Functional screening of an asthma QTL in YAC transgenic mice.

Many quantitative trait loci (QTLs) contributing to genetically complex conditions have been discovered, but few causative genes have been identified. This is mainly due to the large size of QTLs and the subtle connection between genotype and quantitative phenotype associated with these conditions. Transgenic mice have been successfully used to analyse well-characterized genes suspected of contributing to quantitative traits. Although this approach is powerful for examining one gene at a time, it can be impractical for surveying the large genomic intervals containing many genes that are typically associated with QTLs. To screen for genes contributing to an asthma QTL mapped to human chromosome 5q3 (refs 6,7), we characterized a panel of large-insert 5q31 transgenics based on studies demonstrating that altering gene dosage frequently affects quantitative phenotypes normally influenced by that gene. This panel of human YAC transgenics, propagating a 1-Mb interval of chromosome 5q31 containing 6 cytokine genes and 17 partially characterized genes, was screened for quantitative changes in several asthma-associated phenotypes. Multiple independent transgenic lines with altered IgE response to antigen treatment shared a 180-kb region containing 5 genes, including those encoding human interleukin 4 (IL4) and interleukin 13 (IL13 ), which induce IgE class switching in B cells. Further analysis of these mice and mice transgenic for mouse Il4 and Il13 demonstrated that moderate changes in Il4 and Il13 expression affect asthma-associated phenotypes in vivo. This functional screen of large-insert transgenics enabled us to identify genes that influence the QTL phenotype in vivo.

Tangier disease is caused by mutations in the gene encoding ATP-binding cassette transporter 1.

Tangier disease (TD) was first discovered nearly 40 years ago in two siblings living on Tangier Island. This autosomal co-dominant condition is characterized in the homozygous state by the absence of HDL-cholesterol (HDL-C) from plasma, hepatosplenomegaly, peripheral neuropathy and frequently premature coronary artery disease (CAD). In heterozygotes, HDL-C levels are about one-half those of normal individuals. Impaired cholesterol efflux from macrophages leads to the presence of foam cells throughout the body, which may explain the increased risk of coronary heart disease in some TD families. We report here refining of our previous linkage of the TD gene to a 1-cM region between markers D9S271 and D9S1866 on chromosome 9q31, in which we found the gene encoding human ATP cassette-binding transporter 1 (ABC1). We also found a change in ABC1 expression level on cholesterol loading of phorbol ester-treated THP1 macrophages, substantiating the role of ABC1 in cholesterol efflux. We cloned the full-length cDNA and sequenced the gene in two unrelated families with four TD homozygotes. In the first pedigree, a 1-bp deletion in exon 13, resulting in truncation of the predicted protein to approximately one-fourth of its normal size, co-segregated with the disease phenotype. An in-frame insertion-deletion in exon 12 was found in the second family. Our findings indicate that defects in ABC1, encoding a member of the ABC transporter superfamily, are the cause of TD.

Protection against atherogenesis in mice mediated by human apolipoprotein A-IV.

Apolipoproteins are protein constituents of plasma lipid transport particles. Human apolipoprotein A-IV (apoA-IV) was expressed in the liver of C57BL/6 mice and mice deficient in apoE, both of which are prone to atherosclerosis, to investigate whether apoA-IV protects against this disease. In transgenic C57BL/6 mice on an atherogenic diet, the serum concentration of high density lipoprotein (HDL) cholesterol increased by 35 percent, whereas the concentration of endogenous apoA-I decreased by 29 percent, relative to those in transgenic mice on a normal diet. Expression of human apoA-IV in apoE-deficient mice on a normal diet resulted in an even more severe atherogenic lipoprotein profile, without affecting the concentration of HDL cholesterol, than that in nontransgenic apoE-deficient mice. However, transgenic mice of both backgrounds showed a substantial reduction in the size of atherosclerotic lesions. Thus, apoA-IV appears to protect against atherosclerosis by a mechanism that does not involve an increase in HDL cholesterol concentration.

Opposite regulation of human versus mouse apolipoprotein A-I by fibrates in human apolipoprotein A-I transgenic mice.

The regulation of liver apolipoprotein (apo) A-I gene expression by fibrates was studied in human apo A-I transgenic mice containing a human genomic DNA fragment driving apo A-I expression in liver. Treatment with fenofibrate (0.5% wt/wt) for 7 d increased plasma human apo A-I levels up to 750% and HDL-cholesterol levels up to 200% with a shift to larger particles. The increase in human apo A-I plasma levels was time and dose dependent and was already evident after 3 d at the highest dose (0.5% wt/wt) of fenofibrate. In contrast, plasma mouse apo A-I concentration was decreased after fenofibrate in nontransgenic mice. The increase in plasma human apo A-I levels after fenofibrate treatment was associated with a 97% increase in hepatic human apo A-I mRNA, whereas mouse apo A-I mRNA levels decreased to 51%. In nontransgenic mice, a similar down-regulation of hepatic apo A-I mRNA levels was observed. Nuclear run-on experiments demonstrated that the increase in human apo A-I and the decrease in mouse apo A-I gene expression after fenofibrate occurred at the transcriptional level. Since part of the effects of fibrates are mediated through the nuclear receptor PPAR (peroxisome proliferator-activated receptor), the expression of the acyl CoA oxidase (ACO) gene was measured as a control of PPAR activation. Both in transgenic and nontransgenic mice, fenofibrate induced ACO mRNA levels up to sixfold. When transgenic mice were treated with gemfibrozil (0.5% wt/wt) plasma human apo A-I and HDL-cholesterol levels increased 32 and 73%, respectively, above control levels. The weaker effect of this compound on human apo A-I and HDL-cholesterol levels correlated with a less pronounced impact on ACO mRNA levels (a threefold increase) suggesting that the level of induction of human apo A-I gene is related to the PPAR activating potency of the fibrate used. Treatment of human primary hepatocytes with fenofibric acid (500 microM) provoked an 83 and 50% increase in apo A-I secretion and mRNA levels, respectively, supporting that a direct action of fibrates on liver human apo A-I production leads to the observed increase in plasma apo A4 and HDL-cholesterol.

Negative regulation of Apo A-I gene expression by retinoic acid in rat hepatocytes maintained in a coculture system.

Rat hepatocytes cocultured with rat liver epithelial cells (RLEC) were used to investigate the influence of all-trans retinoic acid (RA) on the regulation of apolipoproteins (Apo) A-I and A-II gene expression, the major protein constituent of high-density lipoproteins. In contrast to rat hepatocytes in conventional primary culture, Apo A-I and Apo A-II gene expression remained high and stable for several days in parenchymal cells in coculture. Treatment of cocultured rat hepatocytes with RA resulted in a specific decrease in Apo A-I mRNA levels whereas no marked difference in Apo A-II mRNA levels was observed. Such a negative effect of RA was already detected as early as 2 days of treatment and was effective for the entire experimental period (6 days). As controls, RARbeta mRNA levels increased whereas those of GAPDH mRNA were not affected by the RA treatment. The decrease in Apo A-I mRNA levels was associated with lower amounts of Apo A-I secreted in the culture medium within day 1 of treatment. This effect required active transcription and protein synthesis. These results show that, contrary to primary pure hepatocyte cultures and hepatoma cell lines, cocultures of rat hepatocytes reproduce the in vivo results suggesting that only well differentiated hepatocytes may correctly respond to RA. Furthermore, they demonstrate that RA can directly act on hepatocytes and differently affect Apo A-I and Apo A-II gene expression.

Structural domain of apolipoprotein A-I involved in its interaction with cells.

Apolipoprotein A-I (apo A-I) is the major protein constituent of high-density lipoprotein (HDL), the lipoprotein fraction which mediates the reverse cholesterol transport. This apolipoprotein plays an important role in the binding of HDL to cells and participates in the efflux of cellular cholesterol. We have recently compared six different genetic variants of apo A-I and found that the apo A-I (Pro 165-->Arg) mutant is defective in promoting cellular cholesterol efflux from murine adipocytes and peritoneal macrophages and we have proposed that this region of apo A-I may be involved in their interaction with cells. To confirm this hypothesis, four monoclonal antibodies (mAbs) specific for apo A-I were used to study the inhibition of the interaction of palmitoyloleoylphosphatidylcholine (POPC): apoA-I complexes with HeLa cells and adipocytes. Among these antibodies, the apo A-I epitope recognized by the A44 mAb lies in the COOH terminal region (amino acid residues 149-186) including the proposed region. The antibodies A05, and A03 react with residues 25-82, 135-140, respectively and the A11 mAb corresponds to a discontinuous epitope at residues 99-105 and 126-132. Our results show clearly that the A44 and A05 mAbs reduce both the binding to HeLa cells and the cholesterol efflux from adipocytes. The inhibition of POPC: apoA-I complexes binding to both cell types is more strictly observed with the Fab fragments of monoclonal antibodies A44 and A05. Partial cotitration curves of these mAbs in a solid phase assay (RIA), indicated partial competition between these two antibodies. We propose a structural model for the POPC: apoA-I complexes where the N-terminal domain of one apo A-I molecule is in close spatial relationship with the C-terminal domain of the adjacent apo A-I molecule. We therefore suggest that the domain around amino acid 165 of apo A-I and which is recognized by mAb A44 (149-186) forms or contains some specific regions which mediate selectively the interaction with the binding site of cells and is involved in the efflux of cellular cholesterol.

Characterization of human apolipoprotein A-I expressed in Escherichia coli.

Human apolipoprotein A-I (apoA-I), with an additional N-terminal extension (Met-Arg-Gly-Ser-(His)6-Met) (His-apoA-I), has been produced in Escherichia coli with a final yield after purification of 10 mg protein/1 of culture medium. We have characterized the conformation and structural properties of His-apoA-I in lipid-free form, and in reconstituted lipoproteins containing two apoA-I per particle (Lp2A-I) by both immunochemical and physicochemical techniques. The lipid-free forms of the two proteins present very similar secondary structure and stability, and have also very similar kinetics of association with dimyristoyl phosphatidylcholine. His-apoA-I and native apoA-I can be complexed with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) to form similar, stable, either discoidal or spherical (sonicated) Lp2A-I particles. Lipid-bound native apoA-I and His-apoA-I showed very similar alpha-helical content (69% and 66%, respectively in discoidal Lp2A-I and 54% and 51%, respectively in spherical Lp2A-I). The conformation of His-apoA-I in lipid-free form and in discoidal or spherical Lp2A-I has also been shown to be similar to native apoA-I by immunochemical measurements using 13 monoclonal antibodies recognizing distinct apoA-I epitopes. In the free protein and in reconstituted Lp2A-I, the N-terminal has no effect on the affinity of any of the monoclonal antibodies and minimal effect on immunoreactivity values. Small differences in the exposure of some apoA-I epitopes are evident on discoidal particles, while no difference is apparent in the expression of any epitope of apoA-I on spherical Lp2A-I. The presence of the N-terminal extension also has no effect on the reaction of LCAT with the discoidal Lp2A-I or on the ability of complexes to promote cholesterol efflux from fibroblasts in culture. In conclusion, we show that His-apoA-I expressed in E. coli exhibits similar physicochemical properties to native apoA-I and is also identical to the native protein in its ability to interact with phospholipids and to promote cholesterol esterification and cellular cholesterol efflux.

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways.

Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.

Apolipoprotein A-I (R151C)Paris is defective in activation of lecithin: cholesterol acyltransferase but not in initial lipid binding, formation of reconstituted lipoproteins, or promotion of cholesterol efflux.

ApoA-I(R151)Paris is a natural apolipoprotein (apo) A-I variant that is associated with low levels of high-density lipoprotein cholesterol (HDL-cholesterol) and the partial deficiency of lecithin:cholesterol acyl-transferase (LCAT) in the plasma of heterozygous carriers. We compared the abilities of recombinant normal apoA-I and recombinant apoA-I(R151C)Paris to clear an emulsion of dimyristoylphosphatidylcholine (DMPC), to form reconstituted lipoproteins with dipalmitoylphosphatidylcholine (DPPC), to activate LCAT, and to promote efflux of biosynthetic cholesterol from porcine aortic smooth muscle cells (SMCs) or of exogenous cholesterol from lipid-loaded mouse peritoneal macrophages. Recombinant apoA-I(R151C)Paris occurred in monomeric and dimeric forms at a ratio of 60:40. Normal apoA-I and apoA-I(R151C)Paris cleared DMPC emulsions at equal rates. Both isoforms associated completely with DPPC during cholate dialysis. Normal apoA-I formed one single particle with a mean diameter of 9.3 nm, whereas apoA-I(R151)Paris gave rise to three particles with mean diameters of 9.3 nm (containing 74% of apoA-I), 10.6 nm, and 12.1 nm, respectively. Compared to normal apoA-I, apoA-I(R151C)Paris had a reduced LCAT-cofactor activity with a 60% lower Vmax/Km ratio due to a 50% higher affinity constant, Km. During incubations for 10 min and 360 min, normal apoA-I/DPPC complexes and apoA-I(R151C)Paris/DPPC complexes were equally efficient in releasing biosynthetic cholesterol from SMCs. In the lipid-free form, apoA-I(R151C)Paris induced normal hydrolysis of cholesteryl esters and normal cholesterol efflux from lipid-loaded mouse-peritoneal macrophages. In conclusion, in addition to its ability to form homo- and heterodimers, apoA-I(R151C)Paris is characterized by defective LCAT-cofactor activity but by normal lipid binding and cholesterol-efflux-promoting abilities.

Percutaneous delivery of the gax gene inhibits vessel stenosis in a rabbit model of balloon angioplasty.

OBJECTIVES: The expression of gax, an anti-proliferative homeobox gene, is rapidly downregulated in vascular smooth muscle cells (VSMCs) following arterial injury. Here we performed percutaneous adenovirus-mediated gene transfer into the iliac arteries of normal rabbits using a channel balloon catheter to assess the effects of gax overexpression on neointima formation, lumen diameter, reendothelialization and functional vasomotion. METHODS: A channel balloon catheter was used to perform both the arterial injury and local gene delivery. In each animal both iliac arteries were randomly assigned to receive either an adenovirus expressing the gax gene (Ad-Gax) or the beta-galactosidase gene (Ad-beta gal). In a second group of animals arteries were randomly assigned to receive either Ad-beta gal or saline. RESULTS: At one month, angiography revealed 36% less luminal narrowing in the Ad-Gax-treated arteries relative to the Ad-beta gal-treated control arteries. Histological analysis revealed that the intimal/medial ratio (I/M) was reduced by 56% in the Ad-Gax group. Endothelium-dependent vasomotion was not affected by the gax gene transfer. In the second group, no statistically significant differences were found between the saline and the Ad-beta gal-treated vessels for any of these parameters. CONCLUSIONS: Percutaneous adenovirus delivery of the gax gene to rabbit iliac arteries following endothelial denudation and vessel wall injury reduces neointimal hyperplasia and luminal stenosis, but does not affect endothelium-dependent vasomotion. This study demonstrates that a VSMC transcription factor can potentially be utilized for the development of a molecular therapy for vascular disorders.

Efficient adenovirus-mediated ectopic gene expression of human lipoprotein lipase in human hepatic (HepG2) cells.

Gene therapy to deliver and express a corrective lipoprotein lipase (LPL) gene may improve the lipid profile and reduce the morbidity and potential atherogenic risk from hypertriglyceridemia and dyslipoproteinemia in patients with complete or partial LPL deficiency. We have used an E1-/E3- adenoviral vector, with an RSV-driven human LPL cDNA expression cassette (Ad-RSV-LPL), to achieve high ectopic LPL gene expression in the human hepatoma cell line HepG2, an accepted hepatocellular model of lipoprotein metabolism. Ad-RSV-LPL transduction of HepG2 cells with a multiplicity of infection (moi) between 12.5 and 100 yielded dose-dependent increments in LPL mass and activity. Peak levels of LPL protein of 2,032.1 +/- 274.5 ng/10(5) cells per ml (mol 100) correlated with increased activity of 92.7 +/- 22.6 mU/10(5) cells per ml relative to negligible LPL levels in Ad-RSV-LacZ (beta-galactosidase) controls. Exogenous LPL expression over a 5-day period peaked at day 3. Susceptibility to inhibition by 1 M NaCl and an anti-LPL monoclonal antibody confirmed that lipase activity was indeed derived from human LPL. Hydrolysis, by LPL-overexpressing HepG2 cells, of TG carried in very-low-density lipoprotein (VLDL) showed that greater than 50% of the triglycerides (TG) disappeared after 4 hr of incubation. These results were compatible with FPLC evidence of a marked reduction in VLDL-TG. These results provide strong in vitro evidence that adenoviral-mediated ectopic expression of the human LPL gene could render hepatic cells capable of VLDL catabolism and thus support the possibility for in vivo adenoviral vector-mediated liver-targeted LPL gene therapy.

Chemical synthesis of a gene coding for human angiogenin, its expression in Escherichia coli and conversion of the product into its active form.

A synthetic gene coding for human angiogenin was synthesized by solid support phosphoramidite chemistry as eight long oligodeoxynucleotides which were subsequently assembled and cloned in Escherichia coli. The gene was designed to use codons found in highly expressed E. coli proteins. A pBR322-derived expression vector was constructed containing the E. coli trp promoter, the ribosome-binding site of the bacteriophage lambda cII gene, the angiogenin coding sequence, and the transcription terminator region of the E. coli rrnB operon. Under tryptophan deprivation, angiogenin was strongly expressed in E. coli cells at a yield of 5-10% of total protein. The eukaryotic protein was found to be insoluble but could be easily renatured and purified. The purified angiogenin was demonstrated to be active as an angiogenic factor and exhibited a characteristic RNase activity.

Heterologous protein export in Escherichia coli: influence of bacterial signal peptides on the export of human interleukin 1 beta.

Expression plasmids carrying the coding sequence of mature human interleukin 1 beta (IL 1 beta) linked either to a Met start codon, or fused to different efficient Escherichia coli secretion signal sequences, have been constructed. In the latter case, we used signal peptides derived either from an outer membrane protein (OmpA) or from a periplasmic protein (PhoA). The synthesis of IL1 beta from these fusions was investigated in an otherwise strictly isogenic context using identical conditions of derepression and culture media. The Met-IL1 beta fusion produced a soluble cytoplasmic protein which could be released from the cells by osmotic shock whereas the OmpA and PhoA fusions were always insoluble. The extent of sOmpA-IL1 beta maturation was found to vary from 50 to 100%, mainly depending on the medium used, whereas no significant maturation of the signal peptide could be detected in the case of the sPhoA-IL1 beta fusion. Immuno-electron microscopy revealed that the sOmpA-IL1 beta fusion was targeted to the inner membrane, whereas the sPhoA-IL1 beta fusion remained within the cytoplasm and thus did not appear to enter the secretion pathway. Amplifying the E. coli signal peptidase lep gene on a multicopy plasmid did not improve signal peptide removal from sOmpA-IL1 beta. Moreover, these E. coli secretion vectors allowed us to produce, in high levels, IL1 beta fragments which otherwise could not be stably accumulated within the cytoplasmic compartment.

Clinical and pathologic abnormalities in a family with parkinsonism and parkin gene mutations.

A Dutch family with autosomal recessive early-onset parkinsonism showed a heterozygous missense mutation in combination with a heterozygous exon deletion in the parkin gene. Although the main clinical syndrome consisted of parkinsonism, the proband clinically had additional mild gait ataxia and pathologically showed neuronal loss in parts of the spinocerebellar system, in addition to selective loss of dopaminergic neurons in the substantia nigra pars compacta. Lewy bodies and neurofibrillary tangles were absent, but tau pathology was found.

Tryptophan promoter derivatives on multicopy plasmids: a comparative analysis of expression potentials in Escherichia coli.

A collection of variant plasmids expressing either Escherichia coli galactokinase or human serum albumin under the control of several E. coli trp promoter derivatives were constructed and studied for both efficiency of expression and regulation by tryptophan. Several variables, including the length of the upstream region, tandem duplications of a core promoter, and the insertion of the trp repressor trpR gene onto the expression vector, were studied. It is shown that derivatives containing sequences upstream from the -35 region or multiple copies of the trp promoter produce twofold higher levels of protein than plasmids with a minimal trp promoter truncated at -40. We show that the expression of a heterologous protein such as albumin can be significantly improved (13% vs. 7% of total proteins) if both the upstream trp promoter region, which enhances promoter strength, and an intact trpR gene, are included on the plasmids.

High-cell density fermentation studies of recombinant Escherichia coli strains expressing human interleukin-1 beta.

A high-productivity process has been developed for the production of mature human interleukin-1 beta (IL-1 beta) from recombinant Escherichia coli strains. Conditions were found that allow high IL-1 beta expression levels in high cell density cultures. Improved fed-batch fermentation strategies are described which include maintenance of glucose and acetate concentrations below 1 g/l and sparging the fermentor with an O2-enriched air supply. Using the E. coli tryptophan promoter control of transcription, a 2.2 g/l production level of IL-1 beta was achieved in E. coli B at cell densities of 55 g dry weight per litre. Another genetic construction involving the bacteriophage lambda cIts-PR expression cassette allowed a similar IL-1 beta production level (1.9 g/l) in E. coli E103S, albeit at a lower cell density (30 g/l). A simplified procedure allowing the purification of fully active IL-1 beta is also presented.

Efficient dual transcomplementation of adenovirus E1 and E4 regions from a 293-derived cell line expressing a minimal E4 functional unit.

Transgene expression after the administration of recombinant adenovirus with E1 deleted is constantly transient. It is admitted that E1A-substituting activities of cellular or viral origin allow viral antigen synthesis and trigger cytotoxic lymphocyte-mediated clearance of the recipient cells. Our approach to solving this problem relies on the additional deletion of the E4 region from the vector backbone as this region upregulates viral gene expression at both transcriptional and posttranscriptional levels. As a prerequisite to the construction of E1 E4 doubly defective adenoviruses, we investigated the possibility of transcomplementing both functions within a single cell. In particular, the distal ORF6+ORF7 segment from the E4 locus of adenovirus type 5 was cloned under the control of the dexamethasone-inducible mouse mammary tumor virus long terminal repeat. Following transfection into 293 cells, clone IGRP2 was retained and characterized as it can rescue the growth defect of all E1+ E4- adenoviral deletants tested. DNA and RNA analysis experiments verified that the mouse mammary tumor virus promoter drives the expression of the ORF6+ORF7 unit and permits its bona fide alternative splicing, generating ORF6/7 mRNA in addition to the ORF6-expressing primary transcript. Importantly, IGRP2 cells sustain cell confluence for a period longer than that of 293 parental cells and allow the plaque purification of E1- or E4- defective viruses. The dual expression of E1 and E4 regulatory genes within IGRP2 cells is demonstrated by the construction, plaque purification, and helper-free propagation of recombinant lacZ-encoding doubly defective adenoviruses harboring different E4 deletions. In addition, the emergence, if any, of replicative particles during viral propagation in this novel packaging cell line will be drastically impaired as only a limited segment of E4 has been integrated.

Brain macrophages synthesize interleukin-1 and interleukin-1 mRNAs in vitro.

Amoeboid microglial cells (brain macrophages) were purified from early post-natal mouse brain cultures. The percentage of cells stained with an anti-Mac-1 antibody was greater than 95%. Stimulation of these brain macrophages by lipopolysaccharides induced the synthesis of interleukin-1 (IL-1), which, in part, remained associated with the cell surface and, in part, was released into the culture medium. In contrast, pure primary astrocyte cultures and cell lines of transformed or immortalised astrocytes did not synthesise significant amounts of IL-1, demonstrating that amoeboid microglia and not astrocytes synthesise IL-1 in vitro. These physiological data were confirmed by RNA hybridisation studies showing that, on LPS treatment, brain macrophages synthesise significant amounts of IL-1 alpha and IL-1 beta mRNAs.

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.