Properties of Calcium and Potassium Currents of Clonal Adrenocortical Cells

The ionic currents of clonal Y-1 adrenocortical cells were studied using the whole-cell variant of the patch-clamp technique. These cells had two major current components: a large outward current carried by K ions, and a small inward Ca current. The Ca current depended on the activity of two populations of Ca channels, slow (SD) and fast (FD) deactivating, that could be separated by their different closing time constants (at -80 mV, SD, 3.8 ms, and FD, 0.13 ms). These two kinds of channels also differed in (a) activation threshold (SD, approximately -50 mV; FD, approximately -20 mV), (b) half-maximal activation (SD, between -15 and -10 mV; FD between +10 and +15 mV), and (c) inactivation time course (SD, fast; FD, slow). The total amplitude of the Ca current and the proportion of SD and FD channels varied from cell to cell. The amplitude of the K current was strongly dependent on the internal [Ca2+] and was almost abolished when internal [Ca2+] was less than 0.001 microM. The K current appeared to be independent, or only slightly dependent, of Ca influx. With an internal [Ca2+] of 0.1 microM, the activation threshold was -20 mV, and at +40 mV the half-time of activation was 9 ms. With 73 mM external K the closing time constant at -70 mV was approximately 3 ms. The outward current was also modulated by internal pH and Mg. At a constant pCa gamma a decrease of pH reduced the current amplitude, whereas the activation kinetics were not much altered. Removal of internal Mg produced a drastic decrease in the amplitude of the Ca-activated K current. It was also found that with internal [Ca2+] over 0.1 microM the K current underwent a time-dependent transformation characterized by a large increase in amplitude and in activation kinetics

CSPα, a molecular co-chaperone essential for short and long-term synaptic maintenance

Cysteine string protein a (CSPα) is a vesicle protein located in the presynaptic terminal of most synapses. CSPα is an essential molecular co-chaperone that facilitates the correct folding of proteins and the assembly of the exocytic machinery. The absence of this protein leads to altered neurotransmitter release and neurodegeneration in multiple model systems, from flies to mice. In humans, CSPα mutations are associated with the development of neuronal ceroid lipofuscinosis (NCL), a neurodegenerative disease characterized by intracellular accumulation of lysosomal material. Here, we review the physiological role of CSPα and the pathology resulting from the homozygous deletion of the gene or its mutations. In addition, we investigate whether long-term moderate reduction of the protein produces motor dysfunction. We found that 1-year-old CSPα heterozygous mice display a reduced ability to sustain motor unit recruitment during repetitive stimulation, which indicates that physiological levels of CSPα are required for normal neuromuscular responses in mice and, likely, in humans.Ministerio de Ciencia e Innovación BFU2013–43763-

Carbonic anhydrase inhibitor acetazolamide shifts synaptic vesicle recycling to a fast mode at the mouse neuromuscular junction

Acetazolamide (AZ), a molecule frequently used to treat different neurological syndromes, is an inhibitor of the carbonic anhydrase (CA), an enzyme that regulates pH inside and outside cells. We combined fluorescent FM styryl dyes and electrophysiological techniques at ex vivo levator auris longus neuromuscular junctions (NMJs) from mice to investigate the modulation of synaptic transmission and vesicle recycling by AZ. Transmitter release was minimally affected by AZ, as evidenced by evoked and spontaneous end-plate potential measurements. However, optical evaluation with FM-styryl dyes of vesicle exocytosis elicited by 50 Hz stimuli showed a strong reduction in fluorescence loss in AZ treated NMJ, an effect that was abolished by bathing the NMJ in Hepes. The remaining dye was quenched by bromophenol, a small molecule capable of diffusing inside vesicles. Furthermore, in transgenic mice expressing Synaptophysin-pHluorin (SypHy), the fluorescence responses of motor nerve terminals to a 50 Hz train of stimuli was decrease to a 50% of controls in the presence of AZ. Immunohistochemistry experiments to evaluate the state of the Myosin light chain kinase (MLCK), an enzyme involved in vesicle recycling, demonstrated that MLCK phosphorylation was much stronger in the presence than AZ than in its absence in 50 Hz stimulated NMJs. We postulate that AZ, via cytosol acidification and activation of MLCK, shifts synaptic vesicle recycling to a fast (kiss-and-run) mode, which changes synaptic performance. These changes may contribute to the therapeutic action reported in many neurological syndromes like ataxia, epilepsy, and migraine.Fil: Bertone Cueto, Nicolás Iván. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Groisman, Ayelén Ivana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Mazzone, Graciela Luján. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Cano, Raquel. Universidad de Sevilla; EspañaFil: Tabares, Lucía. Universidad de Sevilla; EspañaFil: Uchitel, Osvaldo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentin

Smn-deficiency increases the intrinsic excitability of motoneurons

During development, motoneurons experience significant changes in their size and in the number and strength of connections that they receive, which requires adaptive changes in their passive and active electrical properties. Even after reaching maturity, motoneurons continue to adjust their intrinsic excitability and synaptic activity for proper functioning of the sensorimotor circuit in accordance with physiological demands. Likewise, if some elements of the circuit become dysfunctional, the system tries to compensate for the alterations to maintain appropriate function. In Spinal Muscular Atrophy (SMA), a severe motor disease, spinal motoneurons receive less excitation from glutamatergic sensory fibers and interneurons and are electrically hyperexcitable. Currently, the origin and relationship among these alterations are not completely established. In this study, we investigated whether Survival of Motor Neuron (SMN), the ubiquitous protein defective in SMA, regulates the excitability of motoneurons before and after the establishment of the synaptic contacts. To this end, we performed patch-clamp recordings in embryonic spinal motoneurons forming complex synaptic networks in primary cultures, and in differentiated NSC-34 motoneuron-like cells in the absence of synaptic contacts. Our results show that in both conditions, Smn-deficient cells displayed lower action potential threshold, greater action potential amplitudes, and larger density of voltage-dependent sodium currents than cells with normal Smn-levels. These results indicate that Smn participates in the regulation of the cell-autonomous excitability of motoneurons at an early stage of development. This finding may contribute to a better understanding of motoneuron excitability in SMA during the development of the disease.This study was supported by Fundación Tatiana Pérez de Guzmán el Bueno, the Spanish Ministry of Science and Innovation/FEDER (BFU2013-43763-P and BFU2016-78934-P), Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI14/00060), Unión Europea, Fondo Europeo de Desarrollo Regional (FEDER) ‘‘Una manera de hacer Europa’’ and Generalitat de Catalunya (SGR740)

Streamlined method for parallel identification of single domain antibodies to membrane receptors on whole cells

Background Owing to their minimal size, high production yield, versatility and robustness, the recombinant variable domains (nanobodies) of camelid single chain antibodies are valued affinity reagents for research, diagnostic, and therapeutic applications. While their preparation against purified antigens is straightforward, the generation of nanobodies to difficult targets such as multi-pass or complex membrane cell receptors remains challenging. Here we devised a platform for high throughput identification of nanobodies to cell receptor based on the use of a biotin handle. Methods Using a biotin-acceptor peptide tag, the in vivo biotinylation of nanobodies in 96 well culture blocks was optimized allowing their parallel analysis by flow cytometry and ELISA, and their direct use for pull-down/MS target identification. Results The potential of this strategy was demonstrated by the selection and characterization of panels of nanobodies to Mac-1 (CD11b/CD18), MHC II and the mouse Ly-5 leukocyte common antigen (CD45) receptors, from a VHH library obtained from a llama immunized with mouse bone marrow derived dendritic cells. By on and off switching of the addition of biotin, the method also allowed the epitope binning of the selected Nbs directly on cells. Conclusions This strategy streamlines the selection of potent nanobodies to complex antigens, and the selected nanobodies constitute ready-to-use biotinylated reagents. General significance This method will accelerate the discovery of nanobodies to cell membrane receptors which comprise the largest group of drug and analytical targets.Fil: Rossotti, Martín. Universidad de la República. Facultad de Química; UruguayFil: Tabares, Sofía. Universidad de la República. Facultad de Química; UruguayFil: Alfaya, Lucía. Universidad de la República. Facultad de Química; UruguayFil: Leizagoyen, Carmen. No especifíca;Fil: Moron, Victor Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: González Sapienza, Gualberto. Universidad de la República. Facultad de Química; Urugua

Calcium is reduced in presynaptic mitochondria of motor nerve terminals during neurotransmission in SMA mice

Spinal muscular atrophy (SMA) is an autosomal recessive degenerative motor neuron disease characterized by symmetrical muscle weakness and atrophy of limb and trunk muscles being the most severe genetic disease in children. In SMA mouse models, motor nerve terminals display neurotransmitter release reduction, endocytosis decrease and mitochondria alterations. The relationship between these changes is, however, not well understood. In the present study, we investigated whether the endocytosis impairment could be related to the functional alteration of the presynaptic mitochondria during action potential (AP) firing. To this aim, we generated a Synaptophysin-pHluorin (SypHy) transgenic mouse, crossed it with Taiwanese SMA mice, and recorded exo- and endocytosis and mitochondria Ca2+ signaling in real-time at ex vivo motor nerve terminals of Taiwanese-SypHy mice. The experiments were performed at the beginning of the motor symptoms to get an integrated view of the nerve terminal’s functional state before degeneration. Our electrophysiological and live imaging results demonstrated that the mitochondria’s capacity to increase matrix-free Ca2+ in SMA mice was significantly limited during nerve AP firing, except when the rate of Ca2+ entry to the cytosol was considerably reduced. These results indicate that both the mitochondrial Ca2+ signaling alterations and the secretion machinery defects are significant players in the dysfunction of the presynaptic terminal in SMA

Homicide mortality trend in seven of the main cities of Colombia between 1985 and 2010

La investigación que se realizó permitió determinar la tendencia real de la mortalidad en Colombia a causa del homicidio en un periodo de tiempo específico. Tuvo en cuenta los siguientes elementos: Se toma la definición de homicidio propuesta por el Centro para el Control y Prevención de Enfermedades (CDC por sus siglas en inglés) que considera a este como : “La muerte resultante del uso de fuerza física o poder, ya sea por amenaza o de hecho, contra otra persona, grupo o comunidad cuando una preponderancia de la evidencia indica que el uso de la fuerza fue intencional” La investigación se desarrolló con siete de las principales ciudades de Colombia, por tanto es importante describir las situaciones de violencia que inciden en los índices de mortalidad por homicidio. Colombia ha sido afectada notablemente por el número de homicidios que se atribuyen a la violencia ocasionada por el narcotráfico, la guerrilla, el paramilitarismo y las bacrim (bandas criminales emergentes), que paradójicamente oscilan sin control de manera alarmante; a través de los medios de comunicación se anuncian cifras de homicidio en Colombia que dejan al país con no muy buenas referencias frente al mundo. El homicidio genera un daño familiar y social, lo cual impacta sobre la salud pública, solo por mencionar algunos de estos daños: Alteración de la salud mental de la población. Desplazamiento forzado. Aumento en gastos económicos de un país. Aumento de la inseguridad en la población. Alteración en la dinámica familiar. Incremento del indicador de años de vida potencialmente perdidos de un país. Con los resultados obtenidos en ésta investigación se podrán formular y desarrollar acciones que permitan el control de éste evento en el país.INTRODUCCIÓN 2 1.OBJETIVOS 5 1.1 Objetivo general 5 1.2 Objetivos específicos 5 2. PREGUNTA DE INVESTIGACIÓN 6 3. MARCO TEORICO Y ESTADO DEL ARTE 7 4. DISEÑO METODOLOGICO 12 4.1 Tipo de estudio 12 4.2 Población de estudio 12 4.3 Tamaño de la muestra 12 4.4 Fuentes de información 12 4.5 Técnicas y procedimientos 13 4.5.1 Preparación de las bases de datos: 13 4.5.2 Construcción de las bases de datos: 13 4.5.3 Métodos de análisis 14 4.5.4 Herramientas de software 18 5. CONSIDERACIONES ETICAS 19 6. ANÁLISIS Y RESULTADOS 20 7. DISCUSIÓN 37 8. CONCLUSIONES 40 9. CRONOGRAMA 41 10. PRESUPUESTO 42EspecializaciónThe investigation that was carried out allowed us to determine the real trend of mortality in Colombia due to homicide in a specific period of time. It took into account the following elements: The definition of homicide proposed by the Center for Disease Control and Prevention (CDC) is taken, which considers it as: "Death resulting from the use of physical force or power, whether by threat or in fact, against another person, group or community when a preponderance of the evidence indicates that the use of force was intentional" The research was carried out with seven of the main cities in Colombia, therefore it is important to describe the situations of violence that affect mortality rates from homicide. Colombia has been notably affected by the number of homicides attributed to violence caused by drug trafficking, the guerrillas, paramilitaries and the bacrim (emerging criminal gangs), which paradoxically oscillate without control in an alarming way; Homicide figures in Colombia are announced through the media, leaving the country with not very good references to the world. Homicide generates family and social damage, which impacts public health, just to mention some of these damages: Alteration of the mental health of the population. Forced displacement. Increase in economic expenses of a country. Increase in insecurity in the population. Alteration in family dynamics. Increase in the indicator of years of life potentially lost in a country. With the results obtained in this investigation, actions can be formulated and developed that allow the control of this event in the country

Health and occupational risks due to pesticide handling among agricultural workers in marinilla, antioquia, 2009

ABSTRACT: The agricultural sector has played a key role in the development of Colombia’s economy and rural communities. The Marinilla municipality, located in the eastern area of the department of Antioquia, bases its economy in agriculture and livestock activities. This requires the use of a wide variety of chemical products that pose health risks. Objective: to describe the occupational and health risks of the farmers who use pesticides in Marinilla. Methodology: a descriptive study with multistage sampling and a sample of 157 farmers. Results: 64.9% of the farmers combine more than two chemicals. 83% of them have received no training on safe handling of agricultural chemical products. Approximately 80% of these individuals do not use the appropriate protective equipment to perform this task. Only 2.5 % of the farmers have occupational risks insurance. The most common disease was acute respiratory infection (78%). 22.3% have suffered from acute intoxication, and 14% (11) of the female farmers who have ever been intoxicated had abortions, premature births, or bore children with some kind of disability. A statistically significant difference was found between the age of farmers and pesticide usage time. No statistical difference was found between low schooling level and knowledge of the risks posed by chemical products. Conclusion: most agricultural workers have not been trained in safety standards; therefore, they disregard all kinds of safety measures during their work, and act without safety in mind and without personal hygiene habits.RESUMEN: El sector agrícola ha representado un importante polo de desarrollo para las comunidades campesinas y la economía colombiana. El municipio de Marinilla de la región del oriente de Antioquia fundamenta su economía en la actividad agropecuaria, actividad que obliga a la utilización de variados productos químicos cuyo empleo conlleva riesgos para la salud. Objetivo: describir los riesgos sanitarios y ocupacionales de los agricultores que usan plaguicidas en Marinilla. Metodología: estudio descriptivo y transversal, con muestreo polietápico y muestra de 157 agricultores. Resultados: 64,9% combinan más de dos químicos; 83% no ha recibido capacitación respecto del manejo seguro de agroquímicos; alrededor del 80% de los agricultores no utilizan equipo de protección adecuado para realizar la labor. Solo 2,5% están afiliados a riesgos profesionales y la morbilidad más frecuente fue infección respiratoria aguda (78%); 22,3% ha sufrido intoxicación aguda y 14% (11) de las mujeres de los campesinos que alguna vez se han intoxicado presentaron abortos, partos prematuros o engendraron niños con algún tipo de discapacidad. Se encontró diferencia estadística significativa entre la edad del agricultor y el tiempo de uso de plaguicida, y no se encontró diferencia estadística entre el bajo nivel educativo y el conocimiento de los riesgos de los químicos. Conclusión: los cultivadores no han sido capacitados en su mayoría y realizan su labor omitiendo todo tipo de normas de seguridad, con actos inseguros y sin uso de hábitos de higiene personal

Resignificación de la noción de fracción a través de los usos con estudiantes de 5° grado (entre los 10 y 12 años de edad)

La investigación parte del reconocimiento de la dificultad que representa la enseñanza y aprendizaje de la Noción de Fracción en el ámbito escolar, por los diferentes matices, significados, notaciones, representaciones y aplicaciones de las mismas. En este mismo sentido, es preciso recordar que ya ha pasado más de una década en la que se viene estudiando este objeto matemático. En la actualidad sigue presente dicha problemática y a pesar de que ya se han formalizado y tomado estudios para mejorar los procesos de enseñanza y aprendizaje, se hace necesario continuar indagando sobre las fracciones con nuevas investigaciones que aporten a la construcción de dicho conocimiento y su funcionalidad en diferentes contextos.The investigation takes place from the acknowledgement of the difficulty that represents the learning of the Notion of Fraction at a school level, with different meanings, notations, representations and applications from these. In additions, it is imperative to remember that it has been more than decade in which this topic has been studied. Nowadays, this problem continues to happen, despite the studies performed to improve the learning methods. It is vital to continue searching in depth about these fractions with new researches that potentially could contribute to the construction of this knowledge and functionality in different contexts.Magíster en EducaciónMaestrí