An approach to the study of G-protein-coupled receptor kinases: an in vitro-purified membrane assay reveals differential receptor specificity and regulation by G beta gamma subunits.

Phosphorylation of GTP-binding-regulatory (G)-protein-coupled receptors by specific G-protein-coupled receptor kinases (GRKs) is a major mechanism responsible for agonist-mediated desensitization of signal transduction processes. However, to date, studies of the specificity of these enzymes have been hampered by the difficulty of preparing the purified and reconstituted receptor preparations required as substrates. Here we describe an approach that obviates this problem by utilizing highly purified membrane preparations from Sf9 and 293 cells overexpressing G-protein-coupled receptors. We use this technique to demonstrate specificity of several GRKs with respect to both receptor substrates and the enhancing effects of G-protein beta gamma subunits on phosphorylation. Enriched membrane preparations of the beta 2- and alpha 2-C2-adrenergic receptors (ARs, where alpha 2-C2-AR refers to the AR whose gene is located on human chromosome 2) prepared by sucrose density gradient centrifugation from Sf9 or 293 cells contain the receptor at 100-300 pmol/mg of protein and serve as efficient substrates for agonist-dependent phosphorylation by beta-AR kinase 1 (GRK2), beta-AR kinase 2 (GRK3), or GRK5. Stoichiometries of agonist-mediated phosphorylation of the receptors by GRK2 (beta-AR kinase 1), in the absence and presence of G beta gamma, are 1 and 3 mol/mol, respectively. The rate of phosphorylation of the membrane receptors is 3 times faster than that of purified and reconstituted receptors. While phosphorylation of the beta 2-AR by GRK2, -3, and -5 is similar, the activity of GRK2 and -3 is enhanced by G beta gamma whereas that of GRK5 is not. In contrast, whereas GRK2 and -3 efficiently phosphorylate alpha 2-C2-AR, GRK5 is quite weak. The availability of a simple direct phosphorylation assay applicable to any cloned G-protein-coupled receptor should greatly facilitate elucidation of the mechanisms of regulation of these receptors by the expanding family of GRKs.

Structural basis for receptor subtype-specific regulation revealed by a chimeric beta 3/beta 2-adrenergic receptor.

The physiological significance of multiple G-protein-coupled receptor subtypes, such as the beta-adrenergic receptors (beta ARs), remains obscure, since in many cases several subtypes activate the same effector and utilize the same physiological agonists. We inspected the deduced amino acid sequences of the beta AR subtypes for variations in the determinants for agonist regulation as a potential basis for subtype differentiation. Whereas the beta 2AR has a C terminus containing 11 serine and threonine residues representing potential sites for beta AR kinase phosphorylation, which mediates rapid agonist-promoted desensitization, only 3 serines are present in the comparable region of the beta 3AR, and they are in a nonfavorable context. The beta 3AR also lacks sequence homology in regions which are important for agonist-mediated sequestration and down-regulation of the beta 2AR, although such determinants are less well defined. We therefore tested the idea that the agonist-induced regulatory properties of the two receptors might differ by expressing both subtypes in CHW cells and exposing them to the agonist isoproterenol. The beta 3AR did not display short-term agonist-promoted functional desensitization or sequestration, or long-term down-regulation. To assign a structural basis for these subtype-specific differences in agonist regulation, we constructed a chimeric beta 3/beta 2AR which comprised the beta 3AR up to proline-365 of the cytoplasmic tail and the C terminus of the beta 2AR. When cells expressing this chimeric beta 3/beta 2AR were exposed to isoproterenol, functional desensitization was observed. Whole-cell phosphorylation studies showed that the beta 2AR displayed agonist-dependent phosphorylation, but no such phosphorylation could be demonstrated with the beta 3AR, even when beta AR kinase was overexpressed. In contrast, the chimeric beta 3/beta 2AR did display agonist-dependent phosphorylation, consistent with its functional desensitization. In addition to conferring functional desensitization and phosphorylation to the beta 3AR, the C-terminal tail of the beta 2AR also conferred agonist-promoted sequestration and long-term receptor down-regulation.

Chromosomal organization of adrenergic receptor genes.

The adrenergic receptors (ARs) (subtypes alpha 1, alpha 2, beta 1, and beta 2) are a prototypic family of guanine nucleotide binding regulatory protein-coupled receptors that mediate the physiological effects of the hormone epinephrine and the neurotransmitter norepinephrine. We have previously assigned the genes for beta 2- and alpha 2-AR to human chromosomes 5 and 10, respectively. By Southern analysis of somatic cell hybrids and in situ chromosomal hybridization, we have now mapped the alpha 1-AR gene to chromosome 5q32----q34, the same position as beta 2-AR, and the beta 1-AR gene to chromosome 10q24----q26, the region where alpha 2-AR is located. In mouse, both alpha 2- and beta 1-AR genes were assigned to chromosome 19, and the alpha 1-AR locus was localized to chromosome 11. Pulsed field gel electrophoresis has shown that the alpha 1- and beta 2-AR genes in humans are within 300 kilobases (kb) and the distance between the alpha 2- and beta 1-AR genes is less than 225 kb. The proximity of these two pairs of AR genes and the sequence similarity that exists among all the ARs strongly suggest that they are evolutionarily related. Moreover, they likely arose from a common ancestral receptor gene and subsequently diverged through gene duplication and chromosomal duplication to perform their distinctive roles in mediating the physiological effects of catecholamines. The AR genes thus provide a paradigm for understanding the evolution of such structurally conserved yet functionally divergent families of receptor molecules.

Inhibition of beta-adrenergic receptor kinase prevents rapid homologous desensitization of beta 2-adrenergic receptors.

Homologous (agonist-specific) desensitization of beta-adrenergic receptors (beta ARs) is accompanied by and appears to require phosphorylation of the receptors. We have recently described a novel protein kinase, beta AR kinase, which phosphorylates beta ARs in vitro in an agonist-dependent manner. This kinase is inhibited by two classes of compounds, polyanions and synthetic peptides derived from the beta 2-adrenergic receptor (beta 2AR). In this report we describe the effects of these inhibitors on the process of homologous desensitization induced by the beta-adrenergic agonist isoproterenol. Permeabilization of human epidermoid carcinoma A431 cells with digitonin was used to permit access of the charged inhibitors to the cytosol; this procedure did not interfere with the pattern of isoproterenol-induced homologous desensitization of beta 2AR-stimulated adenylyl cyclase. Inhibitors of beta AR kinase markedly inhibited homologous desensitization of beta 2ARs in the permeabilized cells. Inhibition of desensitization by heparin, the most potent of the polyanion inhibitors of beta AR kinase, occurred over the same concentration range (5-50 nM) as inhibition of purified beta AR kinase assessed in a reconstituted system. Inhibition of desensitization by heparin was accompanied by a marked reduction of receptor phosphorylation in the permeabilized cells. Whereas inhibitors of beta AR kinase inhibited homologous desensitization, inhibitors of protein kinase C and of cyclic-nucleotide-dependent protein kinases were ineffective. These data establish that phosphorylation of beta ARs by beta AR kinase is an essential step in homologous desensitization of the receptors. They further suggest a potential therapeutic value of inhibitors of beta AR kinase in inhibiting agonist-induced desensitization.

The effect of prenatal or early postnatal irradiation on the production of anti-arsonate antibodies and Cross-reactive idiotypes

Preliminary studies on the long-term effects of prenatal and early postnatal irradiation on the immune response to arsonate were performed using A/J mice. Pregnant mice were irradiated (0·5 Gy, X-rays) or sham-irradiated on a single occasion during gestation (between day 5 and 18 post-conception). Alternatively, newborn mice received the same treatment between day 2 and 7 after birth. Mice were immunized with keyhole limpet haemocyanin-arsonate (KLH-Ars) in adjuvant from 2 months after birth. The levels of specific antibodies to arsonate (anti-Ars) were measured by radioimmunoassay. In addition, the Ars-related cross-reactive idiotype (CRIA) was measured by the haemagglutination technique. In the primary response the titre of anti-Ars was reduced in animals that had been irradiated between day 12 and 15 of gestation. In the second response, in contrast, they had increased levels of anti-Ars. After immunization with KLH-Ars, high levels of CRIA were observed in all groups. However, in mice irradiated 18-20 days after conception the level of CRIA was often much higher than the level of anti-Ars, indicating that a large proportion of the CRIA-positive molecules were not specific for Ars. Thus, in this particular case, some specificity of the immune response was lost after irradiation. The expression of recurrent idiotypes may be a sensitive indicator of immunological perturbations after irradiation. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

El análisis de las redes sociales: una herramienta matemática aplicada a una situación educativa

En este trabajo se presenta una aplicación del Análisis de Redes Sociales (ARS) al estudio de las relaciones entre alumnos de segundo año de una Escuela Técnica. El ARS se apoya en la teoría de grafos cuyo bagaje matemático permite analizar y medir, en términos generales, propiedades de las estructuras sociales en particular la escuela. La vida escolar es una trama compleja de factores que influirían en el rendimiento académico de los alumnos, tales como: tiempo de estudio que comparten, desde cuándo se conocen entre los compañeros, la proximidad de sus domicilios, sexo, edad, entre otros. Los factores sexo y edad no son relevantes dado que el grupo bajo estudio está formado por varones alrededor de los 16 años. En este trabajo se mostrarán los resultados obtenidos por el primer factor mencionado que fueron procesados a través de los software Ucinet 6 y Netdraw.

A Bird’s Eye View of Discard Reforms: Bird-Borne Cameras Reveal Seabird/Fishery Interactions

Commercial capture fisheries produce huge quantities of offal, as well as undersized and unwanted catch in the form of discards. Declines in global catches and legislation to ban discarding will significantly reduce discards, but this subsidy supports a large scavenger community. Understanding the potential impact of declining discards for scavengers should feature in an eco-system based approach to fisheries management, but requires greater knowledge of scavenger/fishery interactions. Here we use bird-borne cameras, in tandem with GPS loggers, to provide a unique view of seabird/fishery interactions. 20,643 digital images (one min 21) from ten bird-borne cameras deployed on central place northern gannets Morus bassanus revealed that all birds photographed fishing vessels. These were large (>15 m) boats, with no small-scale vessels. Virtually all vessels were trawlers, and gannets were almost always accompanied by other scavenging birds. All individuals exhibited an Area-Restricted Search (ARS) during foraging, but only 42% of ARS were associated with fishing vessels, indicating much 'natural' foraging. The proportion of ARS behaviours associated with fishing boats were higher for males (81%) than females (30%), although the reasons for this are currently unclear. Our study illustrates that fisheries form a very important component of the prey-landscape for foraging gannets and that a discard ban, such as that proposed under reforms of the EU Common Fisheries Policy, may have a significant impact on gannet behaviour, particularly males. However, a continued reliance on 'natural' foraging suggests the ability to switch away from scavenging, but only if there is sufficient food to meet their needs in the absence of a discard subsidy.

Mesoscale fronts as foraging habitats: composite front mapping reveals oceanographic drivers of habitat use for a pelagic seabird

The oceanographic drivers of marine vertebrate habitat use are poorly understood yet fundamental to our knowledge of marine ecosystem functioning. Here, we use composite front mapping and high-resolution GPS tracking to determine the significance of mesoscale oceanographic fronts as physical drivers of foraging habitat selection in northern gannets Morus bassanus. We tracked 66 breeding gannets from a Celtic Sea colony over 2 years and used residence time to identify area-restricted search (ARS) behaviour. Composite front maps identified thermal and chlorophyll-a mesoscale fronts at two different temporal scales—(i) contemporaneous fronts and (ii) seasonally persistent frontal zones. Using generalized additive models (GAMs), with generalized estimating equations (GEE-GAMs) to account for serial autocorrelation in tracking data, we found that gannets do not adjust their behaviour in response to contemporaneous fronts. However, ARS was more likely to occur within spatially predictable, seasonally persistent frontal zones (GAMs). Our results provide proof of concept that composite front mapping is a useful tool for studying the influence of oceanographic features on animal movements. Moreover, we highlight that frontal persistence is a crucial element of the formation of pelagic foraging hotspots for mobile marine vertebrates.

Nomenclature and syntaxonomic notes on some high-rank syntaxa of the European grassland vegetation

We present descriptions of a new order (Ranunculo cortusifolii-Geranietalia reuteri and of a new alliance (Stachyo lusitanicae-Cheirolophion sempervirentis) for the herbaceous fringe communities of Macaronesia and of the southwestern Iberian Peninsula, respectively. A new alliance, the Polygalo mediterraneae-Bromion erecti (mesophilous post-cultural grasslands), was introduced for the Peninsular Italy. We further validate and typify the Armerietalia rumelicae (perennial grasslands supported by nutrient-poor on siliceous bedrocks at altitudes characterized by the submediterranean climate of central-southern Balkan Peninsula), the Securigero-Dasypyrion villosae (lawn and fallow-land tall-grass annual vegetation of Italy), and the Cirsio vallis-demoni-Nardion (acidophilous grasslands on siliceous substrates of the Southern Italy). Nomenclatural issues (validity, legitimacy, synonymy, formal corrections) have been discussed and clarified for the following names: Brachypodio-Brometalia, Bromo pannonici-Festucion csikhegyensis, Corynephoro-Plantaginion radicatae, Heleochloion, Hieracio-Plantaginion radicatae, Nardetea strictae, Nardetalia strictae, Nardo-Callunetea, Nardo-Galion saxatilis, Oligo-Bromion, Paspalo-Heleochloetalia, Plantagini-Corynephorion and Scorzoneret alia villosae. 

Art and Social Work: an intervention programme for transforming society’s vision to include people with early-onset dementia and Alzheimer’s

This paper presents a program centred on arts and education as tools in social work for the inclusion of people with earlyonset dementia and Alzheimer’s. The objective of the programme is to eradicate the stigma and myths associated with the disease.The program is part of the Junta de Castilla y León and the European Social Fund’s ARS Project (Arte y Salud Alzheimer; Alzheimer’s Art & Health). The programme presents a series of evaluated artistic and educational activities that can be undertaken by people in the early stages of Alzheimer’s disease and that can also be used by caregivers and family when working with this group of people, with the aim of improving their wellbeing, self-esteem and quality of life.

Social Work and theatre: considering intersections

Theatre is a cultural and artistic form that involves a process of communication between creators and is received in a space and time located in the public sphere, which has meant that, over the centuries, it has acted as a space for expression, exchange and debate regarding all manner of ideas, causes and struggles. Implicit within this process are processes of expression, creation and reception, by way of which people demonstrate, analyse and question ways of seeing and understanding life, and ways of being and existing in the world. This gives rise to educational, cultural, social and political potential, which has been endorsed in numerous studies and investigations. In this work, in which theoretical orientation is established through a review of the relevant literature, we consider different intersections that occur between theatre and social work in order to also show that dramatic and theatrical expression offers substantive methodologies for achieving some objectives of social work, particularly in areas such as critical literacy, reflexivity and recognition, awareness raising, social participation, personal and/or community development, ownership of cultural capital and access to personal and social wellbeing.

La ruptura de una economía moral y las deslegitimación de las deudas hipotecarias

In Spain, during the recent housing bubble, purchasing a home seemed the most advantageous strategy to access housing, and there was a wide social consensus about the unavoidability of mortgage indebtedness. However, such consensus has been challenged by the financial and real-estate crisis. The victims of home repossessions have been affected by the transgression of several principles, such as the fair compensation for effort and sacrifice, the prioritisation of basic needs over financial commitments, the possibility of a second chance for over-indebted people, or the State's responsibility to guarantee its citizens' livelihood. Such principles may be understood as part of a moral economy, and their transgression has resulted in the emergence of a social movement, the Plataforma de Afectados por la Hipoteca (PAH), that is questioning the legitimacy of mortgage debts. The article reflects on the extent to which the perception of over-indebtedness and evictions as unfair situations can have an effect on the reproduction of the political-economic system, insofar the latter is perceived as able or unable to repair injustice.

Reflexiones y representaciones del exilio: de El canto del peregrino (1999) a "El sefardí romántico" (2005)

El exilio es un tema recurrente en la obra de Angelina Muñiz-Huberman, miembro del llamado grupo hispanomexicano. La visión del exilio en su obra incluye no solo el exilio republicano sino también el del pueblo judío. Por eso, diferentes Diásporas históricas se superponen enriqueciendo el significado de la experiencia. En su ensayo El canto del peregrino: hacia una poética del exilio (1999) desarrolla sus pensamientos sobre el exilio y analiza numerosos trabajos de autores exiliados (judíos y republicanos españoles) centrándose en como cada uno reflejó la vivencia del exilio en su trabajo.En mi opinión, las ideas desarrolladas en el ensayo se pueden encontrar en su novela El sefardí romántico: la azarosa vida de Mateo Alemán II (2005). El título de la novela establece una conexión directa con la novela picaresca y la vida judía aludiendo al autor converso de Guzmán de Alfarache (1599-1604). Siguiendo el patrón establecido por la novela picaresca clásica, el protagonista de Muñiz-Huberman viaja por España y Europa denunciando la intolerancia que llevó a la Guerra Civil española y a la II Guerra Mundial. Las circunstancias lo llevan al exilio en México, como en el caso de Mateo Alemán. Según la teoría de Ulrich Wicks, pícaros y exiliados tienen mucho en común en su búsqueda continua de libertad, libertad que el exiliado puede encontrar solo en el idioma, como expone Muñiz-Huberman en su ensayo.

Some thoughts on Juan Antonio Ramírez´s iconologic-paranoid method for the study of Dali´s works

In 1990, Juan Antonio Ramírez made known his iconological paranoiac method to the academic world. It is a system devised to study Dalí´s work, born of happy matrimony between the catalonian artist´s critical paranoiac method and the first iconographic school of Warburg and Panofsky. What, at first sight, seemed to be an extravagant methodology with little academic credibility, turned out to be, by going forward in time, an effective research method when analyzing Dali´s initial works. It is no wonder that, at the beginning of XXI century, this hybridization between Dalí´s and Panofsky’s methods, were regarded as a strange and nonsensical idea. Nevertheless, twenty years after, blatant examples have been noted, which endorse the advantages of this new system for “iconographic” study. As usual, this new contribution by Juan Antonio Ramirez to art history methodology is called upon to have a successful future survival, in forthcoming generations. This article, attempts to analyze this method, inquiring into the validity, effectiveness and practical utility.