Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Aggregative properties of different cell types of the fresh-water spongeEphydatia fluviatilis isolated on ficoll gradients

Summary Cell suspensions of the fresh-watersponge Ephydatia fluviatilis have been fractionated by means ofFicoll gradient centrifugation. Three fractions were isolated. The densest contains archeocyte-like cells only; the intermediate fraction is very rich in choanocytes, and the lightest is a mixture of cell types. Earch fraction shows specificaggregative properties and potentialities to reconstitute functional sponges.It appears that the sequence of reconstitution events can be selectively altered by certain disequilibria in the cell populationThese preliminary results constitute a first approach to the analysis ofcell type specificity in sponges.     

Intact chloroplast electron flow. Effects of ribose 5-phosphate

Additions of ribose 5-phosphate to intact spinach chloroplasts were used to probe the effects of ADP regeneration on pH-gradient formation and electron-transfer reactions. In weakly illuminated chloroplasts, the ATP/ADP ratio dropped by 64% and the transthylakoid pH gradient decreased by a minimum of 0.2 units in response to ribose 5-phosphate. Nitrite reduction increased 2-fold while, under conditions of cyclic electron flow, the half-time for cytochrome f reduction decreased by a factor of two from 4.1 to 1.9 ms. The results suggest that metabolic ATP consumption, during the conversion of ribulose 5-phosphate to ribulose 1,5-bisphosphate, enhances electron transfer between plastohydroquinone and cytochrome f through decreases in the transthylakoid pH gradient caused by phosphorylation of ADP.     

The isolation of Saccharomyces cerevisiae nuclear membranes with nuclease and high-salt treatment

Saccharomyces cerevisiae nuclear membranes were prepared from isolated nuclei by digesting chromatin with deoxyribonuclease and ribonuclease, washing of residual nuclei with 0.5 M MgCl₂, and discontinuous gradient centrifugation in buffered Ficoll solutions. Electron microscopic examination of the preparations showed single membrane and double membrane vesicles and membrane sheets. Pores or residual pores were often visible. In double membrane profiles the two unit membranes were often separated by the remains of the perinuclear cistern. The nuclear membrane fragments contained 58% protein, 23.8% phospholipid, 6% sterols, 7.1% neutral acylglycerols, 4.8% RNA, and 0.3% DNA. The phospholipid content of the membrane preparations was influenced by a phospholipase activity with acidic pH optimum.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

The performance and distribution of species along soil salinity gradients of mangrove swamps in southeastern Nigeria

Based on periodic soil salinity measurements direct gradient analysis procedures were used to relate the performance and distribution of mangrove swamp species to salinity gradients. Variations in soil salinity were due to distance from the coast, tidal incursions and freshwater inputs. All overstorey species showed statistically significant negative correlations with salinity while most groundlayer species were positively correlated with the same factor. Based on ecological group classification, no species was found to occur on the highest values of soil salinity. There were overlapping range of occurrences and ecological optima for most species along the gradients.     

Estimating predation risk in zooplankton communities: the importance of vertical overlap

In order to estimate predation risk in nature, two basic components of predation need to be quantified: prey vulnerability, and density risk. Prey vulnerability can be estimated from clearance rates obtained from enclosure experiments with and without predators. Density risk is a function of predator density, and the spatial and temporal overlap of the predator and prey populations. In the current study we examine the importance of the vertical component of overlap in making accurate estimates of predation risk from the invertebrate predator Mesocyclops edax on rotifer versus crustacean prey. The results indicate that assumptions of uniform predator and prey densities cause a significant underestimation of predation risk for many crustacean prey due to the coincident vertical migration of these prey with the predator. The assumption of uniformity is more reasonable for estimating predation risk for most rotifer prey.     

Excretion of total phosphorus,dissolved reactive phosphorus,ammonia, and urea by Lake MichiganMysis relicta

Rates of nutrient release byMysis relicta in Lake Michigan were measured on five nights at a 45-m station near Milwaukee, Wisconsin, U.S.A., in the summer of 1977. Nocturnal vertical migrations of the mysids were monitored with both echosounder tracings and vertical net tows. Estimates of the total areal dry mass of the mysids ranged from 600 to 1 820 mg m–2. Rates of release of dissolved reactive phosphorus, total phosphorus, ammonia, and urea were measured in dark incubations on shipboard. Excretion experiments were initiated immediately after mysids were collected from each of several vertical net hauls. The depths of maximum mysid densities corresponded approximately with a deep phytoplankton peak located in the vicinity of the thermocline. Semiquantitative demands for N and P by phytoplankton within this peak were obtained from¹⁴C estimates of primary production from a previous study, assuming a constant C:N:P ratio for the algae. These algal nutrient demands were compared to potential N and P release by the mysids to obtain a first approximation of the relative rates of nutrient supply and demand for the field phytoplankton populations. Our analysis indicates that mysids may directly supply about 1–10% of the daily N and P demands of the phytoplankton in the deep peak. However, indirect interactions betweenMysis relicta and other organisms, such as small zooplankton and fishes, could be major factors in nutrient recycling within the metalimnion and subthermocline region of Lake Michigan.Contribution No. 238, Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Wisconsin.Contribution No. 238, Center for Great Lakes Studies, University of Wisconsin-Milwaukee, Wisconsin.     

In vitro maintenance of differentiation marker synthesis by subpopulations of mouse thymocytes

Mouse thymocyte populations enriched in functionally incompetent, “immature” cells on the one hand, or in competent “mature” cells on the other hand, express different steady-state levels of certain surface antigens and marker enzymes. In the cases of the glycoproteins H-2 (K and D), Qa, and TL, and the DNA polymerase terminal deoxynucleotidyl transferase (TdT), these levels reflect different rates of de novo synthesis in the two populations. Thus each population appears to manifest a characteristic pattern of synthetic rates for the various products relative to total protein synthesis. To investigate the maintenance of these patterns, enriched pools of “immature” and “mature” thymocytes were incubated in vitro for 24 h, and the rates of product synthesis before and after culture were compared. H-2 synthesis, initially most rapid in the mature cells, continued to be made at the highest rate in this population. TdT synthesis, a characteristic activity of the immature cells, was not induced in the mature cells, but proceeded at an increased relative rate in the immature population. Therefore, the differences between the rates of H-2 and TdT synthesis were stable properties of the two thymocyte populations. Another marker of immature cells, TL, did not continue to be produced in parallel with TdT. Rather, its synthesis was selectively curtailed in relation to the continuing protein synthesis in the immature cultures. This non-coordinate regulation of TL and TdT production in immature thymocytes may be due to several mechanisms. These are discussed with regard to their implications for pathways of thymocyte maturation.