It was preliminarily revealed by a histogram made from the electron micrographs of acidophils on the tissue sections of anterior pituitaries in normal rats that the two different sizes of storage granules were distributed in high population in many acidophils. These two granules, small and large in size, were isolated from the pellet of isolated acidophils and separated each other by filtrations through membrane filters with various pore sizes and by discontinuous sucrose density gradient centrifugations. The other histogram made from the electron micrographs of these separated granules coincided with the above histogram. ACTH, GH and prolactin activities were bioassayed on the free granules. GH activity was found only in the large granules with diameter between 300 and 450mμ. ACTH activity was detected only in the small granules with diameter between 100 and 200 mμ, though the content of ACTH contained in the isolated granules represented only a few per cent of the total ACTH content of the homogenate of anterior pituitary. No prolactin activity was found in the both kinds of granules. These findings strongly suggested that the two kinds of granules originated from the acidophils of the rat anterior pituitaries were involved in GH and ACTH storage and the acidophils were responsible for ACTH production. The findings were also discussed related to the mode of intracellular localization of the hormones.

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Effects of cortisol administration to adrenalectomized rats on the half-life of RNA in liver, kidney and spleen were examined. The effect of administration of Actinomycin D and cycloheximide was also studied. A significant increase in the half-life of RNA was observed in liver and kidney from cortisol-treated rats but the rate of RNA degradation in spleen was not significantly influenced. Administration of Actinomycin D at a dose which caused 30-50% inhibition of ³H-orotate incorporation into the liver, spleen and kidney also decreased the degradation of labeled RNA in liver and spleen but not in kidney, while cycloheximide, a dose which evoked approximately 40% inhibition of ³H-leucine incorporation into the tissues, exhibited no significant effect on the turnover rate of RNA in all tissues examined.

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Concentration of adenosine 3', 5'-monophosphate (cAMP), binding activity of cAMP to protein and activities of cAMP-dependent and-independent protein kinase were determined in adrenal glands of rats treated with ACTH (Cortrosyn-Z). cAMP was increased rapidly after 6 U. ACTH injection, reached the maximum level (20-30 times the control) at 10min and returned to the control level by 120min. Hypertrophied adrenals of rats given repeated injections of 3 U. ACTH every 12hr contained a significantly higher amount of cAMP than untreated control. Treatment of animals with ACTH did not modify the activity of cAMP-binding protein throughout the experimental period. cAMP-independent activity of protein kinase was not influenced by ACTH but cAMP-dependent activity of the enzyme disappeared completely immediately after ACTH administration and a part of the activity was restored 80min after the treatment. Administration of cycloheximide prior to ACTH treatment did not disturb the effect of ACTH on cAMP formation and cAMP-dependent protein kinase activity.

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Chromophobes isolated by the original method from the rat anterior pituitaries morphologically differentiated into acidophils when cultured in a chemically defined medium, NCTC-109, supplemented with various amounts of CRF (0.15 to 15μg/2ml) and colchicine. An electrophoresis and a paper chromatography revealed that ACTH was a main hormone produced by the cells maintained in the supplemented medium for as long as 12 days. The addition of larger amounts of CRF shortened a duration to reach to a certain maximal level of ACTH concentration in the cells, and this maximal level was equivalent in the cases of the variable amounts of CRF. A maximal release of the ACTH from the cells into the medium took place always 3 days after reaching to a maximal concentration of ACTH in the cells. The chromophobes produced α-MSH only in an earlier phase of differentiation without discharging it into the medium. The intracellular granules isolated from the cells kept in the control medium were equally 100 to 150 mp in diameter and regarded as the common storage carriers of six trophic hormones because they showed commonly all their low activities. Three different granules were successfully isolated from the cells differentiating in the CRF-medium from the chromophobes into the acidophils with negligible cross-contaminations. They were small (100 to 200mμ in diameter), large (300 to 450 mμ) and huge and irregularly shaped granules (450 to 1000 mμ) respectively. All the granules were confirmed by bio-and immuno-assays to contain ACTH activity alone regardless of their dimension.

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A method has been devised for measuring the level of adenosine 3', 5'-monophosphate (cyclic AMP) newly synthesized ruring in vitro incubation of the isolated rat kidney cells with adenine-8-14C. By combined use of the BaSO₄ treatment and the onedimensional ascending cellulose thin-layer chromatography with stepwise development by two solvents, newly formed or labeled cyclic AMP (¹⁴C-cyclic AMP) could completely be separated from the related radioactive metabolites in the cells.
Effect of lysine-vasopressin on the isolated rat kidney cells was studied by using the radioassay method. It was shown that (1) in the presence of theophylline, vasopressin increased ¹⁴C-cyclic AMP level, and (2) the stimulative effect was specific to vasopressin. Moreover, (3) vasopressin was shown to increase ¹⁴C-cyclic AMP level markedly in the medium but little in the cells. It was thus indicated that over-all synthesis of cyclic AMP in the rat kidney cells and consequential excretion of the newly synthesized cyclic AMP into the medium were enhanced by vasopressin.

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The method of simultaneous measurement of total triiodothyronine (T₃) and thyroxine (T₄) in serum has been developed by modifying Sterling's method. More sensitive standard curve was obtained by using Sephadex-charcoal mixture to separate free from bound form, where only one or two ml of serum is required. Also the method of simultaneous measurement of free T₃ and T₄ was described on the basis of the fact that dialyzate of free T3 could be completely precipitated in the same way as the measurement of free T₄. Dialyzable fraction of T₃ (DFT₃) and DFT₄ have become simultaneously obtainable by using ¹²⁵I-T₃ and ¹³¹I-T₄ resulting in saving time and serum.
Total T₃ and total T₄ values (mean±SD) were respectively, 0.31±0.12, 7.5±1.0μg per 100ml in normal, 0.73±0.15, 19.0±2.9μg per 100ml in hyperthyroid, and 0.14±0.06, 1.3±0.4μg per 100ml in hypothyroid. The respective values (mean±SD) for DFT₃ and DFT₄ were 0.53±0.17, 0.044±0.015% in normal, 1.19±0.63, 0.092± 0.031% in hyperthyroid and 0.23±0.04, 0.028±0.006% in hypothyroid. Percentage of DFT₃ was approximately ten times as high as that of DFT₄ in each thyroid state.
The respective values (mean±SD) for the absolute concentration of free T₃ (AFT3) and AFT₄ were 2.21±0.90, 3.60±0.32 mμg per 100ml in normal, 6.70±2.60, 19.6±3.8 mpg per 100ml in hyperthyroid and 0.36±0.04, 0.35±0.02 mpg per 100 ml in hypothyroid. Total T₃ is minimal as compared with total T₄, but AFT₃ is not so different from AFT₄.
The ratios of total T₃ to total T₄ and of AFT₃ to AFT₄ were 0.053±0.012, 0.67± 0.31 in normal, 0.037±0.004, 0.44±0.14 in hyperthyroid and 0.119±0.073, 1.10± 0.67 in hypothyroid.
These ratios increased along with the decrease of thyroid hormone in serum, which suggests that the less are thyroid hormones in serum the more important is T₃.
From these data and kinetic study indicating degradation rate of 51μg per day for T₃ and 75μg per day for T₄, the physiological meaning of T₃ may be more important in health and diseases than considered before.

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A single subcutaneous injection of progesterone, estradiol-17β or oil was given to castrated rats. One and half hr after the injection, a single injection of ³H-leucine was given intraperitoneally to animals. The incorporation of the radioactive material in the brain was studied autoradiographically 2 hr after the injection of ³H-leucine. The radioactivity in various brain areas was measured by counting the number of reduced grains over cell bodies of neurons.
In the castrated controls, high incorporation of ³H-leucine was observed in the supraoptic and paraventricular nuclear regions and Ammon's horn. Treatment with either progesterone or estradiol-17β induced a significant enhancement of the incorporation of radioisotope in the arcuate, ventromedial and supraoptic nuclei, and Ammon's horn. Not estradiol-17β but progesterone caused an increase in the incorporation of ³H-leucine in the suprachiasmatic and anterior periventricular nuclear regions, whereas the incorporation of the isotope in the paraventricular nucleus and the nucleus of mammillary body was enhanced only by estradiol-17β.
Possible reasons for high incorporation of the radioisotope in the supraoptic and paraventricular nuclei, the arcuate and ventromedial nuclei and the suprachiasmatic and anterior periventricular nuclei in hormone-traeted animals were discussed.

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Intraperitonael administration of 0.1 to 0.2gm/kg Na-Sulfacetylthiazole (SAT) twice a week for 4 weeks induced obstructive nephropathy, and bone changes similar to osteoporosis with a decrease in the X-ray density and calcium content of the tail bone and femur, decrease of femoral cortical thickness and an increase in bone resorption cavities in 3 month old rats. Such bone changes, prevented through prior parathyroidectomy, might be due to increased parathyroid hormone activity secondary to SAT-induced renal injury.

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A survey for endemic goiter was carried out on Iban natives (Sea Dayak) in four districts along the Rajang River in Sarawak, Borneo Island.
Among 608 subjects (271 males and 337 females) examined, 134 subjects (22 males and 112 females) were found to be goitrous, the prevalence of goiter being 8.1 for male and 33.2% for female. None were clinically hypo-or hyperthyroid, no cretinism was detected.
Twenty-four hr. thyroidal ¹³¹I-uptake was 54.3±13.1 %(mean±S. D.) in goitrous subjects while 47.1±10.2% in non-goitrous (no statistical significance). Urinary excretion of iodine was 45±27μg per day in goitrous group and 49±25μg in nongoitrous group (no significance). Iodine content of drinking water was extremely low in all four districts, the lowest one being obtained from region in which the prevalence of goiter is highest. Thyroid parameters such as PBI, T₃ resin sponge uptake and cholesterol were all within normal limits. Anti-thyroid antibodies were not demonstrated in sera from either goitrous or nongoitrous subjects. KSCN discharge test was positive in two cases in a single family, among 33 cases studied.
Experimental feeding of cassava on our expedition members for two weeks did not show any change in thyroid parameters including KSCN discharge test.
It is thought that iodine deficiency is probably a major causative factor of endemic goiter in Sarawak, Borneo Island. Other factors such as organification defect or defective thyroxine binding proteins might have some role in individual cases.

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An approach to the comparison of the affinity of thyroxine-binding-globulin (TBG) in normal and hyperthyroid sera was described. The method was based on the measurement of free thyroxine fraction (FT₄F) of sera at a fixed level of thyroxine (T₄) and TBG-capacity. In normal and hyperthyroid sera, the concentration of T₄ was measured with Murphy's method and TBG-capacity with reverse-flow paper electrophhoresis. The TBG-capacity was brought to a fixed level by diluting the sera to be tested with TBGdeficient serum. T₄ was added to the sera at a final concentration of 50μg/100ml. FT₄F in sera pretreated as described above was measured with a modification of Sterling's Mgprecipitation method employing barbital buffer, minimizing the effect of TBPA on FT₄F.
Thus, FT₄F of normal and hyperthyroid sera was compared at essentially the same concentration of T₄ and TBG-capacity. Therefore, the difference of FT₄F must reflect the affinity of T₄-TBG binding. With the method described, TBG-affinity of 6 hyperthyroid sera was compared with that of age-sex matched normal subjects. No significant difference of TBG-affinity was detected.

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Calcitonin activity was demonstrated in the ultimobranchial gland of Anguilla japonica, the Japanese eel, with a wide distribution over an area involving the esophagus pericardium. Total calcitonin content was approximately 10-40 MRC u/kg body weight. Purification of eel calcitonin through gel filtration on Sephadex G-75 and SE Sephadex G-75 yielded a preparation with a potency of 1360 MRC u/mg. While this preparation is already more potent than porcine and other mammalian calcitonin, further purification is indicated.

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A comparision of the effects of intraperitoneal and subcutaneous injections of PMS as an ovulatory gonadotropin was made by using the induced ovulatory response in immature mice pretreated with 2.5 IU of PMS. The intraperitoneal injection increased the sensitivity of the assay by 2.3-fold, as compared to the subcutaneous administration.
The blood levels of PMS after a single intravenous or subcutaneous injection into adult ovariectomized mice were examined. The half-life of PMS after an intravenous injection was shown to be 6 hr in the mouse. After a subcutaneous injection, the plateau period of PMS in the blood lasted 9 hr from 9 to 18 hr after administration, but the level was 25 %, as compared with the initial one after an intravenous injection of the same amount. Either by an intravenous or a subcutaneous injection, the blood level of PMS 24 hr after administration was the same, that is, 12.5%, as compared with the initial one after the intravenous injection.
These results were discussed in relation to various responses to PMS.

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Using an everted sac technique, the intestinal absorption of insulin in the rabbit has been investigated.
Insulin was shown to cross the intestinal wall, as demonstrated by the immunoreactive insulin inside the sacs (serosal fluid). The rate of insulin transference for a period of one hr was small, corresponding to approximately 0.1% of the insulin added to the mucosal fluid. This rate was not inhibited by the addition of 2, 4-dinitrophenol.
The small transference rate would indicate that the submucosal and muscular tissues of the intestinal wall, in the absence of vascular perfusion, present a barrier to the movement of large molecular weight substances.

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