An homologous radioimmunoassay for human carboxyl-terminal parathyroid hormone (PTH) was developed using the antiserum raised against synthetic human PTH (46-84) and its clinical evaluation was attempted. Antisera were produced in guinea pigs by immunization with synthetic human PTH (46-84) conjugated with BSA. One of the antisera was used at a final concentration of 1: 10, 000 in the assay, and synthetic human PTH (53-84) for a standard and ¹²⁵I-Tyr⁴⁵ human PTH (46-84) for a tracer were used. A standard curve was obtained at concentrations from 0.001 to 10 ng per tube of human PTH (53-84). The displacement curves for human PTH (51-84), human PTH (46-84), Tyr⁴⁵ human PTH (46-84) and bovine PTH (1-84) were almost identical with the standard curve made by sythetic human PTH (53-84). This antiserum, however, did not recognize the human PTH (1-34) fragment at all. Using this assay system, circulating immunoreactive PTH was measured in normal subjects and patients with parathyroid disorders. Normal PTH values in serum ranged from 0.47 ng/ml to an undetectable level (undetectable in two out of 32 normal subjects) and serum PTH values in patients with primary or secondary hyperparathyroidism or idiopathic hypoparathyroidism were almost completely discriminated from normal values. These results suggest that this homologous radioimmunoassay is a carboxylterminal specific assay for human PTH and can be quite useful in clinical practice.

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Effects of the neutralization of endogenous progesterone with rabbit antiserum to progesterone (anti-progesterone) on germinal vesicle breakdown of ova in follicles of small (<125 μm), intermediate (125-250 μm) and large (>250 μm) diameter were examined by a quantitative histological technique. Immature rats were treated with 5 IU pregnant mare's serum gonadotropin (PMS) then with 10 IU human chorionic gonadotropin (hCG). Administration of anti-progesterone together with hCG 6 h later significantly decreased the incidence of germinal vesicle breakdown of ova in the large follicles, but not in the intermediate ones. This treatment did not affect the proportion of intermediate to large follicles in the population. Replacement with progesterone 1 h after the simultaneous injection of hCG and anti-progesterone partly reversed the reduced incidence of meiosis. An injection of rabbit antiserum to estrone, in addition to the replacement with progesterone 1 h after the simultaneous injections of hCG and anti-progesterone, restored the incidence of meiosis to a value comparable to the values found for control rats treated sequentially with PMS and hCG. We concluded that the hCG-induced preovulatory rise in progesterone has a limited but definite stimulatory effect on the resumption of meiosis in the ova of large follicles and that it mediates the meiosis-inducing action of hCG.

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Using seventeen human tumor cell lines derived from a variety of tissues, specific binding sites for epidermal growth factor (EGF), a mouse submandibular glandderived growth factor, has been characterized. A significant amount of membranebound EGF receptors, although considerably varied, was demonstrated in all the tumor cell lines studied. Epidermoid carcinoma appeared to have more EGF receptors than adenocarcinoma. One small cell carcinoma of the lung, one choriocarcinoma of the stomach and three bone tumors also possessed EGF recptors comparable to those of epidermoid carcinoma, while one adenoacanthoma of the stomach had less EGF receptors comparable to adenocarcinoma. Among a variety of phorbol esters tested, tetradecanoyl phorbol acetate, a potent tumor promotor, was shown to be the most effective compound in inhibiting ¹²⁵I-labeled EGF binding to its receptors.
Our results indicate that human tumor cells contain varying amounts of membranebound receptors for EGF and that phorbol esters interact with these EGF receptor sites. However, the relationship between EGF receptor sites on tumor cells and cellular proliferation and/or differentiation awaits further study.

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The maxium ouabain binding capacity in erythrocytes from 8 normal subjects and 14 patients with hyperthyroidism was assessed by measuring [³H]-ouabain binding. The mean value for maximum ouabain binding capacity was significantly lower in the patients than in normals (0.422±0.084 vs 0.671±0.095 pmol per 10⁹ cells, p<0.001). Furthermore, a close inverse correlation was found between the ouabain binding capacity and serum T₃ (r=-0.766; p<0.01) or T₄ (r=-0.870; p<0.001) levels. These results suggest that the maximum ouabain binding capacity in erythrocytes may provide a useful index of the peripheral effect of thyroid hormone.

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Effects of a low dose of estradiol on the luteal function were studied in intact pregnant rats. The pregnant rats received daily sc injections of 0.1μg estradiol or sesame oil from day 7 to 14 of pregnancy (day 1=day of insemination). Serum progesterone levels on day 15 were significantly lower in the estrogen-treated group than the oil-treated group. In order to study how estrogen induced luteolysis, the pregnant rats received each of the following treatments on day 7 of pregnancy:(1) The uterus except that containing a single conceptus was removed by hysterectomy (hysterectomy group);(2) All but a single conceptus were removed by aspiration, so that rats carried only a single conceptus with the whole part of the nongravid uterus (aspiration group). Each group of rats received daily sc injections of 0.1μg estradiol or sesame oil from day 7 to 14 of pregnancy. Estradiol treatment caused a significant decline in serum progesterone levels in the aspiration group on day 15, but this was not the case in the hysterectomy group. There was no significant difference in serum LH levels among any of the groups on day 15 of pregnancy. These results indicated that estradiol induced luteolysis in the intact pregnant rats, which would, at least in part, be mediated through the uterus.

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The process of growth of the mammary gland and change in the plasma concentration of progesterone were investigated throughout the course of pregnancy (30 days) in the house musk shrew, Suncus murinus L.
Development of the mammary gland of the musk shrew was limited during thefirst half of pregnancy. Extensive branching of ducts and conspicuous alveolar formation and a marked increase in the DNA content of the gland started between day 15 and 20 of pregnancy, and continued until term. Milk synthesis indicated by accumulation of the secretory fluid in the alveolar lumen and a sudden rise in the RNA/DNA ratio of the gland tissue seemed to be initiated 1 or 2 days before parturition. No lactose was detected in either the mammary tissue or milk of the house musk shrew.
Plasma concentration of progesterone was very low until mid-pregnancy and began to rise after day 15, reaching a peak of 10-13 ng/ml around day 25. The steroid level started to fall shortly before parturition and returned to the basal level in postparturient animals.
Ovariectomy interrupted pregnancy in some animals, but not in others. When pregnancy was maintained, the mammary development and the plasma level of progesterone were normal.

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The present study was undertaken to assess the metabolism of radiolabeled pregnenolone in the placenta throughout pregnancy in organ culture. The product generated from pregnenolone was only progesterone regardless of the weeks of gestation. The amount of progesterone formed by the placenta was 5.60±0.15×10^-11 moles/mg protein/hour in the 1st trimester (n=4), 6.47±0.09×10^-11 moles/mg protein/hour in the 2nd trimester (n=3) and 7.72±0.25×10^-11 moles/mg protein/hour in the 3rd trimester (n=6). The increase in the formation of progesterone as gestation advanced was statistically significant. The increase in the placental weight throughout pregnancy was in proportion to the rise in maternal plasma levels of progesterone. These data indicate that the manifold increase in progesterone in the maternal circulation as gestation advances mainly reflects the increase in the functional mass of the placenta rather than the increased rate of production of progesterone in the placenta.

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Glycosaminoglycan (GAG) was extracted from the porcine thyroid gland with a buffer comprising 5.3 M guanidine-HCl and proteolytic enzyme inhibitors and was fractionated by subsequent isodensity CsCl centrifugation. 60% of uronic acid positive materials was accumulated in the bottom one-fourth fraction with high buoyant density. More than 90% of this uronic acid positive material in the thyroid tissue was heparin or heparan sulfate (sensitive to nitrous acid treatment) and the rest was chondroitin sulfate or dermatan sulfate (sensitive to chondroitinase ABC treatment). When the accumulated high buoyant density GAG was analyzed on a Sepharose CL-6-B column, approximately 14% of the heparin sulfate were in the macromolecular portion as a form of proteoglycan because it was destroyed by the papain digestion or alkaline borohydride treatment which extensively digests protein or releases GAG from protein by the elimination reaction, respectively. This study demonstrates the existence of heparan sulfate proteoglycan in thyroid tissue for the first time.

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We have performed studies on blood hormone dynamics following intravenous administration of acebutolol, a newly synthesized, β-blocker, and its direct action on the adrenal medulla in vitro.
Intravenous injection of acebutolol into anesthetized dogs almost doubled the plasma adrenaline and noradrenaline concentrations within 5 to 15 minutes, while renin activity was reduced to approximately two-thirds of the pre-administration level.
When arrhythmia was induced in dogs with ouabain, the plasma adrenaline and noradrenaline levels increased to 220±109 and 392±84pg/ml, respectively, from the basal levels of 44±24 and 140±43pg/ml. The restoration of sinus rhythm following the administration of acebutolol was accompanied by a further increase in the plasma adrenaline and noradrenaline levels to 797±364 and 1226±263 pg/ml, respectively. A perifusion experiment indicated that acebutolol directly accelerated catecholamine release from the adrenal medulla in pigs.

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A 12-year-old Japanese girl with polyostotic fibrous dysplasia and endocrine concomitants, was treated with elcatonin, a synthetic eel calcitonin analogue, 10 MRC unit/twice a week given by intramuscular injection. Significant decreases in 24 hr urinary content of hydroxyproline and other amino acids from bone collagen were observed during the course of treatment over 5 months. This biochemical result suggests that the synthetic eel calcitonin analogue exhibits the therapeutic effect in patients with polyostotic fibrous dysplasia by inhibiting bone resorption.

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By serial transplantation of CS 1, a subline of Shionogi carcinoma SC 115, to female mice, another subline was obtained and designated CS 2. The subline showed a complete loss of androgen dependency on the growth of the tumor. When male mice bearing the tumor were castrated and treated with testosterone, the activity of RNA polymerase I in isolated nuclei from the tumor hardly varied during the period of the experiments (36 h), while the activity of RNA polymerase II exhibited a transient increase (about 40%) at 6h after the testosterone injection. The results, together with the previous ones showing 80% and 40% increases in RNA polymerase I activity at 24 h after testosterone administration in the case of SC 115 (androgen-dependent tumor) and CS 1 (less androgen-dependent tumor), respectively, indicate that the stimulation of RNA polymerase I activity by androgen in the tumor tissues is closely related to the androgen dependency on the growth of the tumors.

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Free α-subunit in normal term placenta was examined for molecular weight, electric charge and ability to combine with standard hCG-β in comparison with extracellular free α-subunit and standard hCG-α dissociated from urinary hCG in vitro. The gel chromatography on Sephadex G-100 of the placental extract revealed three major immunoreactive hCG-α peaks, designated as Pa-A (Kav=0.32-0.46), Pα-B (0.47-0.58) and Pα-C (0.59-0.70), near the position of standard hCG-α. In the isoelectric focusing, while Pa-A was mainly distributed over the acidic region, the major components of Pa-B and Pa-C were distributed over the basic region. Furthermore, in the combination study with standard hCG-β, such a-subunit with acidic pI scarcely showed any combining activity whereas α-subunit with basic pI revealed significant combining activity.
These results suggest the following possibilities: that 1) the various size species of placental α-subunit may be responsible for the progressive glycosylation; 2) the small α-subunit with basic pI may combine with β-subunit to form immunoreactive hCG ; 3) the α-subunit, which has not associated with β-subunit, may be converted to a large and incombinative form with acidic pI by further glycosylation, followed by secretion as a free α-subunit.

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Aldosterone responsiveness to angiotensin II (A II) was evaluated in 65 diabetic patients with and without various diabetic complications versus 38 age-matched nondiabetic subjects. Plasma aldosterone (PA), together with plasma renin activity (PRA), was low and responded poorly to furosemide (80 mg, orally) plus upright posture (4 hours) stimulation in diabetic patients. When the PA response to stimulation relative to PRA response was estimated from the ratio of PA increase to PRA increase after stimulation (δPA/δPRA), the 38 non-diabetic subjects had ratios more than 3.0. Of the 65 diabetic patients, 48 had normal (δPA/δPRA) ratios (more than 3.0) and 17 had low (δPA/δPRA) ratios (less than 2.9). Graded A II infusions (1, 2, and 4 ng/kg/min each for 30 min) were performed under a low sodium intake (sodium, 120 mEq/day) in 25 of the 65 diabetic patients, whose (δPA/δPRA) ratios were normal in 15 and low in 10, and in 16 non-diabetic subjects. The PA responses to the graded A II infusions in the normal (δPA/δPRA) diabetic patients were similar to those in the non-diabetic subjects. However, the PA responses to the graded A II infusions in the low (δPA/δPRA) diabetic patients were significantly lower.
It is concluded that, although the majority of diabetic patients have normal aldosterone responsiveness to A II, some diabetic patients have blunted aldosterone responsiveness to A II probably attributable to the abnormality of the adrenal cortex in addition to the impaired renin secretion.

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We studied insulin degradation in human cultured lymphocytes (RPMI-1788 line) with a small but significant number of lysosomes under the electron microscope.
Insulin degradation determined by the TCA solubility method was 64.6±1.2%(mean±SEM) at a trace concentration after the incubation with 2.0×10⁷ cells (4.0×10⁷ cells/ml) for 60 min at 37°C. Because insulin degradation was 54.6±7.0 % in the cell-free buffer in which 2.0×10⁷ cells were previously incubated, most of the insulin was degraded outside of the cells. Gel filtration of the radioactive materials also revealed that most of the labeled insulin in the medium was degraded, and the main peak of the cell-associated radioactivities was intact labeled insulin.
Chloroquine, a lysosomotropic agent, failed not only to increase insulin binding but also to decrease the insulin degradation. Other lysosomal protease inhibitors, antipain and leupeptin had also no effect on insulin degradation. In contrast, bacitracin (500μg/ml) significantly decreased the insulin degardation analyzed by TCA solubility, receptor-rebinding, and the gel filtration method.
These results suggest that insulin molecules are degraded by the enzymes leaked from the cells. The non-receptor mediated process, which is the bacitracin sensitive pathway, might be a general mechanism of insulin degradation in human cultured lymphocytes in vitro.

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Responses of plasma ACTH and cortisol to corticotropin-releasing factor (CRF) were evaluated in 31 normal human males. 1.0μg/kg of sterilized synthetic ovine CRF was administered to the subjects, aged 19 to 53 yr and weighing 50 to 78 kg, at between 9: 30 a.m. and 10: 30 a.m. as an intravenous bolus injection after an overnight fast. Blood specimens were drawn before and 15, 30, 60, 90 and 120 min after injection for later determination of plasma ACTH and cortisol concentrations by radioimmunoassays. Plasma ACTH and cortisol levels for all subjects rose significantly (p<0.001) from the basal level (mean±SEM, 26.8±4.5pg/ml and 12.6±0.9μ/dl) to peak levels (58.4±5.5pg/ml and 22.9± 1.0μg/dl) at 30 min and at 60 min, respectively. Although the plasma concentrations of ACTH and cortisol thereafter declined gradually, the levels at 120 min (43.4±5.2pg/ml and 18.9±0.9 μg/ml, respectively) were still significantly higher than the basal levels (p<0.001). Significant inverse correlations were observed between the basal levels of each hormone and the ratio of the peak level to the basal level (p<0.01), and the increases in plasma ACTH and cortisol concentrations were either not significant or much smaller for the individuals in whom the basal levels were higher than 65 pg/ml and 17.0μg/dl, respectively. No serious subjective symptom was observed during the experimental period in any of the subjects. These data indicate that intravenous injection of 1.0μg CRF significantly stimulates ACTH and cortisol secretion in normal subjects and thus appears to be useful in evaluating and investigating various disorders of the hypothalamic-pituitary-adrenal axis. However, the optimal dose and the most adequate time of administration of CRF for these purposes remain to be established.

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The immunoreactivities of 5 hGH preparations were examined in RIA systems for hPRL, hACTH, hLH, hFSH and hTSH. All preparations derived from pituitary extracts showed distinct displacements in almost all RIA systems when added 10³ to 10⁴ times larger amounts than the proper hormones. On the other hand, biosynthetic methionyl-hGH did not manifest any immunoreactivity in any system with comparable amounts. These results indicate that the displacements caused by hGH preparations in RIA systems for other pituitary hormones are solely due to contamination, and not due to intrinsic cross reaction. In in vitro and in vivo studies using extractive preparations of hGH one should be cautious about contamination of other pituitary hormones.

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