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Biomedical subjects

Masaru Harada

Publications and source records attributed to Masaru Harada.

At least 19 recordsLinked to original sources
PubMed2007

Keratin 18 overexpression but not phosphorylation or filament organization blocks mouse Mallory body formation.

UNLABELLED: Several human liver diseases are associated with formation of Mallory body (MB) inclusions. These hepatocyte cytoplasmic deposits are composed primarily of hyperphosphorylated keratins 8 and 18 (K8/K18). Feeding a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-containing diet is a well-established mouse model of MBs. K8 overexpression, and K8-null or K18-null mouse models, indicate that a K8-greater-than-K18 expression ratio is critical for MB formation. We used established transgenic mouse models to study the effect of K18 overexpression and phosphorylation, or keratin filament disorganization, on MB formation. Five mouse lines were used: nontransgenic, those that overexpress wild-type K18 or the K18 phosphorylation mutants Ser33-to-Ala (S33A) or Ser52-to-Ala (S52A), and mice that overexpress K18 Arg89-to-Cys, which causes collapse of the keratin filament network into dots. DDC feeding induced MBs in nontransgenic livers, but MBs were rarely seen in any of the K18 transgenic mice. Wild-type K18 overexpression protected mice from DDC-induced liver injury. CONCLUSION: K18 overexpression protects mice from MB formation and from DDC-induced liver injury, which supports the importance of the K8-to-K18 ratio in MB formation. The effect of K18 on MB formation is independent of hepatocyte keratin filament organization or K18 Ser33/Ser52 phosphorylation. Keratin filament collapse, which is a major risk for acute liver injury, is well tolerated in the context of chronic DDC-mediated liver injury.

Animals↗
PubMed2006

Endothelial progenitor cell transplantation improves the survival following liver injury in mice.

BACKGROUND & AIMS: Neovascularization, which is vital to the healing of injured tissues, recently has been found to include both angiogenesis, which involves in mature endothelial cells, and vasculogenesis, involving endothelial progenitor cells. The aim of this study was to clarify the possible roles of endothelial progenitor cells during postnatal liver regeneration. METHODS: To determine how endothelial progenitor cells participate in liver regeneration, human or mouse endothelial progenitor cells were transplanted into the mice with carbon tetrachloride-induced acute liver injury. Survival rate of the mice in endothelial progenitor cell-transplanted and control groups was calculated. Separately, livers removed temporally from both groups were examined. RESULTS: At an early stage, transplanted human endothelial progenitor cells were seen mainly surrounding hepatic central veins where hepatocytes showed extensive necrosis; later, the transplanted cells formed tubular structures. More of these cells were observed along hepatic sinusoids. Transplantation of human or mouse endothelial progenitor cells improved survival of the mice following liver injury (from 28.6% to 85.7%, P < .0005 and from 33.3% to 80.0%, P < .001, respectively), accompanied by greater proliferation of hepatocytes. Human endothelial progenitor cells produced several growth factors, such as hepatocyte growth factor, transforming growth factor-alpha, heparin-binding epidermal growth factor-like growth factor, and vascular endothelial growth factor, and also elicited endogenous growth factors. CONCLUSIONS: Endogenous and exogenous growth factors and direct neovascularization after endothelial progenitor cell transplantation promoted liver regeneration, thus improving survival after liver injury. Transplantation of endothelial progenitor cells could represent a new therapeutic strategy for promoting liver regeneration.

Animals↗
PubMed2005

Keratin-containing inclusions affect cell morphology and distribution of cytosolic cellular components.

Many neurodegenerative diseases are characterized by the presence of protein aggregates bundled with intermediate filaments (IFs) and similar structures, known as Mallory bodies (MBs), are observed in various liver diseases. IFs are anchored at desmosomes and hemidesmosomes, however, interactions with other intercellular junctions have not been determined. We investigated the effect of IF inclusions on junction-associated and cytosolic proteins in various cultured cells. We performed gene transfection of the green fluorescent protein (GFP)-tagged cytokeratin (CK) 18 mutant arg89cys (GFP-CK18 R89C) in cultured cells and observed CK aggregations as well as loss of IF networks. Among various junction-associated proteins, zonula occludens-1 and beta-catenin were colocalized with CK aggregates on immunofluorescent analyses. Similar results were obtained on immunostaining for cytosolic proteins, 14-3-3 zeta protein, glucose-6-phosphate dehydrogenase and DsRed. E-cadherin, a basolateral membrane protein in polarized epithelia, was present on both the apical and basolateral domains in GFP-CK18 R89C-transfected cells. Furthermore, cells containing CK aggregates were significantly larger than GFP-tagged wild type CK18 (GFP-WT CK18)-transfected or non-transfected cells (P < 0.01) and sometimes their morphology was significantly altered. Our data indicate that CK aggregates affect not only cell morphology but also the localization of various cytosolic components, which may affect the cellular function.

14-3-3 Proteins↗
PubMed2005

A human T-cell lymphotropic virus type-1 (HTLV-1) carrier complicated with various autoimmune diseases including primary biliary cirrhosis.

A 47-year-old woman with primary biliary cirrhosis and scleroderma was examined at our hospital for a 1-week history of non-resolving fever, arthralgia, myalgia, muscle weakness and fatigue. A diagnosis of systemic lupus erythematosus was made based on arthralgia, low leukocyte count, low lymphocyte count, low serum concentration of complements, positive anti-nuclear antibody and positive anti-double-strand-DNA antibody. She was negative for anti-U1RNP antibody, but positive for anti-Jo1 antibody, and her initial serum concentration of creatine phosphokinase was elevated. We diagnosed her as having overlap syndrome with scleroderma, systemic lupus erythematosus and possible polymyositis associated with primary biliary cirrhosis. Prednisolone rapidly improved her symptoms. Lobulated leukocytes were observed in her peripheral blood specimen. She was positive for anti-HTLV-1 antibody, but Southern blot hybridization did not confirm monoclonal integration of HTLV-I proviral DNA in her peripheral blood. This suggests the possibility of a relationship between HTLV-1 infection and various autoimmune disorders including primary biliary cirrhosis.

Journal Article↗
PubMed2005

The Wilson disease protein ATP7B resides in the late endosomes with Rab7 and the Niemann-Pick C1 protein.

Wilson disease is a genetic disorder characterized by the accumulation of copper in the body due to a defect of biliary copper excretion. Although the Wilson disease gene has been cloned, the cellular localization of the gene product (ATP7B) has not been fully clarified. Therefore, the precise physiological action of ATP7B is still unknown. We examined the distribution of ATP7B using an anti-ATP7B antibody, green fluorescent protein (GFP)-ATP7B (GFP-ATP7B) and ATP7B-DsRed in various cultured cells. Intracellular organelles were visualized by fluorescence microscopy. The distribution of ATP7B was compared with that of Rab7 and Niemann-Pick C1 (NPC1), proteins that localize in the late endosomes. U18666A, which induces the NPC phenotype, was used to modulate the intracellular vesicle traffic. GFP-ATP7B colocalized with various late endosome markers including Rab7 and NPC1 but not with Golgi or lysosome markers. U18666A induced the formation of late endosome-lysosome hybrid organelles, with GFP-ATP7B localized with NPC1 in these structures. We have confirmed that ATP7B is a late endosome-associated membrane protein. ATP7B appears to translocate copper from the cytosol to the late endosomal lumen, thus participating in biliary copper excretion via lysosomes. Thus, defective copper ATPase activity of ATP7B in the late endosomes appears to be the main defect of Wilson disease.

Adenosine Triphosphatases↗
PubMed2005

Vascular endothelial growth factor is involved in angioedema associated with eosinophilia.

Angioedema associated with eosinophilia is a disorder characterized by angioedema and eosinophilia. However, the pathogenesis of this disorder has not been fully understood. We experienced 4 patients with angioedema associated with eosinophilia. All patients were young, 3 were female and 1 was male. All patients revealed edema of the limbs and eosinophilia. The symptom was rapidly improved after the initiation of low or medium dose of prednisolone. We evaluated the serum concentration of interleukin 5 (IL-5) and the plasma concentration of vascular endothelial growth factor (VEGF) in 1 patient. Both cytokines were markedly elevated before the treatment and decreased after the treatment. Angioedema associated with eosinophilia is not so rare, and IL-5 and VEGF are involved in the pathogenesis of this disease.

Adult↗
PubMed2005

Scleroderma and repeated spontaneous abortions treated with vitamin E--a case report--.

A 33-year-old woman was referred to our hospital due to repeated spontaneous abortions and positive autoantibodies. She had noticed Raynaud's phenomenon 13 years earlier. We diagnosed scleroderma based on the presence of Raynaud's phenomenon, proximal scleroderma, presence of anti-centromere antibodies, and histological findings on skin biopsy. Neither lupus anticoagulant nor anti-cardiolipin-beta2-glycoprotein 1 antibody was detected. We administered tocopherol nicotinate. Five months after the initiation of the treatment, she became pregnant and later delivered a healthy baby.

Abortion, Habitual↗
PubMed2005

Cholestasis in a rat model of graft-versus-host disease is accompanied by alteration of the expression and distribution of tight-junction-associated proteins.

Intrahepatic cholestasis has been recognized as one of the characteristic features of the hepatic manifestations in graft-versus-host disease (GVHD). Tight junctions (TJs) play crucial roles in bile formation and alterations of TJs in hepatocytes and/or biliary epithelial cells (BECs) that cause intrahepatic cholestasis. To assess the changes of TJs in hepatocytes and BECs in a rat model of GVHD, we examined the localization of TJ-associated proteins, 7H6 and zonula occludens (ZO)-1. GVHD was induced by injecting spleen cells of parental strain rats (Brown Norway) into non-irradiated (Brown Norway x Lewis) F1 hybrid rats. Untreated F1 hybrid rats served as controls. Double-labeled immunofluorescent staining for 7H6 and ZO-1 was performed in liver sections. In control rats, immunostaining for 7H6 and ZO-1 colocalized to the apical site of BECs and was continuous along the bile canaliculi. In GVHD, 7H6 expression was decreased in BECs and was discontinuous along the bile canaliculi. On the other hand, the intensity of ZO-1 staining in hepatocytes increased and did not change in BECs compared with that of control rats. Changes in TJ-associated proteins of both hepatocytes and BECs may reflect the immunopathogenesis of GVHD-associated intrahepatic cholestasis.

Animals↗
PubMed2005

Causal relationship between hepatitis C virus core and the development of type 2 diabetes mellitus in a hepatitis C virus hyperendemic area: a pilot study.

Hepatitis C virus (HCV) infection may contribute to the development of type 2 diabetes mellitus. However, this association at the population level remains unclear. The aim of this pilot study is to examine the relationship between HCV infection and the development of type 2 diabetes mellitus in an HCV hyperendemic area where we conducted health screenings in 1995. After 7 years of follow-up, we evaluated the relative risk of the development of type 2 diabetes mellitus in anti-HCV-inhabitants. Among 71 subjects free of diabetes mellitus in 1995, 7 developed type 2 diabetes mellitus during the 7-year follow-up evaluation. Overall, anti-HCV-positive subjects were nearly 3-fold as likely as anti-HCV-negative subjects to develop diabetes mellitus, but this difference was not statistically significant (p=0.08). After stratification of the anti-HCV-positive group according to serum HCV core titer, a significant increase in the incidence of diabetes was seen in subjects with high titer of HCV core compared to anti-HCV-negative subjects (p=0.02; relative hazard, 5.60; 95% confidence interval, 1.41 to 37.42). In conclusion, HCV infection potentially has a significant role in the development of type 2 diabetes mellitus at the population level. Further large-scale studies are needed to confirm these preliminary findings.

Diabetes Mellitus, Type 2↗
PubMed2004

Aggregation and loss of cytokeratin filament networks inhibit golgi organization in liver-derived epithelial cell lines.

Intermediate filaments are one of the three major cytoskeletons. Some roles of intermediate filaments in cellular functions have emerged based on various diseases associated with mutations of cytokeratins. However, the precise functions of intermediate filament are still unclear. To resolve this, we manipulated intermediate filaments of cultured cells by expressing a mutant cytokeratin. Arginine 89 of cytokeratin18 plays an important role in intermediate filament assembly. The expression of green fluorescent protein-tagged cytokeratin18 arg89cys induced aggregations and loss of the intermediate filament network composed of cytokeratins in liver-derived epithelial cells, Huh7 and OUMS29, but only induced the formation of cytokeratin aggregates and did not affect the intermediate filament network of endogenous vimentin in HEK293. The expression of this mutant affected the distribution of Golgi apparatus and the reassembly of Golgi apparatus after perturbations by nocodazole or brefeldin A in both Huh7 and OUMS29, but not in HEK293. Our data show that loss of the original intermediate filament network, but not the existence of cytokeratin aggregates, induces redistribution of the Golgi apparatus. The original intact intermediate filament network is necessary for the organization of Golgi apparatus.

Arginine↗
PubMed2004

Hepatitis C virus down-regulates insulin receptor substrates 1 and 2 through up-regulation of suppressor of cytokine signaling 3.

The pathogenesis of hepatitis C virus (HCV)-associated insulin resistance remains unclear. Therefore, we investigated mechanisms for HCV-associated insulin resistance. Homeostasis model assessment for insulin resistance was increased in patients with HCV infection. An increase in fasting insulin levels was associated with the presence of serum HCV core, the severity of hepatic fibrosis and a decrease in expression of insulin receptor substrate (IRS) 1 and IRS2, central molecules of the insulin-signaling cascade, in patients with HCV infection. Down-regulation of IRS1 and IRS2 was also seen in HCV core-transgenic mice livers and HCV core-transfected human hepatoma cells. Carbobenzoxy-l-leucyl-l-leucyl-l-leucinal, a potent proteosomal proteolysis inhibitor, blocked down-regulation of IRS1 and IRS2 in HCV core-transfected hepatoma cells. In human hepatoma cells, HCV core up-regulated suppressor of cytokine signaling (SOCS) 3 and caused ubiquitination of IRS1 and IRS2. HCV core-induced down-regulation of IRS1 and IRS2 was not seen in SOCS3(-/-) mouse embryonic fibroblast cells. Furthermore, HCV core suppressed insulin-induced phosphorylation of p85 subunit of phosphatidylinositol 3-kinase and Akt, activation of 6-phosphofructo-2-kinase, and glucose uptake. In conclusion, HCV infection changes a subset of hepatic molecules regulating glucose metabolism. A possible mechanism is that HCV core-induced SOCS3 promotes proteosomal degradation of IRS1 and IRS2 through ubiquitination.

Adult↗
PubMed2003

Proteasome inhibition induces inclusion bodies associated with intermediate filaments and fragmentation of the Golgi apparatus.

The ubiquitin-proteasome system is involved in a variety of biological processes. Inclusion bodies associated with intermediate filaments (IFs) and ubiquitin are observed in various diseases; however, the precise mechanisms of formation and the pathological significance of inclusion bodies have not been fully understood. We examined the effect of proteasome inhibitors on the structure of IF using anti-cytokeratin antibodies or transfection of green fluorescent protein-fused cytokeratin 18 in a hepatoma cell line, Huh7. Intracellular organelles were visualized by immunofluorescent and electron microscopies. Proteasome inhibitors induced IF inclusions associated with ubiquitin. Electron microscopic examination revealed inclusion bodies surrounded by filamentous structures. Autophagic vacuoles and lysosomes were frequently observed, and the organization of the Golgi apparatus was disrupted in these cells. After the removal of the proteasome inhibitors, the IF network and organization of the Golgi apparatus were restored. The IF inclusions could be induced by inhibition of the proteasome function. IF inclusions induced fragmentation of the Golgi apparatus and might inhibit the function of this important station of membrane traffic. The IF inclusions disappeared by restoring proteasome function, and autophagy and lysosomal degradation might be, at least in part, associated with the elimination of inclusion bodies.

Autophagy↗
PubMed2003

Unusual hyperbilirubinemia associated with bacterial pneumonia and acute myeloid leukemia.

A 77-year-old man with pneumonia associated with acute myeloid leukemia was introduced to the hepatology unit at our hospital for hyperbilirubinemia. He had been suffering from a high fever because of pneumonia. He was icteric and his serum concentrations of total and direct bilirubin were 13.1 and 7.9 mg/dl, respectively. However, the other standard biochemical examinations for hepatic function, such as serum concentrations of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase and alkaline phosphatase were normal except for lactate dehydrogenase. Lactate dehydrogenase isoenzyme analysis revealed that the high concentration was derived from leukemia cells. Ultrasonography of the abdomen revealed no abnormality in the liver or biliary tract. Administration of antibiotics for pneumonia decreased the serum bilirubin concentration, however, he died because of respiratory failure caused by the progression of pneumonia at 33 days after the admission. It was suggested that a disturbance in the bilirubin metabolism without hepatocyte necrosis or mechanical cholestasis might be involved in the pathogenesis of hyperbilirubinemia in patients with infectious diseases.

Journal Article↗
PubMed2003

Tumor necrosis factor-alpha and interferon-gamma directly impair epithelial barrier function in cultured mouse cholangiocytes.

BACKGROUND/AIMS: In primary biliary cirrhosis (PBC), cytokines from CD4+ T lymphocytes were suggested to contribute to the intralobular bile duct damage together with cellular immunity by CD8+ T lymphocytes. Recently, we reported that immunolocalization of 7H6--a tight junction (TJ)-associated protein--was significantly diminished in cholangiocytes in the PBC liver. In this study, we examined the direct effects of several cytokines--tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-2 and 4 (IL-2 and 4)--on TJ in immortalized mouse cholangiocytes. Moreover, we examined the inhibitory effect of ursodeoxycholic acid (UDCA) on cytokine-induced changes in paracellular permeability. METHODS: Barrier function of TJ was evaluated by measuring transepithelial electrical resistance (TER) and 3H-inulin flux. We also performed immunostaining and immunoblotting for TJ-associated proteins--claudin-1 and -3, occludin, zonula occluden-1 (ZO-1) and 7H6. RESULTS: TNF-alpha and IFN-gamma, but neither IL-2 nor IL-4, significantly decreased TER (P < 0.005). 3H-inulin flux studies confirmed IFN-alpha-induced increases in paracellular permeability of cholangiocytes (P < 0.001). In immunostaining and immunoblotting studies, TJ-associated proteins were well preserved in TNF-alpha- or IFN-gamma-treated cells. Ursodeoxycholic acid has been found to have no inhibitory effect on increased paracellular permeability induced by TNF-alpha or IFN-gamma. CONCLUSION: These findings show that TNF-alpha and IFN-gamma disrupt barrier function of TJ in cholangiocytes without major structural changes to TJ and suggest that disruption of TJ function and subsequent leakage of the bile constituents may influence the aggravation of cholestasis in PBC.

Animals↗
PubMed2003

Hydrogen peroxide overproduction in megamitochondria of troglitazone-treated human hepatocytes.

Troglitazone has been withdrawn from therapeutic options for diabetes mellitus because of its severe hepatocyte toxicity of unknown pathogenesis. The aim of the present study was to assess both morphologic and functional alterations in the mitochondria of troglitazone-treated hepatocytes. A polarized human hepatocyte cell line, OUMS-29, was used in this study. The mitochondrial volume and the mitochondrial transmembrane potential (DeltaPsi(m)) were examined using flow cytometry with nonylacridine orange (NAO) and rhodamine-123, respectively. An ultrastructural examination of the troglitazone-treated OUMS-29 cells was performed using transmission electron microscopy (TEM). Reactive oxygen species (ROS) production was assessed using flow cytometry with dihydroethidium and 2',7'-dichlorodihydrofluorescein diacetate. A significant increase in the mitochondrial volume of the troglitazone-treated cells was found by the NAO analysis, in comparison with pioglitazone-treated and ciglitazone-treated cells. The increase in volume was due to a marked enlargement in the mitochondria. The markedly enlarged mitochondria with intramitochondrial electron-dense deposits were confirmed on TEM, which showed myelin-like structures, indicating degraded membrane constituents. The troglitazone-treated cells showed a significant decline in the DeltaPsi(m) per unit mitochondrial volume but resulted in no clear cell death. ROS analysis revealed a significant production of hydrogen peroxide in the troglitazone-treated hepatocytes. This production was attenuated using an antioxidant, N-acetyl-L-cysteine. In conclusion, troglitazone caused overproduction of hydrogen peroxide, which deteriorated both mitochondrial membrane structures and mitochondrial function, leading to a possible priming for the severe hepatocyte toxicity.

Acetylcysteine↗