Alteration of ZnT5-mediated zinc import into the early secretory pathway affects the secretion of growth hormone from rat pituitary cells

BACKGROUND Aggregation of growth hormone (GH) required for its proper storage in granules is facilitated by zinc (Zn(2+)) transported by specific zinc transporters in and out of the regulated secretory pathway. Slc30a5 (ZnT5) was reported to have the highest gene expression among all zinc transporters in primary mouse pituitary cells while ZnT5-null mice presented with abnormal bone development and impaired growth compared to wild-type counterparts. METHODS In vitro studies performed in GH3 cells, a rat pituitary cell line that endogenously produces rat GH (rGH), included analysis of: cytoplasmic Zn(2+) pool changes after altering rSlc30a5 expression (luciferase assay), rZnT5 association with different compartments of the regulated secretory pathway (confocal microscopy), and the rGH secretion after rSlc30a5 knock-down (Western blot). RESULTS Confocal microscopy demonstrated high co-localization of rZnT5 with ER and Golgi (early secretory pathway) while siRNA-mediated knock-down of rSlc30a5 gene expression led to a significant reduction in rGH secretion. Furthermore, altered expression of rSlc30a5 (knock-down/overexpression) evoked changes in the cytoplasmic Zn(2+) pool indicating its important role in mediating Zn(2+) influx into intracellular compartments of the regulated secretory pathway. CONCLUSION Taken together, these results suggest that ZnT5 might play an important role in regulated GH secretion that is much greater than previously anticipated.

IGHD II: A Novel GH-1 Gene Mutation (GH-L76P) Severely Affects GH Folding, Stability, and Secretion

CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.

Molecular dissolved organic matter composition and environmental parameters from a laboratory incubation study

Dissolved organic matter (DOM) in the oceans constitutes a major carbon pool involved in global biogeochemical cycles. More than 96% of the marine DOM resists microbial degradation for thousands of years. The composition of this refractory DOM (RDOM) exhibits a molecular signature which is ubiquitously detected in the deep oceans. Surprisingly efficient microbial transformation of labile into RDOM was shown experimentally, implying that microorganisms produce far more RDOM than needed to sustain the global pool. By assessing the microbial formation and transformation of DOM in unprecedented molecular detail for 3 years, we show that most of the newly formed RDOM is molecularly different from deep sea RDOM. Only <0.4% of the net community production was channeled into RDOM molecularly undistinguishable from deep sea DOM. Our study provides novel experimentally derived molecular evidence and data for global models on the production, turnover and accumulation of marine DOM.

Conflito, resistência e philía no Héracles furioso, de Eurípides

En la pieza Heracles Furioso, de Eurípides, el héroe se somete a la prueba definitiva: vencerse a sí mismo, aceptar seguir con vida después de cometer un error irremediable. La acción dramática tiene doble motivación, humana y divina. El conflicto humano requiere capacidad de resistencia de la familia y amigos de Heracles y la oposición de Lico es un elemento clave para la acción. En el enfrentamiento posterior entre Heracles y la diosa Hera, la victoria corresponde a la deidad, motivando a la catástrofe, pero se confirma la heroicidad de Heracles, que se resiste a la tentación de aniquilación tras el asesinato de sus niños. Su amigo, el rey Teseo, le proporciona el apoyo necesario para disuadirle de su propósito de aniquilamiento y fortalece al héroe decaído para la resistencia. La valoración de philia es un elemento importante en la constitución de la significación del texto de Eurípides

Biopoder en los cuerpos

En las últimas fechas, los estudios de biopolítica y biopoder se han elevado significativamente. Es un asunto antiguo, presente en la filosofía griega y en su realidad. Es la vieja discusión de la relación y la intromisión de la política en la vida y la superación del Estado de naturaleza. La actualidad de la biopolítica se refleja en las dimensiones que adquiere la tecnología y en el asedio de los poderes globales contra la democracia. El cuerpo humano, entidad visible y externa, resiente toda clase de intervenciones y sujeciones encaminadas a la individualización y al establecimiento de una realidad totalitaria. La Biopolítica y la Tanatopolítica se entrelazan al administrar la vida y la muerte en una realidad escalofriante donde la técnica introduce el artificio más allá de lo natural. El cuerpo humano como zona fronteriza de lo natural y lo artificial expresa los alcances del post-humanismo. Una cultura física más equilibrada ayudaría a contener la deshumanización tan extendida, que reduce y degrada lo corporal a la vez, mientras los poderes someten, discriminan, intervienen a través de cuerpos cada vez más dóciles

Biopoder en los cuerpos

En las últimas fechas, los estudios de biopolítica y biopoder se han elevado significativamente. Es un asunto antiguo, presente en la filosofía griega y en su realidad. Es la vieja discusión de la relación y la intromisión de la política en la vida y la superación del Estado de naturaleza. La actualidad de la biopolítica se refleja en las dimensiones que adquiere la tecnología y en el asedio de los poderes globales contra la democracia. El cuerpo humano, entidad visible y externa, resiente toda clase de intervenciones y sujeciones encaminadas a la individualización y al establecimiento de una realidad totalitaria. La Biopolítica y la Tanatopolítica se entrelazan al administrar la vida y la muerte en una realidad escalofriante donde la técnica introduce el artificio más allá de lo natural. El cuerpo humano como zona fronteriza de lo natural y lo artificial expresa los alcances del post-humanismo. Una cultura física más equilibrada ayudaría a contener la deshumanización tan extendida, que reduce y degrada lo corporal a la vez, mientras los poderes someten, discriminan, intervienen a través de cuerpos cada vez más dóciles

Conflito, resistência e philía no Héracles furioso, de Eurípides

En la pieza Heracles Furioso, de Eurípides, el héroe se somete a la prueba definitiva: vencerse a sí mismo, aceptar seguir con vida después de cometer un error irremediable. La acción dramática tiene doble motivación, humana y divina. El conflicto humano requiere capacidad de resistencia de la familia y amigos de Heracles y la oposición de Lico es un elemento clave para la acción. En el enfrentamiento posterior entre Heracles y la diosa Hera, la victoria corresponde a la deidad, motivando a la catástrofe, pero se confirma la heroicidad de Heracles, que se resiste a la tentación de aniquilación tras el asesinato de sus niños. Su amigo, el rey Teseo, le proporciona el apoyo necesario para disuadirle de su propósito de aniquilamiento y fortalece al héroe decaído para la resistencia. La valoración de philia es un elemento importante en la constitución de la significación del texto de Eurípides

Conflito, resistência e philía no Héracles furioso, de Eurípides

En la pieza Heracles Furioso, de Eurípides, el héroe se somete a la prueba definitiva: vencerse a sí mismo, aceptar seguir con vida después de cometer un error irremediable. La acción dramática tiene doble motivación, humana y divina. El conflicto humano requiere capacidad de resistencia de la familia y amigos de Heracles y la oposición de Lico es un elemento clave para la acción. En el enfrentamiento posterior entre Heracles y la diosa Hera, la victoria corresponde a la deidad, motivando a la catástrofe, pero se confirma la heroicidad de Heracles, que se resiste a la tentación de aniquilación tras el asesinato de sus niños. Su amigo, el rey Teseo, le proporciona el apoyo necesario para disuadirle de su propósito de aniquilamiento y fortalece al héroe decaído para la resistencia. La valoración de philia es un elemento importante en la constitución de la significación del texto de Eurípides

Biopoder en los cuerpos

En las últimas fechas, los estudios de biopolítica y biopoder se han elevado significativamente. Es un asunto antiguo, presente en la filosofía griega y en su realidad. Es la vieja discusión de la relación y la intromisión de la política en la vida y la superación del Estado de naturaleza. La actualidad de la biopolítica se refleja en las dimensiones que adquiere la tecnología y en el asedio de los poderes globales contra la democracia. El cuerpo humano, entidad visible y externa, resiente toda clase de intervenciones y sujeciones encaminadas a la individualización y al establecimiento de una realidad totalitaria. La Biopolítica y la Tanatopolítica se entrelazan al administrar la vida y la muerte en una realidad escalofriante donde la técnica introduce el artificio más allá de lo natural. El cuerpo humano como zona fronteriza de lo natural y lo artificial expresa los alcances del post-humanismo. Una cultura física más equilibrada ayudaría a contener la deshumanización tan extendida, que reduce y degrada lo corporal a la vez, mientras los poderes someten, discriminan, intervienen a través de cuerpos cada vez más dóciles

(Supplemental Table 2) Plant characteristics, GHG fluxes, and soil chemical and microbial properties in experimental treatments in Alexandra Fiord

We evaluated above- and belowground ecosystem changes in a 16 year, combined fertilization and warming experiment in a High Arctic tundra deciduous shrub heath (Alexandra Fiord, Ellesmere Island, NU, Canada). Soil emissions of the three key greenhouse gases (GHGs) (carbon dioxide, methane, and nitrous oxide) were measured in mid-July 2009 using soil respiration chambers attached to a FTIR system. Soil chemical and biochemical properties including Q10 values for CO2, CH4, and N2O, Bacteria and Archaea assemblage composition, and the diversity and prevalence of key nitrogen cycling genes including bacterial amoA, crenarchaeal amoA, and nosZ were measured. Warming and fertilization caused strong increases in plant community cover and height but had limited effects on GHG fluxes and no substantial effect on soil chemistry or biochemistry. Similarly, there was a surprising lack of directional shifts in the soil microbial community as a whole or any change at all in microbial functional groups associated with CH4 consumption or N2O cycling in any treatment. Thus, it appears that while warming and increased nutrient availability have strongly affected the plant community over the last 16 years, the belowground ecosystem has not yet responded. This resistance of the soil ecosystem has resulted in limited changes in GHG fluxes in response to the experimental treatments.

(Table 1) Effects of experimental warming on plant cover and diversity indices in an evergreen shrub at Alexandra Fiord

1. Identifying plant communities that are resistant to climate change will be critical for developing accurate, wide-scale vegetation change predictions. Most northern plant communities, especially tundra, have shown strong responses to experimental and observed warming. 2. Experimental warming is a key tool for understanding vegetation responses to climate change. We used open-top chambers to passively warm an evergreen-shrub heath by 1.0-1.3 °C for 15 years at Alexandra Fiord, Nunavut, Canada (79 °N). In 1996, 2000 and 2007, we measured height, plant composition and abundance with a point-intercept method. 3. Experimental warming did not strongly affect vascular plant cover, canopy height or species diversity, but it did increase bryophyte cover by 6.3% and decrease lichen cover by 3.5%. Temporal changes in plant cover were more frequent and of greater magnitude than changes due to experimental warming. 4. Synthesis. This evergreen-shrub heath continues to exhibit community-level resistance to long-term experimental warming, in contrast to most Arctic plant communities. Our findings support the view that only substantial climatic changes will alter unproductive ecosystems.

Fabrication of luminescent nanostructures by electron-beam direct writing of PMMA resist

We report on the conversion of non-luminescent conventional poly(methylmethacrylate) (PMMA)-based electron-beam resists into luminescent materials when used as negative-tone resists, that is, when exposed to high electron irradiation doses. Raman spectroscopy reveals the chemical transformation induced by electron irradiation which is responsible for the observed luminescence in the visible (blue) region. The emission intensity from exposed PMMA-based patterns can be controlled by the electron irradiation dose employed to create them.

Análisis de la geometría de Pablo Palazuelo desde la visión del arquitecto

Esta investigación indaga sobre la relación entre el método geométrico empleado por Pablo Palazuelo y el proceso del proyecto arquitectónico. La elección de este pintor y escultor madrileño como hilo conductor de esta tesis no es fortuita, puesto que la arquitectura desempeña una influencia esencial sobre su obra. Un influjo que le llega en parte a través de su formación académica, dado que cursó estudios de arquitectura en la School of Arts and Crafts de la ciudad de Oxford (1933-1936). Así mismo diseñó propuestas estrechamente vinculadas a un lugar construido, con el consiguiente condicionante de las trazas del mismo. La hipótesis de trabajo formulada a partir de textos elaborados por autores como Víctor Nieto Alcaide y Juan Daniel Fullaondo sugería una interconexión con la arquitectura orgánica. Como comprobación del grado de profundidad en otros análisis publicados, se han seleccionado los textos que indagan en el proceso que el artista desarrollaba durante la producción de su obra, y se adentran en cuestiones estructurales que trascienden el ámbito formal. Siguiendo esta pauta, además de una acotación temporal, se han escogido los realizados por Santiago Amón, Carme Bonell, Valerio Bozal, Manuel J. Borja-Villel, Francisco Calvo Serraller, Claude Esteban, Julián Gállego, Teresa Grandas, Max Hölzer, George Limbour, Kevin Power y Carlos Rodríguez-Spiteri. A estos autores se suman las fuentes orales consultadas dentro de un entorno intrínsecamente próximo a sus realizaciones, procedentes de Pere Casanovas y Soledad Lorenzo. Además de personas que en diferentes etapas de su vida coincidieron por distintos motivos con sus realizaciones, como son Ramón Ayerza, Mariano Bayón, José Antonio Corrales, Luis Gordillo, Rafael Moneo, José Rodríguez-Spiteri y Antonio Tornero. A partir del acceso obtenido a los escritos, libros, bocetos y abundante obra gráfica y escultórica que atesora la Fundación del pintor, se ha podido elaborar un andamiaje tanto teórico como geométrico que ha servido de base para confrontar estas premisas. Esta empresa se ha estructurado en una narración que, además de los estudios precedentes citados, comienza con los cimientos del pensamiento de Palazuelo. Elaborada a partir de sus escritos, donde defendía un sincretismo que concilia las visiones de las culturas occidental y oriental. En los siguientes apartados, se han analizado las principales obras gráficas y escultóricas del autor haciendo un especial hincapié en el método productivo. Una gestación que se resiste a una mera enumeración cronológica, por lo que la clasificación que se propone en este trabajo trata de ser lo más fiel posible al espíritu expresado por Palazuelo basado en linajes y coherencias, para desvelar las herramientas empleadas y poder compararlas con el proceso del proyectual. Este recorrido se completa con una última sección se reúnen por primera vez las dieciséis obras y los dieciséis proyectos más representativos que ilustran la aproximación más directa que obró Palazuelo entre sus investigaciones geométricas y un locus determinado. Durante casi cuatro años se desarrolló un inventariado y catalogación pormenorizada de la documentación y piezas sobre papel, lienzo y metal realizadas por Palazuelo. Esta indagación saca a la luz un conjunto constituido por casi cuatro mil obras, en su mayoría inéditas, que constituyen el archivo de la citada institución. En definitiva, esta investigación construye un tejido gráfico y geométrico referido a uno de los artistas españoles más importantes del siglo XX, entreverado por su pensamiento teórico y realizaciones en dibujos, maquetas, esculturas y propuestas arquitectónicas. Las cuales permiten establecer los acuerdos y desacuerdos con el proceso de la arquitectura para proponer una nueva aproximación geométrica interdisciplinar. ABSTRACT This research investigates the relationship between the geometric method used by Palazuelo and the architectural design’s process. Choosing this Spanish painter and sculptor as thread of this thesis is not fortuitous, since the architecture has an essential influence on his work. An influx that arrives in part through his academic training, as he was an architecture student at the School of Arts and Crafts of the city of Oxford (1933-1936). Furthermore his proposals designed closely linked to a built place, therefore conditioned by its traces. The working hypothesis formulated from texts written by authors like Victor Nieto Alcaide and Juan Daniel Fullaondo suggested an interconnection with organic architecture. As a check on the degree of depth in other published reviews, articles that explore the process that the artist developed during the production of his work, and penetrate into structural issues beyond formal domain have been selected. Following this pattern, along with a temporal dimension, assays by Santiago Amón, Carme Bonell, Valerio Bozal, Manuel J. Borja-Villel, Francisco Calvo Serraller, Claude Esteban, Julián Gállego, Teresa Grandas, Max Hölzer, George Limbour, Kevin Power and Carlos Rodriguez-Spiteri have been selected. Oral sources within an inherently environment close to his achievements, as Pere Casanovas and Soledad Lorenzo, are also added. In addition to people coincided with his accomplishments, such as Ramón Ayerza, Mariano Bayón, José Antonio Corrales, Luis Gordillo, Rafael Moneo, José Rodríguez-Spiteri and Antonio Tornero. From obtained access to the writings, books, sketches and abundant graphic and sculptural work that holds the Foundation painter, it has been able to develop a theoretical and geometric framework that have served as the basis for confronting these premises. This dissertation has been structured in a narrative that ⎯in addition to the previously mentioned studies⎯, begins with the foundations of Palazuelo thought. A structure built from his writings, where he defended a syncretism that reconciles the views of Western and Eastern cultures. In the following sections, his main graphic and sculptural works have been analyzed with particular emphasis on the productive method. A process that resists mere chronological enumeration, so the classification proposed in this investigation tries to be as faithful as possible to the spirit expressed by Palazuelo, based on bloodlines and coherences, to uncover the tools he used and to compare them with the architectural design process. This tour is completed with a final chapter that gathers the sixteen proposals and sixteen works most representative projects that illustrate the more direct approach that Palazuelo worked between geometric investigations and a given locus. For nearly four years, a detailed inventory and cataloguing of documents and works on paper, canvas and metal made by Palazuelo was developed. This research brings to light a set consisting of nearly four thousand works, mostly unpublished, that constitute the current archive of the aforementioned institution. Ultimately, this research builds a graph and geometric fabric referred to one of the most important Spanish artists of the twentieth century, interspersed by his theoretical thinking and achievements in drawings, models, sculptures and architectural proposals. Which allow establishing agreements and disagreements with the process of architecture to propose a new geometric interdisciplinary approach.

Transmembrane Insertion of the Toxoplasma gondii GRA5 Protein Occurs after Soluble Secretion into the Host Cell

The intracellular parasite Toxoplasma gondii resides within a specialized compartment, the parasitophorous vacuole (PV), that resists fusion with host cell endocytic and lysosomal compartments. The PV is extensively modified by secretion of parasite proteins, including the dense granule protein GRA5 that is specifically targeted to the delimiting membrane of the PV (PVM). We show here that GRA5 is present both in a soluble form and in hydrophobic aggregates. GRA5 is secreted as a soluble form into the PV after which it becomes stably associated with the PVM. Topological studies demonstrated that GRA5 was inserted into the PVM as a transmembrane protein with its N-terminal domain extending into the cytoplasm and its C terminus in the vacuole lumen. Deletion of 8 of the 18 hydrophobic amino acids of the single predicted transmembrane domain resulted in the failure of GRA5 to associate with the PVM; yet it remained correctly packaged in the dense granules and was secreted as a soluble protein into the PV. Collectively, these studies demonstrate that the secretory pathway in Toxoplasma is unusual in two regards; it allows soluble export of proteins containing typical transmembrane domains and provides a mechanism for their insertion into a host cell membrane after secretion from the parasite.

The leucine zipper may induce electrophoretic mobility anomalies without DNA bending

Numerous proteins bend DNA upon binding, a phenomenon of potential significance for regulation of gene expression and chromatin. DNA bending is commonly predicted from the presence of electrophoretic mobility anomalies in protein–DNA complexes. However, as compared with electrophoretic methods, several DNA binding oncoprotein families do not display comparable evidence of DNA bends in x-ray structural studies. Herein, circularization kinetics and affinity measurements with prebent DNA templates were employed to assess bending and DNA structural preferences for Max and other basic helix–loop–helix/leucine zipper proteins. In this way, proteins in the Myc/Max basic helix–loop–helix/leucine zipper family were found not to bend DNA in solution but to actually stabilize DNA in an unbent configuration that resists circularization. The mobility anomaly was found to be induced by the leucine zipper protein motif, rather than structural distortions of DNA. Thus rigid protein domain structures may induce anomalous electrophoretic mobility. Moreover, the energetic preference of non-DNA bending proteins for unbent templates suggests mechanisms whereby chromatin structure may regulate transcription.