Biomedical subjects

L Brissette

Publications and source records attributed to L Brissette.

29 records · Page 2Linked to original sources

PubMed1991

Differences between the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and low density lipoprotein receptor in human hepatoma cells and fibroblasts reside primarily at the translational and post-translational levels.

We have carried out parallel analyses of the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGr) and low density lipoprotein receptor (LDLr) in two highly differentiated human hepatoma cell lines, HepG2 and Hep3B, and primary cultures of human fibroblasts. Analyses of the levels of HMGr and LDLr mRNAs under a variety of culture conditions that perturb intracellular sterol metabolism, or which differ in the levels of extracellular sterols, indicated that the hepatoma cells and fibroblasts responded similarly in terms of the repression or induction ratios of both mRNAs. However, the absolute levels of the mRNAs were severalfold higher in the hepatoma cells. The major difference between the responses of the hepatoma cells and fibroblasts involved the increase in expression of LDLr which occurred upon shifting the cells from complete to lipoprotein-depleted serum. Under these conditions, the 3-fold increase in rate of synthesis of LDLr in the hepatomas was closely matched by increases in the level of its mRNA. In the case of fibroblasts, a 10-fold increase in translational efficiency was required to explain the 30-fold change in rate of synthesis of LDLr. Polysome profiles from both hepatoma cells and fibroblasts suggest that the rate of elongation or termination on LDLr mRNA is relatively low in the presence of reconstituted complete serum, and that it increases in fibroblasts upon lipoprotein depletion, but not in the hepatoma cells. These data indicate that hepatic expression of LDLr may be relatively refractory to induction by decreased circulating levels of lipoprotein when compared with peripheral tissues.

Carcinoma, Hepatocellular↗

PubMed1991

Outgrowth of stable class I major histocompatibility complex-expressing subsets from immunogenic variants of a murine mammary carcinoma: association with a differentially staining region on chromosome 9.

We have examined interactions among intratumor subpopulations during the rejection of immunogenic variants of a murine mammary carcinoma (SPI) and in the outgrowth of tumorigenic "revertant" subsets. Analysis of subclones isolated during the early phase of rejection of one immunogenic variant revealed extensive cellular heterogeneity of tumor-forming ability and class I major histocompatibility complex (MHC) expression. Two main categories of subclones were identified. One set expressed high levels of class I MHC (MHCH) and grew poorly or not at all in normal syngeneic mice. The second set of clones expressed generally low levels of class I MHC (MHCL) and exhibited progressive growth in vivo, similar to the parent tumor. The steady-state mRNA levels for class I MHC and beta 2-microglobulin were constitutively elevated in MHCH clones compared to MHCL clones or the parent tumor. However, in vivo tumorigenic outgrowths from immunogenic variants always expressed the MHCH phenotype. A cytogenetic analysis was carried out to determine the clonal origin and lineage relationship of in vivo selected tumor outgrowths. Surprisingly, tumor outgrowths from mixtures of karyotypically distinct MHCH and MHCL subclones were derived from one lineage within the MHCH subset, despite the fact that MHCH subclones exhibited slower growth in vivo than MHCL subsets when analyzed individually. These results suggest that in polyclonal populations the various subsets sometimes interact in a way that overrides the influence of immunogenic and MHC phenotypes of individual subclones.

Adenocarcinoma↗

PubMed1991

Expression of recombinant human apolipoprotein A-I in Chinese hamster ovary cells and Escherichia coli.

Human recombinant apolipoprotein (apo) A-I was produced by Chinese hamster ovary (CHO) cells and Escherichia coli with expression vectors containing cDNAs encoding preproapoA-I or apoA-I, respectively. The apoA-I from CHO cells was purified from the culture medium by ammonium sulfate precipitation, phenyl-Sepharose chromatography, and affinity purification on anti-apoA-I immunoabsorber. Human apoA-I was produced in E. coli as a fusion protein with glutathione S-transferase. A four amino acid linker, which separated the two proteins, was specifically recognized and cut by Factor Xa. The purification was accomplished by chromatography of E. coli extracts on glutathione-Sepharose and digestion with Factor Xa. The highest production level was found to be 0.5 micrograms/ml of culture medium per 48 h for a clone of stable transformant of CHO cells, whereas E. coli could produce as much as 20 micrograms/ml of bacterial culture. These apoA-I forms were compared in terms of molecular weight, isoelectric point, and expression of several epitopes. Recombinant apoA-I obtained from CHO cells appears intact and its isoelectric point is compatible with that of the mature form and the proform of apoA-I, whereas a part of the apoA-I produced by E. coli does not contain the COOH-terminus. Also, two of six epitopes are expressed to a greater extent in apoA-I obtained from E. coli than in apoA-I obtained from human plasma.

Animals↗

PubMed1991

The disappearance rate of human versus rat intermediate density lipoproteins from rat liver perfusion.

The aim of this work was to compare the disappearance rate of human and rat intermediate density lipoproteins (IDL) using the rat liver perfusion system. Human and rat IDL were produced in vitro by incubating human or rat very low density lipoproteins (VLDL) with either rat post-heparin plasma (method I) or a resolubilized isopropanol precipitate of rat post-heparin plasma (method II). With both methods, the degree of triacylglycerol lipolysis was approximately 55%. The different preparations of IDL were labelled with 125I and added to perfusates of rat livers. The disappearance rates of 125I-labelled IDL were monitored by measuring the radioactivity associated with apolipoprotein (apo) B in the perfusate during a 15-min period. Both human and rat IDL prepared with method I had an increased apoE to apoC ratio as compared with their native counterparts. Furthermore, human IDL had a significantly higher apoE to apoC ratio than rat IDL. However, when IDL were produced in the absence of exchangeable apolipoproteins (method II), no change in the apoE to apoC ratios was observed for the transformation of VLDL to IDL and the ratios were similar for human and rat IDL. Despite these differences, human IDL were always removed at a lower rate than rat IDL. The only striking difference between the two types of IDL made by method II was that the apoB100 to apoB48 ratio was considerably higher in human than in rat IDL. These results suggest that the apoB100 to apoB48 ratio is likely to be responsible for the observed differences in liver uptake between rat and human IDL.

Animals↗

PubMed1989

Differential induction of the serum amyloid A gene family in response to an inflammatory agent and to amyloid-enhancing factor.

The murine serum amyloid A1 (SAA1), SAA2, and SAA3 genes are expressed in various tissues in response to acute inflammation. Prolonged expression may be accompanied by amyloid deposition in liver, spleen, and kidney. Shortly before and during deposition, an amyloid-enhancing factor (AEF) can be extracted from these tissues which accelerates amyloid formation when administered with an inflammatory agent. We have investigated the ability of liver AEF to alter expression of the three SAA genes in liver, spleen, and kidney when administered to normal mice or to mice in which inflammation was created with the injection of silver nitrate. In liver, both AEF and silver nitrate induce SAA1 and SAA2 mRNA accumulation. However, AEF elicits a more rapid response and also acts as a potent inducer of hepatic SAA3 mRNA. Silver nitrate does not induce any SAA mRNA species in kidney, whereas AEF induces all three species. In contrast, AEF induces only SAA3 mRNA in the spleen. We also show that the elevation in hepatic SAA mRNA levels induced by either AEF or silver nitrate is associated with a transient increase in the length of the poly(A) tail.

Animals↗

PubMed1988

Binding of intermediate density lipoproteins rich or poor in high molecular weight apolipoprotein B to rat liver membranes.

Very low density lipoproteins rich or poor in high molecular weight apolipoprotein B (Bh-rich or Bh-poor VLDL, respectively) were prepared from rats fasted for 2 days and animals fasted and then refed for 2 days, respectively. Bh-rich or Bh-poor VLDL remnants (IDL) were also prepared by in vitro lipolysis of the corresponding VLDL preparations, and their apolipoprotein (apo) profile and lipid composition determined. Bh-rich IDL are richer in esterified cholesterol than Bh-poor IDL, but poorer in apoC and triglycerides. The binding of 125I-labeled Bh-rich IDL and 125I-labeled Bh-poor IDL to rat liver membranes was assessed by saturation-curve studies. Both types of IDL bound to high- and low-affinity sites on rat liver membranes. There were no significant differences between the binding of IDL produced from Bh-rich or Bh-poor VLDL to either the high- or low-affinity sites. However, by masking the low-affinity binding sites with saturating amounts of human high density lipoproteins 3 (HDL3), we were able to demonstrate that Bh-rich IDL bound to high-affinity binding sites with five times less affinity than Bh-poor IDL. These results show that saturating the low-affinity binding sites of rat liver membranes reveals differences in the binding abilities of lipoproteins to the high-affinity sites. Also, an analysis of apo and lipid compositions of the two types of IDL reveals that the apoBh contribution is likely to be responsible for differences in affinities of IDL for the high-affinity binding sites of rat liver membranes.

Animals↗

PubMed1986

The effects of liposome-reconstituted apolipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes.

Rat intermediate density lipoproteins (IDL) bind specifically to high and low affinity binding sites on rat liver membranes. In a recent paper (Brissette, L., and Noël, S.-P. (1986) J. Biol. Chem. 261, 6847-6852), we have demonstrated that human low density lipoproteins and high density lipoproteins-3 can totally prevent the specific binding of rat IDL to the low affinity binding sites. The aim of the present studies was to determine the effects of apoA-I, apoC, and apoE, reconstituted into liposomes, on the binding of rat iodinated IDL to rat liver membranes. We found that a 50-, 100-, or 300-fold excess of liposome-reconstituted apoE, apoC, or apoA-I, respectively, abolished the specific binding of IDL to the low affinity binding sites. Only apoE liposomes had an effect on the high affinity component; at a 100-fold excess no specific binding of IDL could be detected. Liposomes by themselves or associated with erythrocyte membrane proteins had virtually no effect on the binding of IDL. Taken together our results suggest that apoE is the only ligand that can compete efficiently for the sites that bind rat IDL with a high affinity. These sites may be the expression of both the remnant and the LDL receptors. The binding to the low affinity component probably represents weak interactions between IDL and "unspecified-lipoprotein binding sites," which can be entirely masked by human low density lipoproteins, high density lipoproteins-3, or liposome-reconstituted apoA-I, apoE, or apoC at appropriate concentrations.

Animals↗

PubMed1986

The effects of human low and high density lipoproteins on the binding of rat intermediate density lipoproteins to rat liver membranes.

Upon incubation with rat liver membranes, radioiodinated rat intermediate density lipoproteins (IDL) interacted with at least two binding sites having a low and a high affinity as demonstrated by the curvilinear Scatchard plots obtained from the specific binding data. The purpose of our work was to identify the nature of these binding sites. Human low density lipoproteins (LDL), contain apolipoprotein B only, and human high density lipoproteins (HDL3), containing neither apolipoprotein B nor E, were both capable of decreasing the specific binding of rat 125I-IDL. The Scatchard analysis clearly revealed that only the low affinity component was affected by the addition of these human lipoproteins. In fact, the low affinity binding component gradually decreased as the amount of human LDL or HDL3 increased in the binding assay. At a 200-fold excess of human LDL or HDL3, the low affinity binding was totally masked, and the Scatchard plot of the specific 125I-IDL binding became linear. Only the high affinity binding component was left, enabling a precise measurement of its binding parameters. In a series of competitive displacement experiments in which the binding assay contained a 200-fold excess of human LDL or HDL3, only unlabeled rat IDL effectively displaced the binding of rat 125I-IDL. We conclude that the low affinity binding of rat IDL to rat liver membranes is due to weak interactions with unspecified lipoprotein binding sites. The camouflage of these sites by human lipoproteins makes possible the study of IDL binding to the high affinity component which likely represents the combined effect of IDL binding to both the remnant and the LDL receptors.

Animals↗

PubMed1984

An in vitro model to study lipolysis of rat hepatic very low density lipoprotein using cardiac lipoprotein lipase.

The purpose of this work was to design an in vitro model for studying the lipolysis of very low density lipoprotein (VLDL) under conditions that approximate those likely to exist in the rat circulation. We first measured the total lipase activity available in the circulation of normal fasted rats. Knowing the VLDL-triacylglycerol concentration and the circulating time of VLDL in rat serum, we calculated that incubating 80 mU of lipoprotein lipase activity per milligram of VLDL triacylglycerols at 37 degrees C, for 50 min, approximated the in vivo conditions of lipolysis in normal fasted rats. Under these conditions, different concentrations of albumin were tested. The degree of lipolysis gradually increased from 25 +/- 10% without albumin to 75 +/- 5% (mean +/- SD, n = 3) with 4% albumin. No further increase occurred above 4% albumin. The rate of triacylglycerol hydrolysis was faster with VLDL having a high triacylglycerol/protein ratio. However after 50-60 min of incubation, the extent of hydrolysis tended to become similar for all VLDL (75-90%). This suggests that the rate of VLDL hydrolysis differs from one particle to another (depending on the size), but the extent of hydrolysis ends up being approximately the same. Furthermore, addition of high density lipoprotein to the incubation medium did not affect the rate nor the extent of triacylglycerol hydrolysis. We conclude that a large proportion of VLDL triacylglycerols can be hydrolyzed in the normal fasted rat if there is no limitation in the capacity of fatty acid removal from the lipolysis site. The physiological significance of these results obtained in vitro is discussed.

Animals↗

PubMed1983

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart.

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.

Animals↗

PubMedDate not supplied

Analysis of the lipoprotein binding site of rat liver membranes.

Intermediate density lipoproteins (IDL) were shown to bind to high- and low-affinity binding sites on rat liver membranes. The low-affinity sites were named lipoprotein binding sites (LBS), since they bind all classes of lipoproteins. This study was undertaken to further characterize the interaction of 125I-labelled IDL with the LBS of rat liver membranes to determine the chemical nature of the LBS. We found that the binding of IDL to the LBS is insensitive to EDTA and sensitive to heparin and that it is present on plasma membranes. Also, membranes were pretreated with various enzymes that have an effect on the membrane constituents, and the activity of the LBS on these treated membranes was determined. Our results reveal that the LBS of rat liver membranes is insensitive to heparinase I, chondroitinase ABC, and phospholipase C, while it is partially sensitive to phospholipase A2 and sensitive to proteases and heat. Rat liver membrane proteins were solubilized with Triton X-100, reconstituted in liposomes, and analyzed for their ability to bind lipoproteins. 125I-labelled IDL were shown to bind to high- and low-affinity sites that are similar, in affinity and specificity, to the ones observed with intact rat liver membranes, indicating that a LBS activity is detectable on these liposomes. We found that the binding capacity of low-affinity sites in liposomes containing either no protein or containing proteins solubilized from Escherichia coli membranes is five times weaker than low-affinity sites in liposomes containing liver membrane proteins. Thus, a protein solubilized from rat liver membranes has LBS activity when reconstituted in liposomes. Taken altogether our results provide new information on the binding of IDL to the LBS and indicate that the LBS activity is in part mediated by a protein. Thus, the LBS appears to be a bona fide receptor.

Animals↗