Gene therapy for mitochondrial disease by delivering restriction endonucleaseSmaI into mitochondria

The restriction endonucleaseSmaI has been used for the diagnosis of neurogenic muscle weakness, ataxia and retinitis pigmentosa disease or Leigh's disease, caused by the Mt8993TG mutation which results in a Leu156Arg replacement that blocks proton translocation activity of subunit a of F₀F₁-ATPase. Our ultimate goal is to applySmaI to gene therapy for this disease, because the mutant mitochondrial DNA (mtDNA) coexists with the wild-type mtDNA (heteroplasmy), and because only the mutant mtDNA, but not the wild-type mtDNA, is selectively restricted by the enzyme. For this purpose, we transiently expressed theSmaI gene fused to a mitochondrial targeting sequence in cybrids carrying the mutant mtDNA. Here, we demonstrate that mitochondria targeted by theSmaI enzyme showed specific elimination of the mutant mtDNA. This elimination was followed with repopulation by the wild-type mtDNA, resulting in restoration of both the normal intracellular ATP level and normal mitochondrial membrane potential. Furthermore, in vivo electroporation of the plasmids expressing mitochondrion-targetedEcoRI induced a decrease in cytochromec oxidase activity in hamster skeletal muscles while causing no degenerative changes in nuclei. Delivery of restriction enzymes into mitochondria is a novel strategy for gene therapy of a special form of mitochondrial diseases.     

Waking Action of Ursodeoxycholic Acid (UDCA) Involves Histamine and GABA(A) Receptor Block

Since ancient times ursodeoxycholic acid (UDCA), a constituent of bile, is used against gallstone formation and cholestasis. A neuroprotective action of UDCA was demonstrated recently in models of Alzheimer''s disease and retinal degeneration. The mechanisms of UDCA action in the nervous system are poorly understood. We show now that UDCA promotes wakefulness during the active period of the day, lacking this activity in histamine-deficient mice. In cultured hypothalamic neurons UDCA did not affect firing rate but synchronized the firing, an effect abolished by the GABA_AR antagonist gabazine. In histaminergic neurons recorded in slices UDCA reduced amplitude and duration of spontaneous and evoked IPSCs. In acutely isolated histaminergic neurons UDCA inhibited GABA-evoked currents and sIPSCs starting at 10 µM (IC₅₀ = 70 µM) and did not affect NMDA- and AMPA-receptor mediated currents at 100 µM. Recombinant GABA_A receptors composed of α1, β1–3 and γ2L subunits expressed in HEK293 cells displayed a sensitivity to UDCA similar to that of native GABA_A receptors. The mutation α1V256S, known to reduce the inhibitory action of pregnenolone sulphate, reduced the potency of UDCA. The mutation α1Q241L, which abolishes GABA_AR potentiation by several neurosteroids, had no effect on GABA_AR inhibition by UDCA. In conclusion, UDCA enhances alertness through disinhibition, at least partially of the histaminergic system via GABA_A receptors.     

Biochemical Properties and Inhibition Kinetics of Phosphatase from Wheat Thylakoid Membranes

A phosphetase that hydrolyses phosphate monoesters has been Isolated from wheat thylakold membranes. Biochemical properties and inhibition kinetics of the phosphatase were Investigated using several Ions, organlc solvents, and Inhlbltors. Wheat （Trltlcum aestivum L. cv. PH82-2-2） thylakold membrane phosphatase activity was activated by Mg^2＋, Ca^2＋, and Fe^2＋ and was inhibited by Mn^2＋ and Cu^2＋. For example, enzyme activity was acUvated 34.81% by 2 mmol/l. Mg^2＋, but was Inhibited 22.3% and 8.5% by 2 and 1 mmol/L Cu^2＋, respectively. Methanol, ethanol and glycol were all able to activate enzyme activity. Enzyme activity was activated 58.5%, 48.2%, and 8.7% by 40% ethanol, methanol and glycol, respectively. From these results, It can be seen that the degree of actlvetlon of the phosphetase was greatest for ethanol and the type of acUvatlon was uncompetltlve. Moreover, the activity of the thylakold membrane phosphetase was Inhibited by molybdate, vanadete, phosphate, and fluoride and the type of Inhibition produced by these elements was uncompetltlve, non-competitive, competltlve and mixed, respectively.     

基于基因型选择提高QTL作图的精度——以一个RIL群体为例

以PCR为基础的分子标记以及其他检测技术的发展,使得大规模的标记分析成为现实。这也为通过大群体标记分析,然后基于基因型选择挑选合适的小群体,从而提高QTL定位准确性和精度提供了可能。以一个包含294个家系的重组自交系（RIL）群体为例,通过基因型选择和随机选择的办法产生了一系列大小不等的亚群体,比较了两类群体QTL定位的结果。分析表明：相同大小的基因型选择群体与随机群体相比性状的表型分布都符合正态分布;标记的偏分离情况也没有明显的差别,都随着群体大小的增大,偏分离的比例也逐渐增大。但同等大小的基因型选择群体比随机群体的交换富集率（CE）要大,且随着选择强度的增大不断增大,如群体大小为270时,CE=1．04,群体大小为30时,CE=1．45。总体上,随着群体大小的增加,不管是随机群体还是选择群体,其QTL检测能力、灵敏性和特异性也随之增加,但选择群体的检测能力、灵敏性和特异性总体上要好于随机群体。当群体大于或等于240时,其QTL检测能力基本没有差别;群体大小大于或等于210时,其QTL检测的灵敏性和特异性也没有什么差别。这也说明：选择强度越大,效果越明显。以QTLI—LOD区间作为衡量QTL精度的一个指标,结果显示所有基因型选择群体都比相同大小随机群体的QTL定位精度高。目前QTL定位研究中,基因型数据较表型数据而言更容易准确获得,因此通过基因型选择可以更好的优化群体结构,减少田间实验的工作量,提高全基因组水平QTL作图的精度,为随后的QTL辅助选择和精细定位以及克隆提供帮助。     

斑头大翠鸟在我国的分布

斑头大翠鸟(Alcedo hercules)在我国的分布以往仅知见于西藏东南部、云南南部和海南岛的局部地区,自20世纪80年代后期以来,于广东北部(车八岭)和江西西南部(九连山)相继出现采集记录和连续的目击记录,本世纪初又有报道该种见于广西东北部(花坪)、中南部(十万大山)和西南部的中越边境地带,并于近期发现其分布远及福建西北部的邵武。据此,可以认为斑头大翠鸟在我国南方相当一些地区也应有分布。     

玉米油脂代谢相关基因的全基因组挖掘、表达谱及与籽粒油份QTL共定位分析

油脂是植物中广泛存在且参与众多生物学过程的一类物质,具有重要的生物学功能。本研究从拟南芥和水稻油脂代谢基因出发,基于序列相似和功能域相同原则,克隆并注释了1003个玉米油脂代谢相关基因,其中,774个从GenBank中直接注释,229个为新克隆基因。电子表达谱分析发现97个基因在玉米籽粒、根、叶或分生组织中特异高表达。整合已发表籽粒油分相关QTL,在玉米基因组中发现70个控制籽粒油份的QTL富集区（分布于34％的基因组区域）,其中59个QTL富集区内存在油脂代谢候选基因147个。在玉米籽粒中特异高表达的13个脂肪酸合成代谢基因都落在QTL富集区。本研究挖掘的油脂代谢相关基因信息,电子表达谱数据及整合的油份QTL结果,为玉米油份QTL的进一步精细定位和克隆、玉米籽粒油份形成的分子生物学基础研究提供了有益信息。     

Nucleotide diversity and molecular evolution of the PSY1 gene in Zea mays compared to some other grass species

Phytoene synthase (PSY), which is encoded by the phytoene synthase 1 (PSY1) gene, is the first rate-limiting enzyme in the plant carotenoid biosynthetic pathway. In order to examine the genetic diversity and evolution pattern of PSY1 within the Andropogoneae, sequences of 76 accessions from 5 species (maize, teosinte, tripsacum, coix, and sorghum) of the Andropogoneae were tested, along with 4 accessions of rice (Oryza sativa L.) included as outliers. Both the number and the order of exons and introns were relatively conserved across the species tested. Three domains were identified in the coding sequence, including signal peptide (SP), PSY, and highly conserved squalene synthase (SQS) domain. Although no positive selection signal was detected at an overall coding level among all species tested, the SP domain and the region upstream of the SQS–PSY domain appear to have undergone rapid evolution, as evidenced by a high d _N/d _S ratio (>1.0). At the nucleotide level, positive selection and balancing selection were detected only among the yellow maize germplasm and the white maize germplasm, respectively. The phylogenetic tree based on full-length sequences of PSY1-like regions supported the monophyletic theory of the Andropogoneae and the closest relationship between Zea and Tripsacum among the Andropogoneae. Coix, which was theorized to have a closer relationship with maize due to similarities in morphology and chromosome number, has been shown in this study to have diverged relatively early from the other Andropogoneae, including maize.     

玉米淀粉生物合成及其遗传操纵

淀粉是许多植物重要的储藏物质。淀粉突变体以及转基因植物中淀粉变异的特点使我们对淀粉生物合成的过程有了较深入的了解,许多研究的结果揭示了玉米淀粉的生物合成涉及4类酶--ADPG焦磷酸化酶、淀粉合成酶、淀粉分支酶和去分支酶。随着编码这些酶的基因的克隆,利用转基因技术对淀粉合成过程进行遗传操纵业已成为可能,并且在提高淀粉产量以及不同特性淀粉品质的种质资源创新等方面展示出巨大的潜力。 Abstract:Starch is the most important source of calories and a vital storage component in plants.The characterization and production of starch variants from mutation and with transgenic technology has improved our understanding of the synthesis of starch granule.In starch biosynthesis in plants,four enzymes,including ADP-glucose pyrophosphorylase,starch synthase,starch branching enzyme and starch debranching enzyme,are widely accepted from an enormous amount of research aimed primarily at enzyme characterization.As many genes encoding the enzymes and their multiple isoforms in starch biosynthesis pathway have been isolated,genetic manipulation of the starch biosynthesis pathway shows to be a practical way by which starch quantity is increased and starch with novel properties can be created.     

刺槐掌舟蛾的生物学特性及防治

刺槐掌舟蛾是为害刺槐的重要食叶害虫。在山东荣成市１年发生１代,以蛹在土中越冬。每年６～９月为害,各虫态极不整齐。本文记述了该虫的生活年史、发育历期、生活习性、发生与环境的关系及天敌情况,提出了防治建议。     

Histamine Receptor Expression, Hippocampal Plasticity and Ammonia in Histidine Decarboxylase Knockout Mice

Genetic ablation of the histamine producing enzyme histidine decarboxylase (HDC) leads to alteration in exploratory behaviour and hippocampus-dependent learning. We investigated how brain histamine deficiency in HDC knockout mice (HDC KO) affects hippocampal excitability, synaptic plasticity, and the expression of histamine receptors. No significant alterations in: basal synaptic transmission, long-term potentiation (LTP) in the Schaffer collateral synapses, histamine-induced transient changes in the CA1 pyramidal cell excitability, and the expression of H1 and H2 receptor mRNAs were found in hippocampal slices from HDC KO mice. However, when compared to WT mice, HDC KO mice demonstrated: 1. a stronger enhancement of LTP by histamine, 2. a stronger impairment of LTP by ammonia, 3. no long-lasting potentiation of population spikes by histamine, 4. a decreased expression of H3 receptor mRNA, and 5. less potentiation of population spikes by H3 receptor agonism. Parallel measurements in the hypothalamic tuberomamillary nucleus, the origin of neuronal histamine, demonstrated an increased expression of H3 receptors in HDC KO mice without any changes in the spontaneous firing of “histaminergic” neurons without histamine and their responses to the H3 receptor agonist (R)-α-methylhistamine. We conclude that the absence of neuronal histamine results in subtle changes in hippocampal synaptic transmission and plasticity associated with alteration in the expression of H3 receptors.