Identification and Characterization of a Pepsin- and Chymotrypsin-Resistant Peptide in the α Subunit of the 11S Globulin Legumin from Common Bean (Phaseolus vulgaris L.).

The 11S globulin legumin typically accounts for approximately 3% of the total protein in common beans (Phaseolus vulgaris). It was previously reported that a legumin peptide of approximately 20 kDa is resistant to pepsin digestion. Sequence prediction suggested that the pepsin-resistant peptide is located at the C-terminal end of the α-subunit, within a glutamic acid-rich domain, overlapping with a chymotrypsin-resistant peptide. Using purified legumin, the peptide of approximately 20 kDa was found to be resistant to pepsin digestion in a pH-dependent manner, and its location was determined by two-dimensional gel electrophoresis and LC-MS-MS. The location of the chymotrypsin-resistant peptide was confirmed by immunoblotting with peptide-specific polyclonal antibodies. The presence of a consensus site for proline hydroxylation and arabinosylation, the detection of hydroxyproline residues, purification by lectin affinity chromatography, and a difference in electrophoretic migration between the chymotrypsin- and pepsin-resistant peptides suggest the presence of a large O-glycan within these peptides.

Calcium binds and rigidifies the dysferlin C2A domain in a tightly coupled manner.

The membrane protein dysferlin (DYSF) is important for calcium-activated plasma membrane repair, especially in muscle fibre cells. Nearly 600 mutations in the DYSF gene have been identified that are causative for rare genetic forms of muscular dystrophy. The dysferlin protein consists of seven C2 domains (C2A-C2G, 13%-33% identity) used to recruit calcium ions and traffic accessory proteins and vesicles to injured membrane sites needed to reseal a wound. Amongst these, the C2A is the most prominent facilitating the calcium-sensitive interaction with membrane surfaces. In this work, we determined the calcium-free and calcium-bound structures of the dysferlin C2A domain using NMR spectroscopy and X-ray crystallography. We show that binding two calcium ions to this domain reduces the flexibility of the Ca2+-binding loops in the structure. Furthermore, calcium titration and mutagenesis experiments reveal the tight coupling of these calcium-binding sites whereby the elimination of one site abolishes calcium binding to its partner site. We propose that the electrostatic potential distributed by the flexible, negatively charged calcium-binding loops in the dysferlin C2A domain control first contact with calcium that promotes subsequent binding. Based on these results, we hypothesize that dysferlin uses a 'calcium-catching' mechanism to respond to calcium influx during membrane repair.

Evaluación de agroecosistemas mediante indicadores de sostenibilidad en san josé de las lajas, provincia de mayabeque, cuba / Agroecosystems assessment by sustainability indicators in san josé de las lajas, mayabeque province, cuba

La presente investigación tiene como propósito la elaboración de un set de indicadores de sostenibilidad para tres (3) agroecosistemas localizados en el municipio de San José de Las Lajas (Cuba). Estos agroecosistemas fueron seleccionados por sus altos niveles de biodiversidad, por implementar técnicas agroecológicas, y por su fácil acceso y abierta disposición para participar en este proyecto. Metodológicamente se utilizaron principios de investigación acción participativa (IAP) y se analizaron muestras de agua y suelo en laboratorio. La construcción de los indicadores se hizo a partir de la metodología Marco para la evaluación de sistemas de manejo de recursos naturales incorporando indicadores de sustentabilidad (MESMIS) y en total se obtuvieron 34 indicadores para estimar la sostenibilidad de los agroecosistemas. De los resultados obtenidos se resalta que los tres agroecosistemas ostentan niveles de sostenibilidad relativamente altos (>0.7), donde la finca La Joya es la que presenta el mayor índice (0.86) como resultado de las prácticas agroecológicas implementadas.

The present research aims to elaborate a set of sustainability indicators for three (3) agroecosystems in the municipality of San José de las Lajas (Cuba). These agroecosystems were selected for its high levels of biodiversity, by they implement agroecological techniques and its easy access and open willingness to participate in this project. About the methodology, principles from Participatory Action Research (PAR) were used, and water and soil samples were analyzed. The construction of indicators was attained based on the Framework for the Evaluation of Natural Resource Management Systems Incorporating Sustainability Indicators (in Spanish, MESMIS) and, overall, 34 indicators were achieved to estimate the agroecosystem sustainability. From the results obtained, it should be emphasized that the three agroecosystems hold relative high levels of sustainability (>0.7), where La Joya's farm shows the highest rate (0.86) as a result of the agro-ecological practices implemented.