Cell type-specific regulation of choline acetyltransferase gene expression - Role of the neuron-restrictive silencer element and cholinergic-specific enhancer

This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer

Beyond the cell surface: new mechanisms of receptor function.

a b s t r a c t The text book view of cell surface receptors depicts them at the top of a vertical chain of command that starts with ligand binding and proceeds in a lineal fashion towards the cell nucleus. Although pedagogically useful, this view is incomplete and recent findings suggest that the extracellular domain of cell surface receptors can be a transmitter as much as a receiver in intercellular communication. GFRa1 is a GPI-anchored receptor for GDNF (glial cell line-derived neurotrophic factor), a neuronal growth factor with widespread functions in the developing and adult nervous system. GFRa1 partners with transmembrane proteins, such as the receptor tyrosine kinase RET or the cell adhesion molecule NCAM, for intracellular transmission of the GDNF signal. In addition to this canonical role, GFRa1 can also engage in horizontal interactions and thereby modify the function of other cell surface components. GFRa1 can also function as a ligand-induced adhesion cell molecule, mediating homophilic cell-cell interactions in response to GDNF. Finally, GFRa1 can also be released from the cell surface and act at a distance as a soluble factor together with its ligand. This plethora of unconventional mechanisms is likely to be a feature common to several other receptors and considerably expands our view of cell surface receptor function

RET-independent signaling by GDNF ligands and GFRα receptors

The discovery in the late 1990s of the partnership between the RET receptor tyrosine kinase and the GFRα family of GPI-anchored co-receptors as mediators of the effects of GDNF family ligands galvanized the field of neurotrophic factors, firmly establishing a new molecular framework besides the ubiquitous neurotrophins. Soon after, however, it was realized that many neurons and brain areas expressed GFRα receptors without expressing RET. These observations led to the formulation of two new concepts in GDNF family signaling, namely, the non-cell-autonomous functions of GFRα molecules, so-called trans signaling, as well as cell-autonomous functions mediated by signaling receptors distinct from RET, which became known as RET-independent signaling. To date, the best studied RET-independent signaling pathway for GDNF family ligands involves the neural cell adhesion molecule NCAM and its association with GFRα co-receptors. Among the many functions attributed to this signaling system are neuronal migration, neurite outgrowth, dendrite branching, spine formation, and synaptogenesis. This review summarizes our current understanding of this and other mechanisms of RET-independent signaling by GDNF family ligands and GFRα receptors, as well as their physiological importance.Fil: Ibáñez, Carlos F.. Karolinska Huddinge Hospital. Karolinska Institutet; SueciaFil: Paratcha, Gustavo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; ArgentinaFil: Ledda, Maria Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Drosophila RET contains an active tyrosine kinase and elicits neurotrophic activities in mammalian cells

AbstractThe RET receptor tyrosine kinase controls kidney organogenesis and development of subpopulations of enteric and sensory neurons in different vertebrate species, including humans, rodents, chicken and zebrafish. RET is activated by binding to a ligand complex formed by a member of the glial cell line-derived neurotrophic factor (GDNF) family of neurotrophic factors bound to its cognate GFRα GPI-linked co-receptor. Despite the absence of GDNF or GFRα molecules in the Drosophila genome, a RET orthologue (dRET) has recently been described in this organism and shown to be expressed in subpopulations of cells of the excretory, digestive and nervous systems, thus resembling the expression pattern of RET in vertebrates. In this study, we report on the initial biochemical and functional characterization of the dRET protein in cell culture systems. Full-length dRET could be produced in mammalian and insect cells. Similar to its human counterpart (hRET), overexpression of dRET resulted in its ligand-independent tyrosine phosphorylation, indicating that it bears an active tyrosine kinase. Unlike hRET, however, the extracellular domain of dRET was unable to interact with mammalian GDNF and GFRα1. Self association between dRET molecules could neither be detected, indicating that dRET is incapable of mediating cell adhesion by homophilic interactions. A chimeric molecule comprising the extracellular domain of hRET and the kinase domain of dRET was constructed and used to probe ligand-mediated downstream activities of the dRET kinase in PC12 cells. GDNF stimulation of cells transfected with the hRET/dRET chimera resulted in neurite outgrowth comparable to that obtained after transfection of wild-type hRET. These results indicate significant conservation between the biological effects elicited by the human and Drosophila RET kinases, and suggest functions for dRET in neuronal differentiation in the fly

Biology of GDNF and its receptors — Relevance for disorders of the central nervous system

a b s t r a c t a r t i c l e i n f o Article history: Received 7 December 2015 Revised 14 January 2016 Accepted 25 January 2016 Available online xxxx A targeted effort to identify novel neurotrophic factors for midbrain dopaminergic neurons resulted in the isolation of GDNF (glial cell line-derived neurotrophic factor) from the supernatant of a rat glial cell line in 1993. Over two decades and 1200 papers later, the GDNF ligand family and their different receptor systems are now recognized as one of the major neurotrophic networks in the nervous system, important for the development, maintenance and function of a variety of neurons and glial cells. The many ways in which the four members of the GDNF ligand family can signal and function allow these factors to take part in the control of multiple types of processes, from neuronal survival to axon guidance and synapse formation in the developing nervous system, to synaptic function and regenerative responses in the adult. In this review, we will briefly summarize basic aspects of GDNF signaling mechanisms and receptor systems and then review our current knowledge of the physiology of GDNF activities in the central nervous system, with an eye to its relevance for neurodegenerative and neuropsychiatric diseases

Hybrid Biomass-Wind Power Plant For Reliable Energy Generation

[EN] Massive implementation of renewable energy resources is a key element to reduce CO2 emissions associated to electricity generation. Wind resources can provide an important alternative to conventional electricity generation mainly based on fossil fuels. However, wind generators are greatly affected by the restrictive operating rules of electricity markets because, as wind is naturally variable, wind generators may have serious difficulties on submitting accurate generation schedules on a day ahead basis, and on complying with scheduled obligations in real-time operation. In this paper, an innovative system combining a biomass gasification power plant, a gas storage system and stand-by generators to stabilize a generic 40 MW wind park is proposed and evaluated with real data. The wind park power production model is based on real data about power production of a Spanish wind park and a probabilistic approach to quantify fluctuations and so, power compensation needs. The hybrid wind-biomass system is analysed to obtain main hybrid system design parameters. This hybrid system can mitigate wind prediction errors and so provide a predictable source of electricity. An entire year cycle of hourly power compensations needs has been simulated deducing storage capacity, extra power needs of the biomass power plant and stand-by generation capacity to assure power compensation during critical peak hours with acceptable reliability.Pérez-Navarro, A.; Alfonso-Solar, D.; Álvarez, C.; Ibáñez, F.; Sánchez-Diaz, C.; Segura Heras, I. (2010). Hybrid Biomass-Wind Power Plant For Reliable Energy Generation. Renewable Energy. 35(7):1436-1443. https://doi.org/10.1016/j.renene.2009.12.0181436144335

Plataformas de sensado basadas en nanopartículas monometálicas y aleaciones

El objetivo general del plan de tesis es la síntesis, caracterización y aplicación de nanopartículas (NPs) metálicas protegidas con grupos orgánicos. Nuestro enfoque está en el estudio de los comportamientos físico-químicos de tales sistemas y el aprovechamiento de los mismos para la construcción de sensores de gas, principalmente en la detección de H2, así como también la posibilidad de volcar la plataforma de sensado a aplicaciones en catálisis. En esta etapa de la investigación se ensayaron distintas rutas de síntesis para la obtención de distintas nanopartículas aleadas (Pd-Au; Ag-Pd) en medio acuoso. El método de síntesis consiste en la obtención de nanopartículas protegidas por citrato por reducción de un precursor metálico con NaBH4 en presencia de Citrato de sodio (CitNa3) en medio acuoso, estas nanopartículas (5-10 nm)  pueden luego ser crecidas (20-40 nm). Con el fin de obtener estructuras core-shell, las nanopartículas crecidas son sometidas a intercambio galvánico utilizando un precursor metálico adecuado (Ej: Pdº NPs/AuCl4- ; Agº NPs/PdCl4=). Con el fin de obtener nanopartículas dispersas en medio orgánico (tolueno) se realizaron ensayos de transferencia interfacial (tolueno/agua) con octilamina (C8H17-NH2), este grupo orgánico no solo protege al core metálico (mono metálico o aleado) sino que además permite la transferencia de las nanopartículas en medio acuoso a medio no polar. Tanto los ensayos de intercambio galvánico como los ensayos de trasferencia de fase fueron seguidos por espectroscopia UV-vis, se pudo constatar el intercambio galvánico en el sistema Pdº NPs/AuCl4-  por la desaparición de los picos de absorción correspondientes al AuCl4-, la aparición de picos de absorción asignables a especies de PdCl4=  y la aparición de una banda de absorción alrededor de los 500 nm asignable al plasmón de Auº. En el sistema Agº NPs/ PdCl4= el intercambio galvánico fue evidenciado por la disminución en la intensidad del plasmon de Agº centrada en los 400 nm y la ausencia de los picos de absorción del PdCl4=.Facultad de Ciencias Exacta

Plataformas de sensado basadas en nanopartículas monometálicas y aleaciones

El objetivo general del plan de tesis es la síntesis, caracterización y aplicación de nanopartículas (NPs) metálicas protegidas con grupos orgánicos. Nuestro enfoque está en el estudio de los comportamientos físico-químicos de tales sistemas y el aprovechamiento de los mismos para la construcción de sensores de gas, principalmente en la detección de H2, así como también la posibilidad de volcar la plataforma de sensado a aplicaciones en catálisis. En esta etapa de la investigación se ensayaron distintas rutas de síntesis para la obtención de distintas nanopartículas aleadas (Pd-Au; Ag-Pd) en medio acuoso. El método de síntesis consiste en la obtención de nanopartículas protegidas por citrato por reducción de un precursor metálico con NaBH4 en presencia de Citrato de sodio (CitNa3) en medio acuoso, estas nanopartículas (5-10 nm)  pueden luego ser crecidas (20-40 nm). Con el fin de obtener estructuras core-shell, las nanopartículas crecidas son sometidas a intercambio galvánico utilizando un precursor metálico adecuado (Ej: Pdº NPs/AuCl4- ; Agº NPs/PdCl4=). Con el fin de obtener nanopartículas dispersas en medio orgánico (tolueno) se realizaron ensayos de transferencia interfacial (tolueno/agua) con octilamina (C8H17-NH2), este grupo orgánico no solo protege al core metálico (mono metálico o aleado) sino que además permite la transferencia de las nanopartículas en medio acuoso a medio no polar. Tanto los ensayos de intercambio galvánico como los ensayos de trasferencia de fase fueron seguidos por espectroscopia UV-vis, se pudo constatar el intercambio galvánico en el sistema Pdº NPs/AuCl4-  por la desaparición de los picos de absorción correspondientes al AuCl4-, la aparición de picos de absorción asignables a especies de PdCl4=  y la aparición de una banda de absorción alrededor de los 500 nm asignable al plasmón de Auº. En el sistema Agº NPs/ PdCl4= el intercambio galvánico fue evidenciado por la disminución en la intensidad del plasmon de Agº centrada en los 400 nm y la ausencia de los picos de absorción del PdCl4=.Facultad de Ciencias Exacta

Thermal effects on the drilling performance of a limestone: relationships with physical and mechanical properties

This work evaluates the effect of high temperatures and cooling methods on the drillability of Prada limestone. Samples from boreholes drilled during the design stage of the Tres Ponts Tunnel in the Catalan south Pyrenean zone (Spain) were subjected to temperatures of 105, 200, 300, 400, and 600 °C, and then cooled at a slow rate or by quenching. Sievers’ J-value (SJ) and brittleness (S20) were determined on thermally treated samples, and the drilling rate index (DRI) was calculated for each temperature. The results show that thermal treatment implied a sustained increase in the drillability of the rock of up to 40% at 600 °C and a change in the drillability category (from medium to high). At 600 °C, SJ and S20 tripled and doubled, respectively, the initial values obtained for the intact rock. The results were inconclusive about the influence of the cooling method on the drilling performance of Prada limestone for the tested range of temperatures. The substantial improvement observed in the drillability of Prada limestone when heated, measured in terms of DRI, could help in the development of novel thermally-assisted mechanical excavation methods. Additionally, strong correlations between drillability variables (i.e., SJ and S20) and physical and mechanical variables of Prada limestone (i.e., P- and S-wave velocities, uniaxial compression strength, elastic modulus, and Poisson’s ratio) are proposed. Correlations will help make preliminary predictions of drillability based on properties such as uniaxial compression strength and ultrasound wave velocities.The authors wish to acknowledge David Benavente and Juan Carlos Canaveras from the University of Alicante, for their valuable help on mineralogical and petrographic description of the rock. Additionally, Kreum SA, Ayesa SA, Infraestructures de la Generalitat de Catalunya, S.A.U., and the Lleida regional roads authority (Servei Territorial de Carreteres de Lleida, Generalitat de Catalunya) for providing rock samples. This work was supported by the Department of Geological and Geotechnical Engineering, Universitat Politecnica de Valencia

Cross-talk between the Notch and TGF-β signaling pathways mediated by interaction of the Notch intracellular domain with Smad3

The Notch and transforming growth factor-β (TGF-β) signaling pathways play critical roles in the control of cell fate during metazoan development. However, mechanisms of cross-talk and signal integration between the two systems are unknown. Here, we demonstrate a functional synergism between Notch and TGF-β signaling in the regulation of Hes-1, a direct target of the Notch pathway. Activation of TGF-β signaling up-regulated Hes-1 expression in vitro and in vivo. This effect was abrogated in myogenic cells by a dominant-negative form of CSL, an essential DNA-binding component of the Notch pathway. TGF-β regulated transcription from the Hes-1 promoter in a Notch-dependent manner, and the intracellular domain of Notch1 (NICD) cooperated synergistically with Smad3, an intracellular transducer of TGF-β signals, to induce the activation of synthetic promoters containing multimerized CSL- or Smad3-binding sites. NICD and Smad3 were shown to interact directly, both in vitro and in cells, in a ligand-dependent manner, and Smad3 could be recruited to CSL-binding sites on DNA in the presence of CSL and NICD. These findings indicate that Notch and TGF-β signals are integrated by direct protein–protein interactions between the signal-transducing intracellular elements from both pathways