RESUMEN
Infection of human tissues by Candida albicans has been reported to cause the release of arachidonic acid (AA), eicosanoids and other proinflammatory mediators from host cells. Therefore, we investigated the interaction of this pathogen with AA. AA stimulated cell growth at micromolar concentrations when used as a sole carbon source. Moreover, it selectively inhibited the antimycin A-resistant alternative oxidase. [1-(14)C]AA was completely metabolised by C. albicans. Only one-seventh of the radioactivity metabolised was found in CO(2), whereas two-thirds occurred in carbohydrates suggesting a predominant role of the glyoxalate shunt of citrate cycle. About 1% of radioactivity was found in polar lipids including eicosanoids. A novel AA metabolite, which revealed immunoreactivity with an antibody against 3(R)-hydroxy-oxylipins, was identified as 3, 18-dihydroxy-5,8,11,14-eicosatetraenoic acid. Using immunofluorescence microscopy, endogenous 3(R)-hydroxy-oxylipins were found in hyphae but not in yeast cells. Such compounds have recently been shown to be connected with the sexual stage of the life cycle of Dipodascopsis uninucleata. Together, we propose that infection-mediated release of AA from host cells may modulate cell growth, morphogenesis and invasiveness of C. albicans by several modes. A better understanding of its role is thus promising for novel approaches towards the treatment of human mycoses.
Asunto(s)
Ácido Araquidónico/farmacología , Candida albicans/efectos de los fármacos , Ácido Araquidónico/metabolismo , Candida albicans/crecimiento & desarrollo , Candida albicans/metabolismo , Radioisótopos de Carbono , División Celular/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Ácidos Hidroxieicosatetraenoicos/metabolismo , Ácido Linoleico/farmacología , Microscopía Fluorescente , Proteínas Mitocondriales , Oxidorreductasas/antagonistas & inhibidores , Proteínas de Plantas , Ácido Salicílico/farmacologíaRESUMEN
Dipodascopsis uninucleata has been recently shown to produce 3-hydroxy polyenoic fatty acids from several exogenous polyenoic fatty acids. In order to examine whether endogenous 3-hydroxy fatty acids (3-OH-FA) may be implicated in the developmental biology of this yeast, we mapped by immunofluorescence microscopy their occurrence in fixed cells with or without cell walls using an antibody raised against 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid (3R-HETE), the biotransformation product from arachidonic acid (AA). This antibody turned out to cross-react with other 3-OH-FA. 3-OH-FA were detected in situ in gametangia, asci, as well as between released ascospores, and proved to be associated with the sexual reproductive stage of the life cycle of the yeast. Acetylsalicylic acid (1 mM), which is known to suppress the formation of 3-OH-FA from exogenous polyenoic fatty acids, inhibited the occurrence of immunoreactive material as well as the sexual phase of the life cycle suggesting a prominent regulatory role of 3-OH-FA for the latter.
Asunto(s)
Ascomicetos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Animales , Anticuerpos/inmunología , Ascomicetos/fisiología , Ascomicetos/ultraestructura , Reacciones Cruzadas , Ácidos Grasos Insaturados/inmunología , Ácidos Grasos Insaturados/fisiología , Ácidos Hidroxieicosatetraenoicos/inmunología , Microscopía Fluorescente , Conejos , ReproducciónRESUMEN
When arachidonic acid (AA) is added to the yeast Dipodascopsis uninucleata UOFS Y128, one of the major metabolites isolated and purified with the help of thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) is 3-hydroxy-5,8,11,14-eicosatetraenoic acid (3-HETE). The structure of this new AA metabolite was elucidated mainly by electron impact (EI) mass spectrometry (MS). Strikingly, the formation of this new metabolite was found to be inhibited by aspirin.
Asunto(s)
Ácidos Araquidónicos/metabolismo , Ácidos Hidroxieicosatetraenoicos/aislamiento & purificación , Saccharomycetales/metabolismo , Ácido Araquidónico , Aspirina/farmacología , Cromatografía Líquida de Alta Presión , Cromatografía en Capa Delgada , Ácidos Hidroxieicosatetraenoicos/química , Espectrometría de Masas , Saccharomycetales/efectos de los fármacos , EspectrofotometríaRESUMEN
It was discovered that the addition of 10 g/l acetate to a medium containing 30 g/l sunflower oil caused a drastic increase in citric acid production by Yarrowia lipolytica UOFS Y-1701 i.e. from 0.5 g/l in the absence of acetate to 18.7 g/l in the presence of acetate. Similarly, the ratio of citric acid:isocitric acid increased significantly from 1.7:1 in the absence of acetate to 3.7:1 in the presence of acetate after 240 h of growth.
Asunto(s)
Acetatos/farmacología , Ácido Cítrico/metabolismo , Aceites de Plantas/metabolismo , Yarrowia/efectos de los fármacos , Yarrowia/metabolismo , Isocitratos/metabolismo , Aceite de GirasolRESUMEN
In order to develop a rapid yeast identification technique using fatty acid profiles, an extensive survey has been conducted in our laboratory on the long-chain fatty acid composition (CLFAC) of yeasts representing the endomycetous and basidiomycetous yeast domain. It was accomplished by cultivating the yeast strains under standardized conditions in a synthetic liquid medium. When stationary phase was reached the cells of each culture were freeze-dried and the CLFAC was examined by gas chromatography. It was found that the fatty acid profile obtained for each strain was reproducible. However, as work progressed, it became clear that variation exists within species and that the relative percentages of some strains from different species may overlap. Identification of species could therefore not be achieved in all attempts, even when the resolution of the fatty acid analyses was enhanced by using capillary columns, useful for the detection of minor fatty acids. When used in isolation, CLFAC analyses is therefore not a generally applicable identification technique for yeast species. However, the technique was found to be a valuable chemotaxanomical tool to distinguish between strains of certain species, species of certain genera and species from particular environments. The technique currently finds application in the South African food and beverage industry as a quick, cheap and easy way to distinguish between strains of Saccharomyces cerevisiae. It is also used by an industry which produces bioprotein from Geotrichum candidum, to determine fungal contaminants in a quality control process.
Asunto(s)
Ácidos Grasos/análisis , Microbiología de Alimentos , Levaduras/química , Levaduras/clasificación , Control de CalidadRESUMEN
Various fatty acids were fed to the yeast Dipodascopsis uninucleata UOFS Y 128, and the extracted samples were analyzed for the accumulation of 3-hydroxy metabolites with the help of electron impact gas chromatography-mass spectrometry. Fatty acids containing of 5Z,8Z-diene system (5Z,8Z,11Z-eicosatrienoic, 5Z,8Z,11Z,14Z-eicosatetraenoic, and 5Z,8Z,11Z,14Z,17Z-eicosapentaenoic acids) yielded the corresponding 3-hydroxy-all-Z-eicosapolyenoic acids. Moreover, linoleic acid (9Z,12Z-octadecadienoic acid) and 11Z,14Z,17Z-eicosatrienoic acid were converted to the 3-hydorxylated metabolites of shorter chain length, e,g., 3-hydroxy-5Z,8Z-tetradecadienoic acid and 3-hydroxy-5Z,8Z,11Z-tetradecatrienoic acid, respectively. In contrast, no accumulation of a 3-hydroxy metabolite was observed with oleic acid (9Z-octadecenoic acid), linolelaidic acid (9E,12E-octadecadienoic acid), gamma-linolenic acid (6Z,9Z,12Z-octadecatrienoic acid), and eicosanoic acid as substrate. These findings pinpoint that the 3-hydroxylation of a fatty acid in Dipodascopsis uninucleata requires a 5Z,8Z-diene system either directly or following initial incomplete beta-oxidation. Following analysis of the enantiomer composition, the arachidonic acid metabolite was identified as 3R-hydroxy-5Z,8Z,11Z,14Z-eicosatetraenoic acid, which rules out a normal beta-oxidation as biosynthetic route to this new class of oxylipins.
Asunto(s)
Ascomicetos/metabolismo , Ácidos Grasos Insaturados/metabolismo , Ácidos Araquidónicos/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Ácidos Hidroxieicosatetraenoicos/metabolismo , Hidroxilación , Estructura Molecular , Oxidación-Reducción , EstereoisomerismoAsunto(s)
Cacao/química , Hongos/química , Lípidos/farmacología , N-Formilmetionina Leucil-Fenilalanina/farmacología , Neutrófilos/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Técnicas In Vitro , Lípidos/química , Neutrófilos/efectos de los fármacos , Fosfatidilinositol 3-Quinasas , Proteína Quinasa C/antagonistas & inhibidores , Estallido Respiratorio/efectos de los fármacos , Estaurosporina/farmacología , Superóxidos/metabolismoAsunto(s)
Ácidos Grasos Insaturados/química , Ácidos Grasos Insaturados/metabolismo , Saccharomycetales/metabolismo , Señalización del Calcio/efectos de los fármacos , Quimiotaxis de Leucocito/efectos de los fármacos , Ácidos Grasos Insaturados/biosíntesis , Humanos , Ácidos Hidroxieicosatetraenoicos/farmacología , Técnicas In Vitro , Espectrometría de Masas , Estructura Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Saccharomycetales/crecimiento & desarrolloAsunto(s)
Ácidos Grasos Insaturados/biosíntesis , Saccharomycetales/metabolismo , Ácidos Grasos Insaturados/química , Ácidos Hidroxieicosatetraenoicos/biosíntesis , Ácidos Hidroxieicosatetraenoicos/química , Microscopía Electrónica de Rastreo , Saccharomycetales/crecimiento & desarrollo , Saccharomycetales/ultraestructura , Esporas Fúngicas/metabolismo , Esporas Fúngicas/ultraestructuraRESUMEN
Interesting distribution patterns of acetylsalicylic acid (ASA, aspirin) sensitive 3-hydroxy (OH) oxylipins were previously reported in some representatives of the yeast genus Eremothecium--an important group of plant pathogens. Using immunofluorescence microscopy and 3-OH oxylipin specific antibodies in this study, we were able to map the presence of these compounds also in other Eremothecium species. In Eremothecium cymbalariae, these oxylipins were found to cover mostly the spiky tips of narrowly triangular ascospores while in Eremothecium 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. Our results suggest that 3-OH oxylipins, produced by mitochondria through incomplete beta-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 a target for ASA antifungal action.
Asunto(s)
Aspirina/farmacología , Fungicidas Industriales/farmacología , Saccharomycetales/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Microscopía Electrónica de Rastreo , Microscopía Fluorescente , Mitocondrias/metabolismo , Saccharomycetales/metabolismo , Saccharomycetales/ultraestructuraRESUMEN
Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography-mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing 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.
Asunto(s)
Ácidos Grasos/metabolismo , Saccharomycetales/metabolismo , Esporas Fúngicas/metabolismo , Ácidos Grasos/aislamiento & purificación , Cromatografía de Gases y Espectrometría de Masas , Microscopía Confocal , Microscopía Electrónica de Rastreo , Saccharomycetales/ultraestructura , Esporas Fúngicas/ultraestructuraRESUMEN
Using transmission electron microscopy with glutaraldehyde and osmium tetroxide as chemical fixatives, hat-shaped ascospores with two brims each were uncovered in the yeast Ambrosiozyma platypodis. This is the first report on such structures.
Asunto(s)
Saccharomycetales/citología , Esporas Fúngicas/aislamiento & purificación , Esporas Fúngicas/ultraestructura , Pared Celular/ultraestructura , Citoplasma/ultraestructura , Microscopía Electrónica de Transmisión , Saccharomycetales/ultraestructuraRESUMEN
Ungerminated ascospores of Dipodascopsis uninucleata contained 18 times more lipid (5.5 % dry wt) than germinated cells; the lipid comprised 58 % (w/w) glycolipids, 28 % (w/w) neutral lipids (mainly triacylglycerols)and 14 % (w/w) phospholipids (mainly phosphatidylcholine and phosphatidylethanolamine). During germination the absolute amounts of all three lipid fractions fell sharply but, during the subsequent initiation of hyphal growth,the amount of phospholipids increased. As these hyphae began to differentiate for the sexual stage of the life cycle,the amount of neutral lipid then increased. The fatty acyl groups of the glyco-, neutral and phospholipid fractions throughout the life cycle were mainly palmitate (16:0), oleate (18: 1) and linoleate (18:2). The percentage of 16:O remained constant during the life cycle while the relative amounts of 18:2 plus alpha-linolenate (18:3) in the glyco-,neutral and phospholipid fractions first increased during initiation of growth and then decreased during the onset of differentiation. The opposite trend occurred with 18: 1. When [(3)H]arachidonic acid (ARA) and [1-(14)C]181: were fed separately to D. uninucleata, both were rapidly incorporated into phospholipids. Highest incorporation of ARA was in the growth phase; during the onset and remainder of the differentiation phase, the amount of ARA decreased in this fraction. Incorporation of 18: 1 increased during growth and differentiation, with a significant proportion(49% to 57%) being incorporated into triacylglycerols compared to a much smaller proportion (12% to 17%) of ARA.
Asunto(s)
Ácido Araquidónico/metabolismo , Metabolismo de los Lípidos , Levaduras/metabolismo , Germinación , Esporas Fúngicas/crecimiento & desarrollo , Esporas Fúngicas/metabolismo , Levaduras/química , Levaduras/crecimiento & desarrolloRESUMEN
Using radioimmunoassay, blood platelet aggregation studies and GC-MS the existence of prostaglandins in the endomycetalean yeast Dipodascopsis uninucleata was confirmed by our group. These findings triggered the search for similar eicosanoids in the rest of the Endomycetales. We commenced by scanning for the easily detectable precursors of eicosanoids, linoleic- and linolenic acid. We selected two families (i.e. Lipomycetaceae and Dipodascaceae), both producing these precursors, for further investigation. Representative strains of the two families were tested for their ability to grow in the presence of 1 mM aspirin, a specific inhibitor of prostaglandin biosynthesis. In contrast to the lipomycetaceous species the dipodascaceous species were insensitive to this drug. These results were verified when representative strains of both families were investigated for their ability to produce eicosanoids from externally fed radio-labeled arachidonic acid along an aspirin sensitive pathway. Thin layer chromatography of culture extracts, followed by autoradiography, showed that while none of the Dipodascaceae produced aspirin sensitive arachidonic acid metabolites, the members of the Lipomycetaceae tested positive for these metabolites. These findings supported the separation of the lipomycetaceous yeast Dipodascopsis from the Dipodascaceae. The findings also correlate with the delimitation of these yeasts in two families (i.e. Dipodascaceae and Lipomycetaceae). Further investigation indicated that prostaglandin production by the genus Dipodascopsis is mainly associated with ascosporogenesis. Thin layer chromatography of cell extracts from Dipodascopsis tóthii, followed by scintillation counting, indicated the presence of PGF2 alpha and PGE2 during ascosporogenesis.
Asunto(s)
Eicosanoides/análisis , Ácidos Grasos/análisis , Saccharomycetales/química , Ácido Araquidónico/metabolismo , Aspirina/farmacología , Cromatografía en Capa Delgada , Dinoprost/análisis , Dinoprost/fisiología , Dinoprostona/análisis , Dinoprostona/fisiología , Eicosanoides/fisiología , Cromatografía de Gases y Espectrometría de Masas , Radioinmunoensayo , Saccharomycetales/clasificación , Saccharomycetales/efectos de los fármacos , Saccharomycetales/fisiología , Esporas Fúngicas/fisiología , Ubiquinona/metabolismoRESUMEN
The cellular long-chain fatty acids of 36 strains representing 18 genera of the Saccharomycetace, Endomycetaceae, Metchnikowiaceae, Saccharomycodaceae, Schizosaccharomycetaceae and Dipodascaceae were extracted and analyzed as methyl esters by gas chromatography. On the basis of their fatty acid content the set of strains was divided into 6 groups, coinciding with the above families. The members of the Saccharomycetaceae (group I) had a high percentage of oleic acid while the strains classified under the Endomycetaceae (group II) and Metchnikowiaceae (group III) were characterized by oleic acid and linoleic acid as major fatty acids. The Saccharomycodaceae (group IV) had the highest percentage of palmitoleic acid. The Schizosaccharomycetaceae (group V) had the highest percentage of oleic acid, while the Dipodascacea (group VI) were characterized by a high percentage of linoleic acid.
Asunto(s)
Ácidos Grasos Monoinsaturados , Ácidos Grasos/análisis , Saccharomycetales/clasificación , Cromatografía de Gases , Ácidos Linoleicos/análisis , Ácidos Oléicos/análisis , Ácidos Palmíticos/análisis , Saccharomycetales/análisisRESUMEN
Hydroxy long-chain fatty acids occur widely in animals and plants and have important physiological activities in these eukaryotes. There are indications that these compounds are also common and important in fungi. The occurrence of hydroxy-polyunsaturated fatty acids (hydroxy-PUFAs) is of biotechnological importance, because these compounds are potentially high-value lipid products with medical applications. This review pays particular attention to the production of hydroxy-PUFAs by yeasts and other fungi. Hydroxy-PUFAs derived from lipoxygenase activity appear to be present in most fungi, while hydroxy-PUFAs from cyclooxygenase activity (i.e. prostaglandins) have mainly been implicated in the Oomycota and in yeasts from the genus Dipodascopsis. The occurrence of other hydroxy long-chain fatty acids in fungi is also discussed briefly; these include hydroxy fatty acids that are generally associated with cytochrome P-450 monooxygenase activity (i.e. terminal and sub-terminal hydroxy acids and diols derived from the corresponding epoxides) as well as 2-hydroxy-fatty acids and 3-hydroxy-fatty acids.
RESUMEN
The long-chain fatty acid compositions of 22 species of Candida were determined, and compared with the fatty acid compositions of 10 species of the genus Pichia that contain coenzyme Q9. The long-chain fatty acid results were also compared with other phenotypic criteria (i.e. assimilation of carbon sources, coenzyme Q type, G + C content and proton magnetic resonance spectra) in order to establish possible anamorph/teleomorph relations. Close correlations were found between known perfect/imperfect states. The results suggest that C. cacaoi and P. farinosa, and C. maltosa and P. etchellsii, also have anamorph/teleomorph relationships.
Asunto(s)
Candida/metabolismo , Ácidos Grasos/metabolismo , Pichia/metabolismo , Saccharomycetales/metabolismo , Candida/enzimología , Fenotipo , Pichia/enzimología , Ubiquinona/metabolismoRESUMEN
The occurrence of proton symport mechanisms for the transport of glucose, galactose, fructose, raffinose and sucrose in 21 yeast strains representing the species of the genus Kluyveromyces was surveyed. Proton symport of one or more sugars occurred in 57% of the strains. Similarly, all the sugars investigated were transported by symports by several strains. Symport systems for non-utilisable sugars were rare. Starvation of cells frequently resulted in the appearance of a symport absent in non-starved glucose-grown cells, indicating that repression of proton symports by glucose and subsequent derepression by starvation is a general phenomenon in members of Kluyveromyces. The addition of a sugar to cell suspensions resulted in acidification in 80% of cases, indicating the activity of a membrane-bound ATPase. Acidification was also observed with a number of sugars that cannot be utilised by the particular species. Interesting correlations between the number of proton symports and the abundance of other phenotypic characteristics in members of the genus emerged. Most members of the infertile group of species showing an increase in the number of small chromosomes, inability to produce well-developed pseudomycelium, linoleic and linolenic acid, a decrease in the number of carbon compounds utilised and inability to utilise ethylamine also had no proton symports, whereas most members of the interfertile species produced one or more proton symports. It was concluded that the distribution of the number of proton symports amongst Kluyveromyces species coincided with that of other positive characteristics and may therefore be of taxonomic value.