Cloning of TSHR gene and some subsequent studies using the gene were described. Enormous numbers of studies have been performed since the cloning of TSHR gene. Recent molecular studies on TSH receptor and TSHRAb gave various impacts on thyroidology and are resolving past problems. We mainly focused on regulation, processing and glycosylation, TSH- and TSHRAb binding sites, T cell epitopes, and signal transduction of TSHR. Furthermore, we isolated and characterized TSHRAb genes using lymphocytes producing monoclonal TSHRAb obtained from patients with Graves' disease and primary hypothyroidism. Thus, both antigen and antibody genes are cloned. Combined use of these genes will help to investigate the interactions between TSHR and TSHRAb, and may be expected to contribute to the understanding of molecular mechanisms underlying the pathogenesis of autoimmune thyroid disease as well as the physiology of the thyroid gland.
Glucocorticoids regulate the levels of their cognate receptors in a number of target tissues and in many different cell lines. We have compared the effect of three glucocorticoids, cortisol and its synthetic derivatives, prednisolone and dexamethasone, on the levels of glucocorticoid receptor (GR) mRNA in HeLa cells. Clinically, the synthetic derivatives are more active in hormonal action and have a longer half-life than cortisol. In the present study, the amounts of GR mRNA in HeLa cells were examined by Northern blot hybridization after treatment with cortisol, prednisolone or dexamethasone. These glucocorticoids decreased GR mRNA levels differently. After 24h treatment with 1×10-5M cortisol, GR mRNA levels were only marginally suppressed (90% of the control), while prednisolone and dexamethasone suppressed GR mRNA levels to 67 and 57%, respectively. These differences may relate to the biological activities of these glucocorticoids. In time course studies, GR mRNA levels of the cells treated with cortisol and prednisolone decreased to the minimum levels within 4h and then recovered gradually, while those treated with dexamethasone reached the minimum level at 8h and remained suppressed for more than 24h. These differences may relate to the biological half-lives of these glucocorticoids.
A 29-year-old woman with deoxycorticosterone (DOC)-producing adrenocortical adenoma had hypertension and hypokalemia but without Cushingoid features. Plasma renin activity and the aldosterone concentration were low, while the DOC concentration was high (6.10-10.3ng/ml; normal range 0.03-0.33). Plasma cortisol, androgens, and estrogens as well as urinary 17-OHCS and 17-KS were within normal limits. Furosemide administration and two hours upright posture resulted in a 3-fold increase in plasma DOC, but the administration of ACTH, dexamethasone, or angiotensin III had no effect on plasma DOC. Following resection of a right adrenal tumor weighing 70g, the hypertension and hypokalemia disappeared. DOC content in the tumor was high. On light microscopic examination, the tumor was encapsulated, composed of cells with clear cytoplasm and large nuclei and there were extensive areas of fibrosis and infiltration of lymphocytes. According to Weiss's criteria, the tumor was considered to be an adrenocortical adenoma. Immunohistochemically, P450scc, 3βHSD, P450C21 and P45011β were positive with heterogeneity of intra-tumoral expression. No immunoreactivity for P45017α in this adenoma was detected. This is different from a previous report in which a relatively small number of cells in DOC-secreting adrenocortical carcinoma were positive for P45017α.
We have characterized the androgen receptor in a Japanese infant with complete androgen insensitivity syndrome (or androgen resistance), and have investigated the molecular basis. Androgen binding was undetectable in cultured genital skin fibroblasts from this patient by whole-cell androgen receptor binding assay. Sequence analysis of the entire coding region of the androgen receptor gene from this patient revealed a single nucleotide substitution (G→T) at nucleotide position 2676 in exon E (or 5), resulting in conversion of glutamine codon (GAG) to amber stop codon (TAG) at amino acid position 772 within the hormone-binding domain of the androgen receptor. This premature termination mutation (or nonsense mutation), introducing a truncated androgen receptor that lacks most of its androgen binding capacity, is thought to cause the receptor-negative form of complete androgen insensitivity syndrome in this patient.
We examined the expression of reg protein in neonatal rat pancreas treated with streptozotocin (STZ) by means of the immunohistochemical technique and northern blotting. Seven days after STZ injection, the plasma glucose levels in STZ-treated neonatal rats were significantly higher than those in control rats. Scattered distribution of reg protein in pancreatic islet cells was clearly observed in STZ-treated rats, but not in control rats. On the other hand, reg protein was positively stained in the exocrine cells in both groups of rats. Northern blot analyses revealed that the expression of insulin mRNA markedly decreased in STZ-treated rat pancreas, but a significant increase in reg mRNA expression was recognized in the STZ-treated rat pancreas compared with that of control rats. Rats treated with STZ during the neonatal period have been used as a model of non-insulin-dependent diabetes mellitus (NIDDM) and beta cell regeneration. Thus, the increased reg gene expression in neonatal STZ-treated rat pancreas was therefore described for the first time, and this would be a useful model for studying the relationship between NIDDM and beta cell regeneration or reg gene protein.
A rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.
We examined the effects of the long-term administration to mice of thyroid hormone or propylthiouracil (PTU) on lymphocyte subsets in spleens, and thymuses to clarify whether hyperthyroxinemia itself causes the changes in lymphocyte subsets, such as the marked increase in CD5+ B cells and decrease in natural killer (NK) cells, observed in hyperthyroid Graves' disease. Both the number and proportion of splenic NK (Thy-1+ asialo GM1+) cells were increased in hyperthyroxi nemic mice treated with thyroxine (T4) for both short and long terms (8 and 32 weeks, respectively), those of splenic and thymic T (CD5+ sIgM-) cells were increased only in hyperthyroxinemic mice treated for 32 weeks, and those of splenic B (sIgM+) cells and CD5- B cells were increased only in hypothyroxinemic mice treated with PTU for 32 weeks, compared with those in euthyroid mice. These data indicate that 1) long-term hyperthyroxinemia increases splenic and thymic T cells and splenic NK cells, but not CD5+ B cells, in mice, 2) long-term hypothyroxinemia induced by PTU treatment increases splenic B cells and CD5- B cells, and 3) hyperthyroxinemia itself does not cause the changes in CD5+ B cells and NK cells, which are observed in hyperthyroid Graves' disease, in mice.
The effects of infusion of clonidine, an α2-adrenergic agonist, on plasma insulin-like growth factor-I (IGF-I) concentrations were investigated with a single growth hormone (GH) injection in pigs. Four barrows were subjected to four treatments: saline infusion with a vehicle injection, clonidine infusion (0.5nmol/kg/min for 8h) with a vehicle injection, saline infusion with a bovine GH (bGH:100 μg/kg) injection, and clonidine infusion with a bGH injection. Infusion was started 1h before the injection. Plasma IGF-I, bGH, porcine GH (pGH), insulin, glucose, non-esterified fatty acid (NEFA), and blood urea nitrogen (BUN) concentrations were measured. Plasma IGF-I concentrations during saline infusion increased after a bGH injection (P<0.05). However, the IGF-1 concentrations during clonidine infusion did not increase after the bGH injection. Plasma endogenous GH (pGH) was not increased during clonidine infusion. The plasma glucose concentration was noticeably increased during clonidine infusion and moderately increased after the GH injection. Despite the extreme increase in plasma glucose during clonidine infusion, plasma insulin did not change. Neither plasma NEFA nor BUN was changed by these treatments. These results demonstrate that the α2-adrenergic agonist clonidine altered the action of GH to increase the plasma IGF-I concentrations.
HPLC-purified glycosaminoglycans (hpGAG) prepared from extracts of non-luteal mouse (JcL: ICR strain) ovaries were assayed for neovascularization by implanting Elvax films, containing test samples, on the lateral wall of the sheath of m. rectus abdominis in adult female mice of the same strain. Neovascularization occurred in a dose-dependent manner, and was characterized by capillary outgrowth extending into the tissue surrounding the implant. The single major peak of purified GAG on a column of TSK gel DEAE got out of order after treatment with streptococcal hyaluronidase or nitrous acid. The activity of this fraction was also greatly reduced when treated with streptococcal hyaluronidase or nitrous acid. When hpGAG was embedded in the implant with 17α-hydroxyprogesterone at a dose of 20μg/film, neovascularization induced by means of hpGAGs was suppressed. Progesterone at a dose of 50μg/film did not suppress the neovascularization induced by ovarian hpGAG. These findings suggest that 17α-hydroxyprogesterone suppresses the angiogenic activity of hyaluronic acid-like hpGAG in the ovary.
Seventeen consecutive patients (3 men and 14 women, aged 14-75 years) with a hemorrhagic degeneration of the thyroid nodule, which was confirmed by both ultrasonography and either reddish or brown fluid evacuated by fine-needle aspiration, were classified as either acute type with an episode of abrupt painful swelling of the thyroid (n=4), or chronic type in which a painless thyroid nodule was incidentally found (n=13). One of the four acute type patients demonstrated subacute thyroiditis-like symptoms and laboratory findings including transient painful thyrotoxicosis associated with high serum levels of thyroid hormones and thyroglobulin (Tg), a suppressed serum TSH level, a low thyroidal radioactive iodine uptake (RAIU), and an accelerated erythrocyte sedimentation rate (ESR). In the other three acute type patients the serum level of Tg increased markedly, the serum thyroid hormones level increased in one, the thyroidal RAIU was low in two, and the ESR was accelerated in one. In the thirteen chronic type patients, the serum levels of the thyroid hormones and the thyroidal RAID were within the normal range, and few inflammatory signs were observed. These findings suggest that acute hemorrhagic degeneration of the thyroid nodule may thus cause transient subacute thyroiditis-like symptoms and laboratory findings.
The purpose of this study is to find out whether hypercalcemia and hyperparathyroidism are rare or not in manic-depressive patients taking lithium carbonate. The subjects were 13 patients receiving lithium and 19 healthy subjects not receiving it as normal controls. Serum ionized calcium (Ca++), serum parathyroid hormone (PTH), urinary calcium and cyclic AMP (CAMP) were measured. Cervical ultrasonographic examination was also performed. The mean serum Ca++ level in the lithium administered group was significantly higher than that in the control group (P<0.02). There was no significant difference between the serum PTH levels in the two groups. The mean urinary calcium level in the lithium administered group was below the normal range, but the mean urinary cAMP level was within the normal range. Although a parathyroid cyst was found in one lithium administered patient on ultrasonographic examination, no swelling of the parathyroid gland was observed in the other patients in the lithium administered group or in any of the control subjects. In the present study, no distinct hyperparathyroidism was found in the patients in the lithium administered group. Lithium administration affects calcium metabolism in manic-depressive patients and hypercalcemia seems to be one of the complications needing attention at the time of lithium administration.
We treated a patient who was hyperthyroid due to Graves' disease and strongly resistant to methimazole (MMI): in spite of good compliance, she needed 150mg of MMI daily to control her hyperthyroidism. To elucidate the reasons of resistance to MMI, her serum and intrathyroidal MMI concentrations were determined by high pressure liquid chromatography (HPLC). After taking a 30mg dose of MMI, she had a similar serum MMI concentration-time curve to that of a normal subject: drug malabsorption and rapid drug metabolism were not evident when studied after surgical treatment. After her serum containing MMI was incubated with Protein G, the MMI concentration of the fraction not bound to Protein G did not change significantly from that of untreated serum: the possibility of anti-MMI IgG antibody production was considered unlikely. Furthermore, the intrathyroidal concentration of MMI in a surgically obtained tissue specimen was 3μ/g wet tissue and appeared to be comparable with those of other Graves' tissues reported. Considering that the patient had been taking 150mg per day of MMI by the time of thyroidectomy, her intrathyroidal MMI concentration was relatively low, suggesting possible impairment of intrathyroidal MMI accumulation. The possibilities of impaired intrathyroidal actions and the severity of hyperthyroidism, especially high T3 levels, also remained as possible causes. In conclusion, here was a severely hyperthyroid patient who was poorly responsive to conventional doses of MMI, and impairment of thyroid uptake of MMI or of pathways after uptake were considered as possible mechanisms.
Effects of ethanol treatment on Leydig cell NADPH-generating enzymes and lipid profiles were studied. Ethanol treatment (3.0g/kg. b.wt.) twice daily as a 25% (v/v) aqueous solution given to adult Wistar rats reduced the body weight, testis weight and relative weights of the seminal vesicles and ventral prostate. Serum LH and testosterone were also decreased. Similarly, the NADPH-generating enzymes such as G-6-PDH, 6-PGDH, NADP+-ICDH were reduced, but malic enzyme was unaltered. Leydig cell total lipid was decreased: neutral lipids such as esterified cholesterol and triacyl glycerol were decreased but free cholesterol and diacyl glycerol were increased. The reduction in total phospholipid was contributed to by fractions such as phosphatidyl inositol, phosphatidyl serine, phosphatidyl choline and phosphatidyl ethanolamine. Withdrawal of ethanol treatment for 30 days restored these to the normal level. The present findings suggest that the ethanol treatment impairs Leydig cellular NADPH generation which may be one of the biochemical mechanisms mediating the direct and indirect effects of ethanol resulting in hypoandrogenization.
The prevalence of thyroid dysfunction in the elderly is reported to be markedly high, at least in some Western countries in which iodine intake is sufficient or increased because of recent supplementation of iodine for public health. We therefore wished to investigate the prevalence of thyroid dysfunction among elderly people in an endemic goiter area. The study included 198 subjects over the age of 55 years. It was carried out in two towns 20-30km, south of Kayseri, Central Anatolia. Questioning on medical history, physical examination and grading of thyroid gland size were performed. Serum TSH was measured by a sensitive immunoradiometric assay. Serum free thyroid hormones and thyroid autoantibodies were measured in the subjects with TSH concentrations below 0.4 μU/ml or above 4.5μIU/ml on the initial screen. Drinking water was also analyzed for iodine content. Twenty-five (12.6%) subjects had either elevated (6.5%) or suppressed (6.1%) serum TSH levels. No patient had clinical hypothyroidism (high TSH and low free thyroxine and free triiodothyronine). Three (1.5%) subjects had clinical hyperthyroidism (low TSH and high free thyroxine and free triiodothyronine). Only one subject was positive for antimicrosomal and antithyroglobulin antibodies. The prevalence of goiter was 25.8%. The iodine level in drinking water was found to be 3μg/L. In conclusion, we believe that the prevalence of thyroid dysfunction in the elderly may depend on the iodine status in the environment. We think that hyperthyroidism due to multinodular goiter is more important than hypothyroidism for elderly people living in an endemic goiter area, probably due to the low frequency of autoimmune thyroid disorders.
The effects of hypothalamic peptides vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), growth hormone (GH)-releasing hormone (GHRH) and somatostatin (SRIF) on GH release from cultured bovine adenohypophysial cells were studied. The cells were incubated for 2h with the peptides after preincubation for 3.5 days. At doses from 10-9 to 10-7M VIP, the amount of GH released was significantly greater than in the controls (P<0.05 to P<0.001). PHI (10-10 to 10-7 M) did not alter the bovine GH concentration in the media. Incubation with the media containing 10-7 M GHRH, 10-7 M VIP, and combined treatment with the VIP plus GHRH increased GH by 186, 40 and 182%, respectively (P<0.001). Furthermore, although VIP-induced GH release was significantly decreased by SRIF compared with the treatment with VIP alone (P<0.001), the VIP significantly blunted the inhibitory effect of the SRIF on GH release by 24% when compared with that of the SRIF plus GHRH without the VIP (P<0.05). GH release in combined treatments with VIP, GHRH and SRIF was significantly less than that of the VIP plus GHRH (P<0.001), but it was a significant 29% increase compared with the SRIF plus GHRH (P<0.05). The combined effects of the VIP (10-7 M) with GHRH (10-7, 10-8 and 10-10M) significantly induced GH release compared with the controls (P<0.001), but no additive effect was not observed when compared with the GHRH alone. The results indicate that VIP, but not PHI, acts directly on cultured adenohypophysial cells to induce GH release in cattle.
Although c-kit proto-oncogene product is known to be weakly expressed on normal thyrocytes, its function is unclear. In order to investigate the significance of thyroid c-kit, c-kit gene expression in 37 various thyroid tissues was analyzed by comparing c-kit gene expression with the mRNA expression of three thyroid-specific genes: thyroid peroxidase, thyroglobulin, and thyroid stimulating hormone receptor. c-kit mRNA was hardly detected by the usual northern blot method in 2 of 7 follicular carcinomas, 11 of 12 papillary carcinomas, and a medullary carcinoma. On the other hand, a high level of c-kit mRNA expression was found in all 17 benign thyroid tissues (4 normal thyroid tissues, 4 Graves' disease, 2 adenomatous goiters, and 7 follicular adenomas). This study found that c-kit proto-oncogene is more likely to lose expression in differentiated thyroid carcinoma than any thyroid-specific gene. Decreased c-kit gene expression may serve as an indicator for the de-differentiation of thyrocytes.