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Biomedical subjects

U Zimmermann

Publications and source records attributed to U Zimmermann.

At least 253 records · Page 14Linked to original sources
PubMed1981

Fusion of Avena sativa mesophyll cell protoplasts by electrical breakdown.

Studies with the light microscope were carried out on mesophyll cell protoplasts of Avena sativa which had been made to undergo fusion by reversible electrical breakdown of the cell membrane. In order to establish close membrane contact between the cells, an important prerequisite for fusion, a method known as dielectrophoresis was used. In an inhomogeneous alternating electrical field the protoplasts adhere to the electrodes and to each other in the direction of the field lines. The cells which were thus brought into close contact with each other could be made to fuse by the application of a field pulse of high amplitude (about 750 V/cm) and short duration (20-50 mus). The field strength required for fusion exceeds the value necessary for the electrical breakdown of the cell membrane. Fusion took place within some minutes and led to a high yield of fused protoplasts. The fusion of cells being in the electric field occurred in a synchronous manner. In some of the fusion experiments part of the protoplasts of A. sativa were stained with neutral red. When these cells were fused with unstained protoplasts, the vacuoles from the different cells within the fused aggregate could be shown to remain separate for quite some time.

Cell Fusion↗
PubMed1981

The resealing process of lipid bilayers after reversible electrical breakdown.

The resealing process of lipid bilayer membranes after reversible electrical breakdown was investigated using two voltage pulses switched on together. Electrical breakdown of the membranes was induced with a voltage pulse of high intensity and short duration. The time course of the change in membrane conductance after the application of the high (short) voltage pulse was measured with a longer voltage pulse of low amplitude. The decrease in membrane conductance during the resealing process could be fitted to a single exponential curve with a time constant of 10--2 micros in the temperature range between 2 and 20 degrees C. The activation energy for this exponential decay process was found to be about 50 kJ/mol, which might indicate a diffusion process. Above 25 degrees C the resealing process is controlled by two exponential processes. The data obtained for the time course of the resealing process can be explained in terms of pore formation in the membrane in response to the high electrical field strength. A radius of about 4 nm is calculated for the initial pore size. From the assumed exponential change of the pore area with progressive resealing time a diffusion constant of 10(-8) cm2/s for lateral lipid diffusion can be estimated.

Animals↗
PubMed1981

Electrically Stimulated Fusion of Different Plant Cell Protoplasts : MESOPHYLL CELL AND GUARD CELL PROTOPLASTS OF VICIA FABA.

Cell fusion is induced between guard cell and mesophyll cell protoplasts of Vicia faba by electrical field application. The process of fusion is initiated by electrical breakdown of the cell membrane. Prior to the application of an external electrical field pulse which brings about reversible breakdown of the membrane, the cells (suspended in a low-conducting medium) are brought into close contact with one another by exposing them to an external alternating, nonuniform field (5 volts, electrode distance, 200 micrometers; 500 kiloHertz). During this process, they form "pearl chains" which may become sufficiently long to form bridges between the electrodes. The process is reversible as long as this voltage is not exceeded. Cell fusion is initiated as a result of an electrical field pulse of 50 microseconds duration and of sufficiently high intensity to induce reversible electrical breakdown of the membranes. The process of fusion is completed within 40 minutes or less in the case of guard cell protoplasts, as well as in the case of fusion between guard cell and mesophyll cell protoplasts. The fused cells are spherical in shape, if the fusion product consists only of two or three cells.

Journal Article↗
PubMed1981

Water Relations of Leaf Epidermal Cells of Tradescantia virginiana.

Water-relation parameters (cell turgor pressure [P], volumetric elastic modulus [epsilon] and hydraulic conductivity [Lp]) of individual leaf epidermal cells of Tradescantia virginiana have been determined with the pressure-probe technique. Turgor was 4.5 +/- 2.1 [41] bar (mean +/- sd; in brackets the number of cells) and ranged from 0.9 to 9.6 bar. By vacuum infiltration with nutrient solution, it was raised to 7.5 +/- 1.5 [5] bar (range: 5.3-8.8 bar). There was a large variability in the absolute value of epsilon of individual cells. epsilon ranged from 40 to 360 bar; mean +/- sd: 135 +/- 83 bar; n = 50 cells. epsilon values of individual cells seemed to be rather independent of changes in cell turgor. A critical assessment of the errors incurred in determining epsilon by the technique is included. The half-times of water exchange of individual cells ranged from 1 to 35 seconds, which gave values of 0.2 to 11 x 10(-6) centimeters per second per bar for Lp (mean +/- sd: 3.1 +/- 2.3 x 10(-6) centimeters per second per bar; n = 39 cells). The large range in Lp and epsilon is believed to be due to the difficulties in determining the effective surface area of water exchange of the cells. Lp is not influenced by active salt pumping driven by respiration energy inasmuch as it was not altered by 0.1 millimolar KCN. The temperature dependence of Lp (T((1/2))) was measured for the first time in individual higher-plant cells. Lp increased by a factor of 2 to 4, when the temperature was increased by 10 C. The activation energy of water exchange was found to be between 50 and 186 kilojoules per mole. Within the large range of variation it was found that T((1/2)), Lp, and epsilon did not change under various experimental conditions (intact and excised tissue, water content and turgidity, age, etc.). Similar results were obtained for the epidermal cells of Tradescantia andersoniana. The measurements suggest that the entire epidermis would respond very rapidly (i.e. with a half-time of 1 to 30 s) to a demand for water from the stomata.

Journal Article↗
PubMed1980

Immobilisation and mechanical, support of individual protoplasts.

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl2. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca2+ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4 degrees C in the matrix.

Alginates↗
PubMed1980

Electro-mechanical properties of human erythrocyte membranes: the pressure-dependence of potassium permeability.

Electrical breakdown of cell membranes is interpreted in terms of an electro-mechanical model. It postulates for certain finite membrane areas that the actual membrane thickness depends on the voltage across the membrane and the applied pressure. The magnitude of the membrane compression depends both on the dielectric constant and the compressive, elastic modulus transverse to the membrane plane. The theory predicts the existence of a critical absolute hydrostatic pressure at which the intrinsic membrane potential is sufficiently high to induce "mechanical" breakdown of the membrane. The theoretically expected value for the critical pressure depends on the assumption made both for the pressure-dependence of the elastic modulus of the membrane and of the intrinsic membrane potential. It is shown that the critical pressure is expected at about 65 M Pa. The prediction of a critical pressure could be verified by subjecting human erythrocytes to high pressures (up to 100 M Pa) in a hyperbaric chamber. The net potassium efflux in dependence on pressure was used as an criterion for breakdown. Whereas the potassium net efflux was linearly dependent on pressure up to 60 M Pa, a significant increase in potassium permeability was observed towards higher pressure in agreement with the theory. The increase in the net potassium efflux above 60 M Pa was reversible, as indicated by measurements in which the same erythrocyte sample was subjected to several consecutive pressure pulses. Temperature changes in the erythrocyte suspension during compression and decompression were so small (less than 2 degrees C) that they could not account for the observed effects.

Erythrocyte Membrane↗
PubMed1980

Pulse-length dependence of the electrical breakdown in lipid bilayer membranes.

Charge-pulse experiments were performed on artificial lipid bilayer membranes with charging times in the range between 10 ns and 10 mus. If the membranes are charged to voltages in the order of 100 mV, the membrane voltage at the end of the charge pulse is a linear function of the injected charge. However, if the membranes are charged to voltages in the range of 1V, this relationship no longer holds and a reversible high conductance state occurs. This state is defined as an electrical breakdown and it does not allow the membranes to charge to higher voltages than the breakdown voltage, Vc. Between charging times of 300 ns and 5 mus at 25 degrees C and between 100 ns and 2 mus at 40 degrees C, Vc showed a strong dependence on the charging time of the membrane and decreased from 1.2 to 0.5 V (25 degrees C) and from 1 to 0.4 V (40 degrees C). For other charging times below and above these ranges, the breakdown voltage seemed to be constant. The results indicate that the breakdown phenomenon occurs in less than 10 ns. The pulse-length dependence of the breakdown voltage is consistent with the interpretation of the electrical breakdown mechanism in terms of the electromechanical model. However, it seems possible that below a charging time of the membrane of 300 ns (25 degrees C) and 100 ns (40 degrees C) other processes (such as the Born energy) become possible.

Cell Membrane↗
PubMed1980

Erythrocytes and lymphocytes as drug carrier systems: techniques for entrapment of drugs in living cells.

Mouse thymocytes and erythrocytes are loaded electrically with drugs in isotonic solution. The loaded cells are used for targeting the drugs to specific sites in the organism in order to achieve a controlled drug release in time and space. The field technique used for the loading of the cells is based on the dielectric breakdown of the cell membrane which is observed when cell suspensions are subjected to external field pulses of 2-20 kV/cm for short time intervals (ns to microseconds). When an apparent membrane potential of about 1 V is reached in response to the external field, the membrane breaks down reversibly. The breakdown of the membrane is associated with a remarkable and reversible permeability increase of the cell membrane. The increase in permeability depends on the strength and the duration of the field pulse.

Animals↗
PubMed1979

Reversible electrical breakdown of lipid bilayer membranes: a charge-pulse relaxation study.

Charge-pulse experiments were performed with lipid bilayer membranes from oxidized cholesterol/n-decane at relatively high voltages (several hundred mV). The membranes show an irreversible mechanical rupture if the membrane is charged to voltages on the order of 300 mV. In the case of the mechanical rupture, the voltage across the membrane needs about 50-200 musec to decay completely to zero. At much higher voltages, applied to the membrane by charge pulses of about 500 nsec duration, a decrease of the specific resistance of the membranes by nine orders of magnitude is observed (from 10(8) to 0.1 omega cm2), which is correlated with the reversible electrical breakdown of the lipid bilayer membrane. Due to the high conductance increase (breakdown) of the bilayer it is not possible to charge the membrane to a larger value than the critical potential difference Vc. For 1 M alkali ion chlorides Vc was about 1 V. The temperature dependence of the electrical breakdown voltage Vc is comparable to that being observed with cell membranes. Vc decreases between 2 and 48 degrees C from 1.5 to 0.6 V in the presence of 1 M KCl. Breakdown experiments were also performed with lipid bilayer membrane composed of other lipids. The fast decay of the voltage (current) in the 100-nsec range after application of a charge pulse was very similar in these experiments compared with experiments with membranes made from oxidized cholesterol. However, the membranes made from other lipids show a mechanical breakdown after the electrical breakdown, whereas with one single membrane from oxidized cholesterol more than twenty reproducible breakdown experiments could be repeated without a visible disturbance of the membrane stability. The reversible electrical breakdown of the membrane is discussed in terms of both compression of the membrane (electromechanical model) and ion movement through the membrane induced by high electric field strength (Born energy).

Cell Membrane↗
PubMed1979

Theoretical and experimental exclusion of errors in the determination of the elasticity and water transport parameters of plant cells by the pressure probe technique.

The volumetric elastic modulus of the cell wall and the hydraulic conductivity of the cell membranes were measured on ligatured compartments of different sizes of Chara corallina internodes using the pressure probe technique. The ratio between intact cell surface area and the area of puncture in the cell wall and membrane introduced by the microcapillary of the pressure probe was varied over a large range by inserting microcapillaries of widely varying diameters in different sized compartments. The relationship of the elastic modulus and the hydraulic conductivity to turgor pressure was independent of the ratio of intact cell surface area to the area of injury. The increase in the hydraulic conductivity below 2 bar turgor pressure and the volume dependence of the elastic modulus were shown to be the same as those observed in intact nonligatured cells. Theoretical considerations of the possible influence of injury of the cell wall and cell membrane around the inserted microcapillary on the measurement of the water transport and cell wall parameters do not explain the experimental findings. Thus, mechanical artifacts, if at all present, are too small to account for the observed dependence of the hydraulic conductivity and the elastic modulus on turgor pressure. The pressure probe technique thus represents an accurate method for measuring water transport parameters in both giant algal cells and in tissue cells of higher plants.

Journal Article↗
PubMed1978

Use of glyceraldehyde-3-phosphate dehydrogenase-depleted human erythrocyte ghosts as specific high affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from various tissues.

Human erythrocyte ghosts depleted of glyceraldehyde-3-phosphate dehydrogenase are used as specific high-affinity adsorbents for the purification of glyceraldehyde-3-phosphate dehydrogenase from mouse muscle, liver, kidney and brain. On incubation with the crude tissue homogenates, the depleted ghosts bind glyceraldehyde-3-phosphate dehydrogenase, aldolase, and a few other proteins. Washing the incubated ghosts several times with 5 mM phosphate buffer(pH 8.0) removed several of the non specifically bound proteins. Aldolase can be eliminated from the membrane by incubating the ghosts for 30 min in 5 mM phosphate buffer (pH 8.0)/2mM fructose 1,6-biphosphate, and then washing with the same solution. Glyceraldehyde-3-phosphate dehydrogenase can then be specifically eluted from the ghosts by incubating them with 2 mM NADH in 5mM phosphate buffer (pH 8.0). Although the enzyme from brain appears to bind less strongly to the ghosts it was possible, using this procedure, to purify glyceraldehyde-3-phosphate dehydrogenase from all the tissues investigated. The purified enzyme exhibits high specific activity and migrates as a single band (during SDS polyacrylamide gel electrophoresis) which corresponds to a protomer molecular weight of 37 000.

Animals↗