Ultracytochemical localization of dihydroorotate dehydrogenase in mitochondria and vacuoles ofSaccharomyces cerevisiae

The coenzyme-independent dihydroorotate dehydrogenase (EC 1.3.3.1) linking the pyrimidine biosynthetic pathway to the respiratory chain, was ultracytochemically localized by the tetrazolium method in derepressed exponential-phase cultures ofSaccharomyces cerevisiae. Biochemical analysis showed a considerable variation of this enzyme activity in inverse proportion to the aeration of the yeast cultures. The assay also showed that after prefixation of yeast cells with 1% glutaraldehyde at 0°C for 20 min, approximately one-half of the enzyme activity was preserved. The cytochemical reaction mixture contained dihydroorotate (2 mmol/L), thiocarbamyl nitroblue tetrazolium (0.44 mmol/L), phenazine methosulfate (0.16 mmol/L) and KCN (1.7 mmol/L) in Tris-HCl buffer (100 mmol/L) of pH 8.0. The osmicated formazan deposits featured envelopes of mitochondria and of nuclei and were prominent in the mitochondrial inclusions and in the vacuolar membranes. The latter sites of dihydroorotate dehydrogenase activity represent biosynthetic activity in yeast vacuoles, still generally assumed to function as yeast lysosomes and storage organelles. In the light of the generally observed invasions of juvenile yeast vacuoles into mitochondria, the enzymic sites observed in mitochondrial inclusion were considered as evidence of the interactions of yeast vacuoles and mitochondria. Transfer of vacuolar membranes with dihydroorotate dehydrogenase activity into mitochondrial matrix is suggested.     

Ontogenetic change in effectiveness of chemical defence against different predators in Oxycarenus true bugs

Many prey species change their antipredator defence during ontogeny, which may be connected to different potential predators over the life cycle of the prey. To test this hypothesis, we compared reactions of two predator taxa – spiders and birds – to larvae and adults of two invasive true bug species, Oxycarenus hyalinipennis and Oxycarenus lavaterae (Heteroptera: Oxycarenidae) with life-stage-specific chemical defence mechanisms. The reactions to larvae and adults of both true bug species strikingly differed between the two predator taxa. The spiders were deterred by the defences of adult bugs, but the larval defences were ineffective against them. By contrast, birds attacked the larvae considerably less often than the adult bugs. The results indicate a predator-specific ontogenetic change in defence effectiveness of both Oxycarenus species. The change in defence is likely linked to the life-stage-specific composition of secretions in both species: whereas secretions of larvae are dominated by unsaturated aldehydes, secretions of adults are rich in terpenoids, which probably serve dual function of defensive chemicals and pheromones. Our results highlight the variation in defence between different life stages and the importance of testing responses of different types of predators.     

Glucoamylases encoded by variantSaccharomycopsis fibuligera genes: Structure and properties

The genes of two variant glucoamylases (GLA1 and GLU1) ofSaccharomycopsis fibuligera were expressed inSaccharomyces cerevisiae, and biochemical properties of the secreted enzymes were compared. It was found that three amino acid alterations in the signal peptide and N-terminal regions of the variants had no effect on the levels of the secreted enzymes. Amino acid alterations in the C-terminal region of the mature proteins influenced their specific activity, substrate specificity, as well as temperature and pH optima. Because of the glycosylation heterogeneity, the glucoamylases of each gene variant were isolated and purified in two forms (A and B), which were essentially similar in catalytic and physicochemical properties but differed in their thermal stability and ability to renaturate after thermal denaturation.     

Experimental evidence of size-selective harvest and environmental stochasticity effects on population demography,fluctuations and non-linearity

Theory and analyses of fisheries data sets indicate that harvesting can alter population structure and destabilise non-linear processes, which increases population fluctuations. We conducted a factorial experiment on the population dynamics of Daphnia magna in relation to size-selective harvesting and stochasticity of food supply. Harvesting and stochasticity treatments both increased population fluctuations. Timeseries analysis indicated that fluctuations in control populations were non-linear, and non-linearity increased substantially in response to harvesting. Both harvesting and stochasticity induced population juvenescence, but harvesting did so via the depletion of adults, whereas stochasticity increased the abundance of juveniles. A fitted fisheries model indicated that harvesting shifted populations towards higher reproductive rates and larger-magnitude damped oscillations that amplify demographic noise. These findings provide experimental evidence that harvesting increases the non-linearity of population fluctuations and that both harvesting and stochasticity increase population variability and juvenescence.     

Novel Miscanthus hybrids: Modelling productivity on marginal land in Europe using dynamics of canopy development determined by light interception

New biomass crop hybrids for bioeconomic expansion require yield projections to determine their potential for strategic land use planning in the face of global challenges. Our biomass growth simulation incorporates radiation interception and conversion efficiency. Models often use leaf area to predict interception which is demanding to determine accurately, so instead we use low-cost rapid light interception measurements using a simple laboratory-made line ceptometer and relate the dynamics of canopy closure to thermal time, and to measurements of biomass. We apply the model to project the European biomass potentials of new market-ready hybrids for 2020–2030. Field measurements are easier to collect, the calibration is seasonally dynamic and reduces influence of weather variation between field sites. The model obtained is conservative, being calibrated by crops of varying establishment and varying maturity on less productive (marginal) land. This results in conservative projections of miscanthus hybrids for 2020–2030 based on 10% land use conversion of the least (productive) grassland and arable for farm diversification, which show a European potential of 80.7–89.7 Mt year⁻¹ biomass, with potential for 1.2–1.3 EJ year⁻¹ energy and 36.3–40.3 Mt year⁻¹ carbon capture, with seeded Miscanthus sacchariflorus × sinensis displaying highest yield potential. Simulated biomass projections must be viewed in light of the field measurements on less productive land with high soil water deficits. We are attempting to model the results from an ambitious and novel project combining new hybrids across Europe with agronomy which has not been perfected on less productive sites. Nevertheless, at the time of energy sourcing issues, seed-propagated miscanthus hybrids for the upscaled provision of bioenergy offer an alternative source of renewable energy. If European countries provide incentives for growers to invest, seeded hybrids can improve product availability and biomass yields over the current commercial miscanthus variety.     

Cultivation of lymphatic cells in protein-free medium and chemical study of the products

Rabbits immunized withBrucella suis within a period of one week formed antibodies in high titres. These antibodies were of a macroglobulin character only. Cells from the spleen and popliteal lymph nodes were cultivatedin vitro in protein-free medium, in which the only macromolecular substance was dextran or carbowax. Physico-chemical analysis of secretion products was not succesful, since under the given cultivation conditions the cells started to disintegrate from the outset and the medium contained nucleic acids as well as proteins. Some of these substances were present in particles of the size of different subcellular particles. After cultivation of spleen and lymph node cells, antibodies were detected by an agglutination test in media containing dextran and carbowax. When particles substantially larger than the antibody molecule were removed by ultracentrifugation, the agglutination antibody titre in the medium fell.     

Antibody binding capacity of different peptide chains isolated from digested and purified horse diphtheria antitoxin

In commercial digested and purified horse diphtheria antitoxin, which is formed largely of the gamma globulin fragment with the sedimentation coefficient 5.3 S, the reactive disulphide bonds were destroyed by S-sulphonation. Gel filtration on Sephadex G-100 in 0.05 M formic acid with 6 M urea showed that the molecule of the S-sulphonated preparation dissociated into chains similar in character to the peptide chains of native horse antitoxins. Antibody activity was still partly maintained even after treatment with 6 M urea. On mixing the two types of chains isolated by gel filtration, antibody activity was recovered, the amount of antibody protein determined in the mixed fractions being greater than the sum of the amounts in the separate fractions. The neutralizing activity of the mixed fractions tested against toxinin vivo was also greater than the sum of the activity of the separate fractions.     

Rapid reorganization of microtubular cytoskeleton accompanies early changes in nuclear ploidy and chromatin structure in postmitotic cells of barley leaves infected with powdery mildew

Summary Post-mitotic epidermal cells of barley leaves were found to contain, in addition to cortical microtubules (CMTs), distinct arrays of endoplasmic microtubules (EMTs). These encircle nuclei and continuously merge into the CMT arrays that underly the plasmalemma. Detailed three-dimensional reconstruction of both types of MTs during fungal infection showed that profound and very rapid MT rearrangements occurred especially in the case of incompatible (resistant) barley-powdery mildew genotype combination. The most early MT responses, followed by their subsequent complete disintegration, were recorded around nuclei. These events might be relevant for the induction of such nuclear processes as onset of DNA synthesis and nuclear chromatin condensation. Observed pattern of early infection events, as well as less prominent responses in the case of compatible (susceptible) barley-powdery mildew genotype combination, both findings suggest that rapid reorganization of the MT cytoskeleton could be involved in recognition of the fungus by host cells and in the initiation of resistance responses in barley leaves. We hypothesize that the integrity and dynamics of the MT cytoskeleton, especially of its perinuclear part, might participate in control mechanisms involved in activation of resistance genes.Abbreviations CMTs cortical microtubules - EMTs endoplasmic microtubules - MT microtubules - PI propidium iodide - SC sensitive combination - RC resistant combination     

Genetic transformation of alfalfa somatic embryos and their clonal propagation through repetitive somatic embryogenesis

Genetically transformed alfalfa (Medicago sativa L., cv. Zajearska 83) plantlets were obtained by inoculating somatic embryos with Agrobacterium tumefaciens strains A281/pGA472 and LBA4404/pBI121. Single somatic embryos, 5–7 mm long, were released from a repetitively embryogenic culture, wounded, and cocultivated with the bacteria. The agar-solidified culture medium contained mineral salts, vitamins, 40 g l^–1 sucrose, 1 g l^–1 yeast extract and 0.05 mg l^–1 BA. Five clones, transformed with A281/pGA472, and 4 clones transformed with LBA4404/pBI121, were selected for proliferation by repetitive somatic embryogenesis, on media containing 100 mg l^–1 of kanamycin. The transformation of kanamycin-resistant clones was confirmed by assaying the activity of neomycin phosphotransferase II and/or -glucuronidase enzymes, and by the Southern blot analysis. It is suggested that the transformation/regeneration system based on somatic embryogenesis may be suitable for establishing transgenic alfalfa lines. The relatively low frequency of embryo transformation is compensated for by abundant proliferation in secondary somatic embryogenesis.Abbreviations BA 6-benzyladenine - GUS -glucuronidase - Km kanamycin - NPTII neomycin phosphotransferase II - X-gluc 5-bromo-4-chloro-3-indolyl--glucuronic acid - BM basal medium