We have previously shown that telomerase-negative immortalized cells have extra-chromosomal telomere repeat (ECTR) DNA that appeared to form a complex with telomere-repeat binding proteins. We show here isolation and characterization of ECTR DNA-protein complexes. ECTR DNA was recovered through successive centrifugation processes as DNA-protein complexes with heterogeneous sizes of about that of ribosome small subunit. The complex contained small telomere-repeat DNA (<1kb) and several kinds of proteins including TRF2 that is a typical telomere binding protein.

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Clonal rat pheochromocytoma (PC12) cells have been widely used to study the molecular mechanisms of neurite outgrowth and neurotransmitter release. We obtained neurotransmitterhypersensitive PC12 variant cells (PC12m12) in which neurite outgrowth was strongly stimulated by the neurotransmitters acetylcholine, serotonin and dopamine under the condition of nerve growth factor (NGF) treatment. When acetylcholine and NGF were introduced to the PC12m12 cells, the frequency of neurite outgrowth increased by approximately 14 fold compared to that in the case of treatment with NGF alone. However, effects of acetylcholine and dopamine on neuritogenesis in drug-hypersensitive PC12m3 variant cells were not detected. In neurotransmitter-hypersensitive PC12m12 variant cells, acetylcholine and dopamine activated mitogen-activated protein (MAP) kinase, whereas acetylcholine and dopamine did not activate MAP kinase in PC12m3 cells. Acetylcholine-induced neurite outgrowth was inhibited by an acetylcholine inhibitor, nicardipine, and by a MAP kinase inhibitor, U0126, in PC12m12cells. When dopamine-stimulated PC12m12 cells were simultaneously treated with low-frequency sound wave, neurite outgrowth activity was greatly enhanced. Furthermore, we detected activation of cyclic-AMP responsive element binding protein (CREB) by acetylcholine or low-frequency sound wave in PC12m12 cells. CREB is a transcription factor that is the target of MAP kinase. These findings suggest that neurotransmitters and sound waves induce neurite outgrowth through MAP kinase and CREB pathways in PC12m12 cells.

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生体内において後根神経節細胞は、胎生期に双極細胞から偽単極細胞へと変化する。今回、我々は後根神経節の器官培養にて神経節細胞はどの様な形態変化をとるのか経時的に観察した。胎生14、16、18、20日および生後1日目のラツト後根神経節を培養し、経時的(4-32日)にBodian-大塚の渡銀染色による培養標本を作製した。神経節細胞はMatsuda&Uehara(1984)に従い、5型(1.multipolar cell, 2.spindle shaped bipolar cell, 3.bell-shapedbipolar cell, 4.short-stem unipolar cell, 5.Iong-stem unipolar cell)に分類し、その出現比率を求めた。また、画像解析装置を用い、細胞体の大きさ(細胞直径)を計測した。培養1週では、採取時期の早い神経節ほど偽単極細胞(4、5型)は少ない。培養2-3週目では採取時期による偽単極細胞の出現数の差は徐々に小さくなり、培養4週では、ほとんどの神経節の偽単極細胞は90%前後の出現比率を示した。培養初期にみられる偽単極細胞の出現比率の差は、神経節細胞の成熟度の違いによるによる単極化の速さに依存することが示唆される。また、神経節細胞体の大きさの変化は培養初期では1集団(φ20μm)であったものが、培養4週までにin vivoと同様な2集団(小細胞、大細胞)を形成する傾向にあった。

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