Motomori Izumi

Molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor transcript.

In the present study, we report the molecular cloning and sequencing of an alternatively spliced form of the human thyrotropin receptor (hTSHR) mRNA transcript, which has previously been detected on Northern blot analysis of human thyroid cells. The smaller hTSHR cDNA, designated hTSHR cDNA-I, is approximately 1 kb in size and encodes a protein of 253 amino acids. Comparison of the nucleotide sequence of hTSHR cDNA-I with available hTSHR genomic sequence data reveals that the cDNA-I contains exons 1-8 and unidentified DNA tract, presumably an intron. Thus, the hTSHR cDNA-I encodes for the N-terminal half of the extracellular domain of the hTSHR (approximately 60%). The truncated TSHR-I may be secreted and function as a TSH binding protein.

An enzyme immunoassay for the measurement of anti-thyroglobulin autoantibody in human serum

An enzyme-linked sandwich immunoassay using human thyroglobulin conjugated with beta-D-galactosidase and silicone rods coated with human thyroglobulin was developed for the measurement of circulating anti-thyroglobulin autoantibody. The volume of serum needed for the assay was as little as 5 microliter. The sensitivity of the assay was approximately 7 x 10(-15) mol/tube of antithyroglobulin immunoglobulin G corresponding to 220 ng/ml of serum, which was equal to or rather higher than that of radioimmunoassay. The specificity of the assay was demonstrated by (1) parallelism of the standard curve with dilution of sample sera of patients with thyroid diseases, and (2) non-detectability of anti-thyroglobulin in autoantibody in the sera of normal subjects. The precision of the assay was proven by (1) sufficient recovery of antithyroglobulin immunoglobulin G added to serum, and (2) coefficients of variance within and between assays were 6.5 to 10.3% and 4.9 to 14.1%, respectively. No effect of thyroglobulin on the present assay was observed when the ratio of the amount of thyroglobulin to that of anti-thyroglobulin immunoglobulin G was lower than 1 : 10. Furthermore, a significant correlation was observed between anti-thyroglobulin autoantibody concentrations measured by our enzyme immunoassay and those by tanned red cell hemagglutination (r = 0.78), and between those by the enzyme immunoassay and those by radioimmunoassay (r = 0.80). The application to clinical samples ensured the high sensitivity and the adequate validity of the present assay.

Long-term follow-up studies on Iodine-131 treatment of hyperthyroid graves’ disease based on the measurement of thyroid volume by ultrasonography

In the present series of studies, the long-term (four year) effect of 80 Gy of131I treatment was evaluated in patients with hyperthyroid Graves’ disease whose thyroid volumes have been accurately estimated with a high resolution ultrasound scanner. One year after131I treatment, 23.1 % (3 out of 13 patients) remained hyperthyroid, 69.2% (9 out of 13) became euthyroid, and 7.7% (1 out of 13) were in a hypothyroid state. Since three patients in a hyperthyroid state one year after treatment were subsequently treated with either antithyroid drugs or additional131I treatment, the remaining ten patients (9 euthyroid and 1 hypothyroid patients) have been followed up for three more years. Two patients developed a hypothyroid state three years after treatment and one patient four years after treatment. Overall, 60% (6 out of 10 patients) were in a euthyroid state and 40% (4 out of 10) in a hypothyroid state, four years after 80 Gy131I treatment. There was no significant difference between eu- and hypothyroid groups in the sex ratio, age, radiation dose, therapeutic dose, thyroid gland volume, 24-hr131I uptake, the effective half-life of131I in the thyroid or the duration of hyperthyroidism.In our preliminary studies, the incidence of late hypothyroidism in our131I treatment is similar to those previously reported. These suggest that uncertain factor(s), such as inhomogeneity of iodine distribution in the thyroid, unequal sensitivity of the thyroid cells to the radiation, and/or persistent destructive effects of the autoimmune process may influence the long-term effect of131I treatment of Graves’ disease.

Retinole acid inhibits human thyroid peroxidase and thyroglobulin gene expression in cultured human thyrocytes

The effect of retinoic acid (RA) on thyroid peroxidase (TPO) and thyroglobulin (Tg) gene expression was investigated in cultured human thyrocytes. Thyrocytes dispersed from Graves’ thyroid tissues were incubated with TSH 5mU/ml and RA 0, 0.01, 0.1,1.0 μM for 72 h respectively. The samples were then subjected to Northern gel analysis. Northern gel analysis using the specific cDNA probes showed that RA suppressed the accumulation of TPO and Tg mRNA stimulated by TSH in a time- and dose-responsive manner. Furthermore, RA inhibited forskolin and 8-Bromo-cyclic-AMP-induced TPO and Tg gene expression, suggesting a distal action site for these cAMP mediated gene expressions. Immunoprecipitation analysis using the specific monoclonal antibodies showed that TSH increased newly synthesized 100, 75, 36-kDa [35S] TPO. The increased de novo TPO was markedly inhibited by RA. Tg secretion from monolayer cultures was measured by radioimmunoassay. RA also inhibited TSH-in-duced Tg secretion in a dose dependent manner. RA did not affect [3H] thymidine uptake into primary cultured human thyrocytes. In conclusion, RA inhibits the synthesis of TPO and Tg via the suppression of thyroid-specific gene expression although the exact site of RA action on these genes in human thyroids remains to be further elucidated. These results suggest that RA may play a regulatory role in Tg and TPO gene expression, subsequently resulting in the suppression of thyroid hormone synthesis.

Impairment of the TSH signal transduction system in human thyroid carcinoma cells

In order to further evaluate the role of TSH in the proliferation and the differentiation of human thyroid carcinoma cells, we have analyzed the function of the TSH receptor in the established thyroid carcinoma cell lines NPA and WRO. The TSH signal transduction system in the carcinoma cells was also compared with that in normal thyroid cells. Although unresponsiveness to bovine and human TSH was demonstrated by measurement of cAMP production and [3H]thymidine incorporation after treatment of TSH, cAMP production was induced after stimulation of these cells by forskolin, cholera toxin, and isoproterenol. Specific binding to 125I-TSH was demonstrated in both NPA and WRO cells in addition to the existence of a TSH receptor mRNA and thyroglobulin mRNA species, although thyroid-specific gene expression in these cells was not regulated by TSH. These findings suggest that the unresponsiveness to TSH in these cells may be due to an abnormality of TSH receptor-G protein coupling rather than to a decreased level of TSH-receptor expression or a Gs protein abnormality.

Association between heart rate variability, blood pressure and autonomic activity in cyclic alternating pattern during sleep.

STUDY OBJECTIVES Cyclic alternating pattern (CAP) is frequently followed by changes in heart rate (HR) and blood pressure (BP), but the sequential associations between CAP and autonomic nerve activity have not been studied. The study aimed to reveal the precise changes in heart rate variability (HRV) during phase A of the CAP cycle. DESIGN Polysomnography was recorded according to the CAP Atlas (Terzano, 2002), and BP and electrocardiogram were simultaneously recorded. The complex demodulation method was used for analysis of HRV and evaluation of autonomic nerve activity. SETTING Academic sleep laboratory. PARTICIPANTS Ten healthy males. MEASUREMENTS AND RESULTS The increase in HR (median [first quartile - third quartile]) for each subtype was as follows: A1, 0.64 (-0.30 to 1.69), A2, 1.44 (0.02 to 3.79), and A3, 6.24 (2.53 to 10.76) bpm (A1 vs. A2 P < 0.001, A1 vs. A3 P < 0.001, A2 vs. A3 P < 0.001). The increase in BP for each subtype was as follows: A1, 1.23 (-2.04 to 5.75), A2, 1.76 (-1.46 to 9.32), and A3, 12.51 (4.75 to 19.94) mm Hg (A1 vs. A2 P = 0.249, A1 vs. A3 P < 0.001, A2 vs. A3 P < 0.001). In all of phase A, the peak values for HR and BP appeared at 4.2 (3.5 to 5.4) and 8.4 (7.0 to 10.3) seconds, respectively, after the onset of phase A. The area under the curve for low-frequency and high-frequency amplitude significantly increased after the onset of CAP phase A (P < 0.001) and was higher in the order of subtype A3, A2, and A1 (P < 0.001). CONCLUSIONS All phase A subtypes were accompanied with increased heart rate variability, and the largest heart rate variability was seen in subtype A3, while a tendency for less heart rate variability was seen in subtype A1.

Acromegaly and Pituitary Carcinoma

Excerpt To the Editors:Growth hormone (GH)-secreting adenocarcinoma with clear evidence of distant metastasis has been unequivocally documented in only five patients (1, 2). We report the case of a...

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and Km value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60 degrees C for 10 min.

Multiple endocrine neoplasia type 2 with malignant pheochromocytoma — Long term follow-up of a case by131I-meta-iodobenzylguanidine scintigraphy—

The case of a 33-year-old Japanese man, who has Multiple Endocrine Neoplasia Type 2 (MEN IIa) (Sipple’s syndrome) with malignant pheochromocytoma, is reported. He had survived for twelve years since the initial diagnosis of malignant pheochromocytoma. Within this period, he had undergone131I-meta-iodobenzylguanidine scintigraphy twice, in 1983 and 1990. This is the first case in Japan of a longterm surviving patient with malignant pheochromocytoma followed up by131I-MIBG scintigraphy. Although he had no exacerbation of clinical symptoms or urinary catecholamine levels, second scintigraphy clearly showed an increase in the tumor size, new metastasis of the malignant pheochromocytoma and exacerbation of the medullary thyroid carcinoma. Compared with any other roentgenological device and hormonal data,131I-MIBG scintigraphy was seen to be a good tool for evaluating the localization and the progression of tumors.131I-MIBG scintigraphy is a useful procedure not only for initial diagnosis but also for judging progression in a case of advanced malignant pheochromocytoma.

DHT formation and degradation in cultured human skin fibroblasts: DHT accumulation in the genital skin

The conversion of testosterone to dihydrotestosterone (DHT) by 5 alpha-reductase and the interconversion between DHT and 5 alpha-androstane-3 alpha, 17 beta-diol (3 alpha-diol) by 3 alpha-hydroxy-steroid oxidoreductase (3 alpha-HSOR) were studied in fibroblasts derived from the genital skin of 22 males and 6 females, and from the nongenital skin of 19 males and 9 females with normal gonadal function. The formation of DHT from testosterone (5 alpha-reduction) was significantly greater in fibroblasts from genital skin than in those from nongenital skin in both males (2.15 +/- 1.43 vs 0.81 +/- 0.46 pmol/mg protein/h, mean +/- SD, P less than 0.001) and females (2.52 +/- 1.99 vs 0.69 +/- 0.18, P less than 0.01). Furthermore, DHT formation from 3 alpha-diol (3 alpha-HSOR oxidation) was also significantly greater in genital skin fibroblasts than in nongenital skin fibroblasts of males (5.47 +/- 3.37 vs 2.52 +/- 1.74 pmol/mg protein/h, P less than 0.01). However, the degradation of DHT to 3 alpha- and/or 3 beta-diol (3 alpha- and/or 3 beta-HSOR reductions) was not different between genital and nongenital skin fibroblasts of either males or females. Respective ratios of DHT formation to DHT degradation (5 alpha-reduction/3 alpha-HSOR reduction, 3 alpha-HSOR oxidation/3 alpha-HSOR reduction) were also significantly greater (P less than 0.002) in genital skin fibroblasts than in nongenital skin fibroblasts of males. On the other hand, both DHT formation and degradation were not different between male and female genital skin fibroblasts. These results suggest that the increased production of DHT in genital compared to nongenital skin results from increased 5 alpha-reduction and 3 alpha-HSOR oxidation.