Hydrogen Peroxide and Cell Signaling, Part C

Series Page

METHODS IN ENZYMOLOGY

Contributors

Preface

Volume in Series

Section I: H₂O₂ Regulation of Cell Signaling

Chapter One. The Biological Chemistry of Hydrogen Peroxide

1 Introduction

2 Chemical Properties

3 Antioxidant Defenses Against H₂O₂

4 Kinetics and Identification of Biological Targets for H₂O₂

5 Transmission of Redox Signals Initiated by H₂O₂

6 Diffusion Distances and Compartmentalization

7 Biological Detection of H₂O₂

8 Conclusion

References

Chapter Two. Reactive Oxygen Species in the Activation of MAP Kinases

1 Introduction

2 Reactive Oxygen Species

3 Mitogen-Activated Protein Kinases

4 Roles of ROS in MAPK Activation

5 Summary

Acknowledgment

References

Chapter Three. Hydrogen Peroxide Signaling Mediator in the Activation of p38 MAPK in Vascular Endothelial Cells

1 Introduction

2 Materials and Methods

References

Chapter Four. In Vivo Imaging of Nitric Oxide and Hydrogen Peroxide in Cardiac Myocytes

1 Introduction

2 Isolation and Culture of Adult Mouse Ventricular Cardiac Myocytes

3 Live Cell Imaging of Cardiac Myocytes

4 Imaging Intracellular NO with Cu₂(FL2E) Dye

5 Production and In Vivo Expression of Lentivirus Expressing the HyPer2 H₂O₂ Biosensor

6 Imaging Intracellular H₂O₂ in Cardiac Myocytes and Endothelial Cells Expressing HyPer2

Acknowledgments

References

Chapter Five. Methods for Studying Oxidative Regulation of Protein Kinase C

1 Introduction

2 Materials

3 Direct Oxidative Modification of PKC Isoenzymes by H₂O₂

4 Indirect Cellular Regulation of PKC Isoenzymes by Sublethal Levels of H₂O₂

5 H₂O₂-Induced Signaling in GTPP-Induced Preconditioning for Cerebral Ischemia

6 Summary

Acknowledgments

References

Chapter Six. p66Shc, Mitochondria, and the Generation of Reactive Oxygen Species

Abbreviations

1 Introduction

2 The P66 Gene and Protein

3 The Mitochondrial Function of p66Shc

4 Preparation of Recombinant p66Shc Protein

5 Mitochondrial Swelling Assay

6 Mitochondrial ROS Formation by p66Shc

7 Conclusions: Role of p66Shc ROS

Acknowledgments

References

Chapter Seven. Detecting Disulfide-Bound Complexes and the Oxidative Regulation of Cyclic Nucleotide-Dependent Protein Kinases by H₂O₂

1 Introduction

2 Experimental Considerations and Procedures

3 Summary

Acknowledgments

References

Chapter Eight. Redox Regulation of Protein Tyrosine Phosphatases: Methods for Kinetic Analysis of Covalent Enzyme Inactivation

1 Introduction

2 Rate Expressions Describing Covalent Enzyme Inactivation

3 Ensuring That the Enzyme Activity Assay Accurately Reflects the Amount of Active Enzyme

4 Assays for Time-Dependent Inactivation of PTPs

5 Analysis of the Kinetic Data

6 Obtaining an Inactivation Rate Constant from the Data

7 Summary

References

Section II: H₂O₂ in the Redox Regulation of Transcription and Cell-Surface Receptors

Chapter Nine. Activation of Nrf2 by H₂O₂: De Novo Synthesis Versus Nuclear Translocation

1 Introduction

2 Experimental Conditions and Considerations

3 Pilot Experiments

4 Experimental H₂O₂ Exposure

5 Data Handling and Analysis

6 Summary

Acknowledgments

References

Chapter Ten. H₂O₂ in the Induction of NF-κB-Dependent Selective Gene Expression

1 Introduction

2 Experimental Components and Considerations

3 Pilot Experiments

4 Steady-State Titration Experiments

5 NF-κB Family Protein Levels

6 NF-κB-Dependent Gene Expression

7 Summary

Acknowledgments

References

Chapter Eleven. Detection of H₂O₂-Mediated Phosphorylation of Kinase-Inactive PDGFRα

1 Construction of Kinase-Dead PDGFRα

2 Characterization of the Kinase-Inactive Receptor

3 Detection of H₂O₂-Mediated Phosphorylation of Kinase-Inactive PDGFRα

4 Implication

Acknowledgment

References

Section III: H₂O₂ and Regulation of Cellular Processes

Chapter Twelve. Genetic Modifier Screens to Identify Components of a Redox-Regulated Cell Adhesion and Migration Pathway

1 Introduction

2 Mutations in a D. melanogaster Gene Encoding a Peroxiredoxin Cause Germ Cell Adhesion and Migration Defects

3 Dominant Modifier Screens

4 Conducting a Dominant Modifier Screen to Identify Missing Components of a Redox-Regulated Germ Cell Migration Pathway

5 Limitations to Dominant Modifiers Screens

6 Concluding Remarks

Acknowledgments

References

Chapter Thirteen. Investigating the Role of Reactive Oxygen Species in Regulating Autophagy

1 Introduction

2 Regulation of Autophagy

3 ROS and Autophagy

4 Mechanisms for ROS Regulation of Autophagy

5 Methods for the Detection of Autophagy

6 Consideration When Using Oxidative Stress and Detecting ROS Under Autophagy Conditions

7 Conclusions

Acknowledgment

References

Chapter Fourteen. H₂O₂: A Chemoattractant?

1 Introduction

2 The Zebrafish Tail Fin Wounding Assay

3 Measuring H₂O₂ Signals in Zebrafish

4 Imaging H₂O₂ Production by Wide-Field Microscopy

5 Imaging H₂O₂ Production by Confocal Microscopy

Acknowledgments

References

Chapter Fifteen. Measuring Mitochondrial Uncoupling Protein-2 Level and Activity in Insulinoma Cells

1 Introduction

2 Tissue Culture

3 UCP2 Protein Detection

4 UCP2 Protein Knockdown

5 UCP2 Activity

Acknowledgments

References

Chapter Sixteen. Effects of H₂O₂ on Insulin Signaling the Glucose Transport System in Mammalian Skeletal Muscle

1 Introduction

2 In Vitro Exposure to H₂O₂

3 Effects of H₂O₂ on the Glucose Transport System in Isolated Skeletal Muscle

4 Summary

References

Chapter Seventeen. Monitoring of Hydrogen Peroxide and Other Reactive Oxygen and Nitrogen Species Generated by Skeletal Muscle

1 Introduction

2 Monitoring Extracellular ROS Using Microdialysis Techniques

3 Assessment of Intracellular ROS Activities

4 Concluding Remarks

Acknowledgments

References

Author Index

Subject Index