We examined first, the suitable level concentration of L-ascorbic acid (AsA) and second, the vitamin C activity of 2-O-α-D-glycopyranosyll-ascorbic acid (AA-2G) on the mineralization, and the collagen synthesis of the embryonic chick femurs in 10 days cultivation in organ culture system. In the first experiment, the contents of minerals (Ca, P and Mg) of the cultured femurs reached a maximum at the level of 0.6 mM AsA and decreased at the levels of 1.2 mM and more. The activities of alkaline phosphatase (ALP) and acid phosphatase (ACP) of the cultured femurs showed maximum values at the level of 1.2 mM AsA. The suitable concentration of AsA for the 10-day embryonic chick femurs was 0.6 mM. The second experiment focused the contents of minerals, total protein, collagen, the molar ratio of hydroxyproline to proline and the enzyme activity of the cultured femurs. These three reached a maximum at the level of 0.06 mM AA-2G and showed no difference in the values of analyzed substances except for the collagen at the levels of 0.06 mM and more. Among equimolor amount (0.6 mM) of AsA derivatives, AA-2G showed the vitamin C activity comparable to that of L-ascorbic acid-2-O-phosohate (AA-2P), and had the ability more than that of Ask These results indicate that AA-2G is extremely stable in the culture medium and has the vitamin C activity comparable to that of AA-2P. Therefore it is very useful AsA derivative for bone culture in the medium containing fetal bovine serum.

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The human megakaryoblastic cell line, MEG-01s, released particles identified by a characteristic marginal microtubule bundle and by the localization of platelet-specific glycoprotein (GPIIb/IIIa) in the plasma membrane, detected by an immunofluorescence method. The fluorescence image and size of these particles were similar to these features in human blood platelets. To analyze the regulation of particle release by MEG-01s, the effects of DNA synthesis inhibitors, tumor promoters, and protein kinase inhibitors on these cells were examined by an immunofluorescence method. The release of particles from MEG-Ols cells was enhanced by inhibitors of DNA synthesis: aphidicolin,5-bromodeoxyuridine,5-fluorodeoxyuridine, hydroxyurea, etoposide, and camptothecin. The particle release was specific to the MEG-01s cells, and did not occur in the myeloid leukemic cells, HL-60. These results suggest that the cessation of DNA synthesis may trigger terminal differentiation by synthesis of factors involving particle release by MEG-01s.

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Avian embryonic digestive tract has been used for the analysis of epithelialmesenchymal interactions in the organogenesis. Many tissue recombination experiments carried out so far demonstrated that the morphological differentiation of the epithelium is often determined by the influences of the mesenchyme, whereas the functional, molecular differentiation depends on both the mesenchymal influence and the reactivity of the epithelium. The epithelium of the avian embryonic digestive tract can be classified into two parts: cells in one part can express pepsinogen gene, a marker gene of the proventriculus (glandular stomach) under certain conditions and cells in another part never express it. The expression of ECPg gene in epithelial cells is controlled by the action of the mesenchyme, through 5' promoter region of ECPg gene. The establishment of two quantitatively different parts in digestive tract epithelium occurs just after the formation of endoderm, and is considered to be accompanied by the expression of some homeobox genes. The elucidation of functions of these genes may greatly contribute to the understanding of the mechanisms underlying organogenesis of many organ systems.

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We isolated vascular endothelial cells and smooth muscle cells from human donors of various ages and investigated their ability to secrete ET-1 and that to contract induced by ET-1. Expression of preproET-1 mRNA was enhanced with age of donor and secretion of ET-1 was also increased. Smooth muscle cells in culture contracted with changes of intracellular Ca²⁺ concentration as a response to ET-1 stimulation. Senescent cells less-responded to ET-1 stimulation than young cells. Senescent cells were suggested to have receptors for ET-1 fewer than young. Smooth muscle cells in aorta of the elderly were stained immunohistologically with anti-ET-1 antibody. When analyzed the culture medium of smooth muscle cells, it was found that smooth muscle cells synthesize and secrete ET-1. This suggests the possibility of autocrine action of ET-1 on smooth muscle cells. On the other hand, ET-1 receptor exists on the surface of endothelial cell itself, suggesting the possibility of cross-talk between endothelial cells and smooth muscle cells. Similar possibility can exist in heart: ET-1 stimulates the rhythmic heart-beat of cardiac myocytes and is produced by the cardiac myocyte itself under the condition of hypoxia.

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[in Japanese]

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