A model of structure and catalysis for ketoreductase domains in modular polyketide synthases

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.     

Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division

Mutations in the Drosophila trol gene cause cell cycle arrest of neuroblasts in the larval brain. Here, we show that trol encodes the Drosophila homolog of Perlecan and regulates neuroblast division by modulating both FGF and Hh signaling. Addition of human FGF-2 to trol mutant brains in culture rescues the trol proliferation phenotype, while addition of a MAPK inhibitor causes cell cycle arrest of the regulated neuroblasts in wildtype brains. Like FGF, Hh activates stem cell division in the larval brain in a Trol-dependent fashion. Coimmunoprecipitation studies are consistent with interactions between Trol and Hh and between mammalian Perlecan and Shh that are not competed with heparin sulfate. Finally, analyses of mutations in trol, hh, and ttv suggest that Trol affects Hh movement. These results indicate that Trol can mediate signaling through both of the FGF and Hedgehog pathways to control the onset of stem cell proliferation in the developing nervous system.     

Mechanism of Galpha i-mediated inhibition of type V adenylyl cyclase

The topology of mammalian adenylyl cyclase reveals an integral membrane protein composed of an alternating series of membrane and cytoplasmic domains (C1 and C2). The stimulatory G protein, Galpha(s), binds within a cleft in the C2 domain of adenylyl cyclase while Galpha(i) binds within the opposite cleft in the C1 domain. The mechanism of these two regulators also appears to be in opposition. Activation of adenylyl cyclase by Galpha(s) or forskolin results in a 100-fold increase in the apparent affinity of the two domains for one another. We show herein that Galpha(i) reduces C1/C2 domain interaction and thus formation of the adenylyl cyclase catalytic site. Mutants that increase the affinity of C1 for C2 decrease the ability of Galpha(i) to inhibit the enzyme. In addition, Galpha(i) can influence binding of molecules to the catalytic site, which resides at the C1/C2 interface. Adenylyl cyclase can bind substrate analogs in the presence of Galpha(i) but cannot simultaneously bind Galpha(i) and transition state analogs such as 2'd3'-AMP. Galpha(i) also cannot inhibit the membrane-bound enzyme in the presence of manganese, which increases the affinity of adenylyl cyclase for ATP and substrate analogs. Thus homologous G protein alpha-subunits promote bidirectional regulation at the domain interface of the pseudosymmetrical adenylyl cyclase enzyme.     

Up-Regulation of TREK-2 Potassium Channels in Cultured Astrocytes Requires De Novo Protein Synthesis: Relevance to Localization of TREK-2 Channels in Astrocytes after Transient Cerebral Ischemia

Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K⁺ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.     

Ecological Risk Assessment of a Metal-Contaminated Area in the Tropics. Tier II: Detailed Assessment

This study presents data on the detailed evaluation (tier 2) of a site-specific ecological risk assessment (ssERA) in a former smelter area contaminated with metals (Santo Amaro, Bahia, Brazil). Combining information from three lines of evidence (LoE), chemical (ChemLoE), ecotoxicological (EcotoxLoE) and ecological (EcoLoE), in the Triad approach, integrated risk values were calculated to rank sites and confirm the potential risk disclosed with tier 1. Risk values were calculated for the habitat and for the retention functions in each sampling point. Habitat function included the ChemLoE calculated from total metal concentrations. The EcotoxLoE was based on reproduction tests with terrestrial invertebrates (Folsomia candida, Enchytraeus crypticus, Eisenia andrei), shoot length and plant biomass (Avena sativa, Brassica rapa). For the EcoLoE, ecological parameters (microbial parameters, soil invertebrate community, litter breakdown) were used to derive risk values. Retention function included the ChemLoE, calculated from extractable metal concentrations, and the EcotoxLoE based on eluate tests with aquatic organisms (Daphnia magna reproduction and Pseudokirchneriella subcapitata growth). Results related to the habitat function indicated that the metal residues are sufficient to cause risk to biota, while the low metal levels in extracts and the general lack of toxicity in aquatic tests indicated a high soil retention capacity in most sampling points. Integrated risk of tier 2 showed the same trend of tier 1, suggesting the need to proceed with remediation actions. The high risk levels were related to direct toxicity to organisms and indirect effects, such as failure in the establishment of vegetation and the consequent loss of habitat quality for microorganisms and soil fauna. This study shed some light on the selection of tools for the tier 2 of an ssERA in tropical metal-contaminated sites, focusing on ecological receptors at risk and using available chemical methods, ecological surveys and ecotoxicity tests.     

Impact of sheep grazing on nutrient budgets of dry heathlands

Questions: What effect does sheep grazing have on the nutrient budgets of heathlands? Can grazing compensate for atmospheric nutrient loads in heathland ecosystems? What are the conclusions for heathland management? Location: Lüneburg Heath, NW Germany. Methods: During a one-year grazing experiment (stocking rate 1.1 sheep/ha) nutrient balances for N, Ca, K, Mg and P were calculated by quantifying input rates (atmospheric deposition, sheep excrement) and output rates (biomass removal, leaching). Results: Atmospheric nutrient deposition amounted to 22.8 kg.ha⁻¹.a⁻¹ for N and < 0.2 kg.ha⁻¹.a⁻¹ for P. Sheep excrement increased the inputs for N and P by ca. 3.5 and 0.2 kg.ha⁻¹.a⁻¹, respectively. Grazing reduced N- and P-stores in the above-ground biomass by 25.6 and 1.9 kg.ha⁻¹.a⁻¹, respectively. N-and P-losses via leaching amounted to 2.2 and < 0.2 kg.ha⁻¹.a⁻¹. Output:input ratios for P were high, indicating that grazing severely affected P-budgets of heaths. Conclusions: Our results suggest that sheep grazing has the potential to compensate for atmospheric nutrient loads (particularly for current N deposition rates). However, in the long term the combination of elevated N-deposition and P-loss due to grazing may cause a shift from N-(co-) limited to more P-(co-) limited plant growth. To counteract an aggravation of P-deficiency in the long term, grazing may be combined with management measures that affect P-budgets to a lesser extent (e.g. prescribed burning).     

Differences in control by UV radiation of inflammatory airways disease in na?ve and allergen pre-sensitised mice

Exposure of skin to UV radiation (UVR) prior to allergen exposure can inhibit inflammatory airways disease in mice by reducing effector CD4+ T cells in both the trachea and the airway draining lymph nodes. This study analysed the immunomodulatory properties of UVR delivered to na?ve versus allergen pre-sensitised mice. In a model of inflammatory airways disease, BALB/c mice were sensitised by peritoneal injection of the allergen, ovalbumin (OVA) (20 μg/mouse), in the adjuvant, alum (4 mg/mouse), on days 0 and 14. On day 21, the mice were exposed to aerosolised OVA and 24 h later, proliferative responses by the cells in the airway draining lymph nodes were examined. UVR (8 kJ m(-2)) was administered 3 days prior to first OVA sensitisation (day -3), or OVA aerosol challenge (day 18). UVR before sensitisation reduced immune responses associated with expression of allergic airways disease; seven days after first OVA sensitisation, regulation of OVA-induced proliferation in vitro but not in vivo by CD4+CD25+ cells from UV-irradiated mice was detected. UVR administered to pre-sensitised mice regulated allergen responsiveness by cells from the airway draining lymph nodes only with a sensitisation protocol involving allergen and adjuvant at 5% strength of the original dose (1 μg OVA in 0.2 mg alum/mouse). These results suggest that UVR may modulate allergic airways disease by two mechanisms. The first, and more potent, is by reducing effector cells in respiratory tissues and requires UV delivery prior to sensitisation. The second, associated with administration to pre-sensitised mice, is weaker and is detected when the mice are sensitised with lower levels of allergen and adjuvant.