Biomedical subjects

Marcio L Rodrigues

Publications and source records attributed to Marcio L Rodrigues.

23 records · Page 2Linked to original sources

PubMed2003

Cleavage of human fibronectin and other basement membrane-associated proteins by a Cryptococcus neoformans serine proteinase.

The interaction between the fungal pathogen Cryptococcus neoformans and human fibronectin (HFN) was examined in this study. Polypeptides from cryptococcal whole homogenates and cell wall with molecular masses of 25 and 35 kDa, respectively reacted with HFN. The relevance of the occurrence of these proteins in intact cells was uncertain, since yeast cells from different strains and serotypes of C. neoformans did not significantly adhere to soluble or solid-phased HFN. In contrast, an exocellular proteolytic activity that cleaves HFN was suggested. Degradation of HFN by culture supernatant fluids was demonstrated by Western blotting using a monoclonal anti-HFN antibody. Several fragments of lower molecular weights were observed which reacted with the antibody. Proteolysis was mediated by a serine protease activity, since HFN cleavage was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), aprotinin, and N-tosyl-L-phenylalanyl chloromethylketone (TPCK), but not by inhibitors of metalo, cysteine, or aspartyl proteases. Similar results were obtained when the fluorogenic peptide carbobenzoxy-phenylalanyl-arginyl-7-amido-4-methylcoumarin (CBZ-Phe-Arg-NHmet-C) was used as substrate. The cryptococcal supernatant also cleaved laminin and type IV collagen, as demonstrated by polyacrylamide gel electrophoresis with co-polymerized proteins. The hydrolysis of these proteins was mediated by a single cryptococcal protease with a molecular mass of 75 kDa. The cleavage of key host components of the basement membrane and extracellular matrix by C. neoformans may be a relevant factor in the process of fungal invasion.

Aprotinin↗

PubMed2002

Local antilaminin antibody treatment alters the rejection pattern of murine cardiac allografts: correlation between cellular infiltration and extracellular matrix.

BACKGROUND: Emerging data place extracellular matrix (ECM) proteins as important elements in lymphocyte positioning and effector function in alloreactive responses. Using a non-vascularized model of allogeneic heart transplantation in Swiss mice, we have observed a correlation between the cellular infiltration and ECM deposition towards the interior of the graft during the kinetics of rejection. METHODS: To confirm the importance of ECM during the rejection process in this model, we treated the transplanted animals with local injections of antilaminin monoclonal antibody and analyzed, by histology and immunohistochemistry, the grafts on day 15, which corresponds to the peak of cellular infiltration and ECM deposition. RESULTS: The treatment with mAb antilaminin decreased the cellular infiltrate and ECM deposition within the grafts, as compared to controls. Moreover, we found a diminished IFN-gamma, TNF-alpha and IL-2 deposition in the transplant area, and a reduced co-localization of these cytokines with laminin. By contrast, the antilaminin treatment increased tenascin deposition, a molecule with immunosuppressive properties, and also caused an increase in apoptosis of the cellular infiltrate. CONCLUSIONS: These data hallmark the importance of laminin, in distinct aspects concerning the events leading to allograft rejection, and also reinforce this molecule as a potential target for immune intervention in organ transplantation.

Animals↗

PubMed2002

Glycosphingolipids from Magnaporthe grisea cells: expression of a ceramide dihexoside presenting phytosphingosine as the long-chain base.

Magnaporthe grisea is a fungal pathogen that infects rice leaves and causes rice blast, a devastating crop disease. M. grisea produces active elicitors of the hypersensitive response in rice that were previously identified as ceramide monohexosides (CMHs). Using several chromatographic approaches, mass spectrometry, and nuclear magnetic resonance, we identified ceramide mono- and dihexosides (CDH) in purified lipid extracts from M. grisea cells. As described by other authors, CMH consists of a ceramide moiety containing 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecenoic or 2-hydroxyhexadecenoic acids and a carbohydrate segment consisting of one residue of glucose. CDHs, however, contain beta-galactose (1-->4)-linked to beta-glucose as sugar units and phytosphingosine as the long-chain base, bound to a C24 alpha-hydroxylated fatty acid. To our knowledge, this is the first report on the occurrence of CDH in a fungal species and illustrates the existence of an alternative path of ceramide glycosylation in fungal cells.

Carbohydrate Conformation↗

PubMed2002

Sialylglycoconjugates and sialyltransferase activity in the fungus Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen associated with systemic mycoses in up to 10% of AIDS patients. C. neoformans yeasts express sialic acids on the cell wall, where they play an anti-phagocytic role, and may represent a virulence factor at the initial phase of infection. Since the nature of the sialic acid-carrying components is undefined in C. neoformans, our aim in the present work was to identify sialylated molecules in this fungus and study the sialylation process. C. neoformans yeast forms were cultivated in a chemically defined medium free of sialic acids, to search for autologous sialylglycoconjugates. Sialylated glycolipids were not detected. Two glycoproteins with molecular masses of 38 and 67 kDa were recognized by Sambucus nigra agglutinin, an alpha2,6-sialic acid-specific lectin. The 67 kDa glycoprotein also interacted with Influenza C virus, but not with Limax flavus agglutinin, suggesting the presence of the 9-O-acetylated sialic acid derivative as a constituent of the oligosaccharide chains. A partially purified protein fraction from cryptococcal yeast forms was able to transfer sialic acid from CMP-Neu5Ac to both N-(acetyl-1-(14)C)-lactosamine and asialofetuin. Additional evidence for a sialyltransferase in C. neoformans was obtained through the reactivity of fungal proteins with rabbit anti-rat alpha2,6 sialyltransferase polyclonal antibody. Our results indicate that sialic acids in C. neoformans are linked to glycoproteins, which are sialylated by the action of a fungal sialyltransferase. This is the first demonstration of this biosynthetic step in pathogenic fungi.

Acquired Immunodeficiency Syndrome↗

PubMed2002

Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation.

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.

Cell Differentiation↗