MAB379 Sigma-AldrichAnti-Glutamate Receptor 5, 6, 7 Antibody, clone 4F5

The dose-response relationship is a fundamental pharmacological parameter necessary to determine therapeutic thresholds. Epi-allelic hypomorphic analysis using RNA interference (RNAi) can similarly correlate target gene dosage with cellular phenotypes. This however requires a set of RNAi triggers empirically determined to attenuate target gene expression to different levels.In order to improve our ability to incorporate epi-allelic analysis into target validation studies, we developed a novel flow cytometry-based functional screening approach (CellSelectRNAi) to achieve unbiased selection of shRNAs from high-coverage libraries that knockdown target gene expression to predetermined levels. Employing a Gaussian probability model we calculated that knockdown efficiency is inferred from shRNA sequence frequency profiles derived from sorted hypomorphic cell populations. We used this approach to generate a hypomorphic epi-allelic cell series of shRNAs to reveal a functional threshold for the tumor suppressor p53 in normal and transformed cells.The unbiased CellSelectRNAi flow cytometry-based functional screening approach readily provides an epi-allelic series of shRNAs for graded reduction of target gene expression and improved phenotypic validation.

Neurotoxicity induced in fish by domoic acid (DA) was assessed with respect to occurrence of neurotoxic signs, lethality, and histopathology by light microscopy. Sparus aurata were exposed to a single dose of DA by intraperitoneal (i.p.) injection of 0, 0.45, 0.9, and 9.0 mg DA kg(-1) bw. Mortality (66.67 ± 16.67%) was only observed in dose of 9.0 mg kg(-1) bw. Signs of neurological toxicity were detected for the doses of 0.9 and 9.0 mg DA kg(-1) bw. Furthermore, the mean concentrations (±SD) of DA detected by HPLC-UV in extracts of brain after exposure to 9.0 mg DA kg(-1) bw were 0.61 ± 0.01, 0.96 ± 0.00, and 0.36 ± 0.01 mg DA kg(-1) tissue at 1, 2, and 4 hours. The lack of major permanent brain damage in S. aurata, and reversibility of neurotoxic signs, suggest that lower susceptibility to DA or neuronal recovery occurs in affected individuals.

Domoic acid (DA), a potent neurotoxin produced by select species of algae and diatoms, kills neurons bearing kainic acid-type glutamate receptors. Studies have shown that DA bioaccumulates in invertebrates and fish that consume the diatoms. In every vertebrate species tested or observed in the wild, dietary or systemic DA causes neuronal damage or clinical signs of neurotoxicity. Sharks, like marine birds and mammals, are exposed to DA through their diet; however, no research has demonstrated the effect of DA on shark behavior or physiology. In this study, juvenile leopard sharks (Triakis semifasciata) were given DA by intracoelomic injection at doses of 0, 1, 3, 9, and 27 mg/kg and observed for 7 days. The sharks failed to demonstrate behavioral or histological changes in response to the toxin. We identified putative brain glutamate receptors by probing western blots with an antibody specific for kainic acid-type glutamate receptors and demonstrated receptor localization in the cerebellum with immunohistochemistry. Blood levels of DA in three sharks dosed at 9 mg/kg fell rapidly within 1.5h of injection. We show that leopard sharks possess the molecular target for DA but are resistant to doses of DA known to be toxic to other vertebrates.