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J L Ellsworth

Publications and source records attributed to J L Ellsworth.

29 records · Page 2Linked to original sources
PubMed1988

A method for the preparation of metaphase chromosomes from rat small intestine.

A major obstacle to successful cytogenetic analysis of small intestinal crypt cells is the acquisition of a sufficient number of high-quality metaphases. A squash procedure has been developed for analysis of metaphase chromosomes from rat small intestine that largely circumvents this difficulty. The method involves a schedule of hypotonic treatment, fixation in ethanol: acetic acid, followed by maceration of the intestinal tissue in 3.5 N HCl. The procedure resulted in large numbers of well-spread, cytoplasm-free metaphases.

Animals↗
PubMed1988

Defining cellular senescence in IMR-90 cells: a flow cytometric analysis.

Using multiparameter flow cytometric analysis, we find that senescent cells accumulate in a unique cell-cycle compartment characterized in cell-cycle arrest in G1 and a significantly reduced nucleocytoplasmic ratio (genome size/cell mass) relative to cycling cells. With respect to gross cellular phenotype, the quiescent state of senescent cells differs from quiescence induced by density inhibition; the former is associated with a reduction in the nucleocytoplasmic ratio, while the latter is associated with an increase in the nucleocytoplasmic ratio. Senescent cells were present at all passages examined. The frequency of senescent cells was low in early-passage cultures and increased with passage number. Senescence of populations of IMR-90 cells reflects change in the relative frequency of these cells. The frequency of cells with karyotypic changes increased with the progressive accumulation of out-of-cycle cells.

Cell Cycle↗
PubMed1987

Human plasma lipid transfer protein catalyzes the speciation of high density lipoproteins.

The role of purified plasma lipid transfer protein complexes in determining the particle size distribution of human plasma high density lipoproteins (HDL) was examined in vitro. Incubation of HDL2 or HDL3, isolated from normolipemic subjects with very low density lipoproteins (VLDL) or VLDL-remnants and lipid transfer protein complex had little or no effect on HDL particle size. In contrast, HDL isolated from patients with hypertriglyceridemia, designated HDL3D, showed speciation of particle size distribution when incubated with VLDL-remnants and the transfer protein. Incubation of HDL3D with VLDL-remnants and lipid transfer complex resulted in the production of two particles of radius 4.3 and 3.7 nm; incubation with VLDL or in the absence of the transfer protein did not result in a redistribution of particle size. We suggest that the action of lipid transfer protein complex on triacylglycerol-rich lipoprotein remnants and HDL accounts for the low levels of HDL-cholesterol observed in subjects with severe hypertriglyceridemia.

Carrier Proteins↗
PubMed1987

Transport of beta-very low density lipoproteins and chylomicron remnants by macrophages is mediated by the low density lipoprotein receptor pathway.

The receptor-mediated uptake of rat hypercholesterolemic very low density lipoproteins (beta VLDL) and rat chylomicron remnants was studied in monolayer cultures of the J774 and P388D1 macrophage cell lines and in primary cultures of mouse peritoneal macrophages. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was reduced 80-90% in the presence of high concentrations of unlabeled human low density lipoproteins (LDL). Human acetyl-LDL did not significantly compete at any concentration tested. Uptake of 125I-beta VLDL and 125I-chylomicron remnants was also competitively inhibited by specific polyclonal antibodies directed against the estrogen-induced LDL receptor of rat liver. Incubation in the presence of anti-LDL receptor IgG, but not nonimmune IgG, reduced specific uptake greater than 80%. Anti-LDL receptor IgG, 125I-beta VLDL, and 125I-chylomicron remnants bound to two protein components of apparent molecular weights 125,000 and 111,000 on nitrocellulose blots of detergent-solubilized macrophage membranes. Between 70-90% of 125I-lipoprotein binding was confined to the 125,000-Da peptide. Binding of 125I-beta VLDL and 125I-chylomicron remnants to these proteins was competitively inhibited by anti-LDL receptor antibodies. Comparison of anti-LDL receptor IgG immunoblot profiles of detergent-solubilized membranes from mouse macrophages, fibroblasts, and liver, and normal and estrogen-induced rat liver demonstrated that the immunoreactive LDL receptor of mouse cells is of a lower molecular weight than that of rat liver. Incubation of J774 cells with 1.0 micrograms of 25-hydroxycholesterol/ml plus 20 micrograms of cholesterol/ml for 48 h decreased 125I-beta VLDL uptake and immuno- and ligand blotting to the 125,000- and 111,000-Da peptides by only 25%. Taken together, these data demonstrate that uptake of beta VLDL and chylomicron remnants by macrophages is mediated by an LDL receptor that is immunologically related to the LDL receptor of rat liver.

Animals↗
PubMed1986

Immunoaffinity purification of the lipid transfer protein complex directly from human plasma.

The human cholesteryl ester (CE) and triglyceride (TG) exchange protein (denoted LTC or lipid transfer complex) was isolated in a single step from plasma using immunoaffinity batch extraction. Antibodies were raised against two preparations of conventionally purified LTC. LTC-I and LTC-II (purified 20,000-fold and 3500-fold, respectively) were used as immunogens. The antiLTC antibodies were isolated by anion-exchange chromatography and coupled to Affi-Gel 10. Chromatography of plasma on antiLTC Affi-Gel removed all of the CE and TG transfer activity. Moreover, LTC prepared from both antiLTC-I and antiLTC-II-Affi-Gel matrices were identical when analyzed by sodium dodecyl sulfate-polyacrylamide gel LTC electrophoresis. LTC exhibited two protein bands of Mr (apparent) 67,000 and 58,000 and a broad, faintly staining region at greater than 150,000. Analysis of LTC by immunoblotting indicated that both antiLTC-I and antiLTC-II antibodies recognized the same LTC proteins. Isoelectric focussing of LTC gave two pI values, 5.2 and 8.7. These data suggest that LTC is a complex of specific proteins and perhaps lipid. Specific CE and TG exchange activities of immunoaffinity-purified LTC were comparable, although the activities were low with respect to that of the antigen used to generate antiLTC-I. This is not due to contamination of LTC by albumin, lecithin:cholesterol acyltransferase, or apolipoproteins AI, AII, B, CIII, D, or E.

Adult↗
PubMed1986

Very low and low density lipoprotein synthesis and secretion by the human hepatoma cell line Hep-G2: effects of free fatty acid.

The liver is a major source of the plasma lipoproteins; however, direct studies of the regulation of lipoprotein synthesis and secretion by human liver are lacking. Dense monolayers of Hep-G2 cells incorporated radiolabeled precursors into protein ([35S]methionine), cholesterol ([3H]mevalonate and [14C]acetate), triacylglycerol, and phospholipid ([3H]glycerol), and secreted them as lipoproteins. In the absence of free fatty acid in the media, the principal lipoprotein secretory product that accumulated had a density maximum of 1.039 g/ml, similar to serum low density lipoprotein (LDL). ApoB-100 represented greater than 95% of the radiolabeled apoprotein of these particles, with only traces of apoproteins A and E present. Inclusion of 0.8 mM oleic acid in the media resulted in a 54% reduction in radiolabeled triacylglycerol in the LDL fraction and a 324% increase in triacylglycerol in the very low density lipoprotein (VLDL) fraction. Similar changes occurred in the secretion of newly synthesized apoB-100. The VLDL contained apoB-100 as well as apoE. In the absence of exogenous free fatty acid, the radiolabeled cholesterol was recovered in both the LDL and the high density lipoprotein (HDL) regions. Oleic acid caused a 50% decrease in HDL radiolabeled cholesterol and increases of radiolabeled cholesterol in VLDL and LDL. In general, less than 15% of the radiolabeled cholesterol was esterified, despite the presence of cholesteryl ester in the cell. Incubation with oleic acid did not cause an increase in the total amount of radiolabeled lipid or protein secreted. We conclude that human liver-derived cells can secrete distinct VLDL and LDL-like particles, and the relative amounts of these lipoproteins are determined, at least in part, by the availability of free fatty acid.

Acetates↗
PubMed1986

Evidence that chylomicron remnants and beta-VLDL are transported by the same receptor pathway in J774 murine macrophage-derived cells.

To characterize lipoprotein uptake by macrophages, we studied J774 murine macrophage-derived cells. Uptake of 125I-labeled beta-VLDL and 125I-labeled chylomicron remnants was saturable, specific, and of high affinity. Maximal specific uptake and the concentration at which half-maximal uptake occurred were similar for both beta-VLDL and chylomicron remnants. Specific uptake of 125I-labeled chylomicrons was only 1/5 that of the other two lipoproteins. Cholesterol loading decreased 125I-labeled chylomicron remnant and 125I-labeled beta-VLDL uptake by 25%. Chylomicron remnants and beta-VLDL were equipotent in cross-competition studies; acetyl-LDL did not compete, and human LDL was a poor competitor. Although the amounts of cell-associated lipoproteins were similar, beta-VLDL and chylomicron remnants had different effects on cellular lipid metabolism. beta-VLDL produced a threefold stimulation while chylomicron remnants caused a decrease in [3H]oleate incorporation into cholesteryl ester. beta-VLDL had no effect while chylomicron remnants caused a threefold increase in [3H]oleate incorporation into triacylglycerol. beta-VLDL produced a 44% suppression and chylomicron remnants produced a 78% increase in HMG-CoA reductase activity. In summary, J774 macrophages express a receptor site that recognizes both beta-VLDL and chylomicron remnants; however, these lipoproteins exhibit strikingly different effects on intracellular lipid metabolism.

Animals↗
PubMed1982

Interaction of a human plasma lipid transfer protein complex with lipid monolayers.

The interaction of a purified human plasma lipid transfer complex with cholesteryl ester, triacylglycerol and phosphatidylcholine in binary and ternary lipid monolayers was investigated. The lipid transfer complex, designated LTC, catalyzes the removal of cholesteryl oleate and triacylglycerol from phosphatidylcholine monolayers. Preincubation of LTC with p-chloromercuriphenyl sulfonate inhibits LTC-catalyzed removal of triacylglycerol; cholesteryl ester removal is not affected. The rate of LTC-facilitated removal of cholesteryl oleate from a phosphatidylcholine monolayer depends on the amount of LTC added to the subphase up to 100 micrograms protein. In addition, the rate of the LTC-catalyzed transfer of cholesteryl oleate to the subphase increases linearly as the amount of cholesteryl oleate in the monolayer increases to 6 mol%. LTC also removes cholesterol from phosphatidylcholine-cholesterol monolayers, albeit at a rate which is 15% of that for removal of cholesteryl oleate. The ability of LTC to facilitate triacylglycerol and cholesteryl ester removal depends on the composition of the monolayer. Phosphatidylcholine supports cholesteryl ester transfer whereas sphingomyelin-cholesteryl ester monolayers are almost refractory to LTC. In contrast, LTC removes triacylglycerol from either a phosphatidylcholine or a sphingomyelin monolayer. The results suggest the existence of at least two lipid transfer proteins, one of which catalyzes the removal of cholesteryl ester and the other triacylglycerol. The role of these proteins as they relate to lipoprotein metabolism is discussed.

Carrier Proteins↗
PubMed1982

Plasma protein-facilitated coupled exchange of phosphatidylcholine and cholesteryl ester in the absence of cholesterol esterification.

A protein(s) which catalyzes the exchange of phosphatidylcholine and cholesteryl ester between plasma lipoproteins has been purified 10,000-fold from lipoprotein-free human plasma. The apparent molecular weight of the protein of the active fraction, designated lipid transfer complex (LTC), is approximately 61,000; when electrophoresed in 6 M urea, 0.1% sodium dodecyl sulfate on a 3-20% polyacrylamide gradient, the protein appears as a doublet of molecular weights 58,000 and 63,000. The active material is a glycoprotein which binds to concanavalin A. Human LTC is a lipid-protein complex with phospholipid, cholesterol, cholesteryl ester, and glyceride comprising 7% of the total mass. A similar glycoprotein (or glycoproteins) exists in rat plasma, although the fold-purification thus far achieved is low: about 500-fold. Moreover, the rat preparation enhances exchange of phosphatidylcholine, but does not appreciably enhance exchange of cholesteryl ester. Partially purified LTC (less than or equal to 3500-fold) exists in a complex with lecithin: cholesterol acyltransferase. Active lecithin: cholesterol acyltransferase is not, however, required for exchange of phosphatidylcholine or cholesteryl ester facilitated by human LTC. The rates of exchange of phosphatidylcholine and cholesteryl ester facilitated by human LTC are equal. Coupled lipid exchange occurs at all stages of LTC purification, at values of pH between 5 and 10, and at ionic strengths as great as 0.9. Moreover, phosphatidylcholine and cholesteryl ester are exchanged with 1:1 stoichiometry in the presence of thiol group reagents such as 5,5'-dithiobis-(2-nitrobenzoic acid). Both lipid exchange activities are relatively resistant to elevated temperatures. Coupled exchange of phospholipid and neutral lipid is not dictated by the nature of the lipoprotein donor and acceptor substrates: bovine liver phospholipid exchange protein catalyzes exchange of phosphatidylcholine but not cholesteryl ester between low and high density lipoproteins under conditions identical with those in which human LTC facilitates exchange of both lipids.

Animals↗
PubMed1982

Human plasma lipid exchange protein(s): a method for separation of donor and acceptor lipoproteins by heparin-Sepharose chromatography.

The transfer or exchange of cholesteryl esters, triglycerides, and phospholipids between plasma very low (VLDL), low (LDL), and high (HDL) density lipoproteins is facilitated by specific lipid transfer proteins. The present report describes a method to separate donor and acceptor lipoprotein pools used in assays for lipid exchange activities. The method is based on the differential binding of lipoproteins to immobilized heparin. At 50 mM NaCl concentration, VLDL and LDL bind to heparin-Sepharose whereas greater than 85% of HDL is unretained; VLDL and LDL are than eluted with 300 mM NaCl, 2% sodium dodecyl sulfate with a recovery greater than 85%. The procedure is rapid and quantitative, as judged by a comparison to ultracentrifugation.

Biological Transport, Active↗