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Biomedical subjects

Y K Ho

Publications and source records attributed to Y K Ho.

17 recordsLinked to original sources
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↗
PubMed1976

Binding, internalization, and hydrolysis of low density lipoprotein in long-term lymphoid cell lines from a normal subject and a patient with homozygous familial hypercholesterolemia.

Long-term established human lymphoid cells were shown to possess high affinity cell surface receptors for low density lipoprotein (LDL), the major cholesterol-carrying protein in human plasma. Binding of LDL to these receptors was followed by internalization of the lipoprotein and hydrolysis of its protein and cholesteryl ester components. Cultured lymphocytes from a patient with the homozygous form of familial hypercholesterolemia lacked cell surface LDL receptors and therefore failed to take up and degrade the lipoprotein with high affinity. Cultured human lymphocytes should prove useful for further studies of: (a) the relation between cholesterol metabolism and cellular function and (b) the mechanism by which LDL binding at the cell surface leads to internalization of the lipoprotein.

Apoproteins↗
PubMed1976

The low-density lipoprotein pathway in human fibroblasts: relation between cell surface receptor binding and endocytosis of low-density lipoprotein.

The studies reported here, coupled with our previous studies, indicate that LDL is ingested by cultured human fibroblasts in a process that resembles adsorptive endocytosis. The critical step is the binding of the lipoprotein to a high-affinity cell surface receptor. Uptake of LDL by this receptor-mediated process permits the cell to aquire cholesterol from the lipoprotein, and this acquisition, in turn, suppresses the cell's own cholesterol synthesis and activates the cell's system for reesterification and storage of the incoming cholesterol. In cells from patients with the receptor-negative form of homozygous FH, the cell surface receptor is functionally absent. The absence of high-affinity binding to this receptor produces a defective uptake of LDL and prevents the normal process of feedback regulation of cholesterol synthesis by the lipoprotein. The physiologic importance of the LDL pathway is indicated by the fact that patients who lack the LDL receptor (FH homozygotes) develop both profound hyper-cholesterolemia and fulminant atherosclerosis. It is likely that other defects in the LDL pathway account for other forms of hypercholesterolemia and atherosclerosis in man.

Binding Sites↗
PubMed1976

Differential effects of 5-methylmercapto-2'-deoxyuridine on the replication of herpes simplex virus type 1 in two cell systems.

5-Methylmercapto-2'-deoxyuridine (MeMUdR), a structural analogue of thymidine (TdR), inhibits herpes simplex virus type 1 production in mouse L (Lb) cells at concentrations that are not inhibitory to viral growth in monkey kidney (CV-1) cells. It is moderately toxic to Lb cells but not to CV-1 cells at a concentration that causes 95% inhibition of viral replication in Lb cells. MeMUdR is incorporated into cellular and viral deoxyribonucleic acid (DNA) in both systems, but to a significantly higher level (compared with thymidine) in Lb cells. These results indicate that MeMUdR is a substrate for enzymes leading to DNA synthesis and suggest that the biological function of herpes simplex virus type 1 DNA is impaired only when the incorporation of MeMUdR into the DNA reaches a relatively high level.

Animals↗
PubMed1976

Regulation of low density lipoprotein receptor activity in freshly isolated human lymphocytes.

Circulating human lymphocytes freshly isolated from venous blood of 15 normal subjects exhibited a low capacity to bind, take up, and degrade 125I-labeled low density lipoprotein (LDL). However, when these cells were incubated for 72 h in the absence of lipoproteins, they gradually acquired in increased number of high affinity cell surface receptors for LDL. The increase in the number of LDL receptors was associated with a 16-fold increase in the rate at which the cells were able to take up and degrade the lipoprotein. The LDL binding and degradation processes that developed in normal lymphocytes exhibited the following characteristics; (a) high affinity (saturation was achieved at LDL concentrations below 50 mug protein/ml); (b) specificity (unlabeled LDL was much more effective than human high density lipoprotein or other plasma proteins in competing with 125I-LDL for binding to the LDL receptor); and(c) feedback regulation (the increase in the number of LDL receptors that appeared after incubation of freshly isolated lymphocytes in lipoprotein-deficient medium was prevented by exposure of the cells to either LDL or a mixture of 25-hydroxycholesterol plus cholesterol but not to HDL). Freshly isolated lymphocytes obtaine from three subjects with the homozygous form of familial hypercholesterolemia failed to develop normal amounts of LDL receptor activity when incubated in medium devoid of lipoproteins. The current data indicate: (a) that the LDL receptors that appear on the surface of cholesterol-deprived, normal human lymphocytes are genetically identical to the previously characterized LDL receptors of cultured human fibroblasts and long-term lymphoid cells and (b) that at least one cell type in the human body, the circulating human lymphocyte, has the capacity to produce a high affinity LDL receptor that mediates the cellular uptake and degradation of plasma LDL.

Adolescent↗