蛋白质组学在去泛素化酶研究中的应用

泛素-蛋白酶体系统是细胞内蛋白质特异性降解的主要途径,参与并调控细胞周期、免疫应答、信号传递和DNA修复等真核生物体内几乎所有的生命活动。去泛素酶的存在使泛素化修饰成为可逆过程,保证了泛素系统及其相关生理过程的动态平衡,其表达紊乱也是诱发多种疾病的主要原因。对去泛素化酶进行系统、全面的研究是理解其作用机制并将其作为治疗药物靶点的前提。蛋白质组学技术的快速发展为系统深入研究去泛素化酶提供了条件,特别是在去泛素化酶的相互作用网络和底物特异性研究等方面发挥了独特的作用。因此,文中结合课题组研究工作,对去泛素化酶的分类及功能进行介绍并总结了蛋白质组学在去泛素化酶研究中的应用进展。     

趋磁细菌多样性与应用研究进展 *

趋磁细菌是一类可以沿磁场方向进行运动的微生物统称,在细胞内合成由生物膜包被、链状排列、纳米级、单磁畴的磁铁矿 (Fe₃O₄) 或胶黄铁矿 (Fe₃S₄) 的磁小体颗粒。趋磁细菌在自然界分布广泛且多样性丰富,不仅在水环境和沉积环境的铁、硫、碳、氮、磷等元素生物地球化学循环中发挥重要作用,而且在污染治理、疾病诊断和治疗等方面有较好的应用。趋磁细菌磁小体由生物膜包被并在细胞调控下合成,是一类新型的生物源磁性纳米材料。相比常规化学合成的磁性纳米颗粒,磁小体具有大小均一、生物相容性高、兼具化学修饰和基因工程修饰功能等特点,在磁性分离、固定化酶、食品检测、环境监测、医学诊断、磁共振成像、磁热疗和靶向治疗等方面具有广阔的应用前景。在介绍趋磁细菌多样性研究的基础上,综述了趋磁细菌和磁小体的制备、修饰及其应用的最新进展,并对未来的研究进行了展望。     

Ultrasensitive nucleic acid biosensors for early cancer diagnostics

We have developed ultrasensitive nucleic acid detection systems involving an amplification step where the analytical signal correlates directly to the amount of nucleic acid in the solution So far, we have performed nucleic acid quantification on several breast cancer susceptibility genes and were able to detect nucleic acid amounts that ranged from 0.1–1.0 fg of nucleic acid, which is at least 1000 times more sensitive than conventional fluorescent detection methods. The biosensors are so sensitive that they can be used for direct detection of breast cancer susceptibility genes in mRNA without involving a PCR step.     

蛇毒类凝血酶calobin在毕赤酵母中的表达

蛇毒类凝血酶是临床上防治血栓栓塞性疾病的有效药物。参照朝鲜蝮蛇(Agkistrodon caliginosus,Korean Viper)类凝血酶calobin基因序列(GenBank AccessionNo.U32937.1),将人工合成的calobin基因克隆到酵母表达载体pPICZαA,于毕赤酵母中表达,得到了分子量约为32kD的重组calobin蛋白,经甲醇诱导培养,表达产物可获得3.5g/L的高表达量。重组蛋白经过阴离子交换柱Q-Sepharose Fast Flow和分子筛Sephacryl-S-100凝胶过滤层析等纯化步骤进行了初步纯化。纯化后的重组calobin可以在纤维蛋白原平板上形成水解圈,经SDS-PAGE实验显示,重组蛋白能水解纤维蛋白原的Aα链,产生一条约40kD左右的降解带。在实验中未能发现重组calobin对纤维蛋白原的凝固作用。     

极端耐盐放线菌白色普氏菌YIM 90005T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆

摘要：【目的】 为了研究耐盐放线菌对高盐环境的适应机理。【方法】 用HPLC定量检测了极端耐盐、丝状产孢放线菌——白色普氏菌（Prauserella alba） YIM 90005T在不同盐浓度下胞内相容性溶质的种类和含量。【结果】 结果发现,四氢嘧啶和5-羟基四氢嘧啶是其主要的相容性溶质。在培养基NaCl浓度为10%时,四氢嘧啶在胞内累积浓度最大,为18.77 μg/mg干菌体重。之后随NaCl浓度的升高,胞内的四氢嘧啶含量逐渐减少,而5-羟基四氢嘧啶的含量逐渐增加,在该菌耐受的最高NaCl浓度下（24% w/v）,胞内5-羟基四氢嘧啶含量达到最大值,为22.98 μg/mg干菌体重。设计兼并引物,利用染色体步移,克隆得到四氢嘧啶及5-羟基四氢嘧啶合成相关基因ectABCD。序列分析表明,ectABCD位于一个操纵子中。进一步对不同NaCl浓度培养条件下ectB,D的表达量进行定量分析,结果表明该基因簇表达量随着培养基中NaCl浓度的增加而增大。【结论】 研究结果证实5-羟基四氢嘧啶是P. alba YIM 90005T在极高盐浓度条件下起渗透调节及保护的相容性溶质。     

Putative phosphorylation sites on WCA domain of HA2 is essential for <Emphasis Type="Italic">Helicoverpa armigera</Emphasis> single nucleopolyhedrovirus replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPv) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Tbr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.     

Spindlin1, a novel nuclear protein with a role in the transformation of NIH3T3 cells

spindlin1, a novel human gene recently isolated by our laboratory, is highly homologous to mouse spindlin gene. In this study, we cloned cDNA full-length of this novel gene and send it to GenBank database as spindlin1 (Homo sapiens spindlin1) with Accession No. AF317228. In order to investigate the function of spindlin1, we studied further the subcellular localization of Spindlin1 protein and the effects of spindlin1 overexpression in NIH3T3 cells. The results showed that the fusion protein pEGFP-N1-spindlin1 was located in the nucleus and the C-terminal is correlated with nuclear localization of Spindlin1 protein. NIH3T3 cells which could stably express spindlin1 as a result of RT-PCR analysis compared with the control cells displayed a complete morphological change; made cell growth faster; and increased the percentage of cells in G2/M and S phase. Furthermore, overexpressed spindlin1 cells formed colonies in soft agar in vitro and formed tumors in nude mice. Our findings provide direct evidence that spindlin1 gene may contribute to tumorigenesis.     

miR-10a Regulates Proliferation of Human Cardiomyocyte Progenitor Cells by Targeting GATA6

microRNAs (miRNAs) play essential roles in cardiogenesis. The altered expression of miRNAs can result in cardiac malformations by inducing abnormalities in the behavior of cardiac cells. However, the role of miR-10a in the regulation of cardiomyocyte progenitor cells (CMPCs) remains undetermined. In the present study, we found that up- or down-regulation of miR-10a inhibited or promoted the proliferation of human CMPCs, respectively, without affecting their differentiation toward cardiomyocytes. miR-10a bound to GATA6 directly and reduced GATA6 expression. Over-expression of GATA6 greatly attenuated the miR-10a-mediated inhibitory effect on the proliferation of human CMPCs. Thus, our results indicate that miR-10a could effectively modulate the proliferation of human CMPCs by targeting GATA6. The finding provides novel insights into the potency of miR-10a during heart development.     

人骨髓间充质干细胞在成年大鼠脑内的迁移及分化

骨髓间充质干细胞 (mesenchymalstemcells,MSCs)是目前备受关注的一类具有多向分化潜能的组织干细胞 ,体外可以分化为骨、软骨、脂肪等多种细胞。因此 ,MSCs是细胞治疗和基因治疗的种子细胞之一。为了探索MSCs的迁移和分化趋势 ,为帕金森病 (Parkinsondisease,PD)的干细胞治疗提供理论和实验依据 ,本实验将体外扩增并转染增强型绿色荧光蛋白 (enhancedgreenfluorescentprotein ,EGFP)的人骨髓MSCs注入PD大鼠脑内纹状体 ,观察了人骨髓MSCs在大鼠脑内的存活、迁移、分化以及注射MSCs前后大鼠的行为变化。结果表明 ,人骨髓MSCs在大鼠脑内可存活较长时间 ( 10周以上 ) ;随着时间的延长 ,MSCs迁移范围扩大 ,分布于纹状体、胼胝体、皮质以及脑内血管壁 ;免疫组化法检测证实MSCs在大鼠脑内表达人神经丝蛋白 (neurofilament,NF)、神经元特异性烯醇化酶 (neuron specificeno lase,NSE)以及胶质原纤维酸性蛋白 ( glialfibrillaryacidprotein ,GFAP) ;PD大鼠的异常行为有所缓解 ,转圈数由 8 86±2 0 9r/min下降到 4 87± 2 0 6r/min ,统计学分析P <0 0 5为差异显著。以上观察结果表明 ,骨髓MSCs有望成为治疗PD的种子细胞     

17个新的C2H2型锌指基因片段的分离与克隆

按照Ｃ２Ｈ２型锌指基因保守结构域的ＤＮＡ序列设计一对简并引物,以人基因组ＤＮＡ为模板进行ＰＣＲ同源扩增,将由此获得的锌指基因片段为探针,从人胎肾、骨骼肌、骨骼组织的ｃＤＮＡ分子库中筛选到２２个Ｃ２Ｈ２型锌指蛋白ｃＤＮＡ片段,经国际ＮＣＢＩ数据库查询检索,其中１７个为新的锌指基因片段。对从胎肾ｃＤＮＡ分子库中分离到的Ｋ３－４和Ｋ５－１２克隆进行了表达谱分析,发现Ｋ３－４在肾脏中的表达量明显高于其他几