Name: a method for extracting pigments from squid doryteuthis pealeii
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The authors gratefully acknowledge the use of facilities at the University of New Hampshire including the University Instrumentation Center. This work was supported by the University of New Hampshire, Department of Chemistry.

los estudios en animales invertebrados realizadas en este documento no están regulados en los Estados Unidos; Por lo tanto, el Comité de Cuidado y Uso de Animales institucional no tiene autoridad para la revisión de dichos protocolos. En lugar de estos no cae bajo la jurisdicción de la regulación en los Estados Unidos, los autores declaran que estos estudios fueron realizados con un esfuerzo sincero hacia el uso ético, el cuidado y el tratamiento de estos animales, se redujo al mínimo el número de individuos y estos esfuerzos son consistentes con la Declaración de Basilea y el Consejo Internacional para la Ciencia Animal de Laboratorio (ICLAS) las directrices éticas. 1. Doryteuthis pealeii (pealeii D.) La disección <ol><li> Compra de calamar decapitado D. pealeii del Departamento de Recursos Marinos en el Laboratorio de Biología Marina en Woods Hole, MA o de cualquier mercado de pescado fresco. No utilice muestras que han sido previamente fileteados o alterados con el fin de aplicar este procedimiento. Específicamente, el cromacapa tophore todavía debe estar intacto en la muestra. <ol><li> Si los animales no se utilizan inmediatamente, almacenar a -20 ° C hasta su uso. Para descongelar las muestras, colocarlos en agua a temperatura ambiente durante 1 hora antes de la disección. </li></ol></li><li> El uso de acero inoxidable tijeras de disección de punta fina rectas, cortadas a lo largo de la parte posterior del animal. Comience en la base del manto ventral, y terminará en la región de la aleta. </li><li> Eliminar todos los órganos internos (estómago, las branquias, glándula de tinta, órganos reproductivos, corazón sistémico, y el corazón braquial) levantándolas con pinzas de disección y la ruptura de su tejido conjuntivo con tijeras de disección. Desechar los órganos en una bolsa de muestras. </li><li> Utilice la disección de T-alfileres para fijar el manto (lado de las aletas hacia arriba) en un estándar de 11-1 / 2 &quot;x 7-1 / 2&quot; x 1-1 / 2 &quot;pan disección de aluminio que contiene una almohadilla de disección flexible. Sumergir el tejido en 250 ml de agua de mar que se ha filtrado a través de un sistema de filtración a vacío desechable de poliestirenoque contiene 0,2 micras tamaños de poro. </li><li> Retirar con cuidado la capa epidérmica de la piel (color opaco) con pinza de disección, manteniendo intacta la capa de piel que contiene chromatophore. Una vez que se retira la capa epidérmica, desprenderse de ella en una bolsa de muestras. </li><li> Diseccionar pequeños (1 cm x 1 cm) de las secciones de la capa de chromatophore con unas pinzas y tijeras de disección. Recoger muestras en tubos de 1,5 ml de microcentrífuga (llene la mitad de cada tubo de vacío con el tejido diseccionado). </li><li> Añadir 500 l de agua de mar filtrada al tejido que contiene tubos. Las muestras se pueden almacenar durante la noche a 4 ° C. </li></ol> 2. Aislamiento de chromatophore pigmento gránulos <ol><li> La digestión del tejido <ol><li> Preparación de papaína / colagenasa <ol><li> Pesar 30 mg de colagenasa y 5 mg de papaína utilizando una &quot;x 4&quot; bajo nitrógeno 4 Documento de pesar. Transferirlas a un tubo de centrífuga de polipropileno de 50 ml con tapón de rosca. </li><li> Medir 10 ml de deionized agua usando una pipeta graduada. Añadir directamente al vial que contiene papaína / colagenasa. Vórtice a la máxima temperatura hasta que la solución es clara. </li></ol></li><li> La eliminación de tejido extracelular <ol><li> Centrifugar el tejido chromatophore recogido de la etapa 1.7 durante 5 minutos a 14.000 x g. Desechar la capa superior de agua de mar en un cubo de residuos plásticos. </li><li> Añadir 500 l de la solución de papaína / colagenasa al tejido usando una micropipeta de volumen variable solo canal P1000. Vortex cada muestra durante 1-2 minutos en la posición más alta. </li><li> Sonicar las muestras durante 5 min a una frecuencia de 40 kHz para disociar las células del tejido circundante. </li><li> Centrifugar durante 5 min a 14.000 x g. Descartar el sobrenadante en un cubo de residuos plásticos. </li><li> Centrifugar el tejido chromatophore recogido durante 5 min a 14.000 x g. Desechar la capa superior de agua de mar en un cubo de residuos plásticos. Repetir una vez más. </li><li> quitar manualmente cualquier tejido seg gran restantelas que no digieren en este proceso con unas pinzas antes de proceder al siguiente paso. </li></ol></li></ol></li><li> El aislamiento del pigmento gránulos <ol><li> Preparación de tampón de homogeneización <ol><li> Pesar 2,38 g de (4- (2-hidroxietil) -1-piperazinetanosulfónico) (HEPES, 100 mM), 0,095 g de cloruro de magnesio (10 mM), 0,856 g de potasio-aspartato (50 mM), y 0,015 g de ditiotreitol (1 mM). Transferir a un recipiente de poliestireno 150 ml con un tapón de rosca. Añadir un inhibidor de la proteasa de mini tableta comercial. </li><li> Medir 100 ml de agua desionizada usando un cilindro graduado. Añadir directamente al recipiente que contiene las sales de homogeneización. Vortex hasta que la solución es clara. </li></ol></li><li> La homogeneización de gránulos de pigmento <ol><li> Centrifugar cada muestra de la etapa 2.1 a 14.000 xg durante 5 min. Descartar el sobrenadante en un cubo de residuos plásticos. Esta muestra debe ser nula de tejido extracelular. </li><li> Añadir 500 y# 181; l de tampón de homogeneización a cada tubo y agitar durante 1-2 minutos cada uno para mezclar bien las muestras. Sonicar las muestras para 30 min (~ 40 kHz) y seguir con 5 min de centrifugación a 14.000 x g. </li><li> Descartar el sobrenadante en un cubo de residuos de plástico, y repetir el paso anterior 2-3 veces para asegurar la digestión completa de las correas de sujeción sáculo chromatophore y proteínas. La solución restante debe contener un sobrenadante de color amarillo claro y un sedimento de color rojo que contiene los gránulos de pigmento aisladas. </li></ol></li></ol></li></ol> 3. Pigmento Extracción <ol><li> Preparación de metanol ácido (HCl-MeOH) <ol><li> Añadir cuidadosamente 25 l de concentrado (36,5 a 38% (w / w)) de ácido clorhídrico (HCl) a 5 ml de metanol (MeOH). Almacenar la solución de HCl-MeOH en un vial de muestra de vidrio tapón de rosca. Agitar suavemente para mezclar uniformemente la solución. NOTA: La más ácida la solución, la más pigmento se extrajo durante la fiextracción de primera. </li></ol></li><li> Extracción de Pigmento <ol><li> Centrifugar la suspensión de gránulos de pigmento (de 2.2.2) durante 5 min a 14.000 xg y descartar el sobrenadante en un cubo de residuos de plástico. </li><li> Añadir 500 l de la solución de HCl-MeOH y agitar cada muestra para ~ 3-4 min. Sonicar las muestras para 10 min (~ 40 kHz), centrifugar durante 5 min a 14.000 x g. </li><li> Usando una pipeta de transferencia, recoger el sobrenadante en un vial de vidrio con tapa de rosca 1 dram. El sobrenadante debería ser de color rojo oscuro. </li><li> 3.2.2 Repetir la etapa de extracción hasta que ya no se extrae el color (~ 4-5 lavados). NOTA: Cada extracción producirá ~ 1,5 ml de pigmento. El sedimento restante es incoloro los gránulos de pigmento extraído. NOTA: Los dos gránulos incoloros y pigmentos extraídos se pueden almacenar en agua a 4 ° C hasta su uso posterior. </li></ol></li></ol> 4. Caracterización largo proceso de extracción <ol><li> El Escaneoectron Microscopía <ol><li> La imagen de gránulos (sin reaccionar de la etapa 2.2.1 y pigmento extraído de la etapa 3.2.3) usando microscopía electrónica de barrido (SEM) para verificar la pureza del proceso de extracción. </li><li> Para preparar las muestras de los gránulos para formación de imágenes, primero centrífuga tanto gránulos que no han reaccionado y los gránulos de pigmento se extrajo durante 5 minutos a 14.000 xg, y descartar el sobrenadante en un cubo de residuos de plástico. Vuelva a suspender estos en 1 ml de etanol, y depósito de 2-3 gotas de la suspensión de gránulos Onto trozos de aluminio SEM utilizando una pipeta de transferencia. </li><li> Después de secar las muestras durante la noche, revestir con recubrimiento de oro-platino durante 3 minutos para lograr un revestimiento de 15 nm. </li><li> Imagen utilizando un SEM con un emisor Schottky y un detector de electrones secundarios. Utilice un voltaje del haz de 5-7 kV y una distancia de trabajo entre 8 y 9 mm a imagen de estos materiales. </li></ol></li><li> Las mediciones espectrofotométricas ultravioleta-visible (UV-VIS) <ol><li> Supervisar el pro absorbanciaarchivos para las 3 condiciones (gránulo sin reaccionar de 2.2.2, 3.2.3 pigmento extraído de, y pigmentos extraídos de gránulos 3.2.4) utilizando un espectrofotómetro de doble haz de 200-800 nm. </li><li> Se recoge una medida en blanco usando agua desionizada en una cubeta de 1 ml para los gránulos sin reaccionar y los gránulos de pigmento extraído. Recoger los espectros de absorción UV-Vis para ambas muestras. </li><li> Se recoge una medida en blanco utilizando la solución de metanol ácido en una cubeta de 1 ml para el pigmento extraído. Recoger espectro de absorción UV-Vis para la muestra. </li><li> Determinar la máxima longitud de onda de cada muestra mediante la identificación de la longitud de onda a la que el pico de absorbancia alcanza su altura máxima relativa. </li></ol></li></ol>

<ol>
	<li>Bell, G. R. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatophore+radial+muscle+fibers+anchor+in+flexible+squid+skin.">Chromatophore radial muscle fibers anchor in flexible squid skin.</a> Invertebr Biol. 132 (2), 120-132 (2013).</li><li>Crookes, W. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflectins:+The+unusual+proteins+of+squid+reflective+tissues.">Reflectins: The unusual proteins of squid reflective tissues.</a> Science. 303 (5655), 235-238 (2004).</li><li>Deravi, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure-function+relationships+of+a+natural+nanoscale+photonic+device+in+cuttlefish+chromatophores.">The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.</a> J. R. Soc. Interface. 11 (93), 1-9 (2014).</li><li>M&#228;thger, L. M., Denton, E. J., Marshall, N. J., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+behavioural+functions+of+structural+coloration+in+cephalopods.">Mechanisms and behavioural functions of structural coloration in cephalopods.</a> J. R. Soc. Interface. 6 (2), 149-163 (2009).</li><li>M&#228;thger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+basis+for+camouflaged+polarized+light+communication+in+squid.">Anatomical basis for camouflaged polarized light communication in squid.</a> Biol. Lett. 2 (4), 494-496 (2006).</li><li>M&#228;thger, L. M., Hanlon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Malleable+skin+coloration+in+cephalopods:+selective+reflectance,+transmission+and+absorbance+of+light+by+chromatophores+and+iridophores.">Malleable skin coloration in cephalopods: selective reflectance, transmission and absorbance of light by chromatophores and iridophores.</a> Cell Tissue Res. 329 (1), 179-186 (2007).</li><li>Cloney, R. A., Brocco, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatophore+Organs,+Reflector+Cells,+Iridocytes+and+Leucophores+in+Cephalopods.">Chromatophore Organs, Reflector Cells, Iridocytes and Leucophores in Cephalopods.</a> Amer. Zool. 23 (3), 581-592 (1983).</li><li>Hanlon, R. T., Messenger, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+coloration+in+young+cutttlefish+(Sepia+officinalis+L)-+The+morphology+and+development+of+body+patterns+and+their+relation+to+behaviour.">Adaptive coloration in young cutttlefish (Sepia officinalis L)- The morphology and development of body patterns and their relation to behaviour.</a> Philos Trans R Soc Lond B Biol Sci. 320 (1200), 437-487 (1988).</li><li>Sutherland, R. L., M&#228;thger, L. M., Hanlon, R. T., Urbas, A. M., Stone, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cephalopod+coloration+model.+II.+Multiple+layer+skin+effects.">Cephalopod coloration model. II. Multiple layer skin effects.</a> J. Opt. Soc. Am. 25 (8), 2044-2054 (2008).</li><li>Florey, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrastructure+and+function+of+cephalopod+chromatophores.">Ultrastructure and function of cephalopod chromatophores.</a> Amer. Zool. 9 (2), 429-442 (1969).</li><li>Florey, E., Kriebel, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+and+mechanical+responses+of+chromatophore+muscle+fibers+of+squid,+Loligo+opalescens,+to+nerve+stimulation+and+drugs.">Electrical and mechanical responses of chromatophore muscle fibers of squid, Loligo opalescens, to nerve stimulation and drugs.</a> Z. Vergl. Physiol. 65 (1), 98-130 (1969).</li><li>Williams, T. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contributions+of+Phenoxazone-Based+Pigments+to+the+Structure+and+Function+of+Nanostructured+Granules+in+Squid+Chromatophores.">Contributions of Phenoxazone-Based Pigments to the Structure and Function of Nanostructured Granules in Squid Chromatophores.</a> Langmuir. , (2016).</li><li>Nijhout, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ommochrome+pigmentation+of+the+linea+and+rosa+seasonal+forms+of+Precis+coenia+(Lepidoptera:+Nymphalidae).">Ommochrome pigmentation of the linea and rosa seasonal forms of Precis coenia (Lepidoptera: Nymphalidae).</a> Arch. Insect. Biochem. Physiol. 36 (3), 215-222 (1997).</li><li>Van Den Branden, C., Decleir, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Study+of+the+Chromatophore+Pigments+in+the+Skin+of+the+Cephalopod+Sepia+Officinalis.">A Study of the Chromatophore Pigments in the Skin of the Cephalopod Sepia Officinalis.</a> L. Biol. Jb. Dodonaea. 44 (2), 345-352 (1976).</li><li>Aubourg, S., Torres-Arreola, W., Trigo, M., Ezquerra-Brauer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Jumbo+Squid+Skin+Pigment+Extract+and+its+Antioxidant+Potential+ina+Marine+Oil+System.">Characterization of Jumbo Squid Skin Pigment Extract and its Antioxidant Potential ina Marine Oil System.</a> Eur. J. Lipid Sci. Technol. 118, (2016).</li></ol>

We have nothing to disclose.

Hemos demostrado un método para extraer pigmentos de gránulos cromatóforos calamar. Al dirigirse específicamente a los gránulos, nuestro objetivo es determinar su papel en la mediación de la coloración de adaptación. Este método difiere de informes anteriores diseñados para caracterizar los pigmentos de cefalópodos usando muestras de tejido a granel 14 o liofilizado piel 15. Mientras que este protocolo es eficaz en la extracción de pigmentos cromatóforos, s...

Cefalópodos como el calamar, la sepia, el pulpo y tienen la capacidad de alterar su apariencia dinámica para camuflar y señalización. 1-6 Esta capacidad es apoyado en parte por la expansión de área selectiva de órganos pigmentadas conocidas como cromatóforos. 4,7-9 Cromatóforos son actuadores blandos que contienen una red de gránulos de pigmento nanoestructurados confinados dentro de un sáculo cytoelastic que está anclado radialmente por las fibras musculares. 1,3 a medida que se accionan, cromatóforos expanden por 500% de la superficie presentado la distribución de los gránulos de todo el órgano. 3,7 10,11 Cuando esta acción es concertada a través de una serie de cromatóforos, se cambia la coloración general del animal. Aunque se sabe que los gránulos de pigmento contribuyen a este cambio de color, su composición sigue siendo desconocido. Se describe un procedimiento para aislar y purificar los pigmentos cromatóforos que pueden ser adaptados para futuros estudios de composición. 3,12 chromatophore tejido que contiene se cosecha a través de la extirpación cuidado del cefalópodo. Un proceso de la digestión y la homogeneización se utiliza a continuación para disociar el tejido circundante y separar las células cromatóforos. Los gránulos nanoestructurados son entonces aislado y purificado a partir de los restantes cromatóforos utilizando sonicación y centrifugación repetidas. Tras la purificación, los pigmentos se extraen de los gránulos en un proceso que es una adaptación de la extracción de color visible de alas de mariposa usando soluciones de metanol ácidos. Microscopía electrónica 13 de barrido (SEM) y espectrofotometría se utilizan para confirmar que los pigmentos cromatóforos se extraen con éxito utilizando este proceso. Este método describe el aislamiento de gránulos cromatóforos que se utiliza para explorar las contribuciones moleculares acoloration en cefalópodos. 12 extracciones de moléculas de animales enteros a menudo puede ser un proceso largo y tedioso. El objetivo es informar a los futuros investigadores de un protocolo eficaz y fácil para la adquisición de los pigmentos de los gránulos nanoestructurados en los cefalópodos.

<table><tbody><tr><td>Collagenase</td><td>Alfa Aesar</td><td>9001-12-1</td><td>No hazard</td></tr><tr><td>Dithiothreitol</td><td>Sigma-Aldrich</td><td>3482-12-3</td><td>Irritant, acute toxicity</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>7365-45-9</td><td>Mild irritant</td></tr><tr><td>Hydrochloric acid</td><td>EMD Chemicals</td><td>7647-01-0</td><td>Corrosive</td></tr><tr><td>k-Aspartate</td><td>Sigma-Aldrich</td><td>1115-63-5</td><td>Reacts violently with oxidants</td></tr><tr><td>Magnesium Chloride</td><td>Sigma-Aldrich</td><td>7786-30-3</td><td>Mild eye irritant</td></tr><tr><td>Methanol</td><td>Pharmco-AAPER</td><td>67-56-1</td><td>Highly flammable</td></tr><tr><td>Mini tablet prtoease inhibitor</td><td>Sigma-Aldrich</td><td>469315-90-01</td><td>Corrosive to metal and skin</td></tr><tr><td>Papain</td><td>Sigma-Aldrich</td><td>9001-73-4</td><td>Irritant&amp;nbsp;</td></tr></tbody></table>

a method for extracting pigments from squid doryteuthis pealeii

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities are supported in part by the areal expansion and retraction of pigmented organs known as chromatophores. While it is known that the chromatophores contain a tethered network of pigmented granules, their structure-function properties have not been fully detailed. We describe a method for isolating the nanostructured granules in squid Doryteuthis pealeii chromatophores and demonstrate how their associated pigments can be extracted in acidic solvents. To accomplish this, the chromatophore containing dermal layer is first manually isolated using a superficial dissection, and the pigment granules are removed using sonication, centrifugation, and washing cycles. Pigments confined within the purified granules are then extracted via acidic methanol solutions, leaving nanostructures with smaller diameters that are void of visible color. This extraction procedure produces a 58% yield of soluble pigments isolated from granules. Using this method, the composition of the chromatophore pigments can be determined and used to provide insight into the mechanism of adaptive coloration in cephalopods.

Cromatóforos se disecan de la D. pealeii dorsal manto (Figura 1A, 1B). Una vez que se quitan, cromatóforos se lisan y se purificaron usando los ciclos de centrifugación y lavado para aislar los gránulos pigmentados (Figuras 2A, 2B). Soluciones de metanol ácido (HCl-MeOH) se utilizan para extraer el pigmento de los gránulos (Figura 2C), produciendo un extracto de pigmento soluble y gránulo...

Watch this Scientific Journal Video about A Method for Extracting Pigments from Squid Doryteuthis pealeii at JoVE.com

A Method for Extracting Pigments from Squid Doryteuthis pealeii

Se presenta un protocolo para la extracción de pigmentos a partir de los gránulos nanoestructurados en Doryteuthis pealeii cromatóforos calamar.

Un método para extraer pigmentos de calamar<em&gt; Pealeii Doryteuthis</em

Cephalopods can undergo rapid and adaptive changes in dermal coloration for sensing, communication, defense, and reproduction purposes. These capabilities ...

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Research

JoVE Journal

Biology

A Method for Extracting Pigments from Squid Doryteuthis pealeii

9.3K vistas.  University of New Hampshire.  Se presenta un protocolo para la extracción de pigmentos a partir de los gránulos nanoestructurados en Doryteuthis pealeii cromatóforos calamar. 

Video: A Method for Extracting Pigments from Squid Doryteuthis pealeii

A Fish-feeding Laboratory Bioassay to Assess the Antipredatory Activity of Secondary Metabolites from the Tissues of Marine Organisms

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with the goal of examining the ecological functions of secondary metabolites from the tissues of marine organisms. The result has been a progression of protocols that have increasingly refined the ecological relevance of the experimental approach. Here we present the most up-to-date version of a fish-feeding laboratory bioassay that enables investigators to assess the antipredatory activity of secondary metabolites from the tissues of marine organisms. Organic metabolites of all polarities are exhaustively extracted from the tissue of the target organism and reconstituted at natural concentrations in a nutritionally appropriate food matrix. Experimental food pellets are presented to a generalist predator in laboratory feeding assays to assess the antipredatory activity of the extract. The procedure described herein uses the bluehead, Thalassoma bifasciatum, to test the palatability of Caribbean marine invertebrates; however, the design may be readily adapted to other systems. Results obtained using this laboratory assay are an important prelude to field experiments that rely on the feeding responses of a full complement of potential predators. Additionally, this bioassay can be used to direct the isolation of feeding-deterrent metabolites through bioassay-guided fractionation. This feeding bioassay has advanced our understanding of the factors that control the distribution and abundance of marine invertebrates on Caribbean coral reefs and may inform investigations in diverse fields of inquiry, including pharmacology, biotechnology, and evolutionary ecology.

This bioassay employs a model predatory fish to assess the presence of feeding-deterrent metabolites from organic extracts of the tissues of marine organisms at natural concentrations using a nutritionally comparable food matrix.

Un pez de amamantar Laboratorio Bioensayo para evaluar la actividad antidepredación de metabolitos secundarios de los tejidos de organismos marinos

Marine chemical ecology is a young discipline, having emerged from the collaboration of natural products chemists and marine ecologists in the 1980s with ...

Investigación

Medio ambiente

Quantifying Abdominal Pigmentation in Drosophila melanogaster

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for understanding the development and evolution of morphological phenotypes. Abdominal pigmentation in Drosophila melanogaster has been particularly useful, allowing researchers to identify the loci that underlie inter- and intraspecific variations in morphology. Hitherto, however, D. melanogaster abdominal pigmentation has been largely assayed qualitatively, through scoring, rather than quantitatively, which limits the forms of statistical analysis that can be applied to pigmentation data. This work describes a new methodology that allows for the quantification of various aspects of the abdominal pigmentation pattern of adult D. melanogaster. The protocol includes specimen mounting, image capture, data extraction, and analysis. All the software used for image capture and analysis feature macros written for open-source image analysis. The advantage of this approach is the ability to precisely measure pigmentation traits using a methodology that is highly reproducible across different imaging systems. While the technique has been used to measure variation in the tergal pigmentation patterns of adult D. melanogaster, the methodology is flexible and broadly applicable to pigmentation patterns in myriad different organisms.

Quantifying Abdominal Pigmentation in Drosophila melanogaster

This work presents a method to quickly and precisely quantify the abdominal pigmentation of Drosophila melanogaster using digital image analysis. This method streamlines the procedures between phenotype acquisition and data analysis and includes specimen mounting, image acquisition, pixel value extraction, and trait measurement.

Cuantificación de la Pigmentación Abdominal en<em&gt; Drosophila melanogaster</em

Pigmentation is a morphologically simple but highly variable trait that often has adaptive significance. It has served extensively as a model for ...

Genética

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

Emulsiones agua en aceite: Un nuevo sistema para el ensamblaje de las proteínas de unión de clorofila-solubles en agua con pigmentos hidrófobos

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Química

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy transfer processes in the thylakoid membrane of chloroplasts. The light harvesting antenna of diatoms, the so called fucoxanthin chlorophyll a/c binding proteins (FCP), are required for the light absorption and efficient transfer to the photosynthetic reaction centers as well as for photo-protection from excessive light. The switch between these two functions is a long-standing matter of research. Many of these studies have been carried out with FCP in detergent micelles. For interaction studies, the detergents have been removed, which led to an unspecific aggregation of FCP complexes. In this approach, it is hard to discriminate between artifacts and physiologically relevant data. Hence, more valuable information about FCP and other membrane bound light harvesting complexes can be obtained by studying protein-protein interactions, energy transfer and other spectroscopic features if they are embedded in their native lipid environment. The main advantage is that liposomes have a defined size and a defined lipid/protein ratio by which the extent of FCP clustering is controlled. Further, changes in the pH and ion composition that regulate light harvesting in vivo can easily be simulated. In comparison to the thylakoid membrane, the liposomes are more homogenous and less complex, which makes it easier to obtain and understand spectroscopic data. The protocol describes the procedure of FCP isolation and purification, liposome preparation, and incorporation of FCP into liposomes with natural lipid composition. Results from a typical application are given and discussed.

Isolating and Incorporating Light-Harvesting Antennas from Diatom Cyclotella Meneghiniana in Liposomes with Thylakoid Lipids

Here, we present a protocol to isolate fucoxanthin chlorophyll a/c binding proteins (FCP) from diatoms and incorporate them into liposomes with natural lipid compositions to study excitation energy transfer upon ion composition changes.

Aislamiento y la incorporación de recolección de luz las antenas de la diatomea Cyclotella Meneghiniana en liposomas con lípidos de tilacoides

The photosynthetic performance of plants, algae and diatoms strongly depends on the fast and efficient regulation of the light harvesting and energy ...

Bioquímica

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri (Uljanin 1884) for Experimental Studies

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, and trophic interactions primarily due to methodological challenges, including the ability to culture them. This is particularly true for the doliolid, Dolioletta gegenbauri. D. gegenbauri commonly occurs in productive subtropical continental shelf systems worldwide, often at bloom concentrations capable of consuming a large fraction of daily primary production. In this study, we describe cultivation approaches for collecting, rearing, and maintaining D. gegenbauri for the purpose of conducting laboratory-based studies. D. gegenbauri and other doliolid species can be captured live using obliquely towed conical 202 &micro;m mesh plankton nets from a drifting ship. Cultures are most reliably established when water temperatures are below 21 &deg;C and are started from immature gonozooids, maturing phorozooids, and large nurses. Cultures can be maintained in rounded culture vessels on a slowly rotating plankton wheel and sustained on a diet of cultured algae in natural seawater for many generations. In addition to the ability to establish laboratory cultures of D. gegenbauri, we demonstrate that the collection condition, algae concentration, temperature, and exposure to naturally conditioned seawater are all critical to the culture establishment, growth, survival, and reproduction of D. gegenbauri.

Cultivation of the Marine Pelagic Tunicate Dolioletta gegenbauri Uljanin 1884 for Experimental Studies

Doliolids, including the species Dolioletta gegenbauri, are small gelatinous marine zooplankton of ecological significance found on productive subcontinental shelf systems worldwide. The difficulty of culturing these delicate organisms limits their investigation. In this study, we describe cultivation approaches for collecting, rearing, and maintaining the doliolid Dolioletta gegenbauri.

Cultivo del Tenicato Pelágico Marino Dolioletta gegenbauri (Uljanin 1884) para Estudios Experimentales

Gelatinous zooplanktons play a crucial role in ocean ecosystems. However, it is generally difficult to investigate their physiology, growth, fecundity, ...

Large-Scale Purification of Porcine or Bovine Photoreceptor Outer Segments for Phagocytosis Assays on Retinal Pigment Epithelial Cells

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer segments (POS) can be performed in vitro. Photoreceptor outer segments with stacks of membranous discs containing the phototransduction machinery are continuously renewed in the retina. Spent POS are eliminated daily by RPE cells. Rodent, porcine/bovine and human RPE cells recognize POS from various species in a similar manner. To facilitate performing large series of experiments with little variability, a large stock of POS can be isolated from porcine eyes and stored frozen in aliquots. This protocol takes advantage of the characteristic of photopigments that display an orange color when kept in the dark. Under dim red light, retinae are collected in a buffer from opened eyecups cut in halves. The retinal cell suspension is homogenized, filtered and loaded onto a continuous sucrose gradient. After centrifugation, POS are located in a discrete band in the upper part of the gradient that has a characteristic orange color. POS are then collected, spun, resuspended sequentially in wash buffers, counted and aliquoted. POS obtained this way can be used for phagocytosis assays and analysis of protein activation, localization or interaction at various times after POS challenge. Alternatively, POS can be labeled with fluorophores, e.g., FITC, before aliquoting for subsequent fluorescence quantification of POS binding or engulfment. Other possible applications include the use of modified POS or POS challenge combined with stress conditions to study the effect of oxidative stress or aging on RPE cells.

This article describes the protocol for the purification of photoreceptor outer segment fragments (POS) via ultracentrifugation from porcine/bovine retinae using homogenization and sucrose gradient centrifugation. This protocol allows the preparation of large stocks of POS aliquots, labeled or unlabeled, that can then be stored at -80 &#176;C.

A gran escala de purificación de porcino o bovino fotorreceptor Outer Segmentos para Fagocitosis ensayos sobre células pigmento retiniano epitelial

Analysis of one of the vital functions of retinal pigment epithelial (RPE) cells, the phagocytosis of spent aged distal fragments of photoreceptor outer ...

Inmunología e Infección

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host's innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.

Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria

This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.

La obtención de hemocitos de la hawaiana Squid Bobtail Euprymna scolopes Y la observación de su adhesión a las bacterias simbióticas y no simbióticas-

Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made ...

Biología

Adsorptive Bioprocess Improves Yield of Melanin from Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as UV protective agents, skincare, cosmetics etc. A marine Pseudomonas stutzeri produces melanin without the addition of tyrosine. The feedback inhibition was observed by melanin in the culture of a melanin-producing marine bacterium, Pseudomonas stutzeri. Melanin also demonstrated microbial growth inhibition. The Han-Levenspiel model-based analysis identified uncompetitive type product inhibition of melanin on the cell growth. Tyrosinase enzyme, which produces melanin, was inhibited by melanin. The double reciprocal plot of the enzymatic reaction in the presence of different melanin concentrations revealed uncompetitive product inhibition. An adsorbent-based adsorptive bioprocess was developed to reduce the feedback inhibition by melanin. Different adsorbents were screened to select the best adsorbent for melanin adsorption. Dosage amount and time were optimized to develop the adsorptive bioprocess, which resulted in an 8.8-fold enhancement in melanin production by the marine bacteria Pseudomonas stutzeri (153 mg/L to 1349 mg/L) without supplementation of tyrosine and yeast extract.

<meta charset="utf8"></head><body>El bioproceso adsorbente mejora el rendimiento de melanina de Pseudomonas stutzeri

Here we present a protocol on adsorptive bioprocess to produce a biological product, especially a pigment that inhibits its own biosynthesis and inhibits the growth of the microorganism that produces it.

El bioproceso adsorbente mejora el rendimiento de melanina de Pseudomonas stutzeri

Melanins are natural pigments, and the presence of indole ring and numerous functional groups makes melanin an ideal choice for many applications such as ...

Ingeniería

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls and carotenoids. In vivo, these proteins are folded with pigments to form complexes which are inserted in the thylakoid membrane of the chloroplast. The high similarity in the chemical and physical properties of the members of the family, together with the fact that they can easily lose pigments during isolation, makes their purification in a native state challenging. An alternative approach to obtain homogeneous preparations of LHCs was developed by Plumley and Schmidt in 19871, who showed that it was possible to reconstitute these complexes in vitro starting from purified pigments and unfolded apoproteins, resulting in complexes with properties very similar to that of native complexes. This opened the way to the use of bacterial expressed recombinant proteins for in vitro reconstitution. The reconstitution method is powerful for various reasons: (1) pure preparations of individual complexes can be obtained, (2) pigment composition can be controlled to assess their contribution to structure and function, (3) recombinant proteins can be mutated to study the functional role of the individual residues (e.g., pigment binding sites) or protein domain (e.g., protein-protein interaction, folding). This method has been optimized in several laboratories and applied to most of the light-harvesting complexes. The protocol described here details the method of reconstituting light-harvesting complexes in vitro currently used in our laboratory, and examples describing applications of the method are provided.

In Vitro Reconstitution of Light-harvesting Complexes of Plants and Green Algae

This protocol details the reconstitution of light-harvesting complexes in vitro. These integral membrane proteins coordinate chlorophylls and carotenoids and are responsible for harvesting light in higher plants and green algae.

En Vitro Reconstitución de complejos captadores de luz de Plantas y Algas

In plants and green algae, light is captured by the light-harvesting complexes (LHCs), a family of integral membrane proteins that coordinate chlorophylls ...

Un método para extraer pigmentos de calamar

Resumen

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Cuantificación de la Pigmentación Abdominal en

Emulsiones agua en aceite: Un nuevo sistema para el ensamblaje de las proteínas de unión de clorofila-solubles en agua con pigmentos hidrófobos

Aislamiento y la incorporación de recolección de luz las antenas de la diatomea Cyclotella Meneghiniana en liposomas con lípidos de tilacoides

Cultivo del Tenicato Pelágico Marino Dolioletta gegenbauri (Uljanin 1884) para Estudios Experimentales

A gran escala de purificación de porcino o bovino fotorreceptor Outer Segmentos para Fagocitosis ensayos sobre células pigmento retiniano epitelial

La obtención de hemocitos de la hawaiana Squid Bobtail Euprymna scolopes Y la observación de su adhesión a las bacterias simbióticas y no simbióticas-

El bioproceso adsorbente mejora el rendimiento de melanina de Pseudomonas stutzeri

El bioproceso adsorbente mejora el rendimiento de melanina de Pseudomonas stutzeri

En Vitro Reconstitución de complejos captadores de luz de Plantas y Algas