Hydrogen Peroxide and Cell Signaling, Part C - 1st Edition - ISBN: 9780124058811, 9780124055407

Hydrogen Peroxide and Cell Signaling, Part C, Volume 528

1st Edition

Serial Volume Editors: Enrique Cadenas
Series Volume Editors: Lester Packer
eBook ISBN: 9780124055407
Hardcover ISBN: 9780124058811
Imprint: Academic Press
Published Date: 29th July 2013
Page Count: 384
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Table of Contents

Series Page




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


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



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

1 Introduction

2 Materials and Methods


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



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



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


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



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



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


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



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



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



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



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



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



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



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


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



Author Index

Subject Index


This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This is the third of three volumes on hydrogen peroxide and cell signaling, and includes chapters on such topics as the biological chemistry of hydrogen peroxide, reactive oxygen species in the activation of MAP kinases, and investigating the role of reactive oxygen species in regulating autophagy.

Key Features

  • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
  • Covers hydrogen peroxide and cell signaling
  • Contains chapters on such topics as the biological chemistry of hydrogen peroxide, reactive oxygen species in the activation of MAP kinases, and investigating the role of reactive oxygen species in regulating autophagy


Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.


No. of pages:
© Academic Press 2013
Academic Press
eBook ISBN:
Hardcover ISBN:

Ratings and Reviews

About the Serial Volume Editors

Enrique Cadenas Serial Volume Editor

ENRIQUE CADENAS, MD, PhD, received his PhD in biochemistry from the University of Buenos Aires, School of Medicine. He is professor of pharmacology and pharmaceutical sciences at the University of Southern California School of Pharmacy and of biochemistry and molecular biology at the University of Southern California Keck School of Medicine, and doctor honoris causa (medicine) at the University of Linköping, Sweden. Cadenas was president of the Society for Free Radical Research International (SFRRI) and is fellow of the Society for Free Radical Biology & Medicine. He served the scientific community by participating on NIH study sections (2002-2006; chair 2006-2008). His research interests include energy and redox metabolism in brain aging and the coordinated inflammatory-metabolic responses in brain and neurodegenerative diseases.

Affiliations and Expertise

Pharmacology & Pharmaceutical Sciences, School of Pharmacy, University of Southern California, USA

About the Series Volume Editors

Lester Packer Series Volume Editor

Lester Packer received a PhD in Microbiology and Biochemistry in 1956 from Yale University. In 1961, he joined the University of California at Berkeley serving as Professor of Cell and Molecular Biology until 2000, and then was appointed Adjunct Professor, Pharmacology and Pharmaceutical Sciences, School of Pharmacy at the University of Southern California.

Dr Packer received numerous distinctions including three honorary doctoral degrees, several distinguished Professor appointments. He was awarded Chevalier de l’Ordre National du Merite (Knight of the French National Order of Merit) and later promoted to the rank of Officier. He served as President of the Society for Free Radical Research International (SFRRI), founder and Honorary President of the Oxygen Club of California.

He has edited numerous books and published research; some of the most cited articles have become classics in the field of free radical biology:

Dr Packer is a member of many professional societies and editorial boards. His research elucidated - the Antioxidant Network concept. Exogenous lipoic acid was discovered to be one of the most potent natural antioxidants and placed as the ultimate reductant or in the pecking order of the “Antioxidant Network” regenerating vitamins C and E and stimulating glutathione synthesis, thereby improving the overall cellular antioxidant defense. The Antioxidant Network is a concept addressing the cell’s redox status. He established a world-wide network of research programs by supporting and co-organizing conferences on free radical research and redox biology in Asia, Europe, and America.

Affiliations and Expertise

Department of Molecular Pharmacology and Toxicology, School of Pharmaceutical Sciences, University of Southern California, USA