FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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Hydrogen bonding involving side chain exchangeable groups stabilizes amyloid quarternary structure
Agarwal, V., Linser, R., Dasari, M., Fink, U., del Amo, J. M.; Reif, B.
Phys Chem Chem Phys, 15:12551-12557

Tags: Solid-State NMR Spectroscopy (Reif)

Abstract: The amyloid beta-peptide (Abeta) is the major structural component of amyloid fibrils in the plaques of brains of Alzheimer's disease patients. Numerous studies have addressed important aspects of secondary and tertiary structure of fibrils. In electron microscopic images, fibrils often bundle together. The mechanisms which drive the association of protofilaments into bundles of fibrils are not known. We show here that amino acid side chain exchangeable groups like e.g. histidines can provide useful restraints to determine the quarternary assembly of an amyloid fibril. Exchangeable protons are only observable if a side chain hydrogen bond is formed and the respective protons are protected from exchange. The method relies on deuteration of the Abeta peptide. Exchangeable deuterons are substituted with protons, before fibril formation is initiated.

Dynamic nuclear polarization of spherical nanoparticles
Akbey, Ü., Altin(*), B., Linden, A., Ozcelik(*), S., Gradzielski(*), M.; Oschkinat, H.
Phys Chem Chem Phys, 15:20706-20716

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Spherical silica nanoparticles of various particle sizes (~10 to 100 nm), produced by a modified Stoeber method employing amino acids as catalysts, are investigated using Dynamic Nuclear Polarization (DNP) enhanced Nuclear Magnetic Resonance (NMR) spectroscopy. This study includes ultra-sensitive detection of surface-bound amino acids and their supramolecular organization in trace amounts, exploiting the increase in NMR sensitivity of up to three orders of magnitude via DNP. Moreover, the nature of the silicon nuclei on the surface and the bulk silicon nuclei in the core (sub-surface) is characterized at atomic resolution. Thereby, we obtain unique insights into the surface chemistry of these nanoparticles, which might result in improving their rational design as required for promising applications, e.g. as catalysts or imaging contrast agents. The non-covalent binding of amino acids to surfaces was determined which shows that the amino acids not just function as catalysts but become incorporated into the nanoparticles during the formation process. As a result only three distinct Q-types of silica signals were observed from surface and core regions. We observed dramatic changes of DNP enhancements as a function of particle size, and very small particles (which suit in vivo applications better) were hyperpolarized with the best efficiency. Nearly one order of magnitude larger DNP enhancement was observed for nanoparticles with 13 nm size compared to particles with 100 nm size. We determined an approximate DNP penetration-depth (~4.2 or ~5.7 nm) for the polarization transfer from electrons to the nuclei of the spherical nanoparticles. Faster DNP polarization buildup was observed for larger nanoparticles. Efficient hyperpolarization of such nanoparticles, as achieved in this work, can be utilized in applications such as magnetic resonance imaging (MRI).

A Floquet description of phase alternated sequences for efficient homonuclear recoupling in solid perdeuterated systems
Jayanthi(*), S., Akbey, Ü., Uluca(*), B., Oschkinat, H.; Vega(*), S.
Journal of Magnetic Resonance, 234:10-20

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: A Floquet description of a phase alternated homonuclear recoupling scheme for perdeuterated systems is presented. As a result, we demonstrate improvements in the recoupling efficiency of the DOuble Nucleus Enhanced Recoupling [DONER; J. Am. Chem. Soc. 131 (2009) 170541 technique by utilizing Phase Alternated Recoupling Irradiation Schemes [PARIS; Chem. Phys. Lett. 469 (2009) 342]. The effect of proton and deuterium radio frequency irradiation during recoupling has been systematically studied and theoretical observations have been verified experimentally using a deuterated model compound, L-Alanine, at 10 and 20 kHz magic angle spinning frequency. Experimental results are well in agreement with theoretical observations, thereby significantly increasing the recoupling efficiency of conventional DONER in perdeuterated systems. (C) 2013 Elsevier Inc. All rights reserved.

CRIS-A Novel cAMP-Binding Protein Controlling Spermiogenesis and the Development of Flagellar Bending
Krähling(*), A. M., Alvarez(*), L., Debowski(*), K., Van(*), Q., Gunkel(*), M., Irsen(*), S., Al-Amoudi(*), A., Strünker(*), T., Kremmer(*), E., Krause, E., Voigt(*), I., Wortge(*), S., Waisman(*), A., Weyand(*), I., Seifert(*), R., Kaupp(*), U. B.; Wachten(*), D.
Plos Genet, 9

Tags: Mass Spectrometry (Krause, E.)

Abstract: The second messengers cAMP and cGMP activate their target proteins by binding to a conserved cyclic nucleotide-binding domain (CNBD). Here, we identify and characterize an entirely novel CNBD-containing protein called CRIS (cyclic nucleotide receptor involved in sperm function) that is unrelated to any of the other members of this protein family. CRIS is exclusively expressed in sperm precursor cells. Cris-deficient male mice are either infertile due to a lack of sperm resulting from spermatogenic arrest, or subfertile due to impaired sperm motility. The motility defect is caused by altered Ca2+ regulation of flagellar beat asymmetry, leading to a beating pattern that is reminiscent of sperm hyperactivation. Our results suggest that CRIS interacts during spermiogenesis with Ca2+-regulated proteins that-in mature sperm-are involved in flagellar bending.

PI4K2beta/AP-1-based TGN-endosomal sorting regulates Wnt signaling
Wieffer, M., Cibrian Uhalte(*), E., Posor, Y., Otten(*), C., Branz, K., Schütz, I., Mössinger, J., Schu(*), P., Abdelilah-Seyfried(*), S., Krauss, M.; Haucke, V.
Curr Biol, 23:2185-2190

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2beta (PI4K2beta) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2beta and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2beta or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2beta-AP-1 complex formation and PI4K2beta-mediated PI(4)P synthesis. Furthermore, PI4K2beta binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2beta and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

KSHV ORF67 encoded lytic protein localizes on the nuclear membrane and alters emerin distribution
Farina(*), A., Santarelli(*), R., Bloise(*), R., Gonnella(*), R., Granato(*), M., Bei(*), R., Modesti(*), A., Cirone(*), M., Bengtsson, L., Angeloni(*), A.; Faggioni(*), A.
Virus Res, 175:143-150

Tags: Physiology and Pathology of Ion Transport (Jentsch)

Abstract: p29, a newly identified Kaposi's sarcoma-associated herpesvirus (KSHV) protein, is the product of ORF67, the positional homolog of the conserved herpesvirus protein UL34. Like its homologues in other herpesviruses, p29 is expressed early during viral lytic cycle, and is localized on the nuclear rim. Upon chemical induction of viral replication in primary effusion lymphoma cells, p29 interacts with p33, encoded by ORF69, the positional homolog of the conserved herpesvirus protein UL31, and both proteins colocalize on the nuclear membrane. IFA and biochemical analysis of infected or transfected cells showed that p29 expression resulted in delocalization and hyperphosphorylation of emerin, whereas other nuclear lamin associated proteins, such as LUMA, LB1 and LBR were not affected. Mislocalization of emerin was robustly increased upon combined expression of p29 and p33, suggesting that emerin destabilization might represent the first step in nuclear lamina disassembling, a process necessary for nucleocapsid maturation. (C) 2013 Elsevier B.V. All rights reserved.

Crimean-Congo hemorrhagic fever virus utilizes a clathrin- and early endosome-dependent entry pathway
Garrison(*), A. R., Radoshitzky(*), S. R., Kota(*), K. P., Pegoraro(*), G., Ruthel(*), G., Kuhn(*), J. H., Altamura(*), L. A., Kwilas(*), S. A., Bavari(*), S., Haucke, V.; Schmaljohn(*), C. S.
Virology, 444:45-54

Tags: Molecular Pharmacology and Cell Biology (Haucke)

Abstract: The early events in Crimean-Congo hemorrhagic fever virus (CCHFV) have not been completely characterized. Earlier work indicated that CCHFV likely enters cells by clathrin-mediated endocytosis (CME). Here we provide confirmatory evidence for CME entry by showing that CCHFV infection is inhibited in cells treated with Pitstop 2, a drug that specifically and reversibly interferes with the dynamics of clathrin-coated pits. Additionally, we show that CCHFV infection is inhibited by siRNA depletion of the clathrin pit associated protein AP-2. Following CME entry, we show that CCHFV has a pH-dependent entry step, with virus inactivation occurring at pH 6.0 and below. To more precisely define the endosomal trafficking of CCHFV, we show for the first time that overexpression of the dominant negative forms of Rab5 protein but not Rab7 protein inhibits CCHFV infection. These results indicate that CCHFV likely enters cells through the early endosomal compalunent. Published by Elsevier Inc.

Characterization of a chip-based bioreactor for three-dimensional cell cultivation via Magnetic Resonance Imaging
Gottwald(*), E., Kleintschek(*), T., Giselbrecht(*), S., Truckenmuller(*), R., Altmann(*), B., Worgull(*), M., Döpfert, J., Schad(*), L.; Heilmann(*), M.
Z Med Phys, 23:102-110

Tags: Molecular Imaging (Schröder)

Abstract: We describe the characterization of a chip-based platform (3(D)-KITChip) for the three-dimensional cultivation of cells under perfusion conditions via magnetic resonance imaging (MRI). Besides the chip, the microfluidic system is comprised of a bioreactor housing, a medium supply, a pump for generating active flow conditions as well as a gas mixing station. The closed circulation loop is ideally suited for a characterization via MRI since the small bioreactor setup with active perfusion, driven by the pump from outside the coils, not only is completely MRI-compatible but also can be transferred into the magnetic coil of an experimental animal scanner. We have found that the two halves of the chip inside the bioreactor are homogeneously perfused with cell culture medium both with and without cells inside the 3(D)-KITChip. In addition, the homogeneity of perfusion is nearly independent from the flow rates investigated in this study, and furthermore, the setup shows excellent washout characteristics after spiking with Gadolinium-DOTA which makes it an ideal candidate for drug screening purposes. We, therefore, conclude that the 3(D)-KITChip is well suited as a platform for high-density three-dimensional cell cultures, especially those requiring a defined medium flow and/or gas supply in a precisely controllable three dimensional environment, like stem cells.

Electrospray Ionization Mass Spectrometry Reveals an Unexpected Coupling Product in the Copper-Promoted Synthesis of Pyrazoles
Hyvl(*), J., Agrawal, D., Pohl(*), R., Suri(*), M., Glorius(*), F.; Schröder(*), D.
Organometallics, 32:807-816

Tags: Chemical Biology II (Hackenberger)

Abstract: The reaction mechanism of the intermolecular oxidative formation of pyrazole 2 via a C-C/N-N bond-formation cascade of the enaminone 1 is investigated by means of ESI-MS. No direct reaction intermediates are observed; however, the formation of an unexpected imidazolid-3-one derivative X is observed that involves an oxidative dimerization of 1 in the presence of dioxygen. The derivative X is isolated and characterized by means of multidimensional H-1 and C-13 NMR spectroscopy.

Lipophilic prodrugs of a triazole-containing colchicine analogue in liposomes: Biological effects on human tumor cells
Kuznetsova(*), N. R., Svirshchevskaya(*), E. V., Sitnikov(*), N. S., Abodo(*), L., Sutorius(*), H., Zapke, J., Velder(*), J., Thomopoulou(*), P., Oschkinat, H., Prokop(*), A., Schmalz(*), H. G., Fedorov(*), A. Y.; Vodovozova(*), E. L.
Russ J Bioorg Chem+, 39:543-552

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Colchicine site binders-blockers of tubulin polymerization-are potential antimitotic agents for anticancer therapy. To reduce their systemic toxicity and improve biodistribution, encapsulation in nanosized liposomes may be employed. Liposomes present a convenient means for preparation of injectable for-mulations of hydrophobic compounds, however colchicine as such is known to leak through the lipid bilayer. In this study, newly synthesized triazole-containing analogues of colchicine and allocolchicine, and their palmitic and oleic esters (lipophilic prodrugs) were tested for anti-proliferative activity and apoptosis-inducing potential. In contrast to colchicine conjugates, whose activities ranged with those of colchicine, allocolchicine derivatives exhibited drastically lower effects and were discarded. Liposomes of about 100 nm in diameter composed of egg phosphatidylcholine-yeast phosphatidylinositol-palmitic or oleic prodrug, 8: 1: 1, by mol, were prepared by standard extrusion technique and tested in a panel of four human tumor cell lines. Liposome formulations preserved the biological activities of the parent colchicinoid the most towards human epithelial tumor cells. Moreover, liposomal form of the oleoyl bearing colchicinoid inhibited cell proliferation more efficiently than free lipophilic prodrug. Due to substantial loading capacity of the liposomes, the dispersions contain sufficient concentration of the active agent to test wide dose range in experiments on systemic administration to animals.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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