Isopycnic Density Gradient Ultracentrifugation Research Articles

Solution-Processed 2D Materials for Electronic Applications

Toward practical demonstration of high-quality two-dimensional (2D) nanomaterial-based electronics, solution-based processing has been considered as a promising route of scalable synthesis for more than a decade due to the capability of mass production without process complexity. However, in the earlier stage, solution-processed 2D nanomaterials were not desirable for scalable electronic applications due to their structural limitations including a small lateral dimension and polydispersity in thickness, which hinder formation of electronically active percolated thin-film networks. To overcome this limitation, various strategies have been reported including isopycnic density gradient ultracentrifugation to minimize thickness distribution and alkali-metal intercalations to increase exfoliation yield. Along this line, molecular intercalation-based electrochemical exfoliation has been reported as an emerging solution-based approach by resolving the well-known limitations of conventional solution processing. The resulting 2D nanomaterials with highly desired morphologies can be successfully utilized to form electronically active ultra-thin-film networks over a large area, and thus, gate-tunable thin-film electronics can be achieved with decent device performance. In this Spotlight article, we provide a chronological introduction of solution-based processing of 2D nanomaterials and their applications in electronics. Also, we discuss the remaining challenges and opportunities of solution-based processing of 2D nanomaterials for electronics.

Towards safer stable isotope probing - effect of formamide on the separation of isotope-labeled and unlabeled Escherichia coli RNA by isopycnic density ultracentrifugation.

RNA-based stable isotope probing (RNA-SIP) is used in molecular microbial ecology to link the identity of microorganisms in a complex community with the assimilation of a distinct substrate. The technique is highly dependent on a reliable separation of isotopic-labeled RNA from unlabeled RNA by isopycnic density gradient ultracentrifugation. Here we show that 13C-labeled and unlabeled Escherichia coli RNA can be sufficiently separated by isopycnic ultracentrifugation even in the absence of formamide. However, a slightly lower starting density is needed to obtain a distribution pattern similar to that obtained when formamide was used. Hence, the commonly used addition of formamide to the centrifugation solution might not be needed to separate 13C-labeled RNA from unlabeled RNA, but this must be verified for more complex environmental mixtures of RNA. Clearly, an omission of formamide would increase the safety of RNA-SIP analyses.

RNA-Based Stable Isotope Probing Suggests Allobaculum spp. as Particularly Active Glucose Assimilators in a Complex Murine Microbiota Cultured In Vitro.

RNA-based stable isotope probing (RNA-SIP) and metabolic profiling were used to detect actively glucose-consuming bacteria in a complex microbial community obtained from a murine model system. A faeces-derived microbiota was incubated under anaerobic conditions for 0, 2, and 4 h with 40 mM [U13C]glucose. Isopycnic density gradient ultracentrifugation and fractionation of isolated RNA into labeled and unlabeled fractions followed by 16S rRNA sequencing showed a quick adaptation of the bacterial community in response to the added sugar, which was dominated by unclassified Lachnospiraceae species. Inspection of distinct fractions of isotope-labeled RNA revealed Allobaculum spp. as particularly active glucose utilizers in the system, as the corresponding RNA showed significantly higher proportions among the labeled RNA. With time, the labeled sugar was used by a wider spectrum of faecal bacteria. Metabolic profiling indicated rapid fermentation of [U13C]glucose, with lactate, acetate, and propionate being the principal 13C-labeled fermentation products, and suggested that “cross-feeding” occurred in the system. RNA-SIP combined with metabolic profiling of 13C-labeled products allowed insights into the microbial assimilation of a general model substrate, demonstrating the appropriateness of this technology to study assimilation processes of nutritionally more relevant substrates, for example, prebiotic carbohydrates, in the gut microbiota of mice as a model system.

Layer-by-Layer Sorting of Rhenium Disulfide via High-Density Isopycnic Density Gradient Ultracentrifugation

Isopycnic density gradient ultracentrifugation (iDGU) has been widely applied to sort nanomaterials by their physical and electronic structure. However, the commonly used density-gradient medium iodixanol has a finite maximum buoyant density that prevents the use of iDGU for high-density nanomaterials. Here, we overcome this limit by adding cesium chloride (CsCl) to iodixanol, thus increasing its maximum buoyant density to the point where the high-density two-dimensional nanomaterial rhenium disulfide (ReS2) can be sorted in a layer-by-layer manner with iDGU. The resulting aqueous ReS2 dispersions show photoluminescence at ∼1.5 eV, which is consistent with its direct bandgap semiconductor electronic structure. Furthermore, photocurrent measurements on thin films formed from solution-processed ReS2 show a spectral response that is consistent with optical absorbance and photoluminescence data. In addition to providing a pathway for effective solution processing of ReS2, this work establishes a general methodology for sorting high-density nanomaterials via iDGU.

Lifestyle intervention enhances high-density lipoprotein function among patients with metabolic syndrome only at normal low-density lipoprotein cholesterol plasma levels

Metabolic syndrome (MetS) is associated with altered lipoprotein metabolism and impairment in the functionality of small, dense high-density lipoprotein (HDL) particles secondary to compositional alterations. The objective of this study was to investigate the capacity of a lifestyle program to improve the composition and antioxidative function (AOX) of small dense HDL3c in MetS. Patients with MetS (n=33) not taking lipid-lowering drugs were recruited to follow a 12-week educational program to reduce caloric intake and to increase physical activity. HDL subfractions were preparatively isolated by isopycnic density-gradient ultracentrifugation. AOX of HDL3c was assessed as its capacity to inhibit low-density lipoprotein oxidation induced by an azoinitiator. AOX of HDL3c was significantly improved (mean reduction in the propagation rate of low-density lipoprotein oxidation by HDL3c, -6.8%, P=.03) and systemic oxidative stress, assessed as plasma levels of 8-isoprostanes, tended to decrease in normocholesterolemic MetS patients (low-density lipoprotein cholesterol [LDL-C] < 130mg/dL) but not in patients with elevated LDL-C levels and in the whole study population. In both the whole study population and the normocholesterolemic subgroup, lifestyle intervention resulted in a significant degree of normalization of HDL3c composition, (enrichment in apolipoprotein A-I and cholesteryl esters, depletion in triglycerides), which was more pronounced at LDL-C < 130mg/dL. In patients with MetS, a lifestyle program improves AOX of small, dense HDL in subjects with normal LDL-C levels. Correction of HDL composition, involving partial normalization of apoA-I content and core lipid composition, 2 central features of the lipid hydroperoxide-inactivating capacity of HDL, may account for this effect.

Solution-Processed Dielectrics Based on Thickness-Sorted Two-Dimensional Hexagonal Boron Nitride Nanosheets.

Gate dielectrics directly affect the mobility, hysteresis, power consumption, and other critical device metrics in high-performance nanoelectronics. With atomically flat and dangling bond-free surfaces, hexagonal boron nitride (h-BN) has emerged as an ideal dielectric for graphene and related two-dimensional semiconductors. While high-quality, atomically thin h-BN has been realized via micromechanical cleavage and chemical vapor deposition, existing liquid exfoliation methods lack sufficient control over h-BN thickness and large-area film quality, thus limiting its use in solution-processed electronics. Here, we employ isopycnic density gradient ultracentrifugation for the preparation of monodisperse, thickness-sorted h-BN inks, which are subsequently layer-by-layer assembled into ultrathin dielectrics with low leakage currents of 3 × 10(-9) A/cm(2) at 2 MV/cm and high capacitances of 245 nF/cm(2). The resulting solution-processed h-BN dielectric films enable the fabrication of graphene field-effect transistors with negligible hysteresis and high mobilities up to 7100 cm(2) V(-1) s(-1) at room temperature. These h-BN inks can also be used as coatings on conventional dielectrics to minimize the effects of underlying traps, resulting in improvements in overall device performance. Overall, this approach for producing and assembling h-BN dielectric inks holds significant promise for translating the superlative performance of two-dimensional heterostructure devices to large-area, solution-processed nanoelectronics.

Thickness sorting of two-dimensional transition metal dichalcogenides via copolymer-assisted density gradient ultracentrifugation.

Two-dimensional transition metal dichalcogenides have emerged as leading successors to graphene due to their diverse properties, which depend sensitively on sample thickness. Although solution-based exfoliation methods hold promise for scalable production of these materials, existing techniques introduce irreversible structural defects and/or lack sufficient control over the sample thickness. In contrast, previous work on carbon nanotubes and graphene has shown that isopycnic density gradient ultracentrifugation can produce structurally and electronically monodisperse nanomaterial populations. However, this approach cannot be directly applied to transition metal dichalcogenides due to their high intrinsic buoyant densities when encapsulated with ionic small molecule surfactants. Here, we overcome this limitation and thus demonstrate thickness sorting of pristine molybdenum disulfide (MoS2) by employing a block copolymer dispersant composed of a central hydrophobic unit flanked by hydrophilic chains that effectively reduces the overall buoyant density in aqueous solution. The resulting solution-processed monolayer MoS2 samples exhibit strong photoluminescence without further chemical treatment.

Abstract 627: A Deleterious Mutation in SCARB1 Significantly Alters High-Density Lipoprotein Level, Composition and Biological Function in Humans

The membrane protein Scavenger Receptor Class B, Type 1 (SR-BI), coded by the gene SCARB1, facilitates the selective uptake of cholesteryl esters from HDL in the liver. Its impact on human lipoprotein metabolism however is not fully understood. Our laboratory has recently identified two homozygotes and 16 heterozygotes for a coding mutation in SCARB1 (c.1127C&gt;T, p.Pro376Leu). We assessed the hypothesis that this SCARB1 mutation significantly increases HDL-C and alters HDL composition and biological function. Plasma from subjects homozygous (n=2) or heterozygous (n=8) for the SR-BI Pro376Leu mutation was compared with that of normolipidemic controls. Five major HDL subfractions were isolated from EDTA-treated plasma using isopycnic density gradient ultracentrifugation and their chemical composition was assayed using commercial enzymatic assay kits. Quantification of &gt;160 molecular species of HDL phospho- and sphingolipids was accomplished via a novel LC-ESI/MS/MS based analysis. Cholesterol efflux capacity, measured from THP-1 macrophages, was determined in total HDL. We identified a &gt;2-fold increase in HDL-C in homozygotes and a &gt;1.5-fold increase in heterozygotes compared to controls, owing mainly to an increase in large, light HDL2 particles. The ApoA-I/ApoA-II ratio was increased in a gene-dose dependent fashion. HDL isolated from heterozygotes mediated 13.5% less efflux when compared to particles from control subjects. Analysis of the HDL phosphosphingolipidome revealed a 23% decrease in phosphatidylinositol content between carriers and controls. A gene-dose dependent trend towards diminishment of phosphatidylserine and increased ceramide was noticeable. In conclusion, the SR-BI Pro376Leu significantly increases HDL levels, but potentially reduces the capacity of HDL to mediate cellular cholesterol efflux. This may be due in part to a decrease in minor, negatively charged phospholipid subclasses including PI and PS, which have previously been shown to play a functional role in stimulating cellular cholesterol efflux.

Abstract 414: In Hyperalphalipoproteinemic Subjects, the Presence of Coronary Artery Disease is Associated With Marked Changes in the High-Density Lipoprotein Phosphosphingolipidome

Plasma concentrations of HDL-C have long been shown to correlate inversely with risk of coronary artery disease (CAD), however the causal nature of this association has not been established. Here we examine the chemical composition and phosphoshingolipidome of HDL in a cohort of subjects with premature CAD, despite having HDL levels ≥90th percentile. We hypothesized that HDL from hyperalphalipoproteinemic subjects with CAD would have distinct compositional changes compared to healthy hyperalphalipoproteinemic subjects, which may relate a reduction in HDL functionality and a pro-atherogenic state. Subjects with HDL ≥90th percentile and CAD (HHDL+CAD, n=25) were compared to healthy subjects with HDL ≥90th percentile (HHDL, n=23). A group of healthy controls with HDL between the 25th and 75th percentile (n=11) was used as reference. HDL subfractions were isolated from EDTA plasma using isopycnic density gradient ultracentrifugation and their chemical composition was assayed using commercial enzymatic assay kits. Quantification of &gt;160 molecular species of total HDL phospho- and sphingolipids was accomplished via a novel method using LC-ESI/MS/MS analysis. The most striking differences were observed in total HDL phospho- and sphingolipid subclasses between groups. When expressed as a percentage of total phosphosphingolipid, phosphatidylcholine (PC) and phosphatidylinositol (PI) were depleted in HHDL+CAD group compared to HHDL, while sphingomyelin (SM) was increased, resulting in a lower PC/SM ratio. The HDL PC/SM ratio has previously been shown to correlate with HDL surface fluidity and antioxidative activity. Additionally, enrichment of PI, a minor, negatively charged phospholipid, has been shown to stimulate cholesterol efflux to reconstituted HDL. Reduction in these two metrics, observed in the HHDL+CAD group, may indicate functionally deficient HDL and may represent pro-atherogenic, HDL-associated biomarkers of CAD in hyperalphalipoproteinemia.

Morphology and structure of lipoproteins revealed by an optimized negative-staining protocol of electron microscopy

Plasma lipoprotein levels are predictors of risk for coronary artery disease. Lipoprotein structure-function relationships provide important clues that help identify the role of lipoproteins in cardiovascular disease. The compositional and conformational heterogeneity of lipoproteins are major barriers to the identification of their structures, as discovered using traditional approaches. Although electron microscopy (EM) is an alternative approach, conventional negative staining (NS) produces rouleau artifacts. In a previous study of apolipoprotein (apo)E4-containing reconstituted HDL (rHDL) particles, we optimized the NS method in a way that eliminated rouleaux. Here we report that phosphotungstic acid at high buffer salt concentrations plays a key role in rouleau formation. We also validate our protocol for analyzing the major plasma lipoprotein classes HDL, LDL, IDL, and VLDL, as well as homogeneously prepared apoA-I-containing rHDL. High-contrast EM images revealed morphology and detailed structures of lipoproteins, especially apoA-I-containing rHDL, that are amenable to three-dimensional reconstruction by single-particle analysis and electron tomography.

Proteomic Analysis of Defined HDL Subpopulations Reveals Particle-Specific Protein Clusters

Recent proteomic studies have identified multiple proteins that coisolate with human HDL. We hypothesized that distinct clusters of protein components may distinguish between physicochemically-defined subpopulations of HDL particles, and that such clusters may exert specific biological function(s). We investigated the distribution of proteins across 5 physicochemically-defined particle subpopulations of normolipidemic human HDL (HDL2b, 2a, 3a, 3b, 3c) fractionated by isopycnic density gradient ultracentrifugation. Liquid chromatography/electrospray mass spectrometry identified a total of 28 distinct HDL-associated proteins. Using an abundance pattern analysis of peptide counts across the HDL subfractions, these proteins could be grouped into 5 distinct classes. A more in-depth correlational network analysis suggested the existence of distinct protein clusters, particularly in the dense HDL3 particles. Levels of specific HDL proteins, primarily apoL-I, PON1, and PON3, correlated with the potent capacity of HDL3 to protect LDL from oxidation. These findings suggest that HDL is composed of distinct particles containing unique (apolipo)protein complements. Such subspeciation forms a potential basis for understanding the numerous observed functions of HDL. Further work using additional separation techniques will be required to define these species in more detail.

Small, dense HDL 3 particles attenuate apoptosis in endothelial cells: pivotal role of apolipoprotein A‐I

Plasma high-density lipoproteins (HDLs) protect endothelial cells against apoptosis induced by oxidized low-density lipoprotein (oxLDL). The specific component(s) of HDLs implicated in such cytoprotection remain(s) to be identified. Human microvascular endothelial cells (HMEC-1) were incubated with mildly oxLDL in the presence or absence of each of five physicochemically distinct HDL subpopulations fractionated from normolipidemic human plasma (n= 7) by isopycnic density gradient ultracentrifugation. All HDL subfractions protected HMEC-1 against oxLDL-induced primary apoptosis as revealed by nucleic acid staining, annexin V binding, quantitative DNA fragmentation, inhibition of caspase-3 activity and reduction of cytoplasmic release of cytochrome c and apoptosis-inducing factor. Small, dense HDL 3c displayed twofold superior intrinsic cytoprotective activity (as determined by mitochondrial dehydrogenase activity) relative to large, light HDL 2b on a per particle basis (P < 0.05). Equally, all HDL subfractions attenuated intracellular generation of reactive oxygen species (ROS); such anti-oxidative activity diminished from HDL 3c to HDL 2b. The HDL protein moiety, in which apolipoprotein A-I (apoA-I) predominated, accounted for ∼70% of HDL anti-apoptotic activity. Furthermore, HDL reconstituted with apoA-I, cholesterol and phospholipid potently protected HMEC-1 from apoptosis. By contrast, modification of the content of sphingosine-1-phosphate in HDL did not significantly alter cytoprotection. We conclude that small, dense, lipid-poor HDL 3 potently protects endothelial cells from primary apoptosis and intracellular ROS generation induced by mildly oxLDL, and that apoA-I is pivotal to such protection.

Soy protein reduces hepatic lipotoxicity in hyperinsulinemic obese Zucker fa/fa rats

Hepatic steatosis is commonly present during the development of insulin resistance, and it is a clear sign of lipotoxicity attributable in part to an accelerated lipogenesis. There is evidence that a soy protein diet prevents the overexpression of hepatic sterol-regulatory element binding protein-1 (SREBP-1), decreasing lipid accumulation. Therefore, the aim of the present work was to study whether a soy protein diet may prevent the development of fatty liver through the regulation of transcription factors involved in lipid metabolism in hyperinsulinemic and hyperleptinemic Zucker obese fa/fa rats. Serum and hepatic cholesterol and triglyceride levels, as well as VLDL-triglyceride and LDL-cholesterol, were significantly lower in rats fed soy protein than in rats fed a casein diet for 160 days. The reduction in hepatic cholesterol was associated with a low expression of liver X receptor-alpha and its target genes, 7-alpha hydroxylase and ABCA1. Soy protein also decreased the expression of SREBP-1 and several of its target genes, FAS, stearoyl-CoA desaturase-1, and delta5 and delta6 desaturases, decreasing lipogenesis even in the presence of hyperinsulinemia. Reduction in SREBP-1 was not associated with the presence of soy isoflavones. Finally, soy protein reduced SREBP-1 expression in adipocytes, preventing hypertrophy, which also helps prevent the development of hepatic lipotoxicity.

Effect of Pioglitazone on Lipids in Well Controlled Patients with Diabetes Mellitus Type 2 - Results of a Pilot Study

Patients with diabetes mellitus type 2 are characterized by a typical dyslipoproteinemia. Improvement in glucose control usually also ameliorates this dyslipoproteinemia. It is unclear whether different antidiabetic strategies differ in their effects on the lipid profile. Particularly, it is unknown whether glitazones improve lipid values independently of their effects on glucose metabolism. Ten patients well controlled on sulfonylureas (HbA1c 6.9 +/- 0.5 %) with diabetic dyslipoproteinemia were treated with additional pioglitazone (30 mg/d) for 3 months. Every 4 weeks the sulfonylurea dose was adjusted to keep HbA1c and fasting glucose constant. Before and after 3 months of pioglitazone therapy lipid metabolism was determined in detail (cholesterol, triglyceride, LDL-cholesterol, HDL-cholesterol, VLDL-cholesterol, VLDL-triglycerides, lipoprotein(a), LDL-subtype distribution by isopycnic density gradient ultracentrifugation). Although glucose control remained unchanged (HbA1c 6.9 +/- 0.5 % vs. 6.8 +/- 0.6 %; fasting glucose concentration 7.7 +/- 1.1 vs. 7.3 +/- 1.3 mmol/l) we observed a significant reduction in triglyceride concentration (1.9 +/- 0.6 vs. 1.4 +/- 0.5 mmol/l, - 26 %, p < 0.01), a significant increase in HDL-cholesterol concentration (1.2 +/- 0.2 vs. 1.4 +/- 0.2 mmol/l, + 14 %, p < 0.05), a significant decrease in LDL/HDL-ratio (3.03 +/- 0.77 vs. 2.51 +/- 0.61, - 24 %, p < 0.05) and non-significant improvements in total cholesterol, LDL-cholesterol, VLDL-triglycerides, and VLDL-cholesterol concentrations. The LDL-subtype profile improved (significant reduction [- 20 %] in small dense LDL). This pilot study indicates that at comparable fasting glucose concentration and at comparable HbA1c value pioglitazone is superior to sulfonylureas concerning the improvement of diabetic dyslipoproteinemia. Whether this relates to indirect effects (improvement in insulin sensitivity) or direct effects (stimulation of PPARalpha) remains to be determined.

Ultracentrifugation-based approaches to study regulation of Sec6/8 (exocyst) complex function during development of epithelial cell polarity

The Sec6/8 (exocyst) complex is an essential component of the exocytic apparatus and plays an evolutionarily conserved role in polarized membrane growth. During development of epithelial cell polarity, this cytosolic protein complex is recruited to plasma membrane sites of cell–cell contact, where it facilitates exocytosis to the lateral membrane domain. However, the identity of membrane binding sites for Sec6/8 complex, mechanisms regulating association of Sec6/8 complex with these sites, and the precise function of the complex in polarized trafficking are not known. Biochemical strategies involving differential, rate-zonal, and isopycnic density gradient ultracentrifugation are providing clues to these questions.

Effects of obesity on lipid profiles in neutered male and female cats.

To examine whether obese cats, compared with lean cats, have alterations in lipoprotein metabolism that might lead to a decrease in glucose metabolism and insulin secretion. 10 lean and 10 obese adults cats (5 neutered males and 5 neutered females each). Intravenous glucose tolerance tests with measurements of serum glucose, insulin, and nonesterified fatty acid (NEFA) concentrations were performed. Lipoprotein fractions were examined in serum by isopycnic density gradient ultracentrifugation. Obese cats had insulin resistance. Plasma triglyceride and cholesterol concentrations were significantly increased in obese cats, compared with lean cats. Very low density lipoprotein (VLDL) concentrations were increased in obese cats, compared with lean cats; however, the composition of various fractions remained unchanged between obese and lean cats, indicating greater synthesis and catabolism of VLDL in obese cats. Serum high density lipoprotein (HDL) cholesterol concentrations were increased in obese cats, compared with lean cats. Serum NEFA concentrations were only significantly different between obese and lean cats when separated by sex; obese male cats had higher baseline serum NEFA concentrations and greater NEFA suppression in response to insulin, compared with lean male cats. Lipid metabolism changes in obese cats, compared with lean cats. The increase in VLDL turnover in obese cats might contribute to insulin resistance of glucose metabolism, whereas the increase in serum HDL cholesterol concentration might reflect a protective effect against atherosclerosis in obese cats.

LDL particle size: Relation to risk factors and subclinical atherosclerosis measured by coronary calcium

The effect of ciprofibrate treatment on the atherogenic profile of low-density lipoprotein (LDL) subspecies in combined hyperlipidemia (CHL) has been investigated in six patients displaying elevated plasma triglyceride and cholesterol levels ( > 200 and > 250 mg/dl, respectively). The E2E2 phenotype was excluded; four patients possessed familial antecedents of premature coronary heart disease (CHD). Analysis of five LDL subclasses separated by isopycnic density gradient ultracentrifugation showed a predominance of dense LDL subspecies (LDL-4 and LDL-5, d 1.039–1.063 g/ml; 51% of total LDL mass) in the asymmetric LDL density profile characteristic of CHL patients at baseline. Ciprofibrate treatment (100 mg/day for 1 month) effected marked reductions in both total plasma LDL and apo B-100 levels ( ∼ 19% and ∼ 23%, respectively). Equally, the plasma profile of LDL subspecies was normalised to a significant degree as a result of preferential reduction in the elevated levels of both dense subspecies (LDL-4 and LDL-5; −43% and −54%, respectively; P < 0.03 and P < 0.06, respectively). The circulating concentrations of light LDL (LDL-1, d 1.019-1.023 g/ml) were also diminished significantly by ciprofibrate (−30%; P < 0.06). Furthermore, ciprofibrate not only effected reductions in the elevated triglyceride content of the hydrophobic core of all LDL subspecies but also normalised their common deficiency in free cholesterol. In addition, the abnormally small particle diameters of LDL-4 and −5 were increased to normal. Plasma levels of both apo B-100 and triglycerides were significantly and positively correlated with those of LDL-4 and LDL-5, suggesting not only that the degree of triglyceride elevation is intimately linked to the extent of shift in LDL subclass profile towards denser subspecies, but also that triglyceride reduction upon treatment strongly influences LDL-4 and LDL-5. In conclusion, our findings indicate that ciprofibrate treatment in combined hyperlipidemia results in marked reduction in plasma triglyceride levels (-33%), and that such reduction is intimately linked to normalisation of both the qualitative and quantitative features of the atherogenic LDL subspecies profile typical of this disorder.

Cholesteryl ester hydroperoxide lability is a key feature of the oxidative susceptibility of small, dense LDL.

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.

O-Glycosylated MUC2 Monomer and Dimer from LS 174T Cells Are Water-soluble, whereas Larger MUC2 Species Formed Early during Biosynthesis Are Insoluble and Contain Nonreducible Intermolecular Bonds

The MUC2 mucin is the major gel-forming mucin in the small and large intestine. Due to its sequence similarities with the von Willebrand factor, it has been suggested to dimerize in the endoplasmic reticulum and polymerize in the trans-Golgi network. Using an O-glycosylation-sensitive MUC2 antiserum, a dimerization has been shown to occur in the endoplasmic reticulum of LS 174T cells (Asker, N., Axelsson, M. A. B., Olofsson, S.-O., and Hansson, G. C. (1998) J. Biol. Chem. 273, 18857-18863). Using an antiserum immunoprecipitating O-glycosylated MUC2 mucin, monomers and dimers were shown to occur in soluble form in the lysate of LS 174T cells. The amount of O-glycosylated dimer was small, and no larger species were found even after long chase periods. However, most of the labeled MUC2 mucin was found in pelleted debris of the cell lysate. This insoluble MUC2 mucin was recovered by immunoprecipitation after reduction of disulfide bonds. Analysis by agarose gel electrophoresis revealed two bands, of which the smaller migrated as the O-glycosylated monomer and the larger migrated as the O-glycosylated dimer of the cell lysis supernatant. Mucins insoluble in 6 M guanidinium chloride could also be obtained from LS 174T cells. Such mucins have earlier been found in the small intestine (Carlstedt, I., Herrmann, A., Karlsson, H., Sheehan, J., Fransson, L. -A., and Hansson, G. C. (1993) J. Biol. Chem. 268, 18771-18781). Reduction of the mucins followed by purification by isopycnic density gradient ultracentrifugation and analysis by agarose gel electrophoresis revealed two bands reacting with an anti-MUC2 tandem repeat antibody after deglycosylation. These bands migrated identically to the bands shown by metabolic labeling, and they could also be separated by rate zonal ultracentrifugation. These results suggest that the MUC2 mucin is forming nonreducible intermolecular bonds early in biosynthesis, but after initial O-glycosylation.

Tumour growth modifies intravascular polyamine transport by plasma lipoproteins in the mouse

Polyamines are polycationic compounds which are implicated in cell division and tumor growth. We have evaluated the potential role of plasma lipoproteins in the transport of major polyamines, spermine, spermidine and putrescine, and the effect of tumor growth on such transport. Plasmas of healthy male BL6/DBA2 mice and of mice bearing Lewis lung carcinoma (3LL) were fractionated by isopycnic density gradient ultracentrifugation, and polyamine content determined in lipoprotein fractions. Spermidine was the most abundant polyamine in the lipoproteins of both control and tumor-bearing mice and was principally associated with HDL (d: 1.046–1.136 g/ml); approx. 40% of total plasma polyamines was lipoprotein-associated in control mice and 60% in cancerous mice. Only minor amounts were transported by LDL (<10% of total lipoprotein-associated polyamines), while VLDL were devoid of these substances. Marked elevations of circulating levels of LDL were found in 3LL grafted mice: in these particles however, the contents of spermidine and spermine were significantly reduced. A preferential uptake of polyamines by red blood cells could in part explain this marked reduction of LDL polyamine content, but the consequence of this reduction on the net electrical charge and biochemical function of LDL remains unknown. Elevations of plasma LDL and HDL levels in 3LL-grafted mice underlie the finding that only minor modification was detected in the putrescine content of these particles. However, it is evident that elevated total amounts of putrescine were present in the plasma of such animals. Finally, the density profile of polyamines was modified in cancerous mice in which a shift to transport in lighter apo.AI-containing HDL particles was observed for spermidine; an even more marked shift was found for spermine. In conclusion, our data demonstrate that HDL particles constitute the major plasma vehicle for polyamine transport in both control and in tumor-bearing mice. © 1997 Elsevier Science B.V. All rights reserved.