ETHANOL AND CELL TYROSINE KINASE

乙醇会影响细胞信号传递。多种细胞功能包括 受这些机制的制约。在这个项目中，目标是 研究一种新发现的乙醇对细胞酪氨酸激酶的影响。 你的假设是“乙醇调节细胞酪氨酸激酶和 A-431细胞，一种人类表皮样细胞，具有 丰富的酪氨酸激酶和周皮生长因子(EGF) 感受器。EGF受体是一种酪氨酸激酶(EGF-RTK)。基于 初步结果，将以A-431细胞为模型发起这一 项目。随后，还将对其他系统进行测试。以下两个 目标将检验这一假设。 目的一：测定和表征乙醇对小鼠心脏功能的影响。 酪氨酸激酶及其细胞底物。 [A]A-431细胞的细胞膜将用于研究乙醇的作用， 以及其他脂肪醇对基础和EGF刺激的酪氨酸的影响 使用合成的13个氨基酸的多肽底物用于其测定。 随后，用[32P]标记的完整的A-431细胞将被处理 用乙醇，然后用EGF挑战。[32P]蛋白磷酸化， [32P]磷酸氨基酸和EGF-R的自磷酸化 特色化的。作为一种功能性反应，离子传输将受到监测 并与乙醇对酪氨酸的影响相关 激活剂。 [B]乙醇对细胞信号成分的影响(例如，PLC-伽马， Pp60、GAP、PI-3激酶)，这些都是酪氨酸激酶的靶点 用特定的抗体确定的。 目标II.确定长期接触乙醇的影响 细胞酪氨酸激酶。 [A]A-431细胞将持续暴露在乙醇中一段时间 从2小时到1周不等，它们的膜酪氨酸激酶和 酪氨酸激酶底物的磷酸化(SEEI)将被监测 并与对照孵化进行了系统的比较。 [B]将使用其他细胞模型来提供进一步的证据 乙醇对酪氨酸激酶的影响。原代培养和其他细胞 脑(NG 108)、肝(Hep-1或ARL)和肾脏(OK-1或MDCK)起源 将特别被选中，因为这些器官受到深刻的影响 通过酒精摄取。 这项探索性拨款将为乙醇对环境的影响提供确凿证据 细胞酪氨酸激酶及其某些底物上。IT职能协作- 与离子传输的关系将会出现。这种影响在中国的普遍程度 不同的型号也将公之于众。该提案提供了一个新的维度 乙醇在细胞信号水平上的作用机制。此外 它在信号转导中的作用，酪氨酸激酶也作为一种 在细胞增殖、生长和发育过程中必不可少的成分。这些 因此，研究将对我们的长期评估产生影响。 在这些过程受到影响的地方，乙醇的有害影响。

Ethanol affects cell signalling. A variety of cellular functions are regulated by these mechanisms. In this project, the aim is to investigate a newly discovered effect of ethanol on cell tyrosine kinase. Thy hypothesis is that "ethanol modulates cell tyrosine kinase and the consequential response." A-431 cells, a human epidermoid cell, have an abundance of tyrosine kinase and peridermal growth factor (EGF) receptors. EGF receptor is a tyrosine kinase (EGF-RTK). Based on preliminary results, A-431 cells will be used as a model to initiate this project. Subsequently, other systems will be tested. The following two objectives will test this hypothesis. Objective I: To determine and characterized the effects of ethanol on tyrosine kinase and its cellular substrates. [A] Membranes of A-431 cells will be used to study the effect of ethanol, as well as other aliphatic alcohols, on basal and EGF stimulated tyrosine kinase using a synthetic 13 amino acid peptide substrate for its assay. Subsequently, intact A-431 cells, labelled with [32P], will be treated with ethanol and then challenged with EGF.[32P] Protein phosphorylation, [32P] phosphoamino acids, and autophosphorylation of EGF-R will be characterized. As a functional response, ion transport will be monitored in these studies and correlated with the effect of ethanol in tyrosine kinase. [B] Effects of ethanol on cell signalling components (e.g., PLC-gamma, pp60, GAP, PI-3 kinase), which are targets of tyrosine kinase will be determined using specific antibodies. Objective II. To determine the effects of long-term exposure to ethanol on cell tyrosine kinase. [A] A-431 cells will be exposed continuously to ethanol for periods ranging from 2 hrs to 1 week and their membrane tyrosine kinase and phosphorylation of tyrosine kinase substrates (seeI) will be monitored systematically and compared with control incubations. [B] Other cell models will be used to provide further evidence for the ethanol effect on tyrosine kinase. Primary cultures and other cell of brain (NG 108), liver (HEP-1 or ARL), and kidney (OK-1, or MDCK) origin will be particularly selected since these organs are profoundly affected by alcohol ingestion. This exploratory grant will provide firm evidence for ethanol affects on cell tyrosine kinase and on some of its substrates. It functional co- relationship to ion transport will emerge. Prevalence of this effect in different models will also be known. The proposal offers a new dimension into the mechanism of action of ethanol at cell signalling level. Besides it role in signal transduction, tyrosine kinase is also emerging as an essential component in cell proliferation, growth and development. These studies will therefore have an impact on our assessment of the long term detrimental effects of ethanol where these processes are affected.