Ret：一种受体酪氨酸激酶及其基因突变与疾病

RET是一个在转化中发生重排的原癌基因,且因此行为而得名.它编码细胞膜受体酪氨酸激酶,初步研究表明它介导的信号转导途径较为独特.RET基因突变与人类4种癌症的发生相关：甲状腺乳头状腺癌存在RET基因与其他基因多种重排;多发性内分泌腺瘤2型,家族遗传甲状腺髓样癌等存在7个位点点突变;先天巨结肠疾病与RET基因缺失相关.因此近年来备受关注.对Ret蛋白的结构功能,RET基因突变对Ret蛋白功能的影响及与人类相关疾病的关系作一综述.     

脾脏酪氨酸激酶促进肝肺综合征大鼠肺血管新生

本研究旨在研究脾脏酪氨酸激酶(spleen tyrosine kinase,Syk)在肝肺综合征(hepatopulmonary syndrome,HPS)血管新生过程中的作用及其机制.Sprague Dawley(SD)大鼠随机分为3组:假手术组(Sham组)、胆总管结扎(common bile duct ligat...     

小鼠再生肝胞浆磷酸酪氨酸蛋白磷酸酶的纯化和性质研究

以~(32)P(Tyr)-Poly Glu,Tyr(4:1)为底物,用于研究小鼠再生肝胞浆磷酸酪氨酸蛋白磷酸酶(PTPP)的分离纯化和性质。再生肝胞浆经60％饱和度硫酸铵盐析,二次DEAE纤维素层析,Sephadex G-200柱层析和Poly Glu,Tyr-Sepha-rose 4B亲和层析后,得到的PTPP分子量为67000,纯度提高1123倍,活性回收率为28％,对~(32)P(Tyr)-Poly Glu,Tyr有很高的活力,对~(32)P(Ser/Thr)-Casein(酪蛋白)和PNPP(对硝基苯酚磷酸盐)没有作用,其最适pH为6.8～7.1,对热不稳定。EDTA对酶有激活作用,Zn~(2+)、PNPP、P-Tyr、多胺化合物、焦磷酸根、钼酸根、柠檬酸根对酶有明显的抑制作用。酶对Na_3VO_4不敏感。碱性蛋白质组蛋白、鱼精蛋白对酶活力有抑制作用,酸性蛋白质酪蛋白和酸性多糖物质肝素对酶活力有激活作用,且后者能减弱前者的抑制作用。     

鲤科鱼类胰岛素受体基因IRa和IRb酪氨酸激酶区CDS的分子变异和进化

胰岛素受体基因在调控细胞生长、分化和个体发育方面具有重要的生物学功能,研究基因功能区变异和分子进化对于理解鱼类不同物种之间胰岛素受体基因表达调控和个体尺寸控制具有重要意义.本研究在鲤科鱼类中选择37种代表性物种和5种鲤科外的鲤形目鱼类作为外类群,通过PCR扩增、克隆和测序,获得它们的IRa和IRbDNA序列,并提取CDS序列.分别基于IRa和IRb酪氨酸激酶区CDS序列,分析其分子变异和进化,以理解IR基因在鲤科鱼类中的基因分化特征.结果显示,采用UPGMA方法对IR基因家族构建的基因树中,鱼类IRa和IRb基因分别聚为单系群,说明鲤科鱼类IR基因复制后IRa和IRb基因分别进化为两类明显不同的基因.分子进化分析显示,基于IRa或IRb单个基因构建的鲤科鱼类MP树中各系群间进化速率无显著性差异,说明基因IRa在鲤科鱼类不同系群间受到相似的进化压力,基因IRb也是如此.尽管由IRa和IRbCDS推导出的酪氨酸激酶区氨基酸序列高度保守,但序列变异特征可清楚地显示该区中氨基酸序列的差异,蛋白质IRa和IRb酪氨酸激酶区中呈现出来的这种差异,预示着胰岛素受体IRa和IRb在信号传导中扮演不同的角色.     

大鼠8种肝脏细胞中缺血反应相关基因与肝再生的作用分析

为了解8种肝脏细胞的缺血反应相关基因与大鼠肝再生的相关性, 用percoll密度梯度离心和免疫磁珠方法分离大鼠部分肝切除后不同时间(0, 2, 6, 12, 24, 30, 36, 72, 120和168 h)再生肝中的8种细胞, 用Rat Genome 230 2.0芯片等方法检测上述8种细胞的缺血反应相关基因在大鼠肝再生中表达变化, 用生物学和系统生物学等方法分析上述基因与大鼠肝再生的相关性. 结果显示, 缺血反应主要在肝再生启动阶段及进展阶段前期发挥作用, 且上调占优势, 可刺激il6, tnf等肝再生关键基因表达; 肝星形细胞、树突状细胞中的缺血反应相关基因具有表达的相似性. 缺血反应能推动肝再生的顺利进行, 但胆管上皮细胞的基因表达情况特殊, 值得进一步研究.     

大鼠再生肝中表达上调基因的筛选与鉴定

采用新发展的抑制差减杂交技术（suppression subtractive hybridization,SSH）在基因组水平筛选再生肝中高表达基因。大鼠肝部分切除后24h的再生杆组织来源的cDNA作为受检者（tester）,正常肝组织的cDNA作为驱动者（driver）,进行差减杂交,获得一900个克隆的差减杂交库,随后对差减克隆进行了差异筛选,得到50个在再生肝中高表达的强阳性克隆,序列测定和同源比较表明这些克隆代表了37个基因,其中13个与已报道的肝再生相关的基因同源,15个为忆知基因但首次发现与肝再生相关,9个为新的基因（EST）已被GenBank收录。制备了标准化RNA点杂交膜,通过对上述部分基因的RNA点杂交分析,不但确认了这些基因在再生肝中表达水平的升高,同时发现它们在肝再生过程中有不同的表达模式。实验结果提示这些基因在肝再生过程中具有重要功能。     

细胞连接相关基因在大鼠肝再生中表达模式

细胞连接是组织、器官形成的基础。为在基因转录水平了解紧密连接、粘附连接、粘着斑和间隙连接相关基因在肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome 230 2.0芯片检测它们在大鼠再生肝中表达情况,将3次检验结果相同或相似、在肝再生中发生有意义表达变化、真手术组和假手术组表达差异显著的基因视为肝再生相关基因。初步证实上述4种细胞连接中79、53、109和53个基因与肝再生相关。其中,肝再生启动(部分肝切除后0.5～4h)、G0/G1过渡(PH后4～6h)、细胞增殖(部分肝切除后6～66h)、细胞分化和组织结构功能重建(部分肝切除后72～168h)等4个阶段起始表达的基因数和基因的总表达次数为124、43、122、10和249、145、957、306。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共上调972次,下调540次,表明肝再生中大多数细胞连接相关基因表达加强,少数基因表达降低。它们表达的相似性分为均上调、上调占优势、均下调、下调占优势、上调和下调相近等5类,涉及102、38、73、27和16个基因,它们表达的时间相关性分为0.5和1h、2h、4和6h、8和12h、16h、18和48h、24h、30和42h、36h、54和60h、66和72h、96h、120h、144和168h等14组,表明肝再生中细胞生理生化活动具有阶段性。它们的表达模式分为41类,表明肝再生中细胞生理生化活动具有多样性和复杂性。根据肝再生中基因表达变化和表达模式推测,肝再生早期和前期间隙连接形成增强,晚中期和后期间隙连接形成减少;早期、前期和后期粘着斑形成增强;紧密连接和粘附连接的形成贯穿于整个肝再生。     

脂肪细胞分化相关基因在大鼠再生肝中表达变化

肝脏由多种细胞构成,肝再生与细胞分化密切相关,细胞分化受基因转录水平调控。为在基因转录水平了解脂肪细胞分化基因在大鼠肝再生中作用,本文用搜集网站资料和查阅相关论文等方法获得上述基因,用Rat Genome2302.0芯片检测它们在大鼠肝再生(liver regeneration,LR)中表达情况,将三次检验结果相同或相似、在肝再生中表达变化2倍以上、真手术组和假手术组相比差异显著的基因视为肝再生相关基因。初步证实上述基因中75个基因与肝再生相关。肝再生启动(PH后0.5-4h)、G0/G1过渡(PH后4-6h)、细胞增殖(PH后6-66h)、细胞分化和组织结构功能重建(PH后72-168h)等四个阶段起始表达的基因数为44、13、30和1;基因的总表达次数为88、58、302和90。表明相关基因主要在肝再生启动阶段起始表达,在不同阶段发挥作用。它们共表达上调313次、下调167次,分为43种表达方式。表明肝再生中脂肪细胞发生和分化相关基因活动多样和复杂。根据本文研究结果推测,上述基因不仅调节脂肪细胞分化,而且参与肝再生的生理生化活动。     

Isolation and characterization of a human putative receptor protein kinase cDNA STYK1*

Protein kinases (PKs) represent a well studied but most diverse protein superfamily. The covalent, reversible linkage of phosphate to serine, threonine, and tyrosine residues of substrate proteins by protein kinases is probably ubiquitous cellular mechanism for regulation of physiological processes. It is known to us that most signaling pathways impinge at some point on protein kinases. Here we report a human putative receptor protein kinase cDNA STYK1. The STYK1 cDNA is 2749 base pairs in length and contains an open reading frame encoding 422 amino acids. The STYK1 gene is mapped to human chromosome 12p13 and 11 exons were found. RT-PCR showed that STYK1 is widely expressed in human tissues.     

PKC site mutations reveal differential modulation by insulin of NMDA receptors containing NR2A or NR2B subunits

Insulin modulates N-methyl-d-aspartate (NMDA) receptors in the CNS and potentiates currents of recombinant NMDA receptors in a subunit-specific manner in Xenopus oocytes. Previously we identified two sites in the NR2B C-terminus as targets for direct phosphorylation by C-type protein kinases (PKCs). Mutating these sites reduced insulin potentiation of currents by one half, reflecting the PKC-mediated portion of the NR2B insulin effect. The PKC-proline rich tyrosine kinase (Pyk2)-Src family kinase pathway may also mediate insulin potentiation. A dominant negative Pyk2 mutant significantly reduced insulin potentiation when co-expressed with NR2B-containing receptors, suggesting that Pyk2 and downstream Src-family tyrosine kinases are involved, along with PKCs, in insulin potentiation of NR2B. The NR2A C-terminus contains two residues homologous to the NR2B PKC targets. Mutating both these sites eliminated insulin potentiation of NR2A-containing receptors, while co-expression of dominant negative Pyk2 had no effect. Together, these data indicate that PKCs alone mediate the NR2A insulin effect. When tested individually for importance in insulin potentiation, the two PKC sites showed an additive effect in potentiation of NR2A-containing receptors. Insulin modulation of NR2A-containing receptors is mediated solely by PKCs, whereas insulin modulation of NR2B-containing receptors is mediated by PKCs and tyrosine kinases (PTKs).     

Specific desensitization of the epidermal growth factor receptor by pp60v-src

BALB/c 3T3 cells infected with a temperature-sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v-src tyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v-src exhibits a very rapid activation t1/2 (less than 5 min) of protein kinase activity on decreasing the temperature from 40 degrees C to 35 degrees C. This change in temperature was also found to induce a very rapid decrease in the affinity for 125I-EGF of receptors on the RSV-LA90-infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of 3H-bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v-src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactivation of pp60v-src.     

Influence of sustained mechanical stress on Egr-1 mRNA expression in cultured human endothelial cells

Restenosis after initially successful balloon angioplasty of coronary artery stenosis remains a major problem in clinical cardiology. Previous studies have identified pathogenetic factors which trigger cell proliferation and vascular remodeling ultimately leading to restenosis. Since there is evidence that endothelial cells adjacent to the angioplasty wound area synthesize factors which may initiate this process, we investigated the effects of mechanical stimulation on endothelial gene expression in vitro and focussed on the influence of sustained mechanical stress on expression of immediate early genes which have previously been shown to be induced in the vascular wall in vivo. Primary cultured human umbilical vein endothelial cells (HUVEC) and the human endothelial cell line EA.hy 926 were plated on collagen-coated silicone membranes and subjected to constant longitudinal stress of approximately 20% for 10 min to 6 h. Total RNA was isolated and the expression of the immediate early genes c-Fos and Egr-1 was studied by Northern blot analysis. We found a rapid upregulation c-Fos and Egr-1 mRNA which started at 10 min and reached its maxima at 30 min. HUVEC lost most of their stretch response after the third passage whereas immediate early gene expression was constantly in EA.hy 926 cells. Using specific inhibitors we investigated the contribution of several signal transduction pathways to stretch-activated Egr-1 mRNA expression. We found significant suppression of stretch-induced Egr-1 mRNA expression by protein kinase C (PKC) inhibition (p < 0.05) and by calcium depletion (EA.hy926, p < 0. 05; HUVEC, p = 0.063). No effect on stretch-activated Egr-1 mRNA expression was detected by inhibition of protein kinase A, blockade of stretch-activated cation channels or inhibition of microtubule synthesis. We conclude that sustained mechanical strain induces Egr-1 mRNA expression by PKC- and calcium-dependent mechanisms.     

The protein kinases tightly bound to DNA are present in normal tissues and in regenerating liver, but strongly decreased in hepatomas

We have previously described in rat liver two protein kinases tightly bound to DNA, one is serine-specific, the other arginine-specific. In this work we show that both enzymes are present in various rat tissues and in liver from various species. Both kinase specific activities are strongly decreased in methyl-DBA-induced hepatomas and in HTC cells but not in regenerating liver after hepatectomy. This decrease is then not related to cell proliferation.     

Regulation of immediate-early gene expression in rat retinal ganglion cells after axotomy and during regeneration through a peripheral nerve graft

To determine mechanisms of structural plasticity in adult CNS neurons, we investigated the expression of immediate early genes (IEGs) in the rat retina. Gene products of different IEG families (JUN and FOS proteins) and cAMP-responsive element binding protein (CREBP) were examined by immunohistochemistry under three different paradigms. Normal rats which were not axotomized were compared with axotomized animals, where retinal ganglion cells (RGCs) were axotomized by intraorbital optic nerve cut and retrogradely labeled with fluorogold (FG). Under these circumstances, RGCs show only transient sprouting, followed by continuous retrograde RGC degeneration. In the third group, after the optic nerve lesion, adult rats additionally received a sciatic nerve graft to the transected optic nerve stump. This allows some RGCs to regenerate an axon into the grafted nerve. In both groups, the time course of RGC survival and JUN, CREB, and FOS protein expression was monitored. In normal animals, JUN-Immunoreactivity (JUN-Ir) was not detectable in the retinal ganglion cell layer. JUN-Ir was induced in about 70% of all FG-positive RGCs 5 days after axotomy. The expression of JUN-Ir started to decline 8 days after axotomy. Only a few JUN-Ir-positive RGCs were found after 2 weeks. In transplanted animals, however, the numbers of JUN-Ir-positive RGCs were significantly higher 2 and 3 weeks after transplantation compared to animals that exclusively received axotomy. Furthermore, in grafted rats about 70% of the regenerating RGCs expressed JUN-Ir 2 weeks after grafting as compared to only 38% JUN-positive RGCs among the surviving but not regenerating RGCs. In normal animals CREBP-Ir was constitutively expressed in nearly all cells of the retinal ganglion cell layer. The decline in number of CREBP-Ir-positive cells paralleled the axotmy-induced RGC death. FOS-Ir-positive cells were not found in the ganglion cell layer at any time. These results demonstrate a selective and transient JUN expression of RGCs after axotomy which is sustained during axonal regeneration. This suggests that sciatic nerve grafts are able to regulate the expression of JUN proteins in axotomized RGCs of adult rats. 1994 John Wiley & Sons, Inc.