Ken Kubota

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.

Chronic Effect of TSH on Human Thyroid Tissue in Organ Culture

Abstract The chronic effect of TSH on thyroidal cAMP concentrations and release of thyroid hormones was investigated using human thyroid tissue in organ culture. Normal human thyroid slices were placed in HAMs F-10 synthetic culture medium in Falcon organ tissue culture dishes, and incubated at 37° in a humidified atmosphere of 5% CO2 in air. Medium was changed everyday and daily T3 or T4 release was determined using concentration of T3 or T4 in the medium. After incubation, slices were transferred to the medium containing 10 mM theophylline and incubated without TSH for an additional 30 min to determine thyroidal cAMP concentrations. Thyroidal cAMP concentrations in slices incubated with 10 mU/ml of TSH increased significantly at 2, 6, and 24 hr and even on the 6th day of incubation. Daily T3 release was significantly increased above control from the 3rd day and daily T4 release from the 4th day to the 11th day of incubation with 10 mU/ml of TSH. Histologically, almost all follicles were structurally maintained even on the 11th day of incubation. These results suggest that both thyroidal cAMP concentrations and release of thyroid hormones are stimulated chronically by TSH. This organ culture system is useful for investigating chronic effects of various materials on human thyroid tissue.

Changes in Thyrotropin Binding Inhibiting Immunoglobulins (TBII) in sera of patients with Graves’ disease at the time of relapse or exacerbation

Thyrotropin Binding Inhibiting Immunoglobulins (TBII) were measured in sera of 240 patients with Graves’ disease who were followed 0–25 yr as a cross-sectioned study (21 untreated, 189 under therapy and 30 T3-suppressible and drug-discontinued patients) by using solubilized porcine thyroid TSH receptor. Assays were performed by using 50 μl of serum. All untreated 21 patients showed positive TBII. Frequency of positive patients decreased yearly with treatment although 36% of patients remained positive after 6 yr of therapy. After that time TBII were positive in 61 % of follow-up patients and in 16 positive patients who have been treated for more than 10 yr, drug therapy could not be stopped because of recurrence. TBII were positive in 6 of 30 T3-suppressible patients. As a longitudinal study changes in TBII were studied in 10 patients at the time of relapse or exacerbation. TBII increased in parallel with increases in thyroid hormone concentrations in 3 of 10 patients. Six of the others showed earlier or later TBII increases than those in thyroid hormones. One patient did not show any change in TBII, albeit thyroid hormone concentrations were found to be increased. Our observations suggest that abnormal IgGs detected as TBII in sera of patients with Graves’ disease by the present method do not explain the occurrence of hyperthyroidism.