ABSTRACT
Abstract Background: Endogenous phospholipase A2 inhibitors from snake blood (sbPLIs) have been isolated from several species around the world, with the primary function of self-protection against the action of toxic phospholipases A2. In American snakes, sbPLIs were solely described in pit vipers, in which the natural protection role is justified. In this study, we described a sbPLI in Boa constrictor (popularly known as jiboia), a non-venomous snake species from America. Methods: PLA2 inhibitory activity was tested in the blood plasma of B. constrictor using C. d. terrificus venom as the enzyme source. Antibodies developed against CNF, a sbγPLI from Crotalus durissus terrificus, were used to investigate the presence of homologues in the blood plasma of B. constrictor. A CNF-like molecule with a PLA2 inhibitory activity was purified by column chromatography. The encoding gene for the inhibitor was cloned from B. constrictor liver tissue. The DNA fragment was cloned, purified and sequenced. The deduced primary sequence of interest was aligned with known sbγPLIs from the literature. Results: The blood plasma of B. constrictor displayed PLA2 inhibitory activity. A CNF-like molecule (named BcNF) was identified and purified from the blood plasma of B. constrictor. Basic properties such as molecular mass, composing amino acids, and pI were comparable, but BcNF displayed reduced specific activity in PLA2 inhibition. BcNF showed highest identity scores (ISs) with sbγPLIs from pit vipers from Latin America (90-100%), followed by gamma inhibitors from Asian viperid (80-90%). ISs below 70% were obtained for BcNF and non-venomous species from Asia. Conclusion: A functional sbγPLI (BcNF) was described in the blood plasma of B. constrictor. BcNF displayed higher primary identity with sbγPLIs from Viperidae than to sbγPLIs from non-venomous species from Asia. The physiological role played by sbγPLIs in non-venomous snake species remains to be understood. Further investigation is needed.(AU)
Subject(s)
Animals , Snakes , Viperidae , Elapid Venoms , Phospholipases A2 , Phospholipase A2 InhibitorsABSTRACT
DNA amplification by the polymerase chain reaction (PCR) was applied in the investigation of the presence of Leishmania (Kinetoplastida: Trypanosomatidae) parasites in single phlebotomine sandflies. Three phlebotomine/parasite pairs were used: Lutzomyia longipalpis/Leishmania chagasi, Lutzomyia migonei/Leishmania amazonensis and Lutzomyia migonei/Leishmania braziliensis, all of them incriminated in the transmission of visceral or cutaneous leishmaniasis. DNA extraction was performed with whole insects, with no need of previous digestive tract dissection or pooling specimens. The presence of either mouse blood in the digestive tract of the sandflies or the digestive tract itself did not interfere in the PCR. Infection by as few as 10 Leishmania sp. per individual were sufficient for DNA amplification with genus-specific primers. Using primers for L. braziliensis and L. mexicana complexes, respectively, it was possible to discriminate between L. braziliensis and L. amazonensis in experimentally infected vectors (L. migonei)
Subject(s)
Animals , Female , Mice , Leishmania , Leishmaniasis , Polymerase Chain Reaction , Psychodidae , Deer , DNA, Protozoan , Insect VectorsABSTRACT
O flebotomineo Lutzomyia longipalpis tem sido incriminado como vetor da leishmaniose visceral americana, causada pelo protozoario Leishmania chagasi. Entretanto, tem-se acumulado evidências que sugerem a existência de um complexo e näo apenas uma espécie de L. longipalpis na natureza. Nosso trabalho teve como objetivo comparar, ao nível molecular, quatro populaçöes de L. longipalpis de referência, utilizando especimens criados em laboratório, provenientes de regiöes geograficamente distintas, através de RAPD-PCR (reaçäo de polimerase em cadeia com amplificaçäo por iniciadores ao acaso). Para isso, o DNA genomico de grupos de flebotomineos foi amplificado com iniciadores decamericos unicos com sequencia de nucleotideos arbitraria, na tentativa de se detectar sitios polimorficos...