A device for a discontinuous fractionation of liquid density gradients yielding high resolution

When a liquid density gradient is fractionated through the bottom of a tube turbulences occur near the hole resulting in a mixing of closely spaced bands. To prevent this mixing the gradient is pumped upward and, in conventional instruments, guided through tubings into a fraction collector. Since mixing of bands in the tubings may occur, the advantage of pumping the gradient upward is diminished. To prevent this mixing an apparatus is described which, with the help of a peristaltic pump, collects fractions discontinuously after the gradient has been pumped upward thus preventing a mixing in instruments used for analysis and tubings attached to the fractionator.     

Fractionation of Different Life Cycle Stages of Microsporidia Nosema grylli from Crickets Gryllus bimaculatus by Centrifugation in Percoll Density Gradient for Biochemical Research

ABSTRACT. A new method of fractionation and purification of different life cycle stages of microsporidia Nosema grylli , parasitizing the fat body of cricket Gryllus bimaculatus , by centrifugation in Percoll density gradient is elaborated. The whole procedure can be summarized as: 1) infected fat body preparation, 2) homogenization in buffer and filtration through cotton wad and filter paper, 3) first centrifuging, resulting in the separation of the pellet into three layers containing different life cycle stages, 4) second centrifuging of the chosen layer in Percoll density gradient, 5) washing out the Percoll from the fraction under study. After centrifugation in Percoll density gradient, meronts and early sporonts form a band in the area corresponding to density 1.016 g/ml. Mature spores form the pellet at the bottom of centrifuge tube, while immature spores are distributed throughout the layer of 1.016 g/ml up to the bottom of the centrifuge tube, according to their buoyant densities. The offered technique is simple, it takes about one hour and may become a routine procedure for biochemical studies on microsporidia.     

一种简便、高效、经济的从凝胶中回收DNA的方法

目的:尝试一种简便、高效的从凝胶中回收DNA的方法。方法:在Eppendorf管的管底用注射器扎孔,将一小团用Eppendorf管融化后拉成的细丝放入管中,把含有DNA的凝胶放在细丝上,离心,收集从管底流出的液体,经酚氯仿抽提后用乙醇沉淀DNA。结果:经过简单的离心即可近乎100%地回收凝胶中的DNA。结论:使用该方法从琼脂糖凝胶回收DNA,操作简单,回收率高,无其他试剂污染。     

Rapid DNA amplification in a capillary tube by natural convection with a single isothermal heater

Herein we describe a simple platform for rapid DNA amplification using convection. Capillary convective PCR (CCPCR) heats the bottom of a capillary tube using a dry bath maintained at a fixed temperature of 95°C. The tube is then cooled by the surrounding air, creating a temperature gradient in which a sample can undergo PCR amplification by natural convection through reagent circulation. We demonstrate that altering the melting temperature of the primers relative to the lowest temperature in the tube affects amplification efficiency; adjusting the denaturation temperature of the amplicon relative to the highest temperature in the tube affects maximum amplicon size, with amplicon lengths of ≤500 bp possible. Based on these criteria, we successfully amplified DNA sequences from three different viral genomes in 30 min using CCPCR, with a sensitivity of ~30 copies per reaction.     

Density gradient system. I. Formation and fractionation of density gradients

The instrumentation of a precise yet flexible system for the formation and fractionation of linear density gradients is described. The use of a special peristaltic pump and gradient-forming chambers permits the rapid formation of both dilute and concentrated solutions into ca. 5 to 300 ml of gradient. This gradient can be delivered to a single centrifuge tube or equally apportioned to as many as 24 tubes.The special peristaltic pump, which is described in the paper that follows, delivers the concentrated solution into the gradient-forming chamber as well as conveying the gradient from the chamber to the special centrifuge tube. A capillary in the wall of the tube conveys the gradient to the bottom, where it is dispersed into 18 equidistant orifices. This dispersion permits the rapid formation and fractionation of the gradient without disruption. A presettable counter coupled to the peristaltic pump permits the gradients to be apportioned into equal volume fractions.Results obtained with both dilute and concentrated gradients are presented.     

Probe for production and measurement of acute mitral regurgitant flow in dog.

A probe for production and measurement of acute mitral regurgitation in dogs is described. It consists of a tube that is introduced into the mitral valve through the left atrial appendage. Regurgitant flow through the tube is measured by an electromagnetic device. Variation of flow and zero flow are achieved by narrowing or occluding the tube with a rubber cuff. In animals weighing 30-50 kg, the probe does not produce significant mitral stenosis and the mitral leaflets fit closely around the probe during ventricular systole. The instantaneous relationship between mitral regurgitant flow (MRF) and the gradient between left ventricular and left atrial pressure shows a marked delay of MRF at the beginning and end of regurgitation. This delay can be attributed to some extent to electrical phase lag and to the small movement of the probe relative to the mitral valve during the cardiac cycle. Measurement of regurgitant stroke volume is affected by this movement only to a small extent.     

A New Procedure for Quantitative Measurements of Virus Particles in Crude Preparations

A new method is described for the quantitative measurement of virus concentration in crude preparations by density gradient centrifugation and electron microscopy. The centrifugation is carried out in a specially designed centrifuge tube which permits separation and sedimentation of virus particles at different levels according to their sedimentation velocity. The gradient of a mixture of heavy and normal water (D(2)O-H(2)O) is designed to sediment the virus particles with constant velocity so that the optimal time of centrifugation can easily be calculated. The virus particles are collected on carbon-coated nickel grids floating on mercury at the bottom of the centrifuge tube and are counted by means of electron microscopy. The efficiency of the method is demonstrated with a crude plant extract of tobacco mosaic virus.     

Prediction of centrifugation times for equilibrium and velocity sedimentation on various gradients

Computer and calculator programs have been prepared which can predict sedimentation times for a significant fraction of the kinds of density gradient centrifugation experiments currently being carried out in biochemistry. Gradients of sucrose, glycerol, or CsCl were accommodated for linear or several forms of convex or concave gradient profiles. Times of sedimentation to various uniformly spaced points between the meniscus and the bottom of the tube, or to a point near the isopycnic density, are predicted.     

Heterogeneity of the gradients performed by the freeze-thaw method

Gradients produced by the freeze-thaw method were analyzed at various rates of freezing. The shape of the gradient depended on the rate of freezing. At high rates the pattern did not change but at low rates the steepness of the gradient increased with time. Solutes concentrated at the bottom in a fashion which depended on the density of the solvent and on the rate of freezing. It should also be noted that the gradient was not uniform over the entire surface as the concentration of solute increased near the wall of the test tube.     

Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E

A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.     

Zonal electrophoresis and isoelectric focusing of proteins and virus particles in density gradients of small volume

Proteins and virus particles were separated by zonal electrophoresis or isoelectric focusing in glass tubes of small volume. The tubes were covered at the bottom with dialysis membranes and sucrose gradients containing either buffer or ampholytes were generated directly into them. When ampholytes were employed, reproducible pH gradients were generated during electrophoresis. After the separations were finished, dense sucrose was pumped into the bottom of each tube and the gradient was fractionated from the top; the recovery of virus was nearly complete.     

Measurement of oscillatory flow pressure gradient in an elastic artery model

In vitro experiments were conducted to measure the oscillatory flow pressure gradient along an elastic tube in order to assess the recent nonlinear theory of Wang and Tarbell. According to this theory, in an elastic tube with oscillatory flow, the mean (time-averaged) pressure gradient cannot be calculated using Poiseuille's law. The effect of wall motion creates a nonlinear convective acceleration, and an induced mean pressure gradient is required to balance the convective acceleration. The induced mean pressure gradient depends on the diameter variation over a cycle, the pulsatility and unsteadiness of the flow, and the phase difference between the pressure wave form and the flow wave form. The amplitude of the pressure gradient also depends on these parameters and may deviate significantly from Womersley's rigid tube theory. A flow loop was constructed to produce oscillatory flow in an elastic tube. Flow wave forms were measured with an ultrasonic flow probe, and ultrasonic diameter crystals were used to measure wall movement. A special device for pressure drop measurement was constructed using Millar catheter tip transducers to obtain both forward and backward pressure drops that were then averaged. This averaging method eliminated the static error of the pressure transducers. The pressure-flow phase angle was varied by clamping a distal elastic section at various locations. Pressure gradients were obtained for a range of phase angles between −55 ° and +49 °. The mean and amplitude of the measured pressure gradient were compared to theoretical values. Both positive and negative induced mean pressure gradients were measured over the range of phase angles. The measured pressure gradient amplitudes were always lower than predicted by Womersley's rigid tube theory. The experimental means and amplitudes are in good agreement with the elastic tube theoretical values. Thus, the experiments verify the theory of Wang and Tarbell.     

Rapid purification of plasmid DNA by a single centrifugation in a two-step cesium chloride-ethidium bromide gradient

A procedure for rapid, preparative purification of plasmid DNA is described and compared with a conventional equilibrium centrifugation method. A discontinuous, two-step CsCl-ethidium bromide gradient is used, with the starting position of the plasmid-containing extract being at the bottom of the tube. During centrifugation in a fixed angle rotor, covalently closed circular plasmid DNA is separated from contaminating protein, RNA, and chromosomal DNA in 5 hr. Plasmids purified by this method are considerably less contaminated with RNA than when purified by a 48-hr equilibrium run in a homogeneous gradient, as determined by agarose gel electrophoresis and 5'-end-labeling studies. Plasmid DNA purified in two-step gradients can be used directly for restriction endonuclease analysis and DNA sequencing.     

Effects of sedimentation of small red blood cell aggregates on blood flow in narrow horizontal tubes

The flow properties of aggregating red cell suspensions flowing at low flow rates through horizontal tubes are analyzed using a theoretical model. The effects of sedimentation of small aggregates, which will be formed at comparatively high flow rates, on the relative apparent viscosity are considered. In the case in which a large number of small aggregates are formed in a suspension flowing through a horizontal tube, it seems that red cells are transported as a concentrated suspension through the bottom part of the tube because of sedimentation of aggregates. A two-layer flow model is used for the distribution of red cells. It consists of plasma in the upper part and a concentrated red cell suspension in the bottom part of the tube divided by a smooth and horizontal interface. It is assumed that the suspension is a Newtonian fluid whose viscosity increases exponentially with hematocrit. The velocity distribution, the relative apparent viscosity and the flux of red cells are calculated as functions of width of plasma layer for a different discharge hematocrit. The theoretical results are compared with the results obtained from experimental data. The relative apparent viscosity increases rapidly with an increasing degree of sedimentation over a wide range of plasma layer widths.     

Interaction of peristaltic flow with pulsatile flow in a circular cylindrical tube

The effect of pulsatile flow on peristaltic transport in a circular cylindrical tube is analysed. The flow of a Newtonian viscous incompressible fluid in a flexible circular cylindrical tube on which an axisymmetric travelling sinusoidal wave is imposed, is considered. The initial flow in the tube is induced by an arbitrary periodic pressure gradient. A perturbation solution with amplitude ratio (wave amplitude/tube radius) as a parameter is obtained when the frequency of the travelling wave and that of the imposed pressure gradient are equal. The interaction effects of periodic wall induced flow and periodic pressure imposed flow are visualized through the presence of substantially different components of steady and higher harmonic oscillating flow in the first order flow solution. Numerical results show a strong variation of steady state velocity profiles with boundary wave number and Reynolds number and a strong phase shift behaviour of the flow in the radial direction.     

钙在被子植物受精过程中的作用

近年来,花粉管中的钙信号和生理功能的研究取得了明显的进展,同时在雌蕊系统中有关钙分布的研究也初步显示了其时、空特征与被子植物的受精作用密切相关。该文总结了花粉萌发和花粉管生长过程中外源钙和内源钙的作用机制,结合雌蕊组织中钙分布的特征,进一步探讨了钙在被子植物受精过程中的功能。     

GABA,a new player in the plant mating game

Pollen tubes are guided through female tissues to deliver sperms to the embryo sac. A recent study reveals a GABA gradient along the pollen tube path, which, together with guidance defects in GABA-overaccumulating mutants, implies a role for GABA in regulating pollen tube growth and guidance.     

Isolation and characterization of proteoglycans from porcine lungs.

Proteoglycans were extracted from porcine lungs with 4 M guanidinium chloride. The extract was subjected to associative density gradient centrifugation, and four equal fractions, labeled A1 through A4 from the bottom to the top of the gradient, were obtained. The pooled A1 fractions containing proteoglycan aggregates were further fractionated by dissociative density gradient centrifugation to yield four equal fractions labeled A1D1 through A1D4 from the bottom to the top of the gradient. These fractions were analyzed for their protein, uronic acid, glucosamine, galactosamine, hexose, and sialic acid content. The fraction A1D1 with the highest buoyant density had the highest content of uronic acid and galactosamine, and lowest content of protein, indicating the enrichment of proteoglycan monomers at the bottom of the dissociative density gradient. As the density of the gradient decreased, the protein, hexoses, and sialic acid content increased, whereas uronic acid and galactosamine content decreased. The amino acid analysis showed similar composition for all four fractions with aspartic acid, serine, glutamic acid, proline, glycine, alanine, valine, and leucine as the major constituent amino acids. No hydroxyproline was detected in any of the fractions. As the buoyant density of the fractions decreased, the aspartic acid content increased and glycine content decreased.     

Plant reproduction: GABA gradient, guidance and growth

How a pollen tube manages to navigate through the female tissues during plant reproduction has been a mystery. A new analysis of an Arabidopsis mutant has provided the strongest evidence yet that a GABA gradient may be a critical signal for correct targeting of the pollen tube.