マウス歯発生時におけるインシュリン様成長因子ファミリーの遺伝子発現(Gene expression of insulin-like growth factor family during tooth development of the mouse)

マウス臼歯発生時におけるインシュリン様成長因子(IGF)とIGF結合蛋白質(IGFBP)の発現の時間的・空間的パターンを調べた。IGFとIGFBP発現はin situハイブリダイゼーションにて決定した。E14マウス胚の帽状期、E15胚の帽状期後期、E17-P0の鐘状期、P0-P13のクラウン形成期、P5-P13のルート形成期につき、IGF-I、IGF-II、IGFBP-2、-3、-4、-5、IGF-I受容体(IGF-IR)の発現部位と時期を網羅した。IGFおよびその作用調節因子は歯の成長分化の局所仲介因子となり、歯胚の上皮と間充織で局所的に発現するIGFBP-2、-3、-4、-5は歯の成長分化に役割を果たす可能性が示唆された。マウス臼歯発生時におけるインシュリン様成長因子(IGF)とIGF結合蛋白質(IGFBP)の発現の時間的・空間的パターンを調べた。IGFとIGFBP発現はin situハイブリダイゼーションにて決定した。E14マウス胚の帽状期、E15胚の帽状期後期、E17-P0の鐘状期、P0-P13のクラウン形成期、P5-P13のルート形成期につき、IGF-I、IGF-II、IGFBP-2、-3、-4、-5、IGF-I受容体(IGF-IR)の発現部位と時期を網羅した。IGFおよびその作用調節因子は歯の成長分化の局所仲介因子となり、歯胚の上皮と間充織で局所的に発現するIGFBP-2、-3、-4、-5は歯の成長分化に役割を果たす可能性が示唆された

Image-based quantitative determination of DNA damage signal reveals a threshold for G2 checkpoint activation in response to ionizing radiation

Background: Proteins involved in the DNA damage response accumulate as microscopically-visible nuclear foci on the chromatin flanking DNA double-strand breaks (DSBs). As growth of ionizing radiation (IR)-induced foci amplifies the ATM-dependent DNA damage signal, the formation of discrete foci plays a crucial role in cell cycle checkpoint activation, especially in cells exposed to lower doses of IR. However, there is no quantitative parameter for the foci which considers both the number and their size. Therefore, we have developed a novel parameter for DNA damage signal based on the image analysis of the foci and quantified the amount of the signal sufficient for G2 arrest.Results: The parameter that we have developed here was designated as SOID. SOID is an abbreviation of Sum Of Integrated Density, which represents the sum of fluorescence of each focus within one nucleus. The SOID was calculated for individual nucleus as the sum of (area (total pixel numbers) of each focus) x (mean fluorescence intensity per pixel of each focus). Therefore, the SOID accounts for the number, size, and fluorescence density of IR-induced foci, and the parameter reflects the flux of DNA damage signal much more accurately than foci number. Using very low doses of X-rays, we performed a "two-way" comparison of SOID of Ser139-phosphorylated histone H2AX foci between G2-arrested cells and mitosis-progressing cells, and between mitosis-progressing cells in the presence or absence of ATM or Chk1/2 inhibitor, both of which abrogate IR-induced G2/M checkpoint. The analysis revealed that there was a threshold of DNA damage signal for G2 arrest, which was around 4000~5000 SOID. G2 cells with < 4000 SOID were neglected by G2/M checkpoint, and thus, the cells could progress to mitosis. Chromosome analysis revealed that the checkpoint-neglected and mitosis-progressing cells had approximately two chromatid breaks on average, indicating that 4000~5000 SOID was equivalent to a few DNA double strand breaks.Conclusions: We developed a novel parameter for quantitative analysis of DNA damage signal, and we determined the threshold of DNA damage signal for IR-induced G2 arrest, which was represented by 4000~5000 SOID. The present study emphasizes that not only the foci number but also the size of the foci must be taken into consideration for the proper quantification of DNA damage signal

A Possible Role of Stress-Induced Premature Senescence, SIPS, as a Producer of the Stress-Resistant Microenvironment

The microenvironment is consisted both of soluble factors involving growth factors and of insoluble factors. Stroma cells contribute to form the microenvironment through a secretion of these factors. Fibroblast, which is known as stroma cells, also secretes various soluble/ insoluble factors, but the secretion level is significantly up-regulated when they reach to a finite replicative lifespan. Recent accumulating studies not only in vitro but also in vivo provide us that secreted proteins from senescent cells promote pro-survival pathway in bystander cells, especially tumor cells rather than normal cells. Since various stresses including ionizing radiation (IR) prematurely induces cellular senescent stage, called Stress-Induced Premature Senescence (SIPS), there is the possibility that the secretion pathway in cells undergoing SIPS is also activated. Here, we propose that pro-survival factor is secreted from SIPS cells to provide the stress-resistant microenvironment

発生中および除神経された味蕾におけるβ-カテニンの発現と活性化(Expression and activation of β-catenin in developing and denervated taste buds)

出生後発育期間または神経除去後期間の味蕾における活性化β-カテニンの発現の発現機構を明らかにすることを目的として、マウス味蕾におけるβ-カテニン、Wnt10b、Wnt5aおよびWntのレセプターであるFrizzled(Fzd)の発現パターンを調べた。これらパターンはin situハイブリダイゼーションおよび免疫組織化学法を用いて測定した。活性化β-カテニンの細胞質内の蓄積および核内への移行をソニックヘッジホッグ(Shh)免疫反応性基底細胞および味蕾内細胞において観察し、WntおよびそのレセプターであるFzd-1および-3の細胞内挙動を追跡した。これらの結果から、発生および再生の初期段階におけるβ-カテニン転写増大が基底細胞および未熟細胞の味蕾細胞への分化を促進すること、およびマウス味蕾細胞におけるWnt10bおよびFzd-1および-3はWnt/β-カテニン経路を構成し、その経路がβ-カテニンを活性化して、発生および再生の初期段階におけるβ-カテニン遺伝子の発現を上方調節する役割を持つことが示唆された。出生後発育期間または神経除去後期間の味蕾における活性化β-カテニンの発現の発現機構を明らかにすることを目的として、マウス味蕾におけるβ-カテニン、Wnt10b、Wnt5aおよびWntのレセプターであるFrizzled(Fzd)の発現パターンを調べた。これらパターンはin situハイブリダイゼーションおよび免疫組織化学法を用いて測定した。活性化β-カテニンの細胞質内の蓄積および核内への移行をソニックヘッジホッグ(Shh)免疫反応性基底細胞および味蕾内細胞において観察し、WntおよびそのレセプターであるFzd-1および-3の細胞内挙動を追跡した。これらの結果から、発生および再生の初期段階におけるβ-カテニン転写増大が基底細胞および未熟細胞の味蕾細胞への分化を促進すること、およびマウス味蕾細胞におけるWnt10bおよびFzd-1および-3はWnt/β-カテニン経路を構成し、その経路がβ-カテニンを活性化して、発生および再生の初期段階におけるβ-カテニン遺伝子の発現を上方調節する役割を持つことが示唆された

Biological Significance of DNA Damage Checkpoint and the Mode of Checkpoint Signal Amplification

It is generally accepted that DNA damage checkpoint is the mechanism that allows time for DNA damage repair. However, several lines of evidence challenge this paradigm, especially, in the case of G1 checkpoint. The first evidence is the complete difference between the repair kinetics of DNA double-strand breaks (very rapid) and the timing of G1 checkpoint induction (very slow) after ionizing radiation. The second evidence is that inactivation of p53, which is a central player of G1 checkpoint, does not render cells radiosensitive, rather, such cells become radioresistant. Moreover, it was shown that G1 arrest persists almost permanently after irradiation, until the time when most of the initial damage should be repaired and disappear. Therefore, cells should have a mechanism to maintain G1 checkpoint signaling by amplifying the signal from a limited number of damage. In this review, we discuss what is the bona fide role of G1 arrest and how G1 checkpoint signal is maintained long after irradiation

免疫不全ブタ開発に関する研究

学位の種別: 論文博士審査委員会委員 : (主査)東京大学准教授 山内 啓太郎, 東京大学教授 松本 芳嗣, 東京大学教授 内藤 邦彦, 東京大学准教授 田中 智, 東京大学准教授 角田 茂University of Tokyo(東京大学

Fabrication of Bi2212 Cross Whiskers Junction

An intrinsic Josephson junction has been successfully fabricated without any micro-fabrication technique. Two Bi2212 whiskers were crossed with one another and joined by post-annealing. The inter-whisker electrical transport properties were measured by the four-probe method. The temperature dependence of resistance exhibited metallic behavior above TC. The resistance decreased to zero around 80K, corresponding to the superconducting transition. The current-voltage characteristics at 5K exhibited a small hysteresis and voltage jump, which can be explained by the intrinsic Josephson effect.Comment: 3 page PDF fil