MALLORY BODIES INDUCED BY ALCOHOL AND DRUGS

长期目标：确定Mallory小体(MB)的发病机制 酒精性肝病在肝脏中的形成。假设是 测试表明，MBS在肝细胞中以不溶性蛋白质复合体的形式存在 这是对细胞压力的类似热休克反应的结果。这 假说认为，MBS的形成是后翻译的结果 引起细胞角蛋白(CK)构象变化的事件 蛋白。推测导致这一现象的热休克相关蛋白 变化包括泛素、转谷氨酰胺酶和热休克蛋白(HSP) 70.已知这些蛋白质会改变蛋白质的构象。 在经历了细胞应激的细胞中，热休克蛋白的反应。至 实现这一目标，具体目标如下：1)建立 MB形成的实验模型，以研究其 使用饲喂灰黄霉素或DDC的小鼠的发病机制。在……诱导之后 饲喂、热休克或热休克反应诱导剂4个月时的MBS 如高剂量酒精或砷将在体内和体内应用 体外处理的小鼠肝脏和对照。随后给小鼠喂食 GRAS或DDC，然后停用药物一个月，以允许MBS 消失，将在体外或体外受到挑战以诱导MB 队形。2)将利用分子生物学技术 1)使用原代组织培养和活体肝脏的实验模型 转谷氨酰胺酶、细胞角蛋白基因表达的切片制备 49和55，泛素和热休克蛋白70。3)合成率和转化率 ~(35)S标记蛋氨酸掺入CK 49和55的CK蛋白 将通过凝胶电泳分离和放射自显影进行研究。 4)CK 49和55的相对合成速率将在#年研究 用电子显微镜放射自显影技术研究中间纤维和微球 使用(3)H-蛋氨酸制作细胞骨架的薄片。5) 细胞角蛋白和MBS的磷酸化将用~(32)P研究。 2D电泳放射自显影检测掺入。6)MB 高压傅里叶变换用于蛋白质构象测定 红外光谱分析。因此，通过研究热休克蛋白的作用 在MB的形成过程中，我们希望表明翻译后的改变 在细胞角蛋白构象中导致MBS的形成和 不溶，对细胞角蛋白的正常周转有抵抗力。至 进一步证实热休克样反应与MBS的关系 我们将研究MBS消失的恢复情况，看看热冲击是否会 蛋白质表达和其他翻译后改变也会恢复 在肝细胞的组织培养中恢复到正常。

Long term objectives: To determine the pathogenesis of Mallory body (MB) formation in the liver of alcoholic liver disease. The hypothesis to be tested is that MBs form as an insoluble protein complex in liver cells as the result of a heat shock-like response to cellular stress. This hypothesis suggests that MBs form as a result of post translational events which cause conformational changes in the cytokeratins (CK) protein. The heat shock associated proteins postulated to cause this change include ubiquitin, transglutaminase and heat shock protein (hsp) 70. These proteins are known to alter protein conformation as a result of the hsp response in cells which have undergone cellular stress. To meet this goal, the following specific aims are: 1) establish experimental models for MB formation in order to investigate their pathogenesis using mice fed griseofulvin or DDC. After the induction of MBs at 4 months of feeding, heat shock or heat shock response inducers such as high dose alcohol or arsenic will be applied in vivo and in in vitro to pretreated mouse livers and controls. Subsequently mice fed gris or DDC, then withdrawn from the drugs for a month to allow MBs to disappear, will be challenged in vitro or in vitro to induce MB formation. 2) Molecular biology technology will be employed using the experimental models in 1) using primary tissue culture and in vivo liver slice preparations for mRNA expression for transglutaminase, cytokeratins 49 and 55, ubiquitin and hsp 70. 3) Rate of synthesis and turn over of CK proteins using (35)S labeled methionine incorporation in CK 49 and 55 will be studied by gel electrophoresis separation, and autoradiography. 4)Relative rates of synthesis of CK 49 and 55 will be studied in intermediate filaments and MBs using electron microscopy autoradiography of thin sections of cytoskeletons using (3)H-methionine. 5) Phosphorylation of the cytokeratins and MBs will be studied using (32)P incorporation detected by 2D electrophoresis autoradiography. 6) MB protein conformation determination using high pressure fourier transform intrared spectroscopy. Thus, by studying the role of heat shock proteins during MB formation we hope to show that post translational alteration in cytokeratin protein conformation causes MBs to form and become insoluble and resistant to normal turnover of cytokeratin proteins. To further substantiate the relationship of heat shock-like response and MBs we will study the recovery where MBs disappear to see if the heat shock proteins expression and other post translational alterations also revert to normal in tissue cultures of hepatocytes.