eIF4E在非小细胞肺癌耐药性中的作用

探讨eIF4E对厄洛替尼耐药细胞株细胞增殖的影响及作用机制.在配对的厄洛替尼敏感和耐药细胞株(HCC827-EP、HCC827-ER)上,采用实时定量PCR检测mRNA水平的变化,Western Blot检测蛋白水平的变化;通过SRB检测细胞的生长和药物的抑制率的变化.以及用脂质体转染eIF4E的小干扰RNA,用上述方法检测多种信号分子mRNA、蛋白水平的变化,及对细胞生长和对药物抑制率的影响.结果显示,与HCC827-EP相比,eIF4E在HCC827-ER上高表达;HCC827-ER转染eIF4E siRNA的细胞生长速度明显减慢,从第3 d起差异具有统计学意义(P0.05),在相同浓度的厄洛替尼作用下,转染eIF4E siRNA的细胞具有更高的抑制率,差异具有统计学意义(P0.05).结论:下调eIF4E的表达能够增强厄洛替尼对NSCLC的抑制作用,eIF4E的高表达参与了厄洛替尼获得性耐药的产生.     

番茄真核翻译起始因子4E基因RNA干涉及其抗病毒特性研究

植物真核翻译起始因子4E（eIF4E）在蛋白质翻译过程中发挥重要作用．最近研究表明eIF4E在参与植物病毒互作,影响病毒在寄主中复制和侵染过程．文中通过RNA干涉调控番茄eIF4E表达,鉴定eIF4E在植物病毒互作中的作用．根据数据库假设性一致序列（TC171564,TC170275）分别克隆了番茄“中蔬五号”SleIF4E基因及其异构体基因SleIF（iso）4E．构建了SleIF4ERNAi抑制表达载体pSL4D,通过农杆菌介导的方法导入番茄品种“中蔬五号”基因组．PCR检测和Southern blot结果表明,目标基因已整合到番茄基因组中．通过半定量RT-PCR对转基因番茄进行eIF4E表达分析,表明pSL4D转化番茄中eIF4E得到不同程度抑制．转基因植株分别接种PVY和CMV两种病毒,病毒ELISA测定和病毒RNA积累量分析表明,pSL4D转化植株相对于对照组均获得不同程度的病毒抗性,番茄eIF4E参与PVY的复制或侵染寄主过程．就不同病毒而言,RNAi对PVY抗性的调控效果比CMV调控效果好,抑制PIF4E表达可提高植物对PVY的抗性．     

长非编码RNA与SAFB1相互作用中RNA二级结构的作用

为探究RNA与SAFB1蛋白相互作用过程中RNA二级结构的作用.采用足迹法确定SAFB1与LINC-PINT-1特定的结合区域,在此基础上根据RNA结构分析研究SAFB1结合区域所具有特定的二级结构.最后通过凝胶迁移率实验论证该二级结构在RNA与SAFB1蛋白结合中的作用.结果显示:在针对LINC-PINT-1的亚克隆发现只有包含30～55 nt区域的亚克隆才能维持RNA结合区域的二级结构,并且证实了LINC-PINT-1与SAFB1蛋白相互作用依赖于特定的RNA二级结构.结果表明:长非编码RNA与SAFB1蛋白相互作用或依赖于特定的RNA二级结构,本研究为RNA与蛋白质相互作用的分子机制研究提供了有力的理论实验支撑.     

人PSF蛋白结合长非编码RNA的筛选及鉴定

通过RNA亲和层析（RNA affinity chromatograph）,4种可能与人PSF（human polypyrimidine tractbinding proteinassociated splicing factor,hPSF）蛋白结合的长非编码RNA（long noncoding RNA,lncRNA）片段在人黑色素瘤细胞yusac细胞核RNA文库中被筛选得到. 它们分别定位于人内源性逆转座子L1PA16、MER11C、非编码基因MALAT1以及一个未知基因(unknown g     

SARS-CoVS基因表达及纯化

严重急性呼吸道综合症是由一种新的冠状病毒SARS-CoY引起的．作者通过PCR扩增得到了S蛋白的6个编码片段，并利用表达载体pET28a( )在E．coli BL21中进行了原核表达．通过亲和层析纯化了包含大部分ACE2结合区域的S蛋白片段(S4)．ELISA分析结果表明S4与SARS病人恢复期血清具有良好的反应能力．     

家蚕新的非编码RNA功能初探

构建了家蚕50～500 nt非编码RNA的cDNA文库,发现了189个新的ncRNA,其中一个小RNA-Bm-86在家蚕幼虫和蛹中的表达量显著高于卵和成虫.利用生物信息学软件对该ncRNA的特性及其与上下游基因的关系进行了深入分析,并利用Northern杂交和半定量RT-PCR技术对该ncRNA与其宿主基因在家蚕不同组织的表达情况进行了研究.结果发现,该ncRNA属于H/ACA box snoRNA家族,是由家蚕eIF5A基因的第二个内含子转录产生的,且在家蚕中没有明显的靶标位点.分析其上下游基因结构发现,在其上游263 bp处存在着一个可能的启动子位点,表明其有独立转录的倾向.进一步分析Bm-86与其宿主基因eIF5A在家蚕不同组织部位的表达情况发现,二者在表达上存在着负相关的关系,且该现象在丝腺中尤其明显.推测Bm-86可能通过影响eIF5A基因的表达参与到家蚕丝腺发育调控过程中.     

高低危型HPV E7的分析以及58型E7结构与结合位点预测

 对18种HPV E7蛋白进行生物信息学分析,同时对HPV58 E7进行结构以及可结合位点的预测.从NCBI数据库中获取蛋白序列,通过ExPASy Protparam进行生物信息分析,Clustal W进行多序列比对,MEGA软件构建进化树,利用Zhang lab QUARK以及Q-SiteFinder能量依赖的检测对HPV58 E7进行结构预测与结合位点预测.得到了E7生物信息,多序列比对数据,构建成进化树,获得HPV58 E7蛋白结构,并且找到pRb与E7结合的三维位点.不同类型HPV E7蛋白存在进化差异,得到的HPV58型结构以及与pRb的可以结合位点,为抑制HPV58 E7蛋白功能的相关实验提供了理论依据.     

草酸青霉外切葡聚糖酶CBHⅠ基因的克隆

以草酸青霉SJ1菌株的基因组DNA和总RNA,利用PCR和RT-PCR技术进行扩增得到外切葡聚糖酶CBHⅠ(ExocellobiohydrolaseⅠ)基因的序列.CBHⅠ基因DNA序列全长1 638 bp,无内含子,编码545个氨基酸的多肽,推测其蛋白分子大小约为57 ku,蛋白的p I值为4.90.预测结果表明,CBHⅠ是一种很强疏水性的蛋白,具有43个磷酸化修饰位点,蛋白质三级结构以β-折叠为主,根据蛋白质序列比对分析,推测E236、D238和E241这3个氨基酸为酶的活性位点,为后续利用分子改造方法提高青霉产纤维素酶的能力提供理论依据.     

乳腺癌中肿瘤相关巨噬细胞的浸润与eIF4E表达的关系

目的:探讨乳腺癌组织中CD163~+肿瘤相关巨噬细胞的浸润情况与eIF4E表达及其与临床病理特征的关系,并分析两者的相关性.方法:收集乳腺癌组织92例及癌旁正常乳腺组织74例,应用Maxvision法进行免疫组化染色检测eIF4E及CD163表达,并分析其与乳腺癌临床病理特征的关系.结果:乳腺癌组织中的CD163表达量高于癌旁正常组织(P0.001),乳腺癌组织中的eIF4E表达量高于癌旁正常乳腺组织(P0.001),CD163的表达与ki67表达相关(P0.05),CD163与eIF4E的表达呈正相关(r=0.273,P0.05).结论:乳腺癌中CD163和eIF4E的表达明显高于癌旁组织,CD163的过表达与ki67的表达相关,同时与eIF4E呈正相关,提示CD163与eIF4E的共同作用可能参与了乳腺癌的发生与发展.     

植物类病毒研究进展

类病毒是一类小的单链环状RNA分子,侵染植物引起严重病害。类病毒虽然不编码任何蛋白质,但这类亚病毒病原可通过基因组RNA中的功能域利用寄主蛋白在植物中进行自我复制和传播。本文概述了类病毒的结构与分类、复制机理和策略、结构与功能的关系、类病毒运动与类病毒结合蛋白,以及植物RNA沉默机制对类病毒RNA的影响。     

An oxygen-regulated switch in the protein synthesis machinery

Protein synthesis involves the translation of ribonucleic acid information into proteins, the building blocks of life. The initial step of protein synthesis is the binding of the eukaryotic translation initiation factor 4E (eIF4E) to the 7-methylguanosine (m(7)-GpppG) 5'?cap of messenger RNAs. Low oxygen tension (hypoxia) represses cap-mediated translation by sequestering eIF4E through mammalian target of rapamycin (mTOR)-dependent mechanisms. Although the internal ribosome entry site is an alternative translation initiation mechanism, this pathway alone cannot account for the translational capacity of hypoxic cells. This raises a fundamental question in biology as to how proteins are synthesized in periods of oxygen scarcity and eIF4E inhibition. Here we describe an oxygen-regulated translation initiation complex that mediates selective cap-dependent protein synthesis. We show that hypoxia stimulates the formation of a complex that includes the oxygen-regulated hypoxia-inducible factor 2α (HIF-2α), the RNA-binding protein RBM4 and the cap-binding eIF4E2, an eIF4E homologue. Photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) analysis identified an RNA hypoxia response element (rHRE) that recruits this complex to a wide array of mRNAs, including that encoding the epidermal growth factor receptor. Once assembled at the rHRE, the HIF-2α-RBM4-eIF4E2 complex captures the 5'?cap and targets mRNAs to polysomes for active translation, thereby evading hypoxia-induced repression of protein synthesis. These findings demonstrate that cells have evolved a program by which oxygen tension switches the basic translation initiation machinery.     

Translation factors promote the formation of two states of the closed-loop mRNP

Efficient translation initiation and optimal stability of most eukaryotic messenger RNAs depends on the formation of a closed-loop structure and the resulting synergistic interplay between the 5' m(7)G cap and the 3' poly(A) tail. Evidence of eIF4G and Pab1 interaction supports the notion of a closed-loop mRNP, but the mechanistic events that lead to its formation and maintenance are still unknown. Here we use toeprinting and polysome profiling assays to delineate ribosome positioning at initiator AUG codons and ribosome-mRNA association, respectively, and find that two distinct stable (resistant to cap analogue) closed-loop structures are formed during initiation in yeast cell-free extracts. The integrity of both forms requires the mRNA cap and poly(A) tail, as well as eIF4E, eIF4G, Pab1 and eIF3, and is dependent on the length of both the mRNA and the poly(A) tail. Formation of the first structure requires the 48S ribosomal complex, whereas the second requires an 80S ribosome and the termination factors eRF3/Sup35 and eRF1/Sup45. The involvement of the termination factors is independent of a termination event.     

Regulation of cap-dependent translation by eIF4E inhibitory proteins

Eukaryotic messenger RNAs contain a modified guanosine, termed a cap, at their 5' ends. Translation of mRNAs requires the binding of an initiation factor, eIF4E, to the cap structure. Here, we describe a family of proteins that through a shared sequence regulate cap-dependent translation. The biological importance of this translational regulation is immense, and affects such processes as cell growth, development, oncogenic transformation and perhaps even axon pathfinding and memory consolidation.     

非编码RNA调控心脏重构与再生

近年来, 越来越多的证据表明, 大量的非编码RNA(non-coding RNAs, ncRNAs)在基因的表达调控、细胞和机体的生理功能维持与病理环境调节方面都有重要作用, 其中主要包括微小RNA(microRNAs, miRNAs) 和长链非编码RNA(long non-coding RNAs, lncRNAs).心脏重构与再生是心血管疾病领域的关键问题, 其调控过程非常复杂, 包括表观遗传、转录、转录后及翻译水平的调控. 大量研究发现在转录后水平, miRNAs 通过负性调节靶标的表达调控心脏发育、疾病及再生进程. 近期研究揭示, lncRNAs 在心脏发育和疾病中具有潜在的作用, 可通过表观遗传、转录及转录后水平发挥作用. lncRNAs 已成为继miRNAs 之后的又一重要的调节性非编码RNA. 就非编码RNA 在心脏重构及再生进程中的调控作用进行综述.     

原位聚合PAM改性环氧树脂Ⅰ.合成与表征

以对苯二甲醛（ＰＤＡ）和对苯二胺（ＴＰＡ）为原料在环氧树脂（ＥＰ）基体中原位聚合聚甲亚胺（ＰＡＭ）,得到ＰＡＭ分散良好的改性ＥＰ体系。用红外光谱、紫外光谱对位聚合的结果进行了表征,并用ＧＰＣ测定了原位聚合生成的ＰＡＭ的相对分子质量。以三乙烯四胺（ＴＥＴＡ）固化原位聚合ＰＡＭ改性的ＥＰ,原位聚合过程中未加稀释的改性体系与纯ＥＰ相比力学性能变化不明显;原有ＰＡＭ改性的ＥＰ,原位聚合过程中未加稀释剂的改     

Eukaryotic initiation factor 6 is rate-limiting in translation, growth and transformation

Cell growth and proliferation require coordinated ribosomal biogenesis and translation. Eukaryotic initiation factors (eIFs) control translation at the rate-limiting step of initiation. So far, only two eIFs connect extracellular stimuli to global translation rates: eIF4E acts in the eIF4F complex and regulates binding of capped messenger RNA to 40S subunits, downstream of growth factors, and eIF2 controls loading of the ternary complex on the 40S subunit and is inhibited on stress stimuli. No eIFs have been found to link extracellular stimuli to the activity of the large 60S ribosomal subunit. eIF6 binds 60S ribosomes precluding ribosome joining in vitro. However, studies in yeasts showed that eIF6 is required for ribosome biogenesis rather than translation. Here we show that mammalian eIF6 is required for efficient initiation of translation, in vivo. eIF6 null embryos are lethal at preimplantation. Heterozygous mice have 50% reduction of eIF6 levels in all tissues, and show reduced mass of hepatic and adipose tissues due to a lower number of cells and to impaired G1/S cell cycle progression. eIF6(+/-) cells retain sufficient nucleolar eIF6 and normal ribosome biogenesis. The liver of eIF6(+/-) mice displays an increase of 80S in polysomal profiles, indicating a defect in initiation of translation. Consistently, isolated hepatocytes have impaired insulin-stimulated translation. Heterozygous mouse embryonic fibroblasts recapitulate the organism phenotype and have normal ribosome biogenesis, reduced insulin-stimulated translation, and delayed G1/S phase progression. Furthermore, eIF6(+/-) cells are resistant to oncogene-induced transformation. Thus, eIF6 is the first eIF associated with the large 60S subunit that regulates translation in response to extracellular signals.     

Synthetic RNA-dependent DNA polymerase activity in normal rat liver and hepatomas

Normal adult rat liver contains a high level of a synthetic RNA-dependent DNA polymerase activity, which is distinct from cellular DNA-dependent polymerases. It uses poly(dT).poly(rA) and poly(rA).poly(rU) as templates but has little or no response to DNA or several single-stranded RNAs.