Search PubMed⌕ Search

Biomedical subjects

G A Coetzee

Publications and source records attributed to G A Coetzee.

At least 91 records · Page 5Linked to original sources
PubMed1987

The surface distribution of low density lipoprotein receptors on cultured fibroblasts and endothelial cells. Ultrastructural evidence for dispersed receptors.

The distribution of low density lipoprotein (LDL) receptors marked with colloidal gold-conjugated low density lipoproteins has been mapped on the surfaces of cultured human skin fibroblasts and bovine aortic endothelial cells viewed whole in the transmission electron microscope. A dispersed or scattered population of LDL receptors, in addition to and clearly distinct from clustered receptors was detected on the surfaces of both fibroblasts and dividing endothelial cells. No LDL receptors could be detected on contact-inhibited endothelial cells. Clustered receptors imaged in whole-mount preparations were often arranged in rings with an approximate diameter of 250 nm. In ultra-thin sections of marked cells, clustered receptors were localised in coated pits while the few dispersed receptors seen were restricted to non-coated membrane regions. Clustered receptors often appeared localised on the rims of coated pits whose central areas were not marked. The dispersed population of receptors was usually distributed diffusely amongst the clusters on dividing endothelial cells and normal fibroblasts. Only the dispersed population appeared on LDL receptor internalisation-defective mutant fibroblasts. The marginal zones of both fibroblasts and dividing endothelial cells were populated by dispersed receptors. Clusters appeared further "inland" and were rarely seen near the cell margins. These results indicate that LDL receptors on dividing endothelial cells and fibroblasts may be dispersed on the cell surface upon or soon after their insertion during recycling.

Animals↗
PubMed1986

Serum amyloid A-containing human high density lipoprotein 3. Density, size, and apolipoprotein composition.

Serum amyloid A protein (apo-SAA), an acute phase reactant, is an apolipoprotein of high density lipoproteins (HDL), in particular the denser subpopulation HDL3. The structure of HDL3 isolated from humans affected by a variety of severe disease states was investigated with respect to density, size, and apolipoprotein composition, using density gradient ultracentrifugation, gradient gel electrophoresis, gel filtration, and solid phase immunoadsorption. Apo-SAA was present in HDL particles in increasing amounts as particle density increased. Apo-SAA-containing HDL3 had bigger radii than normal HDL3 of comparable density. Purified apo-SAA associated readily with normal HDL3 in vitro, giving rise to particles containing up to 80% of their apoproteins as apo-SAA. The addition of apo-SAA resulted in a displacement of apo-A-I and an increase in particle size. Acute phase HDL3 represented a mixture of particles, polydisperse with respect to apolipoprotein content; for example, some particles were isolated that contained apo-A-I, apo-A-II, and apo-SAA, whereas others contained apo-A-I and apo-SAA but no apo-A-II. We conclude that apo-SAA probably associates in the circulation of acute phase patients with existing HDL particles, causing the remodeling of the HDL shell to yield particles of bigger size and higher density that are relatively depleted of apo-A-I.

Adult↗
PubMed1985

Standardisation of the quantitation of serum amyloid A protein (SAA) in human serum.

An adequate method for standardising the quantitation of serum amyloid A protein (SAA) in human serum was developed. Acute phase high density lipoprotein3 (HDL3) was used as a standard. The concentration of the SAA in the standard was determined by the use of purified SAA. After protein determination, various concentrations of purified SAA were run on SDS-polyacrylamide gel together with the HDL3 standard containing an unknown amount of SAA amongst the apolipoproteins. From the standard curve obtained by pyridine extraction (Coomassie blue colour yield at A605 nm) the concentration of SAA in the HDL3 standard was determined. An established immunoradiometric assay (IRMA) for SAA was standardised with the HDL3. SAA concentrations in normal and acute phase sera were determined.

Amyloid↗
PubMed1985

Native and acetylated low density lipoprotein metabolism in proliferating and quiescent bovine endothelial cells in culture.

Lipoprotein binding and metabolism in actively dividing (sparse) and quiescent (confluent) bovine aortic endothelial cells (EC) were compared quantitatively using 125I-labelled lipoproteins. The amounts of receptor-bound low density lipoproteins (LDL) decreased five- to ten-fold as the cultures progressed from sparse to confluent morphology. High affinity receptor-bound LDL levels were extremely low in confluent EC and accounted for the inability of confluent EC to internalize and degrade significant amounts of LDL. Conversely, the amounts of acetylated LDL (acLDL) bound and degraded via distinct sites increased at least five-fold during EC growth to confluence. LDL binding and metabolism in individual cells was assessed by fluorescence microscopy using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-labelled lipoproteins or fluorescein-conjugated antibodies. LDL and acLDL bound to the surfaces of sparse EC, at either 4 degrees or 37 degrees C, in a random distribution of fine punctate foci, contrary to a previous report. EC therefore appear to resemble fibroblasts in their distribution of surface LDL receptors. No binding or uptake of LDL was seen in confluent EC. Patterns of acLDL binding and uptake in confluent EC resembled those of LDL in sparse EC. Intracellular LDL and acLDL occurred as perinuclear accumulations of large fluorescent foci in sparse EC. Regeneration experiments were carried out in artificially wounded confluent cultures and renewed LDL receptor activity was shown in actively-dividing cells which had migrated into the "wounded" areas. We conclude that quiescent endothelial cells metabolize little LDL via the LDL-receptor pathway due to a drastically reduced number of receptors in confluent cells. This contrasts with the ability of confluent cells to metabolize relatively large amounts of acLDL via a receptor-mediated mechanism.

Acetylation↗
PubMed1984

Metabolic fate of low density lipoprotein and high density lipoprotein labeled with an ether analogue of cholesteryl ester.

Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled with a nonhydrolyzable cholesteryl ether analogue, 3H-cholesteryl linoleyl ether (CLE). The 3H-CLE-LDL was taken up by the apo-B, E receptor mediated endocytosis similar to 125I-labeled LDL. This was shown by saturation kinetics of uptake with respect to 3H-CLE-LDL concentration and very low uptake of 3H-CLE-LDL by receptor negative cell strains. When injected (CE)-LDL were cleared at equal rates and about 30% of the injected LDL was recovered in the liver. Treatment with ethinyl estradiol resulted in a three-fold increase in 3H-CLE-LDL uptake by the liver. The liver is also the major site of uptake of 3H-CLE-high density lipoprotein (HDL) (40%-45% of the injected dose) but its uptake by the liver increased only by 20% with estradiol treatment. As 3H-CLE-HDL was cleared from the circulation at a somewhat faster rate than 125I-HDL it appeared that some dissociation in the tissue uptake of the protein and CE moieties occurs.

Adrenal Glands↗
PubMed1983

Scavenger lipoprotein receptors are more effective in ligand internalization than low density lipoprotein receptors in human monocyte-macrophages.

Human monocyte-macrophages in culture express specific receptors for low density lipoproteins (LDL receptor) and human acetylated LDL (AcLDL receptors or scavenger receptors). After 24 h in lipoprotein-deficient serum, the cells expressed 2-3 fold more AcLDL receptors than LDL receptors as measured by trypsin releasable radioactivity after exposure to 125I-LDL or 125I-AcLDL at 37 degrees C. The efficiency of intracellular ligand delivery by the two receptors was evaluated as an internalization index (defined as intracellular + degraded/bound ligand). This index was several fold greater for 125I-AcLDL than for 125I-LDL, in the same cells exposed to either ligand under identical conditions. These results suggest that the scavenger receptors recycle more rapidly than do LDL receptors.

Cell Adhesion Molecules↗
PubMed1983

Ascorbate increases the number of low density lipoprotein receptors in cultured arterial smooth muscle cells.

Receptor-mediated catabolism of low density lipoprotein (LDL) was increased 2-3-fold in down-regulated smooth muscle cells when the culture medium was supplemented with physiological concentrations of sodium ascorbate for 24 h. The enhanced degradation of LDL was associated with increased LDL receptor activity and LDL uptake. The increase in receptor activity was rapid, transient and inhibited by cycloheximide. Kinetic analysis of saturable binding indicated that ascorbate increased the number of LDL receptors but had no effect on the affinity of the lipoprotein for its receptor. Our data indicate that ascorbic acid may play a role in the regulation of plasma cholesterol levels by influencing LDL receptor number.

Animals↗
PubMed1982

The degradation of endogenous and exogenous proteins in cultured smooth muscle cells.

The pathways of degradation followed by endogenous proteins in cultured smooth muscle cells were compared with the well-characterized lysosomal pathway involved in the degradation of apolipoprotein B of endocytosed LDL. Under conditions in which lysosomal activity towards 125I-labeled LDL was almost completely inhibited by chloroquine and/or ammonium chloride, the degradation of short-lived and abnormal proteins, assessed by the release of [3H]phenylalanine, was reduced by only 10-17%. The basal rate of degradation of long-lived proteins was reduced by about 30% by the same inhibitors while the accelerated proteolysis found under nutrient-poor conditions could be completely accounted for by the lysosomal system as defined by these lysosomotrophic agents. Temperature studies indicated differences between the mechanisms involved in the degradation of long-lived proteins (Ea = 18 kcal/mol) and short-lived proteins (Ea = 10 kcal/mol). Arrhenius plots for the degradation of endogenous proteins showed no transitions between 15 and 37 degrees C in contrast to the breakdown of LDL which ceased below 20 degrees C. The results indicate that the degradation of rapid-turnover proteins is largely extralysosomal and that a significant breakdown of long-lived proteins occurs also outside lysosomes.

Animals↗
PubMed1981

Retro-endocytosis of low density lipoprotein by cultured bovine aortic smooth muscle cells.

Cultured bovine aortic smooth muscle cells, pretreated with 125I-labelled low density lipoprotein (LDL), rapidly released significant amounts of the lipoprotein as trichloroacetic acid-precipitable material during a subsequent chase period. The time and temperature dependence of this release process and its insensitivity to heparin-pretreatment of equilibrated cells suggest that LDL was regurgitated from cells by a rapid process that we have termed 'retro-endocytosis'. The total amount of lipoprotein released from cells equilibrated at 37 degrees C with 125I-labelled LDL was approximately 20% of the amount degraded, pointing to the existence of a small pool of material which was distinct from the lysosomal pathway. To quantify the flux of LDL through retro-endocytosis, the fate of surface-bound lipoproteins was analyzed. Cells, pretreated with 125I-labelled LDL at 4 degrees C, regurgitated about 50% of the initial surface-bound LDL during a chase period at 37 degrees C and degraded the remainder more slowly through the lysosomal pathway. The involvement of LDL-receptors was implicated because retro-endocytosis was a saturable process and was affected by up- and down-regulation. The apolipoprotein of the released LDL showed little proteolytic modification as analyzed by gel filtration. We conclude that in a steady-state situation the fraction of LDL that passes through the retro-endocytosis pathway is of the same order as that which is directed through the lysosomal system.

Animals↗
PubMed1981

Degradation of apolipoprotein B of low density lipoprotein by cultured bovine smooth muscle cells. Accumulation of intermediates in the presence of protease inhibitors.

The incubation of bovine aortic smooth muscle cells with 125I-labeled low density lipoprotein in the presence of protease inhibitors resulted in the significant intracellular accumulation of intact apolipoprotein B, as well as a number of high molecular weight degradation intermediates. This effect was brought about both by leupeptin, a specific inhibitor of thiol proteases (40% inhibition of degradation), as well as by the more general lysosomotrophic inhibitors, chloroquine and NH4Cl. Qualitatively identical spectra of degradation intermediates were formed in the presence of chloroquine and NH4Cl as determined by autoradiography of SDS-polyacrylamide gel electrophoretic fractions, ranging from the apolipoprotein B band (Mr = 340,000) to bands with molecular weights of less than 14,000. The effect of NH4Cl was reversible and release of inhibition resulted in the sequential loss of intermediates from the cells; those species having higher molecular weights disappearing first. Inhibition by leupeptin was associated with the accumulation of degradation products in the lower molecular weight range only (Mr less than or equal to 72,000). These results provide evidence that apolipoprotein B proteolysis progresses in distinct stages via specific breakdown products and suggest that the thiol cathepsins become more active later in the degradation pathway.

Ammonium Chloride↗
PubMed1981

Metabolism of cytoplasmic triacylglycerol in cultured aortic smooth muscle cells.

A turnover of cytoplasmic triacylglycerol was studied in cultured rat, rabbit, and bovine aortic smooth muscle cells. Cytoplasmic triacylglycerol was labeled with [3H]glycerol in the presence of oleic acid in the medium and its loss from the cell was studied in the presence of carrier glycerol. Multiple additions of Isuprel or dibutyryl cyclic AMP during the chase period did not enhance the loss of labeled triacylglycerol. The rate of hydrolysis of cellular triacylglycerol was unchanged in the absence or in the presence of 100 microM chloroquine. Modulation of cellular cholesterol content by addition of low density lipoprotein or high density apolipoprotein--sphingomyelin liposomes did not affect the residence time of the cellular triacylglycerol. We conclude that cytoplasmic triacylglycerol in cultured aortic smooth muscle cells is metabolized by an extralysosomal enzyme which is neither catecholamine responsive nor affected by modulation of cellular cholesterol.

Animals↗
PubMed1980

Modulation of cytoplasmic cholesteryl ester of smooth muscle cells in culture derived from rat, rabbit and bovine aorta.

Esterification of cholesterol in smooth muscle cells, isolated from rat, rabbit and bovine aorta, was achieved by incubation with cholesterol enriched medium containing [7(n)-3H]cholesterol. The newly formed cholesteryl ester was readily hydrolyzed when the cells were post-incubated with medium containing lipoprotein deficient serum. The rate of loss of labeled cholesteryl ester was not inhibited by the presence of 100 microM chloroquine. Addition of LDL to the post-incubation medium retarded the decrease in labeled cellular cholesteryl ester in rat smooth muscle cells and this effect of LDL was abolished by chloroquine. In bovine and rabbit smooth muscle cells, enriched in cholesteryl ester, addition of LDL to post-incubation medium resulted in an increase in labeled cholesteryl ester and in cholesteryl ester mass. Retardation in the loss of labeled cellular cholesteryl ester occurred also on addition of oleic acid to the post-incubation medium. In the presence of HDL and especially of high density apolipoprotein-sphingomyelin liposomes there was an efflux of cellular free cholesterol and a reduction in cholesteryl ester. These findings indicate that the catabolism of cytoplasmic cholesteryl ester in aortic smooth muscle cells is catalyzed by extralysosomal enzymes. The cytoplasmic cholesteryl ester hydrolase is apparently not activated by cyclic AMP. The intracellular availability of unesterified cholesterol, which can be modulated by plasma lipoproteins, may determine the residence time of cellular cholesteryl ester. Thus under pathological conditions an increase in extracellular LDL accompanied by a reduction in HDL would prolong the residence time of cholesteryl esters and thus promote their intracellular accretion.

Animals↗
PubMed1980

Cathepsin-D-dependent initiation of the hydrolysis by lysosomal enzymes of apoprotein B from low-density lipoproteins.

The degradation of 135I-apoprotein B of human low-density lipoprotein by cell extracts of cultured bovine aortic smooth muscle cells was determined by measuring the formation of acid-soluble products and by analyzing the electrophoretic patterns of digested apoprotein in gels containing sodium dodecyl sulfate. Degradation resulted in an initial rapid accumulation of a limited number of distinct smaller fragments. Two products with apparent molecular weights of 220,000 and 200,000 predominated. Pepstatin inhibited proteolysis almost completely, as measured by either assay. Leupeptin decreased hydrolysis to acid-soluble products by approximately 50%, but had no effect on the initial cleavage of intact apoprotein B. Similar results were found in the case of extracts from cultured human skin fibroblasts and from adult bovine arterial smooth muscle. Leupeptin inhibited intracellular degradation of 125I-apoprotein B in cultured cells by approximately 50%. It is concluded that the intralysosomal degradation of apoprotein B involves an initial limited endoproteolytic attack at susceptible sites by cathepsin D. This and other enzymes, including cathepsin B, then act synergistically to bring degradation to completion.

Animals↗
PubMed1980

Plasmin-treated low density lipoproteins: polypeptide analyses and metabolism by cultured smooth muscle cells.

Plasmin, generated by the interaction of urokinase with plasminogen, degraded the apoprotein B moiety of human low density lipoprotein to yield distinct high moleculr weight intermediates under conditions where only a small fraction (less than 3%) of the protein was hydrolyzed to trichloroacetic acid-soluble products. The molecular weights of these intermediates were between 60 000 and 200 000 as estimated by SDS-polyacrylamide electrophoresis. Trypsin treatment yielded fragments of similar size to those obtained with plasmin. When enzyme-treated low density lipoproteins were added to bovine aortic smooth muscle cells in culture, the receptor-binding, and rates of internalization and degradation were no different from those obtained in the case of native low density lipoproteins.

Animals↗
PubMed1979

Cardiac myofibrillar phosphorylation and adenosine triphosphatase activity.

When myofibrils from rat hearts were dissolved in concentrated salt solutions and reprecipitated by dilution, they contained both protein kinase (partly cyclic 3':5'-AMP-dependent) and protein phosphatase activities. Troponin-I was the major protein to be phosphorylated by the endogenous myofibril-associated kinase and by added protein kinase. Approximately 1 mole of phosphate per mole of troponin-I was incorporated from radioactive ATP, but the extent of troponin-I phosphorylation could be varied experimentally. An inverse correlation was found between protein phosphorylation and the maximum Ca2+-stimulated myofibrillar Mg2+-ATPase activity, while the amout of calcium required for half-maximum activation was proportional to the extent of protein phosphorylation. The changes in Mg2+-ATPase activity produced in vitro by protein phosphorylation were reproduced in isolated perfused rat hearts treated for short periods with L-noradrenaline (10(-6)M). The changes in myofibrillar function brought about as the result of the phosphorlyation by cAMP-dependent protein kinase suggest that the contractile response is desensitized in order to cope with the rise in intracellular Ca2+ which results from the action of catecholamines on cardiac ventricular cells.

Adenosine Triphosphatases↗