Introduction: Oncolytic virus therapy is currently considered as a promising therapeutic approach for cancer treatment. Adenovirus is well-known and extensively characterized as an oncolytic agent. The increasing number of clinical trials using this virus generates the demand for the development of a well-established purification approach.
View Article and Find Full Text PDFWestern blotting is a widely used method for analyzing specific target proteins in complex protein samples. Housekeeping proteins are often used for normalization to correct for uneven sample loads, but these require careful validation since expression levels may vary with cell type and treatment. We present a new, more reliable method for normalization using Cy5-prelabeled total protein as a loading control.
View Article and Find Full Text PDFA pre-labeling protocol based on Cy5 N-hydroxysuccinimide (NHS) ester labeling of proteins has been developed for one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. We show that a fixed amount of sulfonated Cy5 can be used in the labeling reaction to label proteins over a broad concentration range-more than three orders of magnitude. The optimal amount of Cy5 was found to be 50 to 250pmol in 20μl using a Tris-HCl labeling buffer at pH 8.
View Article and Find Full Text PDFWestern blotting is a powerful and widely used method, but limitations in detection sensitivity and specificity, and dependence upon high quality antibodies to detect targeted proteins, are hurdles to overcome. The in situ proximity ligation assay, based on dual antibody recognition and powerful localized signal amplification, offers increased detection sensitivity and specificity, along with an ability to identify complex targets such as phosphorylated or interacting proteins. Here we have applied the in situ proximity ligation assay mechanism in Western blotting.
View Article and Find Full Text PDFSurface proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of almost all intracellular signaling. Moreover, they play an important role in environmental adaptation and drug treatment, and are often involved in disease pathogenesis and pathology (1).
View Article and Find Full Text PDFGlucuronyl C5-epimerase catalyzes the conversion of d-glucuronic acid to l-iduronic acid units in heparan sulfate biosynthesis. Substrate recognition depends on the N-substituent pattern of the heparan sulfate precursor polysaccharide and requires the adjacent glucosamine residue toward the non-reducing end to be N-sulfated. Epimerization of an appropriately N-sulfated substrate is freely reversible in a soluble system, with equilibrium favoring retention of d-gluco configuration (Hagner-McWhirter, A.
View Article and Find Full Text PDFThe glycosaminoglycan, heparan sulfate (HS), binds proteins to modulate signaling events in embryogenesis. All identified protein-binding HS epitopes contain l-iduronic acid (IdoA). We report that targeted disruption of the murine d-glucuronyl C5-epimerase gene results in a structurally altered HS lacking IdoA.
View Article and Find Full Text PDFIn the biosynthesis of heparin and heparan sulphate, D-glucuronic acid residues are converted into L-iduronic acid (IdoA) units by C-5 epimerization, at the polymer level. The reaction catalysed by the epimerase occurs by reversible abstraction and readdition of a proton at C-5 of target hexuronic acid residues, through a carbanion intermediate, with or without an inversion of configuration at C-5 [Prihar, Campbell, Feingold, Jacobsson, Jensen, Lindahl and Rodén (1980) Biochemistry 19, 495-500]. Incubation of chemically N-sulphated capsular polysaccharide from Escherichia coli K5 ([4GlcAbeta1-4GlcNSO(3)alpha1-](n)), or of O-desulphated heparin (predominantly [4IdoAalpha1-4GlcNSO(3)alpha1-](n)) with purified C-5 epimerase from bovine liver, resulted in the interconversion of glucuronic acid and IdoA residues, which reached equilibrium (30-40% IdoA/total hexuronic acid) after approx.
View Article and Find Full Text PDFThe D-glucuronyl C5-epimerase involved in the biosynthesis of heparin and heparan sulfate was investigated with focus on its substrate specificity, its kinetic properties, and a comparison of epimerase preparations from the Furth mastocytoma and bovine liver, which synthesize heparin and heparan sulfate, respectively. New substrates for the epimerase were prepared from the capsular polysaccharide of Escherichia coli K5, which had been labeled at C5 of its D-glucuronic and N-acetyl-D-glucosamine moieties by growing the bacteria in the presence of D-[5-(3)H]glucose. Following complete or partial ( approximately 50%) N-deacetylation of the polysaccharide by hydrazinolysis, the free amino groups were sulfated by treatment with trimethylamine.
View Article and Find Full Text PDFGlucuronyl C5-epimerases catalyze the conversion of D-glucuronic acid (GlcUA) to L-iduronic acid (IdceA) units during the biosynthesis of glycosaminoglycans. An epimerase implicated in the generation of heparin/heparan sulfate was previously purified to homogeneity from bovine liver (Campbell, P., Hannesson, H.
View Article and Find Full Text PDFThe capsular polysaccharide from Escherichia coli K4 consists of a chondroitin ([GlcA(beta 1-->3)GalNAc(beta 1-->4)]n) backbone, to which beta-fructofuranose units are linked to C-3 of D-glucuronic acid (GlcA) residues. Removal of the fructose units by mild acid hydrolysis provided a substrate for the GlcA C-5 epimerase, which is involved in the generation of L-iduronic acid (IdoA) units during dermatan sulphate biosynthesis. Incubation of this substrate with solubilized fibroblast microsomal enzyme in the presence of 3H2O resulted in the incorporation of tritium at C-5 of hexuronyl units.
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