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RISS 인기검색어
- 검색키워드
- 저자 : David J. Harvey (검색결과 9 건)
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The dynamics of long-term transgene expression in engrafted neural stem cells
Lee, Jean-Pyo,Tsai, David J.,In Park, Kook,Harvey, Alan R.,Snyder, Evan Y. Wiley Subscription Services, Inc., A Wiley Company 2009 Journal of comparative neurology Vol.515 No.1
<P>To assess the dynamics and confounding variables that influence transgene expression in neural stem cells (NSCs), we generated distinct NSC clones from the same pool of cells, carrying the same reporter gene transcribed from the same promoter, transduced by the same retroviral vector, and transplanted similarly at the same differentiation state, at the same time and location, into the brains of newborn mouse littermates, and monitored in parallel for over a year in vivo (without immunosuppression). Therefore, the sole variables were transgene chromosomal insertion site and copy number. We then adapted and optimized a technique that tests, at the single cell level, persistence of stem cell-mediated transgene expression in vivo based on correlating the presence of the transgene in a given NSC's nucleus (by fluorescence in situ hybridization [FISH]) with the frequency of that transgene's product within the same cell (by combined immunohistochemistry [IHC]). Under the above-stated conditions, insertion site is likely the most contributory variable dictating transgene downregulation in an NSC after 3 months in vivo. We also observed that this obstacle could be effectively and safely counteracted by simple serial infections (as few as three) inserting redundant copies of the transgene into the prospective donor NSC. (The preservation of normal growth control mechanisms and an absence of tumorigenic potential can be readily screened and ensured ex vivo prior to transplantation.) The combined FISH/IHC strategy employed here for monitoring the dynamics of transgene expression at the single cell level in vivo may be used for other types of therapeutic and housekeeping genes in endogenous and exogenous stem cells of many organs and lineages. J. Comp. Neurol. 515:83–92, 2009. © 2009 Wiley-Liss, Inc.</P>
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Kim, Yun-Gon,Harvey, David J.,Yang, Yung-Hun,Park, Chung-Gyu,Kim, Byung-Gee John Wiley Sons, Ltd. 2009 Journal of mass spectrometry Vol.44 No.10
<P>Glycosphingolipid (GSL) is a major component of the plasma membrane in eukaryotic cells that is involved directly in a variety of immunological events via cell-to-cell or cell-to-protein interactions. In this study, qualitative and quantitative analyses of GSL-derived glycans on endothelial cells and islets from a miniature pig were performed and their glycosylation patterns were compared. A total of 60 and 47 sialylated and neutral GSL-derived glycans from the endothelial cells and islets, respectively, were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and collision-induced fragmentation using positive-ion electrospray ionization (ESI) ion-trap tandem mass spectrometry (MS/MS). In accordance with previous immunohistochemistry studies, the α-Gal-terminated GSL was not detected but NeuGc-terminated GSLs were newly detected from miniature pig islets. In addition, the neutral GSL-derived glycans were relatively quantified by derivatization with carboxymethyl trimethylammonium hydrazide (so called Girard's T reagent) and MALDI-TOF MS. The structural information of the GSL-derived glycans from pig endothelial cells and islets suggests that special attention should be paid to all types of glycoconjugates expressed on pig tissues or cells for successful clinical xenotransplantation. Copyright © 2009 John Wiley & Sons, Ltd.</P>
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Yun-Gon Kim,Dong-Sik Shin,David J. Harvey,Hee-Jin Jeong,Kyoung-Soon Jang,Yung-Hun Yang,Chung-Gyu Park,Pauline M. Rudd,Raymond A. Dwek,Yoon-Sik Lee,Byung-Gee Kim 한국당과학회 2008 한국당과학회 학술대회 Vol.2008 No.1
Glycan recognitionleading to cell-cell interactions, signaling, and immune responses is mediated by various glycan-binding proteins (GBPs) showing highly diverse ligand specificities. We describe here a rapid glycan immobilization technique via 4-hydrazinobenzoic acid (HBA)-functionalized beads and its application to high-throughput screening of miniature pig kidney N-glycan-binding proteins by using a mass-spectrometric approach. Firstly, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney were qualitatively and quantitatively identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N-glycans, including sialylated and neutral types, were identified. Without any derivatization steps, the characterizedpig kidney N-glycans were directly immobilized on to HBA-functionalized beads and subsequently used to identify GBPs from human serum. This screening method showed remarkable performance for identifying potential GBPs closely involved in pig-to-human xenograft rejection mediated by human serum, including antibodies, cytokines, complement components, siglec, and CD antigens. Thus, these results demonstrate that the GBP screening method was firmly established by one-step immobilization of the N-glycans on to microsphere and highly sensitive mass-spectrometric analysis.
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Kim, Yun-Gon,Gil, Geun-Cheol,Harvey, David J.,Kim, Byung-Gee WILEY-VCH Verlag 2008 Proteomics Vol.8 No.13
<P>The major barrier in transplantation of pig organs into humans is the presence of surface carbohydrate antigens (e.g., the Galα1-3Galβ1-4GlcNAc-R (α-Gal) epitope) expressed on pig endothelial cells. In this study, total N-glycans from membrane glycoproteins derived from specific pathogen-free miniature pig kidney are identified by MALDI-TOF, negative ion ESI MS/MS and normal-phase HPLC (NP-HPLC) combined with exoglycosidase digestion. Over 100 N-glycans, including sialylated and neutral types, were identified. As well as the known α-Gal antigens, some of these glycans contained novel non-Gal carbohydrate antigens such as (Neu5Gc-Gal-GlcNAc) and Galα1-3Lewis<SUP>x</SUP> (Gal-Gal-(Fuc)GlcNAc) which have not been reported before in N-glycans from pig organs. The ability of MALDI, ESI, and HPLC to measure the relative proportions of the glycans was evaluated. The HPLC resolution was insufficient for accurate work and some minor differences were noted in the ionization efficiencies of different glycan groups when measured by the two mass spectrometric techniques. However, the results indicated that the relative quantity of α-Gal epitope was in the region of 50% of the complex glycans. High-mannose type glycans were also abundant (35–43%) but appeared to be ionized more efficiently than the complex glycans by ESI than by MALDI.</P>
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Kim, Han Ie,Saldova, Radka,Park, Jun Hyoung,Lee, Young Hun,Harvey, David J.,Wormald, Mark R.,Wynne, Kieran,Elia, Giuliano,Kim, Hwa-Jung,Rudd, Pauline M.,Lee, Seung-Taek American Chemical Society 2013 JOURNAL OF PROTEOME RESEARCH Vol.12 No.8
<P>Tissue inhibitor of metalloproteinases-1 (TIMP-1) inhibits matrix metalloproteinases (MMPs) by binding at a 1:1 stoichiometry. Here we have shown the involvement of <I>N</I>-glycosylation in the MMP inhibitory ability of TIMP-1. TIMP-1, purified from HEK 293 cells overexpressing TIMP-1 (293 TIMP-1), showed less binding and inhibitory abilities to MMPs than TIMP-1 purified from fibroblasts or SF9 insect cells infected with TIMP-1 baculovirus. Following deglycosylation of TIMP-1, all forms of TIMP-1 showed similar levels of MMP binding and inhibition, suggesting that glycosylation is involved in the regulation of these TIMP-1 activities. Analysis of the <I>N</I>-glycan structures showed that SF9 TIMP-1 has the simplest <I>N</I>-glycan structures, followed by fibroblast TIMP-1 and 293 TIMP-1, in order of increasing complexity in their <I>N</I>-glycan structures. Further analyses showed that cleavage of outer arm fucose residues from the <I>N</I>-glycans of 293 TIMP-1 or knockdown of both FUT4 and FUT7 (which encode for fucosyltransferases that add outer arm fucose residues to <I>N</I>-glycans) enhanced the MMP-binding and catalytic abilities of 293 TIMP-1, bringing them up to the levels of the other TIMP-1. These results demonstrate that the ability of TIMP-1 to inhibit MMPs is at least in part regulated by outer arm fucosylation of its <I>N</I>-glycans.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/jprobs/2013/jprobs.2013.12.issue-8/pr400276r/production/images/medium/pr-2013-00276r_0011.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/pr400276r'>ACS Electronic Supporting Info</A></P>
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Kim, Yun-Gon,Kim, Sun-Young,Hur, Young-Mi,Joo, Hwang-Soo,Chung, Junho,Lee, Dong-Sup,Royle, Louise,Rudd, Pauline M.,Dwek, Raymond A.,Harvey, David J.,Kim, Byung-Gee WILEY-VCH 2006 Proteomics Vol. No.
<P>The immunogenic nonhuman carbohydrate sequences in membrane proteins from porcine kidney were identified and characterized using MALDI-TOF MS and ESI-QTOF-MS. The MALDI profile, investigated by incubation with exoglycosidases, showed a series of about 40 carbohydrates that were identified as high mannose glycans (Man<SUB>3–9</SUB>GlcNAc<SUB>2</SUB>) and complex bi-, tri-, and tetra-antennary glycans with and without core fucose. The antennae of many of the complex glycans were terminated with α-galactose residues, with the numbers of these residues ranging from one up to the number of antennae. Negative ion ESI-MS/MS spectra confirmed the location of the α-galactose residues on the ends of the antennae. This total glycan profile of the membrane proteins from porcine kidney will thus provide important information for the study of molecular interactions between antigenic carbohydrates and proteins in xenotransplantation.</P>
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Kim, Yun-Gon,Gil, Geun-Cheol,Jang, Kyung-Soon,Lee, Sukmook,Kim, Hyoung-Il,Kim, Jung-Sik,Chung, Junho,Park, Chung-Gyu,Harvey, David J.,Kim, Byung-Gee John Wiley Sons, Ltd. 2009 Journal of mass spectrometry Vol.44 No.7
<P>N-glycan structures released from miniature pig endothelial and islet cells were determined by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), negative ion electrospray ionization (ESI) MS/MS and normal-phase high performance liquid chromatography (NP-HPLC) combined with exoglycosidase digestion. Totally, the identified structures were 181 N-glycans including 129 sialylated and 18 α-galactosylated glycans from pig endothelial cells and 80 N-glycans including 41 sialylated and one α-galactosylated glycans from pig islet cells. The quantity of the α-galactosylated glycans from pig islet cells was certainly neglectable compared to pig endothelial cells. A number of NeuGc-terminated N-glycans (80 from pig endothelial cells and 13 from pig islet cells) are newly detected by our mass spectrometric strategies. The detailed structural information will be a matter of great interest in organ or cell xenotransplantation using α 1,3-galactosyltransferase gene-knockout (GalT-KO) pig. Copyright © 2009 John Wiley & Sons, Ltd.</P>
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