Quasielastic light scattering from migrating chemotactic bands of Escherichia coli. II. Analysis of anisotropic bacterial motions.

Chemotactic effects of dissolved oxygen on motions of Escherichia coli in a motility buffer solution have been studied by measurements of quasielastic light scattering. Under conditions where the bacteria form a sharp band in an oxygen concentrations gradient created by their metabolism, components of motions along the direction of the gradient and perpendicular to it were studied separately at each point within the band profile. A theoretical model for bacterial self correlation function based on two-state motions has been developed to extract the mean square speed of run motion and the relative probability of twiddle vs. run at each point of the band profile. A combined novel experimental set-up and new data analysis method allowed us to extract also the mean square displacements at short times along and perpendicular to the direction of the gradient. Parameters extracted from the measured correlation functions have been discussed in the framework of the established picture of bacterial motions under chemotaxis.     

A numerical method for the continuous spectrum biphasic poroviscoelastic model of articular cartilage

A method for numerical solution of the continuous spectrum linear biphasic poroviscoelastic (BPVE) model of articular cartilage is presented. The method is based on an alternate formulation of the continuous spectrum stress-strain law that is implemented using Gaussian quadrature integration combined with quadratic interpolation of the strain history. For N time steps, the cost of the method is O(N). The method is applied to a finite difference solution of the one-dimensional confined compression BPVE stress-relaxation problem. For a range of relaxation times that are representative of articular cartilage, accuracy of the method is demonstrated by direct comparison to a theoretical Laplace transform solution.     

Density gradient system: II. A 50 channel programmable undulating diphragm peristaltic pump

A special peristaltic pump is described that has functioned as part of a system for density gradient formation and fractionation. Twenty-five pumping tubes are actuated on both the top and bottom of the pump. A Mylar diaphragm interposed between the rollers and the pumping tubes filters out the horizontal, stretching component of the forces imparted to the tubes. This greatly prolongs tube life, increases the allowable pressures that can be achieved with such a pump, and thus permits accurate delivery of viscous solutions.An explanation of the cause of the pulsations produced by peristaltic pumps is presented and the virtual elimination of these pulsations is demonstrated.Both velocity and direction of flow of the pump are controlled. By means of two independent bidirectional digital counters, preset volumes of fluid can be delivered and the total volume of liquid determined.Studies demonstrating the relative independence of fluid volume delivered at a preset count versus flow velocity and composition are presented.Other possibilities for use of the pump in automating density gradient analysis are discussed. Possibilities for employment of the pump for autoanalysis and in artificial organs are indicated.     

Simultaneous control of turbidity and dilution rate through adjustment of medium composition in semi-continuous Chlamydomonas cultures

For production of starch in algal cultures, a growth rate limited by a nutrient is an important factor. Under phototrophic conditions, turbidity must be also paid attention, as the shading effect may affect its productivity. Semi-continuous cultivation methods, which enable control of turbidity and dilution rate (D) at the same time, have been developed for evaluation of those factors on starch production in Chlamydomonas sp. A specific feature of the methods is in a process of alternately feeding medium adjusted at two different nitrogen (N) concentrations. In the turbidostat-based method, a turbidostat culture was operated repeating three steps of determining D within a preset interval, alternating media by comparing the D with a preset value, and adjusting D in the next interval by feeding the selected medium. In the chemostat-based method, turbidity of a chemostat culture was controlled by repeating two steps of alternating media by comparing transmitted photon flux intensity (I) with a preset value and adjusting I by feeding the selected medium. D controlled by the turbidostat-based method reached quickly a preset value as low as 0.010/h, and then it was dispersed around but above the preset value. On the other hand, mean N concentrations of fed media formed a plateau. In the chemostat-based method, I was well controlled to a preset value while the mean N concentrations were a bit fluctuated. Starch concentration varied from 0.052 to 0.41 g/L with turbidity and D defined by these methods.     

A fast quadrature-based numerical method for the continuous spectrum biphasic poroviscoelastic model of articular cartilage

A new and efficient method for numerical solution of the continuous spectrum biphasic poroviscoelastic (BPVE) model of articular cartilage is presented. Development of the method is based on a composite Gauss–Legendre quadrature approximation of the continuous spectrum relaxation function that leads to an exponential series representation. The separability property of the exponential terms in the series is exploited to develop a numerical scheme that can be reduced to an update rule requiring retention of the strain history at only the previous time step. The cost of the resulting temporal discretization scheme is O(N) for N time steps. Application and calibration of the method is illustrated in the context of a finite difference solution of the one-dimensional confined compression BPVE stress-relaxation problem. Accuracy of the numerical method is demonstrated by comparison to a theoretical Laplace transform solution for a range of viscoelastic relaxation times that are representative of articular cartilage.     

On using a double-exponential model for describing faecal marker concentration curves

A mathematical and numerical appraisal of the double-exponential model is presented, with particular reference to its ability to reproduce preset values of biological measures such as rumen and whole-tract mean retention times and faecal production rate. A complete, formal mathematical derivation is provided, with consideration given to a solution to the model when the two smallest rate constants are equal. It is concluded that any error introduced by using an approximation in deriving the double-exponential form is negligible in practice, and that the double exponential in logarithmic form is able to determine the relevant biological measures quite satisfactorily and speedily.     

Generating controlled molecular gradients in 3D gels

A new method for producing molecular gradients of arbitrary shape in thin three dimensional gels is described. Patterns are produced on the surface of the gel by printing with a micropump that dispenses small droplets of solution at controlled rates. The molecules in the solution rapidly diffuse into the gel and create a smooth concentration profile that is independent of depth. The pattern is relatively stable for long times, and its evolution can be accurately described by finite element modeling of the diffusion equation. As a demonstration of the method, direct measurements of protein gradients are performed by quantitative fluorescence microscopy. A complementary technique for measuring diffusion coefficients is also presented. This rapid, flexible, contactless approach to gradient generation is ideally suited for cell culture experiments to investigate the role of gradients of diffusible substances in processes such as chemotaxis, morphogenesis, and pattern formation, as well as for high-throughput screening of system responses to a wide range of chemical concentrations.     

Separation of bacterial cells by isoelectric focusing, a new method for analysis of complex microbial communities.

A simple isoelectric focusing (IEF) method for whole bacterial cells was developed. In a pH gradient of 2 to 10 and an electric field of 11.5 V cm-1, mixtures of cells from the three different bacterial strains Chlorobium limicola 6230, Pseudomonas stutzeri DSM 50227, and Micrococcus luteus DSM 20030 could be separated. A density gradient of Ficoll prevented convective currents in the system. The method was tested with a concentrated mixture of bacteria from a shallow eutrophic lake and yielded up to 10 different bands. Species composition in each IEF band was analyzed by PCR plus denaturing gradient gel electrophoresis (DGGE). Each IEF band exhibited a different species composition. After the separation of cells by IEF three times more 16S ribosomal DNA signals could be detected by DGGE than in the unfractionated natural bacterial community. It is concluded that the resolution of these molecular biological methods is significantly enhanced if cells are first separated by IEF. At the same time, the IEF fractions are enriched for certain species, which can be used in subsequent cultivation experiments.     

A simple and rapid method for the quantitative isolation of proteins from polyacrylamide gels

A simple method for protein recovery from gel slices following polyacrylamide gel electrophoresis is proposed which requires neither additional devices nor much skill. The method is based on reversed electrophoresis using a discontinuous conductivity gradient which is in turn maintained by a gradient in density. Protein recovery is possible within a small volume and approaches the theoretical yield. A large number of slices can be processed simultaneously.     

小麦高分子量麦谷蛋白亚基分离方法的研究

小麦高分子量麦谷蛋白亚基（ＨＭＷ－ＧＳ）与小麦面包烘烤质量和面粉的加工特性密切相关,ＳＤＳ－ＰＡＧＥ是其常用的分离方法之一。ＳＤＳ－ＰＡＧＥ方法一般分为２类：第一类采用１１％和５％浓度的胶,后者用于分离２亚基和２＾＊亚基,该种方法常使用碱性提取液,需要２次电泳过程,且在５％浓度的胶中ＨＭＷ－ＧＳ易于和麦醇蛋白混淆;另外一类ＳＤＳ－ＰＡＧＥ采用梯度胶,配合使用银染方法,制梯度胶则使用梯度仪及磁力搅拌     

"Blind focusing": an objective method for avoiding errors of focus at the television texture analysis system (TAS).

The amount of light energy a particle absorbs does not depend upon correct focus. The change in the path of light rays brought about by defocusing causes absorbing areas to be registered as areas of higher transmittance than when in focus. Already wellknown in photometry, this effect is put to use by the "blind focus" method at the television texture analysis system (TAS, Leitz). Some chromophores within the object to be measured are compared to a preset value of transmittance, for example T = 0.40. Only the area representing the structures as dense or denser than the preset density are registered. If the structures are out of focus the size of the registered area is too low, since by defocusing, structures to be measured become pale and diffuse, the correct focus corresponds to the largest area to a preset value of transmittance.     

Tracking single quantum dot and its spectrum in free solution with controllable thermal diffusion suppression

Thermal motions of semiconductor quantum dots (QDs) are suppressed on a dehydrated agarose-modified surface. The diffusion coefficients (D) of particles can be controlled by modifying the surface with an appropriate agarose concentration. The value of D is more than 100 times lower than the theoretical value when the dried agarose surface is made with an 8% agarose solution. This makes it possible to real-time record the diffusion process of single particles and single molecules in low-viscosity solution. A transmission grating installed in front of the charge-coupled device separates the QD fluorescence into the zeroth-order and first-order spectrum. Therefore, the spectrum of dynamic QDs is tracked on the modified surface. Tracking the dynamic QD spectral image is a promising method to explore the process of the molecular interactions in the physiological buffer.     

Isoelectric focusing of proteins using a pH gradient created by a concentration gradient of nonelectrolytes in solution.

A new method of producing a stable pH gradient in buffer solutions is suggested, obtained by the concentration gradient of a nonelectrolyte in buffer solutions as a result of the gradual change in the dielectric properties of the solution. The maximal concentrations of nonelectrolyte which do not influence the protein configuration allow a pH gradient with a range of two pH units to be produced. It is suggested that the properties of some polyols (i.e. glycerol or mannitol) be used to change the pH of the borate buffer for the production of greater pH gradient with a range of up to 4-5 pH units. Creating the gradient of concentration of polyols, one can obtain a pH gradient in borate buffer solutions. Though the polylydroxyl compound-borate complexes posses mobility in an electric field, a stable pH gradient can be achieved during 12 days of electrophoresis. The isoelectric focusing of haemoglobin, human serum albumin and immunoglobulins was carried out in both systems suggested. These findings were compared with isoelectric focusing in Ampholines. There was a good agreement between the methods compared. The possible differences are discussed.     

A Molecular Dynamics Simulation Study of Rigid and Non-rigid Hard Dumb-bells

Abstract

We present a comparative study, using molecular dynamics, of systems of diatomic, hard dumb-bell, molecules in which the interatomic distance is either constrained to a fixed value or is allowed to vary freely between preset limits. A significant improvement in simulation effciency can be attained by allowing the bond length to vary. We find that thermodynamic properties, and some time correlation functions, are only slightly affected by the removal of the rigid bond-length constraint. The atomic velocity correlation function responds dramatically at short times to changes in the degree of non-rigidity, but at long times these differences are much less important.     

A rapid electrophoretic procedure for the detection of SDS-released oncorna-viral RNA using polyacrylamide-agarose gels

A rapid method for analyzing SDS-released viral RNA on acrylamideagarose gels is described. Studies carried out on Rauscher leukemia virus and Mason-Pfizer monkey virus utilizing this method show that the distribution and recovery of the RNA components are similar for phenol-extracted or SDS-released viral RNA. The method is approximately 20 times more sensitive than sucrose gradient analysis and can be utilized in cases where virus concentration (EM particle count or radioactivity) is low.Data presented illustrate the versatility of this method.     

Isolation of oligodendroglial cells from young and adult whole rat brains using an in situ generated percoll density gradient

A method for the isolation of oligodendroglial cells from young and adult whole rat brains, using a Percoll density gradient is presented. The minced tissue, incubated in a balanced salt solution containing 0.1% trypsin is further dissociated by forcing it through nylon screens to 145 and 74 μm pore size. The crude suspension is then mixed with an isosmotic Percoll solution and centrifuged for 15 min. An in situ generated density gradient allows the separation of five bands, only one of which (Band C) lying between δ1.050 and δ1.062 contains cellular elements. The isolated cells show the typical morphological characteristics of oligodendroglia.A detailed morphological study of the cells isolated from whole brains of 10-, 30- and 120-day old rats is presented for the first time in the literature and immunocytochemical characterization is carried out using specific (antigalactocerebroside) and non specific (anti-glial fibrillary acidic protein) anti-sera.The method is simple and rapid and isosmotic conditions are maintained throughout, resulting in a better preservation of cell integrity. It represents an improvement over the two previous methods described in the literature and will be useful for studying different developmental events (biochemical and morphological) occurring in oligodendroglial cells at early stages of myelin formation.     

Foam separation of DNA and proteins from solutions

Foam separation on BSA-DNA (bovine serum albumine/deoxyribonucleic acid) and Lysozyme-DNA systems is performed. The separation of the total protein from DNA is evaluated for dissociated chromatin solution. Foam separation for the same systems is done also by a new method of creating a pressure gradient in the Plateau-Gibbs borders in the foam and obtaining a "dry" foam. It is shown that the effectiveness of the foam separation can be improved significantly by the application of the latter method. Some factors (pH, initial concentration of the solution, expansion factor of the foam) influencing the separation of proteins from DNA in the foam and in the residual solution are studied as well.     

Determination of the novel non-peptidic HIV-protease inhibitor tipranavir by HPLC-UV after solid-phase extraction

An HPLC method previously described for the assay of amprenavir (APV), ritonavir (RTV), indinavir (IDV), saquinavir (SQV), nelfinavir (NFV), lopinavir (LPV), atazanavir (ATV), nevirapine (NVP) and efavirenz (EFV) can be also conveniently applied, with minor gradient program adjustment, for the determination of the novel non-peptidic HIV protease inhibitor tipranavir (TPV) in human plasma, by off-line solid-phase extraction (SPE) followed by HPLC coupled with UV-diode array detection (DAD). After viral inactivation by heat, the plasma is diluted with phosphate buffer (pH 7), and subjected to a SPE on a C18 cartridge. Matrix components are eliminated with a solution of 0.1% H3PO4 solution neutralised to pH 7, and TPV is eluted with MeOH. The resulting eluate is evaporated and reconstituted in 100 microl MeOH/H2O 50/50. A 40 microl volume is injected onto a Nucleosil C18 AB column and TPV is analysed by UV detection at 201 nm using a gradient elution program constituted of MeCN and phosphate buffer adjusted to pH 5.12 and containing 0.02% sodium heptanesulfonate. The calibration curves are linear up to 75 microg/ml, with a lower limit of quantification of 0.125 microg/ml. The mean absolute recovery of TPV is 77.1+/-4.0%. The method is precise with mean inter-day coefficient of variations (CVs) within 2.2-3.4%, and accurate (range of inter-day deviations from 0.7 to 1.2%). The method has been validated and is currently applied to the monitoring of TPV plasma levels in HIV patients.     

A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid

In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented.Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative–dissociative cesium chloride density gradient centrifugation (A1D1–D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions.The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800–1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample.This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.     

Responding phospholipid membranes--interplay between hydration and permeability.

Osmotic forces are important in regulating a number of physiological membrane processes. The effect of osmotic pressure on lipid phase behavior is of utmost importance for the extracellular lipids in stratum corneum (the outer part of human skin), due to the large gradient in water chemical potential between the water-rich tissue on the inside, and the relative dry environment on the outside of the body. We present a theoretical model for molecular diffusional transport over an oriented stack of two-component lipid bilayers in the presence of a gradient in osmotic pressure. This gradient serves as the driving force for diffusional motion of water. It also causes a gradient in swelling and phase transformations, which profoundly affect the molecular environment and thus the local diffusion properties. This feedback mechanism generates a nonlinear transport behavior, which we illustrate by calculations of the flux of water and solute (nicotine) through the bilayer stack. The calculated water flux shows qualitative agreement with experimental findings for water flux through stratum corneum. We also present a physical basis for the occlusion effect. Phase behavior of binary phospholipid mixtures at varying osmotic pressures is modeled from the known interlamellar forces and the regular solution theory. A first-order phase transformation from a gel to a liquid--crystalline phase can be induced by an increase in the osmotic pressure. In the bilayer stack, a transition can be induced along the gradient. The boundary conditions in water chemical potential can thus act as a switch for the membrane permeability.