Ca²⁺-dependent neutral proteases in hog thyroid cytosol were found to digest thyroglobulin. The protease activity was divided into two peaks by DEAE-cellulose column chromatography. Peak I was eluted at 0.2M NaCl and required only a micromolar range of Ca²⁺ for its 50% activation, while peak II, which was eluted at about 0.4M NaCl, displayed little activity until the Ca²⁺ concentration was increased at more than 10^-4M. Among various inhibitors used, thiol protease inhibitors (leupeptin, E-64 and monoiodoacetic acid) were the most effective, whereas a calmodulin antagonist (trifluoperazine) and serine protease inhibitors (phenylmethyl-sulfony-fluoride and pepstatin A) were not effective, indicating that these Ca²⁺-dependent proteases corresponded to calpains 1 and 2. Among the substrates tested, casein was the best and thyroglobulin was also a good for calpain 2. By using immunoblotting procedure with anti-thyroglobulin antibody, it has been found that calpain 2 degrades thyroglobulin to yield67K and46K thyroglobulin and further that it also degrades 40K thyroglobulin.

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In this report on two cases of anorexia nervosa associated with Graves' disease, metabolism and the relationship between the two illness are considered. Case 1 was a 25-year-old female. Anorexia was associated with a stressful life situation following marriage. One year after the onset of anorexia, her condition was diagnosed as Graves' disease. In spite of high levels of serum thyroid hormone, she did not show the clinical signs and symptoms of hyperthyroidism. The hypermetabolic state of Graves' disease seems to be suppressed by the hypometabolism of anorexia. Case 2 was a 17-year-old female whose body weight, due to anorexia, at one time had decreased from 55kg to 35.2kg. A rebound from anorexia to bulimia increased her body weight to 80kg in spite of an association with the hypermetabolic state of Graves' disease. In light of the abovementioned cases, it seems that the clinical picture of Graves' disease is usually hidden by the clinical symptoms of anorexia nervosa.

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We tried to improve the sensitivity of a radioreceptor assay for thyrotropinbinding inhibitor immunoglobulins (TBII) by modifying assay conditions. About a twofold increase in sensitivity without a loss of reproducibility was obtained by prolonging the incubation of the receptors with test serum from 15 to 120 min before the addition of ¹²⁵I-labeled thyrotropin. In 20 untreated, 49 treated patients with Graves' disease and19patients with euthyroid Graves' disease, TBII activities obtained using 120 min preincubation were significantly higher than those obtained using15min preincubation (p<0.005). No significant increase in TBII activities was observed in the presence of sera from patients with primary hypothyroidism (n=17), simple goiter (n=7), adenomatous goiter (n=11), thyroid adenoma (n=11) or cancer (n=12). TBII were detectable in 15 (47%) of32triiodothyronine-nonsuppressible Graves' patients who were receiving maintenance antithyroid drug therapy using 120min preincubation, while they were positive in only6patients (19%) using 15min preincubation. The assay using a longer preincubation period was found to be sensitive, specific and useful for diagnosis and follow-up of Graves' disease.

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This study was designed to investigate the effects of progesterone on the gonadotrophin rise after bilateral salpingo-oophorectomy (BSO). Twenty-eight regularly menstruating women underwent hysterectomy and BSO during the follicular phase of the menstrual cycle. They were divided into5groups depending on the treatment after BSO. Plasma LH and FSH were studied serially for 14 days after BSO and the patterns of LH and FSH rises were contrasted to those observed in the control group which received neither progesterone nor estrogen. LH and FSH levels in the group which were given low dose progesterone only, rose consistently after BSO and these patterns were similar to those seen in the control group. However, the addition of estrogen reduced gonadotrophin rises significantly more than estrogen did alone. Further, the luteal phase level of progesterone solely has a suppressive effect on the gonadotrophin rises after BSO. Our observations suggest that synergism of progesterone with estrogen may exist in suppressing gonadotrophin secretion in the normal luteal phase and should help in understanding why gonadotrophin levels in the luteal phase are lower than those in the follicular phase of the menstrual cycle.

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We have investigated the effect of a single oral administration of 600mg ketoconazole, an imidazole derivative used in clinical practice as an antimycotic agent, on the response of plasma adrenocortical steroids to 1-24 ACTH in 5 normal male subjects pretreated with dexamethasone. The 2mg of dexamethasone was administered orally at 2300h on the preceding night, and then a rapid ACTH test was started at 0830h. After a 1 week interval, the ACTH test was repeated in the same manner under the same dexamethasone pretreatment, but 600mg of ketoconazole was given orally at 0500h. The absolute plasma concentration and the increase over the basal level of each steroid after ACTH were evaluated and compared in both conditions with and without ketoconazole administration. A single ingestion of ketoconazolecaused a decrease in both indices of the responsiveness of plasma dehydroepiandrosterone and a rise in the plasma level of 17α-hydroxypregnenolone. The response of plasma aldosterone was clearly blunted by ketoconazole administration, whereas that of plasma corticosterone was clearly increased. Ketoconazole also blocked the response of plasma cortisol with concomitantly increased responses of plasma 11-deoxycortisol and11-deoxycorticosterone. The increased response of plasma corticosterone seemed to be likely due to the severe inhibitory action of ketoconazole on the conversion of corticosterone to aldosterone. These results imply the inhibitory effect of ketoconazole on C17-20-lyase activity and on the conversion of corticosterone to aldosterone, and suggest an inhibitory action on 11β-hydroxylase activity. The effects of ketoconazole on the other enzyme activities in adrenal steroid biosynthesis were also discussed.

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The effect of a high plasma progesterone level on the PRL releasing mechanism was investigated in rats of both sexes. Progesterone levels were maintained by implanting silicone tubes filled with the steroid. In the intact female, 6 progesterone tubes (inner diameter2mm; outer diameter3mm; length40mm) were implanted subcutaneously on the estrous day. With2-to5-day latent periods, the daily rise in the plasma PRL level was observed coincident with the time of nocturnal surge in the pseudopregnant rats induced by cervical stimulation. The same treatment applied to ovariectomized rats induced diurnal and nocturnal surges. The peak height was lower in ovariectomized rats than that in intact or normal pseudopregnant rats, and was restored to almost the normal range by concomitant implantation of estradiol with progesterone. This latter protocol, however, did not induce any PRL surge in chronically orchidectomized rats.
These results suggest that1) chronically elevated progesterone levels can induce such PRL surges as are observed in pseudopregnant rats, 2) estradiol enhances the magnitude of the PRL surge, and3) the progesterone sensitive central mechanism, controlling the PRL surge, does not exist in adult male rats.

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Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26.
Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52nM). After ECP treatment, there was a tendency for translocation in all4tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES.
DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.

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Pouch granuloma formation induced by Freund's complete adjuvant containing 0.1% croton oil was studied in both normal and alloxan-induced diabetic mice, and was found to be significatly suppressed in the diabetic group. Insulin repeatedly injected into the pouch facilitated granuloma formation dose-dependently, especially in the diabetic mice. The topical insulin treatment did not affect blood glucose levels. The suppression of granuloma formation by diabetes and its reverse by insulin treatment were verified by histological findings in the granuloma pouch wall. Characteristic changes in neovascularization occurred in the pouch wall. The hydroxyproline content in the granuloma tissue in diabetic mice was not significantly enhanced by the insulin treatment. This indicates that differences in collagen production in normal and diabetic mice were not the critical factor affecting granuloma formation. It was concluded that the decreased granuloma formation in the diabetic state was due to the lack of insulin as a growth factor, and that angiogenesis induced by insulin preceding collagen fiber formation may play an essential role in granuloma formation.

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An adrenocortical carcinoma (15.5g) secreting excessive amounts of steroids with weak mineralocorticoid activity in a 25-year-old woman was studied with particular reference to its in vivo and in vitro secretions of steroids. Severe hypertension, occasional low serum potassium and suppressed PRA were the major clinical findings, and were improved with removal of the tumor. In the preoperative stage, plasma levels of 11-deoxycorticosterone, 18-hydroxy-11-deoxycorticosterone, corticosterone and 18-hydroxycorticosterone were all increased. However, the plasma level of aldosterone was repeatedly normal. Although plasma levels of pregnenolone, 17-hydroxypregnenolone, progesterone and 17-hydroxyprogesterone were very high, those of other late step steroids, i. e. 11-deoxycortisol, cortisol, dehydroepiandrosterone, androstenedione and testosterone were almost normal. From these findings, a major etiological role of weak mineralocorticoids such as 11-deoxycorticosterone, 18-hydroxycorticosterone and corticosterone in her hypertension was suggested. Pregnenolone and 17-hydroxypregnenolone in tumor tissue were increased, but 11-deoxycorticosterone, corticosterone, aldosterone, cortisol and adrenal androgens such as dehydroepiandrosterone, androstenedione and testosterone were below normal or low normal. In vitro production of 11-deoxycorticosterone, aldosterone or cortisol by the tumor tissue slices was very low and scarcely responded to synthetic ACTH.

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The mechanism of induction of micromelia in6-day-old chick embryo by 6-aminonicotinamide (6-AN) was investigated. Six-day-old chick embryo exposed to 6-AN did not show micromelia when tenfold excess of nicotinamide over 6-AN was co-administered. The ability of nicotinamide to prevent the induction of micromelia was partially offset after 4hr of exposure to 6-AN and completely disappeared after6hr. The length of time necessary for the induction of micromelia was not affected by the concentration of 6-AN. These results indicate that exposure to 6-AN for only a short period of 6 hr is sufficient to commit the limb to micromelia and that cellular components involved in the induction of micromelia alter during this period. During this period, newly synthesized proteoglycan monomers typical of cartilage decreased in average molecular size, and isolated femora did not respond to parathyroid hormone (PTH) but to dibutyryl cyclic AMP to stimulate growth of cartilage in organ culture.

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Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562cells) were identified and characterized.[¹²⁵I] IGF-I specifically bound to K-562cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7ng/ml IGF-I.[¹²⁵I] IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were10, and100times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562cells. When the cells were differentiated by hemin (40μM), specific binding of [¹²⁵I] IGF-I to the cells was decreased to 56.8±5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells.
These data demonstrate that K-562cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.

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Calcium ions have been shown to play a mojor regulatory role in the release of various hormones from a wide variety of endocrine organs. More recently, in vitro evidence suggests that a calcium-binding protein, calmodulin, is also involved in the release of many hormones. So we examined the effects of several types of calmodulin antagonists on TSH-stimulated thyroid hormone release in vitro. Mouse thyroid lobes (one thyro-tracheal unit/tube) were incubated in Krebs-Ringer bicarbonate buffer at37°C for 4h. Free thyroxine (fT₄) released in the incubation medium, thyroidal cAMP and calmodulin content were measured by RIA. TSH (5mU/ml) and dibutyryl cAMP (DBC)(200μg/ml) caused a 2-4 fold increase in thyroidal release of fT₄. The stimulatory effects of TSH on fT₄ release were significantly inhibited by trifluoprazine and prenylamine lactate at the concentration of5×10^-5M. More specific calmodulin antagonists, W-7and W-13, were also shown to inhibit TSH stimulation of fT₄ release at the concentration of5×10^-5M. In contrast, TSH stimulation of fT₄ release was not depressed by non-specific antagonists, W-5or W-12, at the same concentration as5×10^-5M. Further, W-13also markedly inhibited DBC-stimulated fT₄ release. Neither TSH nor PGI₂ altered the thyroidal calmodulin content, dissociating with a marked increase in the cAMP concentration. These results suggest that calmodulin plays an important role in TSH-stimulated thyroid hormone release and further that this mechanism exists, at least in part, at the site subsequent to the generation of cAMP.

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Plasma1, 25-dihydroxyvitamin D (1, 25-(OH) ₂D) level, which is considered to be an indicator of parathyroid function, is possibly modified by the level of vitamin D.
In the present study, we have investigated parathyroid function in terms of enhancement of the plasma levels of1, 25-(OH) ₂D after oral administration of100μg of 25-hydroxyvitamin D₃ (25OHD₃) in 9 cases of primary hyperparathyroidism (1°HPT), 7cases of hypoparathyroidism (HP), 2cases of pseudohypoparathyroidism (PHP) and6normal subjects.
The plasma levels of25-hydroxyvitamin D (250HD) increased and reached a peak at 6-12hours after the administration of250HD₃. The plasma levels of1, 25-(OH) ₂D slightly increased but remained within the normal range after 250HD₃ administration in3of the normal subjects whose basal levels were rather low, but the increase in plasma1, 25-(OH) ₂D in control subjects was not statistically significant. In cases of1HPT, the plasma1, 25-(OH) ₂D level rose significantly in all cases (P<0.05), although the pattern of the increase was not uniform. These increases were remarkable in the patients whose basal levels were low. On the other hand, an increase in the level was rarely observed in any of the cases of HP and in one of the cases of PHP. In another case, normocalcemic PHP, the plasma1, 25-(OH) ₂D level rose.
The ratios of peak values of the plasma1, 25-(OH) ₂D to that of 25OHD were within a narrow range, 0.65-1.20 (×10^-3), in the normal subjects, but were high, over 2.0, in all cases of1°HPT except for one mild case (MEN type I). In contrast, such a ratio in cases of HP remained below 1.18 in all cases. These results suggest that parathyroid function is closely related to a rise in plasma levels of 1, 25-(OH) ₂D after oral administration of 25OHD₃ and that this loading test can possibly be used as a parathyroid function test.

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Modes of inhibition and binding of ketoconazole, an orally antimycotic agent, to NADPH-cytochrome P-450dependent enzymes were investigated using subcellular fractions of human and rat testes, human adrenocortical adenoma tissue and rat adrenals and livers. Ketoconazole competitively inhibited the activities of steroid17α-hydroxylase and C17-20lyase in rat and human testes, 16α-hydroxylase in human testes and 21-hydroxylase in rat adrenal glands. Ki values were in the order of10^-8M for human testicular enzymes, while the order was10^-7-10^-6M for rat adrenal and testicular enzymes. Kinetic studies indicated that ketoconazole bound to cytochrome P-450and not to other components of monooxygenase systems. Spectrophotometric studies also revealed direct binding of ketoconazole to cytochrome P-450 component by inducing type II difference spectra in all tissue preparations examined, indicating that ketoconazole is possibly a universal inhibitor of NADPH-cytochrome P-450dependent monooxygenases which are involved in metabolism of many substances including steroids, toxins, carcinogens and others.

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Synthetic human GRF (hGRF (1-44) NH₂; SM-8144) was administered as an iv bolus to141normal children of short stature (NSC), 73 patients with severe idiopathic GH deficiency (IGD; group A), 30patients with mild idiopathic GH deficiency (IGD; group B), 29patients with secondary GH deficiency, 3patients with primary hypothyroidism, 21patients with Turner's syndrome and25patients with various other diseases. Their height was below normal for their age and sex, and they were all below25years old without obesity. The maximal GH responses (M±SEM) were 39.5±2.2, 7.2±0.9, 27.2±3.7, 5.2±0.8, 9.7±4.4, 25.1±2.8 and32.3±4.8ng/ml, respectively (significance from the NSC, ; p<0.05, ; p<0.001). The GH responses to hGRF were greater than those elicited by standard pharmacological tests. There was a negative correlation between bone age and peak plasma GH response to hGRF in patients with idiopathic GH deficiency (IGD) but not in normal children (NSC). In twenty-two percent of the patients with IGD in group A the response was above10ng/ml and in 57% of the patients with IGD in group B the response was above 20ng/ml, suggesting that a large percentage of patients with idiopathic GH deficiency lack hypothalamic GRF. The side effect of flushing was observed in 15.2% of all subjects.
These results indicate the potential usefulness of hGRF (1-44) NH₂ (SM-8144) in inducing GH release from the pituitary.

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The 24-h secretory pattern of GH was evaluated in normal women (n=4) and hyperprolactinemic-amenorrheic women with pituitary microadenoma (n=6). Serum GH concentrations significantly differed according to the time of day, and there were nocturnal rises in normal women and hyperprolactinemic women. However, episodic fluctuations in serum GH levels were almost absent during the day time in hyperprolactinemic women, and the mean 24-h GH concentration was significantly lower in these women than in normal women. Thus, GH secretion was diminished in hyperprolactinemic women with pituitary microadenoma, though a nocturnal increase in GH secretion continued.

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A radioimmunoassay (RIA) for human pro-LRF (14-69) OH was developed with an antiserum, generated in a rabbit, to [Tyr⁶⁷] pro-LRF (47-67) NH₂ conjugated to BSA. This antiserum bound 28-32% of [¹²⁵I] pro-LRF (14-69) 0H at a final dilution of 1:2500 and the binding was inhibited by pro-LRF (14-69) OH in a dose-dependent manner. The sensitivity of the RIA was 31.2-62.5 pg and the dose that inhibited 50% of the binding to the tracer was 280-320 pg. Intra-and inter-assay coefficients of variation at 50% inhibition were 8 and 12%, respectively. Neither LRF nor pro-LRF (14-37) OH was recognized by the antiserum. The dilution curve generated with human hypothalamic extract was parallel to that of pro-LRF (14-69) OH. In addition the extract yielded a major immunoreactive peak emerging in elution volumes concordant with [¹²⁵I] pro-LRF (14-69) OH on Sephadex G-SO chromatography.

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To investigate the effect of prolactin (PRL) on testicular function, especially on spermatogenesis, testicular inhibin content in male rats treated with PRLwas compared with those treated with HMG and testosterone. Mature Wistar male rats were given 10 or 50 IU of ovine PRL, 10 IU of HMG and 5 mg of testosterone, i. m. for 5 consecutive days and testes were removed for assessing inhibin content. Inhibin content was measured by a FSH suppressing activity in cultured rat anterior pituitary cells using aquous extract of testes. Five days' treatment with PRL, HMG, or testosterone did not influence testicular inhibin content in male rats. The possibility that these treatments had transiently affected testicular inhibin content, or that inhibin content did not reflect inhibin production was not ruled out.

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A 17 year-old boy was admitted to the hospital because of thirst, polyuria (5-61/day), delayed sexual development and muscle weakness. He appeared obese, had an eunuchoidal body habitus and was excessively tall. Chromosomal analysis revealed a 47XXY karyotype. Serum cortisol was 1.3μg/dl, LH, 10.4 mIU/ml, FSH, 2.0 mIU/ml, and testosterone, 10 ng/dl. Endocrinological dynamic tests indicated diabetes insipidus and hypopituitarism of a hypothalamic type. Brain CT disclosed the existence of a tumor shadow around the calcified pineal body, extending towards the suprasellar region. Replacement therapy with glucocorticoid and DDAVP was started. The patient complained of a headache and plasma AFP and hCG concentrations were 868 ng/ml and 68.6 IU/ml respectively. A hCG- and AFP- producing germ cell tumor was suspected and radiation therapy with ⁶⁰Co was performed. Plasma AFP and hCG were decreased with significant clinical improvement. Soon after irradiation, he started to complain of a headache and had elevated AFP and hCG levels. Right hemiparesis and unconsciousness suddenly appeared and he died of left thalamic bleeding. This is the first case of Klinefelter's syndrome associated with intracranial germ cell tumor. Plasma testosterone levels fluctuated in parallel with the change in plasma hCG levels. This shows that the Leydig cells in this patient could respond to some extent to tumor-producing hCG.

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