Name: quantification of hypopigmentation activity in vitro
Uploaded: 2019-03-06T12:30:00
Description: Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Este trabajo fue apoyado por la Corea Instituto de planificación y evaluación de tecnología en alimentos, agricultura y silvicultura (IPET) a través del programa de desarrollo de tecnología de Agri-bioindustria, financiado por el Ministerio de agricultura, alimentación y asuntos rurales (MAFRA ) (116159-02-2-WT011) y escuela de Ciencias de la vida y biotecnología BK21 Plus, Universidad de Corea.

1. preparación del medio, compuestos y reactivos
<ol><li>Preparar el medio completo de crecimiento B16F10. Suplemento Dulbecco había modificado medio de águila (DMEM) con 10% suero bovino fetal (FBS) y 1% de penicilina/estreptomicina (plagas).<ol>
<li>Para hacer 500 mL de medio completo, mezclar 445 mL de medio de Eagle modificado de Dulbecco (DMEM) con 50 mL de SBF y 5 mL de plagas en una botella esterilizada de vidrio de 1 L. Después de mezclar suavemente, filtrar el medio a través de un filtro de 0.2 μm superi...

<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+melanogenesis+and+mechanisms+of+skin-lightening+agents+-+existing+and+new+approaches.">The melanogenesis and mechanisms of skin-lightening agents - existing and new approaches.</a> International Journal of Cosmetic Science. 33 (3), 210-221 (2011).</li><li>Passeron, T., Coelho, S. G., Miyamura, Y., Takahashi, K., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemistry+and+in+situ+hybridization+in+the+study+of+human+skin+melanocytes.">Immunohistochemistry and in situ hybridization in the study of human skin melanocytes.</a> Experimental Dermatology. 16 (3), 162-170 (2007).</li><li>Lee, J. 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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=Dual+inhibitions+of+lemon+balm+(Melissa+officinalis)+ethanolic+extract+on+melanogenesis+in+B16-F1+murine+melanocytes:+inhibition+of+tyrosinase+activity+and+its+gene+expression.">Dual inhibitions of lemon balm (Melissa officinalis) ethanolic extract on melanogenesis in B16-F1 murine melanocytes: inhibition of tyrosinase activity and its gene expression.</a> Food Science and Biotechnology. 20 (4), 1051-1059 (2011).</li><li>Jun, H. 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=p-Coumaric+acid+inhibition+of+CREB+phosphorylation+reduces+cellular+melanogenesis.">p-Coumaric acid inhibition of CREB phosphorylation reduces cellular melanogenesis.</a> European Food Research and Technology. 235 (6), 1207-1211 (2012).</li><li>Jun, H. 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=Dual+inhibition+of+&#947;-oryzanol+on+cellular+melanogenesis:+inhibition+of+tyrosinase+activity+and+reduction+of+melanogenic+gene+expression+by+a+protein+kinase+a-dependent+mechanism.">Dual inhibition of &#947;-oryzanol on cellular melanogenesis: inhibition of tyrosinase activity and reduction of melanogenic gene expression by a protein kinase a-dependent mechanism.</a> Journal of Natural Products. 75 (10), 1706-1711 (2012).</li><li>Choi, Y. M., 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=Effects+of+the+isoflavone+puerarin+and+its+glycosides+on+melanogenesis+in+B16+melanocytes.">Effects of the isoflavone puerarin and its glycosides on melanogenesis in B16 melanocytes.</a> European Food Research and Technology. 231 (1), 75-83 (2010).</li><li>Cho, B. R., Jun, H. J., Thach, T. T., Wu, C., Lee, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaine+reduces+cellular+melanin+content+via+suppression+of+microphthalmia-associated+transcription+factor+in+B16-F1+murine+melanocytes.">Betaine reduces cellular melanin content via suppression of microphthalmia-associated transcription factor in B16-F1 murine melanocytes.</a> Food Science and Biotechnology. 26 (5), 1391-1397 (2017).</li><li>Overwijk, W. W., Restifo, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=B16+as+a+mouse+model+for+human+melanoma.">B16 as a mouse model for human melanoma.</a> Current Protocols in Immunology. , (2001).</li><li>Virador, V. M., Kobayashi, N., Matsunaga, J., Hearing, V. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+standardized+protocol+for+assessing+regulators+of+pigmentation.">A standardized protocol for assessing regulators of pigmentation.</a> Analytical Biochemistry. 270 (2), 207-219 (1999).</li><li>D'Mello, S. A. N., Finlay, G. J., Baguley, B. C., Askarian-Amiri, 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=Signaling+pathways+in+melanogenesis.">Signaling pathways in melanogenesis.</a> International Journal of Molecular Sciences. 17 (7), 1144 (2016).</li><li>Hou, L., Panthier, J. J., Arnheiter, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+the+neural+crest-derived+melanocyte+lineage:+interactions+between+KIT+and+MITF.">Signaling and transcriptional regulation in the neural crest-derived melanocyte lineage: interactions between KIT and MITF.</a> Development. 127 (24), 5379-5389 (2000).</li><li>Hedley, S. J., Gawkrodger, D. J., Weetman, A. P., MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#945;-MSH+and+melanogenesis+in+normal+human+adult+melanocytes.">&#945;-MSH and melanogenesis in normal human adult melanocytes.</a> Pigment Cell Research. 11 (1), 45-56 (1998).</li><li>Hu, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodology+for+evaluation+of+melanin+content+and+production+of+pigment+cells+in+vitro.">Methodology for evaluation of melanin content and production of pigment cells in vitro.</a> Photochemistry and Photobiology. 84 (3), 645-649 (2008).</li><li>Carriel, V. 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Se han presentado protocolos para la evaluación de la actividad de hipopigmentación de prueba compuestos con melanocitos cultivados. Resultados representativos mostraron el efecto de hipopigmentación de la arbutina, un inhibidor de la tirosinasa que inhibió el contenido de melanina de celular y actividad de tirosinasa. Estos métodos son ampliamente utilizados en la investigación de actividad anti-melanogenic. Mediante estos ensayos, que con éxito hemos identificado varios compuestos bioactivos que tienen efectos i...

Melanina juega un papel fundamental en la fisiología, patología y toxicología de los varios órganos incluyendo la piel, ojos y cerebro1. Principales funciones de la melanina son efectos foto-proyección y bioquímicos. Melanina absorbe la luz infrarroja y visible, así como la radiación ultravioleta (UV), con tasas de aumento de la absorción en longitudes de onda más cortas de la luz; así, melanina protege los tejidos contra daños causados por la luz visible o UV radiación2. Melanina es un antioxidante y tiene una afinidad por los metales y otras sustancias químicas tóxicas; por lo tanto, puede proteger los tejidos del estrés oxidativo y química3. Sin embargo, la producción excesiva de melanina provoca problemas dermatológicos.
La cantidad y calidad de melanina en la piel y el iris son los más importantes determinantes del color del iris y la piel. Los individuos pueden tener preferencias de color de piel diferente; algunos favorecen la piel bronceada, mientras que otros favorecen colores de piel más claros. Dependiendo de estos perfiles de consumidor, hipopigmentación cosmética se han desarrollado para satisfacer mercados individuales de piel favorece colores4. En consecuencia, estudios de actividad hipopigmentación y anti-melanogenic son importantes científicamente y prácticamente.
Melanogénesis es el complejo proceso de biosíntesis de la melanina mediante una serie de reacciones químicas enzimáticas y espontáneas en los melanocitos. Un melanocito es rodeado por aproximadamente 36 queratinocitos y melanocitos son las fábricas de la síntesis de melanina que distribuyen su producto a los queratinocitos vecinos. En la piel, la melanina producida y almacenada en el compartimiento melanosomal de melanocitos es transportada a vecinos queratinocitos en las epidermis a través de las dendritas.
L-tirosina sirve como sustrato inicial para la melanogénesis y la tirosinasa enzima cataliza dos reacciones consecutivas que transforman la L-tirosina en 3, 4-dopa (DOPA) y luego en dopaquinona. Estas reacciones son el paso tarifa-limitador en la melanogénesis5,6. Por consiguiente, hipopigmentación actividad primero puede medirse determinando la actividad de la tirosinasa celular directamente. Para ello, extractos de melanocitos que contiene tirosinasa se incuban con la DOPA y la dopaquinona producida en las muestras se puede medir por espectrofotometría a 475 nm. Los valores se normalizan por las concentraciones de proteína de las muestras y sustancias con hipopigmentación actividad dan como resultado menos formación de dopaquinona en comparación con controles.
En segundo lugar, actividad de hipopigmentación puede cuantificarse midiendo directamente la melanina en los melanocitos cultivados. Después de tratar las células con el material de prueba, la melanina se extrae en condiciones alcalinas y el contenido de melanina se cuantifica por espectrofotometría a 400 nm. Un agente de la hipopigmentación se traducirá en un menor contenido de melanina que la de controles7.
Por último, la actividad de hipopigmentación puede cuantificarse por coloración de Fontana-Masson melanina y análisis de imagen posterior. En la tinción de Fontana-Masson, gránulos de la melanina reducen nitrato de amoníaco-plata en un visible estado metálico negro y las áreas negras de las células en los imágenes microscópicas representan la cantidad de melanina.
Un agente de hipopigmentación generalmente da resultados consistentes y comparables con estos tres métodos, que confirma que la actividad de la sustancia es válida. Alternativamente, puede ser útil medir la expresión de genes clave y proteínas en la melanogénesis en respuesta a una sustancia de prueba para examinar la actividad de hipopigmentación. Además de tirosinasa, proteínas relacionadas con la tirosinasa (TRP-1) y dopachrome tautomerase (TRP-2) son enzimas críticas en la melanogénesis8. El factor de transcripción factor transcripción asociada a microftalmia (MITF) es un regulador principal en la melanogénesis y cuantificación de sus niveles de expresión del gen de la proteína o un análisis de la actividad de promotor puede utilizarse también para evaluar actividad de hipopigmentación.

<table>
	<tbody>
		<tr>
			<td>3,4-Dihydroxy-l-phenylalanine</td>
			<td>Sigma</td>
			<td>D9628</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution</td>
			<td>Sigma</td>
			<td>#320145</td>
			<td></td>
		</tr>
		<tr>
			<td>Arbutin</td>
			<td>Fluka</td>
			<td>#10960</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>HyClone</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>HyClone</td>
			<td>SH30084.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin</td>
			<td>Yakuri Pure Chemicals</td>
			<td>#16223</td>
			<td>37%</td>
		</tr>
		<tr>
			<td>Gold chloride</td>
			<td>American MasterTech</td>
			<td>AHG0226</td>
			<td>0.10%</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Samchun chemical</td>
			<td>H0255</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bio basic Canada Inc.</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>#60218</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>J.T.Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Duksan pure chemicals</td>
			<td>#81</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma</td>
			<td>655104</td>
			<td></td>
		</tr>
		<tr>
			<td>Nuclear fast red</td>
			<td>Merck</td>
			<td>100121</td>
			<td>Nuclear fast red 0.1% in 5% aluminum sulfate.</td>
		</tr>
		<tr>
			<td>Penicillin and streptomycin solution</td>
			<td>HyClone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#900</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Na2HPO4)</td>
			<td>J.T. Baker</td>
			<td>#3817-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic (Na2HPO4)</td>
			<td>Sigma</td>
			<td>S5011</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium thiosulfate pentahydrate</td>
			<td>Duksan Pure Chemicals</td>
			<td>#2163</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(hydroxymethyl)aminomethane</td>
			<td>Bio Basic Canada</td>
			<td>TB0196</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Union Carbide</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr>
			<td>Tyrosinase from mushroom</td>
			<td>Sigma</td>
			<td>T3824</td>
			<td>25 KU</td>
		</tr>
	</tbody>
</table>

quantification of hypopigmentation activity in vitro

Este estudio presenta los métodos de laboratorio para la cuantificación de hipopigmentación de la actividad in vitro. Melanina, el pigmento principal en los melanocitos, se sintetiza en respuesta a múltiples factores ambientales y celulares. Melanina, protege las células de la piel contra el daño ULTRAVIOLETA, pero también tiene funciones biofísicas y bioquímicas. Producción excesiva o acumulación de melanina en los melanocitos puede causar problemas dermatológicos, tales como pecas, manchas, melasma y moles. Por lo tanto, el control de la melanogénesis con hipopigmentación agentes es importante en personas con necesidades clínicas o cosméticas. Melanina se sintetiza principalmente en los melanosomas de melanocitos en un proceso bioquímico complejo llamado melanogénesis, influenciado por extrínsecos y factores intrínsecos, como las hormonas, inflamación, edad y exposición a la luz ultravioleta. Se describen tres métodos para determinar la actividad de hipopigmentación de productos químicos o sustancias naturales en los melanocitos: medición de la actividad de la tirosinasa 1) celular y contenido de melanina 2) y melanina celular 3) tinción y cuantificación con imagen Análisis.
En la melanogénesis, tirosinasa cataliza el paso de limitación de velocidad que convierte la L-tirosina en 3, 4-dopa (l-dopa) y luego en dopaquinona. Por lo tanto, la inhibición de la tirosinasa es un mecanismo primario de hipopigmentación. En los melanocitos cultivados, actividad de la tirosinasa puede cuantificarse por la adición de l-dopa como sustrato y dopaquinona producción de medición por espectrofotometría. Melanogénesis puede medirse también cuantificando el contenido de melanina. La fracción celular que contiene melanina es extraída con NaOH y melanina se cuantifica espectrofotométricamente. Finalmente, el contenido de melanina puede ser cuantificado por análisis de imagen después de la coloración de Fontana-Masson de melanina. Aunque los resultados de estos ensayos in vitro no siempre se pueden reproducir en la piel humana, estos métodos son ampliamente utilizados en la investigación de la melanogénesis, especialmente como el paso inicial para identificar la potencial actividad de hipopigmentación. Estos métodos pueden utilizarse también para evaluar la diferenciación, crecimiento y actividad de melanocitos. Resultados consistentes con los tres diferentes métodos de aseguran la validez de los efectos.

Resultados representativos para la actividad de hipopigmentación de la arbutina, un anti-melanogenic compuesto, en B16F10 melanocitos se muestran a continuación. Figura 1A muestra que arbutin suprimió significativamente la actividad de la tirosinasa celular en comparación con el control tratados con vehículo. Del mismo modo, el contenido de melanina de las células estimuladas con arbutin se redujo significativamente en comparación con los controles (<s...

Watch this Scientific Journal Video about Quantification of Hypopigmentation Activity In Vitro at JoVE.com

Quantification of Hypopigmentation Activity In Vitro

Se describen tres métodos experimentales para la evaluación de la actividad de hipopigmentación de químicos in vitro: cuantificación del contenido 2) melanina y actividad tirosinasa 1) celular y 3) medición de la melanina por análisis de imagen y coloración celular melanina.

Cuantificación de hipopigmentación de la actividad In Vitro

This study presents laboratory methods for the quantification of hypopigmentation activity in vitro. Melanin, the major pigment in melanocytes, is ...

Este método puede ser útil en el desarrollo de cosméticos funcionales y agentes de la piel con actividades anti-melanogénicas. Está ahí para realizar y el resultado se puede obtener rápidamente. Además, este método puede ser útil para los investigadores, que quieren identificar o investigar los efectos o los compuestos o investigar las actividades antimelanogénicas o promelanogénicas.Para medir la actividad tirosinasa comenzar por la siembra de melanicidas B16-F10 a cinco veces 10 a las cuartas células por pozo de una concentración de placa de 24 pozos en medio completo durante 24 horas en una incubadora de cultivo celular. Al día siguiente, reemplazar el sobrenadante en cada pozo con DMEM complementado con compuesto de prueba recién preparado y control de inhibidores o un control negativo y devolver la placa a la incubadora durante otras 72 horas. Al final del tratamiento enjuague los pozos dos veces con 300 microlitros de pbs fríos por pozo y coloque la placa sobre hielo.A continuación, agregue 300 microlitros de búfer de lelisis a cada pozo usando un rascador de celda para recoger las lesates celulares después de cinco minutos. Transfiera las suspensiones de partículas celulares resultantes a tubos individuales de micro centrífuga de 1,5 mililitros. Y homogeneizar los lysates tres veces durante dos segundos a 14.500 rpm en hielo para liberar la tirosinasa intercelular.Pele el pelado de los desechos celulares por centrifugación y transfiera los sobrenadantes a tubos individuales de centrífuga de 1,5 mililitros sobre hielo. Asigne 70 microlitros de cada sobrenadante a pozos individuales de una microplaca clara de 96 pozos de poliestireno y agregue 140 microlitros de solución de sustrato de tirosinasa a cada pozo de sobrenadante. Agitar la placa suavemente para mezclar el contenido del pozo.Luego incubar la placa a 37 grados centígrados durante dos horas. Y mida la actividad de la tirosinasa a 475 nanómetros en un lector de microplacas. Medir el contenido de melanina de las células después de recoger el el izado de las células tratadas como se demuestra.Recoger los lysates por centrifugación y transferir los sobrenadantes a nuevos tubos. Añadir 300 microlitros de un hidróxido de sodio normal a cada pellet para una incubación de una hora a 60 grados centígrados. Seguido por centrifugación de las suspensiones de células disueltas.A continuación, transfiera 200 microlitros de los sobrenadantes a cada pozo de una placa de 96 pozos. Y mida el contenido de melanina en un lector de microplacas a una longitud de onda de 400 nanómetros. Para la tinción Fontana-Masson, lave las células tratadas dos veces con 300 microlitros de pbs fríos y fije las células con 200 microlitros de 10% de formol durante una hora a cuatro grados centígrados.Enjuague las células gruesas con agua destilada y agregue 200 microlitros de 58 a 68 grados centígrados Solución de trabajo de plata amoniacal a cada pozo. Durante una hora de incubación a 37 grados centígrados o hasta que las células se vuelvan de color amarillo-marrón. Enjuague las células manchadas con agua destilada seguida de una incubación de dos minutos en cloruro de oro al 0,1% a temperatura ambiente.Enjuague las células tratadas con cloruro de oro con agua destilada y agregue 200 microlitros de solución de tiosulfato sódico a las células. Después de dos minutos enjuague las células de nuevo. Y etiqueta las células con 200 microlitros de rojo rápido nuclear por pozo durante cinco minutos.A continuación, lave las células manchadas con agua del grifo antes de tomar imágenes bajo un microscopio de luz. Para el análisis de umbral, abra las imágenes en la imagen J y seleccione imagen, ajuste y umbral de color. Para medir el área teñida con Fontana Masson, establezca la barra de parámetros de brillo en cero y ajuste el brillo hasta el punto en el que se incluyen todas las celdas manchadas negras o marrones.Seleccione Analizar y analizar partículas y marque la casilla de resumen en la ventana Analizar partículas y, a continuación, haga clic en Aceptar para obtener un resumen de los resultados y copiar y pegar los datos en una hoja de cálculo. El tratamiento con arbutina suprime significativamente la actividad celular de la tirosinasa en comparación con el control de las células tratadas por el vehículo. Del mismo modo, el contenido de melanina de las células estimuladas con Arbutin se reduce significativamente en comparación con los controles.Aunque las células son morfológicamente similares entre los grupos de tratamiento, Arbutin disminuye el área del pigmento negro en comparación con las células tratadas por control. Este método es útil para la selección de actividades utilizando nuestra biblioteca compuesta. Hay más de cientos de muestras para ser probadas rápidamente.Además, este método también se puede utilizar para investigar el mecanismo que implica melanogénesis. Después de identificar los compuestos de los candidatos bioactivos, por su testimonio al estudio de mecanismos biológicos o aplicaciones comerciales.

quantification-of-hypopigmentation-activity-in-vitro

Research

JoVE Journal

Biochemistry

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many of these conditions cause protein unfolding in the cell and have detrimental impact on the survival of the organism. A group of unrelated, stress-specific molecular chaperones have been shown to play essential roles in the survival of these stress conditions. While fully folded and chaperone-inactive before stress, these proteins rapidly unfold and become chaperone-active under specific stress conditions. Once activated, these conditionally disordered chaperones bind to a large number of different aggregation-prone proteins, prevent their aggregation and either directly or indirectly facilitate protein refolding upon return to non-stress conditions. The primary approach for gaining a more detailed understanding about the mechanism of their activation and client recognition involves the purification and subsequent characterization of these proteins using in vitro chaperone assays. Follow-up in vivo stress assays are absolutely essential to independently confirm the obtained in vitro results.
This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

Detection of the pH-dependent Activity of Escherichia coli Chaperone HdeB In Vitro and In Vivo

This study describes biophysical, biochemical and molecular techniques to characterize the chaperone activity of Escherichia coli HdeB under acidic pH conditions. These methods have been successfully applied for other acid-protective chaperones such as HdeA and can be modified to work for other chaperones and stress conditions.

Detección de la actividad dependiente del pH de<em&gt; Escherichia coli</em&gt; Acompañante HdeB<em&gt; In Vitro</em&gt; y<em&gt; En Vivo</em

Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

Investigación

Bioquímica

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. The relative levels of different tRNAs, and the ratio of aminoacylated tRNA to total tRNA, known as the charging level, are important factors in determining the accuracy and speed of translation. Therefore, the abundance and charging levels of tRNAs are important variables to measure when studying protein synthesis, for example under various stress conditions. Here, we describe a method for harvesting tRNA and directly measuring both the relative abundance and the absolute charging level of specific tRNA species in Escherichia coli. The tRNA is harvested in such a way that the labile bond between the tRNA and its amino acid is preserved. The RNA is then subjected to gel electrophoresis and Northern blotting, which results in separation of the charged and uncharged tRNAs. The levels of specific tRNAs in different samples can be compared due to the addition of spike-in cells for normalization. Prior to RNA purification, we add 5% of E. coli cells that overproduce the rare tRNAselC to each sample. The amount of the tRNA species of interest in a sample is then normalized to the amount of tRNAselC in the same sample. Addition of spike-in cells prior to RNA purification has the advantage over addition of purified spike-in RNAs that it also accounts for any differences in cell lysis efficiency between samples.

Quantification of the Abundance and Charging Levels of Transfer RNAs in Escherichia coli

Here we present a method for directly measuring transfer RNA charging levels from purified Escherichia coli RNA as well as a way to compare relative levels of transfer RNA, or any other short RNA, across different samples based on the addition of spike-in cells expressing a reference gene.

Cuantificación de la abundancia y la carga de los niveles de ARN de transferencia en Escherichia coli

Transfer RNA (tRNA) is an essential part of the translational machinery in any organism. tRNAs bind and transfer amino acids to the translating ribosome. ...

Mapping Metabolism: Monitoring Lactate Dehydrogenase Activity Directly in Tissue

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ metabolic activity assays can provide information about the spatial distribution of metabolic activity within a tissue. We provide here a detailed protocol for monitoring the activity of the enzyme lactate dehydrogenase directly in tissue samples. Lactate dehydrogenase is an important determinant of whether consumed glucose will be converted to energy via aerobic or anaerobic glycolysis. A solution containing lactate and NAD is provided to a frozen tissue section. Cells with high lactate dehydrogenase activity will convert the provided lactate to pyruvate, while simultaneously converting provided nicotinamide adenine dinucleotide (NAD) to NADH and a proton, which can be detected based on the reduction of nitrotetrazolium blue to formazan, which is visualized as a blue precipitate. We describe a detailed protocol for monitoring lactate dehydrogenase activity in mouse skin. Applying this protocol, we found that lactate dehydrogenase activity is high in the quiescent hair follicle stem cells within the skin. Applying the protocol to cultured mouse embryonic stem cells revealed higher staining in cultured embryonic stem cells than mouse embryonic fibroblasts. Analysis of freshly isolated mouse aorta revealed staining in smooth muscle cells perpendicular to the aorta. The methodology provided can be used to spatially map the activity of enzymes that generate a proton in frozen or fresh tissue.

We describe a protocol for mapping the spatial distribution of enzymatic activity for enzymes that generate nicotinatmide adenine dinucleotide phosphate (NAD(P)H) + H+ directly in tissue samples.

Asignación de metabolismo: Supervisar la actividad de la deshidrogenasa del lactato directamente en el tejido

Mapping enzymatic activity in space and time is critical for understanding the molecular basis of cell behavior in normal tissue and disease. In situ ...

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

Un ensayo In Vitro para detectar actividad de transferasa de Isopentenyl tRNA

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Calibración-libre In Vitro cuantificación de proteína Homo-Oligomerización utilizando instrumentación comercial y Software de análisis de brillo libre de código abierto

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

Un ensayo colorimétrico de la actividad de citrato sintasa en Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). Endocannabinoids are conserved lipid mediators that regulate multiple biological processes in a variety of organisms. In C. elegans, 2-AG has been found to possess different roles, including modulation of dauer formation and cholesterol metabolism. This report describes a method to overcome the difficulties associated with the costs and stability of deuterated standards required for 2-AG quantification. The procedure for the synthesis of the standard is simple and can be performed in any laboratory, without the need for organic synthesis expertise or special equipment. In addition, a modification of Folch&#39;s method to extract the deuterated standard from C. elegans culture is described. Finally, a quantitative and analytic method to detect 2-AG using the stable isotopically labeled analog 1-AG-d5 is described, which provides reliable results in a fast-chromatographic run. The procedure is useful for studying the multiple roles of 2-AG in C. elegans while also being applicable to other studies of metabolites in different organisms.

Synthesis of a Deuterated Standard for the Quantification of 2-Arachidonoylglycerol in Caenorhabditis elegans

This work describes a robust and straightforward method to detect and quantify the endocannabinoid 2-arachidonoylglycerol (2-AG) in C. elegans. An analytical deuterated standard us prepared and used for the quantification of 2-AG by isotopic dilution and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).

Síntesis de un estándar deuterated para la cuantificación de 2-Arachidonoylglycerol en Caenorhabditis elegans

This work presents a method to prepare an analytical standard to analyze 2-arachidonoyl glycerol (2-AG) qualitatively and quantitatively by liquid ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

Cuantificación de la dinámica de la red de interacción de proteínas mediante coinmunoprecipitación multiplexada

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Quantification of Coenzyme A in Cells and Tissues

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. Measurement of cellular CoA is a challenge due to its relatively low abundance and the dynamic conversion of free CoA to CoA thioesters that, in turn, participate in numerous metabolic reactions. A method is described that navigates through potential pitfalls during sample preparation to yield an assay with a broad linear range of detection that is suitable for use in many biomedical laboratories.

This method describes sample preparation from cultured cells and animal tissues, extraction and derivatization of coenzyme A in the samples, followed by high pressure liquid chromatography for purification and quantification of the derivatized coenzyme A by absorbance or fluorescence detection.

Cuantificación de la coenzima A en células y tejidos

Emerging research has revealed that the cellular coenzyme A (CoA) supply can become limiting with a detrimental impact on growth, metabolism and survival. ...

Cuantificación de alto rendimiento de la síntesis y distribución de ADNmt

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells produce most of their ATP in the mitochondria by oxidative phosphorylation. Mitochondria are unique organelles because they have their own genome that is replicated and passed on to the next generation of cells. In contrast to the nuclear genome, there are multiple copies of the mitochondrial genome in the cell. The detailed study of the mechanisms responsible for the replication, repair, and maintenance of the mitochondrial genome is essential for understanding the proper functioning of mitochondria and whole cells under both normal and disease conditions. Here, a method that allows the high-throughput quantification of the synthesis and distribution of mitochondrial DNA (mtDNA) in human cells cultured in vitro is presented. This approach is based on the immunofluorescence detection of actively synthesized DNA molecules labeled by 5-bromo-2'-deoxyuridine (BrdU) incorporation and the concurrent detection of all the mtDNA molecules with anti-DNA antibodies. Additionally, the mitochondria are visualized with specific dyes or antibodies. The culturing of cells in a multi-well format and the utilization of an automated fluorescence microscope make it easier to study the dynamics of mtDNA and the morphology of mitochondria under a variety of experimental conditions in a relatively short time.

Autor destacado: Cuantificación basada en imágenes de alto rendimiento de la síntesis y distribución del ADN mitocondrial  

A procedure for studying the dynamics of mitochondrial DNA (mtDNA) metabolism in cells using a multi-well plate format and automated immunofluorescence imaging to detect and quantify mtDNA synthesis and distribution is described. This can be further used to investigate the effects of various inhibitors, cellular stresses, and gene silencing on mtDNA metabolism.

Cuantificación basada en imágenes de alto rendimiento de la síntesis y distribución del ADN mitocondrial

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...