Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles

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dc.contributor.advisorSmith, Peter Jen_ZA
dc.contributor.authorSaliba, Kevin Johnen_ZA
dc.date.accessioned2017-12-14T09:35:03Z
dc.date.available2017-12-14T09:35:03Z
dc.date.issued1997en_ZA
dc.description.abstractDue to the development of drug-resistance by Plasmodium falciparum, the utilisation of chloroquine, a cheap and effective antimalarial has become limited. The mechanism of chloroquine-resistance is, at best, unresolved. This thesis describes an investigation of chloroquine accumulation in pure and intact Plasmodium falciparum isolated digestive vacuoles, the site of chloroquine accumulation and action. Marker enzymes and gel electrophoresis were used to demonstrate purity, and electron microscopy to verify integrity of isolated vacuoles. Using this method, vacuoles were isolated in a yield high enough to enable characterisation of chloroquine accumulation in this organelle in terms of time-, temperature-, Mg²⁺-, pH-, ATP- and other nucleotide-dependence. The chloroquine accumulating capabilities of vacuoles isolated from chloroquine-resistant and chloroquine-sensitive parasites were compared. At an external chloroquine concentration of 100 and 250nM vacuoles isolated from two chloroquine-sensitive strains accumulated significantly more chloroquine (± 3 x) than those isolated from three of the four chloroquine-resistant strains of Plasmodium falciparum tested. Although it is often suggested that the parasite digestive vacuole is involved in the mechanism of chloroquine-resistance, this is the first direct evidence to suggest this. Vacuolar proton pump inhibitors, proton gradient dissipaters, P-glycoprotein ATPase- and drug transport-inhibitors, weak bases, and structural analogues of chloroquine were used to examine the driving force of chloroquine accumulation in the isolated food vacuole. A pH gradient between the vacuole and cytoplasm is necessary to retain chloroquine in this organelle, but a chloroquine transport mechanism appears to be the main driving force in chloroquine accumulation. A polyclonal antibody directed at Pgh1, a Plasmodium falciparum homologue of P-glycoprotein, confirmed its presence on the vacuole, but was unable to inhibit chloroquine accumulation in isolated vacuoles. This thesis also shows that agents, such as verapamil, which are able to reverse chloroquine-resistance by increasing chloroquine accumulation in parasitised erythrocytes, are unable to increase chloroquine accumulation in the isolated vacuole, suggesting the involvement of an alternate site for the reversal of chloroquine-resistance.en_ZA
dc.identifier.apacitationSaliba, K. J. (1997). <i>Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles</i>. (Thesis). University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Pharmacology. Retrieved from http://hdl.handle.net/11427/26631en_ZA
dc.identifier.chicagocitationSaliba, Kevin John. <i>"Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles."</i> Thesis., University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Pharmacology, 1997. http://hdl.handle.net/11427/26631en_ZA
dc.identifier.citationSaliba, K. 1997. Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles. University of Cape Town.en_ZA
dc.identifier.ris TY - Thesis / Dissertation AU - Saliba, Kevin John AB - Due to the development of drug-resistance by Plasmodium falciparum, the utilisation of chloroquine, a cheap and effective antimalarial has become limited. The mechanism of chloroquine-resistance is, at best, unresolved. This thesis describes an investigation of chloroquine accumulation in pure and intact Plasmodium falciparum isolated digestive vacuoles, the site of chloroquine accumulation and action. Marker enzymes and gel electrophoresis were used to demonstrate purity, and electron microscopy to verify integrity of isolated vacuoles. Using this method, vacuoles were isolated in a yield high enough to enable characterisation of chloroquine accumulation in this organelle in terms of time-, temperature-, Mg²⁺-, pH-, ATP- and other nucleotide-dependence. The chloroquine accumulating capabilities of vacuoles isolated from chloroquine-resistant and chloroquine-sensitive parasites were compared. At an external chloroquine concentration of 100 and 250nM vacuoles isolated from two chloroquine-sensitive strains accumulated significantly more chloroquine (± 3 x) than those isolated from three of the four chloroquine-resistant strains of Plasmodium falciparum tested. Although it is often suggested that the parasite digestive vacuole is involved in the mechanism of chloroquine-resistance, this is the first direct evidence to suggest this. Vacuolar proton pump inhibitors, proton gradient dissipaters, P-glycoprotein ATPase- and drug transport-inhibitors, weak bases, and structural analogues of chloroquine were used to examine the driving force of chloroquine accumulation in the isolated food vacuole. A pH gradient between the vacuole and cytoplasm is necessary to retain chloroquine in this organelle, but a chloroquine transport mechanism appears to be the main driving force in chloroquine accumulation. A polyclonal antibody directed at Pgh1, a Plasmodium falciparum homologue of P-glycoprotein, confirmed its presence on the vacuole, but was unable to inhibit chloroquine accumulation in isolated vacuoles. This thesis also shows that agents, such as verapamil, which are able to reverse chloroquine-resistance by increasing chloroquine accumulation in parasitised erythrocytes, are unable to increase chloroquine accumulation in the isolated vacuole, suggesting the involvement of an alternate site for the reversal of chloroquine-resistance. DA - 1997 DB - OpenUCT DP - University of Cape Town LK - https://open.uct.ac.za PB - University of Cape Town PY - 1997 T1 - Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles TI - Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles UR - http://hdl.handle.net/11427/26631 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/26631
dc.identifier.vancouvercitationSaliba KJ. Chloroquine accumulation in Plasmodium falciparum isolated digestive vacuoles. [Thesis]. University of Cape Town ,Faculty of Health Sciences ,Division of Clinical Pharmacology, 1997 [cited yyyy month dd]. Available from: http://hdl.handle.net/11427/26631en_ZA
dc.language.isoengen_ZA
dc.publisher.departmentDivision of Clinical Pharmacologyen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.subject.otherPharmacologyen_ZA
dc.titleChloroquine accumulation in Plasmodium falciparum isolated digestive vacuolesen_ZA
dc.typeDoctoral Thesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnamePhDen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceThesisen_ZA
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