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revealed that all isolates clustered within genotype 3 but differed in the subtype depending 36 on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one 37 population HEV isolates were closely related, but social groups of animals in close 38 proximity might be infected with different subtypes. Two full-length genomes of subtypes 39 3i and 3e from two different geographic regions were generated. The wild boar is discussed 40 as one of the main sources of human autochthonous infections in Germany. 41 42

Real time biomolecular interaction analysis based on surface plasmon resonance has been proven useful for studying protein-protein interaction but has not been extended so far to investigate enzyme-enzyme interactions, especially as pertaining to regulation of metabolic activity. We have applied BIAcore technology to study the regulation of enzyme-enzyme interaction during mitochondrial cysteine biosynthesis in Arabidopsis thaliana. The association of the two enzyme subunits in the hetero-oligomeric cysteine synthase complex was investigated with respect to the reaction intermediate and putative

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The Hedgehog proteins are potent organizers of animal development. They carry a cholesterol ester at the C terminus of their signaling domain. The membrane anchoring mediated by this lipophilic modification was studied by means of an approach integrating cell biology, biochemistry, biophysics, and organic chemistry techniques. Sterol-modified and fluorescent-labeled Hedgehog-derived peptides and proteins were synthesized and investigated in biophysical and cell-biological assays. These experiments revealed that cholesterol alone anchors proteins to membranes with significant strength and half-times for spontaneous desorption of several hours. Its membrane anchoring ability is comparable to dual lipidation motifs such as double geranylgeranylation or S-palmitoylation plus S-farnesylation found in other lipidated proteins. The experiments also demonstrate that membrane binding changes dramatically if short lipidated peptides are equipped with a large protein. These data suggest that for Hedgehog release and subsequent signaling an interaction partner such as the Dispatched protein is necessary. In addition to these findings the described approach allows one to correlate biophysical data obtained with model peptides with data determined with fully functional proteins and to combine results from in vitro and in vivo experiments. It should be generally applicable to other membrane anchors and proteins.M any proteins involved in key processes of cell growth and differentiation embody lipid modifications that are essential for their biological activity. These modifications serve in most cases as anchoring groups for targeting the proteins to a certain membrane or submembrane compartment. In addition, they may mediate controlled release of proteins from membrane regions to form stable gradients. This is particularly true for the Hedgehog protein (Hh), which is among the key players in patterning numerous types of tissues. Mutations in Hh and its downstream signaling molecules are also associated with numerous oncogenic and disease states.The Hh family of molecules consists of secreted proteins that undergo several posttranslational modifications to gain full activity. In a maturation process they perform an autocatalytic cleavage, generating an N-terminal polypeptide (Hh-Np) containing all of the signaling functions (1-4). During this cleavage process, a cholesterol moiety is attached covalently by an ester function to the C-terminal glycine of the signaling domain (5). The hydrophobicity of the protein is further increased by the addition of a palmitic acid residue to the N terminus of the cleavage product (6, 7).In Drosophila, forms of Hh in which the C terminus has been deleted have much more potent and seemingly longer-ranging signaling activity than the cholesterol-modified forms. This observation led to the idea that the cholesterol moiety acts as an anchor to the cell membrane and limits the spread of Hh (1-3, 8). However, in vertebrate Sonic Hedgehog (Shh) the cholesterol may be required for correct biological ...

Small molecule kinase inhibitors are an attractive means to modulate kinase activities in medicinal chemistry and chemical biology research. In the physiological setting of a cell, kinase function is orchestrated by a plethora of regulatory processes involving the structural transition of kinases between inactive and enzymatically competent conformations and vice versa. The development of novel kinase inhibitors is mainly fostered by high-throughput screening initiatives where the small molecule perturbation of the phosphorylation reaction is measured to identify inhibitors. Such setups require enzymatically active kinase preparations and present a risk of solely identifying classical ATP-competitive Type I inhibitors. Here we report the high-throughput screening of a library of approximately 35000 small organic molecules with an assay system that utilizes enzymatically inactive human p38alpha MAP kinase to detect stabilizers of the pharmacologically more desirable DFG-out conformation. We used protein X-ray crystallography to characterize the binding mode of hit compounds and reveal structural features which explain how these ligands stabilize and/or induce the DFG-out conformation. Lastly, we show that although some of the hit compounds were confirmed by protein X-ray crystallography, they were not detected in classic phosphorylation assays, thus validating the unique sensitivity of the assay system used in this study and highlighting the potential of screening with inactive kinase preparations.

The generation of entanglement between two oscillators that interact via a common reservoir is theoretically studied. The reservoir is modeled by a one-dimensional harmonic crystal initially in thermal equilibrium. Starting from a separable state, the oscillators can become entangled after a transient time, that is of the order of the thermalization time scale. This behaviour is observed at finite temperature even when the oscillators are at a distance significantly larger than the crystal's interparticle spacing. The underlying physical mechanisms can be explained by the dynamical properties of the collective variables of the two oscillators which may decouple from or be squeezed by the reservoir. Our predictions can be tested with an ion chain in a linear Paul trap.

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk for lung cancer and an aberrant microbiota of the lung. Microbial colonization contributes to chronic neutrophilic inflammation in COPD. Nontypeable Haemophilus influenzae (NTHi) is frequently found in lungs of stable COPD patients and is the major pathogen triggering exacerbations. The epithelial cytokine interleukin-17C (IL-17C) promotes the recruitment of neutrophils into inflamed tissues. The purpose of this study was to investigate the function of IL-17C in the pulmonary tumor microenvironment. We subjected mice deficient for IL-17C (IL-17C) and mice double deficient for Toll-like receptor 2 and 4 (TLR-2/4) to a metastatic lung cancer model. Tumor proliferation and growth as well as the number of tumor-associated neutrophils was significantly decreased in IL-17C and TLR-2/4 mice exposed to NTHi. The NTHi-induced pulmonary expression of IL-17C was dependent on TLR-2/4. In vitro, IL-17C increased the NTHi- and tumor necrosis factor-α-induced expression of the neutrophil chemokines keratinocyte-derived chemokine and macrophage inflammatory protein 2 in lung cancer cells but did not affect proliferation. Human lung cancer samples stained positive for IL-17C, and in non-small cell lung cancer patients with lymph node metastasis, IL-17C was identified as a negative prognostic factor. Our data indicate that epithelial IL-17C promotes neutrophilic inflammation in the tumor microenvironment and suggest that IL-17C links a pathologic microbiota, as present in COPD patients, with enhanced tumor growth.