Lipid-Mediated Signaling (Methods in Signal Transduction Series)

Lipid-Mediated Signaling......Page 2
Methods In Signal Transduction Series......Page 3
Lipid-mediated signaling......Page 4
Dedication......Page 6
Contents......Page 8
Series Preface......Page 12
Preface......Page 14
Editors......Page 16
Contributors......Page 18
Part I Enzyme Systems Involved in Lipid-Mediated Signaling......Page 22
Contents......Page 24
1.3.1 STRUCTURE, EXPRESSION, AND MECHANISM OF ACTION......Page 25
1.3.2 FUNCTION......Page 28
1.4.1 STRUCTURE, EXPRESSION, AND MECHANISM OF ACTION......Page 29
1.4.2 FUNCTION......Page 30
1.5.1 STRUCTURE, EXPRESSION, AND MECHANISM OF ACTION......Page 31
1.5.2 FUNCTION......Page 32
1.6 INHIBITION OF PHOSPHOLIPASE A2 ACTIVITY......Page 33
1.6.2 PHARMACOLOGIC INHIBITORS DESIGNED......Page 34
1.6.4 ALTERNATIVES......Page 35
1.7 MEASUREMENT OF PHOSPHOLIPASE A2 ACTIVITY......Page 36
1.7.1 PAF- AH ASSAY......Page 41
LIST OF ABBREVIATIONS......Page 42
REFERENCES......Page 43
CONTENTS......Page 48
2.1.2 THE PTDINS- SPECIFIC PLC FAMILY......Page 49
2.1.3 CHARACTERISTIC FUNCTIONAL UNIT STRUCTURES FOUND IN THE PTDINS-SPECIFIC PLC FAMILY......Page 52
2.1.4 ACTIVATION AND REGULATION OF PTDINS-SPECIFIC PLC......Page 54
2.2.1 RNA ANALYSIS......Page 55
2.2.2 RT- PCR......Page 56
2.2.3 IN SITU HYBRIDIZATION......Page 58
2.2.4 IMMUNOHISTOCHEMICAL ANALYSIS......Page 59
2.2.5 IMMUNOHISTOCHEMICAL METHODS......Page 60
2.3 PHOSPHOLIPASE C ASSAYS......Page 61
2.3.1 HOMOGENIZATION OF TISSUE FOR PLC ASSAYS......Page 63
2.3.3 ASSAY OF PTDINS-SPECIFIC PLC......Page 64
2.3.5 INHIBITORS OF PHOSPHOLIPASE C ACTIVITY......Page 65
2.4 CONCLUSIONS......Page 67
APPENDIX: COMMONLY USED REAGENTS AND BUFFERS......Page 68
REFERENCES......Page 70
CONTENTS......Page 76
3.1.1 IDENTIFICATION OF PLD ACTIVITY......Page 77
3.1.3 THE PLD SUPERFAMILY......Page 78
3.1.4 PLD PRIMARY STRUCTURE......Page 79
3.2.1 SMALL GTPASES......Page 80
3.2.4 POSTTRANSLATIONAL MODIFICATION AND LIPIDATION......Page 81
3.3.4 CYTOSKELETON ORGANIZATION......Page 82
3.3.5.3 The Role of PLD in Regulation of Metastasis......Page 83
3.4.1 EXPRESSION OF PLD ISOFORMS IN MAMMALIAN TISSUES AND CELL LINES......Page 84
3.4.2.1.2 Headgroup Release......Page 85
3.4.2.2.2 Transphosphatidylation Assay......Page 87
3.4.2.2.4 Conductimetric/Charge-Dependent Assays......Page 88
REFERENCES......Page 89
CONTENTS......Page 98
4.2.1 PHOSPHATIDYLINOSITOL TURNOVER AND DIACYLGLYCEROL......Page 99
4.2.2 DISCOVERY OF DIACYLGLYCEROL KINASE ACTIVITY......Page 100
4.2.3 SUBSTRATE SPECIFICITY TOWARD ARACHIDONOYL DIACYLGLYCEROL......Page 101
4.3.1 PROPERTIES OF DIACYLGLYCEROL......Page 102
4.4.2 MAMMALIAN DIACYLGLYCEROL KINASE FAMILY......Page 103
4.4.3 DIACYLGLYCEROL KINASES IN OTHER SPECIES......Page 105
4.5.1 SUBCELLULAR LOCALIZATION AND TRANSLOCATION......Page 106
4.5.3 METABOLISM OF DIACYLGLYCEROL IN THE NUCLEUS......Page 107
4.5.4.1.1 Cerebral Ischemia and Infarction......Page 109
4.5.4.1.3 Emotion......Page 111
4.5.4.2.1 Cardiac Hypertrophy......Page 112
4.5.4.2.2 Myocardial Infarction......Page 113
4.5.4.3 Knockout Mice......Page 114
4.6.1 MEASUREMENT OF DIACYLGLYCEROL KINASE ACTIVITY......Page 116
4.6.2 ACTIVITY OF ARACHIDONOYL DIACYLGLYCEROL KINASE......Page 117
LIST OF ABBREVIATIONS......Page 118
REFERENCES......Page 119
CONTENTS......Page 130
5.1.1 BIOSYNTHESIS OF ENDOCANNABINOIDS......Page 131
5.1.1.1 Biosynthesis of AEA......Page 132
5.1.1.2 Biosynthesis of 2-AG......Page 133
5.1.2 ENDOCANNABINOID DEGRADATION......Page 134
5.1.3 PHYSIOPATHOLOGICAL FUNCTION OF THE ENDOCANNABINOID SYSTEM......Page 136
5.2 EXTRACTION, PURIFICATION, AND QUANTIFICATION OF ENDOCANNABINOIDS......Page 137
5.4 NAPE- PLD AND NAT ACTIVITY EVALUATION......Page 140
5.5 FAAH ACTIVITY EVALUATION......Page 143
5.6 DAGL ACTIVITY EVALUATION......Page 144
5.7 MAGL ACTIVITY EVALUATION......Page 147
5.8 IMMUNOHISTOCHEMICAL METHODS USED TO STUDY THE EXPRESSION OF ENDOCANNABINOID METABOLIC ENZYMES......Page 148
5.9 QUANTITATIVE METHODS TO STUDY THE......Page 149
5.9.1 N-ACYL-PHOSPHATIDYLETHANOLAMINE-HYDROLYZING PHOSPHOLIPASE D......Page 150
5.9.3 DIACYLGLYCEROL LIPASE......Page 154
5.9.4 FATTY ACID AMIDE HYDROLASE......Page 156
LIST OF ABBREVIATIONS......Page 157
REFERENCES......Page 159
Part II Mass Spectrometry Methods for Lipid Analysis......Page 170
CONTENTS......Page 172
6.1 INTRODUCTION TO MULTIDIMENSIONAL MASS SPECTROMETRY– BASED SHOTGUN LIPIDOMICS......Page 173
6.2.2 INTERNAL STANDARDS AND GENERAL LIPID EXTRACTION......Page 176
6.2.3 SAMPLE PREPARATION......Page 177
6.2.5 MASS SPECTROMETRY......Page 178
6.3.1 ANIONIC GLYCEROPHOSPHOLIPIDS......Page 179
6.3.2 CHOLINE GLYCEROPHOSPHOLIPID......Page 181
6.3.3 ETHANOLAMINE GLYCEROPHOSPHOLIPID......Page 183
6.3.4 SULFATIDE......Page 184
6.3.5 SPHINGOMYELIN......Page 187
6.3.7 CERAMIDE......Page 188
6.3.8 CHOLESTEROL......Page 189
6.4.1 CAREFUL EXTRACTION......Page 190
6.4.2 ANALYSIS......Page 191
6.4.4 AT LEAST ONE INTERNAL STANDARD......Page 192
LIST OF ABBREVIATIONS......Page 193
REFERENCES......Page 194
CONTENTS......Page 198
7.1.1 OVERVIEW AND BACKGROUND......Page 199
7.1.1.1 Sphingolipid Metabolism: A Foundation of Structural Complexity......Page 200
7.1.3 GENERAL CLASSIFICATION......Page 202
7.1.3.2 Sphingolipids......Page 203
7.2 LIPIDOMICS APPROACH AND MASS SPECTROMETRY......Page 204
7.3 SAMPLE PREPARATION......Page 205
7.4.1 MASS SPECTROMETRY......Page 207
7.4.2 MS SCAN MODES......Page 208
7.4.2.1.2 Neutral Loss......Page 209
7.4.3 SPL IDENTIFICATION......Page 210
7.4.4 HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY/MASSSPECTROMETRY METHODOLOGY......Page 211
7.4.5.1 Selection of Internal Standards......Page 212
7.4.5.2 Calibration Curves......Page 213
7.5 ALTERNATIVE METHODOLOGY......Page 214
LIST OF ABBREVIATIONS......Page 215
REFERENCES......Page 216
CONTENTS......Page 220
8.1 INTRODUCTION......Page 221
8.2.1 HANDLING AND STORAGE OF BIOLOGICAL FLUIDS AND TISSUES......Page 224
8.2.2 EXTRACTION AND HYDROLYSIS OF F2-ISOP- OR F4-NP-CONTAININGPHOSPHOLIPIDS IN TISSUES AND BIOLOGICAL FLUIDS......Page 225
8.3 PURIFICATION, DERIVATIZATION, AND QUANTIFICATION OF FREE F2-ISOP......Page 226
8.4 PURIFICATION, DERIVATIZATION, AND QUANTIFICATION OF FREE F4- NP......Page 227
8.5 F2- ISOP AND F4- NP AS INDEXES OF OXIDANT STRESS IN VIVO......Page 230
LIST OF ABBREVIATIONS......Page 231
REFERENCES......Page 232
Part III Lipid-Mediated Regulation of Gene Expression......Page 234
CONTENTS......Page 236
9.1.1 CLASSIFICATION OF NUCLEAR RECEPTORS......Page 238
9.1.2 ROLE OF LIPID SENSORS......Page 243
9.1.3 TRANSCRIPTIONAL MECHANISMS OF LIPID SENSORS......Page 245
9.2 POSSIBLE EXPERIMENTAL APPROACHES......Page 249
9.3.1 CELLULAR MODELS......Page 254
9.3.2 TRANSFECTION OF CULTURED CELLS......Page 255
9.3.3 HETEROLOGOUS CELL LINES......Page 257
9.3.4 CELLULAR GENE KNOCKDOWN......Page 259
9.3.5 CELL TREATMENT STRATEGIES......Page 261
9.4.1 POSSIBLE ANIMAL MODELS......Page 263
9.4.2 TRANSGENIC ANIMALS......Page 265
9.4.3 GENE KNOCKOUT......Page 266
9.4.4 ANIMAL TREATMENT STRATEGIES......Page 268
9.5.1 SAMPLE PREPARATION......Page 270
9.5.2 LABELING OF PROBES AND PROTEIN PREPARATION......Page 271
9.5.3.1 In Situ Hybridization......Page 272
9.5.3.3 PCR-Based RNA Analysis......Page 273
9.5.3.4 Real-Time RT-PCR......Page 274
9.5.4 PROTEIN ANALYSIS......Page 276
9.5.4.3 Western Blot......Page 277
9.6 PURE IN VITRO APPROACHES......Page 278
9.6.1.1 Reporter Gene Assays......Page 279
9.6.1.2 Electrophoretic Mobility Shift Assay......Page 282
9.6.1.3 Footprinting Methods......Page 283
9.6.1.5 Fluorescence Resonance Energy Transfer......Page 286
9.6.2.1 Microarray......Page 289
9.6.2.2 ChIP-on-Chip......Page 290
9.6.2.3 Serial Analysis of Gene Expression......Page 291
9.6.2.4 Gene Trapping......Page 292
9.6.3.3 GAL4 Two Hybrid Systems......Page 293
9.6.3.4 GST Pull-Down Assay......Page 294
9.6.3.5 Co-Immunoprecipitation......Page 295
9.6.4 ANALYSIS OF LIPID SENSOR–LIGAND INTERACTIONS......Page 297
9.6.4.1 Radioligand-Binding Assay......Page 298
9.6.4.3 Fluorescence Resonance Energy Transfer......Page 299
9.6.4.6 GAL4 One-Hybrid System......Page 300
9.7.1 LIPID SENSOR DATABASES......Page 301
9.7.2 FUNCTIONAL BIOINFORMATICS......Page 302
9.8 CONCLUSION......Page 303
LIST OF ABBREVIATIONS......Page 304
REFERENCES......Page 307
CONTENTS......Page 320
10.1 INTRODUCTION......Page 321
10.1.2 RECONCILIATION OF IN VIVO AND IN VITRO FINDINGSADDRESSING THE HYPOTHESIS......Page 322
10.2.2 CELL SUSPENSION METHODS— FLUORESCENCE SPECTROSCOPY......Page 326
10.2.3 SINGLE CELL METHODS— FLUORESCENCE MICROSCOPY......Page 327
10.2.3.2 Intracellular Transport/Diffusion......Page 328
10.2.4 PROS......Page 329
10.3.1 INTRODUCTION......Page 330
10.3.2 DIRECT BINDING ASSAYS OF FLUORESCENT LIPID ANALOGUES......Page 331
10.3.4 FLUORESCENCE LIFETIME AND ANISOTROPY ASSAYS......Page 334
10.3.5 FLUORESCENCE RESONANCE ENERGY TRANSFER......Page 340
10.3.6 PROS AND CONS......Page 342
10.4.2 NUCLEIC ACID STAINS......Page 343
10.4.3 NUCLEAR LOCALIZATION......Page 344
10.4.4.1 Fusion of Fluorescent Expression Proteins......Page 346
10.4.4.2 Recombinant Proteins Covalently Labeledwith Small Fluorescent Tags......Page 347
10.4.4.3 Immunofl uorescence Labeling of Fixed Cells or Tissues......Page 348
10.4.5 PROS AND CONS......Page 349
10.5.1 INTRODUCTION......Page 351
10.5.2 DIRECT BINDING ASSAYS OF FLUORESCENT LIPID ANALOGUES......Page 352
10.5.3 DISPLACEMENT ASSAYS......Page 353
10.5.4 QUENCHING OF INTRINSICALLY FLUORESCENT AMINO ACIDS......Page 354
10.5.6 PROS AND CONS......Page 356
10.6.2 FLUOROPHORE CHOICES......Page 357
10.6.3 SPECTROSCOPY FRET IN VITRO......Page 358
10.6.4 CONFOCAL MICROSCOPY— COLOCALIZATION......Page 360
10.6.6 IMMUNOGOLD ELECTRON MICROSCOPY......Page 362
10.6.8 PROS......Page 363
LIST OF ABBREVIATIONS......Page 364
REFERENCES......Page 365
Part IV Methods to Assess Fatty Acid and Phospholipid Metabolism......Page 370
CONTENTS......Page 372
11.1 INTRODUCTION......Page 374
11.2.1 DERIVATIZATION BEFORE GLC ANALYSIS......Page 375
11.2.1.1 Acid-Catalyzed Esterifi cation......Page 376
11.2.1.3 Methylation Using Diazomethane......Page 377
11.3.1 FATTY ACID AUTOXIDATION......Page 378
11.3.4 EXTRACTION FROM TLC PLATES......Page 379
11.4.1 PHENACYL ESTERS AND RELATED DERIVATIVES......Page 381
11.4.2 ANTHRONYL DERIVATIVES......Page 382
ACKNOWLEDGMENTS......Page 383
Protocol A: Acid-catalyzed esterifi cation......Page 384
Protocol B: Base- catalyzed transesterifi cation......Page 385
Protocol C: Elution of lipids from silica......Page 387
Protocol E: Phenacyl derivatives......Page 388
REFERENCES......Page 389
12.1 INTRODUCTION......Page 394
12.1.1 IMPORTANCE......Page 395
12.1.2 FACTORS REGULATING FATTY ACID UPTAKE......Page 396
12.1.3 WHY CONCENTRATION MATTERS......Page 397
12.1.4 CELL CULTURES......Page 398
12.2.1 CELL INCUBATION WITH FATTY ACIDS AND CELL LIPID EXTRACTION......Page 399
12.2.2 PULSE EXPERIMENTS AND WHY TIME IS IMPORTANT......Page 401
12.3 FATTY ACID UPTAKE IN TISSUES......Page 402
12.3.1 TRACER PREPARATION, SURGERY, AND INFUSION......Page 403
12.3.2 TISSUE AND PLASMA EXTRACTION......Page 404
12.3.4 ASSESSING FATTY ACID UPTAKE AND TARGETING......Page 405
ACKNOWLEDGMENTS......Page 406
Protocol A: Cell extraction of lipids and proteins......Page 407
Protocol B: Silica columns for separation of neutral lipids and phospholipids......Page 408
Protocol C: Folch extraction: Two- phase extraction ( radioactive preparation)......Page 409
LIST OF ABBREVIATIONS......Page 410
REFERENCES......Page 411
CONTENTS......Page 416
13.2 STEADY- STATE INCORPORATION KINETICS......Page 417
13.2.1 FATTY ACIDS AND STEADY-STATE RADIOACTIVITY......Page 419
13.2.2 SURGICAL CONSIDERATIONS AND CANNULA PLACEMENT......Page 420
13.2.2.1 Surgical Placement of Catheters......Page 422
13.2.3 PLASMA CURVES AND INPUT FUNCTIONS......Page 423
13.2.4.1 Brain and Plasma Lipid Extraction and Chromatography......Page 424
13.2.4.4 Long-Chain Fatty Acyl-CoA Analysis......Page 425
13.3 CALCULATING RATES OF INCORPORATION AND FATTY ACID TURNOVER......Page 426
13.4 CONCLUSIONS......Page 427
APPENDIX: COMMONLY USED REAGENTS AND PROTOCOLS......Page 429
REFERENCES......Page 433
Color Insert......Page 436