KINDAI UNIVERSITY


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YOSHIDA Koji

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FacultyDepartment of Biomedical Engineering / Graduate School of Biology-Oriented Science and Technology
PositionProfessor
Degree
Commentator Guidehttps://www.kindai.ac.jp/meikan/1351-yoshida-koji.html
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Last Updated :2020/09/10

Education and Career

Education

  •  - 1990 , Kinki University, Graduate School, Division of Medicine
  •  - 1984 , Kinki University, Faculty of Medicine
  •   1978 04  - 1984 03 , Kindai University, Faculty of Medicine
  •   1986 04  - 1990 03 , Kindai University, Graduate School of Medical Sciences

Academic & Professional Experience

  •   2015 04 ,  - 現在, Faculty of Biology-Oriented Science and Technology, Department of Biomedical Engineering, Kindai University
  •   2007 04 ,  - 2015 03 , Faculty of Medicine, Kindai University
  •   2001 04 ,  - 2007 03 , Faculty of Medicine, Kindai University
  •   1990 04 ,  - 2001 03 , Faculty of Medicine, Kindai University

Research Activities

Research Areas

  • Life sciences, Pathobiochemistry
  • Life sciences, Medical biochemistry
  • Life sciences, Molecular biology

Research Interests

  • Molecular Biology, Pathological Medical Chemistry, Biochemistry

Published Papers

  • Inhibition by Epigallocatechin Gallate of IL-1-Induced Urokinase-Type Plasminogen Activator Expression and Collagen Degradation by Corneal Fibroblasts, Sugioka K, Yoshida K, Murakami J, Itahashi M, Mishima H, Nishida T, Kusaka S, Invest Ophthalmol Vis Sci., Invest Ophthalmol Vis Sci., 60(8), 2895 - 2903, Jul. 2019 , Refereed
  • Extracellular Collagen Promotes Interleukin-1 beta-Induced Urokinase-Type Plasminogen Activator Production by Human Corneal Fibroblasts, Koji Sugioka, Aya Kodama-Takahashi, Koji Yoshida, Keiichi Aomatsu, Kiyotaka Okada, Teruo Nishida, Yoshikazu Shimomura, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 58(3), 1487 - 1498, Mar. 2017 , Refereed
    Summary:PURPOSE. Keratocytes maintain homeostasis of the corneal stroma through synthesis, secretion, and degradation of collagen fibrils of the extracellular matrix. Given that these cells are essentially embedded in a collagen matrix, keratocyte-collagen interactions may play a key role in regulation of the expression or activation of enzymes responsible for matrix degradation including urokinase-type plasminogen activator (uPA), plasmin, and matrix metalloproteinases (MMPs). We examined the effect of extracellular collagen on the production of uPA by corneal fibroblasts (activated keratocytes) stimulated with the proinflammatory cytokine interleukin-1 beta (IL-1 beta). METHODS. Human corneal fibroblasts were cultured either on plastic or in a three-dimensional gel of type I collagen. Plasminogen activators were detected by fibrin zymography, whereas the IL-1 receptor (IL-1R) and MMPs were detected by immunoblot analysis. Collagen degradation by corneal fibroblasts was assessed by measurement of hydroxyproline in acid hydrolysates of culture supernatants. RESULTS. Collagen and IL-1 beta synergistically increased the synthesis and secretion of uPA in corneal fibroblasts. Collagen also upregulated IL-1R expression in the cells in a concentration-dependent manner. The conversion of extracellular plasminogen to plasmin, as well as the plasminogen-dependent activation of MMP-1 and MMP-3 and degradation of collagen apparent in three-dimensional cultures of corneal fibroblasts exposed to IL-1 beta, were all abolished by a selective uPA inhibitor. CONCLUSIONS. Collagen and IL-1 beta cooperate to upregulate uPA production by corneal fibroblasts. Furthermore, IL-1 beta-induced collagen degradation by these cells appears to be strictly dependent on uPA expression and mediated by a uPA-plasmin-MMP pathway.
  • Involvement of Heat Shock Protein A4/Apg-2 in Refractory Inflammatory Bowel Disease, Teppei Adachi, Toshiharu Sakurai, Hiroshi Kashida, Hiromasa Mine, Satoru Hagiwara, Shigenaga Matsui, Koji Yoshida, Naoshi Nishida, Tomohiro Watanabe, Katsuhiko Itoh, Jun Fujita, Masatoshi Kudo, INFLAMMATORY BOWEL DISEASES, INFLAMMATORY BOWEL DISEASES, 21(1), 31 - 39, Jan. 2015 , Refereed
    Summary:Background: Expression of heat shock protein A4 (HSPA4, also called Apg-2), a member of the HSP110 family, is induced by several forms of stress. The physiological and pathological functions of HSPA4 in the intestine remain to be elucidated. Methods: We assessed HSPA4 expression and function by generating HSPA4-deficient mice and using 214 human intestinal mucosa samples from patients with inflammatory bowel disease (IBD). Results: In the colonic mucosa of patients with IBD, a significant correlation was observed between the expression of HSPA4 and antiapoptotic protein Bcl-2, a T-cell-derived cytokine IL-17 or stem cell markers, such as Sox2. In refractory ulcerative colitis, a condition associated with increased cancer risk, expression of HSPA4 and Bcl-2 was increased in inflammatory cells of colonic mucosae. HSPA4 was overexpressed both in cancer cells and immune cells of human colorectal cancers. Patients with high expression of HSPA4 or Bmi1 showed significantly lower response rates upon subsequent steroid therapy as compared with patients with low expression of each gene. HSPA4-deficient mice exhibit more apoptosis and less expression of IL-17/IL-23 in inflammatory cells and less number of Sox(2+) cells after administration of dextran sodium sulfate than control mice. Transduction of HspaA4(+/-) bone marrow into wild-type mice reduced the immune response. Conclusions: Upregulation of Bcl-2 and IL-17 by HSPA4 would control apoptosis of inflammatory cells and immune response in the gut, which might develop treatment resistance in IBD. HSPA4 and Bmi1 would be a useful biomarker for refractory clinical course and a promising approach for a therapeutic strategy in patients with IBD.
  • fibrosisCIB1と integrin α11 は結合し、肺線維症に関わっている可能性がある。, Advances in Biological Chemistry, Advances in Biological Chemistry, 4, 59 - 66, Feb. 2014 , Refereed
  • EGCG suppresses TGF-bete signaling by interacting with TGF-beta type II receptor., Tabuchi M, Hayakawa S, Honda E, Ooshima K, Itoh I, Yoshida K, Park AM, Higashino H, Isemura M, Munakata H, World Journal of Experimental Medicine, World Journal of Experimental Medicine, 3(4), 100 - 107, Nov. 2013 , Refereed
  • TGF-beta 2 promotes RPE cell invasion into a collagen gel by mediating urokinase-type plasminogen activator (uPA) expression, Koji Sugioka, Aya Kodama, Kiyotaka Okada, Mihoko Iwata, Koji Yoshida, Shunji Kusaka, Chota Matsumoto, Hiroshi Kaji, Yoshikazu Shimomura, EXPERIMENTAL EYE RESEARCH, EXPERIMENTAL EYE RESEARCH, 115, 13 - 21, Oct. 2013 , Refereed
    Summary:Transforming growth factor-beta (TGF-beta) is one of the main epithelial mesenchymal transition (EMT)-inducing factors. In general, TGF-beta-induced EMT promotes cell migration and invasion. TGF-beta also acts as a potent regulator of pericellular proteolysis by regulating the expression and secretion of plasminogen activators. Urokinase-type plasminogen activator (uPA) is a serine protease that binds to its cell surface receptor (uPAR) with high affinity. uPA binding to uPAR stimulates uPAR's interaction with transmembrane proteins, such as integrins, to regulate cytoskeletal reorganization and cell migration, differentiation and proliferation. However, the influence of TGF-beta and the uPA/uPAR system on EMT in retinal pigment epithelial (RPE) cells is still unclear. The purpose of this study was to determine the effect of TGF-beta 2, which is the predominant isoform in the retina, and the uPA/uPAR system on RPE cells. In this study, we first examined the effect of TGF-beta 2 and/or the inhibitor of uPA (u-PA-STOP (R)) on the proliferation of a human retinal pigment epithelial cell line (ARPE-19 cells). Treatment with TGF-beta 2 or u-PA-STOP (R) suppressed cell proliferation. Combination treatment of TGF-beta 2 and u-PA-STOP (R) enhanced cell growth suppression. Furthermore, western blot analysis, fibrin zymography and real-time reverse transcription PCR showed that that TGF-beta 2 induced EMT in ARPE-19 cells and that the expression of uPA and uPAR expression was up-regulated during EMT. The TGF-beta inhibitor SB431542 suppressed TGF-beta 2-stimulated uPA expression and secretion but did not suppress uPAR expression. Furthermore, we seeded ARPE-19 cells onto Transwell chambers and allowed them to invade the collagen matrix in the presence of TGF-beta 2 alone or with TGF-beta 2 and u-PA-STOP (R). TGF-beta 2 treatment induced ARPE-19 cell invasion into the collagen gel. Treatment with a combination of TGF-beta 2 and the uPA inhibitor strongly inhibited ARPE-19 cell invasion compared with treatment with TGF-beta 2 alone. Furthermore, the interaction between uPA and ARPE-19 cells was analyzed using a surface plasmon biosensor system. The binding of uPA to ARPE-19 cells was observed. In addition, TGF-beta 2 significantly promoted the binding activity of uPA to ARPE-19 cells in a time-dependent or cell-number-dependent fashion. These results indicate that TGF-beta-induced EMT-associated phenotype changes in ARPE-19 cells and the invasiveness of ARPE-19 cells into a collagen gel matrix are mediated, at least in part, by uPA. (C) 2013 Elsevier Ltd. All rights reserved.
  • Epigallocatechin-3-gallate suppresses transforming growth factor-beta signaling by interacting with the transforming growth factor-beta type Ⅱ receptor., World J Exp Med, World J Exp Med, 3(4), 100 - 107, Aug. 2013 , Refereed
  • 線溶系遺伝子欠損マウスを用いた角膜上皮創傷治癒過程の検討, 児玉 彩, 杉岡 孝二, 三島 弘, 吉田 浩二, 岡田 清孝, 下村 嘉一, 日本眼科学会雑誌, 日本眼科学会雑誌, 116(臨増), 362 - 362, Mar. 2012
  • Transforming Growth Factor-beta Upregulates the Expression of Integrin and Related Proteins in MRC-5 Human Myofibroblasts, Eiko Honda, Koji Yoshida, Hiroshi Munakata, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, 220(4), 319 - 327, Apr. 2010 , Refereed
    Summary:Myofibroblasts are defined as fibroblasts that express certain features of smooth muscle differentiation. Activation of myofibroblasts plays a central role in fibrosis. Transforming growth factor-beta (TGF-beta) is a potent activator of myofibroblasts; namely, TGF-beta causes changes in myofibroblast phenotypes including morphological alterations and the expression of alpha-smooth muscle actin (alpha-SMA), a marker of myofibroblasts. Because it has been well known that humoral factors, especially, TGF-beta, and extracellular matrix components cause myofibroblast activation, we examined the expression of integrin and related proteins during activation of MRC-5 human myofibroblasts with TGF-beta. Western blot analysis revealed that TGF-beta treatment for 3 days increased the expression of alpha-SMA, which was also immunocytochemically observed as actin stress fibers. In the early phase of TGF-beta treatment, fibronectin expression was greatly increased, followed by the increased expression of integrin alpha v and alpha 11 and integrin beta 1 and beta 3. Co-immunoprecipitation assays revealed that the integrin av subunit was co-precipitated with integrin beta 1 and beta 3, and that integrin beta 1 was co-precipitated with all, alpha v, alpha 2, and alpha 5. The expression of focal adhesion kinase and integrin-linked kinase proteins was also upregulated by treatment with TGF-beta. In addition, the expression of type I collagen mRNA was increased by TGF-beta, but not type III collagen mRNA, as judged by real-time PCR analysis. These results suggest the possibility that TGF-beta induces fibronectin expression in MRC-5 cells, which subsequently induces the expression of integrin receptors, alpha v beta 3, alpha v beta 1, and alpha 11 beta 1. This report also shows that expression of integrin alpha 11 is upregulated during the TGF-beta-mediated activation of myofibroblasts.
  • 角膜上皮細胞の接着、伸長に対するCTGFとフィブロネクチンの相互作用, 杉岡 孝二, 児玉 彩, 吉田 浩二, 三島 弘, 下村 嘉一, 日本眼科学会雑誌, 日本眼科学会雑誌, 113(臨増), 281 - 281, Mar. 2009
  • Aggrecanase-1 (ADAMTS-4) interacts with alpha 1-antitrypsin, K Yoshida, Y Suzuki, A Saito, K Fukuda, C Hamanishi, H Munakata, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1725(2), 152 - 159, Sep. 2005 , Refereed
    Summary:In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1.(ADAMTS-4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha 1-antitrypsin, a member of the family of plasma serine protemase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha 1-antitrypsin were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length aggrecanase-1, and alpha 1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of otiantitrypsin against elastase, and a 1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha 1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha 1-antitrypsin remains unclear. (c) 2005 Elsevier B.V. All rights reserved.
  • Guinea pig alpha(1)-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 122(2), 165 - 172, Feb. 1999 , Refereed
    Summary:cDNA encoding alpha(1)-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavin porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gin rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change. (C) 1999 Elsevier Science Inc. All rights reserved.
  • cDNA sequencing of guinea pig alpha(2)-HS glycoprotein, its expression in various tissues and acute phase expression, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, BIOLOGICAL CHEMISTRY, BIOLOGICAL CHEMISTRY, 380(1), 95 - 99, Jan. 1999 , Refereed
    Summary:cDNA encoding alpha(2)-HS glycoprotein was amplified from guinea pig liver mRNA by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, cloned and sequenced. By RT-PCR and nested PCR, alpha(2)-HS glycoprotein mRNA was detected not only in liver tissue but also in pancreas, stomach, small intestine, colon, spleen, kidney, testis, skeletal muscle, brain, heart and leukocytes, but not in the lung. The alpha(2)-HS glycoprotein mRNA levels in the liver were reduced to half at 48 h after subcutaneous injection of turpentine oil.
  • MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF CDNA-ENCODING PLASMA ALPHA-1-ANTIPROTEINASE FROM SYRIAN-HAMSTER - IMPLICATIONS FOR THE EVOLUTION OF RODENTIA, T NAKATANI, Y SUZUKI, K YOSHIDA, H SINOHARA, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1263(3), 245 - 248, Sep. 1995 , Refereed
    Summary:Complementary DNA clones encoding plasma alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) were isolated from Syrian hamster liver cDNA library and sequenced. The deduced amino acid sequence of putative reactive site (P3-P'3) was Ile-Pro-Met-Ser-Val-Pro, characteristic of alpha-1-antiproteinase of orthodox type (Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932). A molecular phylogenetic tree of all known orthologous proteins was constructed based on the synonymous substitution rate. The result shows that the hamster has branched off first before the divergence among mice, rats, and gerbils, and that the rabbit is the closest relative of the guinea pig which is separated from the rodents. Although this tree differs largely from the classical phylogeny based on the morphology (hamsters and gerbils belong to the same family, Cricetidae, and the guinea pig belongs to the order Rodentia), it lends support to recent concepts that the hamster and guinea pig differ, in a number of biochemical features, not only from each other but also from mice and rats, and that the guinea pig may belong to an order distinct from Rodentia.
  • PLASMA ALPHA-1-ANTIPROTEINASE FROM THE MONGOLIAN GERBIL, MERIONES-UNGUICULATUS - ISOLATION, PARTIAL CHARACTERIZATION, SEQUENCING OF CDNA, AND IMPLICATIONS FOR MOLECULAR EVOLUTION, K GOTO, Y SUZUKI, K YOSHIDA, K YAMAMOTO, H SINOHARA, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 116(3), 582 - 588, Sep. 1994 , Refereed
    Summary:alpha-1-Antiproteinase (also called alpha-1-proteinase inhibitor or alpha-1-antitrypsin) with a molecular mass of 56 kDa was purified from plasma of the Mongolian gerbil, Meriones unguiculatus, to apparent homogeneity. It inhibited trypsin, chymotrypsin, elastase, and plasmin, but not kallikrein or thrombin. Eight cDNA clones coding for this protein were isolated from a liver cDNA library and sequenced. They contained the same coding regions consisting of a 24-residue signal peptide and a 382-residue mature protein. The reactive site sequence (P3-P'3) was Val-Pro-Met-Ser-Ile-Pro, characteristic of alpha-1-antiproteinase of orthodox type [Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932]. A molecular phylogenetic tree of 11 orthologous inhibitors, constructed on the basis of the synonymous substitution rate, shows (i) that the reactive site region is highly conserved as compared to the other part of the molecule, which contrasts with the generally accepted view that the reactive site region of serpins is strongly hypervariable, and (ii) that the myomorphs (gerbil, rat, and two species of mouse, i.e. Mus domesticus and Mus caroli) and the caviomorph (guinea pig) fail to consist of a monophyletic order, which also contradicts the traditional taxonomy based on the morphology. In the present tree, the guinea pig joins the lagomorph (rabbit), and is rather widely separated from the myomorph branch. The result, however, supports the recent hypothesis based on the molecular evolution of several other proteins that the guinea pig does not belong to the same order as the myomorph, and the caviomorphs should be elevated in taxonomic rank and conferred an ordinal status distinct from the rodents.
  • Molecular cloning and sequence analysis of cDNAs coding for guinea pig α1-antiproteinases S and F and contrapsin, Yasuyuki Suzuki, Koji Yoshida, Eiko Honda, Hyogo Sinohara, Journal of Biological Chemistry, Journal of Biological Chemistry, 266(2), 928 - 932, Jan. 15 1991 , Refereed
    Summary:The cDNAs encoding two isoforms, S (slow) and F (fast), of α1-antiproteinase (also referred to as α1-antitrypsin or α1-proteinase inhibitor) as well as contrapsin were obtained by screening λgt11 cDNA library prepared from inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of α1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own α1-antiproteinases F and S, rat α1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of α1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of α1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P′3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple α1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin superfamily.
  • TRYPSIN-INHIBITORS IN GUINEA-PIG PLASMA - ISOLATION AND CHARACTERIZATION OF CONTRAPSIN AND 2 ISOFORMS OF ALPHA-1-ANTIPROTEINASE AND ACUTE PHASE RESPONSE OF 4 MAJOR TRYPSIN-INHIBITORS, Y SUZUKI, K YOSHIDA, T ICHIMIYA, T YAMAMOTO, H SINOHARA, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 107(2), 173 - 179, Feb. 1990 , Refereed
  • A Case of Waldenstrom Macroglobulinemia with Temporary Appearance of 7S IgM Half Molecule, Mayumi Imoto, Koji Yoshida, Yasuhiro Maeda, Ken-Ichi Nakae, Masatoshi Kudo, Ikunosuke Sakurabayashi, Toshiyuki Yamada, Toshinori Kamisako, CLINICAL LABORATORY, CLINICAL LABORATORY, 63(5-6), 983 - 989, 2017 , Refereed
    Summary:Background: We encountered a rare case of Waldenstrom macroglobulinemia with temporary appearance of 7S IgM half molecule and with monoclonal proteins binding to agarose gel. Methods: The patient's serum and urine were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. The N-terminal amino acid sequences of the IgM with abnormal mass (68 kDa) were determined and compared with those of known immunoglobulin. Results: The 68 kDa IgM consisted of a defective Et chain (36 kDa) and an intact kappa chain. N-terminal amino acid sequence analysis demonstrated that the defective It chain had the variable region of IgM. The agarose gel-binding ability of the IgM-w M-protein was lost after reduction or alkaline treatment of serum. Conclusions: The 7S half molecule IgM in the present case may miss a large part of the constant region of the mu chain.
  • The Roles of Urokinase-Type Plasminogen Activator in Leukocyte Infiltration and Inflammatory Responses in Mice Corneas Treated With Lipopolysaccharide, Koji Sugioka, Aya Kodama, Koji Yoshida, Kiyotaka Okada, Hiroshi Mishima, Keiichi Aomatsu, Osamu Matsuo, Yoshikazu Shimomura, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE, 55(8), 5338 - 5350, Aug. 2014 , Refereed
    Summary:PURPOSE. Urokinase-type plasminogen activator (u-PA) plays an important role in corneal wound healing, yet its role in corneal inflammation remains poorly understood. We investigated the role of u-PA in a murine model of lipopolysaccharide (LPS)-induced corneal inflammation. METHODS. The corneal epithelium was scraped and LPS was applied to u-PA wild-type (u-PA(+/+)) and u-PA-deficient (u-PA(-/-)) mice. Corneal re-epithelialization and opacity were measured by stereomicroscopy. Fibrin zymography was performed to detect plasminogen activators in corneas from u-PA(+/+) and u-PA(-/-) mice. Neutrophil, macrophage, and u-PA receptor (u-PAR) expression were determined by immunohistochemistry. Gene expression of corneal macrophage chemoattractant protein (MCP)-1 and macrophage inflammatory protein (MIP)-2 was assessed with reverse transcription-polymerase chain reaction. The in vitro effects of endogenous u-PA on MCP-1, MIP-2, matrix metalloproteinase (MMP)-2, and MMP-9 expression, and macrophage migration activity in mouse ocular fibroblasts stimulated by LPS, were examined. RESULTS. The u-PA(+/+) mice showed enhanced corneal inflammation as compared with u-PA(-/-) mice. The u-PA expression was increased by LPS stimulation. Immunohistochemical analyses indicated that more neutrophils and macrophages were present in corneas from u-PA(-/-) mice than u-PA(-/-)mice. The u-PAR expression was detected in inflammatory cells and in the leading edges of the epithelial migrating cells. Enhanced mRNA expression of MCP-1 and MIP-2 was observed in corneas from u-PA(+/+) mice compared to u-PA(-/-) mice. Macrophage chemoattractant protein-1, MIP-2, and MMP-9, but not MMP-2, significantly increased in corneal fibroblasts from u-PA(+/+) mice compared with u-PA(-/-) mice. CONCLUSIONS. These data indicate that u-PA promotes LPS-induced leukocyte infiltration in cornea and that u-PA is an important component in LPS-induced corneal inflammatory responses.
  • Myofibroblasts: Biochemical and Proteomic Approaches to Fibrosis, Eiko Honda, Ah-Mee Park, Koji Yoshida, Masaki Tabuchi, Hiroshi Munakata, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, 230(2), 67 - 73, Jun. 2013 , Refereed
    Summary:Fibrosis is a state, in which excess amounts of extracellular matrix are deposited in the tissue. Fibrosis can occur in various organs, including the liver, lung, kidney and heart. The progression of fibrosis involves interstitial hypercellularity, accumulation of extracellular matrix, and atrophy of epithelial structures, resulting in a loss of normal function. Myofibroblasts play a crucial role in the development and progress of fibrosis. When stimulated, myofibroblasts actively synthesize connective tissue components and cause organ fibrosis. As a result, the process and the mechanism of myofibroblast activation represent a target for antifibrotic treatment. As yet, however, an effective treatment has not been developed, and new treatment modalities are expected. Because activation of myofibroblasts is a key event during fibrosis development, there is great interest in identifying and characterizing proteins whose expression is changed after this activation. In this review, fibrosis is outlined and the role of myofibroblasts in this disorder is described. Furthermore, the search for candidate proteins to target for treatment and the prospects of antifibrotic therapy are discussed.
  • Immunohistochemical localization of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and alpha 2-antiplasmin in human corneal perforation: a case report, Koji Sugioka, Aya Kodama, Koji Yoshida, Kiyotaka Okada, Masahiko Fukuda, Yoshikazu Shimomura, BMC OPHTHALMOLOGY, BMC OPHTHALMOLOGY, 12(1), Nov. 2012 , Refereed
    Summary:Background: Corneal ulceration leading to perforation is associated with infectious and non-infectious destructive conditions in the cornea. The fibrinolytic (plasminogen/plasmin) system is considered to contribute to tissue remodeling in the wound healing process and it is believed to play an important role in proteolysis and fibrosis. To determine the localization of urokinase-type plasminogen activator (u-PA), u-PA receptor (u-PAR) and alpha 2-antiplasmin (alpha 2AP) in the tissue of a corneal perforation, we investigated immunohistochemical expressions of u-PA, u-PAR, alpha 2AP, CD68, and alpha-smooth muscle actin (alpha-SMA) in a patient with corneal perforation that developed from an ulcer of no clear cause. Case presentation: The patient was a 77-year-old woman who presented with a perforated corneal ulcer in her right eye. The cause of her corneal ulcer was unknown. Double immunohistochemistry was performed for the combinations of u-PA with u-PAR, CD68 or alpha-SMA and alpha 2AP with CD68 or alpha-SMA to detect the localization of u-PA and alpha 2AP. u-PA and u-PAR co-localization was seen in the corneal ulceration area. u-PA was mainly observed in CD68-positive cells and in some alpha-SMA positive cells. On the other hand, alpha 2AP was not expressed in CD68-positive cells, but was expressed in alpha-SMA positive cells. Conclusion: We identified expression of the u-PA/u-PAR complex and alpha 2AP in a patient with a corneal ulcer. These two molecules are believed to play a crucial role in inflammatory cell recruitment, ECM synthesis and degradation during corneal wound healing.
  • Conditioned media from lung cancer cell line A549 and PC9 inactivate pulmonary fibroblasts by regulating protein phosphorylation, Ah-Mee Park, Sumio Hayakawa, Eiko Honda, Yoshihiro Mine, Koji Yoshida, Hiroshi Munakata, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 518(2), 133 - 141, Feb. 2012 , Refereed
    Summary:Pulmonary fibrosis is a devastating condition resulting from excess extracellular matrix deposition that leads to progressive lung destruction and scarring. In the pathogenesis of fibrotic diseases, activation of myofibroblasts by transforming growth factor-beta (TGF-beta) plays a crucial role. Since no effective therapy for pulmonary fibrosis is currently recognized, finding an effective antifibrotic agent is an important objective. One approach might be through identification of agents that inactivate myofibroblasts. In the current study we examined the potential of conditioned medium obtained from several types of cells to exhibit myofibroblast inactivating activity. Conditioned media from lung cancer cell lines A549 and PC9 were found to have this action, as shown by its ability to decrease alpha-smooth muscle actin expression in MRC-5 cells. Subsequently the inhibitory factor was purified from the medium and identified as 5'-deoxy-5'-methylthioadenosine (MTA), and its mechanism of action elucidated. Activation of protein kinase A and cAMP responsive element binding protein (CREB) were detected. MTA inhibited TGF-beta-induced mitogen-activated protein kinase activation. Furthermore, the gain-of-function mutant CREB caused inactivation of myofibroblasts. These results show that A549 and PC9 conditioned media have the ability to inactivate myofibroblasts, and that CREB-phosphorylation plays a central role in this process. (C) 2011 Elsevier Inc. All rights reserved.
  • Connective Tissue Growth Factor Cooperates with Fibronectin in Enhancing Attachment and Migration of Corneal Epithelial Cells, Koji Sugioka, Koji Yoshida, Aya Kodama, Hiroshi Mishima, Kosuke Abe, Hiroshi Munakata, Yoshikazu Shimomura, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, TOHOKU JOURNAL OF EXPERIMENTAL MEDICINE, 222(1), 45 - 50, Sep. 2010 , Refereed
    Summary:Corneal wound healing is a complex process involving the integrated actions of various growth factors, cytokines and extracellular matrix produced by corneal cells and inflammatory cells. Connective tissue growth factor (CTGF) has been linked to wound healing, and fibronectin (FN) is a major component of the extracellular matrix. However, the functions of CTGF and FN in corneal epithelial cells are not well understood. We therefore investigated the coordinated function of CTGF and FN in the attachment and migration of corneal epithelial cells. Treatment of human corneal epithelial cells (HCECs) with transforming growth factor (TGF) beta 1 up-regulated the expression of CTGF, but did not noticeably affect FN expression, as judged by immunoblot analysis of cell lysates. In contrast, the amount of FN accumulated in the cultured media was increased in a time-dependent manner, but CTGF was undetectable in the cultured media. The expression level of FN was decreased by the knockdown of CTGF expression with a specific short hairpin RNA, indicating that CTGF acts as an upstream mediator of FN expression. CTGF augmented the FN-mediated increase in the attachment of HCEC by about twofold, although CTGF alone did not influence the attachment. Moreover, the migration assay with rabbit corneal blocks revealed that CTGF (390 nM) alone or in combination of FN (10 mu g/mL) promoted corneal epithelial migration; the mean migration distances of control, CTGF, and CTGF + FN were 272, 325, and 626, pm, respectively. In conclusion, CTGF cooperates with FN in enhancing the attachment and migration of corneal epithelial cells.
  • Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin, Koji Yoshida, Hiroshi Munakata, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1770(4), 672 - 680, Apr. 2007 , Refereed
    Summary:Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role. (c) 2006 Elsevier B.V. All rights reserved.
  • Oxidized LDL binding to LOX-1 upregulates VEGF expression in cultured bovine chondrocytes through activation of PPAR-gamma, Sohya Kanata, Masao Akagi, Shunji Nishimura, Sumio Hayakawa, Kohji Yoshida, Tatsuya Sawamura, Hiroshi Munakata, Chiaki Hamanishi, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 348(3), 1003 - 1010, Sep. 2006 , Refereed
    Summary:It has been reported that vascular endothelial growth factor (VEGF) and its receptors play an important role in the destruction of articular cartilage in ostcoarthritis through increased production of matrix metalloproteinases. We investigated whether the oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) upregulates VEGF expression in cultured bovine articular chondrocytes (BACs). Ox-LDL markedly increased VEGF mRNA expression and protein release in time- and dose-dependent manners, which was significantly suppressed by anti-LOX-1 antibody pretreatment. Activation of peroxisome proliferator-activated receptor (PPAR)-gamma was evident in BACs with ox-LDL addition and was attenuated by anti-LOX-1 antibody. The specific PPAR-gamma inhibitor GW9662 suppressed ox-LDL-induced VEGF expression. These results suggest that the ox-LDL/LOX-1 system upregulates VEGF expression in articular cartilage, at least in part, through activation of PPAR-gamma and supports the hypothesis that ox-LDL is involved in cartilage degradation via LOX-1. (c) 2006 Elsevier Inc. All rights reserved.
  • Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized LDL receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis, Masao Akagi, Shunji Nishimura, Kohji Yoshida, Takumi Kakinuma, Tatsuya Sawamura, Hiroshi Munakata, Chiaki Hamanishi, JOURNAL OF ORTHOPAEDIC RESEARCH, JOURNAL OF ORTHOPAEDIC RESEARCH, 24(8), 1782 - 1790, Aug. 2006 , Refereed
    Summary:Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 mu g/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 mu g/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 mu g/mL ox-LDL significantly suppressed chondrocytes viability (84.6% 3.4% and 80.9% 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% 7.1% and 85.7% 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 mu g/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% 4.9% and 48.7% 6.7% of control at 24 h, respectively (n 3; p < 0.01 when compared with individual application of cyclic loading or 10 mu g/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis. (c) 2006 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.
  • Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase, H Munakata, JY Sun, K Yoshida, T Nakatani, E Honda, S Hayakawa, K Furuyama, N Hayashi, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 136(2), 233 - 238, Aug. 2004 , Refereed
    Summary:5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (AIAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.
  • Hypoxia-induced nitric oxide protects chondrocytes from damage by hydrogen peroxide, T Matsushita, K Fukuda, K Yamazaki, N Yamamoto, S Asada, K Yoshida, H Munakata, C Hamanishi, INFLAMMATION RESEARCH, INFLAMMATION RESEARCH, 53(8), 344 - 350, Aug. 2004 , Refereed
    Summary:Objective:Because articular cartilage has no vascular supply, chondrocytes are hypoxic under normal physiological conditions. Nitric oxide (NO) plays an important role in chondrocyte damage, such as apoptosis. Although oxygen stress with hydrogen peroxide was found to cause chondrocyte damage, these data were obtained under normoxic (21% O-2) conditions. We investigated the effects of hypoxia on hydrogen peroxide-induced chondrocyte damage Methods:Bovine articular chondrocytes were used in this study. Proteoglycan (PG) synthesis and the induction of apoptosis were analyzed with [S-35]-sulfate incorporation and annexin V staining, respectively. The induction of NO was examined using a fluorescent probe and RT-PCR. Results:Cells maintained at 5% O-2 had the maximum PG synthesis. Under normoxic conditions, hydrogen peroxide inhibited PG synthesis and induced annexin V positive cells in a dose-dependent fashion. However, in those cells cultured under hypoxic (5%) conditions, the hydrogen peroxide-induced annexin V expression was attenuated. Chondrocytes exposed to hypoxia showed induction of NO. When the hypoxia-induced NO was inhibited, the hypoxia-enhanced PG synthesis was abolished and hydrogen peroxide clearly induced cell damage. Conclusions:Endogenous NO induced by hypoxia protects chondrocytes from apoptosis induced by an oxidative stress.
  • Oxidized low-density lipoprotein (ox-LDL) binding to lectin-like ox-LDL receptor-1 (LOX-1) in cultured bovine articular chondrocytes increases production of intracellular reactive oxygen species (ROS) resulting in the activation of NF-kappa B, S Nishimura, M Akagi, K Yoshida, S Hayakawa, T Sawamura, H Munakata, C Hamanishi, OSTEOARTHRITIS AND CARTILAGE, OSTEOARTHRITIS AND CARTILAGE, 12(7), 568 - 576, Jul. 2004
    Summary:Objective: To examine the effect of oxidized low-density lipoprotein (ox-LDL) on the intracellular production of reactive oxygen species (ROS) in bovine articular chondrocytes (BACs) and to investigate whether this increase occurs through binding to the receptor lectin-like ox-LDL receptor-1 (LOX-1). Furthermore, to ascertain whether the binding of ox-LDL to LOX-1 results in NF-kappaB activation. Design: BACs were preincubated with 2',7'-dichlorofluorescin diacetate (DCFH-DA), a dye that allows the monitoring of intracellular ROS production for DCF by spectrofluorometry. BACs were incubated with native LDL and ox-LDL (10, 50, and 100 mug/ml) for 5 min at 37degreesC and DCF formation was observed. BACs were also preincubated with anti-LOX-1 mAb (40 mug/ml) or ascorbic acid (10 t!V!). Nuclear extracts from BACs treated for the indicated periods with 50 mug/ml ox-LDL, and preincubated with anti-LOX-1 mAb or ascorbic acid, were prepared and analyzed by electrophoretic mobility shift assay (EMSA). Results: ox-LDL induced a significant dose-dependent increase in ROS production after 5-min incubation with BACs (P<0.001). ROS formation was markedly reduced in BACs preincubated with anti-LOX-1 mAb and ascorbic acid (P<0.001). Activation in BACs of the transcription factor NF-κB was evident after 5-min incubation with ox-LDL and was attenuated by anti-LOX-1 mAb and ascorbic acid. Conclusion: ox-LDL binding to LOX-1 in BACs increased the production of intracellular ROS and activated NF-κB. Reduction of NF-κB activation by ascorbic acid indicates that the activation, at least in part, is ROS-dependent. These observations support the hypothesis that hypercholesterolemia is one of several risk factors for arthritis, and that lipid peroxidation products such as ox-LDL are involved in cartilage matrix degradation. © 2004 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
  • Enhancement of nitric oxide and proteoglycan synthesis due to cyclic tensile strain loaded on chondrocytes attached to fibronectin, M Matsukawa, K Fukuda, K Yamasaki, K Yoshida, H Munakata, C Hamanishi, INFLAMMATION RESEARCH, INFLAMMATION RESEARCH, 53(6), 239 - 244, May 2004 , Refereed
    Summary:Objective:Mechanical stress is an essential factor in the pathogenesis of osteoarthrosis. We sought to determine whether the strain-mediated alteration in proteoglycan (PG) synthesis was modulated by nitric oxide (NO) synthesis. Methods:Cyclic tensile strain was applied to bovine articular chondrocytes. PG and NO synthesis were determined by [S-35] sulfate incorporation and chemiluminescence analysis, respectively. To determine the expression of inducible NO synthase (iNOS), quantitative RT-PCR was used. Results:Enhanced PG and NO synthesis were evident when cyclic tensile strain was applied to chondrocytes seeded on fibronectin-coated plates. When NO production was inhibited, PG synthesis was further enhanced. Conclusions:Cyclic tensile strain loaded on the chondrocytes enhanced NO synthesis and this enhanced NO inhibited PG synthesis.
  • Cyclic tensile stretch loaded on bovine chondrocytes causes depolymerization of hyaluronan - Involvement of reactive oxygen species, K Yamazaki, K Fukuda, M Matsukawa, F Hara, T Matsushita, N Yamamoto, K Yoshida, H Munakata, C Hamanishi, ARTHRITIS AND RHEUMATISM, ARTHRITIS AND RHEUMATISM, 48(11), 3151 - 3158, Nov. 2003 , Refereed
    Summary:Objective. We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. Decreased size of hyaluronan (HA), the major macromolecule in synovial fluid, to which it imparts viscosity, is reported in patients with arthritis. The purpose of this study was to determine the alteration in the molecular weight range of HA as a result of mechanical deformation loaded on the chondrocytes, as well as the involvement of ROS in this action. Methods. ROS were generated via the oxidation of hypoxanthine by xanthine oxidase. Cyclic tensile stretch was loaded using a vacuum-operated instrument. Levels of HA were measured using a sandwich enzyme-binding assay. Superoxide dismutase (SOD) activity and ROS were measured using water-soluble tetrazolium and a chemiluminescent probe, respectively. Results. ROS depolymerized HA molecules. Cyclic tensile stretch depolymerized HA and induced ROS. SOD inhibited not only ROS induction but also HA depolymerization caused by the mechanical stress. Conclusion. ROS play an important role in mechanical stress-induced HA depolymerization.
  • Reactive oxygen species depolymerize hyaluronan: involvement of the hydroxyl radical, Kenji Yamazaki, Kanji Fukuda, Masataka Matsukawa, Fumihiko Hara, Koji Yoshida, Masao Akagi, Hiroshi Munakata, Chiaki Hamanishi, Pathophysiology, Pathophysiology, 9(4), 215 - 220, Sep. 2003 , Refereed
  • Leucine-rich repeat region of decorin binds to filamin-A, K Yoshida, Y Suzuki, E Honda, K Amemiya, T Nakatani, M Ebina, K Narumi, K Satoh, H Munakata, BIOCHIMIE, BIOCHIMIE, 84(4), 303 - 308, Apr. 2002 , Refereed
    Summary:Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane. (C) 2002 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.
  • Molecular cloning and sequence analysis of C57BL/6 mouse contrapsin cDNA, K Yoshida, Y Suzuki, H Sinohara, DNA SEQUENCE, DNA SEQUENCE, 12(4), 289 - 291, 2001 , Refereed
    Summary:Contrapsin is a member of the serpin superfamily and inhibits trypsin much more strongly than alpha(1)-antiproteinase. Mouse and rat contrapsins, however, have similarity in sequence to human alpha(1)-antichymotrypsin. In order to test the hypothesis that reactive site regions of contrapsin family evolved under strong selective pressure, cDNA sequence of C57BL/6 mouse contrapsin was determined and compared with that of ICR mouse. The cDNA sequence of C57BL/6 mouse contrapsin was found to contain an open reading frame encoding polypeptide consisting of 418 amino acid residues. The work reported in this paper shows that the reactive site is not hypervariable as compared with the rest of molecule.
  • Molecular cloning and sequence analysis of C57BL/6 mouse contrapsin cDNA, Koji Yoshida, Yasuyuki Suzuki, Hyogo Sinohara, Mitochondrial DNA, Mitochondrial DNA, 12(4), 289 - 291, 2001 , Refereed
    Summary:Contrapsin is a member of the serpin superfamily and inhibits trypsin much more strongly than αi-antiproteinase. Mouse and rat contrapsins, however, have similarity in sequence to human α1 -antichymotrypsin. In order to test the hypothesis that reactive site regions of contrapsin family evolved under strong selective pressure, cDNA sequence of C57BL/6 mouse contrapsin was determined and compared with that of ICR mouse. The cDNA sequence of C57BL/6 mouse contrapsin was found to contain an open reading frame encoding polypeptide consisting of 418 amino acid residues. The work reported in this paper shows that the reactive site is not hypervariable as compared with the rest of molecule. © 2001 OPA (Overseas Publishen Aswciatinn) N V.
  • Molecular evolution in the hypervariable regions of fetuin: Comparison between human and African green monkey fetuin, K Yoshida, Y Suzuki, K Yamamoto, K Matsuura, M Watanabe, H Sinohara, BIOLOGICAL CHEMISTRY, BIOLOGICAL CHEMISTRY, 381(8), 773 - 776, Aug. 2000 , Refereed
    Summary:Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding alpha(1)-antitrypsin, beta-actin and the sequences of intron 4 of alpha(1)-antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.
  • Two type of aggregate in the cerebral cortex of a seizure-sensitive strain of the Mongolian gerbil, A Seto-Ohshima, M Katoh, S Yokota, N Karasawa, N Kawamura, S Kitajima, M Tsuzuki, K Yoshida, M Oh-Ishi, YL Murashima, M Onozuka, M Kishikawa, NEUROSCIENCE LETTERS, NEUROSCIENCE LETTERS, 277(3), 177 - 180, Dec. 1999 , Refereed
    Summary:A 70-kDa protein, P70, found mostly in the pyramidal cells of the cerebral cortex of cobalt-induced epileptogenic rats, has been-implicated in epileptogenesis. The presence of a P70-like substance was searched for immunohistochemically in the cerebral cortex of MGS/ldr, a seizure-sensitive strain of the Mongolian gerbil (Meriones unguiculatus) that we previously established. Immunoreactive aggregates were observed in the pyramidal neurons of the motor cortex and the primary somatosensory cortex. Analysis using confocal laser:scanning microscopy revealed that the aggregates were often colocalized with a second type of aggregate with red autofluorescence at the marginal zone of the cell somata. Both aggregates appeared and increased before the appearance of generalized tonic-clonic convulsion. These may be involved in some change of physiological function of the cerebral cortex but their presence itself is not enough to determine the occurrence of epileptic seizure because the gerbils that showed no such seizure had both aggregates. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • DNA-hydrolyzing activity of Bence Jones proteins, K Matsuura, S Ikoma, K Yoshida, H Sinohara, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 243(3), 719 - 721, Feb. 1998 , Refereed
    Summary:Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status. (C) 1998 Academic Press.
  • Molecular cloning and sequencing of cDNA encoding plasma countertrypin, a member of mammalian fetuin family, from the mongolian gerbil, Meriones unguiculatus, K Goto, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, JOURNAL OF BIOCHEMISTRY, JOURNAL OF BIOCHEMISTRY, 121(3), 619 - 625, Mar. 1997 , Refereed
    Summary:Complementary DNA clones coding for countertrypin were isolated from a liver cDNA library of the Mongolian gerbil, and sequenced, They contained one open reading frame encoding 348 amino acid residues, which were assigned to consist of an 18-residue signal peptide and a 330-residue mature protein, The amino acid sequence was about 74% identical with mouse countertrypin and rat fetuin, 60% with bovine fetuin, and 55% with human alpha(2)HS glycoprotein, indicating that this protein belongs to the mammalian fetuin family, The members of this family are known to consist of three domains, i.e., two tandemly arranged cystatin domains (D1 and D2) and an unrelated domain (D3) located at the C-terminal region, When compared with the other members of this family, D3, especially its N-terminal half, varies greatly with deletion or insertion as well as nucleotide substitutions even among three rodent species, i.e., gerbil, rat, and mouse, The sequence comparison also suggests that the conformation of human alpha(2)HS glycoprotein differs greatly from that of other members of this family, A molecular phylogenetic tree of 7 members, constructed on the basis of the synonymous substitution rate of D1 and D2, shows that the gerbil gene diverged prior to the separation of mouse and rat.
  • Sequencing of cDNA encoding serum albumin and its extrahepatic synthesis in the mongolian gerbil, Meriones unguiculatus, Koji Yoshida, Akiko Seto-Ohshima, Hyogo Sinohara, DNA Research, DNA Research, 4(5), 351 - 354, 1997 , Refereed
    Summary:We have sequenced serum albumin cDNA from liver of the Mongolian gerbil, Memories unguiculatus. The deduced amino acid sequence showed 82.6% and 73.6% identity with the corresponding proteins from rats and humans, respectively. Identical cDNA was detected in pancreas by reverse transcription followed by polymerase chain reaction (RT-PCR). Further amplification of cDNA by nested PCR revealed the presence of the same cDNA in the brain and kidney. These results indicate that serum albumin is expressed in some extrahepatic tissues. In rats, an albumin-related 70-kDa protein (P70) has been proposed to be associated with cobalt-induced epilepsy (Onozuka et al. (1995) Neurochem. Res., 20, 901-905). We intensively searched for a P70-like protein in the brain of an epilepsy-prone gerbil strain, MGS/Idr, by the RT-PCR and nested PCR using several pairs of primers based on the albumin cDNA sequence. However, we found only mRNA for albumin itself.
  • Cystatin-like domain of mouse countertrypin, a member of mammalian fetuin family, is responsible for the inhibition of trypsin. Evidence from site-directed mutagenesis, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, 39(5), 1023 - 1028, Aug. 1996 , Refereed
    Summary:Members of mammaliar, fetuin family, such as human alpha(2)HS glycoprotein and bovine fetuin, consist of three domains, two tandemly arranged cystatin-like domains and an unrelated domain, but they have no inhibitory activity against cysteine proteinases. We found that countertrypin, a novel trypsin inhibitor, is mouse counterpart of human alpha(2)HS glycoprotein, and that human alpha(2)HS glycoprotein and bovine fetuin which were prepared without use of ethanol are capable of inhibiting trypsin (Yamamoto, K. and Sinohara, H. (1993) J. Biol. Chem, 268, 17750-17753). In the present study, cDNA encoding countertrypin was isolated and sequenced, and evidence is presented, based on the site-directed mutagenesis, that lysine-231 in the second cystatin domain is the P1 site for trypsin inhibition.
  • SAME SEQUENCE BETWEEN OSTEOPONTIN AND URINARY STONE PROTEIN, K KOHRI, Y SUZUKI, K YOSHIDA, N AMASAKI, T YAMATE, T UMEKAWA, M IGUCHI, H SINOHARA, T KURITA, UROLITHIASIS 2, UROLITHIASIS 2, 281 - 283, 1994 , Refereed
  • SEQUENCING OF A URINARY STONE PROTEIN, IDENTICAL TO ALPHA-ONE ANTITRYPSIN, WHICH LACKS 22 AMINO-ACIDS, T UMEKAWA, K KOHRI, N AMASAKI, T YAMATE, K YOSHIDA, K YAMAMOTO, Y SUZUKI, H SINOHARA, T KURITA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 193(3), 1049 - 1053, Jun. 1993 , Refereed
  • MOLECULAR-CLONING AND SEQUENCING OF CDNA-ENCODING URINARY STONE PROTEIN, WHICH IS IDENTICAL TO OSTEOPONTIN, K KOHRI, Y SUZUKI, K YOSHIDA, K YAMAMOTO, N AMASAKI, T YAMATE, T UMEKAWA, M IGUCHI, H SINOHARA, T KURITA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 184(2), 859 - 864, Apr. 1992 , Refereed
  • ISOLATION AND PARTIAL CHARACTERIZATION OF A NOVEL FORM OF LOW-MOLECULAR WEIGHT KININOGEN FROM GUINEA-PIG PLASMA, K YOSHIDA, A SAITO, H SINOHARA, BIOCHEMISTRY INTERNATIONAL, BIOCHEMISTRY INTERNATIONAL, 19(6), 1339 - 1344, Dec. 1989 , Refereed

Conference Activities & Talks

  • Epigallocatechin Gallate inhibits IL-1-Induced Urokinase-Type Plasminogen Activator Expression and Collagen Degradation in Corneal Fibroblasts,   2020 09 14
  • Investigation of the effects of EGCG on Epithelial-Mesenchymal Transition (EMT) in Human Corneal Epithelial Cell stably overexpressing urokinase-type plasminogen activator receptor, Yuta Kimura, Kimi Sakata, Shota Hamano, Koji Yoshida, The 51st Annual Meeting of the Japanese Society for Matrix Biology and Medicine,   2019 05 31
  • The pathophysiology of lifestyle-related diseases,   2018 08 24
  • Investigation of the effects of epigallocatechin gallate on transforming growth factor-induced epithelial mesenchymal transition in renal tubular epithelial cells, SAKATA Kimi, HAMANO Shota, YOSHIDA Koji, The 50th Annual Meeting of the Jaapanese Society for Matrix Biology and Medicine,   2018 06 30
  • Investigation of the effects of Epigallocatechin gallate on Epithelial Mesenchymal Transition in Human Corneal Epithelial Cells, YOSHIDA Koji, HAMANO Shota, SAKATA Kimi, The 50th Annual Meeting of the Japanese Society for Matrix Biology and Medicine,   2018 06 30
  • Investigation of the effects of epigallocatechin gallate on transforming growth factor-beta - induced epithelial mesenchymal transition in human corneal epithelial cells, HAMANO Shota, SAKATA Kimi, YOSHIDA Koji, ConBio2017,   2017 12 07
  • Investigation of the effects of epigallocatechin gallate on, SAKATA Kimi, HAMANO Shota, YOSHIDA Koji, ConBio2017,   2017 12 07
  • The pathophysiology of lifestyle-related diseases, YOSHIDA Koji,   2017 08 24
  • Let's understand organ fibrosis - good and bad aspects of fibrosis, YOSHIDA Koji, BOST Science Café,   2017 06 24
  • TGF-β2 enhances urokinase type plasminogen activator (uPA), uPA receptor (uPAR) expression and binding activity of uPA to reinal pigment epithelial (RPE) cells and promotes RPE cell invasion into collagen gels by mediating uPA expression., ARVO 2013 Annual Meeting,   2013 05 , The Association for Reserch
  • Matrix metalloproteinase-2 cleaves α2-antiplasmin,   2012 12
  • Integrin alpha11 interacts with calcium and integrin binding protein 1,   2010 12
  • Role of Connective Tissue Growth Factor (CTGF) in Corneal Epithelial Migration, 19th Biennial Meeting of the International Society for Eye Research,   2010 07 , International society for eye research
  • Interaction between Connective Tissue Growth Factor and Fibronectin in Attachment and Migration of Corneal Epithelial Cells,   2009 04
  • Activation of myofibroblast (MRC-5) by TGF-b,   2008 12
  • The effects of transgelin knockdown on MRC-5 cells,   2008 12
  • Connective Tissue Growth Factor (CTGF) Effectively Facilitates TGF-β-induced Fibronectin Production in Human Corneal Fibroblasts, Kodama A, Sugioka K, Mishima H, Yoshida K, Shimomura Y, ARVO Annual Meeting,   2008 04
  • Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin,   2007 12
  • The interaction between aggrecanase-1 (ADAMTS-4) and α1-antitrypsin, 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress,   2006 06 , 20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
  • Connective tissue growth factor interacts with fibronectin,   2005 10
  • Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase, Munakata H, Sun JY, Yoshida K, Nakatani T, Honda E, Hayakawa S, Furuyama K, Hayashi N, First Congress of of the International Bioiron Society (IBIS),   2005 05
  • Aggrecanase-1 interacts with α1-antitrypsin,   2004 10
  • The search for the protein interacting with aggrecanase-1, HUPO 2nd Annual & IUBMB XIX World Congress,   2003 10 , HUPO 2nd Annual & IUBMB XIX World Congress
  • Leucine-rich repeat region of decorin binds to filamin-A,   2002 10
  • Inhibition by heme of mitochondrial import of 5-aminolevulinate synthase,   2001 10
  • The search for the protein interacting with aggrecanase-1,   2001 10
  • The search for the protein interacting with leucine-rich repeat region of decorin,   2000 10
  • FTY720 prolongs renal allograft survival in a rat model,   1998 06
  • Sequencing of cDNA encoding α-1-antiproteinase from Syrian hamster: its implication for the evolution of rodents, Tatsuya Nakatani, Atsuo Suzuki, Koji Yoshida, Hyogo Sinohara, 2nd World Congress on Inflammation,   1995 09
  • Cloning and sequence analysis of cDNAs encoding plasma α-macroglobulin and murinoglobulin from guinea pig, Hiromitsu Iwasaki, Atsuo Suzuki, Koji Yoshida, Hyogo Sinohara, 2nd World Congress on Inflammation,   1995 09
  • Isolation and sequence analysis of cDNAs coding for plasma α-macroglobulins from guinea pig and Syrian hamste, Hiromitsu Iwasaki, Atsuo Suzuki, Koji Yoshida, Hyogo Sinohara, Yoshimasa Miyake, 16th International Congress of Biochemistry,   1994 09
  • Plasma α-1-antiproteinases from Syrian hamster and Mongorian gerbil: isolation, partial characterization and sequence analysis of cDNA, Tatsuya Nakatani, Kana Goto, Koji Yoshida, Atsuo Suzuki, Syuji Amemiya, Kazuhuko Yamamoto, Hyogo Sinohara, 16th International Congress of Biochemistry,   1994 09
  • Sama sequence between osteopontin and urinary stone protein, Ⅶth International Symposium on Urolithiasis,   1992 08
  • Acute phase response of eight plasma proteinase inhibitors in the guinea pig. comparison with other mammals, Koji Yoshida, Yasuyuki Suzuki, Hyogo Sinohara, 15th International Congress of Biochemistry,   1991 08
  • Acute phase response of α-1-antiproteinase and kininogen in the guinea pig, YOSHIDA KojiAkio Saito, Yasuyuki Suzuki, Tetsuya Yamamoto, Hyogo Sinohara, 5th the Federation of Asian and Oceanian Biochemists Congress,   1989 08

Misc

  • The role of the interaction between TGF-β and integrin in the eye wound healing, YOSHIDA Koji,   2014 04
    Summary:When corneal fibroblast (keratocyte) was stimulated by TGF-beta, the keratocytes showed myofibroblast-like morphology. In addition, the expression of integrin alpha11 (ITGA11), alpha-smooth muscle actin (alpha-SMA), fibronectin in keratocytes was increased by TGF-beta treatment. Sensitivity to TGF-beta was up-regulated in the cells constitutively expressing ITGA11 or calcium-and integrin-binding protein 1. It was suggested that ITGA11 and CIB1 could be the target protein of the organ fibrosis treatment. Furthermore, we found that the green tea component epigallocatechin-3-gallate (EGCG) binds to TGF-beta type II receptor and inhibits the action of TGF-beta.
  • Roles of cytoskeletal proteins in organic fibrosis and their application of therapy in fibrotic diseases,   2011 03
  • 角膜上皮細胞の伸長、接着に対するconnective tissue growth factorとfibronectinの相互作用, 杉岡 孝二, 児玉 彩, 吉田 浩二, 三島 弘, 阿部 考助, 宗像 浩, 下村 嘉一, 日本生化学会大会・日本分子生物学会年会合同大会講演要旨集, 83回・33回, 1P, 0469,   2010 12
  • Basic approaches to the protective effect of decorin on pulmonary fibrosis, 39, 43,   2002
  • 5-アミノレブリン酸合成酵素のミトコンドリア移行のヘムによる阻害, 宗像 浩, 仲谷 達也, 本田 映子, 雨宮 科名, 吉田 浩二, 孫 継英, 古山 和道, 林 典夫, 生化学, 73, 8, 780, 780,   2001 08
  • デコリンの肺線維化抑制機能の基礎的研究, EBINA MASAHITO, MUNAKATA HIROSHI, NAKATANI TATSUYA, YOSHIDA KOJI, SAITO AKIO, NARUMI KO, SATO KEN, 臓器線維症における線維化抑制物質の誘発を活用した治療法開発に関する研究班 平成11年度研究報告書, 39, 43,   2000 , http://jglobal.jst.go.jp/detail.php?from=API&JGLOBAL_ID=200902103336535900
  • モルモットα2-HS glycoproteinのcDNA塩基配列決定と解析, 吉田 浩二, 鈴木 康之, 山本 和彦, 篠原 兵庫, 生化学, 70, 8, 813, 813,   1998 08
  • Connective tissue growth factor binds to fibronectin through the type I repeat modules and enhances the affinity of fibronectin to fibrin, Koji Yoshida, Hiroshi Munakata, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1770, 4, 672, 680,   2007 04 , 10.1016/j.bbagen.2006.11.010
    Summary:Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role. (c) 2006 Elsevier B.V. All rights reserved.
  • Aggrecanase-1 (ADAMTS-4) interacts with alpha 1-antitrypsin, K Yoshida, Y Suzuki, A Saito, K Fukuda, C Hamanishi, H Munakata, BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS, 1725, 2, 152, 159,   2005 09 , 10.1016/j.bbagen.2005.06.009
    Summary:In degradative articular diseases such as rheumatoid arthritis and osteoarthritis, loss of the extracellular matrix occurs, resulting in the destruction of joint cartilage. Proteolysis of aggrecan is one of the early events that leads to breakdown of the extracellular matrix. Aggrecanase-1.(ADAMTS-4) is considered to play a pivotal role in the abrasion of cartilage aggrecan in rheumatoid arthritis and osteoarthritis. To identify an endogenous inhibitor of aggrecanase-1, we performed a yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait and transformed an EGY48 yeast strain carrying the bait plasmid with a human liver cDNA library plasmid. This screen identified alpha 1-antitrypsin, a member of the family of plasma serine protemase inhibitors, as a prey. Recombinant aggrecanase-1 and alpha 1-antitrypsin were expressed in mammalian cells and used in coimmunoprecipitation experiments, which showed that full-length aggrecanase-1, and alpha 1-antitrypsin are also associated in vivo. However, aggrecanase-1 did not interfere with the inhibitory activity of otiantitrypsin against elastase, and a 1-antitrypsin had no effect on the proteolytic activity of aggrecanase-1. Taken together, these data suggest that aggrecanase-1 and alpha 1-antitrypsin bind in vivo, although the physiological significance of the interaction between aggrecanase-1 and alpha 1-antitrypsin remains unclear. (c) 2005 Elsevier B.V. All rights reserved.
  • Role of the heme regulatory motif in the heme-mediated inhibition of mitochondrial import of 5-aminolevulinate synthase, H Munakata, JY Sun, K Yoshida, T Nakatani, E Honda, S Hayakawa, K Furuyama, N Hayashi, JOURNAL OF BIOCHEMISTRY, 136, 2, 233, 238,   2004 08 , 10.1093/jb/mvh112
    Summary:5-Aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS1) is regulated by heme through a feedback mechanism. A short amino acid sequence, the heme regulatory motif (HRM), is known to be involved in the regulatory function of heme. To determine the role of the HRM in the heme-regulated transport of the nonspecific and erythroid forms of ALAS in vivo, we constructed a series of mutants of rat ALAS1, in which the cysteine residues in the three putative HRMs in the N-terminal region of the enzyme were converted to serine ones by site-directed mutagenesis. The wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, and the mitochondrial import of these enzymes was examined in the presence of hemin. Hemin inhibited the mitochondrial import of wild-type ALAS1, but this inhibition was reversed on the mutation of all three HRMs in the enzyme, indicating that the HRMs are essential for the heme-mediated inhibition of ALAS1 transport in the cell. By contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS isoform (AIAS2) under the same experimental conditions. These results may reflect the difference in the physiological functions of the two ALAS isoforms.
  • Cyclic tensile stretch loaded on bovine chondrocytes causes depolymerization of hyaluronan - Involvement of reactive oxygen species, K Yamazaki, K Fukuda, M Matsukawa, F Hara, T Matsushita, N Yamamoto, K Yoshida, H Munakata, C Hamanishi, ARTHRITIS AND RHEUMATISM, 48, 11, 3151, 3158,   2003 11 , 10.1002/art.11305
    Summary:Objective. We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. Decreased size of hyaluronan (HA), the major macromolecule in synovial fluid, to which it imparts viscosity, is reported in patients with arthritis. The purpose of this study was to determine the alteration in the molecular weight range of HA as a result of mechanical deformation loaded on the chondrocytes, as well as the involvement of ROS in this action. Methods. ROS were generated via the oxidation of hypoxanthine by xanthine oxidase. Cyclic tensile stretch was loaded using a vacuum-operated instrument. Levels of HA were measured using a sandwich enzyme-binding assay. Superoxide dismutase (SOD) activity and ROS were measured using water-soluble tetrazolium and a chemiluminescent probe, respectively. Results. ROS depolymerized HA molecules. Cyclic tensile stretch depolymerized HA and induced ROS. SOD inhibited not only ROS induction but also HA depolymerization caused by the mechanical stress. Conclusion. ROS play an important role in mechanical stress-induced HA depolymerization.
  • Reactive oxygen species depolymerize hyaluronan: involvement of the hydroxyl radical, Kenji Yamazaki, Kanji Fukuda, Masataka Matsukawa, Fumihiko Hara, Koji Yoshida, Masao Akagi, Hiroshi Munakata, Chiaki Hamanishi, Pathophysiology, 9, 4, 215, 220,   2003 09 , 10.1016/s0928-4680(03)00024-5
  • Leucine-rich repeat region of decorin binds to filamin-A, K Yoshida, Y Suzuki, E Honda, K Amemiya, T Nakatani, M Ebina, K Narumi, K Satoh, H Munakata, BIOCHIMIE, 84, 4, 303, 308,   2002 04 , 10.1016/S0300-9084(02)01391-3
    Summary:Decorin is a member of the family of small leucine-rich proteoglycans found in the extracellular matrix and has an important role in promoting fiber formation and in controlling cell proliferation. Here, we have investigated whether the leucine-rich repeat (LRR) region of decorin interacts with proteins from human lung fibroblasts by using a yeast two-hybrid assay. We report that the LRR region of decorin interacts with the cytoskeletal protein, filamin-A (ABP-280), a peripheral cytoplasmic protein. This interaction is dependent on the 288 carboxyl-terminal amino acids of filamin-A, which correspond to repeats 22-24 of its conserved beta-sheet structure. We also show that the recombinant LRR region of decorin binds to filamin-A in vitro, and that the deglycosylated core protein of decorin coprecipitates with filamin-A, whereas intact decorin does not. Together, these results suggest that proteins containing the LRR motif that interact with filamin-A may be present in the cytoplasm or at the plasma membrane. (C) 2002 Societe francaise de biochimie et biologie moleculaire/Editions scientifiques et medicales Elsevier SAS. All rights reserved.
  • Molecular cloning and sequence analysis of C57BL/6 mouse contrapsin cDNA, K Yoshida, Y Suzuki, H Sinohara, DNA SEQUENCE, 12, 4, 289, 291,   2001 , 10.3109/10425170109025005
    Summary:Contrapsin is a member of the serpin superfamily and inhibits trypsin much more strongly than alpha(1)-antiproteinase. Mouse and rat contrapsins, however, have similarity in sequence to human alpha(1)-antichymotrypsin. In order to test the hypothesis that reactive site regions of contrapsin family evolved under strong selective pressure, cDNA sequence of C57BL/6 mouse contrapsin was determined and compared with that of ICR mouse. The cDNA sequence of C57BL/6 mouse contrapsin was found to contain an open reading frame encoding polypeptide consisting of 418 amino acid residues. The work reported in this paper shows that the reactive site is not hypervariable as compared with the rest of molecule.
  • Molecular evolution in the hypervariable regions of fetuin: Comparison between human and African green monkey fetuin, K Yoshida, Y Suzuki, K Yamamoto, K Matsuura, M Watanabe, H Sinohara, BIOLOGICAL CHEMISTRY, 381, 8, 773, 776,   2000 08 , 10.1515/BC.2000.099
    Summary:Sequences of fetuin cDNA and its deduced amino acid residues from the African green monkey cell line Vero were found to differ by 7.3% and 12.9%, respectively, from the corresponding human sequences. Most amino acid substitutions were clustered within a small segment of the third domain (D3). Calculations of nonsynonymous and synonymous nucleotide substitution rates suggest that this small segment was mutated under positive selection. cDNAs encoding alpha(1)-antitrypsin, beta-actin and the sequences of intron 4 of alpha(1)-antitrypsin gene in human liver and Vero cells were also investigated. The results substantiated the positive selection imposed on the D3 segment.
  • Two type of aggregate in the cerebral cortex of a seizure-sensitive strain of the Mongolian gerbil, A Seto-Ohshima, M Katoh, S Yokota, N Karasawa, N Kawamura, S Kitajima, M Tsuzuki, K Yoshida, M Oh-Ishi, YL Murashima, M Onozuka, M Kishikawa, NEUROSCIENCE LETTERS, 277, 3, 177, 180,   1999 12 , 10.1016/S0304-3940(99)00873-3
    Summary:A 70-kDa protein, P70, found mostly in the pyramidal cells of the cerebral cortex of cobalt-induced epileptogenic rats, has been-implicated in epileptogenesis. The presence of a P70-like substance was searched for immunohistochemically in the cerebral cortex of MGS/ldr, a seizure-sensitive strain of the Mongolian gerbil (Meriones unguiculatus) that we previously established. Immunoreactive aggregates were observed in the pyramidal neurons of the motor cortex and the primary somatosensory cortex. Analysis using confocal laser:scanning microscopy revealed that the aggregates were often colocalized with a second type of aggregate with red autofluorescence at the marginal zone of the cell somata. Both aggregates appeared and increased before the appearance of generalized tonic-clonic convulsion. These may be involved in some change of physiological function of the cerebral cortex but their presence itself is not enough to determine the occurrence of epileptic seizure because the gerbils that showed no such seizure had both aggregates. (C) 1999 Elsevier Science Ireland Ltd. All rights reserved.
  • Guinea pig alpha(1)-microglobulin/bikunin: cDNA sequencing, tissue expression and expression during acute phase, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY, 122, 2, 165, 172,   1999 02 , 10.1016/S0305-0491(98)10149-9
    Summary:cDNA encoding alpha(1)-microglobulin/bikunin (AMBP) was amplified from guinea pig (Cavin porcellus) liver mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends methods, cloned and sequenced. The deduced amino acid sequence was found to be homologous to the sequence of AMBP of other mammals (69-76% amino acid identity). It has two Kunitz-type trypsin inhibitor domains in the bikunin part as reactive sites, one in the N-terminal region and another in the C-terminal region. The N-terminal inhibitor domain sequence is well-conserved, but the P1 residue of the C-terminal inhibitor domain sequence was found to be Gin rather than Arg, a residue highly conserved in the AMBP of seven other mammals examined to date. By RT-PCR and nested PCR, AMBP mRNA was detected not only in liver tissue, previously known to be a site of its synthesis, but also in pancreas, stomach, small intestine, colon, lung, spleen, kidney, testis, skeletal muscle, and leukocytes, but not in brain or heart. We examined the AMBP mRNA levels in guinea pig liver by RT-PCR, comparing normal levels and those in a state of inflammation. The mRNA levels, however, did not significantly change. (C) 1999 Elsevier Science Inc. All rights reserved.
  • Cloning and comparative sequence analysis of Xenopus laevis α1-antiproteinase, J. Biochem. Mol. Biol. Biophys., 3, 1, 59, 63,   1999
  • cDNA sequencing of guinea pig alpha(2)-HS glycoprotein, its expression in various tissues and acute phase expression, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, BIOLOGICAL CHEMISTRY, 380, 1, 95, 99,   1999 01 , 10.1515/BC.1999.013
    Summary:cDNA encoding alpha(2)-HS glycoprotein was amplified from guinea pig liver mRNA by reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends, cloned and sequenced. By RT-PCR and nested PCR, alpha(2)-HS glycoprotein mRNA was detected not only in liver tissue but also in pancreas, stomach, small intestine, colon, spleen, kidney, testis, skeletal muscle, brain, heart and leukocytes, but not in the lung. The alpha(2)-HS glycoprotein mRNA levels in the liver were reduced to half at 48 h after subcutaneous injection of turpentine oil.
  • DNA-hydrolyzing activity of Bence Jones proteins, K Matsuura, S Ikoma, K Yoshida, H Sinohara, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 243, 3, 719, 721,   1998 02 , 10.1006/bbrc.1998.8164
    Summary:Of 18 monoclonal Bence Jones proteins purified from urine of patients with multiple myeloma, 4 were found to have a DNA-nicking activity. In contrast, all Bence Jones proteins tested showed detectable amidase activity against carbobenzoxy-L-valyl-glycyl-L-arginine p-nitroanilide. No correlation between the two activities was found. Four patients excreting Bence Jones protein with the DNA-nicking activity showed somewhat severe symptoms, suggesting that this activity may be related to the progressive deterioration of clinical status. (C) 1998 Academic Press.
  • Molecular cloning and sequencing of cDNA encoding plasma countertrypin, a member of mammalian fetuin family, from the mongolian gerbil, Meriones unguiculatus, K Goto, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, JOURNAL OF BIOCHEMISTRY, 121, 3, 619, 625,   1997 03
    Summary:Complementary DNA clones coding for countertrypin were isolated from a liver cDNA library of the Mongolian gerbil, and sequenced, They contained one open reading frame encoding 348 amino acid residues, which were assigned to consist of an 18-residue signal peptide and a 330-residue mature protein, The amino acid sequence was about 74% identical with mouse countertrypin and rat fetuin, 60% with bovine fetuin, and 55% with human alpha(2)HS glycoprotein, indicating that this protein belongs to the mammalian fetuin family, The members of this family are known to consist of three domains, i.e., two tandemly arranged cystatin domains (D1 and D2) and an unrelated domain (D3) located at the C-terminal region, When compared with the other members of this family, D3, especially its N-terminal half, varies greatly with deletion or insertion as well as nucleotide substitutions even among three rodent species, i.e., gerbil, rat, and mouse, The sequence comparison also suggests that the conformation of human alpha(2)HS glycoprotein differs greatly from that of other members of this family, A molecular phylogenetic tree of 7 members, constructed on the basis of the synonymous substitution rate of D1 and D2, shows that the gerbil gene diverged prior to the separation of mouse and rat.
  • Sequencing of cDNA encoding serum albumin and its extrahepatic synthesis in the mongolian gerbil, Meriones unguiculatus, Koji Yoshida, Akiko Seto-Ohshima, Hyogo Sinohara, DNA Research, 4, 5, 351, 354,   1997 , 10.1093/dnares/4.5.351
    Summary:We have sequenced serum albumin cDNA from liver of the Mongolian gerbil, Memories unguiculatus. The deduced amino acid sequence showed 82.6% and 73.6% identity with the corresponding proteins from rats and humans, respectively. Identical cDNA was detected in pancreas by reverse transcription followed by polymerase chain reaction (RT-PCR). Further amplification of cDNA by nested PCR revealed the presence of the same cDNA in the brain and kidney. These results indicate that serum albumin is expressed in some extrahepatic tissues. In rats, an albumin-related 70-kDa protein (P70) has been proposed to be associated with cobalt-induced epilepsy (Onozuka et al. (1995) Neurochem. Res., 20, 901-905). We intensively searched for a P70-like protein in the brain of an epilepsy-prone gerbil strain, MGS/Idr, by the RT-PCR and nested PCR using several pairs of primers based on the albumin cDNA sequence. However, we found only mRNA for albumin itself.
  • Cystatin-like domain of mouse countertrypin, a member of mammalian fetuin family, is responsible for the inhibition of trypsin. Evidence from site-directed mutagenesis, K Yoshida, Y Suzuki, K Yamamoto, H Sinohara, BIOCHEMISTRY AND MOLECULAR BIOLOGY INTERNATIONAL, 39, 5, 1023, 1028,   1996 08
    Summary:Members of mammaliar, fetuin family, such as human alpha(2)HS glycoprotein and bovine fetuin, consist of three domains, two tandemly arranged cystatin-like domains and an unrelated domain, but they have no inhibitory activity against cysteine proteinases. We found that countertrypin, a novel trypsin inhibitor, is mouse counterpart of human alpha(2)HS glycoprotein, and that human alpha(2)HS glycoprotein and bovine fetuin which were prepared without use of ethanol are capable of inhibiting trypsin (Yamamoto, K. and Sinohara, H. (1993) J. Biol. Chem, 268, 17750-17753). In the present study, cDNA encoding countertrypin was isolated and sequenced, and evidence is presented, based on the site-directed mutagenesis, that lysine-231 in the second cystatin domain is the P1 site for trypsin inhibition.
  • MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF CDNA-ENCODING PLASMA ALPHA-1-ANTIPROTEINASE FROM SYRIAN-HAMSTER - IMPLICATIONS FOR THE EVOLUTION OF RODENTIA, T NAKATANI, Y SUZUKI, K YOSHIDA, H SINOHARA, BIOCHIMICA ET BIOPHYSICA ACTA-GENE STRUCTURE AND EXPRESSION, 1263, 3, 245, 248,   1995 09
    Summary:Complementary DNA clones encoding plasma alpha-1-antiproteinase (also called alpha-1-antitrypsin or alpha-1-proteinase inhibitor) were isolated from Syrian hamster liver cDNA library and sequenced. The deduced amino acid sequence of putative reactive site (P3-P'3) was Ile-Pro-Met-Ser-Val-Pro, characteristic of alpha-1-antiproteinase of orthodox type (Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932). A molecular phylogenetic tree of all known orthologous proteins was constructed based on the synonymous substitution rate. The result shows that the hamster has branched off first before the divergence among mice, rats, and gerbils, and that the rabbit is the closest relative of the guinea pig which is separated from the rodents. Although this tree differs largely from the classical phylogeny based on the morphology (hamsters and gerbils belong to the same family, Cricetidae, and the guinea pig belongs to the order Rodentia), it lends support to recent concepts that the hamster and guinea pig differ, in a number of biochemical features, not only from each other but also from mice and rats, and that the guinea pig may belong to an order distinct from Rodentia.
  • PLASMA ALPHA-1-ANTIPROTEINASE FROM THE MONGOLIAN GERBIL, MERIONES-UNGUICULATUS - ISOLATION, PARTIAL CHARACTERIZATION, SEQUENCING OF CDNA, AND IMPLICATIONS FOR MOLECULAR EVOLUTION, K GOTO, Y SUZUKI, K YOSHIDA, K YAMAMOTO, H SINOHARA, JOURNAL OF BIOCHEMISTRY, 116, 3, 582, 588,   1994 09
    Summary:alpha-1-Antiproteinase (also called alpha-1-proteinase inhibitor or alpha-1-antitrypsin) with a molecular mass of 56 kDa was purified from plasma of the Mongolian gerbil, Meriones unguiculatus, to apparent homogeneity. It inhibited trypsin, chymotrypsin, elastase, and plasmin, but not kallikrein or thrombin. Eight cDNA clones coding for this protein were isolated from a liver cDNA library and sequenced. They contained the same coding regions consisting of a 24-residue signal peptide and a 382-residue mature protein. The reactive site sequence (P3-P'3) was Val-Pro-Met-Ser-Ile-Pro, characteristic of alpha-1-antiproteinase of orthodox type [Suzuki, Y. et al. (1991) J. Biol. Chem. 266, 928-932]. A molecular phylogenetic tree of 11 orthologous inhibitors, constructed on the basis of the synonymous substitution rate, shows (i) that the reactive site region is highly conserved as compared to the other part of the molecule, which contrasts with the generally accepted view that the reactive site region of serpins is strongly hypervariable, and (ii) that the myomorphs (gerbil, rat, and two species of mouse, i.e. Mus domesticus and Mus caroli) and the caviomorph (guinea pig) fail to consist of a monophyletic order, which also contradicts the traditional taxonomy based on the morphology. In the present tree, the guinea pig joins the lagomorph (rabbit), and is rather widely separated from the myomorph branch. The result, however, supports the recent hypothesis based on the molecular evolution of several other proteins that the guinea pig does not belong to the same order as the myomorph, and the caviomorphs should be elevated in taxonomic rank and conferred an ordinal status distinct from the rodents.
  • SEQUENCING OF A URINARY STONE PROTEIN, IDENTICAL TO ALPHA-ONE ANTITRYPSIN, WHICH LACKS 22 AMINO-ACIDS, T UMEKAWA, K KOHRI, N AMASAKI, T YAMATE, K YOSHIDA, K YAMAMOTO, Y SUZUKI, H SINOHARA, T KURITA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 193, 3, 1049, 1053,   1993 06 , 10.1006/bbrc.1993.1731
  • MOLECULAR-CLONING AND SEQUENCING OF CDNA-ENCODING URINARY STONE PROTEIN, WHICH IS IDENTICAL TO OSTEOPONTIN, K KOHRI, Y SUZUKI, K YOSHIDA, K YAMAMOTO, N AMASAKI, T YAMATE, T UMEKAWA, M IGUCHI, H SINOHARA, T KURITA, BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 184, 2, 859, 864,   1992 04 , 10.1016/0006-291X(92)90669-C
  • MOLECULAR-CLONING AND SEQUENCE-ANALYSIS OF CDNAS CODING FOR GUINEA-PIG ALPHA-1-ANTIPROTEINASE-S AND ALPHA-1-ANTIPROTEINASE-F AND CONTRAPSIN, Y SUZUKI, K YOSHIDA, E HONDA, H SINOHARA, JOURNAL OF BIOLOGICAL CHEMISTRY, 266, 2, 928, 932,   1991 01
    Summary:The cDNAs encoding two isoforms, S (slow) and F (fast), of alpha1-antiproteinase (also referred to as alpha-1-antitrypsin or alpha-1-proteinase inhibitor) as well as contrapsin were obtained by screening lambda-gt 11 cDNA library prepared from inflamed guinea pig liver. The sequence analyses of these cDNAs and NH2-terminal peptides of the purified proteins revealed that both isoforms of alpha-1-antiproteinase consist of 405 amino acid residues including a signal peptide of 24 residues and that contrapsin consists of 410 amino acid residues with the same length of the signal peptide. Guinea pig contrapsin had 89, 88, 62, 42, and 41% homology to its own alpha-1-antiproteinases F and S, rat alpha-1-antiproteinase, mouse and rat contrapsins, respectively. This suggests that guinea pig contrapsin is not orthologous to mouse and rat contrapsins and that it developed from a much later duplication of alpha-1-antiproteinase gene after the guinea pig had diverged from the murine lineage. The available data suggest that the reactive site region of alpha-1-antiproteinase can be categorized into orthodox and unorthodox types: the former has P3-P'3 consensus sequence of Xaa-Pro-Met-Ser-Xaa-Pro, where Xaa is Leu, Ile, Val, or Met, while the latter, which occurs in species having multiple alpha-1-antiproteinase isoforms, has the sequence whose P1 Met has changed to other amino acids. Thus, the reactive site region of the orthodox type, which occurs in all seven mammals examined to date, is highly conserved. This is in marked contrast to the fact that the same region is hypervariable among the paralogous proteins belonging to the serpin super-family.
  • TRYPSIN-INHIBITORS IN GUINEA-PIG PLASMA - ISOLATION AND CHARACTERIZATION OF CONTRAPSIN AND 2 ISOFORMS OF ALPHA-1-ANTIPROTEINASE AND ACUTE PHASE RESPONSE OF 4 MAJOR TRYPSIN-INHIBITORS, Y SUZUKI, K YOSHIDA, T ICHIMIYA, T YAMAMOTO, H SINOHARA, JOURNAL OF BIOCHEMISTRY, 107, 2, 173, 179,   1990 02
  • ISOLATION AND PARTIAL CHARACTERIZATION OF A NOVEL FORM OF LOW-MOLECULAR WEIGHT KININOGEN FROM GUINEA-PIG PLASMA, K YOSHIDA, A SAITO, H SINOHARA, BIOCHEMISTRY INTERNATIONAL, 19, 6, 1339, 1344,   1989 12

Research Grants & Projects

  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), The role of the interaction between integrin and TGF-beta in the eye wound healing, When corneal fibroblast (keratocyte) was stimulated by TGF-beta, the keratocytes showed myofibroblast-like morphology. In addition, the expression of integrin alpha11 (ITGA11), alpha-smooth muscle actin (alpha-SMA), fibronectin in keratocytes was increased by TGF-beta treatment. Sensitivity to TGF-beta was up-regulated in the cells constitutively expressing ITGA11 or calcium-and integrin-binding protein 1. It was suggested that ITGA11 and CIB1 could be the target protein of the organ fibrosis treatment.Furthermore, we found that the green tea component epigallocatechin-3-gallate (EGCG) binds to TGF-beta type II receptor and inhibits the action of TGF-beta.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Roles of cytoskeletal proteins in organic fibrosis and their application of therapy in fibrotic diseases, Activation of myofibroblasts was inhibited when the expression of transgelin was knocked down. When human lung fibroblasts were stimulated by TGF-β, the integrin α11 (ITGA11) expression was markedly increased. Calcium and integrin binding protein 1 (CIB1) bound to the intracellular domain of ITGA11. The expression of CIB1 increased in human lung fibroblasts treated with TGF-β and in bleomycin-treated mice pulmonary tissues. These results suggest that CIB1 may be involved in the fibrosis.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Proteomic a-myofibroblastnalysis of activated- and inactivated, Previously we showed that the expression of integrin aω, β1, β3 in MRC-5 cells increased by TGF-β. Increased expression of fibronectin and focal adhesion kinase was also shown. Here, we show the results of proteomics analysis of activation and inactivation of myofibroblast cell line, MRC-5. Transforming growth factor-b stimulated MRC-5 cell extract was compared with that from non-stimulated cells. Several proteins differentially expressed between stimulated and non-stimulated cells including actin-bindin proteins and molecular chaperones.As previously reported the culture supernatant of A-549 inactivates myofibroblast MRC-5. RNA was obtained from MRC-5 cells after inactivation by the conditioned medium of A-549 and the expressions were analyzed using low density arrays. Expression of some proteins concerned in cell protrusions was increased. The cells treated with forskolin were subjected to proteome analysis. Inceased expression of actin capping protein was observed.These results, together with previous our findings, suggests that signal transduction mediated by interantions between extracellular matrix and integrins are important in activation and inactivation of myofibroblasts.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Research for the medical treatment of organic fibrosis by controlling the protein interacting with connective tissue growth factor, Connective tissue growth factor (CTGF) is a member of the CCN family of the cysteine-rich proteins and involved in wound healing and fibrosis. We have previously shown a biochemical interaction between the CTGF and fibronectin (FN) using the yeast two-hybrid system. In this study, we confirmed the interaction between the CTGF and FN using the surface plasmon resonance (SPR) and solid-phase binding analysis. Our results show that the regions containing the FN type I repeat modules (the N-terminal fibrin, the gelatin-collagen and the C-terminal fibrin binding domains) of FN and the C-terminal domain of CTGF are required for the interaction. We also demonstrated that CTGF enhances the affinity of FN to fibrin. It appears that CTGF contributes to the extracellular matrix accumulation in wound healing and tissue fibrosis by enhancing the affinity of FN to fibrin. Because CTGF is up-regulated during the tissue repair and in coagulation cascade-associated fibrotic disorders, the new function of CTGF found in this study is consistent with its physiological role."
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Research for the medical treatment of rheumatoid arthritis by endogenous inhibitor of aggrecanase, Aggrecan is greatly responsible for bad-bearing and shock-absorbing junction of the joint cartilage. In degradative articular diseases such as rheumatoid arthritis(RA) and osteoarthritis(OA), loss of extracellular matrices occurs resulting in the destruction of joint cartilage. Aggrecan proteolysis is one of the early events leading to the breakdown of the extracellular matrix. Matrix metalloproteinases have been reported to be the key enzymes in cartilage degeneration. Recent studies show that aggrecanase-1 and aggrecanase-2 are members of the ADAMTS(a disintegrin and metalloproteinase with thrombospondin motifs) family, and these enzymes are considered to play a pivotal role in the abrasion of cartilage aggrecan in RA or OA. Tissue inhibitor of matrix metalloproteinase-3(TIMP-3) is a potent inhibitor of aggrecanase-1 and -2, but whether TIMP-3 is the only inhibitor of aggrecanases remains unknown. The identification of a specific endogenous aggrecanase inhibitor therefore would be of critical importance in considering new medication for degradative joint diseases. The aims of the current study were to identify the endogenous inhibitor of aggrecanase-1 and to characterize the mechanism of interaction between aggrecanase-1 and its endogenous inhibitor We performed the yeast two-hybrid screen using the catalytic domain of human aggrecanase-1 as a bait, and an EGY48 yeast strain containing the bait plasmid was transformed with a human liver cDNA library plasmid. We identified alpha-1-antitrypsin and alpha-1-antichymotrypsin, members of plasma serine proteinase inhibitors, as preys.
  • Ministry of Education, Culture, Sports, Science and Technology, Grants-in-Aid for Scientific Research(基盤研究(C)), Molecular Mechanism of Posttranscriptional modification by Heme, 5-aminolevulinate synthase (ALAS) is a mitochondrial enzyme that catalyzes the first step of the heme biosynthetic pathway. The mitochondrial import, as well as the synthesis, of the nonspecific isoform of ALAS (ALAS-N) is regulated by heme. Recently, a short amino acid sequence, the heme regulatory motif (HRM), has been shown to be involved in the hemin inhibition of protein transport in vitro. To elucidate the role of HRM in the heme regulation of ALAS transport in vivo, we constructed a series of mutants of rat ALAS-N in which the specific cysteine residues within the HRMs were converted to serines by site-directed mutagenesis. Wild-type and mutant enzymes were expressed in quail QT6 fibroblasts through transient transfection, followed by analyses of the mitochondrial import of the enzymes. The heme inhibition, which was observed in the wild-type ALAS-N, abolished completely when all the three HRMs in the enzyme were mutated, indicating that the HRM is actually required for the heme inhibition of ALAS-N transport within the cells. In contrast, exogenous hemin did not affect the mitochondrial import of the erythroid-specific ALAS (ALAS-E) under the comparable experimental conditions. Mitochondrial import of the deletion mutant of ALAS-N in which 40 amino acids between HRM2 and HRM3 are removed was not affected by heme. Then, fusion protein of presequnce of ALAS-E and mature enzyme of ALAS-N was made. Heme inhibited the mitochondrial import of the fusion enzyme. These results may reflect the difference in the physiological function between two ALAS isoforms.
  • The search for the protein interacting with aggrecanase
  • The function of decorin