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 Subsequently mice fed gris or DDC, then withdrawn from the drugs for a month to allow MBs to disappear, will be challenged in vitro or in vitro to induce MB formation. 2) Molecular biology technology will be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.
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