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Biomedical subjects

Y Sasaguri

Publications and source records attributed to Y Sasaguri.

At least 55 records · Page 3Linked to original sources
PubMed1997

The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast.

To investigate the biological function of hepatitis C virus (HCV)-NS5A, the NS5A was fused at its N-terminus with the DNA binding domain (DBD) of yeast transcriptional activator GAL4 (GAL4-DBD). The GAL4-DBD alone had no transcriptional activation function. However, a mutant of the GAL4-DBD/NS5A fusion protein, in which 129 amino acid residues were deleted from the N-terminus of NS5A, exhibited strong transcriptional activation in yeast cells, bearing the Escherichia coli lacZ reporter gene encoding the beta-galactosidase under the transcriptional control of GAL4 promoter and TATA box. Further mutational analysis of NS5A revealed that the region between the amino acid residues 130 to 352 were critical for optimal level of transactivation. This region includes two acidic domains and one proline-rich region which have been shown to be involved in the function of several transcriptional activators.

Gene Expression Regulation, Viral↗
PubMed1997

Expression of stem cell factor in human aortic endothelial and smooth muscle cells.

It has been confirmed that the receptor protein encoded by the c-kit proto-oncogene is expressed by cells of the hematopoietic, gonadal, pigment, and mast cell lineages and that its ligand, stem cell factor (SCF), is mainly expressed in their microenvironment. In a previous study we investigated the expression of the c-kit gene in human aortic endothelial cells (EC). In the present study we investigated the expression of SCF in human aortic EC and smooth muscle cells (SMC). Reverse transcription (RT)-PCR and Northern blot analyses showed that both human arterial EC and SMC expressed mRNA specific for the SCF gene. In addition, tissue-specific expression of the SCF gene was confirmed by in situ hybridization in the EC and the SMC. Western blot analysis and immunocytochemistry showed evidence of production of SCF protein in both the EC and the SMC. These results indicate the existence of mast cell-SMC interaction and of an autocrine loop of c-kit and its ligand on the surface of EC, suggesting that the interaction between c-kit protein and SCF may play an important role in metabolism of arterial wall and in the pathogenesis of atherosclerosis in the arterial intima.

Animals↗
PubMed1997

Apoptosis induced by cadmium.

Cell death resulting from cadmium (Cd) intoxication has been confirmed to occur through apoptosis by morphological and biochemical studies. However it is still not clear whether Cd itself or metallothionein (MT) induced by Cd is the major factor responsible for the apoptosis. Although apoptosis is inducible by exposure of cells to various stimuli, the common pathway involved is generally accepted to be activation of endonucleases that induce internucleosomal cleavage of DNA, resulting in the 'ladder' formation observed upon agarose gel electrophoresis and the chromatin condensation seen by electron microscopy. Cd does not seem to activate the endonuclease in vitro. However, Cd itself can be associated with apoptosis through indirect oxidative stress by inhibition of antioxidant enzymes and possible interaction with zinc finger protein. In addition to the direct effect of Cd, MT appears to play dual roles in apoptosis induction: one as a Cd carrier by which Cd accumulates in the nucleus, and the other as an inhibitor of zinc finger proteins, which include transcriptional factors related to apoptosis such as the product of the apoptosis resistance gene A20. In this review, we demonstrated that the mode of cell death following Cd exposure is associated with intracellular movement of Cd and MT. A possible mechanism for Cd-induced apoptosis is also discussed.

Journal Article↗
PubMed1996

Characterization of the nuclear localization signal and subcellular distribution of hepatitis C virus nonstructural protein NS5A.

Hepatitis C virus (HCV) has a positive strand RNA genome that codes for a polyprotein that is processed co-translationally and post-translationally into three structural and at least seven nonstructural (NS) proteins. To investigate the function of NS5A, a recombinant vaccinia virus was constructed in which the NS5A gene was cloned under the control of T7 promoter and encephalomyocarditis virus 5'-untranslated region (EMCV-UTR) for cap-independent translation in mammalian cells. In addition, the NS5A gene was also cloned under the control of cytomegalovirus (CMV) early promoter. The NS5A expressed in monkey kidney (CV-1) cells was located predominantly in the cytoplasm. Using immunohistochemical analysis, the subcellular distribution of NS5A in liver biopsy samples from chronic HCV-infected patients was also found to be in the cytoplasm. However, the NS5A protein has a stretch of positively charged domain in the vicinity of proline and valine residues, (PPRKKRTVV), characteristic of a nuclear localization signal (NLS), in the COOH-terminal half of the protein. To investigate whether the putative NLS of NS5A is functional, chimeric expression plasmids were constructed in which regions containing the NLS were fused to the N-terminus of the E. coli beta-galactosidase (E. coli beta-Gal). The expression of the fusion proteins in CV-1 cells resulted in their nuclear localization, indicating that the putative NLS is functional in targeting the heterologous protein, E. coli beta-Gal, to the nucleus, although the native NS5A is retained in the cytoplasm.

Animals↗
PubMed1996

A possible mechanism for the stimulation of metalloproteinase production in human aortic intimal smooth muscle cells by linoleic acid hydroperoxide.

To approach the mechanism of the stimulating effect of linoleic acid hydroperoxide on the production of matrix metalloproteinases (MMPs) in human aortic intimal smooth muscle cells (ISMC), we investigated the effect of the hydroperoxide on the cytosolic level of Ca2+. Linoleic acid hydroperoxide provoked an increase in the cytosolic Ca2+ level, but it had no effect on the level of inositol phosphates (IPs) in these cells, in contrast with the effect of platelet-derived growth factor (PDGF), which elevated the level of both Ca2+ and IPs in these cells. A23187, a calcium ionophore, stimulated ISMC to produce matrix prometalloproteinase 1. These results indicate that linoleic acid hydroperoxide stimulates the production of MMPs in ISMC by elevation of the cytosolic Ca2+ level without the intervention of IPs. In addition, we found that the hydroperoxide has no effect on the binding of PDGF to its specific receptor on ISMC.

Aorta, Thoracic↗
PubMed1996

Degradation of interleukin 1beta by matrix metalloproteinases.

Matrix metalloproteinases (MMPs) and interleukin 1 (IL-1) are implicated in inflammation and tissue destruction, where IL-1 is a potent stimulator of connective tissue cells to produce the extracellular matrix-degrading MMPs. Here, we report that IL-1beta, but not IL-1alpha, is degraded by MMP-1 (interstitial collagenase), MMP-2 (gelatinase A), MMP-3 (stromelysin 1), and MMP-9 (gelatinase B). This degradation was effectively blocked by tissue inhibitor of metalloproteinases (TIMP)-1. When IL-1beta was treated with MMPs it lost the ability to enhance the synthesis of prostaglandin E2 and pro-MMP-3 in human fibroblasts. The primary cleavage site of IL-1beta by MMP-2 was identified at the Glu25-Leu26 bond. These results suggest that IL-1beta stimulates connective tissue cells to produce MMPs, but activated MMPs in turn negatively regulate the activity of IL-1beta.

Amino Acid Sequence↗
PubMed1996

Apoptosis of human kidney 293 cells is promoted by polymerized cadmium-metallothionein.

Transformed human kidney cells (293 cells) exposed to 12.5 to 37.5 microM CdCl(2) showed apoptosis as confirmed by characteristic electron microscopic features, a ladder on gel electrophoresis of extracted DNA, and fragmentation of nucleosomes as detected by enzyme linked immunosorbent assay (ELISA). Higher concentrations of Cd were less effective in inducing apoptosis. Furthermore, addition of the protein extract from the serum-free medium used for Cd-exposure promoted apoptosis exhibiting the same features as that after Cd-exposure. The apoptosis induced by the protein was dose-dependent. The molecular weight of the protein (Cd-protein) was shown to be 40 kDa by gel filtration. Two-dimensional electrophoresis revealed the Cd-protein as a single spot with a molecular weight of 6 kDa and pI of 4.5. Competitive ELISA showed that the Cd-protein reacted with anti-metallothionein antibody. The present findings suggest that apoptosis is induced not only by Cd itself but also by polymerized metallothionein molecules (MT) released from cells into the medium.

Animals↗
PubMed1996

Morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate.

The purpose of this study was to investigate the mechanism of cell death in chondrocytes of the growth plate. In the degenerative chondrocyte zone of the growth plate, apoptotic chondrocytes were defeated by the in situ nick end labelling method, by DNA analysis in agarose gel, and by electron microscopy. The results of the in situ nick end labelling method and the occurrence of a ladder pattern of DNA in agarose gel analysis indicated the activation of endogenous endonucleases, resulting in DNA fragmentation. Electron micrographs showed the early morphological changes associated with apoptosis. This report presents both morphological and biochemical evidence for apoptosis in the terminal hypertrophic chondrocytes of the growth plate. These data suggest that apoptosis of degenerative chondrocytes may play an important role in the control of normal and pathological endochondral ossification.

Animals↗
PubMed1996

The significance of epidermal growth factor receptor and matrix metalloproteinase-3 in squamous cell carcinoma of the oral cavity.

Surgical specimens from 65 patients with squamous cell carcinoma (SCC) of the oral cavity were examined immunohistochemically. The clinicopathological significance of the expression of epidermal growth factor receptor (EGFR) and matrix metalloproteinase-3 (MMP-3) was assessed. Among the 65 tumours, 20(30.8%) and 37(56.9%) tested positively for EGFR and MMP-3, respectively. A positive correlation between the expression of EGFR and MMP-3 was found. The expression of EGFR in oral SCCs was associated with an advanced T stage of the primary tumour, an advanced pathological stage, and a high incidence of neck metastasis. In addition, MMP-3 was primarily expressed at the advancing front of cancer with a diffuse invasive mode. Thus, overexpression of MMP-3 was associated with an advanced pathological stage, a diffuse invasive mode, and a high incidence of neck metastasis. The analysis of MMP-3 expression is useful to evaluate the pathological status of tumours. Because EGFR-overexpressed tumour should produce larger amounts of MMP-3 in vivo, a close examination of oral SCC for expression of EGFR and MMP-3 should be helpful to predict their malignant potential.

Adult↗
PubMed1996

Interleukin-4 stimulates rheumatoid synovial fibroblasts to express matrix metalloproteinase-1 (tissue collagenase) and histamine H1 receptor mRNA.

In the present study, we investigated the effect of interleukin-4 (IL-4) on metalloproteinase 1 (MMP-1/tissue collagenase) production in human rheumatoid synovial fibroblasts. Northern blot analysis revealed that addition of IL-4 with or without histamine stimulated the cells to increase the amount of proMMP-1 mRNA, and the IL-4 with histamine addition resulted in a 3.3-fold increase compared with histamine only. Furthermore, IL-4 itself stimulated the expression of histamine H1 receptor mRNA. These results suggest that IL-4 may play an important role in joint destruction in RA.

Arthritis, Rheumatoid↗
PubMed1996

Endometrial stromal sarcoma of the uterus with rhabdoid features.

A case of endometrial stromal sarcoma of the uterus with rhabdoid features, occurring in a 57 year old woman is reported. Electron microscopy and immunohistochemistry revealed that the rhabdoid cells contained intermediate filaments which were positive for vimentin, cytokeratin, alpha-smooth muscle actin, and muscle specific actin, but not for myoglobin and desmin. This indicated that the tumor in this case differed somewhat from the three rhabdoid tumors and an endometrial stromal sarcoma with rhabdoid differentiation previously reported and that, therefore, these tumors were heterogeneous.

Biomarkers, Tumor↗
PubMed1996

Intracranial plasma-cell granuloma: a case report.

A 57-year-old woman presented with right-sided ptosis and diplopia. CT scans showed a tumor in the right cerebellopontine angle attached to the tentorium. Subtotal resection was conducted prior to local irradiation (20 Gy). Histological examination of the resected specimen indicated a diagnosis of plasma-cell granuloma. Diffuse thickening of the tentorium and falx were seen on follow-up MR imaging six years after the operation. Only eleven cases of intracranial plasma-cell granuloma have been reported in the literature. This paper reports a new case with unusual recurrence on neuroimagings.

Brain↗
PubMed1995

Ovarian mucinous cystadenocarcinoma with sarcoma-like mural nodules.

A rare case of ovarian mucinous cystadenocarcinoma with sarcoma-like mural nodules is reported. Macroscopically, nodular or granular lesions were scattered over the inner surface of a huge, multilocular tumor in the left ovary. The histopathological features were consistent with those of previously documented cases, except significant squamous metaplasia in the present case. The pleomorphic sarcoma-like cells showed a positive reaction for vimentin and alpha-antichymotrypsin but were negative for carcinoembryonic antigen and common epithelial membrane antigen by immunohistochemistry. Electron microscopically, the tumor cells had neither desmosomes nor secretory granules, but they possessed abundant intermediate filaments. Both immunostaining and ultrastructure suggested that the sarcoma-like nodules were derived from reactive proliferation of fibrohistiocytic cells. The fibrohistiocytic reaction seemed to occur in response to hemorrhage, because hemorrhage was reported to be associated with the mural nodules in most of the previous cases as well as ours.

Cystadenocarcinoma, Mucinous↗
PubMed1995

Mild emphysema: a novel method using formalin-fixed lungs for computed tomography and pathological analyses.

We evaluated a novel method of computed tomography (CT) analysis using formalin-fixed lungs of autopsy cases with mild emphysema. Eight formalin-inflated lungs (FILs) obtained at autopsy were examined using CT after draining off the formalin and air inflation with an air pump, and subjected to pathological study including pathological scoring of emphysema and microscopic image analysis (MIA). Satisfactory CT examination was carried out within 5 h of lung fixation. The mean alveolar area determined by MIA correlated highly with the lung volume (r = 0.845) and CT score (r = 0.722). This method is simple compared with conventional polyethylene glycol fixation for CT and enables CT examination of resected lungs without anxiety about biohazards, Mild emphysema can be detected by MIA.

Aged↗
PubMed1995

Histamine-stimulated production of matrix metalloproteinase 1 by human rheumatoid synovial fibroblasts is mediated by histamine H1-receptors.

The purpose of this study was to investigate the role of histamine in human rheumatoid synovial fibroblasts in the production of factors responsible for tissue remodelling and cartilage breakdown in rheumatoid arthritis. We examined the effects of histamine of tritiated thymidine incorporation, production of matrix metalloproteinase-1 (MMP-1), histamine H1-receptor expression, phosphoinositide metabolism and intracellular calcium ion concentration ([Ca2+]i) in human rheumatoid synovial fibroblasts. Tritiated thymidine incorporation studies demonstrated that histamine markedly stimulated the proliferation of rheumatoid synovial fibroblasts. Immunofluorescence and Northern blot analyses revealed that proMMP-1 production was also stimulated by histamine. The levels of inositol phosphates and [Ca2+]i in the cells were elevated in response to histamine, indicating that the cells expressed histamine H1-receptors; and Northern blot analysis indicated that these H1-receptors were up-regulated by histamine. In in situ hybridization, large amounts of histamine H1-receptor mRNA were also detected in rheumatoid synovial tissue. These results suggest that the interaction between H1-receptor expression in rheumatoid synovial fibroblasts and histamine secretion by mast cells and macrophages in the affected sites is an important event responsible for tissue remodelling and joint destruction in rheumatoid arthritis.

Arthritis, Rheumatoid↗
PubMed1995

Anti-proliferative effect of 2-methoxyestradiol on cultured smooth muscle cells from rabbit aorta.

The proliferation of cultured smooth muscle cells from rabbit aorta was inhibited markedly by 2-methoxyestradiol, considerably by 2-hydroxyestradiol, and only slightly by 17 beta-estradiol. The effects at doses from 1 to 40 nmol/ml were cytostatic rather than cytocidal. 2-Methoxyestradiol could be useful to suppress excessive proliferation of arterial smooth muscle cells, and thus to prevent the progression of atherosclerosis.

2-Methoxyestradiol↗
PubMed1995

Atherogenic change in the thoracic aorta of the senescence-accelerated mouse.

Biochemical and histological changes in the thoracic aorta of the senescence-accelerated prone mouse were examined in comparison with those of the senescence-accelerated resistant mouse. At 3 and 5 months of age, the lipid peroxide level in the thoracic aorta was higher in the former than in the latter. At 5 months of age, levels of both total cholesterol and triglycerides in the aorta were higher in the former than in the latter, and vice versa for the level of aortic phospholipids. At 5 months of age, the prone mice showed macrophage invasion beneath the endothelial cells of the thoracic aorta, whereas the resistant ones did not.

Aging↗