Elsevier S & T

Small GTPases in Disease, Part B

Balch, W. E.

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Table of contents
  • Contentsv
  • Contributorsxv
  • Methods in Enzymologyxxv
  • Chapter 1: Human Pancreatic Duct Epithelial Cell Model for KRAS Transformation1
  • 1. Introduction2
  • 2. Description of Methods3
  • 3. Concluding Remarks11
  • Acknowledgment12
  • References12
  • Chapter 2: Mouse Model for NRAS-Induced Leukemogenesis15
  • 1. Introduction16
  • 2. Modeling NRAS Leukemogenesis in Mice Using the BMT Method18
  • 3. Characterization of Diseased NRAS Mice22
  • Acknowledgment127
  • References127
  • Chapter 3: Inducible BRAF Suppression Models for Melanoma Tumorigenesis25
  • 1. Introduction26
  • 2. Materials and Methods31
  • 3. Conclusion37
  • Acknowledgment127
  • References127
  • Chapter 4: A Method to Generate Genetically Defined Tumors in Pigs39
  • 1. Introduction40
  • 2. Overview41
  • 3. Derivation of Cell Line from Swine Ear Notches41
  • 4. Creation of Six Gene-Transformed Cells43
  • 5. Injecting Cells into Pigs48
  • 6. Concluding Remarks51
  • References127
  • Chapter 5: Tools to Study the Function of the Ras-Related, Estrogen-Regulated Growth Inhibitor in Br53
  • 1. Introduction54
  • 2. Molecular Constructs56
  • 3. Cell Culture and Expression Vector Transfection and Infection58
  • 4. Cell Growth and Invasion Assays59
  • 5. Generation and Validation of Rerg Antibodies for Western Blotting and Immunohistochemistry60
  • 6. Transient and Stable Expression of Wild-Type and Mutant Rerg Proteins63
  • 7. Establishment of a Tamoxifen-Inducible Rerg Protein Expression System64
  • 8. Suppression of Rerg Expression by RNA Interference68
  • 9. Summary70
  • Acknowledgments127
  • References127
  • Chapter 6: K-Ras-Driven Pancreatic Cancer Mouse Model for Anticancer Inhibitor Analyses73
  • 1. Introduction74
  • 2. Pancreatic Ductal Adenocarcinoma Models75
  • 3. Imaging76
  • 4. Drug Delivery78
  • 5. End Point Analysis79
  • 6. Model Fidelity for Preclinical Testing82
  • 7. Novel Therapeutics and Preclinical Trial Design82
  • 8. Conclusions84
  • References127
  • Chapter 7: Analysis of K-Ras Phosphorylation, Translocation, and Induction of Apoptosis87
  • 1. Introduction88
  • 2. Analysis of K-Ras Phosphorylation by Metabolic Labeling with 32P89
  • 3. Phoshorylation of Endogenous K-Ras in T Cells92
  • 4. Analysis of K-Ras Farnesyl-Electrostatic Switch by Live Cell Imaging94
  • 5. Targeting of PKC to Activated K-Ras with the Ras Binding Domain of Raf-1 (RBD)98
  • 6. Single-Cell Fluorescent Analysis of Apoptosis: YFP-Caspase Sensor99
  • References127
  • Chapter 8: Regulation of RhoBTB2 by the Cul3 Ubiquitin Ligase Complex103
  • 1. Introduction104
  • 2. Methods105
  • References127
  • Chapter 9: Characterization of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPase111
  • 1. Introduction112
  • 2. Experimental Procedures114
  • 3. Concluding Remarks126
  • Acknowledgments127
  • References127
  • Chapter 10: Analysis of Rho-GTPase Mimicry by a Family of Bacterial Type III Effector Proteins131
  • 1. Introduction132
  • 2. Analysis of WxxxE Effectors as Rho-GTPase Mimics in Mammalian Cells133
  • 3. Analysis of Small G-Protein Mimicry Using Yeast as a Model System138
  • 4. Concluding Remarks142
  • Acknowledgments
  • References
  • Chapter 11: Investigating the Function of Rho Family GTPases during Salmonella/Host Cell Interaction145
  • 1. Introduction146
  • 2. Determining Salmonella-Induced Rho GTPase Activation147
  • 3. Determining the Role of Rho GTPases in S. typhimurium-Induced Actin Remodeling150
  • 4. Determining the Role of Rho GTPases in S. typhimurium Internalization154
  • 5. Summary156
  • Acknowledgment
  • References
  • Chapter 12: Contribution of Cdc42 to Cholesterol Efflux in Fibroblasts from Tangier Disease and Wern159
  • 1. Introduction160
  • 2. cDNA Subtraction Technique Reveals Decreased Expression of Cdc42 in Macrophages from a Patient wi161
  • 3. Cdc42 Plays Some Role in Cholesterol Efflux from Cells162
  • 4. Cdc42 Binds with a Gate Player for Cholesterol Efflux, ABCA1163
  • 5. Role of Cdc42 in Intracellular Lipid Transport164
  • 6. The Significance of Cdc42 in Werner Syndrome166
  • 7. Summary166
  • Acknowledgments
  • References
  • Chapter 13: Rac and Nuclear Translocation of Signal Transducers and Activators of Transcription Fact171
  • 1. Introduction172
  • 2. Rac1 and MgcRacGAP Regulate Nuclear Accumulation of p-STATs172
  • 3. Protocol for Purification of Flag-Tagged Recombinant Proteins Using Sf-9 cells174
  • 4. Rac1 and MgcRacGAP Serve as a Nuclear Chaperone for p-STATs174
  • 5. Protocol for Nuclear Import Assays with Digitonin-Permeabilized Cells Using Purified Flag-Tagged177
  • References
  • Chapter 14: A Method for Measuring Rho Kinase Activity in Tissues and Cells181
  • 1. Introduction182
  • 2. Downstream Targets of ROCK183
  • 3. Measurement of ROCK Activity184
  • 4. Cultured Cells184
  • 5. Leukocytes186
  • 6. Tissues187
  • 7. Summary187
  • References188
  • Chapter 15: Rho Kinase-Mediated Vasoconstriction in Rat Models of Pulmonary Hypertension191
  • 1. Introduction192
  • 2. Rat Models of Severe Pulmonary Hypertension194
  • 3. Acute Hemodynamic Effects of Selective Rho Kinase Inhibitors196
  • 4. Dephospholyration of MYPT1197
  • 5. Ca2+ Sensitization199
  • 6. RhoA Activity200
  • References
  • Chapter 16: Epithelial Rho GTPases and the Transepithelial Migration of Lymphocytes205
  • 1. Introduction206
  • 2. Culture of Bronchial Epithelial Cells208
  • 3. Measurement of TER and Permeability to FITC-D extran 40 KDa209
  • 4. Culture of Primary Human T Lymphocytes210
  • 5. Development of an Assay for Lymphocyte Transepithelial Migration211
  • Acknowledgments
  • References
  • Chapter 17: Invasion and Metastasis Models for Studying RhoGDI2 in Bladder Cancer219
  • 1. Introduction220
  • 2. Methods222
  • 3. Evaluating Lung Metastasis Using the Classic "Experimental Metastasis" Assay224
  • 4. Creating Increasingly Lung Metastatic Bladder Cancer Cell Lines229
  • Acknowledgments
  • References
  • Chapter 18: Characterization of the Roles of Rac1 and Rac2 GTPases in Lymphocyte Development235
  • 1. Introduction236
  • 2. Analysis of B-Cell Development237
  • 3. Analysis of T-Cell Development246
  • Acknowledgment
  • References
  • Chapter 19: Characterization of Oligophrenin-1, a RhoGAP Lost in Patients Affected with Mental Retar255
  • 1. Introduction: Rho GTPases, Synaptic Structure and Function256
  • 2. Preparation of Organotypic Brain Slice Cultures257
  • 3. Lentivirus Preparation and Injection260
  • 4. Conclusions264
  • Acknowledgments264
  • References
  • Chapter 20: Rho GTPases and Hypoxia in Pulmonary Vascular Endothelial Cells267
  • 1. Introduction
  • 2. Purification and Culture of Primary Pulmonary Artery Endothelial Cells
  • 3. Hypoxic Exposure and Reoxygenation
  • 4. Studying Endothelial Integrity
  • 5. Transendothelial Permeability Assays273
  • 6. Localization of F-actin and VE-cadherin
  • 7. Investigating the Mechanism of Hypoxia/Reoxygenation-Induced Changes: The Role of Rho GTPases275
  • 8. Measuring Rho GTPases Activity275
  • 9. Analysis of Rho GTPase Function in Hypoxia/Reoxygenation-Induced Changes in Endothelial Permeabil278
  • 10. Effects of Changes in NADPH-Mediated ROS Production on the Activity of Rho GTPases and Endotheli279
  • 11. Measurement of ROS281
  • References
  • Chapter 21: Role of Rho GTPases in the Morphogenesis and Motility of Dendritic Spines285
  • 1. Introduction286
  • 2. Methods288
  • 3. Imaging292
  • 4. Analysis294
  • 5. Role of RhoA and Rho Kinase295
  • 6. Role of Rac1295
  • Acknowledgments
  • References
  • Chapter 22: Rho GTPases in Alveolar Macrophage Phagocytosis303
  • 1. Introduction304
  • 2. Alveolar Macrophages304
  • 3. Rho GTPase Activation in Macrophage Phagocytosis305
  • 4. Rho GTPases and Alveolar Macrophage Phagocytosis306
  • 5. Methods and Materials308
  • 6. Conclusions311
  • Acknowledgment
  • References
  • Chapter 23: Analysis of the Elp Complex and Its Role in Regulating Exocytosis315
  • 1. Introduction315
  • 2. Generation of Polyclonal Antibodies against Elp1p316
  • 3. Antigen Injections and IgY Purification318
  • 4. Antibody Testing Procedure319
  • 5. Genetic Analysis to Identify Negative Regulators of Exocytosis321
  • References
  • Chapter 24: Rab-Regulated Membrane Traffic between Adiposomes and Multiple Endomembrane Systems327
  • 1. Introduction328
  • 2. Rab-Mediated Adiposome-Endosome Interaction in vitro329
  • 3. Conclusion336
  • Acknowledgments
  • References
  • Chapter 25: Detection of Compounds That Rescue Rab1-Synuclein Toxicity339
  • 1. Introduction340
  • 2. Strains341
  • 3. Methods for Detecting Compounds That Rescue a-Synuclein-Induced Toxicity341
  • 4. Primary Assays343
  • 5. Secondary Assays346
  • 6. Conclusion349
  • References350
  • Chapter 26: Analysis of Rab GTPase and GTPase-Activating Protein Function at Primary Cilia353
  • 1. Introduction353
  • 2. Methods355
  • References
  • Chapter 27: Rho GTPases and Regulation of Hematopoietic Stem Cell Localization365
  • 1. Basic Mechanisms of Hematopoietic Stem Cell and Progenitor (HSC/P) Homing and Retention in Bone M366
  • 2. Basic Mechanisms of HSC/P Mobilization and Trafficking368
  • 3. Rho GTPases370
  • 4. Role of Rac1 and Rac2 GTPases in Hematopoiesis373
  • 5. Rac3 GTPase, a Newly Defined Member of the Rac Family Cloned from a Bcr-abl Transformed Cell Line374
  • 6. Cdc42 in Hematopoiesis375
  • 7. RhoA in Hematopoiesis376
  • 8. RhoGTPase in Human Diseases377
  • 9. Summary and Perspectives383
  • References
  • Chapter 28: In Vitro and In Vivo Assays to Analyze the Contribution of Rho Kinase in Angiogenesis395
  • 1. Introduction396
  • 2. In Vitro Assays Used to Analyze the Contribution of Rho Kinase in Angiogenesis397
  • 3. In Vivo Assays Used to Analyze the Contribution of Rho Kinase in Angiogenesis407
  • 4. Concluding Remarks409
  • Acknowledgments
  • References
  • Chapter 29: Analysis of Cell Migration and Its Regulation by Rho GTPases and p53 in a Three-Dimensio413
  • 1. Introduction414
  • 2. Experimental Procedure415
  • 3. Concluding Remarks421
  • Acknowledgments
  • References
  • Chapter 30: Use of Caenorhabditis elegans to Evaluate Inhibitors of Ras Function In Vivo425
  • 1. Introduction426
  • 2. Experimental Protocol430
  • 3. Experimental Procedure438
  • 4. Scoring Wild-Type versus Muv Phenotypes441
  • 5. Proof-of-Principle Experiment444
  • 6. Conclusions and Future Directions446
  • Acknowledgments
  • References
  • Chapter 31: Ras-Driven Transformation of Human Nestin-Positive Pancreatic Epithelial Cells451
  • 1. Introduction452
  • 2. Isolation and Immortalization of Primary Pancreatic Ductal Cells453
  • 3. Additional Genetic Steps Required to Transform Pancreatic Duct-Derived Cells455
  • 4. Analysis of K-Ras Effector Pathways in Pancreatic Cell Transformation459
  • 5. Discussion462
  • Acknowledgments463
  • References463
  • Chapter 32: The Ras Inhibitor Farnesylthiosalicylic Acid (Salirasib) Disrupts the Spatiotemporal Loc467
  • 1. Introduction468
  • 2. Methods469
  • 3. Disruption of Ras Localization as a Potential Method for Cancer Treatment474
  • Acknowledgments483
  • References484
  • Chapter 33: Quantitative High-Throughput Cell-Based Assays for Inhibitors of ROCK Kinases491
  • 1. Introduction492
  • 2. Materials and Methods493
  • 3. Results and Discussion495
  • References499
  • Chapter 34: Tandem Affinity Purification of the BBSome, a Critical Regulator of Rab8 in Ciliogenesis501
  • 1. Introduction502
  • 2. Materials503
  • 3. Procedures505
  • Acknowledgments
  • References
  • Author Index515
  • Subject Index549
Book details
  • Vendor Elsevier S & T
  • SKU 9780123743114
  • ISBN-13 9780080570075
  • Author Balch, W. E.
  • Category Medical
  • Subject Biochemistry

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This volume is the second of two planned volumes on the topic of small GTPases and their role in disease.