Functional Glycomics

Series Editors
......Page 1
Copyright page......Page 2
Contributors......Page 3
Preface......Page 8
Volume in Series
......Page 9
beta1,4-Galactosyltransferase V: A Growth Regulator in Glioma......Page 37
Overview......Page 38
Cell culture and transfection......Page 39
Survival assay......Page 40
Dual luciferase assay......Page 41
Elevated expression of beta1,4GalT V in glioma......Page 42
Function and mechanism of beta1,4GalT V in glioma process......Page 44
Transcriptional regulation of beta1,4GalT V......Page 45
Recent research on beta1,4GalT V in glioma-initiating cell......Page 50
Conclusion and Future Direction......Page 52
References......Page 56
Roles of Polysialic Acid in Migration and Differentiation of Neural Stem Cells......Page 58
Overview......Page 59
Polysialic acid-deficient mice......Page 60
Method for preparation of neurosphere cells......Page 61
In vitro migration assay......Page 62
In vitro differentiation assay......Page 63
Lentivirus generation......Page 66
References......Page 67
Structural and Functional Analysis of Chondroitin Sulfate Proteoglycans in the Neural Stem Cell Niche......Page 70
Overview......Page 72
Immunohistochemistry of Embryonic and Postnatal Mouse Brain Sections......Page 75
Method to Stain Embryonic Sections for NSPC Markers......Page 76
Method to cultivate NSPCs from adult neurogenic niches......Page 77
Method for immuncytochemistry performed on neural cell monolayers......Page 79
Isolation of NSPCs by Immunopanning or by Immunoisolation Using Paramagnetic Beads (EasySep)......Page 80
Method for immunopanning of NSPCs with MAb 473HD......Page 81
Neurosphere Cultures and Various Methods for Their Analysis......Page 82
Method to perform a differentiation assay with neurospheres......Page 83
Method for immunoblot analysis of neurospheres for biochemical analysis......Page 85
Method for the partial purification and identification of CSPGs from the conditioned neurosphere culture medium.........Page 86
Method for the suppression of sulfation in NSPC cultures......Page 88
Analysis of CSPG Functions in NSPCs Using Chondroitinase ABC Treatment in Culture......Page 89
Method for the treatment of neurosphere cultures with ChABC......Page 90
Method for intracerebroventricular injections in utero......Page 91
RT-PCR and Semiquantitative Analysis of the Synthetic Machinery for Glycosaminoglycans......Page 92
Method for the amplification of distinct sulfotransferases using RT-PCR......Page 94
Method for the in situ hybridization of sulfotransferase mRNA......Page 96
References......Page 99
Transcript Analysis of Stem Cells......Page 105
Introduction......Page 106
Materials and equipment......Page 107
Primer design......Page 108
Primer validation......Page 109
RNA isolation......Page 111
qRT-PCR data analysis......Page 112
Display of qRT-PCR data......Page 113
Examples of the Applications of qRT-PCR Transcript Analysis to Investigate Changes in Glycan-Related Gene Expression in Ste......Page 115
qRT-PCR analysis of glycosaminoglycan biosynthetic genes in pluripotent and differentiated murine embryonic stem cells.........Page 116
Transcript analysis of genes involved in N- and O-linked glycan biosynthesis......Page 117
Comparison transcript abundance of pluripotency and differentiation marker genes in human ES cells......Page 118
References......Page 120
Directing Stem Cell Trafficking via GPS......Page 124
Overview......Page 125
Rationale for GPS......Page 126
Guiding Principles and Method for GPS......Page 128
Method of α(1,3)-Fucosylation of Cell Surface Using FTVI......Page 130
Detection of E-Selectin Ligand Expression Following GPS......Page 131
Testing for E-Selectin Ligand Activity Using E-Selectin-Ig Chimera (Three-Step Method for Flow Cytometry)......Page 132
Summary......Page 133
References......Page 134
Functional Assays for the Molecular Chaperone Cosmc......Page 137
Overview......Page 138
T-Synthase and Cosmc......Page 140
T-Synthase Activity Assay......Page 143
Cell Lines and Preparation of Cell Extracts......Page 144
Assay Procedure for T-Synthase and Calculation of Total Product......Page 145
Generating an Expression Construct for Cosmc......Page 146
Analyzing the Activity of Cosmc in the Baculovirus System......Page 147
Analyzing Cosmc Activity in Mammalian Cells......Page 148
Conclusion and Future Directions......Page 149
References......Page 150
Core 3-Derived O-Glycans Are Essential for Intestinal Mucus Barrier Function......Page 153
Introduction......Page 154
Reverse transcription (RT)-PCR analysis of C3GnT transcripts......Page 156
PCR genotyping......Page 157
Intestinal glycan structure analysis......Page 158
Results and analysis......Page 159
Immunohistochemical staining for intestinal Muc2......Page 161
Examine bacterial translocation in colonic mucosa by real-time PCR......Page 162
Dextran sodium sulfate-induced colitis model......Page 163
Immunohistochemistry......Page 165
Results and analysis......Page 166
Conclusion and Future Direction......Page 168
References......Page 170
Core3 Glycan as Tumor Suppressor......Page 172
Overview......Page 173
Generating Core3 Glycan Expressing Cell Lines......Page 174
FACS analysis......Page 175
Determination of Major Integrin Using Functional Blocking Antibodies......Page 176
Orthotopic tumor cell injection......Page 178
Western blotting for α2 and beta1 detection......Page 179
Heterodimerization Assay......Page 180
FAK Signaling......Page 181
References......Page 182
Characterization of Mice with Targeted Deletion of the Gene Encoding Core 2 beta1,6-N-Acetylglucosaminyltransferas......Page 184
Introduction......Page 185
PCR genotyping of Gcnt3f/f and Gcnt3Delta/Delta mice......Page 186
Core 2 and Core 4 enzyme assays......Page 188
Mass spectrometry......Page 189
C2GnT2 KO mice have reduced mucosal barrier function......Page 190
Quantification of immunoglobulin isotypes in sera......Page 191
Quantification of IgA in fecal samples......Page 193
C2GnT2 KO mice developed exaggerated pathogenesis in the DSS-induced colitis model......Page 194
C2GnT2 KO mice do not have altered levels of Muc2......Page 195
Conclusions and Future Directions......Page 197
References......Page 199
Analyzing Physiological Function of Polypeptide GalNAcT-1-Deficient Mice in Humoral Immunity......Page 202
Assay for In Vitro B Lymphocyte Activation......Page 203
Assay for In Vivo Antibody Production......Page 204
Immunohistochemical Staining of Frozen Sections......Page 206
Apoptosis Detection Using Antibody Against Caspase 3 Active Form......Page 208
Apoptosis Detection by TUNEL System......Page 211
References......Page 212
beta3GnT2 (B3GNT2), a Major Polylactosamine Synthase: Analysis of B3gnt2-Deficient Mice......Page 214
Glycogenes for Polylactosamine Synthesis (i.e., beta1,3-N-Acetylglucosaminyltransferase Genes)......Page 215
N-Glycan Polylactosamine is Greatly Reduced in B3gnt2-/- Mice......Page 216
Phenotype of B3gnt2-/- Lymphocytes Lacking Polylactosamine on N-Glycans......Page 222
Genotyping of B3gnt2-deficient (B3gnt2-/-) mice......Page 226
beta3GnT In vitro assays......Page 227
LEL Lectin-blot analysis......Page 228
Immunoprecipitation and lectin microarray analysis of immunoprecipitated glycoproteins......Page 229
Lymphocyte isolation and proliferation assays......Page 230
References......Page 231
Targeted Genetic Inactivation of N-Acetylglucosaminyltransferase-IVa Impairs Insulin Secretion from Pancreatic beta Cells and Evokes
Type 2 Diabetes......Page 234
Overview......Page 235
Engineering GnT-IVa-Deficient Mice......Page 236
GnT-IV Enzymology......Page 238
Glucose and Insulin Homeostasis......Page 239
Immunohistochemical Analysis......Page 240
Islet Cell Preparation and Culture......Page 243
Pulse-Chase Labeling......Page 244
Cell Surface Half-Life Time of GLUT2......Page 245
GLUT2 Glycan Analysis by Lectin Blot......Page 246
Cell-Surface Protein Cross-Linking......Page 247
References......Page 249
The Ashwell-Morell Receptor......Page 252
Overview......Page 253
Endogenous Ligands of the AMR......Page 256
Hepatocytes in Molecular Clearance Mechanisms of the Liver......Page 257
Genotyping HL-1- or HL-2-Deficient Mice......Page 258
Method for measuring platelet levels and glycosylation by lectin binding......Page 259
Antithrombin activity assay......Page 260
Protein S antigen assay......Page 261
Fibrinogen activity assay......Page 262
Factor X antigen assay......Page 263
Von Willebrand factor lectin-binding assay......Page 264
References......Page 265
Roles of GlcNAc-6-O-Sulfotransferases in Lymphoid and Nonlymphoid Tissues......Page 271
Overview......Page 272
Establishment of a Mouse Colon Adherent Cell Line and Cell Culture......Page 274
RT-PCR......Page 275
Histology and Immunostaining......Page 276
Preparation of Colonic-Mucin-Enriched Fraction......Page 277
Carbohydrate Structural Analysis......Page 278
Induction of Colitis by Dextran Sulfate Sodium......Page 282
References......Page 283
Core O-Glycans Required for Lymphocyte Homing: Gene Knockout Mice of Core 1 beta1,3-N-Acetylglucosaminyltransfera......Page 285
Introduction......Page 286
Lymphocyte Homing Assay......Page 288
Preparation of fluorescence-labeled lymphocytes......Page 289
Intravenous injection......Page 290
Materials......Page 291
Preparation and fixation of frozen sections......Page 292
Probing of L-Selectin Ligands on Membrane Filters with L- and E-selectin-IgM Chimeric Proteins......Page 293
Materials......Page 294
Conclusions......Page 295
References......Page 297
Abstract......Page 299
Overview......Page 300
Immunohistochemical Analysis Using Conventional Immunostaining......Page 302
Methods......Page 303
Quantification of HEV-like vessels......Page 304
Practical example 1......Page 305
Practical example 3......Page 306
Immunohistochemical Analysis Using Multiple Immunostaining......Page 307
Materials......Page 308
Methods......Page 309
Immunohistochemical Analysis Using L-SelectinIgM Chimera Binding......Page 311
Preparation of the L-selectinIgM chimera......Page 312
L-selectinIgM chimera in situ binding assay......Page 313
Practical example......Page 314
References......Page 315
Abstract......Page 318
Overview......Page 319
Mouse Models of Muscular Dystrophy......Page 321
Approach to Phenotype Analysis in Mouse Muscular Dystrophy Models......Page 333
References......Page 339
Abstract......Page 350
Overview......Page 351
Northern blot analysis......Page 352
RT-qPCR......Page 353
Preparation of the α-DG-GST fusion protein substrate......Page 354
Assay for POMT activity......Page 355
Expression of murine Pomt1 during embryo development......Page 356
Generation and genotyping of knockout mice......Page 360
Characterization of extracellular components in Pomt1-/- embryos......Page 363
Discussion/Conclusions......Page 364
Acknowledgments......Page 366
References......Page 367
Abstract......Page 370
Overview......Page 371
Cell Culture and Preparation of Microsomal Membrane Fraction......Page 372
GnT1 activity......Page 373
POMT activity......Page 374
Procedures for Enzymatic Activity and Mutation Search......Page 375
References......Page 377
Abstract......Page 380
Overview......Page 381
Analysis of α-DG Glycosylation and Laminin Binding by Western Blot......Page 382
Histological Analysis of POMGnT1 Knockout Brain......Page 383
Lamination Defects in the Neocortex of POMGnT1 Knockout Mice......Page 384
Analysis of the Pial Basement Membrane by Laminin Immunostaining......Page 386
Analysis of the Pial Basement Membrane by Transmission Electron Microscopy......Page 387
Analysis of the Glia Limitans by GFAP Immunofluorescence Staining......Page 389
Acknowledgments......Page 391
References......Page 392
Abstract......Page 394
Overview......Page 395
Human LARGE and Relevance to Disease......Page 396
Identification of Mice Carrying Mutations in Large......Page 397
Veils and enr-two additional mutant alleles of Large......Page 399
Loss of Functional Large Protein Results in Hypoglycosylation of α-Dystroglycan......Page 400
Embryonic phenotypes......Page 401
Muscle phenotype......Page 402
Central nervous system......Page 403
Ocular defects......Page 404
Expression of Large in adult CNS by in situ hybridization......Page 405
Does Large Encode a Functional Glycosyltransferase?......Page 406
References......Page 408
Abstract......Page 414
Background......Page 415
Cell sorting and flow cytometry......Page 416
Laminin-binding assay......Page 417
Colony formation assay......Page 419
Tumor invasion assay......Page 420
Orthotopic prostate tumor formation......Page 421
References......Page 422
Abstract......Page 424
Overview......Page 425
Analgesia and Anesthesia in Mice......Page 426
IV Injection into the Tail Vein......Page 428
IV Tumor Formation Assay Using Immune-Deficiency Mice......Page 430
SC Inoculation......Page 432
FP Inoculation......Page 434
Prostate Inoculation......Page 435
References......Page 438
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