Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase.

INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

IgE antibodies from schistosomiasis patients to recognize epitopes in potato apyrase

Abstract INTRODUCTION: High percentages of structural identity and cross-immunoreactivity have been reported between potato apyrase and Schistosoma mansoni ATP diphosphohydrolase (SmATPDases) isoforms, showing the existence of particular epitopes shared between these proteins. METHODS: Potato apyrase was employed using ELISA, western blot, and mouse immunization methods to verify IgE reactivity. RESULTS: Most of the schistosomiasis patient's (75%) serum was seropositive for potato apyrase and this protein was recognized using western blotting, suggesting that parasite and plant proteins share IgE-binding epitopes. C57BL/6 mice immunized with potato apyrase showed increased IgE antibody production. CONCLUSIONS: Potato apyrase and SmATPDases have IgE-binding epitopes.

Chromatographic fingerprint analysis and effects of the medicinal plant species Mitracarpus frigidus on adult Schistosoma mansoni worms.

The aims of this work were to evaluate the in vitro and in vivo schistosomicidal properties of the methanolic extract of the aerial parts of Mitracarpus frigidus (MFM) and to determine its HPLC profile. For the in vitro experiment, four pairs of adult worms, obtained from infected mice, were exposed to different concentrations of MFM (100 to 400 µg/mL) for 24 and 48 h and analyzed under an inverted microscope. For the in vivo experiment, mice were inoculated with cercariae and, 20 days after infection, MFM (100 and 300 mg/kg) was administered orally for the following 25 days. Mice were euthanized after 60 days. MFM showed in vitro schistosomicidal activity, exhibiting the opening of the gynaecophoral canal of some male schistosomes, the presence of contorted muscles, vesicles, and the darkening of the paired worms skin. In vivo experiments showed that MFM treatments significantly reduced total worm count, as praziquantel, showing a decrease in liver and spleen weight. Also, a significant reduction in granuloma density was observed. MFM treatment did not cause alterations in the liver function of either infected or noninfected mice. The HPLC chromatogram profile showed the presence of kaempferol-O-rutinoside, rutin, kaempferol, psychorubrin, and ursolic acid.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients.

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80%) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

Detection of IgG1 and IgG4 subtypes reactive against potato apyrase in schistosomiasis patients

In this paper, we showed for the first time that the conserved domains within Schistosoma mansoni ATP diphosphohydrolase isoforms, shared with potato apyrase, possess epitopes for the IgG1 and IgG4 subtypes, as 24 (80 percent) of the 30 schistosomiasis patients were seropositive for this vegetable protein. The analyses for each patient cured (n = 14) after treatment (AT) with praziquantel revealed variable IgG1 and IgG4 reactivity against potato apyrase. Different antigenic epitopes shared between the vegetable and parasite proteins could be involved in susceptibility or resistance to S. mansoni AT with praziquantel and these possibilities should be explored.

A new series of schistosomicide drugs, the alkylaminoalkanethiosulfuric acids, partially inhibit the activity of Schistosoma mansoni ATP diphosphohydrolase.

The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100-1000 microM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[(tert-butyl)amino]-1-ethanethiosulfuric acid (100 microM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-(sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited <10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs.