Este estudio fue apoyado por una carrera NSF Grant (MCB0954016) y una Beca de Investigación de Bioenergía del DOE (DEFG0208ER64694).

1. De cultivo de células (Figura 1) <ol><li> Se cultivan las células en medio mínimo con los elementos traza, sales, vitaminas, y específicamente etiquetados sustratos de carbono que son los mejores para la investigación de la vía. Utilizar cualquiera de frascos de agitación o bioreactores para el cultivo celular. Los nutrientes orgánicos, tales como extracto de levadura, puede interferir con la medición de etiquetado de aminoácidos y por lo tanto no pueden estar presentes en el medio de cultivo. </li><li> Controlar el crecimiento celular por la densidad óptica de la cultura en una longitud de onda óptima (por ejemplo, OD 730 para Cyanothece 51.142) con un espectrofotómetro UV / Vis. </li><li> Las primeras células pueden ser cultivadas en un medio no-etiquetado. El crecimiento de las células en fase de registro medio se prefiere que se utiliza para la inoculación (3% (v / v) por la relación de volumen de inoculación) del medio de la etiqueta. La cultura de la etiqueta deben ser sub-cultivados (3% v / v relación de inoculación) en el mismo medio la etiqueta para evitar la introducción de la etiqueta de carbono del inóculo inicial. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Extracción de aminoácidos <ol><li> Cosecha sub-cultivos de células (10 ml) en la fase de crecimiento media sesión por centrifugación (10 min, 8000 × g). </li><li> Resuspender el precipitado en 1,5 ml de HCl 6M y transferirlo a un vidrio transparente, con tapa de rosca vial GC. Cap de los viales y colóquelos en un horno de 100 ° C durante 24 horas para hidrolizar las proteínas de la biomasa en los aminoácidos. La hidrólisis de pellets de biomasa puede producir 16 de los 20 aminoácidos comunes (Figura 2) 5. Cisteína y triptófano se degrada, y la glutamina y asparagina se convierten en el glutamato y el aspartato, respectivamente. </li><li> Centrifugar la solución de aminoácidos a 20000 × g durante 5 minutos con 2 ml tubos Eppendorf, y la transferencia de los sobrenadantes a los nuevos viales de GC. Este paso elimina las partículas sólidas en la solución de hidrólisis. </li><li> Quitar las tapas vial GC y secar las muestras completamente bajo una corriente de aire con un Thermo Scientific Reacti-Vap evaporador (nota: un secador de congelación también se puede utilizar to seca las muestras). Este paso se puede hacer durante la noche. </li></ol> 3. Derivatización de aminoácidos y las condiciones de GC-MS Análisis de aminoácidos o carga / alta de metabolitos polares a través de GC requiere que estos metabolitos se derivatizado, a fin de que los aminoácidos son volátiles y se pueden separar por cromatografía de gases 2. <ol><li> Disuelva las muestras secas con 150 l de tetrahidrofurano (THF) y 150 l de N-(terc-butildimetilsilil)-N-methyltrifluoroacetamide reactivo de derivatización. </li><li> Incubar todas las muestras en un horno o un baño de agua entre 65 y 80 ° C durante 1 hora. Vortex ocasionalmente para asegurarse de que los metabolitos en el vial se disuelven. </li><li> Centrifugar las muestras a 20000 × g durante 10 minutos, y luego transferir el sobrenadante a los nuevos viales de GC. El sobrenadante debe ser una solución clara y amarillenta. Debido a la saturación de los detectores, GC-MS precisión de la medición puede verse afectada por la alta concentration de inyección TBDMS derivado de aminoácidos (las muestras a menudo muestra de color marrón oscuro), por lo tanto, hay que diluir las muestras con THF antes de GC-MS medición 6. </li><li> Analizar las muestras por GC-MS (usar un split 1:5 o 1:10, el volumen de inyección = 1 l, de transporte de gas helio = 1,2 ml / min). Utilice el siguiente programa de GC temperatura: mantener a 150 º C durante 2 minutos, el aumento de 3 ° C por minuto hasta 280 ° C, aumentando a 20 ° C por minuto hasta 300 ° C y mantenga durante 5 minutos. Espera para el solvente se puede establecer como ~ 5 minutos (para una columna de 30 metros GC). El rango de la masa de carga (m / z) en la EM se puede establecer entre 60 y 500. </li></ol> 4. GC-MS análisis de datos <ol><li> TBDMS derivado de medición de aminoácidos también pueden verse afectados por la discriminación de isótopos en la separación de GC. Isótopos ligeros se mueven ligeramente más rápido que levantar los isótopos en la columna GC. Para reducir el potencial de sesgo de medición, es posible que la media del espectro de masas de todo elaminoácido rango máximo 6 </li><li> La GC y los espectros de MS de metabolitos TBDMS derivado se ha informado antes de las 7. El tiempo de retención GC y los picos únicos m / z para cada aminoácido se ilustra en la Figura 3. </li><li> Derivatización de aminoácidos o de metabolitos central introduce grandes cantidades de isótopos naturales marcado, entre ellos 13 C (1,13%), 18 S (0,20%), de 29 años Si (4,70%), y 30 Si (3,09%). La medición del ruido de los isótopos naturales en el espectro de masa isotopomer primas pueden ser corregidos mediante el uso de software publicado 5, 8. Los datos finales de etiquetado isotópico se presentan como fracciones de masa, por ejemplo, M 0, M 1, M 2, M 3 y M 4 (en representación de los fragmentos que contienen cero a cuatro átomos de carbono marcados 13 C). </li><li> La medición de los aminoácidos pueden proporcionar información sobre el etiquetado isotópico ocho metabolitos precursores crucial: 2-oxo-glutarcomió, 3-P-glicerato, acetil-CoA, eritrosa-4-P, oxalacetato, fosfoenolpiruvato, piruvato y la ribosa-5-P. Los patrones de etiquetado de estos metabolitos pueden ser utilizados para identificar varias rutas metabólicas centrales (Figura 2) 9. El resultado de los experimentos de etiquetado puede ser confirmado mediante otros métodos bioquímicos (por ejemplo, RT-PCR). </li></ol> 5. Vía de análisis utilizando datos de la etiqueta de aminoácidos Al investigar sólo algunos ácidos clave amino producidos a partir de experimentos bien diseñados 13 C del marcador, que puede revelar varias vías únicas o las actividades de la enzima sin necesidad de realizar sofisticados 13 C-metabólico análisis de flujo del metabolismo central de todo. <ol><li> Entner-Doudoroff vía: [1 - 13 C] glucosa puede ser utilizada como fuente de carbono. Si la vía está activa, el etiquetado serina será significativamente menor que el etiquetado de la alanina 10. </li><li> Ciclo ramificada TCA: [1 - 13 C]piruvato puede ser utilizado como fuente de carbono. Si el ciclo de TCA se ha roto, aspartato pueden ser etiquetados por dos átomos de carbono, mientras que el glutamato es etiquetado con sólo un átomo de carbono 11, 12. </li><li> Fijación de CO 2 por Calvin-Benson-Bassham ciclo en el metabolismo mixotróficos: no marcado y etiquetado de CO 2 sustratos de carbono son utilizados como fuentes de carbono. Si el ciclo de Calvin es funcional, serina y etiquetado histidina será significativamente diluida, en comparación con otros aminoácidos. Tal método puede determinar la relación CO 2 cuando la fijación de fuentes orgánicas de carbono están presentes en el medio 13. </li><li> Vía oxidativa de las pentosas fosfato: [1 - 13 C] glucosa puede ser utilizada como fuente de carbono. Si la vía está activa, la alanina sin etiqueta será&gt; 50% 12. </li><li> Anapleróticas vía (por ejemplo, PEP + CO 2 oxalacetato a): 13 CO 2 y no de carbono marcado sustratos (por ejemplo, el glicerol o pyruvate) se puede utilizar como fuente de carbono. Si la vía está activa, el etiquetado aspartato se ha enriquecido considerablemente, en comparación con alanina y serina 13. </li><li> Re-citrato sintasa: [1 - 13 C] piruvato puede ser utilizado como fuente de carbono. Si la enzima está activa, el glutamato es etiquetada en β-carboxilo grupo de 3, 4. </li><li> Vía Citramalate: [1 - 13 C] piruvato, [2 - 13 C] glicerol, o [1 - 13 C] acetato se puede utilizar como fuente de carbono. Si la vía está activo, las cantidades de etiquetado leucina e isoleucina son idénticos 14. </li><li> Serina-isocitrato liasa ciclo: [1 - 13 C] piruvato o [1 - 13 C] lactato puede ser utilizado como fuente de carbono. Si la vía está activa, la tercera posición de carbono en la serina se etiquetarán 15. </li><li> Utilización de los nutrientes (es decir, aminoácidos exógenos): un medio de cultivo con sustratos de carbono completamente etiquetados y no etiquetados aminoácidosse puede utilizar. Si las células de forma selectiva utilizar estos nutrientes complementa los no etiquetados, vamos a ver una dilución significativa de etiquetado de estos aminoácidos en la biomasa. Este método puede ser utilizado para investigar lo que los suplementos de nutrientes son los preferidos por la celda 16. </li></ol> 6. Resultados representante Estudios recientes han reavivado el interés de bioenergía en el uso de nuevos microorganismos fototróficas para la producción de bioenergía y la captura de CO 2. En los últimos años, un buen número de metabolismo de la 13C-análisis, incluyendo avanzados análisis de flujo metabólico-13C (13C-AMF), se han aplicado para investigar el metabolismo central en bacterias fototróficas, porque el conocimiento bioquímico de las rutas metabólicas centrales no está bien fundada en estos organismos que no son modelo 10, 11, 17-20. Aquí, se presenta un ejemplo del descubrimiento de una vía alternativa en isoleucina Cyanothece 51.142 21. Cianothece 51.142 no contiene la enzima (EC 4.3.1.19, treonina amonio-liasa), que cataliza la conversión de treonina a 2-cetobutirato en la vía de síntesis de isoleucina típico. Para resolver la vía isoleucina, crecemos Cyanothece 51.142 (20 ml) en medio ASP2 22 con glicerol de 54 mm (2-13C,&gt; 98%). Cyanothece 51142 utiliza 2 ª posición de glicerol etiquetados como fuente de carbono principal. Observamos que la treonina y alanina tienen una etiqueta de carbono, mientras que la isoleucina es etiquetado con tres carbonos. Por lo tanto, la síntesis de Cyanothece 51142 no se puede derivar de la ruta treonina empleado por la mayoría de los organismos (Figura 4). Por otro lado, leucina e isoleucina tienen patrones idénticos de etiquetado basado en fragmentos (M-15) y el fragmento + (M-159) +. Por ejemplo, los datos isotopomer de [M-15] + (que contiene los aminoácidos no fragmentado) muestran el etiquetado idénticas de leucina (M0 = 0,01, M1 = 0,03, M2 = 0.21, M3 = 00.69) e isoleucina (M0 = 0,01, M1 = 0,03, M2 = 0.24, M3 = 0,67). Así, leucina e isoleucina se sintetiza a partir de los precursores del mismo (es decir, el piruvato y el acetil-CoA). Esta observación es consistente con la transición de carbono marcado en la vía citramalate para la síntesis de isoleucina. Para confirmar esta vía, que buscar en la base de datos Joint Genome Institute y encontrar la presencia de una sintasa citramalate Cima (cce_0248) en Cyanothece. <img src="/files/ftp_upload/3583/3583fig1.jpg" alt="Figura 1"/> Figura 1. El 13 C con ayuda de los pasos de análisis vía. <img src="/files/ftp_upload/3583/3583fig2.jpg" alt="Figura 2"/> Figura 2. Los aminoácidos utilizados para la adquisición del patrón de etiquetado de sus precursores metabólicos. ACOA, la acetil-CoA, AKG, α-cetoglutarato; C5P, ribosa 5-fosfato, CIT, citrato; E4P, eritrosa 4-fosfato, G6P, glucosa 6-fosfato, OAA,<html> oxalacetato, PEP, fosfoenolpiruvato, PGA, 3-fosfoglicerato, PYR, el piruvato. <img src="/files/ftp_upload/3583/3583fig3.jpg" alt="Figura 3"/> Figura 3. Picos GC de 16 aminoácidos. TBDMS aminoácidos derivatizados están agrietadas por los Estados miembros en dos fragmentos: (M-57) +, que contiene el ácido amino todo, y (M-159) +, que carece del grupo α carboxilo del aminoácido. De leucina e isoleucina, el + (M-57) fue solapado por los picos de masas. Sugerimos el uso de fragmentos (M-15) + para analizar el etiquetado completo de aminoácidos. El grupo (F302) + se detecta en la mayoría de los aminoácidos, que contiene sólo el primero (α-carboxilo del grupo) y los carbones segundo en una red troncal de aminoácidos. Debido a que este pico de MS a menudo ha de ruido a la señal de ratios, (F302) + no se recomienda para analizar cuantitativamente los flujos metabólicos 7. igura 4 &quot;/&gt; Figura 4. Etiquetado de las transiciones en las vías de isoleucina en Cyanothece 51.142 (modificado a partir de nuestro trabajo anterior) 21.

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Este protocolo consiste en alimentar la celda con un sustrato marcado y medición de los patrones resultantes isotópica de etiquetado en los aminoácidos a través de GC-MS. Desde MS de datos (m / z ratios) dar la cantidad total de etiquetado de los iones de MS, tenemos que evaluar las distribuciones de isotopomer de aminoácidos mediante el examen de la m / z ratios de ambos no fragmentado (M-57) + y fragmentado aminoácidos (es decir, (M-159) y + (F302) +). Además, podemos realizar v...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> TBDMS </td><td> Sigma-Aldrich </td><td> 19915 </td><td> - </td></tr><tr><td> THF </td><td> Sigma-Aldrich </td><td> 34865 </td><td> - </td></tr><tr><td> De carbono marcado sustrato </td><td> Cambridge Laboratorios de isótopos </td><td> Dependen del requisito experimental </td><td> Sitio Web: <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td> Cromatógrafo de gases </td><td> Agilent Technologies </td><td> Hewlett-Packard, modelo 7890A </td><td> - </td></tr><tr><td> GC Columnas </td><td> J &amp; W Scientific, Folsom, CA </td><td> DB5 (30) </td><td> - </td></tr><tr><td> Espectrómetro de masas </td><td> Agilent Technologies </td><td> 5975C </td><td> - </td></tr><tr><td> Reactivación-Vap EvaporAtor </td><td> Thermo Scientific </td><td> TS-18825 </td><td> Para el secado de las muestras de aminoácidos </td></tr></tbody></table>

metabolic pathway confirmation and discovery through 13c-labeling of proteinogenic amino acids

Los microbios tienen complejos procesos metabólicos que pueden ser investigados con métodos genómica bioquímica y funcional. Una técnica importante para examinar el metabolismo celular central y descubrir nuevas enzimas es de 13 C con ayuda de análisis de metabolismo 1. Esta técnica se basa en el etiquetado isotópico, en el que los microbios se alimentan con un 13 sustratos C etiquetados. Al trazar las rutas de transición entre el átomo de metabolitos en la red bioquímica, podemos determinar las vías funcionales y descubrir nuevas enzimas. Como un método complementario a la transcriptómica y proteómica, los enfoques de isotopomer análisis asistido de las vías metabólicas contiene tres pasos principales 2. En primer lugar, crecen las células con 13 substratos C etiquetados. En este paso, la composición del medio y la selección de sustratos marcados son dos factores clave. Para evitar los ruidos de medición de carbono no llevan la etiqueta en los suplementos nutricionales, Un medio mínimo con una única fuente de carbono es necesario. Además, la elección de un sustrato marcado se basa en la eficacia con que aclarará el camino que se analiza. Debido a nuevas enzimas a menudo implican estereoquímica reacción diferente o productos intermedios, en general, solo etiquetados sustratos de carbono son más informativos para la detección de nuevas vías que los etiquetados de manera uniforme para la detección de tres nuevas vías, 4. En segundo lugar, se analizan los patrones de amino ácido etiquetado con GC -MS. Los aminoácidos son abundantes en proteínas y por lo tanto se puede obtener a partir de la hidrólisis de la biomasa. Los aminoácidos pueden ser derivado de N-(terc-butildimetilsilil)-N-methyltrifluoroacetamide (TBDMS) antes de la separación GC. TBDMS aminoácidos derivado puede ser fragmentado por los Estados miembros y dar lugar a matrices diferentes de fragmentos. Sobre la base de la masa de carga (m / z) La proporción de aminoácidos fragmentados y no fragmentados, podemos deducir los posibles patrones de los metabolitos de la etiqueta central que seprecursores de los aminoácidos. En tercer lugar, se traza la transición 13C de carbono en las vías propuestas y, en base a los datos isotopomer, confirme si estas vías están activas 2. La medición de aminoácidos proporciona información sobre el etiquetado isotópico ocho metabolitos precursores crucial en el metabolismo central. Estos nodos clave del metabolismo pueden reflejar las funciones de los asociados vías centrales. 13 C-análisis asistido por el metabolismo de los aminoácidos a través de proteinogenic puede ser ampliamente utilizado para la caracterización funcional del metabolismo microbiano caracterizado mal 1. En este protocolo, vamos a utilizar Cyanothece 51142 como la cepa modelo para demostrar el uso de sustratos de carbono marcado por el descubrimiento de nuevas funciones enzimáticas.

Watch this Scientific Journal Video about Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids at JoVE.com

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

13 C-etiquetado de isótopos es una técnica útil para determinar el metabolismo de la célula central para varios tipos de microorganismos. Después las células han sido cultivadas con un sustrato marcado, GC-MS medición puede revelar las vías metabólicas funcional basada en los patrones de etiquetado único en proteinogenic aminoácidos.

Confirmación vía metabólica y el descubrimiento a través de 13 C-etiquetado de proteinogenic Aminoácidos

Microbes have complex metabolic pathways that can be investigated using biochemistry and functional genomics methods. One important technique to examine ...

metabolic-pathway-confirmation-discovery-through-13c-labeling

Research

JoVE Journal

Biology

Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

24.1K vistas.  Washington University. 13 C-etiquetado de isótopos es una técnica útil para determinar el metabolismo de la célula central para varios tipos de microorganismos. Después las células han sido cultivadas con un sustrato marcado, GC-MS medición puede revelar las vías metabólicas funcional basada en los patrones de etiquetado único en proteinogenic aminoácidos.

Video: Metabolic Pathway Confirmation and Discovery Through 13C-labeling of Proteinogenic Amino Acids

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Utilizando el sistema de fraccionamiento GELFREE 8100 de peso molecular basado en Fraccionamiento con la recuperación de la fase líquida

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Investigación

Biología

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

The urease method of sample preparation for GC/MS analysis of intermediary metabolites is presented by its inventor. The method allows one-step follow-up of newborn screening for inborn errors by tandem mass spectrometry by quantifying carbohydrates, organic and amino acids all in a single process.

De un solo paso metabolómica: Hidratos de carbono, ácidos orgánicos y aminoácidos cuantificados en un procedimiento único

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on ...

NanoDrop Microvolume Quantitation of Nucleic Acids

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional cuvette-based instruments for nucleic acid quantitation for those that can perform microvolume quantitation.
The NanoDrop microvolume sample retention system (Thermo Scientific NanoDrop Products) functions by combining fiber optic technology and natural surface tension properties to capture and retain minute amounts of sample independent of traditional containment apparatus such as cuvettes or capillaries. Furthermore, the system employs shorter path lengths, which result in a broad range of nucleic acid concentration measurements, essentially eliminating the need to perform dilutions. Reducing the volume of sample required for spectroscopic analysis also facilitates the inclusion of additional quality control steps throughout many molecular workflows, increasing efficiency and ultimately leading to greater confidence in downstream results.
The need for high-sensitivity fluorescent analysis of limited mass has also emerged with recent experimental advances. Using the same microvolume sample retention technology, fluorescent measurements may be performed with 2 &mu;L of material, allowing fluorescent assays volume requirements to be significantly reduced. Such microreactions of 10 &mu;L or less are now possible using a dedicated microvolume fluorospectrometer.
Two microvolume nucleic acid quantitation protocols will be demonstrated that use integrated sample retention systems as practical alternatives to traditional cuvette-based protocols. First, a direct A260 absorbance method using a microvolume spectrophotometer is described. This is followed by a demonstration of a fluorescence-based method that enables reduced-volume fluorescence reactions with a microvolume fluorospectrometer. These novel techniques enable the assessment of nucleic acid concentrations ranging from 1 pg/ &mu;L to 15,000 ng/ &mu;L with minimal consumption of sample.

The use of NanoDrop microvolume systems as practical and efficient alternatives to traditional nucleic acid quantitation methodology is described through the demonstration of two microvolume nucleic acid quantitation protocols.

NanoDrop microvolumen cuantificación de ácidos nucleicos

Biomolecular assays are continually being developed that use progressively smaller amounts of material, often precluding the use of conventional ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Análisis del perfil metabólico de embriones de pez cebra

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

La medición de la tasa metabólica en<em&gt; Drosophila</em&gt; Mediante respirometría

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Determinación basada en el crecimiento y en la confirmación bioquímica de Requisitos Genéticos para la degradación de proteínas en<em&gt; Saccharomyces cerevisiae</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

El desarrollo de inhibidores de interacciones proteína-proteína a través REEMPLAZAR: Aplicación al Diseño y Desarrollo para no ATP inhibidores de CDK Competitiva

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an Escherichia coli Cell-free Transcription-translation System

An easy-to-use, cell-free expression protocol for the residue-specific incorporation of noncanonical amino acid analogs into proteins, including downstream analysis, is presented for medical, pharmaceutic, structural and functional studies.

La incorporación de residuos específicos de no canónicos aminoácidos en las proteínas modelo usando una<em&gt; Escherichia coli</em&gt; Sistema de transcripción-traducción libre de células

The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

El aislamiento de filtración de ácidos nucleicos: A Extracción de ADN método sencillo y rápido

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells

The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.

This protocol describes the isolation and culture of mixed murine small intestinal cells. These primary intestinal cultures enable the investigation of signaling pathways underlying gut peptide secretion using a number of techniques.

Mezcla de cultivos primarios de murino intestino delgado previstos para el Estudio de Gut Hormone secreción y Imágenes de células vivas de células enteroendocrinas