Mouse L-cell Fibroblasts Research Articles

Glucose regulates fatty acid binding protein interaction with lipids and peroxisome proliferator-activated receptor α

Although the pathophysiology of diabetes is characterized by elevated levels of glucose and long-chain fatty acids (LCFA), nuclear mechanisms linking glucose and LCFA metabolism are poorly understood. As the liver fatty acid binding protein (L-FABP) shuttles LCFA to the nucleus, where L-FABP directly interacts with peroxisome proliferator-activated receptor-α (PPARα), the effect of glucose on these processes was examined. In vitro studies showed that L-FABP strongly bound glucose and glucose-1-phosphate (K(d) = 103 ± 19 nM and K(d) = 20 ± 3 nM, respectively), resulting in altered L-FABP conformation, increased affinity for lipid ligands, and enhanced interaction with PPARα. In living cells, glucose stimulated cellular uptake and nuclear localization of a nonmetabolizable fluorescent fatty acid analog (BODIPY C-16), particularly in the presence of L-FABP. These data suggest for the first time a direct role of glucose in facilitating L-FABP-mediated uptake and distribution of lipidic ligands to the nucleus for regulation of PPARα transcriptional activity.

Sterol carrier protein-2 (SCP-2) involvement in cholesterol hydroperoxide cytotoxicity as revealed by SCP-2 inhibitor effects

Sterol carrier protein-2 (SCP-2) plays an important role in cholesterol trafficking and metabolism in mammalian cells. The purpose of this study was to determine whether SCP-2, under oxidative stress conditions, might also traffic hydroperoxides of cholesterol, thereby disseminating their cytotoxic effects. Two inhibitors, SCPI-1 and SCPI-3, known to block cholesterol binding by an insect SCP-2, were used to investigate this. A mouse fibroblast transfectant clone (SC2F) overexpressing SCP-2 was found to be substantially more sensitive to apoptotic killing induced by liposomal 7α-hydroperoxycholesterol (7α-OOH) than a wild-type control. 7α-OOH uptake by SC2F cells and resulting apoptosis were both inhibited by SCPI-1 or SCPI-3 at a subtoxic concentration. Preceding cell death, reactive oxidant accumulation and loss of mitochondrial membrane potential were also strongly inhibited. Similar SCPI protection against 7α-OOH was observed with two other types of SCP-2-expressing mammalian cells. In striking contrast, neither inhibitor had any effect on H(2)O(2)-induced cell killing. To learn whether 7α-OOH cytotoxicity is due to uptake/transport by SCP-2, we used a fluorescence-based competitive binding assay involving recombinant SCP-2, NBD-cholesterol, and SCPI-1/SCPI-3 or 7α-OOH. The results clearly showed that 7α-OOH binds to SCP-2 in SCPI-inhibitable fashion. Our findings suggest that cellular SCP-2 not only binds and translocates cholesterol but also cholesterol hydroperoxides, thus expanding their redox toxicity and signaling ranges under oxidative stress conditions.

Use of Cholesterol in Sperm Cryopreservation: Present Moment and Perspectives to Future

Sperm cryosurvival rates are not optimal for most species. Therefore, new cryopreservation strategies are needed with the objective of increasing the number of surviving sperm and the quality of those sperm after thawing. Cholesterol plays important roles in many sperm functions, including effects on membrane properties. One of these effects is to stabilize membranes at low temperatures. Thus, species that produce sperm which possess high membrane cholesterol : phospholipid ratios are more resistant to cold shock than sperm with low cholesterol : phospholipid ratios. Therefore, increasing the cholesterol content of sperm membranes may be a strategy that can improve sperm quality after freeze-thawing. In this review, information is presented related to using cyclodextrins pre-loaded with cholesterol for cryopreserving sperm from different species. The topics discussed include both in vitro and in vivo assessments of sperm quality after cryopreservation, as well as how increasing sperm cholesterol content affects other sperm functions.

Three-dimensional structure/function analysis of SCP-2-like2 reveals differences among SCP-2 family members

Mosquito sterol carrier protein-2 (AeSCP-2) and sterol carrier protein-2-like2 (AeSCP-2L2) are members of the SCP-2 protein family with similar expression profiles in the mosquito life cycle. In an effort to understand how lipids can be transported by different SCP-2 proteins, the three-dimensional crystal structure of AeSCP-2L2 was solved at 1.7 A resolution. AeSCP-2L2 forms a dimer and binds three fatty acids, one of which resides in a position within the internal cavity at a right angle to the others. This first report of ligand-bound dimerized protein in the SCP-2 protein family indicates that the family has a much more divergent mode of interaction with ligands than previously reported. The potential function of AeSCP-2L2 was investigated via in vivo incorporation of [(3)H]cholesterol and [3H]palmitic acid. Overexpression of AeSCP-2L2 in mosquito cells leads to an increased uptake of free fatty acid, whereas knockdown of AeSCP-2L2 in adult females decreases the accumulation of free fatty acid in the fat body from a blood meal. In contrast, overexpression or knockdown of AeSCP-2L2 has no effect on cholesterol uptake. Our results suggest that the main function of AeSCP-2L2 is as a general intracellular fatty acid carrier, as opposed to having a dedicated role in cholesterol transport.

Sterol carrier protein-2 expression alters sphingolipid metabolism in transfected mouse L-cell fibroblasts

The influence of sterol carrier protein-2 (SCP-2) on the cellular metabolism of sphingolipids was examined in control mouse L-cells and stably transfected clones expressing the protein SCP-2. Three approaches were used to examine for differences; (1) compositional analysis of endogenous sphingolipid classes, (2) metabolism of NBD-ceramide, and (3) live cell labelling via endocytic uptake of BODIPY-sphingomyelin. SCP-2 over expression significantly altered the endogenous levels of both neutral and acidic sphingolipid classes. Among the neutral sphingolipids, expression of SCP-2 induced a 1.7-fold increase in the level of lactosylceramide (LacCer, p < 0.05) and a similar fold decrease in the level of the higher-order neutral glycosylceramides (p < 0.05). Among the acidic sphingolipids, SCP-2 resulted in a 5.2-fold decrease in the endogenous plasma membrane level of ganglioside GM1 (p < 0.03). Incubation of both control and transfected cell lines with NBD-ceramide resulted in the rapid establishment of a steady-state distribution of NBD-labelled sphingomyelin (NBD-SM) and glucosylceramide (NBD-GlcCer). In the SCP-2 expressing clones the conversion of NBD-Cer to NBD-GlcCer was 30% lower during incubation periods between 5 and 30 min (p < 0.025). Inspection of the cells by fluorescence microscopy after incubation with BODIPY labelled sphingomyelin (BODIPY-SM) revealed similar punctuated patterns with no distinguishable differences between the cell types. These results imply that SCP-2 plays a role in modulating enzymatic steps involved in metabolism of sphingolipid homeostasis.

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Isoform-Specific Differences in the Size of Desmosomal Cadherin/Catenin Complexes

Effects of Antiandrogens on Chromatin Remodeling and Transcription of the Integrated Mouse Mammary Tumor Virus Promoter

Inhibition of the ligand-activated androgen receptor (AR) by antiandrogens plays an important role in the treatment of various hyperandrogenic disorders including prostate cancer. However, the molecular mechanisms of antiandrogen activity in vivo remain unclear. In this study we analyzed the effects of cyproterone acetate (CPA), flutamide (F), and hydroxyflutamide (OHF) on transcriptional activation and chromatin remodeling of the genomically integrated mouse mammary tumor virus (MMTV) promoter. This promoter has provided an excellent model system to study the impact of steroid hormones on transcriptional activation in the context of a defined chromatin structure. The MMTV hormone response element is positioned on a phased nucleosome, which becomes remodeled in response to steroids. We utilized this model system in mouse L-cell fibroblasts that contain a stably integrated MMTV promoter. In these cells, dihydrotestosterone (DHT) induced a large increase of AR protein levels that correlated with transcriptional activation and chromatin remodeling of the MMTV promoter. Coadministration of DHT and CPA or DHT and OHF in these cells inhibited the increase of AR levels, which resulted in a strong blockage of transcriptional activation and chromatin remodeling of the MMTV promoter. In contrast, F had no significant influence on these activities. We conclude that a major portion of the antiandrogenic effects of CPA and OHF in vivo are mediated by the reduction of AR levels.

Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts

Although the sterol carrier protein 2 (SCP-2) gene encodes for two proteins, almost nothing is known of the function and potential processing of the larger transcript corresponding to the 58 kDa sterol carrier protein-2/3-oxo-acyl-CoA thiolase (SCP-x), in intact cells. L-cell fibroblasts transfected with cDNA encoding for the 58 kDa SCP-x protein had a 4.5-fold increase in SCP-x mRNA transcript levels. Western blot analysis showed SCP-x protein expression reached 0.011% of total protein, representing a 4.1-fold increase over basal levels. Surprisingly, the 13.2 kDa SCP-2 protein also increased 2-fold in the transfected cells. This was consistent with part of the 58 kDa SCP-x being proteolytically processed to 13.2 kDa SCP-2 as there was no evidence of an mRNA transcript corresponding to a 13.2/15.2 kDa gene product in the transfected L-cell clones. Confocal immunofluorescence microscopy of transfected L-cells showed that SCP-x/SCP-2 co-localized in highest concentration with catalase in peroxisomes, but significant amounts appeared extra-peroxisomal. Overexpression of SCP-x significantly altered cholesterol uptake and metabolism. Uptake of exogenous [3H]cholesterol and total cholesterol mass were increased 1.9- and 1.4-fold, respectively, in SCP-x expressors. Although cholesterol ester mass was unaltered, incorporation of exogenous [3H]cholesterol and [3H]oleic acid into cholesteryl esters increased 2.3- and 2.5-fold, respectively. These results from intact cells suggest the 13.2 kDa SCP-2 can arise from the larger SCP-2 gene product and indicate a role for the 58 kDa SCP-x protein in cholesterol uptake and intracellular cycling.—Atshaves, B. P., A. D. Petrescu, O. Starodub, J. B. Roths, A. B. Kier, and F. Schroeder. Expression and intracellular processing of the 58 kDa sterol carrier protein-2/3-oxoacyl-CoA thiolase in transfected mouse L-cell fibroblasts. J. Lipid Res. 1999. 40: 610–622.

Dietary modification of high density lipoprotein phospholipid and influence on cellular cholesterol efflux

African green monkeys fed fat-specific diets served as a model to investigate the effect of phospholipid acyl chain modification on high density lipoprotein (HDL)-mediated cellular cholesterol efflux. Diets enriched in saturated, monounsaturated, n–6 polyunsaturated, or n–3 polyunsaturated fats were provided during both low cholesterol and cholesterol-enriched stages; sera and HDL3 samples were obtained at specific points during the treatment period. Analysis of the HDL phospholipid composition revealed significant acyl chain modification, consistent with the respective fat-specific diet. Cholesterol efflux from mouse L-cell fibroblasts to HDL3 isolated from the specific diet groups was measured and revealed no differences in the abilities of the particles to accept cellular cholesterol; determination of the bidirectional flux of cholesterol between the cells and HDL3 species further demonstrated no effect of phospholipid acyl chain modification on this process. The effects of dietary modification of phospholipid acyl chains on cellular cholesterol efflux were directly examined by isolating the HDL phospholipid and combining it with human apolipoprotein A-I to form well-defined reconstituted HDL particles. These complexes did not display any differences with respect to their ability to stimulate cellular cholesterol efflux. Incubations with 5% sera further confirmed that the fat-specific diets do not influence cholesterol efflux. These results suggest that the established influences of specific dietary fats on the progression of atherosclerosis are due to effects on cholesterol metabolism other than the efflux of cellular cholesterol in the first step of reverse cholesterol transport.—Gillotte, K. L., S. Lund-Katz, M. de la Llera-Moya, J. S. Parks, L. L. Rudel, G. H. Rothblat, and M. C. Phillips. Dietary modification of high density lipoprotein phospholipid and influence on cellular cholesterol efflux. J. Lipid Res. 1998. 39: 2065–2075.

Receptors for the chemoattractants C5a and IL-8 are clustered on the surface of human neutrophils.

We have used high-resolution field emission scanning electron microscopy with backscatter electron imaging to detect immunogold-labeled C5a and interleukin-8 (IL-8) receptors on human blood neutrophils. The receptors were labeled with receptor-specific antibodies in combination with secondary antibody conjugated to immunogold. When neutrophils were isolated in a "nonactivated" state, both of these receptor populations were expressed primarily in clusters on nonprojecting domains of the cell membrane. When these cells were double labeled for C5a and IL-8 receptors, intermixing of these receptor species in a common cluster was not found. When neutrophils were isolated in an "activated" state, by mixing the blood with N-formylmethionyl-leucyl-phenylalanine, the cells were seen to be elongated and ruffled at their anterior pole, but the C5a receptors did not disperse or redistribute on the surface of the peptide-activated cells. Analysis of the distribution of human C5a receptors expressed by transfected mouse L-cell fibroblasts showed the C5a receptors to be clustered, but expressed on nonprojecting and projecting domains of the cell surface. These observations provide new information on the topographical expression of leukocyte receptors involved in directing cell migration.

Metallothionein-IIAPromoter Induction Alters Rat Intestinal Fatty Acid Binding Protein Expression, Fatty Acid Uptake, and Lipid Metabolism in Transfected L-Cells

Mouse L-cell fibroblasts, transfected with the cDNA encoding for rat intestinal fatty acid-binding protein (I-FABP) under the control of the human metallothionein-IIApromoter, were tested for their protein inducibility by the heavy metals cadmium (Cd2+) and zinc (Zn2+). I-FABP levels were quantitated by Western immunoblotting. Expression of I-FABP in all transfected cell lines tested was induced several-fold by optimized levels of Cd2+and Zn2+. Induction conditions had no effect on cell growth rates or cell densities for any of the cell lines. Induction of high I-FABP-expressing cells (H141) decreased the initial rate and extent of uptake ofcis-parinaric acid, a nonmetabolizable fatty acid, and of [3H]oleic acid, an esterifiable fatty acid. These effects of induction were specific for I-FABP-expressing cells since they were not observed in control cells or cells expressing a high level of liver (L-) FABP. Induction of H141 cells also significantly altered the esterification and distribution of exogenous [3H]oleic acid, especially among triglycerides and phosphatidylcholine, but less so among other glycerophospholipids, cholesteryl esters, and phosphatidylethanolamine. Induction of H141 cells normalized [3H]oleic acid esterification into cholesteryl esters, phosphatidylcholine, total neutral lipids, and total phospholipids such that they no longer differed from control levels. In contrast, induction did not normalize [3H]oleic acid esterification into triacylglycerols and phosphatidylethanolamine to control levels in H141 cells; both remained significantly increased over control cells. Therefore, promoter induction levels of Cd2+and Zn2+enhanced I-FABP expression in H141 cells, thereby modulating both fatty acid uptake and intracellular esterification into neutral and phospholipids.

Intestinal fatty acid-binding protein expression stimulates fibroblast fatty acid esterification

The effect of intestinal fatty acid binding protein (I-FABP) expression on cell growth and cell lipid content is not known. Therefore, mouse L-cell fibroblasts were transfected with the cDNA encoding for I-FABP. The high expression clones expressed 0.35% of the total cytosolic proteins as I-FABP. Mock transfected L-cells did not differ from control L-cells in any properties tested. Neither the growth rate, maximal cell density, nor [ 3H]oleic acid uptake differed in I-FABP expressing as compared to control cells. In contrast, I-FABP expression increased triacylglycerol and cholesteryl ester mass (nmol/mg protein) by 63% and 25%, respectively. Phospholipid mass was unchanged in I-FABP expressing cells. The initial [ 3H]oleic acid esterification into triacylglycerols and cholesteryl esters was increased 3.9- and 2.5-fold in I-FABP expressing cells. Although, the initial [ 3H]oleic acid esterification into total phospholipids was unchanged, within the phospholipid fraction the initial [ 3H]oleic acid esterification into phosphatidylethanolamine was increased 70% and decreased 50% in phosphatidylcholine in I-FABP expressing cells. These observed differences suggest a distinct role for I-FABP in stimulating net formation, and not just turnover, of triacylglycerides and cholesteryl esters in transfected L-cell fibroblasts.

Apolipoprotein A-I Structural Modification and the Functionality of Reconstituted High Density Lipoprotein Particles in Cellular Cholesterol Efflux

The role of HDL and its major protein constituent, apolipoprotein (apo) A-I, in promoting the removal of excess cholesterol from cultured cells has been well established; however, the mechanisms by which this occurs are not completely understood. To address the effects of apoA-I modification on cellular unesterified (free) cholesterol (FC) efflux, three recombinant human apoA-I deletion mutants and plasma apoA-I were combined with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) and FC to make reconstituted high density lipoprotein (rHDL) discoidal complexes. These particles were characterized structurally and for their efficiency as acceptors of mouse L-cell fibroblast cholesterol. The deletion mutant proteins lacked NH2-terminal (apoA-I (Delta44-126)), central (apoA-I (Delta139-170)), or COOH-terminal (apoA-I (Delta190-243)) domains of apoA-I. The three deletion mutants all displayed lipid-binding abilities and formed discoidal complexes that were similar in major diameter (13.2 +/- 1.5 nm) to those formed by human apoA-I when reconstituted at a 100:5:1 (POPC:FC:protein) mole ratio. Gel filtration profiles indicated unreacted protein in the preparation made with apoA-I (Delta190-243), which is consistent with the COOH terminus portion of apoA-I being an important determinant of lipid binding. Measurements of the percent alpha-helix content of the proteins, as well as the number of protein molecules per rHDL particle, gave an indication of the arrangement of the deletion mutant proteins in the discoidal complexes. The rHDL particles containing the deletion mutants had more molecules of protein present than particles containing intact apoA-I, to the extent that a similar number of helical segments was incorporated into each of the discoidal species. Comparison of the experimentally determined number of helical segments with an estimate of the available space indicated that the deletion mutant proteins are probably more loosely arranged than apoA-I around the edge of the rHDL. The abilities of the complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts. All four discoidal complexes displayed similar abilities to remove FC from the plasma membrane of L-cells when compared at an acceptor concentration of 50 microg of phospholipid/ml. Thus, none of the deletions imposed in this study notably altered the ability of the rHDL particles to participate in cellular FC efflux. These results suggest that efficient apoA-I-mediated FC efflux requires the presence of amphipathic alpha-helical segments but is not dependent on specific helical segments.

Sterol carrier protein-2 expression in mouse L-cell fibroblasts alters cholesterol uptake

Despite the progress made on the possible functions of sterol carrier protein (SCP-2) using assays in vitro, very little is known regarding the role of SCP-2 in intact cells. To further elucidate this role, mouse L-cell fibroblasts were transfected with the cDNA encoding for mouse 15 kDa or 13.2 kDa SCP-2. The data show for the first time, that SCP-2 expression increases cholesterol uptake into transfected L-cell fibroblasts. Untransfected L-cells expressed SCP-2 at levels near or below the lower limit of detectability. SCP-2 immunoreactive protein levels were 0.030 ± 0.004% and 0.036 ± 0.002% of total cytosolic proteins in the 15 and 13.2 kDa stable transfectants, respectively. Both the 15 and 13.2 kDa SCP-2 expressions products were found as 13.2 kDa proteins, consistent with rapid post-translational cleavage of the putative amino terminal mitochondrial targeting sequence from the 15 kDa SCP-2. The effect of expressing either form of SCP-2 on [ 3H]cholesterol uptake was determined. Expression of the 15 kDa form, but not the 13.2 kDa form of SCP-2, enhanced the rate and extent of [ 3H]cholesterol uptake compared to control or mock-transfected L-cells. The [ 3H]cholesterol uptake rate in 15 kDa SCP-2 expressing cells was increased 1.3-fold, while the extent of [ 3H]cholesterol uptake was increased 1.4-fold after 12 h of uptake compared to control L-cells. The differences in cholesterol uptake between the cells expressing the 13.2 versus the 15 kDa protein, suggest that the 15 kDa form of SCP-2 is functionally localized within the cell, while the 13.2 kDa product is not.

Intestinal and liver fatty acid binding proteins differentially affect fatty acid uptake and esterification in L-cells.

Differential effects of intestinal (I-FABP) or liver (L-FABP) fatty acid binding proteins on fatty acid uptake and esterification were examined using transfected mouse L-cell fibroblasts. L-FABP, but not I-FABP, expression increased the initial rate and extent of cis-parinaric acid uptake by 50 and 29%, respectively, compared to control cells. I-FABP and L-FABP expression preferentially increased [3H]-oleic acid incorporation into triacylglycerols by 5.5-fold and 3.8-fold, respectively. While both L-FABP and I-FABP increased esterification of [3H]-oleic acid into ethanolamine glycerophospholipids, these proteins had opposite effect on esterification into choline glycerophospholipids. These data show for the first time that distinct FABP differentially affect both fatty acid uptake and intracellular esterification.

Inhibition of cellular cholesterol efflux by 25-hydroxycholesterol.

The effect of oxysterols on efflux of cholesterol from mouse L-cell fibroblasts, rat Fu5AH hepatoma cells, J774 macrophages, and human EA.hy 926 endothelial cells was studied. Cells were preincubated with 25-hydroxycholesterol (25-OHC) either during labeling of the cells with [3H]cholesterol or during equilibration after labeling. Subsequently, the release of [3H]cholesterol into medium containing 0.2 mg HDL3/ml was measured and the fractional release of cellular [3H]cholesterol was calculated. Pretreatment with 25-OHC (1 microgram/ml) caused a 30% reduction in [3H]cholesterol efflux from L-cells during 8 h of incubation with HDL3. 25-OHC also inhibited cholesterol efflux from Fu5AH and J774 cells, but the effect was less marked. There was only a small, nonsignificant reduction of efflux from EA.hy 926 cells. The mechanisms of 25-OHC-induced inhibition of cellular cholesterol efflux was further studied in L-cells, because of their sensitivity to 25-OHC treatment. The effect of 25-OHC on cholesterol efflux was dose-dependent, with significant effects seen at 25-OHC concentrations as low as 50 ng/ml. The half-time for cholesterol efflux from 25-OHC-treated cells (5 micrograms/ml) was 13.0 +/- 3.3 h compared to 5.7 +/- 1.0 in control cells, corresponding to a 55% reduction in the rate of cholesterol release. Other oxysterols, including 7-ketocholesterol, 7 alpha- and 7 beta-hydroxycholesterol, and 22(S)-hydroxycholesterol also inhibited [3H]cholesterol efflux from L-cells significantly, but to a lesser degree. 25-Hydroxycholesterol (5 micrograms/ml) reduced efflux from both normal and cholesterol-enriched cells by 31 and 14%, respectively. Inhibition of efflux was similar when reconstituted HDL3-apolipoprotein/phosphatidylcholine particles or small unilamellar phosphatidylcholine vesicles were used as cholesterol acceptors instead of HDL3. The content of phospholipids, cholesterol and the FC/PL ratio of intact cells and from isolated plasma membrane vesicles were the same for control and 25-OHC-treated cells. Efflux of [3H]cholesterol from plasma membranes isolated from 25-OHC-treated cells was 20% less than efflux from membranes from control cells. The difference in efflux observed in intact cells is partially explained by the reduction in efflux from the plasma membrane. In conclusion, our studies suggest that oxysterols, especially 25-hydroxycholesterol, can reduce cellular cholesterol efflux in vitro. Therefore oxysterols, either endogenous or derived from the diet, may influence cellular cholesterol efflux in vivo, the first step in reverse cholesterol transport.

The influence of apolipoprotein structure on the efflux of cellular free cholesterol to high density lipoprotein.

The influence of apolipoprotein conformation on the ability of high density lipoprotein (HDL) to remove cellular free cholesterol (FC) has not been studied in detail. To address the effects of amphipathic alpha-helix structure on cellular FC efflux, three class A helical peptides and apolipoprotein (apo) AI were complexed to dimyristoyl phosphatidylcholine (DMPC) to make discoidal complexes that were used as acceptors of cell cholesterol. The peptides consisted of an 18-amino acid, amphipathic, alpha-helical peptide with the sequence DWLKAFYDKVAEKLKEAF (18A), a dimer of 18A covalently linked by a proline residue (37pA), and acetyl-18A-amide (Ac-18A-NH2) that has a higher alpha-helix content than the unblocked 18A molecule. The three peptides strongly mimic the lipid-binding characteristics of the amphipathic segments of apolipoproteins and form discoidal complexes with DMPC that are similar in diameter (11-12 nm) to those formed by human apoAI when reconstituted at a 2.5:1 (w:w) phospholipid to protein ratio. The abilities of these complexes to remove radiolabeled FC were compared in experiments using cultured mouse L-cell fibroblasts; efflux of FC from both the plasma membrane and the lysosomal pools was examined. For each of the acceptors, the removal of cholesterol from the plasma membrane and lysosomal pools was equally efficient. All four discoidal complexes were equally efficient cell membrane FC acceptors when compared at saturating acceptor concentrations of > 200 micrograms of DMPC/ml of medium. However, at the same lipid concentration, protein-free DMPC small unilamellar vesicles (SUV) were significantly less efficient. The initial rates of FC removal from cells at saturating concentrations of acceptor particles (Vmax) were 12, 10, 10, and 11% per h, respectively, for the complexes containing either 18A, Ac-18A-NH2, 37pA, or apoAI, but only 1% cellular FC per h for the DMPC SUV. The 10-fold higher Vmax for the apoprotein/peptide-containing acceptors was likely due to a reversible interaction of apoprotein or peptide with the plasma membrane that changed the lipid packing characteristics in such a way as to increase the rate of FC desorption from the cell surface. This interaction required amphipathic alpha-helical segments, but it was not affected by the length, number, or lipid-binding affinity of the helices. Furthermore, the efflux efficiency was not dependent on the amino acid sequence of the helical segments which suggests that this interaction is not mediated by a specific cell surface binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

The murine myeloperoxidase promoter contains several functional elements, one of which binds a cell type-restricted transcription factor, myeloid nuclear factor 1 (MyNF1).

The myeloperoxidase (MPO) gene is expressed specifically in myeloid cells. There is significant homology between the murine and human MPO genes in the 1.6-kb region located upstream of the murine MPO transcription initiation sites. 5',3', and internal deletions of this DNA segment localized several cis-acting DNA elements in the murine MPO promoter which are functional in 32D cl3 cells, a murine myeloblast cell line which expresses MPO. These DNA elements did not function well in mouse L-cell fibroblasts. Additional mutagenesis of the most active promoter region allowed the delimitation of a functional 20-bp segment. Mutation of the enhancer core motif within this segment was functionally deleterious, and an oligonucleotide containing these base pairs increased the activity of a minimal promoter. This same oligonucleotide, but not a mutant variant, could bind a set of nuclear proteins, myeloid nuclear factors 1 alpha and 1 beta (MyNF1 alpha and -1 beta), present in 32D cl3 cells but absent from L cells, murine erythroleukemia cells, and SP2 lymphoid cells.

Mechanisms controlling competence gene expression in murine fibroblasts stimulated with minimally modified LDL.

Mildly oxidized low density lipoprotein (minimally modified low density lipoprotein [MM-LDL] is capable of inducing gene expression in cells of the artery wall. In this study, we investigated the mechanisms that control the mRNA expression of JE, KC, c-myc, and c-fos in quiescent mouse L-cell fibroblasts stimulated with MM-LDL. The data demonstrate that MM-LDL induces increases greater than or equal to 20-fold in the levels of transcripts of these genes within 15-60 minutes. Of the four genes examined, JE and KC mRNA showed the greatest response to MM-LDL. The pattern of induction by MM-LDL is distinct from that observed in fibroblasts stimulated with serum, a known inducer of these genes. Treatment with cycloheximide (10 micrograms/ml) did not block the MM-LDL-induced increase in the mRNA levels of these genes. The increase of JE and KC mRNA levels in response to MM-LDL could be blocked by treatment with actinomycin D (5 micrograms/ml). In nuclear runoff studies, MM-LDL increased the transcription rate of JE and KC at 4 hours by 13-fold and fivefold, respectively. Small but reproducible stimulations of c-fos and c-myc transcription by MM-LDL were also observed. In addition, the half-life of JE mRNA was increased after addition of MM-LDL to fibroblasts, suggesting that the MM-LDL-induced accumulation of these mRNAs might be accomplished by both transcriptional and posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

The B-cell surface protein CD72/Lyb-2 is the ligand for CDS

The glycoprotein CD5 is expressed on the surface membrane of all mature T cells and a small proportion of B lymphocytes. Its exact role in immune interactions is still unknown. Studies indicate that CD5 functions both in mice and humans as a receptor, delivering co-stimulatory signals to T cells in a manner similar to CD2 (ref. 11) and CD28 (ref. 12). Anti-CD5 antibodies stimulate both T-cell proliferation mediated by CD3 in association with the T-cell receptor and secretion of interleukin-2 and expression of its receptor, as well as inducing an increase in intracellular Ca2+ concentration (refs 5-10). To identify the ligand for CD5 we purified the human CD5 protein, labelled it with biotin and used it as a probe. Here we report that CD5 specifically interacts with the cell-surface protein CD72 exclusive to B cells. This interaction is blocked by anti-CD72 antibodies, but not by any other anti-B-cell antibodies. Moreover, non-B cells (mouse L-cell fibroblasts and human Jurkat T cells) expressing a transfected human CD72 complementary DNA could bind to the CD5-biotin conjugate. The results demonstrate that the B-cell surface protein CD72 (Lyb-2 in mice) is the ligand for CD5.