Masters Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Subject "Acetylsalicylic acid"
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Item Open Access Oxylipin distribution in Eremothecium(University of the Free State, 2006-11) Leeuw, Ntsoaki Joyce; Kock, J. L. F.; Pohl, C. H.; Van Wyk, P. W. J.English: In the early 1990’s, Kock and co-workers discovered acetylsalicylic acid (ASA)-sensitive oxylipins in yeasts. It was also reported that the site of production of these compounds may serve as important targets to control fungal infections. In 2004, researchers exposed another function for these oxylipins – they may act as lubricants during spore release from enclosed asci. Since oxylipin production in only a limited number of species representing Eremothecium was thus far studied, it became the aim of this project to further extend this study and to determine the type and distribution of 3- hydroxy (OH) oxylipins in the remaining species i.e. Eremothecium coryli, E. cymbalariae and E. gossypii. In addition, the possible functions of these oxylipins as well as ascospore shape and ornamentations were assessed. Finally, the antifungal activity of ASA was also investigated in this group of important plant pathogens as well as other yeasts. Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. In this study, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluoresence confocal laser scanning microscopy it was found that hydrophobic 3-OH oxylipins cover the surfaces of these ascospores. Using gas chromatography-mass spectrometry, only the oxylipin 3-OH 9:1 (a monounsaturated fatty acid consisting of a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies, it was possible to map the presence of these compounds also in other Eremothecium species. In E. cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in E. gossypii, oxylipins covered the whole spindle-shaped ascospore with terminal appendages. The presence of these oxylipins was confirmed by chemical analysis. When ASA, a 3-OH oxylipin inhibitor, was added to these yeasts in increasing concentrations, the sexual stage was found to be the most sensitive. Results suggest that 3-OH oxylipins, produced by mitochondria through incomplete β-oxidation, are associated with the development of the sexual stages in both yeasts. Strikingly, preliminary studies on yeast growth suggest that yeasts, characterized by mainly an aerobic respiration rather than a fermentative pathway, are more sensitive to ASA than yeasts characterized by both pathways. These data further support the role of mitochondria in sexual as well as asexual reproduction of yeasts and its role to serve as target for ASA antifungal action.Item Open Access Oxylipins in automictic yeast life cycles(University of the Free State, 2007-11) Swart, Chantel Wendy; Kock, J. L. F.; Van Wyk, P. W. J.; Pohl, C. H.English: 3-OH oxylipins are saturated and unsaturated oxidized fatty acids which are produced in mitochondria via incomplete b-oxidation or fatty acid synthesis type II. These compounds possibly play an important role in the sexual cycle of yeasts by assisting with ascospore liberation. Literature suggests that 3-OH oxylipins act as a lubricant during ascospore liberation, thereby ensuring efficient release of ascospores. Since the discovery of oxylipins i.e. 3-hydroxy (OH) oxylipins in the early 1990’s, these compounds were found to be distributed in various species of the fungal domain. Studies performed thus far, however, focused mainly on non-fermenting yeast species such as Ascoidea, Dipodascopsis and Eremothecium. According to various studies performed so far, 3-OH oxylipins were found to accumulate specifically in the sexual structures (asci and surrounding ascospores) of various nonfermenting yeasts. These studies also revealed that the sexual stages of nonfermenting yeasts are most susceptible to acetylsalicylic acid (ASA), a known mitochondrial (respiration and 3-OH oxylipin production) inhibitor. No information regarding oxylipin accumulation in asci or ASA-sensitivity of fermentative yeasts, however, has so far been reported. Using confocal laser scanning microscopy in combination with an oxylipin probe for 3-OH oxylipins and coupled to a fluorescing secondary antibody, the accumulation of these oxylipins was discovered in the asci of the following fermentative yeasts i.e. Pichia anomala, Pichia farinosa and Schizosaccharomyces octosporus. Interestingly, no 3-OH oxylipin accumulation was observed in the asci of the fermenting yeast Zygosaccharomyces bailii. This could be ascribed to the fact that this yeast depends more on a fermentative pathway for growth and sexual reproduction, than on mitochondrial respiration. Since 3-OH oxylipins are produced in the mitochondria, it is expected that there should be an increase in mitochondrial activity associated with these sexual structures. Using confocal laser scanning microscopy and a mitochondrial fluorescing probe (Rhodamine 123), an increase in mitochondrial activity was also observed in the asci of the fermenting yeasts tested, again with the exception of Z. bailii. Furthermore, during this study links between yeast sexual reproduction, 3-OH oxylipin accumulation/production, mitochondrial activity and oxygen requirement were established. This study revealed that fermenting yeasts are more resistant to ASA than non-fermenting yeasts when grown in liquid media. This is probably due to the fact that these yeasts can use either aerobic respiration or a fermentative pathway for growth and reproduction. This research prompted the development of a bio-assay that may find application in screening for effective antimitochondrial antifungals.Item Open Access Oxylipins in the yeast genus Ascoidea(University of the Free State, 2007-05) Ncango, Desmond Mbulelo; Kock, J. L. F.; Van Wyk, P. W. J.; Pohl, C. H.; Joseph, M.English: Acetylsalicylic acid (ASA)-sensitive 3-hydroxy (3-OH) oxylipins were uncovered in 1991 in the yeast Dipodascopsis uninucleata. Since then, various similar oxylipins were found to be widely distributed in fungi. Interestingly, 3-OH oxylipins were reported to play a role in ascospore release from enclosed asci, where they are involved in assisting nano-scale gear-like (D. uninucleata); sliding (Dipodascus); drilling (Eremothecium sinecaudum) and piercing movements (E. ashbyi and E. coryli). In Ascoidea africana, a 3- OH 10:1 oxylipin was found to be associated with hat-shaped ascospores carried inside ellipsoidal asci. However, in this study no function was proposed for this oxylipin. Since only one species representing the genus Ascoidea was studied, it became the aim to further expand this study to also include A. corymbosa and A. rubescens. Using confocal laser scanning microscopy (CLSM) on cells stained with fluorescein-coupled 3-OH oxylipin specific antibodies, this study suggests that oxylipins are specifically associated with ascospores and not vegetative cells of A. corymbosa and A. rubescens. Using gas chromatography - mass spectrometry (GC-MS) the oxylipin, 3-OH 17:0, was identified in A. corymbosa. Here, oxylipin-coated razor sharp ascospore brims may play a role in rupturing the ascus to affect forced release of hat-shaped ascospores. Literature suggests that 3-OH oxylipins are produced by β-oxidation or fatty acid synthesis in mitochondria of yeasts. Since these oxylipins accumulate in sexual cells (asci), increased mitochondrial activity is therefore expected in these structures. Strikingly, this assumption is supported in this study. Using selective fluorescence mitochondrial staining and CLSM, evidence is provided that mitochondrial function is much higher in asci containing increased amounts of 3-OH oxylipins compared to the corresponding asexual vegetative cells. Furthermore, when ASA, a mitochondrial inhibitor, was added in increased concentrations to cultures of Ascoidea, the sexual stage was found to be more sensitive. Ascospore liberation from asci was first inhibited followed by asci formation while some vegetative growth could still be observed.