Overexpression of human hepatic lipase and ApoE in transgenic rabbits attenuates response to dietary cholesterol and alters lipoprotein subclass distributions.
(17/6488)
The effect of the expression of human hepatic lipase (HL) or human apoE on plasma lipoproteins in transgenic rabbits in response to dietary cholesterol was compared with the response of nontransgenic control rabbits. Supplementation of a chow diet with 0.3% cholesterol and 3.0% soybean oil for 10 weeks resulted in markedly increased levels of plasma cholesterol and VLDL and IDL in control rabbits as expected. Expression of either HL or apoE reduced plasma cholesterol response by 75% and 60%, respectively. The HL transgenic rabbits had substantial reductions in medium and small VLDL and IDL fractions but not in larger VLDL. LDL levels were also reduced, with a shift from larger, more buoyant to smaller, denser particles. In contrast, apoE transgenic rabbits had a marked reduction in the levels of large VLDLs, with a selective accumulation of IDLs and large buoyant LDLs. Combined expression of apoE and HL led to dramatic reductions of total cholesterol (85% versus controls) and of total VLDL+IDL+LDL (87% versus controls). HDL subclasses were remodeled by the expression of either transgene and accompanied by a decrease in HDL cholesterol compared with controls. HL expression reduced all subclasses except for HDL2b and HDL2a, and expression of apoE reduced large HDL1 and HDL2b. Extreme HDL reductions (92% versus controls) were observed in the combined HL+apoE transgenic rabbits. These results demonstrate that human HL and apoE have complementary and synergistic functions in plasma cholesterol and lipoprotein metabolism. (+info)
A genome search identifies major quantitative trait loci on human chromosomes 3 and 4 that influence cholesterol concentrations in small LDL particles.
(18/6488)
Small, dense LDL particles are associated with increased risk of cardiovascular disease. To identify the genes that influence LDL size variation, we performed a genome-wide screen for cholesterol concentrations in 4 LDL size fractions. Samples from 470 members of randomly ascertained families were typed for 331 microsatellite markers spaced at approximately 15 cM intervals. Plasma LDLs were resolved by using nondenaturing gradient gel electrophoresis into 4 fraction sizes (LDL-1, 26.4 to 29.0 nm; LDL-2, 25.5 to 26.4 nm; LDL-3, 24.2 to 25.5 nm; and LDL-4, 21.0 to 24.2 nm) and cholesterol concentrations were estimated by staining with Sudan Black B. Linkage analyses used variance component methods that exploited all of the genotypic and phenotypic information in the large extended pedigrees. In multipoint linkage analyses with quantitative trait loci for the 4 fraction sizes, only LDL-3, a fraction containing small LDL particles, gave peak multipoint log10 odds in favor of linkage (LOD) scores that exceeded 3.0, a nominal criterion for evidence of significant linkage. The highest LOD scores for LDL-3 were found on chromosomes 3 (LOD=4.1), 4 (LOD=4.1), and 6 (LOD=2.9). In oligogenic analyses, the 2-locus LOD score (for chromosomes 3 and 4) increased significantly (P=0.0012) to 6.1, but including the third locus on chromosome 6 did not significantly improve the LOD score (P=0.064). Thus, we have localized 2 major quantitative trait loci that influence variation in cholesterol concentrations of small LDL particles. The 2 quantitative trait loci on chromosomes 3 and 4 are located in regions that contain the genes for apoD and the large subunit of the microsomal triglyceride transfer protein, respectively. (+info)
Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.
(19/6488)
Abundant evidence has been provided to substantiate the elevated cardiovascular risk associated with small, dense, low density lipoprotein (LDL) particles. The diminished resistance of dense LDL to oxidative stress in both normolipidemic and dyslipidemic subjects is established; nonetheless, the molecular basis of this phenomenon remains indeterminate. We have defined the primary molecular targets of lipid hydroperoxide formation in light, intermediate, and dense subclasses of LDL after copper-mediated oxidation and have compared the relative stabilities of the hydroperoxide derivatives of phospholipids and cholesteryl esters (CEs) as a function of the time course of oxidation. LDL subclasses (LDL1 through LDL5) were isolated from normolipidemic plasma by isopycnic density gradient ultracentrifugation, and their content of polyunsaturated molecular species of phosphatidylcholine (PC) and CE and of lipophilic antioxidants was quantified by reverse-phase high-performance liquid chromatography. The molar ratio of the particle content of polyunsaturated CE and PC species containing linoleate or arachidonate relative to alpha-tocopherol or beta-carotene did not differ significantly between LDL subspecies. Nonetheless, dense LDL contained significantly less polyunsaturated CE species (400 mol per particle) compared with LDL1 through LDL4 (range, approximately 680 to 490 mol per particle). Although the formation of PC-derived hydroperoxides did not vary significantly between LDL subspecies as a function of the time course of copper-mediated oxidation, the abundance of the C18:2 and C20:4 CE hydroperoxides was uniquely deficient in dense LDL (23 and 0.6 mol per particle, respectively, in LDL5; 47 to 58 and 1.9 to 2.3 mol per particle, respectively, in other LDL subclasses) at propagation half-time. When expressed as a lability ratio (mol hydroperoxides formed relative to each 100 mol of substrate consumed) at half-time, the oxidative lability of CE hydroperoxides in dense LDL was significantly elevated (lability ratio <25:100) relative to that in lighter, larger LDL particle subclasses (lability ratio >40:100) throughout the oxidative time course. We conclude that the elevated lability of CE hydroperoxides in dense LDL underlies the diminished oxidative resistance of these particles. Moreover, this phenomenon appears to result not only from the significantly elevated PC to free cholesterol ratio (1.54:1) in dense LDL particles (1.15:1 to 1.25:1 for other LDL subclasses) but also from their unique structural features, including a distinct apoB100 conformation, which may facilitate covalent bond formation between oxidized CE and apoB100. (+info)
Kinetics and mechanism of exchange of apolipoprotein C-III molecules from very low density lipoprotein particles.
(20/6488)
Transfer of apolipoprotein (apo) molecules between lipoprotein particles is an important factor in modulating the metabolism of the particles. Although the phenomenon is well established, the kinetics and molecular mechanism of passive apo exchange/transfer have not been defined in detail. In this study, the kinetic parameters governing the movement of radiolabeled apoC molecules from human very low density lipoprotein (VLDL) to high density lipoprotein (HDL3) particles were measured using a manganese phosphate precipitation assay to rapidly separate the two types of lipoprotein particles. In the case of VLDL labeled with human [14C]apoCIII1, a large fraction of the apoCIII1 transfers to HDL3 within 1 minute of mixing the two lipoproteins at either 4 degrees or 37 degrees C. As the diameter of the VLDL donor particles is decreased from 42-59 to 23-25 nm, the size of this rapidly transferring apoCIII1 pool increases from about 50% to 85%. There is also a pool of apoCIII1 existing on the donor VLDL particles that transfers more slowly. This slow transfer follows a monoexponential rate equation; for 35-40 nm donor VLDL particles the pool size is approximately 20% and the t1/2 is approximately 3 h. The flux of apoCIII molecules between VLDL and HDL3 is bidirectional and all of the apoCIII seems to be available for exchange so that equilibrium is attained. It is likely that the two kinetic pools of apoCIII are related to conformational variations of individual apo molecules on the surface of VLDL particles. The rate of slow transfer of apoCIII1 from donor VLDL (35-40 nm) to acceptor HDL3 is unaffected by an increase in the acceptor to donor ratio, indicating that the transfer is not dependent on collisions between donor and acceptor particles. Consistent with this, apoCIII1 molecules can transfer from donor VLDL to acceptor HDL3 particles across a 50 kDa molecular mass cutoff semipermeable membrane separating the lipoprotein particles. These results indicate that apoC molecules transfer between VLDL and HDL3 particles by an aqueous diffusion mechanism. (+info)
Recombinant human type II collagens with low and high levels of hydroxylysine and its glycosylated forms show marked differences in fibrillogenesis in vitro.
(21/6488)
Type II collagen is the main structural component of hyaline cartilages where it forms networks of thin fibrils that differ in morphology from the much thicker fibrils of type I collagen. We studied here in vitro the formation of fibrils of pepsin-treated recombinant human type II collagen produced in insect cells. Two kinds of type II collagen preparation were used: low hydroxylysine collagen having 2.0 hydroxylysine residues/1,000 amino acids, including 1.3 glycosylated hydroxylysines; and high hydroxylysine collagen having 19 hydroxylysines/1,000 amino acids, including 8.9 glycosylated hydroxylysines. A marked difference in fibril formation was found between these two kinds of collagen preparation, in that the maximal turbidity of the former was reached within 5 min under the standard assay conditions, whereas the absorbance of the latter increased until about 600 min. The critical concentration with the latter was about 10-fold, and the absorbance/microgram collagen incorporated into the fibrils was about one-sixth. The morphology of the fibrils was also different, in that the high hydroxylysine collagen formed thin fibrils with essentially no interfibril interaction or aggregation, whereas the low hydroxylysine collagen formed thick fibrils on a background of thin ones. The data thus indicate that regulation of the extents of lysine hydroxylation and hydroxylysine glycosylation may play a major role in the regulation of collagen fibril formation and the morphology of the fibrils. (+info)
A physiological barrier distal to the anatomic blood-brain barrier in a model of transvascular delivery.
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BACKGROUND AND PURPOSE: Osmotic disruption of the blood-brain barrier (BBB) provides a method for transvascular delivery of therapeutic agents to the brain. The apparent global delivery of viral-sized iron oxide particles to the rat brain after BBB opening as seen on MR images was compared with the cellular and subcellular location and distribution of the particles. METHODS: Two dextran-coated superparamagnetic monocrystalline iron oxide nanoparticle contrast agents, MION and Feridex, were administered intraarterially in rats at 10 mg Fe/kg immediately after osmotic opening of the BBB with hyperosmolar mannitol. After 2 to 24 hours, iron distribution in the brain was evaluated first with MR imaging then by histochemical analysis and electron microscopy to assess perivascular and intracellular distribution. RESULTS: After BBB opening, MR images showed enhancement throughout the disrupted hemisphere for both Feridex and MION. Feridex histochemical staining was found in capillaries of the disrupted hemisphere. Electron microscopy showed that the Feridex particles passed the capillary endothelial cells but did not cross beyond the basement membrane. In contrast, after MION delivery, iron histochemistry was detected within cell bodies in the disrupted hemisphere, and the electron-dense MION core was detected intracellularly and extracellularly in the neuropil. CONCLUSION: MR images showing homogeneous delivery to the brain at the macroscopic level did not indicate delivery at the microscopic level. These data support the presence of a physiological barrier at the basal lamina, analogous to the podocyte in the kidney, distal to the anatomic (tight junction) BBB, which may limit the distribution of some proteins and viral particles after transvascular delivery to the brain. (+info)
Functional and morphological correlates of connexin50 expressed in Xenopus laevis oocytes.
(23/6488)
Electrophysiological and morphological methods were used to study connexin50 (Cx50) expressed in Xenopus laevis oocytes. Oocytes expressing Cx50 exhibited a new population of intramembrane particles (9.0 nm in diameter) in the plasma membrane. The particles represented hemichannels (connexin hexamers) because (a) their cross-sectional area could accommodate 24 +/- 3 helices, (b) when their density reached 300-400/microm2, they formed complete channels (dodecamers) in single oocytes, and assembled into plaques, and (c) their appearance in the plasma membrane was associated with a whole-cell current, which was activated at low external Ca2+ concentration ([Ca2+]o), and was blocked by octanol and by intracellular acidification. The Cx50 hemichannel density was directly proportional to the magnitude of the Cx50 Ca2+-sensitive current. Measurements of hemichannel density and the Ca2+-sensitive current in the same oocytes suggested that at physiological [Ca2+]o (1-2 mM), hemichannels rarely open. In the cytoplasm, hemichannels were present in approximately 0.1-microm diameter "coated" and in larger 0.2-0.5-microm diameter vesicles. The smaller coated vesicles contained endogenous plasma membrane proteins of the oocyte intermingled with 5-40 Cx50 hemichannels, and were observed to fuse with the plasma membrane. The larger vesicles, which contained Cx50 hemichannels, gap junction channels, and endogenous membrane proteins, originated from invaginations of the plasma membrane, as their lumen was labeled with the extracellular marker peroxidase. The insertion rate of hemichannels into the plasma membrane (80, 000/s), suggested that an average of 4,000 small coated vesicles were inserted every second. However, insertion of hemichannels occurred at a constant plasma membrane area, indicating that insertion by vesicle exocytosis (60-500 microm2 membranes/s) was balanced by plasma membrane endocytosis. These exocytotic and endocytotic rates suggest that the entire plasma membrane of the oocyte is replaced in approximately 24 h. (+info)
Particle deposition in the trachea: in vivo and in hollow casts.
(24/6488)
The pattern of deposition within the respiratory tract of potentially harmful particulates is a major factor in assessing any risk from individual and community exposures. Although the trachea is the most easily observed of the conductive airways, very little information concerning its particle collection characteristics is available, information which is essential for a complete and realistic description of particle deposition patterns within the entire respiratory tract. Data on tracheal deposition are also needed for development of accurate predictive models for particle deposition. The pattern of particle deposition in the trachea, and its relation to air flow, was studied in a hollow cast of the human larynx-tracheobronchial tree. Results were compared with data obtained in humans in vivo and from previous studies in hollow casts. In addition, the relevance of tracheal deposition in the hollow cast test system to deposition in vivo was examined by a direct comparison of deposition in a cast prepared from the lungs of donkeys previously studied in a series of in vivo tests. The disturbance of the air flow within the trachea caused by the larynx promoted the deposition of suspended particulates throughout the length of the trachea, and especially in proximal regions. This proximal deposition was due both to direct impaction from the air jet coming from the glottis and to effects of the tubulent flow. Turbulence produced inhomogenous deposition patterns within the trachea for particles of all sizes, although its effect was more pronounced as size decreased. Tracheal deposition in the human cast was within the range of normal in vivo tracheal depostion only when a larynx was used during cast test exposures; this emphasizes the need for the use of realistic experimental test systems for the study of particle deposition patterns. The relative patterns of deposition in casts of the donkey trachea and in the same tracheas in vivo were similar. (+info)