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.     

Precipitation of protein by ultracentrifuge with angle rotor

Precipitation of a protein by ultracentrifuge with an angle rotor was investigated experimentally. Experimental data for Lysozyme and Bovine serum albumin were compared with the calculated distribution of concentration, and an agreement between calculated curves and experimental data during the course of ultracentrifugation was obtained. From the first approximated model presented in our previous paper, it is observed that the precipitation of a protein by ultracentrifuge with an angle rotor proceeds more rapidly than that with a swing rotor. Part 1 of this article was published in Vol. 5. No. 2     

An optical pulse modulation system for laser interference studies in the analytical ultracentrifuge

The mode of operation and construction of a high-speed laser light modulator is presented that is designed for use with the analytical ultracentrifuge. The system may be used with continuous wave lasers having optical powers not exceeding 100 W and has an optical bandwidth from 200 nm to 800 nm. The electronic modulation circuitry described is capable of producing optical pulses of 0.8 μsec duration at a firing frequency in excess of 100,000 pps and is designed to permit the laser source to operate in either a pulsed or nonpulsed mode. The unit is extremely versatile, and its characteristics permit full advantage to be taken of pulsed laser interferometry in the ultracentrifuge.     

Fluorescence detection for the XLI analytical ultracentrifuge

Analytical ultracentrifugation (AUC) provides first-principle hydrodynamic and thermodynamic information concerning the size, shape and interactions of macromolecules. The fundamental measurement needed in AUC is the macromolecular concentration as a function of radial position and time. Currently, the Beckman Coulter XLI analytical ultracentrifuge may be equipped with absorbance and refractive detectors, which provide complementary concentration determinations. For detecting trace quantities of materials, fluorescence detection offers unique advantages over either absorbance or interference detection. A prototype fluorescence detector for the XLI analytical ultracentrifuge has been developed and its characteristics determined. An Ar(+) laser provides a continuous 488-nm excitation beam. Radial resolution is achieved by scanning the focused beam along a radial axis. Detection of the fluorescence signal uses a co-axial, front-face optical configuration to reduce inaccuracies in the concentration caused by inner filter effects. A high-speed A/D data acquisition system allows the fluorescence intensity to be monitored continuously and at a sufficiently high angular resolution so that at any radial position the intensities from all of the samples may be acquired at each revolution. The fluorescence detector is capable of detecting concentrations as low as 300 pM for fluorescein-like labels. The radial resolution of the fluorescence detector is comparable to that of the absorbance system. Both sedimentation velocity and sedimentation equilibrium measurements may be made with the fluorescence detector. Results are presented comparing data acquired using the fluorescence with those acquired using the absorbance detector.     

Quantifying and Optimizing Single-Molecule Switching Nanoscopy at High Speeds

Single-molecule switching nanoscopy overcomes the diffraction limit of light by stochastically switching single fluorescent molecules on and off, and then localizing their positions individually. Recent advances in this technique have greatly accelerated the data acquisition speed and improved the temporal resolution of super-resolution imaging. However, it has not been quantified whether this speed increase comes at the cost of compromised image quality. The spatial and temporal resolution depends on many factors, among which laser intensity and camera speed are the two most critical parameters. Here we quantitatively compare the image quality achieved when imaging Alexa Fluor 647-immunolabeled microtubules over an extended range of laser intensities and camera speeds using three criteria – localization precision, density of localized molecules, and resolution of reconstructed images based on Fourier Ring Correlation. We found that, with optimized parameters, single-molecule switching nanoscopy at high speeds can achieve the same image quality as imaging at conventional speeds in a 5–25 times shorter time period. Furthermore, we measured the photoswitching kinetics of Alexa Fluor 647 from single-molecule experiments, and, based on this kinetic data, we developed algorithms to simulate single-molecule switching nanoscopy images. We used this software tool to demonstrate how laser intensity and camera speed affect the density of active fluorophores and influence the achievable resolution. Our study provides guidelines for choosing appropriate laser intensities for imaging Alexa Fluor 647 at different speeds and a quantification protocol for future evaluations of other probes and imaging parameters.     

Variation and sedimentation coefficient with rotor velocity

The sedimentation coefficients of three proteins, which appeared homogeneous in the ultracentrifuge, have been determined at different rotor velocities. Two of the proteins, ovalbumin and human gamma-globulin, showed sedimentation coefficients that were approx. 10% higher at rotor velocities below 20000rev./min. than at 56100rev./min. The third protein, haemocyanin of Jasus lalandii, could not be investigated over the same rotor-velocity range, but it showed a similar effect.     

Theoretical Fraunhofer light diffraction patterns calculated from three-dimensional sarcomere arrays imaged from isolated cardiac cells at rest.

Sarcomere striation positions have been obtained throughout the volumes of calcium-tolerant resting heart cells by direct computer interfaced high-resolution optical imaging. Each sarcomere position is stored in a three-dimensional (3-D) matrix array from which Fraunhofer light diffraction patterns have been calculated using numerical methods based on Fourier transforms. Diffraction patterns have been calculated from heart cell data arrays oriented normal to a theoretical laser beam. Twelve characteristic features have been identified and described from these diffraction patterns that correlate to diffraction phenomena observed from both cardiac and skeletal muscle. This numerical approach provides the means to directly assess diffraction pattern formulation, the precision of layer line angular separation, layer-line intensity and angular asymmetries, line widths and fine structures in terms of the known diffracting source structures. These results confirm that theoretical calculations can predict real muscle diffraction patterns and their asymmetries.     

A proposal for optical diagnostics through the enhancement of diffraction patterns using thin-film interference filters

Coarse clumping of solid materials within diseased biological cells can have a marked influence on the light scattering pattern. Perturbations in refractive index lead to distinct variations in the cytometric signature, especially apparent over wide scattering angles. The large dynamic range of scattering intensities restricts collection of data to narrow angular intervals believed to have the highest potential for medical diagnosis. We propose the use of an interference filter to reduce the dynamic range. Selective attenuation of scattering intensity levels is expected to allow simultaneous data collection over a wide angular interval. The calculated angular transmittance of a commercial shortwave-pass filter of cut-off wavelength 580 nm indicates significant attenuation of scattering peaks below ∼10°, and reasonable peak equalization at higher angles. For the three-dimensional calculation of laser light scattered by cells we use a spectral method code that models cells as spatially varying dielectrics, stationary in time. However, we perform preliminary experimental testing with the interference filter on polystyrene microspheres instead of biological cells. A microfluidic toolkit is used for the manipulation of the microspheres. The paper intends to illustrate the principle of a light scattering detection system incorporating an interference filter for selective attenuation of scattering peaks.     

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.     

Acid-induced dimerization of skeletal troponin C

We have investigated pH-dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low-speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20 degrees C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20 degrees C, and was completely prevented at a rotor speed of 40,000 rpm and 4 degrees C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met-25 and Cys-98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH-dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The proton-induced increase in the energy transfer distance is related to the acid-induced dimerization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anomalies in the sedimentation behavior of high molecular weight DNA isolated from Proteus mirabilis

The sedimentation properties of a P. mirabilis DNA sample have been investigated at different concentrations and rotor speeds. Pronounced speed effects occurred at high angular velocities. The s value evaluated from low-speed experiments amounts to 61 S., indicating a mean molecular weight of 105 million. Anomalous concentration distributions in the ultracentrifuge cell have been observed at low speeds. At the boundary, a pile-up of concentration occurs which exceeds the total plateau concentration. The concentration elevation decreases with increasing time due to convection which is caused by the existence of a negative density gradient. Despite this convection, accurate mean sedimentation coefficients could be obtained even at extremely low concentrations. A careful analysis of sedimentation coefficient distributions shows, however, that the lending and trailing tails of the boundaries are disturbed by convection. Thus it may be expected that the effect produces difficulties in determining true sedimentation coefficient distributions of polydisperse DNA samples of very high molecular weight.     

Whisker exploration behaviours in the 5xFAD mouse are affected by sex and retinal degeneration

Active whisking in mice and rats is one of the fastest behaviours known in mammals and is used to guide complex behaviours such as exploration and navigation. During object contact, whisker movements are actively controlled and undergo robust changes in timing, speed and position. This study quantifies whisker movements in 6‐ to 7‐month old male and female 5xFAD mice, and their C57/SJL F1 wild‐type (WT) controls. As well as genotype, we examined sex differences and the effects of retinal degeneration (rd). Mice were filmed using a high‐speed video camera at 500 frames per second (fps), under infrared light while behaving freely in three tasks: object exploration, sequential object exploration and tunnel running. Measures of whisker position, amplitude, speed and asymmetry were extracted and analysed for each task. The 5xFAD mice had significantly altered whisker angular positions, amplitude and asymmetry during object contacts and female 5xFAD mice with rd had lower mean angular positions during object contact. There were no significant effects of genotype on sequential object exploration or on tunnel running but differences due to sex and rd were found in both tasks, with female mice making larger and faster whisker movements overall, and mice with rd making larger and faster whisker movements during object contact. There were sex differences in whisker movements during sequential object exploration and females with rd had higher whisker retraction speeds in tunnel running. These data show that measuring whisker movements can quantify genotype and sex differences and the effects of rd during exploratory behaviour in these mice.     

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.     

Ultracentrifugation in the Concentration and Detection of Enteroviruses

Ultracentrifugation has been evaluated as a method of concentrating enteroviruses from suspensions whose initial titers ranged from 1.7 × 10⁸ to 1.6 × 10^-2 plaque-forming units (PFU) per ml. A technique employing a “trap” of 0.1 ml of 2% gelatin solution at the point at which the pellet forms in tubes for the number 30 and number 50 rotors of the Spinco model L preparative ultracentrifuge has been tested and found to have a number of advantages. Qualitative studies have been performed to determine the sensitivity of the ultracentrifuge technique in detecting the presence of enteroviruses in very dilute suspensions. There was found to be at least a 50% probability of detecting virus present initially at levels as low as 0.12 PFU per ml by means of the number 50 rotor. The input level for similar results with the number 30 rotor was found to be 0.025 PFU per ml.     

The Open AUC Project

Progress in analytical ultracentrifugation (AUC) has been hindered by obstructions to hardware innovation and by software incompatibility. In this paper, we announce and outline the Open AUC Project. The goals of the Open AUC Project are to stimulate AUC innovation by improving instrumentation, detectors, acquisition and analysis software, and collaborative tools. These improvements are needed for the next generation of AUC-based research. The Open AUC Project combines on-going work from several different groups. A new base instrument is described, one that is designed from the ground up to be an analytical ultracentrifuge. This machine offers an open architecture, hardware standards, and application programming interfaces for detector developers. All software will use the GNU Public License to assure that intellectual property is available in open source format. The Open AUC strategy facilitates collaborations, encourages sharing, and eliminates the chronic impediments that have plagued AUC innovation for the last 20 years. This ultracentrifuge will be equipped with multiple and interchangeable optical tracks so that state-of-the-art electronics and improved detectors will be available for a variety of optical systems. The instrument will be complemented by a new rotor, enhanced data acquisition and analysis software, as well as collaboration software. Described here are the instrument, the modular software components, and a standardized database that will encourage and ease integration of data analysis and interpretation software.     

Optimizing the rotor design for controlled-shear affinity filtration using computational fluid dynamics

Controlled shear affinity filtration (CSAF) is a novel integrated processing technology that positions a rotor directly above an affinity membrane chromatography column to permit protein capture and purification directly from cell culture. The conical rotor is intended to provide a uniform and tunable shear stress at the membrane surface that inhibits membrane fouling and cell cake formation by providing a hydrodynamic force away from and a drag force parallel to the membrane surface. Computational fluid dynamics (CFD) simulations are used to show that the rotor in the original CSAF device (Vogel et al., 2002) does not provide uniform shear stress at the membrane surface. This results in the need to operate the system at unnecessarily high rotor speeds to reach a required shear stress of at least 0.17 Pa at every radial position of the membrane surface, compromising the scale-up of the technology. Results from CFD simulations are compared with particle image velocimetry (PIV) experiments and a numerical solution for low Reynolds number conditions to confirm that our CFD model accurately describes the hydrodynamics in the rotor chamber of the CSAF device over a range of rotor velocities, filtrate fluxes, and (both laminar and turbulent) retentate flows. CFD simulations were then carried out in combination with a root-finding method to optimize the shape of the CSAF rotor. The optimized rotor geometry produces a nearly constant shear stress of 0.17 Pa at a rotational velocity of 250 rpm, 60% lower than the original CSAF design. This permits the optimized CSAF device to be scaled up to a maximum rotor diameter 2.5 times larger than is permissible in the original device, thereby providing more than a sixfold increase in volumetric throughput.     

STUDIES ON ISOLATED CELL COMPONENTS : XVI. The Distribution of Acid Phenyl Phosphatase Activities in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge has been used to separate the major components of rat liver brei (soluble phase, ribosomes, microsomes, mitochondria, membranous fragments, and nuclei) during one centrifugation, by using a 1200 ml sucrose gradient varying linearly with radius from 17 to 55 per cent (w/w) with a "cushion" of 66 per cent sucrose at the rotor edge at speeds up to 30,000 RPM. Liver brei was found to contain a family of phosphatases (phenol disodium phosphate substrate, sodium malonate buffers and Turgitol NPX, a non-ionic detergent). Activity maxima at pH 4.1 and 5.9 were observed in untreated brei prepared in 0.25 M sucrose. The addition of the non-ionic detergent Turgitol NPX selectively caused the release of considerable additional activity between these optima. The activity measured at pH 4.1 was primarily associated with the cytoplasmic granules, while the activities at pH 4.8, 5.4 and 5.9 were found in both soluble phase and particulate-mitochondria and membranous fractions. The activities present beyond the region of the gradient occupied by the soluble phase (sample layer) were all bound to particles sedimentable at 105,536 g (average) in the preparative ultracentrifuge. The data suggest that the different activities are not similarly distributed between soluble phase and particulate fractions. When the data are expressed in terms of specific activity, the area in the gradient between the microsomes and mitochondria now appears richest in all the acid phenyl phosphatase activities measured, while the soluble phase and larger particulate fractions appear relatively poor in activity. This part of the gradient is occupied by small, dense granules which may be the so called lysosomes. Pretreatment of the brei with Turgitol NPX prior to fractionation in the zonal ultracentrifuge resulted in the solubilization of acid phenyl phosphatase activities (almost all the activity was in the sample zone of the gradient) and the non-specific destruction of the formed elements of the brei. Essentially all of the activities present in the original brei measured under these conditions were recovered after zonal ultracentrifuge fractionations.     

The organization and repair of DNA in the mammalian chromosome. II. A gradient-shape-induced speed dependence affecting DNAs larger than T4 DNA

The addition of a lytic layer to a preformed linear sucrose gradient induces a temporary (up to half a day) initial gradient of considerable steepness which retards the sedimentation of large (>T₄) DNAs. Centrifugation at sufficiently slow angular velocities permits the temporary initial gradient to disappear and therefore the sedimentation distance increases, yielding a rotor speed dependent effect on sedimentation distance. Gradients which are free of this effect are described and shown to permit mouse leukemia cell DNA to sediment independently of rotor speed (5–30 krpm).     

Required ultracentrifugal time for condensing solution by ultracentrifuge

Required ultracentrifugal time for condensing a solution by ultracentrifuge with an angle rotor or a swing rotor was estimated by considering the calculated distribution of concentration in an ultracentrifugal tube. From the calculated distribution of the concentration a free boundary between solvent and solution was found in the ultracentrifugal tube under the condition that t^*>0.25. A condense constant was newly defined as the ratio of the mean concentration in the ultracentrifugal tube after removing the solvent to the concentration of initial solution. The equation determining the required ultracentrifugal time for condensing a solution up to a given condense constant is derived and solved numerically. The required ultracentrifugal time for an angle rotor was shorter than that for a swing rotor.     

Steps in the Bacterial Flagellar Motor

The bacterial flagellar motor is a highly efficient rotary machine used by many bacteria to propel themselves. It has recently been shown that at low speeds its rotation proceeds in steps. Here we propose a simple physical model, based on the storage of energy in protein springs, that accounts for this stepping behavior as a random walk in a tilted corrugated potential that combines torque and contact forces. We argue that the absolute angular position of the rotor is crucial for understanding step properties and show this hypothesis to be consistent with the available data, in particular the observation that backward steps are smaller on average than forward steps. We also predict a sublinear speed versus torque relationship for fixed load at low torque, and a peak in rotor diffusion as a function of torque. Our model provides a comprehensive framework for understanding and analyzing stepping behavior in the bacterial flagellar motor and proposes novel, testable predictions. More broadly, the storage of energy in protein springs by the flagellar motor may provide useful general insights into the design of highly efficient molecular machines.