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Single-cell amperometry is a powerful technique that permits the detection of electrochemically active transmitters, such as catecholamines, histamine, or serotonin, released by exocytosis from secretory cells.Amperometry has two main characteristics that make it ideal for the study of exocytosis at the single-cell level with single-vesicle resolution quantal release. (i) It is noninvasive. The carbon fiber microelectrode can be carefully positioned on plasma membrane of a single cell, allowing the detection of the oxidation current of the secreted molecules. (ii) High temporal resolution and sensitivity. Exocytosis can be monitored with a real-time resolution that allows the determination of the kinetics release with an attomol detection sensitivity, which ensures an accurate calculation of the amount of transmitter released.Here, we compile some recommendations and advices to perform amperometry quantal analysis.

Neuropeptides are essential signaling molecules transported and secreted by dense‐core vesicles (DCVs), but the number of DCVs available for secretion, their subcellular distribution, and release probability are unknown. Here, we quantified DCV pool sizes in three types of mammalian CNS neurons in vitro and in vivo . Super‐resolution and electron microscopy reveal a total pool of 1,400–18,000 DCVs, correlating with neurite length. Excitatory hippocampal and inhibitory striatal neurons in vitro have a similar DCV density, and thalamo‐cortical axons in vivo have a slightly higher density. Synapses contain on average two to three DCVs, at the periphery of synaptic vesicle clusters. DCVs distribute equally in axons and dendrites, but the vast majority (80%) of DCV fusion events occur at axons. The release probability of DCVs is 1–6%, depending on the stimulation. Thus, mammalian CNS neurons contain a large pool of DCVs of which only a small fraction can fuse, preferentially at axons.

Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba2+ > Sr2+ ≥ Ca2+, contrasting with that elicited by these cations in permeabilized cells (Ca2+ > Sr2+ > Ba2+). Current-clamp recordings show that extracellular Sr2+ and Ba2+ cause membrane depolarization and action potentials, which are not blocked by Cd2+ but that can be mimicked by tetra-ethyl-ammonium. When applied intracellularly, only Ba2+ provokes action potentials. Voltage-clamp monitoring of Ca2+-activated K+ channels (KCa) shows that Ba2+ reduces outward currents, which were enhanced by Sr2+. Extracellular Ba2+ increases cytosolic Ca2+ concentrations in Fura-2-loaded intact cells, and it induces long-lasting catecholamine release. Conversely, amperometric recordings of permeabilized cells show that Ca2+ promotes the longest lasting secretion, as Ba2+ only provokes secretion while it is present and Sr2+ induces intermediate-lasting secretion. Intracellular Ba2+ dialysis provokes exocytosis at concentrations 100-fold higher than those of Ca2+, whereas Sr2+ exhibits an intermediate sensitivity. These results are compatible with the following sequence of events: Ba2+ blocks KCa channels from both the outside and inside of the cell, causing membrane depolarization that, in turn, opens voltage-sensitive Ca2+ channels and favors the entry of Ca2+ and Ba2+. Although Ca2+ is less permeable through its own channels, it is more efficient in triggering exocytosis. Strontium possesses both an intermediate permeability and an intermediate ability to induce secretion.

Resumen Objetivo Analizar la utilidad percibida por los gestores de centros de atencion primaria respecto a las herramientas utilizadas para su gestion (presupuesto y cuadro de mando integral [CMI]) y su impacto sobre la motivacion del personal. Diseno Estudio cualitativo de casos basado en teoria fundamentada, realizado entre enero y junio de 2014. Emplazamiento Unidades de Gestion Clinica de Atencion Primaria (UGCAP) del Area de Gestion Sanitaria metropolitana de Sevilla. Participantes Directores de UGCAP y gestores del Area. Metodo Los datos se recogieron en 8 entrevistas semiestructuradas mediante muestreo intencional no probabilistico con criterios de representatividad y suficiencia del discurso. Las entrevistas fueron grabadas, transcritas y analizadas mediante codigos in-vivo. Resultados Ambas herramientas estan implementadas en los centros de atencion primaria, con usos diferentes. El presupuesto se percibe como una herramienta coercitiva, impuesta para el control del gasto, y poco util para la gestion diaria. Aunque el CMI es mas flexible y permite identificar los problemas financieros y asistenciales del centro, los limites presupuestarios reducen enormemente el margen para implementar soluciones. Ademas, la politica de incentivos es insuficiente, generando problemas de motivacion. Conclusiones Este estudio muestra que las tensiones presupuestarias que afectan a la atencion primaria han reducido al minimo la autonomia de los centros de salud, imponiendo una toma de decisiones centralizada en la que prima el ahorro en costes sobre la calidad asistencial, generando tensiones con el personal. Se plantea la necesidad de incrementar la autonomia de los centros y mejorar el compromiso del personal mediante programas de formacion y desarrollo profesional.

Adrenal chromaffin cells release epinephrine (EPI) and norepinephrine (NE) into the bloodstream as part of the homeostatic response to situations like stress. Here we utilized EPI-deficient mice generated by knocking out (KO) the phenylethanolamine N-methyltransferase (Pnmt) gene. These Pnmt-KO mice were bred to homozygosis but displayed no major phenotype. The lack of EPI was partially compensated by an increase in NE, suggesting that EPI storage was optimized in adrenergic cells. Electron microscopy showed that despite the lack of EPI, chromaffin granules retain their shape and general appearance. This indicate that granules from adrenergic or noradrenergic cells preserve their characteristics even though they contain only NE. Acute insulin injection largely reduced the EPI content in wild-type animals, with a minimal reduction in NE, whereas there was only a partial reduction in NE content in Pnmt-KO mice. The analysis of exocytosis by amperometry revealed a reduction in the quantum size (−30%) and Imax (−21%) of granules in KO cells relative to the wild-type granules, indicating a lower affinity of NE for the granule matrix of adrenergic cells. As amperometry cannot distinguish between adrenergic or noradrenergic cells, it would suggest even a larger reduction in the affinity for the matrix. Therefore, our results demonstrate that adrenergic cells retain their structural characteristics despite the almost complete absence of EPI. Furthermore, the chromaffin granule matrix from adrenergic cells is optimized to accumulate EPI, with NE being a poor substitute. (Figure presented.). Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/.

La presente investigación analizará el uso político de la comunicación 2.0, específicamente de la red de microblogging Twitter. El actor político elegido es la Presidenta de la Nación, Cristina Fernández de Kirchner durante el período agosto de 2011 y octubre de 2011 . Se realizará un análisis sobreconstrucción de la subjetividad política en las redes sociales, las concepciones sobre: la comunicación masiva, el uso de las redes sociales, los debates políticos y la interacción con los usuarios de redes sociales. Además, se realizará una comparación entre el tratamiento de la información electoral de uno de los medios masivos de comunicación mas tradicionales de alcance nacional, Diario La Nacion y el material político, de opinión o de gestión publicado por el actor político seleccionado., Facultad de Periodismo y Comunicación Social

Deviation from optimum percentage (DOP) and Diagnosis and Recommendation Integrated System (DRIS) are methodologies for leaf analysis interpretation. DOP calculates the deviation as a percentage for each essential element while DRIS calculates the average deviation for different ratios that include each element. A vine survey was undertaken for 11 years in La Rioja, northeastern Spain, to establish nutritional references for cv. ‘Tempranillo’ (Vitis vinifera L.). Diagnosis of nitrogen, phosphorus, potassium, calcium, and magnesium were performed by DRIS and DOP on the dataset for leaf blade and petiole, at both flowering (n = 684) and veraison (n = 801) with a moderate-yield criterion (3,800–10,000 kg·ha). Important differences between DOP and DRIS were found when a diagnosis using the same dataset was performed. In spite of the differences, the regression equations obtained are an acceptable adjustment between both methods for phosphorus (P) and magnesium (Mg) in blades and petioles, and for potassium (K) in blades., This study was supported by the National Institute of Agricultural Research (Spain), the European Social Fund, and the Regional Government of La Rioja (Spain) Projects INIA-SC00-016, PR-01-03, PR-01-04, and PR-03-05, among others.

Three divalent cations can elicit secretory responses in most neuroendocrine cells, including chromaffin cells. The extent to which secretion is elicited by the cations in intact depolarized cells was Ba

Large dense core vesicles and chromaffin granules accumulate solutes at large concentrations (for instance, catecholamines, 0.5–1 M; ATP, 120–300 mM; or Ca2+, 40 mM (12)). Solutes seem to aggregate to a condensed protein matrix, which is mainly composed of chromogranins, to elude osmotic lysis. This association is also responsible for the delayed release of catecholamines during exocytosis. Here, we compile experimental evidence, obtained since the inception of single-cell amperometry, demonstrating how the alteration of intravesicular composition promotes changes in the quantum characteristics of exocytosis. As chromaffin cells are large and their vesicles contain a high concentration of electrochemically detectable species, most experimental data comes from this cell model.

Chromogranins are acidic proteins; both chromogranins A and B constitute the main protein component in the vesicular matrix of large dense core vesicles. Chromogranins are a natural source of peptides with different physiological activities that have been associated with vascular and neurological diseases. We have used three different genetic mutant models of mice lacking chromogranin A, chromogranin B and both all on the same C57BL/6J background, to characterize the physiological roles of these proteins using metabolic, cardiovascular and behavioural tests. In mice from 3 to 18 months of age, the lack of any chromogranin promoted age-dependent hypersensitivity to insulin, while the lack of both chromogranins provoked progressive lack of response to stress, as restriction did not promote tachycardia in old mice. Moreover, the lack of chromogranin B produced a depressive-like and aggressive phenotype, while the lack either or both chromogranins increased barbering behaviour. In addition, we observed no effects on light–dark box or RotaRod tests. Mice lacking chromogranin B exhibited lower exploratory activity. Based on this extensive phenotyping with more than 2800 mice, these findings support roles of chromogranins, or the peptides derived from them, in the control of aggressive behaviour along with changes in their metabolic profile beyond their previously described activities in the secretory pathway.

Este artículo analiza cómo, durante la historia del arte, diferentes dispositivos han ayudado a infundir la figura del artista como genio, basándose principalmente en la mitificación de elementos biográficos y en la concepción del artista tocado por la divinidad. Este estudio previo sirve posteriormente para analizar dos periodos concretos y antagónicos de la historia del siglo XX: la modernidad, que ayudó a corroborar tales construcciones a través de la disolución entre artista y público; y la aparición del arte conceptual durante la posmodernidad como paradigma del cuestionamiento de prácticas como la desmaterialización y la desespecificación, que ayudaron a cuestionar las ideas de autoría, originalidad y genialidad. This article analyses how, throughout art history, different devices have helped to arose the figure of the artist as a genius, fundamentally by the mystification of some biographic elements and the conception of the artist as a divine entity. The initial study will subsequently help to examine two very precise and antagonistic periods during the XX century: Modernism, which helped to promote those conceptions by the dissolution between the artist and the audience; and the birth of conceptual art during Post‐modernism as a paradigm from which to question artistic practices such dematerialization and de‐specialisation, helping to wonder about authority, originality and genius.

Chromogranins (Cgs) are the most abundant intravesicular proteins of chromaffin granules. Using Cgs knockout mice, we found that the lack of chromogranin A (CgA), chromogranin B (CgB) or both drastically reduce the vesicular content of catecholamines (CA), impair its accumulation in granules and largely affect the kinetics of exocytosis. Conversely, the overexpression of CgA induces the genesis of vesicles, increases their quantal content and even transforms non-secretory in cells capable to secrete substances. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and regulates the exocytotic process.

Chromogranins (Cgs) are acidic proteins that have been described in the large, dense core vesicles (LDCVs) of adrenal chromaffin cells and that have been shown to promote LDCV formation, even in nonsecretory cells. Catecholamines (CAs) are adsorbed by Cgs in vitro, and the absence of Cgs modifies the storage and exocytosis of CAs in chromaffin cells. In this study, we set out to assess the role of CgA in the accumulation and exocytosis of CAs in cells when the levels of CgA and CA are manipulated. We overexpressed CgA in nonsecretory HEK293 cells and in secretory PC12 cells, to study the formation, movement, and exocytosis of newly formed granules by evanescent wave microscopy. We analyzed the association of Cgs/CA by HPLC and amperometry and their role in the accumulation and exocytosis of amines, both under resting conditions and after l-DOPA overloading. To our knowledge, this is the first demonstration that CgA expression in a nonsecretory cell line facilitates the storage and exocytosis of CA. In addition, CgA overexpression causes a doubling of the accumulation of CA, although it slows down exocytosis in PC12 cells. We propose a model to explain how the CgA/CA complex governs the accumulation and exocytosis of secreted amines.

Backgrounds and Aims Leaf blade and petiole of the grapevine at different sampling times are normally employed for nutritional diagnosis. Both tissues were studied to determine their reliability for diagnosis at different stages of development and, also, the period of time, around the phenological stage of a reference, in which an analysis could be compared with this reference. Methods and Results Blades and petioles of Garnacha tinta grapevine were collected throughout the growing season for analysis of ten essential elements. In general, petioles showed greater variability between replicates for nitrogen, phosphorus, potassium, manganese and zinc, while iron, copper and boron presented greater variability in blades. Differences between consecutive sampling times suggested that references for complete cap-fall will be valid only until fruitset. Furthermore, petiole references at any time within veraison are valid at least until the beginning of ripening, while blade references for veraison are valid from the end of berry development until the advanced ripening stage for most nutrients. Conclusions Diagnosis of nitrogen, phosphorus, potassium, manganese and zinc is preferable in the blade at complete cap-fall, fruitset and veraison, while the petiole is a better choice for iron and boron at both complete cap-fall and fruitset, and for boron at veraison. Calcium and magnesium at flowering or veraison, and iron at veraison, showed similar reliability for both tissues. Significance of the Study This study contributes to identification of improved sampling strategies that improve the accuracy of grapevine nutrient diagnosis based on tissue analysis.

Chromogranins (Cgs) are acidic proteins that have been implicated in several physiological processes such as vesicle sorting, the production of bioactive peptides and the accumulation of soluble species inside large dense core vesicles (LDCV). They constitute the main protein component in the vesicular matrix of LDCV. This latter characteristic of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca(2+). It is likely that Cgs are behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion, due to their low affinity and high capacity to bind solutes present inside LDCV. The recent availability of mouse strains lacking Cgs, combined with the arrival of several techniques for the direct monitoring of exocytosis, have helped to expand our knowledge about the mechanisms used by granins to concentrate catecholamines and Ca(2+) in LDCV, and how they affect the kinetics of exocytosis. We will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

J. Neurochem. (2010) 114, 335–343. Abstract Chromogranins (Cgs) constitute the main protein component in the vesicular matrix of large dense core vesicles (LDCV). These acidic proteins have been implicated in several physiological processes such as vesicle sorting, the generation of bioactive peptides and the accumulation of soluble species inside LDCV. This latter feature of Cgs accounts for the ability of vesicles to concentrate catecholamines and Ca2+. Indeed, the low affinity and high capacity of Cgs to bind solutes at the low pH of the LDCV lumen seems to be behind the delay in the neurotransmitter exit towards the extracellular milieu after vesicle fusion. The availability of new mouse strains lacking Cgs in combination with the arrival of several techniques for the direct monitoring of exocytosis (like amperometry, patch-amperometry and intracellular electrochemistry), have helped advance our understanding of how these granins concentrate catecholamines and Ca2+ in LDCV, and how they influence the kinetics of exocytosis. In this review, we will discuss the roles of Cgs A and B in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from adrenal chromaffin cells.

© 2015 INIA. An adequate nutritional state of a crop can be kept by means of a well-designed fertilization plan based on the assessment of the nutrient availability throughout the growing season. The objective of this study was to determine the reliability of leaf blade and petiole diagnosis and the period of validity of their references at both flowering and veraison by means of systematic monitoring throughout the complete growing season. The study was carried out in two plots planted with Vitis vinifera L. cv. Graciano within the AOC Rioja (Spain). Blades and petioles were collected throughout a growing season (2006) and total N, P, K, Ca, Mg, Fe, Mn, Zn, Cu and B concentrations were analyzed in both tissues. Results suggest, in general, that petioles have higher variability and lower analysis reproducibility than blades. Therefore, blade could be a more appropriate tissue to evaluate N, P, K, Ca, and Mg at both flowering and veraison in this variety. Micronutrients in blade and petiole showed different variability behaviour in each of the vineyards studied, therefore, based on our results, it was difficult to determine which one could be the best tissue for the nutritional diagnosis of the ‘Graciano’ variety. Seasonal changes of nutrient concentration in both tissues also confirmed the need for reference values for each tissue and each phenological stage.

Deviation from optimum percentage (DOP) is a diagnosis methodology for leaf analyses which expresses the deviation for each element with respect to its optimal concentration. This deviation is an individual index for each nutrient and allows the sorting of all the analyzed nutrients according to their limitations. A nutritional survey was undertaken over eleven years in La Rioja (Spain), to establish reference concentrations for the nutritional diagnosis of Vitis vinifera L., cv. 'Tempranillo' grafted on Richter-110. Reference concentrations for DOP methodology are proposed, and sensibility for the nutritional diagnosis was evaluated for blade and petiole analysis of N, P, K, Ca, Mg, Fe, Mn, Zn, Cu, and B at flowering and veraison phenological stages by comparison between DOP and sufficiency ranges (SR) methods. Results suggest that petiole has lower sensibility than blade to detect deficiencies or excesses of N, P, K, Ca, Mg, Zn, and Mn at veraison. In addition, petiole is a better tissue than blade to detect Fe and B deficiencies or excesses. Therefore, our results make possible the right choice between tissues, leaf blade or petiole, for a general nutritional diagnosis of 'Tempranillo' grapevines. On the other hand, it is possible to evaluate the status of each nutrient in each phonological stage analyzing both tissues and comparing the nutrient status to its references, DOP or SR, in the most adequate tissue., This study was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA, Spain), the European Social Fund and the Regional Government of La Rioja (Spain): Projects INIA-SC00-016, PR-01-03, PR-01-04, and PR-03-05, among others.

Chromogranins (Cgs) are acidic proteins implicated in several physiological processes, including the biogenesis and sorting of secretory vesicles, the generation of bioactive peptides, and the accumulation of soluble species inside large dense core vesicles (LDCV). Indeed, Cgs are the main protein component of the vesicular matrix in LDCV, and they are involved in the concentration of soluble species like neurotransmitters and calcium. Experiments using electrochemical techniques such amperometry, patch amperometry, and intracellular electrochemistry have clarified the functional roles of Cgs in the accumulation and release of catecholamines. We have focused this review at a single event of exocytosis of chromaffin cells from three mouse strains lacking Cgs. Accordingly, in this brief review, we will focus on the role of Cgs in maintaining the intravesicular environment of secretory vesicles and in exocytosis, bringing together the most recent findings from studies on adrenal chromaffin cells.

Plant nutritional diagnosis requires specific reference levels for each grape variety to effectively reflect the nutritional status of the crop. A vine nutritional survey was undertaken in the Rioja A.O.C., northeastern Spain. The survey method was used to establish the preliminary norms in the application of the Diagnosis and Recommendation Integrated System (DRIS) to the nutritional diagnosis of leaf blade and petiole tissues of Vitis vinifera L., cv. Tempranillo, on Richter-110 rootstock. These DRIS norms were generated from data obtained during the course of 11 years (2000¿10) from 166 vineyards trained on both Gobelet and Vertical Shoot Position (VSP) pruned Guyot and Double Cordon Royat systems. The database obtained includes information from different altitudes, soil types, cultural practices, and climatic conditions within the Rioja appellation. The majority of the vineyards had no irrigation and soil was tilled mechanically. Nitrogen, phosphorus, potassium, calcium, and magnesium were analyzed in leaf blade and petiole dry tissues sampled at both flowering and veraison. Vineyards with phytosanitary damages or flowering problems were discarded. Ratios among nutrients were established for the blade and petiole at both flowering and veraison by comparing two subpopulations of high and low quality. Both subpopulations were selected from a limited production criteria by a Student¿s t-test mean comparison. Finally, DRIS norms for leaf blade and petiole at both flowering and veraison are proposed for the nutrition diagnosis of the cv. Tempranillo in the Rioja A.O.C., This study was supported by the National Institute of Agricultural Research (Spain), the European Social Fund, and by the Regional Government of La Rioja (Spain), including Projects INIA-SC00-016, PR-01-03, PR-01-04, and PR-03-05, among others.

Plant nutritional diagnosis requires specific reference levels for each variety to effectively reflect the nutritional status of the crop. A vine nutritional survey was undertaken in the Rioja A.O.C., northeastern Spain. The survey method was used to establish the preliminary reference levels for the nutritional diagnosis of leaf blade and petiole tissues of Vitis vinifera L., cv. Tempranillo on Richter-110 rootstock. These references were generated from data obtained during the course of 11 years (2000¿10) from 166 vineyards trained on Gobelet and Vertical Shoot Position (VSP), pruned in Guyot or Double Cordon Royat, systems. The database obtained included information from different altitudes, soil types, cultural practices, and climatic conditions within the Rioja appellation. The majority of the vineyards had no irrigation and soil was tilled mechanically. Nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), iron (Fe), manganese (Mn), zinc (Zn), copper (Cu), and boron (B) were analyzed in leaf blade and petiole dry tissues sampled at both flowering and veraison. Vineyards with phytosanitary damages, flowering problems, and data with extreme nutrient values were discarded. Data were statistically analyzed to obtain the normal average values considering the (¿ ± 0.25¿) of the central data as optimal levels and the tails values (¿ ± 0.84¿) of the population as excessive or deficient levels. The statistical procedure included the Kolmogorov¿Smirnov nonparametric test for normality, data transformations for normality, and the calculation of percentiles when those transformations to normal distributions become unlikely (P20, P40, P60, and P80). Finally, reference levels for leaf blade and petiole at both flowering and veraison are proposed for the nutrition diagnosis of the cv. Tempranillo grapevine in the Rioja A.O.C., This study was supported by the National Institute of Agricultural Research (Spain), the European Social Fund, and the Regional Government of La Rioja (Spain) with Projects INIA-SC00-016, PR-01-03, PR-01-04, and PR-03-05, among others.

Este libro forma parte de la colección Libros de Cátedra que lanzó la Universidad Nacional de La Plata. Como tal, es un compilado de artículos académicos cuyos autores son docentes de la cátedra Metodología de la Investigación Social, y cada texto surgió como planteo teórico/metodológico en cada reunión de cátedra, cada clase y fundamentalmente como parte de la respuesta docente a las necesidades que expresaron nuestros alumnos a lo largo de los últimos nueve años. Asimismo, consideramos la importancia de contar con aportes metodológicos propios de la comunicación social, abordando cada etapa de la investigación desde nuestra propia disciplina y desde nuestro posicionamiento teórico/ideológico, que busca profundizar la mirada latinoamericana en nuestros objetos de estudio. Cabe en estas líneas agradecer a todos los compañeros docentes que formaron parte de la cátedra en los últimos años y contribuyeron de manera esencial en los debates que derivaron en el presente libro: Celina Artigas, Alcira Martínez y Santiago Puca Molina., Facultad de Periodismo y Comunicación Social

Catecholamines (CAs) and granin peptides are costored in dense-core vesicles within the chromaffin cells of the adrenal medulla and in other endocrine organs and neurons. Granins play a major functional and structural role in chromaffin cells but are ubiquitous proteins, which are present also in secretory cells of the nervous, endocrine, and immune systems, where they regulate a number of cellular functions. Furthermore, recent studies also demonstrate that granin-derived peptides can functionally interact with CA to modulate key physiological functions such as lipolysis and blood pressure. In this chapter, we will provide a brief update on the interaction between CA and granins at the cellular and organ levels. We will first discuss recent data on the regulation of exocytosis of CA and peptides from the chromaffin cells by the sympathetic nervous system with a specific reference to the prominent role played by splanchnic nerve-derived pituitary adenylate cyclase-activating peptide (PACAP). Secondly, we will discuss the role of granins in the storage and regulation of exocytosis in large dense-core vesicles. Finally, we will provide an up-to-date review of the roles played by two granin-derived peptides, the chromogranin A-derived peptide catestatin and the VGF-derived peptide TLQP-21, on lipolysis and obesity. In conclusion, the knowledge gathered from recent findings on the role played by proteins/peptides in the sympathetic/target cell synapses, discussed in this chapter, would contribute to and provide novel mechanistic support for an increased appreciation of the physiological role of CA in human pathophysiology.

Cultured chromaffin cells have been used for almost 40 years in the study of different cell functions using biochemical, electrophysiological, pharmacological, and toxicological approaches. Chromaffin cells are essentially secretory cells that are used to model sympathetic neurons or neuroendocrine cells. In this chapter, we describe the most common methods currently used to isolate and culture chromaffin cells from the animals used most commonly: cows, rats, and mice. We also provide some advice on the use of these cells in the laboratory.

Cultured chromaffin cells have been used for almost 40 years in the study of different cell functions using biochemical, electrophysiological, pharmacological, and toxicological approaches. Chromaffin cells are essentially secretory cells that are used to model sympathetic neurons or neuroendocrine cells. In this chapter, we describe the most common methods currently used to isolate and culture chromaffin cells from the animals used most commonly: cows, rats, and mice. We also provide some advice on the use of these cells in the laboratory.

Chromogranins A (CgA) and B (CgB) are the main soluble proteins of large dense-core secretory vesicles (LDCVs). Using CgA- and CgB-knockout (KO) mice, we found that the absence of chromogranins A and B induces significant changes in catecholamine (CA) accumulation and the kinetics of exocytosis. By crossing these two knockout strains, we generated a viable and fertile double CgA/B-KO mouse in which the catecholamine content in chromaffin LDCVs was halved, and the secretory response significantly reduced. Incubating cells with l-DOPA increased the vesicular CA content in wild-type (WT) but not in Cg-KO cells, which was not due to changes in amine transport, or in the synthesis or degradation of cytosolic amines. Electron microscopy revealed the presence of giant secretory vesicles exhibiting significant alterations, with little or no electrodense inner matrix. Proteomic analysis confirmed the absence of CgA and B, and revealed small changes in SgII in the LDCV-enriched fraction, as well as the overexpression of fibrinogen and other proteins. In summary, our findings indicate that the mechanisms responsible for vesicular accumulation of CA are saturated in Cgs-KO cells, in contrast to the ample capacity for further accumulation in WT cells. We conclude that Cgs contribute to a highly efficient system that directly mediates monoamine accumulation and exocytosis in LDCVs.

Secretory vesicles of chromaffin cells are acidic organelles that maintain an increasing pH gradient towards the cytosol (5.5 vs. 7.3) that is mediated by V-ATPase activity. This gradient is primarily responsible for the accumulation of large concentrations of amines and Ca(2+), although the mechanisms mediating Ca(2+) uptake and release from granules, and the physiological relevance of these processes, remain unclear. The presence of a vesicular matrix appears to create a bi-compartmentalised medium in which the major fractions of solutes, including catecholamines, nucleotides and Ca(2+), are strongly associated with vesicle proteins, particularly chromogranins. This association appears to be favoured at acidic pH values. It has been demonstrated that disrupting the pH gradient of secretory vesicles reduces their rate of exocytosis and promotes the leakage of vesicular amines and Ca(2+), dramatically increasing the movement of secretory vesicles and triggering exocytosis. In this short review, we will discuss the data available that highlights the importance of pH in regulating the association between chromogranins, vesicular amines and Ca(2+). We will also address the potential role of vesicular Ca(2+) in two major processes in secretory cells, vesicle movement and exocytosis.

[EN] [Aim] To obtain specific references for the nutritional diagnosis of ten essential nutrients for leaf blade and petiole of ‘red Grenache’ (Vitis vinifera L.)., [Methods and results] Leaf blades and petioles from 36 vineyards of ‘red Grenache’ (Vitis vinifera L.) grafted on Richter 110 were collected and analyzed at flowering and veraison between 1992 and 2008. Using the compiled data bank, nutritional references for ten elements (N, P, K, Ca, Mg, Fe, Mn, Zn, Cu and B were calculated. Optimal values were those around the central data (μ ± 0.25σ), while excessive and deficient values were those beyond the tails of the distribution (μ ± 0.84σ). Percentile calculation was performed when transformations to normal distributions became unlikely., [Conclusion] References for Sufficiency Ranges (SR) and Deviation from Optimum Percentage (DOP) methods were obtained for those ten nutrients studied., [Significance and impact of the study] The proposed ‘red Grenache’ references for leaf blade and petiole contribute to the improvement of the accuracy of ‘red Grenache’ grapevine nutrient diagnosis based on tissue analysis. These references are a guide to assess the nutritional status of ‘red Grenache’ grapevine around the world in general and, with higher accuracy, for the Rioja region and areas with similar vineyard conditions., [FR] [Objectif] Obtenir des références spécifiques pour le diagnostic nutritionnel de dix éléments nutritifs essentiels au limbe et pétiole de ‘Grenache noir’ (Vitis vinifera L.)., [Méthodes et résultats] Les limbes et pétioles des feuilles provenant de 36 vignobles de ‘Grenache noir’ (Vitis vinifera L.) greffés sur Richter 110 ont été recueillis et analysés à la floraison et à la véraison entre 1992 et 2008. À partir de la banque de données, des références nutritionnelles pour dix éléments (N, P, K, Ca, Mg, Fe, Mn, Zn, Cu et B) ont été calculées. Les valeurs optimales étaient celles autour de la valeur centrale de données (μ ± 0.25σ), tandis que les valeurs excessives et déficientes étaient celles au-delà des extrémités de la distribution (μ ± 0.84σ). Lorsque les valeurs n’étaient pas conformes à une distribution normale et que la transformation logarithmique n’était pas efficace, les percentiles correspondants ont été calculés., [Conclusion] Les références pour les méthodes Gammes de Suffisance et Déviation du Pourcentage Optimal ont été obtenues pour les dix nutriments étudiés., [Signification et impact de l’étude] Les références pour le limbe et le pétiole de ‘Grenache noir’ contribuent à l’amélioration du diagnostic de nutriments. Ces références sont un guide pour évaluer l’état nutritionnel des vignes de ‘Grenache noir’ dans le monde et, avec une plus grande précision, dans la région de Rioja et dans d’autres régions similaires., This study was supported by the National Institute of Agricultural Research (Spain) and the Regional Government of La Rioja (Spain) with Projects INIA-SC00-016, PR-01-03, PR-01-04 and PR-03-05, among others. We also thank Mrs. M. Carmen Arroyo, the staff at the Regional Laboratory of La Grajera (La Rioja), the staff at the Viticulture and Oenology Section (SIDTA-ICVV), and the vine growers who helped develop the database.