http://hdl.handle.net/10386/70 Tue, 21 May 2013 15:38:46 GMT 2013-05-21T15:38:46Z http://hdl.handle.net/10386/806 Title: Proteomic approaches to profiling of cysteine proteases expressed in leaves and root nodules during natural senescence of the soybean plant Authors: Karumazondo, Rumbidzai Patience Abstract: Soybean is one of the most cultivated legume plants in developing countries. Nodule senescence is a major limitation in producing high yields of soybean as it coincides with the pod filling stage. Delaying nodule senescence could be a way of increasing the yield of soybean therefore determination of the role of cysteine protease in soybean is of vital importance. In this study, soybean plants were grown under controlled temperature and light conditions. Leaves and root crown nodules were collected at 4, 6, 10, 12 and 16 weeks of age. In a comparative 1-dimensional SDS-PAGE analysis of soybean nodule proteomes as the plant matured, it showed differences in proteins expressed as shown by different banding patterns with less variation between the younger soybean nodule extracts (4, 6 and 10 weeks old) as compared to the older ones (12 and 16 weeks old). As determined by azocasein assay and protease zymography, the protease activity of the nodule extracts generally decreased with an increase in the age of the nodules whereas that of the leaves increased as the plants grew older. Cysteine proteases in the soybean nodule extracts readily cleaved the Z-Arg-Arg-AMC substrate with the highest activity shown in the younger nodules as compared to the older ones. In the leaf extracts, cysteine protease activity increased with age of the leaves. DCG-04, a biotinylated irreversible inhibitor, proved to be an effective label in profiling of activity of cysteine proteases in 1-dimensional and 2-dimensional systems. The labelling was inhibited specifically by cysteine protease inhibitor, E-64. In root nodules, the DCG-04 probing demonstrated that the expression of cysteine proteases is higher in early stages of development of the soybean nodules as compared to the later stages whereas in the leaves, there is higher expression of cysteine proteases in the old leaves (16 weeks). Using 2-dimensional polyacrylamide gel electrophoresis, five cysteine protease isoforms were visualised with the size ranging from approximately 25 to 30 kDa and a pI range of 4-6. In older nodules (12 and 16 weeks old) the higher pI isoforms are down-regulated with the 26 kDa and pI 4.5 protease being the predominant isoform. Affinity precipitation of the cysteine proteases yielded a strong band with the size of about 26 kDa. All assays used show that while in leaves, the expected trend of high expression of cysteine proteases in senescing leaves is observed, in soybean nodules the expression of cysteine proteases decreases with senescence. There is, therefore, no correlation between senescence and cysteine proteases in nodules. The highly expressed cysteine protease in young nodules could play a developmental or regulatory role during the early stages of development. Description: Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2011 Sat, 01 Jan 2011 00:00:00 GMT http://hdl.handle.net/10386/806 2011-01-01T00:00:00Z http://hdl.handle.net/10386/790 Title: Development of chromatographic bioseparations based on lectins and supermacroporous affinity cryogels Authors: Raletjena, Moloko Ivonne Abstract: Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about the changes in the expression of plasma membrane glycoproteins during apoptosis, and whether these changes could be used for detection of apoptosis. A critical element of this study was to purify lectins from crude homogenate on glycoprotein-cryogel affinity matrices, and later use the lectins to detect changes on the cell surface of apoptotic cells. Pterocarpus angolensis seed lectin was extracted and fractionated using ammonium sulphate precipitation. The 60 % ammonium sulphate pellet was dissolved in saline azide and purified using Sephadex G-75 affinity chromatography. A 28 kDa lectin was retarded within the column and appeared as a short and broad peak on the chromatogram. Traditionally, Sephadex G-75 column are used predominantly for size exclusion, in this study, the column was used in a non-traditional way for affinity chromatography, as the purified protein is able to bind sugar moieties existing in the structure of Sephadex G-75. A single-step purification of P. angolensis seed lectin was achieved by directly applying unclarified P. angolensis crude extract to the pAAm-cryogel using fetuin as the affinity ligand. Pterocarpus angolensis extract fractionated into 2 peaks, which revealed a highly concentrated band on SDS-PAGE. The results also revealed that an increased binding of the lectin to the fetuin-cryogel matrices was also dependent on the time of incubation. This study suggested very low capacities of the cryogels for the protein due to low coupling sites on the matrix. Taking into account that lectins serve as invaluable tools in diverse area of biomedical research, this study proposed using specific plant lectins to follow the expression of plasma membrane glycoproteins during programmed cell death. Treatment of HL-60 cells with lithium and actinomycin D confirmed a time- and dose-dependent inhibition of proliferation and a decrease in proliferation, which suggest cell death of the treated cells. The observed cell death was further investigated for cellular and biochemical hallmark features of apoptosis, which has shown preferential binding of annexin V-FITC to phosphatidylserine and low molecular DNA ladder. Several FITC labelled lectins were used to detect changes in cell surface glycosylation that accompany apoptosis. This study xvii has shown amongst several FITC-labelled lectins that T. vulgaris lectin could intensively stain the membrane area of apoptotic cells suggesting that the expression of N-acetylglucosamine was significantly increased during actinomycin D induced apoptosis of HL-60 cells. Binding was shown to be specific because it was blocked by the corresponding inhibitory sugar. Thus, the method described in this study could be suitable for the detection of very early stages of apoptosis by recognizing the cell surface carbohydrates of apoptosis. Description: Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2012 Sun, 01 Jan 2012 00:00:00 GMT http://hdl.handle.net/10386/790 2012-01-01T00:00:00Z http://hdl.handle.net/10386/757 Title: The anti-proliferative, antioxidative and anti-inflammatory properties of the D2 fraction and HPLC semi-purified sub-fractions of dicerocaryum senecioides Authors: Chokoe, Pirwana Kholofelo Abstract: Dicerocaryum senecioides is a crawling herb that is found growing mostly in sandy areas of southern and south-eastern Africa and its small, hairy leaves have been used over the years as food, shampoo, and for treatment of various ailments. In this study, the dichloromethane (D2) fraction was prepared from a crude methanol extract of D. senecoides leaves, and its effect on the proliferation of RAW 264.7 murine macrophages was investigated. Treatment of the macrophages with the extract resulted in a dose- and time-dependent decrease in cell viability as determined by the MTT assay and real time cell analysis. Cytotoxicity of the D2 fraction on the macrophages was demonstrated to be due to apoptosis by staining the cells with DAPI nucleic acid stain. Anti-inflammatory activity of D2 fraction on RAW cells was determined by evaluating intracellular ROS production by the DCFH-DA fluorescent assay. Cells treated with the D2 fraction and stimulated with PMA were found to have a lower fluorescence intensity compared to untreated, stimulated cells; thus mimicking the response observed in the resting cells. The percentage fluorescence in untreated, stimulated cells doubled, while no significant change was observed in the D2-treated cells. The effect of the D2 fraction on iNOS activity was also assessed. The fraction reduced the NO synthesised by iNOS in cells treated with the D2 fraction and stimulated with LPS dose-dependently. The D2 fraction was further fractionated by semi-preparative HPLC; and thin layer chromatography was used to analyse phytocompounds of the 96 HPLC sub-fractions as well as to screen these sub-fractions for anti-oxidative activity. Sub-fractions 1-7 and 33-39 showed an intensely pronounced DPPH-scavenging compound and this scavenging ability was confirmed by a quantitative DPPH assay that provided parallel results. The reducing potential of the sub-fractions was assessed by evaluating their Fe3+-reducing ability through the FRAP assay. Sub-fractions 1-7 and 33-39 displayed remarkable reducing potential. Taken together with the DPPH-scavenging activity, these findings suggest that HPLC sub-fractions 1-7 and 33-39 possess a compound(s) with impressive antioxidant activity. These findings merit the D2 fraction as an extract that can be used to control chronic inflammation as it does not only inhibit free radical production, but also scavenges excessive ROS and has the ability to induce apoptosis in the macrophages responsible for dysregulated production of the free radicals. The extract also has commendable chemoprotective and chemotherapeutic potential as it demonstrated pro-apoptotic activity along with prevention of excess free-radical production. Description: Thesis (M.Sc. (Biochemistry)) --University of Limpopo, 2011 Thu, 01 Sep 2011 00:00:00 GMT http://hdl.handle.net/10386/757 2011-09-01T00:00:00Z http://hdl.handle.net/10386/751 Title: Interactions of ethanol and chloroquine in the protein-mulnourished male sprague dawley rats : haemotological, biochemical and testicular effects Authors: Mbajiorgu, Ejikeme Felix Abstract: Refere to document Description: Thesis (PH.D. (Medical Sciences)) --University of Limpopo, 2010 Fri, 01 Jan 2010 00:00:00 GMT http://hdl.handle.net/10386/751 2010-01-01T00:00:00Z