Hepatic cholesterol and lipoprotein conference. Report of a conference.
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Animals↗
Biomedical subjects
P A Edwards
Publications and source records attributed to P A Edwards.
Changes in in vitro growth behaviour of the mammary epithelial cell line NMuMG caused by the v-fos oncogene.
A defective retrovirus was constructed to investigate the effect of the expression of the v-fos oncogene from FBJ-MSV on the in vitro growth properties of the mammary epithelial cell line NMuMG. Clearly visible areas of overgrowth in monolayer cultures of NMuMG were seen in cells infected with the v-fos-containing retrovirus but not in cells infected with control virus which did not contain an oncogene. Two cell lines, representing two morphological types of infected cell, were isolated from a morphologically altered region and further characterized. Fos.3.1.NMuMG grew as very spindly cells, achieving a higher density than control cells in 5% foetal calf serum (FCS) but growing very poorly in 1% FCS or in soft agar. Fos.3.3.NMuMG grew to a high density in 5% FCS and to a limited extent in low serum. This cell line also grew in soft agar. Fos.3.3.NMuMG seemed to be more transformed than fos.3.1.NMuMG using the criteria of growth in soft agar and low serum. All the cells used in this study were shown to retain epithelial characteristics by staining for cytokeratins and to contain at least one viral genome by Southern blotting. fos mRNA expression was raised over control levels in the two transformed cell lines.
Animals↗
Growth retardation in asthmatic children treated with inhaled beclomethasone dipropionate.
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Asthma↗
Physically disabled parents and their normal children: assessment, diagnosis, and intervention.
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Persons with Disabilities↗
Macrophage lipoprotein receptors.
Macrophages possess a number of surface receptors that are capable of mediating the internalization of lipoproteins. The low-density lipoprotein (LDL) receptor of human monocyte macrophages recognizes apolipoprotein B-100 and apolipoprotein E and is rapidly regulated in response to changes in intracellular cholesterol levels. In contrast, in J774 macrophages LDL receptor regulation is defective and LDL can cause massive cholesterol accumulation. The beta migrating very low density lipoprotein (beta-VLDL) receptor is poorly regulated by cellular cholesterol concentrations, readily recognizes apolipoprotein E, poorly recognizes apolipoprotein B-100, and is immunologically related to the LDL receptor. The scavenger receptor (acetyl-LDL receptor) appears to have a molecular weight of 250,000 and is not regulated by cellular cholesterol levels. This receptor recognizes LDL that has been chemically or biologically altered. LDL complexes can also enter macrophages and cause cholesterol accumulation. Examples of such complexes are LDL-dextran sulphate complexes, LDL-proteoglycan aggregates, LDL-mast cell granule complexes, LDL-heparin-fibronectin-denatured collagen complexes, and LDL-antibody complexes. The entry of lipoprotein into macrophages by a pathway that is poorly regulated or is not regulated by cellular cholesterol concentrations appears to be a prerequisite for the formation of arterial foam cells.
Animals↗
Reproducibility study on the Scheimpflug Cataract Video Camera.
The Zeiss Scheimpflug Cataract Video Camera was designed to photograph, store and analyze cataracts in a semi-automated fashion for cross-sectional and longitudinal studies. We conducted a reproducibility study of this system. Twenty-four normal and 61 cataractous eyes were photographed twice by each of two of the authors in the 90 degree meridian and microdensitometry was performed on each of the stored images. Reproducibility was then determined using the intraclass correlation coefficient to determine whether or not the differences encountered were due to variability in the system or due to actual differences among the images. The intraclass correlation in the lens nucleus was 0.995 with 95% confidence limits of .992-.996. Therefore, reproducibility was 99.5%. In the anterior cortex, intraclass correlation was .941 with 95% confidence limits of .919-.959. In the posterior cortex intraclass correlation was .905 with 95% confidence limits of .870-.932. Reproducibility with this instrument was therefore excellent and with certain limitations, this may be a useful instrument in monitoring lens changes in certain diseases and the effects of anti-cataract agents.
Cataract↗
Localization of 3-hydroxy-3-methylglutaryl CoA reductase and 3-hydroxy-3-methylglutaryl CoA synthase in the rat liver and intestine is affected by cholestyramine and mevinolin.
In the normal fed rat, both 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) synthase and HMG-CoA reductase are found in high concentrations in hepatocytes that are localized periportally. The majority of the liver cells show little or no evidence of either enzyme. Addition of cholestyramine and mevinolin to the diet resulted in all liver cells showing strong positive staining for both HMG-CoA reductase and HMG-CoA synthase. These two drugs increased the hepatic HMG-CoA reductase and HMG-CoA synthase activities 92- and 6-fold, respectively, and also increased the HMG-CoA reductase activity in intestine, heart, and kidney 3- to 15-fold. We used immunofluorescence and avidin-biotin labeled antibody to localize HMG-CoA reductase in the rat intestine. In rats fed a normal diet, the most HMG-CoA reductase-positive cells were the villi of the ileum greater than jejunum greater than duodenum. Crypt cells showed no evidence of HMG-CoA reductase. Addition of cholestyramine and mevinolin to the diet led to a dramatic increase in the concentration of HMG-CoA reductase in the apical region of the villi of the ileum and jejunum and in the crypt cells of the duodenum. Hence these two drugs affected both the relative concentration and distribution of intestinal HMG-CoA reductase. Cholestyramine and mevinolin feeding induced in the liver, but not intestine, whorls of smooth endoplasmic reticulum that were proximal to the nucleus and contained high concentrations of HMG-CoA reductase. Administration of mevalonolactone led to the rapid dissolution of the hepatic whorls within 15 min, at a time when there is little or no change in the mass of HMG-CoA reductase. We conclude that the whorls are present in the livers of rats fed cholestyramine and mevinolin because the cells are deprived of a cellular product normally synthesized from mevalonate.
Animals↗
Alteration of morphogenesis by the v-myc oncogene in transplants of mammary gland.
To see if individual oncogenes can alter three-dimensional growth in vivo, we have inserted the v-myc oncogene into transplants of mouse mammary gland. Primary cultures of mammary epithelial cells were infected with helper-free retrovirus to insert v-myc oncogenes, and transplanted into cleared mammary fat pads (mammary glands from which the natural epithelium had been removed) of host mice. Uninfected transplants, and transplants infected with retrovirus constructs that carry no oncogene, grew to form an epithelial 'tree' resembling normal mammary gland, as expected. Transplants infected with retroviruses carrying v-myc oncogenes grew to form a characteristic, abnormal (hyperplastic) pattern in which the ducts were more densely packed than normal. Integration of the retrovirus in the transplants was demonstrated. The effect of the oncogene was local, not systemic, as some transplants showed adjacent areas of normal and hyperplastic growth. Thus v-myc can alter morphogenesis without growth becoming disorganized.
Animals↗
A comparative study between the PAM and the laser interferometer in cataracts.
We conducted a prospective study on 35 consecutive eyes undergoing cataract extraction to compare the Guyton-Minkowski Potential Acuity Meter (PAM) and the Laser Interferometer (LI) in determining potential visual acuity. The eyes were divided into: group A, which had good visualization of optic disc details, and group B, whose media allowed only disc outline or less to be seen. We found that in group A eyes, both instruments were reliable (with accuracies of 94% for the PAM and 88% for the LI). However, in group B eyes (advanced cataracts), both instruments were unreliable (accuracies of 33% for the PAM and 53% for the LI). Interestingly, in a subgroup of high myopes (axial length greater than 29 mm) with moderate cataracts and poor visual acuties, both instruments were very reliable and helpful in determining how much of the vision loss was due to the cataracts. We therefore strongly recommend potential acuity testing in high myopes with moderate cataracts and poor visual acuity.
Aged↗
Ameloblastic carcinoma: a clinicopathologic study and assessment of eight cases.
The term ameloblastic carcinoma is differentiated from the term malignant ameloblastoma and is defined as an ameloblastoma in which there is histologic evidence of malignancy in the primary tumor or the recurrent tumor (or metastasis), regardless of whether it has metastasized. Eight cases of ameloblastic carcinoma from the Armed Forces Institute of Pathology (AFIP) are reported. The mean age of patients was 30.1 years, with no sex predilection noted. Seven cases involved the mandible and one involved the maxilla, with the posterior regions favored. The most common sign was swelling, although pain, rapid growth, trismus, and dysphonia also occurred. Lesions characteristically were evident as ill-defined destructive radiolucencies, with occasional radiopacities noted. Histologic features generally resembled those of conventional ameloblastoma but with cytologic features of epithelial malignant disease. The clinical course was uniformly aggressive with extensive local destruction and spread, frequent recurrences, and one case of neck node metastasis. The nomenclature and classification of odontogenic carcinomas are discussed, as well as entities that should be included in the differential diagnosis. Further reporting of ameloblastic carcinoma is encouraged.
Adolescent↗
Molecular cloning and sequence of a cholesterol-repressible enzyme related to prenyltransferase in the isoprene biosynthetic pathway.
Differential hybridization and molecular cloning have been used to isolate CR39, a cDNA which hybridizes to a 1.2-kilobase (kb) mRNA in rat liver. The level of CR39 mRNA was increased seven- to ninefold over normal levels by dietary cholestyramine and mevinolin and decreased about fourfold compared with normal levels by cholesterol feeding or administration of mevalonate. Similar changes in the mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and HMG-CoA synthase were observed under the various conditions. In vitro translation of either CR39 hybrid selected RNA or 1.2-kb CR39 RNA generated by an SP6 in vitro transcription system produced a polypeptide of 39,000 daltons. As deduced from the nucleotide sequence of a full-length CR39 cDNA, the rat CR39 polypeptide contained 344 amino acids and had a molecular weight of 39,615. The predicted amino acid composition and submit molecular weight of the rat CR39 were very similar to those of prenyltransferases isolated from chicken, pig, and human. The sequence of amino acid residues 173 through 203 in the rat CR39 polypeptide showed that 17 out of 30 matched an active-site peptide of avian liver prenyltransferase. Thus, alterations in the rate of cholesterogenesis resulted in the coordinate regulation of three mRNAs encoding HMG-CoA reductase, HMG-CoA synthase, and CR39, the latter being tentatively identified as prenyltransferase.
Amino Acid Sequence↗
In vivo studies on cataracts using the Scheimpflug slit lamp camera.
We conducted reproducibility studies on an in vivo objective method of documenting cataracts: Scheimpflug photography using the Topcon SL 45 camera. Normal and cataractous lenses were photographed and the photographs digitized and analyzed using a Perkin Elmer microdensitometer attached to a PDP 11 and Vax 11/780 computer. Linear densitometry was performed through the center of the lens. We found very good reproducibility. The intraclass correlation for the mean densities in the nucleus was 0.95: that is, intra- and interobserver variability accounted for only 5% of the overall variability. For the anterior cortex, intraclass correlation was 0.88 and for posterior cortex it was 0.84. This method may be useful, within limits, in future clinical trials of anticataract medications.
Cataract↗
The role of lipoproteins and receptor-mediated endocytosis in the transport of bacterial lipopolysaccharide.
The addition of bacterial lipopolysaccharide (LPS) from Escherichia coli 0111:B4 to human monocyte-macrophages cultured in serum results in suppression of scavenger receptor activity. The present studies were performed to examine if the effect on scavenger receptor activity was mediated by LPS alone or by LPS in association with lipoproteins. Radioiodinated LPS (125I-LPS) was added to human plasma in vitro and to normal and hyperlipidemic rabbit plasma in vitro and in vivo to determine the distribution of 125I-LPS among the lipoprotein classes. It was found that all lipoprotein classes bound LPS in direct proportion to their plasma cholesterol concentration. LPS alone was compared to LPS bound to low density lipoprotein (LDL), high density lipoprotein, or reductively-methylated LDL for their abilities to suppress scavenger receptor activity in monocyte-macrophages in lipoprotein-free serum. Only LPS bound to LDL (LPS-LDL) demonstrated an effect similar to that observed when LPS was added to cells in serum. Either unlabeled LDL or unlabeled LPS-LDL complexes competed with the uptake of 125I-LPS-LDL complexes, which appeared to proceed by receptor-mediated endocytosis. In contrast to the uptake of 125I-LDL, the uptake of 125I-LPS-LDL by cultured monocyte-macrophages was not followed by its hydrolysis and the release of its radioactive degradation products into the medium. The association of LPS with lipoproteins was very stable and appeared to be mediated by a lipid-lipid interaction. We hypothesize that LPS bound to lipoproteins may be transported into the artery wall and may initiate the atherosclerotic reaction.
Animals↗
Rabbit beta-migrating very low density lipoprotein increases endothelial macromolecular transport without altering electrical resistance.
Rabbit aortic endothelial cells (RAEC) were grown on micropore filters in a new device. This system allowed in situ measurement of transendothelial electrical resistance (TEER). The monolayers demonstrated a TEER of 14 +/- 1 omega X cm2 at confluence. No difference was seen in the transport of low density lipoproteins (LDL) across endothelial cell monolayers obtained from normal or Watanabe heritable hyperlipidemic rabbits, indicating that the LDL receptor was not involved in the LDL transport. TEER was inversely correlated with 22Na transport (r2 = 0.93, P = less than 0.001) but not with 125I-LDL transport. The amount of LDL transported at 15 degrees C or across glutaraldehyde-fixed monolayers was half that of the controls at 37 degrees C. Preincubation of the monolayers with rabbit beta-migrating very low density lipoproteins (beta-VLDL) increased cholesterol content by 65%, and the transport of albumin and LDL doubled without a change in TEER. Removal of beta-VLDL from the culture medium resulted in the return of cellular cholesterol content and LDL transport to control values. We conclude that preincubation of RAEC with beta-VLDL resulted in an increased permeability to LDL and albumin, and that beta-VLDL may promote increased transendothelial transport of macromolecules in cholesterol-fed rabbits.
Albumins↗
Clonal analysis of expression of epithelial antigens in cultures of normal human breast.
Cells from normal human breast epithelium were cloned in monolayer culture and the clones were stained with monoclonal antibodies. Tissue was from reduction mammoplasty operations. Cloning efficiencies were 5-30%. Two types of clone were identified: 10 to 30% were of relatively spread cells whose boundaries were often difficult to see by phase-contrast microscopy but where they were visible they appeared as dark lines. The edges of the clones usually appeared to be under tension. These clones were stained by two monoclonal antibodies, LICR-LON-M8 and M24, that stain luminal epithelial cells in the intact tissue, but not myoepithelial or stromal cells. Within a clone the cells showed a full range of antigenic phenotypes. This was confirmed for clones grown from single cells that had been isolated manually. The second type of clone was more compact with little evidence of tension at the edges, and cell boundaries were clearly visible and bright under phase contrast. These clones were not stained by antibodies M8 or M24. Both types of clone stained with a third monoclonal antibody that is specific for luminal epithelial cells in the intact tissue, LICR-LON-M18, but the distribution of staining was different in the different types of clone. The simplest interpretation of the two types of clone is that luminal epithelial cells give rise to the spread type of clone while the myoepithelial cells give rise to the more abundant and vigorous compact clones. Alternatively, the compact clones may be from luminal epithelial cells that have lost differentiated characteristics.
Antibodies, Monoclonal↗
Inhibition of lysosomal protein degradation inhibits the basal degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
The effect of inhibiting lysosomal protein degradation on the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was determined using a mouse mammary cell line (TS-85) which expresses a temperature-sensitive mutation in the ubiquitin degradative pathway. Incubating cells for 18 hr in medium containing 20 mM NH4Cl did not alter total protein synthesis or cell growth, but it did inhibit the rate of total protein degradation by 19%, which is consistent with the known inhibitory effect of NH4Cl on lysosomal protein degradation. NH4Cl treatment also resulted in an increase (81% +/- 20) in HMG-CoA reductase activity. The increase in reductase activity was not correlated with changes in the phosphorylation state of the enzyme or with alteration in the relative rate of reductase synthesis. However, the basal degradation rate of the reductase was significantly inhibited, and after NH4Cl treatment, the half-life of the enzyme increased from 4.0 +/- 0.4 hr to 8.3 +/- 0.8 hr. The change in the rate of reductase degradation can account completely for the increase in reductase activity observed in NH4Cl-treated cells. The accelerated degradation of HMG-CoA reductase induced by 25-hydroxycholesterol treatment was not affected by either NH4Cl or by inactivation of the ubiquitin degradative pathway. Therefore, two different mechanisms may be responsible for the accelerated degradation and basal degradation of HMG-CoA reductase. The latter can be inhibited by NH4Cl and may imply that under basal conditions the enzyme may be degraded in lysosomes.
Ammonium Chloride↗
The effect of glucagon on the synthesis and degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
Incubation of rat hepatocytes with glucagon results in a time- and dose-dependent decrease in the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase. We demonstrate, using immunoprecipitation of radiolabeled enzyme, that 10 nM glucagon inhibits the synthesis of the enzyme by approximately 50%, but that the apparent rate of degradation of the enzyme is not affected by the hormone. We also demonstrate that the intact reductase polypeptide contained phosphoserine. We conclude that glucagon inhibits the activity of the reductase by inhibition of enzyme synthesis.
Animals↗
Transcriptional regulation of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver.
This study addresses whether transcriptional control of the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene in rat liver plays a role in determining the level of reductase mRNA. Isolated rat liver nuclei were allowed to elongate nascent RNA transcripts in the presence of [alpha-32P]CTP, and radiolabeled nuclear reductase RNA was quantitated by filter hybridization. Rats fed a diet supplemented with the drugs cholestyramine and mevinolin and having 20-60-fold induced levels of reductase mRNA exhibited levels of reductase transcription which were 20-fold higher than in rats fed an unsupplemented diet. Over 90% of the transcription of the reductase gene was inhibited by concentrations of alpha-amanitin which selectively inhibit RNA polymerase II. Administration of mevalonolactone (the end product of the reaction catalyzed by reductase) to rats fed cholestyramine and mevinolin caused an 80% decrease in the rate of reductase transcription by approximately 1 h. We conclude that under these conditions changes in reductase transcription are primarily responsible for the regulation of reductase mRNA levels.
Amanitins↗