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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The dorsal root ganglion cell in fetal stage changes from bipolar cell to the pseudounipolar cell in vivo. We observed what kind of morphological change the ganglion cell took on timedependent. Rat dorsal root ganglia in embryonal days 14,16,18,20 and 1 postnatal day were cultured, and cultures were stained by silver-impregnated method in time-dependent. The stained ganglion cell was classified into 5 types with Matsuda and Uehara (1984), and the frequency of appearance in each cell type was obtained in each stage. In addition, the size (the cell diameter) of the cell body was measured using the image analysis equipment. In the culture of 1 week, the unipolar cell (type 4 or 5) as a ganglion in early stage is few. In culture of 4 weeks, the appearance ratio of unipolar cells was about 90%. It is suggested that the difference of appearance ratio of unipolar cell in early stages of culture is dependent on the difference in the degree of maturity of dorsal root ganglion cell.

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