Modulation of prostaglandin E2, F2 alpha, I2 content and synthesis by thyrotropin in cultured porcine thyroid cells and intact rat thyroid glands.

Porcine thyroid gland contains prostaglandin E2, prostaglandin F2 alpha and prostacyclin (prostaglandin I2) (measured as an end metabolite, 6-keto prostaglandin F1 alpha) and it contains more prostaglandin I2 than prostaglandin E2 or F2 alpha. Cultured porcine thyroid cells contain prostaglandin E2, F2 alpha and I2. When cultured in the presence of thyroid stimulating hormone (TSH), thyroid cells contain more prostaglandin I2 than prostaglandin E2 or F2 alpha but when cultured in its absence, they contain more prostaglandin E2 or F2 alpha than prostaglandin I2. Thyroid cells synthesize prostaglandin E2, F2 alpha and I2; when cultured in the presence of TSH, they synthesize more prostaglandin I2 than prostaglandin E2 or F2 alpha but when cultured in its absence, they synthesize more prostaglandin E2 or F2 alpha than prostaglandin I2. When cultured in the presence of TSH, thyroid cells take up iodide and organify it but when cultured in its absence, they do not take up iodide. When cultured in the presence of TSH, thyroid cells synthesize prostaglandin I2 and take up iodide, indicating that in the physiological conditions, prostaglandin I2 plays a more important role than prostaglandin E2 or F2 alpha. Rat thyroid gland contains prostaglandin E2, F2 alpha and I2. Endogenous increase in serum TSH through goitrogen treatment and exogenous TSH administration augment prostaglandin I2 contents and depress prostaglandin E2 and F2 alpha contents. Suppression of endogenous TSH by thyroxine treatment depresses prostaglandin I2 contents and augments prostaglandin E2 and F2 alpha contents. Chronic exposure to TSH augments prostaglandin I2 synthesis and depresses prostaglandin E2 and F2 alpha syntheses but chronic nonexposure to TSH depresses prostaglandin I2 synthesis and augments prostaglandin E2 and F2 alpha synthesis in in vivo rat thyroid glands and in vitro cultured porcine thyroid cells.

Intracellular localization of group II phospholipase A2 in rat vascular smooth muscle cells and its possible relationship to eicosanoid formation.

We investigated the localization of group II phospholipase A2 (PLA2-II) in rat vascular smooth muscle cells (VSMCs) by applying immunofluorescence and immunoelectron microscopy with its polyclonal antibody. In unstimulated cells, no immunolabelling was detected in the cells. On the other hand, in the cells stimulated with tumor necrosis factor (TNF) and/or forskolin (FK), intense fluorescence was detected in the cytoplasm. The immunoperoxidase reactions were detected in the cisternae of rough endoplasmic reticulum (rER), trans-cisternae of Golgi apparatus, and small vesicles beneath the plasma membrane. Western blot analysis showed VSMCs secrete PLA2-II after stimulation. Secreted PLA2-II was associated with the plasma membrane and extracellular matrix. Colchicine inhibited PLA2-II synthesis and its secretion to the extracellular space. These observations indicate that in VSMCs PLA2-II is synthesized at rER. transported to Golgi apparatus, discharged into extracellular space via the small vesicles, and microtubules may concern with its process. Furthermore, in VSMCs treated with TNF or TNF + FK, prostaglandin E2 formation was also increased. Actinomycin D and cycloheximide inhibited the potentiation of the prostaglandin E2 formation induced by TNF or TNF + FK, indicating that both RNA and protein synthesis are required for the potentiation. These results suggest an involvement of PLA2-II in the prostaglandin formation.

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by epidermal growth factor.

We studied the effects of epidermal growth factor (EGF), thyroid-stimulating hormone (TSH) and amiloride on cytoplasmic pH (pHi) in cultured porcine thyroid cells. We used 2',7'-bis(2-carboxyethyl)-5- (and 6-)carboxyfluorescein (BCECF), an internalized fluorescent pH indicator, to measure pHi. EGF stimulated thyroid cell alkalinization and proliferation, which were blocked by amiloride. EGF-stimulated thyroid cell alkalinization depended on extracellular Na+ concentrations. EGF stimulation resulted in an activation of Na+/H+ exchange, which alkalinized the cells. The results indicated that Na+/H+ exchange or cell alkalinization might function as a transmembrane signal transducer in the action of EGF. In the present system, TSH did not stimulate alkalinization or proliferation.

Streptozocin- and alloxan-induced H2O2 generation and DNA fragmentation in pancreatic islets. H2O2 as mediator for DNA fragmentation.

Streptozocin (STZ) and alloxan (ALX) exhibit the most potent diabetogenicity and are used for induction of experimental diabetes mellitus. An understanding of the mechanisms of action of the typical diabetogenic agents is important for elucidating the causes of diabetes. Okamoto proposed a model in which DNA fragmentation plays an important role in the development of diabetes. DNA fragmentation supposedly results from the accumulation of superoxide or hydroxyl radicals. However, direct evidence for this accumulation is lacking. With isolated rat pancreatic islets in vitro, we demonstrated that STZ and ALX stimulated H2O2 generation and caused DNA fragmentation. Addition of STZ or ALX resulted in an increase in H2O2 generation. On DNA analysis, when incubated without STZ or ALX, DNA sedimented as a single peak; when incubated with STZ or ALX, DNA sedimented slower as a broad peak and was fragmented. Graded doses of STZ and ALX stimulated H2O2 generation and induced DNA fragmentation; their effects on H2O2 generation and DNA fragmentation were evident at a concentration of 0.1 mM and were maximal at 1 mM. Administration of STX or ALX to rats in vivo stimulated H2O2 generation and caused DNA fragmentation in pancreatic islets. H2O2 itself also induced DNA fragmentation. These findings may support Okamoto's proposal that STZ and ALX induce diabetes through the following biochemical events: STZ and ALX----H2O2 generation----DNA fragmentation----beta-cell destruction. This study may constitute the first demonstration of STZ- and ALX-stimulated H2O2 generation, which probably acts as a mediator of STZ- and ALX-induced DNA fragmentation.

A missense mutation of Pax4 gene (R121W) is associated with type 2 diabetes in Japanese.

Pax4 is one of the transcription factors that play an important role in the differentiation of islet beta-cells. We scanned the Pax4 gene in 200 unrelated Japanese type 2 diabetic patients and found a missense mutation (R121W) in 6 heterozygous patients and 1 homozygous patient (mutant allele frequency 2.0%). The mutation was not found in 161 nondiabetic subjects. The R121W mutation was located in the paired domain and was thought to affect its transcription activity through lack of DNA binding. Six of seven patients had family history of diabetes or impaired glucose tolerance, and four of seven had transient insulin therapy at the onset. One of them, a homozygous carrier, had relatively early onset diabetes and slowly fell into an insulin-dependent state without an autoimmune-mediated process. This is the first report of a Pax4 gene mutation that exhibits loss of function and seems to be associated with type 2 diabetes. This work provides significant implications for the Pax4 gene as one of the predisposing genes for type 2 diabetes in the Japanese.

Enhanced insulin response relates to acetylcholine-induced vasoconstriction in vasospastic angina.

OBJECTIVES: This study investigated whether insulin response to an oral glucose load correlates to acetylcholine-induced coronary vasoconstriction in subjects with vasospastic angina. BACKGROUND: It has been suggested that coronary vasospasm is caused by augmented vascular responsiveness possibly exerted by atherosclerosis. Recently, insulin resistance syndrome has been proposed as a major promotor of atherosclerotic disease, potentially enhancing vascular smooth muscular tone. METHODS: Among subjects with angiographically smooth coronary arteries, we selected 14 subjects with vasospastic angina and 14 age- and gender-matched subjects with atypical chest pain. We compared coronary vasomotor response to acetylcholine infusion, glucose and insulin responses to an oral glucose load (75 g), serum lipid concentrations, obesity, heart rate, blood pressure and smoking habits in both groups. RESULTS: Fasting serum insulin concentrations and insulin response were higher in subjects with vasospastic angina than in those with atypical chest pain; however, glucose tolerance, obesity, heart rate, blood pressure and smoking habits did not differ between groups. In subjects with vasospastic angina, nearly all coronary segments, except distal segments of the left circumflex coronary artery, were constricted at peak acetylcholine infusion (20 to 100 micrograms), whereas all segments were dilated in subjects with atypical chest pain. Regression analysis for both groups demonstrated a correlation between coronary vasoconstriction and fasting serum insulin concentrations (r = 0.52, p < 0.01), insulin response (r = 0.71, p < 0.001), serum triglyceride concentrations (r = 0.51, p < 0.05) and atherogenic index (r = 0.44, p < 0.05). CONCLUSIONS: Results show that acetylcholine-induced coronary vasoconstriction in subjects with vasospastic angina correlates with hyperinsulinemia and enhanced insulin response, suggesting insulin resistance syndrome as a feature of vasospastic angina.

The Tokyo subway sarin attack--lessons learned.

The sarin gas attack in the Tokyo subway system is reviewed from a clinical toxicology perspective. Based on the lessons learned from this attack, the following areas should be addressed on a global scale. First, an adequate supply of protective equipment is required, including level B protective equipment with a pressure demand breathing apparatus. In addition, a system should be established that enables a possible cause to be determined based on symptoms, physical findings, general laboratory tests, and a simple qualitative analysis for poisonous substances. If an antidote is needed, the system should enable it to be administered to the victims as quickly as possible. Preparation for a large-scale chemical attack by terrorists requires the prior establishment of a detailed decontamination plan that utilizes not only mass decontamination facilities but also public facilities in the area. A system should be established for summarizing, evaluating, and disseminating information on poisonous substances. Finally, a large-scale scientific investigation of the Tokyo sarin attack should be conducted to examine its long-term and subclinical effects and the effects of exposure to asymptomatic low levels of sarin.

A polymorphic marker in the leptin gene associated with Japanese morbid obesity.

The prevaleance of morbid obesity (body mass index of 35.0 or greater) is low in Japan (0.2-0.3%), and little systematic investigation of its cause in this population has been carried out. Leptin plays a central role in regulation of body weight; mice deficient in leptin develop marked obesity. We sought mutations in the leptin gene in 53 morbidly obese Japanese (maximum body mass index 35-60) including 46 with type 2 diabetes. Direct DNA sequencing was performed following polymerase chain reaction amplification. Apart from a silent mutation at codon 25 (CAA/CAG, glutamine) detected in eight subjects, no mutations were detected. We found a significantly higher prevalence of the variant leptin 25CAG allele among the 53 obese subjects (0.085) studied than in 132 nonobese control subjects (0.011, P<0.001). In Japanese populations mutations in the protein coding sequence of the leptin gene are unlikely to be a major cause of morbid obesity. However, the leptin 25CAG allele may be linked to morbid obesity in this population. Specifically, genetic variation located near the leptin gene may be involved in pathogenesis. The leptin polymorphism 25CAG appears to be a new genetic marker for obesity susceptibility, at least in Japanese.

Association of CTLA-4 gene A/G polymorphism in Japanese type 1 diabetic patients with younger age of onset and autoimmune thyroid disease.

OBJECTIVE: We studied the association between type 1 diabetes with autoimmune thyroid disease (AITD) and A/G allele polymorphism in exon 1 of the CTLA-4 gene in a Japanese population. RESEARCH DESIGN AND METHODS: We studied 74 Japanese type 1 diabetic patients with or without AITD and 107 normal subjects to identify the association between CTLA-4 polymorphism and type 1 diabetes using polymerase chain reaction-restriction fragment length polymorphism analysis. RESULTS: The frequency of the CTLA-4 G allele differed significantly between the type 1 diabetic patients (61%) and the normal control subjects (48%) (P = 0.016). The difference in the CTLA-4 G allele became greater between patients with a younger age of onset of type 1 diabetes (age at onset <30 years) and the normal control subjects (64% and 48%, respectively). However, the frequency of the CTLA-4 G allele did not differ between type 1 diabetic patients with younger and older age of onset (64% vs. 57%). The G allele frequencies in the patients with younger-onset type 1 diabetes and AITD increased more than in the control patients (P = 0.025). These differences reflected a significant increase in the frequency of G/G genotype--that is, 54% in those with younger-onset type 1 diabetes and AITD, 39% in those without AITD, and 28% in control subjects. CONCLUSIONS: An association was detected between the CTLA-4 gene polymorphism and younger-onset type 1 diabetes with AITD. The G variant was suggested to be genetically linked to AITD-associated type 1 diabetes of younger onset in this apanese population. The defect in these patients presumably lies in a T-cell-mediated autoimmune mechanism.

Polarized thyroid cells in monolayers cultured on collagen gel: their cytoskeleton organization, iodine uptake, and resting membrane potentials.

Porcine thyroid cells were cultured on collagen gel-coated cover glasses. They were reorganized into polarized monolayer cells; the basal cell membranes were in contact with the collagen gel, and the apical ones faced the culture medium. We studied cytoskeleton organization, resting membrane potentials, and iodine uptake of these cells. The quick-freezing and deep-etching replica method provided three-dimensional images of the cytoskeleton organization. Networks of microfilaments were observed under the apical cell membrane. In the deep cytoplasm and near the basal cell membranes, intermediate filaments predominated and were interlinked with the microfilaments. When the cells were cultured in the presence of TSH, TSH induced the formation of microvilli at the apical cell membranes and the accumulation of microfilaments under these membranes; in the deep cytoplasm, the intermediate filaments were more closely interlinked with the microfilaments. The microfilaments were immunostained with antiactin antibody. Thus, collagen is a factor in determining the cell polarity, and TSH further augments polarization through reorganizing the cytoskeletons. Electrophysiological study revealed that the resting membrane potential of cells cultured in the absence of TSH was -46 mV, and that of cells cultured in the presence of TSH was -58 mV. TSH hyperpolarized resting membrane potentials. These cells took up iodine. TSH in the medium augmented this uptake. TSH augments thyroid cell polarization through reorganizing the cytoskeletons and hyperpolarizing the resting membrane potentials and enhances iodine uptake by the cells.

Requirements of follicle structure for thyroid hormone synthesis; cytoskeletons and iodine metabolism in polarized monolayer cells on collagen gel and in double layered, follicle-forming cells.

Thyroid cells take up iodine and synthesize thyroid hormones. Thyroid cell polarity plays an important role in the uptake of iodine. However, we do not know whether polarity itself is enough for thyroid hormone synthesis or whether follicle structure is required for it. Using polarized monolayer porcine thyroid cells, cultured on collagen-coated filters, and double layered, follicle-forming cells, we analyzed the relationships of iodine metabolism and cell polarity (and follicle formation). We demonstrated that follicle structure was required for thyroid hormone synthesis. A quick-freezing and deep-etching method revealed the three-dimensional ultrastructures of cytoskeletons in the thyroid cells. On the collagen gel, the thyroid cells are reorganized into polarized monolayer cells; the basal cell membranes are in contact with the collagen gel and the apical ones face the culture medium. Actin microfilaments predominate under the apical cell membranes and intermediate filaments in the basal cytoplasm. The arrangement of these cytoskeletons determines the polarity of the cells. When the cells are cultured as double layers, follicle structures are reconstructed between the two monolayers. When apical cell membranes are in contact with other apical ones or when the cells are cultured as double layers, the cells are reorganized into follicles; the basal cell membranes are in contact with the collagen gel, and the apical ones face the follicle cavities. Actin microfilaments predominate at the apical cell membranes and intermediate filaments in the basal cytoplasm. Polarized thyroid cells transport iodine from the basal compartments to the apical ones, but cannot organify iodine into thyroid hormones. However, follicle-forming cells, cultured as double layers, take up iodine and organify it into thyroid hormones. Polarity is important for iodine uptake, and follicle structure is required for thyroid hormone synthesis.

Thyrotropin-stimulated electrical excitation and its refractoriness in cultured porcine thyroid cells.

This report demonstrates TSH-stimulated electrical excitation in the thyroid and its refractoriness after exposure to TSH. TSH depolarizes the membrane potentials and causes action potentials. TSH-induced electrical activity is characterized by a latent period, rapid depolarization, action potentials (usually two spikes were observed), and then repolarization to the potential level of the silent phase. This TSH-induced electrical excitation is associated with iodide discharge. Previous exposure to TSH induces refractoriness of electrophysiological excitation and iodide discharge to further TSH stimulation.

Effects of iodide on thyroid follicle structure and electrophysiological potentials of cultured thyroid cells.

In cultured porcine thyroid cells, exposure to iodide induces morphological and electrophysiological changes in the cells and suppresses the iodine uptake and organification activities of the cells. NaI affects thyroid structures: after exposure to 10(-7), 10(-6), and 10(-5) M NaI, the follicles first lose their typical roundness, and then the numbers of microvilli decrease. NaI (10(-6) and 10(-5) M) decreases the thyroid electrical membrane potentials. NaI induces suppression of iodine uptake and organification: exposure to 10(-6) and 10(-5) M NaI suppresses subsequently determined iodine uptake and organification. This iodide-induced suppression of iodine uptake and organification may be related to the iodide-induced morphological and electrophysiological changes. The iodide-induced changes and suppression of iodide uptake and organification are reversible. They are observed when thyroid cells are cultured in the presence of TSH.

Mastoparan-induced hormone release from rat pancreatic islets.

Mastoparan, a tetradecapeptide purified from wasp venom, stimulates insulin and glucagon release by rat pancreatic islets in a dose-related manner. In perifusion experiments, mastoparan produces monophasic hormone release, which ceases within 10 min of removal of the peptide. After exposure of the isles to mastoparan, glucose-induced insulin release is clearly retained. In incubation experiments, mastoparan-induced insulin release is greatly blocked by pretreatment of the islets with pertussis toxin or neomycin (inhibitor of phosphoinositide turnover) or by lowering the ambient temperature to 17 C. Pretreatment of the islets with nifedipine (calcium channel blocker), H-7 (inhibitor of A- and C-kinase), somatostatin, or divalent cation-free medium does not affect the response to mastoparan. Pretreatment with parabromophenacylbromide (phospholipase-A2 inhibitor) does not block the response induced by a high concentration of (58 microM) mastoparan. The peptide does not stimulate insulin synthesis during 30 min of incubation. Mastoparan raises the cytosolic free Ca2+ concentration, measured by fura-2, in isolated islet cells at normal (1.9 mM) and very low (6.5 microM) extracellular Ca2+ concentrations. Intravenous administration of mastoparan in rats causes a significant elevation of both insulin and glucagon. Together with the previous data, we conclude that mastoparan stimulates islet hormone release through a temperature-dependent process mediated by pertussis toxin-sensitive GTP-binding protein(s). Activation of phospholipase-C and liberation of intracellular Ca2+ are likely to be coupled to exocytosis. Ca2+ influx through the Ca2+ channel and protein kinase-A and -C appear not to be involved in mastoparan's hormone-releasing action. Phospholipase-A2 may be involved in the hormone release induced by low, but not high, concentrations of the peptide.

Transient neonatal hypothyroidism due to maternal immunoglobulins that inhibit thyrotropin-binding and post-receptor processes.

Transient neonatal hypothyroidism was found in a daughter of a 25-yr-old mother, who was receiving treatment for primary hypothyroidism due to Hashimoto's thyroiditis. During the neonatal period the infant had antithyroid microsomal and antithyroglobulin antibodies and TSH-receptor antibodies. The daughter recovered spontaneously from the hypothyroid state and the antithyroid antibodies disappeared from her serum. The mother's serum contained the same antibodies, and immunoglobulin G (IgG) from maternal serum blocked TSH binding to its receptors, TSH-stimulated cAMP responses, and cAMP-stimulated iodine uptake and organification in cultured thyroid cells. The latter finding suggests that the IgG had a postreceptor locus of action as well as inhibiting TSH binding to its receptor. The presence of such IgGs might have induced hypothyroidism both in the mother and in the daughter.

Pituitary-thyroid feedback regulation in patients with Graves' disease during antithyroid drug therapy.

In an attempt to study the mode of normalization of thyroid function in patients with Graves' disease, a study was made on 140 patients with Graves' disease who were eumetabolic after appropriate therapy with antithyroid drugs for more than 9 months. T3 administration failed to suppress thyroidal radioiodine uptake and serum T4 in patients with TRH-unresponsive TSH secretion. In addition, exogenous TSH failed to elevate serum levels of T4 and T3. In patients with TRH-responsive pituitaries, T3 administration uniformly made serum TSH undetectable but produced various effects (unsuppressible, partially suppressible, and suppressible) on radioiodine uptake and serum T4. The magnitude of suppression of radioiodine uptake paralleled that of serum T4. In patients with unsuppressible or partially suppressible thyroids, exogenous and endogenous TSH were less effective in elevating serum T4 and T3. In patients with suppressible thyroids, T3 administration depressed radioiodine uptake and serum T4; the magnitudes of depression were comparable to those found in normal subjects. Exogenous and endogenous TSH elevated serum T4 and T3 in patients with suppressible thyroids. Here again, the magnitudes of elevation were comparable to those found in the normal subjects. The serum T3 to T4 ratio was high before treatment, but decreased significantly during antithyroid drug therapy. The magnitude of decrease was roughly proportional to the degree of T3 suppressibility.

Reappraisal of the 3,5,3'-triiodothyronine-suppression test in the prediction of long term outcome of antithyroid drug therapy in patients with hyperthyroid Graves' disease.

Thyroidal suppressibility by exogenous T3 in terms of both radioiodine uptake (RAIU) and serum T4 was evaluated in 115 hyperthyroid patients treated with methimazole for 2 yr and followed for an additional 2 yr to study the rate of recurrence. Various other serum parameters including serum thyroglobulin concentrations, thyroid autoantibody, and TSH receptor antibody titers, and thyroidal responses to TRH-induced TSH elevation were also determined. After 2 yr of methimazole therapy, thyroidal RAIU was not suppressible (RAIU less than 12%/4 h was defined as suppressible) in 50 of 115 patients (group I). Of 65 patients with suppressible thyroid RAIU, serum T4 was significantly reduced (less than 60% of pre-T3 level) by T3 administration in only 43 patients (group III) but not in the remainder (group II). Antithyroid drug therapy was discontinued in the group II and III patients, and 7 of the patients had recurrence of hyperthyroidism within 2 yr of follow-up. All of them were from group II. The thyroidal response to TSH was greater in group III patients than in group II patients. During antithyroid drug therapy, decrease of microsomal antibody titer was more likely to occur in group III patients than in those of group II. Serum thyroglobulin concentrations were uniformly normal in treated patients irrespective of T3 suppressibility. TSH receptor antibody was positive in all 13 untreated patients with Graves' disease but was negative in treated patients regardless of their T3 suppressibility. Measurement of both thyroidal RAIU and serum T4 after administration of T3 improves the reliability of T3-suppression testing as a predictor of the remission of Graves' disease.

Effect of thyrotropin-releasing hormone on serum thyroid hormones: a study in the patients with untreated and treated Graves' disease and subacute thyroiditis.

In order to investigate the extrapituitary action of TRH on the thyroid, serum T3, T4, and TSH levels after im administration of TRH were analyzed in 63 patients with untreated hyperthyroid Graves' disease, in 60 euthyroid patients with treated Graves' disease, in 8 patients with subacute thyroiditis, and in 140 healthy subjects. TRH administration in the healthy subjects resulted in a significant increase in serum T3 and T4 levels after 2 h. However, in the patients with untreated hyperthyroid Graves' disease, a significant decrease in serum T3 and T4 levels with undetectable TSH was found 2 h after TRH administration. In the patients with subacute thyroiditis, serum T3 levels also significantly decreased after TRH administration. When a decrease in serum T3 and T4 levels after TRH administration in the patients with hyperthyroid Graves' disease was analyzed in terms of thyroid microsomal antibody and thyroglobulin antibody, a decrease in serum T3 and T4 levels was largest in patients with thyroid microsomal antibody and thyroglobulin antibody. In contrast, an increase in serum T3 and T4 levels in response to TRH in the euthyroid patients with Graves' disease was largest in patients without thyroid autoantibodies. It is concluded that TRH acts directly on the thyroid to suppress the thyroid hormone secreting activity in the absence of circulating TSH and that thyroid autoantibodies affect thyroidal response after TRH administration.

Seventeen alpha-hydroxylase deficiency with one base pair deletion of the cytochrome P450c17 (CYP17) gene.

Mutation of the cytochrome P450c17 (CYP17) gene causes 17 alpha-hydroxylase deficiency (17OHD). Recently, several researchers have elucidated the molecular basis of 17OHD by gene analysis. We experienced a case of 17OHD and intended to reveal the abnormality of the CYP17 gene in this Japanese female with 17OHD. Leukocytes were obtained from the patient, her mother and sister, and normal control subjects. We amplified the CYP17 gene using polymerase chain reaction and performed the sequence analysis using the dideoxy terminator method and restriction enzyme analysis. We found that the patient had one base-pair deletion at the position of amino acid 438. An identical result was obtained with restriction enzyme analysis. This G deletion altered the reading frame and resulted in a premature stop codon at position 443; the ligand of heme iron (Cys: cystine 442) was absent. This small mutation may account for the patient's clinical manifestations of 17OHD. This is the first case of 17OHD with only one base pair deletion of the CYP17 gene.