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Biomedical subjects

S K Basu

Publications and source records attributed to S K Basu.

At least 127 records · Page 7Linked to original sources
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

Solubilization of the low density lipoprotein receptor.

The low density lipoprotein (LDL) receptor was solubilized from membranes of bovine adrenal cortex and cultured human cells by incubation with the nonionic detergent octyl-beta-D-glucoside. Receptor activity released into the 100,000 x g supernatant was assayed by a solid-phase procedure: an aliquot of the soluble extract was removed, the detergent was diluted below its critical micellar concentration, causing the receptor to precipitate as a lipid-protein aggregate; the precipitate was collected by centrifugation and incubated with (125)I-labeled LDL ((125)I-LDL); and the receptor-bound (125)I-LDL was separated from free (125)I-LDL by filtration. The (125)I-LDL binding site that was precipitated from the soluble extract of bovine adrenocortical membranes appeared to be the same as the functional LDL receptor of cultured bovine adrenocortical cells and human fibroblasts. It exhibited high affinity and specificity (affinity for LDL more than 200-fold greater than for acetylated LDL, methylated LDL, or high density lipoprotein), dependence on calcium, and susceptibility to destruction by Pronase. The amount of (125)I-LDL binding activity in solubilized membranes from cultured cells was proportional to the number of receptors on the surface of the intact cells. Thus, the number of solubilized receptors was 1/20th of normal in mutant fibroblasts from a subject with homozygous familial hypercholesterolemia and was 1/4th of normal in human epithelioid carcinoma A-431 cells when they were grown in the presence of 25-hydroxycholesterol plus cholesterol. While in the soluble form in the presence of octyl-beta-D-glucoside, the LDL receptor can be carried through several steps of purification.

Adrenal Cortex↗
PubMed1979

Low density lipoprotein receptors in bovine adrenal cortex. II. Low density lipoprotein binding to membranes prepared from fresh tissue.

Low density lipoprotein (LDL)-binding activity was measured in whole homogenates and membranes prepared from fresh bovine adrenal cortex by an ultracentrifugation assay. The binding site for 125I-labeled LDL in isolated membranes shared the properties of the LDL receptor previously demonstrated in intact monolayers of cultured bovine adrenocortical cells. The amount of high affinity [125I]iodo-LDL-binding activity in the adrenal cortex was 6- to 12-fold higher than in the medulla of the same glands. Large amounts of high affinity [125I]iodo-LDL-binding activity were also present in the ovarian corpus luteum but not in the ovarian interstitium. Lesser amounts of high affinity binding activity were observed in 14 other bovine tissues. These results lend support to the concept that cells in the bovine adrenal cortex can obtain cholesterol for steroid hormone synthesis through the receptor-mediated uptake of plasma LDL.

Adrenal Cortex↗
PubMed1978

Characterization of the low density lipoprotein receptor in membranes prepared from human fibroblasts.

An ultracentrifugation assay has been developed to measure low density lipoprotein (LDL) receptor activity in membranes prepared from cultured human fibroblasts. The binding site for 125I-labeled LDL in isolated membranes reflected the properties of the LDL receptor previously demonstrated in intact fibroblasts. It exhibited high affinity (Kd approximately 4 microgram of LDL protein/ml), specificity (LDL approximately 400-fold more effective than high density lipoprotein in competing with 125I-LDL for the binding site), dependence on calcium, and susceptibility to destruction by pronase. The number of LDL receptors detected in the in vitro membrane binding assay was similar to the number detected in intact cells. The number of receptors was reduced in membranes from fibroblasts that were grown in the presence of 25-hydroxycholesterol plus cholesterol and in fibroblast membranes from a subject with homozygous familial hypercholesterolemia, two situations in which the number of LDL receptors in intact fibroblasts is known to be reduced. The availability of a membrane binding assay that faithfully reflects the properties of the physiologic LDL receptor of intact cells should permit the characterization of this receptor in organs from intact humans and animals.

Cell Membrane↗
PubMed1978

Inhibition of the binding of low-density lipoprotein to its cell surface receptor in human fibroblasts by positively charged proteins.

A group of proteins and polyamino acids with positively charged domains were shown to inhibit the binding of 125I-LDL to its receptor on the surface of human fibroblasts. The list of inhibitory proteins included platelet factor 4 (which has a cluster of lysine residues at its carboxyl terminus), two lysine-rich histones, poly-L-lysines of chain length greater than 4, and protamine. These proteins were effective in the concentration range of 5--10 microgram/ml. Two other positively charged proteins, lysozyme and avidin, did not inhibit 125I-LDL binding. Kinetic studies suggested that protamine was not acting simply as a competitive inhibitor with regard to the LDL receptor. In light of previous data showing that polyanions such as heparin and polyphosphates also inhibit 125-I-LDL binding to its cell surface receptor, the current findings suggest that charge interactions are important in this binding reaction. In a related series of studies, a number of glycoproteins and their asialo derivatives as well as a number of sugar phosphates failed to inhibit 125I-LDL binding to its receptor in fibroblasts.

Blood Coagulation Factors↗
PubMed1977

Metabolism of cationized lipoproteins by human fibroblasts. Biochemical and morphologic correlations.

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 mug/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.

Binding Sites↗
PubMed1977

Electrolytes in surgical patients: the effect of pre-operative starvation and environmental temperature.

Serum sodium, potassium and chloride values were measured before and after pre-operative starvation and after premedication in healthy subjects under going routine surgery, during both temperate and hot weather. No significant change in serum electrolytes occurred during temperate weather either after starvation or after premedication. In hot weather, when the subjects were sweating, a rise in serum electrolytes occurred, indicating fluid deficit of about 1-8 litres after a mean period of starvation of 11 hours; premedication with atropine and diazepam in these subjects was followed by a significant decrease in the serum electrolytes from the previous raised level after pre-operative starvation.

Adolescent↗
PubMed1977

Mutations in prophage phi11 that impair the transducibility of their Staphylococcus aureus lysogens for methicillin resistance.

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sjöström et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sjöström and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.

DNA, Viral↗
PubMed1977

Overloading human aortic smooth muscle cells with low density lipoprotein-cholesteryl esters reproduces features of atherosclerosis in vitro.

Human aortic smooth muscle cells accumulate only small amounts of cholesteryl esters in tissue culture, even when incubated for prolonged periods with high levels of plasma low density lipoprotein (LDL). This failure to overaccumulate LDL-cholesteryl esters is due to an LDL-mediated feedback suppression of the activity of the cell surface LDL receptor, a regulatory action that limits the rate at which the cells take up LDL. This regulatory system can be bypassed by incubating smooth muscle cells with LDL that has been given a strong positive charge by covalent linkage with N,N-dimethyl-1,3-propanediamine (DMPA-LDL). The unregulated uptake of DMPA-LDL produces a massive deposition of cholesteryl esters in the form of inclusions within the cell. These inclusions take up lipid stains and exhibit positive birefringence with formée crosses that are typical of liquid crystals of cholesteryl esters. By electron microscopy, the cholesteryl ester inclusions appear as homogeneous gray cytoplasmic lipid droplets. The current studies demonstrate that the unregulated uptake of LDL-cholesteryl esters by human aortic smooth muscle cells can reproduce in vitro the major biochemical and morphological alterations that occur within smooth muscle cells in vivo during the process of atherosclerosis.

Aorta, Thoracic↗