Biomedical subjects

V P Tsibulsky

Publications and source records attributed to V P Tsibulsky.

14 recordsLinked to original sources

PubMed1998

Binding of apoB-containing lipoproteins from unfractionated human blood sera to immobilized LDL receptor.

Binding of apoB-containing lipoproteins from unfractionated human blood sera to the immobilized bovine receptor of low density lipoproteins (LDL receptor) was studied. Peroxidase-labeled anti-human apoB antibodies were used to evaluate the lipoprotein binding. The equilibrium dissociation constant (Kd) of the interaction between apoB-containing lipoproteins from unfractionated human sera from healthy donors and the immobilized LDL receptor varied from 1 to 20 microg apoB/ml. To describe the binding of lipoproteins to the LDL receptor, a parameter of relative binding affinity (RBA) was used. RBA is inversely related to value of Kd and equal to unity for the standard serum. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera to the immobilized LDL receptor were found to correlate with the RBA values for the binding of isolated VLDL (r = 0.76, p < 0.001) and fail to correlate with the RBA values for the binding of isolated LDL. The RBA values for the binding of apoB-containing lipoproteins from unfractionated sera correlated with the RBA values for the binding of apoE-containing lipoproteins from unfractionated sera (r = 0.92, p < 0.001) and with values of triglyceride concentration in the sera (r = 0.93, p < 0.001). The RBA values for the binding of apoB-containing lipoproteins from sera of patients with FDB whose LDL were unable to bind to the LDL receptor did not significantly differ from the RBA values for the normal sera. However, the removal of VLDL from the normal sera significantly decreased the RBA values for the binding of apoB-containing lipoproteins from unfractionated sera. The results indicate that the different binding of apoB-containing lipoproteins to the immobilized LDL receptor mainly depended on the different binding of VLDL and not of LDL.

Animals↗

PubMed1998

Immunoreactivity of apolipoprotein B-100 and binding to LDL-receptor of phospholipase A2-treated low density lipoproteins.

Lipid--protein particles were obtained by treatment of low density lipoproteins (LDL) with phospholipase A2 from bee venom. Under these conditions, half of the phosphatidylcholine (PC) of LDL was changed to lysophosphatidylcholine (LPC). At the same time, the composition of other lipids and the apoprotein structure were unaffected. Three monoclonal antibodies (MAbs) against various apo B epitopes were used to test immunoreactivity of phospholipase A2-treated LDL (pl-LDL). The apo B epitope interacting with MAb 4C11 (amino acid residues 2377-2658) showed significantly decreased immunoreactivity. Increase in MAb 4C11 binding was demonstrated to depend on oxidation degree of LDL. Thus, changing of half of PC to LPC modified apo B translocation in the lipoprotein globule in an opposite manner as compared with changes induced by oxidative modification. A minor increase in immunoreactivity of pl-LDL with 1D1 MAb against a large middle part of apo B (residues 1297-3249) may be due to the effect of the change of surface lipid composition on the extent of immersion of apo B into the hydrophobic phase. No changes in the interaction of pl-LDL with MAb 2G8 (residues 3748-4306) were observed in comparison with native LDL. This fact demonstrates that 50% phospholipolysis of LDL does not affect the expression of apo B C-terminal residues in pl-LDL. Twofold increase in pl-LDL affinity to immobilized LDL-receptor was shown in contrast to LDL. The data indicate that LPC accumulation in LDL results in better elimination of LDL from the blood stream than in case of accumulation of oxidative products.

Antibodies, Monoclonal↗

PubMed1997

Immunoenzyme assessment of human apoB-lipoprotein binding to immobilized receptor of low density lipoproteins. 1. Preparation of anti-receptor monoclonal antibodies.

The preparation and properties of V5 monoclonal antibody to low density lipoprotein receptor (LDL-receptor) from bovine adrenal cortex membranes are described. The monospecific V5 antibody recognizes the LDL-receptor (the only protein with molecular mass of 140 kD) in bovine adrenal cortex membranes. V5 antibody fails to compete with human low density lipoproteins (LDL) for binding to the LDL-receptor. After absorption in standard 96-well polystyrene plates, V5 antibody efficiently binds the affinity-purified LDL-receptor from the solution and the subsequent binding of the LDL-receptor with human LDL was determined using peroxidase-labelled antibodies to apolipoprotein B. The LDL-receptor immobilized by V5 antibodies is suggested for use in studies on the binding of human lipoproteins to the LDL-receptor.

Adrenal Cortex↗

PubMed1997

Immunoenzyme assessment of human apoB-lipoprotein binding to immobilized receptor of low density lipoproteins. 2. Binding of isolated lipoproteins.

The receptor of low density lipoproteins (LDL-receptor) from bovine adrenal cortex membranes was immobilized in standard 96-well polystyrene plates using monoclonal V5-antibodies to the LDL-receptor. The binding of the immobilized LDL-receptor with human low density lipoproteins (LDL) and very low density lipoproteins (VLDL) was determined using peroxidase-labelled antibodies to human apoB. The value of Kd for the interaction of LDL with the immobilized LDL-receptor for 40 samples of LDL was found to be from 5 to 20 micrograms apoB per ml. The immobilized LDL-receptor failed to bind LDL modified by acetylation or malonic dialdehyde, while the binding of non-modified LDL to the immobilized LDL-receptor was inhibited in the presence of EDTA, which is known to be specific for the interaction of LDL with the LDL-receptor. Unlike LDL, VLDL were more variable in the binding to the LDL-receptor. The value of Kd for the interaction of VLDL with the LDL-receptor for 40 samples of VLDL was found to be from 0.5 to 10 micrograms apoB per ml. Thus, the described method is suggested to study the interaction of apoB-containing lipoproteins with the LDL-receptor.

Acetylation↗

PubMed1989

Binding and uptake of native and modified low-density lipoproteins by human hepatocytes in primary culture.

The binding and uptake of native low-density lipoproteins and malondialdehyde-treated low density lipoproteins by human hepatocytes in primary culture has been analyzed. Experiments with 125I-labeled malondialdehyde-treated low-density lipoproteins showed that cultured liver cells took up and degraded malondialdehyde-treated low-density lipoproteins, but the cell type(s) responsible for this action remain unclear. Immunofluorescent visualization of receptor-bound low-density lipoproteins revealed that low-density lipoprotein binding sites were distributed on the surface of nearly all cells of the culture. Binding sites for malondialdehyde-treated low-density lipoproteins were found in only 5% of the cultured cells, and these cells differed from hepatocytes in shape and size. Cultured hepatocytes internalized and native low-density lipoproteins, but not malondialdehyde-treated low-density lipoproteins, labeled with the fluorescent dye 3',3'-dioctadecylindocarbocyanine. About 15% of the cells that take up 3',3'-dioctadecylindocarbocyanine-labeled malondialdehyde-treated low-density lipoproteins could be identified as liver endothelial cells and macrophages, since they internalized formaldehyde-treated human albumin and fluorescent carboxylated microspheres. Our results indicate that human hepatocytes in primary culture express surface receptors for native low-density lipoproteins but not for modified low-density lipoproteins.

Cells, Cultured↗

PubMed1988

Use of enzyme label for quantitative evaluation of liposome adhesion on cell surface: studies with J774 macrophage monolayers.

A method for quantitation of cell surface-bound liposomes utilizing J774 macrophage monolayers is developed. Surface-bound biotinyl-containing and 125I-labeled liposomes were quantified with avidin-peroxidase in an ELISA-like assay. Peroxidase substrate absorbance values were recalculated into the absolute amount of liposomal lipid using a special calibration plot. Total liposome uptake by macrophages was determined following the binding of 125I radioactivity. The approach suggested allows quantitative evaluation of the changes in the content of surface-adhered liposomes during their interaction with cells in vitro.

Adsorption↗

PubMed1988

Identification of intimal subendothelial cells from human aorta in primary culture.

Subendothelial cells (SEC) were obtained from the inner intimal layer of adult human aorta by collagenase treatment. SEC were identified in primary culture either as smooth muscle cells by staining with FITC-labeled antisera against human smooth muscle myosin or as macrophages, foam cells and contaminating endothelial cells by their uptake of malondialdehyde treated low density lipoproteins labeled with fluorescent dye 3,3'-dioctadecylindocarbocyanine. Between 1 and 5 days in culture, along with smooth muscle cells (SMC, 38-82%), endothelial cells (0-9%), macrophages and foam cells (2-32%), one more type of cell was found. This cell type resembled SMC in size and shape, but was not stained by antisera to SMC myosin. By ultrastructural criteria these cells were characterized as modulated SMC for they contained prominent rough endoplastic reticulum and Golgi complex together with basement membrane and a large number of plasmalemmal vesicles. Like SMC they reacted with phalloidin and were stained by anti-vimentin but not by anti-desmin monoclonal antibodies. The proportion of such cells varied from 5 to 33% of total cell number and increased in parallel to macrophages and foam cells in vessels with well developed atherosclerotic lesions. We conclude that the applied technique may be used for identification of cultured vascular cells including modulated SMC.

Aorta, Thoracic↗

PubMed1986

Enzyme immunoassay of the receptors for modified low density lipoprotein.

Mouse macrophages (line J 774) were incubated with monospecific goat anti-low density lipoprotein antibodies, which were conjugated to horseradish peroxidase (AB-HRP). Addition of low density lipoprotein (LDL) modified by treatment with malondialdehyde to cultures of these cells resulted in a dose-dependent increase in the amount of cell-associated enzyme activity. The concentration curve was hyperbolic with half-saturation of modified LDL at a concentration of about 3 micrograms/ml. This effect was completely blocked by polyinosinic acid and was not observed in experiments with human fibroblasts, which do not exhibit high affinity binding sites that recognize chemically modified LDL. Our data indicate that receptor-mediated endocytosis of AB-HRP in the presence of native or modified LDL may be used as very simple, efficient, and sensitive assay for investigation of the scavenger receptors for modified LDL.

Animals↗

PubMed1986

Apolipoprotein B release from activated human platelets.

Apolipoprotein B (apoB) release from activated washed human platelets was measured by enzyme-linked immunosorbent assay (ELISA) using monospecific rabbit antibodies to human low density lipoprotein (LDL). Activation of platelets with thrombin, Ca2+-ionophore A23187 or stable analogue of prostaglandin endoperoxides U46619 stimulated release of approximately 20 ng apoB/10(8) platelets. Thrombin-induced apoB release was inhibited by the prostacyclin analogue carbacyclin. Dose-response curves of thrombin stimulation and carbacyclin inhibition of apoB and beta-thromboglobulin (beta-TG) release were very similar. Treatment of platelets with heparin did not remove significant amounts of apoB or affect the subsequent release of apoB induced by thrombin. The results of density gradient ultracentrifugation indicated that most of the apoB was released in the LDL density range. These data suggest that human platelets contain immunoreactive apoB, which can be released during platelet activation.

Apolipoproteins B↗

PubMed1986

Specific high affinity binding and degradation of high density lipoproteins by isolated epithelial cells of human small intestine.

The interaction of high density lipoproteins (HDL) with isolated epithelial cells of human small intestine (enterocytes) was studied. 125I-HDL3 (density = 1,125 to 1,126 g/cm3) exhibit a high-affinity (Kd = 8.3 X 10(-8). Bmax = 886 ng/mg cell protein), saturable and reversible binding to isolated enterocytes. In the presence of excess unlabeled HDL3, the cell surface-bound 125I-HDL3 are released into the medium nondegraded. Treatment of cells with pronase does not affect 125I-HDL3 binding. The binding is accompanied with internalization and degradation of 125I-HDL3. Chloroquine inhibits the degradation and increases 125I-HDL3 uptake. A threefold excess of HDL3 and HDL2 inhibits the binding and degradation of 125I-HDL3 by 60%, whereas a 20-fold excess of low density lipoproteins (LDL), only by 20%. HDL3 (20 to 1,000 micrograms/mL) stimulates the synthesis of sterols and inhibits sterol ester synthesis in enterocytes. The obtained results make it possible to assume that epithelial cells of the small intestine may participate in the catabolism of HDL in human organism.

Acetates↗

PubMed1985

Cholesterol can stimulate secretion of apolipoprotein B by cultured human hepatocytes.

During a 5 day cultivation of human hepatocytes in a primary culture the secretion of apolipoprotein B was measured by enzyme-linked immunosorbent assay. Density-gradient ultracentrifugation demonstrated that the majority of the secreted apolipoprotein B was associated with the very-low-density lipoprotein fraction. Exposure of the cells to cholesterol (5-100 micrograms/ml) resulted in a dose-dependent increase in apolipoprotein B secretion rate.

Adult↗

PubMed1985

Enzyme-linked immunoreceptor assay of low-density-lipoprotein receptors.

A new modification of enzyme immunoassay-enzyme-linked-immunoreceptor assay (ELIRA) was used to study low-density lipoprotein (LDL) binding by cultured human skin fibroblasts. Time-course and concentration curves obtained by this method were typical of LDL binding by cultured fibroblasts. According to ELIRA, fibroblasts bound native LDL 10-20 times more effectively than reductively methylated LDL or native LDL in the presence of heparin. Receptor-specific Km and maximum binding capacity calculated from these data were 5.4 micrograms/ml and 177.5 ng/mg of cell protein, respectively. Receptor-specific Km and maximum binding capacity for surface 125I-LDL binding measured in the same experiment were 11.0 micrograms/ml and 138.0 ng/mg of cell protein. Incubation of cells with isolated LDL or with unfractionated serum containing the same amount of apoB yielded similar concentration curves for lipoprotein binding. These data indicate that the ELIRA can be used for investigation of receptor-mediated lipoprotein binding without purification of lipoproteins.

Apolipoproteins B↗

PubMed1985

Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins.

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of malondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified LDL.

Antibodies↗

PubMed1985

High-affinity association and degradation of 125I-labelled low density lipoproteins by human hepatocytes in primary culture.

Catabolism of homologous low density lipoproteins (LDL) was studied in primary culture of human hepatocytes (HH). The cell association and degradation of 125I-labeled LDL (125I-LDL) were curvilinear functions of substrate concentration. Cell association and degradation of 125I-LDL were inhibited by excess unlabeled LDL. Reductive methylation of unlabeled LDL abolished its ability to complete with 125I-LDL for cell association and degradation. Preincubation of HH with unlabeled LDL caused a 63% inhibition of the 125I-LDL degradation. It is concluded that the catabolism of LDL by HH proceeds in part through a receptor-mediated pathway similar to that demonstrated on extrahepatic cells.

Adult↗