No description available.

No description available.

Malaria causes nearly 3 million deaths annually. The parasite species responsible for the most lethal forms of malaria is P. falciparum (Miller et.al., 2002). Its destructive effect is most evident in the developing African countries, which lack the infrastructure and financial support to effectively control the disease. The only viable means of control at this stage is the use of antimalarial chemotherapy and –prophylaxis, but these drugs are losing their effectivity due to parasite resistance. This underlines the need for new, safe, efficient and cheap drugs as a solution to the African malaria problem. Within the validated folate metabolic pathway of P. falciparum, the identification of three new genes has provided new options for drug inhibition (Lee et.al., 2001). One of these genes encodes the bifunctional dihydrofolate synthase-folylpolyglutamate synthase (DHFS-FPGS), which is unique to P. falciparum (Salcedo et.al., 2001). When compared to human folylpolyglutamate synthase (FPGS), the parasite enzyme is an attractive drug target for selective inhibition due to the additional DHFS activity and low sequence similarity. However, to assess the value of DHFS-FPGS as a drug target and rationally design new drugs against the enzyme, large amounts of enzyme are needed for activity studies and structural determination. The heterologous expression of malaria genes often result in low expression levels, due to its high A+T content and codon bias. To circumvent this problem, a modified P. falciparum dhfs-fpgs, adapted to E.coli codon preferences and with a lower A+T content was synthesised in this study for increased expression. A two-step overlap-extension PCR method was optimised for the synthesis of the 1586bp dhfs-fpgs from only 1 pmol each of partially overlapping oligonucleotides. The use of partially overlapping oligonucleotides, lower amounts of starting material and fewer PCR cycles cut the costs of gene synthesis and the optimisation increased PCR efficiency, when compared to other gene synthesis reports (Carpenter et al., 1999; Zhang et al., 2002). The correct sequences could be obtained from the sequencing of as little as three clones. The successfully constructed dhfs-fpgs gene was expressed in a variety of E. coli expression hosts and vector systems. In all the systems, expression levels of the synthetic gene were much higher than for the native P. falciparum gene. Functional complementation of a DHFS-FPGS deficient E. coli strain verified that the DHFS and FPGS activities were encoded by the synthetic gene, that complementation was achieved to a greater extent than for native P. falciparum dhfs-fpgs and that a synthetic tagless and C-terminal Histidine-tagged DHFS-FPGS had the highest levels of DHFS and FPGS activity. Preliminary purification studies for these two constructs were performed for optimised enzyme isolation, to be followed by activity assays. These optimisations will also serve as basis for future large-scale isolation strategies to obtain sufficient amounts of protein for the structural determination of the enzyme, which would be vital to drug target verification, drug development and –design, thus paving the way for a new generation of antifolate malaria therapy. / Dissertation (MSc(Biochemistry))--University of Pretoria, 2006. / Biochemistry / unrestricted

No description available.

Malaria remains one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite. / Thesis (PhD (Biochemistry))--University of Pretoria, 2002. / Biochemistry / unrestricted

Malaria is a devastating parasitic disease in humans caused by species in the genus Plasmodium. With over 100 million cases and at least 1.5 million fatalities each year, the disease accounts for 4-5% of all fatalities in the world. A recent increase in the number of malaria cases in South Africa has imposed severe costs on the economy and public health. Immunity to malaria is a multi-component system involving both B and T celllymphocytes. Pc96 is a 96 kDa antigen identified in the mouse malaria model Plasmodium chabaudi adami. It is known to be associated with the outer membrane of mouse erythrocytes infected with the parasite and has shown protective roles in mice challenged with P. chabaudi adami. A specific T cell clone has been identified that adoptively provides protection to athymic mice infected with P. chabaudi adami. Antibodies raised against Pc96 identified proteins that induced the proliferation of the protective T cell clones. At least four other antigens of different species of. malaria share at least one cross-reactive epitope. In an attempt to identify a Plasmodiumfalciparum homologue ofPc96, the amino-acid sequence was used in a BLAST search of the P. falciparum genome database, identifying a 403 kDa protein with a high degree of homology to Pc96. Sequence alignments indicated a region spanning 90 amino acids in Pf403 that overlaps the Pc96 amino acid sequence. A 178 kDa protein in P. yoelii yoelii (Pyy178) was shown to be highly similar to Pc96. Tvcell epitope prediction programs identified putative T cell epitopes in Pc96 which appear to be conserved in Pf403 and Pyy178. A casein kinase IT phosphorylation site was also identified in this region and is conserved in both sequences. PCR primers were designed to amplify regions of the MAL3P6.11 gene coding for Pf403 from P.falciparum genomic DNA. An 817 bp region in the MAL3P6.11 gene was amplified. This codes for the region ofPf403 that shows high homology to Pc96 and contains the conserved T cell epitopes and casein kinase phophorylation site. A BamHI site was incorporated into the forward primer to facilitate in-frame ligation with cloning vectors. The PCRproduct obtained was verified by restriction analysis using HindIII and EcoRI sites within the fragment. The 817 bp peR product was cloned into the pMOSBlue vector using a blunt-endedPCR cloning kit, and transformed into MOSBlue competent cells. Recombinants were identified using the uIV complementation system, and verified by PCR, plasmid DNA isolation, and restriction digestion analysis. The insertDNA in pMOSBlue was cut out with BamHI and sub-cloned into the BamHI site in the pMAL-C2x expression vector. Sequencing ofthe construct confirmed the identity of the cloned insert and showed the sequence to be in frame with the malE gene coding for maltose binding protein (MBP). The fusion protein, MBP-Pf32 .5, was induced and expressed as a 75 kDa protein comprising ofthe 32.5 kDa region ofPf403, and MBP (42.5 kDa) and was detected by anti-MBP antibodies, by western blotting. This recombinant protein has many applications for further studies involving the characterisation of the Pf403 protein, and the determination of possible roles that the protein may have in stimulating an immune response during human malaria infections. / Thesis (M.Sc.) - University of Natal, Pietermaritzburg, 2003.

Petroleum ether, dichloromethane, and 80% ethanol extracts of 15 plant species collected in Nigeria were screened for in vitro antibacterial, anti-inflammatory and antimalarial activities. Antibacterial activity was tested using the agar diffusion method, while the minimum inhibitory concentrations (MIC) of the active extracts were determined using the microtitre serial dilution method. Most antibacterial activity detected was against Gram-positive bacteria with Staphylococcus aureus being the most susceptible. The highest activity was found in petroleum ether and dichloromethane leaf extracts of Mallotus oppositifolius; petroleum ether, dichloromethane and ethanolic root extracts of Newbouldia laevis; and ethanolic root extracts of Morinda lucida and Canthium subcordatum. Against the Gram-negative bacterium Escherichia coli, the highest activity was found in dichloromethane leaf extracts of Newbouldia laevis, ethanolic root extracts of Phyllanthus amarus, Mallotus oppositifolius, and Canthium subcordatum. A total of 60 plant extracts were screened for antiplasmodial activity. A chloroquine sensitive strain of Plasmodium falciparum (D10) was used. In the assay, the parasite lactate dehydrogenase (pLDH) activity was used to measure parasite viability. About 11 extracts showed promising activity with an IC₅₀ ranging from 2.5 to 13.4 µg/ml. The petroleum ether leaf extract of Hyptis suaveolens had the highest activity (IC₅₀ = 2.5 µg/ml). The cyclooxygenase (COX-1 and COX-2) assays were used to test for anti-inflammatory activity. All the plant species, with the exception of Hedranthera barteri and Picralima nitida showed anti-inflammatory activity. Apart for a few ethanolic extracts, all the activities were recorded with petroleum ether and dichloromethane extracts. Employing bioassay-guided activity fractionation, an antibacterial anthraquinone identified as emodin was isolated from ethanolic root extract of Senna occidentalis. Although this compound had been isolated from other sources, this was the first report of isolation from Senna occidentalis. Using a similar approach a novel antimalarial diterpenoid was isolated from the petroleum ether leaves extract of Hyptis suaveolens. It had IC₅₀ of 0.1 µg/ml. This new compound is worthy of further investigation and may act as an important lead compound for future antimalarial drugs. / Thesis (Ph.D.)-University of KwaZulu-Natal, 2005.