Biomedical subjects

Masakatsu Takanashi

Publications and source records attributed to Masakatsu Takanashi.

10 recordsLinked to original sources

PubMed2006

Generation of the novel monoclonal antibody against TLS/EWS-CHOP chimeric oncoproteins that is applicable to one of the most sensitive assays for myxoid and round cell liposarcomas.

The fusion oncoproteins, TLS-CHOP and EWS-CHOP, are characteristic markers for myxoid and round cell liposarcomas (MLS/RCLS). Especially, the peptide sequence of 26 amino acids corresponding to the normally untranslated CHOP exon 2 and parts of exon 3 (5'-UTR) is a unique structure for these chimeric proteins. In this report, we have generated monoclonal antibodies against the unique peptide sequence of TLS/EWS-CHOP oncoproteins. These antibodies reacted with TLS-CHOP fusion protein, but not reacted with normal TLS and CHOP proteins by Western blot analysis. In addition, one of the antibodies also recognized the chimeric oncoprotein in archival paraffin-embedded tissue samples of MLS/RCLS. The oncoprotein was detectable by the antibody even in the paraffin-embedded tissue samples whose mRNAs were too degraded to be detected by a nested reverse transcription-polymerase chain reaction-based assay. Thus, the molecular assay using the novel antibody is expected to be one of the most sensitive diagnostic assays for MLS/RCLS.

Adult↗

PubMed2005

Detection of fusion genes in sarcomas using paraffin-embedded tissues.

Many sarcomas are characterized by specific recurrent chromosomal translocations resulting in gene fusions. The genes involved in almost all of these translocations have been cloned, greatly changing sarcoma diagnosis. At the biological level, these chromosomal translocations produce highly specific fusion genes that encode key molecules for tumor development. The clinical correlation between these translocation-derived genetic markers and discrete histopathological entities has been remarkable. Today, detection of fusion genes plays a crucial role in the diagnosis of sarcomas that harbor atypical clinical or pathological presentations. The focus of this brief review is the recent impact that cytogenetic and molecular detection of these translocations has had on sarcoma diagnosis using paraffin-embedded sections.

Animals↗

PubMed2004

Myocardial fibrosis and diastolic dysfunction in deoxycorticosterone acetate-salt hypertensive rats is ameliorated by the peroxisome proliferator-activated receptor-alpha activator fenofibrate, partly by suppressing inflammatory responses associated with the nuclear factor-kappa-B pathway.

OBJECTIVES: We sought to clarify that a peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activator inhibits myocardial fibrosis and its resultant diastolic dysfunction in hypertensive heart disease, as well as to investigate whether inflammatory mediators through the nuclear factor (NF)-kappa-B pathway are involved in the effects. BACKGROUND: Patients with hypertensive heart disease often have diastolic heart failure without systolic dysfunction. Meanwhile, it has been well established in atherosclerosis that PPAR-alpha activation negatively regulates early inflammation. In hypertensive hearts, however, it is still unclear whether PPAR-alpha activation inhibits inflammation and fibrosis. METHODS: Twenty-one rats were randomly separated into the following three groups: deoxycorticosterone acetate (DOCA)-salt hypertensive rats treated with a PPAR-alpha activator, fenofibrate (80 mg/kg/day for 5 weeks); DOCA-salt rats treated with vehicle only; and uni-nephrectomized rats as normotensive controls. RESULTS: Fenofibrate significantly inhibited the elevation of left ventricular end-diastolic pressure and the reduction of the magnitude of the negative maximum rate of left ventricular pressure rise and decline, corrected by left ventricular pressure (-dP/dt(max)/P), which are indicators of diastolic dysfunction. Next, fenofibrate prevented myocardial fibrosis and reduced the hydroxyproline content and procollagen I and III messenger ribonucleic acid expression. Finally, inflammatory gene expression associated with NF-kappa-B (interleukin-6, cyclooxygenase-2, vascular cell adhesion molecule-1, and monocyte chemoattractant protein-1), which is upregulated in DOCA-salt rats, was significantly suppressed by fenofibrate. Activation of NF-kappa-B and expression of I-kappa-B-alpha in DOCA-salt rats were normalized by fenofibrate. CONCLUSIONS: A PPAR-alpha activator reduced myocardial fibrosis and prevented the development of diastolic dysfunction in DOCA-salt rats. The effects of a PPAR-alpha activator may be mediated partly by prevention of inflammatory mediators through the NF-kappa-B pathway. These results suggest that treatment with PPAR-alpha activators will improve diastolic dysfunction in hypertensive heart disease.

Animals↗

PubMed2004

Endothelin-1-induced cardiac hypertrophy is inhibited by activation of peroxisome proliferator-activated receptor-alpha partly via blockade of c-Jun NH2-terminal kinase pathway.

BACKGROUND: Peroxisome proliferator-activated receptor-alpha (PPAR-alpha) is a lipid-activated nuclear receptor that negatively regulates the vascular inflammatory gene response by interacting with transcription factors, nuclear factor-kappaB, and AP-1. However, the roles of PPAR-alpha activators in endothelin (ET)-1-induced cardiac hypertrophy are not yet known. METHODS AND RESULTS: First, in cultured neonatal rat cardiomyocytes, a PPAR-alpha activator, fenofibrate (10 micromol/L), and PPAR-alpha overexpression markedly inhibited the ET-1-induced increase in protein synthesis. Second, fenofibrate markedly inhibited ET-1-induced increase in c-Jun gene expression and phosphorylation of c-Jun and JNK. These results suggest that this PPAR-alpha activator interferes with the formation and activation of AP-1 protein induced by ET-1 in cardiomyocytes. Third, fenofibrate significantly inhibited the increase of ET-1 mRNA level by ET-1, which was also confirmed by luciferase assay. Electrophoretic mobility shift assay revealed that fenofibrate significantly decreased the ET-1-stimulated or phorbol 12-myristate 13-acetate-stimulated AP-1 DNA binding activity, and the nuclear extract probe complex was supershifted by anti-c-Jun antibody. Fourth, 24 hours after aortic banding (AB) operation, fenofibrate treatment significantly inhibited left ventricular hypertrophy and hypertrophy-related gene expression pattern (ET-1, brain natriuretic peptide, and beta-myosin heavy chain mRNA) in AB rats. CONCLUSIONS: These results suggest that PPAR-alpha activation interferes with the signaling pathway of ET-1-induced cardiac hypertrophy through negative regulation of AP-1 binding activity, partly via inhibition of the JNK pathway in cultured cardiomyocytes. We also revealed that fenofibrate treatment inhibited left ventricle hypertrophy and phenotypic changes in cardiac gene expression in AB rats in vivo.

Animals↗

PubMed2004

Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats.

Exercise training improves the aging-induced downregulation of myosin heavy chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in the regulation of cardiac contraction and relaxation. Thyroid hormone receptor (TR), a transcriptional activator, affected the regulation of gene expression of MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling contributes to a molecular mechanism of exercise training-induced improvement of MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged heart are affected by exercise training, using the hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo old, swimming training for 8 wk). Trained aged rats showed improvement in cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart were significantly lower in sedentary aged rats than in sedentary young rats and were significantly higher in trained aged rats than in sedentary aged rats. The activity of TR DNA binding to the transcriptional regulatory region in the alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and thyroxine levels were altered in association with changes in the myocardial TR protein levels. These findings suggest that exercise training improves the aging-induced downregulation of myocardial TR signaling-mediated transcription of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of cardiac function in trained aged hearts.

Aging↗

PubMed2004

The peroxisome proliferator-activated receptor alpha activator fenofibrate inhibits endothelin-1-induced cardiac fibroblast proliferation.

Endothelin-1 has been known to promote tissue fibrosis. We previously reported in our animal experiments that a peroxisome proliferator-activated receptor alpha (PPARalpha) inhibited cardiac fibrosis with suppression of endothelin-1 production, and it was also reported that PPARalpha activation suppressed the production of c-jun, which is a component of activator protein-1. The objective of this study is to clarify on the in vitro level that PPARalpha activators inhibited cardiac fibroblast proliferation via their suppressive action on c-jun expression. We investigated the effects of the PPARalpha activator fenofibrate (10 microM) on DNA synthesis in neonatal rat cardiac fibroblasts by [H]thymidine incorporation. The [H]thymidine incorporation in cardiac fibroblasts showed an increase of 1.1-fold by endothelin-1 (10(-8) M) stimulation. Fenofibrate treatment showed significant inhibition of [3H]thymidine incorporation in both endothelin-1-stimulated and non-stimulated fibroblasts. Additionally, we also evaluated mRNA expressions of c-jun and c-fos in the fibroblasts by the reverse transcription-polymerase chain reaction method. Fenofibrate treatment markedly reduced c-jun mRNA expression, whereas it did not affect c-fos mRNA expression. In conclusion, we demonstrated that the PPARalpha activator fenofibrate inhibited endothelin-1-induced proliferation of cardiac fibroblasts and also inhibited non-stimulated proliferation. This inhibition of proliferation may be caused by up-regulation of p27 by suppressing c-jun expression.

Animals↗

PubMed2004

Establishment of hypoxia inducible factor-1alpha overexpressing cells that produce endothelin-1.

Cardiomyocytes produce endothelin-1. Upregulation of endothelin-1 is induced under hypoxic conditions. It has been reported that tissue hypoxia induces transcriptional factor hypoxia inducible factor-1alpha (HIF-1alpha) expression. It has also been reported that cellular hypoxia leads to HIF-1alpha activation. Previously, we reported that HIF-1alpha bound to the site in the ET-1 gene promoter region. HIF-1alpha is a master transcriptional factor that controls transcriptional activation of a number of genes responsive to cellular hypoxia, including endothelin-1, vascular endothelial growth factor, erythropoietin and glycolytic enzymes. We aimed to establish the HIF-1alpha-overexpressing cardiomyocytes. Because it has been reported that HIF-1alpha is destroyed by the ubiquitin proteasome pathway under normoxic conditions, we constructed the point-mutated HIF-1alpha that was exchanged at 564 proline with alanine. The point-mutated HIF-1alpha is not destroyed by ubiquitin proteasome, because ubiquitin ligase cannot bind to the point-mutated area. This mutated HIF-1alpha was transfected to rat cardiomyocytes. Using the protein extraction from the mutated HIF- 1alpha transfected cells under normoxic conditions, western blot methods with anti-HIF-1alpha antibody showed that the band was detected at 120 kDa, which is of identical size to HIF-1alpha. In the normoxic conditions of the control cardiomyocyte cells, the band was undetectable. The endothelin-1 mRNA was significantly altered in HIF-1alpha-overexpressing cells. These results indicate that we have established the cells overexpressing-HIF-1alpha, and that these cells produce endothelin-1 mRNA.

Animals↗

PubMed2004

Effects of endothelin-1 eyedrops on the retina in rats.

Eye disorder accompanied with chronic retinal microvascular obstruction, such as diabetic retinopathy, exists in many diseases. However, it is difficult to produce this model experimentally in the animal eye. Endothelin-1 eyedrops were prepared in order to examine whether the eyedrops affect the rat retina and whether we can produce an obstruction model. Endothelin-1 eyedrops diluted by artificial tears in seven stages from 4 x 10(-5) M to 4 x 10(-11) M were arranged. We administered this solution three times a day in the left eye of male Sprague-Dawley rats. Artificial tears alone were applied to the right eye as a control vehicle. After 2 weeks, rats were sacrificed under anesthesia and the retinal tissues were isolated. As an index to the action of endothelin- 1 eyedrops to the retina, the expressions of endothelin-A (ETA) and endothelin-B (ETB) receptors in the retina were compared in both eyes. Frozen sections of the retina were immunostained to reveal the distribution of the ETA and ETB receptors. We also examined ETA and ETB mRNA expression by quantitative real-time polymerase chain reaction. As a result, the expressions of ETA and ETB receptors are reduced with both immunostaining and the mRNA levels in the left eye, in which endothelin-1 eyedrops were applied at 4 x 10(-5) M. It is suggested that endothelin-1 eyedrops affected the retina and the possibility of producing the experimental model of chronic microvascular obstruction in the rat retina.

Administration, Topical↗

PubMed2003

Overexpression of cadherins suppresses pulmonary metastasis of osteosarcoma in vivo.

Osteosarcoma by nature shows aggressive pulmonary metastasis; however, the underlying molecular mechanisms remain unclear. We previously showed that N-cadherin and cadherin-11 (OB-cadherin), which are highly expressed in normal osteoblasts, are anomalously expressed in human osteosarcoma (Kashima et al., Am J Pathol 1999;155:1549-55). In the present study, we examined the role of cadherins in osteosarcoma metastasis using the mouse osteosarcoma cell line Dunn and its highly metastatic subline LM8. Oligonucleotide array and RT-PCR analyses demonstrated that Dunn and LM8 cells did not express appreciable levels of several members of the cadherin family, and Western blot analysis confirmed that Dunn and LM8 cells did not express P-cadherin, E-cadherin, N-cadherin or cadherin-11 protein. We therefore investigated the functional consequences of cadherin overexpression on cell migration and in vivo metastatic potential of LM8 cells. Several LM8 clones were isolated which expressed exogenous N-cadherin and cadherin-11 localized to the cell membrane and able to bind to beta-catenin. Overexpression of N-cadherin or cadherin-11 in LM8 cells did not affect cell proliferation but caused an inhibitory effect on cell migration in vitro. In vivo analysis showed that N-cadherin- and cadherin-11-overexpressing cells exhibited a marked reduction in their ability to form pulmonary metastases, with significant decreases in lung weight and the number and weight of metastatic lesions, as well as the size and weight of primary lesions at the s.c.-inoculated site. These observations demonstrate that disruption of N-cadherin- and cadherin-11-mediated cell-cell adhesion is critical in the pulmonary metastasis of osteosarcoma.

Animals↗

PubMed2002

Aging-induced decrease in the PPAR-alpha level in hearts is improved by exercise training.

Peroxisome proliferator-activated receptor (PPAR)-alpha, a transcriptional activator, regulates genes of fatty acid (FA) metabolic enzymes. To study the contribution of PPAR-alpha to exercise training-induced improvement of FA metabolic capacity in the aged heart, we investigated whether PPAR-alpha signaling and expression of its target genes in the aged heart are affected by exercise training. We used hearts of sedentary young rat (4 mo old), sedentary aged rat (23 mo old), and swim-trained aged rat (23 mo old, training for 8 wk). The mRNA and protein expression of PPAR-alpha in the heart was significantly lower in the sedentary aged rats compared with the sedentary young rats and was significantly higher in the swim-trained aged rats compared with the sedentary aged rats. The activity of PPAR-alpha DNA binding to the transcriptional regulating region on the FA metabolic enzyme genes, the mRNA expression of 3-hydroxyacyl CoA dehydrogenase (HAD) and carnitine palmitoyl transferase-I, which are PPAR-alpha target genes, and the enzyme activity of HAD in the heart altered in association with changes of the myocardial PPAR-alpha mRNA and protein levels. These findings suggest that exercise training improves aging-induced downregulation in myocardial PPAR-alpha-mediated molecular system, thereby contributing to the improvement of the FA metabolic enzyme activity in the trained-aged hearts.

3-Hydroxyacyl CoA Dehydrogenases↗