PubMed · 6964058
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
D C McNamara, B F Stone, P K Lee. 1982. Acrylic occlusal splints.. https://pubmed.ncbi.nlm.nih.gov/6964058/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
Here we show that biotinylated polyacrylamide-type glycoconjugates which contain sialyl Lewis X (sLex-polymer) or sialyl Lewis A (sLea-polymer) are ligands for E-selectin. sLea-polymer bound E-selectin with higher affinity than sLex-polymer. Based on this property we used the sLea-polymer to establish a sensitive cell-free binding assay for the characterization of E-selectin antagonists. The assay involves complexation of the biotinylated sLea-polymer with streptavidin-peroxidase. This complex is incubated with E-selectin mouse Ckappa fusion protein immobilized onto microtiter plates. Bound complex is detected by the peroxidase reaction. sLea-polymer bound in a Ca2+-dependent manner consistent with the function of E-selectin as a C-type lectin. Control glycoconjugates with sialic acid (alpha-Neu5Ac), Lewis A (Lea), or beta-D-glucose residues instead of sLea failed to interact with the E-selectin. Neutralizing anti-E-selectin antibodies blocked completely binding to E-selectin. This demonstrates specificity of the assay system. sLex blocked binding of the sLea-polymer to E-selectin by 50% at a concentration of 550 microM (IC50). The assay was used to characterize sLea-polymers with differing sLea content as multivalent inhibitors of E-selectin binding. The inhibitory activity of these polymeric forms of sLea increased with their sLea content up to IC50s in the low micromolar range. The binding assay described is sensitive, rapid, and simple and of low variability. Therefore it should be advantageous for the identification and characterization of novel E-selectin antagonists.
In the previous paper, we reported a sensitive method for detection of protein kinase activities in gels after SDS-polyacrylamide gel electrophoresis (Kameshita, I., and Fujisawa, H. (1989) Anal. Biochem. 183, 139-143). This method is useful for the detection of various protein kinase activities toward protein substrates included in gels, but inapplicable to oligopeptide substrates because most of the oligopeptides eluted from the gel matrix during electrophoresis. The present study describes a new procedure for the detection of protein kinase activities toward synthetic oligopeptides in the gel. The oligopeptides which were linked to amino acid polymers such as poly-L-lysine through their amino-terminal cysteinyl residue by a heterobifunctional reagent were efficiently retained in the gel matrix and served as substrates for the protein kinases. As little as 2.5 pg of the catalytic subunit of cAMP-dependent protein kinase was detected by this in-gel assay method using a synthetic peptide as a substrate. This technique can be used for selective and sensitive detection of various protein kinases in crude tissue extracts.
N,N'-Diethyldithiocarbamate has been shown to be an analytical stain for copper in native polyacrylamide gels of the copper-zinc superoxide dismutase from purified preparations as well as from crude red cell extracts, i.e., lysates from which hemoglobin has been removed (Jewett, S. L., and Rocklin, A. M. (1994) Anal. Biochem. 217, 236-240). Applying this methodology, it was found that the relative amounts of copper-containing forms of copper-zinc superoxide dismutase (EC 1.15.1.1) from bovine red cell extracts did not change significantly with either the age or with hydrogen peroxide treatment of the red cells. Furthermore, no significant changes were seen in the specific activity of the dismutase in either type of experiment. These observations for both types of experiments are contrary to what was expected from similar studies reported in the literature. However, discrepancies may be accounted for by hemoglobin interference in indirect dismutase assays of the previous work. In the case of the peroxide treatment of red cells, however, there is an additional factor in that the dismutase is protected from peroxide-mediated changes in copper content and heterogeneity by the hemoglobin present. This protection was demonstrated in in vitro experiments using only a 24-fold excess of hemoglobin over the dismutase.