Name: preparation of quality inositol pyrophosphates
Uploaded: 2011-09-03T14:00:00
Duration: 10 min 34 s
Description: Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Damos las gracias a A. Riccio por sus valiosos comentarios y de leer el manuscrito. Este trabajo fue financiado por el Medical Research Council (MRC) de financiamiento a la Unidad de Biología Celular y por un ser humano Frontier Science Programa de Subvenciones (RGP0048/2009-C).

1. La reacción enzimática - día 1 (1 hora de la tarde) <ol><li> El primer paso es preparar a los 10-20 reacciones enzimáticas independientes en el que 6 de IP K1 o VIP1 convertir 6 de IP a las isoformas pyrophosphorylated. </li><li> Nosotros usamos su IP-6 K1 y las enzimas GST-VIP1 purificada de E. coli según el protocolo descrito anteriormente 17,18. </li><li> Preparar 50 l reacciones que contiene 1X tampón de reacción (30 mM Hepes pH 6,8, NaCl 50 mM, 6 mM MgSO 4, 1 mM DTT), 6 mM fosfocreatina (PCr), 25 U / mL creatinfosfoquinasa (CPK), 5 mM ATP (Mg sal), 0,3 mM IP6, 0,05-0,1 mg Su IP-6 K1 o GST VIP1. Ajustar el volumen con MiliQ ddH 2 O. </li><li> En pocas palabras girar la reacción y se incuba a 37 ° C durante la noche con la rotación. </li></ol> 2. Poliacrilamida fundición de gel y la carga - día 2 (4 horas por la tarde) <ol><li> El gel de poliacrilamida se prepara utilizando placas de 24 cm de largo, 18 cm de ancho y 1,5 de vidrio espaciadores mm de ancho. Por lo general, un carril de 16 o con un peine de preparación de un solo carril se utiliza. </li><li> Prepare una mezcla de (50 ml / gel) que contenga lo siguiente: 35,5% (w / v) acrilamida: bis-acrilamida 19:1, 1X Tris / Borato / EDTA (TBE), 0,05% (w / v) persulfato de amonio (APS ), un 0,05% (w / v) TEMED. Vierta la mezcla entre las placas de vidrio pre-fundido, insertar el peine y dejar polimerizar durante 30-60 minutos a temperatura ambiente. </li><li> Una vez que el gel se polimeriza, la transferencia del aparato a la sala de frío y pre-run en TBE 1X durante unos 30-60 minutos a 200-300 voltios. </li><li> Añadir 1X de OrangeG tinte (10 mM Tris-HCl pH 7,0, 1 mM EDTA, 30% de glicerol, 0,1 OrangeG%) a cada reacción. Prepare una muestra que contiene 2 nmol de 6 de IP para cargar en un control estándar. </li><li> Lave bien a fondo con cada funcionamiento de amortiguación con una jeringa y una aguja de 21G para eliminar cualquier precipitado, a continuación, cargar el gel. Evitar la colocación de los pozos secundarios. </li><li> Dejar correr el gel durante la noche a 450-550 voltios (7 mAmp / gel), hasta que la banda tinte OrangeG está dentro de los últimos 10 cm del fondo del gel. </li></ol> 3. El aislamiento de IP 7 - 3 días (4 horas) y el día 4 (07.06 horas SpeedVac proceso de secado) <ol><li> Desmontar el aparato del gel y retirar con cuidado una placa de vidrio dejando el gel en la otra. Cortar una porción pequeña del gel de poco más de la banda tinte OrangeG a la parte inferior que contiene el 6 de IP estándar y un carril de la muestra, como se muestra en la Figura 1. </li><li> Mancha la parte cortada del gel con azul de toluidina (0,1% (w / v) azul de toluidina, el 20% (w / v) de metanol, el 2% (w / v) de glicerol) por unos minutos (1-3 min) o hasta que la banda inositol pirofosfato parece. Coloque la placa de vidrio previamente eliminado de nuevo en la parte superior del gel para evitar que el gel se seque sin mancha. </li></ol> La IP de 7 bandas deben ser visibles, ya que corre más lento que el 6 de IP estándar. ATP, que se ejecuta más rápido que el 6 de IP, también debe ser visible (Figura 1). La transferencia de la parte manchada del gel en una solución de la tinción (20% (w / v) metanol) durante unos minutos, lavar el exceso de azul de toluidina y la posición del gel con el gel sin mancha. Si la visualización de mayor isoformas pyrophosphorylated inositol (IP IP 8 y 9) es necesario, manchar el gel con una solución de tinción con azul de toluidina durante 20 minutos a temperatura ambiente. Posteriormente, lavar el azul de toluidina con la solución de tinción de unos 15 minutos. <ol start="3"><li> Con una navaja cortar la IP de 7 bandas en la parte sin teñir el gel usando como referencia la posición de la migración de IP 7 determinado con el gel teñido (Figura 1). </li><li> Ponga la IP de 7 bandas que se cortó del gel en un tubo de 15 ml y agregar 10 ml de MilliQ ddH 2 O. Poner los tubos en rotación durante 10 minutos a temperatura ambiente. Deseche el líquido para eliminar el exceso de partículas de acrilamida TBE y microscópicas. </li><li> Posteriormente, realizar dos ciclos de hidratación-deshidratación. Añadir 5 ml de 50% (w / v) de metanol al tubo con el gel que contiene 7 IP y rotar a temperatura ambiente durante 2 horas. La transferencia de la porción de gel en un tubo de 15 ml nuevo que contiene 5 ml de MilliQ ddH2O y rotar a temperatura ambiente durante 2 horas. No deseche el metanol y MilliQ H 2 O de los tubos. Repita el ciclo de deshidratación-hidratación, una vez más por volver a transferir el gel de poliacrilamida en tubos de 15 ml utilizados anteriormente. Uno de los lavados se pueden realizar durante la noche. </li><li> Para concentrar la IP eluye 7, secar el de 10 ml junto (5 ml MilliQddH 2 O y 5 ml de metanol al 50% (w / v)) con un SpeedVac calienta a 60 ° C. </li><li> Una vez que las muestras son casi seco, transferir el líquido restante (300 a 600 l) a A1.5 tubo de centrífuga mL y girar durante 2 minutos a 5000 rpm. </li><li> Recoger el líquido sobrenadante y transferir a un nuevo tubo de centrífuga de 1.5 ml, dejarla parte inferior 20 a 30 l, ya que puede contener partículas de acrilamida. </li><li> Si es necesario continuar con el proceso de secado mediante un SpeedVac sin calefacción. La recuperación de la propiedad intelectual 7 se reduce drásticamente si las muestras se seque por completo, por lo tanto, terminar el proceso de secado cuando las muestras de alcanzar el volumen de 100-300 mL. </li></ol> 4. Determinación de la IP 7 de concentración y la pureza. <ol><li> 5.2 l uso de la IP se recuperó 7 muestra a correr en un gel de PAGE, de manera similar a las secciones 2.2-2.5. Cargar varias diluciones de 6 de IP (es decir, 0,5, 1, 2, 4 nmol) como estándar de concentración y 4 nmol de Poly-P marcador. Después de ejecutar el gel, visualizar las isoformas inositol pirofosfato de manchas y de-tinción del gel con una solución completa de toluidina, siguiendo el procedimiento descrito en la Sección 3.2 (Figura 2). </li><li> Después de la tinción de toluidina, la concentración se puede determinar mediante el análisis del gel y la comparación de las diferencias de intensidad entre IP 6 y 7 IP, utilizando el software de imagen como la imagen-J, como se muestra en la Figura 2B. </li></ol> 5. Los resultados representativos: La conversión de preparación enzimática de 6 de IP a IP 7 usando enzimas IP 6 K1 y VIP1 puede ser fácilmente resuelto mediante análisis PAGE (Figura 1). La carga de 6 de IP como un control de tamaño, junto con la tinción de toluidina azul gel permite la identificación de los derivados pyrophosphorylated, ya que correr más lento, dependiendo del número de grupos fosfatos presentes en el anillo de inositol. El procedimiento descrito anteriormente permite la purificación fácil de IP 7. El análisis del pirofosfato purificada inositol por PAGE reveló la pureza de nuestra IP 7 (Figura 2A). Curiosamente, el isómero 1/3PP-IP 5 de 7 productos IP de VIP1 desplaza ligeramente más lento que la 5PP-IP 5 isómero de IP 7, que es generado por la IP 6 K1. El uso de IP 6 normas permiten una sencilla cuantificación de la concentración de la propiedad intelectual purificada 7 (Figura 2B). Antes de utilizar 7 IP para nuevos experimentos, su actividad biológica puede ser evaluado (Figura 3). 5PP-IP 5 se incuba con VIP1 y con la IP 7 fosfatasa DDP1 (phosphohydrolase diphosphoinositol polifosfato). Rutinariamente, el IP purificada 7 se convierte en IP 8 por VIP1 y 6 de IP por DDP1 (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Figura 1:. Tinción de toluidina de PAGE y el aislamiento de la IP de 7 bandas La porción del gel que contiene la norma (IP 6) fue cortado y teñido con una solución de azul de toluidina. Las tres bandas representan (de arriba a abajo) IP 7, 6 de IP y ATP. La parte manchada del gel se alineó entonces con el resto del gel. Esto permite la localización de la porción del gel que contiene 7 IP, que puede ser cortado y purificado (cuadro de líneas discontinuas). <img alt="Figura 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Figura 2: PAGE análisis de productos de la reacción IP 6 K1 y VIP1. A) Análisis de las 6 de IP (4, 2, 1, 0,5 nmol) por tinción con azul de toluidina se utilizó para determinar la concentración de propiedad intelectual 7 purificada a partir de los 6 de IP K1 (5PP IP-5) y VIP1 (1/3PP-IP 5) las reacciones. B) Análisis Gráfico de dispersión para determinar la concentración de la propiedad intelectual purificada 7. Las concentraciones se determinaron de acuerdo a la intensidad de la banda, calculada utilizando el software ImageJ, en comparación con cantidades predeterminadas de 6 de IP. El eje X representa la intensidad, el eje Y representa la concentración expresada en nmol. <img alt="Figura 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Figura 3:. Análisis de IP 7 actividad biológica para determinar la calidad de la IP purificada siete se incubaron 5PP IP-5 (IP 6 K1 IP generadas 7) con VIP1 o con la IP 7 fosfatasa DDP1 y luego resolver la reacción en la página. El DAPI y toluidina reveló la producción esperada de 8 por VIP1 IP y la conversión de IP a IP 7 6 por DDP1.

<ol>
	<li>Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+metabolism+and+signaling.">Inositol pyrophosphates: metabolism and signaling.</a> Cell Mol Life Sci. 63, 552-564 (2006).</li><li>Burton, A., Hu, X., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+inositol+pyrophosphates+signalling+molecules?.">Are inositol pyrophosphates signalling molecules?.</a> J Cell Physiol. 220, 8-15 (2009).</li><li>Barker, C. J., Illies, C., Gaboardi, G. C., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+structure,+enzymology+and+function.">Inositol pyrophosphates: structure, enzymology and function.</a> Cell Mol Life Sci. 66, 3851-3871 (2009).</li><li>Shears, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphosphoinositol+polyphosphates:+metabolic+messengers.">Diphosphoinositol polyphosphates: metabolic messengers.</a> Mol Pharmacol. 76, 236-252 (2009).</li><li>Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+diphosphoinositol+pentakisphosphate+by+a+newly+identified+family+of+higher+inositol+polyphosphate+kinases.">Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.</a> Curr Biol. 9, 1323-1326 (1999).</li><li>Draskovic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+hexakisphosphate+kinase+products+contain+diphosphate+and+triphosphate+groups.">Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.</a> Chem Biol. 15, 274-286 (2008).</li><li>Mulugu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+enzymes+that+phosphorylate+inositol+hexakisphosphate.">A conserved family of enzymes that phosphorylate inositol hexakisphosphate.</a> Science. 316, 106-109 (2007).</li><li>Fridy, P. C., Otto, J. C., Dollins, D. E., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+two+human+VIP1-like+inositol+hexakisphosphate+and+diphosphoinositol+pentakisphosphate+kinases.">Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.</a> J Biol Chem. 282, 30754-30762 (2007).</li><li>Illies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+inositol+pyrophosphates+for+full+exocytotic+capacity+in+pancreatic+beta+cells.">Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.</a> Science. 318, 1299-1302 (2007).</li><li>Saiardi, A., Resnick, A. C., Snowman, A. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+cell+death+and+telomere+length+through+phosphoinositide+3-kinase-related+protein+kinases.">Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.</a> Proc Natl Acad Sci U S A. 102, 1911-1914 (2005).</li><li>York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+diphosphate+signaling+regulates+telomere+length.">Inositol diphosphate signaling regulates telomere length.</a> J Biol Chem. 280, 4264-4269 (2005).</li><li>Luo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+mediate+chemotaxis+in+Dictyostelium+via+pleckstrin+homology+domain-PtdIns(3,4,5)P3+interactions.">Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions.</a> Cell. 114, 559-572 (2003).</li><li>Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+endocytic+trafficking.">Inositol pyrophosphates regulate endocytic trafficking.</a> Proc Natl Acad Sci U S A. 99, 14206-14211 (2002).</li><li>Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plc1p,+Arg82p,+and+Kcs1p,+enzymes+involved+in+inositol+pyrophosphate+synthesis,+are+essential+for+phosphate+regulation+and+polyphosphate+accumulation+in+Saccharomyces+cerevisiae.">Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.</a> J Biol Chem. 280, 25127-25133 (2005).</li><li>Azevedo, C., Burton, A., Ruiz-Mateos, E., Marsh, M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphate+mediated+pyrophosphorylation+of+AP3B1+regulates+HIV-1+Gag+release.">Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.</a> Proc Natl Acad Sci U S A. 106, 21161-21166 (2009).</li><li>Bhandari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+pyrophosphorylation+by+inositol+pyrophosphates+is+a+posttranslational+event.">Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.</a> Proc Natl Acad Sci U S A. 104, 15305-15310 (2007).</li><li>Azevedo, C., Burton, A., Bennett, M., Onnebo, S. M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+InsP7+by+the+Inositol+Hexakisphosphate+Kinase+1+(IP6K1).">Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).</a> Methods Mol Biol. 645, 73-85 (2010).</li><li>Otto, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+inositol+phosphate+kinases.">Biochemical analysis of inositol phosphate kinases.</a> Methods Enzymol. 434, 171-185 (2007).</li><li>Losito, O., Szijgyarto, Z., Resnick, A. C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

No hay conflictos de interés declarado.

El uso de pirofosfato de inositol en la bioquímica está severamente limitada por la falta de disponibilidad comercial de estos compuestos y la escasa sensibilidad de los métodos de detección existentes. La combinación de la página, que permite la separación de las moléculas que poseen distinto número de grupos fosfato, y el azul de toluidina (Figura 1), un colorante metacromática que se une a los grupos de fosfatos, permite la fácil detección de las isoformas de pyrophoshate inositol apertura de nuevas vías de investigación 20. El uso de la tecnología descrita PAGE para purificar productos inositol pirofosfato de la reacción enzimática llevado outby ya sea IP 6 K1 o VIP1 es un método sencillo, económico y fiable que permite la producción de grandes cantidades de IP de alta calidad 7. El método descrito anteriormente no se limita a la simple purificación de IP 7, pero pequeñas modificaciones del protocolo descrito podría permitir la purificación de un rango diferente de pirofosfatos de inositol. Isoformas inositol fosforilado mayor pirofosfato, que contiene más de ocho grupos de fosfato pueden ser detectados utilizando IP 7 o diferentes cantidades de 6 de IP como un sustrato de 20,6. Estos pirofosfatos inositol puede ser detectado por el aumento de la duración del procedimiento de tinción y posteriormente purificado (sección 3.2). Por otra parte, el uso de IP 5 como sustrato para la reacción enzimática que permite la purificación del PP-5 de IP y otros pirofosfatos inositol que contiene un grupo hidroxilo en el anillo de inositol. En conclusión, este método permite que poco exigente para la purificación confiable de cantidades de miligramos de pirofosfatos inositol con instrumentos ampliamente disponible, lo que abre nuevas vías para este campo de investigación apasionante.

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th></tr><tr><td> El ácido fítico (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poli-P (hexametafosfato de sodio) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + sal </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> La fosfocreatina (PCr) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> CreatinePhospho quinasa (CPK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-VIP1 </td><td> 17 </td><td> 17 </td></tr><tr><td> Su-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> Su-Ddp1 </td><td> Disponibles en el laboratorio </td><td> Disponibles en el laboratorio </td></tr><tr><td> Acrilamida: bis-acrilamida 19:1 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Persulfato de amonio (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / Borato / EDTA (TBE) </td><td> Sigma-Aldrich </td><td> T 9060 </td></tr><tr><td> TEMED </td><td> BDH </td><td> 43083G </td></tr><tr><td> Azul de toluidina </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Cristo </td><td> 100218 </td></tr><tr><td> Gel aparato </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Vacío en el colector </td><td> Cristo </td><td> Alfa 2-4 </td></tr><tr><td> Bomba de vacío </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol está presente en la naturaleza ya sea modificado o en derivados más complejos fosforilada. De los últimos, los más abundantes en las células eucariotas son dos pentaquifosfato inositol (IP 5) y hexakisfosfato inositol (ácido fítico o IP 6). IP 5 y 6 de IP son los precursores de las moléculas de pirofosfato de inositol que contienen uno o más bonos de un pirofosfato. La fosforilación de 6 de IP genera diphoshoinositolpentakisphosphate (IP 7 o 5 PP-PI) y bisdiphoshoinositoltetrakisphosphate (IP 8 o (PP) 2-IP 4). Pirofosfatos inositol se han aislado de todos los organismos eucariotas estudiados hasta ahora. Además, las dos clases distintas de las enzimas responsables de la síntesis de pirofosfato inositol son altamente conservado durante la evolución 2-4. El 6 de IP quinasas (IP 6 Ks) posee una flexibilidad enorme catalizador, la conversión de IP 5 y 6 de IP a IP PP-4 y 7, respectivamente, IP y, posteriormente, mediante el uso de estos productos como sustratos, promover la generación de moléculas más complejas 5,6 . Recientemente, una segunda clase de enzimas generadoras de pirofosfato fue identificado en la forma de la proteína de levadura VIP 1 (también conocido como PP-IP 5 K), que es capaz de convertir 6 de IP a IP e IP 7 8 7,8. Pirofosfatos inositol regulan muchos procesos celulares diferentes, tales como la secreción de insulina 9, 10,11 longitud de los telómeros, la quimiotaxis 12, el tráfico vesicular 13, 14 y homeostasis del VIH-1 gag versión 15. Dos mecanismos de acción han sido propuestos para esta clase de moléculas. Pueden afectar a la función celular por alostéricamente interactuar con proteínas específicas como AKT 16. Por otra parte, el grupo pirofosfato puede donar un grupo fosfato a la pre-fosforilados proteínas 17. El enorme potencial de este campo de investigación se ve obstaculizada por la ausencia de una fuente comercial de pirofosfatos de inositol, que está impidiendo que muchos científicos del estudio de estas moléculas y esta nueva modificación después de la traducción. Los métodos actualmente disponibles para aislar pirofosfatos inositol requieren sofisticados aparatos de cromatografía 18,19. Estos procedimientos utilizan condiciones ácidas que pueden conducir a la degradación del pirofosfato de inositol 20 y por lo tanto a una mala recuperación. Además, el engorroso columna post-procedimientos de desalinización restringir su uso a laboratorios especializados. En este estudio se describe un método poco exigente para la generación, el aislamiento y la purificación de los productos de las 6 de IP-cinasa y PP-IP reacciones 5-quinasas. Este método fue posible gracias a la capacidad de la electroforesis en gel de poliacrilamida (PAGE) para resolver los polifosfatos inositol altamente fosforilada 20. Después de 6 de IP K1 y PP-IP 5 K reacciones enzimáticas con 6 de IP como el sustrato, PAGE se utiliza para separar los pirofosfatos genera inositol que se eluyen posteriormente en el agua.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

Pirofosfatos inositol juegan un papel importante en patologías humanas del cáncer, la diabetes y la obesidad, sin embargo, el mecanismo exacto de acción es un asunto de disputa. La falta de pirofosfatos inositol disponibles en el mercado hace que los estudios detallados problemática. A continuación se describe un protocolo sencillo para producir y aislar miligramos de pirofosfatos de inositol.

Preparación de la Calidad pirofosfatos Inositol

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Research

JoVE Journal

Biology

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarrays de ADN: Control de calidad de la muestra, la hibridación de la matriz y de escaneo

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Investigación

Biología

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

Un cabello no invasiva técnica de muestreo para obtener ADN de alta calidad de esquiva Pequeños Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Qué hacer y qué no hacer de crio-microscopía electrónica: Una introducción a la preparación de muestras y de alta calidad de recopilación de datos para la Reconstrucción de Macromoléculas 3D

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

Una herramienta web para los procesos biológicos Generación de alta calidad legibles por máquina

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Recogida de esperma de calidad diferencial mediante centrifugación de gradiente de densidad

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Identificación de campo de Matricaria chamomilla utilizando un sistema qPCR portátil

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Un ensayo de quimiotaxis mejorado para la identificación rápida de quimioatrayentes rizobacterianos en exudados radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

El cultivo, crecimiento y viabilidad de las bacterias del ácido láctico: una perspectiva de control de calidad

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

Optimización de la calidad de los productos probióticos secados por pulverización mediante el desarrollo de procesos

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

Autor destacado: Desarrollo de procesos para el secado por pulverización de bacterias probióticas y evaluación de la calidad del producto  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Desarrollo de procesos para el secado por atomización de bacterias probióticas y evaluación de la calidad del producto

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...