Isolation of plasma lipoproteins by a combination of differential and density gradient ultracentrifugation

Summary A method for preparative isolation of serum lipoproteins by a combination of differential and density gradient ultracentrifugation is presented. Total plasma lipoproteins are first isolated in a concentrated form by ultracentrifugation in a fixed angle rotor at a plasma background density of 1.21 kg/l. Subsequently, the various lipoprotein classes are separated by density gradient ultracentrifugation in a swinging bucket rotor. The procedure requires only two ultracentrifugation steps and combines advantages of both ultracentrifugation techniques.

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Isolierung von Plasmalipoproteinen durch eine Kombination von Differential- und Dichtegradient-Ultrazentrifugation

Abbreviations VLDL very low density lipoproteins - LDL low density lipoproteins - HDL high density lipoproteins - VHDL very high density lipoproteins     

Ultracentrifugation Studies of Polyelectrolytes in Polyglucose Density Gradient

An ideal solute for density gradient ultracentrifugation of polymers in aqueous solution should be inert and readily soluble in water to form an extended range of solution densities of low viscosity. High molecular weight is an added attraction because osmotic effects are minimized. Highly branched spherical synthetic polysaccharides fulfill these requirements. High degree of branching is a consequence of the condensation of polyfunctional monomers. Density and relative viscosity of solutions of polyglucose, sucrose, and of a natural sucrose polymer, Ficoll, are compared. The behavior of various polyelectrolytes was studied in low viscosity polyglucose density gradients in equilibrium buoyant density measurement in the ultra-centrifuge. Macromolecules or macromolecular complexes attain low apparent equilibrium buoyant density, probably because of an excluded volume effect of the solute. This allows sedimentation to isopycnic position of complex biopolymers in inert polyglucose solutions, which otherwise can be attempted only in concentrated solutions of heavy salts (such as CsCI or Cs₂SO₄). Such salts, however, may salt out, or through osmotic effects degrade or alter the properties of certain biologic macromolecular complexes.     

Native agarose gel electrophoresis and electroelution: A fast and cost‐effective method to separate the small and large hepatitis B capsids

Hepatitis B core antigen (HBcAg) expressed in Escherichia coli is able to self‐assemble into large and small capsids comprising 240 (triangulation number T = 4) and 180 (triangulation number T = 3) subunits, respectively. Conventionally, sucrose density gradient ultracentrifugation and SEC have been used to separate these capsids. However, good separation of the large and small particles with these methods is never achieved. In the present study, we employed a simple, fast, and cost‐effective method to separate the T = 3 and T = 4 HBcAg capsids by using native agarose gel electrophoresis followed by an electroelution method (NAGE‐EE). This is a direct, fast, and economic method for isolating the large and small HBcAg particles homogenously based on the hydrodynamic radius of the spherical particles. Dynamic light scattering analysis demonstrated that the T = 3 and T = 4 HBcAg capsids prepared using the NAGE‐EE method are monodisperse with polydispersity values of ～15% and ～13%, respectively. ELISA proved that the antigenicity of the capsids was not affected in the purification process. Overall, NAGE‐EE produced T = 3 and T = 4 capsids with a purity above 90%, and the recovery was 34% and 50%, respectively (total recovery of HBcAg is ～84%), and the operation time is 15 and 4 times lesser than that of the sucrose density gradient ultracentrifugation and SEC, respectively.     

Numerical analysis of density gradient centrifugation profiles from eukaryotic DNA

A numerical method for the deconvolution of superimposed Gaussian distributions with a unique solution has been proposed by Medgyessy [10]. We have tested the usefulness of this method for the analysis of density gradient centrifugation profiles from eukaryotic DNA, which are normally composed from overlapping Gaussian distributed profiles of several subcomponents with different mean buoyant densities. From the analysis of human DNA and from model calculations we conclude that major subcomponents can be identified by this method, if they differ in their buoyant density by approximatly 0.005 g/ml. Minor components can only be identified if the total DNA has been fractionated according to buoyant density and the analysis is performed on the density profiles of the subfractions. This procedure represents a quick method to determine a reliable minimum number of subcomponents of DNA.This paper was presented at the VI. Symposium on Analytical Ultracentrifugation, Marburg, FRG, February 16–17, 1989.     

Adsorption behavior of cesium on montmorillonite-type clay in the presence of potassium ions

The adsorption behavior of Cs ions on a montmorillonite-type clay was investigated in the presence of potassium ions, using a radiotracer technique. The initial concentration of K⁺ added to the CsCl was between 10^-4 and 10^-1 mol/l. The addition of K⁺ to the CsCl solution at different concentrations (10^-6-10^-2 mol/l) reduced the amount of Cs⁺ adsorbed on clay. The maximum ratio of Cs⁺ exchanged, calculated from a linearized form of Langmuir plot was in agreement with the ion exchange isotherms of Cs-K ions. Sorption energy evaluated from the graph of corrected selectivity coefficients vs. equivalent fraction of Cs in the solid phase was compared to the energy values obtained from Dubinin-Radushkevich (D-R) isotherms. Freundlich isotherm parameters were used to characterize a site distribution function, which provides information about the affinity ratio of the adsorption sites of Cs⁺ and K⁺ ions. The ion exchange isotherm of Cs-K systems exhibited a Langmuir type curve for all K⁺ concentrations.     

Expression,Purification, and Characterization of Hepatitis B Virus Surface Antigens (HBsAg) in Yeast <Emphasis Type="Italic">Pichia Pastoris</Emphasis>

Prevention of the prevalence of HB depends upon the development of efficient diagnostic reagent and preventive vaccine. Pichia pastoris offers many advantages over the other expression systems in the production of recombinant HBsAg. In this study, we reported that the recombinant P. pastoris strains were cultured in shake flasks and then scaled up in a 5.0-l bioreactor: approximately 27 mg/l of the protein and the maximal cell OD at 600 nm of 310 were achieved in the bioreactor. The recombinant HBsAg was purified by three steps of purification procedures. SDS-PAGE showed that the purified recombinant HBsAg constituted only one homogeneous band of ~24 kDa. CsCl density gradient ultracentrifugation assay indicated that the density of the HBsAg was 1.2 mg/ml, which was in agreement with the natural HBsAg, the HBsAg expressed in Saccharomyces cerevisiae and in mammalian cells. Electron microscope observation revealed that the purified recombinant HBsAg was homogeneous 22-nm particles, suggesting the HBsAg expressed in P. pastoris was self-assembled to virus-like structures. Competitive ELISA indicated that P. pastoris-derived HBsAg possessed the excellent immunoreaction with anti-HBsAg. Animal immunization showed that the immunogenicity of P. pastoris-derived HBsAg was superior to that of S. cerevisiae-derived HBsAg. Together, our results demonstrated that the recombinant HBsAg expressed in P. pastoris could provide promising, inexpensive, and large-scale materials for the diagnostic reagent and vaccine to prevent HBV infection. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Rushi Liu and Qinlu Lin contributed equally to this work.     

Physical,chemical and immunological characteristics of continuous-density lipoproteins isolated by single-spin gradient ultracentrifugation

The isolation of various lipoproteins of very low, intermediate, low and high density from 5 ml of serum was obtained by single-step ultracentrifugation in a potassium bromide density gradient obtained by smoothing a discontinuous gradient during the run (120 000g, 48 h, 4°C; Beckman SW27 rotor). The density profiles were reproducible. The lipoproteins were characterized by electron microscopy, fluorescence polarization, polyacrylamide gel electrophoresis and complete chemical assays. The composition of the entire lipoprotein spectrum was in accordance with previous results. The lipoprotein molecular mass, particle size distribution, Apo AI/AII ratio and microviscosity varied according to the density range.     

Advances in the Synthesis and Characterization of Polypeptide-Based Hybrid Block Copolymers

Linear polystyrene-block-poly(Z-L-lysine) copolymers with a very narrow molecular weight distribution (polydispersity index < 1.03) could be obtained via the ring-opening polymerization of Z-L-lysine-N-carboxyanhydride using ω-(primary amino hydrochloride)-polystyrenes as macroinitiators in N,N-dimethylformamide as the solvent at 40-80 °C. The block copolymer samples were analyzed by means of NMR, size exclusion chromatography, and analytical ultracentrifugation.     

Volumetric Properties and Volumetric Interaction Parameters of the CsCl‐saccharides (D‐glucose,D‐fructose)‐water Solutions at 298.15 K

Densities have been measured for the CsCl‐saccharide (D‐glucose, D‐fructose)‐water systems at 298.15 K. These data were used to calculate the apparent molar volume of CsCl (V_φ,E) and the saccharides (V_φ,S), and the infinite dilution apparent molar volume V_φ,E⁰ and V_φ,S⁰ in the studied solutions. In addition, the standard transfer volume Δ_tV_φ,E⁰ of CsCl from water to aqueous saccharides solutions, and Δ_tV_φ,S⁰ of saccharides from water to CsCl solutions have been evaluated and discussed using the structural interaction model. The volumetric interaction parameters for CsCl with saccharide in water were obtained and analyzed by the group additivity principle and the stereochemistry of the saccharide molecules.     

Potential energy surfaces for Rh?CO from DFT calculations

We present potential energy surfaces for Rh? CO obtained from density functional theory for two electronic states of Rh? CO. We have performed local spin-density calculations including relativistic as well as gradient corrections. The construction of a reasonably accurate atom–atom potential for Rh? CO is not possible. We were much more successful in constructing the potential energy surfaces by representing the potential as a spherical expansion. The expansion coefficients, which are functions of the distance between the rhodium atom and the carbon monoxide center of mass, can be represented by Lennard-Jones, Buckingham, or Morse functions, with an error of the fit within 10 kJ/mol. The potential energy surfaces, using Morse functions, predict that the electronic ground state of Rh? CO is ²Σ⁺ or ²Δ. This is a linear structure with an equilibrium distance of rhodium to the carbon monoxide center of mass of 0.253 nm. The bonding energy is ?184 kJ/mol. Further, Morse functions predict that the first exicted state is ⁴A′. This is a bent structure (∠Rh? CO = 14°) with an equilibrium distance of rhodium to the carbon monoxide center of mass of 0.298 nm. The bonding energy of this state is ?60 kJ/mol. Both these predictions are in good agreement with the actual density functional calculations. We found 0.250 nm with ?205 kJ/mol for ²Σ⁺ and 0.253 nm with ?199 kJ/mol for ²Δ. For ⁴A′, we found 0.271 nm, ∠Rh? CO = 30°, with ?63 kJ/mol. The larger deviation for ⁴A′ than for ²Σ⁺ or ²Δ is a consequence of the fact that the minimum for ⁴A′ is a very shallow well. © 1994 by John Wiley & Sons, Inc.     

New method for the computation of ionic distribution around rod-like polyelectrolytes with the helical distribution of charges. I. General approach and a nonlinearized Poisson–Boltzmann equation

This article is a first step in an attempt to reevaluate the relative role of different contributions to the energetics of DNA in salt solutions. To identify individual terms yielding such contributions a new derivation is given of the generalized Poisson–Boltzmann equation, which includes correlation effects, and explicitly shows terms ignored in the regular Poisson–Boltzmann approach. A general method based on the Boundary Element Technique is discussed, which can be used to evaluate these terms in the next steps of the reevaluation. An implementation of this method for the solution of the nonlinear Poisson–Boltzmann equation is described in detail, and is used to compute the ionic atmosphere around DNAs modeled as cylinders with helical distributions of charges. In the B-type DNA models, it is found that the ion densities in the minor and major grooves near the DNA surface differ by up to threefold. This difference is ca. 10-fold for Z-type DNA models. There are 20–25% differences in the magnitude of the maximum ionic charge density between DNA models of the same type. The addition of excess salt (up to 0.15 M) changes this maximum by only 10–15%. This change is not proportional to the concentration of excess salt. The contributions of different factors to the stabilization of alternative forms of DNA are evaluated. These factors are: (1) interactions between the phosphates, (2) interactions of phosphates with water, (3) interactions of phosphates with the ionic cloud, (4) interactions within the ionic cloud, (5) entropy of the ionic cloud. It is found that regardless of large variations in the counterion distributions around different DNAs, energetic contributions from these distributions are similar (?12.65 ± 0.6 kcal/mol · cell). The calculated change in stabilization per unit cell of models of B and Z-type DNAs due to 0.15 M excess NaCl is only ?0.56 ± 0.02 kcal/mol, indicating no tendency toward B-Z transition in this concentration range. Significantly larger variations of the order of 10 kcal/mol per unit cell can result from factors 1–2. Possible effects of the realistic DNA-solvent boundaries on the energetics of DNA solutions are discussed.     

Influence of local chain dynamics on diffusion of gases in polymers as determined by pulsed field gradient NMR

Diffusion of gases in polymers below the glass transition temperature, T_g, is strongly modulated by local chain dynamics. For this reason, an analysis of pulsed field gradient (PFG) nuclear magnetic resonance (NMR) diffusion measurements considering the viscoelastic behavior of polymers is proposed. Carbon‐13 PFG NMR measurements of [¹³C]O₂ diffusion in polymer films at 298 K are performed. Data obtained in polymers with T_g above (polycarbonate) and below (polyethylene) the temperature set for diffusion measurements are analyzed with a stretched exponential. The results show that the distribution of diffusion coefficients in amorphous phases below T_g is wider than that above it. Moreover, from a PFG NMR perspective, full randomization of the dynamic processes in polymers below T_g requires long diffusion times, which suggests fluctuations of local chain density on a macroscopic scale may occur. © 2009 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 48: 231–235, 2010     

The very-high-temperature pyrolysis of ethane: Evidence against high rates for dissociative recombination reactions of methyl radicals

The pyrolysis of 1 and 2% ethane in krypton has been studied in shock waves by the laser-schlieren technique over 1700–4800 K. For 2400–2800 K an effective zero density gradient is seen following the rapid dissociation of the ethane. Through simulation with various mechanisms it is evident that the high rates for the dissociative recombination reactions of methyl radicals obtained in recent shock-tube studies, are incompatible with this observation; these rates must be reduced at least an order of magnitude. On the basis of theory and previous low-temperature (T) measurements, k = 7.8 × 10¹¹ (-6562/T) (cm³/mol s) is recommended for the second of these reactions.     

Fluorinated hydrazonoyl chlorides as precursors for synthesis of antimicrobial azoles

Hydrazonoyl halides have been known for their ability to react with different reagents to afford wide range of bioactive heterocyclic systems as thiazoles and imidazopyrazoles. This research work focused on the synthesis of two new fluorinated hydrazonoyl chlorides and used them in synthesis of novel series of thiazole derivatives and two imidazopyrazole systems. The mechanistic pathways and the structures of all synthesized derivatives were discussed and assured based on the available spectral data. The results of antimicrobial activity of the tested thiazoles and imidazopyrazoles showed that some derivatives have moderate to no activity against tested fungi and bacteria strains. Only one derivative namely 2-(2-(3-fluoro-4-methylphenyl)hydrazono)-7-(2-oxoindolin-3-ylidene)-2,7-dihydro-3H-imidazo[1,2-b]pyrazole-3,6(5H)-dione is the most active against Candida albicans (CA) with IZD = 20 mm, which was equipotent to ketoconazole. Furthermore, docking simulation was carried out to predict the binding pattern of the new compounds in the ATP binding site of the DNA gyrase B enzyme. The results of the docking simulation revealed that compounds 9a-e , 12 , and 13a,b fit well in the ATP binding site of DNA gyrase B with docking score values ranging from −5.883 to −6.833 kcal/mol.     

Synthesis and discovery of N-(1-methyl-4-oxo-4,5-dihydro-1H-imidazol-2-yl)-[1,1′-biphenyl]-2-carboxamide derivatives as antimicrobial agents

Novel dihydro-1H-imidazole-2-yl)-[1,1′-biphenyl]-2-carboxamides ( 4a-l ) was achieved using a three-step synthesis process and evaluated as antimicrobial agents. These compounds were characterized through FTIR, NMR, LCMS and evaluated for DNA gyrase inhibition potentials and antimicrobial properties against Gram-negative bacteria Escherichia coli (MTCC 443), Pseudomonas aeruginosa (MTCC 424), Klebsiella pneumoniae MTCC 530 and Gram-positive bacteria Staphylococcus aureus (MTCC 3160), Corynebacterium diphtheriae (MTCC 116) and Streptococcus pyogenes (MTCC 442). Excellent DNA gyrase inhibition exhibited by compound 4f (IC₅₀ 0.2 μM and relative percentage activity 96.24%). A broad spectrum of antimicrobial activity showed by compounds 4d , 4f and 4 k with a Minimal Inhibitory Constant (MIC) of 1.05, 1.35 and 1.25 μg mL⁻¹, respectively.     

Particle size measurement of lipoprotein fractions using diffusion-ordered NMR spectroscopy

The sizes of certain types of lipoprotein particles have been associated with an increased risk of cardiovascular disease. However, there is currently no gold standard technique for the determination of this parameter. Here, we propose an analytical procedure to measure lipoprotein particles sizes using diffusion-ordered nuclear magnetic resonance spectroscopy (DOSY). The method was tested on six lipoprotein fractions, VLDL, IDL, LDL₁, LDL₂, HDL₂, and HDL₃, which were obtained by sequential ultracentrifugation from four patients. We performed a pulsed-field gradient experiment on each fraction to obtain a mean diffusion coefficient, and then determined the apparent hydrodynamic radius using the Stokes–Einstein equation. To validate the hydrodynamic radii obtained, the particle size distribution of these lipoprotein fractions was also measured using transmission electron microscopy (TEM). The standard errors of duplicate measurements of diffusion coefficient ranged from 0.5% to 1.3%, confirming the repeatability of the technique. The coefficient of determination between the hydrodynamic radii and the TEM-derived mean particle size was r ² = 0.96, and the agreement between the two techniques was 85%. Thus, DOSY experiments have proved to be accurate and reliable for estimating lipoprotein particle sizes.     

L.D.A. Simultaneous Measurements of Local Density and Velocity Distribution of Particles in Plasma Fluidized Bed at Atmospheric Pressure

Laser Doppler anemometry (L.D.A.) is an efficient and nonintrusive technique. Today, improved in its configuration, the L.D.A. has been applied even in flowing plasmas. ^(1,2) In-flight simultaneous measurements were performed for local density and velocity of particle distribution. The measurements provide an insight into thermal and mass transfer, chemical reactivity, and the distribution of residence times of particles in a plasma fluidized bed. The difficulties of L.D.A. in a plasma fludized bed such as high emission intensity of the plasma torch, high temperature, high particle density, and large distribution of particle granulometry were overcomed in the present investigation. The aims achieved were the characterization of the plasma fluidized bed distribution together with accurate measurements of local particle density and velocity as measured by L.D.A.     

Isolation and characterization of native nuclear deoxyribonucleic acid from tobacco leaves]

Tobacco nuclear DNA (nDNA) was isolated from tobacco leaf nuclei which were prepared according to our previously published procedure [8]. The nDNA was characterized by base analysis, absorption spectrophotometry, analytical CsCl density gradient equilibrium centrifugation and by its melting behaviour. The results show that the isolated tobacco nDNA is native, high molecular weight DNA, which is free of detectable amounts of chloroplast DNA, RNA, protein and polysaccharides. From its melting behaviour it was concluded that tobacco nDNA can be placed close to calf thymus DNA with respect to intramolecular heterogeneity. Experiments on the partial and complete denaturation of tobacco nDNA and its ability to renature are also reported.