Glucose uptake in the mammalian stages of Trypanosoma cruzi

Trypanosoma cruzi, the agent of Chagas` disease, alternates between different morphogenetic stages that face distinct physiological conditions in their invertebrate and vertebrate hosts, likely in the availability of glucose. While the glucose transport is well characterized in epimastigotes of T cruzi, nothing is known about how the mammalian stages acquire this molecule. Herein glucose transport activity and expression were analyzed in the three developmental stages present in the vertebrate cycle of T cruzi. The infective trypomastigotes showed the highest transport activity (V(max) = 5.34 +/- 0.54 nmol/min per mg of protein: K(m) = 0.38 +/- 0.01 mM) when compared to intracellular epimastigotes (V(max) = 2.18 +/- 0.20 nmol/min per mg of protein; K(m) = 0.39 +/- 0.01 mM). Under the conditions employed no transport activity could be detected in amastigotes. The gene of the glucose transporter is expressed at the mRNA level in trypomastigotes and in intracellular epimastigotes but not in amastigotes, as revealed by real-time PCR. In both trypomastigotes and intracellular epimastigotes protein expression could be detected by Western blot with an antibody raised against the glucose transporter correlating well with the transport activity measured experimentally. Interestingly, anti-glucose transporter antibodies showed a strong reactivity with glycosome and reservosome organelles. A comparison between proline and glucose transport among the intracellular differentiation forms is presented. The data suggest that the regulation of glucose transporter reflects different energy and carbon requirements along the intracellular life cycle of T cruzi. (C) 2009 Elsevier B.V. All rights reserved.

Multiple stages of detergent-erythrocyte membrane interaction-A spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate- and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9 mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5 mM detergent. Between 3.2 and 10 mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10 mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs: the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5 mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111). (C) 2010 Elsevier B.V. All rights reserved.