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C A Reardon

Publications and source records attributed to C A Reardon.

32 records · Page 2Linked to original sources
PubMed1994

Metabolic mechanisms for responses to dietary cholesterol and fat in high and low LDL responding baboons (Papio sp.).

These studies were conducted to determine how plasma low density lipoprotein (LDL) cholesterol levels respond to dietary cholesterol, fed in increasing amounts with either corn oil or coconut oil diets, in high as compared to low LDL responding baboons; and to determine how apolipoprotein (apo) B transcription levels are modulated in response to dietary lipids. Eight high and eight low LDL responding pedigreed adult baboons, balanced for sire, age, sex, and weight, were challenged for successive 7-week periods with increasing levels of dietary cholesterol combined with either coconut oil or corn oil. At the end of each dietary period, plasma and lipoprotein lipids, apoB, apoA-I, and hepatic mRNA levels for apolipoproteins were measured. As dietary cholesterol increased, plasma cholesterol concentrations (mostly LDL cholesterol) increased in both phenotypes and with both types of fat, but phenotypic differences were greater with coconut oil. There was not a consistent dose-response relationship of plasma or LDL cholesterol levels to increasing intakes of dietary cholesterol. Neither dietary cholesterol, type of dietary fat, nor LDL phenotype affected hepatic apoB or apoE mRNA levels. In a second experiment to resolve the inconsistent dose-response to dietary cholesterol, we fed the animals varying levels of dietary cholesterol combined with coconut oil, and separated the challenge periods with intervening 12-week chow periods. Plasma and LDL cholesterol and apoB concentrations rose consistently with increasing dietary cholesterol, and the slope of the increase diminished at the higher doses. The results suggest that genetic differences in the initial response of LDL cholesterol to dietary cholesterol and saturated fatty acids are not due to the differences in hepatic transcription of apoB, and that the preceding dietary intake of cholesterol and saturated fatty acids is a major determinant of the response of plasma lipids and the associated metabolic processes to a dietary challenge. The response of baboon plasma LDL cholesterol concentrations to dietary cholesterol, when fed with saturated fatty acids, is similar to that of humans.

Animals↗
PubMed1993

Inhibition of apolipoprotein E degradation in a post-Golgi compartment by a cysteine protease inhibitor.

In our prior studies on lipoprotein stimulation of apolipoprotein E (apoE) secretion in HepG2 cells, it became clear that a proportion of the newly synthesized apoE was degraded intracellularly (Ye, S. Q., Olson, L. M., Reardon, C. A., and Getz, G. S. (1992) J. Biol. Chem. 267, 21961-21966). The present study was designed to determine the nature of the proteases and the intracellular sites involved in newly synthesized apoE degradation. The effect of seven protease inhibitors on total apoE levels was examined. Only N-acetyl-leucyl-leucyl-norleucinal (ALLN), a cysteine protease inhibitor, significantly blocked apoE degradation in HepG2 cells. The amount of total apoE from cells chased with ALLN for 4 h was increased by 1.58 +/- 0.05-fold relative to the controls (n = 11, p < 0.01). ALLN extended the half-life of apoE from 2.61 h to 4.38 h (p < 0.01). This effect occurs in a post-Golgi compartment since in the presence of brefeldin A, ALLN had no effect on intracellular apoE levels. Chloroquine and NH4Cl significantly reduced apoE degradation; however, ALLN plus either of these reagents appear to have an additive effect. The amount of apoE in cells chased in Ca(2+)-free medium was significantly higher than that in cells chased in Ca(2+)-containing medium (1.70 +/- 0.07-fold, n = 6, p < 0.01). ALLN plus Ca(2+)-free medium had no additive effect. ALLN had no significant influence on the degradation of albumin but had a similar effect on transfected apoE in Chinese hamster ovary cells. Overall, these data suggest that apoE may be degraded in a post-Golgi compartment of HepG2 and Chinese hamster ovary cells by lysosomal enzymes and cytosolic Ca(2+)-dependent cysteine proteases. ALLN inhibits the latter.

Albumins↗
PubMed1992

Human plasma lipoproteins regulate apolipoprotein E secretion from a post-Golgi compartment.

The molecular regulation of apolipoprotein E (apoE) synthesis and secretion is incompletely understood. In this study, we have examined the effect of human low density lipoprotein (LDL) on apoE mRNA and protein levels in HepG2 and other eukaryotic cells. Exposing HepG2 cells to LDL for times up to 4 h resulted in an increase in 35S-labeled apoE accumulation in the medium by 2.2-fold, relative to serum free controls (n = 10, p < 0.001), with no changes in apoE mRNA levels. Similar observations have been made in JeG-3 cells and Chinese hamster ovary cells stably transfected with human apoE cDNA constructs. These results indicate that the LDL effect operates at a post-transcriptional level. In pulse-chase experiments, the LDL effect on apoE accumulation in the media was observed when it was added only during the chase even in the presence of cycloheximide, indicating that LDL is functioning at a post-translational level. The use of brefeldin A (BFA), an agent that impedes protein transport from the endoplasmic reticulum to the Golgi apparatus, suggests that the LDL effect occurs in a post-Golgi compartment. The addition of protease inhibitors could not duplicate the effects of LDL on the apoE accumulation in the medium. ApoA-I accumulation in the medium of HepG2 cells, but not albumin, was also significantly increased by 1.9-fold (n = 5, p < 0.001).

Albumins↗
PubMed1992

Post-translational regulation of macrophage apoprotein E production.

We have transfected the murine macrophage cell line, J774, which does not express its endogenous apoE gene, with a constitutively expressed human apoE cDNA in order to study post-transcriptional and post-translational control of apoE production in macrophages. Loading cells with cholesterol using preincubations in acetylated low density lipoprotein, previously shown to enhance macrophage apoE gene transcription and apoE synthesis, did not increase apoE synthesis or secretion in constitutively expressing transfected cells, suggesting that sterol control of macrophage apoE production occurs predominantly at a transcriptional locus. However, incubation in human high density lipoprotein (HDL3) stimulated apoE secretion and appeared to inhibit degradation of newly synthesized apoE. This effect could be entirely reproduced by incubation with phosphatidylcholine vesicles which increased apoE accumulation in the medium by 2-6-fold. Pulse-chase experiments indicated that the effect of HDL3 or phospholipid vesicles was very rapid (occurring within 15 min) and was independent of changes in apoE synthesis. Furthermore, the increased apoE which accumulated in the medium in the presence of phospholipid vesicles or HDL3 was not due to altered rates of reuptake of labeled apoE since this difference was completely preserved in the absence of extracellular calcium. These results indicate that alteration of sterol content does not regulate macrophage apoE production at a translational or post-translational locus but that incubation with HDL3 or phospholipid vesicles can enhance apoprotein E production independent of changes in apoE gene transcription or apoE synthesis. The nature of the signal generated by the phospholipid vesicles which leads to inhibition of intracellular apoE degradation and enhanced apoE secretion will require further investigation.

Animals↗
PubMed1991

Influence of dietary lipids on hepatic mRNA levels of proteins regulating plasma lipoproteins in baboons with high and low levels of large high density lipoproteins.

Selective breeding of baboons has produced families with increased plasma levels of large high density lipoproteins (HDL1) and very low (VLDL) and low (LDL) density lipoproteins when the animals consume a diet enriched in cholesterol and saturated fat. High HDL1 baboons have a slower cholesteryl ester transfer, which may account for the accumulation of HDL1, but not of VLDL and LDL. To investigate the mechanism of accumulation of VLDL + LDL in plasma of the high HDL1 phenotype, we selected eight half-sib pairs of baboons, one member of each pair with high HDL1, the other member with little or no HDL1 on the same high cholesterol, saturated fat diet. Baboons were fed a chow diet and four experimental diets consisting of high and low cholesterol with corn oil, and high and low cholesterol with lard, each for 6 weeks, in a crossover design. Plasma lipids and lipoproteins and hepatic mRNA levels were measured on each diet. HDL1 phenotype, type of dietary fat, and dietary cholesterol affected plasma cholesterol and apolipoprotein (apo) B concentrations, whereas dietary fat alone affected plasma triglyceride and apoA-I concentrations. HDL1 phenotype and dietary cholesterol alone did not influence hepatic mRNA levels, whereas dietary lard, compared to corn oil, significantly increased hepatic apoE mRNA levels and decreased hepatic LDL receptor and HMG-CoA synthase mRNA levels. Hepatic apoA-I message was associated with cholesterol concentration in HDL fractions as well as with apoA-I concentrations in the plasma or HDL. However, hepatic apoB message level was not associated with plasma or LDL apoB levels. Total plasma cholesterol, including HDL, was negatively associated with hepatic LDL receptor and HMG-CoA synthase mRNA levels. However, compared with low HDL1 baboons, high HDL1 baboons had higher concentrations of LDL and HDL cholesterol at the same hepatic mRNA levels. These studies suggest that neither overproduction of apoB from the liver nor decreased hepatic LDL receptor levels cause the accumulation of VLDL and LDL in the plasma of high HDL1 baboons. These studies also show that, in spite of high levels of VLDL + LDL and HDL1, the high HDL1 baboons had higher levels of mRNA for LDL receptor and HMG-CoA synthase. This paradoxical relationship needs further study to understand the pathophysiology of VLDL and LDL accumulation in the plasma of animals with the high HDL1 phenotype.

Animals↗
PubMed1988

Identification and characterization of transcriptional regulatory regions associated with expression of the human apolipoprotein E gene.

Multiple cis-acting regulatory elements have been mapped within a 1-kilobase fragment spanning nucleotides -651 through +356 of the human apolipoprotein E gene using a transient expression system based on the chloramphenicol acetyltransferase gene as well as DNase I footprinting techniques. A 651-base pair 5'-flanking region of the human apolipoprotein E gene was capable of directing chloramphenicol acetyltransferase gene expression over a 48-fold range among the various cultured cell lines tested. Deletion analysis of this 651-base pair upstream region linked to either the chloramphenicol acetyltransferase gene or the intact apolipoprotein E structural sequences revealed at least three regulatory domains within the proximal 383 nucleotides. One of these domains contained a GC box transcriptional control element. Further analysis demonstrated that the other two domains contained enhancer-like activity. These enhancer-like elements were located within the nucleotides spanning -366 to -246 and -193 to -124. A third enhancer element was identified in the first intron, within nucleotides +44 to +262. Changing the distance of the three enhancer elements from the transcription start site and reversing their orientation did not significantly alter their effects on transcription rates. However, enhancer activity was influenced by the promoter and cell line that were used. DNase I footprinting assays showed that specific sequences within two of these elements (-193 to -124 and +44 to +262) bind proteins in nuclear extracts from HepG2 and Chinese hamster ovary cells. A protein footprint also was identified for a GC box element at nucleotides -59 to -45. Thus, control of apolipoprotein E gene expression is the result of a complex interaction of several different regulatory elements.

Acetyltransferases↗
PubMed1988

Two copies of the human apolipoprotein C-I gene are linked closely to the apolipoprotein E gene.

The gene for human apolipoprotein (apo) C-I was selected from human genomic cosmid and lambda libraries. Restriction endonuclease analysis showed that the gene for apoC-I is located 5.5 kilobases downstream of the gene for apoE. A copy of the apoC-I gene, apoC-I', is located 7.5 kilobases downstream of the apoC-I gene. Both genes contain four exons and three introns; the apoC-I gene is 4653 base pairs long, the apoC-I' gene 4387 base pairs. In each gene, the first intron is located 20 nucleotides upstream from the translation start signal; the second intron, within the codon of Gly-7 of the signal peptide region; and the third intron, within the codon for Arg39 of the mature plasma protein coding region. The upstream apoC-I gene encodes the known apoC-I plasma protein and differs from the downstream apoC-I' gene in about 9% of the exon nucleotide positions. The most important difference between the exons results in a change in the codon for Gln-2 of the signal peptide region, which introduces a translation stop signal in the downstream gene. Major sequence differences are found in the second and third introns of the apoC-I and apoC-I' genes, which contain 9 and 7.5 copies, respectively, of Alu family sequences. The apoC-I gene is expressed primarily in the liver, and it is activated when monocytes differentiate into macrophages. In contrast, no mRNA product of the apoC-I' gene can be detected in any tissue, suggesting that it may be a pseudogene. The similar structures and the proximity of the apoE and apoC-I genes suggest that they are derived from a common ancestor. Furthermore, they may be considered to be constituents of a family of seven apolipoprotein genes (apoE, -C-I, -C-II, -C-III, -A-I, -A-II, and -A-IV) that have a common evolutionary origin.

Amino Acid Sequence↗
PubMed1986

Expression of the human apolipoprotein E gene in cultured mammalian cells.

The gene for human apolipoprotein (apo-) E was isolated from a human genomic library constructed in the cosmid shuttle vector pCV108. The transient expression of the apo-E gene was examined in cultured mammalian cells 48 h following calcium phosphate-mediated gene transfer. The expression of the cloned human apo-E gene, which contained between 0.7 and 29 kilobases of 5'-flanking DNA, was not restricted to human cells or to cultured cells derived from tissues that have been shown to synthesize apo-E. Several independent mouse L cell stable transfectants with the human apo-E gene integrated into their genome were selected on the basis of G418 resistance, which is conferred by the selectable gene marker in the cosmid vector. The levels of human apo-E mRNA found in the stable transfected mouse L cells ranged from undetectable to a level comparable to that found in the human liver. The size of the apo-E mRNA observed in the stable transfectants was identical to that found in the liver, indicating that the mouse L cells were capable of correctly processing the human apo-E gene transcripts. The integrated human apo-E genes had not undergone major rearrangements or deletions during transfer, and the level of apo-E mRNA found in the different stable transfectants correlated directly with the number of integrated copies of the human apo-E gene. The stable transfected L cells secreted authentic human apo-E into the medium. The secreted protein interacted specifically with antibodies to human plasma apo-E and had an apparent Mr = 35,000 to 36,000, which is slightly larger than that of plasma apo-E. The secreted human apo-E was associated with lipid (presumably phospholipids), floated at d approximately 1.09 g/ml, and bound with high affinity to the apo-B,E(LDL) receptor on fibroblasts.

Animals↗
PubMed1986

The charge polymorphism of rat apoprotein E.

Rat apolipoprotein E (apo-E) exists in plasma as four unique isoelectric forms (designated E-1, E-2, E-3, or E-4 from acidic to basic, respectively). We have examined the processes accounting for this polymorphism using intact rats or cultured rat hepatocytes. Intrahepatic precursors of rat apo-E were isolated and analyzed on isoelectric focusing gels. The primary translation product of rat liver apo-E mRNA focused as two isoproteins with more basic pI values than the isoproteins of plasma apo-E. The microsome-processed translation product also focused as two isoproteins having pI values corresponding to apo-E-4 and apo-E-3 isoproteins of plasma apo-E. Following a bolus injection of [3H]leucine into the portal vein, intrahepatic isoproteins corresponding to plasma apo-E-2 and apo-E-1 isoproteins were first detected in the rough endoplasmic reticulum (RER) and Golgi fractions, respectively. The apparent molecular weight of intrahepatic apo-E increased as it passed from the RER to the Golgi. Only the most acidic isoform, apo-E-1, of plasma apo-E was sensitive to neuraminidase treatment indicating that sialic acid residues are responsible, in part, for the polymorphism of rat apo-E. Using cultured hepatocytes, tunicamycin (1 microgram/ml) inhibited the incorporation of [3H]glucosamine into both molecular weight forms of apolipoprotein B but did not influence the synthesis, glycosylation (as measured by [3H]glucosamine incorporation), or secretion of apo-E. Tunicamycin-inhibited hepatocytes secreted the normal complement of apo-E isoforms including apo-E-1, thus confirming that apo-E-1 is not an N-linked glycoprotein. These results suggest that post-translational modifications involving both RER and Golgi-specific reactions contribute to the polymorphism of rat apo-E.

Animals↗
PubMed1985

Nucleotide sequence and structure of the human apolipoprotein E gene.

The gene for human apolipoprotein E (apo-E) was selected from a library of cloned genomic DNA by screening with a specific cDNA hybridization probe, and its structure was characterized. The complete nucleotide sequence of the gene as well as 856 nucleotides of the 5' flanking region and 629 nucleotides of the 3' flanking region were determined. Analysis of the sequence showed that the mRNA-encoding region of the apo-E gene consists of four exons separated by three introns. In comparison to the structure of the mRNA, the introns are located in the 5' noncoding region, in the codon for glycine at position -4 of the signal peptide region, and in the codon for arginine at position +61 of the mature protein. The overall lengths of the apo-E gene and its corresponding mRNA are 3597 and 1163 nucleotides, respectively; a mature plasma protein of 299 amino acids is produced by this gene. Examination of the 5' terminus of the gene by S1 nuclease mapping shows apparent multiple transcription initiation sites. The proximal 5' flanking region contains a "TATA box" element as well as two nearby inverted repeat elements. In addition, there are four Alu family sequences associated with the apo-E gene: an Alu sequence located near each end of the gene and two Alu sequences located in the second intron. This knowledge of the structure permits a molecular approach to characterizing the regulation of the apo-E gene.

Amino Acid Sequence↗
PubMed1984

Processing of rat liver apoprotein E primary translation product.

The primary translation product of rat liver apoE mRNA was isolated from wheat germ cell-free translation systems. Plasma apoE and the primary translation product migrated similarly on SDS-polyacrylamide gels, had similar partial proteolytic peptide maps, and bound to and coeluted from heparin-Sepharose columns. Comparison of the partial amino acid sequence of the primary translation product with the amino-terminal sequence of plasma apoE indicated that rat apoE is initially synthesized with an 18 amino acid amino-terminal extension. This entire segment was removed cotranslationally by canine microsomes possessing signal peptidase activity. The microsome-processed translation product did not contain an endoglycosidase H-sensitive oligosaccharide, suggesting that rat apoE is O-glycosylated.

Amino Acid Sequence↗
PubMedDate not supplied

Apolipoprotein E and apolipoprotein E receptors modulate A beta-induced glial neuroinflammatory responses.

Large numbers of activated glia are a common pathological feature of many neurodegenerative disorders, including Alzheimer's disease (AD). Several different stimuli, including lipopolysaccharide (LPS), dibutyryl (db)cAMP, and aged amyloid-beta 1-42 (A beta), can induce glial activation in vitro, as measured by morphological changes and the production of pro-inflammatory cytokines and oxidative stress molecules. Only A beta-induced activation is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. In addition, only A beta also induces an increase in the amount of endogenous apoE, the primary apolipoprotein expressed by astrocytes in the brain. The functional significance of the increase in apoE appears to be to limit the inflammatory response. Indeed, compared to wild type mice, glial cells cultured from apoE knockout mice exhibit an enhanced production of several pro-inflammatory markers in response to treatment with A beta and other activating stimuli. The mechanism for both the A beta-induced glial activation and the increase in apoE appears to involve apoE receptors, a variety of which are expressed by both neurons and glia. Experiments using receptor associated protein (RAP), an inhibitor of apoE receptors with a differential affinity for the low-density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), revealed that LRP mediates A beta-induced glial activation, while LDLR mediates the A beta-induced changes in apoE levels. In summary, both an apoE receptor agonist (apoE) and an antagonist (RAP) inhibit A beta-induced glial cell activation. Thus, apoE receptors appear to translate the presence of extracellular A beta into cellular responses, both initiating glial cell activation and limiting its scope by inducing apoE, an anti-inflammatory agent.

Amyloid beta-Peptides↗