Main description:

This detailed volume explores the NADPH oxidase family of enzymes in human physiology and genetic disease, in which early discoveries represent prime examples of the finest translational "from bed to bench and back" studies. Methods are included for testing assembly and function of multicomponent oxidase complexes and for analyzing reactive oxygen species (ROS) generation in different systems by various means, while addressing pitfalls of ROS probes currently being used, as well as protocols on NADPH oxidase regulation and their function in cells. Written in the highly successful Methods in Molecular Biology series format, chapters include introduction to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, NADPH Oxidases: Methods and Protocols will aid researchers working with the NOX/DUOX family in continuing and expanding upon their vital research.

Contents:

Part I: The NADPH Oxidase Family: Overviews

1. Intersecting Stories of the Phagocyte NADPH Oxidase and Chronic Granulomatous Disease

William M. Nauseef and Robert A. Clark

2. Mammalian NADPH Oxidases

Helene Buvelot, Vincent Jaquet, and Karl-Heinz Krause

Part II: Biochemistry and Model Systems

3. Enhanced Immunoaffinity Purification of Human Neutrophil Flavocytochrome B for Structure Determination by Electron Microscopy

Algirdas J. Jesaitis, Marcia Riesselman, Ross Taylor, and Susan Brumfield

4. Purification and Characterization of DUOX Peroxidase Homology Domains (PHDs)

Jennifer L. Meitzler

5. A Close-Up View of the Impact of Arachidonic Acid on the Phagocyte NADPH Oxidase

Tania Bizouarn, Hager Souabni, Xavier Serfaty, Aicha Bouraoui, Rawand Masoud, Gilda Karimi, Chantal Houee-Levin, and Laura Baciou

6. NOX5 Cell-Free Assay for the High-Throughput Screening of Small Molecules

Fiona Augsburger, Delphine Rasti, Yves Cambet, and Vincent Jaquet

7. Spectroscopy of NOX Protein Family Members

Yoko Nakano and William M. Nauseef

8. Soluble Regulatory Proteins for Activation of NOX Family NADPH Oxidases

Hideki Sumimoto, Reiko Minakami, and Kei Miyano

9. Insights into the NOX NADPH Oxidases Using Heterologous Whole Cell Assays

Mary C. Dinauer

10. The X-CGD PLB-985 Cell Model for NOX2 Structure-Function Analysis

Sylvain Beaumel and Marie Jose Stasia

11. Functional Characterization of DUOX Enzymes in Reconstituted Cell Models

Agnieszka Korzeniowska, Agnes P. Donko, Stanislas Morand, and Thomas L. Leto

12. Guidelines for the Detection of NADPH Oxidases by Immunoblot and RT-qPCR

Becky A. Diebold, S. Garrett Wilder, Xavier De Deken, Jennifer L. Meitzler, James H. Doroshow, James W. McCoy, Yerun Zhu, and J. David Lambeth

Part III: Detection of ROS

13. Methods for Detection of NOX-Derived Superoxide Radical Anion and Hydrogen Peroxide in Cells

Fiona Augsburger, Aleksandra Filippova, and Vincent Jaquet

14. HPLC-Based Monitoring of Oxidation of Hydroethidine for Detection of NADPH Oxidase-Derived Superoxide Radical Anion

Jacek Zielonka, Monika Zielonka, and Balaraman Kalyanaraman

15. Visualization of Intracellular Hydrogen Peroxide with the Genetically-Encoded Fluorescent Probe HyPer in NIH-3T3 Cells

Yulia G. Ermakova, Nataliya M. Mishina, Carsten Schultz, and Vsevolod V. Belousov

16. Imaging Intracellular H2O2 with the Genetically-Encoded PerFRET and OxyFRET Probes

Balazs Enyedi and Miklos Geiszt

17. Quantitative Imaging of Endogenous and Exogenous H2O2 Gradients in Live Zebrafish Larvae

Mark Jelcic, Balazs Enyedi, and Philipp Niethammer

18. Kinetic Analysis of Phagosomal ROS Generation

Sophie Dupre-Crochet, Marie Erard, and Oliver Nu e

19. Imaging Intestinal ROS in Homeostatic Conditions Using L-012

Emer Conroy and Gabriella Aviello

20. Hydro-Cy3-Mediated Detection of Reactive Oxygen Species In Vitro and In Vivo

Bejan J. Saeedi, Bindu Chandrasekharan, and Andrew S. Neish

Part IV: Regulation of NADPH Oxidase

21. Phosphorylation of Gp91phox/NOX2 in Human Neutrophils

Houssam Raad, Riad Arabi Derkawi, Asma Tlili, Sahra A. Belambri, Pham My-Chan Dang, and Jamel El-Benna

22. The Molecular Regulation and Functional Roles of NOX5

David J.R. Fulton

23. Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex

Edgar Pick

24. Rational Design and Delivery of NOX-Inhibitory Peptides

Eugenia Cifuentes-Pagano and Patrick J. Pagano

25. High-Throughput Screening of NOX Inhibitors

Jacek Zielonka, Monika Zielonka, Gang Cheng, Micael Hardy, and Balaraman Kalyanaraman

26. Protein-Protein Interaction Assay to Analyze NOX4/p22phox Heterodimerization

Sharon O'Neill and Ulla G. Knaus

Part V: Function of NADPH Oxidase

27. Isolation of Redox Active Endosomes (Redoxosomes) and Assessment of NOX Activity

Weam S. Shahin and John F. Engelhardt

28. Model Systems to Investigate NOX-Dependent Cell Migration and Invasiveness

Howard E. Boudreau and Thomas L. Leto

29. NADPH Oxidases and Aging Models of Lung Fibrosis

Karen Bernard and Victor J. Thannickal

30. Proteomic Methods to Evaluate NOX-Mediated Redox Signaling

Christopher M. Dustin, Milena Hristova, Caspar Schiffers, and Albert van der Vliet

31. Neutrophil Extracellular Traps

Balazs Rada

Part VI: Genetic Disorders

32. Chronic Granulomatous Disease

Dirk Roos

33. Diagnostic Testing for Chronic Granulomatous Disease

Douglas B. Kuhns

34. Gastrointestinal Complications in Chronic Granulomatous Disease

E. Liana Falcone and Steven M. Holland

35. Ex Vivo Models of Chronic Granulomatous Disease

Julie Brault, Benedicte Vigne, and Marie Jose Stasia

36. Gene Editing in Chronic Granulomatous Disease

Colin L. Sweeney, Randall K. Merling, Suk See De Ravin, Uimook Choi, and Harry L. Malech

37. DUOX Defects and Their Roles in Congenital Hypothyroidism

Xavier De Deken and Francoise Miot

38. NAPDH Oxidases in Inflammatory Bowel Disease

Emily Stenke, Billy Bourke, and Ulla G. Knaus