CLONING AND COMPARISON OF THE GENES ENCODING PREPROAPAMIN FROM THE VENOM OF 2 HONEYBEE AND 4 WASP SPECIES

Preproapamin genes were amplified by RT-PCR from total RNA from the venom glands of 2 honeybee species, Apis mellifera, A. cerana cerana, and 4 wasp species, Vespa magnifica, V. velutina nigrothorax and Polistes hebraeus, respectively. Their PCR products were ligated into pGEM -T easy vector and the nucleotide sequences analyzed. The six fragments were all 141?bp in length and contained a n ORF coding the precursor of apamin. The apamin precursors of V. magnifica, V. velutina nigrothorax and P. hebraeus had 95% and 93% similarity with that of A. melliera in nucleotide and amino acid sequences, respectively. That of Vespu la maculifrons was identical to that of A. mellifera in nucleotide and amino acid sequences. Apamin precursors of V. magnifica, P. hebraeus and V. velutina nigrothorax also had the same nucleotide sequences. The nucleotide sequences o f preproapamin genes from the Chinese honeybee, A. cerana cerana and 4 wasp sp ecies are described for the first time. A notable discovery was that the wasps species had exactly same apamins as the honeybees despite the fact they belong to different insect families.

Effects of SKF-96365, a TRPC inhibitor, on melittin-induced inward current and intracellular Ca^2+ rise in primary sensory cells

Objective Melittin （MEL） is a major component of bee venom and can produce both persistent spontaneous nociception and pain hypersensitivity when injected subcutaneously in the periphery. The present study aimed to examine the roles of transient receptor potential canonical （TRPC） channels in mediation of MEL-indueed activation of primary nociceptive cells. Methods Whole-cell patch-clamp and laser scanning confocal calcium detection were used to evalu- ate the effects of SKF-96365, a TRPC inhibitor, applied on the acutely isolated dorsal root ganglion （DRG） cells of rat, on MEL-induced increase in intracellular calcium concentration （[Ca2＋]i） and inward current. Results Under voltage- clamp mode, 43.9% （40/91） DRG cells were evoked to give rise to the inward current by 2 pmol/L MEL, which could be significantly suppressed by 3 doses of SKF-96365 （1, 5 and 10μmol/L） in a dose-dependent manner. Of the other 210 cells, 67.6% responded to MEL with an intracellular Ca2＋ rise, as revealed by confocal calcium imaging. Of these MEL- sensitive cells, 46.5% （66/142） were suppressed by the highest dose of SKF-96365. Conclusion MEL-induced activation of small to medium-sized DRG cells can be suppressed by SKF-96365, suggesting the involvement of TRPC channels in the mediation of MEL-induced activation of primary nociceptive cells.