We designed a simple technique for surgical excision of the pineal body in mice. Such can be accomplished in 10 min by a skilled worker and there are no lesions whatever.
In pinealectomized El-strain mice the appearance of convulsions was inhibited up to 30 days, while intact El-mice were all fallen into convulsions by being shaken up and down on a flat carton. Pentazole-induced convulsions propagate from one dd-mouse to another in an aggregated state, but such propagation did not occur in pinealectomized animals.
Exploratory movements of pinealectomized dd-mice increased as compared to intact animals and the increase was always more extensively observed in El-mice in which, even in the intact animals, the exploratory movements were much more than those of dd-mice.
From the above-mentioned experiment, it is considered that the pineal body plays a role not only in initiating but also in propagating convulsions, and that it may have a depressive action on motor activities in mice.

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The synergetic effects of dietary obesity and stress induced by electrical shocks on insulin secretion from the perfused pancreas, the action of insulin on adipose tissues and glucose tolerance were studied in rats.
The rat was fed either a control (C)(50% starch) or a high fat diet (F)(40% butter) for a period of 12 weeks. Half of the rats on each diet received electrical shocks, one hour per day for the last three weeks of the experiment (group C-S, F-S). The remaining rats were not given any stress sessions (group C-N, F-N). The rats on the high fat diet gained a significant amount of weight at the 8th week, and as determined by the adipocytes, were obese at the end of 12 weeks. However, the high fat diet itself did not have any effect on plasma glucose, plasma insulin and insulin release from the perfused pancreas in response to glucose. It caused glucose intolerance and the insensitivity to insulin of adipose tissue. The rats which received electrical shocks stopped gaining weight when the shock sessions began. Moreover, the size of the adipocytes in F-S group was significantly smaller than that in F-N group, but the insensitivity to insulin of adipose tissue remained. In F-S group, glucose-induced insulin release from the perfused pancreas was signiffcantly diminished in the initial phase of release, and glucose tolerance was much impaired by stress, while in C-S group insulin release increased in the late phase of release, and glucose tolerance was unaffected by stress.
These findings indicate that the ability of glucose to stimulate insulin secretion is decreased by the synergetic effects of a high fat diet and stress induced by electric shock. Thus, it may be concluded that the mechanism which mediates the effects of glucose upon insulin secretion, especially in the initial response phase, is modified by such synergetic effects.

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The effects of ethacrynic acid and furosemide on the adenylate cyclase activity of the hamster kidney and heart were examined. Ethacrynic acid and furosemide suppressed the vasopressin-mediated increment of adenylate cyclase activity much stronger than the basal activity of the renal medulla. Ethacrynic acid and furosemide strongly suppressed the parathyroid hormone-mediated increment of adenylate cyclase activity on the renal cortex, though furosemide could not inhibit the basal activity of the enzyme. Ethacrynic acid and furosemide suppressed the 1-adrenaline-mediated increment of adenylate cyclase activity of the cardiac muscle, while furosemide could not inhibit the basal activity.
Ethacrynic acid and furosemide suppressed the NaF-mediated increment of adenylate cyclase activity of the renal medulla. Thus, these diuretics seem to inhibit the catalytic unit of the enzyme. Ethacrynic acid and furosemide strongly suppressed the hormone-mediated increment of the adenylate cyclase activity, while Ca⁺⁺ could only depress it to the same degree as it did the basal activity. This difference suggests that these diuretics inhibit not only the catalytic unit of the enzyme, but also other steps of the adenylate cyclase system.
Ethacrynic acid and furosemide suppressed the effect of 5'-guanylylimido-diphosphate [Gpp (NH) p] on the adenylate cyclase activation and strongly inhibited the enzyme activity by using the combination of vasopressin and Gpp (NH) p.These results might suggest that ethacrynic acid and furosemide not only affect the catalytic unit of the adenylate cyclase, but also the GTP sensitive site of the enzyme system.

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A sensitive and simplified radioreceptor assay for 1, 25-dihydroxyvitamin D (1, 25-(OH) ₂D) in human plasma was described and applied to preliminary clinical studies. Tritium-labeled 1, 25-(OH) ₂D₃ was produced by incubating chick kidney homoeenate with tritium labeled 25-hydroxyvitamin D₃ (25-OHD₃). A cytosol receptor was obtained from rachitic chick intestine (Kd=5.3 × 10^-11 M). L ipids in 5ml of heparinized human plasma were extracted with dichloromethane, and 1, 25-(OH) 2D was isolated by a Sephadex LH-20 column followed by high pressure liquid column chromatography. Recovery of 1, 25-(OH) 2D3 after the plasma extraction and chromatography ranged from 58 to 100%. The assay was sensitive to 5pg/tube. Diluted plasma from a patient on a high dose of 1 α-OHD₃ showed a dilution curve parallel to the standard curve. The cytosol receptor showed a cross reactivity to various vitamin D₃ metabolites physiologically present in the circulation and it was thought to be essential to eliminate other vitamin D₃ metabolites than 1, 25-(OH) ₂D from plasma samnles by high nressure liauid chromatography. Plasma concentrations of 1, 25-(OH) ₂D were, in the case of most normal subjects, distributed from 7 to 33pg/ml and the ranee of distribution became greater in relation to age, indicating that plasma values should be matched to age. Whereas markedly high values of 1, 25-(OH) 2D in plasma were found in some cases of primary hyperparathyroidism with prominent bone resorption, relatively low values were seen in some patients with chronic renal failure, senile osteoporosis, osteomalacia and hypercalcemia due to bone metastasis.

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Human growth hormone (HGH) response to exercise was studied in 27 normal children and 152 children with a chief complaint of short stature and in 9 patients with HGH deficency. Of the 152 children 107 could be ruled out of HGH deficiency since HGH level increased to more than 5ng/ml during exercise, and of the remaining children of non-responders to exercise, 22 were ruled out of HGH deficiency since bone age to chronological age ratio was more than 0.9 and/or height increment for the past 3 years was more than 15cm. Seventeen patients were hospitalized for further examinations, which resulted in finding normal HGH secretion in response to insulin-induced hypoglycemia, arginine infusion and/or propranolol-glucagon test 12 children and also in finding no HGH deficiency in children higher than-2.0 S. D., but 5 HGH-deficient children in those lower than-2.0 S.D. Exercise can be used as a screening test for ruling out HGH deficiency when HGH secretion in response to exercise is evaluated by height, bone age and height increment of the patients.

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The effect of bonito insulin on insulin release was examined in the monolayer culture of rat pancreatic β-cells. The β-cells were preincubated for 5 to 20hr with or without a small dose (100μU/ml) of bonito insulin in the medium containing 100mg% glucose. And then, they were incubated in 300mg% glucose alone or together with bonito insulin for 5hr. There was no significant difference between the IRI release from these n-cells with or without bonito insulin. The concentration of bonito insulin was augmented from 100μU/ml to 500, 1, 000 and 2, 000μU/ml. A significant inhibitory effect on the glucose-induced insulin release was observed only after the preincubation for 20hr with 2, 000μU/ml of bonito insulin.

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Two angiotensin II analogues, i. e., 1-sarcosine, 8-isoleucine angiotensin II (Sar¹, Ile-AII) and 1-sarcosine, 8-alanine angiotensin II (Sar¹, Ala⁸-AII), are now available in the clinical study. Comparative studies of the antagonistic potency and the agonistic effect of these two AII analogues were made in normal subjects on three soidum balances and in hypertensive patients with various etiologies on sodium depletion. Both AII analogues had an agonistic pressor effect in normal subjects. This effect changed with different sodium balances. In the low sodium state, this agonistic action was minimized. The agonistic pressor effect of Sar¹, IIe⁸-AII was greater than that of Sar¹, Ala⁸-AII in all sodium states. There was found an agonistic activity of both AII analogues not only on blood pressure, but also on renin and aldosterone secretion, and renal vasculature in normal subjects on a regular diet. The antagonistic depressor potency of both compounds was also varied by changing sodium balance, being greatest in the low sodium state. In hypertensive patients on sodium depletion, the blood pressure responses of individual patients to these two AII analogues were significantly correlated (r=0.8, n=20). These results indicate that pretreatment of sodium depletion is necessary to prevent the side effect caused by the agonistic pressor action of AII analogue, and also to predict renin dependency in hypertensive patients efficiently.

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The effect of various steroidal compounds on the binding of the androphilic protein to dihydrotestosterone in the cytosol of tissues of human benign prostatic hypertrophy was examined. Androgens, as well as estrogens and gestagens showed an inhibitory effect on the binding. Non-steroidal anti-androgens were revealed to be weak inhibitors on the binding.
Two androphilic proteins were observed in Sephadex G-200 chromatography in fractions eluted at the void volume and in fractions appearing at the site of IgG, and the rate of inhibition on the binding of both fractions by various steroids was compared. The rate of inhibition by various compounds was generally higher in the IgG fraction than in the void volume fraction. When the ligand and inhibitors were incubated with the cytosol prior to fractionation by Sephadex chromatography, rate of inhibition was lower than that obtained when the ligand and inhibitors were reacted with fractions after chromatography. Implications of the difference observed in these two experiments are not clear at this moment.
The results obtained by the protamine precipitation experiment were almost the same as those by the experiment using the extract of the unfractionated acetone-dried cytosol, therefore, the protamine procedure does not seem to precipitate the tissue specific androphilic protein specifically.

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To investigate the effect of physiological doses of secretin on pancreatic A and B cell functions, secretin was infused at a rate of 0.5, 1.0 and 2.0 clinical unit per kg body weight per hr into eight normal men for 30min or 60min after an overnight fast, and changes in plasma immunoreactive insulin (IRI) and glucagon immunoreactivity (GI) were measured while monitoring circulating secretin levels by a radioimmunoassay. During the infusion of secretin, the plasma immunoreactive secretin (IRS) levels rose to 140-390 pg/ml which was within a range of the physiological fluctuation in plasma secretin levels reported hitherto. No significant alteration of plasma IRI and GI levels could be demonstrated during these simulated physiologic infusions of secretin. These data suggest that, in humans, the physiological dose of secretin does not influence insulin and glucagon secretion from the pancreas in the basal state.

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adrenal glands of the mouse, fixed either in glutaraldehyde followed by osmiumtetroxide or in a mixture of potassium dichromate and glutaraldehyde, and embeddedin Epon 812, were investigated by light and electron microscopy. An argentaffinreaction was applied to semi-thin sections for light microscopy and to ultra-thinsections for electron microscopy. Since the mature secretory granules in the SmallGranule Chromaffin (SGC) cell were argentaffin and were mainly located along thecell membrane, this cell was clearly distinguishable under the light microscope bothfrom the A (adrenaline) cell whose secretory granules were non-argentaffin and fromthe NA (noradrenaline) cell whose cytoplasm was rich and was filled with large, strongly argentaffin granules. Chromaffinity of the SGC cell was demonstrated underthe light microscope. The SGC cell was intensively stained with toluidine bluewithout revealing metachromasia. It was demonstrated at the EM level that notonly the secretory granules but also the synaptic-like vesicles in the SGC cell containedargentaffin substances. Possible functional relationship between the secretory granules and the synaptic-like vesicles was discussed.

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Epinephrine or carbachol, alone or together with parathyroid hormone (PTH) orcalcitonin, was added into the incubation of young rat tibia and cyclic AMP as wellas cyclic GMP in the tissue was determined. Epinephrine induced a dose related andrapid increase of cyclic AMP probably through mechanism different from that ofPTH or calcitonin. The effect of these hormones to increase cyclic AMP was additiveto the effect of maximal dose of epinephrine and, in contrary to epinephrine effect, was not influenced by propranolol. Carbachol induced a dose-related and rapidincrease of cyclic GMP in bone. The effect of carbachol was completely abolishedby adding atropine or tetracaine. A marked increase of cyclic GMP was also inducedby NaN3. There was no reciprocal relationship between the levels of cyclic AMPand cyclic GMP. The increase of cyclic GMP by carbachol was unaffected by theincrease of cyclic AMP induced by PTH, and the increase of cyclic AMP by PTHor calcitonin was the same in the presence or absence of carbachol at the dosemaximally effective to increase cyclic GMP.
The results indicate the presence in bone of adrenergic and cholinergic receptorsites, and possible regulation of bone cell activity by these nervous systems throughcyclic AMP and cyclic GMP. Though the regulation of the cyclic nucleotides contentin bone by these neurotransmitters appeared to be through mechanism independentof PTH or calcitonin action, it seems worth-while to study further how these neurotransmittersby themselves or in concert with these hormones act on the various aspects of bone cell function.

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Described here is a 21-year-old man, who had several episodes of generalized muscle weakness, blepharoptosis and a diffuse goiter. The results of thyroid function tests and histological findings of specimen obtained by thyroid biopsy was compatible with mild Graves' disease. Blepharoptosis was moderately improved immediately after edrophonium injection, thus established the diagnosis of occular type of myasthenia gravis. In addition, the patient had markedly diminished serum potassium level, in association with markedly elevated plasma renin activity (PRA). Plasma aldosterone concentration (PAC) and urinary aldosterone excretion (UAE) were also moderately increased. The insensitivity to pressor effect of infused synthetic angiotensin II and hyperplasia of the juxtaglomerular apparatus of the kidney in histological examination substantiated the diagnosis of Bartter's syndrome complicated with Graves' disease and myasthenia gravis.
In order to investigate the effect of indomethacin, an inhibitor of prostaglandin biosynthesis, on the biochemical abnormalities of Bartter's syndrome, indomethacin (75mg/day) was given with spironolactone and potassium treatment. However, the biochemical abnormalities were not corrected and, moreover, muscle weakness and further decrease in potassium level were observed during the treatment with indomethacin alone. The findings suggest that the abnormalities observed in this patient could not be explained by the overproduction of renal prostaglandin.
The present case with Bartter's syndrome was characterized by complicating of Graves' disease and myasthenia gravis, both of which have been regarded as autoimmune diseases. Immunological studies, including subpopulation of lymphocyte determined by surface immunoglobulin bearing cells, rosette formation and blastoid formation, revealed the suppression of B lymphocyte population in the present case. Although these immunological abnormalities were corrected after administration of azathioprine, an immunosuppressive agent, serum potassium level was not ameliorated. The result suggest that immunological mechanism is not related to the etiology of Bartter's syndrome in the present case.

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The recoveries of human C-peptide and insulin from human serum were determined utilizing the method for extraction of insulin. Serum samples were obtained from healthy subjects before and after glucose loads. The elution profiles from Bio-Gel P-30 of acid-ethanol extracts of these sera revealed that the C-peptide immunoreactivity (CPR) from healthy subjects was eluted at the fractions corresponding to Cpeptide.
First, precipitation of extracted CPR and immunoreactive insulin (IRI) was performed by the addition of 1.5 vol of absolute ethanol and 2.5 vol of ethyl ether to each volume of the extract. The CPR and IRI recovered by this procedure were 32.8 ± 2.8 and 66.0 ± 2.9%(mean ±S. E.), respectively. When gel filtration was employed after the acid-ethanol extraction and the ethanol-ether precipitation, recovery of CPR was calculated as 29.7 ± 1.8%(mean ± S. E.). Differences in recoveries of CPR by these two methods were not significant.
About one third of CPR remained in the supernatant of alcohol-ether precipitation. When the higher proportion of ether to ethanol was adopted to the step of ethanol-ether precipitation, a considerable improvement of recovery of CPR was achieved. The minimum volumes of ethanol and ether to obtain the highest recoveries of CPR and IRI were 1.5 vol of ethanol and 5.0 vol of ether to each volume of the extract. Reextraction of the residue, obtained from the first step of acidethanol extraction, resulted in a considerable improvement of recoveries of these two peptides. The improved recoveries of CPR and IRI by the modified extraction method were calculated as 60.7 ± 1.5 and 83.3 ± 3.4%(mean ± S. E.), respectively.

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The effect of acute administration of L-DOPA on TSH and TRH levels in serum was studied in primary or pituitary hypothyroidism. TRH levels in serum fell and then returned to initial levels after L-DOPA administration in primary or pituitary hypothyroidism. TSH levels in serum fell and then returned. to initial levels after L-DOPA administration in primary hypothyroidism. T₄ and T3 levels in serum did not change after L-DOPA administration in primary or pituitary hypothyroidism. These data suggested that L-DOPA might act directly to hypothalamus.

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The ovulation-inducing effects of synthetic LH-RH and its analogues were studiedin 1) proestrous rats treated with chlorpromazine, 2) persistent estrous rats produced by neonatal treatment with androgen, 3) intact rats at the diestrous stage, and 4) pseudopregnant rats produced by treatment with estrogen, and the gonadotrophic responses of ovaries and follicles in these animals were examined. The results obtained were as followes: 1) Histological examination revealed that theovaries of the animals in the respective groups before treatment with LH-RH contained comparable populations of small- and large-sized follicles, but the population of largesized follicles tended to decrease in the following order: persistent estrous, proestrous, diestrous, and pseudopregnant rats. 2) Both subcutaneousand intravenous injections of human chorionic gonadotrophin into the animals of the respective groups induced ovulation effectively. However, the ovulating responses in terms of the threshold dose differed among the respective groups two-fold in the subcutaneous route and three- to seven-fold in the intravenous route. 3) LH-RH and its analogues administered subcutaneously or intravenously in the animals of the respective groups induced dose-dependent ovulation. Proestrous rats were the most sensitive and pseudopregnant rats were the least sensitive to the subcutaneous or intravenous injection of LH-RH. When the sensitivity of pseudopregnant rats to LH-RH was taken as 1 in terms of the reciprocal of the subcutaneous ED₅₀s, the relative sensitivities of proestrous, persistent estrous, and diestrous animals were 950, 500, and 110, respectively. Among the analogues examined, des-G1y¹⁰-[D-Leu⁶]-LH-RH-ethylamide was the most effective and also much more effective than LH-RH. The relative sensitivities to this analogue in proestrous, persistent estrous, and diestrous rats to pseudopregnant rats through the subcutaneous route were 12.8, 5.4, and 4.3, respectively.

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Prolactin binding in normal mammary glands and pregnancy-dependent mammary tumors and plasma prolactin levels were assayed near parturition in GR/A female mice.Mammary glands and tumors cut into 0.5-mm thick slices were incubated in Medium 199 containing 5ng of ¹²⁵I-labeled ovine prolactin at 37° for 2hr, and specificprolactin binding was expressed in terms of the difference between total and nonspecific bindings per mg tissue. On day 19 of pregnancy, prolactin binding was low in both normal mammary glands and tumors. One day after parturition, the binding of nonlactating mammary gland was as low as that on day 19 of pregnancy. On the other hand, the binding of lactating glands was raised significantly. There was no difference in the binding of tumors between lactating and nonlactating animals and both were significantly higher on day 1 after parturition than on day 19 of pregnancy. The plasma prolactin level assayed by radioimmunoassay was significantly higher on day 19 of pregnancy than on day 1 after parturition regardless of lactation. No relations were observed between the plasma prolactin level and the prolactin binding of either normal or neoplastic mammary glands.

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A simultaneous competitive enzyme immunoassay (SICEIA) for hCG was developed using β-D-galactosidase (β-Gal) as a labelled enzyme and anti-hCG antibody coatedsheep red blood cells (SRBC) as a solid phase. In this report, a new coupling agent, MCAE, was used to couple β-Gal with hCG. The sensitivity was improved to the degree of 2.5mIU/ml, equal to that of RIA. The present procedure was safer and rapider than RIA. The value of hCG in urine by our procedure had good correlation with that by RIA.

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