微囊藻毒素合成酶基因的PCR检测方法

针对微囊藻毒素合成酶基因簇的核酸序列,筛选特异性引物,探索一种适用于自然水样中微囊藻产毒潜能检测的全细胞PCR方法。经灵敏度测试表明,这种PCR方法的检测下限相当于100cells。该方法不需要提取基因组DNA,检测所需水样量少,具有操作简便、快速、成本低、灵敏度高等优点,能应用于水库等饮用水源水体中具有产毒潜能的微囊藻的检测。     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

It has been demonstrated that maternal drinking during pregnancy had serious adverse effects on the health of the newborns. Fetal alcohol syndrome (FAS) is the most important developmental abnormality caused by maternal alcohol abuse during pregnancy. Clinically, it is characterized by head and facial ab-normalities, cardiovascular malformation, and perma-nent nervous system damage[1,2]. A lot of experimental models have been developed to study the ethanol’s effects on embryonic development,…     

Purification and characterization of UDPG:ginsenoside Rd glucosyltransferase from suspended cells of Panax notoginseng

Heterogeneity of ginsenosides is an interesting and important issue because those structure-similar secondary metabolites have different or even totally opposite pharmacological activities. In this work, a new enzyme UDP-glucose:ginsenoside Rd glucosyltransferase (UGRdGT), which catalyzes the formation of ginsenoside Rb₁ from ginsenoside Rd [Biotechnol. Bioeng. 89: 444–52, 2005], was purified approximately 145-fold from suspended cells of Panax notoginseng with an overall yield of 0.2%. Purification to apparent homogeneity, as judged by SDS-PAGE, was successfully achieved by using sequential ammonium sulphate precipitation, anion-exchange chromatography and native PAGE. The enzyme had a molecular mass of 36 kDa, and its activity was optimal at pH 8.5 and 35 °C. The enzyme activity was enhanced by Mn²⁺, Ca²⁺ and Mg²⁺, but strongly inhibited by Zn²⁺, Hg²⁺, Co²⁺, Fe²⁺ and Cu²⁺. The apparent K_m value for UDP-glucose and ginsenoside Rd was 0.32 and 0.14 mM, respectively. The biotransformation yield from ginsenoside Rd to Rb₁ by UGRdGT in 50 mM Tris–HCl buffer at pH 8.5 and 35 °C was over 80%. This work provides a basis for further molecular study on the ginsenoside Rb₁ biosynthesis by P. notoginseng cells and it is also useful for potential application to in vitro biotransformation from ginsenoside Rd to Rb₁.     

鲤鱼中Sox9b基因的克隆和表达

Sox9基因是一个重要的转录调控因子,参与性别决定及软骨等多种组织和器官的发育过程。本研究利用简并引物扩增鲤鱼基因组DNA,首次发现在鲤鱼中存在两种形式的Sox9基因。二者在保守盒区编码的氨基酸序列相同,并都存在一个内含子,但内含子序列差异很大,分别长704bp和616bp。在此基础上采用RACE技术克隆了鲤鱼Sox9b基因的5’端和3’端,通过拼接获得了2447bp 的全长cDNA序列,编码428个氨基酸。其中96-174位共79个氨基酸为HMG保守盒。将鲤鱼Sox9b基因与三刺鱼等九种动物的氨基酸序列相比较发现,它们的同源性高达75%以上,显示Sox9 基因在进化中较保守。应用半定量RT-PCR技术对成体鲤鱼不同组织中Sox9b基因的表达进行了分析,结果表明该基因广泛表达,尤以脑及精巢中表达最为丰富。     

脊椎动物线粒体DNA的基因重排

将GenBank上已公布的321种脊椎动物mtDNA全序列,按纲整理归类,绘制基因排布图并进行比对。比对结果表明：81个物种的mtDNA中观察到基因重排现象,涉及脊椎动物各纲,其中9个物种同时存在基因顺序变化和基因倒置现象,所有的基因重排都涉及tRNA的变化。脊椎动物mtDNA基因顺序变化可分为3类：1)邻接的基因或片段的位置交换;2)接近于控制序列或轻链起始位点的基因或片段的位置变化,有时还伴随着控制序列的倍增;3)I-Q-M区域的变化。所有鸟类、蛇类、鳄类和有袋类的mtDNA具有各自独特的基因排列顺序。基因倒置现象常见于鱼类和哺乳类,且多表现为tRNA从轻链往重链上迁移。本文就这些基因重排现象、发生重排的机制和mtDNA基因重排在系统发生研究中的应用做一简要概述。     

犊牛前脂肪细胞的原代培养

为了建立犊牛前脂肪细胞原代培养模式,以便深入地研究奶牛脂肪组织增生的生物学特征。选用犊牛小肠网膜,采用原代消化细胞培养法培养出梭形细胞;同时以皮肤组织的成纤维细胞培养作为对照。结果显示：培养出的梭形细胞成分均一,增殖旺盛,分化率高。经形态学动态变化的观察,生长曲线及油红O脂肪染色抽取法测定,证明是功能活跃的前脂肪细胞,并在体外重现了其增殖的全过程。因此,在犊牛小肠网膜中存在着可分化成熟的、生成脂肪的前脂肪细胞。为进一步研究与肥胖、胰岛素抵抗相关的疾病如奶牛酮病、脂肪肝等打下了基础。     

Protein nitration increased by simulated weightlessness and decreased by melatonin and quercetin in PC12 cells.

A variety of experiments suggest that space flight is associated with an increase in oxidative stress in organism. To explore the effects of oxidative stress on neuronal cells during microgravity, we used rat pheochromocytoma (PC12) cells as a neuronal cell model, cultured in a clinostat, which could simulate microgravity, to investigate the effects of reactive nitrogen species on protein nitration in PC12 cells during clinorotation. The effects of melatonin and quercetin on protein nitration in PC12 cells were also assayed to evaluate the possible protective role of melatonin or quercetin as an antioxidant. The results of immunological staining showed that after the 3 days' clinorotation the protein expressions of neuronal nitric oxide synthase and inducible nitric oxide synthesis were up-regulated. Our data also reflected that the concentrations of nitric oxide and nitrotyrosine were significantly increased after clinorotation, and they were reduced markedly in cells that were treated with 50 micromol/L melatonin or 0.5 micromol/L quercetin during simulated microgravity, when compared to those of control cells. These results suggest that clinorotation-induced weightlessness increases oxidative stress responses in PC12 cells, and melatonin or quercetin was shown to protect PC12 cells from oxidative damage during simulated weightlessness.     

Deferoxamine inhibits iron induced hippocampal tau phosphorylation in the Alzheimer transgenic mouse brain

Prior work has shown that iron interacts with hyperphosphorylated tau, which contributes to the formation of neurofibrillary tangles (NFTs) in Alzheimer’s disease (AD), whereas iron chelator desferrioxamine (DFO) slows down the clinical progression of the cognitive decline associated with this disease. However, the effects of DFO on tau phosphorylation in the presence or absence of iron have yet to be determined. Using amyloid precursor protein (APP) and presenilin 1 (PS1) double transgenic mouse brain as a model system, we investigated the effects and potential mechanisms of intranasal administration of DFO on iron induced abnormal tau phosphorylation. High-dose iron treatment markedly increased the levels of tau phosphorylation at the sites of Thr205, Thr231 and Ser396, whereas highly induced tau phosphorylation was abolished by intranasal administration of DFO in APP/PS1 transgenic mice. Moreover, DFO intranasal administration also decreases Fe-induced the activities of cyclin-dependent kinase 5 (CDK5) and glycogen synthase kinase 3β (GSK3β), which in turn suppressing tau phosphorylation. Cumulatively, our data show that intranasal DFO treatment exerts its suppressive effects on iron induced tau phosphorylation via CDK5 and GSK3β pathways. More importantly, elucidation of DFO mechanism in suppressing tau phosphorylation may provide insights for developing therapeutic strategies to combat AD.