Chemical and Biochemical Studies on Pathological Aspect of Nitric Oxide

一氧化氮病理学方面的化学和生化研究

• 批准号: 15350099
• 负责人: MAKINO Keisuke
• 金额: $ 9.47万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2005
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-15350099/
• 关键词: Nitric Oxide; Carcinogenesis; Deoxvoxanosine (dOxo); dOxo amidite monomer synthesis; Synthesis of DNA oligomer containing dOxo; Crosslink formation between dOxo in DNA and repair enzymes; Enzymes repairing dOxo in DNA; Recognition of dOxo in DNA by

Endogenous NO, necessary for many physiological functions, is sometimes overproduced in inflammation and therefore carcinogenic, however no clear evidence has been reported. In this study, we obtained important data to answer this essential question.1. Oxanine (Oxa), a damaged base generated from Gua by NO-or HNO2-induced nitrosative deamination, has been considered as a mutagen-potent lesion. In this study, for exploring more detailed properties of Oxa, large-scale preparation of Oxa-containing DNA oligomers (Oxa-ODN) was developed: 2'-deoxynucleoside of Oxa (deoxyoxanosine, dOxo) obtained from dGuo by HNO2-nitrosation was subjected to 5'-0-selective tritylation (yield of DMT-dOxo, 70%). Subsequently DMT-dOxo was converted by conventional phosphoramidation to DMT-dOxo-amidite (yield, 72.5%). The amidite was used for synthesizing Oxa-ODNs: The coupling yields for Oxa incorporation and the total synthesis of 25mer DNA were over 93 and 85 %, respectively.2. Using DNA oligomers with or wi … More thout dOxo as templates and primers, DNA polymerase chain elongation was investigated and we found that in the opposite site to Oxa, T and C are incorporated almost equally, indicating that Oxa is mutagenic.3. We performed screening for the base-excision repair (BER) system to find no enzymic activity for the excision of Oxa, and revealed that Oxa-polyamine adduct is repaired by the nucleotide excision and recombination repair system, indicative of the cellular Oxa formation and that other DNA-relevant enzymes such as ligase and restriction enzymes recognize Oxa as Gua.4. We also found that Oxa-recognizing BER enzymes cross-link with Oxa in the DNA duplex irreversibly and this activity is eliminated by heat, while histones binding DNA nonspecifically also crosslink Oxa in DNA duplexes although slowly. This unique crosslinking will be helpful for fishing Oxa-recognizing and-repairing enzymes.5. NMR analysis for the structure around Oxa in DNA is now being conducted to reveal the mechanism for such unique properties of Oxa. Less

许多生理功能所必需的内源性NO有时在炎症中过量产生，因此具有致癌性，但没有明确的证据报道。在这项研究中，我们获得了回答这个基本问题的重要数据。恶嗪（Oxanine，Oxa）是一种由NO或HNO 2诱导的Gua亚硝化脱氨基作用产生的损伤碱基，被认为是一种致突变性损伤。本研究中，为了探索Oxa的更详细的性质，开发了大规模制备含Oxa的DNA寡聚物（Oxa-ODN）：将通过HNO 2-亚硝化从dGuo中获得的Oxa的2 '-脱氧核苷（deoxyoxanosine，dOxo）进行5'-0-选择性三苯甲基化（DMT-dOxo，产率70%）。随后，通过常规的磷酰胺化将DMT-dOxo转化为DMT-dOxo-amidite（产率，72.5%）。将该亚酰胺用于Oxa-ODNs的合成：Oxa掺入偶联率达93%以上，25聚体DNA的总合成率达85%以上.使用DNA寡聚体与或与 ...更多信息 在不使用dOxo作为模板和引物的情况下，我们研究了DNA聚合酶链的延伸，发现在Oxa的对侧位点，T和C几乎等量掺入，表明Oxa具有致突变性.我们对碱基切除修复（BER）系统进行了筛选，发现Oxa切除没有酶活性，并揭示Oxa-多胺加合物通过核苷酸切除和重组修复系统修复，这表明细胞Oxa形成，并且其他DNA相关酶如连接酶和限制性内切酶将Oxa识别为Gua.4。我们还发现，Oxa识别BER酶与DNA双链体中的Oxa不可逆地交联，并且这种活性通过加热而消除，而非特异性结合DNA的组蛋白也交联DNA双链体中的Oxa，尽管缓慢。这种独特的交联作用将有助于Oxa识别和修复酶的捕捞.现在正在对DNA中Oxa周围的结构进行NMR分析，以揭示Oxa这种独特性质的机制。少

项目成果

A hydrogen peroxide-generating agent, 6-formylpterin, enchances heat-induced apoptosis.

过氧化氢生成剂 6-甲酰蝶呤可增强热诱导的细胞凋亡。

• 发表时间: 2005
• 期刊: Int. J. Hyprethermia 21
• 作者: S.Wada;Z.-G.Cui;T.Kondo;Q.-L.Zhao;R.Ogawa;M.Shoji;T.Arai;K.Makino;I.Furuta
• 通讯作者: I.Furuta

Protective effects of intracellular reactive oxygen species generated by 6-formylpterin on tumor necrosis factor-・-induced apoptotic cell injury in cultured rat hepatocytes

6-甲酰蝶呤产生的细胞内活性氧对肿瘤坏死因子诱导的培养大鼠肝细胞凋亡细胞损伤的保护作用

• 发表时间: 2005
• 期刊: Life Sciences 77
• 作者: H.Ishii;T.Arai;H.Mori;H.Yamada;N.Endo;K.Makino;K.Fukuda
• 通讯作者: K.Fukuda

Differential transcriptional regulation of two distinct S-adenosylmethionine synthetase genes (SAMI and SAM2) of Saccharomyces serevisiae.

酿酒酵母两种不同的 S-腺苷甲硫氨酸合成酶基因（SAMI 和 SAM2）的差异转录调控。

• 发表时间: 2003
• 期刊: Nucleic Acids Res. Supplement 3
• 作者: T.Kodaki;S.Tsuji;N.Otani;D.Yamamoto;K.S.Rao;S.Watanabe;M.Tsukatsune;K.Makino
• 通讯作者: K.Makino

T.Nakano, H.Terato, K.Asagoshi, A.Masaoka, M.Mukuta, Y.Ohyama, T.Suzuki, K.Makino, H.Ide: "DNA-protein cross-link formation mediated by oxanine. A novel genotoxic mechanism of nitric oxide-induced DNA damage"J.Biol.Chem.. 278. 25264-25272 (2003)

T.Nakano、H.Terato、K.Asagoshi、A.Masaoka、M.Mukuta、Y.Ohyama、T.Suzuki、K.Makino、H.Ide：“恶烷介导的 DNA-蛋白质交联形成。一种新颖的

K.Sugimoto, Y.Mori, K.Makino, K.Ohkubo, T.Morii: "Functional reassembly of a split PH domain"J.Am.Chem.Soc.. 125. 5000-5004 (2003)

K.Sugimoto、Y.Mori、K.Makino、K.Ohkubo、T.Morii：“分裂 PH 结构域的功能重组”J.Am.Chem.Soc.. 125. 5000-5004 (2003)