The addition of growth factors and vitamins enhances goat embryonic development in vitro. However, few attempts have been reported trying to identify supplementation regimens for oocyte maturation or embryo culture with additive properties. The present report was aimed to evaluate if retinoids [0.3 μM retinyl acetate (RAc) and 0.5 μM 9-cis-retinoic acid (RA)] supplementation during goat oocyte maturation and retinoids and/or 50 ng mL–1 IGF-I during embryo culture synergically enhanced embryonic development while diminishing the incidence of apoptosis. All combinations of RAc and RA treatment produced blastocysts with similar efficiencies, while IGF-I enhanced embryos yields irrespectively of retinoid addition. Moreover, retinoids and IGF-I supplementation showed similar caspase activity or DNA fragmentation indexes in blastocysts. In conclusion, supplementation with retinoids and IGF-I during goat embryo culture enhances blastocysts development without synergic reduction of apoptosis.

Cyclosporin A (CsA) specifically inhibits the mitochondrial permeability transition pore (mPTP). Opening of the mPTP, which is triggered by high levels of matrix [Ca2+] and/or oxidative stress, leads to mitochondrial dysfunction and thus to cell death by either apoptosis or necrosis. In the present study, we analysed the response of Trypanosoma cruzi epimastigote parasites to oxidative stress with 5 mm H2O2, by studying several features related to programmed cell death and the effects of pre-incubation with 1 μ m of CsA. We evaluated TcPARP cleavage, DNA integrity, cytochrome c translocation, Annexin V/propidium iodide staining, reactive oxygen species production. CsA prevented parasite oxidative stress damage as it significantly inhibited DNA degradation, cytochrome c translocation to cytosol and TcPARP cleavage. The calcein-AM/CoCl2 assay, used as a selective indicator of mPTP opening in mammals, was also performed in T. cruzi parasites. H2O2 treatment decreased calcein fluorescence, but this decline was partially inhibited by pre-incubation with CsA. Our results encourage further studies to investigate if there is a mPTP-like pore and a mitochondrial cyclophilin involved in this protozoan parasite.

Chagas disease chemotherapy is based on drugs that exhibit toxic effects and have limited efficacy, such as Benznidazole. Therefore, research into new chemotherapeutic agents from natural sources needs to be exploited. Apis mellifera venom consists of many biologically active molecules and has been reported to exhibit remarkable anti-cancer effects, often promoting an apoptosis-like death phenotype. This study demonstrates that A. mellifera venom can affect the growth, viability and ultrastructure of all Trypanosoma cruzi developmental forms, including intracellular amastigotes, at concentrations 15- to 100-fold lower than those required to cause toxic effects in mammalian cells. The ultrastructural changes induced by the venom in the different developmental forms led us to hypothesize the occurrence of different programmed cell death pathways. Autophagic cell death, characterized by the presence of autophagosomes-like organelles and a strong monodansyl cadaverine labelling, appears to be the main death mechanism in epimastigotes. In contrast, increased TUNEL staining, abnormal nuclear chromatin condensation and kDNA disorganization was observed in venom-treated trypomastigotes, suggesting cell death by an apoptotic mechanism. On the other hand, intracellular amastigotes presented a heterogeneous cell death phenotype profile, where apoptosis-like death seemed to be predominant. Our findings confirm the great potential of A. mellifera venom as a source for the development of new drugs for the treatment of neglected diseases such as Chagas disease.

The fate of cells in the endosperm of developing wheat kernels was investigated under normal conditions and upon premature slow drying on the cut ear. To follow the changes in membrane integrity and cellular ultrastructure, an electron spin resonance (ESR) spin probe technique and low temperature scanning electron microscopy (LTSEM) were used. ESR data indicated that during development, the relative amount of cells with intact membranes decreased and became almost zero at the stage of mass maturity, i.e. several days before the onset of maturation drying. This suggests that the death of starchy endosperm cells is a developmental phenomenon rather than one induced by water loss. Even at 8 days after anthesis (daa), early in the differentiation phase, a considerable proportion of endosperm cells had already lost plasma membrane integrity. Comparison of ESR data with LTSEM micrographs revealed that the loss of plasma membrane integrity occurred early in the starch accumulation process, from which it was concluded that the programmes of starch accumulation and developmental death are simultaneously switched on. When the differentiation into starchy endosperm was arrested by premature slow drying of kernels on the cut ear, meristematic cells (aleurone initials) acquired desiccation tolerance as assessed by the combination of the spin probe technique and LTSEM. In contrast, fast drying caused immediate death of these cells. Thus, meristematic endosperm cells have the competence to acquire desiccation tolerance. This occurs upon premature slow drying or during normal differentiation into aleurone cells, but is lost when the cells differentiate into starchy endosperm.

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro – activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Until recently it had generally been assumed that apoptosis and other forms of programmed cell death evolved during evolution of the metazoans to regulate growth and development in these multicellular organisms. However, recent research is adding strength to the original phenotypic observations described almost a decade ago which indicated that some parasitic protozoa may have evolved a cell death pathway analogous to the process described as apoptosis in metazoa. Here we explore the implications of a programmed cell death pathway in the African tsetse-transmitted trypanosomes.

Programmed cell death or apoptosis occurs in many tissues during normal development and in the normal homeostasis of adult tissues. Apoptosis also plays a significant role in abnormal development and disease. Increased interest in apoptosis and cell death in general has resulted in the development of new techniques and the revival of old ones. Each assay has its advantages and disadvantages that can render it appropriate and useful for one application, but inappropriate or difficult to use in another. Understanding the strengths and limitations of the assays would allow investigators to select the best methods for their needs.

Although programmed cell death (PCD) has been associated with multicellular organisms, there have been more reports of its presence in some protozoans. Our study shows the existence of PCD in an intestinal protozoan, Blastocystis hominis. Light and electron microscopy, biochemical and flow cytometry studies showed apoptosis-like death in B. hominis cells exposed to a cytotoxic monoclonal antibody (MAb 1D5). B. hominis cells displayed key morphological and biochemical features of apoptosis, namely, nuclear condensation and in situ fragmentation, reduced cytoplasmic volume, some externalization of phosphatidylserine and maintenance of plasma membrane integrity. No oligonucleosomal DNA laddering was observed in gel electrophoresis. This study supports earlier observations that the cellular machinery that is required to carry out PCD may have existed before the advent of multicellularity. Our study also ascribes a novel function for the B. hominis central vacuole in apoptosis; it acts as a repository where apoptotic bodies are stored before being released into the extracellular space.

A previous study had indicated that scavengers of reactive oxygen species (ROS) delayed cell death (the hypersensitive response (HR)) triggered in epidermal cells of intact, resistant, cowpea (Vigna unguiculata (L.) Walp) leaves by the monokaryotic stage of the cowpea rust fungus (Uromyces vignae Barclay race 1). This HR had been monitored by cell autofluorescence, which occurs after protoplast collapse. In the present study, when cytoplasmic disorganization was used to monitor cell death more directly, ROS-scavengers, superoxide dismutase, catalase, horseradish peroxidase, and desferal-Mn(IV) had no effect on HR development. Cytological staining for superoxide or hydrogen peroxide generation also did not reveal the presence of ROS before or during the early stages of the HR, but did, as in the previous study, suggest a role in the autofluorescence and browning of invaded cells that occur following protoplast collapse. Staining of plant mitochondria with nitroblue tetrazolium, possibly attributable to increased dehydrogenase activity but not superoxide generation, occurred transiently around invasion hyphae (monokaryotic stage of the fungus) or haustoria (dikaryotic stage) of the fungus as they entered a cell in the susceptible or resistant cultivar. Around invasion hyphae in epidermal cells in resistant plants, this staining diminished as cytoplasmic streaming stopped, and gradually disappeared as cell death progressed. These data are consistent with other evidence that rust fungi initially negate non-specific defensive responses in both resistant and susceptible cells as part of the establishment of biotrophy. They also suggest that the HR in the cowpea–cowpea rust fungus pathosystem is not triggered by an oxidative burst.