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Biomedical subjects

T Onaya

Publications and source records attributed to T Onaya.

At least 19 recordsLinked to original sources
PubMed1992

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.

Amino Acid Sequence↗
PubMed1992

Heterogeneous responses of recombinant human thyrotropin receptor to immunoglobulins from patients with Graves' disease.

Non-thyroid mammalian cells, CHO-K1 cells, stably expressing human thyrotropin receptor (CHO-TSH-R cells) were used for the assay of thyroid stimulating antibody (TSAb) activities of IgGs from 24 patients with Graves' disease and we compared them with the values obtained in porcine thyroid cells. A significant positive correlation was observed between the results given by CHO-TSH-R cells (hTSAb) and porcine thyrocytes (pTSAb) (r = 0.94, p less than 0.001). However, we found that hTSAb values of IgGs from 5 patients were extremely different from their hTSAb values. Four out of these 5 IgGs showed strong pTSAb activity but exhibited a weak or negative hTSAb activity. Conversely, one out of 5 autoantibodies was very strong for hTSAb but its pTSAb was low. These heterogeneous responses of recombinant hTSH-R to Graves' IgGs suggest that there exist different types of TSAb and also that the epitope(s) for TSAb may be different from case to case.

Animals↗
PubMed1992

Anti-platelet action of isoliquiritigenin, an aldose reductase inhibitor in licorice.

The mechanism was studied by which isoliquiritigenin, a new aldose reductase inhibitor purified from licorice (Glycyrrhizae radix), inhibits platelet aggregation. This new agent significantly inhibited the phosphorylation of 40,000- and 20,000-dalton proteins, and inhibited the formation of 12 (S)-hydroxy-5,8,10-heptadecatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B2. The inhibitory effect of isoliquiritigenin on platelet aggregation in vitro was comparable to that of aspirin. Our findings may indicate that isoliquiritigenin elicits an anti-platelet action by inhibiting not only cyclooxygenase but also lipoxygenase or peroxidase activity in platelets. Isoliquiritigenin also showed an anti-platelet action in vivo. Isoliquiritigenin appears to be the only aldose reductase inhibitor with a significant anti-platelet action. Since the hyperaggregability of platelets has been implicated in the pathogenesis of diabetic complications, isoliquiritigenin may offer a unique benefit as an aldose reductase inhibitor.

Aldehyde Reductase↗
PubMed1992

Glucose modulation of aldose reductase mRNA expression and its activity in cultured calf pulmonary artery endothelial cells.

We examined the effect of glucose on aldose reductase mRNA expression and its activity in calf pulmonary artery endothelial cells. After the cells were exposed to 18 mmol/l glucose, aldose reductase mRNA expression began to increase at 6 h, reached a maximum (about 2.4-fold increase) at 12 h, and thereafter gradually decreased. Aldose reductase activity was found to strongly correlate with aldose reductase mRNA expression after cells were exposed to 18 mmol/l glucose. In contrast, aldose reductase mRNA expression was significantly decreased following exposure to 55 mmol/l glucose. Aldose reductase activity was also decreased at 24 h after 55 mmol/l glucose. The increase in aldose reductase mRNA level caused by glucose was inhibited by 1 microgram/ml of actinomycin D. These phenomena appear to be glucose-specific since neither 3-O-methylglucose nor fructose affected the levels of aldose reductase mRNA. We clearly demonstrate that aldose reductase mRNA level and its activity are modulated by glucose in calf pulmonary artery endothelial cells. Our data suggest that activation of aldose reductase in endothelial cells may contribute to the development of diabetic macroangiopathy.

3-O-Methylglucose↗
PubMed1992

Aldose reductase mRNA expression and its activity are induced by glucose in fetal rat aortic smooth muscle (A10) cells.

The induction of aldose reductase (AR) mRNA expression and its activity by glucose were investigated in fetal rat aortic smooth muscle (A10) cells. The increase in the expression of AR mRNA was observed at 6 h, and reached a maximum (2.3 fold) at 12 h after exposure to 80 mM glucose. On the other hand, increase in AR activity was observed at 12 h and reached a maximum (2 fold) at 48 h after exposure to 80 mM glucose. AR mRNA levels as well as its activity increased almost linearly in a concentration dependent manner up to 80 mM. Although not significant, there was a consistent increase in AR mRNA level and its activity at 23.75 mM glucose. There was a good correlation between AR mRNA level and AR activity. In the presence of 1 microgram/ml actinomycin D, the increase in the expression of AR mRNA level by glucose was almost completely abolished, suggesting its transcriptional regulation by glucose. Other osmolytes were also effective in inducing AR mRNA expression as well as its activity at 80 mOsm/kg. Therefore, the increase in AR mRNA level and its activity by glucose in A10 cells may be a response to the increase in osmolarity.

Aldehyde Reductase↗
PubMed1992

A synthetic analogue of vitamin D3, 22-oxa-1,25-dihydroxy-vitamin D3, stimulates the production of prostacyclin by vascular tissues.

We investigated the effect of 22-oxa-1,25-dihydroxyvitamin D3, a synthetic analogue of vitamin D3, on the production of prostacyclin by vascular tissues using rat aortic rings and A7r5 cells derived from fetal rat aortic smooth muscle. Prostacyclin synthesis by aortic rings of rats treated with 22-oxa-1,25-dihydroxyvitamin D3 was much higher than that of non-treated controls, but did not cause any significant hypercalcemia. Treatment with 22-oxa-1,25-dihydroxyvitamin D3 significantly increased the production of prostacyclin by A7r5 cells for 48 hours in a dose-dependent manner. In time-course studies, cells incubated with 22-oxa-1,25-dihydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 produced prostacyclin progressively over a period of 48 hours. The shortest period of incubation that produced a significant amount of prostacyclin compared with control cultures was 24 hours. We observed that treatment with 22-oxa-1,25-dihydroxyvitamin D3 induced cyclooxygenase mRNA in A7r5 cells. Our data suggest that 22-oxa-1,25-dihydroxyvitamin D3 may possibly be a protective substance against the development of atherosclerosis by modulating prostaglandin metabolism.

6-Ketoprostaglandin F1 alpha↗
PubMed1992

Transcriptional regulation of hexokinase I mRNA levels by TSH in cultured rat thyroid FRTL5 cells.

We investigated the effect of thyroid stimulating hormone (TSH) on the expression of hexokinase I mRNA by cultured rat thyroid FRTL5 cells. TSH stimulated hexokinase I gene expression in a time- and dose-dependent manner. An increase in hexokinase I mRNA was detected after 3 h of incubation with TSH, and a maximum was reached after 12 h showing about 2.5-fold increase at 1 mU/ml TSH. A nuclear run-on transcriptional assay showed that the effect of TSH on hexokinase I mRNA was due to an increase in the rate of gene transcription. (Bu)2cAMP and forskolin also increased hexokinase I mRNA expression to almost the same extent as TSH. These findings suggest that TSH stimulates hexokinase I gene expression at the transcriptional level via the cAMP-dependent pathway.

Animals↗
PubMed1992

Beta 2-adrenergic receptor mRNA is overexpressed in neoplastic human thyroid tissues.

The expression of beta 2-adrenergic receptor (AR) mRNA was investigated in normal and neoplastic human thyroid tissues. A combination of techniques for reverse transcribing mRNA into cDNA and the incorporation of 32P-gamma ATP into the polymerase chain reaction (PCR)-generated fragments allowed us to detect beta 2-AR mRNA in surgically excised thyroid specimens. The levels of beta 2-AR cDNA generated by PCR in thyroid adenomas and cancers were 3.3 and 6.9 times, respectively, as high as that of normal thyroid tissues. These findings suggest that the level of beta 2-AR mRNA is correlated with the extent of differentiation in neoplastic tissues. The present study provides new insights into the relationships between the AR-adenylate cyclase system and the regulation of the growth and differentiation in neoplastic human thyroid tissues.

Base Sequence↗
PubMed1992

The regulation of two distinct glucose transporter (GLUT1 and GLUT4) gene expressions in cultured rat thyroid cells by thyrotropin.

We investigated the glucose transporter mRNAs expressed in FRTL5, a rat thyroid cell line, and their regulation by TSH by means of the polymerase chain reaction. FRTL5 cells as well as rat thyroid tissue expressed three types of glucose transporter mRNAs: GLUT1 or erythrocyte/HepG2/brain isoform, GLUT2 or pancreatic beta-cell/liver isoform, and GLUT4 or muscle/fat isoform. GLUT1 mRNA predominated, GLUT4 mRNA was minor, and GLUT2 mRNA expression was faint. Incubation of FRTL5 cells with TSH induced a time- and concentration-dependent increase in GLUT1 mRNA levels, while GLUT4 mRNA levels were decreased. The response of GLUT1 mRNA to TSH was evident at 3 h, and the maximal response was achieved at 12 h. TSH at a dose of 1 mU/ml elicited an approximately 3-fold increase in GLUT1 mRNA levels. (Bu)2cAMP (1 mM), 8-bromo-cAMP (1 mM), and forskolin (50 microM) mimicked the effect of TSH on GLUT1 and GLUT4 mRNA levels. The increase in GLUT1 mRNA by TSH was correlated with the increase in GLUT1 protein and the increase in 2-deoxyglucose transport activity. These observations suggest that in thyroid cells, TSH stimulates glucose transport at least in part by enhancing GLUT1 gene expression, and that the effect of TSH on GLUT1 and GLUT4 mRNA levels is mediated by a cAMP-dependent pathway.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1992

The role of cyclic adenosine 3',5'-monophosphate and polyol metabolism in diabetic neuropathy.

The effects of a stable prostacyclin analog, Iloprost, and aldose reductase inhibitors (ONO-2235 and isoliquiritigenin) were studied to elucidate the role of cAMP in diabetic neuropathy in relation to polyol metabolism. In in vivo experiments, the cAMP and myoinositol contents in sciatic nerves and motor nerve conduction velocity were significantly reduced in diabetic rats. Iloprost significantly restored the reduced cAMP content in sciatic nerves and improved motor nerve conduction velocity in diabetic rats. However, the contents of sorbitol or myoinositol in sciatic nerves were not affected by Iloprost in diabetic rats. On the other hand, aldose reductase inhibitors significantly reduced the sorbitol content and increased the cAMP and myoinositol contents in the sciatic nerves of diabetic rats. The motor nerve conduction velocity was also slightly but significantly improved by treatment with aldose reductase inhibitors. There was a negative correlation between cAMP and sorbitol in the sciatic nerves of diabetic rats treated with aldose reductase inhibitors and a positive correlation between cAMP and motor nerve conduction velocity. In in vitro experiments, Iloprost significantly increased cAMP, but did not affect the sorbitol content in sciatic nerves. Aldose reductase inhibitors inhibited sorbitol accumulation and increased cAMP in sciatic nerves. Our data suggest that polyol pathway activation somehow results in cAMP reduction in sciatic nerves and that the reduction of cAMP in peripheral nerves may be closely related to the pathogenesis of diabetic neuropathy.

Aldehyde Reductase↗
PubMed1992

Immunization with human thyrotrophin receptor peptide induces an increase in thyroid hormone in rabbits.

Eight rabbits were immunized with a synthetic peptide corresponding to the unique N-terminal region (termed N peptide; amino acid residues 29-57) in the extracellular domain of the human thyrotrophin (TSH) receptor. After 10 weeks, all of the eight rabbits produced anti-N peptide antibodies. Western blot analysis revealed that the antibodies recognized rabbit TSH receptor as an approximately 100 kDa protein. We compared the level of thyroid hormone in serum taken before immunization (preimmune sera) with that of serum taken after immunization (postimmune sera) in these immunized rabbits. Postimmune sera from the eight rabbits had higher mean (+/- S.D.) levels of tri-iodothyronine (T3) and thyroxine (T4) than did preimmune sera (T3, preimmune 0.82 +/- 0.26 micrograms/l vs postimmune 1.33 +/- 0.35, P < 0.01; T4, preimmune 33.7 +/- 10.0 micrograms/l vs postimmune 41.0 +/- 6.0, P < 0.05). T3 levels in four rabbits and T4 levels in four rabbits after immunization were over the normal range obtained from six age-matched control rabbits. Seven rabbits exhibited thyroid-stimulating antibody (TSAb) activity with various degrees (241-545%). The concentration of T3 and T4 did not increase over 10 weeks in either non-immunized rabbits (T3, preimmune 0.89 +/- 0.34 micrograms/l vs postimmune 0.82 +/- 0.22; T4, preimmune 31.1 +/- 7.3 micrograms/l vs postimmune 30.3 +/- 5.1) or other peptide-immunized rabbits (T3, preimmune 0.68 micrograms/l (n = 2) vs postimmune 0.69; T4, preimmune 33.1 micrograms/l vs postimmune 26.4). These results indicate that experimentally produced anti-TSH receptor antibody with TSAb activity induces an increase in thyroid hormone in rabbits.

Animals↗
PubMed1991

Rabbit antibodies toward extracellular loops of the membrane spanning region of human thyrotropin receptor possess thyroid stimulating activities.

We have synthesized three different peptides, E1 (amino acid residues 478-497), E2 (amino acid residues 561-580) and E3 (amino acid residues 649-652), corresponding to the first, the second and the third extracellular loops of the membrane spanning region of human thyrotropin receptor (TSH-R), respectively. We have produced rabbit antibodies toward these peptides and evaluated their thyroid stimulating antibody (TSAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. Although only slight TSAb activity was observed in E1 antibodies, E2 and E3 antibodies possessed strong TSAb activities, the values of which were 1118% and 910%, respectively. None of these antibody had TBII activities. These results suggest that antibodies against the extracellular loops of the TSH-R can stimulate cAMP formation in thyroid cells and that these regions may be one of the candidates for the epitope against autoantibodies from patients with Graves' disease.

Amino Acid Sequence↗
PubMed1991

Thyrotropin receptor non-mediated thyroid stimulating immunoglobulin in Graves' disease.

There exists a consensus that hyperthyroid Graves' disease is caused by thyrotropin receptor (TSH-R) autoantibodies. To test the possibility that the TSH-R is the sole antigen for thyroid stimulating antibodies (TSAb), we compared bioactivities of Graves' IgGs between non-thyroid mammalian cells transfected with human TSH-R cDNA and the reference thyroid bioassay. A Graves' IgG with TSH-binding inhibitor immunoglobulin (TBII) activity (89%) markedly stimulated cAMP formation in both CHO-K1 cells transfected with TSH-R cDNA (340 microU/ml of TSH equivalent) and rat thyroid cells, FRTL-5, (410 microU/ml of TSH equivalent). In contrast, a TBII negative (-1.5%) IgG from another patient with Graves' disease showed a strong thyroid stimulating activity (87 microU/ml of TSH equivalent) when FRTL-5 cells were used for the assay. But no stimulating activity was observed in this IgG when CHO-K1 cells transfected with TSH-R cDNA were used, suggesting a possible existence of TSH-R non-mediated thyroid stimulating immunoglobulin in some cases of Graves' disease.

Animals↗
PubMed1991

Rabbit antibodies against two different extracellular domains of human thyrotropin receptor possess thyroid stimulating activities.

We have produced rabbit antibodies against synthetic peptides corresponding to the mid-region (amino acid residues 172-202, C peptide) and to the unique segment near the transmembrane region (amino acid residues 341-370, P peptide) in the extracellular component of the human thyrotropin (TSH) receptor and evaluated their biological activities. Both anti-C peptide antibodies raised in two rabbits showed strong thyroid stimulating activities (TSAb) (4127% and 2548%). Anti-P peptide antibodies raised in two rabbits were also strongly positive for TSAb activities (359% and 3468%). However, none of these antibodies had TSH-binding inhibitor immunoglobulin (TBII) activities. These results suggest that the domains responsible for TSAb are likely to span the entire extracellular component of the TSH receptor.

Amino Acid Sequence↗
PubMed1991

Role of N-terminal region of the thyrotropin (TSH) receptor in signal transduction for TSH or thyroid stimulating antibody.

To identify the site(s) on the thyrotropin (TSH) receptor that interacts with TSH or thyroid stimulating antibody (TSAb), we examined the effect of the synthetic TSH receptor peptide (termed N2 peptide, No. 35-50) on the cAMP accumulation induced by TSH or TSAb. Preincubation of bovine TSH with N2 peptide resulted in a significant and dose-dependent decrease in cAMP accumulation. This decrease was not observed when bovine TSH was preincubated with P1 peptide, which was used as a control (No. 398-417). In contrast, the N2 peptide did not affect TSAb activity in immunoglobulin fractions from three TSAb-positive patients with Graves' disease. P1 peptide also had no effect on TSAb activity. These results suggest that the N-terminal region of the TSH receptor is important for TSH action, and also that TSAb activity cannot be suppressed only by the application of the synthetic peptide corresponding to the N-terminal region.

Animals↗
PubMed1991

Thyroid stimulating activity of rabbit antibodies toward the human thyrotropin receptor peptide.

We have produced antibodies against a peptide corresponding to the unique N-terminal segment (amino acid residues 29-57) in the extracellular domain of the human thyrotropin (TSH) receptor by immunizing it to rabbits, and evaluated for their thyroid stimulating antibody (TSAb), thyroid stimulation blocking antibody (TSBAb) and TSH-binding inhibitor immunoglobulin (TBII) activities. Antibody raised in rabbit B showed significant TSAb activity but not TSBAb activity. In contrast, antibody raised in rabbit A lacked TSAb activity but possessed TSBAb activity. None of these antibodies had TBII activity. These results indicate that TSH receptor antibody can successfully mimic the action of TSH and also suggest that the N-terminal region of TSH receptor is substantially associated with both TSAb and TSBAb activities, but not parallel to TBII activity.

Amino Acid Sequence↗