Since TPO plays a cardinal role in regulating many cellular processes of thyroid hormone biosynthesis in thyrocytes, it is important to present a summary of the impact that TPO research on the basis of molecular structure has had on our understanding of thyroid diseases. This review has therefore been written to highlight the biochemical, molecular biological, immunological and clinical aspects of TPO in thyroid follicular cells.
As for hormonogenesis, further details of the properties of the superoxide generating system remain unknown, but based on decisive evidence showing what active iodinating species are at initial and principal stages, a novel concept of the mechanism of two electron oxidation has been put forth.
Recent progress in studies of analyses of TPO gene structure has also provided stimuli for renewed investigation into a much wider field of thyroidology, including thyroiditogenesis.

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Insulin-like growth factor-I (IGF-I) is a potent mitogen in many cell systems. In cultured rat granulosa cells, however, IGF-I is known to be an inducer of differentiation. The present study was conducted to identify the factor which determines the direction of IGF-I action: either DNA synthesis or LH receptor expression. When granulosa cells were incubated with IGF-I in the presence of various concentrations of follicle-stimulating hormone (FSH), DNA synthesis as assessed by [³H]thymidine incorporation was increased only in the presence of low doses of FSH. The stimulatory effect of FSH on DNA synthesis was observed in a very narrow range of FSH concentration between 2 and 10ng/ml. At higher concentrations, FSH had little effect on DNA synthesis but instead induced expression of receptors for luteinizing hormone (LH), a marker of granulosa cell differentiation. At 5ng/ml, FSH elicited maximal stimulation of DNA synthesis and simultaneously induced LH receptor expression to some extent. In these cells, DNA synthesis peaked at 36h but expression of LH receptor occurred later than 36h, peaking at 60h. The ability of IGF-I to stimulate DNA synthesis was enhanced by the long term pretreatment with FSH: when FSH was added from the beginning and IGF-I was added after 36h or later, ICE-I-mediated DNA synthesis was approximately twice as great, and was accompanied by a two-fold increase in the number of bromodeoxyuridine-labeled nuclei. Under these conditions, LH receptor expression was reduced to approximately 50%. Finally when cells were incubated for 12h with or without FSH, washed extensively with the medium and then IGF-I was added, DNA synthesis was augmented only in FSH-primed cells. Forskolin, an activator of adenylate cyclase, reproduced the effect of FSH. These results indicate that, in the presence of FSH, IGF-I has the ability to induce both DNA synthesis and differentiation and that FSH determines the action of IGF-I on promotion of either growth or differentiation. Furthermore, priming with FSH renders granulosa cells responsive to IGF-I in terms of DNA synthesis.

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Since a heterozygous missense mutation of the RET proto-oncogene in the germline was found to cause multiple endocrine neoplasia type 2A (MEN 2A) in 1993, some 20 different mutations of this gene have been identified in MEN 2A kindreds. We report an MEN 2A family in which serine (AGC) substitutes for cysteine (TGC) at codon 618 in exon 10 of the RET proto-oncogene. The mutation was identified by sequencing PCR products of exons 10 and 11 in the proband. Since this mutation results in creation of a new cleavage site for Alu I restriction enzyme, most of the other members of the family were screened by digestion of the PCR product of exon 10 with this enzyme. Eleven of 20 subjects across four generations examined have the mutation of the RET proto-oncogene, and all of the adult gene carriers except one woman had MTC. Characteristics of this family are 1) pheochromocytoma has been found in only the proband, 2) no obvious hyperparathyroidism has been observed, and 3) the prognosis is favorable, with nobody dying of MEN 2A itself. Genetic analysis of MEN 2A is definitely useful and essential for screening of a MEN 2A family. It is very important to accumulate cases with MEN 2A and investigate the phenotype and the prognosis in each mutation.

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Our previous study demonstrated that the serum IL-6 correlated inversely with serum T3 and the T3/T4 ratio in children with acute respiratory infection during the acute phase of illness. To investigate whether serum IL-6 inversely correlates with serum thyroid hormone during not only the active stage of the disease but also in the follow-up period in nonthyroidal illness, we measured serum levels of IL-6, T3 and T4 in 31 children from the acute to the convalescent phase. They were divided into 3 groups; 7 patients with Kawasaki disease, 16 patients with infectious disease and 8 patients with non-inflammatory disease. In the follow-up of patients with Kawasaki disease, a marked inverse relationship was observed between serum IL-6 and T3 (r=-0.844, P<0.001) or the T3/T4 ratio (r=-0.863, P<0.001). Serum T4 showed a weak but significant negative correlation with serum IL-6 (r=-0.474, P=0.035) only in this situation. There was also a significant negative correlation between serum IL-6 and T3 (r=-0.582, P<0.001) or the T3/T4 ratio (r=-0.660, P<0.001) during the follow-up of children with infectious disease. In the follow-up study of patients with non-inflammatory disease, however, no significant relationships were observed between serum thyroid hormones and IL-6. IL-6 may be one important factor involved in the decrease in the serum T3 level and the T3/T4 ratio in patients with nonthyroidal illness particularly characterized by strong inflammation and activation of the immune system as observed in Kawasaki and infectious disease.

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Immunoreactive activin A (ir-activin A) release from cultured rat anterior pituitary cells was examined by measuring iractivin A in culture medium by a specific radioimmunoassay. Ir-activin A release into the medium increased over 1-18 days, and reached a maximal level at 12-15 days. The basal levels of ir-activin A in the culture media were 0.70±0.10 (mean±SD), 1.30±0.36 and 1.83±0.44ng/10⁶ cells, when cultured for 6 days with 0, 2 and 10% fetal calf serum, respectively. LHRH induced an approximate 1.4-fold increase in ir-activin A release in contrast to a 40-60% inhibition with FSH, but LH did not affect the activin A release. In the presence of 12-o-tetradecanoylphorbol acetate (TPA), ir-activin A release was enhanced, but no significant effect was induced by forskolin. Activin A was distinctly immunostained in cultured rat anterior pituitary cells. These results suggested that activin A release from the pituitary is modified by FSH and LHRH, and that the activation of protein kinase C may be involved in the action of LHRH.

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Complementary DNA (cDNA) encoding a long form of prolactin receptor (PRL-R_L) was cloned from mouse mammary gland by PCR using primers designed from the noncoding regions of previously reported rat ovarian PRL-R_L cDNA. The nucleotide sequence encoding the extracellular and transmembrane domains of PRL-R_L is completely identical to that of short forms of mouse PRL-R. The amino acid sequence deduced from the nucleotide sequence of mouse PRL-R_L is 91% identical to that of rat PRL-R_L. To address the question of whether or not the cloned mouse PRL-R_L cDNA encodes a functional PRL-R_L we transfected Ba/F3 IL-3-dependent murine pro-B lymphoid cells with the cDNA. By culturing the transfected cells in a medium which contained PRL in place of IL-3, we selected 5 PRL-dependent clones. All of these PRL-dependent clones, BaF/PD cells, expressed PRL-R_L mRNA. In addition, BaF/PD cells expressed mammary-specific γ-casein mRNA in response to PRL and dexamethasone. Based on these results, it was concluded that the mouse mammary PRL-R_L cDNA cloned in this study is functionally active in mediating both PRL-dependent growth and mammary-specific gene expression.

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This paper reports the results of research to examine the possibility of using the differential expression of two enzymes, dipeptidyl peptidase IV (DPPIV/CD26, EC: 3.4.14.5) and thyroid peroxidase (TPO, EC: 1.11.1.7), as histochemical markers histopathologically to diagnose thyroid carcinomas. The research is based on previous reports that DPPIV/CD26 is overexpressed in differentiated thyroid carcinoma tissues, and that TPO activity is very low in thyroid carcinoma tissues. Differential expression of the two enzymes in 32 thyroid tissues of various thyroid diseases was studied by Northern blot analysis and histochemical analysis. On Northern blot analyses, all 14 differentiated thyroid carcinomas (11 papillary carcinomas and 3 follicular carcinomas) overexpressed DPPIV/CD26 mRNA, whereas all 17 benign thyroid tissues (4 normal thyroid tissues, 4 Graves' diseases, 2 adenomatous goiters and 7 follicular adenomas) showed faint mRNA expression of DPPIV/CD26. All 17 benign thyroid tissues expressed high levels of TPO mRNA, whereas all 11 papillary carcinomas strongly underexpressed TPO mRNA. Histochemically, all 17 benign tissues were DPPIV/CD26 negative and strongly TPO positive, while all 11 papillary carcinomas were strongly DPPIV/CD26 positive and TPO negative. Two of 3 follicular carcinomas were histochemically positive for the two enzymes. A medullary carcinoma did not show any mRNA expression of either enzyme. These results suggest that the differential expression of these two enzymes can be applied to study the thyroid tumorigenesis.

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Some patients with Graves' disease respond well to anti-thyroid drug treatment but others do not. Factors determining the patient's responsiveness to the medical treatment are unclear, but the intrathyroidal iodine pool is believed one of the important factors. In this study, we found that Δ radioactive iodine uptake (RAIU) (RAID at 24h-RAIU at 3h) is useful in predicting early response to treatment with methimazole (MMI). Among 32 patients with Graves' disease, who were given 30mg MMI as an initial dose, 11 patients responded quickly to MMI-treatment. Within one month, serum free T₄ levels decreased to below the normal range in 6 patients (<10.3pmol/L) or decreased from beyond the highest level of the assay (>125pmol/L) to the normal range in 5 patients. When these rapid responders (group A) were compared with the remaining 21 patients who showed a more gradual response to MMI-treatment (group B), a different pattern of ¹²³I-thyroid uptake was noted. RAIU at 3h was significantly higher in group A than in group B, while RAIU at 24 h was similar in the two groups. As a result, rapid responders had a significantly lower ΔRAIU value than gradual responders (-0.7± 8.4% in group A, 14.2±8.2 in group B, P<0.01). No significant difference was found between the two groups in various pre-treatment parameters such as severity and duration of thyrotoxicosis, the titer of TSH receptor antibodies (TRAb), frequency of positive antithyroglobulin antibodies (TGHA), urinary excretion of iodine and thyroid volume. The incidence of positive antithyroid microsomal antibodies (MCHA) was higher in group A than in group B, and thyroid ultrasonography showed a tendency to low echogenicity in group A. ΔRAIU was negatively correlated with the reduction in the serum free T₄ level during the first two weeks after MMI-treatment was initiated (r=-0.60, P<0.01). Moreover, ΔRAIU correlated positively with the biological half-life of the intrathyroidal iodine, calculated in a different series of 24 patients with Graves' disease who received radioisotope treatment (r=0.54, P<0.01). The low ΔRAIU value is considered to reflect the rapid turnover of the intrathyroidal iodine, and may be related to the small intrathyroidal iodine pool. ΔRAIU is useful in predicting early responsiveness of patients with Graves' disease to MMI-treatment.

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Serum granulocyte colony-stimulating factor (G-CSF) levels were measured for 24h in 6 patients with hematological disorders and two patients with Graves' disease associated with malignant exophthalmos after iv infusion of high-dose methylprednisolone (HDMP) (20mg/kg BW)for 2h starting at 0900h. Saline solution (500ml) was infused iv for 2h as a control on two days before starting the HDMP therapy. Serum G-CSF levels increased with the mean peak values at 4h after the HDMP therapy (mean±SD: 488.1±125.8pg/ml vs. saline control 74.4±21.9pg/ml, P<0.01). Circulating absolute neutrophil counts (ANC) also increased with the mean peak values at 24h after the HDMP (6, 057±1, 460/μl vs. saline control 2, 007±390/μl, P<0.025). These findings indicate that glucocorticoid has a stimulating effect on G-CSF release and that the increased G-CSF release could be involved, at least partly, in neutrophilia induced by glucocorticoid.

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We treated an 11-year-old boy with a testicular Leydig cell tumor. We analyzed the testosterone production of this tumor by immunolocalization of steroidogenic enzymes and in vitro three-dimensional histoculture. Spermatic venous blood from the tumor bearing testis had noticeably high concentrations of testosterone and androstenedione. The tumor had the characteristic ultras-tructural features of steroid producing cells and was immunoreactive for P450scc (side chain cleavage), 3βHSD (hydroxysteroid dehydrogenase) and P450c17 (17α-hydroxylase). Three-dimensional collagengel-supported histoculture demonstrated that the tumor tissue in the culture maintained its histologic architecture, expression of steroidogenic enzymes, and secretion of testosterone into the medium for up to 7 days in culture. Histoculture preserved in vitro testosterone production in this case of testicular Leydig cell tumor.

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To clarify the precise function of incidentally discovered adrenocortical adenoma, immunohistochemical and dispersed adrenal cell studies were performed. We have recently seen five patients with so-called nonfunctioning adrenocortical adenoma. Diurnal variation in plasma cortisol and suppression of plasma cortisol and urine 17-hydroxycorticosteroids in response to dexamethasone administration revealed adrenocortical function within normal limits in all cases, and no signs or symptoms of adrenal steroid hormone excess were evident. Since a high uptake of iodomethyl-norcholesterol was recognized in each adrenal mass, it was supposed that these adrenal tumors produced steroid hormone to a certain extent, and each patient received unilateral adrenalectomy. P450c17, a key enzyme involved in cortisol production, was expressed in the tumor region in all cases in an immunohistochemical study. Upon in vitro steroidogenesis with dispersed adrenal cells in two cases, all steroid hormones measured except for aldosterone (progesterone, 17α-hydroxyprogesterone, pregnenolone, 17α-hydroxypregnenolone, 11-deoxycortisol, cortisol, 11-deoxycorticosterone, corticosterone, 18-hydroxydeoxycorticosterone, dehydroepiandrosterone and androstenedione) were produced in a culture medium. The results indicated that these tumors possessed the capacity for cortisol production, which was in agreement with the results of an iodomethyl-norcholesterol scintigraphy. All patients with mild hypertension or diabetes mellitus had no signs or symptoms of steroid hormone excess, but they could potentially develop a steroid excess syndrome such as Cushing's syndrome in the future.

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In order to clarify the role of GH on ovarian function, recombinant human GH (rhGH) was administered to adult spontaneous dwarf rats (SDR) for 14 days. The SDR were sacrificed on the day of estrus and the effects of rhGH on ovarian weight, follicular population and the viability estimated by 5-bromodeoxyuridine (BrdU) incorporation, were studied. RhGH administration caused a significant gain in body weight of the SDR, and the ovarian weight of the GH-injected SDR showed a significant increase in comparison with saline injected SDR. The total population of follicles was significantly greater in the GH-treated group than in the control, and the difference was attributed to the increase in the number of large follicles more than 500μm. Total uptake of BrdU into granulosa cells was significantly higher in GH-treated rats than in the control, and, in particular, the intermediate-sized follicles (300-500μm) of the GH-treated rats showed signs of high incorporation of BrdU. These results suggest that hGH acts on intermediate follicles, stimulates their viability and increases the number of growing follicles.

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In order to characterize the dissociation of receptor-bound prolactin (PRL) by mammary cells, cells were prepared from lactating mice by collagenase digestion and loaded with PRL. The dissociation reaction was performed in the presence of PRL. In the concentration range 1ng/ml-1μg/ml examined, PRL at higher than 10ng/ml accelerated the dissociation of PRL in a concentration-dependent manner. The action of PRL on dissociation was completed within a short period. At the end of the 1h-incubation period, the dissociation rate constant (k_-1) was about 2 times larger in the presence of 1μg/ml of PRL than in its absence. The action of PRL occurred predominantly at the level of the plasma membrane receptor. Mammary cells with greater PRL-binding capacities had larger k_-1 in response to PRL. The present data showed that the dissociation of PRL from the receptor was influenced by the concentration of PRL and by the PRL-binding capacity of the cell. The rate of PRL-receptor interaction is expressed by the equation of k_-1(PRL-bound receptors). It is probable that at exceedingly high levels of PRL, the PRL-receptor interaction occurs more frequently in the presence of PRL-dependent dissociation than in its absence.

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The present study was undertaken to determine the pathophysiological role of arginine vasopressin (AVP) in elderly patients with hyponatremia, and the efficacy of fludrocortisone acetate in treating their hyponatremia. Eleven hospitalized patients aged 65 years or older whose serum sodium levels were less than 130mEq/l were examined. The hyponatremic patients included two groups of patients: syndrome of inappropriate secretion of antidiuretic hormone (SIADH) and central salt-wasting syndrome. And 24 healthy, young subjects aged 20 to 34 years, and 24 healthy, elderly subjects aged 65 to 80 years were recruited by community announcement. The elderly subjects had decreased urinary concentrating ability and exaggerated response of AVP secretion to osmotic and nonosmotic stimuli, as compared to the young subjects. All the patients had hyponatremia, with the exaggerated urinary loss of Na. Plasma AVP levels were elevated despite hypoosmolality in all the 2 groups of hyponatremic, elderly patients. Plasma renin activity and plasma aldosterone concentrations were low in the patients with SIADH and central salt-wasting syndrome. Fludrocortisone acetate therapy was effective in the patients with central salt-wasting syndrome and 3 patients with SIADH whose hyponatremia remained unchanged after water restriction. Water restriction therapy normalized serum Na levels in only 3 patients with SIADH. These results indicate that AVP is involved in the mechanism for hyponatremia in the elderly patients with SIADH and central salt-wasting syndrome. Severe hyponatremia associated with SIADH and central salt-wasting syndrome responds well to mineralocorticoid therapy. Both the secretion of AVP and renal sodium handling may be involved in the mechanisms of action of the disorders. The diagnostic criteria for SIADH in the elderly patients may have to be reevaluated and should be considered to indicate fludrocortisone acetate therapy.

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Sixteen sporadic pheochromocytomas, 3 pheochromocytomas in neurofibromatosis 1, and 4 pheochromocytomas in multiple endocrine neoplasia (MEN) 2A or 2B were screened for mutations at codon 768 of the RET proto-oncogene by AluI digestion of polymerase chain reaction (PCR) products and mutations in exon 13 by PCR-single strand conformation polymorphism (SSCP) analysis. Although mutations at codon 768 (GAG→GAC; Glu→Asp) of the RET proto-oncogene were recently reported to be found in 40% of sporadic medullary thyroid carcinomas (MTCs), the absence of missense mutations at codon 768 was confirmed both with PCR-restriction fragment length polymorphism (RFLP) and PCR-SSCP analysis in all examined cases of pheochromocytomas. These results suggest that mutations at codon 768 of the RET proto-oncogene do not represent a frequent mechanism of tumorigenesis for both sporadic and hereditary pheochromocytomas.

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Although it is well recognized that continuous administration of gonadotropin-releasing hormone agonist (GnRHa) induces pituitary desensitization, the precise molecular mechanism of this phenomenon is still unclear. To test the hypothesis that pituitary gonadotroph desensitization is mediated by a change in GnRH receptor (GnRH-R) gene expression, the GnRH-R mRNA concentration was analyzed in immature female rats during GnRHa treatment. Northern blot hybridization was used to determine the GnRH-R mRNA concentration several times after an injection of TAP-144-SR, a slow releasing GnRHa. The GnRH-R mRNA readings were 92.7±9.5%, 49.9±5.0%, 35.7±2.3% and 73.8± 5.7% (Mean±SD) compared to each control value at 1, 2, 4 and 8 weeks, respectively, after a single injection of 0.94mg TAP-144 SR. These changes in GnRH-R mRNA coincided with the changes in gonadotropin secretion and LH-β mRNA in response to GnRH in the results of our previous report. The present results indicate that the reduction of the number of pituitary GnRH-R sites induced by continuous stimulation with GnRHa is regulated at a transcriptional level.

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A decreased concentration of dehydroepiandrosterone sulfate (DHEA-S) in patients with Alzheimer's disease (AD) has been reported but is still controversial. In the present study, serum concentrations of DHEA and DHEA-S were determined in 19 patients with AD, 21 patients with cerebrovascular dementia (CVD) and 45 age- and gender matched elderly control individuals from the Japanese community at large. Serum concentrations of DHEA among controls, patients with AD and patients with CVD did not significantly differ from one another. However, patients with AD and patients with CVD were found to have lower concentrations of serum DHEA-S and a lower DHEA-S/DHEA ratio compared to normal control individuals. No significant difference was observed in the concentration of serum DHEA-S or the DHEA-S/DHEA ratio between patients with AD and those with CVD. These results suggest that reduced concentrations of serum DHEA-S may not be unique to AD, but instead reflect a common phenomenon in dementing diseases. However, since serum concentration of DHEA in these patients remained unchanged, the significance of DHEA in dementia remains unclear.

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In order to evaluate the steady state plasma glucose (SSPG) method by using a new somatostatin derivative, octreotide acetate (Sandostatin^®) instead of somatostatin that we had used for the insulin sensitivity test, we examined whether octreotide was able to suppress C-peptide (CPR), glucagon (IRG), and GH to a similar degree to that achieved with somatostatin. A total of 52 studies were performed in 45 essential hypertensive subjects and 7 healthy subjects. Octreotide was given subcutaneously in a does of 50μg or 100μg 10min before the test (sc 50, sc 100 groups) or intravenously infused over 2h (10μg in bolus followed by a constant infusion, 50, 100, or 150μg/2h: iv 50, iv 100, iv 150 groups). In all of the groups the plasma immunoreactive insulin (IRI) concentration increased gradually after insulin injection and reached the steady state plasma insulin (SSPI) level between 40 and 60μU/ml at 60min through 120min. Plasma CPR at 120min was the most suppressed (by 67% of the basal level in iv 150 group during the study period), but on the other hand in both the sc 100 and iv 100 groups the plasma CPR concentration at 120min was suppressed by nearly 40%, but not significantly suppressed in either the sc 50 or the iv 50 group. Plasma IRG and GH were strongly suppressed after 60min in all the groups during the study period. Plasma glucose had increased significantly at 30min and reached the steady state at 90min through 120min in hypertensive and healthy subjects. The results indicated that the modified SSPG method with continuous intravenous infusion of Octreotide at 150μg/2h was adequate for the measurement of insulin sensitivity.

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