Archive for the ‘Cell’ Category - Part 2

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(9)

Heparin had a more ubiquitous effect and severely decreased binding of all biotinylated carbohydrates tested (P < 0.001, n = 3; see Table 2 and Fig. 8C). Higher levels of glucose in the T+BIC+Hep buffer did not significantly reduce binding of biotinylated carbohydrates (Table 2; P > 0.05). However, the oligosaccharide binding of mannan-BSA-biotin remained […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(8)

In all cases, a strong staining in a rim-like area of the anterior region of the sperm head was observed in approximately 60% of the sperm cells (Fig. 8). The remaining part of the cell was unstained or showed only very weak labeling. This staining pattern was similar but weaker when sperm cells were stained […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(7)

Under all conditions, <5% of total cells showed signs of acrosome disruption with the exception of the T+BIC treatment, where approximately 8% of the total cells showed signs of acrosome disruption detected by the CTC-staining assay. The SPM fractions of these four incubated sperm samples were isolated and immobilized on 96-well plates. Fucose-PAA-biotin showed the […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(6)

Influence of Capacitation on SPM-Carbohydrate Binding The effect of in vitro capacitation on sperm-carbohydrate interaction was studied using bicarbonate-enriched Tyrode media (T + BIC) either supplemented with heparin (T+BIC+Hep) or with heparin and glucose (T+BIC+Hep+Glu) and compared with Tyrode medium without bicarbonate (T—BIC). The subpopulation of sperm cells that showed signs of capacitation following incubation […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(5)

Heparin, a well-described capacitation factor for bovine sperm, strongly inhibits sperm binding to the oviductal epithelial monolayers. Here, the competition of heparin for fucose-PAA-biotin or for mannan-BSA-biotin binding to immobilized SPM was tested. Immobilized SPM was incubated with increasing concentrations of heparin (0.001100 |xg/ml) and a constant concentration of fucose-PAA-biotin or mannan-BSA-biotin (0.25 |xg/ml). Heparin […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(4)

Lower inhibition of fu-cose-PAA-biotin binding was achieved at >10 mM concentrations of maltose, mannose, glucose, and fucose with EC50 values of 31.12 ± 3.6, 218.6 ± 41.4, 85.81 ± 4.2, 162.6 ± 28.1 mM, respectively (Fig. 3). Mannan and fucoidan were the most efficient carbohydrates blocking fucose-PAA-biotin binding to immobilized SPM. Therefore, the binding affinities […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(3)

Carbohydrate Binding Competition Fucose-PAA-biotin binding to immobilized SPM was challenged by preincubating the SPM-coated wells (0.5 |xg SPM per well) with various concentrations (0-100 mM) of carbohydrates before addition of 0.25 |xg/ml fucose-PAA-biotin per well. The competition of fucose-PAA-biotin binding by fucose, glucose, maltose, mannose, galactose, inositol, 1- O-methyl-a-D-glycopyranoside, mannan, and fu-coidan (where mannan and […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(2)

Maximal binding (Bmax) was 4.71 ± 0.48 and 0.40 ± 0.03 (mean arbitrary units ± SD, n = 3; P < 0.001) for fucose-PAA-biotin and LeA-PAA-biotin, respectively. SPM proteins had approximately two times lower affinity for fucose-PAA-biotin when compared with LeA-PAA-biotin. Half maximal binding (Kd values) was obtained at 2.13 ± 0.35 |xg/ml and 1.16 […]

Dynamics of Carbohydrate Affinities at the Cell Surface: RESULTS(1)

Sperm Plasma Membrane Isolation SPMs obtained from fresh sperm immediately after Per-coll washing were purified at least with a factor eight (see Table 1) as the specific activity of alkaline phosphatase, acid phosphatase, and 5′-nucleotidase were respectively 8.5, 8, and 9, times higher in isolated plasma membranes when compared with complete sperm cavitate. The purified […]

Dynamics of Carbohydrate Affinities at the Cell Surface: MATERIALS AND METHODS(13)

Replicate experiments (n = 3) were performed on separate days using fresh sperm from two bulls. Toluidine Blue Staining of the Oviducts Oviducts were obtained from a local slaughterhouse (Gosschalk and Zn BV) and the oviduct connected to the ovary with the dominant follicle was used. The isthmus and ampulla were dissected into 50-mm pieces […]

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