RESUMO
Infections by Candida albicans in immune compromised patients cause significant morbidity and mortality. In the search for potential molecular targets for drug development, the family of agglutinin-like proteins (Als) in C. albicans have been identified due to numerous attributes associated with high virulence, most prominently due to their role in adherence. Here, molecular models of individual members of the Als family illustrated common and unique structure features. Additionally, dynamic simulations were performed to display regions of high mobility. The results showed variations between Als members in the fluctuation of the A1B1 protein loop, which is located at the entrance to the peptide binding cavity, suggesting that this feature may be a factor contributing to observed differences in affinities to ligands and adhesion properties. Molecular docking results further suggested that ligand affinity could be influenced by movements in the A1B1 loop. In addition, a new site was identified in Als in an area adjacent to the peptide binding cavity that could serve as a new binding site for the design of future anti-adhesion ligands that provide increased specificity inhibiting Als proteins from C. albicans.
Assuntos
Aglutininas/química , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/prevenção & controle , Proteínas Fúngicas/química , Aglutininas/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Sítios de Ligação , Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/microbiologia , Proteínas Fúngicas/metabolismo , Humanos , Ligantes , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Ligação Proteica , Domínios Proteicos , VirulênciaRESUMO
Streptococcus mutans is a major etiologic agent of dental caries, a prevalent worldwide infectious disease and a serious public health concern. The surface-localized S. mutans P1 adhesin contributes to tooth colonization and caries formation. P1 is a large (185-kDa) and complex multidomain protein considered a promising target antigen for anticaries vaccines. Previous observations showed that a recombinant P1 fragment (P1(39-512)), produced in Bacillus subtilis and encompassing a functional domain, induces antibodies that recognize the native protein and interfere with S. mutans adhesion in vitro. In the present study, we further investigated the immunological features of P1(39-512) in combination with the following different adjuvants after parenteral administration to mice: alum, a derivative of the heat-labile toxin (LT), and the phase 1 flagellin of S. Typhimurium LT2 (FliCi). Our results demonstrated that recombinant P1(39-512) preserves relevant conformational epitopes as well as salivary agglutinin (SAG)-binding activity. Coadministration of adjuvants enhanced anti-P1 serum antibody responses and affected both epitope specificity and immunoglobulin subclass switching. Importantly, P1(39-512)-specific antibodies raised in mice immunized with adjuvants showed significantly increased inhibition of S. mutans adhesion to SAG, with less of an effect on SAG-mediated bacterial aggregation, an innate defense mechanism. Oral colonization of mice by S. mutans was impaired in the presence of anti-P1(39-512) antibodies, particularly those raised in combination with adjuvants. In conclusion, our results confirm the utility of P1(39-512) as a potential candidate for the development of anticaries vaccines and as a tool for functional studies of S. mutans P1.
Assuntos
Adesinas Bacterianas/imunologia , Anticorpos Antibacterianos/imunologia , Cárie Dentária/prevenção & controle , Proteínas de Membrana/imunologia , Infecções Estreptocócicas/prevenção & controle , Vacinas Estreptocócicas/imunologia , Streptococcus mutans/imunologia , Adesinas Bacterianas/genética , Adjuvantes Imunológicos/administração & dosagem , Aglutininas/metabolismo , Animais , Anticorpos Antibacterianos/sangue , Bacillus subtilis/genética , Aderência Bacteriana , Cárie Dentária/imunologia , Feminino , Proteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Boca/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Saliva/metabolismo , Infecções Estreptocócicas/imunologia , Vacinas Estreptocócicas/administração & dosagem , Vacinas Estreptocócicas/genética , Streptococcus mutans/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
A novel lectin, HGA-2, was isolated from the sea cucumber Holothuria grisea. The protein was isolated by a single chromatographic step using a column of Guar Gum as affinity. HGA-2 showed an apparent molecular mass of 17 kDa and 34 kDa under reducing and nonreducing conditions, respectively. The hemagglutinating activity was specific for rabbit erythrocytes, showing no activity for human blood A, B and O. Its hemagglutinating activity was inhibited by carbohydrates containing galactose, with higher affinity for GalNAc and glycoprotein porcine stomach mucin (PSM). HGA-2 was stable at pH 6-10, significantly declining at pH 5 and a temperature of 40°C, with its activity being abolished at 100 °C. The HGA-2 protein was found to be Ca(2+)-dependent; it was highly toxic against Artemia nauplii and able to recognize and agglutinate cells of Escherichia coli. Amino acid sequences of tryptic peptides of HGA-2 strongly suggest that HGA-2 is a member of the C-type lectin family.
Assuntos
Aglutininas/química , Aglutininas/metabolismo , Escherichia coli/metabolismo , Galactosídeos/metabolismo , Holothuria/química , Lectinas/química , Lectinas/metabolismo , Aglutininas/isolamento & purificação , Aglutininas/toxicidade , Sequência de Aminoácidos , Animais , Hemaglutinação , Testes de Hemaglutinação , Humanos , Concentração de Íons de Hidrogênio , Íons , Lectinas/isolamento & purificação , Lectinas/toxicidade , Lectinas Tipo C , Dados de Sequência Molecular , Coelhos , Alinhamento de Sequência , TemperaturaRESUMO
In cervical cancer, glycosylation has been suggested as being involved in both its carcinogenesis and invasive capacity. In this work, we analyzed mucin type O-glycosylation in biopsies of invasive cervical cancer in FIGO stage II B through histochemistry using lectins specific for O-glycosidically linked glycans. Our results reveal that the lectin Machaerocereus eruca (MeA, specific for Gal in a Fucα1,2 (GalNAcα1,3) Galß1,4) showed increased recognition of tumoral cells and tumoral stroma tissue compared to other lectins with similar specificity; healthy cervical tissue was negative for MeA. Trypsin treatment of recognized tissues abolished MeA's recognition;moreover, interaction of MeA was inhibited with oligosaccharides from mucin. As demonstrated by Western blot of 2-D electrophoresis, MeA recognized ten glycoproteins in the range from 122 to 42 kDa in cervical cancer lysates. The LC-ESI-MS/MS analysis of the MeAs' recognized peptides revealed that the latter matched mainly with the amino acid sequences of lamin A/C, vimentin, elongation factor 2, keratin 1, and beta actin. Our results suggest that MeA recognizes a complex of over-expressed O-glycosidically-linked proteins that play a relevant role in cervical cancer's invasive capacity. O-glycosylation participates in the disassembly of intercellular junctions favoring cancer progression.
Assuntos
Aglutininas/metabolismo , Glicoproteínas/metabolismo , Lectinas de Plantas/metabolismo , Neoplasias do Colo do Útero/patologia , Animais , Cromatografia Líquida , Eletroforese em Gel Bidimensional , Feminino , Fluoresceína-5-Isotiocianato/metabolismo , Glicosilação , Histocitoquímica , Humanos , Immunoblotting , Espectrometria de Massas , Sus scrofa , Tripsina/metabolismoRESUMO
Overexpression and amplification of several genes (MDM2, CDK4 and SAS) located on chromosome 12q13-15 have been noted to occur in various human sarcomas. As a result, two major growth regulation pathways may be inhibited. MDM2 may down regulate the p53-mediated growth control and CDK4 may affect pRB-mediated events. To determine the frequency of alterations in these genes and their correlation with clinicopathologic features, we analyzed the MDM2 and CDK4 protein levels by immunohistochemistry and assessed MDM2, CDK4 and SAS amplification by real-time PCR in nine osteosarcomas of the jaws. Positive staining for CDK4 and MDM2 was observed in eight cases (88.8%) and five cases (55.5%), respectively. Intense CDK4 staining was noted in four cases (two high grade, one intermediate grade and one low grade). Intense MDM2 staining was observed in the same four previous cases, as well as, one additional high-grade tumor. Individual DNA amplification for CDK4, MDM2 and SAS was observed in six cases for each gene. Co-amplification was observed in five cases that showed CDK4 and MDM2 concomitant amplification and four cases that displayed amplification for all of the genes. In addition, among the five cases that presented CDK4 and MDM2 amplification, strong overexpression of CDK4 and MDM2 was observed in three and in four cases, respectively (three high grade and one intermediate grade). These results suggest that 12q13-15 genes are involved in neoplastic disease and concurrent amplification and overexpression of these genes might help to define high-grade tumors.
Assuntos
Biomarcadores Tumorais/metabolismo , Cromossomos Humanos Par 12 , Neoplasias Maxilomandibulares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Osteossarcoma/metabolismo , Adulto , Aglutininas/genética , Aglutininas/metabolismo , Biomarcadores Tumorais/genética , Quinase 4 Dependente de Ciclina , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Feminino , Seguimentos , Amplificação de Genes , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Técnicas Imunoenzimáticas , Neoplasias Maxilomandibulares/genética , Neoplasias Maxilomandibulares/patologia , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Osteossarcoma/genética , Osteossarcoma/patologia , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-mdm2RESUMO
The surface anionogenic groups and sialoglycoconjugate structures of Paracoccidioides brasiliensis yeast forms were analysed by cell microelectrophoresis, binding assays with lectins and viral particles, ultrastructural cytochemistry, enzymic digestion and flow cytofluorimetry. P. brasiliensis yeast forms, particularly the budding primordia, reacted strongly with cationized ferritin. Binding assays showed that the reaction with sialic-acid-specific Limax flavus lectin (LFA) was distributed over the entire P. brasiliensis cell wall. Treatment of yeast forms with neuraminidase significantly reduced their negative surface charge and LFA labelling, which suggests that sialic acid residues are major anionogenic groups exposed on the P. brasiliensis surface. Furthermore, after neuraminidase treatment, labelling with Arachis hypogaea (peanut) agglutinin increased due to unmasking of subterminal beta-D-galactopyranosyl residues. The sialic acid linkages to galactose are alpha 2,6 and alpha 2,3 as assessed, respectively, by fungal attachment to M1/5 and M1/5 HS8 strains of influenza A virus and binding of Sambucus niger and Maackia amurensis agglutinins. The alpha 2,6 linkage clearly predominated in both experiments. Flow cytofluorimetry analysis revealed the heterogenicity of P. brasiliensis yeast cell populations, which comprised young and mature budding yeasts. Both express binding sites to LFA and Limulus polyphemus agglutinin.
Assuntos
Ácido N-Acetilneuramínico/análise , Ácido N-Acetilneuramínico/metabolismo , Paracoccidioides/química , Paracoccidioides/metabolismo , Aglutininas/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Ferritinas/farmacocinética , Citometria de Fluxo , Proteínas Fúngicas/metabolismo , Galactose/metabolismo , Vírus da Influenza A/metabolismo , Lectinas/metabolismo , Microscopia Eletrônica , Neuraminidase/farmacologia , Paracoccidioides/ultraestrutura , Ligação Proteica , Receptores de Superfície Celular/metabolismoRESUMO
We have previously described that normal mouse serum inhibits the PCA reaction mediated by IgE. The present study attempts to characterize this PCA inhibitory factor from the biologically active fraction of the serum. The physicochemical properties of this glycoprotein are the following: it is inactivated at 55 degrees C; it has a molecular weight between 182 kD and 240 kD, determined by gel filtration; it shows affinity to concanavalin A and lentil lectin but not to peanut agglutin; it demonstrates affinity to IgE and, apparently, its carbohydrate moiety is not required for its biological activity. Two bands corresponding to 64.5 kD and 48. 1 kD, which are likely to constitute the biologically active molecule, are observed by SDS-PAGE. These properties are different from those found in factors with IgE affinity involved in IgE synthesis.
Assuntos
Proteínas Sanguíneas/isolamento & purificação , Proteínas Sanguíneas/farmacologia , Glicoproteínas/isolamento & purificação , Glicoproteínas/metabolismo , Anafilaxia Cutânea Passiva/efeitos dos fármacos , Aglutininas/metabolismo , Anafilaxia/prevenção & controle , Animais , Cromatografia de Afinidade , Cromatografia em Gel , Concanavalina A/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicoproteínas/imunologia , Calefação , Imunoglobulina E/imunologia , Imunoglobulina E/metabolismo , Lectinas/metabolismo , Camundongos , Ligação ProteicaRESUMO
Foram desenvolvidas formulaçöes de comprimento de pó de guaraná (Paullinia cupana H.B.K., Sapindaceae), visando facilitar sua administraçäo por via oral, visto que este possui sabor fracamente amargo e adstringente. Foram utilizadas goma de amido, gelatina, sorbitol, xarope simples e goma arábica como aglutinantes, analisando-se em seguida as características físicas dos comprimidos obtidos no comércio. A formulaçäo contendo xarope simples, na concentraçäo de 15,5% apresentou melhor comportameno reológico, capacidade de resistência aos choques e atrito e tempo de desagregaçäo adequado às formulaçöes sólidas de liberaçäo gástrica
Assuntos
Aglutininas/metabolismo , Extratos Vegetais/metabolismo , Comprimidos , Administração Oral , Composição de MedicamentosRESUMO
Labeling of lectin receptors during the cell cycle. (Localizabión de receptores para lectinas durante el ciclo celular). Arch. Biol. Med. Exper. 10: 100-104, 1976. The topographic distribution of specific cell surface receptors for concanavalin A and wheat germ agglutinin was studied by ultrastructural labeling in the course of the cell cycle. C12TSV5 cells were synchronized by double thymidine block or mechanical selection (shakeoff). They were labeled by means of lectin-peroxidase techniques while in G1 S, G2 and M phases of the cycle. The results obtained were similar for both lectins employed. Interphase cells (G1 S, G2) present a stlihtly discontinous labeling pattern that is similar to the one observed on unsynchronized cells of the same line. Cells in mitosis, on the contrary, present a highly discontinous distribution of reaction product. This pattern disappears after the cells enters G1 and is not present on mitotic cells fixed in aldehyde prior to labeling.