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M F Linton

Publications and source records attributed to M F Linton.

43 records · Page 3Linked to original sources
PubMed1993

Transgenic mice expressing high plasma concentrations of human apolipoprotein B100 and lipoprotein(a).

The B apolipoproteins, apo-B48 and apo-B100, are key structural proteins in those classes of lipoproteins considered to be atherogenic [e.g., chylomicron remnants, beta-VLDL, LDL, oxidized LDL, and Lp(a)]. Here we describe the development of transgenic mice expressing high levels of human apo-B48 and apo-B100. A 79.5-kb human genomic DNA fragment containing the entire human apo-B gene was isolated from a P1 bacteriophage library and microinjected into fertilized mouse eggs. 16 transgenic founders expressing human apo-B were generated, and the animals with the highest expression had plasma apo-B100 levels nearly as high as those of normolipidemic humans (approximately 50 mg/dl). The human apo-B100 in transgenic mouse plasma was present largely in lipoproteins of the LDL class as shown by agarose gel electrophoresis, chromatography on a Superose 6 column, and density gradient ultracentrifugation. When the human apo-B transgenic founders were crossed with transgenic mice expressing human apo(a), the offspring that expressed both transgenes had high plasma levels of human Lp(a). Both the human apo-B and Lp(a) transgenic mice will be valuable resources for studying apo-B metabolism and the role of apo-B and Lp(a) in atherosclerosis.

Animals↗
PubMed1993

Four new mutations in the apolipoprotein B gene causing hypobetalipoproteinemia, including two different frameshift mutations that yield truncated apolipoprotein B proteins of identical length.

Familial hypobetalipoproteinemia can be caused by mutations in the apolipoprotein (apo)B gene that interfere with the translation of a full-length apoB molecule. Frequently, a truncated apoB molecule can be detected in the plasma lipoproteins of affected subjects. In this report, we characterize four different apoB gene mutations causing hypobetalipoproteinemia that are associated with the synthesis of truncated apoB proteins. Two of the mutations are nonsense mutations caused by single nucleotide substitutions; these mutations are associated with the production of apoB-32.5 (1473 amino acids) and apoB-82 (3733 amino acids). The other two mutations are single nucleotide deletions (of apoB cDNA nucleotides 7295 and 7359, respectively). The altered reading frames created by these different frameshift mutations terminated with the same stop codon, and both therefore yielded a truncated protein of identical size: apoB-52.8 (2395 amino acids). The two apoB-52.8 proteins differ, however, in the number of novel carboxyl-terminal amino acids introduced by the frameshift. The buoyant density of lipoproteins containing the truncated apoBs was inversely related to the length of the truncated apoB. ApoB-32.5 was present only in high density lipoproteins (HDL) and the d > 1.21 g/ml fraction, whereas apoB-82 was present almost exclusively in very low density lipoproteins (VLDL). ApoB-52.8 was present primarily in VLDL, intermediate density lipoproteins (IDL), and low density lipoproteins (LDL); trace amounts were observed in the HDL.

Adult↗
PubMed1992

Reading-frame restoration with an apolipoprotein B gene frameshift mutation.

We examined a mutant human apolipoprotein B (apoB) allele that causes hypobetalipoproteinemia and has a single cytosine deletion in exon 26. This frameshift mutation was associated with the synthesis of a truncated apoB protein of the predicted size; however, studies in human subjects and minigene expression studies in cultured cells indicated that the mutant allele also yielded a full-length apoB protein. The 1-base-pair deletion in the mutant apoB allele created a stretch of eight consecutive adenines. To understand the mechanism whereby the mutant apoB allele yielded a full-length apoB protein, the cDNA from cells transfected with the mutant apoB minigene expression vector was examined. Splicing of the mRNA was normal; however, 11% of the cDNA clones had an additional adenine within the stretch of eight adenines, yielding nine consecutive adenines. The insertion of the extra adenine, presumably during apoB gene transcription, is predicted to restore the correct apoB reading frame, thereby permitting the synthesis of a full-length apoB protein.

Alleles↗
PubMed1992

Apolipoprotein B gene mutations affecting cholesterol levels.

In the past 5 years, many different mutations in the apolipoprotein (apo) B gene have been described that affect plasma cholesterol levels. More than 20 different mutations in the apoB gene have been shown to cause familial hypobetalipoproteinaemia, a condition characterized by abnormally low plasma concentrations of apoB and LDL cholesterol. Almost all of the mutations are nonsense or frameshift mutations that interfere with the translation of a full-length apoB100 molecule. Many, but not all, of these apoB gene mutations result in the synthesis of a truncated species of apoB that can be detected within the plasma lipoproteins. Familial hypobetalipoproteinaemia heterozygotes are almost always asymptomatic and have LDL cholesterol levels about one-quarter to one-third of those of unaffected family members. Several homozygotes and compound heterozygotes for familial hypobetalipoproteinaemia have been described. In these individuals, the LDL cholesterol levels are extremely low, usually less than 5 or 10 mg dl-1, and the clinical phenotype is variable, ranging from completely asymptomatic to severe problems related to intestinal fat malabsorption. One missense mutation in the apoB gene (an Arg----Gln substitution at apoB amino acid 3500) is associated with very poor binding of apoB100 to the cellular LDL receptor. This syndrome has been designated familial defective apolipoprotein B (FDB). The amino-acid substitution at residue 3500 delays the clearance of LDL from the plasma and results in hypercholesterolaemia. In some Western populations, the frequency of FDB heterozygotes appears to be as high as 1 in 500 individuals.

Apolipoproteins B↗
PubMed1991

Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins.

We examined the relationship between the size of human apolipoprotein (apo) B and the formation and secretion of apoB-containing lipoprotein particles. Stable transformants of the rat hepatoma cell line McA-RH7777 harboring a variety of human apoB cDNA constructs were established, and these produced carboxyl-terminally truncated apoB proteins (apoB18, -B23, -B28, -B31, -B48, and -B53). Immunoblotting of apoB proteins secreted into the culture medium and fractionated by equilibrium density ultracentrifugation revealed that each of the truncated apoB species was secreted from the cells. The peak densities of the apoB-containing particles decreased as the length of the apoB proteins increased. Apolipoproteins B18 and B23 appeared at the bottom of the salt gradient (d = 1.23 g/ml), whereas particles containing apoB28, -B31, -B37, -B48, and -B53 exhibited progressive decreases in density. The density distribution of secreted apolipoproteins was not affected by the expression or secretion of these recombinant apoB species. As determined by nondenaturing gel electrophoresis, apoB28, -B31, -B37, -B48, and -B53 formed their own discrete particles, and there was a direct correlation between the size of the particles and the length of the apoB species. The efficiency and rate of secretion of these truncated forms of apoB were studied by measuring the decrease of immunoprecipitated 35S-labeled apoB proteins in the cells and their accumulation in the medium. Proteins corresponding to apoB28 or larger were rapidly and efficiently secreted, whereas apoB18 and apoB23 were secreted much more slowly. These data imply that the size of these truncated apoB forms governs the lipid content of the apoB-containing lipoproteins formed as well as the kinetics of secretion.

Animals↗
PubMed1991

Phenotypes of apolipoprotein B and apolipoprotein E after liver transplantation.

Apolipoprotein (apo) E and the two B apolipoproteins, apoB48 and apoB100, are important proteins in human lipoprotein metabolism. Commonly occurring polymorphisms in the genes for apoE and apoB result in amino acid substitutions that produce readily detectable phenotypic differences in these proteins. We studied changes in apoE and apoB phenotypes before and after liver transplantation to gain new insights into apolipoprotein physiology. In all 29 patients that we studied, the postoperative serum apoE phenotype of the recipient, as assessed by isoelectric focusing, converted virtually completely to that of the donor, providing evidence that greater than 90% of the apoE in the plasma is synthesized by the liver. In contrast, the cerebrospinal fluid apoE phenotype did not change to the donor's phenotype after liver transplantation, indicating that most of the apoE in CSF cannot be derived from the plasma pool and therefore must be synthesized locally. The apoB100 phenotype (assessed with immunoassays using monoclonal antibody MB19, an antibody that detects a two-allele polymorphism in apoB) invariably converted to the phenotype of the donor. In four normolipidemic patients, we determined the MB19 phenotype of both the apoB100 and apoB48 in the "chylomicron fraction" isolated from plasma 3 h after a fat-rich meal. Interestingly, the apoB100 in the chylomicron fraction invariably had the phenotype of the donor, indicating that the vast majority of the large, triglyceride-rich apoB100-containing lipoproteins that appear in the plasma after a fat-rich meal are actually VLDL of hepatic origin. The MB19 phenotype of the apoB48 in the plasma chylomicron fraction did not change after liver transplantation, indicating that almost all of the apoB48 in plasma chylomicrons is derived from the intestine. These results were consistent with our immunocytochemical studies on intestinal biopsy specimens of organ donors; using apoB-specific monoclonal antibodies, we found evidence for apoB48, but not apoB100, in donor intestinal biopsy specimens.

Adult↗