Construction of Double-Copy Glucose Isomerase Gene Engineering Strain of Streptomyces diastaticus by Homologous Recombination

The plasmid pUT for homologous recombination was constructed by the insertion of the 1.1-kb thiostrepton resistance (tsr ^R) gene into the E. coli plasmid pUB1-GI1. Plasmid pUTK was produced through ligating the cleaved plasmid pUT by KpnI. After pUT and pUTK were introduced into Streptomyces diastaticus No.7 strain M1033 (SM33) by protoplast transformation, a series of tsr^R transformants were obtained, further based on enzyme assays. These results for polymerase chain reaction (PCR), DNA sequencing, restriction enzyme digestion, and recovery of cloned fragments from the transformant chromosome demonstrated the plasmid pUT and pUTK had integrated into the SM33 chromosome in three different patterns of single cross-over by homologous recombination. This directly results in double-copy GI gene in the transformant chromosome, of which one is wild-type GI gene, the other mutant GI (GIG138P, GI1) gene. Among the strains of the three kinds of recombinant patterns, one transformant was chosen and named K1, T2, and T3, respectively. The further identification of the three recombinant strains by PCR, DNA sequencing, restriction enzyme digestion, and Southern hybridization also proved there is a double-copy GI gene within their chromosome. Enzyme activity assay and thermostability analysis indicated that all three engineering strains expressed not only wild-type enzyme but also mutant GI. Received: 9 July 2001 / Accepted: 8 August 2001     

秦岭羚牛高度重复序列DNA片段的分子克隆和序列分析

羚牛（Budorcas taxicolor)属偶蹄目（Artiodactyla)、牛科（Bovidae),为我国一类大型珍贵保护动物。我们从其基因组中克隆得到若干约800bp的BamHI高度重复序列并对部分克隆进行了序列测定,发现它们显示了很高的同源性。利用其中一个单元为探针,对限制酶消化后的羚牛基因组DNA作杂交分析,发现其杂交谱带不具有个体及亚种间特异性,说明该重复序列在羚牛基因组中具有保守的分布和排列。在牛科动物中,羚牛BamHI片段与绵羊属和山羊属的相关序列具有高度同源性,而与水牛和家牛序列差异较大。这些结果为羚牛与羊亚科物种亲源关系较近的分类学观点提供了分子生物学证据。有证据表明,这些片段可能代表羚牛染色体着丝点的卫星DNA单体。     

吗啡对培养海马神经元钙离子作用的机制研究

目的：研究吗啡对海马神经元[Ca^2 ]i影响的机制,为探索吗啡成瘾的神经生物学机制与可能的治疗途径。方法：荧光探针Fluo-4标记细胞内游离钙后,用激光共聚焦显微镜检测吗啡对大鼠原代培养海马神经元[Ca^2 ]i的影响。结果：吗啡急性刺激引起海马神经元[Ca^2 ]i升高,CTOP不能阻断吗啡引起的细胞内[Ca^2 ]i增加,而naltrindole能阻断吗啡引起的细胞内[Ca^2 ]i反应;Thapsigargin预处理阻断吗啡诱导的细胞内[Ca^2 ]i增加,Verapamil预处理不能完全抑制吗啡引起的细胞内[Ca^2 ]i增加;吗啡长时程作用后,海马神经元[Ca^2 ]i升高,加入纳络酮急性戒断后,不能阻断吗啡引起的细胞内[Ca^2 ]i升高,反而引起[Ca^2 ]i异常升高。结论：吗啡急性刺激引起的海马神经元内游离钙增加主要来源于δ2阿片受体介导的IP3敏感的钙库释放。     

Nogo—p4与NgR结合抑制神经干细胞分化为双极形星形胶质细胞的突起生长

目的观察Nogo—p4是否通过与NgR结合的途径对大鼠脊髓来源神经干细胞分化形成双极形星形胶质细胞突起长度产生抑制。方法取4只出生24h内的Wistar大鼠,悬浮培养法培养大鼠脊髓来源的神经干细胞。把神经干细胞分为A、B、C、D四组,A组加入血清,B组加入血清和Nogo—p4,C组神经干细胞经RNA干扰沉默NgR基因后加入血清分化,D组神经干细胞经RNA干扰沉默NgR基因后加入血清和Nogo—p4。分化第7d,GFAP抗体标记星形胶质细胞,使用Image—ProPlus5．0软件测量双极形星形胶质细胞突起长度。结果神经干细胞分化第7d,四组均可形成双极形星形胶质细胞。B组中双极形星形胶质细胞的突起长度明显短于其它各组。A、C、D组中双极形星形胶质细胞的突起长度没有显著差异。结论Nogo—p4经与NgR结合途径显著抑制脊髓来源神经干细胞分化成的双极形星形胶质细胞的突起生长。     

Mitochondrial permeability transition and cytochrome c release induced by selenite

Cytochrome c release and mitochondrial permeability transition (MPT) play important roles in apoptosis. In this study, we found that selenium, an essential trace element, induced mitochondrial membrane potential (Delta psi(m)) loss, swelling, and cytochrome c release in isolated mitochondria. All of the above observations were blocked by cyclosporin A (CsA), which is a specific inhibitor to permeability transition pore (PTP), indicating selenite-induced mitochondrial changes were mediated through the opening of PTP. In physiological concentration, selenite could induce mitochondria at low-conductance PTP 'open' probability, which is correlated to regulate the physiological function, whereas in toxic concentration, induce mitochondria at high-conductance PTP 'open' probability and rapidly undergo a process of osmotic swelling following diffusion toward matrix as for inducer (Ca(2+)/P(i)). Selenite also induced other mitochondrial marker enzymes including monoamine oxidase (MAO) and mitochondria aspartate aminotransferase (mAST). Oligomycin inhibited the selenite-induced cytochrome c release and Delta psi(m) loss, showing that F(0)F(1)-ATPase was important in selenite or Ca(2+)/P(i)-induced MPT.     

Group V phospholipase A2 induces leukotriene biosynthesis in human neutrophils through the activation of group IVA phospholipase A2

We reported previously that exogenously added human group V phospholipase A(2) (hVPLA(2)) could elicit leukotriene B(4) (LTB(4)) biosynthesis in human neutrophils (Han, S. K., Kim, K. P., Koduri, R., Bittova, L., Munoz, N. M., Leff, A. R., Wilton, D. C., Gelb, M. H., and Cho, W. (1999) J. Biol. Chem. 274, 11881-11888). To determine the mechanism of the hVPLA(2)-induced LTB(4) biosynthesis in neutrophils, we thoroughly examined the effects of hVPLA(2) and their lipid products on the activity of group IVA cytosolic PLA(2) (cPLA(2)) and LTB(4) biosynthesis under different conditions. As low as 1 nm exogenous hVPLA(2) was able to induce the release of arachidonic acid (AA) and LTB(4). Typically, AA and LTB(4) were released in two phases, which were synchronized with a rise in intracellular calcium concentration ([Ca(2+)](i)) near the perinuclear region and cPLA(2) phosphorylation. A cellular PLA(2) assay showed that hVPLA(2) acted primarily on the outer plasma membrane, liberating fatty acids and lysophosphatidylcholine (lyso-PC), whereas cPLA(2) acted on the perinuclear membrane. Lyso-PC and polyunsaturated fatty acids including AA activated cPLA(2) and 5-lipoxygenase by increasing [Ca(2+)](i) and inducing cPLA(2) phosphorylation, which then led to LTB(4) biosynthesis. The delayed phase was triggered by the binding of secreted LTB(4) to the cell surface LTB(4) receptor, which resulted in a rise in [Ca(2+)](i) and cPLA(2) phosphorylation through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2. These results indicate that a main role of exogenous hVPLA(2) in neutrophil activation and LTB(4) biosynthesis is to activate cPLA(2) and 5-lipoxygenase primarily by liberating from the outer plasma membrane lyso-PC that induces [Ca(2+)](i) increase and cPLA(2) phosphorylation and that hVPLA(2)-induced LTB(4) production is augmented by the positive feedback activation of cPLA(2) by LTB(4).     

Ginsennoside Rd Attenuates Cognitive Dysfunction in a Rat Model of Alzheimer’s Disease

Alzheimer??s disease is a neurodegenerative disease characterized by the production of ??-amyloid proteins and hyperphosphorylation of tau protein. Inflammation and apoptotic severity were highly correlated with earlier age at onset of Alzheimer??s disease and were also associated with cognitive decline. This study aims to examine whether the traditional Chinese medicine ginsennoside Rd could prevent cognitive deficit and take neuroprotective effects in ??-amyloid peptide 1?C40-induced rat model of Alzheimer??s disease. To produce Alzheimer??s disease animal model, aggregated ??-amyloid peptide 1?C40 injected into hippocampus bilaterally. Ginsennoside Rd protected their cognitive impairment and improved their memory function by daily intraperitoneal injection for 30?days consecutively. In addition, ginsennoside Rd alleviated the inflammation induced by ??-amyloid peptide 1?C40. Furthermore, ginsennoside Rd played a role in the down-regulation of caspase-3 proteins and reduced the apoptosis that normally followed ??-amyloid peptide 1?C40 injection. The results of this study showed that the pretreatment of ginsennoside Rd had neuroprotective effects in ??-amyloid peptide 1?C40-induced AD model rat.     

Symposium 2: The Genetic,Molecular and Cellular Bases of Neurofibromatosis Type 1. A role of NF1 as a tumor suppressor

To gain insight into the role of the NF1 (Neurofibromatosis type 1) gene during neural development and in tumorigenesis, we have utilized the bacteriophage P1, Cre/loxP system to generate a conditional allele at the NF1 locus (NF1 flox) that permits temporal and spatial ablation of function through Cre‐mediated recombination. We have been using these mice to assess the scope of NF1 requirement in distinct cell types. At the center of this approach is to identify the cells that give origin to the tumors most frequently found in NF1 patients: neurofibromas, neurofibrosarcomas, and astrocytomas. We have hypothesized that specific stem cells must lose NF1 by LOH to begin this process. I will discuss the consequences of NF1 loss in neurons, Schwann cells, and neural precursors. Distinct tumor phenotypes appear in each case. In malignant tumors, our mouse models indicate that the p53 pathway must also become mutated to cooperate with loss of NF1. Additionally, we have genetic evidence that the haploin‐sufficient state is essential for tumor appearance. These data suggest that profilactic therapies preceding tumor appearance should be considered for NF1. Acknowledgements: Funded by NINDS, NNFF, and DOD.     

Genetic relationships among twelve Chinese indigenous goat populations based on microsatellite analysis

Twelve Chinese indigenous goat populations were genotyped for twenty-six microsatellite markers recommended by the EU Sheep and Goat Biodiversity Project. A total of 452 goats were tested. Seventeen of the 26 microsatellite markers used in this analysis had four or more alleles. The mean expected heterozygosity and the mean observed heterozygosity for the population varied from 0.611 to 0.784 and 0.602 to 0.783 respectively. The mean F_ST (0.105) demonstrated that about 89.5% of the total genetic variation was due to the genetic differentiation within each population. A phylogenetic tree based on the Nei (1978) standard genetic distance displayed a remarkable degree of consistency with their different geographical origins and their presumed migration throughout China. The correspondence analysis did not only distinguish population groups, but also confirmed the above results, classifying the important populations contributing to diversity. Additionally, some specific alleles were shown to be important in the construction of the population structure. The study analyzed the recent origins of these populations and contributed to the knowledge and genetic characterization of Chinese indigenous goat populations. In addition, the seventeen microsatellites recommended by the EU Sheep and Goat Biodiversity Project proved to be useful for the biodiversity studies in goat breeds.     

鱼类远缘杂交正反交杂种胚胎发育差异的细胞遗传学分析

本文报道了鲤(Cyprinus carpio)×鲢(Hypophthalmichthys molitrix)、鲫(Carassiusauratus)×鲢、白鲫(Carassius auratus cuvieri)×鲢和鲢×鲤、鲢×鲫、鲢×白鲫的正反交试验。在鲤×鲢、鲫×鲢和白鲫×鲢3个正交组中,胚胎发育基本正常,尽管孵出的鱼苗绝大多数生命力弱,但孵化率都在50％左右;而在鲢×鲤、鲢×鲫和鲢×白鲫3个反交组中,胚胎发育均为畸形,不能孵化出苗。 胚胎发育细胞遗传学分析表明,鲤×鲢、鲫×鲢和白鲫×鲢的杂种胚胎几乎都是整倍体,而鲢×鲤、鲢×鲫×鲢×白鲫的杂种胚胎基本上是非整倍体,染色体数变化较大。这些正反交杂种胚胎发育的显著差异可能与其亲本物种间的基因组大小有关。文中还分析讨论了这些正反交差异与天然多倍体物种以及胚胎发育速度的相关性,认为天然多倍体物种可能具有一些不同于普通二倍体物种协调外源基因组的能力。