Agradecemos a A. Riccio pelos comentários úteis e de ler o manuscrito. Este trabalho foi financiado pelo Conselho de Pesquisa Médica de financiamento (MRC) para a Unidade de Biologia Celular e por um Human Frontier Ciência Grant Program (RGP0048/2009-C).

1. Reação enzimática - 1 º dia (1 hora da tarde) <ol><li> O primeiro passo é preparar 20/10 independentes reações enzimáticas em que IP 6 K1 ou VIP1 converter IP 6 e as isoformas pyrophosphorylated. </li><li> Usamos His-IP 6 K1 e GST-Vip1 enzimas purificada de E. coli acordo com o protocolo descrito anteriormente 17,18. </li><li> Prepare 50 mL reações contendo 1X tampão de reação (30 mM Hepes pH 6,8, 50 mM NaCl, 6 mM MgSO 4, 1 mM DTT), 6 mM fosfocreatina (PCr), 25 U / mL CreatinePhosphoKinase (CPK), 5 mM ATP (Mg sal), 0,3 mM IP6, 0,05-0,1 mg His-IP 6 K1 ou GST-Vip1. Ajustar o volume com MiliQ DDH 2 O. </li><li> Brevemente girar a reação e incubar a 37 ° C durante a noite com rotação. </li></ol> 2. Fundição em gel de poliacrilamida e carregamento - dia 2 (4 horas da tarde) <ol><li> O gel de poliacrilamida é preparada usando 24 cm de comprimento, 18 cm de largura e placas de vidro 1,5 milímetros espaçadores de largura. Geralmente uma faixa de 16 ou um preparativo comb única pista é usada. </li><li> Prepare uma mistura (50 mL / gel) contendo o seguinte: 35,5% (w / v) acrilamida: bis-acrilamida 19:1, persulfato de amônio 1X Tris / Borato / EDTA (TBE), 0,05% (w / v) (APS ), 0,05% (w / v) Temed. Despeje a mistura entre o pré-fundido placas de vidro, coloque o pente e deixar polimerizar por 30-60 minutos em temperatura ambiente. </li><li> Uma vez que o gel tem polimerizado, a transferência do aparelho para a sala fria e pré-executado em TBE 1X por cerca de 30-60 minutos a 200-300 Volts. </li><li> Adicionar 1X de OrangeG corante (10 mM Tris-HCl pH 7,0, EDTA 1 mM, glicerol 30%, 0,1 OrangeG%) para cada reação. Preparar uma amostra contendo 2 nmol de IP 6 para carregar como um controle padrão. </li><li> Lavar cada cavidade completamente com tampão de corrida usando uma seringa e uma agulha 21G para remover qualquer precipitado, em seguida, carregar o gel. Evitar carregar nos poços de lado. </li><li> Executar o gel durante a noite a 450-550 volts (7 mAmp / gel), até que a banda corante OrangeG é nos últimos 10 cm da parte inferior do gel. </li></ol> 3. Isolamento do IP 7 - dia 3 (4 horas) e dia 4 (6-7 processo SpeedVac hora de secagem) <ol><li> Desmontar o aparelho gel e remova cuidadosamente uma placa de vidro deixando o gel sobre o outro. Corte uma pequena porção do gel a partir de apenas acima da banda corante OrangeG para o fundo que contém o padrão de 6 IP e uma pista de amostra, conforme mostrado na Figura 1. </li><li> Mancha a parte cortada do gel com Azul de Toluidina (0,1% (w / v) azul de toluidina, 20% (w / v) metanol, 2% (w / v) de glicerol) por alguns minutos (1-3 min) ou até que a banda pirofosfato inositol aparece. Coloque a placa de vidro previamente removido de volta ao topo do gel para evitar o gel imaculada de secagem. </li></ol> A banda 7 IP devem ser visíveis, uma vez que é executado um pouco mais lento do que o padrão de 6 IP. ATP, que corre mais rápido do que 6 IP, também deve ser visível (Figura 1). Transferir a parte manchada do gel em uma solução de coloração (20% (w / v) metanol) por alguns minutos, lave o excesso de azul de toluidina e reposicionar o gel com o gel imaculada. Se a visualização de maior pyrophosphorylated isoformas inositol (IP 8 e IP 9) é exigido, a mancha do gel com azul de toluidina solução corante por 20 minutos em temperatura ambiente. Posteriormente, lavar o azul de toluidina com a solução de coloração para cerca de 15 minutos. <ol start="3"><li> Com uma lâmina de corte da banda 7 IP na parte imaculada do gel utilizando como referência a posição migração IP 7 determinada com o gel corado (Figura 1). </li><li> Colocar a banda 7 IP que foi cortada do gel em um tubo de 15 mL e adicionar 10 mL de MilliQ DDH 2 O. Coloque os tubos em rotação por 10 minutos em temperatura ambiente. Descartar o líquido para remover o excesso de partículas de acrilamida TBE e microscópicas. </li><li> Posteriormente, realizar duas desidratação-hidratação ciclos. Adicionar 5 mL de 50% (w / v) metanol para o tubo com o gel contendo 7 IP e rodar em temperatura ambiente por 2 horas. Transferência do gel para um tubo de 15 mL novo contendo 5 mL de MilliQ DDH 2 O e gire em temperatura ambiente por 2 horas. Não descarte o metanol e MilliQ H 2 O a partir dos tubos. Repita o ciclo de desidratação-hidratação, uma vez mais por re-transferência de gel de poliacrilamida a nos 15 tubos mL usado anteriormente. Uma das lavagens podem ser realizadas durante a noite. </li><li> Para concentrar o IP eluída 7, secar a 10 mL em conjunto (5 mL MilliQddH 2 O e 5 mL de metanol 50% (w / v)), utilizando um SpeedVac aquecida a 60 ° C. </li><li> Uma vez que as amostras são quase seco, transferir o líquido restante (300-600 mL) para A1.5 tubo de centrifugação mL e girar durante 2 minutos a 5000 rpm. </li><li> Recolher o sobrenadante e transferir para um novo tubo de centrífuga de 1,5 mL; deixara 20-30 mL de fundo, pois pode conter partículas de acrilamida. </li><li> Se necessário continuar o processo de secagem usando um SpeedVac sem aquecimento. A recuperação do IP 7 é drasticamente reduzido se as amostras secar completamente, portanto, finalizar o processo de secagem quando as amostras atingir o volume de 100-300 mL. </li></ol> 4. Determinação da concentração de 7 IP e pureza. <ol><li> Use 2-5 mL da amostra recuperada IP 7 para executar em um gel de PAGE, à semelhança do Seções 2.2-2.5. Carregar várias diluições de IP 6 (ie 0,5, 1, 2, 4 nmol) como padrão de concentração e 4 nmol de Poly-P marcador. Depois de executar o gel, visualize as isoformas pirofosfato inositol por coloração e coloração de-gel com toda a solução de toluidina azul, seguindo o procedimento descrito na Seção 3.2 (Figura 2A). </li><li> Após a coloração toluidina, as concentrações podem ser determinadas por varredura do gel e comparar as diferenças de intensidade entre 6 IP e IP 7, utilizando o software de imagem, como J-Imagem, como mostrado na Figura 2B. </li></ol> 5. Resultados representativos: A conversão enzimática de preparativa IP 6 e IP 7 usando enzimas IP 6 K1 e VIP1 pode ser facilmente resolvida através da análise PAGE (Figura 1). O carregamento de seis IP como um controle de tamanho juntamente com Toluidina coloração azul gel permite a identificação dos derivados pyrophosphorylated, uma vez que ficar mais lento, dependendo do número de grupos fosfatos presentes no anel de inositol. O procedimento descrito acima permite a purificação fácil de 7 de IP. A análise do pirofosfato purificada inositol por PAGE revelou a pureza do nosso IP 7 (Figura 2A). Curiosamente, a 1/3PP-IP 5 isômero do produto 7 de IP VIP1 migra ligeiramente mais lento do que o IP 5 5pp-isômero de 7 de IP que é gerado pelo IP 6 K1. Uso de IP 6 padrões permitem uma quantificação mais fácil da concentração do IP purificada 7 (Figura 2B). Antes de usar sete IP para novas experiências, sua atividade biológica pode ser avaliada (Figura 3). 5pp-IP 5 é incubada com VIP1 e com a fosfatase 7 IP DDP1 (diphosphoinositol phosphohydrolase polifosfato). Rotineiramente, o IP purificada sete é convertido para IP 8 por VIP1 e IP 6 por DDP1 (Figura 3). <img alt="Figura 1" src="/files/ftp_upload/3027/3027fig1.jpg" /> Figura 1:. Coloração Toluidina de PAGE e isolamento da banda 7 IP A porção do gel contendo o padrão (IP 6) foi cortado e corado usando uma solução de azul de toluidina. As três bandas representam (de cima para baixo) IP 7, IP 6 e ATP. A parte manchada do gel foi, então, alinhado com o restante do gel. Isso permite que a localização da parte do gel contendo 7 IP, que pode então ser cortada e purificada (caixa tracejada). <img alt="Figura 2" src="/files/ftp_upload/3027/3027fig2.jpg" /> Figura 2: Análise PAGE dos produtos de reacção IP 6 K1 e VIP1. A) Análise de IP 6 (4, 2, 1, 0,5 nmol) pela coloração azul de toluidina foi utilizado para determinar a concentração de 7 IP purificadas de ambas 6 K1 IP (IP-5pp 5) e VIP1 (1/3PP-IP 5) reações. B) Dispersão análise para determinar a concentração do IP purificada 7. As concentrações foram determinadas de acordo com a intensidade da banda, calculado utilizando o software ImageJ, em comparação com quantidades pré-determinadas de 6 de IP. O eixo X representa intensidades, o eixo Y representa a concentração expressa em nmol. <img alt="Figura 3" src="/files/ftp_upload/3027/3027fig3.jpg" /> Figura 3:. Análise da atividade 7 IP biológica Para determinar a qualidade do IP purificada sete nós incubadas 5pp-IP 5 (IP 6 IP K1 gerados 7) com VIP1 ou com a fosfatase 7 IP DDP1 e então resolvida a reação na página. A coloração DAPI e toluidina revelou a produção esperada de 8 por IP VIP1 ea conversão do IP 7 a 6 por IP DDP1.

<ol>
	<li>Bennett, M., Onnebo, S. M., Azevedo, C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+metabolism+and+signaling.">Inositol pyrophosphates: metabolism and signaling.</a> Cell Mol Life Sci. 63, 552-564 (2006).</li><li>Burton, A., Hu, X., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Are+inositol+pyrophosphates+signalling+molecules?.">Are inositol pyrophosphates signalling molecules?.</a> J Cell Physiol. 220, 8-15 (2009).</li><li>Barker, C. J., Illies, C., Gaboardi, G. C., Berggren, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates:+structure,+enzymology+and+function.">Inositol pyrophosphates: structure, enzymology and function.</a> Cell Mol Life Sci. 66, 3851-3871 (2009).</li><li>Shears, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diphosphoinositol+polyphosphates:+metabolic+messengers.">Diphosphoinositol polyphosphates: metabolic messengers.</a> Mol Pharmacol. 76, 236-252 (2009).</li><li>Saiardi, A., Erdjument-Bromage, H., Snowman, A. M., Tempst, P., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+diphosphoinositol+pentakisphosphate+by+a+newly+identified+family+of+higher+inositol+polyphosphate+kinases.">Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases.</a> Curr Biol. 9, 1323-1326 (1999).</li><li>Draskovic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+hexakisphosphate+kinase+products+contain+diphosphate+and+triphosphate+groups.">Inositol hexakisphosphate kinase products contain diphosphate and triphosphate groups.</a> Chem Biol. 15, 274-286 (2008).</li><li>Mulugu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+family+of+enzymes+that+phosphorylate+inositol+hexakisphosphate.">A conserved family of enzymes that phosphorylate inositol hexakisphosphate.</a> Science. 316, 106-109 (2007).</li><li>Fridy, P. C., Otto, J. C., Dollins, D. E., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+characterization+of+two+human+VIP1-like+inositol+hexakisphosphate+and+diphosphoinositol+pentakisphosphate+kinases.">Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases.</a> J Biol Chem. 282, 30754-30762 (2007).</li><li>Illies, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+inositol+pyrophosphates+for+full+exocytotic+capacity+in+pancreatic+beta+cells.">Requirement of inositol pyrophosphates for full exocytotic capacity in pancreatic beta cells.</a> Science. 318, 1299-1302 (2007).</li><li>Saiardi, A., Resnick, A. C., Snowman, A. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+cell+death+and+telomere+length+through+phosphoinositide+3-kinase-related+protein+kinases.">Inositol pyrophosphates regulate cell death and telomere length through phosphoinositide 3-kinase-related protein kinases.</a> Proc Natl Acad Sci U S A. 102, 1911-1914 (2005).</li><li>York, S. J., Armbruster, B. N., Greenwell, P., Petes, T. D., York, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+diphosphate+signaling+regulates+telomere+length.">Inositol diphosphate signaling regulates telomere length.</a> J Biol Chem. 280, 4264-4269 (2005).</li><li>Luo, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+mediate+chemotaxis+in+Dictyostelium+via+pleckstrin+homology+domain-PtdIns(3,4,5)P3+interactions.">Inositol pyrophosphates mediate chemotaxis in Dictyostelium via pleckstrin homology domain-PtdIns(3,4,5)P3 interactions.</a> Cell. 114, 559-572 (2003).</li><li>Saiardi, A., Sciambi, C., McCaffery, J. M., Wendland, B., Snyder, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+regulate+endocytic+trafficking.">Inositol pyrophosphates regulate endocytic trafficking.</a> Proc Natl Acad Sci U S A. 99, 14206-14211 (2002).</li><li>Auesukaree, C., Tochio, H., Shirakawa, M., Kaneko, Y., Harashima, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plc1p,+Arg82p,+and+Kcs1p,+enzymes+involved+in+inositol+pyrophosphate+synthesis,+are+essential+for+phosphate+regulation+and+polyphosphate+accumulation+in+Saccharomyces+cerevisiae.">Plc1p, Arg82p, and Kcs1p, enzymes involved in inositol pyrophosphate synthesis, are essential for phosphate regulation and polyphosphate accumulation in Saccharomyces cerevisiae.</a> J Biol Chem. 280, 25127-25133 (2005).</li><li>Azevedo, C., Burton, A., Ruiz-Mateos, E., Marsh, M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphate+mediated+pyrophosphorylation+of+AP3B1+regulates+HIV-1+Gag+release.">Inositol pyrophosphate mediated pyrophosphorylation of AP3B1 regulates HIV-1 Gag release.</a> Proc Natl Acad Sci U S A. 106, 21161-21166 (2009).</li><li>Bhandari, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+pyrophosphorylation+by+inositol+pyrophosphates+is+a+posttranslational+event.">Protein pyrophosphorylation by inositol pyrophosphates is a posttranslational event.</a> Proc Natl Acad Sci U S A. 104, 15305-15310 (2007).</li><li>Azevedo, C., Burton, A., Bennett, M., Onnebo, S. M., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+InsP7+by+the+Inositol+Hexakisphosphate+Kinase+1+(IP6K1).">Synthesis of InsP7 by the Inositol Hexakisphosphate Kinase 1 (IP6K1).</a> Methods Mol Biol. 645, 73-85 (2010).</li><li>Otto, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+analysis+of+inositol+phosphate+kinases.">Biochemical analysis of inositol phosphate kinases.</a> Methods Enzymol. 434, 171-185 (2007).</li><li>Losito, O., Szijgyarto, Z., Resnick, A. C., Saiardi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inositol+pyrophosphates+and+their+unique+metabolic+complexity:+analysis+by+gel+electrophoresis.">Inositol pyrophosphates and their unique metabolic complexity: analysis by gel electrophoresis.</a> PLoS One. 4, e5580-e5580 (2009).</li></ol>

Não há conflitos de interesse declarados.

O uso de pirofosfato de inositol em bioquímica é severamente limitada pela indisponibilidade comercial de tais compostos ea pouca sensibilidade dos métodos de detecção existentes. A combinação de PAGE, que permite a separação de moléculas que possuem número diferente de grupos fosfato e Azul de Toluidina (Figura 1), um corante metacromática que se liga aos grupos fosfato, permite a fácil detecção de isoformas pyrophoshate inositol abrindo novos caminhos de pesquisa 20. O uso da tecnologia descrita PAGE para purificar produtos pirofosfato inositol da reação enzimática realizada outby ou 6 IP K1 ou VIP1 é um método simples, econômico e confiável, que permite a produção de grandes quantidades de IP de alta qualidade 7. O método descrito acima não se limita à simples purificação do IP 7, mas pequenas modificações do protocolo descrito podem permitir a purificação de uma gama diferente de pirofosfatos inositol. Maior fosforilada isoformas pirofosfato inositol, contendo mais de oito grupos de fosfato pode ser detectada usando IP 7 ou diferentes quantidades de 6 IP como substrato 20,6. Estes pirofosfatos inositol pode ser detectado através do aumento da duração do processo de coloração e posteriormente purificada (seção 3.2). Além disso, o uso de 5 IP como substrato para a reação enzimática permitiria a purificação do PP-IP 5 e outras pirofosfatos inositol contendo um grupo hidroxila no anel inositol. Em conclusão, este método permite pouco exigente para a purificação de confiança de quantidades miligrama de pirofosfatos inositol com instrumentos amplamente disponível, abrindo novos caminhos para esse campo de pesquisa emocionante.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número de catálogo </th></tr><tr><td> Ácido fítico (IP 6) </td><td> Sigma-Aldrich </td><td> P8810 </td></tr><tr><td> Poli-P (hexametafosfato de sódio) </td><td> Sigma-Aldrich </td><td> P8510 </td></tr><tr><td> ATP-Mg 2 + sal </td><td> Sigma-Aldrich </td><td> A9187 </td></tr><tr><td> OrangeG </td><td> Sigma-Aldrich </td><td> O3756 </td></tr><tr><td> Fosfocreatina (PCr) </td><td> Sigma-Aldrich </td><td> P7936 </td></tr><tr><td> Quinase CreatinePhospho (CPK) </td><td> Sigma-Aldrich </td><td> C3755 </td></tr><tr><td> GST-Vip1 </td><td> 17 </td><td> 17 </td></tr><tr><td> His-IP6K1 </td><td> 18 </td><td> 18 </td></tr><tr><td> His-Ddp1 </td><td> Disponível em laboratório </td><td> Disponível em laboratório </td></tr><tr><td> Acrilamida: bis-acrilamida 19:01 (40%) </td><td> Flowgen </td><td> H16972 </td></tr><tr><td> Persulfato de amônio (APS) </td><td> Sigma-Aldrich </td><td> A9164 </td></tr><tr><td> Tris / Borato / EDTA (TBE) </td><td> Sigma-Aldrich </td><td> T 9060 </td></tr><tr><td> Temed </td><td> BDH </td><td> 43083G </td></tr><tr><td> Azul de Toluidina </td><td> Sigma-Aldrich </td><td> 198161 </td></tr><tr><td> SpeedVac </td><td> Cristo </td><td> 100218 </td></tr><tr><td> Aparelho de gel </td><td> Hoefer </td><td> SE600 </td></tr><tr><td> Colector de vácuo </td><td> Cristo </td><td> Alpha 04/02 </td></tr><tr><td> Bomba de vácuo </td><td> ABM Greiffenberger </td><td> 4EKF63CX </td></tr></tbody></table>

preparation of quality inositol pyrophosphates

Myo-inositol está presente na natureza ou não modificado ou mais complexos derivados fosforilados. Do último, os dois mais abundantes nas células eucarióticas são pentakisphosphate inositol (IP 5) e hexakisphosphate inositol (ácido fítico ou IP 6). IP 5 e IP 6 são os precursores de moléculas de pirofosfato inositol que contêm uma ou mais ligações pirofosfato 1. Fosforilação de IP 6 gera diphoshoinositolpentakisphosphate (IP 7 ou PP-PI 5) e bisdiphoshoinositoltetrakisphosphate (IP 8 ou (PP) 2 IP-4). Pirofosfatos inositol foram isolados de todos os organismos eucarióticos, até agora estudados. Além disso, as duas classes distintas de enzimas responsáveis ​​pela síntese de pirofosfato inositol são altamente conservadas ao longo da evolução 2-4. O IP 6 quinases (IP 6 Ks) possuem uma enorme flexibilidade catalítico, convertendo IP 5 e IP 6 a PP-IP 4 e IP 7, respectivamente e, posteriormente, usando estes produtos como substratos, promover a geração de moléculas mais complexas 5,6 . Recentemente, uma segunda classe de enzimas gerando pirofosfato foi identificada na forma da proteína de levedura VIP 1 (também conhecido como PP-PI 5 K), que é capaz de converter IP 6 e IP 7 e 8 IP 7,8. Pirofosfatos inositol regular muitas diferentes processos celulares, tais como a secreção de insulina, 9 de telômeros de comprimento 10,11, quimiotaxia, 12 de tráfico vesicular 13, 14 e homeostase do fosfato HIV-1 gag release 15. Dois mecanismos de ação têm sido propostos para esta classe de moléculas. Eles podem afetar a função celular por alostericamente interagir com proteínas específicas como a AKT 16. Alternativamente, o grupo pirofosfato pode doar um fosfato de pré-fosforilada proteínas 17. O enorme potencial deste campo de pesquisa é dificultada pela ausência de uma fonte comercial de pirofosfatos inositol, que está impedindo que muitos cientistas de estudar essas moléculas e esta modificação pós-translacional novo. Os métodos actualmente disponíveis para isolar pirofosfatos inositol exigem aparato cromatográfico sofisticado 18,19. Estes procedimentos utilizam condições ácidas que podem levar à degradação pirofosfato inositol 20 e, assim, para a recuperação pobres. Além disso, o incômodo procedimentos pós-coluna dessalinização restringir seu uso aos laboratórios especializados. Neste estudo, descrevemos um método pouco exigente para a geração de isolamento e purificação dos produtos do IP 6-quinase e PP-IP 5-quinases reações. Este método foi possível pela capacidade de eletroforese em gel de poliacrilamida (PAGE) para resolver altamente fosforilada polifosfatos inositol 20. Após IP 6 K1 e PP-IP 5 reações enzimáticas K usando 6 IP como substrato, PAGE foi usada para separar o pirofosfatos gerada inositol, que foram posteriormente eluídos em água.

Watch this Scientific Journal Video about Preparation of Quality Inositol Pyrophosphates at JoVE.com

Preparation of Quality Inositol Pyrophosphates

Pirofosfatos inositol desempenhar um papel importante em patologias humanas como câncer, diabetes e obesidade, no entanto, o mecanismo exato de ação é uma questão de disputa. A falta de disponível comercialmente pirofosfatos inositol estudos detalhados torna problemática. Aqui nós descrevemos um protocolo simples para produzir e isolar miligramas de pirofosfatos inositol.

Preparação de Qualidade pirofosfatos Inositol

Myo-inositol is present in nature either unmodified or in more complex phosphorylated derivates. Of the latest, the two most abundant in eukaryotic cells ...

preparation-of-quality-inositol-pyrophosphates

Research

JoVE Journal

Biology

13.0K visualizações.  University College London. Pirofosfatos inositol desempenhar um papel importante em patologias humanas como câncer, diabetes e obesidade, no entanto, o mecanismo exato de ação é uma questão de disputa. A falta de disponível comercialmente pirofosfatos inositol estudos detalhados torna problemática. Aqui nós descrevemos um protocolo simples para produzir e isolar miligramas de pirofosfatos inositol.

Vídeo: Preparation of Quality Inositol Pyrophosphates

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarrays de DNA: Controle de Qualidade da amostra, Hibridação Array e Digitalização

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Biologia

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

A técnica de amostragem não invasiva cabelo obter DNA de alta qualidade a partir de Elusive Pequenos Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A longo prazo de Teste de toxicidade letal com o crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with single-particle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules. 
The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules. 
The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

Do&#039;s and Don&#039;ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

This paper describes the cryo-electron microscopy methodology, which is used to obtain high quality microscopic images of macromolecules in their near-native state. The method yields images suitable for further computerized processing using the single particle approach, devised to generate the 3D reconstruction of a macromolecule.

Fazer e não fazer de Microscopia Cryo-elétron: uma cartilha sobre Preparação de Amostras e coleta de dados de alta qualidade para a Reconstrução Macromolecular 3D

Cryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

A ferramenta Web para caminhos biológicos que gera alta qualidade de leitura mecânica

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

Sperm Collection of Differential Quality Using Density Gradient Centrifugation

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with fertilizing ability are required to interact with a female gamete to ensure fertilization. As such, the fertilizing ability of individual sperm cells is critical for successful reproduction. Density gradient centrifugation has been utilized for several decades as a reproducible, fast, efficient, effective and extremely adaptable method to collect only high-quality sperm to be used in assisted reproductive technology. The protocols we described herein focus on the utilization of the discontinuous Percoll density gradient centrifugation (PDGC) technique to isolate three distinct populations of rooster sperm by their quality. We were able to collect low-, medium- and high-quality sperm. We also describe reproducible protocols that entail determining fertility potential of sperm by assessing their viability, mobility and penetrability. Collection of sperm by their quality using PDGC technique would be useful to accurately and thoroughly characterize sperm with differential fertility potential.

In this paper, we aim to describe the performance of the density gradient centrifugation technique and its application in sperm physiology research.

Coleção de esperma de qualidade diferencial usando centrifugação gradiente de densidade

In sexual reproduction, a male gamete or sperm cell fuses with a female gamete to bring about fertilization. However, a large number of sperm cells with ...

Field Identification of Matricaria chamomilla using a Portable qPCR System

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological assessment and chemical analytical methods. However, the lack of availability of botanists, especially in recent years, coupled with the need to enhance quality control to combat the stresses on the supply chain brought by increasing consumer demand and climate change, necessitates alternative approaches. The goal of this protocol is to facilitate botanical species identification using a portable qPCR system on the field or in any setting, where access to laboratory equipment and expertise is limited. Target DNA is amplified using dye-based qPCR, with DNA extracted from botanical reference materials serving as a positive control. The target DNA is identified by its specific amplification and matching its melting peak against the positive control. A detailed description of the steps and parameters, from hands-on field sample collection, to DNA extraction, PCR amplification, followed by data interpretation, has been included to ensure that readers can replicate this protocol. The results produced align with traditional laboratory botanical identification methods. The protocol is easy to perform and cost-effective, enabling quality testing on raw materials as close to the point of origin of the supply chain as possible.

Field Identification of Matricaria chamomilla using a Portable qPCR System

Presented here is a protocol for field identification of Matricaria chamomilla using a portable qPCR system. This easy-to-perform protocol is ideal as a method to confirm the identity of a botanical species at locations where access to laboratory equipment and expertise is limited, such as farms and warehouses.

Identificação de campo de matricaria chamomilla usando um sistema portátil qPCR

Quality control in botanical products begins with the raw material supply. Traditionally, botanical identification is performed through morphological ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Um ensaio de quimiotaxis melhorado para a identificação rápida de quimioattractants rizobacterianos em Exudates radiculares

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

The Cultivation, Growth, and Viability of Lactic Acid Bacteria: A Quality Control Perspective

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as yogurt and cheese. LAB predominantly produce lactic acid as a major end product of fermentation, and they synthesize important metabolites that impart the organoleptic characteristics of fermented food products. LAB are fastidious bacteria that thrive in many environments when adequate nutritional requirements are fulfilled. The demand for superior LAB dairy starter cultures for fermentation applications in the food and dairy industry, has resulted in the need to provide viable and active cultures for all bioprocessing operations. The development of a standard protocol for ensuring the viability and enhanced functionality of LAB cultures in the laboratory as well as dairy processing environments is thus very critical. In addressing concerns linked to resuscitating weak, stressed, and injured LAB culture cells, a protocol that vividly outlines salient steps to recover, enhance cell regeneration, and improve metabolic functionality of LAB strains is of the utmost importance. The maintenance of culture purity, functionality, and viability for LAB starter cultures is likewise critical. Therefore, adherence to a unique protocol guideline will result in the promotion of fermentation performance for many LAB strains dedicated to fermentation and biotechnology processes. As a result, the Food Microbiology and Biotechnology Laboratory at North Carolina Agriculture and Technical State University has developed a standard protocol for the activation and quality control of selected LAB strains that has resulted in highly functional and viable LAB culture strains employed for fermentation research. The adaptation and recommendation of a protocol such as this for use in the dairy and food industry&#160;will help to ensure LAB viability and functionality for many applications.

The quality control assessment of lactic acid bacteria (LAB) cultures has been confirmed as an effective way to enhance the viability and functionality of LAB strains for fermentation procedures. To buttress this assertion, we developed a protocol that elucidates how LAB cultures are activated and cultivated for fermentation and bioprocessing procedures.

O cultivo, crescimento e viabilidade de bactérias do ácido lático: uma perspectiva de controle de qualidade

Lactic acid bacteria (LAB) are essential dairy starter cultures that are significantly employed for the manufacture of fermented dairy products such as ...

Otimizando a qualidade do produto probiótico seco por pulverização por meio do desenvolvimento de processos

Process Development for the Spray-Drying of Probiotic Bacteria and Evaluation of the Product Quality

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that can confer beneficial effects on human and animal wellbeing, while prebiotics are types of nutrients that feed the beneficial gut bacteria. Powder probiotics have gained popularity due to the ease and practicality of their ingestion and incorporation into the diet as a food supplement. However, the drying process interferes with cell viability since high temperatures inactivate probiotic bacteria. In this context, this study aimed to present all the steps involved in the production and physicochemical characterization of a spray-dried probiotic and evaluate the influence of the protectants (simulated skim milk and inulin:maltodextrin association) and drying temperatures in increasing the powder yield and cell viability. The results showed that the simulated skim milk promoted higher probiotic viability at 80 &#176;C. With this protectant, the probiotic viability, moisture content, and water activity (Aw) reduce as long as the inlet temperature increases. The probiotics&#39; viability decreases conversely with the drying temperature.&#160;At temperatures close to 120 &#176;C, the dried probiotic showed viability around 90%, a moisture content of 4.6% w/w, and an Aw of 0.26; values adequate to guarantee product stability. In this context, spray-drying temperatures above 120 &#176;C are required to ensure the microbial cells&#39; viability and shelf-life in the powdered preparation and survival during food processing and storage.

Autor em destaque: Desenvolvimento de processos para a secagem por pulverização de bactérias probióticas e avaliação da qualidade do produto  

This protocol details the steps involved in the production and physicochemical characterization of a spray-dried probiotic product.

Desenvolvimento de processos para spray-drying de bactérias probióticas e avaliação da qualidade do produto

Probiotics and prebiotics are of great interest to the food and pharmaceutical industries due to their health benefits. Probiotics are live bacteria that ...