When released extracellularly, the purine nucleoside guanosine (Guo) can exert a wide range of physiological effects in vitro and in vivo. Guo can induce the release of neurotrophic factors such as nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) and can initiate the differentiation, growth and proliferation of neurons and glia. While structural and pharmacological evidence support the existence of a putative Guo binding site in the rat brain, there is a paucity of information on the mechanism through which Guo exerts these effects. Through bioinformatic research, our lab has identified an orphan G-protein coupled receptor as the first Guo receptor (termed G1R). The aim of this dissertation is to determine the mechanism of action of Guo using radioligand binding assays. It is hypothesized here that G1R is a distinct purinergic receptor for Guo. Using the calcium phosphate (CaP) co-precipitation (co-i.p.) method, Drosophila Schneider 2 (S2) cells were stably and transiently transfected with G1R recombinant cDNA. A series of binding assays using tritiated Guo ([3H]-Guo) showed no difference in binding between CaP transfection groups and wild S2 controls that do not endogenously express G1R, suggesting that the [3H]-Guo may not have a high binding affinity for the G1R binding site. Preliminary experiments using the Lipofectamine® 3000 to transfect S2 cells showed higher G1R mRNA expression as well as increased binding affinity to Guo when compared to the CaP transfected groups. This suggests that the results in the CaP mediated groups may be due to low transfection efficiency. In conclusion, transfections using the CaP method resulted in too low of a transfection efficiency to see a difference in binding affinity between wild S2 and transfected S2 cells. Findings from this work can be used to further examine the binding relationship of Guo to the G1R and optimize transfections using S2 cells and radioligand binding assays using purine based compounds. / Thesis / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/20626 |
| Date | January 2016 |
| Creators | Mahadeo, Crystal |
| Contributors | Jiang, Shucui, Mishra, Ram, Neuroscience |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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