Este estudo foi suportado por uma carreira NSF Grant (MCB0954016) e um Bioenergia DOE Research Grant (DEFG0208ER64694).

1. Cultura de células (Figura 1) <ol><li> Cultivar células em meio mínimo com oligoelementos, sais, vitaminas e substratos de carbono especificamente rotulados que são melhores para a investigação caminho. Usar qualquer agitação ou biorreatores frascos para cultura de células. Nutrientes orgânicos, tais como extrato de levedura, pode interferir com a medição de rotulagem de aminoácidos e, portanto, não pode estar presente no meio de cultura. </li><li> Monitorar o crescimento de células pela densidade óptica da cultura em um comprimento de onda ideal (por exemplo, OD 730 para Cyanothece 51142), com um espectrofotômetro UV / Vis. </li><li> As células podem ser cultivadas primeiro em um meio não marcado. O meio-log células fase de crescimento são preferidos a serem utilizados para a inoculação (3% (v / v) por razão de inoculação volume) do meio rotulados. A cultura deve ser rotulado sub-cultura (3% v / v relação de inoculação) no mesmo meio rotulado para evitar a introdução de carbono não-marcado a partir do inóculo inicial. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Extração de aminoácidos <ol><li> Colheita sub-cultura de células (10 ml) em fase de crescimento médio log-por centrifugação (10 min, 8000 × g). </li><li> Ressuspender o sedimento em 1,5 ml de HCl 6M e transferi-lo para um vidro transparente, com tampa frasco GC. Cap os frascos e coloque-os em forno a 100 ° C por 24 horas para hidrolisar as proteínas da biomassa em aminoácidos. Hidrólise de pellets de biomassa pode produzir 16 dos 20 aminoácidos comuns (Figura 2) 5. Cisteína e triptofano são degradadas, e glutamina e asparagina são convertidos para o glutamato e aspartato, respectivamente. </li><li> Centrifugar a solução de aminoácidos a 20.000 × g por 5 min em tubos de 2 ml Eppendorf, e transferir o sobrenadante para novos frascos GC. Essa etapa remove partículas sólidas na solução de hidrólise. </li><li> Remover as tampas frasco GC e secar as amostras completamente sob um fluxo de ar através de uma reativação Thermo Scientific-Vap evaporador (nota: um secador de congelamento também pode ser usado tamostras o seco). Esta etapa pode ser feito durante a noite. </li></ol> 3. Derivatização de aminoácidos e GC-MS condições Análise de aminoácidos ou carregadas / altamente metabolitos polares via GC exige que estes metabólitos ser derivatizado, de modo que os aminoácidos são voláteis e podem ser separados por cromatografia em fase gasosa 2. <ol><li> Dissolver as amostras secas com 150 mL de tetrahidrofurano (THF) e 150 mL de N-(terc-butyldimethylsilyl) reagente de derivatização-N-methyltrifluoroacetamide. </li><li> Incubar todas as amostras em um forno ou em banho-maria entre 65 e 80 ° C por 1 hora. Vortex, ocasionalmente, para se certificar de que os metabolitos no frasco são dissolvidos. </li><li> Centrifugar as amostras a 20.000 × g por 10 min, e depois transferir o sobrenadante para novos frascos GC. O sobrenadante deve ser uma solução clara e amarelada. Devido à saturação dos detectores, GC-MS precisão da medição pode ser afetada pela alta concentration de injeção TBDMS derivatizado aminoácidos (estas amostras freqüentemente mostra cor marrom escuro), portanto, devemos diluir essas amostras utilizando THF antes de GC-MS medição 6. </li><li> Análise das amostras por GC-MS (use um 1:05 ou 1:10 dividir relação de volume de injeção, = 1 mL de transporte de gás hélio = 1,2 mL / min). Use o programa de temperatura seguintes GC: mantenha a 150 ° C por 2 minutos, aumento de 3 ° C por min a 280 ° C, aumento de 20 ° C por minuto até 300 ° C e mantenha por 5 minutos. Atraso solvente pode ser definido como ~ 5 minutos (para uma coluna GC 30 metros). O alcance da massa e carga ratio (m / z) em MS pode ser definida entre 60 e 500. </li></ol> 4. GC-MS de análise de dados <ol><li> TBDMS medição de ácido amino derivatizado também podem ser afetados pela discriminação dos isótopos na separação GC. Isótopos luz mover-se ligeiramente mais rápido do que heave isótopos na coluna GC. Para reduzir o viés de aferição potencial, podemos média do espectro de massa do conjuntogama de pico amino ácido 6 </li><li> A GC e MS espectros de metabólitos TBDMS derivatizado foram relatados antes de 7. O tempo de retenção ea única GC m / z picos para cada aminoácido são ilustrados na Figura 3. </li><li> Derivatização de aminoácidos ou metabólitos centrais apresenta quantidades significativas de isótopos naturalmente marcado, incluindo 13 C (1,13%), 18 O (0,20%), 29 Si (4,70%), e 30 Si (3,09%). O ruído de medição de isótopos naturais no espectro de massa crua isotopomer pode ser corrigido usando software publicou 5, 8. A final de dados isotópicos rotulagem são relatadas como frações de massa, por exemplo, M 0, M 1, M 2, M 3 e M 4 (representando fragmentos contendo zero a quatro carbonos marcados 13 C). </li><li> Medição de aminoácidos pode fornecer informações de rotulagem isotópica cerca de oito metabólitos precursor crucial: 2-oxo-glutarcomeu, 3-P-glicerato, acetil-CoA, eritrose-4-P, oxaloacetato, fosfoenolpiruvato, piruvato e ribose-5-P. Os padrões de rotulagem desses metabólitos podem ser usados ​​para identificar várias vias metabólicas central (Figura 2) 9. O resultado dos experimentos rotulagem pode ser confirmado através de métodos bioquímicos outros (por exemplo, RT-PCR). </li></ol> 5. Análise de caminho usando rotulados de dados de aminoácidos Ao investigar apenas alguns aminoácidos-chave produzido a partir de 13 bem concebidos experimentos C traçador, que pode revelar várias vias originais ou atividades enzimáticas sem executar sofisticadas análises de fluxo 13 C-metabólicas do metabolismo central de todo. <ol><li> Entner-Doudoroff caminho: [1 - 13 C] glicose pode ser usado como fonte de carbono. Se o caminho está ativo, rotulagem serina será significativamente menor do que a rotulagem da alanina 10. </li><li> Ciclo TCA ramificada: [1 - 13 C]piruvato pode ser usado como fonte de carbono. Se o ciclo TCA é quebrado, aspartato pode ser rotulado por dois átomos de carbono, enquanto o glutamato é rotulado com apenas um carbono 11, 12. </li><li> CO 2 por fixação de Calvin-Benson-Bassham ciclo em um metabolismo mixotróficas: Non-rotulados CO 2 e rotuladas substratos de carbono são usados ​​como fontes de carbono. Se o ciclo de Calvin é funcional, serina e histidina rotulagem será significativamente diluída, em comparação com outros aminoácidos. Tal método pode determinar a relação CO 2 quando as fontes de fixação de carbono orgânico está presente nos 13 médio. </li><li> Via fosfato pentose oxidativo: [1 - 13 C] glicose pode ser usado como fonte de carbono. Se o caminho está ativo, a alanina não marcado será&gt; 50% 12. </li><li> Pathway Anaplerotic (por exemplo, PEP + CO 2 à oxaloacetato): 13 CO 2 e não-rotulados de carbono substratos (eg glicerol, ou pyruvate) pode ser usado como fonte de carbono. Se o caminho está ativo, rotulagem aspartato será significativamente enriquecido, em comparação com alanina e serina 13. </li><li> Re citrato-sintase: [1 - 13 C] piruvato pode ser usado como fonte de carbono. Se a enzima é ativa, o glutamato é rotulado de β-carboxi grupo 3, 4. </li><li> Pathway Citramalate: [1 - 13 C] piruvato, [2 - 13 C] glicerol, ou [1-13 C] acetato pode ser usado como fonte de carbono. Se o caminho está ativo, quantidades rotulagem leucina e isoleucina são idênticos 14. </li><li> Serina-isocitrato liase ciclo: [1 - 13 C] piruvato ou [1-13 C] lactato pode ser utilizado como fonte de carbono. Se o caminho está ativo, o carbono terceira posição em serina será marcado 15. </li><li> Utilização dos nutrientes (ou seja, aminoácidos exógenos): um meio de cultura com substratos de carbono totalmente rotulados e não rotulados aminoácidospode ser usado. Se as células seletivamente utilizar esses nutrientes suplementada não marcado, vamos ver diluição significativa rotulagem destes aminoácidos na biomassa. Este método pode ser usado para investigar quais suplementos nutricionais são os preferidos pela célula 16. </li></ol> 6. Resultados representante Estudos de bioenergia recentes têm reavivado interesse em usar microorganismos fototróficas novela para a produção de bioenergia e de captura de CO 2. Nos últimos anos, muito poucos 13C metabolismo-análises, incluindo análises avançadas 13C-Metabolic Flux (MFA-13C), têm sido aplicados para investigar o metabolismo central em bactérias fototróficas, porque o conhecimento bioquímicos das vias metabólicas centrais não é bem fundamentada nestes organismos não-modelo de 10, 11, 17-20. Aqui, apresentamos um exemplo da descoberta de um caminho alternativo no isoleucina Cyanothece 51.142 21. Cyanothece 51.142 não contém a enzima (CE 4.3.1.19, treonina amônia-liase), que catalisa a conversão de treonina a 2 cetobutirato-no caminho típico isoleucina síntese. Para resolver o caminho isoleucina, nós crescemos Cyanothece 51.142 (20 mL) em meio ASP2 22 com 54 mM de glicerol (2-13C,%&gt; 98). Cyanothece 51142 utiliza 2 ª posição glicerol rotulados como fonte de carbono principal. Observamos que treonina e alanina tem um escrito de carbono, enquanto isoleucina é rotulado com três carbonos. Portanto, a síntese em Cyanothece 51142 não pode ser derivada a partir da rota de treonina utilizados pela maioria dos organismos (Figura 4). Por outro lado, leucina e isoleucina têm padrões de rotulagem idênticos com base no fragmento (M-15) + e fragmento (M-159) +. Por exemplo, os dados isotopomer a partir de [M-15] + (contendo unfragmented aminoácidos) mostrou marcação idêntica para 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). Assim, leucina e isoleucina deve ser sintetizada a partir de precursores mesma (ou seja, piruvato e acetil-CoA). Esta observação é consistente com a transição de carbono marcado na via citramalate para a síntese de isoleucina. Para confirmar esta via, que busca a Joint Genome Institute banco de dados e encontrar a presença de um sintase citramalate Cima (cce_0248) em Cyanothece. <img src="/files/ftp_upload/3583/3583fig1.jpg" alt="Figura 1"/> Figura 1. O 13 C-assisted etapas da análise caminho. <img src="/files/ftp_upload/3583/3583fig2.jpg" alt="Figura 2"/> Figura 2. Aminoácidos usado para adquirir o padrão de marcação de seus precursores metabólicos. ACOA, acetil-CoA; AKG, α-cetoglutarato; C5P, ribose 5-fosfato; CIT, citrato; E4P, eritrose 4-fosfato; G6P, glicose 6-fosfato; OAA,<html> oxaloacetato; fosfoenolpiruvato PEP,; PGA, 3-fosfoglicerato, PYR, piruvato. <img src="/files/ftp_upload/3583/3583fig3.jpg" alt="Figura 3"/> Figura 3. Picos GC por 16 aminoácidos. TBDMS derivatizado aminoácidos são rachados por MS em dois fragmentos: (M-57) +, contendo o ácido amino inteira, e (M-159) +, o que não tem o grupo α carboxila do aminoácido. Para leucina e isoleucina, a + (M-57) foi sobreposta por picos de massa outros. Sugerimos o uso de fragmento (M-15) + para analisar a rotulagem de ácido amino inteira. O (F302) + grupo é detectada na maioria dos aminoácidos, que contém apenas o primeiro (grupo α-carboxila) e carbonos segundo em um backbone de aminoácidos. Porque esse pico MS muitas vezes tem alto nível de ruído ao sinal-razões, (F302) + não é recomendado para quantitativamente análise dos fluxos metabólicos 7. IGURA 4 &quot;/&gt; Figura 4. Labeling transições em vias de isoleucina na Cyanothece 51.142 (modificado de nosso artigo anterior) 21.

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Este protocolo consiste em alimentar a célula com um substrato rotulados e medir o resultado padrões de rotulagem isotópica na aminoácidos através de GC-MS. Desde MS de dados (m / z rácios) dar apenas o montante global da rotulagem de íons de MS, temos que avaliar as distribuições isotopomer de aminoácidos, examinando as razões m / z de ambos não fragmentado (M-57) + e fragmentado aminoácidos (ie, (M-159) + e (F302) +). Além disso, podemos realizar várias culturas de célu...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th><th> Comments (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> Substrato de carbono marcado </td><td> Cambridge Isotope Laboratories </td><td> Dependem da exigência experimental </td><td> Website: <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td> Cromatógrafo a gás </td><td> Agilent Technologies </td><td> Hewlett-Packard, modelo 7890A </td><td> - </td></tr><tr><td> Colunas GC </td><td> J &amp; W Scientific, Folsom, CA </td><td> DB5 (30m) </td><td> - </td></tr><tr><td> Espectrômetro de massa </td><td> Agilent Technologies </td><td> 5975C </td><td> - </td></tr><tr><td> Reativação-Vap EvaporAtor </td><td> Thermo Scientific </td><td> TS-18825 </td><td> Para a secagem de amostras de aminoácidos </td></tr></tbody></table>

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

Micróbios têm complexo de vias metabólicas que podem ser investigados usando métodos de bioquímica e funcional genômica. Uma técnica importante examinar o metabolismo celular central e descobrir novas enzimas é de 13 C-assistida análise metabolismo 1. Esta técnica é baseada na rotulagem isotópica, em que os micróbios são alimentados com um 13 substratos C rotulados. Ao traçar os caminhos átomo de transição entre metabólitos na rede bioquímicos, podemos determinar caminhos funcional e descobrir novas enzimas. Como um método complementar para transcriptômica e proteômica, abordagens para isotopomer assistida análise de vias metabólicas conter três etapas principais 2. Primeiro, cultivar células com 13 substratos C rotulados. Nesta etapa, a composição do meio e da seleção de substratos marcados são dois fatores-chave. Para evitar ruídos de medição a partir de carbono não-rotulados de suplementos nutricionais, Um meio mínimo com uma única fonte de carbono é necessária. Além disso, a escolha de um substrato marcado é baseada em como efetivamente ele irá elucidar o caminho a ser analisada. Porque muitas vezes envolvem enzimas romance estereoquímica reação diferente ou produtos intermediários, em geral, substratos de carbono individualmente rotulados são mais informativos para a detecção de caminhos novos do que os uniformemente marcado para a detecção de vias novela 3, 4. Em segundo lugar, analisar os padrões de rotulagem de aminoácidos usando GC -MS. Aminoácidos são abundantes em proteínas e, portanto, pode ser obtido a partir da hidrólise da biomassa. Aminoácidos pode ser derivatizado por N-(terc-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) antes da separação GC. TBDMS derivatizado aminoácidos pode ser fragmentado por MS e resultam em diferentes matrizes de fragmentos. Com base na massa à relação de carga (m / z) de fragmentação e não fragmentado aminoácidos, podemos deduzir os padrões possíveis rotulados dos metabólitos que são centraisprecursores dos aminoácidos. Terceiro, traçamos transições de carbono 13C nas vias propostas e, com base nos dados isotopomer, confirmar se estes caminhos são dois ativos. Medição de aminoácidos fornece informações de rotulagem isotópica cerca de oito metabólitos precursor crucial no metabolismo central. Esses nós metabólicos chave podem refletir as funções de associados vias centrais. 13 A análise do metabolismo C-assistida via proteinogénicos aminoácidos pode ser amplamente utilizado para a caracterização funcional de mal-caracterizada metabolismo microbiano 1. Neste protocolo, usaremos Cyanothece 51142 como cepa modelo para demonstrar o uso de substratos de carbono marcado para descobrir novas funções 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-isótopo rotulagem é uma técnica útil para determinar o metabolismo da célula central para vários tipos de microorganismos. Depois que as células foram cultivadas com um substrato específico rotulados, GC-MS medição pode revelar vias metabólicas funcionais com base em padrões de rotulagem única no proteinogénicos aminoácidos.

Confirmação via metabólica e descoberta através 13 C-rotulagem de proteinogénicos 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 visualizações.  Washington University. 13 C-isótopo rotulagem é uma técnica útil para determinar o metabolismo da célula central para vários tipos de microorganismos. Depois que as células foram cultivadas com um substrato específico rotulados, GC-MS medição pode revelar vias metabólicas funcionais com base em padrões de rotulagem única no proteinogénicos aminoácidos.

Vídeo: 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.

Usando o sistema de fracionamento GELFREE 8100 para Fracionamento baseada no peso molecular com Recovery Fase Líquida

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

Biologia

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.

Um passo-metabolômica: carboidratos, ácidos orgânicos e aminoácidos quantificados em um procedimento 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 quantificação Microvolume de Ácidos Nucléicos

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.

Análise do Perfil Metabólico de embriões Zebrafish

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.

A medição da taxa metabólica em<em&gt; Drosophila</em&gt; Usando respirometria

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.

Determinação baseado no crescimento e confirmação bioquímica de Requisitos Genéticos para a degradação de proteínas em<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.

Desenvolvimento de inibidores de interacções proteína-proteína através REPLACE: Aplicação para a concepção e desenvolvimento não-ATP inibidores 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.

Incorporação específico de resíduos de não-canônicos Aminoácidos em proteínas modelo Usando um<em&gt; Escherichia coli</em&gt; Sistema de transcrição-tradução livre-Cell

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.

Isolamento de filtração de Ácidos Nucleicos: Um Método de extracção de DNA simples e 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.

Culturas primárias mistas de Murino Intestino Delgado destinadas ao estudo de Gut hormonal Secreção e imagens de células vivas de Células Enteroendócrinas