Search PubMed⌕ Search

Biomedical subjects

T Nagaya

Publications and source records attributed to T Nagaya.

At least 37 records · Page 2Linked to original sources
PubMed1999

Cleavage of RXRalpha by a lysosomal enzyme, cathepsin L-type protease.

In this study, we characterized a protease responsible for the cleavage of RXRalpha in two human derived cell lines, HepG2 and JEG-3 cells. Electrophoretic mobility shift assay (EMSA) combined with antibody supershift analysis suggested that contamination of cytoplasmic components during nuclear extract preparation could result in complete cleavage of RXRalpha at its N-terminus in JEG-3 cells, while such proteolytic activity was much less evident in HepG2. When the nuclei were purified in the presence of leupeptin, only full-length RXRalpha was found in the extracts prepared from both JEG-3 and HepG2 cells, suggesting a member of cysteine protease family is responsible for the cleavage. The presence of the protease in the cytoplasm, but not in the nucleus, was confirmed by incubating full-length 35S-labeled RXRalpha with each fraction. The cytoplasmic fraction from JEG-3 and HepG2 cells cleaved RXRalpha into smaller sizes with molecular mass of 45, 43, and 31 kD. Immunoprecipitation with antibodies recognizing distinct epitopes indicated that the cleaved RXRalpha with the size of 45 and 43 kD were truncated at N-terminus in which most of the A/B domain was absent. Using a series of protease inhibitors, the enzyme cleaving RXRalpha was characterized as cathepsin L-type protease. The enzyme activity in JEG-3 cells was much higher than that in HepG2 cells. This is the first demonstration that RXRalpha is cleaved by a lysosomal enzyme, cathepsin L-type protease.

Cathepsin L↗
PubMed1999

Dose-response relationships between drinking and serum tests in Japanese men aged 40-59 years.

Alcohol intake per Japanese adult has been increasing year by year. To show biological effects of drinking, the dose-response relationships between alcohol use and serum indices were analyzed in 5919 Japanese men aged 40-59 years. The subjects were classified into nine groups: a nondrinking (ND, n = 1827) group and eight drinking (1D-8D) groups, by self-reported drinking habit. The 1D (the lightest drinking, n = 699), 5D (n = 942), and 8D (the heaviest drinking, n = 46) groups consumed alcohol less than 30 g per week, 25-30 g alcohol per day, and 100 g alcohol per day or more, respectively. Ten serum indices, total cholesterol, HDL cholesterol, triglyceride, total/HDL cholesterol ratio, LDL cholesterol, gamma-glutamyl transpeptidase, aspartate aminotransferase, alanine aminotransferase, glucose, and uric acid, were used. The dose-response analysis was statistically controlled for age, body mass index, smoking, and habitual exercise, and showed that drinking, even a small amount of alcohol, always had both beneficial and adverse effects on humans. However, alcohol less than 30 g per day may be tolerable for middle-aged Japanese men, because it improved serum lipids profile but did not induce apparent liver cell damage, hyperglycemia, or hyperuricemia.

Adult↗
PubMed1999

Body mass index (weight/height2) or percentage body fat by bioelectrical impedance analysis: which variable better reflects serum lipid profile?

We present population data on percentage body fat (%BF) by bioelectrical impedance analysis (BIA) in 12,287 men and 6657 women aged 30-69 y. In addition, we examined relationships among BMI, %BF and serum lipids (total-cholesterol (TC), HDL-cholesterol (HDLC), TC/HDLC ratio, LDL-cholesterol (LDLC) and triglycerides (TG)) in order to determine whether body mass index (BMI, kg/m2) or %BF by BIA better reflected lipid metabolism. Women had larger %BF than men in all age groups. On the other hand, women aged < 60 y had smaller BMI than corresponding men. Regardless of age, BMI was obviously correlated with %BF (r = 0.743-0.924). As previously reported, high BMI and high %BF were strongly associated with high serum TC/HDLC ratio and TG, and low serum HDLC rather than high serum TC and LDLC. Compared with BMI, %BF by BIA was better correlated with the serum indices except for serum HDLC. These results were found in both sexes, and there was a trend in which younger subjects had stronger correlations among BMI, %BF and serum lipids. Consequently, %BF by BIA (an index of body composition) better reflects serum lipid profile than BMI.

Adipose Tissue↗
PubMed1999

Responsiveness to thyroid hormone is enhanced in rat hepatocytes cultured as spheroids compared with that in monolayers: altered responsiveness to thyroid hormone possibly involves complex formed on thyroid hormone response elements.

We previously reported that the expression of type I iodothyronine 5'-deiodinase (5'DI) gene was increased by 3,3,',5-triiodothyronine (T3) in isolated rat hepatocytes when cultured as spherical aggregates (spheroids), whereas this effect was greatly attenuated in conventional monolayer cultures. In the current study, we examined whether the enhanced T3 responsiveness in spheroid cultures extends to other T3-responsive genes. As observed for 5'DI, T3 increased spot 14, malic enzyme and fibronectin messenger RNAs (mRNAs) by fourfold to fivefold in spheroid cultures, while the effect in monolayer cultures was blunted. This difference in T3 responsiveness was also observed when T3-responsive reporters consisting of the luciferase gene under the control of triiodothyronine response element (TRE) were introduced into hepatocytes using a replication-defective adenovirus vector. These results suggest that the factors required for T3-dependent transcriptional activation are preserved in spheroid cultures and that they must exert their effect by interacting with TRE. Maximal binding capacity of nuclear T3 receptor was not different between monolayer and spheroid cultures while the expression of retinoid X receptor-alpha (RXR alpha) mRNA was higher in spheroid cultures compared with that in monolayers. The difference in RXR alpha mRNA expression, together with enhanced proteolytic cleavage in monolayers that we demonstrated recently, may account for the difference in T3 responsiveness between the two hepatocyte culture systems.

Animals↗
PubMed1999

Glucocorticoids increase retinoid-X receptor alpha (RXRalpha) expression and enhance thyroid hormone action in primary cultured rat hepatocytes.

We have previously demonstrated that dexamethasone (DEX) enhances the T3-dependent increase in type I 5'-deiodinase (5'DI) mRNA in primary cultured rat hepatocytes grown as spheroids. Here we report that DEX-enhanced T3-responsiveness also occurs in two other T3-regulated hepatic genes, Spot 14 and malic enzyme. This enhancement was inhibited by pretreatment with cycloheximide and the stability of 5'DI and Spot 14 mRNAs was not affected by DEX. We thus hypothesized that a factor(s) that augments T3-responsiveness is induced by DEX. Among the possible candidates examined, retinoid-X receptor alpha (RXRalpha), which is a main heterodimer partner with T3 receptor, appeared to be involved. Whereas DEX increased the amount of RXRalpha mRNA, it did not affect the expression of other possible factors such as steroid receptor coactivator-1 and the binding protein of cAMP response element-binding protein. Northern and Western blot analysis, and electrophoretic mobility shift assay revealed that DEX increased RXRalpha expression at both the mRNA and protein levels. Maximal increase in RXRalpha protein was achieved with the addition of physiological concentrations of DEX (10(-8) M). To test whether the DEX-induced increase in RXRalpha affects ligand-dependent transcriptional activation through other receptors that form heterodimer with RXR, we examined its effect on the retinoic acid (RA)/RA receptor (RAR) system. Indeed, DEX also enhanced the RA-dependent increase in RARbeta mRNA in a cycloheximide-sensitive manner. Increase in the level of RXRalpha in hepatocytes by infection with the RXRalpha-expressing adenovirus resulted in enhancement of the T3-dependent increase in 5'DI mRNA. These results strongly suggest that the DEX-induced augmentation of T3-responsiveness in cultured hepatocytes is mediated, in part, by the increased expression of RXRalpha.

Animals↗
PubMed1999

Culture in vector-averaged gravity environment in a clinostat results in detachment of osteoblastic ROS 17/2.8 cells.

Studies carried out in space flights and in altered gravitational environments have shown that exposure to altered gravity conditions results in alterations in cellular structure and function. In the present study, we used a clinostat to generate a vector-averaged gravity environment, and evaluated the responses of osteoblast-like ROS 17/2.8 cells subsequent to rotation at 50 r.p.m from 24 to 72 hr. We found that the cells started to detach during the first 24 hr of culture in clinostat, but not in stationary and horizontal rotation (the latter serving as a control for turbulence, shear forces and vibrations). At 24 hr, there was a significant decrease in the number of adherent cells under clino-rotation (2.75 +/- 0.5 x 10(5) in stationary culture versus 2.02 +/- 0.27 x 10(5) under clino-rotation), and 19.8% of adherent cells were trypan-blue positive when cultured in 2% fetal bovine serum. All the detached cells were trypan-blue positive. At 72 hr, the cells became confluent in all three groups. These results suggest that vector-averaged gravity could cause the death of osteoblasts during the first 24 hr of clino-rotation. We hypothesize that this cell death might play a role in the pathogenesis of osteoporotic bone loss as observed in actual space flight.

Animals↗
PubMed1998

Intracellular proteolytic cleavage of 9-cis-retinoic acid receptor alpha by cathepsin L-type protease is a potential mechanism for modulating thyroid hormone action.

We previously reported that the responsiveness of hepatocytes to thyroid hormone is markedly attenuated when they were cultured as monolayers rather than spheroids. To elucidate the mechanisms underlying the altered responsiveness, thyroid hormone receptor auxiliary proteins in the hepatocytes were analyzed by electrophoretic mobility shift assay. The major thyroid hormone receptor auxiliary protein was identified as 9-cis-retinoic acid receptor alpha (RXRalpha) in the hepatocytes regardless of the culture conditions. The cytoplasmic fraction was shown to contain a protease(s) that cleaves RXRalpha at its amino terminus. The presence of the protease in the cytosol, but not in the nucleus, was ascertained by incubating full-length 35S-labeled RXRalpha with each fraction. Using various protease inhibitors, it was shown that cathepsin L-type protease could participate in the cleavage of the RXRalpha. The enzyme activity was much higher in the monolayers than the spheroids. Inhibition of this enzyme activity in the monolayer hepatocyte resulted in the increase of nuclear RXRalpha protein and the augmentation of T3-dependent induction of spot 14 mRNA. These results suggest that the changes in cathepsin L-type protease activity in the cytosol may alter the turnover of RXRalpha in the nucleus and modify the function of steroid receptor superfamilies that heterodimerize with RXRalpha.

Animals↗
PubMed1998

Requirement of corepressor binding of thyroid hormone receptor mutants for dominant negative inhibition.

Resistance to thyroid hormone (RTH) is mainly caused by dominant negative inhibition of wild type thyroid hormone receptor (TR) function due to mutations in the ligand binding domain of the TR beta. Because no RTH mutant was identified in the hinge region of the TR, the contribution of this region for dominant negative inhibition was explored. In transient transfection assays, the dominant negative activity of a RTH-type mutant (P453X) was abolished by an introduction of a mutation into the hinge region (P214R). Although this hinge mutation did not alter homo- or heterodimerization with retinoid X receptor (RXR), its association with nuclear receptor corepressor (N-CoR) was impaired. These results indicate that association of corepressor with the RTH mutants through the hinge region is crucial for their dominant negative activity.

Binding Sites↗
PubMed1998

Inductions of immediate early genes (IEGS) and ref-1 by human chorionic gonadotropin in murine Leydig cell line (MA-10).

The effect of human chorionic gonadotropin (hCG) on the expression of immediate early genes (IEGs) including all members of fos and jun family, and c-myc was studied using mouse Leydig cell line (MA-10 cells) by Northern blot analyses. In addition, the induction of ref-1 which enhances DNA binding of fos/jun proteins was also analyzed. HCG induced a rapid and transient expression of c-fos, fosB, c-jun, junB, junD and c-myc with a peak at 30 min to 1 h. In contrast, induction of fra-1 mRNA was delayed with a peak at 3 hr. However, fra-2 mRNA was immediately increased by hCG with a peak at 1 h. The ref-1 mRNA was expressed before the stimulation and its level was not altered by hCG at least for 8 hr. The differential induction of IEGs and continuous expression of ref-1 mRNA suggest an important role of their gene products on the regulation of Leydig cell function by hCG.

Animals↗
PubMed1998

Activation of transcriptionally active nuclear factor-kappaB by tumor necrosis factor-alpha and its inhibition by antioxidants in rat thyroid FRTL-5 cells.

ABSTRACT Tumor necrosis factor-alpha (TNF-alpha) exerts pleiotropic effects on thyroid follicular cells. However, the intracellular signaling pathway for the TNF-alpha action has not been well elucidated. The present study examined the effects of TNF-alpha on the activation of nuclear factor-kappa B (NF-kappaB) and on the expression of interleukin (IL)-6 gene in rat thyroid FRTL-5 cells. The treatment of the cells with TNF-alpha resulted in the nuclear translocation of p65-p50 heterodimer as well as p50-p50 homodimer NF-kappaBs. The treatment with the antioxidants 20 mM N-acetyl-L-cysteine (NAC) and 10 microM pyrrolidine dithiocarbamate (PDTC) inhibited the TNF-alpha-dependent activation of p65-p50 heterodimer but not the p50-p50 homodimer, indicating that generation of oxidants is required for the activation of the heterodimer NF-kappaB. When the plasmid containing the multimerized NF-kappaB sites upstream of a luciferase reporter gene was transfected into FRTL-5 cells, the treatment with NAC or PDTC prevented the TNF-alpha-dependent increase in the luciferase activities, indicating that the p65-p50 heterodimer is a transcriptionally active NF-kappaB. Accordingly, the TNF-alpha-dependent increase in IL-6 messenger RNA and in secretion of the protein was prevented by the treatment with NAC. These results strongly suggest that TNF-alpha increases the IL-6 gene expression through the activation of NF-kappaB in the thyroid cells, and that antioxidants suppress the TNF-alpha-dependent IL-6 expression by inhibiting the activation of the transcriptionally active NF-kappaB.

Animals↗
PubMed1998

An inhibitory region of the DNA-binding domain of thyroid hormone receptor blocks hormone-dependent transactivation.

We have employed a chimeric receptor system in which we cotransfected yeast GAL4 DNA-binding domain/retinoid X receptor beta ligand-binding domain chimeric receptor (GAL4RXR), thyroid hormone receptor-beta (TRbeta), and upstream activating sequence-reporter plasmids into CV-1 cells to study repression, derepression, and transcriptional activation. In the absence of T3, unliganded TR repressed transcription to 20% of basal level, and in the presence of T3, liganded TRbeta derepressed transcription to basal level. Using this system and a battery of TRbeta mutants, we found that TRbeta/RXR heterodimer formation is necessary and sufficient for basal repression and derepression in this system. Additionally, an AF-2 domain mutant (E457A) mediated basal repression but not derepression, suggesting that interaction with a putative coactivator at this site may be critical for derepression. Interestingly, a mutant containing only the TRbeta ligand binding domain (LBD) not only mediated derepression, but also stimulated transcriptional activation 10-fold higher than basal level. Studies using deletion and domain swap mutants localized an inhibitory region to the TRbeta DNA-binding domain. Titration studies further suggested that allosteric changes promoting interaction with coactivators may account for enhanced transcriptional activity by LBD. In summary, our findings suggest that TR heterodimer formation with RXR is important for repression and derepression, and coactivator interaction with the AF-2 domain may be needed for derepression in this chimeric system. Additionally, there may be an inhibitory region in the DNA-binding domain, which reduces TR interaction with coactivators, and prevents full-length wild-type TRbeta from achieving transcriptional activation above basal level in this chimeric receptor system.

Allosteric Regulation↗
PubMed1998

Advanced onset of menarche and higher bone mineral density depending on vitamin D receptor gene polymorphism.

Bone mineral density (BMD) at distal forearm, and weight and height of healthy Japanese girls aged 18-19 years were measured and their age at menarche was obtained through a questionnaire. A statistically significant association was found between BMD at distal radius and vitamin D receptor (VDR) gene polymorphism at the ApaI site. The age at menarche in the population with Aa genotype was significantly earlier than that in the aa population. In addition, BMD was significantly dependent on the earlier onset of menarche in the population with genotype Aa but not in the population with genotype aa. BMD was also positively associated with the body mass index (BMI) in the population with genotype Aa. Statistical analysis suggested a stronger effect of VDR genotype on age at menarche than on BMI. Thus, we show that VDR gene polymorphism advances the age at menarche and increases BMD in cooperation with age at menarche.

Absorptiometry, Photon↗
PubMed1998

[Functional analysis of nuclear hormone receptors].

Nuclear hormone receptors are transcription factors which regulate gene transcription by lipophilic hormones. The functional domains such as transactivation, DNA binding, ligand binding and dimerization are mapped in the distinct region of the receptors. To analyze these functions, ligand binding assay, gel shift assay, transfection study, GST pull-down method, immunoprecipitation method, two hybrid system and yeast functional assay are employed. These assays have been shown to clarify the molecular basis of the disorders which are due to mutation in the receptors.

Alleles↗
PubMed1998

[Thyroid hormone receptors and the disorders].

Thyroid hormone is essential for normal development and several metabolic pathways. Its action is mediated through its nuclear receptors (thyroid hormone receptors: TRs), which regulate gene transcriptions in a ligand-dependent manner. The abnormality of TR functions is linked with several disorders including resistance to thyroid hormone (RTH), malignancy and euthyroid sick syndrome. For example, mutant TRs were identified to cause RTH and a certain malignancy. The functionally impaired TRs might be related with euthyroid sick syndrome. The analyses of these TRs will elucidate the molecular mechanism or basis for the disorders, and may provide a new insight to find the way of diagnosis and treatment.

Animals↗
PubMed1998

[Pathogenesis of disorder due to thyroid hormone receptor mutation].

Thyroid hormone action is mediated through its nuclear receptor (thyroid hormone receptor; TR). A mutation in the TR causes resistance to thyroid hormone (RTH) with autosomal dominant inheritance. Using T3 binding assay, gel shift assay and transfection study, the pathogenesis of this disorder was elucidated at a molecular level. Mutant TRs lose their T3 binding activity, and inhibit wild type TR action in a dominant negative manner. For this dominant negative inhibition, the preservation of DNA binding and dimerization activities are required. It is indicated that the mutant TR complex competes with wild type TR complex for the DNA binding site to exert dominant negative inhibition. Thus, the functional analyses of mutant TRs are useful to elucidate the molecular basis of this disorder.

Humans↗
PubMed1997

NGFI-B, c-fos, and c-jun mRNA expression in mouse brain after acute carbon monoxide intoxication.

The expression of immediate early genes (IEG) has been documented in the brain after various kinds of insults such as ischemia and hypoxia. To determine whether acute carbon monoxide intoxication (ACOI) might trigger IEG expression, adult ddY mice were subjected to carbon monoxide exposure at a rate of 30 mL/min for 35 seconds. The levels of NGFI-B, c-fos, and c-jun mRNA were determined by Northern blot analysis. A time-course study in the cerebral cortex indicated that the induction of NGFI-B, c-fos, and c-jun mRNA started as early as 15 minutes, reached a peak at 30 minutes, and returned to the basal level at 1 hour after the ACOI. In addition, the temporal feature of the induction of these IEG mRNA in the hippocampus was very similar to that in the cerebral cortex. Examination of brain regions at 30 minutes after the ACOI revealed a significant induction of NGFI-B mRNA in the cerebellum, thalamus-hypothalamus, brainstem. as well as in the cortex and hippocampus, but not in the striatum or olfactory bulb. Furthermore, the neuroanatomical distribution of c-fos mRNA at 30 minutes after the ACOI was very similar to that of the NGFI-B mRNA. The widespread distribution of these IEG in the brain, especially in the cerebellum and brainstem, indicates that the major cause for the triggering of IEG expression in the brain by the ACOI might be a diffuse hypoxia. These findings show for the first time the temporal and spatial expression of IEG in the brain after ACOI.

Acute Disease↗