2009
Saxon
Biotechnology Symposium
May 26, 2009
ABSTRACTS
Editors:
Andrea A. Robitzki
Manfred Blessing
Thole Zuchner
Michael Brand
Francis Stewart
Andreas Beyer
Erik Schaffer
IMPRINT
Editors Prof. Dr. Andrea A. Robitzki
Director
Center for Biotechnology and Biomedicine, Universitat Leipzig
Prof. Dr. Manfred Blessing
Professorship for Molecular Pathogenesis
Center for Biotechnology and Biomedicine, Universitat Leipzig
Dr. Thole Zuchner
Junior Group Leader
Center for Biotechnology and Biomedicine, Universitat Leipzig
Prof. Dr. Michael Brand
Scientific Director
Biotechnology Center, Technische Universitat Dresden
Prof. Dr. Francis Stewart
Professor of Genomics
Biotechnology Center, Technische Universitat Dresden
Dr. Andreas Beyer
Junior Group Leader
Biotechnology Center, Technische Universitat Dresden
Dr. Erik Schaffer
Junior Group Leader
Biotechnology Center, Technische Universitat Dresden
Compilation Dr. Svenne Eichler
Chief Executive Officer
Center for Biotechnology and Biomedicine, Universitat Leipzig
Dr. Sabine Matthia
Head of Administration
Biotechnology Center, Technische Universitat Dresden
Anja Landsmann
Coordinator of the PbF3, Universitat Leipzig
Layout Katrin Giersch, M. A.
Center for Biotechnology and Biomedicine, Universitat Leipzig
Jana Schulze, M. A.
Center for Biotechnology and Biomedicine, Universitat Leipzig
Print Merkur Druck- und Kopierzentrum GmbH
Editorial Deadline March 15, 2009
ISBN 978-3-00-027884-6
Contents
Page
Foreword 31
Program 33
Presentations
1. Non-coding RNAs as Regulators and Tools 37
1 STAT3 regulated non-coding RNAs in tumor cells
Friedemann Horn 39
2 Predicting and modeling microRNA regulation with an
application to stem cell development
Fabian J. Theis, Dominik Lutter, Carsten Marr, Lena Uetzmann,
Heiko Lickert 40
3 Exploring chemical space with aptamers
Michael Famulok 41
4 From RNAi Screens to Molecular Function: A Systematic
Pipeline for Gene Function in Mammalian Cells
Frank Buchholz 42
2. Stem cell therapies for neurological disorders 45
5 Stem Cell Therapy for Parkinsonfs Disease
Patrick Brundin 47
6 Biomaterial assisted cellular self-organization and tissue
program: a new paradigm in regenerative medicine
Carlos E. Semino 48
7 Regenerating the brain with endogenous stem cells
Verdon Taylor, Onur Basak, Claudio Giachino, Philip Knuckles 49
8 Cell Transplantation into the Retina: A Treatment Option
for Photoreceptor Loss?
Marius Ader 50
3. Biophotonics . molecular motors and optical
tweezers 53
9 Single Molecule Biophysics: Soft Matter, Weak Interactions
and Complex Mechanisms
Dario Anselmetti 55
10 Optical trapping and 3D particle tracking: from concept to
versatile applications
Anna Wozniak, Joost van Mameren, Gerd Behme,
Sebastian Roth 56
11 Bioanalytics at the nanometer and attoliter scale
Horst Vogel, Ralf Schmauder 57
12 Single molecule studies in live and reconstituted cellular
systems
Petra Schwille 58
Posters
4. Molecular Medicine 61
13 PDE10A inhibitors for the treatment of schizophrenia and
psychosis
Ghadir Barbar Asskar, Sabine Mann, Karen Nieber, Norbert
Strater, Peter Brust, Detlef Briel 63
14 Aminosulfonate-Modulated pH-Induced Closure of Cx26
Hemichannels Observed by High-Resolution Atomic Force
Microscopy
Christian A. Bippes, Jinshu Yu, Galen M. Hand, Daniel J.
Muller, Gina E. Sosinsky 64
15 THE P53 TRANSCRIPTOME . Discovery of Regulated noncoding
RNA Genes
Levin Bohlig, Antje Kretzschmar, Kristin Reiche, Jorg
Hackermuller, Friedemann Horn, Kurt Engeland 65
16 Interaction between PIP2 and Ceramide in Drosophila
Phototransduction
Salvatore Chiantia, Ujjaini Dasgupta, Acharia Usha,
Petra Schwille 66
17 Wnt signaling in osteogenic specification of embryonic
stem cells
Huawen Ding, Beatrice Kuske, Nicole I. zur Nieden 67
18 Role of FGD6 in Actin Ring Formation and Organelle
Dynamics in Resorbing Osteoclasts
Ana Isabel Espirito Santo, Tobias Heckel, Bernard Hoflack 68
19 Dual role of the EGF-receptor in regulation of glutamate
transporter expression
Darko Glisic, Claudia Lehmann, Jurgen Engele 69
20 Two-dimensional difference in gel electrophoresis (DIGE)
for analyzing ischemic Cardiomyocytes
Sina Haas, Martin von Bergen, Andrea A. Robitzki 70
21 Comprehensive (Molecular) Cytogenetic Characterization
of rare intracranial tumors
Heidrun Holland, Helene Hantmann, Wolfgang Krupp,
Ronald Koschny, Michela Livrea, Ralf Schober, Jurgen
Meixensberger, Peter Ahnert 71
22 The effects of thrombin on RPE cells are mediated by
transactivation of growth factor receptors
Margrit Hollborn, Carola Petto, Peter Wiedemann, Leon
Kohen, Andreas Bringmann 72
23 Copper overload leads to fragmentation of mitochondrial
membrane lipids: implications for the pathogenesis of liver
toxicity in Wilson disease
Dominik Huster, Irina Yurkova, Jurgen Arnhold 73
24 Loss of pluripotency and acquisition of an osteoblast fate
in embryonic stem cells is accompanied by modulated
microRNA expression
Dorota Karniowska, Kristin Reiche, Antje K. Kretzschmar,
Joerg Hackermueller, Nicole I. zur Nieden 74
25 Synthesis of a new class of N1,N3- adamantylated uraciles
and their biological activity as future non-nucleoside
Inhibitors (NNIs)
Matthias Klemm, Kurt Eger, Rudolf Fahrig, Detlef Briel 75
26 Determination of the structure of F1F0 ATP synthase rotor
from Acetobacterium woodii
Adriana Klyszejko, Michael Fritz, Thomas Meier, Volker
Muller, Daniel J. Muller 76
27 The importance of osteoblasts in the stump of the
regenerating tail fin
Franziska Knopf, Christina Hammond, Stefan Schulte-Merker,
Gilbert Weidinger 77
28 Adiponectin Receptor 1 Dimerization is Inhibited by
Adiponectin
David Kosel, John T. Heiker, Cornelia M. Wottawah, Matthias
Bluher, Karin Morl, Annette G. Beck-Sickinger 78
29 Novel Electroactive Nanovalves for a Implantable
Controlled Drug Delivery Device
Randy Kurz, Anselm Sickinger, Andrea A. Robitzki 79
30 Inhibition of dedicated Wnt/ƒÀ-catenin pathway-associated
kinases by natural and chemical modified polyphenoles
with peculiar features
Katja Steffi Lerche, Robert Gunther, Hans-Jorg Hofmann, Rolf
Gebhardt 80
31 The influence of phosphatidylserine content in lipidlayers of
biopolymer-coated CaCO3-particles on phorbol myristate
acetate differentiated U937
Jacqueline Lessig, Uta Reibetanz, Bjorn Neu, Hans-Jurgen
Glander, Jurgen Arnhold 81
32 Critical Role of Granulocyte-Macrophage Colony-
Stimulating-Factor in Ultraviolet B Radiation-Induced Murine
Skin Cancer
Amrit Mann, Kerstin Niekisch, Thorsten Maass, Peter
Schirmacher, Manfred Blessing 82
33 A gene-dosage effect for IL-4Ralpha expression has an
impact on Th2-mediated allergic inflammation during
bronchopulmonary mycosis
Uwe Muller, Werner Stenzel, Gabriele Kohler, Tobias
Polte, Manfred Blessing, Amrit Mann, Daniel Piehler, Frank
Brombacher, Gottfried Alber 83
34 IL-4/IL-13-dependent alternative activation of macrophages
but not microglial cells is associated with uncontrolled
cerebral cryptococcosis
Uwe Muller, Werner Stenzel, Gabriele Kohler, Frank L.
Heppner, Manfred Blessing, Andrew N.J. McKenzie, Frank
Brombacher, Gottfried Alber 84
35 The transcription factor Elf3 in the gastrointestinal tract:
pathomorphological changes in the transgenic mouse
model
Martina Protschka, Manfred Blessing 85
36 Metabolites of flavones . observations and results of
synthesis and tests
Benjamin Reissig, Steffen Rodewald, Detlef Briel, Rolf Gebhardt 86
37 Osteoclasts Control Osteoblast Chemotaxis via PDGF-BB/
PDGF receptor beta Signaling in vitro
Maria Arantzazu Sanchez Fernandez, Bernard Hoflack 87
38 Peptide mediated DNA import into mitochondria
Ingo Schafer, Christian Kukat, Alexandra Kukat, Peter Seibel 88
39 Distribution of mitofusin 2 (Mfn2) after the formation of
megamitochondria
Susanna Schubert, Christian Kukat, Ingo Schafer, Alexandra
Kukat, Peter Seibel 89
40 In the absence of IL-12 protective immunity to infection with
Salmonella Enteritidis depends on Il-23 and is associated
with IL-22 but not IL-17
Silke Mara Schulz, Gabriele Kohler, Alissa A. Chackerian,
Ellen Witte, Kerstin Wolk, Robert Sabat, Yoichiro Iwakura,
Robert A. Kastelein, Gottfried Alber, Christoph Holscher,
Uwe Muller 90
41 Non-protein coding RNAs as highly specific biomarkers
for cancer
Katharina Schutt, Friedemann Horn, Kerstin Ullmann, Antje
K. Kretzschmar, Jorg Hackermuller 91
42 Synthesis of novel PDE10A-Inhibitors
Gregor Schwan, Lenka Kubicova, Karen Nieber, Norbert
Strater, Peter Brust, Detlef Briel 92
43 Effect of purine analogues on macrophage function during
in vitro ischemia
Fritzi Siegert, Karen Nieber 93
44 Cymantrene-peptide bioconjugates: a promising approach
to generate cytostatic compounds
Katrin Splith, Harmel Peindy Nfdongo, Ulrich Schatzschneider,
Ines Neundorf 94
45 Single-cell force spectroscopy reveals s1 -integrin as
central molecule mediating /ABL expression of 32D-BCR/
ABL cells to bone marrow stromal cells
Anna Taubenberger, Fernando A. Fierro, Pierre-Henri Puech,
Gerhard Ehninger, Martin Bornhauser, Daniel J. Muller,
Thomas Illmer 95
46 MicroRNAs lost during prostate carcinoma pathogenesis
cooperatively regulate mRNAs involved in Androgen
Receptor signalling
Kerstin Ullmann, Antje Kretzschmar, Friedemann Horn, Nora
Morbt, Martin von Bergen, Gerald Verhaegh, Jack Schalken,
Katharina Schutt, Jorg Hackermuller 96
47 The Role of extra- and intracellular domains for Y Receptor
Internalisation
Cornelia Walther, Diana Lindner, Ilka Bohme, Annette G.
Beck-Sickinger 97
48 Characterization of substrates and inhibitors for the in
vitro assessment of BCRP mediated drug transport in the
lactating mammary gland of dairy cattle
Luise Wasermann, Stefan Lindner, Kerstin Honscha, Walther
Honscha 98
49 Reconstitution of the Interleukin-4 dependent JAK/STAT
signalling pathway for a detailed analysis by single
molecule fluorescence detection methods in living cells
Thomas Weidemann, Remigiusz Worch, Tibor Szekeres,
Petra Schwille 99
50 Inhibition of matrix metalloproteinases (MMPs) by selected
anthraquinones and synthetic peptides
Claudia Wierzchacz, Rolf Gebhardt 100
51 Identification of the receptor for Pigment Epithelium Derived
Factor on retinal endothelial cells
Xiu Mei Yang, Wolfram Eichler, Johannes Lange, Andreas
Reichenbach, Peter Wiedemann 101
52 Refolding of ecto-nucleotidases
Karen Yates, Norbert Strater 102
53 Structure-based design of inhibitors of human cAMP
specific phosphodiesterase 4A
Michael Zahn, Roy Eylenstein, E. Bartholomeus Kuttner,
Susanne Moschutz, Norbert Strater 103
54 Interfering with Signal Inactivation in Purinergic Signaling
Matthias Zebisch, Norbert Strater 104
5. Bioanalytics 107
55 Quantitative microscopy with trace element sensitivity
Nirav Barapatre, Anja Fiedler, Thomas Arendt, Tilman Butz,
Markus Morawski, Tilo Reinert 109
56 Easy and fast separation of multiple targets from whole
blood with a new bead based cell separation method
Doreen Beck, Christoph Mohr, Berthold Seidel, Iwona
Goczalik, Anne Rasser, Jan-Michael Heinrich 110
57 The structural and functional landscape of the human ADP
receptor P2Y12 . methods for generating mutant libraries
Maxi Coster, Doreen Thor, Holger Rompler, Torsten Schoneberg 111
58 Biological properties of Apidaecin derivatives with
enhanced antimicrobial activities
Patricia Czihal, Laszlo Otvos Jr., Ralf Hoffmann 112
59 Characterization of proteins oxidatively modified in rat
muscles by tandem mass spectrometry
Maria Fedorova, Nadezda Kuleva, Ralf Hoffmann 113
60 Study of toxicological effects of a silicon material on mouse
fibroblast cell line L929
Marco Glas, Andrea A. Robitzki 114
61 A novel system for quantification of peptides bound to
solid surfaces
Rayk Hassert, Annette G. Beck-Sickinger 115
62 Metabolite analysis of adherent-growing cancer cell lines
with GC-MS
Antje Hutschenreuther, Ferdinand Fischer, Gerd Birkenmeier,
Claudia Birkemeyer 116
63 Development of cell type specific microelectrode array
based label-free screening systems
Heinz-Georg Jahnke, Sabine Schmidt, Ronny Azendorf,
Andrea A. Robitzki 117
64 Insights into the Secondary Structure of the N-Terminus of
the Adiponectin Receptor 1
Cathleen Juhl, Annette G. Beck-Sickinger 118
65 Oncocin . A novel proline-rich antimicrobial peptide to
treat multi-resistant Gram-negative bacteria
Daniel Knappe, Anne Hansen, Ralf Hoffmann 119
66 A bond for a lifetime . Using Membrane nanotubes from
living cells to determine receptor ligand kinetics
Michael Krieg, Jonne Helenius, Carl-Philipp Heisenberg,
Daniel J. Muller 120
67 The Core Unit Fluorescence-Technologies in the IZKF
Leipzig
Andreas Losche, Jens Grosche 121
68 Temperature feedback for thermal stabilization in optical
tweezers
Mohammed Mahamdeh, Erik Schafer 122
69 Impedimetric Detection of Transient Receptor Potential
Channel Activity
Oliver Panke, Andrea A. Robitzki 123
70 Epitope mapping of monoclonal anti-Tau antibodies
AT8 and Tau5
Robert Porzig, David Singer, Ralf Hoffmann 124
71 PH sensor-functionalized colloidal DNA-carriers for direct
localisation in cell compartments
Uta Reibetanz, Liaw Zi Yen, Bernice Oh Hui Lin, Averil Chen
Min Hui, Edwin Donath, Bjorn Neu 125
72 A platform for the automatic identification and quantification
of metabolites using Nuclear Magnetic Resonance
Spectroscopy
Frank-Michael Schleif, Thomas Riemer, Uta Boerner, Rudiger
Alt, Lydia Schnapka-Hille, Christoph Wirling, Rolf Herwig,
Stefan Leidich, Michael Cross 126
73 Evaluation of carbon tetrachloride-induced oxidative stress
on rat hepatocytes: Thermoinduced light emission and
biochemical standard methods
Anika Schumann, Alexander Bauer, Matthias Gilbert, Jan G.
Hengstler, Christian Wilhelm 127
74 Human cationic trypsinogen PRSS1: recombinant
expression, purification and subsequent refolding
Peter Simeonov, Katherina Bellmann, Matthias Zebisch, Ralf
Hoffman, Norbert Strater, Thole Zuchner 128
75 Synthesis and mass spectrometrical characterization of
model peptides containing oxidized tryptophan residues
relevant for protein aging
Toni Todorovski, Ralf Hoffmann 129
76 Optical trapping and 3D particle tracking: from concept to
versatile applications
Anna Wozniak, Joost van Mameren, Gerd Behme, Sebastian
Roth 130
77 In vivo biophysical study of fibroblast growth factor
signalling using fluorescence correlation spectroscopy
Shuizi Rachel Yu, Markus Burkhardt, Jonas Ries, Steffen
Scholpp, Peter Schwille, Michael Brand 131
78 Introduction of a peptide-based Immunoassay using a novel
fluorophore and Fluorescence Resonance Energy Transfer
(FRET)
Thomas Zauner, Renate Berger-Hoffmann, Katrin Muller, Ralf
Hoffmann, Thole Zuchner 132
79 High Sensitive Multiplex Protein Quantitation Using Metal
Element Chelating Tags on an LTQ-Orbitrap-ETD Mass
Spectrometer
Nicole Zehethofer, Ralf Hoffmann, Thole Zuchner 133
6. Bioinformatics 135
80 Family-based analysis of genome-wide gene-gene
interactions
Marit Ackermann, Andreas Beyer 137
81 Biomedical word sense disambiguation with ontologies
and metadata
Dimitra Alexopoulou, Bill Andreopoulos, Andreas Doms,
Khaled Khelif, Michael Schroeder 138
82 The nanometis software system for automated analysis of
single molecular force spectroscopy data on membrane
proteins
Bill Andreopoulos, Frank Dressel, Dirk Labudde, Joscha
Koellner, Michael Schroeder 139
83 A systematic approach of osteoclastogenesis
Antigoni Elefsinioti, Angela Simeone, Jacob Michaelson,
Andreas Beyer 140
84 Evolutionary and Functional Analysis of the RedƒÀ/RecT
Single-Strand Annealing Protein Family
Axel Erler, Madeleine Teucher, Marcello Maresca, Vineeth
Surendranath, Bianca Habermann, Youming Zhang, A.
Francis Stewart 141
85 Xenoturbella bocki: Analyses of the mitochondrial genome
and a PCR survey of hox genes
Guido Fritzsch, Marleen Perseke, Bettina Weich, Manja
Boehme, Detlef Bernhard, Thomas Stach, Olle Israelsson,
Mike Thorndyke, Hiroaki Nakano, Thomas Hankeln, Martin
Schlegel, Peter F. Stadler 142
86 Confluent mesenchymal stem cell cultures in silico
Martin Hoffmann, Jens-Peer Kuska, Matthias Zscharnack,
Christian Thummler, Augustinus Bader, Jorg Galle 143
87 Pathway Prediction with eQTL and Gene Interaction
Networks
Jacob Michaelson, Andreas Beyer 144
88 PhenoFam: a web tool for the gene set enrichment analysis
in the protein domain context
Maciej Paszkowski-Rogacz, Maria Teresa Pisabarro, Frank
Buchholz 145
89 GoGene: a search engine for genes and gene-related
information.
Conrad Plake, Loic Royer, Rainer Winnenburg, Michael
Schroeder 146
90 Unraveling protein networks with Power Graph Analysis
Matthias Reimann, Loic Royer, Bill Andreopoulos, Michael
Schroeder 147
91 Studying molecular targets of human FOXP2 in a
neuroblastoma cell line
Sabrina Reimers, Ines Bliesener, Svante Paabo, Wolfgang Enard 148
92 Unraveling protein networks with Power Graph Analysis
Loic Royer, Matthias Reimann, Bill Andreopoulos, Michael
Schroeder 149
93 Fluorine in protein environments: theoretical and
experimental study
Sergey Samsonov, Mario Salwiczek, Gerd Anders, Beate
Koksch, Maria Teresa Pisabarro 150
94 A systems biology approach for analyzing RNAi data
using functional networks
Angela Simeone, Andreas Beyer 151
95 Development of a framework for structure-based functional
protein annotation
Aurelie Tomczak, Jana Sontheimer, Maria Teresa Pisabarro 152
96 Three-dimensional modeling of protein complexes
Anne Tuukkanen, Andreas Henschel, Michael Schroeder 153
97 Utilising mutation tagging with gene identifiers for protein
structure function studies
Rainer Winnenburg, Conrad Plake, Christof Winter, Annalisa
Marsico, Michael Schroeder 154
98 SCOPPI . A Structural Classification of Protein-Protein
Interfaces
Christof Winter, Andreas Henschel, Wan Kyu Kim, Gihan
Dawelbait, Michael Schroeder 155
99 Serum proteomic profiling and targeted metabolomic of
obese: Integrative data analysis of biological profile data
Henry Wirth, Martin von Bergen, Jayaseelan Murugaiyan,
Hans Binder, Andreas Oberbach 156
7. Tissue and Cell Engineering 159
100 Hematopoietic progenitor cells are vulnerable to high
oxygen concentrations
Rudiger Alt, Nicole Noack, Michael Cross 161
101 Fabrication of Microscaffolds by Solid Lipid Templating
Kristina Ambrosch, Michaela Schulz-Siegmund, Michael C.
Hacker 162
102 iPS cells as model system for human virus diseases
Antje Arnold, Claire Fabian, Steven Sauerzweig, Yahaira
Naldijk, Ute Brinkmann, Uwe G. Liebert, Alexandra Stolzing 163
103 Characterization of the early embryo upon loss of histone
methyltransferase Setd1a
Anita Sabine Bledau, Andrew Francis Stewart, Konstantinos
Anastassiadis 164
104 Hepatocytes from human mesenchymal stem cells support
liver regeneration after acute injury
Bruno Christ, Peggy Stock, Hendryk Aurich, Ines Aurich,
David Wohlrab, Werner Hein, Sabine Ebensing, Madlen
Hempel, Matthias M. Dollinger, Wolfgang E. Fleig 165
105 Generation and Characterization of Tumor Spheroids as
Cellular Models for Anti-Cancer Drug Discovery
Anja Drose, Mirjam Ingargiola, Bernd Schwenzer 166
106 The impact of iPS on stem cell legislation and administration
in Germany . points to consider for international stem cell
research cooperation
Timo Faltus 167
107 Isolation and Characterisation of Human Melanocytes
from Hair follicles for Clinical Use
Christina Fieber, Jan C. Simon, Andreas Emmendorffer 168
108 Differential expression of biofunctional GM1 and GM3
gangliosides within the plastic-adherent multipotent
mesenchymal stromal cell population
Daniel Freund, Denis Corbeil 169
109 Telomerase activity and hepatic functions of rat embryonic
liver progenitor cell in nanoscaffold coated small scale
bioreactor
Shibashish Giri, Sanja Pavlica, Mario Keller, Augustinus Bader 170
110 Temporally-controlled site-specific recombination in
zebrafish
Stefan Hans, Jan Kaslin, Dorian Freudenreich, Michael Brand 171
111 Osteoclastic activity of bone morphogenetic protein-2 in
lumbar spinal fusion
Christian Hohaus, Yvonne Minkus , Timothy Ganey, Hans-
Jorg Meisel 172
112 Fetal and adult hematopoiesis requires continuous Mll1
function
Andrea Kranz, Frieder Schwenk, Jost Seibler, Konstantinos
Anastassiadis, A. Francis Stewart 173
113 Plasma membrane mechanics in zebrafish germlayer
progenitor cells
Michael Krieg, Jonne Helenius, Daniel J. Muller, Carl-Philipp
Heisenberg 174
114 DNA repair in aged human MSC
Michela Livrea, Alexandra Stolzing 175
115 Microspherical delivery of a growth factor
Alexander Lochmann, Hagen Nitzsche, Sabrina von Einem,
Elisabeth Schwarz, Karsten Mader 176
116 Electrospun poly(ƒÃ-caprolactone) (PCL) microfiber meshes
with predefined fiber diameters
Tina Loth, Markus Manhardt, Kristina Ambrosch, Michaela
Schulz-Siegmund, Michael C. Hacker 177
117 Surface modification of medical CoCr alloys by a
thermochemical process
Johanna Lutz, Stephan Mandl 178
118 Regeneration of chronic osteochondral defects using
autologous mesenchymal stem cells in a sheep model
Bastian Marquass, Pierre Hepp, Robert Richter, Steffanie
Schmidt, Frank Stein, Augustinus Bader, Christoph Josten,
Matthias Zscharnack, Ronny Schulz 179
119 Intervertebral disc repair using adipose tissue-derived stem
and regenerative cells: experiments in a canine model
Hans Jorg Meisel, Timothy Ganey, Christian Hohaus, William
Hutton, Tim Moseley, Marc Hedrick, Brian Strem 180
120 Conditional Mutagenesis of Histone Methyltransferase
Mll2 in Neural Stem Cells
Katrin Neumann, Maria Rostovskaya, Sandra Lubitz, Andrea
Kranz, A. Francis Stewart, Konstantinos Anastassiadis 181
121 Peptide vectors for siRNA delivery into cells
Ines Neundorf, Anja Tennemann, Robert Rennert 182
122 PEEK-WC-PU membranes for expansion of rat embryonic
liver cells
Sanja Pavlica, Antonella Piscioneri, Frank Peinemann,
Angela Hennig, Javorina Milosevic, Stefania Laera, Piero
Favia, Loredana DeBartolo, Augustinus Bader 183
123 Dynamic Coupling of Pattern Formation and Morphogenesis
in the Developing Vertebrate Retina
Alexander Picker, Florencia Cavodeassi, Anja Machate,
Sabine Bernauer, Stefan Hans, Gembu Abe, Koichi
Kawakami, Stephen Wilson, Michael Brand 184
124 Establishment of risk analysis of bone replacing scaffolds
Heike Schneider, Kathleen Schrock, Manja Kamprad,
Michaela Schulz-Siegmund 185
125 Osteogenic differentiation of human adipose tissue-derived
stem cells: Are the standard ascorbate-2-phosphate
concentrations too high?
Hellen Schneider, Michael Hacker, Michaela Schulz-Siegmund 186
126 Electrospinning parameters critical for the generation of
poly(ƒÃ-caprolactone) (PCL) nanofibers
Maximilian Sperlich, Markus Manhardt, Michaela Schulz-
Siegmund, Michael Hacker 187
127 Controlled formation of embryoid bodies in bioreactors for
reproducible differentiation initiation of mouse and primate
embryonic stem cells
Susanne Trettner, Alexander Seeliger, Nicole I. zur Nieden 188
128 Formation of MMP cleavable hydrogel materials for
the development of novel biohybrid system for tissue
engineering
Mikhail Tsurkan, Kandice Levental, Petra Welzel, Milauscha
Grimmer, Andrea Zieris, Woranan Panyanuwa, Uwe
Freudenberg, Carsten Werner 189
129 3D-cardiomyocyte structures generated from murine
embryonic stem cells . A novel tool for drug screening on
microcavity arrays
Silvia Vinz, Randy Kurz, Andrea A. Robitzki 190
130 Non-invasive acoustical imaging of mesenchymal stem
cells
Moritz von Buttlar, Esam Ahmed Mohamed, Amro
Abdelrahman, Albert Kamanyi, Wolfgang Grill 191
131 Bioreactor with integrated monitoring and mechanical
stimulation for cartilage tissue engineering by collagen
scaffold associated Mesenchymal Stem Cells
Erik von der Burg, Moritz von Buttlar, Wolfgang Grill 192
132 Potential ageing effects in long-term cultured mouse
neurospheres
Vladimir Vukicevic, Anna Jauch, Timo C. Dinger, Linda
Gebauer, Veronika Hornich, Stefan R. Bornstein, Monika
Ehrhart-Bornstein, Albrecht M. Muller 193
133 Spatially confined cell growth
Ronald Werner, Torsten Koal, Heinz Georg Jahnke, Andrea
A. Robitzki, Tilman Butz, Tilo Reinert 194
8. Neuromedicine 197
134 Improved brain outcome by autologous bone marrowderived
mononuclear cells (BMCs) intravenously given 24
hours after stroke in sheep as imaged by PET
Henryk Barthel, Johannes Boltze, Christiane Boltze, Udo
Grosmann, Magnus Kluge, Andreas Schildan, Anita Seese,
Frank Emmrich, Uwe Gille, Osama Sabri 199
135 Saffron extract inhibits the glutamatergic synaptic
transmission on rat cortical neurones
Frauke Berger, Andreas Hensel, Matthias Lechtenberg, Karen
Nieber 200
136 Vascular endothelial growth factor (VEGF) affects processing
of amyloid precursor protein and ƒÀ-amyloidogenesis in
brain slice cultures derived from transgenic Tg2576 mouse
brain
Susanne Burger, Monika Noack, Elena Kouznetsova, Yousef
Yafai, Ludmil Kirazov, Evgeni Kirazov, Reinhard Schliebs 201
137 Isolation of Chromaffin Progenitor Cells from Adult Adrenal
Medulla
Kuei-Fang Chung, Flavie Sicard, Vladimir Vukicevic, Linda
Gebauer, Wieland B. Huttner, Stefan R. Bornstein, Monika
Ehrhart-Bornstein 202
138 Cellular characteristics of neural progenitor cells in the
adult zebrafish telencephalon
Julia Ganz, Jan Kaslin, Heiner Grandel, Michael Brand 203
139 Viral gene transfer of cell cycle inhibitors to slow
down progressive neurodegeneration
Pia Glockner, James Uney, Thomas Arendt, Uwe Ueberham 204
140 A novel fluorescent acetylcholinesterase inhibitor released
from nanoparticles selectively binds hippocampal
ƒÀ-amyloid plaques in triple transgenic mice
Wolfgang Hartig, Johannes Kacza, Bernd-Reiner Paulke, Jens
Grosche, Anke Hoffmann, Paul Wilhelm Elsinghorst, Michael
Gutschow 205
141 Role of purinergic signalling in the development of fibre
projections?
Claudia Heine, Nico Scherf, Jens-Peer Kuska, Ulf-Dietrich
Braumann, Heike Franke 206
142 A study of Imaging Geno-Phenotypes in dyslexia
Holger Kirsten, Carolin Ligges, Arndt Wilcke, Peter Ahnert,
Johannes Boltze 207
143 Depression-like deficits in rats are improved by subchronic
modafinil
Holger Koch, Ralf Regenthal, Christian Kohler, Ute Krugel 208
144 Impedance spectroscopy: A method for developing a labelfree
detection system for neurodegenerative diseases
Dana Krinke, Heinz-Georg Jahnke, Andrea A. Robitzki 209
145 Effects of blue light scleral cross-linking on rabbit
eye growth
Qing Liu, Hans Peter Iseli, Nicole Korber, Niclas Lindqvist,
Martin Gryga, Peter Wiedemann, Andreas Reichenbach,
Mike Francke 210
146 Isolation and biological potential of enteric nervous system
precursors derived from human gut
Marco Metzger, Nikhil Thapar, Lothar Just 211
147 Non-hypoxic stabilization of hypoxia-inducible
factor alpha (HIF-ƒ¿): Relevance in neural progenitor/stem
cells
Javorina Milosevic, Irena Adler, Sigrid C. Schwarz, Gail
Walkinshaw, Alexander Storch, Johannes Schwarz 212
148 Ca2+ Responses of Muller cells induced by light stimulation
of photoreceptor cells
Katja Rillich, Janina Gentsch, Michael Weick, Andreas
Bringmann, Andreas Reichenbach 213
149 Polyethylenimine as a possible gene therapeutical tool
against Alzheimerfs Disease
Susanne Rohn, Thomas Arendt, Uwe Ueberham 214
150 Increase of intracellular Ca2+ by adenine and uracil
nucleotides in human midbrain-derived neuronal precursor
cells
Patrizia Rubini Illes, Johannes Engelhardt, Mahmoud Al-
Khrasani, Javorina Milosevic, Johannes Schwarz, Peter Illes,
Wolfgang Norenberg 215
151 Study of human neural progenitor cell fate after grafting
into rat striatum
Johanna Scheibe 216
152 Organotypic cocultures as an alternative to conventional
animal models
Sabine Schewtschik 217
153 Analysing a potential neuroprotective function of
perineurinal nets by using organotypic slice cultures
Anne Suttkus, Markus Morawski, Gert Bruckner, Thomas Arendt 218
154 Distinct reactions of retinal microglia cells evoked by
various stimuli
Elke Ulbricht, Mike Francke, Ulrike Zeitschel, Andreas
Reichenbach 219
155 Connexins control glial glutamate transporter expression.
Tina Unger, Stefanie Bette, Jurgen Engele 220
156 A new hippocampal ex vivo model to study tauopathies by
label-free impedance spectroscopy
Annett Wegner, Heinz-Georg Jahnke, Till G. A. Mack, Frank
Striggow, Andrea A. Robitzki 221
157 A new mouse model for targeting the astrocytic NADH /
NAD+ redox state in vivo
Franziska Wilhelm, Jan Rillich, Ulrike Winkler, Johannes
Hirrlinger 222
158 Structural plasticity of astrocytes and the impact of the cell
adhesion protein vinculin
Ulrike Winkler, Marcello Sestu, Alice Zemljic-Harpf, Robert
S. Ross, Wolfgang H. Ziegler, Johannes Hirrlinger 223
9. Diagnostics 225
159 Effects of A2A and A2B ligands on ach contraction in
inflamed rat small intestinal preparation
Karen Nieber, Claudia Warstat, Fabien Michel, Luo Yan,
Christa Muller, Sebastian Michael 227
160 Concentration of mucus in gastric juice in normal adult
horses withhold feed and after application of pronurtin
Gerald F. Schusser, Alice Spallek, Stephan Recknagel, Julia
Breuer, Gabor Koller 228
161 Dendritic sugar balls for biological experiments driven by
H-bonds
Dietmar Appelhans, Brigitte Voit 229
162 Development of an ELISA and a Candidate Vaccine for
Pigeon Circovirus Infection
Mohammad Yahya Halami, Wieland Schrodl, Reimar Johne,
Erhard F. Kaleta, Hermann Muller 230
163 Development and fabrication of a novel proteinbased
biosensor for specific detection and immobilisation
of cells
Anja Steude, Oliver Panke, Sabine Schmidt, Matthias Nieber,
Andrea A. Robitzki 231
164 Mycotoxin determination by means of an electronic nose
Anselm Werner, Claudia Winter, Monika Kruger, Andrea
Lindner, Klaus Kruger 232
10. Microfluidics 235
165 Structural levels of organization in the TmHU-DNAcomplex
as studied by optical tweezers assisted Force
spectroscopy
Carolin Wagner, Mathias Salomo, Friedrich Kremer 237
166 Optical tweezers to investigate receptor-ligand interactions
on a single contact level
Carolin Wagner, Mathias Salomo, Friedrich Kremer 238
11. Protein Engineering and Biocatalysis 241
167 Evaluating 3D experiments in optical tweezers
Marcel Ander 243
168 Reconstituting Cytokinesis in Artificial Lipid Systems
Senthil Arumugam 244
169 Variants of Candida antarctica lipase B convert ƒ¿-substituted
substrates
Sally Bayer, Thomas Greiner-Stoffele, Meike Ballschmiter 245
170 Expression, Purification and Characterization of the
Neuropeptide Y Receptor Type 2
Sandra Berndt, Peter Schmidt, Cindy Montag, Christian
Berger, Susann Schimmer, Diana Lindner, Annette G. Beck-
Sickinger, Rainer Rudolph, Daniel Huster 246
171 Construction of a RNaseT1 expression system for Aspergillus
niger
Kathrin Bonsch, Thomas Greiner-Stoffele, Meike Ballschmiter 247
172 Torque Measurements on DNA with Magnetic Tweezers
Hergen Brutzer, Nicholas Luzzietti, Friedrich Schwarz, Ralf Seidel 248
173 Tagging methods for proteomics and regulomics in mouse
embryonic stem cells
Giovanni Ciotta, A. Francis Stewart 249
174 Two ways to screen for new proteases in (meta-) genomic
libraries
Antje Eichler, Thomas Greiner-Stoffele, Meike Ballschmiter 250
175 Pseudomonas putida . development of a heterologous
expression system for complex natural products
Frank Gros, Dominik Pistorius, Youming Zhang, A. Francis
Stewart, Rolf Muller 251
176 Prediction of Flocculation Ability of Brewing Yeast
Inoculates by Flow Cytometry, Proteome Analysis, and
mRNA Profiling
Franziska Heine, Frank Stahl, Heike Strauber, Claudia
Wiacek, Dirk Benndorf, Cornelia Repenning, Frank Schmidt,
Thomas Scheper, Martin von Bergen, Hauke Harms, Susann
Muller 252
177 Reduction of substrate binding pocket of glucose
dehydrogenase B for improved substrate specificity
Michael Hofer, Kathrin Bonsch, Meike Ballschmiter 253
178 Screening for indole hydroxylating variants of P450cam
Gregor Hoffmann, Katrin Bonsch, Meike Ballschmiter 254
179 Interactions in the plant RNase P/ MRP
Mario Krehan, Sebastian Braun, Janine Dahl, Nicolas
Menzel, Christian Heubeck, Astrid Schon 255
180 The crystal structure of arylmalonate decarboxylase
reveals active site flexibility in catalysis
Bartholomeus Kuttner, Markus Kircher, Susann Rosmus, Antje
Keim, Norbert Strater 256
181 RiboxX: RNA-interference in a box !R
Jacques Rohayem, Katrin Jager, Ivonne Robel, Kristin Hille,
Dorothea Kramer, Romy Zieger, Mirko Bergmann, Julia
Gebhardt, Christiane Petzold 257
182 Mutagenesis of the Thermus aquaticus amylomaltase to
produce large cyclic glucans
Christian Roth, Nicole Weizenmann, Wolfgang Zimmermann,
Norbert Strater 258
183 NMR Measurements of a Class A GPCR from Prokaryotic
Expression
Peter Schmidt, Andreas Bunge, Diana Lindner, Sandra Berndt,
Christian Berger, Annette G. Beck-Sickinger, Rainer Rudolph,
Daniel Huster 259
184 Single-Molecule Studies of DNA Translocating Restriction
Enzymes
Friedrich Schwarz, Kara van Aelst, Mark Szczelkun, Ralf Seidel 260
185 Immobilization of Proteins via Cu(I) catalyzed Alkyne Azide
Cycloaddition
Max Steinhagen, Annette G. Beck-Sickinger 261
12. Appendix 263
Center for Biotechnology and Biomedicine (BBZ) 265
The Biotechnology Center (BIOTEC) 268
13. Index 271


FOREWORD
After its great success in Dresden in 2007, the Saxon Biotechnology Symposium series is
being continued in Leipzig in 2009, the year of the 600th anniversary of the foundation of
Leipzig University in 1409.
The Center for Biotechnology and Biomedicine at the University of Leipzig and the
Biotechnology Center of the Technical University of Dresden are continuing cooperation
to hold the joint Biotechnology Symposium in Saxony. The main topics of the conference,
hosted in Leipzig this year, will be eNon-Coding RNAs as Regulators and Toolsf, eStem Cell
Therapies for Neurological Disordersf and eBiophotonics . Molecular Motors and Optical
Tweezersf.
It is the purpose of the 2009 Biotechnology Symposium to further bridge the gap between
basic, applied and integrative research in the fields of nanobiotechnology / nanomedicine
and molecular bioengineering. This will increasingly strengthen regional networks and
make Saxony well-known on a supraregional and also on an international level as a place
where biotechnology ranks high. Saxony is an excellent location for research and a growing
biotechnological industry, providing a platform for the transfer of knowledge and technology
in the fields of molecular design, the development and testing of active substances, genomics,
proteomics, diagnostics, cell techniques, nano-bioelectronics, biophysics, cellular machines,
tissue engineering, and bioinformatics.
Biotechnology is emerging as a key technology for the future, inventions and innovations
being the motors for biotechnological progress. We hope this symposium will give the
Saxon biotechnological sector a boost and encourage the universities, research centers and
companies.
Prof. Dr. Andrea A. Robitzki Prof. Dr. Michael Brand
Director Director
Center for Biotechnology and Biomedicine Biotechnology Center

PROGRAM
9:00 am . 9:30 am Opening
Prof. Dr. Martin Schlegel
Prorektor fur Forschung und wissenschaftlichen Nachwuchs der Universitat
Leipzig
Prof. Dr. Petra Schwille
stellvertretende Direktorin des Biotechnologischen Zentrums der Technischen
Universitat Dresden
9:30 am . 11:30 am Session 1
Non-coding RNAs as regulators and tools
Chair: Prof. Dr. Manfred Blessing/Dr. Andreas Beyer
STAT3 regulated non-coding RNAs in tumor cells
Prof. Dr. Friedemann Horn
Universitat Leipzig, Medizinische Fakultat, Institut fur Klinische Immunologie
Predicting and modeling microRNA regulation with an application to stem
cell development
Dr. Fabian Theis
Helmholtz-Zentrum Munchen, Institut fur Bioinformatik und Systembiologie,
CMB
Exploring chemical space with aptamers
Prof. Dr. Michael Famulok
Universitat Bonn, Life & Medical Sciences (LIMES)-Institut
From RNAi Screens to Molecular Function: A Systematic Pipeline for Gene
Function in Mammalian Cells
Dr. Frank Buchholz
Max-Planck-Institut fur Molekulare Zellbiologie und Genetik Dresden
11:30 am . 1:00 pm Postersession & lunch break
1:00 pm . 3:00 pm Session 2
Stem cell therapies for neurological disorders
Chair: Prof. Dr. Francis Stewart/Dr. Thole Zuchner
Stem cell therapy for Parkinsones disease
Prof. Dr. Patrik Brundin
Wallenberg Neuroscience Center, Department of Experimental Medical
Science Lund, Schweden
Biomaterial assisted cellular self-organization and tissue program: a new
paradigm in regenerative medicine
Prof. Dr. Carlos E. Semino
Universitat Leipzig, Translationszentrum fur Regenerative Medizin
Regenerating the brain with endogenous stem cells
Dr. Verdon Taylor
Max-Planck-Institut fur Immunobiologie, Fachbereich molekulare Embryologie,
Freiburg
Cell Transplantation into the Retina: A Treatment Option for Photoreceptor
Loss?
Dr. Marius Ader
Technische Universitat Dresden, Zentrum fur Regenerative Therapien Dresden
3:00 am . 3:30 pm Postersession & coffee break
3:30 pm . 5:30 pm Session 3
Biophotonics . molecular motors and optical tweezers
Chair: Prof. Dr. Andrea A. Robitzki/Dr. Erik Schaffer
Single molecule biophysics: soft matter, weak interaction and complex Mechanisms
Prof. Dr. Dario Anselmetti
Universitat Bielefeld, Fakultat fur Physik
Optical trapping and 3D particle tracking: from concept to versatile applications
Dr. Anna Wozniak
JPK Instruments AG, Berlin
Bioanalytics at the nanometer and attoliter scale
Prof. Dr. Horst Vogel
Ecole polytechnique federale de Lausanne, Institut des sciences et ingenierie
chimiques, Schweiz
Single molecule studies in live and reconstituted cellular systems
Prof. Dr. Petra Schwille
Technische Universitat Dresden, Biotechnologisches Zentrum
5:30 pm . 6:00 pm Poster awards
6:15 pm . 6:45 pm Evening lecture
Opening by the Minister of the Saxon Ministry of Science and the Fine Arts
Dr. Eva-Maria Stange
Award ceremony of the initiative .Germany . Land of Ideasg
Mike Roseler
Direktor des Investment & FinanzCenter Leipzig-Mitte, Deutsche Bank Privatund
Geschaftskunden AG
6:45 pm . 7:30 pm
3D Micro Chip for pharmacological high throughput studies
Evening lecture from the awardee of the initiative .Germany . Land of Ideasg
Prof. Dr. Andrea A. Robitzki
Universitat Leipzig, Biotechnologisch-Biomedizinisches Zentrum
7:30 pm . 9:30 pm Get-together and buffet
All day Industrial exhibition and information desk of the 3D-Chip
Project

Non-coding RNAs as
Regulators and Tools
1.
Presentations

39
non -coding RNAs as Regulators and tols
1 STAT3 regulated non-coding RNAs in tumor cells
Friedemann Horn
Recent analyses of the human genome revealed the existence of a high number of noncoding
(nc)RNA transcripts. It is increasingly being recognised that these ncRNAs now constitute
an important and complex layer of cellular regulation. In addition, a growing number of
ncRNAs prove to be disease-associated. However, data on how cellular signalling networks
interact with the ncRNA layer are still sparse.
The transcription factor Stat3 was discovered by our group as a central component of
interleukin-6 signalling. Stat3 is involved in differentiation, proliferation and apoptosis
control of many cell types during embryogenesis as well as in adult tissues. In addition, Stat3
is now regarded a highly oncogenic protein, being constitutively active in >70% of human
tumours.
We currently address the question whether in addition to its known functions, the Stat3
pathway is involved in the transcriptional regulation of ncRNAs. We could demonstrate
that Stat3 controls the expression of the anti-apoptotic microRNA miR-21 through a
phylogenetically conserved enhancer. This microRNA is highly expressed in many cancers.
Our results suggest that the oncogenic potential of Stat3 relies at least in part on the induction
of the miR-21 gene.
Furthermore, genome-wide expression analyses provided evidence that Stat3 regulates many
novel ncRNA genes, suggesting an intimate cross-talk between the Stat3 signal pathway and
the unfolding .RNA worldg.
Prof. Dr. Friedemann Horn
Universitot Leipzig
Faculty of Medicine
Institute of clinical immunology
friedemann.horn@medizin.uni-leipzig.de
http://ikit.uniklinikum-leipzig.de/
non -coding RNAs as Regulators and tols
40
2 Predicting and modeling microRNA regulation
with an application to stem cell development
Fabian J. Theis, Dominik Lutter, Carsten Marr, Lena
Uetzmann, Heiko Lickert
The differentiation of embryonic stem (ES) cells during gastrulation into the cell types of the
three germ layers is controlled by multiple interacting gene regulatory mechanisms. In addition
to transcription factor (TF) driven regulations, there is strong evidence that microRNAs play
an important role during stem cell development. The identification of microRNA-controlled
gene regulation using expression data remains difficult, mainly for two reasons: (i) there is
still a lack of microRNA expression data with adequate time resolution, and (ii) the inhibitory
effect of microRNAs on translation stays invisible on the mRNA level. Here we study gene
regulation under the influence of microRNAs controlling differentiation of murine ES cells
into endoderm and mesoderm lineages. For this we use binding site prediction methods
to estimate putative microRNA targets. Moreover, we uncover microRNA-driven gene
regulation by measuring the indirect influence of an intronic microRNA on a microRNA-TFtarget-
gene motif.
Using microarray expression data of differentiating ES cells, we identify a regulatory system
including an intronic microRNA that stabilizes differentiation during development. Through
dynamical modeling we determine a bistable system that can switch between mesoderm
and endoderm state starting from an undifferentiated cell. This sheds light into the potential
cellular mechanisms driving ES cell differentiation.
Dr. Fabian J. Theis
Helmholtz Zentrum Munchen
Institute for Bioinformatics and Systems Biology
Computational modeling in biology
fabian.theis@helmholtz-muenchen.de
www.helmholtz-muenchen.de
41
non -coding RNAs as Regulators and tols
3 Exploring chemical space with aptamers
Michael Famulok
Small molecule inhibitors of proteins are invaluable tools in Chemical Biology. Their
identification can be tedious, because most screening methods have to be tailored to the
corresponding drug target. We have developed modular assays based on aptamer displacement
or protein-dependent reporter ribozymes for the screening of small-molecule inhibitors.
Asaptamers can be generated for virtually any protein, the assay potentially identifies inhibitors
for targets or individual protein domains for which no functional screen is available. Thereby,
chemical space is explored in a rapid, focused, and modular manner, by indirectly taking
advantage of the highest molecular diversity currently amenable to screening, namely that of
1016 different nucleic acid sequences. I will discuss the application of these approaches to
find new inhibitors for target proteins. Examples showing that these modulators can be used
as tools for gaining novel biological insight are provided.
Prof. Dr. Michael Famulok
Universitat Bonn
LIMES Program, Unit Chemical Biology & Medicinal
Chemistry
m.famulok@uni-bonn.de
www.chembiol.uni-bonn.de/
non -coding RNAs as Regulators and tols
42
4 From RNAi Screens to Molecular Function:
A Systematic Pipeline for Gene Function in
Mammalian Cells
Frank Buchholz
RNAi screens typically deliver a large number of candidate genes that play a role in a biological
process. The validation of these candidates and the dissection of the molecular mechanism of
action are often time consuming and cumbersome. We have developed a pipeline using BAC
recombineering technology and tissue culture transgenesis to streamline the analysis of hits
identified in large scale RNAi screens. Examples from this pipeline will be presented.
Dr. Frank Buchholz
Max-Planck-Institute for Molecular Cell Biology and
Genetics, Dresden
buchholz@mpi-cbg.de
www.mpi-cbg.de
43
non -coding RNAs as Regulators and tols

Stem cell therapies
for neurological
disorders
2.
Presentations

47
stem cel therapies for neurological disorders
5 Stem Cell Therapy for Parkinsonfs Disease
Patrick Brundin
The prospect of cell replacement therapy for neurological disorders has generated a great
deal of excitement. Cell transplantation has been tested in, e.g., Parkinsonfs disease (PD),
Huntingtonfs disease, stroke, multiple sclerosis and spinal cord damage. I will primarily
review neural transplantation in PD over the past two decades, and describe the main obstacles
that currently prevent transfer of the technology into an established treatment.
In my presentation I will also discuss our recent observations that grafted neurons can develop
Lewy bodies, which are the neuropathological hallmark of PD, and what the implications of
these findings are for the field of cell transplantation. Further, I will describe our current
attempts to obtain high numbers of dopaminergic neurons from stem cells. For example, we
attempt to control cell division after the stem cell-derived neurons are transplanted, in order
to avoid tumor/teratoma formation. We use knowledge from the developmental biology of
midbrain dopamine neurons both to increase the yield of dopaminergic neurons and to limited
uncontrolled growth of grafts. A safe and reproducible supply of cells is necessary for future
systematic, large-scale clinical trials. Only after such trials will we know whether neural cell
grafting has a place in the therapeutic arsenal against PD.
Prof. Dr. Patrick Brundin
Lund University
Wallenberg Neuroscience Center
Neuronal Survival Unit
Department of Experimental Medical Science
patrik.brundin@med.lu.se
www.med.lu.se/expmed/neuronal_survival_unit
stem cel therapies for neurological disorders
48
6 Biomaterial assisted cellular self-organization and
tissue program: a new paradigm in regenerative
medicine
Carlos E. Semino
Cellular self-organization studies have been mainly focused on models such as Volvox, the
slime mold Dictyostelium discoideum, and animal (metazoan) embryos. Moreover, animal
tissues undergoing regeneration also exhibit properties of embryonic systems such as cellular
dedifferentiation and self-organization processes that ends in rebuilding tissue complexity
and function. We speculate that the recreation in vitro of the biological, biophysical and
biomechanical conditions similar to those of regenerative milieu could elicit the intrinsic
capacity of differentiated cells to proceed to the development of a tissue-like structure. In
this presentation I will show that when primary mouse embryonic fibroblasts are cultured
in a soft nanofiber scaffold they establish a cellular network that causes an organized cell
contraction, proliferation and migration that ends in the formation of a symmetrically
bilateral structure with a distinct central axis. Interestingly, a subset of mesodermal genes
(Brachyury, Sox9 and Runx2) is upregulated during this morphogenetic process. The
expression of Brachyury was localized first at the central axis, extending then to both sides
of the structure. Furthermore, expression of Sox9 and Runx2 was followed by the spontaneous
formation of cartilage-like tissue mainly at the paraxial zone. Since cellular self-organization
is an intrinsic property of the tissues undergoing development this model could bring new
ways to consider tissue regeneration.
Prof. Dr. Carlos E. Semino
Translational Centre for Regenerative Medicine Leipzig
semino@trm.uni-leipzig.de
www.trm.uni-leipzig.de
49
stem cel therapies for neurological disorders
7 Regenerating the brain with endogenous stem
cells
Verdon Taylor, Onur Basak, Claudio Giachino, Philip
Knuckles
The brains of adult mammals contain stem cells that continuously generate neurons throughout
life. The identity of these stem cells and most of the mechanisms regulating their fate are
unresolved. Although cells with stem cell properties can be isolated from the embryonic
and adult brain, and expanded endlessly in vitro, it is unclear what potential these cells
really possess. Transplantation into the postnatal brains of host animals results in extensive
gliogenesis and limited or no neurogenesis, even in lesions. Here we will discuss genetic
evidence for the identity of adult neural stem cells and that the Notch signaling pathway is
critical for neurogenic and regenerative stem cells in the adult brain. In addition, we will
present evidence that adult derived neural stem cells, expanded in vitro retain multipotent
potential when transplanted into the brain, opening up the potential use of these cells for
identifying differentiation cues to generate defined neuronal cell types and thus for human
therapy.
Dr. Verdon Taylor
Max Planck Institute of Immunobiology Freiburg
Department of Molecular Embryology
taylor@immunbio.mpg.de
www.mpg.de
stem cel therapies for neurological disorders
50
8 Cell Transplantation into the Retina: A Treatment
Option for Photoreceptor Loss?
Marius Ader
Impairment of vision and blindness due to loss of photoreceptors, the light-sensitive cells of
the retina, are one of the most prevalent causes of disability in industrialized countries. The
adult mammalian retina lacks endogenous repair mechanisms and thus is unable to regenerate
photoreceptors lost due to injury or inherited diseases.
Possible treatment options for hindering disease progression or regaining sight include
pharmacological interventions, electrical implants, gene therapeutic approaches, or
molecular inhibition methods using antibodies or RNA interference techniques. However,
although promising, most of these treatment options require an early intervention in the
disease development before massive photoreceptor loss has occurred. Thus, cell-based
strategies might represent an important treatment option to replace lost photoreceptors in
retinopathies.
Diverse cell populations were used for transplantation experiments into the retina of rodents
in recent years. However, although some cells showed potential to integrate into the host
tissue and survived for prolonged time periods, differentiation into mature photoreceptors
was rarely observed. In contrast, recently we could demonstrate that integration and
differentiation into mature rod photoreceptors following transplantation into adult mouse
retinas is most successful when primary retinal cells isolated at the peak of rod photoreceptor
generation, i.e. the first postnatal week in mice, are used for grafting. The data implicates
that cells committed to the photoreceptor lineage rather then stem/progenitor cells have the
greatest potential for integration into host retinas and photoreceptor differentiation. Thus,
young photoreceptors represent prime candidates for the development of cell replacement
therapies for diseases characterized by photoreceptor loss.
Dr. Marius Ader
Technische Universitat Dresden
Center for Regenerative Therapies
Cell-based Therapies for the Treatment of Retinopathies
marius.ader@crt-dresden.de
www.crt-dresden.de
51
stem cel therapies for neurological disorders

Biophotonics .
molecular motors and
optical tweezers
3.
Presentations

55
biophotonics . molecular motors and optical twezers
9 Single Molecule Biophysics: Soft Matter, Weak
Interactions and Complex Mechanisms
Dario Anselmetti
Over the last 15 years, novel ultrasensitive biophysical methods have been developed that
allow identification of physical mechanisms, quantitative analysis of cellular processes, and
investigations of individual biomolecules and cells, with respect of protein organisational
structure, functional interplay and temporal dynamics. Namely, atomic force microscopy and
single molecule force spectroscopy (AFM, AFM-FS), optical tweezers, and single molecule
optical microscopy beyond the diffraction limit allow nowadays deep insights into complex
and regulated biological processes.
In my presentation I will report on recent work where we quantitatively investigated effectorstimulated
DNA-protein interaction in bacteria (Sinorhizobium meliloti) , posttranscriptionally
regulated RNA-protein interactions in plants (Arabidopsis thaliana), as well as the reverse
engineering of an artificial single molecule affinity switch in supramolecular host-guest
complexes (calixarenes) with AFM single molecule force spectroscopy. These examples
highlight the possibility to investigate (post)transcriptionally regulated processes at a single
molecule level as well as its synthetic imitation.
The dynamic action of DNA binding ligands can be studied by optical tweezers. The
manipulation of single-molecule DNA molecules allow nanoscreening of potentially
important therapeutic agents in anticancer chemotherapy, where the mechanism of action of
the enzyme topoisomerase I in the presence of inhibitors is investigated. Furthermore, the
threading of single DNA molecules into nanopores can be observed with a novel concept of
a quantitative 3D optical tweezers systems, which will allows the investigation of molecular
translocation binding and dynamic sliding phenomena along DNA.
Prof. Dr. Dario Anselmetti
Bielefeld University
Experimental Biophysics and Applied Nanoscience
Institute for Biophysics and Nanoscience (BINAS)
dario.anselmetti@physik.uni-bielefeld.de
www.physik.uni-bielefeld.de/biophysik
biophotonics . molecular motors and optical twezers
56
10 O ptical trapping and 3D particle tracking: from
concept to versatile applications
Anna Wozniak, Joost van Mameren, Gerd Behme,
Sebastian Roth
In the past decade, experiments involving the manipulation and observation of nanostructures
with light using optical tweezers methodology have developed from proof-of-principle
experiments to an established quantitative technique in fields ranging from (bio)physics to cell
biology. With optical tweezers, microscopically small objects can be held and manipulated.
At the same time, the forces exerted on the trapped objects can be accurately measured.
JPK Instruments has developed a quantitative optical tweezers platform, the NanoTracker.
This platform allows the controlled trapping and accurate tracking of nanoparticles. With its
3D detection system, particle displacements within the trap can be recorded with nanometer
precision. Moreover, dynamic forces acting on the particle (e.g., exerted by motor proteins)
can be measured with better than piconewton resolution on a microsecond time-scale.
Here, we detail these and further features of the NanoTracker platform. Several successful
biophysical applications will be demonstrated. In particular, we show how some of the
hallmarks of single-molecule biophysics, the overstretching transition of DNA and the
famous 8-nm steps and stall forces of kinesin motor proteins, can be studied in a versatile and
operator-friendly manner.
With the NanoTracker, optical tweezers finally transcend from the labs of self-building
scientists who helped the technique mature, to a turn-key system able to serve a much wider
community of researchers in the life sciences.
Dr. Anna Wozniak
JPK Instruments AG
Applications group
nanotracker@jpk.com
www.jpk.com
57
biophotonics . molecular motors and optical twezers
11 Bioanalytics at the nanometer and attoliter scale
Horst Vogel, Ralf Schmauder
Living systems are characterized by spatial compartmentalization to facilitate the co-existing
of highly diverse chemical process. Without the existence of clearly defined borders and
tightly regulated signal transduction across these borders, differentiation and diversity at the
cellular level would not be possible.
In nanobiotechnology and ultrasensitive bioanalytics subdivision of solutions in miniaturized
autonomous units are required to increase the functional complexity of a system, reduce reagent
consumption, and to monitor fast chemical kinetics or even to study single-molecules. Here
we report a biomimetic toolbox to generate functional compartments and nested structures on
a nanoscale and to analyze chemical processes and complexity in them:
We developed a self-assembled nanofluidic system for executing chemical synthesis by
mixing attoliter volumes (released from nanometer-sized lipid vesicles) in a closed femtoliter
reactor vessel (a larger unilamellar lipid vesicle). The number of mixed reactants and their
enzymatic turnover are monitored with single molecule precision in situ by fluorescence
correlation spectroscopy. We demonstrated an approach to use FRET as a molecular amplifier
to selectively and sensitively monitor the reaction states of individual molecules. We report
on the parallel isolation of attoliter (10-18 L) sized artificial and native (cell-derived) vesicles
and their self-positioning with 100-nm precision in ordered arrays on surfaces or free-floating
in solution using multiple laser tweezers. Biological processes, mediated on and across
cellular membranes via trans-membrane can be reported in a parallel fashion in this system.
Prof. Dr. Horst Vogel
Ecole polytechnique federale de Lausanne
Institut des sciences et ingenierie chimiques
horst.vogel@epfl.ch
www.epfl.ch/index.en.html
biophotonics . molecular motors and optical twezers
58
12 Single molecule studies in live and reconstituted
cellular systems
Petra Schwille
Cell and developmental biology are immensely complex and rapidly growing fields that are
particularly in need of quantitative methods to determine their key processes. With all the data
known about protein interactions and interaction networks from biochemical analysis, there
still remains the important task of in situ proteomics, i.e. determining the thermodynamic and
kinetic parameters of certain reactions in the cellular environment. Further, to understand
how cells polarize and develop into organisms, we need quantitative methods to determine
concentration gradients and diffusion coefficients of key factors such as morphogens. In
conjunction with two-photon excitation and spectrally resolved detection, Fluorescence
Correlation Spectroscopy (FCS) is a powerful means for the study of concentrations,
translocation processes, molecular association or enzymatic turnovers. It is fair to state that
this technique raises strong hopes for the possibility of in situ proteomics, but also for a
more quantitative access to developmental processes. During the past years, we applied FCS
to a variety of cell-associated phenomena, among them protein-protein binding, enzymatic
reactions, endocytosis, and gene delivery. To study processes on cell membranes, and to
elucidate the delicate interplay between membrane proteins and the surrounding lipids, we
devised cell-like model membrane systems mimicking the formation of membrane domains
whose cellular counterparts are potentially active as recruitment platforms for signalling
proteins. We established one- and two-photon scanning FCS for processes on membranes
which are too slow for standard FCS observation with a fixed beam. Performing circular
scanning FCS on developing embryos of C.elegans, we show how the motion of labelled
proteins is non-uniformly distributed in the cortex during cell polarization. Additionally,
scanning FCS overcomes the problems of photobleaching and low statistical accuracy
commonly encountered in FCS with fixed measurement volume, when applied to slowly
moving molecules. By using two-photon excitation one additionally benefits from the
possibility of long measurement times without disturbing the embryo development.
Prof. Dr. Petra Schwille
Technische Universitat Dresden
Biotechnological Centre (Biotec)
Biophysics
petra.schwille@biotec.tu-dresden.de
www.biotec.tu-dresden.de
59
biophotonics . molecular motors and optical twezers

4. Molecular Medicine
Posters

63
Molecular Medicine
13 PDE10A inhibitors for the treatment of
schizophrenia and psychosis
Ghadir Barbar Asskar, Sabine Mann, Karen Nieber,
Norbert Strater, Peter Brust, Detlef Briel
Phosphodiesterases (PDEs) are key enzymes in the cyclic nucleotide signalling pathways.
They are a class of enzymes which are able to hydrolyse the 3L-5L phosphodiester bond of
cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP).
Until now 11 families of phosphodiesterases are identified. PDE4, -7 and -8 are selective
for cAMP, PDE5, -6 and -9 are specific for cGMP and PDE1, -2, -3, -10 and -11 hydrolyze
cAMP and cGMP. There are hints that inhibitors of PDE10A may be potential drugs for
various therapeutic fields, e.g. in the treatment of schizophrenia and psychosis. The aim of
this work was to synthesize and characterize the unknown (S)-enantiomer of a known and
already tested PDE10A-selective inhibitor as well as the already described (R)-enantiomer
to investigate whether the enzyme is able to distinguish between the two enantiomers. The
synthesized heterocyclic compound is an analogue of papaverine. Although enantiomers
should have the same physicochemical properties, we found unexpected differences.
In conclusion we were able to synthesize the two entantiomers each with an overall yield of
44% over 4 steps and we were also able to fully characterize both compounds.
Ghadir Barbar Asskar
Universitat Leipzig
Institute of Pharmacy
Department of Pharmacognosy Chemistry
gadeer_barbar@hotmail.com
www.uni-leipzig.de/~pharm
Molecular Medicine
64
14 Aminosulfonate-Modulated pH-Induced Closure of
Cx26 Hemichannels Observed by High-Resolution
Atomic Force Microscopy
Christian A. Bippes, Jinshu Yu, Galen M. Hand, Daniel J.
Muller, Gina E. Sosinsky
Gap junction channels regulate cell-cell communication by passing metabolites, ions
and signaling molecules. Gap junction channel closure in cells by acidification is well
documented; however, it is unknown whether acidification affects connexins or modulating
proteins or compounds that in turn act on connexins. Protonated aminosulfonates directly
inhibit connexin channel activity in an isoform specific manner as shown in previously
published studies. High-resolution atomic force microscopy of force dissected connexin26
gap junctions revealed that in HEPES buffer the pore was closed at pH < 6.5 and opens
reversibly by increasing the pH to 7.6. This pH effect was not observed in non-aminosulfonate
buffers. Increasing the protonated HEPES concentration did not close the pore indicating
that a saturation of the binding sites occurs at 10 mM HEPES. Analysis of the extracellular
surface topographs reveals that the pore diameter increases gradually with pH. The outer
connexon diameter remains unchanged and there is an ~ 6.5‹ rotation in connexon lobes.
These observations suggest that the underlying mechanism closing the pore is different from
an observed Ca2+ induced closure.
Christian A. Bippes
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Machines Group
christian@biotec.tu-dresden.de
www.biotec.tu-dresden.de
65
Molecular Medicine
15 THE P53 TRANSCRIPTOME . Discovery of
Regulated non-coding RNA Genes
Levin Bohlig, Antje Kretzschmar, Kristin Reiche, Jorg
Hackermuller, Friedemann Horn, Kurt Engeland
Non-coding RNA is involved in the regulation of fundamental cellular processes like
transcription, mRNA turnover, translation, dosage compensation and differentiation.
Although some long non-coding RNAs have been identified, the regulation and functional
significance of these novel genes emerges only very recently. In order to identify new noncoding
RNA genes regulated by the tumor suppressor p53 we carried out genome-wide tiling
array experiments on an Affymetrix platform. We compared RNA expression before and after
p53 wild-type expression in cells carrying an inducible p53 system. Using software defining
specific thresholds for transcript length and signal intensity we identified 7845 regions in
the genome differentially expressed after p53 induction. We observed RNAs which were
either up- or downregulated following p53 expression. Among these signals 80% were
located in protein-coding genes. From a set of sixty selected p53-target genes described in
the literature we found 66% differentially regulated in this experiment confirming the quality
of this approach. Furthermore, we developed a bioinformatic tool which allows to select for
potential functional non-coding RNA genes from the group of significant hits. This analysis
led to identification of 1526 hits outside protein-coding genes. We verified several non-coding
RNAs for their regulation in other cell systems. Since p53 is an important cell cycle regulator
the identified ncRNAs may be functionally involved in cell cycle progression.
Levin Bohlig
Universitat Leipzig
University Hospital
Institute for Molecular Oncology
Department of Obstetrics and Gynecology
levin.boehlig@medizin.uni-leipzig.de
www.engeland-research.de
Molecular Medicine
66
16 Interaction between PIP2 and Ceramide in
Drosophila Phototransduction
Salvatore Chiantia, Ujjaini Dasgupta, Acharia Usha, Petra
Schwille
The lipid phosphatidylinositol 4,5-biphosphate (PIP2) is critical in a number of physiological
processes and its lateral segregation in the plasma membrane appears to be important for
several of these spatially localized events. It has recently been shown, for example, that
clustering of PIP2 is necessary for the activity of NORPA -a phospholipase C homolog- in
the context of Drosophila phototransduction. A mutation in ceramide kinase (CERK) and the
consequent accumulation of unphosphorylated ceramide produce lateral reorganization of
PIP2, loss of NORPA activity and failure in light transduction. In this work, we clarified the
interaction between ceramide and PIP2, with a particular focus of PIP2 segregation, using
fluorescence imaging and Image Correlation Spectroscopy (ICS) on supported membranes.
The mechanism of formation of PIP2 domains in vivo is still matter of debate. Although
several experiments argue for the existence of protein-independent PIP2 segregation, an
interesting hypothesis states that certain proteins containing clusters of basic amino acid
residues can mediate PIP2 clustering in cholesterol-rich membrane domains. In order to
clarify the role of CERK and ceramide accumulation in determining the lateral organization of
PIP2, we explored separately both scenarios. Our results show that ceramides can affect PIP2
clustering, either via direct interaction or through ceramide-induced reorganization of raftlike
domains. Therefore, we could validate the model according to which CERK mutation
affect the activity of NORPA due to accumulation of ceramide in the plasma membrane.
Salvatore Chiantia
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Biophysics Group
chiantia@biotec.tu-dresden.de
www.biotec.tu-dresden.de
67
Molecular Medicine
17 Wnt signaling in osteogenic specification of
embryonic stem cells
Huawen Ding, Beatrice Kuske, Nicole I. zur Nieden
The differentiation of embryonic stem (ES) cells offers a powerful approach to study
mechanisms implicated in cell fate decision. To understand pathways controlling normal
bone development is imminent for deducing novel therapeutic targets, which could be aimed
at disease intervention in the clinic.
Wnt signaling has central roles in embryogenesis and human disease including cancer, in
which ƒÀ-catenin is a key mediator. However, several Wnt-mediated pathways have been
proposed to function independent of ƒÀ-catenin. Wnt5a can both inhibit and activate ƒÀ-catenin
activity, depending on which combination of receptors is expressed on the cell surface, which
was previously found to support specific stages of the osteogenic process in our lab.
In order to investigate how Wnt5a is involved in osteogenesis, we examined the RNA
expression pattern of the perspective Wnt5a downstream targets during differentiation.
Our results showed that all have distinct expression patterns during the first 11 days of
differentiation. Furthermore, blocking the Calmodulin dependent protein kinase II (CamKII),
c-Jun terminal kinase (JNK) and protein kinase C (PKC) pathways with specific inhibitors
upregulated ƒÀ-catenin protein expression levels in the nucleus. As a conclusion, Wnt5a seems
to enhance osteogenesis by activating CamKII, JNK, and PKC sub-pathways.
Huawen Ding
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Stem Cell Technology
huawen.ding@izi.fraunhofer.de
www.izi.fraunhofer.de
Molecular Medicine
68
18 Role of FGD6 in Actin Ring Formation and
Organelle Dynamics in Resorbing Osteoclasts
Ana Isabel Espirito Santo, Tobias Heckel, Bernard Hoflack
Osteoclasts (OCs), multinucleated cells derived from the monocyte/macrophage lineage,
are critical for bone remodelling. Their resorption function depends on the organization of
their actin cytoskeleton into a specialized structure . the sealing zone. OCs attach to the
bone matrix at the particular site where the bone will be resorbed, they are activated and
initiate the resorption cycle, that includes the relocalization at the sealing zone of secretory
and endocytic organelles required for the secretion of hydrolytic enzymes or the uptake of
digested material, respectively. The sealing zone is formed by condensing podosomes into a
highly dynamic podosomal belt upon contact with bone surface. Podosome turnover, actin
ring formation and endocytosis are regulated by RhoGTPases, whose activity is catalyzed,
among others, by RhoGEFs in response to extracellular stimuli. We have shown that, during
osteoclastogenesis, 8 RhoGEFs, including FGD6, are drastically upregulated. We show
that FGD6 is a Src target that regulates sealing zone formation and may play a role in the
recruitment of early endosomes (EEs) to the sealing zone. We analyze the different domains
of FGD6 in order to understand how they contribute to actin cytoskeleton organization and
to the recruitment of EEs. Our data indicates that, in HeLa cells and OCs, the PH and FYVE
domains, but not the RhoGEF domain, are sufficient for FGD6 association actin filaments. In
OCs, the PH domain is sufficient for localization to podosomes. FGD6 regulates sealing zone
formation and recruitment of EEs to this area.
Ana Isabel Espirito Santo
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Proteomics Group
ana.sanchez@biotec.tu-dresden.de
www.biotec.tu-dresden.de
69
Molecular Medicine
19 Dual role of the EGF-receptor in regulation of
glutamate transporter expression
Darko Glisic, Claudia Lehmann, Jurgen Engele
Prolonged high glutamate levels in the extracellular space are excitotoxic and are associated
with acute and chronic brain disease conditions, such as traumatic brain injury and hypoxia.
Termination of glutamatergic neurotransmission and prevention of excitotoxicity occurs
through rapid uptake of glutamate by high-affinity glutamate transporters, particularly by the
astrocyte-specific GLT-1. Since glutamate transporter levels decrease under aforementioned
disease conditions revealing the molecular factors that regulate glutamate transporter
expression is of particular interest. Endothelins (ETs) . a family of peptides upregulated in
the injured brain . are potent inhibitors of GLT-1 expression in cultured rat astrocytes. On
the other hand, ligands of the epidermal growth factor receptor (EGF-R) stimulate GLT-1
expression. Preliminary experiments indicate involvement of the EGF-receptor in liganddependent
stimulation as well as in ET-dependent inhibition of GLT-1 expression. EGF and
ETs differentially influence dimerization & subcellular localization of the EGF-R hinting at
a previously unknown, dual role of the EGF-R as a pathway intermediate allowing for the
differential regulation of GLT-1 expression.
Darko Glisic
Universitat Leipzig
Medical Faculty
Institute of Anatomy, Department of Molecular
Neuroanatomy
Glisic@medizin.uni-leipzig.de
www.uni-leipzig.de/~anatomie
Molecular Medicine
70
20 Two-dimensional difference in gel electrophoresis
(DIGE) for analyzing ischemic Cardiomyocytes
Sina Haas, Martin von Bergen, Andrea A. Robitzki
Cardiac diseases and myocardial dysfunctions following ischemia are the leading cause of
mortality in the western industrialized countries. Ischemia and reperfusion injury, resulting
from clinical setting of coronary revascularization in acute myocardial infarction, bypass
surgery and heart transplantation is a demanding issue.
In order to understand the cellular and molecular mechanisms, which are involved in ischemia
and reperfusion injury, ischemia was carried out on vital and contractive HL-1 cardiomyocytes
with glucose-deficient culture medium in the present of hydrogen peroxide. The impact of
ischemia induction on cardiomyocytes has been quantified via DIGE-proteomic analysis
and proteins that are concerned in these processes are identified using MALDI-TOF/TOFMS.
After incubation HL-1 cells with glucose-deficient culture medium multiple changes in
protein expression could be detected in 2D-gel-electrophoresis and proteins, correlated with
ischemic processes, were identified using MALDI-TOF/TOF, e.g. proteins for cytoskeleton
organisation, apoptosis regulation or energy metabolism proteins. Gel analysis after 16h
revitalisation phase showed less difference in protein expression.
By providing new insights into cellular mechanisms involved in ischemia and cardiac
dysfunction proteomics helps to identify new target structures and will contribute to generate
new diagnostic markers and strategies, which could be of great importance for heart attack
and stroke therapy in future.
Sina Haas
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
sina.haas@bbz.uni-leipzig.de
www.uni-lepzig.de/~dmpt
71
Molecular Medicine
21 Comprehensive (Molecular) Cytogenetic
Characterization of rare intracranial tumors
Heidrun Holland, Helene Hantmann, Wolfgang Krupp,
Ronald Koschny, Michela Livrea, Ralf Schober, Jurgen
Meixensberger, Peter Ahnert
Chromosomal aberrations play an important role in tumor formation, as predictors of
clinical outcome and response to therapy. Only a few publications report about (molecular)
cytogenetic investigations of rare brain tumors. Therefore, we performed the first
comprehensive cytogenetic analyses in esthesioneuroblastoma, adult medulloblastoma,
atypical meningioma, and hemangiopericytoma using trypsin-Giemsa staining (GTGbanding),
multicolor fluorescence in situ hybridization (M-FISH) and molecular karyotyping
using single nucleotide polymorphism array (SNP-A). Structural chromosomal aberrations
were found, predominantly located on chromosomes 2q,6q,21q,22q (esthesioneuroblastoma),
4q,9q,10q,11p,20q (adult medulloblastoma), 6q,8q,10q,12p (atypical meningioma), and
8q,10q (hemangiopericytoma). Novel, so far not described chromosomal aberrations were
detected: deletions del(2)(q37),del(21)(q22) [esthesioneuroblastoma], translocations t(4;11)
(q25;p15),t(9;20)(p23;p12) [adult medulloblastoma], t(8;19)(q24;q13), t(10;16)(q22;q12.1)
[atypical meningioma], and (partial) trisomy 8 [hemangiopericytoma]. For the first time,
SNP-A karyotyping revealed partial uniparental disomy on chromosomal regions 1q, 9q
(adult medulloblastoma), 1p31.1,2p16.1,2q23.3,6q14.1,6q21,9p21.1,10q21.1, and 14q23.3
(atypical meningioma). Our study underlines the necessity to apply complementary methods
for a comprehensive cytogenetic analysis of tumor genomes.
Heidrun Holland, Helene Hantmann
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
hhantmann@trm.uni-leipzig.de
www.trm.uni-leipzig.de
Molecular Medicine
72
22 The effects of thrombin on RPE cells are mediated
by transactivation of growth factor receptors
Margrit Hollborn, Carola Petto, Peter Wiedemann, Leon
Kohen, Andreas Bringmann
Purpose: Thrombin has been implicated in VEGF-induced angiogenesis. We
investigated whether thrombin alters the mRNA expression of various cytokines, the
expression of VEGF-A protein, and the proliferation and chemotaxis of RPE cells.
Methods: The mRNA expression was evaluated by real-time PCR. The secreted VEGF
protein was determined by ELISA. Cell proliferation was analyzed by a BrdU-immunoassay,
and chemotaxis was investigated by a Boyden chamber assay. The phosphorylation levels of
ERK1/2, p38, and Akt were investigated by Western blotting.
Results: Acutely isolated and cultured RPE cells expressed mRNAs for the thrombinreceptors
PAR1 and PAR3, as well as for the effector cell protease receptor-1. Exogenous
thrombin significantly stimulated the mRNA expression of PDGF, HB-EGF, bFGF
and VEGF. Thrombin stimulated dose-dependently the secretion of VEGF protein.
This effect was blocked by an anti-TGF-s1 antibody, by hirudin and by selective
inhibitors of ERK1/2, p38, JNK, Akt and mTOR activation. Thrombin increased the
chemotaxis. The thrombin induced chemotaxis is likely mediated by a transactivation
of the PDGF receptor tyrosine kinase and the p38 MAPK signaling pathway.
Conclusion: Thrombin enhances the expression of VEGF and induces chemotaxis in
human RPE cells. Thrombin evokes the activation of several signaling pathways which are
differentially involved in various cellular responses, i.e., migration and VEGF synthesis.
Transactivation of growth factor receptors is involved in these processes. Thrombin may
represent a critical factor that promotes neovascularization and formation of epiretinal
membranes.
Margrit Hollborn
Universitat Leipzig
Medical Faculty
Department of Ophthalmology, Eye Hospital
hollbm@medizin.uni-leipzig.de
www.augenklinik.uniklinikum-leipzig.de/
73
Molecular Medicine
23 Copper overload leads to fragmentation of
mitochondrial membrane lipids: implications
for the pathogenesis of liver toxicity in Wilson
disease
Dominik Huster, Irina Yurkova, Jurgen Arnhold
Mitochondria are targets of oxidative damage under conditions such as Cu overload.
Wilson disease (WD) is characterized by hepatic Cu accumulation due to ATP7B mutations.
Mitochondrial damage occurs in livers of WD patients and mouse models. Oxidative damage
is a proposed mechanism, but experimental evidence is hardly available. Aim of this study
was to examine if Cu affects liver mitochondria by free-radical fragmentation of membrane
lipids.
Mitochondrial lipids (cardiolipin, phosphatidylcholine etc.) were treated with Cu in presence
of H2O2/ascorbate for several times. Furthermore membrane lipids extracted from freshly
isolated wild-type and Atp7b-KO mice liver mitochondria were incubated in a comparable
Cu environment. Reaction products were analyzed by HP-TLC and MALDI-TOF-MS to
identify changes in mitochondrial lipids.
The analysis of lipids treated with Cu showed that lipids containing free hydroxyl group
in its polar part undergo a fragmentation with formation of new lipids. The combined
action of Cu2+/H2O2/ascorbate on cardiolipin-liposomes as well as on lipids extracted from
wild-type mouse liver mitochondria resulted in the formation of phosphatidic acid and
phosphatidylhydroxyacetone. MS-analysis revealed that these newly formed lipids derived
from fragmentation of cardiolipin by HO-radical induced fragmentation. Lipids extracted
from Atp7b-KO mice mitochondria in contrast to wild-type mice contained a new lipid which
was identified as phosphatidic acid.
In conclusion, Cu overload leads to fragmentation of mitochondrial membrane lipids and
induces deleterious effects on mitochondrial integrity of hepatocytes.
Dr. Dominik Huster
Otto-von-Guericke Universitat Magdeburg
Clinic for Gastroenterology, Hepatology and
Infectiology
dominik.huster@med.ovgu.de
www.med.uni-magdeburg.de/Kliniken/Gastroent
Molecular Medicine
74
24 Loss of pluripotency and acquisition of an
osteoblast fate in embryonic stem cells is
accompanied by modulated microRNA expression
Dorota Karniowska, Kristin Reiche, Antje K. Kretzschmar,
Joerg Hackermueller, Nicole I. zur Nieden
The search for general regulators of osteoblast specific gene expression remains a central
challenge in understanding bone formation and devising novel therapies for degenerative
bone disorders. Embryonic stem cells are an ideal model to study the processes of vertebrate
differentiation and development. In vitro osteogenesis in these cells is typically triggered
with vitamin D3. There has been a principal focus on microRNAs, which have emerged
as key negative regulators of osteoblast differentiation. Mature microRNAs are a class of
endogenous, single-stranded RNAs, approximately 19-25 nucleotides in length, which
represses protein synthesis by binding to target mRNAs. MicroRNAs are involved in
post-transcriptional gene silencing using the RNAi (RNA interference) pathway. They act
as adaptors that employ a silencing complex to target mRNAs by selective base-pairing,
primarily in the 3f-UTR region. We used a customized miRNA microarray to screen potential
miRNAs involved in osteogenesis. We found altered miRNA levels following spontaneous
differentiation and vitamin D3 induced differentiation of mESCs. MiRNA profiling during
the proliferation stage and mesenchymal commitment showed up- and down-regulation of
miRNAs that have predicted targets involved in embryonic development and osteoprogenitor
differentiation. Twenty-five miRNAs in particular were significantly expressed during the
first 8 days of ESCs differentiation. The predicted effectors of these miRNAs are involved in
embryonic development and bone formation particularly by regulating the canonical and non
canonical Wnt signalling pathways.
Dorota Karniowska
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Stem Cell Technology
dorota.karniowska@izi.fraunhofer.de
www.izi.fraunhofer.de
75
Molecular Medicine
25 Synthesis of a new class of N1,N3-
adamantylated uraciles and their biological
activity as future non-nucleoside Inhibitors (NNIs)
Matthias Klemm, Kurt Eger, Rudolf Fahrig, Detlef Briel
The antiviral agent (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU/RP101) supports apoptosis
and prevents the acquisition of chemoresistance in cultured human pancreatic tumour cells
via down-regulation of uridine phosphorylase and the DNA repair gene APEX1. Gemcitabine
given in combination with RP101 prevents the decrease of apoptotic effects during the course
of chemotherapy in pancreatic adenocarcinoma. Additionally such a combination inhibits
amplification of chemoresistance genes (e.g. the human multidrug resistance gene MDR1)
and reduces the non-specific toxicity. The aim of this study was to synthesize and test new
substances as future Non-Nucleoside Inhibitors (NNIs). Such novel compounds should
exhibit better chemoresistance and enhance tumor cell apoptosis. Therefore a new class of
N1,N3-adamantylated uraciles has been synthesized. The C5-position which is a key site
for biological activity and the N1,N3-positions were modified with various non-nucleoside
lipophilic groups. Examples include 1,3-bis-(2-adamantan-1-yl-ethyl)-5-fluorouracil and
3-(2-adamantan-1-yl-ethyl)-1-benzhydryl-5-trimethylsilanylethynyluracil. The first series of
biological tests for selected new substances has been accomplished and obtained results will
be presented.
Matthias Klemm
Universitat Leipzig
Institute of Pharmacy
Department of Pharmacognosy Chemistry
klemm@rz.uni-leipzig.de
www.uni-leipzig.de/~pharm
Molecular Medicine
76
26 Determination of the structure of F1F0 ATP synthase
rotor from Acetobacterium woodii
Adriana Klyszejko, Michael Fritz, Thomas Meier, Volker
Muller, Daniel J. Muller
Atomic force microscopy (AFM) is a powerful technique enabling direct observation of the
surface structure of fragile biological specimen under native conditions. AFM imaging allows
resolving structural details of membrane proteins with subnanometer lateral resolution.
In cell metabolism the ATP is regenerated by F1F0 ATP synthase utilizing energy stored
in an electrochemical ion gradient to phosphorylate ADP. The enzyme is localized in the
inner membrane of mitochondria, the thylakoid membrane of chloroplasts and cytoplasmic
membranes of bacteria. It consists of a membrane-embedded rotor (F0) that translocates ions,
and a soluble stator (F1) that performs ATP synthesis/hydrolysis. The hetero-oligomeric motor
Fo is composed of subunits ab2c10-15. The number of c subunits forming the ring in the rotor
structure determines the efficiency of energy conversion by the enzyme.
Unlike other organisms, Acetobacterium woodii produces three types of c subunits (c1, c2 and
c3). The sequence of the subunit c1 equals two c2/3 subunits in length, but contains only one
predicted ion-binding site.
The ATP synthase rotors were purified from plasma membranes and reconstituted into 2D
crystals. AFM imaging was employed to determine their organization into oligomers. AFM
topographs show identical c-rings consisting of 11 helical hairpins. The result was confirmed
by single particle correlation averaging. No distinction between c-type subunits could be
made basing on hairpin size and orientation within the ring. This opens the question how the
ATP synthase of A. woodii is functional lacking sodium ion binding sites.
Adriana Klyszejko
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Machines Group
adriana.klyszejko@biotec.tu-dresden.de
www.biotec.tu-dresden.de
77
Molecular Medicine
27 The importance of osteoblasts in the stump of the
regenerating tail fin
Franziska Knopf, Christina Hammond, Stefan Schulte-
Merker, Gilbert Weidinger
Many invertebrates (e.g. planaria and cnidarians) and some vertebrates such as salamanders
and teleost fish have a remarkable capacity to regenerate injured body parts. Zebrafish can
regenerate their tail fins in less than two weeks after amputation which is accompanied by
growth of fin tissues such as bone, mesenchyme and epidermis. Fin regeneration includes
the formation of a blastema, a mass of pluripotent cells accumulating at the amputation
plane. While proliferation in the blastema has been thoroughly studied, little is known about
proliferation patterns in the stump of the regenerating fin. Here we show that amputation of
the tail fin leads to a significant increase of proliferation in the stump of the fin. Interestingly,
bone forming cells close to the amputation plane become proliferative and change their
morphology. Fin amputation of transgenic reporter fish indicates that the late osteoblast
marker osteocalcin is down-regulated in bone forming cells close to the amputation plane.
Quantitative RT-PCR of stump tissue of amputated fins confirms this result and reveals
that also other bone markers change their expression. Our observations point towards
dedifferentiation of mature osteoblasts into a more proliferative cell type as a consequence to
amputation. Proliferation of these cells might contribute to regenerative fin growth and could
be responsible for the formation of a subpopulation of the blastema.
Franziska Knopf
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Wnt signaling in Development and Regeneration Group
franziska.knopf@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Molecular Medicine
78
28 Adiponectin Receptor 1 Dimerization is Inhibited
by Adiponectin
David Kosel, John T. Heiker, Cornelia M. Wottawah,
Matthias Bluher, Karin Morl, Annette G. Beck-Sickinger
Adiponectin receptors (AdipoR) 1 and 2 are newly discovered members of the huge family of
seven-transmembrane receptors, but both receptors are structurally and functionally different
from G-protein-coupled receptors. Little is known about the oligomerization behaviour of the
AdipoRs. Here, we show the presence of endogenous AdipoR1 dimers in various cell lines
and human femoral muscle tissue. To directly follow the dimerization we applied bimolecular
fluorescence complementation (BiFC) in combination with fluorescence microscopy and flow
cytometry. Indeed, we could visualize and quantify AdipoR1 homodimers in HEK293 cells.
Moreover, we identified a GXXXG dimerization motif in the fifth transmembrane domain of
the AdipoR1. By mutating both glycines to phenylalanine or glutamic acid we were able to
modulate the dimerization of the AdipoR1, implicating the contribution of the GXXXG motif
in AdipoR1 dimerization. We also addressed the question, whether adiponectin as natural
ligand for AdipoR1 has any influence on receptor dimerization. Interestingly, flow cytometry
and Western blot analysis revealed that adiponectin decreases in a concentration dependent
manner for both, the wild-type and mutant receptor. Accordingly, this is the first direct readout
signal of adiponectin at the AdipoR1 receptor and the first report which revealed the
involvement of specific amino acids modulating the quaternary structure of the AdipoR1.
David Kosel
Universitat Leipzig
Faculty of Biosciences, Pharmacy and Psychology
Institute of Biochemistry
dkosel@uni-leipzig.de
www.biochemie.uni-leipzig.de/agbs
79
Molecular Medicine
29 Novel Electroactive Nanovalves for a Implantable
Controlled Drug Delivery Device
Randy Kurz, Anselm Sickinger, Andrea A. Robitzki
The goal of this project was the development of a first demonstrator of an electrically
controlled drug delivery micro-implant for a physiologically mediated in vivo release of
active substances in the case of critical space. The micro-implant was designed for controlled
release of an active substance from a reservoir comprising a thin nanoporous carrier substrate
made of a material that is impermeable with regard to the active substance but that has
adjustable nanopores for substance release. The release mechanism is based on the conducting
polymer polypyrrole (PPy) that is located in the nanopores on a gold layer, as conductor.
The composition of an conducting redox-polymer (PPy) with the charged counterion sodium
dodecylbenzenesulfonate (DBS) allows an overall volume change of this polymer depending
on the redox-state of the PPy.
The project includes the designing, developing and testing of this micro-device, of the drug
immobilisation and release techniques, and of the establishment of in vitro biological test
model for a proof-of-principle according to a controlled drug release. Finally the microsystem
should be available for a controlled release of active substances in applications where
space availability in vivo is critical (e.g. spinal cord, CNS etc.).
Dr. Randy Kurz
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
kurz@uni-leipzig.de
www.uni-leipzig.de/~dmpt
Molecular Medicine
80
30 Inhibition of dedicated Wnt/ƒÀ-catenin pathwayassociated
kinases by natural and chemical
modified polyphenoles with peculiar features
Katja Steffi Lerche, Robert Gunther, Hans-Jorg Hofmann, Rolf
Gebhardt
The Wnt/ƒÀ-catenin signalling pathway is a complex network of proteins which is involved in
normal physiological processes, such as insulin signalling, as well as in the pathophysiology
of cancer. In this work, we have investigated CK-1, GSK-3ƒÀ and the kinase CK-2, which
are quantitatively elevated in most proliferating tissues such as tumor cells. Therefore, the
development of selective cell-permeable inhibitors may represent an important advance for
therapeutic intervention.
With the intention to find inhibitors having a high potency in association with high selectivity,
we were virtually docking various polyphenoles into their crystallographic structures and
analyzed their binding affinities and possible reasons of them. Secondly, we collected in vitro
data of selected hit-compounds, measured by an in vitro kinase phosphorylation assay.
The screening of several flavonoids and chemical modified anthranoid structures showed on
the one hand, in the majority of cases, a selective inhibition of CK-2 in the upper nano molar
range.
On the other hand, one of these compounds, 4-[N-2-(aminoethyl)-amino]-emodin is a
sensitive and potent inhibitor of GSK-3ƒÀ, a key target and effector of downstream insulin
signalling, with interesting biological properties.
Katja Steffi Lerche
Universitat Leipzig
Faculty of Medicine
Institute of Biochemistry
katja.lerche@medizin.uni-leipzig.de
www.uni-leipzig.de/~biochem/
81
Molecular Medicine
31 The influence of phosphatidylserine content in
lipidlayers of biopolymer-coated CaCO3-particles
on phorbol myristate acetate differentiated U937
Jacqueline Lessig, Uta Reibetanz, Bjorn Neu, Hans-Jurgen
Glander, Jurgen Arnhold
Phosphatidylserine (PS) exposure at the outer leaflet of cell membranes can be regarded as
clear apoptotic signal of cells inducing macrophages to phagocytose them and to terminate
the inflammatory process by the release of ant-inflammatory signal molecules. Contrary,
the phagocytosis of necrotic cells are supposed to induce macrophages to release proinflammatory
cytokines, as TNFƒ¿ or IL1ƒÀ causing inflammatory prosecution.
In this study it was our aim to investigate the influence of the PS content in the outer leaflet of
lipid membranes on macrophage signalling after phagocytosis or macropinocytosis. Since it is
known that PS exposure increases with progression of lesion and the intensity of macrophage
macropinocytosis depends on the PS surface amount the terminal coating of biopolymercoated
CaCO3 particles with phospholipids containing increasing PS amounts was used as a
model for apoptotic/necrotic cells. Monocyte-like U937 cells were differentiated with phorbol
myristate acetate to macrophage-like cells and coincubated with these particles to simulate
the influence of apoptotic or necrotic cell material on macrophage functions depending on the
phosphatidylserine concentration. The differentiation of U937 cells into a macrophage-like
morphology could be clearly identified by several methods.
The correlation of the PS-amount in the lipid layer of the model particles and the TGFƒÀ1- and
TNFƒ¿-release by differentiated U937 cells could be demonstrated indicating the importance
of PS exposure for the fate of inflammations.
Jacqueline Lessig
Universitat Leipzig
Faculty of Medicine
Institute of Medical Physics and Biophysics
jacqueline.lessig@medizin.uni-leipzig.de
www.uni-leipzig.de/~biophys/
Molecular Medicine
82
32 Critical Role of Granulocyte-Macrophage Colony-
Stimulating-Factor in Ultraviolet B Radiation-
Induced Murine Skin Cancer
Amrit Mann, Kerstin Niekisch, Thorsten Maass, Peter
Schirmacher, Manfred Blessing
UV-B-radiation is the main causative agent of basal cell carcinoma and squamous cell
carcinoma in humans. According to WHO, 2-3 million human beings worldwide are affected
every year. Role of GM-CSF in skin carcinogenesis has been controversial. Function of GMCSF
in the UV-B-induced skin carcinogenesis was examined in vivo using transgenic mice
which overexpress either GM-CSF or a GM-CSF-antagonist in the skin. These and wildtype
mice were subjected to chronic and acute UV-B irradiation protocols. GM-CSF transgenic
mice exhibited early onset of benign and malignant lesions and higher tumor loads, leading
to a poor constitution and high mortality. GM-CSF seems to facilitate tumor development
in different ways. GM-CSF stimulates and sustains prolonged proliferation of keratinocytes
after UV-B-irradiation, which contributes towards an endogenous tumor promotion. The
antagonist delays onset of proliferation and keratinocytes remain longer in G1-phase of
the cell cycle thus getting more time to repair of DNA-damage caused by UV-B-radiation.
Inability of GM-CSF to activate Langerhans cells to induce antitumor immunity and higher
numbers of mast cells in the skin of these animals probably also contribute towards the
susceptibility for skin carcinogenesis. In addition, mice that overexpress GM-CSF, develop
an environment of antagonistically working cytokines like TNF-ƒ¿, TGF-ƒÀ1, IL-12p40 and
GM-CSF in their skin after UV-B-irradiation. The antagonist on the other hand inhibits the
release of immunosuppressive cytokines and facilitates Th2-development by releasing IL-10
and IL-4, which negatively modulate tumor development.
Dr. Amrit Mann
Unversitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Veterinary Medicine
amrit.mann@bbz.uni-leipzig.de
www.uni-leipzig.de/~blessing
83
Molecular Medicine
33 A gene-dosage effect for IL-4Ralpha expression
has an impact on Th2-mediated allergic
inflammation during bronchopulmonary mycosis
Uwe Muller, Werner Stenzel, Gabriele Kohler, Tobias
Polte, Manfred Blessing, Amrit Mann, Daniel Piehler, Frank
Brombacher, Gottfried Alber
Interleukin (IL)-4 and IL-13 are key factors in the pathogenesis of bronchopulmonary
mycosis induced by infection of mice with Cryptococcus neoformans. Both cytokines use
the IL-4 receptor alpha-chain (IL-4Ralpha). In this study we investigated the role of IL
4Ralpha expression for susceptibility to pulmonary C. neoformans infection. IL-4Ralpha-/-
mice were extremely resistant. To characterize the role of IL-4Ralpha expression level on
disease outcome, we generated IL- 4Ralpha+/- F1 animals. IL-4Ralpha+/- animals showed
intermediate levels of IL-4Ralpha in contrast to higher levels in wild-type and no expression
in IL-4Ralpha-/- mice, indicating bi-allelic expression of the IL-4Ralpha gene. Concomitant
with intermediate IL-4Ralpha expression, F1 mice showed intermediate susceptibility
associated with altered Th2/Th17 cytokine production, decreased IgE levels and reduced
allergic inflammation. This indicates a gene-dosage effect of IL-4Ralpha expression on
susceptibility to bronchopulmonary mycosis. The data provide the basis for novel therapies
antagonising IL-4Ralpha in Th2-related pulmonary infection and possibly also in asthma.
Dr. Uwe Muller
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Immunology, Molecular Pathogenesis
u.mueller@vetmed.uni-leipzig.de
www.uni-leipzig.de/~blessing
Molecular Medicine
84
34 IL-4/IL-13-dependent alternative activation of
macrophages but not microglial cells is associated
with uncontrolled cerebral cryptococcosis
Uwe Muller, Werner Stenzel, Gabriele Kohler, Frank L.
Heppner, Manfred Blessing, Andrew N.J. McKenzie, Frank
Brombacher, Gottfried Alber
IL-4- and IL-13-dependent Th2-mediated immune mechanisms exacerbate murine
Cryptococcus neoformans-induced bronchopulmonary disease. To study the roles of IL-4
and IL-13 in cerebral cryptococcosis, interleukin (IL)-4 receptor ƒ¿-deficient (IL-4Rƒ¿-/-),
IL-4-deficient (IL-4-/-), IL-13-deficient (IL-13-/-), IL-13 transgenic (IL-13T/+), and wildtype
(WT) mice were infected intranasally. IL-13T/+ mice displayed higher fungal brain
burden than WT mice, whereas the brain burden of IL-4Rƒ¿-/-, IL-4-/-, and IL-13-/- mice was
significantly lower as compared to WT mice. Upon infection 68 % of WT mice, and 88 %
of IL-13-overexpressing IL-13T/+ mice developed significant cerebral lesions. In contrast,
only few IL-4Rƒ¿-/-, IL-4-/-, and IL-13-/- mice had small lesions in their brains. Furthermore,
IL-13T/+ mice harbored large pseudocystic lesions in the CNS parenchyma, bordered by
voluminous foamy alternatively activated macrophages (aaMph) containing intracellular
cryptococci, without significant microglial activation. In WT mice, also aaMph tightly
bordered pseudocystic lesions and these mice, in addition, showed microglial cell activation.
Interestingly, in resistant IL-4-/-, IL-13-/-, and IL-4Rƒ¿-/- mice, no aaMph were discernible.
Microglial cells of all mouse genotypes neither internalized cryptococci nor were found to
express markers of alternative activation although they displayed similar IL-4Rƒ¿ expression
as macrophages. These data provide first evidence of the development of aaMph in a CNS
infectious disease model, pointing to distinct roles of macrophages versus microglial cells in
the central nervous system immune response against C. neoformans.
Dr. Uwe Muller
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Immunology, Molecular Pathogenesis
u.mueller@vetmed.uni-leipzig.de
www.uni-leipzig.de/~blessing
85
Molecular Medicine
35 The transcription factor Elf3 in the gastrointestinal
tract: pathomorphological changes in the
transgenic mouse model
Martina Protschka, Manfred Blessing
Aim of this project was to investigate the role of the transcription factor Elf3, a member of
the family of Ets transcription factors, in the gastro-intestinal tract in view of its potential
target genes and influence on the morphology of the gut. Elf3 is specifically expressed in cell
lineages of epithelial origin, above all in enterocytes and is crucial to the function of the gut
epithelia. If both alleles of Elf3 are inactivated by knockout technology, 30% of the fetuses
die in utero and 50% of pups die within three weeks after birth due to severe pathological
intestinal changes, which also occur in human diseases like Crohnes disease, ulcerative colitis
or intestinal epithelial dysplasia.
We generated a transgenic mouse model that conditionally (Cre/loxP-system) expresses
a dominant-negative variant of Elf3 in the gut epithelia. The expression of this dominantnegative
Elf3 results in loss in weight, morphological changes of the gut epithelia and a
significantly reduced expression level of Claudin-7. Claudin-7 is a transmembrane protein
of epithelial cell junctions, and its downregulation could explain the pathomorphological
modifications of the gut epithelia.
Claudin-7 is a target gene of Elf3 and here we could show for the first time that Claudin-7
expression is regulated by Elf3 in the gut.
Martina Protschka
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Molekulare Pathogenese
martina.protschka@bbz.uni-leipzig.de
www.uni-leipzig.de/~blessing
Molecular Medicine
86
36 Metabolites of flavones . observations and results
of synthesis and tests
Benjamin Reissig, Steffen Rodewald, Detlef Briel, Rolf
Gebhardt
A new generation of medication are inhibitors of tyrosine-kinases. Tyrosine-kinases are a
subdivision of protein-kinases. Protein-kinases are enzymes which catalyse the transfer of
phosphates from donor (generally ATP) to hydroxyl-containing side chains of aminoacids.
The phosphorylation of proteins is a relevant post-translational control-mechanism in the cell
signal transduction. Dysfunction of protein kinases cause lots of diseases. Therefore proteinkinases
are attractive targets of the medicinal intervention. Specific inhibitors of proteinkinases
are successful by the treatment of cancer (e.g. Imatinib for the treatment of chronic
myelogenous leukaemia). Tests show that metabolits of flavones are suspected inhibitors of
tyrosin-kinases.
The following paper contains observations and results of synthesis and the test of these
metabolites. The focus of the synthesis is glucuronation und sulphation of the aglyka
Diosmetin, Acacetin und Luteolin. The paper shows deliberations of useful derivates to
improve the effect to tyrosine-kinases as well.
Benjamin Reissig
Universitat Leipzig
Institute of Pharmacy
Pharmaceutic Chemistry
bennyreissig@gmx.de
www.uni-leipzig.de/~pharm/phchem.html
87
Molecular Medicine
37 O steoclasts Control Osteoblast Chemotaxis via
PDGF-BB/PDGF receptor beta Signaling in vitro
Maria Arantzazu Sanchez Fernandez, Bernard Hoflack
Bones undergo remodeling to maintain the mass, the shape and the physical properties of
the skeleton. Two major cell types, the bone-forming osteoblasts and the bone-resorbing
osteoclasts contribute to this process. The balance between bone formation and degradation
is normally tightly controlled but it becomes deregulated, shifting towards more degradation
under pathological conditions or during aging, thereby leading to osteoporosis. This tight
balance implies the existence of mechanisms coordinating the differentiation of osteoblasts
and osteoclasts as well as their migration to locations where they function. Several studies have
now described the molecular mechanisms by which osteoblasts control osteoclastogenesis
and bone degradation. The mechanisms by which osteoclasts influence bone rebuilding are
currently unclear. Using in vitro cell systems of osteoclastogenesis and osteoblastogenesis,
we can show that mature osteoclasts, but not their precursors, secrete chemotactic factors
recognized by both mature osteoblasts and their precursors. Several growth factors whose
expression is upregulated during osteoclastogenesis were identified by DNA microarrays as
candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate
that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression
is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely,
osteoblasts whose platelet-derived growth factor receptor ƒÀ (PDGFR-ƒÀ) expression is
reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted
by osteoclasts.
Dr. Maria Arantzazu Sanchez Fernandez
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Proteomics Group
arantxa.sanchez@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Molecular Medicine
88
38 Peptide mediated DNA import into mitochondria
Ingo Schafer, Christian Kukat, Alexandra Kukat, Peter Seibel
Energy of eukaryotic cells is generated by the oxidative phosphorylation system located in
the mitochondria. The compounds of this system are encoded either in the nuclear or the
mitochondrial genome. Therefore genetic defects within the mitochondrial DNA can cause
disorders in the cellfs energy production and ultimately lead to neuromuscular dysfunctions.
Mitochondrial DNA variations can range from single point mutations to deletions of large
DNA fragments encoding for several genes.
Import of DNA from the cytoplasm into the mitochondrial matrix is an obligatory step for
a site directed mutagenesis or gene therapy approach on mitochondrial DNA diseases. Up
to now, no endogenous system mediating this transfer is known in mammalian cells. To
close this gap we developed peptide conjugated DNA vectors that are capable of delivering
nucleic acids to the mitochondrial matrix. The vector is made up of the mitochondrial signal
peptide of the ornithine transcarbamylase (OTC, a nuclear encoded protein finally located in
mitochondria) which is chemically cross linked to a nucleic acid component harboring the
desired DNA molecule to be located in the mitochondrial matrix. Due to the unique physical
structure of the attached DNA, induction of regulated self-replication is emphasized upon
reaching the final cellular compartment.
At the moment we focus on the composition of the DNA to achieve correct expression of the
gene of interest (EGFP as marker) in the mitochondrial matrix.
Ingo Schafer
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Molecular Cell Therapy
ingo.schaefer@bbz.uni-leipzig.de
www.bbz.uni-leipzig.de
89
Molecular Medicine
39 Distribution of mitofusin 2 (Mfn2) after the
formation of megamitochondria
Susanna Schubert, Christian Kukat, Ingo Schafer, Alexandra
Kukat, Peter Seibel
Extraordinary large mitochondria are known as megamitochondria, the formation of which is
characterized not only by simple swelling as in isolated organelles in hypotonic solutions but
also by additional fusion events.
The accumulation of megamitochondria was detected both in physiological (e.g. mammalian
sperm cells) as well as pathological conditions (e.g. diabetes) and can be induced for instance
by ethanol, chloramphenicol, hydrazine or . as in our study . by valinomycin.
We used valinomycin-induced megamitochondria in human culture cells to examine
mitochondrial fusion events, especially by monitoring the distribution of mitofusin 2
(Mfn2).
Mitofusins are conserved, large GTPases localised to the mitochondrial outer membrane. In
mammals, two closely related but not redundant mitofusin homologs, Mfn1 and Mfn2 can be
found. Both the N- and C-terminal regions of these structural similar proteins protrude from
the mitochondrial outer membrane into the cytosol.
Mitofusins are involved in outer membrane fusion of mitochondria, especially by homoand
heterodimeric interactions via coiled-coil domains. Mutations in the gene for Mfn2 are
known to be involved in the hereditary neuropathy Charcot-Marie-Tooth Type 2.
As an approach to localise Mfn2 in this study we used a plasmid encoding a fusion protein
consisting of EGFP (enhanced green fluorescent protein) and mitofusin 2 (Mfn2). This vector
allowed us to observe the distribution of Mfn2 in living cells with valinomycin-induced
megamitochondria via confocal microscopy.
Susanna Schubert
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Molecular Cell Therapy
susanna.schubert@bbz.uni-leipzig.de
www.uni-leipzig.de/~mct
Molecular Medicine
90
40 In the absence of IL-12 protective immunity to
infection with Salmonella Enteritidis depends on
Il-23 and is associated with IL-22 but not IL-17
Silke Mara Schulz, Gabriele Kohler, Alissa A. Chackerian,
Ellen Witte, Kerstin Wolk, Robert Sabat, Yoichiro Iwakura,
Robert A. Kastelein, Gottfried Alber, Christoph Holscher,
Uwe Muller
IL-12 is essential for protective T cell-mediated immunity against Salmonella infection.
We already have shown the related cytokine IL-23 to be dispensable for protection against
S. Enteritidis when IL-12 is present. Nevertheless, IL-23 is required for T-cell dependent
cytokine responses as we found a defective IL-17A and IL-22 production and also a defect
in recruitment of neutrophils and in delayed-type hypersensitivity responde in p19-/- mice
(lacking IL-23).
To analyze the role of IL-23 in the absence of IL-12, low doses of an attenuated strain of
Salmonella enterica serovar Enteritidis (S. Enteritidis) were administered to p35-/- mice
(lacking IL-12), p35/19-/- mice (lacking IL-12 and IL-23), p35/40-/- mice (lacking IL-12,
IL-23, and homodimeric p40), or p35/IL-17A-/- mice (lacking IL-12 and IL-17A). We found
survival of p35-/- and p35/IL-17A-/- mice, whereas p35/19-/- and p35/p40-/- mice died within
3 - 6 weeks and developed liver necrosis. This indicates that IL-23 but not homodimeric IL-
12p40 is required for protection which, surprisingly, is independent of IL-17A. Moreover,
protection was associated with IL-22 but not IL-17F or IL-21 expression or with neutrophil
recruitment. Finally, anti-IL-22 treatment of S. Enteritidis-infected p35-/- mice resulted in
liver necrosis indicating a central role of IL-22 in hepatocyte protection during salmonellosis.
In conclusion, IL-23-dependent IL-22 but not IL-17A production is associated with protection
against systemic infection with S. Enteritidis in the absence of IL-12.
Silke Mara Schulz
Universitat Leipzig
Faculty of Veterinary Medicine
Institute of Immunology
sischulz@vetmed.uni-leipzig.de
www.uni-leipzig.de/~immun/
91
Molecular Medicine
41 Non-protein coding RNAs as highly specific
biomarkers for cancer
Katharina Schutt, Friedemann Horn, Kerstin Ullmann, Antje
K. Kretzschmar, Jorg Hackermuller
The gcentral dogmah of molecularbiology saying that DNA is transcribed into mRNA, which
is subsequently translated into protein has changed dramatically since transcriptome studies
like the ENCODE-Project showed that nearly the entire genome is actively transcribed,
whereas only 1.5% codes for proteins. The remaining 98.5%, previously named as gjunkh,
is now known as the class of non-protein coding RNAs (ncRNA), which are predominantly
expressed in a highly controlled, cell type and state specific manner. Changes within ncRNA
expression patterns are often associated with diseases or developmental disorders. Therefore
ncRNAs have a great potential to serve as biomarkers for several diseases.
After establishing cell line model systems for two prominent cancerous diseases . prostate
cancer and breast cancer . novel ncRNA candidates could be identified via genome-wide
Tiling array analysis. Those candidates served for the development of the 2nd generation
nONCOchip, a microarray tool to detect novel ncRNA biomarker candidates. Using the
nONCOchip for the analysis of patient samples, we could identify ncRNA candidates, which
have the potential to serve as specific prognostic biomarkers for the respecting cancerous
diseases. Subsequent studies in in vitro and in vivo models will help to functionally
characterize and definitively verify those novel prostate cancer- and breast cancer-specific
biomarkers and will emphasize the great potential which lies in the usability of ncRNAs as
prognostic biomarkers.
Katharina Schutt
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Molecular Biology
Rnomics
katharina.schutt@izi.fraunhofer.de
www.izi.fraunhofer.de/izi_rnomics.html
Molecular Medicine
92
42 Synthesis of novel PDE10A-Inhibitors
Gregor Schwan, Lenka Kubicova, Karen Nieber, Norbert
Strater, Peter Brust, Detlef Briel
The phosphodiesterase(PDE)10A is a key enzyme in cellular signaling pathways. It catalyzes
the transformation of cAMP as well as cGMP to the corresponding non-cyclic nucleotides,
which finally effectuate a neurotransmitter release in striatal neurons. Dysfunctions in this
signaling pathway might lead to neuronal disorders, e.g. schizophrenia. Hence selective and
potent inhibitors of PDE10A are claimed to be qualified therapeutics for CNS-disorders. Also
radio-labeled ligands might be useful diagnostics for these disorders.
Papaverine is known as a potent inhibitor of the PDE10A. Important for ligands with a high
affinity to the PDE10A is a bidentate hydrogen-bonding interaction between Glutamine in the
catalytic center and two methoxy groups, which can also be found in the catecholic partial
structure of Papaverine. The following paper aims to show syntheses of novel and selective
heterocyclic PDE10A-inhibitors based on the structure of Papaverine as a lead compound.
The role of the methoxy groups, especially if changes in this catecholic partial structure have
an influence on affinity to PDE10A is of special interest. Various methods of demethylation
have been tested to synthesize demethylated derivates, e.g. with hydrogen bromide, an
aluminium chloride/methylene chloride-system and a methansulfonic acid/methioninesystem.
Particularly the selectivity of demethylation in a heterocyclic setting is spotlighted
by this paper.
Based on these results it is now possible to synthesize novel derivatives with a change in
substituents of the catecholic structure to specify the conditions for an increase of affinity to
PDE10A.
Gregor Schwan
Universitat Leipzig
Institute of Pharmacy
Department fur Pharmacology and Sciences
gschwan@uni-leipzig.de
www.uni-leipzig.de/~pharm
93
Molecular Medicine
43 Effect of purine analogues on macrophage
function during in vitro ischemia
Fritzi Siegert, Karen Nieber
It has become clear in the latest years that actors of the immune system are involved in
multiple and various neurobiological processes such as cerebral ischemia and neuroprotection.
An immunological approach to cerebral ischemia can distinguish, besides the implication of
inflammation in the development of atherothrombosis thus leading to stroke. Although several
approaches for anti-inflammatory treatment have proven effective in cellular and animal
models clinical trials of immune system modulation therapy have not yet proved successful.
Therefore, the aim of the present study was to investigate whether the activation of adenosine
A1 (A1Rfs) . and adenine receptors may influence protectively the function of macrophages
during glucose deprivation, an in vitro hypoxia model, using specific receptor ligands.
Glucose deprivation decreased time-dependently the cell viability of human monocytederived
macrophages as well as THP-1 cells which have been differentiated into macrophages.
Activation of adenine receptors and A1Rfs for 24 hours by adenine (10 ƒÊM . 10 mM) and
N6-cyclopentyladenosine (CPA, 0.1 . 100 ƒÊM) respectively did not affect the viability
of the cells under normoxic conditions. After 24 hours glucose deprivation adenine in a
concentration range from 10 to 500 ƒÊM protected against the reduced macrophage function
induced by glucose deprivation. Adenine in a concentration of 10 mM drastically reduced
the cell viability. The activation of the A1Rfs by CPA moderately influenced the cell viability
after glucose deprivation.
Fritzi Siegert
Universitat Leipzig
Institute of Pharmacy
Department fur Pharmacology and Sciences
siegert@uni-leipzig.de
www.uni-leipzig.de/~pharm/phfn
Molecular Medicine
94
44 Cymantrene-peptide bioconjugates: a promising
approach to generate cytostatic compounds
Katrin Splith, Harmel Peindy Nfdongo, Ulrich
Schatzschneider, Ines Neundorf
Intracellular delivery of therapeutics is the challenging task in medicinal chemistry research.
One way to transport different cargos inside the cell are so called cell-penetrating peptides
(CPPs), which can internalise without the need of receptors or transporters. Several metalbased
building blocks like metallocenes have promising features for applications in diagnosis
and therapy. But the limitation for using these organometallic compounds in medicine is their
low water solubility and bioavailability. Coupling them to CPPs, could be a possibility to
generate potent drugs.
In this work we coupled cymantrene (CpMn(CO)3), an organometallic marker, to cellpenetrating
peptides based on the antimicrobial peptide cathelicidin (CAP18). Cymantrene
was chosen as a robust and easy-to-functionalise complex. Synthesis was done by solid
phase peptide synthesis using standard Fmoc chemistry and activation by HOBt/DIC. Cell
uptake of the new bioconjugates was investigated using two different methods, fluorescence
microscopy and atomic absorption spectroscopy. Both methods showed high accumulation in
different tumor cells (MCF-7/HT-29). Cell viability assays on MCF-7 cells showed that these
organometallic peptide conjugates are very potent and possess promising anti-proliferative
properties. Interestingly, only minor toxic effects were observed when incubating the
bioconjugates with HT-29 cells. Furthermore, the toxicity could be increased by introducing
different enzymatic cleavage sites between the metal complex and the peptide.
In conclusion, we designed molecules with new features may be interesting as cytostatic
drugs in cancer.
Katrin Splith
Universitat Leipzig
Faculty of Biosciences, Pharmacy and Psychology
Institute of Biochemistry
splith@uni-leipzig.de
www.biochemie.uni-leipzig.de/agbs
95
Molecular Medicine
45 Single-cell force spectroscopy reveals s1 -integrin
as central molecule mediating /ABL expression of
32D-BCR/ABL cells to bone marrow stromal cells
Anna Taubenberger, Fernando A. Fierro, Pierre-Henri Puech,
Gerhard Ehninger, Martin Bornhauser, Daniel J. Muller,
Thomas Illmer
The expression of the fusion protein BCR/ABL is a hallmark of chronic myeloid leukemia.
BCR/ABL is a constitutively active tyrosine kinase influencing cell proliferation, apoptosis,
and differentiation. To which extend and by which mechanisms BCR/ABL affects the
adhesion of leukemic cells to bone marrow stromal cells (BMSC) is discussed controversial.
To characterize adhesion of BCR/ABL transformed 32D cells (32D-BCR/ABL) to the
BMSC line M2-10B4, we applied washing-assays and single-cell force spectroscopy
(SCFS). Compared to control 32D cells (32D-V), 32D-BCR/ABL developed three fold
higher adhesion forces. This enhanced cell adhesion could be reduced to control levels after
specifically inhibiting the activity of the tyrosine kinase BCR/ABL using imatinib mesylate
(IM). SCFS further showed that the adhesion forces of 32D-BCR/ABL were strongest to
fibronectin and collagen type I, suggesting that ƒÀ-integrin plays a major role in mediating
the adhesion of leukemic cells to BMSC. Indeed, the ƒÀ-integrin blocking antibody Ha2/5
abrogated the attachment of 32D-V and 32D-BCR/ABL cells to BMSC. Although 32D-BCR/
ABL cells show significantly increased ƒÀ-integrin expression, no significant differences of
ƒÀ-integrin mRNA levels could be detected, indicating a post-transcriptional regulation of
ƒÀ-integrin by BCR/ABL. The data presented argues that the interaction of ƒÀ-integrin and
extracellular matrix components is functionally important in leukemic cells expressing highlevels
of BCR/ABL and could provide a rationale for the development of optimized targeted
therapies.
Anna Taubenberger
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Machines Group
anna.taubenberger@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Molecular Medicine
96
46 MicroRNAs lost during prostate carcinoma
pathogenesis cooperatively regulate mRNAs
involved in Androgen Receptor signalling
Kerstin Ullmann, Antje Kretzschmar, Friedemann Horn, Nora
Morbt, Martin von Bergen, Gerald Verhaegh, Jack Schalken,
Katharina Schutt, Jorg Hackermuller
MicroRNAs (miRNAs) are class of small non-coding RNAs that have been shown to be
extensively involved in posttranscriptional regulation of mRNAs. MiRNAs have crucial
functions for basic cellular processes, normal development, but also play important roles
in the origin of diseases, especially cancer. Prostate cancer (PCa) is the most frequently
diagnosed malignancy and 2nd leading cause of cancer death in men. Identifying miRNAs
that are deregulated in PCa may help to better understand the etiology of the disease and
might be a promising strategy for finding new targets for the therapy of prostate cancer.
Using microarrays we could identify miRNAs that are differently expressed between
different prostate cancer cell lines and a healthy prostate cell line. To evaluate the function
of these miRNAs in the in vivo situation we analyzed the expression of the miRNAs in
tumor samples of different progression stages and also healthy tissue. qRT-PCR showed
an overall downregulation of the miRNAs in tumor cells compared to healthy tissue with
stronger downregulation in recurrent samples. Re-introduction of these miRNAs into the
PCa cell line LNCaP, which also lacks the expression of the respecting miRNAs, led to a
change in morphology and decreased viability, which was enhanced when the miRNAs were
overexpressed together.To identify candidate target mRNAs of these miRNAs we used the
DIGE technology to identify proteins that are downregulated upon miRNA overexpression.
DIGE experiments revealed several targets that have been reported to be associated with
androgen receptor transactivation and are also predicted by several databases.
Kerstin Ullmann
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Molecular Biology
Rnomics
kerstin.ullmann@izi.fraunhofer.de
www.izi.fraunhofer.de
97
Molecular Medicine
47 The Role of extra- and intracellular domains for Y
Receptor Internalisation
Cornelia Walther, Diana Lindner, Ilka Bohme, Annette G.
Beck-Sickinger
Y receptors belong to the large superfamily of heptahelical G-protein coupled receptors.
Four Y receptor subtypes have been cloned from human tissue (Y1, Y2, Y4 and Y5). These
receptors are activated by the members of the NPY hormone family: neuropeptide Y (NPY),
pancreatic polypeptide (PP) and peptide YY (PYY) . neuroendocrine hormones which consist
of 36 amino acids and are C-terminally amidated.
Agonist stimulation of Y receptors results in the redistribution of the receptor from the cell
surface into intracellular compartments through the process of endocytosis. By using HEK293
cells that transiently express the human Y receptor subtypes hY1R, hY2R, hY4R and hY5R
we were able to gain more insight into receptor internalisation as the rate was receptor
subtype dependent. We could show that the hY2R internalised as fast as the hY1R and the
hY4R whereas the hY5R internalised much slower. In comparison to the other three receptor
subtypes the hY5R has a very long third intracellular loop and a very short C terminus. To
study the influence of these domains novel hY5/hY2 receptor chimera were generated and
we found out that both the C terminus as well as the third intracellular loop contribute to the
reduced internalisation rate of the hY5R.
Besides intracellular domains also extracellular domains are necessary not only for stabilisation
of the receptor structure and therefore membrane integration but also for internalisation. For
the hY2R we could show that the complete deletion of the N terminus resulted in a mutant
that is fully integrated in the membrane and internalised much slower compared to the wild
type receptor.
Cornelia Walther
Universitat Leipzig
Faculty of Biosciences, Pharmacy and Psychology
Institute of Biochemistry
cwalther@uni-leipzig.de
www.biochemie.uni-leipzig.de/agbs
Molecular Medicine
98
48 Characterization of substrates and inhibitors for
the in vitro assessment of BCRP mediated drug
transport in the lactating mammary gland of
dairy cattle
Luise Wasermann, Stefan Lindner, Kerstin Honscha, Walther
Honscha
The presence of drugs or other potential toxic xenobiotics in milk has enormous toxicological
and nutritional consequences for the suckling and the consumers of dairy products. It was
recently demonstrated that the ABC-transporter BCRP is expressed in alveolar epithelial
cells of the mammary gland during pregnancy and lactation, and plays a major role for the
active secretion of a variety of drugs (Ivermectin, Enrofloxacin), toxins (Aflatoxin B1), and
carcinogens (PhIP: Amino-1<-Methyl-6-Phenylimidazo[4,5-b]Pyridin; Trp-P-1: Tryptophan-
P-1) into milk. So far there is little information about the transport activity of BCRP in the
mammary gland of dairy cattle. Therefore we want to establish in vitro cell culture models
expressing the BCRP of dairy cattle in order to characterize their substrate specificity. To
address this issue, mRNA was isolated from the bovine, caprine and ovine mammary gland.
After the identification of BCRP gene specific primers, full length clones were created using
RACE (rapid amplification of cDNA ends) PCR. The final full-length bovine BCRP cDNAclone
was submitted to the NCBI genebank (EU570105). The sequencing of the full-length
ovine and caprine BCRP cDNA clone is under progress. For stable expression of the different
BCPR clones the cell lines MCF7 and MDCK were selected. After the tranfection of the
BCRP full length clones, the efflux ratio of several substrates will be evaluated by monitoring
the basolateral-to-apical as well as the apical-to-basolateral transport using transwell filter
devices.
Luise Wasermann
Universitat Leipzig
Faculty of Veterinary Medicine
Institute of Pharmacology, Pharmacie and Toxicology
wassermann@vetmed.uni-leipzig.de
www.uni-leipzig.de/~vetppt
99
Molecular Medicine
49 Reconstitution of the Interleukin-4 dependent
JAK/STAT signalling pathway for a detailed
analysis by single molecule fluorescence detection
methods in living cells
Thomas Weidemann, Remigiusz Worch, Tibor Szekeres,
Petra Schwille
Cytokine cell signalling via the JAK/STAT pathway is mediated by a sequence of precise
molecular rearrangements: (1) binding of the ligand to corresponding cell surface receptors,
(2) ligand-induced dimerization and/or conformational changes of the receptorfs extracellular
domains, (3) transmembrane Janus kinase (JAK) activation, (4) binding of downstream
factors to the cytoplasmic receptor tails, and (5) dimerization and translocation of a freely
diffusing signal transducer and activator of transcription (STAT) into the nucleus. While the
interaction network of the proteins is well described, individual steps of pathway activation
are difficult to observe in real-time in the living cell. Fluorescence correlation spectroscopy
(FCS) is a non-invasive method with which the diffusion properties and concentrations of
fluorescent particles are extracted from a time-resolved signal detected in a tiny illuminated
spot as e.g. provided by a confocal laser scanning microscope (CLSM). While FCS in the
cytoplasm is now well established, recent variations and advancements of the method like
scanning FCS (SC-FCS) or total internal reflection FCS (TIR-FCS) have great potential to
resolve receptor activation events of at the plasma membrane. The ability to monitor specific
steps in pathway activation will provide both powerful tools to establish modern screening
formats and sensitive systems to validate prospective lead compounds.
Dr. Thomas Weidemann
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Biophysics Group
thomas.weidemann@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Molecular Medicine
100
50 Inhibition of matrix metalloproteinases (MMPs) by
selected anthraquinones and synthetic peptides
Claudia Wierzchacz, Rolf Gebhardt
MMPs are a family of zinc-containing endopeptidases which cleave components of the
extracellular matrix and play a significant role in tissue resorption and remodelling in many
physiological and pathological processes. Their activity is closely balanced and a loss of
control can lead to excessive, as in cancer metastasis, or insufficient matrix degradation, as in
fibrosis. In this work we focus on the effects of selected anthraquinones and small synthetic
peptides on the activity of MMP-13 (Collagenase), MMP-2 and MMP-9 (Gelatinases).
Natural occurring anthraquinones are able to inhibit MMPs in the micro molar range.
Comparisons between chemical structure and inhibitory potential of these compounds
revealed the functional important structural features. On the one hand these compounds
can explain the beneficial properties of some medical plants, and on the other hand there
structures can be used as a starting point for drug discovery.
We also demonstrated that a 20-mer peptide, a fragment of the MMP-2 hemopexin domain,
inhibits the catalytic domains of these MMPs with IC50 values in the lower micro molar range.
To discover the functional relevant residues we inserted single amino acid substitutions into
the peptide sequence. Thus a hydrophobic core could be identified, which is essential for the
inhibitory effect. Similarity searches displayed other proteins with related sequence motifs,
especially other MMPs, which have equivalent inhibitory properties. Since degradation
products of MMPs can occur in vivo, they possibly act as physiological relevant inhibitors.
Claudia Wierzchacz
Universitat Leipzig
Faculty of Medicine
Institute of Biochemistry
claudia.wierzchacz@medizin.uni-leipzig.de
www.uni-leipzig.de/~biochem/bch_cms/
101
Molecular Medicine
51 Identification of the receptor for Pigment
Epithelium Derived Factor on retinal endothelial
cells
Xiu Mei Yang, Wolfram Eichler, Johannes Lange, Andreas
Reichenbach, Peter Wiedemann
Retinal neovascularization (RNV) is the primary cause of blindness in a wide range of ocular
diseases such as proliferative diabetic retinopathy (PDR). Endothelial cells are one of the
most important cell populations in RNV. Pigment epithelium-derived factor (PEDF) is the
most potent natural antiangiogenic factor so far. It has been shown that the different activities
of PEDF are mediated via binding to the cell surface. However, little is known about the
identity of the receptor and molecular mechanism(s) by which PEDF functions to regulate
endothelial cell behavior. In this study, we detected a potential receptor for PEDF using
recombinant human PEDF (rhPEDF).
DNA encoding for human PEDF was amplified from human retinal pigment epithelium (RPE)
cell mRNA and cloned into a plasmid that gives rise to the production of a PEDF fusion
protein. Subsequently, rhPEDF was produced and purified from mammalian cell cultures.
Expression of a putative PEDF receptor on bovine retinal endothelial cells (BRECs), RPE
cells and human umbilical vein endothelial cells (HUVECs) was detected using rhPEDFcoated
magnetic beads, by immunohistochemical staining and Western blotting. In particular,
Western blots were used to assess the molecular weight of the PEDF-binding molecule.
Purified rhPEDF bound to retinal cells and HUVECs. The putative PEDF receptor, with a
molecular weight of about 160 kDa, was localized to the surface of BRECs, HUVECs and
RPE cells.
Retinal cells express a ~160-kDa binding protein that may function as a receptor and probably
accounts for PEDF-mediated effects.
Xiu Mei Yang
Universitat Leipzig
Faculty of Medicine
Department of Ophthalmology, Eye Hospital
XiuMei.Yang@uniklinik-leipzig.de
www.augenklinik.uniklinikum-leipzig.de/
Molecular Medicine
102
52 Refolding of ecto-nucleotidases
Karen Yates, Norbert Strater
In addition to its important cellular functions as an energy carrier, ATP also serves as an
extracellular signalling substance. It acts on P2X and P2Y receptors. These signalling
pathways via ATP and other nucleotides are termed purinergic signalling. Extracellular
nucleotides influence a wide variety of physiological processes, including exocrine and
endocrine secretion, immune responses, inflammation, platelet aggregation, endothelialmediated
vasodilatation as well as cell proliferation, differentiation, migration and death
in development, regeneration and cancer. Extracellular nucleotidases are involved in the
hydrolysis of the nucleotides, including the NTPDases which dephosphorylate ATP via ADP
to AMP and the 5e-nucleotidases, which catalyze the hydrolysis of AMP to adenosine.
The aim of our work is to study the structure and function of ecto-nucleotidases in purinergic
signalling. Most of these extracellular enzymes contain disulfide bridges and are expressed
as inclusion bodies in E.coli. The resolubilised enzymes are purified via Ni-NTA affinity
chromatography and optimal refolding conditions are determined by sparse matrix screens
and systematic screens. A major problem is the separation of the correctly refolded enzyme
from misfolded species via different chromatography techniques, such that a homogenous
protein solution is obtained for crystallization. We also report on the use of the influence of
different variants of the proteins on the refolding yield.
Karen Yates
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Chemistry and Mineralogy
karen.yates@bbz.uni-leipzig.de
www.bbz.uni-leipzig.de
103
Molecular Medicine
53 Structure-based design of inhibitors of human
cAMP specific phosphodiesterase 4A
Michael Zahn, Roy Eylenstein, E. Bartholomeus Kuttner,
Susanne Moschutz, Norbert Strater
The second messenger cyclic nucleotide cAMP plays an important role in intracellular
signal transduction. Induced by an extracellular signal, cAMP is generated and leads to the
activation of further signal cascades thereby triggering cellular responses. The rapid signal
inactivation of the cAMP-mediated cellular response is exclusively provided by cAMPspecific
phosphodiesterases (PDE). PDE4, one of the eleven families of PDEs hydrolyses
the cyclic 3f-5f phosphodiester bond in cAMP to generate the product AMP. Dysfunction of
PDE4 can lead to diseases such as asthma and chronic obstructive pulmonary disease. The
aim of our work is to analyze structure-function relationships for novel PDE4A inhibitors
developed by Curacyte AG, Leipzig.
After expression of the catalytic domain of human PDE4A in E. coli into inclusion bodies,
the protein was renatured, purified and crystallized. Crystal structures of complexes with
nine different synthetic inhibitors were determined. Interestingly, for the structurally related
pharmaceutical inhibitors three different modes of binding were found. These differences in
the binding modes correlate well with the inhibition constants, which were determined by
isothermal titration calorimetry.
Michael Zahn
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Bioanalytical Chemistry
michael.zahn@bbz.uni-leipzig.de
www.bbz.uni-leipzig.de
Molecular Medicine
104
54 Interfering with Signal Inactivation in Purinergic
Signaling
Matthias Zebisch, Norbert Strater
Nucleotides and nucleosides act as extracellular messengers. They mediate short-term
signaling functions as in neurotransmission and long-term signaling functions as in cell
proliferation and differentiation. The nucleotides act on ligand-gated P2X ion channels and
G protein-coupled P2Y receptors. These gpurinergich signals are terminated by the action
of extracellular membrane-bound nucleotidases. Since nucleotide-mediated signaling is
involved in cardiovascular diseases, pain perception, inflammation etc., the receptors as well
as the nucleotidases are interesting pharmacological targets.
We have established a protocol for recombinant production of the catalytic domains of
nucleoside triphosphate diphosphohydrolases (NTPDases), which cleave the gamma and beta
phosphate from extracellular ATP and ADP. After solving the structure of NTPDase2, we
could identify the active site and determine the catalytic mechanism of NTPDases. Together
with high throughput inhibitor screening the structure is now used to guide the development
of highly effective and subtype-specific NTPDase inhibitors.
Dr. Matthias Zebisch
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Chemistry and Mineralogy
matthias.zebisch@bbz.uni-leipzig.de
www.bbz.uni-leipzig.de
105
Molecular Medicine

5. Bioanalytics
Posters

109
Bioanalytics
55 Quantitative microscopy with trace element
sensitivity
Nirav Barapatre, Anja Fiedler, Thomas Arendt, Tilman Butz,
Markus Morawski, Tilo Reinert
What is quantitative trace element microscopy? The Faculty of Physics and Earth Science
runs the high-energy ion nanoprobe LIPSION, which can also serve as a mega-electron volt
proton microscope. The proton microscope uses a focussed proton beam, scanned over the
sample, to induce characteristic X-rays which are a fingerprint of the elemental composition
of the sample (PIXE: proton induced X-ray emission). The intensity of the characteristic
X-rays is a measure of the concentrations. The high sensitivity in the range of a few ƒÊM for
physiologically important trace metals (e.g. Fe, Cu, Zn) is based on the very low background
signal in the case of proton excitation.
We present examples from a study on the intracellular iron distribution in neurons, from a
study on the elemental concentrations of neuromelanin in Parkinson disease, and the elemental
distribution in atherosclerotic lesions in the aortic wall. The examples will demonstrate the
ability to image the elemental distributions, to extract elemental profiles, and to quantitatively
analyse the concentrations in regions of interest.
Nirav Barapatre
Universitat Leipzig
Faculty of Physics and Earth Science
Institute of Experimental Physics II
barapatre@physik.uni-leipzig.de
www.uni-leipzig.de/~nfp/
Bioanalytics
110
56 Easy and fast separation of multiple targets
from whole blood with a new bead based cell
separation method
Doreen Beck, Christoph Mohr, Berthold Seidel, Iwona
Goczalik, Anne Rasser, Jan-Michael Heinrich
Several cell separation methods are currently available, mostly based on magnetic nanometer
sized beads or on physical cell parameters like size or density. They are great tools for
separation and functional analysis of defined cell types, for removal of unwanted cell types
from samples or for the diagnosis of certain diseases. But their shortcoming is the need for
pre-treatment of samples to achieve sufficient purity, especially for whole blood. The removal
of cell debris and other distracting components is often time consuming and error prone.
We are developing a new easy and time saving particle based cell separation method which
combines physically and affinity attributes. It does not acquire any sample pre-treatment.
The system is suitable for simultaneously fractionation of multiple targets, for example cells
and/or proteins, from one sample. The sample could be whole blood or aqueous media. The
targets are captured by specifically affinity functionalized micro particles of distinct sizes.
During 30 minutes of incubation each size of particle binds to its belonging kind of target.
Thus after incubation the captured targets could be separated from each other via cascade wet
sieving by size. The cells can be removed from the micro particles and subsequent cultivation
or analysis is possible.
Doreen Beck
Universitat Leipzig
Faculty of Medicine
Interdisciplinary Centre fur Clinical Research
doreen.beck@medizin.uni-leipzig.de
www.uni-leipzig.de/~izkf/
111
Bioanalytics
57 The structural and functional landscape of
the human ADP receptor P2Y12 . methods for
generating mutant libraries
Maxi Coster, Doreen Thor, Holger Rompler, Torsten
Schoneberg
Signals as diverse as light, odors, hormones and neurotransmitters are transduced into
intracellular responses by members of the superfamily of G protein-coupled receptors
(GPCRs). Understanding the fundamental structure-function relationship of this important
class of membrane receptors is a key goal in fields ranging from evolutionary biology to
pharmacology to physics. Recent crystal structures of a few GPCRs represent a first step in
defining the molecular landscape of these receptors. However, these structures provide only
a single structural snapshot. We combined the information from natural variation in GPCRs
together with high-throughput saturation mutagenesis, in vitro pharmacological testing
and bioinformatic analysis to generate a predictive and dynamic GPCR model. Our model
system is the human ADP receptor P2Y12 which is clinically used as target for prevention of
thrombocyte aggregation. This study will provide a comprehensive picture of the relationship
between P2Y12 sequence and function. We will achieve this goal by testing the biological
relevance of all amino acid positions in this GPCR using saturating mutagenesis, in which
each amino acid position in the human P2Y12 is replaced with all other possible amino
acids. There are different methods for generating mutant libraries. Here we compare three
approaches: mutagenesis via a.) PCR using degenerated primer, b.) PCR using an optimised
primer set and c.) using SlonoMaxTM Libraries.
Maxi Coster
Universitat Leipzig
Faculty of Medicine
Institute of Biochemistry
maxi.coester@medizin.uni-leipzig.de
www.uni-leipzig.de/~biochem/mbch_cms/
Bioanalytics
112
58 Biological properties of Apidaecin derivatives
with enhanced antimicrobial activities
Patricia Czihal, Laszlo Otvos Jr., Ralf Hoffmann
In recent years more and more Gram-positive and Gram-negative bacteria acquired
resistance mechanisms against common antibiotics at alarming and still increasing rates.
Thus, the development of antimicrobial compounds with novel modes of action is a major
focus in current pharmaceutical research. An interesting and promising approach relies on
antimicrobial peptides (AMP) isolated from different species, as bacteria have not been able
to overcome these peptides of the innate immune system. Very promising drug leads are
AMPs from the family of short, proline-rich antibacterial peptides originally isolated from
insects. These peptides represent a viable treatment option for urinary tract infections (UTI),
for example, which are caused in 90 to 95% of all cases by Escherichia coli and Klebsiella
pneumoniae.
We identified several residues in the native sequence of Apidaecin, which are crucial for the
antibacterial activity. These positions were replaced by other natural or non-natural amino
acid derivatives to increase the antibacterial activity against Gram-negative bacteria (e.g. K.
pneumoniae, Pseudomonas aeruginosa) and to extend the activity spectrum to other human
pathogens. Other favourable properties of the current drug leads are: bactericidal activity,
high serum stability, no haemolytic activity, no toxicity against mammalian cell lines, no
resistance induction over 15 passages, and favourable in vivo distributions in mice.
Patricia Czihal
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Chemistry and Mineralogy, Institute of
Bioanalytical Chemistry
patricia.czihal@bbz.uni-leipzig.de
ww.uni-leipzig.de/~bioanaly/
113
Bioanalytics
59 Characterization of proteins oxidatively modified
in rat muscles by tandem mass spectrometry
Maria Fedorova, Nadezda Kuleva, Ralf Hoffmann
A number of human diseases, including Alzheimerfs disease, Parkinsonsfs disease and
diabetes mellitus are strongly connected with oxidative stress caused by reactive oxygen
species (ROS). ROS can oxidize most biomolecules, especially proteins present in high
concentrations in the cells. The resulting oxidative modifications of certain amino acid
residues can alter the protein structure, which results in a loss of functional activity.
To model oxidative stress in vivo, rats were irradiated by X-rays (5 Gy dose). Proteins were
isolated from skeletal muscle tissues 3, 9 or 24 hours after irradiation. These protein extracts
as well as a protein extract from non-irradiated rats were separated by two-dimensional gel
electrophoresis. The proteins were visualized with Coomassie Brilliant Blue and the gel
images at the different time points analyzed for significant changes of the spot intensities.
Protein spots of interest were excised, digested with trypsin, and identified by MALDI-TOF/
TOF mass spectrometry. Peptides containing oxidized residues were sequenced by tandem
mass spectrometry. Several types of modifications were identified, such as different stages
of tryptophan oxidation (kynurenine, hydroxytryptophan, N-formylkynurenine, hydroxyNformylkynurenine),
hydroxyleucine, hydroxyisoleucine, oxidized methionine and cysteic
acid.
Maria Fedorova
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Chemistry and Mineralogy, Institute of
Bioanalytical Chemistry
maria.fedorova@bbz.uni-leipzig.de
www.uni-leipzig.de/~bioanaly/index.html
Bioanalytics
114
60 Study of toxicological effects of a silicon material
on mouse fibroblast cell line L929
Marco Glas, Andrea A. Robitzki
In a technological concept of a bio-hybrid system it is necessary to consider potential
interactions between the material used for building the system and biological samples.
There must be no effect to the bio-material concerning parameters like Proliferation and
Viability after contact with biological samples. In this study we determined the influence of a
silicon elastomer, that is used as a thin membrane at the bottom of cell drums. On this silicon
membrane thin tissue and cell layers are cultured in medium, so that the silicon elastomer
has to be biocompatible. The Proliferation was measured via an EdU-Proliferation-Assay
with following flow-cytometry analysis. For determining Viability we used a XTT-Assay
analyzed by spectrophotometry. The silicon elastomer was extracted in medium for one day.
This medium was used for incubating the mouse fibroblast cell line L929. In this study, our
results show no toxic effects of the silicon elastomer to the L929 cells.
Marco Glas
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
Marco.Glass@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
115
Bioanalytics
61 A novel system for quantification of peptides
bound to solid surfaces
Rayk Hassert, Annette G. Beck-Sickinger
Since the discovery of peptides that are capable of specifically binding to a solid surface
like iron oxide in 1992, this type of molecules have attained considerable attention. By
combinatorial approaches like phage display systems peptides were obtained that bind
specifically to different inorganics. Recent studies have shown that electrostatic interactions
play a key role in peptide-surface-interactions but the impact of peptide folding remains
unclear. In order to address this issue, a reliable assay is required to obtain quantitative data
for binding of short peptides to different surfaces.
Here we present an ELISA-like system based on the biotin/streptavidin complex for
quantification of peptide-surface interactions. Solid surface binding peptides were
synthesized by solid phase peptide synthesis and N-terminally modified with a biotin residue.
After purification and careful analysis the peptides were bound to different materials and
detected by a streptavidin-POD-conjugate. The subsequent oxidation of TMBH2 catalyzed
by the enzyme is measured photometrically at 450 nm. Different materials were investigated
including TiO2, ZnO, SiO2 and glass.
Considering the advantages of an ELISA-like assay with respect to parallel testing of different
peptides on various solid surfaces we are now able to improve the understanding of peptidesurface
interaction. This is necessary to bridge the gap from the common combinatorial
procedure to a more rational approach in the design of peptides binding to solids to obtain
sequences that show a stronger affinity and an improved selectivity of solid-surface binding
peptides.
Rayk Hassert
Universitat Leipzig
Institute of Biochemistry
Research group of Biochemistry and Bioorganic
Chemistry
hassert@uni-leipzig.de
www.biochemie.uni-leipzig.de
Bioanalytics
116
62 Metabolite analysis of adherent-growing cancer
cell lines with GC-MS
Antje Hutschenreuther, Ferdinand Fischer, Gerd Birkenmeier,
Claudia Birkemeyer
Metabolism of cells changes dramatically with cancerogenesis. In particular, glucose uptake
and glycolysis are increased (Warburg effect). A possible correlation between the glycolytic
phenotype (aerobic glycolysis) and the aggressiveness of cancer is under discussion.
Metabolomic approaches are powerful tools to identify biomarkers for different types of
cancer and/or different states in tumour progression and, thereby, improve proper diagnosis.
We used GC-MS to apply metabolite profiling to adherent-growing cancer cell lines. We
compared three different extraction protocols (extraction with methanol, and two different
protocols with methanol/ chloroform/ aqua dest.). Further, we compared lyophilised and
frozen pellets consisting of different cell numbers to optimize adopted protocols. The relative
signal intensities of commonly detected metabolites were evaluated with respect to different
extraction protocols. For systematic analysis, a list with about 150 identified metabolite
signals (identified using customer libraries, J. Kopka, Golm) out of a total number of ~300
peaks was created.
For cell line comparison, we used the breast cancer cell lines, i.e. MCF-7, MDA-MB-231,
MDA-MB-435, MDA-MB-436 and JIMT-1, and the glioblastoma cell line 1321 N1. The
concentration profiles of a subset of common metabolites were evaluated by correlation
analysis and a few of the most variable compounds with respect to the relative intensities of
normalized peak areas were investigated further.
Antje Hutschenreuther
Universitat Leipzig
Faculty of Medicine
Institute of Biochemistry
Antje.Hutschenreuther@medizin.uni-leipzig.de
www.uni-leipzig.de/~biochem
117
Bioanalytics
63 Development of cell type specific microelectrode
array based label-free screening systems
Heinz-Georg Jahnke, Sabine Schmidt, Ronny Azendorf,
Andrea A. Robitzki
Impedance spectroscopy is an excellent method for real-time, non-invasive and label-free
detection of cellular changes. Recognising potential and facilities of this detection method,
instrument developers as well as pharmaceutical industry showing increased interest in
impedance spectroscopy based detection systems. The great challenge for development of
cell based impedimetric assays is the optimisation of the cell-electrode-interface as well
as powerful electronic circuit design capable of multiplexing and measuring cell covered
electrodes with the necessary sensitivity.
In our clean room facility we produced multielectrode array (MEA) prototypes with optimised
microelectrode geometry for different cell types for instance neuronal, cardiac and smooth
muscle cells. Furthermore we tested electrode material like gold, platinum, titannitride,
indium tin oxide or alloys as well as different passivation substrates for optimum cell
adherence and stabilised cell-electrode-interface resulting in more sensitive measurements.
Strongly associated with multielectrode array development, we built efficient multiplexer
circuits fulfilling demands for non-invasive measurements of cells in low voltage range on
microelectrodes. First prototypes for MEA with 60 electrodes and 96 well plate arrays were
designed and produced.
Moreover we developed software for automated system control and data acquisition as well
as data processing and analysing of cellular electric properties. Adjusting all components for
the individual cell and assay demands we can provide optimised and sensitive impedance
spectroscopy based measurement setups.
Dr. Heinz-Georg Jahnke
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
heinz-georg.jahnke@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
Bioanalytics
118
64 Insights into the Secondary Structure of the
N-Terminus of the Adiponectin Receptor 1
Cathleen Juhl, Annette G. Beck-Sickinger
Adiponectin is an adipose tissue derived hormone, which is involved in a multitude of metabolic
pathways. Functions like inhibition of metabolic syndrome, protection of hypertension and
suppression of atherosclerosis have already been described. Until now, the transmembrane
receptors adiponectin-receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2) have been
identified. Both receptors consist of seven transmembrane helices. However, in contrast to
conventional G-Protein coupled receptors, the N-terminus is located intracellular and the
C-terminus is extracellular. For a better understanding of adiponectin signalling it is very
important to get more insights into ligand receptor interactions. Therefore, it might be helpful
to gain detailed structural information.
To investigate the secondary structure of the adiponectin receptor 1 N-terminus, the sequence
was subdivided into overlapping peptides of at least 16 amino acids, which were synthesized
by solid phase synthesis. These peptides were purified, characterized and analyzed by circular
dichroism spectroscopy, which is a useful tool to study peptides and proteins in solution. Hence
we are able to identify an ƒ¿-helical structure of the adiponectin receptor 1 N-terminus.
Cathleen Juhl
Universitat Leipzig
Institute of Biochemistry
Research group of Biochemistry and Biorganic Chemistry
cjuhl@uni-leipzig.de
www.biochemie.uni-leipzig.de
119
Bioanalytics
65 O ncocin . A novel proline-rich antimicrobial
peptide to treat multi-resistant Gram-negative
bacteria
Daniel Knappe, Anne Hansen, Ralf Hoffmann
Antimicrobial host defense peptides are conserved components of the immune system
and have been found across a wide variety of species. In lower organisms they comprise
a major component of the innate immunity, whereas in higher species they are part of the
complex immune system dedicated to form the first line of defense against bacteria, fungi
and viruses. Cationic proline-rich antimicrobial peptides isolated first from insects, such
as drosocin, apidaecin and pyrrhocoricin, have been studied by several research groups
for their antimicrobial properties as possible lead structures for further drug development
programs. This requires to optimize the antimicrobial activity for human pathogens and to
improve the serum stability for systemic applications. If successful, they may represent a
very promising novel class of antibiotic drug leads for treatment of multi-resistant bacteria,
such as Vancomycin-resistant enterococci (VRE), extended-spectrum beta-lactamase hyper
producing (ESBL+) E. coli, beta-lactam resistant and Carbapenem-resistant K. pneumoniae.
Here, we describe a lead optimization process for Oncocin, a novel peptide-based compound
based on a proline-rich antimicrobial peptide isolated from the milkweed bug O. fasciatus.
Oncocin is highly active against E. coli, K. pneumoniae, Salmonella typhimurium and
Pseudomonas aeruginosa with minimal inhibitory concentrations in the low micro molar
range. With a half life of 4 h the peptides are remarkably stable in full mouse serum.
Additionally, the best lead compounds do not penetrate human cell lines and thus are neither
toxic to HeLa and SH-SY5Y cells nor hemolytic.
Daniel Knappe
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute for Bioanalytical Chemistry
daniel.knappe@bbz.uni-leipzig.de
www.uni-leipzig.de/~bioanaly
Bioanalytics
120
66 A bond for a lifetime . Using Membrane
nanotubes from living cells to determine receptor
ligand kinetics
Michael Krieg, Jonne Helenius, Carl-Philipp Heisenberg,
Daniel J. Muller
Cells interact constantly with their environment using various kinds of cell adhesion
receptors. It is natural that each interaction is subjected to a force which alters the lifetime of
this particular interaction. According to the model of Bell [Bell, Science, 1978], the lifetime
of a receptor ligand bond decreases exponentially with increasing loads [Marschall, Nature,
2003]. In this work we implemented a single molecule force spectroscopy method that used
membrane nanotubes (tethers) to excerpt constant forces on a specific biological bond. By
using this cell-made force-clamp instrument we probed the binding kinetics of a fully native
and functional receptor-ligand bond at forces that are determined by plasma-membrane
properties [Krieg, ACIE, 2008]. We demonstrate the utility of this method with the well
characterized ConcanavalinA-N-linked oligosaccharide binding pair.
Michael Krieg
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Machines Group
krieg@biotec.tu-dresden.de
www.biotec.tu-dresden.de
121
Bioanalytics
67 The Core Unit Fluorescence-Technologies in the
IZKF Leipzig
Andreas Losche, Jens Grosche
The overall purpose of the Core Unit Fluorescence-Technologies in the IZKF Leipzig is
to offer access to expert assistance with various techniques in flow cytometry/cell sorting,
slide-based cytometry and laser scanning confocal and multiphoton microscopy. The specific
objectives of this shared resources are to provide the users with a powerful array of cytometric
and microscopic techniques, with expert consultation also in experimental design to optimise
data generation, as well as in data presentation and publication.
Currently the Core Unit houses a BD LSR II digital benchtop analyser, a Laser Scanning
Cytometer, a FACSVantage SE high-speed cell sorter, two confocal Laser Scanning
Microscopes and one multiphoton Laser Scanning Microscope. The cytometers are equipped
with up to four lasers (UV, 405 nm, 488 nm and 633 nm) and up to 12 parameters can
be measured at the same time. With the microscopes six laser lines (364 nm, 405 nm, 458
nm, 488 nm, 543 nm and 633 nm) are available. The Core Unit is open to all scientists
from the Faculty of Medicine and other faculties of the University of Leipzig as well as to
external researchers from other institutions. It is designed to provide services, like training
users to operate the analytical cytometers and the microscopes, performing high-speed cell
sorting by the staff only, ensuring that all instruments are properly calibrated on a daily basis,
advising users concerning the proper settings for their experiments, advising investigators on
experimental design and data analysis and performing further training of graduate students,
postdoctoral fellows and other colleagues.
Dr. Andreas Losche
Universitat Leipzig
Faculty of Medicine
Interdisciplinary Centre for Bioinformatics
andreas.loesche@medizin.uni-leipzig.de
www.izkf-leipzig.de
Bioanalytics
122
68 Temperature feedback for thermal stabilization in
optical tweezers
Mohammed Mahamdeh, Erik Schafer
Single molecule experiments done with optical tweezers usually involve high infrared laser
powers. Due to low IR transmittance, objectives are considerably heated. Such heating leads
to thermal drift and compromise stress free properties of the objective. With mainly focusing
on drift elimination, researchers presented a number of effective solutions. A drawback that
these solutions share is considerable complication of the setup. In this work we present a
practical yet easy to implement temperature feedback that minimizes drift and protects the
objective from heating-cooling cycles. By actively controlling the temperature of the trapping
objective and the condenser objective we were able to reduce low-frequency thermal noise by
2 folds. Also, time required to restore thermal equilibrium after a disturbance (e.g. changing
laser intensity) is shortened. This is due to restoring equilibrium is dependent on the feedback
performance and not thermal properties of the objectives and other parts within the setup.
Mohammed Mahamdeh
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Nanomechanics Group
mohammed.mahamdeh@biotec.tu-dresden,de
www.biotec.tu-dresden.de/schaeffer/nanomech
123
Bioanalytics
69 Impedimetric Detection of Transient Receptor
Potential Channel Activity
Oliver Panke, Andrea A. Robitzki
Transient receptor potential (TRP) channels are non-selective ion channels permeable to
cations including Na+, Ca2+ and Mg2+. They play a unique role as cell sensors and are involved
in many Ca2+-mediated cell functions. The variety of functions to which TRP channels
contribute predict that failure in channel gating will contribute to complex pathophysiological
mechanisms. Dysfunctions of TRP channels cause diseases but are also involved in the
progress of diseases. We present a novel method to screen chemical compounds as potential
activators or inhibitors of TRP channels to provide pharmaceutical tools to regulate channel
activity in disease control. Compared to common methods such as patch clamp or Ca2+
imaging, the presented impedance assay is automatable, experimental less demanding and
not restricted to Ca2+ flow.
We choose TRPA1 from the TRPA (eankyrinf) family as a model which is stimulated by
irritants like allylisothiocyanate (AITC). HEK293 cells, stably transfected with human
TRPA1 cDNA, were grown on microelectrode arrays. Confluent cell layers of high density
were analysed. Impedance spectra of cell-covered and non-covered Pt electrodes revealed a
cell-specific signal at 90 kHz, which was chosen to monitor TRPA1 activity thereupon. An
impedance decrease to 70 . 90 % of its original value was observed after application of 10
ƒÊM AITC indicating an increased conductance of the cell layer mediated by TRPA1. Control
cells lacking TRPA1 showed no changes upon AITC stimuli.
Dr. Oliver Panke
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Molecular Biological-Biochemical Processing Technology
oliver.paenke@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt/
Bioanalytics
124
70 Epitope mapping of monoclonal anti-Tau
antibodies AT8 and Tau5
Robert Porzig, David Singer, Ralf Hoffmann
Alzheimerfs disease (AD) is characterized by two neuropathological hallmarks, the formation
of extracellular senile plaques (SP) and intracellular neurofibrillary tangles (NFT). NFTfs
mostly consist of hyperphosphorylated versions of the microtubule associated protein tau,
which contains more than 30 possible serine and threonine phosphorylation sites. During
disease progression it appears that the balance of kinase and phosphatase activity is disturbed
resulting in aggregation and accumulation of hyperphosphorylated tau, also called paired helical
filaments (PHF-tau). Characterization of the hyperphosphorylated PHF-tau bears the promise
to develop a diagnosis for early AD. Since prognosis is based on phosphorylation-dependent
monoclonal antibodies (mAbs) recognizing disease-specific phosphorylation patterns, here
we present the characterization of two widely used monoclonal anti-tau antibodies. The anti-
PHF-tau mAb AT8 recognizes an epitope phosphorylated at both serine 202 and threonine
205. A third phosphate group at serine 199 did not influence the recognition. However mAb
AT8 was cross-reactive to two neighbored doubly phosphorylated motives containing either
serines 199 and 202 or threonine 205 and serine 208. The epitope of anti-tau mAb Tau5 was
mapped to the human tau sequence 218 to 225, which is not phosphorylated in vivo.
Robert Porzig
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute for Bioanalytical Chemistry
Robert.Porzig@bbz.uni-leipzig.de
www.uni-leipzig.de/~bioanaly
125
Bioanalytics
71 PH sensor-functionalized colloidal DNA-carriers
for direct localisation in cell compartments
Uta Reibetanz, Liaw Zi Yen, Bernice Oh Hui Lin, Averil Chen
Min Hui, Edwin Donath, Bjorn Neu
In recent years colloidal particles and capsules in sub-micrometer dimensions, Layer-by-
Layer (LbL) coated with biocompatible poly-electrolytes, have received much attention as
drug delivery systems. In order to establish such particles as transport systems it is not only
necessary to get a detailed understanding on the fabrication and functionalisation of these
capsules but also on the processing within the cell including the release of the particle from
the endo-lysosome into the cytoplasm and eventually of the active agent.
In this study particles were functionalised with pH sensitive fluorescent dye (FITC) to allow
particle localisation in the endo-lysosome and cytoplasm. A plasmid DNA (pDsRed) reporter
was integrated into the degradable biopolymer multi-layer. The processing and localisation
of the pDsRed containing particles were investigated in HEK293T cells via flow cytometry
and confocal laser scanning microscopy. After particle uptake we found a clear correlation
between the FITC intensity and the DsRed-expression. A low intensity of the FITC signal
was found in cells without expression, indicating exposure to a pH<5 and thus that the
particles remained in the endo-lysosomes. A significantly higher intensity was found in cells
expressing fluorescent proteins suggesting that the carriers are located in the cytoplasm with
pH 7.5. In conclusion this particle design allows the simultaneous study of particle location
and processing within cells and should thus be a useful tool for studying drug release in cells
by poly-electrolyte coated particles and capsules.
Dr. Uta Reibetanz
Nanyang Technological University Singapore
School of Chemical and Biomedical Engineering
Devision of Bioengineering
aureibetanz@ntu.edu.sg
www.ntu.edu.sg
Bioanalytics
126
72 A platform for the automatic identification and
quantification of metabolites using Nuclear
Magnetic Resonance Spectroscopy
Frank-Michael Schleif, Thomas Riemer, Uta Boerner, Rudiger
Alt, Lydia Schnapka-Hille, Christoph Wirling, Rolf Herwig,
Stefan Leidich, Michael Cross
Stem cells therapy is currently at the frontier of biomedical research. A better understanding
of the metabolism of stem cells is necessary to improve and extend initial promising results.
Nuclear Magnetic Resonance Spectroscopy allows for a precise measurement of metabolites
in cell extracts. The identification and quantification of these metabolites is essential to model
cellular metabolic network activity.
To meet clinical standards and high throughput demands a full automatic evaluation is
required. Based on the ongoing project .NMR Metabolic Profiling of the Stem Cell Niche .
METASTEMg, funded by the BMBF-QuantPro initiative, we have established an operational
platform for NMR-spectroscopy based metabolic profiling at the Medical Faculty of the
University of Leipzig.
This platform is currently applied to the qualitative and quantitative, time resolved metabolic
analysis of hematopoietic stem cells and their postulated stem cell niche.
Initial results on murine hematopoietic progenitor cell extracts (FDCPmix cells) and simulated
NMR spectra are shown.
Dr. Frank Michael Schleif
Universitat Leipzig
Institute of Informatics
Medical Department
schleif@informatik.uni-leipzig.de
www.uni-leipzig.de/~compint
127
Bioanalytics
73 Evaluation of carbon tetrachloride-induced
oxidative stress on rat hepatocytes:
Thermoinduced light emission and biochemical
standard methods
Anika Schumann, Alexander Bauer, Matthias Gilbert, Jan G.
Hengstler, Christian Wilhelm
Oxidative stress has become one of the most intensively studied topics in biomedical research
and is an often observed mechanism of non-genotoxic carcinogens like carbon tetrachloride
(CCl4). In this study we tested a new thermoluminescence (TL) based technique. CCl4 was
applied in in vitro cultured primary hepatocytes of rats using an incubation period of 24
hours. In a first step, we identified the range of concentrations that leads to induction of
toxicity in our hepatocyte in vitro model. For that, leakage of lactate dehydrogenase (LDH)
was used as an indicator of cell damage. Furthermore, the cell viability was determined.
Whereas the cell viability of the treated hepatocytes decreased, the LDH leakage increased in
a significant and dose dependent manner. To monitor the oxidative stress status, we compared
TL measurements with biochemical standard methods for oxidative stress markers. In contrast
to biochemical analysis TL measurements can be performed without any time-consuming
extraction procedures by using directly unprocessed cell material. After incubation with CCl4
thermoinduced light emission increased with rising concentration of CCl4 up to eightfold at
10 mM CCl4. Simultaneously, we determined the content of different secondary oxidative
stress products, like the combination of malondialdehyde and 4-hydroxynonenal. The rise
of this biochemical marker complied with the increasing concentration of CCl4. Finally, we
could show that the CCl4 induced increase of oxidative stress markers determined by timeconsuming
biochemical methods perfectly correlates with the signal increase in rapid TL
measurements.
Anika Schumann
Universitat Leipzig
Faculty of Biosciences, Pharmacy and Psychology,
Institute of Biology I
Department of Plant Physiology
anikaschumann@gmx.de
www.uni-leipzig.de/~pflaphys/
Bioanalytics
128
74 Human cationic trypsinogen PRSS1: recombinant
expression, purification and subsequent refolding
Peter Simeonov, Katherina Bellmann, Matthias Zebisch, Ralf
Hoffman, Norbert Strater, Thole Zuchner
We are investigating possibilities to recombinantly overexpress human cationic trypsinogen
(PRSS1) by using different pET vectors and bacterial expression strains as well as to
subsequently refold the protein from the inclusion bodies formed.
After cloning of PRSS1 into pET28b, pET32b, pET41b and subsequent transformation, the
overexpression conditions were optimised. Best overexpression could be achieved by using
the expression system pET28b in E.coli BL21 (D3E) codon plus RIL. Terrific broth medium
with 1% glucose at 37‹C was used, induction occurred for 3 hours and the correct product was
verified by MS-MS/MS-MALDI-TOF/TOF. Samples were denatured using 6 M guanidine-
HCl and the protein was purified via Ni NTA affinity chromatography. A refolding screen
testing several combinations of different additives with several concentrations of oxidized
and reduced glutathione/cysteine was performed afterwards. The yield of correctly folded
human cationic trypsinogen was determined by measuring the proteolytic activity.
In summary, human cationic trypsinogen was overexpressed using the expression system
pET28b in E.coli BL21 codon plus (D3E) RIL and refolding conditions were tested.
Peter Simeonov
Universitat Leipzig
Institute for Bioanalytical Chemistry
Ultrasensitive Protein Detection Unit
p.simeonov@bbz.uni-leipzig.de
www.uni-leipzig.de/uspdu/
129
Bioanalytics
75 Synthesis and mass spectrometrical
characterization of model peptides containing
oxidized tryptophan residues relevant for protein
aging
Toni Todorovski, Ralf Hoffmann
The continuous production of reactive oxygen species (ROS) with cells under physiological
conditions, modifies protein residues prone to oxidation. Besides cysteine, which can be
reversibly oxidized to cystine or sulfenic acid, tryptophan is probably the dominant target
of ROS in proteins. Several oxidation products of tryptophan appear to be associated
with the pathogenesis of age related diseases and cellular aging in general. Despite
the general interest in this area and its clinical importance, both an analytical strategy to
characterize such modifications and a synthetic strategy to produce model peptides to study
functional and structural aspects are still missing. Thus, we synthesized building blocks of
5-hydroxytryptophan and oxindolylalanine to specifically synthesize two model peptides
containing different oxidized tryptophan residues to study their fragmentation characteristics
by MALDI-TOF/TOF-MS (matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry) and ESI-MS/MS (electrospray ionization). Whereas only the peptide sequence
was retrieved from mass spectra acquired in post-source decay (PSD) mode, it was possible
to identify the oxidized tryptophan derivatives by collision induced dissociation (CID) based
on v and w ions characteristic for different oxidation products of tryptophan.
Toni Todorovski
Universitat Leipzig
Faculty of Chemistry and Mineralogy
Institute of Bioanalytical Chemistry
hemton2004@yahoo.com
www.uni-leipzig.de/~bioanaly/
Bioanalytics
130
76 O ptical trapping and 3D particle tracking: from
concept to versatile applications
Anna Wozniak, Joost van Mameren, Gerd Behme,
Sebastian Roth
In the past decade, experiments involving the manipulation and observation of nanostructures
with light using optical tweezers methodology have developed from proof-of-principle
experiments to an established quantitative technique in fields ranging from (bio)physics to cell
biology. With optical tweezers, microscopically small objects can be held and manipulated.
At the same time, the forces exerted on the trapped objects can be accurately measured.
JPK Instruments has developed a quantitative optical tweezers platform, the NanoTracker.
This platform allows the controlled trapping and accurate tracking of nanoparticles. With its
3D detection system, particle displacements within the trap can be recorded with nanometer
precision. Moreover, dynamic forces acting on the particle (e.g., exerted by motor proteins)
can be measured with better than piconewton resolution on a microsecond time-scale.
Here, we detail these and further features of the NanoTracker platform. Several successful
biophysical applications will be demonstrated. In particular, we show how some of the
hallmarks of single-molecule biophysics, the overstretching transition of DNA and the
famous 8-nm steps and stall forces of kinesin motor proteins, can be studied in a versatile and
operator-friendly manner.
With the NanoTracker, optical tweezers finally transcend from the labs of self-building
scientists who helped the technique mature, to a turn-key system able to serve a much wider
community of researchers in the life sciences.
Dr. Anna Wozniak
JPK Instruments AG
Applications group
nanotracker@jpk.com
www.jpk.com
131
Bioanalytics
77 In vivo biophysical study of fibroblast growth
factor signalling using fluorescence correlation
spectroscopy
Shuizi Rachel Yu, Markus Burkhardt, Jonas Ries, Steffen
Scholpp, Peter Schwille, Michael Brand
Fibroblast growth factors (Fgf) are a large family of protein growth factors with important
roles in regulating cell proliferation, migration and differentiation. Fgfs are morphogens
which are capable of assigning cell fate by propagating through developing tissues and
causing differential gene activation in a dosage specific manner. While cellular responses
to Fgfs have been extensively studied, very little is known about the molecular mechanism
of how Fgfs move through tissues and set up concentration gradient. It has been suggested
that endocytosis via cell surface receptors is an important negative regulator of Fgf8 protein
spreading . The restrictive clearance mechanism carefully controls the concentration
of Fgf8 available to the target cells. In this project, single molecule Fluorescence
Correlation Spectroscopy (FCS) is applied to investigate Fgf signaling in living zebrafish
embryos. Genetically encoded fluorescent probes and biosensors are engineered to
enable visualization of protein diffusion and interaction in living embryos. By studying
Fgf movement in living zebrafish embryos using FCS, we can determine local protein
concentrations, directionality of protein movement, and in vivo association and dissociation
constants of protein-protein interactions. The study will promote the understanding
of how cell signaling works dynamically in the context of multicellular tissues.
Shuizi Rachel Yu
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Developmental Genetics Group
rachel.shuizi-yu@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Bioanalytics
132
78 Introduction of a peptide-based Immunoassay
using a novel fluorophore and Fluorescence
Resonance Energy Transfer (FRET)
Thomas Zauner, Renate Berger-Hoffmann, Katrin Muller, Ralf
Hoffmann, Thole Zuchner
Cystic Fibrosis is a genetic disorder mainly affecting the lungs as well as the digestive
system, leading to progressive disability. The determination of the proteolytic activity as
well as the sensitive detection of human Trypsin play an essential role for the diagnosis
of this hereditary disease. Here we present a method based on Fluorescence Resonance
Energy Transfer (FRET) to determine the activity of proteolytic enzymes such as human
Trypsin. This technique includes a novel fluorophore (phen3dtpa) and a quencher molecule
which are covalently bound to the protein or peptide, decreasing the fluorescence intensity
dramatically. After proteolytic digest, the fluorophore and the quencher are separated, which
results in an increased fluorescence signal. Furthermore, our assay is based on time-resolved
fluorescence detection, leading to reduced background and increased signal to noise ratio.
Thus, the application of this technique offers a promising possibility to determine changes in
the proteolytic activity of human Trypsin. Additionally, our method can help to introduce a
suitable diagnostic test for several diseases involving misregulated proteolytic enzymes.
Thomas Zauner
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Faculty of Chemistry and Mineralogy, Institute of
Bioanalytical Chemistry
thomas.zauner@bbz.uni-leipzig.de
www.uni-leipzig.de/uspdu/
133
Bioanalytics
79 High Sensitive Multiplex Protein Quantitation
Using Metal Element Chelating Tags on an LTQOrbitrap-
ETD Mass Spectrometer
Nicole Zehethofer, Ralf Hoffmann, Thole Zuchner
In this work we present a new method for multiplex protein quantification of up to eight
different samples in one analysis step. This is a highly time- and cost-saving approach. The
samples are labeled with DTPA by using the cyclic anhydride derivative. When different
metal ions form complexes with DTPA, this results in the formation of tags with similar
chromatographic behavior but different m/z ratios.
The suggested protocol for protein labeling and digestion can easily be implemented into
commonly used protocols for tryptic digestion of proteins: labeling of the proteins is simply
performed after reduction, alkylation and tryptic digestion. The samples are then separated
using a nanoLC system and analyzed by MS/MS with an LTQ Orbitrap Mass Spectrometer.
When subjected to MS/MS, all labeled proteins produce a similar specific product ion which
can be used to identify them in complex mixtures. Intensity ratios of the corresponding
precursor ions are then used to determine the ratio of a protein of interest in different
samples.
The method presented herein allows relative quantification and identification of proteins
in more than eight different samples in a single LC-MS/MS experiment. The characteristic
product ion of the labeled peptides in combination with the high resolution capabilities of
the LTQ Orbitrap allow the unambiguous identification of labeled proteins in the sample.
Therefore, no purification step prior to LC-MS analysis is required to facilitate complex
mixture and no sample is lost during purification. Preliminary results show detection limits
in the femtomolar range.
Nicole Zehethofer
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Bioanalytical Chemistry, Ultrasensitive Protein
Detection Unit
zehethof@rz.uni-leipzig.de
www.uni-leipzig.de/uspdu

6. Bioinformatics
Posters

137
Bioinformatics
80 Family-based analysis of genome-wide genegene
interactions
Marit Ackermann, Andreas Beyer
Complex diseases are caused by an interplay of several genetic alterations and environmental
factors such as life style. Recent advances in genomics and biotechnology have opened the
gate to the genome-wide genotyping of thousands of possibly related individuals. Such data
can now be used for studying epistatic genetic interactions at a genomic level.
While traditional family-based association or linkage studies are restricted to either a small
number of markers or very specific pedigree structures, new methods for high-throughput
data often disregard the inherent population structure leading to spurious findings of genegene
interactions.
We propose an approach to infer genome-wide genetic interactions by using the genotype
information of parent-child trios. Our method is applicable to very large data samples and a
large number of markers. Instead of using the marginal frequencies of the observed alleles
at two markers, we make use of inheritance patterns to infer the expected allele frequencies.
Since the approach works conditional on ancestral genotype information it drastically reduces
the number of false positive findings due to population effects. Moreover, we correct for the
selection pressure against certain alleles that can also confound the results.
The approach is illustrated using a pedigree of almost $2300$ mice that have been genotyped
at more than $10,000$ SNPs. Some of the results of our analysis and their biological
significance will be discussed.
Marit Ackermann
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Networks Group
marit.ackermann@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Bioinformatics
138
81 Biomedical word sense disambiguation with
ontologies and metadata
Dimitra Alexopoulou, Bill Andreopoulos, Andreas Doms,
Khaled Khelif, Michael Schroeder
Ontology term labels can be ambiguous and have multiple senses. While this is no problem
for human annotators, it is a challenge to automated methods, which identify ontology terms
in text. Classical approaches to word sense disambiguation use co-occurring words or terms.
However, most treat ontologies as simple terminologies, without making use of the ontology
structure or the semantic similarity between terms. Another useful source of information for
disambiguation are metadata. Here, we systematically compare three approaches to WSD,
which use ontologies and metadata, respectively.
The .Closest Sensee method assumes that the ontology defines multiple senses of the term. It
computes the shortest path of co-occurring terms in the document to one of these senses. The
'Term Cooc' method defines a log-odds ratio for co-occurring terms including co-occurrences
inferred from the ontology structure. The .MetaDatae approach trains a classifier on metadata.
It does not require any ontology, but requires training data, which the other methods do not.
To evaluate these approaches we defined a manually curated training corpus of 2600 documents
for seven ambiguous terms from GO and MeSH. All approaches over all conditions achieve
80% success rate on average. MD performed best with 96%, when trained on high-quality
data. Its performance deteriorates as quality of the training data decreases. TC performs better
on GO (92%) than on MeSH (73%) as MeSH is not a strict is-a/part-of, but rather a loose isrelated-
to hierarchy. CS achieves on average 80% success rate.
Dimitra Alexopoulou
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
dimitraa@biotec.tu-dresden.de
www.biotec.tu-dresden.de/schroeder
139
Bioinformatics
82 The nanometis software system for automated
analysis of single molecular force spectroscopy
data on membrane proteins
Bill Andreopoulos, Frank Dressel, Dirk Labudde, Joscha
Koellner, Michael Schroeder
Nowadays, more and more automated machines enable scientists to measure thousands of
force-distance curves within hours. Nevertheless, the analysis is mostly based on manual
peak finding and annotation of the force-distance curves. The manual annotation is timeconsuming
and error-prone. While the machines can measure 40,000 curves in 24 hours, a
human annotator would need months to analyze these 40,000 curves.
The nanometis software system is able to analyze 40,000 curves in about one hour. Nanometis
looks for main peaks and side peaks in force-distance curves, representing modular units of
unfolding events.
Side peaks, corresponding to intermediate states in the unfolding process, occur less
frequently than main peaks. We apply our method to datasets from unfolding experiments
of bacteriorhodopsin (bR) and four bR mutants. While the peaks are clear from our analysis,
we also want to group the curves by their likely unfolding pathways. We used hierarchical
clustering to match curves that have similar sets of detected peaks. We achieved a recall
of 72% in clustering the curves correctly, according to the manual curation of unfolding
pathways.
Besides the large performance benefit, the nanometis software system enables the user to get
an objective evaluation with clearly defined criteria for the quality of the found unfolding
patterns.
Overall, nanometis leads to more consistent and reproducible results paving the way for highthroughput
analysis of structural features of membrane proteins.
Dr. Bill Andreopoulos
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Department of Computer Science and Engineering
williama@biotec.tu-dresden.de
www.cse.yorku.ca/~billa
Bioinformatics
140
83 A systematic approach of osteoclastogenesis
Antigoni Elefsinioti, Angela Simeone, Jacob Michaelson,
Andreas Beyer
During osteoclastogenesis progenitor cells change concentrations of a number of proteins
that are related with the new characteristics of the differentiated osteoclasts. Czupalla et
al. measured the patterns of protein and mRNA expression during osteoclastogenesis. The
authors found two largely distinct groups of genes, the first one altering mRNA concentrations
and the second one changing protein levels during cell differentiation. Surprisingly, these two
gene groups showed very small overlap between them.
In order to better understand the biological mechanisms separating these two groups of genes,
we adopted a network based approach. In our study we mapped the expression measurements
onto a gene interaction network, which was previously derived using independent information.
Next, we applied the TopModule network analysis algorithm to identify network regions
(emodulesf) with specific expression patterns. TopModule identified four distinct network
modules. Two of the four modules were enriched for transcriptionally up- while the other two
for post-transcriptionally down and up-regulated genes.
Our study underlines the well known fact that mRNA concentrations may not be predictive
of protein concentration changes even at the level of entire pathways. The lack of overlap
between the network modules could have different reasons, such like that the modules could
be regulated by different means, or that the timing of up-regulation could be different. Such
hypotheses could be tested by analyzing time-course expression data.
Antigoni Elefsinioti
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Networks and Systems Biology
antigoni.elefsinioti@biotec.tu-dresden.de
www.biotec.tu-dresden.de/beyer
141
Bioinformatics
84 Evolutionary and Functional Analysis of the RedƒÀ/
RecT Single-Strand Annealing Protein Family
Axel Erler, Madeleine Teucher, Marcello Maresca, Vineeth
Surendranath, Bianca Habermann, Youming Zhang, A.
Francis Stewart
DNA single-strand annealing proteins (SSAPs) are primarily of bacteriophage origin and
function in DNA recombination pathways. Recent computational sequence analysis identified
three evolutionarily distinct families, namely RecT/ƒÀ ERF, and RAD52. SSAPs are usually
encoded in predicted operons and neighboring genes therefore represent potential functional
partners. Among these especially RedƒÀ and RecT gained prominence through the development
of the DNA engineering technology known as erecombineeringf or eRed/ETf. Both proteins
are found in operons (red and recET) with a co-operating 5f to 3f exonuclease (Redƒ¿ and
RecE, respectively). Based on the recent increase of fully sequenced bacterial genomes we
re-examined the RecT/RedƒÀ SSAP superfamily and identified 106 individual sequences in 78
host strains, which cluster into four separate subfamilies. Representatives of each subfamily
were functionally investigated based on two in vivo recombination assays, first single-strand
Oligonucleotide Repair (ssOR) and second beta recombination. Our analyses provide an
important source towards the identification of novel recombinases.
Axel Erler
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Genomics Group
axel.erler@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Bioinformatics
142
85 Xenoturbella bocki: Analyses of the mitochondrial
genome and a PCR survey of hox genes
Guido Fritzsch, Marleen Perseke, Bettina Weich, Manja
Boehme, Detlef Bernhard, Thomas Stach, Olle Israelsson,
Mike Thorndyke, Hiroaki Nakano, Thomas Hankeln, Martin
Schlegel, Peter F. Stadler
The phylogenetic position of Xenoturbella bocki has been a matter of controversy since
its description in 1949. Here we report a polymerase chain reaction survey of partial
Hox homeobox sequences and the analysis of two mitochondrial genomes of X. bocki.
Surprisingly,we did not find evidence for more than five Hox genes, one clear labial/PG1
ortholog, one posterior gene most similar to the PG9/10 genes of Ambulacraria, and three
central group genes whose precise assignment to a specific paralog group remains open. Our
results of the analysis of the mitochondrial genomes confirm the deuterostome relationship
of Xenoturbella. However, in contrast to a recently published study (Bourlat et al. in Nature
444:85.88, 2006), our data analysis suggests a more basal branching of Xenoturbella within
the deuterostomes, rather than a sistergroup relationship to the Ambulacraria (Hemichordata
and Echinodermata).
Guido Fritzsch
Universitat Leipzig
Faculty of Medicine
Interdisciplinary Centre for Bioinformatics
fritzsch@izbi.uni-leipzig.de
www.izbi.uni-leipzig.de
143
Bioinformatics
86 Confluent mesenchymal stem cell cultures in silico
Martin Hoffmann, Jens-Peer Kuska, Matthias Zscharnack,
Christian Thummler, Augustinus Bader, Jorg Galle
Mesenchymal stem cell (MSC) cultures typically grow to confluence in monolayers within 1
. 2 weeks after seeding. The adherent cells show a characteristic spindle-shaped, fibroblastoid
morphology and align to each other in a way that is reminiscent of structured fur or spindomains
in magnetic systems.
In the present work we simulate the dynamics of MSC cultures using a new individual-cell
based model that explicitly represents cell podia. It accounts for cell differentiation on the
population level, individual cell-cell interactions and employs podia-generated forces for
cell movement. At the same time it is simple enough to simulate thousands of cells with a
reasonable computational effort.
Our modeling results give deeper insight into how properties of single cells and characteristics
of cell-cell interactions lead to the gemergenth structure and dynamics observed in MSC
cultures.
Dr. Martin Hoffmann
Universitat Leipzig
Interdisciplinary Centre for Bioinformatics
hoffmann@izbi.uni-leipzig.de
www.izbi.uni-leipzig.de
Bioinformatics
144
87 Pathway Prediction with eQTL and Gene
Interaction Networks
Jacob Michaelson, Andreas Beyer
Expression quantitative trait loci (eQTL) have become increasingly popular in recent years
as a technique to discover genetic regulators of gene expression. Several problems plague the
effective interpretation of eQTL results, including noisy data prone to many false positives,
a lack of molecular context for the significant loci, and an inability to identify causal genes
within causal loci, due partially to linkage disequilibrium. To address these challenges, we
developed a simple network analysis method and applied it to eQTL measurements from
mouse BXD strains. The algorithm uses a gene interaction network as a topology, maps eQTL
association scores to the genes in the network, and searches for areas of localized score
enrichment. These areas of score enrichment are compared to an empirical null distribution
to assess significance. The nature of the gene interaction network imparts a molecular context
to sets of eQTL, while aiding in filtering non-causative genes from significant loci.
To benchmark this network technique, we evaluated eQTL measurements from several tissues
from mouse BXD strains. We examined genes whose expression is regulated by well-known
pathways, and assessed the ability of our method to recover upstream pathway members, as
compared to traditional cut-off approaches to eQTL analysis. Our results indicate a marked
improvement over traditional eQTL analysis, and are enriched for known regulatory pathway
members.
Jacob Michaelson
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Networks and Systems Biology
jacob.michaelson@biotec.tu-dresden.de
www.biotec.tu-dresden.de/beyer
145
Bioinformatics
88 PhenoFam: a web tool for the gene set
enrichment analysis in the protein domain context
Maciej Paszkowski-Rogacz, Maria Teresa Pisabarro, Frank
Buchholz
Functional analysis of large gene lists, derived from high-throughput genomic experiments,
is still a challenging and promising field of study. The enrichment analysis is a very powerful
strategy helping researchers in identifying biological processes or other aspects of gene
function related to their studies. Most of available tools search for enrichment of gene ontology
terms in a subset of genes. This approach relies on algorithms selecting genes for the analysis.
Moreover, the experimental result (i.e. level of expression or strength of a phenotype) is not
considered. There are few applications overcoming these limitations by performing the gene
set enrichment analysis (GSEA). They search for gene annotations enriched on the top of a
list of genes ranked by their experimental values. However, to our knowledge, the analyzed
set of annotations is limited to Gene Ontology terms and only few of the tools are available
on-line.
Here we present PhenoFam, a web application for interpreting results of high-throughput
experiments in the protein domain context. Our algorithm associates uploaded experimental
results with protein domains and assesses weather the results associated with individual
domains are drawn randomly from the overall distribution of values. The p-value is calculated
with Mann-Whitney U test and the false discovery rate (FDR) is controlled with Benjamini-
Hochberg procedure. We demonstrate that the tool can give researchers a greater insight into
the protein structure-function relationship and can aid in annotating domains of unknown
function or associating already characterized domains with new processes.
Maciej Paszkowski-Rogacz
Max Planck Institute of Molecular Cell Biology and
Genetics
Research Group of Frank Buchholz
Research topic: Functional Genomics in mammalian cells
paszkows@mpi-cbg.de
www.mpi-cbg.de
Bioinformatics
146
89 GoGene: a search engine for genes and generelated
information.
Conrad Plake, Loic Royer, Rainer Winnenburg, Michael
Schroeder
Each day the literature database PubMed growths by thousands of new publications.
Microarray screens that measure expression levels of thousands of genes at a time are
common practice. All this results in masses of data for which manual investigation is not
feasible anymore. We present the web server GoGene that helps to reduce the time researchers
spend to find gene-related information. GoGene searches the literature for genes and concepts
such as biological processes, molecular functions, diseases, mutations etc. All information
is presented together with already known facts contained in the gene and protein databases
EntrezGene and UniProt. In total, 4.000.000 gene annotations are now available for 10 model
organisms such as human, mouse, rat, zebrafish, and fruit fly. All annotations are summarized
and displayed as a tree following the structure of the Gene Ontology and Medical Subject
Headings that can be seen as a table of contents to quickly find genes related to a category.
GoGene can be queried by keywords, gene lists and nucleotide or amino acid sequences. All
annotations are linked to the relevant literature and database entries. Thus, all information is
made transparent and can be verified by the user.
Availability: http://gopubmed.biotec.tu-dresden.de/gogene/.
Conrad Plake
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
conrad.plake@biotec.tu-dresden.de
www.biotec.tu-dresden.de/schroeder/
147
Bioinformatics
90 Unraveling protein networks with Power Graph
Analysis
Matthias Reimann, Loic Royer, Bill Andreopoulos, Michael
Schroeder
Networks play a crucial role in biology and are often used as a way to represent experimental
results. Yet, their analysis and representation is still an open problem. Recent experimental
and computational progress yields networks of increased size and complexity.
A common way to access the information in a network is though direct visualization, but this
fails as it often just results in 'fur balls' from which little insight can be gathered. On the other
hand, clustering techniques manage to avoid the problems caused by the large number of
nodes and even larger number of edges by coarse-graining the networks and thus abstracting
details. But these fail too since, in fact, much of the biology lies in the details.
Power Graphs are a lossless representation of networks, which reduces network complexity
by explicitly representing re-occurring network motifs. Moreover, power graphs can be
clearly visualized: Power Graphs compress up to 90% of the edges in biological networks and
are applicable to all types of networks such as protein interaction, regulatory, or homology
networks.
Matthias Reimann
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
reimann@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Bioinformatics
148
91 Studying molecular targets of human FOXP2 in a
neuroblastoma cell line
Sabrina Reimers, Ines Bliesener, Svante Paabo, Wolfgang
Enard
Language is an ability which seems to set humans apart from other animals. The transcription
factor FOXP2 is suggested to regulate the development of vocalization and underlying
neuronal circuits in birds, echolocating bats, and in humans. It has also been shown that
FOXP2 underwent recent positive selection in humans which implies that the two humanspecific
amino acids might be responsible for our ability to create speech and language.
To test a species-specific effect of FOXP2 on gene regulation, we stably transfected human
and chimpanzee FOXP2 into neuroblastoma cells and analyzed the changes in gene expression
using Affymetrix Exon Arrays.
Among these genes differentially regulated by human or chimpanzee FOXP2 we might find
an explanation to what sets us apart from our closest living relatives, the chimpanzees.
Sabrina Reimers
Max Planck Institute for Evolutionary Anthropology
Department of Evolutionary Genetics
sabrinareimers@web.de
www.eva.mpg.de/genetics
149
Bioinformatics
92 Unraveling protein networks with Power Graph
Analysis
Loic Royer, Matthias Reimann, Bill Andreopoulos, Michael
Schroeder
Networks play a crucial role in biology and are often used as a way to represent experimental
results. Yet, their analysis and representation is still an open problem. Recent experimental
and computational progress yields networks of increased size and complexity. There are for
example small and large scale interaction networks, regulatory networks, genetic networks,
protein-ligand interaction networks, and homology networks analyzed and published regularly.
A common way to access the information in a network is though direct visualization, but this
fails as it often just results in 'fur balls' from which little insight can be gathered. On the other
hand, clustering techniques manage to avoid the problems caused by the large number of
nodes and even larger number of edges by coarse-graining the networks and thus abstracting
details. But these fail too since, in fact, much of the biology lies in the details. Our PLoS
Computational Biology paper presents a novel methodology for analyzing and representing
networks. Power Graphs are a lossless representation of networks which reduces network
complexity by explicitly representing re-occurring network motifs. Moreover, power graphs
can be clearly visualized: Power Graphs compress up to 90% of the edges in biological
networks and are applicable to all types of networks such as protein interaction, regulatory,
or homology networks. We demonstrate the usefulness of Power Graph Analysis on five
detailed biological examples ranging from protein-ligand binding to regulatory networks and
homology networks.
Loic Royer
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
royer@biotec.tu-dresden.de
www.biotec.tu-dresden.de/schroeder
Bioinformatics
150
93 Fluorine in protein environments: theoretical and
experimental study
Sergey Samsonov, Mario Salwiczek, Gerd Anders, Beate
Koksch, Maria Teresa Pisabarro
Use of non-canonical amino acids is a powerful tool for modification of proteins properties.
In this context fluorinated amino acids have increasingly gained recently in importance.
However, the basic properties of fluorinated chemical groups are not clear yet. The aim of
this study has been to characterize the physico-chemical properties of fluorinated amino acids
by the use of quantum mechanics (QM) and molecular dynamics (MD) approaches. For this,
we have analyzed geometry, charge characteristics and hydrogen bonding abilities of ethane
fluorinated derivatives, which model fluorinated amino acids side chains, by QM. We have
parametrized four fluorinated L-amino acids for the AMBER MD package (three ethyl- and
one propylglycine derivatives), and have characterized them in terms of molecular volumes,
conformations, and energy of hydration. The obtained results show that fluorine and hydrogen
atoms of fluoromethyl groups are weak acceptors and donors of hydrogen bonds. Hydration
of side-chains of the studied fluorinated amino acids was found to be more favorable than for
their non-fluorinated analogues, which is in accordance with experimental data on retention
times for these amino acids. Our results from MD simulations and MM-PBSA analysis of a
parallel coiled-coil system with fluorinated amino acids substitutions agree with experimental
data obtained by CD-spectra and qualitatively reproduce experimentally observed energetic
trends. This study emphasizes the importance of fluorinated amino acids for rational drug
design.
Sergey Samsonov
Technische Universtat Dresden
Biotechnology Center (BIOTEC)
Structural Bioinformatics
sergeys@biotec.tu-dresden.de
www.biotec.tu-dresden.de/pisabarro
151
Bioinformatics
94 A systems biology approach for analyzing RNAi
data using functional networks
Angela Simeone, Andreas Beyer
The improvement and the automation of genome-wide RNAi screens for studying complex
cellular processes provides a huge amount of data turning the data analysis and interpretation
into a bottleneck.
RNA interference (RNAi) is a method used to specifically suppress gene expression by
targeting and degrading mRNA in order to systematically analyse the effects of the loss
of function. RNAi screening data are subject to noise because the suppression may be
inefficient, because the detection of the phenotype can be inaccurate or due to off-target
effects. Moreover, it can be difficult to explain the observed genotype-phenotype association
exclusively based on phenotypic data.
In order to address these issues and to correctly understand the role of each gene/protein
in the specific (emergent) cellular process we integrate the phenotypic information with
independent gene interaction data (functional network). We then screen such network for
identify functional modules associated with respective phenotypic effects.
As a proof of principle we have applied this approach to a recently published RNAi screen,
which aimed at detecting cell-cycle related genes in human cell-lines [Kittler et al. 2007].
When applied to the G1-arrest phenotype our method identified a network module of 106
genes. This analysis successfully detected genes that were truly related to G1-arrest as
confirmed by independent experiments (true positive). Also, the network module contained
genes that did not show a significant phenotype in the primary screen, but which were later
confirmed to be also related to G1-arrest (false negatives).
Angela Simeone
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Networks and Systems Biology
angela.simeone@biotec.tu-dresden.de
www.biotec.tu-dresden.de/beyer
Bioinformatics
152
95 Development of a framework for structure-based
functional protein annotation
Aurelie Tomczak, Jana Sontheimer, Maria Teresa Pisabarro
Despite the availability of the human genome and an increasing number of up-to genomescale
screening data there are still many proteins with unknown function. Classical sequencebased
bioinformatics approaches often fail to predict protein function, in case no homologous
protein can be found. As protein structure is more conserved than sequence, structure-based
methods (e.g. fold recognition) may recognize possible structural similarities even in the
absence of recognizable sequence similarity and thus provide functional clues or confirm
tentative functional assignments. These methods have already proven to be successful for
individual proteins but, due to the large amount of data obtained by sequencing and other
high throughput approaches, automation of structure-based functional annotation of proteins
is needed.
We are developing a framework for automatic structure-based annotation of uncharacterized
proteins with interesting phenotypes obtained by human genome-scale RNAi screenings (e.g.
DNA repair, cell viability). For this we are integrating sequence, structural and functional
information from publicly available resources (i.e. PDB, SwissProt, SCOP), proteomic
data as well as phenotypic information coming from functional RNAi-screenings with fold
recognition and other computational structure-based methods (e.g. protein modelling).
This automatic setup is being used to link phenotypic information to molecular function and
derive functional hypotheses, some of them being tested experimentally to characterize novel
proteins with striking phenotypes.
Aurelie Tomczak, Jana Sontheimer
Technische Universtat Dresden
Biotechnology Center (BIOTEC)
Structural Bioinformatics
aurelie.tomczak@biotec.tu-dresden.de
www.biotec.tu-dresden.de
153
Bioinformatics
96 Three-dimensional modeling of protein complexes
Anne Tuukkanen, Andreas Henschel, Michael Schroeder
Understanding of many cellular processes requires structural knowledge about large
protein assemblies. It is not enough to know the interacting protein components to
understand the function of a large protein complex. Recently there have been several
attempts to produce either atomic models or more coarse-grained architectural models
of large protein complexes. The goal of this work is to combine experimental protein
. protein interaction data of various accuracy and computational interaction modeling
techniques in order to produce reliable structures of large protein complexes. The produced
models, on the other hand, can be used to study interaction interfaces and the amino acids
involved in binding. These predictions can be validated with experimental methods and
the knowledge used to further improve the modeling in an iterative process. The main
idea is to build large protein complexes from structures of individual subunits with the
help of experimental constraints. The developed approach was applied on 3-dimensional
modeling of Set1 histone methyltransferase complex from Saccharomyces cerevisae.
Anne Tuukkanen
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
annet@biotec.tu-dresden.de
www.biotec.tu-dresden.de/schroeder
Bioinformatics
154
97 Utilising mutation tagging with gene identifiers
for protein structure function studies
Rainer Winnenburg, Conrad Plake, Christof Winter,
Annalisa Marsico, Michael Schroeder
The automated retrieval and integration of information about protein point mutations in
combination with structure, domain and interaction data from literature and databases
promises to be a valuable approach to study structure-function relationships in biomedical
data sets. We developed a rule- and regular expression-based protein point mutation retrieval
pipeline for PubMed abstracts, which shows an F-measure of 87% for the mutation retrieval
task on a benchmark dataset. In order to link mutations to their proteins, we utilised a named
entity recognition algorithm for the identification of gene names co-occurring in the abstract,
and established links based on sequence checks. Vice versa, we could show that gene
recognition improved from 77% to 91% F-measure when considering mutation information
given in the text. From 31,000 abstracts, we identified 36,000 unique mutations for almost
6000 genes from the nine model organisms Human, Mouse, Yeast, Rat, Fruit Fly, E.coli,
A.thaliana, C.elegans, and Zebrafish. In comparison to the known variants and mutations
from UniProt KB (version 1.47) we found additional 27,000 mutations. Our data are publicly
accessible via GoGene, a search engine for genes and gene-related information. In addition,
we transfered these data as well as phenotypic descriptions and disease relations extracted
from the corresponding abstracts into a database for subsequent protein structure function
studies. To demonstrate practical relevance, we examined the influence of mutations on
structural protein protein interfaces from the SCOPPI database and structure/sequence motifs
in transmembrane proteins.
Rainer Winnenburg
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Junior Research Group .Bioinformaticsg
rainer.winnenburg@biotec.tu-dresden.de
www.biotec.tu-dresden.de/schroeder
155
Bioinformatics
98 SCOPPI . A Structural Classification of Protein-
Protein Interfaces
Christof Winter, Andreas Henschel, Wan Kyu Kim, Gihan
Dawelbait, Michael Schroeder
SCOPPI, the Structural Classification of Protein-Protein Interfaces, is a comprehensive
database that classifies and annotates domain-domain interactions found in all known protein
structures. SCOPPI applies SCOP domain definitions and a distance criterion to determine
inter-domain interfaces. Interfaces are clustered using geometrical properties and thus
classified. Here, we present several applications for SCOPPI. First, we can predict and model
an interaction between two proteins consistently found deregulated in patients with pancreas
cancer. Using a SCOPPI structural template, we propose a putative inhibition of TMPRSS4,
which is up-regulated in pancreas cancer, by TFPI2, which is down-regulated in pancreas
cancer. Second, we screen gene fusion events of protein complex subunits for conservation
of the binding orientation of the fused proteins. We find a conserved orientation in two out
of three cases. Last, we show how the Baculovirus protein p35 mimics the binding site of
human inhibitor of apopotosis in order to bind to human caspase. Eventually, this can help
the virus to prevent apoptosis by caspase inhibition. Although structurally similiar, there is
no apparent sequence similarity present at the binding site, displaying a fine example of
convergent evolution.
Encoding the sequence information of SCOPPI interfaces in Hidden Markov Models allows
for screening uncharacterised genome sequences and predicting protein binding as well as
ligand binding sites.
SCOPPI is online at http://www.scoppi.org for browsing and querying, and available for
download upon request.
Dr. Christof Winter
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Bioinformatics
winter@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Bioinformatics
156
99 Serum proteomic profiling and targeted
metabolomic of obese: Integrative data analysis
of biological profile data
Henry Wirth, Martin von Bergen, Jayaseelan Murugaiyan,
Hans Binder, Andreas Oberbach
According to the WHO, more than one billion adults are overweight and 300 million are
clinically obese. Obese subjects have a decreased life quality and expectancy as well as an
increased risk of developing comorbidities such as insulin resistance and type 2 diabetes.
Thus, early detection tools for a precise identification of individuals at risk are urgently
needed.
Since biomarkers are often low-molecular-weight proteins secreted into the bloodstream as
a result of the disease process, metabolic and protein profiles may be modified in individuals
with elevated blood glucose. To develop new non-invasive tests for diagnosis of morbide
obesity and pre-diabetes, these profiles have to be processed in statistical analysis.
The purpose of the present study was to evaluate the impact of statistical tools to identify
metabolic and proteomic pathways in serum of obesity population.
Therefore, patients with obesity and lean control subjects, all completely equivalent and
healthy, were selected for metabolic and protein profiling (using gBiocrates Absolute IDQh
resp. 2DE-gels with DIGE labeling after depletion of 20 high-abundance proteins). Based on
this data, analytical methods as t-statistics, discriminant analysis and cluster analysis were
performed and compared for integrated metabolite and protein data.
It has been shown, that the simultaneous application of complementary statistical tools is
necessary to translate purely descriptive proteomic and metabolomic profiles into a functional
context and provide important insights into pathophysiological mechanisms of obesity that
would not have been obtained by other techniques.
Henry Wirth
Universitat Leipzig
Interdisciplinary Centre for Bioinformatics
wirth@izbi.uni-leipzig.de
www.izbi.uni-leipzig.de
157
Bioinformatics

Tissue and Cell
Engineering
7.
Posters

161
Tisue and Cel Enginering
100 Hematopoietic progenitor cells are vulnerable to
high oxygen concentrations
Rudiger Alt, Nicole Noack, Michael Cross
In order to investigate the tolerance of hematopoietic progenitors to hypoxia and the
effects of reducing oxidative stress, the proliferation and clonogenic expansion of a murine
multipotential progenitor cell line (FDCPmix) and of primary CD133+ cells from human
umbilical cord blood (hUCB) were tested at selected culture conditions. FDCPmix and freshly
isolated CD133+ hUCB cells were entered either into liquid cultures or directly into colony
forming (CFU) assay of clonogenic activity at either 1% or 21% oxygen. Both FDCPmix and
CD133+ hUCB cultures expanded well under hypoxic conditions. Strikingly, the clonogenic
development of CD133+ hUCB cells improved markedly from 9.5 % in normoxia to 16.1 %
in hypoxia, the frequency of both erythroid and non-erythroid colonies being significantly
icreased. A comparable increase was observed in FDCPmix CFU-blast assays, when exposed
to hypoxia. Thus, a proportion of colony forming progenitors, which were unable to develop
under conventional oxygen tension did so under hypoxic conditions. In order to test whether
the suppression of this subpopulation of potential CFUs in normoxia might be due to the
generation of reactive oxygen species, we screened the effects of a range of antioxidants
directly in the CFU-assays. Reduced glutathione and cysteine, the active compound of
glutathione, most effectively increased colony counts in normoxia (>25%), while both
oxidized glutathione and cystine were ineffective. We conclude that a subpopulation of
hematopoietic progenitors is sensitive to high oxygen tension, and will not expand in vitro
unless protected by either hypoxia or antioxidants.
Rudiger Alt
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Cell Therapies for Repair and Replacement
ralt@trm.uni-leipzig.de
www.trm.uni-leipzig.de
Tisue and Cel Enginering
162
101 Fabrication of Microscaffolds by Solid Lipid
Templating
Kristina Ambrosch, Michaela Schulz-Siegmund, Michael C.
Hacker
Microscaffolds have gained interest as macroporous particulate cell carriers for different
applications in tissue engineering and regenerative medicine. In an ideal case, their well
interconnected pore network facilitates the transport of nutrients, waste and oxygen
ensuring a sufficient supply throughout the entire volume of the 3-D carrier in suspension
culture. Classical preparation techniques for porous microspheres often do not allow for
the controlled generation of interconnected macropores. Based on our experience with the
solid lipid templating (SLT) technique for the fabrication of conventional scaffolds this
study investigates the implementation of solid lipid particles with defined diameters as pore
forming agents. For this aim SLT was combined with a spraying technique. Therefore, solid
lipid microspheres were dispersed in a viscous poly(lactide-co-glycolide) (PLGA) solution
and small particles were generated using a two-component jet and collected in a non-solvent
for the polymer. After polymer solvent extraction, the hardened particles were leached in
n-hexane to extract the embedded particles to create macroporous networks. Surface structure
and size of the particles were analyzed by light and electron microscopy. Different processing
parameters and their effects on microscaffold structure are reported.
Kristina Ambrosch
Universitat Leipzig
Institute of Pharmacy
Department of Pharmaceutical Technology
ambrosch@uni-leipzig.de
www.uni-leipzig.de/~pharm/phatech/
163
Tisue and Cel Enginering
102 iPS cells as model system for human virus
diseases
Antje Arnold, Claire Fabian, Steven Sauerzweig, Yahaira
Naldijk, Ute Brinkmann, Uwe G. Liebert, Alexandra Stolzing
Human embryonic stem (ES) cells are an excellent model system to study neurodegenerative
diseases and to investigate human viral infections. In the last three years a great deal of
information concerning a new type of stem cells has been discovered called induced
pluripotent stem cells (iPS).
To investigate infections of human pathogenic viruses there are several broadly used model
systems like immortalised cell lines of human or animal origin. Immortalised human cell lines
are often altered in their metabolism and most animal model systems are not the natural host
of the virus studied; therefore there could be differences to the mechanisms of infection. iPS
cells give the opportunity to study the infection of cells during their development and they can
be differentiated into several types of tissues which are afflicted by the infection. Therefore it
is possible to study the infection without the flaws of the above described systems. We were
able to show that the morphology of human fibroblasts and mesenchymal stem cells (MSC)
after transfection and transduction with shuttles/vectors of pluripotency genes changes to ES
cell-like morphology, i.e. growth in defined colonies.
In addition to human used fibroblasts and MSC from Lewis rats were used to establish an in
vitro infection model to study measles. We used qRT-PCR and immunostaining to analyse the
expression of pluripotency genes including nanog, oct4, klf4 and sox-2.
Antje Arnold
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Stem Cell Biology
antje.arnold@izi.fraunhofer.de
www.izi.fraunhofer.de
Tisue and Cel Enginering
164
103 Characterization of the early embryo upon loss of
histone methyltransferase Setd1a
Anita Sabine Bledau, Andrew Francis Stewart, Konstantinos
Anastassiadis
Epigenetics highly determine chromatin structure and enable inheritance of genes in
a temporal and spatial depended manner. During embryonic development, epigenetic
mechanisms are essential to established and further maintain gene expression patterns.
Activation or silencing of a specific gene loci correlates with posttranscriptional modifications
at core histone tails of the eukaryotic chromatin. Among those modifications, histone tail
methylation originating from trithorax group (trxG) protein function have been shown to be
crucial to the developing embryo. These trxG proteins specifically methylate nucleosomes at
their histone tail 3 at lysine residue 4 (H3K4) that is associated with active gene expression.
However, how functional trxG methylation complexes accomplish precise gene activation
ultimately determining cell fate is still unclear. (In particular how is it regulated in a specific
tissue at a particular time?) Complexity increases with the fact that there are six functional
H3K4 histone methyltransferases expressed in embryonic stem (ES) cells, namely Mll1 .
Mll4 and Setd1a and Setd1b. Therefore, our laboratory focuses on conditional mutagenesis
of all six methyltransferases in mouse ES cells. By these means homozygous conditionally
targeted ES cells have been studied to identify specific target genes and to analyze their in
vitro differentiation capacity into all three germ layers. We further focused on analyzing
methyltransferase null embryos that we found to be severely disorganized and lethal at
specific embryonal stages. The ultimate goal is to understand the individual role of each
histone methyltransferase in the process of self -renewal and differentiation of mouse ES
cells and their impact on mouse embryonic development.
Anita Sabine Bledau
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Center for Regenerative Therapies, Genomics
anita.bledau@biotec.tu-dresden.de
www.crt-dresden.de
165
Tisue and Cel Enginering
104 Hepatocytes from human mesenchymal stem cells
support liver regeneration after acute injury
Bruno Christ, Peggy Stock, Hendryk Aurich, Ines Aurich,
David Wohlrab, Werner Hein, Sabine Ebensing, Madlen
Hempel, Matthias M. Dollinger, Wolfgang E. Fleig
Mesenchymal stem cells from human bone marrow (hBM-MSC) have the potential to
differentiate into hepatocyte-like cells in vitro and continue to maintain predominant
hepatocyte functions in vivo after transplantation into host mouse livers. Here, hMSC were
differentiated to hepatocyte-like cells in vitro (hBM-MSC-HC) and transplanted into livers
of immunodeficient Pfp/Rag2-/- mice treated with a single sublethal dose of acetaminophen
(APAP) to induce acute liver injury. APAP induced a time- and dose-dependent damage in the
perivenous areas of the liver. Serum levels of AST elevated after APAP treatment were similar
in mice without or with hBM-MSC-HC transplanted into the livers 24 h after treatment.
Yet, in mouse livers receiving hBM-MSC-HC transplanted cells filled the perivenous areas
damaged by APAP. No further tissue damage was visible in the residual parenchyma and
livers displayed less tissue lesions due to attenuation of hepatocyte apoptosis in response
to APAP treatment. After 7 weeks, hepatic injury had completely recovered in both animals
with or without hBM-MSC-HC. Clusters of transplanted cells appeared predominantly in
the periportal portion of the liver lobule and cells stained positive for human HepPar1 and
albumin featuring prominent qualities of differentiated hepatocytes. Human albumin was
secreted into the serum to amounts comparable to mice transplanted with human hepatocytes.
Thus, hBM-MSC-HC contributed to liver regeneration by the attenuation of drug-induced
hepatic damage. They hence may serve as a novel source of hepatocyte-like cells for the
treatment of acute liver injury.
Bruno Christ
Martin-Luther-Universitat Halle-Wittenberg
Clinic and Policlinic for Internal Medicine I
Research Group of Hepatozytentransplantation
bruno.christ@medizin.uni-halle.de
www.medizin.uni-halle.de/kim1
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166
105 Generation and Characterization of Tumor
Spheroids as Cellular Models for Anti-Cancer Drug
Discovery
Anja Drose, Mirjam Ingargiola, Bernd Schwenzer
Tumor spheroids represent the morphological and functional features of avascular tumors
more realistically than monolayer cultures. The tumor microenvironment which influences
uptake, penetration, action of drugs and the development of resistance is better mimicked.
By using heterologous co-cultures even interactions with stromal cells can be taken into
account.
We established spheroidal systems for different types of tumors as powerful in vitro models
for anti-cancer research by using the methylcellulose technique as culturing method.
After optimization initial cell number, culturing period and methylcellulose concentration
as influencing factors for spheroid formation, spheroids from eight different tumor cell lines
could be generated as well as the corresponding co-cultures with fibroblasts and endothelial
cells. Scanning Electron Microscopy and paraffin sections were used for characterization.
Furthermore the growth parameters spheroid diameter and cell number were determined.
Most notably FaDu, Hep-G2, HT-29 and A-498 form highly compact, perfect spheroidal
aggregates and allow culturing for several weeks rendering them ideal test systems for longterm
studies.
To study potential cell-cell contact effects, the expression of VEGF in monolayer and spheroid
cultures was compared. Our results indicate that tumor spheroid co-culture with endothelial
cells leads to higher gene expression of VEGF, which probably is more consistent with the in
vivo situation in tumor tissue.
Anja Drose
Technische Universitat Dresden
Faculty of Mathematics and Science
Department of Chemistry and Food Chemistry
anja_drose@chemie.tu-dresden.de
www.chm.tu-dresden.de/bc1
167
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106 The impact of iPS on stem cell legislation and
administration in Germany . points to consider
for international stem cell research cooperation
Timo Faltus
In 2008 the German lawmaker has amended the national Stem Cell Act (Stammzellengesetz .
StZG). This reform applies to the provisions for the import of human embryonic stem cells to
Germany and to the provisions for the infringement of the Stem Cell Act. This modernisation
of the German stem cell law coincided with unexpected results of stem cell research in the
field of the creation of ethically unloaded stem cells by techniques of reprogramming. These
techniques lead to so-called induced pluripotent stem cells (iPS). However, the German Stem
Cell Act contains a subsidiarity provision which states that the import of human embryonic
stem cells to Germany is not allowed if there is a scientific alternative for the use of human
embryonic stem cells. Therefore, the scientific and medical success of the reprogramming
research could legally inhibit the further import of and research with human embryonic stem
cells in Germany if iPS were an alternative for the use of human embryonic stem cells.
But, this subsidiarity provision is only applicable if the galternativeh could fully replace
the scientific use of human embryonic stem cells. For this reason, the legal (and scientific)
status of iPS must be clarified. This scientific project gives a broad overview for these topics
and gives answers regarding the legal impact of reprogrammed stem cells in legislation and
administration.
Timo Faltus
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Cell Therapies for Repair and Replacement
tfaltus@trm.uni-leipzig.de
www.trm.uni-leipzig.de
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168
107 Isolation and Characterisation of Human
Melanocytes from Hair follicles for Clinical Use
Christina Fieber, Jan C. Simon, Andreas Emmendorffer
Despite significant progress in tissue engineering and cell biology there is still an unmet need
for treating patients suffering from Vitiligo.Vitiligo (or leukoderma) is an acquired chronic
skin disease that causes loss of pigment, resulting in irregular pale patches of skin. It occurs
when the melanocytes die or are unable to function. The precise pathogenesis of Vitiligo
is multifactorial and not fully understood. There is some evidence suggesting it is caused
by a combination of auto-immune, genetic, and/or environmental factors. The population
incidence worldwide is considered to be between 1% and 2%.
The hair follicle bulge area is an abundant source of actively growing pluripotent adult stem
cells. These cells can be differentiated into various cell lineages, e. g. keratinocytes and
melanocytes amongst others. One important advantage is that the hair follicles are easily
accessible by just plucking the hair follicles from the scalp. Using hair follicles as source for
melanocytic stem cells we are going to develop a causative, non-invasive and autologous cell
therapy for patients suffering from Vitiligo. We are aiming to differentiate and propagate the
hair follicle cells in vitro. This will enable us to treat larger areas of depigmented skin.
However, a crucial prerequisite for use in clinical application is that the growth conditions
have to fulfill current GMP conditions. Very recently, we succeeded in the cultivation of
melanocytes isolated from hair follicles by using culture medium that does not contain any
supplements from animal origin, and therefore fulfills GMP requirements.
Dr. Christina Fieber
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Tissue Engineering and Materials Science
cfieber@trm.uni-leipzig.de
www.trm.uni-leipzig.de
169
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108 Differential expression of biofunctional GM1 and
GM3 gangliosides within the plastic-adherent
multipotent mesenchymal stromal cell population
Daniel Freund, Denis Corbeil
It is unclear whether the plastic-adherent multipotent mesenchymal stromal cells (MSCs)
isolated from human bone marrow represent a homogeneous and stable cell population or
they are heterogeneous in terms of cell surface constituents and hence functionality.
Therefore we investigated the expression profile of certain biofunctional lipids by plasticadherent
MSCs . focusing particularly on two membrane microdomain (lipid raft)-associated
gangliosides, GM1 and GM3. Phenotypically, we could observe a differential expression
where certain MSC subsets express either GM1 or GM3 or both. Furthermore, the GD2
expression increases the complexity of the expression patterns giving rise to seven identifiable
cell phenotypes. In contrast, the binding of various lectins appeared homogenous indicating
that the general glycosylation pattern remains common. As expected the classical CD markers
did not display any differential expression among the MSC population. Morphologically, a
segregation of GM1 and GM3 clusters was observed, GM3 being mostly excluded from
the highly curved plasma membrane protrusions. These data highlight the phenotypic
heterogeneity of plastic-adherent MSCs in terms of certain lipid constituents of the plasma
membrane, and the presence and/or absence of distinct ganglioside-based membrane
microdomains suggest their potential functional diversity.
Dr. Daniel Freund
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Molecular Tissue Engineering
daniel.freund@biotec.tu-dresden.de
www.biotec.tu-dresden.de
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170
109 Telomerase activity and hepatic functions of rat
embryonic liver progenitor cell in nanoscaffold
coated small scale bioreactor
Shibashish Giri, Sanja Pavlica, Mario Keller, Augustinus
Bader
Presently, there has been growing interest on telomerase activity in all cells (somatic cell, stem
cells, cancerous cells and others cells) and this activity is associated with cellular changes
such as proliferation, differentiation, immortalization, cell injury and ageing. Telomerase
activity are absent in most of somatic cell but present in over 90% of cancerous cells and
other immortalized cell lines. In our present study, we cultured rat embryonal liver progenitor
cell line RLC- 18 in self assembly nanostructured scaffold (puramatrix ) coated bioreactor,
collagen coated plates and uncoated plates and evaluated for changes of telomerase activity
by telomerase ELISA, cell proliferation by MTT assay and hepatic functions. We found less
telomerase activity and less cell proliferation but more hepatic functions on nanoscaffold
coated bioreactor than collagen coated plates and uncoated plates. Our data supports the
concept that cell-scaffold interaction may be one of significant roles on telomerase activity as
well as hepatic functions. Although the mechanism involved in telomerase regulation is still
not fully completed. But our result may provide clues to cell differentiation that telomerase
activity may be regulated by cell- scaffold interaction.
Shibashish Giri
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Department of Cell Techniques and Stem Cell Biology
giri.shibashish@bbz.uni-leipzig.de
www.uni-leipzig.de/bbz
171
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110 Temporally-controlled site-specific recombination
in zebrafish
Stefan Hans, Jan Kaslin, Dorian Freudenreich, Michael
Brand
Cre recombinase, an integrase from bacteriophage P1 catalyzes DNA strand exchanges in
DNA target sequences called the locus of crossover (lox site). The target site consists of
34 base-pairs, containing two 13 bp inverted repeats flanking an 8 bp core sequence and
its orientation determines the type of recombination that will occur between the lox sites.
Inverted orientation of two lox sites will produce an inversion whereas two co-aligned lox
sites result in excision and circularization of the DNA between the sites.
In order to establish a conditional cell- or tissue-specific Cre/lox system that could be used for
overexpression studies in zebrafish we tested two different approaches:
I. Tissue-specific effector line crossed with already existing hsp:cre or hsp:egfp-cre lines
(Thummel et al., 2005; Feng et al., 2007).
II. Ubiquitous effector line crossed with tamoxifen-inducible Cre lines.
We will present evidence that approach I can not be used due to basal leakiness of the heat
shock promoter which entails a premature site-specific recombination event. However, these
results indicate that very low amounts of Cre recombinase are sufficient and that Cre is highly
efficient in the zebrafish embryo.
Furthermore, our results show that approach II is applicable to zebrafish leading to a sitespecific
recombination event only after the application of tamoxifen. Kinetics and efficiency
of this conditional Cre/lox system will be presented as well as current problems.
Stefan Hans
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Department of Molecular Developmental Genetics
stefan.hans@biotec.tu-dresden.de
www.biotec.tu-dresden.de/
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172
111 O steoclastic activity of bone morphogenetic
protein-2 in lumbar spinal fusion
Christian Hohaus, Yvonne Minkus , Timothy Ganey, Hans-
Jorg Meisel
The use of biologic technologies for the treatment of degenerative spinal diseases is
undergoing rapid clinical and scientific development. Patients with an instability in the spinal
motion segment profit from stabilisation by dorsal fixation in combination with interbody
fusion. BMP- 2 has gained broad acceptance as an adjuvant to spinal fusion when used with
interbody fusion device to improve the ossification process.
The clinical and surgical experience of patients treated for degenerative lumbar spine disease
has been analysed retrospectively. We included 17 patients with neurological deficits causing
by spinal stenosis and instability after degenerative disc disease. All patients underwent a
posterior lumbar interbody fusion in combination with BMP- 2 filled cages. The 3 year follow
up is monitored retrospectively with clinical examination, radiographs and CT-scans.
All patients showed radiographic evidence of fusion at 6 months. There was evidence of
vertebral endplate osteoclastic activity in the radiographs at 3 months in all patients. None
of the patients were clinically symptomatic; events were radiographic findings. All patients
improved and there was evidence of good ossification of the decalcified areas of the vertebrae
at 6 months. Ectopic ossification was found the 3 year CT-scans in the surgical approach and
around the pedicle screws.
The effects of BMPs on osteoclast activity and ectopic ossification have not been widely
investigated. To evaluate this phenomenon, dose dependency, osteogenic activity, and
associated osteoclastic activity attendant with the use of BMP-2 will be studied.
Christian Hohaus
BG-Clinic Bergmannstrost
Department of Neurosurgery
christian.hohaus@bergmannstrost.com
www.bergmannstrost.com
173
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112 Fetal and adult hematopoiesis requires continuous
Mll1 function
Andrea Kranz, Frieder Schwenk, Jost Seibler, Konstantinos
Anastassiadis, A. Francis Stewart
Mll1 (Mixed lineage leukemia) belongs to the SET1 super family catalyzing the methylation
of H3K4 leading to transcriptional activation. Translocations resulting in fusion proteins of
Mll1 with over 50 different partner genes are known to cause acute lymphocytic leukemia
and acute myeloid leukemia. Understanding the role of Mll1 in the hematopoietic system is
therefore of critical importance.
In order to explore the function of Mll1 we are using a conditional knockout mouse line in
which the gene is ablated according to the knock-out-first strategy. A stop cassette inserted
into the first intron truncates the transcript before the second exon. Removal of this cassette
restores wildtype function. Removal of exon 2 by Cre-mediated recombination in this case
with the tamoxifen inducible ROSACreERT2 line results in a frameshift.
Mll1-/- embryos die before E13.5 and show a characteristic hemorrhage in the abdomen
suggesting a fetal hematopoietic defect, which is currently under investigation.
Acute loss of Mll1 in 12-week-old mice after tamoxifen gavage led to rapid death after
approximately 20 days. The heterozygous control mice that were also tamoxifen treated were
healthy beyond 6 months. Analysis of peripheral blood revealed a decreased hematocrit along
with reduced erythrocyte counts in Mll1-/- mice. Thrombocyte and leukocyte numbers were
also decreased. Blood cell morphology was unchanged determined by measurements of mean
cell volume. Inspection of internal organs revealed a reduction in the size of thymus and
spleen. However the architecture of thymus and spleen was generally maintained. Histological
analysis of paraffin embedded decalcified femur sections revealed a decreased cellularity in
the bone marrow. Flush outs of the femur followed by red blood cell lysis and subsequent cell
counts confirmed this drop in cell number. One mechanism which can account for this bone
marrow failure is the reduced expression of several Mll1 target genes namely hox a7, hox
a9 and hox b4. We assume a cell-intrinsic defect, which will be further investigated by bone
marrow transplantation experiments.
Dr. Andrea Kranz
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Center for Regenerative Therapies, Genomics
andrea.kranz@biotec.tu-dresden.de
www.biotec.tu-dresden.de/
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174
113 Plasma membrane mechanics in zebrafish
germlayer progenitor cells
Michael Krieg, Jonne Helenius, Daniel J. Muller, Carl-Philipp
Heisenberg
Gastrulation is a key process in vertebrate development, where the different germ layers
(ectoderm, mesoderm and endoderm) are formed out of a seemingly unstructured precursor
tissue. The germ layer progenitor cells obtain spatial information from the embryonic
environment and begin to migrate to their target niches. Such migratory behaviour requires
continuous cell shape changes to drive the individual progenitor cells forward. Components
that regulate these cell shape changes in zebrafish have been identified earlier (Link, JCS,
2006) and include members of the Ezrin/Radixin/Moesin (ERM) superfamily. ERM proteins
such as Ezrin are known to bind to both the plasma-membrane and actin-cytoskeleton and
are thought to control cell shape by modulating plasma membrane-cytoskeleton adhesion. To
obtain insights into the role of ERM proteins in regulating germ layer progenitor cell shape,
we use atomic force microscopy based dynamic force spectroscopy (DFS) of single tether
extraction experiments. This allows us to probe the membrane mechanics of the different
germlayer progenitor cell types dependent on ERM function. We show that interfering with
ezrin activity gives rise to pronounced plasma-membrane blebbing in vitro and a reduction in
static tether force also expressible as membrane tension (Sheetz, NRMCB, 2001).
Michael Krieg
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Cellular Machines Group
krieg@biotec.tu-dresden.de
www.biotec.tu-dresden.de
175
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114 DNA repair in aged human MSC
Michela Livrea, Alexandra Stolzing
Cellular replicative senescence in somatic cells was described as the loss of proliferation
potential induced by a variety of internal and external stresses and the loss of telomere
sequences is a major contributing factor. However, senescence is heterogeneous and the rate
of the process can markedly differ not only between different cell lineages but also across
the same cell type depending on the tissue origin, cellular turnover and proliferative history.
This heterogeneity is also related to the progressive accumulation of mitochondrial and
cytoplasmatic damages and increased ROS and RNS production. Mesenchymal stem cells
(hMSCs) are a promising cell source for autologous cell therapies, the purpose of the present
study was to determine the effect of aging in hMSCs by analysing its DNA repair capacity,
telomere shortening and telomerase activity.
We compared telomere length and telomerase activity in human fibroblasts to MSC performing
quantitative PCRs. DNA repair capacity was evaluated using Comet assay at different time
points between 1 h and 24 h. To increase the repair capacity, a cocktail containing vitamins
and small chemical compounds was tested in the Comet assay.
Our results show that MSC from older patients posses shorter telomeres and less telomerase
activity compared to MSCs derived from younger patients. Initial DNA damage in MSC
treated with cocktail were decreased in young and aged MSC and the level of apoptosis was
also decreased in these samples. This suggests that for cell therapies, the donor age must be
considered but can be modulated by chemical factors during the culture phase.
Michela Livrea
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Stem Cell Biology
michela.livrea@izi.fraunhofer.de
www.izi.fraunhofer.de
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176
115 Microspherical delivery of a growth factor
Alexander Lochmann, Hagen Nitzsche, Sabrina von Einem,
Elisabeth Schwarz, Karsten Mader
In critical size defects, the administration of Bone Morphogenetic Proteins (BMPs) has been
shown to be a powerful tool to enable the healing progress. However, there is a strong need to
control its release from the drug carrier in order to prevent serious adverse effects attributed
to high doses of the growth factor.
In order to control the release of rhBMP-2 from an injectable in situ-forming implant,
microspheres were produced. The objective of this study was the production and evaluation
of these particles. They were assessed for their ability to entrap the growth factor and release
it in different profiles. Microspheres of three different polymers, hydrophobic or amphiphilic,
were produced by a double emulsion . solvent evaporation method. They were found to be
round-shaped and smooth upon scanning electron microscopical investigations, the resulting
size differed just insignificantly.
After fluorescent labelling, the distribution of labelled rhBMP-2 within the microspheres was
investigated with confocal laser scanning microscopy, which showed a dependency on the
polymer used and on the presence of polyethylene glycol in the inner phase. Investigations on
the entrapment efficiency of fluorescence labelled rhBMP-2 were carried out by a difference
method and showed higher incorporation yields for the amphiphilic polymers.
Alexander Lochmann
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Regulatory Molecules and Delivery Systems
alochmann@trm.uni-leipzig.de
www.trm.uni-leipzig.de
177
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116 Electrospun poly(ƒÃ-caprolactone) (PCL) microfiber
meshes with predefined fiber diameters
Tina Loth, Markus Manhardt, Kristina Ambrosch, Michaela
Schulz-Siegmund, Michael C. Hacker
Electrospinning has gained increasing interest as a method to produce biodegradable
micro- and nanofiber meshes because of its uncomplicated setup and versatility. In addition,
electrospun meshes structurally mimic natural extracellular matrix and therefore hold promise
as scaffolds for tissue regeneration supporting cell attachment, proliferation and migration.
Although a typical electrospinning setup appears simple, the factors that control fiber size and
morphology are manifold and their interaction is complex. Numerous parameters concerning
properties of the polymer solution, feeding rate, voltage and geometrical set up as well as
ambient conditions have considerable influence.
This study accompanied the set up of an electrospinning apparatus in our lab. Based on
literature reports we systematically varied polymer solvent composition, flow rate, distance
between needle and collector, collector size and voltage for 12% PCL solutions. On the basis
of these findings, the process parameters were adapted to yield fibers of different predefined
sizes. Analysis of fiber diameters was done by scanning electron microscopy (SEM). In
summary, we succeeded in producing microfiber meshes with different predefined fiber
diameters between 2 ƒÊm and 10 ƒÊm. The meshes were characterized by porosities of 80 . 85%
and 85 . 90% as determined by gravimetric measurement and ethanol intrusion, respectively.
Pore size distributions were analyzed using mercury porosimetry and found to correlate with
fiber diameter. Pore sizes suitable for cell seeding were achieved.
Tina Loth
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
loth@rz.uni-leipzig.de
www.trm.uni-leipzig.de
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178
117 Surface modification of medical CoCr alloys by a
thermochemical process
Johanna Lutz, Stephan Mandl
CoCr-alloys are common materials for medical implants. Due to their good mechanical
properties and biocompatibility they are mainly used for prosthetic replacements and
cardiovascular applications. Nevertheless, revision rates of up to 5 . 15%, depending on the
application, are still encountered in clinical use. For minimising these revision rates a costeffective
novel thermochemical surface treatment is employed.
Using energetic ion implantation of either nitrogen or oxygen, the formation of a nanostructure,
hard and wear resistant surface layer is observed. The resulting increase in surface hardness
by a factor of 3 . 5 to 20 . 25 GPa is connected with an improved wear resistance by a
factor of up to ten. Thus, a strongly reduced formation of nanoscopic wear particles with
a very large surface area, prone to corrosion attacks, is observed. The corrosion rate of the
modified surface layer is slightly increased, depending strongly on the process conditions.
However, in conjunction with the wear reduction, a global reduction of the release rate of
Co2+ is realized, allowing a beneficial level of Co2+ in the surrounding tissue. At the same
time, the nanostructured surface will lead to an enhanced cell adhesion and bioactivity.
Johanna Lutz
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Tissue Engineering and Materials Science
jlutz@trm.uni-leipzig.de
www.trm.uni-leipzig.de
179
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118 Regeneration of chronic osteochondral defects
using autologous mesenchymal stem cells in a
sheep model
Bastian Marquass, Pierre Hepp, Robert Richter, Steffanie
Schmidt, Frank Stein, Augustinus Bader, Christoph Josten,
Matthias Zscharnack, Ronny Schulz
The aim was to investigate the reconstruction of a chronic, osteochondral defect of a knee
joint in sheep by using autologous bone marrow-derived mesenchymal stem cells (MSCs)
embedded in a collagen I hydrogel. The issue of whether chondrogenic differentiation of the
MSCs in vitro bears an influence on the regeneration in vivo is also to be addressed.
First, osteochondral defects were created in the medial femoral condyle of 18 sheeps. Isolated
and expanded MSCs were seeded with 4*105 cells/ml in the collagen-I-Matrix and cultivated
for 14 days. One part of the MSC gels was differentiated with chondrogenic medium +10 ng/
ml TGF-s3, whilst the other part of the gels was cultivated with DMEM and 10% autologous
serum. The implantation of the constructs then followed. Explantation and histological
scoring took place after 6 and 12 months. The untreated defects and implanted cell-free gels
were used as control.
An succesfull in-vitro predifferentiation could be demonstrated. After 6 months the
predifferentiated constructs showed the highest histological scoring with 14,3 for ICRS and
17,4 for the OLDriscoll Score. The not predifferentiated MSC and the control groups were
significant lower. Similar results were found after 12 months.
The collagen gel implants seeded with chondrogenically differentiated MSC gels led to
a partial hyaline structure of the regeneration matrix after 6 and 12 months. These results
suggest that chondrogenic differentiation in vitro might be beneficial for regeneration of focal
cartilage defects compared to transplants with undifferentiated MSCs.
Dr. Bastian Marquass
Universitat Leipzig
University Hospital
Department of Trauma, Reconstructive and Plastic
Surgery
Bastian.Marquass@medizin.uni-leipzig.de
www.chirurgie1.uniklinikum-leipzig.de/
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180
119 Intervertebral disc repair using adipose tissuederived
stem and regenerative cells: experiments
in a canine model
Hans Jorg Meisel, Timothy Ganey, Christian Hohaus,
William Hutton, Tim Moseley, Marc Hedrick, Brian Strem
Disc injury can lead to disc degeneration. Our goal was to test the hypothesis that repair of a
damaged disc is possible using autologous adipose tissue derived stem and regenerative cells
(ADRCs).
Twelve dogs underwent a partial nucleotomy at three lumbar levels (L3-L4, L4-L5, L5-L6);
adjacent levels served as non-operated controls. At 6 weeks post-op adipose tissue derived
stem and regenerative cells (ADRCs) were isolated. The three experimental discs were
randomized to receive: 1) ADRCs in hyaluronic acid carrier (Cells/HA); 2) HA only; or 3) No
Intervention. Assessments were made using MRI, radiography,histology and biochemistry.
The animals were euthanized at 6 months, and at 12 months.
Repair in this study was specifically demonstrated through histology and biochemical analysis.
Disc levels receiving ADRCs more closely resembled the healthy controls as evidenced in
matrix translucency, compart-mentalization of the anulus, and in cell density within the
nucleus pulposus. Matrix analysis for Type-II collagen and aggrecan demonstrated evidence
of a statistically better regenerative stimulation to the disc provided by ADRCs.
Autologous adipose tissue derived stem and regenerative cells, as used in this disc injury
model, were effective in promoting disc regeneration, as evidenced by disc matrix production
and overall disc morphology.
Prof. Dr. Hans Jorg Meisel
BG-Clinic Bergmannstrost
Department of Neurosurgery
neurochirurgie@bergmannstrost.com
www.bergmannstrost.com
181
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120 Conditional Mutagenesis of Histone
Methyltransferase Mll2 in Neural Stem Cells
Katrin Neumann, Maria Rostovskaya, Sandra Lubitz, Andrea
Kranz, A. Francis Stewart, Konstantinos Anastassiadis
Post-translational modifications of histone tails act as epigenetic signals maintaining gene
expression patterns during cellular development. Polycomb and trithorax group (trxG)
methyltransferases counteract each other by repressing or preserving gene expression. Mll2
(Wbp7) is a mammalian member of the trxG involved in maintaining gene expression by
methylating lysine 4 of histone 3.
The constitutive Knock-out of Mll2 in mice is embryonic lethal before E11.5 due to widespread
developmental defects and Mll2 knock-out embryonic stem (ES) cells display differentiation
defects. As differentiation to neurons was most severely impaired, this study focuses on the
role of Mll2 in mouse Neural Stem (NS) cells.
NS cells were either generated from ES cells or fetal forebrain. Both sources produced
comparable results. Due to embryonic lethality, a conditional knock-out of Mll2 was performed
using the 4-OH-tamoxifen inducible Cre/loxP site-specific recombinase system. We found
that self-renewal of NS cells was not affected by Mll2 knock-out, but Mll2 deficient cells
exhibited a proliferative defect due to increased apoptosis. During in vitro differentiation
most of the Mll2 deficient cells died. Surviving cells generated only few astrocytes and no
mature neurons. Thus, differentiation deficiency of Mll2 knock-out cells seems to be a general
effect not restricted to ES cells.
These findings indicate a redundancy of Mll2 for maintaining gene expression patterns
in NS cells. However, Mll2 seems to be essential for differentiation events when histone
modification patterns have to be altered.
Katrin Neumann
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Center for Regenerative Therapies, Genomics
katrin.neumann@biotec.tu-dresden.de
www.crt-dresden.de
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182
121 Peptide vectors for siRNA delivery into cells
Ines Neundorf, Anja Tennemann, Robert Rennert
For efficient DNA or RNA delivery nucleic acids have to overcome a series of
extracellular and intracellular barriers that can limit their efficiency. Several techniques
have been developed to address efficient DNA or RNA delivery, including nonviral
methods (by using physical and chemical means) or viral delivery vectors.
However, due to several drawbacks of these strategies like limited efficiencies or
severe cytotoxic effects there is need of the development of alternative vector tools.
One additional possibility to transport oligonucleotides beyond cellular membranes might be
the use of so-called cell-penetrating peptides (CPP). CPP are non-viral transfection agents,
acting as shuttles for a controlled cellular delivery of therapeutics. Several CPP have been
designed and tested so far. We focus on the development of peptide carriers derived from
human calcitonin (hCT). HCT is a peptide hormone that consists of 32 amino acids and is
secreted by the thyroid gland. HCT was found to possess membrane-translocating properties,
since nasal therapeutic application was found to be as effective as i.v. injection. Recently,
truncated fragments of hCT have been synthesized and characterized as efficient transporters
for peptides, proteins and small molecules.
In this work we focused on the application of our peptide carriers for siRNA delivery. We
present data showing that these shuttles work as alternatives to polymer and lipid-based
siRNA delivery systems.
Dr. Ines Neundorf
Universitat Leipzig
Faculty of Biosciences, Pharmacy and Psychology
Institute of Biochemistry
neundorf@uni-leipzig.de
www.biochemie.uni-leipzig.de/agbs
183
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122 PEEK-WC-PU membranes for expansion of rat
embryonic liver cells
Sanja Pavlica, Antonella Piscioneri, Frank Peinemann,
Angela Hennig, Javorina Milosevic, Stefania Laera, Piero
Favia, Loredana DeBartolo, Augustinus Bader
Biomaterials play an important role in directing tissue growth and may provide another
tool to manipulate and control stem cell behavior, having a significant impact on the fields
of regenerative medicine and tissue engineering. Herein, we designed and developed new
bioactive membranes to be used for the expansion of rat embryonic liver cells.
New modified polyetheretherketone PEEK-WC membranes were prepared in hollow fibre
configurations, by phase inversion technique. Their surface was modified by means of different
plasma processes, introducing amino group. The performance of the developed biomaterials
was evaluated by analysis of the expression of the liver specific functions of cells cultured
in the 6-well bioreactor. Liver progenitors on the membranes exhibited higher functional
activities compared to those cultured on conventional plates as demonstrated by higher
albumin and urea production. They showed gene expression of alpha-fetoprotein and albumin
in a time-dependent manner of the hepatic differentiation process. LDH assay revealed that
a high number of viable liver stem cells attached to the membranes. Unexpectedly, liver
progenitors cultured on membrane bioreactors had higher telomerase activity than ones in the
plates. Further, FACS analyses showed that cells grown on membranes had longer G1 phase
while S phase was shortened. Thus, membrane bioreactors are able to sustain the same in vivo
liver functions in vitro and to allow the expansion of stem cells.
Sanja Pavlica, Ph. D.
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Department of Cell Techniques and Stem Cell Biology
sanja.pavlica@bbz.uni-leipzig.de
www.uni-leipzig.de/~bader/
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184
123 Dynamic Coupling of Pattern Formation and
Morphogenesis in the Developing Vertebrate
Retina
Alexander Picker, Florencia Cavodeassi, Anja Machate,
Sabine Bernauer, Stefan Hans, Gembu Abe, Koichi
Kawakami, Stephen Wilson, Michael Brand
During embryonic development, pattern formation must be tightly synchronized with tissue
morphogenesis to coordinate the establishment of spatial cell identities with cell movements.
In the vertebrate retina, patterning along the dorsal-ventral and nasal-temporal (anteriorposterior)
axis is required for correct spatial representation in the retinotectal map. But
it is unknown how specification of axial cell positions in the retina can occur during the
complex process of early eye morphogenesis. Studying zebrafish embryos, we show how
the morphogenetic tissue re-arrangements during eye evagination result in progenitor cells
in the nasal half of the retina primordium being brought into proximity to the sources of
three fibroblast growth factor molecules, Fgf8/3/24, outside the eye. Triple mutant analysis
shows that this combined Fgf signal fully controls nasal retina identity by regulating the nasal
transcription factor Foxg1. Surprisingly, nasal-temporal axis specification occurs very early
along the dorsal-ventral axis of the evaginating eye. By in vivo imaging GFP-tagged retinal
progenitor cells in transgenic embryos, we find that subsequent eye morphogenesis requires
gradual tissue compaction in the nasal half and directed cell movements into the temporal
half of the retina. Balancing these processes drives the progressive alignment of the nasaltemporal
retina axis with the anterior-posterior body axis and is controlled by a feed-forward
effect of Fgf signaling on Foxg1-mediated cell cohesion.
Alexander Picker
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Molecular Developmental Genetics
alexander.picker@biotec.tu-dresden.de
www.biotec.tu-dresden.de
185
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124 Establishment of risk analysis of bone replacing
scaffolds
Heike Schneider, Kathleen Schrock, Manja Kamprad,
Michaela Schulz-Siegmund
In the field of regenerative and translational medicine it is a main goal to find and characterize
new biodegradable and biomimetic materials that support endogenous repair of bone tissue
for improved physiological functions.
Here, we present a method to assess material biosafety in vitro prior to testing in animal
models. Therefore, in the present study different methods for evaluation of biological risk
factors of 3D materials including immunological compatibility, thrombolytic and haemolytic
activities were established. 3D poly(lactic-co-glycolic acid) (PLGA) cell carriers with high
porosity and pore interconnectivity served as scaffold material.
Immunological compatibility was tested by culturing peripheral blood mononuclear cells
in presence of the material. The activation of lymphocytes was examined by measuring
concentrations of the cytokines IFN-gamma, IL-2, TNF-alpha, IL-4, IL-5 and IL-10 in culture
supernatant by specific cytometric bead arrays. Thrombolytic activity was determined by
FACS analysis of CD62P, an activation dependent surface marker, after incubation with the
material. Furthermore, haemolytic activity was analysed by measuring optical density of free
haemoglobin in the supernatant of whole blood incubated with scaffold material.
In summary, PLGA scaffolds revealed no significant differences in cytokine secretion
of lymphocytes compared to control cultures without scaffold material, indicating the
immunological compatibility of PLGA. Furthermore, no haemolytic activity could be
observed. A slight increase in the amount of CD62P, however, was detected after incubation
with the scaffold material.
Heike Schneider
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
hschneider@trm.uni-leipzig.de
www.trm.uni-leipzig.de
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186
125 O steogenic differentiation of human adipose
tissue-derived stem cells: Are the standard
ascorbate-2-phosphate concentrations too high?
Hellen Schneider, Michael Hacker, Michaela Schulz-
Siegmund
Adipose tissue-derived stem cells (ADSCs) offer a high potential for use in regenerative
medicine of bone. There are different protocols for the osteogenic differentiation of ADSCs
concerning ascorbate-2-phosphate (A2P) concentrations suggesting 15 to 150 ƒÊM, next
to other supplements. We investigated the influence of different amounts of A2P during
differentiation to find optimal conditions in our lab for proliferation and mineralization of
the cell culture.
Adipose tissue was obtained by lipoaspiration and ADSCs were isolated using collagenase
digestion. Cells from two patients (male, female) were grown in DMEM with 10% FBS
over 3 passages and finally cryopreserved. For differentiation, cells from passages 4 and 5
were seeded in 12-well plates (5000 cells/cm2). We tested 4 different A2P concentrations (10
ƒÊM, 50 ƒÊM, 150 ƒÊM, 250 ƒÊM) supplemented to DMEM/10 % FBS based osteogenic media
containing glycerol-2-phosphate (10 mM). The different A2P conditions were combined with
2 levels of dexamethasone (DEX) (10, 100 nM). Calcium (Ca) content was determined by
a colorimetric assay on days 28 and 36. Cell numbers were measured using Pico-Green on
day 28.
Independent of DEX concentration, the gender of the donor or the number of passages we
reproducibly found decreased calcium deposition with increasing A2P concentrations at day
36, suggesting improved osteogenic differentiation with A2P concentrations as low as 10
ƒÊM compared to standard protocols often involving concentrations of 50 ƒÊM or higher. Cell
numbers, however, were lowest in samples with 10 ƒÊM which may be a consequence of
higher mineralization.
Hellen Schneider
Universitat Leipzig
Institute of Pharmacy
Department of Pharmaceutical Technology
Hellen@rz.uni-leipzig.de
www.uni-leipzig.de/~pharm/phatech/
187
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126 Electrospinning parameters critical for the
generation of poly(ƒÃ-caprolactone) (PCL)
nanofibers
Maximilian Sperlich, Markus Manhardt, Michaela Schulz-
Siegmund, Michael Hacker
In the last years electrospinning has gained increasing interest as a versatile method to produce
non-woven fiber meshes with fibers in the low micrometer and nanometer scale. This study
deals with evaluation and optimization of process parameters for production of nanofibers. A
standard electrospinning setup consisting of a polymer solution (PCL in chloroform/DMF)
containing syringe, a blunt needle, a copper ring electrode and a collector plate was used.
In a previous study, parameters for the fabrication of meshes with predefined homogenous
fiber diameters between 3 and 9 ƒÊm were established. The effects of PCL concentration,
solvent properties, flow rate, applied voltage and distance between copper ring and collector
plate on fiber diameter were identified. Approaching the nanometer scale the process becomes
increasingly critical and prone to beading, which describes the formation of large beads along
the developing fibers. In order to identify suitable conditions, polymer concentration and
solvent composition, which were determined as decisive parameters were systematically
varied. Reducing polymer concentration in a chloroform/DMF (50:50) solution from 15 to
5% led to decreasing fiber diameters until beading occurred below a defined concentration
(Cb). Below Cb, beading increased with decreasing polymer concentration. Further solvent
compositions were investigated and the effects of solvent composition and polymer
concentration on fiber diameter, beading and Cb are described. We also intent to correlate the
findings with the viscosity and electrical properties of the electrospun solutions.
Maximilian Sperlich
Universitat Leipzig
Institute of Pharmacy
Department of Pharmaceutical Technology
sperlich@uni-leipzig.de
www.uni-leipzig.de/~pharm/phatech/
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188
127 Controlled formation of embryoid bodies in
bioreactors for reproducible differentiation
initiation of mouse and primate embryonic stem
cells
Susanne Trettner, Alexander Seeliger, Nicole I. zur Nieden
Many in vitro systems have been developed to steer embryonic stem cell differentiation into
specific cell types, including hepatocytes, neural precursors, cardiomyocytes and osteoblasts.
Typically, differentiation is initiated by the formation of embryoid bodies (EBs). Methods to
generate EBs have been improved over the years, including single cell suspension cultures
in low adherent culture plates, EB formation in multi-well plates and the hanging drop
protocol. The first method leads to EBs, which are different in size and shape. Although the
advantage of the other two methods is the more uniform size distribution of EBs, they are
labor and material intensive. In order to apply ESCs in high-throughput screens, such as for
the evaluation of toxic properties of pharmacological compounds, differentiations need to
be cost-effective and fast. Here, the first step is to scale up and reduce the costs of the initial
stage of differentiation . the EB formation. Similarly, the reproducibility of EB formation has
to be guaranteed in order to standardize these screens. In the present study, we generated EBs
of mouse and primate embryonic stem cells in scalable, controlled suspension bioreactors and
examined the influence of different agitation rates to the formation of EBs.
Susanne Trettner
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Stem Cell Technology
susanne.trettner@izi.fraunhofer.de
www.izi.fraunhofer.de
189
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128 Formation of MMP cleavable hydrogel materials
for the development of novel biohybrid system
for tissue engineering
Mikhail Tsurkan, Kandice Levental, Petra Welzel, Milauscha
Grimmer, Andrea Zieris, Woranan Panyanuwa, Uwe
Freudenberg, Carsten Werner
The development of artificial materials that mimic extracellular matrices has a great potential
to provide the mechanical support and chemical signals that direct cell adhesion, proliferation
and differentiation. This presentation outlines current progress in the development of
biomaterial scaffolds toward fabrication a novel modular system for tissue engineering.
In this approach, a new class of biodegradeble materials was prepared by combining
Polyethylene glycol (PEG), heparin and matrix metalloproteinase (MMP) cleavable peptide
molecules into a well defined hydrogen network. A MMP cleavable peptide sequence was
included into the PEG-Heparin hydrogel network in order to introduce a dynamic reciprocal
responce of the material to the cell prolifiration (expansion). In first step of this approach,
MMP cleavable peptide GPQGIWGQ is directly attached to star PEG by applying click
chemistry. The formed sPEG-peptide hybrid is then coupled to heparin molecules through
standard EDC/NHS chemistry in order to form a hydrogel network. The sensitivity of the
material to matrix metalloproteinases is dependant on the presence of cleavable crosslinking
with in the hydrogel material and can be controled through variation of the peptide sequence.
The hydrogel can be fine-tuned in terms of mechanical (swelling, stiffness, meshsize) and
biofunctional (incorporation of growth factors and adhesive ligands) characteristics. These
series of biomaterials with defined chemical modifications and topographies are screened for
their ability to trigger extra cellular matrix (ECM) reorganisation.
Dr. Mikhail Tsurkan
Leibniz Institute of Polymer Research Dresden
Center for Regenerative Therapies
Max Bergmann Center of Biomaterials
tsurkan@ipfdd.de
www.mbc-dresden.de
Tisue and Cel Enginering
190
129 3D-cardiomyocyte structures generated from
murine embryonic stem cells . A novel tool for
drug screening on microcavity arrays
Silvia Vinz, Randy Kurz, Andrea A. Robitzki
It is well known that three-dimensional cell or tissue structures are closer to the in vivo situation
than cell monolayers. For that reason drug testing with such artificial structures could provide
more reliable data. An important criterion for the exclusion of a drug candidate is a possible
influence on the electrophysiology of the heart. Primary cardiomyocytes from neonatal rats
can be used to form three-dimensional beating structures where such effects can be detected,
but due to the preparation procedure they can not be standardised. An alternative source for
these structures are embryonic stem cells, which can differentiate into cardiomyocytes under
appropriate conditions via the formation of aggregates called embryoid bodies (EBs).
For the generation of 3D-cardiomyocyte structures we use the murine embryonic stem cell
line ES-D3. Following the formation of EBs from the stem cells they are left in a resting
suspension culture for further differentiation to cardiomyocytes. After differentiation EBs
that show spontaneous contractions are selected for electrophysiological measurements. For
this purpose the EBs are placed in the cavities of a microcavity chip that was developed in our
group. Via extracellular recording of action potentials with gold electrodes on the walls of the
cavities the contraction rates of the EBs can be determined. So we could show the influence
of cardioactive drugs on the electrophysiology of these differentiated cardiomyocytes. Thus
these 3D-cardiomyocyte structures in combination with microcavity arrays are a novel
promising tool for in vitro drug screening.
Silvia Vinz
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
silvia.vinz@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
191
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130 Non-invasive acoustical imaging of mesenchymal
stem cells
Moritz von Buttlar, Esam Ahmed Mohamed, Amro
Abdelrahman, Albert Kamanyi, Wolfgang Grill
Time-lapsed imaging of mesenchymal stem cells in culture is performed with a phase contrast
scanning acoustic microscope operating at 1.2 GHz. The observed mesenchymal stem cells
are adherent to a cover glass inside a life support system with gas and temperature control.
Images of 1024 times 512 pixels acquired with the acoustic microscope in magnitude and
phase contrast are collected with 1.5 frames per minute. The averaged power of acoustic
waves delivered to the cells and the surrounding fluid of 1 ƒÊW and the extremely low
quantum energy of acoustic phonons at 1.2 GHz do both not harm the living cells and allow
non-invasive imaging for extended times. For the chosen substrate with high reflectivity the
phase contrast is dominated by the difference of the velocity of acoustic waves in the cell
with respect to the surrounding fluid. The variation of the time-of-flight of the acoustic waves
derived from the phase contrast and the extinction, both observed confocal in reflection, are
the basis for subsequent data and image processing implemented to deliver a pseudo threedimensional
(time laps) movie of the cells moving on the substrate within the observation
area of 0.3 mm times 0.2 mm. The acoustic microscope is combined with a confocal laser
scanning microscope (ZEISS LSM510) for simultaneous multi-contrast imaging. Cell
locomotion, endocytosis, and exocytosis have been observed based exclusively on noninvasive
monitoring for extended times with the developed combined scanning confocal
acoustic and laser microscope.
Support by the BMBF, grant 0313836 (MS CartPro), is gratefully acknowledged.
Moritz von Buttlar
Universitat Leipzig
Faculty of Physics and Earth Science
Institute of Experimental Physics II
vbuttlar@physik.uni-leipzig.de
www.uni-leipzig.de/~fko
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192
131 Bioreactor with integrated monitoring and
mechanical stimulation for cartilage tissue
engineering by collagen scaffold associated
Mesenchymal Stem Cells
Erik von der Burg, Moritz von Buttlar, Wolfgang Grill
A novel bioreactor with integrated non-destructive and non-invasive monitoring for the in
situ measurements of ultrasonic and rheological parameters has been developed. The device
is suitable for the production of collagen scaffolds with bone marrow derived Mesenchymal
Stem Cells (MSC) for the regeneration of articular cartilage for transplantation. Actuators
for physiological stimulation in the aseptic environment of our bioreactor are included in
order to enhance perfusion and chondrocytic differentiation. From the measurement with the
integrated load cells during deployment of physiological stimuli rheological parameters of the
scaffold during the two weeks long cultivation period of the scaffolds in the bioreactor can
be monitored continuously. Additionally implemented ultrasonic monitoring in transmission
allows further mechanical sample characterization. By a continuous measurement of the
longitudinal sound velocity in the monitored samples under variable compressional stress
and by ultrasonic characterization of the culture medium, information on the consistency
and permeability of the scaffold for the culture fluid can be derived. This information can
be employed to drive a feedback controlled stimulation program in the bioreactor. This is
currently used to stabilize the initial growth phase of the scaffolds with the seeded MSCLs.
Presented are first results of the rheological and ultrasonic monitoring of scaffolds with ovine
MSCLs for extended times during the culturing phase.
Erik von der Burg
Universitat Leipzig
Faculty of Physics and Earth Science
Institute of Experimental Physics II
evdb@uni-leipzig.de
www.uni-leipzig.de/~fko/
193
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132 Potential ageing effects in long-term cultured
mouse neurospheres
Vladimir Vukicevic, Anna Jauch, Timo C. Dinger, Linda
Gebauer, Veronika Hornich, Stefan R. Bornstein, Monika
Ehrhart-Bornstein, Albrecht M. Muller
Neural cells are isolated from forebrain of 14.5 days old mouse embryos. In selective
conditions these cells cluster forming sphere-like structures . neurospheres. Neurospheres
are heterogeneous structures containing 2 . 5% of neural stem cells (NSCs). In fact, it is
generally assumed that progenitors and stem cells remain intact in long-term culture and
therefore convenient for potential transplantation therapy. In spite, we hypothesized that
potential ageing effects of overall sphere certainly will impact the fraction of stem cells and
progenitors. Therefore, this assumption was tested exploring distinct aspects of ageing in
long-term NSC culture.
Potential alterations that might occur due to ageing were monitored within 1 . 16 weeks
of culturing. Initially, tremendous structural and numerous chromosomal aberrations were
observed upon 16 weeks of culturing such as chromsome 1 gain. This was accompanied
with upregulated Hmga2 which controls self-renewal. Indeed, neural cells unexpectedly selfrenewed
displaying a capacity to form spheres after 16 weeks of culturing even when seeded
at low cell density (10 . 20 cells/well). Increased capacity to form spheres was accompanied
with significantly decreased ability to differentiate into neural lineages. Telomere shortening
is considered as indication of ageing. Significant telomere length erosion was found in
transition between 4 and 5 weeks of culturing.
Genetic instability and diminished differentiation capacity seem to be a consequence of long
term culturing implying potential transformation.
Dr. Vladimir Vukicevic
Technische Universitat Dresden
Carl Gustav Carus University Clinic, Medical Clinic III
Molecular Endocrinology
vladimir.vukicevic@uniklinikum-dresden.de
www.tu-dresden.de
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194
133 Spatially confined cell growth
Ronald Werner, Torsten Koal, Heinz Georg Jahnke, Andrea
A. Robitzki, Tilman Butz, Tilo Reinert
Spatially confined cell growth is very useful to study network behaviour, cellular motility
or cell-cell communication. Therefore we wrote structures in PMMA, SU8 and agar. The
experiments show that agar is an excellent material for confined cell growth. Agar is a waterinsoluble
polysaccharide which prevents cell adhesion. The proton beam irradiation destroys
the polysaccharide into water-soluble monosaccharides. With this technique compartments
in multi-electrode-arrays were produced. They allow investigations of the irradiation induced
Bystander Effects with exclusion of cell communication by Gap-junctions.
First Petri dishes were cleaned and disinfected. A solution of 0,65% AGAR (w/w) in distilled
deionised water, was heated to boiling point at 95‹C. 2 ml of this solution was applied to
the Petri dish. After 1 minute the agar solution is removed leaving behind a thin film. This
film solidifies after 12 h. The agar is subsequently irradiated by a focused beam with energy
of 2.25 MeV protons. With proton beam writing we can create any possible 2 dimensional
structure with a maximum size of 2~2 mm2 with an accuracy of < 2 ƒÊm. After the irradiation
the Petri dish is developed in PBS solution and disinfected with 70% alcohol. EaHy cells
then were seeded into the Petri dish. After 2 hours the sample is washed twice with growth
medium and the rest of the cells were removed. Only cells in the written structure, where the
agar was removed adhere on the Petri dish.
Ronald Werner
Universitat Leipzig
Faculty of Physics and Earth Science
Institute of Experimental Physics II
r.werner@physik.uni-leipzig.de
www.uni-leipzig.de/~nfp
195
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8. Neuromedicine
Posters

199
Neuromedicine
134 Improved brain outcome by autologous bone
marrow-derived mononuclear cells (BMCs)
intravenously given 24 hours after stroke in
sheep as imaged by PET
Henryk Barthel, Johannes Boltze, Christiane Boltze, Udo
Grosmann, Magnus Kluge, Andreas Schildan, Anita Seese,
Frank Emmrich, Uwe Gille, Osama Sabri
In acute ischemic stroke, there is new optimism that cell therapies could overcome the
therapeutic dilemma evident for most patients. Recently, a new large animal model has been
introduced for testing of new stroke therapies close to the human situation. In the present
study, we investigated by means of positron emission tomography (PET) the effect of BMCs
on stroke outcome in this new sheep model.
Adult Merino rams underwent permanent middle cerebral artery occlusion (pMCAO). 24 h
after pMCAO, 9 sheep were i.v. administered with 4 . 6 x 106 MBCs per kg bodyweight.
5 untreated sheep served as controls. In all sheep, cerebral blood flow (CBF) PET ([15O]
H2O, ECAT EXACT HR+) was carried out 20 h, 2 wks and 6wks after pMCAO. Further,
6 wks after pMCAO, the cerebral metabolic rate of glucose (CMRGlu) was measured with
[18F]FDG and PET. The PET imaging was paralleled by stroke-specific MRI and scoring of
neurological symptoms.
2 wks after pMCAO, no treatment effects on the CBF were observed. In contrast, after 6 wks
a decrease of the CBF deficit volumes was observed only in the treatment group (73}9% as
compared to baseline vs. 135}9% for controls, p=0.002). Also, the CMRGlu deficit volumes
6 wks after pMCAO were smaller for the treatment as compared to the control group (4.6}0.8
vs. 10.7}0.9ml, p=0.002). In parallel to these PET findings, the final degree of stroke-related
brain atrophy in MRI and neurological symptom scores were lower (p<0.01) for the treatment
group.
Our PET results provide evidence for a beneficial effect of BMCs on brain outcome in acute
stroke, encouraging clinical trials.
Dr. Henryk Barthel
Universitat Leipzig
Faculty of Medicine
Department of Nuclear Medicine
henryk.barthel@medizin.uni-leipzig.de
http://nuklmed.uniklinikum-leipzig.de/
Neuromedicine
200
135 Saffron extract inhibits the glutamatergic synaptic
transmission on rat cortical neurones
Frauke Berger, Andreas Hensel, Matthias Lechtenberg,
Karen Nieber
Crocus sativus L., commonly known as saffron, is a small perennial plant from the Iridaceae
family. It has been shown that an ethanolic (80 vol.-%) saffron extract interacts with the
PCP binding site of the NMDA receptor. The aim of the present study was to examine the
influence of the ethanolic saffron extract (CSE) on the glutamatergic synaptic transmission
in rat cortical brain slices. Postsynaptic potentials (PSPs) were elicited by electrical field
stimulation in pyramidal cells of the cingulate cortex and recorded using intracellular placed
microelectrodes. PSPs are caused by glutamate released from presynaptic terminals which
activates postsynaptic NMDA and non-NMDA receptors. Additionally, glutamate induces
a membrane depolarisation when applied directly to the brain slices. CSE (100 ƒÊg/ml)
decreased the glutamate-induced membrane depolarisation and inhibited the evoked PSPs.
To specify the receptor subtype involved in the inhibition, the non-NMDA component of the
PSPs was separated by application of the NMDA receptor antagonist APV (10 ƒÊM) and the
NMDA component by application of the non-NMDA receptor antagonist CNQX (10ƒÊM).
Under these conditions CSE (100 ƒÊg/ml) decreased the isolated non-NMDA and NMDA
components of the PSPs. Our results indicate that CSE, beside of its antagonistic effect at
NMDA receptors, also acts antagonistic at non-NMDA receptors. To clarify which of the
non-NMDA receptors contribute to the effect AMPA (1 ƒÊM) was applied. CSE (100 ƒÊg/ml)
did not influence the AMPA-induced membrane depolarisation. This result indicates that CSE
has no antagonistic effect at AMPA receptors.
Frauke Berger
Universitat Leipzig
Institute of Pharmacy
Department fur Pharmacology and Sciences
fberger@uni-leipzig.de
www.uni-leipzig.de/~pharm/phfn
201
Neuromedicine
136 Vascular endothelial growth factor (VEGF) affects
processing of amyloid precursor protein and
ƒÀ-amyloidogenesis in brain slice cultures derived
from transgenic Tg2576 mouse brain
Susanne Burger, Monika Noack, Elena Kouznetsova, Yousef
Yafai, Ludmil Kirazov, Evgeni Kirazov, Reinhard Schliebs
The upregulation of the angiogenic vascular endothelial growth factor (VEGF) in brains of
Alzheimer patients in close relationship to ƒÀ-amyloid (AƒÀ) plaques, suggests a link of VEGF
action and processing of the amyloid precursor protein (APP). To reveal whether VEGF may
affect APP processing, brain slices derived from 16-month-old transgenic Tg2576 mice were
exposed with 1ng/ml VEGF for 6, 24, and 72 hours, followed by assessing cytosolic and
membrane-bound APP expression, level of both soluble and fibrillar AƒÀ-peptides, as well as
activities of ƒ¿- and ƒÀ-secretases in brain slice tissue preparations.
VEGF exposure of brain slices for 6 h reduced the formation of soluble, SDS extractable AƒÀ(1-
40) and AƒÀ(1-42) as compared to brain slice cultures incubated in the absence of any drug,
while the fibrillar AƒÀ peptides did not change significantly. This effect was less pronounced
24 h after VEGF exposure, but was no longer detectable when slices were exposed by VEGF
for 72 h, indicating adaptive responses to chronic VEGF exposure. The VEGF-mediated
reduction in AƒÀ formation was accompanied by a transient decrease in ƒÀ-secretase activity
peaking 6h after VEGF exposure. Incubation of AƒÀ preparations obtained from Tg2576 mouse
brain cortex, in the presence of VEGF slightly decreased the fibrillar content with increasing
incubation time up to 72 h, suggesting a role of VEGF in inhibition of AƒÀ fibrillogenesis. The
data demonstrate that VEGF may affect APP processing, at least in vitro, suggesting a role of
VEGF in the pathogenesis of AlzheimerLs disease.
Supported by Alzheimer Forschung Initiative (AFI) to R. Schliebs.
Susanne Burger
Universitat Leipzig
Paul-Flechsig-Institute for Brain Research
Devision of Molecular Imaging
Susanne.Buerger@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
Neuromedicine
202
137 Isolation of Chromaffin Progenitor Cells from
Adult Adrenal Medulla
Kuei-Fang Chung, Flavie Sicard, Vladimir Vukicevic, Linda
Gebauer, Wieland B. Huttner, Stefan R. Bornstein, Monika
Ehrhart-Bornstein
Chromaffin cells of the adrenal medulla are neural crest derived cells that are able to
proliferate throughout life, unlike the closely- related sympathetic neurons. It is suggested
that a subpopulation of proliferation-competent cells exists even in the adult. In the present
study proliferation-competent cells were isolated from the bovine adrenal medulla. Similar
to neurospheres, these cells, when prevented from adherence to the culture dish, grow in
spheres, which we named chromospheres. The sphere-forming cells had self-renewing
capacity and differentiated into neuronal and endocrine cell lineages. In accordance with
these differentiation properties, genes specific to differentiated chromaffin cells, such as
the epinephrine synthesizing enzyme PNMT were dramatically downregulated while the
expression of progenitor markers nestin, Musashi1, Sox1 and Sox9 were upregulated. Cells
from chromospheres were able to differentiate. Dexamethasone induced the expression of
PNMT which was very low in chromospheres. Nerve growth factor or bone morphogenetic
protein 4, on the other hand, induced the formation of neurite-like extrusions, positive for
s-III-tubulin by immuno-fluorescent staining. This study provides evidence that neural
crest derived proliferation and differentiation competent chromaffin progenitor cells can be
isolated from adult adrenal medulla that might harbour the potential for the treatment of
neurodegenerative diseases, e.g. Parkinsonfs Disease.
Kuei-Fang Chung
Technische Universitat Dresden
Carl Gustav Carus University Clinic
Department of Medicine III
Molecular Endocrinology
Kuei-Fang.Chung@uniklinikum-dresden.de
www.uniklinikum-dresden.de/
203
Neuromedicine
138 Cellular characteristics of neural progenitor cells
in the adult zebrafish telencephalon
Julia Ganz, Jan Kaslin, Heiner Grandel, Michael Brand
In all vertebrate species examined neurogenesis takes place not only at embryonic stages,
but also during adult life. In contrast to mammals, new neurons are produced continuously in
many regions of the adult central nervous system of non-mammalian vertebrates. In zebrafish,
there are different characteristic proliferation zones along the whole rostro-caudal axis of the
adult brain. Colocalisation studies with neuronal markers additionally demonstrate that those
proliferation zones are mainly neurogenic showing that there is widespread neurogenesis
in the adult zebrafish brain. In addition we can show that there are label-retaining, actively
cycling cells that remain in the proliferation zones. We are currently analyzing in detail the
composition of the telencephalic proliferation zones focusing both on glial cells and the
characteristics of the label-retaining cells in vivo and in vitro. Additionally, we compare the
situation in the zebrafish telencephalon with the adult neurogenic regions in the mammalian
brain.
Julia Ganz
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Department of Molecular Developmental Genetics
Julia.Ganz@biotec.tu-dresden.de
www.biotec.tu-dresden.de/brand
Neuromedicine
204
139 Viral gene transfer of cell cycle inhibitors to slow
down progressive neurodegeneration
Pia Glockner, James Uney, Thomas Arendt, Uwe Ueberham
Alzheimerfs disease (AD) is the most common neurodegenerative disorder of humans with
an enormous socio-economic burden on the aging society. Currently, causes of the disease
are still unknown and there is neither an effective prevention nor a therapy treatment. To
prevent neuronal cell death we want to develop a new gene therapeutic tool, which ensures
long-lasting, safe, neuron-specific and regulated transgene expression. The concept is based
on neuroprotective effects of cell cycle inhibitors (cdkis) to block the cell cycle re-entry
of neurons. In previous transgenic experiments we could show, that overexpression of the
inhibitor p16INK4a has neuroprotective effects in vivo models of neurodegeneration. Our new
strategy is based on non-integrating (NI) lentiviral vectors, which can regulable express
physiological cell cycle inhibitors of the INK4 and Cip/Kip family. These vectors can deliver
8kb . 10kb transgene sequences, they have the ability to infect specific neuronal cells, induce
no or low immune response and the normal cell functions are not negative affected. The
neuron specific expression enabled by application of neuron specific promoters. The cdki
expression will be regulated by Tet-On / Tet-Off system. All these properties create them
to a powerful tool for gene delivery in the nervous system. We have established expression
vectors for p15Ink4b, p16Ink4a, p18Ink4c, p19Ink4d, p19ARFInk4d, p21Wafl/Cip1,p27Kip1 and p57Kip2 and
analysed their effects in vitro. Currently, we are focused on the generation of the viral vectors
with the specific cdkis.
Pia Glockner
Universitat Leipzig
Faculty of Medicine
Paul Flechsig Institute for Brain Research
gloeck@rz.uni-leipzig.de
www.uni-leipzig.de/~pfi
205
Neuromedicine
140 A novel fluorescent acetylcholinesterase inhibitor
released from nanoparticles selectively binds
hippocampal ƒÀ-amyloid plaques in triple
transgenic mice
Wolfgang Hartig, Johannes Kacza, Bernd-Reiner Paulke,
Jens Grosche, Anke Hoffmann, Paul Wilhelm Elsinghorst,
Michael Gutschow
A hallmark of Alzheimerfs disease (AD) is the drastic loss of cholinergic projection neurons in
the basal forebrain. Frequently applied drugs for the treatment of dementia include inhibitors
of the acetylcholine-degrading enzyme, acetylcholinesterase (AChE). This protein is known
to act as a ligand of s-amyloid (As) in senile plaques, a neuropathological sign of AD. Here,
we present histochemical data on the novel strongly fluorescent, heterodimeric AChE inhibitor
PE154. Advantageous spectroscopic properties of PE154 . a sharp excitation peak with a
maximum at 405 nm and an emission maximum at 517 nm . allow for its combined use with
immunolabelling based on fluorescent carbocyanines Cy3 and Cy5. As deposits were screened
both in fixed cortical tissue sections from an autoptic case with verified AD and in triple
transgenic (TTG) mice with age-dependent s-amyloidosis and tau hyperphosphorylation, an
established animal model for aspects of AD. Numerous plaques double-stained for PE154 and
As-immunoreactivity were revealed by confocal laser-scanning microscopy. Additionally,
we were able to visualize the targeting of As-immunopositive plaques with PE154 in vivo
three days after injection into the hippocampi of 13 . 20-months-old TTG mice. Furthermore,
PE154 labelled selectively As, but not phospho-tau in aged TTG mice after intrahippocampal
injection of biodegradable core-shell polybutylcyanoacrylate/polystyrene nanoparticles
releasing the fluorescent marker in vivo. These data suggest that nanoparticles are promising
carriers of AChE inhibitors and other drugs for the treatment of AD.
Dr. Wolfgang Hartig
Universitat Leipzig
Faculty of Medicine
Paul Flechsig Institute for Brain Research
hartig@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
Neuromedicine
206
141 Role of purinergic signalling in the development
of fibre projections?
Claudia Heine, Nico Scherf, Jens-Peer Kuska, Ulf-Dietrich
Braumann, Heike Franke
A possible role of purines in development, regeneration and on neurite outgrowth has been
reported. Using organotypic tissue slice co-cultures including the ventral tegmental area/
substantia nigra (VTA/SN)-complex and the prefrontal cortex (PFC) the expression of P2
receptors and the growth promoting effect of different P2 receptor agonists was shown.
In this study we focused on the characterization of the fibres interconnecting the dopaminergic
VTA/SN-complex with the PFC using fluorescence labelling with antibodies against various
neuronal and glial structures. Additionally, after substance application (P2 receptor agonist
and/or antagonist) and biocytin tracing, the fibre outgrowth was quantified and characterized
with the help of an image processing procedure. After segmentation of single fibre tracts
detailed information about their growth were gained together with global characteristics of
the specimen.
Using immunocytochemistry the fibres in the border region were identified e.g. as TH-,
GABA-, PSA-NCAM- and P2Y1 receptor-positive projections. The incubation with different
P2 receptor agonists induced an increase in axonal outgrowth and fibre density, which could
be inhibited by pre-treatment with the P2 receptor antagonist. Besides, after P2 receptor
antagonist treatment, the fibres showed different growth characteristics, in that they tend to
grow in a more linear way and developed fewer branches in comparison to the treatment with
P2 agonists.
In conclusion, our results indicate a stimulating capacity of ATP on fibre outgrowth, suggesting
an important role of purinergic signalling in the developing brain.
Dr. Claudia Heine
Universitat Leipzig
Faculty of Medicine
Rudolf-Boehm-Institute for Pharmacology and Toxicology
heinec@medizin.uni-leipzig.de
www.uni-leipzig.de/~pharma/
207
Neuromedicine
142 A study of Imaging Geno-Phenotypes in dyslexia
Holger Kirsten, Carolin Ligges, Arndt Wilcke, Peter Ahnert,
Johannes Boltze
Analysis of Imaging Geno-Phenotypes (IGP) is a highly promising new method to identify
new putative risk genes for neurological conditions. The aim of this study is to identify new
putative risk genes for dyslexia. In addition, genetic influence on phonological processing,
auditory processing, and visual processing will be assessed.
A group of ca. 20 dyslexic German children and ca. 20 matched controls performed controlled
auditory, visual and phonological tasks in an fMRI study. In addition, EEG data was also
recorded. Genetic variation is analyzed using Affymetrix Genome-Wide Human SNP Arrays
6.0.
For each of the three tested paradigms, networks of brain regions relevant for processing these
controlled tasks have been described in literature. This processing seems to occur differently
in dyslexia. IGPs are analyzed for each of these three performed controlled tasks.
Analysis of data of the phonological task could confirm differences in processing between
dyslexics and controls within a network consisting of the inferior temporal, inferior frontal,
and the superior temporal region. The study is still in progress. Analysis of imaging genophenotypes
will comprise all genetic data but will also focus on genomic regions known to be
related to dyslexia. Comparison of IGP resulting from the auditory, visual, and phonological
tasks may reveal differences in the genetic basis of these three dyslexia relevant aspects.
IGP offer a promising new approach for identification of new candidate genes. We are
adopting this approach in order to identify new candidate genes in dyslexia.
Holger Kirsten
Fraunhofer Institut for Cell Therapy and Immunology (IZI)
Cell Therapy
Neurorepair
hkirsten@medizin.uni-leipzig.de
www.izi.fraunhofer.de
Neuromedicine
208
143 Depression-like deficits in rats are improved by
subchronic modafinil
Holger Koch, Ralf Regenthal, Christian Kohler, Ute Krugel
Attentional and sensorimotor gating deficits in human depression are observed as residual
symptoms irrespective of antidepressant treatment. Clinical studies point to a benefit of
modafinil in depression. No data are available on modafinil effects in depression-like animal
models. We investigated effects of modafinil on attention and sensorimotor gating after
subchronic treatment during restraint stress inducing depression-like changes in rats. Effects of
modafinil were investigated acutely in the forced swim test (FST) 1 hour after administration
of drug or placebo and in a further experiment on cognition-related behaviour in rats after
induction of depression-like changes using a restraint stress protocol for 15 days. Beginning
from day 10, one restrained and one non-restrained group were treated with modafinil and
two respective groups with placebo. At the end behavioural testing was performed under
conditions of nearly drug-free plasma. Depression-like behaviour was examined in the FST.
Selective attention and sensorimotor gating were investigated as social novelty discrimination
(SND) and prepulse inhibition (PPI) of acoustic startle response. Restraint led to reduced
body weight, decreased mobility in the FST and impaired cognitive capabilities in the SND
and the PPI. Subchronic modafinil treatment reversed restraint induced deficits in the FST, the
SND and PPI, whereas it was without effect on body weight. The improvement of impaired
attentional and information processing functions under depression-like conditions suggests a
benefit of modafinil in treatment of cognitive residual symptoms in affective disorders.
Holger Koch
Universitat Leipzig
Faculty of Medicine
Rudolf-Boehm-Institute of Pharmacology and Toxicology
holger.koch@medizin.uni-leipzig.de
www.uni-leipzig.de/~pharma
209
Neuromedicine
144 Impedance spectroscopy: A method for
developing a label-free detection system for
neurodegenerative diseases
Dana Krinke, Heinz-Georg Jahnke, Andrea A. Robitzki
Tauopathies are characteristic for broad range neurodegenrative diseases which have as
a common pathological feature the presence of intracellular accumulations of abnormal
filaments of hyperphosphorylated tau protein in neurofibrillary tangles. Alzheimerfs disease
(AD) is a member of this group and because of the complex pathological mechanism,
we want to develop an in vitro AD model, where pathological consequences of induced
hyperphosphorylation can be detected quantitatively and label-free by multielectrode array
based impedance spectroscopy. For the establishment of the in vitro model the pathologic
tau mutants K257T, K280, P301L, V337M and R406W as well as combinations of these
mutations were generated by site-directed mutagenesis. The neuronal cell line SH-SY5Y
was transfected with the EGFP tau mutants. Afterwards we were able to isolate single stable
clones of EGFP-control and EGFP-tau P301L mutants. The obtained single SH-SY5Y
clones were treated with okadaic acid for induction of hyperphosphorylation. Analysis of
tau expression and phosphorylation pattern by western blot showed differences between the
generated clones. Moreover first impedimetric measurements with EGFP-control and EGFPtau
P301L expressing SH-SY5Y cells on multielectrode arrays could be correlated with
molecular biological results. Our first results provide a novel SH-SY5Y cell-based real-time
screening system for testing active pharmaceutical ingredients against tauopathies.
Dana Krinke
Universitat Leipzig
Centre for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
dana.krinke@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
Neuromedicine
210
145 Effects of blue light scleral cross-linking on rabbit
eye growth
Qing Liu, Hans Peter Iseli, Nicole Korber, Niclas Lindqvist,
Martin Gryga, Peter Wiedemann, Andreas Reichenbach,
Mike Francke
Scleral cross-linking with riboflavin and blue light might increase the scleral strength and
such treatment was proposed to reduce the axial elongation during progressive myopia. We
investigate the effects of blue light scleral cross-linking on eye growth as well as possible
side effects on retinal tissue.
The posterior scleral of 2-week-old rabbits were unilaterally treated with riboflavin and
blue light with intensities of 40 and 1000mW/cm2. Two weeks after treatment, eye size
was measured by A-scan ultrasonography and vernier caliper. Retinal and scleral histology
were examined by light microscopy and immunohistology. Possible retinal injuries were
investigated by detecting Muller cell gliosis and immune cells activation. Muller cell density
was calculated to monitor retinal shrinkage.
The eye size was not significantly changed after treatment with riboflavin/blue light of 40mW/
cm2, whereas eyes treated with 1000mW/cm2 were significantly smaller compared to control
eyes. Activated microbial cells and increased GFAP expression were detected in retinas of
1000mw/cm2 treated eyes, but were not observed in retinas of control and 40mw/cm2 treated
eyes. Scleral collagen fibers were more irregularly shaped after high intesity treatment. Muller
cell density was found to be similar in control, 40 and 1000 mW/cm2 treated eyes.
Scleral collagen cross-linking with riboflavin and blue light is effective to arrest eye growth.
Effective and safe irradiation dose of the blue light should be in the range of 40 . 1000mW/
cm2.
Qing Liu
Universtiat Leipzig
Faculty of Medicine
Paul-Flechsig-Institute for Brain Research
Department of Neurophysiology
Liu.Qing@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
211
Neuromedicine
146 Isolation and biological potential of enteric
nervous system precursors derived from human
gut
Marco Metzger, Nikhil Thapar, Lothar Just
A number of gut motility disorders are caused by abnormalities of the enteric nervous system
(ENS), and stem cell-based therapies may offer the potential of replacing defective, damaged,
or missing neural elements within the bowel. Here, we describe a method suitable for the
preparation of ENS stem cells from human postnatal and adult gut tissue and assess their
transplantation potential.
Human gut tissue was obtained from colonic biopsy samples and surgical resection specimens.
We established isolation and cultivation protocols to generate neurospheres, which were
injected into recipient aganglionic chick or human gut tissues, respectively. At both pre- and
post-transplantation stages cell proliferation and differentiation along with integration of
neurosphere-derived cells were analyzed by immunohistochemistry, in situ hybridisation and
electrophysiology.
After in vitro differentiation mature neuronal and glial cells could be generated as demonstrated
by the expression of a variety of phenotypic markers and clearly distinguishable sodium
currents. When implanted into aganglionic gut, neurosphere-derived cells integrated into the
recipient tissues and differentiated appropriately into ENS components.
This study provides a significant and necessary first step for the development of enteric neural
cell transplantation for the treatment of a specific group of gastrointestinal disorders but,
importantly, has wider applicability for the establishment of neural components within the
many facets of Regenerative Medicine.
Marco Metzger
Universitat Leipzig
Translational Center for Regenerative Medicine (TRM)
Cell Therapies for Repair and Replacement
mmetzger@trm.uni-leipzig.de
www.trm.uni-leipzig.de/html/en/index.php
Neuromedicine
212
147 Non-hypoxic stabilization of hypoxia-inducible
factor alpha (HIF-ƒ¿): Relevance in neural
progenitor/stem cells
Javorina Milosevic, Irena Adler, Sigrid C. Schwarz, Gail
Walkinshaw, Alexander Storch, Johannes Schwarz
Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC)
propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxiainducible
factor 1 alpha (HIF-1ƒ¿) is rapidly degraded via the prolyl hydroxylase (PHD)/
VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1ƒ¿ in
NPCs and influence the transcription of HIF-regulated genes. Here we investigate various
hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine
(DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl2) with respect to
their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation
of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to
stabilize HIF-1ƒ¿, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl2
was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497
enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both
CoCl2 and FG-4497 were protective to human dopaminergic neurons. These findings suggest
that several HIF stabilizing agents can rescue impaired neurons and promote neurogenesis
in vitro.
Dr. Javorina Milosevic
Universitat Leipzig
Translational Centre for Regenerative Medicine (TRM)
Department of Neurology
jmilosevic@trm.uni-leipzig.de
www.trm.uni-leipzig.de
213
Neuromedicine
148 Ca2+ Responses of Muller cells induced by light
stimulation of photoreceptor cells
Katja Rillich, Janina Gentsch, Michael Weick, Andreas
Bringmann, Andreas Reichenbach
Muller glial cells respond with an intracellular calcium rise to light stimulation of the retina.
Under dark adapted conditions this very slow calcium rise applies to all Muller cells and
starts right after the beginning of the light stimulation. After further strong light stimulation
Muller cells react with a second faster calcium rise which starts at the level of the ganglion
cell layer. It originates in the smooth endoplasmatic reticulum of the Muller glial cells, which
could be shown by the application of cyclopiazonic acid. Cyclopiazonic acid reduces the
number of Muller cells displaying fast calcium rises to zero, but leaves the first slow calcium
rise of the cells unaltered.
Pharmacological experiments so far showed for the slow calcium rise that the signal transfer
from neurons to Muller cells occurs at the level of the photoreceptor cells and is dependent
on a transmitter release of the photoreceptors. Glutamate does have an effect on the signal
transduction, but neither via ionotropic nor via metabotropic glutamate receptors, but rather
via glutamate transporters. Zinc, which is co-released with glutamate by the photoreceptors,
also seems to be involved in the signal transduction to the Muller cells.
Dr. Katja Rillich
Universitat Leipzig
Faculty of Medicine
Paul-Flechsig-Institute for Brain Research
Katja.Rillich@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
Neuromedicine
214
149 Polyethylenimine as a possible gene therapeutical
tool against Alzheimerfs Disease
Susanne Rohn, Thomas Arendt, Uwe Ueberham
Alzheimerfs Disease (AD) is an age-associated incurable neurodegenerative disorder with
specific characteristics like neurofibrillary tangles, amyloid deposition and disturbances in
the expression of cell cycle proteins followed by neuronal dedifferentiation and apoptosis.
Probably, neurons can re-enter the cell cycle but cannot pass through it completely. We
suggest that the inhibition of re-entry could be achieved by repression of the cyclin dependent
kinase 4 and 6 activity with ectopic expression of their physiological inhibitors. This requires
a specific application procedure.
Here we present a unique modified polycation polyethylenimine (PEI) as a therapeutical
tool for neuron-specific gene transfer. PEI can be coupled to different ligands possessing
high affinity to surface receptors of the target cell and has also successfully been applied
to the CNS. By application of inhibitor-DNA complexed with antibody (ab)-coupled PEI
conjugates through drug delivery and the use of neuron-specific promoters this tool could
prevent or even slow down the progression of neurodegeneration.
In experiments two different antibodies were coupled to PEI, purified by FPLC and the
conjugates were analysed by Ninhydrin assay. The complexation of PEI/ab-PEI with DNA
was determined by transfection studies in vitro and gel retardation assay using GFP. Optimal
N/P ratios between PEI/ab-PEI and DNA were also identified.
Susanne Rohn
Universitat Leipzig
Faculty of Medicine
Paul Flechsig Institute for Brain Research
Susanne.rohn@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
215
Neuromedicine
150 Increase of intracellular Ca2+ by adenine and
uracil nucleotides in human midbrain-derived
neuronal precursor cells
Patrizia Rubini Illes, Johannes Engelhardt, Mahmoud Al-
Khrasani, Javorina Milosevic, Johannes Schwarz, Peter Illes,
Wolfgang Norenberg
Membrane currents of human midbrain-derived neuronal precursor cells (hmNPCs)
were measured by means of the whole-cell patch-clamp technique. None of the cells was
sensitive to ATP, although AMPA caused consistent membrane currents. Increases in the
intracellular Ca2+ concentration ([Ca2+]i) were determined by the Fura-2 method. Various
nucleotide agonists concentration-dependently increased [Ca2+]i, with the rank order of
potency ATP>ADP.UTP>UDP. Although UTP acted only at higher concentrations than
ATP, the mode of action of these agonists were almost indistinguishable. A Ca2+-free external
medium moderately decreased, whereas a depletion of the intracellular Ca2+ storage sites by
cyclopiazonic acid markedly depressed the [Ca2+]i transients induced by either nucleotide.
Further, the P2Y1 receptor antagonists, PPADS and MRS 2179, as well as the nucleotide
catalyzing enzyme apyrase, all abolished the ATP and UTP effects. However, the P2Y1,2
selective antagonist suramin only slightly blocked the action of ATP, but strongly inhibited
that of UTP. In agreement with this finding, UTP released ATP from hmNPCs in a suramin-,
but not PPADS-sensitive manner. Immunocytochemistry indicated the co-localization of
P2Y1,2,4-immunoreactivities (IR) with nestin-IR at these cells. In conclusion, UTP may release
ATP from hmNPCs via P2Y2 receptor-activation and thereby induces [Ca2+]i transients by
stimulating a P2Y1-like receptor.
Dr. Patrizia Rubini Illes
Universitat Leipzig
Rudolf Boehm Institute of Pharmacology and Toxicology
patrizia.rubini@medizin.uni-leipzig.de
www.uni-leipzig.de
Neuromedicine
216
151 Study of human neural progenitor cell fate after
grafting into rat striatum
Johanna Scheibe
Mesencephalic neural progenitor cells (hNPCs) are a promising source for therapeutic
approaches, especially for cell replacement therapy in Parkinsones disease. Until now these
cells are expanded and differentiated in vitro for following transplantation studies in rats.
Grafting outcome was very limited, since only one of eight transplanted 6-Hydroxdopaminelesioned
rats showed behavioral improvement by reconstruction of destroyed nigrostriatal
projections and well integrated dopaminergic (DA) neurons. Therefore, it is necessary to
examine cell fate after transplantation using quantitative real time PCR. This method allows
specific discrimination of grafted hNPCs and rat tissue using human or rat specific primers.
In this study, human-specific markers for proliferation, differentiation and apoptosis have
been determined. Furthermore, different inflammatory markers were tested in rat tissue. Our
preliminary results indicate that only glial cells survive over a three week period. DA-neurons
stayed vitally only for three days after the transplantation. From the third day to the seventh
day caspase activity increased, indicating apoptosis of DA-neurons. Concerning the tested
inflammation markers, no increased immune reaction was observed. Hence, apoptosis of DA
neurons seems to be the key reason for poor graft outcome. Further studies are necessary to
prevent or inhibit apoptosis in DA neurons after transplantation.
Johanna Scheibe
Universitat Leipzig
Clinic for Neurology
johanna.scheibe@medizin.uni-leipzig.de
www.neurologie.uniklinikum-leipzig.de
217
Neuromedicine
152 O rganotypic cocultures as an alternative to
conventional animal models
Sabine Schewtschik
Cell replacement therapies are a promising strategy to compensate cell loss appearing in
the progress of neurodegenerative disorders e.g. ParkinsonLs disease. In line with preclinical
studies, it is of high importance to follow the cells fate post transplantation. Since the use
of conventional animal models are often complex, time consuming and realtime analyses
of the cells are unfeasible the aim of this study was to generate an alternative test system.
Therefore, we established so called organotypic cocultures comprising parasagital mouse
brain slices and eGFP-transfected human neural precursor cells (hNPCs) transplanted on the
tissues top. This system models the in vivo situation while containing the same cells as in
a living animal under retention of native cell contacts and projections. In vitro cultivation
indeed enables the possibility to control and manipulate basic culture conditions. So far, we
achieved a good slice vitality and structural preservation for up to 5 months. Transplanted
eGFP-positive hNPCs are easy to recover so that it is possible to follow the cells fate on the
tissues top in realtime by fluorescence microscopy. Further investigations, e.g. determination
of differentiation status of the cells by immunocytochemistry or quantitative realtime PCR,
should complete the system to be a good alternative to conventional animal models.
Sabine Schewtschik
Universitat Leipzig
Clinic for Neurology
sabine.schertschik@medizin.uni-leipzig.de
www.neurologie.uniklinikum-leipzig.de
Neuromedicine
218
153 Analysing a potential neuroprotective function
of perineurinal nets by using organotypic slice
cultures
Anne Suttkus, Markus Morawski, Gert Bruckner, Thomas
Arendt
Perineuronal nets (PNs) are a special form of extracellular matrix and consist of large
aggregating chondroitin sulphate proteoglycans connected to hyaluronan and stabilized
by the link protein 1 as main components. PNs surround different types of neurons in the
brain of many vertebrate species including man. Due to their highly negatively charged
character, which is caused by their glycosaminoglycan and hyaluronan components, the PNs
might be involved in local ion homeostasis. PNs might also potentially be able to scavenge
and bind redox-active iron, and thus reduce the local oxidative potential in the neuronal
microenviroment and may provide some neuroprotection to net-associated neurons. Here,
we investigate whether neurons enwrapped by a PN are less vulnerable against iron-induced
oxidative processes. We prepared organotypic slice cultures of postnatal day 1 mouse brains.
After 3 weeks of cultivation we vitally stained the fully developed PNs with the lectin
Wisteria floribunda agglutinin. To mimic oxidative stress we applied either FeSO4 (Fe2+) or
FeCl3 (Fe3+) via injection into the slices or by adding the solution to the culture media. For
evaluating the effect of the two iron solutions we fixed the slices and treated them with
Hoechst dye to assess the state of nuclear fragmentation which is a widely used indication
of cell injury. We could demonstrate that neurons ensheathed by a PN only rarely show
fragmented nuclei, indicating a low vulnerability and a protective role of PNs against ironinduced
damage.
Anne Suttkus
Universitat Leipzig
Faculty of Medicine
Paul-Flechsig-Institute for Brain Research
anne.suttkus@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
219
Neuromedicine
154 Distinct reactions of retinal microglia cells evoked
by various stimuli
Elke Ulbricht, Mike Francke, Ulrike Zeitschel, Andreas
Reichenbach
There are two classes of glial cells in the central nervous system (CNS), namely macroglia
and microglia. Microglial cells are the immunocompetent cells in the immune privileged
space of the retina. Microglial cells become activated in every retinal disease as well as
Muller glia. The interaction of microglia in retinal diseases is not yet well understood. To
optimize therapeutic interventions a better knowledge of microglia functionality is required
in retina. Two different models were used; LPS-induced microglia activation and application
of a microglia-specific immunotoxin in the guinea pig eye to examine microglia reaction.
After intravitreal injection of LPS, microglial cells become activated. This LPS-induced
activation is gradually downregulated after a few days, first noticeable by re-changing lectin
staining profile. No gliotic changes of Muller cells were found. Microglia reaction is obviously
not causal for activation of Muller glia in every case. Therefore, signals of degenerating
neurons might be elementary. For the signaling of microglia and neurons a direct as well as
indirect pathway might be possible. The established models in this study provide a valuable
opportunity to investigate those signaling pathways more intensely. The immunotoxin Mac1-
Saporin is selectively inducing apoptosis in vitro. Intravitreal application of the immunotoxin
evokes a strong microglial reaction and remarkable morphological alterations which may
refer to dystrophic changes. The immunotoxin might be a useful tool for inducing artificial,
dystrophic modifications in microglial functionality.
Elke Ulbricht
Universitat Leipzig
Faculty of Medicine
Paul-Flechsig Institute for Brain Research
elke.ulbricht@medizin.uni-leipzig.de
www.uni-leipzig.de/~pfi
Neuromedicine
220
155 Connexins control glial glutamate transporter
expression
Tina Unger, Stefanie Bette, Jurgen Engele
Glutamate is the major excitatory neurotransmitter in the mammalian nervous system. At
high extracellular levels, glutamate represents an extremely potent neurotoxin, leading to
excitotoxic neuronal cell death. Both the termination of glutamatergic neurotransmission
and the prevention of toxic extracellular glutamate concentrations are achieved by the rapid
removal of glutamate from the extracellular space by a family of high-affinity, sodiumdependent
glutamate transporters. The family member, GLT-1, which prevails in astroglia,
is currently regarded as the most important glutamate transporter subtype. By using cultured
rat astrocytes as an assay system, we recently obtained evidence for a direct control of GLT-1
expression by connexin43 (Cx43), the major gap junctional protein of astrocytes, in terms
that inhibition of Cx43 leads to a decline in GLT-1 expression. We now extend these findings
by demonstrating that a similar decline in GLT-1 expression occurs under in vivo conditions.
In fact, animals with conditional knockouts of both Cx43 and connexin30 (Cx30) in GFAPexpressing
astrocytes exhibit reduced expression levels of GLT-1. Additional experiments,
aimed at characterizing the molecular mechanisms underlying this connexin-dependent
control of glial glutamate transporter expression, unravelled that over-expression of the
cytoplasmic C-terminus of the Cx43 protein is sufficient to promote GLT-1 expression in
cultured astrocytes. Since it is well documented that over-expression of the Cx43 C-terminus
does not affect cellular coupling, we conclude that Cx43 controls GLT-1 expression by a
C-terminal mechanism.
Tina Unger
Universitat Leipzig
Faculty of Medicine
Institute for Anatomy
Department of Molecular Neuroanatomy
Tina.Unger@medizin.uni-leipzig.de
www.uni-leipzig.de/~anatomie/
221
Neuromedicine
156 A new hippocampal ex vivo model to study
tauopathies by label-free impedance spectroscopy
Annett Wegner, Heinz-Georg Jahnke, Till G. A. Mack, Frank
Striggow, Andrea A. Robitzki
Tauopathies are histopathologically characterized by a specific type of slow and progressive
neurodegeneration, which involves the abnormal hyperphosphorylation of the microtubule
associated protein (MAP) tau. The hippocampal organotypic slice culture model can be used
for a high throughput screening of new drug candidates for tauopathies.
In a first step, we optimized preparation and culturing of neonatal rat hippocampal slices as
an ex vivo model. After 7 days in vitro cultivation, pathological hyperphosphorylation was
induced by treading the slices with okadaic acid (OA). Molecular methods (western blotting,
immunocytochemistry) were used to analyze the different hyperphosphorylation pattern of
the tau protein in slices treated with OA and control slices.
After evaluation of our ex vivo model we will now optimize hippocampal slice cultivation on
multielectrode arrays for an impedance spectroscopy based label-free screening system. Finally
the capabilities of the novel screening system will be demonstrated by testing of reference
compounds like kinase inhibitors as well as novel active pharmaceutical ingredients.
Annett Wegner
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
annett.wegner@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
Neuromedicine
222
157 A new mouse model for targeting the astrocytic
NADH / NAD+ redox state in vivo
Franziska Wilhelm, Jan Rillich, Ulrike Winkler, Johannes
Hirrlinger
Astrocytes are a vital part of the neural communication system. They take up neurotransmitters,
modulate the extracellular ion balance, actively modulate signalling events e.g. by the release
of gliotransmitters and provide metabolic support to neurons. The most prominent model of
such a metabolic interaction is the astrocyte-to-neuron lactate-shuttle. Astrocytes metabolize
glucose via glycolysis yielding lactate which is then transferred to neurons and used for
oxidative metabolism. By the conversion of pyruvate to lactate (astrocyte) and vice versa
(neuron), this shuttle is directly linked to the NADH / NAD+ redox state in both types of
cells. Additional evidence suggests that neural signalling also influences these redox states
in astrocytes and neurons and that several proteins are able to sense them (like sirtuins,
several transcription factors, IP3-receptors). However, the role of changed levels in NADH
or NAD+ within this metabolic-signalling network in vivo is largely unknown. To address
these questions, a new model system that enables a cell-type specific and inducible targeting
of the NADH / NAD+ redox state in mice in vivo is introduced.
Franziska Wilhelm
Universitat Leipzig
Faculty of Medicine
Interdisciplinary Centre fur Clinical Research,
Junior Research Group .Neurale Plasticityg
happylino@hotmail.com
www.uni-leipzig.de/~izkf
223
Neuromedicine
158 Structural plasticity of astrocytes and the impact
of the cell adhesion protein vinculin
Ulrike Winkler, Marcello Sestu, Alice Zemljic-Harpf, Robert
S. Ross, Wolfgang H. Ziegler, Johannes Hirrlinger
In this study, we attempt to elucidate the mechanisms controlling the structural plasticity of
astrocytes in vivo. Vinculin is a central component of the cell adhesion complex that links
structurally and functionally cell adhesion receptors to the actin cytoskeleton. In murine brain
slices using immunohistochemistry and confocal microscopy, we observed that vinculin was
localised in the astrocytic soma as well as in processes. Vinculin revealed a punctate staining
pattern as expected for adhesion site localisation. By using two-photon laser scanning
microscopy, we were able to confirm the presence of motile processes in acutely isolated
brain slices. Furthermore, we generated a mouse line with an astrocyte-specific conditional
and inducible knock-out of vinculin by using the GFAP-promoter driven tamoxifen-inducible
Cre-ERT2 and a LoxP-flanked vinculin-allele. Preliminary results indicate successful
recombination of the vinculin gene in astrocytes of tamoxifen-treated mice, which will
allow us to directly study consequences of vinculin-deficiency on astrocytic morphology and
motility in brain slices.
Ulrike Winkler
Universitat Leipzig
Faculty of Medicine
Interdisciplinary Centre fur Clinical Research
Ulrike.Winkler@medizin.uni-leipzig.de
www.uni-leipzig.de/~izkf/

9. Diagnostics
Posters

227
Diagnostics
159 Effects of A2A and A2B ligands on ach contraction in
inflamed rat small intestinal preparation
Karen Nieber, Claudia Warstat, Fabien Michel, Luo Yan,
Christa Muller, Sebastian Michael
Adenosine can show anti-inflammatory as well as pro-inflammatory activities, and the
contribution of specific adenosine receptor subtypes in various organs is complex. We
examined the effect of the adenosine A2A receptor agonist CGS 21680 and the A2B antagonist
PSB-1115 on acute inflammation induced by TNBS on rat ileum/jejunum preparations.
Preincubation of the tissue segment with TNBS for 30 min resulted in a concentrationdependent
inhibition of ACh-induced contraction. Pharmacological activation of the A2A
receptor with CGS 21680 (0.1 . 10 ƒÊM) preincubated simultaneously with TNBS (0.01M)
restored concentration-dependently the TNBS-induced inhibition of the ACh-contractions.
Stimulation of A2B receptors with the selective agonist BAY 60-6583 (10 ƒÊM) did neither
result in an increase nor in a further decrease of ACh-induced contraction compared to the
TNBS-induced inhibition. The simultaneous preincubation of the ileum/jejunum segments
with TNBS (0.01 M) and the selective A2B antagonist PSB-1115 (100 ƒÊM) inhibited the
contraction-decreasing effect of TNBS. A significant amelioration of the TNBS-diminished
contractility was found by the combination of the A2AR agonist CGS 21680 and the A2B
antagonist PSB-1115 at subthreshold concentrations of both agents, which was in the same
range as the effect induced by 1 ƒÊM methotrexate. Our results demonstrate that the activation
of A2A or the blockade of A2B receptors can decrease the inflammation-induced disturbance
of the ACh-induced contraction in TNBS pretreated small intestinal preparations. The
combination of both may be useful for the treatment on inflammation bowel diseases.
Prof. Dr. Karen Nieber
Universitat Leipzig
Institute of Pharmacy
Department fur Pharmacology and Sciences
nieber@rz.uni-leipzig.de
www.uni-leipzig.de/~pharm
Diagnostics
228
160 Concentration of mucus in gastric juice in normal
adult horses withhold feed and after application
of pronurtin
Gerald F. Schusser, Alice Spallek, Stephan Recknagel, Julia
Breuer, Gabor Koller
Mucus is composed of water, electrolytes and glycoproteins. Mucus is different in all parts
of the oroesophagogastrointestinal tract. The important characteristics of mucus are an
excellent lubricant and a protectant for the wall of the gut. Mucus produced in goblet cells of
tubular glands in the fundus region of the stomach protects the gastric mucosa against acid
and pepsin. Ulceration of the gastric mucosa results when mucus is reduced. The aim of this
study was the measurement of concentration of total mucus in saliva and gastric juice in adult
horses withholding feed.
Six normal adult horses were withhold of feed over a period of 12 h. Gastric juice was collected
through an indwelling nasogastric tube before (prae) and hourly after (post) application of
50 g PronutrinR (Boehringer, Ingelheim, Germany) per 100 kg b.w. through the nasogastric
tube. Mucus in saliva and gastric juice was measured spectralfotometricly using mucus of
pigs as positive controls. Saliva was collected with cotton swabs at the same time as the prae
gastric juice sample. Values were expressed as mg/ml and medians.
Saliva prae 1 h post 2 h post 3 h post 4 h post 5 h post 6 h post 7 h post
49.555 1.9 16.855 7.9 8.52 8.71 4.74 1.73 1.1
The pH of the gastric juice before application of PronutrinR was 3.47 which was measured
in horses withholding feed.
The mucus concentration of saliva is significant higher than in gastric juice collected from
horses withholding feed. Pronutrin is able to increase the total mucus concentration in gastric
juice over a period of five hours after application. The defensive factor mucus could be
increased by Pronutrin (pectin lecithin complex) to prevent in the grandular region of horses
withhold of feed during transportation for instance.
Prof. Dr. Gerald F. Schusser
Universitat Leipzig
Faculty of Veterinary Medicine
Department of Large Animal Medicine
koeller@vetmed.uni-leipzig.de
www.vetmed.uni-leipzig.de/ik/wmedizin/
229
Diagnostics
161 Dendritic sugar balls for biological experiments
driven by H-bonds
Dietmar Appelhans, Brigitte Voit
Recently, dendrimers and hyperbranched polymers with various (oligo-)saccharide
architectures are widely used as multifunctional materials for potential biological, (bio-)
medical and pharmaceutical applications such as e.g. DNA carrier and drug carrier. The
establishment of such (oligo-)saccharide units on dendritic surface also resulted in the desired
requirement of enhanced biocompatibility, including reduced toxicity.
Here, we report on the bio-interaction of PPI dendrimers towards biomolecules and
-macromolecules (HSA protein and prion peptide 185-208) which is compared with their
cationic counterparts. In all cases, one common, but also surprising result exhibited that
(A) the strength of interaction towards the HSA protein is on the same level for the parent
and modified PPI dendrimers and (B) the maltose-modified PPI dendrimers can also act as
potential anti-prion agent. Although the size, molecular weight and charge (density) of the
oligosaccharide-modified derivatives are very different from their cationic counterparts for the
HSA and prion peptide interaction, similar results can be realized. Further, also a generationdependent
bio-interaction of the maltose-modified PPI dendrimers was observed where only
the 4th and 5th generation can undergo bio-interactions. Thus, one tendency was received
from the various experiments that the biological properties of cationic dendritic polymers
can be substituted by the dominant non-specific H-bonding properties of the oligosaccharidemodified
counterparts. Also, latest results will be presented and discussed.
Dr. Dietmar Appelhans
Leibniz Institute of Polymer Research Dresden
Institute of Makromolecular Chemistry
Department of Polymer Structure
applhans@ipfdd.de
www.ipfdd.de
Diagnostics
230
162 Development of an ELISA and a Candidate Vaccine
for Pigeon Circovirus Infection
Mohammad Yahya Halami, Wieland Schrodl, Reimar
Johne, Erhard F. Kaleta, Hermann Muller
Pigeon circovirus (PiCV) is the causative agent of young pigeon disease syndrome, a
multifactorial disease complex associated with severe immunosuppression, high morbidity
and variable mortality rates. PiCV cannot be propagated in vitro, thus rendering antigen
production for immunodiagnostic tests and vaccines rather difficult.
In order to develop reliable diagnostic tools and vaccines, a truncated PiCV capsid protein
C1 was expressed in E. coli and purified using a histidine tail. The N-terminal truncation was
introduced to increase the yield of the recombinant protein. For detection of PiCV-specific
antibodies, an ELISA protocol was developed using the purified PiCV C1 protein as antigen
and anti-pigeon IgY antiserum elicited in rabbits as a secondary antibody. Testing of field sera
derived from pigeon in the years 1982, 1993 and 2006 revealed a relative constant proportion
of positive sera, thus indicating circulation of PiCV in pigeon aviaries for at least 25 years.
The immunogenicity of the antigen was evaluated by inoculation of pigeons with the purified
PiCV C1 protein and seroconversion could be demonstrated using the developed ELISA.
It is concluded that the recombinantly expressed truncated C1 protein of PiCV can successfully
be used for serodiagnosis of PiCV infections and has a good immunogenicity in pigeons.
Further investigations will focus on the testing of this protein in a vaccine against the severe
disorder caused by PiCV, which is urgently needed.
Mohammad Yahya Halami
Universitat Leipzig
Faculty of Veterinary Medicine
Institute for Virology
halami@vetmed.uni-leipzig.de
www.vmf.uni-leipzig.de/ik/wvirologie/index
231
Diagnostics
163 Development and fabrication of a novel proteinbased
biosensor for specific detection and
immobilisation of cells
Anja Steude, Oliver Panke, Sabine Schmidt, Matthias
Nieber, Andrea A. Robitzki
Electrochemical biosensors, using multielectrode arrays with immobilised proteins as
recognition layer, constitute a promising tool for diagnostics. Compared to conventional
biochemical assays, electrochemical methods offer essential advantages, such as label-free,
real-time, and non-destructive detection of the analytes. By applying multielectrode arrays,
which were analysed by impedance spectroscopy and cyclic voltammetry, high-throughput
measurements become feasible. The presented project follows a twofold aim: the development
of a novel protein-based biosensor for the detection of marker proteins of specific target cells,
and secondly the selective immobilisation of target cells from a biological sample, which
permits further analyses. A first prototype was designed, consisting of nine gold working
electrodes and nine platinum auxiliary electrodes in a 96-well scale. For its fabrication,
methods of photolithography, alternating current sputtering, and etching techniques were
applied. Nine separate measurement chambers were implemented as well as an Ag/AgCl
reference electrode. The validation of the multielectrode array and the reference electrode
was carried out using impedance spectroscopy and cyclic voltammetry. Furthermore a
surface modification of the working electrode with thiols could be achieved and detected with
these measuring techniques. The next milestone will be a covalent immobilisation of specific
antibodies on the working electrode using the thiol self-assembled-monolayer as an interlink.
With this antibody-based biosensor the capture of specific cells will be investigated.
Anja Steude
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Division of Molecular Biological-Biochemical Processing
Technology
anja.steude@bbz.uni-leipzig.de
www.uni-leipzig.de/~dmpt
Diagnostics
232
164 Mycotoxin determination by means of an
electronic nose
Anselm Werner, Claudia Winter, Monika Kruger, Andrea
Lindner, Klaus Kruger
Intoxications by Fusarium associated mycotoxins increase worldwide and lead to diseases,
reduced feed consumptions and animal yielding. Therefore the early identification of the
mycotoxin loads of grains is very important information for the feed industry and the farmers.
The aim of our investigations was to identify volatile substances produced by F. graminearum
during mycotoxin generation analysable by electronic nose to select and exclude such
grains.
Washed and in aqua dest until saturation soaked specimens of wheat, maize and rice were
autoclaved and inoculated with conidia of three different F. graminearum strains. Weekly until
4 weeks of cultivation specimens were investigated for Deoxynivalenon (DON), Fumonisin
(FUM) and Zearalenon (ZEA) by ELISA and 0, 5 . 1 ml of gas phase of these cultures were
taken off and analysed by electronic nose (PEN3, Airsense) with 10 semiconductive sensors
to get analyses about volatile components correlating with mycotoxin expression. Until now
we can show that there is not only the possibility to discriminate between spoiled and not
spoiled cereals but also between mycotoxin and non mycotoxin contaminated probes in a
however weak manner. Further studies are necessary to optimise the method and to confirm
the capacity of discrimination.
Anselm Werner
Universitat Leipzig
Faculty of Veterinary Medicine
Institute for Bacteriology und Mykology
anselm_werner@yahoo.de
www.vmf.uni-leipzig.de/ik/wbakteriologie
233
Diagnostics

10. Microfluidics
Posters

237
Microfluidics
165 Structural levels of organization in the TmHUDNA-
complex as studied by optical tweezers
assisted Force spectroscopy
Carolin Wagner, Mathias Salomo, Friedrich Kremer
The interaction of the histone-like protein TmHU (from Thermotoga maritima) to DNA is
analyzed on a single molecule level by use of optical tweezers. This technique provides a
nm-resolution in positioning a micron-sized colloid and an accuracy of } 50 fN in measuring
the forces acting on it. As a further refinement, our set-up is now accomplished with a fast
feed-back loop (regulation frequency: 30 Hz) which allows to carry out the experiment under
conditions of a constant and adjustable force. The proceeding of the condensation and its
dependence on the applied force (2 . 40 pN) is investigated. At a pre-stretching of 2 pN the
length of the DNA is reduced by about 80%. At higher forces, the reaction is disrupted at
an incomplete level. The process shows two distinct regimes that can be related to different
organizational levels. The condensation also shows a pronounced dependence on the
concentration. By stretching the TmHU/DNA-complex, it is possible to disrupt the proteins
from the DNA. The length of the smallest event conforms with the results of a simulated
rupture.
Carolin Wagner
Universitat Leipzig
Faculty for Physics and Earth Sciences
Department of Molecular Physics
Wagner.Carolin@gmx.net
www.uni-leipzig.de/~mop/
Microfluidics
238
166 O ptical tweezers to investigate receptor-ligand
interactions on a single contact level
Carolin Wagner, Mathias Salomo, Friedrich Kremer
The extraordinary features of optical tweezers having a nm-resolution in positioning a
micron-sized colloid and an accuracy of (} 50 fN) in measuring the forces acting on it, enable
one to study the interaction within a single receptor/ligand-contact. We introduce a newly
developed assay using optical tweezers to investigate the interactions between Protein A from
Staphylococcus aureus and Immunoglobulin G from rabbit serum (RIgG). The the rupture
forces depend on the loading rate and on the sodium chloride concentration. The measured
loading rate effect is well known in the literature and the data we obtained were found to be in
good agreement with an already published theoretical model. The dependence of the rupture
forces on the salt concentration demonstrates the influence of hydrophobic interactions on
the bond strength. Our experimental setup can probe the interaction between a single receptor
and its specific ligand under changing conditions and hence offers manifold applications in
single molecule biotechnology.
Carolin Wagner
Universitat Leipzig
Faculty for Physics and Earth Sciences
Department of Molecular Physics
Wagner.Carolin@gmx.net
www.uni-leipzig.de/~mop/
239
Microfluidics

Protein Engineering
and Biocatalysis
11.
Posters

243
Protein enginering & Biocatalysis
167 Evaluating 3D experiments in optical tweezers
Marcel Ander
Our setup comprises a single optical trap which works close to the surface of a microfluidic cell
allowing the surface to be coated with a large number of sample molecules. These molecules
can be readily scanned simplifying the task of finding a sample. We currently look at DNA
molecules and tether them covalently to a microsphere and via a Biotin-Neutravidin-complex
to the surface of the microfluidic cell. As a consequence, all three dimensions of space need to
be considered during stretching experiments. We found a practical method which allows for
most of the side effects observed during 3D stretching. We show this by simulating a DNAstretching
experiment based on published DNA parameters: Considering solely constants
obtained by calibration we get a worthwhile agreement between simulated and measured
data. This shows that discarding dimensions for experimental handling can be circumvented
and, thus, an optical tweezerse potential for 3D manipulation and 3D measurement can be
fully utilized and evaluated without additional detection components.
Utilizing this method and robustness of covalent coupling, we conducted repeated stretching
experiments with DNA. The force necessary to move the microsphere handle to the same
position again and again has a tendency to remain constant or decrease due to force-induced
DNA-strand dissociation events. However, when the homologous-recombination protein
RedƒÀ is present a significant increase of more than 10 pN can be found.
Marcel Ander
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Nanomechanics Group
marcel.ander@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Protein enginering & Biocatalysis
244
168 Reconstituting Cytokinesis in Artificial Lipid
Systems
Senthil Arumugam
To quantitatively understand cellular processes, bottom-up approaches involving cell-free
reconstitution of a minimal set of individual building blocks are powerful. They offer a very
simple, interference free system to exclusively look at dynamics, activity and interaction of
specific proteins or molecules. In the niche of membrane protein interactions, where it is
often difficult to tease apart the functions of individual proteins owing to complex factors and
molecular players involved, the bottom-up approach has been most befitting and rewarding.
The process of bacterial cytokinesis still remains elusive to a great extent. One of the
main problems of studying the proteins involved in the process is the size of bacteria. The
reconstitution approach therefore offers a practical solution to this. The various approaches
taken to reconstitute the process in a suitable lipid system is presented.
Senthil Arumugam
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Biophysics Group
senthil.arumugam@biotec.tu-dresden.de
www.biotec.tu-dresden.de
245
Protein enginering & Biocatalysis
169 Variants of Candida antarctica lipase B convert
ƒ¿-substituted substrates
Sally Bayer, Thomas Greiner-Stoffele, Meike Ballschmiter
The lipase B (CalB) from Candida antarctica is one of the best analyzed and industrially
most used enzymes. This protein catalyzes a great number of different reactions, including
enantio- and regioselective conversions. CalB is applied in the modification of fats and oils.
Furthermore it is used in the synthesis of structured lipids and expands in the production of
biodiesel. In addition to its natural substrates, CalB synthesizes a very broad range of synthetic
products applied in flavour and fragrance industries. The most impressive application of
CalB is the enantioselective resolution of racemic substrates whereupon this lipase shows
outstanding qualities concerning substrate spectrum and conversion rates.
The combination of selectivity and a broad substrate range is attributed to structural
aspects like the available space in the active site pocket of CalB. The aim of this study
was to enlarge the active site pocket of CalB in order to change the enzymes selectivity
regarding the conversion of ƒ¿-substituted substrates. For this purpose the effect of
mutating several amino acids close to the active centre was investigated. By rational
protein design of CalB two variants were found converting ƒ¿-substituted substrates.
Enhancing the hydrolysis of ƒ¿-substituted substrates is most interesting in regard of the
conversion of ibuprofen, since only the S-enantiomer is responsible for analgic effects.
Sally Bayer
Universitat Leipzig
Institute of Biochemistry
Junior Research Group .Protein Engineeringg
sbayer@rz.uni-leipzig.de
www.biochemie.uni-leipzig.de
Protein enginering & Biocatalysis
246
170 Expression, Purification and Characterization of
the Neuropeptide Y Receptor Type 2
Sandra Berndt, Peter Schmidt, Cindy Montag, Christian
Berger, Susann Schimmer, Diana Lindner, Annette G. Beck-
Sickinger, Rainer Rudolph, Daniel Huster
The Neuropeptide Y Receptor type 2 (Y2R) is a G protein-coupled receptor (GPCR). GPCRs
are integral membrane proteins with 7 transmembrane helices and they are the key players in
signal transduction. Over 50 % of all modern drugs interact with these receptors. It is necessary
for the regulation of pain, food intake, the tendency for alcohol and cocaine dependence and
also for the neurotransmitter release.
We are able to produce large amounts (~25 mg/l) of the target receptor in a prokaryotic
expression system as inclusion bodies. These protein aggregates were isolated, solubilized
in SDS-micelles and purified by gelfiltration and affinity chromatography. Then the receptor
has to be refolded in a functional state. Various parameters were optimized in refolding, such
as mix of detergents, additives, redox-shuffling system and concentrating strategy. So far we
achieve a final concentration of 54 ƒÊM pure and refolded receptor. With CD measurements
we could show that the receptor has a high ƒ¿-helical content in the folded as well as in the
unfolded state.
To proof the functionality of the refolded receptor we use 3H labeled NPY for ligand-binding
assays. Here we could determine an IC50 value of (8.5 } 2) nM and a KD value of (3.8 } 0.9)
nM. Further with SDS-PAGE, Western-Blot and analytical gelfiltration we could show that
the receptor forms mainly dimers upon folding. Until now, we donft know if this dimerization
is necessary for functionality. This will be clarified by ligand binding experiments with the
separated fractions.
Sandra Berndt
Universitat Leipzig
Faculty of Medicine
Medical Physics and Biophysics
sandra.berndt@student.uni-halle.de
www.uni-leipzig.de/~biophys
247
Protein enginering & Biocatalysis
171 Construction of a RNaseT1 expression system for
Aspergillus niger
Kathrin Bonsch, Thomas Greiner-Stoffele, Meike Ballschmiter
Industrial enzymes and proteins produced by genetic engineering are used in a wide field, e.g.
medical and industrial applications. Thus, recombinant proteins have brought modern society
many benefits. Various protein expression systems for microorganism, for insect cells and
for plant hosts were developed in order to produce such valuable proteins more efficiently.
Filamentous fungi, including members of the genus Aspergillus, are considered an attractive
resource as expression host as well. They are capable to produce and secrete large amounts
of specific proteins and they perform posttranslational modifications, which are essential for
a many proteins. For commercial processes, yields of >30 g/l of a specific protein are not
uncommon.
In our group we designed a new expression system for the fungus Aspergillus niger, including
the development of high transformation methods and the application of different promoters.
The A. niger systems were tested by the heterologous expression of ribonuclease (RNase) T1
from Aspergillus oryzae. Often the yields of secreted recombinant proteins are low compared
with the yields of homologous proteins. In most cases the expression level does not exceed
a few milligrams per liter of culture medium. In order to overcome this bottleneck we have
additionally developed various improvement strategies for the overproduction of RNaseT1 in
A. niger and we will further establish a fermentation protocol.
Dr. Kathrin Bonsch
Universitat Leipzig
Institute of Biochemistry
Junior Research Group .Protein Engineeringg
boensch@rz.uni-leipzig.de
www.biochemie.uni-leipzig.de/nwg_wb
Protein enginering & Biocatalysis
248
172 Torque Measurements on DNA with Magnetic
Tweezers
Hergen Brutzer, Nicholas Luzzietti, Friedrich Schwarz, Ralf
Seidel
In contrast to its well-characterized stretching and bending behavior, the response of DNA
upon twisting is less understood. A complete description of DNA mechanics must also
consider the effects of torque. Therefore we are developing a technique which allows us to
measure the torque acting on a single DNA molecule while simultaneously monitoring the
applied force. Using a magnetic tweezers setup to manipulate the DNA molecule, the torque is
directly calculated from the angular motion of a fluorescent particle internally attached to the
DNA. The required internal modification of the double-stranded DNA is realized by nicking
one strand at multiple sites and replacing this region with a biotinylated oligonucleotide.
The fluorescent bead is bound via streptavidin to the DNA and visualized in the magnetic
tweezers setup with a TIRF (total internal reflection fluorescence) microscope. Besides DNA
supercoiling, we will study the torque acting during unfolding of single chromatin fibers.
Hergen Brutzer
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
DNA Motors Group
Hergen.Brutzer@biotec.tu-dresden.de
www.biotec.tu-dresden.de/seidel
249
Protein enginering & Biocatalysis
173 Tagging methods for proteomics and regulomics
in mouse embryonic stem cells
Giovanni Ciotta, A. Francis Stewart
Proteomic approaches in mammalians require simple and specific protein purification
methodologies that are amenable to high-throughput approaches for the isolation of protein
complexes. The most prominent technique used to pull down protein complexes is the
tandem affinity purification (TAP) tag method, so far successfully applied in yeast but still
inefficient in mammalians. Here, we describe an approach for a single-step purification of
protein complexes based on Green Fluorescent Protein (GFP). The GFP tag was fused to the
C-terminus of Ash2l, a component of histone H3K4 methyltransferase complexes. The fusion
protein was expressed in the E14tg2A mouse embryonic stem cell line. From our results we
can conclude that the GFP tag altered neither the factorfs protein interactions or DNA binding
properties in vivo nor its sub-nuclear distribution.
Therefore, GFP tag provides a promising basis for the analysis of the mammalian proteome.
Giovanni Ciotta
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Genomics Group
giovanni.ciotta@biotec.tu-dresden.de
www.biotec.tu-dresden.de
Protein enginering & Biocatalysis
250
174 Two ways to screen for new proteases in (meta-)
genomic libraries
Antje Eichler, Thomas Greiner-Stoffele, Meike Ballschmiter
Proteases are a ubiquitous enzyme family with the property to degrade proteins into small
peptide fragments or to activate protein precursor sequences. They have a wide range of
applications in industrial processes like food, beverage and detergent industry or in the
medical sector. For this reason there is the need to search for proteases with specific activities
and new characteristics for industrial applications. One way to screen for new and unknown
proteases is the cluster screening system [1]. The system is able to test hundreds of clones
simultaneously for the desired activity.
Proteases are often toxic and thus their expression is potentially lethal for expression hosts like
Escherichia coli. A system to circumvent this problem is the in vitro expression of proteins. In
vitro transcription/translation avoids insoluble inclusion bodies, reduced expression of toxic
proteins or the need for a functional secretion system. Another way to screen for proteases is
the expression and secretion of proteins in Bacillus subtilis. Expressed proteins are secreted
into the surrounding media and directly detected in a protease specific assay. This way the
formation of inclusion bodies and the potential host cell death are avoided. Both systems are
used in combination with the cluster screening approach for the screening of (meta-) genomic
libraries to find and identify proteases.
Antje Eichler
Universitat Leipzig
Institute of Biochemistry
Junior Research Group .Protein Engineeringg
eichler@uni-leipzig.de
www.biochemie.uni-leipzig.de
251
Protein enginering & Biocatalysis
175 Pseudomonas putida . development of a
heterologous expression system for complex
natural products
Frank Gros, Dominik Pistorius, Youming Zhang, A. Francis
Stewart, Rolf Muller
Microorganisms produce an immense number of secondary metabolites, which are often
used as therapeutics in medicine or agrochemicals. These secondary metabolites span a wide
range of chemical classes in which polyketides and nonribosmal peptides are two prominent
representatives. Both types of natural products are synthesized by megasynthases, polyketide
synthases and nonribosomal peptide synthetases, respectively, which show an analogous
enzyme build up and reaction scheme.
In recent years the growing number of sequenced microorganisms revealed a large number of
gene clusters, which are not expressed under conditions so far administered in the laboratory.
In addition to these, one has also to consider that the number of microorganisms we can
cultivate in the lab is believed less than 1% of the existing species. Their biosynthetic potential
can be discovered using metagenomics.
In both cases there is a need for suitable heterologous expression systems. We chose
Pseudomonas putida as a heterologous host because the genus itself is a producer of
secondary metabolites; it harbors the necessary PPTase with broad substrate specificity
for posttranslational activation of PKS and NRPS and has a similar GC content to the
two prominent bacterial families producing secondary metabolites streptomycetes and
myxobacteria.
We already developed a strain harboring the methylmalonyl-CoA biosynthesis genes
of Sorangium cellulosum So ce56 and showed itfs value by expressing the myxothiazol
biosynthetic gene cluster in this strain. We improved this strain and present first results for
the new strains regarding their biosynthesis capacity.
Dr. Frank Gros
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Genomics Group
frank.gross@biotec.tu-dresden.de
www.biotec.tu-dresden.de/stewart
Protein enginering & Biocatalysis
252
176 Prediction of Flocculation Ability of Brewing Yeast
Inoculates by Flow Cytometry, Proteome Analysis,
and mRNA Profiling
Franziska Heine, Frank Stahl, Heike Strauber, Claudia
Wiacek, Dirk Benndorf, Cornelia Repenning, Frank Schmidt,
Thomas Scheper, Martin von Bergen, Hauke Harms, Susann
Muller
The ability of brewing yeast to flocculate is an important feature for brewing of qualitatively
good beer. Flocculation involves two main cell wall structures, which are the flocculation
proteins (flocculins) and mannans, to which these flocculins bind. Unfortunately, in practice,
the flocculation ability may get lost after several repitches. Flow cytometry was employed
to analyze glucose and mannose structures of the cell surface by application of fluorescent
lectins. Validation of the expression of the flocculin genes Lg-FLO1, FLO1, FLO5, and FLO9
was carried out using microarray techniques. SDS-PAGE, western blot, and ESI-MS/MS
analyses served to isolate and determine yeast cell flocculins. Mannose and glucose labeling
with fluorescent lectins allowed differentiating powdery and flocculent yeast cells under
laboratory conditions. Using microarray techniques and proteomics, the four flocculation
genes Lg-FLO1, FLO1, FLO5, FLO9, and the protein Lg-Flo1p were identified as factors of
major importance for flocculation. The expression of the genes was several times higher in
flocculent yeast cells than in powdery ones. Flow cytometry is a fast and simple method to
quantify the proportions of powdery and flocculent yeast cells in suspensions under defined
cultivation conditions. However, differentiation under industrial conditions will require
mRNA and protein expression profiling.
Franziska Heine
Helmholtz Centre for Environmental Research (UFZ)
Environmental Microbiology
Franziska.Heine@ufz.de
www.ufz.de
253
Protein enginering & Biocatalysis
177 Reduction of substrate binding pocket of glucose
dehydrogenase B for improved substrate
specificity
Michael Hofer, Kathrin Bonsch, Meike Ballschmiter
The enzyme gPyrroloquinoline quinone dependent glucose dehydrogenase Bh (PQQ GDH B)
is frequently used as an enzymatic biosensor for measuring blood glucose levels. Compared
with other available enzymes for enzymatic glucose biosensors, PQQ GDH B has a high
turnover number and is not influenced by oxygen, which makes it faster and more reliable.
Beside these beneficial properties, there are still some features of PQQ GDH B which are
not perfect for its use as enzymatic glucose biosensor. One of these features is the substrate
specificity of PQQ GDH-B. The wild type enzyme of PQQ GDH-B isolated from Acinetobacter
calcoaceticus, shows a broad substrate specificity. Besides monosaccharides like glucose or
D-xylose there are also several disaccharides which are recognized as substrate. Especially
the maltose activity, leads to imprecise blood glucose measurements. Several efforts have
already been made to improve substrate specificity of PQQ GDH B.
We focus on designing new variants of PQQ GDH B with reduced maltose affinity. Therefore
we constructed a so called structure library of PQQ GDH B where we tried to minimize the
substrate binding pocket, so that glucose can still bind but not maltose. Amino acids which
form the substrate binding pocket of PQQ GDH B were exchanged by amino acids which are
larger or have side chains, able to interfere with the substrate. This library was then screened
with the patented cluster screening approach for PQQ GDH B variants with reduced maltose
affinity.
Michael Hofer
Universitat Leipzig
Institute of Biochemistry
hofer@uni-leipzig.de
www.biochemie.uni-leipzig.de
Protein enginering & Biocatalysis
254
178 Screening for indole hydroxylating variants of
P450cam
Gregor Hoffmann, Katrin Bonsch, Meike Ballschmiter
P450 monooxygenases catalyse the stereo- and regiospecific insertion of one molecule of
oxygen at inactivated carbon atoms, opening new and interesting prospects for the application
of P450 in chemical industries. But just for a minor part of possible tasks the established P450
enzymes offer adequate solutions. There are two ways to find a proper catalyst. One way is
the extensive search for new P450-activities in the overwhelming variety of uncultivable
microorganisms promising to deliver an enzymatic solution for nearly every chemical
problem. The second way is to use direct evolution methods to generate a library of variants
of a already known P450 and to alter the specificity for a non-natural substrate.
In our group both paths were taken. Here we present the direct evolution of camphor
hydroxylating P450 from Pseudomonas putida to new variants able to hydroxylate indole. A
specificity library was constructed by selecting nine residues in the active site and exchanging
them randomly for three to six defined amino acids. This resulted in a library of approximately
300.000 variants. The hydroxylation of indole leads to the spontaneous formation of indigoid
pigments. We established a screening system based on solid media. The necessary electron
transport proteins putidaredoxin reductase and putidaredoxin were coexpressed in the E. coli
screening host. Positive colonies were identified by there brownish to bluish colour. Hits
were sequenced, expressed, purified by his tag affinity chromatography and characterised by
determining the specific activity, coupling efficiency and product spectra.
Gregor Hoffmann
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Biochemistry
Junior Research Group .Protein Engineeringg
gregor.hoffmann@bbz.uni-leipzig.de
www.biochemie.uni-leipzig.de
255
Protein enginering & Biocatalysis
179 Interactions in the plant RNase P/ MRP
Mario Krehan, Sebastian Braun, Janine Dahl, Nicolas
Menzel, Christian Heubeck, Astrid Schon
RNase P is the enzyme responsible for the 5f maturation of all pre tRNAs and thus essential
for cellular protein biosynthesis. This ribonucleoprotein enzyme is composed of one RNA
and several proteins in all eukaryote organisms.
In plants no RNase P RNA but two RNase MRP RNA variants and several RNase P protein
subunits could be identified in silico. The RNase MRP is a narrow relative to RNase P and
shares up to 8 protein subunits with it but cleaves other RNA substrates.
Our aim is to understand composition and function of RNase P and MRP in plants. We have
therefore identified and cloned a number of protein subunits and the two RNase MRP RNA
variants from the plant model organism A. thaliana. Using a binding assay, we could analyse
the interactions between these two RNAs and the RNase P/MRP proteins ATPOP1p and
ATRpp38p, respectively. Interaction studies with other putative RNase P/MRP proteins, as
well as with substrate pre-tRNA, are in progress.
Mario Krehan
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Molecular Cell Therapy
mario.krehan@bbz.uni-leipzig.de
www.mitonet.de
Protein enginering & Biocatalysis
256
180 The crystal structure of arylmalonate
decarboxylase reveals active site flexibility in
catalysis
Bartholomeus Kuttner, Markus Kircher, Susann Rosmus, Antje
Keim, Norbert Strater
The enzyme arylmalonate decarboxylase (AMDase, molecular weight of 24 kDa) from the
soil bacterium Alcaligenes bronchisepticus catalyzes the stereoselective decarboxylation of
arylmalonates to carbon dioxide and arylpropionates in a cofactor independent reaction. In the
active site, cysteine 188 is responsible for the enantioselective decarboxylation yielding the R
form propionates. The decarboxylation reaction of AMDase is of interest for the synthesis of
fine chemicals. Therefore, elucidating the three-dimensional crystal structure is a vital point
for rational enzyme design.
AMDase shows a typical ATC (aspartate transcarbamoylase)-like fold. In all monomers
two (148,188) of four cysteine residues are covalently modified by solvent molecule
ƒÀ-mercaptoethanol. The four monomers in the asymmetric unit show large deviations of
more than 10 A in loops surrounding the active site which indicates structural transition
intermediates in the catalytic cycle. Database searches revealed the protein ST0656 from
Sulfolobus tokodaii (PDB: 2DGD) and aspartate racemase from Pyrococcus horikoshii (PDB:
1JFL) as closest structural relatives.
Dr. Bartholomeus Kuttner
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Bioanalytical Chemistry
kuettner@uni-leipzig.de
www.uni-leipzig.de/bbz/~straeter
257
Protein enginering & Biocatalysis
181 RiboxX: RNA-interference in a box!R
Jacques Rohayem, Katrin Jager, Ivonne Robel, Kristin Hille,
Dorothea Kramer, Romy Zieger, Mirko Bergmann, Julia
Gebhardt, Christiane Petzold
The RNA-interference Technology (RNAi-Technology) is one of the major biotechnologies
of the 21st century, being broadly used in life sciences. The RNAi-Technology allows the
specific and efficient knockdown of gene expression. So far, scientists using the RNAi-
Technology rely on companies offering synthesis, purification and analysis of the RNAimolecules
with a variable quality and at elevated costs. This leads in turn to dissatisfied
scientists being however totally dependent on the .good willg of commercial suppliers as to
purity, quality and quantity of the RNAi-molecules, and paying a lot for a product they cannot
even assess by themselves.
RiboxXR offers life scientists for the first time the possibility to design, synthesize, purify and
analyse RNAi-molecules easily by themselves, with high quality and flexibility. The RiboxXproducts
are RNAi-tools offered as toolboxes or gkitsh. RiboxX offers a complete solution
of RNAi-tools encompassing synthesis, purification and analysis of RNAi-molecules. The
RiboxX-products are easy to handle, flexible, robust and highly qualitative. The slogan of
RiboxX is .RNAi-SYNTHESIS + PURIFICATION + ANALYSIS = DO IT YOURSELF
AND BETTER!g
PD Dr. Jacques Rohayem
Technische Universitat Dresden
Institute for Virology
Jacques.Rohayem@tu-dresden.de
www.tu-dresden.de/medviro
Protein enginering & Biocatalysis
258
182 Mutagenesis of the Thermus aquaticus
amylomaltase to produce large cyclic glucans
Christian Roth, Nicole Weizenmann, Wolfgang
Zimmermann, Norbert Strater
Cyclodextrins are cyclic glucans with a degree of polymerization (dp) ranging from 4 to
100.
They are used in numerous applications, for example in cosmetics as stabilizing agents,
odour suppressants or flavoring agents. They are also in widespread use in pharmacy as
complexing agents for hydrophobic pharmaceuticals to improve their bioavailability and
controlled release. Commonly used cyclodextrins are the ƒ¿-, ƒÀ- and ƒÁ-subtypes because they
can be produced easily by enzymatic treatment of starch. So far it is difficult to produce
cyclodextrins with a dp higher than the ƒÁ-subtype in sufficient quantities for industrial
purposes. The amylomaltase from T. aquaticus is able to produce cyclodextrins with a dp
up to 70. Such CDs provide larger or multiple cavities and could enhance may complex
larger guestmolecules. Crystallographic studies have suggested structural determinants
for the formation of large cyclodextrins by T. aquaticus amylomaltase. Variants should be
generated to identify important factors influencing the cyclization reaction and a variant with
improved cyclization activity shall be developed. We have generated several variants with
point mutations, insertions and deletions. All mutants have been cloned and overexpressed in
E.coli Bl21. All mutants show heat resistance comparable to the wildtype enzyme indicating
the stability of the core fold. The deletion variants 250 and 460 were purified and crystallized.
For both mutants a crystallographic dataset was collected. The structure for the 250-variant
was determined and structural features which might responsible for the reduced activity were
identified.
Christian Roth
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
Institute of Bioanalytical Chemistry
Christian.roth@bbz.uni-leipzig.de
www.uni-leipzig.de/bbz
259
Protein enginering & Biocatalysis
183 NMR Measurements of a Class A GPCR from
Prokaryotic Expression
Peter Schmidt, Andreas Bunge, Diana Lindner, Sandra
Berndt, Christian Berger, Annette G. Beck-Sickinger, Rainer
Rudolph, Daniel Huster
G protein-coupled receptors (GPCRs) are a class of membrane proteins that represent a major
target for pharmacological developments. However, there is still little knowledge about GPCR
structure and dynamics since high-level expression and characterization of active GPCRs in
vitro is extremely complicated.
Here, we show solution and solid-state NMR measurements from the GPCR neuropeptide
Y receptor type 2 in comparison to measurements of bovine rhodopsin. After high yield
expression in E. coli as inclusion bodies 15N labelled receptor was solubilized with the strong
ionic detergents SDS. In this micellear state the receptor shows no binding to its natural ligand
NPY. However, it is already structured very well, what is shown in 1H-HSQC spectra, but has
a lower dispersion than active rhodopsin. After refolding and reconstitution into lipid bilayer
high specific binding of the ligand to the receptor was detectable. Also, in cp spectra could be
shown that the dispersion of the reconstituted receptor in the 15N projection is comparable
to active rhodopsin. From that it can be concluded, that the receptor is properly structured
in its native form. Since the receptor is stable for at least 12 days and highly concentrated in
lipid bilayer with a protein/lipid ration of 1/200 structure determination experiments may be
performed with 13C labeled GPCR.
Peter Schmidt
Universitat Leipzig
Faculty of Medicine
Medical Physics and Biophysics
peter.schmidt@biochemtech.uni-halle.de
www.uni-leipzig.de/~biophys
Protein enginering & Biocatalysis
260
184 Single-Molecule Studies of DNA Translocating
Restriction Enzymes
Friedrich Schwarz, Kara van Aelst, Mark Szczelkun, Ralf
Seidel
Restriction enzymes (REs) are the central part of the bacterial defence system against
invading viruses. These protein complexes recognize viral DNA by the methylation state of
their target sequence and destroy it by cleaving it into pieces. For this, the majority of REs
need to interact with two distant target sites. This long-range inter-site communication can
be accomplished either by passive 3D diffusive looping or by 1D motion along the DNA
contour. Among the different classes of REs, Type I and Type III play a special role due to
their helicase domains, which are key to the inter-site communication.
For Type I REs it is established that the helicase domain acts as a dsDNA translocating motor.
Cleavage is triggered after a pure 1D communication process, when two translocating motors
from distant target sites collide. However details of the actual cleavage-collision process
still remain unclear. In comparison, the communication mechanism for Type III REs has not
been accurately defined and conflicting models including 3D diffusion and 1D translocation
have been proposed. Our recent findings suggest that Type III REs move along DNA by
diffusion.
In order to explore the cleavage-collision process and to test the diffusion hypothesis we
started to track the movement of Type I and III REs along DNA using a setup combining
magnetic tweezers with single-molecule fluorescence.
Friedrich Schwarz
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
DNA Motors Group
friedrich.schwarz@biotec.tu-dresden.de
www.biotec.tu-dresden.de
261
Protein enginering & Biocatalysis
185 Immobilization of Proteins via Cu(I) catalyzed
Alkyne Azide Cycloaddition
Max Steinhagen, Annette G. Beck-Sickinger
The 1,3 dipolar cycloaddition between alkynes and azides was first developed by Huisgen
and coworkers. In 2002 the group of Meldal reports on the stereoselective formation of the
1,4 isomer by using Cu(I) as catalyst. The usage of Cu(I) decreases the reaction time, leads
to only one product and can be used with mild reaction conditions (aqueous buffer, room
temperature). Therefore this reaction was described as the first gclick reactionh.
We expressed the proteins aldo-keto reductase 1A1 (AKR1A1) and the green fluorescent
protein (GFP) as fusion proteins in E.coli by using the IMPACTR system (Intein mediated
purification with an affinity chitin-binding tag). Furthermore, the short tripeptide Cys-Lys-
Pra (Pra = propargylglycine) was synthesized by using solid phase peptide synthesis (SPPS).
After cleavage of the target proteins from the chitin beads the resulting thioesters were ligated
to the tripeptide via native chemical ligation (NCL). The alkyne function of the C-terminal
propargylglycine was then used to gclickh the ligation product to an azided PEGA-resin by
CuAAC. The proteins were characterized by MALDI-TOF analysis, kinetic assay and SDSPAGE.
The characterization of the peptide was realized by MALDI-TOF and HPLC. The
immobilized proteins could be identified by ELISA and tryptic digest followed by peptide
Mascot fingerprint analysis.
We could achieve a successful immobilization of the proteins AKR1A1 and GFP on the
PEGA resin. Accordingly, CuAAC was identified as an additional ligation method.
Max Steinhagen
Universitat Leipzig
Institute of Biochemistry
Research group of Biochemistry and Biorganic
Chemistry
msteinha@uni-leipzig.de
www.biochemie.uni-leipzig.de

12. Appendix

265
Center for Biotechnology and Biomedicine (BBZ)
At the Center for Biotechnology and Biomedicine (BBZ) new methods and technologies
are combined at the interface of molecular cell biology and genetics with nanotechnology,
biophysics, (nano) medicine, pharmacy, biochemistry, bioinformatics and bio medical
engineering. Multi-, trans- and interdisciplinary groups of researchers with scientific excellence
and expertise address current and future-oriented aspects in the area of nanobiotechnology and
biomedicine. During the last few years innovations were implemented by the establishment
of technology lines and research projects focussing on therapies and diagnostic methods,
bioinstruments, biophysical test procedures and tissue replacement. New approaches are found
not only in the traditional fields of biology, biochemistry, bioinformatics and biophysics, but
also in the evolving areas at the edge of these classical disciplines. Scientists of different
fields pay attention for example to protein engineering for tumor therapy, the development of
in vivo disease models, the biosensor technology for diagnostics and active substance testing
as well as the bio-reactor development for tissue and organ engineering. Beside the versatile
expertise in the red biotechnology and biomedicine, the white biotechnology (bio catalysis)
is an established second main focus. The expertise of scientists in Leipzig in the protein
technology (protein expression, structure analytics, protein modification, bioanalytics, protein
detection and protein design) plays thereby a substantial role as a link between the combined
development of red and white biotechnology.
At the end of 2007 the Saxon Ministry of Science and the Fine Arts agreed with the University
of Leipzig upon the further infrastructural extension and the realization of projects and
investments with the focus topic 'THERANOSTIK . therapy and diagnostics of the future
with specialization, visualization and miniaturization: Active agents and cells as products and
instruments'. The BBZ is responsible for the coordination of the THERANOSTIK project.
The agreement defines topic-bound goals, which are to be accomplished until 2013. Innovative
and cooperative joint ventures started in 2008 on the following areas:
. Research and development and validating of tools and technologies for high throughput
screening/- diagnostics and rational active agent identification
. Development of bioactive, intelligent (micro) implants and cell transplants for repair,
regeneration and control of biological processes
. genetic reprogramming of cells, cell lines and stem cells for the treatment of inherited
or acquired diseases
. Investigation of molecular causes and development of therapy strategies for infectious
and neurodegenerative diseases, in particular Alzheimerfs disease
266
The BBZ harbors the following members:
From the Faculty of Biology, Pharmacy and Psychology:
- Prof. Dr. Annette Beck-Sickinger, Biochemistry / Bioorganic Chemistry
- Prof. Dr. Sunna Hauschildt, Immunbiology
- Prof. Dr. Karen Nieber, Pharmacology for Natural Scientists
- Prof. Dr. Andrea Robitzki, Molecular Biological-Biochemical Process Technology
(BBZ Chair)
- Prof. Dr. Martin Schlegel, Molecular Evolution and Animal Systematics with focus on
Molecular Phylogeny
- Prof. Dr. Christian Wilhelm, Plant Physiology
From the Faculty of Chemistry and Mineralogy:
- Prof. Dr. Stefan Berger, Analytical Chemistry
- Prof. Dr. Athanassios Giannis, Organic Chemistry / Natural Products Chemistry
- Prof. Dr. Evamarie Hey-Hawkins, Inorganic Chemistry
- Prof. Dr. Ralf Hoffmann, Bioanalytics (BBZ Chair)
- Prof. em. Dr. Helmut Papp, Technical Chemistry / Heterogeneous Catalysis
- Prof. Dr. Norbert Strater, Structural Analysis of Biopolymers (BBZ Chair)
- Dr. Thole Zuchner, Ultrasensitive Protein Detection Unit (BBZ Junior Research Group)
From the Faculty of Mathematics and Computer Science:
- Prof. Dr. Martin Middendorf, Parallel Computing and Complex Systems
- Prof. Dr. Erhard Rahm, Informatics
- Prof. Dr. Gerik Scheuermann, Image and Signal Processing
- Prof. Dr. Peter Stadler, Bioinformatics
From the Faculty of Medicine:
- Dr. Peter Ahnert, Molecular Diagnostics - Microarray Techniques (BBZ Junior Research
Group)
- Prof. Dr. Thomas Arendt, Neuroanatomy
- Prof. Dr. Augustinus Bader, Cell Techniques and Applied Stem Cell Biology (BBZ Chair)
- Prof. Dr. Frank Emmrich, Clinical Immunology
- Prof. Dr. Kurt Engeland, Molecular Oncology
- Prof. Dr. Friedemann Horn, Immunology
- Prof. Dr. Markus Loffler, Medical Informatics, Statistics and Epidemiology
- Prof. Dr. Andreas Reichenbach, Neurophysiology
- PD Dr. Astrid Schon, Molecular Cell Therapy
- Prof. Dr. Peter Seibel, Molecular Cell Therapy (BBZ Chair)
- Prof. Dr. Jan C. Simon, Dermatology and Allergology
From the Faculty of Veterinary Medicine:
- Prof. Dr. Gottfried Alber, Immunology
- Prof. Dr. Manfred Blessing, Molecular Pathogenesis (BBZ Chair)
267
- Prof. Dr. Almuth Einspanier, Endocrinology
- Prof. Dr. Walther Honscha, Veterinary Toxicology
- Prof. Dr. Hermann Muller, Veterinary Virology including Diagnostics
From the Faculty of Physics and Earth Science:
- Prof. Dr. Josef Alfons Kas, Experimental Physics / Soft Matter Physics with focus on
Cellbiophysics
- Prof. Dr. Friedrich Kremer, Experimental Physics, Molecular Physics, Polymer Physics,
Material Science
From Scientific and Research Institutes in cooperation with the Universitat Leipzig:
- Prof. Dr. Jorg Steinbach, Institute of Interdisciplinary Isotope Research
Prof. Dr. Andrea A. Robitzki (Director)
Dr. Svenne Eichler (Chief Executive Officer)
Universitat Leipzig
Center for Biotechnology and Biomedicine (BBZ)
E-Mail: kontakt@bbz.uni-leipzig.de
www.bbz.uni-leipzig.de
268
The Biotechnology Center (BIOTEC)
The Biotechnology Center (BIOTEC) is a unique interdisciplinary center focusing on research
and teaching in molecular bioengineering. The term molecular bioengineering describes the
interdisciplinary combination of modern cell biology and genetics with traditional material
and engineering sciences. The center hosts 13 top international research groups with about
200 employees out of 30 nations dedicated to genomics, proteomics, biophysics, cellular
machines, tissue engineering, and bioinformatics. All relevant activities of the region are
bundled and coordinated in the BIOTEC. It offers its competences to many users and is
supporting startups in this field. Essential elements for accomplishing these tasks are the
central technology platforms the BIOTEC offers. As technological and intellectual basic
conditions are required in the field of biotechnology this central appropriation of latest
devices and cutting edge technology for research groups and companies describes a so far
unique feature. Scientists and entrepreneurs have access to different microscopy and imaging
facilities, genomic engineering facilities, mass spectrometry facilities, zebrafish facility and
they take advantage of the media kitchen service.
Laying in the center of the young biotechnology cluster BIOPOLIS Dresden the
BioInnovationCenter (BIOZ) with its unique concept Economy and Science consolidated
hosts the BIOTEC as central scientific unit of the Technische Universitat Dresden and many
companies working in the field of medical technology, computer software, characterization
of agents and genes for drugs, development of biomaterials, organic light emitting diodes,
researches on cell membranes e.g. for treating influenca. Through this grouping of scientific
and economic competences the BIOTEC Dresden has set the stage to furthermore transfer
results from the basic research into startups and marketable products. This concept leads to a
union of latest research and successful companies as well as founders of new businesses. Up
to now 3 spin-offs have been set up by professors of the BIOTEC . Genebridges by Francis
Stewart, nAmbition by Daniel J. Muller and Transinsight by Michael Schroeder.
Biotechnology in Dresden is a young and dynamic economic sector with an already existing
international reputation. An outstanding feature of the Dresden Biotechnology is the strong
network of high-level research institutes like Max Planck-Institutes, Leibniz and Fraunhofer
Institutes or the Technische Universitat Dresden which cover a large scientific spectrum and
achieve groundbraking results through an efficient interdisciplinary cooperation.
The following heads of research groups are working in the BIOTEC:
- Dr. Konstantinos Anastassiadis (Stem Cell engineering)
- Dr. Andreas Beyer (Systems Biology and cellular Networks)
- Prof. Dr. Michael Brand (Director BIOTEC, Developmental Genetics)
- Dr. Denis Corbeil (Tissue Engineering)
- Prof. Dr. Bernhard Hoflack (Proteomics)
- Prof. Dr. Daniel J. Muller (Cellular Machines)
269
- Dr. Maria Teresa Pisabarro (Structural Bioinformatics)
- Dr. Erik Schaffer (Nanomechanics)
- Prof. Dr. Michael Schroeder (Bioinformatics)
- Prof. Dr. Petra Schwille (Biophysics)
- Dr. Ralf Seidel (DNA Motors)
- Prof. Dr. Francis Stewart (Genomics)
- Dr. Gilbert Weidinger (Wnt signaling in Development and Regeneration)
Prof. Dr. Michael Brand
Technische Universitat Dresden
Biotechnology Center (BIOTEC)
Email: katrin.grosser@biotec.tu-dresden.de
www.biotec.tu-dresden.de

13. Index

273
A
Abdelrahman, Amro 191
Abe, Gembu 184
Ackermann, Marit 137
Ader, Marius 47
Adler, Irena 212
Ahnert, Peter 71, 207
Alber, Gottfried 83, 84, 90
Alexopoulou, Dimitra 138
Al-Khrasania, Mahmoud 215
Alt, Rudiger 126, 161
Ambrosch, Kristina 162, 177
Anastassiadis, Konstantinos
164,173, 181
Ander, Marcel 243
Anders, Gerd 150
Andreopoulos, Bill
138, 139, 147, 149
Anselmetti, Dario 55
Appelhans, Dietmar 229
Arendt, Thomas 109, 204, 214, 218
Arnhold, Jurgen 73, 81
Arnold, Antje 163
Arumugam, Senthil 244
Aurich, Hendryk 165
Aurich, Ines 165
Azendorf, Ronny 117
B
Bader, Augustinus
143, 170, 179, 183
Ballschmiter, Meike
245, 247, 250, 253, 254
Barapatre, Nirav 109
Barbar Asskar, Ghadir 63
Barthel, Henryk 199
Basak, Onur 50
Bauer, Alexander 127
Bayer, Sally 245
Beck, Doreen 110
Beck-Sickinger, Annette G.
78, 97, 115, 118, 246, 259, 261
Behme, Gerd 58, 130
Bellmann, Katherina 128
Benndorf, Dirk 252
Berger, Frauke 200
Berger, Christian 246, 259
Berger-Hoffmann, Renate 132
Bergmann, Mirko 257
Bernauer, Sabine 184
Berndt, Sandra 246, 259
Bernhard, Detlef 142
Bette, Stefanie 220
Beyer, Andreas 137, 140, 144, 151
Binder, Hans 156
Bippes, Christian 64
Birkenmeier, Gerd 116
Birkenmeyer, Claudia 116
Bledau, Anita Sabine 164
Blessing, Manfred 82, 83, 84, 85
Bliesener , Ines 148
Bluher, Matthias 78
Boerner, Uta 126
Bohlig, Levin 65
Bohme, Ilka 97
Bohme, Manja 142
Boltze, Johannes 199, 207
Boltze, Christian 199
Bonsch, Kathrin 247, 253, 254
Bornhauser, Martin 95
Bornstein, Stefan R. 193, 202
Brand, Michael 131, 171, 184, 203
Braumann, Ulf-Dietrich 206
Braun, Sebastian 255
Breuer, Julia 228
Briel, Detlef 63, 75, 86, 92
Bringmann, Andreas 72, 213
Brinkmann, Ute 163
Brombacher, Frank 83, 84
Bruckner, Gert 218
Brundin, Patrick 48
Brust, Peter 63, 92
Brutzer, Hergen 248
Buchholz, Frank 39, 145
274
Bunge, Andreas 259
Burger, Susanne 201
Burkhardt, Markus 131
Butz, Tilman 109, 194
C
Cavodeassi, Florencia 184
Chackerian, Alissa A. 90
Chen Min Hui, Averil 125
Chiantia, Salvatore 66
Christ, Bruno 165
Chung, Kuei-Fang 202
Ciotta, Giovanni 249
Corbeil, Denis 169
Coster, Maxi 111
Cross, Michael 126, 161
Czihal, Patricia 112
D
Dahl, Janine 255
Dasgupta, Ujjaini 66
Dawelbait, Gihan 155
DeBartolo, Loredana 183
Ding, Huawen 67
Dinger, Timo C. 193
Dollinger, Matthias M. 165
Doms, Andreas 138
Donath, Edwin 125
Dressel, Frank 139
Drose, Anja 166
E
Ebensing, Sabine 165
Eger, Kurt 75
Ehninger, Gerhard 95
Ehrhart-Bornstein, Monika 193, 202
Eichler, Antje 250
Eichler, Wolfram 101
Elefsinioti, Antigoni 140
Elsinghorst, Paul Wilhelm 205
Emmendorffer, Andreas 168
Emmrich, Frank 199
Enard, Wolfgang 148
Engeland, Kurt 65
Engele, Jurgen 69, 220
Engelhardt, Johannes 215
Erler, Axel 141
Espirito Santo, Ana Isabel 68
Eylenstein, Roy 103
F
Fabian, Claire 163
Fahrig, Rudolf 75
Faltus, Timo 167
Famulok,Michael 40,
Favia, Piero 183
Fedorova, Maria 113
Fieber, Christina 168
Fiedler, Anja 109
Fierro, Fernando A. 95
Fischer, Ferdinand 116
Fleig, Wolfgang E. 165
Francke, Mike 210, 219
Franke, Heike 206
Freudenberg, Uwe 189
Freudenreich, Dorian 171
Freund, Daniel 169
Fritz, Michael 76
Fritzsch, Guido 142
G
Galle, Jorg 143
Ganey, Timothy 172, 180
Ganz, Julia 203
Gebauer, Linda 193, 202
Gebhardt, Rolf 80, 86, 100
Gebhardt, Julia 257
Gentsch, Janina 213
Giachino, Claudio 50
Gilbert, Matthias 127
Gille, Uwe 199
Giri, Shibashish 170
Glander, Hans-Jurgen 81
275
Glas, Marco 114
Glisic, Darko 69
Glockner, Pia 204
Goczalik, Iwona 110
Grandel, Heiner 203
Greiner-Stoffele, Thomas 245, 247,
250
Grill, Wolfgang 191, 192
Grimmer, Milauscha 189
Grosche, Jens 121, 205
Gros, Frank 251
Grosmann, Udo 199
Gryga, Martin 210
Gunther, Robert 80
Gutschow, Michael 205
H
Haas, Sina 70
Habermann, Bianca 141
Hacker, Michael C. 162, 177, 186
Hackermuller, Jorg 65, 74, 91, 96
Halami, Mohammad Yahya 230
Hammond, Christina 77
Hand, Galen M. 64
Hankeln, Thomas 142
Hans, Stefan 171, 184
Hansen, Anne 119
Hantmann, Helene 71
Harms, Hauke 252
Hartig, Wolfgang 205
Hassert, Rayk 115
Heckel, Tobias 68
Hedrick, Marc 180
Heiker, John T. 78
Hein, Werner 165
Heine, Claudia 206
Heine, Franziska 252
Heinrich, Jan-Michael 110
Heisenberg, Carl-Philipp 120, 174
Helenius, Jonne 120, 174
Hempel, Madlen 165
Hengstler, Jan G. 127
Hennig, Angela 183
Henschel, Andreas 153, 155
Hensel, Andras 200
Hepp, Pierre 179
Heppner, Frank L. 84
Herwig, Rolf 126
Heubeck, Christian 255
Hille, Kristin 257
Hirrlinger, Johannes 222, 223
Hofer, Michael 253
Hoffmann, Martin 143
Hoffmann, Gregor 254
Hoffmann, Ralf 112, 113, 119, 124,
128, 129, 132, 133
Hoffmann, Anke 205
Hoflack, Bernhard 68, 87
Hofmann, Hans-Jorg 80
Hohaus, Christian 172, 180
Holland, Heidrun 71
Hollborn, Margrit 72
Holscher, Christoph 90
Honscha, Kerstin 98
Honscha, Walther 98
Horn, Friedemann 41, 65, 91, 96
Hornich, Veronika 193
Huster, Dominik 73
Huster, Daniel 246, 259
Hutschenreuther, Antje 116
Huttner, Wieland B. 202
Hutton, William 180
I
Illes, Peter 215
Illmer, Thomas 95
Ingargiola, Mirjam 166
Iseli, Hans Peter 210
Israelson, Olle 142
Iwakura, Yoichiro 90
276
J
Jager, Katrin 257
Jahnke, Heinz-Georg
117, 194, 209, 221
Jauch, Anna 193
Johne, Reimar 230
Josten, Christoph 179
Juhl, Cathleen 118
Just, Lothar 211
K
Kacza, Johannes 205
Kaleta, Erhard F. 230
Kamanyi, Albert 191
Kamprad, Manja 185
Karniowska, Dorota 74
Kaslin, Jan 171, 203
Kastelein, Robert A. 90
Kawakami, Koichi 184
Keller, Mario 170
Keim, Antje 256
Khelif, Khaled 138
Kirazov, Ludmil 201
Kirazov, Evgeni 201
Kircher, Markus 256
Kirsten, Holger 207
Klemm, Matthias 75
Kluge, Magnus 199
Klyszejko, Adriana 76
Knappe, Daniel 119
Knopf, Franziska 77
Knuckles, Philip 50
Koal, Torsten 194
Koch, Holger 208
Kohen, Leon 72
Kohler, Gabriele 83, 84, 90
Kohler, Christian 208
Koksch, Beate 150
Koller, Gabor 228
Kollner, Joscha 139
Korber, Nicole 210
Koschny, Ronald 71
Kosel, David 78
Kouznetsova, Elena 201
Kramer, Dorothea 257
Kranz, Andrea 173
Krehan, Mario 255
Kremer, Friedrich 237, 238
Kretzschmar, Antje K. 65, 74, 91, 96
Krieg, Michael 120, 174
Krinke, Dana 209
Krugel, Ute 208
Kruger, Monika 232
Krupp, Wolfgang 71
Kruger, Klaus 232
Kubicova, Lenka 92
Kukat, Christian 88, 89
Kukat, Alexandra 88, 89
Kuleva, Nadezda 113
Kurz, Randy 79, 190
Kuska, Jens-Peer 143, 206
Kuske, Beatrice 67
Kuttner, E. Bartholomeus 103, 256
Kyu Kim, Wan 155
L
Labudde, Dirk 139
Laera, Stefania 183
Lange, Johannes 101
Lechtenberg, Matthias 200
Lehmann, Claudia 69
Leidich, Stefan 126
Lerche, Katja Steffi 80
Lessig, Jacqueline 81
Levental, Kandice 189
Lickert, Heiko 42
Liebert, Uwe G. 163
Ligges, Carolin 207
Lindner, Diana 97, 246, 259
Lindner, Stefan 98
Lindner, Andrea 232
Lindqvist, Niclas 210
Liu, Qing 210
Livrea, Michela 71, 175
Lochmann, Alexander 176
277
Losche, Andreas 121
Loth, Tina 177
Lubitz, Sandra 181
Lutter, Dominik 42
Lutz, Johanna 178
Luzzietti, Nicholas 248
M
Maass, Torsten 82
Machate, Anja 184
Mack, Till G. A. 221
Mader, Karsten 176
Mahamdeh, Mohammed 122
Mandl, Stephan 178
Manhardt, Markus 177, 187
Mann, Amrit 82, 83
Mann, Sabine 63
Maresca, Marcello 141
Marquass, Bastian 179
Marr, Carsten 42
Marsico, Annalisa 154
McKenzie, Andrew N. J. 84
Meier, Thomas 76
Meisel, Hans-Jorg 172, 180
Meixensberger, Jurgen 71
Menzel, Nicolas 255
Metzger, Marco 211
Michael, Sebastian 227
Michaelson, Jacob 140, 144
Michel, Fabien 227
Milosevic, Javorina 183, 212, 215
Minkus, Yvonne 172
Mohamed, Esam Ahmed 191
Mohr, Christoph 110
Montag, Cindy 246
Morawski, Markus 109, 218
Morbt, Nora 96
Morl, Karin 78
Moschutz, Susanne 103
Moseley, Tim 180
Muller, Uwe 83, 84, 90
Muller, Daniel J. 64
Muller, Katrin 132
Muller, Christa 227
Muller, Hermann 230
Muller, Susann 252
Muller, Daniel J. 76, 95, 120, 174
Muller, Albrecht M. 193
Muller, Rolf 251
Muller, Volker 76
Murugaiyan, Jayaseelan 156
N
NLdongo, Harmel Peindy 94
Nakano, Hiroaki 142
Naldijk, Yahaira 163
Neu, Bjorn 81, 125
Neumann, Katrin 181
Neundorf, Ines 94, 182
Nieber, Karen 63, 92, 93, 200, 227
Nieber, Matthias 231
Nieden, Nicole I. zur 67, 74, 188
Niekisch, Kerstin 82
Nitzsche, Hagen 176
Noack, Nicole 161
Noack, Monika 201
Norenberg, Wolfgang 215
O
Oberbach, Andreas 156
Oh Hui Lin , Bernice 125
Otvos Jr., Laszlo 112
P
Paabo, Svante 148
Panke, Oliver 123, 231
Panyanuwa, Woranan 189
Paszkowski-Rogacz, Maciej 145
Paulke, Bernd-Reiner 205
Pavlica, Sanja 170, 183
Peinemann, Frank 183
Perseke, Marleen 142
Petto, Carola 72
Petzold, Christiane 257
278
Picker, Alexander 184
Piehler, Daniel 83
Piscioneri, Antonella 183
Pissabarro, Maria Teresa
145, 150, 152
Pistorius, Doninik 251
Plake, Conrad 146, 154
Polte, Tobias 83
Porzig, Robert 124
Protschka, Martina 85
Puech, Pierre-Henri 95
R
Rasser, Anne 110
Recknagel, Stephan 228
Regenthal, Ralf 208
Reibetanz, Uta 81, 125
Reiche, Kristin 65, 74
Reichenbach, Andreas
101, 210, 213, 219
Reimann, Matthias 147, 149
Reimers, Sabrina 148
Reinert, Tilo 109, 194
Reissig, Benjamin 86
Rennert, Robert 182
Repenning, Cornelia 252
Richter, Robert 179
Riemer, Thomas 126
Ries, Jonas 131
Rillich, Katja 213
Rillich, Jan 222
Robel, Ivonne 257
Robitzki, Andrea A. 70, 79, 114, 117,
123, 190, 194, 209, 221, 231
Rodewald, Steffen 86
Rohayem, Jacques 257
Rohn, Susanne 214
Rompler, Holger 111
Rosmus, Susann 256
Ross, Robert S. 223
Rostovskaya, Maria 181
Roth, Christian 258
Roth, Sebastian 58, 130
Royer, Loic 146, 147, 149
Rubini Illes, Patrizia 215
Rudolph, Rainer 246, 259
S
Sabat, Robert 90
Sabri, Osama 199
Salomo, Mathias 237, 238
Salwiczek, Mario 150
Samsonov, Sergey 150
Sanchez Fernandez, Maria A. 87
Sauerzweig, Steven 163
Schafer, Ingo 88, 89
Schafer, Erik 122
Schalken, Jack 96
Schatzschneider, Ulrich 94
Scheibe, Johanna 216
Scheper, Thomas 252
Scherf, Nico 206
Schewtschik, Sabine 217
Schildan, Andreas 199
Schimmer, Susann 246
Schirmacher, Peter 82
Schlegel, Martin 142
Schleif, Frank-Michael 126
Schliebs, Reinhard 201
Schmauder, Ralf 57
Schmidt, Peter 246, 259
Schmidt, Sabine 117, 231
Schmidt, Steffanie 179
Schmidt, Frank 252
Schnapka-Hille, Lydia 126
Schneider, Heike 185
Schneider, Hellen 186
Schober, Ralf 71
Scholpp, Steffen 131
Schon, Astrid 255
Schoneberg, Torsten 111
Schrock, Kathleen 185
Schrodel, Wieland 230
Schroeder, Michael 138, 139, 146,
147, 149, 153, 154, 155
279
Schubert, Susanna 89
Schulte-Merker, Stefan 77
Schulz, Silke Mara 90
Schulz, Ronny 179
Schulz-Siegmund, Michaela 162,
177, 185, 186, 187
Schumann, Anika 127
Schusser, Gerald F. 228
Schutt, Katharina 91, 96
Schwan, Gregor 92
Schwarz, Friedrich 248, 260
Schwarz, Elisabeth 176
Schwarz, Sigrid C. 212
Schwarz, Johannes 212, 215
Schwenk, Frieder 173
Schwenzer, Bernd 166
Schwille, Petra 56, 66, 99, 131
Seeliger, Alexander 188
Seese, Anita 199
Seibel, Peter 88, 89
Seibler, Jost 173
Seidel, Berthold 110
Seidel, Ralf 248
Semino, Carlos E. 49
Sestu, Marcello 223
Sicard, Flavie 202
Sickinger, Anselm 79
Siegert, Fritzi 93, 260
Simeone, Angela 140, 151
Simeonov, Peter 128
Simon, Jan C. 168
Singer, David 124
Sontheimer, Jana 152
Sosinsky, Gina E. 64
Spallek, Alice 228
Sperlich, Maximilian 187
Splith, Katrin 94
Stach, Thomas 142
Stadler, Peter F. 142
Stahl, Frank 252
Stein, Frank 179
Steinhagen, Max 261
Stenzel, Werner 83, 84
Steude, Anja 231
Stewart, A. Francis
141, 164, 173, 249, 251
Stock, Peggy 165
Stolzing, Alexandra 163, 175
Storch, Alexander 212
Strater, Norbert 63, 92, 102, 103,
104, 128, 256, 258
Strauber, Heike 252
Strem, Brian 180
Striggow, Frank 221
Surendranath, Vineeth 141
Suttkus, Anne 218
Szczelkun, Mark 260
Szekeres, Tibor 99
T Taubenberger, Anna 95
Taylor, Verdon 50
Tennemann, Anja 182
Teucher, Madeleine 141
Thapar, Nikhil 211
Theis, Fabian J. 42,
Thor, Doreen 111
Thorndyke, Mike 142
Thummler, Christian 143
Todorovski, Toni 129
Tomczak, Aurelie 152
Trettner, Susanne 188
Tsurkan, Mikhail 189
Tuukkanen, Anne 153
U
Ueberham, Uwe 204, 214
Uetzmann, Lena 42
Ulbricht Elke 219
Ullmann, Kerstin 91, 96
Uney, James 204
Unger, Tina 220
Usha, Acharia 66
280
V
van Aelst, Kara 260
Verhaegh, Gerald 96
Vinz, Silvia 190
Vogel, Horst 57
Voigt, Brigitte 229
von Bergen, Martin 70, 96, 156, 252
von Buttlar, Moritz 191, 192
von der Burg, Erik 192
von Einem, Sabrina 176
von Mameren, Joost 58, 130
Vukicevic, Vladimir 193, 202
W Wagner, Carolin 237, 238
Walkinshaw, Gail 212
Walther, Cornelia 97
Warstat, Claudia 227
Wasermann, Luise 98
Wegner, Annett 221
Weich, Bettina 142
Weick, Michael 213
Weidemann, Thomas 99, 101
Weidinger, Gilbert 77
Weizenmann, Nicole 257
Welzel, Petra 189
Werner, Ronald 194
Werner, Anselm 232
Werner, Carsten 189
Wiacek, Claudia 252
Wiedemann, Peter 72, 210
Wierzchacz, Claudia 100
Wilcke, Arndt 207
Wilhelm, Franziska 222
Wilhelm, Christian 127
Wilson, Stephen 184
Winkler, Ulrike 222, 223
Winnenburg, Rainer 146, 154
Winter, Christof 154, 155
Winter, Claudia 232
Wirling, Christoph 126
Wirth, Henry 156
Witte, Ellen 90
Wohlrab, David 165
Wolk, Kerstin 90
Worch, Remigiusz 99
Wottawah, Cornelia M. 78
Wozniak, Anna 58, 130
Y Yafai, Yousef 201
Yan, Luo 227
Yang, Xiu Mei 101
Yates, Karen 102
Yu, Shuizi Rachel 131
Yu, Jinshu 64
Yurkova, Irina 73
Z
Zahn, Michael 103
Zauner, Thomas 132
Zebisch, Matthias 104, 128
Zehethofer, Nicole 133
Zeitschel, Ulrike 219
Zemljic-Harpf, Alice 223
Zhang, Youming 141, 251
Zi Yen, Liaw 125
Zieger, Romy 257
Ziegler, Wolfgang H. 223
Zieris, Andrea 189
Zimmermann, Wolfgang 258
Zscharnack, Matthias 143, 179
Zuchner, Thole 128, 132, 133
281


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