Native crystal structure of a nitric oxide-releasing lectin from the seeds of Canavalia maritima

Here, we report the crystallographic study of a lectin from Canavalia maritima seeds (ConM) and its relaxant activity on vascular smooth muscle, to provide new insights into the understanding of structure/function relationships of this class of proteins. ConM was crystallized and its structure determined by standard molecular replacement techniques. The amino acid residues, previously suggested incorrectly by manual sequencing, have now been determined as I17, I53, S129, S134, G144, S164, P165, S187, V190, S169, T196, and S202. Analysis of the structure indicated a dimer in the asymmetric unit, two metal binding sites per monomer, and loops involved in the molecular oligomerization. These confer 98% similarity between ConM and other previously described lectins, derived from Canavalia ensiformis and Canavalia brasiliensis. Our functional data indicate that ConM exerts a concentration-dependent relaxant action on isolated aortic rings that probably occurs via an interaction with a specific lectin-binding site on the endothelium, resulting in a release of nitric oxide. (C) 2005 Elsevier B.V. All rights reserved.

Comportamiento agronomico y productivo de nueve leguminosas herbaceas forrajeras, en el municipio de Muy Muy, Matagalpa

Las dos grandes fuentes de alimentación animal son: energética que comúnmente esta dada por las gramíneas y proteica, la cual puede ser suplementada por diferentes vías, pero las mas simple y que la naturaleza la provee es a través de las leguminosas, producto de la asociación con bacterias del género Rhizobium, que les permite fijar nitrógeno atmosférico. Los estudio hechos de leguminosas al momento han sido muy pocos, y cuando se han hecho se ha centrado únicamente en zonas del caribe con alta precipitación y suelos ácidos. Por lo que con el objeto de conocer el comportamiento de adaptación agronómico y productivo de 9 especies de leguminosas consideradas como forrajeras en el municipio de Muy Muy, se realizó el presente estudio en la finca “La Cruz” propiedad del señor Santiago Espino, ubicada en el km 153 de la carretera Muy Muy-Matiguás, en la comunidad de “Aguas Calientes”, zona baja del municipio de Muy Muy, Matagalpa, localizado en las coordenadas geográficas 12º 45 ́48” latitud Norte y 85º 37 ́36”longitud Oeste, a una altitud de unos 286msnmy con una temperatura promedio anual de 25.0°C. se estableció el experimento de campo el 4 de Junio del 2007. Utilizándose un Diseño de Bloques Completos al Azar (BCA), con nueve tratamientos (especies evaluadas: Centrosema plumieri cv DICTA, Clitoria ternatea cv CEVAS, Canavalia brasiliensis CIAT 1700, Clitoria ternatea CV DICTA, Canavalia ensiformis, Lablab purpureus, Vigna unguiculata CIAT9611, Vigna unguiculata CIAT390-2 y Stylosanthes guianensis CIAT2243). Se midieron 9 variables: sobrevivencia, altura, vigor, cobertura, cobertura de maleza, suelo descubierto, incidencia de plagas, incidencia de enfermedades y producción de biomasa seca, además se realizó análisis de la composición química a ocho de las especies en estudio. S.guianensis 2243 presentó el mejor comportamiento agronómico, superando al resto de especies en casi todas las variables en estudio, salvo el caso de altura donde fue superada por C. brasiliensis 1700 con 95.5 cm y en suelo descubierto por C. plumieri DICTA con 4.38%. En sobrevivencia Stylo alcanzó un 100%, con cobertura de 76.25%, menor incidencia de plagas y enfermedades (0%) y ponderación en vigor de 4.9. Además S.guianensis 2243 presentó el mejor comportamiento en producción de biomasa seca con 3717.02 kg ha-1, seguida de C. plumieriDICTA con 1486.38 kg ha-1. La especie de menor comportamiento adaptativo fue V. unguiculata 9611, y en producción de biomasa L. purpureus con 206.82 kg ha-1. C. ensiformis presento mejor contenido proteico con 26.20% y C. ternateaCEVAS en la porción digerible con 85.71%, la de menor calidad fue S.guianensis 2243 con 13.26% de proteína y 54.59% de porción digerible. Aun con los resultados de calidad se considera que S. guianensis2243 es una especie promisoria para estas condiciones por su a daptabilidad y producción de biomasa, y calidad y a las especies C. plumieriDICTA y C. ternateaCEVAS por su calidad nutritiva, adaptación y producciónde biomasa.

Microbial response to restoration of tantalite mine soils in western Rwanda

Artisanal columbite-tantalite (coltan) mining has had negative effects on the rural economy in the great Lakes region of Africa through labor deficits, degradation and loss of farmland, food insecurity, high cost of living, and reduced traditional export crop production alongside secondary impacts that remotely affect the quality of air, water, soil, plants, animals, and human wellbeing. The situation is multifaceted and calls for a holistic approach for short and long-term mitigation of such negative effects. This study focuses on the effects of mine land restoration on soil microbiological quality in the Gatumba Mining District of western Rwanda. Some coltan mine wastelands were afforested with pine and eucalyptus trees while farmers directly cultivated others due to land scarcity. Farmyard manure (FYM) is the sole fertilizer applied on the wastelands although it is insufficient to achieve the desired crop yields. Despite this, several multi-purpose plants such as Tithonia diversifolia, Markhamia lutea, and Canavalia brasiliensis thrive in the area and could supplement FYM. The potential for these “new” amendments to improve soil microbial properties, particularly in the tantalite mine soils was investigated. The specific objectives of the study were to: (a) evaluate the effects of land use on soil microbial indices of the tantalite mine soils; (b) investigate the restorative effects of organic amendments on a Technosol; and (c) estimate the short-term N and P supply potential of the soil amendments in the soils. Fresh soils (0-20 cm) from an unmined native forest, two mine sites afforested with pine and eucalyptus forests (pine and eucalyptus Technosols), an arable land, and two cultivated Technosols (Kavumu and Kirengo Technosols) were analyzed for the physicochemical properties. Afterwards, a 28-day incubation (22oC) experiment was conducted followed by measurements of mineral N, soil microbial biomass C, N, P, and fungal ergosterol contents using standard methods. This was followed by a 12-week incubation study of the arable soil and the Kavumu Technosol amended with FYM, Canavalia and Tithonia biomass, and Markhamia leaf litter after which soil microbial properties were measured at 2, 8, and 12 weeks of incubation. Finally, two 4-week incubation experiments each were conducted in soils of the six sites to estimate (i) potential mineralizable N using a soil-sand mixture (1:1) amended with Canavalia and goat manure and (ii) P mineralization mixtures (1:1) of soil and anion exchange resins in bicarbonate form amended with Tithonia biomass and goat manure. In study one, afforestation increased soil organic carbon and total N contents in the pine and eucalyptus Technosols by 34-40% and 28-30%, respectively of that in the native forest soil. Consequently, the microbial biomass and activity followed a similar trend where the cultivated Technosols were inferior to the afforested ones. The microbial indices of the mine soils were constrained by soil acidity, dithionite-extractable Al, and low P availability. In study two, the amendments substantially increased C and N mineralization, microbial properties compared with non-amended soils. Canavalia biomass increased CO2 efflux by 340%, net N mineralization by 30-140%, and microbial biomass C and N by 240-600% and 240-380% (P < 0.01), respectively after four weeks of incubation compared with the non-amended soils. Tithonia biomass increased ergosterol content by roughly 240%. The Kavumu Technosol showed a high potential for quick restoration of its soil quality due to its major responses to the measured biological parameters. In study three, Canavalia biomass gave the highest mineralizable N (130 µg g-1 soil, P < 0.01) in the Kavumu Technosol and the lowest in the native forest soil (-20 µg g-1 soil). Conversely, the mineralizable N of goat manure was negative in all soils ranging from -2.5 µg N g-1 to -7.7 µg N g-1 soil except the native forest soil. However, the immobilization of goat manure N in the “cultivated soils” was 30-70% lower than in the “forest soils” signifying an imminent recovery of the amended soils from N immobilization. The mineralization of goat manure P was three-fold that of Tithonia, constituting 61-71% of total P applied. Phosphorus mineralization slightly decreased after four weeks of incubation due to sulfate competition as reflected in a negative correlation, which was steeper in the Tithonia treatment. In conclusion, each amendment used in this research played a unique role in C, N, and P mineralization and contributed substantially to microbial properties in the tantalite mine soils. Interestingly, the “N immobilizers” exhibited potentials for P release and soil organic carbon storage. Consequently, the combined use of the amendments in specific ratios, or co-composting prior to application is recommended to optimize nutrient release, microbial biomass dynamics and soil organic matter accrual. Transport of organic inputs seems more feasible for smallholder farmers who typically manage small field sizes. To reduce acidity in the soils, liming with wood ash was recommended to also improve P availability and enhance soil biological quality, even if it may only be possible on small areas. Further, afforestation with mixed-species of fast-growing eucalyptus and legume or indigenous tree species are suggested to restore tantalite mine wastelands. It is emphasized most of this research was conducted under controlled laboratory conditions, which exclude interaction with environmental variables. Also fine fractions of the amendments were used compared with the usual practice of applying a mixture of predominantly coarser fractions. Therefore, the biological dynamics reported in the studies here may not entirely reflect those of farmers’ field conditions.

Efeitos de geada no desenvolvimento do feijoeiro em sucessão a espécies vegetais e adubação nitrogenada

O objetivo deste trabalho foi verificar o efeito de geada no desenvolvimento do feijoeiro em experimentação de cultivo em sucessão a espécies de cobertura e adubação nitrogenada, em delineamento experimental de blocos ao acaso em esquema de parcelas subdivididas, com quatro repetições. As parcelas foram constituídas pelas espécies Sorghum bicolor tipo guinea, Canavalia brasiliensis, Sorghum bicolor, Crotalaria juncea, Pennisetum glaucum e uma testemunha mantida no limpo, enquanto as subparcelas por doses de nitrogênio em cobertura (0, 25, 50 e 75 kg ha-1 de N), utilizando-se como fonte a uréia. Menores danos e severidade da geada foram constatados em plantas de feijão cultivadas após as espécies Canavalia brasiliensis e Crotalaria juncea, devido à menor quantidade de massa seca residual em relação à das culturas de gramíneas. Produtividades superiores de grãos de feijoeiro após geada foram obtidas na sucessão com Crotalária juncea e C. brasiliensis e com a aplicação de doses entre 44,5 e 52,0 kg ha-1 de N, em cobertura.

Sucessão de cultivo de feijão-arroz com doses de adubação nitrogenada após adubação verde, em semeadura direta

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Differential effect of plant lectins on mast cells of different origins

Histamine release induced by plant lectins was studied with emphasis on the carbohydrate specificity, external calcium requirement, metal binding sites, and mast cell heterogeneity and on the importance of antibodies bound to the mast cell membrane to the lectin effect. Peritoneal mast cells were obtained by direct lavage of the rat peritoneal cavity and guinea pig intestine and hamster cheek pouch mast cells were obtained by dispersion with collagenase type IA. Histamine release was induced with concanavalin A (Con A), lectins from Canavalia brasiliensis, mannose-specific Cymbosema roseum, Maackia amurensis, Parkia platycephala, Triticum vulgaris (WGA), and demetallized Con A and C. brasiliensis, using 1-300 µg/ml lectin concentrations applied to Wistar rat peritoneal mast cells, peaking on 26.9, 21.0, 29.1, 24.9, 17.2, 10.7, 19.9, and 41.5%, respectively. This effect was inhibited in the absence of extracellular calcium. The lectins were also active on hamster cheek pouch mast cells (except demetallized Con A) and on Rowett nude rat (animal free of immunoglobulins) peritoneal mast cells (except for mannose-specific C. roseum, P. platycephala and WGA). No effect was observed in guinea pig intestine mast cells. Glucose-saturated Con A and C. brasiliensis also released histamine from Wistar rat peritoneal mast cells. These results suggest that histamine release induced by lectins is influenced by the heterogeneity of mast cells and depends on extracellular calcium. The results also suggest that this histamine release might occur by alternative mechanisms, because the usual mechanism of lectins is related to their binding properties to metals from which depend the binding to sugars, which would be their sites to bind to immunoglobulins. In the present study, we show that the histamine release by lectins was also induced by demetallized lectins and by sugar-saturated lectins (which would avoid their binding to other sugars). Additionally, the lectins also released histamine from Rowett nude mast cells that are free of immunoglobulins.

Histamine release induced by glucose (mannose)-specific lectins isolated from Brazilian beans. Comparison with concanavalin A

The histamine releasing properties of glucose (mannose)-specific lectins isolated from Brazilian beans was examined. The Canavalia brasiliensis, Dioclea rostrata, and Dioclea virgata lectins induced histamine release in rat peritoneal mast cells similar to concanavalin A. Less potency and efficacy was observed for Canavalia maritima, Dioclea guianensis, and Dioclea violacea while very low activities were seen for the lectins from Dioclea grandiflora, Canavalia bonariensis, and Cratylia floribunda. The histamine releasing effect was quenched by higher doses of D. virgata lectin similar to what was reported for concanavalin A. This effect was abrogated by increasing the concentration of calcium in the incubating medium. As these above proteins have sites that bind calcium, higher doses of the lectins might withdraw the calcium which is essential for the mast cell secretion.

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.

ANTIFUNGAL ACTIVITY OF LECTINS AGAINST YEAST OF VAGINAL SECRETION

Lectins are carbohydrate-binding proteins of non-imune origin. This group of proteins is distributed widely in nature and they have been found in viruses, microorganisms, plants and animals. Lectins of plants have been isolated and characterized according to their chemical, physical-chemical, structural and biological properties. Among their biological activities, we can stress its fungicidal action. It has been previously described the effect of the lectins Dviol, DRL, ConBr and LSL obtained from the seeds of leguminous plants on the growth of yeasts isolated from vaginal secretions. In the present work the experiments were carried out in microtiter plates and the results interpreted by both methods: visual observations and a microplate reader at 530nm. The lectin concentrations varied from 0.5 to 256 mu g/mL, and the inoculum was established between 65-70% of trammitance. All yeast samples isolated from vaginal secretion were evaluated taxonomically, where were observed macroscopic and microscopic characteristics to each species. The LSL lectin did not demonstrate any antifungal activity to any isolate studied. The other lectins DRL, ConBr and DvioL, showed antifungal potential against yeast isolated from vaginal secretion. These findings offering offer a promising field of investigation to develop new therapeutic strategies against vaginal yeast infections, collaborating to improve women's health.

Towards an assessment of on-farm niches for improved forages in Sud-Kivu, DR Congo

Inadequate quantity and quality of livestock feed is a persistent constraint to productivity for mixed crop-livestock farming in eastern Democratic Republic of Congo. To assess on-farm niches of improved forages, demonstration trials and participatory on-farm research were conducted in four different sites. Forage legumes included Canavalia brasiliensis (CIAT 17009), Stylosanthes guianensis (CIAT 11995) and Desmodium uncinatum (cv. Silverleaf), while grasses were Guatemala grass (Tripsacum andersonii), Napier grass (Pennisetum purpureum) French Cameroon, and a local Napier line. Within the first six months, forage legumes adapted differently to the four sites with little differences among varieties, while forage grasses displayed higher variability in biomass production among varieties than among sites. Farmers’ ranking largely corresponded to herbage yield from the first cut, preferring Canavalia, Silverleaf desmodium and Napier French Cameroon. Choice of forages and integration into farming systems depended on land availability, soil erosion prevalence and livestock husbandry system. In erosion prone sites, 55–60% of farmers planted grasses on field edges and 16–30% as hedgerows for erosion control. 43% of farmers grew forages as intercrop with food crops such as maize and cassava, pointing to land scarcity. Only in the site with lower land pressure, 71% of farmers grew legumes as pure stand. When land tenure was not secured and livestock freely roaming, 75% of farmers preferred to grow annual forage legumes instead of perennial grasses. Future research should develop robust decision support for spatial and temporal integration of forage technologies into diverse smallholder cropping systems and agro-ecologies.

Cover Crops and Nitrogen Fertilization Effects on Nitrogen Soil Fractions under Corn Cultivation in a No-Tillage System.

ABSTRACT: The use of cover crops has recently increased and represents an essential practice for the sustainability of no-tillage systems in the Cerrado region. However, there is little information on the effects of nitrogen fertilization and cover crop use on nitrogen soil fractions. This study assessed changes in the N forms in soil cropped to cover crops prior to corn growing. The experiment consisted of a randomized complete block design arranged in split-plots with three replications. Cover crops were tested in the plots, and the N topdressing fertilization was assessed in the subplots. The following cover species were planted in succession to corn for eight years: Urochloa ruziziensis, Canavalia brasiliensis M. ex Benth, Cajanus cajan (L.) Millsp, and Sorghum bicolor (L.) Moench. After corn harvesting, the soil was sampled at depths of 0.00-0.10 and 0.10-0.20 m. The cover crops showed different effects at different soil depths. The soil cultivated with U. ruziziensis showed higher contents of total-N and particulate-N than the soil cultivated with C. cajan. Particulate-N was the most sensitive to changes in the soil management among the fractions of N assessed. The soil under N topdressing showed a lower content of available-N in the 0.10-0.20 m layer, which may be caused by the season in which the sampling was conducted or the greater uptake of the available-N by corn.

Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Determinação de motivos de ligação à quitina em vicilinas de Canavalia ensiformis e Vigna unguiculata através de métodos in silico e relação com suas toxicidades para o bruquídeo Callosobruchus maculatus (Coleoptera:Bruchidae)

Chitin is an important structural component of the cellular wall of fungi and exoskeleton of many invertebrate plagues, such as insects and nematodes. In digestory systems of insects it forms a named matrix of peritrophic membrane. One of the most studied interaction models protein-carbohydrate is the model that involves chitin-binding proteins. Among the involved characterized domains already in this interaction if they detach the hevein domain (HD), from of Hevea brasiliensis (Rubber tree), the R&R consensus domain (R&R), found in cuticular proteins of insects, and the motif called in this study as conglicinin motif (CD), found in the cristallography structure of the β-conglicinin bounded with GlcNac. These three chitin-binding domains had been used to determine which of them could be involved in silico in the interaction of Canavalia ensiformis and Vigna unguiculata vicilins with chitin, as well as associate these results with the WD50 of these vicilins for Callosobruchus maculatus larvae. The technique of comparative modeling was used for construction of the model 3D of the vicilin of V. unguiculata, that was not found in the data bases. Using the ClustalW program it was gotten localization of these domains in the vicilins primary structure. The domains R&R and CD had been found with bigger homology in the vicilins primary sequences and had been target of interaction studies. Through program GRAMM models of interaction ( dockings ) of the vicilins with GlcNac had been gotten. The results had shown that, through analysis in silico, HD is not part of the vicilins structures, proving the result gotten with the alignment of the primary sequences; the R&R domain, although not to have structural similarity in the vicilins, probably it has a participation in the activity of interaction of these with GlcNac; whereas the CD domain participates directly in the interaction of the vicilins with GlcNac. These results in silico show that the amino acid number, the types and the amount of binding made for the CD motif with GlcNac seem to be directly associates to the deleterious power that these vicilins show for C. maculatus larvae. This can give an initial step in the briefing of as the vicilins interact with alive chitin in and exert its toxic power for insects that possess peritrophic membrane

Evaluación del efecto de incluir los granos de leguminosas (Canavalia ensiformis, vigna Sinensis y stizolobium deeringianum) en la suplementación concentrada de vacas lecheras

Se realizó un experimento con nueve vacas lecheras raza Brahman Pardo Suizo en diferentes períodos de lactancia superior a los tres meses, diferentes números de partos, con una producción promedio de 2.35 litros/vaca/día y un peso vivo promedio de 350 kg. La duración del experimento fue de 45 días (15 días por periodo) con el objetivo de evaluar el efecto de incluir los granos de leguminosas canavalia, terciopelo y caupi sobre la producción de leche y estimar la utilidad económica. Las vacas se establecieron en un solo bloque donde los tres tratamientos se asignaron al azar mediante el modelo esta dístico (Swich back). No se encontró diferencias significativas (P < 0.05) para los tres tratamientos que estaban constituidas por TA (20.13 % de terciopelo v 79.87% por otros ingredientes), el TB (23.66% de canavalia y 76.34 % otros ingredientes) y el TC (25.35 de Caupi y 74.7% otros ingredientes lográndose establecer tres raciones isoprotéicas e isoenergéticas. Los promedios de producción de leche obtenidos fueron de 4.11, 3.29 y 3.5 Kg para el TA. TB y TC respectivamente. El análisis económico evidencia que las mejores utilidades se obtuvieron con el TA con C$290.04 y C$ 320.08. Se concluye que se puede suministrar los granos de leguminosas como ingrediente en la suplementación concentrada a vacas lecheras como una alternativa de alimentación en los meses de verano.