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Biomedical subjects

G A Coetzee

Publications and source records attributed to G A Coetzee.

99 records · Page 6Linked to original sources
PubMed1979

Uptake and degradation of low density lipoproteins (LDL) by confluent, contact-inhibited bovine and human endothelial cells exposed to physiological concentrations of LDL.

Metabolism of low density lipoproteins (LDL) was studied in cultures of endothelial cells derived from bovine aorta or heart and from human umbilical veins. At low LDL concentrations nonconfluent cultures of bovine endothelial cells catabolized more LDL protein than contact-inhibited confluent cultures but this difference was reduced at high LDL concentrations. Nonconfluent human endothelial cells displayed also a higher rate of LDL degradation than their contact-inhibited counterparts, but this difference was less pronounced than in the bovine cells. Bovine endothelial cells grown in the presence of fibroblast growth factor metabolized less LDL than those cultured without fibroblast growth factor (FGF), but this difference was not consistent in the human endothelial cells. The data presented provide evidence that contact-inhibited confluent human endothelial cells are capable of catabolizing LDL when exposed to physiological concentrations of this lipoprotein.

Animals↗
PubMed1979

Modulation by sodium ascorbate of the effect of chloroquine on low density lipoprotein retention and degradation in cultured human skin fibroblasts.

Human skin fibroblasts in culture were incubated for 48 h with 125I-labelled low density lipoprotein and chloroquine in the presence and absence of sodium ascorbate. Pretreatment of the cells for 3 days with sodium ascorbate and addition of the vitamin during incubation resulted in a decrease in cellular retention and an increase in degradation of the labelled low density lipoprotein. Similar results were obtained when the cells were pretreated for 3 days but the vitamin was not added during the final 48 h of incubation. Pretreatment of the cells with dithiothreitol, butylated hydroxy-toluene, beta-mercaptoethanol and D-alpha-tocopherol had a similar effect to that of ascorbate, i.e. reduction in low density lipoprotein retention and increase in degradation. Neither ascorbate nor the other reducing agents affected low density lipoprotein catabolism in control cells not treated with chloroquine. Sodium ascorbate pretreatment resulted also in a slight but significant alleviation of the chloroquine-induced inhibition of hydrolysis of cholesterol linoleate. It is proposed that sodium ascorbate by virtue of its reducing properties provides some protection to the intralysosomal hydrolases against the inhibitory action of chloroquine. If cholesterol accumulation in human and experimental atheroma is caused by partial inhibition of lysosomal enzymes, sodium ascorbate could play a role in the alleviation of such an inhibition.

Adult↗
PubMed1977

Effects of 5-bromo-2'-deoxyuridine on beating heart cell cultures from neonatal hamsters.

1. Primary heart cell cultures from neonatal hamsters yielded a heterogeneous cell population, containing muscle cells undergoing progressive differentiation, as well as non-muscle cells. 2. Addition of 5-bromo-2'-deoxyuridine, at an early stage, to such cultures enhanced the formation of beating sheets of differentiated muscle cells. Accumulation of myosin heavy chains and creatine kinase also occurred in the presence of the analogue. 3. To obtain these effects, the analogue had to be added during the initial rapid growth phase of the cells. Division of the treated cells then ceased when the cell numbers had approximately doubled. 4. Similar results were obtained with other inhibitors of DNA synthesis. Thus improved muscle cell cultures can be obtained by preventing non-muscle cells from overgrowing the cultures. 5. One effect caused only by 5-bromo-2'-deoxyuridine was a large increase in the Ca2+-stimulated ATPase (adenosine triphosphatase) activity which sedimented at low ionic strength. This increase was not due to a greater content of myofibrillar myosin, or to myosin isoenzyme changes, because purified myosin prepared from treated and untreated cultures did not exhibit the increased Ca2+-stimulated ATPase activity.

Adenosine Triphosphatases↗
PubMed1977

5-bromo-2'-deoxyuridine-stimulated calcium ion- or magnesium ion-dependent ecto-(adenosine triphosphatase) activity of cultured hamster cardiac cells.

1. Treatment of hamster heart cells in primary culture with 5-bromo-2'-deoxyuridine resulted in the greatly increased activity of a particulate Ca2+- or Mg2+-dependent ATPase (adenosine triphosphatase). 2. 5-Bromo-2'-deoxyuridine exerted these effects only when it was incorporated into cellular DNA, and then in a concentration-dependent manner. 3. Serially replated cells contained less of the activity (expressed as a function of total cell protein) than did the primary cultures, but the stimulation caused by 5-bromo-2'-deoxyuridine addition was much greater. 4. The affected enzyme was apparently localized in the plasma membrane of the cells with its active centre exposed to the outer environment [ecto-(ATPase) dependent on Ca2+ or Mg2+].5. The activity was unaffected by treatment with p-chloromercuriphenylsulphonate, ouabain andverapamil. 6. Ecto (5'-nucleotidase) activity was not increased by 5-bromo-2'-deoxyuridine treatment of cells, and ecto-(p-nitrophenyl phosphatase) activity was only slightly enhanced.

Adenosine Triphosphatases↗
PubMedDate not supplied

Low density lipoprotein metabolism in cultured fibroblasts from a new group of patients presenting clinically with homozygous familial hypercholesterolemia.

The metabolism of low density lipoproteins (LDL) was studied in cultured fibroblasts obtained from five local patients diagnosed, on the basis of clinical features and serum cholesterol concentrations, as having the homozygous form of familial hypercholesterolemia. LDL receptor function was assessed by measuring the binding, internalization, and degradation of 125I-labeled LDL, and by measuring the stimulation of cellular acyl-CoA cholesterol acyltransferase (ACAT) activity which followed exposure to LDL. Fibroblasts from two cases (CF and GM) showed receptor activities which were approximately 10% of the values obtained with normal cells, while ACAT stimulation by LDL was very low. These two patients were classified as homozygous for a receptor-defective abnormality. However, fibroblasts from the other three patients (JG, ES, and TT) showed greater than 25% of normal receptor activity, as assessed by 125I-LDL binding and catabolism. ACAT stimulation by LDL in cells from JG and ES was within the range of values shown by cells previously characterized as heterozygous for a receptor-negative mutation. Cells from ES behaved atypically: a low, but nonsaturable, activation by LDL was evident. ACAT stimulation by LDL was normal in cells from TT. Receptor activities of the cells from the available parents, assessed on the basis of LDL binding and degradation or of ACAT stimulation, were not clearly distinguishable from those of normal cells. These results add to the growing evidence of genetic heterogeneity underlying the clinical picture associated with familial hypercholesterolemia in different geographical distributions.

Adolescent↗
PubMedDate not supplied

In vitro metabolism of LDL labeled with a nondegradable cholesteryl ester analogue.

Low density lipoprotein (LDL) metabolism by human skin fibroblasts was studied using LDL labeled in the neutral lipid fraction with a nonhydrolysable cholesteryl ester analogue 3H-cholesteryl linoleyl ether (3H-CLE-LDL). LDL uptake could be quantitated accurately using 3H-CLE-LDL, since the label accumulated intracellularly due to its resistance to hydrolysis. 3H-CLE-LDL was taken up via apo B,E receptor-mediated endocytosis in a manner similar to 125I-labeled LDL. This was demonstrated by similar rates of uptake of the two differently labeled LDL preparations, saturation kinetics of uptake with respect to 3H-CLE-LDL concentration, regulation of 3H-CLE-LDL uptake by procedures that up-regulate or down-regulate the number of apo B,E receptors, and negligible uptake of 3H-CLE-LDL by receptor-negative cell strains. The major difference between the handling of 125I-LDL and 3H-CLE-LDL was the finding that, while the amount of trypsin-releasable (surface) 3H-CLE increased progressively over a 24-hour experimental period, trypsin-releasable 125I-LDL reached a maximum within 30 minutes. After 24 hours of incubation, the 3H radioactivity released by brief trypsinization was three to four times higher than could be accounted for by 125I radioactivity released by a similar treatment. Possible reasons for this behavior are discussed.

Cholesterol↗
PubMedDate not supplied

Low density lipoprotein receptor mutations in South African homozygous familial hypercholesterolemic patients.

The binding and catabolism of low density lipoprotein (LDL) were studied in cultured skin fibroblasts and/or Epstein-Barr virus-transformed lymphoblastoid cells from 20 familial hypercholesterolemic (FH) homozygotes of 14 Afrikaner kindreds. Cells from available heterozygotes were also analyzed. Good resolution between the normal, heterozygote, and homozygote groups was obtained on the basis of these assays using either cell type. Results obtained with fibroblasts allowed the classification of 13 of these kindreds as being typically receptor-defective, and one kindred with two homozygotes as being receptor-negative. Fibroblasts from homozygotes of the receptor-defective class expressed LDL receptor activities which varied between 3% and 25% of normal. Where two homozygotes from the same family were available for assays (six families), both yielded similar activities. In contrast to fibroblasts, all the lymphoblastoid cells derived from FH homozygotes yielded LDL receptor activities equal to or less than 10% of normal; in a number of cases no significant 125I-LDL binding was detectable. The use of lymphoblastoid cells provides a convenient means for screening for FH at the cellular level. Our results indicate a predominance of a receptor-defective type of abnormality, which is consistent with a founder gene effect in the Afrikaner population.

Adolescent↗
PubMedDate not supplied

Retro-endocytosis of low density lipoprotein by cultured human skin fibroblasts.

A fraction of the low-density lipoprotein (LDL) internalized by cells via receptor-mediated endocytosis follows a short-circuit pathway, termed "retro-endocytosis," that results in the rapid exocytosis of ligand. Results from the current study suggest that retro-endocytosis of LDL in human fibroblasts is caused by resurfacing of endocytotic vesicles that contain both free and receptor-bound ligand, resulting in discharge of vesicular contents and in spontaneous dissociation of LDL from its receptor. The bulk of the released LDL particles had the same size, density, and immunogenic properties as native LDL, indicating that they were discharged intact. Some of the retro-endocytosed LDL was larger than native LDL, and some exhibited altered sedimentation properties. When fusion of endosomes with lysosomes was inhibited by chilling cells to 18 degrees C, the proportion of intracellular LDL subsequently released was unaffected, suggesting that retro-endocytosis does not require lysosomal participation. Furthermore, the shorter the internalization phase the greater was the proportion of LDL subsequently released, suggesting that LDL was discharged from compartments formed early in endocytosis. Retro-endocytosis of LDL was stimulated by agents that neutralize acid intracellular compartments, such as ionophores (monensin) and weak bases (chloroquine and methylamine). Monensin increased the proportion of intracellular LDL released, suggesting that it had a direct effect on retro-endocytosis. The effect of weak bases appeared to be secondary to their ability to promote cellular accumulation of undegraded LDL. Thus, retro-endocytosis of LDL becomes a major pathway when intracellular compartments fail to maintain a low pH or where the intracellular concentration of LDL reaches abnormal levels.

Cells, Cultured↗