RESUMO
Protozoan parasites of the genus Leishmania cause a number of important human diseases. One of the key determinants of parasite infectivity and survival is the surface glycoconjugate lipophosphoglycan (LPG). In addition, LPG is shown to be useful as a transmission blocking vaccine. Since culture supernatant of parasite promastigotes is a good source of LPG, we made attempts to characterize functions of the culture supernatant, and membrane LPG isolated from metacyclic promastigotes of Leishmania major. The purification scheme included anion-exchange chromatography, hydrophobic interaction chromatography and cold methanol precipitation. The purity of supernatant LPG (sLPG) and membrane LPG (mLPG) was determined by SDS-PAGE and thin layer chromatography. The effect of mLPG and sLPG on nitric oxide (NO) production by murine macrophages cell line (J774.1A) was studied. Both sLPG and mLPG induced NO production in a dose dependent manner but sLPG induced significantly higher amount of NO than mLPG. Our results show that sLPG is able to promote NO production by murine macrophages.
Assuntos
Camundongos , Animais , Óxido Nítrico/análise , Camundongos Endogâmicos BALB C , Macrófagos/efeitos dos fármacos , Leishmania major/química , Glicoesfingolipídeos/isolamento & purificação , Endotoxinas/análise , Eletroforese em Gel de Poliacrilamida , Meios de Cultura , Cromatografia em Camada Fina/métodos , Membrana Celular/química , Linhagem CelularRESUMO
Two glycoinositol phospholipids (GIPL A and GIPL B) have been purified from epimastigotes of Trypanosoma cruzi at the logarithmic phase of growth (2 days). The GIPLs differ mainly in the lipid moiety and are similar to the lipopeptidophosphoglycan (LPPG) previously isolated from epimastigotes at the stationary phase (4-5 days). [3H]-palmitic acid was incorporated into 1-O-hexadecyl-2-O-palmitoylglycerol in GIPL A and into a sphinganine ceramide with palmitic acid and lignoceric acid as the fatty acids in GIPL B. The lipids could be released by incubation with phosphatidylinositol-specific phospholipase C (PI-PLC) or glycosylphosphatidylinositol phospholipase D (GPI-PLD) from rat serum. The oligosaccharides share the common core structure of the glycosylphosphatidilinositol (GPI) membrane anchors. Microheterogeneity was demonstrated, as well as substitution by galactose, which is mainly in the furanose configuration as was previously described for the LPPG. However, methylation analysis indicated that 20 percent of the galactose is present as terminal pyranose units. In infective trypomastigotes, [3H]-palmitic acid was incorporated into the anchor of the Tc-85 glycoprotein. The lipid cleaved by phospholipase C digestion was identified as 1-O-hexadecylglycerol and the main oligosaccharide has the structure of the conserved core of all GPI anchors. [3H]-palmitic acid-labelled Tc-85 released into the culture medium as membrane vesicles showed 80 percent resistance to the action of PI-PLC. However, after mild alkaline hydrolysis, part of the radioactivity was released by the enzyme
Assuntos
Animais , Ratos , Glicoesfingolipídeos/química , Oligossacarídeos/química , Trypanosoma cruzi/química , Sequência de Carboidratos , Ácidos Graxos , Glicoesfingolipídeos/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos/análise , Oligossacarídeos/isolamento & purificação , Ácidos Palmíticos , Peptidoglicano/química , Peptidoglicano/isolamento & purificação , Fosfolipídeos/química , Fosfolipídeos/isolamento & purificação , Fosfolipases Tipo CRESUMO
Glycoinositolphospholipids (GIPLs) were extracted from the trypanosomatid Leishmania adleri by hot phenol extraction and the carbohydrate moieties isolated after base cleavage. Purification of the crude oligosaccharides by high performance anion exchange (HPAE) chromatography yielded four fractions whose structures were determined by a combination of methylation analysis, fast atom bombardment (FAB) mass spectrometry and two-dimensional nuclear magnetic resonance (NMR) spectroscopy
Assuntos
Animais , Glicoesfingolipídeos/química , Oligossacarídeos/química , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Trypanosomatina/química , Sequência de Carboidratos , Glicoesfingolipídeos/isolamento & purificação , Dados de Sequência Molecular , Oligossacarídeos/isolamento & purificação , Espectroscopia de Ressonância MagnéticaRESUMO
Nuclear magnetic resonance (NMR) spectroscopy provides an extremely powerful technique to determine the structure of oligosaccharides, particularly when used in conjuction with other physical techniques such as methylation analysis and fast atom bombardment mass spectroscopy (FAB-MS). This brief review describes the application of NMR to the determination of the structure of an oligosaccharide isolated from the glycophosphosphingolipid (GPS) from the monogenetic trypanosomatid Leptomonas samueli. Where ambiguities arise in the NMR interpretation, the use of other data will be discussed