RESUMO
Galactoxylomannans (GalXMs) are a mixture of neutral and acidic capsular polysaccharides produced by the opportunistic fungus Cryptococcus neoformans that exhibit potent suppressive effects on the host immune system. Previous studies describing the chemical structure of C. neoformans GalXMs have reported species without O-acetyl substituents. Herein we describe that C. neoformans grown in capsule-inducing medium produces highly O-acetylated GalXMs. The location of the O-acetyl groups was determined by nuclear magnetic resonance (NMR) spectroscopy. In the neutral GalXM (NGalXM), 80% of 3-linked mannose (α-Manp) residues present in side chains are acetylated at the O-2 position. In the acidic GalXM also termed glucuronoxylomannogalactan (GXMGal), 85% of the 3-linked α-Manp residues are acetylated either in the O-2 (75%) or in the O-6 (25%) position, but O-acetyl groups are not present at both positions simultaneously. In addition, NMR spectroscopy and methylation analysis showed that ß-galactofuranose (ß-Galf) units are linked to O-2 and O-3 positions of nonbranched α-galactopyranose (α-Galp) units present in the GalXMs backbone chain. These findings highlight new structural features of C. neoformans GalXMs. Among these features, the high degree of O-acetylation is of particular interest, since O-acetyl group-containing polysaccharides are known to possess a range of immunobiological activities.
Assuntos
Cryptococcus neoformans/química , Polissacarídeos Fúngicos/química , Polissacarídeos/químicaRESUMO
One of the main obstacles to the treatment of Chagas disease is the genetic and phenotypical variance displayed by T. cruzi strains, resulting in differences in morphology, virulence, pathogenicity and drug susceptibility. To better understand the role of glycoconjungates in Chagas disease, we performed the molecular characterization of the O-linked chains from mucins and glycoinositolphospholipids (GIPLs) of the Silvio X10 clone 1 strain. We demonstrated the presence of a ß-galactofuranose (ß-Galf) unity linked to the O-4 position of the α-N-acetylglucosamine (α-GlcNAc)O-4 in Tc-mucins. GIPLs analysis showed that the lipidic portion is exclusively composed of ceramide and the PI-oligossacharidic portion contains the Man4(AEP)GlcN-Ins-PO4 core, substituted by ethanolamine-phosphate (EtNP) on the third distal mannose from inositol, which may or may not have a terminal ß Galf unity. These results confirm the classification of the Silvio X10/1 strain in group T. cruzi I. Again, it is noted that the study of T. cruzi surface glycoconjugates confirm the molecular results and the hypothesis that surface glycoconjugates may be interesting biomarker for the differentiation of trypanosomatid strains.
Assuntos
Glicoconjugados/química , Glicolipídeos/química , Mucinas/química , Fosfolipídeos/química , Trypanosoma cruzi/química , Trypanosoma cruzi/classificação , GenótipoRESUMO
BACKGROUND: Propofol (2,6-diisopropylphenol) has been shown to protect several organs, including the kidneys, from ischemia-reperfusion (I-R)-induced injury. Although propofol affects adenosine triphosphate-sensitive potassium (K(ATP)) channels in nonrenal tissues, it is still not clear by which mechanisms propofol protects renal cells from such damage. In this study, we investigated whether propofol induces renal preconditioning through renal K(ATP) channels. METHODS: A reversible ATP depletion (antimycin A) followed by restoration of substrate supply in LLC-PK1 cells was used as an in vitro model of renal I-R. Cell viability was assessed by dimethylthiazol-diphenyltetrazol bromide and trypan blue dye exclusion test assays. Apoptosis was evaluated by annexin V-fluorescein isothiocyanate staining by flow cytometry and immunofluorescence. Propofol treatments were initiated at various time intervals: 1 or 24 h before ischemia, only during ischemia, or only during reperfusion. To evaluate the mechanisms of propofol protection, specific K(ATP) channel inhibitors or activators were used in some experiments during propofol pretreatment. RESULTS: Propofol attenuated I-R injury on LLC-PK1 cells when present either 1 or 24 h before initiated I-R, and also during the recovery period, but not when added only during ischemia. Propofol pretreatment significantly protected LLC-PK1 from I-R-induced apoptosis. The protective effect of propofol was prevented by glibenclamide (a sarcolemmal ATP-dependent K(+) channel blocker) and decreased by 5-hydroxidecanoic acid (a mitochondrial ATP-dependent K(+) channel blocker), but it was not modified by diazoxide (a selective opener of ATP-sensitive K(+) channel). CONCLUSION: Propofol protected cells against apoptosis induced by I-R. This protection was probably due to a preconditioning effect of propofol and was, at least in part, mediated by K(ATP) channels.
Assuntos
Canais KATP/agonistas , Nefropatias/prevenção & controle , Rim/efeitos dos fármacos , Propofol/farmacologia , Substâncias Protetoras/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Trifosfato de Adenosina/deficiência , Animais , Antimicina A/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citoproteção , Ácidos Decanoicos/farmacologia , Diazóxido/farmacologia , Glibureto/farmacologia , Hidroxiácidos/farmacologia , Canais KATP/metabolismo , Rim/metabolismo , Rim/patologia , Nefropatias/metabolismo , Nefropatias/patologia , Células LLC-PK1 , Necrose , Bloqueadores dos Canais de Potássio/farmacologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Suínos , Fatores de TempoRESUMO
Glucuronoxylomannogalactans (GXMGals) are characteristic capsular polysaccharides produced by the opportunistic fungus C. neoformans, which are implicated in cryptococcal virulence, via impairment of the host immune response. We determined for the first time the structure of a lipoglucuronomannogalactan (LGMGal), isolated from the surface of a mutant C. neoformans carrying a deletion in the UDP-GlcA decarboxylase gene. Monosaccharide composition and methylation analyses, as well as nuclear magnetic resonance spectroscopy were employed in discerning the structure. Our results show that the polysaccharide structure of the LGMGal differs from GXMGal by the absence of xylose and 2-O-acetylated mannose residues. LGMGal consists of a galactan main chain -[-6-α-Gal-]-, where every second Gal residue is substituted at O-3 with an oligosaccharide α-Man6OAc-3-α-Man-4-(ß-GlcA-3)-ß-Gal-; components in italic being non-stoichiometric. The substitution rate of ß-Galp units by GlcpA is 35%. Additionally, we determined that the glycolipid anchor of the LGMGal is based on an myo-inositol phosphoceramide composed of C18-phytosphingosine and monohydroxylated lignoceric acid (2OHC24:0 fatty acid).
Assuntos
Parede Celular/química , Cryptococcus neoformans/química , Cryptococcus neoformans/citologia , Polissacarídeos/isolamento & purificação , Acetilação , Ressonância Magnética Nuclear Biomolecular , Polissacarídeos/químicaRESUMO
Trans-sialidase from Trypanosoma cruzi (Tc-TS) belongs to a superfamily of proteins that may have enzymatic activity. While enzymatically active members (Tc-aTS) are able to transfer sialic acid from the host cell sialyl-glycoconjugates onto the parasite or to other molecules on the host cell surface, the inactive members (Tc-iTS) are characterized by their lectinic properties. Over the last 10 years, several papers demonstrated that, individually, Tc-aTS or Tc-iTS is able to modulate several biological events. Since the genes encoding Tc-iTS and Tc-aTS are present in the same copy number, and both proteins portray similar substrate-specificities as well, it would be plausible to speculate that such molecules may compete for the same sialyl-glycan structures and govern numerous immunobiological phenomena. However, their combined effect has never been evaluated in the course of an acute infection. In this study, we investigated the ability of both proteins to modulate the production of inflammatory signals, as well as the homing of T cells to the cardiac tissue of infected mice, events that usually occur during the acute phase of T. cruzi infection. The results showed that the intravenous administration of Tc-iTS, but not Tc-aTS protected the cardiac tissue from injury caused by reduced traffic of inflammatory cells. In addition, the ability of Tc-aTS to modulate the production of inflammatory cytokines was attenuated and/or compromised when Tc-iTS was co-injected in the same proportions. These results suggest that although both proteins present structural similarities and compete for the same sialyl-glycan epitopes, they might present distinct immunomodulatory properties on T cells following T. cruzi infection.
RESUMO
Trypanosoma cruzi, the etiological agent of Chagas disease exhibits multiple mechanisms to guarantee its establishment and persistence in the infected host. It has been well demonstrated that T. cruzi is not able to synthesize sialic acids (Sia). To acquire the monosaccharide, the parasite makes use of a multifunctional enzyme called trans-sialidase (Tc-TS). Since this enzyme has no analogous in the vertebrate host, it has been used as a target in drug therapy development. Tc-TS preferentially catalyzes the transfer of Sia from the host glycoconjugates to the terminal ß-galactopyranosyl residues of mucin-like molecules present on the parasite's cell surface. Alternatively, the enzyme can sialylate/re-sialylate glycoconjugates expressed on the surface of host cells. Since its discovery, several studies have shown that T. cruzi employs the Tc-TS activity to modulate the host cell sialoglycophenotype, thus favoring its perpetuation in the infected vertebrate. In this review, we summarize the dynamic of host/parasite sialoglycophenotype modulation, highlighting its role in the subversion of host immune response in order to promote the establishment of persistent chronic infection.
Assuntos
Adenocarcinoma Mucinoso/secundário , Neoplasias do Colo/diagnóstico , Neoplasias do Colo do Útero/secundário , Adenocarcinoma Mucinoso/diagnóstico , Adenocarcinoma Mucinoso/terapia , Biópsia , Colectomia , Neoplasias do Colo/cirurgia , Terapia Combinada , Feminino , Humanos , Pessoa de Meia-Idade , Metástase Neoplásica , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/terapiaRESUMO
AIMS: Glutathione (GSH) plays an important role in protecting cells against oxidative damage. ABCC1 protein transports GSH. Although this protein is largely studied in cancer, due to multidrug resistance phenotype, its role in the tubular cells of the kidney is unknown. The goal of this study was to find out whether ABCC1 has a role in protecting cells from the distal nephron against the stress caused by high medullar osmolality. MAIN METHODS: MA104 cells were treated with high concentrations of sodium chloride, urea, or both to raise the osmolality of the culture medium. Cell viability was accessed by MTT and trypan blue assays. ABCC1 expression and extrusion of carboxi-fluorescein (CF), a fluorescent ABCC1 substrate, were measured by flow cytometry. KEY FINDINGS: Incubation of MA104 cells in a high sodium concentration medium resulted in changes in cell granularity and altered expression and activity of ABCC1. Urea did not alter ABCC1 expression or activity, but reversed the observed NaCl effects. High sodium concentrations also had a negative effect on cell viability and urea also protected cells against this effect. SIGNIFICANCE: Our findings demonstrate that ABCC1 plays a significant role in the protection of kidney epithelial cells against the stress caused by high sodium environment present in renal medulla.