Institute For Physiologische Chemie

Hannover, Germany

Institute For Physiologische Chemie

Hannover, Germany
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Ribeiro J.P.,French National Center for Scientific Research | Diercks T.,CIC bioGUNE | Jimenez-Barbero J.,CIC bioGUNE | Jimenez-Barbero J.,Ikerbasque | And 3 more authors.
Biomolecules | Year: 2015

The characterization of the binding of reducing carbohydrates present as mixtures of anomers in solution to a sugar recepor (lectin) poses severe difficulties. In this situation, NMR spectroscopy enables the observation of signals for each anomer in the mixture by applying approaches based on ligand observation. Saturation transfer difference (STD) NMR allows fast and efficient screening of compound mixtures for reactivity to a receptor. Owing to the exceptionally favorable properties of 19F in NMR spectroscopy and the often complex 1H spectra of carbohydrates, 19F-containing sugars have the potential to be turned into versatile sensors for recognition. Extending the recently established 1H → 1H STDre19F-NMR technique, we here demonstrate its applicability to measure anomeric selectivity of binding in a model system using the plant lectin concanavalin A (ConA) and 2-deoxy-2-fluoro-D-mannose. Indeed, it is also possible to account for the mutual inhibition between the anomers on binding to the lectin by means of a kinetic model. The monitoring of 19F-NMR signal perturbation disclosed the relative activities of the anomers in solution and thus enabled the calculation of their binding affinity towards ConA. The obtained data show a preference for the α anomer that increases with temperature. This experimental approach can be extended to others systems of biomedical interest by testing human lectins with suitably tailored glycan derivatives. © 2015 by the authors; licensee MDPI, Basel, Switzerland.


Meyer W.,Anatomisches Institute | Schoennagel B.,Anatomisches Institute | Busche R.,Institute For Physiologische Chemie | Steinhagen D.,Abteilung Fischkrankheiten und Fischhaltung
Kleintierpraxis | Year: 2010

The study describes the keratinisation of the oesophageal epithelium of fish from a comparative point of view, as related to the nutritional biology of three species (carnivorous: Rainbow trout [Oncorhynchus mykiss]; plantivorous: Common rudd [Scardinius ertythropthalmus]; omnivorous: Common carp [Cyprinus carpid]), based on histological and different histochemical methods (demonstration of glyco-conjugates, and thiol and disulfide groups; immunohistochemical demonstration of proliferation proteins and cytokeratins). Independent of this approach, the basic structure of the epithelium is also analysed and discussed. The oesophageal epithelium exhibited a similar basic structure in all three species; namely, that of a highly prismatic monolayer system. The trout showed only one cell type with a large basal cytoplasm and a small apical mucous zone, whereas the rudd and the carp had two different cell types (slender highly prismatic cells with a border of microvilli and thin goblet cells). The trout produced a somewhat neutral mucus, while the goblet cells of rudd and carp had a predominantly acid mucus. The tunica mucosa generally had high longitudinal folds. A lamina propria was only distinct in the trout in addition to a strong lamina muscularis mucosae.The tunica submucosa was indistinct in both the rudd and carp, though the carp had a very prominent tunica muscularis. The highly prismatic epithelial cells reacted ubiquitously with a weak to medium staining for thiol groups, producing a homogeneous cytoplasmic staining. The reactions for disulfide groups were negative.The immunohistochemical demonstration of PCNA (proliferating cell nuclear antigen) resulted in 0.5-1% positive nuclei in the cells of the stratum basale in both the carnivorous trout and the omnivorous carp, while there were 2-8% in the plantivorous rudd. The KI-67 reaction (a cellular marker for proliferation) was only positive in the rudd, whereby the nuclei in cells near the lumen and in the tips of the intestinal villi were stained. The demonstration of pan-keratin produced a medium to very strongly positive cellular reaction in all three species. In the trout, the cytoplasm near the cell membrane was strongly stained. In comparison, the epithelial cells of the rudd showed a strongly reacting apical border region, whereas the cytoplasm in the carp appeared homogeneously darkly stained. All three species reacted positively for cytokeratin (CK6), with the strongest staining in the basal cytoplasm of the trout. The plantivorous rudd showed only a weak reaction, while the omnivorous carp reacted very strongly positive for this cytokeratin. A positive CK6 staining was also obvious in the muscle cells of the lamina muscularis mucosae of the trout. The carp, in contrast, showed a reaction only in the tunica muscularis. CK13 was negative in all three species. The study's results are discussed regarding the different aspects of the nutritional biology of the species studied, whereby a close relationship to the main type of feeding is obvious. The highly prismatic cells of the oesophageal epithelium of fishes have to endure strongly variable burdens caused by the specific type of nutrition. The associated mechanical stress may, at the same time, activate proliferative and anti-apoptotic cell signals.


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.


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.


PubMed | Institute For Physiologische Chemie, University of Bari and University of Catanzaro
Type: | Journal: Data in brief | Year: 2016

The data reported herein are related to the article entitled: The switching mechanism of the mitochondrial ADP/ATP carrier explored by free-energy landscapes (Pietropaolo et al., 2016) [1]. We report the coordinates of the ADP/ATP carrier (AAC2) in the presence and absence of adenine and guanine nucleotides in the c-, intermediate- and m-states obtained from the free-energy simulations and corresponding to the free-energy minima.


PubMed | Institute For Physiologische Chemie, University of Bari and University of Catanzaro
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2016

The ADP/ATP carrier (AAC) of mitochondria has been an early example for elucidating the transport mechanism alternating between the external (c-) and internal (m-) states (M. Klingenberg, Biochim. Biophys. Acta 1778 (2008) 1978-2021). An atomic resolution crystal structure of AAC is available only for the c-state featuring a three repeat transmembrane domain structure. Modeling of transport mechanism remained hypothetical for want of an atomic structure of the m-state. Previous molecular dynamics studies simulated the binding of ADP or ATP to the AAC remaining in the c-state. Here, a full description of the AAC switching from the c- to the m-state is reported using well-tempered metadynamics simulations. Free-energy landscapes of the entire translocation from the c- to the m-state, based on the gyration radii of the c- and m-gates and of the center of mass, were generated. The simulations revealed three free-energy basins attributed to the c-, intermediate- and m-states separated by activation barriers. These simulations were performed with the empty and with the ADP- and ATP-loaded AAC as well as with the poorly transported AMP and guanine nucleotides, showing in the free energy landscapes that ADP and ATP lowered the activation free-energy barriers more than the other substrates. Upon binding AMP and guanine nucleotides a deeper free-energy level stabilized the intermediate-state of the AAC2 hampering the transition to the m-state. The structures of the substrate binding sites in the different states are described producing a full picture of the translocation events in the AAC.


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.


PubMed | Institute For Physiologische Chemie, Brandeis University and University Paris Diderot
Type: Journal Article | Journal: 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.


Hanke N.,Zentrum Physiologie | Kubis H.-P.,Bangor University | Scheibe R.J.,Institute For Physiologische Chemie | Berthold-Losleben M.,Zentrum Physiologie | And 3 more authors.
American Journal of Physiology - Cell Physiology | Year: 2010

We have studied the mechanism by which a previously described primary muscle culture growing on microcarriers predominantly expresses fast myosin heavy chain (MHC) IId/x. We have measured MHC IId/x mRNA and protein levels, mRNA of MHC I and markers of muscle metabolism, insulin-like growth factor (IGF)-1 and mechano-growth factor (MGF) transcripts, indicators of the activation of the Akt-mammalian target of rapamycin (mTOR) axis, the p38-, ERK1/2-, and JNK-mitogen-activated protein kinase (MAP) kinase pathways, and of protein phosphatase PP2A, and we have assessed the involvement of integrin. By placing the culture flasks on a rotary shaker, we induce a continuous motion of the culture medium in which the carrier-myotube aggregates are suspended. This motion exerts passive forces on the myotubes that are decisive for the predominance of MHC II expression. These forces act via integrin, which transduces the mechanical signal into activation of PP2A and of p38 MAP-Kinase. The latter presumably is directly responsible for a drastic upregulation of MHC IId/x, whereas MHC I and metabolic markers remain unaffected. At the same time, despite an elevated level of IGF-1 transcription under passive forces, the IGF-1 receptor-Akt-mTOR axis is switched off as evident from the lack of an effect of inhibition of the IGF-1 receptor and from the PP2A-mediated low degree of phosphorylation of Akt and 4E-BP1. Similarly, the ERK1/2- and JNK-MAP kinase pathways are repressed. We conclude that passive stretch exerted on the myotubes by the rotary fluid motion induces a rather selective upregulation of fast MHC II, which goes along with a mild muscle hypertrophy as judged from the amount of protein per cell and is caused by p38 MAP kinase activity elevated via integrin sensing. The direct link between passive stretch and MHC II expression constitutes a novel mechanism, which is expected to become effective physiologically under passive stretch and eccentric contractions of skeletal muscles. Copyright © 2010 the American Physiological Society.


PubMed | Institute For Physiologische Chemie
Type: Comparative Study | Journal: Shock (Augusta, Ga.) | Year: 2012

Lactated Ringer (LR) is a widely used resuscitation fluid that is known to mediate beneficial effects on acid-base balance when compared with normal saline. We here compared LR with the more physiological Ringer solution (RS) regarding acid-base status, hemodynamics, survival, and organ injury following fluid resuscitation subsequent to severe hemorrhagic shock. Anesthetized rats were hemorrhaged to a mean arterial blood pressure of 25 to 30 mmHg within 30 min. After 60 min, they were resuscitated with either RS or LR (three times the shed blood volume) or with RS or LR plus blood (shed blood plus twice its volume) within 30 min. Subsequently, the animals were observed for further 150 min. When the rats were resuscitated with pure LR or RS, all animals of the shock/LR group, but only three of eight shock/RS group rats were dead 100 min later (median survival, 50 13.1 vs. 120 14.1 min; P < 0.05). Coadministration of the shed blood with RS or LR increased the survival rates to 100%. In these blood-resuscitated groups, organ injury, especially of the kidney, was diminished by the use of RS compared with LR. Time-matched acid-base parameters were not different in all shock groups until death of the animals or euthanasia at the end of experimental time. We conclude that, in severe hemorrhagic shock, resuscitation with RS leads to an improved outcome compared with resuscitation with LR, regardless whether blood is coadministered or not.

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