Plasmodium falciparum: genome wide perturbations in transcript profiles among mixed stage cultures after chloroquine treatment.

A genomic approach was taken to study the effect of chloroquine (CQ) on Plasmodium falciparum cultures in multiple cell states, following short and long exposures to drug at varying concentrations. Six hundred genes from numerous functional groups were responsive to CQ amongst all cell states assayed in a micro-array analysis; however, the amplitude of fold-change was low in the majority of cases. Moreover, alterations in specific, functionally related cascades could not be discerned, leading us to believe there is no single signature response to CQ at the transcript level in P. falciparum. Instead, cell cycle changes appear to have a more pronounced effect on gene expression; only a fraction of the drug responsive loci (approximately 5%) were shared between two separate starting cultures that varied in staging profile in the current study, as well as a previous published analysis using SAGE technology [Gunasekera, A.M., Patankar, S., Schug, J., Eisen, G.,Wirth, D.F., 2003. Drug-induced alterations in gene expression of the asexual blood forms of Plasmodium falciparum. Molecular Microbiology 50, 1229-1239]. These findings are important to report, given the striking contrast to similar studies in other model eukaryotic organisms.

Rapid test for the evaluation of the activity of the prodrug hydroxymethylnitrofurazone in the processing of Trypanosoma cruzi messenger RNAs.

No fully effective treatment has been developed since the discovery of Chagas' disease by Carlos Chagas in 1909. Since drug-resistant Trypanosoma cruzi strains are occurring and the current therapy is effectiveness in the acute phase but with various adverse side effects, more studies are needed to characterize the susceptibility of T. cruzi to new drugs. Many natural and/or synthetic substances showing trypanocidal activity have been used, even though they are not likely to be turned into clinically approved drugs. Originally, drug screening was performed using natural products, with only limited knowledge of the molecular mechanism involved in the development of diseases. Trans-splicing, which is unusual RNA processing reaction and occurs in nematodes and trypanosomes, implies the processing of polycistronic transcription units into individual mRNAs; a short transcript spliced leader (SL RNA) is trans-spliced to the acceptor pre-mRNA, giving origin to the mature mRNA. In the present study, permeable cells of T. cruzi epimastigote forms (Y, BOL and NCS strains) were treated to evaluate the interference of two drugs (hydroxymethylnitrofurazone - NFOH-121 and nitrofurazone) in the trans-splicing reaction using silver-stained PAGE analysis. Both drugs induced a significant reduction in RNA processing at concentrations from 5 to 12.5 microM. These data agreed with the biological findings, since the number of parasites decreased, especially with NFOH-121. This proposed methodology allows a rapid and cost-effective screening strategy for detecting drug interference in the trans-splicing mechanism of T. cruzi.

Cloning and characterization of a novel serine/threonine protein phosphatase type 5 from Trypanosoma brucei.

Reversible protein phosphorylation is essential for the regulation of numerous cellular functions and differentiation. The haemo-flagellated parasitic protozoan Trypanosoma brucei, the causative agent for African trypanosomiasis undergoes various stages of cellular differentiation during its digenetic life cycle. A complete cDNA of a unique serine/threonine phosphatase type five (TbPP5) was cloned and characterized from T. brucei. TbPP5 contains an open reading frame of 1416 bp that encodes a protein of about 53 kDa and exists as a single copy gene in the T. brucei genome. The deduced amino acid sequence showed 45-48% overall identity and 60-65% similarity with protein phosphatase 5's (PP5) from different species. Analysis of the primary sequence revealed that TbPP5 contains three TPR motifs at the N-terminal region (amino acid residues 7-107) while the phosphatase catalytic domain occurs in the C-terminal region (amino acid residues 210-410). A TbPP5 cDNA hybridized with a transcript of 2.5 kb which is present at similar levels in the procyclic and the bloodstream forms. However, the level of expression of the TbPP5 protein (52 kDa) detected by an antibody developed against a recombinant protein produced in E. coli was about 2-fold higher in the procyclic than the bloodstream form. The TbPP5 transcript level gradually decreased in cells grown in the logarithmic phase to the stationary phase in culture. Moreover, 18 h serum starvation of the procyclic forms decreased the level of the specific transcript about 3-fold suggesting that this protein may play a role during the active growth phase of the organism. The recombinant protein showed phosphatase activity which was stimulated about 2.6-fold by arachidonic acid with half-maximal activity at 75 microM. Indirect immuno-fluorescence of permeabilized cells revealed that the protein is localized in the cytosol and the nucleus This is the first report for the identification of a type 5 serine/threonine protein phosphatase in an ancient eukaryote such as T. brucei.

Action of new organometallic complexes against Leishmania donovani.

The action of 16 newly synthesized metal complexes having the general structure cis-Pt-(II)-Xn-Ln have been tested in vitro against the promastigote forms of Leishmania donovani. The metal complexes at 24 h and maximum dosages inhibited growth from 0%, e.g. in cis-Pt-nifurtimox, to 100%, e.g. in cis-Pt-(2,3,4,5,6-pentafluoroaniline)2Br2 or cis-Pt-pentamidine-I2. A study of the cytotoxicty of these latter complexes on the phagocytic cell line J-774 showed neither high cytotoxicity nor cytolysis. At the maximum dosage after 24 h of permanent contact with the cells (extreme, non-physiological conditions), cytolysis did not exceed 30%. For most of the compounds, cytolysis ranged from 0%, for cis-Pt-oxamniquine-Cl2 to 27.7%, for cis-Pt-pentamidine-I2. The compound cis-Pt-(2,3,4,5,6-pentafluoroaniline)2-Br2 caused up to 1.4% cytolysis under the above conditions. Parasites exposed to cis-Pt-pentamidine-I2 showed notably reduced DNA, RNA and protein synthesis, unlike those exposed to other compounds. Parasites examined by electron microscopy showed effects mainly on the nucleus, though in some cases the mitochondria were affected, altering the internal membranes of the cytoplasmic organelles. The in-vivo activity of the complex cis-Pt-guanethidine-Cl2 was evaluated in parasitized Wistar rats, in which the number of amastigotes per gram of spleen was reduced by 75% compared with controls.