12 research outputs found
The Molecular Mechanism of Gephyrin Binding to the Glycine Receptor: Regulation, Binding Sites and Novel Model Systems
Identification of a new genomic hot spot of evolutionary diversification of protein function.
Establishment of phylogenetic relationships remains a challenging task because it is based on computational analysis of genomic hot spots that display species-specific sequence variations. Here, we identify a species-specific thymine-to-guanine sequence variation in the Glrb gene which gives rise to species-specific splice donor sites in the Glrb genes of mouse and bushbaby. The resulting splice insert in the receptor for the inhibitory neurotransmitter glycine (GlyR) conveys synaptic receptor clustering and specific association with a particular synaptic plasticity-related splice variant of the postsynaptic scaffold protein gephyrin. This study identifies a new genomic hot spot which contributes to phylogenetic diversification of protein function and advances our understanding of phylogenetic relationships
Personas con discapacidad : su abordaje desde miradas convergentes
Necchi, S., Suter, M., Gaviglio, A. (2015). Personas con discapacidad: su abordaje desde miradas convergentes. Bernal, Argentina : Universidad Nacional de Quilmes.Personas con discapacidad: su abordaje desde miradas convergentes es una compilación de artículos pensada para acompañar desde la Universidad ese proceso de reconocimiento de derechos y construcción de conocimiento sobre las singularidades y potencialidades del sector. El presente de la educación y el trabajo, el rol de comunicación y del turismo, así como aspectos legales y filosóficos están presentes en estas páginas para quienes conocen del tema o buscan acercarse a él. Reflexiones, preguntas actualizadas y búsqueda de respuestas de una sociedad que debe abrazar a todos, y no al revés.Introducción / Silvia Necchi -- Filosofía sobre las personas con discapacidad / Luisa Ripa -- Los derechos de las personas con discapacidad / Luisa Ripa -- Aspectos legales que regulan los derechos de las personas con discapacidad / Liliana Bastons -- Los medios de comunicación y la discapacidad / Nancy Díaz Larrañaga -- Programas de discapacidad del Ministerio de Desarrollo Social de la provincia de Buenos Aires: su surgimiento y estado actual / Andrea Gaviglio -- Discapacidad y trabajo / María Esther Fernández -- Consideraciones en educación para personas con discapacidad / Susana Haddad -- Personas con discapacidad: algunos conceptos sobre el clivaje arte-subjetivación / Mónica Bottini -- Accesibilidad al medio: de la eliminación de barreras al diseño universal / Nora Demarchi -- Turismo accesible / Luis Grünewald y Agueda Fernández -- Convención sobre los derechos de las personas con discapacidad -- Protocolo facultativo de la Convención sobre los derechos de las personas con discapacidad -- Declaración de los derechos de las personas con retraso mental -- Los derechos humanos de las personas con discapacidades distintas (análisis del Movimiento de los Pueblos para la Educación de los Derechos Humanos) -- Convención interamericana para la eliminación de todas las formas de discriminación contra las personas con discapacidad -- Ratificación argentina de la Convención Interamericana sobre los Derechos de las Personas con Discapacidad: ley nacional Nº 25.280 -- Ley nacional Nº 22.431/81: Sistema de protección integral de los discapacitados -- Reglamentación de la ley nacional Nº 22.431 -- Ley Nº 10.592 de la Provincia de Buenos Aires: Régimen jurídico básico e integral para las personas discapacitadas
Gephyrin splice variant-specific recruitment of GlyR α1-βE9A-3.
<p>(A-E) Images of transfected primary hippocampal neurons show HA-α1-βE9A-3 and EGFP-tagged gephyrin splice variants. (A-D) No or apparently occasional overlap between HA-α1-βE9A-3 clusters and large aggregates of the over-expressed denoted different gephyrin splice variants was observed (arrowheads in grey scale high-power views). (E) In contrast, GlyR HA-α1-βE9A-3 co-localized with the G2-gephyrin splice variant (arrows). Scale bars, 10 μm and 2 μm. (F) GST co-sedimentation of gephyrin with the indicated large cytosolic GlyR loops between transmembrane domains 3 and 4 out of 350 μg mouse brain extract. GST alone was used as control. The western blot was probed with the monoclonal mAb3B11 antibody. Note that the GlyR βE9A-3-Δβgb loop was not detected in the pellet fraction, whereas GlyR β-βgb and βE9A-3-βgb loops co-sedimented with gephyrin.</p
Species-specific alternative RNA splicing of GlyR β exon 9A.
<p>(A) The scheme illustrates four alternative splicing patterns regarding novel exon 9A in mice. Grey boxes denote known exons (which use splice donor “GT<sub>E9</sub>” and acceptor “AG<sub>E10</sub>” sites), and red boxes mark the three new variants of alternative exon 9A (depending on use of AG<sub>9A2-4</sub> and corresponding to predicted GlyR β-X2-4 protein variants). Note that the splice donor site only exists in mouse and bushbaby, whereas in all other investigated species this site is disrupted due to G/T substitution (“(T)”, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125413#pone.0125413.s003" target="_blank">S3 Fig</a> for sequence details). Also note that the splice acceptor site of E9A-3 is disrupted in rat due to additional G/A substitution (“(A)”). (B) Relative frequency of the four splicing patterns in embryonic (E17, open bars) and adult (P84-112, filled bars) mouse cerebral cortex. Apparently, splicing of exons 9A2-4 is not developmentally regulated. However, splicing of E9A-3 is significantly stronger compared to expression of mRNA with E9A-2 or E9A-4 splice variants. The numbers in brackets indicate the number of RNA sequencing replicates. (C) The donor site of the novel exon 9A (GT<sub>E9A</sub>) is as potent as that of the upstream exon (GT<sub>E9</sub>). The acceptor sites of the novel exon 9A (AG<sub>E9A-2</sub>, AG<sub>E9A-3</sub> and AG<sub>E9A-4</sub>) are much weaker than that of the downstream exon 10 (AG<sub>E10</sub>), and the acceptor site of the most prevalent splice variant X3 is the weakest of the new acceptor sites. This suggests that inclusion of exon 9A is first defined by its strong donor site followed by selecting one of the three acceptor sites, with the preference of the AG<sub>E9A-3</sub> acceptor site which is in nearest distance to the alternative splice donor site.</p
The exon 9A-3-coding sequence facilitates postsynaptic GlyR localization in the somatodendritic compartment of primary spinal cord neurons.
<p>(A-C) Spinal cord neurons were transfected with plasmids coding for the chimeric GlyR α1 with the new GlyR β insert (A, HA-α1-βE9A-3), the established gephyrin-binding variant (B, HA-α1-βgb), or without any additional sequence (C, HA-α1). (D-F) Quantitative analysis of postsynaptic GlyR α1-βE9A-3 (HA-signal peaks) and postsynaptic gephyrin revealed strong positive correlation of fluorescence intensities (R = 0.71, D). The established gephyrin-binding GlyR α1-βgb produced comparable results (R = 0.65, E). In contrast, postsynaptic gephyrin-associated clustering of GlyR α1 without any additional sequence in the large cytosolic loop was not evident (R = 0.16, F). (G-K) Histograms show the distributions of postsynaptic HA-signals in relation to locally corresponding gephyrin fluorescence intensities (HA / gephyrin fluorescence intensity ratio, bin size 0.2). Note that a value of 1 indicates perfect overlap. Note the different scale of the X-axis in panel K, and that values are mostly below or above the value of 1 in the case of gephyrin-independent postsynaptic clustering of GlyR HA-α1. Scale bars: 10 μm and 2 μm.</p
RT-PCR analysis of GlyR β RNA splicing.
<p>(A) PCR using oligonucleotides spanning the large cytosolic loop between transmembrane domains 3 and 4 revealed an additional band (arrow). The additional band seems to be specific for mouse cDNA samples (e.g. brain [hippocampus] and spinal cord) as it was not detected in human brain (“Human PM”: human postmortem hippocampus; provided by Clontech “Human TLE #030310”: human cortex from epilepsy patient #030310; “Human TLE #040310”: human cortex from epilepsy patient #040310, see [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125413#pone.0125413.ref032" target="_blank">32</a>] for medical history) or rat spinal cord. (B) Molecular cloning and sequence analysis of the additional PCR band identified a new GlyR β variant which contains 16 additional amino acids (bold and underlined). The new insert is located upstream of the established gephyrin binding sequence (βgb) of the GlyR β subunit (underlined). (C, D) Blast search revealed 100% homology with the depicted mouse genome region on chromosome 3, downstream of exon 9 and upstream of exon 10 (C). Actually, two additional splice acceptor sites (X4 and X2) are located upstream of the splice acceptor site X3 which gives rise to the cloned splice variant (D, sequence in bold and underlined).</p