PREPARATION AND IN VITRO CHARACTERIZATION OF RESEALED ERYTHROCYTES CONTAINING TMR-DEXTRAN FOR DETERMINATION OF HEMOGLOBIN UPTAKE AND TRANSFER BY THE MALARIA PARASITEAbstract
The hemoglobin uptake and transport by the malaria parasite, Plasmodium falciparum are preferable targets for antimalarial drug development. There is a need for alternative approaches to investigate the endocytic process of live and intact cells under non-disruptive conditions as previous findings were mostly based on morphological analysis of thin-section electron micrographs. In the present study, resealed erythrocytes containing TMR-dextran, the host cell cytoplasm marker, were prepared and characterized. Fresh erythrocytes were collected in EDTA anticoagulant vacutainer tubes, which proved to maintain a normal biconcave disk shape of the cells. Using a modified hypotonic dilution method, washed erythrocytes were lysed in 3 volumes of hemolysis buffer, which permitted the retention of 33.56 ± 7.84% of the original hemoglobin content of the cells. Resealed erythrocytes containing TMR-dextran were invaded by the parasites with similar efficiency to unlabeled cells, and able to support the parasite growth and development. Live cell fluorescence imaging of the endocytic process revealed the appearance of TMR-dextran-containing small endocytic vesicles with intense signals at early ring stage. These compartments coalesced in the early trophozoite stage to form a central digestive vacuole. A larger spherical structure within TMR-dextran labelled ring and trophozoite stage parasites was often observed, which is likely to resemble the previously described “Big Gulp”. It is therefore concluded that resealed erythrocytes incorporated with TMR-dextran, which were proved their biological applicability, can be a model for the endocytic study of P. falciparum.
N. H. Mohd-Zamri, N. J. Sinin and N. Abu-Bakar *
School of Health Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia.
12 September, 2016
06 November, 2016
07 December, 2016
01 March, 2017