Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.     

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

Role of endothelial cells and their products in the modification of low-density lipoproteins

A majority of the LDL preparations from various donors could be modified by incubation with endothelial cells from human arteries, veins and microvessels. These alterations comprise changes in electrophoretic mobility, buoyant density and lipid composition of LDL, the generation of thiobarbituric acid reactive substances in the medium, and a decrease in primary amino groups of LDL. Furthermore, the association of endothelial cell proteins with LDL was demonstrated by [35S]methionine incorporation and trichloroacetic acid precipitation of reisolated endothelial cell-modified LDL. After SDS-polyacrylamide gel electrophoresis of the reisolated modified LDL particles, radioactivity was mainly found at a molecular mass of 48 kDa and at one or two bands with a molecular mass of more than 100 kDa. The 48 kDa protein was identified as a latent plasminogen activator inhibitor. Cell viability was necessary for the cell-mediated LDL modification, which indicates that endothelial cells are actively involved in this process. The Ca2+ ionophore A23187 and monensin did not influence LDL modification. LDL modification was markedly inhibited by antioxidants. It was not prevented by cyclooxygenase and lipoxygenase inhibitors, which indicates that non-enzymatic lipid peroxidation is involved. Transition metal- (copper-) induced lipid peroxidation results in similar physiochemical alterations of the LDL particle as found with endothelial cells; it is prevented by the presence of superoxide dismutase. In contrast, endothelial cell LDL modification was not influenced by superoxide dismutase. Catalase or singlet oxygen and hydroxyl radical scavengers also did not affect it. We suggest that yet unidentified radicals or lipid peroxides are generated in the cells or on the cell membrane and that these reactive molecule(s) will react with LDL after leaving the cell. HDL and lipoprotein-depleted serum prevented LDL modification markedly, and to a larger extent than that by copper ions. We speculate that LDL modification by endothelial cells will only occur under those conditions in which the balance between the generation of reactive oxygen molecules and the cellular protection against these reactive species is disturbed.     

Excess amino acid polymorphism in mitochondrial DNA: contrasts among genes from Drosophila, mice, and humans

Recent studies of mitochondrial DNA (mtDNA) variation in mammals and Drosophila have shown an excess of amino acid variation within species (replacement polymorphism) relative to the number of silent and replacement differences fixed between species. To examine further this pattern of nonneutral mtDNA evolution, we present sequence data for the ND3 and ND5 genes from 59 lines of Drosophila melanogaster and 29 lines of D. simulans. Of interest are the frequency spectra of silent and replacement polymorphisms, and potential variation among genes and taxa in the departures from neutral expectations. The Drosophila ND3 and ND5 data show no significant excess of replacement polymorphism using the McDonald-Kreitman test. These data are in contrast to significant departures from neutrality for the ND3 gene in mammals and other genes in Drosophila mtDNA (cytochrome b and ATPase 6). Pooled across genes, however, both Drosophila and human mtDNA show very significant excesses of amino acid polymorphism. Silent polymorphisms at ND5 show a significantly higher variance in frequency than replacement polymorphisms, and the latter show a significant skew toward low frequencies (Tajima's D = -1.954). These patterns are interpreted in light of the nearly neutral theory where mildly deleterious amino acid haplotypes are observed as ephemeral variants within species but do not contribute to divergence. The patterns of polymorphism and divergence at charge-altering amino acid sites are presented for the Drosophila ND5 gene to examine the evolution of functionally distinct mutations. Excess charge-altering polymorphism is observed at the carboxyl terminal and excess charge-altering divergence is detected at the amino terminal. While the mildly deleterious model fits as a net effect in the evolution of nonrecombining mitochondrial genomes, these data suggest that opposing evolutionary pressures may act on different regions of mitochondrial genes and genomes.      

Isolation and characterization of Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor.

Escherichia coli K-12 F- mutants defective in conjugation with an I-type donor (ConI-) were isolated and characterized. These mutants are specific in that they are conjugation proficient with other types of donor strains. They have an altered susceptibility to phages and detergents. Chemical analysis of the cell envelopes of mutant strains has shown that the lipopolysaccharide (LPS) is altered and that one major outer-membrane protein is absent. Conjugation experiments in which LPS from wild-type cells was added to a mating mixture, made up with wild-type donor and recipient cells, showed inhibition in transconjugant formation when an I-type donor, but not an F-type donor, was used. This strongly suggests that LPS of the recipient cell is directly involved in the ability to mate with an I-type donor but not with an F-type donor. The mutations are located in the 78- to 82-min region of the E. coli map, with one exception where the mutation maps near or in the galactose operon.     

meoA is the structural gene for outer membrane protein c of Escherichia coli K12

Summary The isolation and characterization of two mutants of Escherichia coli K12 with an altered outer membrane protein c is described. The first mutant, strain CE1151, was isolated as a bacteriophage Mel resistant strain which contains normal levels of protein c. Mutant cells adsorbed the phage with a strongly decreased rate. Complexes of purified nonheat modified wild type protein c and wild type lipopolysaccharide inactivated phage Me1, indicating that these components are required for receptor activity for phage Me1. When wild type protein c was replaced by protein c of strain CE1151, the receptorcomplex was far less active, showing that protein c of strain CE1151 is altered. The second mutant produces a protein c with a decreased electrophoretic mobility, designated as protein c^*. An altered apparent molecular weight was also observed for one or more fragments obtained after fragmentation of the mutant protein with cyanogen bromide, trypsin and chymotrypsin. Alteration of protein c was not accompanied by a detectable alteration in protein b or its fragments. Both mutations are located at minute 48 of the Escherichia coli K12 linkage map. The results strongly suggest that meoA is the structural gene for protein c.     

The binding of human lipoprotein lipase treated VLDL by the human hepatoma cell line HepG2.

It has been suggested that besides the LDL-receptor, hepatocytes possess an apo E or remnant receptor. To evaluate which hepatic lipoprotein receptor is involved in VLDL remnant catabolism, we studied the binding of VLDL remnants to HepG2 cells. Native VLDL was obtained from type IIb hyperlipidemic patients and treated with bovine milk lipoprotein lipase (LPL). This LPL-treated VLDL (LPL-VLDL) was used as representative for VLDL remnants. Our results show that LPL-VLDL binds with high affinity to HepG2 cells. Competition experiments showed that the binding of 125I-labelled LPL-VLDL is inhibited to about 30% of the control value by the simultaneous addition of an excess of either unlabelled LDL or LPL-VLDL. Preincubation of HepG2 cells with LDL resulted in a reduction of the binding of LDL and LPL-VLDL to 34 and 55% of the control value, whereas preincubation of the cells with heavy HDL (density between 1.16 and 1.21 g/ml) stimulated the binding of LDL and LPL-VLDL to about 230% of the control value. Preincubation of the cells with insulin (250 nM/l) also stimulated the binding of both LDL and LPL-VLDL (175 and 143% of the control value, respectively). We conclude that LPL-VLDL binds to the LDL-receptor of HepG2 cells and that no evidence has been obtained for the presence on HepG2 cells of an additional receptor that is involved in the binding of VLDL remnants.     

Immunological discrimination between the human apolipoprotein E2(Arg158----Cys) and E3 isoforms.

A specific anti-apoE2(Arg158----Cys) monoclonal antibody was raised by means of immunization of mice with a variant specific synthetic peptide. The peptide sequences used were homologous to apolipoprotein E of human and mouse. Consequently, the mouse immune system was tolerant to most of the selected sequences. Immunization with only one of selected peptides (amino acids 154-172) evoked an anti-peptide and anti-native protein response. Surprisingly, this peptide was predicted to have a low antigenicity index, in contrast to the other used peptides. The variant specific anti-peptide MAb that was generated with this sequence, recognizes apoE2(Arg158----Cys) and not apoE3. We here describe a sensitive, time saving, and simple immunoblot assay to detect apoE2(Arg158----Cys) in human sera without prior isoelectric focusing of serum proteins.