[en] Methods for the purification of the low-density lipoprotein (LDL) receptor from various sources are described. These methods are designed to enable researchers to obtain chemical amounts from a rich source, the adrenal cortex, and to obtain smaller amounts of radiolabeled LDL receptor from less abundant sources, including cultured cells. Procedures for the purification of bovine adrenal receptors include solubilization, DEAE-cellulose chromatography, and affinity chromatography. LDL receptors of cultured cells can be biosynthetically labelled with [³⁵S]methionine and then purified by immunoaffinity chromatography

[en] 3-Hydroxy-3-methylglutaryl coenzyme A synthase (hydroxymethylglutaryl-CoA synthase, EC 4.1.3.5) is a negatively regulated enzyme in the synthetic pathway for cholesterol, isopentenyl tRNA, and other isoprenoids. The 5'-untranslated region of the mRNA for Chinese hamster hydroxymethylglutaryl-CoA synthase contains an optional exon of 59 nucleotides located 10 nucleotides upstream of the translation start site. About 50% of the mRNAs contain this exon, and the other 50% lack it owing to differential intron splicing. The authors show that the two transcripts are found in similar ratios in multiple tissues of the Syrian hamster, including the brain. The relative amounts of the two transcripts in brain and liver are constant from day 0 to day 75 of life. A similar alternative splicing pattern for hydroxymethylglutaryl-CoA synthase was observed in three human tissues: cultured fibroblasts, fetal adrenal gland, and fetal liver. A cDNA for human synthase had 90% homology to the hamster sequence in the region corresponding to the optional exon. This sequence contains a 20 out of 26 nucleotide match with the sequence immediately upstream of the initiator AUG codon in the mRNA for hamster hydroxymethylglutaryl-CoA reductase, the enzyme that follows the synthase in the isoprenoid biosynthetic pathway. These findings raise the possibility that the optional exon plays an important, conserved functional role in humans and hamsters

[en] Pharmacologic doses of 17 α-ethinyl estradiol are known to increase the number of low density lipoprotein (LDL) receptors in livers of rats, thereby producing a profound fall in plasma cholesterol levels. The authors report that ethinyl estradiol exerts the same effect in livers of male and female rabbits and that the increase in receptor number is correlated with a 6- to 8-fold increase in the levels of receptor mRNA. Receptor protein was measured by ligand blotting, and mRNA levels were measured by a quantitative solution hybridization/S1 nuclease protection assay using uniformly ³²P-labeled single-stranded cDNA probes. These experiments demonstrate that pharmacologic induction of the mRNA for the LDL receptor in liver can lead to increased LDL receptor levels and a fall in plasma cholesterol in experimental animals

[en] Hybridization studies with [³²P]cDNA probes revealed detectable amounts of mRNA for the low density lipoprotein (LDL) receptor in the central nervous system (CNS) of rabbits. mRNA levels were highest in the medulla/pons and spinal cord, which were the most heavily myelinated regions that were studied. Lower, but detectable levels were present in cerebral cortex, hypothalamus, thalamus, midbrain, and cerebellum. In the medulla/pons and spinal cord, the levels of receptor mRNA were in a range comparable to that detected in the liver. The levels of receptor mRNA in whole brain were constant from 3 days of age to adulthood and, thus, did not vary in proportion to the rate of myelin synthesis. LDL receptor mRNA in the CNS was produced by the same gene that produced the liver and adrenal mRNA as revealed by the demonstration of a deletion in the neural mRNA of Watanabe-heritable hyperlipidemic (WHHL) rabbits identical to the deletion in the LDL receptor gene of these mutant animals. Using antibodies directed against the bovine LDL receptor, the authors showed that LDL receptor protein is present in the medulla/pons of adult cows. The cell types that express LDL receptors in the CNS and the functions of these receptors are unknown

[en] The 5'-flanking region of the gene for hamster 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is shown to contain promoter sequences that drive transcription in vitro in the presence of a HeLa whole-cell extract. DNase I protection studies revealed at least six different regions within the 277-base-pair (bp) promoter that bind nuclear proteins and produce footprints. The functional significance of these sequences was determined through transcriptional analysis of a series of substitution mutations that scrambled short sequences throughout this region. Two of the footprint sequences were crucial for transcription in vitro; one of these contains a match in 6 of 6 bp, with a sequence in the adenovirus type 2 major late promoter that is know to be required for transcription. Scrambling a 26-bp sequence in a third footprint led to a consistent 2-fold increase in transcription, suggesting that this sequence might be a site for negative regulation. These studies define three regions that play a role in regulating transcription of the gene for HMG-CoA reductase, a negatively regulated enzyme in the cholesterol biosynthetic pathway

[en] A bundle of 1-mm-diam fused silica optical fibers on an existing TFTR diagnostic has been exposed to 11 high-power DT discharges. Each shot subjected the fibers to a peak fast (14.7 MeV) neutron flux of ∼2x10¹² n/cm²/s and a γ-dose rate of 500 rad(Si)/s for 0.75--1.0 s. The total fast-neutron fluence for these shots was ∼5x10¹² n/cm². A 15-m-long section of the bundle ran along the tokamak's toroidal field coils and the remaining 15 m ran radially away from the reactor. Fiber luminescence at 660 nm was ∼10¹⁰ photons/s/sr/cm²/A for the above flux (∼5%--10% of the bremsstrahlung emission), and varied linearly with DT neutron rate. Luminescence at 530 nm was 50% stronger, consistent with a Cerenkov radiation spectrum. Sensitivity to 3.5 MeV DD neutrons was ∼1/3 to 1/2 of that for DT neutrons. Fiber transmission decreased with the time integral of the neutron source rate and was reduced by 4% for the above flux. The fiber recovered rapidly: within 10 s, the transmission loss was only 2.5%. Shortly thereafter, the rate of recovery slowed to ∼0.05% per minute, but was sufficient to restore 75% of the transmission loss within two to four discharges. Recovery continued at ∼0.1% per hour and slowed overnight to ∼0.1% per day. Within the relative error of <0.2%, full transmission was recovered after five days

[en] A bundle of 1-mm-diam fused silica optical fibers on an existing TFTR diagnostic has been exposed to 11 high-power DT discharges. Each shot subjected the fibers to a peak fast (14.7 MeV) neutron flux of ∼2x10¹² n/cm²/s and a γ-dose rate of 500 rad(Si)/s for 0.75--1.0 s. The total fast-neutron fluence for these shots was ∼5x10¹² n/cm². A 15-m-long section of the bundle ran along the tokamak's toroidal field coils and the remaining 15 m ran radially away from the reactor. Fiber luminescence at 660 nm was ∼10¹⁰ photons/s/sr/cm²/A for the above flux (∼5%--10% of the bremsstrahlung emission), and varied linearly with DT neutron rate. Luminescence at 530 nm was 50% stronger, consistent with a Cerenkov radiation spectrum. Sensitivity to 3.5 MeV DD neutrons was ∼1/3 to 1/2 of that for DT neutrons. Fiber transmission decreased with the time integral of the neutron source rate and was reduced by 4% for the above flux. The fiber recovered rapidly: within 10 s, the transmission loss was only 2.5%. Shortly thereafter, the rate of recovery slowed to ∼0.05% per minute, but was sufficient to restore 75% of the transmission loss within two to four discharges. Recovery continued at ∼0.1% per hour and slowed overnight to ∼0.1% per day. Within the relative error of <0.2%, full transmission was recovered after five days

[en] A protein in the sarcoplasmic reticulum of rabbit skeletal and cardiac muscle was identified because of its ability to bind 125I-labeled low density lipoprotein (LDL) with high affinity after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This protein, referred to as the 165-kDa protein, is restricted to striated muscle. It was not detected in 14 other tissues, including several that contain smooth muscle, but it appears in rat L6 myoblasts when they differentiate into myocytes. Immunofluorescence and immunoelectron microscopic studies revealed that the protein is present throughout the sarcoplasmic reticulum and the terminal cisternae. It binds 45Ca2+ on nitrocellulose blots and stains metachromatically with Stains-all, a cationic dye that stains Ca2+-binding proteins. It does not appear to be a glycoprotein, and it appears slightly larger than the 160-kDa glycoprotein previously described in sarcoplasmic reticulum. The 165-kDa protein binds LDL, beta-migrating very low density lipoprotein, and a cholesterol-induced high density lipoprotein particle that contains apoprotein E as its sole apoprotein with much higher affinity than it binds high density lipoprotein. The protein is stable to boiling and to treatment with sodium dodecyl sulfate, but it becomes sensitive to these treatments when its cystine residues are reduced and alkylated. The protein was purified 1300-fold to apparent homogeneity from rabbit skeletal muscle membranes. It differs from the cell surface LDL receptor in that (1) its apparent molecular weight is not changed by reduction and alkylation; (2) it is present in Watanabe-heritable hyperlipidemic rabbits, which lack functional LDL receptors; (3) binding of lipoproteins is not inhibited by EDTA; and (4) it is located within the lumen of the sarcoplasmic reticulum where it has no access to plasma lipoproteins

[en] Cultured amniotic-fluid cells from a fetus at risk for homozygous familial hypercholesterolaemia (F.H.) almost completely lacked cell-surface receptors for plasma low-density lipoprotein (L.D.L.), as evidenced by direct measurement of binding, uptake, and degradation of ¹²⁵ I-L.D.L. Functional consequences of L.D.L. binding to the receptor - i.e., suppression of 3-hydroxy-3-methylglutaryl coenzyme A reductase and stimulation of cholesterol esterification - were proportionately reduced when compared with results in cultured amniotic cells from two control fetuses. On the basis of these findings, homozygous F.H. was diagnosed and the pregnancy was terminated at the 20th week. The diagnosis of homozygous F.H. was confirmed by a serum-cholesterol of the aborted fetus of 279 mg/dl, a value 9 times the mean of four control fetuses of similar gestational age. More than 80% of the serum-cholesterol of the affected fetus was contained within L.D.L. Prenatal diagnosis of homozygous F.H. now seems practical; moreover, the finding of a raised serum-L.D.L. in the affected fetus indicates that the L.D.L. receptor is normally functional as early as the 20th week of fetal life. (author)