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Guo R.,Tokyo Medical University | Hirano T.,Toda Chuo General Hospital | Kawakami T.,Tokyo Medical University | Kawakami T.,Medical ProteoScope Company | And 7 more authors.
Japanese Journal of Lung Cancer | Year: 2010

Objective. There is a need to establish clinically useful serum biomarkers for effective lung cancer screening. We investigated extracellular proteins secreted or shed from surgically resected lung adenocarcinoma tissue into its primary culture medium. Methods. In 12 lung adenocarcinoma cases, protein specimens from both culture supernatants of adenocarcinoma tissue and the normal peripheral lung (NPL) tissue were labeled with different fluorescent dyes (CyDye™: Cy2, Cy3 and Cy5) and co-resolved in single two-dimensional (2-D) gels. Quantitative protein profiles were obtained by differential fluorescence imaging of the gels. Results. The twelve pairs of 2-D gel images of both adenocarcinoma and NPL specimens revealed 34 distinct protein spots, detected in at least 5 gel images and highly concentrated in the adenocarcinoma cultures, on average more than double the concentration of analysis identified all of these protein spots, including napsin A and pulmonary surfactant-associated protein A which are responsible for the development of type II pneumocytes, and are the proteins commonly upregulated in tumor cells. Conclusion. These results suggest the usefulness of the tissue culture method to identify potential serum biomarkers to enable the early detection of lung adenocarcinoma. © 2010 The Japan Lung Cancer Society. Source

Kohroki J.,Tokyo University of Science | Kuroda S.,Tokyo University of Science | Takiguchi E.,Tokyo University of Science | Nakamura T.,Tokyo University of Science | And 5 more authors.
Biochemical and Biophysical Research Communications | Year: 2011

An alternative splicing variant of E3 ubiquitin ligase ASB2, termed ASB2a, has a distinct N-terminal sequence containing a ubiquitin-interacting motif (UIM) consensus sequence. Examination of the minimal essential region for binding to polyubiquitinated proteins indicated that the UIM consensus sequence (residues 26-41) alone is not enough, and that amino acids 12-41 from the N-terminus of ASB2a is essential for binding. ASB2a(12-41) peptide was chemically synthesized and coupled to Sepharose 4B via disulfide bonds. This ASB2a(12-41) peptide-coupled affinity resin bound both K48- and K63-linked polyubiquitinated proteins in cell lysates and comprehensively captured polyubiquitinated proteins, including polyubiquitinated β-catenin, I-κB, and EGF receptor, which were eluted with 2-mercaptoethanol under non-denaturing conditions. These results indicate that this UIM affinity purification (designated as ubiquitin-trapping) is a useful method to discover polyubiquitinated proteins and their associated proteins. © 2011 Elsevier Inc. Source

Nagata K.,Yokohama City University | Kawakami T.,Yokohama City University | Kawakami T.,Medical ProteoScope Company | Kurata Y.,Yokohama City University | And 6 more authors.
Journal of Proteomics | Year: 2015

Mutations in the Kit receptor tyrosine kinase gene (. KIT), which result in constitutive activation of the protein (KIT), are causally related to the development of gastrointestinal stromal tumors (GISTs). Imatinib, a targeted anticancer drug, exerts a therapeutic effect against GISTs by repressing the kinase activity of KIT. Long-term administration of this drug, however, causes the emergence of imatinib-resistant GISTs. We performed quantitative phosphoproteome analysis using a cell-based GIST model system comprising an imatinib-sensitive GIST cell line (GIST882), GIST882 under treatment with imatinib (GIST882-IM), and secondary imatinib-resistant GIST882 (GIST882-R). Phosphorylated peptides were purified from each cell line using titania-based affinity chromatography or anti-phosphotyrosine immunoprecipitation, and then subjected to LC-MS/MS based quantitative phosphoproteome analysis. Using this method we identified augmentation of the kinase activities of multiple elements of the signal transduction pathway, especially KIT and EGFR. Although, these elements were up-regulated in GIST882-R, no additionally mutated KIT mRNA was found in secondary imatinib-resistant GIST cells. Treatment of GIST882-R with imatinib in combination with gefitinib, an EGFR inhibitor, partially prevented cell growth, implying that EGFR may be involved in acquisition of secondary imatinib resistance in GIST. Biological significance: In this study, we performed a quantitative phosphoproteome analysis using a cell culture-based GIST model system. The goal of the study was to investigate the mechanism of acquired resistance in GISTs against imatinib, a molecularly targeted drug that inhibits kinase activity of the KIT protein and that has been approved for the treatment of GISTs. In imatinib-resistant GIST cells, we observed elevated expression of KIT and restoration of its kinase activity, as well as activation of multiple proliferative signaling pathways. Our results indicate that the effects of even so-called 'molecularly targeted' drugs, are broad rather than convergent, and that the mechanisms of action of such drugs during continuous administration are extremely complex. © 2014 Elsevier B.V. Source

Nishiyama T.,Tokyo University of Science | Kuroda S.,Tokyo University of Science | Takiguchi E.,Tokyo University of Science | Nakamura T.,Tokyo University of Science | And 7 more authors.
Biochemical and Biophysical Research Communications | Year: 2012

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2β. ASB2β has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2β but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2β and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2β protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2β, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2β is produced in cells. ASB2β with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2β M1) or a Ser38Ala mutation, (ASB2β M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2β monoubiquitination. Furthermore, an ASB2β mutant that lacked a SOCS box (ASB2β ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2β. In contrast, an ASB2β mutant that lacked the UIM-containing domain (ASB2β ΔN) was not monoubiquitinated. These results suggest that ASB2β but not ASB2α might be monoubiquitinated and that the ASB2β UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination. © 2012. Source

Sato K.,Central Institute for Experimental Animals | Oiwa R.,Central Institute for Experimental Animals | Kumita W.,Central Institute for Experimental Animals | Henry R.,Horizon Discovery | And 23 more authors.
Cell Stem Cell | Year: 2016

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the . IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications. In this article, Sato and colleagues describe the generation of an immunodeficient nonhuman primate model. By optimizing ZFN and TALEN targeting of the . IL2RG gene in marmoset fibroblasts and embryos, and using blastomere splitting as a proxy for rapid genome editing to reduce mosaicism, the authors generated founder animals with an immunodeficient phenotype similar to that of human patients with X-SCID. © 2016 Elsevier Inc. Source

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