Phosphodiesterase Methods and Protocols / Edition 1

by Claire Lugnier

Overview

The multigenic family of cyclic nucleotide phosphodiesterases (PDEs) is now known to offer many new therapeutic possibilities through their influence over intracellular signaling, though their precise cell mechanisms to modulating that process remain uncertain. In Phosphodiesterase Methods and Prools, a panel of research leaders in the PDE field describes new concepts and techniques for investigating the role of PDEs in orchestrating normal and pathophysiological responses. Presented in step-by-step detail, these readily reproducible methods allow the measurement of cyclic nucleotide variations in living cells, as well as their visualization in a spatiotemporal manner, the localization and characterization of their activities in tissues and living cells, and the assessment of targeted PDEs in creating specific tools and drugs. Additional chapters discuss the generation of PDE4 knockout mice to demonstrate not only the potential role of targeted PDEs, but also their use in further studies of the central role of PDE regulation in intracellular signaling control. These techniques offer clinicians a way to find new therapies for numerous pathologies whose molecular origins are unknown and whose treatment is symptomatic. Alterations of intracellular signaling related to PDE deregulation in such pathologies as inflammation, neurodegeneration, and cancer may explain the difficulties observed in their prevention and treatment. The prools follow the successful Methods in Molecular Biology™ series format, each offering step-by-step laboratory instructions, an introduction outlining the principle behind the technique, lists of the necessary equipment and reagents, and tips on troubleshooting and avoiding known pitfalls.

Path-breaking and highly practical, Phosphodiesterase Methods and Prools offers cell biologists, molecular biologists, pharmacologists, and medicinal chemists a broad ranging survey of the advanced tools and concepts needed to understand therole of PDEs in physiological functions, their implications in several critical pathologies, and the opportunities they offer for new drug design.

Product Details

ISBN-13:	9781588293145
Publisher:	Springer-Verlag New York, LLC
Publication date:	06/22/2005
Series:	Methods in Molecular Biology , #307
Edition description:	2005
Pages:	336
Product dimensions:	6.10(w) x 9.25(h) x 0.04(d)

Table of Contents

Study of Cyclic Adenosine Monophosphate Microdomains in Cells.- High-Resolution Measurements of Cyclic Adenosine Monophosphate Signals in 3D Microdomains.- Cygnets.- High-Throughput Screening of Phosphodiesterase Activity in Living Cells.- Assessment of Phosphodiesterase Isozyme Contribution in Cell and Tissue Extracts.- Localization of the Cyclic Guanosine 3?,5'-Monophosphate- Hydrolyzing Phosphodiesterase Type 9 in Rat Brain by Nonradioactive In Situ Hybridization.- Determination of Ca2+/Calmodulin-Stimulated Phosphodiesterase Activity in Intact Cells.- Adenovirus-Mediated Overexpression of Murine Cyclic Nucleotide Phosphodiesterase 3B.- Identification of Promoter Elements in the 5'-Flanking Region of Murine Cyclic Nucleotide Phosphodiesterase 3B Gene.- Purification of PDE6 Isozymes From Mammalian Retina.- Cyclic Guanosine 5'-Monophosphate Binding to Regulatory GAF Domains of Photoreceptor Phosphodiesterase.- Renaturation of the Catalytic Domain of PDE4A Expressed in Escherichia coli as Inclusion Bodies.- Determining the Subunit Structure of Phosphodiesterases Using Gel Filtration and Sucrose Density Gradient Centrifugation.- Crystallization of Cyclic Nucleotide Phosphodiesterases.- Generation of PDE4 Knockout Mice by Gene Targeting.- Immunoprecipitation of PDE2 Phosphorylated and Inactivated by an Associated Protein Kinase.- Investigation of Extracellular Signal-Regulated Kinase 2 Mitogen-Activated Protein Kinase Phosphorylation and Regulation of Activity of PDE4 Cyclic Adenosine Monophosphate-Specific Phosphodiesterases.- Radiolabeled Ligand Binding to the Catalytic or Allosteric Sites of PDE5 and PDE11.- Analysis of Dimerization Determinants of PDE6 Catalytic Subunits.- Interactions Between Catalytic and Inhibitory Subunits of PDE6.- Purification,Reconstitution on Lipid Vesicles, and Assays of PDE6 and Its Activator G Protein, Transducin.

Editorial Reviews

Reviewer: Bruce A. Fenderson, PhD (Thomas Jefferson University)
Description: Cells regulate a wide range of physiological functions by flooding intracellular compartments with second messengers such as calcium, IP3, and cyclic nucleotides. In this manner, cells are able to activate or suppress a wide array of biochemical pathways in concert. The basal levels of cyclic nucleotides (cAMP, cGMP) are controlled by a multigenic family of phosphodiesterases. These enzymes play a critical role in regulating basal levels and episodic changes in cyclic nucleotide concentration within cells. As stated in the foreword, this book aims to provide a "palette of conceptual and technical approaches designed to solve questions" concerning the role of phosphodiesterases in normal functions and disease. The book's 21 chapters focus on topics such as cyclic nucleotide microdomains, determination of phosphodiesterase activity, crystallization of enzymes, and analysis of dimerization determinants in catalytic subunits.
Purpose: The editor has brought together a group of experts in various aspects of phosphodiesterase biochemistry. Their goals are to provide bench researchers with a useful source of research methods for studying phosphodiesterase structure and function. A theme that runs through this book and lends it excitement, is that phosphodiesterases may be therapeutic targets. The discovery of new synthetic compounds that block the activity of these enzymes may have significant clinical benefit.
Audience: The book is written for basic science researchers and clinicians at all levels of training who are interested in the control of intracellular signaling cellular physiology. This group of researchers includes biochemists, molecular biologists, cell biologists and pharmacologists "who wish to initiate or deepen studies" in the phosphodiesterase field.
Features: This excellent guide to current research techniques in the field of phosphodiesterase biochemistry is part of the popular Methods in Molecular Biology series. Each chapter includes detailed, step-by-step protocols for measuring specific enzymes or testing specific research questions. Each chapter includes a brief introduction and a list of essential references. The authors also include a list of "notes" that provide the interested reader with further details and tips for success in the laboratory.
Assessment: One of the major strengths of the book is that many diverse topics are threaded together. For example, there are chapters on in vivo imaging techniques, as well as on methods for over-expressing specific phosphodiesterase genes and crystallizing proteins. The topics are interesting and thought provoking, but technically challenging. The book is written primarily for experts interested in the field of phosphodiesterase biochemistry. One minor criticism is that the authors do not provide the nonexpert with enough background information to evaluate the importance of past discoveries and future directions in this field. The line drawings and photomicrographs are helpful, but simple summary figures are generally lacking. Despite these minor criticisms, I enjoyed this book primarily for the breadth of coverage and attention to scientific detail.

Doody's Review Service

3 Stars from Doody

"...interesting and thought provoking, but technically challenging...excellent guide to current research techniques in the field of phosphodiesterase." - Doody's Health Sciences Book Review Journal

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