Advanced Methods to Investigate the Role of
PDEs in Intracellular Signaling
Research leaders in the PDE field describe 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 spatio-temporal 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.
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.
