RESUMO
To investigate the peculiar absence of phospholipases A(2) (PLA(2)s) in most Crotalus horridus (CH) venom, we cloned and sequenced the venom PLA(2)s of three CH specimens from different regions. The results revealed that all the venom glands contained mRNAs that encoded an acidic PLA(2) (designated as either CH-E6 or CH-E6'). The predicted CH-E6 from the Iowan CH and CH-E6' from the South Carolinian CH differed by only one amino acid residue, while the PLA(2) cDNA cloned from the Kentuckian CH contained an early stop codon instead of a Tyr(22) codon. Only the individual South Carolinian CH venom was found to contain the CH-E6' protein whose mass was confirmed by MALDI-TOF analysis. Our results suggest that low PLA(2) expression levels in most CH venom can be attributed to translation blockage. We also purified two acidic PLA(2)s and canebrake toxin from the pooled venom of Crotalus horridus atricaudatus (neurotoxic CH subspecies). One of the acidic PLA(2)s was identical to CH-E6 and showed high lipolytic activity and weak anti-platelet activities. The possibility that C. h. atricaudatus could be a hybrid between CH and Crotalus scutulatus is discussed.
Assuntos
Anticoagulantes , Códon de Terminação , Venenos de Crotalídeos/enzimologia , Crotalus/genética , Regulação Enzimológica da Expressão Gênica , Fosfolipases A2 Secretórias/genética , Proteínas de Répteis/genética , Sequência de Aminoácidos , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/metabolismo , Anticoagulantes/farmacologia , Sequência de Bases , Coagulação Sanguínea/efeitos dos fármacos , Venenos de Crotalídeos/genética , DNA Complementar/química , Glândulas Exócrinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neurotoxinas/isolamento & purificação , Fosfolipases A2 Secretórias/química , Fosfolipases A2 Secretórias/isolamento & purificação , Fosfolipases A2 Secretórias/metabolismo , Filogenia , RNA Mensageiro/isolamento & purificação , RNA Mensageiro/metabolismo , Proteínas de Répteis/química , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/metabolismo , Alinhamento de Sequência , Mordeduras de Serpentes/enzimologia , Mordeduras de Serpentes/fisiopatologia , Especificidade da EspécieRESUMO
Interleukin-15 (IL-15) signaling has pleiotropic actions in many cell types during development and has been best studied in cells of immune system lineage, where IL-15 stimulates proliferation of cytotoxic T cells and induces maturation of natural killer cells. A few reports have indicated that IL-15 and the IL-15 receptor are expressed in central nervous system tissues and neuronal cell lines. Because this aspect of IL-15 action is poorly studied, we used cultured rat neural stem cells (NSCs) to study IL-15 signal transduction and activity. Primary cultures of rat NSCs in culture will form neurospheres and will differentiate into neuron, astrocyte, and oligodendrocyte progenitors under permissive conditions. We found by immunofluorescence that the IL-15Ralpha subunit of the IL-15 receptor was expressed in NSCs and differentiating neurons, but not astrocyte or oligodendrocyte progenitors. We also showed that IL-15 treatment reduced MAP-2 protein levels in neurons and could reduce neurite outgrowth in differentiating neurons but did not affect NSC proliferation, and cell proportions and viability of the corresponding lineage cells. In the presence of a STAT3 inhibitor, Stattic, IL-15 no longer reduced MAP-2 protein levels. IL-15 treatment caused STAT3 phosphorylation. Furthermore, using anti-IL-15Ralpha antibody to block IL-15 signaling completely inhibited IL-15-induced phosphorylation of STAT3 and prevented IL-15 from decreasing neurite outgrowth. In conclusion, IL-15 may influence neural cell differentiation through a signal transduction pathway involving IL-15Ralpha and STAT3. This signal transduction modifies MAP-2 protein levels and, consequently, the differentiation of neurons from NSCs, as evidenced by reduced neurite outgrowth.
