Mizuho Harashima

Nippon Bunri University, Edo, Tōkyō, Japan

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Publications (9)15.12 Total impact

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    ABSTRACT: In the present study, we investigated the role of p16(INK4a) in the inhibition of DNA synthesis stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) using RNA interference in primary cultured rat hepatocytes. The transfection of small interfering RNAs targeting p16(INK4a) reduced the corresponding mRNA and protein expression by more than approximately 90% and 50%, respectively, at 24 h after transfection. In the cells transfected with p16(INK4a) small interfering RNA, control, HGF, and EGF-stimulated DNA synthesis as assessed by (3)H-thymidine incorporation increased by approximately 1.5-fold, 1.6-fold, and 1.7-fold, respectively, compared with that in the control small interfering RNA-transfected cells. These findings indicate that p16(INK4a) plays a significant role in the inhibition of DNA synthesis.
    Biomedical Research 01/2013; 34(5):269-73. · 1.15 Impact Factor
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    ABSTRACT: RNA interference (RNAi) is the process of sequence-specific gene silencing, initiated by small double-stranded RNA homologous in sequence to the target gene. Various factors involved in the regulation of hepatocyte function have been identified using RNAi, indicating that RNAi is a useful strategy for characterization. There has been some success in treating experimental liver dysfunction using RNAi in several model systems, suggesting a promising new therapeutic strategy. A number of groups have also demonstrated that RNAi can interfere with hepatitis C virus and hepatitis B virus gene expression and replication in several model systems, suggesting a new approach for the treatment of these viral diseases. This review summarizes studies of hepatocytes using RNAi.
    07/2009; 3(3):164-182.
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    Shingo Niimi, Mizuho Harashima, Masashi Hyuga
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    ABSTRACT: Therapeutic angiogenesis, stimulated growth of new vasculature to compensate for tissue ischemia, has been studied in a number of clinical trials in patients with various ischemic vascular diseases. These clinical trials include growth factor protein and gene therapy, as well as cell therapy. However, almost randomized clinical trials using vascular endothelial growth factor and fibroblast growth factor families, delivered as either recombinant protein or gene therapy, have failed to demonstrate improvement in patients with coronary artery or peripheral artery disease until now. However, randomized clinical trials using bone marrow-derived cells demonstrated modest but some significant benefit in patients with myocardial infarction. This report reviews the current status of randomized clinical trials and some non-randomized clinical trials using these therapies, plus related potential problems.
    Current Drug Therapy. 01/2009; 4(3).
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    ABSTRACT: Annexin (Anx) A3 increases and plays important roles in the signalling cascade in hepatocyte growth in cultured hepatocytes. However, no information is available on its expression and role in rat liver regeneration. In the present study, AnxA3 expression was investigated to determine whether it also plays a role in the signalling cascade in rat liver regeneration. AnxA3 protein and mRNA level both increase in liver after administration of carbon tetrachloride (CCl4) or 70% partial hepatectomy. AnxA3 protein level increases in isolated parenchymal hepatocytes, but not in non-parenchymal liver cells, in these rat liver regeneration models. AnxA3 mRNA increases in hepatocytes after CCl4 administration. Anti-hepatocyte growth factor antibody suppresses this increase in AnxA3 mRNA level. These results demonstrate that AnxA3 expression increases in hepatocytes through a hepatocyte growth factor-mediated pathway in rat liver regeneration models, suggesting that AnxA3 plays an important role in the signalling cascade in rat liver regeneration.
    Journal of Biochemistry 05/2008; 143(4):537-45. · 3.07 Impact Factor
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    ABSTRACT: We have recently reported that annexin (Anx) A3 expression is necessary for hepatocyte growth in cultured rat hepatocytes seeded at half the subconfluent density on collagen. In the present study, we investigated the effects of various regulatory factors of hepatocyte growth on AnxA3 expression. AnxA3 expression was significantly reduced in hepatocytes cultured under various growth inhibitory conditions such as presence of dexamethasone, culture at subconfluent cell density, and on EHS-Matrigel and lactose-carrying styrene polymer. On the other hand, hepatocyte growth factor and epidermal growth factor, stimulators of hepatocyte growth, significantly increased AnxA3 expression in hepatocytes cultured on EHS-Matrigel. These results show close correlation between known stimulatory or inhibitory actions of various factors to hepatocyte growth and increase or decrease in AnxA3 expression, and suggest the involvement of AnxA3 in their regulation of hepatocyte growth.
    Biological & Pharmaceutical Bulletin 08/2006; 29(7):1339-43. · 1.85 Impact Factor
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    ABSTRACT: Thrombomodulin (TM) is a thrombin receptor on the surface of endothelial cells that converts thrombin from a procoagulant to an anticoagulant. Thrombin promotes invasion by various tumor cells, and positive or negative correlations are found between the expression of TM and tumorigenesis in some patients. In this study, we used an invasion assay to investigate the effect of TM on the invasive activity of a mouse mammary tumor cell line, MMT cells, and the effects of TM were compared with those of thrombin as a positive control. In the presence of 1% fetal calf serum (FCS), TM significantly stimulated MMT cell invasion in a dose-dependent manner, resulting in an approximately 3-fold increase at 1-10 pg/ml over the untreated control. Thrombin also caused a similar degree of stimulation at 50 ng/ml. Since thrombin activity was detected in the components of the assay system, an invasion assay was also performed in a thrombin-activity-depleted assay system constructed to eliminate the effect of thrombin activity; TM (10 pg/ml) plus thrombin (1 pg/ml) stimulated invasion by approximately 3.5-fold in this assay system. Hirudin, a specific thrombin inhibitor, inhibited stimulation by TM as well as by thrombin in both the presence and absence of 1% FCS. Investigations of the effects of TM on proliferation, adhesion and chemotaxis to clarify the mechanism of stimulation by TM revealed that TM does not affect proliferation or adhesion in the presence of 1% FCS, but stimulates chemotaxis by approximately 2.3-fold. Similar results were obtained in experiments using thrombin. TM (10 pg/ml) plus thrombin (1 pg/ml), on the other hand, stimulated chemotaxis by approximately 2.3-fold in the thrombin-activity-depleted assay system. Binding studies using [125I]-thrombin revealed that the cells have specific saturable binding sites for thrombin. These results show that TM stimulates the invasive activity of MMT cells, probably by acting as a cofactor for the thrombin-stimulated invasion of the cells via its receptor and lowering the effective concentration of thrombin. The findings also indicate that the stimulation of invasive activity in the presence of 1% FCS and in the thrombin-activity-depleted assay system may mainly be mediated by the stimulation of chemotaxis.
    Journal of Biochemistry 06/2005; 137(5):579-86. · 3.07 Impact Factor
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    ABSTRACT: Annexin A3 is a member of the lipocortin/annexin family, which binds to phospholipids and membranes in a Ca(2+)-dependent manner. Although annexin A3 has various functions in vitro, its cellular significance is completely unknown. Annexin A3 is not found in rat liver in vivo. In the present study, we investigated the expression of annexin A3 in primary cultured parenchymal rat hepatocytes. Annexin A3 protein was detected in 48-h, but not 2.5-h, cultured hepatocytes using Western blot analysis. The annexin A3 level further increased after an additional 24 h of culture. Annexin A3 mRNA was not detected in 2.5-h cultured hepatocytes but was detected 22 h after the start of culture by RT-PCR analysis, reaching a maximum value after 48 h of culture. To define the role of Annexin A3 in DNA synthesis, RNA interference was used to reduce annexin III gene expression in hepatocytes. The transfection of small interfering RNAs targeting annexin A3 in the hepatocytes reduced the corresponding mRNA and protein expression by approximately 80% and more than 90%, respectively, at 24 h after transfection. In the annexin A3 small interfering RNAs-transfected cells, DNA synthesis, as assessed by [3H]thymidine incorporation, decreased by approximately 70% not only in the control cultures, but also in the hepatocyte growth factor- or epidermal growth factor-treated cells. These findings show that annexin A3 is expressed in primary cultured parenchymal rat hepatocytes and that the suppression of annexin A3 expression using RNA interference inhibits DNA synthesis.
    Biological & Pharmaceutical Bulletin 04/2005; 28(3):424-8. · 1.85 Impact Factor
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    ABSTRACT: We recently showed that annexin III is expressed in isolated small rat hepatocytes but, not in parenchymal hepatocytes. In the present study, we used reverse transcription polymerase chain analysis to examine the annexin III mRNA level in isolated small rat hepatocytes and parenchymal hepatocytes. Annexin III mRNA was detected in isolated small hepatocytes, but not in isolated parenchymal hepatocytes, confirming the presence of annexin III expression in isolated small rat hepatocytes at the mRNA level and indicating that the absence of annexin III expression in isolated parenchymal hepatocytes is due to the absence of annexin III mRNA. Furthermore, we examined the mRNA level of tyrosine aminotransferase and tryptophan oxygenase, two terminally differentiated hepatocyte markers. mRNA for these markers was detected in both parenchymal hepatocytes and small hepatocytes.
    Biological & Pharmaceutical Bulletin 12/2004; 27(11):1864-6. · 1.85 Impact Factor
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    ABSTRACT: Small hepatocytes are cells that express characteristic phenotypes such as a high growth potential and differentiation capacity. In order to identify rat-small-hepatocyte specific proteins, we separated the cellular proteins of isolated small and parenchymal hepatocytes by 2D polyacrylamide gel electrophoresis. Comparison of their profiles revealed a protein with a molecular mass of 37 kDa in the small hepatocytes that was not present in the parenchymal hepatocytes. Proteolytic peptide mass fingerprinting was used to identify the protein and it was found to be annexin III. The validity of the identification was confirmed by Western blot analysis with anti-annexin III antibody.
    Biochemical and Biophysical Research Communications 02/2003; 300(3):770-4. · 2.28 Impact Factor