Shoot differentiation, regeneration of cauliflower and analysis of somaclonal variation by RAPD

Our major goal in this study was to establish and characterize an efficient callus induction and shoot regeneration protocol for cauliflower (Brassica oleracea var. botrytis). The effects of induction medium, growth regulator combination, organic component, AgNO(3), genotype and explants type on shoot differentiation on the cauliflower regeneration process were taken in account and hereby evaluated. The optimal media for shoot differentiation and rooting were modified MS medium (MS with PG-96 organic components) supplemented with NAA at 0.5 mg l(-1), TDZ at 0.25 mg l(-1), BA at 3.0 mg l(-1), AgNO(3) at 2.0 mg l(-1) and MS supplemented with IBA at 0.4 mg l(-1), respectively. Among the four varieties tested, Saixue gave the best shoot differentiation response (average over 18 shoots per explant) from the upper section of the hypocotyls. We have so far obtained over 500 regenerated plants under this improved protocol. We have further analyzed the somaclonal variation of regenerated plants at the DNA level by using the RAPD molecular markers. By PCR amplification, we were able to amplify 75 scoreable bands from 15 primers out of 40 arbitrary primers screened, where 35 of them were monomorphic and 40 polymorphic bands (53.3%) in four varieties studied. The absence of polymorphism among regenerated plants from the same variety indicated the conformity of the regeneration protocol.     

Multivariate analysis of protein profiles of metal hyperaccumulator Thlaspi caerulescens accessions

Thlaspi caerulescens is increasingly acknowledged as one of the best models for studying metal hyperaccumulation in plants. In order to study the mechanisms underlying metal hyperaccumulation, we used proteomic profiling to identify differences in protein intensities among three T. caerulescens accessions with pronounced differences in tolerance, uptake and root to shoot translocation of Zn and Cd. Proteins were separated using two-dimensional electrophoresis and stained with SYPRO Orange. Intensity values and quality scores were obtained for each spot by using PDQuest software. Principal component analysis was used to test the separation of the protein profiles of the three plant accessions at various metal exposures, and to detect groups of proteins responsible for the differences. Spot sets representing individual proteins were analysed with the analysis of variance and non-parametric Kruskal-Wallis test. Clearest differences were seen among the Thlaspi accessions, while the effects of metal exposures were less pronounced. The 48 tentatively identified spots represent core metabolic functions (e.g. photosynthesis, nitrogen assimilation, carbohydrate metabolism) as well as putative signalling and regulatory functions. The possible roles of some of the proteins in heavy metal accumulation and tolerance are discussed.     

Effects of Management on Economic Profitability of Forest Biomass Production and Carbon Neutrality of Bioenergy Use in Norway Spruce Stands Under the Changing Climate

We analyzed the effects of management on the economic profitability of forest biomass production and carbon neutrality of bioenergy use in Norway spruce (Picea abies L. Karst) stands under the changing climate. We employed a forest ecosystem model and life cycle assessment tool. In particular, we studied the effects of thinning, nitrogen fertilization, and rotation length on: (1) the production of timber and energy biomass, and its economic profitability (net present value), (2) carbon stock in the forest ecosystem and carbon balance in forestry, and (3) carbon dioxide (CO₂) emissions from the use of biomass in energy production. Results showed that the current Finnish baseline management with and without nitrogen fertilization resulted in the highest mean annual timber production and net present value (NPV) for long rotations (60 to 80 years), regardless of climate scenario. Mean annual production of energy biomass was enhanced by increasing stocking by 20–30 % compared to the baseline management, and/or use of nitrogen fertilization. Such management gave lower CO₂ emissions per unit of energy compared to the baseline management, as the carbon stock in the forest ecosystem and the carbon balance in forestry increased. Overall, the carbon neutrality and net present value were, on average, the highest in the baseline management or with a 20 % increase in stocking, with nitrogen fertilization and 60- to 80-year rotation lengths, regardless of the climate applied. However, it was not possible to simultaneously maximize the NPV of forest biomass production and the carbon neutrality of bioenergy use.     

Effects of climate change and management on net climate impacts of production and utilization of energy biomass in Norway spruce with stable age‐class distribution

We studied the effects of climate change and forest management scenarios on net climate impacts (radiative forcing) of production and utilization of energy biomass, in a Norway spruce forest area over an 80‐year simulation period in Finnish boreal conditions. A stable age‐class distribution was used in model‐based analyses to identify purely the management effects under the current and changing climate (SRES B1 and A2 scenarios). The radiative forcing was calculated based on an integrated use of forest ecosystem model simulations and a life cycle assessment (LCA) tool. In this work, forest‐based energy was used to substitute coal, and current forest management (baseline management) was used as a reference management. In alternative management scenarios, the stocking was maintained 20% higher in thinning compared to the baseline management, and nitrogen fertilization was applied. Intensity of energy biomass harvest (e.g. logging residues, coarse roots and stumps) was varied in the final felling of the stands at the age of 80 years. Also, the economic profitability (NPV, 3% interest rate) of integrated production of timber and energy biomass was calculated for each management scenario. Our results showed that compared to the baseline management, climate benefits could be increased by maintaining higher stocking in thinning over rotation, using nitrogen fertilization and harvesting logging residues, stumps and coarse roots in the final felling. Under the gradually changing climate (in both SRES B1 and A2), the climate benefits were lower compared to the current climate. Trade‐offs between NPV and net climate impacts also existed.     

Maximum growing depth of submerged macrophytes in European lakes

Submerged macrophytes are important elements for the structure and functioning of lake ecosystems. In this study, we used chemical and maximum colonisation depth (C_max) data from 12 European countries in order to investigate how suitable C_max may describe the impact by eutrophication. The analyses include data from 757 lakes and 919 lake years covering oligotrophic to eutrophic lakes. Overall, C_max was closely related to Secchi depth (R ² = 0.58) and less closely to chlorophyll a (R ² = 0.31), TP (R ² = 0.31) and total nitrogen, TN (R ² = 0.24). The low coefficients of determination between C_max and nutrient concentrations suggest that other response factors than nutrient-phytoplankton-light conditions are important for C_max and that it will be difficult to establish strong relationships between external nutrient loading and C_max. Yearly monitoring for 13–16 years in eight Danish lakes showed considerable year-to-year variability in C_max, which for the individual lakes only related weakly to changes in Secchi depth. The use of C_max as an eutrophication indicator is especially relevant in not very shallow lakes (maximum depth >4–5 m), not too turbid lakes (C_max >1 m) and not very humic lakes (colour <60 mg Pt/l).     

Structure of a Microbial Community in Soil after Prolonged Addition of Low Levels of Simulated Acid Rain

Humus samples were collected 12 growing seasons after the start of a simulated acid rain experiment situated in the subarctic environment. The acid rain was simulated with H₂SO₄, a combination of H₂SO₄ and HNO₃, and HNO₃ at two levels of moderate acidic loads close to the natural anthropogenic pollution levels of southern Scandinavia. The higher levels of acid applications resulted in acidification, as defined by humus chemistry. The concentrations of base cations decreased, while the concentrations of exchangeable H⁺, Al, and Fe increased. Humus pH decreased from 3.83 to 3.65. Basal respiration decreased with decreasing humus pH, and total microbial biomass, measured by substrate-induced respiration and total amount of phospholipid fatty acids (PLFA), decreased slightly. An altered PLFA pattern indicated a change in the microbial community structure at the higher levels of acid applications. In general, branched fatty acids, typical of gram-positive bacteria, increased in the acid plots. PLFA analysis performed on the bacterial community growing on agar plates also showed that the relative amount of PLFA specific for gram-positive bacteria increased due to the acidification. The changed bacterial community was adapted to the more acidic environment in the acid-treated plots, even though bacterial growth rates, estimated by thymidine and leucine incorporation, decreased with pH. Fungal activity (measured as acetate incorporation into ergosterol) was not affected. This result indicates that bacteria were more affected than fungi by the acidification. The capacity of the bacterial community to utilize 95 different carbon sources was variable and only showed weak correlations to pH. Differences in the toxicities of H₂SO₄ and HNO₃ for the microbial community were not found.     

Probiotic Strains and Their Combination Inhibit <Emphasis Type="Italic">In Vitro</Emphasis> Adhesion of Pathogens to Pig Intestinal Mucosa

The aim of this study was to investigate in vitro the protective effect of commercial probiotic strains (Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus LGG) alone and in combination on the adhesion of pathogenic strains as Salmonella, Clostridium, and Escherichia coli to pig intestinal mucus obtained from different intestinal regions. In combination, probiotic strains enhanced each other’s adhesion, mainly in large intestinal mucus. Treatment of intestinal mucus with Bb12 and LGG, alone or in combination, significantly reduced (P < 0.05) the adhesion of the tested pathogens. The ability to inhibit pathogen adhesion appears to depend on the specific probiotics and pathogens and on the mucosal site. B. lactis Bb12 and L. rhamnosus LGG in combination revealed a better ability to inhibit adhesion of all pathogens tested to pig intestinal mucus than probiotic strains. Probiotic combinations could be useful for counteracting disease-associated aberrations in intestinal microbiota. Specific protective probiotics could be selected for particular pig pathogens. Probiotic strains from human origin and intended for human use also adhere to pig intestinal mucus and are able to displace and inhibit pathogens.     

Anion inhibition studies of the α-carbonic anhydrase from the protozoan pathogen Trypanosoma cruzi,the causative agent of Chagas disease

The protozoan pathogen Trypanosoma cruzi, the causative agent of Chagas disease, encodes an α-class carbonic anhydrase (CA, EC 4.2.1.1), TcCA, which was recently shown to be crucial for its life cycle. Thiols, a class of strong TcCA inhibitors, were also shown to block the growth of the pathogen in vitro. Here we report the inhibition of TcCA by inorganic and complex anions and other molecules interacting with zinc proteins, such as sulfamide, sulfamic acid, phenylboronic/arsonic acids. TcCA was inhibited in the low micromolar range by iodide, cyanate, thiocyanate, hydrogensulfide and trithiocarbonate (K_Is in the range of 44–93 μM), but the best inhibitor was diethyldithiocarbamate (K_I = 5 μM). Sulfamide showed an inhibition constant of 120 μM, but sulfamic acid was much less effective (K_I of 10.6 mM). The discovery of diethyldithiocarbamate as a low micromolar TcCA inhibitor may be useful to detect leads for developing anti-Trypanosoma agents with a diverse mechanism of action compared to clinically used drugs (benznidazole, nifurtimox) for which significant resistance emerged.     

Brood size manipulations within the natural range did not reveal intragenerational cost of reproduction in the Willow Tit Parus montanus

To test for the existence of a reproductive cost, we manipulated brood sizes (-2 and +2 nestlings) over 6 years in a northern population of Willow Tits Parus montanus breeding in natural holes. Possible effects were sought in subsequent survival and fecundity of the parents. Parents given extra chicks made more feeding visits than did parents with reduced and control broods. However, this was not reflected in differences in parental body-weight between groups at the end of the nestling period. Brood size manipulation did not significantly affect female or male survival. In 4 out of 6 years, there was a weak and nonsignificant effect on male survival, consistent with a cost of reproduction. Female and male fecundity in the year following the experiment was not affected by the manipulations. Thus, the data do not give evidence of an intragenerational cost of reproduction in the Willow Tit. Parents appeared unwilling to increase their breeding effort to a level which jeopardized their own survival or future breeding success. It is possible that, because of the time constraints in northern latitudes, females work under their capacity and lay smaller clutches than would otherwise be most profitable. Thus, no costs to the parents would be expected as a consequence of manipulations. These results suggest that the current reproductive rate is not maintained by reproductive cost in the Willow Tit. However, the results do not rule out the possibility that selection has operated outside the current range of reproductive rates during evolutionary history of the species.     

An anion and small molecule inhibition study of the β-carbonic anhydrase from Staphylococcus aureus

Pathogenic bacteria resistant to most antibiotics, including the methicillin-resistant Staphylococcus aureus (MRSA) represent a serious medical problem. The search for new antiinfectives, possessing a diverse mechanism of action compared to the clinically used antibiotics, has become an attractive research field. S. aureus DNA encodes a β-class carbonic anhydrase, SauBCA. It is a druggable target that can be inhibited by certain aromatic and heterocyclic sulphonamides. Here we investigated inorganic anions and some other small molecules for their inhibition of SauBCA. The halides, nitrite, nitrate, bicarbonate, carbonate, bisulphite, sulphate, stannate, and N,N-diethyldithiocarbamate were submillimolar SauBCA inhibitors with K_Is in the range of 0.26 − 0.91 mM. The most effective inhibitors were sulfamide, sulfamate, phenylboronic acid, and phenylarsonic acid with K_Is of 7 − 43 µM. Several interesting inhibitors detected here may be considered lead compounds for the development of even more effective derivatives, which should be investigated for their bacteriostatic effects.