RESUMO
Chronic Chagas disease cardiomyopathy, caused by Trypanosoma cruzi infection, is a major cause of heart failure in Latin America. Galectin-3 (Gal-3) has been linked to cardiac remodeling and poor prognosis in heart failure of different etiologies. Herein, we investigated the involvement of Gal-3 in the disease pathogenesis and its role as a target for disease intervention. Gal-3 expression in mouse hearts was evaluated during T. cruzi infection by confocal microscopy and flow cytometry analysis, showing a high expression in macrophages, T cells, and fibroblasts. In vitro studies using Gal-3 knockdown in cardiac fibroblasts demonstrated that Gal-3 regulates cell survival, proliferation, and type I collagen synthesis. In vivo blockade of Gal-3 with N-acetyl-d-lactosamine in T. cruzi-infected mice led to a significant reduction of cardiac fibrosis and inflammation in the heart. Moreover, a modulation in the expression of proinflammatory genes in the heart was observed. Finally, histological analysis in human heart samples obtained from subjects with Chagas disease who underwent heart transplantation showed the expression of Gal-3 in areas of inflammation, similar to the mouse model. Our results indicate that Gal-3 plays a role in the pathogenesis of experimental chronic Chagas disease, favoring inflammation and fibrogenesis. Moreover, by demonstrating Gal-3 expression in human hearts, our finding reinforces that this protein could be a novel target for drug development for Chagas cardiomyopathy.
Assuntos
Cardiomiopatia Chagásica/metabolismo , Galectina 3/metabolismo , Miocardite/metabolismo , Miocárdio/patologia , Acetilgalactosamina/farmacologia , Animais , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Doença Crônica , Colágeno Tipo I/biossíntese , Fibrose/etiologia , Fibrose/metabolismo , Galectina 3/antagonistas & inibidores , Transplante de Coração , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Miocardite/etiologia , Miocárdio/metabolismo , Miofibroblastos/metabolismo , Linfócitos T/metabolismoRESUMO
This work addresses the synthesis and biological evaluation of glycosyl diketopiperazines (DKPs) cyclo[Asp-(αGalNAc)Ser] 3 and cyclo[Asp-(αGalNAc)Thr] 4 for the development of novel anti-trypanosomal agents and Trypanosoma cruzi trans-sialidase (TcTS) inhibitors. The target compounds were synthetized by coupling reactions between glycosyl amino acids αGalNAc-Ser 7 or αGalNAc-Thr 8 and the amino acid (O-tBu)-Asp 17, followed by one-pot deprotection-cyclisation reaction in the presence of 20% piperidine in DMF. The protected glycosyl amino acid intermediates 7 and 8 were, in turn, obtained by α-selective, HgBr2-catalysed glycosylation reactions of Fmoc-Ser/Thr benzyl esters 12/14 with αGalN3Cl 11, being, subsequently, fully deprotected for comparative biological assays. The DKPs 3 and 4 showed relevant anti-trypanosomal effects (IC50 282-124 µM), whereas glycosyl amino acids 1 and 2 showed better TcTS inhibition (57-79%) than the corresponding DKPs (13-25%).
Assuntos
Acetilgalactosamina/química , Acetilgalactosamina/farmacologia , Dicetopiperazinas/química , Dicetopiperazinas/farmacologia , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Acetilgalactosamina/síntese química , Animais , Linhagem Celular , Células Cultivadas , Doença de Chagas/tratamento farmacológico , Doença de Chagas/parasitologia , Dicetopiperazinas/síntese química , Glicoproteínas/antagonistas & inibidores , Glicoproteínas/metabolismo , Glicosilação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mucinas/química , Mucinas/farmacologia , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Tripanossomicidas/síntese química , Trypanosoma cruzi/enzimologiaRESUMO
This work aimed at describing the first biochemical and structural data of a lectin belonging to Swartzieae, a primitive Legume Taxa. A lactose-binding seed lectin (SLL) was purified by affinity chromatography of crude saline extracts of Swartzia laevicarpa on immobilized lactose. The SLL agglutinated rabbit erythrocytes but not rat or human (A, B, O) erythrocytes. Lectin activity was retained after heating at 100 �C for 15 min and was best inhibited by Nacetylgalactosamine, lactose and galactose. The lectin exhibited a single electrophoretic pattern that corresponded to a molecular mass of 29,000 Da, which was confirmed by MS analysis. In addition, the lectin reacted positively with Schiff's reagent. The unique N-terminal amino acid sequence (39 residues) and the internal peptide sequence were determined by Edman degradation and MS/MS, respectively. The sequencing revealed complete homology of the SLL with legume lectins belonging to primitive groups (Dalbergieae and Sophoreae). The SLL (at 1 mg/ml) did not exhibit antifungal activity against various phytopathogens or cytotoxicity (at 100 µg/ml) towards different cancer cell lines.
Assuntos
Fabaceae/química , Fungos/efeitos dos fármacos , Hemaglutinação/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Lectinas de Plantas/farmacologia , Acetilgalactosamina/farmacologia , Sequência de Aminoácidos , Animais , Morte Celular , Cromatografia de Afinidade , Eletroforese , Galactose/farmacologia , Testes de Hemaglutinação , Humanos , Lactose/metabolismo , Dados de Sequência Molecular , Peso Molecular , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Lectinas de Plantas/isolamento & purificação , Coelhos , Ratos , Sementes/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Células Tumorais CultivadasRESUMO
Sperm binding to oviductal epithelium would be involved in sperm reservoir formation in the utero tubal junction (UTJ). Although in other mammals sperm-oviduct interaction has been proved to be mediated by carbohydrate-recognition mechanisms, the factors implicated in the sperm adhesion to oviductal epithelium of llama are still unknown. In order to assess the role of carbohydrates present in the mucosa surface, we examined the distribution of glycoconjugates in the llama oviduct by confocal lectin-histochemistry. Mannosyl, glucosyl, N-acetylglucosaminyl, galactosyl, N-acetylgalactosaminyl and sialic acid residues were detected in the oviductal mucose glycocalyx. By incubation of UTJ oviductal explants with LCA, DBA, UEA-1 or PNA lectin previous to co-culture with sperm, we observed a significant decrease in sperm binding only with LCA lectin. In the mucosa surface there were numerous d-glucosyl and D-manosyl residues, which were spotted by this lectin. Probably, this fact promotes the whole covering of the oviduct luminal surface by the sugar-lectin complex, preventing sperm access and adhesion of further residues. However, sperm incubation with mannose or glucose does not significantly prevent binding, which means that glucose and mannose would not be involved in a specific sperm-oviduct interaction. On the other hand, we observed a high reduction in sperm binding to UTJ explants with N-acetylgalactosamine and galactose (p<0.001). Coincidentally, binding sites for N-acetylgalactosamine-PAA-FITC conjugate were observed on the whole surface of the sperm, supporting the concept that llama sperm have lectin-like molecules in their surface, as is the case in other mammals. Probably, these lectin-like molecules, by means of N-acetylgalactosamine and galactose recognition, could link the sperm to the oviductal mucosa with the purpose of forming storing sites in the UTJ. Our results support the idea that more than one carbohydrate could participate in sperm reservoir formation in the llama UTJ oviductal segment.
Assuntos
Camelídeos Americanos/fisiologia , Carboidratos/fisiologia , Tubas Uterinas/metabolismo , Lectinas/metabolismo , Espermatozoides/metabolismo , Acetilgalactosamina/farmacologia , Animais , Carboidratos/análise , Adesão Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Tubas Uterinas/química , Tubas Uterinas/citologia , Feminino , Galactose/farmacologia , Glucose/farmacologia , Glicoconjugados/análise , Histocitoquímica , Lectinas/análise , Lectinas/farmacologia , Masculino , Manose/farmacologia , Microscopia de Fluorescência , Mucosa/química , Espermatozoides/química , Técnicas de Cultura de Tecidos/veterináriaRESUMO
Escherichia coli producing heat-labile enterotoxin is responsible for numerous cases of diarrhea worldwide, leading to considerable morbidity and mortality. The B subunits of this toxin are responsible for the binding to the receptor, the complex ganglioside GM1 which has galactose as its terminal sugar. In this study we showed that analogs of galactose (gal) and N-acetylgalactosamine (GalNAc) interfere with the binding of heat-labile toxin to GM1. Antibodies to lectins which mimic sugar structures and neoglycoprotein were employed. These compounds were able to inhibit heat-labile toxin activity efficiently in Vero cells: 37 microg of IgG-enriched fraction from an antiserum inhibited up to 70% of this activity, and 50% of the binding of heat-labile toxin to GM1. Neoglycoprotein was more efficient than antibodies, since 2.5 microg of this ligand completely abolished the activity of heat-labile toxin on Vero cells. These data suggest that these molecules could be developed for prophylaxis and diagnosis of diarrhea caused by heat-labile toxin.
Assuntos
Anticorpos/farmacologia , Toxinas Bacterianas/antagonistas & inibidores , Enterotoxinas/antagonistas & inibidores , Proteínas de Escherichia coli , Escherichia coli , Glicoproteínas/farmacologia , Receptores de Superfície Celular/metabolismo , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/farmacologia , Animais , Toxinas Bacterianas/metabolismo , Chlorocebus aethiops , Enterotoxinas/metabolismo , Galactose/análogos & derivados , Galactose/farmacologia , Lectinas/imunologia , Células VeroRESUMO
An enzyme-linked immunoabsorbent assay was developed to quantify the lectin present in the hemolymph of the freshwater prawn Macrobrachium rosenbergii. This method involves the use of murine monoclonal IgG1 with kappa light chain (designated as 3G1) antibodies raised against the purified lectin, the assay that we developed recognized as little as 30 ng/ml of lectin, and was used to measure the lectin concentration in animals at different maturation stages. The highest concentration of lectin was identified in the hemolymph from post-larval prawns and the lowest in molt stage adult animals. The hemagglutination activity of the lectin was four-fold higher in adult than in juvenile specimens, although in all cases N-acetylated sugar residues, such as N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and N-acetyl-D-neuraminic acid were inhibitors of the lectin activity, suggesting that lectin plays a role in the transport of N-acetylated sugar in juvenile prawns. Our results indicate that lectin concentration and hemagglutinating activity could be influenced by developmental conditions of the freshwater prawn.
Assuntos
Ensaio de Imunoadsorção Enzimática/métodos , Lectinas/análise , Lectinas/metabolismo , Acetilgalactosamina/farmacologia , Acetilglucosamina/farmacologia , Animais , Anticorpos Monoclonais/metabolismo , Relação Dose-Resposta a Droga , Hemolinfa/metabolismo , Hibridomas/metabolismo , Lectinas/fisiologia , Camundongos , Ácido N-Acetilneuramínico/farmacologia , Palaemonidae , Fatores de TempoRESUMO
Haemagglutination of purified F42 fimbriae was found to be inhibited by N-acetyl-galactosamine. Purified F42 fimbrial adhesin reacted with distinct membrane components from chicken erythrocytes (35, 37 and 40 kDa) in immunoblot analysis, suggesting that the binding occurred to proteins or glycoproteins.
Assuntos
Eritrócitos/metabolismo , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Receptores de Superfície Celular/metabolismo , Acetilgalactosamina/farmacologia , Animais , Anticorpos Antibacterianos/imunologia , Galinhas , Membrana Eritrocítica/metabolismo , Glicoproteínas/metabolismo , Testes de Inibição da Hemaglutinação , Immunoblotting , Ligação ProteicaRESUMO
1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli.
Assuntos
Candida albicans/imunologia , Escherichia coli/imunologia , Lectinas Tipo C , Lectinas de Ligação a Manose , Fagocitose/imunologia , Receptores Mitogênicos/imunologia , Acetilgalactosamina/farmacologia , Animais , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Depressão Química , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Humanos , Soros Imunes/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Mananas/farmacologia , Receptor de Manose , Camundongos , Fagocitose/efeitos dos fármacos , Receptores de Superfície Celular/imunologia , Receptores Mitogênicos/efeitos dos fármacosRESUMO
1. Ingestion of enteropathogenic Escherichia coli or Candida albicans by thioglycollate-elicited macrophages and polymorphonuclear leukocytes was investigated in vitro, 2. Goat antiserum against mannose receptors caused about 50% inhibition of E. coli phagocytosis and about 90% inhibition of C. albicans phagocytosis. 3. E. coli and C. albicans uptake was inhibited by about 60% and 98%, respectively, by plating the macrophages onto substrates coated with poly-L-lysine-mannan. Further addition of 50 mM mannose to the medium significantly increased the inhibition of phagocytosis of E. coli by macrophages from 60.7 +/- 1.5 to 79.8 +/- 13.1 and by polymorphonuclear cells from 58.9 +/- 3.7 to 88.7 +/- 4.9. 4. Preincubation of phagocytic cells with antiserum against substance A of human erythrocytes reduced E. coli ingestion by 95%, but this inhibition was not observed when the antiserum was incubated with N-acetylgalactosamine (50 mM) before being added to the phagocytes. The phagocytosis of C. albicans was not inhibited by anti-substance A antiserum. 5. The phagocytosis of E. coli was inhibited by about 25% by the addition of 7.8 micrograms/ml soluble mannan to the medium, and by about 50% by the addition of 50 mMN-acetylgalactosamine; when both substances were added to the medium, an additive inhibition of about 75% was observed. 6. These results indicate that mannose receptors on the surface of phagocytic cells mediate E. coli or Candida albicans uptake and that the binding of bacteria to N-acetylgalactosamine residues from the membrane of phagocytes is also involved in the phagocytosis of E. coli
Assuntos
Animais , Humanos , Candida albicans/imunologia , Escherichia coli/imunologia , Fagocitose/imunologia , Receptores Mitogênicos/imunologia , Acetilgalactosamina/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Aderência Bacteriana/imunologia , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Depressão Química , Eritrócitos/efeitos dos fármacos , Eritrócitos/imunologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/patogenicidade , Soros Imunes/farmacologia , Macrófagos Peritoneais/efeitos dos fármacosAssuntos
Acetilgalactosamina/farmacologia , Colostro/imunologia , Entamoeba histolytica/fisiologia , Galactosamina/análogos & derivados , Imunoglobulina A Secretora/imunologia , Animais , Antígenos de Protozoários/imunologia , Adesão Celular/efeitos dos fármacos , Depressão Química , Entamoeba histolytica/efeitos dos fármacos , Entamoeba histolytica/imunologia , Eritrócitos/fisiologia , Humanos , Ratos , Formação de RosetaRESUMO
Protectins and agglutinins in several organs, fluids and spawn from Argentine terrestrial and fresh-water gastropod species were examined. Differences or analogies with vertebrate immunoglobulin serological behaviour are summarized. Individual or group variability and the evolutionary meaning of the reproductive system-linked and the Ca++ ion-linked protectins are discussed.