Neurotoxic activity of ringhalexin

All animal experiments were conducted according to the protocol (021/07a) approved by the Institutional Animal Care and Use Committee of the National University of Singapore.

Ringhalexin protein (200 μl in 0.9% saline) was injected intraperitoneally (i.p.) into male Swiss albino mice at doses of 10 and 100 mg/kg (n = 2) and the symptoms were observed. The control group was injected with 200 μl of 0.9% saline (n = 2).

Isolated tissue experiments were conducted as described previously^6,52 using a conventional organ bath (6 ml) containing physiological Krebs-Henseleit buffer of the composition (in mM): 118 NaCl, 4.8 KCl, 1.2 KH₂PO₄, 2.5 CaCl₂, 2.4 MgSO₄, 25 NaHCO₃ and 11 D-(+) glucose), pH 7.4. Organ bath chambers were continuously aerated with carbogen (5% carbon dioxide in oxygen) and maintained at 37 °C throughout the experiment. The resting tension of the isolated tissues was maintained between 1–2 g tension and the tissues were allowed to equilibrate for 30–45 min before the start of an experiment. Electrical field stimulation (EFS) was carried out through platinum ring electrodes using a Grass stimulator S88 (Grass instruments, West Warwick, RI, USA). The magnitude of the contractile responses was measured in gram tension. Data were continuously recorded on PowerLab LabChart 6 data acquisition system using a force displacement transducer (Model MLT0201) (ADInstruments, Bella Vista, New South Wales, Australia).