Nicotinamide/nicotinate adenine dinucleotide (NAD+/NaAD) performs essential functions in cell metabolism and energy production due to its redox properties. The nicotinamide/nicotinate mononucleotide adenylyltransferase (NMNAT, EC 2.7.7.1/18) enzyme catalyses the key step in the biosynthesis of NAD+. Previously, the enzyme NMNAT was identified in Trypanosoma cruzi (TcNMNAT), a pathogenic agent with epidemiological importance in Latin America. To continue with the functional characterisation of this enzyme, its subcellular location and its possible post-translational modifications were examined in this study. For this, polyclonal antibodies were generated in mice, with soluble and denatured recombinant protein being used to detect the parasite’s NMNAT. Immunodetection assays were performed on whole extracts of T. cruzi, and an approximation of its intracellular location was determined using confocal microscopy on wild and transgenic parasites, which revealed the cytosol distribution patterns. This localisation occurs according to the needs of the dinucleotides that exist in this compartment. Additionally, a bioinformatics study was performed as a first approach to establish the post-translational modifications of the enzyme. Possible phosphorylation events were experimentally analysed by western blot, highlighting TcNMNAT as a potential target for serine kinases.

Introducción. El empleo etnofarmacológico de plantas en el manejo de procesos inflamatorios crónicos y la necesidad su caracterización farmacológica, promueven la evaluación de actividad antiinflamatoria de sustancias en modelos in vivo. Materiales y métodos. Evaluación de extractos y fracciones de Calea prunifolia (CP), Curatella americana (CA), Myrcianthes leucoxila (ML) y Physalis peruviana (PP) sobre los modelos edema auricular por acetato de tetradecanoilforbol (TPA) en ratón albino ICR y edema plantar por carragenina en ratas Wistar, seleccionando un extracto para valorar su actividad antiartrítica en el modelo artritis inducida por colágeno en ratones DBA. Resultados. Las fracciones con mayor porcentaje de inhibición del edema en el modelo edema auricular por TPA fueron ML etanólica total (82±6%), CP rica en terpenos (81±6%) y CA rica en terpenos (81±7%) (P<0,05). No se obtuvo actividad antiinflamatoria significativa sobre el modelo edema plantar por carragenina. Se evaluó la actividad antiartrítica de la fracción rica en terpenos de ML sobre el modelo artritis inducida por colágeno, sin encontrarse efecto significativo sobre edema de patas traseras, peso corporal, escala histopatológica de severidad de artritis ni inmunohistoquímica para factor de necrosis tumoral alfa (P>0,05). Discusión. La actividad antiinflamatoria en el modelo de inflamación aguda edema auricular por TPA para los extractos y fracciones de CP, CA y ML se puede relacionar con la afectación de mediadores relacionados con fosfolipasa A2 dado el nivel de efecto similar a indometacina encontrado. La fracción terpénica de ML no mostró actividad antiartrítica ni modificó la expresión de TNF-a en el modelo de artritis crónica autoinmune empleado, por lo cual no posee actividad inmunomoduladora ni antiinflamatoria en la dosis evaluada. Conclusión: Las fracciones terpénicas de los extractos de CA y CP y los extractos metanólicos de ML mostraron una actividad antiinflamatoria significativa en el edema auricular inducida por TPA. Estos extractos tuvieron poca actividad sobre el edema inducido por carragenina en la pata. La fracción terpénica del extracto ML no presentó actividad antiartrítica en el modelo de artritis inducido por el colágeno.

Introduction. Ethnopharmacological use of plants in management of chronic inflammatory diseases and the need to have their pharmacological characterization promote the evaluation of anti-inflammatory activity over in vivo models. Materials and methods. Evaluation of extracts and fractions of Calea prunifolia (CP), Curatella americana (CA), Myrcianthes leucoxila(ML) and Physalis peruviana (PP) on auricular edema by tetradecanoylphorbol acetate (TPA)-in ICR albino mice and carrageenan-induced leg edema in Wistar rats selecting an extract to evaluate its anti-arthritic activity in collagen-induced arthritis model in DBA mice. Results. The fractions with greater edema inhibition percentage on TPA-induced ear edema included whole ethanolic fraction of ML (82±6%), CP terpenes rich fraction (81±6%) and CA terpenes rich fraction (81±7%) (P<0.05). Significant antiinflamatory activity was not obtained on the carrageenan-induced leg edema. Evaluation of antiarthritic activityof the ML terpenes rich fraction was carried out on collagen induced arthritis. Without finding any significant effect on back leg edema, corporal weight, arthritis histopathology severity scale or immunohistochemical tumoral necrosis factor alfa immunohistochemical evaluation (P>0.05). Discussion. Anti-inflammatory activity in TPAinduced acute ear edema model for CP, CA, and ML extracts and fractions can be interfered by phospholipase A2 related mediators due to similar effect with indomethacin was found. The ML terpens rich fraction neither show any anti-arthritic activity nor affected TNF-a expression on the autoimmune chronic arthritis model used, reason why it does not have immunomodulatory/anti-inflammatory effect on the evaluated dose. Conclusion. CA and CP terpenic rich fractions and ML ethanol extract showed significant anti-inflammatory activity in TPA-induced ear edema. They did have some activity in carrageenan-induced leg edema. The ML terpenic rich fraction did not have anti-arthritic activity in the collagen-induced arthritis model.