110 lines
No EOL
21 KiB
XML
110 lines
No EOL
21 KiB
XML
<?xml version="1.0" encoding="utf-8"?>
|
||
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
|
||
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">
|
||
|
||
<head><meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
|
||
<!-- AppResources meta begin -->
|
||
<meta name="paf-app-resources" content="" />
|
||
<script type="text/javascript">var ncbi_startTime = new Date();</script>
|
||
|
||
<!-- AppResources meta end -->
|
||
|
||
<!-- TemplateResources meta begin -->
|
||
<meta name="paf_template" content="" />
|
||
|
||
<!-- TemplateResources meta end -->
|
||
|
||
<!-- Logger begin -->
|
||
<meta name="ncbi_db" content="books" /><meta name="ncbi_pdid" content="book-part" /><meta name="ncbi_acc" content="NBK594037" /><meta name="ncbi_domain" content="glycopodv2" /><meta name="ncbi_report" content="printable" /><meta name="ncbi_type" content="fulltext" /><meta name="ncbi_objectid" content="" /><meta name="ncbi_pcid" content="/NBK594037/?report=printable" /><meta name="ncbi_app" content="bookshelf" />
|
||
<!-- Logger end -->
|
||
|
||
<title>Enzyme assay of glycolipid glycosyltransferases: GM2/GD2 synthase (β1,4-GalNAc transferase, B4GALNT1) - Glycoscience Protocols (GlycoPODv2) - NCBI Bookshelf</title>
|
||
|
||
<!-- AppResources external_resources begin -->
|
||
<link rel="stylesheet" href="/core/jig/1.15.2/css/jig.min.css" /><script type="text/javascript" src="/core/jig/1.15.2/js/jig.min.js"></script>
|
||
|
||
<!-- AppResources external_resources end -->
|
||
|
||
<!-- Page meta begin -->
|
||
<meta name="robots" content="INDEX,FOLLOW,NOARCHIVE" /><meta name="citation_inbook_title" content="Glycoscience Protocols (GlycoPODv2) [Internet]" /><meta name="citation_title" content="Enzyme assay of glycolipid glycosyltransferases: GM2/GD2 synthase (β1,4-GalNAc transferase, B4GALNT1)" /><meta name="citation_publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="citation_date" content="2022/03/22" /><meta name="citation_author" content="Koichi Furukawa" /><meta name="citation_author" content="Keiko Furukawa" /><meta name="citation_author" content="Yuhsuke Ohmi" /><meta name="citation_author" content="Robiul Bhuiyan" /><meta name="citation_pmid" content="37590759" /><meta name="citation_fulltext_html_url" content="https://www.ncbi.nlm.nih.gov/books/NBK594037/" /><meta name="citation_keywords" content="glycolipid" /><meta name="citation_keywords" content="complex ganglioside" /><meta name="citation_keywords" content="membrane preparation" /><meta name="citation_keywords" content="sugar nucleotides" /><meta name="citation_keywords" content="UDP-GalNAc" /><link rel="schema.DC" href="http://purl.org/DC/elements/1.0/" /><meta name="DC.Title" content="Enzyme assay of glycolipid glycosyltransferases: GM2/GD2 synthase (β1,4-GalNAc transferase, B4GALNT1)" /><meta name="DC.Type" content="Text" /><meta name="DC.Publisher" content="Japan Consortium for Glycobiology and Glycotechnology" /><meta name="DC.Contributor" content="Koichi Furukawa" /><meta name="DC.Contributor" content="Keiko Furukawa" /><meta name="DC.Contributor" content="Yuhsuke Ohmi" /><meta name="DC.Contributor" content="Robiul Bhuiyan" /><meta name="DC.Date" content="2022/03/22" /><meta name="DC.Identifier" content="https://www.ncbi.nlm.nih.gov/books/NBK594037/" /><meta name="description" content="Since molecular cloning of glycosyltransferase enzymes was successfully achieved, it has become possible to directly observe the reaction (1), substrate specificity (2), enzyme kinetics, and interaction with other molecules. Here, methods for measuring glycosyltransferase activity of GM2/GD2 synthase (β1,4-GalNAc transferase) were described using in vitro enzyme reaction using membrane preparation from cultured cells and tissues. This protocol applies not only to GM3 as an acceptor but also to GD3 and lactosylceramide. These methods were reported in the past articles (1–3)." /><meta name="og:title" content="Enzyme assay of glycolipid glycosyltransferases: GM2/GD2 synthase (β1,4-GalNAc transferase, B4GALNT1)" /><meta name="og:type" content="book" /><meta name="og:description" content="Since molecular cloning of glycosyltransferase enzymes was successfully achieved, it has become possible to directly observe the reaction (1), substrate specificity (2), enzyme kinetics, and interaction with other molecules. Here, methods for measuring glycosyltransferase activity of GM2/GD2 synthase (β1,4-GalNAc transferase) were described using in vitro enzyme reaction using membrane preparation from cultured cells and tissues. This protocol applies not only to GM3 as an acceptor but also to GD3 and lactosylceramide. These methods were reported in the past articles (1–3)." /><meta name="og:url" content="https://www.ncbi.nlm.nih.gov/books/NBK594037/" /><meta name="og:site_name" content="NCBI Bookshelf" /><meta name="og:image" content="https://www.ncbi.nlm.nih.gov/corehtml/pmc/pmcgifs/bookshelf/thumbs/th-glycopodv2-lrg.png" /><meta name="twitter:card" content="summary" /><meta name="twitter:site" content="@ncbibooks" /><meta name="bk-non-canon-loc" content="/books/n/glycopodv2/g67-assayb4galnt1/" /><link rel="canonical" href="https://www.ncbi.nlm.nih.gov/books/NBK594037/" /><link rel="stylesheet" href="/corehtml/pmc/css/figpopup.css" type="text/css" media="screen" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books.min.css" type="text/css" /><link rel="stylesheet" href="/corehtml/pmc/css/bookshelf/2.26/css/books_print.min.css" type="text/css" /><style type="text/css">p a.figpopup{display:inline !important} .bk_tt {font-family: monospace} .first-line-outdent .bk_ref {display: inline} </style><script type="text/javascript" src="/corehtml/pmc/js/jquery.hoverIntent.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/common.min.js?_=3.18"> </script><script type="text/javascript">window.name="mainwindow";</script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/book-toc.min.js"> </script><script type="text/javascript" src="/corehtml/pmc/js/bookshelf/2.26/books.min.js"> </script>
|
||
|
||
<!-- Page meta end -->
|
||
<link rel="shortcut icon" href="//www.ncbi.nlm.nih.gov/favicon.ico" /><meta name="ncbi_phid" content="CE8CAD227D6689010000000000BC0096.m_5" />
|
||
<meta name='referrer' content='origin-when-cross-origin'/><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3852956/3985586/3808861/4121862/3974050/3917732/251717/4216701/14534/45193/4113719/3849091/3984811/3751656/4033350/3840896/3577051/3852958/3984801/12930/3964959.css" /><link type="text/css" rel="stylesheet" href="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/css/3411343/3882866.css" media="print" /></head>
|
||
<body class="book-part">
|
||
<div class="grid no_max_width">
|
||
<div class="col twelve_col nomargin shadow">
|
||
<!-- System messages like service outage or JS required; this is handled by the TemplateResources portlet -->
|
||
<div class="sysmessages">
|
||
<noscript>
|
||
<p class="nojs">
|
||
<strong>Warning:</strong>
|
||
The NCBI web site requires JavaScript to function.
|
||
<a href="/guide/browsers/#enablejs" title="Learn how to enable JavaScript" target="_blank">more...</a>
|
||
</p>
|
||
</noscript>
|
||
</div>
|
||
<!--/.sysmessage-->
|
||
<div class="wrap">
|
||
<div class="page">
|
||
<div class="top">
|
||
|
||
<div class="header">
|
||
|
||
|
||
</div>
|
||
|
||
|
||
|
||
<!--<component id="Page" label="headcontent"/>-->
|
||
|
||
</div>
|
||
<div class="content">
|
||
<!-- site messages -->
|
||
<div class="container content">
|
||
<div class="document">
|
||
<div class="pre-content"><div><div class="bk_prnt"><p class="small">NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.</p><p>Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet]. Saitama (JP): Japan Consortium for Glycobiology and Glycotechnology; 2021-. </p></div></div></div>
|
||
<div class="main-content lit-style" itemscope="itemscope" itemtype="http://schema.org/CreativeWork"><div class="meta-content fm-sec"><h1 id="_NBK594037_"><span class="title" itemprop="name">Enzyme assay of glycolipid glycosyltransferases: GM2/GD2 synthase (β1,4-GalNAc transferase, B4GALNT1)</span></h1><div class="contrib half_rhythm"><span itemprop="author">Koichi Furukawa</span>, M.D., Ph.D.<div class="affiliation small">Department of Biomedical Sciences, Chubu University College of Life and Health Sciences<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.ubuhc.csi@ihciok" class="oemail">pj.ca.ubuhc.csi@ihciok</a></div></div><div class="small">Corresponding author.</div></div><div class="contrib half_rhythm"><span itemprop="author">Keiko Furukawa</span>, Ph.D.<div class="affiliation small">Department of Biomedical Sciences, Chubu University College of Life and Health Sciences<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.ubuhc.csi@ufokiek" class="oemail">pj.ca.ubuhc.csi@ufokiek</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Yuhsuke Ohmi</span>, Ph.D.<div class="affiliation small">Department of Medical Technology, Chubu University College of Life and Health Sciences<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="pj.ca.ubuhc.csi@28imuoO" class="oemail">pj.ca.ubuhc.csi@28imuoO</a></div></div></div><div class="contrib half_rhythm"><span itemprop="author">Robiul Bhuiyan</span>, Ph.D.<div class="affiliation small">Department of Biomedical Sciences, Chubu University College of Life and Health Sciences<div><span class="email-label">Email: </span><a href="mailto:dev@null" data-email="moc.liamg@97ibortsimehcoib" class="oemail">moc.liamg@97ibortsimehcoib</a></div></div></div><p class="small">Created: <span itemprop="datePublished">January 8, 2022</span>; Last Revision: <span itemprop="dateModified">March 22, 2022</span>.</p></div><div class="body-content whole_rhythm" itemprop="text"><p>
|
||
<b>Introduction</b>
|
||
</p><p>Since molecular cloning of glycosyltransferase enzymes was successfully achieved, it has become possible to directly observe the reaction (<a class="bk_pop" href="#g67-assayb4galnt1.REF.1">1</a>), substrate specificity (<a class="bk_pop" href="#g67-assayb4galnt1.REF.2">2</a>), enzyme kinetics, and interaction with other molecules. Here, methods for measuring glycosyltransferase activity of GM2/GD2 synthase (β1,4-GalNAc transferase) were described using <i>in vitro</i> enzyme reaction using membrane preparation from cultured cells and tissues. This protocol applies not only to GM3 as an acceptor but also to GD3 and lactosylceramide. These methods were reported in the past articles (<a class="bk_pop" href="#g67-assayb4galnt1.REF.1">1</a>–<a class="bk_pop" href="#g67-assayb4galnt1.REF.3">3</a>).</p><div id="g67-assayb4galnt1.Protocol"><h2 id="_g67-assayb4galnt1_Protocol_">Protocol</h2><p>In this chapter, the <i>in vitro</i> enzyme assay protocol will be described for determining the enzyme activity of GM2/GD2 synthase (β1,4GalNAc transferase) using membrane preparation of cultured cells or animal tissues (<a class="bk_pop" href="#g67-assayb4galnt1.REF.2">2</a>). Although this enzyme is responsible for the synthesis of GA2, GM2, GD2, and GT2 (<a class="figpopup" href="/books/NBK594037/figure/g67-assayb4galnt1.F1/?report=objectonly" target="object" rid-figpopup="figg67assayb4galnt1F1" rid-ob="figobg67assayb4galnt1F1">Figure 1</a>), this protocol will be described using GM3 as a representative acceptor (<a class="bk_pop" href="#g67-assayb4galnt1.REF.4">4</a>).</p><div id="g67-assayb4galnt1.Materials"><h3>Materials</h3><p>1. Sodium cacodylate (Wako Pure Chemical Industries Ltd., Osaka, Japan)</p><p>2. CDP-choline (Kohjin, Tokyo, Japan)</p><p>3. UDP-GalNAc (Sigma-Aldrich, St. Louis, MO)</p><p>4. GM3 (or GD3) as an acceptor (Sigma-Aldrich)</p><p>5. [<sup>14</sup>C]-UDP-GalNAc (NEN/PerkinElmer, Waltham, MA)</p><p>6. Extracts as an enzyme source</p><p>7. Triton CF54 (Sigma-Aldrich)</p></div><div id="g67-assayb4galnt1.Instruments"><h3>Instruments</h3><p>1. N2 cavitation apparatus</p><p>2. Water bath</p><p>3. N2 evaporator</p><p>4. Thin-layer chromatography (TLC) plate</p><p>5. Imaging analyzer</p><p>6. Ultracentrifuge and swing-type bucket</p><p>7. SepPak C18 cartridge (Waters Corp., Milford, MA)</p></div><div id="g67-assayb4galnt1.Methods"><h3>Methods</h3><dl class="temp-labeled-list"><dt>1.</dt><dd><p class="no_top_margin">Preparation of membrane fraction</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">N2 cavitation of cell pellets at 400 psi on ice for 30 min.</p></dd><dt>b.</dt><dd><p class="no_top_margin">Centrifuge at 1,000 rpm for 10 min at 4°C.</p></dd><dt>c.</dt><dd><p class="no_top_margin">Ultracentrifuge the supernatant at 34 K (Beckman, SW 55Ti) for 1 h at 4°C.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Resuspend the pellets in 0.1 M cacodylate buffer, pH 7.2.</p></dd></dl></dd><dt>2.</dt><dd><p class="no_top_margin">Enzyme assay</p><dl class="temp-labeled-list"><dt>a.</dt><dd><p class="no_top_margin">Evaporate the following items in a glass tube.</p><p>UDP-GalNAc (400 μM)</p><p>GM3 (or GD3) (325 μM)</p><p>[<sup>14</sup>C]-UDP-GalNAc (3.5 × 10<sup>5</sup> dpm)</p><p>CDP-choline (10 mM)</p></dd><dt>b.</dt><dd><p class="no_top_margin">Dissolve in 0.1 M cacodylate buffer (adjust to make final volume 50 μL).</p></dd><dt>c.</dt><dd><p class="no_top_margin">Add 10 mM of MnCl<sub>2</sub> and 0.3% Triton CF54 in cacodylate buffer.</p></dd><dt>d.</dt><dd><p class="no_top_margin">Sonicate for 10 s.</p></dd><dt>e.</dt><dd><p class="no_top_margin">Add membrane proteins (200 μg).</p></dd><dt>f.</dt><dd><p class="no_top_margin">Incubate at 37°C for 2–3 h with shaking.</p></dd><dt>g.</dt><dd><p class="no_top_margin">Add 1 mL of distilled water to stop the reaction.</p></dd><dt>h.</dt><dd><p class="no_top_margin">Separate the products by SepPak C18 column.</p></dd><dt>i.</dt><dd><p class="no_top_margin">Dry with N<sub>2</sub> stream.</p></dd><dt>j.</dt><dd><p class="no_top_margin">Count radioactivity of one-fifth of the products using a scintillation counter (<b>Note 1</b>).</p></dd><dt>k.</dt><dd><p class="no_top_margin">Analyze four-fifths of the products in TLC and autoradiography (<b>Note 2</b>).</p></dd></dl></dd></dl></div><div id="g67-assayb4galnt1.Notes"><h3>Notes</h3><p>1. Discussion</p><p>The enzyme activity is usually expressed as unit (pmol/mg/h) and corresponds well with the glycolipid expression on the cell surface (<a class="bk_pop" href="#g67-assayb4galnt1.REF.4">4</a>).</p><p>2. Low levels of the enzyme activity should be confirmed using autofluorography (<a class="bk_pop" href="#g67-assayb4galnt1.REF.4">4</a>).</p></div></div><div id="g67-assayb4galnt1.References"><h2 id="_g67-assayb4galnt1_References_">References</h2><dl class="temp-labeled-list"><dt>1.</dt><dd><div class="bk_ref" id="g67-assayb4galnt1.REF.1">Yamashiro S, Ruan S, Furukawa K, Tai T, Lloyd KO, Shiku H, Furukawa K. Genetic and enzymatic basis for the differential expression of GM2 andGD2 gangliosides in human cancer cell lines. <span><span class="ref-journal">Cancer Res. </span>1993 Nov 15;<span class="ref-vol">53</span>(22):5395–400.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/8221677" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 8221677</span></a>]</div></dd><dt>2.</dt><dd><div class="bk_ref" id="g67-assayb4galnt1.REF.2">Furukawa K, Tsuchida A, Okajima T, Furukawa K. Glycoconjugate glycosyl-transferases. <span><span class="ref-journal">Glycoconj J. </span>2009 Nov;<span class="ref-vol">26</span>(8):987–98.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/18683045" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 18683045</span></a>] [<a href="http://dx.crossref.org/10.1007/s10719-008-9156" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd><dt>3.</dt><dd><div class="bk_ref" id="g67-assayb4galnt1.REF.3">Furukawa K, Furukawa K. Enzyme assay of glycolipid glycosyltransferases ~GM2/GD2 synthase (β1,4GalNAc transferase). GlycoPOD, 2013. <a href="https://jcggdb.jp/GlycoPOD/protocolShow.action?nodeId=t54" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">https://jcggdb<wbr style="display:inline-block"></wbr>.jp/GlycoPOD/protocolShow<wbr style="display:inline-block"></wbr>.action?nodeId=t54</a>.</div></dd><dt>4.</dt><dd><div class="bk_ref" id="g67-assayb4galnt1.REF.4">Bhuiyan RH, Ohmi Y, Ohkawa Y, Zhang P, Takano M, Hashimoto N, Okajima T, Furukawa K, Furukawa K. Loss of enzyme activity of mutated <em>B4GALNT1</em> gene products found in patients with hereditary spastic paraplegia induces relatively mild neurological disorders: Similarity with phenotypes of <em>B4galnt1</em> knockout mice. <span><span class="ref-journal">Neuroscience. </span>2019 Jan 15;<span class="ref-vol">397</span>:94–106.</span> [<a href="https://pubmed.ncbi.nlm.nih.gov/30521973" ref="pagearea=cite-ref&targetsite=entrez&targetcat=link&targettype=pubmed">PubMed<span class="bk_prnt">: 30521973</span></a>] [<a href="http://dx.crossref.org/10.1016/j.neuroscience.2018.11.034" ref="pagearea=cite-ref&targetsite=external&targetcat=link&targettype=uri">CrossRef</a>]</div></dd></dl></div><h2 id="NBK594037_footnotes">Footnotes</h2><dl class="temp-labeled-list small"><dt></dt><dd><div id="g67-assayb4galnt1.FN1"><p class="no_top_margin">The authors declare no competing or financial interests.</p></div></dd></dl><div class="bk_prnt_sctn"><h2>Figures</h2><div class="whole_rhythm bk_prnt_obj bk_first_prnt_obj"><div id="g67-assayb4galnt1.F1" class="figure bk_fig"><div class="graphic"><img src="/books/NBK594037/bin/g67-assayb4galnt1-Image001.jpg" alt="Figure 1: . B4GALNT1 is a key enzyme for the synthesis of complex gangliosides." /></div><h3><span class="label">Figure 1: </span></h3><div class="caption"><p>B4GALNT1 is a key enzyme for the synthesis of complex gangliosides.</p><p>Loss of B4GALNT1 results in the loss of all complex gangliosides (<a class="bk_pop" href="#g67-assayb4galnt1.REF.4">4</a>).</p></div></div></div></div><div id="bk_toc_contnr"></div></div></div>
|
||
<div class="post-content"><div><div class="half_rhythm"><a href="/books/about/copyright/">Copyright Notice</a><p class="small">Licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 Unported license. To view a copy of this license, visit <a href="http://creativecommons.org/licenses/by-nc-nd/4.0/" ref="pagearea=meta&targetsite=external&targetcat=link&targettype=uri">http://creativecommons.org/licenses/by-nc-nd/4.0/</a>.</p></div><div class="small"><span class="label">Bookshelf ID: NBK594037</span><span class="label">PMID: <a href="https://pubmed.ncbi.nlm.nih.gov/37590759" title="PubMed record of this page" ref="pagearea=meta&targetsite=entrez&targetcat=link&targettype=pubmed">37590759</a></span></div><div style="margin-top:2em" class="bk_noprnt"><a class="bk_cntns" href="/books/n/glycopodv2/">Contents</a><div class="pagination bk_noprnt"><a class="active page_link prev" href="/books/n/glycopodv2/g66-assayb3galnt2/" title="Previous page in this title">< Prev</a><a class="active page_link next" href="/books/n/glycopodv2/g68-assayfut3/" title="Next page in this title">Next ></a></div></div></div></div>
|
||
|
||
</div>
|
||
</div>
|
||
</div>
|
||
<div class="bottom">
|
||
|
||
<div id="NCBIFooter_dynamic">
|
||
<!--<component id="Breadcrumbs" label="breadcrumbs"/>
|
||
<component id="Breadcrumbs" label="helpdesk"/>-->
|
||
|
||
</div>
|
||
|
||
<script type="text/javascript" src="/portal/portal3rc.fcgi/rlib/js/InstrumentNCBIBaseJS/InstrumentPageStarterJS.js"> </script>
|
||
</div>
|
||
</div>
|
||
<!--/.page-->
|
||
</div>
|
||
<!--/.wrap-->
|
||
</div><!-- /.twelve_col -->
|
||
</div>
|
||
<!-- /.grid -->
|
||
|
||
<span class="PAFAppResources"></span>
|
||
|
||
<!-- BESelector tab -->
|
||
|
||
|
||
|
||
<noscript><img alt="statistics" src="/stat?jsdisabled=true&ncbi_db=books&ncbi_pdid=book-part&ncbi_acc=NBK594037&ncbi_domain=glycopodv2&ncbi_report=printable&ncbi_type=fulltext&ncbi_objectid=&ncbi_pcid=/NBK594037/?report=printable&ncbi_app=bookshelf" /></noscript>
|
||
|
||
|
||
<!-- usually for JS scripts at page bottom -->
|
||
<!--<component id="PageFixtures" label="styles"></component>-->
|
||
|
||
|
||
<!-- CE8B5AF87C7FFCB1_0191SID /projects/books/PBooks@9.11 portal105 v4.1.r689238 Tue, Oct 22 2024 16:10:51 -->
|
||
<span id="portal-csrf-token" style="display:none" data-token="CE8B5AF87C7FFCB1_0191SID"></span>
|
||
|
||
<script type="text/javascript" src="//static.pubmed.gov/portal/portal3rc.fcgi/4216699/js/3879255/4121861/3501987/4008961/3893018/3821238/3400083/3426610.js" snapshot="books"></script></body>
|
||
</html> |