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发布于:2019-5-9 23:38:11  访问:61 次 回复:0 篇
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AlthParasitesTissue culture-derived trypomastigotes in the CL-Brener strain of T. cruzi had been
To label the parasites with Neu5Az, they were incubated for 30min in PBS Dimethocainemechanism of action containing 1mM Neu5AzLac or Neu5AzGal as donor and 10mM 2-deoxi-Glucose (Sigma, MO). Soon after completely washing, parasites had been incubated overnight with Phosphine compounds or for 30min with DBCO compounds, which have quicker reactionPLOS Pathogens | DOI:10.1371/journal.ppat.1005559 April 8,17 /T. cruzi Surface GlycosylationFig 8. The membrane of trypomastigotes is complicated to the nanometer scale. Membrane model for T. cruzi trypomastigotes. The surface is packed in microdomains of distinct size, shape, lipid composition and embedded proteins. Some of these domains are detergent resistant, having said that this does not imply a functional profile. Mucins are incorporated in DRMs whereas TS is just not, thus becoming ZylofuramineCOA segregated within the membrane of trypomastigotes. This challenges the membrane bound TS as the sialylating element for mucins, a role proposed for the shed TS as an alternative. DRMs embed distinctive proteins, several of them localized for the flagellum. Neu5Az-LacOMe (Neu5AzLac) and Neu5Az-GalOMe (Neu5AzGal) had been synthesized [67]. The Neu5Az sialic acid was reacted with many compounds: Phos-FLAG, Phos-(PEG)3-Biotin (Thermo Scientific, Waltham, MA) DBCO(PEG)4-Biotin and DBCO-FLAG (Jena Bioscience, Germany) [30, 31]. All compounds had been applied at saturating circumstances. Equivalent labeling in between compounds was verified by Western blot and immunofluorescence. Phos-FLAG or DBCO-FLAG had been preferred for imaging because they showed considerable lower background. To label the parasites with Neu5Az, they had been incubated for 30min in PBS containing 1mM Neu5AzLac or Neu5AzGal as donor and 10mM 2-deoxi-Glucose (Sigma, MO). The enzymatic labeling was accomplished by the endogenous parasite-derived TS inside the case of trypomastigotes or by the addition of recombinant TS (10ng/ 106 cells) for epimastigotes. Soon after completely washing, parasites had been incubated overnight with Phosphine compounds or for 30min with DBCO compounds, which have faster reactionPLOS Pathogens | DOI:ten.1371/journal.ppat.1005559 April eight,17 /T. cruzi Surface GlycosylationFig eight. The membrane of trypomastigotes is complicated for the nanometer scale. Membrane model for T. cruzi trypomastigotes. The surface is packed in microdomains of diverse size, shape, lipid composition and embedded proteins. A few of these domains are detergent resistant, however this will not imply a functional profile. Mucins are incorporated in DRMs whereas TS just isn‘t, thus getting segregated in the membrane of trypomastigotes. This challenges the membrane bound TS as the sialylating element for mucins, a role proposed for the shed TS as an alternative. DRMs embed distinct proteins, several of them localized for the flagellum. Flagellum domains are likely to be smaller sized and closer with each other than these inside the cell body and recommend an association to the flagellar cytoskeleton. Mucins and TS are shed to the extracellular environment integrated in microvesicles almost certainly resulting from membrane budding and fission events. Additionally, TS is shed associated to vesicles instead of as a soluble protein. No hydrolysis in the GPI-anchors happens within the trypomastigote stage. doi:10.1371/journal.ppat.1005559.gPLOS Pathogens | DOI:10.1371/journal.ppat.1005559 April eight,18 /T. cruzi Surface Glycosylationkinetics [30, 31]. As controls, trypomastigotes were incubated with 3‘sialyllactose (Carbosynth, UK) and labeled via exactly the same procedure.Recombinant protein expression and purificationRecombinant TS was expressed in PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28497120 bacteria and purified as described elsewhere [3].
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