Cyclic nucleotide specific phosphodiesterases of Leishmania major

Background Leishmania represent a complex of important human pathogens that belong to the systematic order of the kinetoplastida. They are transmitted between their human and mammalian hosts by different bloodsucking sandfly vectors. In their hosts, the Leishmania undergo several differentiation steps, and their coordination and optimization crucially depend on numerous interactions between the parasites and the physiological environment presented by the fly and human hosts. Little is still known about the signalling networks involved in these functions. In an attempt to better understand the role of cyclic nucleotide signalling in Leishmania differentiation and host-parasite interaction, we here present an initial study on the cyclic nucleotide-specific phosphodiesterases of Leishmania major. Results This paper presents the identification of three class I cyclic-nucleotide-specific phosphodiesterases (PDEs) from L. major, PDEs whose catalytic domains exhibit considerable sequence conservation with, among other, all eleven human PDE families. In contrast to other protozoa such as Dictyostelium, or fungi such as Saccharomyces cerevisiae, Candida ssp or Neurospora, no genes for class II PDEs were found in the Leishmania genomes. LmjPDEA contains a class I catalytic domain at the C-terminus of the polypeptide, with no other discernible functional domains elsewhere. LmjPDEB1 and LmjPDEB2 are coded for by closely related, tandemly linked genes on chromosome 15. Both PDEs contain two GAF domains in their N-terminal region, and their almost identical catalytic domains are located at the C-terminus of the polypeptide. LmjPDEA, LmjPDEB1 and LmjPDEB2 were further characterized by functional complementation in a PDE-deficient S. cerevisiae strain. All three enzymes conferred complementation, demonstrating that all three can hydrolyze cAMP. Recombinant LmjPDEB1 and LmjPDEB2 were shown to be cAMP-specific, with Km values in the low micromolar range. Several PDE inhibitors were found to be active against these PDEs in vitro, and to inhibit cell proliferation. Conclusion The genome of L. major contains only PDE genes that are predicted to code for class I PDEs, and none for class II PDEs. This is more similar to what is found in higher eukaryotes than it is to the situation in Dictyostelium or the fungi that concomitantly express class I and class II PDEs. Functional complementation demonstrated that LmjPDEA, LmjPDEB1 and LmjPDEB2 are capable of hydrolyzing cAMP. In vitro studies with recombinant LmjPDEB1 and LmjPDEB2 confirmed this, and they demonstrated that both are completely cAMP-specific. Both enzymes are inhibited by several commercially available PDE inhibitors. The observation that these inhibitors also interfere with cell growth in culture indicates that inhibition of the PDEs is fatal for the cell, suggesting an important role of cAMP signalling for the maintenance of cellular integrity and proliferation.

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions--for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine--which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes' affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO's widespread occurrence in the fungal kingdom.

Effects of forest management on the diversity of deadwood-inhabiting fungi in Central European forests

Land use and land use change affect deadwood amount, quality and associated biodiversity in forest ecosystems. Old growth or virgin forests, which are exceptionally rare in temperate Europe harbor more deadwood and associated fungal species than managed forests. Whether and how more recent abandonment of management, to reestablish more natural forests, affects deadwood amount and fungal diversity on deadwood is unknown. Our main aim was to compare deadwood amount, characteristics and deadwood inhabiting fungi in differently managed forest types typical for large areas of Central Europe. We sampled deadwood inhabiting fungi on 27 forest plots of 400 m2 each in three geographically distant regions in Germany. Three forest management types, namely managed coniferous, managed deciduous and unmanaged deciduous forests, were represented by nine plots each. In autumn 2008 we collected all fungal fruiting bodies on deadwood >7 cm of diameter. We found deadwood amounts and fungal species numbers in unmanaged forests to be lower than in managed forests, which we attributed to the lack of natural tree death during the short time since management abandonment of usually 10–30 years. However, rarefaction analysis among deadwood items in forest plots indicated a slightly higher species density in unmanaged forests, which may be the first signal of a positive effect on fungal species richness on deadwood after management was abandoned. Although the three study regions span a large geographical gradient, we did not detect differences in the fungal species composition or in deadwood amounts and patterns, which reflects the wide distribution of this group of organisms and points to consistent management procedures among study regions. A very clear composition difference however occurred between deciduous and coniferous wood showing species substrate specialization. We conclude that the amount of deadwood is the main driver of deadwood fungal species richness, and substrate diversity in terms of various decay degrees, deadwood tree species and deadwood size are also important. Thus, to promote species richness of deadwood fungi it is vital to enhance deadwood amounts and diversity

P450 oxidoreductase deficiency: a new disorder of steroidogenesis affecting all microsomal P450 enzymes

Combined partial deficiency of 17alpha-hydroxylase and 21-hydroxylase activities was first described in 1985; however the genes for P450c17 and P450c21 in these patients lack mutations. In 1986 we postulated that this disorder might be due to mutations in P450 oxidoreductase (POR), the flavoprotein that donates electron to these and all other microsomal P450 enzymes, but this hypothesis was not tested until the POR gene sequence became available through the genome database. We found five POR missense mutations in our first four patients. In vitro assays of the activities of these mutations showed that the standard assay of POR activity, reduction of cytochrome c, correlated poorly with the patients' phenotypes, but that assays of POR-supported 17alpha-hydroxylase and 17,20 lyase activities correlated well. POR deficiency is a new disorder of adrenal and gonadal steroidogenesis that affects all microsomal cytochrome P450 enzymes, hence may have important implications for genetic differences in drug metabolism.

Soil property and management effects on grassland microbial communities across a latitudinal gradient in Germany

There is much interest in the identification of the main drivers controlling changes in the microbial community that may be related to sustainable land use. We examined the influence of soil properties and land-use intensity (N fertilization, mowing, grazing) on total phospholipid fatty acid (PLFA) biomass, microbial community composition (PLFA profiles) and activities of enzymes involved in the C, N, and P cycle. These relationships were examined in the topsoil of grasslands from three German regions (Schorfheide-Chorin (SCH), Hainich-Dun (HAI), Schwabische Alb (ALB)) with different parent material. Differences in soil properties explained 60% of variation in PLFA data and 81% of variation in enzyme activities across regions and land-use intensities. Degraded peat soils in the lowland areas of the SCH with high organic carbon (OC) concentrations and sand content contained lower PLFA biomass, lower concentrations of bacterial, fungal, and arbuscular mycorrhizal PLFAs, but greater enzyme activities, and specific enzyme activities (per unit microbial biomass) than mineral soils in the upland areas of the HAI and ALB, which are finer textured, drier, and have smaller OC concentrations. After extraction of variation that originated from large-scale differences among regions and differences in land-use intensities between plots, soil properties still explained a significant amount of variation in PLFA data (34%) and enzyme activities (60%). Total PLFA biomass and all enzyme activities were mainly related to OC concentration, while relative abundance of fungi and fungal to bacterial ratio were mainly related to soil moisture. Land-use intensity (LUI) significantly decreased the soil C:N ratio. There was no direct effect of LUI on total PLFA biomass, microbial community composition, N and P cycling enzyme activities independent of study region and soil properties. In contrast, the activities and specific activities of enzymes involved in the C cycle increased significantly with LUI independent of study region and soil properties, which can have impact on soil organic matter decomposition and nutrient cycling. Our findings demonstrate that microbial biomass and community composition as well as enzyme activities are more controlled by soil properties than by grassland management at the regional scale. (C) 2013 Elsevier B.V: All rights reserved.

Induction of cytochrome P450 enzymes in primary equine hepatocyte culture

In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC50=1.20 μM and 6.06 μM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system.

Unraveling environmental drivers of a recent increase in Swiss fungi fruiting

Disentangling biotic and abiotic drivers of wild mushroom fruiting is fraught with difficulties because mycelial growth is hidden belowground, symbiotic and saprotrophic supply strategies may interact, and myco-ecological observations are often either discontinuous or too short. Here, we compiled and analyzed 115 417 weekly fungal fruit body counts from permanent Swiss inventories between 1975 and 2006. Mushroom fruiting exhibited an average autumnal delay of 12 days after 1991 compared with before, the annual number of fruit bodies increased from 1801 to 5414 and the mean species richness doubled from 10 to 20. Intra- and interannual coherency of symbiotic and saprotrophic mushroom fruiting, together with little agreement between mycorrhizal yield and tree growth suggests direct climate controls on fruit body formation of both nutritional modes. Our results contradict a previously reported declining of mushroom harvests and propose rethinking the conceptual role of symbiotic pathways in fungi-host interaction. Moreover, this conceptual advancement may foster new cross-disciplinary research avenues, and stimulate questions about possible amplifications of the global carbon cycle, as enhanced fungal production in moist mid-latitude forests rises carbon cycling and thus increases greenhouse gas exchanges between terrestrial ecosystems and the atmosphere.

Are ectomycorrhizal fungi alleviating or aggravating nitrogen limitation of tree growth in boreal forests?

•Symbioses between plant roots and mycorrhizal fungi are thought to enhance plant uptake of nutrients through a favourable exchange for photosynthates. Ectomycorrhizal fungi are considered to play this vital role for trees in nitrogen (N)-limited boreal forests. •We followed symbiotic carbon (C)–N exchange in a large-scale boreal pine forest experiment by tracing 13CO2 absorbed through tree photosynthesis and 15N injected into a soil layer in which ectomycorrhizal fungi dominate the microbial community. •We detected little 15N in tree canopies, but high levels in soil microbes and in mycorrhizal root tips, illustrating effective soil N immobilization, especially in late summer, when tree belowground C allocation was high. Additions of N fertilizer to the soil before labelling shifted the incorporation of 15N from soil microbes and root tips to tree foliage. •These results were tested in a model for C–N exchange between trees and mycorrhizal fungi, suggesting that ectomycorrhizal fungi transfer small fractions of absorbed N to trees under N-limited conditions, but larger fractions if more N is available. We suggest that greater allocation of C from trees to ectomycorrhizal fungi increases N retention in soil mycelium, driving boreal forests towards more severe N limitation at low N supply.