Norio Sasaki

Characterization of receptor for granulocyte colony-stimulating factor on human circulating neutrophils

We have made a mutein of human G-CSF with more stable, and potent biological activity. Using 125 I-labeled mutein human G-CSF, high affinity binding sites were identified on human circulating neutrophils. Receptor number per cell was 560 with a Kd of 250 pM. The human G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000.

Effects of estrogen replacement on insulin-like growth factor I concentrations in serum and bone tissue and on interleukin 1 secretion from spleen macrophages in oophorectomized rats

SummaryOophorectomy (OOX) has been known to increase bone turnover, but its precise mechanism is not fully understood. In order to further investigate the mechanism, we determined insulinlike growth factor I (IGF-I) concentrations in serum and bone tissue and interleukin 1 (IL-1) release from spleen macrophages in oophorectomized rats because it has been demonstrated that IGF-I stimulates bone formation and IL-1 stimulates bone resorption.Female 8-week-old Wistar rats were divided into four groups: (1) control, (2) OOX, (3) OOX given estradiol, and (4) control given estradiol. Ten μg/kg of 17β-estradiol was given daily by subcutaneous injection. After 5 weeks of treatment, IGF-I concentrations in the extract from right femur and in serum were determined by specific radioimmunoassay. IL-1 activity released from lipopolysaccharide (LPS)-stimulated spleen macrophages was determined by bioassay. IGF-I contents in the femur and IGF-I concentrations in serum in oophorectomized rats were significantly higher than those in control rats. Treatment by estradiol inhibited the increase in IGF-I concentrations both in femur and in serum. IL-1 release from LPS-stimulated spleen macrophages in oophorectomized rats was increased, and treatment by estradiol also inhibited the stimulated IL-1 release. The ash weights and the calcium contents of left femur in oophorectomized rats were lower than those in control rats. These results suggest that both IGF-I and IL-1 may be involved in the mechanism of the regulation of bone turnover in oophorectomized rats.

Stimulation of human chorionic gonadotropin and progesterone secretion by tumor promoters, phorbol ester and teleocidin B, in cultured choriocarcinoma cells

Both 12-O-tetradecanoyl-phorbol-1-acetate and teleocidin B stimulated the secretion of human chorionic gonadotropin by cultured choriocarcinoma cells. These tumor promoters also stimulated production of progesterone in the cells. However, the 2 tumor promoters did not exert a marked effect on the cellular binding of epidermal growth factor that also had a stimulatory effect on production of these hormones.

Characterization of Receptors for Insulin-Like Growth Factors in Human Brain

The structural properties of receptors for insulin-like growth factors (IGFs) in human brain were studied. Brain membranes were incubated with 125I-IGF-I or II, cross-linked with disuccinimidyl suberate and subjected to electrophoresis under reducing conditions and autoradiography. Two proteins with apparent molecular weights of 120 and 220 kD were specifically labeled. The labeled proteins were immunoprecipitated with monoclonal antibody to type IIGF receptors, indicating that they represent alpha-subunit and its dimer of type IIGF receptor. The size of brain alpha-subunit was smaller than placental alpha-subunit (130 kD). Treatment with N-glycosidase F reduced the brain alpha-subunit from 120 to 95 kD and the placental alpha-subunit from 130 to 105 kD. Neuraminidase decreased the placental alpha-subunit from 130 to 125 kD, but it had no effect on the mobility of the brain alpha-subunit. Solubilized IGF-I receptors from placenta were retained by wheat germ agglutinin and concanavalin A columns, and eluted with the specific sugars. In contrast, solubilized IGF-I receptors from brain did not bind to these columns. These results indicate that human brains have only type IIGF receptors and that the molecular size of the alpha-subunit in brain receptors is smaller than in placental receptors. The size discrepancy may result from the differences in both protein and carbohydrate moieties.