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Biomedical subjects

J M Hoeg

Publications and source records attributed to J M Hoeg.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of LDL receptor, apoB, and apoE protein and mRNA in Hep G2 cells.

The regulation of low-density lipoprotein (LDL) receptor activity, protein synthesis, and cellular mRNA content was evaluated in the human hepatoma cell line Hep G2. Incubation of the cells with LDL led to a complete downregulation of LDL receptor mRNA and LDL receptor protein synthesis. This LDL regulation of the LDL receptor and its mRNA was both time- and concentration-dependent. In contrast to protein synthesis and cellular mRNA concentrations of the LDL receptor, which were reduced to undetectable levels by prolonged incubation in the presence of LDL, LDL receptor activity was reduced to only 44% of preincubation levels. These findings support the presence of a second metabolic pathway for LDL uptake in human hepatocytic cells. The effect of LDL on cellular LDL receptor expression was specific for LDL because incubation in the presence of HDL did not affect any of these study end points. The potential coordinate regulation of the expression of the LDL receptor with its principal ligands, apolipoproteins (apo) B and E, was also investigated. In contrast to the LDL receptor mRNA downregulation with LDL incubation, cellular apoB and apoE mRNA concentrations were not affected by either LDL or HDL. Secretion of apoB, however, was significantly increased by incubating Hep G2 cells with LDL. These findings indicate that, in contrast to LDL receptor which is regulated at the mRNA level, the ligands for the LDL receptor are regulated either co- or post-translationally.

Apolipoproteins B↗
PubMed1992

Ontogenetic regulation of apolipoprotein B mRNA editing during human and rat development in vivo.

The solubilization and delivery of lipids in plasma rely on both forms of apolipoprotein B (apo B): apo B-100 and apo B-48. Apo B-48 is the translational product of apo B-100 mRNA that undergoes peritranscriptional conversion of C----U, replacing codon CAA (glutamine 2,153) with the inframe stop codon (UAA). We examined mRNA editing activity in the human and the rat by reverse transcription-polymerase chain reaction primer-extension analysis of intestine and liver total RNA. In rat intestine the percentage of apo B transcripts that undergo editing increases dramatically the day before birth (from approximately 1% to 80%), whereas the rat liver acquires an adult level of editing activity during the third postnatal week (rising from approximately 8% to 30%), when weaning is completed, bile acid composition matures, and plasma thyroid hormone levels peak. In contrast to the rat, the human intestine acquires adult levels of apo B mRNA editing relatively early in fetal development, rising from 10% at 10 weeks to approximately 80% by the end of the second trimester. Our results establish that apo B mRNA editing is 1) developmentally regulated in a tissue- and species-specific manner; 2) fully developed prenatally in both human and rat intestine, suggesting a crucial role of apo B-48 in mammalian fetal adaptation to extrauterine life; and 3) acquired early in human fetal intestine, implying a potential role for apo B-48 in prenatal lipid metabolism.

Animals↗
PubMed1991

Apolipoprotein B upstream suppressor site: identification of an element which can decrease apolipoprotein B transcription.

Elevated plasma levels of apolipoprotein B (apoB) may predispose to development of premature coronary atherosclerosis. We have identified the first well localized domain of the apoB gene which can effect negative regulation of its transcription. This region binds trans-activating factors present only in apoB producing cell lines. Mutagenesis of this region causes up-regulation of its transcriptional activity. We have termed this element apoB upstream suppressor site (aBUSS) and its trans-activators the apoB repressor proteins (ARP). aBUSS and ARP may play important roles in the transcriptional modulation of apoB.

Animals↗
PubMed1991

Clinical and laboratory findings in the oculocerebrorenal syndrome of Lowe, with special reference to growth and renal function.

BACKGROUND: The oculocerebrorenal syndrome of Lowe is an X-linked disorder whose clinical manifestations include congenital cataracts, mental retardation, and renal tubular dysfunction. We investigated growth, renal function, and serum chemistry values in patients with the oculocerebrorenal syndrome to determine the natural history of the disorder and its heterogeneity with respect to these characteristics. METHODS: Twenty-three patients with the oculocerebrorenal syndrome, ranging in age from 4 months to 31 years, were examined. Height was compared with bone age. Renal function was assessed by measurements of proteinuria, urinary volume, and fractional excretions of potassium, phosphate, carnitine, and amino acids. Creatinine clearance was determined as a measure of glomerular function. RESULTS: In the oculocerebrorenal syndrome, linear growth decreases after one year of age; bone age lies between chronologic age and height age. Renal dysfunction occurs in the first year of life, characterized by proteinuria (mean [+/- SD], 1.38 +/- 0.77 g of urinary protein per square meter of body-surface area per day; normal, less than or equal to 0.10), generalized aminoaciduria (mean, 686 +/- 505 mumol of urinary amino acid per kilogram of body weight per day; normal, 94 +/- 45), carnitine wasting (mean fractional excretion, 0.10 +/- 0.05; normal, 0.03 +/- 0.01), and phosphaturia progressing into the third decade. Urinary wasting of individual amino acids is milder than in cystinosis, and branched-chain amino acids are relatively spared. Reciprocal serum creatinine levels fall linearly with age, predicting renal failure in the fourth decade. Concentrations of the muscle enzymes creatine kinase, aspartate aminotransferase, and lactate dehydrogenase, as well as of total serum protein, serum alpha 2-globulin, and high-density lipoprotein cholesterol, are elevated. CONCLUSIONS: Renal glomerular deterioration is slowly progressive in the oculocerebrorenal syndrome. Renal tubular dysfunction begins early and persists; most patients require alkalinization therapy, and many benefit from supplemental potassium, phosphate, calcium, or carnitine. Serum enzyme elevations suggest muscle involvement in the oculocerebrorenal syndrome.

Adolescent↗
PubMed1991

Recent advances in lipoprotein metabolism and the genetic dyslipoproteinemias.

The elucidation of the structure and function of the plasma apolipoproteins has provided the unique opportunity to understand the physiological pathways for the transport and cellular metabolism of the plasma lipoproteins. The complexity of the individual density classes of plasma lipoproteins has been revealed by a detailed analysis of the apolipoprotein composition of the individual lipoprotein particles. In addition, the elucidation of the molecular defects in patients with dyslipoproteinemias has now permitted the understanding of the defects at the level of the apolipoprotein gene. The ability to define the genetic defect in individuals at risk for the development of premature cardiovascular disease provides the unique opportunity to now identify these individuals at an earlier age, and to initiate therapy to prevent the development of early heart disease.

Apolipoprotein A-I↗
PubMed1991

Retroviral vector-mediated in vivo expression of low-density-lipoprotein receptors in the Watanabe heritable hyperlipidemic rabbit.

We have achieved in vivo expression of recombinant low-density-lipoprotein (LDL) receptors in the Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for the human disease familial hypercholesterolemia. A retroviral vector was constructed containing the human LDL receptor cDNA and was used to stably transduce primary skin fibroblasts from WHHL rabbits. The integrity and function of the introduced LDL receptor was established by immunoprecipitation, by a fluorescent LDL binding assay, and by the ability of the transduced cells to suppress 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase activity in response to exogenous cholesterol. Autologous transduced fibroblasts were reimplanted into donor rabbits; in vivo LDL receptor expression and the survival of the transduced cells were analyzed by immunohistochemistry and by LDL binding assays performed on cells recovered from the implants. LDL receptor-bearing cells could be identified on tissue sections and recovered from implants for up to four weeks. Total and LDL cholesterol levels decreased significantly after implantation of the transduced cells; however, control experiments indicated that the decreases were not mediated through the recombinant LDL receptor. While in vivo stable expression of recombinant LDL receptors in Watanabe rabbits is possible, consequent changes in lipid levels must be interpreted with caution. This system of site-specific in vivo expression of recombinant LDL receptors permits further evaluation of the role of LDL receptor-gene replacement in the therapy of hypercholesterolemia.

Animals↗
PubMed1991

Pharmacologic and surgical treatment of dyslipidemic children and adolescents.

A wide variety of treatment modalities have been used in children with dyslipidemias to reduce the concentrations of atherogenic lipoprotein particles. Most of the published experience has focused upon children with familial hypercholesterolemia (FH). A variety of pharmacologic regimens have been utilized with variable degrees of success. The bile acid sequestrants colestipol and cholestyramine, lovastatin, pantethine, paraminosalicylic acid, and fenofibrate have all been successful in reducing total blood cholesterol concentrations by 18-24% in hypercholesterolemic children. Of these medications, only the bile acid sequestrants are not absorbed into the circulation. This theoretic advantage is paralled by long-term safety studies which indicate the absence of serious adverse effects with bile acid sequestrant therapy. Therefore, the bile acid sequestrants represent the drugs of choice in treating severely dyslipidemic children. In selected cases of profoundly dyslipidemic children, other therapeutic strategies have been utilized. Most of these efforts have been directed in the treatment of the child homozygous for FH. Despite the lipid lowering effects of partial ileal bypass surgery in hypercholesterolemic adults, homozygous familial hypercholesterolemic children are not adequately treated by this approach. Portacaval shunt has reduced the total cholesterol concentrations by 20-35% in homozygous FH children without having a negative impact on growth and development. These children have, however, gone on to develop atherosclerotic cardiovascular disease despite therapy. Liver transplantation has led to virtual normalization of the plasma lipoprotein concentrations in 3 children homozygous for familial hypercholesterolemia, and there is evidence for regression of vascular lesions in the coronary arteries in one of these children. However, considering the expense, the difficulty in posttransplantation management, and the irreversible nature of the therapy, liver transplantation should be reserved as the therapy of last resort for homozygous FH. The best therapy for FH homozygotes is the frequent removal of the atherogenic lipoproteins by one of the several apheresis procedures currently available. Total plasma exchange, immunoadsorption, membrane filtration, dextran sulfate adsorption, and heparin extracorporeal precipitation have all been used successfully in significantly reducing the concentrations of total and low-density lipoprotein cholesterol. Studies currently under way will more extensively evaluate the long-term safety as well as the efficacy of apheresis procedures.

Adolescent↗
PubMed1991

Differential tissue-specific expression of human apoA-I and apoA-II.

To evaluate the sources of high density lipoprotein (HDL) particles containing only apolipoprotein A-I (apoA-I), the synthesis of apoA-I and apolipoprotein A-II (apoA-II) was examined in human liver and small intestine as well as the human intestinally derived cell line, Caco-2. Human liver contained apoA-I, apoA-II as well as apolipoprotein B (apoB) mRNA. In contrast, human adult small intestine total and polyA+ RNA had little or no apoA-II despite the presence of apoA-I and apoB. Intestinal biopsies from normal individuals failed to show de novo apoA-II protein synthesis in the media of organ cultures during [35S]methionine pulse-chase labeling, whereas apoA-I could be readily detected. Caco-2 cells contained apoA-II mRNA and secreted apoA-II protein into the tissue culture media. These data indicate that the primary site of human apoA-II synthesis is in the liver and that the small intestine secretes apoA-I-containing high density lipoproteins.

Apolipoprotein A-I↗
PubMed1990

Cholesteryl ester storage disease: hepatopathology and effects of therapy with lovastatin.

We describe three patients with cholesteryl ester storage disease. Diagnosis was confirmed by demonstrating a deficiency in lysosomal acid cholesteryl hydrolase activity in cultured skin fibroblasts from each of these patients. All had hepatomegaly, elevated serum aminotransferase activities and hyperlipoproteinemia. Histological examination of liver biopsy specimens before treatment revealed accumulation of fat within hepatocytes, bile duct epithelium and endothelial and Kupffer cells. Cholesterol crystals were recognized by their birefringence in frozen sections. A striking feature was the presence of markedly hypertrophied Kupffer cells and portal macrophages with foamy, tan-colored cytoplasm that stained readily with the periodic acid-Schiff reagent and aldehyde fuchsin. Periportal fibrosis was noted in all cases; incomplete cirrhosis was present in one case. Distinctive and hitherto undescribed lysosomal accumulations of triglyceride and cholesterol crystals were noted. The patients were treated with lovastatin, a cholesterol-lowering agent, for at least 12 mo. No significant changes were seen in serum lipoprotein concentrations or liver histopathology after therapy. Thus lovastatin did not have an obviously beneficial effect on abnormal lipid metabolism in these patients.

Biopsy↗
PubMed1990

Both apolipoproteins B-48 and B-100 are synthesized and secreted by the human intestine.

Apolipoprotein B (apoB), an apolipoprotein associated with very low density lipoproteins and the atherogenic low density lipoproteins (LDL), directs the metabolism of lipoprotein particles in plasma by interacting with the LDL receptor. Utilizing human intestinal biopsy organ cultures, we have studied the synthesis of intestinal apoB in man. Intestinal organ cultures from normal adults (n = 6) were incubated in the presence of protease inhibitors in media supplemented with [35S]methionine. Media from these cultures were evaluated by sequential NaDodSO4 polyacrylamide gel electrophoresis, radioautography, and Western blot analyses, and intestinal biopsies were studied using immunohistochemistry. The relative abundance of apoB-100 and apoB-48 mRNA was assessed using reverse transcriptase-polymerase chain reaction followed by primer extension. Although apoB-48 was the principal isoprotein that was newly synthesized by intestinal organ cultures, apoB-100 was also synthesized and secreted by human intestinal organ cultures with 16 +/- 3% of the intestinal apoB mRNA coding for apoB-100. These results establish that apoB-100 is produced by the human intestine. The synthesis of the atherogenic apoB-100 by the intestine has pathophysiologic implications for the development of diet-induced atherosclerosis.

Adult↗
PubMed1990

A genetic therapy for familial hypercholesterolemia.

The WHHL rabbit serves as an animal model for the human genetic disease, familial hypercholesterolemia. In initial studies aimed at the development of a genetic therapy for familial hypercholesterolemia (i.e., introduction of a normal LDL receptor gene), WHHL rabbit skin fibroblasts were transduced with a retroviral vector expressing a normal human LDL receptor. Correction of the WHHL rabbit genetic defect in vitro was confirmed. Autologous fibroblasts expressing LDL receptors were reimplanted in donor rabbits and were found to survive and express the recombinant receptor in vivo for up to 4 weeks. In vivo LDL receptor expression by autologous cells stably transduced with functioning LDL receptors is possible. Transduction of greater numbers of cells along with increased cell survival in vivo may eventually lead to a specific and effective genetic therapy for familial hypercholesterolemia.

Animals↗
PubMed1990

Detection and evaluation of dyslipoproteinemia.

Screening for dyslipoproteinemias should be undertaken in all individuals older than 20 years of age at least once every 5 years. The initial screening, as recommended by the Adult Treatment Guidelines Panel of the National Cholesterol Education Program, is to determine the concentration of total blood cholesterol. This initial determination can be made on blood obtained in the nonfasting state. Further evaluation of the patient's lipoprotein concentrations is dependent upon the presence of other cardiovascular risk factors. in the absence of definite coronary heart disease, hypertension, diabetes mellitus, a family history of coronary artery disease, cigarette smoking, or severe obesity, the patient with a total blood cholesterol concentration less than 200 mg/dL requires no specific instruction and should have a repeated screening performed within 5 years. Patients with blood cholesterol concentrations greater than 200 mg/dL should have their lipoprotein profiles determined if they have atherosclerotic cardiovascular disease or two other cardiovascular disease risk factors. The lipoprotein profile includes the determination of fasting cholesterol and triglyceride and HDL cholesterol concentrations. From these values, the LDL cholesterol concentration can be calculated. This LDL cholesterol concentration is central in selecting the appropriate therapy. HDL cholesterol concentrations may be useful in evaluating patients with ischemic heart disease. Concentrations of HDL cholesterol less than 35 mg/dL are associated with increased risk for coronary artery disease. Although there is currently no convincing evidence that support the specific treatment of depressed HDL cholesterol concentrations, therapy directed to modulating lipoprotein metabolism in patients with heart disease and low HDL concentrations may be of benefit. Patients with recurrent abdominal pain, pancreatitis, and eruptive xanthomatosis frequently have fasting hypertriglyceridemia concentrations exceeding 1000 mg/dL. These patients should be identified in order to effectively reduce their triglyceride concentrations, which can prevent these complications.

Adult↗
PubMed1989

Homozygous hypobetalipoproteinemia: transcriptional regulation and 5'-flanking sequence analysis in an apolipoprotein B deficiency state.

Apolipoprotein (apo) B is the principal apolipoprotein of chylomicrons, very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL). Patients with homozygous hypobetalipoproteinemia (HBL), characterized by apoB deficiency, have markedly decreased levels of hepatocyte mRNA as well as intracellular B apolipoprotein, and a virtual absence of plasma apoB. We have cloned, sequenced and analyzed the 5' regulatory region of the human apoB gene from -899 to +121 bp in normal and hypobetalipoproteinemic subjects. TATA and CAAT boxes were located at -30 and -61, respectively, and two GC-like boxes were identified at positions +56 and +108. The analysis of the HBL sequence revealed two substitutions at positions -838 and -517, when compared to the normal sequence. These substitutions were not present in any known apoB regulatory elements. The transcriptional activities of the homozygous hypobetalipoproteinemic and normal regulatory regions were compared by chloramphenicol acetyltransferase (CAT) assays in Hep G2 cells, and were found to be the same. Therefore, we conclude that the 5' regulatory region of the HBL apoB gene in this kindred is normal, and the two base substitutions do not affect promoter activity of the apoB gene. These studies suggest that a coding region abnormality in the apoB gene may lead to HBL.

Apolipoproteins B↗
PubMed1989

Human apolipoprotein A-I. Post-translational modification by covalent phosphorylation.

In vitro phosphorylation of purified human plasma apolipoprotein A-I (apoA-I) by a recently characterized Ca2+/calmodulin-dependent kinase (Beg, Z. H., Stonik, J. A., and Brewer, H. B., Jr. (1987) J. Biol. Chem. 262, 13228-13240) was time-, Ca2+-, and calmodulin-dependent. Maximal phosphorylation of human apoA-I revealed a stoichiometry of approximately 1 mol of PO4/mol of apoA-I. Phosphorylation of apoA-I resulted in an increase of two negative charges and consequently a shift to a more acidic pI for each apoA-I isoform following isoelectrofocusing. Dephosphorylation of 32P-apoA-I with either phosphatase I or a Ca2+/calmodulin-dependent phosphatase was associated with a virtually complete loss of 1 mol of 32PO4/mol of apoA-I. Phosphoamino acid analysis of a purified 32P-peptide established that the phosphorylation occurred on a single serine residue. Automated Edman degradation of the purified 32P-peptide revealed a single amino acid sequence and indicated that phosphorylation occurred on the serine at residue 201 in the apoA-I sequence. ApoA-I was shown to be secreted as a phosphoapolipoprotein by HepG-2 cells as well as primary human hepatocytes. Analysis of HepG-2 cells established that intracellular apoA-I, like secreted apoA-I, is phosphorylated. Dephosphorylation of both secreted and intracellular 32P-apoA-I revealed the loss of radioactivity in the apoA-I protein bands. These data provide the initial description of a post-translational modification involving reversible phosphorylation of extracellular as well as intracellular apoA-I on a serine residue. These combined results suggest that synthesis and secretion of apoA-I as a phosphoapolipoprotein in HepG-2 cells as well as primary human hepatocytes may play an important role in lipoprotein assembly, intracellular transport as well as processing, and lipoprotein secretion.

Adenosine Triphosphate↗
PubMed1989

Effect of low density lipoproteins, high density lipoproteins, and cholesterol on apolipoprotein A-I mRNA in Hep G2 cells.

We have utilized the human hepatocellular carcinoma cell line, Hep G2, to study the effects of low density lipoproteins (LDL), high density lipoproteins (HDL), and free cholesterol on apolipoprotein (apo) A-I mRNA levels. Incubation of the Hep G2 cells with LDL and free cholesterol led to a significant increase in the cellular content of cholesterol without any effect on the yield of total RNA or in the cellular protein content. Our studies established that incubation with LDL or free cholesterol increased the relative levels of apoA-I mRNA in the Hep G2 cells. In contrast with cholesterol loading, HDL had the effect of lowering the levels of apoA-I mRNA. These results indicate the LDL and HDL pathways as well as intracellular cholesterol may be important in apoA-I gene expression and regulation.

Apolipoprotein A-I↗
PubMed1988

Tissue-specific expression of apolipoprotein A-I (ApoA-I) is regulated by the 5'-flanking region of the human ApoA-I gene.

We have isolated and characterized a 2.5-kilobase pairs genomic DNA fragment which includes the 5'-flanking region and the first and second exons of the human apolipoprotein (apo) A-I gene. The major transcriptional start site was determined by primer extension analysis and is 235 base pairs (bp) upstream from the AUG translational start codon in liver and 234 bp upstream in the intestine. TATA box-like and CAT box-like sequences and two GC box sequences are present in the intestine 30, 108, 220, and 440 bp upstream, respectively, from the transcriptional start site. Fragments of 570 bp (-487 to +71) and 2.15 kilobase pairs (-2067 to +99) containing the 5'-flanking region of the apoA-I gene were fused upstream to the bacterial chloramphenicol acetyltransferase (CAT) gene. These constructs, designated pA-I(0.6)CAT and pA-I(2.2)CAT, respectively, were introduced into human oral epithelial cells (KB), mouse NIH 3T3 cells, Chinese hamster ovary (CHO) cells, human hepatoma cells (Hep G2), human duodenal epithelial cells (Hutu 80), and human colonic epithelial cells (Caco-2) by calcium phosphate coprecipitation. When compared with control vectors, highly efficient CAT expression of both the pA-I(0.6)CAT and pA-I(2.2)CAT constructs were observed only in cells derived from the liver (Hep G2) and intestine (Caco-2), which is consistent with the tissue specificity of expression of the native gene. Analysis of deletion mutants of the human apoA-I 5'-flanking region revealed that: 1) the region from -250 to -199 bp, from -487 to -413 bp, and -1021 to -691 bp upstream from the transcriptional start site contain sequences required for maximum gene expression; and 2) the regions from -2067 to -1476 bp and -199 to -80 bp contain the sequences required for tissue-specific repression of apoA-I gene expression in non-apoA-I producing cells.

Animals↗