Apolipoprotein E and A-IV Polymorphisms in Ethnic Russians Living in Estonia

To determine the distribution of genetic variations in apolipoprotein E (apoE) and apolipoprotein A-IV (apoA-IV) genes, 137 Russians living in Estonia was screened by isoelectric focusing and immunoblotting procedures. The apoA-IV-2 allele and apoE4 allele frequency of the Russians tended to be lower than in most other European populations.     

Apolipoprotein E and A-IV polymorphisms in ethnic Russians living in Estonia

137 Russians living in Estonia was screened by isoelectric focusing and immunoblotting procedures to determine the distribution of genetic variations in apolipoprotein E (apoE) and apolipoprotein A-IV (apoA-IV) genes. The apoA-IV-2 allele and epsilon4 allele frequency of the Russians tended to be lower than in most other European populations.     

Effect of a neutralizing monoclonal antibody to cholesteryl ester transfer protein on the redistribution of apolipoproteins A-IV and E among human lipoproteins

The effect of inhibiting cholesteryl ester transfer protein (CETP) on the in vitro redistribution of apolipoproteins(apo) A-IV and apoE among lipoproteins in whole plasma was studied in seven normal male subjects. Plasmas were incubated in the presence of a purified monoclonal antibody TP2 (Mab TP2) that neutralizes the activity of CETP. Mab TP2 had no effect on lecithin:cholesterol acyltransferase (LCAT) activity. Prior to and following a 6-h incubation at 37 degrees C in the presence of Mab TP2 or a control mouse myeloma immunoglobulin (IgG), plasmas were gel-filtered on Sephacryl S-300 and the distribution of apoA-IV and apoE among lipoproteins was determined by radioimmunoassay. Incubation (i.e., with active LCAT and CETP) increased the amount of apoA-IV associated with lipoproteins by 240%. When CETP activity was inhibited during incubation, the amount of apoA-IV that became lipoprotein-associated was significantly increased (315% of basal). Plasma incubation also caused a redistribution of apoE from high density lipoproteins (HDL) to larger lipoproteins (131% of basal); however, when CETP was inhibited, significantly greater amounts of apoE became associated with the larger particles (155% of basal). These effects were observed in all seven subjects. Increased movement of apoE from HDL to triglyceride-rich particles was not due to displacement by apoA-IV since loss of apoE from HDL was still observed when no movement of apoA-IV onto HDL occurred, such as during LCAT or combined LCAT and CETP inhibition. We speculate that low CETP activity (e.g., in species such as rats) may lead to an increased content of HDL apoA-IV and also to apoE enrichment of triglyceride-rich lipoproteins, augmenting their clearance.     

Transport of lipid and apolipoproteins A-I and A-IV in intestinal lymph of the rat

Intestinal lipid absorption is associated with marked increases in the synthesis and secretion of apolipoprotein A-IV (apoA-IV) by the small intestine. Whether the increased intestinal apoA-IV synthesis and secretion results from increased fat uptake, increased cellular triglyceride (TG) content, or increased secretion of TG-rich lipoproteins by the enterocytes is unknown. Previous work from this laboratory has shown that a hydrophobic surfactant, Pluronic L-81 (L-81), is a potent inhibitor of intestinal formation of chylomicrons (CM), without reducing fat uptake or re-synthesis to TG. Furthermore, this inhibition can be reversed quickly by the cessation of L-81 infusion. Thus L-81 offers a unique opportunity to study the relationship between lymphatic TG, apoA-I and A-IV secretion. In this study, we studied the lymphatic transport of TG, apoA-I, and apoA-IV during both the inhibitory phase (L-81 infused together with lipid) and the subsequent unblocking phase (saline infusion). Two groups of lymph fistula rats were used, the control and the experimental rats. In the experimental rats, a phosphate-buffered taurocholate-stabilized emulsion containing 40 mumol [3H]triolein, 7.8 mumol of phosphatidylcholine, and 1 mg L-81 per 3 ml was infused at 3 ml/h for 8 h. This was then replaced by glucose-saline infusion for an additional 12 h. The control rats received the same lipid emulsion as the experimental rats, but without L-81 added, for 8 h. Lymph lipid was determined both by radioactivity and by glyceride-glycerol determination, and the apoA-I and apoA-IV concentrations were determined by rocket electroimmunophoresis assay. L-81 inhibited the rise in lymphatic lipid and apoA-IV output in the experimental rats after the beginning of lipid + L-81 infusion. Upon cessation of L-81 infusion, the mucosal lipid accumulated as a result of L-81 treatment was rapidly cleared into lymph as CM. This was associated with a marked increase in apoA-IV output; the maximal output was about 3 times that of the fasting level. There was a time lag of 4-5 h between the peak lymph lipid output and the peak lymph apoA-IV output during the unblocking phase in the experimental rats. There was also a comparable time lag between the maximal lipid and apoA-IV outputs in the control animals. Incorporation studies using [3H]leucine showed that apoA-IV synthesis was not stimulated during lipid + L-81 infusion, perhaps explaining the lack of increase in lymphatic A-IV secretion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic studies of human apolipoproteins. VII. Population distribution of polymorphisms of apolipoproteins A-I, A-II, A-IV, C-II, E, and H in Nigeria.

Familial porphyria cutanea tarda (PCT) results from a generalized deficiency of uroporphyrinogen decarboxylase (URO-D) activity. The molecular defect responsible for this disorder has not been characterized. To determine whether decreased levels of URO-D mRNA are responsible for subnormal URO-D activity, steady-state levels of URO-D mRNA in lymphoblastoid cells were determined. Northern blots were hybridized with a URO-D cDNA probe and quantified by densitometry. No difference in the levels of URO-D mRNA was detected between affected individuals and their normal relatives. Thus, the deficiency of URO-D activity in two familial PCT pedigrees characterized here does not arise from a deficiency of URO-D mRNA.     

Effects of apolipoproteins A-IV and A-I on the uptake of phospholipid liposomes by hepatocytes

We examined the effects of apolipoproteins A-IV and A-I on the catabolism of whole particles by hepatoma G2 cells and cultured primary hepatocytes. For this type of experiment, high density lipoprotein is unsuitable, because all of its lipid and protein components independently dissociate and exchange and hence poorly trace whole particle catabolism. We therefore used phosphatidylcholine liposomes with radioactive tracers entrapped within their aqueous cores. Apolipoproteins A-IV, A-I, or E added to liposomes became liposome-associated and produced no detectable release of encapsulated label. As a positive control, apolipoprotein E doubled the uptake of labeled liposomes by hepatoma cells, compared to apolipoprotein-free controls, and this increase could be blocked by the addition of excess unlabeled low density lipoprotein. Degradation of labeled liposomes by hepatoma cells was increased 6-fold by the addition of apolipoprotein E. In contrast, neither apolipoprotein A-IV nor A-I increased cellular uptake or degradation of the particles. Similar results were obtained with primary hepatocytes. In studies using apolipoprotein combinations, apolipoproteins A-IV and A-I were each able to displace apolipoprotein E from liposomes and thereby reduce cellular uptake. Our data indicate that apolipoproteins A-IV and A-I do not facilitate uptake or degradation of whole particles by liver-derived cells in vitro. However, these apolipoproteins may modulate receptor-mediated uptake of particles by reducing the amount of particle-bound apolipoprotein E.     

Electroimmunoassay of rat apolipoproteins A-I, A-IV, and E. A procedure for sample treatment to increase the sensitivity in diluted fractions

Methods for the quantitative determination of rat apolipoproteins A-I, A-IV, and E by electroimmunoassay are described. Apolipoproteins present in diluted samples of biological fluids (approx. 2 ml) were concentrated by precipitation with deoxycholate and trichloroacetic acid. The protein pellets were solubilized in 0.1 ml of 0.5 M NaOH and these samples were delipidated with tetramethylurea and assayed. This protocol enables the measurement of apolipoprotein concentrations that are at least 10 times lower than normally detectable; 0.2 micrograms of apolipoprotein A-IV, 0.2 micrograms of apolipoprotein A-I, and 0.8 micrograms of apolipoprotein E can be easily detected in samples of 2 ml.     

Polymorphisms in the aurora-A gene is not associated with lung cancer in the Turkish population

Lung cancer, a complex neoplasm of lung tissue, is influenced by several environmental and genetic factors which could be changed in each individual. Aurora-A gene is related to mitotic events such as: chromosome instability, cell cycle regulation, spindle formation, and kinetechore-microtubule connections. This centrosomic serine/threonine kinase provides a strong connection between mitotic errors and carcinogenesis. The genomic alterations such as single nucleotide polymorphisms (SNPs) can exist in molecular pathways of lung cancer. Therefore, we evaluated the role of genetic polymorphisms of Aurora-A gene in the lung cancer in the Turkish population. Genotypes of five Aurora-A polymorphisms (F31I, V57I, 6328G/A, P50L, and S104L) were determined in 102 healty controls and 102 new diagnosed lung cancer cases. All samples were genotyped with DNA sequence technique. There were not any genotype variations in P50L, S104L, and 6328G/A polymorphisms. The frequencies of both genotypes F31I and V57I in lung cancer patients were not significantly different from those in controls (p > 0.05). A multivariable logistic regression analysis including patient characteristics, such as age and gender, did not change the results.     

Genetic studies of human apolipoproteins. I. Polymorphism of apolipoprotein A-IV.

Genetic polymorphism of human apolipoprotein A-IV has been detected by means of a simple and rapid one-dimensional isoelectric-focusing technique followed by immunoblotting. In plasma samples of normal U.S. whites and blacks, the specificity and sensitivity of the technique have been demonstrated to elucidate biochemical and genetic variation present in the APO A-IV molecule. Two common alleles, APO A-IV 1 and APO A-IV 2, have been observed with respective frequencies of .909 and .088 in whites and .961 and .035 in blacks. In addition, the products of two rare alleles designated APO A-IV 3 and APO A-IV 4 also have been observed. Family studies show autosomal codominant transmission of four alleles coded by a single structural locus.     

In vivo metabolism of apolipoproteins A-IV and A-I associated with high density lipoprotein in normolipidemic subjects

The kinetics of apolipoprotein A-IV associated with high density lipoproteins (HDL) of plasma from fasting human subjects was followed for 15 days in five healthy normolipidemic volunteers. Purified apoA-IV and apoA-I were radioiodinated, respectively, with 125I and 131I, incubated in vitro with normal HDL, isolated at density 1.250 g/ml, and finally reinjected intravenously as HDL-125I-labeled apoA-IV and HDL-131I-labeled apoA-I. Blood samples were withdrawn at regular intervals for 15 days, and 24-h urine samples were collected. More than 93% (93.5 +/- 0.9%) of apoA-IV was recovered in apoA-I-containing lipoprotein particles after affinity chromatography on an anti-apoA-I column and 69.7 +/- 4.8% was bound to apoA-II in apoA-I:A-II particles separated on an anti-apoA-II column. 125I-labeled apoA-IV showed a much faster decay than 131I-labeled apoA-I for the first 5 days and thereafter the curves became parallel. Urinary/plasma ratios (U/P) for the 125I-labeled parallel. Urinary/plasma ratios (U/P) for the 125I-labeled apoA-IV were much higher than those for 131I-labeled apoA-I for the first days, but the U/P curves became parallel for the last 7 days, suggesting heterogeneity of apoA-IV metabolism. A heterogeneous multicompartmental model was constructed to describe the metabolism of lipoprotein particles containing apoA-IV and apoA-I and to calculate the kinetic parameters, fitting simultaneously all plasma and urine data for both tracers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of human apolipoprotein A-IV: a distinct domain architecture among exchangeable apolipoproteins with potential functional implications

Apolipoprotein A-IV (apoA-IV) is an exchangeable apolipoprotein that shares many functional similarities with related apolipoproteins such as apoE and apoA-I but has also been implicated as a circulating satiety factor. However, despite the fact that it contains many predicted amphipathic alpha-helical domains, relatively little is known about its tertiary structure. We hypothesized that apoA-IV exhibits a characteristic functional domain organization that has been proposed to define apoE and apoA-I. To test this, we created truncation mutants in a bacterial system that deleted amino acids from either the N- or C-terminal ends of human apoA-IV. We found that apoA-IV was less stable than apoA-I but was more highly organized in terms of its cooperativity of unfolding. Deletion of the extreme N and C termini of apoA-IV did not significantly affect the cooperativity of unfolding, but deletions past amino acid 333 on the C terminus or amino acid 61 on the N terminus had major destabilizing effects. Functionally, apoA-IV was less efficient than apoA-I at clearing multilamellar phospholipid liposomes and promoting ATP-binding cassette transporter A1-mediated cholesterol efflux. However, deletion of a C-terminal region of apoA-IV, which is devoid of predicted amphipathic alpha helices (amino acids 333-376) stimulated both of these activities dramatically. We conclude that the amphipathic alpha helices in apoA-IV form a single, large domain that may be similar to the N-terminal helical bundle domains of apoA-I and apoE but that apoA-IV lacks the C-terminal lipid-binding and cholesterol efflux-promoting domain present in these apolipoproteins. In fact, the C terminus of apoA-IV appears to reduce the ability of apoA-IV to interact with lipids and promote cholesterol efflux. This indicates that, although apoA-IV may have evolved from gene duplication events of ancestral apolipoproteins and shares the basic amphipathic helical building blocks, the overall localization of functional domains within the sequence is quite different from apoA-I and apoE.     

Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I

In this report, we have identified two apolipoproteins (apo), apoD and apoA-IV, that, together with the previously identified apoA-I and apoE, accumulate in the regenerating peripheral nerve. These four apolipoproteins were identified in regenerating rat sciatic nerves by their molecular weights, their isoelectric points, and their recognition by specific antibodies. Antibodies were also used to document the changing concentrations of these apolipoproteins in homogenates of regenerating sciatic nerves collected 1 day to 6 weeks after a denervating crush injury. By 3 weeks after injury, at their peak accumulation, apoA-IV and apoA-I had increased 14- and 26-fold, respectively, relative to their concentrations in the normal nerve. Apolipoproteins D and E, in contrast, increased over 500- and 250-fold, respectively, by 3 weeks. These same apolipoproteins also accumulated in the regenerating sciatic nerves of two other species, the rabbit and the marmoset monkey. Immunocytochemistry showed that apoD was produced by astrocytes and oligodendrocytes in the normal central nervous system, and by neurolemmal or fibroblastic cells in the normal peripheral nervous system. Metabolic labeling of both apoD and apoE by [35S]methionine during an in vitro incubation of regenerating rat sciatic nerve segments confirmed that these apolipoproteins are synthesized by the nerve. Neither apoA-IV nor apoA-I was metabolically labeled, however, suggesting that they enter the nerve from the plasma. The results from this study provide evidence that several different apolipoproteins from various sources may play a role in lipid transport within neural tissues.     

Purification of recombinant apolipoproteins A-I and A-IV and efficient affinity tag cleavage by tobacco etch virus protease

The expression of recombinant apolipoproteins provides experimental avenues that are not possible with plasma purified protein. The ability to specifically mutate residues or delete entire regions has proven to be a valuable tool for understanding the structure and function of apolipoproteins. A common feature of many recombinant systems is an affinity tag that allows for straightforward and high-yield purification of the target protein. A specific protease can then cleave the tag and yield the native recombinant protein. However, the application of this strategy to apolipoproteins has proven somewhat problematic because of the tendency for these highly flexible proteins to be nonspecifically cleaved at undesired sites within the native protein. Although systems have been developed using a variety of proteases, many suffer from low yield and, especially, the high cost of the enzyme.We developed a method that utilizes the tobacco etch virus protease to cleave a histidine-tag from apolipoproteins A-I and A-IV expressed in Escherichia coli. This protease can be easily and inexpensively expressed within most laboratories. We found that the protease efficiently cleaved the affinity tags from both apolipoproteins without nonspecific cleavage. All structural and functional measurements showed that the proteins were equivalent to native or previously characterized protein preparations. In addition to cost-effectiveness, advantages of the tobacco etch virus protease include a short cleavage time, low reaction temperature, and easy removal using the protease''s own histidine-tag.     

Free apolipoproteins A-I and A-IV present in human plasma displace high-density lipoprotein on cultured bovine aortic endothelial cells

Adult bovine aortic endothelial (ABAE) cells, exposed to serum-free medium, specifically bind 125I-labeled human high-density lipoprotein (125I-HDL). Addition of human lipoprotein-deficient serum (LPDS) reduces the specific binding of 125I-HDL in a concentration-dependent manner, such that LPDS at a concentration of 6 mg protein/ml almost completely inhibits the specific binding of 125I-HDL. ABAE cultures exposed to 125I-labeled LPDS (125I-LPDS) specifically bind two peptides, which appear as minor iodinated components in 125I-LPDS. The binding of these two components is abolished in the presence of excess amounts of unlabeled LPDS or HDL. Preincubation of ABAE cells with 25-hydroxycholesterol (25-HC) results in an increase in the binding of the two 125I-LPDS components, similar to the increase observed in 125I-HDL binding in the presence of 25-HC. These two LPDS components comigrate on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) with apolipoproteins A-I and A-IV of molecular masses 28 kDa and 43 kDa respectively. Furthermore, these two proteins were transferred from the SDS gel to nitrocellulose paper and interacted specifically with anti-(A-I) and anti-(A-IV) sera respectively. When ABAE cultures, pretreated with 25-HC in the presence of LPDS, are subjected to cell-surface iodination, the A-IV appears as one of the major proteins on the cell surface accessible to iodination. The interaction of A-IV with the cell surface of 25-HC-treated cells is not specific to ABAE cells and appears also in human skin fibroblasts. Analysis of the relative amounts of various apolipoproteins in the 125I-HDL bound to ABAE cells demonstrates a decrease in the relative amount of iodinated A-II concomitant with increase in the relative amounts of the other iodinated apolipoproteins, when compared to the composition of the native 125I-HDL. These changes are similar whether the binding is done in the presence or absence of LPDS. It indicates that the decrease in 125I-HDL binding in the presence of LPDS is not due to displacement of the iodinated apolipoproteins A-I and A-IV in the 125I-HDL by unlabeled A-I and A-IV present in LPDS. The results indicate that free apolipoproteins A-I and A-IV, present in LPDS, can displace HDL on the cell surface of ABAE cells. Thus, free A-I and A-IV, present in plasma, control the binding of HDL to endothelial cells and may regulate the process of cholesterol removal from the cells performed by HDL.     

Polymorphisms of alcohol metabolizing enzyme and cytochrome P4502E1 genes in mongolian population

Although the genetic polymorphism of the alcohol-metabolizing enzymes was extensively studied at the molecular level by many investigators, the genetic polymorphism studies for ethanolmetabolizing enzymes in Mongolians are very rare. The present study was therefore performed to determine the genetic distribution of various forms of alcohol-metabolizing enzymes such as alcohol dehydrogenase 2 (ADH2, currently accepted nomenclature ADH1B), ADH3 (ADH1C), aldehyde dehydrogenase 2 (ALDH2) and cytochrome P4502E1 (CYP2E1) in 300 healthy Mongolian males. Genetic polymorphisms were determined by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) methods. The allele frequencies ofADH2 *1 andADH2 *2 were 0.24 and 0.76;ADH3 *1 andADH3 *2 were 0.92 and 0.08;ALDH2 *1 andALDH2 *2 were 0.96 and 0.04; andCYP2E1 *C andCYP2E1 *D were 0.15 and 0.85, respectively. Compared to the results reported by other investigators, the allele frequencies ofALDH2 *2 andCYP2E1 *C among Mongolian subjects were much lower than among East Asians (Korean, Japanese, and/or Han-Chinese), while those ofADH2 andADH3 were more similar. Interestingly, this study shows that the ineffectiveALDH2 gene (ALDH2*2 allele) among Mongolians is not as common as among East Asians.     

Genetic polymorphism of cytochrome P450 2E1 in the Turkish population.

CYP 2E1 is involved in metabolic activation of carcinogenic N-nitrosamines, benzene, urethane and other low molecular weight compounds. CYP2E1 gene is present in the population in various polymorphic forms. We detected the RFLP of the human CYP2E1 gene with the restriction endonuclease PstI, RsaI and DraI in a group of 153 Turkish individuals. According to the results of the PstI/RsaI analysis, 96.07% of the subjects were of the c1/c1 genotype, and 3.93% were of the c1/c2 genotype. In the DraI RFLP analysis, 84.30% DD genotype, 15.03% CD genotype and 0.66% CC genotype were determined. The data obtained may be useful in epidemiological studies of the influence of CYP2E1 polymorphism on carcinogenesis.     

Distribution of apolipoproteins A-I and A-IV among lipoprotein classes in rat mesenteric lymph, fractionated by molecular sieve chromatography

The distribution of apolipoproteins A-I and A-IV among lymph lipoprotein fractions was studied after separation by molecular sieve chromatography, avoiding any ultracentrifugation. Lymph was obtained from rats infused either with a glucose solution or with a triacylglycerol emulsion. Relative to glucose infusion, triacylglycerol infusion caused a 20-fold increase in the output of triacylglycerol, coupled with a 4-fold increase in output of apolipoprotein A-IV. The output of apolipoprotein A-I was only elevated 2-fold. Chromatography on 6% agarose showed that lymph apolipoproteins A-I and A-IV are present on triacylglycerol-rich particles and on particles of the size of HDL. In addition, apolipoprotein A-IV is also present as 'free' apolipoprotein A-IV. The increase in apolipoprotein A-I output is caused by a higher output of A-I associated with large chylomicrons only, while the increase in apolipoprotein A-IV output is reflected by an increased output in all lymph lipoprotein fractions, including lymph HDL and 'free' apolipoprotein A-IV. The increased level of 'free' A-IV, seen in fatty lymph, may contribute to, and at least partly explain, the high concentrations of 'free' apolipoprotein A-IV present in serum obtained from fed animals.     

Role of apolipoproteins E and C in type V hyperlipoproteinemia

Type V hyperlipoproteinemia is characterized by elevations of chylomicron (CM) and very low density lipoprotein (VLDL) triglycerides. The development of this lipid disorder involves a multitude of metabolic derangements including deficient clearance of triglycerides and/or their increased output aggravated by obesity, diabetes, alcohol intake, or use of some hormones. Some studies have suggested that the apolipoprotein E4 phenotype is involved in this dyslipoproteinemia but this concept is still a matter of controversy. Therefore, we determined the apoE phenotype in 21 patients with severe hypertriglyceridemia classified as type V. Their apoE4 gene frequency was 0.595 which is 2.6-fold higher (P less than 0.001) than that in the Finnish population. Correspondingly, their apoE3 gene frequency was lower than that in the normal population. No differences were noted in plasma lipoproteins of the apoE4 phenotypes and the other type V subjects. The apolipoprotein C-II and C-III distribution was similar to that in normolipidemic subjects. The results suggest that apoE4 may be involved in the development of type V hyperlipoproteinemia.     

Apolipoprotein A-IV polymorphism in the Finnish population: gene frequencies and description of a rare allele

The apolipoprotein A-IV (apoA-IV) allele frequencies were determined in 387 adult Finns by immunoblotting after isoelectric focusing of serum. The gene frequencies were: A-IV1 = 0.942 and A-IV2 = 0.058. The phenotypes of 147 mother-child pairs studied were in accordance with the two allelic modes of inheritance. In 2 subjects, a rare apoA-IV variant was found.