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At least 451 records · Page 25Linked to original sources
PubMed1975

Distribution studies of proteoglycan aggregates by ultracentrifugation.

The tendency forcartilage proteoglycans to aggregate or disperse in ultracentrifugation at high ionic strength is influenced by cations. Guanidinum, Li+ and Ca++ promote disaggregation and Na+, K+ and Cs+ support aggregation. The process of disaggregation is fundamentally the separation of low density from high density proteoglycans, the density being an inverse function of the protein content. PPL 5 pellet formation is a packing or aggregation phenomenon occurring well below the isopycnic density of proteoglycan which accounts for its difference from the isopycnic density equilibrated complex. Cations which promote disaggregation result in shifts in distribution of the proteoglycans on equilibrated density gradients. Comparisons of the desulfated products of the high density aggregated and disaggregated complexes and of more selective high and low density proteoglycan fractions clearly show the heterogeneity of proteoglycans related to protein content. It is suggested that the nature of the variable population of proteoglycan macromolecules permits its existence as a self aggregating system.

Adhesiveness↗
PubMed1976

[Micromethod of ultracentrifugation in cesium chloride].

The micromethod of ultracentrifugation in cesium chloride is described. Band destributions have been analysed by the direct scanning of microtubes with the differential doublewave micro-spectrophotometer at 260 nm. The 10(-8)-10(-9) g of DNA or ribosomes are enough for one analysis, tube volume is 2 mul. The method described permits the simultaneous centrifugation of several probe scores. The error of the beyoant density determination is 0.001 g/sm3 relatively internal standard. DNA's from 5 types of Acetabularia were analysed by the developed technique.

Acetabularia↗
PubMed1979

[Cryoglobulin analysis by ultracentrifugation in density stabilized gradient followed by immunodiffusion (author's transl)].

A technique is described which combines ultracentrifugation in a density gradient stabilized by gelification, with agar gel immunodiffusion. The use of this procedure to determine sedimentation parameters and antigenic determinations is presented and illustrated with the diagramms obtained in the identification of various cryoglobulins.

Centrifugation, Density Gradient↗
PubMed1992

A procedure for large-scale plasmid isolation without using ultracentrifugation.

An expedient procedure for large-scale plasmid isolation from Escherichia coli strains without using ultracentrifugation or special setups or reagents is described. The protocol, which utilizes a modified alkaline extraction procedure as well as differential precipitations by isopropanol and lithium chloride, is simple and rapid and yet produces plasmid DNA with a yield of about 2 mg/liter culture. The isolated plasmids consisted of mostly monomeric and dimeric covalently closed circular DNA. The plasmids could be digested by various restriction endonucleases and were compatible with gene cloning, transfection-gene expression, and viral production.

1-Propanol↗
PubMed1991

A centrifugation method for the isolation of plasmid DNA without the use of an ultracentrifuge.

This paper describes the use of a high-performance high-speed centrifuge, a Sorvall RC-28S, for the preparation of plasmid DNA using standard CsCl-ethidium bromide gradients. If the sample is layered into the bottom of the gradient, the plasmid DNA can be banded in 24 hours; this is comparable with the time required for ultracentrifuge separations. Alternatively, plasmids can be banded in a fixed-angle rotor in 36 hours when the sample is mixed throughout the gradient solution.

Centrifugation↗
PubMed1991

Single-spin density-gradient ultracentrifugation vs gradient gel electrophoresis: two methods for detecting low-density-lipoprotein heterogeneity compared.

Single-spin density-gradient ultracentrifugation (DUC) has proven to be a reproducible method for detection of low-density-lipoprotein (LDL) heterogeneity. Recently another method has been described for this: gradient gel electrophoresis (GGE) of serum, a method that might be more suitable for screening. To gain insight into the relationship of GGE to DUC and into their reproducibility, we determined LDL heterogeneity by DUC and GGE in 41 healthy individuals. In 90.2% (n = 37) of the subjects, the number of LDL subfractions found by both methods agreed. In addition, the density and the relative migration distance of the predominant LDL subfraction observed with the respective methods showed a strong correlation (Pearson correlation, r = 0.85, P less than 0.0001). Although it was not possible to compare for all aspects of LDL heterogeneity, these data suggest that GGE is a valid method of analysis for LDL heterogeneity. In screening programs, it may be necessary to store samples. Therefore, we studied in 24 sera the influence of storage at -80 degrees C for one, four, and 12 weeks on the LDL subfraction distribution detected by each method. LDL heterogeneity was maintained during storage under these conditions.

Adult↗
PubMedDate not supplied

[Study of lipoprotein changes in the vessel wall by ultracentrifugation of aortic extracts in discontinuous density gradients].

As shown by stepwise density gradient ultracentrifugation, atherogenic lipoproteins pentrating into aorta wall, underwent conversions, which were accompanied by alterations in their hydratation density. The data obtained suggest that complex formation of lipoproteins with components of connective tissue matrix is one of important steps responsible for conversion of atherogenic lipoproteins in vessel wall.

Aged↗
PubMed1989

Relationships between LDL density and kinetic heterogeneity in subjects with normolipidemia and familial combined hyperlipidemia using density gradient ultracentrifugation.

The metabolism of heterogeneous subpopulations of low density lipoprotein (LDL) apoB100 was examined in three normolipidemic and two familial combined hyperlipidemic subjects. Autologous radioiodinated plasma LDL (1.019 less than d less than 1.063 g/ml) were injected into each subject and the disappearance and appearance of radiolabeled lipoproteins into various LDL subpopulations were examined using density gradient ultracentrifugation. Eleven to 13 fractions (-320 microliter each) were collected within LDL defined uniquely in each subject. In all subjects, the disappearance of radiolabeled LDL from plasma was biexponential. However, changes with time in the distribution of radiolabeled LDL among the various LDL density subpopulations revealed complex metabolic behavior that differed among the subjects. When the relationships between density and kinetic characteristics were examined in more detail by following the disappearance of individual fractions defining LDL in each subject, the data suggested that: 1) the kinetic behavior of the LDL fractions was more complex than suggested by the disappearance of radiolabeled LDL from plasma: 2) certain fractions within specific density ranges were kinetically similar; 3) distinct differences in the disappearance curves among the fractions occurred within narrow density ranges; and 4) precursor-product relationships were seen among specific LDL density fractions and varied from subject to subject. These studies underscore the complexities of plasma LDL apoB-100 metabolism. More detailed characterizations of the kinetic behavior of various LDL subpopulations should help in our understanding of the origin(s) and potential physiological consequences of different LDL subpopulations.

Adult↗
PubMed1989

Isolation of high molecular weight mucins from human whole saliva by ultracentrifugation.

A high molecular weight mucin fraction was prepared from human whole saliva using relatively mild conditions. The method involves ultracentrifugation of human whole saliva in the presence of 7.2 M urea and 0.5 M sodium chloride. The resulting preparation consists of a highly purified salivary mucin fraction, as judged by several purity criteria: molecular weight analysis of the final preparation by gel electrophoresis and analytical gel filtration indicated an apparent molecular weight greater than 10(6). Analytical isopycnic density centrifugation demonstrated that the preparation consisted of a mucin fraction with a buoyant density of approximately 1.47 g/ml. The final preparation comprised 12.8% protein, 31.8% N-acetylglucosamine, 11.5% N-acetylgalactosamine, 9.5% fucose, 21.4% galactose, 0.8% mannose, 10.3% N-acetylneuraminic acid, 1.7% sulphate and 0.16% fatty acids.

Centrifugation, Isopycnic↗
PubMed1974

Separation of the main lipoprotein density classes from human plasma by rate-zonal ultracentrifugation.

The major lipoprotein density classes (chylomicrons-VLDL, LDL, HDL(2) and HDL(3)) were isolated from human plasma in a two-step ultracentrifugal procedure using the Ti-14 zonal rotor. The isolation of the two major high density lipoprotein subclasses (HDL(2) and HDL(3)) was achieved in a 24-hr run using a nonlinear NaBr gradient in the density range of 1.00-1.40. The lipoproteins with a density < 1.063 found in the rotor's center were isolated in a second run of 140 min duration using a continuous linear NaBr gradient in the density range of 1.00-1.30. The isolated lipoproteins were analyzed for chemical composition and for electrophoretic mobility; purity of isolated fractions was checked by immunochemistry. The lipoproteins exhibited flotation rates, chemical compositions, and molecular weights similar to those found with the common sequential procedures in angle-head rotors. The amount of lipoprotein lipids in the bottom fraction of the zonal rotor was comparable to that of the angle-head rotor. The described method yields the main lipoprotein density classes free from albumin in a very short running time; compared with the rate-zonal techniques already in use, this method allows the quantitative separation of an additional lipoprotein density class (HDL(2)) without increasing the running time. Furthermore, this procedure proved to be suitable for isolation of plasma lipoproteins from subjects with various types and varying degrees of hyperlipoproteinemia.

Adolescent↗
PubMed1979

Fc-mediated immune precipitation. II. Analysis of precipitating immune complexes by rate-zonal ultracentrifugation.

The formation of immune complexes was studied by analytical rate-zonal ultracentrifugation using isomolar solutions of rabbit anti-human serum albumin IgG and of the corresponding F(ab')2 fragments. The F(ab')2 fragments retained full ability to react with the antigenic determinants and to form genuine antigen-antibody complexes. Thus, the difference between the IgG and the F(ab')2 systems was supposed solely to reflect the lack of the Fc portion. The complexes formed with F(ab')2 fragments in the zone of low and moderate antigen excess were found to be distinctively mor soluble than those formed with intact IgG. The data indicated that there were two kinds of precipitating immune complexes, namely antibody-rich and antigen-rich complexes. In the antibody-excess zone and the first part of the equivalence zone the immune complexes precipitated due to their antibody richness. Antigen-rich complexes formed in the zone of low antigen excess precipitate only in the presence of antibody-rich insoluble complexes. It is believed that this type of precipitation was due to an Fc-Fc interaction. This new function of the Fc portion of IgG was designated Fc-mediated immune precipitation.

Animals↗
PubMed1984

Density ultracentrifugation of sickle cells during and after pain crisis: increased dense echinocytes in crisis.

An increase in the number of irreversibly sickled cells (ISCs) in pain crisis has been found by some investigators but not others. We have used the technique of discontinuous arabinogalactan density-gradient ultracentrifugation of whole blood to study ISCs from patients with sickle cell anemia (SCA) during pain crisis and again when pain free (5-331 days after crisis). Nine patients have been studied through ten episodes of pain crisis. Five layers with densities from 1.128 g/mL to 1.158 g/mL have been used. Careful classification of the cells using Nomarsky optics demonstrated highly significant changes occurring in the layers of the gradient. The changes involve the appearance of an increased percentage of echinocytic ISCs and echinocytic cells that were not ISCs, especially in the denser gradient layers, during crisis, and their replacement by normal-appearing discocytes in the pain-free state. There was no change in ISCs that were not echinocytic. Data collected previously demonstrated that reduced glutathione activity correlated with increased echinocytosis in our gradient layers. This indicates that the echinocytic change may occur as a result of oxidant stress. Hemoglobin F levels and the percentage of hemoglobin F from reticulocytes showed no consistent change to coincide with the rise in normal-appearing discocytes in the lightest layers after crisis. Our data indicate that pain crisis occurs in association with an echinocytic change, which may be induced by oxidant injury. The rise in normal-appearing cells after crisis may reflect increased hemoglobin F production in some patients but mainly relates to the disappearance of these echinocytic erythrocytes.

Adult↗
PubMed1983

Proteoglycans from rabbit articular and growth plate cartilage. Ultracentrifugation, gel chromatography, and electron microscopy.

Proteoglycans are considered to be important for the mechanical properties of articular cartilage and growth plate and for the regulation of calcification of growth plate. We have used ultracentrifugation, gel chromatography, and electron microscopy to characterize and compare proteoglycans isolated from rabbit articular cartilage, uncalcified growth plate, and calcified cartilage. We found that proteoglycans from articular cartilage were more resistant to extraction than were proteoglycans from uncalcified growth plate and calcified cartilage. Long term neutral decalcification of calcified cartilage resulted in degraded proteoglycans. The chondroitin sulfate chains from all three tissues had similar size distribution. Gel chromatography and electron microscopy of proteoglycan monomers suggested that those from uncalcified growth plate were largest, those from articular cartilage intermediate, and those from calcified cartilage smallest. Proteoglycan aggregates from articular cartilage were longer than those from uncalcified growth plate. Both biochemical and quantitative electron microscopic data suggested the existence in mineralizing cartilage of at least two different sized populations of proteoglycan aggregates.

Animals↗
PubMed1982

Complement-mediated inhibition of immune precipitation. II. Analysis by sucrose density gradient ultracentrifugation.

The factors influencing the ultracentrifugation characteristics of immune complexes generated in the presence of fresh normal human serum have been analysed. In the absence of alternative pathway factors B, D or Properdin, the size of complexes was increased. When classical pathway function was blocked, in C1q deficient serum or in the presence of Mg EGTA, although the proportion of complexes remaining in solution were reduced their size was similar to those formed in normal human serum. In C2 deficient serum, a heterogeneous population of complexes was generated. In all instances repletion with the appropriate missing complement component reversed the abnormality. We conclude that there is normally a rapid sequential process of classical followed by alternative pathway activation leading to stable soluble complexes. In the absence of C1 activation the alternative pathway process requires precipitation of the antigen-antibody aggregates whereas in normal serum these events occur in the fluid phase. We suggest that in C2 deficient serum the C1 and/or C4 reacted complexes fail to activate the alternative pathway efficiently.

Animals↗
PubMed1982

High-density lipoprotein subfractions as measured by differential polyanionic precipitation and rate zonal ultracentrifugation.

Recent interest in the putative protective role of high-density lipoprotein (HDL) and its subfractions against atherosclerosis has highlighted the need for a rapid, simple subfractionation procedure. Here we compared HDL subfractionation by two recently developed polyanionic-precipitation methods with the values obtained by rate zonal ultracentrifugation. A similar result for total HDL cholesterol was obtained by all three methods. However, HDL2 cholesterol as measured by the precipitation procedures was significantly higher than the zonal value, and HDL3 was lower. This reflects the different underlying principles involved in the separations and highlights the need for a clearer understanding of the functional roles of the HDL fractions.

Adult↗
PubMed1982

[Rapid visualization and separation of serum lipoproteins of the rat by ultracentrifugation in saline density gradients].

A method is presented for the visualization and location of lipoprotein bands in density gradient ultracentrifugation. A 3 hour spin results in a good separation of the major rat serum lipoprotein classes: very low, low and high density lipoproteins, by using small centrifuge tubes and highspeed rotors. In addition, the concentration of cholesterol, triglyceride, phospholipid and protein in the lipoprotein fractions obtained by this technique was also determined.

Animals↗
PubMed1980

Preparative and quantitative isolation of plasma lipoproteins: rapid, single discontinuous density gradient ultracentrifugation in a vertical rotor.

A rapid method has been developed for separation of the major plasma lipoproteins from up to 96 ml of plasma by a single ultracentrifugation step. This separation was achieved by a discontinuous density gradient centrifugation between the density range of 1.006 and 1.30 g/ml in Sorvall vertical rotors. Each lipoprotein fraction was sharply banded with VLDL at the top, LDL in the upper middle, and HDL in the lower middle portion of the tube. Use of authentic 125I-labeled lipoproteins showed that complete separation of the three major classes and partial separation of HDL2 and HDL3 was achieved. The lipoprotein fractions prepared by this technique have properties indistinguishable from those isolated by the sequential flotation method in regard to their equilibrium banding density, electrophoretic mobility, and apolipoprotein composition. This method is suitable for the preparative isolation of lipoproteins as well as for quantitative clinical determinations of cholesterol and triglycerides in VLDL, LDL and HDL fractions of plasma. Used as an analytical tool this method allows samples as small as 1 ml of plasma and spin times as short as 45 min. Cholesterol levels in HDL fractions separated by this method have significantly lower values (P less than 0.05) than those estimated by the heparin-manganese chloride precipitation method.

Centrifugation, Density Gradient↗