Symposium:_Other (free) topic_

Jürgen Alves, Dorothee von Witzendorff, Minh-Cam Ha-Thi, Susanne von Pall deTolna, Gabriele GrabowskiRestriction endonuclease EcoRI - fusions for a change of specificity

Stefanie BarbirzThe tailspike from E.Coli H bacteriophage HK620: Another Parallel beta-HelixProtein?

Mike Beck, Kieran Brickley, Miriam Smith, Seema Sharma, Helen Wilkinson, F.Anne StephensonGRIF-1 - a novel GABAA receptor associated protein

Daniela BesongGenexpression of the human membrane-associated Progesterone Receptor(hmPR) in HepG2 cells

Susanne Brakmann, Sylvia Löbermann, Petra NieckchenOptimal enzymes for single molecule sequencing

Peter Rehling, Peter Kovermann, Richard Wagner, Nikolaus Pfanner, Kaye, N.Truscott, Katrin BrandnerThe TIM22 translocase mediates insertion of carrier precursors into themitochondrial inner membrane via a two-step process

S. Breidert, R. Jacob, A. Ngezahajo, H.-A. Kolb, H. NaimTrafficking pathways of connexin49-GFP in living mammalian cells

Claudia Loske, Gerald Münch, Björn Kuhla, Sladjana Dukic-StefanovicDifferential effects of "advanced glycation endproducts" and ß-amyloid peptideon glucose utilization and ATP levels in the neuronal cell line SH-SY5Y

Sladjana Dukic-Stefanovic, Jovana Gasic-Milenkovic, Winnie Deuther-Conrad,Gerald MünchProinflammatory signal transduction pathways in N11 mouse microglial cell lineactivated by "Advanced Glycation Endproducts"

Johanna Mansfeld, Renate Ulbrich-Hofmann, Peter DürrschmidtIdentification of an intermediate in the unfolding of neutral protease fromBacillus stearothermophilus by fluorescence spectroscopy

Matthias Eckhardt, Simon Fewou, Volkmar GieselmannEffect of N-glycan removal on catalytic activity of cerebroside sulfotransferase

Jörg Bär, Jürgen Kirchberger, Anke EdelmannInteractions between the two types of subunits forming an enzymatically activeoligomeric phosphofructokinase-1 from Saccharomyces cerevisiae

Ralph-Peter ElsnerSolution structure of the Ras binding domain of AF6

Jochen Frenzel, Jan Richter, Klaus EschrichNitrogen oxide-induced cell death in rabbit cultured Müller cells is modulated byglucose

Leopold FlohéThe element of the moon moonlighting for fertility

Bernd Moritz, Gudrun Franke, Martin Siemann, Matthias Reuss, Helmut E. MeyerQuantitative Proteomics with Escherichia coli

Alexander Freiberg, Jan Lengefeld, Matthias Machner, Wolfgang Pfeil, DirkHeinz, Robert SecklerStability and Folding of Internalin B - A Biophysical Characterization

Susana Gariba de Garcia, Gerald Münch, Gabriele Oehme

"Carbonyl stress” by methylglyoxal causes mitochondrial dysfunction, ATPdepletion and NMDA receptor overstimulation in neurons

Jovana Gasic-Milenkovic, Sladjana Dukic-Stefanovic, Winnie Deuther-Conrad,Gerald Münchß-amyloid peptide potentiates inflammatory responses induced bylipopolysaccharide, interferon-gamma and "advanced glycation endproducts" ina mouse microglia cell line

Klaus Golka, Seidel Thilo, Roetzel Claudia, Geller Frank, Bolt Hermann M,Dietrich Holger, Foth Heidi, Thier RicardaEnzym polymorphisms and risk factors in bladder cancer patients in LutherstadtWittenberg

Oliver Ohlenschläger, Jens Wöhnert, Enrico Bucci, Ramadurai Ramachandran,Roland Zell, Matthias GörlachStructure of an RNA Involved in Enteroviral Replication

Anke Hannemann, Marina Bigl, Frank Gaunitz, Klaus EschrichCharacterization of the human platelet-type 6-phosphofructo-1-kinase genepromoter

Monika Walter, Robert Seckler, Benjamin HeinzFolding Kinetics of Pectate Lyase from Bacillus subtilis

Susanne Ammon, Peter Mayer, Volker HölltMicroarray analysis fo genes expressed in the frontal cortex of rats chronicallytreated with morphine

Gudrun Horn, Daniel Weinfurtner, Roland Hofweber, Robert Macek, Robert Gail,Till Maurer, Hans Robert KalbitzerOptimization of intein based isotope labeling methods for NMR investigations ofproteins

Franziska Bleichert*, J.P. Warnke**, Klaus Eschrich*, Renate Keßler*Expression of ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatasein intracranial neoplasms and non-tumorous brain tissue

V. KrügelInhibition of LiCl-induced induction of glutamine synthetase in HepG2 cells byflavonoids and anthraquinone derivatives is mediated by CK I

Peter KühlWho discovered the Michaelis-Menten hyperbola?

Antonios Kyriakopoulos, Barbara Hoppe, Alexandra Graebert, MarcusKühbacher, Dietrich BehneSelenoproteins in the perinuclear structures of rat kidney Dedt. Molecular TraceElement research in the Life Sciences Hahn- Meitner-Istitut, Glienicker Str.100,14109 Berlin

Carsten Skarke, Helmut Schmidt, Jürgen Liefhold, Gerd Geisslinger, Jörn LötschA mathematical model to predict plasma concentrations of morphine and itsactive metabolite morphine-6-glucuronide in healthy young persons

Johanna Mansfeld, Eva Petermann, Renate Ulbrich-HofmannGeneration of catalytically active neutral protease from Bacillusstearothermophilus does not require the presence of the propeptide

Walter-Vesely Seb. MeisterPolyvinylnucleobases: Synthesis, characterization and applications innanotechnology

Nils Hanekop, Susanne Wied, Stefan Petry, Norbert Tennagels, Wendelin Frick,Günter MüllerA Receptor for Glycolipid Headgroups Mediates Insulin-mimetic Signaling in RatAdipocytes

Pavel I. Nedvetsky, Alexander Y. Kots, Helmut Müller, Ferid Murad, HaraldH.H.W. SchmidtHsp90 effects on the regulation of soluble guanylyl cyclase

Ellen Niederberger, Achim Schmidtko, Jeffrey D. Rothstein, Gerd Geisslinger,Irmgard TegederModulation of spinal nociceptive processing through the glutamate transporterGLT-1

Kathrin Fuchs, Diana Kühn, Johannes Schmitt, Joachim Nöller�Membrane affinity and pore forming properties of proteins/peptides - Easy andfast by using solid supported lipid bilayers.�

Ingo Paarmann, Manfred KonradStructural requirements for calmodulin binding to membrane-associatedguaylate kinases

Thomas Schneider, Gerhard Braus, Gabriele Heinrich, Markus Hartmann, AndreaPfeilEvolution of Feedback-inhibited (&beta/&alpha)8 Barrel Isoenzymes by GeneDuplication and A Single Mutation

Gia Anh Bao PHAN, Detlef PIETROWSKI, Christoph KECKEXPRESSION OF A NEW SPLICE VARIANT OF IGFBP-7/MAC25 TUMORSUPPRESSOR GENE IN HUMAN GRANULOSA CELLS LACKING THE IGFBP MOTIF(GCGCCXXC)

Paola Pocar, Robert Augustin, Bernd FischerInduction of apoptotic cell death in bovine cumulus-oocyte complexes aftertreatment with Aroclor-1254 during in vitro maturation.

Albert Sickmann, Stefan Lehr, Armin Herkner, Dirk Müller-Wieland, Helmut E.Meyer, Jörg ReindersIdentification of the Major Tyrosin Phosphorylation Sites of Human Gab-1

Jens Wulfaenger, Alexander Navarrete Santos, Tanja Blosz, Juergen Langner,Dagmar RiemannThe intracellular N-terminus of aminopeptidase N/CD13 affects association withcaveolae

Ina Wittko, Sigrid Saaler-ReinhardtInvolvement of La protein in alternative translation processes in neuralprogenitor cells

Polin Mahjour, Katja Mühlberg, Andreas Hagendorff, Stefan Dhein, DietrichPfeiffer, Aida SalamehDifferential regulation of gap junction protein expression in cardiomyocytes

Albert Sickmann, Marion Herrmann, Joachim Klose, Helmut E. Meyer, HeikeSchaeferAnalysis of Posttranslational Modifications of alpha-A-Crystallin during Aging ofthe Eye Lens

Martin Haßler, Edgar Brändle, Joachim Greven, Jan Henrik SchlattjanInteraction of the organic base cimetidine with the renal basolateralp-aminohippurate (PAH) transport system

Heide Schmid, Thai-Hoa Nguyen-Xuan, Minh-Huy Tran, Gerold Schwarz, HubertKalbacher, Heinrich WiesingerThe endosomal/lysosomal distribution of cathepsins (cat) B, L and S andN-acetyl-ß-D-glucosaminidase (NAG) is differentially modulated in murine celllines of microglial and monocytic origin

Marcel Schmitt, Georg Gellert, Bettina Kirberg, Jost Ludwig, HellaLichtenberg-FratéCyGene: Eine neue hefebasierte Methode zur Bestimmung des cyto- undgenotoxischen Potentials von Umweltgiften im Wasserbereich. CyGene: A NewYeast Based Method for the Detection of Cyto- and Genotox

Reinhild Tenderich, Brigitte SchmitzFunction of the PEST sequence of NCAM 140

Astrid Schön, Olaf Gimple, Christian HeubeckThe evolution of RNase P in eukaryotic organelles

Lorenz Trümper, Frank Griesinger, Christa Fonatsch, Astrid Heutelbeck, DetlefHaase, Ernst Hallier, Thomas SchulzGLUTATHIONE S-TRANSFERASE GSTP1 POLYMORPHISMS IN DE NOVO ANDTHERAPY-INDUCED HEMATOLOGIC MALIGNANCIES

Gabi Bauer-Manz, Andreas Kuhn, Lambertus van den Berg, Justyna SerekThe E.coli membrane insertase YidC: A Chaperone for Protein Folding into theMembrane

Michael Spoerner, Guido Steiner, Thomas Prisner, Alfred Wittinghofer,Hans-Robert KalbitzerAre GTP-analogs really good analogs?

Anna Stepczynska, Jakob Troppmair, Thomas Voegel, Monika Poppe, KlausSchulze-Osthoff, Michael SchwarzBcl-2 antagonist of cell death (Bad) retains its proapoptotic activity despiteproteolytic cleavage by caspase-3

Stefan Henrich, Robert Huber, Albert Ries, Karlheinz Mann, Klaus Kühn, RupertTimpl, Gleb P. Bourenkov, Hans D. Bartunik, Wolfram Bode, Manuel E. ThanCrystal structure of the noncollagenous (NC1) domain of human placentacollagen IV: Stabilization via a novel type of covalent Met-Lys cross-link

Cornelia Körting, Alexander Froschauer, Reinhold Hanel, Jean-Nicolas Volff,Anne-Marie VeithDMRT Genes and Sex Determination in the Fish Xiphophorous

Hubert Zipper, Katrin Lämmle, Christiane Buta, Herwig Brunner, JürgenBernhagen, Frank VitzthumInvestigations on the binding of SYBR Green I to double-stranded (ds)DNA

Markus von Nickisch-Rosenegk, Jenny Steffen, Frank F. BierTranscription of Reporter Genes with Immobilized Templates

Melanie Wagner, Reinhard Reents, David Owen, Herbert Waldmann, AlfredWittinghofer, Jürgen KuhlmannLocalisation of fluorescently labelled Ras protein in the living cell

Albert Sickmann, Helmut E. Meyer, Yvonne WagnerTwo-dimensional chromatography in protein analysis

Robert Seckler, Monika WalterPectate Lyase from Bacillus subtilis (BsPel): Mutations in the asparagine laddercause a temperature-sensitive folding phenotype

Monika WalterPectate Lyase from Bacillus subtilis (BsPel): Mutations in the asparagine laddercause a temperature-sensitive folding phenotype

Sabine Werner, Stefan Kuklinski, Brigitte SchmitzAltered O-GlcNAc level after neuronal differentiation of PC12 cells

Barth Holger, Klaus Aktories, Christian WildeClostridium botulinum C2 toxin as a protein transport system

Klaus Aktories, Christian WildeClostridium botulinum C3 exoezyme like ADP-ribosyltransferases

Jens Wulfänger, Tanja Blosz, Alexander Navarrete Santos, Jürgen Langner,Dagmar RiemannAminopeptidase N/CD13 is associated with Lubrol rafts

Ulrike Sattler, Ingrid Koziollek-Drechsler, Danuta Dormann, Christina ZechelPutative role of the nuclear factor NCNF in neuronal differentiation

Suisheng Zhang, Carsten Köhler, Frank GrossePHYSICAL INTERACTION BETWEEN NUCLEAR DNA HELICASE II AND WRNHELICASE STIMULATES WRN´S EXONUCLEASE ACTIVITY

Jürgen Alves, Dorothee von Witzendorff, Minh-Cam Ha-Thi, Susanne von Pall deTolna, Gabriele Grabowski

Restriction endonuclease EcoRI - fusions for a change of specificity

Restriction enzymes very accurately cleave DNA at a specific nucleotidesequence. Recognition of this sequence by amino acid side chains is highlyredundant. Therefore, all attempts to change sequence specificity have failed sofar.In order to widen the recognition sequence of the restriction endonuclease EcoRIwe have inserted short DNA binding domains into the enzyme structure. The tipsof the outer or inner arm were chosen as insertion points to place the additionaldomains above the major groove. One zinc finger of Zif268 or the third repeat ofthe Myb oncogene were used as DNA binding modules which are likely to have astable fold. In a site selection assay one of the fusion mutants showed apreference for the sequence AAGGAATTCCTA. Although the EcoRI sequenceGAATTC in varying sequence contexts was still cleaved with low velocity.We also created fusions of the two monomers of EcoRI. These fused dimers areeven more toxic to cells than the wild type enzyme. This sheds light on a hithertounrecognized evolutionary advantage of the dimeric state for restrictionenzymes.

contact:

Prof. Jürgen AlvesMedizinische Hochschule HannoverBiophysikalische Chemiealves@bpc.mh-hannover.deCarl-Neuberg-Str. 130625 Hannover (Deutschland)

Stefanie Barbirz

The tailspike from E.Coli H bacteriophage HK620: Another Parallel beta-HelixProtein?

Recently, sequencing of the E.Coli H phage HK620resulted in cloning of the gene for its tailspike protein,HK620 TSP. The first about 100 amino acids of the 710residue protein exhibit high sequence homology in itsN-terminal capsid binding domain with the tailspikes of twoother phages of the Podoviridae family, Salmonella phageP22 and Shigella phage Sf6. However, sequence homologybetween the three tailspikes is completly lacking in the majorC-terminal part which forms a large parallel-b-helix domain inP22 TSP. In spite of the completely differing sequences wepropose the same parallel b-helix fold for HK620 TSP. Tosupport this, we present here the spectroscopical andbiochemical characterization of HK620 TSP.The protein was recombinantly expressed in E.coli and purified.HK620 TSP is a SDS-resistant thermostable trimer with anmolecular weight of 230 kDa as shown by size-exclusionchromatography. The secondary structure of the proteinis similar to that of P22 TSP as shown by circular dichroismand FT-IR spectroscopy. The N-terminal capsid binding domaincan be deleted without affecting the ability of the protein toform a trimer. This truncated protein is resistant to proteasesand shows thermostability comparable to P22 TSP. It waspredominantly used for crystallization experiments. Conclusiveevidence for the similarity of the two tailspike structures canonly be provided by crystallographic data once available.However, biochemical and spectroscopical characterization stronglysuggest that HK620 tailspike protein joins the family of trimeric parallelbeta-helix-proteins as a third member together with P22 and Sf6 tailspikes.

contact:

Stefanie BarbirzUniversität PotsdamBiologie und Biochemiebarbirz@rz.uni-potsdam.deKarl-Liebknechtstr. 24-25, Haus 2514476 Golm (Deutschland)

Mike Beck, Kieran Brickley, Miriam Smith, Seema Sharma, Helen Wilkinson, F.Anne Stephenson

GRIF-1 - a novel GABAA receptor associated protein

The yeast two-hybrid (Y2H) assay was used to identify a novel 913 amino acidprotein, GABAA receptor interacting factor-1 (GRIF-1), that specifically associateswith the intracellular loop of the GABAA receptor β2 subunit. GRIF-1aprotein is expressed in the brain and excitable tissues (heart and skeletal muscle)as protein with Mr of 115 and 106kDa detectable in soluble andmembrane-associated fractions.GRIF-1 has no homology with proteins of known function. However, it is evidentthat it belongs to an evolutionary conserved gene family with two knownmembers in mammals and a single homologue in Drosophila. One of theconserved features of this family is the presence two coiled:coil motifs containedwithin a region which has significant aa similarity to huntingtin-associated protein(HAP-1). Part of this region (GRIF-1 aa 124-283) is also responsible for bindingto the ²2 subunit (aa 324-394) as mapped utilizing the Y2H system.Furthermore, it was shown that GRIF-1 strongly interacts with itself and that itsbiological active form most likely represents a dimer. Screening of a Y2H ratbrain cDNA library revealed that GRIF-1 interacts with the O-GlcNac transferaseOGT-1.

We propose a possible role for GRIF-1 in GABAA receptor ²2 subunit traffickingand receptor assembly.

Literature(1) Beck et al. 2002 J.Biol. Chem. in press

contact:

Dr. Mike BeckUniversity of LondonThe School of Pharmacymike.beck@gmx.co.uk29/39 Brunswick Sq.WC1N 1AX London (UK)

additional information

present address:MPI for Molecular Cell Biology and GeneticsPfotenhauerstr. 108D-01307 DresdenGermany

Daniela Besong

Genexpression of the human membrane-associated Progesterone Receptor(hmPR) in HepG2 cells

Hmpr is a putative membrane associated progesterone binding protein, thatcould play an important role in nongenomic effects of hormones (Wehling, 1997;Falkenstein et al., 2000). In this study the transcriptional regulation of the hmprgene in the human hepatoma cell line, HepG2, was characterised by evaluatingthe transient expression of a luciferase reporter gene. The 5´flanking region(-1698 to +195) of the hmpr gene showed an adequate transcriptional activity.The maximum activity was in a region between –309 and +110, which containstwo arylhydrocarbon receptor and the Ahr nuclear translocator (Ahr/Arnt), a RasResponsive element, an upstream and downstream AP2, two downstream SP1,three GC-Boxes and an HNF4 binding sites. Transient expression assay withvarious deletion mutants showed that the SP1 and the GC-Box binding site arecrucial for the promoter activity and that the upstream Ahr/Arnt binding site hasa potent enhancer activity. No activity was detected when the 5´flanking regionof the hmpr gene was inserted in the reverse orientation. Gel mobility shiftassays with various DNA fragments indicated that the GC-Box, SP1, Ahr/Arntbinding sites interact specifically with nuclear protein (s) of HepG2 cells.These results suggest that the GC-Boxes and SP1 binding sites are the corepromoter, while the Ahr/Arnt binding sites act as enhancer in HepG2 cells.

LiteratureWehling, M. (1997) Specific, nongenomic actions of steroid hormones. Annu RevPhysiol, 59, 365-393Falkenstein, E.et al (2001) Multiple actions of steroid hormones-a focus on rapidnongenomic effects. Pharmakol Rev, 52, in Press

contact:

Daniela BesongUniversitätsklinikum MannheimKlinische Pharmakologiedaniela.besong@kpha.ma.uni-heidelberg.deTheodor Kutzer Ufer68167 Mannheim (Baden-Würtemberg)

Susanne Brakmann, Sylvia Löbermann, Petra Nieckchen

Optimal enzymes for single molecule sequencing

Over the past 15 years, there has been a group of chemists who haveworked to develop a novel strategy for sequencing DNA based on single-molecule detection techniques. In single-molecule sequencing, it isproposed that a strand of DNA is synthesized with each nucleotidelabeled with a characteristic fluorescent tag. An exonucleasewould then be used to successively cleave nucleotides from the labeledstrand, and the cleaved nucleotide would be detected using anultrasensitive fluorescence spectrometer.

There are two challenging biochemical steps involving hitherto unknownenzymatic activities that are required for the realization ofsingle-molecule DNA sequencing. First, a strand of DNA must be synthesizedthat carries a fluorescently modified base at each position. This step requiresthat a DNA polymerase is found which can incorporate the sterically andelectronically demanding nucleotides during the synthesis of acomplementary strand of DNA. Recently, we identified a natural polymerasewhich retained activity in the sole presence of rhodamine-modifiednucleotides and furthermore, we reported the preparation of DNA inwhich one strand is synthesized with complete substitution of thepyrimidine nucleotides (T, C) by their rhodamine-labeled analogs.

The second challenging step in single-molecule sequencing is theenzymatic digestion of the labeled strand which must be performed byan exonuclease, that is, directly from one end, sequentially processingalong the strand. Endonuclease activity is undesired, which woulddigest nucleotides at random positions within the strand. Recently, wealso discovered that a natural exonuclease degrades a DNA duplexwherein one strand is synthesized with rhodamine-labeled cytosineand thymine - despite some unusual physical characteristics of thesubstrate DNA. We also performed a series of experiments to demonstratethat the activity is processive under the conditions chosen so far.

contact:

Dr. Susanne BrakmannUniversität LeipzigInstitut für Zoologie/Angewandte Molekulare Evolutionsbrakma@rz.uni-leipzig.deTalstrasse 3304103 Leipzig (Deutschland)

Peter Rehling, Peter Kovermann, Richard Wagner, Nikolaus Pfanner, Kaye, N.Truscott, Katrin Brandner

The TIM22 translocase mediates insertion of carrier precursors into themitochondrial inner membrane via a two-step process

The vast majority of mitochondrial proteins are nuclear-encoded and synthesizedin the cytosol. Depending on their final destination their import may requiretranslocation machinery present in both the outer and inner membranes. Specifictargeting signals, contained within preproteins, direct them to the mitochondrialsurface where they are imported into or through the lipid bilayer by thetranslocase of outer membrane (TOM). Two distinct translocases of the innermitochondrial membrane (TIM), facilitate import of preproteins into or across thisinternal lipid bilayer. Preproteins with positively charged presequences at theirN-terminus are inserted via the TIM23 complex, whereas preproteins withinternal targeting information such as carrier proteins are imported via the TIM22complex. In both cases a membrane potential across the inner membrane isessential for their import.

Carrier preproteins are imported into mitochondria in five defined stages: stage I– binding to cytosolic chaperones; stage II – recognition by receptors on theouter membrane surface; stage III –transport through the outer membrane;stage IV – insertion into the inner membrane; stage V – dimerisation. At stage IVcarrier preproteins are directed to the TIM22 complex (formed by Tim54, Tim22,Tim18 and small Tims) where insertion into the inner membrane is mediated bythe pore forming protein Tim22. We have examined the requirements for stageIV import using the functionally intact purified TIM22 complex that associate in astable manner with carrier translocation intermediates. Electrophysiologicalmeasurements with the isolated TIM22 complex and in vitro import assays withcarrier preproteins together indicate that carrier membrane insertion is atwo-step process intimately linked to the energy source, the membrane potential.

contact:

Dipl.-Ing. (FH) Katrin BrandnerUniversität Freiburg, PfannerInstitiut f. Biochemie und Molekularbiologiekatrin.brandner@biochemie.uni-freiburg.deHermann-Herder 779104 Freiburg (Deutschland)

additional information

Brandner, K.1, Rehling, P.1, Kovermann, P.2, Wagner, R.2, Pfanner, N.1 &Truscott, K. N.1

1Institut für Biochemie und Molekularbiologie, Universität Freiburg,Hermann-Herder-Strabe 7, D-79104 Freiburg, Germany, 2Biophysik, UniversitätOsnabrück, FB Biologie/Chemie, D-49034 Osnabrück, Germany.

S. Breidert, R. Jacob, A. Ngezahajo, H.-A. Kolb, H. Naim

Trafficking pathways of connexin49-GFP in living mammalian cells

The present study examined the trafficking pathways of connexin49 (Cx49)labelled with green fluorescent protein (GFP) in polarized and nonpolarizedeukaryotic cell lines. Connexin proteins are thought to follow several transportpathways including subcellular compartments as well as cytosolic transportroutes towards the plasma membrane. There, they are integrated to form gapjunction plaques between adjacent cells.Cx49 was cloned from ovine lens by RT-PCR. The Cx49 cDNA sequence was fusedin frame to GFP (povCx49-GFP) and transfected into several eukaryotic cell lines.Synthesis, assembly, degradation and removal of povCx49-GFP was determinedby pulse-chase experiments in nonpolar COS cells. The trafficking pathways weremonitored by confocal microscopy. Fusion proteins were localized in subcellularcompartments including ER, ERGIC, Golgi, trans Golgi network (TGN)as well as intransport vesicles travelling towards the plasma membrane. Gap junction plaqueassembly resembling the classical punctuate distribution pattern could bedemonstrated for nonpolar COS-7 and polar MDCK cells. A basolateraldistribution of povCx49-GFP became apparent in polar MDCK cells indicating aspecific sorting behaviour for polar organized cells. The results gained by confocalmicroscopy and pulse-chase experiments provide a conclusive evidence for atransport competent conformation of povCx49-GFP in all examined cell lines.

contact:

Prof. Dr. Hassan NaimTierärztliche Hochschule HannoverInstitut für Physiologische ChemieHassan.Naim@tiho-hannover.deBünteweg 1730559 Hannover (Deutschland)

additional information

PD Dr. R. JacobInstitut für Physiologische ChemieTierärztliche Hochschule HannoverBünteweg 17e-mail:Ralf.Jacob@tiho-hannover.de

Prof. Dr. H.-A. KolbDr. A. NgezahajoDipl.-Bio. S. BreidertInstitut für BiophysikUniversität Hannovere-mail: kolb@mbox.biophysik.uni-hannover.de

Claudia Loske, Gerald Münch, Björn Kuhla, Sladjana Dukic-Stefanovic

Differential effects of "advanced glycation endproducts" and ß-amyloid peptide onglucose utilization and ATP levels in the neuronal cell line SH-SY5Y

"Advanced glycation endproducts" (AGEs) and beta-amyloid peptide (A-beta) areboth structural components of the senile plaques in the Alzheimer's disease brain[1], and, among other ligands such as amphoterin, bind to the receptor foradvanced glycation endproducts (RAGE) [2]. Since AGEs and A-beta wereproposed to exert their redox-sensitive neurotoxic properties through the samereceptor [3], we compared their effects on parameters of cellular energymetabolism including cell viability, glucose consumption, lactate production andATP level in the human neuroblastoma cell line SH-SY5Y. In this cell line, RAGEexpression was detected the mRNA level (by qualitative RT-PCR); and on theprotein level (by Western blotting). AGEs (BSA-AGE) and A-beta ("fibrillar")dose-dependently decreased cell viability measured by the MTT assay, but onlyAGEs decreased the actual number of cells. Normalised, cell number-basedenergetic parameters show that both AGEs and A-beta increased glucose uptakebut reduced ATP levels in a dose-dependent manner. Furthermore, only AGE (butnot A-beta) caused a significant (> 6 fold) increase in lactate production. Thesedata indicate, that both AGEs and A-beta cause differential disturbances inneuronal metabolism, which might contribute to the pathophysiological findingsin Alzheimer’s disease. However, their signaling pathways appear to be quitediverse, a fact which should stimulate a more detailed investigation of theirsignaling pathways, e.g. for the purpose of a rational design of potential"neuroprotective" RAGE antagonists.

Literature[1] Wong A, Lüth HJ, Arendt Th, Münch G (2001) Advanced glycationendproducts co-localize with inducible nitric oxide synthase in Alzheimer’sdisease. Brain Res 920: 32-40.

[2] Sajithlal G, Huttunen H, Rauvala H, Münch G (2002) Receptor for advancedglycation end products (RAGE) plays a more important role in cellular survivalthan neurite outgrowth during retinoic acid-induced differentiation ofneuroblastoma cells. J Biol Chem 277: 6888-97

[3] Loske C, Neumann A, Nichols K, Cunningham AM, Schinzel R, Riederer P,Münch G (1998) Cytotoxicity of advanced glycation endproducts - protection byantioxidants. J Neural Transm 105: 1005-15

contact:

PD Gerald MünchIZKF LeipzigNeuroimmunoloische Zellbiologiemueg@medizin.uni-leipzig.deJohannisallee 30 a04103 Leipzig (Germany)

Sladjana Dukic-Stefanovic, Jovana Gasic-Milenkovic, Winnie Deuther-Conrad,Gerald Münch

Proinflammatory signal transduction pathways in N11 mouse microglial cell lineactivated by "Advanced Glycation Endproducts"

Deposition of AGE-crosslinked insoluble protein aggregates (amyloids) ischaracteristic for many degenerative diseases of the elderly includingAlzheimer?s disease. Microglial activation and neuroinflammatory processes havebeen shown to play a supporting role in functional degeneration as well as celldeath of neurons. "Advanced Glycation Endproducts? (AGEs) activate specificpro-inflammatory signal transduction pathways, resulting in the up-regulation ofcytokines (ILs, TNF-alpha) and inducible nitric oxide synthase (iNOS), possibly bytheir interaction with AGE-specific receptors [1, 2]. Our goal was to delineate theAGE-activated signal transduction pathways involved in the induction ofpro-inflammatory markers (TNF-alpha, NO) in the murine microglial cell lineN-11.Chicken egg albumin-AGE (CEA-AGE), used as a model AGE, induces both NOand TNF-alpha production is a time- and dose dependent manner. The AGEreceptor, RAGE, appears to be the starting point for both pathways, since bothNO and TNF-a production can be inhibited by a neutralising RAGE antibody. NOand TNF-a production are regulated independently, since NOS inhibitors did notdecrease TNF-alpha secretion and a neutralising TNF-alpha antibody did notreduce NO production. Inhibition of the MEK and PI3K pathway, but not of theMAPK-p38 and SAPK/JNK pathways, reduced NO and TNF-alpha significantly,suggesting that simultaneous activation of the first two pathways is necessary forthe AGE-induced induction of these pro-inflammatory stimuli.

Literature[1] Neumann A, Schinzel R, Palm D, Riederer R, Münch G (1999) High molecularweight hyaluronic acid inhibits advanced glycation endproduct-induced NF-kBactivation and cytokine expression. FEBS Lett 453: 283-287.

[2] Wong A, Schinzel R, Wiesinger H, Riederer P, Münch G (2001)Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxidesynthase mediated by advanced glycation endproducts in murine microglia. Eur JNeurosci 14: 1-8

contact:

Sladjana Dukic-StefanovicIZKF LeipzigNeuroimmulogical cell biologyduks@medizin.uni-leipzig.deJohannisallee 30A04103 Leipzig (germany)

Johanna Mansfeld, Renate Ulbrich-Hofmann, Peter Dürrschmidt

Identification of an intermediate in the unfolding of neutral protease from Bacillusstearothermophilus by fluorescence spectroscopy

In the thermolysin-like neutral protease from B. stearothermophilus thesurface-located region around the residues 56-69 is important for thermostabilityof the enzyme, as shown by mutational studies (1). The introduction of twocysteines into the positions 8 and 60 of the molecule resulted in the spontaneousformation of a disulfide bond causing an extreme stabilization of the proteaseagainst thermal inactivation (2). Changes in one calcium binding site which is inthe vicinity of this region are probably involved in this stabilizing effect (3). Inthe present paper the influence of calcium ions on the kinetics of guanidinehydrochloride (GdnHCl) induced unfolding was investigated by fluorescencemeasurements. Under strong denaturing conditions (> 7M GdnHCl) unfolding canbe observed without interference of autoproteolysis and the observed rateconstants (k) yield linear chevron plots (log k vs. [GdnHCl]). In lower GdnHClconcentrations (< 6M) autoproteolysis occurred in significant degree andincreased k tenfold, yielding deviations from the linear chevron-plots. Addition ofisopropanol which acts as an inhibitor on the enzyme activity abolished thiseffect. Also with increasing concentrations of calcium ions chevron plots becamelinear. The influence of calcium ions is higher for the wild type than for themutant enzyme. These results can be interpreted by assuming a native-likeintermediate which is susceptible to autoproteolysis. In the disulfide stabilizedmutant enzyme, the unfolding route via this intermediate is less predominant.

Literature1. Eijsink, V. G. H., Veltman, O. R., Aukema, W., Vriend, G., and Venema, G.(1995). Nat. Struct. Biol. 2, 374-379.2. Mansfeld, J., Vriend, G., Ulbrich-Hofmann, R., and Eijsink, V. G. H. (1997). J.Biol. Chem. 272, 11152-11156.3. Veltman, O. R., Vriend, G., Berendsen, H. J. C., Van den Burg, B., Venema,G., and Eijsink, V. G. H. (1998). Biochemistry 37, 5312-5319.

contact:

Peter DürrschmidtUni-HalleBiotechnologieduerrschmidt@biochemtech.uni-halle.deKurt-Mothes-Str. 306120 Halle (Deutschland)

Matthias Eckhardt, Simon Fewou, Volkmar Gieselmann

Effect of N-glycan removal on catalytic activity of cerebroside sulfotransferase

3-O-sulfogalactosyl ceramide (sulfatide) is a major lipid component of myelinmembranes and is required for proper myelin formation. Sulfatide is synthesizedin the Golgi apparatus by galactocerebroside sulfotransferase (CST; EC 2.8.2.11).Murine and human CST contain two putative N-glycosylation sites (Asn 66 andAsn 312). The second site is conserved among all Gal-3-O sulfotransferasescloned to date. To study the functional relevance of N-glycosylation, wegenerated epitope-tagged CST and soluble protein A-CST fusion proteins lackingboth N-glycosylation sites, separately or in combination. Our results show thatboth sites become glycosylated when expressed in CHO or COS cells. Moreover,transfecting CST mutants lacking both N-glycosylation sites or only Asn-312significantly reduced the amount of sulfatide synthesized, whereas changing Asn66 to Gln did not. In contrast, in vitro activity of mutant N66Q was reduced byabout 50% and was almost undetectable in N312Q and N66/312Q transfectants.Furthermore, CST expressed in the presence of tunicamycin was almost inactiveand accumulated in transfected cells. Expression of fully active CST in a CHOglycosylation mutant lacking N-acetylglucosaminyltransferase I demonstrate thatcondensation of the N-linked pentamannosyl-core structure is sufficient to form afully active enzyme.

contact:

Dr. Matthias EckhardtUniversität BonnPhysiologische Chemieeckhardt@institut.physiochem.uni-bonn.deNussallee 1153115 Bonn (Germany)

Jörg Bär, Jürgen Kirchberger, Anke Edelmann

Interactions between the two types of subunits forming an enzymatically activeoligomeric phosphofructokinase-1 from Saccharomyces cerevisiae

Phosphofructokinase-1 (Pfk-1) from yeast is a hetero-octamer (alpha4beta4, 835kDa). The subunits are encoded by two independent genes, PFK1 and PFK2.Pfk-1 was used as a model protein to study the process of folding and assemblygenerating a functionally oligomeric enzyme.We have investigated the generation of Pfk-1 in a cell-free system (in vitrotranslation) and the reactivation of the chemically denatured enzyme. Moreover,we compared the structure of genetically modified Pfk-1 with that of an enzymeform obtained by limited proteolysis.Our results can be summarised as follows:* The correct folding of the two types of subunits is independent of each other.* Truncation of the polypeptide chains in vivo and in vitro results in differentenzyme forms. Genetic deletion influences mainly the folding of the subunits andyields a reduced level of this polypeptide chain. The truncated subunit is still ableto assemble forming an active hetero-octamer. In contrast, limited proteolysis ofthe native Pfk-1 results in a tetrameric enzyme without loss of activity.* The correct assembly of the subunits proceeds via intermediates and isATP-dependent.* The ability of separately folded polypeptides to assemble is time limited.* Subunits, which are not able to assemble, are proteolytically degraded oraggregate. The aggregation can be reduced by artificial chaperones in vitro.* Fructose 6-phosphate and ATP stabilise the octameric structure of Pfk-1.* The formation of an active hetero-octameric yeast Pfk-1 is also possible in amammalian system, although a homo-tetrameric enzyme is characteristic forthese cells.

LiteratureBär et al., 2000, Biochem. 39, 6960-6968Edelmann et al., 2000, Eur. J. Biochem. 267, 4825-4830

contact:

Dr. rer.nat. Anke EdelmannUniversität LeipzigInstitut für Biochemiescha@medizin.uni-leipzig.deLiebigstraße 1604103 Leipzig (BRD)

Ralph-Peter Elsner

Solution structure of the Ras binding domain of AF6

1Guido Steiner, 1Werner Kremer, 1Ralph-Peter Elsner, 2Thomas Linnemann,2Christian Herrmann, 1Michael Wenzler, 1Gudrun Horn and 1Hans RobertKalbitzer

1Institut für Biophysik und physikalische Biochemie, Universität Regensburg,93040 Regensburg2Max-Planck-Institut für molekulare Physiologie, Otto-Hahn-Str. 11, 44227Dortmund

AF6, a protein with high sequence homology to Drosophila CANOE, was originallyidentified as a fusion partner of ALL-1 in acute myeloid leucaemia [1]. In 1996,Kuriyama et al. reported the discovery of a Ras binding protein in bovine brainextract [2]. The sequence of this protein with a molecular mass of 180 kDaturned out to be almost identical to human AF6. Thus, AF6 was identified as aputative effector in a Ras modulated signalling cascade. Binding of AF6 was alsoobserved to ZO-1, a protein involved in the formation of tight junctions inepithelial cells [3]. It could be shown that a null mutation in the murine AF6 locusleads to abnormal development of cell-cell-junctions in early embryogenesis, withlethal consequences for the mouse embryo [4].The amino terminus of AF6 (residues 1-141) adopts a stable fold and wascharacterized as the minimal Ras binding domain (RBD) [5]. AF6-RBD exhibitsRas binding properties similar to other effectors such as Raf and RalGDS [5]. Onthe other hand, AF6-RBD is significantly larger than those.Here we present the three dimensional structure of AF6-RBD in solution whichwas determined by high resolution NMR spectroscopy. The structure showed tobe widely identical to other known Ras-binding domains. The fold of AF6 exhibitsa five-stranded mixed ²-sheet complemented by two ±-helices, which is knownas a ubiquitin ±/²-roll.In comparison to other known RBD structures we observe n-terminal anadditional &alpha; helix and a longer loop region, thus extending the commonfold of RBD´s.

Literature[1] Prasad, R., Gu, Y., Alder, H., Nakamura, T., Canaani, O., Saito, H., Huebner,K., Gale, R. P., Nowell, P. C., Kuriyama, K., Miyazaki, Y., Croce, C. M., Canaani,E., (1993) Cancer Res. 53, 5624

[2] Kuriyama, M., Harada, N., Kuroda, S., Yamamoto, T., Nakafuku, M.,Iwamatsu, A., Yamamoto, D., Prasad, R., Croce, C., Canaani, E., Kaibuchi, K.(1996) J. Biol. Chem. 271, 607

[3] Yamamoto, T., Harada, N., Kano, K., Taya, S., Canaani, E., Matsuura, Y.,Mizoguchi, A., Ide, C., Kaibuchi, K. (1997) J. Cell. Biol. 139, 785

[4] Zhadanov, A., Provance, D. W., Speer, C. A., Coffin, J. D., Goss, D., Blixt, J.A., Reichert, C. M., Mercer, J. A. (1999) Current Biology 9, 880

[5] Linnemann, T., Geyer, M., Jaitner, B. K., Block, C., Kalbitzer, H. R.,Wittinghofer, A., Herrmann, C., (1999) J. Biol. Chem. 274, 13556

contact:

Ralph-Peter ElsnerUni RegensburgBiophysik/physikalische Biochemieralph-peter.elsner@biologie.uni-regensburg.deUniversitätsstr. 3193053 Regensburg (Deutschland)

Jochen Frenzel, Jan Richter, Klaus Eschrich

Nitrogen oxide-induced cell death in rabbit cultured Müller cells is modulated byglucose

Müller cells are the principal glia in the mammalian retina. Incubation of isolatedand cultured rabbit Müller cells with Papanonoate, a donor for exogenousnitrogen oxide, decreased the intracellular gluthathione content to 70% within 2h. In consequence, cells with significantly enhanced content of reactive oxygenspecies increased 1.5-fold between 0.5 and 2 h when the cells were analyzed byflow cytometry using the non-polar dye dichlorodihydrofluorescein-diacetate (1).Approx. 20% of the cells showed apoptosis after staining with Hoechst No. 33258and propidium iodide. Deprivation of glucose results in a more drastic decrease ofthe cellular glutathione content to 50% followed by a twofold increase of cellscontaining excess reactive oxygen species. The extent of dead cells did notchange although cell death was now nearly exclusively caused by necrosis.

Literature1) Frenzel, et al. (2002), Biochim. Biophys. Acta. 1542, 82-94.

contact:

Prof. Dr. Klaus EschrichUniversität LeipzigInstitut für Biochemieeschrich@uni-leipzig.deLiebigstr. 1604103 Leipzig (Germany)

additional information

J. Richter: Institut für Klinische Immunologie und Transfusionsmedizin,Universitätsklinikum Leipzig, Johannisallee 30, D-04103 Leipzig, Germany

Leopold Flohé

The element of the moon moonlighting for fertility

Selenium is contained in spermatozoa primarily as an integral part ofphospholipid hydroperoxide glutathione peroxidase (GPx-4). In testis, GPx-4 isexpressed as a cytosolic, a mitochondrial and a nuclear variant, most abundantlyin spermatids. The differentiation of spermatids to mature spermatozoa isaccompanied by a shift of the redox equilibrium characterized by a loss of GSHand oxidation of protein SH groups. In parallel, GPx-4 is transformed from anactive peroxidase into an enzymatically inactive structural protein that representsthe major component of the mitochondrial capsule.1 Biochemically, thistransformation of GPx-4 is explained by the use of protein SH groups as alternatesubstrates, which results in protein corss-linking via Se-S bonds. The clinicalrelevance of this “moonlighting” process for male fertility is underscored bysignificant correlations between GPx-4 content of sperm and fertility-relatedparameters such as sperm motility and morphological integrity.2 Further,clustered single base polymorphisms were detected in gpx-4 of patients withidiopathic infertility.3 It is concluded that selenium deficiency may compromisemale fertility by impairing GPx-4 synthesis, yet infertility may equally be due togenetic gpx-4 variants or any other disturbance of the time- and site-specificgpx-4 expression or the GPx-4 transformation process in late spermatogenesis.

Literature1F. Ursini, S. Heim, M. Kiess, M. Maiorino, A. Roveri, J. Wissing, L. Flohé,Science 285, 1393 (1999) ;2C. Foresta, L. Flohé, A. Garolla, A. Roveri, F. Ursini, M. MaiorinoBiol. Reprod. (2002), in press3M. Maiorino, V. Bosello, F. Ursini, C. Foresta, M. Scapin, L. FlohéBiol. Reprod. submitted

contact:

Prof. Dr. Leopold FlohéUniversität BraunschweigInstitut für Biochemielfl@gbf.deMascheroder Weg 138124 Braunschweig (Deutschland)

additional information

L. Flohé,* M. Maiorino,** S. Pilawa,* H. Stzajer,* and F. Ursini,*** Dept. Biochemistry, Technical University of Braunschweig, D-38124Braunschweig;** Dept. Biological Chemistry, University of Padova, I-35121 Padova

Bernd Moritz, Gudrun Franke, Martin Siemann, Matthias Reuss, Helmut E.Meyer

Quantitative Proteomics with Escherichia coli

Mathematical formulations of metabolic pathways contain metabolite and proteinconcentrations as variables [1]. Therefore, the combination of quantitativemetabolite pool analysis with mass spectrometric determination of proteinconcentration ratios is a promising tool for getting a deeper insight into centralregulation structures. Proteins with altered expression levels in diseased tissuesare possible drug targets, which stresses the importance of quantitativeproteomics in medicine.Quantitative proteomics can be done after in vitro or in vivo labelling. In vivolabelling is done by cell cultivation in 15N labelled ammonium-sulfate, whereas invitro labelling by deuterated N-Acetoxysuccinimid or by tryptic digestion in H2

18Ois carried out after cell lysis. In vivo labelling of microorganisms is an accuratemethod [2], but in case of tissue samples only error-prone in vitro labellingtechniques or gel-spot intensity measurements are applicable. Therefore, acomparison and statistical analysis of these techniques with a relatively simplesystem like E. coli is a prerequisitefor the analysis of tissue samples.

Literature[1] Moritz B.et al. (2000), Eur J Biochem. 267(12), 3442-52.[2] Oda Y. et al. (1999), Proc. Natl. Acad. Sci. 96, 6591-96.

contact:

Dipl.-Chem. Gudrun FrankeRuhr-Universität BochumMedical Proteom-Centergudrun.franke@ruhr-uni-bochum.deUniversitätsstr. 15044780 Bochum (Germany)

additional information

B. Moritz, H.E. Meyer, Medical Proteom-Center, Ruhr-Universität Bochum, 44780Bochum, GermanyM. Siemann, M. Reuss, Institute of Biochemical Engineering, Universität Stuttgart,70569 Stuttgart, Germany

Alexander Freiberg, Jan Lengefeld, Matthias Machner, Wolfgang Pfeil, DirkHeinz, Robert Seckler

Stability and Folding of Internalin B - A Biophysical Characterization

Internalin B belongs to the internalin family of Listeria monocytogenes andpromotes entry by phagocytosis of the Gram-positive pathogen into manynormally non-phagocytic cells. The N-terminal fragment of Internalin B (residues36-211, InlB241) (Schubert et al. 2001) used in this study consists of aleucine-rich repeat domain that is flanked by an N-terminal cap domain. Eachsingle leucine-rich repeat consists of 22 amino-acid residues folded into a shortbeta-strand and an opposing 310-helix. The beta-strands and helices areconnected to each other by turns. Our Internalin B fragment is a monomericprotein, with a molecular mass of 23 kDa and without disulfide bonds.

In the present study, we characterized the spectroscopic properties, the stability,and the folding of this leucine-rich repeat protein using different biophysicalprobes. The pronounced circular dichroism spectra of InlB241 exhibit no finestructure in the near-UV. In contrast, the far-UV CD spectra display a maximumat 185 nm and minima at 219 and 199 nm. The latter may be characteristic for310-helix. The single tryptophane residue at position 124 exhibits maximumfluorescence around 348 nm, compatible with its solvent exposure.InlB241 shows reversible folding behaviour after chemical denaturation measuredby fluorescence at 10 µg/ml. The midpoint of the unfolding transition is at 0.63 Mguanidinium chloride. Fitting the guanidinium chloride induced unfoldingtransition with a two-state model, yielded a free energy of stabilization deltaG(H2O) of - 4.8 ± 0.2 kcal/mol. The relative high denaturation m-value of 7.5 ±0.3 kcal/(mol M) is indicative of the absence of intermediates and, thus, supportsthe two-state analysis. By contrast, thermal denaturation is not reversible butresults in aggregation. The native structure of InlB241 is stable in the pH rangefrom 6 to 10. Below pH 4, the maximum fluorescence emission wavelength isblue-shifted to 333 nm, suggesting the presence of a collapsed, non-native state.Renaturation kinetics indicate the presence of at least one folding intermediateforming on the stopped-flow time scale.

LiteratureSCHUBERT, W.-D., GÖBEL, G., DIEPHOLZ, M., DARJI, A., KLOER, D., HAIN, T.,CHAKRABORTY, T., WEHLAND, J., DOMANN, E., and HEINZ, D. W.: Internalinsfrom the Human Pathogen Listeria monocytogenes Combine Three Distinct Foldsinto a Contiguous Internalin Domain. J. Mol. Biol. 312, 783-794 (2001)

contact:

Alexander FreibergUniversität PotsdamInstitut für Biochemie und Biologiefreiberg@rz.uni-potsdam.deKarl-Liebknecht-Strasse 24 - 25, Haus 2514476 Golm (Deutschland)

Susana Gariba de Garcia, Gerald Münch, Gabriele Oehme

"Carbonyl stress” by methylglyoxal causes mitochondrial dysfunction, ATPdepletion and NMDA receptor overstimulation in neurons

Advanced glycation endproducts (AGEs), di(carbonyl)-derived compounds, arefound in "neurofibrillary tangles", characteristic intraneuronal protein deposits ofAlzheimer‘s disease (AD), suggesting that crosslinking-reactive dicarbonylcompounds such as methylglyoxal (MG) are elevated in affected neurons [1]. MGis a very reactive dicarbonyl compound, and is formed by non-enzymaticdegradation of triosephosphates. It is accumulated under conditions of decreasedmetabolic utilisation of triosephosphates, or by impaired MG detoxificationoccurring under conditions of GSH depletion [2].The aim of this study was to investigate cellular effects of MG using a modelneuronal cell line (SH-SY5Y neuroblastoma cells, which are, like many cancercells particularly sensitive to MG toxicity). Furthermore, protective effects ofcarbonyl scavengers, antioxidants, nitric oxide synthase inhibitors and NMDAreceptor antagonists were tested.MG (applied in concentrations ranging from 70 - 2500 µM) caused cell death, adecrease in intracellular ATP content, mitochondrial membrane depolarizationand increased formation of mitochondrial reactive oxygen and nitrogen species ina concentration-dependent manner. MG induced reduction in cell number wasaccompanied by a decrease in the number and length of neurites. ATP depletioncould be significantly prevented by the carbonyl scavengers aminoguanidine,tenilsetam, carnosine; and by the NMDA receptor antagonists MK-801,Memantine, and D-AP7. Thus, scavenging of dicarbonyl compounds orinterference with their downstream pathways may provide new therapeuticopportunities to reduce the pathophysiological changes associated withdicarbonyl derived neurodegeneration.

Literature[1] Wong A, Lüth HJ, Arendt Th, Münch G (2001) Advanced glycationendproducts co-localize with inducible nitric oxide synthase in Alzheimer’sdisease. Brain Res 920: 32-40.

[2] Thornalley PJ, Jahan I, Ng R. (2001) Suppression of the accumulation oftriosephosphates and increased formation of methylglyoxal in human red bloodcells during hyperglycaemia by thiamine in vitro. J Biochem (Tokyo) 129: 543-9.

contact:

PD Dr. Gerald MünchIZKF LeipzigNeuroimmunologische Zellbiologiemueg@medizin.uni-leipzig.deJohannisallee 30a04103 Leipzig (Germany)

Jovana Gasic-Milenkovic, Sladjana Dukic-Stefanovic, Winnie Deuther-Conrad,Gerald Münch

ß-amyloid peptide potentiates inflammatory responses induced bylipopolysaccharide, interferon-gamma and "advanced glycation endproducts" in amouse microglia cell line

ß-amyloid peptide (Aß) and "Advanced glycation endproducts" (AGEs), proteinbound oxidation products of sugars, accumulate in the senile plaques inAlzheimer’s disease (AD) brain. Activated microglial and astroglial cells areassociated with the senile plaques, produce free radicals and inflammatorycytokines, and are suggested to accelerate the neurodegenerative process [1].We hypothesized that Aß needs a pre-stimulated environment to exert itspro-inflammatory potential. Since AGEs accumulate in the AD brain over time, agradual increase in the level of AGEs may gradually induce a low-level ofneurotoxicity and chronic inflammation in the ageing brain [2]. Thus, we testedwhether Aß acts as an amplifier of a sub-threshold pro-inflammatory responseinitiated by exposure to AGEs as the AD-specific physiological stimulus usingchicken egg albumin-AGE (CEA-AGEs), or the established stimuli LPS orIFN-gamma. Synthetic Aß (1-40) was used to produce three different samples(large sedimentable Aß aggregates, small non-sedimentable Aß aggregates andAß-AGE) which were further characterised for fibril content by Thioflavin-T assayand light microscopy. M-CSF, NO and TNF-alpha production were measured asmarkers of microglial activation. All three Aß preparations could not induce adetectable microglial response on their own. However, the combination of Aßpreparations with CEA-AGEs, LPS or IFN-gamma provoked a strong microglialresponce. Aß-AGE and the small non-sedimentable Aß aggregates were the mostpotent co-inducers of the pro-inflammatory response. Thus, we can assume thatAß induces significant microglial activation only in the presence of some otherinflammatory stimulus.

Literature[1] Wong A, Schinzel R, Wiesinger H, Riederer P, Münch G (2001)Anti-inflammatory antioxidants attenuate the expression of inducible nitric oxidesynthase mediated by advanced glycation endproducts in murine microglia. Eur JNeurosci 14: 1-8

[2] Gasic-Milenkovic J, Loske C, Münch G (2001) "AGEing" of a protein – thecytotoxicity of a glycated protein increases with its degree of AGE modification. ZGerontol Geriatrie 34: 457-60

contact:

Dr Jovana Gasic-MilenkovicIZKF LeipzigNeuroimmunological Cell Biologygasj@medizin.uni-leipzig.deJohannisallee 30a04103 Leipzig (Germany)

Klaus Golka, Seidel Thilo, Roetzel Claudia, Geller Frank, Bolt Hermann M,Dietrich Holger, Foth Heidi, Thier Ricarda

Enzym polymorphisms and risk factors in bladder cancer patients in LutherstadtWittenberg

Several occupational bladder carcinogens are metabolized by polymorphicenzymes. Therefore, the distribution of the polymorphic enzymesN-acetyltransferase 2 (NAT2; substrates: e.g., aromatic amines), glutathioneS-transferase MI (GSTM1; substrates: e.g., reactive metabolites of polycyclicaromatic hydrocarbons), and glutathione S-transferase T1 (GSTT1; substrates:small molecules with 1-2 carbon atoms) were investigated in bladder cancerpatients in the former center of the chemical industry in Halle-Wittenberg in theformer German Democratic Republic. Occupations performed for more than 6months by 136 patients of the urological clinic in Wittenberg were examined witha questionnaire. All patients had a histologically approved transitional cell cancerof the urinary bladder. In addition, several occupational and non-occupationalrisk factors were considered. The genotypes of NAT2, GSTM1, and GSTT1 weredetermined from leucocyte DNA by PCR. Compared to the general population inCentral Europe, in the entire group the percentage of GSTT1 negative persons(22.1 %) was ordinary, the percentages of slow acetylators (59.8 %) and ofGSTM1 negative persons (58.8 %) were slightly elevated. Shifts in thedistribution of the genotypes were observed in subgroups who had been exposedto asbestos (6/6 GSTM1 negative, 5/6 slow acetylators), chlorinated solvents(9/15 GSTM1 negative) or who did work in rubber manufacturing (8/10 GSTM1negative). The overrepresentation of GSTM1 negative bladder cancer patients inthe industrialized area, which was more pronounced in several occupationallyexposed subgroups, points to chemoprotection by the enzyme GSTM1 in bladdercarcinogenesis.

contact:

PD Dr. med. Klaus Golka

Institut für Arbeitsphysiologie an der Universität Dortmundgolka@ifado.deArdeystr. 6744139 Dortmund (Deutschland)

Oliver Ohlenschläger, Jens Wöhnert, Enrico Bucci, Ramadurai Ramachandran,Roland Zell, Matthias Görlach

Structure of an RNA Involved in Enteroviral Replication

Enteroviruses are human pathogens responsible for a number of acute andchronic diseases. The initiation of enteroviral positive-strand synthesis requires aribonucleoprotein complex consisting of a cloverleaf-like RNA structure at the5´-end of the viral positive-strand RNA, and a set of proteins including the viralprotease 3Cpro. The 5´-cloverleaf RNA comprises one stem (A) and threestemloop subdomains (B, C, D). In vitro binding experiments using the 3Cpro ofcoxsackievirus B3 revealed that the subdomain D of the 5´-cloverleaf isnecessary and sufficient for specific 3Cpro binding. Binding analysis using a set ofcloverleaf and stemloop D mutants, together with CD spectroscopy, suggestedthat the apical loop of subdomain D is a major determinant for the specificRNA-protein interactions here (1). To elucidate the structural basis of therecognition of subdomain D by 3Cpro we have determined the structure of a30-mer RNA from subdomain D by heteronuclear multidimensional NMRspectroscopy. Hydrogen bonds indicating the presence of canonical base pairsand one unusual U:C base pair have been directly detected by the HNN-COSYexperiment. Resonance assignments have been obtained using standard tripleresonance experiments and a new experiment to link the exchangeable with theH5/H6 protons in pyrimidine bases simultaneously. The stem of stemloop D is inan all-helical conformation and includes a central base paired triple pyrimidine(U:U-U:C-U:U) mismatched region. The apical tetraloop has a defined structureclosed by a U:G base pair. Even though the sequence and the closing base pair ofthe apical tetraloop differ from known stable tetraloops its structure issurprisingly similar to the structure of one of the known stable tetraloops.

Literature(1) Zell, Sidigi, Bucci, Stelzner and Görlach, RNA (2002), 8:188-201.

contact:

Dr. Matthias GörlachInstitut für Molekulare BiotechnologieAbt. Mol. Biophys. / NMR-Spektroskopiemago@imb-jena.deBeutenbergstr. 1107745 Jena (Deutschland)

additional information

J. Wöhnert: Institut für Organische Chemie, J.W.G.-Universität, FrankfurtEnrico Bucci: Istituto di Biostrutture e Bioimmagini, Naples, ItalyRoland Zell: Institut für Virologie, Friedrich-Schiller-Universität, Jena

Anke Hannemann, Marina Bigl, Frank Gaunitz, Klaus Eschrich

Characterization of the human platelet-type 6-phosphofructo-1-kinase genepromoter

In humans three isoforms of 6-phosphofructo-1-kinase (PFK) exist. Thepromoters of muscle and liver-type PFK have been partially characterized byothers. We cloned bases -1697 to -1 relative to the translational start site ofhuman platelet-type PFK and investigated for promoter activity by deletionanalysis with a luciferase reporter. The cloned fragment showed a GC-content of75% with a predicted CpG-island ranging from -1164 to -48. Promoter activitywas assayed in SH-SY5Y (human neuroblastoma) and 1321N1 (humanastrocytoma) cells. The region from -113 to -1 was sufficient for promoteractivity and fragment -201 to -1 showed the highest promoter activity whileactivity decreased in longer fragments. Deletion fragments ranging from -113 to-201 relative to the translational start site showed an increasing promoteractivity with higher values in SH-SY5Y cells than in 1321N1 cells. This promoterregion lacks a TATA-box but has four putative Sp-family binding sites, a GC-boxand a putative NF-Y binding site.

contact:

Anke HannemannUniversität LeipzigInstitut für Biochemiehanna@medizin.uni-leipzig.deLiebigstr. 1604103 Leipzig (Germany)

Monika Walter, Robert Seckler, Benjamin Heinz

Folding Kinetics of Pectate Lyase from Bacillus subtilis

Pectate lyase from Bacillus subtilis belongs to the class of proteins with a parallelbeta-helix folding motiv. The denaturation of the protein follows amonoexponential time course. The renaturation is at least biphasic with theslowest phase coinciding with the reactivation of the enzymatic activity of theprotein and appears to be dependend on proline isomerisation. BsPel possesses14 prolines, one of which forms a cis-peptide bond. Random mutations of thecis-proline to different amino acids produce mutants with renaturation kineticssimilar to the wild-type, but faster denaturation.

contact:

Benjamin HeinzUniversität PotsdamPhysikalische BiochemieBen.Heinz@web.deKarl-Liebknechtstr. 24-25, Haus 2514476 Golm (Deutschland)

Susanne Ammon, Peter Mayer, Volker Höllt

Microarray analysis fo genes expressed in the frontal cortex of rats chronicallytreated with morphine

Opioid dependence may be associated with adaptive changes in gene expressionin brain. In the present study we used DNA microarrays (U34A; Affymetrix) toanalyse the expression of about 8000 genes in the frontal cortex of ratschronically treated with morphine and in rats after naloxone precipitatedwithdrawal. Chronic treatment for 10 days with ascending doses of morphine (10to 50 mg/kg bid) morphine resulted in a more than twofold induction of 14genes. The majority of these genes code for heat shock proteins (hsp70, hsp 27,hsp 40, hsp 105, GRP78 etc.). The expression of the heat shock genes wasreversed in the morphine-treated animals after withdrawal precipitated bynaloxone (10 mg/kg). The opioid antagonist, in turn, increased the expression ofa set of genes which are predominantly transcription factors (krox20, CREM,NGFI-B, IkappaB etc). Few genes only remained increased after naloxoneapplication. Such persistently changed genes code for arc, acytoskeleton-associated protein which is induced by synaptic activity, ania-3, asplice variant of the Homer 1 protein which is critically involved inactivity-dependent alterations of synaptic function and per2, a protein regulatingcircadian rhythmicity. For selected genes the changes in gene expression wereconfirmed by quantitative real time PCR and by in situ hybridization. Thesefindings indicate that the persistent changes in long-lasting plasticity duringopiate dependence do not primarily depend on an increased expression levels ofneurotransmitter, receptor and/or ion channel proteins, but rather on alteredpattern of synaptic connectivity.

contact:

Prof. Dr. Volker HölltOtto-von-Guericke-Universität MagdeburgInstitut für Pharmakologie und Toxikologiehoellt@medizin.uni-magdeburg.deLeipziger Strasse 4439291 Magdeburg (Deutschland)

Gudrun Horn, Daniel Weinfurtner, Roland Hofweber, Robert Macek, Robert Gail,Till Maurer, Hans Robert Kalbitzer

Optimization of intein based isotope labeling methods for NMR investigations ofproteins

Gudrun Horn, Daniel Weinfurtner, Roland Hofweber,Robert Macek, Robert Gail,Till Maurer, and Hans Robert KalbitzerInstitute of Biophysics and Physical Biochemistry. University of Regensburg,93040 Regensburg, Germany

With the advent of the TROSY-spectroscopy the NMR structure determination oflarger than 30 kDa is in principle feasable. However, with the size of the proteinthe number of resonance lines and thus the signal overlap increases and makesthe assignment of resonances extremlydifficult. Specific isotope labeling candecrease the number of signals and facilitate the spectral assignement.Regiospecific labeling by intein trans-splicing represents a powerful newmethod.[1-3].Inteins are able to enzymatically cut themselves out of the precursor protein andto subsequently splice the flanking residues by a peptide bond. For practicalapplication in NMR spectroscopy a genetically engineered split intein is used todifferently labelling of proteins.The N-terminal and the C-terminal part of a protein are fused to one fragment ofa split intein.Then the fragments can be produced separately in E. coli expressionsystem in culture media with and without isotopes. The splice reaction takesplace in vitro where the intein can fuse and reasssemble a functional proteindomain containing a protein which is isotope enriched in one part of the sequenceand unlabelled in the otherone.We tested the intein system in different proteins involved in the cellular signaltransduction, the guanine nucleotide dissociation stimulator of the small GTPaseRAL, RalGEF, the Ran protein and the Ran-binding domain of RanBP2. Thespecific problems associated with the expression of the intein constructs, thepurification and the splicing reaction are discussed.

LiteratureLiterature1. Maurice W. Southworth et al.(1998) Control of Protein splicing by inteinfragment reassemly. The EMBO Journal 17, 918-9262. Francine B. Perler (1998) Protein Splicing of Inteins and HedgehogAutoproteolysis: Structure, Function and Evolution, CELL, 92, 1-43. Yamazaki et al. (1998) Segmental Isotope Labeling for Protein NMR usingpeptide splicingJ.Am.Chem. Soc. 120, 5591-5592

contact:

Dr. Gudrun HornUniversität RegensburgInstitut für Biophysik und Physikalische Biochemiegudrun.horn@biologie.uni-regensburg.deUniversitätsstr.93040 Regensburg (Germany)

Franziska Bleichert*, J.P. Warnke**, Klaus Eschrich*, Renate Keßler*

Expression of ubiquitous 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatasein intracranial neoplasms and non-tumorous brain tissue

In eukaryotic cells, the bifunctional6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2)catalyses the production and degradation of fructose-2,6-bisphosphate, the mostpotent activator of the key glycolytic enzyme 6-phosphofructo-1-kinase.PFK2/FBPase2 is assumed to be essential for tumor cell proliferation (1). Wehave identified six splice variants of the ubiquitous PFK-2/ FBPase-2 in humanbrain (2). To investigate the role of the ubiquitous FBPase-2 in intracranialneoplasms, we analysed the mRNA splice pattern of ubiquitous PFK-2/FBPase-2in human gliomas of different WHO-grades, meningiomas, and intracranialmetastases as well as in non-tumorous brain tissue. Furthermore, the expressionof the corresponding proteins were studied by Western blot analysis.

Literature1) Chesney, J. et al. (1999). Proc. Natl. Acad. Sci. USA 96, 3047-52.2) Kessler, R. and Eschrich, K. (2001). Mol. Brain Res. 87, 190-95.

contact:

Dr. rer. nat. Renate KeßlerUniversität LeipzigInstitut für Biochemie, Medizinische Fakultätkessr@medizin.uni-leipzig.deLiebigstrasse 1604103 Leipzig (Deutschland)

additional information

*Institut für Biochemie, Medizinische Fakultät, Universität Leipzig, Liebigstraße 16,04103 Leipzig

**Neurochirurgische Abteilung, Paracelsus Klinik, Werdauer Straße 68, 08008Zwickau

V. Krügel

Inhibition of LiCl-induced induction of glutamine synthetase in HepG2 cells byflavonoids and anthraquinone derivatives is mediated by CK I

Induction of glutamine synthetase (GS) in liver tumor cells is associated withaggressive tumor growth (1). Recently, we found that GS can be induced inHepG2 cells by LiCl through a signaling pathway involving beta-catenin.Activation of beta-catenin may explain the association of GS overexpression inselective tumors. In this study, a screening of different inhibitors of GS inductionwas performed, in order to learn more about possible kinases participating in thissignaling.Among various inhibitors tested, the flavonoid apigenin and the anthraquinonederivative emodin showed a potent inhibition of LiCl-dependent GS induction inHepG2 cells. Since both compounds are known as inhibitors of casein kinase II(CK II) a direct assay of this kinase was applied. The EC50-value for inhibiton byemodin was around 2 microM, while those of some newly synthesizedemodin-derivatives were in the nanomolar range. Indeed, these compoundsinhibited GS-induction also more effectively as emodin. These results suggestthat CK II is an essential component of the signaling pathway regulating GSexpression in hepatoma cells.

Literature(1) (1) Gebhardt R, Williams G. Carcinogenesis 1995; 16: 1673-1681

contact:

V. KrügelUniversität LeipzigInstitut für Biochemiergebhardt@medizin.uni-leipzig.deLiebigstr. 1604103 Leipzig (D)

additional information

V. Krügel, F. Gaunitz, R. Gebhardt, Institute of Biochemistry, K. Daub, L. Teich, K.Eger, Institute of Pharmacy, University of Leipzig

Peter Kühl

Who discovered the Michaelis-Menten hyperbola?

There exist a number of eponyms which carry the names of L. Michaelis(1875-1949) and M.L. Menten (1879-1960) and derive from their sole jointpaper(1), e.g., Michaelis-Menten [MM] complex, MM constant, MM equation, MMhyperbola, MM hypothesis, MM mechanism, MM plot, MM theory. All theseeponyms – with one exception, the MM constant Km – are incorrect (or ofquestionable legitimation) because they give M & M undeserved credit for theoriginality and priority of concepts which M & M never claimed for themselves;actually, M & M adopted expressis verbis the ideas of V. Henri(2) (1872-1940)and corroborated them by an improved experimental technique. Concerning the“MM hyperbola” it may also be mentioned that M & M(1) – contrary to previousenzyme kineticists(2,3) – neither used the term “hyperbola” or “hyperbolic” nordid they present their kinetic data as hyperbolic graphs. Clearly, the discovery ofhyperbolic enzyme kinetics and its mass-action theoretical foundation belong tothe achievements of Henri, not to those of M & M; for this reason, the usage ofthe misnomers “MM hyperbola” and “MM kinetics” should be discontinued.To eradicate or change an established eponym is difficult but not impossible asan example in physics illustrates: the unit of radioactivity was changed in 1975from curie to becquerel in order to honour the true discoverer of radioactivity. Sowhy not rename “MM kinetics” after the true discoverer and founder of hyperbolickinetics into “Henri kinetics”?

Literature1) Michaelis, L., and Menten, M. L. (1913). Biochem. Z. 49, 333-369.2) Henri, V. (1903). Lois générales de l’action des diastases (Paris: LibrairieScientifique A. Hermann).3) Philoche, C. (1908). J. Chim. Phys. 6, 212-293 and 355-423.

contact:

Dr. Peter KühlUniversity of BaselInstitute of Theoretical Biologypeter-w.kuehl@unibas.chSchaulistr. 24142 Münchenstein BL (Switzerland)

Antonios Kyriakopoulos, Barbara Hoppe, Alexandra Graebert, MarcusKühbacher, Dietrich Behne

Selenoproteins in the perinuclear structures of rat kidneyDedt. Molecular Trace Element research in the Life SciencesHahn- Meitner-Istitut, Glienicker Str.100, 14109 Berlin

The essentiality of the tracer element selenium was shown to be due to thebiological functionof several selenoproteins. In the selenoproteins identified so far the element ispresent as sele-nocysteine and in this form is part of the active site of some enzymes.For the determination of the selenium laves instrumental neutron activationanalysis (INAA)via 75Se was used (1) Information on the membrane-bound selenoproteins wasobtained by labellingof rats in vivo with 75Se-selenite, subcellular fractionation of the tissuehomogenates, separationof the proteins by SDS-PAGE, two-dimensional electrophoresis and seleniumdetection by autoradiography (2).In this way more than 30 selenium-containing proteins were detected (3). Ofthose six selenium-containingproteins with the approximately molecular masses of 60kDa, 38kDa, 28kDa,23-25kDa, 20kDa and 16kDawere detected in the perinuclear structures fraction.The 75Se-labeled protein bands of 60kDa, 38 kDa ,23-25 kDa and 16kDa aftertreatment with detergents,protein separation by SDS-PAGE and detection of selenium by autoradiographyare present in the endoplasmaticreticulum fraction (ER) again.This finding is of great interest regarding the function of the cellular organells.Therefore several experiments have been curried out in order to characterizetheseselenium-containing proteins.

Literature1. Behne, D., Kyriakopoulos, A., Weiss-Nowak, C,. Kalcklösch, M. Westphal, C.,Gessner, H.Newly found selenium-containing proteins in the tissues of the rat.Biol. Trace Element Res. 55 (1996) 99-110.2. Kyriakopoulos, A., Röthlein, D., Pfeifer, H., Bertelsmann, H , Behne, D.Detection of small selenium-containing proteins in tissues of the rat.J. Trace Elements. Med. Biol. 14 (2000) 179-183.3. Behne, D., Scheid, S., Hilmert, H.Gessner, H., Gawlik, D and Kyriakopoulos, A.ombination of neutron activation analysis, tracer techniques, and biochemicalmethods in the investigation of selenium metabolism.Biological Trace element Research (1990) 439-447.

contact:

Dr. Antonios KyriakopoulosHGFHahn-Meitner-InstitutKyriakopoulos@hmi.deGlienicker Str. 10014109 Berlin (D)

Carsten Skarke, Helmut Schmidt, Jürgen Liefhold, Gerd Geisslinger, Jörn Lötsch

A mathematical model to predict plasma concentrations of morphine and itsactive metabolite morphine-6-glucuronide in healthy young persons

Aim: To develop a mathematical model that describes the plasma concentrationsof morphine and its active metabolite morphine-6-glucuronide (M6G) and to testits prediction performanceMethods: Population compartmental pharmacokinetic modeling using NONMEMwas applied to plasma concentration versus time data of morphine and M6Gobtained from eight healthy volunteers (4 men, 4 women, aged 23 – 30 years)after intravenous bolus injection of 5.64 mg morphine base (7.5 mg morphinesulfate) and of 1 mg deuterized M6G. The prediction performance was tested byapplying the obtained model to morphine and M6G plasma concentrationsavailable from eight other healthy young subjects.Results: Two models were identified that described the plasma concentrationversus time courses of morphine and M6G after administration of morphine. Thefirst model consisted of a standard three-compartment model for morphine, anda standard two-compartment model for M6G, with input into and output from thecentral compartments. An alternative model assigned the formation of M6Gamong the first peripheral compartment of morphine and the centralcompartment of M6G, while morphine and M6G were again described by threeand two compartment models, respectively. Both models predicted morphine andM6G plasma concentrations available from an independent study with acceptableaccuracy and without bias.Conclusions: Two models were obtained that predict plasma concentrations ofmorphine and M6G with acceptable accuracy in healthy young volunteers.

Acknowledgement: DFG Lo 612/2-1

pharmazentrum frankfurt, Department of Clinical Pharmacology, JohannWolfgang Goethe-University, Theodor Stern Kai 7, D-60590 Frankfurt, Germany

Mundipharma GmbH, Mundipharma-Strasse 2-6, D-65549 Limburg (Lahn),Germany

contact:

Priv.-Doz. Dr. med Jörn LötschUniversität Frankfurtpharmazentrum frankfurt, Institut für Klinische Pharmakologiej.loetsch@em.uni-frankfurt.deTheodor-Stern-Kai 760590 Frankfurt (Deutschand)

Johanna Mansfeld, Eva Petermann, Renate Ulbrich-Hofmann

Generation of catalytically active neutral proteasefrom Bacillus stearothermophilus does notrequire the presence of the propeptide

The thermolysin-like neutral protease from Bacillus stearothermophilus (TLP-ste)belongs - like neprilysin, matrilysin, or angiotensin-converting enzyme - to thephysiologically important group of metalloendoproteinases, which participate invarious mportant physiological processes.TLP-ste and their mutants are usually produced extracellularly in Bacillus subtilis,where they are expressed as pre-proenzymes and subsequently processed in anautocatalytical, intramolecular process.Therefore, the presence of the prosequence is assumed to be necessary forcorrect folding of the enzyme. With the aim to elaborate a strategy which allowsto produce also inactive mutants for stability studies without theinterference of autoproteolysis, we investigated the expression of TLP-ste in E.coli. The coexpression of TLP-ste and propeptide were studied with one- andtwo-vector constructs. While the propeptide was expressed inhigh amounts, the expression of the mature sequence was only successfull whenthe N-terminus of TLP-ste was modified by an N-terminal His-tag or the fusionwith GST.In this case, however, TLP-ste accumulated in inclusion bodies, whereas thepropeptide was localized in the cytosol. Surprisingly, renaturation succeededwithoutthe presence of the propeptide. Therefore, the propeptide is not required for thegeneration of catalytically active TLP-ste.

contact:

Dr. rer. nat. Johanna MansfeldMartin-Luther-Universität HalleBiotechnologiemansfeld@biochemtech.uni-halle.deKurt-Mothes-Straße 306120 Halle (Deutschland)

Walter-Vesely Seb. Meister

Polyvinylnucleobases: Synthesis, characterization and applications innanotechnology

Artificial homopolymeric polyvinylnucleobases with a polyvinyl backbone mimicnative RNA, show the ability to hybridize to their complementary native nucleicacid strands, forming so-called "semiplastic" double- and triple-stranded RNAanalogues. Many of such pure and peptide complexed more nuclease-resistantnucleic acid arrangements with better immunomodulatory activities have beendeveloped. But their structure-phase behaviour as well as their structure-functionrelationship remain open problems. The studies of physico-chemical andbiological behaviour of these compounds are basic goals of our approach.

AFM-studies of surface immobilization of the first artificial nucleobase compoundvia thiol-gold reaction or silylation procedure show formation of uniform layers ofmolecules. We polymerized the artificial strands on gold and silicon wafers usingchemical methods of chain extension. The wafer fixation of these biologicallyactive and self-organizing structures stimulates new applications for"regenerable" nucleic acid drugs and nucleic acid computing.

LiteratureS. Hoffmann, W. Witkowski in: Mesomorphic order in polymers andpolymerization in liquid crystalline media (A. Blumstein, ed.) ACS-Symp. Ser. 74(1978) 178-236.B. Glück, W.-V. Meister, W. Witkowski, A. Stelzner, S. Hoffmann, J. BasicMicrobiol. 28 (1988) 501-507.W.-V. Meister, S. Lindau, A.L. Hauser, Ch. Bohley, U. Gromann, St. Naumann, M.Madre, L. Kovalenko, G. Bischoff, R. Zhuk, S. Hoffmann, Journal of BiomolecularStructure & Dynamics 18(3) (2000) 385-392.W.-V. Meister, S. Lindau, Anton L. Hauser, E. Birch-Hirschfeld, J. Reinert, K.Friese, C. Bohley, S.I. Kargov, U. Gromann, S. Hoffmann, Surface and InterfaceAnalysis 33(2) (2002) 137-145.

contact:

Dr. Walter-Vesely Seb. Meister

BioMera@webPOBox 900-239 (HPA)06054 Halle /Saale (Germany)

Nils Hanekop, Susanne Wied, Stefan Petry, Norbert Tennagels, Wendelin Frick,Günter Müller

A Receptor for Glycolipid Headgroups Mediates Insulin-mimetic Signaling in RatAdipocytes

The phosphoinositolglycan(-peptide)(PIG-P) portion ofglycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins isrecognized by a binding protein which is located in high-cholesterol-containingdetergent-insoluble glycolipid-enriched raft domains (hcDIGs) of the adipocyteplasma membrane. In isolated rat adipocytes, synthetic PIG molecules induce theredistribution of GPI proteins from hcDIGs to low-cholesterol-containing DIGs(lcDIGs) and concomitantly provoke insulin-mimetic signaling and metabolicaction. Using a set of synthetic PIG(-P) derivatives we demonstrate here thattheir specific binding to hcDIGs and their insulin-mimetic signaling/metabolicactivity strictly correlate with respect to (i) translocation of the GPI proteins,Gce1 and 5'-nucleotidase, from hcDIGs to lcDIGs, (ii) dissociation of thenon-receptor tyrosine kinase, pp59Lyn, from caveolin residing at DIGs (1), (iii)translocation of pp59Lyn from hcDIGs to lcDIGs, (iv) activation of pp59Lyn, (v)tyrosine phosphorylation of insulin receptor substrate proteins (IRS-1/2)(2), andfinally (vi) stimulation of glucose transport. The natural PIG(-P) derived from thecarboxy-terminal tripeptide of Gce1, YCN-PIG, exhibits the highest potencyfollowed by combination of the separate peptidylethanolamidyl and PIGconstituents. We conclude that efficient cross-talk of PIG(-P) to the insulinsignaling cascade requires a receptor protein for PIG(-P) at hcDIGs, which mayretain GPI proteins within hcDIGs in the unstimulated state but upon occupancyby soluble PIG(-P) release them for lateral movement to lcDIGs which initiatesthe DIGs-caveolin-pp59Lyn-IRS pathway (3,4)

Literature1) Schlegel, A., Volonte, D., and Engelmann, J.A. (1999) Cell. Signal. 10,457-463.2) Nystrom, F.H., and Quon, M.J. (1999) Cell. Signal. 11, 563-574.3) Müller, G., Jung, C., Wied, S., Welte, S., and Frick, W. (2001) Biochemistry40, 14603-14620.4) Müller, G., Jung, C., Wied, S., Welte, S., Jordan, H., and Frick, W. (2001) Mol.Cell. Biol. 21, 4553-4567.

contact:

Dr Günter MüllerAventis Pharma Deutschland GmbHDG Metabolic DiseasesGuenter.Mueller@aventis.comIndustriepark Höchst Bldg. H82565926 Frankfurt am Main (Deutschland)

Pavel I. Nedvetsky, Alexander Y. Kots, Helmut Müller, Ferid Murad, HaraldH.H.W. Schmidt

Hsp90 effects on the regulation of soluble guanylyl cyclase

Soluble guanylyl cyclase (sGC) is one of the most important receptors for nitricoxide (NO), participating in regulation of many physiological processes.Recently, an interaction between sGC and heat shock protein 90 (Hsp90) hasbeen demonstrated in endothelial cells. In the present study we haveinvestigated the role of Hsp90 in regulation of sGC. Hsp90 increasedNO-stimulated activity of purified sGC. This can be due to sGC stabilization andprotection from oxidative damage of the enzyme. Short (up to 1 h) treatment ofendothelial cells with a Hsp90 inhibitor, geldanamycin (GA; up to 1 &muM) hadno effect on the NO donor-stimulated cGMP accumulation in the cells. Longexposure of the cells to GA or one other Hsp90 inhibitor (radicicol), producedtime- and concentration-dependent loss of sGC protein. Both substances reducedsGC&alpha1 and sGC&beta1 protein levels to about 40-30% of control levels.Half-life time for these effects was between 4 and 6 h for both inhibitors. Tostudy if down-regulation of sGC expression is responsible for the effects observedwe have used the translation inhibitor, DRB. DRB alone produced much slowerdecrease in sGC protein level than Hsp90 inhibitors. This suggests that inhibitionof Hsp90 affects protein stability rather then expression. Indeed, proteasomeinhibitor, MG132, reversed GA-induced sGC decrease. In conclusion, Hsp90 isrequired to maintain sGC in endothelial cells. Inhibition of this chaperon inducedproteasome-mediated degradation of sGC.

contact:

Pavel NedvetskyJustus-LIebig-Universität GiessenRudolf-Buchheim-Institutpavel.nedvetsky@pharma.med.uni-giessen.deFrankfurter Str. 10735392 Giessen (Germany)

additional information

Affiliation of A.Y.K. and F.M.:Department of Integrative Biology and Pharmacology, UT-Houston Medical School,Houston, USA

Ellen Niederberger, Achim Schmidtko, Jeffrey D. Rothstein, Gerd Geisslinger,Irmgard Tegeder

Modulation of spinal nociceptive processing through the glutamate transporterGLT-1

GLT-1 is the predominant glutamate transporter in most brain regions andtherefore plays a major role in terminating synaptic transmission and protectingneurons from glutamate neurotoxicity. In the present study we assessed (i) theregulation of GLT-1 expression in the spinal cord after peripheral nociceptivestimulation and (ii) the nociceptive behaviour of rats following inhibition ortransient knockdown of spinal GLT-1. Formalin injection into one hindpaw causeda rapid transient upregulation of GLT-1 protein expression in the spinal cordwhich did not occur when rats were pretreated with morphine (10 mg/kg, i.p.)suggesting that the nociceptive input specifically caused the increase of GLT-1transcription. Inhibition of GLT-1 by the transportable inhibitortrans-pyrrolidine-2,4-dicarboxylic acid (t-PDC) resulted in a significant reductionof nociceptive behaviour in the rat formalin assay. Similar results were obtainedwith a transient reduction of GLT-1 protein expression by antisenseoligonucleotides. These data suggest that inhibition of GLT-1 activity orexpression reduces excitatory synaptic efficacy and thereby nociception.Mechanisms that might explain this phenomenon include activation of inhibitorymetabotropic glutamate receptors, postsynaptic desensitization or disturbance ofglutamate recycling.

contact:

Dr. Ellen NiederbergerKlinikum der Johann Wolfgang Goethe Universität Frankfurtpharmazentrum frankfurt, Inst. f. klinische Pharmakologiee.niederberger@em.uni-frankfurt.deTheodor-Stern-Kai7/Haus7560590 Frankfurt (Hessen)

Kathrin Fuchs, Diana Kühn, Johannes Schmitt, Joachim Nöller

�Membrane affinity and pore forming properties of proteins/peptides - Easy andfast by using solid supported lipid bilayers.�

Binds a protein to a biological membrane and form parts/domains of thisprotein a pore after incorporating in a plasma membrane of a cell ? Toanswer these questions we have developed an easy and quick assay toquantify membrane affinity and to analyse pore formation usingTRANSIL®[1] in a micro well plate. In this assay the protein/peptide ofinterest is incubated with a very well defined single lipid bilayer on asolid support shortly. Separation of the bound/unbound fractions occursin a low speed centrifugation step and for quantification e.g. a westernblot is used. Pore formation will be analysed by ion sensitivefluorescence dyes beneath the lipid bilayer in a fluorescencespectrometer. Taking advantage of the broad spectrum of possible lipidcompositions, the identification of binding partners or binding conditionsof a protein will be accessible. Our examples include gramicidin A, alphahemolysin, hrpZ and MRP.

Literature[1] Loidl-Stahlhofen et al., � Solid-Supported Biomolecules on Modified SilicaSurfaces � A Tool for Fast Physiocochemical Characterization andHigh-Throughput Screening.� 2001, Adv. Mater. 13, 1829 - 1834

contact:

Dr. Joachim NöllerNIMBUS Biotechnologie GmbH

jnoeller@nimbus-biotech.comEilenburger Str. 404317 Leipzig (Germany)

Ingo Paarmann, Manfred Konrad

Structural requirements for calmodulin binding to membrane-associated guaylatekinases

Membrane-associated guanylate kinases (MAGUKs) have been identified atcell-cell contact sites in organisms from Drosophila to man. In mammals,MAGUKs such as SAP90, SAP97, CASK, and p55 constitute a novel superfamily ofmultidomain proteins that are localized at synapses and participate in theformation of the membrane cytoskeleton. MAGUKs contain at least one PDZdomain, an SH3 domain, a HOOK region, and a domain with striking sequencehomology to the known low molecular mass guanylate kinases (GKs). In MAGUKproteins, the SH3 domain interacts intramolecularly with the GK domain. Wehave analyzed the functional role of the HOOK region localized between the SH3and the GK domains. Our results clearly show that (Ca2+)4-calmodulin binds tothe HOOK regions of both SAP97 and CASK belonging to two different MAGUKsubfamilies preferentially under conditions where the intramolecular interactionbetween the SH3 and the GK domains can occur. Calmodulin binding mightstabilize this interaction and serve as an effector molecule to modulate theassociation of various domain-specific binding partners.

contact:

Dr. Ingo PaarmannMax-Planck-Institut für biophysikalische ChemieAbteilung für Molekulare Genetikipaarma1@gwdg.deAm Fassberg 11, Turm 537077 Göttingen (Germany)

Thomas Schneider, Gerhard Braus, Gabriele Heinrich, Markus Hartmann, AndreaPfeil

Evolution of Feedback-inhibited (&beta/&alpha)8 Barrel Isoenzymes by GeneDuplication and A Single Mutation

The (&beta/&alpha)8 barrel is the common protein fold of numerous enzymesand was recently proposed to be the result of gene duplication and fusion of anancient half-barrel1. The initial enzyme of shikimate biosynthesis possesses theadditional feature of feedback-regulation. The loop connecting the twohalf-barrels and structural elements added to the barrel are prerequisites forregulation and form a cavity on the N-terminal face of the (&beta/&alpha)8barrel. In vitro evolution shows that after gene duplication a single mutationwithin the internal extra element was the crucial evolutionary event creatingdifferently feedback-inhibited isoenzymes.

Literature1. D. Lang et al., Science 289, 1546 (2000).

contact:

Dipl.Biol. Andrea PfeilGeorg-August-University GöttingenInstitute for Microbiology and Geneticsapfeil@gwdg.deGrisebachstr. 837077 Göttingen (Deutschland)

Gia Anh Bao PHAN, Detlef PIETROWSKI, Christoph KECK

EXPRESSION OF A NEW SPLICE VARIANT OF IGFBP-7/MAC25 TUMORSUPPRESSOR GENE IN HUMAN GRANULOSA CELLS LACKING THE IGFBP MOTIF(GCGCCXXC)

Introduction: IGF binding proteins are important regulators of IGF action bymodulating IGF binding to its receptor. They share a common 8 amino acid motif(GCGCCXXC) supposed to be involved in IGF binding which is located on theN-terminus of the protein. They are grouped into high affinity IGF bindingproteins and the more recently detected low affinity IGF binding proteins. Mac 25(Meningioma-associated complimentary DNA) is a 282 amino acid protein andbelongs to the low affinity IGF binding proteins. It is involved in growthregulation and has been shown to play a role in the senescence of varioustissues. Additionally, it was demonstrated to be a potential tumor suppressorgene in prostate cancer, liver cancer, and breast cancer. Whereas the function ofhigh affinity IGFBPs is presumed to protect IGF peptides from degradation, thefunctions of the low affinity IGFBPs are more complex and not completelyunderstood.Here we analyse the expression of Mac25 and a newly detected splice variant ofMac25 in tumorous as well as in non tumorous cells.

Material and methods: Human granulosa cells (GC) were isolated from follicularfluid of patients undergoing IVF therapy and then cultured. The followingcarcinoma cell lines HeLa, SW756, Caski, MDA-MB-453 were given by otherresearching groups in the University Women's Hospital Freiburg. RNA wasisolated by the method of Chomczynski and Sacchi (1987) and messenger RNAexpression of mac25 was determined by RT-PCR. Cloning and sequencing wereperformed using standard protocols according to manufacturers instructions.Sequence alignments were done with the SIM 4 computerprogramm package(http://pbil.univ-lyon1.fr/sim4.html)

Results: We could demonstrate that GC and tumor cells express mac 25.Interestingly in GC an additional fragment of smaller size was amplified. Cloningand sequencing this fragment revealed that it is a deleted splice variant of themac25 mRNA. Sequence alignment with known mac25 mRNA demonstrated thatthe deletion occurs in the first exon of mac25 and contains the typical IGFBPmotif. The deleted form is 165 nucleotides shorter than the full length mac25mRNA. Interestingly, we could show that the short version is expressed inprimary cells but not in tumorigenic Hela cells.

Conclusion: We detected for the first time a splice variant of the IGFBP mac25.The splicing seems to occur on cryptic splice sites within the first exon of thegene containing the IGFBP motif. These findings point to novel functions ofmac25 beside its IGF binding properties. We hypothesize that both the completeform of mac25 and its deleted form could be involved in growth modulatingaction in different ways in various tissues.

contact:

Dr.med. Gia Anh Bao PHANUniversity of Freiburg- GermanyUniversitäts- Frauenklinikbphan@frk.ukl.uni-freiburg.deHugstetterstrasse 5579106 Freiburg im Breisgau (Germany)

Paola Pocar, Robert Augustin, Bernd Fischer

Induction of apoptotic cell death in bovine cumulus-oocyte complexes aftertreatment with Aroclor-1254 during in vitro maturation.

Polychlorinated biphenyls (PCB) are persistent environmental contaminantsexhibiting, among others, reproductive toxicity. Previous studies have shown asAroclor-1254, a technical mixture of PCB, induces adverse effects on the in vitromaturation of bovine oocytes and on their subsequent development(Pocar et al,2001). It has been demonstrated that an increase in cumulus cell apoptosisresults in a deterioration of oocyte quality, thus reducing fertilization rates andembryo development (Lee et al, 2001). In the present study the apoptotic indexwas quantified by the TUNEL assay and alterations in expression of several Bcl-2family members induced by Aroclor-1254 were investigated in bovinecumulus-oocyte complexes (COCs). Bovine COCs were cultured for 24 h in thepresence or absence of 1 µg/ml of Aroclor-1254. The results show that theapoptotic index was increased in PCB-treated COCs compared to the control(10.67 vs. 2.80 % of total cells). Furthermore, despite semi-quantitative RT-PCRwas unable to detect any alterations in the expression of Bcl-2, Bcl-XL(antiapoptotic), Bax and Bak (proapoptotic) genes when singly considered, asignificative increase in the Bax/Bcl-2 ratio was observed. In conclusion, thisstudy suggests that apoptosis may be a possible mechanism involved in the toxiceffects of PCBs in bovine oocytes. The alteration of the Bax/Bcl-2 ratio points toan involvement of BCL-2 family members in PCB-induced apoptotic death incumulus cells.

LiteratureLee KS, Joo BS, Na YJ, Yoon MS, Choi OH, Kim WW. (2001) Cumulus cellsapoptosis as an indicator to predict the quality of oocytes and the outcome ofIVF-ET. J Assist Reprod Genet 18:490-8Pocar P, Perazzoli F, Luciano AM, Gandolfi F (2001) In vitro reproductive toxicityof polychlorinated biphenyls: effects on oocyte maturation and developmentalcompetence in cattle. Mol Reprod Dev 58(4):411-6

contact:

DVM, PhD Paola PocarMartin Luther Universität HalleInstitut Anatomie u. Zellbiologiepaola.pocar@medizin.uni-halle.deGrosse Steinstrasse, 5206097 Halle (Saale) (Germany)

Albert Sickmann, Stefan Lehr, Armin Herkner, Dirk Müller-Wieland, Helmut E.Meyer, Jörg Reinders

Identification of the Major Tyrosin Phosphorylation Sites of Human Gab-1

Signal transduction by receptor tyrosine kinases involves phosphorylation ofdifferent signaling proteins and their interaction via specificphosphotyrosine-recognizing protein domains. Therefore, intracellular signalingappears to be determined by a complex network of protein-protein interactionsregulated by tyrosine phosphorylation. Recently, a novel member of amultisubstrate docking protein, called Gab-1 has been characterized. Gab-1 isexpressed in many tissues and there is some evidence that it is part of signalingpathway related to cell growth, transformation and apoptosis. Gab-1 isphosphorylated upon stimulation with insulin and several other growth factors.Therefore, as a multifunctional adapter protein Gab-1 can converge differentextracellular stimuli and recruit various intracellular signaling proteins.The autophosphorylated GST-hGab-1 was in-vitro-phosphorylated by the purifiedrIR kinase using [g-32P]ATP. The phosphorylation mixture was separated bySDS-PAGE and Coomassie stained, the radioactivity containing bands wereexcised and tryptically in-gel-digested. The peptides were eluted from the gel andseparated using a high resolution two-dimensional HPLC. Fractions containing32P were analysed by ESI-MS, ESI-MS/MS and MALDI-MS as well as MALDI-PSD.It is possible to localize eight different tyrosine phosphorylation sites in hGab-1using the described techniques in contrast to the unseparated digest wherein nota single phosphorylation site was detectable.

contact:

Jörg ReindersRuhr-Universität BochumMedizinisches Proteom-Center, ZKF 01-142Joerg.Reinders@ruhr-uni-bochum.deUniversitätsstrasse 15044780 Bochum (Germany)

additional information

Address of Armin Herkner, Stefan Lehr and Dirk Müller Wieland:

Klinik II and Poliklinik fuer Innere Medizin, Zentrum fuer Molekulare Medizin,Universität Köln.

Jens Wulfaenger, Alexander Navarrete Santos, Tanja Blosz, Juergen Langner,Dagmar Riemann

The intracellular N-terminus of aminopeptidase N/CD13 affects association withcaveolae

Aminopeptidase N (APN)/CD13 is a zinc-dependent metalloprotease withubiquitous expression and a broad functional repertoire. It is a type II-integralmembrane protein with a short (8-10 amino acids) cytoplasmic domain. APNmodulates signal transduction by processing bioactive peptides (enkephalins,kinins, chemokines). Otherwise, APN ligation via antibodies is sufficient toactivate intracellular signalling in monocytes (increase in [Ca2+]i, MAP kinaseactivation). A possible explanation could be the association of APN in specializedmembrane microdomains, also referred to as rafts or caveolae, which areinvolved in signal transduction. To investigate the role of the intracellular part inthe signalling via APN, a mutant lacking the intracellular tail was constructed andtransiently transfected in CHO-K1 cells. Mutation did not change neithermembrane localization nor enzyme activity of APN. However, raft association wasstrongly diminished in the tailless variant. Further studies have to deal with thefunction of the intracellular part in signal transduction.

contact:

Dr. med. Dagmar RiemannMartin-Luther-University Halle/WittenbergInstitute of Med. Immunologydagmar.riemann@medizin.uni-halle.deMagdeburger Str. 206097 Halle (Germany)

Ina Wittko, Sigrid Saaler-Reinhardt

Involvement of La protein in alternative translation processes in neuralprogenitor cells

In contrast to its known physiological localization and function, we observed thatthe nuclear housekeeping protein La is exclusively expressed in the cytoplasm ofmultipotential neural precursor cells. In the present study we examined theexpression of La in the developing mouse brain using the specific anti-Lamonoclonal antibody 5B9. Proliferating precursor cells of different brain regionsexpress La protein in the cytoplasm until they exit the cell cycle and startmigration. At this important developmental checkpoint the localization of Laswitches from cytoplasmic to nuclear in postmitotic neurons. Therefore, thecytoplasmic expression of La seems to have an important physiological functionpossibly in terms of cell cycle control on the level of translation in the course ofneural differentiation.

contact:

PD Dr. Sigrid Saaler-ReinhardtJohannes Gutenberg-Universität MainzInstitut für Physiologische Chemiereinhard@mail.uni-mainz.deDuesbergweg 655099 Mainz (Deutschland)

Polin Mahjour, Katja Mühlberg, Andreas Hagendorff, Stefan Dhein, DietrichPfeiffer, Aida Salameh

Differential regulation of gap junction protein expression in cardiomyocytes

Gap junction form the basis of intercellular electrical communication in the heartand are made of connexins. However little is known about the differentialregulation of the main cardiac connexins CX43 and Cx40 by pathophysiologicallyrelevat stimuli such as TNFa, cAMP or direct activators of protein kinase C oradenylyl cyclase. Thus, we wanted to investigate the influence of these stimuli oncardiac gap junction protein expression. Therefore, confluent cultures of neonatalrat cardiomyocytes were incubated 24hours with various concentrations of eitherTNFa (1-100U/ml), forskolin (0,1-10µM), db-cAMP (10-1000µM) or the PK Cactivator PDD (1nM-10µM) in absence or presence of the p38 inhibitor SB203580(10µM).Connexin expression was investigated by Western blotting and PCR. 24hours incubation with either TNFa, forskolin and db-cAMP led to a significantincrease in Cx43 protein expression as revealed by Western blot and byimmunohistology. These results were also confirmed by PCR, indicating de-novosynthesis. In contrast Cx40 was not altered by these stimuli. However the PK Cactivator PDD enhanced both connexins Cx43 and Cx40 significantly. Additionalincubation with SB203580 completely inhibited Cx43 enhancement by TNFa,PDD, forskolin and db-cAMP but not the PDD-induced increase in Cx40 proteinexpression. The ERK 1/2 inhibitor PD98059 (10µM) did not influence Cx43increase. From these results we conclude, that (a) the cardiac connexins Cx43and Cx40 are differentially regulated, (b) protein kinase C seems to regulate bothCx40 and Cx43 expression and from the PCR results (c) that the upregulation inCx43 seems to be attributable to enhanced de-novo sythesis. Thus, gap junctionexpression in cardiomyocytes seems to be highly dynamic.

contact:

Dr.med. Aida SalamehUniversität LeipzigMed. Klinik I Abt. Kardiologiesala@medizin.uni-leipzig.deJohannisalle 3204103 Leipzig (Germany)

Albert Sickmann, Marion Herrmann, Joachim Klose, Helmut E. Meyer, HeikeSchaefer

Analysis of Posttranslational Modifications of alpha-A-Crystallin during Aging ofthe Eye Lens

The eye lens allows to focus pictures on the retina. It consists of two types ofcells, epithelial and fiber cells [1]. The lens grows throughout life, maintainingtransparency without significant turnover of its densely packed proteins [2].Mainly the crystallins are responsible for the transparency. They comprise morethan 90% of the lens proteins. During aging these proteins are posttranslationallymodified, i.e. phosphorylated. We report the identification of differentposttranslationally modified alpha-A-Crystallin after 2D-PAGE described by Kloseet al. [3] of eye lenses (mus musculus) of different ages.The number of detected protein spots of alpha-A-Crystallin in the gel raised from3 protein spots in the embryo state to 22 protein spots in the gel of the lensproteins analyzed from the oldest mouse (100 weeks). This let to the assumptionthat the number of modifications in the alpha-A-Crystallin accumulates with age.Using nano-LC-MS/MS alpha-A-Crystallin could be identified with a sequencecoverage 50% in all protein spots. Furthermore, some posttranslationalmodifications, like phosphorylation of specific serine residues could be detected.A set of these protein spots was also analyzed by MALDI-TOF-MS. With theseaddditional measurements the data of the LC-MS/MS could be verified andcomplemented.This work demonstrates that LC-ESI-MS/MS and MALDI-TOF-MS are powerfulcomplementary tools for the identification of posttranslational modifications fromsingle 2D-PAGE proteinspots.

Literature[1] Colvis, C. M. et al., Electrophoresis 2000, 21, 2219-2227.[2] Jaenicke, R., Naturwissenschaften 1994, 81, 423-429.[3] Klose, J. et al., Electrophoresis 1995, 16, 1034-1059.

contact:

Heike SchaeferRuhr-Universitaet BochumMedizinisches Proteom-CenterHeike.Schaefer@ruhr-uni-bochum.deUniversitaetsstr. 15044780 Bochum (Deutschland)

Martin Haßler, Edgar Brändle, Joachim Greven, Jan Henrik Schlattjan

Interaction of the organic base cimetidine with the renal basolateralp-aminohippurate (PAH) transport system

Our previous studies have shown that cimetidine is secretedin the kidney by the transport system for organic cations (1).However, we could also demonstrate that cimetidine mayinteract with the renal basolateral transport system fororganic anions (PAH transport system) because cimetidineinhibited tubular uptake of p-aminohippurate (PAH) (2).But it was not clear from these studies whether cimetidine istransported by the basolateral PAH transport system into thetubular cells. To address this question, experiments onnon-perfused proximal tubular S2-segments microdissectedfrom rabbit kidneys were performed. The basolateral PAHtransporter in proximal S2-segments acts as a countertransporter which exchanges PAH with dicarboxylatese. g. glutarate. Hence, from the effect of a drug on the14C-glutarate efflux rate of 14C-glutarate preloadedS2-segments it can be deduced whether the drug istranslocated across the basolateral cell membrane by thePAH transporter. The results revealed that the proximalS2 segments markedly accumulated 14C-glutarate: Aftera 20 min incubation period the cell to bath 14C- glutarateconcentration ratio amounted to 157 ± 14 (n=24).14C-glutarate efflux was linear within the first 60 s.In the presence of PAH (160 µM), efflux rate significantly(p< 0.01) increased from 1.21 ± 0.10 to 2.29 ± 0.48pmol/nl/min (n=17). However, cimetidine (100 µM) did notsignificantly affect tubular 14C-glutarate efflux: Efflux ratechanged from 1.42 ± 0.09 pmol/nl/min with controlconditions to 1.47 ± 0.14 pmol/nl/min (n=17) aftercimetidine. It is concluded that - albeit cimetidine mayinteract with the PAH binding site of the PAH transporter -it is not translocated by this transport system into theproximal tubular cells.

Literature(1) Brändle and Greven, J. Pharm.and Exp. Therapeutics, 1991, 258: 1038-1045(2) Brändle and Greven, Pharmacology, 1992, 45: 221-340

contact:

Prof. Dr. med. Joachim GrevenUniversitätsklinikum AachenInstitut für Pharmakologie und Toxikologiejgreven@ukaachen.deWendlingweg 252074 Aachen (BRD)

Heide Schmid, Thai-Hoa Nguyen-Xuan, Minh-Huy Tran, Gerold Schwarz, HubertKalbacher, Heinrich Wiesinger

The endosomal/lysosomal distribution of cathepsins (cat) B, L and S andN-acetyl-ß-D-glucosaminidase (NAG) is differentially modulated in murine celllines of microglial and monocytic origin

Lysosomal hydrolases are involved in turnover of cellular constituents andtherefore highly regulated by metabolic conditions. Moreover, inimmunocompetent cells like phagocytes endosomal cathepsins act asantigen-processing enzymes. In order to assess such functional differences inmurine microglial (N11) and monocytic (RAW 264.7) cell lines, the catalyticactivity of the lysosomal marker enzyme NAG was compared with that ofcathepsins B, L and S in crude organelle preparations, endosomal and lysosomalfractions under different culture conditions. In contrast to RAW 264.7 cells N11cells exhibited higher endosomal than lysosomal activities of all four enzymeswhen cultured in DMEM with 10 % FCS. 24 h of culture in DMEM with 1 % FCSand 1 mM L-NAME resulted in reduced organellar enzyme activities of both celllines with the exception of cat L and cat S in N11 cells, however, with lysosomalenzyme activities exceeding endosomal activities. Simulation ofimmunomodulatory conditions with interferon-&gamma; (200 U/ml) reduced theratio of endosomal versus lysosomal enzyme activities in N11 cells, but increasedthis ratio in RAW 264.7 cells. Thus this cytokine may convert N11 cells into amore immunogenic state, RAW 264.7 cells, however, into a more degradativestate.

contact:

Dr. rer.nat. Heide SchmidUniversitätsklinikumInstitut für Pathologieheschmid@med.uni-tuebingen.deLiebermeisterstr. 872076 Tübingen (Deutschland)

additional information

H. Schmid, T.-H. Nguyen-Xuan, M.-H. Tran, Institut für Pathologie,Liebermeisterstr. 8, G. Schwarz, H. Kalbacher, H. Wiesinger,Physiologisch-chemisches Institut, Hoppe-Seyler-Str. 4, Universität Tübingen,D-72076 Tübingen

Marcel Schmitt, Georg Gellert, Bettina Kirberg, Jost Ludwig, HellaLichtenberg-Fraté

CyGene: Eine neue hefebasierte Methode zur Bestimmung des cyto- undgenotoxischen Potentials von Umweltgiften im Wasserbereich.CyGene: A New Yeast Based Method for the Detection of Cyto- and Genotox

The evaluation of toxic effects is an important tool in the characterization ofenvironmental samples. In particular, the detection of potential genotoxic, i. e.DNA-integrity damaging activity has recently received increasing attention.Currently, standardized prokaryotic genotoxicity procedures include the Amestest (Maron et al., 1983; Gee et al., 1994), the umuC test (Oda et al., 1985) andthe SOS Chromotest (Quillardet & Hofnung, 1985) which are based on geneticallyengineered bacterial Salmonella typhimurium strains. The Escherichia coli basedassay involves Lac+ reversion of a F' plasmid borne lacZ gene (Ohta et al. 1999).A miniaturised short-term in vivo genotoxicity screening assay based ongenetically modified Saccharomyces cerevisiae cells was performed to explorethe capacity of this eukaryotic organism to detect the presence of genotoxicactivity of soluble water contents. An increased sensitivity of yeast cells wasobtained by using a strain being deleted in the prominent pleiotropic drugresistance mediating efflux transporters. The RAD54 promoter, fused to yeastenhanced green fluorescent protein served in the sensitised strain as indicator toinvestigate the activity of potential effluent induced DNA-damage. Uponapplication of various model substances pure compounds including the antiknockmethyl tertiary-butyl ether (MTBE) which is used as oxygenated fuel additive, thepreservative triphenyltin (TPhT) and the known direct acting genotoxinmethyl-N-nitro-N-nitrosoguanidine (MNNG) the results point out the sufficiency ofboth, the sensitivity of the yeast cells and the intensity of the fluorescence signalfor the assessment of potential genotoxic contamination.Since there are limited data available for the chronic toxicity of MTBE in thisstudy we examined the effect of MTBE. All model substances were examined ongrowth of the sensitised S. cerevisiae strain in correlation to enhancedfluorescence development as indicator for putative genotoxic effects. Incombination with growth inhibition assays to assess chronic toxicity and bypartial automation of the system to microtitration scale this bioassay offers asensitive, meaningful pre-screening test to prove toxic effects of numeroussamples.

LiteratureGee, P. Maron, D.M., Ames, B.N. (1994). Detection and classification ofmutagens: A set of base-specific Salmonella tester strains. Proc. Natl. Acad. Sci.91, 11606-11610Maron, D.M. and Ames, B.N. (1983). Revised methods for the Salmonellamutagenicity test. Mutat. Res. 113, 173-215Oda, Y., Nakamura, S., Oki, I., Kato, T. and Shinagawa, H. (1985). Evaluation ofthe new system (umu-test) for the detection of environmental mutagens andcarcinogens. Mutat Res. 147, 219-229.Ohta, T., Watanabe-Akanuma, M., Tokishita, S. and Yamagata H. (1999).Mutation spectra of chemical mutagens determined by Lac+ reversion assay withEscherichia coli WP3101P-WP3106P tester strains. Mutat Res. 440, 59-74.Quillardet, P., & Hofnung, M. (1985). The SOS Chromotest, a colorimetricbacterial assay for genotoxins: procedures. Mutat Res. 147, 65-78.

contact:

Dr. Hella Lichtenberg-FratéUniversität BonnBotanisches Instituth.lichtenberg@uni-bonn.deKirschallee 153115 Bonn (D)

Reinhild Tenderich, Brigitte Schmitz

Function of the PEST sequence of NCAM 140

The neural cell adhesion molecule NCAM is a member ofthe immunoglobulin superfamily and plays an importantrole during the development of the nervous system, inregeneration and synaptic processes of the adultnervous system.Since the PEST sequence of apCAM in aplysia -a homologue of NCAM - plays a key role in endocytosis(Bailey et al, 1997) we investigated whether the PESTsequence of human NCAM 140 is also important for itsinternalization, which has been implicated in neuriteoutgrowth (Schmid et al, 1998).Therefore, cDNA constructs of human NCAM 140 withdifferent mutations of the PEST sequence were created.NCAM negative B35 neuroblastoma cells were transfectedwith either wild type cNDA or the different mutant cDNAsof NCAM 140 and their internalization was studied byconfocal microscopy.

LiteratureBailey et al, 1997, Neuron, Vol. 18, 913-924Schmid et al, 1999, J Neurobiol., 38, 542-58

contact:

Dipl. Oectroph. Reinhild TenderichUni BonnFür Anatomie der Haustierer.tenderich@uni-bonn.deKatzenburgweg 9a53115 Bonn (Deutschland)

Astrid Schön, Olaf Gimple, Christian Heubeck

The evolution of RNase P in eukaryotic organelles

Many essential metabolic reactions are dependent on nucleic acids as cofactors orcatalysts. Examples are reactions in nucleic acid biosynthesis, RNA-dependentconversions of small metabolites, and RNA processing reactions. Many of of thesepathways are highly conserved in extant organisms, suggesting an ancient originin an "RNA world".The essential tRNA processing enzyme RNase P is a striking example for enzymeevolution: it is a ribonucleoprotein (RNP) in bacteria, archea, and eukaryoticnuclei, with an essential RNA component. This RNA is a true ribozyme in bacteriaand some archaea. In contrast, no RNA component exists in the mitochondriaand chloroplasts of multicellular organisms, posing the question of enzymeevolution in these bacteria-derived organelles. The primitive plastids from certainunicellular algae, however, contain an RNP-type RNase P. Their essential RNAcomponents are similar to those of cyanobacteria, and show high structuraldiversity.The overall composition of the plastid RNase P, with a rather large proteincontribution, is more similar to the eukaryote- than to the bacterial-typeenzymes. However, as in nuclear RNase P, the RNA is an essential enzymecomponent, but is not catalytically active as a ribozyme.Surprisingly, a fully active RNase P holoenzyme can be reconstituted from thecorresponding organelle RNAs and cyanobacterial protein subunits. This suggeststhat, despite the observed structural diversity, the catalytic activity of theenzyme still resides in the RNA subunit.

LiteratureBaum, M., Cordier, A. and Schön, A. (1996). J. Mol. Biol. 257, 43-52.Cordier, A. and Schön, A. (1999). J. Mol. Biol. 289, 9-20.Heubeck, C. and Schön, A. (2001). In: Meth. Enzymol. 342, 118-134.Gimple, O. and Schön, A. (2001). Biol. Chem. 382, 1421-1429.

contact:

Dr. Astrid SchönUniversity of LeipzigBiochemistryschoena@uni-leipzig.deTalstr.3304109 Leipzig (Germany)

additional information

Adress of Co-Authors:Institute of Biochemistry, Biozentrum, Am Hubland, 97074 Würzburg

Lorenz Trümper, Frank Griesinger, Christa Fonatsch, Astrid Heutelbeck, DetlefHaase, Ernst Hallier, Thomas Schulz

GLUTATHIONE S-TRANSFERASE GSTP1 POLYMORPHISMS IN DE NOVO ANDTHERAPY-INDUCED HEMATOLOGIC MALIGNANCIES

T.G. Schulz, D. Haase, A. Heutelbeck, C. Fonatsch, F. Griesinger, L. Trümper, E.Hallier, Department of Occupational Health and Social Medicine, University ofGöttingen, Waldweg 37, 37073 Göttingen, GermanyGlutathione S-transferases (GST) metabolize exogenous and endogenousmutagenic compounds. Recently, GSTT1-defeciency has been reported to be arisk factor for myelodysplastic syndrome (MDS). It was the aim of our study todetermine the incidence of two GSTP1 polymorphisms in patients with de novoand therapy-induced hematologic malignancies. Blood samples or bone marrowof 299 patients were examined by PCR/RFLP analysis. Data were compared withthe results of 266 healthy controls.There were no significant differences found between patients with AML/MDS andhealthy controls.We conclude that the GSTP1 polymorphisms are not associated with the risk ofthe development of AML or MDS.

contact:

PD Dr. Thomas SchulzUniversität GöttingenArbeitsmedizintschulz1@gwdg.deWaldweg 3737073 Göttingen (Deutschland)

Gabi Bauer-Manz, Andreas Kuhn, Lambertus van den Berg, Justyna Serek

The E.coli membrane insertase YidC:A Chaperone for Protein Folding into the Membrane

YidC, the homologe of the mitochondrial inner membrane protein Oxa-1 has beencloned from the E.coli chromosome, modified with a his-tag and purified tohomogeneity. The 60kD protein was reconstituted with E. coli phospholipids forproteoliposomes. These proteoliposomes were capable in supporting the insertionofpurified membrane proteins in chemical amounts. We suggest that YidC is amembrane insertase that promotes membrane protein insertion.

The two model proteins we tested for membrane insertion in vitro, were thesingle-spanning Pf3 coat protein and the double-spanning M13 procoat protein.Both havebeen recently shown to depend on YidC for membrane insertion in vivo (1,2).The Pf3 coat protein was purified by reverse phase and size exclusionchromatography and soluble in 100mM Tris-HCl pH 8,5, 10% isopropanol. TheM13 procoat proteinwas purified by affinity chromatography.

When the proteins were added to the YidC containing proteoliposomes theproteins readily integrated in a transmembrane configuration. This was tested byadditionof protease to the outside showing that the integrated proteins were protectedfrom proteolysis. In the absence of YidC both proteins bind to the membranesurfacebut only minute amounts are resistant to the protease and therefore translocatedacross the membrane.

Since the YidC protein catalytically supports the integration of the two modelproteins we propose that it acts similar to a chaperone by binding thenon-translocatedform of the substrate and releasing the translocated membrane protein.

Literature1.Samuelson, J.C., F. Jiang, L. Yi, M.Chen, A.Kuhn and R.E.Dalbey (2001).Funktion of YidC for the insertion of M13 procoat protein in E. coli:Translocationof mutantsthat show differences in their membrane potential dependence andSec-requirement. J. Biol Chem.276,34847-34852.2.Chen, M., J. samuelson, F.Jiang, M. Müller, A.Kuhn and R.Dalbey (2002)Direct interaction of YidC with the Sec-independent Pf3 coat protein during istmembrane insertion. J.Biol.Chem. 277, 7670-7675.

contact:

Justyna SerekUniversität HohenheimMikrobiologie und Molekularbiologiejusserek@uni-hohenheim.deGarbenstr.3070599 Stuttgart (Baden Württemberg)

Michael Spoerner, Guido Steiner, Thomas Prisner, Alfred Wittinghofer,Hans-Robert Kalbitzer

Are GTP-analogs really good analogs?

For structural and kinetic investigations of Guanine nucleotide binding proteins inthe active state different slowly-hydrolyzing GTP-analogs are commonly used. Inour group, recently it was shown by 31P NMR, that Ras(wt).GppNHp exists in twodifferent conformations (state 1 and state 2), which are in a dynamicalequilibrium(1). Binding of effector-proteins stabilizes state 2. Whereas state 2seems to be very similar to the effector bound state, structural and kineticstudies indicated a disordered, highly dynamic switch I region for state 1 (2).Here we find similar results for the analog GppCH2p. 31P NMR spectra show alsosplitting of the Pa and Pß signal in the Ras(wt).GppCH2p complex with similarequilibrium constant and rate constants of exchange. In contrast the GTP-analogGTP³S only shows one conformation if it is bound to Ras protein.Replacing the bound Mg2+ in the active site of Ras by Mn2+ allowshighfield-EPR-investigations of the Ras-nucleotide complex. Recently we showeda correlation between different conformations seen by 31P NMR and thecoordination of ligands of metal ion bound to Ras.GDP (3). Similar results areobtained for the active state of Ras bound to different GTP-analogs. The twoconformational states observed by 31P NMR spectroscopy appear to be closelylinked to changes in the coordination sphere of the metal ion.We can show, that GppNHp and GppCH2p change the dynamical behaviour ofRas in the active state by destabilizing the conformation necessary for effectorbinding defined by the equilibrium constant K = [2]/[1] by a factor of 2. Thisindicates that the dynamic behaviour of Ras.GTP is highly sensitive to slightmodifications in the active site and that this in turn affects interaction of Ras witheffectors.

Literature(1) Geyer, M., Schweins, T., Herrmann, C., Prisner, T., Wittinghofer, A., andKalbitzer, H.R. (1996) Biochemistry 35, 10308(2) Spoerner, M., Herrmann, C., Vetter, I.R., Kalbitzer H.R., and Wittinghofer, A.(2001) Proceedings of the National Academy of Sciences USA 98, 4944(3) Rohrer, M., Prisner, T.F., Brügmann, O., Käss, H., Spoerner, M., Wittinghofer,A., and Kalbitzer, H.R. (2001) Biochemistry 40, 1884

contact:

Michael SpoernerUniversität RegensburgInstitut für physikalische Biochemie und Biophysikmichael1.spoerner@biologie.uni-regensburg.de9304093053 Regensburg (Germany)

Anna Stepczynska, Jakob Troppmair, Thomas Voegel, Monika Poppe, KlausSchulze-Osthoff, Michael Schwarz

Bcl-2 antagonist of cell death (Bad) retains its proapoptotic activity despiteproteolytic cleavage by caspase-3

Bcl-2 family member, Bad, promotes death by neutralising the protective effectof Bcl-Xl and Bcl-2. The phosphorylation status of Bad determines whether themolecule is sequestered in cytosol by 14-3-3 protein family members or whetherit dimerizes with Bcl-xL or Bcl-2 and thereby releases block on cytochrome crelease. We demonstrate that overexpression of Bad in MCF7 cell line is sufficientto trigger cytochrome c release and sensitizes the MCF7/casp-3 cells tostaurosporine induced apoptosis. We detect cleavage of endogenous Bad duringCD95 and staurosporine-induced apoptosis in Jurkat cell and identify the SATDsequence in murine Bad as caspase-3 substrate site. According to our analysis ofapoptotic nuclei and cell morphology cleavage of Bad at this site does notimprove Bad death promoting activity. Since we observe that overexpression oftruncated Bad can induce formation of apoptotic nuclei in vivo, we support viewthat death promoting ability-of Bad relies mainly on the intact BH3 domain thatenables heterodimerization among Bcl-2 family members and is not subjected tothe proteolytic control of caspases.

contact:

Dr.rer.nat. Anna StepczynskaUniversity of TübingenPharmacology and Toxicologyanna.stepczynska@uni-tuebingen.deWilhelmstr. 5672074 Tübingen (Germany)

additional information

J.T. is at the Inst. of Medical Radiation and Cell Research, University of WürzburgT.V is at the Dept. of Experimental Dermatology, University of MünsterM.P. is at the Div. of Apoptosis and Cell Death, University of MünsterK.S. is at the Dept. of Immunology and Cell Biology, University of Münster

Stefan Henrich, Robert Huber, Albert Ries, Karlheinz Mann, Klaus Kühn, RupertTimpl, Gleb P. Bourenkov, Hans D. Bartunik, Wolfram Bode, Manuel E. Than

Crystal structure of the noncollagenous (NC1) domain of human placentacollagen IV: Stabilization via a novel type of covalent Met-Lys cross-link

Triple-helical collagen IV protomers associate through their N- and C-terminiforming a three-dimensional network, which provides basement membranes withan anchoring scaffold and mechanical strength. The non-collagenous (NC1)domain of the C-terminal junction between two adjacent collagen IV protomersfrom human placenta was crystallized and its 1.9 Å structure was solved by MADphasing. This hexameric NC1 particle is composed of two trimeric caps, whichinteract through a large planar interface. Each cap is formed by two &Alpha;1and one &Alpha;2 fragments with a similar novel fold, segmentally arrangedaround an axial tunnel. Each monomer chain folds into two structurally verysimilar subdomains, which each contain a finger-like hairpin loop that inserts intoa 6-stranded &Beta;-sheet of the neighbouring subdomain of the same or theadjacent chain. Thus each trimer forms a quite regular, but non-classical, 6-foldpropeller. The trimer-trimer interaction is further stabilized by a novel type ofcovalent cross-link between the side chains of a Met and a Lys residue of the&Alpha;1 and &Alpha;2 chains from opposite trimers, but not by interchaindisulfide exchange as proposed previously. This cross-link, explaining previousfindings of non-reducible cross-links in NC1, was supported by crystallographicand biochemical data. This structure explains the stability of the dimericprotomer interaction within the collagen IV networt and provides new insightsinto NC1-related diseases such as Goodpasture and Alport syndromes.

LiteratureThan, M. E., Henrich, S., Huber, R., Ries, A., Mann,K., Kühn, K., Timpl, R., Bourenkov, G. P., Bartunik, H. D.& Bode, W. (2002) Proc. Natl. Acad. Sci, 99, 6607-6612

contact:

Dr. Manuel ThanMPI für BiochemieAbt. Strukturforschungthan@biochem.mpg.deAm Klopferspitz 18A82152 Martinsried (Germany)

Cornelia Körting, Alexander Froschauer, Reinhold Hanel, Jean-Nicolas Volff,Anne-Marie Veith

DMRT Genes and Sex Determination in the Fish Xiphophorous

Almost nothing is known about the genetic basis of sex determination in fish.Fishes display many different types of sex determination systems, fromhermaphroditism to gonochorism, and from environmental sex determination togenetic sex determination including male and female heterogamety, polyfactorialsystems and autosomal influences. In the platyfish Xiphophorus maculatus, thesex-determining region on the sex chromosomes is flanked by two relatedreceptor tyrosine kinase genes, Xmrk and egfr-b. The primary sex-determininggene remains to be identified in the platyfish and in the great majority of otherfish species.Some members of the DMRT (Doublesex and MAB-3-related transcription factors)family like doublesex in Drosphila melanogaster, mab-3 in Caenorhabiditiselegans and DMRT1 in vertebrates function in sex determination. In animals,DMRT genes can be considered as an interface between variable, lineage-specificupstream cascades of regulatory genes activated by primary sex-determiningsignals, and downstream structural genes responsible for sex differentiation. Onthe other hand, some DMRT genes might function as primary sex-determininggenes in certain vertebrate lineages.In order to understand the function and evolution of DMRT genes in sexdetermination in fish, a platyfish bacterial artificial chromosome (BAC) genomiclibrary was screened under low stringency using a heterologous DMRT probe fromthe medaka fish Oryzias latipes. Six different groups of DMRT-containing BACclones potentially containing a total of eight different DMRT genes were isolated.As observed in mammals, medaka and pufferfish, DMRT1 and DMRT2 arephysically clustered. Novel fish DMRT genes were identified as well. Interestingly,two ancient DMRT duplicates orthologous to one unique mammalian gene weredetected in the platyfish. These duplicates might be remnants of the proposedwhole genome duplication having arisen early during fish evolution. Differentialevolution of paralogous sex-determining genes in different fish lineages aftergenome duplication might be involved in the amazing variability ofsex-determining systems observed in fish.

contact:

Dipl. Anne-Marie VeithUniversität WürzburgPhysiologische Chemie Iveith@biozentrum.uni-wuerzburg.deAm Hubland97074 Würzburg (Deutschland)

additional information

Biofuture Research Group "Evolutionary Fish Genomics"

Funded by the Biofuture program of the Bundesministerium für Bildung undForschung

Hubert Zipper, Katrin Lämmle, Christiane Buta, Herwig Brunner, JürgenBernhagen, Frank Vitzthum

Investigations on the binding of SYBR Green I to double-stranded (ds)DNA

The fluorescent dsDNA stain SYBR Green I (SGI) is used in many molecularbiology applications, including the detection of nucleic acids in gels, dsDNAquantification in solution, and real-time PCR [1,2]. Although SGI is a commonlyapplied stain, its binding to dsDNA has not been studied in detail, becauseinformation on the structure and concentration of SGI are not available [3]. Wedetermined the concentration of SGI in the reagent to be 19.6 mM using ionchromatography. Mass spectroscopy revealed a relative molecular mass of 509.3g/mol, which exactly corresponds to the mass of Molecular Probes cyanine dye937 [4]. On the basis of the identified structure of SGI, we prepared SGI/dsDNAcomplexes with defined dye/base-pair (dye/bp) ratios and performed bindingstudies by fluorescence and differential spectroscopy [5]. The studies revealedthat a significant fluorescence intensity increase was only observed, when thedye/bp ratio exceeded 0.15. After reaching a value of about 2, fluorescenceintensity quickly approached a maximum. Consequently, linearity of dsDNAquantification is sustained as long as the dye/bp ratio is above 2. Viscometricstudies of SGI/dsDNA complexes in comparison to the intercalator ethidiumbromide and the groove-binding dye Hoechst 33258 suggested that SGI initiallyintercalates into dsDNA. This intercalation is probably followed by groove binding,when the dye/bp ratio exceeds 0.15.

Literature[1] Vitzthum, F., and Bernhagen, J. (2002). SYBR Green I: an ultrasensitivefluorescent dye for double-stranded DNA quantification in solution and otherapplications. In: Recent Research Developments in Analytical Biochemistry,Pandalai, S.G., ed. (Transworld Research Network), in press.[2] Haugland, R.P. (2001) Handbook of Fluorescent Probes and ResearchChemicals., Molecular Probes, Inc., Eugene, OR.[3] Vitzthum, F., Geiger, G., Bisswanger, H., Brunner, H., and Bernhagen, J.(1999). A quantitative fluorescence-based microplate assay for the determinationof double-standed DNA using SYBR Green I and a standard UV transilluminatorgel imaging system. Anal. Biochem. 276, 59-64.[4] Yue, S.T., Singer, V.L., Roth, B.L., Mozer, T.J., Millard, P.J., Jones, L.J., Jin,X., and Haugland, R.P. (1997). Substituted unsymmetrical cyanine dyes withselected permeability, US-patent 5658751.[5] Geiger, G., Bernhagen, J., Wagner, E., Bisswanger, H., Brunner, H., andVitzthum, F. (2001). Standardized measurements and differential spectroscopy inmicroplates. Anal. Biochem. 296, 29-40.

contact:

Dr. rer. nat. Frank VitzthumUniversität StuttgartInstitut für Grenzflächenverfahrenstechnikfvi@igb.fhg.deNobelstr. 1270569 Stuttgart (Deutschland)

Markus von Nickisch-Rosenegk, Jenny Steffen, Frank F. Bier

Transcription of Reporter Genes with Immobilized Templates

Our first attempt regarding the reconstruction of cellular mechanisms on biochips(e.g. proteine synthesis) was the construction and the immobilization oftranscriptionable DNA.Therefore we chose the EGFP gene which could report itself in further translationexperiments.In a PCR reaction we amplified the flourescent protein gene using a forwardprimer which contains a T7 promotor region followed by a Shine-Delgardo-motivupstream to the startcodon of the EGFP gene primer sequence and afluorescein-modification (FAM) at the 5‘-end. The reverse primer was modifiedwith biotin at its 5‘-end and has a poly-T spacer linked to the stopcodon of thegene.The amplicons were spotted on a neutravidin-covered glaschip and immobilizedvia the biotin molecule of the reverse primer. After a wash step the spots couldbe verified by UV excitation an the resulting fluorescence of the FAM molecule.These fixed DNA molecules were incubated with a reaction mixture containingT7-RNA-Polymerase for in vitro on-chip-transcription.The synthesized RNA was verified by DNA digestion using DNAse I followed byRT-PCR or by an incorporation of FITC-dUTP during the RNA synthesis. A thirdmethod was an incorporation of Cy5 dye-labeled dNTPs during a first strandcDNA synthesis.

contact:

Dr. Markus von Nickisch-RosenegkFraunhofer GesellschaftIBMTmarkus.nickisch@ibmt.fhg.deArthur-Scheunert-Allee 114-11614558 Bergholz-Rehbrücke (D)

Melanie Wagner, Reinhard Reents, David Owen, Herbert Waldmann, AlfredWittinghofer, Jürgen Kuhlmann

Localisation of fluorescently labelled Ras protein in the living cell

The small GTPase binding proteins of the Ras superfamily are involvedin regulation of cell differentiation and proliferation. Posttranslationalmodifications direct the protein to the plasma membrane, whereit fulfills its biological function. Combination of bacterial expressionof truncated Ras protein and organo-chemical synthesis of the lipid-modifiedC-terminus of Ras results in biological active constructs (Bader et al., Kuhn etal.).This approach allows the insertion of other than the natural occuring lipidmodificationsand substitution of other posttranslational modifications. Small fluorophors wereattached to the lipid moiety or the very C-terminal end of the Ras peptide.

LiteratureBader et al.Nature 403 (2000) 223-226Kuhn et al. J. Am. Chem. Soc. 123:1023-1035 (2001)

contact:

Melanie Wagner

Max-Planck-Institut für molekulare Physiologiemelanie.wagner@mpi-dortmund.mpg.deOtto-Hahn-Str. 1144227 Dortmund (Deutschland)

Albert Sickmann, Helmut E. Meyer, Yvonne Wagner

Two-dimensional chromatography in protein analysis

The analysis of complex protein mixtures like ribosomes, centrosomes,peroxisomes and further multi protein complexes is still a difficult problem. Ingeneral such analyses are done by a high-resolution technique, e.g. 2D gelelectrophoresis (2D-PAGE), followed by a protein identification via massspectrometry. The 2D-PAGE is a time-consuming method for the separation andis not suitable for all kinds of proteins, e.g. hydrophobic proteins, basic proteins,very large or very small proteins. To overcome this problem we validatedmethods for the analysis of complex protein mixtures that manage with verysmall amounts of protein. The separation is done either with 2D-nano-HPLC or1D-nano-HPLC. Both methods were validated by separation of yeast-ribosomes.The amount of the digested ribosomes was less than 1 µg. For 2D-HPLC anorthogonal system was used consisting of a capillary ion exchangechromatography for the first dimension and a nano-reversed phasechromatography for the second one. Between both dimensions the sample iscleaned up by a preconcentration step. Furthermore, the reversed phase columnwas coupled with an ion trap mass spectrometer (ESI-MS) recording the massspectra automatically. A comparison between the two-dimensionalchromatography and a one-dimensional separation showed a larger sum ofidentified proteins and furthermore a better sequence coverage within thetwo-dimensional system using the same protein amount. In addition thepossibility of combining different one-dimensional separation methods adapt thistechnique to almost any biological sample.

contact:

Yvonne WagnerRuhr-Universität BochumMedical Proteom-CenterYvonne.Wagner@ruhr-uni-bochum.deUniversitätsstr. 15044780 Bochum (Germany)

Robert Seckler, Monika Walter

Pectate Lyase from Bacillus subtilis (BsPel): Mutations in the asparagine laddercause a temperature-sensitive folding phenotype

Pectate lyases are microbial extracellular enzymes that are important duringplant pathogenesis. The enzymes cleave a-1,4 linked galacturonic acid by abeta-elimination. Calcium is essential for the enzymatic activity.The main structural building block of pectate lyases is a right-handed paralllelbeta-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has beensolved in complex with calcium (Pickersgill et al. 1994). A characteristic featureof parallel beta-helices is the occurence of extensive stacks of polar andhydrophobic side chains filling the interior of the protein core. In Bacillus subtilispectate lyase (BsPel), three types of side chain stacks are observed: an aliphaticstack, an aromatic stack and an asparagine ladder. Such an asparagine ladder isa conserved feature of many monomeric beta-helices. It is discussed to be criticalfor stability and may consitute a nucleus for folding (Jenkins et. al. 1998). Theeffect of disruption of the asparagine ladder was tested in this mutagenesisstudy. The middle asparagine (Asn271) of the ladder was selected andexchanged for various residues (Arg, Thr, Leu, Pro, Asp, Glu, Gly). All mutantswere expressed at 25°C as soluble proteins, although with significantly reducedyields. The specific activity of the mutants was comparable to that of the wildtype. The mutants N271T and N271A were selected for a detailed study of theirstability and folding in comparison to the wild type protein.Unfolding of BsPel is not freely reversible and transition curves display apparenthysteresis, connencted with a dramatic decrease of refolding rates atintermediate denaturant concentrations. Refolding, as measured by fluorescence,is at least biphasic with the slowest phase coinciding with reactivation. Refoldingof both mutants also follows a biexponential time course. At 10°C and pH 7 thefolding rate constants of both mutants were identical within experimental errorand very similar to wild type. At pH 7.5 and 25°C, however, refolding of bothmutants was very slow even at low guanidinium chloride concentrations andthere were considerable differences between the two mutants. Reactivation ofboth mutants was only possible up to 30°C, whereas the wild type was able torefold up to 40°C. The temperature-sensitive folding phenotype of the mutantswas even more pronounced at higher pH. Unfolding of both mutants under theseconditions (25°C, pH 7.5) was identical and only 2-fold faster than unfolding ofthe wild type. Thus, the disruption of the asparagine ladder does not appear todrastically destabilize the native structure of BsPel, but the mutations destabilizean essential folding intermediate formed in the fast kinetic phase of refolding.

LiteratureJenkins, J., O. Mayans, and R. Pickersgill. 1998. Structure and evolution ofparallel beta-helix proteins. J Struct Biol. 122:236-46.Pickersgill, R., J. Jenkins, G. Harris, W. Nasser, and J. Robert-Baudouy. 1994.The structure of Bacillus subtilis pectate lyase in complex with calcium. NatStruct Biol. 1:717-23.

contact:

Monika WalterUni-PotsdamPhysikalische Biochemiemwalter@rz.uni-potsdam.deKarl-Liebknecht Str. 24-25 / Haus 2514476 Golm (Germany)

Monika Walter

Pectate Lyase from Bacillus subtilis (BsPel): Mutations in the asparagine laddercause a temperature-sensitive folding phenotype

Pectate lyases are microbial extracellular enzymes that are important duringplant pathogenesis. The enzymes cleave a-1,4 linked galacturonic acid by ab-elimination. Calcium is essential for the enzymatic activity.The main structural building block of pectate lyases is a right-handed paralllelb-helix. The structure of pectate lyase from Bacillus subtilis (BsPel) has beensolved in complex with calcium (Pickersgill et al. 1994). A characteristic featureof parallel b-helices is the occurence of extensive stacks of polar and hydrophobicside chains filling the interior of the protein core. In Bacillus subtilis pectate lyase(BsPel), three types of side chain stacks are observed: an aliphatic stack, anaromatic stack and an asparagine ladder. Such an asparagine ladder is aconserved feature of many monomeric b-helices. It is discussed to be critical forstability and may consitute a nucleus for folding (Jenkins et. al. 1998). The effectof disruption of the asparagine ladder was tested in this mutagenesis study. Themiddle asparagine (Asn271) of the ladder was selected and exchanged forvarious residues (Arg, Thr, Leu, Pro, Asp, Glu, Gly). All mutants were expressedat 25°C as soluble proteins, although with significantly reduced yields. Thespecific activity of the mutants was comparable to that of the wild type. Themutants N271T and N271A were selected for a detailed study of their stabilityand folding in comparison to the wild type protein.Unfolding of BsPel is not freely reversible and transition curves display apparenthysteresis, connencted with a dramatic decrease of refolding rates atintermediate denaturant concentrations. Refolding, as measured by fluorescence,is at least biphasic with the slowest phase coinciding with reactivation. Refoldingof both mutants also follows a biexponential time course. At 10°C and pH 7 thefolding rate constants of both mutants were identical within experimental errorand very similar to wild type. At pH 7.5 and 25°C, however, refolding of bothmutants was very slow even at low guanidinium chloride concentrations andthere were considerable differences between the two mutants. Reactivation ofboth mutants was only possible up to 30°C, whereas the wild type was able torefold up to 40°C. The temperature-sensitive folding phenotype of the mutantswas even more pronounced at higher pH. Unfolding of both mutants under theseconditions (25°C, pH 7.5) was identical and only 2-fold faster than unfolding ofthe wild type. Thus, the disruption of the asparagine ladder does not appear todrastically destabilize the native structure of BsPel, but the mutations destabilizean essential folding intermediate formed in the fast kinetic phase of refolding.

LiteratureJenkins, J., O. Mayans, and R. Pickersgill. 1998. Structure and evolution ofparallel beta-helix proteins. J Struct Biol. 122:236-46.Pickersgill, R., J. Jenkins, G. Harris, W. Nasser, and J. Robert-Baudouy. 1994.The structure of Bacillus subtilis pectate lyase in complex with calcium. NatStruct Biol. 1:717-23.

contact:

Monika WalterUni-PotsdamPhysikalische Biochemiemwalter@rz-uni-potsdam.deKarl_liebknechtstrasse 24-25 / Haus 2514476 Golm (Germany)

Sabine Werner, Stefan Kuklinski, Brigitte Schmitz

Altered O-GlcNAc level after neuronal differentiation of PC12 cells

O-linked N-acetylglucosamine (O-GlcNAc) is an ubiquitousand abundant post-translational modification found onmany cytosolic proteins, nuclear proteins and also on thecytosolic tail of transmembrane proteins. It is adynamically regulated modification similar tophosphorylation and plays a role in regulation of cellularprocesses (Zachara and Hart, 2002). To assess potentialcell cycle functions of O-GlcNAc we induced thedifferentiation of PC12 cells by treatment with nervegrowth factor (NGF). The O-GlcNAc level decreasedsignificantly after differentiation. This observationindicates that a high O-GlcNAc level of proteins may bemore important for dividing cells than for fullydifferentiated cells. An increased O-GlcNAc level has alsobeen detected in Alzheimer`s disease (Griffith andSchmitz, 1995), which may be linked to thereactivation of the cell cycle, a process currently discussedto be implicated in the pathogenesis ofneurodegenerative diseases like Alzheimer`s disease.

LiteratureGriffith, L and Schmitz, B. (1995), Biochem Biophys ResCommun 213 (2), 424-431Zachara, N. and Hart G.W. (2002), Chem Rev 102 (2),431-438

contact:

Dipl. Biologin Sabine WernerUni-BonnFür Anatomie und Physiologie der Haustierewerner@uni-bonn.deKatzenburgweg 9a53115 Bonn (Deutschland)

Barth Holger, Klaus Aktories, Christian Wilde

Clostridium botulinum C2 toxin as a protein transport system

Clostridium botulinum C2 toxin as a protein transport systemHolger Barth, Christian Wilde and Klaus Aktories

C2 toxin from Clostridium botulinum is a binary toxin, i. e. it is composed of twoindividual proteins, the binding and transport component C2II and the enzymecomponent C2I. Proteolytically activated C2IIa forms ring-shaped heptamers,which bind to a carbohydrate receptor on surface of target cells and mediateuptake of C2I. The toxin complex is taken up via receptor-mediated endocytosisand C2I escapes from an acidic endosomal compartment into the cytoplasm.There, C2I ADP-ribosylates G-actin at arginine-177 thereby inhibiting assembly ofactin filaments. Dissociation of actin filaments leads to a breakdown of the actincytoskeleton. Cells treated with C2 toxin round up within a few hours.

The C2 toxin was used as a cell delivery system for foreign proteins. TheN-terminal domain of C2I (amino acid residues 1-225) was used as an adaptorfor interaction with C2II and for translocation of constructed fusion toxins intothe cytoplasm of target cells. In the C2I1-225-C3 fusion toxin C3 exoenzymefrom Clostridium limosum was fused C-terminally to C2I1-225. C3 exoenzyme isan ADP-ribosyltransferase, which modifies the small GTPase Rho and therebyinactivates it. C3 exoenzyme is poorly taken up into cells. Further analysis of theN-domain of C2I revealed that amino acid residues 1-87 are sufficient fordelivery of C3 transferase into cells.

The C2I-C3 fusion toxins were used for a rapid intoxication of various cell lines aswell as primary cell cultures with C3 transferase. C2I-C3 fusion toxin was used tostudy the role of Rho in various cellular signal transduction pathways.

Institut für Experimentelle und Klinische Pharmakologie und Toxikologie derAlbert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25,D-79104 Freiburg, Germany

contact:

Prof. Dr. Dr. Klaus AktoriesUniversität FreiburgInstitut für experimentelle Pharmakologie und ToxikologieKlaus.Aktories@pharmakol.uni-freiburg.deAlbertstrasse 2579104 Freiburg (Deutschland)

Klaus Aktories, Christian Wilde

Clostridium botulinum C3 exoezyme like ADP-ribosyltransferases

Seven related Rho-modifying ADP-ribosyltransferases from Clostridium botulinum(C3bot1 and 2), C. limosum (C3lim), Staphylococcus aureus (C3stau1-3) andBacillus cereus (C3cer) have been described. The transferases C3stau1, 2 and 3from S. aureus, (also known as EDIN A, B and C) are 65 to 80% identicalbetween each other but only 30% identical with the other C3-likeADP-ribosyltransferases.

The C3-like exoenzymes modify the eukaryotic target proteins RhoA, B and C,which are involved in extracellular regulation of the actin cytoskeleton and ofseveral other signal processes. The ADP-ribosylation renders the Rho proteinsfunctionally inactive. Therefore, C3-like ADP-ribosyltransferases are frequentlyused as pharmacological tools. C3stau2 additionally ADP-ribosylates RhoE/Rnd3.Both GTP-binding proteins are involved in the rearrangement of the actincytoskeleton and supposed to act as antagonists of RhoA activity.

Recent crystal structure analysis of C3 from C. botulinum (Han et al. J.Mol.Biol.(2001) 305, 95-107) identified a novel structural motif termed “ARTT”(ADP-ribosylating toxin turn-turn motif) to be crucial for enzymatic activity andsubstrate recognition. We studied the role of the ARTT-motif in C3stau2 bymutational analysis. Exchange of Glu180 to leucine caused a complete loss bothADP-ribosyltransferase activity and NAD glycohydrolase activity. Mutation of aglutamine178 to lysine blocked ADP-ribosyltransferase activity without majorchanges in NAD glycohydrolase activity. NAD- and RhoA-binding of this mutantprotein were comparable to that of wild-type protein. Exchange of Tyr175, whichis located in the first half of the ARTT-motif to alanine or lysine led to a slightincrease in NAD glyco-hydrolase activity. In contrast, these mutants exhibited nodetectable ADP-ribosyltransferase activity and were not precipitated bymatrix-bound RhoA. The findings support the significance of the ARTT-motif forenzymatic activity of C3-like ADP-ribosyltransferases.

contact:

Dr. Christian WildeUniversität FreiburgInstitut für experimentelle Pharmakologie und Toxikologiewildec@gmx.deAlbertstrasse 2579104 Freiburg (Deutschland)

Jens Wulfänger, Tanja Blosz, Alexander Navarrete Santos, Jürgen Langner,Dagmar Riemann

Aminopeptidase N/CD13 is associated with Lubrol rafts

Prominin is a pentaspan membrane glycoprotein associated with a novel type ofcholesterol-based lipid rafts [1], which are located especially in structuresprotruding from the planar regions of the plasmalemma (microvilli, filopodia).Whereas the ‘classical’ cholesterol-sphingolipid rafts are characterized by theirinsolubility in the non-ionic detergent Triton X-100 at 4 °C, prominin was foundto be completely soluble in this detergent, but can be preserved using Lubrol WXas another non-ionic detergent [2].Earlier studies of our group showed that the membrane-associated ectoenzymeaminopeptidase N/CD13 is partially located in rafts in monocytes [3] and in raftsand caveolae in fibroblast-like synoviocytes [4]. Now, we show that APN ispredominantly associated with Lubrol rafts in myeloid cell lines and fibroblasts.Our results point to a special role for APN in filopodia of cells.

Literature[1] Corbeil D et al Traffic 2 (2001) 82-91[2] Roeper K et al Nature Cell Biol 2 (2000) 582-592[3] Navarrete Santos A et al Biochem Biophys Res Commun 269 (2000) 143-148[4] Riemann D et al Biochem J 354 (2001) 47-55

contact:

Dipl. biochem. Jens WulfängerMartin Luther University Halle-WittenbergInstitute of Med. Immunologyjens.wulfaenger@medizin.uni-halle.deMagdeburger Straße 206097 Halle (Germany)

Ulrike Sattler, Ingrid Koziollek-Drechsler, Danuta Dormann, Christina Zechel

Putative role of the nuclear factor NCNF in neuronal differentiation

Previously, we cloned a nuclear receptor from a cDNA library of neuronalderivatives of retinoic-acid induced embryonic carcinoma cells. This factor wasdesignated NCNF (neuronal cell nuclear factor), and is identical to the germ cellnuclear factor GCNF (also named RTR), which had been cloned from murinetestis. NCNF/GCNF is an orphan receptor which most likely functions byrepressing transcription. NCNF/GCNF was proposed to be a regulator of (i) germcell development and (ii) neurogenesis and/or neuronal differentiation. Moreover,data published by the O'Malley group indicate that NCNF/GCNF is (i) required formaintenance of somatogenesis and posterior development and is (ii) essential forembryonic survival.We are interested in the role of NCNF in neurogenesis. To elucidate whetherNCNF/GCNF could play a role in neuronal differentiation, we generated transgenicPCC7-Mz1 embryonic carcinoma cells. The PCC7-Mz1 cell line is a model ofembryonic mouse brain development in that a similar pattern of neuralphenotypes, i.e. neurons, astroglia, and fibroblasts, is produced in a comparabletime frame, following exposure to retinoic acid (RA). Firstly, we stablytransfected the uninduced cells with the tetracycline-repressor expressing vectorpcDNA6TR (T-Rex system; InVitrogen), resulting in the subclone Mz1-TRep.Retinoic acid-induced neural differentiation of Mz1Trex1 was indistinguishablefrom the parental PCC7-Mz1 cells. The clone was then stably transfected withpcDNA4TO vectors containing a sense or antisense insertion of a FLAG-taggedNCNF cDNA. Fate decision was not influenced by the stable transfection of theNCNF-FLAG construct per se, since both, sense- and antisense clonesdifferentiated indistinguishable from the parental PCC7-Mz1 cells. Ectopicexpression of sense NCNF-FLAG mRNA influenced stem cell morphology andfavoured the formation of neurons during the course of RA-induced neuronaldifferentiation. Neutralization of endogenous NCNF mRNA by the expression ofantisense NCNF-FLAG mRNA downregulated MAP2 expression in maturingneurons and changed the pattern of neural phenotypes. In particular, fibroblastsdeveloped much more rapidly and the ratio of neurons to fibroblasts to glial cellsdiffered significantly from the parental PCC7 cell line. This data establishes forthe first time that NCNF is indeed involved in RA-induced neural patternformation and neuron maturation.

contact:

Dr. Christina ZechelJohannes Gutenberg Universität MainzInstitut für Physiologische Chemie und Pathobiochemiezechel@mail.uni-mainz.deDuesberg Weg 655099 Mainz (Germany)

Suisheng Zhang, Carsten Köhler, Frank Grosse

PHYSICAL INTERACTION BETWEEN NUCLEAR DNA HELICASE II AND WRNHELICASE STIMULATES WRN´S EXONUCLEASE ACTIVITY

The progeriatric Werner syndrome is characterized by a molecular defect of theWRN gene. WRN codes for a 3´-5´-directed DNA helicase that contains anadditional 3´-5´ exonucelase activity at its N-terminus. Here we show that WRNhelicase forms a physical complex with another intranuclear helicase, i.e. nuclearDNA helicase (NDH II), which is also known as RNA helicase A. Moreover, theearlier described NDH II-copurifying enzyme NDH I was identical to WRNhelicase. NDH II and WRN copurify over several chromatographic steps, such asBio-Rex 70, DEAE Sepharose and phosphocellulose. A high salt elution fractionfrom the latter column did not only contain NDH II and WRN helicase but alsoDNA polymerase delta and RPA. This suggests that both WRN helicase and NDHII may be involved in DNA replication and/or DNA repair. NDH II and WRNhelicase hardly showed a synergistic effect upon the unwinding of a 3´-taileddouble-stranded DNA. However such an effect may be overridden by the strong3´-5´ exonuclease activty of WRN´s helicase. Indeed, NDH II increased WRN´sexonuclease activity when the unwinding activity was halted by omitting anecessary nucleotide cofactor. These data are in agreement with a model whereNDH II unwinds DNA, most likely in the course of transcription. Whenever NDH IIis arrested, it may recruit WRN helicase, RPA, and DNA polymerase delta toperform DNA repair synthesis.

contact:

Dr. Suisheng Zhang

Institut für Molekulare Biotechnologie - Jenaszhang@Beutenbergstraße 1107745 JENA (Deutschland)