Doctoral Degrees (Biochemistry)

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Anti-c-myc cholesterol-based lipoplexes: development, characterisation and evaluation as Onconanotherapeutic agents in vitro.
(2018) Habib, Saffiya.; Singh, Moganavelli.
Strategies aimed at inhibiting the expression of the c-myc oncogene could provide the basis for alternative cancer treatment. In this regard, silencing c-myc expression using small interfering RNA (siRNA) is an attractive option. However, the development of a clinically viable, siRNAbased, c-myc silencing system is largely dependent upon the design of an appropriate siRNA carrier that can be easily prepared. Nanostructures formed by the electrostatic association of siRNA and cationic lipid vesicles represent uncomplicated, well-recognised siRNA delivery systems. Therefore, this study has focused on traditional cationic liposomes as the foundation for the development of a simple, but effective anti-c-myc onconanotherapeutic agent. Novel liposome formulations contained equimolar quantities of the cytofectin, N,Ndimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09), and cholesterol (Chol); with or without 2 mol % pegylation. Liposomes which contained dioleoylphosphatidylethanolamine (DOPE) as the co-lipid were included for comparative purposes. Pegylated and non-pegylated MS09/Chol (1:1) suspensions were reproducibly prepared by lipid film hydration to give unilamellar vesicles that were stable for at least 10 months at 4 ˚C. Liposomes successfully bound siRNA to form lipoplexes of less than 200 nm in size, with zeta potentials between -16 and -44 mV. These assumed globular and bilamellar structures in which siRNA was partially protected. Although all formulations were well tolerated at ≤14 nM siRNA, pegylation severely inhibited siRNA delivery in cancer cell lines, MCF-7 and HT-29, which overexpress c-myc. The non-pegylated MS09/Chol (1:1) lipoplex, at the MS09:siRNA (w /w) ratio of 16:1, was most effectively taken up by MCF-7 and HT-29 cells, with negligible effect in non-transformed cells when applied at 12 nM siRNA. Lipoplexes directed against the c-myc transcript (anti-c-myc siRNA), mediated a dramatic reduction in c-myc mRNA and protein levels. This was accompanied by a loss of migratory potential and apoptotic cell death. Moreover, oncogene knockdown and anti-cancer effects were superior to that of a commercially available transfection reagent, Lipofectamine™ 3000. Although the DOPE-containing counterpart performed with iii comparable efficacy under standard in vitro conditions, it was incapable of siRNA delivery at physiological serum concentration. Hence, the anti-c-myc MS09/Chol (1:1) lipoplex reported exemplifies a straightforward anti-cancer agent that warrants further investigation in vivo.
Antibody-mediated inhibition of proteases of African trypanosomes.
(2006) Huson, Laura.; Coetzer, Theresa Helen Taillefer.
The protozoan parasites Trypanosoma congolense and T. vivax cause trypanosomosis in cattle. The major lysosomal cysteine proteinase of T. congolense, congopain, may contribute to pathogenesis of the disease, and antibody-mediated inhibition of this enzyme may contribute to mechanisms of trypanotolerance. Oligopeptidase B, a trypanosomal serine peptidase, is also a potential virulence factor in African trypanosomes because it is released into the host circulation by dead or dying parasites, where it retains catalytic activity due to the enzyme's insensitivity to serum protease inhibitors. The vaccine potential of the catalytic domain of congopain, C2, and oligopeptidase B complexed with 0'2-macroglobulin (0'2M) was evaluated by producing antibodies in rabbits. Inhibition of congopain and oligopeptidase B activity by these antibodies was assessed. The oligopeptidase B open reading frame from T. congolense and T. vivax was cloned and expressed in Escherichia coli, from which active recombinant enzymes were purified. These recombinant enzymes exhibited trypsin-like specificity for peptide substrates, cleaving on the carboxy side of basic amino acid residues such as arginine and lysine. Enzymes were found to be optimally active between pH 8 and 10, optimally stable at pH 6, and showed activation by reducing agents and sensitivity to ionic strength. The enzymes showed typical oligopeptidase B-like inhibitor profiles, except that they were not inhibited by thiol sensitive inhibitors such as iodoacetamide and Nethylmaleimide. High yields of bovine and rabbit 0'2M were isolated by a three-step procedure of fractionation by PEG 6000, and zinc chelate and Sephacryl S-300 HR chromatography. Congopain, its catalytic domain C2, papain and cathepsin L all cleaved the bait region of bovine 0'2M and became trapped inside the 0'2M molecule, where their activity against large molecular weight substrates was inhibited. C2 could thus be complexed with 0'2M directly or used to form C2-0'2M-oligopeptidaseB complexes for immunisation purposes. iv The catalytic domain of congopain, C2, was used to immunise rabbits either without adjuvant, as a water-in-oil emulsion with Freund's adjuvant, or in a complex with either bovine or rabbit U2M. Freund's adjuvant elicited the highest anti-C2 antibody response. However, the greatest inhibition, 65%, of C2 activity against Z-Phe-Arg-AMC was obtained with antibodies produced by rabbits receiving C2-U2Mcomplexes. In a second study, C2 and oligopeptidase B were used to immunise rabbits , either in alum, or complexed to bovine U2M. Anti-C2 antibody levels were highest in rabbits immunised with the free proteins in alum, whereas anti-oligopeptidase B antibody levels were comparable for each adjuvant system. Anti-oligopeptidase antibodies produced with alum gave 100% inhibition of oligopeptidase B activity. In contrast, antibodies produced against C2-u2M-oligopeptidase B complexes had little effect on oligopeptidase B activity. However, these antibodies inhibited 55% of C2 activity. Alum was a slightly less efficient adjuvant for C2 and 50% inhibition of C2 activity was observed. It appeared that immunisation of rabbits with C2 complexed to U2M resulted in the production of antibodies that were better able to neutralise the proteolytic activity of C2 and congopain in vitro than that with conventional adjuvants . The immunisation of C2 complexed to bovine u2-macroglobulin therefore has the potential to neutralise parasite congopain in vivo, and may contribute to an anti-disease vaccine against African trypanosomosis. Complexation of oligopeptidase B to u2M offers no benefit, since antibodies produced against this complex are not able to inhibit the activity of oligopeptidase B. Immunisation with oligopeptidase B in alum is sufficient to produce efficient enzyme-inhibiting antibodies in the context of an anti-disease vaccine against African trypanosomosis.
Antidiabetic and toxicological properties of some African medicinal plants used in the treatment of diabetes and its complications.
(2018) Erukainure, Ochuko Lucky.; Islam, Mohammad Shahidul.
This study investigated the antioxidant, antidiabetic and toxicity properties of antidiabetic medicinal plants comprising of Vernonia amygdalina, Cola nitida, Raffia palm (Raphia hookeri) wine, Phaseolus lunatus, Dacryodes edulis, and Clerodendrum volubile using in vitro, ex vivo, in silico and in vivo models. The leaves of V. amygdalina and D. edulis, as well as C. volubile flower were sequentially extracted with solvents of increasing polarity to yield ethyl acetate, ethanol and aqueous extracts. Cola nitida and V. amygdalina were infused in hot water to yield infusion extracts. Phaseolus lunatus was subjected to aqueous extraction to yield aqueous extract, while Raffia palm wine was concentrated to yield the concentrate. The extracts and concentrate were screened for their in vitro and ex vivo antioxidant activities, as well as their inhibitory effect on α-glucosidase, α-amylase and pancreatic lipase activities, and their ability to stimulate muscle glucose uptake and inhibit intestinal glucose absorption in vitro. The ethanol extracts of D. edulis, C. volubile and V. amygdalina were subjected to GC-MS analysis, while the aqueous extract of P. lunatus, palm wine concentrate and the infusions were analyzed with LC-MS to elucidate the active compounds that may be responsible for their bioactivities. The ethanol extracts of C. volubile and D. edulis were further subjected to liquid-liquid fractionation to yield the hexane, dichloromethane, ethyl acetate, butanol and aqueous fractions. These fractions were also assayed for their antioxidant and antidiabetic properties in vitro and ex vivo. The dichloromethane, ethyl acetate and butanol fractions were subjected to GC-MS analysis to elucidate their active compounds. The identified compounds were molecularly docked with the test enzymes in silico to further validate their bioactivities. The antidiabetic properties of palm wine concentrate, C. nitida infusion, and D. edulis butanol fraction were investigated in a type 2 diabetes rat model. The in vivo study revealed a potent hypoglycemic activity, with concomitant amelioration of oxidative stress in the serum, pancreas, testes and brain. This was further substantiated by the downregulation of Nrf2 expressions in the pancreas and brain. These results further validate the use and safety of these plants in diabetes management.
Antidiabetic properties of centella asiatica in type II diabetic rats.
(2016) Oyenihi, Ayodeji Babatunde.; Masola, Bubuya.; Mukaratirwa, Samson.
Abstract available in PDF file.
Antioxidative and antidiabetic effects of some African medicinal plants.
(2016) Mohammed, Aminu.; Islam, Mohammad Shahidul.
Three (3) medicinal plants [Aframomum melegueta K. Schum., Xylopia aethiopica (Dunal.) A. Rich. and Capsicum annuum L.] were selected based on their traditional uses in the treatment of diabetes in Africa. Various crude extracts and fractions from different parts of the plants were screened using several anti-oxidative and anti-diabetic tests in vitro. Most active fractions from each plant were used to examine in vivo anti-diabetic activity in type 2 diabetes (T2D) rat model. Additionally, possible bioactive compounds from most active extracts and fractions were analyzed by using GC-MS, TLC and NMR spectroscopy. The results showed that ethanolic extracts derived from the fruits of the plants demonstrated excellent anti-oxidative and anti-diabetic activities in vitro compared to other extracts from the same or different parts of these plants. After fractionation, ethyl acetate fraction from A. melegueta and acetone fractions from X. aethiopica and C. annuum exhibited strong radical scavenging (IC₅₀: 1-120 μg/mL) activity, inhibition of hemoglobin glycation (IC₅₀: 100-150 μg/mL), α-amylase (IC₅₀: 50-170 μg/mL) and α-glucosidase (IC₅₀: 40-87 μg/mL) activities hence were used for the in vivo study. The GC-MS analysis of the three (3) most active fractions revealed the presence of mostly phenolic compounds of 4-hydroxy-3-methoxyphenyl derivatives. Furthermore, the data of the in vivo study showed that oral intervention of the fractions (150 and 300 mg/kg bw) for 4 weeks demonstrated potent anti-diabetic actions via improving body weight gain, reducing feed and fluid intake and hyperglycemia, improving glucose tolerance ability, insulin sensitivity, amelioration of pancreatic β-cell histology and β-cell functions, improving dyslipidemia in a T2D rat model. Additionally, the pancreatic histopathological damages and other oxidative damages caused by the induction of diabetes were attenuated to near normal in the liver, kidney, heart and pancreas of the treated animals. The bioassay-guided fractionations lead to the isolation of 3 arylalkanes (6-paradol (1), 6-shagaol (2), and 6- gingerol (3)) and oleanolic acid (4) from A. melegueta fruits, when oleanolic acid (4) was the first to be isolated from A. melegueta. Moreover, 6-gingerol (3) and oleanolic acid (4) were similarly isolated for the first time from X. aethiopica fruits as well. These compounds have exhibited significant inhibitions against the α-amylase and α-glucosidase actions and thus are possible anti-diabetic agents and the anti-diabetic action of A. melegueta and X. aethiopica fruits is attributed to the presence of these compounds. This study also confirmed the use of these plants in African anti-diabetic traditional medicines by traditional healers. However, further clinical study is required to confirm these effects in human subjects.
Assessment of lysine damage during food processing.
(1985) Anderson, Trevor Ryan.; Quicke, George Venn.
The fluorodinitrobenzene (FONB), succinic anhydride (SA), dansyl chloride (DAN), dye-binding lysine (OBL), total lysine (TL), ninhydrin (NIN) and Tetrahymena lysine (TET) methods were compared for their ability to assess available lysine in soyaprotein heated in the absence or presence of glucose, lactose or xylose and in formaldehyde-treated lactalbumin. The reactive lysine methods showed comparable sensitivity to lysine damage in soyaprotein heated in the absence of sugar, the results indicating the presence of acid labile isopeptides and unidentified acid stable derivatives. Results for soyaprotein heated with glucose, lactose or xylose showed that the type of sugar and the extent of heat treatment has a strong influence on the progress of the Maillard reaction. Furthermore since fructoselysine (F-L) and lactulosyl-lysine (L-L) are colourless up to 30% loss of available lysine can occur without any change in product colour. The FONB method is the most sensitive for mildly damaged glucose-soya samples followed by DAN or OBL, SA and TL whereas for mildly damaged lactose-soya samples the order is OBL, FONB, SA, TL and DAN. For severely damaged samples the DAN or SA methods were the most sensitive followed by OBL, FONB and TL. Formylation of lactalbumin occurred more readily at higher formaldehyde concentrations. Exposure time had less effect while pH (5 and 9) had no effect. Methylene derivatives reached maximum levels sooner than the methylol compounds. Lysine and tyrosine but not histidine formed methylene bridges while tyrosine was found to condense with free formaldehyde during acid hydrolysis raising questions as to the interpretation of similar studies reported in the literature. The FONB, OBL and DAN methods were all very sensitive to this type of damage with the NIN and TL methods being less sensitive and the SA method being completely unsuitable. The TET assay is unsuitable for 'early' Maillard damage since at low sample-N levels growth is stimulated by its ability to utilise unavailable F-L and L-L while at higher N-levels growth is inhibited. No single method is most suitable for all types of damage. Furthermore, all except DAN and DBL are either too long, rather complicated, require expensive equipment or involve the use of dangerous chemicals. The DAN method appears promising but the problem of converting arbitrary fluorescence units to lysine values needs to be overcome. The DBL is recommended for routine analysis since it is simple, economical and highly sensitive to all lysine damage provided care is taken to optimise dye-binding for each type of material analysed.
A biochemical and immunological comparison of the Jaagsiekte and two related retroviruses.
(1987) York, Denis Francis.; Verwoerd, D. W.; Dennison, Clive.
Jaagsiekte is a contagious cancer affecting the lungs of sheep. Although the etiological agent is Jaagsiekte retrovirus (JSRV), two other retroviruses viz South African maedi visna virus ( SA - OMVV) and a novel Bovine retrovirus (BRV) have been associated with or implicated in the jaagsiekte disease complex. JSRV was sufficiently purified from lung rinse material using a Freon extraction, Percoll density gradient centrifugation and chranatography on a Sephacryl column, its polypeptide composition was studied by gel electrophoresis and its morphology observed electron microscopically. Monoclonal antibodies were made against purified preparations of the virus. Two hybridomas were isolated that produced MAbs which appear to be tumour cell specific. A third hybridoma, called 4A1O, produces antibodies considered to be viral specific. These MAbs have been used in the development of JS specific immunoassays. A cross reaction between JSRV and a polyclonal serum against Mason Pfizer monkey virus (MPMV) was confirmed and used in a Western blot technique to identify, monitor and differentiate JSRV from other viruses. During the study of JSRV it became apparent that another retrovirus was often present in JS infected lungs. This virus, referred to as SA - OM1V I, is a novel South African isolate of maedi visna virus (MVV). As SA - OM1V I has physicochemical characteristics similar to JSRV, it was often found in purified JSRV preparations. Being a retrovirus it is also detected by the reverse transcriptase assay which was the only method used to assay and monitor for JSRV during the early stages of our work. Using a Westen blot technique and sera against MVV and MPMV it was possible to simultaneously detect and differentiate JSRV from SA - OMVV I. A method was also developed whereby the two viruses could be separated from each other during purification. The information gained and techniques developed whilst studyiing JSRV were also used to isolate and characterize BRV. This novel virus originated from bovine cells that had been co-cultivated with white blood cells from an ox suffering from malignant catarrhal fever. Three out of four sheep inoculated with BRV developed JS. It therefore had to be· ascertained whether this virus was related to JSRV or not. The comparative study revealed that BRV was biochemically and morphologically quite different fran JSRV. Interestingly, it was shown that serum against MPMV cross reacted with a 32 kd protein of BRV indicating a serological relationship between JSRV, MPMV and BRV. The possible role of BRV in the etiology of jaagsiekte remains to be elucidated.
The biochemistry and medical aspects of naturally occurring toxins.
(1999) Dutton, Michael Francis.
The work presented here represents research done on mycotoxins and plant toxins by the author and his postgraduate students over a period from 1964 to date. The first phase, which ends at 1980, mainly addresses the biosynthesis of the aflatoxins. The involvement of anthraquinone derivatives in this process was investigated and the role of versicolorin A and its derivatives was partially elucidated. Novel active enzymes systems were derived from protoplasts and used in these studies. The period lasting from 1980 to 1992 concentrates on the occurrence of mycotoxins in agricultural commodities and effects on animals and their systems. Over 7000 samples were analysed using a multimycotoxin analytical method and a fungal screen. The most common mycotoxin found was aflatoxin B₁ and prevalent fungus was Fusarium moniliforme. Later work is indicating that fumonisin B₁ is the most commonly occurring mycotoxin. As this was only discovered in 1988, its presence was only looked from 1995 onwards. It was also found that rumen fluid could metabolise trichothecenes. During this period (1980-1992) further work on aflatoxin metabolism was done and a novel dehydrogenase involved in aflatoxin B₁ was isolated and characterised. An Elisa assay was developed for atractyloside, a toxin found in a plant (Callilepis laureola) used in tradition medicine. The site of atractyloside storage was found to be in the plant vacuole. The final period covers 1992 to the present, where the occurrence and effects of mycotoxins in human disease were studied. The major and most important finding is that fumonisin B₁ is present in the blood and tissues of many of the Black population examined in Kwazulu Natal. This includes, oesophageal cancer patients, eclamptic patients, school children and members of the rural population. A similar circumstance also appertains for the presence of aflatoxin B₁. It seems likely from these results that chronic mycotoxicoses are a common occurrence, particularly in the Black rural population and are not the sporadic rare event that is found in the first world countries.
Biochemistry students' difficulties with the symbolic and visual language used in molecular biology.
(2007) Gupthar, Abindra Supersad.; Anderson, Trevor Ryan.
This study reports on recurring difficulties experienced by undergraduate students with respect to understanding and interpretation of certain symbolism, nomenclature, terminology, shorthand notation, models and other visual representations employed in the field of Molecular Biology to communicate information. Based on teaching experience and guidelines set out by a four-level methodological framework, data on various topic-related difficulties was obtained by inductive analyses of students’ written responses to specifically designed, free-response and focused probes. In addition, interviews, think-aloud exercises and student-generated diagrams were also used to collect information. Both unanticipated and recurring difficulties were compared with scientifically correct propositional knowledge, categorized and subsequently classified. Students were adept at providing the meaning of the symbol “Δ” in various scientific contexts; however, some failed to recognize its use to depict the deletion of a leucine biosynthesis gene in the form, Δ leu. “Hazard to leucine”, “change to leucine” and “abbreviation for isoleucine” were some of the erroneous interpretations of this polysemic symbol. Investigations on these definitions suggest a constructivist approach to knowledge construction and the inappropriate transfer of knowledge from prior mental schemata. The symbol, “::”, was poorly differentiated by students in its use to indicate gene integration or transposition and in tandem gene fusion. Idiosyncratic perceptions emerged suggesting that it is, for example, a proteinaceous component linking genes in a chromosome or the centromere itself associated with the mitotic spindle or “electrons” between genes in the same way that it is symbolically shown in Lewis dot diagrams which illustrate covalent bonding between atoms. In an oligonucleotide shorthand notation, some students used valency to differentiate the phosphite trivalent form of the phosphorus atom from the pentavalent phosphodiester group, yet the concept of valency was poorly understood. By virtue of the visual form of a shorthand notation of the 3,5 phosphodiester link in DNA, the valency was incorrectly read. VSEPR theory and the Octet Rule were misunderstood or forgotten when trying to explain the valency of the phosphorus atom in synthetic oligonucleotide intermediates. Plasmid functional domains were generally well-understood although restriction mapping appeared to be a cognitively demanding task. Rote learning and substitution of definitions were evident in the explanation of promoter and operator functions. The concept of gene expression posed difficulties to many students who believed that genes contain the entity they encode. Transcription and translation of in tandem gene fusions were poorly explained by some students as was the effect of plasmid conformation on transformation and gene expression. With regard to the selection of transformants or the hybridoma, some students could not engage in reasoning or lateral thinking as protoconcepts and domain-specific information were poorly understood. A failure to integrate and reason with factual information on phenotypic traits, media components and biochemical pathways were evident in written and oral presentations. DNA-strand nomenclature and associated function were problematic to some students as they failed to differentiate coding strand from template strand and were prone to interchange the labelling of these. A substitution of labels with those characterizing DNA replication intermediates demonstrated erroneous information transfer. DNA replication models posed difficulties integrating molecular mechanisms and detail with line drawings, coupled with inaccurate illustrations of sequential replication features. Finally, a remediation model is presented, demonstrating a shift in assessment score dispersion from a range of 0 - 4.5 to 4 - 9 when learners are guided metacognitively to work with domain-specific or critical knowledge from an information bank. The present work shows that varied forms of symbolism can present students with complex learning difficulties as the underlying information depicted by these is understood in a superficial way. It is imperative that future studies be focused on the standardization of symbol use, perhaps governed by convention that determines the manner in which threshold information is disseminated on symbol use, coupled by innovative teaching strategies which facilitate an improved understanding of the use of symbolic representations in Molecular Biology. As Molecular Biology advances, it is likely that experts will continue to use new and diverse forms of symbolic representations to explain their findings. The explanation of futuristic Science is likely to develop a symbolic language that will impose great teaching challenges and unimaginable learning difficulties to new generation teachers and learners, respectively.
Biosystematic studies in Southern African species of Strychnos L. (Loganiaceae)
(2014) Adebowale, Adekunle.; Nicholas, Ashley.; Lamb, Jennifer Margaret.; Naidoo, Yougasphree.
Strychnos L. is the largest genus of the pantropical or subtropical family Loganiaceae with about 200 species. Their habits range from trees and shrubs in open areas to lianas in rain forests. The genus is well-known as a source of alkaloids such as strychnine and brucine and other allied compounds, all of which have been used medicinally and in curare formulation for centuries. While taxonomic circumscription of the genus has never been contentious, there is no consensus about infrageneric affiliations, the latest of which recognises 12 sections based on morphological characters. Recent molecular evaluation of the genus on a global scale with the internal transcribed spacer (ITS) marker suggests that many of the currently recognised sections are not monophyletic. An understanding of regional patterns of evolution, which is relevant for biodiversity conservation, requires an in-depth study of the focus group on a regional scale. Using a multiplicity of approaches from morphological and molecular to biogeographical, this study is an attempt at elucidating diversity patterns at different levels among the southern African species of Strychnos. Various combinations of morphological attributes from branches, leaves, flowers and fruits distinguish seemingly homologous clusters of species, sometimes supported by molecular data. A lack of molecular support for a hypothetical relationship may viii indicate case(s) of convergent evolution in these features across the taxa involved. Molecular phylogenies based on the ITS and chloroplast markers confirm the nonmonophyletic nature of all but section Spinosae. Proposals for sectional recircumscriptions of the genus are provided. Patterns of speciation within Strychnos suggest a Miocene origin in the rain forests along the South America/Guinea-Congolian axis. Within the southern African subcontinent, the evolution of the genus carries a strong ecological signature along either the forest or savanna biome, with many accompanying morphological adaptations for the respective habitats. The non-synonymy of S. gerrardii with S. madagascariensis is demonstrated with multiple sources of data, as a case of integrative taxonomy succeeding where single-source data approaches might have failed. Routes to current distribution of the genus in southern Africa are hypothesised to involve a combination of palaeo-climatic oscillations and allopatric speciation, consistent with the process indicated in many other plant groups for the region. The findings are discussed in the wider context of their implications for taxonomy and biodiversity conservation in the face of climate change, food security and other relevant issues in systematics.
A carboxymethylcellulase and a xylanase from Sclerotium rolfsii.
(1983) Lindner, William Andrew.; Dennison, Clive.; Dekker, Robert F. H.
No abstract available.
Changes in endosome-lysosome pH accompanying pre-malignant transformation.
(2005) Jackson, Jennifer Gouws.; Elliott, Edith.
The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype.
Characterisation of infectious bursal disease virus (IBDV) polyprotein processing.
(2011) Vukea, Phillia Rixongile.; Coetzer, Theresa Helen Taillefer.
Infectious bursal disease virus (IBDV) is a birnavirus that infects the B-cells in the bursa of Fabricius of young chickens, causing Gumboro disease. The IBDV 114 kDa polyprotein (NH2-pVP2-VP4-VP3-COOH) is thought to be processed at 512Ala-Ala513 and 755Ala-Ala756 through the proteolytic activity of VP4, a serine protease which uses a Ser/Lys catalytic dyad, to release pVP2, VP4 and VP3. Precursor VP2 (pVP2) is further processed at its C-terminus to generate VP2 and structural peptides through the cleavage of the 441Ala-Phe442, 487Ala-Ala488, 494Ala-Ala495 and 501Ala-Ala502 peptide bonds to release VP2 and four structural peptides, pep46, pep7a, pep7b and pep11. While the processing at the 441Ala-Phe442 site was shown to be mediated by the endopeptidase activity of VP2, the processing at the other two sites is not well understood. The products resulting from the processing of the IBDV polyprotein were previously identified by anti-VP2 and anti-VP3 antibodies. The present study used anti-VP4 peptide antibodies to identify products resulting from the IBDV polyprotein processing. It was hypothesised that VP4 exists in two forms, the embedded form which exists as an integral part of the polyprotein and a mature form which is released after the processing. In order to characterise the two forms of VP4, six different fragments i.e. full-length polyprotein (Met1-Glu1012), truncated polyprotein (Ile227-Trp891), VP4-RA (Arg453-Ala755), VP4-RK (Arg453-Lys722), VP4-ΔVP3 (Ala513-Trp891, called VP4-AW for the sake of simplicity) and VP4-AA (Ala513-Ala755) were amplified from the IBDV dsRNA, cloned into a T-vector and sub-cloned into several expression vectors. The constructs were sequenced prior to expression. The sequence of the polyprotein coding region was used to determine the pathotype of the isolate used for viral dsRNA isolation. This isolate was from IBDV-infected bursae harvested from commercial chickens during an IBD outbreak in KwaZulu-Natal, South Africa in 1995, thus naming the isolate SA-KZN95. The comparison of the deduced amino acid sequence of SA-KZN95 polyprotein with 52 sequences of other IBDV strains highlighted 21 residues which could be molecular markers of different IBDV pathotypes. The residues of SA-KZN95 were identical to those of the Malaysian very virulent UPM94/273 strain. The constructs representing the embedded and mature forms of VP4 were recombinantly expressed. Processing was observed from the expression of the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, but not from VP4-AA expression. The mutation of the Ser/Lys catalytic dyad in the full-length polyprotein, truncated polyprotein, VP4-RA, VP4-RK and VP4-AW, prevented processing thus verifying that the proteolytic activity was due to VP4. Anti-VP4 peptide antibodies were raised in chickens for the identification of the polyprotein cleavage products. The anti-VP4 peptide antibodies detected more cleavage products than expected from the polyprotein, suggesting that additional or different cleavage sites may be used. The characterisation of the cleavage products suggested that the processing for the release of VP4 occurs either at the 487Ala-Ala488 or the 512Ala-Ala513 site in a single polyprotein molecule. Ultimately, an IBDV polyprotein processing strategy that would explain the release of the additional products was proposed in the present study. The present study also illustrated the importance of Pro377 in the processing of the polyprotein where its replacement with Leu induced a prominent change in polyprotein processing. The mutation seemed to induce structural changes that may possibly affect the cleavage sites. Although no autocatalytic activity was observed during the expression of VP4-AA (mature form), it cleaved mutant VP4-RK in trans. It seemed to be active as a dimer on a gelatine gel but no activity was observed against a dialanyl fluorogenic peptide substrate. It also appeared to form peptidase-inhibitor complexes with anti-thrombin III. The present study also describes attempts to detect native VP4 in IBDV-infected bursa homogenates by anti-VP4 peptide antibodies on a western blot and by proteolytic activity determination on gelatine-containing SDS-PAGE gels. The findings of the study provide new information that may contribute to the development of anti-viral agents. These anti-viral agents may target polyprotein processing, capsid assembly and thus prevent virus replication during IBDV infection.
Comparative antidiabetic effects and mechanisms of actions of five Chinese and South African indigenous teas.
(2020) Xiao, Xin.; Islam, Shahidul.
The present thesis assessed the in vitro, ex vivo and in vivo anti-oxidative and antidiabetic activities of five teas which are widely consumed in China or South Africa. Three of the selected five teas are from South Africa, namely red rooibos (Aspalathus linearis), green rooibos (Aspalathus linearis) and red honeybush (Cyclopia genistoides) tea. The remaining two from China are jasmine green (Camellia sinensis) and zhengshanxiaozhong (ZSXZ) black tea (Camellia sinensis). The different sequential solvent extracts following increasing polarity index (dichloromethane, ethyl acetate, ethanol, and water) and hot water extract of different teas were evaluated at in vitro and ex vivo conditions for their antioxidant properties, inhibitory potentials on α-glucosidase, α-amylase and pancreatic lipase, effects on ameliorating Fe2+- induced oxidative pancreatic or hepatic injury, as well as the glucose absorption inhibition in small intestine and the glucose uptake stimulation in isolated psoas muscle of rats. Possible bioactive components responsible for the activities of the extracts were identified by using Gas Chromatography-Mass Spectrometry (GC-MS) analysis or liquid chromatograph-mass spectrometry (LC-MS) analysis. In vitro and ex vivo tests presented promising antioxidant and antidiabetic activities of these five teas. The red honeybush, jasmine green and green rooibos teas, were further subjected to an in vivo intervention trial in a fructose-streptozotocin (STZ) induced T2D model of Sprague-Dawley rats. Assays were carried out to reveal the effects of these teas on lowering blood glucose level, improving oral glucose tolerance ability, stimulating insulin secretion and hepatic glycogen synthesis and ameliorating some diabetes related parameters such as serum lipid profile, hepatic and renal function tests and calculated insulin resistance (HOMA-IR), β-cell function (HOMA-β) from the blood glucose and serum insulin data. Furthermore, in vivo oxidative stress markers such as reduced glutathione, superoxide dismutase and catalase activity and lipid peroxidation were analysed in harvested organs (liver, kidney, heart and pancreas). The results of in vivo tests demonstrated that high dose of jasmine green tea showing the best activity followed by the high dose of red honeybush tea, low dose of jasmine green tea, high dose of green rooibos tea, low dose of red honeybush tea, when lowest activity was observed for the low dose green rooibos tea. The results of this study indicated promising anti-T2D properties of the above-mentioned teas. However, further clinical trials are needed to ascertain the results of these in vitro, ex vivo and in vivo studies.
Comparative immunochemical studies on normal and monoclonal immunoglobulin M.
(1973) Conradie, Jan Dirk.; Visser, Leon.
No abstract available.
A comparative investigation of transgene expression and gene silencing of non-functionalized layered double hydroxides versus amino-acid functionalized hydrotalcites.
(2017) Nundkumar, Nirasha.; Singh, Moganavelli.; Singh, Sooboo.
Abstract available in PDF file.
A comparative study of three toxic legume glycoproteins.
(1973) Dennison, Clive.; Quicke, George Venn.; Visser, Leon.
No abstract available.
A contribution to the biochemistry of Erwinia chrysanthemi.
(1985) Gray, James Steward Sanders.; Dutton, Michael Francis.
No abstract available.
The design and synthesis of gold nanoparticles and its interaction with mammalian cells in culture.
(2014) Lazarus, Geraldine Genevive.; Singh, Moganavelli.
Cancer is a disease characterized by accelerated cell growth, resulting in healthy tissue being destroyed by the processes of invasion and metastasis. Non-viral gene delivery approaches have been extensively studied as a basic tool for intracellular gene transfer and gene therapy especially for genetic aberrations including cancer. Gold nanoparticles have attracted strong biomedical interest for drug delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. In the present study the synthesis of photoluminescent nanoparticles consisting of a gold core coated with polyethyleneimine, poly-L-lysine, cysteine and chitosan is reported. These functionalized gold nanoparticles (FAuNPs) were investigated at different pH values and ionic strength to identify the optimum conditions to produce stable monodisperse nanoparticles. FAuNPs showed good stability at low ionic strength which is important for the flexibility of the polymer chain. All nanoparticle/polymer formulations remained in the size range 11.9-195 nm with narrow particle distributions and low PDI (<1.2). TEM images revealed nanoparticles that were spherical and monodispersed. Nanoparticle and pDNA complexation was efficiently demonstrated in the band shift and ethidium bromide intercalation assays respectively. The serum nuclease digestion assay revealed that the nanoparticles provided partial protection to the complexed plasmid DNA (pCMV-luc). MTT cytotoxicity experiments indicated that the FAuNPs elicited a dose dependent cytotoxic effect with the four mammalian cell lines (HepG2, HEK293, HeLa and Caco2) tested responding differently. Au-PEI/pDNA maintained over 80% cell viability across all cell lines, while the Au-cys/pDNA exhibited a significant 91.8% (p<0.001) in Caco2 cells, Au-Chit/pDNA 126% (p<0.01) in HepG2 cells and Au-PLL/pDNA 104% in Hela cells. Transfection studies were accomplished using the luciferase reporter gene assay. Results showed that the FAuNPs produced greater transgene activity than the cationic polymer/DNA complexes on their own. This was evident for the Au-PEI/pDNA complex which produced a 12 fold increase in the HEK293 cells and a 9 fold increase in the HepG2 cells, compared to the PEI/pDNA complexes alone. The results of this study suggest that FAuNPs low cytotoxicity coupled with the ability to parametrically control particle size and surface properties, make these nanoparticles suitable non-viral gene delivery vectors. However further engineering and modifications of the FAuNPs may be required to enable in vivo gene delivery.
Developing antibodies against Plasmodium lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase and phosphoethanolamine-N-methyltransferase
(2016) Krause, Robert Gerd Erich.; Goldring, James Philip Dean.
Abstract available in PDF file.

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