4B). Moreover, there was no significant difference in the extent of SNAP-25 hydrolysis between the BoNT/A poisoned cells without or with DDV-Mas-7 treatment. These findings suggested that SNAP-25 cleavage may not necessarily be correlated with the inhibition of neurotransmitter

release due to BoNT/A. Alternatively, factors other than SNAP-25 hydrolysis might be responsible for PI3K cancer BoNT/A inhibition of stimulated neurotransmitter release and its rescue by a pharmacological agent such as Mas-7. Mas is a wasp venom derived peptide known to be a phospholipase A2 (PLA2) activator. Previously, we had shown that Mas effectively stimulates acetylcholine exocytosis in PC12 cells in a SNAP-25 independent manner (Ray et al., 1997). In our effort to establish a therapeutic approach to treat botulinum poisoning, we had demonstrated the validity of a drug delivery vehicle (DDV) approach utilizing the BoNT/A rHC as a neuron specific targeting molecule conjugated via a disulfide linkage to a drug delivery platform (10 kD dextran) (Zhang et al., 2009). Therapeutic DNA-PK inhibitor targeting is important for two main reasons: (a) minimizing systemic toxicity, if any, due to treatment compounds, and (b) delivering

an effective high concentration of a therapeutic compound to the site of toxicity, e.g., nerve terminals for botulism. We had demonstrated that the targeting component of the DDV, i.e., rHC was nontoxic (Zhang et al., 2009). Glycine is a major inhibitory neurotransmitter in the mammalian spinal cord where, by acting at strychnine-sensitive receptors, it plays important roles in a variety of motor and sensory functions. Within the spinal cord, glycine can be released from nerve endings of glycinergic neurons

as well as from nerve terminals of interneurons in which glycine and GABA are co-stored in the same vesicles and from which the two amino acid transmitters are co-released onto motoneurons (Chaudhry et al., 1998 and Jonas et al., 1998). In addition to the inhibitory functions, glycine is involved in excitatory neurotransmission (Supplisson and Tau-protein kinase Roux, 2002). Therefore, glycine is an important neurotransmitter in the spinal cord. 3[H]-glycine release assay was initially used as an indicator of spinal cord function for evaluation of Tetanus toxin (Williamson et al., 1992) and botulinum neurotoxin (Williamson et al., 1996). In this assay, 3[H]-glycine was taken up by cells in physiological solution and released by depolarization with 56 mM K+ in the presence of 2 mM Ca2+. Since the assay is relatively simple and reliable, it is accepted by researchers in Clostridial neurotoxins studies. In the present study, the objective was to test the utility of a DDV-Mas-7 construct as a viable therapeutic against botulism in a peripheral neuronal model.