Iodixanol Density Gradient Research Articles

Biochemical properties of bull spermatozoa separated in iodixanol density solution

Bull spermatozoa samples contain variable portion of motile and normal morphology spermatozoa along with spermatozoa incapable of fertilization due to their pathologic changes. As semen quality is influenced by biochemical and morphological characteristics of all spermatozoa, the aim of the study was to separate spermatozoa in discontinuous iodixanol density gradient solution and to determine their cholesterol, phospholipid, triacylglycerol and lipid peroxide concentrations and creatine kinase activity. The study was performed in winter and included seven Simmental bulls aged 1.5–3.5years. Semen samples were collected by use of artificial vagina. Upon evaluation of semen quality (volume, concentration and progressive sperm motility), the samples were centrifuged in iodixanol density solution to obtain two sperm fractions. The two fractions included sperms with progressive motility greater than 90% and less than 20%, respectively. A statistically significantly higher lipid peroxide concentration was determined in sperm fraction with <20% progressive motility. Different sperm subpopulations can be obtained by separating bull spermatozoa in different iodixanol density gradient solutions, while monitoring their biochemical properties can help assess the sperm quality.

Insulin resistance and low-density apolipoprotein B-associated lipoviral particles in hepatitis C virus genotype 1 infection

BackgroundThe density of hepatitis C virus (HCV) in plasma is heterogeneous but the factors which influence this are poorly understood. Evidence from animal models and cell culture suggest that low-density...

The Arabidopsis Peroxisomal ABC Transporter, Comatose, Complements the Saccharomyces cerevisiae pxa1 pxa2Δ Mutant for Metabolism of Long-chain Fatty Acids and Exhibits Fatty Acyl-CoA-stimulated ATPase Activity

The Arabidopsis ABC transporter Comatose (CTS; AtABCD1) is required for uptake into the peroxisome of a wide range of substrates for β-oxidation, but it is uncertain whether CTS itself is the transporter or if the transported substrates are free acids or CoA esters. To establish a system for its biochemical analysis, CTS was expressed in Saccharomyces cerevisiae. The plant protein was correctly targeted to yeast peroxisomes, was assembled into the membrane with its nucleotide binding domains in the cytosol, and exhibited basal ATPase activity that was sensitive to aluminum fluoride and abrogated by mutation of a conserved Walker A motif lysine residue. The yeast pxa1 pxa2Δ mutant lacks the homologous peroxisomal ABC transporter and is unable to grow on oleic acid. Consistent with its exhibiting a function in yeast akin to that in the plant, CTS rescued the oleate growth phenotype of the pxa1 pxa2Δ mutant, and restored β-oxidation of fatty acids with a range of chain lengths and varying degrees of desaturation. When expressed in yeast peroxisomal membranes, the basal ATPase activity of CTS could be stimulated by fatty acyl-CoAs but not by fatty acids. The implications of these findings for the function and substrate specificity of CTS are discussed.

OP14 Measurement of low density apolipoprotein B associated hepatitis C virus lipoviral particles in genotype 1 infection is more clinically relevant than total viral load

IntroductionThe density of hepatitis C virus (HCV) in plasma is heterogeneous but the factors that influence this are poorly understood. Evidence from animal models and cell culture suggest that low-density...

Self-assembly of Severe Acute Respiratory Syndrome Coronavirus Membrane Protein

Coronavirus membrane (M) protein can form virus-like particles (VLPs) when coexpressed with nucleocapsid (N) or envelope (E) proteins, suggesting a pivotal role for M in virion assembly. Here we demonstrate the self-assembly and release of severe acute respiratory syndrome coronavirus (SARS-CoV) M protein in medium in the form of membrane-enveloped vesicles with densities lower than those of VLPs formed by M plus N. Although efficient N-N interactions require the presence of RNA, we found that M-M interactions were RNA-independent. SARS-CoV M was observed in both the Golgi area and plasma membranes of a variety of cells. Blocking M glycosylation does not appear to significantly affect M plasma membrane labeling intensity, M-containing vesicle release, or VLP formation. Results from a genetic analysis indicate involvement of the third transmembrane domain of M in plasma membrane-targeting signal. Fusion proteins containing M amino-terminal 50 residues encompassing the first transmembrane domain were found to be sufficient for membrane binding, multimerization, and Golgi retention. Surprisingly, we found that fusion proteins lacking all three transmembrane domains were still capable of membrane binding, Golgi retention, and interacting with M. The data suggest that multiple SARS-CoV M regions are involved in M self-assembly and subcellular localization.

An efficient process for the purification of helper-dependent adenoviral vector and removal of helper virus by iodixanol ultracentrifugation

The preparation of large amount of purified helper-dependent adenoviral vector material is hampered by the lack of development of downstream processes with proven records on separation and recovery efficiencies. In order to facilitate the use of clinical-grade helper-dependent virus material for large-scale in vivo studies, a three-step purification scheme consisting of (1) an anion-exchange chromatography for initial capturing of virus, (2) a shallow iodixanol density gradient ultracentrifugation for the removal of helper virus from helper-dependent virus, and (3) a size-exclusion chromatography for the removal of iodixanol and residual protein contaminants as a polishing step was developed. The use of a fast iodixanol density ultracentrifugation step was highly effective in separating infectious helper-dependent virus from contaminating helper virus. The overall downstream processing scheme gave 80% infectious particle yield. The contamination ratio of helper virus in the helper-dependent virus preparation are reduced from 2.57 to 0.03% corresponding to a reduction of helper virus by factors of 85 by two iodixanol purification steps. It was also demonstrated that size-exclusion chromatography is an excellent step for the removal of iodixanol and polishing of the final helper-dependent virus preparation.

Improved, Low-Cost Methods for Pancreatic Islet Purification in Rats

Objective Density gradient separation of islets from exocrine tissue is usually performed using Ficoll. However, this reagent significantly increases the cost of isolation. The aim of the present study was to investigate the effects on islet preparations of purification methods using Lymphoprep and Iodixanol (OptiPrep) density gradients. Methods Pancreata were procured from 46 Wistar rats, loaded with collagenase V (Sigma), and mechanically dissociated using standard procedures. After the digestion phase, the islets purified by 3 methods—Ficoll, Lymphoprep, and Iodixanol (OptiPrep)—were assessed for yields, purity, morphology, and in vitro function. Result We expressed the yields as islet equivalents (IEQ, diameter standardizing to 150 μm), showing no significant differences. Compared with the Ficoll group, the purity was significantly higher in the Lymphoprep ( P = .005) and Iodixanol (OptiPrep) groups ( P = .011). While the viability was >90% in all 3 groups, the viability in the Lymphoprep Group and OptiPrep groups was significantly higher than that of the Ficoll group ( P < .001). In vitro islet function did not differ among the 3 experimental groups. Conclusion Lymphoprep and Iodixanol were as effective as Ficoll in terms of islet yield and in vitro function. High-purity and high-viability islet cells were obtained using improved Lymphoprep-based or Iodixanol (OptiPrep)-based density gradient methods, potential low-cost substitutes for Ficoll.

Potential abnormalities in iron metabolism in hyperlipidemia patient fibroblasts

Mucolipidosis type IV (MLIV) is an autosomal recessive neurodegenerative disorder that results from a mutation in mucolipin 1, a 580 amino acid non‐selective cation channel present on lysosomal membranes. This mutation disrupts sorting, transport and/or fusion of endosomes and lysosomes, and subjects suffer from iron deficiency anemia. To establish whether lysosomal turnover of endogenous ferritin was disrupted, fibroblasts from normal and MLIV subjects were pretreated with 59Fe‐labeled ferric ammonium citrate (180 uM) for 24h to produce ferritin (Ft) and then with desferioxamine to induce Ft turnover in lysosomes (Kidane et al, Am J Physiol 291: C445, 2006). No defects in Ft turnover and Fe release were observed, as demonstrated with iodixanol density gradients separating cytoplasmic and lysosomes/endosomal Ft and its iron. However, when exogenous (cationized) horse spleen Ft was administered, turnover of this iron was slowed and more was held in lysosomes and endosomes in the case of cells from diseased subjects. Accumulation of cytoplasmic Ft protein (measured by ELISA) was also reduced. We conclude that a reduced rate of processing of iron entering by endocytosis may slow recycling of red cell iron and contribute to the development an iron deficiency‐like anemia. Supported in part by PHS Grant No. RO1 HD 46949.

Lipoprotein separation in a novel iodixanol density gradient, for composition, density, and phenotype analysis

Separation of lipoproteins by traditional sequential salt density floatation is a prolonged process ( approximately 72 h) with variable recovery, whereas iodixanol-based, self-generating density gradients provide a rapid ( approximately 4 h) alternative. A novel, three-layered iodixanol gradient was evaluated for its ability to separate lipoprotein fractions in 63 subjects with varying degrees of dyslipidemia. Lipoprotein cholesterol, triglycerides, and apolipoproteins were measured in 21 successive iodixanol density fractions. Iodixanol fractionation was compared with sequential floatation ultracentrifugation. Iodixanol gradient formation showed a coefficient of variation of 0.29% and total lipid recovery from the gradient of 95.4% for cholesterol and 84.7% for triglyceride. Recoveries for VLDL-, LDL-, and HDL-cholesterol, triglycerides, and apolipoproteins were approximately 10% higher with iodixanol compared with sequential floatation. The iodixanol gradient effectively discriminated classic lipoproteins and their subfractions, and there was evidence for improved resolution of lipoproteins with the iodixanol gradient. LDL particles subfractionated by the gradient showed good correlation between density and particle size with small, dense LDL (<25.5 nm) separated in fractions with density >1.028 g/dl. The new iodixanol density gradient enabled rapid separation with improved resolution and recovery of all lipoproteins and their subfractions, providing important information with regard to LDL phenotype from a single centrifugation step with minimal in-vitro modification of lipoproteins.

Proteomic Analysis of Highly Purified Peroxisomes from Etiolated Soybean Cotyledons

To identify previously unknown peroxisomal proteins, we established an optimized method for isolating highly purified peroxisomes from etiolated soybean cotyledons using Percoll density gradient centrifugation followed by iodixanol density gradient centrifugation. Proteins in highly purified peroxisomes were separated by two-dimensional PAGE. We performed peptide mass fingerprinting of proteins separated in the gel with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and used the peptide mass fingerprints to search a non-redundant soybean expressed sequence tag database. We succeeded in assigning 92 proteins to 70 sequences in the database. Among them, proteins encoded by 30 sequences were judged to be located in peroxisomes. These included enzymes for fatty acid beta-oxidation, the glyoxylate cycle, photorespiratory glycolate metabolism, stress response and metabolite transport. We also show experimental evidence that plant peroxisomes contain a short-chain dehydrogenase/reductase family protein, enoyl-CoA hydratase/isomerase family protein, 3-hydroxyacyl-CoA dehydrogenase-like protein and a voltage-dependent anion-selective channel protein.

Potential chitinase activating factor from yeast cells of Candida albicans.

Microsomal chitinase from yeast and hyphal cells of Candida albicans was activated endogenously by incubation at 30 degrees C and exogenously by trypsin. The putative activating factor of yeast cells was separated from chitinase activity by fractionation of lysed protoplasts on an Iodixanol density gradient. The vacuole fraction contained no significant chitinase activity, but was enriched in chitinase activating factor. Activity of microsomal chitinase increased upon incubation with this, but no other gradient factor. Results suggest that the regulatory system governing microsomal chitinase activity, like that governing chitin synthase, involves a 'vacuolar' activating factor in Candida albicans.

Isolation of chicken follicular dendritic cells

The aim of the present study was to isolate chicken follicular dendritic cells (FDC). A combination of methods involving panning, iodixanol density gradient centrifugation, and magnetic cell separation technology made it possible to obtain functional FDC from the cecal tonsils from chickens, which had been infected with Eimeria tenella. CD45− dendritic cells were selected using the specific monoclonal antibody against chicken CD45, which is a marker for chicken leukocytes, but is not expressed on chicken FDC. Isolated FDC were characterized morphologically, phenotypically and functionally. The phenotype of the selected cells was consistent with FDC in that they expressed IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1, but lacked cell surface markers characteristic of macrophages, T-, and B cells. Transmission electron microscopy confirmed their characteristic dendritic morphology. In addition, the identity of the FDC was further confirmed by their ability to trap chicken immune complexes (ICs) on their surface, whereas they did not trap naive antigen (ovalbumin) or ICs generated with mammalian immunoglobulins. Co-culturing allogeneic or autologous isolated FDC with B cells resulted in enhanced B cell proliferation and immunoglobulin production. The lack of MHC restriction, a functional characteristic feature of FDC, further reinforces the identity of the isolated cells as chicken FDC.

SorLA/LR11 Regulates Processing of Amyloid Precursor Protein via Interaction with Adaptors GGA and PACS-1

SorLA has been recognized as a novel sorting receptor that regulates trafficking and processing of the amyloid precursor protein (APP) and that represents a significant risk factor for sporadic Alzheimer disease. Here, we investigated the cellular mechanisms that control intracellular trafficking of sorLA and their relevance for APP processing. We demonstrate that sorLA acts as a retention factor for APP in trans-Golgi compartments/trans-Golgi network, preventing release of the precursor into regular processing pathways. Proper localization and activity of sorLA are dependent on functional interaction with GGA and PACS-1, adaptor proteins involved in protein transport to and from the trans-Golgi network. Aberrant targeting of sorLA to the recycling compartment or the plasma membrane causes faulty APP trafficking and imbalance in non-amyloidogenic and amyloidogenic processing fates. Thus, our findings identified altered routing of sorLA as a major cellular mechanism contributing to abnormal APP processing and enhanced amyloid beta-peptide formation.

Purification of human respiratory syncytial virus by ultracentrifugation in iodixanol density gradient

Ultracentrifugation in sucrose density gradient remains the most commonly used technique for hRSV purification. However, the high viscosity and hyper-osmotic property of sucrose can cause damage to the extremely labile virus leading to loss of infectivity. To overcome these limitations, an alternative purification technique was developed using iodixanol as gradient medium, incorporating MgSO4 as a stabilizing agent and EDTA to disaggregate the virus prior to infectivity assay. Virus particles were banded at the 20–36% interface after purification of polyethylene glycol-concentrated viruses by rate zonal ultracentrifugation on a 20–52% discontinuous iodixanol gradient. The presence of the virus was confirmed by viral fusion glycoprotein content using ELISA. After further purification by buoyant density ultracentrifugation on a 20–52% continuous gradient, the virus was recovered in the region of density 1.15–1.19g/ml and this was confirmed by the coincidence of the infectivity titre, viral genome and fusion glycoprotein peaks. Analysis of recovery rates showed that the use of iodixanol increased the virus yield up to 69%. Iodixanol was also found to be non-toxic to HeLa cells used in infectivity assay, eliminating the need of its downstream removal by dialysis.

Quantum Yield Heterogeneities of Aqueous Single-Wall Carbon Nanotube Suspensions

Aqueous suspensions of single-wall carbon nanotubes (SWNTs) prepared by traditional methods from the supernatant of ultracentrifuged micellar SWNT mixtures are shown to be strongly heterogeneous with respect to the quantum yield of their constituents. The heterogeneities are isolated using preparative ultracentrifugation in iodixanol density gradients and are optically characterized by photoluminescence and linear absorption spectroscopy. We find that the most buoyant fractions with a density of 1.055 ± 0.005 g·cm-3 have the highest photoluminescence quantum yield η of 1.1%, a factor of 5 higher than that of the supernatant. Denser fractions with lower η make up for about 2/3 of these samples and contain mostly small ropes in which the quantum yield is reduced by nonradiative decay through coupling to metallic tubes.

Ultracentrifugation of Serum Samples Allows Detection of Hepatitis C Virus RNA in Patients with Occult Hepatitis C

Occult hepatitis C virus (HCV) infection of patients with abnormal liver function tests of unknown origin who are anti-HCV and serum HCV RNA negative but who have HCV RNA in the liver has been described. As HCV replicates in the liver cells of these patients, it could be that the amount of circulating viral particles is under the detection limit of the most sensitive techniques. To prove this hypothesis, serum samples from 106 patients with occult HCV infection were analyzed. Two milliliters of serum was ultracentrifuged over a 10% sucrose cushion for 17 h at 100,000 x g(av), where av means average, and HCV RNA detection was performed by strand-specific real-time PCR. Out of the 106 patients, 62 (58.5%) had detectable serum HCV RNA levels after ultracentrifugation, with a median load of 70.5 copies/ml (range, 18 to 192). Iodixanol density gradient studies revealed that HCV RNA was positive at densities of 1.03 to 1.04 and from 1.08 to 1.19 g/ml, which were very similar to those found in the sera of patients with classical chronic HCV infection. Antigenomic HCV RNA was found in the livers of 56 of 62 (90.3%) patients with detectable serum HCV RNA levels after ultracentrifugation, compared to 27 of 44 (61.4%) negative patients (P < 0.001). No differences in the median loads of antigenomic HCV RNA between patients with an those without serum HCV RNA (4.5 x 10(4) [range, 7.9 x 10(2) to 1.0 x 10(6)] versus 2.3 x 10(4) [range, 4.0 x 10(2) to 2.2 x 10(5)]) were found. Alanine aminotransferase and gamma-glutamyl transpeptidase levels, liver necroinflammatory activity, and fibrosis did not differ between both groups. In conclusion, HCV RNA can be detected in the sera of patients with occult HCV infection after circulating viral particles are concentrated by ultracentrifugation.

Alzheimer’s presenilin 1 modulates sorting of APP and its carboxyl‐terminal fragments in cerebral neurons in vivo

Studies in continuously cultured cells have established that familial Alzheimer's disease (FAD) mutant presenilin 1 (PS1) delays exit of the amyloid precursor protein (APP) from the trans-Golgi network (TGN). Here we report the first description of PS1-regulated APP trafficking in cerebral neurons in culture and in vivo. Using neurons from transgenic mice or a cell-free APP transport vesicle biogenesis system derived from the TGN of those neurons, we demonstrated that knocking-in an FAD-associated mutant PS1 transgene was associated with delayed kinetics of APP arrival at the cell surface. Apparently, this delay was at least partially attributable to impaired exit of APP from the TGN, which was documented in the cell-free APP transport vesicle biogenesis assay. To extend the study to APP and carboxyl terminal fragment (CTF) trafficking to cerebral neurons in vivo, we performed subcellular fractionation of brains from APP transgenic mice, some of which carried a second transgene encoding an FAD-associated mutant form of PS1. The presence of the FAD mutant PS1 was associated with a slight shift in the subcellular localization of both holoAPP and APP CTFs toward iodixanol density gradient fractions that were enriched in a marker for the TGN. In a parallel set of experiments, we used an APP : furin chimeric protein strategy to test the effect of artificially forcing TGN concentration of an APP : furin chimera that could be a substrate for beta- and gamma-cleavage. This chimeric substrate generated excess Abeta42 when compared with wildtype APP. These data indicate that the presence of an FAD-associated mutant human PS1 transgene is associated with redistribution of the APP and APP CTFs in brain neurons toward TGN-enriched fractions. The chimera experiment suggests that TGN-enrichment of a beta-/gamma-secretase substrate may play an integral role in the action of mutant PS1 to elevate brain levels of Abeta42.

The Use of Iodixanol for the Purification of Rat Pancreatic Islets

Transplantation of pancreatic islets is a promising therapeutic treatment for type 1 diabetes mellitus. For clinical and experimental transplantation, a large number of pure pancreatic islets are required for transplantation. Thus, the improvement of islet isolation and purification techniques are crucial. In this context, iodixanol-based solution, successfully used for the purification of porcine islets, seems to be a possible alternative to Ficoll for purification of islets. The aim of this study was to test the efficacy of iodixanol compared with Ficoll density gradients for the purification of rat pancreatic islets. Twelve Wistar rats were used for isolation and purification of pancreatic islets. Pancreata were digested with Liberase R1 and islets purified by two gradients: Ficoll or iodixanol gradient. The number and the purity of the pancreatic islets were assessed. To analyze the response of isolated pancreatic islet to glucose challenge, in vitro experiments were performed by measuring the insulin concentration in the Supernatant. The results demonstrated that the iodixanol gradient provided a higher purity of pancreatic islets compared to the Ficoll gradient. In addition, the rat islet yield by iodixanol gradient was significantly higher compared to a Ficoll gradient (751 ± 16 versus 464 ± 19 pancreatic islets, respectively; P < .001). The viability of pancreatic islets isolated by an iodixanol gradient was confirmed by high glucose challenge, with more than twofold higher increase in insulin secretion. The present study demonstrated that iodixanol density gradient overcomes Ficoll density gradient, providing a greater number of pure and functional rat pancreatic islets.

Insulin-stimulated exocytosis of GLUT4 is enhanced by IRAP and its partner tankyrase

The glucose transporter GLUT4 and the aminopeptidase IRAP (insulin-responsive aminopeptidase) are the major cargo proteins of GSVs (GLUT4 storage vesicles) in adipocytes and myocytes. In the basal state, most GSVs are sequestered in perinuclear and other cytosolic compartments. Following insulin stimulation, GSVs undergo exocytic translocation to insert GLUT4 and IRAP into the plasma membrane. The mechanisms regulating GSV trafficking are not fully defined. In the present study, using 3T3-L1 adipocytes transfected with siRNAs (small interfering RNAs), we show that insulin-stimulated IRAP translocation remained intact despite substantial GLUT4 knockdown. By contrast, insulin-stimulated GLUT4 translocation was impaired upon IRAP knockdown, indicating that IRAP plays a role in GSV trafficking. We also show that knockdown of tankyrase, a Golgi-associated IRAP-binding protein that co-localizes with perinuclear GSVs, attenuated insulin-stimulated GSV translocation and glucose uptake without disrupting insulin-induced phosphorylation cascades. Moreover, iodixanol density gradient analyses revealed that tankyrase knockdown altered the basal-state partitioning of GLUT4 and IRAP within endosomal compartments, apparently by shifting both proteins toward less buoyant compartments. Importantly, the afore-mentioned effects of tankyrase knockdown were reproduced by treating adipocytes with PJ34, a general PARP (poly-ADP-ribose polymerase) inhibitor that abrogated tankyrase-mediated protein modification known as poly-ADP-ribosylation. Collectively, these findings suggest that physiological GSV trafficking depends in part on the presence of IRAP in these vesicles, and that this process is regulated by tankyrase and probably its PARP activity.

Establishment of subcellular fractionation techniques to monitor the intracellular fate of polymer therapeutics II. Identification of endosomal and lysosomal compartments in HepG2 cells combining single-step subcellular fractionation with fluorescent imaging

As they are often designed for lysosomotropic, endosomotropic and/or transcellular delivery, an understanding of intracellular trafficking pathways is essential to enable optimised design of novel polymer therapeutics. Here, we describe a single-step density gradient subcellular fractionation method combined with fluorescent detection analysis that provides a new tool for characterisation of endocytic traffic of polymer therapeutics. Hepatoma (HepG2) cells were used as a model and cell breakage was optimised using a cell cracker to ensure assay of the whole cell population. After removal of unbroken cells and nuclei, the cell lysate as a post-nuclear supernatant (PNS) was layered onto an iodixanol (OptiPrep™) density gradient optimised to 5–20%. Early endosomes, late endosomes and lysosomes were identified from gradient fractions by immunoblotting for marker proteins early endosome antigen 1 (EEA 1) and lysosomal associated membrane protein 1 (LAMP 1) using horseradish peroxidase or fluorescently-labelled secondary antibodies. Lysosomes were also detected using N-acetyl-β-glucosamindase (Hex A) activity. In addition, cells were incubated with Texas-red labelled transferrin (TxR-Tf) for 5 min to specifically label early endosomes and this was directly detected from SDS-PAGE gels. Internalised macromolecules and colloidal particles can potentially alter vesicle buoyant density. To see if typical macromolecules of interest would alter vesicle density or perturb vesicle traffic, HepG2 cells were incubated with dextran or a polyethyleneglycol (PEG)-polyester dendron G4 (1 mg/ml for 24 h). The PEG-polyester dendron G4 caused a slight redistribution of endocytic structures to lower density fractions but immunofluorescence microscopy showed no obvious dendron effects. In conclusion, the combined subcellular fractionation with fluorescent imaging approach described here can be used as a tool for both fundamental cell biology research and/or the quantitative localisation of polymer therapeutics in the endocytic pathway.