Screening of microbes for novel acidic cutinases and cloning and expression of an acidic cutinase from Aspergillus niger CBS 513.88

Isolates from gardening waste compost and 38 culture collection microbes were grown on agar plates at pH 4.0 with the cutinase model substrate polycaprolactone as a carbon source. The strains showing polycaprolactone hydrolysis were cultivated in liquid at acidic pH and the cultivations were monitored by assaying the p-nitrophenyl butyrate esterase activities. Culture supernatants of four strains were analyzed for the hydrolysis of tritiated apple cutin at different pHs. Highest amounts of radioactive hydrolysis products were detected at pHs below 5. The hydrolysis of apple cutin by the culture supernatants at acidic pH was further confirmed by GC–MS analysis of the hydrolysis products. On the basis of screening, the acidic cutinase from Aspergillus niger CBS 513.88 was chosen for heterogeneous production in Pichia pastoris and for analysis of the effects of pH on activity and stability. The recombinant enzyme showed activity over a broad range of pHs with maximal activity between pH 5.0 and 6.5. Activity could be detected still at pH 3.5.     

Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata

Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O¹⁴CH₃]-labeled β-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% ¹⁴CO₂ of the applied ¹⁴C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of ¹⁴CO₂ in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O¹⁴CH₃]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.     

Comparison of sensitivity between gas chromatography–low-resolution mass spectrometry and gas chromatography–high-resolution mass spectrometry for determining metandienone metabolites in urine

In doping control laboratories the misuse of anabolic androgenic steroids is commonly investigated in urine by gas chromatography–low-resolution mass spectrometry with selected ion monitoring (GC–LRMS–SIM). By using high-resolution mass spectrometry (HRMS) detection sensitivity is improved due to reduction of biological background. In our study HRMS and LRMS methods were compared to each other. Two different sets were measured both with HRMS and LRMS. In the first set metandienone (I) metabolites 17α-methyl-5β-androstan-3α,17β-diol (II), 17-epimetandienone (III), 17β-methyl-5β-androst-1-ene-3α,17α-diol (IV) and 6β-hydroxymetandienone (V) were spiked in urine extract prepared by solid-phase extraction, hydrolysis with β-glucuronidase from Escherichia coli and liquid–liquid extraction. In the second set the metabolites were first spiked in blank urine samples of four male persons before pretreatment. Concentration range of the spiked metabolites was 0.1–10 ng/ml in both sets. With HRMS (resolution of 5000) detection limits were 2–10 times lower than with LRMS. However, also with the HRMS method the biological background hampered detection and compounds from matrix were coeluted with some metabolites. For this reason the S/N values of the metabolites spiked had to be first compared to S/N values of coeluted matrix compounds to get any idea of detection limits. At trace concentrations selective isolation procedures should be implemented in order to confirm a positive result. The results suggest that metandienone misuse can be detected by HRMS for a prolonged period after stopping the intake of metandienone.     

Average niche breadths of species in lake macrophyte communities respond to ecological gradients variably in four regions on two continents

Different species’ niche breadths in relation to ecological gradients are infrequently examined within the same study and, moreover, species niche breadths have rarely been averaged to account for variation in entire ecological communities. We investigated how average environmental niche breadths (climate, water quality and climate–water quality niches) in aquatic macrophyte communities are related to ecological gradients (latitude, longitude, altitude, species richness and lake area) among four distinct regions (Finland, Sweden and US states of Minnesota and Wisconsin) on two continents. We found that correlations between the three different measures of average niche breadths and ecological gradients varied considerably among the study regions, with average climate and average water quality niche breadth models often showing opposite trends. However, consistent patterns were also found, such as widening of average climate niche breadths and narrowing of average water quality niche breadths of aquatic macrophytes along increasing latitudinal and altitudinal gradients. This result suggests that macrophyte species are generalists in relation to temperature variations at higher latitudes and altitudes, whereas species in southern, lowland lakes are more specialised. In contrast, aquatic macrophytes growing in more southern nutrient-rich lakes were generalists in relation to water quality, while specialist species are adapted to low-productivity conditions and are found in highland lakes. Our results emphasise that species niche breadths should not be studied using only coarse-scale data of species distributions and corresponding environmental conditions, but that investigations on different kinds of niche breadths (e.g., climate vs. local niches) also require finer resolution data at broad spatial extents.     

CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.     

Topical superantigen exposure induces epidermal accumulation of CD8+ T cells, a mixed Th1/Th2-type dermatitis and vigorous production of IgE antibodies in the murine model of atopic dermatitis

Patients with atopic dermatitis (AD) have repeated cutaneous exposure to both environmental allergens and superantigen-producing strains of Staphylococcus aureus. We used a murine model of AD to investigate the role of staphylococcal enterotoxin B (SEB) in the modulation of allergen-induced skin inflammation. Mice were topically exposed to SEB, OVA, a combination of OVA and SEB (OVA/SEB), or PBS. Topical SEB and OVA/SEB exposure induced epidermal accumulation of CD8+ T cells and TCRVbeta8+ cells in contrast to OVA application, which induced a mainly dermal infiltration of CD4+ cells. SEB and OVA/SEB exposure elicited a mixed Th1/Th2-associated cytokine and chemokine expression profile within the skin. Restimulation of lymph node cells from OVA- and OVA/SEB-exposed mice with OVA elicited strong production of IL-13 protein, whereas substantial amounts of IFN-gamma protein were detected after SEB stimulation of cells derived from SEB- or OVA/SEB-exposed mice. Topical SEB treatment elicited vigorous production of SEB-specific IgE and IgG2a Abs and significantly increased the production of OVA-specific IgE and IgG2a Abs. The present study shows that topical exposure to SEB provokes epidermal accumulation of CD8+ T cells, a mixed Th2/Th1 type dermatitis and vigorous production of specific IgE and IgG2a Abs, which can be related to the chronic phase of atopic skin inflammation.     

Food‐web structure and mercury dynamics in a large subarctic lake following multiple species introductions

相似文献   

Net climate impacts of forest biomass production and utilization in managed boreal forests

In this work, we studied the potentials offered by managed boreal forests and forestry to mitigate the climate change using forest‐based materials and energy in substituting fossil‐based materials (concrete and plastic) and energy (coal and oil). For this purpose, we calculated the net climate impacts (radiative forcing) of forest biomass production and utilization in the managed Finnish boreal forests (60°–70°N) over a 90‐year period based on integrated use forest ecosystem model simulations (on carbon sequestration and biomass production of forests) and life‐cycle assessment (LCA) tool. When studying the effects of management on the radiative forcing in a system integrating the carbon sink/sources dynamics in both biosystem and technosystem, the current forest management (baseline management) was used a reference management. Our results showed that the use of forest‐based materials and energy in substituting fossil‐based materials and energy would provide an effective option for mitigating climate change. The negative climate impacts could be further decreased by maintaining forest stocking higher over the rotation compared to the baseline management and by harvesting stumps and coarse roots in addition to logging residues in the final felling. However, the climate impacts varied substantially over time depending on the prevailing forest structure and biomass assortment (timber, energy biomass) used in substitution.     

Fine root foraging strategies in Norway spruce forests across a European climate gradient

Fine root acclimation to different environmental conditions is crucial for growth and sustainability of forest trees. Relatively small changes in fine root standing biomass (FRB), morphology or mycorrhizal symbiosis may result in a large change in forest carbon, nutrient and water cycles. We elucidated the changes in fine root traits and associated ectomycorrhizal (EcM) fungi in 12 Norway spruce stands across a climatic and N deposition gradient from subarctic‐boreal to temperate regions in Europe (68°N–48°N). We analysed the standing FRB and the ectomycorrhizal root tip biomass (EcMB, g m^?2) simultaneously with measurements of the EcM root morphological traits (e.g. mean root length, root tissue density (RTD), N% in EcM roots) and frequency of dominating EcM fungi in different stands in relation to climate, soil and site characteristics. Latitude and N deposition explained the greatest proportion of variation in fine root traits. EcMB per stand basal area (BA) increased exponentially with latitude: by about 12.7 kg m^?2 with an increase of 10° latitude from southern Germany to Estonia and southern Finland and by about 44.7 kg m^?2 with next latitudinal 10° from southern to northern Finland. Boreal Norway spruce forests had 4.5 to 11 times more EcM root tips per stand BA, and the tips were 2.1 times longer, with 1.5 times higher RTD and about 1/3 lower N concentration. There was 19% higher proportion of root tips colonized by long‐distance exploration type forming EcM fungi in the southern forests indicating importance of EcM symbiont foraging strategy in fine root nutrient acquisition. In the boreal zone, we predict ca. 50% decrease in EcMB per stand BA with an increase of 2 °C annual mean temperature. Different fine root foraging strategies in boreal and temperate forests highlight the importance of complex studies on respective regulatory mechanisms in changing climate.     

Filopodia and adhesion in cancer cell motility

Slender bundled actin containing plasma membrane protrusions, called filopodia, are important for many essential cellular processes like cell adhesion, migration, angiogenesis and the formation of cell-cell contacts. In migrating cells, filopodia are the pioneers at the leading edge which probe the environment for cues. Integrins are cell surface adhesion receptors critically implicated in cell migration and they are transported actively to filopodia tips by an unconventional myosin, myosin-X. Integrin mediated adhesion stabilizes filopodia and promotes cell migration even though integrins are not essential for filopodia initiation. Myosin-X binds also PtdIns(3,4,5)P₃ and this regulates its activation and localization to filopodia. Filopodia stimulate cell migration in many cell types and increased filopodia density has been described in cancer. Furthermore, several proteins implicated in filopodia formation, like fascin, are also relevant for cancer progression. To investigate this further, we performed a meta-analysis of the expression profiles of 10 filopodia-linked genes in human breast cancer. These data implicated that several different filopodia-inducing genes may contribute in a collective manner to cancer progression and the high metastasis rates associated with basal-type breast carcinomas.Key words: filopodia, integrins, migration, cancer