DNA damage and repair in cardiovascular disease

(1)氧化应激诱导血管平滑肌细胞DNA损伤与修复：应用免疫细胞化学技术发现，氧化应激(H_2O_2)可诱导核内胸腺嘧啶二醇和8-氧基胍的积聚。在30μ的H_2O_2作用下，胸腺嘧啶二醇和8-氧基胍在H_2O_2作用15分钟后在细胞核内积聚，30分钟达到高峰。在这种氧化应激诱导的DNA损伤后，DNA修复机制，如Rad 51焦点形成和BRCA1积累，被激活。RAD51的靶点形成与BRCA1的积聚不是共定位的。(2)氧化应激使S移位到细胞核的新蛋白的鉴定。我们发现已知的参与真核翻译的MAPK整合蛋白1(Mnk1)通过氧化应激转移到细胞核。在H_2O_2作用30分钟后，Mnk1被磷酸化，并与sc35共定位，表明斑点处存在磷酸化(活化)的Mnk1。为了研究修复基因的多态性与心血管疾病的关系，从有无心血管疾病患者的血液中提取基因组DNA，用聚合酶链式反应-限制性片段长度多态性方法检测XRCC3基因的多态性。大约1000名患者同意了知情同意，并进行了DNA分析。日本人的等位基因分布似乎与西方人不同。目前正在分析XRCC3基因多态与心血管疾病的关系。

(1)Oxidative stress-induced DNA damage and repair in vascular smooth muscle cells.We found that oxidative stress (H_2O_2) induced the accumulation of thymine glycol and 8-oxo-guanidine in the nucleus, by using immunocytochemical technique. With as little as 30 μM H_2O_2, thymine glycol and 8-oxo-guanidine accumulated in the nucleus 15 minutes after H_2O_2 exposure with a peak at 30 minutes. Upon this oxidative stress-induced DNA damage, DNA repair machinery, such as Rad 51 focus formation and BRCA1 accumulation, was activated. Focus formation of Rad51 did not co-localize with BRCA1 accumulation.(2)Identification of the novel protein(s) that translocate(s) to nucleus by oxidative stress.We found that MAP kinase integrating kinase 1 (Mnk1), which is known to be involed in eukaryotic translation, translocates to nucleus by oxidative stress. Mnk1 is phosphorylated 30 minutes after the exposure to H_2O_2, and co-localized with sc35, showing phosphorylated (activated) Mnk1 is present at the speckles. We are now analyzing the significance of Mnk1 phosphorylation and speckle localization in the nucleus.(3)Polymorphism in genomic repair gene and cardiovascular disease.To examine the association between polymorphism of repair gene and cardiovascular disease, genomic DNA was purified from the blood of patients with or without cardiovascular disease and polymorphism of XRCC3 gene was determined with PCR-RFLP method. About 1000 patients were agreed with the informed consent and DNA analysed. The distribution of the alleles in Japanese seems different from that in Western people. The association between polymorphism of XRCC3 and cardiovascular disease is now being analysed.