Study of Trypanosoma cruzi epimastigote cell death by NMR-visible mobile lipid analysis.
ABSTRACT Cell death mechanisms in Trypanosoma cruzi have not been disclosed in detail though different conventional techniques have been used in the classification of parasite-cell death type. Nuclear magnetic resonance (NMR) has successfully been used as a tool to evaluate the onset of apoptosis in a number of higher eukaryote-cell models analysing the ratio of CH(2)/CH(3) integration from the visible mobile lipids (VML). Surprisingly, this versatile non-invasive spectroscopy technique has never been employed with this purpose in T. cruzi. In the present study it is shown that under different parasite death-conditions the ratio CH(2)/CH(3) varied drastically. Thus, T. cruzi epimastigotes in apoptotic conditions increase significantly this ratio while in necrotic as well as in autophagic situations the parasites maintain the VML, CH(2)/CH(3) ratio, in normal values. Additionally, other VML markers commonly used in these studies, such as the change in the region of methyl-choline moiety, -N(+)(CH(3))(3), exhibited different particular patterns according to the type of cell death. Our results suggest that the (1)H NMR-VML technique is an adequate tool to discriminate different T. cruzi death pathways.
- SourceAvailable from: Victor M. Mikhailenko[show abstract] [hide abstract]
ABSTRACT: Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH(2) at 1.3 ppm) to methyl (CH(3) at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance ((1)H NMR). We studied CH(2) and CH(3) resonances in cultured cell lines treated with etoposide and fludarabine or bioflavonoid quercetin. Etoposide treatment (10 microM, 18 h) resulted in 3.3 fold increase of the CH(2)/CH(3) signal intensity ratio and 6.4 fold decrease in choline signal of MT4 cells. Incubation of Namalwa cells with fludarabine (3 microM, 72 h) increased the CH(2)/CH(3) signal intensity ratio by 2.4 fold and choline resonance intensity was unchanged. Quercetin treatment (30 microM, 1.5 month) increased CH(2)/CH(3) ratio by 2.1 fold. Necrotic cell death upon ethanol (20%) or DMSO (30%) treatment did not change the CH(2)/CH(3) signal intensity ratio. (1)H NMR-based study of mobile lipid domains is sensitive for detection of early engagement into apoptosis.Cell Biology International 02/2005; 29(1):33-9. · 1.64 Impact Factor
Article: Necrotic death as a cell fate.[show abstract] [hide abstract]
ABSTRACT: Organismal homeostasis depends on an intricate balance between cell death and renewal. Early pathologists recognized that this balance could be disrupted by the extensive damage observed in internal organs during the course of certain diseases. This form of tissue damage was termed "necrosis", derived from the Greek "nekros" for corpse. As it became clear that the essential building block of tissue was the cell, necrosis came to be used to describe pathologic cell death. Until recently, necrotic cell death was believed to result from injuries that caused an irreversible bioenergetic compromise. The cell dying by necrosis has been viewed as a victim of extrinsic events beyond its control. However, recent evidence suggests that a cell can initiate its own demise by necrosis in a manner that initiates both inflammatory and/or reparative responses in the host. By initiating these adaptive responses, programmed cell necrosis may serve to maintain tissue and organismal integrity.Genes & Development 02/2006; 20(1):1-15. · 12.44 Impact Factor
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ABSTRACT: We report here the combined application of (1)H magic angle spinning (MAS) and high-resolution NMR spectroscopy and pattern recognition methods to study the effects of a model toxin (D-galactosamine) in liver spheroid cultures. (1)H NMR spectra of metabolic profiles of spheroids showed closer similarities to intact liver spectra than those of isolated hepatocytes, suggesting their superiority as an in vitro model system. Batches of spheroids were prepared from male Sprague Dawley rat livers and incubated in control hepatocyte medium or medium containing D-galactosamine (4 or 20 mM) for 4 or 24 h. Intact spheroids were packed into rotors and analyzed using MAS-NMR spectroscopy or homogenized and analyzed using conventional (1)H NMR spectroscopy. Principal components analysis, (PCA), of the NMR data revealed separation of control and D-galactosamine-treated spheroids based on changes in the concentrations of the triglycerides and elevations in cholesterol and esters. The absence of cholesterol in hepatocytes and the relative under-representation of the lipid resonances offer an important advantage of spheroids over hepatocytes for the (1)H NMR studies of fatty liver. Orthogonal signal correction (OSC) was used as a data filter to remove non-dose-dependent variation from the NMR spectra, improving the classification of treated spheroids and controls. This work shows that useful metabolic information can be obtained on drug toxicity by the use of combined MAS-NMR and high-resolution NMR of liver spheroids and that such studies may enhance the validation of in vitro techniques against in vivo models for metabolic profiling.Chemical Research in Toxicology 12/2002; 15(11):1351-9. · 3.67 Impact Factor