Chemokines, Part A

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ISBN: 9780123749086 | 0123749085
Cover: Hardcover
Copyright: 5/27/2009

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The understanding of chemokines, the proteins that control the migration of cells, and their receptors, is critical to the study of causes and therapies for a wide range of human diseases and infections, including certain types of cancer, inflammatory diseases, HIV, and malaria. This volume, focusing on chemokines as potential targets for disease intervention, and its companion volume (Methods in Enzymology volume 462, focusing on chemokine structure and function, as well as signaling) provide a comprehensive overview and time-tested protocols in this field, making it an essential reference for researchers in the area. Gathers tried and tested methods and techniques from top players in the field Provides an essential reference for the experienced research scientist

Contributors	p. xiii
Preface	p. xxi
Volumes in Series	p. xxiii
Chemokines and Receptors in Disease	p. 1
Chemokines in Human Breast Tumor Cells: Modifying Their Expression Levels and Determining Their Effects on the Malignancy Phenotype	p. 3
Introduction	p. 4
Modifying Chemokine Expression in Breast Tumor Cells	p. 5
Establishment of Primary Local Breast Tumors and Pulmonary Metastases	p. 9
Acknowledgments	p. 15
References	p. 15
CCR5 Pharmacology Methodologies and Associated Applications	p. 17
Introduction	p. 18
CCR5 Signaling Assays and Application to Quantify and Characterize Ligand-Dependent Agonism, Antagonism, and Inverse Agonism	p. 19
CCR5-Associated Ligand-Binding Assays	p. 33
Surrogate In Vitro Antiviral Assays	p. 38
CCR5 Site-Directed Mutagenesis and Ligand Docking Studies	p. 43
Non-HIV Indications-Associated Studies, Human CCR5 Knock-In Mice	p. 48
References	p. 52
CXCR4 and Mobilization of Hematopoietic Precursors	p. 57
Introduction	p. 58
HSPC Mobilizing Agents that Target the CXCL12/CXCR4 Axis	p. 59
Donor Selection for HSPC Mobilization	p. 64
Flow Cytometric Enumeration of Mobilized HSPCs	p. 65
Dosing and Kinetics of HSPC Mobilization by G-CSF and Plerixafor	p. 66
Flow Cytometric Analysis of CXCR4 Expression on Human CD34⁺ Subsets	p. 69
Functional Characterization of Mobilized HSPCs	p. 73
Concluding Remarks	p. 80
Acknowledgment	p. 80
References	p. 80
Double-Label Nonradioactive In Situ Hybridization for the Analysis of Chemokine Receptor Expression in the Central Nervous System	p. 91
Introduction	p. 92
Basic Protocol for ISH (Using Digoxygenin-Labeled Probe)	p. 93
Comments	p. 101
References	p. 102
Expression of Chemokines and Chemokine Receptors in Human Colon Cancer	p. 105
Introduction	p. 106
Materials and Methods	p. 108
Results	p. 110
Discussion	p. 117
Acknowledgments	p. 119
References	p. 119
Chemokine Related Proteins from Pathogens	p. 123
Kaposi's Sarcoma Virally Encoded, G-Protein-Coupled Receptor: A Paradigm for Paracrine Transformation	p. 125
Introduction	p. 127
Cloning of vGPCR	p. 128
Assaying vGPCR Transforming Activity In Vitro	p. 130
vGPCR Transforming Activity In Vivo Using Xenograft Systems	p. 130
In Vivo Targeted Infection Using the TVA-RCAS System	p. 131
vGPCR-Induced Paracrine Transformation	p. 134
Characterization of vGPCR-Induced Molecular Signaling	p. 135
Evaluation of Activation of Second-Messenger-Generating Systems	p. 136
Activation of Signal-Transducing Protein Kinases and Small GTPases	p. 137
Akt Kinase Assay	p. 137
vGPCR Stimulated Activation of Rac1-Pulldown Assays	p. 138
Western Blotting Using Phospho-Specific Antibodies	p. 140
Activation of Transcription Factors	p. 141
Global Changes in Gene Expression: Microarray Analysis	p. 141
NFkB Luciferase Assays	p. 143
NFkB Binding Assays	p. 144
Nuclear Translocation of NFkB	p. 146
Acknowledgments	p. 147
References	p. 147
Pharmacological and Biochemical Characterization of Human Cytomegalovirus-Encoded G Protein-Coupled Receptors	p. 151
Introduction	p. 152
Virally Encoded GPCR Engineering	p. 154
vGPCR Expression, Trafficking, and Radioligand Binding	p. 156
vGPCR-Induced Signal Transduction	p. 159
vGPCR-Induced Oncogenesis	p. 162
Generation of Recombinant HCMV Strains by Markerless Bacterial Artificial Chromosome Mutagenesis	p. 165
Conclusions	p. 167
Acknowledgments	p. 167
References	p. 167
Identification and Characterization of Virus-Encoded Chemokine Binding Proteins	p. 173
Introduction	p. 174
Methods for Studying Chemokine-Binding Proteins	p. 175
Acknowledgments	p. 189
References	p. 189
The Chemokine-Binding Protein M3 as a Tool to Understand the Chemokine Network In Vivo	p. 193
Introduction	p. 193
Generation of Transgenic Mice Expressing M3 in Insulin-Producing b Cells	p. 195
M3 Expression in Islets of Langerhans Blocks CCL2-, CCL21-, and CXCL13-Induced Migration of Cells to Islets	p. 198
M3 Expression in b Cells Blocks Cellular Infiltration and Prevents Diabetes Development	p. 199
Generation of a Conditional Transgenic System for Expression of M3	p. 202
Concluding Remarks	p. 204
References	p. 205
M-T7: Measuring Chemokine-Modulating Activity	p. 209
Introduction	p. 210
Protein Expression	p. 213
Quantifying the Effects of M-T7 In Vitro and Ex Vivo	p. 216
Quantifying the Effects of M-T7 on Vascular Inflammatory Responses in Rodent Vascular Transplant Models	p. 220
Preclinical Toxicity Testing	p. 225
References	p. 227
Atypical and Novel Chemoattractants and Receptors	p. 229
Role of the Chemokine Scavenger Receptor D6 in Balancing Inflammation and Immune Activation	p. 231
Introduction	p. 232
Methods	p. 232
Results	p. 235
Discussion	p. 236
References	p. 241
Structure-Function Dissection of D6, an Atypical Scavenger Receptor	p. 245
Introduction	p. 246
Acknowledgments	p. 260
References	p. 260
Modeling Small Molecule-Compound Binding to G-Protein-Coupled Receptors	p. 263
Introduction	p. 264
Similarity and Differences in the Crystal Structures of Class-A GPCRs Solved to Date	p. 265
GPCR Modeling Methods	p. 266
Computational Methods for Receptor Flexibility and Ligand-Induced Conformational Changes in GPCRs	p. 271
Validation of GPCR-Ligand Models	p. 274
Conclusions	p. 284
References	p. 286
Elucidation of Chemerin and Chemokine-Like Receptor-1 Function in Adipocytes by Adenoviral-Mediated shRNA Knockdown of Gene Expression	p. 289
Introduction	p. 290
The 3T3-L1 Cell Model for Adipogenesis and Adipocyte Metabolism	p. 292
RNA Interference	p. 293
Methods for Adenoviral shRNA Knockdown of Chemerin and CMKLR1 in 3T3-L1 Cells	p. 297
Concluding Remarks	p. 309
Acknowledgments	p. 309
References	p. 309
Chemokine Signaling	p. 313
Characterization of Chemokine Receptor CXCR2 Interacting Proteins Using a Proteomics Approach to Define the CXCR2 "Chemosynapse"	p. 315
Introduction	p. 316
Validation of the Interaction of Novel Proteins with CXCR2	p. 320
Mutational Analysis of Residues at Interactive Interface of CXCR2 and CXCR2-Binding Proteins	p. 324
Radioactive Phosphorylation of CXCR2 Interacting Proteins	p. 325
Chemotaxis Assay	p. 326
Conclusions	p. 329
Acknowledgments	p. 329
References	p. 329
Phosphoproteomic Analysis of Chemokine Signaling Networks	p. 331
Introduction	p. 332
Methods	p. 334
Summary	p. 343
Acknowledgments	p. 344
References	p. 345
Monitoring NF-kB Mediated Chemokine Transcription in Tumorigenesis	p. 347
Introduction	p. 348
Development of NF-kB Reporter Model for Tumors	p. 348
Bioluminescent Imaging of Intratumor Signaling of Anesthetized Mice	p. 349
Cell-Based Assays for Kinase and Transcriptional Activity In Vitro	p. 352
Peripheral Spying of Intratumoral Signaling of Conscious Mice	p. 353
Acknowledgments	p. 354
References	p. 354
Analysis of Chemokine Receptor Endocytosis and Intracellular Trafficking	p. 357
Introduction	p. 358
Receptor Detection	p. 360
Cells	p. 362
Monitoring Receptor Endocytosis	p. 364
Monitoring Receptor Recycling	p. 366
Monitoring Receptor Degradation	p. 368
Electron Microscopy Analysis of Receptor Internalization	p. 371
Acknowledgments	p. 375
References	p. 375
Measuring the Proximity of T-Lymphocyte CXCR4 and TCR by Fluorescence Resonance Energy Transfer (FRET)	p. 379
Introduction	p. 380
Assaying CXCR4-TCR Proximity via the PE/APC mAb FRET Approach	p. 386
Assaying CXCR4-TCR Proximity via the CFP/YFP Fusion Protein Approach	p. 392
Concluding Remarks	p. 396
References	p. 396
Expression of CXCR4, a G-Protein-Coupled Receptor for CXCL12 in Yeast: Identification of New-Generation Inverse Agonists	p. 399
Introduction	p. 400
Methods and Discussion	p. 402
Summary	p. 408
References	p. 410
Ubiquitination of Chemokine Receptors	p. 413
Introduction	p. 414
Cell Culture and Transfections	p. 416
Agonist Treatment and Ubiquitination Assay	p. 417
E3 Ubiquitin Ligase AIP4 Mediates Ubiquitination of CXCR4	p. 419
References	p. 421
Author Index	p. 423
Subject Index	p. 451
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