A separation chamber to sort cells and cell organelles by weak physical forces: III. Preparative electrophoresis of cells in stationary density gradients at low electric field strength

An apparatus was designed for preparative density gradient electrophoresis of mammalian cells. In a low conductivity isotonic Ficoll density gradient of 1.5 cm length, human erythrocytes treated with neuraminidase were separated from untreated erythrocytes at an electric field strength of approximately 2.7 v/cm. Within 5 min two bands of erythrocytes were visible. Electrophoretic separation was completed within 25 min. The fractionation is performed in a design consisting of three Perspex circular plates, bottom and top plates of which can be displaced simultaneously relative to the stationary middle plate by a worm-gear mechanism. The middle plate contains a cylindrical separation chamber of 50 cm² and 1.5 cm high. Top and bottom plates contain cones and flow deflectors for the undisturbed thin layering of cell suspensions and for introduction of the density gradient. Also present in top and bottom plates are electrode compartments containing a large platinum electrode and a cellophane membrane that isolates the separation chamber hydrodynamically but not electrically from the electrode compartment. The electrode compartments were flushed with electrophoresis buffer to remove products of electrophoresis as well as the (low) generated Joule heat.     

Electrophoretic affinity chromatography: method validation

A new method for preparative-scale separation of biomolecules, electrophoretic affinity chromatography (EAC), is proposed in this paper. Separation by EAC is carried out in a long and ribbon-like multicompartment electrolyser separated by membranes, in which the two central compartments are used for packing the gel matrix and for sample loading respectively. Next to the central compartments are the elution compartments and electrode compartments. The electric field is applied perpendicular to the fluid flow in the compartments. Adsorption and desorption steps may both be carried out in the presence of an electric field, which transports the target components into the gel compartment for adsorption and the impurities into the elution compartments for washing. After the adsorption step an elution solution is introduced and the product is released from the gel matrix and washed out. Separation of human serum albumin (HSA) from human serum gives HSA product of high purity, as demonstrated by isoelectric focusing analysis. The characteristics of electrophoretic binding of HSA on Blue Sepharose Fast Flow are examined. The preliminary results show that this new method has advantages in terms of high rate of mass transfer and ease of scaling up, which are of particular interest when large-scale separation of biomolecules is considered.     

Comparison of two test systems for measuring plant phosphorus uptake via arbuscular mycorrhizal fungi

 Plant phosphorus uptake via external hyphae of arbuscular mycorrhizal fungi has been measured using compartmented systems where a hyphal compartment is separated from a rooting compartment by a fine mesh. By labelling the soil within the hyphal compartment with a radioactive phosphorus (P) isotope, hyphal uptake of P into the plant can be traced. The objective of this growth chamber study was to test two hyphal compartments of different design with respect to their suitabilities for measurement of hyphal P uptake. One hyphal compartment was simply a nylon mesh bag filled with ³²P-labelled soil. The labelled soil in the other hyphal compartment was completely surrounded by an 8–10 mm layer of unlabelled soil that served as a buffer zone. Mycorrhizal and non-mycorrhizal subterranean clover plants were grown in pots with a centrally positioned hyphal compartment. Uptake of radioactive P by non-mycorrhizal control plants was 25% of that by mycorrhizal plants with the mesh bag but only 3% when including the buffer zone. Based on this good control of non-mycorrhizal P uptake from within the hyphal compartment and its greater ease of handling once produced, we judged the hyphal compartment including a buffer zone to be superior to the mesh bag. Accepted: 15 September 1998     

Interaction Between Two Perpendicular Fabry–Perot-Like Resonances of the Antenna–Dielectric–Slit Structure and Their Influences on the Transmission Enhancement

The interaction between the two perpendicular Fabry–Perot-like resonances of the antenna–dielectric–slit structure and their influences on the transmission enhancement are investigated with a finite-difference time-domain method. The transmission enhancement is found with the antenna width corresponding to a Fabry–Perot-like resonance condition in the antenna–dielectric–slit structure; otherwise, there is no such an enhancement even when the slit is positioned under the magnetic field maximum. On the other hand, the resonance characteristics of the vertical slit can also modify the field distribution in the horizontal cavity by changing the phase difference at the two antenna ends. It is shown that the enhanced transmission can be realized in a wide range of incident wavelengths from the visible to near-infrared regime for different slit geometries. The physical mechanism of extraordinary optical transmission is discussed with a theoretical dispersion relationship of surface plasmon polaritons based on a metal–insulator–metal cavity model.     

Theory of the near-wall conductivity

A model describing the phenomenon of the electron current in a plane plasma slab bounded by parallel dielectric walls in the presence of a homogeneous magnetic field perpendicular to the walls and an electric field oriented along the walls is presented. The current flows along the electric field because of the electron collisions with diffusely scattering walls. The model takes into account the presence of Debye layers and the non-Maxwellian character of the electron distribution function. Collisions in the plasma volume are ignored.     

Preference for structured environment in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis)

Information about the welfare and husbandry of pet and laboratory fish is scarce although millions of fish are sold in pet shops and used in laboratory research every year. Inadequate housing conditions can cause behavioural problems also in fish since they are complex animals with sophisticated behaviour. In this study, we investigated the influence of environmental complexity on compartment preference and behaviour in zebrafish (Danio rerio) and checker barbs (Puntius oligolepis). For the preference test, large aquaria were divided by two semi-transparent walls of Plexiglas into an empty compartment, a structured compartment enriched with plants and clay pots, and a smaller compartment in-between, where food was provided. For observation, the empty and structured compartments were divided into six zones of similar size by defining three vertical layers and two horizontal areas (back vs. front area). Seven groups of six to nine zebrafish and seven groups of seven or eight checker barbs were observed on four days each (within a time period of ten days) to assess compartment use and activity, and to assess behavioural diversity and use of zones within compartments. Both zebrafish and checker barbs showed a significant preference for the structured compartment. Nevertheless, in neither species did behavioural diversity differ between the empty and structured compartment. Zebrafish used all zones in both compartments to the same extent. Checker barbs, however, used the structured compartment more evenly than the empty compartment, where they mainly used the lower and middle zones. These results suggest that zebrafish and checker barbs have a preference for complex environments. Furthermore, they indicate that the behavioural and ecological needs of fish may vary depending on species, and recommendations for husbandry should be specified at species level.     

A Slow Rotary Shaker for Embedding in Viscous Media

Embedding vessels are carried by a metal tray which has a tilt of about 20° to the horizontal plane. The tray is supported by a shaft attached perpendicular to its bottom. The shaft passes loosely through a hole in a rigid plate and is attached by a ball joint to a crank, which is driven at about 20 rev/min by a geared-down electric motor. Thus a combined tilting and rotary motion is imparted to the tray to produce a continuous flow of the embedding medium about the specimens.     

Structural Difference Between Walls from Ends and Sides of the Rod-Shaped Bacterium Bacillus subtilis

Electron microscopy studies show that walls from ends of Bacillus subtilis cells are more resistant to autolytic degradation that walls from sides of the same cells. This observation suggests that these two parts of the cell are structurally different. A method is available for the isolation of walls from ends of cells.     

Hox genes and morphological identity: axial versus lateral patterning in the vertebrate mesoderm

The successful organization of the vertebrate body requires that local information in the embryo be translated into a functional, global pattern. Somite cells form the bulk of the musculoskeletal system. Heterotopic transplants of segmental plate along the axis from quail to chick were performed to test the correlation between autonomous morphological patterning and Hox gene expression in somite subpopulations. The data presented strengthen the correlation of Hox gene expression with axial specification and focus on the significance of Hox genes in specific derivatives of the somites. We have defined two anatomical compartments of the body based on the embryonic origin of the cells making up contributing structures: the dorsal compartment, formed from purely somitic cell populations; and the ventral compartment comprising cells from somites and lateral plate. The boundary between these anatomical compartments is termed the somitic frontier. Somitic tissue transplanted between axial levels retains both original Hox expression and morphological identity in the dorsal compartment. In contrast, migrating lateral somitic cells crossing the somitic frontier do not maintain donor Hox expression but apparently adopt the Hox expression of the lateral plate and participate in the morphology appropriate to the host level. Dorsal and ventral compartments, as defined here, have relevance for experimental manipulations that influence somite cell behavior. The correlation of Hox expression profiles and patterning behavior of cells in these two compartments supports the hypothesis of independent Hox codes in paraxial and lateral plate mesoderm.     

A continuous,multistage tower fermentor. I. Design and performance tests

The design of a continuous column fermentor with a multiple staging effect is described. The column is divided into four compartments by horizontal perforated plates and is provided with a central agitator shaft driving an impeller in each compartment. A tube at the center of each plate forms a liquid seal around the shaft and also acts as a “downcomer.” The fermentor is normally operated with counter-current flow of gas and medium. Fresh medium is added to the top stage and product is withdrawn from the bottom. The effect of plate and agitator design on fermentor performance was studied in terms of factor such as oxygen transfer rate, gas holdup, and interstage mixing. By proper choice of the design parameters, the fermentor was made to approximate a perfect four-stage cascade in terms of reactor performance. Preliminary experiments were performed with air-water systems, but a more realistic picture of fermentor performance was obtained in experience involving propagation of Escherichia coli. Data for business and substrate concentrations in each stage confirmed the staging effect of the apparatus. The fermentor operated in a stable manner for periods of more than two weeks.     

Effects of prolonged omnilateral gravistimulation on the ultrastructure of statocytes and on the graviresponse of roots

Statocytes of vertically growing roots of Lepidium sativum L. exhibit a strict polarity: The nucleus is positioned near the proximal periclinal cell wall, amyloplasts are sedimented on a complex of rough endoplasmic reticulum (ER) consisting of parallel cisternae near the distal periclinal cell wall.When 24 h old, vertically grown roots are rotated for an additional 20 h on a horizontal clinostat, this polarity is destroyed. Furthermore, the prolonged omnilateral stimulation leads to a damage of the statocytes, which in some cases ends in the self-destruction of the sensitive cells. The different components of the ultrastructural respones of the statocytes are: Displacement of the nucleus; changes in amount and distribution of the ER; loss of amyloplast starch; confluence of lipid droplets to large aggregates: a considerable increase of the lytic compartment. In addition, even anticlinal cell walls may be lysed up to small stumps. As all these effects are clearly restricted to the statocytes, only these cells are able to respond to the continuously changing direction of the gravity vector, thus perceiving gravity as such.After being exposed horizontally, the graviresponse of rotated roots is delayed as compared to the controls. About 20% of the rotated roots do not respond (curve) at all, but grow perpendicular in relation to the gravity vector. Perception of gravity is inevitably correlated with the polarity and the integrity of the statocytes.Abbreviation ER endoplasmic reticulum A preliminary report was presented at the Fall Meeting of the German Society for Cell Biology in Salzburg, Austria, September 1979 (Hensel and Sievers 1979)This paper represents part of a dissertation (D 5) of W. H.     

Isoelectric focusing using non-amphoteric buffers in free solution: I. Determination of stable concentration profiles

For large-scale separations of proteins, the use of simple non-amphoteric buffers in free solution and in multicompartment electrolyzers seems promising for industrial applications. The stabilization of a pH profile with this type of buffer requires the strict observation of two conditions: choice of an adequate buffer; stationary profiles of concentrations. During electrolysis in free solution, the ions of the buffer are displaced across the compartments by migration and by diffusion. To keep a stationary composition, the inflow and outflow of all individual ionic species through each compartment must be identical. At high current, diffusion may be neglected against migration and the ionic flows will be identical if the transport number of each ion is constant at each location within the cell. In these conditions, stationary compositions will be independent of the electric current. This condition of constant transport numbers implies the use of profiles of buffer concentrations different from those published up to now. The new equations for these profiles of concentrations are given in the present paper. The constant migration of the ions must be compensated in the end compartments of the isoelectric focusing cell to provide a stable steady state. Two methods are proposed in the literature: the buffer renewal method and the external recycling method (rheoelectrolysis). Here modified buffer renewal method is proposed. Using stationary mass balances, analytical equations are given to calculate the flows and the composition of the solutions to be recycled or added. Using these equations and the profiles of concentrations to keep constant transport numbers, it is demonstrated that only a renewal of the buffers in the end compartments may lead to stable pH profiles and thus to valid conditions of separation.     

Action of a 50 Hz magnetic field on proliferation of cells in culture

Proliferation of SV40-3T3 mouse fibroblasts and human HL-60 promyelocytes was studied after treatment with a sinusoidal 2 mT_rms 50 Hz magnetic field. A single exposure of 60 minutes caused quasicyclic changes in the cell number of SV40-3T3 cultures as function of time after treatment, which was interpreted to be due to the induction of chronobiological mechanisms by the field. Moreover, small variations in cell cycle distribution were measured during postexposure incubation for both cell lines. To discriminate between the effect of the magnetic vector and the induced electric field, HL-60 cell exposure was also performed on organ culture dishes. These dishes consist of two coaxially centered, isolated compartments in which different electric field levels are induced in the medium during treatment. Cell growth was affected in the outer compartment only where the induced electric field ranged from 8 to 12 mV_peak/meter at 2 mT, but it was not affected in the inner compartment (field range 0–4 mV_peak/meter). This suggests that the effects on cell growth are due to the induced electric field and are expressed only above a threshold of between 4 and 8 mV_peak/meter. Bioelectromagnetics 18:177–183, 1997. © 1997 Wiley-Liss, Inc.     

New system for preparative electrochromatography of proteins

Electrochromatography employs an axial electric field across a chromatographic stationary phase to separate proteins and other molecules based on differences in electrophoretic mobility. Because the separation is electrically driven, the need for additional chemical reagents is reduced. Two major impediments to scale-up of electrochromatography columns, removal of heat and electrolysis gases, have historically limited the diameter of packed columns to 2.5 cm ID with volumes of approximately 55 mL. We report a novel electrochromatography column that effectively removes electrolysis gases and minimizes heating. A vital component of this system is a new electrode design that couples a platinum gauze with an ultrafiltration membrane across both ends of the column. Use of a methacrylate base stationary phase enabled axial voltage gradients of 10 to 20 V/cm. Thermocouples inserted radially in the column at four axial positions showed that the flow of a 4 degrees C mobile phase coupled with heat conduction through the column walls controlled the temperature to 28 degrees C. The new column design, with dimensions of 3.81 cm ID x 38.1 cm long and bed volume of 400 mL, was demonstrated by separating mixtures of BSA and myoglobin. The column was operated in a horizontal position with radial sample injection and withdrawal at the ends of the packed bed. These experiments are a first step in demonstrating that scale-up of electrochromatography columns can be achieved by choosing appropriate flow rates, voltage gradients, and stationary phase.     

A calcium requirement for electric field-induced cell shape changes and preferential orientation

C3H/10T1/2 mouse embryo fibroblasts stimulated by a steady electric field (10 V/cm) for 30 min exhibited lamellar retraction on the sides facing the electrodes. Some cells elongated and preferentially oriented with their long axis perpendicular to the field direction. Depletion of external calcium or blockage of calcium influx with lanthanum or the calcium channel blocker D-600 resulted in a reduction of the field-induced response. When external calcium was elevated stepwise from 0 to 10 mM, the field-induced response increased correspondingly. Electric stimulation in the presence of the calcium ionophore A23187 resulted in an increase of spindle-shaped cells with no preferential orientation. This response was blocked by calcium depletion and lanthanum, but not by D-600. The anticalmodulin drug W-13 inhibited the field-induced responses observed in normal buffer as well as in the presence of A23187. Some cell death resulted from prolonged electric field exposure, and the mortality was reduced by calcium depletion, lanthanum or D-600, but was not affected by W-13. We postulate that local calcium influx through channels opened by the electric field produces areas of high intracellular calcium which stimulate the cytoskeletal network to induce lamellar retraction. Prolonged field-induced calcium influx may eventually overcome the cell's mitochondrial calcium-buffer system, leading to necrotic calcification.     

Dynamic transition of microbial communities in response to acidification in fixed-bed anaerobic baffled reactors (FABR) of two different flow directions

Two five-compartment fixed-bed anaerobic baffled reactors (FABRs) were operated under deteriorative and stable conditions. The FABRs were identical except for flow direction: one was horizontal (H-reactor) and the other was vertical (V-reactor). The microbial community dynamics in 1, 3 and 5 compartments were analyzed using denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone library screening and quantitative PCR. After start-up, the Methanomicrobiales were typically dominant in adhering sludge of 5th compartments of two reactors. Because methanogenesis mainly occurred in the latter compartment, Methanomicrobiales were likely to play important roles in FABRs. FABRs recovered from performance deterioration very quickly. Meanwhile, methanogens and dominant methanogens greatly increased in every compartment of two reactors. Our results indicated that 16S rRNA levels of methanogens in the adhering sludge were higher than those in the deposited sludge and the adhering fraction of V-reactor held up more acid-resistant bacteria and methanogens than that of H-reactor.     

Modeling two-component isoelectric focusing buffers in a vortex-stabilized electrophoresis apparatus

Defined buffer systems have been considered by researchers as replacements for synthetic carrier ampholyte mixtures in IEF in order to reduce both costs and deleterious protein-ampholyte interactions. In this paper, a mathematical model for the behavior of two-component pH gradients during IEF is applied to a vortex-stabilized electrophoresis apparatus. Equations for mass conservation, molar fluxes, and the electric field describe the behavior of the components in the electric field. Equilibrium constants are used in the model to account for interconversion between the positive, negative, and neutral states for each component. The model was applied to predict pH gradients using two different defined buffer systems. The model's predictions fell within the 95% confidence interval of a least-squares fit to the experimental pH gradients except in the regions near the ends of the pH gradients, where large pH excursions occurred during the experiments.     

Organisation of Xenopus egg cytoplasm: response to simulated microgravity

The cytoplasm of Xenopus fertilised eggs appears to be organised into three major compartments based primarily on the uneven distribution of yolk platelets. There is a shift of these yolk compartments during the first cell cycle that is thought to be involved in the dorsal/ventral morphogenesis of the embryo. The involvement of gravity in Xenopus cytoplasmic organisation and in compartment shifts was addressed by examining, cytologically, the yolk compartments in embryos that developed under the simulated microgravity conditions of the horizontal clinostat. The cytoplasmic organisation into yolk compartments was found to be maintained, and the asymmetric movements of compartments still occurred in eggs that developed on the clinostat. It is suggested that the organisation of Xenopus egg cytoplasm into discrete compartments relies on forces other than those involving gravity (i.e., not density differences), and that the compartment shifts that take place during the first cell cycle are active movements. The variation in compartment size and composition observed from batch to batch of eggs, and to a lesser extent from egg to egg, during this study was addressed.     

Modeling the electromobility of ions in a target tissue

Electroporation is a clinical and laboratory technique for the delivery of molecules to cells. This method imposes electric fields onto cells or tissues through the use of electrodes and a set of electrical parameters to ultimately incorporate molecules into the cells. Clinical applications may include using directional fields to bring therapeutics to the target tissues before triggering an electroporation event. The choice of applicator may also have a significant influence on this molecular flow. Modeling ionic flow in tissues will yield insight into selecting the appropriate parameters or electroporation signature for a desired target application. In this paper, the motion of tissue injected ions was modeled for two common electroporation applicator configurations-the parallel plate, and the four needle electrodes. This electric field induced fluid flow model predicts that the parallel plate applicator ultimately directs the movement of an ionic therapeutic in a forward manner with side motion due only to obstruction, while the four-needle applicator directs anisotropic flow within the field ultimately forcing the therapeutic into a mound at the fringes of the induced electric field.     

Split flexor carpi radialis muscle

A detailed anatomic and intramuscular neural staining study in 22 human and 5 monkey upper limbs revealed that the flexor carpi radialis can be raised on its proximal neurovascular pedicle and that the muscle can be split along its tendon into two independently functioning neuromuscular compartments, each with its own nerve and blood supply. A study of the muscle architecture in the human specimens found the radial compartment to have significantly longer fiber length and a larger physiologic cross-sectional area than the ulnar compartment. Independence of function of each compartment was demonstrated in electrical stimulation studies in six monkeys (Macaca fascicularis), but no significant difference was noted in the peak isometric load between the two compartments (p = 0.68) in the monkey. The extra functioning muscle units become important in local transfers for restoring function in multiple nerve palsies as in Hansen's disease, severe traumatic loss of muscle in crush injuries and compartment syndromes, and after wide resection in infective and neoplastic conditions in the forearm and hand.