Nuclear and cytosolic thioredoxin redox state

产品说明：（改编自申请人的摘要）：活性氧物质（ROS）和内毒素（LPS）诱导的细胞因子应答与酒精性肝病的发病机制有关。ROS信号传导中的一个关键问题是当转录激活被氧化剂抑制时，氧化剂信号如何影响转录激活。如果细胞核的氧化还原独立于细胞质的氧化还原而受到控制，则会发生这种情况。然而，流行的文献表明，细胞核的GSH池是类似的细胞质池，因为核孔是可渗透的小分子量的化合物。我建议使用硫氧还蛋白（Trx）来研究细胞核中的氧化还原。我已经建立了使用新的蛋白质印迹技术测量Trx的氧化还原的方法。我的假设是，细胞核Trx的氧化还原状态将不同于细胞质Trx的生理和毒理学刺激诱导的氧化条件下。我将通过测量细胞核和细胞质Trx的氧化还原和细胞内GSH/GSSG的氧化还原在人单核细胞系（THP-1）在分化剂（维生素D3和佛波酯）和LPS的反应来测试这一假设。我将确定是否孤立的细胞核有能力独立调节Trx的氧化还原状态和核Trx的氧化还原状态是否影响NF-κ B依赖的基因表达。这项研究应该提供了一个重要的知识进步，也为我未来的职业生涯奠定了坚实的基础。

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.