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Biomedical subjects

P A Edwards

Publications and source records attributed to P A Edwards.

At least 145 records · Page 8Linked to original sources
PubMed1983

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages.

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.

Adult↗
PubMed1983

Erythroid properties of K562 cells. Effect of hemin, butyrate and TPA induction.

Two sublines of the human leukemia cell line K562 including the original cell line and three clones have been investigated for their erythroid features. All of them produce embryonic and fetal hemoglobins, glycophorin A, spectrin and true acetylcholinesterase, but to a varying extent among the cell lines. The Hb and glycophorin contents were correlated in the different K562 cell lines, whereas acetylcholinesterase was independently expressed from these two other erythroid markers. Hb accumulation is enhanced by exposure of the cells to 100 microM hemin without a significant modification of the expression of the other erythroid markers. Butyrate greatly increased the activity of acetylcholinesterase, slightly enhanced the production of hemoglobin, but did not modify the expression of glycophorin and spectrin. 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced an almost complete disappearance of glycophorin, reduced the synthesis of Hb by K562 cells and also abolished the action of hemin on Hb accumulation. Therefore, all the different K562 cell lines exhibit clear erythroid features including acetylcholinesterase. Butyrate or hemin did not induce terminal differentiation of K562 cells, whereas TPA significantly diminished the erythroid phenotype.

Acetylcholinesterase↗
PubMed1983

Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase mRNA levels in rat liver.

Addition of cholestyramine or cholestyramine plus mevinolin to the diet has been reported to increase the activity and mass of rat liver 3-hydroxy-3-methylglutaryl-CoA reductase. The present data show that these same dietary manipulations cause an induction of functional reductase mRNA. RNA was isolated from rat livers and added to an in vitro translation system, and the reductase was immunoprecipitated and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. One protein was specifically immunoprecipitated and was found to have a Mr of 90,000 on 0.5 M urea/sodium dodecyl sulfate/polyacrylamide gels and a Mr of 94,000 on 8 M urea/sodium dodecyl sulfate/polyacrylamide gels. In animals fed rat chow supplemented with 5% cholestyramine and 0.1% mevinolin, reductase mRNA levels were 5.7-fold higher than in animals fed rat chow with 5% cholestyramine and were 16-fold higher than in animals fed rat chow with 5% cholestyramine and given mevalonolactone by stomach intubation. RNA isolated from animals fed a normal diet and killed at the nadir of the diurnal cycle of enzyme activity contained no detectable amounts of reductase mRNA as determined by this assay.

Animals↗
PubMed1983

Lymphokines secreted by an established lymphocyte line modulate receptor-mediated endocytosis in macrophages derived from human monocytes.

As little as 1 microliter of serum-free supernatant from Mo(t), an established lymphocyte line, when added to a 500-microliters incubation of macrophages derived from human monocytes, significantly decreased the receptor-mediated uptake and degradation of three cholesterol-rich molecules: low density lipoprotein (LDL); LDL complexed to dextran sulfate; and LDL modified by malondialdehyde (MDA-LDL). In contrast, the receptor-mediated uptake and degradation of mannosyl bovine serum albumin was increased three-fold. The Mo(t) supernatant did not contain competitive inhibitors of the cholesterol-rich ligands, and it did not alter macrophage receptor-independent endocytosis, protein synthesis, or phagocytosis of heat-killed yeast. The effect of the Mo(t) supernatant was specific for macrophages and was abolished by preincubation of the supernatant with trypsin, which indicates that the active substances are protein in nature. The decrease in the uptake and degradation of MDA-LDL induced by preincubating the macrophages with Mo(t) supernatant appeared to result from a decrease in the number of receptors for this ligand at the cell surface. The isolation of these lymphokines should offer new insights into macrophage receptor-mediated endocytosis, and may yield substances useful in preventing foam cell formation in atherosclerosis.

Cell Differentiation↗
PubMed1983

Monoclonal antiglycophorin as a probe for erythroleukemias.

A monoclonal antibody (LICR.LON.R10) specific for the major sialoglycoprotein of the erythroid cell membrane, glycophorin A (alpha), has been used to test the possibility that "cryptic" erythroleukemia may be diagnosed as acute lymphoblastic leukemia (ALL) or acute myeloblastic leukemia (AML). In addition to 27 overt erythroleukemias, 724 leukemias, including 329 ALL (103 in relapse), 205 AML, and 109 blast crises of Ph1-positive chronic myeloid leukemia, were analyzed. Twenty cases with a significant proportion of glycophorin-A-positive (gA+) cells were found; 8 of these (5 AML and 3 blast crises of chronic myeloid leukemia, CML) had an obvious erythroid component, but 12 others were diagnosed as AML (2), AMML (1), CML in myeloid blast crisis (4) or megakaryoblastic blast crisis (1), acute megakaryoblastic leukemia (2), or acute lymphoblastic leukemia (2). The latter two patients had no immunologic evidence supporting a diagnosis of ALL and were resistant to chemotherapy. We conclude that AML and ALL only very rarely express gA, and these are probably genuine "cryptic" erythroleukemias. Other gA+ leukemias (megakaryoblastic and CML blast crises) may arise from bi- or pluripotent stem cells and contain distinct and separable blast cell populations.

Antibodies, Monoclonal↗
PubMed1982

Analysis of regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase in a somatic cell mutant auxotrophic for mevalonate.

Regulation of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and synthesis by 25-hydroxycholesterol is aberrant in a somatic cell mutant of the Chinese hamster ovary K1 cell auxotrophic for mevalonate by virtue of a defect in HMG-CoA synthase activity. Normal regulation of HMG-CoA reductase activity and synthesis in this mutant by 25-hydroxycholesterol can be restored by simultaneous incubation with a small (0.4 mM) mevalonate supplement. Normal regulation of HMG-CoA reductase is also observed in a revertant of the mutant cell with normal HMG-CoA synthase activity.

Animals↗
PubMed1982

Monoclonal antibodies to human erythrocytes.

Eight monoclonal antibodies from mouse hybridomas raised to normal human erythrocytes were tested with a panel of null-type erythrocytes, enzyme-treated normal cells, and by inhibition with human erythrocyte sialoglycoproteins. Two antibodies reacted poorly or not at all with RhNULL cells. These antibodies are of considerable interest since it may be possible to use them to elucidate the chemical nature of the antigens of the Rhesus blood group system. Four other antibodies were inhibited by sialoglycoprotein preparations. The antigens recognized were, respectively, two different determinants on the major sialoglycoprotein alpha (glycophorin A) and one determinant which is probably common to sialoglycoproteins alpha and delta (glycophorins A and B). Another antibody had anti-Wrb specificity. One of these antibodies is of considerable potential value for the further characterization of erythrocyte sialoglycoproteins.

Antibodies, Monoclonal↗
PubMed1982

A human-hybridoma system based on a fast-growing mutant of the ARH-77 plasma cell leukemia-derived line.

A rapidly-growing, HAT-sensitive cell line LICR-LON-HMy2 has been derived from the ARH-77 human plasma cell leukemia-derived line. It lacks the enzyme hypoxanthine phosphoribosyltransferase (EC 2.4.2.8). Hybrids have been reproducibly made for more than a year and in independent laboratories with lymphocytes from tonsils, lymph nodes and peripheral blood and tonsil lymphocytes cultured with antigens. The parent line and hybrids are very robust in culture and double in 20-30 h. Hybrids clone easily and have stable karyotypes, most with modal numbers in the sixties. Stable Ig secretion patterns have been observed over 20-30 passages, and after cloning. It was estimated that about half the hybrids produce new immunoglobulin chains in addition to the parent cell line's IgG1 (kappa light chain). High, but not limiting, density hybridoma cultures (approximately 5 x 10(5) cells/ml) typically produce 0.25-2 micrograms/ml immunoglobulin per day, but some hybrids produce more. A high-secreting variant of LICR-LON-HMy2 has been derived. The LICR-LON-HMy2 line is human and is distinct from the HAT-sensitive human B cell-lines SKO-007 and GM 1500-6TG Al 2. It is available for distribution.

Antibodies, Monoclonal↗
PubMed1982

Monoclonal antibodies to the human mammary gland. I. Distribution of determinants in non-neoplastic mammary and extra mammary tissues.

Mouse monoclonal antibodies have been raised to the human milk fat globule membrane. The distribution of the antigens detected by four of the antibodies has been examined in formalin-fixed paraffin-embedded human tissues by light microscopic immunocytochemistry. The four antibodies stain lactating breast and normal resting breast. Two exclusively stain the luminal membranes of breast epithelial cells. A third antibody stains in addition the lateral membranes of duct epithelial cells. The fourth antibody stains both epithelial and myoepithelial cells. None of the antibodies is breast specific, nor do they stain every epithelial cell within the breast. Instead, each antibody reveals a complex and heterogeneous distribution of staining throughout the normal tissues. Within the breast, the staining by a given antibody is usually segmental and conforms to secretory units and their associated ducts. Similarly heterogeneous patterns of staining are also observed in the extramammary normal tissues. Despite the apparent morphological identity between breast epithelial cells when examined by conventional light microscopy, the hitherto unrecognised "functional" heterogeneity, which has been revealed by the monoclonal antibodies could have importance in understanding the biology of the normal breast and the pathology of breast cancer.

Antibodies, Monoclonal↗
PubMed1982

Monoclonal antibodies to the human mammary gland. II. Distribution of determinants in breast carcinomas.

A range of primary and metastatic human breast carcinomas has been examined with respect to the staining by four monoclonal antibodies which were raised to the human milk fat globule membrane. Within the normal breast the luminal epithelial cells expressing the antigens detected by the monoclonal antibodies were heterogeneous in their distribution. The heterogeneity was not only confined to single cells, but also to regions within the breast. The breast carcinomas also expressed the antigens in a variable manner. Morphological differentiation and functional differentiation, defined by the monoclonal antibodies, were not invariably coincident. Lymph node metastases gave similar results to the primary carcinomas. The monoclonal antibodies have revealed a heterogeneity, with respect to surface antigenic expression, within the normal and neoplastic breast epithelium. This cellular heterogeneity of breast carcinomas, may have significant prognostic and therapeutic implications in the management of primary breast cancer.

Antibodies, Monoclonal↗
PubMed1982

Lymphocyte-conditioned medium protects human monocyte-macrophages from cholesteryl ester accumulation.

Exposure of human monocyte-macrophages to as little as 50 microliters of cultured medium from lymphocytes stimulated by concanavalin A (Con A) resulted in a dramatic decrease in the activities of the low density lipoprotein (LDL) receptor pathway, the LDL-dextran sulfate pathway, and the scavenger receptor pathway. This effect was not seen when the monocyte-macrophages were exposed to culture medium from lymphocytes cultured without Con A or with Con A together with alpha-methyl mannoside or control medium without lymphocytes. The activity of 3-hydroxy-3-methyglutaryl-coenzyme A reductase also decreased in monocyte-macrophages exposed to culture medium from stimulated lymphocytes. Acyl-CoA:cholesterol O-acyltransferase activity, protein synthesis, protein content, phagocytosis of heat-killed yeast, and non-receptor-mediated endocytosis were not inhibited. Monocyte-macrophages exposed to malondialdehyde altered-LDL in the presence of stimulated lymphocyte culture medium accumulated substantially less cholesteryl esters than did cells in control medium. We propose that substances produced by stimulated lymphocytes may be useful in protecting macrophages from cholesteryl ester accumulation.

Cells, Cultured↗
PubMed1982

Specificity of receptor-mediated recognition of malondialdehyde-modified low density lipoproteins.

Blood-borne human monocytes and macrophages derived from human monocytes in vitro express an active low density lipoprotein (LDL) receptor and an active receptor for negatively charged proteins, the scavenger receptor. When less than 15% of the lysine residues of human LDL were modified by malondialdehyde while the lipoprotein was in solution, recognition and uptake of the modified lipoprotein occurred via the LDL receptor. Further modification resulted in threshold recognition and uptake by the scavenger receptor with concomitant loss of recognition by the LDL receptor. The rate of degradation via the LDL receptor pathway was inversely related to the degree of modification whereas that mediated by the scavenger receptor was independent of the extent of incorporation of malondialdehyde once threshold recognition was achieved. In contrast to the interaction of LDL with malondialdehyde in solution, modification of less than 15% of the lysine residues of LDL adsorbed to heparin-Sepharose resulted in recognition and uptake by the scavenger receptor. The scavenger receptor-mediated uptake of malondialdehyde-modified LDL may be dependent on formation of recognition sites involving specific modified lysine residues or changes in the conformation of LDL induced by neutralization of specific lysine residues of the apoB polypeptides or both.

Apolipoproteins↗
PubMed1982

The effect of cholestyramine and Mevinolin on the diurnal cycle of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Rats were fed powdered rat chow or a rat chow diet containing 5% cholestyramine or 5% cholestyramine with Mevinolin (112 mg/100 g food, 200 mg/kg body weight per day). The specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase was determined at different times during the diurnal cycle of the enzyme. Animals fed cholestyramine had higher specific activities of HMG-CoA reductase at all time points tested when compared to controls. The specific activity at the peak in the diurnal cycle was approximately 8-fold higher in cholestyramine-treated animals. Rats administered the cholestyramine-Mevinolin diet had higher specific activities of the enzyme than either cholestyramine-treated or control animals. In the cholestyramine-Mevinolin-treated animals the peak in the diurnal cycle was shifted to D-12 (12th hour of the dark cycle) and the specific activity at this point was approximately 133-fold greater than the basal (L-6) activity in control animals. Optimal conditions for immunotitration studies were determined such that valid conclusions could be drawn from these data. Based on immunotitration experiments, the increased hepatic HMG-CoA reductase activity in cholestyramine-treated animals resulted in part from a 3-fold activation of the enzyme, while the increased specific activity in the cholestyramine-Mevinolin-treated animals was due solely to increased enzyme mass. Hence the administration of Mevinolin blocked the activation of the enzyme induced by feeding cholestyramine alone. This was confirmed by purifying the enzyme to apparent homogeneity from cholestyramine-Mevinolin-treated animals; the specific activity of this enzyme was 4,000-7,700 nmol NADPH oxidized/min per mg protein. Cholestyramine-Mevinolin treatment affords a novel system for generating milligram quantities of rat hepatic HMG-CoA reductase.-Tanaka, R. D., P. A. Edwards, S. Lan, E. M. Knöppel, and A. M. Fogelman. The effect of cholestyramine and Mevinolin on the diurnal cycle of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Animals↗
PubMed1982

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase from human liver was purified to homogeneity. Electrophoresis of the purified human enzyme on non-denaturing gels revealed a single protein-staining band that co-migrated with reductase activity and could be selectively removed by treatment with cross-reacting antibody prepared to the purified reductase from rat liver. On SDS polyacrylamide gel electrophoresis, the purified reductase showed only one protein-staining band corresponding to a molecular weight of approximately 52,000. Based on the sedimentation rate during sucrose density gradient centrifugation, most of the enzyme from crude solubilized preparations had an apparent molecular weight of approximately 104,000. The human reductase, like the rat liver enzyme, was dimeric and composed of subunits of approximately 50,000 molecular weight.

Animals↗