How suspension cultures of Catharanthus roseus respond to oxygen limitation: small-scale tests with applications to large-scale cultures

The effect of oxygen supply on the growth of suspension cultures of Catharanthus roseus in Erlenmeyer flasks was investigated. Below a critical oxygen supply rate the culture could not survive. By increasing the oxygen supply, a point is reached where the culture survives but no growth is possible. At higher oxygen supply rates there is a regime where both growth rate and the maximum biomass concentration increase with oxygen supply. Eventually there comes a point where no further increase in biomass is achieved, probably due to the depletion of the sugars; however, the growth rate continues to increase with oxygen supply until a maximum growth rate is obtained. The ratio of fresh to dry weight at maximum fresh weight increased with shaker table speed of rotation accompanied by a greater rate of sugar depletion.     

Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets

Conditions necessary for the isolation and culture of protoplasts from suspension cultures of sugar, fodder and garden beets were investigated. Good yields of protoplasts were obtained by treating cells with a mixture of cellulase, Macerozyme and Driselase enzymes. Nutritional requirements of beet protoplasts were found to be quite simple: protoplasts could be cultured in MS, B₅ or PGo based media with 0.4 M glucose with the optimum result being produced on KM8p medium. Plating efficiency (P.E) was genotype-dependent with the sugar beet giving better P.E. than the fodder or garden beets used, and higher values being achieved with the use of desalted Driselase for isolation followed by culture on KMBp medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP N⁶ benzylaminopurine - NAA naphthaleneacetic acid - P.E. plating efficiency - * University of Birmingham beet germplasm accession number     

A technique for bulk production of cytoplasts and miniprotoplasts from suspension culture-derived protoplasts

Protoplasts isolated from suspension cultures of atrazine resistant black nightshade (Solanum nigrum L.) a weed biotype, were enucleated by centrifugation through a stepwise mannitol/sucrose gradient. Two cytoplast, enucleated subprotoplast, bands were routinely formed: one, a minor band at the 6.4%/18.2% mannitol border containing highly vacuolate cytoplasts with 95%+ enucleation; secondly a major cytoplast band at the 18.2% mannitol/33% sucrose border containing 90%+ enucleated protoplasts in quantities up to 4 million per 50 ml gradient tube. Efficient production of cytoplasts depended on the subculture procedures used for the cell suspensions. Optimal cytoplast yield (44%) occurred for protoplasts isolated three days after subculture. The vigor of the donor suspension cultures as visually monitored had to be controlled in order to obtain consistently high enucleation percentages.Abbreviations CPW Cell and Protoplast Wash Solution - 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO Dimethylsulfoxide - FDA Fluorescein diacetate - MS Murashige and Skoog medium (1962) - UM Uchimiya and Murashige medium (1976)     

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High-performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl-sol-uble proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27-kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27-kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27-kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg .     

Meiotic variation in an intergenomic autopolyploid series. I. Chiasma frequency.

Variation in chiasma frequency has been studied in PMCs of diploids and C0 autotetraploids of seven Lathyrus species exhibiting a range of genome size (10.8-19.9 pg DNA/2C). Variation in chiasma frequency showed no relation to changes in genome size, either between species or between disomic sets within nuclei. Mean chiasma frequency of the tetraploids showed a 75% increase over that in the diploids. Half of this increase represents an additive effect of chromosome doubling. Total variance in chiasma frequency of autotetraploids increases by 80% over that in diploids, in line with the square of the multiplicative effect of chromosome doubling. At the diploid level, interspecific differences account for the major component of variance (63.1%). Phenotypic variation in chiasma frequency was apparent in all seven species but represented the smallest component of variance (2.8%). Chromosome doubling results in an eightfold increase in the absolute size of the phenotypic component of variance in chiasma frequency and a threefold increase in that of the cellular component. It has no effect on the absolute size of the interspecific component.     

Preparation of protoplasts from the green alga Enteromorpha intestinalis (L.) Link

Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase. The viability of purified protoplast fractions was assessed by staining and measurements of O₂ uptake and evolution.Abbreviations MES 2-(N-morpholino) ethanesulphonic acid - TES N-tris(hydoxymethyl) methyl-2 aminoethanesulphonic acid     

Peroxidases in Relation to Removal of Dormancy and Germination of Apple Embryos

The changes in germination, peroxidase activity and isoperoxidase spectrum have been studied in apple embryos at 5°C (stratification) and at 20°C in the presence or absence of seed coats. The embryo dormancy is progressively released at 5°C, but not at 20°C. The peroxidase activity in embryos covered with seed coats is very low at 5°C as well as at 20°C which corresponds to a restricted number of isoenzymes. In isolated embryos the peroxidase activity increases significantly. This is due to an increase in both the number and the activity of the isoperoxidases and it is more pronounced at 20°C than at 5°C. The obtained results suggest that the soluble peroxidases are not involved in the process of the release of embryo dormancy. The variations observed are attributed to the growth process following germination, which can occur even at low temperature.     

Dissociation of intracellular lysosomal rupture from the cell death caused by silica

The relationship between intracellular lysosomal rupture and cell death caused by silica was studied in P388d(1) macrophages. After 3 h of exposure to 150 μg silica in medium containing 1.8 mM Ca(2+), 60 percent of the cells were unable to exclude trypan blue. In the absence of extracellular Ca(2+), however, all of the cells remained viable. Phagocytosis of silica particles occurred to the same extent in the presence or absence of Ca(2+). The percentage of P388D(1) cells killed by silica depended on the dose and the concentration of Ca(2+) in the medium. Intracellular lyosomal rupture after exposure to silica was measured by acridine orange fluorescence or histochemical assay of horseradish peroxidase. With either assay, 60 percent of the cells exposed to 150 μg silica for 3 h in the presence of Ca(2+) showed intracellular lysosomal rupture, was not associated with measureable degradation of total DNA, RNA, protein, or phospholipids or accelerated turnover of exogenous horseradish peroxidase. Pretreatment with promethazine (20 μg/ml) protected 80 percent of P388D(1) macrophages against silica toxicity although lysosomal rupture occurred in 60-70 percent of the cells. Intracellular lysosomal rupture was prevented in 80 percent of the cells by pretreatment with indomethacin (5 x 10(-5)M), yet 40-50 percent of the cells died after 3 h of exposure to 150 μg silica in 1.8 mM extracellular Ca(2+). The calcium ionophore A23187 also caused intracellular lysosomal rupture in 90-98 percent of the cells treated for 1 h in either the presence or absence of extracellular Ca(2+). With the addition of 1.8 mM Ca(2+), 80 percent of the cells was killed after 3 h, whereas all of the cells remained viable in the absence of Ca(2+). These experiments suggest that intracellular lysosomal rupture is not causally related to the cell death cause by silica or {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. Cell death is dependent on extracellular Ca(2+) and may be mediated by an influx of these ions across the plasma membrane permeability barrier damaged directly by exposure to these toxins.     

A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.