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Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Glycosaminoglycans (GAGs) are a heterogeneous family of unbranched polysaccharides that exist in either a free state or attached to proteins and are found on the cell surface as well as in the extracellular matrix. GAGs play essential roles in cellular and tissue homeostasis, and their metabolism is altered in response to several pathological conditions. Despite strong experimental evidence supporting the function of GAGs in various diseases, little is known about the regulation of GAG biosynthesis via pharmacological intervention. In recent studies, the effects of several experimental drugs on GAG biosynthesis in animal models of disease were examined and key enzymes involved in GAG biosynthesis were found to be druggable. In addition to experimental small-molecule drugs that alter GAG biosynthesis, a number of clinically approved drugs modulate GAG metabolism, contributing to the therapeutic benefits associated with the use of these drugs. In this review article, we propose a classification scheme for drugs affecting GAG biosynthesis. Our goal is to present a rational approach to investigate the pharmacological regulation of these important biological molecules.

Glycosaminoglycans (GAGs) are charged polysaccharides ubiquitously present at the cell surface and in the extracellular matrix. GAGs are crucial for cellular homeostasis, and their metabolism is altered during pathological processes. However, little consideration has been given to the regulation of the GAG milieu through pharmacological interventions. In this review, we provide a classification of small molecules affecting GAG metabolism based on their mechanism of action. Furthermore, we present evidence to show that clinically approved drugs affect GAG metabolism and that this could contribute to their therapeutic benefit.

Several lines of evidence support the link between air pollution exposure and accelerated atherosclerosis. The chronic actions of air particulate matter upon the cardiovascular system, such as the enhancement of atherosclerosis, are most likely to be induced through development of a chronic pro-inflammatory and pro-oxidant state. Oxidative stress is pro-inflammatory, and many of the pro-inflammatory genes are oxidative stress responsive. In the lung, inhalation of polluted air creates an oxidative stress condition that reverberates systemically in the different body organs. The vessels are particularly sensitive to the insult. An atherogenic cascade may be initiated by the reactive species, involving oxidized lipoproteins, cytokines, and growth factors generated or evoked as a result of the stress condition. In this review, we survey the available information regarding the atherosclerotic process as modified by exposure to air particulate matter.

Spontaneous pneumomediastinum (SPM) is an uncommon disease defined by the presence of free air in the mediastinal cavity. Its pathogenesis is characterized by all the processes that cause a sudden increase of intrathoracic pressure. Triggering factors are: lower airway infections, asthma, esophageal rupture, foreign body aspiration and diabetic ketoacidosis. When a cause is not found, SPM is defined idiopathic. The abnormal presence of air in mediastinum can cause subcutaneous emphysema (SE) in the neck and upper part of the thorax. The aim of this report was to describe a patient with an underlying genetic disorder (Pierre Robin syndrome) and SPM with consequent SE. A multidisciplinary approach in our Accident and Emergency department was important for the right management.

The enzyme α-glucosidase has attracted interest owing to its involvement in the digestive process of carbohydrate, its role in intracellular glycoprotein trafficking, tumorigenesis and viral infection. In this study, several members of a new family of N-heteroarylmethyl substituted azasugars were synthesized and evaluated as α-glucosidase inhibitors. We systematically investigated the effect of different N-substituents as well as the role of hydroxyl and carboxylate moieties on the piperidine ring. The compounds N-heteroarylmethyl-5-hydroxy-1,2,5,6-tetrahydropyridine-3-carboxylic acid emerged as potent α-glucosidase inhibitors. Unlike Acarbose and other clinically relevant α-glucosidase inhibitors, these compounds act through a reversible uncompetitive mechanism of inhibition which make them attractive candidates for drug development.

The structural maintenance of chromosome protein SMC3 is a component of the cohesin complex that mediates sister chromatid cohesion and segregation in prokaryotes and eukaryotes. It is also present extracellularly in the form of a chondroitin sulfate proteoglycan known as bamacan. We have found previously that SMC3 expression is elevated in a large fraction of human colon carcinomas. The additional finding that the protein is significantly increased in the intestinal polyps of ApcMin/+ mice has led us to hypothesize that SMC3 expression is linked to activation of the APC/beta-catenin/TCF4 pathway. The immunohistochemical analysis of colon adenocarcinomas from clinical specimens revealed that beta-catenin and SMC3 antigens co-localize with maximal stain intensity within the transformed areas. Cloning and sequencing of 1578 bp of the human SMC3 promoter unveiled the presence of seven putative consensus sequences for beta-catenin/TCF4 binding, two of which are conserved in the mouse Smc3 promoter. Transient transfection experiments in HCT116 and SW480 human colon carcinoma cells using deletion and mutated promoter constructs in luciferase reporter vectors confirmed that the putative sites, the first located at -48 bp and the second located at -701 bp, are susceptible to beta-catenin/TCF4 transactivation. Co-transfection with a beta-catenin expression vector enhanced the promoter activity whereas E-cadherin had the opposite effect. Binding of beta-catenin/TCF4 complexes from SW480 nuclear extracts to these sequences was confirmed by electrophoretic shift and supershift mobility assays. Altogether these results are consistent with the idea that the beta-catenin/TCF4 transactivation pathway contributes to SMC3 overexpression in intestinal tumorigenesis.

Incubation of human HDL (d = 1.063-1.21 g/ml) with monocyte-derived elastase causes selective proteolysis of apoA-II and apoA-I apolipoproteins. We have found that elastase-digested HDL (ED-HDL) bind to J774-A1 murine macrophages with enhanced affinity and are internalized and degraded at a rate threefold higher than that of native HDL. Unlike oxidized LDL and HDL and proteolytically modified LDL, the uptake of ED-HDL lipoproteins does not affect the cellular lipid biosynthesis nor modify the cell lipid content. The cell surface binding of (125)I-ED-HDL can be competed by native HDL but not by acetylated LDL, consistent with the idea that ED-HDL are recognized by the class B type I scavenger receptor. The liberation of elastase by lipid-engorging macrophages is regarded as an important event during atherogenesis. By enhancing the cellular uptake of HDL this process can lead to a local decrease of antiatherogenic HDL particles.

Bamacan is a chondroitin sulfate proteoglycan that abounds in basement membranes. To gain insights into the bamacan gene regulation and transcriptional control, we examined the genomic organization and identified the promoter region of the mouse bamacan gene. Secondary structure analysis of the protein reveals a sequential organization of three globular regions interconnected by two α-helix coiled-coils. The N- and the C-terminal ends carry a P-loop and a DA box motif that can act cooperatively to bind ATP. These features as well as the high sequence homology with members of the SMC (structural maintenance ofchromosome) protein family led us to conclude that bamacan is a member of this protein family. The gene comprises 31 exons and is driven by a promoter that is highly enriched in GC sequences and lacks TATA and CAAT boxes. The promoter is highly functional in transient cell transfection assays, and step-wise 5′ deletions identify a strong enhancer element between −659 and −481 base pairs that includesJun/Fos proto-oncogene-binding elements. Using backcrossing experiments we mapped the Bam gene to distal chromosome 19, a locus syntenic to human chromosome 10q25. Bamacan is differentially expressed in mouse tissues with the highest levels in testes and brain. Notably, bamacan mRNA levels are low in normal cells and markedly reduced during quiescence but are highly increased when cells resume growth upon serum stimulation. In contrast, in all transformed cells tested, bamacan is constitutively overexpressed, and its levels do not change with cell cycle progression. These results suggest that bamacan is involved in the control of cell growth and transformation.

Rabbits fed a wheat starch casein diet develop hypercholesterolaemia characterized by the plasma elevation of low density lipoprotein (LDL) that is caused by oversecretion of apoB-100 containing lipoproteins by the liver and by the suppression of the EDTA-sensitive hepatic beta- very low density lipoprotein (VLDL)-LDL receptor. In this study, the effect of FCE 27677 ((-)N-[2,6-bis(1-methylethyl)phenyl]-N'-[(4R,5R)-2-(4-dimethylaminoph eny l)-4,5-dimethyl-dioxolan-2-yl]methylurea) a novel potent systemic acylCoA:cholesterol acetyltransferase (ACAT, EC 2.3.1.26) inhibitor, has been evaluated. When New Zealand White rabbits were fed with casein for 4 weeks, LDL cholesterol increased from 14 +/- 3 mg/dl-1 to 77 +/- 6 mg/dl-1. By contrast the animals receiving FCE 27677 (10 mg kg-1 d-1) mixed with the casein diet maintained a normal LDL concentration (22 +/- 3 mg dl-1). This hypolipidaemic effect was also observed when rabbits previously made hypercholesterolaemic by being fed casein for 4 weeks were then treated for a month with FCE 27677. [125I]LDL plasma turnover studies and [125I]LDL binding studies to liver membranes were carried out with the purpose of investigating the mechanism of action of the drug. The LDL apoB-100 production rate in chow-fed, casein-fed, and casein-fed rabbits receiving FCE 27677, was respectively 10.5, 22.4, and 12.5 mg kg-1 d-1. The turnover rate of [125I]LDL in the animals receiving the drug was not, however, different from that in the rabbits fed the casein diet alone (2.381 vs 2.079 pools d-1). Both values were lower than that in chow-fed animals (3.271 pools d-1). FCE 27677 did not normalize the activity of the hepatic beta-VLDL-LDL EDTA-sensitive receptor which is suppressed by casein feeding. Altogether the results are consistent with the idea that FCE 27677 by acting through inhibition of the cholesterol esterification in the liver normalizes the LDL synthetic rate. ACAT inhibitors may be useful drugs for the treatment of human dyslipoproteinaemia secondary to derangement of the apoB-100 synthetic rate.

There is evidence that the overproduction of apoB-100-containing lipoproteins by the liver is the underlying event in some forms of dyslipoproteinemia. This metabolic status is associated to an increased risk of developing premature coronary artery disease CAD. The conclusions from previous studies suggested that the availability to the hepatocytes of cholesterol that is readily esterified is an important determinant for VLDL and LDL secretion. In the present study, we set out to investigate the effect of the specific stimulation and inhibition of the rate-limiting enzyme of the cholesterol esterification, acyl-CoA:cholesterol acyltransferase (ACAT, E.C. 2.3.1.26), on the lipid and on the apoB-100 secretion rate from a human hepatoma cell line (HepG2). When the specific ACAT inhibitor FCE 27677 (10-5 M) was added to the cultures, a decrease of the cellular cholesteryl ester content and at the same time a significant reduction of the neutral lipids and of the apoB-100 secretion rate were noticed. The stimulation of ACAT by 25-hydroxycholesterol (20 microgram/ml) caused a 4-fold increase of the cellular cholesteryl ester content and a 2-fold increase of the lipoprotein secretion rate. FCE 27677 (10-5 M to 10-7 M) prevented the effects elicited by the oxysterol. On the contrary, lovastatin (10-6 M) and gemfibrozil (10-6 M) had no effect. The analysis of the lipid and of the apolipoprotein composition of the lipoproteins secreted in the medium revealed that ACAT inhibition had the dual effect of both decreasing the number of apoB-100-containing lipoproteins secreted as well as their cholesteryl ester load. Altogether, these data support the idea of a close relationship between ACAT activation, leading to increased cholesteryl ester availability, and apoB-100-containing lipoprotein secretion. It is speculated that ACAT inhibitors may prove useful for the treatment of human dyslipoproteinemias caused by the hepatic overproduction of apoB-100-containing lipoproteins.

The synthesis and the pharmacological evaluation of FCE 28654, a novel water-soluble ACAT inhibitor, are reported. The compound positively modifies blood and hepatic cholesterol levels when orally or intravenously administered to hypercholesterolemic animals.

A decreased plasma concentration of high-density lipoprotein (HDL) cholesterol is associated with a higher incidence of coronary artery disease in populations. Therefore, there is intense investigation into the mechanisms responsible for the regulation of HDL cholesterol concentration in plasma. Insulin has a potent effect on HDL cholesterol, but it is unclear whether this is mediated by the primary effect insulin has on plasma triglycerides (TG). In this study, the question of the relationship between glucose, insulin, and HDL cholesterol has been addressed by investigating a cohort of nondiabetic normolipidemic men living in the Venice, Italy, area. One hundred twenty-eight men aged 30 to 69 years were initially recruited. The following parameters were measured: fasting plasma cholesterol, TG, HDL cholesterol, glucose, and insulin. One hundred seventeen of these subjects underwent an oral glucose tolerance test (OGTT), and the glucose and insulin responses were assessed. The final statistical analysis was performed on 98 nondiabetic individuals with plasma lipid levels within the 75th percentile for cholesterol and TG concentrations of the general population of the same age. The insulin response was a positive independent variable for plasma TG (P < .005) and HDL cholesterol (P < .005). On the other hand, HDL cholesterol was negatively associated with plasma TG. This relationship remained significant (P < .0001) also after controlling for age, body mass index (BMI), and glucose- and insulin-related measurements. Consistent with these results, both a stepwise variable selection analysis and a stratification analysis of the data indicated that the plasma TG concentration is the major determinant of HDL cholesterol level. The insulin response appears to play a less important role. No independent relationship could be found between fasting plasma glucose and insulin, or the glucose response and HDL cholesterol.

Elevation of blood cholesterol, low-density lipoproteins (LDL) and apolipoprotein B (apoB) are hallmarks of familial hypercholesterolemia (FH), a genetic condition caused by the defective functioning of the cellular receptor for apoB-100 in LDL. ApoB-100 is also present in lipoprotein(a) (Lp(a)). In this lipoprotein, apoB-100 is linked to a plasminogen-like protein called apo(a). By direct comparison of the Lp(a) and apoB plasma concentrations in 28 affected and 31 unaffected members of seven families carrying the FH trait and without history of coronary artery disease, we reached the conclusion that LDL receptor activity is not a major determinant of the Lp(a) plasma levels in these subjects. This suggests that the molecular determinants for catabolism of Lp(a) are not the same as those for LDL. Consistent with this view is our observation that the turnover rate of Lp(a) and of LDL apoB, calculated from their rate of reappearance in plasma following Lp(a)/LDL apheresis, differ greatly.

Uptake of modified lipoproteins by resident arterial monocytes/macrophages is believed to be a key event in the formation of foam cells and thus in the early phases of atherosclerosis. Low-density lipoproteins (LDLs) that undergo oxidative changes become suitable for uptake by macrophages through a specific scavenger receptor that leads to cholesteryl ester accumulation. Because the interaction of other oxidized lipoproteins with macrophages has been poorly investigated, we studied the effect of oxidatively modified high-density lipoproteins (HDLs) on the sterol metabolism of J774-A1 macrophages. Unlike native HDLs, oxidized HDLs caused a concentration-dependent accumulation of unesterified cholesterol and decreased [14C]oleate incorporation into steryl esters. Oxidized HDLs also decreased [14C]acetate incorporation into newly synthesized sterols. Cell surface binding of 125I-oxidized HDLs to the macrophages was saturable, with an apparent dissociation constant (Kd) of 0.96 nmol/mL. Both oxidized and acetylated LDLs but not native lipoproteins could compete for binding of 125I-oxidized HDL. The data support the conclusion that the effects elicited by oxidized HDLs on the sterol metabolism of macrophages are significantly different from those of native HDLs. The binding of oxidized HDLs to macrophages occurs at sites that are likely the same as those for modified LDLs. We speculate that, if occurring in vivo, HDL oxidation would generate modified lipoproteins capable of modulating the cholesterol homeostasis of macrophages.

The association between low-density lipoprotein (LDL) and a series of well characterized dermatan and chondroitin sulfates has been investigated by means of the fluorescence anisotropy technique with competition experiments using a fluorescein-labeled high LDL-affinity heparin fraction as a reference. Preparations of glycosaminoglycan (GAG) with sulfation degrees varying over a wide range, as obtained by fractionation or by chemical modification, were chosen for this study. The influence of chain length, which had been found sizeable in a former study of heparin affinity for LDL, was taken into account with an empirical correction of dissociation constants. After this correction, a linear relationship was found between the logarithm of dissociation constants and the number of sulfate groups per disaccharide unit, ns, both for dermatan and chondroitin sulfates, and for heparins. At comparable ns values, however, dermatan sulfates and heparins, which contain L-iduronic acid in their backbone, show higher LDL-affinity than chondroitin sulfates, which contain only D-glucuronic acid. Though confirming a non-specific, predominantly electrostatic interaction between GAGs and LDL, these results indicate modulation of LDL affinity by the polysaccharide backbone.

The synthesis and the pharmacological evaluation of FCE 28654, a novel water-soluble ACAT inhibitor, are reported. The compound positively modifies blood and hepatic cholesterol levels when orally or intravenously administered to hypercholesterolemic animals.

Hypoalphalipoproteinemia (plasma HDL-cholesterol concentration at or below 35 mg/dl as reported in the National Cholesterol Education Program Guidelines) is a well known risk factor for premature coronary artery disease (CAD). In hypertriglyceridemic patients, hypoalphalipoproteinemia is commonly believed to be linked to the derangement of triglyceride metabolism. In this study the occurrence of primary hypoalphalipoproteinemia has been investigated in a cohort of hypertriglyceridemic patients whose plasma triglyceride concentration had been normalized either through diet or diet plus drug treatment. Following the initial visit, 115 hypertriglyceridemic patients received dietary advice and returned for the second visit four months later. Diet reduced plasma triglycerides in all the patients. HDL-cholesterol increased in 76 patients whereas in the others, it remained unchanged or even decreased. Plasma triglyceride concentration was normalized (less than 200 mg/dl) in 54 patients by diet alone, but among these 11 remained hypoalphalipoproteinemics. Patients in whom, despite dietary restrictions, triglycerides exceeded 200 mg/dl, were considered for pharmacological treatment with Bezafibrate (300 mg t.i.d.) for 4 months. Thirty-nine concluded the study. Treatment significantly decreased plasma triglyceride concentration in all the subjects. Normalization was achieved in 32 patients. Four of them, however, remained hypoalphalipoproteinemic. These results indicate that a subgroup of hypertriglyceridemic patients remained hypoalphalipoproteinemic even after normalization of triglyceride levels. In these patients hypertriglyceridemia and hypoalphalipoproteinemia may occur as expression of two distinct primary metabolic defects.

Background Heparan sulfate (HS) is an ubiquitous component of the extracellular matrix that binds and modulates the activity of growth factors, cytokines and proteases. Animals with defective HS biosynthesis display major developmental abnormalities however the processes that are affected remain to be defined. D-glucuronyl-C5-epimerase (Glce) is a key HS chain modifying enzyme that catalyses the conversion of glucuronic acid into iduronic acid, a biosynthetic step that enhances HS biological activity. In this study the role of Glce during early zebrafish development has been investigated. Results Two Glce-like proteins (Glce-A and -B) are expressed in zebrafish at all times. They are the products of two distinct genes that, based on chromosomal mapping, are both orthologues of the same single human gene. Transcripts for both proteins were detected in fertilized zebrafish embryos prior to the onset of zygotic transcription indicating their maternal origin. At later developmental stages the epimerases are expressed widely throughout gastrulation and then become restricted to the hindbrain at 24 h post-fertilization. By monitoring the expression of well characterized marker genes during gastrulation, we have found that misexpression of Glce causes a dose-dependent expansion of the ventral structures, whereas protein knockdown using targeted antisense morpholino oligonucleotides promotes axis dorsalization. The ventralizing activity of Bmp2b is enhanced by Glce overexpression whereas Glce knockdown impairs Bmp2b activity. Conclusion Glce activity is an important determinant of of dorso-ventral axis formation and patterning in zebrafish. In particular Glce acts during gastrulation by affecting Bmp-mediated cell specification. The results obtained further corroborate the concept that HS encodes information that affect morphogenesis during early vertebrate development.

Heparan sulphate (HS) is a ubiquitous constituent of the extracellular matrix that is required for the biological activity of circulating soluble and insoluble extracellular ligands. GLCE (D-glucuronyl C5-epimerase), an enzyme responsible for the epimerization of D-glucuronic acid into L-iduronic acid of HS, endows the nascent polysaccharide chain with the ability to bind to growth factors and cytokines. In order to examine the mechanism of regulation of GLCE expression, the functional organization of the human GLCE gene promoter has been investigated. Studies utilizing stepwise deleted and site-directed mutagenized promoter constructs have shed light on the functional relevance of two cis-acting binding elements for the β-catenin–TCF4 complex (where TCF4 stands for T-cell factor 4) that are located in the enhancer region of the promoter. The ability of the putative binding sequences to bind the β-catenin–TCF4 complex has been confirmed through electrophoretic mobility-shift and supershift analyses. We have found that, in a set of human colon carcinoma cell lines, the expression of GLCE correlates with the degree of activation of the β-catenin–TCF4 transactivation complex. Furthermore, the ectopic expression of β-catenin–TCF4 in cells that constitutively express low levels of the transactivation complex produces a significant increase of GLCE transcript level and, at the same time, enhances the rate of D-glucuronic acid epimerization in HS. The data obtained are consistent with the idea that the β-catenin–TCF4 transactivation pathway plays a major role in modulating GLCE expression, thus contributing to the regulation of HS biosynthesis and its structural organization.

The structural maintenance of chromosome 3 protein (SMC3) is a component of the multimeric cohesin complex that holds sister chromatids together and prevents their premature separation during mitosis. By screening a human cDNA library for interacting proteins we have established that the proto-oncogene RET finger protein (RFP) interacts with SMC3. The sites of interaction map to part of the central coiled coil region of RFP and to the C-terminal region of the SMC3 globular hinge domain. SMC3/RFP interaction was confirmed in vivo by co-immunoprecipitation studies and by performing mammalian two-hybrid interaction assays. Cytoimmunolocalization experiments showed that SMC3 and RFP co-localize in the same cell substructures. Overexpression of RFP in NIH3T3 cells significantly increased the fraction of SMC3 recovered in the nucleus supporting the idea that RFP regulates the intracellular distribution of SMC3. These studies identify a novel SMC3-interacting protein that may affect SMC3 availability to complex with its cohesin partners.

Background The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. Results In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. Conclusions Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

Objective— Epidemiological studies have demonstrated that moderate alcohol consumption reduces mortality associated with coronary artery disease. The protective effect is correlated with the amount of ethanol consumed but is unrelated to the form of alcoholic beverage. Adoption of a favorable lipoprotein profile accounts for about half of the protective action of alcohol, but the remaining causative factors remain conjectural. Fibroblast growth factors (FGFs) play important roles in mediating smooth muscle cell (SMC) proliferation and migration, which are key factors in the atherosclerotic process. In the present study, we examined the effect of ethanol on FGF-mediated SMC growth and signaling. Methods and Results— Pharmacologically relevant concentrations of ethanol inhibited the proliferation of a rat aortic SMC line (SV40LT-SMCs) in response to FGF1 and FGF2. Human aortic SMC growth was similarly inhibited by ethanol. Transition into the G 2 /M phase was specifically affected. FGF-mediated phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) c-Raf, MAP kinase kinase kinase, MEK1/2 MAP kinase, kinase, stress-activated protein kinase/c-Jun–NH 2 -terminal kinase, and p38 MAPK were variably reduced by ethanol. The inhibition of intracellular signaling by ethanol was correlated with inhibition of FGF receptor autophosphorylation. By contrast, neither epidermal growth factor receptor autophosphorylation nor epidermal growth factor–mediated p42/p44 MAPK activation was affected by ethanol. Conclusions— The findings identify the FGF receptor as an inhibitory target for ethanol, which could account in part for the inhibitory actions of ethanol on SMC proliferation observed in vivo.

Bamacan can occur in certain cell types as either a secreted proteoglycan assembled into basement membranes or as an intracellular protein known as structural maintenance of chromosome 3 (SMC3). To assess the role of this protein in tumorigenesis, we investigated whether induced overexpression of bamacan/SMC3 could transform normal fibroblasts. We generated a full-length cDNA encoding the entire mouse bamacan/SMC3 and demonstrated appropriate transcription and translation into a 146-kDa protein. All the NIH and Balb/c 3T3 murine fibroblasts overexpressing this bamacan/SMC3 transgene generated foci of transformation and acquired anchorage-independent growth. The increased levels of bamacan/SMC3 expression achieved in the transfected fibroblasts were the same as those detected in a series of spontaneously transformed murine and human colon carcinoma cells. Moreover, a 3-4-fold overexpression of bamacan/SMC3 was detected in approximately 70% of human colon carcinoma specimens from matched pairs (n = 19, p0.0002) and in a cohort of intestinal tumors from Apc-deficient Min/+ mice. These results support the concept that deregulated expression of bamacan/SMC3 is involved in cell transformation.

Heparan sulfate is thought to regulate the biological activities of several proteins implicated in the pathogenesis of atherosclerosis. While the interactions of heparan sulfate with lipoprotein lipase and various growth factors have been actively studied, little is known of the cellular regulation of heparan sulfate biosynthesis in response to lipid accumulation. We have investigated heparan sulfate biosynthesis during conversion of murine J774 macrophages into lipid-laden foam cells. Such conversion is shown to accelerate the rate of glycosaminoglycan synthesis and the transport of newly synthesized proteoglycans into the medium. Moreover, the structure of heparan sulfate is specifically altered due to an approximately 30% increase in the 6-O-sulfation of glucosamine residues within the N-sulfated heparan sulfate domains, whereas the sulfation of chondroitin sulfate remains unaffected. These results suggest a selective effect of foam cell conversion on the biosynthesis of heparan sulfate.

The proliferation of smooth muscle cells (SMC) in the arterial wall is a key event in the development of atherosclerosis. The locally formed mitogen bFGF is likely to play an important role in initiating and in maintaining the SMC proliferative state. A distinguishing characteristic of bFGF is its strong interaction with the sulfated glycosaminoglycans. In this study the ability of a series of chemically modified heparins and heparan sulfates to compete with the cell surface glycosaminoglycans for 125I-bFGF binding has been investigated. Rat aortic SMC between the 8th and the 18th passage were used. Within the heparin series the order of potency was: Heparin, 6-O-desulfated > 2-O desulfated >> N-acetylated. However, when the activity of the series of heparins for the inhibition of cell growth was assessed, 6-O sulfation was as important as 2-O and N- sulfation. Heparan sulfates gave comparable results. The data presented are consistent with the concepts that: 1) specific structural features are involved in the interaction of SMC glycosaminoglycans with 125I-bFGF, and 2) antimitogenic activity of heparin has structural requirements different than those for bFGF binding.

FCE 27677 ([(-)N-[2,6-bis(1-methylethyl)phenyl]-N'-[(4R,5R)-2- (4-dimethylaminophenyl)-4,5 dimethyl-dioxolan-2-yl]methylurea) is a new systemically available ACAT inhibitor belonging to the class of ketalic disubstituted ureas. When tested in microsomes from rabbit intestine, aorta and liver, it inhibited the enzyme with IC50 of 9.31, 6.99 and 92.2 nM, respectively. It had no effect on plasma LCAT and intestinal cytosolic cholesterol esterases and, when tested in a tissue culture system, it did not interfere with the synthesis of cholesterol, triglycerides, and phospholipids. Enzyme inhibition kinetics indicated that FCE 27677 is a non-competitive inhibitor of the enzyme with respect to acylCoA and to cholesterol. When administered mixed to a 1.5% cholesterol and 0.5% sodium cholate-enriched diet to rats, it prevented the development of hypercholesterolemia with ED50 of 0.35 mg kg-1 day-1. Given in a single oral dose to hypercholesterolemic rats it significantly reduced both the plasma lipid levels and the hepatic cholesteryl ester content within 6 h from gavage. VLDL and LDL levels and composition were also significantly affected. Similar effects were observed when the drug was given mixed to a regular chow diet for 4 weeks to hypercholesterolemic rabbits. These results are consistent with the idea that systemically available ACAT inhibitors can affect the composition and the metabolism of the atherogenic cholesteryl ester-rich VLDL and LDL. ACAT inhibitors appear promising for the correction of dyslipoproteinemias secondary to lipoprotein overproduction, and in reducing the atherogenic index of apoB-100 containing lipoproteins.

BACKGROUND: The vascular endothelium produces nitric oxide, which has vasodilatory properties. It has been postulated that some lipoproteins may increase arterial vascular tone by decreasing the availability of endothelium-derived nitric oxide. The mechanism underlying this effect, however, is still poorly understood. METHODS: We investigated the effect of native and oxidized human low- and high-density lipoproteins on the nitric oxide synthetic activity of an endothelioma cell line (bEnd.4). Oxidized lipoproteins were obtained by incubation with CuSO4. The production of nitric oxide by the cells was monitored by quantifying the nitrite concentration in the medium using Greiss reagent. RESULTS: The synthesis of nitric oxide by the bEnd.4 cell line was calcium-dependent and was abolished by a selective inhibitor of the constitutive nitric oxide synthase. Incubation with oxidized lipoproteins caused a time- and dose-dependent inhibition of nitric oxide synthetic activity. At a concentration of 100 micrograms/ml cholesterol, oxidized low- and high-density lipoproteins inhibited the production of nitric oxide by 27 and 51%, respectively, within 6h. The lipid fraction obtained from the native or the oxidized lipoproteins mimicked the effect of the intact lipoproteins. CONCLUSION: These results support the involvement of oxidized lipoproteins in the modulation of endothelial functions relevant to the pathogenesis of cardiovascular disease.

Several biological properties of lipoproteins are modified by oxidative reactions. Modified lipoproteins are rapidly degraded by macrophages, and this is likely to be a major pathway for the formation of foam cells in the early phases of atherosclerosis. The effect of modification on other aspects of cholesterol homeostasis has, however, received lesser attention. In this study, the influence of copper ion- as well as rat aortic smooth muscle cell-oxidation-modified high density lipoprotein (HDL) on cholesterol biosynthesis in human skin fibroblasts has been investigated. Modified lipoproteins eluted at higher ionic strength than did control HDL on a Mono-Q 5/5 anion-exchange column. However, only copper ion-modified HDLs displayed greater electrophoretic mobility than did control lipoproteins on agarose gel electrophoresis. Both control and modified HDLs decreased cholesterol esterification in fibroblasts. On the other hand, whereas control HDLs were virtually ineffective in modulating cholesterol biosynthesis, modified HDLs had a significant suppressing effect. This was observed in normal as well as low density lipoprotein (LDL) receptor-defective fibroblasts, which are unresponsive to the LDL-mediated downregulation of cholesterol synthesis. These results are consistent with the concept that oxidative modification of HDLs drastically alters their effect on cholesterol homeostasis in fibroblasts. The data furthermore suggest the existence of a lipoprotein pathway for cholesterol biosynthesis regulation that is independent of the LDL receptor-mediated pathway. Downregulation of cholesterol biosynthesis would be a new function for oxidatively modified lipoproteins.

Familial Hypercholesterolemia (FH) is a condition characterized by markedly elevated blood cholesterol, low-density lipoproteins (LDL), and apolipoprotein B-100 (apo B). The molecular basis of this monogenic disease is the defective functioning of the cellular receptor for LDL that recognizes apo B. Lipoprotein(a) [Lp(a)] is a circulating lipoprotein that is structurally related to LDL, as it also contains apo B. To assess the impact of the LDL receptor deficiency on the plasma Lp(a) concentration, we measured Lp(a) in 28 FH patients and in 31 unaffected relatives. Because elevation of Lp(a) concentration in plasma of patients with coronary artery disease (CAD) appears to occur independently from plasma cholesterol levels, to avoid potentially confounding problems, members of the families chosen had no history for the disease. Whereas apo B clearly showed a bimodality of distribution by being significantly higher in the FH patients (166 ± 38 mg/dL) than in the unaffected relatives (92 ± 18 mg/dL), Lp(a) concentration did not differ in the two groups of patients (30 ± 24 mg/dL in the FH patients v 31 ± 23 in the normolipidemic relatives). Similar results were obtained when only siblings were further considered. We conclude that although Lp(a) is closely related to LDL structurally, its level in plasma is not significantly affected by the LDL receptor activity.

Binding between low-density lipoproteins (LDL) and fluorescein-labeled heparin was studied quantitatively with a modified form of a published procedure [Cardin, A. D., Randall, C. I., Hirose, N., & Jackson, R. L. (1987) Biochemistry 26, 5513-5518], using fluorescence anisotropy titrations. Assumption of binding site equivalence satisfactorily interpreted experimental data. Accordingly, the apparent total capacity, n, and the average dissociation constant, Kd, were estimated as n approximately 24 disaccharides per LDL particle and Kd approximately 4 microM in 0.05 M HEPES/0.1 M NaCl, pH 7.4, 22 degrees C. Competition experiments with unlabeled heparins were exploited for the quantitative study of Kd as a function of heparin chain length and sulfation degree (ns = sulfate groups per disaccharide). The former parameter was investigated with a series of bovine lung heparin fractions with Mw ranging from 1,800 to 21,000 and constant sulfation degree (ns = 2.8 +/- 0.1). A series of physically fractionated or chemically modified heparins having 1.2 less than ns less than 3.5 were used to explore the dependence on sulfation degree. LDL affinity was found to increase with increasing both ns and Mw: an empirical Mw-1.6 dependence represented very well the chain length data set; a linear dependence was observed for log Kd as a function of ns, after appropriate allowance was made for chain length differences among samples. This regularity confirmed that LDL-heparin binding is mainly driven by electrostatic forces.(ABSTRACT TRUNCATED AT 250 WORDS)

Cholesterol accumulation in macrophages that have migrated in the subintimal space leads to foam cell formation, which is believed to be one of the initiating events in atherosclerosis. In this study we investigated the effect of cholesterol feeding on peritoneal monocyte/macrophage cholesterol content and peritoneal cavity lipoprotein composition in rats. A cholesterol (2%) and cholic acid (1%) diet caused significant hypercholesterolemia in plasma, and at the same time the cholesterol content of peritoneal monocytes/macrophages was increased. At day 7, the cellular cholesteryl ester content had risen to 30.1 micrograms/mg cellular protein from a baseline value of 9.2 micrograms/mg. The unesterified cholesterol content also increased by 56%. At this time, acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity was doubled, whereas neutral and acidic cholesteryl ester hydrolase activities were unchanged. Reversal to the regular chow diet after 7 days of the cholesterol-enriched diet normalized plasma cholesterol levels as well as peritoneal monocyte/macrophage cholesteryl ester content. ACAT activity also decreased toward normal levels. Analysis of the d less than 1.21 g/ml peritoneal lipoproteins isolated by ultracentrifugation revealed the presence, in both normal and hypercholesterolemic rats, of apolipoprotein A-I-rich lipid complexes with pre-beta mobility on agarose gel electrophoresis. The size of the peritoneal lipoproteins was smaller than that of plasmatic high density lipoproteins, and their chemical composition was also different from that of the major plasma lipoproteins. The cholesteryl ester content of peritoneal lipoproteins increased after feeding of the cholesterol-enriched diet. In conclusion, our results show that cholesterol feeding leads to rapid accumulation of cholesteryl esters in monocytes/macrophages. As soon as plasma cholesterol levels are returned to normal, cellular cholesterol content is also normalized.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-IV (apo A-IV) is present in plasma associated to both HDL and as a complex with lipids that cannot be flotated by ultracentrifugation at 1.21 g/ml density. Apo A-IV is likely an important molecular determinant in HDL binding to the liver. In this communication, data are presented supporting the view that a specific liver plasma membrane protein of Mr 95000 is a constituent of the apo A-IV binding site. The protein was solubilized with CHAPS from purified rat liver plasma membranes and subjected to SDS-PAGE. Transblotted to nitrocellulose sheet could be identified as recognizing 125I-apo A-IV-DMPC by autoradiography. 125I-apo A-I-DMPC and radioiodinated rat apo E-poor HDL, also bound to the protein. Apo B-100 (as human LDL) and apo C-III did not bind. The protein identified is likely to be the same that has been previously identified by Graham and Oram ((1987) J. Biol. Chem. 262, 7439–7442) as ‘HDL receptor protein’.

The term reverse cholesterol transport first introduced by Glomset (1), identifies a process where there is net flux of cholesterol from the peripheral tissues to the liver. Because the liver is the single organ capable of significant cholesterol elimination from the body via bile acids peripheral tissues must utilize this system for carrying excess cholesterol (both the newly synthesized and that derived from plasma) to the liver. Since the first inception of the idea it was held that circulating HDL were the plasma elements mainly implicated in reverse cholesterol transport. They would pick up cholesterol from extrahepatic cells through a process amplified by continous esterification of this cholesterol by lecithin cholesterol acyl transferase (LCAT), and final delivery to the liver. It has been within this conceptual framework that investigations on reverse cholesterol transport have developed in the last twenty years. The picture have become more complex with evidences for specific receptorial processes, protein(s) facilitating cholesteryl ester transfer between lipoproteins, and a number of other circulating putative tissue cholesterol acceptors/carriers in addition to HDL. The following is a brief account of the current status on reverse cholesterol transport research with some emphasis on the role of apolipoprotein A-IV (apoA-IV) which we have investigated more closely. The three main metabolic compartment composing the reverse cholesterol system, i.e. the peripheral tissue, the plasma compartment, and the liver, are discussed individually.

The structural maintenance of chromosome 3 (SMC3) protein is a constituent of a number of nuclear multimeric protein complexes that are involved in DNA recombination and repair in addition to chromosomal segregation. Overexpression of SMC3 activates a tumorigenic cascade through which mammalian cells acquire a transformed phenotype. This has led us to examine in depth how SMC3 level affects cell growth and genomic stability. In this paper the effect of SMC3 knockdown has been investigated. Mammalian cells that are SMC3 deficient fail to expand in a clonal population. In order to shed light on the underlying mechanism, experiments were conducted in zebrafish embryos in which cell competence to undergo apoptosis is acquired at specific stages of development and affects tissue morphogenesis. Zebrafish Smc3 is 95% identical to the human protein, is maternally contributed, and is expressed ubiquitously at all developmental stages. Antisense-mediated loss of Smc3 function leads to increased apoptosis in Smc3 expressing cells of the developing tail and notocord causing morphological malformations. The apoptosis and the ensuing phenotype can be suppressed by injection of a p53-specific MO that blocks the generation of endogenous p53 protein. Results in human cells constitutively lacking p53 or BAX, confirmed that a p53-dependent pathway mediates apoptosis in SMC3-deficient cells. A population of aneuploid cells accumulated in zebrafish embryos following Smc3-knockdown whereas in human cells the transient downregulation of SMC3 level lead to the generation of cells with amplified centrosome number. Smc3 is required for normal embryonic development. Its deficiency affects the morphogenesis of tissues with high mitotic index by triggering an apoptotic cascade involving p53 and the downstream p53 target gene bax. Cells with low SMC3 level display centrosome abnormalities that can lead to or are the consequence of dysfunctional mitosis and/or aneuploidy. Collectively the data support the view that SMC3 deficiency affects chromosomal stability leading to the activation of p53-dependent mitotic checkpoint.

The structural maintenance of chromosome proteins SMC1 and SMC3 play an important role in the maintenance of chromosomal integrity by preventing the premature separation of the sister chromatids at the onset of anaphase. The two proteins are constitutive components of the multimeric complex cohesin and form dimers by interacting at their central globular regions. In order to identify proteins that by binding to SMC3 may interfere with the protein dimerization process, a human cDNA library was screened by the yeast two-hybrid system by using the hinge region of SMC3 as bait. This has lead to the identification of Hinderin, a novel five domains protein including two coiled-coil motifs and sharing a strikingly structural similarity to the SMC family of proteins. Hinderin is ubiquitously expressed in human tissues. Orthologue forms of the protein are present in other vertebrates but not in lower organisms. A mapping of the interaction sites revealed that the N- and C-terminal globular domains mediate the binding of Hinderin to SMC3. Hinderin/SMC3 complexes could be recovered by immunoprecipitation from cell lysates using an anti-SMC3 antibody, thus demonstrating that the two proteins interact in vivo. On the contrary, Hinderin did not interact with SMC1. In vivo the rate of SMC1/SMC3 interaction was decreased by the ectopic expression of Hinderin. Hinderin is a novel binding partner of SMC3. Based on its ability to modulate SMC1/SMC3 interaction we postulate that Hinderin affects the availability of SMC3 to engage in the formation of multimeric protein complexes.

Background The Structural Maintenance of Chromosome 3 protein (SMC3) plays an essential role during the sister chromatid separation, is involved in DNA repair and recombination and participates in microtubule-mediated intracellular transport. SMC3 is frequently elevated in human colon carcinoma and overexpression of the protein transforms murine NIH3T3 fibroblasts. In order to gain insight into the mechanism of SMC3-mediated tumorigenesis a gene expression profiling was performed on human 293 cells line stably overexpressing SMC3.

Rat apoA-IV complexes with dimyristoyl phosphatidylcholine (apoA-IV-DMPC) have been prepared and their ability to bind to purified rat liver plasma membranes investigated. Binding equilibrium at 37 degrees C was reached in 30 minutes. Saturation binding experiments and subsequent analysis of the results with Scatchard plots gave results consistent with the presence of a single saturable binding site. DMPC or POPC unilamellar vesicles could not compete with apoA-IV-DMPC for binding; apoA-I-DMPC competed only partially. ApoE-poor HDL effectively competed with apoA-IV-DMPC. The fact that binding could be greatly reduced (greater than 70%) by preincubating the membrane with pronase (18 micrograms/ml), supports the conclusion that a membrane protein is involved in binding. Based on these results, we speculate that the rapid catabolism of apoA-IV in plasma may be mediated by a specific uptake mechanism in the liver. The implications of these results support the hypothesis that apoA-IV is involved in reverse cholesterol transport.

Apolipoprotein E (apoE) circulates as a mixture of sialilated and asialilated forms. In this study the catabolic fate and the plasma turnover rate of the different apoE forms have been investigated in vivo in humans. Asialo apoE (E) and doubly sialiated apoE (Ess) were isolated by preparative isoelectrofocusing from the VLDL of subjects homozygous for the E3 allele. 131E3 and 125E3ss were injected simultaneously into three hypertriglyceridemic subjects, and plasma samples were collected up to the sixth day. VLDL were isolated by ultracentrifugation, and the apoE forms were separated by isoelectrofocusing. Gel bands corresponding to E3 and E3ss were cut out and counted for the associated radioactivity. Residence times in plasma for 131E3 and 125E3ss were 0.95 ± 0.16 and 0.74 ± 0.16 days, respectively. As determined from the gel count distribution up to 24 hours, no conversion of the injected sialilated form to the correspondent asialilated form was detected.

As assessed by molecular sieve chromatography and quantitation by a specific radioimmunoassay, apoA-IV is associated in plasma with the triglyceride-rich lipoproteins, to a high density lipoprotein (HDL) subfraction of smaller size than HDL3, and to the plasma lipoprotein-free fraction (LFF). In this study, the turnover of apoA-IV associated to the triglyceride-rich lipoproteins, HDL and LFF was investigated in vivo in normal volunteers. Human apoA-IV isolated from the thoracic duct lymph chylomicrons was radioiodinated and incubated with plasma withdrawn from normal volunteers after a fatty meal. Radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, HDL, and LFF were then isolated by chromatography on an AcA 34 column. Shortly after the injection of the radioiodinated apoA-IV-labeled triglyceride-rich lipoproteins, most of the radioactivity could be recovered in the HDL and LFF column fractions. On the other hand, when radioiodinated apoA-IV-labeled HDL or LFF were injected, the radioactivity remained with the originally injected fractions at all times. The residence time in plasma of 125I-labeled apoA-IV, when injected in association with HDL or LFF, was 1.61 and 0.55 days, respectively. When 125I-labeled apoA-IV was injected as a free protein, the radioactivity distributed rapidly among the three plasma pools in proportion to their mass. The overall fractional catabolic rate of apoA-IV in plasma was measured in the three normal subjects and averaged 1.56 pools per day. The mean degradation rate of apoA-IV was 8.69 mg/kg X day. The results are consistent with the conclusions that: apoA-IV is present in human plasma in three distinct metabolic pools; apoA-IV associated with the triglyceride-rich lipoproteins is a precursor to the apoA-IV HDL and LFF pools; apoA-IV in LFF is not a free protein and its turnover rate is faster than that of apoA-IV in HDL; since no transfer of apoA-IV from the HDL or the LFF occurs, these pools may represent a terminal pathway for the catabolism of apoA-IV; and the catabolism of apoA-IV in HDL is dissociated from that of apoA-I although both apoproteins may reside on the same lipoprotein particles.

A variant of apolipoprotein E, denoted E Bethesda, has been identified in the plasma of a 72-year-old woman with type III hyperlipoproteinemia. An offspring of the proband also has this variant and type III hyperlipoproteinemia. Apolipoprotein E Bethesda was isolated by preparative isoelectrofocusing followed by preparative SDS-polyacrylamide gel electrophoresis from the very low density lipoproteins of the proband's son. The purity and the identity of the preparation were analyzed by analytical SDS-polyacrylamide gel electrophoresis, two-dimensional gel electrophoresis and by immunochemical analysis. Apolipoprotein E Bethesda migrates in the E 1 position and its electrophoretic mobility is not affected by neuraminidase treatment. The protein is shifted to the E3 position after cysteamine treatment. The amino acid composition revealed the presence of two cysteine residues. These data support the concept that the apolipoprotein E Bethesda allele is derived from a mutation of the E2 or E2* allele.

The effect of L-propranolol on the receptor mediated uptake of human LDL by fibroblasts was investigated. When L-propranolol was tested between 10 −6 and 10 −4 M at a 125 I LDL concentration ranging from 6.25 to 198 μg/ml, an increase in binding, internalization and degradation of the labelled lipoproteins was demonstrated with a maximal effect respectively of 52%, 680% and 105% observed with L-propranolol at 10 −4 M and a lipoprotein concentration of 37.5 μg/ml. The effect occurs rapidly and is seen 2 hrs after the addition of the drug. Lower drug concentrations, although less effective, still produced significant increase in lipoprotein uptake and degradation. Procaine, a local anesthetic, used at 10 −4 M has no effect on 125 I-LDL binding and internalization. D-propranolol, another local anesthetic and L-propranolol enantiomer, is also without effect. This, excluding a drug induced stabilization of the membrane as the responsible mechanism of action, indicates that stereospecificity is important.

Apolipoprotein E (apoE) is associated with plasma lipoproteins and is important in modulating the catabolism of remnants of triglyceride-rich lipoproteins. ApoE is a polymorphic protein, and homozygosity for the E2 allele is associated with type III hyperlipoproteinemia. A variant of apoE, apoEBethesda, was found that migrates in the E1 position on isoelectric focusing. Evaluation of the proband and her family showed that both individuals with apoEBethesda had type III hyperlipoproteinemia, and the analysis was consistent with the concept that apoEBethesda was coded by a new E allele and that this allele was expressed in a codominant fashion. The discovery of apoEBethesda and its association with type III hyperlipoproteinemia provides additional evidence for the genetic heterogeneity of the E allele and suggests that a number of different structural mutations of the E apolipoprotein may be associated with the type III phenotype.