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  • 1
    Description: This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a short illness. Victor is best known for his landmark advances in the subcellular fractionation of brain tissue which led to the isolation of synaptosomes and subsequently synaptic vesicles at the beginning of the 1960s and for the cellular and molecular analysis of the cholinergic synapse. This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a short illness. Victor is best known for his landmark advances in the subcellular fractionation of brain tissue which led to the isolation of synaptosomes and subsequently synaptic vesicles at the beginning of the 1960s and for the cellular and molecular analysis of the cholinergic synapse.
    ISSN: 0022-3042
    E-ISSN: 1471-4159
    Source: John Wiley & Sons, Inc.
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  • 2
    Language: English
    In: Journal of neurochemistry, October 2016, Vol.139(2), pp.333-335
    Description: This Obituary honors Victor P. Whittaker, one of the pioneers in the field of neurochemistry. Victor Whittaker died on 5th July 2016 aged 97 in Cambridge (UK) after a short illness. Victor is best known for his landmark advances in the subcellular fractionation of brain tissue which led to the isolation of synaptosomes and subsequently synaptic vesicles at the beginning of the 1960s and for the cellular and molecular analysis of the cholinergic synapse.
    Keywords: Neurochemistry -- History
    E-ISSN: 1471-4159
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  • 3
    Language: English
    In: The journal of physical chemistry. B, 16 June 2011, Vol.115(23), pp.7561-7
    Description: In this article, we present quantum chemical density functional theory (DFT) calculations of the NMR (13)C chemical shift (CS) tensors in 2,3,6,7,10,11-hexahexylthiotriphenylene (HHTT). The DFT calculations are performed on a smaller model molecule where the hexyl chains were reduced to methyl groups (HMTT). These tensors are compared with our previously reported experimental results carried out under magic-angle spinning (MAS) conditions. The phase diagram of HHTT is K ↔ H ↔ D(hd) ↔ I, where H is a helical phase and D(hd) is a columnar liquid crystal. The motivation for the present study was to explain experimentally observed and puzzling thermal history effects, which resulted in different behavior in the helical phase upon cooling and heating. In particular, the CS tensors for the aromatic carbons measured in the helical phase upon heating from the solid phase were essentially unaffected, while the cooling from the columnar liquid crystal resulted in a significant averaging. We investigate the effect on the CS tensors of (i) conformational transitions, and (ii) relative molecular orientations within the columns for dimer and trimer configurations. Finally a motional wobbling (PIZZA) model for the dynamic averaging of the CS tensor in the helical phase is suggested.
    Keywords: Liquid Crystals -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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  • 4
    Language: English
    In: The journal of physical chemistry. B, 16 June 2011, Vol.115(23), pp.7621-8
    Description: Isotope-labeled riboflavin in DMSO was employed in conjunction with femtosecond time-resolved infrared vibrational spectroscopy and quantum chemical calculations to analyze and assign the electronically excited state vibrational modes of the isoalloxazine unit as a prototype for the cofactors in flavin binding blue-light receptors. Using the riboflavin (13)C-analogues RF-2-(13)C and RF-4,10a-(13)C, the carbonyl vibrations, in particular, were studied. Various quantum chemical models were applied that take into account a polarizable environment or the impact of hydrogen bonds. The CIS quantum-chemistry method was successfully applied to describe the lowest singlet excited electronic state in riboflavin. The experimentally observed frequencies and isotope-shifts as well as their variability in the diverse model calculations are discussed. On these grounds, a consistent assignment of the electronic ground and excited state vibrations is presented.
    Keywords: Vibration ; Dimethyl Sulfoxide -- Chemistry ; Riboflavin -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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  • 5
    Language: English
    In: Analytical Biochemistry, Feb 1, 2014, Vol.446, p.53(6)
    Description: To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ab.2013.10.012 Byline: Marianne Freundlieb, Herbert Zimmermann, Christa E. Muller Abstract: Ecto-5'-nucleotidase (eN) is a membrane-bound enzyme that hydrolyzes extracellular nucleoside-5'-monophosphates yielding the respective nucleoside and phosphate. Increased levels of eN expression have been observed in many cancer cells. By increasing extracellular adenosine concentrations, they contribute to their proliferative, angiogenic, metastatic, and immunosuppressive effects. Therefore, eN is of considerable interest as a novel drug target for the treatment of cancer as well as of inflammatory diseases. In this study, we developed, optimized, and applied a highly sensitive radiometric assay using [.sup.3H]adenosine-5'-monophosphate (AMP) as a substrate. The reaction product [.sup.3H]adenosine was separated from [.sup.3H]AMP by precipitation of the latter with lanthanum chloride and subsequent filtration through glass fiber filters. Conditions were optimized to reproducibly collect the [.sup.3H]adenosine-containing filtrate used for quantitative determination. Validation of the assay yielded a mean Z' factor of 0.73, which demonstrates its suitability for high-throughput screening. The new assay shows a limit of detection that is at least 30-fold lower than those of common colorimetric methods (e.g., optimized malachite green assay and capillary electrophoresis-based assay procedures), and it is also superior to a recently developed luciferase-based assay. Article History: Received 22 July 2013; Revised 5 October 2013; Accepted 8 October 2013
    Keywords: Nucleotidases ; Precipitation (Meteorology)
    ISSN: 0003-2697
    Source: Cengage Learning, Inc.
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  • 6
    Language: English
    In: Journal of Neurochemistry, August, 2011, Vol.118(4), p.558(13)
    Description: To authenticate to the full-text of this article, please visit this link: http://dx.doi.org/10.1111/j.1471-4159.2011.07344.x Byline: Joern Barth, Herbert Zimmermann, Walter Volknandt Keywords: novel protein; synaptic vesicle; synaptic vesicle membrane protein; transporter Abstract: J. Neurochem. (2011) 118, 558-570. Abstract We recently identified in a proteomic screen a novel synaptic vesicle membrane protein of 31 kDa (SV31) of unknown function. According to its membrane topology and its phylogenetic relation SV31 may function as a vesicular transporter. Based on its amino acid sequence similarity to a prokaryotic heavy metal ion transporter we analyzed its metal ion-binding properties and show that recombinant SV31 binds the divalent cations Zn.sup.2+ and Ni.sup.2+ and to a minor extent Cu.sup.2+, but not Fe.sup.2+, Co.sup.2+, Mn.sup.2+, or Ca.sup.2+. Zn.sup.2+-binding of SV31 in viable cells was verified following heterologous transfection of pheochromocytoma cells 12 (PC12) with recombinant red fluorescent SV31 (SV31-RFP) and the fluorescent zinc indicator FluoZin-3. Sucrose density gradient fractionation of SV31-RFP-transfected PC12 cells revealed a partial overlap of SV31-RFP with synaptic-like vesicle markers and the early endosome marker rab5. Immunocytochemical analysis demonstrated a punctuate distribution in the cell soma and in neuritic processes and in addition in a compartment in vicinity to the plasma membrane that was immunopositive also for synaptosomal-associated protein 25 (SNAP-25) and syntaxin1A. Our data suggest that SV31 represents a novel Zn.sup.2+-binding protein that in PC12 cells is targeted to endosomes and subpopulations of synaptic-like microvesicles. Article History: Received April 8, 2011; revised manuscript received June 6, 2011; accepted June 6, 2011. Article note: Address correspondence and reprint requests to Joern Barth, JW-Goethe University, Biocenter, Institute of Cell Biology and Neuroscience, Neurochemistry, Max-von-Laue-Stra[sz]e 9, D-60438 Frankfurt/Main, Germany. E-mail: j.barth@em.uni-frankfurt.de
    Keywords: Protein Binding -- Chemical Properties ; Protein Binding -- Analysis ; Zinc Compounds -- Chemical Properties ; Zinc Compounds -- Analysis ; Cells (Biology) -- Chemical Properties ; Cells (Biology) -- Analysis
    ISSN: 0022-3042
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  • 7
    Language: English
    In: The journal of physical chemistry. B, 28 February 2013, Vol.117(8), pp.2280-93
    Description: The organization and orientation of membrane-inserted helices is important for better understanding the mode of action of membrane-active peptides and of protein-membrane interactions. Here we report on the application of ESEEM (electron spin-echo envelope modulation) and DEER (double electron-electron resonance) techniques to probe the orientation and oligomeric state of an α-helical trans-membrane model peptide, WALP23, under conditions of negative mismatch between the hydrophobic cores of the model membrane and the peptide. Using ESEEM, we measured weak dipolar interactions between spin-labeled WALP23 and (2)H nuclei of either the solvent (D2O) or of lipids specifically deuterated at the choline group. The ESEEM data obtained from the deuterated lipids were fitted using a model that provided the spin label average distance from a layer of (2)H nuclei in the hydrophilic region of the membrane and the density of the (2)H nuclei in the layer. DEER was used to probe oligomerization through the dipolar interaction between two spin-labels on different peptides. We observed that the center of WALP23 does not coincide with the bilayer midplane and its N-terminus is more buried than the C-terminus. In addition, the ESEEM data fitting yielded a (2)H layer density that was much lower than expected. The DEER experiments revealed the presence of oligomers, the presence of which was attributable to the negative mismatch and the electrostatic dipole of the peptide. A discussion of a possible arrangement of the individual helices in the oligomers that is consistent with the ESEEM and DEER data is presented.
    Keywords: Lipid Bilayers -- Chemistry ; Peptides -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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  • 8
    Language: English
    In: The journal of physical chemistry. B, 20 October 2011, Vol.115(41), pp.11950-63
    Description: We consider the state-of-the-art capabilities and future perspectives of electron-spin triangulation by high-field/high-frequency dipolar electron paramagnetic resonance (EPR) techniques designed for determining the three-dimensional structure of large supra-molecular complexes dissolved in disordered solids. These techniques combine double site-directed spin labeling (SDSL) with orientation-resolving pulsed electron-electron double resonance (PELDOR) spectroscopy. In particular, we appraise the prospects of angular triangulation, which extends the more familiar distance triangulation. As a model case for spin-labeled proteins, the three-dimensional structures of two nitroxide biradicals with rather stiff bridging blocks and deuterated nitroxide headgroups have been derived. To this end we applied 95 GHz high-field electron dipolar EPR spectroscopy with the microwave pulse-sequence configurations for PELDOR and relaxation-induced dipolar modulation enhancement (RIDME). Various specific spectroscopic strategies are discussed to overcome the problems of overlapping spectra of the chemically identical nitroxide labels when attached to macromolecular systems. We conclude that due to the high detection sensitivity and spectral resolution the combination of SDSL with high-field RIDME/PELDOR stands out as an extremely powerful tool for 3D structure determination of large disordered systems. The approach compares favorably with other structure-determining magnetic-resonance methods. This holds true both for stable and transient radical-pair states. Angular constraints are provided in addition to distance constraints obtained for the same sample. Thereby, the number of necessary distance constraints is strongly reduced. Since each measurement of a distance constraint requires an additional doubly spin-labeled sample, the reduction of necessary distance constraints is another appealing aspect of orientation-resolving EPR spin triangulation for protein structure determination.
    Keywords: Electron Spin Resonance Spectroscopy ; Nitric Oxide -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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  • 9
    Language: English
    In: The journal of physical chemistry. B, 12 January 2012, Vol.116(1), pp.179-88
    Description: Studies of membrane peptide interactions at the molecular level are important for understanding essential processes such as membrane disruption or fusion by membrane active peptides. In a previous study, we combined several electron paramagnetic resonance (EPR) techniques, particularly continuous wave (CW) EPR, electron spin echo envelope modulation (ESEEM), and double electron-electron resonance (DEER) with Monte Carlo (MC) simulations to probe the conformation, insertion depth, and orientation with respect to the membrane of the membrane active peptide melittin. Here, we combined these EPR techniques with cryogenic transmission electron microscopy (cryo-TEM) to examine the effect of the peptide/phospholipid (P/PL) molar ratio, in the range of 1:400 to 1:25, on the membrane shape, lipids packing, and peptide orientation and penetration. Large unilamellar vesicles (LUVs) of DPPC/PG (7:3 dipalmitoylphosphatidylcholine/egg phosphatidylglycerol) were used as model membranes. Spin-labeled peptides were used to probe the peptide behavior whereas spin-labeled phspholipids were used to examine the membrane properties. The cryo-TEM results showed that melittin causes vesicle rupture and fusion into new vesicles with ill-defined structures. This new state was investigated by the EPR methods. In terms of the peptide, CW EPR showed decreased mobility, and ESEEM revealed increased insertion depth as the P/PL ratio was raised. DEER measurements did not reveal specific aggregates of melittin, thus excluding the presence of stable, well-defined pore structures. In terms of membrane properties, the CW EPR reported reduced mobility in both polar head and alkyl chain regions with increasing P/PL. ESEEM measurements showed that, as the P/PL ratio increased, a small increase in water content in the PL headgroup region took place and no change was observed in the alkyl chains part close to the hydrophilic region. In terms of lipid local density, opposite behavior was observed for the polar head and alkyl chain regions with increasing P/PL; while the DPPC density increased in the polar head region, it decreased in the alkyl chain region. These results are consistent with disruption of the lipid order and segregation of the PL constituents of the membrane as a consequence of the melittin binding. This work further demonstrates the applicability and potential of pulse EPR techniques for the study of peptide-membrane interactions.
    Keywords: Electron Spin Resonance Spectroscopy ; Microscopy, Electron, Transmission ; Melitten -- Chemistry ; Unilamellar Liposomes -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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  • 10
    Language: English
    In: The journal of physical chemistry. B, 20 October 2011, Vol.115(41), pp.11793-804
    Description: The conformation and interconversion dynamics of two derivatives of the 18-membered hexathia metacyclophane 1 and 2 were studied by (1)H NMR spectroscopy in isotropic solvents and by (2)H NMR in chiral liquid crystalline (CLC) solutions, as well as by molecular structure computations. For the analysis of the dynamic effects, we made use of the concepts of "average symmetry" and "isodynamic groups", introduced by Altmann (Altmann, Proc. R. Soc.1967, 184, A298). Compound 1, which is unsubstituted in the inner aromatic site, has, according to the NMR and molecular force field calculations, a boat shaped ground conformation with C(2) symmetry. It is highly flexible and in the NMR spectrum exhibits two successive dynamic processes. There is a low temperature (170-210 K, E(a) = 10.5 kcal/mol) alternate "wing flipping", which corresponds to interchange between pairs of enantiomers and results, in the fast exchange limit, in an average prochiral molecule with C(2v) symmetry. This process is followed, at higher temperatures (290-320 K, E(a) = 28.5 kcal/mol), by an umbrella flipping type inversion with an average structure of D(2h) symmetry. This second process involves averaging of effective enantiotopic into homotopic sites and can only be studied in chiral solvents. The origin of the chiral discrimination and of their stepwise averaging is discussed. Compound 2, which is substituted with methoxy groups at the inner sites of the benzene rings, is much less flexible and exhibits dynamic effects in the NMR spectrum only at temperatures above 370 K. We were able to study the kinetic parameters of this process in isotropic solvents (E(a) = 21.4 kcal/mol). As for 1, the detailed mechanism of this process can in principle be established using dynamic NMR in CLC; however, experimental limitation precluded us from doing so. Possible alternatives and their effect on the 1D and 2D exchange spectra in CLC are discussed in a concluding section.
    Keywords: Magnetic Resonance Spectroscopy ; Liquid Crystals -- Chemistry ; Macrocyclic Compounds -- Chemistry
    ISSN: 15206106
    E-ISSN: 1520-5207
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