Questo studio è stato supportato da una carriera NSF Grant (MCB0954016) e un DOE Bioenergy Research Grant (DEFG0208ER64694).

1. Coltura cellulare (Figura 1) <ol><li> Crescere le cellule in terreno minimo con oligoelementi, sali, vitamine e substrati di carbonio specificatamente etichettati che sono i migliori per via di indagini. Utilizzare contenitori agitazione o bioreattori per colture cellulari. Nutrienti organici, come ad esempio estratto di lievito, può interferire con la misura di etichettatura di aminoacidi e quindi non può essere presente nel terreno di coltura. </li><li> Monitorare la crescita delle cellule per la densità ottica della cultura alle lunghezze d&#39;onda ottimali (ad esempio, OD 730 per Cyanothece 51142) con uno spettrofotometro UV / Vis. </li><li> Le cellule possono essere coltivate prima in un mezzo non etichettati. Il centro-log cellule fase di crescita sono preferiti da utilizzare per l&#39;inoculazione (3% (v / v) da rapporto di volume inoculazione) del mezzo etichettati. La cultura dovrebbe essere etichettati sotto-cultura (3% v / v rapporto inoculazione) sullo stesso supporto etichettati in modo da evitare l&#39;introduzione della non-etichettati carbonio dal inoculo iniziale. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. Amino acid estrazione <ol><li> Harvest sub-colture di cellule (10 ml) al centro-log fase di crescita mediante centrifugazione (10 min 8000 × g). </li><li> Risospendere il pellet in 1,5 ml di HCl 6M e trasferirlo ad un vetro trasparente, con tappo a vite fiala GC. Cap le fiale e metterli in un forno a 100 ° C per 24 ore di idrolizzare le proteine ​​in amminoacidi biomassa. Idrolisi di pellet biomassa può produrre 16 dei 20 comuni amminoacidi (Figura 2) 5. Cisteina e triptofano sono degradati, e glutammina e asparagina vengono convertiti in glutammato e aspartato, rispettivamente. </li><li> Centrifugare la soluzione di amminoacidi a 20.000 g per 5 minuti con 2 ml provette Eppendorf, e trasferire il surnatante alle nuove fiale GC. Questo passaggio rimuove le particelle solide nella soluzione idrolisi. </li><li> Rimuovere i coperchi fiala GC e asciugare i campioni completamente sotto un flusso di aria con un Thermo Scientific riattiva Vap evaporatore (nota: un liofilizzatore può essere utilizzato anche tcampioni o secco). Questo passo può essere fatto dall&#39;oggi al domani. </li></ol> 3. Derivatizzazione amino acidi e GC-MS condizioni Analisi di aminoacidi o di carica / altamente metaboliti polari via GC richiede che questi metaboliti essere derivatizzati, in modo che gli aminoacidi sono volatili e possono essere separati mediante gascromatografia 2. <ol><li> Sciogliere l&#39;campioni essiccati con 150 ml di tetraidrofurano (THF) e 150 ml di N-(terz-butyldimethylsilyl)-N-methyltrifluoroacetamide reagente derivatizzazione. </li><li> Incubare tutti i campioni in un forno o un bagno d&#39;acqua compresa tra 65 e 80 ° C per 1 ora. Vortice di tanto in tanto per assicurarsi che i metaboliti nel flacone sono dissolti. </li><li> Centrifugare i campioni a 20.000 × g per 10 minuti, quindi trasferire il surnatante alle nuove fiale GC. Il surnatante deve essere una soluzione chiara e giallognola. A causa della saturazione dei rivelatori, GC-MS precisione della misura può essere influenzata dal conce altantration di iniezione TBDMS derivatizzati amino acidi (questi campioni presenta spesso colore marrone scuro), quindi, dovremmo diluire i campioni con THF prima di GC-MS di misura 6. </li><li> Analizzare i campioni da GC-MS (usare una divisione 01:05 o rapporto 1:10, volume di iniezione = 1 ml, trasporto di gas elio = 1,2 mL / min). Utilizzare il seguente programma di temperatura GC: tenere a 150 ° C per 2 minuti, aumentare a 3 ° C al minuto a 280 ° C, aumentare a 20 ° C al minuto a 300 ° C, e quindi tenere per 5 minuti. Termine solvente può essere impostato come ~ 5 minuti (per una colonna di 30 metro GC). La gamma della massa per caricare il rapporto (m / z) in MS può essere impostata tra 60 e 500. </li></ol> 4. GC-MS analisi dei dati <ol><li> TBDMS misura derivatizzati aminoacidi può anche essere influenzata dalla discriminazione isotopica nella separazione GC. Isotopi luce muoversi leggermente più veloce di tirare isotopi nella colonna GC. Per ridurre il bias potenziale misura, possiamo media lo spettro di massa di tutta lapicco di acido gamma amino 6 </li><li> Il GC e MS spettri di metaboliti TBDMS derivatizzati sono stati segnalati prima del 7. Il tempo di ritenzione GC e l&#39;unico m / z picchi per ogni amminoacido sono illustrati nella Figura 3. </li><li> Derivatizzazione degli aminoacidi o metaboliti centrale introduce una notevole quantità di isotopi naturalmente marcato, tra cui 13 C (1,13%), 18 O (0,20%), 29 Si (4,70%), e 30 Si (3,09%). Della misura del rumore da isotopi naturali nello spettro prime isotopomero di massa può essere corretta utilizzando il software pubblicato 5, 8. I dati finali etichettatura isotopici sono segnalati come frazioni di massa, ad esempio, M 0, M 1, M 2, M 3 e M 4 (che rappresentano frammenti contenenti da zero a quattro atomi di carbonio 13 C etichettati). </li><li> Misurazione di aminoacidi in grado di fornire informazioni isotopica etichettatura circa otto metaboliti cruciale precursore: 2-oxo-glutarmangiavano, 3-P-glicerato, l&#39;acetil-CoA, erythrose-4-P, ossaloacetato, fosfoenolpiruvato, piruvato e ribosio-5-P. Gli schemi di etichettatura di questi metaboliti possono essere usati per individuare alcuni percorsi metabolici centrale (Figura 2) 9. Il risultato degli esperimenti di etichettatura può essere ulteriormente confermata con metodi biochimici (ad esempio, RT-PCR). </li></ol> 5. Percorso di analisi dei dati utilizzando l&#39;etichetta di aminoacidi Investigando solo pochi amino acidi chiave prodotto da ben progettato 13 C esperimenti tracciante, si può rivelare diversi percorsi unici o attività enzimatiche senza eseguire sofisticate 13 C-metabolica analisi del flusso di tutto il metabolismo centrale. <ol><li> Entner-Doudoroff percorso: [1 - 13 C] glucosio può essere utilizzato come fonte di carbonio. Se il percorso è attivo, l&#39;etichettatura serina sarà significativamente inferiore a quella di etichettatura alanina 10. </li><li> Ramificato TCA ciclo: [1 - 13 C]piruvato può essere utilizzato come fonte di carbonio. Se il ciclo TCA è rotto, aspartato può essere etichettata da due atomi di carbonio, mentre il glutammato viene etichettato con un solo carbonio 11, 12. </li><li> CO 2 fissazione di Calvin-Benson-Bassham ciclo in un metabolismo mixotrophic: non etichettati CO 2 ed etichettato substrati di carbonio sono entrambi utilizzati come fonti di carbonio. Se il ciclo di Calvin è funzionale, serina e istidina etichettatura sarà notevolmente diluita, rispetto ad altri aminoacidi. Tale metodo può determinare la relativa fissazione di CO 2 quando le fonti di carbonio organico sono presenti in media 13. </li><li> Ossidativo via dei pentoso fosfati: [1 - 13 C] glucosio può essere utilizzato come fonte di carbonio. Se il percorso è attivo, alanina non etichettati sarà&gt; 50% 12. </li><li> Anaplerotiche percorso (ad esempio, PEP + CO 2 ossalacetato à): 13 CO 2 e non etichettati carbonio substrati (per esempio, glicerolo o pyruvate) può essere utilizzato come fonte di carbonio. Se il percorso è attivo, l&#39;etichettatura aspartato sarà notevolmente arricchito, rispetto al alanina e serina 13. </li><li> Re-citrato sintasi: [1 - 13 C] piruvato può essere utilizzato come fonte di carbonio. Se l&#39;enzima è attivo, il glutammato è etichettato in β-carbossi gruppo 3, 4. </li><li> Percorso Citramalate: [1 - 13 C] piruvato, [2 - 13 C] glicerolo, o [1 - 13 C] acetato può essere utilizzato come fonte di carbonio. Se il percorso è attivo, gli importi etichettatura leucina e isoleucina sono identici 14. </li><li> Serina-isocitrato ciclo liasi: [1 - 13 C] piruvato o [1 - 13 C] lattato può essere utilizzato come fonte di carbonio. Se il percorso è attivo, il carbonio terza posizione in serina saranno etichettate 15. </li><li> Utilizzo di sostanze nutritive (ad esempio, amminoacidi esogeni): un terreno di coltura con substrati di carbonio completamente etichettati e non etichettati aminoacidipuò essere utilizzato. Se le cellule selettivamente utilizzare questi nutrienti integrato non etichettati, vedremo una significativa diluizione etichettatura di questi aminoacidi nella biomassa. Questo metodo può essere utilizzato per indagare quali supplementi nutrizionali sono preferiti dalla cella 16. </li></ol> 6. Rappresentante Risultati Studi recenti hanno ripreso la bioenergia interessi nell&#39;utilizzo microrganismi fototrofi romanzo per la produzione di bioenergia e cattura di CO 2. Negli ultimi anni, un bel po &#39;13C metabolismo analisi, tra cui all&#39;avanguardia 13C-Metabolica Analisi Flux (13C-MAE), sono stati applicati per indagare il metabolismo centrale nei batteri fototrofi, perché la conoscenza biochimica delle vie metaboliche centrale non è ben fondata in questi organismi non-modello 10, 11, 17-20. Qui, presentiamo un esempio della scoperta di un percorso isoleucina si alternano in Cyanothece 51.142 21. Cianoothece 51142 non contiene l&#39;enzima (CE 4.3.1.19, treonina ammoniaca-liasi), che catalizza la trasformazione del treonina a 2-ketobutyrate nel percorso tipico sintesi isoleucina. Per risolvere il percorso isoleucina, si cresce Cyanothece 51142 (20 ml) in ASP2 medio 22 con 54 mM glicerolo (2-13C,&gt; 98%). Cyanothece 51142 utilizza 2 ° posto glicerolo etichettata come la principale fonte di carbonio. Osserviamo che treonina e alanina hanno una etichetta carbonio, mentre isoleucina è etichettato con tre atomi di carbonio. Pertanto, in sintesi Cyanothece 51142 non possono essere derivati ​​dal percorso treonina utilizzati dalla maggior parte degli organismi (Figura 4). D&#39;altra parte, leucina e isoleucina hanno modelli identici etichettatura basato su frammento (M-15) + e frammento (M-159) +. Per esempio, i dati isotopomero da [M-15] + (contenente non frammentato aminoacidi) mostrano l&#39;etichettatura identico per leucina (M0 = 0,01, M1 = 0,03, = 0,21 M2, M3 = 0.69) E isoleucina (M0 = 0,01, M1 = 0,03, = 0,24 M2, M3 = 0,67). Così leucina e isoleucina devono essere sintetizzata dai precursori stesso (cioè, piruvato e acetil-CoA). Questa osservazione è coerente con la transizione carbonica marcata nella via citramalate per la sintesi isoleucina. A conferma di questa via, si cerca il comune banca dati Genome Institute e trovare la presenza di un sintasi citramalate Cima (cce_0248) in Cyanothece. <img src="/files/ftp_upload/3583/3583fig1.jpg" alt="Figura 1"/> Figura 1. Il 13 C-assistita fasi di analisi via. <img src="/files/ftp_upload/3583/3583fig2.jpg" alt="Figura 2"/> Figura 2. Aminoacidi utilizzato per acquisire il modello di etichettatura dei loro precursori metabolici. ACoA, l&#39;acetil-CoA, AKG, α-chetoglutarato; C5P, ribosio 5-fosfato, CIT, citrato; E4P, erythrose 4-fosfato; G6P, glucosio-6-fosfato; OAA,<html> ossalacetato; PEP, fosfoenolpiruvato, PGA, 3-fosfoglicerato, PYR, piruvato. <img src="/files/ftp_upload/3583/3583fig3.jpg" alt="Figura 3"/> Figura 3. Picchi GC di 16 aminoacidi. TBDMS acidi derivatizzati aminoacidi sono incrinate da MS in due frammenti: (M-57) +, che contiene l&#39;acido intero aminoacidi, e (M-159) +, che manca il gruppo α carbossile l&#39;aminoacido. Per leucina e isoleucina, la (M-57) + è stato sormontato da picchi di massa. Ti consigliamo di utilizzare frammento (M-15) + per analizzare l&#39;etichettatura intero aminoacido. L&#39;(f302) gruppo + viene rilevato nella maggior parte dei aminoacidi, che contiene solo il primo (α-carbossilico gruppo) e carboni secondo una dorsale di aminoacidi. Perché questo picco MS è spesso elevato rumore a segnale rapporti, (f302) + non è raccomandato per analizzare quantitativamente i flussi metabolici 7. IGURA 4 &quot;/&gt; Figura 4. Etichettatura transizioni nei percorsi isoleucina in Cyanothece 51142 (modificato dal nostro precedente articolo) 21.

<ol>
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Questo protocollo è costituito da alimentare la cella con un substrato etichettati e misurare i modelli con conseguente etichettatura isotopica in aminoacidi via GC-MS. Dal momento che MS dati (m / z rapporti) fare solo l&#39;importo complessivo di etichettatura di ioni MS, dobbiamo valutare le distribuzioni isotopomero di aminoacidi esaminando la m / z rapporti di entrambi non frammentato (M-57) + e frammentato aminoacidi (cioè, (M-159) + e (f302) +). Inoltre, siamo in grado di esegui...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th><th> Commenti (opzionale) </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 di carbonio etichettati </td><td> Cambridge Isotope Laboratories </td><td> Dipendono dal requisito sperimentale </td><td> Sito web: <a href="http://www.isotope.com">http://www.isotope.com</a> </td></tr><tr><td> Gascromatografo </td><td> Agilent Technologies </td><td> Hewlett-Packard, modello 7890A </td><td> - </td></tr><tr><td> GC colonne </td><td> J &amp; W Scientific, Folsom, CA </td><td> DB5 (30m) </td><td> - </td></tr><tr><td> Spettrometro di massa </td><td> Agilent Technologies </td><td> 5975C </td><td> - </td></tr><tr><td> Riattiva Vap Evaporator </td><td> Thermo Scientific </td><td> TS-18825 </td><td> Per l&#39;essiccazione dei campioni di amminoacidi </td></tr></tbody></table>

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

I microbi sono complesse vie metaboliche che possono essere indagati con metodi biochimica e genomica funzionale. Una tecnica importante esaminare il metabolismo centrale cellulare e scoprire nuovi enzimi è di 13 C-assistiti di analisi del metabolismo 1. Questa tecnica si basa sulla etichettatura isotopica, in cui sono alimentati i microbi con 13 substrati C etichettati. Tracciando i percorsi di transizione tra atomo metaboliti della rete biochimica, possiamo determinare i percorsi funzionali e scoprire nuovi enzimi. Come metodo complementare alla trascrittomica e la proteomica, approcci per isotopomero assistita analisi delle vie metaboliche contengono tre fasi principali 2. Per prima cosa, crescere le cellule con 13 substrati C etichettati. In questa fase, la composizione del mezzo e la selezione dei substrati marcati sono due fattori chiave. Per evitare rumori di misura dal carbonio non etichettati negli integratori di nutrienti, Un terreno minimo con una unica fonte di carbonio è necessario. Inoltre, la scelta di un substrato marcato è basato su come effettivamente sarà chiarire la via oggetto di analisi. Perché nuovi enzimi spesso coinvolgono diversi stereochimica di reazione o prodotti intermedi, in generale, substrati di carbonio singolarmente etichettati sono più informativi per la rilevazione di nuovi percorsi, di quelli etichettati in modo uniforme per la rilevazione di nuovi percorsi, 3, 4. In secondo luogo, analizziamo amino acido utilizzando schemi di etichettatura GC -MS. Gli aminoacidi sono abbondanti di proteine ​​e quindi può essere ottenuto da idrolisi di biomassa. Gli aminoacidi possono essere derivatizzati da N-(terz-butyldimethylsilyl)-N-methyltrifluoroacetamide (TBDMS) prima della separazione GC. TBDMS derivatizzati amino acidi può essere frammentato dagli Stati membri e determinare la disposizione degli frammenti. Funzione della massa di carica (m / z) rapporto di aminoacidi frammentato e non frammentati, possiamo dedurre i modelli possibili etichetta dei metaboliti centrali che sonoprecursori degli aminoacidi. In terzo luogo, abbiamo traccia transizioni di carbonio 13C nei percorsi proposti e, sulla base dei dati isotopomero, confermare se queste vie sono attivi 2. Misurazione di aminoacidi fornisce informazioni isotopica etichettatura circa otto metaboliti precursore fondamentale nel metabolismo centrale. Questi nodi chiave del metabolismo possono riflettere le funzioni associate di vie centrali. 13 C-assistita di analisi del metabolismo tramite proteinogenici aminoacidi può essere ampiamente usato per la caratterizzazione funzionale di mal caratterizzati metabolismo microbico 1. In questo protocollo, useremo Cyanothece 51142 come il ceppo modello per dimostrare l&#39;uso di substrati carbonica marcata per scoprire nuove funzioni enzimatiche.

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-isotopo etichettatura è una tecnica utile per determinare il metabolismo cellula centrale per vari tipi di microrganismi. Dopo che le cellule sono state coltivate con un substrato specifico etichettato, GC-MS di misurazione in grado di rivelare vie metaboliche funzionali basati su modelli di etichettatura unico nel proteinogenici aminoacidi.

Percorso di conferma metaboliche e scoperta attraverso 13 C-etichettatura dei proteinogenici Aminoacidi

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 Views.  Washington University. 13 C-isotopo etichettatura è una tecnica utile per determinare il metabolismo cellula centrale per vari tipi di microrganismi. Dopo che le cellule sono state coltivate con un substrato specifico etichettato, GC-MS di misurazione in grado di rivelare vie metaboliche funzionali basati su modelli di etichettatura unico nel proteinogenici aminoacidi.

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.

Utilizzando il sistema GELFREE 8100 Frazionamento per peso molecolare basata su Frazionamento con recupero della fase liquida

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

Ricerca

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.

One-step Metabolomica: carboidrati, acidi organici e aminoacidi quantificati in una procedura unica

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 microvolumi quantificazione degli acidi nucleici

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.

Analisi Profilo metabolico di embrioni di 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.

Misurazione del tasso metabolico in<em&gt; Drosophila</em&gt; Utilizzando 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.

Determinazione base alla crescita e conferma biochimica dei requisiti genetici per Protein Degradazione in<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.

Sviluppo di inibitori di interazioni proteina-proteina attraverso REPLACE: Applicazione alla progettazione e allo sviluppo non ATP inibitori CDK competitivo

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.

L&#39;incorporazione specifici residuo di non canonici amminoacidi in proteine ​​modello utilizzando un<em&gt; Escherichia coli</em&gt; Trascrizione-traduzione sistema non cellulare

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.

L&#39;isolamento di filtrazione di acidi nucleici: DNA metodo di estrazione semplice e veloce

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.