Phosphodiesterase Methods and Protocols

2005
ISBN: 978-1-58829-314-5
Verlag: Humana Press

Buch, Englisch, Band 307, 336 Seiten, Format (B × H): 160 mm x 241 mm, Gewicht: 738 g

Reihe: Methods in Molecular Biology

Adenosine 3 ,5 -cyclic monophosphate (cAMP) and guanosine 3 ,5 -cyclic monophosphate are ubiquitous nucleotides that have been described as the first and second messengers. In concert with intracellular calcium and IP3, they play a major role in the control of intracellular signaling, which orchestrates normal and pathophysiological responses. Downstream from the cyclic nucleotide synthesis by adenylyl and guanylyl cyclases, the multigenic family of cyclic nucleotide phosphodiesterases (PDEs), by specifically hydrolyzing cyclic nucleotides, controls cAMP and cGMP levels to maintain a basal state. Their critical role in intracellular sign- ing has recently designated them as new therapeutical targets. Several leading pharmaceutical companies are searching and developing new therapeutic agents that would potently and selectively inhibit PDE isozymes, notably PDE4 and PDE5. Nevertheless, the precise mechanism and the contribution of the various PDE isozymes in modulating intracellular signaling remain to be established. The aim of Phosphodiesterase Methods and Protocols is to provide a palette of a variety of conceptual and technical approaches designed to solve qu- tions concerning the role of PDEs, and ultimately of their different variants, in physiological functions as well as their implications in several pathologies.

Zielgruppe

Research

Autoren/Hrsg.

Lugnier, Claire

Fachgebiete

• Medizin | Veterinärmedizin Medizin | Public Health | Pharmazie | Zahnmedizin Medizinische Fachgebiete Pharmakologie, Toxikologie
• Naturwissenschaften Biowissenschaften Biochemie (nichtmedizinisch)
• Naturwissenschaften Biowissenschaften Enzymologie
• Naturwissenschaften Biowissenschaften Molekularbiologie

Inhaltsverzeichnis

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.