Amino acid residues constituting the agonist binding site of the human P2X3 receptor

Homomeric P2X3 receptors are present in sensory ganglia and participate in pain perception. Amino acid (AA) residues were replaced in the four supposed nucleotide binding segments (NBSs) of the human (h) P2X3 receptor by alanine, and these mutants were expressed in HEK293 cells and Xenopus laevis oocytes. Patch clamp and two-electrode voltage clamp measurements as well as the Ca(2+) imaging technique were used to compare the concentration-response curves of the selective P2X1,3 agonist α,β-methylene ATP obtained at the wild-type P2X3 receptor and its NBS mutants. Within these NBSs, certain Gly (Gly-66), Lys (Lys-63, Lys-176, Lys-284, Lys-299), Asn (Asn-177, Asn-279), Arg (Arg-281, Arg-295), and Thr (Thr-172) residues were of great importance for a full agonist response. However, the replacement of further AAs in the NBSs by Ala also appeared to modify the amplitude of the current and/or [Ca(2+)](i) responses, although sometimes to a minor degree. The agonist potency decrease was additive after the simultaneous replacement of two adjacent AAs by Ala (K65A/G66A, F171A/T172A, N279A/F280A, F280A/R281A) but was not altered after Ala substitution of two non-adjacent AAs within the same NBS (F171A/N177A). SDS-PAGE in the Cy5 cell surface-labeled form demonstrated that the mutants appeared at the cell surface in oocytes. Thus, groups of AAs organized in NBSs rather than individual amino acids appear to be responsible for agonist binding at the hP2X3 receptor. These NBSs are located at the interface of the three subunits forming a functional receptor.     

High-Altitude Rock Shelters and Settlements in an African Alpine Ecosystem: The Bale Mountains National Park,Ethiopia

This first survey of rock shelters and settlements in the Bale Mountains in Ethiopia is a baseline assessment for further research into the settlement history of Africa’s largest alpine highlands. Extensive GPS-based mapping and interviews resulted in two detailed maps, a catalogue of profiles, and complete photographic documentation. In total, 331 rock shelters (four permanently inhabited, 51 seasonally inhabited, and 276 currently uninhabited) and 870 settlements (207 permanently inhabited, 449 seasonally inhabited, 214 uninhabited) were recorded together with information about the activities and livelihoods of the inhabitants of the current settlements. This 2015 study was part of the Ethiopian-European research project “The mountain exile hypothesis – how humans benefited from and re-shaped African high-altitude ecosystems during Quaternary climate changes” (DFG FOR 2358). It was designed to support future management plans in this internationally important conservation area that has recently faced increasing land-use pressure and the threat of degradation.     

Biochemical and structural analysis of the molybdenum cofactor biosynthesis protein MobA

Molybdopterin guanine dinucleotide (MGD) is the form of the molybdenum cofactor that is required for the activity of most bacterial molybdoenzymes. MGD is synthesized from molybdopterin (MPT) and GTP in a reaction catalyzed by the MobA protein. Here we report that wild type MobA can be copurified along with bound MPT and MGD, demonstrating a tight binding of both its substrate and product. To study structure-function relationships, we have constructed a number of site-specific mutations of the most highly conserved amino acid residues of the MobA protein family. Variant MobA proteins were characterized for their ability to support the synthesis of active molybdenum enzymes, to bind MPT and MGD, to interact with the molybdenum cofactor biosynthesis proteins MobB and MoeA. They were also characterized by x-ray structural analysis. Our results suggest an essential role for glycine 15 of MobA, either for GTP binding and/or catalysis, and an involvement of glycine 82 in the stabilization of the product-bound form of the enzyme. Surprisingly, the individual and double substitution of asparagines 180 and 182 to aspartate did not affect MPT binding, catalysis, and product stabilization.     

Large Scale Relationship between Aquatic Insect Traits and Climate

Climate is the predominant environmental driver of freshwater assemblage pattern on large spatial scales, and traits of freshwater organisms have shown considerable potential to identify impacts of climate change. Although several studies suggest traits that may indicate vulnerability to climate change, the empirical relationship between freshwater assemblage trait composition and climate has been rarely examined on large scales. We compared the responses of the assumed climate-associated traits from six grouping features to 35 bioclimatic indices (~18 km resolution) for five insect orders (Diptera, Ephemeroptera, Odonata, Plecoptera and Trichoptera), evaluated their potential for changing distribution pattern under future climate change and identified the most influential bioclimatic indices. The data comprised 782 species and 395 genera sampled in 4,752 stream sites during 2006 and 2007 in Germany (~357,000 km^² spatial extent). We quantified the variability and spatial autocorrelation in the traits and orders that are associated with the combined and individual bioclimatic indices. Traits of temperature preference grouping feature that are the products of several other underlying climate-associated traits, and the insect order Ephemeroptera exhibited the strongest response to the bioclimatic indices as well as the highest potential for changing distribution pattern. Regarding individual traits, insects in general and ephemeropterans preferring very cold temperature showed the highest response, and the insects preferring cold and trichopterans preferring moderate temperature showed the highest potential for changing distribution. We showed that the seasonal radiation and moisture are the most influential bioclimatic aspects, and thus changes in these aspects may affect the most responsive traits and orders and drive a change in their spatial distribution pattern. Our findings support the development of trait-based metrics to predict and detect climate-related changes of freshwater assemblages.