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Abstract

The effect of interferon-g (rIFN-g) in the control of Toxoplasma gondii and Leishmania donovani replication in cells of human origin, is due, in part, to the induction of indoleamine 2,3-dioxygenase (INDO), enzyme that degrades the essential amino acid tryptophan to kynurenine. Ceravolo e cols. (1996) showed that the treatment with rIFN-g had no effect on the replication of T. cruzi (Y strain) in human fibroblasts. The authors showed that rIFN-g induced significant amount of INDO mRNA in the human fibroblasts and that T. cruzi was not sensible to tryptophan degradation catalyzed by this enzyme. In this study we have investigated the participation of tryptophan metabolism in the resistance of T. cruzi to rIFN-g stimulation in human fibroblasts. The INDO activity of the cells was determinated by high-performance liquid chromatography (HPLC). The levels of tryptophan and kynurenine were analyzed in acid-soluble supernatants of fibroblasts activated by rIFN-g and infected by T. cruzi. Infected cells showed levels of kynurenine lower than the non infected ones. The presence of the parasites reduced in 35% the levels of kynurenine in the supernatants of the activated cells. This reduction was time-dependent with the parasitecell contact. The addition of soluble protein extract of tissue culture trypomastigotes reduced in 47% the production of the kynurenine in cells stimulated with rIFN-g. These results suggest that the parasite may be able to produce a partial inhibition of the enzyme INDO. This inhibiton may explain part of the T. cruzi resistance to tryptophan degradation induced by rIFN-g in human fibroblasts.Supported by CNPq, Papes/Fiocruz and Fapemig. BC-2 TRYPANOSOMA CRUZI GENOME PROJECT: CONSTRUCTION OF METACYCLIC AND TISSUE CULTURE-DERIVED TRYPOMASTIGOTE CDNA LIBRARIESFernandes M, Soares MB*, Bonaldo MF*, Rondinelli E, Ürményi TP Instituto de Biofísica Carlos Chagas Filho, CCS, Universidade Federal do Rio de Janeiro, 21949-900 Rio de Janeiro, RJ, Brasil *The University of Iowa, Iowa City, IA, USA A joint effort of several laboratories is in progress to map and sequence the entire Trypanosoma cruzi genome. Complete genomic nucleotide sequences allow whole genome studies, which include large scale genome organization, sistematic knockout to determine gene function, characterization of the complete set of expressed sequences of a given cell type (sometimes called a transcriptome) and evolutionary comparisons between organisms. Genome projects are greatly aided by the sistematic, large-scale sequencing of randomly picked clones of cDNA libraries, which generates expressed sequence tags (ESTs) for the mapping effort and facilitates gene discovery. We have constructed four directionally cloned, oligo(dT)-primed cDNA libraries of poly(A)+ RNA from clone CL Brener epimastigotes: two standard, non-normalized libraries, a library enriched with abundant clones and a normalized cDNA library. The frequency of individual cDNA clones in a normalized library are brought within a narrow range, thereby greatly increasing the...