Full details of the purification procedure are available online as Supplementary material to this article. The molecular mass of the toxin assessed by tricine SDS-PAGE (Schägger and von Jagow, 1987) and mass spectrometry, as well as the identification of tryptic fragments by MALDI-TOF mass spectrometry confirmed that the toxin was Bbil-TX (Carregari et al., in press). Chicks were killed with isoflurane and the biventer cervicis muscles were removed and mounted under a resting tension of 1 g in a 5 ml organ bath (Panlab, Spain) containing aerated (95% O2 and 5% CO2) Krebs solution (composition, in mM: NaCl 118.7, KCl 4.7, CaCl2 1.88, KH2PO4

1.17, MgSO4 1.17, NaHCO3 25.0 and glucose 11.65, pH 7.5) at 37 °C, as described by Ginsborg and Warriner Selleckchem Galunisertib (1960). Stimuli (0.1 Hz, 0.2 ms) were delivered to the nerve from an LE 12406 TC stimulator (Panlab, Spain) and the muscle twitches were recorded using a TRI201AD force displacement transducer coupled to a Quad Bridge Amp and LabChart 6.0 software (all from ADInstruments Pty Ltd., Australia). Contractures to exogenous acetylcholine (ACh, 110 μM) and potassium selleck chemicals chloride

(KCl, 40 mM) were obtained in the absence of stimulation, before and after the addition of peaks P1–P3 or Bbil-TX, to test for myotoxic and neurotoxic activities (Harvey et al., 1994). After the initial tests with ACh and KCl, the preparations were washed and electrical stimulation was recommenced, with the preparations

being allowed to stabilize Phenylethanolamine N-methyltransferase for at least 20 min before the addition of peaks P1–P3 (a single concentration of 10 μg/ml) or Bbil-TX (0.5, 1, 5 or 10 μg/ml). Muscle twitches were recorded for up to 120 min or until complete blockade. Some experiments were done using preparations incubated with d-tubocurarine (d-Tc, 10 μg/ml) to examine the effect of Bbil-TX (10 μg/ml) on muscle responses to direct stimulation with supramaximal pulses (0.1 Hz, 2 ms). Other preparations were incubated at 22–24 °C to assess the influence of temperature on Bbil-TX-induced (5 μg/ml) neuromuscular blockade. In some experiments, the PLA2 activity of Bbil-TX was inhibited by pretreating the toxin with p-bromophenacyl bromide (p-BPB; 0.6 μM, 24 h, 23 °C) essentially as described elsewhere ( Rodrigues-Simioni et al., 2011) and then testing for neuromuscular activity. The diaphragm and its phrenic nerve were dissected from male Swiss mice killed with isoflurane. The preparations were mounted under a resting tension of 5 g in a 5 ml organ bath containing aerated (95% O2 and 5% CO2) Tyrode solution (composition, in mM: NaCl 137, KCl 2.7, CaCl2 1.8, MgCl2 0.49, NaH2PO4 0.42, NaHCO3 11.9 and glucose 11.1) at 37 °C, as described by Bülbring (1946). Supramaximal stimuli (0.1 Hz and 0.2 ms for indirect stimulation) were delivered from a Grass S88 stimulator (Grass Instrument Co.