Chemo Sero Therapeutic Research Institute

Kikuchi, Japan

Chemo Sero Therapeutic Research Institute

Kikuchi, Japan
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Shin Y.,Japan National Cardiovascular Center Research Institute | Shin Y.,Kogakuin University | Akiyama M.,Japan National Cardiovascular Center Research Institute | Kokame K.,Japan National Cardiovascular Center Research Institute | And 2 more authors.
Journal of Biochemistry | Year: 2012

The metalloprotease ADAMTS13 affects platelet adhesion and aggregation through depolymerization of von Willebrand factor (VWF) multimers. Identification of ADAMTS13-binding proteins would reveal the hitherto unrecognized mechanisms underlying microvascular thrombus. To identify ADAMTS13-binding proteins, we performed a yeast two-hybrid screen using the Cys-rich and spacer domains of ADAMTS13, the critical regions for the binding and cleavage of VWF, as a bait region. We identified Lys-plasminogen, an amino-terminal truncated form of plasminogen, as the binding protein to ADAMTS13. Intact Glu-plasminogen did not bind to ADAMTS13. Active-site blocked Lys-plasmin bound to ADAMTS13. Domain truncation of ADAMTS13 and elastase digest of plasminogen indicated that the Cys-rich and spacer domains of ADAMTS13 and the kringle 5 and protease domains of plasminogen served as the main binding sites. Biacore measurements revealed that Lys-plasminogen bound to ADAMTS13 with a Kd of 1.9 ± 0.1 × 10-7 M and Glu-plasminogen exhibited a significantly lower affinity to ADAMTS13. Specific activity measurements revealed that ADAMTS13 and Lys-plasmin were still active even after the binary complex was formed. The binding of ADAMTS13 to Lys-plasminogen may play an important role to localize these two proteases at sites of thrombus formation or vascular injury where the fibrinolytic system is activated. © 2012 The Authors.

PubMed | Monash University, Australian National University, Chemo Sero Therapeutic Research Institute, University of Birmingham and 2 more.
Type: | Journal: Journal of thrombosis and haemostasis : JTH | Year: 2017

Platelet GPVI binding to subendothelial collagen exposed upon blood vessel injury initiates thrombus formation. Dimeric GPVI has high affinity for collagen and occurs constitutively on resting platelets.To identify higher order oligomerisation (clustering) of pre-existing GPVI-dimers upon interaction with collagen as a mechanism to initiate GPVI-mediated signalling.GPVI was located using fluorophore-conjugated GPVI-dimer-specific Fab (antigen-binding fragment). Tested substrates include Horm collagen I fibres, soluble collagen III, GPVI-specific collagen peptides and fibrinogen. GPVI-dimer clusters on the platelet surface interacting with these substrates were visualised using complementary imaging techniques: Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor real time interactions and direct Stochastic Optical Reconstruction Microscopy (dSTORM), providing relative quantification of GPVI cluster size and density. Confocal microscopy was used to locate GPVI-dimer clusters, GPIb, integrin 21, and phosphotyrosine.Upon platelet adhesion to all collagenous substrates, GPVI-dimers coalesced to form clusters; notably clusters formed along the fibres of Horm collagen. dSTORM revealed that GPVI density within clusters depended on the substrate, collagen III being most effective. Clusters on fibrinogen-adherred platelets were much smaller and more numerous; whether these are pre-existing oligomers of GPVI-dimers or fibrinogen-induced is not conclusive. Some GPVI-dimer clusters colocalized with areas of phosphotyrosine, indicative of signalling activity. Integrin 21 localized to collagen fibres close to GPVI-dimer clusters. GPVI-clustering depends on a dynamic actin cytoskeleton.Platelet adhesion to collagen induces GPVI-dimer clustering. GPVI-clustering increases both avidity for collagen and proximity of GPVI-associated signalling molecules, which may be crucial for initiation and persistence of signalling. This article is protected by copyright. All rights reserved.

Takeda S.,Osaka National Research Institute | Takeya H.,Tottori University | Iwanaga S.,Chemo Sero Therapeutic Research Institute
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2012

Metalloproteinases are among the most abundant toxins in many Viperidae venoms. Snake venom metalloproteinases (SVMPs) are the primary factors responsible for hemorrhage and may also interfere with the hemostatic system, thus facilitating loss of blood from the vasculature of the prey. SVMPs are phylogenetically most closely related to mammalian ADAM (a disintegrin and metalloproteinase) and ADAMTS (ADAM with thrombospondin type-1 motif) family of proteins and, together with them, constitute the M12B clan of metalloendopeptidases. Large SVMPs, referred to as the P-III class of SVMPs, have a modular architecture with multiple non-catalytic domains. The P-III SVMPs are characterized by higher hemorrhagic and more diverse biological activities than the P-I class of SVMPs, which only have a catalytic domain. Recent crystallographic studies of P-III SVMPs and their mammalian counterparts shed new light on structure-function properties of this class of enzymes. The present review will highlight these structures, particularly the non-catalytic ancillary domains of P-III SVMPs and ADAMs that may target the enzymes to specific substrates. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. © 2011 Elsevier B.V. All rights reserved.

Masaki T.,Chemo Sero Therapeutic Research Institute | Masaki T.,Saga University | Ohkusu K.,Gifu University | Ezaki T.,Gifu University | Miyamoto H.,Saga University
Journal of Infection and Chemotherapy | Year: 2012

Nocardia elegans infection in humans is rare and is predominantly associated with pulmonary infections. We describe the first case of N. elegans infection associated with purulent arthritis in humans. The patient was a 66-year-old woman without underlying disease. She had swelling in her left ankle that was increasing in size, but it did not cause the patient substantial pain. Punctual discharge was collected for Gram staining and Kinyoun's acid-fast staining. The results of microscopic findings were suggestive of the genus Nocardia. The 16S rRNA sequence of the isolate was completely identical (100%) with that of N. elegans, indicating that the isolate was N. elegans. All the previously reported 4 cases of N. elegans infection in humans were associated with respiratory infections; we present the first case of the infection involving purulent arthritis. © Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2011.

Mochizuki S.,Keio University | Soejima K.,Chemo Sero Therapeutic Research Institute | Shimoda M.,Keio University | Abe H.,Keio University | And 4 more authors.
Journal of the National Cancer Institute | Year: 2012

Background A disintegrin and metalloproteinase 28 (ADAM28) is implicated in tumor growth and metastasis in human breast and non-small cell lung carcinomas. We explored the mechanism of ADAM28-mediated metastasis by searching for new substrates of ADAM28.MethodsWe used a yeast two-hybrid system to screen the human lung cDNA library for ADAM28-binding proteins and identified von Willebrand factor (VWF) as a potential candidate. Binding was confirmed using yeast two-hybrid and protein binding assays, and ADAM28-mediated cleavage of VWF was analyzed by immunoblotting. Exogenous VWF-induced apoptosis in vitro was examined in human lung carcinoma (PC-9 and Calu-3), breast carcinoma (MDA-MB231 and MCF-7), renal cell carcinoma (Caki-2 and 769P), and hepatocellular carcinoma (HepG2) cells, and expression of ADAM28 was assessed by reverse transcription-polymerase chain reaction and immunoblotting. Effect on lung metastasis of PC-9 and MDA-MB231 cells was assessed by knockdown of ADAM28 expression using short hairpin RNAs (ADAM28-shRNA) and small interfering RNAs (ADAM28-siRNA), and inhibition of activity using neutralizing anti-ADAM28 antibody, in a mouse xenograft model by in vivo imaging (n = 9 mice per group). All statistical tests were two-sided. Results ADAM28 could bind to and cleave native VWF. Cells with very low ADAM28 expression (MCF-7, 769P, and HepG2) were susceptible to VWF-induced apoptosis, whereas cells with high expression (PC-9, Calu-3, MDA-MB231, and Caki-2) were resistant. Knockdown of ADAM28 expression in PC-9 and MDA-MB231 cells by shRNA showed increased carcinoma cell apoptosis mainly in lung blood vessels and statistically significantly decreased lung metastasis at week 3 after injection (PC-9-control [n = 9 mice] vs PC-9-ADAM28-shRNA [n = 9 mice]: mean count = 198 × 10 6 vs 37 × 10 6 photons/s, difference = 161 × 10 6 photons/s, 95% confidence interval = 134 × 10 6 to 188 × 10 6 photons/s, P < .001). Similar inhibition of lung metastasis was observed with ADAM28-siRNA and anti-ADAM28 antibody. Conclusion ADAM28 cleaves and inactivates proapoptotic VWF in carcinoma cells and enhances lung metastasis probably by promoting carcinoma cell survival within the blood vessels. © 2012 The Author.

Higuchi A.,Keio University | Takahashi K.,Hokkaido University | Hirashima M.,Chemo Sero Therapeutic Research Institute | Kawakita T.,Keio University | Tsubota K.,Keio University
PLoS ONE | Year: 2010

The ocular surface is always attacked by oxidative stress, and cornea epithelial cells are supposed to have their own recovery system against oxidative stress. Therefore we hypothesized that tears supply key molecules for preventing oxidative stress in cornea. The potential target key molecule we focused is selenoprotein P (SeP). SeP is a carrier of selenium, which is an essential trace element for many animals, for oxidative stress metabolism in the organism, and was extremely expressed in lacrimal gland. An experiment was performed with SeP eye drops in a rat dry eye model, prepared by removing the lacrimal glands. The anticipated improvement in corneal dry eye index and the suppression of oxidative stress markers were observed in SeP eye drop group. Furthermore, the concentration of SeP was significantly higher in dry eye patients compared with normal volunteers. Collectively, we concluded that tear SeP is a key molecule to protect the ocular surface cells against environmental oxidative stress. © 2010 Higuchi et al.

Yasuda H.,Hokkaido University | Kuroda S.,Hokkaido University | Shichinohe H.,Hokkaido University | Kamei S.,Chemo Sero Therapeutic Research Institute | And 2 more authors.
Journal of Neurosurgery | Year: 2010

Object. In this study the authors' aim was to assess whether fibrin matrix could act as an injectable, valuable scaffold in bone marrow stromal cell (BMSC) transplantation for injured CNS tissue. Methods. Both clotting time and 3D structure of fibrin matrix were analyzed with various concentrations of fibrinogen and CaCl2. The BMSCs were harvested from green fluorescent protein-transgenic mice and cultured. A cortical lesion was produced in rats by application of a very cold rod to the right cerebral hemisphere. The BMSCs, fibrin matrix, or BMSC-fibrin matrix complex was transplanted into the lesion though a small bur hole 7 days after the insult. Using immunohistochemical analysis, the authors evaluated the survival, migration, and differentiation of the transplanted cells 4 weeks after transplantation. Results. Based on in vitro observations, the concentrations of fibrinogen and CaCl2 were fixed at 2.5 mg/ml and 2 μM in animal experiments, respectively. Fibrin matrix almost completely disappeared 4 weeks after transplantation. However, immunohistochemical analysis revealed that fibrin matrix exclusively enhanced the retention of the transplanted cells within the lesion, migration toward the lesion boundary zone, and differentiation into the neurons and perivascular cells. Conclusions. Injectable fibrin matrix enhanced the survival, migration, and differentiation of the BMSCs transplanted into the cortical lesion in rats. The authors believe that it is one of the promising candidates for a potential, minimally invasive scaffold for CNS disorders. The present findings strongly suggest that such a strategy of tissue engineering could be a therapeutic option for CNS regeneration in patients with CNS injuries.

Ahnstrom J.,Imperial College London | Andersson H.M.,Imperial College London | Hockey V.,Imperial College London | Meng Y.,Imperial College London | And 4 more authors.
Blood | Year: 2012

Protein S is a cofactor for tissue factor pathway inhibitor (TFPI) that critically reduces the inhibition constant for FXa to below the plasma concentration of TFPI. TFPI Kunitz domain 3 is required for this enhancement to occur. To delineate the molecular mechanism underlying enhancement of TFPI function, in the present study, we produced a panel of Kunitz domain 3 variants of TFPI encompassing all 12 surface-exposed charged residues. Thrombin-generation assays in TFPI-depleted plasma identified a novel variant, TFPI E226Q, which exhibited minimal enhancement by protein S. This was confirmed in purified FXa inhibition assays in which no protein S enhancement of TFPI E226Q was detected. Surface plasmon resonance demonstrated concentration- dependent binding of protein S to wildtype TFPI, but almost no binding to TFPI E226Q. We conclude that the TFPI Kunitz domain 3 residue Glu226 is essential for TFPI enhancement by protein S. © 2012 by The American Society of Hematology.

Kaminaka K.,Chemo sero therapeutic Research Institute | Matsuda J.-I.,Chemo sero therapeutic Research Institute | Nozaki C.,Chemo sero therapeutic Research Institute
Viral Immunology | Year: 2013

The amino acid sequence of the extracellular domain of matrix protein 2 (M2e) is conserved among all subtypes of influenza A viruses. Therefore, the M2e peptide can be considered as a target antigen for the development of a universal influenza vaccine. We evaluated the effects of adding cysteine residues to a peptide of amino acids 2-24 of M2e. Mice immunized with some of these peptides containing one, two, three, four, or five extra cysteines displayed enhanced antibody titers to M2e. In addition, immunization with a peptide containing three extra cysteines, along with an aluminum adjuvant, protected mice more effectively against a lethal influenza virus challenge than the original M2e peptide. These results indicated that an M2e peptide containing additional cysteine residues could be a universal influenza vaccine candidate even without the addition of strong adjuvants. © 2013, Mary Ann Liebert, Inc.

Hori Y.,Chemo Sero Therapeutic Research Institute | Hayakawa M.,Chemo Sero Therapeutic Research Institute | Isonishi A.,Chemo Sero Therapeutic Research Institute | Soejima K.,Chemo Sero Therapeutic Research Institute | And 2 more authors.
Transfusion | Year: 2013

Background Thrombotic thrombocytopenic purpura (TTP) is characterized by deficient ADAMTS13 activity. Treatment involves plasma exchange (PE). Both fresh-frozen plasma (FFP) and cryosupernatant (CSP) are used, but it remains to be determined which is more effective. Study Design and Methods To analyze the interaction between von Willebrand factor (VWF) and ADAMTS13, we used large-pore isoelectric focusing (IEF) analysis followed by detection with anti-ADAMTS13 monoclonal antibody. FFP, CSP, cryoprecipitate (CP), and purified ADAMTS13 were analyzed for their effects on high shear stress-induced platelet aggregation (H-SIPA). Results IEF analysis of normal plasma revealed three groups of ADAMTS13 bands with pI of 4.9 to 5.6, 5.8 to 6.7, and 7.0 or 7.5. Two band groups (pI 4.9-5.6 and 5.8-6.7) were found in plasma of a patient with Type 3 von Willebrand disease, in which VWF is absent, whereas no bands were found in plasma of a patient with congenital ADAMTS13 deficiency. Mixing these plasmas generated the bands at pI 7.0 or 7.5, representing the VWF-ADAMTS13 complex; these bands were absent in CSP. FFP and purified ADAMTS13 down regulated H-SIPA in a dose-dependent manner. However, CP did not inhibit H-SIPA in the initial phase, and the degree of inhibition at the endpoint was almost indistinguishable from those of the other two plasma products. Conclusion Both plasma products (FFP and CSP) are effective for PE in TTP patients. However, CSP may be more favorable, because it has lower levels of VWF and almost normal ADAMTS13 activity, but lower levels of ADAMTS13 in complex with larger VWF multimers. © 2013 American Association of Blood Banks.

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