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論文

Order effect of repair processes to clustered DNA damage

白石 伊世; 鈴木 雅雄*; 鹿園 直哉; 藤井 健太郎; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i92 - i93, 2014/03

In a living cell, cluster DNA damage is thought to be processed by several different pathways simultaneously or sequentially. Under this situation the cellular response to cluster DNA might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or excision activities of latter proteins. In the present study, we investigate whether initial enzymatic repair affects latter processes. Plasmid DNA exposed to C ion is treated with two base excision repair enzymes, Nth and Fpg, which convert pyrimidine and purine lesions to a SSB, respectively. Obtained results show that the amount of enzymatically induced SSB is very slightly less in DNA sample treated with Nth first and then Fpg than that of other treatments. These results indicate that the configuration-change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme.

論文

Dependence of the yields of AP sites and AP clusters produced in plasmid DNA on scavenging capacity and LET

椎名 卓也*; 渡辺 立子; 鈴木 雅雄*; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i15 - i16, 2014/03

BB2013-0476.pdf:0.21MB

The object of the study is to reveal whether the induction process of an abasic site (AP site) and the clustered DNA damage which contain AP sites (AP cluster) depends on ionizing density of radiation. In order to clarify the relation between track structure of C ions and the induction processes of an AP site or AP cluster, we measure the yield of AP using Nfo protein as an enzymatic probe, which converts an AP site to detectable single strand break. Several scavenging capacities in the samples are tested to estimate the effect of OH radicals. We compare those experimental yields with theoretical ones that are obtained from Monte Carlo track simulation to develop a model of AP site induction by irradiation. The yields of AP sites analyzed by the simulation are in good agreement with experimental ones. As a result, we newly determined the branching ratio of induction of the AP the site in the simulation.

論文

Live-cell imaging study of mitochondrial morphology in mammalian cells irradiated

嘉成 由紀子; 野口 実穂; 神長 輝一; 坂本 由佳; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i129 - i130, 2014/03

In this study, we report a new approach of tracing the mitochondrial morphological change of the cells exposed to high LET radiation using a live-cell imaging technique. We used the NMuMG (Normal murine mammary gland)-FUCCI2 cell line. Using the FUCCI2 expressing cells, we can observe red fluorescence in the nuclei of G1 phase cells and green fluorescence in the nuclei of S/G2/M phase cells. We labeled mitochondria by Mitotracker Red. Kinetics of mitochondrial morphology was analyzed by the live-cell imaging technique using a fluorescence microscope. Mitochondrial images were captured for 4 days after irradiation. In a preliminary study, we found that X-ray irradiation of cells caused mitochondrial fragmentation, and proportion of cell population with fragmented mitochondria increased with increasing dose and with time after irradiation. It is demonstrated that the method proposed in this study works successively to trace cytoplasmic effects by high LET irradiation.

論文

Visualization of cell-cycle modification by ionizing irradiation in single HeLa cells using fluorescent ubiquitination-based cell-cycle indicator

神長 輝一; 坂本 由佳; 嘉成 由紀子; 野口 実穂; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i127 - i128, 2014/03

Fluorescent ubiquitination-based cell cycle indicator (FUCCI) HeLa cells are one of useful model cell lines to visualize a cell-cycle because their nuclei show different colors; orange indicating G1; green indicating S/G2. In order to establish a novel assay system to study cell-cycle modification by high LET irradiation such as ion beams for cancer therapy we have observed time-lapse images of HeLa-FUCCI cells irradiated as a preliminary experiment using conventional X-rays instead of high LET ion beams. The cell-cycle was strongly arrested by irradiation at S/G2 and never progressed to G1. In contrast, cells irradiated at G1 progress to S/G2 with a similar time course as non-irradiated control cells. These results show that single FUCCI cell exposure and live cell imaging are a powerful method to trace the single cell effect of high LET irradiation on the cell-cycle in future.

論文

Three-dimensional culture of HeLa-FUCCI cells for study of bystander cell cycle effect of high LET particles

坂本 由佳; 神長 輝一; 嘉成 由紀子; 野口 実穂; 横谷 明徳

Journal of Radiation Research, 55(Suppl.1), p.i120 - i121, 2014/03

In order to examine bystander effects in the 3D cell system, we have developed a FUCCI-HeLa spheroid system. FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator) cells show specific colors of cell nuclei depending on a cell cycle. Thus we can easily trace cell cycle modifications by irradiation. We observed bystander cell-cycle delay as preliminary tests using monolayer culture of the HeLa-FUCCI cells. It will be very interesting to examine whether the cell-cycle effect also appears in the 3D cell system exposed to single high LET particles. We have determined suitable conditions for the spheroid culture, such as size of spheroids and methods of stable fixing a spheroid in a dish to perform the microbeam irradiation, and observation of the cell cycles of each cell in a spheroid after irradiation using a time-lapse micro-imaging technique.

論文

Radiation-quality-dependent bystander effects induced by the microbeams with different radiation sources

鈴木 雅雄*; Autsavapromporn, N.*; 宇佐美 徳子*; 舟山 知夫; Plante, I.*; 横田 裕一郎; 武藤 泰子*; 鈴木 芳代; 池田 裕子; 服部 佑哉; et al.

Journal of Radiation Research, 55(Suppl_1), P. i54, 2014/03

It is essentially important for evaluating risk such a low-dose-rate exposure as the accident of Fukushima Daiichi Nuclear Power Plants to examine bystander effects induced by low-LET electromagnetic radiations, such as X or $$gamma$$ rays. We have been studying the cellular responses in normal human fibroblasts by targeted cell nucleus irradiations with monochromatic X-ray microbeams (5.35 keV) produced by Photon Factory in High Energy Accelerator Research Organization. The results indicated that the bystander effect in cell- killing effect was observed in the targeted cell nucleus irradiation, not in the random irradiation containing both cell nucleus and cytoplasm by Poisson distribution. The results suggest that energy deposition in cytoplasm is an important role of inducing bystander effects in case of low-LET radiations. We have also been investigating high-LET-radiation induced bystander effects using the heavy-ion microbeams at Takasaki Ion Accelerators for Advanced Radiation Application in Japan Atomic Energy Agency. Only 0.04% of the total numbers of normal human fibroblasts were irradiated with C-ion (220 MeV), Ne-ion (260 MeV) and Ar-ion (460 MeV) microbeams collimated at 20 micro meter in diameter. Cell-killing effect and gene mutation at HPRT locus in the cells irradiated with C ions were higher beyond our expectations and returned the estimated values that only 0.04% of the total cells were irradiated when using the specific inhibitor of gap junctions. On the other hand, no induced biological effects were observed in Ne and Ar ions whether the inhibitor was applied or not. The result suggested that the C-ion microbeam was capable of inducing bystander cellular effects via gap junction mediated cell-cell communication. There is clear evidence that bystander cellular effects are dependent on radiation quality.

口頭

Systematic measurements of lineal energy distributions of various energetic heavy ion beams using a wall-less tissue equivalent proportional counter

津田 修一; 佐藤 達彦; 渡辺 立子; 高田 真志*

no journal, , 

重粒子線に対する生物効果を実験的に評価するうえで、重粒子線の飛跡及びその近傍における詳細なエネルギー付与分布データは重要である。本研究では機構で開発されたエネルギー付与分布計算モデルの精度検証を行うために、壁なし型の組織等価比例計数管を用いて、重粒子ビームの飛跡沿いに生成される高エネルギー電子を含む線エネルギー(y)分布をさまざまなエネルギーの重粒子ビーム種に対して系統的に取得してきた。これまでに陽子,炭素等について幅広い照射ビーム条件においてy分布データを取得し、yはエネルギー付与の指標として適していることを示した。今回、入射重粒子ビームと二次粒子の寄与をより詳細に調べるために、ペンシル状のビームを用いてy分布を測定した結果、y分布の形状に関して実験結果とPHITSコードのエネルギー分布計算結果に違いがあることがわかった。

口頭

Astronaut dose estimation using the PHITS code in combination with realistic models of the Kibo module and the MATROSHKA phantom

佐藤 達彦; 永松 愛子*; 武田 和雄*; 仁井田 浩二*; Puchalska, M.*; Sihver, L.*; Reitz, G.*

no journal, , 

宇宙飛行士の被ばく線量評価は、長期宇宙滞在を計画するうえで極めて重要となる。そのため宇宙航空研究開発機構(JAXA)は、欧州宇宙機構(ESA)などと協力して、国際宇宙ステーションきぼうモジュール内に人体ファントムMATROSHKAを設置し、その被ばく線量を計測した。本研究では、原子力機構が中心となって開発している粒子線輸送計算コードPHITSに、JAXAが開発したきぼうモジュールモデル及びChalmers工科大学が開発したMATROSHKAファントム数値モデル(NUNDO)を組合せ、測定結果のシミュレーションによる再現精度を検証した。その結果、臓器線量などの測定値と計算値は比較的よく一致することがわかり、PHITSが宇宙飛行士被ばく線量に適応できることを実証した。

口頭

Exploration of the site responsible for the radiation response of the salt chemotaxis learning in ${it C. elegans}$ using heavy-ion microbeam

坂下 哲哉; 鈴木 芳代; 武藤 泰子*; 服部 佑哉; 池田 裕子; 横田 裕一郎; 舟山 知夫; 浜田 信行*; 深本 花菜*; 小林 泰彦

no journal, , 

神経系のモデル生物として知られる線虫を用いて、これまでに、化学走性学習に対する放射線の影響を調べた結果、全身照射した線虫の化学走性学習が特定の条件下においてのみ一時的に亢進することを明らかにした。しかし、線虫のどの部位における放射線応答が、化学走性学習の亢進を誘導するかは明らかでない。そこで、マイクロビームを用いて、線虫の化学走性学習に対する直接的な放射線の影響部位を明らかにすることを目的とした。炭素イオンマイクロビームを、シリコン樹脂製小動物用マイクロデバイスを用いて、非麻酔下の線虫の頭部(哺乳類での中枢神経に相当する神経環がある部位),腸部,尾部に照射し、化学走性学習への影響を調べた。その結果、線虫の頭部と尾部への照射により、化学走性学習の有意な亢進が観察された。この結果は、全身照射実験で明らかとなったGPC-1タンパク質の局在性(頭部,尾部)と関係している可能性がある。線虫で見いだされた神経機能への放射線影響メカニズムは、ヒトなど高等生物の脳神経系機能に対する放射線照射の影響の解明に役立つ可能性がある。

口頭

Construction of plasmid containing clustered DNA damage to analyze its in vivo repair

高橋 桃子*; 鹿園 直哉

no journal, , 

We constructed lesion-containing plasmids using a novel approach in order to analyze repair of clustered DNA damage. Single stranded circular DNA (the 1st strand) was synthesized in vitro from DNA oligonucleotide as a primer. The template, which is single-stranded circular DNA containing uracil, was then digested with UDG and nucleases. From the resulting 1st strand, the 2nd strand was again synthesized in vitro from a complementary DNA oligonucleotide. As DNA lesions could be placed at any position in the DNA oligonucleotide on either strand, the method allows us to construct a clustered DNA damage site in a plasmid. We have confirmed that the double-stranded plasmid DNA could be constructed with DNA lesions. The constructed plasmid would be highly useful in analyzing in vivo processing of clustered DNA damage. The level of repair of lesions within clustered DNA damage in E. coli is discussed.

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