Trypanosoma brucei: Maintenance of concentrated suspensions of bloodstream trypomastigotes in vitro using continuous dialysis for measurement of endocytosis |
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Authors: | AH Fairlamb IBR Bowman |
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Institution: | Department of Biochemistry, The Medical School, University of Edinburgh, Teviot Place, Edinburgh, Scotland, United Kingdom |
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Abstract: | A continuous dialysis technique capable of maintaining concentrated suspensions of bloodstream Trypanosoma brucei (up to 4 × 108/ml) at 25 C for 2 hr without loss of viability has been developed in order to measure endocytosis under controlled conditions in vitro. Using this technique, the kinetics and mechanism of uptake of the metabolically inert macromolecule, 125I-labelled polyvinylpyrrolidone (125I-PVP), have been investigated. Binding to the plasma membrane and the rate of uptake of 125I-PVP from the extracellular medium by the trypanosome are both decreased by the addition of unlabelled PVP and human serum albumin. A mechanism for uptake of 125I-PVP by a combination of fluid-phase and adsorptive endocytosis from the flagellar pocket of the trypanosome is proposed. In the presence of serum albumin and unlabelled PVP, endocytosis of 125I-PVP occurs in the fluid phase only, with endocytic indices of 14.5 ± 0.9 and 54.1 ± 11.3 nl/hr/mg protein in vitro at 25 C and in vivo at 37 C, respectively. |
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Keywords: | Protozoa parasitic Endocytosis Flagellar pocket Nutrition Drug targeting |
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