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J K Boyles

Publications and source records attributed to J K Boyles.

28 records · Page 2Linked to original sources
PubMed1987

Lipoprotein B37, a naturally occurring lipoprotein containing the amino-terminal portion of apolipoprotein B100, does not bind to the apolipoprotein B,E (low density lipoprotein) receptor.

In 1979, Steinberg and colleagues described a unique kindred with familial hypobetalipoproteinemia (Steinberg, D., Grundy, S. M., Mok, H. Y. I., Turner, J. D., Weinstein, D. B., Brown, W. V., and Albers, J. J. (1979) J. Clin. Invest. 64, 292-301). Recently, we demonstrated the existence of an abnormal species of apolipoprotein (apo-) B, apo-B37 (Mr = 203,000) in nine members of that kindred (Young, S. G., Bertics, S. J., Curtiss, L. K., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1831-1841; Young, S. G., Bertics, S. J., Curtiss, L. K., Dubois, B. W., and Witztum, J. L. (1987) J. Clin. Invest. 79, 1842-1851). Apolipoprotein B37 contains only the amino-terminal portion of apo-B100. In affected individuals most of the apo-B37 is contained in the high density lipoprotein (HDL) fraction (d = 1.063-1.21 g/ml), where it is the principal apolipoprotein in a unique lipoprotein (Lp) particle, Lp-B37, which contains little, if any, apo-A-I. However, the most abundant lipoprotein in the HDL density fraction is a smaller particle, which contains apo-A-I, but no apo-B. The Lp-B37 particles were isolated from the HDL of affected individuals by immunoabsorption of apo-B37. Selected affinity antibodies specific for apo-B37 were used to prepare an anti-apo-B37-Sepharose 4B column. Lipoproteins not bound by the column (unbound HDL fraction) contained apo-A-I, but no apo-B. The Lp-B37, which was eluted from the column with 3 M KI, contained apo-B37 and trace amounts of apo-A-I, but no apo-B100. Over a 4-h period, normal human fibroblasts degraded 10-fold more 125I-low density lipoprotein (LDL) than 125I-Lp-B37. Also, whereas addition of excess unlabeled LDL markedly reduced degradation of 125I-LDL, it did not significantly reduce the degradation of 125I-Lp-B37. Unlabeled Lp-B37 did not inhibit uptake and degradation of 125I-LDL by fibroblasts. These data suggest that the amino-terminal portion of apo-B100, when expressed on a naturally occurring lipoprotein particle, does not contain a functional apo-B,E(LDL) receptor binding domain.

Apolipoprotein B-100↗
PubMed1987

Lipoproteins and their receptors in the central nervous system. Characterization of the lipoproteins in cerebrospinal fluid and identification of apolipoprotein B,E(LDL) receptors in the brain.

This study was undertaken to determine if apolipoprotein (apo) E-containing lipoproteins and their receptors could provide a system for lipid transport and cholesterol homeostasis in the brain, as they do in other tissues. To accomplish this goal, the lipoproteins in human and canine cerebrospinal fluid (CSF) were characterized, and rat brain and monkey brain were examined for the presence of apoB,E(LDL) receptors. Apolipoprotein E and apoA-I were present in human and canine CSF, but apoB could not be detected. Apo-lipoprotein E and apoA-I were both present on lipoproteins with a density of approximately 1.09 to 1.15 g/ml. In human CSF, the lipoproteins were primarily spherical (approximately 140 A), whereas in canine CSF the lipoproteins were a mixture of discs (200 x 65 A) and spheres (approximately 130 A). Apolipoproteins E and A-I were contained primarily in separate populations of lipoproteins. Although the apoE of CSF was more highly sialylated than plasma apoE, the apoE-containing lipoproteins in canine CSF competed as effectively as canine plasma apoE HDLc for binding of 125I-LDL to the apoB,E(LDL) receptors on human fibroblasts. The presence of apoB,E(LDL) receptors in both rat and monkey brain was demonstrated by immunocytochemistry. Astrocytes abutting on the arachnoid space and pial cells of the arachnoid itself, both of which contact CSF, expressed apoB,E(LDL) receptors. Relatively few receptors were present in the cells of the gray matter of the cortex. Receptors were more prominent on the astrocytes of white matter and in the cells of the brain stem. The expression of apoB,E(LDL) receptors by brain cells and the presence of apoE- and apoA-I-containing lipoproteins in CSF suggest that the central nervous system has a mechanism for lipid transport and cholesterol homeostasis similar to that of other tissues.

Adult↗
PubMed1987

Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein E-containing lipoproteins.

We have previously demonstrated that astrocytes synthesize and secrete apolipoprotein E in situ. In the present work, primary cultures of rat brain astrocytes were used to study apolipoprotein E synthesis, secretion, and metabolism in vitro. The astrocytes in culture contained immunoreactive apolipoprotein E in the area of the Golgi apparatus. Incubation of the astrocytes with [35S]methionine resulted in the secretion of labeled immunoprecipitable apolipoprotein E, which constituted 1-3% of the total secreted proteins. The apolipoprotein E secreted in culture and the apolipoprotein E in rat brain extracts differed from serum apolipoprotein E in two respects: both had a slightly higher apparent molecular weight (approx. 36,000) and more acidic isoforms than serum apolipoprotein E. Sialylation of the newly secreted apolipoprotein accounted for the difference in both the apparent molecular weight and isoelectric focusing pattern of newly secreted apolipoprotein E and plasma apolipoprotein E. The astrocytes possessed apolipoprotein B,E(LDL) receptors capable of binding and internalizing apolipoprotein E-containing lipoproteins. The uptake of lipoproteins by the cells led to a reduction in the number of cell surface receptors and to the intracellular accumulation of cholesteryl esters. Since apolipoprotein E is present within the brain, and since brain cells can express apolipoprotein B,E(LDL) receptors, apolipoprotein E-containing lipoproteins may function to redistribute lipid and regulate cholesterol homeostasis within the brain.

Animals↗
PubMed1987

Apolipoprotein E synthesis in neurofibrosarcoma and schwannoma cell cultures from two individuals with neurofibromatosis.

Apolipoprotein E is expressed in neurofibrosarcoma and Schwannoma cell cultures derived from two patients with different types of neurofibromatosis, but not in six cell cultures derived from the benign neurofibromatosis neurofibromas. In addition, a cell culture derived from a nonneurofibromatosis human malignant astrocytoma showed apolipoprotein E expression. Although all cells in either the neurofibrosarcoma or Schwannoma cultures appeared morphologically similar (suggesting homogeneity), apolipoprotein E was immunochemically detected in the perinuclear region of only half of the cells. Thus, production of apolipoprotein E in neurofibromatosis-associated neurofibroma tumors may be a marker for a specific subclass of transformed cells. The expression of apolipoprotein E in glial cell neoplasms is possibly related to an alteration in their lipid metabolism.

Adult↗
PubMed1987

Receptor binding activity of lipid recombinants of apolipoprotein B-100 thrombolytic fragments.

Apolipoprotein (apo) B-100, the protein constituent of low density lipoproteins (LDL), is the determinant responsible for LDL binding to the apoB,E(LDL) receptor on cells. The current study was designed to identify the region(s) of apoB-100 that interact with the apoB,E(LDL) receptor. Apolipoprotein B-100 was fragmented by thrombin digestion, and the isolated fragments (T2, T3, T4) were recombined with cholesterol-induced canine high density lipoproteins (HDLc). Before the recombination, the receptor binding activity of apoE of the HDLc was abolished by reductive methylation and extensive trypsin treatment. This treatment permitted almost complete replacement of the small residual apoE fragments by the large apoB fragments. Recombinant apoB particles were isolated by ultracentrifugation and tested for binding to receptors on cultured human fibroblasts. The recombinant particles had chemical and physical properties similar to those of native HDLc. Recombinants of both the whole thrombolytic digest and of isolated fragments displayed specific binding to the apoB,E (LDL) receptor. Anti-apoB,E(LDL) receptor antibodies abolished 90% of the binding, and there was almost no specific binding to receptor-negative fibroblasts or to cells in which the receptors had been down-regulated. The binding of apoB-100 recombinants to the receptor also demonstrated calcium dependency; in addition, the surface binding of the recombinants was released by polyanionic compounds. All these recombinants had binding affinities comparable to one another but less than that of native LDL. Although T2, T3 and T4 recombinants can all bind specifically to the apoB,E(LDL) receptor, it remains to be established whether their activity represents physiologically relevant binding. Nevertheless, the present findings illustrate the potential of the recombinant method using HDLc lipids to reconstitute biological activity.

Apolipoprotein B-100↗
PubMed1985

Apolipoprotein E associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system.

The plasma protein apolipoprotein (apo) E is an important determinant of lipid transport and metabolism in mammals. In the present study, immunocytochemistry has been used to identify apo E in specific cells of the central and peripheral nervous systems of the rat. Light microscopic examination revealed that all astrocytes, including specialized astrocytic cells (Bergmann glia of the cerebellum, tanycytes of the third ventricle, pituicytes of the neurohypophysis, and Müller cells of the retina), possessed significant concentrations of apo E. In all of the major subdivisions of the central nervous system, the perinuclear region of astrocytic cells, as well as their cell processes that end on basement membranes at either the pial surface or along blood vessels, were found to be rich in apo E. Extracellular apo E was present along many of these same surfaces. The impression that apo E is secreted by astrocytic cells was confirmed by electron microscopic immunocytochemical studies, which demonstrated the presence of apo E in the Golgi apparatus. Apo E was not present in neurons, oligodendroglia, microglia, ependymal cells, and choroidal cells. In the peripheral nervous system, apo E was present within the glia surrounding sensory and motor neurons; satellite cells of the dorsal root ganglia and superior cervical sympathetic ganglion as well as the enteric glia of the intestinal ganglia were reactive. Apo E was also present within the non-myelinating Schwann cells but not within the myelinating Schwann cells of peripheral nerves. These results suggest that apo E has an important, previously unsuspected role in the physiology of nervous tissue.

Animals↗
PubMed1984

Actin filament content and organization in unstimulated platelets.

The extent of actin polymerization in unstimulated, discoid platelets was measured by DNase I inhibition assay in Triton X-100 lysates of platelets washed at 37 degrees C by gel filtration, or in Triton X-100 lysates of platelets washed at ambient temperatures by centrifugation in the presence of prostacyclin. About 40% of the actin in the discoid platelets obtained by either method existed as filaments. These filaments could be visualized by electron microscopy of thin sections. Similar results were obtained when the actin filament content of discoid platelets was measured by sedimentation of filaments from Triton X-100 lysates at high g forces (145,000 g for 45 min). However, few of these filaments sedimented at the lower g forces often used to isolate networks of actin filaments from cell extracts. These results indicate that actin filaments in discoid cells are not highly crosslinked. Platelets isolated by centrifugation in the absence of prostacyclin were not discoid, but were instead irregular with one or more pseudopodia. These platelets also contained approximately 40-50% of their actin in a filamentous form; many of these filaments sedimented at low g forces, however, indicating that they were organized into networks. The discoid shape of these centrifuged platelets could be restored by incubating them for 1-3 h at 37 degrees C, which resulted in the reversal of filament organization. High g forces were then required for the sedimentation of the actin. Approximately 80-90% of the actin in platelets washed at 4 degrees C was filamentous; this high actin filament content could be attributed to actin polymerization during the preparation of the platelets at low temperatures. These studies show that platelet activation involves mechanisms for the structural reorganization of existing filaments, in addition to those previously described for mediating actin polymerization.

Actins↗
PubMedDate not supplied

Isolation and characterization of plasma lipoproteins of common marmoset monkey. Comparison of effects of control and atherogenic diets.

This study examines the potential of the common marmoset monkey (Callithrix jacchus) to serve as a model for human lipoprotein metabolism and atherosclerosis. The lipoproteins of animals fed a low-fat, low-cholesterol diet and a high-fat (12% wt/wt lard), high-cholesterol (0.34% wt/wt) diet were characterized by the combination of sequential ultracentrifugation and Pevikon block electrophoresis. Based on chemical and physical properties, equivalents of human very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL), including and HDL-with apolipoprotein E subclass, were demonstrated. In control animals, whose plasma cholesterol concentration was 140.1 +/- 20.2 mg/dl (means +/- SD), approximately 40% of the plasma cholesterol was transported by LDL as compared with approximately 70% in humans. The cholesterol-fed marmosets segregated into two groups: hypo- and hyper-responders. The hyper-responders had plasma cholesterol levels of 450 to 970 mg/ml. The hypercholesterolemia was associated with elevated concentrations of VLDL, intermediate density lipoproteins, and LDL; in addition, these lipoproteins were enriched in cholesteryl esters relative to lipoproteins isolated from control animals. The HDL (d greater than 1.09 g/ml) levels did not change in response to cholesterol feeding, although the HDL-with apolipoprotein E found in the d = 1.02 to 1.09 g/ml fraction increased approximately fivefold. Based on immunological characteristics and sialic acid content, the common marmoset appeared to lack a lipoprotein(a) equivalent. The results of a short-term feeding study (11 months) suggest that this monkey was susceptible to the development of diet-induced atherosclerosis. The hyper-responsive animals developed foam cell lesions and moderately proliferative intimal lesions, predominantly within the thoracic aorta. In summary, the results of our studies indicate that the common marmoset monkey potentially is a useful model for the study of both lipoprotein metabolism and diet-induced atherosclerosis.

Animals↗
PubMedDate not supplied

Metabolism of canine beta-very low density lipoproteins in normal and cholesterol-fed dogs.

Cholesteryl ester-rich beta-very low density lipoproteins (beta-VLDL) are beta-migrating lipoproteins that accumulate in the plasma of cholesterol-fed animals and of patients with type III hyperlipoproteinemia. There are two distinct fractions: fraction I beta-VLDL are chylomicron remnants of intestinal origin, and fraction II beta-VLDL are cholesterol-rich VLDL of hepatic origin. The liver rapidly clears fraction I beta-VLDL from the plasma of both normal and cholesterol-fed dogs. The liver also clears fraction II beta-VLDL rapidly and efficiently from the plasma of normal dogs by receptor-mediated uptake. In cholesterol-fed dogs the clearance is biphasic: an initial rapid die-away of about 30% to 40% of the injected dose within 5 minutes, followed by a slow clearance of plasma radioactivity (a half-life of more than 20 hours). The rapid, initial phase of fraction II beta-VLDL clearance appears to be related to sequestration of the lipoproteins presumably on endothelial cells and is apparently associated with lipolytic processing. Treatment of the fraction II beta-VLDL with lipoprotein lipase abolishes this rapid phase. In the cholesterol-fed dog, the slow, late phase of clearance corresponds to the conversion of fraction II beta-VLDL to the smaller, denser intermediate and low density lipoproteins (IDL and LDL), which are slowly cleared from the plasma. It is concluded that fraction II beta-VLDL are catabolized in the normal dog by rapid uptake mediated at least in part by the apo B,E(LDL) receptor of hepatic parenchymal cells. In cholesterol-fed dogs, in which these receptors are markedly down-regulated, fraction II beta-VLDL are apparently initially bound to endothelial cells and converted to IDL and LDL by lipolytic processing.

Animals↗