Showing papers by "Yoshiki Yamaguchi published in 2013"

A Single Dose of Lipopolysaccharide into Mice with Emphysema Mimics Human Chronic Obstructive Pulmonary Disease Exacerbation as Assessed by Micro-Computed Tomography

Abstract: Chronic obstructive pulmonary disease (COPD), manifested as emphysema and chronic airway obstruction, can be exacerbated by bacterial and viral infections. Although the frequency of exacerbations increases as the disease progresses, the mechanisms underlying this phenomenon are largely unknown, and there is a need for a simple in vivo exacerbation model. In this study, we compared four groups of mice treated with PBS alone, elastase alone, LPS alone, and elastase plus LPS. A single intratracheal administration of LPS to mice with elastase-induced emphysema provoked infiltration of inflammatory cells, especially CD8(+) T cells, into alveolar spaces and increased matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1, and perforin production in bronchoalveolar lavage fluid at the acute inflammatory phase compared with the other groups. We also measured the percentage of low-attenuation area (LAA%) in the above mice using micro-computed X-ray tomography. The LAA% was the most sensitive parameter for quantitative assessments of emphysema among all the parameters evaluated. Using the parameter of LAA%, we found significantly more severe alveolar destruction in the group treated with elastase plus LPS compared with the other groups during long-term longitudinal observations. We built three-dimensional images of the emphysema and confirmed that the lungs of elastase plus LPS-treated mice contained larger emphysematous areas than mice treated with elastase alone. Although human exacerbation of COPD is clinically and pathologically complicated, this simple mouse model mimics human cases to some extent and will be useful for elucidating its mechanism and developing therapeutic strategies.

Expression and structural characterization of anti-T-antigen single-chain antibodies (scFvs) and analysis of their binding to T-antigen by surface plasmon resonance and NMR spectroscopy

Abstract: T-antigen (Galβ1-3GalNAcα-1-Ser/Thr), also known as Thomsen-Friedenreich antigen (TF antigen), is an oncofetal antigen commonly found in cancerous tissues. Availability of anti-T-antigen human antibodies could lead to the development of cancer diagnostics and therapeutics. Four groups of single-chain variable fragment (scFv) genes were previously isolated from a phage library (Matsumoto-Takasaki et al. (2009) Isolation and characterization of anti-T-antigen single chain antibodies from a phage library. BioSci Trends 3:87-95.). Here, four anti-T-antigen scFv genes belonging to Group 1-4 were expressed and produced in a Drosophila S2 cell expression system. ELISA and surface plasmon resonance (SPR) analyses confirmed the binding activity of 1E8 scFv protein to various T-antigen presenting conjugates. NMR experiments provided evidence of the folded nature of the 1E8 scFv protein. ScFv-ligand contact was identified by STD NMR, indicating that the galactose unit of T-antigen at the non-reducing end was primarily recognized by 1E8 scFv. This thus provides direct evidence of T-antigen specificity.

Lectin-dependent inhibition of antigen-antibody reaction: application for measuring α2,6-sialylated glycoforms of transferrin.

Abstract: We developed a high-throughput Enzyme-linked immunosorbent assay (ELISA) for measuring α2,6-sialylated transferrin (Tf), based on inhibition of anti-Tf antibody binding to α2,6-sialylated Tf by a lectin, Sambucus sieboldiana Agglutinin (SSA). The inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform specific. This finding was applied to an automated latex-agglutination immunoassay, using SSA lectin as an inhibitor (SSA-ALI). The concentration of α2,6-sialylated Tf measured by SSA-ALI in human cerebrospinal fluid was correlated with that of ELISA (r2 = 0.8554), previously developed for measuring α2,6-sialylated Tf.

The Fifth ACGG-DB Meeting report : towards an international glycan structure repository

Abstract: Kiyoko FAoki-Kinoshita1, Hiromichi Sawaki2, Hyun Joo An3, Matthew Campbell4, Qichen Cao5, Richard Cummings6, Daniel K Hsu7, Masaki Kato8, Toshisuke Kawasaki9, Kay-Hooi Khoo7, Jaehan Kim3, Daniel Kolarich10, Xianyu Li5, Mingqi Liu11, Masaaki Matsubara12, Shujiro Okuda9,13, Nicolle H Packer4, Rene Ranzinger14, Huali Shen11, Toshihide Shikanai2, Daisuke Shinmachi2, Philip Toukach15, Issaku Yamada12, Yoshiki Yamaguchi8, Pengyuan Yang11, Wantao Ying5, Jong Shin Yoo16, Yan Zhang17, Yang Zhang11, and Hisashi Narimatsu2 Soka University, Tokyo, Japan; National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan; Chungnam National University, Daejeon, Korea; Macquarie University, Sydney, NSW, Australia; Beijing Institute of Radiation Medicine, Beijing, China; Emory University, Atlanta, GA, USA; Academia Sinica, Taipei, Taiwan; RIKEN Global Research Cluster, Wako-shi, Saitama, Japan; Ritsumeikan University, Kusatsu, Shiga, Japan; Max Planck Institute of Colloids and Interfaces, Potsdam, Germany; Fudan University, Shanghai, China; The Noguchi Institute, Tokyo, Japan; Niigata University, Niigata, Japan; The University of Georgia, Athens, GA, USA; Zelinsky Institute of Organic Chemistry, Moscow, Russia; Korea Basic Science Institute, Daejeon, Korea; and Shanghai Jiao Tong University, Shanghai, China

Structural Analysis of N-glycans attached to Pig Kidney Na+/K+-ATPase

Abstract: Na+/K+-ATPase is a membrane glycoprotein composed of α, β, and γ subunits, generating ion gradients across plasma membranes. Ion pumping is mainly accomplished by the α subunit, while the glycosylated β subunit binds tightly to the α subunit to assemble the pump and plays an essential role in the stabilization and maturation of Na+/K+-ATPase. Accumulating evidence suggests that the N-glycans of the β subunit contribute to cell-cell interaction and the tightness of cell contacts is modulated by N-glycan branching. However, N-glycan function is not fully understood due to a lack of detailed information on the oligosaccharide structure. We, here, perform glycosylation profi ling of the N-glycans attached to pig kidney Na+/K+-ATPase in order to better understand the mechanism of Na+/K+-ATPase-mediated cell adhesion. We purifi ed Na+/K+-ATPase from pig kidney outer medulla to homogeneity and solubilized it with the detergent C12E8. The enzyme thus obtained was identifi ed as α1β1 subtype by LC-MS/MS analysis of tryptic digests. During the course of MS analysis, we found that Lys456 of the α subunit was partially modifi ed with 4-hydroxynonenal, an aldehydric lipid peroxidation product. Three N-glycosylation sites on the β1 subunit were confi rmed to be fully occupied by time course analysis of enzymatic deglycosylation monitored by SDS-PAGE. HPLC profi ling of pyridylaminated oligosaccharides derived from Na+/K+-ATPase showed that high-mannose type oligosaccharides predominate while most of the less abundant complex-type oligosaccharides are capped with galactose residues. No glycans could be detected on the four consensus N-glycosylation sites on the α1 subunit. We briefl y discuss the possibility that oligomerization of Na+/K+- ATPase via β-β interactions assembles the N-glycans and promotes cell-cell adhesion.