Nuclear and cytosolic thioredoxin redox state

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DESCRIPTION: (Adapted from the Applicant's Abstract): Reactive oxygen species (ROS) and endotoxin (LPS)-induced cytokine responses have been implicated in the pathogenesis of alcoholic liver disease. A key question in ROS signaling is how an oxidant signal can effect transcriptional activation when the latter is inhibited by oxidants. This can occur if the nuclear redox is controlled independently of the cytoplasmic redox. However, the prevailing literature indicates that the nuclear GSH pool is similar to the cytoplasmic pool because nuclear pores are permeable to small molecular weight compounds. I propose to use thioredoxin (Trx) to study redox in the nucleus. I have established methods to measure the redox of Trx using a novel Western blot technique. My hypothesis is that the redox state of nuclear Trx will differ from cytoplasmic Trx under oxidative conditions induced by physiologic and toxicologic stimuli. I will test this hypothesis by measuring redox of nuclear and cytoplasmic Trx and redox of cellular GSH/GSSG in a human monocyte cell line (THP-1) in response to differentiating agents (Vit D3 and phorbol ester) and LPS. I will determine whether isolated nuclei have the capacity to independently regulate Trx redox state and whether nuclear Trx redox state affects NF-kB-dependent gene expression. The research should provide an important advance in knowledge and also a solid foundation for my future career.