Finally, Zobel-Thropp et al. (2012) reported the cloning and heterologous expression of a phospholipase-D from L. arizonica. Additionally, proteomic analysis of Loxosceles
species venoms demonstrated the existence of eleven isoforms of related phospholipase-D proteins in Loxosceles gaucho venom ( Machado et al., 2005) and at least seven related phospholipase-D proteins in L. intermedia ( dos Santos et al., 2009). The toxins characterized as phospholipase-D proteins have been grouped into a family ( Kalapothakis et al., selleck compound 2007). The noxious effects induced by crude Loxosceles venom may be a result of synergism among these toxins, strengthening the biological importance of these molecules in the biological cycle of Loxosceles spiders ( Kalapothakis et al., 2007). Here, using a recombinant isoform of L. intermedia phospholipase-D selleck chemical (LiRecDT1)
and related biotools, such as polyclonal antibodies against LiRecDT1 and GFP-LiRecDT1 ( Chaim et al., 2006; Kusma et al., 2008; Chaves-Moreira et al., 2011), we achieved immune detection of several expressed phospholipase-D isoforms in crude venom and found that they exhibit modulatory activities, such as affecting membrane binding, phospholipid hydrolysis, calcium influx and proliferative activity, in the mouse melanoma cell line B16-F10. The results open the possibility of using this toxin as an exogenous biotool to modulate cellular processes and in studies addressing calcium and phospholipid metabolism. Polyclonal antibodies against recombinant phospho-lipase-D (LiRecDT1) were produced in rabbits following the procedure in Chaim et al. (2006). Adult rabbits weighing approximately 3 kg from the Central
Animal House of the Federal University of Paraná were used. All experimental protocols involving animals were performed according to the Principles of Laboratory Animal Care (NIH Glutathione peroxidase Publication n° 85-23, revised 1985), Brazilian Federal Laws, and ethical committee agreement number 566 of the Federal University of Paraná. Crude L. intermedia venom was extracted from wild-caught spiders following Feitosa et al. (1998), in accordance with the Brazilian Federal System for Authorization and Information on Biodiversity (SISBIO-ICMBIO, N° 29801-1). DAPI, AlexaFluor-conjugated anti-rabbit IgG, Fluo-4 AM and the CyQUANT Cell proliferation assay kit were purchased from Molecular Probes (Eugene, Oregon, USA). A venom gland cDNA library was constructed previously (Chaim et al., 2006; Gremski et al., 2010). The GenBank designation for the deposited data on the cloned L. intermedia LiRecDT1 cDNA sequence is DQ218155.1. The cDNA sequence corresponding to the mature phospholipase-D LiRecDT1 protein was amplified via PCR.