Event Title

Optimization of a Phosphodiesterase (PDE) Assay to Determine the Effects of Vindeburnol on cAMP Levels

Session Number

Q21

Advisor(s)

David Braun, University of Illinois at Chicago
Douglas Feinstein, University of Illinois at Chicago

Location

B-131 Grainger

Start Date

28-4-2016 9:15 AM

End Date

28-4-2016 9:40 AM

Disciplines

Neuroscience and Neurobiology

Abstract

Derived from the vincamine plant, vindeburnol is a drug that has been shown to enhance release of noradrenaline, a key chemical in the regulation of neuroinflammation, in locus coeruleus neurons. Evidence shows that vincamine modulates cAMP levels in neurons, suggesting that vindeburnol is able to as well. Since the enzyme phosphodiesterase (PDE) breaks down cAMP, we use the PDE- Glo™ Assay to explore the idea that vindeburnol is a direct inhibitor of PDE, increasing cAMP levels. The assay is comprised of two separate reactions. In the first, PDE converts cAMP into AMP. In the second, AMP is converted into ATP, which is quantified as light, and is proportional to the amount of cAMP converted. Completed optimization of the second reaction shows that consistent peak light levels can be obtained for up to 20 uM AMP using a pH 7.8 buffer with 5mM Mg after 30 minutes of incubation at room temperature. We are currently optimizing the first reaction with respect to the concentration of cAMP, incubation time, and temperature. Following optimization, we will use similar conditions to thoroughly determine the effects of vindeburnol on PDE, and how its derivatives may work in animal models of Alzheimer’s disease and multiple sclerosis.


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Apr 28th, 9:15 AM Apr 28th, 9:40 AM

Optimization of a Phosphodiesterase (PDE) Assay to Determine the Effects of Vindeburnol on cAMP Levels

B-131 Grainger

Derived from the vincamine plant, vindeburnol is a drug that has been shown to enhance release of noradrenaline, a key chemical in the regulation of neuroinflammation, in locus coeruleus neurons. Evidence shows that vincamine modulates cAMP levels in neurons, suggesting that vindeburnol is able to as well. Since the enzyme phosphodiesterase (PDE) breaks down cAMP, we use the PDE- Glo™ Assay to explore the idea that vindeburnol is a direct inhibitor of PDE, increasing cAMP levels. The assay is comprised of two separate reactions. In the first, PDE converts cAMP into AMP. In the second, AMP is converted into ATP, which is quantified as light, and is proportional to the amount of cAMP converted. Completed optimization of the second reaction shows that consistent peak light levels can be obtained for up to 20 uM AMP using a pH 7.8 buffer with 5mM Mg after 30 minutes of incubation at room temperature. We are currently optimizing the first reaction with respect to the concentration of cAMP, incubation time, and temperature. Following optimization, we will use similar conditions to thoroughly determine the effects of vindeburnol on PDE, and how its derivatives may work in animal models of Alzheimer’s disease and multiple sclerosis.