The post‐translational attachment of biotin and lipoic acid to specific lysine residues displayed in protruding β‐turns in homologous biotinyl and lipoyl domains of their parent enzymes is catalysed ...by two different ligases. We have expressed in Escherichia coli a sub‐gene encoding the biotinyl domain of E.coli acetyl‐CoA carboxylase, and by a series of mutations converted the protein from the target for biotinylation to one for lipoylation, in vivo and in vitro. The biotinylating enzyme, biotinyl protein ligase (BPL), and the lipoylating enzyme, LplA, exhibited major differences in the recognition process. LplA accepted the highly conserved MKM motif that houses the target lysine residue in the biotinyl domain β‐turn, but was responsive to structural cues in the flanking β‐strands. BPL was much less sensitive to changes in these β‐strands, but could not biotinylate a lysine residue placed in the DKA motif characteristic of the lipoyl domain β‐turn. The presence of a further protruding thumb between the β2 and β3 strands in the wild‐type biotinyl domain, which has no counterpart in the lipoyl domain, is sufficient to prevent aberrant lipoylation in E.coli. The structural basis of this discrimination contrasts with other forms of post‐translational modification, where the sequence motif surrounding the target residue can be the principal determinant.
Los objetivos de esta investigación son: (i) describir el comportamiento de las variables Player Load y AcelT y (ii) cuantificar la carga neuromuscular endiferentes puntos anatómicos (espalda, centro ...de masas, rodilla y tobillo) durante un test incremental en rampa en tapiz rodante. Veintitrés jugadores semiprofesionales de fútbol varones participaron voluntariamente en este estudio (edad: 22,56±4,8 años; masa corporal: 75,5±5,5 kg; altura: 1,79±0,5 m). Ambas variables se registraron empleando 4 dispositivos inerciales WIMU PROTM. Los principales resultados indican que: (1) existe una correlación casi perfecta entre ambas variables (r=0,931), (2) los mayores valores en ambas variables se han encontrado en la rodilla (PL = 8,01±2,76; AcelT = 2,70±0,50) y el tobillo (PL = 7,85±2,27; AcelT = 2,87±0,49) y (3) existe una amplia variabilidad intersujeto. En conclusión, Player Load y AcelT son dos indicadores válidos para el análisis y cuantificación de las demandas neuromusculares.
Este estudio analiza el comportamiento táctico en Juegos Reducidos (JRs) a través de las variables área y centroide en función de: (1) tecnología empleada: Sistema de Posicionamiento Global (GPS) y ...Ultra-Banda Ancha (UWB); (2) fase de juego (ataque y defensa); y (3) objetivo del JRs. Dieciséis jugadores semiprofesionales de fútbol participaron en esta investigación (Edad: 23.6±3.3 años; Peso: 78.1±5.2 kg; Altura: 1.8±0.1 metros). Para el registro se utilizaron dispositivos inerciales WIMU PROTM(RealTrack System, Almería, España). Los principales resultados muestran diferencias significativas: (1) entre tecnologías de seguimiento (GPS y UWB) en la variable área, (2) entre fases de juego, y (3) en función del objetivo de los JRs. En conclusión, los datos obtenidos por ambas tecnologías no pueden compararse debido a las diferencias encontradas, siendo muy importante el análisis en función del objetivo y la fase de juego de los JRs por su influencia en la disposición táctica para conseguir una mejora en el rendimiento deportivo.
One hundred and sixty strains of Pseudomonas syringae subsp. savastanoi from Olea europaea, Olea europaea var. sylvestris, Nerium oleander, Fraxinus angustifolia and Retama sphaerocarpa, and four ...type strains of other pathovars were studied, investigating 102 phenotypic traits, among which we include biochemical characteristics, assimilation of different carbon sources, sensitivity or resistance to antibiotics and indoleacetic acid (IAA) production. Results were analysed with an affinity dendrogram via the Jaccard coefficient. They indicate an influence of environmental factors on the formation of the 15 phenons obtained, since isolated (knot) strains from the same species but different geographical areas are segregated. Segregation, also detected in strains from different hosts within the same area, added to the pathogenicity test helps to characterise these strains as different pathovars.PUBLICATION ABSTRACT
ADP-ribosyltransferases (ADPRTs) form an interesting class of enyzmes with well-established roles as potent bacterial toxins and metabolic regulators. ADPRTs catalyze the transfer of the ADP-ribose ...moiety from NAD
+ onto specific substrates including proteins. ADP-ribosylation usually inactivates the function of the target. ADPRTs have become adapted to function in extra- and intracellular settings. Regulation of ADPRT activity can be mediated by ligand binding to associated regulatory domains, proteolytic cleavage, disulphide bond reduction, and association with other proteins. Crystallisation has revealed a conserved core set of elements that define an unusual minimal scaffold of the catalytic domain with remarkably plastic sequence requirements—only a single glutamic acid residue critical to catalytic activity is invariant. These inherent properties of ADPRTs suggest that the ADPRT catalytic fold is an attractive, malleable subject for protein design.
Immunoinformatics is an emerging new field that benefits from computational analyses and tools that facilitate the understanding of the immune system. A large number of immunoinformatics resources ...such as immune-related databases and analysis software are available through the World Wide Web for the benefit of the research community. However, immunoinformatics developments have sometimes remained isolated from mainstream bioinformatics. Therefore, there is clearly a need for integration, which will empower the exchange of data and annotations within the scientific community in a quick and efficient fashion. Here, we have chosen the Distributed Annotation System (DAS), for integrating in house annotations on experimental and predicted HLA I-restriction elements of CD8 T-cell epitopes with sequence and structural information.
Biotin-dependent enzymes contain a biotinyl-lysine residue in a conserved sequence motif, MKM, located in a surface hairpin turn in one of the two beta-sheets that make up the domain. A sub-gene ...encoding the 82-residue C-terminal biotinyl domain from the biotin carboxy carrier protein of acetyl-CoA carboxylase from Escherichia coli as a fusion protein with glutathione S-transferase was created and over-expressed in E. coli. The biotinyl domain was readily released by cleavage with thrombin. Five mutant domains were created in which the conserved MKM motif was systematically replaced: by MAK and KAM, in which the target lysine is moved one place; by KKM and MKK, in which a second potential site for biotinylation is introduced; and by DKA, the motif found in the correspondingly conserved site of lipoylation in the structurally related lipoyl domains of 2-oxo acid dehydrogenase multienzyme complexes. No biotinylation of the MAK or KAM mutants was observed in vivo or by purified biotinyl protein ligase in vitro; in the KKM and MKK mutants, only one lysine residue, presumed to be that in its native position in the hairpin turn, was found to be biotinylated in vivo and in vitro. The DKA mutant was not biotinylated in vivo, but was partly lipoylated and octanoylated. It was also a poor substrate for lipoylation in vitro catalysed by the E. coli lipoyl protein ligase encoded by the lplA gene. The flanking sequence in the MKM motif is important, but not crucial, and appears to have been conserved in part to be compatible with the subsequent carboxylation reactions of biotin-dependent enzymes. The DKA motif, displayed in the hairpin loop, is sufficient to address lipoylation in E. coli but probably by a pathway different from that mediated by the lplA-dependent ligase. The recognition of the structurally homologous lipoyl and biotinyl domains by the appropriate ligase evidently has a major structural component to it, notably the positioning of the target lysine residue in the exposed hairpin loop, but there appear to be additional recognition sites elsewhere on the domains.
During development, thymocytes carrying TCRs mediating low-affinity interactions with MHC-bound self-peptides are positively selected for export into the mature peripheral T lymphocyte pool. Thus, ...exogenous administration of certain altered peptide ligands (APL) with reduced TCR affinity relative to cognate Ags may provide a tool to elicit maturation of desired TCR specificities. To test this "thymic vaccination" concept, we designed APL of the viral CTL epitopes gp33-41 and vesicular stomatitis virus nucleoprotein octapeptide N52-59 relevant for the lymphocytic choriomeningitis virus-specific P14- and vesicular stomatitis virus-specific N15-TCRs, respectively, and examined their effects on thymocytes in vivo using irradiation chimeras. Injection of APL into irradiated congenic (Ly-5.1) mice, reconstituted with T cell progenitors from the bone marrow of P14 RAG2(-/-) (Ly-5.2) or N15 RAG2(-/-) (Ly-5.2) transgenic mice, resulted in positive selection of T cells expressing the relevant specificity. Moreover, the variants led to export of virus-specific T cells to lymph nodes, but without inducing T cell proliferation. These findings show that the mature T cell repertoire can be altered by in vivo peptide administration through manipulation of thymic selection.
PDF
