Functional Glycomics

Front......Page 1
Table of Contents......Page 2
Contributors to Volume 417......Page 5
Preface......Page 9
Previous Titles of Methods in Enzymology......Page 11
Introduction......Page 35
Methods......Page 36
Mammary Tumor Cells from Polyomavirus Middle T Transgenic Mice......Page 37
Nuclear Translocation Assay......Page 38
Notes......Page 41
References......Page 42
Overview......Page 43
Lacking Core Fucosylation on TGF-beta Receptor Type II Leads to Emphysema-Like Changes in Fut8-null Mice Lung......Page 46
Core Fucosylation Regulates EGF Receptor-Mediated Intracellular Signaling......Page 47
Gene Targeting......Page 49
Establishment of Embryonic Fibroblasts......Page 50
EGF Binding Assay......Page 51
References......Page 52
Overview......Page 55
RT-PCR Analysis......Page 56
Western Blot Analysis......Page 58
Method for Polysialic Acid Staining of Frozen Sections......Page 59
5-Bromo-2'-Deoxyuridine (BrdU)-Labeling......Page 60
Method for BrdU Detection in Paraffin Sections......Page 61
Primary Cell Culture and Neurite Outgrowth Assay......Page 62
References......Page 65
[4] Roles of Glycolipids in the Development and Maintenance of Nervous Tissues......Page 67
Overview......Page 68
Overexpression of Glycolipids in Transgenic Mice......Page 69
Knock-Out Strategy......Page 70
Hot Plate Test......Page 75
Rota-Rod Test......Page 76
Hypoglossal Nerve Resection System......Page 77
Morphological Approaches......Page 79
Detection of Low-Level Glycolipids: Histology and Immunohistochemistry......Page 80
References......Page 81
Further Reading......Page 0
[5] Analysis of Neural Cell Functions in Gene Knockout Mice: Electrophysiological Investigation of Synaptic Plasticity in Acute......Page 82
Overview......Page 83
Materials for Slice Preparation......Page 85
Method for Slice Preparation......Page 86
Materials for Extracellular Recordings of fEPSPs......Page 87
Method for Extracellular Recordings of fEPSPs......Page 88
Baseline Recording......Page 89
Induction of LTP by High-Frequency Stimulation......Page 91
Measurements and Calculation of Parameters......Page 93
References......Page 95
Introduction......Page 96
Day 8 Neurological Screen......Page 99
Day 16 Pulmonary Function......Page 100
Day 1-3 (4-6) Metabolism Chambers......Page 101
Day 8 Neurological Screen......Page 102
Day 9 Motor and Nociception Tests......Page 103
Days 11-12 Memory: Fear Conditioning......Page 104
PPI: Prepulse Inhibition Is a Measure of Sensorimotor Gating and Deficits Suggest Abnormal Processing of Incoming Sensory Stimuli (Geyer and Braff, 1987; Geyer et al., 2002)......Page 105
Day 17 Social Dominance......Page 106
Days 24-26 Blood Pressure......Page 107
References......Page 108
Overview......Page 110
Solutions......Page 111
Preparation of Phospholipid......Page 112
Chloriding the Electrodes......Page 113
Materials for Tip-Dip Bilayer Method......Page 114
Tip-Dip bilayer Method......Page 115
Acknowledgment......Page 119
References......Page 120
Overview......Page 121
Analysis of O-Fucose and O-Glucose Glycan Structure by Metabolic Radiolabeling Coupled with Alkali-Induced beta-Elimination......Page 123
Standards......Page 124
Metabolic Radiolabeling......Page 125
Fluorography......Page 126
Oligosaccharide Structural Analysis by Size-Exclusion Chromatography and Exoglycosidase Digestion......Page 127
Oligosaccharide Charge Analysis......Page 128
Glycosylation Site Mapping by Mass Spectrometry......Page 129
Stock Solutions......Page 130
Sample Preparation......Page 131
In-Gel Digestions......Page 132
LC-MS of the Digested Sample......Page 133
References......Page 136
[9] Roles of O-Fucosyltransferase 1 and O-Linked Fucose in Notch Receptor Function......Page 139
Overview......Page 140
Enzyme Assay for O-Fucosyltransferase 1 Activity......Page 141
Methods......Page 142
Preparation of dsRNA for Ofut1......Page 143
Materials......Page 144
Materials......Page 145
Notch Secretion Assay......Page 146
Western Blotting for Notch......Page 147
Notch-Ligand Binding Assay......Page 148
Preparation of N:AP Conditioned Medium......Page 149
Preparation of N-EGF:FLAG Beads......Page 150
Binding Reaction......Page 151
Materials......Page 152
References......Page 153
Overview......Page 155
Glycosylation of Notch Receptors and Notch Ligands......Page 156
Pofut1 Is an Essential Component of the Canonical Notch Signaling Pathway......Page 157
Fringe Is a Modulator of Notch Signaling......Page 158
Mutations in Other Glycosyltransferase Genes......Page 161
References......Page 162
Overview......Page 165
Materials......Page 167
Method for Purification of alpha-DG......Page 168
Materials......Page 169
Reagents......Page 170
Method for Preparation of Membrane Fraction from HEK293T Cells......Page 171
Preparation of Membrane Fraction for Enzyme Sources from Brain......Page 172
Method for Preparation of GST-alpha-DG......Page 173
Reagents......Page 174
Method for POMT Assay......Page 175
POMGnT1 Assay......Page 176
Solutions......Page 177
POMGnT1 Assay......Page 178
References......Page 179
Overview......Page 181
Isolation of PtdGlc from Developing Brains......Page 182
TLC Immunostaining......Page 183
Materials for Purification......Page 184
Aquasil Chromatography Using HPLC......Page 185
Identification of PtdGlc by Normal-phase HPLC/Electrospray Ionization Ion-trap Mass Spectrometry......Page 186
Elution Solvents......Page 187
Analysis of PtdGlc......Page 189
References......Page 192
Further Reading......Page 193
Overview......Page 194
Cell Culture......Page 200
EGFR Autophosphorylation Assay......Page 201
Phospho-p44/42 MAP Kinase Assays......Page 202
125I-EGF Binding......Page 203
Measurement of Effects of Cellular Ganglioside Enrichment on Cell Proliferation......Page 204
Results and Conclusion......Page 205
References......Page 209
Overview......Page 211
Valpha14i NKT Cell Isolation......Page 213
Method 1 (Rapid Method)......Page 214
Method......Page 215
Purification of Valpha14i NKT Cells by Cell Sorting......Page 216
Method......Page 217
Method......Page 218
Method......Page 220
In Vivo iNKT Cell Activation Assay......Page 221
Solutions......Page 222
Biochemical Assays......Page 223
Method......Page 224
References......Page 225
Overview......Page 228
Microplate (ELISA)-Based Recognition......Page 229
Surface Plasmon Resonance Spectroscopy......Page 230
Microplate (ELISA)-Based Recognition......Page 231
Thin Layer Chromatography......Page 233
TLC Overlay......Page 235
TLC Blotting......Page 236
Liposome Immobilization and SPR......Page 237
Notes......Page 238
References......Page 242
Overview......Page 244
Marine Sponges......Page 245
Synthesis of Spacer-Containing Oligosaccharides......Page 247
Synthesis of Gold Nanoparticles......Page 248
Analysis of Gold Glyconanoparticles......Page 251
Monosaccharide Analysis......Page 253
1H-NMR Spectroscopy......Page 255
Atomic Force Microscopy......Page 258
Force Curve Measurements......Page 259
References......Page 262
Overview......Page 267
Chemicals and Reagents......Page 268
Preparation of ECM from Cultured Cells......Page 269
Coating Matrigel with Galectin-1......Page 270
CFSE-labeled Cell Adhesion Assay......Page 271
Cell Migration through ECM......Page 272
Assessing Cell Death on Matrigel......Page 273
Conclusions......Page 274
References......Page 275
[18] Galectins in Apoptosis......Page 276
Galectin-1......Page 277
Other Galectins......Page 279
Galectin-3......Page 280
Other Galectins......Page 282
Purification of Endotoxin-free Galectin-3......Page 283
Affinity Chromatography of Galectin-3......Page 284
Overexpression of Galectin-3 in Eukaryotic Cells......Page 285
Construction of the Expression Vectors pEF1 and pEF1-Neo......Page 286
Transfection of Mammalian Cells......Page 287
References......Page 288
Overview......Page 293
Galectins and Integrin-Related Signaling......Page 295
Galectins and Growth Factor Receptor-Related Signaling......Page 297
Galectins and Ras Signaling......Page 298
Galectins and TNF-Related Signaling......Page 299
Galectins and NF-kappaB Signaling......Page 300
Galectin-3 and Phosphorylation......Page 301
Preparation of Recombinant Galectin-3......Page 302
Binding Analysis of Extracellular Galectins to Specific Ligands......Page 303
Phosphorylation of Galectin-3......Page 304
GSK-3beta Kinase Assay......Page 305
References......Page 306
Overview......Page 310
Characterization of Bacterial Adherence by In Vitro Binding to Host Tissue......Page 311
Glycoprotein Array for Screening and Identification of Adhesin Binding Properties......Page 314
Spotting of Glycoprotein Arrays......Page 315
Bacterial Overlay on Nitrocellulose Membranes......Page 316
125I Labeling of Glycoconjugate (Hunter and Greenwood, 1962)......Page 317
FITC Labeling of Glycoconjugates......Page 318
Affinity Analysis by RIA According to Scatchard......Page 319
Practical Considerations......Page 320
Surface Labeling of H. pylori Bacterial Cells......Page 322
ELISA Binding Assay......Page 323
Receptor Activity-directed Affinity Tagging (Retagging) Technique for Adhesin Protein Identification......Page 324
Methods......Page 326
Method......Page 327
Method for Magnetic-bead Enrichment of Biotin-labeled Adhesin......Page 328
Practical Considerations of Retagging such as Modifications for SabA......Page 329
Phylogenetic Methods for Detecting Adaptive Change......Page 330
Estimation of dN and dS between Homologous Sequences......Page 331
Molecular Adaptation in H. pylori Adhesin Gene, babA......Page 333
Knock-out of Adhesin Gene: General Strategy for Genetic Constructions by PCR without Recombinant DNA Cloning......Page 335
PCR Condition for Initial DNA Segment (A, B, C) Amplification......Page 336
Primers (numbers in parentheses refer to primers used in Figs. 4 and 5)......Page 338
Materials......Page 339
Solutions......Page 340
Solutions......Page 341
Oligonucleotide PROBES for H. pylori......Page 342
Detection of H. pylori Gene Expression......Page 343
Controls......Page 344
Instrumentation......Page 345
Biological Model System......Page 346
Conclusions......Page 348
Role of Adhesins in Activation of Human Neutrophils......Page 350
Isolation of Human Neutrophil Granulocytes......Page 351
Acknowledgments......Page 352
References......Page 353
Overview......Page 357
C. elegans Chemical Mutagenesis......Page 358
Section 2: EMS Mutagenesis......Page 359
ENGIC Plate Method for Cry5B-resistant Mutants......Page 360
Plate Media......Page 361
Isolation of Glycosphingolipids from C. elegans by Svennerholm Partitioning......Page 362
Section 1: Preparing Worms......Page 363
Section 2: Lipid Extraction......Page 364
Section 3: Purification......Page 365
Analysis of Glycolipids by Thin Layer Chromatography......Page 366
Section 1: Resolving Sample by TLC......Page 367
Section 2: Orcinol Staining Resolved Glycolipids......Page 368
Section 3: Preparing Protein Probe for Overlay Analysis......Page 369
Section 4: Preparing Resolved Samples and Protein Overlay Analysis......Page 370
Section 5: Polystyrene Microplate Overlay......Page 371
Section 1: Preparation of a 4-ml Silica Gel Column......Page 372
Section 3: Enzymatic Release of a Purified Glycolipid Carbohydrate......Page 373
References......Page 374
Overview......Page 376
The Biology of Caenorhabditis elegans......Page 377
The Structure and Biosynthesis of GlcNAcbeta1-N-Asn Glycans (N-Glycans) in C. elegans......Page 378
The Function of N-Glycans in C. elegans......Page 382
General Techniques......Page 383
Life Span of C. elegans Growing on Bacterial Pathogens......Page 384
Production of gly-13 and gly-14 Phenotypes by RNA-mediated Interference (RNAi)......Page 385
Rescue of the Mutant gly-13 Phenotype by a Wild-type gly-13 Extrachromosomal Array......Page 386
Effects of E. coli OP50 on PGS Medium on the Survival of C. elegans......Page 388
Effects of Pseudomonas aeruginosa Strain 14 on PGS Medium on the Survival of C. elegans......Page 391
Production of gly-13 and gly-14 Phenotypes by RNA-mediated Interference (RNAi)......Page 394
Discussion......Page 396
Acknowledgments......Page 399
References......Page 400
[23] Detection of Cytoplasmic Glycosylation Associated with Hydroxyproline......Page 406
Overview......Page 407
Choice of Inhibitors......Page 409
Interpretation......Page 410
Metabolic Labeling and Preparation of the Cytosolic Extract......Page 411
Interpretation......Page 412
Preparation of Dictyostelium His10Skp1......Page 414
Reaction Labeling Conditions......Page 415
Interpretation......Page 416
Example in Toxoplasma gondii......Page 417
References......Page 420
Author Index......Page 422
Subject Index......Page 450