purification-microrna-profiling-exosomes-derived-from-blood-culture

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Drexel University College of Medicine

A putative Î²-hydroxybutyrate dehydrogenase (Î²HBDH) ortholog was identified in Trypanosoma brucei, the unicellular eukaryotic parasite responsible for causing African Sleeping Sickness. The trypanosome enzyme has greater sequence similarity to bacterial sources of soluble Î²HBDH than to membrane-bound Type I Î²HBDH found in higher eukaryotes. The Î²HBDH gene was cloned from T. brucei genomic DNA and active, recombinant His-tagged enzyme (His(10)-TbÎ²HBDH) was purified to approximate homogeneity from E. coli. Î²HBDH catalyzes the reversible NADH-dependent conversion of acetoacetate to D-3-hydroxybutyrate. In the direction of D-3-hydroxybutyrate formation, His(10)-TbÎ²HBDH has a k(cat) value of 0.19 s(-1) and a K(M) value of 0.69 mM for acetoacetate. In the direction of acetoacetate formation, His(10)-TbÎ²HBDH has a k(cat) value of 11.2 s(-1) and a K(M) value of 0.65 mM for D-3-hydroxybutyrate. Cofactor preference was examined and His(10)-TbÎ²HBDH utilizes both NAD(H) and NADP(H) almost equivalently, distinguishing the parasite enzyme from other characterized Î²HBDHs. Furthermore, His(10)-TbÎ²HBDH binds NAD(P)(+) in a cooperative fashion, another unique characteristic of trypanosome Î²HBDH. The apparent native molecular weight of recombinant His(10)-TbÎ²HBDH is 112 kDa, corresponding to tetramer, as determined through size exclusion chromatography. RNA interference studies in procyclic trypanosomes were carried out to evaluate the importance of TbÎ²HBDH in vivo. Upon knockdown of TbÎ²HBDH, a small reduction in parasite growth was observed suggesting Î²HBDH has an important physiological role in T. brucei.

The characterization of a unique Trypanosoma brucei Î²-hydroxybutyrate dehydrogenase.

Un putatif &beta;-hydroxybutyrate d&eacute;shydrog&eacute;nase (&beta;HBDH) orthologue a &eacute;t&eacute; identifi&eacute; &agrave; Trypanosoma brucei, parasite unicellulaire eucaryote responsable de la maladie du sommeil africaine. L&#39;enzyme trypanosome a une s&eacute;quence plus grande similarit&eacute; &agrave; des sources bact&eacute;riennes de &beta;HBDH soluble que de membranaires de Type I &beta;HBDH trouv&eacute; chez les eucaryotes sup&eacute;rieurs. Le g&egrave;ne &beta;HBDH a &eacute;t&eacute; clon&eacute; &agrave; partir d&#39;ADN g&eacute;nomique de T. brucei et active, recombinant enzyme His-tag (His(10)-Tb&beta;HBDH) a &eacute;t&eacute; purifi&eacute;e jusqu&#39;&agrave; homog&eacute;n&eacute;it&eacute; approximative d&#39;e. coli. &Beta;HBDH catalyse la conversion de NADH-d&eacute;pendante r&eacute;versible d&#39;ac&eacute;toac&eacute;tate en D-3-hydroxybutyrate. Dans la direction de la formation de D-3-hydroxybutyrate, son 10-Tb&beta;HBDH a une valeur de k(cat) de s(-1) 0.19 et une valeur K(M) de 0,69 mM pour ac&eacute;toac&eacute;tate. Dans le sens de formation de l&#39;ac&eacute;toac&eacute;tate, son 10-Tb&beta;HBDH a une valeur de k(cat) de s(-1) 11,2 et une valeur K(M) de 0,65 mM pour D-3-hydroxybutyrate. Pr&eacute;f&eacute;rence de cofacteur a &eacute;t&eacute; examin&eacute; et ses 10-Tb&beta;HBDH utilise les NAD et le NADP (h), de fa&ccedil;on presque &eacute;quivalente, distinguant l&#39;enzyme parasite des autres &beta;HBDHs caract&eacute;ris&eacute;es. En outre, son 10-Tb&beta;HBDH lie NAD(P)(+) de mani&egrave;re coop&eacute;rative, une autre caract&eacute;ristique unique du trypanosome &beta;HBDH. Le poids mol&eacute;culaire natif de recombinant sa 10-Tb&beta;HBDH est de 112 kDa, correspondant &agrave; t&eacute;tram&egrave;re, telle que d&eacute;termin&eacute;e par chromatographie d&#39;exclusion. RNA interference &eacute;tudes en trypanosomes procycliques ont &eacute;t&eacute; r&eacute;alis&eacute;es pour &eacute;valuer l&#39;importance des Tb&beta;HBDH in vivo. &Agrave; la pr&eacute;cipitation de Tb&beta;HBDH, une petite r&eacute;duction de croissance du parasite a &eacute;t&eacute; observ&eacute;e sugg&eacute;rant &beta;HBDH a un r&ocirc;le physiologique important dans T. brucei.

La caract&eacute;risation d&#39;une unique Trypanosoma brucei &beta;-hydroxybutyrate d&eacute;shydrog&eacute;nase.

Un putativo &beta;-idrossibutirrato deidrogenasi (&beta;HBDH) ortholog &egrave; stato identificato in Trypanosoma brucei, il parassita eucarioti unicellulare responsabile di malattia del sonno africana. L&#39;enzima trypanosome ha maggiore sequenza somiglianza alle fonti batteriche di &beta;HBDH solubile rispetto a membrana-limiti di tipo &beta;HBDH trovato negli eucarioti superiori. Il gene &beta;HBDH &egrave; stato clonato da T. brucei DNA genomic e attivo, ricombinante His-Tag enzima (His(10)-Tb&beta;HBDH) &egrave; stato purificato di omogeneit&agrave; approssimativa da e. coli. &Beta;HBDH catalizza la conversione di NADH-dipendente reversibile di acetoacetato in D-3-idrossibutirrato. Nella direzione della formazione D-3-idrossibutirrato, sua 10-Tb&beta;HBDH ha un valore di k(cat) di s(-1) 0,19 e un valore K(M) di 0,69 mM per acetoacetato. In direzione di formazione acetoacetato, sua 10-Tb&beta;HBDH ha un valore di k(cat) di s(-1) 11,2 e un valore K(M) di 0,65 mM per D-3-idrossibutirrato. Preferenza cofattore &egrave; stato esaminato e suo 10-Tb&beta;HBDH utilizza sia NAD(H) che NADP(H) quasi equivalentemente, distinguere l&#39;enzima parassita da altri &beta;HBDHs caratterizzato. Inoltre, sua 10-Tb&beta;HBDH lega NAD(P)(+) in maniera cooperativa, un&#39;altra caratteristica unica di trypanosome &beta;HBDH. Il peso molecolare apparente nativo ricombinante suo 10-Tb&beta;HBDH &egrave; 112 kDa, corrispondenti a tetramero, come determinato tramite cromatografia di esclusione di formato. Studi interferenza RNA in prociclico tripanosomi sono stati effettuati per valutare l&#39;importanza di Tb&beta;HBDH in vivo. Su atterramento di Tb&beta;HBDH, &egrave; stata osservata una piccola riduzione nella crescita del parassita che suggerisce &beta;HBDH ha un ruolo fisiologico importante nel T. brucei.

Kathryn E. Capasso

Department of Pharmacology & Physiology

Drexel University College of Medicine

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media