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Doo Chol Han, Kanji Sato, Yuko Fujii, Minoru Ozawa, Hidehito Imamura, Toshio Tsushima and Kazuo Shizume

Abstract. To elucidate the effect of rT3 on iodothyronine-5′-deiodinating activity (I-5′-DA) in the liver of neonatal mice, rT3 was injected sc on the 5–8th day after birth and I-5′-DA in the liver was determined. A single injection of rT3 (0.01–1 μg/g) inhibited the ontogenetically developing I-5′-DA in a dose- and time-dependent manner. The inhibitory effect was reversible and specific for I-5′-DA. Lineweaver-Burk analysis revealed that the time- and dose-dependent decrease in the enzyme activity was due to a decrease in Vmax with no alteration in Km values (5 × 10−8 mol/l). The maximal inhibitory effect was observed at a dose of 1 μg rT3/g, whereas the inhibitory effect was diminished at greater doses (4–10 μg/g), probably owing to a contamination with T4 of the rT3 preparation administered. Furthermore, consistent with our previous in vitro findings, rT3 inhibited the I-5′-DA induced by T3 in the liver of neonatal mice. These findings suggest that rT3 inhibited I-5′-DA in the liver of neonatal mice by decreasing the amount of enzyme available to the substrate and that rT3 also elicited an antagonistic effect against T3 in the induction of I-5′-DA in vivo.

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Kanji Sato, Doo Chol Han, Minoru Ozawa, Yuko Fujii, Toshio Tsushima and Kazuo Shizume

Abstract. A highly sensitive bioassay for PTH was developed by using rat osteosarcoma cells (ROS 17/2.8). By limiting dilution, ROS cells were subcloned and the subclonal cell line (ROS 17/2.8–5) most responsive to PTH was selected. When subconfluent ROS 17/2.8–5 cells were treated with hydrocortisone for 3 days and then incubated with PTH, the cAMP response was significant at 10–40 ng/l hPTH (1–34) (4 ∼ 16 × 10−12 mol/l). Osteoclast activating factors such as human interleukin I alpha and beta, and tumour necrosis factor alpha did not stimulate cAMP production, whereas a conditioned medium of oesophageal carcinoma cells established from a patient with humoral hypercalcaemia stimulated cAMP production. By selecting PTH-responsive subclonal cells and treating them with hydrocortisone, the sensitivity for detecting PTH was improved approximately 15 times. This method will be useful in the characterization and purification of PTH-like factors produced by malignant tumours from hypercalcaemic patients.