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Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.
In this study we compared the phylogeographic patterns of two Rusa species, Rusa unicolor and Rusa timorensis, in order to understand what drove and maintained differentiation between these two geographically and genetically close species and investigated the route of introduction of individuals to the islands outside of the Sunda Shelf. We analyzed full mitogenomes from 56 archival samples from the distribution areas of the two species and 18 microsatellite loci in a subset of 16 individuals to generate the phylogeographic patterns of both species. Bayesian inference with fossil calibration was used to estimate the age of each species and major divergence events. Our results indicated that the split between the two species took place during the Pleistocene, similar to 1.8Mya, possibly driven by adaptations of R. timorensis to the drier climate found on Java compared to the other islands of Sundaland. Although both markers identified two well-differentiated clades, there was a largely discrepant pattern between mitochondrial and nuclear markers. While nDNA separated the individuals into the two species, largely in agreement with their museum label, mtDNA revealed that all R. timorensis sampled to the east of the Sunda shelf carried haplotypes from R. unicolor and one Rusa unicolor from South Sumatra carried a R. timorensis haplotype. Our results show that hybridization occurred between these two sister species in Sundaland during the Late Pleistocene and resulted in human-mediated introduction of hybrid descendants in all islands outside Sundaland.
There is growing interest in biological control as a sustainable and environmentally friendly way to control pest insects. Aphids are among the most detrimental agricultural pests worldwide, and parasitoid wasps are frequently employed for their control. The use of asexual parasitoids may improve the effectiveness of biological control because only females kill hosts and because asexual populations have a higher growth rate than sexuals. However, asexuals may have a reduced capacity to track evolutionary change in their host populations. We used a factorial experiment to compare the ability of sexual and asexual populations of the parasitoid Lysiphlebus fabarum to control caged populations of black bean aphids (Aphis fabae) of high and low clonal diversity. The aphids came from a natural population, and one-third of the aphid clones harbored Hamiltonella defensa, a heritable bacterial endosymbiont that increases resistance to parasitoids. We followed aphid and parasitoid population dynamics for 3months but found no evidence that the reproductive mode of parasitoids affected their effectiveness as biocontrol agents, independent of host clonal diversity. Parasitoids failed to control aphids in most cases, because their introduction resulted in strong selection for clones protected by H.defensa. The increasingly resistant aphid populations escaped control by parasitoids, and we even observed parasitoid extinctions in many cages. The rapid evolution of symbiont-conferred resistance in turn imposed selection on parasitoids. In cages where asexual parasitoids persisted until the end of the experiment, they became dominated by a single genotype able to overcome the protection provided by H.defensa. Thus, there was evidence for parasitoid counteradaptation, but it was generally too slow for parasitoids to regain control over aphid populations. It appears that when pest aphids possess defensive symbionts, the presence of parasitoid genotypes able to overcome symbiont-conferred resistance is more important for biocontrol success than their reproductive mode.
Germination, a crucial phase in the life cycle of a plant, can be significantly influenced by competition and facilitation. The aim of this study was to test whether differences in cover of surrounding vegetation can lead to population differentiation in germination behaviour of an annual grassland species, and if so, whether such a differentiation can be found in the native as well as in the introduced range. We used maternal progeny of Erodium cicutarium previously propagated under uniform conditions that had been collected in multiple populations in the native and two introduced ranges, in populations representing extremes in terms of mean and variability of the cover of surrounding vegetation. In the first experiment, we tested the effect of germination temperature and mean cover at the source site on germination, and found interlinked effects of these factors. In seeds from one of the introduced ranges (California), we found indication for a 2-fold dormancy, hindering germination at high temperatures even if physical dormancy was broken and water was available. This behaviour was less strong in high cover populations, indicating cross-generational facilitating effects of dense vegetation. In the second experiment, we tested whether spatial variation in cover of surrounding vegetation has an effect on the proportion of dormant seeds. Contrary to our expectations, we found that across source regions, high variance in cover was associated with higher proportions of seeds germinating directly after storage. In all three regions, germination seemed to match the local environment in terms of climate and vegetation cover. We suggest that this is due to a combined effect of introduction of preadapted genotypes and local evolutionary processes.
Müller, Sandra Marie ; Ebert, Franziska ; Raber, Georg ; Meyer, Sören ; Bornhorst, Julia ; Hüwel, Stephan ; Galla, Hans-Joachim ; Francesconi, Kevin A. ; Schwerdtle, Tanja
Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood–brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood–brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood–brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood–brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.
Retrieval of water constituents from hyperspectral in-situ measurements under variable cloud cover
(2018)
Göritz, Anna ; Berger, Stella A. ; Gege, Peter ; Grossart, Hans-Peter ; Nejstgaard, Jens C. ; Riedel, Sebastian ; Röttgers, Rüdiger ; Utschig, Christian
Remote sensing and field spectroscopy of natural waters is typically performed under clear skies, low wind speeds and low solar zenith angles. Such measurements can also be made, in principle, under clouds and mixed skies using airborne or in-situ measurements; however, variable illumination conditions pose a challenge to data analysis. In the present case study, we evaluated the inversion of hyperspectral in-situ measurements for water constituent retrieval acquired under variable cloud cover. First, we studied the retrieval of Chlorophyll-a (Chl-a) concentration and colored dissolved organic matter (CDOM) absorption from in-water irradiance measurements. Then, we evaluated the errors in the retrievals of the concentration of total suspended matter (TSM), Chl-a and the absorption coefficient of CDOM from above-water reflectance measurements due to highly variable reflections at the water surface. In order to approximate cloud reflections, we extended a recent three-component surface reflectance model for cloudless atmospheres by a constant offset and compared different surface reflectance correction procedures. Our findings suggest that in-water irradiance measurements may be used for the analysis of absorbing compounds even under highly variable weather conditions. The extended surface reflectance model proved to contribute to the analysis of above-water reflectance measurements with respect to Chl-a and TSM. Results indicate the potential of this approach for all-weather monitoring.
Heg1 and Ccm1/2 proteins control endocardial mechanosensitivity during zebrafish valvulogenesis
(2018)
Donat, Stefan ; Lourenco, Marta Sofia Rocha ; Paolini, Alessio ; Otten, Cecile ; Renz, Marc ; Abdelilah-Seyfried, Salim
Endothelial cells respond to different levels of fluid shear stress through adaptations of their mechanosensitivity. Currently, we lack a good understanding of how this contributes to sculpting of the cardiovascular system. Cerebral cavernous malformation (CCM) is an inherited vascular disease that occurs when a second somatic mutation causes a loss of CCM1/KRIT1, CCM2, or CCM3 proteins. Here, we demonstrate that zebrafish Krit1 regulates the formation of cardiac valves. Expression of heg1, which encodes a binding partner of Krit1, is positively regulated by blood-flow. In turn, Heg1 stabilizes levels of Krit1 protein, and both Heg1 and Krit1 dampen expression levels of klf2a, a major mechanosensitive gene. Conversely, loss of Krit1 results in increased expression of klf2a and notch1b throughout the endocardium and prevents cardiac valve leaflet formation. Hence, the correct balance of blood-flow-dependent induction and Krit1 protein mediated repression of klf2a and notch1b ultimately shapes cardiac valve leaflet morphology.
The Amyloid-precursor-like protein 1 (APLP1) is a neuronal type I transmembrane protein which plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1 trans interactions have never been directly observed in higher eukaryotic cells. Here, we investigate APLP1 interactions and dynamics directly in living human embryonic kidney (HEK) cells, using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy (sFCS) and Number&Brightness (N&B). Our results show that APLP1 forms homotypic trans complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of trans binding and strongly reduced dynamics. Further evidence from Giant Plasma Membrane Vesicles and live cell actin staining suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced cis multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1 direct trans interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and provide a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms. Further, they show that fluorescence fluctuation spectroscopy techniques are useful tools for the investigation of protein-protein interactions at cell-cell adhesion sites.
Modulating the Molybdenum Coordination Sphere of Escherichia coli Trimethylamie N-Oxide Reductase
(2018)
Kaufmann, Hans Paul ; Duffus, Benjamin R. ; Mitrova, Biljana ; Iobbi-Nivol, Chantal ; Teutloff, Christian ; Nimtz, Manfred ; Jaensch, Lothar ; Wollenberger, Ulla ; Leimkühler, Silke
The well-studied enterobacterium Escherichia coli present in the human gut can reduce trimethylamine N-oxide (TMAO) to trimethylamine during anaerobic respiration. The TMAO reductase TorA is a monomeric, bis-molybdopterin guanine dinucleotide (bis-MGD) cofactor-containing enzyme that belongs to the dimethyl sulfoxide reductase family of molybdoenzymes. We report on a system for the in vitro reconstitution of TorA with molybdenum cofactors (Moco) from different sources. Higher TMAO reductase activities for TorA were obtained when using Moco sources containing a sulfido ligand at the molybdenum atom. For the first time, we were able to isolate functional bis-MGD from Rhodobacter capsulatus formate dehydrogenase (FDH), which remained intact in its isolated state and after insertion into apo-TorA yielded a highly active enzyme. Combined characterizations of the reconstituted TorA enzymes by electron paramagnetic resonance spectroscopy and direct electrochemistry emphasize that TorA activity can be modified by changes in the Mo coordination sphere. The combination of these results together with studies of amino acid exchanges at the active site led us to propose a novel model for binding of the substrate to the molybdenum atom of TorA.