Methods in molecular biology
-
cAMP-dependent protein kinase (PKA) is tethered at different subcellular locations by A-kinase anchoring proteins (AKAPs). AKAPs present amphipathic helices that bind to the docking and dimerization (D/D) domain of PKA regulatory subunits. ⋯ Accordingly, here we describe a bacteriophage screening procedure for identifying variants of PKA regulatory subunit D/D domains that bind selectively to individual AKAPs. This procedure can be adapted for engineering specificity into other shared protein interfaces.
-
Procalcitonin is the prohormone of calcitonin and present in minute quantities in health. However, during infection, its levels rise considerably and are correlated with the severity of the infection. ⋯ Procalcitonin levels have also been successfully used to guide antibiotic administration. However, procalcitonin is not specific for sepsis, and values need to be interpreted in the context of a full clinical examination and the presence of other signs and symptoms of sepsis.
-
Pain assessment in animal models of osteoarthritis is integral to interpretation of a model's utility in representing the clinical condition, and enabling accurate translational medicine. Here we describe two methods for behavioral pain assessments available for use in animal models of experimental osteoarthritic pain: Von Frey filaments and spontaneous activity monitoring.
-
Synthesis of the second messenger cAMP activates a variety of signaling pathways critical for all facets of intracellular regulation. Protein kinase A (PKA) is the major cAMP-responsive effector. Where and when this enzyme is activated has profound implications on the cellular role of PKA. ⋯ A popular means of evaluating the impact of these anchored signaling events is to biochemically interfere with the PKA-AKAP interface. Hence, peptide disruptors of PKA anchoring are valuable tools in the investigation of local PKA action. This article outlines the development of PKA isoform-selective disruptor peptides, documents the optimization of cell-soluble peptide derivatives, and introduces alternative cell-based approaches that interrogate other aspects of the PKA-AKAP interface.
-
Before there was access to space, all experiments on plant tropisms were conducted upon the background of gravity. The gravity vector could be disrupted, such as with clinorotation and random positioning machines, and by manipulating incident angles of root growth with respect to gravity, such as with Darwin's plants on slanted plates, but gravity could not be removed from the experimental equation. Access to microgravity through spaceflight has opened new doors to plant research. Here we provide an overview of some of the methodologies of conducting plant research in the unique spaceflight environment.