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Al-Samir S.,Zentrum Physiologie | Papadopoulos S.,Universitatsklinikum Cologne | Scheibe R.J.,Institute For Physiologische Chemie | Meissner J.D.,Zentrum Physiologie | And 5 more authors.
Journal of Physiology | Year: 2013

We have investigated the previously published 'metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3 - permeability sensitive to inhibition by 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII-AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII-AE1 metabolon to erythroid AE1-mediated HCO3 - transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3 - and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H+ makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm. © 2013 The Authors. Source

Michael G.B.,Institute For Nutztiergenetik | Freitag C.,Institute For Nutztiergenetik | Fessler A.T.,Institute For Nutztiergenetik | Wendlandt S.,Institute For Nutztiergenetik | And 8 more authors.
Berliner und Munchener Tierarztliche Wochenschrift | Year: 2014

In the light of frequent discussions about the correct performance of in vitro susceptibility testing and the interpretation of the results obtained, the aim of the present report is to summarize basic facts that may facilitate the understanding of this complex topic. For this, the terms „antimicrobial resistance“, „ESBL“, and „MRSA“ are defined. Besides the statements on antimicrobial resistance, information on intrinsic and acquired resistance properties as well as basic rules for the correct performance of antimicrobial susceptibility testing in routine diagnostics are presented. Moreover, the two groups of interpretive criteria – clinical breakpoints and epidemiological cut-off values – including their applications are explained in detail. Furthermore, currently valid diagnostic procedures – as published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI) – for the screening of ESBL-producing Enterobacteriaceae and MRSA as well as for the confirmation of suspicious isolates are presented and compared. Based on the information given, it becomes obvious that the correct performance of the diagnostic tests, which includes strict following the performance standards and the detailed information given therein, is an indispensable prerequisite for a standardized and harmonized in vitro susceptibility testing and – as a consequence – for the determination of valid and reliable susceptibility data in routine diagnostics. This is of utmost importance since the susceptibility data based on the use of clinical breakpoints often represent the basis for therapeutic interventions. © 2014 Schlütersche Verlagsgesellschaft mbH & Co. KG. Source

Sikorra S.,Institute For Physiologische Chemie | Litschko C.,Institute For Physiologische Chemie | Muller C.,Institute For Physiologische Chemie | Thiel N.,Institute For Physiologische Chemie | And 3 more authors.
Journal of Molecular Biology | Year: 2016

Botulinum neurotoxins (BoNTs) are highly potent bacterial proteins that block neurotransmitter release at the neuromuscular junction by cleaving SNAREs (soluble N-ethyl maleimide sensitive factor attachment protein receptors). However, their serotype A (BoNT/A) that cleaves SNAP-25 (synaptosomal-associated protein of 25 kDa) has also been an established pharmaceutical for treatment of medical conditions that rely on hyperactivity of cholinergic nerve terminals for 25 years. The expansion of its use to a variety of further medical conditions associated with hypersecretion components is prevented partly because the involved SNARE isoforms are not cleaved. Therefore, we examined by mutational analyses the reason for the resistance of human SNAP-23, an isoform of SNAP-25. We show that replacement of 10 SNAP-23 residues with their SNAP-25 counterparts effects SNAP-25-like cleavability. Conversely, transfer of each of the replaced SNAP-23 residues to SNAP-25 drastically decreased the cleavability of SNAP-25. By means of the existing SNAP-25-toxin co-crystal structure, molecular dynamics simulations, and corroborative mutagenesis studies, the appropriate binding pockets for these residues in BoNT/A were characterized. Systematic mutagenesis of two major BoNT/A binding pockets was conducted in order to adapt these pockets to corresponding amino acids of human SNAP-23. Human SNAP-23 cleaving mutants were isolated using a newly established yeast-based screening system. This method may be useful for engineering novel BoNT/A pharmaceuticals for the treatment of diseases that rely on SNAP-23-mediated hypersecretion. © 2015 Elsevier Ltd. All rights reserved. Source

Willjes G.,Institute For Physiologische Chemie | Mahrhold S.,Institute For Physiologische Chemie | Strotmeier J.,Institute For Toxikologie | Eichner T.,Brandeis University | And 2 more authors.
Biochemistry | Year: 2013

Botulinum neurotoxins (BoNTs) block neurotransmitter release by proteolyzing SNARE proteins in peripheral nerve terminals. Entry into neurons occurs subsequent to interaction with gangliosides and a synaptic vesicle protein. Isoforms I and II of synaptotagmin were shown to act as protein receptors for two of the seven BoNT serotypes, BoNT/B and BoNT/G, and for mosaic-type BoNT/DC. BoNT/B and BoNT/G exhibit a homologous binding site for synaptotagmin whose interacting part adopts helical structure upon binding to BoNT/B. Whereas the BoNT/B-synaptotagmin-II interaction has been elucidated in molecular detail, corresponding information about BoNT/G is lacking. Here we systematically mutated the synaptotagmin binding site in BoNT/G and performed a comparative binding analysis with mutants of the cell binding subunit of BoNT/B. The results suggest that synaptotagmin takes the same overall orientation in BoNT/B and BoNT/G governed by the strictly conserved central parts of the toxins' binding site. The surrounding nonconserved areas differently contribute to receptor binding. Reciprocal mutations Y1186W and L1191Y increased the level of binding of BoNT/G approximately to the level of BoNT/B affinity, suggesting a similar synaptotagmin-bound state. The effects of the mutations were confirmed by studying the activity of correspondingly mutated full-length BoNTs. On the basis of these data, molecular modeling experiments were employed to reveal an atomistic model of BoNT/G-synaptotagmin recognition. These data suggest a reduced length and/or a bend in the C-terminal part of the synaptotagmin helix that forms upon contact with BoNT/G as compared with BoNT/B and are in agreement with the data of the mutational analyses. © 2013 American Chemical Society. Source

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