Workshop at Castle Rauischholzhausen

on May 15th and 16th, 2008

Conference of the German Society of Biochemistry

and Molecular Biology, Study Group Biomembranes,

and the German Society of Experimental and Clinical Pharmacology and Toxicology

Representatives of the Study Group Biomembranes of the German Society for Biochemistry and

Molecular Biology and

Organizing Committee

Prof. Dr. Ernst Petzinger Justus-Liebig-University of Gießen

Institute of Pharmacology and Toxicology Frankfurter Str. 107

35392 GIESSEN

Prof. Dr. Thomas Pomorski Humboldt University of Berlin

Faculty of Mathematics and Natural Science I Institute of Biology

Invalidenstr. 42 10115 BERLIN

Prof. Dr. Robert Tampé Johann-Wolfgang Goethe University of Frankfurt

Institute of Biochemistry, Biocenter Max-von-Laue-Str. 9

60438 FRANKFURT am Main

Acknowledgements: The organizing committee would like to express its gratitude to the following persons for their efforts in supporting the organizers: Dorothee von Schnakenburg, Dr. Jörg Alber, Christoph Zimmermann, and Gary Grosser.

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Castle Rauischholzhausen Workshop 15th to 16th May 2008

GBM-Workshop Method Course on Proteins

organized by E. Petzinger, Justus-Liebig-University, Giessen,

T. Pomorski, Humboldt University, Berlin, R. Tampé, Johann Wolfgang Goethe-University, Frankfurt/M.

PROGRAM 15th, May 17.00 till 18.30 Arrival at Castle Rauischholzhausen D-35085 Ebsdorfergrund/Rauischholzhausen Registration and Rooms, Payment of Fees Welcome: E. Petzinger (Giessen) Dinner 18.30 - 20.00 Evening Lecture 20.00

Böttcher, Christoph Cryo-TEM in Three Dimensions Get together-Party 16th, May Chairman: T. Pomorski (Berlin) 9.00 - 9.15 Geyer, Joachim (Giessen) Searching for New Carriers: From Genes to Biology

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9.15 – 9.30 Koepsell, Hermann (Würzburg) Site-Directed Mutagenesis in Organic Cation Transporter 1 9.30 – 9.45 Brandsch, Matthias (Halle) Transwell Systems in Transport Studies: Cell Culture, Flux Measurements, Data Processing 9.45 – 10.00 Fricker, Gert (Heidelberg) Drug Transport to the Central Nervous System – Models of the Blood/CNS Bar-riers 10.00 – 10.15 Pohl, Peter (Linz) Planar Membranes and Reconstituted Membrane Proteins 10.15 - 10.30 Lochnit, Günter (Giessen) Separation of Membrane Proteins by Two-Dimensional Electrophoresis using Cationic Rehydrated Strips 10.30 – 11.00 Coffee Break and Industry Exhibitions Chairman: P. Pohl (Linz) 11.00 – 11.15 Bernhard, Frank (Frankfurt/M.) Cell-free Expression of Membrane Proteins 11.15 – 11.30 Pomorski, Thomas (Berlin) Functional Expression of Heterologous Proteins in Yeast: Tracking down Lipid Flippases and their Biological Functions 11.30 – 11.45 Herrmann, Andreas (Berlin) FLIM - Fluorescence Life Time Imaging Microscopy

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11.45 – 12.00 Wehner, Frank (Dortmund) Subunits α, β and γ of the Epithelial Na+ Channel (ENaC) are Functionally Related to the Hypertonicity-Induced Cation Channel (HICC) in Rat Hepatocytes LUNCH TIME 12.15 – 14.00 AFTER LUNCH WALK in PARK 13.30 – 14.00 Method Administrations and Industry ExhibitionsDiscussion with Applicants and Industry Representatives

Chairman: F. Wehner (Dortmund) 14.00 – 14.20 Pehl, Ulrich (Frankfurt/M.) IONGATE Advances in Transporter Screening: Automated Biosensor Array Workstations for Functional Transport Protein Analysis 14.20 – 14.40 Stölzle, Sonja (Munich) NAN(I)ON The Port-a-Patch: the World´s Smallest Patch Clamp Rig 14.40 – 15.00 Weiss, Eike (Saarbrücken) KIBERO Time Resolved Acoustic Microscopy 15.00 – 15.20 Krämer, Benedikt (Berlin) PICOQUANT Instruments for Time-Resolved Fluorescence Spectroscopy - Advances in Fluo-rescence Lifetime Microscopy 15.20 – 15.50 Coffee Break Additional Demonstrations at Tables Chairman: G. Fricker (Heidelberg) 15.50 - 16.10 Heinemann, Christian (Lambrecht, Pfz.) HEKA Ionovation Compact: Precise Measurement of Ion Channel, Transporter or Pore Activity

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16.10. – 16.30 Sutija, Margareta (Schiphol-Rijk) CORNING Transwell® Permeable Support 16.30 – 16.50 Bleisteiner, Monika (St.Leon-Rot) FERMENTAS Fermentas – a Leader in Molecular Biology Products Concluding remarks: T. Pomorski (Berlin)

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Christoph Böttcher Cryo-TEM in three dimensions In recent years the cryo-TEM technique has become one of the most im-portant tools in order to directly characterize natural or synthetic su-pramolecular assemblies in the native environment of the solvent. With the general availability of dedicated computers the most challenging goal of three-dimensional structure determination of such assemblies at near atomic resolution appears to be possible in the very near future. This aim is especially most desirable in those cases where the growth of crystals for X-ray analysis fails as is often the case in the vast expanding field of syn-thetic nanostructures. Here, the method of choice for 3D data analysis is by now the so called single particle approach, which offers the possibility to extract three-dimensional structure information from randomly distributed entities (“particles”). As each two-dimensional projection image which is created by the TEM presents the object in a different spatial orientation the method offers an “a posteriori” way to determine the relative angle orien-tation of the particles. The three-dimensional density distribution is then calculated in a very similar way as is used in a tomographic approach. This pincipal route is complemented by alignment and classification algorithms which help to improve the signal-to-noise ratio of the normally very noisy and low-contrast data. As the resolution of a 3D-reconstruction achievable by the method is in the first place limited by statistical parameters, e.g. by the number of the particles used, the success of the method appears for the most part only to be restricted by the capacity of the computational environment. CB acknowledges support by the DFG. Contact: PD Dr. Christoph Böttcher Free University of Berlin Institute of Chemistry and Biochemistry – Research Centre of Electron Microscopy christoph.boettcher@fzem.fu-berlin.de Fabeckstr. 36a D-14195 Berlin (Germany)

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Joachim Geyer and Barbara Döring Searching for new carriers: from genes to biology Solute Carriers (SLCs) are one of the largest families of membrane bound proteins in the human genome and the number of newly discovered SLC sequences is still increasing. Currently, 46 SLC families were classified in-cluding more than 360 transporter genes in man. Most of these trans-porter genes have not been identified by experimental methods including expression cloning or cDNA library screening methods. As today along with the almost finished human genome, a large number of EST and full-length cDNA sequences are available, mostly from large-scale projects. Many of these genes were identified by using bioinformatic methods. Although functional characterization of many of the recently identified SLC transporters was successful due to a high level of functional overlap be-tween individual members of a particular SLC family, about 20% of all SLC transporters still remain marginally characterized or are orphan at all. Thus, the key questions in the „post genome sequencing era“ that need to be answered to determine the biological activity of a gene product are as follows: (1) What biological activity does the gene product have, and how does the cell react to changes of protein levels or transport activity, (2) in which tissue and cell types is the gene expressed, and to which compart-ment in the cell is the gene product sorted, (3) when is the gene ex-pressed during growth and development, (4) is the gene differentially ex-pressed in healthy and diseased tissue, (5) what is the biological context in which the protein acts, and what are the interaction partners, (6) how is the transport activity of the carrier protein be regulated, and (7) what are the critical structures and single amino acids within the protein which determine substrate recognition and translocation. In combination, these questions are central toward the identification of disease-related genes as well as the definition of potential drug targets. To this end, in silico analysis of gene and protein sequences is not suffi-cient. Additional, in vitro and in vivo studies are mandatory to be carried out to understand the biological activity and context of a novel carrier pro-tein. Contact Prof. Dr. Joachim Geyer Justus-Liebig-University of Giessen Institute of Pharmacology und Toxicology Juniorprofessor for Pharmacogenetic and Pharmacogenomics Joachim.M.Geyer@vetmed.uni-giessen.de Frankfurter Str. 107 D-35392 Giessen (Germany)

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Hermann Koepsell1, Christopher Volk1, Thomas Müller2, Valentin Gorboulev1 Site-directed mutagenesis in organic cation transporter 1 Organic cation transporters of the SLC22 family are responsible for up-take, excretion and tissue distribution of various endogeneous cations and cationic drugs (1). Medical treatment of diseases may be significantly in-fluenced by mutations in these transporters. The SLC22 transporter family contains organic cation transporters (OCTs) that are facilitative diffusion systems, transporters that mediate sodium-carnitine cotransport as well as sodium independent cation transport (OCTNs), and organic anion transporters (OATs) that operate as anion exchangers. Three OCT sub-types (OCT1-3) have been cloned that are supposed to operate in a simi-lar manner. They translocate structurally different organic cations in both directions across the plasma membrane and transfer positive electrical charge. Translocation of organic cations is driven by substrate gradients and membrane potentials. We performed extensive mutagenesis in rat OCT1. The mutants were characterized for changes of transport proper-ties, changes of selectivity, and changes of movement of a fluorescent-labeled amino acid in the transporter (2, 3, 4). Using the crystal structure of the inward-open conformation of lactose permease from Escherichia coli and the biochemically characterized outward-open conformation of lactose permease, we modeled tertiary structures of OCT1. The models of OCT1 were validated by mutagenesis experiments. The data strongly suggest that OCT1 contains a large substrate binding pocket that may be present in an outward-facing or inward-facing configuration. They suggest that the binding pockets contain partially overlapping interaction domains that may mediate high and low affinity cation binding. Amino acids within the in-nermost parts of the binding pockets may be accessible from the extracel-lular and intracellular side of the plasma membrane. Support by the DFG (SFB487/A4) is acknowledged. (1). Koepsell, H., Lips, K. and C. Volk (2007). Pharm Res 24: 1227-1251. (2). Gorboulev, V., Shatskaya, N., Volk, C. and H. Koepsell (2005). Mol Pharmacol 67: 1612-1619. (3). Popp, C., Gorboulev, V., Müller, T.D., Gorbunov, D., Shatskaya, N. and H. Koepsell (2005). Mol Pharmacol 67: 1600-1611. (4). Gorbunov, D., Gorboulev, V., Shatskaya, N., Mueller, T., Bamberg, E., Friedrich, T. and H. Koepsell (2008). Mol Pharmacol 73: 50-61. 1 Universität Würzburg, Fachbereich Medizin, Institut für Anatomie und Zellbiologie, Koel-

likerstr. 6, 97070 Würzburg, Germany 2 Universität Würzburg, Fachbereich Biologie, Molekulare Pflanzenphysiologie und Biophy-

sik, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany Contact: Prof. Dr. Hermann Koepsell University of Würzburg Department of Anatomy and Cell Biology Hermann@Koepsell.uni-wuerzburg.de Koellikerstr. 6 D-97070 Würzburg (Germany)

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Matthias Brandsch1, Linda Metzner1

Transwell systems in transport studies: cell culture, flux meas-urements, data processing The transfer of substances across epithelial barriers can occur via paracel-lular or transcellular routes. Paracellular simple diffusion is very often re-stricted by tight cell junctions. The transcellular route requires transport across two different cell membranes, i.e. the apical and the basolateral membrane. The extent of transcellular simple diffusion of compounds de-pends on their size, charge and lipophilicity. Large proteins are translo-cated across cell layers mainly by specialized transcytotic processes. For most smaller inorganic and organic solutes, passage is mediated by more or less specific translocators. To study total net transepithelial passage of any given compound, epithe-lial cells can be seeded on permeable filters in so-called Transwell- or Snapwell-systems for time periods determined by cell type specific pa-rameters. During cell culture, the transepithelial electrical resistance (TEER) is monitored. The integrity of the monolayers has to be controlled microscopically and by measuring flux of labelled space markers. Provided that the cells on the filter become tightly connected, the systems allow separate or simultaneous measurement of uptake into the cells from apical and basolateral side or flux measurements in apical-to-basolateral and basolateral-to-apical direction. Modifying experimental conditions such as driving forces or the substrate concentrations, the technique allows as-signment of specific translocators. The aim of our presentation is to sum-marize specifics of the cell culture technique, the experimental flux proto-cols and ways to calculate and to compare data and parameters. We will illustrate this overview with examples from studies on peptide, amino acid and drug flux measurements (1-4). (1) Bretschneider, B., Brandsch, M. and Neubert, R. (1999). Pharm Res 16: 55-61. (2) Neumann, J. and Brandsch, M. (2003) J Pharmacol Exp Ther 305: 219-224. (3) Metzner, L., Kalbitz, J. and Brandsch, M. (2004) J Pharmacol Exp Ther 309: 28-35. (4) Neumann, J., Bruch, M., Gebauer, S. and Brandsch, M. (2004) Eur J Biochem 271: 2012-2017. 1 Membrane Transport Group, Biozentrum, Martin-Luther-University Halle-Wittenberg,

Germany Contact: PD Dr. habil. Matthias Brandsch Biocentrum of the Martin-Luther-University Halle-Wittenberg matthias.brandsch@biozentrum.uni-halle.de Weinbergweg 22 D-06120 Halle (Germany)

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Gert Fricker1,2, S. Burgard2, A. Mahringer2, M. Ott2, J. Parmentier2, I. Reimhold2, V. Reichel2 Drug transport to the central nervous system – models of the Blood/CNS barriers Two barriers – the blood brain barrier (BBB) formed by capillary endothe-lial cells and the epithelial cells of the choroid plexus separate blood circu-lation and central nervous system and protect the brain from xenobiotics and potentially toxic metabolites. Overcoming these barriers without de-structing them represents one of the big challenges in CNS-drug develop-ment. Prerequisite to study the mechanisms of transport is the establish-ment of predictive in vitro models, such as isolated primary cells, immor-talized cell lines or functionally active ex vivo preparations of both tissues. The models have to be carefully characterized with respect to carrier pro-tein equipment, metabolic capacity and protein expression. Representing a barrier tissue cells can be cultured as monolayers on filter supports allow-ing to study substance flux from blood to brain an vice versa. A key element of the BBB is the expression of export proteins, mainly p-glycoprotein (ABCB1, P-GP, MDR1-gene product), but also breast cancer resistance protein (ABCG2, BCRP) and multidrug resistance related pro-teins (MRPs, ABCC). Within the past years several assays have been de-veloped to assess drug/carrier interactions including fluorescence based test systems, ATP-measurements or flux measurements. Inhibition of P-GP is one possibility to overcome the BBB. E.g., coadminis-tration of a potent P-GP blocker, with Paclitaxel to nude mice bearing a glioblastoma led to a significantly increased accumulation of the otherwise impermeable cytostatic drug in the brain. As a consequence, a dramatic size-reduction of the brain tumour could be observed in these animals1. Another possibility to by-pass export proteins is the use of colloidal drug delivery systems consisting of polymeric nanoparticles or liposomes. Sur-face coating and attachment to target seeking structures enables these systems to enrich in or even to cross the blood brain barrier and to accu-mulate in the brain. In vitro as well as in vivo studies have clearly demon-strated that impermeable drugs may accumulate in the CNS and exert their pharmacological effects after administration of colloidal carriers2. GF, AM and VR acknowledge support by the DFG and NIH, respectively. (1) Fellner et al., (2002) J. Clin. Invest. 110(9):1309-18. (2) Ambruosi et al. (2006) J Microencapsul. 23(5):582-92 1 Steinbeis Transferzentrum Biopharmazie und Analytik, INF 366, 69120 Heidelberg 2 Institut für Pharmazie und Molekulare Biotechnologie, INF 366, 69120 Heidelberg Contact: Prof. Dr. Gert Fricker Ruprecht-Karls-University of Heidelberg, Institute for Pharmacy and Molecular Biotechnology Gert.fricker@uni-hd.de Im Neuenheimer Feld 366 D-69120 Heidelberg (Germany)

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Peter Pohl Planar membranes and reconstituted membrane proteins Functional reconstitution of purified proteins into artificial membranes en-ables the posing of molecular questions about channel or transporter be-haviour, questions that cannot be asked in the complicated cellular envi-ronment. I am going to present two different motivations for this radically reductionist approach: (i) Solving the sealing mechanism of the protein translocation channel; (ii) Determining the stoichiometry of gas transport through aqueous channels. The translocon, formed by the Sec61p com-plex in eukaryotes and the SecY complex in bacteria and archaea, is re-sponsible for the transport of many proteins across the membrane of the endoplasmic reticulum (ER) or across the bacterial plasma membrane dur-ing or after their biosynthesis. It opens across the membrane to enable hydrophilic polypeptide segments to cross the lipid bilayer. At the same time, the channel has to prevent small molecules from crossing the mem-brane. We have tested the exclusion mechanism by reconstituting the channel complex into planar bilayers and measuring ion and water fluxes (1). We have also investigated the opposite phenomenon: the optimiza-tion of turnover rates for small molecules by membrane channels. Results from water and gas flux measurements through reconstituted aquaporins (2) are presented. Support by the Austrian Science Fund (FWF) is gratefully acknowledged. 1. Saparov SM, Erlandson K, Cannon K, Schaletzky J, Schulman S,

Rapoport TA, Pohl P (2007) Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell 26:501-509.

2. Saparov SM, Liu K, Agre P, Pohl P (2007) Fast and selective ammonia transport by aquaporin-8. J. Biol. Chem. 282:5296-5301.

Contact: Prof. Dr. Peter Pohl Johannes Kepler University of Linz Institute for Biophysics peter.pohl@jku.at Altenberger Str. 69 A-4040 Linz (Austria)

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Günter Lochnit1, J. Grabitzki1, M. Ahrend2

Separation of membrane proteins by two-dimensional electropho-resis using cationic rehydrated strips Due to their poor solubility during IEF membrane proteins can not be separated and analyzed satisfactorily with classical 2-DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl-n-hexadecylammonium chloride (16-BAC)/SDS-PAGE, but the corresponding protocol is intricate and time-consuming. We now developed an easy-to-handle electrophoresis method in connec-tion with a novel device which enables reproducible separation of ionic solubilized membrane proteins. It was our aim to combine the advantages of a first dimensional electro-phoresis performed in prepared plastic sheet gel strips] for IEF and the high resolution of hydrophobic membrane proteins of a 2-D 16-BAC/SDS combination. Therefore, we developed an electrophoresis technique, based on a novel device which realizes separation of ionic solubilized proteins (e.g., with 16-BAC) in precasted and individually rehydrated gel strips. These strips can be transferred easily to the top edge of an SDS gel for the second dimension, according to IEF/SDS protocols. This technique re-quires a new device consisting of a horizontal running tray for an electro-phoretic run of gel strips. Contact of the electrodes to the gel strips is re-alized by buffer-filled electrode blocks offering high buffer capacity. The suitability of this new method was demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells (1). (1) B. Wenge, H. Bönisch, J. Grabitzki, G. Lochnit, B. Schmitz, M.J. Ahrend, Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips. Electrophoresis (2008) in press. 1 Institute of Biochemistry, Faculty of Medicine, University of Giessen, Giessen, Germany 2 Institute of Animal Sciences, Department of Biochemistry, University of Bonn, Bonn,

Germany Contact: PD Dr. Günter Lochnit Justus-Liebig-University of Giessen Institute of Biochemistry Protein Analytics Guenter.Lochnit@biochemie.med.uni-giessen.de Friedrichstr. 24 D-35392 Giessen (Germany)

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Frank Bernhard, D. Schwarz, F. Junge, B. Schneider, S. Reckel, S. Sobhanifar, F. Durst, and V. Doetsch Cell-free expression of membrane proteins Cell-free expression techniques have emerged in recent times as powerful tools for the fast and efficient production of membrane proteins. Most cen-tral problems associated with conventional cellular expression systems are eliminated and the direct synthesis into defined artificial hydrophobic envi-ronments like detergents or liposomes enables completely new strategies for membrane protein production. In addition, reaction protocols of cell-free expression systems can be individualized according to the specific re-quirements of particular target proteins. This exceptional versatility guar-antees high success rates for the production of even complex membrane proteins. We have established throughput strategies for the optimization of cell-free reaction protocols for the expression of membrane proteins as integrated processes with robotic platforms. Different reaction modes have been de-veloped for the optimized production of functionally folded membrane pro-teins. The efficiencies of our techniques are demonstrated by the expres-sion of representative sets of > 100 membrane proteins involved in trans-port, efflux, signalling, metabolism or biosynthesis in mg amounts in a single ml of cell-free reaction mixtures. The quality of selected membrane proteins including eukaryotic solute carriers, G-protein coupled receptors and diverse transporters is evaluated by a number of complementary techniques comprising functional assays as well as structural and bio-physical approaches. We demonstrate the preparative scale production of pharmaceutical important targets such as G-protein coupled receptors in high quality in less than 24 hours and we present new strategies for their specific labelling and their functional as well as structural evaluation in particular by NMR spectroscopy. References: Schwarz et al., (2007). Nat Protocols 2: 2945-57 Klammt et al., (2007). FEBS J 274: 3257-69

Klammt et al., (2007). J Struct Biol 158: 482-493 Schwarz et al., (2007). Methods 41: 355-69

Klammt et al., (2006). Meth Mol Biol 375: 57-78 Klammt et al., (2005). FEBS J 272: 6024-38

Trbovic et al., (2005). J Am Chem Soc 127: 13504-5 Klammt et al., (2004). Eur J Biochem 271: 568-80

Contact: Prof. Dr. Frank Bernhard Johann Wolfgang Goethe-University Frankfurt/M. Institute of Biophysical Chemistry fbern@bpc.uni-frankfurt.de Max-von-Laue Str. 9 D-60438 Frankfurt/M. (Germany)

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Thomas Pomorski

Functional expression of heterologous proteins in yeast: tracking down lipid flippases and their biological functions The baker’s yeast Saccharomyces cerevisiae is a versatile and widely used model organism. It offers a compact and fully sequenced genome, tracta-ble genetics, simple and inexpensive culturing conditions, and, impor-tantly, a conservation of basic cellular machinery with higher eukaryotes. The utility of the yeast system is demonstrated by rapid advances in the study of specific membrane proteins, termed lipid flippases that are re-quired for lipid flip-flop across cellular membranes (1). Our studies led to the identification of two novel P4 ATPases that are required for the en-ergy-coupled, inward transport of specific phospholipids across the yeast plasma membrane with unexpected and important functions in vesicular traffic: their activities are required to support vesicle formation in the sec-retory and endocytic pathways (2). Putative flippases in the newly se-quenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of yeast strains lacking endogenous lipid flippases (3, 4). Functional expression of plant P4 ATPase in yeast has provided insight into the physiology and biochemical characteristics of this pump (4). Future studies will help identifying side chains critical for lipid transport and selectivity and facilitate structure-function analysis. TP acknowledges support by the DFG and Robert Bosch Foundation. (1). Pomorski, T. and Menon, A. K. (2006). CMLS 63, 2908-2921. (2). Pomorski, T., Lombardi, R., Riezman, H., Devaux, P.F., van Meer, G. and Holthuis, J.C.M. (2003). Mol. Biol. Cell 14, 1240-1254. (3). Castanys-Muñoz, E., Alder-Baerens, N., Pomorski, T., Gamarro, F. and Castanys, S. (2007). Mol. Microbiol. 64, 1141-1153. (4). Poulsen, L.R., López-Marqués, R.L., McDowell, S.C., Okkeri, J., Licht, D., Pomorski, T., Harper, J.F. and Palmgren, M.G. (2008). Plant Cell, in press. Contact: Prof. Dr. Thomas Pomorski Humboldt University of Berlin Faculty of Mathematics and Natural Science I Institute of Biology thomas.pomorski@rz.hu-berlin.de Invalidenstr. 42 D-10115 Berlin (Germany)

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Andreas Herrmann FLIM - Fluorescence Life Time Imaging Microscopy Fluorescence life time of a fluorophor is sensitive to physical (e.g. lipid packing) and physico-chemical properties (e.g. pH) of its local environ-ment. Furthermore, protein-protein, protein-lipid as well as lipid-lipid in-teractions can be characterized by life time employing Förster-Resonance-Energy Transfer (FRET) between donor and acceptor fluorophors. The life time of the donor fluorophor becomes shorter in case of FRET. Modern fluorescence microscopy allows life time imaging (FLIM) in living cells with rather high spatial resolution. Based on variation of lipid composition lipid packing differs between cellu-lar membranes. This can be visualized by FLIM of fluorescent lipid ana-logues. However, differences in lipid composition and organization exist not only between cellular membranes. Lipids of plasma membranes are inhomogenously organized. While the transversal lipid asymmetry is well established, the size and dynamics of lipid domains in membrane leaflets and their function are still elusive. FLIM provides a basis to study the presence of lipid domains and the recruitment of distinct membrane pro-teins to those domains. For example, we could allocate different lifetime components of a fluorescent lipid analogue to either so called liquid disor-dered or liquid ordered domains by FLIM. Tagging membrane proteins with GFP-variants appropriate for FRET we could study whether lipid do-mains could function as a platform for enrichment of distinct proteins. AH acknowledges support by the DFG and EU. Contact: Prof. Dr. Andreas Herrmann Humboldt University of Berlin Department of Biology Molecular Biophysics andreas.herrmann@rz.hu-berlin.de Invalidenstr. 42 D-10115 Berlin (Germany)

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Sandra Plettenberg1, Eike C. Weiss2, Robert Lemor3, Frank Wehner1

Subunits α, β and γ of the epithelial Na+ channel (ENaC) are func-tionally related to the hypertonicity-induced cation channel (HICC) in rat hepatocytes It became quite evident in recent years that mechanisms of cell volume regulation are employed in a variety of physiological processes in addition to mere the maintenance of cell homoeostasis. Among these are the coor-dination of transport across epithelia, the control of cell metabolism and gene expression and, most notably, the triggering of cell proliferation and apoptosis. Whenever analysed in a quantitative fashion, hypertonicity-induced cation channels (HICCs) were shown to be the main mechanism of the regulatory volume increase (RVI) of a shrunken cell. Despite the obvious significance of HICCs for cell volume control and functioning, however, very little is known so far as for the actual molecular architecture of these channels. One of the very few exceptions is the sensitivity of the hepatocyte HICC to µM concentrations of amiloride, the classic blocker of the epithelial Na+ channel (ENaC). In the present study, specific siRNA constructs were used to test for the functional relation of subunits α, β and γ of the ENaC to the hypertonicity-induced cation channel (HICC) in confluent primary rat hepatocytes. In current-clamp recordings, hypertonic stress (300 → 400 mosM) increased membrane conductance from 75.4 ± 9.4 to 91.1 ± 11.2 pS (p < 0.001). The effect was completely blocked by 100 µM amiloride and reduced to 46, 30 and 45 % of the control value by anti-α, anti-β and anti-γ-rENaC siRNA, respectively. Scanning acoustic-microscopy (as a novel technique in cell biology) revealed an initial shrinkage of cells from 6.98 ± 0.45 pl to 6.03 ± 0.43 pl within 2 minutes. This passive response was then followed by a regulatory volume increase (RVI) by 0.42 ± 0.05 pl (p < 0.001). With anti-α, anti-β and anti-γ-rENaC siRNA, the volume response was reduced to 31, 31 and 36 %, of the reference level. It is concluded that, in rat hepatocytes, all three subunits of the ENaC are functionally related to HICC activation and RVI. Furthermore, scanning-acoustic microscopy is introduced as a novel and non-invasive technique which allows the determination of cell volumes over periods up to several hours (without any staining of cells) and at a high temporal and spatial resolution. 1Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str. 11, 44227 Dortmund 2kibero GmbH, Bismarckstr. 56, 66121 Saarbrücken 3Fraunhofer Institut Biomedizinische Technik, Ensheimer Str. 48, 66386 St. Ingbert Contact: Prof. Dr. Frank Wehner Max-Planck-Institute of Institute of Molecular Physiology Department of Systemic Cell Biology frank.wehner@mpi-dortmund.mpg.de Otto-Hahn-Str. 11 D-44227 Dortmund (Germany)

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Ulrich Pehl, P. Sörensen, R. Krause, J. Englert, F. Czepan, B. Kelety, and W. Dörner Advances in transporter screening: automated biosensor array workstations for functional transport protein analysis Transporters are increasingly recognized as an important target class in various disease areas like CNS or metabolic disorders, not to mention their relevance for ADME. Although screening technologies for transporter targets have improved within recent years, data quality remained poor in many cases. IonGate has developed a cell-free electrophysiological tech-nology suitable for high quality screening of transporters using sensors based on solid-supported membranes (SSM). The SURFE2R (SURFace Electrogenic Event Reader) Technology is a label-free method to investigate ion transporters and channels in membrane fragments or vesicles from various sources, e.g. cultured mammalian cells, native tissue or subcellular organelles as well as liposomes. These protein-containing membrane entities are adsorbed onto sensors. Subse-quently, protein-specific charge movements resulting from rapid solution exchanges can be detected as transient electrical currents in a highly sen-sitive manner. Current amplitudes reflect the effect of added substrates and/or modulators on the transporter activity, allowing the quantitative determination of dose-response relationships. SURFE2R One, the first commercially available device utilizing IonGate´s SURFE2R Technology, is a single channel, bench-top device aimed at aca-demic and industrial research applications where high information content is prevalent. Recent technological advances led to the development of the SURFE2R Workstation product family that combines a versatile liquid han-dling system with a 96-well biosensor array detection unit and a high de-gree of automation. These systems have been designed for high through-put applications in drug discovery. To prove applicability, different transport proteins were characterized thoroughly focussing on general quality benchmarks such as signal-to-noise ratio, baseline RMS and signal reproducibility. In summary, the SURFE2R Technology enables screening of focused librar-ies and secondary screening with high quality data for transporter targets.

Contact: Dr. Ulrich Pehl IonGate Biosciences GmbH Assay Development Ulrich.Pehl@iongate.de Industriepark Hoechst D 528 D-65926 Frankfurt/M.(Germany)

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Sonja Stoelzle1, Cecilia Farre1, Claudia Haarmann1, Ali Haythornthwaite1, Michael George1, Jürgen Steindl1, Matthias Beckler1, Andrea Brueggemann1 & Niels Fertig1

The Port-a-Patch: the world´s smallest patch clamp rig The Port-a-Patch is a one-cell-at-a-time system, which is surprisingly small for a complete patch clamp rig. Patch clamp recordings in the whole cell configuration, and single channel recordings in the cell attached con-figuration, can be performed with planar patch clamp chips. Manual liquid-handling is combined with computer controlled suction supply for auto-mated sealing and whole cell access. The Port-a-Patch uses planar, microstructured borosilicate glass chips for patch clamping of cells where giga seals and whole-cell recordings are ob-tained with high success rates (80%). Temperature control as well as automated external solution exchange are also possible. The system also has an add-on for the automated perfusion of the internal side of the membrane. This way, both external and internal components can be ex-changed and controlled throughout the experiment. These features, and also the ease of use, make the Port-a-Patch a very well suited device for research grade electrophysiological analysis of ion channels as well as drug screening on ion channels.

1 Nanion Technologies GmbH, Erzgiessereistr. 4, 80335 München, Germany. Contact: Dr. Sonja Stoelzle Nanion Technologies sonja@nanion.de Erzgiessereistr. 4 D-80335 München (Germany)

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Eike Christian Weiss Time resolved acoustic microscopy kibero GmbH was founded in 2007 as a spin-off from the Fraunhofer Institut fuer Biomedizinische Technik (IBMT) by employees of the Department of Ultrasound. kibero's focus in research development and products lies in the application of very high frequency ultrasound to the field of life sciences. The company is located in Saarbruecken in the South West of Germany. kibero produces the SASAM® acoustic microscope series which was especially designed for life sciences applications. With superior signal to noise ratio and the use of time resolved operation throughout the whole frequency range from 50-2000 MHz it is now possible to detect even the very small variations in the local mechanical properties that individual living cells or tissue exhibit. SASAM® Technology allows the characterization of these variations with a depth resolution of less than 100 nm and lateral resolution of better than 1 µm. Acoustic microscopy as an imaging method provides several key advantages. It is non invasive due to the low energy of the used sound wave and not dependent on any staining procedures thus allowing observation of cells over extended periods of time. Acoustic Microscopy is inherently three-dimensional and sensitive to the local mechanical properties of the cell, a parameter not easily obtained by other methods. kibero works in close collaboration with the group of Professor Frank Wehner at the Max-Plank-Institute for Molecular Physiology in Dortmung to exploit the unique properties of acoustic microscopy and the SASAM® Technology for cell volume measurements.

Contact: Dr. Eike Christian Weiss kibero GmbH eike.weiss@kibero.comBismarckstr. 56 D-66121 Saarbrücken (Germany)

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Benedikt Krämer1, V. Buschmann1 , P. Kapusta1 , S. Tannert1

Instruments for time-resolved fluorescence spectroscopy - Advances in fluorescence lifetime microscopy Time-resolved techniques to measure the fluorescence lifetime can reveal important information about the local environment of a given fluorescent probe, help to distinguish fluorophores with similar spectral properties or reveal different conformations of a single fluorophore. We have developed a stable and easy to use upgrade for standard laser scanning confocal mi-croscopes of all major companies towards a time-resolved system, which is based on pulsed lasers, fast detectors and sophisticated single photon counting electronics. The upgrade kit is very flexible and permits to record fluorescence lifetime images (FLIM) as well as to perform fluorescence correlation spectroscopy (FCS). We demonstrate the capabilities of the time-resolved approach by using fluorescence lifetime measurements to detect fluorescence resonance en-ergy transfer (FRET) in living cells. The results show that different FRET-efficiencies can be spatially resolved within a single cell, as shown on U2OS cells transfected with Cerulean and YFP-constructs of the nuclear proteins CENP-B and CENP-A, respectively (sample courtesy of Sandra Orthaus, Fritz-Lipmann Institute, Jena). A fundamental feature of the upgrade kits is the usage of an unrestricted single photon data acquisition approach, which allows to store the full photon information in a generalized format (Time-Tagged Time Resolved Detection - TTTR). With this approach, not only Fluorescence Lifetime Im-aging Microscopy (FLIM) with single molecule sensitivity is realized, but the provided information can also be combined with other techniques such as Fluorescence Correlation Spectroscopy (FCS). This opens the way to complete new analysis and measurement schemes like Fluorescence Life-time Correlation Spectroscopy (FLCS) or Pulsed Interleaved Excitation (PIE). FLCS can, for example, be used to remove the influence of detector afterpulsing, which is classically still done by a cross correlation between two detectors. With FLCS, however, a single detector is sufficient, and it is possible to simultaneously remove non-correlated background, which en-ables quantitative concentration measurements at very low concentra-tions.

Contact: Dr. Benedikt Krämer PicoQuant GmbH kraemer@picoquant.com Rudower Chaussee 29 D-12489 Berlin (Germany)

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Christian Heinemann1, K. Gall2

Ionovation compact: precise measurement of ion channel, trans-porter or pore activity Electrophysiological investigation is one of the central tools for studying pore forming proteins. In the past, standard technological approaches have primarily consisted of various alterations of the patch clamp tech-nique of membranes and cells. Here, we introduce an alternative to con-ventional methods: The Ionovation Compact – a highly flexible bench top system based on the bilayer technique. The bilayer technique is a method to record electrical currents at the single channel level. The bi-layer forms a gigaohm resistance between two saline-buffer filled cham-bers. After incorporation of pore forming proteins (ion channels, solute channels, carriers or pumps), protein mediated currents or membrane po-tentials can be recorded at high resolution. Stable, low noise, Ag/AgCl electrodes with salt bridges allow recordings in a wide current and salt concentration range. The readily mounted bilayer chamber consists of a 25 µm thick Teflon foil with an aperture of about 50 µm diameter separat-ing two polycarbonate compartments of about 3 ml volume each. The Ionovation Compact system is integrated with the well-known EPC 10 Patch Clamp Amplifier for low noise recording and data acquisition. The Ionovation Compact is controlled by the user-friendly software PATCH-MASTER providing full-automated instrument operations, i.e. bilayer pro-duction and validation, capacitance control of bilayer integrity (visual con-trol of the bilayer also possible), and perfusion of both membrane sites. A user defined experimental workflow with predefined protocols allows the system to be run in a self-controlled way. 1 HEKA Elektronik Dr. Schulze GmbH, Wiesenstraße 71, 67466 Lambrecht, Germany 2 Ionovation GmbH, Westerbreite 7, 49084 Osnabrück, Germany Contact: Dr. Christian Heinemann, PhD HEKA Elektronik Dr. Schulze GmbH sales@heka.com Wiesenstraße 71 D-67466 Lambrecht (Germany)

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Margareta Sutija1, L. Hartwieg1, J. Philips2

Transwell® permeable support Transwell® cell culture inserts are permeable support devices with a mi-croporous membrane, resembling as closely as possible the in vivo envi-ronment, and are suitable for the study of both anchorage-dependant and anchorage-independent cell lines. The inserts provide independent access to both sides of a monolayer, thus providing a versatile tool to study cellu-lar functions such as: metabolic activities, chemotaxis, drug transport, cell polarity, invasion and co-culture in vitro. Standard Transwell inserts are available in three membrane materials: polycarbonate (PC), polyester (PET) and collagen-coated polytetrafluoroethylene (PTFE). Polycarbonate and polyester membranes are tissue culture treated for optimal cell at-tachment, and additionally can be coated with collagen, or other biological coatings. Collagen-coated PTFE membrane has an equimolar mixture of types I and III collagen derived from bovine placentas, thus promoting cell attachment. The Corning coating process results in biologically stable collagen that covers every fibril of the membrane thereby retaining mem-brane porosity. Polycarbonate and polyester inserts are available in differ-ent pore sizes, 0.4-8 µm and different insert diameters, 6.5, 12, 24, and 75mm. Additionally, HTS- Transwell in 24 and 96 well formats are avail-able for robotic handling, in which the inserts are connected by a rigid tray that enables handling of the multi-well Transwell as a single unit. The lat-est transwell family product from Corning is Transwell® Permeable Sup-port Invasion Inserts Coated with BME, which are originally created in an effort to accelerate the screening process for compounds that influence cellular invasion through extracellular matrices, which is fundamental to angiogenesis, embryonic development, immune responses, and tumor cell metastasis. ®Corning and Transwell are a registered trademarks of Corning, Incorporated, Corning, NY. Corning Incorporated, One Riverfront Plaza, Corning, NY 14831-0001 1Corning Life Sciences B.V., Koolhovenlaan 12 1119 NE Schiphol-Rijk The Netherlands 2Corning Incorporate Life Sciences 45 Nagog Park, Acton, MA 01720 U.S.A.

Contact: Dr. Margareta Sutija Corning Life Sciences B.V. SutijaM@corning.comKoolhovenlaan 12 NL-1119 NE Schiphol-Rijk (The Netherlands)

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Monika Bleisteiner

Fermentas – a leader in molecular biology products Fermentas is a corporation specializing in the discovery, production and marketing of high quality molecular biology products and providing total custom manufacturing solutions for life science research. As a dynamically evolving biotech company, Fermentas strives to make vital contributions to the rapidly growing fields of genomics, proteomics and microfluidics. Solid scientific and R&D background, product develop-ment experience, flexibility and strong team spirit makes Fermentas an ideal research and development partner. Core Competences During 30 years of its activity Fermentas has developed a strong and broad expertise in several fields of molecular biology.

• Restriction enzymes • Gene engineering • Protein purification • Nucleic acid purification • Transfection • In vitro protein evolution Reliable Manufacturing All Fermentas products are manufactured in new class D clean room facili-ties. These facilities together with Fermentas quality assurance system operating according to ISO9001 quality and ISO14001 environmental management systems enable the company to guarantee the industrie’s highest quality and performance for the entire product line. Fermentas Lecture: “Get your clone in 3 days” Due to years of experience, the Fermentas technical service team is famil-iar with a broad range of methods in molecular biology. On this congress, Product Manager Dr. Monika Bleisteiner will give you an overview about problems and solutions concerning molecular cloning - "get your clone in three days". Contact: Dr. Monika Bleisteiner Fermentas GmbH monika.bleisteiner@fermentas.de Opelstr. 9 68789 St. Leon-Rot (Germany)

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Organizers:

Prof. Dr. Ernst Petzinger Justus-Liebig-University of Giessen Institute of Pharmacology and Toxicology Ernst.Petzinger@vetmed.uni-giessen.de Frankfurter Str. 107 D-35392 Giessen (Germany)

Organizing secretary Dorothee von Schnakenburg 0641-9938401 06424-301-107 (during congress)

Prof. Dr. Thomas Pomorski Humboldt University of Berlin Faculty of Mathematics and Natural Science I Institute of Biology Thomas.Pomorski@rz.hu-berlin.de Invalidenstr. 42 D-10115 Berlin (Germany)

Prof. Dr. Robert Tampé Johann-Wolfgang Goethe University Frankfurt, Biocenter Institute of Biochemistry Tampe@em.uni-frankfurt.de Max-von-Laue-Str. 9 D-60438 Frankfurt/M. (Germany)

List of speakers:

Dr. Frank Bernhard Johann Wolfgang Goethe University of Frankfurt Institute of Biophysical Chemistry fbern@bpc.uni-frankfurt.de Max-von-Laue Str. 9 D-60438 Frankfurt/M. (Germany)

Dr. habil. Matthias Brandsch Biozentrum Martin-Luther-University of the Halle-Wittenberg Matthias.Brandsch@biozentrum.uni-halle.de Weinbergweg 22 D-06120 Halle (Germany)

Dr. Monika Bleisteiner Fermentas GmbH Monika.Bleisteiner@fermentas.de Opelstr. 9 D-68789 St. Leon-Rot (Germany)

Prof. Dr. Gert Fricker Ruprecht-Karls-University of Heidelberg, Institute for Pharmacy and Molecular Biotechnology Gert.Fricker@uni-hd.de Im Neuenheimer Feld 366 D-69120 Heidelberg (Germany)

Dr. Christoph Böttcher Free University of Berlin Institute of Chemistry and Biochemistry – Researchcentre of Electron Microscopy Christoph.Boettcher@fzem.fu-berlin.de Fabeckstr. 36a D-14195 Berlin (Germany)

Prof. Dr. Joachim Geyer Justus-Liebig-University of Giessen Institute of Pharmacology und Toxicology Juniorprofessor for Pharmacogenetic and Pharmacogenomics Joachim.M.Geyer@vetmed.uni-giessen.de Frankfurter Str. 107 D-35392 Giessen (Germany)

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Dr. Christian Heinemann, PhD HEKA Elektronik Dr. Schulze GmbH sales@heka.com Wiesenstraße 71 D-67466 Lambrecht (Germany)

Prof. Dr. Peter Pohl Johannes Kepler University Linz Institute for Biophysics Peter.Pohl@jku.at Altenberger Str. 69 A-4040 Linz (Austria)

Prof. Dr. Andreas Herrmann Humboldt University Berlin Department of Biology Molecular Biophysics Andreas.Herrmann@rz.hu-berlin.de Invalidenstr. 42 D-10115 Berlin (Germany)

Prof. Dr. Thomas Pomorski Humboldt University Berlin Institute of Biology Thomas.Pomorski@rz.hu-berlin.de Invalidenstr. 42 D-10115 Berlin (Germany)

Prof. Dr. Hermann Koepsell University of Würzburg Department of Anatomy and Cell Biology Hermann@Koepsell.uni-wuerzburg.de Koellikerstr. 6 D-97070 Würzburg (Germany)

Dr. Sonja Stoelzle Nanion Technologies Sonja@nanion.de Erzgiessereistr. 4 D-80335 München (Germany)

Dr. Benedikt Krämer PicoQuant GmbH kraemer@picoquant.com Rudower Chaussee 29 D-12489 Berlin (Germany)

Dr. Margareta Sutija Corning life Sciences B.V. SutijaM@corning.comKoolhovenlaan 12 NL-1119 NE Schiphol-Rijk (The Netherlands)

PD Dr. Günter Lochnit Justus-Liebig-University of Giessen Institute of Biochemistry Protein Analytics Guenter.Lochnit@biochemie.med.uni-giessen.de Friedrichstr. 24 D-35392 Giessen (Germany)

Prof. Dr. Frank Wehner Max-Planck-Institute of Institute of Molecular Physiology Department of Systemic Cell Biology Frank.Wehner@mpi-dortmund.mpg.de Otto-Hahn-Str. 11 D-44227 Dortmund (Germany)

Dr. Ulrich Pehl IonGate Biosciences GmbH Assay Development Ulrich.pehl@iongate.de Industrial Parc Hoechst D528 D-65926 Frankfurt/M. (Germany)

Dr. Eike Christian Weiss kibero GmbH eike.weiss@kibero.comBismarckstr. 56 D-66121 Saarbrücken (Germany)

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GBM-Geschäftsstelle Mörfelder Landstr. 125 D-60598 Frankfurt/M. (Germany) www.gbm-online.de

Deutsche Gesellschaft für experimentelle und klinische Pharmakologie und Toxikologie Achenbachstr. 43 D-40237 Düsseldorf (Germany) www.dgpt-online.de

sanofi-aventis in Deutschland Gebäude F821 Industriepark Höchst D-65926 Frankfurt/M. www.sanofi-aventis.de

Bayer Vital GmbH Tiergesundheit Gebäude D 162 D-51368 Leverkusen www.tiergesundheit.bayervital.de

Corning B.V. Life Sciences Koolhovenlaan 12 NL-1119 NE Schiphol-Rijk www.corning.com/lifesciences

Fermentas GmbH Opelstr. 9 D-68789 St. Leon-Rot www.fermentas.de

Heka Elektronik Dr. Schulze GmbH Wiesenstr. 71 D-67466 Lambrecht/Pfalz www.heka.com

IonGate Biosciences GmbH Industriepark Hoechst D 528 D-65926 Frankfurt/M., http://www.iongate.de

Kibero GmbH Bismarckstr. 56 D-66121 Saarbrücken (Germany) www.kibero.com

CEO Nanion Technologies GmbH Erzgiessereistr. 4 D-80335 Munich (Germany) www.nanion.de

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PicoQuant Rudower Chaussee 29 D-12489 Berlin (Germany) www.picoquant.com

Steinbeis-Transfer-Zentrum Biopharmazie und Analytik Professor Dr. Gert Fricker Im Neuenheimer Feld 366 D-69120 Heidelberg (Germany) www.uni-heidelberg.de/institute/fak14/ipmb/phazt/steinbeis/stz_bio.htm

DRG Instruments GmbH Marketing Manager Frauenbergstraße 18 35039 Marburg (Germany)www.drg-diagnostics.de

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