盐酸埃克替尼通过激活蛋白激酶C诱导Tca8113细胞表达iNOS和凋亡

体外培养Tca8113细胞,随机分为4组,(1)对照组;(2)埃克替尼处理组(20μmo1/L);(3)PKC抑制剂Ro31-8220(3μmol/L)处理组;(4)埃克替尼+Ro31-8220组(20μmo1/L Icotinib,3μmol/L Ro31-8220),不同干预因素处理细胞4h.Western blot检测不同处理组的胞膜内的PKC-α的表达水平,iNOS ELISA检测试剂盒测定细胞的iNOS含量,Griess方法测定Tca8113细胞产生的NO水平,用DNA fragmentation检测Tca8113细胞的凋亡情况.结果表明埃克替尼组DNA fragmentation细胞凋亡及上清液的iNOS,NO较对照组均明显升高;埃克替尼组细胞膜的PKC-α蛋白表达量明显增加.用埃克替尼与PKC的抑制剂Ro31-8220共同处理Tca8113细胞后,胞膜的PKC-α,iNOS的蛋白表达水平及产生的NO能被Ro31-8220显著抑制,且PKC抑制剂Ro31-8220能逆转埃克替尼引起的Tca8113细胞凋亡.埃克替尼能诱导Tca8113细胞iNOS表达和NO生成增加并能诱导Tca8113细胞凋亡;埃克替尼能促进细胞膜内的PKC-α表达增加;埃克替尼诱导的Tca8113细胞iNOS表达和凋亡与PKC有关.     

家蝇精氨酸激酶基因克隆及其在害虫防治上的应用

从家蝇转录组数据库中筛选到一条精氨酸激酶同源序列,将其命名为家蝇精氨酸激酶(Musca domestica arginine kinase,简称:MdAK).通过RT-PCR克隆得到MdAK cDNA序列,长2 007bp,编码356个氨基酸残基,编码蛋白的分子质量为40.0ku.利用RNAi技术敲低家蝇幼虫精氨酸激酶的表达后,其生长发育受到影响,死亡率明显升高.认为精氨酸激酶可以作为害虫生物防治的潜在靶点.     

p38信号通路参与P19CL6细胞早期心肌分化

以P19CL6小鼠畸胎瘤细胞心肌分化为模型,研究了p38信号通路在干细胞心肌分化早期阶段的作用.在诱导剂二甲基亚砜作用下,P19CL6细胞分化为自发跳动的心肌细胞,表达心肌标志性基因.在诱导分化过程中,p38信号通路活化.采用特异性抑制剂SB203580在早期分化阶段封闭p38信号通路,结果发现:高剂量SB203580诱导细胞凋亡;低剂量SB203580抑制心肌祖细胞标志基因GATA4和Nkx2.5的表达,并显著下调生心性信号分子BMP2和BMP4的表达.而过表达p38α质粒则能促进P19CL6细胞表达GATA4和Nkx2.5.表明p38信号通路正性调控P19CL6细胞的早期心肌分化,并维持细胞的增殖和存活能力.     

两种MAP激酶共同调节蚕豆气孔保卫细胞中ROS信号途径研究

运用表皮条生物学分析、激光共聚焦扫描及膜片钳技术,在MEK1/2和p38MAP激酶专一性抑制剂PD98059和SB203580处理下,观察促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAP激酶)家族成员介导蚕豆保卫细胞中氧化信号机制.结果表明,PD98059能阻断并逆转ABA和H2O2抑制质膜内向K 电流、ABA诱导H2O2产生及气孔关闭,SB203580也表现相似的作用.因此,两种MAP激酶可能共同调节ABA诱导ROS产生和气孔关闭.     

HeLa细胞中激酶NUAK1调控Tuberiun的磷酸化

NUAK1是LKB1的下游激酶之一,可被LKB1磷酸化而激活,但对其在LKB1相关信号通路中的功能仍缺乏了解.本研究发现在HeLa细胞中重建LKB1表达后NUAK1与tuberin蛋白免疫共沉淀,提示在野生型LKB1存在时NUAK1与tuberin可能存在直接相互作用.进一步的激酶活性测定和in vivo蛋白磷酸化实验表明:在HeLa细胞中葡萄糖匾乏条件下,野生型LKB1可显著激活NUAK1的激酶活性;而被激活的NUAK1可明显提高tuberin 的磷酸化水平,使用NUAK1 siRNA pool干扰NUAK1的表达则几乎将tuberin的磷酸化水平降低为零.上述结果表明NUAK1可能介导了LKB1对tuberin磷酸化的调节,进而下调mTOR通路,抑制蛋白质合成与细胞生长增殖.     

用点突变方法研究海参精氨酸激酶的活性位点

为研究动物能量代谢,用点突变的方法从双亚基海参(Stichopusjaponicus)精氨酸激酶中得到了3个突变体:C274A、R283G和H287A,并通过大肠杆菌E.coli表达。大部分精氨酸激酶都以包涵体的形式存在。经过纯化之后对3种突变体的催化活性和一些结构特征进行了分析。这3种突变体丧失了绝大部分催化活力,特别是突变体C274A,与野生型相比除了活性完全丧失之外,结构却几乎没有变化。这3个残基对海参精氨酸激酶结构或功能的保持有着重要作用,Cys274残基可能是海参精氨酸激酶的活性位点。     

不同的外界环境及生长阶段绿豆黄化幼苗肌醇磷脂代谢的变化

以双酰甘油（DG）和蛋白激酶C（PKC）作为指标研究绿豆黄化幼苗肌醇磷脂代谢．结果表明不同浓度的玉米素、低温（0℃）和高温（35℃）胁迫均会引起DG水平的变化,而且,适当浓度（10-6mol·L－1）的玉米素与温度胁迫可协同促进肌酸磷脂代谢．外源或内源DG对PKC活性的影响均表现为低浓促进、高浓抑制．PKC活性与绿豆黄化幼苗下胚轴的伸长率呈正相关．     

Cytoskeleton-associated protein tyrosine phosphorylation involved in induction of differentiation in mouse melanoma cells

The malignancy of a cancer is due partly to its poor differentiation. Genistein, a protein tyrosine kinase inhibitor, is found to induce the highly malignant B16-BL6 mouse melanoma cells to differentiate to mature phenotypes. When Triton X-100 insoluble fraction of the differentiated cells is prepared and analyzed, tyrosine phosphorylation levels of three cytoskeleton-associated proteins (65, 60 and 53 ku respectively) are found to decrease dramatically. But no any change is found when phosphotyrosine contents of the cytosol fraction or the total cellular protein preparations are evaluated. It is concluded that cytoskeleton-associated protein tyrosine phosphorylation may be involved in the control of differentiation of cancer cells. The decrease of phosphotyrosine contents of cytoskeleton-associated proteins may be one of the important mechanisms underlying the differentiation induction of cancer cells by anticancer agents.     

一个新的人类精子发生相关蛋白激酶cDNA的克隆

蛋白磷酸化被认为在精子发生过程中起重要作用,从人类胎脑cDNA文库中得到2个克隆,分别长1840bp和1571bp,后者仅比前者在3非翻译区少了约300个核苷酸,二者拟编码322个氨基酸残基,基序分析发现其中含有1段明显的蛋白激酶其序,Northern杂交显示其在睾丸中有显著表达,暂命名为精了发生相关蛋白激酶,原位杂交表明精子发生相关蛋白激酶基因在性成熟小鼠睾丸中主要表达在生精小管的外层细胞中,而这一层细胞主要包含分裂的精原细胞的初级精母细胞,提示精子发生相关蛋白激酶可能在精子发生过程中起着比较重要的作用。     

Chromatin assembly during S phase: contributions from histone deposition, DNA replication and the cell division cycle

During S phase of the eukaryotic cell division cycle, newly replicated DNA is rapidly assembled into chromatin. Newly synthesised histones form complexes with chromatin assembly factors, mediating their deposition onto nascent DNA and their assembly into nucleosomes. Chromatin assembly factor 1, CAF-1, is a specialised assembly factor that targets these histones to replicating DNA by association with the replication fork associated protein, proliferating cell nuclear antigen, PCNA. Nucleosomes are further organised into ordered arrays along the DNA by the activity of ATP-dependent chromatin assembly and spacing factors such as ATP-utilising chromatin assembly and remodelling factor ACF. An additional level of controlling chromatin assembly pathways has become apparent by the observation of functional requirements for cyclin-dependent protein kinases, casein kinase II and protein phosphatases. In this review, we will discuss replication-associated histone deposition and nucleosome assembly pathways, and we will focus in particular on how nucleosome assembly is linked to DNA replication and how it may be regulated by the cell cycle control machinery.