Hybrid formation and metabolism of plant cell protoplasts under microgravity.
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Cell Fusion↗
Biomedical subjects
R Hampp
Publications and source records attributed to R Hampp.
A dot-blot assay for quantitation of nanogram amounts of protein in the presence of carrier ampholytes and other possibly interfering substances.
A method for protein determination in one- and two-dimensional electrophoresis sample buffer is presented. Accurate quantitation of protein in two-dimensional electrophoresis sample buffer (9.5 M urea, 2% Nonidet P-40, 2% carrier ampholytes, and 5% 2-mercaptoethanol) required removal of carrier ampholytes prior to the assay. This was made possible by taking advantage of the mutual solubility/insolubility of carrier ampholytes/proteins in saturated ammonium sulfate solution. In addition, improvement of protein determination in denaturing electrophoresis sample buffer containing the anionic detergent sodium dodecyl sulfate and the reducing agent 2-mercaptoethanol was achieved. The assay covers a range of sensitivity from 40 ng to 20 micrograms of protein. The procedure is applicable to large numbers of samples.
Amanita↗
Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts.
Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.
Cell Membrane Permeability↗
Appearance of photochemical function in prothylakoids during plastid development.
1. A method to separate the vesicles of prothylakoids from prolamellar body preparations obtained from etiolated and rapidly greening Avena laminae (0.25--4 h illumination ) is described. The prothylakoid preparations were found to be free from contaminating prolamellar bodies but enriched prolamellar body preparations (enriched prolamellar body preparations) still contained some adhering prothylakoid material. 2. Only existing beta-carotene appears to be transferred from the prolamellar bodies to the prothylakoids during early development and this ceases when freshly synthesized beta-carotene becomes available. 3. Prolamellar body structures proper show no positive association of existing or developing photochemical activities; these are only to be found in the developing prothylakoids. 4. Using methylviologen-linked electron transport-dependent oxygen consumption, Photosystem I activities may be detected with added diaminodurene within 15 min of illumination and within 30 min and 1 h with added tetramethylphenylenediamine and dichlorophenolindophenol, respectively. 5. During the 2nd, and 3rd. h of greening, proton-pumping capability and later ATP formation increased in prothylakoids in the presence of diaminodurene. 6. The first indications of Photosystem II activity using diphenylcarbazide as electron donor are shown at a similar time (2 h) with prothylakoids. The last photochemical activity to appear is the capacity to split water (3 h) and consequently the diphenylcarbazide activity diminished to zero before 8 h of illumination have passed. 7. The lack of effect of uncouplers such as NH4+ prior to 2 h suggests that in spite of some proton-pumping ability there is the possibility of proton-leaky areas existing within prothylakoids. This lack of a persistent proton gradient before 2 h of illumination may explain the different starting times of phenazine methosulfate- and diaminodurene-dependent photophosphorylation (0.25 and 2 h, respectively).
Carotenoids↗
Changes in plastid and mitochondrial envelopes during greening.
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Aminolevulinic Acid↗
Uptake of mevalonate and acetate during plastid development.
1. The envelopes of etioplasts and 1-2h etiochloroplasts are permeable to mevalonate, but plastids from etiolated tissue illuminated for longer than 4h show progressive impermeability towards mevalonate. 2. Acetate permeates the envelopes of 1-4h etiochloroplasts but does not significantly cross the envelopes of etioplasts or 8-24h etiochloroplasts. 3. A translocator system exists within the plastid envelopes for mevalonate which relies on malate as a counter-exchange anion.
Acetates↗
Effect of zinc and cadmium on delta-aminolevulinate dehydratase of red blood cells in protecting against enzyme losses during storage.
The effect of zinc and cadmium on delta-aminolevulinate dehydratase of bovine erythrocytes stored at -30 degrees C for different times was determined. The results show a. storage of erythrocytes leads to an enhancement of the enzyme activity, which after six weeks is 165% (500 muM ZnCl2) respectively 220% (100 muM CdCl2) for red blood cells of calves, and after four weeks in 420% respectively 450% (same concentrations) for red blood cells of adult cattle, b. the older the samples are, the higher is the metal concentrations, needed for activation.
Animals↗