Acta Medica Okayama 2000 新規アシルCoA : コレステロールアシルトランスフｪラーゼ阻害薬 NTE-122 の薬理学的特性に関する研究 EN No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2000 Development of nitrogen application curve in spray chrysanthemums grown in hydroponic system and its practical use in NFT system EN Hae suk Yoon The Graduate School of Natural Science and Technology, Okayama University No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2000 STUDIES ON TUMOR NECROSIS FACTOR-α AND ITS RECEPTOR IN BOVINE CORPUS LUTEUM EN Ryosuke Sakumoto The objective of this study was to investigate the physiological roles of tumor necrosis factor α (TNFα) in bovine corpus luteum (CL) function throughout the estrous cycle and the entire gestation period. In the first series of experirnents, the expression of TNFα, the presence of functional TNFα receptors, and the expression of TNF receptor type I (TNF-RI) mRNA in the CL during different stages of the estrous cycle were examined. RT-PCR showed no difference in TNFα mRNA expression during the estrous cycle. Concentrations of TNFα in the CL tissue increased significantly from the mid- to the late luteal stage and　decreased thereafter. A RT-PCR　analysis showed higher levels of TNF -RI mRNA in CL of　days 3-7 than in other stages. (125)I-TNFα binding to the membranes of bovine CL was maximal after incubation at 38 C for 48 h. The binding was much greater for TNFα than for related peptides. A Scatchard analysis revealed the presence of a high-affinity binding site in the CL membranes collected at each phase of the estrous cycle (dissociation constant (Kd); 3.60±0.58 - 5.79±0.19 nM). In contrast to TNF-RI mRNA expression, the levels of receptor protein were similar at each stage in the estrous cycle. When cultured cells of all luteal stages were exposed to TNFα (0.06-6 nM), TNFα stimulated prostaglandin (PG) F2α and PGE2 secretion by the cells in a dose-dependent fashion, especially during the early luteal phase, although it did not affect progesterone secretion. In the second series of experiments, the presence of TNFα mRNA and TNFα receptors in the bovine CL during the gestation period were investigated. The presence of TNFα mRNA and TNFα receptors on bovine CL from pregnant cows was investigated at three stages: trimesters I, II and III. TNFα mRNA was detected by an RT-PCR analysis in the CL of all stages of gestation. A Scatchard analysis revealed the presence of a high-affinity binding site (Kd; 5.1-6.9 nM) in the CL membranes collected at each stage of gestation. Furthermore, the concentrations of TNFα receptors in the CL of trimesters I (24.0± 1.95 pmol/mg protein) and III (21.6±2.39 pmol/mg protein) of gestation were significantly higher than the concentration in trimester II (14.9±2.07 pmol/mg protein). In the third series of experiments, I investigated the presence of functional TNFα receptors on the microvascular endothelial cells derived from developing bovine CL. TNFα receptors were analyzed by a radioreceptor assay using (125)I-labeled TNFα on two types of cultured endothelial cells. One has a cobblestone appearance (CS cells), and the other has a tube-like structure (TS cells). (125)I-labeled TNFα binding was maximal after incubation for 30 h at 37 C, and the specificity of binding was confinned. A Scatchard analysis showed the presence of two binding sites (high- and low- affinity) for TNFα receptors on both CS and TS cells. The Kd values and concentrations of the high-affinity binding sites for TNFα receptors were similar between CS and TS cells. However, Kd values and concentrations of the low-affinity binding sites in CS cells were significantly higher than those in TS cells. The expression of TNF-RI mRNA was determined in both cell types. Furthermore, TNFα significantly stimulated PGE2 and endothelin-1 secretion by both CS and TS cells. The final series of experiments were conducted to clarify the intracellular signaling pathway of TNFα to stimulate PGF2α production in cultured bovine luteal cells. Bovine luteal cells that were obtained from mid- (days 8-12 after ovulation) CL were incubated with TNFα (0.6 nM) and/or various compounds as follows: U-73122 (a phospholipase (PL) C inhibitor), ACA (a PL-A2 inhibitor), H-89 (a protein kinase (PK) A inhibitor), calphostin C (a PK-C inhibitor), L-NAME/L-NORG (a nitric oxide synthase inhibitor), PD98059 (a mitogenactivated protein kinase (MAPK) kinase inhibitor). U-73122 (0.1-10 μM), H-89 (0.1-10 μM), calphostin C (0.01-1 μM) and L-NAME/L-NORG (1-100 μM) did not affect TNFα-induced PGF2α secretion by the cultured cells. In contrast, ACA (1-100 μM) and PD98059 (0.1-100 μM) inhibited TNFα-stimulated PGF2α secretion by the cells in a dose-dependent fashion. The overall results in the present study indicate the local production of TNFα and the presence of functional TNF-RI in bovine CL throughout the estrous cycle and entire gestation period, and suggest that TNFα plays some roles as a paracrine factor in regulating bovine CL function. Furthermore, the present results indicate the presence of two types of TNF receptors and the expression of TNF-RI mRNA in the endothelial cells derived from bovine CL, suggesting TNFα plays two or more roles in regulating the secretory function of the endothelial cells. Finally, the present study also showed that the stimulatory effect of TNFα on PGs secretion by bovine luteal cells might be mediated via activation of the MAPK and PL-A2 pathways. No potential conflict of interest relevant to this article was reported.

Acta Medica Okayama 2000 Studies on the Synthesis of Highly Functionalized Molecules through Chemo-Enzymatic Methodology EN Koichi Mitsukura No potential conflict of interest relevant to this article was reported.