Small GTPases in Disease, Part B

Name: Small GTPases in Disease, Part B
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Published on 1. April 2008

616 pages

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978-0-08-057007-5 (ISBN)

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Cover
Contents
Contributors
Methods in Enzymology
Chapter 1: Human Pancreatic Duct Epithelial Cell Model for KRAS Transformation
1. Introduction
2. Description of Methods
3. Concluding Remarks
Acknowledgment
References
Chapter 2: Mouse Model for NRAS-Induced Leukemogenesis
1. Introduction
2. Modeling NRAS Leukemogenesis in Mice Using the BMT Method
3. Characterization of Diseased NRAS Mice
Acknowledgment
References
Chapter 3: Inducible BRAF Suppression Models for Melanoma Tumorigenesis
1. Introduction
2. Materials and Methods
3. Conclusion
Acknowledgment
References
Chapter 4: A Method to Generate Genetically Defined Tumors in Pigs
1. Introduction
2. Overview
3. Derivation of Cell Line from Swine Ear Notches
4. Creation of Six Gene-Transformed Cells
5. Injecting Cells into Pigs
6. Concluding Remarks
References
Chapter 5: Tools to Study the Function of the Ras-Related, Estrogen-Regulated Growth Inhibitor in Breast Cancer
1. Introduction
2. Molecular Constructs
3. Cell Culture and Expression Vector Transfection and Infection
4. Cell Growth and Invasion Assays
5. Generation and Validation of Rerg Antibodies for Western Blotting and Immunohistochemistry
6. Transient and Stable Expression of Wild-Type and Mutant Rerg Proteins
7. Establishment of a Tamoxifen-Inducible Rerg Protein Expression System
8. Suppression of Rerg Expression by RNA Interference
9. Summary
Acknowledgments
References
Chapter 6: K-Ras-Driven Pancreatic Cancer Mouse Model for Anticancer Inhibitor Analyses
1. Introduction
2. Pancreatic Ductal Adenocarcinoma Models
3. Imaging
4. Drug Delivery
5. End Point Analysis
6. Model Fidelity for Preclinical Testing
7. Novel Therapeutics and Preclinical Trial Design
8. Conclusions
References
Chapter 7: Analysis of K-Ras Phosphorylation, Translocation, and Induction of Apoptosis
1. Introduction
2. Analysis of K-Ras Phosphorylation by Metabolic Labeling with 32P
3. Phoshorylation of Endogenous K-Ras in T Cells
4. Analysis of K-Ras Farnesyl-Electrostatic Switch by Live Cell Imaging
5. Targeting of PKC to Activated K-Ras with the Ras Binding Domain of Raf-1 (RBD)
6. Single-Cell Fluorescent Analysis of Apoptosis: YFP-Caspase Sensor
References
Chapter 8: Regulation of RhoBTB2 by the Cul3 Ubiquitin Ligase Complex
1. Introduction
2. Methods
References
Chapter 9: Characterization of EHT 1864, a Novel Small Molecule Inhibitor of Rac Family Small GTPases
1. Introduction
2. Experimental Procedures
3. Concluding Remarks
Acknowledgments
References
Chapter 10: Analysis of Rho-GTPase Mimicry by a Family of Bacterial Type III Effector Proteins
1. Introduction
2. Analysis of WxxxE Effectors as Rho-GTPase Mimics in Mammalian Cells
3. Analysis of Small G-Protein Mimicry Using Yeast as a Model System
4. Concluding Remarks
Acknowledgments
References
Chapter 11: Investigating the Function of Rho Family GTPases during Salmonella/Host Cell Interactions
1. Introduction
2. Determining Salmonella-Induced Rho GTPase Activation
3. Determining the Role of Rho GTPases in S. typhimurium-Induced Actin Remodeling
4. Determining the Role of Rho GTPases in S. typhimurium Internalization
5. Summary
Acknowledgment
References
Chapter 12: Contribution of Cdc42 to Cholesterol Efflux in Fibroblasts from Tangier Disease and Werner Syndrome
1. Introduction
2. cDNA Subtraction Technique Reveals Decreased Expression of Cdc42 in Macrophages from a Patient with TD
3. Cdc42 Plays Some Role in Cholesterol Efflux from Cells
4. Cdc42 Binds with a Gate Player for Cholesterol Efflux, ABCA1
5. Role of Cdc42 in Intracellular Lipid Transport
6. The Significance of Cdc42 in Werner Syndrome
7. Summary
Acknowledgments
References
Chapter 13: Rac and Nuclear Translocation of Signal Transducers and Activators of Transcription Factors
1. Introduction
2. Rac1 and MgcRacGAP Regulate Nuclear Accumulation of p-STATs
3. Protocol for Purification of Flag-Tagged Recombinant Proteins Using Sf-9 cells
4. Rac1 and MgcRacGAP Serve as a Nuclear Chaperone for p-STATs
5. Protocol for Nuclear Import Assays with Digitonin-Permeabilized Cells Using Purified Flag-Tagged Proteins
References
Chapter 14: A Method for Measuring Rho Kinase Activity in Tissues and Cells
1. Introduction
2. Downstream Targets of ROCK
3. Measurement of ROCK Activity
4. Cultured Cells
5. Leukocytes
6. Tissues
7. Summary
References
Chapter 15: Rho Kinase-Mediated Vasoconstriction in Rat Models of Pulmonary Hypertension
1. Introduction
2. Rat Models of Severe Pulmonary Hypertension
3. Acute Hemodynamic Effects of Selective Rho Kinase Inhibitors
4. Dephospholyration of MYPT1
5. Ca2+ Sensitization
6. RhoA Activity
References
Chapter 16: Epithelial Rho GTPases and the Transepithelial Migration of Lymphocytes
1. Introduction
2. Culture of Bronchial Epithelial Cells
3. Measurement of TER and Permeability to FITC-D extran 40 KDa
4. Culture of Primary Human T Lymphocytes
5. Development of an Assay for Lymphocyte Transepithelial Migration
Acknowledgments
References
Chapter 17: Invasion and Metastasis Models for Studying RhoGDI2 in Bladder Cancer
1. Introduction
2. Methods
3. Evaluating Lung Metastasis Using the Classic "Experimental Metastasis" Assay
4. Creating Increasingly Lung Metastatic Bladder Cancer Cell Lines
Acknowledgments
References
Chapter 18: Characterization of the Roles of Rac1 and Rac2 GTPases in Lymphocyte Development
1. Introduction
2. Analysis of B-Cell Development
3. Analysis of T-Cell Development
Acknowledgment
References
Chapter 19: Characterization of Oligophrenin-1, a RhoGAP Lost in Patients Affected with Mental Retardation: Lentiviral Injection in Organotypic Brain Slice Cultures
1. Introduction: Rho GTPases, Synaptic Structure and Function
2. Preparation of Organotypic Brain Slice Cultures
3. Lentivirus Preparation and Injection
4. Conclusions
Acknowledgments
References
Chapter 20: Rho GTPases and Hypoxia in Pulmonary Vascular Endothelial Cells
1. Introduction
2. Purification and Culture of Primary Pulmonary Artery Endothelial Cells
3. Hypoxic Exposure and Reoxygenation
4. Studying Endothelial Integrity
5. Transendothelial Permeability Assays
6. Localization of F-actin and VE-cadherin
7. Investigating the Mechanism of Hypoxia/Reoxygenation-Induced Changes: The Role of Rho GTPases
8. Measuring Rho GTPases Activity
9. Analysis of Rho GTPase Function in Hypoxia/Reoxygenation-Induced Changes in Endothelial Permeability
10. Effects of Changes in NADPH-Mediated ROS Production on the Activity of Rho GTPases and Endothelial Barrier Function
11. Measurement of ROS
References
Chapter 21: Role of Rho GTPases in the Morphogenesis and Motility of Dendritic Spines
1. Introduction
2. Methods
3. Imaging
4. Analysis
5. Role of RhoA and Rho Kinase
6. Role of Rac1
Acknowledgments
References
Chapter 22: Rho GTPases in Alveolar Macrophage Phagocytosis
1. Introduction
2. Alveolar Macrophages
3. Rho GTPase Activation in Macrophage Phagocytosis
4. Rho GTPases and Alveolar Macrophage Phagocytosis
5. Methods and Materials
6. Conclusions
Acknowledgment
References
Chapter 23: Analysis of the Elp Complex and Its Role in Regulating Exocytosis
1. Introduction
2. Generation of Polyclonal Antibodies against Elp1p
3. Antigen Injections and IgY Purification
4. Antibody Testing Procedure
5. Genetic Analysis to Identify Negative Regulators of Exocytosis
References
Chapter 24: Rab-Regulated Membrane Traffic between Adiposomes and Multiple Endomembrane Systems
1. Introduction
2. Rab-Mediated Adiposome-Endosome Interaction in vitro
3. Conclusion
Acknowledgments
References
Chapter 25: Detection of Compounds That Rescue Rab1-Synuclein Toxicity
1. Introduction
2. Strains
3. Methods for Detecting Compounds That Rescue a-Synuclein-Induced Toxicity
4. Primary Assays
5. Secondary Assays
6. Conclusion
References
Chapter 26: Analysis of Rab GTPase and GTPase-Activating Protein Function at Primary Cilia
1. Introduction
2. Methods
References
Chapter 27: Rho GTPases and Regulation of Hematopoietic Stem Cell Localization
1. Basic Mechanisms of Hematopoietic Stem Cell and Progenitor (HSC/P) Homing and Retention in Bone Marrow (BM)
2. Basic Mechanisms of HSC/P Mobilization and Trafficking
3. Rho GTPases
4. Role of Rac1 and Rac2 GTPases in Hematopoiesis
5. Rac3 GTPase, a Newly Defined Member of the Rac Family Cloned from a Bcr-abl Transformed Cell Line
6. Cdc42 in Hematopoiesis
7. RhoA in Hematopoiesis
8. RhoGTPase in Human Diseases
9. Summary and Perspectives
References
Chapter 28: In Vitro and In Vivo Assays to Analyze the Contribution of Rho Kinase in Angiogenesis
1. Introduction
2. In Vitro Assays Used to Analyze the Contribution of Rho Kinase in Angiogenesis
3. In Vivo Assays Used to Analyze the Contribution of Rho Kinase in Angiogenesis
4. Concluding Remarks
Acknowledgments
References
Chapter 29: Analysis of Cell Migration and Its Regulation by Rho GTPases and p53 in a Three-Dimensional Environment
1. Introduction
2. Experimental Procedure
3. Concluding Remarks
Acknowledgments
References
Chapter 30: Use of Caenorhabditis elegans to Evaluate Inhibitors of Ras Function In Vivo
1. Introduction
2. Experimental Protocol
3. Experimental Procedure
4. Scoring Wild-Type versus Muv Phenotypes
5. Proof-of-Principle Experiment
6. Conclusions and Future Directions
Acknowledgments
References
Chapter 31: Ras-Driven Transformation of Human Nestin-Positive Pancreatic Epithelial Cells
1. Introduction
2. Isolation and Immortalization of Primary Pancreatic Ductal Cells
3. Additional Genetic Steps Required to Transform Pancreatic Duct-Derived Cells
4. Analysis of K-Ras Effector Pathways in Pancreatic Cell Transformation
5. Discussion
Acknowledgments
References
Chapter 32: The Ras Inhibitor Farnesylthiosalicylic Acid (Salirasib) Disrupts the Spatiotemporal Localization of Active Ras: A Potential Treatment for Cancer
1. Introduction
2. Methods
3. Disruption of Ras Localization as a Potential Method for Cancer Treatment
Acknowledgments
References
Chapter 33: Quantitative High-Throughput Cell-Based Assays for Inhibitors of ROCK Kinases
1. Introduction
2. Materials and Methods
3. Results and Discussion
References
Chapter 34: Tandem Affinity Purification of the BBSome, a Critical Regulator of Rab8 in Ciliogenesis
1. Introduction
2. Materials
3. Procedures
Acknowledgments
References
Author Index
Subject Index

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Small GTPases in Disease, Part B

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