Studies on the cell cycle of Myxobacter AL-1

Myxobacter AL-1 was synchronized by size fractionation of asynchronous cultures on a 5–20% equivolumetric sucrose gradient in a Ti—15 zonal rotor. The degree of synchronization obtained by this method was determined 1. by a comparison of the size distribution of an asynchronous culture to the distribution of cell numbers in the fractions of the zonal run, 2. by studying the size distributions in the different fractions obtained with the zonal rotor and 3. by following the growth and size distribution of a fraction from a zonal run during one division cycle. The results indicate that this method is suitable for the study of the cell cycle of Myxobacter AL-1.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Respiratory Syncytial Virus Isolation by Combined Continuous Flow-Isopycnic Banding Centrifugation

A new zonal centrifuge rotor (B-IX) which combines continuous sample flow centrifugation with isopycnic banding has been used to isolate and concentrate respiratory syncytial virus from liter volumes of culture fluid. This isolation technique utilizes a sucrose density gradient to trap and isopycnically band the virus particles, and permits recovery of the particles from the rotor in an unaggregated condition.     

Purification of the protein crystal from Bacillus thuringiensis by zonal gradient centrifugation.

A method is described for the large-scale purification of the Bacillus thuringiensis protein crystal by zonal gradient centrifugation. NaBr gradients are employed in a Beckman J21-B centrifuge equipped with a JCF-Z rotor.     

Biohazards Assessment in Large-Scale Zonal Centrifugation

A study was conducted to determine the biohazards associated with use of the large-scale zonal centrifuge for purification of moderate risk oncogenic viruses. To safely and conveniently assess the hazard, coliphage T₃ was substituted for the virus in a typical processing procedure performed in a National Cancer Institute contract laboratory. Risk of personnel exposure was found to be minimal during optimal operation but definite potential for virus release from a number of centrifuge components during mechanical malfunction was shown by assay of surface, liquid, and air samples collected during the processing. High concentration of phage was detected in the turbine air exhaust and the seal coolant system when faulty seals were employed. The simulant virus was also found on both centrifuge chamber interior and rotor surfaces.     

A Zonal Density Gradient Centrifuge for Separating Cytoplasmic Granules from Trypanosoma brucei

SYNOPSIS. A small zonal density gradient centrifuge has been designed and constructed for the separation of cell granules from Trypanosoma brucei. Two distinct bands were obtained on a simple sucrose density gradient of the following granules which were found in corresponding fractions of the effluent by electron microscopy: Type I granules (lysosomes) which formed a clear-cut band toward the periphery of the rotor, and Type II (endoplasmic reticular) granules which formed a more diffuse band and showed great diversity of size, shape and state of aggregation. This aggregation is related to the structure of these granules which are formed in the endoplasmic reticular tubules and are therefore not easily dispersed during rupture of the cell body.     

Separation of plant cell organelles by zonal centrifugation in reorienting density gradients

Preparative separations of plant cell organelles (glyoxysomes, proplastids, mitochondria, and endoplasmic reticulum) in an ordinary refrigerated centrifuge were obtained by sucrose density gradient centrifugation in a zonal rotor loaded and unloaded in the static manner. The quality of the separation which was monitored by marker enzymes and electron microscopy compares to analytical separations in swinging-bucket rotors. Membrane alterations observed in glyoxysomes and mitochondria are traced back to sucrose as a major component of the homogenization and density gradient medium.     

THE RESOLUTION OF MIXTURES OF VIABLE MAMMALIAN CELLS INTO HOMOGENEOUS FRACTIONS BY ZONAL CENTRIFUGATION

Large-scale separation of mixtures of mammalian cells was obtained with the A-1X zonal centrifuge rotor and density gradients consisting of Ficoll dissolved in modified Eagle's MEM suspension-culture medium. The cells remained viable as tested by plating efficiency or by motility observed with time-lapse photography. Rabbit thymocyte and HeLa cell mixtures were separated with 99 and 89 per cent purity, respectively. Mixtures of thymocytes and suspension-cultured, human acute leukemia cells (Roswell Park strain LKID) were separated with 93 and 91% purity, respectively. HeLa cells were isolated 92% pure from a mixture with horse leukocytes. A book of charts giving the sedimentation position and velocity versus time of cells in the A rotor under standard conditions of gradient composition, angular velocity, and temperature was prepared with the use of a computer program based on the differential sedimentation equation. The charts are used to estimate the centrifugation time necessary for maximum separation of cells. The success achieved in separating mixtures of cells points to the future possibility of large-scale fractionation of solid tissues, especially tumor tissues, into preparations cf viable cells of a single type.     

Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae.

To study nuclear and mitochondrial deoxyribonucleic acid (DNA) synthesis during the cell cycle, a 15N-labeled log-phase population of Saccharomyces cervisiae was shifted to 14N medium. After one-half generation, the cells were centrifuged on a sorbitol gradient in a zonal rotor to fractionate the population according to cell size and age into fractions representing the yeast cell cycle. DNA samples isolated from the zonal rotor cell samples were centrifuged to equilibrium in CsC1 in an analytical ultracentrifuge to separate the nuclear and mitochondrial DNA components. The amount of 14N incorporated into each 15N-labeled DNA species was measured. The extent of nuclear DNA replication per sample was obtained by measuring the amount of hybrid DNA. The percentage of hybrid nuclear DNA increased from 6 to 68% and then decreased to 44% during the cell cycle. Upon ultracentrifugation, mitochondrial DNA banded as a unimodal peak in all zonal rotor samples. Mitochondrial DNA replication could be ascertained only by the 14N level in each mitochondrial peak and not, as with nuclear DNA, by hybrid DNA level. In contrast to the nuclear incorporation pattern, the 14N percentage in mitochondrial DNA remained effectively constant during the cell cycle. Comparison of the data to theoretical distributions showed that nuclear DNA was replicated discontinuously during the cell cycle, whereas mitochondrial DNA was replicated continuously throughout the entire mitotic cycle.     

Enzyme content of plant microbodies as affected by experimental procedures

The content of specific enzymes in microbodies isolated from tobacco Nicotiana rustica, L. leaves may vary according to the procedure followed during the isolation of the organelles. The type of homogenizing medium, its ionic components and the ratio of medium to tissue during homogenization, affect the over-all yield and relative distribution of each microbody enzyme in different ways. The type of density gradient and the initial acceleration of the centrifuge rotor affect also the enzyme content of sedimenting microbodies. These observations explain some of the conflicting results obtained on the determination of the intracellular location of several enzymes.     

10. Purification of Viruses by Cenlrifugation in Gradients of Inert Substances

ABSTRACT

By using a reorienting gradient centrifuge rotor cut from a block of Nylon and fitted with eight septae, it was possible to separate the components of the haemolymph of the mollusc Turbo sarmaticus into three fractions in a sucrose gradient held in the bowl of the rotor. The fractions were (108 and 98)S, 44S and 16-22S. The success of the experiment was due to the large differences in the sedimentation coefficients of the components. When the rotor was applied to the natural mixture of the five viruses of the caterpillars of Nudaurelia cytheria only the main component could be isolated in a pure state. The viruses were separated by isopycnic centrifugation in “self formed” caesium chloride gradients, using a Beckman Model E analytical centrifuge in which a separation cell fitted with a centerpiece with two perforated partitions was used.

Centrifugation in gradients of inert substances is useful for the separation of components in a mixture¹. There are two principles involved in this type of separation. One, termed reorienting gradient centrifugation (reograd) relies on the differences in masses or, better still on the sedimentation coefficients of the different components in the mixture and the second, termed isopycnic centrifugation², on the densities or specific gravities of the different entities.     

Effect of large refractive index gradients on the performance of absorption optics in the Beckman XL-A/I analytical ultracentrifuge: an experimental study

An analytical centrifuge cell was modified to detect refraction of light transmitted through the cell caused by refractive index gradients formed by sedimenting solute during centrifugation. Sedimentation velocity and sedimentation equilibrium experiments were carried out in this cell on solutions containing high concentrations of protein and polysaccharide in a Beckman-Coulter XLA analytical ultracentrifuge. Analysis of the results indicates that in the absence of an optical artifact easily recognized as a "black band," the dependence of apparent absorbance upon radial position reported by the instrument may be considered a reliable measure of the solute concentration gradient.     

Linear and nonlinear analyses of pulsatile blood flow in a cylindrical tube

A non-Newtonian shear-thinning constitutive relation is proposed to study pulsatile flow of whole blood in a cylindrical tube. The constitutive relation, which satisfies the principle of material frame indifference, is derived from viscometric data obtained from whole blood over a range of hematocrits. Assuming axisymmetric flow in a rigid cylindrical tube of constant diameter, a second-order, nonlinear partial differential equation governing the axial velocity component is obtained. Imposing a periodic pressure gradient, the governing equation was solved numerically using finite difference methods over a range of Stokes values and hematocrits. For a forcing frequency of 1 Hz, results are presented over tube diameters ranging between 0.1 and 2 cm and over hematocrits ranging between 10 and 80%. For a given hematocrit, velocity profiles predicted for the non-Newtonian model under sinusoidal forcing reveal attenuated volume flow rate and enhanced vorticity transport over the tube cross-section relative to a Newtonian fluid having a viscosity corresponding to the high shear-rate limit. For moderate to high Stokes numbers, consistent with flow in large arteries, our results revealed a viscosity distribution that was nearly time invariant. An analytic solution was obtained for a fluid having arbitrarily prescribed radially varying, temporally invariant viscosity and density distributions under arbitrary periodic pressure forcing. Close agreement was observed between our numerical and analytical results when the imposed viscosity distribution was chosen to approximate the time-averaged viscosity distribution predicted by the shear-thinning non-Newtonian model. For St > or approximately= 100, the disparity between our results and those of a Newtonian fluid of constant viscosity grows with a decreasing ratio of the DC to AC components of the pressure-gradient amplitude below 50%. In particular, for any purely oscillatory pressure-gradient (vanishing DC component), the Womersley solution is a particularly poor predictor of the amplitude and phase of wall shear rate for over half of the flow cycle. Under such circumstances, the analytical models presented here provide a simple and accurate means of estimating instantaneous wall shear rate, knowing only the pressure gradient and hematocrit.     

Synchronization of mammalian cells by selection and additional chemical block studied by DNA distribution analysis and BrdUrd-Hoechst 33258-technique

Abstract. Ehrlich ascites tumour cells growing in vitro in suspension culture were separated according to volume by the technique of velocity sedimentation in a zonal rotor with a reorienting gradient. Using DNA distribution analysis the sedimentation pattern of the cells could be analysed in detail. With appropriate conditions it was possible to separate pure G1 cells. Samples could also be obtained which were enriched in S or G2 + M cells. The main limitation of the selection in this type of rotor was the reorientation of the gradient which caused disturbances during deceleration of the rotor. The synchronous growth of selected G1 cells has been studied in detail to investigate the reasons for the rather poor synchrony of these cells. The poor synchrony was found to be caused mainly by the small volume of the selected G1 cells compared with the normal volume of G1 cells in an asynchronous population. The synchronization of these cells could be essentially improved by a short treatment with excess thymidine causing a metabolic block at the G1/S border. The duration of this treatment could be minimized using DNA distribution analysis of growing cells after releasing of the block. The durations of the cell cycle phases in synchronized cells agreed with the values calculated in asynchronous cells by DNA distribution analysis and the BrdUrd-Hoechst 33258-technique.     

Evaluation of a class III biological safety cabinet for enclosure of an ultracentrifuge

An evaluation of a special safety cabinet housing a high-speed centrifuge was made. The cabinet enclosed both the top access port and the drive and pumping machinery of the centrifuge. A titanium rotor was loaded with tubes containing a bacterial culture, weakened, and driven until rotor rupture occurred. There were several bent and broken components in the centrifuge, and bacteria leaked from the vacuum chamber. Although the forces were sufficient to displace the cabinet, none of the test bacteria were found outside the cabinet.     

Zonal centrifuge profiles of rat brain homogenates: Instability in sucrose,stability in iso-osmotic Ficoll-sucrose

By substituting iso-osmotic Ficoll-sucrose for hyperosmotic sucrose between the densities of 1.043 and 1.088 in sucrose density gradients in the B-XV rotor of an Anderson-NIH-AEC zonal centrifuge system, it was possible to stabilize the zonal centrifuge absorbancy profiles of adult rat brain homogenates. The reason for the instability in ordinary sucrose gradients was found to be the interaction of myelin with other brain structures in hyperosmotic sucrose. No such interaction occurred in isoosmotic sucrose (0.32 M) with or without Ficoll. In Ficoll-sucrose, myelin was separated at three reproducible densities of 1.054, 1.060, and 1.066 gm/ml. No myelin appeared at a density if 1.094 gm/ml, which represented the main collection point in ordinary sucrose. Synaptosomes were separated at peak densities of 1.072 and 1.152 gm/ml. Mitochondria were obtained at a density of 1.176 gm/ml. Areas under zonal centrifuge absorbancy profiles of rat brain homogenates were found to be constant regardless of the values of ω²t that were reached.     

一次密度梯度超速离心分离人血清的VLDL、LDL、HDL_2、HDL_3及无脂血清

本文将密度梯度、离心力和离心时间作适当的组合和配比:即不连续密度梯度1.000—1,400g/mlNaBr溶液、离心力168,000g、22小时,10℃,在Beckman L8-80型、区带头Ti-14一次超速离心将血清中四种主要脂蛋白和无脂血清相互分开,获得五个明显的蛋白峰。前四个峰经琼脂糖电泳,聚丙烯酰胺电泳,免疫双扩散和分析超速离心鉴定分别为VLDL、LDL、HDL_2和HDL_3,不含清蛋白。峰五为无脂血清,仅含0.046mg/dl胆固醇和0.2mg/dl甘油三酯,本法重复性佳,分离样品多(50ml),效果好,操作简单,并可延长离心机头的使用期限。已用于研究各种因素对脂蛋白含量的影响及其代谢间的相互关系。     

Generation of density gradients. Approximations to equivolumetric gradients for zonal rotors

The use of a “density gradient generating function” allows the concentration profile of a density gradient to be written explicitly in terms of the required distribution of sedimentation coefficients in place of the previous implicit formulations. This function, which is easily implemented in a computer program, permits calculation of density gradients for a number of applications. This approach is applied to computation of a variety of equivolumetric gradients of sucrose for zonal rotors and yields a formula for the calibration of such gradients. An accurate approximation has been found which allows the generation of virtually all equivolumetric gradients of sucrose for a given rotor using a single program for the gradient generator employed; the adjustment for different particle densities and for different concentrations at the top of the gradient is made by varying only the initial and final concentrations of sucrose used.     

Boundary analysis in sedimentation transport experiments: a procedure for obtaining sedimentation coefficient distributions using the time derivative of the concentration profile.

A procedure is described for computing sedimentation coefficient distributions from the time derivative of the sedimentation velocity concentration profile. Use of the time derivative, (delta c/delta t)r, instead of the radial derivative, (delta c/delta r)t, is desirable because it is independent of time-invariant contributions to the optical baseline. Slowly varying baseline changes also are significantly reduced. An apparent sedimentation coefficient distribution (i.e., uncorrected for the effects of diffusion), g*(s), can be calculated from (delta c/delta t)r as [formula: see text] where s is the sedimentation coefficient, omega is the angular velocity of the rotor, c0 is the initial concentration, r is the radius, rm is the radius of the meniscus, and t is time. An iterative procedure is presented for computing g*(s)t by taking into account the contribution to (delta c/delta t)r from the plateau region to give (delta c/delta t)corr. Values of g*(s)t obtained this way are identical to those of g*(s) calculated from the radial derivative to within the roundoff error of the computations. Use of (delta c/delta t)r, instead of (delta c/delta r)t, results in a significant increase (greater than 10-fold) in the signal-to-noise ratio of data obtained from both the uv photoelectric scanner and Rayleigh optical systems of the analytical ultracentrifuge. The use of (delta c/delta t)r to compute apparent sedimentation coefficient distributions for purposes of boundary analysis is exemplified with an antigen-antibody system.