De las redes de informacion celular a la medicina molecular digital / From cellular information networks to digital molecular medicine

La transformacion que experimenta la biotecnologia con la informacion biologica a escala de sistemas es de tal magnitud y profundidad, que solo a traves de redes globales de cooperacion es posible realizar investigacion competitiva. Las aplicaciones que ahora emergen, aparte de las biofarmaceuticas y agroindustriales que ya entran en su madurez, dan lugar a una nueva medicina molecular, predictiva, preventiva, personalizada, participativa y accesible en forma digital. Una combinacion sin precedentes de tecnicas genomicas y proteomicas permite seguir el curso de la expresion de todos los genes y proteinas de cada individuo asi como la vida social de tales moleculas informacionales, que es donde radican los eventos cruciales para la prediccion del riesgo y la deteccion precoz de enfermedades comunes. Esa sociedad de bioinformacion a escala molecular sigue el mismo tipo de patrones de redes complejas propias de los sistemas sociales y ecologicos, que se repiten en forma fractal a toda escala incluyendo las redes neuronales, genomicas, proteomicas y metabolomicas. A cada nivel de complejidad aparece la firma indeleble de la autoorganizacion, redes entrelazadas cuyos nodos se conectan entre si de acuerdo con una ley de potencia con una distribucion desigual de enlaces entre los nodos, donde muy pocos dominan todas las conexiones y, por ende, la red completa. La importancia practica de esos super nodos, genes y proteinas, se aprecia a partir de 2007 con mas de una docena de publicaciones que demuestran la asociacion de variaciones genomicas con enfermedades comunes. El valor predictivo de estas asociaciones genomicas, no necesariamente deterministas, se incrementa notablemente al combinarlas con otros factores de riesgo de tipo clinico y metabolico haciendo posible una determinacion mas precisa del riesgo, la prevencion y atencion personalizada de la enfermedad.

Cloning and sequence analysis of a trypanosoma cruzi alpha-butulin cDNA

A cDNA clone derived from the Trypanosoma cruzi alpha-tubulin gene was isolated and sequenced (Tc alpha tub; L37345). Tc alpha tub revealed an 87.79 percent and an 85.36 percent identity with the DNA sequence of T. brucei and Leishmania, respectively. This clone was used to study, by Northern blots, alpha-tubulin gene expression in epimastigotes, cell-cultured derived trypomastigotes and extracellular amastigotes. alpha-tubulin MRNA levels were the same in epimastigotes and trypomastigotes, however, there was a drastic decrease in amastigotes. This clone could be useful to elucidate the regulatory mechanisms of alpha-tubulin gene expression during the differentiation of T. cruzi

cAMP receptor protein from Trypanosoma cruzi: purification and cloning of a short sequence of the corresponding cDNA

cAMP is involved in the differentiation of Trypanosoma cruzi, the causative agent of Chagas' disease. cAMP levels are elevated in the infective, non-dividing metacyclic trypomastigote stage, with respect to the non-infective, proliferative, epimastigote stage. In both stages three is a cAMP receptor protein (CARPT), with unique properties that differentiate it from the regulatory subunits of the cAMP-dependent protein kinase (RI and RII). The CARPT from T. cruzi epimastigotes was purified using ion-exchange chromatography, affinity chromatography and gel filtration. After the final step of purification, two protein bands were obtained, p89 and p70, corresponding to the intact CARPT and its proteolytic product. These two CARPT polypeptides were utilized to prepare polyclonal antibodies in rabbits. Previous results from our laboratory showed that CARPT cross-reacts with polyclonal antibodies prepared against the regulatory subunit (RII) of the cAMP-dependent protein kinase (PKA). As expected from these results, the anti-CARPT antibody recognized purified RII protein in an ELISA assay. The anti-CARPT antibodies were used for immunoblot analyses of epimastigote lysates. The two bands corresponding to the CARPT (p89 and p70), as well as a p40 band, were recognized. Immunoscreening of a T. cruzi lambda ZAP cDNA library with these anti-CARPT polyclonal antibodies yielded one positive clone (pBSCARPT) which contained a 540 bp insert. Northern analyses using the pBSCARPT clone as a probe, showed a 5.2 kb mRNA band in epimastigotes, which were grown in culture from 2 to 10 days in LIT medium. Sequence analyses of the 540 bp insert have failed to show homology to other gene sequences in the database.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential turn-over of beta-tubulin during the cell differentiation of Trypanosoma cruzi

We investigated the expression of beta-tubulin during the differentiation of non-infective epimastigotes to infective metacyclics of Trypanosoma cruzi to underlay some of the regulatory mechanisms of the gene expression in this pathogenic parasite. Given the strong evolutionary conservation of tubulin, it was possible to study its translational and transcriptional products with heterologous probes. Quantitative Western blotting with specific monoclonal antibodies against beta-tubulin revealed an increase in the relative amounts of this protein in metacyclics with respect to epimastigotes. Pulse-chase experiments with radioactive methionine followed by immunoprecipitation and polyacrylamide gel electrophoresis showed that beta-tubulin has a slower degradation in metacyclics, which may contribute to its relative higher abundance in these parasite forms. In contrast with these results, both in vitro translation of poly (A+) mRNA in a wheat germ system and Northern blots of total and poly (A+) mRNA with a heterologous DNA probe from Leishmania enriettii, revealed a significant decrease (5 fold) in the specific transcripts of beta-tubulin in the metacyclics with respect to epimastigotes. It thus appeared that after differentiation of T. cruzi the translational machinery for a key protein such as beta-tubulin is shut off by a decrease in its specific message. The protein levels of this protein are maintained, however, by a compensatory mechanism that involves a slower turn-over of the synthesized protein

Estudios moleculares sobre el receptor de AMP ciclico del tripanozoma cruzi / Molecular studies about ciclic AMP receptor of the trypanosoma cruzi

Como parte de nuestros estudios sobre las señales intracelulares potencialmente importantes, para la diferenciación celular del Trypanosoma cruzi, determinados varias de las propiedades de las proteínas ligantes de AMP cíclico (cAMP) de epimastigotes así como su posible asociación con proteínas quinasas dependientes de nicleóticos cíclicos. Los resultados señalan lo siguiente: (1) La actividad ligante de cAMP en extractos crudos de T. cruzi, subestima hasta por 10 veces cuando se comparan los valores obtenidos por el método de Gilman que es el más generalmente utilizado, en relación a los resultados de la técnica de Doskeland y Ueland. Una respuesta similar a la T. cruzi también se obtuvo con la actividad ligante de cAMP presente en homogenizados de otros Tripanosomatidae, tales como T. brucei, L. tropica, y C. luciliae. Este comportamiento distingue a las proteínas ligantes de cAMP de Tripanosomatidae de sus contraparte de otros eucariotes inferiores o superioes, cuyas actividades son subestimadas solo 2 veces