Spatial-temporal expression of pum1 and pum2 in medaka Oryzias latipes

Two pumilios, pum1 and pum2, were identified in medaka Oryzias latipes. Oryzias latipes pum1 and pum2 are ubiquitous in the adult tissues but with specific expression in the germ cells of gonads, ovary and testis. Pum1 is expressed in the spermatogonia to spermatocytes whilst pum2 presents in spermatocytes of testis only. Oryzias latipes pum1 and pum2 are maternally supplied RNA with ubiquitous expression in the early stages, and embryonic expression of pum1 and pum2 may begin from early gastrula. Both pum1 and pum2 are expressed in the tissues including brain, eye and trunk, and both are expressed in the gonads after hatching. Taken together, Pum1 and Pum2 may play important roles in embryonic and germ cell development of O. latipes.     

Effects of Fragmentation of Evergreen Broad-leaved Forests on Genetic Diversity of Ardisia Crenata var. Bicolor (Myrsinaceae)

Due to the long generation times and high densities, dominant tree species usually did not respond consistently with theoretical predictions to the recent fragmentation. Genetic structures of shrubs and herbs, especially those with low densities, may be more sensitive to forest fragmentation. We studied the genetic structure of a self-compatible subshrub, Ardisia crenata var. bicolor (Myrsinaceae) in a recently fragmented landscape. Ten RAPD primers used for analysis generated a total of 76 bands. We found that A. c. var. bicolor had relatively low species-level (P₉₅ = 63.2%; H = 0.106; Shannon diversity index (SI) = 0.246) and within-population diversity (P₉₅ = 5.3−46.1%; H = 0.026−0.175; SI = 0.032−0.253), and significant population differentiation (G_ST = 0.445). Significantly positive relationships were found between measures of diversity (P₉₅, H and SI) and the log of estimated population size. No significant relationship was observed between Nei's genetic distance and spatial distance of pairwise populations, indicating no isolation-by-distance. Given most species of forests are shrubs and herbs with short generation times, our observation indicated that distinct genetic consequences of recent fragmentation may be expected for quite a number of plant species.     

转基因植物中标记基因的安全性新策略

转基因植物中的除草剂和抗生素抗性标记基因潜在的生态环境和食用安全性令人担忧。解决转基因植物中抗性标记基因安全性问题有两种途径：一是转化时仍使用抗性标记基因,转基因植物再生成功后,在释放大田前将标记基因剔除;二是发展安全性标记基因用于植物遗传转化。本文综述了三种标记基因剔除系统和几种安全性标记基因在植物转化中的应用进展。     

小鼠泛素结合酶UBE2W的抗体制备及组织表达谱分析

泛素结合酶(E2)是蛋白泛素化修饰所需的第二个连接酶, 在泛素转移和底物特异性识别过程中发挥着重要作用。UBE2W是一种新发现的E2酶, 其果蝇属同源蛋白可能在光转导或视网膜变性过程中发挥作用, 鼠和人同源蛋白功能未见报道。生物信息学分析UBE2W鼠源氨基酸序列, 发现UBE2W具有典型的UBC结构域并在多种物种高度保守。通过构建UBE2W原核表达质粒, 在大肠杆菌中表达并纯化了GST-UBE2W融合蛋白。以此纯化蛋白作为抗原免疫新西兰白兔制备抗UBE2W多抗血清, 并利用制备的UBE2W抗原柱亲和纯化UBE2W多抗。为了检测纯化抗体特异性, 在真核细胞中瞬时表达了myc-UBE2W融合蛋白, 分别用myc单抗和UBE2W多抗进行Western blotting分析, 结果表明获得了特异性的UBE2W抗体。利用此特异性抗体在小鼠脑、心脏、肾脏、肝脏、肺、肌肉、脾脏和睾丸等组织中均检测到了UBE2W的表达, 且在小鼠睾丸中成年期表达最高。     

氯化钠密度梯度离心法制备用于显微注射的外源DNA片段

DNA显微注射是生产转基因动物最可靠和最常使用的一种方法,外源DNA的纯度对显微注射的成功起着至关重要的作用。本文介绍用氯化钠密度梯度离心的方法制备用于显微注射的外源DNA片段。与传统的琼脂糖凝胶回收的方法相比较,用此方法制备的外源DNA片段对小鼠受精卵进行显微注射后,受卵体母鼠的胚胎存活率,以及子代小鼠的外源基因整合率均有明显的提高。这一方法可为进一步提高转基因动物的成功率,提供方法学上的参考。 Abstract:DNA microinjection is the most popular and reliable method of producing transgenic animals.The purity of foreign DNA plays an important role for the success of microinjection.In this study,we introduced the use of sodium chloride step gradients in fractionating foreign DNA fragment for microinjection.The data demonstrated that,compared with the conventional agarose gel extraction method,NaCl purification scheme of toreign DNA could improve the treated embryo survival and foreign DNA intergration rate markedly.     

Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers

Human embryonic stem (hES) cells are typically maintained on mouse embryonic fibroblast (MEF) feeders or with MEF-conditioned medium. However, these xenosupport systems greatly limit the therapeutic applications of hES cells because of the risk of cross-transfer of animal pathogens. Here we showed that the bone morphogenetic protein antagonist noggin is critical in preventing differentiation of hES cells in culture. Furthermore, we found that the combination of noggin and basic fibroblast growth factor (bFGF) was sufficient to maintain the prolonged growth of hES cells while retaining all hES cell features. Since both noggin and bFGF are expressed in MEF, our findings suggest that they may be important factors secreted by MEF for maintaining undifferentiated pluripotent hES cells. Our data provide new insight into the mechanism how hES cell self-renewal is regulated. The newly developed feeder-free culture system will provide a more reliable alternative for future therapeutic applications of hES cells.     

免疫刺激复合物疫苗制备及其对小鼠免疫功能的影响

确定了免疫刺激复合物(ISCOM)疫苗的安全有效剂量及其对小鼠免疫功能的影响。选取体重25 g左右的昆明小白鼠60只,分为12组,每组5只,腹腔分别注射不同剂量(5-200μg)的ISCOM疫苗,观察小鼠健康状态。选取体重28 g左右的昆明小白鼠60只,分为4组,分别在腹腔注射相同剂量的灭菌生理盐水,Lipase+生理盐水,空ISCOM,Lipase+ISCOM疫苗,利用间接ELISA检测血清中特异性抗体效价。结果表明,当注射剂量为5-25μg/只时,小鼠无任何异常症状。免疫后8 d,小鼠血清特异性抗体效价(log2)可以达到10.5,显著高于对照组。因此,ISCOM疫苗能有效引起小鼠的免疫反应。     

Enhancement of hyperthermia-induced apoptosis by local anesthetics on human histiocytic lymphoma U937 cells

The combined effects of hyperthermia at 44 degrees C and local anesthetics on apoptosis in human histiocytic lymphoma U937 cells were investigated. When the cells were exposed to hyperthermia for l0 min marginal DNA fragmentation and nuclear fragmentation were observed. In the presence of amide-type local anesthetics further enhancement was found depending on concentration. The order of the concentration required for maximum induction was the reverse order of the lipophilicity (prilocaine > lidocaine > bupivacaine). Western blotting revealed that in hyperthermia there was initial release of Ca(2+) from the intracellular store site as indicated by increased expression of the type 1 inositol-1,4,5-trisphosphate receptor. However, the combination with lidocaine did not induce any further enhancement. Lidocaine enhanced the decrease in ATP content and the increase in intracellular Ca(2+) concentration in individual cells induced by hyperthermia. In addition, superoxide formation, decrease in the mitochondrial membrane potential, and activation of intracellular caspase-3 were found in the cells treated with hyperthermia and lidocaine. All of these were suppressed in part in the presence of the intracellular Ca(2+) ion chelator BAPTA-AM (bis-(O-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl). The present results indicate that local anesthetics at optimal concentrations enhance hyperthermia-induced apoptosis via Ca(2+)- and mitochondria-dependent pathways. Initial release of Ca(2+) from intracellular store sites caused by hyperthermia and followed by the subsequent increase in the intracellular Ca(2+) concentration and the additional activation of the mitochondrial caspase-dependent pathway (partly regulated by intracellular Ca(2+) concentration) plays a crucial role in the enhancement of apoptosis induced by the combination of hyperthermia and lidocaine.