Phosphodiesterase Methods and Protocols

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Claire Lugnier
Springer Science & Business Media, 2005 - 324 Seiten
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 Protocols, 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 protocols 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 Protocols offers cell biologists, molecular biologists, pharmacologists, and medicinal chemists a broad ranging survey of the advanced tools and concepts needed to understand the role of PDEs in physiological functions, their implications in several critical pathologies, and the opportunities they offer for new drug design.
 

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Inhalt

HighResolution Measurements of Cyclic Adenosine Monophosphate
15
In Vivo Characterization of Novel cGMP Indicators
27
HighThroughput Screening of Phosphodiesterase Activity
44
Assessment of Phosphodiesterase Isozyme Contribution in Cell
63
Localization of the Cyclic Guanosine 35Monophosphate
75
Determination of Ca2+CalmodulinStimulated
85
AdenovirusMediated Overexpression of Murine Cyclic Nucleotide
93
Identification of Promoter Elements in the 5Flanking Region
109
Determining the Subunit Structure of Phosphodiesterases Using
167
Crystallization of Cyclic Nucleotide Phosphodiesterases
181
Generation of PDE4 Knockout Mice by Gene Targeting
191
Immunoprecipitation of PDE2 Phosphorylated and Inactivated
211
Investigation of Extracellular SignalRegulated Kinase 2 MitogenActivated
225
Radiolabeled Ligand Binding to the Catalytic or Allosteric
239
Analysis of Dimerization Determinants of the PDE6
263
Interaction Between Catalytic and Inhibitory Subunits of PDE6
277

Purification of PDE6 Isozymes From Mammalian Retina
125
Cyclic Guanosine 5Monophosphate Binding to Regulatory
141
Renaturation of the Catalytic Domain of PDE4A Expressed
155
Purification Reconstitution on Lipid Vesicles and Assays
289
Index
315
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