Moreover, the generation time of the final product (ceramide 1-phosphate), is compatible with the cellular early response (before 30 min) observed

for Calcium influx. Finally, the activity of recombinant brown spider phospholipase-D on B16-F10 cells was further confirmed by its ability to stimulate cell Roxadustat proliferation in a concentration and time-dependent manner. Additionally, the increases in the cell proliferation rate in B16-F10 cells following LiRecDT1 exposure were higher when the cells were incubated in the presence of exogenous sphingomyelin (which was, as reported, a good substrate for recombinant phospholipase-D). A putative explanation for this event is that exogenous sphingomyelin increases the concentration and accessibility of enzyme substrates, generating bioactive lipid mediators following treatment with recombinant brown spider proteins (such as ceramide 1-phosphate or interconvertible lipids such as ceramide, sphingosine and sphingosine

HKI-272 1-phosphate), compared to offering lipid substrates organized in the lipid bilayer of cell membranes. The results described herein indicated that B16-F10 melanoma cells subjected to exogenous treatment with a recombinant phospholipase-D from brown spider venom (LiRecDT1) bound phospholipase-D at the cell surface, did not suffer changes in viability, ID-8 experienced metabolism of their phospholipids by the enzyme, generated metabolically bioactive lipids, triggered Calcium mobilization inside the cytosol, and had their proliferation stimulated, especially in the presence of exogenous sphingomyelin.

The data indicated that venom phospholipase-D, which is also referred to as “dermonecrotic toxin” because it is directly involved on gangrenous and necrotic loxoscelism due to generating bioactive lipids such as lysophosphatidic acid and/or ceramide 1-phosphate, can also modulate membrane phospholipid metabolism, regulate tumor cell proliferation, and modulate the cytosolic Calcium influx, opening the possibility of using this enzyme as a novel biotool in studies addressing phospholipid and calcium metabolism. This work was supported by grants from CAPES, CNPq, Fundação Araucária-PR and the Secretaria de Estado de Ciência, Tecnologia e Ensino Superior do Paraná, Brazil. “
“TTX, a specific blocker of voltage-gated sodium channels of excitable membranes of muscle and nerve tissues (Colquhon et al., 1972; Narahashi, 2001), and one of the most potent neurotoxins, was long believed to occur exclusively in pufferfish.