Refine
Year of publication
- 2014 (48) (remove)
Document Type
- Article (31)
- Doctoral Thesis (14)
- Conference Proceeding (3)
Language
- English (48) (remove)
Has Fulltext
- yes (48)
Is part of the Bibliography
- no (48) (remove)
Keywords
- 1,2,3-triazole-acyclonucleosides (1)
- 1,2,4-thiadiazoles (1)
- 3D EM (1)
- ABC Transporter (1)
- Adherens junctions (1)
- Antigen Processing (1)
- Archaea (1)
- Arduengo-type carbene (1)
- C-H...Br hydrogen bond (1)
- CCR5 (1)
Institute
- Biochemie und Chemie (48) (remove)
Heme-copper oxidases (HCOs) are the terminal enzymes of the aerobic respiratory chain in the inner mitochondrial membrane or the plasma membrane in many prokaryotes. These multi-subunit membrane protein complexes catalyze the reduction of oxygen to water, coupling this exothermic reaction to the establishment of an electrochemical proton gradient across the membrane in which they are embedded. The energy stored in the electrochemical proton gradient is used e.g. by the FOF1-ATP synthase to generate ATP from ADP and inorganic phosphate. The superfamily of HCOs is phylogenetically classified into three major families: A, B and C. The A-family HCOs, represented by the well-studied aa3-type cytochrome c oxidases (aa3-CcOs), are found in mitochondria and many bacteria. The B-family of HCOs contains a number of bacterial and archaeal oxidases. The C-family comprises only the cbb3-type cytochrome c oxidase (cbb3-CcO) and is most distantly related to the mitochondrial respiratory oxidases.
Background: Advanced non-small cell lung cancer (NSCLC) represents a significant unmet medical need. Despite advances with targeted therapies in a small subset of patients, fewer than 20% of patients survive for more than two years after diagnosis. Cancer vaccines are a promising therapeutic approach that offers the potential for durable responses through the engagement of the patient's own immune system. CV9202 is a self-adjuvanting mRNA vaccine that targets six antigens commonly expressed in NSCLC (NY ESO-1, MAGEC1, MAGEC2, 5 T4, survivin, and MUC1).
Methods/Design: The trial will assess the safety and tolerability of CV9202 vaccination combined with local radiation designed to enhance immune responses and will include patients with stage IV NSCLC and a response or stable disease after first-line chemotherapy or therapy with an EGFR tyrosine kinase inhibitor. Three histological and molecular subtypes of NSCLC will be investigated (squamous and non-squamous cell with/without EGFR mutations). All patients will receive two initial vaccinations with CV9202 prior to local radiotherapy (5 GY per day for four successive days) followed by further vaccinations until disease progression. The primary endpoint of the study is the number of patients experiencing Grade >3 treatment-related adverse events. Pharmacodynamic analyses include the assessment of immune responses to the antigens encoded by CV9202 and others not included in the panel (antigen spreading) and standard efficacy assessments.
Discussion: RNActive self-adjuvanted mRNA vaccines offer the potential for simultaneously inducing immune responses to a wide panel of antigens commonly expressed in tumors. This trial will assess the feasibility of this approach in combination with local radiotherapy in NSCLC patients.
The asymmetric unit of the title compound, C28H42N2O5·H2O, consists of one half of the organic molecule and one half-molecule of water, both of which are located on a mirror plane which passes through the central C atoms and the hydroxyl group of the heterocyclic system. The hydroxyl group at the central ring is disordered over two equally occupied positions. The six-membered ring adopts a chair conformation, and the 2-hydroxybenzyl substituents occupy the sterically preferred equatorial positions. The aromatic rings make dihedral angles of 75.57 (9)° with the mean plane of the heterocyclic ring. The dihedral angle between the two aromatic rings is 19.18 (10)°. The molecular structure features two intramolecular phenolic O-H...N hydrogen bonds with graph-set motif S(6). In the crystal, molecules are connected via O-H...O hydrogen bonds into zigzag chains running along the a-axis direction.
Na+/H+ antiporters are integral membrane proteins that are present in almost every cell and in every kingdom of life. They are essential for the regulation of intracellular pH-value, Na+-concentration and cell volume. These secondary active transporters exchange sodium ions against protons via an alternating access mechanism, which is not understood in full detail. Na+/H+ antiporters show distinct species-specific transport characteristics and regulatory properties that correlate with respective physiological functions. Here we present the characterization of the Na+/H+ antiporter NhaA from Salmonella enterica serovar Thyphimurium LT2, the causing agent of food-born human gastroenteritis and typhoid like infections. The recombinant antiporter was functional in vivo and in vitro. Expression of its gene complemented the Na+-sensitive phenotype of an E. coli strain that lacks the main Na+/H+ antiporters. Purified to homogeneity, the antiporter was a dimer in solution as accurately determined by size-exclusion chromatography combined with multi-angle laser-light scattering and refractive index monitoring. The purified antiporter was fully capable of electrogenic Na+(Li+)/H+-antiport when reconstituted in proteoliposomes and assayed by solid-supported membrane-based electrophysiological measurements. Transport activity was inhibited by 2-aminoperimidine. The recorded negative currents were in agreement with a 1Na+(Li+)/2H+ stoichiometry. Transport activity was low at pH 7 and up-regulation above this pH value was accompanied by a nearly 10-fold decrease of KmNa (16 mM at pH 8.5) supporting a competitive substrate binding mechanism. K+ does not affect Na+ affinity or transport of substrate cations, indicating that selectivity of the antiport arises from the substrate binding step. In contrast to homologous E. coli NhaA, transport activity remains high at pH values above 8.5. The antiporter from S. Typhimurium is a promising candidate for combined structural and functional studies to contribute to the elucidation of the mechanism of pH-dependent Na+/H+ antiporters and to provide insights in the molecular basis of species-specific growth and survival strategies.
Bacteria have adapted their NhaA Na(+)/H(+) exchangers responsible for salt homeostasis to their different habitats. We present an electrophysiological and kinetic analysis of NhaA from Helicobacter pylori and compare it to the previously investigated exchangers from Escherichia coli and Salmonella typhimurium. Properties of all three transporters are described by a simple model using a single binding site for H(+) and Na(+). We show that H.pylori NhaA only has a small acidic shift of its pH-dependent activity profile compared to the other transporters and discuss why a more drastic change in its pH activity profile is not physiologically required.
The title solvated salt, C29H41N2+·Br-·2CH2Cl2 was obtained from the reaction of the Arduengo-type carbene 1,3-bis(2,6-diisopropylphenyl)-1,3-dihydro-4,5-dimethyl-2H-imidazol-2-ylidene with Si2Br6 in dichloromethane. The complete cation is generated by a crystallographic mirror plane and the dihedral angle between the five-membered ring and the benzene ring is 89.8 (6)°; the dihedral angle between the benzene rings is 40.7 (2)°. The anion also lies on the mirror plane and both dichloromethane molecules are disordered across the mirror plane over two equally occupied orientations. In the crystal, the cations are linked to the anions via C-H...Br hydrogen bonds.
pH and Na+ homeostasis in all cells requires Na+/H+ antiporters. The crystal structure, obtained at pH 4, of NhaA, the main antiporter of Escherichia coli, has provided general insights into an antiporter mechanism and its unique pH regulation. Here, we describe a general method to select various NhaA mutants from a library of randomly mutagenized NhaA. The selected mutants, A167P and F267C are described in detail. Both mutants are expressed in Escherichia coli EP432 cells at 70–95% of the wild type but grow on selective medium only at neutral pH, A167P on Li+ (0.1 M) and F267C on Na+ (0.6 M). Surprising for an electrogenic secondary transporter, and opposed to wild type NhaA, the rates of A167P and F267C are almost indifferent to membrane potential. Detailed kinetic analysis reveals that in both mutants the rate limiting step of the cation exchange cycle is changed from an electrogenic to an electroneutral reaction.
Correlative microscopy incorporates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Several approaches exist for correlative microscopy, most of which have used the green fluorescent protein (GFP) as the label for light microscopy. Here we use chemical tagging and synthetic fluorophores instead, in order to achieve protein-specific labeling, and to perform multicolor imaging. We show that synthetic fluorophores preserve their post-embedding fluorescence in the presence of uranyl acetate. Post-embedding fluorescence is of such quality that the specimen can be prepared with identical protocols for scanning electron microscopy (SEM) and transmission electron microscopy (TEM); this is particularly valuable when singular or otherwise difficult samples are examined. We show that synthetic fluorophores give bright, well-resolved signals in super-resolution light microscopy, enabling us to superimpose light microscopic images with a precision of up to 25 nm in the x-y plane on electron micrographs. To exemplify the preservation quality of our new method we visualize the molecular arrangement of cadherins in adherens junctions of mouse epithelial cells.
In the search for novel organic charge transfer salts with variable degrees of charge transfer we have studied the effects of two modifications of the recently synthesized donor–acceptor system [tetramethoxypyrene (TMP)]–[tetracyanoquinodimethane (TCNQ)]. One is of chemical nature by substituting the acceptor TCNQ molecules by F4TCNQ molecules. The second consists in simulating the application of uniaxial pressure along the stacking axis of the system. In order to test the chemical substitution, we have grown single crystals of the TMP–F4TCNQ complex and analyzed its electronic structure via electronic transport measurements, ab initio density functional theory (DFT) calculations and UV/VIS/IR absorption spectroscopy. This system shows an almost ideal geometrical overlap of nearly planar molecules stacked alternately (mixed stack) and this arrangement is echoed by a semiconductor-like transport behavior with an increased conductivity along the stacking direction. This is in contrast to TMP–TCNQ which shows a less pronounced anisotropy and a smaller conductivity response. Our band structure calculations confirm the one-dimensional behavior of TMP–F4TCNQ with pronounced dispersion only along the stacking axis. Infrared measurements illustrating the C[triple bond, length as m-dash]N vibration frequency shift in F4TCNQ suggest however no improvement in the degree of charge transfer in TMP–F4TCNQ with respect to TMP–TCNQ. In both complexes about 0.1e is transferred from TMP to the acceptor. Concerning the pressure effect, our DFT calculations on the designed TMP–TCNQ and TMP–F4TCNQ structures under different pressure conditions show that application of uniaxial pressure along the stacking axis of TMP–TCNQ may be the route to follow in order to obtain a much more pronounced charge transfer.
5-Lipoxygenase (5LO) is a key enzyme in biosynthesis of leukotrienes (LTs), lipid mediators of inflammation. To study the roles of the 5LO accessory proteins coactosin-like protein (CLP) and 5LO-activating protein (FLAP), we knocked down these proteins in human monocytic cells. Our results show that expression of CLP was required for full cellular 5LO activity when cells were activated with Ca2+ ionophore, as well as with a physiological stimulus (lipopolysaccharide followed by N-formylmethionyl-leucyl-phenylalanine). During LT biosynthesis in stimulated cells, 5LO typically translocates to the nuclear membrane. This redistribution, from cytosolic to perinuclear, was clearly compromised in both CLP- and FLAP-deficient cells. Our results suggest that the CLP–5LO interaction may be a target for reduced LT production.