M Ohishi

Kurume University, Куруме, Fukuoka, Japan

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Publications (18)80.52 Total impact

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    ABSTRACT: The process of receptor-mediated endocytosis is common to a variety of species and cell types. One of the best characterized receptor-ligand systems is the hepatocyte receptor for asialoglycoproteins. We investigated the morphological features of the uptake and intracellular transport of gold-conjugated asialofetuin in isolated rat hepatocyte couplets. We assessed the effects of colchicine, lumicolchicine, cytochalasin B, and chloroquine on the uptake and intracellular transport of asialoglycoproteins. Isolated rat hepatocyte couplets were incubated with gold-conjugated asialofetuin, and transmission electron micrographs of these cells were analyzed to determine the density and distribution of gold particles in the peripheral and pericanalicular areas. Results were analyzed morphometrically. Colchicine significantly inhibited the uptake and intracellular transport of asialoglycoproteins, but did not affect membrane fusion of endocytic compartments in the peripheral area. Lumicolchicine and cytochalasin B had minimal effects on these processes. Chloroquine inhibited the uptake of asialoglycoproteins, but did not affect the intracellular transport of asialoglycoproteins. Results suggest that the microtubule is essential for intracellular movement of endocytosed asialoglycoproteins and receptor recycling, and that endocytic structures in the peripheral regions can fuse in the absence of intact microtubules. We also found that uptake and intracellular transport of asialoglycoproteins were independent of the microfilaments, and the pH gradient in endocytic compartments was important in receptor-mediated endocytosis of asialoglycoproteins.
    Hepatology 12/2005; 21(5):1413 - 1421. · 12.00 Impact Factor
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    ABSTRACT: To identify injured cells in the liver of patients with primary biliary cirrhosis (PBC) and to determine the effects of ursodeoxycholic acid (UDCA) on these cells, we examined the cellular expression of heat shock proteins (HSPs) in PBC both before and after treatment with UDCA. Expression of HSP70 and ubiquitin in PBC livers (n=34) was evaluated immunohistochemically as well as by immunoblot analysis, and compared with chronic viral hepatitis type C (n= 9), primary sclerosing cholangitis (n=8), and controls (n=7). Immunoblot analysis demonstrated a marked expression of HSP70 and ubiquitin in PBC. Immunohistochemical staining for both HSP70 and ubiquitin was observed to be strong in biliary epithelial cells (BECs) and moderate in both hepatocytes and arteries in PBC. Cellular labelling rates for HSP70 and ubiquitin of bile ducts in PBC were significantly higher (p<0.01) than those in chronic viral hepatitis type C, primary sclerosing cholangitis, or controls. The labelling rates for HSP70 and ubiquitin in bile ducts and in hepatocytes were significantly decreased (p<0.01) after treatment with UDCA in PBC. The present data suggest that BECs and hepatocytes significantly express HSPs even in the early stages of PBC, and that UDCA treatment significantly improves their condition. The immunohistochemical evaluation of HSPs is a valid and sensitive means to identify injured cells in PBC.
    Liver International 03/2000; 20(1):78-87.
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    ABSTRACT: Background/Aims: To identify injured cells in the liver of patients with primary biliary cirrhosis (PBC) and to determine the effects of ursodeoxycholic acid (UDCA) on these cells, we examined the cellular expression of heat shock proteins (HSPs) in PBC both before and after treatment with UDCA. Methods: Expression of HSP70 and ubiquitin in PBC livers (n=34) was evaluated immunohistochemically as well as by immunoblot analysis, and compared with chronic viral hepatitis type C (n=9), primary sclerosing cholangitis (n=8), and controls (n=7). Results: Immunoblot analysis demonstrated a marked expression of HSP70 and ubiquitin in PBC. Immunohistochemical staining for both HSP70 and ubiquitin was observed to be strong in biliary epithelial cells (BECs) and moderate in both hepatocytes and arteries in PBC. Cellular labelling rates for HSP70 and ubiquitin of bile ducts in PBC were significantly higher (p<0.01) than those in chronic viral hepatitis type C, primary sclerosing cholangitis, or controls. The labelling rates for HSP70 and ubiquitin in bile ducts and in hepatocytes were significantly decreased (p<0.01) after treatment with UDCA in PBC. Conclusions: The present data suggest that BECs and hepatocytes significantly express HSPs even in the early stages of PBC, and that UDCA treatment significantly improves their condition. The immunohistochemical evaluation of HSPs is a valid and sensitive means to identify injured cells in PBC.
    Liver International 01/2000; 20(1):78 - 87.
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    ABSTRACT: Although hepatitis C virus (HCV) is a major causative agent of hepatocellular carcinoma (HCC), the role of this virus in carcinogenesis is not fully understood. We studied HCV infection and replication by detecting plus- and minus-strand HCV RNA by in situ hybridization (ISH) in surgically resected HCCs and adjacent non-cancerous tissue obtained from 15 anti-HCV antibody-positive patients with HCC. Plus-strand HCV RNA was found in 9 of 15 HCCs. Both plus- and minus-strand HCV RNA were detected in four of these nine patients. In non-cancerous tissues obtained adjacent to the HCC, plus-strand HCV RNA was found in 10 of 15 patients, whereas both plus- and minus-strand HCV RNA were detected in 7 of these 10 patients. The degree of staining by ISH did not correlate with the differentiation of the HCC, histologic classification of the non-cancerous tissue, serum HCV RNA levels, or serum transaminase levels. HCV can be found in and replicates in both hepatoma cells and in non-cancerous hepatocytes in anti-HCV antibody-positive patients with HCC. The direct effects of HCV viral gene products on normal hepatocytes in the development of HCC require additional study.
    Scandinavian Journal of Gastroenterology 05/1999; 34(4):432-8. · 2.33 Impact Factor
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    ABSTRACT: Cyclooxygenase (COX) is a key enzyme in the synthesis of prostanoids. Two isoforms of this enzyme have been identified: COX-1 and COX-2. Recent studies have suggested that COX-2, but not COX-1, may play a role in colorectal tumorigenesis. In the present study, we investigated the expression of COX-2 as well as COX-1 in human hepatocellular carcinoma (HCC) tissues using immunohistochemistry and immunoblotting. Forty-four surgically resected HCC tissues with adjacent nontumorous livers (NTs), involving 17 cases of chronic viral hepatitis and 27 cases of cirrhosis, and 7 surgically resected, histologically normal liver tissues were used. The well-differentiated HCC expressed COX-2 more frequently and strongly than less-differentiated HCC or hepatocytes of NTs. Less-differentiated HCCs expressed less COX-2 than hepatocytes of NTs, which showed scattered, strong COX-2 expression. Histologically normal liver was weakly positive for COX-2. The expression of COX-1 was weaker than that of COX-2 in hepatic neoplastic and non-neoplastic parenchymal cells. An enhanced expression of COX-1 was not observed in well-differentiated HCCs. Immunoblotting also confirmed up-regulation of COX-2, but not COX-1, in well-differentiated HCCs. The present study is the first to demonstrate a high expression of COX-2 in well-differentiated HCC and a low expression in advanced HCC, in contrast to its continuous expression during colorectal carcinogenesis. These findings suggested that COX-2 may play a role in the early stages of hepatocarcinogenesis, but not in the advanced stages, and may consequently be related to HCC dedifferentiation.
    Hepatology 04/1999; 29(3):688-96. · 12.00 Impact Factor
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    ABSTRACT: Asialoglycoproteins are internalized via specific receptors localized on the hepatic parenchymal cells. The role of vacuolar type H+-ATPase (v-ATPase) and vacuolar pH in intracellular transport of asialoglycoproteins was investigated in isolated rat hepatocyte couplets. Two specific inhibitors of v-ATPase, bafilomycin A1 and concanamycin B, were used to inhibit acidification of the vacuolar system. Intracellular transport of asialoglycoproteins was investigated using gold-conjugated asialofetuin with electron microscopy. Fluorescent staining by acridine orange showed that bafilomycin A1 and concanamycin B each inhibited acidification of endocytic compartments. Electron microscopy demonstrated that the number of autophagic vacuoles was increased, and the uptake of gold-conjugated asialofetuin was significantly inhibited by these agents. Vesicle budding and membrane fusion between endocytic structures in the peripheral area and intracellular transport of endocytosed asialofetuin to lysosomes were inhibited by these agents. These results suggest that the intracellular transport of endocytosed asialoglycoproteins to lysosomes depends on acidification of the vacuolar system (vacuolar pH) in hepatocytes.
    Hepatology Research 01/1999; 14(2):105-118. · 2.07 Impact Factor
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    ABSTRACT: The role of vacuolar type H+-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of horseradish peroxidase, a mannose-terminated glycoprotein, was investigated morphologically in isolated rat sinusoidal endothelial cells. Toward this purpose, a specific inhibitor of v-ATPases, bafilomycin A1, was used to inhibit v-ATPases in the vacuolar system. Uptake of horseradish peroxidase was examined by electron microscopy. Fluorescent staining by acridine orange showed that bafilomycin A1 inhibited the acidification of the endocytic compartments. Horseradish peroxidase was taken up via mannose receptors and was distributed in the endocytic structures in the isolated sinusoidal endothelial cells. Uptake of horseradish peroxidase was significantly inhibited by bafilomycin A1, and this finding was confirmed by morphometrical analysis. These results suggest that: a) v-ATPases are necessary for acidification of the endocytic compartments in the sinusoidal endothelial cells and b) the pH gradient between the endocytic compartments and the cytoplasm that is generated by v-ATPases is necessary for the receptor-mediated endocytosis of a mannose-terminated glycoprotein, horseradish peroxidase.
    Liver International 11/1997; 17(5):244-50.
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    ABSTRACT: It is uncertain whether or not apoptosis is involved in the pathogenesis of primary biliary cirrhosis (PBC). The aims of this study were to assess the nuclear DNA fragmentation and expression of Bcl-2 in the biliary epithelial cells (BECs) and in the hepatocytes of PBC. Additionally, the effects of ursodeoxycholic acid (UDCA) on DNA fragmentation and Bcl-2 expression in PBC were evaluated. Liver tissue specimens from 35 PBC patients were examined by in situ nick-end labeling to detect any nuclear DNA fragments, and by immunohistochemistry for Bcl-2. Ten of these patients underwent a second liver biopsy after the treatment with UDCA. Sixteen histologically normal liver tissues and 17 chronic viral hepatitis C (CVHC) samples were chosen as controls. DNA fragmentation in BECs was more frequently found in PBC than in CVHC and more frequently than in the normal controls (both P < .05), and fragmentation in the hepatocytes of PBC more frequently than in normal controls (P < .01). Bcl-2 expression was more frequently found in both the BECs and hepatocytes of PBC than in the controls. UDCA significantly decreased the DNA fragmentation in BECs (P < .05) and the positivity for Bcl-2 in BECs (P < .01), although no significant decrease was found in hepatocytes. In conclusion, de novo Bcl-2 expression in hepatocytes of PBC was shown, and the increased nuclear DNA fragmentation and the Bcl-2 expression both in BECs and in hepatocytes may reflect apoptotic stress, although nuclear DNA fragmentation in BECs did not necessarily represent apoptosis. UDCA showed a potential effect of reducing nuclear DNA fragmentation in BECs.
    Hepatology 05/1997; 25(5):1077-84. · 12.00 Impact Factor
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    ABSTRACT: The role of vacuolar type H(+)-ATPases (v-ATPases) and pH gradient between the endocytic compartments and cytoplasm in the endocytosis of asialoglycoproteins was morphologically investigated in isolated rat hepatocytes using bafilomycin A1, a specific inhibitor of v-ATPases. Fluorescent staining by acridine orange showed that bafilomycin A2 inhibited the acidification of the endocytic compartments. Uptake of gold-conjugated asialofetuin was significantly inhibited by bafilomycin A1. However, bafilomycin A1 did not significantly inhibit uptake of a fluid phase marker, horseradish peroxidase. The number of autophagic vacuoles increased after the bafilomycin A1 treatment. However, materials in the autophagic vacuoles were rapidly degraded after the removal of bafilomycin A1. Results suggest that: (a) v-ATPases are necessary for acidification of the endocytic compartments; (b) the pH gradient between the endocytic compartments and the cytoplasm which is generated by v-ATPases is necessary for the receptor-mediated endocytosis of asialoglycoproteins, and (c) v-ATPases may contribute to the degradation of the materials in autophagic vacuoles.
    Journal of Hepatology 06/1996; 24(5):594-603. · 9.86 Impact Factor
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    ABSTRACT: Four patients with inflammatory myopathy positive for anti-hepatitis C virus are described. One male and three females are included in this report. Mean age of the four patients was 50 years (range 42–60 years). They were referred to our hospital with muscle weakness or myalgia. Active hepatitis C virus infection was confirmed by the detection of hepatitis C virus RNA with the polymerase chain reaction method in sera of these patients. Serum muscle enzymes, such as creatine kinase, lactate dehydrogenase, aspartate aminotransferase, and aldolase were elevated. Inflammation in the muscle tissue was demonstrated in the muscle biopsy specimens from these patients. The patients received prednisolone and symptoms and elevated muscle enzymes were improved. It is suggested that hepatitis C virus might be one of the possible etiologic factors responsible for inflammatory myopathy.
    International Hepatology Communications 12/1995;
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    ABSTRACT: A 25-year-old woman, diagnosed with Sjögren's syndrome at age 20, presented with painful edema of her left neck. Three days later, she additionally complained of bilateral auricular pain, and her nasal cartilage was tender to palpation. She was diagnosed as having phlegmon on the basis of her neck findings. Anti-human cartilage antibodies were demonstrated by indirect immunofluorescence, and the diagnosis of relapsing polychondritis was established. The patient was administered antibiotics and a non-steroidal anti-inflammatory drug, and her symptoms gradually improved. Relapsing polychondritis is one of the possible complications of autoimmune diseases, and infection might be a precipitating factor for this disease.
    Internal Medicine 09/1995; 34(8):768-71. · 0.97 Impact Factor
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    ABSTRACT: A 47-year-old man with primary amyloidosis was admitted with abdominal pain. A new radionuclide liver imaging technique using Technetium-99m diethylenetriamine-pentaacetic acid-galactosyl human serum albumin, was performed and the serum hyaluronate concentration was measured. Although ordinary laboratory tests revealed only slight abnormalities, the uptake of the radiolabelled ligand for hepatocyte asialo-glycoprotein receptors was decreased, and marked hepatomegaly was revealed. Furthermore, the serum hyaluronate level was elevated. Histological examination of a hepatic needle biopsy specimen revealed a marked deposition of amyloid in the hepatic perisinusoidal spaces. These results indicate that this new radionuclide liver imaging technique (using Technetium-99m diethylenetriamine-pentaacetic acid-galactosyl human serum albumin) and the measurement of serum hyaluronate may be useful supplementary tools for identifying amyloid deposition in the hepatic perisinusoidal spaces in patients with amyloidosis.
    Journal of Gastroenterology 07/1995; 30(3):403-7. · 3.79 Impact Factor
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    ABSTRACT: The process of receptor-mediated endocytosis is common to a variety of species and cell types. One of the best characterized receptor-ligand systems is the hepatocyte receptor for asialoglycoproteins. We investigated the morphological features of the uptake and intracellular transport of gold-conjugated asialofetuin in isolated rat hepatocyte couplets. We assessed the effects of colchicine, lumicolchicine, cytochalasin B, and chloroquine on the uptake and intracellular transport of asialoglycoproteins. Isolated rat hepatocyte couplets were incubated with gold-conjugated asialofetuin, and transmission electron micrographs of these cells were analyzed to determine the density and distribution of gold particles in the peripheral and pericanalicular areas. Results were analyzed morphometrically. Colchicine significantly inhibited the uptake and intracellular transport of asialoglycoproteins, but did not affect membrane fusion of endocytic compartments in the peripheral area. Lumicolchicine and cytochalasin B had minimal effects on these processes. Chloroquine inhibited the uptake of asialoglycoproteins, but did not affect the intracellular transport of asialoglycoproteins. Results suggest that the microtubule is essential for intracellular movement of endocytosed asialoglycoproteins and receptor recycling, and that endocytic structures in the peripheral regions can fuse in the absence of intact microtubules. We also found that uptake and intracellular transport of asialoglycoproteins were independent of the microfilaments, and the pH gradient in endocytic compartments was important in receptor-mediated endocytosis of asialoglycoproteins.
    Hepatology 06/1995; 21(5):1413-21. · 12.00 Impact Factor
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    ABSTRACT: We have investigated the localization of HCV in the liver and the expression of Fas antigen, involved in hepatocellular death through apoptosis, to examine the relation between HCV infection and Fas antigen cellular expression. The localization of HCV was investigated by in situ hybridization (ISH), and the expression of Fas antigen was determined by immunohistochemistry using an anti-Fas monoclonal antibody, in 20 surgically-resected liver specimens from anti-HCV positive patients. The Methylgreen/Pyronin Y technique clearly showed well-preserved tissue RNA in 8 of 20 livers. ISH determined the co-localization of signals for plus and minus strands of HCV-RNA in the cytoplasm of hepatocytes. Fas antigen was more strongly expressed on the plasma membrane of the periportal hepatocytes than on the plasma membrane of the centrilobular hepatocytes, even when HCV-RNA was found to be widely distributed in the hepatocytes throughout the liver lobule. Furthermore, Fas antigen positive hepatocytes were frequently observed close to the area of piecemeal necrosis associated with a high incidence of apoptosis. The results suggested that the expression of Fas antigen does not occur by HCV infection alone, but probably requires several other factors
    Acta histochemica et cytochemica official journal of the Japan Society of Histochemistry and Cytochemistry 01/1995; · 1.48 Impact Factor
  • International Hepatology Communications - INT HEPTAOL COMM. 01/1995; 3.
  • Hepatology 01/1995; 22(4). · 12.00 Impact Factor
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    ABSTRACT: To assess whether the hepatocyte asialoglycoprotein receptors are affected in patients with autoimmune hepatitis, the function of the hepatocyte asialoglycoprotein receptor was investigated in patients with autoimmune hepatitis or chronic hepatitis C. A new radionuclide liver imaging technique, Technetium-99m diethylenetriamine-pentaacetic acid-galactosyl human serum albumin, was applied to evaluate the function of the receptor. A Receptor Index (LHL 15) was calculated by dividing the radioactivity of the liver region-of-interest (ROI) by that of the liver plus heart ROI 15 min after radiolabeled ligand injection. Serum albumin, prothrombin time, hepaplastin test and plasma retention rate of indocyanine green at 15 min were not significantly different between patients in the autoimmune hepatitis group and chronic hepatitis C group. In addition, the Receptor Index of patients with autoimmune hepatitis, in whom the asialoglycoprotein receptor is a candidate target antigen for autoimmune response, was similar to that of patients with chronic hepatitis C. These results indicate that the hepatocyte receptor for asialoglycoproteins is not affected in patients with autoimmune hepatitis.
    The Kurume Medical Journal 02/1994; 41(3):131-6.
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    ABSTRACT: The function of hepatocyte asialoglycoprotein receptors was analyzed in normal subjects, in patients with Wilson's disease and in patients with chronic hepatitis C. A new radionuclide liver imaging, using Technetium-99m diethylenetriamine-pentaacetic acid-galactosyl human serum albumin, was applied to evaluate the function of the receptor, and a Receptor Index, LHL 15, which was calculated by dividing the radioactivity of the liver region-of-interest (ROI) by that of the liver plus heart ROI at 15 min after the radiolabeled ligand injection, was evaluated. The values were similar between the patients with Wilson's disease and patients with chronic hepatitis C. Hepatocyte receptors for asialoglycoproteins were not affected specifically in patients with Wilson's disease, suggesting that the defect in patients with Wilson's disease involves steps other than receptor-mediated endocytosis of asialoceruloplasmin via asialoglycoprotein receptor.
    The Kurume Medical Journal 02/1994; 41(3):137-41.