Membrane microdomain components of Histoplasma capsulatum yeast forms, and their role in alveolar macrophage infectivity

Analysis of membrane lipids of Histoplasma capsulatum showed that similar to 40% of fungal ergosterol is present in membrane microdomain fractions resistant to treatment with non-ionic detergent at 4 degrees C. Specific proteins were also enriched in these fractions, particularly Pma1p a yeast microdomain protein marker (a plasma membrane proton ATPase), a 30 kDa laminin-binding protein, and a 50 kDa protein recognized by anti-alpha 5-integrin antibody. To better understand the role of ergosterol-dependent microdomains in fungal biology and pathogenicity, H. capsulatum yeast forms were treated with a sterol chelator, methyl-beta-cyclodextrin (m beta CD). Removal of ergosterol by m beta CD incubation led to disorganization of ergosterol-enriched microdomains containing Pma1p and the 30 kDa protein, resulting in displacement of these proteins from detergent-insoluble to -soluble fractions in sucrose density gradient ultracentrifugation. m beta CD treatment did not displace/remove the 50 kDa alpha 5-integrin-like protein nor had effect on the organization of glycosphingolipids present in the detergent-resistant fractions. Ergosterol-enriched membrane microdomains were also shown to be important for infectivity of alveolar macrophages; after treatment of yeasts with m beta CD, macrophage infectivity was reduced by 45%. These findings suggest the existence of two populations of detergent-resistant membrane microdomains in H. capsulatum yeast forms: (i) ergosterol-independent microdomains rich in integrin-like proteins and glycosphingolipids, possibly involved in signal transduction; (ii) ergosterol-enriched microdomains containing Pma1p and the 30 kDa laminin-binding protein; ergosterol and/or the 30 kDa protein may be involved in macrophage infectivity. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Div Glycoconjugate Immunochem, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Div Glycoconjugate Immunochem, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Immunol & Parasitol, Escola Paulista Med, BR-04023900 São Paulo, BrazilWeb of Scienc

Structural diversity and biological significance of glycosphingolipids in pathogenic and opportunistic fungi

Glycosphingolipids (GSLs) are ubiquitous membrane components and have key roles in biological systems, acting as second messengers or modulators of signal transduction by affecting several events, ranging from cell adhesion, cell growth, cell motility, regulation of apoptosis and cell cycle. Over the last 20 years our laboratory and other research groups determined the glycan and ceramide structures of more than 20 GSLs from several pathogenic/opportunistic fungi, using a combination of gas chromatography, mass spectrometry, nuclear magnetic resonance as well as other immunochemical and biochemical techniques. Fungal GSLs can be divided in two major classes: neutral GSLs, galactosyl- and glucosylceramide (GIcCer), and acidic GSLs, the glycosylinositol-phosphorylceramides (GIPCs). Glycosyl structures in fungal GIPCs exhibited significant structural diversity and distinct composition when compared to mammalian GSLs, e.g., the expression of inositol-mannose and inositol-glucosamine cores and the terminal residue of beta-D-galactofuranose which are absent in mammalian cells. Studies performed by our group demonstrated that GIPC (Galf beta 6[Man alpha 3]Man alpha 2InsPCer) elicited in patients with paracoccidioidomycosis an immune response with production of antibodies directed to the terminal residue of beta-D-galactofuranose. Further studies also showed that inhibition of GIcCer biosynthetic pathways affects fungal colony formation, spore germination and hyphal growth, indicating that enzymes involved in GIcCer biosynthesis may represent promising targets for the therapy of fungal infections. Recently, it was shown that GIcCer and GIPCs are preferentially localized in membrane microdomains and monoclonal antibodies directed to these GSLs interfere in several fungal biological processes such as growth and morphological transition. This review focuses on glycan structures carried on sphingolipids of pathogenic/opportunistic fungi, and aspects of their biological significance are discussed.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, Lab Glycoconjugate Immunochem, BR-04023900 São Paulo, BrazilUniv Santa Cecilia, Dept Pharmaceut Sci, Lab Nat Prod, Santos, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biochem, Lab Glycoconjugate Immunochem, BR-04023900 São Paulo, BrazilWeb of Scienc

Differential regulation of the release of tumor necrosis factor-alpha and of eicosanoids by mast cells in rat airways after antigen challenge.

BACKGROUND: Rat trachea display a differential topographical distribution of connective tissue mast cells (CTMC) and mucosal mast cells (MMC) that may imply regional differences in the release of allergic mediators such as tumor necrosis factor-alpha (TNF-alpha) and eicosanoids. AIM: To evaluate the role of CTMC and MMC for release of TNF-alpha and eicosanoids after allergenic challenge in distinct segments of rat trachea. MATERIALS AND METHODS: Proximal trachea (PT) and distal trachea (DT) from ovalbumin (OVA)-sensitized rats, treated or not with compound 48/80 (48/80) or dexamethasone, were incubated in culture medium. After OVA challenge, aliquots were collected to study release of TNF-alpha and eicosanoids. RESULTS: Release of TNF-alpha by PT upon OVA challenge peaked at 90 min and decayed at 6 and 24 h. Release from DT peaked at 30-90 min and decayed 6 and 24 h later. When CTMC were depleted with 48/80, OVA challenge exacerbated the TNF-alpha release by PT at all time intervals, while DT exacerbated TNF-alpha levels 6 and 24 h later only. Dexamethasone reduced TNF-alpha production after 90 min of OVA challenge in PT and at 3 and 6h in DT. OVA challenge increased prostaglandin D2) in DT and leukotriene B4 in both segments but did not modify prostaglandin E2 and leukotriene C4 release. CONCLUSION: OVA challenge induces TNF-alpha release from MMC, which is negatively regulated by CTMC. The profile of TNF-alpha and eicosanoids depends on the time after OVA challenge and of the tracheal segment considered

Disruption of the glucosylceramide biosynthetic pathway in Aspergillus nidulans and Aspergillus fumigatus by inhibitors of UDP-Glc : ceramide glucosyltransferase strongly affects spore germination, cell cycle, and hyphal growth

The opportunistic mycopathogen Aspergillus fumigatus expresses both glucosylceramide and galactosylceramide (GlcCer and GalCer), but their functional significance in Aspergillus species is unknown. We here identified and characterized a GlcCer from Aspergillus nidulans, a non-pathogenic model fungus. Involvement of GlcCer in fungal development was tested on both species using a family of compounds known to inhibit GlcCer synthase in mammals. Two analogs, D-threo-1-phenyl-2-palmitoyl-3-pyrrolidinopropanol (P4) and D-threo-3',4'-ethylenedioxy-P4, strongly inhibited germination and hyphal growth. Neutral lipids from A. fumigatus cultured in the presence of these inhibitors displayed a significantly reduced GlcCer/GalCer ratio. These results suggest that synthesis of GlcCer is essential for normal development of A. fumigatus and A. nidulans. (C) 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.Univ New Hampshire, Dept Chem, Durham, NH 03824 USAUniv Georgia, Dept Bot, Athens, GA 30602 USAUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilUniv Michigan, Med Ctr, Dept Internal Med, Div Nephrol, Ann Arbor, MI 48109 USAUniv Georgia, Complex Carbohydrate Res Ctr, Athens, GA 30602 USAUniv Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USAUniversidade Federal de São Paulo, Dept Biochem, Escola Paulista Med, BR-04023900 São Paulo, BrazilWeb of Scienc

Heparina: ontogenia, correlação com mastócitos, propriedades físico-químicas e atividades farmacológicas

BV UNIFESP: Teses e dissertaçõe

Heparam sulfato como possível polímero código das interaçöes celulares - variaçäo das quatro unidades estruturais em funçäo do tecido e espécie de origem: estudos biossintéticos

BV UNIFESP: Teses e dissertaçõe

STRUCTURAL DIFFERENCES of HEPARAN SULFATES ACCORDING TO the TISSUE and SPECIES of ORIGIN

ESCOLA PAULISTA MED,DEPT BIOQUIM,BR-04023 São Paulo,SP,BRAZILESCOLA PAULISTA MED,DEPT BIOQUIM,BR-04023 São Paulo,SP,BRAZILWeb of Scienc

A monoclonal antibody directed to terminal residue of beta-galactofuranose of a glycolipid antigen isolated from Paracoccidioides brasiliensis; Cross-reactivity with Leishmania major and Trypanosoma cruzi

A mouse monoclonal antibody, MEST-1, was produced against Band 1 glycolipid antigen of Paracoccidioides brasiliensis, the glycan structure of Band 1 antigen was recently elucidated and the monosaccharides sequence was defined as: Galf beta 1-->6(Manp alpha 1-->3)Manp beta 1-->2Ins. the reactivity of MEST-1 MAb was determined by solid-phase radioimmunoassay and high performance thin layer chromatography immunostaining. Selective oxidation of galactofuranose residues and inhibition assays with different methyl-glycosides, revealed that MAb MEST-1 is directed against the terminal residue of beta-D-gaiactofuranose of Band 1, a phosphoglyceroglycolipid antigen of P.brasiliensis. By indirect immunofluorescence, it was observed that the epitope recognized by MEST-1 is accessible to the antibody in yeast forms of this fungus. Reactivity of MEST-1 with parasites known to express galactofuranose containing glycoconjugates was also analyzed by indirect immunofluorescence. A positive fluorescence was observed with promastigotes of Leishmania major and epimastigotes of Trypanosoma cruzi, GIPL-1 was identified as the antigen recognized by MEST-1 in Leishmania major, indicating that the MAb MEST-1 recognizes terminal galactofuranose residue in either beta 1-->6 or beta 1-->3 linkage to the mannose.Universidade Federal de São Paulo,DEPT BIOCHEM,EPM,BR-04023900 São Paulo,BRAZILUniversidade Federal de São Paulo,DEPT BIOCHEM,EPM,BR-04023900 São Paulo,BRAZILWeb of Scienc