Mitochondria are specialized organelles that control energy metabolism and also activate a multiplicity of pathways that modulate cell proliferation and mitochondrial biogenesis or, conversely, promote cell arrest and programmed cell death by a limited number of oxidative or nitrative reactions. Nitric oxide (NO) regulates oxygen uptake by reversible inhibition of cytochrome oxidase and the production of superoxide anion from the mitochondrial electron transfer chain. In this sense, NO produced by mtNOS will set the oxygen uptake level and contribute to oxidation-reduction reaction (redox)-dependent cell signaling. Modulation of translocation and activation of neuronal nitric oxide synthase (mtNOS activity) under different physiologic or pathologic conditions represents an adaptive response properly modulated to adjust mitochondria to different cell challenges.
Trypanosoma cruzi, the etiological agent of Chagas' disease, is a parasitic protozoan with a digenetic life cycle involving an insect vector and a mammalian host. The parasite undergoes major morphological and biochemical changes during the different stages of its life cycle. The epimastigote form is noninfective and proliferates extracellularly in the insect gut where it differentiates into metacyclic trypomastigotes, which can then infect the mammalian host cells and replicate intracellularly after transforming into amastigotes [1][2][3][4].Epimastigotes from different wild-type strains of T. cruzi are able to grow continuously in vitro in a rich culture medium [5], but proliferation stops after a few passages in a semidefined medium, which contains only traces of polyamines [6,7]. T. cruzi remain viable for several weeks in the defined medium and are able to resume normal growth only upon the addition of exogenous polyamines to the culture [7]. These results confirm previous reports from our and other laboratories indicating that T. cruzi epimastigotes are unable We have previously demonstrated that wild-type Trypanosoma cruzi epimastigotes lack arginine decarboxylase (ADC) enzymatic activity as well as its encoding gene. A foreign ADC has recently been expressed in T. cruzi after transformation with a recombinant plasmid containing the complete coding region of the oat ADC gene. In the present study, upon modulation of exogenous ADC expression, we found that ADC activity was detected early after transfection; subsequently it decreased to negligible levels between 2 and 3 weeks after electroporation and was again detected 4 weeks after electroporation. After this period, the ADC activity increased markedly and became expressed permanently. These changes of enzymatic activity showed a close correlation with the corresponding levels of ADC transcripts. To investigate whether the genome organization of the transgenic T. cruzi underwent any modification related to the expression of the heterologous gene, we performed PCR amplification assays, restriction mapping and pulse-field gel electrophoresis with DNA samples or chromosomes obtained from parasites collected at different time-points after transfection. The results indicated that the transforming plasmid remained as free episomes during the transient expression of the foreign gene. Afterwards, the free plasmid disappeared almost completely for several weeks and, finally, when the expression of the ADC gene became stable, two or more copies of the transforming plasmid arranged in tandem were integrated into a parasite chromosome (1.4 Mbp) bearing a ribosomal RNA locus. The sensitivity of transcription to a-amanitin strongly suggests involvement of the protozoan RNA polymerase I in the transcription of the exogenous ADC gene.Abbreviations ADC, arginine decarboxylase; G418, geneticin; ODC, ornithine decarboxylase; PFGE, pulse-field gel electrophoresis.
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