RESUMO
Glucosylceramides (GlcCer) were extracted from the plant pathogen Colletotrichum gloeosporioides and purified by several chromatographic steps. By using electrospray ionization mass spectrometry and nuclear magnetic resonance, GlcCer from C. gloeosporioides were identified as N-2'-hydroxyoctadecanoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-beta-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against these structures were produced and used as tools for the evaluation of the role of GlcCer in the morphological transition of C. gloeosporioides. In the presence of antibodies to GlcCer, the differentiation of conidia into mycelia was blocked. Since GlcCer is present in several plant pathogens, the inhibitory activity of external ligands recognizing these structures may be applicable in other models of fungal infections.
Assuntos
Colletotrichum/química , Colletotrichum/citologia , Glucosilceramidas/isolamento & purificação , Glucosilceramidas/fisiologia , Anticorpos Monoclonais/biossíntese , Cerebrosídeos/imunologia , Cerebrosídeos/isolamento & purificação , Colletotrichum/crescimento & desenvolvimento , Glucosilceramidas/imunologia , Espectroscopia de Ressonância Magnética , Microscopia de Fluorescência , Morfogênese , Micélio/citologia , Espectrometria de Massas por Ionização por Electrospray , Esporos Fúngicos/citologiaRESUMO
Eight glucosylceramides (1-8) were isolated from the water-insoluble lipid fraction of a methylene chloride/methanol/water extract of the Patagonian starfish Anasterias minuta. One of the constituents was identified as a new glucosylceramide, anasterocerebroside A (1), while the known glucosylceramide 7 was isolated and characterized for the first time as a pure compound. The structures of 1 and 7 were established by spectroscopic and chemical methods.
Assuntos
Cerebrosídeos/química , Glucosilceramidas/química , Estrelas-do-Mar/química , Animais , Argentina , Cerebrosídeos/isolamento & purificação , Glucosilceramidas/isolamento & purificação , Glicoesfingolipídeos/química , Glicoesfingolipídeos/isolamento & purificação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Extratos de Tecidos/químicaRESUMO
From the water-insoluble lipid fraction of the chloroform/methanol/water extract of the gonads and body walls of the Patagonian starfish Allostichaster inaequalis, two new glucosylceramides (4 and 7) were isolated together with the known phalluside-1 (1) and two glucosylceramides (2 and 3) previously isolated from the starfish Cosmasterias lurida. The new compounds were characterized as (2S,3R,4E,8E,10E)-1-(beta-D-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxy-15-tetracosenoyl] amino-4,8,10-octadecatriene (4) and (2S,3R,4E,15Z)-1-(beta-D-glucopyranosyloxy)-3-hydroxy-2-[(R)-2-hydroxyhexadecanoyl] amino-4,15-docosadiene (7) by means of spectroscopic and chemical methods.
Assuntos
Glucosilceramidas/isolamento & purificação , Gônadas/química , Animais , Cromatografia Líquida de Alta Pressão , Glucosilceramidas/química , Ressonância Magnética Nuclear Biomolecular , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Espectrofotometria Ultravioleta , Estrelas-do-MarRESUMO
Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.
Assuntos
Diferenciação Celular/fisiologia , Glucosilceramidas/química , Pseudallescheria/química , Cromatografia Líquida de Alta Pressão , Cromatografia Gasosa-Espectrometria de Massas , Glucosilceramidas/isolamento & purificação , Glucosilceramidas/fisiologia , Microscopia de Fluorescência , Monossacarídeos/química , Ressonância Magnética Nuclear Biomolecular , Pseudallescheria/citologiaRESUMO
From the water-insoluble lipid fraction of the methylene chloride/methanol extract of the starfish Cosmasterias lurida, two new glucosylceramides together with a known glucosylceramide, ophidiacerebroside E, were isolated by chromatographic procedures and characterized by spectroscopic (1H and 13C nuclear magnetic resonance, mass spectrometry) methods. The new compounds were identified as (2S,3R,4E,8E,10E)-1-(beta-D-glucopyranosyloxy)-3 -hydroxy-2-[(R)-2-hydroxyheptadecanoyl)amino]-9-methyl-4,8,10-o ctadecatriene (3) and (2S,3R,4E,8E,10E)-1-(beta-D-glucopyranosyloxy)-3 -hydroxy-2-[(R)-2-hydroxyoctadecanoyl)amino]-9-methyl-4,8,10-oc tadecatriene (4).
Assuntos
Glucosilceramidas/química , Glucosilceramidas/isolamento & purificação , Estrelas-do-Mar/química , Animais , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão , Dados de Sequência Molecular , Ressonância Magnética Nuclear BiomolecularRESUMO
Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated from mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of 1H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcp beta 1-->Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.
Assuntos
Glucosilceramidas/isolamento & purificação , Paracoccidioides/química , AnimaisRESUMO
Twelve different species of neutral monohexosyl ceramides (CMHs) and two species of neutral monohexosyl ceramides were isolated form mycelium and yeast forms of Paracoccidioides brasiliensis, respectively, by a combination of ion-exchange chromatography, HPLC, and HPTLC. The glucosylceramides did not react with sera from patients with paracoccidioidomycosis (PCM). Carbohydrate analysis indicated that CMHs contain glucose. Analysis of (1)H-NMR and mass spectrometry data suggest that the structure of the CMHs is Glcpbeta1(Cer (mycelium forms present 12 different ceramides and yeast forms present 2 different ceramides). The composition of the lipid moieties was analyzed by negative fast atom bombardment mass spectrometry. No glycosphingolipid other than glucosylceramide was detected in P. brasiliensis.