Purificación de una fracción de antígenos de tripomastigotes de Trypanosoma cruzi para el diagnóstico de la enfermedad de Chagas / Purification of a trypomastigote antigen fraction from Trypanosoma cruzi for Chagas’ disease diagnosis

Los antígenos excretados/secretados por las formas tripomastigotes de T. cruzi (antígenos TESA) pertenecen a la familia de las transialidasas, las cuales son responsables de la transferencia de ácido siálico exógeno a moléculas aceptadoras en la superficie de los tripomastigotes. En el presente trabajo se purifican varias proteínas de los antígenos TESA utilizando cromatografía de afinidad con resina de sefarosa B4-concanavalina A, con la intención de ser utilizados en el diagnóstico de la enfermedad de Chagas. El buffer de elución contenía una mezcla de α-D-manopiranósido y α-D-glucopiranósido. Se realizó electroforesis unidimensional en gel con poliacrilamida para identificar las bandas purificadas y la prueba de inmunoelectrotransferencia para visualizar las bandas reactivas con el pool de sueros de individuos con infección por T. cruzi. El gel teñido con azul de Coomassie coloidal permitió visualizar 3 bandas de aproximadamente 220, 170, y 20 kDa. La inmunoelectrotransferencia utilizando un pool de sueros positivos, confirmados para la infección por T. cruzi, reveló 5 bandas inmunogénicas de 220, 120, 85, 50 y 32 kDa mientras que el revelado con diaminobenzidina permitió observar las bandas de 220, 120, 85, 50 32 y 20 kDa. Asimismo las bandas purificadas no fueron reconocidas en la inmunoelectrotransferencia por el pool de sueros confirmados como negativos. Estos resultados sugieren el potencial de estas proteínas purificadas de TESA para ser usadas como nueva herramienta para el diagnóstico de la enfermedad de Chagas.

Trypanosoma cruzi excreted/secreted antigens (TESA) belong to the transialidase family, which are responsible for the transfer of exogenous sialic acid to accepting molecules at the trypomastigote surface. In the present study we purified several proteins from TESA antigens using affinity chromatography with sepharose B4-concanavalin A resin, with the purpose of using them for Chagas’ disease diagnosis. The elution buffer contained a mixture of α-D-manopiranosid and α-D-glucopiranosid. A unidimensional electrophoresis in polyacrilamide gel to identify the purified bands, and an immunoelectrotransference test with a pool of sera from T. cruzi infected individuals to visualize the reactive bands were carried out. The colloidal Coomassie blue stained gel allowed visualizing 3 bands of approximately 220, 170 and 20 kDa. The immunoelectrotransference using a pool of positive sera with confirmed T. cruzi infection showed 5 immunogenic 220, 120, 50 and 32 kDa bands, while a developing with diaminobenzidine showed 220, 120, 85, 50, 32 and 20 kDa bands. The purified bands were not recognized in an immunoelectrotransference test when a pool of confirmed negative sera was used. These results suggest the potential of these TESA purified proteins for using them as a new tool for Chagas’ disease diagnosis.

Estudio del mecanismo de fosforilación de proteínas como respuesta celular hepática inducida por adriamicina, con o sin protección con L carnitina / Study of protein phosphorylation mechanism induced by adriamycin in hepatic tissue with or without L-carnitine

Los cambios en los estados de fosforilación de proteínas han sido asociados a numerosas patologías de diferentes orígenes y severidad, y estas alteraciones, pueden estar vinculadas a estrés oxidativo y modificaciones en proteínas quinasas y fosfatasas. En este sentido, la terapia con adriamicina ha sido vinculada con estrés oxidativo cardiaco y hepático con subsecuente disfunción de tales órganos. Adicionalmente, al estrés cardiaco por adriamicina, el hígado podría representar otro blanco tóxico de la droga. Sin embargo, las alteraciones hepáticas han sido pobremente estudiadas. En este trabajo se estudio el patrón de fosforilación de proteínas de tejido hepático, ante la administración de adriamicina. Ratas Sprague Dawley se distribuyeron en cuatro grupos al azar: control, adriamicina, carnitina y adriamicina carnitina. Los tratamientos administrados por via intravenosa (VI) cada tres días/3 dosis fueron: 5 mg/Kg de peso de ADR y 20mg/Kg de peso de carnitina y combinando ambos agentes. Los animales se sacrificaron, tomándose el lóbulo hepático medio para ensayos de: fosforilación con [ã 32 -P] ATP, inmunodetección de fosfoproteínas en serina y tirosina, proteína JNK y C-jun. Los patrones de fosforilación de proteínas entre los grupos fueron diferentes observándose mayor expresión de proteínas fosforiladas en los grupos adriamicina. La carnitina revierte el efecto sobre la fosforilación comportándose como hepatoprotector ante la droga.

The changes in the pattern of protein phosphorylation have been associated to numerous pathologies of differentorigins and severity; these alterations can be linked to oxidative stress and subsequent modifications in the protein kinases and phosphatases. In this regard, adriamycin therapy have been related to the heart and liver oxidative stress and organ disfunction. Therefore, in addition to the heart, the liver might be another adriamycin toxic target. However, adriamycin liver alterations have been poorly studied. The aim of this work was to determined liver protein phosphorylation before and after adriamycin administration. Female Sprague-Dawley rats (n=3), 40-60g body weight, were randomized into four groups: control, adriamycin, carnitine and adriamycin-carnitine. Saline adriamycin (15mg/Kg body weight) and carnitine (20 mg before adriamycin) were given intravenously (0,1 ml). Samples from the medium liver lobe were taken for biochemical experiments including phosphorylation with [ã 32 -P] ATP, inmunodetection of phosphoproteins in serine and tirosine, JNK and C-jun proteins. The protein phosphorylation was different between the groups studied. The greater expression of protein phosphorylates was determined in the adriamycin group. We suggest that there is a relationship between carnitine administration and decreased expression of protein phosphorylates. Carnitine may be a hepatoprotector.

Intracellular calcium homeostasis in Leishmania mexicana. Identification and characterization of a plasma membrane calmodulin-dependent Ca(2+)-ATPase

The intracellular Ca2+ concentration in different trypanosomatids is about 50 nanomolar, which concentration in different trypanosomatids is about 50 nanomolar, which is 4 orders of magnitude lower than in the extracellular milieu. This fact implies the existence of well developed mechanisms for the maintenance of such a high calcium gradient. In higher eukaryotics a number of different structures have been implicated in this function. Some of them are located in intracellular organelles, and others in the plasma membrane. Since intracellular organelles are limited by their storage capacity, long-term Ca2+ homeostasis resides solely in the plasma membrane. In higher eukaryotics, a calcium pump or Ca(2+)-ATPase located in the plasma membrane, because of its high Ca2+ affinity, has been proposed as the structure responsible for the maintenance of the cytoplasmic Ca2+ concentration at the submicromolar level. The presence of a Ca(2+)-ATPase in trypanosomatids has been debated. While some groups have reported its absence, others have reported the existence of an enzyme which is Mg(2+)-independent or even inhibited by Mg2+. On the other hand, in none of these reports any correlation was shown between the Ca(2+)-ATPase activity observed and the Ca2+ transport function attributed to this enzyme. We have previously shown that a calmodulin-stimulated Mg(2+)-dependent Ca(2+)-ATPase is present in the plasma membrane of Leishmania braziliensis and of Trypanosoma cruzi. Plasma membrane vesicles from these parasites are able to accumulate Ca2+ in the presence of the ATP-Mg complex. The similarities found between the kinetics parameters and other properties of the Ca(2+)-ATPase and the Ca2+ transport activity strongly suggest a common molecular entity. The stoichiometry calculated from these parameters approaches the 1:1 stoichiometry for Ca2+ and ATP, as reported for the Ca2+ pump from higher eukaryotic cells. In this report we show that plasma membrane vesicles from Leishmania mexicana possess a Ca(2+)-ATPase with characteristics which are similar to that reported by us for other trypanosomatids. Thus, the enzyme has a high Ca2+ affinity which is further increased upon addition of calmodulin. The maximal velocity is also increased by calmodulin...

Phosphorylation of proteins in virulent promastigotes from Leishmania major

Protein kinases are present in the plasma membrane of the human parasite Leishmania. A marked increase in enzyme activity has been detected as cultures entered into the stationary phase of growth. Since avirulent parasites can be separated from virulent forms by the peanut agglutinin (PNA), we have examined the change in the protein kinase activity of L. major during growth in vitro and the difference in phosphorylation with virulent promastigotes (PNA-) of L. major. Marked similarities were found between the phosphorylation patterns of the logarithmic and stationary phase promastigotes of L. major. On the other hand, when the phosphorylation pattern of those proteins, shared by both the metacyclic (PNA-) promastigotes and the stationary phase cells, was examined, a marked increase in both the total number of phosphoproteins and the extent of their phosphorylation was observed in PNA-. Both the increase in protein kinase activity in the stationary phase parasites and the marked changes in phosphorylation in the highly infective promastigotes, may provide a clue as to the adaptative mechanism which enable promastigotes to survive within the vertebrate host

Extracellular dephosphorylation in the parasite, leishmania major / Desfosforilacion extracelular en el parasito, leishmania major

Leishmania major promastigotes were analyzed for the presence of protein phosphatase activity in intact cells and membrane-enriched fractions. Parasite phosphoprorylated in live cells with {*-32P} adenosine 5'-triphosphate (ATP) and an edogemous leishmanial ectokinase, were dephosphorylated by endogenous protein phosphatase like activity in intact cells and membrane-rich fractions. An alkaline phosphatase-like activity was also identified using the artificial substrate, p-nitrophenyl phosphate (pNPP). This activity was localized on the extracellular membrane of intact parasite, as well as in the particulate fraction of lysed cells. The phosphatase activity measure using pNPP had inhibition properties and a pH profile between protein phosphatases and general alkaline phosphatase. This study supports the observation that there is extracellular protein phosphorylation/dephosphorylation in L.major which may play a significant role in host-cell-parasite recognition and infection