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Biomedical subjects

O Stein

Publications and source records attributed to O Stein.

At least 73 records · Page 4Linked to original sources
PubMed1989

Lack of effect of probucol on atheroma formation in cholesterol-fed rabbits kept at comparable plasma cholesterol levels.

Rabbits were fed cholesterol for 14 weeks to study the effect of probucol on atheroma formation. Three groups of animals were investigated: group CHOL was fed 1% cholesterol and served as control for group P + CHOL. fed 1% cholesterol and 1% probucol from the onset till the end of the experiment: group CHOL + P received 1% cholesterol throughout the experiment and 1% probucol during the last 4 weeks only. Plasma cholesterol concentrations were monitored at frequent intervals and were modulated by dietary perturbations so that the areas under the curve expressing plasma cholesterol changes with time, were similar in probucol and non-treated rabbits. The efficacy of long-term probucol treatment was evidenced by a significant reduction in plasma apolipoprotein A-I throughout the experiment and lower plasma TBARs during the first 6 weeks, when the hypocholesterolemic effect of probucol was also seen. Two weeks prior to the termination of the experiment, the rabbits were injected with rabbit plasma labeled with [3H]cholesteryl linoleyl ether [( 3H]CLE). Aortic atheromatosis was quantified by determination of total and cholesteryl ester (CE). The aortic cholesterol content was related to the arch, thoracic and abdominal segments, to the surface area of each segment or its dry defatted weight. Total and esterified cholesterol were highest in the aortic arch in all 3 groups when related to any of the above mentioned parameters. No statistically significant difference in aortic total cholesterol and CE content was seen among the three groups studied. The [3H]CLE recovered in the aortic segment correlated with the CE content and the [3H]CLE (dpm)/mg CE in all segments was similar. No statistically significant difference in the [3H]CLE recovered in the aortic segments among the 3 groups was seen. We conclude that in cholesterol-fed rabbits, in which the plasma cholesterol levels were maintained at comparable levels, probucol treatment did not affect plasma CE influx into the aorta and did not attenuate development of aortic atherosclerosis.

Animals↗
PubMed1988

Tissue distribution of [3H]cholesteryl linoleyl ether-labeled human Lp(a) in different rat organs.

The sites of tissue uptake of human lipoprotein(a) (Lp(a] were studied in rats using [3H]cholesteryl linoleyl ether [( 3H]CLE) as a marker. Since rat plasma has no cholesteryl ester transfer activity, the amount of label in various tissues should reflect the quantitative uptake of Lp(a). Isolated Lp(a) was labeled with [3H]CLE by incubation overnight of Lp(a), a source of cholesteryl ester transfer activity (1.23 g/ml infranate of human plasma), and [3H]CLE-labeled Intralipid. Following labeling, the homogeneity and integrity of Lp(a) was shown by agarose electrophoresis and immunoblotting. Intact Lp(a) was injected via the tail vein of rats (120-170 g, n = 4 at each time point), and tissues were collected at various times thereafter (4-48 h). The disappearance curve of [3H]CLE-labeled Lp(a) from rat plasma was bimodal and had an initial rapid t1/2 of 1.8 h followed by a slower component, t1/2 = 13.3 h. Tissue uptake at all sampling times was greatest in liver (28.5% at 48 h of total dpm injected), followed by the intestine (9-12%), with less than 3% uptake by spleen. The small intestine was divided into four segments, and while the 3H radioactivity was similar in the proximal segments, a time-related increase in [3H]CLE was seen in its most distal portion. These studies indicate that the tissue sites of degradation in the rat of human Lp(a) are similar to human low-density lipoproteins (LDL); the increase in label in the distal portion of the small intestine with time may represent [3H]CLE excreted through the bile and absorbed by the mucosal cells.

Animals↗
PubMed1988

Modulation of lipoprotein lipase activity in mouse peritoneal macrophages by recombinant human tumor necrosis factor.

Thioglycollate-elicited mouse peritoneal macrophages spontaneously secrete lipoprotein lipase during culture. Exposure of the cultures to 50 ng/ml of recombinant human tumor necrosis factor (rTNF) for 48 h resulted in a 69% reduction in lipoprotein lipase activity in the culture medium with a concomitant decrease in cellular enzyme activity. The decrease in enzyme activity was not the result of rTNF-dependent reduction in the total protein synthesis, since the presence of rTNF did not affect [3H]leucine incorporation into cellular proteins. The effect of rTNF on lipoprotein lipase was reversible; upon TNF withdrawal, enzyme activity returned to basal levels after 60 h. The reduction of lipoprotein lipase in rTNF-treated cultures could be completely prevented by preincubation with a specific antiserum against recombinant human TNF. The late onset of decrease of lipoprotein lipase (LPL) activity suggests that rTNF might induce a mediator, which in turn suppresses LPL production. While rTNF was very effective in reducing lipoprotein lipase activity in mouse peritoneal macrophages, it did not affect lipoprotein lipase activity when added to the murine J774 cell line and to CT2 macrophage-like cells, a variant of the J774 cell line.

Animals↗
PubMed1988

Sphingomyelin liposomes with defined fatty acids: metabolism and effects on reverse cholesterol transport.

Small unilamellar liposomes prepared from sphingomyelins with defined 14C-labeled fatty acids were studied after injection into rats. The liposomes contained trace amounts of [3H]cholesteryl linoleyl ether (CLE), which served as a nonexchangeable and nonhydrolyzable marker. The liposomes were cleared from the circulation with an initial t1/2 of about 90 min. [14C]18:0- and [14C]18:1-containing sphingomyelins were cleared at a similar rate, but [14C]18:2-sphingomyelin disappeared much faster. The liver accounted for up to 70% of [3H]cholesteryl ether injected with 18:0-sphingomyelin liposomes, and for up to 50% with liposomes prepared from 18:1 or 18:2-sphingomyelin. The initial uptake of the liver appeared to be of the entire particle, and the loss of 14C label with time indicated metabolism of the sphingomyelins. With [14C]18:0-sphingomyelin liposomes, up to 8% of liver radioactivity was recovered in neutral lipids 6 h after injection, and this value was 17 and 22% with [14C]18:2- and [14C]18:1-sphingomyelins, respectively. The recovery in 'carcass' of [3H]cholesteryl ether 3 h after injection of [14C]18:2-sphingomyelin liposomes was 33% and of 14C label, 21%. Injection of 18:1- or 18:2-sphingomyelin liposomes (5.4 mumol/100 g body weight) resulted in a 2-fold increase of plasma unesterified cholesterol; a 30% increase was seen with 18:0 liposomes (2.63 mumol/100 g body weight). In experiments with cultured cells, the unsaturated sphingomyelin liposomes alone enhanced cholesterol efflux more extensively than the saturated ones, but their efficacies became similar when mixed with apoprotein (apo) A-I. At equimolar concentration, apo C-III1 or C-III2 had a smaller effect than apo A-I. It is concluded that 18:1- or 18:2-sphingomyelin tends to form small unilamellar liposomes which may reach also extrahepatic tissues. The liposomes able to enhance cholesterol release in vitro and in vivo. Since they are not a substrate for lecithin-cholesterol acyltransferase, they should be able to deliver the free cholesterol to the liver, where they are also rapidly metabolized.

Adrenal Glands↗
PubMed1988

Tunicamycin-treated rat heart cell cultures synthesize an inactive nonreleasable lipoprotein lipase.

Cells isolated from newborn rat hearts were cultured in the presence of 100 mM Hepes (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). Lipoprotein lipase activity was present in an intracellular and heparin-releasable pool and was also secreted into the culture medium. Treatment of the cultures with 5 micrograms/ml tunicamycin caused almost complete loss of lipoprotein lipase activity in all three compartments. In control cultures, immunoblotting of lipoprotein lipase derived from all three pools revealed a single band of lipoprotein lipase with an apparent Mr of 56,000. In the tunicamycin-treated cultures, the enzyme appeared only intracellularly and had an apparent Mr of 49,000. No immunoreactive enzyme was found in the medium. Thus, glycosylation of lipoprotein lipase in heart cell cultures is mandatory for enzyme activity and translocation from an intracellular to the heparin-releasable pool and for secretion into the medium.

Animals↗
PubMed1988

Lipoprotein lipase in heart cell cultures is suppressed by bacterial lipopolysaccharide: an effect mediated by production of tumor necrosis factor.

Exposure of rat heart cell cultures, consisting mainly of nonbeating mesenchymal cells, to 50 ng/ml of bacterial lipopolysaccharide (LPS) for 24 h resulted in a more than 80% reduction in lipoprotein lipase activity. The loss of enzymic activity was accompanied by a concomitant reduction in enzyme protein, as shown by immunoblotting. Addition of LPS to the culture medium resulted also in the production of tumor necrosis factor (TNF), and the fall in lipoprotein lipase in LPS-treated cultures could be prevented by an antibody to TNF. Addition of recombinant human TNF to the heart cell cultures also depressed lipoprotein lipase activity. LPS treatment of preadipocytes in culture resulted in a fall in lipoprotein lipase activity and TNF production. Since TNF is known as a macrophage product, the cultures were tested for phagocytic capacity, and only 0.2-1.3% of the cells were shown to engulf Staphylococcus albus. Immunofluorescent staining with monoclonal antibodies OX-1, which identify leukocyte common antigen, was negative, and only 0.1 +/- 0.07% of the cells were positive after staining with OX-42 antibody to iC3b receptor. Both antibodies stained more than 98% of rat peritoneal macrophages used as controls. Since LPS treatment of macrophages at numbers comparable to or exceeding the number of phagocytic cells present in the heart cell cultures did not induce measurable amounts of TNF, it is suggested that in the heart cell cultures, TNF may be produced by cells other than macrophages.

Animals↗
PubMed1988

Preferential metabolism by macrophages of conditioned rabbit hypercholesterolemic remnant lipoproteins.

Rabbits were fed a diet containing 1% cholesterol, and lipoproteins were isolated from their plasma by sequential ultracentrifugation. Lipoprotein remnants (d = 1.019-1.063) were conditioned by incubation with bovine aortic smooth muscle cells for 24 h and then incubated for 4 h with J774 cells or mouse peritoneal macrophages. Conditioning of remnants and low-density lipoproteins resulted in enhancement of their uptake and metabolism by the macrophages in culture. The macrophages metabolized conditioned remnant lipoproteins more extensively than controls at all concentrations tested. Addition of 10% safflower oil or 10% butter to the 1% cholesterol diet resulted in a molar ratio of linoleic/oleic acid of 1.62 and 0.62 in plasma neutral lipids and of 5.7 and 2.5 in plasma phospholipids. Conditioned remnants, derived after safflower oil feeding, were metabolized more extensively by macrophages than those obtained after butter feeding. This was also true for control remnants (preincubated without cells). Thiobarbituric acid-reactive substances were higher in remnants from safflower oil-fed than butter-fed rabbit plasma prior to and after conditioning. Mouse peritoneal macrophages metabolized remnant lipoproteins more extensively than low-density lipoproteins. The present results indicate that modification of remnant lipoproteins, the major atherogenic fraction of cholesterol-fed rabbit plasma, results in their enhanced metabolism by macrophages, and that an increase in the linoleic/oleic acid ratio in these lipoproteins might enhance their susceptibility to peroxidative modification.

Animals↗
PubMed1987

Metabolic fate and effect on cholesterol removal of liposomes prepared from 1,3-di-O-octadecenylglycero-2-phosphocholine studied in vivo and in vitro.

Available methodology was adapted to synthesize a labeled diether analog of 2-phosphatidylcholine (1,3-di-O-9'-cis-[9',10' (n)-3H]octadecenylglycero-2-phosphocholine [( 3H]DOE-2-PC). Unilamellar liposomes prepared by sonication from this phospholipid were injected into rats and, 4 h later, 65-78% of injected label was recovered in the liver. Thereafter, liver radioactivity disappeared with a half-life of 2-3 days. The radioactivity lost from the liver was recovered in the feces and in bile. Analysis of liver radioactivity showed that at all time intervals examined (4 h to 3 days after injection), 90% of the label remained as phospholipid. These findings provide evidence that this structural isomer is not readily metabolized, but is fairly rapidly eliminated from the liver. Of the 10% recovered as neutral lipid, 70% comigrated with diacylglycerol and 30% with triacylglycerol. Similar results were obtained when human hepatoma G2 cells in culture were incubated with [3H]DOE-2-PC liposomes. Following incubation of liposomes with liver homogenates, up to 10% conversion of [3H]DOE-2PC to neutral lipid occurred at pH 4.6, but not at pH 7.4. These data show that conversion of [3H]DOE-2-PC to dialkenylglycerol is catalyzed by a lysosomal enzyme. In separate experiments with cultured cells, sonicated dispersions of DOE-2-PC were mixed with high-density apolipoprotein and were shown to enhance markedly cellular cholesterol efflux. This novel diether phospholipid fulfills some of the criteria required of liposomes for their ability to remove cholesterol from the periphery as well as for drug delivery to the liver, i.e., stability in the circulation, marked hepatic uptake, slow metabolism, and elimination from the body.

Animals↗
PubMed1987

Preferential binding of [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal membranes.

Membranes isolated from bovine adrenal cortex, incubated with human high-density lipoproteins (HDL3), labeled with 125I and [3H]cholesteryl linoleyl ether, showed preferential binding of [3H]cholesteryl linoleyl ether. The preferential binding was Ca2+ independent, temperature sensitive and was slightly increased after phospholipase C or pronase treatment. Reduction of membrane phosphatidylcholine by phospholipase A2 resulted in a marked increase in the binding of the entire HDL3 particle and a relative decrease in preferential binding of [3H]cholesteryl linoleyl ether. These findings suggest that the presence of intact phospholipid in the membrane plays an important role in the magnitude of the preferential binding.

Adrenal Cortex↗
PubMed1987

Enhanced release and synthesis of lipoprotein lipase in rat heart cell cultures exposed to high concentrations of Hepes.

While attempting to optimize conditions for synthesis of lipoprotein lipase by cultured heart cells, we encountered an unexpected rise in enzyme activity when media were supplemented inadvertently with 100 mM Hepes buffer (4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid). This finding was further investigated and optimal results were obtained at pH 7.0-7.2. The increase in lipoprotein lipase activity was time dependent; after 3-6 h there was a rise in medium activity but cellular activity increased only after 24 h. The increased enzyme activity was defined as lipoprotein lipase by inhibition with antiserum to rat adipose tissue lipoprotein lipase. A 72-h exposure to Hepes resulted in a 30% increase in the incorporation of [35S]methionine into cellular proteins and a 2-fold increase into heparin-releasable proteins. Using heparin Sepharose chromatography and stepwise elution, a lipoprotein lipase enriched fraction was recovered with 2 M NaCl. The amount of [35S]methionine and [3H]galactose incorporated into protein of this fraction derived from Hepes-treated cells was 2-6-fold that of controls. A 4-fold increase in cellular lipoprotein lipase mass in Hepes-treated cells was shown by immunoblotting. Results obtained with Hepes-conditioned medium suggest the presence of cell-derived compounds that enhance release and subsequent synthesis of lipoprotein lipase. The effect of Hepes-conditioned medium on lipoprotein lipase resembled to some extent that of the addition of heparin. Therefore, it appears that when Hepes is first added to the culture medium, it might promote a release of heparan sulfate or related compounds, possibly by virtue of its negatively charged sulfonic acid residue. The accumulated heparan sulfate could then promote a sustained release of lipoprotein lipase into the culture medium which in turn leads to increased enzyme synthesis.

Animals↗
PubMed1987

Studies on the in vivo transfer of retinoids from parenchymal to stellate cells in rat liver.

Studies were conducted to examine the in vivo transfer of chylomicron (dietary) retinoid from rat liver parenchymal to stellate cells. We specifically addressed the question of whether chylomicron retinyl ester is transferred directly from hepatic parenchymal to stellate cells without first undergoing hydrolysis. [14C]Retinyl palmitate and its non-hydrolyzable ether analog, retinyl [3H]hexadecyl ether, were utilized to answer this question. Chylomicrons labeled with these retinoids were injected intravenously into rats. Liver cell fractions, highly enriched in parenchymal or in stellate cells, were isolated 0.5 h, 4.5 h and 24 h after chylomicron injection. The ratio of 3H: 14C found in parenchymal cell preparations 4.5 h after injection was 1.8 times the ratio for the injected chylomicrons, and 24 h postinjection the ratio had increased to 2.5 times that of the chylomicrons. In the stellate-cell-enriched preparations the 3H: 14C ratio was found to be 0.39, 0.29, and 0.23 times the ratio found in the injected labeled chylomicrons at 0.5 h, 4.5 h and 24 h after injection respectively. From the levels of 14C observed in the isolated stellate cells, it is estimated that 0.5 h postinjection the stellate cells contained approximately 34% of the 14C (i.e. the retinol injected as chylomicron retinyl ester) present in the liver. By 4.5 h the 14C present in isolated stellate cells had risen to approximately 41% of that present in the total liver, and 24 h after injection approximately 55% of hepatic total 14C was found in the stellate cells. These findings suggest that chylomicron retinyl ester is not transferred directly from the parenchymal to stellate cells without first undergoing hydrolysis to retinol.

Animals↗
PubMed1987

Preferential uptake of cholesteryl ester-HDL by cultured macrophages.

The interaction between HDL and macrophages in culture was studied using HDL labeled with 125I and with [3H]cholesteryl linoleyl ether. Mouse peritoneal macrophages and the macrophage-like cell lines J-774 and CT2, of mouse origin, took up and metabolized rat HDL and human HDL3. In all 3 cell types using both rat and human HDL, the uptake of the cholesteryl ester moiety as measured with the nondegradable cholesteryl ether analog, was 2-5-fold higher when compared to the protein moiety. Modulation of the cholesterol content of the cultured macrophages affected the uptake of both protein and lipid moieties of HDL to the same extent. When the macrophages had interacted with the labeled HDL for 5 h and were post-incubated for 20 h, the amount of [125I]HDL which reappeared in the post incubation medium was twice that of [3H]cholesteryl linoleyl ether-HDL. The site from which the HDL may have returned to the culture medium was tentatively localized to the trypsin-releasable, cell surface-related compartment. The present results indicate that interaction between macrophages and HDL may result in some loss of cholesteryl ester and possibly render the particle more receptive for cellular cholesterol removal.

Animals↗
PubMed1986

The role of apolipoproteins of HDL in the selective uptake of cholesteryl linoleyl ether by cultured rat and bovine adrenal cells.

Rat adrenal cells in culture were used to study the uptake of cholesteryl linoleyl ether [( 3H]cholesteryl linoleyl ether), a nonhydrolyzable analog of cholesteryl ester. When [3H]cholesteryl linoleyl ether was added in the form of liposomes, its uptake was enhanced by adrenocorticotropin (ACTH) and by addition of milk lipoprotein lipase and interfered by heparin. When the adrenal cells were incubated with homologous [3H]cholesteryl linoleyl ether-HDL, ACTH treatment also resulted in an increase in [3H]cholesteryl linoleyl ether uptake. The uptake of [3H]cholesteryl linoleyl ether was in excess of the uptake and metabolism of 125I-labeled HDL protein and was not sensitive to heparin. Unlabeled HDL or delipidated HDL reduced very markedly the uptake of [3H]cholesteryl linoleyl ether, while addition of phosphatidylcholine liposomes had little effect. Attempts were made to deplete and enrich the adrenal cells in cholesterol and, while depletion resulted in a decrease in [3H]cholesteryl linoleyl ether-HDL uptake, enrichment of cells with cholesterol had no effect. Among the individual apolipoproteins tested, apolipoprotein A-I and the C apolipoproteins reduced [3H]cholesteryl linoleyl ether uptake, while apolipoprotein E was not effective. Since the labeled ligand studied was a lipid, these effects could not be due to an exchange of apolipoproteins, but indicated competition for binding sites. Preferential uptake of human [3H]cholesteryl linoleyl ether-HDL3 by bovine adrenal cells was found when compared to the uptake and metabolism of 125I-labeled HDL. The present results suggest that the preferential uptake of HDL cholesteryl ester (as studied with [3H]cholesteryl linoleyl ether) requires an interaction between the apolipoproteins of HDL and cell surface components.

Adrenal Glands↗
PubMed1986

The role of apolipoprotein A-IV in reverse cholesterol transport studied with cultured cells and liposomes derived from an ether analog of phosphatidylcholine.

Cholesterol efflux was studied in a model system in culture using apolipoproteins and phospholipids added in the form of liposomes at concentrations expected to be present in the extracellular fluid. Fibroblasts were seeded in medium containing [3H]cholesterol-labeled serum, grown till confluent, and the [3H]cholesterol efflux was studied in serum-free medium. Addition of delipidated HDL apolipoprotein resulted in a very low release of [3H]cholesterol, which did not increase with time of exposure or concentration of apolipoproteins. Addition of increasing amounts of HDL apolipoprotein to liposomes prepared from either dioleoylphosphatidylcholine (PC) or its nonhydrolysable ether analog, dioleylphosphatidylcholine (DOEPC) resulted in a 3-5-fold increase of [3H]cholesterol efflux, over that achieved with liposomes alone. This model system permitted the test of the putative role of apolipoprotein A-IV in cholesterol removal from cells. The ability of apolipoprotein A-IV to enhance [3H]cholesterol efflux from cells by DOEPC liposomes was compared to that of apolipoproteins A-I, E and C, which were added at equimolar concentrations. At nM concentrations, apolipoproteins A-IV, A-I and E were equally able to enhance cholesterol efflux, while C apolipoproteins were less effective at these low concentrations. Mixtures prepared from apolipoprotein A-IV, A-I and E and PC or DOEPC liposomes were equally effective in cholesterol removal, while phosphatidylethanolamine liposome apolipoprotein mixtures had a much lower capacity. The present study provides the first evidence that apolipoprotein A-IV can play a role in reverse cholesterol transport as was suggested on the basis of high concentrations of this apolipoprotein in nonlipoprotein form in plasma and extracellular fluid. The efficacy of DOEPC liposomes to serve as cholesterol acceptors might be of potential value for enhancement of reverse cholesterol transport in vivo.

Apolipoprotein A-I↗
PubMed1986

Beta-adrenergic stimulation enhances translocation, processing and synthesis of lipoprotein lipase in rat heart cells.

Cells isolated from newborn rat hearts were cultured for 10-14 days, and lipoprotein lipase activity was present in an intracellular and heparin-releasable pool. Treatment of the cultures with 10(-7) M isoproterenol for 3 min resulted in a 3-fold increase in heparin-releasable lipoprotein lipase and a concomitant decrease in residual cellular enzyme activity. Similar results were obtained by treatment with dibutyryl cAMP. Treatment with isoproterenol or dibutyryl cAMP for 2 h affected glycosylation of immunoadsorbable lipoprotein lipase, so that the ratio of [3H]galactose to [14C]mannose in the heparin-releasable enzyme increased from 3.8 (control) to 13.0 (isoproterenol-treated). The change in the ratio of the sugars in the cellular fraction of the enzyme was from 3.1 to 9.9. 2 h treatment with isoproterenol did not enhance new enzyme synthesis, as determined by incorporation of [3H]leucine into immunoadsorbable lipoprotein lipase. 24 h after addition of either isoproterenol or dibutyryl cAMP to the culture medium, stimulation of enzyme synthesis was demonstrated. The present results permit three effects of isoproterenol on lipoprotein lipase to be distinguished: stimulation of translocation from a cellular to heparin-releasable pool; enhanced processing of mannose residues and terminal glycosylation; stimulation of synthesis of enzyme protein.

Animals↗
PubMed1986

Characterization of the lipoprotein lipase in the functional pool of rat heart by immunoblotting.

Rat hearts were perfused with heparin for 2 min at 4 degrees C. The lipoprotein lipase activity in the perfusate was inhibited by antiserum to rat adipose tissue lipoprotein lipase. By immunoblotting, the lipoprotein lipase derived from the functional pool of the heart was found to be a protein with an apparent Mr of 69 000. After incubation of the perfusate at 37 degrees C for 24 h an immunologically reactive protein with an apparent Mr of 28 000 was found. This protein is not a physiological derivative of the enzyme but a degradation product.

Animals↗