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Biomedical subjects

N Sawada

Publications and source records attributed to N Sawada.

At least 109 records · Page 6Linked to original sources
PubMed1996

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1).

The LEC rat is an inbred mutant strain which spontaneously develops liver injury and subsequent liver cancer. Liver injury in LEC rats has recently been shown to be closely related to abnormal copper accumulation in the liver. Previously, we reported that LEC rat hepatocytes lose their growth potential, probably allowing selective growth of preneoplastic cells. In this study, to elucidate the effects of copper accumulation on the growth activity of LEC rat hepatocytes, we examined the growth activity and the expression of p53 and p21(waf 1/cip 1) in the livers of LEC rats fed on either a control or a low-copper diet. Potential for cell proliferation of hepatocytes obtained from normal diet fed LEC rats was almost comparable to that of the cells from age-matched Sprague-Dawley (SD) rats. Northern blot analysis showed that the expression of p53 and p21(waf 1/cip 1) was significantly high in the livers of LEC rats fed a control diet, while the expression of p53 and p21(waf 1/cip 1) in the LEC rats fed a low-copper diet was as low as that of SD rat livers. Western blot analysis consistently showed that the amount of p21(waf 1/cip 1) bound to the nuclear matrix scaffold of the LEC rat liver was reduced by feeding a low-copper diet. These findings suggest that abnormal accumulation of copper induced the expression of p53 and p21(waf 1/cip 1), resulting in the inhibition of cell proliferation of LEC rat hepatocytes.

Animals↗
PubMed1996

Expression of p21(waf-1/cip-1) is significantly induced in the livers of LEC rats with chronic liver injury.

It is reported that hepatocytes isolated from LEC rats with chronic liver injury show reduced growth activity in primary culture. To elucidate the molecular basis of this phenomenon, we examined expression of p21(waf-1/ciP-1) and p27, cyclin-dependent kinase inhibitors, by northern blot analysis. The expression of p21(waf-1/cip-1 ) in the LEC rat liver was 3-fold higher than that of age-matched SD rat liver, while there was no significant difference in p27 expression level. Western blot analysis also revealed a significant increase in p21(waf-1/cip-1) in the nuclear matrix fraction of the LEC rat liver. Immunohistochemically, p21(waf-1/cip-1) was detected in the nuclei of normal LEC rat hepatocytes, but not in those of hepatocellular carcinoma cells, suggesting selective growth of neoplastic hepatocytes.

Animals↗
PubMed1996

Comparison between the distribution of 7H6 tight junction-associated antigen and occludin during the development of chick intestine.

7H6 antigen is a novel tight junction-associated protein first identified in our laboratory with a monoclonal antibody generated by immunizing mice with a bile canaliculus-rich membrane fraction obtained from adult rat liver. Although the antigen is found preferentially at the tight junction of epithelial cells in various organs in adult mammals, the relationship between 7H6 antigen and other tight junction proteins remains obscure. In the present study we examined the relationship between the immunohistochemical distribution of 7H6 antigen and occludin in chick intestinal epithelial cells during development in vivo, since occludin is the only known membrane protein composing tight junctions which is found thus far exclusively in avian tissues. 7H6 antigen was found as coarse dots along the basolateral cell membrane of intestinal epithelium in the chick embryo, whereas it was found as a fine dashed line along the cell border in the chicken. In contrast, occludin was found linearly together with ZO-1 at the cell border of intestinal epithelia both in the chick embryo and chicken. The possible significance of such different localization is briefly discussed.

Animals↗
PubMed1996

[Long-term survivor (15 years) following central nervous system involvement in B-cell lymphoma].

A 61-year-old man with multiple subcutaneous, ileocecal and neurologic manifestations was reported. Histological examination of subcutaneous and ileocecal mass showed non-Hodgkin's lymphoma (diffuse medium cell type [LSG classification]), B-cell type. Headache, somnolence and incontinence of urine were considered owing to the CNS involvement by lymphoma cells. The cranial CT findings showed diffuse spread involving the lateral and third ventricle and hypothalamus with adjacent edema. Then he was treated by VEMP regimen and repeated intrathecal MTX injections. The neurologic remission with improvement of cranial CT findings was obtained and he has been free of the disease for 15 years.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1995

Transforming growth factor-beta 1 downregulates dexamethasone-induced tetranectin gene expression during the in vitro mineralization of the human osteoblastic cell line SV-HFO.

In the present study, we examined the regulation of tetranectin gene expression using a human osteoblastic cell line, SV-HFO, that undergoes mineralization upon treatment with dexamethasone. We found that the expression of tetranectin and alkaline phosphatase mRNA was induced by dexamethasone treatment as evidenced by Northern blotting. When transforming growth factor-beta 1 (TGF-beta 1) was added together with dexamethasone to the SV-HFO cell cultures, the mineralization process was markedly suppressed and the expression of tetra nectin and alkaline phosphatase was downregulated in a dose-dependent manner. These results demonstrate that the expression of tetranectin in these osteoblastic cells is regulated by dexamethasone and TGF-beta 1 and that tetranectin expression is tightly linked to the process of mineralization.

Biomarkers, Tumor↗
PubMed1995

Development of the blood-retinal barrier in vitro: formation of tight junctions as revealed by occludin and ZO-1 correlates with the barrier function of chick retinal pigment epithelial cells.

To elucidate the molecular mechanisms of the blood-retinal barrier (BRB), we examined chick retinal tissues histochemically using antibodies against tight junction proteins such as ZO-1, 7H6 antigen, and occludin. Retinal pigment epithelial (RPE) cells in situ in chickens and late chick embryos expressed all of the tight junctional proteins examined, showing that tight junctions seal the cell borders of chick RPE cells in vivo. On the other hand, RPE cells isolated from late chick embryos and transferred in vitro did not express occludin, ZO-1 and 7H6 antigen. The effects of differentiation-inducing agents, such as retinoic acid, dexamethasone and dimethyl sulfoxide (DMSO) were tested. Only DMSO induced an increase in transepithelial electrical resistance (TER) in a time-dependent manner. Under supplementation with DMSO, immunofluorescently demonstrable occludin and ZO-1 were induced progressively at cell borders in parallel with the increase in TER that occurred with decreases in inulin and dextran permeability. Electron microscopically tight junction-like junctional apparatus were induced in RPE cells. These results indicated that tight junctions of RPE cells play an important role in the formation of the BRB.

Animals↗
PubMed1995

Estrogen-depleted condition induces apoptosis of rat mammary cancer cells after entering the S-phase of the cell cycle.

To elucidate the relationship of estrogen-depleted condition to apoptosis and tumor regression, 7,12-dimethylbenz[a]anthracene-induced mammary cancers of Sprague-Dawley rats were ovariectomized or treated with the anti-estrogenic agent epitiostanol after which proliferative activity and the incidence of apoptosis were investigated using the nick end labeling method, agarose gel electrophoresis of DNA, electron microscopy, the BrdU-labeling method and mitotic count. Tumor regression was found after 7-day treatment, and apoptosis induced by the agent on the 3rd day was clearly shown in both agarose gel electrophoresis of DNA and electron microscopy, which are two major methods used to judge apoptosis. The incidence of apoptosis revealed by the nick end labeling method reached its maximum, about threefold the control level, on the 3rd day of epitiostanol treatment compared with control tumors (P < 0.01). The incidence of the cells incorporating BrdU reached its maximum of 9.7% on the 2nd day of the treatment, while the incidence in tumors without treatment was 7.5% (P < 0.05). Subsequently, the incidence of apoptosis was reduced after 7-day treatment, and the incidence of BrdU-positive cells was significantly reduced to about 3% after 5-day treatment. The incidence of mitosis did not change until the 3rd day of the treatment and was reduced after 5-day treatment. Similarly, chronological changes of the incidences of BrdU-labeled cells, apoptotic cells and mitosis were observed in the tumors after ovariectomy. BrdU-labeled apoptotic bodies were detected in the tumors on the 3rd day in epitiostanol-treated rats that received a 6-hr bolus of BrdU before sacrifice.(ABSTRACT TRUNCATED AT 250 WORDS)

Androstanols↗
PubMed1995

Glucocorticoids induce mineralization coupled with bone protein expression without influence on growth of a human osteoblastic cell line.

Mineralization was induced by glucocorticoid treatment in a human osteoblastic cell line derived from normal bone in vitro, designated SV-HFO, immortalized with simian virus 40 (CHIBA, H. et al. (1993). Jpn. J. Cancer Res., 84: 290-297). Mineralization was revealed by electron microscopy, von Kossa staining and electron spectroscopic analysis, which indicated that the Ca/P ratio was approximately 1.70, corresponding to the value of hydroxyapatite. The effect was dose- (10(-8)-10(-6) M) and time-dependent (days 7-28), was greatest at day 28, and was preceded by expression of alkaline phosphatase (ALP) and osteopontin (OPN). The ALP activity induced was highest at day 7, whereas OPN reached its highest level at day 28. When the induction of ALP activity was inhibited by 10(-4) M levamisole, mineralization of SV-HFO cells by glucocorticoid treatment was markedly reduced, suggesting that elevated ALP activity in the early phase is important in the mineralization of human osteoblastic cells. Glucocorticoid treatment did not alter cell proliferation. These results indicated that glucocorticoids play crucial roles in the formation of mineralized matrix in human osteoblasts by inducing differentiation of SV-HFO cells without modulating their proliferative activity.

Alkaline Phosphatase↗
PubMed1994

Detection of the c-met proto-oncogene product in normal skin and tumours of melanocytic origin.

The proto-oncogene c-met product (c-MET) is a receptor tyrosine kinase and functions as a receptor for hepatocyte growth factor (HGF). Although the function of c-MET has yet to be fully clarified, HGF stimulates the phosphorylation of tyrosyl residues on c-MET and triggers the signal transduction pathways, resulting in a contribution to the malignant progression of melanonocytes with synergic factors such as basic fibroblast growth factor and mast cell growth factor. Using immunohistochemical methods, we have studied the localization of c-MET in normal skin and various melanocytic tumours. c-MET was detected in keratinocytes, melanocytes, sebaceous cells, and other cells of the skin. In particular, basal melanocytes almost always showed nuclear labelling. Melanocytic naevi generally revealed predominantly nuclear staining of cells in the epidermis, whereas only a few cases showed a distinct cytoplasmic localization of c-MET in dermal naevus cells. The distribution pattern of c-MET in melanoma cells was basically similar to that of benign lesions, although the numbers tested were small. Cultured human melanoma cells also showed predominantly nuclear labelling, but were unresponsive to exogenous c-MET ligand HGF. Treatment with the glucosidase inhibitor castanospermine caused accumulation of protein at 220 kD, without diminishing the amount of normally-processed 190-kD c-MET. Although there was no significant difference in c-MET distribution between benign and malignant melanocytic lesions, it is suggested that malignant transformation of melanocytes may be associated with loss of response to HGF or other growth-regulating factors.

Blotting, Western↗
PubMed1994

Localization of the 7H6 antigen at tight junctions correlates with the paracellular barrier function of MDCK cells.

An important function of the tight junction is to act as a selective barrier to ions and small molecules, although no molecule responsible for the barrier function has been identified. Here we report evidence that the localization of the 7H6 tight junction-associated antigen identified in our laboratory at tight junctions correlates with the barrier function of MDCK cells. MDCK cells in a confluent monolayer possessed a polarized morphology, having an apical plasma membrane and a basolateral membrane, which is separated from the former by tight junctions. MDCK cells expressed both ZO-1 and 7H6 antigen at tight junctions, which maintain a tight barrier as determined by resistance to lanthanum permeation and high transepithelial electrical resistance (TER, 1500 ohm-cm2). The 7H6 antigen disappeared as tight junctions became permeable to lanthanum with a decrease in TER (below 100 ohm-cm2) due to treatment with metabolic inhibitors (10 microns antimycin A and 10 mM 2-deoxyglucose) for 30 min, while leaving ZO-1 at the cell border. The 7H6 antigen appeared at tight junctions again as TER recovered to a high level (1500 ohm-cm2) within 3 h after withdrawal of metabolic inhibitors. In addition, we found that 7H6 antigen is a phosphorylated protein and that phosphorylation is closely related to the localization of 7H6 antigen in the area of tight junctions.

Adenosine Triphosphate↗
PubMed1994

Sequential changes in intercellular junctions between hepatocytes during the course of acute liver injury and restoration after thioacetamide treatment.

Sequential changes of gap junctions (GJs), tight junctions (TJs) and desmosomes (DSs) between hepatocytes during restorative proliferation were studied in rats after a single intraperitoneal administration of 200 mg/kg thioacetamide (TAA). Antibody against connexin 32 was used to demonstrate GJs; simultaneously the changes in TJs and DSs were studied using antibodies against 7H6 protein and desmoplakins. Propidium iodide and bromodeoxyuridine were used to recognize necrotic and proliferative cells. GJs were evenly distributed in early necrotic hepatocytes at 16 h after TAA treatment, then disappeared from necrotic and surrounding cells at 24 h. At 48 h, GJs had disappeared completely from hepatocytes in whole liver lobules, while many hepatocytes were heavily labelled with BrdU. At 72 h, GJs reappeared, firstly in perinecrotic areas. At 96 h after treatment, when the injured areas had disappeared and restorative proliferation ceased, GJs were distributed evenly throughout the lobules. Immunohistochemical observation of GJs in centrilobular, perinecrotic and periportal areas after TAA-induced hepatic necrosis was confirmed by counting the number of connexin-32-positive spots in the respective areas. TJs and DSs disappeared from necrotic cells at 24 h, but then increased between 24 and 48 h in perinecrotic areas, though the increased intensity of these junctions was more evident at 48 h. At 72 h, localization of TJs and DSs returned to normal. These results suggest that during the course of acute hepatic injury, GJs (cell-cell communication) behave differently from other intercellular junctions.

Animals↗
PubMed1994

Rapid appearance of connexin 26-positive gap junctions in centrilobular hepatocytes without induction of mRNA and protein synthesis in isolated perfused liver of female rat.

In the adult rat liver, the gap junction protein connexin 32 (Cx32) is evenly distributed in hepatocytes within the liver lobules, while connexin 26 (Cx26) is preferentially localized in hepatocytes in periportal zones. We report here that Cx26-positive gap junctions rapidly appear in the centrilobular hepatocytes of adult female rat livers during a 30 minute perfusion of the liver through the hepatic portal vein with a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and oxygen transport FC-43 fluid at a physiological flow rate without any changes in the distribution of Cx32. The change in the localization of Cx26 was closely related to that of E-cadherin, and there was no significant increase in the amounts of Cx26 protein and mRNA. The appearance of Cx26 in the centrilobular hepatocytes was inhibited by treatment with cytoskeleton disruptors such as colchicine and cytochalasin B, and intracytoplasmic transport inhibitors such as brefeldin A. The liver perfusion induced the appearance of Cx26 in the centrilobular hepatocytes only in female rats. Estrogen treatment of ovariectomized rats caused the appearance of both Cx26 and E-cadherin in centrilobular hepatocytes not only in the perfused liver but also in the non-perfused liver. Our results indicate that in the rat liver: (a) the localization of Cx26 can be modulated by a post-translational mechanism; (b) E-cadherin may play an important role in the formation of gap junctions composed of Cx26; and (c) the formation of gap junctions is regulated by female steroid hormones.

Animals↗
PubMed1994

Cellular polarity correlates with vimentin distribution, but not to keratin, in human renal cell carcinoma cells in vitro.

To investigate the relationships among vimentin, keratin and cellular polarity, reorganized glands composed of renal cell carcinoma cells were investigated in vitro. We employed two different three-dimensional collagen gel culture methods, the "floating sandwich method (FSM)" and the "dispersed embedding method (DEM)." The cells composed of reorganized glands formed by FSM culture showed distinct polarity. In contrast, the cellular polarity of the cells formed by DEM culture was less distinct. Keratin was evenly distributed throughout the cytoplasm regardless of the culture method. In contrast, in reorganized glands obtained by FSM culture, vimentin was distinctly polarized at the basal pole while glands obtained by DEM culture showed random distribution of vimentin. These results suggest that there is a close relationship between cell polarity and intracellular localization of vimentin, and that there may be different mechanisms controlling the organization of the two intermediate filament (IF) networks.

Carcinoma, Renal Cell↗
PubMed1994

Hormonal regulation of connexin 43 expression and gap junctional communication in human osteoblastic cells.

We have recently shown that connexin 43 (Cx43), a major gap junction protein in osteoblasts, is expressed with an increase in cell density (CHIBA, H. et al. (1993). Cell Struc. Funct., 18: 419-426). In the present study, we examined what kinds of hormones and cytokines regulate the gap junction protein in osteoblastic cells, using a human osteoblastic cell line (SV-HFO) after reaching a confluent density to avoid influence of cell proliferation. Either retinoic acid (RA) or transforming growth factor-beta 1 (TGF-beta 1) induced the Cx43 expression of SV-HFO cells, as revealed by Northern blot analysis and immunocytochemistry. These modulators also increased gap junctional intercellular communication, in terms of the extent of dye transfer. On the other hand, 1 alpha, 25-dihydroxyvitamin D3 did not influence the Cx43 expression and gap junctional intercellular communication of the cells. These results suggest that RA and TGF-beta might maintain bone tissue as an organized tissue in vivo by increasing intercellular communication of osteoblastic cells.

Calcitriol↗
PubMed1994

The LEC rat: a model for human hepatitis, liver cancer, and much more.

The LEC rat is an inbred mutant strain with spontaneous hepatitis isolated from Long-Evans rats. Since approximately 40% of LEC rats die of fulminant hepatitis, the rat serves an animal model for studying the pathogenesis and treatment of human fulminant hepatitis. The remaining 60% of LEC rats survive and develop chronic (prolonged) hepatitis and subsequently develop liver cancer. Therefore, the LEC rat serves an important animal model for studying the significance of chronic hepatitis in the development of human liver cancer, which often develops in association with chronic hepatitis. The LEC rat can also be used as an animal model of Wilson's disease, since recent studies have disclosed high copper accumulation in the liver and low ceruloplasmin concentration in the serum of this mutant rat.

Animals↗
PubMed1993

Endothelin-1 induces contraction of bile canaliculi in isolated rat hepatocytes.

To examine the effects of endothelin-1(ET-1) on bile canalicular contractions, rat hepatocytes were isolated by a collagenase perfusion technique and cultured on p-n-p-vinylbenzenyl-D-lactonamide-coated dishes under serum-free conditions. The frequency of bile canalicular contractions was expressed by the percentage of the number of bile canaliculi(BC) that contracted compared to the total number of BC observed for 10 min. The treatment of the hepatocytes with ET-1 increased the frequency of contraction of the BC in a dose-dependent manner up to 2nM, control: 10.4%; 0.5nM ET-1:31.6%; 1nM ET-1:54.0%; 2nM ET-1:58.0%; 4nM ET-1:58.9%. The contraction, once it occurred, lasted for rest of the observation period. We also observed a transient increase in the concentration of intracellular Ca in cultured hepatocytes upon the addition of ET-1 to the medium. These results suggest that ET-1 plays an important role in the mortility of BC in vivo.

Animals↗
PubMed1993

Increase in portal flow induces c-myc expression in isolated perfused rat liver.

We examined expression of the c-myc oncogene in isolated perfused livers to elucidate the mechanisms involved in triggering the proliferation of hepatocytes after partial hepatectomy (PH). During perfusion with a 1:1 mixture of Dulbecco's modified Eagle's medium and the oxygen transport fluid FC-43, rat livers were two-thirds resected (PH), and further perfused for 1 1/2 hours at the physiological portal flow throughout the perfusion. Expression of c-myc in the perfused livers with PH(+) was ten times higher than in those with PH(-). Furthermore, expression of c-myc in the PH(-) livers perfused with a threefold volume of the physiological portal flow was 5-10 times higher than that in the livers perfused with the physiological portal flow. The perfusates that passed through the livers did not induce DNA synthesis of primary cultured hepatocytes. These results suggest that an increase in the portal flow volume may act as a trigger for hepatocyte proliferation after PH.

Animals↗