Anti-FLAG tag antibody, clone 7G8 [R-PE]

Background: It has been demonstrated that kappa-opioid receptor agonists can reduce hypoxia-ischemia brain injury in animal models. However, it is unclear how the kappa-opioid receptor responds to hypoxia-ischemia. In the current study, the authors used an in vitro model of oxygen-glucose deprivation and reoxygenation to explore how kappa-opioid receptors respond to hypoxia and reoxygenation. Methods: Mouse neuroblastoma Neuro2A cells were stably transfected with mouse kappa-opioid receptor-tdTomato fusion protein or Flag-tagged mouse kappa-opioid receptor, divided into several groups (n = 6 to 12), and used to investigate the kappa-opioid receptor movement. Observations were performed under normal oxygen, at 30 min to 1 h after oxygen-glucose deprivation and at 1 h after reoxygenation using high-resolution imaging techniques including immunoelectronmicroscopy in the presence and absence of kappa-opioid receptor antagonist, dynamin inhibitors, potassium channel blockers, and dopamine receptor inhibitor. Results: Hypoxic conditions caused the kappa-opioid receptor to be internalized into the cells. Inhibition of dynamin by Dyngo-4a prevented the receptor internalization. Interestingly, a specific kappa-opioid receptor antagonist norbinaltorphimine blocked internalization, suggesting the involvement of activation of a specific kappa-opioid receptor. kappa-Opioid receptor internalization appears to be reversed by reoxygenation. Quantities of intracellular kappa-opioid receptor-associated gold particles as demonstrated by immunoelectron microscopy were increased from 37 to 85% (P < 0.01) after oxygen-glucose deprivation. Potassium channel blockers and dopamine receptor inhibitor failed to block hypoxia-induced kappa-opioid receptor internalization. Conclusions: Hypoxia induces reversible kappa-opioid receptor internalization, which was inhibited by selective kappa-opioid receptor antagonists or dynamin inhibitor, and can be reversed by reoxygenation in neuroblastoma cells, indicating the modulating effects between kappa-opioid receptor and hypoxia via kappa-opioid receptor activation and the dynamin-dependent mechanism.

Tryptophan hydroxylase-type 2 (Tph2) is the first rate-limiting step in the biosynthesis of serotonin (5-HT) in the brain. The ophthalmic administration (Op-Ad) is a non-invasive method that allows delivering genetic vehicles through the eye and reaches the brain. Here, the murine Tph2 gene was cloned in a non-viral vector (pIRES-hrGFP-1a), generating pIRES-hrGFP-1a-Tph2, plus the FLAG-tag. Recombinant Tph2-FLAG was detected and tested in vitro and in vivo, where 25 mu g of pIRES-hrGFP-1a-Tph2-FLAG was Op-Ad to mice. The construct was capable of expressing and producing the recombinant Tph2-FLAG in vitro and in vivo. The in vivo assays showed that the construct efficiently crossed the Hemato-Ocular Barrier and the Blood-Brain Barrier, reached brain cells, passed the optical nerves, and transcribed mRNA-Tph2-FLAG in different brain areas. The recombinant Tph2-FLAG was observed in amygdala and brainstem, mainly in raphe dorsal and medial. Relative Tph2 expression of threefold over basal level was recorded three days after Op-Ad. These results demonstrated that pIRES-hrGFP-Tph2-FLAG, administrated through the eyes was capable of reaching the brain, transcribing, and translating Tph2. In conclusion, this study showed the feasibility of delivering therapeutic genes, such as the Tph2, the first enzyme, rate-limiting step in the 5-HT biosynthesis.