Phosphodiesterase Methods and ProtocolsClaire Lugnier Springer Science & Business Media, 04.02.2008 - 336 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 BiologyTM 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. |
Inhalt
xii | |
Purification of PDE6 Isozymes From Mammalian Retina | 10 |
HighResolution Measurements of Cyclic Adenosine Monophosphate | 15 |
and Wolfgang R G Dostmann 4 HighThroughput Screening of Phosphodiesterase Activity | 27 |
in Living Cells | 44 |
Assessment of Phosphodiesterase Isozyme Contribution in Cell | 63 |
Localization of the Cyclic Guanosine 35Monophosphate | 75 |
Determination of Ca2+CalmodulinStimulated | 85 |
Renaturation of the Catalytic Domain of PDE4A Expressed | 155 |
Determining the Subunit Structure of Phosphodiesterases Using | 167 |
Hengming Ke Qing Huai and Robert X Xu 15 Generation of PDE4 Knockout Mice by Gene Targeting | 181 |
S L Catherine Jin Anne M Latour and Marco Conti 16 Immunoprecipitation of PDE2 Phosphorylated and Inactivated | 191 |
Investigation of Extracellular SignalRegulated Kinase 2 MitogenActivated | 225 |
Radiolabeled Ligand Binding to the Catalytic or Allosteric | 239 |
Interaction Between Catalytic and Inhibitory Subunits of PDE6 | 277 |
315 | |
AdenovirusMediated Overexpression of Murine Cyclic Nucleotide | 93 |
Beverly A Valeriani and Rick H Cote 11 Cyclic Guanosine 5Monophosphate Binding to Regulatory GAF | 125 |
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3H]cGMP 3H]sildenafil acid adenovirus allosteric antibody approx Beavo Biochem Biol bovine buffer Ca2+ cAMP signals cDNA cells centrifugation cGMP binding cGMP-dependent protein kinase Chem chromatography cloned CNG channels coli concentration containing crystallization cyclic GMP cyclic nucleotide cyclic nucleotide phosphodiesterases described in Subheading diluted dimerization dish EDTA elution enzyme expression extract filter fluorescence forskolin fractions gel filtration gradient guanosine homogenizer IBMX immunoprecipitate incubate infection intracellular ligand Lugnier medium membrane method mg/mL MgCl2 molecular weight mPDE3B gene mutant NaCl Note PDE activity PDE inhibitors PDE1 PDE2 PDE3 PDE4 PDE5 PDE6 PDE6C PDE6R pellet phosphorylation photoreceptor pipet plasmid PMSF primers probe protein kinase Protocols purified reaction recombinant refolding resin retinal room temperature sample SDS-PAGE sequence Sigma Chemical Corporation sildenafil solution subunits sucrose supernatant tissue transducin transfection Tris-HCl trypsin tubes vector wash Western blotting zaprinast µg/mL