Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)
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Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Subject "3-HETE"
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Item Open Access The production of 3-hydroxy fatty acids by the yeast Dipodascopsis uninucleata and its implications(University of the Free State, 1999-11) Venter, Pierre; Kock, J. L. F.; Coetzee, D. J.English: In 1991, a novel eicosanoid namely 3-hydroxy-5, 8, 11, 14-eicosatetraenoic acid (3-HETE) was uncovered in the yeast Dipodascopsis uninucleata by van Dyk and co-workers. Strikingly, the production of this compound was found to be sensitive to low concentrations of aspirin and indomethacin. With this as background, a study was conducted that unveiled the possible biochemical pathway used by this yeast for the production of 3-HETE. Here, various fatty acids were fed to D. uninucleata, and the extracted samples analysed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography - mass spectrometry. It was found that 3-hydroxylation of fatty acids in D. uninucleata requires a 5Z, BZ - diene system either directly or following initial incomplete l3-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy - 5Z, BZ, 11Z, 14Z - eicosatetraenoic acid (3R - HETE), which rules out normal l3-oxidation as a biosynthetic route. Consequently, studies on the biological dynamics and distribution of 3-hydroxy oxylipins in D. uninucleata followed. The occurrence of oxylipins was mapped by immunofluorescence microscopy (IF) in fixed cells, with or without cell walls, using an antibody raised against 3R-HETE. This antibody turned out to cross-react with other 3-hydroxy oxylipins. These compounds were detected in situ in gametangia, asci, as well as between released aggregating ascospores. Aspirin (1mM), which is known to suppress the formation of 3-hydroxy oxylipins from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as cell aggregation, suggesting a prominent regulatory role of 3-hydroxy oxylipins for the latter. Since these oxylipins are associated with the aggregation of sexual cells in D. uninuc/eata, the next step was to screen for the presence of these compounds in aggregating cells of other yeasts such as the biotechnological important Saccharomyces cerevisiae. It was found that oxylipins such as 3-hydroxy-8:0 and 3-hydroxy-10:0 are produced over the growth cycle of the flocculating yeast Saccharomyces cerevisiae ATCC 26602. Using oxylipin specific antibodies in IF studies, it was demonstrated that these compounds are synthesized continuously from an early stage of growth and are associated with the cell wall and are present between flocculating cells. Similar results were obtained with a NewFlo phenotype flocculent brewing yeast strain. This implicated the involvement of oxylipins in cell aggregation. Further investigations using scanning- and transmission electronmicroscopy, indicated that changes in the depositing of lipid rich osmiophilic layers in the yeast followed the same pattern as the IF results. Immunogold studies verified the presence of oxylipins in these osmiophilic layers. It was uncovered that the oxylipin containing osmiophilic layers play an important role in cell aggregation. Surprisingly, further investigations implicated the presence of aspirin sensitive 3-hydroxy oxylipins in the LPS layer of the Gram-negative bacterium Escherichia co/i. In this study it was found that aspirin, at moderate concentrations, influences the biosynthesis of the endotoxic 3-hydroxylated myristic acid in the Lipid A of Gram-negative bacteria. This discovery therefore suggests an important role for aspirin as a therapeutic agent in the treatment of LPS mediated diseases