In vitro testing of rat and porcine islets microencapsulated in barium alginate beads.
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Alginates↗
Biomedical subjects
U Zimmermann
Publications and source records attributed to U Zimmermann.
Selection of hybrid plants obtained by electrofusion of vacuolated x evacuolated plant protoplasts in hypo-osmolar solution.
Vacuolated and evacuolated tobacco mesophyll protoplasts were electrically fused in hypo-osmolar media by using an alternating field of modulated amplitude for alignment. The vacuolated fusion partner was isolated from Nicotiana tabaccum L. cv Xanthi and the evacuolated one from the streptomycin-resistant strain Nicotiana tabaccum L. cv Petit Havana SR1. The field and osmolarity conditions used ensured relatively high yields of heterologous fusion products despite the differences in density and size of the parental cells. After removal of the evacuolated, streptomycin-resistant fused and unfused protoplasts by flotation of vacuole-containing cells on iso-osmolar sucrose medium, the cybrids and hybrids were cultured in 25 microliters drops of agarose. During the first 5 weeks the non-fused Xanthi-protoplasts were used as a nurse culture. After addition of streptomycin to the growth media, cybrids and hybrids were successfully selected whereas fused and unfused vacuole-containing protoplasts died within 6 days. Only the streptomycin-resistant cybrids and hybrids developed into whole plants. On average a yield of 0.025% of streptomycin-resistant plants (referred to the total number of parental cells) was obtained. Polyacrylamide gel electrophoresis of leaf extracts of these plants showed that at least 50% of the streptomycin-resistant plants had a hybrid-esterase isoenzyme pattern. The protocol can be generalised by fusion of iodoacetamide-inactivated vacuolated protoplasts with meristematic (or evacuolized) protoplasts carrying no genetic marker. Use of evacolated protoplasts for electrofusion with vacuole-containing protoplasts therefore offers a way of overcoming the lack of suitable genetic markers for hybrid selection.
Electric Stimulation↗
Divalent cation-sensitive pores formed by natural and synthetic melittin and by Triton X-100.
Leakage of ions and low-molecular-weight metabolites from Lettre cells is induced by synthetic melittin, as effectively as by melittin isolated from bee venom; in each case leakage is inhibited by Ca2+, Zn2+ or H+. Inhibition of leakage by divalent cations is reversible in that Lettre cells incubated with melittin (or with Triton X-100) in the presence of inhibitory amounts of Zn2+, when freed of Zn2+ by EGTA or by centrifugation, begin to leak (in Zn2(+)-sensitive manner). Electrorotation of Lettre cells is altered by melittin, compatible with membrane permeabilization; melittin plus Zn2+ does not alter electrorotation until Zn2+ (and unbound melittin) are removed. Melittin or Triton X-100 added to calcein-loaded liposomes induces leakage of calcein; divalent cations inhibit. Energy transfer between liposome-associated melittin and 2-, 7- or 12-(9-anthroyloxy)stearate (AS) is maximal with 12-AS; addition of Zn2+ has little effect. Circular dichroism spectra of melittin plus liposomes are unaffected by Zn2+. These results show that the formation of divalent cation-sensitive pores is not dependent on the presence of endogenous membrane proteins and that the action of divalent cations is not by displacement of melittin (or Triton) from the lipid bilayer.
Animals↗
Influence of intratracheal application of fluorocarbon 72 and different lipid-mixtures on mechanical behavior of isolated immature pig lungs.
Substitution of surfactant in immature lungs has two functional targets: the reduction of the overall alveolar surface tension and the mechanical stabilization of the system of alveoli having different diameters. Indeed, the lowering of the surface tension facilitates the inflation of the lungs, but according to Laplace's law small and large alveoli are not in pressure equilibrium as long as the surface tension is equal in both small and large alveoli. In the present work, we tried to stabilize the lungs and to compare the effect of bolus surfactant substitution with the two-step substitution of fluorocarbons and surfactant. In all, 24 fetal immature lungs were used. For our experiments we used fluorocarbon 72 (FC-72) with a surface tension of 12 mN/m. In groups 1 and 2, a mixture of dipalmitoylphosphatidylcholine (DPPC): cholesterol 9:1 (molar ratio) or DPPC: phosphatidylglycerol (PG) 9:1 (molar ratio) was administered intratracheally as a bolus. In the case of groups 3 and 4, the immature lungs were rinsed first with FC-72. After removing the fluorocarbon, the lungs were artificially ventilated and the DPPC: cholesterol 9:1 (group 3) or DPPC:PG 9:1 mixture (group 4) was given in aerosol form. Static pressure-volume curves (p-v) of the mean values of the 6 lungs in each group were registered at the beginning (0 min) and after 20 and 40 min of artificial ventilation. Airway opening pressure, weight-specific end-inspiratory lung compliance, and phospholipid contents were investigated.(ABSTRACT TRUNCATED AT 250 WORDS)
1,2-Dipalmitoylphosphatidylcholine↗
Empirical evidence against the 'cycle time dependency' assumption.
The interpretation of the 'cardiac cycle time effect', also named 'time-dependent primary bradycardia' by the Laceys, who first observed it, has been controversial in psychophysiology. Unconfounded evidence for the dependence of a vagal effect of psychological stimuli on time of stimulation within the cardiac cycle has been missing to date. An experiment in which the subjects could not anticipate the occurrence of the stimuli (short tones of a specific frequency that had to be counted) was performed. The data reduction procedure secured unambiguous interpretation of the data with respect to time-dependency or no time-dependency. No indication of any kind of cycle time dependency of the vagal effect was found.
Acoustic Stimulation↗
Impact of the perfluorochemical FC43 on function of isolated islets: a preliminary report.
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Animals↗
Motile responses of vestibular hair cells following caloric, electrical or chemical stimuli.
Isolated, living vestibular hair cells (VHCs) from the guinea pig are capable of producing self-movements. Current injections and extracellular alternating current (a.c.) fields evoked mechanical responses of cell body or sensory hairs. Furthermore, superfusion in the presence of kainic acid or slow depolarizations by K-/gluconate evoked reversible slow motile responses of solitary vestibular sensory cells. If present in vivo, active VHC mechanics will influence the mechano-sensitive stereocilia and modulate stiffness and compliance of the receptor structure and its cupular or macular relationship. A tonic VHC motility might directly influence the displacement configuration in the cupula and macula organs and thus the micromechanical operating conditions of these sensory organs. Active mechanical events could contribute to adaptation processes (automatic gain control) and micromechanical non-linearities of stereociliary displacements. In addition, we demonstrate that direct caloric stimulation of isolated, living type I VHCs from guinea pig elicited mechanical responses of the sensory cells. This mechanism could contribute to the caloric induction of a nystagmus both under terrestrial and microgravitational conditions.
Animals↗
Parameters to enhance human hybridoma formation with hypoosmolar electrofusion.
Hypoosmolar conditions have permitted the development of electrofusion techniques capable of producing human hybridomas from as few as 10(5) B cells. A hybridoma formation efficiency of one hybrid for each 125 input B cells has been achieved with Epstein-Barr virus--activated B cells and mouse-human heteromyelomas. This is at least 100-fold higher in efficiency than with polyethylene glycol-induced cell fusion, as well as a 50- to 100-fold decrease in the required number of human B cells. The ability to fuse a small number of input B cells should lead to a greater success rate in immortalizing the rare antigen-specific B cells. The critical parameters include fusion voltages, the composition and number of wash steps used in cell preparation, the composition and duration of exposure to hypoosmolar fusion medium, fusion ratio, plating density, the use of growth medium without pH indicator, and the use of an irradiated human fibroblast feeder layer. By manipulating these parameters, a high hybrid yield can be achieved with different mouse-human heteromyelomas and Epstein-Barr virus-activated B cells.
B-Lymphocytes↗
Sources of reactive oxygen species during multiple organ removal, preservation, and liver transplantation.
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Adenine Nucleotides↗
Development of microfusion techniques to generate human hybridomas.
The rarity of antigen-specific B cells in peripheral blood and lymphoid tissues is a major limitation in the production of human monoclonal antibodies. This has led to a requirement for techniques capable of fusing small numbers of cells and achieving a higher hybridoma formation efficiency than currently is possible. The approach used in these studies to generate human hybridomas is based on the observation that under hypo-osmolar conditions electric field induced cell fusion or electrofusion is facilitated. Electrofusion parameters have been defined in strongly hypo-osmolar solutions which have resulted in a hybridoma formation efficiency greater than 5 X 10(-3) under optimal conditions. Furthermore, this has been accomplished with total input B cells of 1-2 X 10(5). This is a ten-fold reduction in the required number of input B cells and is associated with a hybridoma formation efficiency at least equal to that achieved with a higher input B cell number. An important factor in the development of this microfusion technique appears to be the duration of exposure to the hypo-osmolar solution by B cells to be immortalized. Other parameters which may affect hybridoma yield include the electrical field strength used for cell alignment and membrane breakdown, ratio of human B cells to fusion partner, washing procedure, post-fusion incubation time, and the elimination of toxic molecules.
Antibodies, Monoclonal↗
Efficient hybridization of mouse-human cell lines by means of hypo-osmolar electrofusion.
The fusion of a mouse-human heteromyeloma with a mouse hybridoma is used as a model to define parameters to generate human hybridomas. Electrofusion of these cells in 300 mosM and 75 mosM solutions showed that strong hypo-osmolar conditions resulted in a dramatic increase in the efficiency of hybridoma formation. In contrast to iso-osmolar electrofusion, a high hybrid yield could be obtained by injection of only a single field pulse. The field strength was adjusted in proportion to the increased size of the cells in hypo-osmolar solutions. Hypo-osmolar electrofusion allowed the generation of approximately 0.45% hybrids at a suspension density of 1.75 X 10(5) mouse-human cells/ml corresponding to an input number of 3.5 X 10(4) mouse-human cells. A further increase in the efficiency of hybridoma formation to about 0.6% was achieved by cell alignment in an alternating field of modulated field strength. Experiments in which the total cell number per fusion chamber was decreased at constant optimum suspension density showed that a further increase in the efficiency of hybridoma formation in hypo-osmolar solution was not possible because of the increasing influence of the heterogeneity of the cell lines with decreasing cell number. The results allow the conclusion that hypo-osmolar electrofusion is a potential tool to enhance successful immortalisation of human B lymphocytes.
Animals↗
Biphasic voltage relaxation pattern observed in cells of Eremosphaera viridis after injection of charge-pulses of short duration: detection of tip clogging of intracellular microelectrodes by charge-pulse technique.
Charge pulse experiments performed on the peat-bog alga Eremosphaera viridis revealed an unusual voltage relaxation behaviour. Injection of charge pulses of 1 microseconds duration resulted in an immediate charging of the membranes (time constant of the order of 40 ns). Nevertheless, the potential-measuring microelectrode recorded an exponential increase in membrane voltage with a time constant of about 1.3 ms. The maximum voltage value was recorded after about 3 ms, followed by an exponential decay with a time constant of about 9.6 ms. This biphasic time course was independent of the amplitude of the injected charge and of the location of the impaled microelectrodes in the vacuole. Centrifuged cells in which the chloroplasts and the other organelles were pelleted in one part of the cells showed the same electrical response. Electrical breakdown of the cell membranes resulted in the disappearance of the biphasic voltage response. In this case only the decaying relaxation process could be recorded with a time constant of 3 ms. After resealing of the membranes the original biphasic relaxation response was restored. Increasing concentrations of KCl in the bathing medium reduced both time constants almost correspondingly. The experimental findings were evaluated with an electrical equivalent circuit. Theoretical analysis with reference to the experimental data suggested that the delayed voltage response of the potential-recording electrode resulted from a membrane seal across the tip of this electrode. The resistance of this seal was calculated to be about 400 M omega. The specific resistances and capacitances of tonoplast and plasmalemma membranes were calculated from the decaying part of the biphasic relaxation curves. The average values were found to be 2.58 omega.m2 and 5 mF.m-2. The investigations reported here suggest that charge pulse experiments can be generally used for the detection of membrane and cytoplasmic material clogging of the tip of intracellular microelectrodes, a problem with which most electrophysiologists are faced when interpreting data obtained from impaled microelectrodes.
Cell Membrane↗
Identification of a periplasmic C-type cytochrome as electron donor to the plasma membrane-bound cytochrome oxidase of the cyanobacterium Nostoc Mac.
Photoautotrophically grown cyanobacterium Nostoc sp. strain Mac (PCC 8009) released up to about 10 nmol of a c-type cytochrome per ml packed cells after treatment with EDTA under conditions that left the plasma membrane absolutely intact as judged from the absence of cytosolic proteins in the supernatant. Spectra of the ascorbate reduced cytochrome revealed peaks at 553, 522 and 416 nm. The protein was purified to an A-553/A-275 ratio of 0.8. Midpoint potential (at pH 7), isoelectric point and apparent molecular weight of the cytochrome were +0.35 V, 8.6, and around 10,500, respectively. The cytochrome proved to be an excellent electron donor to the aa3-type cytochrome oxidase in both plasma and thylakoid membranes isolated and purified from Nostoc Mac. Chemoheterotrophic growth of the cells increased the level of periplasmic cytochrome c up to 10-fold and cytochrome oxidase activity of plasma membranes up to 90-fold. The periplasmic cytochrome also transferred electrons to photosystem I in illuminated thylakoid membranes. We conclude that cyanobacteria contain a periplasmic c-type cytochrome presumably identical to so-called cytochrome c6 or c-553 which has long been known as a photosynthetic (i.e. thylakoid-associated) redox protein in these organisms, and which is capable of donating electrons (from the periplasmic space) to the cytochrome oxidase in the plasma membrane and (from the thylakoid lumen) to both P700 and cytochrome oxidase in the thylakoid membrane.
Cell Membrane↗
Transmission of killer activity into laboratory and industrial strains of Saccharomyces cerevisiae by electroinjection.
The killer character was electrically introduced into protoplasts of three yeast strains. These were the killer-negative variant of the K1 killer strain Saccharomyces cerevisiae T 158 C (his-); the killer-sensitive laboratory strain S. cerevisiae AH 215 (leu-, his-); and the killer-sensitive industrial strain S. cerevisiae AS 4/H2 (rho-). The killer dsRNA used for electroinjection was isolated from the super-killer strain S. cerevisiae T 158 C. Optimum numbers of transformed cells were obtained after regeneration and selection in appropriate media if the protoplasts were exposed to three exponentially decaying field pulses of 18.2 kV/cm strength and 40 microseconds duration at 4 degrees C. In the case of the killer-negative variant of S. cerevisiae T 158 C the majority of the protoplasts were transformed, whereas in the case of the two other strains the yield of transformed clones was much less. This latter result is expected if the expression of the electroinjected dsRNA was diminished in these two strains. Gel electrophoresis of the dsRNA of the clones of the three strains supported the conclusion that the transformed clones exhibited killer activity. The transformed clones of all three species were stable.
Electrochemistry↗
Facilitated electrofusion of vacuolated x evacuolated oat mesophyll protoplasts in hypo-osmolar media after alignment with an alternating field of modulated strength.
Electrofusion of evacuolated and vacuolated oat leaf protoplasts is difficult because of the different size and density of these cells which results in separation of the two fusion partners during dielectrophoresis. The fusion yield of this cell system was considerably enhanced by electrofusion in hypo-osmolar media containing 0.4 M mannitol, 0.1 mM calcium acetate and 0.1% bovine serum albumin. This increase in yield was only achieved if the dielectrophoretically induced membrane contact between the two fusion partners was enhanced by an initial short 'burst' of higher field strength (500 V/cm, peak to peak, for 5 s followed by a reduction of to 90 V/cm, peak to peak, for 20 s, frequency 1 MHz). Due to the high field strength of the alternating field at the beginning of cell chain formation separation of fusion partners of different size and density was mainly avoided. Simultaneously, the short duration of this high field 'burst' avoided the generation of lethal effects in the cell membranes. The subsequent low field strength of the alternating field was sufficient to keep the aligned cells in position. Optimum fusion was induced by a single square pulse of 750 V/cm and 30 musec duration. The time required for rounding up of the heterologous fusion products decreased with decreasing osmolarity. Fusion resulted in a 5.7 +/- 1.2% yield of heterologous fusion products (compared to 0.7% using the conventional electrofusion protocol) as determined by flow cytometric assay. About 50% of the vacuolated oat protoplasts and 20-50% of the heterologous fusion products regenerated their cell walls within 5 days after hypo-osmolar treatment, but no cell divisions could be observed. Evacuolated oat protoplasts died after 2-3 days in culture without any detectable cell wall regeneration.
Edible Grain↗
Alterations in the electrical properties of T and B lymphocyte membranes induced by mitogenic stimulation. Activation monitored by electro-rotation of single cells.
Stimulation of either B or T lymphocytes using specific mitogens results in changes in the passive electrical properties of the cell surface. These effects can be related to growth and secretion. This was possible because the high resolution of the contra-field electro-rotation method, combined with the use of very low conductivity media, allowed accurate and analytically-derived values for the cell surface properties. Increases in effective CM (membrane capacity) and changes in apparent membrane conductivity (reflecting the additive effects of true membrane conductivity GM and surface conductance KS) were measured. After 72 h treatment with concanavalin A, thymocyte CM had increased from 0.76 muF/cm2 to 1.24-1.46 muF/cm2 (7.6 to 12.4-14.6 mF/m2). Allowing for the stimulation-induced size increase (cell radius increased from 2.8 to 4.4 micron) these data imply that the plasma membrane area per cell increases 5-fold during stimulation. Stimulation of B cells (by 3 days incubation with bacterial lipopolysaccharide) increased CM from 0.93 to 1.6-1.7 muF/cm2 (9.3 to 16-17 mF/m2). Incubation without mitogen gave no significant increase in CM or in radius. Control cells of different sizes showed no difference in membrane properties. The increases in effective CM are argued to reflect an increase in membrane ramification (microvilli, folding, etc.). The apparent membrane conductivity of T cells also increased during stimulation, from 5 to 21 mS/cm2 (50 to 210 S/m2). This increase is proportionately much greater than that in CM or in membrane area. It seems to be due to a real increase in GM, but a small increase in KS may also occur. The earliest changes in apparent membrane conductivity were evident between 3 and 5 h after stimulation, before the cells increased in size. This response parallels increases in transmembrane transport reported to follow mitogenic stimulation.
Animals↗
Free-flow electrophoresis under microgravity: evidence for enhanced resolution of cell separation.
A mixture of fixed rabbit, guinea pig and rat erythrocytes, suspended in a relatively conductive solution, was separated by means of continuous free flow electrophoresis (CFFE) under 1 g- and microgram- conditions using a specially designed electrophoretic module. Short duration microgram conditions were realized on board a sounding rocket. Due to the energy input and the associated thermal convection a separation of the three differently charged cell types in distinct peaks was not possible under 1 g-conditions as shown by reference experiments on the ground before launch. In contrast to the poor resolution under 1 g-conditions, clear separation of the cell mixture could be recorded after lift-off of the rocket under microgram-conditions. Repeated measurements demonstrated that the separation profile was completely stable during the entire microgram-phase of about 6 min. Since the CFFE experiment in space was an exact replica of the ground reference experiments, the results demonstrated unambiguously the potential of CFFE for cell separation under microgram-conditions in media of high ionic strength.
Animals↗
Mechanisms of electrostimulated uptake of macromolecules into living cells.
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Animals↗