DNA damage and repair in cardiovascular disease

（1）氧化应激诱导的血管平滑肌细胞DNA损伤与修复我们用免疫细胞化学技术发现，氧化应激（H_2O_2）诱导胸腺嘧啶乙二醇和8-氧代胍在细胞核内积累。在30 μM H_2O_2条件下，胸腺嘧啶乙二醇和8-氧代胍在H_2O_2作用15分钟后在细胞核内积累，30分钟时达高峰。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BRCA 1积累被激活。Rad 51的病灶形成与BRCA 1的积聚不共定位。（2）新的氧化应激核转位蛋白的鉴定：我们发现，参与真核生物翻译的MAP激酶整合激酶1（MAP kinase integrating kinase 1，Mnk 1）在氧化应激条件下能够转位到核内。H_2O_2作用30分钟后，Mnk 1磷酸化，并与sc 35共定位，表明磷酸化（活化）的Mnk 1存在于斑点中。我们现在正在分析Mnk 1磷酸化和斑点定位在细胞核中的意义。（3）基因组修复基因多态性与心血管疾病的关系为探讨修复基因多态性与心血管疾病的关系，从心血管疾病患者和非心血管疾病患者的血液中提取基因组DNA，采用PCR-RFLP方法检测XRCC 3基因多态性。约1000例患者同意知情同意书和DNA分析。日本人的等位基因分布似乎与西方人不同。XRCC 3基因多态性与心血管疾病之间的关系正在分析中。

(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.