Crotacetin, a novel snake venom C-type lectin homolog of convulxin, exhibits an unpredictable antimicrobial activity

Snake venom (sv) C-type lectins encompass a group of hemorrhagic toxins that are capable of interfering with blood stasis. A very well-studied svC-type lectin is the heterodimeric toxin, convulxin (CVX), from the venom of South American rattlesnake Crotalus durissus terrificus. CVX is able to activate platelets and induce their aggregation by acting via p62/GPVI collagen receptor. By using polymerase chain reaction homology screening, we have cloned several cDNA precursors of CVX subunit homologs. One of them, named crotacetin (CTC) beta-subunit, predicts a polypeptide with a topology very similar to the tridimensional conformations of other subunits of CVX-like snake toxins, as determined by computational analysis. Using gel permeation and reverse-phase high-performance liquid chromatography, CTC was purified from C. durissus venoms. CTC can be isolated from the venom of several C. durissus subspecies, but its quantitative predominance is in the venom of C. durissus cascavella. Functional analysis indicates that CTC induces platelet aggregation, and, importantly, exhibits an antimicrobial activity against Gram-positive and -negative bacteria, comparable with CVX.

Quartz Crystal Microbalance monitoring the real-time binding of lectin with carbohydrate with high and low molecular mass

Quartz Crystal Microbalance (QCM) was used to monitor the mass changes on a quartz crystal surface containing immobilized lectins that interacted with carbohydrates. The strategy for lectin immobilization was developed on the basis of a multilayer system composed of Au-cystamine-glutaraldehyde-lectin. Each step of the immobilization procedure was confirmed by FTIR analysis. The system was used to study the interactions of Concanavalin A (ConA) with maltose and Jacalin with Fetuin. The real-time binding of different concentrations of carbohydrate to the immobilized lectin was monitored by means of QCM measurements and the data obtained allowed for the construction of Langmuir isotherm curves. The association constants determined for the specific interactions analyzed here were (6.4 +/- 0.2) X 10(4) M-1 for Jacalin-Fetuin and (4.5 +/- 0.1) x 10(2) M-1 for ConA-maltose. These results indicate that the QCM constitutes a suitable method for the analysis of lectin-carbohydrate interactions, even when assaying low molecular mass ligands such as disaccharides. Published by Elsevier B.V.

Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8% (w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 angstrom resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

Crystallization and preliminary X-ray diffraction analysis of an anti-H(O) lectin from Lotus tetragonolobus seeds

The seed lectin from Lotus tetragonolobus (LTA) has been crystallized. The best crystals grew over several days and were obtained using the vapour-diffusion method at a constant temperature of 293 K. A complete structural data set was collected at 2.00 angstrom resolution using a synchrotron-radiation source. LTA crystals were found to be monoclinic, belonging to space group P2(1), with unit-cell parameters a = 68.89, b = 65.83, c = 102.53 angstrom, alpha = gamma = 90, beta = 92 degrees. Molecular replacement yielded a solution with a correlation coefficient and R factor of 34.4 and 51.6%, respectively. Preliminary analysis of the molecular-replacement solution indicates a new quaternary association in the LTA structure. Crystallographic refinement is under way.

Genotypes of the mannan-binding lectin gene and susceptibility to visceral leishmaniasis and clinical complications

Background. Visceral leishmaniasis (VL) is almost always lethal if not treated, but most infections with the causative agents are clinically silent. Mannan-binding lectin (MBL), an opsonin, is a candidate molecule for modifying progression to VL because it may enhance infection with intracellular pathogens. Mutations in the MBL2 gene decrease levels of MBL and may protect against development of VL. This case-control study examines genotypes of MBL2 and levels of MBL in individuals presenting with different outcomes of infection with Leishmania chagasi.Methods. Genotypes for MBL2 and levels of serum MBL were determined in uninfected control subjects (n=76) and in individuals presenting with asymptomatic infection (n=90) or VL (n=69).Results. Genotypes resulting in high levels of MBL were more frequent (odds ratio [OR], 2.5 [95% confidence interval {CI}, 1.3-5.0]; P=.006) among individuals with VL than among those with asymptomatic infections and were even more frequent (OR, 3.97 [95% CI, 1.10-14.38];P=.043) among cases of VL presenting with clinical complications than among those with uneventful courses. Serum levels of MBL were higher (P=.011) in individuals with VL than in asymptomatic infections.Conclusions. Genotypes of the MBL2 gene predict the risk for developing VL and clinical complications in infections with L. chagasi.

Effects of a lectin-like protein isolated from Acacia farnesiana seeds on phytopathogenic bacterial strains and root-knot nematode

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purification, Characterization, and Preliminary X-Ray Diffraction Analysis of a Lactose-Specific Lectin from Cymbosema roseum Seeds

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 A mu g mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A....

Characteristics of the histamine release from hamster cheek pouch mast cells stimulated by lectins from Brazilian beans and concanavalin A

We have characterized the histamine releasing effects of lectins extracted from Brazilian beans, in comparison to concanavalin A, in hamster cheek pouch cell suspensions containing mast cells. The lectins from Dioclea virgata, Canavalia brasiliensis, and Dioclea rostrata induce histamine release in a similar manner to concanavalin A, but appear to differ in potency and efficacy. The effects depended on the temperature, pH, and metabolic energy, demonstrating the non-cytotoxic nature of the histamine release. It is suggested that the lectins studied act by the same mechanism as concanavalin A (interacting with sugars in the antibodies bound to the mast cells), since high concentrations of glucose inhibit the histamine release. The lectins at high concentrations quench the histamine release. This suppression is reversed by increasing calcium concentration, suggesting that the lectins bind to the calcium that is essential for the secretion, thereby confirming and extending our previous data using the lectin from Dioclea virgata in rat peritoneal mast cells.

Inhibitory effects of jackbean (Canavalia ensiformis L.) leaf residues on germination and vigour of crops and weeds

The effects of jackbean leaf residues incorporated in the soil on germination and seedlings growth of cucumber, radish and some weeds was examined. Trials were carried out under greenhouse conditions to (a) determine the amount of incorporated residue that is inhibitory to two test plants, (b) to determine if decomposition time changes the inhibitory levels of jackbean residues on test plants and (c) to determine the amount of residue that is inhibitory to the weed species. Jackbean leaf residues incorporated in soil at concentration of 2% or higher and allowed to decompose for a period of 0 to 2 weeks before sowing, reduced the initial growth of cucumber and radish and at different concentrations, reduced germination and growth of three weed species. These results suggest the presence of allelopathic components in Jackbean leaves that could affect seed germination and seedling development.

Activity of Extracts and Fatty Acids of Canavalia ensiformis (Leguminosae) Against the Symbiotic Fungus of the Leaf-Cutting Ants Atta sexdens

The inhibitory effect of leaves extracts of Carnavalia ensiformis on the development of the symbiotic fungus of the leaf-cutting ants Atta sexdens (Forel) was evaluated. The hexane extract showed highest activity at concentration of 1000 μg/mL. Chromatographic separations of this extract have led to the isolation of a mixture of fatty acids which showed the same activity of the crude extract.

Topical application of the lectin Artin M accelerates wound healing in rat oral mucosa by enhancing TGF-β and VEGF production

The lectin Artin M has been shown to accelerate the wound-healing process. The aims of this study were to evaluate the effects of Artin M on wound healing in the palatal mucosa of rats and to investigate the effects of Artin M on transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) secretion by rat gingival fibroblasts. A surgical wound was created on the palatal mucosa of 72 rats divided into three groups according to treatment: C - Control (nontreated), A - Artin M gel, and V - Vehicle. Eight animals per group were sacrificed at 3, 5, and 7 days postsurgery for histology, immunohistochemistry and determination of the levels of cytokines, and growth factors. Gingival fibroblasts were incubated with 2.5 μg/mL of Artin M for 24, 48, and 72 hours. The expression of VEGF and TGF-β was determined by enzyme-linked immunosorbent assay. Histologically, at day 7, the Artin M group showed earlier reepithelialization, milder inflammatory infiltration, and increased collagen fiber formation, resulting in faster maturation of granular tissue than in the other groups (p < 0.05). Artin M-induced cell proliferation in vivo and promoted a greater expression of TGF-β and VEGF in both experiments (p < 0.05). Artin M was effective in healing oral mucosa wounds in rats and was associated with increased TGF-β and VEGF release, cell proliferation, reepithelialization, and collagen deposition and arrangement of fibers. © 2013 by the Wound Healing Society.

O plantio associado entre banana (Musa acuminata Cultivar Cavendish "Grande Naine") e feijão-de-porco (Canavalia ensiformis (L.) D. C.): uma avaliação agronômica dos efeitos da competição por água, nitrogênio e energia luminosa

Nas regiões de clima tropical, o monocultivo da banana vem causando conseqüências ambientais desastrosas e, muitas vezes, impedindo uma exploração continuada de uma mesma área. A redução do rendimento é devido principalmente as limitações físico-químicas do solo e a rápida degradação do sistema radicular, agravada pela ação de parasitas do solo (nematóides, fungos, etc.). Em virtude destas limitações, várias iniciativas vem sendo buscadas para a minimização das perdas agronômicas e ambientais, destacando-se o melhoramento e a modificação genética, e a associação deste cultivo com espécies leguminosas. Porém uma das grandes dificuldades de avaliarmos os novos sistemas de cultivo alternativos concentra-se na falta de referenciais agronômicos relacionados principalmente com o funcionamento de sistemas de cultivos associados, especialmente relacionados aos fatores e condições que interferem diretamente na definição do rendimento da espécie principal. O presente estudo testou , em campo experimental, o uso de plantas de serviço associada a bananeira e seus efeitos na produção de biomassa durante seu o ciclo vegetativo. Isto porque é durante esta fase que a bananeira constrói sua capacidade de reservas de fotoassimilados e, consequentemente, define o potencial de produção e enchimento dos frutos. Além do monocultivo, definiu-se mais duas parcelas associadas com o feijão-de- porco: 1) o plantio simultâneo das duas espécies e; 2) o plantio de feijão-de-porco e, após 2 meses, a introdução da banana. Além de acompanhamento semanal das parcelas, realizou-se, bimensalmente, coletas destrutivas de dados sobre produção de matéria seca, superfície foliar e análise nutricional das plantas. Após a análise agronômica da fase vegetativa, aplicou-se a modelização dos sistemas de cultivo estudados e comparou-se os possíveis cenários sobre o rendimento final da bananeira, além de outros indicadores sobre os fatores de crescimento das plantas. Após o acompanhamento dos 7 primeiros meses do ciclo vegetativo, concluiu-se que a data de estabelecimento da associação foi determinante para o sucesso do cultivo associado. Podemos destacar que a associação entre a bananeira e o feijão-de-porco não causou limitações na produção de biomassa (4,2 ton/ha), quando comparada com o monocultivo (4,5 ton/ha). A redução do número de capinas também foi um indicador animador deste sistema de cultivo alternativo. Por outro lado, quando a bananeira foi plantada 60 dias após a leguminosa, a mesma representou uma séria limitação na produção de biomassa (2,7 ton/ha). Esta limitação deveu-se ao estado de forte competição devido a agressividade com que o feijão-de-porco recobria toda a parcela e alcançando uma altura (74 cm) superior que a muda de banana (29 cm). Em relação a primeira parte da metodologia aplicada - o diagnóstico agronômico -, a mesma foi eficiente para a avaliação do ciclo vegetativo da associação estudada, ficando a necessidade da continuidade do acompanhamento do ciclo reprodutivo, para a confirmação dos resultados em termos de formação e produção de frutos. Na fase de modelização, chegou-se a uma leitura dos resultados próxima dos resultados obtidos no campo. Em termos de rendimento em frutos, o monocultivo com adubação (400 kg/ha de nitrogênio) e irrigação (133 mm) teve um aumento na ordem de 50% no rendimento final (28 ton/ha) Quando comparada com a parcela nas condições reais do experimento (19,6 ton/ha). Já o rendimento em frutos da associação, apresentou o mesmo resultado com e sem adubação e irrigação (16 ton/ha). No tocante a contrução dos cenários, confirmou-se novamente algumas das vantagens da associação, principalmente na redução da adubação nitrogenada aplicada nos sistemas convencionais de cultivo. Finalmente, podemos imaginar a construção de várias formas de testar e otimizar o uso destes sistemas associados (cenários). Porém, confirma-se que a construção de novos referenciais agronômicos sobre sistemas de cultivo mais complexos (os cultivos associados) torna-se ainda muito necessário para a realização de avaliações mais precisas sobre estas alternativas. E, com estes novos referenciais técnicos, podemos imaginar, a médio e longo prazo, alguns dos benefícios das leguminosas sobre as propriedades físico-químicas do solo cultivado (cobertura viva, adubo verde, redução de adventícias, etc) e sobre a manutenção do rendimento dos cultivos (adubação verde).

Characterization of mannose-binding lectin plasma levels and genetic polymorphisms in HIV-1-infected individuals

INTRODUÇÃO: O presente estudo investigou a associação entre o polimorfismo no gene da lectina ligante de manose (MBL) e os níveis séricos da proteína com a infecção pelo HIV-1. MÉTODOS: As amostras de sangue (5mL) foram coletadas de 97 indivíduos infectados pelo HIV-1 residentes em Belém, Estado do Pará, Brasil, que frequentavam a Unidade de Referência Especial para Doenças Infecciosas e Parasitárias Especiais (URE-DIPE). Os níveis de linfócitos T CD4+ e da carga viral plasmática foram quantificados. Um fragmento de 349pb do exon 1 da MBL foi amplificado via PCR, utilizando DNA genômico extraído das amostras controles e dos indivíduos portadores do HIV-1, seguindo protocolos previamente estabelecidos. O nível plasmático de MBL nos pacientes foi quantificado usando kit de ensaio imunoenzimático. RESULTADOS: Dois alelos foram observados - MBL*O, com uma frequência de 26,3% em indivíduos infectados e o alelo selvagem MBL*A (73,7%). Frequências similares foram observadas no grupo controle (p > 0,05). As frequências genotípicas estavam em equilíbrio de Hardy-Weinberg em ambos os grupos. A média dos níveis plasmáticos MBL variou por genótipo, com diferenças significativas entre os genótipos AA e AO (p < 0,0001), e AA e OO (p < 0,001), mas não entre AO e OO (p=0,17). Além disso, os linfócitos T CD4+ e os níveis plasmáticos de carga viral não diferiram significativamente de acordo com o genótipo (p>0,05). CONCLUSÕES: Os resultados deste estudo não apoiam a hipótese de que o polimorfismo no gene MBL ou baixa concentração plasmática de MBL poderia ter uma influência direta sobre a infecção pelo HIV-1, embora um estudo com número maior de pacientes seja necessário.

Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects

ABSTRACT: The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

ABSTRACT: The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < IC95% < 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.