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PubMed1984

[Possibilities for using the analytical ultracentrifugation method for determining circulating immune complexes in rheumatic diseases].

Analytical ultracentrifugation was used to study circulating immune complexes in 78 patients suffering from different rheumatic diseases. Using this method immune complexes were mostly detectable in patients with rheumatoid arthritis. This correlated with the disease activity, roentgenological stage of articular involvement, development of the systemic manifestations associated with rheumatoid vasculitis. Demonstration of immune complexes by analytical ultracentrifugation was found to correlate on the whole with the presence in sera of high titers of rheumatoid factors and immune complexes according to the PEG method data.

Adolescent↗
PubMed1984

Evidence for the nucleus-centriole association in living cells obtained by ultracentrifugation.

The association of centrioles with the interphase nuclei of L- and PE-cells has been studied using ultracentrifugation of a cell monolayer in a culture medium at 20 and 37 degrees C. Ultracentrifugation at 10 000 to 40 000 gav for 15 to 60 min did not cause any changes in the cell length or in the size of the nucleus, but entailed delocalization of nuclei. The distance between it and the centrioles hardly ever changes. Any nucleus-delocalizing centrifugation of non-treated cells also resulted in the centrioles being shifted towards the centripetal nucleus pole. After 30 to 60 min, at 40 000 gav, the cells remained viable and capable of mitosis. More intensive centrifugation (15 min at 70 000 gav) proved to be fatal to the cells. The distance between the centrioles and the nucleus became greater in the cells which were centrifuged after incubation with cytochalasin B. The results are interpreted as lending support to the previously demonstrated [28] association between the centrioles and the nucleus in the interphase cells.

Animals↗
PubMed1983

Serum lipoprotein measurement--liquid chromatography and sequential floatation (ultracentrifugation) compared.

An HPLC method [J. Biochem. (Tokyo) 91:1381, 1982] was used for evaluating serum lipoproteins, with on-line monitoring of either cholesterol or phospholipids. Five well-distinguished lipoprotein fractions were observed, based on their particle sizes. Serum of 15 normal persons, 12 subjects with various types of hyperlipidemia, 20 patients with various liver diseases, and two cases of familial LCAT deficiency were examined and the results compared with those by a sequential ultracentrifugal floatation technique. In the normal group, the amounts of fractions 2, 3, and 4 by the HPLC method correlated well with concentrations of the LDL, HDL2, and HDL3 fractions as measured by the ultracentrifugal method, respectively. In the hyperlipidemic group, similar good correlations were observed between fractions 1, 2, 3, 4 and chylomicrons + VLDL, LDL, HDL2, and HDL3 fractions, respectively. For those with liver diseases or LCAT deficiency, the corresponding fractions correlated less well, and characteristically the elution profile of lipoproteins in these groups showed heterogeneity of particle size within each lipoprotein density class, especially in LDL and HDL2.

Cholesterol↗
PubMed1981

Separation of serum high-density lipoprotein for cholesterol determination: ultracentrifugation vs precipitation with sodium phosphotungstate and magnesium chloride.

We compared two methods for separation of serum high-density lipoprotein: selective precipitation with sodium phosphotungstate and magnesium chloride, and ultracentrifugation in sodium chloride solution (relative density 1.063). When the cholesterol content (determined enzymically with a centrifugal analyzer) of fractions obtained by each method was compared (ultracentrifugation = x), the correlation coefficient was 0.97; y = 1.01x - 11.2 mg/L; p less than 0.05; n = 54. The within-day and between-day coefficients of variation for this method were 1.1 and 4.0%, respectively. Reference intervals for high-density lipoprotein cholesterol in subpopulations categorized by age and sex were based on data obtained from volunteer blood donors.

Chemical Precipitation↗
PubMed1976

Ultracentrifugal behavior of apolipoprotein A-I of rhesus monkey (Macaca mulatta).

The sedimentation equilibrium and sedimentation velocity of apolipoprotein AI (apo-A-I) from high density lipoproteins of rhesus monkey plasma were studied in aqueous solutions of 0.02 M EDTA, pH 8.6, as a function of protein concentration. The sedimentation equilibrium results showed that, in the concentration range between 9 and 36 muM (0.25 to 1.0 g/liter), apo-A-I behaved as a single species of molecular weight 28,200. This molecular weight corresponds to that of the monomeric form as previously estimated from electrophoretic and chemical data. As the apo-A-I concentration was increased above 36 muM, the log c versus r2 plots from sedimentation equilibrium experiments became curvilinear, suggesting self-association. An analysis of these data generated nonlinear, nonoverlapping molecular weight versus concentration plots pointing at the existence of a heterogeneous population of apo-A-I. By sedimentation velocity studies apo-A-I at concentrations between 68 and 330 muM exhibited two distinct components, with S0/20,w = 1.9 and S0/20,w = 3.9. When separated by gel permeation chromatography, these two components had an identical amino acid composition and retained similar S20,w values as before column fractionation. The slow component had a diffusion coefficient of 8.27 X 10(-7) cm2/s and, assessed by the criteria of sedimentation equilibrium ultracentrifugation, remained monomeric even at concentrations of 125 muM. The fast component, when analyzed by low speed sedimentation equilibrium, behaved as a self-associating system which could best be fitted into a monomer-hexamer model in a rapid equilibrium. The equilibrium constant for this association was found to be 9.5 X 10(23) M(-5). Thus, rhesus apo-A-I, dissolved in 0.02 M EDTA, pH 8.6, consisted of two distinct species, one monomeric over a relatively wide range of concentrations, the other readily self-associating. Their structural relationship remains to be established. This ultracentrifugal behavior of rhesus apo-A-I differs markedly from that reported for human apo-AI (Vitello, L. B., and Scanu, A. M. (1976) J. Biol. Chem. 251, 1131-1136).

Animals↗
PubMed1993

Sequential ultracentrifugation micromethod for separation of serum lipoproteins and assays of lipids, apolipoproteins, and lipoprotein particles.

We describe a fast sequential separation of very-low-density, low-density, and high-density lipoproteins from 400 microL of serum, using the Beckman TL100 ultracentrifuge. The cumulative centrifugation time is 9.5 h. The purity of lipoprotein fractions was verified by a gel-filtration procedure. The major contaminant is the serum albumin, which can be eliminated by a second centrifugation at the same density. Enzymatic measurement of lipids shows good recovery (> 91%) and weak within-sample variation (< 7%). In comparison with a density-gradient procedure, the deleterious effects on the lipoprotein structure appear to be limited, as shown by the low concentrations of apolipoprotein (apo) E and apo A-I in the fraction > 1.21 kg/L. Furthermore, the micro-ultracentrifugation also gives a better recovery rate. Finally, we have studied the distribution of lipids, apolipoproteins, and lipoprotein particles (LpA-I:A-II, LpB:C-III, LpB:E) in each fraction separated from 10 serum samples from healthy subjects.

Adult↗
PubMed1975

Identification of strains of herpes simplex virus by comparison of the density of their DNA using the preparative ultracentrifuge.

The buoyant densities of the DNA of herpes simplex virus type 1, type 2 and Pseudorabies virus, as determined in the analytical ultracentrifuge, are 1.725, 1.727 and 1.731 correlating with G+C contents of 67, 69 and 73 per cent respectively. The density differences for the DNA's of type 1 and type 2 herpes simplex viruses have been confirmed in experiments with isotopically labelled DNA from four type 1 and six type 2 strains by preparative CsCl gradient ultracentrifugation. The DNA of all the type 2 strains was denser than that of any of the type 1 strains examined. Despite these differences in DNA base composition of type 1 and type 2 strains, nearest neighbour analysis of their DNA's disclosed no obvious differences in doublet pattern or general design.

Base Sequence↗
PubMed1996

An improved ultracentrifugation method for the separation of cholesterol carriers in bile.

Vesicles and micelles, the major carriers of cholesterol in bile, play a role in the formation of cholesterol gallstones. A simple and rapid ultracentrifugation method was developed to isolate these biliary cholesterol carriers when only microliter amounts of bile were available. The proposed method employs a 46 to 0% sucrose density gradient, a NVT90 near-vertical rotor, and a centrifugation time of one hour. As little as 25 microL of bile can be used with no disruption of the carriers. The method was validated by comparison with gel filtration column chromatography using 6 mM taurocholate in the elution buffer. The sucrose linear density gradient ultracentrifugation procedure described here is simple, fast, and compares favorably with the gel filtration chromatography method for the separation of cholesterol carriers from bile.

Animals↗
PubMed1985

Isolation of serum chylomicrons prior to density gradient ultracentrifugation of other serum lipoprotein classes.

A method for the removal of serum chylomicrons before density gradient ultracentrifugation of the other serum lipoproteins using an SW 41 swinging bucket rotor is presented. In a preliminary spin, the chylomicrons with an Sf greater than 400 X 10(-13) s float to the top of the gradient, whereas the other lipoproteins are retained in the infranatant fraction. After removal of the chylomicrons, the other serum lipoproteins are subsequently fractionated by isopycnic density gradient ultracentrifugation. Analysis of the separated lipoprotein fractions suggested that this procedure permits isolation of a chylomicron fraction consisting solely of chylomicrons but that the very low density lipoprotein fraction subsequently isolated also contains chylomicrons or chylomicron remnants with an Sf less than 400 X 10(-13) s, and that there is considerable overlap in flotation rate and particle size of very low density lipoproteins and chylomicrons.

Centrifugation, Density Gradient↗
PubMed1986

An automated method for determining buoyant density of nucleic acids using a preparative ultracentrifuge.

We present a technique for analytical buoyant density sedimentation of nucleic acids which is performed in a preparative ultracentrifuge, in contrast to an analytical ultracentrifuge. Following centrifugation in a preparative rotor, small cylindrical quartz tubes are optically scanned; upon completion of the scan the data are processed immediately by a microcomputer and the buoyant density of the nucleic acid is calculated. Experimental data are presented employing several different deoxyribonucleic acids banded in neutral and alkaline cesium sulfate. Results are independent of rotor speed, location of bands within the gradient, and loading density of the cesium sulfate solution. Derived buoyant density values agree within 0.5% of previously published values.

Centrifugation, Density Gradient↗
PubMed1987

Purification of very high density lipoproteins by differential density gradient ultracentrifugation.

Differential density gradient ultracentrifugation procedures, utilizing a vertical rotor, were developed for the preparative purification of very high density lipoproteins (VHDL, density greater than 1.21 g/ml). The VHDLs of several insect species were purified as follows. An initial density gradient ultracentrifugation step removed lipoproteins of lower density from the VHDL-fraction, which partially separated from the nonlipoproteins present in the infranatant. A complete separation was achieved by a second centrifugation step employing a modified gradient system. The use of a vertical rotor and specially designed discontinuous gradients allows a relatively fast, efficient, and economical isolation of the class of very high density lipoproteins. Similar gradient systems should be useful for the detection and purification of VHDLs from other sources.

Animals↗
PubMed1990

Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge.

Commonly, subcellular organelles such as nuclei, mitochondria, lysosomes, and Golgi membranes are isolated first by differential centrifugation in low-speed or high-speed centrifuges and then purified by gradient centrifugation in ultracentrifuges. We have prepared these organelles using a new high-speed centrifuge (28,000 rpm max) which allows the generation of higher radial centrifugal forces (rcfs) than are available in standard machines. We have shown that most subcellular organelles can be purified by using low-viscosity Nycodenz gradients at rcfs lower than those normally used in ultracentrifuges, without increasing the time of centrifugation. Use of Nycodenz also allows rapid harvesting of material from gradients and we have adapted a number of enzyme assays to facilitate gradient analysis.

Animals↗
PubMed1988

Lipid-induced aggregation of phospholipase A2: sucrose density gradient ultracentrifugation and crosslinking studies.

The aggregation behavior of cobra venom (Naja naja naja) phospholipase A2 in the presence of lipids and Ca2+ was examined using ultracentrifugation and crosslinking techniques. Velocity sedimentation experiments were performed in sucrose gradients. The sedimentation coefficients of the cobra phospholipase A2 and various controls, including bovine serum albumin (BSA), malate dehydrogenase, carbonic anhydrase and pancreatic phospholipase A2, were calculated both in the presence and absence of ligands. The monomeric phospholipid, diheptanoylphosphatidylcholine, and the phospholipid analogue, dodecylphosphocholine (DPC), increased the sedimentation coefficient of the cobra phospholipase A2 from 2.2 S to 2.9 S, a value that is consistent with the formation of an enzyme dimer. The control proteins were unaffected by the presence of phospholipid, except for BSA, which apparently binds large amounts of DPC. Crosslinking experiments with glutaraldehyde showed that in the presence of diheptanoylphosphatidylcholine or DPC, the amount of crosslinked enzyme increased. Ca2+ had no effect on the aggregation state of the enzyme as measured by either technique. Both the ultracentrifugation data and crosslinking data are consistent with the hypothesis that the cobra venom phospholipase A2 exists as a dimer or higher-order aggregate in the presence of lipid substrate, although it is yet to be determined whether the functional subunit is a monomer, dimer or higher-order oligomer.

Barium↗
PubMed1986

DNA-repair studies with sodium fluoride: comparative evaluation using density gradient ultracentrifugation and autoradiography.

Sodium fluoride (NaF) was assayed for the induction of DNA-repair synthesis in WI-38 human diploid fibroblasts and in primary cultures of rat hepatocytes. DNA-repair synthesis in non-replicating DNA was measured by ultracentrifugation of density-labeled DNA in CsCl gradients. When this method was used, NaF did not induce DNA-repair synthesis in either of these cell types. However, when NaF was assayed for induction of unscheduled DNA synthesis (UDS) in rat hepatocytes by autoradiography, an increased net nuclear grain count was observed. Because the autoradiographic results were not confirmed by density-gradient ultracentrifugation of hepatocyte DNA, which is a more definitive technique, it is doubtful whether the autoradiographic results actually represent DNA-repair synthesis. Modifications of the UDS/autoradiography protocol to include more extensive washing resulted in no UDS response. Published reports (Hellung-Larsen and Klenow, 1969; Srivastava et al., 1981) describe the formation of precipitable complexes of Mg2+, F-, and [3H]thymidine triphosphate which suggests that autoradiographic measurement of UDS may lead to artifacts when testing NaF unless extensive washing of the cultures is employed.

Animals↗
PubMed1992

Simulation of the time course of macromolecular separations in an ultracentrifuge. II. Controlling the solute concentrations.

We describe algorithms, based on a simulation described in Part I of this series of papers, for the control of the distribution of one or more solutes in preparative or analytical ultracentrifuges equipped with programmable speed control. All of the methods involve the determination, during numerical integration of the Lamm equation, of protocols for continuously varying the rotor speed. We show one such protocol that has been used for the prevention of cesium chloride crystallization during DNA plasmid purification in the preparative ultracentrifuge. Other protocols that are described involve the in situ controlled mixing of two solutes. Limitations of the method owing to problems with input parameter imprecision, fundamental physical constraints, and mechanical limitations are discussed.

Algorithms↗
PubMed2002

A technique for isolation of rubella virus-like particles by sucrose gradient ultracentrifugation using Coomassie brilliant blue G crystals.

An improved method for the isolation of rubella virus-like particles (RVLP) from cell culture supernatant of transfected Chinese hamster ovary (CHO24S) cells is described. It employs a combination of membrane filtration with sucrose gradient ultracentrifugation. It was found that staining the RVLP band with Coomassie brilliant blue G (CBB) resulted in the CBB crystals adsorbing RVLP. After ultracentrifugation (25,000 rpm, 3h, 4 degrees C) a sharp blue band with crystals (diameter 30-40 microm) was observed (at a density of 1.250 g/ml at 25 degrees C) in a 30-60% sucrose gradient. Using a combination of SDS-PAGE and Western blotting techniques, E1 rubella virus structural protein was detected only in the solutions derived from the sharp blue band. A decrease in crystal concentration a few millimeters above or below the main band was associated with a decrease in protein concentration. By dilution with a saturated ice-cold 30% sucrose solution it was possible to pellet the crystals by centrifugation (15,000 rpm, 10 min). SDS-PAGE showed a much higher concentration of RVLP structural protein in the pellet than in the supernatant. This RVLP-containing material is especially suitable for the preparation of rubella virus immunoblot stripes.

Animals↗