Evaluation of the role of lipoprotein metabolism genes in systemic cationic liposome-mediated gene transfer in vivo.

Germ line gene disruption and gene insertion are often used to study the function of selected genes in vivo. We used selected knockout and transgenic mouse models to attempt to identify lipoprotein-related genes and gene products that regulate the process of intravenous cationic liposome-DNA complex (CLDC)-based gene delivery. Several observations suggested that proteins involved in lipoprotein metabolism might be important in influencing the delivery and/or expression of CLDC. First, in vitro transfection of either K562 or CHO cells by CLDCs was enhanced by the presence of a functional low-density lipoprotein receptor (LDLR). Second, pretreatment of mice with 4-aminopyrazolopyrimidine (4APP), an agent that alters lipoprotein profiles in mice, significantly decreased expression of luciferase (luc) after intravenous injection of CLDC-luc complexes in mice. Therefore, we tested mouse model systems either deficient for, or overexpressing, selected genes involved in lipoprotein metabolism, for their potential to regulate intravenous, CLDC-based gene delivery. Although homozygous knockout mutation in the apoE gene caused a significant decrease in gene expression in many tissues of apoE-deficient mice, mice with homozygous deletion of both the apoE and LDLR genes showed wild-type levels of gene transfer efficiency. Thus, a secondary event, produced by homozygous deletion of apoE, but compensated for by the concomitant deletion of LDLR, and/or effects resulting from strain-related, genetic background differences, appeared to play a significant role in mediating intravenous, CLDC-based gene delivery. Secondary alterations resulting from germ line knockouts, as well as epigenetic effects produced by strain differences, may limit the ability to assign specific, gene transfer-related functions to the deleted gene.

Two distinct apolipoprotein B alleles in mice generated by a single 'in-out' targeting.

'In-out' gene targeting using a hypoxanthine phosphoribosyltransferase (HPRT) minigene was applied to generate two new alleles in the gene (Apob) coding for apolipoprotein B (apo B) in murine embryonic stem (ES) cells. Homologous integration of the targeting vector during the 'in step' disrupted the Apob gene leading to an allele encoding apo B81, having a 19% carboxyl-terminal truncation. All six targeted cells obtained had more than one insert at the locus, and the chromosomal target sequence in four of them was changed during the recombination. These results suggest that concatenation of the targeting vector prior to insertion was needed to generate sufficient gene product to yield the HPRT+ phenotype, and that recombination between the concatenated DNA and endogenous DNA was a gene replacement more frequently than a simple insertion. The 'out step' recombination event which occurs between sequences duplicated in the 'in step', was planned to replace the sequences encoding the putative LDL receptor-binding domains of apo B100 with sequences encoding human beta-globin peptides (designated apo B100-beta). 6-Thioguanine (6-TG) resistant colonies were obtained from all the 'in-step' cell lines tested at frequencies of 10(-5) to 10(-4), but the frequency of physical loss of the HPRT sequences accompanied by retention of the modified Apob sequence was variable, indicating that mechanisms other than a simple excision are responsible for the generation of 6-TG resistance. Mice from the 'in-step' produce apo B81 and display characteristics of familial hypobetalipoproteinemia; some homozygotes develop hydrocephaly or exencephaly. Mice from the 'out-step' produce apo B100-beta and secrete lipoprotein particles containing the modified protein; their phenotypic changes are subtle, suggesting the lack of the putative LDL receptor-binding domains is not sufficient to increase the steady-state level of apo B100-beta particles above that of apo B100 particles in control mice.

Mild dyslipidemia in mice following targeted inactivation of the hepatic lipase gene.

In order to gain better understanding of the function of hepatic lipase (HL) in vivo, we have generated mice that lack HL using gene targeting in embryonic stem cells. No mRNA for HL was detected in the liver of homozygous mutants, and no HL activity was detected in their plasma. Total cholesterol levels in plasma of mutant mice were increased by about 30% compared with wild type animals. Plasma phospholipids and high density lipoprotein (HDL) cholesterol were also increased, but plasma levels of triglycerides were not altered. Analysis of density fractions of plasma lipoproteins revealed that HDL1 (d = 1.02-1.04) was increased in homozygous mutants fed regular chow. In response to a diet containing high fat and high cholesterol, HDL cholesterol was doubled in the mutants, but was slightly decreased in the wild type mice. These results clearly demonstrate the importance of HL in HDL remodeling and metabolism in vivo. Various earlier studies suggested a role of HL in metabolism of triglyceride-rich particles, but the mutant mice appear to have no impairment in clearing them; the mutants clear exogenously introduced chylomicrons from plasma at a normal rate, and they tolerate acute fat loading as well as normal animals unless the loading is extreme. These differences may reflect species differences. However, it is also possible that the consequence of absence of HL as in our mutants is different from the consequence when nonfunctional HL protein is present as in the human HL-deficient patients and in rats treated with HL antibodies. We hypothesize that absence of HL in mutant mice allows other lipases to bind to the sites in the liver normally occupied by HL and facilitate the clearance of triglyceride-rich particles in these mice.

Identification of apolipoprotein B-100 low density lipoproteins, apolipoprotein B-48 remnants, and apolipoprotein E-rich high density lipoproteins in the mouse.

Plasma lipoprotein fractions from inbred C57BL/6J mice and outbred ICR mice were prepared by sequential density ultracentrifugation using density ranges that were optimized for separating mouse lipoproteins, or by Superose 6 HR10/30 fast performance liquid chromatography (FPLC). The lipoproteins were characterized by migration behavior in agarose, apolipoprotein (apo) composition, lipid composition, and particle size distribution. Both sequential density ultracentrifugation and Superose 6 FPLC were adapted for the separation of lipoproteins from a single mouse. In the plasma of ICR and C57BL/6J mice, in contrast to human plasma, alpha-migrating high density lipoproteins (HDL) and beta-migrating low density lipoproteins (LDL) had overlapping density ranges. For example, beta-migrating apoB-100 LDL, slow pre-beta-migrating apoB-48 remnants, and alpha-migrating HDL1 were found together in the d 1.02-1.04 g/ml fraction. The d 1.04-1.06 g/ml fraction contained beta-migrating apoB-100 LDL and alpha-migrating HDL1. Large HDL1 that were found at d 1.02-1.06 g/ml were apoE-rich HDL1, characteristic of cholesteryl ester transfer protein-deficient mammals. The d 1.10-1.21 g/ml fraction, in addition to alpha-migrating HDL, included unique slow beta-migrating particles that contained apoE and apoA-I but was deficient in neutral lipids. These slow beta-HDL eluted in the same FPLC fractions as dense alpha-migrating HDL. Compared to ICR mouse plasma, C57BL/6J mouse plasma contained more LDL and less HDL1, which might contribute to the susceptibility of C57BL/6J and the resistance of ICR mice to the development of aortic fatty streak lesions when challenged with an atherogenic diet.

Overexpression of human apolipoprotein C-III in transgenic mice results in an accumulation of apolipoprotein B48 remnants that is corrected by excess apolipoprotein E.

Overexpression of human apolipoprotein (apo) C-III in the plasma of transgenic mice results in hypertriglyceridemia, with up to a 20-fold elevation in plasma triglyceride. Nearly all of the triglyceride accumulates in the d < 1.006 g/ml lipoprotein fraction, which consists predominantly of apoB48-containing particles having a low apoE:apoB48 ratio in contrast to normal mice. The transgenic and nontransgenic d < 1.006 g/ml lipoproteins are similar in size, and they are equivalent substrates for lipoprotein lipase in vitro. Total apoB100 levels are similar in transgenic and normal plasma, but apoB48 levels are increased in transgenic mice. The transgenic d < 1.006 g/ml particles are poor competitors for the binding of low density lipoproteins to the low density lipoprotein receptor in vitro, which is corrected by the addition of exogenous apoE. The rate of clearance of labeled chylomicron remnants in apoC-III-transgenic mice was about half that in nontransgenic mice. The lipoprotein alterations are accompanied by up to a 5-fold increase in circulating nonesterified fatty acids, which may be the cause of fatty livers and increased liver triglyceride production also observed in the transgenic mice. These observations indicate that the primary defect leading to hypertriglyceridemia in apoC-III overexpressers is an impaired clearance of apoB48 remnants due to apoE insufficiency. Therefore, transgenic mice that overexpressed human apoE were cross-bred with the apoC-III overexpressers. Transgenic progeny that produced both human apoE and human apoC-III had normal levels of plasma triglyceride and normal amounts of apoB48 remnants. Thus, our studies suggest that a function of apoC-III is to modulate the apoE-mediated clearance of lipoproteins, and that the concentration of apoC-III relative to apoE is a key determinant of triglyceride levels in plasma.

Purification and characterization of apolipoprotein J.

Apolipoprotein J (apoJ), a unique 70-kDa component of high density lipoproteins in human plasma, consists of two disulfide-linked subunits designated apoJ alpha (34-36 kDa), and apoJ beta (36-39 kDa) which share pI values of 4.9-5.4 and which are recognized by a monoclonal antibody (mAb) 11. ApoJ and its subunits were purified to homogeneity from plasma by a combination of immunoaffinity chromatography, using mAb11 linked to Affi-Gel, and reverse-phase high performance liquid chromatography. ApoJ alpha and apoJ beta are both glycoproteins. When deglycosylated, the molecular mass of apoJ alpha is 24 kDa and that of apoJ beta is 28 kDa, suggesting that approximately 30% of the mass of each subunit is carbohydrate. The amino acid compositions of apoJ alpha and apoJ beta are very similar; however, the sequences of the first 30-amino acid residues are distinct. A comparison of peptide maps suggests that apoJ alpha and apoJ beta are not identical but share limited regions of homology. This possibility is supported by immunochemical data. Five additional mAb specific for apoJ were characterized. One of the mAb, like mAb11, reacts with both apoJ alpha and apoJ beta; the others react with apoJ alpha only. All mAb, including those which recognize both apoJ alpha and apoJ beta and those which recognize apoJ alpha only, immunoprecipitate a approximately 50-kDa protein synthesized from a liver mRNA template translated in a rabbit reticulocyte lysate. We propose that the apoJ alpha and apoJ beta subunits, which have limited homology, are derived by proteolytic cleavage of a common precursor.

A 70-kDa apolipoprotein designated ApoJ is a marker for subclasses of human plasma high density lipoproteins.

A new apolipoprotein, termed apolipoprotein J (apoJ), was purified from human plasma by immunoaffinity chromatography. ApoJ is a glycoprotein consisting of disulfide-linked subunits of 34-36 and 36-39 kDa. Each subunit is glycosylated and has a pI range of 4.9-5.4. ApoJ exists in the plasma associated with high density lipoproteins (HDL) and specifically with subclasses of HDL which also contain apoAI and cholesteryl ester transfer protein activity. Immunoaffinity purified apoJ-HDL subclasses have apparent molecular masses of 80, 160, 240, 340, and 520 kDa, as determined by gradient gel electrophoresis. By negative staining electron microscopy, apoJ-HDL range in diameter from 5 to 16 nm. Fractionation of plasma by vertical gradient density centrifugation revealed apoJ-HDL in HDL2 (d 1.063-1.125 g/ml) with the majority overlapping HDL3 (d 1.125-1.21 g/ml) and very high density lipoprotein (d 1.21-1.25 g/ml). The bimodal density distribution of apoJ-HDL suggests that these subclasses have a unique metabolic relationship and may play a role in the transport of cholesterol from peripheral tissues to the liver.

Apolipoprotein J: structure and tissue distribution.

The primary structure of apolipoprotein J (apoJ) was deduced by the combined strategies of protein sequencing and cDNA cloning and sequencing. ApoJ, an apolipoprotein associated with discrete subclasses of high-density lipoproteins, is encoded by a single gene in both the human and mouse genomes. ApoJ is synthesized as a 427 amino acid polypeptide that is posttranslationally cleaved at an internal bond between Arg-205 and Ser-206. The subunits of apoJ are designated apoJ alpha, corresponding to residues 1-205, and apoJ beta, corresponding to resides 206-427. The subunits are associated through disulfide bonds. Analysis of the primary structure of apoJ predicts the existence of amphiphilic helices, which may account for the association of apoJ with lipoproteins, and heparin-binding motifs in both subunits. ApoJ appears to be the human analogue of a rat protein present in high concentrations in the testis, sulfated glycoprotein 2. ApoJ mRNA (1.9 kb) is expressed in all but one tissue examined. The mRNA is present in relatively high levels in brain, ovary, testis, and liver, is less abundant in heart, spleen, lung, and breast, and is absent in T-lymphocytes. ApoJ is unique among previously characterized human apolipoproteins in its structure and tissue distribution.

Effects of the antimutagen cinnamaldehyde on reversion and survival of selected Salmonella tester strains.

The antimutagenic activity of trans-cinnamaldehyde (C6H5CH = CHCHO) on chemically induced mutagenesis has been shown in E. coli. Using the Ames Salmonella typhimurium tester strains TA1535 (hisG46 uvrB rfa) and TA100 (TA1535/pKM101), the effects of cinnamaldehyde on spontaneous reversions and reversions induced by 4-nitroquinoline-N-oxide(4NQO) and ethyl methanesulfonate (EMS) have been examined. To observe the effect of cinnamaldehyde in the absence of functional muc genes, a third strain, TA1535/pGW201 (pKM101 muc140: :Tn5) was included in the testing. Modifications of the standard Ames test procedures and direct-plating techniques were employed to study the "antimutagenic" response exerted by cinnamaldehyde. In all strains tested, concentrations of cinnamaldehyde up to 25 micrograms/ml slightly decreased the number of spontaneous reversions and induced reversions were more markedly reduced. The decreases in the numbers of 4NQO-induced revertants were greater than those decreases which occurred for EMS-induced reversions. There was no effect on viability in 1% (v/v) nutrient broth supplemented minimal medium containing 5-25 micrograms/ml of cinnamaldehyde. Cinnamaldehyde did not display any mucAB dependent or independent specificity against the mutagens used. On minimal medium supplemented with histidine and biotin, concentrations of cinnamaldehyde above 10 micrograms/ml were lethal for the strains tested. When the test medium was supplemented with 1-5% (v/v) liquid nutrient broth, viability was not affected at concentrations up to 25 micrograms/ml. For both TA100 and TA1535 the presence of 20 micrograms/ml of cinnamaldehyde in 1% (v/v) liquid nutrient broth-supplemented minimal glucose broth extended the lag phase for 2-4 h with no effect on survival. Depending on the test procedure employed, decreases in numbers of revertants may reflect lethality rather than antimutagenesis. When used to test for antimutagenesis rather than mutagenesis, modifications of the standard Ames test procedure may mimic an antimutagenic response due to a decrease in the total number of revertants seen even though enough cells survive to produce a background lawn.