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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. Source

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. Source

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. Source

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. Source

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. Source

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