requires a decrease in glycolytic flux which is brought about, at least in part, by a marked decrease in the content of F2,6P₂ in flight muscle (by 80% within 15 min of flight). The synthesis of F2,6P₂ in flight muscle can be stimulated by the nervous system via the biogenic amine octopamine. Octopamine and F2,6P₂ seem to be part of a mechanism to control the rate of carbohydrate oxidation in flight muscle and thus function in the metabolic integration of insect flight.Dedicated to Dr. Ernst Zebe, Emeritus Professor of Zoology (University of Münster) on the occasion of his 70th birthday.     

A new selective insecticidal uncoupler of oxidative phosphorylation

Summary A new aryl hydrazone structure with high insecticidal activity against the Australian sheep blowfly,Lucilia cuprina, was shown to have a higher activity as an uncoupler of oxidative phosphorylation in insect compared to mammalian mitochondrial preparations. This compound possesses the requirements of other uncouplers in its measured pKa and lipid solubility. However, when compared to a closely related structure with similar physicochemical properties, its insecticidal and insect mitochondrial uncoupling activities are greater and it exhibits decreased mammalian toxicity corresponding to this differential biochemical selectivity.Acknowledgment. We thank Mr K. Rihs for preparation of the hydrazones and Prof. Dr K.H. Büchel for supply of hydrazone III.     

Advances in the study of helminth mitochondrial genomes and their associated applications

Helminths, including flatworms and roundworms, are abundant organisms that have a variety of life histories. Of these, the genera Schistosoma, Echinococcus, Trichinella are notable parasites of veterinary and medical importance, and cause substantial socio- economic losses throughout China and the rest of the world. Genetic markers in the mitochondrial (mt) genome have proven use- ful for systematic, ecological, evolutionary and population studies, and the growth of mt genomic research has increased in the last two decades. Technological improvements, such as the long-polymerase chain reaction method and high-throughput se- quencing have allowed minute amounts of DNA from single worms, biopsy samples or microscopic organisms to be used for whole mt genome characterization. To facilitate the retrieval, annotation and analyses of mitochondrial features, multiple data- bases and specific software have also been designed and established. This review focuses on current progress, applications and perspectives regarding helminth mt genomics. To date, the complete mt genomes for 93 species of helminths have been sequenced and analyzed. Analyses of the mt genes, including gene content, arrangement, composition and variation have revealed unique features among the helminths when compared with other metazoans. This provides important data concerning their functional and comparative mitochondrial genomics, molecular taxonomy and characterization, population genetics and systematics, and evolu- tionary history. Moreover, mt genome data for parasitic helminths are important for diagnosis, epidemiology and ecology of in- fections. Mitochondrial genome data offer a rich source of markers for the systematics and population genetics of socioeconomi- cally important parasitic helminths of humans and other animals.     

Synonymy of Rhynchophorus ferrugineus (Olivier), 1790 and R. vulneratus (Panzer), 1798 (Coleoptera,Curculionidae, Rhynchophorinae)

Morphological, molecular-genetic and breeding data were collected to investigate the species status of the Asian palm weevils, Rhynchophorus ferrugineus (Olivier) and R. vulneratus (Panzer) (Coleoptera: Curculionidae). These weevils are distinguished by characteristic colouring of the pronota and elytra, but naturally occurring colour intermorphs were observed. Contrary to the literature, quantitative measurements of the concavity of subgenal sutures and of pronotal shape indicated no differences between the two species. Larvae did not differ significantly in labral characteristics. Random amplified polymorphic DNA (RAPD) banding patterns were identical for nine of 14 primers, indicating that these weevils are very closely related. Sequences of the cytochrome oxidase gene for 201 base pairs read were identical for R. ferrugineus and R. vulneratus, but the congener R. bilineatus differed from them by 10%, suggesting divergence of these lineages about 5?million years ago. Hybrid F1s were obtained from all heterospecific crosses, and one surviving hybrid F1 female produced viable eggs. Previous studies have revealed no pheromonal differences. On the basis of this evidence, R. ferrugineus and R. vulneratus should be considered colour morphs of the same species and be synonymized under the name Rhynchophorus ferrugineus (Olivier), with the common name Asian palm weevil.