链球菌蛋白G的构建、重组表达及鉴定

化学合成链球茵蛋白G的C3D基因片段,通过分子生物学的方法对蛋白G（proteinG）的C3片段进行PCR扩增,拼接形成含有两个和三个重复C3片段的重组链球茵蛋白G,C3片段间以链接区D连接,即形成C3DC3和C3DC3DC3的形式,进而克隆到质粒pET21中,在大肠杆菌BL21（DE3）中表达。重组表达的蛋白经过DEAE—Sepharose和IgG—Sepharose纯化,得到纯化的重组蛋白。采用非竞争性酶免疫法对重组蛋白与不同来源IgG的结合常数进行测定,实验结果显示两种重组链球茵蛋白G均可有效地与小鼠、兔及山羊等多种不同来源抗体特异性结合。这些实验结果为下一步研究奠定了基础。     

Erythropoietin attenuates the sequels of ischaemic spinal cord injury with enhanced recruitment of CD34+ cells in mice

Erythropoietin has been shown to promote tissue regeneration after ischaemic injury in various organs. Here, we investigated whether Erythropoietin could ameliorate ischaemic spinal cord injury in the mouse and sought an underlying mechanism. Spinal cord ischaemia was developed by cross-clamping the descending thoracic aorta for 7 or 9 min. in mice. Erythropoietin (5000 IU/kg) or saline was administrated 30 min. before aortic cross-clamping. Neurological function was assessed using the paralysis score for 7 days after the operation. Spinal cords were histologically evaluated 2 and 7 days after the operation. Immunohistochemistry was used to detect CD34(+) cells and the expression of brain-derived neurotrophic factor and vascular endothelial growth factor. Each mouse exhibited either mildly impaired function or complete paralysis at day 2. Erythropoietin-treated mice with complete paralysis demonstrated significant improvement of neurological function between day 2 and 7, compared to saline-treated mice with complete paralysis. Motor neurons in erythropoietin-treated mice were more preserved at day 7 than those in saline-treated mice with complete paralysis. CD34(+) cells in the lumbar spinal cord of erythropoietin-treated mice were more abundant at day 2 than those of saline-treated mice. Brain-derived neurotrophic factor and vascular endothelial growth factor were markedly expressed in lumbar spinal cords in erythropoietin-treated mice at day 7. Erythropoietin demonstrated neuroprotective effects in the ischaemic spinal cord, improving neurological function and attenuating motor neuron loss. These effects may have been mediated by recruited CD34(+) cells, and enhanced expression of brain-derived neurotrophic factor and vascular endothelial growth factor.     

Cannabinoid Receptor 1 Signaling in Embryo Neurodevelopment

In utero exposure to tetrahydrocannabinol, the psychoactive component of marijuana, is associated with an increased risk for neurodevelopmental defects in the offspring by interfering with the functioning of the endocannabinoid (eCB) system. At the present time, it is not clearly known whether the eCB system is present before neurogenesis. Using an array of biochemical techniques, we analyzed the levels of CB1 receptors, eCBs (AEA and 2‐AG), and the enzymes (NAPE‐PLD, DAGLα, DAGLβ, MAGL, and FAAH) involved in the metabolism of the eCBs in chick and mouse models during development. The findings demonstrate the presence of eCB system in early embryo before neurogenesis. The eCB system might play a critical role in early embryogenesis and there might be adverse developmental consequences of in utero exposure to marijuana and other drugs of abuse during this period.     

Mammalian PIK3C3/VPS34: the key to autophagic processing in liver and heart

PIK3C3/Vps34 is the class III PtdIns3K that is evolutionarily conserved from yeast to mammals. Its central role in mammalian autophagy has been suggested through the use of pharmacological inhibitors and the study of its binding partners. However, the precise role of PIK3C3 in mammals is not clear. Using mouse strains that allow tissue-specific deletion of PIK3C3, we have described an essential role of PIK3C3 in regulating autophagy, and liver and heart function.     

与细胞周期G2/M期进程相关的H2AX磷酸化

γH2AX焦点(foci)被普遍当做DNA双链断裂（DSB）损伤的分子标志物.为探 讨细胞周期进程相关的H2AX磷酸化规律特征,采用胸腺嘧啶双阻滞结合噻氨酯哒唑(nocodazole)的后续处理,将HeLa细胞同步于有丝分裂的前中期．然后,用流式细胞仪检测细胞周期、Western印迹和免疫荧光法,观察γH2AX表达和γH2AX焦点的形成．结果显示,细胞进入G2/M期和有丝分裂过程中,γH2AX水平显著增加 ;在无DNA DSB发生的情况下,部分M期细胞中也存在大量的γH2AX焦点．随着细 胞完成有丝分裂从M期退出再进入G1期,γH2AX的表达水平逐渐降低．这种 γH2AX表达变化特征与G2/M期密切关联的PLK1和Cyclin B1的表达规律相类似． 在4 Gy大剂量照射下,HeLa细胞于照后8 到12 h出现明显的G2/M期阻滞．γH2AX 焦点数在照后1 h达高峰,随后降低,照后8 h又上升,出现了第2个峰值．与之不同的是,在1 Gy低剂量照射下,细胞的G2/M期阻滞微弱,γH2AX焦点数在照后 0.5 h最高,随后下降,且无反弹,符合DNA DSB的修复动力学特征．因此,将γ H2AX当做DNA DSB分子标志物时,还需要考虑细胞周期变化的影响．γH2AX适合 作为1 Gy以下照射的DNA双链断裂损伤的分子标志．     

不同方法处理供核细胞对细胞周期和重构胚发育的影响

利用流式细胞仪和细胞染色体核型分析技术,比较奶牛的转基因体细胞和正常细胞经血清饥饿、抑制培养周期同步化处理后的G0/G1期细胞比例;并将同步化处理的核供体细胞进行核移植,然后统计囊胚发育率.结果表明,血清饥饿和抑制培养均能获得较高比例的G0/G1期细胞,两组间差异不显著(P>0.05),但均显著高于未处理对照组(P<0.05);血清饥饿组的囊胚率显著高于抑制培养组和非处理对照组(P<0.05);但细胞同步化处理6 d后细胞染色体核型异常率增加.因此,要获得正常核型的G0/G1核移植供体细胞和较高的囊胚率,同步化处理时间以不超过4 d为宜.     

A multivariate test of evolutionary constraints for thermal tolerance in Drosophila melanogaster

Exposure to extreme temperatures is increasingly likely to impose strong selection on many organisms in their natural environments. The ability of organisms to adapt to such selective pressures will be determined by patterns of genetic variation and covariation. Despite increasing interest in thermal adaptation, few studies have examined the extent to which the genetic covariance between traits might constrain thermal responses. Furthermore, it remains unknown whether sex‐specific genetic architectures will constrain responses to climatic selection. We used a paternal half‐sibling breeding design to examine whether sex‐specific genetic architectures and genetic covariances between traits might constrain evolutionary responses to warming climates in a population of Drosophila melanogaster. Our results suggest that the sexes share a common genetic underpinning for heat tolerance as indicated by a strong positive inter‐sexual genetic correlation. Further, we found no evidence in either of the sexes that genetic trade‐offs between heat tolerance and fitness will constrain responses to thermal selection. Our results suggest that neither trade‐offs, nor sex‐specific genetics, will significantly constrain an evolutionary response to climatic warming, at least in this population of D. melanogaster.     

基于SVM 的药物靶点预测方法及其应用

目的:基于已知药物靶点和潜在药物靶点蛋白的一级结构相似性,结合SVM技术研究新的有效的药物靶点预测方法。方法:构造训练样本集,提取蛋白质序列的一级结构特征,进行数据预处理,选择最优核函数,优化参数并进行特征选择,训练最优预测模型,检验模型的预测效果。以G蛋白偶联受体家族的蛋白质为预测集,应用建立的最优分类模型对其进行潜在药物靶点挖掘。结果:基于SVM所建立的最优分类模型预测的平均准确率为81.03%。应用最优分类器对构造的G蛋白预测集进行预测,结果发现预测排位在前20的蛋白质中有多个与疾病相关。特别的,其中有两个G蛋白在治疗靶点数据库(TTD)中显示已作为临床试验的药物靶点。结论:基于SVM和蛋白质序列特征的药物靶点预测方法是有效的,应用该方法预测出的潜在药物靶点能够为发现新的药靶提供参考。     

不同浓度葡萄糖对β-TC3 细胞Gα表达的影响

目的:研究不同浓度葡萄糖对β-TC3(小鼠胰岛β细胞瘤细胞)细胞Gα(G蛋白α亚基)表达的影响。方法:β-TC3细胞在正常葡萄糖浓度下(11.1 mM)适应性生长两天后,将细胞暴露于不同葡萄糖浓度(25、50 mM),分别于24、48、96h收集细胞,并行蛋白质印迹检测Gα的表达。结果:加入葡萄糖后24、48、96h,Gα的表达明显下降(相对对照组)。结论:持续高糖刺激,可导致β-TC3细胞Gα表达的下调,从而影响细胞信号转导,致使胰岛素抵抗的发生。