The Uptake of GABA in Trypanosoma cruzi

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na⁺, K⁺, and H⁺ on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na⁺ dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na⁺/GABA symporter energized by Na⁺‐exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.     

Effect of 3,4-ethylenedioxy-extension of thiophene core on the DNA/RNA binding properties and biological activity of bisbenzimidazole amidines

Novel bisbenzimidazoles (4–6), characterized by 3,4-ethylenedioxy-extension of thiophene core, revealed pronounced affinity and strong thermal stabilization effect toward ds-DNA. They interact within ds-DNA grooves as dimmers or even oligomers and agglomerate along ds-RNA. Compounds 4–6 have shown moderate to strong antiproliferative effect toward panel of eight carcinoma cell lines. Compound 5 displayed the best inhibitory potential and in equitoxic concentration (IC₅₀ = 1 × 10^?6 M) induced accumulation of cells in G2/M phase after 48 h of incubation. Fluorescence microscopy showed that 5 entered into live HeLa cells within 30 min, but did not accumulate in nuclei even after 2.5 h. Compound 5 inhibited the growth of Trypanosome cruzi epimastigotes (IC₅₀ = 4.3 × 10^?6 M).     

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.     

The Trypanosoma cruzi proteins TcCox10 and TcCox15 catalyze the formation of heme A in the yeast Saccharomyces cerevisiae

Trypanosoma cruzi, the etiologic agent for Chagas’ disease, has requirements for several cofactors, one of which is heme. Because this organism is unable to synthesize heme, which serves as a prosthetic group for several heme proteins (including the respiratory chain complexes), it therefore must be acquired from the environment. Considering this deficiency, it is an open question as to how heme A, the essential cofactor for eukaryotic CcO enzymes, is acquired by this parasite. In the present work, we provide evidence for the presence and functionality of genes coding for heme O and heme A synthases, which catalyze the synthesis of heme O and its conversion into heme A, respectively. The functions of these T. cruzi proteins were evaluated using yeast complementation assays, and the mRNA levels of their respective genes were analyzed at the different T. cruzi life stages. It was observed that the amount of mRNA coding for these proteins changes during the parasite life cycle, suggesting that this variation could reflect different respiratory requirements in the different parasite life stages.     

Air pollution-associated fly ash particles induce fibrotic mechanisms in primary fibroblasts

Air pollution is associated with a variety of respiratory and cardiovascular disorders, including fibrosis. To understand the possible molecular mechanisms underlying this observation, we examined the effect of particulate matter on primary fibroblasts, the key regulators of the extracellular matrix. Fly ash collected in an experimental waste incinerator was used as model particles for fine and ultrafine pollution components. Brief treatment of fibroblasts isolated from adult male Wistar rat hearts with fly ash triggered the immediate formation of intracellular reactive oxygen species (ROS). Using phospho-specific antibodies we observed activation of p38 MAP kinase, p44/42 MAP kinase (ERK1/2) and p70(S6) kinase. Prolonged incubation with fly ash increased the expression of collagen 1 and TGF-beta1, but decreased mRNA levels of MMP9 and TNF-alpha. Cell proliferation was inhibited at high concentrations of fly ash. An increase in the level of advanced glycation endproduct (AGE) modification of various cellular proteins after long-term treatment of cultured fibroblasts with fly ash was observed. The results of our study demonstrate that direct activation of fibroblasts by combustion-derived particles is a mechanism that may contribute to the adverse health effects of particulate air pollution.     

Sedentary life style of Neotropical sedge wrens promotes song imitation

To what extent has the style of song development among songbirds coevolved with other life history strategies? Among Cistothorus wrens in North America, it seems that sedentary or site-faithful habits of marsh wrens, C. palustris, favour song imitation, but seminomadic habits of sedge wrens, C. platensis, favour song improvisation, whereby each male generates a large but unique song repertoire. In this study, we tested whether more sedentary populations of sedge wrens in the Neotropics would imitate songs. At our primary study site near Cartago, Costa Rica, breeding birds were colour-banded during 1995 and 1996, and follow-up surveys revealed that the birds remained at this site the year round. Extensive tape recording and analysis of songs showed that males had large song repertoires (200-300+ songs), and that many songs were shared among neighbouring males. In addition, males only 27 km distant, at La Pastora, used different songs. Furthermore, matched countersinging, in which two males answer each other with identical song types, was recorded near Brasilia, in Brazil. The sharing of songs among permanent neighbours, microgeographical variation in song, and matched countersinging can be achieved only through song imitation, thus revealing a striking difference in the style of song development among different populations of the sedge wren. In the Neotropics, having predictable neighbours throughout life appears to have favoured song imitation, so that individuals can interact using a common, learned code typical of the local population; among more mobile populations in North America, however, individuals improvise large repertoires of species-typical songs, thereby enabling singing males to communicate with any individual, no matter what the population of origin. Strategies of song development must correlate with life history features, and further surveys are needed to make sense of the great diversity of singing behaviours among songbirds. Copyright 1999 The Association for the Study of Animal Behaviour.     

Arginine kinase overexpression improves Trypanosoma cruzi survival capability

Arginine kinase catalyzes the reversible transphosphorylation between adenosine diphosphate (ADP) and phosphoarginine, which is involved in temporal and spatial adenosine triphosphate (ATP) buffering. Here we demonstrate that the homologous overexpression of the Trypanosoma cruzi arginine kinase improves the ability of the transfectant cells to grow and resist nutritional and pH stress conditions. The stable transfected parasites showed an increased cell density since day 10 of culture, when the carbon sources became scarce, which resulted 2.5-fold higher than the control group on day 28. Additional stress conditions were also tested. We propose that arginine kinase is involved in the adaptation of the parasite to environmental changes.     

Mutational analysis of the human FATE gene in 144 infertile men

The FATE gene maps to Xq28 where one case of a translocation breakpoint has been found in an infertile man. Moreover, the FATE promoter contains a putative SF-1-binding site, and FATE has been proposed as representing a target gene of SF-1 in testicular development or germ cell differentiation. This study presents a complete mutational screening of the FATE gene in a random group of 144 infertile males. Four polymorphisms and two mutations were found. Three of the polymorphisms, viz., 741CT, 905AC, and 3985CT, occurred in exon 5 and intron 2 and did not alter the deduced polypeptide. One polymorphism resulted in the conservative amino acid exchange, A10 V, in 16.0% of the patients. This substitution occurred with similar frequencies in the control groups, indicating that the mutation does not affect fertility in men or women. The two mutations caused the non-conservative amino acid substitutions S125R (patient 1) and I34T (patient 2). A family study (patient 1) revealed, however, that S125R was inherited and that a fertile male family member carried the mutation. Patient 2 did not have relevant family members who could be examined. Thus, this study has shown that only 1.4% of infertile men have mutations in the FATE gene, and that some of these mutations do not singly cause infertility. Hence, FATE may not play an important role in the disease-state of infertile men attending fertility clinics. However, FATE mutations cannot be excluded as being a contributing factor in some cases of male infertility.     

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.