Masters Degrees (Biochemistry)

Permanent URI for this collectionhttps://hdl.handle.net/10413/8002

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Analyses of lipidic bodies from green microalgae.
(2014) Pather, Verushka.; Gupthar, Abindra Supersad.; Bux, Faizal.
This study presents the analyses of oil body components in microalgae which may be involved in oil droplet assembly including certain triacylglycerol precursors which can be processed to biodiesel, an alternative fuel source. Stress induction of microalgae, Chlorella vulgaris CCAP 211/11B and Dunaliella primolecta CCAP 11/34 was achieved by exclusion of nitrates in growth media. Contrary to other forms of nitrogen depletion, this condition did not greatly enhance lipid biosynthesis in the microalgae. Confocal microscopy and fluorescent dyes nile red and bodipy were employed for the visualization of lipidic body components. The fluorescence hues emitted by neutral lipids and phospholipids were differentiated from those due to autofluorescence and chlorophyll using ZEN software to analyse images from a Zeiss LSM 710 confocal microscope. Oil from both algae, which were subjected to transesterification and gas chromatography, revealed a predominant fatty acid, namely palmitic acid (C16:0). D. primolecta produced linolelaidic acid (C18:2n6t) under growth conditions involving both nitrate supplementation and exclusion; whilst the longest fatty acid, docosanoic acid (C22:0 chain) was produced by the alga C. vulgaris only under conditions of nitrate supplementation. Nitrate limitation had minimal effect on the oil hydrocarbon yield which increased only 0.02% and 0.01% for C. vulgaris and D. primolecta, respectively. The highest biodiesel yield of 26.11 % was recorded from D. primolecta when grown under conditions of nitrate exclusion. The protein concentrations extracted from oil of the former alga ranged from 1.87 - 1.95 Gg/ml when grown under nitrate supplemented conditions and 1.74 - 1.90 Gg/ml when nitrate was excluded from the media. The protein concentrations extracted from oil of D. primolecta ranged from 1.91 - 2.23 Gg/ml and 1.88 - 1.98 Gg/ml, respectively, when the algae were grown in the presence and exclusion of nitrates. In the adaptation of protocols for protein extraction from oil, sunflower and salmon oils were initially used. Sunflower oil extracts produced by 10% (w/v) SDS treatment, yielded protein bands of 198, 96, 70 and 58 KDa on 10% (w/v) polyacrylamide gels while 6M urea treatment yielded a band of 200 KDa. Salmon oil treated with 10% (w/v) SDS and 6 M urea yielded bands of 195 and 27 KDa, and 198 KDa, respectively, as well as common bands of 68 and 64 KDa. In comparison, the extraction of discrete proteins from algal oil proved to be difficult as the extractants SDS and urea could have denatured protein components into subunit structure.
Analysis of the Mycoplasma hominis hsp70 gene and development of a PCR ELISA assay.
(1998) Shearer, Nicollette.; Hastings, John W.
Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive.
Antidiabetic activity of Warburgia salutaris (Bertol. f.) Chiov. (Canellaceae).
(2017) Msomi, Nontokozo Zimbili.; Simelane, Mthokozisi Blessing Cedric.; Murambiwa, Pretty.
Abstract available in PDF file.
Antioxidative and antidiabetic activity and phytochemicals analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Idris Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antioxidative and antidiabetic activity and phytochemicals, analysis of some selected Sudanese traditional medicinal plants.
(2021) Idris, Almahi Mohamed.; Islam, Shahidul.
This study was conducted to evaluate the antioxidant and anti-diabetic properties of selected traditional Sudanese medicinal plants (Cyperus rotundus, Nauclea latifolia, and Hibiscus sabdariffa) using in vitro, ex vivo, and in silico experimental models. The crude extracts (ethyl acetate, ethanol, and aqueous) were screened in vitro for their antioxidant activities using ferricreducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and nitric oxide radical (NO) scavenging activities, as well as their carbohydrate digesting enzyme inhibitory activities for antidiabetic evaluation. Subsequently, the extracts were subjected to Gas Chromatography-Mass Spectrometry (GC-MS) analysis to elucidate their possible bioactive compounds. Additionally, ex vivo studies was conducted to investigate their capability to promote muscle glucose uptake and suppress glucose absorption in the intestine as well as to analyze antioxidative effects in iron–induced oxidative stress in hepatic tissue. Molecular docking was carried out to determine the probable enzymes' inhibitory mode of action by ligands identified through GC-MS. This study indicates that these traditional Sudanese medicinal plants have remarkable antioxidant and antidiabetic activities, which may help to ameliorate oxidative stress and diabetes. Therefore, these plants may be considered a natural source of bioactive compounds beneficial for human health, particularly for managing diabetes and oxidative stress-related metabolic disorders.
Antiplasmodial activity of Warburgia salutaris (Bertol. F.) Chiov. (Cannelaceae).
(2017) Nyaba, Zoxolo Nokulunga.; Simelane, Mthokozisi Blessing Cedric.; Murambiwa, Pretty.
Abstract available in PDF file.
Apoptosis in peripheral blood mononuclear cells of human immunodeficiency virus (HIV) infected patients undergoing highly active antiretroviral therapy.
(2008) Karamchand, Leshern.; Chuturgoon, Anil Amichund.; Dawood, Halima.
Highly active antiretroviral therapy (HAART) is currently the only treatment that effectively reduces the morbidity and mortality of individuals infected with Human Immunodeficiency Virus-1 (HIV-1). Standard HAART regimens typically comprise 2 nucleoside reverse transcriptase inhibitors and either one non-nucleoside reverse transcriptase inhibitor or a protease inhibitor. These drugs bind to and inhibit the HIV-1 Reverse Transcriptase and Protease enzymes respectively, thereby suppressing viral replication. The nucleoside reverse transcriptase inhibitors promote mitochondrial (mt) dysfunction by strongly inhibiting mt polymerase gamma (Pol-y) and subsequently, mtDNA replication. In contrast, the non-nucleoside reverse transcriptase inhibitors, efavirenz (EFV) and nevirapine (NVP) do not inhibit Pol-y although EFV has been shown to induce mt depolarisation ( mlow) in vitro at supra-therapeutic concentrations. However, the capacity of non-nucleoside reverse transcriptase inhibitor drugs to induce mt toxicity in vivo previously remained undetermined. The objective of this study was to determine the influence of EFV and NVP on peripheral lymphocyte mt transmembrane potential (Avj/m) and apoptosis in HIV-1-infected patients treated with these non-nucleoside reverse transcriptase inhibitors. Thirty-two HIV-1-infected patients on HAART between 4 and 24 months (12 on EFV, 20 on NVP) and 16 HAART-naive HIV-1-infected patients were enrolled into this study. All participants were black South African patients. Spontaneous peripheral lymphocyte apoptosis and mlow were measured ex vivo by flow cytometry for all patients. CD4 T-helper apoptosis for the EFV and NVP cohorts was 19.38% ± 2.62% and 23.35% ± 1.51% (mean ± SEM), respectively, whereas total lymphocyte mlow was 27.25% ± 5.05% and 17.04% ± 2.98%, respectively. Both parameters for each cohort were significantly lower (P < 0.05) than that of the HAART-naive patients. The NVP cohort exhibited both a significant time dependent increase in peripheral lymphocyte ö¿mlow (P = 0.038) and correlation between Thelper apoptosis and low (P = 0.0005). These trends were not observed in the EFV cohort. This study provides evidence that both EFV and NVP induce peripheral lymphocyte ö¿ m low in HIV-1-infected patients on non-nucleoside reverse transcriptase inhibitor-based HAART, which in the case of NVP is sufficient to induce the apoptosis cascade.
Apoptosis, redox stress and cancer.
(2000) Moodley, Thunicia.; Elliott, Edith.
Apoptosis is a regulated "programme" by which cells are induced to die in a manner which does not result in pathological inflammatory reactions, and involves dismantling of the cell into membrane-bound fragments that are removed by phagocytosis. This process is induced in order to remodel tissues and maintain homeostasis in cell numbers. Apoptosis may be induced via many pathways, many of which are redox-regulated, and is dysregulated in cancer cells, mainly due to mutational inactivation of certain pathways. Cancer cells also have a non-linear response to redox imbalance, a potentially exploitable characteristic for the therapeutic selective induction of apoptosis in cancer cells in mixed cell populations. Model cell culture systems are required for the selective toxicity testing of anti-cancer drugs, many of which work by inducing redox stress. In the current study, hydrogen peroxide was selected as the redox stress-inducing agent, and the test cells were an immortal, non-invasive breast epithelial cell line (MCFlOA) and its rastransfected, pre-malignant derivative (MCF10AneoT). A reliable, sensitive, cost effective and least time-consuming system for detection of apoptosis in such a system was sort and two novel methods, cytochrome c release and caspase-3 activity assays, were finally selected and compared with results seen by conventional DNA laddering and morphological examination at the light and electron microscopic level. No single procedure was found to be reliable individually. For the model system used, a combination of electron microscopy and DNA laddering was sufficient for simply detecting apoptotic cell death and necrosis. The caspase activity assay distinguished between apoptosis and necrosis, and cytochrome c release proved the most sensitive indicator of cell response. However, since cytochrome c release may be reversible and may not necessarily proceed to the downstream events of apoptosis in the time frame used in the current assays, it is not certain that cytochrome c release ultimately leads to apoptosis. However, three forms of cytochrome c were observed on western blots, the nature and significance of which remains to be determined. A comparison of the results of different methods allowed a model for the sequence of specific apoptotic events to be proposed.
The application of layered double hydroxide nanoparticles (LDHs) as potential anticancer drug delivery systems.
(2016) Mncwabe, Zoleka.; Singh, Moganavelli.
Chemotherapy being one of the principle techniques used in cancer treatment, has been applied in the treatment of a wide spectrum of cancers. However, this mode of treatment is fraught with a myriad of challenges, reducing its effectivity and inducing the need for repeated treatments. Poor drug delivery systems or lack thereof, have led to patients suffering unpleasant side effects that not only cause collateral damage to their bodies but also reduces the quality of their lives. The current array of chemotherapeutic drugs available may be effective in certain cancers, nevertheless the need for their optimization is still necessary for better safety, stability and efficiency of treatment. Thus the current study was designed to investigate the potential of layered double hydroxide (LDH) nanoparticles in the delivery of the broad spectrum anticancer drug, 5-Fluorouracil (5-Fu). Four LDH nanoparticles, MgAl 2:1, MgAl 3:1, ZnAl 2:1and ZnAl 3:1 were successfully synthesized and intercalated with 5-Fu using the calcination reconstruction process to form nanohybrids. The LDHs and their nanohybrids, MgAl 2:1-5-Fu, MgAl 3:1-5-Fu, ZnAl 2:1-5-Fu and ZnAl 3:1-5-Fu were structurally confirmed using XRD, FTIR, UV-Vis, ICP-OES; with size, zeta potential and ultrastructural morphology investigated using nanoparticle tracking analysis (NTA) and electron microscopy (TEM and SEM). LDHs were characteristically hexagonal in shape with sizes ranging from 100 -150 nm, and high zeta potentials enforcing their colloidal stability. The successful intercalation of 5-Fu was confirmed from drug encapsulation efficiency studies to be between 40-60% in the respective LDHs. Furthermore, drug release studies revealed a steady controlled release of the drug over a 7-hour period at pH 4-7, with more than 60% of the drug being released by the end of this period. In vitro MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and SRB (Sulphorhodamine B) cytotoxicity studies on free 5-Fu and LDH bound 5-Fu in human cell lines, breast adenocarcinoma cell line (MCF-7), hepatocellular carcinoma (HepG2), colorectal adenocarcinoma (CaCo-2) and embryonic kidney (HEK293), showed a dose dependent cytotoxicity profile with the free 5-Fu being more toxic to the cells than the bound drug. This was further confirmed in fluorescent apoptotic studies (dual acridine orange and ethidium bromide staining method), where free 5-Fu had a higher apoptotic index than the LDH bound 5-Fu
Assessment of genetic diversity and DNA fingerprinting of the Cape parrot (Poicephalus robustus) using randomly amplified polymorphic DNA (RAPD)
(2004) Blue, Gillian Margaret.; Fossey, Annabel.; Perrin, Michael Richard.
The Cape parrot (Poicephalus robustus) is South Africa's only endemic parrot. It has become increasingly rare in recent years, with fewer than 500 birds left in the wild, and is now regarded as endangered. Possible factors contributing to this rapid decline in numbers include habitat loss, food shortage, disease and illegal trafficking and trading in the species. Habitat loss and food shortage have been brought about by the rapid destruction of the yellowwood trees in the afromontane forests in South Africa and have played a role in reducing the population numbers. The Psittacine beak and feather disease virus (PBFDV) has also contributed to the loss of some individuals, however it is the illegal trafficking of this rare and valuable species that has become of great concern. As the Cape parrot is becoming increasingly rare and therefore highly sought after, its commercial value has multiplied to the extent that illegal black market trapping is on the rise. The industry involved in breeding and conservation of endangered bird species, has a need for the proper establishment of studbooks, containing all available information on captive as well as tagged birds. Most of the information found in studbooks is based on morphological attributes of individual birds. Although this is useful, there is a need to add molecular information in order for complete identification of individuals, particularly in a species threatened by illegal trading and theft. A preliminary analysis of the amount of variation present in the population of interest is therefore required so that appropriate methods and techniques can be developed to identify individual birds. A RAPD analysis was conducted to assess the amount of variation in the Cape parrot and lay the foundations for the establishment of individual identification in the species. Blood samples from 30 parrots, consisting of both related and unrelated individuals, were obtained from three separate locations: Amazona in Assagay, Rehoboth Farm in Dargle, as well as from the Eastern Cape. 15 random primers were selected and used to conduct a randomly amplified polymorphic DNA (RAPD) analysis. RAPDs are extremely useful in situations where relatively inexpensive first approximations of the genetic variation are needed, such as in rare and endangered species. After successful optimisation of the technique in the species, the 15 primers were screened for all 30 individuals and the individual DNA fingerprints, analysed. Clear, distinctive and reliable DNA fingerprints were obtained for all individuals however, it was interesting to note despite the analysis of 85 loci using the 15 primers almost identical DNA fingerprints were produced between the individual birds. A population analysis into the amount of variation present between and within the three populations, as well as for the representative population as a whole, was conducted. Using various statistical programmes such as POPGENE and ARLEQUIN, heterozygosities, genetic distance measures, diversity indices, Wright's fixation index and AMOVAs were estimated. The amount of polymorphism detected in this investigation was 33 % and the heterozygosity, 0.37, which is a relatively high value for the uniformity displayed in the DNA profiles. The high GC content of the primers however, could be a possible explanation thereof. Relationship and kinship determination, sex determination as well as population assignment was possible despite not being able to identify each individual based on unique DNA fingerprints. The AMOVA analysis indicated significant variation on both the between (5.59 %) and within (94.41 %) levels of analysis. Little variation or differentiation was observed between the three subpopulations, which was confirmed with an FST value of 0.056. The variation experienced within each subpopulation was analysed using Shannon's index of phenotypic diversity. The Amazona population displayed the most variation with a value of 0.286 and the Rehoboth population, the least with 0.195. This was expected, with the individuals from the latter population comprising one extended family. Nei's measures of genetic identity revealed that the individuals from Amazona were more similar to the Eastern Cape population, which was again expected with regular exchanging of chicks between the two breeders. RAPD technology was successful in laying the foundations for individual identification in the Cape parrot. It was also successful in producing reproducible DNA fingerprints in the species that were able to determine relatedness to some extent, determine the sex of individuals and identify individuals from a particular subpopulation. Furthermore RAPD analysis gave a good indication of the variation found in the Cape parrot population, which is important for conservation purposes. In order to maximize conservation efforts and strategies in an endangered species, determining the level of genetic diversity and variation found in the remaining individuals of the population is of great importance. This information could provide powerful insight for conservation purposes and depending on the level of diversity detected, appropriate breeding programmes could be set up in order to increase the genetic variation and thereby reduce the chance of extinction of the species. The following important findings emerged from this investigation: • RAPD technology, once optimised for the species of interest, is successful in producing clear and reliable DNA fingerprints, provided the same protocol is followed carefully throughout the investigation. • An optimised protocol for fingerprinting the Cape parrot using RAPDs was established. • Possible sex identification, population assignment and a degree of kinship determination was determined using RAPDs. • Little variation was found within the representative Cape parrot population as a whole due to small population size and possible inbreeding. • As expected for an avian species, little genetic sub-division or differentiation was observed between the three populations analysed.
Assessment of hypoxoside and its derivatives as anti-cancer drugs.
(2013) Xulu, Bongiwe Ziphelele.; Elliott, Edith.; Drewes, Siegfried Ernst.; Van Heerden, Fanie Retief.
Extracts of the African potato have long been believed to have anti-cancer properties. The aim of the current research was to isolate hypoxoside (HYP) from Hypoxis hemerocallidea (African potato) and synthesize the dimethyl (DMH) and decaacetyl (DAH) derivatives and to test their selective cytotoxicity on a model consisting of a normal (MCF10A) and premalignant, invasive breast epithelial cells (MCF10A-NeoT). Hypoxoside was extracted from the H. hemerocallidea corms using ethanol, purified using a C-18 reverse phase column and the compound examined by nuclear magnetic resonance (NMR) spectroscopy and high-resolution mass spectrometry and found to be of high purity. This was also the case for the synthesized compounds. To assess possible selective effects (cytotoxicity) of derivatized and underivatized hypoxoside, effects on the metabolism of premalignant and normal cells were assessed using the 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. Effects on cell number (total counts) and cell death [trypan blue and propidium iodide (PI) staining for dead cells versus a lack of staining for live cells] were, thereafter, assessed. Imaging of live adherent cells was also carried out using acridine orange (AO) and PI for live and dead cells (respectively). Propidium iodide staining of detached cells was carried out for flow cytometric determination of cell death (PI indicating early apoptotic or late apoptotic/necrotic cells). After treatment of normal (MCF10A) breast epithelial cells and premalignant cHa-rastransfected (MCF10A-NeoT) derivative breast epithelial cells with HYP, DMH and the DAH derivative, the MTS assay and the Duncan‟s multiple range, analysis of variance (ANOVA) post hoc analysis of the MTS results revealed that only the 150 and 300 µM DAH derivative had a statistically significant effect on the metabolic activity of the abnormal cell line relative to the dimethyl sulfoxide (DMSO) and revealed no significant effect on the normal MCF- 10A cell line after treatment with any of the test compounds. Supravital PI staining of adherent cells seemed to indicate a far higher rate of induction of cell death in abnormal cells than evident in the MTS assay and the PI-based flow cytometry or the trypan blue exclusion assays and need re-investigating, though result trends were similar. Total cell counts, show that HYP and its derivatives appear to increase both cancer and normal cell proliferation significantly, except in the case of DAH at 150 and 300 μM in the MCF10A-NeoT, without affecting the MCF-10A cell line. The trypan blue method for detection of cell death, together with total cell counts, the Duncan‟s analysis of MTS results and a 24 hour exposure to test compounds, seems to constitute an optimal system for drug screening and indicates the statistically significant selective toxicity of the DAH compound at 150 and 300 μM in the MCF10A-NeoT, suggesting that the DAH derivative at 150 and 300 µM would have significant, selective therapeutic potential on Ras-related malignancies.
Bio-guided isolation of biologically active compounds from seeds of selected South African medicinal plants.
(2016) Perumal, Amanda.; Govender, Patrick.; Naidoo, Sershen.; Pillay, Karen.
Abstract available in PDF file.
Biochemical and microbiological changes in sugarcane stalks during a simulated harvest-to-crush delay.
(2008) Martin, Lauren Anne.; Hunter, Charles Haig.; Watt, Derek Alexander.
Post-harvest cane deterioration in the South African sugar industry results in significant revenue loss that is estimated to be in the region of ZAR 60 million per annum. Despite these large losses, precise biological data relating to the process of cane deterioration under South African conditions is limited. Severity of deterioration is influenced by a number of factors, including the length of the harvest-to-crush delay (HTCD), ambient temperature and harvesting practices. For example, burning of cane prior to harvest may result in rind splitting, which provides entry for microbes, particularly Leuconostoc mesenteroides that may exacerbate deterioration. The effect of these factors on deterioration was examined by quantifying the biochemical and microbiological changes that occur in sugarcane stalks after harvest, with the influence of length of HTCD, degree of L. mesenteroides infection and ambient temperature receiving attention. The primary novelty of the work resides in the analysis of deterioration under tightly regulated temperatures, which were designed to reflect diurnal variations typically experienced during summer and winter in the South African sugar belt. In addition, inoculation of mature internodes with a consistent titre of L. mesenteroides was used as a means to mimic a consistent level of infection of harvested stalks by the bacterium. Metabolites selected for analysis were those both native to the stalk and produced as by-products of microbial metabolism, viz. sucrose, glucose, fructose, ethanol, lactic acid, dextran and mannitol. Simulated HTCDs under summer temperatures resulted in increasing glucose and fructose levels with time, which contrasted to the approximately constant levels of these hexose sugars under winter conditions. Commonly referred to as ‘purity’ in an industrial context, precise determination of the concentration of these hexoses in cane consignments could potentially indicate the extent of deterioration. Despite the detection of a basal concentration of lactic acid in unspoiled cane, the observed increase in concentration of this organic acid over the simulated summer HTCD suggests that this metabolite could also potentially serve as an indicator for postharvest deterioration. In contrast, the investigation indicated that ethanol was an unsuitable biochemical marker for deterioration of L. mesenteroides infected cane. An inability to detect dextran and mannitol in the samples, combined with consistent sucrose levels and variable mill room data, suggest that extreme proliferation of L. mesenteroides is facilitated primarily by in-field practices, particularly the manner in which cane is prepared prior to harvest and transport to the mill. Bacterial proliferation and infection by L. mesenteroides of inoculated stalks were monitored by standard selective culturing techniques. Despite the limited detection of L. mesenteroides-associated metabolites, culture-based analyses revealed that the bacterium was the dominant bacterial species within the samples. A number of other bacterial species were isolated and identified, however the extent to which the total number of microorganisms proliferated was limited to a maximum of 1 x 105 colony forming units per gram of fresh tissue. In conjunction with these analyses, a molecular approach known as Polymerase Chain Reaction-Mediated Denaturing Gradient Gel Electrophoresis (PCR-DGGE) was undertaken to investigate the bacterial diversity patterns associated with deteriorating sugarcane stalks throughout the delay period. In contrast to the results obtained by means of the culture-based assays, PCR-DGGE revealed that L. mesenteroides was not the dominant bacterial population, and showed that the level of bacterial diversity was relatively consistent across the differing treatments and with time. The use of complimentary culture-dependent and cultureindependent analyses thus permitted the detection of this discrepancy and indicated the utility of PCR-DGGE in the determination of bacterial community structure of postharvest sugarcane tissue. The biology of post-harvest deterioration of green sugarcane stalks is highly complex, even under rigorously controlled temperature and infection regimens. The results of this study emphasize the important effects that harvest method and environmental conditions have on post-harvest sugarcane deterioration. Towards the formulation of industry-relevant recommendations for combating post-harvest deterioration, future work will strive to mimic the effects that harsh harvesting and transport practices have on the severity of the problem.
Biochemical and structural characterization of ClpK from Klebsiella pneumoniae.
(2022) Motiwala, Tehrim .; Khoza, Thandeka.
Abstract available in PDF.
The biochemical effects of Sutherlandia Frutescens in cultured H9 cancerous T cells and normal human T lymphocytes.
(2008) Ngcobo, Mlungisi.; Chuturgoon, Anil Amichund.
Indigenous plants have long been used by African populations in their cultural lives and health care. Sutherlandia frutescens (SF) is a popular traditional medicinal plant found in various parts of southern Africa and used for treatment or management of different diseases, including cancer and HIV/AIDS. In this study, the biochemical effects of various dilutions (1/50, 1/150, 1/200, and 1/300) of SF 70% ethanol (SFE) and deionised water (SFW) extracts in cancerous H9 and normal T cells were examined. Untreated, 70% ethanol-treated and camptothecin (CPT, 20jiiM) treated cells were used as reference samples for comparison. Cytotoxicity, apoptotic enzymes activity, oxidant scavenging and antioxidant promoting abilities, cellular morphology and cytokine signalling effects were assessed using the methylthiazol tetrazolium (MTT) assay, adenosine triphosphate (ATP) assay, caspase-3/-7 activity assay, thiobarbituric acid reactant substance (TBARS) and glutathione (GSH) assays, fluorescence microscopy and an ELISAbased cytokine analyses assay respectively. Sutherlandia frutescens ethanol and water extract dilutions (1/50 and 1/200) were shown to be cytotoxic to H9 T cells in a dose- and time-dependent manner with the SFE extract having an average IC50 of 1/40 after 24 hours while SFW extract reached a similar IC50 only after 48 hours. In normal T cells, the SFE extract induced proliferation after 24 hours but this was reverse after 48 hours. The SFW extract dilutions did not significantly change cell viability after 24 hours but significantly increased cell viability after 48 hours. Both SFE and SFW extracts dilutions induced a dose- and time-dependent inhibition of caspase-3/-7 activity in both H9 and normal T cells. Both types of extracts were also shown to efficiently remove lipid peroxides from supernatants of treated cell lines, with SFW extract having a more lasting effect. In the GSH assay, the SFE and SFW extract dilutions reduced GSH levels in H9 T cells, with the SFW extract dilutions being more effective. In normal T cells, the higher dilutions (1/150 and 1/300) of SFW extract increased GSH levels significantly while lower dilutions (1/50) of both SFE and SFW extracts significantly inhibited GSH levels. Lower dilutions (1/50) of SFE and SFW extracts induced chromatin condensation in both H9 and normal T cells after 48 hours incubation. Using treated peripheral blood mononuclear cells (PBMCs) supernatants, SFE and SFW extract dilutions were shown to reduce the levels of pro-inflammatory cytokines IL 1 p and TNF-a in a dose-dependent manner. These results further confirmed the anticancer abilities of SF and showed that higher concentrations of this medicinal plant can be toxic to normal T cells in vitro while lower concentrations can stimulate the immune cells. Therefore further studies should be conducted with regards to the effects of SF on the immune system in both in vitro and in vivo systems.
Biochemical studies on trypanosomal prolyl oligopeptidase family pathogenic factors.
(2014) Ndlovu, Sijabulisiwe Faith.; Coetzer, Theresa Helen Taillefer.
African Animal trypanosomosis, also known as Nagana, is a parasitic disease which affects many mammalian species, mainly livestock such as cattle, sheep and goats. The disease also affects humans (Human African Trypanosomosis) and in this case is referred to as sleeping sickness. Nagana is caused by the Trypanosoma parasite, which is transmitted to the host by a bite from the tsetse fly (Glossina spp). The Trypanosoma causing trypanosomosis in animals are Trypanosoma congolense, T. vivax and T. brucei brucei. Vaccine development has been unsuccessful, due to the presence of the variant surface glycoprotein on the surface of parasites which undergoes antigenic variation therefore enabling the parasite to avoid detection by vaccines. A chemotherapeutic drug such as isometamidium chloride combined with diminazene and suramin have also had little success due to the increase in drug resistance. During infection of the host, trypanosomal parasites utilise various proteolytic enzymes such as the oligopeptidases, which hydrolyse important host factors such as peptide hormones. These proteolytic enzymes are thus considered to be pathogenic factors which contribute to the manifestation of various trypanosomosis symptoms such as anaemia, fever, paralysis and disturbances in sleep cycle patterns. It is these pathogenic factors which are now being considered as drug targets in the hope to eradicate the spread or continuous advancement of trypanosomosis. Three trypanosomal pathogenic factors, which are serine oligopeptidases which belong to the prolyl oligopeptidase family of serine proteases (Clan SC in subfamily S9) were the focus of this study, namely, prolyl oligopeptidase (POP) from T. b. brucei (TbPOP) and T. congolense (TcoPOP) as well as oligopeptidase B (OPB) from T. congolense (TcoOPB). The full length TbPOP gene was cloned into pTZ57R/T cloning vector and successfully sub-cloned into pET32a expression vector and recombinantly expressed in its insoluble form at a size of approximately 100 kDa using the Escherichia coli BL21 DE3 expression system. TbPOP expression was confirmed by western blot probed with anti-His tag antibodies. Expression of TbPOP was optimised under varying temperatures and IPTG concentrations in an attempt to solubilise the inclusion bodies. However, the protein was expressed as part of inclusion bodies. Therefore, urea denaturation was used for its solubilisation. Following solubilisation, recombinant TbPOP was partially purified on a Ni2+ affinity resin. Further attempts to purify TbPOP by molecular exclusion chromatography (MEC) were unsuccessful, this could be due to aggregation of the protein during the refolding step. Therefore refolding by a Sephadex G-25 desalting column was attempted as it removes some impurities. However, further purification by MEC and ion exchange chromatography (IEC) were unsuccessful. The full-length TcoPOP gene was successfully cloned into pGEM-T® cloning vector and subsequently sub-cloned into pET32a expression vector. However, upon sequencing of the plasmid DNA, it was discovered that a mutation had occurred in the recombinant TcoPOP DNA sequence forming the stop codon “TAG” which resulted in the termination of protein expression, therefore, further work on TcoPOP was not pursued. TcoOPB was successfully recombinantly expressed in pET28a using the E. coli BL21 DE3 expression system. The protein had a size of approximately 80 kDa. The protein was affinity purified using a Ni2+ affinity resin. Expression of TcoOPB was confirmed by western blot using chicken raised anti-TcoOPB antibodies. Cross-reactivity of chicken anti-TcoOPB antibodies with TbPOP was also assessed and no cross-reactivity was found which was expected as POP and OPB only share 25% sequence identity. In order to determine the biochemical characteristics of TbPOP and TcoOPB, various activity assays and kinetics studies were conducted. It was found that TbPOP was able to hydrolyse type I collagen from rat tail. In contrast however, TbPOP was unable to digest gelatin which is a denatured form of collagen. Upon further analysis of TbPOP with the synthetic peptide substrate Z-Gly-Pro-AMC, it was found not to have activity as it was unable to hydrolyse the substrate, this is thought to be due to the misfolding of the protein during the refolding step. TcoOPB on the other hand was unable to hydrolyse either collagen or gelatin. Further biochemical analysis of TcoOPB was conducted using synthetic peptide substrates, the kinetic parameters of TcoOPB Km, kcat/Km were determined and it was found that OPB had a high affinity for the substrates Z-Arg-Arg-AMC, Z-Gly-Gly-Arg-AMC, H-Ala-Phe-Lys-AMC and Z-Pro-Arg-AMC and lower affinity for the substrates H-Pro-Phe-Arg-AMC, H-D-Val-Leu-Lys-AMC, Z-Gly-Pro-AMC, Suc-Ala-Phe-Lys-AMC,Boc-Leu-Gly-Arg-AMC. OPB was also found to have an optimal pH of 8 – 9 and retained 79% of its optimal activity at the physiological pH of 7.4. TcoOPB was found not to have good diagnostic potential as an indirect ELISA revealed that the antigen was unable to detect antibodies in T. congolense infected cattle sera. This study laid the foundation to conduct further studies on TbPOP, TcoPOP and TcoOPB as chemotherapeutic and diagnostic targets for Nagana.
Bioremediation of soil contaminated with a mixture of chlorinated aliphatic hydrocarbons.
(2008) Rajpal, Deseree A.
Chlorinated aliphatic hydrocarbons (CAH’s) are a diverse group of industrial chemicals that play a significant role as pollutants of soil and groundwater. They are recalcitrant and resist degradation in most waste treatment systems. Furthermore, physical removal techniques used for CAHs are often very expensive, labour intensive and time consuming. Microbial communities native to contaminated areas are known to participate in biodegradation of these CAHs to an extent. The main focus of this study was therefore to investigate the bioremediation of soil contaminated with a mixture of CAHs, namely carbon tetrachloride (CCl4), dichloromethane (DCM) and 1, 2 dichloroethane (1, 2-DCA). Two different laboratory-scale microcosm types, a stationary microcosm (Type S) and microcosms that received a continuous circulation of groundwater (Type C) were used to determine the effects of 3 different bioremediation approaches, viz, biostimulation, bioaugmentation and a combination of biostimulation and bioaugmentation on the degradation process. For both microcosm types, gas chromatography analysis revealed that the greatest decreases in CAH concentrations occurred in soil that was biostimulated. 1, 2-DCA was rapidly biodegraded in Type C microcosms that contained glucose, with a 57% net degradation in 15 days. Consortia comprising of aerobic Bacillus and Alcaligenes sp. were used for bioaugmenting contaminated soil. However, this approach did not promote biodegradation as significantly as biostimulation experiments. A combination of biostimulation and bioaugmentation revealed that the addition of nutrients was still unable to induce the degradative ability of the introduced microorganisms to produce degradation values comparable to those of biostimulated soil microcosms. Common intermediates of CAH metabolism viz., chloroform, dichloromethane and carbon dioxide were detected by gas chromatography/mass spectrometry. The detection of chloroform and dichloromethane is sufficient evidence to assume that anaerobic conditions had developed, and that biodegradation was occurring under oxygen-limiting or oxygen-free conditions. An aerobic environment was initially created, but soil microbial respiration had probably led to the rapid development of anaerobic conditions and in all likelihood, enhanced degradation. The prevalence of anaerobic conditions can also account for the lack of appreciable degradation by the bacterial consortium used during bioaugmentation. Phospholipid phosphate analysis was conducted and used as an indicator of microbial biomass. It was noted that phospholipid phosphates did not always correlate with the degradation of CAHs in some microcosms. In this regard, different patterns were noted for Type S and Type C microcosms. Microbial biomass patterns for Type C biostimulated and bioaugmented soil microcosms increased within the first 5 days of sampling. This could have been as a result of the larger volume of groundwater required for the circulating microcosm possibly concealing actual CAH concentrations. In contrast, in Type S microcosms, for most treatments, a sharp decline in biomass within the first week was observed. This study clearly demonstrates that the bioremediation of certain chlorinated solvents can be a function of their water solubility. It must also be emphasized that the biodegradation of some CAHs in a mixture can affect the concentrations of others present in the mixture as well, warranting further study with mixtures of CAHs. Furthermore, the development and use of bioreactors, similar to the Type C microcosm can provide novel, simple ways to hasten remediation of chlorinated solvents like 1, 2-DCA.
Biotyping of clinical mycobacterium tuberculosis isolates using MALDI-TOF MS.
(2013) Myende, Pride Siyanda.; Govender, Patrick.
Tuberculosis continues to be a major threat in public health; 8.8 million incidence of TB has been reported and 2 million deaths every year. Diagnosis of TB is impeded by slow growth of an organism with a generation time of 21 days. The emergence of multidrug-resistant TB isolates which are resistant to rifampicin and isoniazid worsened the treatment programme. Furthermore, surfacing of extensively drug-resistant TB isolates especially in HIV positive patients has led to a treatment failure. Currently available diagnostic methods are time consuming and laborious. Polymerase chain reaction-based assay proved to have a better resolution for TB strain discrimination, nevertheless are costly and cannot be routinely employed in resource poor settings. As a result there is an urgent need of cheap, cost effective and rapid diagnostic methods that will reduce a turnaround time. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry potentially offers an alternative rapid and cheaper method for discrimination of TB isolates. Proper discrimination of TB isolates depends on the sample preparation method that is capable of yielding high protein content. Conventional formic/ethanol sample preparation was investigated for mycobacteria MALDI-TOF mass spectrometric analysis. Poor quality of spectra was obtained due to a complex cell wall structure of mycobacteria which released less amounts of proteins. Further attempts were made to optimize the sample preparation method by introducing glass beads for maximum cell wall disruption. Non-consistent spectra were obtained in some mycobacterial strain; therefore it was not used as a method of choice. Introduction of delipidation step using chloroform/methanol (1:1, v/v) before formic/ethanol sample preparation step, led to a generation of reproducible and consistent spectra. This newly developed method was selected to extract protein content from large number of clinical TB isolates. With MALDI-TOF MS and chloroform/methanol-based method, all mycobacterial isolates used in the proof-of-concept were correctly identified and clustered. Fifty six of sixty clinical TB isolates were correctly identified using Biotyper software. Four were incorrectly identified; it might be possible that they carry mutations in unknown regions in their genome which led to a translation of proteins that affected the overall spectra profile. MALDI-TOF MS showed the potential to be used in the clinical laboratories for discrimination of TB isolates at lower costs.
Canine anti-endotoxin immunotherapy in cranial mesenteric arterial occlusion shock and canine parvovirus disease endotoxaemia.
(1986) Wessels, Brian C.; Gaffin, Stephen L.
Endotoxin (LPS, lipopolysaccharide) forms an integral part of the outer cellular membrane of gram negative bacteria (GNB). The canines' intestine always contains large amounts of GNB, and hence LPS. If these GNB with their LPS, remain within the intestinal lumen, they are not harmful to the host. When GNB do gain entry into a hosts' circulation a bacteraemia will occur with a concurrent endotoxaemia. In the past, it had been accepted that GNB were, themselves, primarily responsible for the mortality and morbidity of bacteraemic and septicaemic patients. Evidence has emerged to indicate that this is not altogether true as isolated LPS, without the presence of GNB, can also lead to fatalities. Circulating LPS is exceptionally chemically stable and highly toxic to host cells. Antimicrobial chemotherapy can destroy GNB, but this therapy does not reduce the toxicity of LPS, nor does it clear LPS from the circulation. Destruction of the GNB by certain antibiotics can, in fact, increase the concentration of circulating plasma LPS in a host. The functional integrity of the intestinal wall is highly dependent upon an adequate blood supply, and the mucosal cells acts as the primary defence against the potentially pathogenic, endogenous and exogenous GNB and LPS. Once these pathogens become intravascular then the liver is the next most important organ of defence. Shock, irrespective of its aetiology, without adequate therapy, leads to reduced micro-vascular circulation, and thus a state of either localised or generalised hypoxia occurs. Partial or complete intestinal vascular ischaemia will produce a state of regional hypoxia, and lead to damage of the intestinal wall allowing GNB, with their LPS, or LPS by itself, to enter into the hosts' blood circulation. Therefore, an aetiology that gives rise to any type of "classified shock," may eventually give rise to concurrent endotoxaemia. In clinical practice there are numerous different diseases, physical onslaughts, and either acquired or congenital anatomical defects, that can give rise to intestinal vascular ischaemia, and hence, endotoxaemia. Many treatment regimens to combat the effects of an endotoxaemia have been advocated over the years, but this problem still has an unacceptably high mortality and morbidity index, probably because almost all such therapeutic regimens fail to destroy the LPS molecule. Recent clinical studies have shown that immunotherapy is effective in combating gram negative bacteraemia and septicaemia in humans and animals. Research workers have been able to produce a "broad- spectrum" or "polyvalent" equine, hyperimmune, anti-endotoxir, antibody-enriched plasma (ANTI- LPS), with favourab"^ responses recorded when this plasma was used to treat a variety of experimentally-induced endotoxin-shocked subjects. ANTI-LPS significantly reduced the mortality in experimentally produced superior mesenteric arterial occlusion endotoxaemia in rabbits, presumably by neutralizing and opsonizing the circulating plasma LPS. Equine practitioners have reported successful results when ANTI-LPS was incorporated into the treatment of certain medical and surgical equine endotoxic related problems. A ^/ery recent, independent, Canadian study showed the effectivness of ANTI-LPS, where this preparation was tested against other anti-LPS products, to treat experimentally-induced sepsis in rats. The polyvalent equine ANTI- LPS was the most effective, in that its use resulted in the longest survival. In order to establish the generality of the use of equine ANTI-LPS plasma, I have extended these studies to the canine, since an abdominal vascular ischaemia carries a serious, high-risk, surgical emergency with unsatisfactorily high mortality rates, despite successful surgical intervention with concurrent supportive medical therapy. Twenty healthy dogs were divided into four groups; a control group (n=5) and three experimentally treated groups (n=5 in each group). All twenty dogs were subjected to the well-documented cranial (superior) mesenteric arterial occlusion (CMAO) shock model. The three experimental groups received the polyvalent equine, ANTI-LPS at different times and by two different routes, with no side effects being observed in any of these dogs. One group (n=5)received ANTI-LPS s.c. before CMAO was performed, a second group (n= 5) received their dosage of ANTI-LPS i.v. during the three-hour occlusion period, and a third group (n=5) received their dose s.c, within three minutes after the CMAO was released. Survival was recorded when any dog lived for a minimum of 14 days after the occluded vessel was released. All 5/5 (100%) controls died within 17 hours after the release of the occluded vessel, whereas only one of the 15 (6,5%) experimentally ANTI-LPS treated dogs died (P
Cationic liposome mediated targeted gene delivery with and without pegylated accessories.
(2009) Narainpersad, Nicolisha.; Singh, Moganavelli.; Ariatti, Mario.
As a consequence of safety issues encountered by the use of viral vectors in gene therapy, there has been a steady increase in the development and application of non-viral vectors, especially liposomes. Cationic liposome mediated delivery is one of the most promising nonviral delivery methods. These liposomes are prepared from synthetic lipids, are positively charged and interact favourably with DNA through electrostatic interactions. Cationic liposomes have also shown immense potential in the targeting of specific cell types such as HepG2 (hepatocellular carcinoma) cells, a model in vitro gene delivery system for the study of hepatocyte function. However, these liposomes also have a number of limitations in vivo. In an attempt to overcome these restrictions, a hydrophilic polymer, polyethylene glycol (PEG) is incorporated into the cationic liposome. This covalent attachment of (PEG) to the liposomal surface is thought to sterically stabilise liposomes, promote biological stability, inhibit aggregation, decrease toxicity and immunogenicity, prevent interaction with serum proteins and complement and thus prolonging the circulation time of liposomes in vivo. The versatility and simplicity of cationic liposomes have made them vitally significant non-viral gene delivery vehicles for human gene therapy. In this investigation novel untargeted and targeted glycosylated liposomes with and without PEG were synthesised to evaluate their gene transfer activities in vitro to potentially develop a suitable gene delivery system for future in vivo applications. A constant molar quantity of the cationic cholesterol derivative, 3 [N-(N’, N’-dimethylaminopropane)-carbamoyl] cholesterol (CHOL-T) was mixed with dioleoylphosphatidylethanolamine (DOPE) and a galactose/glucose derivative to produce targeted cationic liposomes. PEG liposomes were prepared in the same way with the addition of distearoylphosphoethanolamine polyethylene glycol 2000 (DPSE-PEG2000), 2% on a molar basis. Supported by transmission electron microscopy characterisation, we present evidence that the pegylation of liposomes affects the DNA binding capability and transfection efficiencies of the cationic liposomes in addition to protecting the plasmid DNA in lipoplexes from serum nuclease degradation. Optimal DNA : liposome binding ratios were obtained from gel retardation studies and confirmed by ethidium bromide intercalation assays. These complexes were then tested on the human hepatoma cell line, HepG2, to determine toxicity and assess transfection efficiencies. From results obtained in this study, it appears that both cationic and pegylated cationic liposomes are well tolerated by cells in vitro. The results further suggest that targeting by use of glycolipids incorporated into the structure of the liposome increases transfection, while pegylation of cationic liposomes marginally decreases the transfection efficiency of the lipoplexes to HepG2 cells in vitro.

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