Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006-1.031 g/ml; LDL-3: d = 1.034-1.037 g/ml; LDL-6: d = 1.044-1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangement in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.     

The pattern of apolipoprotein B100 containing lipoprotein subclasses produced by the isolated visceral rat yolk sac depends on developmental stage and fatty acid availability

Explants of visceral rat yolk sacs from gestational days 16, 18 and 22 were used for studying developmental changes of secretion and density distribution of lipoproteins, particularly of those containing apoB. Moreover, the influence of fatty acid supply on the amount and density distribution of secreted apolipoproteins was studied on day 18 of gestation. Active lipoprotein production was observed in yolk sacs taken on days 16 and 18 of gestation. It declined considerably on day 22 of gestation in parallel with the production of total protein, triacylglycerols and cholesterol. On all gestational days, apoB floated mainly in the LDL range (⩾ 70%) with differences in the distribution pattern of LDL subclasses. The lowest density of secreted LDL was found on day 18 of gestation (peak at d = 1.025 g/ml) followed by day 16 (peak at d = 1.035 g/ml) and day 22 of gestation (peak at d = 1.045 g/ml). ApoIV, apoE and apoAI floated exclusively in the HDL range with a peak at d = 1.089 g/ml independently of the gestational day. After incubation of yolk sacs from the 18th day of gestation with 0.4 mM or 0.8 mM oleate, the density of secreted apoB containing particles was decreased (peaks in the VLDL and IDL density range), whereas palmitate in the same concentrations caused a redistribution of secreted apoB toward higher densities (peaks at d ≥ g/ml). Taken together, the data provide evidence that the density of L.DL subclasses produced by isolated yalk sacs between days 16 and 22 of gestation depended on the gestational stage. Moreover, addition of unsaturated or saturated fatty acids to the organ culture differently affected the secretory rate and the density of lipoproteins delivered by yolk sacs on day 18 of gestation.     

Crystallization and preliminary X-ray diffraction data of two different human low-density lipoprotein (LDL) subfractions

Human LDL subfractions LDL-2 (d = 1.031–1.034 g/ml) and LDL-5 (d = 1.040–1.044 g/ml) were crystallized in two different crystal forms by using polyethylene glycol as a precipitant. Both fractions were from one donor. Crystals of LDL-5 were yellow, hexagonal, and showed no dichroism. Crystals of LDL-2 were of the same color, had a rodlike shape with notches at both ends, and were highly dichroitic. LDL-2 crystals diffracted to a resolution of 29 Å by using synchrotron radiation. Indexing in P1 resulted in preliminary parameters for the reduced cell of a = 171 Å, b = 438 Å, c = 519 Å, α = 102°, β = 99°, γ = 91. These dimensions are consistent with the size of LDL particles. Using Fourier transform infrared spectroscopy (FTIR) and agarose gel electrophoresis, we could further confirm that the crystals consist of LDL. The FTIR spectrum showed bands characteristic for lipids and protein. Dissolved crystals exhibited a mobility similar to native LDL in agarose gels and could be stained with anti-human apolipoprotein B (apoB). Proteins 28:293–297, 1997. © 1997 Wiley-Liss Inc.     

Discrete subspecies of human low density lipoproteins are heterogeneous in their interaction with the cellular LDL receptor.

The low density lipoproteins (LDL) of human plasma consist of a series of discrete particle subspecies of distinct physicochemical, immunological, and hydrodynamic properties. Such structural differences are intimately linked to the metabolic heterogeneity of circulating LDL in vivo. The current studies were designed to evaluate and compare the interaction of discrete LDL subspecies from normolipidemic subjects with the LDL receptor. Plasma LDL of d 1.019-1.063 g/ml from healthy males were fractionated into 15 subspecies of defined physicochemical characteristics by isopycnic density gradient ultracentrifugation as described earlier (Chapman et al., J. Lipid Res. 1988. 29: 442-458). The major LDL subspecies, LDL-5 to LDL-10, exhibited an overall range in density from 1.0244 to 1.0435 g/ml; individual subspecies increased in density by increments of 0.027 (LDL-5), 0.026 (LDL-6), 0.030 (LDL-7), 0.031 (LDL-8), 0.035 (LDL-9), and 0.042 g/ml (LDL-10), respectively. Taken together, these subspecies accounted for approximately 70% of the total mass of LDL of d 1.019-1.063 g/ml; their cholesterol: protein ratios decreased from 1.70 to 1.12 and particle size from 275 to 260 A with increase in density. ApoB-100 was the unique protein component in subspecies 5-8, with trace amounts (less than 0.2% of apoLDL) of both apoA-I and apoE in subspecies 9 and 10. The interaction of individual LDL subspecies with the LDL receptor on cultured human U-937 monocyte-like cells was compared by determining receptor binding affinities at 4 degrees C. Scatchard analysis of specific binding curves demonstrated a single class of binding site for each subspecies. The lowest dissociation constants were displayed by LDL subspecies 6 (Kd 5.71 nM), 7 (Kd 5.24 nM) and 8 (Kd 4.67 nM), while subspecies 5, 9, and 10 displayed significantly higher Kd values (8.35, 7.20, and 6.87 nM, respectively). Competitive displacement studies at 4 degrees C, in which unlabeled subspecies from the same gradient series competed for binding with 125I-labeled LDL subspecies, confirmed the relative binding affinities of these subspecies. As the hydrophobic lipid core of LDL undergoes a thermotropic transition at approximately 37 degrees C, which may in turn influence the surface structure of the particle, internalization and degradation studies were performed at 37 degrees C. No effect of temperature was detectable; again, LDL subspecies at each extreme of the density distribution (LDL-5 and LDL-10) displayed significantly lower binding affinities for the LDL receptor than that from the peak region (LDL-7).(ABSTRACT TRUNCATED AT 400 WORDS)     

Surface structure of the two porcine low-density lipoprotein subclasses — a spin labelling study

Porcine low-density lipoprotein (LDL) subclasses were spin-labelled at both protein and lipid sites. The surface structures of the two subclasses were compared. E.s.r. spectra of stearic acid spin-labels [I(m/n)] and of androstanol spin-label indicated more restricted motion of the labels in LDL₂ (d = 1.063?1.090 g/ml) than in LDL₁ (d = 1.020?1.063 g/ml). Thermotropic change in the surface structure was found in both subclasses by both protein and lipid spin-labels, but at lower temperature in LDl₁, than in LDL₂. These results indicate the relationship between the size and the dynamics of the lipid components in the surface layer of the LDL subclasses.     

Enhancing the contrast of ApoB to locate the surface components in the 3D density map of human LDL

A 26 Å resolution map of the structure of human low-density lipoprotein (LDL) was obtained from electron cryomicroscopy and single-particle image reconstruction. The structure showed a discoidal-shaped LDL particle with high-density regions mainly distributed at the edge of the particle and low-density regions at the flat surface that covers the core region. To determine the chemical components that correspond to these density regions and to delineate the distribution of protein and phospholipid located at the particle surface at the resolution of the map, we used Mono-Sulfo-NHS-Undecagold labeling to increase preferentially the contrast of the apolipoprotein B component on the LDL particle. In the three-dimensional map from the image reconstruction of the undecagold-labeled LDL particles, the high-density region from the undecagold label was distributed mainly at the edge of the particle, and lower density regions were found at the flat surfaces that cover the neutral lipid core. This suggests that apolipoprotein B mainly encircles LDL at the edge of the particle and the phospholipid monolayers are located at the flat surfaces, which are parallel to the cholesterol ester layers in the core and may interact with the core lipid layers through the acyl chains.     

Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC

Low density lipoproteins (LDL), collected from 32 normal male subjects (aged 30-60), were subfractionated by high resolution ion exchange chromatography (IE-HPLC). By this procedure two LDL subfractions were eluted. The first corresponds to normal LDL (nLDL); while the second one corresponds to a more electronegative subfraction, called LDL-. The mean percentage contribution of LDL- to native plasma LDL was of 3.9% (range 0.5-9.8%). The percentage concentration of LDL- in total native LDL did not correlate with plasma total cholesterol, triglycerides, and LDL cholesterol, whereas a significant negative correlation with high density lipoprotein cholesterol was found (r = -.38; p less than .05). LDL- was negatively correlated with LDL phospholipids (r = -.59; p less than .001), and with the LDL vitamin E content (r = -.63; p less than .001), and positively correlated with LDL proteins (r = -.35; p less than .05) and the content of thiobarbituric acid reactive substances (TBARS) in total LDL (r = .43; p less than .05). The TBARS molar content of LDL- was three times higher than in nLDL, with a mean concentration in LDL- of 7.3 mol/mol lipoprotein. By preparative IE-HPLC significant differences of the LDL- chemical composition were observed. The percentage content of cholesterol esters and of phospholipids was decreased, whereas proteins and free cholesterol were increased. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that besides apolipoprotein B-100 there was evidence of peptides with a higher molecular weight in LDL-.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of human serum lipoproteins in solution. II. Small-angle x-ray scattering study of HDL and LDL.

Two human serum lipoprotein particles, HDL₃ and LDL, were studied in solution in solvents of variable density (NaBr in water) by small-angle X-ray scattering using a position-sensitive proportional counter. The data were analysed using the theoretical approach outlined in the accompanying paper (Luzzati et al., 1976). The structures of the particles were found to be independent of the salt concentration of the solution (i.e. the particles are impenetrable to NaBr). Density heterogeneities are negligible and size and shape heterogeneities appear to be small.The particle structures could be quantitatively described in terms of a set of parameters and of a few one-dimensional functions. The parameters are the volume, radius of gyration and surface area of the shape functions; the second moment and square average of the electron density contrast at buoyancy; the electron density level, volume, radius of gyration and surface area of the hydrocarbon and polar regions. The one-dimensional functions are: the distribution of chords, the spherical average of the shape function and of the electron density at buoyancy, and the fraction of each spherical shell occupied by the hydrocarbon and polar regions. These parameters and functions are internally consistent and agree with the chemical data confirming the assumptions made in their derivation.The results are compatible with the shape of the particle being compact and quasi-spherical although with deeply convoluted surfaces. They also indicate that the outer layers of the particles are occupied by the proteins and the polar groups of phospholipids and free cholesterol, and the cores by neutral lipids. The maximum diameters of the particles are 130Å and 280Å for HDL₃ and LDL, respectively, while the hydrocarbon cores have diameters of 80Å and 230Å, respectively. The solvent is considered to penetrate to 25Å from the center of the HDL₃ particle with a minimum solvation at a radius of 45Å. In the case of LDL, the solvent penetrates to 55Å from the center of the particle. The lipids in the cores of the particles, particularly the cholesterol esters, appear to display a micelle-like organization with the steroid nuclei segregated in regions distinct from those occupied by the hydrocarbon chains.Although the data are consistent with several aspects of previously proposed models, they indicate that the structures of the HDL₃ and LDL particles are more complex than previously believed.     

Dynamic structure of the lower density lipoproteins. II. Deuterium NMR studies of the monolayer of very low and low density lipoproteins

The order of phosphatidylcholine (PC) acyl chains in the surface monolayer of very low density lipoproteins (VLDL) and low density lipoproteins (LDL) has been determined from 2H nuclear magnetic resonance order parameters, SCD, using selectively deuterated PC or palmitic acids. From the computer simulated line shapes, we find two distinct phospholipid domains within the amphiphilic monolayer of both VLDL and LDL. In the more ordered domain of LDL, SCD was approximately 0.3 for the "plateau" chain region. The SCD values of VLDL particles are similar to those of LDL for the 5,6- and 11,12-positions, hence we suggest the organization of the more ordered region of VLDL and LDL are similar. The domain of low order in LDL comprises less than 10% of the phospholipid molecules (we do not distinguish between PC and sphingomyelin), having approximately the same order (SCD less than 0.1) as egg PC - sphingomyelin unilamellar vesicles. In VLDL, the domain of low order comprises between approximately 10 and approximately 20% of the phospholipid molecules and the entire acyl chain is in an essentially isotropic environment (SCD less than 0.02). We prepared VLDL-sized microemulsions composed of egg PC, deuterated PC, and triolein to test whether the apoproteins were responsible for creating the two differently organized domains in VLDL and LDL. Surprisingly, these protein-free particles also showed two domains of different order at two temperatures. The high order region, however, is less ordered than in VLDL and LDL. We explain two surface domains of PC in terms of lipid organization and the unique interactions of lipids in the various lipoprotein particles.     

Binding of lipopolysaccharide and complexes of lipopolysaccharide to serum low density lipoproteins to liver macrophages

Binding of [³H]-lipopolysaccharide toxin (LPS) and complexes of LPS with serum [¹²⁵I]-labeled low density lipoproteins (LDL) to primary culture of rat liver macrophages (Kupffer cells) has been studied. Total, specific and nonspecific binding was determined. The receptor interaction was shown to dominate for both LPS and LDL-LPS complexes, representing 70–77% and 80–85%, respectively. The Scatchard plot was essentially non-linear for LPS binding but linear for the LDL-LPS complexes. At the Scatchard graph of LPS binding, however, two regions approximately fitting the linear regression could be identified. These regions correspond to two different types of specific binding sites: the first is for lower toxin concentrations of 0.25–0.50 μg/ml with K _d = 0.75 μg/ml; while the second is for higher LPS concentrations of 7.5–15 μg/ml with K _d = 5.39 μg/ml. For LDL-LPS complexes only K _d of 2.80 μg/ml was obtained. The LDL-LPS complexes significantly blocked the LPS binding (?40%) while acetylated or oxidized LDLs exerted a less pronounced effect. LPS inhibited binding of LDL-LPS complexes (?60%), while acetylated or oxidized LDLs suppressed interaction of LDL-LPS complexes with Kupffer cells insignificantly. It is suggested that, while binding to the Kupffer cell surface, a substantial portion of both LPS and LDL-LPS complexes share the same scavenger receptors with which, however, modified LDLs interact weakly. The LDL-LPS complexes can interact, apart from receptors common with LPS, with other receptors exhibiting similar binding parameters, with the apo-B/E receptors playing an inessential role.     

Polyclonal antibodies against iduronate 2-sulphate sulphatase from human urine

Low-density lipoproteins (density = 1.019-1.063 g/ml) were isolated in 10 subjects with type V hyperlipoproteinemia by ultracentrifugation in a zonal rotor under rate flotation conditions. Plasma LDL concentrations in these patients were extremely reduced, as well as being heterogeneous, and two different subclasses consisting of LDL2 (density = 1.019-1.045 g/ml) and LDL3 (density = 1.045-1.063 g/ml) were observed. LDL2 and LDL3 have similar electrophoretic mobilities in beta position in agarose gel, and their diameters, calculated from gel filtration studies, were inversely proportional to their densities. LDL2 and LDL3 have a mean hydrated density of 1.034 and 1.054 g/ml, respectively. In comparison with normal LDL2, the LDL2 and LDL3 of hypertriglyceridemic subjects are particularly rich in triacylglycerols and poor in cholesteryl esters and free cholesterol, while they have an increasing amount of proteins. The protein moiety is composed almost exclusively of apolipoprotein B-100 in IDL, LDL2 and LDL3 ; in addition, IDL also contain apolipoprotein C peptides. This characterization of LDL heterogeneity in type V hyperlipoproteinemia should be considered in interpreting kinetic data in human normal and pathological lipid metabolism and in evaluating the atherogenic risk of hypertriglyceridemia.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8 degrees C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37 degrees C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37 degrees C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

Physicochemical characterization of soluble complexes of human serum low density lipoprotein with heparin

Soluble complexes of low density lipoproteins (LDL) with heparin (HEP) and chondroitin sulphate (CS) in the absence of divalent cations have been studied by means of a micro-rolling-ball ciscometer to obtain information about molecular size and structure of the aggregates. The rheological results were supplied and corroborated by light scattering measurements, electrophoresis and analytical ultracentrifugation. Molar binding ratios were measured using gel filtration assays and ultracentrifugation. At a certain weight ratio of LD To HEP the solutions showed a significant viscosity maximum. At this weight ratio 2–3 LDL particles are held together 1–2 HEP chains. The hydrodynamic radius R_H of this complex is about 16.3 ± 0.90 nm and the rotational diffusion constant is > 7.1 × 10³ s^?1. With excess HEP the radius of the aggregates is almost the same as that of free LDL (R_H = 11.9 ± 0.70 nm). Quantitative binding studies revealed that in this case 1–2 HEP molecules are bound to a single LDL particle. An interaction was also found with CS and LDL but complex formation in this case showed different characteristics. Very low density lipoproteins (VLDL) and high density lipoproteins (HDL) gave no rheologically effective aggregates with HEP.     

Physico-chemical properties of low density lipoproteins isolated in an equilibrium density gradient

The tryptophanyls of total low density lipoproteins (LDL) (1.006-1.063 g/ml) from coronary heart disease (CHD) patients and subjects without CHD signs had different accessibility to fluorescence quenchers (I-and acrylamide). LDL were separated into subfractions in equilibrium density gradient. The coefficient of extinction , quantum yield and other spectral characteristics of LDL intrinsic fluorescence, rotational mobility of maleimide spin labels and fatty acid spin probe were different in LDL subfractions from healthy subjects. LDL subfractions with hydrated density 1.045-1.05 g/ml bound to B,E-receptors of cultured fibroblasts more effectively than did subfractions with density 1.01-1.03 g/ml. Structural differences of apo-B in the particles with different lipid to protein ratio are supposed.     

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages

Summary The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4° C some LDL-or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8° C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37° C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37° C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.     

The visceral yolk sac--an important site of synthesis and secretion of apolipoprotein B containing lipoproteins in the feto-placental unit of the rat.

Rat fetuses exhibit a high serum LDL concentration at term. Delivery caused a marked decrease of the LDL apolipoprotein (apo) B concentration independent of whether this occurred on days 21, 22 or 23 of gestation. The interruption of the yolk sac circulation by a ligature in situ for 6 h led to the same alterations of the LDL-apo B concentration as Caesarean section. Immunoelectronmicroscopic studies provided evidence that the epithelial cells of the visceral yolk sac exhibited electron dense LDL-sized and apo B containing particles which were localized over the compartments of the Golgi complexes, endoplasmatic reticulum, secretory vesicles and intercellular spaces, but not over the cell nuclei, mitochondria or lysosomes. ApoB containing LDL-sized particles could be obtained by ultracentrifugation from the disrupted material of the microsomal fraction of yolk sac homogenates. Isolated segments of the yolk sac membranes were capable to secrete apoB containing lipoproteins floating in the d less than 1.020 g/ml as well as in the d = 1.020-1.064 g/ml fraction with a 10-fold higher amount of apoB in the higher density class. Incorporation experiments with [35S] methionine gave evidence that these lipoproteins were at least partially provided with newly synthesized apoB predominantly found in the LDL fraction. The size of the negatively stained particles in the d = 1.020-1.064 g/ml fraction secreted from yolk sac segments corresponded to that of LDL from fetal rat serum. In contrast their acylglycerol content was significantly higher, whereas the percentage contribution of total cholesterol and protein was markedly reduced in comparison with serum LDL of the fetus. In summary, biochemical and ultrastructural studies provide clear cut evidence that the rat yolk sac is able to synthesize and to deliver apo B containing lipoproteins in the density ranges of VLDL, IDL and particular of LDL thus contributing to the supply of serum lipoproteins in the rat fetus. By recalculation of recent tracer kinetic data (Plonné et al. (1990) J. Lipid Res. 31, 747) using a mathematical step function model it was possible to assess the contribution of the rat yolk sac to the LDL influx into the fetal serum.     

Structure of the dimyristoylphosphatidylcholine vesicle and the complex formed by its interaction with apolipoprotein C-III: X-ray small-angle scattering studies.

Single bilayer vesicles of dimyristoylphosphatidylcholine have been investigated by small-angle X-ray scattering at 28 degrees C. The results indicate that these vesicles are hollow spherical shell structures with an outer radius of approximately 12 nm and a molecular weight of (3.2 +/- 0.5) X 10(6). The shell was found to be 4.4 +/- 0.2 nm thick with a cross-sectional electron-density profile characteristic for a single phospholipid bilayer. Upon interaction of these vesicles with apolipoprotein C-III from human very low density lipoproteins at a protein/lipid ratio greater than 0.08 (g/g), a complex containing 0.25 g of protein/g of lipid, with molecular weight of (3.9 +/- 0.4) X 10(5), is formed. The shape analysis indicates a highly asymmetric particle with an internal partition of low and high electron density resembling that produced by a bilayer structure. Model calculations and curve-fitting procedures show good agreement between the experimental scattering curve and that computed for an oblate ellipsoidal structure with dimensions of 17 X 17 X 5 nm and a 1 nm thick shell of high electron density surrounding the core of low electron density.     

Synthesis,secretion and immunoelectron microscopic demonstration of apolipoprotein B-containing lipoprotein particles in the visceral rat yolk sac

Summary Electron microscopic investigations on the involvement of the fetal membranes of the rat (visceral yolk sac) in the lipid metabolism revealed the occurrence of lipoprotein-sized particles located in cisternal Golgi stacks, Golgi vesicles and secretory vesicles of the cells of the visceral yolk sac epithelium as well as in distended areas of the intercellular space between adjacent epithelial cells. Application of the protein A-gold technique with specific anti-apoB antiserum resulted in a specific location of immunogold both over the different compartments of the lipoprotein pathway (RER, Golgi complex, secretory vesicles) as well as over the distended intercellular spaces, thus confirming these particles to be lipoproteins in nature. Isolated visceral epithelial cells prepared by a tryptic digestion method exhibited some ultrastructural alterations, such as a loss of apical brush border, a change from columnar to spherical cell shape, a decrease in phagolysosomes, but an increase in autophagosomal structures after 6 h incubation at a vitality rate of at least 85%. Within this period the epithelial cells secreted measurable amounts of apoB-containing lipoproteins into the medium floating in the density classes d<1.006 g/ml, d=1.006–1.020 g/ml and d=1.020–1.064 g/ml. The production of the lipoproteins was partly inhibited by cycloheximide indicating the secretion of particles with preformed as well as newly synthesized apoB. Negative staining of the particles revealed an average diameter of 34 nm of VLDL, 31 nm of IDL and 24 nm of LDL. In summary, our studies demonstrate that in the feto-placental unit of the rat the fetal membranes are capable of synthesizing and secreting lipoproteins. The cells of the visceral yolk sac epithelium were shown to be the producers of apoB-containing particles.Abbreviations apo apolipoprotein - ER endoplasmic reticulum - IDL intermediate density-lipoprotein - LDL low density-lipoprotein - VLDL very low density-lipoprotein - PBS phosphate-buffered salt solution - RER rough endoplasmic reticulum - TEM transmission electron microscopy     

A gene score of nine LDL and HDL regulating genes is associated with fluvastatin-induced cholesterol changes in women

While conventional pharmacogenetic studies have considered single gene effects, we tested if a genetic score of nine LDL- and HDL-associated single nucleotide polymorphisms, previously shown to predict cardiovascular disease, is related to fluvastatin-induced lipid change. In patients with asymptomatic plaque in the right carotid artery, thus candidates for statin therapy, we related score LDL [APOB(rs693), APOE(rs4420638), HMGCR(rs12654264), LDLR(rs1529729), and PCSK9(rs11591147)] and score HDL [ABCA1(rs3890182), CETP(rs1800775), LIPC(rs1800588), and LPL(rs328)] as well as the combined score LDL+HDL to fluvastatin-induced LDL reduction (± metoprolol) (n = 395) and HDL increase (n = 187) following 1 year of fluvastatin treatment. In women, an increasing number of unfavorable alleles (i.e., alleles conferring higher LDL and lower HDL) of score LDL+HDL (P = 0.037) and of score LDL (P = 0.023) was associated with less pronounced fluvastatin-induced LDL reduction. Furthermore, in women, both score LDL+HDL (P = 0.001) and score HDL (P = 0.022) were directly correlated with more pronounced fluvastatin-induced HDL increase, explaining 5.9–11.6% of the variance in treatment response in women. There were no such associations in men. This suggests that a gene score based on variation in nine different LDL- and HDL-associated genes is of importance for the magnitude of fluvastatin HDL increase in women with asymptomatic plaque in the carotid artery.     

Heterogeneity in branching of amylopectin

The angular dependence of scattered light from amylopectin and its β-limit dextrin, the mean square radius of gyration and the molecular weights M_w and M_n have been calculated on the basis of the cascade branching theory for the homogeneously branched model by Meyer &; Bernfeld (1940) (Model I) and for the two heterogeneously branched structures suggested by French (1972) (Model II) and by Robin et al. (1974, 1975) (Model III). The calculations take into account the particularities of topology in branched molecules and the experimentally determined ratio of the number of A- and B-chains, A/B = 1. Furthermore, an average branching density of 4% and an interconnecting chain length of ovbar|n_i2 = 22, found by gel permeation chromatography (GPC) after debranching, were used. The constraints lead to the conclusion that amylopectin is heterogeneously branched. Densely branched clusters containing 3·22 branching units are interconnected by longer chains of 22 units in length. Comparison of the calculated angular dependence of light scattering with measurements from a maize amylopectin β-limit dextrin in 1 n NaOH solution gives strong evidence for a modified Robin-Mercier model. The modification consists of the conclusion that the interconnecting chains are preferentially B-chains, such that these chains carry on the average 1·4 clusters, while Robin and Mercier assume exactly 2 clusters. Our result is in agreement with the distribution of chain length found after debranching the amylopectin β-limit dextrin.