Characterization of an Invertebrate-Type Dopamine Receptor of the American Cockroach,Periplaneta americana

We have isolated a cDNA coding for a putative invertebrate-type dopamine receptor (Peadop2) from P. americana brain by using a PCR-based strategy. The mRNA is present in samples from brain and salivary glands. We analyzed the distribution of the PeaDOP2 receptor protein with specific affinity-purified polyclonal antibodies. On Western blots, PeaDOP2 was detected in protein samples from brain, subesophageal ganglion, thoracic ganglia, and salivary glands. In immunocytochemical experiments, we detected PeaDOP2 in neurons with their somata being located at the anterior edge of the medulla bilaterally innervating the optic lobes and projecting to the ventro-lateral protocerebrum. In order to determine the functional and pharmacological properties of the cloned receptor, we generated a cell line constitutively expressing PeaDOP2. Activation of PeaDOP2-expressing cells with dopamine induced an increase in intracellular cAMP. In contrast, a C-terminally truncated splice variant of this receptor did not exhibit any functional property by itself. The molecular and pharmacological characterization of the first dopamine receptor from P. americana provides the basis for forthcoming studies focusing on the significance of the dopaminergic system in cockroach behavior and physiology.     

Characterisation of CYP102A25 from Bacillus marmarensis and CYP102A26 from Pontibacillus halophilus: P450 Homologues of BM3 with Preference towards Hydroxylation of Medium‐Chain Fatty Acids

Cytochrome P450 monooxygenases are highly desired biocatalysts owing to their ability to catalyse a wide variety of chemically challenging C?H activation reactions. The CYP102A subfamily of enzymes are natural catalytically self‐sufficient proteins consisting of a haem and FMN‐FAD reductase domain fused in a single‐component system. They catalyse the oxygenation of saturated and unsaturated fatty acids to produce primarily ω?1, ω?2 and ω?3 hydroxy acids. These monooxygenases have potential applications in biotechnology; however, their substrate range is still limited and there is a continued need to add diversity to this class of biocatalysts. Herein, we present the characterisation of two new members of this class of enzymes, CYP102A25 (BMar) from Bacillus marmarensis and CYP102A26 (PHal) from Pontibacillus halophilus, both of which express readily in a recombinant bacterial host. BMar exhibits the highest activity toward myristic acid and shows moderate activity towards unsaturated fatty acids. PHal exhibits broader activity towards mid‐chain‐saturated (C₁₄–C₁₈) and unsaturated fatty acids. Furthermore, PHal shows good regioselectivity for the hydroxylation of myristic acid, targeting the ω?2 position for C?H activation.     

Aerobic biodegradation of polyethylene glycols of different molecular weights in wastewater and seawater

In order to distinguish between aerobic biodegradation of synthetic polymers in fresh and seawater, polyethylene glycols (PEGs) were systematically and comparatively investigated in inocula from municipal wastewater and seawater aquarium filters for the first time. The molecular weight (MW) of the PEGs, (HO(CH(2)CH(2)O)(n)H, n=3-1350) as representatives of water-soluble polymers, ranged from 250 to 57,800Da. The biodegradation was observed by removal of dissolved organic carbon and carbon dioxide production by applying standardized ISO and OECD test methods. Specific analyses using liquid chromatography mass spectrometry (LC-MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were performed. All PEGs selected were completely biodegradable in freshwater media within 65d. PEGs with an MW up to 14,600Da have a similar degradation pathway which is characterized by gradual splitting of C(2)-units off the chain resulting in formation of short-chain PEGs. In artificial seawater media, full biodegradation of PEGs up to 7400Da required more time than in freshwater. PEGs with MW 10,300 and 14,600Da were only partially degraded whereas PEGs with MW 26,600 and 57,800Da were not degraded for a period of 135d. The biodegradation pathway of PEG 250 and PEG 970 in seawater is similar to that for freshwater. For PEGs having an MW from 2000 to 10,300Da, the degradation pathway in seawater differs from the pathway of the shorter PEGs.     

Doxycycline Decreases Atherosclerotic Lesions in the Aorta of ApoE-⁄- and Ovariectomized Mice with Correlation to Reduced MMP-2 Activity

Atherogenic events promote changes in vessel walls, with alteration of the redox state, and increased activity of matrix metalloproteinases (MMPs). Thus, this study aims to evaluate aortic remodeling, MMP activity, and reactive oxygen species (ROS) levels after treatment with doxycycline in ApoE^-⁄- and ovariectomized mice (OVX). Female ApoE^-⁄--knockout mice (5 weeks) were submitted to ovariectomy surgery to induce experimental menopause. They then received chow enriched with 1% cholesterol to induce hypercholesterolemia. The animals were divided into two experimental groups: ApoE^-⁄-/OVX vehicle and ApoE^-⁄-/OVX doxycycline (30 mg/kg) administered by gavage once a day for 28 days (15th to the 18th week of life). Blood samples were collected to measure total cholesterol and fractions. The aorta was used for morphometry and to measure the activity and expression of MMP-2 and ROS levels. The ApoE^-⁄-/OVX doxycycline group showed no change in total and fraction cholesterol levels. However, there was a reduction in ROS levels, MMP-2 expression, and activity that correlated with a decrease in atherosclerotic lesions relative to the ApoE^-⁄-/OVX vehicle (p > 0.05). Therefore, we conclude that doxycycline in ApoE^-⁄-/OVX animals promotes a reduction in atherosclerotic lesions by reducing ROS and MMP-2 activity and expression.     

Atomic Friction Investigations on Ordered Superstructures

We review recent friction measurements on ordered superstructures performed by atomic force microscopy. In particular, we consider ultrathin KBr films on NaCl(001) and Cu(001) surfaces, single and bilayer graphene on SiC(0001), and the herringbone reconstruction of Au(111). Atomically resolved friction images of these systems show periodic features spanning across several unit cells. Although the physical mechanisms responsible for the formation of these superstructures are quite different, the experimental results can be interpreted within the same phenomenological framework. A comparison between experiments and modeling shows that, in the cases of KBr films on NaCl(001) and of graphene films, the tip-surface interaction is well described by a potential with the periodicity of the substrate which is modulated or, respectively, superimposed with a potential with the symmetry of the superstructure.     

Neuregelungen in Vorarlberg zu Spielräumen

Ein Ma?nahmenpaket, bestehend aus dem Spielraumgesetz, dem Beschluss von entsprechenden F?rderungsrichtlinien sowie Novellierungen des Baugesetzes und der Kinderspielplatzverordnung und einer überarbeitung der Wohnbaufondsrichtlinie, soll dazu beitragen, dass sich das Bundesland Vorarlberg als besonders kinder- und jugendfreundliche Region etabliert. Hiefür gilt es, ?ffentliche und private Spielpl?tze und Freir?ume bedarfsgerecht zu erhalten, zu sanieren, neu zu errichten und untereinander zu vernetzen.     

Mechanistic Insights into Cyclic Peptide Generation by DnaE Split-Inteins through Quantitative and Structural Investigation

Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction.