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dc.contributor.advisorKock, J. L. F.
dc.contributor.advisorCoetzee, D. J.
dc.contributor.authorVenter, Pierre
dc.date.accessioned2018-07-23T09:06:04Z
dc.date.available2018-07-23T09:06:04Z
dc.date.issued1999-11
dc.identifier.urihttp://hdl.handle.net/11660/8912
dc.description.abstractEnglish: 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 diseasesen_ZA
dc.description.abstractAfrikaans: 'n Nuwe eikosanoïed genaamd 3-hidroksie 5, B, 11, 14 eikosatetraenoësuur (3-HETE) is in 1991 deur Van Dyk en medewerkers in die gis Dipodascapsis uninueleata ontdek. Dit was opvallend dat die produksie van hierdie komponent sensitief was vir lae konsentrasies aspirien en indometasien. Met hierdie inligting as agtergrond is 'n studie uitgevoer wat 'n moontlike biochemiese weg uitwys vir die produksie van 3-HETE deur hierdie gis. In hierdie studie is verskeie vetsure vir D. uninueleata gevoer en die geëkstraheerde monsters geanaliseer vir die teenwoordigheid van 3-hidroksiemetaboliete m.b.v. elektron impak gaschromatografie - massaspektrometrie. Daar is bevind dat 3-hidroksielering van vetsure in D. uninucleata, 'n 5c, Be - diëen sisteem benodig en deur direkte of opvolgende onvoliedige B-oksidasle plaasvind. Analises aangaande die enantiomeer-samestelling het gevolg en die aragidoonsuur-metaboliet is geïdentifiseer as 3R-hidroksie - 5c, Bc, 11c, 14e - eikosatetraenoësuur (3R-HETE) wat normale -oksidasie as 'n biosintetiese weg onwaarskynlik maak. Gevolglik is studies rakende die biologiese dinamika en distribusie van 3-hidroksie-oksilipiene in D. uninueleata gedoen. Immunofluoresensie mikroskopie (IF) is gebruik om die posisie van oksilipiene in gefikseerde selie, met en sonder selwande, vas te stel deur gebruik te maak van 'n teenliggaampie wat opgewek is teen 3R-HETE. Gevolglik is ook vasgestel dat hierdie teenliggaampie met ander 3-hidroksie-oksilipiene kruisreageer. Hierdie verbindings is waargeneem in situ in gametangia, askusse, asook tussen vrygestelde aggregerende askospore. Aspirien (1mM), wat daarvoor bekend is dat dit die produksie van 3-hidroksieoksilipiene vanaf eksogeniese poliënoïese vetsure onderdruk, het ook die teenwoordigheid van immunoreaktiewe materiaal en selaggregasie onderdruk wat daarop wys dat 3-hidroksie-oksilipiene 'n prominente regulerende rol by laasgenoemde speel. Siende hierdie oksilipiene geassosieer is met die aggregerende seksuele selle van D. uninucleata, was die volgende stap om te bepaal of hierdie verbindings ook teenwoordig is in aggregerende selle van ander giste soos die biotegnologies-belangrike Saccharomyces cerevisiae. Dit is bevind dat oksilipiene soos 3-hidroksie-8:0 en 3-hidroksie-10:0 geproduseer word tydens die groeistadia van· die flokkulerende gis Saccharomyces cerevisiae ATCC 26602. Deur gebruik te maak van die oksilipien-spesifieke teenliggaampies in IF studies, is gedemonstreer dat hierdie verbindings voortdurend gesintetiseer word vanaf die vroeë stadia van groei en later geassosieer is met die selwand. Die laasgenoemde is ook teenwoordig tussen flokkulerende selle. Soortgelyke resultate is verkry met 'n "NewFlo" fenotiepe flokkulerende brouersgis-stam. Dit impliseer ook die betrokkenheid van oksilipiene by selaggregasie. Verdere ondersoeke is ook uitgevoer met die skandeer- en transmissie elektronmikroskoop. Dit het uitgewys dat veranderinge in die neerslag van lipiedryke osmiofiliese lae in die gis dieselfde patroon as die IF resultate volg. Immunogoud-studies het die teenwoordigheid van oksilipiene in hierdie osmiofiliese lae bevestig. Dit is dus duidelik dat die osmiofiliese lae 'n belangrike rol by selaggregasie speel. Verdere navorsing het, bo-verwagting, die. teenwoordigheid van aspiriensensitiewe 3-hidroksie-oksilipiene in die LPS laag van die Gram-negatiewe . bacterium Escherichia coli uitgewys. Dit is bevind dat aspirien, teen matige konsentrasies, die biosintese van die endotoksiese 3-hidroksie-miristiensuur in die Lipied A van Gram-negatiewe bakteriëe beïnvloed. Die laasgenoemde bevinding is van uiterste belang, veral in die behandeling van LPS-bemiddelde siekte toestande. Die bevinding wys daarop dat aspirien 'n belangrike rol kan speel as 'n geneesmiddel in bogenoemde siekte toestande.en_ZA
dc.description.sponsorshipFoundation for Research Development, South Africaen_ZA
dc.description.sponsorshipVolkswagen Foundation, Germany (1/74643)en_ZA
dc.language.isoenen_ZA
dc.publisherUniversity of the Free Stateen_ZA
dc.subjectArachidonic aciden_ZA
dc.subjectDipodascopsis uninucleataen_ZA
dc.subjectFlocculationen_ZA
dc.subjectGram negative bacteriaen_ZA
dc.subject3-HETEen_ZA
dc.subject3-Hydroxy fatty acidsen_ZA
dc.subjectImmunofluorescenceen_ZA
dc.subjectNSAIDsen_ZA
dc.subjectβ-oxidationen_ZA
dc.subjectSaccharomyces cerevisiaeen_ZA
dc.subjectYeast fungien_ZA
dc.subjectFatty acidsen_ZA
dc.subjectEicosanoic aciden_ZA
dc.subjectThesis (Ph.D. (Microbiology and Biochemistry))--University of the Free State, 1999en_ZA
dc.titleThe production of 3-hydroxy fatty acids by the yeast Dipodascopsis uninucleata and its implicationsen_ZA
dc.typeThesisen_ZA
dc.rights.holderUniversity of the Free Stateen_ZA


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