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Intact rat thyroid lobes incubated in vitro release recently synthesized thyroglobulin (Tg) into the media at a faster rate than they release thyroglobulin stored in follicular structures. Differential release of this Tg fraction cannot be explained by morphological alterations in thyroid architecture during incubation. This rapidly excreted fraction exhibits a low density on rubidium chloride gradients characteristic of poorly sialylated and poorly iodinated thyroglobulin, comigrating on rubidium chloride gradients with thyroglobulin isolated from tunicamycin treated glands. This poorly sialylated and poorly iodinated thyroglobulin is itself unaffected in its density or release into the media by tunicamycin treatment. Tg isolated from the media of tunicamycin treated glands has nearly the same low iodine and low sialic acid content as rat serum thyroglobulin and does not incorporate radiolabelled glucosamine. This fraction thus appear to duplicate properties of low glycosylated-low iodinated thyroglobulin released from thyroid cells in organisms that have no follicular structures and no follicular storage process as well as from thyroid tissue in patients with thyroid disease states, particularly thyroid tumors. Thus it is proposed a "short loop" pathway of low-glycosylated low-iodinated thyroglobulin directly into circulation, that bypasses and is not stored in the follicular lumen, the "long loop".

Thyroglobuline (Tg) biosynthesis has been studied in an adult fresh water lamprey Lampetra planeri (Bloch), by injection in coelomic cavity or by in vitro incorporation in follicles of branchial region of various radioactive labels: 125I-, 3H-leucine, 3H-mannose, 3H-galactose, N-acetyl mannosamine and 3H-N-acetylglucosamine. Labelling by all theses substances, present in Tg of mammals, proceeds efficiently in two hours. It has been demonstrated that the cyclostome studied synthetizes a 18 S iodinated glycoprotein analogous to Tg of higher vertebrates by its molecular size. It contains all the same hexose and N-acetyl derivatives of these in its sugar moiety and the same iodinated aminoacids. Purified 18 S Tg of the lamprey has been prepared; it is very poor in iodine (127I) and has a very low T3 and T4 content.

Three monoclonal anti-insulin receptor antibodies have been labelled with 125I according to various methods (Cloramine T, Lactoperoxidase and IODO-GEN). The effect of labelling on antibody structure and function has been characterized using the following parameters: a) specific activity obtained in four different labelling procedures, at least; b) TCA labelled antibody precipitable 90 days after labelling; c) interaction between labelled antibodies and the insulin receptor; d) ability of antibodies to inhibit insulin-stimulated receptor auto-phosphorylation. Cloramine T method produced labelled antibody with constant specific activity; however, some preparations were unstable and showed reduced capacity to recognize the insulin receptor. Lactoperoxidase method produced stable antibodies; however, specific activity was highly variable and antibodies had low capacity to interact with the insulin receptor. The IODO-GEN method produced antibodies with constant specific activity, stable, high capacity to interact with the insulin receptor, and, moreover, maintaining in full the capacity to inhibit the insulin-stimulated auto-phosphorylation of the insulin receptor, since it does not induce antibody alterations which in turn affect antibody-receptor interaction biological action.

Studies have been carried out with labeled substrates on isolated adipocytes of male rats injected with sulpho-conjugated estrogens. Three days treatment did not affect insulin receptor concentration or affinity in the cells. Conversely glucose uptake is reduced in the absence of insulin, or by reduced responsiveness in the presence of it. After ten days treatment, insulin binding impaired on account of reduced receptor concentration, whereas glucose uptake was normal in the absence of insulin, increased at low and decreased at high insulin concentration, on account of both decreased responsiveness and increased sensibility to insulin. Thus it is suggested that estrogens acts on glucose uptake prior to act on the insulin receptor in the adipocytes.

Thyroglobulin biosynthesis was studied in thyroid glands of rats treated during 30 days with a daily dose of propranolol, a non-selective beta-adrenergic blocking drug. Studies were carried out by extraction of soluble proteins from homogenates after incubation of the glands in presence of [3H]-4,5-L-leucine and [3H]-D-1-galactose. Thyroglobulin 19S, 12S and 4-8S soluble proteins were separated and identified by ultracentrifugation in a saccharose gradient. The glands of rats treated with propranolol showed a decreased amount of soluble proteins as well as a decreased incorporation of [3H] labeled markers. 19S thyroglobulin is poorly represented, but very large amounts of the 12S monomer are present; this suggests an impairment in the dimerization of the 12S subunit, absent in normal controls. This impairment could be due to a structure modification. Propranolol reduces the biosynthesis of thyroglobulin in thyroid gland as well as the formation of T3 from T4 in peripheral tissues; its therapeutical use against thyrotoxicosis is thus justified. The study of its action on the dimerisation of the 12S subunit could be of interest to clear the mechanism of thyroglobulin biosynthesis.

The effect of naloxone (opioid receptor blocker) on the impairment of growth hormone (GH) release after clonidine (alfa 2-adrenergic agonist) was investigated in 10 volunteer obese subjects. The patients (4 males and 6 females, 16-22 year old) with fat excess (15 +/- 2 kg) estimated by bioelectrical impedance analysis (BIA) were studied repeatedly. The patients, were perfused by a slow saline infusion. 30 min later they received a bolus dose of clonidine (150 micrograms p.o.), followed 30 min later by a bolus dose of naloxone (10 mg i.v.) or a corresponding volume of isotonic sodium cloride (I.S.) for control. No significant changes occurred in blood GH concentration after clonidine administration and naloxone did not induce GH response at clonidine. These results suggest that in obese subjects the impairment of GH release after clonidine is not mediated via receptors sensitivity to naloxone.

Carbohydrate chains formation into thyroglobulin (Tg) is a prerequisite for thyroid hormones formation and completeness of carbohydrates chains is necessary for secretion of Tg into the follicles. Tg biosynthesis has been investigated by in vitro experiments, incubating rat thyroid glands with labeled amino-acid and carbohydrate in the presence of tunicamycin, a specific inhibitor of protein glycosylation. Tunicamycin inhibit Tg biosynthesis which is impaired in carbohydrate chains addition but slightly in the polypeptide synthesis, as shown by inhibition of 3H-glucosamine incorporation. Thus tunicamycin inhibits carbohydrate incorporation into Tg without affecting the polypeptide chain growth and decreases its secretion into the follicles.

Thyroid biosynthesis in glassed eels (Anguilla anguilla L.) was studied to establish whether salinity changes could affect it, when they live in sea water or in fresh water containing 125I. Aqueous extrait of homogenized cephalic heads of glassed eels contains an iodinated protein 17-19 S having thyroglobulin-like properties and including iodotyrosins (MIT and DIT) and thyroid hormones (3 and T4). Biosynthesis of this proteins is roughly twice more important in fresh water than in sea water at 19-21 degrees C and its specific radioactivity (125I) is practically double in fresh water.

The biosynthesis of thyroglobulin (Tg) in larva of a fresh-water lamprey, Lampetra planeri B1. has been established. This glycoprotein presents the same characters as in thyroid follicles of adult lampreys, as shown by its 18-19 S sedimentation coefficient and by the incorporation (in vivo and in vitro experiments of 4, 12, 72 h) of 125I, 3H-leucine and 3H-galactose. 3-8 S fractions and a 12 S monomer are the precursors of the 18-19 S protein. Total I % of Tg is very low (0.002 %) ; about 5 % of 125I are present in thyroid hormones (T3 and T4) in the 125I-labeled protein. The biosynthesis of 18-19 S Tg proceeds in larvs before the morphological differentiation of thyroid cells and follicles after metamorphosis. However, the biosynthesis of this protein is much slower in the endostyle of larvs, in which a primitive mechanism of storage is poorly efficient, compared to the accumulation of Tg in the colloid of the follicles of adults.

Groups of rats have been treated for one month with a daily dose of 50 mg/kg of body weight/day of isoniazide a product used therapeutically in man against tuberculosis. The animals submitted to this experiment showed an increase of thyroid weight (39%) and a decrease of its content in water soluble proteins, including thyroglobulin and precursors. These proteins have been labeled in vitro by [3H]-4,5-L-leucine and by [3H]-galactose, by incubation of isolated glands during 4 h in a medium containing this labeled marker. The decrease of thyroglobulin-like proteins was of 20-37% for [3H]-D-1-galactose, labeled fraction and of 46-53% for the [3H]-leucine labeled fraction. Two thirds of the radioactive protein sedimented by ultracentrifugation in a sucrose gradient were present in a 12S monomer of thyroglobulin. This suggests an impairment of biosynthesis of 19S thyroglobulin, in which two of the subunit are associated. Under experimental conditions adopted this 19S protein was labeled by [3H]-leucine, but not by [3H]-galactose. Two other antituberculous agents studied, ethambutol and rifamizine, exerted not the same effects on thyroid gland in rats. Therefore a control of thyroid function appear to be necessarily only in man treated by isoniazine.

Unlabelled: In order to correlate possible alterations of cell-mediated immune response with the evolutive phases of Plummer Adenoma (P. A.), T lymphocytes subpopulations in FNA samples and in peripheral blood lymphocytes (PBL) have been studied in 5 patients with autonomous nodules. The lymphocyte component in FNA and peripheral blood has been isolated by Lymphoprep gradient centrifugation; the analysis of T helper and T suppressor subpopulations was made by indirect immunofluorescence with OKT8 and OKT4 monoclonal antibodies. Our results show a reduction in OKT4/OKT8 ratio in cytological samples compared with PBL in patients with P. A., while in control subjects there was not statistically significant difference. In the patients with P. A., the relative increase of OKT8 lymphocytes in FNA compared with PBL is correlated with the functional state, that is toxic adenomas have a lower OKT4/OKT8 ratio compared with nodules in pre-toxic phase., In Conclusion: T lymphocyte subpopulations typing in FNA demonstrate that, even in this type of hyperthyroidism, immune response disorders are present and consist of relative increase of suppressor/cytotoxic T cells, compared to T helper cells.

The effects of endogenous opiates on insulin response to oral glucose load were studied in obese subjects and in lean healthy volunteers. None of these having a family diabetes. After 3 days on an 1,800 cal./m2, 40% carbohydrate diet all subjects underwent two standard 75 g oral glucose tolerance tests (OGTT), one of which was accompanied by an i. v. administration of 10 mg of, an antagonist of opiates, the naloxone. In one group of obese impaired oral glucose tolerance test occurred. All obese, but not the lean healthy volunteers, showed: 1) increased basal plasma insulin levels, 2) higher insulin response to OGTT, 3) a decrease in insulin response to OGTT after naloxone administration, with significant differences at 60 min (p less than 0.01) and 90 min (p less than 0.025). In none of the subjects significant differences were observed in blood glucose levels after OGTT plus naloxone administration. These data suggest that increased endogenous opiates may affect insulin response to glucose in obese with impaired or normal oral glucose tolerance test. At present there seems to be no satisfactory explanation for unchanged blood glucose levels during OGTT with and without naloxone despite a decrease in insulin secretion in the obese patients.

The ACTH test is important when hirsutism occurs in women with a slight 21-hydroxylase deficiency, and normal basal 17-OH Progesterone (17-OH-P/plasma levels). Extensive hormonal assays: LH, FSH, Prolactin, 17 beta-estradiol (E2), Estrone, 17OH-P, Androstenedione, Testosterone, Cortisol (C), Dehydroepiandrosterone-S (DEA-S) were carried out in 36 hirsute women. 13 of these presented hormone levels as found in polycystic ovary syndrome (PCOS), 6 women presented a slight 21-hydroxylase deficiency (increased plasma 17-OH-P and decreased C after ACTH test with significant, p less than 0.01, increase of 17-OH-P/C and 17 women presented idiopathic hirsutism (IH). The hormonal pattern, in the basal condition, is not different in IH or in slight 21-hydroxylase deficiency. The ACTH test is able to differentiate between IH and adrenal hirsutism.

1. The effect of ouabain on the molecular properties of (Na+/K+)-ATPase has been studied in purified preparations of the enzyme, isolated from the microsomal fraction of outer red medulla of porcine kidney, according to a modification of the method described by Jorgensen. 2. Ouabain, a specific inhibitor of (Na+/K+)-ATPase, binds at the potassium site of the enzyme, thus generating an increase in its stability towards the common denaturing agents, such as exposure to different concentration of guanidinium chloride (GdmC1) or to acidic solutions.