Gradient Medium Research Articles

Density gradients prepared from colloidal silica particles coated by polyvinylpyrrolidone (Percoll)

A new gradient medium (Percoll) for density gradient centrifugation of cells and subcellular particles is described. It consists of colloidal silica particles which have been firmly coated with a layer of polyvinylpyrrolidone. The particle population is polydisperse, and the average diameter is approximately 17 nm. Both the electrophoretic mobility of the particles and the conductivity of the solution are low, indicating a low surface charge. The colloid has a high solubility and forms clear solutions. It can be used in concentrations which give solution densities between 1.00 and 1.20 g/ml. The solutions have a low osmolality and can be mixed with electrolytes of physiological pH and ionic strength. The sedimentation properties of the particles are described. Density gradients can be formed by highspeed centrifugation. The colloid has been shown to be nontoxic for a number of cells and cell organelles.

Fractionation of unfixed chromatin by buoyant-density centrifugation in gradients containing 3-iodo-1,2-propanediol and metrizamide.

Buoyant-density centrifugation of unfixed chromatin has been performed in a newly devised medium containing 3-iodo-1,2-propanediol and metrizamide. Chromatins were obtained from isotopically labeled mouse hepatoma cells in suspension culture, either grown normally or density labeled in a medium containing bromodeoxyuridine, by mild digestion of isolated nuclei with micrococcal nuclease. When a mixture of normal and density labeled chromatin, marked with [14C]thymidine and [3H]bromodeoxyuridine, respectively, was centrifuged in the medium, chromatin peaks represented by labeled DNA were resolved to the extent expected from their separate banding profiles. Centrifugation of an equivalent chromatin mixture labeled with [14C] and [3H]lysine, respectively, also yielded resolution of chromatin peaks represented by labeled proteins. Only small amounts of labeled proteins were dissociated from chromatin in the gradient medium. Labeled proteins recovered from the gradient fractions were analyzed by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The results suggested that most of the histones remained associated with the original stretches of DNA during the centrifual fractionation period. Essentially all of the dissociated proteins were found to be non-histone proteins.

The viability of cells grown or centrifuged in a new density gradient medium, Percoll(TM)

A new density gradient medium, Percoll (a modified colloidal silica), has been tested for toxicity in primary cultures of rat liver and calf testicle cells, and in continuous cultures of pig kidney and HeLa cells. The presence of Percoll did not appreciably affect the growth or viability of the cells as judged from cell counts and morphology. The various cells were also centrifugea in gradients of Percoll and subsequently cultured. The in vitro growth of the cells was similar to that of untreated cells. Rat liver cells were labelled in vivo with [ 125I]asialoceruloplasmin (parenchymal cells) or heat-denatured [ 125I]albumin (non-parenchymal cells). After dispersion of the cells and iso-pycnic centrifugation in Percoll the non-parenchymal cells banded preferentially at a lower density (1.04−1.05 g/ml) than parenchymal cells (1.07−1.09 g/ml). The two types of cells showed very different morphology in cell culture. The non-parenchymal cells retained their phagocytic properties during culture. Injured cells and cell debris band at the top of the Percoll gradients in contrast to their behaviour in gradients containing low molecular weight substances. Centrifugation in Percoll can be used to enrich viable cells.

Transport of L-proline and D-glucose in luminal (brush border) and contraluminal (basal-lateral) membrane vesicles from the renal cortex

The uptakes of L-proline and D-glucose by brush border and basal-lateral membranes, derived from the rabbit renal cortex tubular cell plasma membrane, represented mostly transport into an intravesicular space rather than membrane binding. A Na+ electrochemical gradient (medium>vesicle) stimulated the initial rates of uptake of both the imino acid and sugar, effecting their transient movements into the membrane vesicle against concentration gradients. The uptakes of L-proline and D-glucose by basal-lateral membranes were less responsive to Na+ and the relatively small stimulation could largely be accounted for by contamination of the basal-lateral membrane preparation with brush border membranes. These findings are consistent with the hypothesis that the transcellular “active” transports of L-proline and D-glucose consist of a Na+ electrochemical gradient-dependent uphill influx at the luminal brush border end and a Na+-independent downhill efflux at the basal-lateral region of the tubule epithelial cell.

SV 40 Nucleoprotein complexes: Structural modifications after isopycnic centrifugation in metrizamide gradients

Nucleoprotein complexes (NPCs) containing viral DNA and cellular histones except H 1 can be isolated from permissive cells infected with SV 40 [ 11 or polyoma virus [2]. Similar complexes can be obtained by mild dissociation of SV 40 or polyoma virions [3,4]. Electron microscopic studies of these complexes showed the presence of 2 1 * 2 v bodies or nucleosomes interconnected by naked DNA filaments [5,6]. This beaded structure is very similar to that of cellular chromatin [7,8]. The purification of viral NPCs, essentially limited to sucrose density gradients, is insufficient. Buoyant density centrifugations of irreversibly fixed NPCs have been described [91 l] , but the fixation process might introduce artifacts, and the material so purified is of little use for further biochemical studies. Metrizamide, a new density gradient medium [ 121, was recently used for the purification of chromatin in the absence of aldehyde fixation [ 131 , for the isolation of reconstituted DNA-histone complexes [ 141, for the purification of ribonucleoprotein particles [ 151, and for biochemical studies on polyoma NPCs [16]. We have isolated SV 40 nucleoprotein complexes by sedimentation on sucrose gradients, and further purified them by isopycnic centrifugation in metrizamide gradients. Detailed electron microscopic studies of SV 40 NPCs show that although the beaded circular structure is generally well preserved

Energetics of the Na+-dependent transport of D-glucose in renal brush border membrane vesicles.

The energetics of the Na+-dependent transport of D-glucose into osmotically active membrane vesicles, derived from the brush borders of the rabbit renal proximal tubule, was studied by determining how alterations in the electrochemical potential of the membrane induced by anions, ionophores, and a proton conductor affect the uptake of the sugar. The imposition of a large NaCl gradient (medium is greater than vesicle) resulted in the transient uptake of D-glucose into brush border membranes against its concentration gradient. In the presence of Na+ salts of isethionate or sulfate, both relatively impermeable anions, there was no accumulation of D-glucose above the equilibrium value. With Na+ salts of two highly permeable lipophilic anions, NO3- and SCN-, the transient overshoot was enhanced relative to that with Cl-. With Na+ salts whose mode of membrane translocation is electroneutral, i.e. acetate, bicarbonate, and phosphate, no overshoot was found. These findings suggest that only anions which penetrate the brush border membrane and generate an electrochemical potential, negative on the inside, permit the uphill Na+-dependent transport of D-glucose.

Selective retention and formation of a delta5-androstenediol-receptor complex in cell nuclei of the rat vagina.

Cellular protein binding of a number of androstene and androstane derivatives that promote the growth of the vagina in rats has been studied. It was found that cell nuclei of the rat vagina contain a tissue-specific protein that binds 3beta,17beta-dihydroxy-androst-5-ene (delta5-androstenediol), a unique steroid causing growth and keratinization of the vaginal epithelium. The formation of the steroid-protein complex can be demonstrated by the administration of delta5-[3H]androstenediol to ovariectomized rats or by the incubation of minced vagina with the radioactive steroid. The steroid can interact with purified vaginal cell nuclei even in the absence of a cytosol preparation, forming the same steroid-protein complex. The formation of the complex is temperature-dependent; it occurs much more readily at 37 degrees than at 0 degrees. The delta5-[3H]androstenediol-protein complex migrated as about 4 S in a sucrose gradient medium containing 0.4 M KCl. A similar complex can be detected when nuclei of vaginal cells are incubated with 3alpha,17beta-dihydroxy-5alpha-androstane, 3beta,17beta-dihydroxy-5alpha-androstane, and 3beta-hydroxy-androst-5-en-17-one which also have the capability of stimulating vaginal epithelium, although in somewhat different ways. These steroids may bind to different groups of chromatin-bound receptor proteins in various layers of vaginal epithelium. The delta5-androstenediol binding protein is not found in the vaginal cytosol fraction that contains receptor proteins for estrogens and progestins, nor in the cytosol or nuclei of rat uterus cells, but not in muscle, brain, kidney, or liver. Testosterone and 5alpha-dihydrostestosterone bind weakly to the protein, whereas cortisol, androstenedione, 17beta-estradiol, and progesterone do not bind to the same protein by any significant extent.

Density gradient fractionation of dentine and bone powder

In order to obtain enough material to analyse the organic matrix of mineralizing tissue a technique for preparative separation according to the degree of mineralization was developed. The method employs ultracentrifugation of powdered material in density gradients made from acetone and bromoform. The density range of the fractions is checked by refractive index measurements of the gradient medium. The amount of the material in the fractions is checked by weighing and their degree of mineralization is estimated by determining the Ca/N and P/N ratios. The homogeneity of the fractions is determined by soft X-ray microscopy. Isolated dissected microscopic bone structure (osteones and lamellar bone fragments) with different degrees of mineralization were fractionated in this way. Chromatography on Sepharose 2B of proteoglycans from costal cartilage exposed to an acetone-bromoform gradient revealed no effect of the gradient medium on the molecular size of the proteoglycans.

The use of metrizamide as a gradient medium for isopycnic separation of rat liver cells

The use of metrizamide as a gradient medium for isopycnic separation of rat liver cells

Ray Delay in Gradient Waveguides with Arbitrary Symmetric Refractive Profile

This paper deals with the investigation of the ray delay in gradient waveguides with any transverse refractive profile. A perturbation method is used for the calculations. It becomes apparent that the delay differences in the pure gradient medium are very small for any decreasing profile and that they are of an order of magnitude of those found in a monomode waveguide (material dispersion). Larger delay differences do, however, occur when the ray paths are to some extent determined by an outer cladding. These influences must be eliminated to allow the excellent delay time properties of a gradient medium to really be utilized.

Demonstration of Electrogenic Na + -dependent D-glucose Transport in Intestinal Brush Border Membranes

Na(+)-coupled D-glucose transport was studied in isolated membrane vesicles from intestinal brush borders. Concentration gradients of SCN(-), K(+), and H(+) were established between the intravesicular solution and the incubation medium and their influence on D-glucose uptake from the medium was measured. A gradient (medium > vesicle) of NaSCN, but not of KSCN, produced a transient overshoot of D-glucose uptake above the equilibrium level. Similarly, an increase of the membrane conductance with valinomycin (K(+)-conductance) or with uncoupling agents of oxidative phosphorylation (H(+)-conductance) induced an overshooting D-glucose uptake, provided a (vesicle > medium) K(+)-gradient or a H(+)-gradient, respectively, was present in each case. The transient overshoot is evidence that D-glucose was taken up against its concentration gradient (up to 10-fold). The gradients of SCN(-), K(+) (in the presence of valinomycin), and H(+) (in the presence of uncouplers) are thought to contribute to the "driving" force for this "active" D-glucose transport by changing the electrical potential across the vesicle membrane and thus making the inside more negative (with respect to the medium). These experiments, therefore, provide evidence that the Na(+)-coupled D-glucose translocation across the brush border membrane is an electrogenic process, i.e., the positive charge associated with Na(+) is not compensated by the co-movement of an anion or the counter-movement of a cation via the glucose "carrier". The results imply that an electrical potential across the brush border membrane may play an important role in determining the transport of D-glucose by intact cells.

Separation of HeLa cells by colloidal silica density gradient centrifugation. I. Separation and partial synchrony of mitotic cells.

Using a colloidal silica density gradient, HeLa cells in mitosis were found to have a density of 1.040-1.046 g/cc, lighter than the remaining interphase cells. The mitotic cells could be harvested and cultured after centrifugation, showing growth synchrony by measurement of a peak in mitotic index 21 hr after establishing the culture. By using Colcemid or vinblastine sulfate, HeLa cells were arrested in metaphase and centrifuged on the colloidal silica density gradient. The blocked metaphase cells were lighter in density than the interphase cells but somewhat more dense than untreated cells selected by the density gradient centrifugation. Near-equilibrium conditions were established during the centrifugation of cells so that cell density measurements could be made, and the gradient medium employed was not measurably toxic to those cells tested.

Transport Phenomena in Zonal Centrifuge Rotors. V. Dispersion Coefficient of Sample in Gradient Solutions

Abstract Dispersion coefficients of a polystyrene latex bead (diameter: 0.091 ± 0.0058 μ) and bovine serum albumin in sucrose and Ficoll (polysucrose by Pharmacia Fine Chemicals, Sweden) gradient solutions were measured in an Oak Ridge B-XV zonal centrifuge rotor. Measurements were analyzed by the moment method. Dispersion coefficients were then correlated in a reduced form in terms of Schmidt number as a function of reduced centrifugal force field strength, Taylor number, reduced sedimentation coefficient, density ratio between particle and gradient medium, and reduced time. The correlation is also given in a formula which fits all the experimental data points with an average deviation of 2.5% and a maximum deviation of 6.3%. Some information on the eddy and molecular components of the dispersion coefficients is also presented.

Signal Distortion in a Constant Gradient Medium is Continuous and Bounded

In an unbounded constant gradient medium, the distortion of an arbitrary signal φ is necessarily bounded and continuous, even when φ is unbounded and discontinuous. The distortion, which goes to zero as time goes to infinity, goes uniformly to zero as a parameter (gradient squared multiplied by travel time) goes to zero.

Distortion of Some “Squarish Pulses” in a Constant Gradient Medium

The exact responses to some “squarish pulses” from a point source are found by using an integral representation due to R. R. Goodman. The resulting distortion is shown to depend only on the dimensionless parameter travel time multiplied by the magnitude of the gradient.

Rate zonal centrifugation in a ficoll gradient

Rate sedimentation has been employed in an increasingly broad variety of separation problems, including macromolecules (1, 2)) viruses (3-5)) bacteria (6), cell organelles (7, 8), yeast (9, lo), and mammalian cells (11). Recent experimental work in our laboratory has required the development of a method for resolving mixtures of viable mammalian cells by zonal centrifugation. For this purpose we have used a Ficol12 (polysucrose) solution at 4°C. For this work it is ,desirable to be able to predict the rate of separation of the cells as a function of the density gradient and other known physical parameters. Diffusion effects are generally negligible for particles the size of cells, hence one can write an ordinary differential equation for the radial position of a cell as a function of time. The differential equation is a nonlinear one since both the density and viscosity of the gradient medium depend on position in the gradient. In earlier work on sedimentation through a sucrose gradient, McEwen found it necessary to assemble a large table to facilitate solution of the equation of motion (2). In the present paper we will show that Ficoll has the desirable property that both its viscosity and its density have a simple functional dependence on concentration, permitting a much simpler solution to be obtained. The equation of motion for a spherical particlc in the absence of diffusion is (9) :

Studies on the Proteinaceous Subcellular Particles in Rice Endosperm: Electron-Microscopy and Isolation

Electron-microscopic examination of rice endosperm revealed the existence of protein-aceous subcellular particles, 1 to 4 µ in diameter and spherical or oval in shape. Isolation of the particles was effected by differential centrifugation in density gradient medium after mechanical or enzymic disintegration of endosperm cells. The isolated particles were predominantly composed of protein, and residual constituents were mainly lipid and carbohydrate. Their shape and behaviors were similar to those found in the endosperm. These facts show that the subcellular particles concerned are “protein bodies” There seemed to be several kinds of protein bodies different with respect to their protein and lipid contents.

Effect of density stratification on sound waves

The theory of sound propagation in mediums with continuous density stratification suggests that a number of interesting effects should occur. For example, acoustic waves will be totally reflected from a medium of strong density gradient, even in the absence of sound velocity contrasts. In this paper the magnitude of such effects is determined and discussed in connection with (1) a transition layer at the water-sediment interface on the ocean bottom and (2) certain properties of atmospheric sound.

A Designing Method of Reflector and Projector Lamps

A designing method of reflector or lens contours for reflector and projector lamps is attemped. It has been customary, in illumination engineering, to use simple second-degree or composed surfaces for these designs, but this time we introduce a new method to calculate directly matched surfaces in the caces of various reflector type lamps and desirable light distribution.The reflecting or refracting law exists at any arbitrary points on the unknown desirable surface, and the differential eqation between directions of light and the function of surface can be composed by the medium of gradient of normal line at these points. If the relation between light source and desirable light distribution is assumed, this eqation can be solved by formal or numerical integration.Generally, this solution is different from second-degree surfaces. It is comparatively easy to solve this eqation in the case of a point source and uniform flood lighting. The photographs of a wide flood type sealed-beam lamp designed by this method and its light pattern compared with a medium flood type one are indicated.