An ATP diphosphohydrolase (EC 3.6.1.5) activity was identified in a Leishmania (Viannia) braziliensis promastigotes preparation (Lb). Ultrastructural cytochemical microscopy showed this protein on the parasite surface and also stained a possible similar protein at the mitochondrial membrane. Isolation of an active ATP diphosphohydrolase isoform from Lb was obtained by cross-immunoreactivity with polyclonal anti-potato apyrase antibodies. These antibodies, immobilized on Protein A-Sepharose, immunoprecipitated a polypeptide of approximately 48 kDa and, in lower amount, a polypeptide of approximately 43 kDa, and depleted 83% ATPase and 87% of the ADPase activities from detergent-homogenized Lb. Potato apyrase was recognized in Western blots by IgG antibody from American cutaneous leishmaniasis (ACL) patients, suggesting that the parasite and vegetable proteins share antigenic conserved epitopes. Significant IgG seropositivity in serum samples diluted 1:50 from ACL patients (n=20) for Lb (65%) and potato apyrase (90%) was observed by ELISA technique. Significant IgG antibody reactivity was also observed against synthetic peptides belonging to a conserved domain from L. braziliensis NDPase (80% seropositivity) and its potato apyrase counterpart (50% seropositivity), in accordance with the existence of shared antigenic epitopes and demonstrating that in leishmaniasis infection the domain r82-103 from L. braziliensis NDPase is a target for the human immune response.
An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.
Background: Eugenol shows both antibacterial and antiparasitic activities, suggesting that it might be evaluated as an option for the treatment of praziquantel-resistant schistosome. Methods: The in vitro activities of three eugenol derivatives (FB1, FB4 and FB9) on adult worms from Schistosoma mansoni were examined by fluorescence and scanning electron microscopy to analyze effects on the excretory system and integument damage, respectively. Biochemical tests with verapamil (a calcium channel antagonist) and ouabain (a Na + /K + -ATPase pump inhibitor) were used to characterize eugenol derivative interactions with calcium channels and the Na + /K + -ATPase, while in silico analysis identified potential Na + /K + -ATPase binding sites. Results: The compounds showed effective doses (ED 50 ) of 0.324 mM (FB1), 0.167 mM (FB4), and 0.340 mM (FB9). In addition, FB4 (0.322 mM), which showed the lowest ED 50, ED 90 and ED 100 (p < 0.05), caused the most damage to the excretory system and integument, according to both fluorescence and scanning electron microscopy analysis. The death of adult worms was delayed by ouabain treatment plus FB1 (192 versus 72 hours) and FB9 (192 versus 168 hours), but the response to FB4 was the same in the presence or absence of ouabain. Besides, no changes were noted when all of the eugenol derivatives were combined with verapamil. Moreover, FB1 and FB9 inhibited
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