Experiments were designed to make the hormone-free serum to serve for a standard curve of radioimmunoassay. The adsorption rate of hormone by dextran-coated charcoal (D. C. C.) was analysed using radioactive hormones such as 3, 5, 3' triiodothyronine (T₃), thyroxine (T₄), insulin, corticosterone, angiotensin I and II, ACTH, growth hormone (GH), TSH and HCG in pooled human serum. In general, the hormone of a small molecular weight was easily adsorbed into D. C. C., but the hormone of a large molecular weight needed a large amount of D. C.C. to remove hormone. Using the solution of high concentrated D. C. C.(20% charcoal and 2% dextran), T₃, insulin, corticosterone and angiotensin I and II were adsorbed by more than 80% into a small amount of D. C. C. solution (0.1ml), T₄, GH and ACTH were adsorbed by more than 80% into a relatively large amount of D. C. C. solution (1ml), and TSH and HCG were removed by 75% into a greater amount of D. C. C. solution (2ml) under 1ml of pooled serum. In this treatment of D. C. C. solution, serum albumin was removed slightly into charcoal.
The standard curve of radioimmunoassay for TSH and GH was figured using the panhypopituitary patient serum (PPS) or PPS treated by D. C. C. The standard curve of TSH or GH using PPS did not remarkably differ from that of PPS treated by D. C. C. The hormone-free serum that was made by D. C. C. treatment is available for the standard serum for the double antibody radioimmunoassay of these hormones.

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The author analyzed the secretion pattern of human placental lactogen (hPL) to understand the endocrine properties of trophoblastic cells and to know the clinical usefulness of assay of this hormone in patients of trophoblastic neoplasia with systematic serial estimation in comparison with the secretory behaviour of human chorionic gonadotropin (hCG). And the following conclusion was extracted.
Neoplastic trophoblastic cells might maintain a certain high activity in hCGsecretion and a low activity in hPL-secretion along with the degree of malignancy.
In clinical practice, measurement of hPL was a very sensitive parameter to understand the completeness of treatment, especially in cases of hydatidiform mole.
Serum hPL disappeared very quickly because of its very short half life in vivo, if the removal of the tumor was complete, or the treatment was sufficient.

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Further examination weie made on the double isotope derivative dilution method by the use of thiosemicarbazide-³⁵S as the labeled reagent and ³H-labeled corticosteroid for correcting the loss during extraction and separation procedure, and fractional determination of free and protein-bound cortisol and corticosterone.Δ⁴-3-Ketosteroids with similar chemical structure present in plasma, namely cortisol, corticosterone, aldosterone, deoxycorticosterone, and cortisone can be separated by repeating thinlayer chromatography at a low temperature of 10 °C, more than four times. Cortisol and corticosterone can be determined correctly and with good precision, relative standard deviation being 1-2%. Other three corticosteroids can be determined correctly and with good precision, relative standard deviation being 3-6%.
It was found that the protein-bound cortisol and corticosterone in plasma are dissociated during solvent extraction and the total amount is determined in free form. It was also found that the protein-bound and free cortisol and corticosterone can be separated by gel filtration, and the combination of gel filtration and the present method of determination has made it possible to carry out fractional determination of bound and free forms of cortisol and corticosterone. From its results, the quantity per 100ml of plasma was found to be 1.83 and 0.18 μg of free cortisol and corticosterone, and 17.45 and 1.75 μg of protein-bound cortisol and corticosterone, respectively.

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To elucidate the physiological role of cAMP-dependent and -independent protein kinase and cAMP-binding protein in regulation of cellular function and metabolism, activity of the protein kinases and capacity of the cAMP-binding protein were assayed in the ventral prostate and the uterus from rats of various hormonal statuses. Castration caused a significant elevation in the activity of both cAMP-dependent and -independent protein kinase and administration of testosterone to castrated rats abolished the effect of castration on the enzyme activity. In the uterus, administration of estradiol to immature rats evoked a significant decrease in the cAMP-dependent activity but not in the cAMP-independent activity of the enzyme. Deprival of the sex hormones caused an increase in the cAMP-binding capacity in both the ventral prostate and uterus. Physiological implications of these observations were discussed.

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Effects of native ACTH and various synthetic ACTH analogues on water balance alcohol-anesthesized polyuria rats were examined. Purified porcine ACTH had an apparent anti-diuretic action on the rats. Synthetic ACTH peptides consisting of 27, 24, 17, and 16 amino acids were biphasic, initially diuretic then anti-diuretic, and the peptides having 14, 13, and 10 amino acids were only diuretic. Small ACTH fragments consisting of 8, 6, 5, and 4 amino acids were inactive in the doses so far examined. In analogues consisting of 18 amino acids, replacement of N-terminal amino acid with β-alanine orα-aminoisobutyric acid (Ibu) and protection of the C-terminal carboxyl group by amide formation enhanced the anti-diuretic action. Purified TSH, LH, FSH, and prolactin probably inert under the experimental condition. Similar biphasic effect of the Ibu¹-ACTH (1-18)-amide was also observed in hypophysectomized or adrenalectomized rats with almost the same potency as that in nonoperated rats. Weak diuretic action of Ibu¹-ACTH (1-18)-amide was also demonstrated in water-loaded intact rats.

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Fluctuations in the urinary LH levels were determined by radioimmunoassay during treatment with cyclophenyl (F6066) and clomiphene in healthy subjects and women with various anovulatory conditions in order to elucidate the effect of these compounds on the hypothalamico-pituitary center. Except Sheenhan's syndrome, Chiari-Frommel syndrome and a few cases of amenorrheic patients where the cause of the disorder was located in hypothalamico-pituitary axis, an increase of LH was observed in all cases. The available evidence suggest that these compounds stimulate the hypothalamico-pituitary regulatory centers and release LH. One of the prime objectives of an ovulation inducer is to stimulate the production of gonadotropins in the pituitary.
An analysis of the LH excretion pattern in anovulatory women during the treatment with clomiphene and F6066, therefore, may serve as a useful tool as a test of the functional status of the hypothalamico-pituitary regulatory centers.

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The anterior pituitary glands of sterilized rats secured by subcutaneous injection of 50μg estradiol benzoate contained a higher content of FSH and a little lower content of LH than those of normal proestrous glands. Serum contents of these hormones were not so different from those in normal proestrus and estrus. Electrical stimulations (0.1ms, 100Hz, 100μA) through chronically implanted electrodes into the medial preoptic area and medial part of amygdala induced the increases of serum LH and FSH. Stimulation into the dorsal hippocampus increased serum and pituitary concentrations of FSH and serum concentration of LH. Serum and pituitary concentrations of prolactin in the estrogenized rat were almost equivalent to those in normal proestrus and estrus, and stimulation of the hippocampus induced an increase of serum one. These results show that the brain of estrogenized rat differs in the function of hippocampus from that of normal cycling rat and in the function of amygdala from that of androgenized rat. However, the dramatic increase of serum and pituitary concentrations of both LH and FSH in response to bilateral ovariectomy indicates the right existence of steroid sensitive component in the brain.

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A newly improved method for obtaining a highly active human chorionic gonadotropin (HCG) has been developed. A crude HCG with an activity of about 3, 000 IU per mg was applied to an Amberlite CG-50 column equilibrated with 0.01 M ammonium acetate buffer, pH 5.0. This first purification procedure yielded two separate peaks. The pooled fraction under the 2nd peak was concentrated and further purified by the chromatography on Sephadex G-100 which was pretreated with 0.1 N hydrochloric acid. An activity of more than 20, 000 IU per mg of protein was obtained through this simple purification procedure. A homogeneous ultracentrifuge pattern was observed with our purified product, however, faint-stained minor components still remained in the disc gel electrophoresis for further investigation.

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The prolactin secretory activity of anterior pituitary during estrous cycle was determined from ¹⁴C-leucine incorporation in vitro into the hormone, using IVCS mice which showed regular 4-day-cycles. Prolactin in the anterior pituitary and in the incubation medium was fractionated by disc electrophoresis on polyacrylamide gel.
The synthesis and release of prolactin were significantly lower at estrus than at proestrus and diestrus. The release rate was higher at estrus than at diestrus.
It was demonstrated that the mouse did not show the similar patterns to the rat (Ieiri et al., 1971) in the changes of pituitary prolactin secretory activity during estrous cycle.

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In an attempt to analyze the pathogenesis of hereditary vasopressin-resistant diabetes insipidus (DI) in mice associated with oligosyndactily (Os), possible participation of adenyl cyclase of the kidney cells in the mechanism of diuresis was examined.
In DI Os/+ mice with severe diuresis intraperitoneal injection of adenosine 3', 5'-monophosphate (cyclic AMP) but not exogenous vasopressin delayed the onset of diuretic response to water load, while the reverse was true in DI +/+ mice with mild diuresis. Urinary osmolarity, moreover, was increased by cyclic AMP but not by vasopressin in Os/+ mice, whereas the reverse was the case with +/+ sibs. Although lack or defect of vasopressin-sensitive adenyl cyclase of the kidney cells might be assumed in the severely diuretic mice, both the basal and vasopressin-activated adenyl cyclase activities in vitro were similar between Os/+ and +/+ animals. The defect of vasopressin-sensitive adenyl cyclase in the kidney thus does not seem to be responsible for the hereditary nephrogenic diuresis of mice.

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The effect of dexamethasone pretreatment on TSH release induced by synthetic TRF was investigated in rats.
Plasma TSH levels were measured by means of McKenzie's method with a slight modification.
Dexamethasone was injected 4 hours before TRF administration. Blood samples were withdrawn from the jugular vein 10 min after TRF injection.
In a group received single dexamethasone pretreatment, TSH release following the TRF administration was significantly enhanced when compared with those observed in the saline pretreated group.
However, the increase in TSH release was not observed in the rats treated chronically by dexamethasone for 15 days.
The mechanism responsible for this dexamethasone effect on TSH secretion induced by TRF exists at hypothalamic level.

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As a supplement to our previous study, a renewal approach to the comparison of TBG-affinity in normal and hyperthyroid sera was described, based on the measurement of FT₄F at the condition in which a fixed concentration of T₄ (10 μg/100ml) and a fixed TBG-capacity were established.
FT₄F as an indicator of TBG-affinity was compared in 7 hyperthyroid patients and 7 normal controls. Significant difference of TBG-affinity was not detected, thus, making the possibility unlikely that the decreased affinity is one of the cause of the elevation of FT₄F in thyrotoxic sera.

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Salmon calcitonin was effective in preventing restraint-induced gastric ulcer production in rats. Moreover, calcitonin was also effective in preventing progress of ulcer formation even when calcitonin was administered 2 hr after initiation of restraint.

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