Effects of transforming growth factor beta 1 and L-ascorbate on synthesis and distribution of proteoglycans in murine osteoblast-like cells.

Proteoglycans synthesized by osteoblasts are incorporated into bone matrix and thought to play a role in bone metabolism. Transforming growth factor (TGF) beta affects the synthesis of matrix proteins, including proteoglycans, in various stromal cells, and proteoglycans, especially decorin, are associated with matrix collagen. In the present study, the effects of TGF-beta 1 and L-ascorbate, a factor essential for collagen synthesis, on the synthesis and distribution of proteoglycans were examined using murine osteoblast-like MC3T3-E1 cells. TGF-beta 1 stimulated the synthesis of proteoglycans in MC3T3-E1 cells. Among various proteoglycans, the synthesis of decorin was preferentially enhanced by TGF-beta 1, and the effect was more pronounced on secreted decorin compared to that associated with the cell/matrix layer. TGF-beta 1 also stimulated the initiation and elongation of the dermatan sulfate glycosaminoglycan chain, resulting in a larger molecular size of decorin. TGF-beta 1 influenced the synthesis of a heparan sulfate proteoglycan only slightly. L-ascorbate had no effect on the synthesis of proteoglycans, but increased those associated with the cell/matrix layer. Furthermore, when L-ascorbate was added to the culture along with TGF-beta 1, the percentage of proteoglycans associated with the cell/matrix layer increased from 25.8 +/- 1.0 to 41.0 +/- 0.5%. These data demonstrate that TGF-beta 1 markedly stimulates the synthesis of proteoglycans, especially decorin, mainly as a secreting form, that the accumulation of decorin into matrix is enhanced by L-ascorbate, and that the effects of TGF-beta 1 and L-ascorbate are additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of sulfhydryl reagents on the conversion of thyroxine to 3,5,3'-triiodothyronine: direct action on thyroxine molecules.

Sulfhydryl reagents are good stimulators of the enzymatic conversion of T4 to T3. Previous studies have suggested that the underlying mechanism for this property is that these reagents may serve as a cofactor in the reaction, or they maintain the enzyme in its active reduced form. We examined another possibility, namely that sulfhydryl reagents affect T4 itself and hence increase T3 generation by the enzyme. The substrate T4 and sulfhydryl reagents dithiothreitol, mercaptoethanol, and reduced glutathione were incubated at 37 C for 1 h and then combined with rat liver homogenate and incubated for additional 15 min to permit the enzymatic formation of T3. For control, substrate T4 was not preincubated with reagents, and those reagents were added just before the incubation. Preincubation of T4 with these reagents markedly enhanced subsequent T3 generation by the homogenate. Without preincubation, however, reduced glutathione and mercaptoethanol did not enhance T3 generation by simultaneous incubation with the homogenate. Dithiothreitol increased T3 generation when introduced into the incubation medium simultaneously with the homogenate and incubated only 15 min. However, the magnitude of stimulation was far less than that obtained from T4 preincubated with this reagent. These procedures did not alter the nonenzymatic generation of T3 significantly. These results suggest that sulfhydryl reagents can affect T4 itself and make it more susceptible to enzymatic outer ring monodeiodination.

LATS-protector activity in thyrotoxicosis measured by thyroidal intracellular colloid droplet formation.

A modified assay method of LATS and LATS protector (LATSP) was devised employing murine thyroidal intracellular colloid droplet formation. This method is 4- to 8-fold more sensitive to stimulators than is the regular McKenzie bioassay. For the assay of LATSP, IgG to be tested was incubated with human thyroid homogenate, then LATS-IgG was added and it was further incubated. The potency of LATS in the mixture was assayed. The adequacy of the present method for the assay of LATSP was supported by the facts that the LATSP activity was roughly related to the dose of IgG tested and that the inactivation of LATSP was observed when the IgG was pretreated with human thyroid particulate fraction and then assayed. With this method, 7 out of 32 patients with thyrotoxicosis showed LATS and 18 showed LATSP. Changes in LATSP were followed up in 7 cases out of the 18. LATSP activity decreased and then disappeared in 6 out of the 7 cases, when they had been euthyroid for a certain period of time. At 12-20 months after the treatment either by thionamide medication or by subtotal thyroidectomy. LATSP and 131I-thyroid uptake were measured in these 6 patients. In all of the 6, LATSP was negative regardless of thyroid suppressibility.

Discrepancy between thyroid-stimulating and thyrotropin-binding inhibitory activities of Graves' immunoglobulin Gs assessed in the mouse.

TSH receptor binding-inhibiting (TBI) and thyroid-stimulating (TSI) activities of Graves' immunoglobulin Gs (IgGs) were measured using the murine TSH receptor (mTBI) and the McKenzie LATS bioassay. In contrast to the expectation that only the IgGs positive for LATS showed mTBI activity, mTBI activity was closely correlated with TBI activity measured at the human TSH receptor regardless of LATS potency (r = 0.79; P less than 0.01), indicating a lack of species specificity of IgG in this particular aspect. In 7 to 15 IgGs positive for both mTBI and LATS, there was a significant correlation between the 2 parameters (r = -0.89; P less than 0.01). However, 4 IgGs among the 15 were positive for mTBI but negative for LATS even when tested at 3- to 7-fold concentrations of IgG.

Endocrine and morphological study of a clinically silent somatotroph adenoma of the human pituitary.

Silent somatotroph adenomas are defined as tumors showing morphological features consistent with GH production, but no clinical evidence of GH excess. We report here the case of a 46-yr-old woman with a large pituitary macroadenoma, slightly elevated serum GH levels, high serum insulin-like growth factor-I levels, and abnormal GH dynamics, but no acromegaly. The endocrinological abnormalities receded after transphenoidal surgery despite tumor persistence, as shown by neuroimaging. The reverse hemolytic plaque assay, performed for the first time in a silent GH cell adenoma, demonstrated that the number of GH cells releasing GH and the amount of GH discharged from individual cells were less than those in clinically functioning somatotroph adenomas. Thus, it is conceivable that this tumor secreted only small quantities of GH and for only short periods, providing an explanation for the lack of acromegaly. It appears that silent somatotroph adenomas do not represent a distinct entity. It is more likely that there is a continuous spectrum from clinically functioning, sparsely granulated somatotroph adenomas with high serum GH levels to silent somatotroph adenomas with normal serum GH levels. The cause of the lack of GH oversecretion in silent GH cell adenomas has yet to be elucidated.

The presence of myoglobin in human thyroid tissue.

In the present study we sought to determine the presence of myoglobin in human thyroid tissue. When reacted with antihuman myoglobin antibody on the Ouchterlony plate, homogenates of human thyroid tissue formed a precipitation line. When the human thyroid extract was included in human myoglobin RIA, the dilution curve of thyroid extract was parallel to the standard curve of myoglobin. When the myoglobin immunoreactivity in thyroid extract was fractionated with Sephadex G 75 column, the immunoreactivity was eluted in a peak identical with authentic myoglobin. The position of the peak was different from that of thyroglobulin. Myoglobin concentration in thyroid tissue was estimated to be 0.7-110 mg/g wet wt, being about 1/6000 to 1/40 of that in skeletal muscle. Histochemical studies demonstrated the presence of myoglobin immunoreactivity in thyroid tissue, especially in the apical border of thyroid epithelial cells, implying a functional role in iodinating process or exocytotic-endocytotic process.

Analysis of proteoglycan synthesis by retro-ocular tissue fibroblasts under the influence of interleukin 1 beta and transforming growth factor-beta.

Retro-ocular tissue fibroblasts are supposed to be responsible for the deposition of glycosaminoglycan in Graves' ophthalmopathy. We have reported in a preliminary fashion that interleukin 1 beta (IL-1 beta) and transforming growth factor-beta (TGF-beta) increased the rate of [35S]sulfate incorporation into proteoglycans two to five times the control in culture of retro-ocular tissue fibroblasts. The increase in the rate of [35S]sulfate incorporation into proteoglycan will occur as a result of: (a) net increase of proteoglycan synthesis; (b) elongation of glycosaminoglycan chains; (c) increased number of glycosaminoglycan chains; (d) oversulfation of glycosaminoglycan chains; (e) increase in cell number; (f) decreased rate of degradation. We have analyzed which mechanism is important for the increase of [35S]sulfate into proteoglycans observed in human retro-ocular tissue fibroblasts under the influence of cytokines. The last two possibilities (e, f) were ruled out because during the observation period there was no consistent proliferation of the cells and no decrease in the rate of degradation of proteoglycan examined by pulse-chase experiment. Cytokines did not change the size of glycosaminoglycan chains released from proteoglycan as measured by alkaline borohydride treatment, ruling out (b). Disaccharide analysis by HPLC after chondroitin sulfate ABC digestion revealed that glycosaminoglycan mainly contains monosulfated chondroitin disaccharides and that oversulfation was not observed under the influence of IL-1 beta or TGF-beta, ruling out (d). The capacity to synthesize glycosaminoglycan chain in the presence of an artifical acceptor of chain elongation, beta-D-xylodide, was increased significantly by IL-1 beta but not obviously so by TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)

Bisphenol-A, an environmental estrogen, activates the human orphan nuclear receptor, steroid and xenobiotic receptor-mediated transcription.

BACKGROUND: There is increasing concern about endocrine-disrupting chemicals (EDCs) which may produce adverse health effects in humans and other species. One such chemical, bisphenol-A (BPA), a monomer of polycarbonate plastics, is widely used in consumer products; it has been reported to contain estrogenic activity through binding to estrogen receptors. Cytochrome P450 mono-oxygenase 3A4 (CYP3A4) is one of the key enzymes for the metabolism of endogenous steroids and foreign chemicals in liver. The orphan nuclear receptor, steroid and xenobiotic receptor (SXR/PXR), has recently been isolated. A variety of known inducers of CYP3A4 bind to SXR/PXR, and stimulate transcription on xenobiotic-response elements (XREs) located in the promoter region of the CYP3A4 gene. Recent study has shown that EDCs, diethylhexylphthalate (DEHP) and nonylphenol, but not BPA, induce mouse SXR/PXR-mediated transcription. However, it is known that species differences in SXR alter CYP3A inducibility. OBJECTIVE: To test whether BPA stimulates human SXR/PXR-mediated transcription using reporter gene assays. METHODS: Transfection assays were performed with human SXR/PXR expression plasmid and a reporter plasmid containing the XREs in the CYP3A4 gene promoter in HepG2 cells. BPA-induced interaction of human SXR/PXR with steroid receptor coactivator-1 (SRC-1) was analyzed by mammalian two-hybrid assays. RESULTS: BPA, as well as DEHP, activated human SXR-mediated transcription on the XREs. In mammalian two-hybrid assays, BPA recruited SRC-1 to the ligand-binding domain of human SXR/PXR. CONCLUSIONS: Our observations have indicated that BPA may be a human-specific inducer of the CYP3A4 gene, and may influence the metabolism of endogenous steroids, drugs, and other xenobiotics.

Growth hormone and prolactin gene expression in human densely and sparsely granulated somatotroph adenomas by in situ hybridization with digoxigenin-labeled probes.

We studied growth hormone (GH) and prolactin (PRL) messenger ribonucleic acids (mRNAs) in 14 cases with densely granulated somatotroph (DG) adenomas and 10 cases with sparsely granulated somatotroph (SG) adenomas using in situ hybridization with digoxigenin-labeled probes and correlated these data with their immunohistochemical results. A good correlation between in situ hybridization results and immunohistochemical data was found in most cases examined. The DG adenomas generally had a diffuse and intense GH immunoreactivity and GH hybridization signal, whereas in SG adenomas the number of cells exhibiting a GH mRNA signal and the strength of the GH mRNA signal in these cells were relatively lower than those of DG adenomas, indicating that lower expression of the GH mRNA signal is responsible for lower GH production in SG adenomas. In addition, PRL mRNA expression differed in the two types of adenoma; in DG adenomas, seven cases (50%) expressed PRL mRNA signal with a focal or scattered distribution despite normal serum PRL levels, but two showed no PRL immunoreactivity. In contrast, in SG adenomas, only one case contained a few cells possessing a PRL mRNA signal despite having no PRL immunoreactivity. It can be concluded that DG and SG adenomas, which have been considered variants of the same tumor, display definite differences as to GH or PRL gene expression in each type of adenoma.

Growth hormone-producing pituitary adenomas: correlations between clinical characteristics and morphology.

In this study, we compared the clinical and endocrinological characteristics, neuroimaging findings, surgical outcome, and conventional histological findings (including immunohistochemistry) with the electron microscopic appearance of 31 growth hormone (GH)-producing adenomas. By electron microscopy, these 31 tumors were divided into 23 densely granulated somatotroph adenomas (DG adenomas) and 8 sparsely granulated somatotroph adenomas (SG adenomas). SG adenomas more frequently affected younger women, but no significant correlation was found between the adenoma type and the characteristic signs and symptoms of acromegaly, the incidence of diabetes mellitus or hypertension, or the basal serum GH and insulin-like growth factor I levels. A distinct response of GH to thyrotropin-releasing hormone, bromocriptine, or GH-releasing hormone was significantly more common in patients with DG adenomas than in those with SG adenomas, whereas the incidence of a response to gonadotropin-releasing hormone or oral glucose was not significantly different between the two groups. An analysis of neuroimaging findings and surgical results indicated that SG adenomas were more likely to be macroadenomas with suprasellar extension or invasive tumors and had a lower surgical cure rate. However, postoperative radiotherapy seemed to be similarly effective in both types of adenoma to prevent a tumor recurrence and to reduce postoperative GH basal level in serum. Light microscopy showed that DG adenomas were mainly acidophilic and were immunopositive not only for GH but also for prolactin (43%), the beta subunit of thyroid-stimulating hormone (26%), and the alpha subunit of glycoprotein hormone (87%), whereas SG adenomas were almost all chromophobic and only revealed immunopositivity for GH.(ABSTRACT TRUNCATED AT 250 WORDS)

Gonadotroph adenoma of the pituitary mimicking a prolactinoma.

In a 41-year-old woman with mild hyperprolactinemia and amenorrhea, preoperative hormonal and neuroradiological findings suggested the diagnosis of a macroprolactinoma. She underwent transsphenoidal surgery since the tumor size had not changed in spite of bromocriptine administration for 5 months. Consequently, this case was diagnosed as a female-type gonadotroph adenoma on the basis of its characteristic ultrastructural features including a honeycomb Golgi complex, even though endocrinological and immunohistochemical findings were not those of a typical gonadotroph adenoma.

Postoperative prognostic indicators of control of acromegaly.

Changes in growth hormone basal levels and growth hormone dynamic tests such as thyrotropin-releasing hormone and luteinizing hormone-releasing hormone tests, and the glucose tolerance test were evaluated during a follow-up period of 1-17 years in 17 acromegalic patients. In these cases, mean growth hormone basal levels obtained 1-2 months after surgery had dropped to levels below 5 ng/mL. In 11 of the patients, the 1-2 months postoperative mean growth hormone basal levels were reduced to less than 3 ng/mL. In these patients, abnormal growth hormone responses to these dynamic tests were not present and mean growth hormone basal levels remained below 3.0 ng/mL during the follow-up period of 1-6 years. In contrast, the six other patients' 1-2 months postoperative mean growth hormone basal levels were between 3 and 5 ng/mL. Of these, four presented abnormal responses in at least one of these three dynamic tests 1-2 months postoperatively, and three exhibited a rise in growth hormone basal levels after intervals of 1-3.5 years after surgery. It is concluded that the restoration of normal growth hormone response to the dynamic tests is better related to the value of the mean growth hormone basal levels obtained 1-2 months postoperatively being less than 3 ng/mL rather than it is to the conventional criterion of the growth hormone basal levels being less than 5 ng/mL.

Elevation of serum aminotransferase as a sign of multiorgan-disorders in severely emaciated anorexia nervosa.

To clarify the clinical significance of elevation of serum aminotransferase levels in anorexia nervosa, we analyzed the relationships of serum aminotransferase levels to other serum biochemistry and physical conditions before and during refeeding therapy in 101 patients with anorexia nervosa. Before refeeding therapy, body mass index (BMI) was distributed from 9.9 to 16.4 kg/m2 (13.2 +/- 1.3, mean +/- SD), and 29 patients (28.7%) showed abnormally high aminotransferase levels. Among 17 patients with a BMI of less than 12 kg/m2, the aminotransferase level was abnormally high in 13 patients (76%). Incidence and severity of serum aminotransferase elevation were greater in the patients with lower BMI. The groups with high serum aminotransferase levels had a lower body temperature, lower pulse rate, and higher incidence of other biochemical abnormalities than the group with normal serum aminotransferase levels. These findings indicate that aminotransferase elevation develops at a high incidence in anorectic patients with a critically life-threatening condition, and it is a sign of multiorgan failure requiring urgent calorie repletion. This type of aminotransferase elevation is to be distinguished from refeeding-induced aminotransferase elevation.

Successful treatment by surgical removal of bone metastasis producing PTH: new approach to the management of metastatic parathyroid carcinoma.

A 62-year-old woman had primary hyperparathyroidism due to parathyroid carcinoma. In spite of surgical removal of the enlarged parathyroid gland, intractable hypercalcemia persisted. 99mTc-methoxyisobutylisonitrile scanning revealed bone metastatic lesions, although conventional 201T1-chloride scanning failed to demonstrate those lesions. Octreotide inhibited parathyroid hormone (PTH) secretion, although various means such as bisphosphonates, calcitonin and hydration were effective to reduce serum calcium level to some extent but failed to reduce PTH secretion at all. The CT-guided transcutaneous tumor marking facilitated the subsequent orthopedic surgery for successful removal of the metastatic focus of PTH secretion from the iliac bone.

Development of Graves' ophthalmopathy and uveitis after radioiodine therapy for Graves' disease in a patient with HTLV-I associated myelopathy (HAM).

HTLV-I carriers or patients with HTLV-I associated myelopathy (HAM) are prone to immune-mediated inflammatory disorders. We present a 44-year-old female with HAM who developed Graves' disease. She developed severe Graves' ophthalmopathy shortly after 131I therapy, concurrently with a remarkable increase in TSH-receptor antibody titer. Ophthalmopathy was aggravated in spite of prednisolone therapy and euthyroidism being maintained by thyroxine replacement. Uveitis also developed after 131I therapy and iridocyclitis finally required trabeculotomy. This case suggests that HAM patients may have a higher risk of immune-mediated Graves' ophthalmopathy after 131I therapy.

Disseminated cryptococcosis presenting with adrenal insufficiency and meningitis: resistant to prolonged antifungal therapy but responding to bilateral adrenalectomy.

A case of disseminated cryptococcosis with features of primary adrenal insufficiency and meningitis in an immunocompetent host is presented. Despite antifungal chemotherapy, neither meningitis nor bilateral adrenal gland enlargement was improved. Aspiration biopsy of the adrenal gland revealed necrotic tissue with numerous fungi, suggesting that the adrenal glands were the focus of the persistent fungemia. Removal of bilateral adrenal glands led to improvement by making the patient more sensitive to antifungal chemotherapy.

CT and MR appearance of multiple intracranial lesions associated with suprasellar dermoid cyst.

Multiple lesions associated with suprasellar dermoid cyst are well demonstrated preoperatively on computed tomography (CT) and magnetic resonance (MR) imaging. Although the presence of multiple fatty globules in ventricles and/or subarachnoid space has been thought to be evidence of ruptured dermoid cyst, there are no signs or symptoms, including operative findings, attributable to dermoid cyst rupture in our case. The distinctive CT and MR appearance is described and possible etiology of the multiple lesions is discussed.

[99mTc-MIBI scintigraphy for the detection of parathyroid lesions in patients with hyperparathyroidism].

The usefulness of 99mTc-MIBI scintigraphy for the detection of parathyroid lesions was evaluated in 17 patients with hyperparathyroidism. Delayed image was used to evaluate the lesions. Detectability of MIBI for parathyroid lesions was 86% (18/21). The smallest lesion detected was parathyroid hyperplasia weighted 270 mg. Ectopic parathyroid adenoma and bone metastases of parathyroid carcinoma were clearly demonstrated. Detectability of MIBI scintigraphy for the lesions including ectopic and metastatic lesions was the highest among those of ultrasonography, CT and MRI methods. MIBI scintigraphy was thought to be useful for the detection of parathyroid lesions, especially for ectopic and metastatic lesions.

[Current status of clinical and molecular-biological research on familial periodic paralysis].

Types of periodic paralysis seen in Japan are numerous: the one most frequently seen is hypokalemic periodic paralysis. Among them, approximately 50% are secondary to thyrotoxicosis. Number of families of familial hyperkalemic periodic paralysis have also been reported so far. Several cases of hyperkalemic periodic paralysis secondary to thyrotoxicosis have also been reported exclusively from Japan. As the pathogenesis of hypokalemic periodic paralysis, depolarization block induced by membrane permeability change in the face of hypokalemia triggered by excess insulin was strongly suggested and supported experimentally in part. Recent linkage analysis on familial hypokalemic periodic paralysis revealed that the abnormality is linked to a mutation in voltage-gated Ca channel. The difficulty remains how to explain the cause of hypokalemia which is almost always preceding the attack of periodic paralysis of this type. The cause of hyperkalemic periodic paralysis was shown to be the mutation in voltage-gated Na channel. Failure of inactivation of the channel causes an increase in inward sodium current which results in depolarization and accumulation of potassium. The explanation of the pathogenesis of paralysis is straight-forward when compared to that of hypokalemic periodic paralysis.