Fukuda Functional Glycomics

1. Auflage 2010
ISBN: 978-0-12-380998-8
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 479, 496 Seiten

Reihe: Methods in Enzymology

In this 3 volume collection focusing on glycomics, readers will appreciate how such discoveries were made and how such methods can be applied for readers' own research efforts - Each chapter has been designed so that enough scientific background will be given in each chapter for further development of methods by readers themselves - Useful for all levels of scientists starting from the last years of colleges, graduate students, postdoctoral fellows to professors and to all levels of scientists in research institutes including industry

Autoren/Hrsg.

Fukuda, Minoru

Inhaltsverzeichnis

1;Front Cover;1
2;Methods in Enzymology: Functional Glycomics;4
3;Copyright Page;5
4;Contents;6
5;Contributors;14
6;Preface;20
7;Methods in Enzymology;22
8;Section one: Stem Cell;50
8.1;Chapter One: beta1,4-Galactosyltransferase V: A Growth Regulator in Glioma;52
8.1.1;1. Overview;53
8.1.2;2. Experimental;54
8.1.3;3. Results;57
8.1.4;4. Conclusion and Future Direction;67
8.1.5;Acknowledgments;71
8.1.6;References;71
8.2;Chapter Two: Roles of Polysialic Acid in Migration and Differentiation of Neural Stem Cells;74
8.2.1;1. Overview;75
8.2.2;Acknowledgments;83
8.2.3;References;83
8.3;Chapter Three: Structural and Functional Analysis of Chondroitin Sulfate Proteoglycans in the Neural Stem Cell Niche;86
8.3.1;1. Overview;88
8.3.2;2. Immunohistochemistry of Embryonic and Postnatal Mouse Brain Sections;91
8.3.3;3. Method to Stain Embryonic Sections for NSPC Markers;92
8.3.4;4. Analysis of the Adult Neurogenic Niche and SVZ-Derived Cells by Immunocytochemistry;93
8.3.5;5. Immunocytochemistry of Acutely Dissociated Cells;95
8.3.6;6. Isolation of NSPCs by Immunopanning or by Immunoisolation Using Paramagnetic Beads (EasySep);96
8.3.7;7. Neurosphere Cultures and Various Methods for Their Analysis;98
8.3.8;8. Disaccharide Analysis of CS/DS Chains from Embryonic Brain and Conditioned Neurosphere Culture Media and Effect of Sodium C;104
8.3.9;9. Analysis of NSPC-Proliferation In Vitro and In Vivo;105
8.3.10;10. Analysis of CSPG Functions in NSPCs Using Chondroitinase ABC Treatment in Culture;105
8.3.11;11. Analysis of Chondroitin Sulfate Functions in the Neural Stem Cell Niche;107
8.3.12;12. RT-PCR and Semiquantitative Analysis of the Synthetic Machinery for Glycosaminoglycans;108
8.3.13;13. In Situ Hybridization of Sulfotransferases in Tissue and Neurosphere Sections;112
8.3.14;14. Microscopy;115
8.3.15;15. Conclusion and Outlook;115
8.3.16;Acknowledgments;115
8.3.17;References;115
8.4;Chapter Four: Transcript Analysis of Stem Cells;122
8.4.1;1. Introduction;123
8.4.2;2. qRT-PCR as a Tool for Determining Transcript Analysis for Glycan-Related Gene Expression;124
8.4.3;3. Examples of the Applications of qRT-PCR Transcript Analysis to Investigate Changes in Glycan-Related Gene Expression in Ste;132
8.4.4;Acknowledgments;137
8.4.5;References;137
8.5;Chapter Five: Directing Stem Cell Trafficking via GPS;142
8.5.1;1. Overview;143
8.5.2;2. Rationale for GPS;144
8.5.3;3. Guiding Principles and Method for GPS;146
8.5.4;4. Method of a(1,3)-Fucosylation of Cell Surface Using FTVI;148
8.5.5;5. Detection of E-Selectin Ligand Expression Following GPS;149
8.5.6;6. Testing for E-Selectin Ligand Activity Using E-Selectin-Ig Chimera (Three-Step Method for Flow Cytometry);150
8.5.7;7. Summary;151
8.5.8;Acknowledgments;152
8.5.9;References;152
8.6;Chapter Six: Functional Assays for the Molecular Chaperone Cosmc;156
8.6.1;1. Overview;157
8.6.2;2. T-Synthase and Cosmc;159
8.6.3;3. T-Synthase Activity Assay;162
8.6.4;4. Assay Activity for Function of Cosmc;165
8.6.5;5. Conclusion and Future Directions;168
8.6.6;Acknowledgments;169
8.6.7;References;169
8.7;Chapter Seven: Core 3-Derived O-Glycans Are Essential for Intestinal Mucus Barrier Function;172
8.7.1;1. Introduction;173
8.7.2;2. Generation of C3GnT-/- Mice;175
8.7.3;3. Disruption of the C3GnT Gene Eliminates Core 3-Derived O-Glycans and Exposes the Tn Antigen in Murine Colon;177
8.7.4;4. Deficiency of C3GnT Results in Reduced Muc2 Expression in Colon and Impaired Mucosal Integrity;180
8.7.5;5. C3GnT-/- Mice are Highly Susceptible to Dextran Sodium Sulfate-Induced Colitis;182
8.7.6;6. Conclusion and Future Direction;187
8.7.7;Acknowledgments;189
8.7.8;References;189
8.8;Chapter Eight: Core3 Glycan as Tumor Suppressor;192
8.8.1;1. Overview;193
8.8.2;2. Generating Core3 Glycan Expressing Cell Lines;194
8.8.3;3. Detection Methods of Core3 Expression;195
8.8.4;4. Migration Assay Using Attractant;196
8.8.5;5. Determination of Major Integrin Using Functional Blocking Antibodies;196
8.8.6;6. Tumor Formation Assay;198
8.8.7;7. Western Blotting and Lectin Blotting;199
8.8.8;8. Heterodimerization Assay;200
8.8.9;9. FAK Signaling;201
8.8.10;References;202
8.9;Chapter Nine: Characterization of Mice with Targeted Deletion of the Gene Encoding Core 2 beta1,6-N-Acetylglucosaminyltransferas;204
8.9.1;1. Introduction;205
8.9.2;2. Generation and Genotyping of C2GnT2 KO Mice;206
8.9.3;3. Mice Lacking C2GnT2 Have Reduced Core 2 and no Core 4 Enzyme Activity and Altered Glycosylation;208
8.9.4;4. Phenotyping C2GnT2 KO Mice;210
8.9.5;5. Conclusions and Future Directions;217
8.9.6;Acknowledgments;219
8.9.7;References;219
8.10;Chapter Ten: Analyzing Physiological Function of Polypeptide GalNAcT-1-Deficient Mice in Humoral Immunity;222
8.10.1;1. Overview;223
8.10.2;2. Assay for In Vitro B Lymphocyte Activation;223
8.10.3;3. Assay for In Vivo Antibody Production;224
8.10.4;4. Immunohistochemical Staining of Frozen Sections;226
8.10.5;5. Apoptosis Detection Using Antibody Against Caspase 3 Active Form;228
8.10.6;6. Apoptosis Detection by TUNEL System;231
8.10.7;Acknowledgment;232
8.10.8;References;232
8.11;Chapter Eleven: beta3GnT2 (B3GNT2), a Major Polylactosamine Synthase: Analysis of B3gnt2-Deficient Mice;234
8.11.1;1. Overview;235
8.11.2;2. Glycogenes for Polylactosamine Synthesis (i.e., beta1,3-N-Acetylglucosaminyltransferase Genes);235
8.11.3;3. N-Glycan Polylactosamine is Greatly Reduced in B3gnt2-/- Mice;236
8.11.4;4. Phenotype of B3gnt2-/- Lymphocytes Lacking Polylactosamine on N-Glycans;242
8.11.5;5. Protocols;246
8.11.6;Acknowledgments;251
8.11.7;References;251
8.12;Chapter Twelve: Targeted Genetic Inactivation of N-Acetylglucosaminyltransferase-IVa Impairs Insulin Secretion from Pancreatic be;254
8.12.1;1. Overview;255
8.12.2;2. Engineering GnT-IVa-Deficient Mice;256
8.12.3;3. GnT-IV Enzymology;258
8.12.4;4. Glucose and Insulin Homeostasis;259
8.12.5;5. Immunohistochemical Analysis;260
8.12.6;6. Islet Cell Preparation and Culture;263
8.12.7;7. Pulse-Chase Labeling;264
8.12.8;8. Cell Surface Half-Life Time of GLUT2;265
8.12.9;9. GLUT2 Glycan Analysis by Lectin Blot;266
8.12.10;10. Cell-Surface Protein Cross-Linking;267
8.12.11;Acknowledgment;269
8.12.12;References;269
8.13;Chapter Thirteen: The Ashwell-Morell Receptor;272
8.13.1;1. Overview;273
8.13.2;2. Endogenous Ligands of the AMR;276
8.13.3;3. Hepatocytes in Molecular Clearance Mechanisms of the Liver;277
8.13.4;4. Genotyping HL-1- or HL-2-Deficient Mice;278
8.13.5;5. Hematology and Coagulation Analyses Methods (Partly Adapted from Ellies et al., 2002; Grewal et al., 2008; Wang et al., 200;279
8.13.6;Acknowledgments;285
8.13.7;References;285
8.14;Chapter Fourteen: Roles of GlcNAc-6-O-Sulfotransferases in Lymphoid and Nonlymphoid Tissues;292
8.14.1;1. Overview;293
8.14.2;2. Establishment of a Mouse Colon Adherent Cell Line and Cell Culture;295
8.14.3;3. Treatment of CAdC1 Cells with SCFAs;296
8.14.4;4. RT-PCR;296
8.14.5;5. Histology and Immunostaining;297
8.14.6;6. Preparation of Colonic-Mucin-Enriched Fraction;298
8.14.7;7. Carbohydrate Structural Analysis;299
8.14.8;8. Induction of Colitis by Dextran Sulfate Sodium;303
8.14.9;Acknowledgments;304
8.14.10;References;304
8.15;Chapter Fifteen: Core O-Glycans Required for Lymphocyte Homing: Gene Knockout Mice of Core 1 beta1,3-N-Acetylglucosaminyltransfera;306
8.15.1;1. Introduction;307
8.15.2;2. Lymphocyte Homing Assay;309
8.15.3;3. Staining of Lymph Nodes by L- and E-Selectin-IgM Chimeric Proteins after Glycosidase Treatment;312
8.15.4;4. Probing of L-Selectin Ligands on Membrane Filters with L- and E-selectin-IgM Chimeric Proteins;314
8.15.5;5. Conclusions;316
8.15.6;Acknowledgments;318
8.15.7;References;318
8.16;Chapter Sixteen: Immunohistochemical Analysis of Carbohydrate Antigens in Chronic Inflammatory Gastrointestinal Diseases;320
8.16.1;1. Overview;321
8.16.2;2. Immunohistochemical Analysis Using Conventional Immunostaining;323
8.16.3;3. Immunohistochemical Analysis Using Multiple Immunostaining;328
8.16.4;4. Immunohistochemical Analysis Using L-SelectinIgM Chimera Binding;332
8.16.5;Acknowledgments;336
8.16.6;References;336
8.17;Chapter Seventeen: Genetic Defects in Muscular Dystrophy;340
8.17.1;1. Overview;341
8.17.2;2. Mouse Models of Muscular Dystrophy;343
8.17.3;3. Approach to Phenotype Analysis in Mouse Muscular Dystrophy Models;355
8.17.4;4. Summary;361
8.17.5;Acknowledgments;361
8.17.6;References;361
8.18;Chapter Eighteen: POMT1 is Essential for Protein O-Mannosylation in Mammals;372
8.18.1;1. Overview;373
8.18.2;2. Experimental;374
8.18.3;3. Discussion/Conclusions;386
8.18.4;Acknowledgments;388
8.18.5;References;389
8.19;Chapter Nineteen: POMGnT1, POMT1, and POMT2 Mutations in Congenital Muscular Dystrophies;392
8.19.1;1. Overview;393
8.19.2;2. Methods;394
8.19.3;3. Procedures for Enzymatic Activity and Mutation Search;397
8.19.4;Acknowledgment;399
8.19.5;References;399
8.20;Chapter Twenty: Cellular and Molecular Characterization of Abnormal Brain Development in Protein O-Mannose N-Acetylglucosaminyltr;402
8.20.1;1. Overview;403
8.20.2;2. Analysis of a-DG Glycosylation and Laminin Binding by Western Blot;404
8.20.3;3. Histological Analysis of POMGnT1 Knockout Brain;405
8.20.4;4. Lamination Defects in the Neocortex of POMGnT1 Knockout Mice;406
8.20.5;5. Analysis of the Pial Basement Membrane by Laminin Immunostaining;408
8.20.6;6. Analysis of the Pial Basement Membrane by Transmission Electron Microscopy;409
8.20.7;7. Analysis of the Glia Limitans by GFAP Immunofluorescence Staining;411
8.20.8;Acknowledgments;413
8.20.9;References;414
8.21;Chapter Twenty-One: Investigating the Functions of LARGE: Lessons from Mutant Mice;416
8.21.1;1. Overview;417
8.21.2;2. Human LARGE and Relevance to Disease;418
8.21.3;3. Identification of Mice Carrying Mutations in Large;419
8.21.4;4. Loss of Functional Large Protein Results in Hypoglycosylation of a-Dystroglycan;422
8.21.5;5. Phenotypes of Mice with Mutations in Large;423
8.21.6;6. Expression of LARGE Genes;427
8.21.7;7. Does Large Encode a Functional Glycosyltransferase?;428
8.21.8;8. Largemyd Mice as a Model for Therapeutic Approaches to Dystroglycanopathy;430
8.21.9;Acknowledgments;430
8.21.10;References;430
8.22;Chapter Twenty - Two: A Tumor Suppressor Function of Laminin-Binding a-Dystroglycan;436
8.22.1;1. Background;437
8.22.2;2. Methods;438
8.22.3;Acknowledgment;444
8.22.4;References;444
8.23;Chapter Twenty - Three: Tumor Formation Assays;446
8.23.1;1. Overview;447
8.23.2;2. Animal Care and Protocol Approval;448
8.23.3;3. Disinfection of Mice;448
8.23.4;4. Analgesia and Anesthesia in Mice;448
8.23.5;5. IP Injection;450
8.23.6;6. IV Injection into the Tail Vein;450
8.23.7;7. IV Tumor Formation Assay Using Immune-Deficiency Mice;452
8.23.8;8. SC Inoculation;454
8.23.9;9. FP Inoculation;456
8.23.10;10. Testicular Inoculation;457
8.23.11;11. Prostate Inoculation;457
8.23.12;Acknowledgment;460
8.23.13;References;460
9;Author Index;462
10;Subject Index;488
11;Color Plates;498