Quantitation and Visualization of Ultraviolet Induced DNA Damage Using Specific Antibodies

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of UV or sunlight is essential to characterize DNA photoproducts and their biologlcal effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis uslng an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scannlng confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containlng DNA photoproducts, and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repalr kinetics followlng irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigrlented cells and in human epidermis

Spatial and temporal cellular responses to single-strand breaks in human cells

DNA single-strand breaks (SSB) are one of the most frequent DNA lesions produced by reactive oxygen species and during DNA metabolism, but the analysis of cellular responses to SSB remains difficult due to the lack of an experimental method to produce SSB alone in cells. By using human cells expressing a foreign UV damage endonuclease (UVDE) and irradiating the cells with UV through tiny pores in membrane filters, we created SSB in restricted areas in the nucleus by the immediate action of UVDE on UV-induced DNA lesions. Cellular responses to the SSB were characterized by using antibodies and fluorescence microscopy. Upon UV irradiation, poly(ADP-ribose) synthesis occurred immediately in the irradiated area. Simultaneously, but dependent on poly(ADP-ribosyl)ation, XRCC1 was translocated from throughout the nucleus, including nucleoli, to the SSB. The BRCT1 domain of XRCC1 protein was indispensable for its poly(ADP-ribose)-dependent recruitment to the SSB. Proliferating cell nuclear antigen and the p150 subunit of chromatin assembly factor 1 also accumulated at the SSB in a detergent-resistant form, which was significantly reduced by inhibition of poly(ADP-ribose) synthesis. Our results show the importance of poly(ADP-ribosyl)ation in sequential cellular responses to SSB

Oil Fog Lubrication of High Speed Ball Bearing

In pursuit of information concerning performances of the preloaded ball bearing operating under the oil fog lubrication at high speed, the coefficient of friction and the temperature rise have been measured for the angular-contact-type ball bearing. In the practice of the oil fog lubrication, the influences of oil consumption, air consumption, viscosity and additive of oil, preload and rotating speed are investigated and compared with those of the ordinary oil lubrication. It is possible to lessen remarkably the friction of high speed ball bearing by the aid of the oil fog lubrication with less supply of oil. Even in that case, the oil fog lubrication is mostly the hydrodynamical lubrication ; consequently the friction of ball bearing under the fog lubrication depends principally on the viscosity of oil. The cooling effect of air is fairly great, but the effect of air on the bearing temperature rise is less than that of oil under the definite ratio of oil and air. Though the temperature rise and frictional moment increase proportionally to preload, the increase of preload does not influence so much upon the temperature and friction when oil is supplied sufficiently. As the rotating speed increases, the bearing temperature rises very rapidly, on the contrary the coefficient of friction increases gradually

Quantitative analysis of UV photolesions suggests that cyclobutane pyrimidine dimers produced in mouse skin by UVB are more mutagenic than those produced by UVC

International audienc

紫外線による細胞致死の主因となるDNA損傷は(6ー4)photoproductか

奈良県立医科大学医学部 / 金沢大学医学部色素性乾皮症患者由来細胞(XPーA),XPーAからの復帰突然変異細胞(XPーrevertant)および正常細胞の紫外線線量ー生存率曲線を描いた結果、XPーrevertantはCleaverらによって示されたように、正常細胞に匹敵する紫外線抵抗性を獲得していることが確認された。紫外線抵抗性とDNA損傷の修復との関係を調べるために、10J/m^2の紫外線を照射した3種の細胞におけるシクロブタン型ダイマ-と(6ー4)photoproductの修復動態を新しく樹立に成功したモノクロ-ナル抗体(TDMー2と64Mー2)を用いた酵素標識免疫法(ELISA)で検討した。(6ー4)photoproductの修復では、XPーAが照射後24時間でも20%しか修復できないのに対し、正常細胞では3時間以内に90%を越す修復がみられた。また、XPーrevertantは正常細胞にきわめて近い修復を示した。一方、シクロブタン型ダイマ-の修復においては、XPーA,XPーrevertantおよび正常細胞は照射後24時間後にそれぞれ20%,40%および60%の修復を示した。これらの結果は、XPーrevertantは(6ー4)photoproductの修復は正常であるが、シクロブタン型ダイマ-に修復欠陥を示すという点でCleaverらの結果と一致した。しかしながら、シクロブタン型ダイマ-の修復欠陥はあくまで部分欠陥であり、完全欠陥であるとするCleaverらのものとは異なる結果となった。そこで2J/m^2といった低線量の紫外線を用いることにより高い修復率が期待できる条件下で、再度シクロブタン型ダイマ-の修復実験を試みた。その結果、新しい実験条件下でもXPーrevertantはXPーAと正常細胞の中間の修復能を示し、部分的修復欠陥であることが再度確認された。また、この修復結果は紫外線照射後に複製された全DNA量で補正されても変化が見られなかった。この結果より、紫外線による細胞死の原因損傷は、(6ー4)photpproductに加えてシクロブタン型ダイマ-の可能性も除去できないことが明白となった。Cleaver et al. have established UV-resistant xeroderma pigmentosum (XP) revertant cells from UV-sensitive XP-A cells by chemical treatment. Surprisingly, UV-resistant XP revertant cells are still deficient in the repair of cyclobutane pyrimidine dimers, although they have recovered the repair ability of (6-4) photoproducts, suggesting that (6-4) photoproducts are the main UV-induced lethal lesions. To confirm these results, I examined UV-induced cytotoxicity in XP-A, XP revertant and normal human cells by colony formation method. XP-A cells were 10 times as UVsensitive as normal cells. I found that XP revertant cells had obtained almost normal UV sensitivity as reported by Cleaver et al. Next, the repair of two types of DNA damage (cyclobutane dimers and (6-4) photoproducts) was examined by the sensitive ELISA using monoclonal antibodies, which I had newly established, against photolesions. In the repair of (6-4) photoproducts, XP-A repaired only 30% within 24 hr after irradiation (10 J/m^2), while normal cells repaired more than 90% within 3 hr. XP revertant showed almost normal repair pattern. In the repair of cyclobutane dimers, XP-A, XP revertant and normal cells repaired 20%, 40% and 60% within 24 hr after irradiation, respectively. I confirmed Cleaver\u27s results showing that XP-revertant had almost normal repair on (6-4) photoproducts, but had reduced repair on cyclobutane dimers. However, I found that XP revertant did have reduced repair on cyclobutane dimers, but not completely inhibited repair reported by Cleaver et al. The results were confirmed by the repair experiment using low UV dose (2 J/m^2). These results suggest that XP revertant cells still have some residual repair capacity for cyclobutane dimers. Thus, these results suggest that cyclobutane pyrimidine dimers are not excluded as a candidate for the main UV-induced lethal damage.研究課題/領域番号:02680167, 研究期間(年度):1990 – 1991出典：研究課題「紫外線による細胞致死の主因となるDNA損傷は(6ー4)photoproductか」課題番号02680167（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-02680167/026801671991kenkyu_seika_hokoku_gaiyo/）を加工して作

モノクローナル抗体法による細胞DNA損傷のin situ検出と定量化

金沢大学薬学部研究課題/領域番号:58780193, 研究期間(年度):1983出典：研究課題「モノクローナル抗体法による細胞DNA損傷のin situ検出と定量化」課題番号58780193（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-58780193/）を加工して作

紫外線損傷の細胞内消長と細胞動態の関連性のモノクローナル抗体法による解析

金沢大学薬学部研究課題/領域番号59780217, 研究期間(年度):1984出典：研究課題「紫外線損傷の細胞内消長と細胞動態の関連性のモノクローナル抗体法による解析」課題番号59780217（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-59780217/）を加工して作

Chromosome 8-14 translocation in a non-African Burkitt's lymphoma with leukemic conversion.

A specific chromosome translocation, t(8q-; 14q+), was observed in a 43-year-old female with non-African Burkitt's lymphoma in which leukemic conversion had occurred. The chromosome studies used cells from ascites. The ascites was apparently the result of a primary tumor involving the ovaries and contained 68% of lymphoma cells. The frequent occurrence of abnormalities related to chromosomes 1, 8 and 14 in African and non-African Burkitt's lymphomas was emphasized.</p