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Carbon monoxide (CO) is a therapeutic gas with therapeutic potential in intestinal bowel disease. Therapeutic efficacy in the gastrointestinal tract (GIT) must be paired with safe and convenient use. Therefore, we designed an oral CO releasing system (OCORS) pairing tunable CO release into the GIT while preventing the release of any other molecule from within the device, causing safety concerns. The dimensions of the device, which is manufactured from 3D printed components, are within compendial limits. This is achieved by controlling CO decarbonylation from a molybdenum complex with a FeCl

Cognitive performance may be impaired in MS even at the earliest stages of disease. We tested whether brain-derived neurotrophic factor and neurofilament light chain levels in serum and cerebrospinal fluid (CSF) samples (sNfL/cNfL/sBDNF/cBDNF) collected at the time of diagnosis are associated with cognitive performance.We measured sNfL/cNfL/sBDNF/cBDNF using single-molecule array (Simoa) in 47 newly diagnosed patients (32 relapsing-remitting MS/6 primary progressive MS/9 clinically isolated syndrome). Partial correlations between average z-score on neuropsychological tests and sNfL/sBDNF/cNfL/cBDNF were computed after adjusting for covariates. Multivariate analysis of covariance determined the effect of cognitive status on biomarker levels. A composite measure of NfL and BDNF was submitted to similar exploratory analysis.Cognitive performance correlated inversely with cNfL (r=-0.451/q=0.032) and cBDNF (r=-0.406/q=0.034). Impairment in at least two different tests was linked to higher cNfL (p=0.011) and cBDNF (p=0.035) levels compared to impairment in only one test and for cNfL also compared to no impairment at all (p=0.01). Composite CSF biomarker measure accounting for both cNfL and cBDNF correlated more strongly with tests of information processing (p=0.048) and verbal learning/memory consolidation (p = 0.02) as compared to the single CSF biomarkers.CSF BDNF and NfL levels measured at the time of diagnosis are inversely associated with cognitive performance in MS. Our findings suggest that CSF biomarkers linked to different pathophysiological processes reflect neuropsychological impairment in the earliest stages of the disease. Combining different CSF measures might facilitate the developing of a better biomarker of cognition in MS.

Magnetic resonance imaging (MRI) is the gold standard imaging technique for diagnosis and monitoring of many neurological diseases. However, the application of conventional MRI in clinical routine is mainly limited to the visual detection of macroscopic tissue pathology since mixed tissue contrasts depending on hardware and protocol parameters hamper its application for the assessment of subtle or diffuse impairment of the structural tissue integrity. Multiparametric quantitative (q)MRI determines tissue parameters quantitatively, enabling the detection of microstructural processes related to tissue remodeling in aging and neurological diseases. In contrast to measuring tissue atrophy via structural imaging, multiparametric qMRI allows for investigating biologically distinct microstructural processes, which precede changes of the tissue volume. This facilitates a more comprehensive characterization of tissue alterations by revealing early impairment of the microstructural integrity and specific disease-related patterns. So far, qMRI techniques have been employed in a wide range of neurological diseases, including in particular conditions with inflammatory, cerebrovascular and neurodegenerative pathology. Numerous studies suggest that qMRI might add valuable information, including the detection of microstructural tissue damage in areas appearing normal on conventional MRI and unveiling the microstructural correlates of clinical manifestations. This review will give an overview of current qMRI techniques, the most relevant tissue parameters and potential applications in neurological diseases, such as early (differential) diagnosis, monitoring of disease progression, and evaluating effects of therapeutic interventions.

Öffentliche Aufträge, die auf der Grundlage staatlich eingestufter Informationen ausgeschrieben werden, setzen beim Auftragnehmer eine vorhandene gesetzeskonforme Infrastruktur voraus, um die geforderte Vertraulichkeit zu gewährleisten. Vor diesem Hintergrund gestaltet sich die IT-Bearbeitung solcher Informationen als sehr aufwändig und kostenintensiv. In diesem Bericht werden die allgemeinen Trends und Entwicklungen bei der Bearbeitung staatlich eingestufter Informationen unter dem speziellen Aspekt der Informationstechnologie dargestellt und mögliche Probleme sowie deren Lösungsmöglichkeiten aufgezeigt.

In order to perform biochemical and pharmacological characterization of CXCR1, we designed several CXCR1 constructs. All constructs, including a CXCR1-G(i2)alpha fusion protein, were produced in insect cells after infection with recombinant baculovirus. The recombinant receptors exhibited specific high-affinity binding of (125)I-labelled interleukin-8, and Scatchard transformation of the binding data indicated the presence of a population of single homogenous binding sites. Furthermore, the pharmacological profiles for the different CXCR1 constructs produced in the baculovirus-infected insect cells were almost identical to those reported for CXCR1 on human neutrophils. Interestingly, when the CXCR1 constructs were coproduced with G(i2) protein as a result of coinfection with baculoviruses encoding the G(i2)alpha-, the beta- and the gamma- subunits, the B(max) values were significantly increased. Hence, the level of FlagCXCR1Bio, after coproduction with G(i2) protein, was found to be almost 10 times higher than that of the FlagCXCR1Bio alone. However, no differences in the K(i) values were observed of the receptor constructs produced either after single infection or coinfection of insect cells. The addition of guanyl-5'-yl imidodiphosphate resulted in a dramatic reduction of the number of binding sites; however, the K(i) values remained unchanged, indicating coupling of the receptor to the guanine nucleotide-binding protein.

To investigate the impact of aromatic residues within transmembrane helix 6 (TMH6) of the human gonadotropin-releasing hormone receptor (GnRH-R) on agonist and antagonist binding, residues Y 2 8 3 , Y 2 8 4 , W 2 8 9 , Y 2 9 0 , W 2 9 1 , and F 2 9 2 were exchanged to alanine and analyzed comprehensively in functional reporter gene and ligand binding assays. Whereas receptor mutants Y 2 8 3 A, Y 2 8 4 A, and W 2 9 1 A were capable of neither ligand binding nor signal transduction, mutants W 2 8 9 A, Y 2 9 0 A. and F 2 9 2 A were functional: the F 2 9 2 A mutant behaved like wild-type receptor, while mutants W 2 8 9 A and Y 2 9 0 A differentiated between agonistic and antagonistic ligands. On the basis of the high-resolution X-ray structure of bovine rhodopsin as well as available data on GnRH-R mutants, models for ligand-receptor interactions are proposed. The model for D-Trp 6 -GnRH (Triptorelin) binding, representing a superagonistic ligand, is in full accordance to available data. Furthermore, new interactions are proposed: pGlu' interacts with N 2 1 2 in transmembrane helix 5, Tyr 5 with Y 2 9 0 , and D-Trp 6 with W 2 8 9 , The binding behavior of mutants W 2 8 9 A and Y 2 9 0 A corresponds to the proposed binding model for the antagonist Cetrorelix. In summary, our data as presented indicate that Y 2 9 0 plays a key function in agonist but not antagonist binding.

Most of the completely sequenced prokaryotic genomes contain genes of potassium channel homologues, but there is still not much known about the role of these proteins in prokaryotes. Here we describe the large-scale overproduction and purification of a prokaryotic voltage-gated potassium channel homologue, Kch, from Escherichia coli. After successful overproduction of the protein, a specific increase in the potassium permeability of the cells was found. Kch could be purified in large amounts using classical purification methods to prevent aggregation of the protein. The physiological state of the protein was revealed to be a homotetramer and the protein was shown to be localized to the cytoplasmic membrane of the cells. In the course of the localization studies, we found a specific increase in the density of the cytoplasmic membrane on Kch production. This was linked to the observed increase in the protein to lipid ratio in the membranes. Another observed change in the membrane composition was an increase in the cardiolipin to phosphatidylglycerol ratio, which may indicate a specific cardiolipin requirement of Kch. On the basis of some of our results, we discuss a function for Kch in the maintenance of the membrane potential in E. coli.

The hydrophobic human β 2 adrenergic receptor was produced in fusion to the hydrophilic maltose-binding protein ( Mal E) in Escherichia coli . Photoaffinity labeling with the adrenergic ligand [ 125 I]cyanopindolole-diazirine indicated that the majority of the protein was proteolyzed in the intergenic region between the fusion partners after production in E. coli . The simple and fast genetics of the bacterium enabled us to engineer a linker with an increased proteolytic stability. The fusion protein produced in E. coli was fully functional with respect to binding of adrenergic ligands and coupling to stimulatory GTP-binding protein. The production level with 3 pmol receptor fusion protein per mg membrane protein in a crude membrane preparation was significantly higher than those reported for other β 2 adrenergic receptor constructs in E. coli . After solubilization with dodecanoyl sucrose, the fusion protein was purified to near homogeneity by affinity chromatography on immobilized Ni 2+ ions (binding to a C-terminal His 6 -tag) and on crosslinked amylose (binding to the MalE). In order to achieve higher production levels, the fusion protein preceded by an insect signal peptide was produced in baculovirus-infected insect cells. As expected, the production level with about 17 pmol receptor per mg membrane protein was higher in the insect cells than in E. coli . The receptor fusion protein produced in the insect cells bound adrenergic ligands and activated heterotrimeric GTP-binding proteins with biochemical properties comparable to that of the unfused receptor.

The human CC chemokine macrophage inflammatory protein-1alpha (MIP-1alpha) was produced at a high level in Pichia pastoris under transcriptional control of the highly inducible alcohol oxidase 1 promoter. To ensure proper folding and secretion of the recombinant polypeptide, the MIP-1alpha gene had been fused to the Saccharomyces cerevisiae alpha-factor prepropeptide. As was revealed by analysis of the cell culture supernatant of recombinant Pichia pastoris, MIP-1alpha was efficiently secreted. Immunoblot analysis of secreted proteins from recombinant clones using a polyclonal antibody directed against MIP-1alpha revealed an apparent molecular mass of 8 kDa for the recombinant polypeptide. Up to 70 mg of MIP-1alpha was purified from 1 liter of yeast culture supernatant by a single chromatography step. Biological activity of recombinant MIP-1alpha was shown in a chemotaxis assay. Here, the polypeptide specifically induced migration of U937 cells expressing the CCR1 (MIP-1alpha receptor). Also, in competition binding assays the recombinant MIP-1alpha displayed high affinity binding.

To establish a procedure for reproducible production of a soluble enzymatically active form of rat ecto-5'-nucleotidase, we heterologously expressed the polypeptide of the mature protein in insect cells. An expression construct was created consisting of this polypeptide and glutathione-S-transferase from Schistosoma japonicum as a fusion partner. Although infected insect cells did not secrete the enzyme into the medium, considerable amounts of recombinant protein were detected in the whole cell extract. A fraction of soluble protein yielding 5'-nucleotidase activity could be purified from cells infected with recombinant baculovirus bearing the glutathione-S-transferase fusion construct. The catalytic properties of this recombinant protein correspond to those of the native protein isolated from animal tissues. Potential agonists and inhibitors of P2 receptors can function as inhibitors of ecto-5'-nucleotidase. The glutathione-S-transferase fusion protein may be used in experiments for drug screening, for the study of the interaction of immobilized ecto-5'-nucleotidase with other proteins, or for application in tissue culture experiments.

Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N- and C-terminally tagging with Flag and His6 epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [3H]MDL105,519 with high affinity (Kd = 5.22 +/- 0. 13 nM) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and D-serine but not by L-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptor in vivo.