New mitochondrial DNA homoplasmic mutations associated with Japanese patients with type 2 diabetes.
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Adult↗
Biomedical subjects
T Onaya
Publications and source records attributed to T Onaya.
Transcription of the thyroid transcription factor-1 (TTF-1) gene from a newly defined start site: positive regulation by TTF-1 in the thyroid.
Regulation of the thyroid transcription factor-1 (TTF-1) gene expression in the thyroid was investigated. We identified a new transcription start site as nucleotide (nt) -1917, 1700 bp upstream of previously described site, and the region encompassing nt -1242 to -14 as the first intron. Although a probe targeting exon 2 hybridized to both 3.7 and 2.7 kp transcripts, a probe targeting newly identified exon 1 mainly reacted with 3.7 kb transcript, indicating that there exsits a transcript from -1917. Chloramphenicol acetyltransferase (CAT) reporter gene assays demonstrated that 5'-flanking region of the start site exhibited promoter activity in FRTL-5 cells but not in rat liver cells, suggesting that this region confers the thyrocyte-selective expression of the gene. Two consensus TTF-1 binding motifs were detected in this promoter region, and electrophoretic mobility-shift assays showed that oligonucleotide probes, each containing one of these motifs, formed a complex with the recombinant TTF-1 homeodomain. Moreover, recombinant TTF-1 increased the transcriptional activity in FRT cells which do not express TTF-1. These results suggest that transcription from the newly identified start site in the TTF-1 gene is positively regulated by TTF-1 in the thyroid.
Animals↗
Structural identification of the myo-inositol 1,4,5-trisphosphate-binding domain in rat brain inositol 1,4,5-trisphosphate 3-kinase.
A series of key amino acids involved in Ins(1,4,5)P3 (InsP3) binding and catalytic activity of rat brain InsP3 3-kinase has been identified. The catalytic domain is at the C-terminal end and restricted to a maximum of 275 amino acids [Takazawa and Erneux (1991) Biochem. J. 280, 125-129]. In this study, newly prepared 5'-deletion and site-directed mutants have been compared both for InsP3 binding and InsP3 3-kinase activity. When the protein was expressed from L259 to R459, the activity was lost but InsP3 binding was conserved. Another deletion mutant that had lost only four amino acids after L259 had lost InsP3 binding, and this finding suggests that these residues (i.e. L259DCK262) are involved in InsP3 binding. To further support the data, we have produced two mutants by site-directed mutagenesis on residues C261 and K262. The two new enzymes were designated M4 (C261S) and M5 (K262A). M4 showed similar Vmax and Km values for InsP3 and ATP to wild-type enzyme. In contrast, M5 was totally inactive but had kept the ability to bind to calmodulin-Sepharose. C-terminal deletion mutants that had lost five, seven or nine amino acids showed a large decrease in InsP3 binding and InsP3 3-kinase activity. One mutant that had lost five amino acids (M2) was purified to apparent homogeneity: Km values for both substrates appeared unchanged but Vmax was decreased approx. 40-fold compared with the wild-type enzyme. The results indicate that (1) a positively charged amino acid residue K262 is essential for InsP3 binding and (2) amino acids at the C-terminal end of the protein are necessary to act as a catalyst in the InsP3 3-kinase reaction.
Amino Acid Sequence↗
Mitochondrial DNA mutations in Japanese detected by polymerase chain reaction--restriction fragment--single strand conformation polymorphism analysis.
PCR-restriction fragment-SSCP (PCR-RF-SSCP) analysis of mitochondrial DNA by HhaI/HincII in Japanese revealed 46 polymorphic patterns. The determinations of nucleotide sequence of these 46 patterns revealed 56 mutations compared with the Cambridge Sequence.
DNA Mutational Analysis↗
Mouse glycine N-methyltransferase is sexually dimorphic and regulated by growth hormone.
The secretion pattern of putuitary growth hormone (GH) in rodents is sexually dimorphic and regulates the expression of some hepatic genes. We report cloning of mouse glycine N-methyltransferase (GNMT) cDNA. The mouse GNMT mRNA is expressed at much lower levels in male than in female livers. Hypophysectomy in male mice resulted in as a high level of the expression as in female mice. Treatment with GH of hypophysectomized male mice twice a day for 5 days decreased the expression. These results indicate that the sex difference of the mouse GNMT expression is regulated by GH secretion patterns.
Amino Acid Sequence↗
Transforming growth factor-beta1 suppresses thyrotropin-induced Na+/I- symporter messenger RNA and protein levels in FRTL-5 rat thyroid cells.
Iodide transport into the thyroid catalyzed by the Na+/I- symporter (NIS), is the first and main rate-limiting step in thyroid hormone synthesis. Recently, we have demonstrated that thyrotropin (TSH) increases NIS messenger RNA (mRNA) and protein levels, as well as iodide uptake activity. Although transforming growth factor-beta1 (TGFbeta1) is known to affect thyroid cell function, it is still unclear how TGFbeta1 regulates TSH-stimulated iodide accumulation. Therefore, the effects of TGFbeta1 on TSH-stimulated NIS mRNA and protein levels were examined in FRTL-5 rat thyroid cells by Northern and Western blot analyses, and iodide uptake was assessed. Northern blot analysis revealed that TGFbeta1 suppressed TSH-stimulated NIS mRNA levels in a dose- and time-dependent manner. Western blot analysis demonstrated that TGFbeta1 suppressed TSH-stimulated NIS protein levels. TGFbeta1 also suppressed (Bu)2 cyclic adenosine monophosphate (cAMP)- and forskolin-stimulated NIS mRNA and protein levels, indicating a role for TGFbeta1 downstream of cAMP production. As predicted, TGFbeta1 inhibited TSH-stimulated iodide uptake activity. These results suggest that the inhibitory effect of TGFbeta1 on TSH-stimulated iodide uptake is at least in part due to a suppression of NIS specific transcription. Therefore, TGFbeta1 may act as an autocrine or paracrine local modulator of thyroid hormone synthesis by influencing NIS mRNA levels in the thyroid.
Animals↗
Iodide uptake and experimental 131I therapy in transplanted undifferentiated thyroid cancer cells expressing the Na+/I- symporter gene.
131I therapy is a widely accepted treatment for differentiated thyroid cancers which can accumulate iodide. We evaluated the efficiency of 131I therapy against tumors which are transfected with the Na+/I- symporter (NIS) gene. We transfected the rat NIS cDNA expression vector into malignantly transformed rat thyroid cells (FRTL-Tc) which do not concentrate iodide. The resultant cell line (Tc-rNIS) accumulated 125I 60-fold in vitro. The FRTL-Tc cells formed solid tumors after injection of cells into subcutaneous tissues of Fischer 344 rats. Tumors formed with Tc-rNIS cells accumulated up to 27.3% of total 125I administered, and concentrated 125I 11 to 27-fold in the tumors. Extracorporeal measurement of radioactivity in the tumors revealed that 125I accumulation peaked at 90 min, and decreased to half levels 6 h after the injections. To investigate the effect of 131I administration on the tumor growth, we injected Na131I 2 and 3 weeks after the transplantation of the cells. The Na131I did not change the tumor volume significantly in either the FRTL-Tc or the Tc-rNIS-induced tumors. The short (6 h) effective half life of 131I in the tumors diminished the radiation dose to the tumor cells. However, this approach may prove beneficial in the treatment of radiosensitive cancers, and could be employed diagnostically.
Animals↗
Thyroid-stimulating hormone-induced down-regulation of thyroid transcription factor 1 in rat thyroid FRTL-5 cells.
Thyroid transcription factor 1 (TTF-1) is thought to play an important role in the expression of genes that encode thyroid-specific proteins such as thyroglobulin, thyroid peroxidase, and TSH-receptor. The role of TSH in the regulation of TTF-1 messenger RNA (mRNA) and protein abundance was investigated in rat thyroid FRTL-5 cells. Northern blot analysis revealed that TSH reduced TTF-1 mRNA abundance in a dose- and time-dependent manner. Immunoblot analysis with rabbit antibodies prepared against a recombinant fragment of TTF-1 expressed in bacteria showed that TSH also reduced the amount of TTF-1 protein in FRTL-5 cells. Whereas the effect of TSH on TTF-1 mRNA was apparent after 3 h, the effect on TTF-1 protein was not apparent until 12 h after TSH addition to the cells. Both TTF-1 mRNA and protein were significantly decreased after the addition of (Bu)2 cAMP or forskolin for 24 h, whereas they were not decreased by 12-O-tetradecanoyl-phorbol-13-acetate. These results indicate that TSH down-regulates TTF-1 expression in FRTL-5 cells via the cAMP pathway.
Animals↗
Regulation of thyrotropin receptor gene expression in 3T3-L1 adipose cells is distinct from its regulation in FRTL-5 thyroid cells.
We previously have demonstrated that rat adipose tissue expresses TSH receptor (TSHR) messenger RNAs (mRNAs) at levels approaching those detected in the thyroid. Furthermore, we recently reported that TSHR mRNA is detected in fibroblast-like 3T3-L1 cells after their hormone-induced differentiation into adipocytes. TSH induces cAMP formation and lipolysis in differentiated 3T3-L1 cells. We now show that, in Northern blot analyses, TSH-induced down-regulation of TSHR mRNA levels, which can be duplicated by forskolin and dibutylyl cAMP, i.e. which is cAMP-mediated. We also have demonstrated that a beta-adrenergic stimulant, which stimulates cAMP formation in adipocytes, induces a down-regulation of TSHR mRNA levels in 3T3-L1 adipocytes. Nuclear run-on assays show that the ability of TSH/cAMP to decrease TSHR mRNA levels in 3T3-L1 cells reflects transcriptional regulation. This report also demonstrates that TSHR gene expression in 3T3-L1 adipocytes is regulated in a manner distinct from that observed in thyroid cells. Thus, in fully differentiated 3T3-L1 adipocytes, TSH-induced down-regulation of TSHR mRNA levels is evident within 1 h and is near maximum within 4 h after addition of TSH. A transient increase of TSHR gene expression, which has been demonstrated in FRTL-5 thyroid cells, was not observed in 3T3-L1 adipocytes. The down-regulation of TSHR gene expression induced by TSH/cAMP in 3T3-L1 cells is cycloheximide-insensitive, suggesting that continuous protein synthesis is not required for this process. In contrast, the down-regulation of TSHR gene expression observed in FRTL-5 cells is sensitive to cycloheximide. In both FRTL-5 thyroid cells and 3T3-L1 adipocytes, insulin or serum increased TSHR mRNA levels. Although insulin or serum was required for the TSH-induced down-regulation of TSHR mRNA levels in FRTL-5 thyroid cells, neither insulin nor serum was required for TSHR down-regulation in 3T3-L1 adipocytes. These findings demonstrate that TSH/cAMP regulates TSHR mRNA levels in adipocytes via a regulatory system distinct from that used in FRTL-5 cells. This report further demonstrates that adipose cells do not express thyroid transcription factor-1, which interacts with the TSHR promoter region in FRTL-5 cells, and that 3T3-L1 nuclear extracts exhibit a different binding activity to the cAMP-response element-like element in the TSHR promoter region compared with extracts from FRTL-5 cells.
3T3 Cells↗
Regulation by thyroid-stimulating hormone of sodium/iodide symporter gene expression and protein levels in FRTL-5 cells.
To investigate the mechanism of I- transport stimulation by TSH, we studied the effects of TSH on Na+/I- symporter (NIS) messenger RNA (mRNA) and protein levels in FRTL-5 cells and correlated these with I- transport activity. When 1 mU/ml TSH was added to quiescent FRTL-5 cells, a 12-h latency was observed before the onset of increased I- transport activity, which reached a maximum [approximately 27 times basal (5H medium) levels] at 72 h. In contrast, Northern blot analysis, using rat NIS complementary DNA as a probe, revealed that addition of TSH to these cells significantly increased NIS mRNA at 3-6 h, reaching a maximum after 24 h (approximately 5.9 times basal levels). Forskolin and (Bu)2cAMP mimicked this stimulatory effect on both the I- transport activity and mRNA levels. D-ribofranosylbenzimidazole, a transcription inhibitor, almost completely blocked TSH-induced stimulation of I- transport and NIS mRNA levels. Western blot analysis demonstrated that TSH increased NIS protein levels at 36 h, reaching a maximum at 72 h, in parallel with the kinetics of TSH-induced I- transport activity. However, it also showed that the amount of NIS protein already present in FRTL-5 cell membranes before the addition of TSH was about one third of the maximum level induced by TSH. These results indicate that stimulation of I- transport activity by TSH in thyrocytes is partly due to a rapid increase in NIS gene expression, followed by a relatively slow NIS protein synthesis. However, the existence of an abundant amount of protein in quiescent FRTL-5 cells with very low I- transport activity also suggests that this activity is controlled by another TSH-regulated factor(s).
Animals↗
Increased expression of the Na+/I- symporter in cultured human thyroid cells exposed to thyrotropin and in Graves' thyroid tissue.
The Na+/I- symporter (NIS) is important in hormone synthesis in the thyroid gland. NIS activity, as reflected by I- uptake, was increased by TSH (1 mU/mL) or forskolin (10 mumol/L) in primary cultured human thyroid cells. Northern blot analysis revealed that incubation of these cells with TSH or forskolin for 24 h increased the abundance of NIS messenger ribonucleic acid (mRNA) 2.3- and 2.5-fold, respectively. Immunoblot analysis revealed 2.7- and 2.4-fold increases, respectively, in the amount of NIS protein after 48 h, suggesting that elevated levels of intracellular cAMP induced the expression of NIS in human thyrocytes. We then studied the levels of NIS mRNA and protein in Graves' thyroid tissue and found that the amount of NIS mRNA in thyroid tissue from individuals with Graves' disease (n = 5) was 3.8 times that in normal thyroid tissue (n = 5). The abundance of NIS mRNA was significantly correlated with that of thyroid peroxidase or thyroglobulin mRNAs, but not with that of TSH receptor mRNA, in the Graves' and normal thyroid tissue specimens. The amount of NIS protein was also increased 3.1-fold in Graves' thyroid tissue compared with that in normal thyroid tissue. The increased expression of NIS may thus contribute to the development of Graves' disease.
Blotting, Northern↗
Thyroid transcription factor-1 activates the promoter activity of rat thyroid Na+/I- symporter gene.
We have cloned 15 kbp of rat thyroid Na+/I- symporter gene from liver genomic library, which contains 6 kbp upstream sequence from the translation initiation site. Southern blot analysis of the genomic DNA from the liver has revealed that thyroid Na+/I- symporter gene is the single gene in the rat. To study the tissue-selective expression mechanism of the gene, we at first determined the transcriptional start site of the gene. Results of a rapid amplification of cDNA end procedure as well as that of primer extension analysis indicated that the transcriptional start sites clustered between -96, -95, and -93 bp of the gene (A in ATG is designated as +1). Chimeras containing 1.9 kbp (-1967 to -46 bp) of the 5'-flanking sequence of the Na+/I- symporter gene and luciferase gene expressed significant enzyme activity when transfected into a rat thyroid cell line, FRTL-5, but little activity was observed in BRL-3A rat liver cells. Deletion analysis of the constructs indicated that a minimal region, exhibiting promoter activity and cell specificity, is located between -291 and -134 bp of the gene. Deoxyribonuclease I footprinting shows that nuclear extracts from FRTL-5, but not BRL-3A, cells protect a region between -245 and -230 bp. Electrophoretic mobility shift assays have demonstrated that nuclear extracts from FRTL-5 cells formed a specific DNA-protein complex with an oligonucleotide probe corresponding to -250 to -211 bp of the gene, but that from BRL-3A cells did not, suggesting that thyrocyte-selective nuclear factors bind to the region. When the nuclear extracts from FRTL-5 cells were preincubated with antibody against thyroid transcription factor-1 (TTF-1), homeodomain containing nuclear protein, formation of the complex was abolished and the band was supershifted. We also found that the probe formed a DNA-protein complex with the recombinant TTF-1 homeodomain, and mutations of the binding site eliminated factor binding. When pRc/CMV-TTF-1 was cotransfected with the minimal promoter fragment of thyroid Na+/I- symporter gene into FRT cells, which express no TTF-1, it caused a significant increase in the transcriptional activity of the reporter construct, but not of the construct having mutated TTF-1-binding element. These results suggest that TTF-1 confers the cell-selective expression of Na+/I- symporter gene in thyrocytes.
Animals↗
Phosphorylation of myosin light chain in resting platelets from NIDDM patients is enhanced: correlation with spontaneous aggregation.
Platelet function in patients with NIDDM is enhanced. We have found that spontaneous aggregation (i.e., the formation of small-sized aggregates in the absence of agonist stimulation) occurs at a high rate in platelets from NIDDM patients. We then investigated basal myosin light chain 20 (MLC) phosphorylation, which plays a key role in platelet shape change and aggregation, using a monoclonal antibody against a phosphorylation site (serine 19 residue) in the MLC molecule in platelets from these patients. Standard calibration curves obtained from purified MLC or the phosphorylated form of myosin light chain 20 (MLC-P) were linear within the range of 0-150 ng for MLC and 0-3 ng for MLC-P. The amount of MLC or MLC-P in platelets was estimated, and basal MLC phosphorylation was calculated. Platelets were obtained from 9 young healthy control subjects, 13 age- and sex-matched nondiabetic control subjects, and 13 patients with NIDDM. The basal MLC phosphorylation in platelets was significantly higher in the NIDDM patients than in the control subjects, irrespective of age. These findings suggest that platelets from NIDDM patients are activated in vivo. Platelets obtained from NIDDM patients generated spontaneous aggregation, the degree of which was significantly higher than that in control subjects. Platelet spontaneous aggregation correlated well with basal MLC phosphorylation. These findings suggest that increases in basal MLC in platelets may be one factor leading to hyperaggregability of platelets in these patients.
Adult↗
Autoantibody against thyroid iodide transporter in the sera from patients with Hashimoto's thyroiditis possesses iodide transport inhibitory activity.
Recently we have newly identified an autoantibody against thyroid iodide transporter (TIT) in the sera from patients with autoimmune thyroid disease. In order to study the function of these autoantibodies, we established CHO-KI cells stably expressing recombinant rat TIT (CHO-TIT cells), and the effect of IgGs from the patients with Hashimoto's thyroiditis on iodide uptake activity of CHO-TIT cells was investigated. We found that 4 out of 34 sera from patients with Hashimoto's thyroiditis strongly recognized TIT by Western blot analysis. These 4 IgGs, purified by protein A column chromatography, caused 14 to 62% inhibition of I- accumulation in CHO-TIT cells. Next, using synthetic peptides, we determined the recognition site of the autoantibody on the TIT molecule. The autoantibody reacted with the synthetic peptide corresponding to the 6th extracellular loop of the TIT molecule. These results suggest that autoantibody against TIT in the sera from patients with Hashimoto's thyroiditis binds to the 6th extracellular loop of TIT protein and inhibits I- transport into the thyrocytes. Anti-TIT autoantibody might participate in the pathogenesis of Hashimoto's thyroiditis and modulate thyroid function of patients with the disease.
Animals↗
Autoantibody against Na+/I- symporter in the sera of patients with autoimmune thyroid disease.
Using recombinant rat Na+/I- symporter (NaIS) protein, we have immunochemically searched for the autoantibody in the sera from patients with autoimmune thyroid disease. We found that 22 out of 26 sera (84%) from patients with Graves' disease and 3 out of 20 sera (15%) from patients with Hashimoto's thyroiditis recognized it. By Western blot, these IgGs reacted with 80 kDa protein in FRTL-5 cell membrane, which co-migrated with the band stained by rabbit antibody toward NaIS. These results indicate that autoantibody against NaIS, newly identified antibody, frequently exists in patients with autoimmune thyroid disease, especially in Graves' disease.
Animals↗
Functional expression of thyrotropin receptor in differentiated 3T3-L1 cells: a possible model cell line of extrathyroidal expression of thyrotropin receptor.
Thyrotropin receptor (TSHR) in extrathyroidal tissue, especially fat tissue, is supposed to have important roles in the development of extrathyroidal manifestations of Graves' disease. However, the molecular mechanism of TSHR expression is not known. Expression of TSHR mRNA and TSH-dependent cAMP production were observed in differentiated but not in undifferentiated 3T3-L1 cells. Maximal expression was obtained when the cells were differentiated in the presence of insulin, dexamethasone, and isobutylmethylxanthine (IBMX). Dexamethasone and IBMX were indispensable for the first three days. On the other hand, after day 4, insulin was indispensable for the expression of TSHR. 3T3-L1 cell is the first non-thyroidal cell line reported that expresses TSHR and whose expression can be induced. 3T3-L1 cell can be a good model to investigate the mechanism of expression of TSHR and extrathyroidal manifestations of Graves' disease.
1-Methyl-3-isobutylxanthine↗
Modulation of basal nitric oxide-dependent cyclic-GMP production by ambient glucose, myo-inositol, and protein kinase C in SH-SY5Y human neuroblastoma cells.
Defective tissue perfusion and nitric oxide production and altered myo-inositol metabolism and protein kinase C activation have been invoked in the pathogenesis of diabetic complications including neuropathy. The precise cellular compartmentalization and mechanistic interrelationships of these abnormalities remain obscure, and nitric oxide possesses both neurotransmitter and vasodilator activity. Therefore the effects of ambient glucose and myo-inositol on nitric oxide-dependent cGMP production and protein kinase C activity were studied in SH-SY5Y human neuroblastoma cells, a cell culture model for peripheral cholinergic neurons. D-Glucose lowered cellular myo-inositol content, phosphatidylinositol synthesis, and phosphorylation of an endogenous protein kinase C substrate, and specifically reduced nitric oxide-dependent cGMP production a time- and dose-dependent manner with an apparent IC50 of approximately 30 mM. The near maximal decrease in cGMP induced by 50 mM D-glucose was corrected by the addition of protein kinase C agonists or 500 microM myo-inositol to the culture medium, and was reproduced by protein kinase C inhibition or downregulation, or by myo-inositol deficient medium. Sodium nitroprusside increased cGMP in a dose-dependent fashion, with low concentrations (1 microM) counteracting the effects of 50 mM D-glucose or protein kinase C inhibition. The demonstration that elevated D-glucose diminishes basal nitric oxide-dependent cGMP production by myo-inositol depletion and protein kinase C inhibition in peripheral cholinergic neurons provides a potential metabolic basis for impaired nitric oxide production, nerve blood flow, and nerve impulse conduction in diabetes.
Base Sequence↗
A case of small cell carcinoma of the uterine cervix presenting Cushing's syndrome.
A proportion of small cell carcinoma of the uterine cervix is known to secrete a neuroendocrine substance. However, cases presenting Cushing's syndrome due to ACTH secreted from a cervical small cell carcinoma are extremely rare. Here, we report a case of small cell carcinoma of the uterine cervix that ectopically secreted ACTH and presented Cushing's syndrome. A 42-year-old woman was initially admitted to the endocrine unit for investigation of ectopic ACTH syndrome. Serum cortisol and plasma ACTH levels were elevated with no diurnal variation. After gynecologic examination, her original disease was found to be a stage IIb cervical small cell carcinoma. She underwent a radical hysterectomy and pelvic lymphadenectomy. The entire tumor was excised and post-TNM classification was T2bN1M0. Immunohistochemically, tumor cells stained strongly for ACTH and chromogranin. Electronmicroscopic pictures showed typical neurosecretory granules. Although plasma ACTH returned to normal after surgery, liver metastasis appeared during the course of postoperative irradiation. She died 9 months after operation in spite of vigorous systemic chemotherapy.
Adrenocorticotropic Hormone↗