Hydrogen Peroxide and Cell Signaling, Part B

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METHODS IN ENZYMOLOGY

Contributors

Preface

Methods in Enzymology

Section I: H₂O₂ Metabolism: Determination of a Cellular Steady-State

Chapter One. The Cellular Steady-State of H₂O₂: Latency Concepts and Gradients

1 Introduction

2 Experimental Components and Considerations When Measuring the H₂O₂ Gradient in S. cerevisiae Cells

3 Experimental Components and Considerations When Measuring the H₂O₂ Gradient in Mammalian Cell Lines

4 Data Handling/Processing

5 Summary

Acknowledgments

References

Chapter Two. Evaluating Peroxiredoxin Sensitivity Toward Inactivation by Peroxide Substrates

1 Introduction

2 Materials

3 Measuring Inactivation Sensitivity by Steady-State NADPH-Linked Assays

4 Measuring Inactivation Sensitivity by Multiturnover Cycling with ROOH and DTT Followed by Mass Spectrometry Analysis

5 Measuring Inactivation Sensitivity of Prx1/AhpC Prxs Under Single Turnover Conditions Followed by Gel Electrophoresis

6 Conclusions/Summary

Acknowledgment

References

Chapter Three. Peroxiredoxins as Preferential Targets in H₂O₂-Induced Signaling

1 Introduction

2 Reaction of H₂O₂ with Cellular Thiols

3 H₂O₂ Diffusion Versus Reaction with Cellular Thiols

4 Sulfenic Acids as Signal Transduction Intermediates

5 Prxs as Preferential Targets for H₂O₂

6 Prxs as Primary H₂O₂ Sensors and Transducers

7 Prx–Protein Interactions are Needed to Transmit the Signal

8 Posttranslational Regulation of Prxs

9 Summary

Acknowledgments

References

Chapter Four. Selenium in the Redox Regulation of the Nrf2 and the Wnt Pathway

1 Some Historical Background for Introduction

2 Selenium Status and Selenoprotein Synthesis

3 Selenium Status and the Keap1/Nrf2 System

4 Selenium and the Wnt Pathway

5 Common Players and Events in Nrf2 and Wnt Signaling

6 How Does Selenium Come into Play?

References

Chapter Five. Selenoprotein W as Biomarker for the Efficacy of Selenium Compounds to Act as Source for Selenoprotein Biosynthesis

1 Introduction

2 Experimental

3 Results

4 Discussion

5 Conclusions

Acknowledgments

References

Chapter Six. Peroxiredoxins and Sulfiredoxin at the Crossroads of the NO and H₂O₂ Signaling Pathways

1 Introduction

2 The Effect of NO on the Level of 2-Cys-Prx Overoxidation

3 Detection of Srx

4 Comments

Acknowledgments

References

Chapter Seven. Glutathione and γ-Glutamylcysteine in Hydrogen Peroxide Detoxification

1 Introduction

2 Materials

3 Previous Considerations

4 Procedure with Purified Enzymes and Substrates

5 Analysis in Biological Samples

6 Further Applications: H₂O₂ Produced by NOS

7 Conclusions

Acknowledgments

References

Chapter Eight. Peroxiredoxin-6 and NADPH Oxidase Activity

1 Introduction

2 Experimental Components and Considerations

3 Peroxiredoxin Activity of Prdx6

4 Effect of Prdx6 on NADPH Oxidase Activity

5 Summary

References

Chapter Nine. Study of the Signaling Function of Sulfiredoxin and Peroxiredoxin III in Isolated Adrenal Gland: Unsuitability of Clonal and Primary Adrenocortical Cells

1 Introduction

2 Hyperoxidation of PrxIII by H₂O₂ Generated During Corticosterone Synthesis

3 Induction of Srx by ACTH

4 Unsuitability of Clonal and Primary Adrenocortical Cells for Studies of the Srx–PrxIII Regulatory Pathway

5 Adrenal Gland Organ Culture as an In Vitro Model for the Srx–PrxIII Regulatory Pathway

6 Concluding Remarks

Acknowledgment

References

Section II: H₂O₂ in the Regulation of Cellular Processes in Plants

Chapter Ten. The Use of HyPer to Examine Spatial and Temporal Changes in H₂O₂ in High Light-Exposed Plants

1 Introduction

2 Experimental Procedures

3 Pilot Experiments Using HL Stress

4 Conclusions

Acknowledgments

References

Chapter Eleven. A Simple and Powerful Approach for Isolation of Arabidopsis Mutants with Increased Tolerance to H₂O₂-Induced Cell Death

Abbreviations

1 Introduction

2 Generation and Isolation of Mutants More Tolerant to H₂O₂-Induced Oxidative Stress

3 Identification of Mutations in the Genome

4 Analysis of the Mutants with Enhanced Tolerance to H₂O₂-Induced Oxidative Stress

5 Conclusion

Acknowledgments

References

Chapter Twelve. Analysis of Environmental Stress in Plants with the Aid of Marker Genes for H₂O₂ Responses

1 Introduction

2 Experimental Materials and Procedures

3 Example of Analysis

References

Chapter Thirteen. The Role of Plant Bax Inhibitor-1 in Suppressing H₂O₂-Induced Cell Death

1 Introduction

2 Morphological Changes of Mitochondria Under ROS Stress

3 Assay for Inhibitory Effect of BI-1 on ROS Stress-Induced Cell Death Using Heterologous Expression System in Suspension Cultured Cells

4 Summary

References

Chapter Fourteen. Comparative Analysis of Cyanobacterial and Plant Peroxiredoxins and Their Electron Donors: Peroxidase Activity and Susceptibility to Overoxidation

1 Introduction

2 Expression and Purification of Recombinant Prxs and Thioredoxins

3 Prx Activity Assays In Vitro

4 Peroxide Decomposition in Cyanobacteria In Vivo

5 Overoxidation of Plant and Cyanobacterial 2-Cys Prx

6 Concluding Remarks

References

Chapter Fifteen. Using Hyper as a Molecular Probe to Visualize Hydrogen Peroxide in Living Plant Cells: A Method with Virtually Unlimited Potential in Plant Biology

1 Introduction

2 NADPH Oxidase in Plant Cells

3 Plant Cells Respond to External and Internal Stimuli

4 Visualizing Hydrogen Peroxide in Living Plant Cells

5 Hyper as a New Genetically Encoded Probe

6 Vector Description and Plant Transformation

7 Preparation and Sterilization of Modified Petri Dishes for Growing Arabidopsis Plants for Microscopy Analysis

8 Seeds Sterilization and Stratification

9 Growth Conditions

10 Image Acquisition and Processing

Acknowledgments

References

Author Index

Subject Index