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J Chambaz

Publications and source records attributed to J Chambaz.

At least 37 records · Page 2Linked to original sources
PubMed1997

Repression of apoC-III gene expression by TNFalpha involves C/EBPdelta/NF-IL6beta via an IL-1 independent pathway.

Apolipoproteins A-II and C-III, which participate in the control of cholesterolemia and triglyceridemia, are negative acute phase proteins. Treatment of HepG2 cells with TNFalpha showed that apoA-II and apoC-III mRNA levels were decreased. Using transient transfection, we found that apoC-III gene expression is controlled at the transcriptional level. By competition and supershift experiments, we demonstrate that TNFalpha-induced complexes were related to C/EBPdelta/NF-IL6beta and p50 and that overexpression of C/EBPdelta was able to reproduce the inhibitory effect of TNFalpha on the apoC-III promoter. RT-PCR failed to detect the IL-1 transcript in TNFalpha-treated HepG2 cells, suggesting that activation of C/EBPdelta by TNFalpha is not related to the IL-1-signalling pathway.

Apolipoprotein A-II↗
PubMed1997

Activation of CAAT enhancer-binding protein delta (C/EBPdelta) by interleukin-1 negatively influences apolipoprotein C-III expression.

Tissue-specific transcription of the apolipoprotein C-III (apoC-III) gene is mainly regulated by synergistic interactions between the liver-enriched transcription factor HNF-4, which binds to the proximal promoter, and ubiquitous factors, which bind to the upstream enhancer region. Here we show that apoC-III expression in HepG2 cells is negatively regulated in response to interleukin-1 (IL-1), and this inhibition is mainly due to transcriptional repression. CAAT enhancer-binding protein delta (C/EBPdelta) was found to be the main mediator of IL-1-induced suppression. Analysis of the apoC-III promoter revealed two IL-1 response elements. The first is located in the proximal promoter region D and the second in the distal enhancer region I. Proximal element D is a high affinity binding site for C/EBPdelta, while the enhancer element I is not directly recognized by this transcription factor. Functional analysis of different combinations of homologous and heterologous promoter constructs revealed that indirect interaction of C/EBPdelta with site I, in the context of the full promoter, leads to repression. C/EBPdelta is activated by phosphorylation during IL-1-induced signal transduction pathway. This modification is important for both DNA binding activity and indirect transrepression of the apoC-III promoter.

Animals↗
PubMed1996

Lipid-binding properties of synthetic peptide fragments of human apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.

Amino Acid Sequence↗
PubMed1996

The proximal element of the human apolipoprotein A-II promoter increases the enhancer activity of the distal region.

We have previously shown that human apolipoprotein A-II (apoA-II) transcription is controlled by a complex set of regulatory elements. In this study, we demonstrate that the distal region of the apoA-II promoter (-911/-614) acts as an enhancer and results in a 6-fold increase in activity when the proximal AB element is inserted between this enhancer and a TATA box. The AB element alone does not display any transcriptional activity. The combination of the proximal AB element and the enhancer is sufficient to activate transcription to the same level as that achieved with the full-length promoter. DNA binding and competition assays, and binding interference experiments allowed us to identify two adjacent binding sites within the AB element. These bind activities designated CIIIB1 and AIIAB3/4, respectively. Mutation on each of these sites showed that the binding site of CIIIB1 within the AB element played a major role in the interaction with the enhancer. Whereas transcriptional activation of other apolipoprotein genes involves the binding of the liver-enriched hepatocyte nuclear factor 4 (HNF4) on their proximal promoter, the present study demonstrates that neither HNF4 nor ApoA-I regulatory protein 1, its antagonistic orphan receptor, was able to bind the AB element. Instead, apoA-II transcription was driven by the interaction of apoA-II enhancer with proximal AB element, which involves an unidentified activity, CIIIB1.

Animals↗
PubMed1994

Purification and characterization of recombinant human apolipoprotein A-II expressed in Escherichia coli.

We have expressed recombinant human apolipoprotein A-II (apoA-II) in Escherichia coli, as a fusion protein with Schistosoma japonicum glutathione-S-transferase (GST). The GST-AII fusion protein was recovered by affinity chromatography using glutathione as a ligand. After thrombin cleavage and removal of the GST carrier, recombinant apoA-II was obtained in a highly purified form and was exclusively composed of dimeric apoA-II. Kinetics of association to dimyristoylglycerophosphocholine (Myr2GroPCho) vesicles showed that recombinant apoA-II exhibited the same pattern of association as human plasma apoA-II. Electron microscopic analysis of the complexes showed a typical pattern of rouleaux, characteristic of stacked discs, with a diameter similar to that determined by gradient-gel electrophoresis. Circular dichroism measurements showed that the alpha-helical content of both plasma and recombinant apoA-II increased similarly when the proteins associated with Myr2GroPCho vesicles, at the expense of a random-coil structure. Lipid-bound apoA-II consisted of 70-72% alpha helices, suggesting the presence of three 18-residue alpha helices/apoA-II monomer. Cross-linking experiments indicated that Myr2GroPCho complexes contained two molecules dimeric apoA-II/vesicle. Recombinant apoA-II was as efficient as plasma apoA-II in associating with HDL subclasses, and in displacing apoA-I from dipalmitoylglycerophosphocholine/cholesterol/apoA-I complexes, most likely due to its highly ordered secondary structure when associated with Myr2GroPCho vesicles. These findings demonstrate that recombinant apoA-II exhibits the same structural and functional properties as human plasma apoA-II. Thus, the expression system utilized is appropriate to produce mutagenized forms to further structure/function analysis.

Amino Acid Sequence↗
PubMed1994

Purification and characterization of nuclear factors binding to the negative regulatory element D of human apolipoprotein A-II promoter: a negative regulatory effect is reversed by GABP, an Ets-related protein.

We have previously shown that transcription of the human apolipoprotein A-II (apoA-II) gene is controlled by a complex set of regulatory elements [Cardot et al. (1993) Biochemistry 32, 9080-9093]. We have also identified previously described, as well as new activities which bind to these elements and influence to varying degrees the transcription of the apoA-II gene. DNA binding and competition assays indicated that element D binds three new activities, designated AIID1, AIID2, and AIID4, as well as an activity related to C/EBP. Activities AIID1, AIID2, and AIID4 were purified and characterized further in order to determine their function on the transcriptional regulation of human apoA-II gene. SDS-PAGE analysis as well as photoaffinity cross-linking of the affinity-purified AIID2 showed that it consists of three proteins with molecular masses ranging between 54 and 63 kDa. The amino acid sequence of tryptic peptides obtained from AIID2 protein bands revealed that it is homologous to GABP, an Ets-related protein. Similar analysis showed that affinity-purified AIID4 has an apparent molecular mass of 130 kDa. AIID1 activity was purified partially; in addition to binding to the apoA-II promoter, AIID1 also binds to the regulatory element C of apoCIII and may play a role in the transcriptional regulation of both genes. Methylation interference of G residues and permanganate modification of T residues indicated that the binding sites of AIID2 and AIID4 were contiguous on element D. However, the binding site of AIID1 overlaps with the binding sites of both AIID2 and AIID4. This suggests that the binding of AIID1 and AIID2 or of AIID1 and AIID4 may be mutually exclusive, whereas AIID2 and AIID4 may bind simultaneously. Transcription from a minimal promoter containing elements AB, C, and D of apoA-II increased 1.5- to 1.6-fold when element D is deleted, as well as by promoter mutations which eliminated the binding of both AIID1 and/or AIID4 to element D, but permitted the binding of AIID2/GABP. The findings suggest that element D has a negative regulatory role on apoA-II gene transcription when it is occupied by protein AIID1 and/or AIID4. This negative effect is reversed when element D is occupied only by the regulatory factor AIID2/GABP.

Amino Acid Sequence↗
PubMed1994

Intracellular early and late modifications of human apolipoprotein A-II. Effect of glutamine- +1 to leucine substitution.

It has been shown previously that apoA-II undergoes several intracellular modifications in HepG2 cells (Hussain & Zannis, 1990). In the present study, we have generated permanent cell lines in mouse C127 cells which express the normal apoA-II gene and a mutated form in which Gln+1 was substituted with Leu (Leu+1). This modification was designed to prevent cyclization of the N-terminal glutamine of apoA-II and thus identify the isoproteins which are precursors and products of the N-terminal cyclization reaction. The C127-expression cells were also utilized to study the cellular compartments where the apoA-II modifications occur as well as the importance of the modifications for apoA-II trafficking and secretion. We have found that apoA-II (Gln+1) synthesized by C127 and HepG2 cells had similar isoproteins. In both cell types, unmodified pro-apoA-II, designated isoprotein 3, had a similar isoelectric point as the cell-free translation product of apoA-II mRNA, suggesting that isoprotein 3 results from cleavage of the signal peptide. Isoprotein 3 represents an unmodified apoA-II isoprotein and undergoes an early modification into a more acidic isoprotein 1, which differs from isoprotein 3 by two negative charges. Brefeldin A treatment of the cells did not prevent the formation of isoprotein 1, suggesting that this modification occurs in a pre-Golgi compartment. Neuraminidase treatment of secreted apoA-II isoproteins did not affect isoprotein 1, indicating that it is not sialylated isoprotein. Isoprotein 1 undergoes further modifications which are consistent with cleavage of the propeptide, N-terminal cyclization and sialylation most likely resulting from O-glycosylation.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-II↗
PubMed1993

Factors participating in the liver-specific expression of the human apolipoprotein A-II gene and their significance for transcription.

We have shown previously that the hepatic and intestinal transcription of the human apolipoprotein A-II (apoA-II) gene in cell cultures is controlled by a complex set of regulatory elements A-N [Chambaz, et al. (1991) J. Biol. Chem. 266, 11676-11685; Cardot, et al. (1991) J. Biol. Chem. 266, 24460-24470]. In the present communication, we have assessed the functional importance of each of the regulatory elements. In addition, we have used DNA binding and competition assays and protein fractionation to identify the hepatic nuclear activities which are involved in the regulation of the human apoA-II gene. Such activities may be of general importance for the regulation of liver-specific genes. The DNA binding and competition analysis showed that the regulatory elements M, D, and F bind new activities which have not been identified in apolipoprotein or other liver-specific promoters. These activities have been designated AIIM1 and AIIM2 for element M, AIID1 and AIID2 for element D, and AIIF2 for element F. The activity AIIM2 is present in liver, but absent in CaCo-2 cells. A set of regulatory elements binds activities which resemble liver-enriched or ubiquitous factors previously shown to play important roles in the regulation of their target genes. Thus, element I binds to activities related to NF1, and elements L, C, D, G, AB, and F bind to C/EBP alpha as well as other heat-stable activities. The affinity of the bacterially expressed C/EBP alpha for the various apoA-II regulatory regions follows the order: AIIL approximately AIIC > AIID > AIIF > AIIG > AIIAB. Protein fractionation showed that element J binds at least three hepatic nuclear activities and is also recognized by members of the nuclear receptor family, HNF4, EAR2, EAR3, and ARP1. Another liver-enriched factor, HNF1, was shown previously to bind to element H. Despite the importance of HNF1, HNF4, NF1, and C/EBP alpha in the regulation of numerous other target genes, deletion of the HNF1, NF1, and HNF4 and several C/EBP binding sites did not drastically affect the hepatic transcription of the apoA-II gene. Rather, the hepatic and intestinal transcription is affected severely by deletion of elements A, B, K, L, and N. In addition, the intestinal transcription is affected by deletion of elements C, J, and M. The in vivo physiological importance of these elements will require analysis of their function in transgenic animals. This analysis establishes the organization of several nuclear activities on the human apoA-II promoter.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Lipid synthesis and apolipoprotein gene expression in hepatocytes in primary culture from (puromycin-induced) nephrotic rats.

Primary culture of hepatocytes from puromycin aminonucleoside-induced nephrotic rats were used to discriminate between the hepatic and extra-hepatic contribution to the hyperlipidemia occurring in the nephrotic syndrome. De novo lipogenesis and utilization of exogenous fatty acids were not modified in nephrotic hepatocytes as compared to controls. In contrast 2.2 and 5.3-fold more triacylglycerol and phospholipids were secreted respectively by nephrotic hepatocytes than by controls. Triacylglycerol overproduction was not associated with an increase either in apo B mRNA level or in apo B synthesis or secretion measured by [35S]-methionine incorporation and immunoprecipitation. We also observed a significant increase in apo AI and apo E synthesis and secretion by nephrotic hepatocytes. This increase was correlated with a greater amount of apo AI and apo E mRNA than in controls. The overproduction of apo AI and apo E by nephrotic hepatocytes might intervene in the clearance of plasma lipoproteins and the redistribution of plasma cholesterol.

Albumins↗
PubMed1992

[Nutritional regulation of apolipoprotein genes. Effect of dietary carbohydrates and fatty acids].

The effect of nutritional factors on apolipoprotein gene expression by rat liver were studied. Dietary carbohydrates or fatty acids regulate the expression of apo E gene, by altering either gene transcription or mRNA stability. Conversely, apo A1 regulation occurs at a post transcriptional level. In vivo and in vitro experiments gave contradictory results concerning apo B gene expression. The more dramatic changes in plasma lipids and apolipoproteins are obtained under dietary fish oil. Hepatocytes from fish oil-fed rats retain for several days modification in fatty acid metabolism, i.e. a shift in oleic acid channeling towards oxidation at the expense of esterification and a reduced ability to synthesize and secrete triacylglycerol. These modifications are paralleled with a decrease in the synthesis and in the secretion of apo Bs. Hepatocytes from fish oil fed rats secrete degradative forms of apo B which might result from either a sluggish VLDL synthesis and secretion or a more specific effect of n-3 long chain polyunsaturated fatty acid peroxidative products. Hepatocytes from fish oil fed rats exhibit a reduced ability to synthesize cholesterol, associated with a decrease in apo A1 synthesis and secretion without any modification in apo A1 mRNA. In contrast, the hepatocytes exhibit a concomitent decrease in apo E synthesis and secretion and in cellular apo E mRNA levels.

Animal Nutritional Physiological Phenomena↗
PubMed1991

Regulation of the human ApoA-II gene by the synergistic action of factors binding to the proximal and distal regulatory elements.

We have recently shown that the human apoA-II promoter contains a set of 11 distal regulatory elements between nucleotides -903 and -255 and three proximal regulatory elements between nucleotides -126 and -33 that are essential for hepatic and intestinal transcription of the apoA-II gene (Chambaz, J., Cardot, P., Pastier, D., Zannis, V. I., and Cladaras, C. (1991) J. Biol. Chem. 266, 11676-11685). Deletion or nucleotide substitution analysis has shown that alterations in elements L (nucleotides -803 to -773) and K (nucleotides -760 to -743) reduced hepatic transcription to 25 and 20% and intestinal transcription to 8 and 4% of control, respectively, as measured by chloramphenicol acetyltransferase assays, indicating that these elements play an important regulatory role. Nucleotide substitutions in element AB (nucleotides -65 to -33) reduced hepatic and intestinal transcription to 60 and 36% of control, respectively. The factors that recognize regulatory regions L, K, and AB were analyzed by DNA binding gel electrophoretic and competition assays. This analysis has shown that elements AB, K, and L bind with different affinities to a newly characterized heat-stable factor, CIIIB1, which is a transcriptional activator of the human apoC-III gene (Ogami, K., Kardassis, D., Cladaras, C., and Zannis, V. I. (1991) J. Biol. Chem. 266, 9640-9646). In addition, elements AB and K bind a heat-labile activity, designated AIIAB1, and element L binds to several CCAAT box binding activities. Mutations in domain L that prevented the binding of CCAAT box binding activities reduced both hepatic and intestinal transcription to 30% of control, indicating the importance of these factors in transcription. Simultaneous nucleotide substitutions that prevented the binding of CIIIB1 activity in elements AB, K, and L reduced hepatic and intestinal transcription to 7 and 6% of control, respectively, suggesting that the synergistic interaction of CIIIB1 (bound to the proximal and distal regulatory elements) with CCAAT box proteins (bound to element L) can modulate the level of transcription of the human apoA-II gene.

Animals↗
PubMed1991

Effect of simvastatin on the synthesis and secretion of lipoproteins in relation to the metabolism of cholesterol in cultured hepatocytes.

In primary culture of rat hepatocytes, simvastatin, a powerful HMGCoA reductase inhibitor, inhibited acetate incorporation into cellular and secreted cholesterol and cholesteryl-esters, without any significant effect on triacylglycerol synthesis and secretion. When applied to the culture for 24 h at 10(-7) M, a concentration shown to inhibit cholesterol synthesis by 61%, simvastatin increased apolipoprotein BH and BL synthesis and secretion and strongly decreased apolipoprotein AI synthesis and secretion whereas apolipoprotein AIV remained unaffected. The synthesis and secretion of apolipoprotein E was only slightly affected in contrast with other situations where cholesterol synthesis decreased. All of these modifications occurred at a post-transcriptional level, as the corresponding messenger RNAs of the apolipoproteins did not vary. These results suggest that either the drug itself or variations in cholesterol synthesis might be involved in apo B and apo AI synthesis and secretion.

Animals↗
PubMed1991

Promoter elements and factors required for hepatic transcription of the human ApoA-II gene.

The apolipoprotein A-II (apoA-II) gene regulatory region -911 to +29 strongly promotes the transcription of the promotorless chloramphenicol acetyltransferase (CAT) gene in cells of hepatic (HepG2) and intestinal (CaCo2) origin but not in HeLa cells. Deletion of nucleotides -911 to -860 decreased the hepatic and intestinal transcription to 7% and 18% of control, respectively. Further progressive deletions extending to nucleotides -614, -440, -230, and -80 abolished both hepatic and intestinal transcription, indicating that the distal promoter region -911 to -614 contains regulatory elements that are essential for intestinal and hepatic transcription. An internal deletion of the -614 to -230 region decreased hepatic transcription 60% while it increased intestinal transcription 140% of control indicating that the elements which control hepatic and intestinal transcription of the apoA-II gene may be different within this regulatory region. DNase I footprinting analysis with rat liver nuclear extracts identified 14 protected regions: A, -40 to -33; B, -65 to -42; C, -126 to -110; D, -276 to -255; E, -377 to -364; F, -404 to -384; G, -468 to -455; H, -573 to -554; I, -706 to -680; J, -734 to -716; K, -760 to -743; L, -803 to -773; M, -853 to -829, and N, -903 to -879, as the DNA binding sites for nuclear factors. Five of the regions (B, C, G, H, and K) bind to heat-stable factors. DNA binding gel electrophoretic assays indicated that region N (-903 to -879), which is essential for efficient transcription, binds predominantly a nuclear activity designated AIIN3. This activity is present in cells of hepatic and intestinal origin but absent in HeLa cells. Similar analysis showed that region H (-573 to -554) binds to the liver-specific factor HNF1/LFB1. Deletion of this region decreased hepatic and intestinal transcription 80 and 64% of control, respectively, suggesting that HNF1/LFB1 or a related activity contributes to optimal transcription but is not essential for the tissue-specific expression of the human apoA-II gene.

Apolipoprotein A-II↗
PubMed1991

Effect of dietary fish oil and corn oil on lipid metabolism and apolipoprotein gene expression by rat liver.

A 3-week fish oil diet induced in weanling rats a decrease in plasma lipids and liver triacylglycerol, and an increase in insulinemia, compared to a corn oil diet. At the same time, plasma apolipoprotein (apo) A-I was slightly lower and plasma heavy apo B/light apo B ratio was higher in fish-oil-fed than in corn-oil-fed rats. Hepatocytes obtained from fish-oil-fed and corn-oil-fed rats were used to examine how fish oil affects lipid and apolipoprotein synthesis and secretion. Primary culture of hepatocytes from fish-oil-fed rats displayed a lower ability to synthesize and secrete triacylglycerol than hepatocytes from corn-fed rats, as measured by mass determination or [U-14C]glycerol incorporation. Hepatocytes from fish-oil-fed rats exhibited a lower synthesis of cholesterol, measured by [14C]acetate incorporation, than hepatocytes from corn-oil-fed rats. This impairment was associated with an increase in beta-oxidation, a higher channeling of oleic acid into phospholipids, and a lower triacylglycerol/diacylglycerol ratio in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats. Incorporation of [35S]methionine into secreted apoB was reduced in hepatocytes from fish-oil-fed rats, but was not paralleled by a decrease in apo B mRNA. The appearance of degradative forms of apo B suggest an increase in apo B degradation in hepatocytes from fish-oil-fed rats. Incorporation of [35S]methionine into cellular and secreted apo A-I was lower in hepatocytes from fish-oil-fed rats than in hepatocytes from corn-oil-fed rats, and was not paralleled by any difference in the apo A-I mRNA level. Finally, [35S]methionine incorporation into cellular and secreted forms of apo E and apo A-I mRNA were reduced in hepatocytes from fish-oil-fed rats, compared with hepatocytes from corn-oil-fed rats. These combined data show that fish oil diet reduces triacylglycerol synthesis and secretion and affects apo B synthesis at a post-transcriptional level, and reduces cholesterol synthesis and affects apo E and apo A-I synthesis at a transcriptional and a post-transcriptional level.

Animals↗
PubMed1991

Nutritional regulation of apolipoprotein genes: effect of dietary carbohydrates and fatty acids.

The effect of nutritional factors on apolipoprotein gene expression by rat liver were studied. Dietary carbohydrates or fatty acids regulate the expression of apo E gene, by altering either gene transcription or mRNA stability. Conversely, apo AI regulation occurs at a post transcriptional level. In vivo and in vitro experiments gave contradictory results concerning apo B gene expression. The more dramatic changes in plasma lipids and apolipoproteins are obtained under dietary fish oil. Hepatocytes from fish oil-fed rats retain for several days modification in fatty acid metabolism, i.e. a shift in oleic acid channeling towards oxidation at the expense of esterification and a reduced ability to synthesize and secrete triacylglycerol. These modifications are paralleled with a decrease in the synthesis and in the secretion of apo Bs. Hepatocytes from fish oil fed rats secrete degradative forms of apo B which might result from either a sluggish VLDL synthesis and secretion or a more specific effect of n-3 long chain polyunsaturated fatty acid peroxidative products. Hepatocytes from fish oil fed rats exhibit a reduced ability to synthesize cholesterol, associated with a decrease in apo AI synthesis and secretion without any modification in apo AI mRNA. In contrast, the hepatocytes exhibit a concomitent decrease in apo E synthesis and secretion and in cellular apo E mRNA levels.

Animals↗
PubMed1990

Partial apolipoprotein E-beta-galactosidase fusion protein expressed in Escherichia coli retains binding activity to the LDL(B/E) receptor.

A partial rat apo E-beta-galactosidase fusion protein was produced in Escherichia coli Y1089 infected with recombinant lambda GT11 obtained by immunoscreening of a rat liver cDNA library with an anti-rat LDL antiserum. Partial cDNA overlapped the apo E mRNA sequence coding for apo E binding domain towards the LDL(B/E) receptor up to codon for Arg-139. Fusion protein specifically bound to human fibroblasts. The high-affinity component exhibited a Kd of 5 x 10(-8) M and 4.1 x 10(5) sites per cell. Fusion protein binding to fibroblasts was mediated by their apo E moiety and not by beta-galactosidase since: (1) specific binding of fusion protein was competed out by human LDL; (2) beta-galactosidase did not compete with fusion protein binding; and (3) human fibroblasts from a patient with familial hypercholesterolemia, deficient in LDL(B/E) receptor, bound fusion protein 10-times lower than control fibroblasts. It was demonstrated that partial fusion protein retained the functional activity of the native apo E. However, compared to full-length native or engineered apo E, fusion protein was able to bind fibroblasts without being complexed with phospholipids. Fusion proteins might be a useful tool for studying the functional efficiency of the LDL(B/E) receptor and for mapping residues and domains involved in the binding process.

Apolipoproteins E↗
PubMed1990

Eicosanoid synthesis in children with cholestatic disease.

The possibility of malabsorption of triglycerides contained in the diets of children with cholestasis suggests a deficiency of essential fatty acids and, therefore, probable effects on eicosanoid metabolism. Children with either biliary atresia (BA) or syndromatic paucity of interlobular bile ducts (PILBD) were evaluated as to plasma and platelet total lipid fatty acid composition and synthesis of prostaglandins (PG) E1, PGE2, PGI2, PGF2, and thromboxane (TXB2) by whole blood incubated at 37 degrees C for 10 min. In both diseases linoleate deficiency was present as shown by low 18:2 fatty acids in plasma lipids. The children with BA had lower plasma arachidonate than controls but normal eicosanoid synthesis except for excess PGI2. Those with PILBD had low platelet arachidonate and were severely deficient in TXB2 synthesis (less than 10% of controls). Three children with PILBD were fed a supplement of structured triglyceride (Captex 810) intended to provide as much as 10% of energy as linoleate for 2-3 months. Results for these three cases suggested that insufficient linoleate was absorbed to increase plasma linoleate and differences in eicosanoids could not be attributed to linoleate supplementation.

Arachidonic Acid↗