indicates origin

indicates origin. extracts with mAb F77 followed by gradient SDS-PAGE revealed a carbohydrate-rich component (<5 kDa) that was not stained by Coomassie Blue (6). A dose-dependent decrease of F77 antigen expression was observed in PC3 and DU 145 cells after treatment with the glycolipid synthase inhibitor 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol but not with the protein Nonsecretor refers to the lack of blood group A, B, or H antigens in donor saliva or cystadenoma fluid (recorded in archives). Samples 1C17 and 23 were lyophilized ovarian cystadenoma fluids from the collection of Winifred Watkins and Walter Morgan at the former Lister Institute. They were enriched for mucin-type glycoproteins by treatment at 37 C for up to 16 h with trypsin (Sigma, T1426, l-1-tosylamide-2-phenylethyl chloromethyl ketone-treated) or Pronase (Roche Applied Science, 10165921001). After the reaction, the samples were centrifuged (4000 for 10C20 min); the supernatants were lyophilized, taken up in 3.5 mg/ml sodium acetate, and precipitated with ethanol, 80% (v/v). Sample 23 had been further purified by phenol extractions (53). Trp, Pro, and Pep refer to trypsin, Pronase, or pepsin enzyme treatments for solubilizing mucin-type glycoproteins. Samples 18C22 were from meconium and enriched after Pronase digestion and ethanol precipitation (54). Samples 24C27 were purified ovarian cystadenoma glycoproteins from your Elvin A. Kabat collection (Columbia Medical Center, New York). These had been pepsin-treated and precipitated with numerous concentrations of ethanol (55, 56). Oligosaccharides The carbohydrate sequences of these oligosaccharides are explained under Results. The following oligosaccharides were from Elicityl (Crolles, France): lacto-microarray analyses of mAbs F77, anti-B (89-F), anti-A (T36), and UEA-I lectin with mucin-type glycoproteins. The descriptions of the glycoproteins are in Table 1. Results are the means of fluorescence intensities of duplicate spots printed at 150 pg of glycoprotein per spot. The symbolize half of the difference between the two values. gel filtration chromatography of the products of reductive alkaline hydrolysis from PSM. is the initial chromatography profile using a Bio-Gel P4 column (1.6 90 cm) eluted with H2O. The shows the profile of portion a from your Bio-Gel P4 column, chromatographed using a Bio-Gel P6 column (1.6 90 cm) eluted with H2O. is the total volume; glucose models 8C11 indicate positions of elution of oligosaccharides with degrees of polymerization 8C11 in an acid hydrolysate of dextran. designate the pooled fractions that were converted to NGLs. binding of mAb F77 to NGLs derived from the MK-8719 indicates the origin. In small level experiments, agglutinin, UEA-I (Vector Laboratories), a lectin with blood group H activity, was examined at 50 g/ml followed by Alexa Fluor-647-labeled streptavidin. Unless otherwise specified, the analyses were performed at 20 C. Imaging and data analysis were as explained (24, 26). Binding signals were RLC probe dose-dependent. Results shown are at 5 fmol/spot for lipid-linked probes and 150 pg per spot for the glycoprotein microarray. Hemagglutination Assays mAb F77 (2.75 mg/ml) was diluted at 1:500 to 1 1:20,000 (5.5 g/ml to 137.5 ng/ml) in 0.9% (w/v) NaCl containing 6% (w/v) human serum albumin. For the hemagglutination gel card column assays (ID-Micro Typing System, Ortho-Clinical Diagnostics, Raritan, NJ), 50 l of 0.8% suspension of MK-8719 adult red cells of blood groups A, B, or O or cord blood cells of blood group O were mixed with 25 l of diluted antibody. After incubation at ambient heat or at 37 C for 2 min, the gel cards were spun at 90 in an ID-Micro Typing System centrifuge MK-8719 for 10 min at ambient heat. The degree of cell agglutination was assessed by the distance of cell sedimentation through the gel and visually scored as 4+, 3+, 2+, and poor. Unagglutinated cells settle at the bottom. End point was taken as 2+. RESULTS The glycolipid extract from PC3 cells was resolved by HPTLC and stained with primulin to detect the lipid moieties. Numerous primulin-stained components were revealed (Fig. 1). However, only minor components were bound by mAb F77, as indicated by the lack MK-8719 of obvious primulin staining corresponding to their positions of migration. As the amounts of glycolipids that can be obtained in PC3 cell extracts are very limited and not readily amenable to detailed characterization, two other approaches were explored toward elucidating the carbohydrate sequence of the F77 antigen. The first approach was to perform microarray analysis with existing sequence-defined oligosaccharide probes. The second approach, based on the fact that indicates the origin. Unpredicted mAb F77 Binding Detected Two Branched Poly-N-acetyllactosamine-based Blood Group B Glycolipids by Microarray Screening Analysis with Sequence-defined Oligosaccharide Probes Microarray analyses of mAb F77 binding using 492 sequence-defined oligosaccharide probes (supplemental Table S1).