Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22 degrees C) to the temperature of the mammalian host (37 degrees C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34 degrees C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [alpha 32P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms.

Phytochemical study of Chromolaena hirsuta (Hook. & Arn.) R. King & H. Robinson crude extract (collected in Furnas, MG-Brasil) produced fifteen flavonoids, two triterpenes and five steroids. IR, UV,(1)H and (13)C NMR spectroscopy and GC analysis were used for the identification of these compounds. The dichloromethanic and ethanolic crude extracts (flowers and leaves) and six flavonoids isolated from this specie have been assayed for antiprotozoal activity against tripomastigote forms of Trypanosoma cruzi and promastigote forms of Leishmania amazonensis. The crude extracts significantly reduced the viability of T. cruzi and Leishmania amazonensis, and the six flavonoids showed considerable antiproliferative effect of development of two parasites evaluated. This is the fi rst report of antiprotozoal activity of extracts of C. hirsuta.

Leishmaniasis is a neglected disease caused by a trypanosomatid protozoan of the genus Leishmania. Most drugs used to treat leishmaniasis are highly toxic, and the emergence of drug‐resistant strains has been observed. Therefore, new therapeutic targets against leishmaniasis are required. Several isoprenoid compounds, including dolichols or ubiquinones, have been shown to be important for cell viability and proliferation in various trypanosomatid species. Here, we detected the biosynthesis of tocopherol in Leishmania (L.) amazonensis promastigotes in vitro through metabolic labelling with [1‐(n)‐3H]‐phytol. Subsequently, we confirmed the presence of vitamin E in the parasite by gas chromatography–mass spectrometry. Treatment with usnic acid or nitisinone, inhibitors of precursors of vitamin E synthesis, inhibited growth of the parasite in a concentration‐dependent manner. This study provides the first evidence of tocopherol biosynthesis in a trypanosomatid and suggests that inhibitors of the enzyme 4‐hydroxyphenylpyruvate dioxygenase may be suitable for use as antileishmanial compounds. Database The amino acid sequence of a conserved hypothetical protein [Leishmania mexicana MHOM/GT/2001/U1103] has been deposited in GenBank ()

Leishmania braziliensis strain M2903 was adapted for growth and serially maintained as amastigotes at 34 degrees C in modified UM-54 medium, with growth curves exhibiting typical log and stationary phases. In late passages, amastigote growth took place in the absence of supplementary haemin and was unaffected when the initial medium pH was adjusted between 5.4 and 6.3. In contrast to promastigotes, which were elongated and exhibited very long free flagella endowed with the paraflagellar rod (PFR), axenic amastigotes were rounded to ovoid and displayed a short flagellum restricted to the pocket area. The absence of PFR in axenic amastigotes was confirmed in Western blots and confocal immunofluorescence microscopy, by lack of reactivity with mAb 1B10. The antibody, which specifically labelled the paraflagellar structure, recognized a 70/72 kDa doublet in Trypanosoma cruzi epimastigotes and two 70/74 kDa related proteins in L. braziliensis promastigotes. Surface 125I-labelling experiments identified promastigote-specific components (> 100, 74, 45/47 and 28 kDa) and at least 1, a 76 kDa polypeptide was specific for the amastigote stage. While axenic amastigotes were agglutinated by both peanut (PNA) and Lens culinaris (LCA) agglutinins, respectively at 50 and 12.5 micrograms/ml, promastigotes were not agglutinated by PNA and agglutinated in the presence of LCA at concentrations of 100 micrograms/ml and higher. Axenic amastigotes infected rat bone marrow-derived macrophages and were avidly taken up by J774 cells, from which numerous organisms, able to proliferate at 34 degrees C in UM-54 medium, could be recovered 48 h later.

Apoptosis and/or programmed cell death have been described in examples ranging from fungi to man as gene-regulated processes with roles in cell and tissue physiopathology. These processes require the operation of an intercellular communicating network able to deliver alternative signals for cells with different fates and is thus considered a prerogative of multicellular organisms. Promastigotes from Leishmania (Leishmania) amazonensis, when shifted from their optimal in vitro growth temperature (22 degrees C) to the temperature of the mammalian host (37 degrees C), die by a calcium-modulated mechanism. More parasites die in the presence of this ion than in its absence, as detected by a colorimetric assay based on the activity of mitochondrial and cytoplasmic dehydrogenases which measures cell death, independently of the process by which it occurs. A heat shock, unable to induce detectable parasite death (34 degrees C for 1 h), is able to significantly raise the concentration of intracellular free calcium in these cells. Heat-shocked parasites present ultrastructural and molecular features characteristic of cells dying by apoptosis. Morphological changes, observed only in the presence of calcium, are mainly nuclear. Cytoplasmic organelles are preserved. Heat shock is also able to induce DNA cleavage into an oligonucleosomal ladder detected in agarose gels by ethidium bromide staining and autoradiography of [alpha 32P]ddATP-labeled fragments. These results indicate that death by apoptosis is not exclusive of multicellular organisms.