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Y K Ho

Publications and source records attributed to Y K Ho.

At least 91 records · Page 5Linked to original sources
PubMed1981

Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E.

Monolayers of mouse peritoneal macrophages were shown to synthesize and secrete a protein that resembles apoprotein E (apoE), a normal constituent of plasma lipoproteins. Synthesis and secretion were studied by incubation of macrophages with L-[35S]methionine and analysis of the 35S-labeled proteins secreted into the culture medium. The 35S-labeled protein resembling apoE showed the following properties: (i) it floated in the ultracentrifuge at a density less than 1.215 g/ml, indicating that it was associated with lipid; (ii) by NaDodSO4/polyacrylamide gel electrophoresis, its Mr of 35,000 was identical to that of authentic apoE obtained from mouse plasma very low density lipoprotein; (iii) its isoelectric point of 5.4 was the same as that of authentic mouse apoE; (iv) it comigrated with authentic mouse apoE after two-dimensional isoelectric focusing/NaDodSO4/polyacrylamide gel electrophoresis; and (v) it was quantitatively precipitated by a monospecific antibody directed against rat apoE. Synthesis and secretion of the apoE-like protein was stimulated 3- to 8-fold when the macrophages were loaded with cholesterol by incubation with either acetylated low density lipoprotein (acetyl-LDL) or beta-migrating very low density lipoprotein from cholesterol-fed rabbits. When the cells were incubated with acetyl-LDL, the apoE-like protein composed approximately 2% of the total 35S-labeled protein synthesized by the cells and approximately 10% of the total 35S-labeled protein secreted into the medium. The current findings suggest a role for apoE in the plasma transport of cholesterol excreted from cholesterol-loaded macrophages.

Animals↗
PubMed1980

Enzymatic synthesis of polyuridylic acid containing modified bases.

5'Mercaptouridine-5'-diphosphate (hs5UDP) has been synthesized and investigated as a substrate of the polynucleotide phosphorylase of Micrococcus luteus. While hs5UDP is not utilized alone, it can be copolymerized with UDP; however, unusually for this enzyme, the ratio of 5'mercaptouridylate vs. uridylate residues in the polynucleotide product (MPU) is always lower than the ratio of hs5UDP v. UDP in the substrate mixture. Furthermore, hs5UDP decreases the rate of the enzymic polymerization reaction. The MPU product forms two-stranded and three-stranded complexes with poly(A). The circular dichroic spectra of these complexes are similar to those formed between poly(U) and poly(A), but their melting profiles indicate somewhat lower stability. The physicochemical and biochemical properties of the enzymic product are qualitatively similar to those of MPU prepared by chemical modification; both are potent inhibitors of a DNA-dependent RNA polymerase.

Circular Dichroism↗
PubMed1980

Cholesteryl ester accumulation in macrophages resulting from receptor-mediated uptake and degradation of hypercholesterolemic canine beta-very low density lipoproteins.

The synthesis and accumulation of cholesteryl esters by monolayers of mouse peritoneal macrophages was stimulated 20- to 160-fold by incubation with beta-migrating very low density lipoproteins (beta-VLDL, density less than 1.006 g/ml) isolated from the plasma of cholesterol-fed dogs. Three other cholesterol-rich lipoprotein fractions obtained from the plasma of the same hypercholesterolemic dogs, including low density lipoprotein (LDL), cholesterol-induced high density lipoprotein (HDLc), and apo-E HDLc, had little to no stimulatory effect. Plasma VLDL (density less than 1.006 g/ml) from normal dogs did not increase cholesteryl ester formation in macrophages. The enhancement in cholesteryl ester synthesis and accumulation by hypercholesterolemic canine beta-VLDL was due to the presence of a high affinity binding site on the macrophage cell surface that mediated the uptake and lysosomal degradation of the beta-VLDL. Competition studies with fucoidin and dextran sulfate indicated that the receptor for canine beta-VLDL was different from that previously described for human acetylated low density lipoprotein (acetyl-LDL). Prior incubation of macrophage monolayers with either unlabeled canine beta-VLDL or human acetyl-LDL, both of which raised the cellular content of cholesteryl esters, reduced the ability of the cells to degrade 125I-labeled beta-VLDL, suggesting that the receptor for beta-VLDL is subject to regulation. The current findings indicate: 1) that macrophages possess a high affinity receptor that recognizes one of the four cholesterol-rich lipoproteins present in the plasma of cholesterol-fed dogs, beta-VLDL, and 2) that the receptor-mediated ingestion of beta-VLDL leads to cholesteryl ester deposition in these cells.

Animals↗
PubMed1980

The scavenger cell pathway for lipoprotein degradation: specificity of the binding site that mediates the uptake of negatively-charged LDL by macrophages.

Macrophages isolated from a variety of organs in several animal species exhibit high affinity binding sites that recognize chemically modified proteins. One of these binding sites recognizes human plasma low density lipoprotein (LDL) in which the positive charges on the epsilon-amino groups of lysine have been removed or neutralized by chemical modification, thus giving the protein an enhanced negative charge. Effective treatments include reaction of LDL with organic acid anhydrides (acetylation or maleylation) and reaction with aldehydes, such as treatment with malondialdehyde. After the negatively-charged LDL binds to the surface receptor sites, it is rapidly internalized by the macrophages by endocytosis and hydrolyzed in lysosomes. The liberated cholesterol is reesterified in the cytoplasm, producing massive cholesteryl ester deposition. The binding sites for negatively-charged LDL has been demonstrated so far only on macrophages and other scavenger cells. It is not expressed in cultured fibroblasts, smooth muscle cells, lymphocytes, or adrenal cells. In addition to its affinity for acetylated LDL and malondialdehyde-treated LDL, the macrophage site binds a variety of polyanions. It exhibits a particularly high affinity for certain sulfated polysaccharides (dextran sulfate and fucoidin), certain polynucleotides (polyinosinic acid and polyguanylic acid), polyvinyl sulfate, and maleylated albumin. It is possible that the site that binds negatively-charged LDL may be responsible for the massive accumulation of cholesteryl esters that occurs in vivo in macrophages and other scavenger cells in patients with high levels of circulating plasma LDL.

Acetylation↗
PubMed1980

Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.

The ability of mouse peritoneal macrophages to hydrolyze and excrete cytoplasmic cholesteryl ester droplets was studied. The macrophages were loaded with cholesteryl esters by incubation with acetylated low density lipoprotein (acetyl-LDL), which is internalized by adsorptive endocytosis. The cholesteryl esters of acetyl-LDL are hydrolyzed within lysosomes and the liberated cholesterol is re-esterified in the cytoplasm where it accumulates as cytoplasmic cholesteryl ester droplets. Hydrolysis and excretion of these stored cholesteryl esters were quantified by gas-liquid chromatographic measurement of the content of free and esterified cholesterol in cells and in medium. After removal of acetyl-LDL from the culture medium, the cytoplasmic cholesteryl esters were rapidly hydrolyzed and large amounts of free cholesterol were excreted from the cells. Hydrolysis and excretion required a cholesterol acceptor in the culture medium. The following agents were shown to be effective as cholesterol acceptors: high density lipoprotein (HDL), whole serum, the density > 1.215 g/ml fraction of whole serum, intact erythrocytes, casein, and thyroglobulin. The following agents did not promote the hydrolysis and excretion of cholesteryl esters under these experimental conditions: LDL, serum albumin, serum gamma-globulins, and phosphatidylcholine/sphingomyelin liposomes. The results indicate that net hydrolysis of cytoplasmic cholesteryl esters in macrophages is coupled to the process of cholesterol excretion and that net hydrolysis does not occur unless an effective cholesterol acceptor is present in the culture medium.-Ho, Y. K., M. S. Brown, and J. L. Goldstein. Hydrolysis and excretion of cytoplasmic cholesteryl esters by macrophages: stimulation by high density lipoprotein and other agents.

Animals↗
PubMed1980

Cholesteryl ester synthesis in macrophages: stimulation by beta-very low density lipoproteins from cholesterol-fed animals of several species.

Animals fed cholesterol accumulate several types of cholesterol-rich lipoproteins in their plasma and ultimately develop cholesteryl ester deposition in tissue macrophages. Previous studies in the cholesterol-fed dog have shown that one class of cholesterol-rich lipoproteins. beta-migrating very low density lipoproteins (beta-VLDL, density < 1.006 g/ml), possesses a unique ability to produce cellular cholesteryl ester accumulation when incubated with mouse peritoneal macrophages in vitro. This accumulation results from the receptor-mediated uptake of beta-VLDL with subsequent lysosomal hydrolysis of the lipoprotein and re-esterification of the liberated cholesterol. In the current studies, we demonstrate that beta-VLDL obtained from cholesterol-fed animals of several other species, including monkeys, rabbits, and rats, also causes cholesteryl ester accumulation in monolayers of mouse peritoneal macrophages, as monitored by an increase in the rate at which the cells incorporate exogenous [14C]oleate into cholesteryl [14C]oleate. Like canine beta-VLDL, the beta-VLDL from these three other species were effective at low concentrations and exhibited saturation kinetics, suggesting that they, too, entered macrophages by receptor-mediated endocytosis. Very low density lipoprotein (VLDL) from normal animals and low density lipoprotein (LDL) from normal and cholesterol-fed monkeys, rats, and rabbits did not stimulate cholesteryl ester synthesis in mouse peritoneal macrophages. In addition to their effects on mouse macrophages, the beta-VLDL from cholesterol-fed dogs and rabbits stimulated cholesteryl ester synthesis in cultured human monocytes. The current findings suggest that beta-VLDL from cholesterol-fed animals has the general property of stimulating cholesteryl ester synthesis and accumulation in macrophages.

Animals↗
PubMed1979

Light dependence of the decay of the proton gradient in broken chloroplasts.

The initial rates and steady-state values of proton uptake by broken chloroplasts have been measured as functions of light intensity at various concentrations of chlorophyll, pyocyanine, supporting electrolyte, buffer, as well as pH and temperature. Kinetics analysis of the data shows that the rate of decay of proton gradient due to backward leakage depends on light intensity. Under steady illumination, the decay constant kL is equal to kD + mR0, where R0 is the initial rate of proton uptake which is a function of light intensity, kD is the decay constant in the dark and m is a parameter which is independent of light intensity. Treatment of chloroplasts with lysolecithin, neutral detergent, 2,4-dinitrophenol, or valinomycin in the presence of K+ increases kD without affecting m. Treatment with N,N'-dicyclohexylcarbodiimide or adenylyl imidodiphosphate under appropriate conditions decreases m without affectsity and hence m = 0. These results suggest that the light-dependent part (mR0) of kL is due to leakage of protons through the coupling factor (CF1-CF0) complex which can open or close depending on light intensity and that the light independent part (kD) of the decay constant kL is due to proton leakage elsewhere.

Adenylyl Imidodiphosphate↗
PubMed1979

Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition.

Resident mouse peritoneal macrophages were shown to take up and degrade acetylated (125)I-labeled low density lipoprotein ((125)I-acetyl-LDL) in vitro at rates that were 20-fold greater than those for the uptake and degradation of (125)I-LDL. The uptake of (125)I-acetyl-LDL and its subsequent degradation in lysosomes were attributable to a high-affinity, trypsin-sensitive, surface binding site that recognized acetyl-LDL but not native LDL. When (125)I-acetyl-LDL was bound to this site at 4 degrees C and the macrophages were subsequently warmed to 37 degrees C, 75% of the cell-bound radioactivity was degraded to mono[(125)I]iodotyrosine within 1 hr. The macrophage binding site also recognized maleylated LDL, maleylated albumin, and two sulfated polysaccharides (fucoidin and dextran sulfate) indicating that negative charges were important in the binding reaction. A similar binding site was present on rat peritoneal macrophages, guinea pig Kupffer cells, and cultured human monocytes but not on human lymphocytes or fibroblasts, mouse L cells or Y-1 adrenal cells, or Chinese hamster ovary cells. Uptake and degradation of acetyl-LDL via this binding site stimulated cholesterol esterification 100-fold and produced a 38-fold increase in the cellular content of cholesterol in mouse peritoneal macrophages. Although the physiologic significance, if any, of this macrophage uptake mechanism is not yet known, we hypothesize that it may mediate the degradation of denatured LDL in the body and thus serve as a "backup" mechanism for the previously described receptor-mediated degradation of native LDL that occurs in parenchymal cells. Such a scavenger pathway might account for the widespread deposition of LDL-derived cholesteryl esters in macrophages of patients with familial hypercholesterolemia in whom the parenchymal cell pathway for LDL degradation is blocked, owing to a genetic deficiency of receptors for native LDL.

Acetylation↗
PubMed1979

Reversible accumulation of cholesteryl esters in macrophages incubated with acetylated lipoproteins.

Mouse peritoneal macrophages accumulate large amounts of cholesteryl ester when incubated with human low-density lipoprotein that has been modified by chemical acetylation (acetyl-LDL). This accumulation is related to a high-affinity cell surface binding site that mediates the uptake of acetyl-LDL by adsorptive endocytosis and its delivery to lysosomes. The current studies demonstrate that the cholesteryl ester accumulation can be considered in terms of a two-compartment model: (a) the incoming cholesteryl esters of acetyl-LDL are hydrolyzed in lysosomes, and (b) the resultant free cholesterol is re-esterified in the cytosol where the newly formed esters are stored as lipid droplets. The following biochemical and morphologic evidence supports the hydrolysis-re-esterification mechanism: (a) Incubation of macrophages with acetyl-LDL markedly increased the rate of cholesteryl ester synthesis from [14C]oleate, and this was accompanied by an increase in the acyl-CoA:cholesteryl acyltransferase activity of cell-free extracts. (b) When macrophages were incubated with reconstituted acetyl-LDL in which the endogenous cholesterol was replaced with [3H]-cholesteryl linoleate, the [3H]cholesteryl linoleate was hydrolyzed, and at least one-half of the resultant [3H]cholesterol was re-esterified to form [3H]cholesteryl oleate, which accumulated within the cell. The lysosomal enzyme inhibitor chloroquine inhibited the hydrolysis of the [3H]cholesteryl linoleate, thus preventing the formation of [3H]cholesteryl oleate and leading to the accumulation of unhydrolyzed [3H]cholesteryl linoleate within the cells. (c) In the electron microscope, macrophages incubated with acetyl-LDL had numerous cytoplasmic lipid droplets that were not surrounded by a limiting membrane. The time course of droplet accumulation was similar to the time course of cholesteryl ester accumulation as measured biochemically. (d) When acetyl-LDL was removed from the incubation medium, biochemical and morphological studies showed that cytoplasmic cholesteryl esters were rapidly hydrolyzed and that the resultant free cholesterol was excreted from the cell.

Acetylation↗
PubMed1979

Effects of partially thiolated polycytidylic acid on the clonogenicity of murine leukemic stem cells.

The effect of partially thiolated polycytidylic acid (MPC) on the colony-forming ability of the progenitor cells (CFUC) of RF/Un leukemic mice was investigated using the plasma clot method in order to study the mode of action of the modified polynucleotide. The results showed that MPC inhibited the CFUC in a dose-dependent and time-dependent manner. Once a maximum level of inhibition of CFUC (approximately 40%) was observed, no further inhibition occurred whether the concentration of MPC was increased or whether the duration of incubation was lengthened. High-specific-activity [3H]thymidine, an S-phase-specific agent, showed a similar inhibition profile on the CFUC as did MPC. When MPC and high-specific-activity [3H]thymidine were incubated together with the bone marrow cells, there was no additive or synergistic inhibitory effect on the CFUC. Thus, it appears that MPC is an S-phase-specific agent. When injected i.v. into the mice, MPC decreased the number of CFUC of both the bone marrow and the spleen significantly.

Animals↗
PubMed1979

Studies on the mode of action of partially thiolated polycytidylic acid (MPC), a novel type of antineoplastic agent.

Partially thiolated polycytidylic acid (MPC), a representative member of the "antitemplate" class of novel chemotherapeutic agents, is a potent inhibitor of the E. coli DNA-dependent RNA polymerase. It inhibited 50% of the enzymic reaction at a concentration of 6 micrometers. Kinetic studies indicated that MPC had no effect on the chain elongation of the transcription process, but it appeared to inhibit the initiation of RNA synthtesis presumably by competing with the DNA template for binding to the RNA polymerase. Binding studies, using a gel filtration method, showed that MPC and the RNA polymerase formed a stable complex which was not dissociated by 0.3 M NaCl. It is inferred that mixed disulfide linkage(s) might have been formed between the enzyme and MPC. The implications of these findings are discussed.

Antineoplastic Agents↗
PubMed1978

Genetics of the low density lipoprotein receptor. Diminished receptor activity in lymphocytes from heterozygotes with familial hypercholesterolemia.

Using circulating mononuclear cells as a readily available tissue and using the rate of high affinity degradation of 125-I-labeled low density lipoprotein (LDL) as an index of cell surface LDL receptor activity, we have measured receptor activity in cells from 53 individuals. This group includes 32 healthy subjects, 15 subjects with the heterozygous form of familial hypercholesterolemia, and 6 subjects with hyperlipidemic disorders other than familial hypercholesterolemia. 7 of the healthy subjects and 10 of the heterozygotes were members of a single large kindred with five-generation transmission of the mutant familial hypercholesterolemia gene. LDL receptor activity was assayed in blood mononuclear cells under two sets of conditions. First, 125I-LDL degradation was measured in purified lymphocytes that had been incubated for 3 days in the absence of lipoproteins so as to induce a high level of LDL receptor activity. Phase-contrast autoradiograms of cells incubated with 125I-LDL and electron micrographs of cells incubated with ferritin-labeled LDL confirmed the existence of LDL receptors on lymphocytes. Second, 125I-LDL degradation was measured in mixed mononuclear cells (85-90% lymphocytes and 5-15% monocytes) immediately after their isolation from the bloodstream. This assay represented an attempt to assess the number of receptors actually expressed on the cells when they were in the circulation. Under both sets of conditions, cells from the familial hypercholesterolemia heterozygotes expressed an average of about one-half the normal number of LDL receptors. The current findings are consistent with the conclusion that heterozygotes with familial hypercholesterolemia possess only one functional allele at the LDL receptor locus and that the consequent deficiency of LDL receptors produces the clinical syndrome of heterozygous familial hypercholesterolemia.

Adhesiveness↗
PubMed1977

Regulation of cholesterol synthesis by low density lipoprotein in isolated human lymphocytes. Comparison of cells from normal subjects and patients with homozygous familial hypercholesterolemia and abetalipoproteinemia.

The rate of cholesterol synthesis from [14C]acetate was low in circulating blood lymphocytes freshly isolated from 17 normal subjects and 4 subjects with homozygous FH. On the other hand, the rate of cholesterol synthesis was two to fourfold above normal in freshly isolated lymphocytes from two subjects with abetalipoproteinemia. When the lymphocytes from subjects with all three genotypes were incubated for 48-72 h in the absence of lipoproteins, the rate of cholesterol synthesis increased by 5-15-fold. The subsequent addition of plasma LDL, but not HDL, rapidly suppressed cholesterol synthesis in the lymphocytes from normal subjects. In contrast, lymphocytes from the FH homozygotes, which have been shown previously to be deficient in cell surface LDL receptors, were resistant to LDL-mediated suppression of cholesterol synthesis. In addition to its ability to suppress cholesterol synthesis after it had been elevated by incubation of the cells in the absence of lipoproteins, LDL was able to suppress the induction of the enhanced rate of sterol synthesis when added to normal lymphocytes immediately after their isolation from the bloodstream. In contrast to the former action of LDL, the latter action of LDL-i.e., the suppression of induction of sterol synthesis-also occurred to a limited extent in lymphocytes from FH homozygotes. However, the FH lymphocytes, but not the normal cells, could be made resistant to this action of LDL by inclusion in the incubation medium of lipoprotein-deficient serum (30 percent, vol/vol) plus HDL (1 mg protein/ml). Considered together with previous data demonstrating a deficiency of LDL receptors in freshly isolated lymphocytes from FH homozygotes, the current studies provide evidence in support of the hypothesis that the interaction of plasma LDL with its cell surface receptor serves to regulate cholesterol synthesis in human lymphocytes.

Abetalipoproteinemia↗