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SSB 2013
8th International Conference
Structure and Stability of Biomacromolecules
Book of Contributions
Department of Biophysics
Institute of Experimental Physics Slovak Academy of Sciences
in cooperation with
Slovak Physical Society
Slovak Biophysical Society
September 10 – 13, 2013, Košice, Slovakia
Department of Biophysics, Institute of Experimental Physics Slovak Academy of Sciences
in cooperation with
Slovak Physical Society and
Slovak Biophysical Society
September 10 – 13, 2013Košice, Slovakia
8th International Conference
Structure and Stabilityof Biomacromolecules
SSB 2013
Book of Contributions
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Book of Contribution, 8th International Conference Structure and Stability of BiomacromoleculesSSB2013, September 10-13, 2013, Košice, Slovakia
Editors: Ing. Jaroslava Bágeľová, CSc., RNDr. Diana Fedunová, PhD., RNDr. Zuzana Gažová, CSc.Reviewers: Doc. MUDr. Marek Dudáš, PhD., Doc. RNDr. Erik Sedlák, PhD.© Institute of Experimental Physics, Slovak Academy of Sciences
ISBN: 978-80-89656-01-1EAN: 9788089656011
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ORGANIZATION
ORGANIZING COMMITTEE M. Antalík T. Kožár J. Bágeľová A. Musatov D. Fedunová Z. Tomori Z. Gažová
TECHNICAL ASSISTANCE E. Demjén I. Hrmo M. Reiffers D. Sedláková K. Šipošová D. Švarcbergerová
CONFERENCE SUBJECTS
structural aspects aggregation activity factors influencing stability ligand binding and inhibition protein modifications and engineering experimental and computational strategies for structural and conformational studies bioinformatics, molecular and nanodesign applications of biomacromolecules in medicine, bio- and nanotechnologies
CONFERENCE VENUE Institute of Experimental Physics, Slovak Academy of Sciences, Lecture Hall, Watsonova 47, Košice, Slovak Republic
REVIEWERS Doc. MUDr. Marek Dudáš, PhD. Doc. RNDr. Erik Sedlák, PhD.
EDITORS D. Fedunová Z. Gažová J. Bágeľová
ACKNOWLEDGEMENTS
The Organizing Committee of SSB 2013 would like to express appreciations and thanks to the
following institutions and companies for their generous support.
ITES
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CONTENTS
SCIENTIFIC PROGRAM
Program SCHEDULE.......................................................................................... 7
Plenary Lectures.................................................................................................... 13
Short Communications............................................................................….... 16
Posters............................................................................................................. 18
CONTRIBUTIONS ……………..................................................................... 25
PARTICIPANTS ADDRESSES.......................................................................... 141
AUTHORS INDEX............................................................................................... 147
SPONSORS........................................................................................................... 153
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SCIENTIFIC PROGRAM
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PROGRAMME SCHEDULE
Tuesday, September 10, 2013
14:00 – 20:00 RegistrationSite: Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, Košice
16:00 – 16:10 Conference Opening
Chairman: Z. Gažová
16:10 – 16:55PL1 Parvalbumin as a Model Calcium Binding Protein
E. A. PERMYAKOV, A. G. BAKUNTS, A. A. VOLOGZHANNIKOVA, S. E. PERMYAKOV
16:55 – 17:40 PL2 HAMLET: To Be, or Not To Be
S. E. PERMYAKOV, E. L. NEMASHKALOVA, E. A. PERMYAKOV
17:40 – 18:00 Coffee break
Chairman: T. Kožár18:00-18:45 PL3 What can we expect from new therapeutic strategies in
nanopharmacology and nano-medicine? H.-C. SIEBERT
18:45-19:30 PL4 New features and improvements in carbohydrate 3D structure
validationD. MOKROS, R.P. JOOSTEN, A. DOMINIK, G. VRIEND, T. LÜTTEKE
19:30-19:50 SC1 Activation of vertebrate transglutaminases; What can learn from
molecular dynamics simulations? I. KOMÁROMI, A. FEKETE, D. MUCS, L. MUSZBEK
19:50 - 20:30 Refreshment “Welcome in Košice” held at lobby of the lecture room
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Wednesday, September 11, 2013
Chairman: A. Musatov 8:30 – 9:15 PL5 The effect of flavonoids in mammary and epidermoid tumor cells
with ErbB proteins overexpression M. M. MOCANU, P. NAGY, L. GEORGESCU, T. VARADI, D. SHRESTHA, I. BARAN, E. KATONA, J. SZÖLLOSI, C. GANEA
9:15 - 9:35 SC2 Factors of virulence of C. burnetii, the etiological agent of Q fever
Ľ. ŠKULTÉTY, R. TOMAN, G. FLOREZ-RAMIREZ 9:35 - 10:20 PL6 Targeting Fas-mediated Apoptosis Through Human Herpesvirus 8
Oncoprotein K1-derived Peptides Z. BERKOVA, S. WANG, U. DANILUK, C. KERROS, J.F. WISE, F. SAMANIEGO
10:20 - 10:50 Coffee break
Chairman: Ľ. Urbániková 10:50 - 11:35 PL7 Modern Trends in Biomolecular Simulations
J. HRITZ 11:35 - 12:20 PL8 Quantitative Analysis of Scanning Tunneling Microscopy Images of
Mixed Ligand Functionalized Nanoparticles F. BISCARINI, Q. K. ONG, C. ALBONETTI, F. LISCIO, M.
LONGOBARDI, K. S. MALI, A. CIESIELSKI, J. REGUERA, C. RENNER, S. De FEYTER, P. SAMORI, F. STELLACCI
12:20 - 12:40 SC3 Multi-view 3D reconstruction of microscopic objects
R. GARGALIK, Z. TOMORI 12:40 -13:00 SC4 The Influence of Quercetin on Lipid Membranes with Cholesterol
D. IONESCU
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13:00 – 13:30 Lunch
Chairman: P. Miškovský14:00 - 14:45 PL9 Mutations in proteolytic side of human mitochondrial Lon protease
uncover the direct connection between proteolytic and ATPase domainsL. AMBRO, V. PEVALA, J. BAUER, G. ONDROVIČOVÁ, E. KUTEJOVÁ
14:45 - 15:05 SC5 How hydrogen peroxide is broken down by oxidized cytochrome c
oxidase D. JANCURA, J. STANICOVA, G. PALMER, M. FABIAN
15:05 - 15:25 SC6 Hypothesis: Mitochondrial Electron-Transfer Proteins are a Part of
Defense Mechanism Against Oxidative Damage E. SEDLAK, R. VARHAC, A. MUSATOV
15:25 - 15:45 Coffee break
Chairman: D. Jancura 15:45 - 16:30 PL10 Directed evolution of new protein therapeutics - DARPins
E. SEDLAK
16:30 – 17:15 PL11 Force as a Single Molecule Probe of Multidimensional Protein
Energy LandscapesG. ŽOLDÁK, B. PELZ, M. RIEF
17:15 - 18:00 Poster session
19:00 - Reception
Thursday, September 12, 2013
Chairman: M. Antalík8:30 - 9:15 PL12 Rich Phase Behavior of Protein Solutions Induced by Multivalent
Ions: Reentrant Condensation and Liquid-Liquid Phase SeparationF. ROOSEN-RUNGE, M. WOLF, A. SAUTER, F. ZHANG, F. SCHREIBER
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9:15 – 10:00PL13 Understanding Charge-Driven Protein Aggregation and
CrystallizationF. ZHANG, A. SAUTER, F. ROOSEN-RUNGE, F. SCHREIBER
10:00– 10:20 SC7 Structure of tau protein in the complexes with monoclonal
antibodies studied by X-ray crystallography R. SKRABANA, O. CEHLAR, R. DVORSKY, B. KOVACECH, A. KOVAC, J. SEVCIK, M. NOVAK
10:20 – 10:40 Coffee break
Chairman: M. Fabian 10:40 - 11:25 PL14 Towards increased selectivity of cancer treatment by Photodynamic
therapy: Development of selective nano-delivery system and detection of therapeutically active form of drugs in cellP. MISKOVSKY
11:25-11:45 SC8 Non-trivial decisioning in Bcl-2 family-mediated regulation of
Apoptosis T. TOKAR
11:45 – 12:05SC9 Galectins-1 and -3 differently modulate wound microenviroment with
different impact on incisional and excisional skin wound healing in rats P. GÁL, V. PERŽEĽOVÁ, T. VASILENKO, J. JAKUBČO, H.-J. GABIUS, B. DVOŘÁNKOVÁ, F. SABOL, K. SMETANA JR
12:30 Trip to Stara Lubovna Castle - departure from Conference venue. Estimated arrival to Košice between 22:00-23:00
Friday, September 13, 2013
Chairman: E. Kutejová8:30 – 9:15 PL15 Role of Membrane Fluidity in Modulation of Function of Subcellular
Membrane Systems in Health and Disease: Relevance to Heart
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Sarcolemma and Mitochondria in Hypoxia, Ischemia, Diabetes, Hypertension, Irradiation and seasonal changes. A. ZIEGELHOFFER, M. FERKO, I. WACZULÍKOVÁ, J. MUJKOŠOVÁ, M. MURÁRIKOVÁ, B. ZIEGELHÖFFER, T. RAVINGEROVÁ, J. SLEZÁK
9:15-10:00 PL16 The study of CE16 acetyl esterase from fungus Hypocrea jecorina
Ľ. URBÁNIKOVÁ, B. VIDOVÁ, A. GODÁNY, P. BIELY
10:00– 10:20 SC10 Using protein engineering to understand the molecular mechanism
underlying enzyme deficiency in clinical mutants of G6PDM. A. HAMZA, A. A.-T. IDRIS, A. MUSTAFA, M. ALMAHREB
10:20 – 10:50 Coffee break
Chairman: E. A. Permyakov10:50 – 11:35 PL17 Computer-aided drug design: basic concepts and applications to
influenza virus and Alzheimer’s diseaseM. S. LI
11:35 – 12:20 PL18 Glassy behavior of proteins
CH. K. HU
12:20-12:40 SC11 Aliskiren loaded PLA nanospheres: preparation, physicochemical
characterization, in vitro release and in vivo effect I. ANTAL, M. KUBOVČÍKOVÁ, O. PECHANOVÁ, A. BARTA, M.
CEBOVÁ, M. KOVÁCSOVÁ, M. KONERACKÁ, V. ZÁVIŠOVÁ, A. JURÍKOVÁ, P. KOPČANSKÝ
12:40-13:00 SC12 Magnetosomes - a new type of magnetic nanoparticles. The methods
of preparation, characterization, and their applicationsM. MOLČAN, A. HASHIM, J. KOVÁČ, P. KOPČANSKÝ, H. GOJZEWSKI, A. SKUMIEL, M. TIMKO
13:00 Conference concluding remarks
13:00 – 13:30 Lunch
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LIST OF PLENARY LECTURES
PL1. Parvalbumin as a model calcium binding protein E. A. PERMYAKOV, A. G. BAKUNTS, A. A. VOLOGZHANNIKOVA, S. E. PERMYAKOV Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
PL2. HAMLET: To be, or not to beS. E. PERMYAKOV, E. L. NEMASHKALOVA, E.A. PERMYAKOV Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
PL3. What can we expect from new therapeutic strategies in nanopharmacology and nano-medicine? H.-C. SIEBERTResearch Institute for Bioinformatics and Nanotechnology (RI-B-NT) Kiel, Germany
PL4. New Features and Improvements in Carbohydrate 3D Structure Validationa D. MOKROS, b,c R.P. JOOSTEN, a A. DOMINIK,c G. VRIEND, d T. LÜTTEKEaUniversity of Applied Sciences, Giessen-Friedberg, Germany bNKI, Amsterdam, The Netherlands cRadboud-University Nijmegen Medical Centre, Nijmegen, The Netherlands
dJustus-Liebig-University Giessen, Germany
PL5. The effect of flavonoids in mammary and epidermoid tumor cells with ErbB proteins overexpression aM.M. MOCANU, bP. NAGY, aL. GEORGESCU, bT. VARADI, bD. SHRESTHA, aI. BARAN, aE. KATONA, b, cJ. SZÖLLŐSI, aC. GANEA aDepartment of Biophysics, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania, bDepartment of Biophysics and Cell Biology, Medical and Health Science Center, University of Debrecen, Nagyerdei krt. 98, H-4032, Debrecen, Hungary, cMTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Nagyerdei krt. 98, H-4032, Debrecen, Hungary
PL6. Targeting Fas-mediated Apoptosis Through Human Herpesvirus 8 Oncoprotein K1-derived Peptides aZ. BERKOVA, aS. WANG, bU. DANILUK, aC. KERROS, aJ.F. WISE, aF. SAMANIEGOaDepartment of Lymphoma and Myeloma, The University of Texas - MD Anderson Cancer Center, Houston, TX; bDepartment of Pediatrics, Gastroenterology and Allergology, Medical University of Bialystok, Bialystok, Poland
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PL7. Modern Trends in Biomolecular Simulations
J. HRITZ Department of Structural Biology, Masaryk University, CEITEC, Brno, Czech Republic
PL8. Quantitative Analysis of Scanning Tunneling Microscopy Images of Mixed
Ligand Functionalized Nanoparticles aF. BISCARINI, bQ.K. ONG, cC. ALBONETTI, dF. LISCIO, eM. LONGOBARDI, fK. S. MALI, gA. CIESIELSKI, bJ. REGUERA, eC. RENNER, fS. DE FEYTER, gP. SAMORI, bF. STELLACCI aDip. Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 183, 41125
Modena, Italy bInstitute of Materials, École Politechnique Fédérale de Lausanne, Lausanne, Switzerland cConsiglio Nazionale delle Ricerche, Istituto per
lo Studio dei Materiali Nanostrutturati Materiali Nanostrutturati (CNR-ISMN), Via P. Gobetti 101, I-40129 Bologna, Italy dConsiglio Nazionale delle Ricerche, Istituto di Microelettronica e Microsistemi (CNR-IMM), Via P. Gobetti 101, I-40129 Bologna, Italy eDepartment of Condensed Matter Physics, NCCR Materials with Novel Electronic Properties, University of Geneva, 24 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland fDepartment of Chemistry, Division of Molecular Imaging and Photonics, KU Leuven - University of Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium gLaboratoire de Nanochimie, ISIS & icFRC, Université de Strasbourg & CNRS, 8 allée Gaspard Monge, 67000 Strasbourg, France
PL9. Mutations in proteolytic side of human mitochondrial Lon protease uncover the
direct connection between proteolytic and ATPase domains aL’. AMBRO, aV. PEVALA, aJ. BAUER, aG. ONDROVIČOVÁ, a,bE. KUTEJOVÁ aDepartment of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia bInstitute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-142 20 Prague 4, Czech Republic
PL10. Directed evolution of new protein therapeutics – DARPins
E. SEDLÁK Department of Biochemistry, P.J.Šafárik University, Košice, Slovakia, Institute of interdisciplinary biosciences, P.J.Šafárik University, Košice, Slovakia
PL11. Force as a Single Molecule Probe of Multidimensional Protein Energy Landscapes
aG. ŽOLDÁK, aB. PELZ, a,b M. RIEF
aPhysik Department E22, Technische Universität München, 85748 Garching, Germany; bMunich Center for Integrated Protein Science, 81377 München, Germany
PL12. Rich Phase Behavior of Protein Solutions Induced by Multivalent Ions: Reentrant Condensation and Liquid-Liquid Phase Separation
F. ROOSEN-RUNGE, M. WOLF, A. SAUTER, F. ZHANG, F. SCHREIBER Institute for Applied Physics, University of Tübingen, Germany
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PL13. Understanding Charge-Driven Protein Aggregation and Crystallization F. ZHANG, A. SAUTER, F. ROOSEN-RUNGE, F. SCHREIBERInstitute for Applied Physics, University of Tübingen, Germany
PL14. Towards increased selectivity of cancer treatment by Photodynamic therapy: Development of selective nano-delivery system and detection of therapeutically active form of drugs in cell P. MISKOVSKYDepartment of Biophysics and Center for Interdisciplinary Biosciences, P. J. Safarik University, Kosice, Slovakia
PL15. Role of Membrane Fluidity in Modulation of Function of Subcellular Membrane Systems in Health and Disease: Relevance to Heart Sarcolemma and Mitochondria in Hypoxia, Ischemia, Diabetes, Hypertension, Irradiation and seasonal changes aA. ZIEGELHÖFFER, aM. FERKO, bI. WACZULÍKOVÁ, aJ. MUJKOŠOVÁ, aM. MURÁRIKOVÁ, cB. ZIEGELHÖFFER, aT. RAVINGEROVÁ, aJ. SLEZÁKaInstitute for Heart Research, Centre of Excelence SAS NOREG, Bratislava, SR bDepartment of Nuclear Physics and Biophysics, Division of Biomedical Physics, Faculty of Mathematics, Physics & Informatics, Comenius University, Bratislava, SR cHeart Center Leipzig, University of Leipzig, Leipzig, Germany
PL16. The Study of CE16 Acetyl Esterase from Fungus Hypocrea jecorina aL. URBANIKOVA, aB. VIDOVA, aA. GODANY, bP.BIELYaInstitute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovak Republic bInstitute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 945 38 Bratislava, Slovak Republic
PL17. Computer-aided drug design: basic concepts and applications to influenza virus and Alzheimer’s disease
M. S. LIInstitute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland
PL18. Glassy behavior of proteins C.-K. HUInstitute of Physics of Academia Sinica, Taipei 11529, Taiwan
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LIST OF SHORT COMMUNICATIONS
SC1. Activation of vertebrate transglutaminases; What we can learn from molecular dynamics simulations? a,bI. KOMÁROMI, bA. FEKETE, bD. MUCS and a,bL. MUSZBEK aHaemostasis, Thrombosis and Vascular Biology Research Group of the HungarianAcademy of Sciences bClinical Research Center, University of Debrecen, Medical and Health Science Center, Debrecen, Hungary
SC2. Factors of virulence of C. burnetii, the etiological agent of Q fever a,b,cL. SKULTETY, aR. TOMAN, aG. FLOREZ-RAMIREZ aInstitute of Virology, Slovak Academy of Sciences, Bratislava, Slovaki, bCentre of Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia cInstitute of Microbiology, Academy of Science of Czech Republic, Prague, Czech Republic.
SC3. Multi-view 3D reconstruction of microscopic objectsaR. GARGALIK, bZ. TOMORI aInstitute of Computer Science, Faculty of Natural Sciences UPJŠ, Košice bInstitute of Experimental Physics SAS, Košice
SC4. The influence of quercetin on lipid membranes with cholesterolD. IONESCUDepartment of Biophysics, "Carol Davila" University of Medicine and Pharmacy, Bucharest, 050474, Romania
SC5. How hydrogen peroxide is broken down by oxidized cytochrome c oxidaseaD. JANCURA, bJ. STANICOVA, cG. PALMER and c,dM. FABIANa Department of Biophysics, University of P. J. Safarik, Kosice b Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine, Kosice c Department of Biochemistry and Cell Biology, Rice University, Houston
dCenter of Interdisciplinary Biosciences, University of P. J. Safarik, Kosice
SC6. Hypothesis: Mitochondrial Electron-Transfer Proteins are a Part of Defense Mechanism Against Oxidative Damage a,bE. SEDLÁK, a,bR. VARHAČ and a,cA. MUSATOVaDepartment of Biochemistry, The University of Texas Health Science Center at San Antonio, Texas, USA bDepartment of Biochemistry, University of P. J. Safarik, Kosice, Slovakia cDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Science, Kosice, Slovakia
SC7. Structure of tau protein in the complexes with monoclonal antibodies studied by X-ray crystallography a,bR. SKRABANA, aO. CEHLAR, cR. DVORSKY, a,bB. KOVACECH, a,bA. KOVAC,
dJ. SEVCIK, a,bM. NOVAK
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aInstitute of Neuroimmunology SAS, Bratislava, Slovakia bAxon Neuroscience SE, Bratislava, Slovakia cMax Planck Institute for Molecular Physiology, Dortmund, Germany dInstitute of Molecular Biology SAS, Bratislava, Slovakia
SC8. Non-trivial decisioning in Bcl-2 family-mediated regulation of Apoptosis
T. TOKAR, J. ULICNY Dept. of Biophysics, Faculty of Science, P. J. Safarik University, Kosice, Slovakia
SC9. Galectins-1 and -3 differently modulate wound microenvironment with different
impact on incisional and excisional skin wound healing in rats aP. GÁL, bV. PERŽEĽOVÁ, bT. VASILENKO, bJ. JAKUBČO, cH.-J. GABIUS, dB. DVOŘÁNKOVÁ, eF. SABOL AND dK. SMETANA JR. aInstitute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovak Republic bFaculty of Medicine, Pavol Jozef Šafárik University, Košice, Slovak Republic cFaculty of Veterinary Medicine, Ludwig-Maximilian University, Munich, Germany d1st Faculty of Medicine, Charles University, Prague, Czech Republic eEast-Slovak Institute of Cardiovascular Diseases, Košice, Slovak Republic
SC10. Using protein engineering to understand the molecular mechanism underlying
enzyme deficiency in clinical mutants of G6PD M. A. HAMZA, A. A.-T. IDRIS, A. MUSTAFA, M. AL-MAHAREB Faculty of Medicine, King Fahad Medical City, King Saud Bin Abdul Aziz University for Health Sciences, Riyadh, KSA
SC11. Aliskiren loaded PLA nanospheres: preparation, physicochemical
characterization, in vitro release and in vivo effect aI. ANTAL, aM. KUBOVCIKOVA, bO. PECHANOVA, bA. BARTA, bM. CEBOVÁ, bM. KOVÁCSOVÁ, aM. KONERACKA, aV. ZAVISOVA, aA. JURIKOVA, aP. KOPCANSKY aInstitute of Experimental Physics Slovak Academy of Sciences, 040 01 Kosice, Slovakia bInstitute of Normal and Pathological Physiology Slovak Academy of Sciences, Bratislava
SC12. Magnetosomes - a new type of magnetic nanoparticles. The methods of
preparation, characterization, and their applications aM. MOLCAN, aA. HASHIM, aJ. KOVAC, aP. KOPCANSKY, bH. GOJZEWSKI, cA. SKUMIEL, aM. TIMKO aInstitute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Košice,Slovakia bInstitute of Physics, Poznan University of Technology, Nieszawska 13A, 60-965 Poznań, Poland cInstitute of Acoustics, Adam Mickiewicz University, Umultowska 85, 61-614 Poznań, Poland
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POSTER LIST
PO1. Application of surface-enhanced Raman spectroscopy for the detection of trace amounts of pesticides aldrin, α-endosulfan and lindane aJ. KUBACKOVA, aP. MISKOVSKY, aG. FABRICIOVA, bS. SANCHEZ-CORTES, aD. JANCURAaDepartment of Biophysics, Faculty of Science, P.J. Safarik University, Kosice, Slovakia b Instituto de Estructura de la Materia, CSIC, Madrid, Spain
PO2. Adsorption and detection of anthraquinone drug parietin on Ag nanoparticles by surface-enhanced Raman scattering aG. FABRICIOVÁ, bE. LOPEZ-TOBAR, bM. V. CAÑAMARES, cM. BAČKOR, bS. SANCHEZ-CORTES aDepartment of Biophysics, Faculty of Science P. J. Šafárik University, Košice bInstituto de Estructura de la Materia, CSIC, Madrid, Spain cDepartment of Botany, Faculty of Science P. J. Šafárik University, Košice
PO3. Preliminary results of in vitro cytotoxicity testing of bacterial magnetic nanoparticles aZ. VARCHULOVÁ NOVÁKOVÁ, bM. TIMKO, bA. HASHIM, bM. MOLČAN, aM.KUNIAKOVÁ, a L. ORAVCOVÁ, cM. CSÖBÖNYEIOVÁ, aD. BÖHMER, aĽ.DANIŠOVIČ aInstitute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, SK-811 08 Bratislava, Slovakia; bInstitute of Experimental Physics, SAS, Watsonova 47, 040 04 Košice, Slovakia, cInstitute of Histology and Embryology, Sasinkova 4, , 811 08 Bratislava
PO4. Activated Charcoals Supporting Nanomagnets with Antimicrobial Character aE. VALUŠOVÁ, bP. PRISTAŠ, bP. JAVORSKÝ, a, cM. ANTALÍK, aM. TIMKOaDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, Košice, 040 01, Slovakia bInstitute of Animal Physiology Slovak Academy of Sciences, Šoltésovej 4-6, 040 01 Košice, Slovakia cDepartment of Biochemistry, Faculty of Science, P. J. Šafárik University, Moyzesova 11, Košice, 040 01, Slovakia
PO5. Use of the modified glassy carbon CuO/ graphene electrode for the glucose determination aP. KEŠA, bI. HRMO, a,bM. ANTALÍKaDepartment of Biochemistry, Faculty of Natural Science UPJŠ, KošicebInstitute of Experimental Physics SAS, Košice
PO6. Change of fluorescence characteristis Ca2+-independent discharged photoprotein obelin under expose to 40°CaR. ALIEVA, a,bN. BELOGUROVA, aA. PETROVA, a,bN. KUDRYASHEVA aSiberian Federal University, Krasnoyarsk, Russia bInstitute of Biophysics SB RAS, Krasnoyarsk, Russia
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PO7. Fluorescence properties of Ca2+- discharged obelin in the presence of ethanol and glycerine aА.S. PETROVA, bN.V. BELOGUROVA, aR.R. ALIEVA aSiberian Federal University, Krasnoyarsk bInstitute of Biophysics SB RAS, Krasnoyarsk
PO8. Temperature effects on optical absorption, fluorescence and circular dichroism of heparin-safranin T complex aJ. KUDLÁČOVÁ, a,bM. ANTALÍKaDepartment of Biochemistry, Faculty of Natural Sciences UPJŠ, Košice bInstitute of Experimental Physics SAS, Košice
PO9. Interaction between carbosilane dendrimers and α-synucleinaK. MILOWSKA, aE. BARTCZAK, aM. BRYSZEWSKA, bR. GOMEZ, bJ. DE LA MATA, cJ.P. MAJORAL, aT. GABRYELAKaDepartment of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland bLaboratoire de Chimie de Coordination CNRS, Toulouse, France cDepartamento de Quimica Inorganica, Universidas de Alcala, Alcala, Spain
PO10. Influence of PAMAM dendrimers on human insulin aggregation processes O. NOWACKA, M. BRYSZEWSKA Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland
PO11. Formation of cytochrome c fibrils as a result of heme destruction K. GARAJOVÁ, R. VARHAČDepartment of Biochemistry, Faculty of Science, Pavol Jozef Šafárik University in Košice
PO12. Effect of Nanospheres on Insulin Fibril Formation a,bZ. BEDNARIKOVA, a M. KONERACKA, a V. ZAVISOVA, a K. SIPOSOVA, a M.
KUBOVCIKOVA, a P. KOPCANSKY, c V. GIRMAN, a Z. GAZOVA aInstitute of Experimental Physics SAS, Košice, Slovakia bDepartment of Biochemistry
Faculty of Science, Safarik University, Košice, Slovakia cDepartment of Condensed Matter Physics, Faculty of Science, Safarik University, Košice, Slovakia
PO13. Experimental conditions of in vitro lysozyme amyloid fibrillization determine their properties aA. ANTOSOVA,a,bZ. BEDNARIKOVA, aK. SIPOSOVA, aE. DEMJEN, aJ. MAREKaZ. GAZOVA aDepartment of Biophysics, Institute of Experimental Physics SAS, Kosice, Slovakia bDepartment of Biochemistry, Faculty of Science, P. J. Safarik University, Kosice, Slovakia
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PO14. Utilization of imidazolium-based ionic liquids as trigger for lysozyme amyloid
fibrillization aD. FEDUNOVÁ, aA ANTOŠOVÁ, aJ. MAREK, aE. DEMJÉN, a,bZ. BEDNÁRIKOVÁ, aK. ŠIPOŠOVÁ, aJ. BÁGEĽOVÁ, aZ. GAŽOVÁ aDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia bDepartment of Biochemistry, Faculty of Science, P. J. Šafárik University, Košice, Slovakia
PO15. Effect of small molecule spirobrassinin and magnetite nanoparticle on ThT
fluorescence in cerebrospinal fluid from patients with Alzheimer disease and multiple sclerosis aZ. KRISTOFIKOVA, bK. SIPOSOVA, a,cA. BARTOS, cJ. KOTOUCOVA, aD. RIPOVA, dZ. GAZOVA aPrague Psychiatric Centre, Alzheimer Disease Centre, Ustavni 91, 181 03 Prague 8, Czech Republic bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia cCharles Universtiy in Prague, Third Faculty of Medicine, University Hospital Kralovske Vinohrady, Department of Neurology, Srobarova 50, 100 34 Prague 10, Czech Republic dDepartment of Medical and Clinical Biochemistry and LABMED, Faculty of Medicine, Safarik University, Kosice, Slovakia
PO16. Inhibition of Aβ peptide fibrillization by tripeptides – importance of Proline
aM. H. VIET, bK. SIPOSOVA, aM. SUAN LI, b,cZ. BEDNARIKOVA, dT. TRANG NGUYEN, b,eZ. GAZOVA aInstitute of Physics, Polish Academy of Sciences Al. Lotnikow 32/46, 02-668 Warsaw, Poland bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia cDepartment of Biochemistry, Faculty of Science, Safarik University, Kosice, Slovakia dInstitute for Computational Science and Technology, 6 Quarter, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam eDepartment of Medical and Clinical Biochemistry and LABMED, Faculty of Medicine, Safarik University, Kosice, Slovakia
PO17. Morphological diversity of lysozyme amyloid fibrils induce different cytotoxicity
aM.-M. MOCANU, aC. GANEA, b,cK. SIPOSOVA, aA. FILIPPI, dE. RADU, bE. DEMJEN, bJ. MAREK, b,cZ. BEDNARIKOVA, bA. ANTOSOVA, b,e *Z. GAZOVA aDepartment of Biophysics "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Kosice, Slovakia cDepartment of Biochemistry, Faculty of Science, Safarik University, Kosice, Slovakia dDepartment of Cellular and Molecular Medicine, "Carol Davila" University of Medicine and Pharmacy, Bucharest eDepartment of Medical and Clinical Biochemistry and LABMED, Faculty of Medicine, Safarik University, Kosice, Slovakia
PO18. Preparation of recombinant fragments of human cardiac ryanodine receptor
J. NOVAKOVA, V. BAUEROVA-HLINKOVA, E. SCHILLEROVA, J. GASPERIK, E. HOSTINOVA, L. BORKO, A. ZAHRADNIKOVA, E. KUTEJOVA, J. SEVCIK Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia
21
PO19. The proteolytic reaction of papain; ONIOM-type hybrid QM/MM study aA. FEKETE, a,bL. MUSZBEK and a,bI. KOMÁROMIaClinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary and bVascular Biology, Haemosthasis and Thrombosis Research Group of The Hungarian Academy of Sciences and University of Debrecen, Debrecen, Hungary
PO20. Mechanism of the irreversible inhibition of human cyclooxygenase-1 by aspirin as predicted by QM/MM calculations aL. TÓTH, a,bL. MUSZBEK, bI. KOMÁROMIaClinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, bVascular Biology, Thrombosis and Haemostasis Research Group of the Hungarian Academy of Sciences at the University of Debrecen, Debrecen, Hungary
PO21. GLYCOGRID Initiative for Computational Studies of Glycan-Protein InteractionsT. KOZARDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01 Kosice, Slovakia
PO22. Primary processes in photosynthetic bacterial reaction centers:initial terms M. PUDLÁK, R.PINČÁK Institute of Experimental Physics SAS, Košice, Slovakia
PO23. Different effects of low-level laser therapy at 635 and 670 nm on the healing of excisional and incisional skin wounds in rats aP. GÁL, bM. MOKRÝ, bB. VIDINSKÝ, bT. VASILENKO, bK. LACJAKOVÁ,bM. SLEZÁK, bM. POLÁKOVÁ, I. bKOVÁČ, aZ. TOMORI
aInstitute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovak Republic bFaculty of Medicine, Pavol Jozef Šafárik University, Košice, Slovak Republic
PO24. DNA-DOPE-Gemini Surfactants Complexes: Structure and Transfection Activity aL. HUBČÍK, a,bP. PULLMANNOVÁ, cĽ. LACINOVÁ, cZ. SULOVÁ, dS.S. FUNARI,
aF. DEVÍNSKY, aD. UHRÍKOVÁaFaculty of Pharmacy, Comenius University, Bratislava, bDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Hradec Králové, cInstitute of Molecular Physiology and Genetics SAV, Bratislava, dHASYLAB at DESY, Hamburg
PO25. Singlet oxygen luminescence and mitochondrial autofluorescence after illumination of Hyp/mitochondria complex aZ. NADOVA, aD. PETROVAJOVA, aD. JANCURA, bD. CHORVAT, bA.CHORVATOVA, cX. RAGAS, cM. GARCIA-DIAZ, cS. NONELL, aP. MISKOVSKYaDepartment of Biophysics, UPJS Kosice, Slovakia bInternational Laser Centre, Bratislava, Slovakia cInstitut Quimic de Sarria, Universitat Ramon LLull, Spain
22
PO26. New bis-tacrine derivatives as topoisomerase I inhibitors aJ. JANOČKOVÁ, bS. HAMUĽÁKOVÁ, cJ. KORÁBEČNÝ, dK. KUČA, aM.
KOŽURKOVÁa Department of Biochemistry, b Department of Organic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic, c Department of Toxicology, d
Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Králové, Czech Republic
PO27. Investigation of DNA binding activities with new oxime-type ligands aJ. JANOČKOVÁ, b-c K. MUSILEK, cK. KUČA, aM. KOŽURKOVÁ
a Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic, b Department of Toxicology, c Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec Králové, Czech Republic
PO28. Interaction of bis-3,6-alkylamido-acridines with calf thymus DNA aD. SABOLOVÁ, aJ. KUDLÁČOVÁ, bL. JANOVEC, bJ. IMRICH aDepartment of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 04167 Košice, Slovak Republic bDepartment of Organic Chemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 04167 Košice, Slovak Republic
PO29. The effect of new acridine derivatives – alkyl (2E)-3-(acridin-9-yl)-prop-2-enoates - on DNA aO. SALEM, bM. VILKOVÁ, bM. PROKAIOVÁ, bJ. IMRICH, aM. KOŽURKOVÁaDepartment of Biochemistry, bDepartment of Organic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic
PO30. Determination of interaction mode and binding constant of emodin incorporated into DNA Z. VOJNOVÁ, V. VEREBOVÁ, J. STANIČOVÁ
Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Košice, Slovakia
PO31. Selected antioxidant properties of known or commonly used plant extracts A. FEJERČÁKOVÁ, K. KREMPASKÁ, J. VAŠKOVÁ, L. VAŠKO Department of Medical and Clinical Biochemistry and LABMED ojsc., Faculty of Medicine, UPJŠ, Košice
PO32. Comparison of the effects of some natural and synthetic hydroxyl substituted chalcones against hydroxyl radical
aK. KREMPASKÁ, aJ. VAŠKOVÁ, aA. FEJERČÁKOVÁ, aL. VAŠKO, bP. PERJÉSIaDepartment of Medical and Clinical Biochemistry and LABMED ojsc., Faculty of Medicine, UPJŠ, Košice, Slovak Republic bInstitute of Pharmaceutical Chemistry, Faculty of Medicine, University of Pécs, Pécs, Hungary
23
PO33. The effect of model carcinogen on the antioxidant enzyme status in rats aĽ. LOHAJOVÁ, bA. SOBEKOVÁa Institute of Biophysics, b Institute of Medical Chemistry, Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice
25
PLENARY LECTURES abstracts
27
Parvalbumin as a model calcium binding protein
E. A. PERMYAKOV, A. G. BAKUNTS, A. A. VOLOGZHANNIKOVA, S. E. PERMYAKOV
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino,
Moscow region 142290, Russia
Parvalbumin (PA) is a vertebrate-specific cytosolic small acidic protein (106–113
residues) of the EF-hand superfamily of Ca2+-binding proteins, which is expressed in fast-
twitch muscles, specific neurons, certain kidney and endocrine glands cells, Corti’s cells, and
others [1]. While function of PA in non-muscle tissues is not firmly established, multiple
studies showed that PA serves as a soluble relaxing factor accelerating the Ca2+-mediated
relaxation in the fast muscles. Though molecular details of this process are not fully clear, the
kinetics of Ca2+ and Mg2+ association and dissociation of their complexes with PA are
believed to reflect the functional status of the protein. Furthermore, PA is a major fish
allergen. In spite that structural and physico-chemical properties of several PAs are well
documented [1], simplicity of PA structure make it an attractive model Ca2+-binding protein.
Our recent studies of PA revealed some unexpected properties important for understanding of
structural aspects of Ca2+-binding and other proteins.
Conformational behavior of five PAs from northern pike (α and β isoforms), Baltic cod, and
rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS
fluorescence [2]. The mechanism of the temperature-induced denaturation of these proteins
depends dramatically on both the peculiarities of their amino acid sequences and on their
interaction with metal ions. A peculiar feature of pike α-PA is that depending on its
interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an
ordered protein of mesophilic (Na+-bound state), thermophilic (Mg2+-form), or even of the
hyperthermophilic origin (Ca2+-form). The melting of Ca2+-loaded pike α-PA can be
described by two successive two-state transitions with mid-temperatures of 90°C and 120°C,
suggesting the presence of two thermodynamic domains. The intermediate state populated at
the end of the first transition was shown to bind Ca2+ ions, and was characterized by the
largely preserved secondary structure and increased solvent exposure of hydrophobic groups.
Mg2+- and Na+-loaded forms of pike α-PA demonstrated a single two-state transition. Overall,
the mechanism of the PA thermal denaturation is controlled by metal binding: it ranged from
the absence of detectable first-order transition (apo-form of pike PA) to the two-state
transition (e.g., Mg2+- and Na+-loaded forms of pike α-PA) and to the more complex
mechanisms (Ca2+-loaded PAs) involving at least one intermediate state. Analysis of isolated
cavities in the protein structures revealed that the interface between the CD and EF
subdomains of Ca2+- loaded pike α-PA is much more loosely packed compared with Pas
PL1
28
manifesting single heat-sorption peak. The impairment of interactions between CD and EF
subdomains may cause a loss of structural cooperativity and appearance of two separate
thermodynamic domains.
The effect of alpha-N-acetylation (Nt-acetylation) on the properties of PA has been
explored [3]. Intact PA contains an N-terminal acetyl group which is absent in the protein
expressed in Escherichia coli (rWT), as confirmed by mass spectrometry. Compared to intact
pike α-PA, its rWT form exhibits essentially altered profile of thermal unfolding, lowered α-
helicity, and decreased affinities to Ca2+ and Mg2+. The structural destabilization of the rWT
protein results in lowered resistance to chymotryptic digestion and increased propensity to
oligomerization. The rate constants of Ca2+ dissociation from the rWT PA are markedly
increased, which indicates that Nt-acetylation modifies functional status of the protein. Rat α-
PA demonstrates similar properties for intact and rWT forms. The drastic difference in the
effects induced by Nt-acetylation in the PA orthologs can be rationalized by higher disorder
level of AB domain in pike PA. Though evolution of PA’s genes resulted in the protein
sequences with highly divergent properties, Nt-acetylation unifies their functional properties.
The structural stability conferred to PA by Nt-acetylation may contribute to its allergenicity.
Overall, Nt-acetylation is shown to be a prerequisite for maintenance of structural and
functional status of some parvalbumins.
References
1. Permyakov, E.A., and Kretsinger, R.H. (2011) Calcium Binding Proteins. A John
Wiley & Sons, Inc., Hoboken, New Jersey
2. Permyakov, S.E., Bakunts, A.G., Permyakova, M.E., Denesyuk, A.I., Uversky, V.N.,
and Permyakov, E.A. (2009) Cell Calcium 46, 163-175.
3. Permyakov, S.E., Vologzhannikova, A.A., Emelyanenko, V.I., Knyazeva, E.L.,
Kazakov, A.S., Lapteva, Y.S., Permyakova, M.E., Zhadan, A.P., and Permyakov, E.A.
(2012) Cell Calcium 52, 366-376.
29
HAMLET: To be, or not to be
S. E. PERMYAKOV, E. L. NEMASHKALOVA, E. A. PERMYAKOV
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
Some natural proteins induce tumor-selective apoptosis. α-Lactalbumin (α-LA), a milk
calcium-binding protein, is converted into an antitumor form, called HAMLET/BAMLET
(Human/Bovine Alpha-lactalbumin Made LEthal to Tumor cells), via partial unfolding and
association with oleic acid (OA) [1]. Besides triggering multiple cell death mechanisms in
tumor cells, HAMLET exhibits bactericidal activity against Streptococcus pneumoniae. The
ability of HAMLET to induce cell death with selectivity for tumor and undifferentiated cells
is partly mediated by interaction of HAMLET with histone proteins, resulting in irreversible
disruption of chromatin structure. We have shown that α-LA in the absence of fatty acids is
also able to bind efficiently to the primary target of HAMLET, histone HIII, regardless of
Ca2+ content [2]. The experiments with poly-Lys and poly-Arg confirm the suggestion that
interaction between α-LA and histone is due to compensation of excess negative charge of α-
LA’s Ca2+-binding loop by positively charged residues of the histone. Overall, the
modification of α-LA by OA is not required for its electrostatically driven interaction with
histones.
The existing methods for preparation of active complexes of α-LA with OA employ
neutral pH solutions, which greatly limit water solubility of OA. Therefore, these methods
suffer from low scalability and/or heterogeneity of the resulting α-LA-OA samples. We have
developed a novel method for preparation of α-LA-OA complexes using alkaline conditions
that favor aqueous solubility of OA [3]. The unbound OA molecules are removed by
precipitation under acidic conditions. The resulting sample, bLA-OA-45, bears 11 OA
molecules and exhibits physico-chemical properties similar to those of BAMLET. Cytotoxic
activities of bLA-OA-45 against human epidermoid larynx carcinoma (HEp-2) and S.
pneumoniae D39 cells are close to those of HAMLET. Treatment of S. pneumoniae with
bLA-OA-45 or HAMLET induces depolarization and rupture of the membrane. The cells are
markedly rescued from death upon pretreatment with an inhibitor of Ca2+ transport. Hence,
the activation mechanisms of S. pneumoniae death are analogous for these two complexes.
The developed express method for preparation of active α-LA - OA complex is high-
throughput and suited for development of other protein complexes with low-molecular-weight
amphiphilic substances possessing valuable cytotoxic properties.
To assess the contribution of the proteinaceous component to cytotoxicity of
HAMLET, OA complexes with proteins structurally and functionally distinct from α-LA were
PL2
30
prepared using the developed alkaline procedure [4]. Similar to HAMLET, the OA complexes
with bovine β-lactoglobulin (bLG) and pike α-parvalbumin (pPA) (bLG-OA-45 and pPA-OA-
45, respectively) induced S. pneumoniae D39 cell death. The activation mechanisms of S.
pneumoniae death for these complexes were analogous to those for HAMLET, and the
cytotoxicity of the complexes increased with OA content in the preparations. The half-
maximal inhibitory concentration for HEp-2 cells linearly decreased with rise in OA content
in the preparations, and OA concentration in the preparations causing HEp-2 cell death was
close to the cytotoxicity of OA alone. Hence, the cytotoxic action of these complexes against
HEp-2 cells is induced mostly by OA. Overall, the proteinaceous component of HAMLET-
like complexes studied is not a prerequisite for their activity; the cytotoxicity of these
complexes is mostly due to the action of OA. This conclusion raises concerns on the future
development of HAMLET and HAMLET-like complexes as antitumor drugs.
References
1. Permyakov, E.A. (2009) Metalloproteomics. A John Wiley & Sons, Inc., Hoboken,
New Jersey.
2. Permyakov, S.E., Pershikova, I.V., Khokhlova, T.I., Uversky, V.N., and Permyakov,
E.A. (2004) Biochemistry 43, 19, 5575-5582.
3. Permyakov, S.E., Knyazeva, E.L., Leonteva, M.V., Fadeev, R.S., Chekanov, A.V.,
Zhadan, A.P., Håkansson, A.P., Akatov, V.S., and Permyakov, E.A. (2011) Biochimie
93, 1495-1501.
4. Permyakov, S.E., Knyazeva, E.L., Khasanova, L.M., Fadeev, R.S., Zhadan, A.P.,
Roche-Hakansson, H., Håkansson, A.P., Akatov, V.S., Permyakov, E.A. (2012) Biol.
Chem. 393, 85-92.
31
What can we expect from new therapeutic strategies in nanopharmacology and nano-medicine?
H.- C. SIEBERT
Research Institute for Bioinformatics and Nanotechnology (RI-B-NT) Kiel, Germany
When talking about nano-pharmacology and nano-medicine one can expect
fundamental new solutions in the broad fields of pharmacy as well as human and veterinary
medicine. However, it is not only the precise knowledge about the molecular interactions on a
nanoscale level. Furthermore, it is essential to understand and apply the sub-molecular based
survival strategies which e.g. marine organisms are using for more than a billion years. The
arsenal of methods used in the Research Institute for Bioinformatics and Nanotechnology (RI-
B-NT) for the functional and structural elucidation of bioactive molecules is applied on
various molecules from plant and animal origin. Beside clinical studies and cell biological
techniques, NMR, molecular modeling (especially quantum chemical calculations), mass
spectrometry, atom force microscopy, surface plasmon resonance techniques also
measurement with a quartz crystal microbalance are carried out. This combination of methods
is extremely suited for the analysis of various processes in the extracellular matrix, which are
related to nerve cell repair, antimicrobial processes and cancer therapy [1-7]. One main
research project of our institute concerns bio-active molecules in Cnidaria (e.g. jellyfishes).
Jellyfishes are a rich source of interesting collagen variants, lectins, defensins and
proteoglycans. We are testing these molecules after detailed structural analysis in various cell
cultures as well as in clinical studies.
References
1. Schadow, S., et al. (2013) PLOS ONE 8, 1, e53955.
2. Stötzel, S., et al. (2012) Chem.Phys.Chem. 13, 3117- 3125.
3. Eckert, T., et al. (2012) O. J. Phys. Chem. 2, 123-133.
4. Tsvetkov, Y. E., et al. (2012) J. Am. Chem. Soc. 134, 426-435.
5. Siebert, H.-C., et al. (2010) Protein & Cell 1(4), 393-405.
6. Bhunia, A., et al. (2010) J. Am. Chem. Soc.132, 96-105.
7. Siebert, H.-C., et al. (2009) Carbohydr. Res. 344, 1515-1525.
PL 3
32
New Features and Improvements in Carbohydrate 3D Structure Validation
a D. MOKROS, b,c R.P. JOOSTEN, a A. DOMINIK,c G. VRIEND, d T. LÜTTEKE
aUniversity of Applied Sciences, Giessen-Friedberg, Germany bNKI, Amsterdam, The
Netherlands cRadboud-University Nijmegen Medical Centre, Nijmegen, The Netherlands
dJustus-Liebig-University Giessen, Germany
More than 5000 entries in the Protein Data Bank (PDB, www.pdb.org, the largest
collection of biomolecular 3D-structures) contain carbohydrates. This makes the PDB a
valuable resource not only for proteomics but also for glycoscience. Unfortunately, the quality
of the carbohydrate moieties is significantly lower than that of the protein parts of
glycoproteins or protein-carbohydrate complexes in the PDB; many entries contain errors [1].
There are several reasons for this, one of them being the lack of validation software [2]. Only
recently crystallographers became aware of this problem and started to use tools such as PDB
Carbohydrate Residue check (pdb-care) [3] to examine the carbohydrate parts. Here we
present an updated version of pdb-care, which in addition to the residue notation checks
already performed by the former version also detects other problems such as invalid residues
within the N- -D- -D-GlcpNAc), missing
LINK records, which often result in “1-deoxy” sugars, or superfluous atoms within glycosidic
linkages. The results are presented via a new, clearly arranged web interface for human
readability, or as a computer-readable xml file to aid automatic validation routines.
Suggestions how to correct errors are also included in many instances. These are used e.g.
within the PDB_REDO project (www.cmbi.ru.nl/pdb_redo/) [4] to enable an automatic
correction of some of the problems. The interface to pdb-care is available at
www.glycosciences.de/tools/pdb-care2/.
References
1. Lütteke, T. (2009) Acta Cryst. D 47, 655-685.
2. Read, R.J., et al. (2011) Structure 19, 1395-1412.
3. Lütteke, T., von der Lieth, C.W. (2004) BMC Bioinformatics 5, 69.
4. Joosten, R.P., Joosten, K., Murshudov, G.N., Perrakis, A. (2011) Acta Cryst.D 68,
484-496.
PL4
33
The effect of flavonoids in mammary and epidermoid tumor cells with ErbB proteins overexpression
aM.M. MOCANU, bP. NAGY, aL. GEORGESCU, bT. VARADI, bD. SHRESTHA, aI. BARAN, aE. KATONA, b, c, *J. SZÖLLŐSI, aC. GANEA
aDepartment of Biophysics, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania, bDepartment of Biophysics and Cell Biology, Medical and Health Science Center,
University of Debrecen, Nagyerdei krt. 98, H-4032, Debrecen, Hungary, cMTA-DE Cell Biology and Signaling Research Group, University of Debrecen, Nagyerdei krt. 98, H-4032,
Debrecen, Hungary
Overexpression of ErbB receptors is a common feature in various cancers. These proteins
are associated with abnormality in signaling pathways linked to apoptosis, tumor cell
proliferation, and metastasis. Several lines of evidence documented the localization of ErbB
proteins in sphingolipids and cholesterol rich regions in the plasma membrane, also called as
lipid-rafts. However, such associations between ErbB proteins and lipid-rafts are entirely
dependent on the types of ErbB receptors. ErbB1 is either localized in lipid rafts in case of
overexpression in tumor cells or is induced to coalesce with lipid-rafts upon ligand binding. In
contrast, ErbB2, an orphan ligand receptor, has a constitutive active conformation which
makes the association of the receptor with lipid-rafts a feature of the tumor cells with ErbB2
overexpression [1, 2].
Despite the ongoing progress regarding the therapies in cancer, such therapies have led to
secondary effects and multidrug resistance which need solutions before therapies are
considered safe. An alternative would be to use natural compounds which are valuable
resources and easily available. Flavonoids, a group of secondary plant metabolites, are one
such compounds commonly found in the daily diet. (–)-Epigallocatechin-3-gallate (EGCG), a
catechin from the green tea and genistein, a natural isoflavone from soybeans has been proven
to exhibit anti-tumor activities, but their mechanisms of action remain to be elucidated.
EGCG displayed pro-apoptosis, anti-proliferative and anti-metastatic effects, whereas
genistein exhibited anti-phosphorylation activity on receptor tyrosin kinases in tumor cells [3,
4].
In our research, we tested the hypothesis that EGCG and genistein mediated pro-apoptotic
and anti-proliferative effects in mammary (SK-BR-3) and epidermoid (A-431) tumor cells
with ErbB protein overexpression are due to disruption of lipid-rafts and the dissipation of
receptors localized in these microdomains. Our results demonstrated that EGCG and to a
lesser extent genistein have pro-apoptotic/ necrotic effects and these features were augmented
by serum starvation of tumor cells. The effect of EGCG was cell line dependent and it was
mediated through a 67 kDa laminin receptor (67LR) on both tumor cell lines. Interestingly,
genistein, but not EGCG, had pronounced effect on cell cycle progression, exhibiting G2/M
PL5
34
phase arrest of tumor cell lines. In addition EGCG, but not genistein, was able to
downregulate and internalize ErbB proteins from the surface of mammary and epidermoid
tumor cells. The downregulation of ErbB proteins was demonstrated by both flow cytometry
and confocal microscopy techniques. EGCG did not interfere with the binding of fluorescent
EGF thus the effect did not seem to be mediated through EGF binding sites in ErbB1
receptors. Instead, reduction in GM1 positive areas and in cluster sizes of GFP-
glycosylphosphatidylinositol (GPI) anchored proteins, two markers used for lipid-raft
labeling, was observed in the plasma membrane due to EGCG treatment.
In conclusion, we demonstrated that the effect of EGCG, on cell death was mediated by its
interaction with 67LR at the plasma membrane level in tumor cells with ErbB proteins
overexpression. At the same time, our data suggest that EGCG acts via mechanisms related
to lipid-raft disruption, but not by virtue of binding to ErbB1 proteins. The present mechanism
of action in case of EGCG could be additionally utilized to develop further therapeutic
approaches against cancer disease.
Acknowledgement. This work was supported by the projects: The grants from the Romanian
National Authority for Scientific Research CNCS–UEFISCDI, projects number PN-II-RU-TE-
2011-3-0204, PNII-IDEI-PCE-2011-3-0800 and VEGA 0181, APVV SK-RO-0016-12, SK-
RO-0016-12, APVV-0171-10, 0526-11, ESF 26220120021, 26220220005.
References
1. Hynes, N.E., Lane, H.A. (2005) Nat. Rev. Cancer 5, 341-54.
2. Nagy, P., Claus, J., Jovin, T.M., Arndt-Jovin, D.J. (2010) PNAS 107, 16524-16529.
3. Yang, C.S., Wang, X., Lu, G., and Picinich, S.C. (2009) Nat. Rev. Cancer 9, 429–439.
4. Park, S.J., Kim, M.J., Kim, Y.K., Kim, S.M., Park, J.Y., Myoung, H. (2010). Cancer
Lett. 292,54-63.
35
Targeting Fas-mediated Apoptosis Through Human Herpesvirus 8 Oncoprotein K1-derived Peptides
aZ. BERKOVA, aS. WANG, bU. DANILUK, aC. KERROS, aJ.F. WISE, aF. SAMANIEGO
aDepartment of Lymphoma and Myeloma, The University of Texas - MD Anderson Cancer Center, Houston, TX; bDepartment of Pediatrics, Gastroenterology and Allergology,
Medical University of Bialystok, Bialystok, Poland
Human herpesvirus 8 (HHV-8), known as Kaposi’s sarcoma-associated herpesvirus
(KSHV), is causally associated also with primary effusion lymphoma and some cases of
Castleman disease.
The transmembrane protein K1 of HHV-8 contains an extracellular immunoglobulin-like (Ig-
like) domain and a cytoplasmic immunoreceptor tyrosine-based activation motif (ITAM). The
ITAM of K1 was shown previously to be involved in a constitutive activation of nuclear
factor kappa B (NF-kB). We have shown that the Ig-like domain of K1 binds Fas and
prevents binding of agonistic antibodies and Fas ligand (FasL) to Fas receptor and thus,
prevents activation of Fas-mediated apoptotic signaling [1].
Fas apoptotic signaling is crucial for the proper development of B-cells. A long-term
expression of K1 in transgenic mice, driven by a ubiquitous SV40 promoter, lead to
development of lymphoid hyperplasia and lymphomas [2]. The phenotype of transgenic
animals resembled the fasfl/lpr CD-19-cre mice with specific inactivation of Fas in B-cells.
Splenocytes of K1 transgenic mice were resistant to Fas-mediated apoptosis when compared
to K1-negative littermates. In addition, K1 transgenic mice were protected from a lethal
challenge with Fas agonistic antibody. Hydrodynamic expression of K1-wt, K1del-Ig, and
K1del-ITAM mutants revealed that this protection was mediated by Ig-like domain of K1 -
mice expressing K1 lacking ITAM domain were protected from Fas lethal challenge whereas
mice expressing K1 lacking Ig-like domain were not [1].
To sensitize K1-expressing cells to Fas-mediated apoptosis, we designed and tested synthetic
peptides derived from the Ig-like domain of K1 [3]. One of the tested peptides killed
lymphoma-derived cell lines, independently of K1 expression, without a significant effect on
the B-cells isolated from healthy donors. Surprisingly, this peptide killed also Daudi cells that
do not express Fas receptor on their surface.
Members of the tumor necrosis factor receptor (TNFR) family and their ligands, such as TNF
and FasL, share a significant degree of structural similarity allowing a certain degree of ligand
promiscuity. Thus, our peptide, originally designed to target Fas receptor, was also able to
bind and activate TNF receptor.
The structural features of the predicted Ig-domain reveal a unique feature of the peptide; a
loop (centered at conserved glycine residue) linking 2 beta sheets. A truncated versions of the
PL6
36
peptide, representing the first beta sheet and the loop and the second beta sheet, respectively,
lacked cell killing properties. On the other hand, a TCR-derived peptide sharing 5 structure-
defining residues with our peptide also showed some cell-killing activity in vitro and in vivo,
suggesting that the biological effect of our peptide might be related to its structure. Seemingly
contradictory effects of the whole Ig-like domain within K1 protein and our peptide on Fas
signaling may also be explained by the structure-function relationship.
Acknowledgement. This work was supported by the research grants from the American
Cancer Society (MRSG-10-052-01-LIB to ZB), the National Institutes of Health (CA1206173,
CA153170, CA158692, and DK091490 to FS), and the Leukemia & Lymphoma Society
(R6132-06 and R6187-09 to FS).
References
1. Berkova, Z., Wang, S., Wise, J.F., Maeng, H., Ji, Y., and Samaniego, F. (2009) J.
Nat.l Cancer Inst. 101, 399-411.
2. Berkova, Z., Wang, S., Jung, J., Samaniego, F. (2007) Blood 110, 2290.
3. Daniluk, U., Kerros, C., Tao, R-H., Wise, J.F., Ao, X., Berkova, Z., Samaniego, F.
(2012) J. Exp. Clin. Cancer Res. 31, 69-80.
37
Modern Trends in Biomolecular Simulations
J. HRITZ
Department of Structural Biology, Masaryk University, CEITEC, Brno, Czech Republic
For deeper understanding of biological processes in a cell, calculation of binding
affinities of protein-ligand, protein-protein interactions and induced conformational changes
is of vital importance. Biomolecular simulations are very useful tools for the rationalization of
experimental findings and for directing the future experiments. Computational simulations
offer atomic and intra-atomic energy contributions at a femtosecond timescale that are usually
beyond experimental means. Relative free energy differences (ΔG) govern important
properties such as binding affinities, populations of different conformers, protein stability,
solubility, etc. The efficient and accurate calculation of ΔG thus represents the holy grail in
thermodynamically oriented computational chemistry. Our recent developments will be
presented for the case of C8-substituted GTP analogs binding to the FtsZ protein.
The FtsZ protein plays a central role in bacterial cell division. Several C8-
substituted GTP analogs are known to inhibit the polymerization of FtsZ proteins by
competing with the natural ligand, GTP, at the same binding site. C8-substituted nucleotides
exhibit high energy barriers between the anti and syn conformations of the base and therefore
represent challenging targets for free energy calculations due to sampling limitations of
conformational space.[1] We tackled this problem and found a highly efficient way for
calculating the relative free energies of FtsZ-bound and free nucleotide in explicit water
solvent using one-step (OS) and enhanced sampling OS (ES-OS) perturbation methods.[2]
The calculated values of the relative binding affinities agree well with the available
experimental data.
The main contribution to the calculated binding affinities arises from conformational
restriction of the ligands. It is known that in water the dihedral angle distributions around the
glycosidic bond (dihedral angle ) for these compounds is highly variable and depend on
physico-chemical properties of the C8 substituent.(Figure 1) However, when bound to the
FtsZ protein, only negligible influences on the dihedral angle distributions are seen and all
angles reside in the narrow region of for the compounds investigated here.[3] The
corresponding ensemble averaged 3J(C4,H1’) coupling constants were calculated to be
2.95±0.1Hz and the conformational selection of the GTP analogues upon binding was
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38
Figure 1. Normalized dihedral angle ( ) distributions for 8-substituted analogs of GTP as
calculated by ES-OS in water environment (solid lines) and by OS in bound state to FtsZ
protein (dashed lines). Ensemble averages of 3J(C4,H1’) values in Hz are listed in the
legend above individual line symbols. Anti and syn conformational regions are indicated by
dotted lines. Note that the dihedral angle in the protein simulations remains in the region
-140°, -90° .
quantified from the corresponding populations.(Figure 1) The calculated free energy values
follow the same trend as the relative binding affinities of the nucleotides to the FtsZ
protein.[3]
Acknowledgment. This work was supported by the International Outgoing Fellowship of the
European Community program Support for Training and Career Development of Researchers
(Marie Curie), under contract no. PIOF-GA-2009-235902.
References
1. Hritz, J., Oostenbrink, C. (2008) J. Chem. Phys. 128, 144121.
2. Hritz, J., Oostenbrink, C. (2009) J. Phys. Chem. B 113, 12711-12720.
3. Hritz, J., Läppchen, T., Oostenbrink, C. (2010) Eur. Biophys. J. 39, 1573-1580.
39
Quantitative Analysis of Scanning Tunneling Microscopy Images of Mixed Ligand Functionalized Nanoparticles
aF. BISCARINI, bQ.K. ONG, cC. ALBONETTI, dF. LISCIO, eM. LONGOBARDI, fK. S. MALI, gA. CIESIELSKI, bJ. REGUERA, eC. RENNER, fS. DE FEYTER,
gP. SAMORI, bF. STELLACCI
aDip. Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 183, 41125 Modena, Italy bInstitute of Materials, École Politechnique Fédérale de Lausanne,
Lausanne, Switzerland cConsiglio Nazionale delle Ricerche, Istituto per lo Studio dei Materiali Nanostrutturati (CNR-ISMN), Via P. Gobetti 101, I-40129 Bologna, Italy
dConsiglio Nazionale delle Ricerche, Istituto di Microelettronica e Microsistemi (CNR-IMM), Via P. Gobetti 101, I-40129 Bologna, Italy eDepartment of Condensed Matter Physics,
NCCR Materials with Novel Electronic Properties, University of Geneva, 24 Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland fDepartment of Chemistry, Division of Molecular
Imaging and Photonics, KU Leuven - University of Leuven, Celestijnenlaan 200F, 3001 Leuven, Belgium gLaboratoire de Nanochimie, ISIS & icFRC, Université de Strasbourg
& CNRS, 8 allée Gaspard Monge, 67000 Strasbourg, France
Ligand-protected gold nanoparticles exhibit large local curvatures, features rapidly
varying over small scales, and chemical heterogeneity. Their imaging by Scanning Tunneling
Microscopy (STM) can, in principle, provide direct information on the architecture of their
ligand shell, yet STM images require laborious analysis and are challenging to be interpreted.
Here, we report a straightforward, robust and rigorous method for the quantitative analysis of
the multiscale features contained in STM images of samples consisting of functionalized Au
nanoparticles deposited onto Au/mica. The method relies on the analysis of the topographical
power spectral density (PSD), and allows us to extract the characteristic length scales of
the features exhibited by nanoparticles in STM images. For the mixed-ligand protected Au
nanoparticles analyzed here the characteristic length scale is 1.2±0.1 nm, whereas for the
homoligand Au NPs this scale is 0.75±0.05 nm. These length scales represent spatial
correlations independent of scanning parameters, and hence the features in the PSD can be
ascribed to a fingerprint of the STM contrast of ligand-protected nanoparticles. PSD spectra
from images recorded at different laboratories using different microscopes and operators can
be overlapped across most of the frequency range, proving that the features in the STM
images of nanoparticles can be compared and reproduced.
PL8
40
Mutations in proteolytic side of human mitochondrial Lon protease uncover the direct
connection between proteolytic and ATPase domains
aL’. AMBRO, aV. PEVALA, aJ. BAUER, aG. ONDROVIČOVÁ, a,bE. KUTEJOVÁ
aDepartment of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak
Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovakia bInstitute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, CZ-
142 20 Prague 4, Czech Republic
ATP-dependent protease Lon is present in all kingdoms of life [1, 2]. In eukaryotes it
functions in mitochondria, peroxisomes and chloroplasts. Mitochondrial Lon proteases
possess not only proteolytic activity but also function as chaperones and are important for
mtDNA stability [1, 2, 4, 5]. Lon proteases catalyze two reactions in a linked manner: ATP to
ADP and peptide bond hydrolysis. ATP hydrolysis is needed for the digestion of larger, intact
proteins, but small peptides can be cleaved without hydrolysis of ATP but requires only ATP
binding [1, 3]. Presence of protein substrates accelerated both ATPase and peptidase activity.
Moreover, presence of ATP is important for stabilization of this protease [6]. Lon protease is
highly flexible and needs of all three domains – N-terminus, ATPase and proteolytic - for its
proper function. This makes teasing out the physical mechanism by which all these events
take place very difficult. We have tried to take advantage of analysis of crystal structure of
proteolytic domain of human Lon protease [7] for design mutations that enable better
understand its mechanism of action. A loop containing residues 891–894 lies to one side of
the proteolytic active site of human Lon. Two of its residues, Gly-893 and Gly-894, are
universally conserved across all Lon homologues. The presence of these two conserved
glycine residues strongly suggests that the flexibility of this loop is important for its function.
To explore its importance, we prepared a series of mutants of these two residues and
evaluated the biochemical characteristics of the resulting mutants. We also explored the
effects of mutations in certain conserved sites adjacent to this loop, namely Trp-770 and Thr-
880. Our results show that two of these mutants, G893P and G893P-G894A (along with a
K529A control mutant) exhibited peptidase activity, but undetectable protease activity. On the
other hand, a G894P mutant supported a low level of protease activity, but exhibited almost
no peptidase activity while three others, G894S, T880V, and W770A, had less than 10% of
the wild-type peptidase activity, but nearly wild-type levels of protease activity (two other
mutants exhibited no peptidase activity, but also displayed low, but detectible levels of
protease activity). In addition, we also found that under some conditions, the G894A, G894S,
and G894P mutants failed to show the expected peptidase stimulation in the presence of β-
PL9
41
casein; these three mutants also showed elevated levels of basal ATPase activity, but no
increase in the stimulated ATPase activity (the G894P mutant was ≈15% lower). The double
glycine mutants typically behaved much like the corresponding Gly-894 mutant, suggesting
that this glycine has a dominant effect on the position of the loop. These results suggest that
the flexibility of this loop is important for proper functioning of the Lon protease and
peptidase activity. The effects of the Gly-894 mutants on the ATPase activity and on the
stimulation of both activities by β-casein suggest that this loop is somehow involved in the
process by which both the ATPase and peptidase activities are regulated.
Acknowledgement. This work was supported by the research grants from the Slovak Grant
Agency VEGA No. 2/0122/11 and the Slovak Research and Development Agency Grant No.
APVV-0123-10.
References
1. Ondrovičová, G., Hlinková, V., Bauer, J., Kutejová, E. (2008) in ATP-Dependent
Proteases (Kutejová E., Ed.) Research Signpost, T.C. 37/661(2), Fort P.O.,
Trivandrum-695 023, Kerala, India, pp. 1–40.
2. Ambro, L., Pevala, V., Bauer, J., Kutejová, E. (2012) J. Struct. Biol. 179, 181-192.
3. Ondrovičová, G., Liu, T., Singh, K., Tian, B., Li, H., Gakh, O., Perečko, D., Janata, J.,
Granot, Z., Orly, J., Kutejová, E., Suzuki C.K. (2005) J. Biol. Chem. 280, 25103–
25110.
4. Liu, T., Lu, B., Lee, I., Ondrovičová, G., Kutejová, E., Suzuki, C.K. (2004) J. Biol.
Chem. 279, 13902–13910.
5. Lu, B., Yadav, S., Liu, T., Tian, B., Pukszta, S., Villaluna, N., Kutejová, E., Newlon,
C.S., Santos, J.H., Suzuki, C.K. (2007) J. Biol. Chem. 282, 17363–17374.
6. Stahlberg, H., Kutejová, E., Suda, K., Wolpensinger, B., Lustig, A., Schatz, G., Engel,
A., Suzuki, C.K. (1999) Proc. Natl. Acad. Sci. USA 96, 6787–6790.
7. García-Nafría, J., Ondrovičová, G., Blagova, E., Levdikov, V.M., Bauer, J.A., Suzuki,
C.K., Kutejová, E., Wilkinson, A.J., Wilson, K.S. (2010) Protein Sci. 19, 987–999.
42
Directed evolution of new protein therapeutics – DARPins
E. SEDLÁK
Department of Biochemistry, P.J.Šafárik University, Košice, Slovakia,
Institute of interdisciplinary biosciences, P.J.Šafárik University, Košice, Slovakia
To date, none of the approximately 60 anticancer drugs used in conventional
chemotherapy exhibits a pronounced selective uptake in a tumor tissue and generally only a
very small fraction of the administered dose reaches the tumor site. The problem of low-
molecular-weight anticancer therapeutics is that due to an unfavorable biodistribution and a
lack of accumulation in tumor tissue, they exhibit poor therapeutic indices and tumor
remissions are often not achieved. Moreover, many undesirable side effects sometimes
encompass cancer chemotherapy. For these reasons, the targeted drug delivery belongs
between the most important field in the oncology research. In other words, any means of
transporting and delivering anticancer drugs in higher concentrations to the tumor over a long
period of time whilst sparing healthy tissues is a step to a more effective cancer therapy.
Recognition of molecular diversity of cell surface proteomes in disease is essential for
the development of targeted therapies.
Progress in targeted therapeutics requires
establishing effective approaches for high-
throughput identification of agents specific
for clinically relevant cell surface markers.
Over the past ~15 years, a number of
platform strategies have been
developed to screen polypeptide
libraries for ligands targeting specific
receptors. These so called display
technologies are indispensable part of directed evolution methods and include such methods
as phage display, yeast display, and ribosome display.
Antibodies are, currently, a major driver of the pharmaceutical industry with several
blockbuster drugs on the market and many more in clinical development. The key for this
success is that antibodies can be selected to bind to virtually any given target with high
affinity and specificity, thereby displaying neutralizing or cytotoxic functions with very
limited side effects (Schrama et al. 2006). Antibodies, however, suffer from clear limitations:
they are expensive to produce, difficult to formulate, show low tissue penetration, feature a
complex architecture, bind their target bivalently, and their commercial use is often blocked
PL10
Figure 1. Ribbon diagram of DARPin with three internal repeats/modules. The terminal and internal repeats are in magenta and green, respectively. Taken from Merz et al. (2008).
43
by intellectual property restrictions. To address some of the shortcomings of antibodies,
several alternative non-immunoglobulin based scaffolds have been brought into attention of
researchers (Binz et al. 2005). Arguably, the most promising group of proteins with
alternative scaffold is designed ankyrin repeat proteins (DARPins).
DARPins show extreme potential for numerous therapeutic applications (Stumpp et al.
2008, Tamaskovic et al. 2012). Advantages of DARPins in comparison with antibodies are:
(i) high tissue penetration, (ii) absence of effector function, (iii) adjustable pharmacokinetics,
(iv) allosteric inhibitions, and (v) new administration routes.
Acknowledgement. This work was supported by grants obtained by laboratory of Prof. Dr.
Andreas Plückthun.
References
1. Schrama, D., Reisfeld, R.A., Becker, J.C. (2006) Nat. Rev. Drug Discov. 5, 147-159.
2. Binz, H.K., Amstutz, P., Plückthun, A. (2005) Nat. Biotechnol. 23, 1257-1268.
3. Stumpp, M.T., Binz, H.K., Amstutz, P. (2008) Drug Discov., Today 13, 695-701.
4. Tamaskovic, R., Simon, M., Stefan, N., Schwill, M., Plückthun, A. (2012) Methods
Enzymol. 503, 101-134.
5. Merz, T., Wetzel, S.K., Firbank, S., Plückthun, A., Grütter, M.G., Mittl, P.R.E., (2008)
J. Mol. Biol. 376, 232–240.
44
Force as a Single Molecule Probe of Multidimensional Protein Energy Landscapes
aG. ŽOLDÁK, aB. PELZ, a,b M. RIEF
aPhysik Department E22, Technische Universität München, 85748 Garching, Germany; bMunich Center for Integrated Protein Science, 81377 München, Germany
Proteins are fascinating molecules involved in nearly all biological processes acting as
metabolic enzymes or required as scaffolds for cell. The variety of function is possible due to
the large repertoire of three-dimensional structures they can achieve during the process of
protein folding. Protein folding is a spontaneous process and all information is encoded solely
by the amino acids sequence. Although much of what we know about protein folding has been
obtained from ensemble experiments, single
molecule force spectroscopy (SMFS) has
evolved into an indispensable tool for studying
folding and dynamics of proteins in real time
[1]. In SMFS, force acts as a denaturant along
the pulling axis. The combination of the
force/distance information makes single
molecule force spectroscopy unique for the
study of protein folding process. Atomic force
microscopy (AFM) and optical tweezers (laser
traps) are two key techniques for SMFS.
Because of the high sensitivity and range
accessibility at low forces (1-50 pN), optical
tweezers are better suited for real-time
observation of protein folding /unfolding
under load.
In my talk, I will give a short
overview of experimental techniques and
kinetic models used in single molecule force spectroscopy. Next I will focus on the folding
dynamics of the DnaK chaperone.
DnaK is an Hsp70 chaperone of E. coli which consists of an N-terminal nucleotide
binding domain (NBD) and a C-terminal substrate binding domain (SBD). Central to the
biological function of this chaperone is an allosteric coupling between functional states and
ligand binding. A single charge reversal mutation in the linker that connects both domains
abolishes the allosteric communication and impairs in vivo function of this heat shock protein
[2]. Peptides or client substrate proteins bind to the SBD depending on the nucleotide status of
PL11
Fig. 1. (A) Sketch of a dual trap with differential detection and a construct used for optical trapping with DnaK connected to DNA handles attached to functionalized silica beads. The terminal cysteine residues are shown as dots. The construct for optical trapping is schematically shown below. (B) Constant distance time trace of the folding/unfolding of the helical lid. The fluctuations are suddenly stopped after 62 s and the protein is trapped in the quenched state (likely post-hydrolysis state ADP+Pi). (C) The SBD structures in the open and the closed state were taken from 2kho.pdb and 4b9q.pdb.
45
the NBD. In the ADP state, the protein substrates bind to the SBD with slow dynamics and
high affinity whereas in the ATP state, substrate affinity is decreased several fold. Many
biophysical/biochemical experiments have provided insight into this allosteric effect which is
accompanied by the large conformational changes triggered by the binding of ATP [3]. In
order to observe such large conformational change in real-time at the single molecule level,
we have prepared several variants of the full-length DnaK and its individual domains for
single molecule force spectroscopy. Stretching curves of the single molecule DnaK displayed
two unfolding events corresponding to the unfolding of the SBD and NBD. Their mechanical
stabilities and folding/unfolding kinetics in the absence of ligands resemble very closely the
properties of the isolated domains. In the presence of Mg2+ATP, however, the folding
dynamics of the helical lid of SBD increases dramatically which was not seen before in the
individual domains (Fig.1b). The fast folding/unfolding kinetics of the lid subdomain could be
ascribed to the disruption of the otherwise mechanically stable interface between the lid and
-subdomain of the SBD (Fig.1c). Real-time observation of force fluctuations of the lid
conformation provides a sensitive single-molecule monitor of the ATP state of DnaK.
Acknowledgements. We thank the members of our group for suggestions and comments. This
work was supported by the SFB 863/A2 and ZO 291/1-1 projects of Deutsche
Forschungsgemeinschaft
References
1. Zoldák G., and Rief, M. (2013) Curr. Opin. Struct. Biol. 23, 48-57.
2. Vogel, M., Mayer, M.P., and Bukau, B. (2006) J. Biol. Chem. 281, 38705-38711.
3. Kityk, R., Kopp, J., Sinning, I., and Mayer, M.P. (2012) Mol. Cell. 48, 863-874.
46
Rich Phase Behavior of Protein Solutions Induced by Multivalent Ions:
Reentrant Condensation and Liquid-Liquid Phase Separation
F. ROOSEN-RUNGE, M. WOLF, A. SAUTER, F. ZHANG, F. SCHREIBER
Institute for Applied Physics, University of Tübingen, Germany
Multiple environmental factors such as temperature, pH and salt ions affect the
stability and phase behavior of protein solutions. Here, we present a rich phase diagram for
globular proteins in the presence of multivalent cations. For a broad range of acidic globular
proteins and multivalent cations, a reentrant condensation is found, i.e. protein solutions are
clear and stable at low and high salt concentration, while they phase separate and precipitate
at intermediate salt concentration [1,2]. Importantly, the phase behavior can be consistently
rationalized with protein interactions that are governed by the ion-protein interaction. In
particular, the reentrant behavior is related to an inversion of the protein charge due to binding
of multivalent counterions to surface groups [1,3]. The ion binding is affected by both the pH
and monovalent salt content of the solution [3,4]. While a counterion-induced increase of
attraction seems essential for the low-salt transition, the high-salt transition is dominated by
charge stabilization of the protein solution [4].
The phase separated regime at intermediate salt concentration contains both cluster and
amorphous aggregates as well as a metastable closed-loop liquid-liquid phase coexistence,
being consistent with theoretical predictions [5]. This observation opens the field for
systematic studies of structure formation and nucleation in protein solutions.
References
1. Zhang, F., Skoda, M.W.A., et al. (2008) Phys. Rev. Lett. 101, 148101.
2. Zhang, F., Weggler, S.,et al. (2010) Proteins 78, 3450-3457.
3. Roosen-Runge, F., Heck, B.S., et al. (2013) J. Phys. Chem. B 117, 5777-5787.
4. Jordan, E., Roosen-Runge, F., et al. in preparation.
5. Zhang, F., Roth, R., et al. (2012) Soft Matter 8, 1313-1316.
PL12
47
Understanding Charge-Driven Protein Aggregation and Crystallization
F. ZHANG, A. SAUTER, F. ROOSEN-RUNGE, F. SCHREIBER
Institute for Applied Physics, University of Tübingen, Germany
Multivalent counterions have been found to induce a rich phase behavior of protein
solutions, including a reentrant condensation and a metastable closed-loop liquid-liquid phase
separation (LLPS) [1]. Interestingly, the LLPS can be used to optimize conditions for protein
crystallization and play a role in its prediction [2,3]. Different crystallization processes can be
identified throughout the phase diagram, leading to differing crystal morphologies [1,4].
Interestingly, high-resolution crystal structures show that the trivalent ion Yttrium acts as an
ion-bridge between two protein molecules in the crystal [4]. Crystallization close to the first
transition seems to follow the classical one-step nucleation process directly from a
homogeneous solution. Under other conditions around the LLPS, crystallization rather
follows a two-step nucleation process with an intermediate precursor state. Protein cluster
seem to play an important role for the nucleation [5], while crystallization inside dense
droplets is seldom, although preferable from the viewpoint of classical nucleation theory,
suggesting that arrested phase behavior obstructs reordering to a crystal nucleus [5]. Evidence
for the formation of protein clusters upon addition of multivalent ions is provided by both
small angle scattering and dynamic light scattering [5,6,7]. We also attempt to elucidate the
role of different specific ions in this process.
References
1. Zhang, F., Roth, R., et al. (2012) Soft Matter 8, 1313-1316.
2. ten Wolde, P. R., Frenkel, D. (1997) Science 277, 1975-1978.
3. Vekilov, P.G. (2004) Cryst. Growth Des. 4, 671-685.
4. Zhang, F., Zocher, G., et al. (2011) J. Appl. Cryst. 44, 755-762.
5. Zhang, F., Roosen-Runge, F., et al. (2012) Faraday Disc. 159, 313-325.
6. Soraruf, D., Roosen-Runge F., et al. in preparation.
7. Sauter, A., Zhang, F., et al. in preparation.
PL13
48
Towards increased selectivity of cancer treatment by Photodynamic therapy: Development of selective nano-delivery system and detection of therapeutically active
form of drugs in cell
P. MISKOVSKY
Department of Biophysics and Center for Interdisciplinary Biosciences, P. J. Safarik University, Kosice, Slovakia
The efficacy of photosensitier (pts) in inducing either apoptosis or necrosis after
photodynamic action critically depends upon different parameters including in particular: i)
selective pts accumulation in tumor cells and, ii) dynamics of sub-cellular distribution of
therapeutically active form of drug.
Low-density lipoproteins (LDL) play a key role in the delivery of hydrophobic pts to tumor
cells in PDT, including hypericin (Hyp). Construction and characterization of new LDL-based
selective nano-delivery system is discussed in the presentation.
Dynamics of organelle specific sub-cellular redistribution of Hyp plays important role in its
photodynamic activity. In this presentation, we report the development and characterization of
micrometer-sized dielectric beads with nano-metal structures attached to their surface. The
metalized beads are sufficiently transparent enabling optical trapping while the presence of
metal nano-islands provided the SERS. This highly efficient probe can be placed and scanned
with nano-metric accuracy in living cells. Application of such optical nano-sensors is
demonstrated on: i) detection of low quantities of photoactive drug emodine, (ii) study the
kinetics of drug diffusion through the cellular membrane towards specific cell organelles.
Acknowledgement. This work was supported by the (i) Agency of the Ministry of Education of
Slovak Republic for the Structural funds of the European Union, Operational program
Research and Development (Contracts: Doctorand, ITMS code: 26110230013) (20%),
NanoBioSens ITMS code: 26220220107 (40%), SEPO II ITMS code: 26220120039 (20%)
and CEVA ITMS code: 26220120040 (20%), (ii) Slovak Research and Development Agency
under the contract s APVV-0242-11 and (iii) Scientific Grant Agency of the Ministry of
Education of Slovak Republic under the grant VEGA No., 1/1246/12, and FP7 EU project
CELIM 316310.
PL14
49
Role of Membrane Fluidity in Modulation of Function of Subcellular Membrane Systems in Health and Disease: Relevance to Heart Sarcolemma and Mitochondria in
Hypoxia, Ischemia, Diabetes, Hypertension, Irradiation and seasonal changes
aA. ZIEGELHÖFFER, aM. FERKO, bI. WACZULÍKOVÁ, aJ. MUJKOŠOVÁ, aM. MURÁRIKOVÁ, cB. ZIEGELHÖFFER, aT. RAVINGEROVÁ, aJ. SLEZÁK
aInstitute for Heart Research, Centre of Excelence SAS NOREG, Bratislava, SR bDepartment of Nuclear Physics and Biophysics, Division of Biomedical Physics, Faculty of Mathematics,
Physics & Informatics, Comenius University, Bratislava, SR cHeart Center Leipzig, University of Leipzig, Leipzig, Germany
Background: Membrane fluidity (MF) represents a widely recognized biophysical variable
providing information about structural organization of the subcellular membranes which
exhibit the characteristics of liquid crystals. The term „fluidity“ reflects in this case the
tightness in packing of acyl parts of membrane phosholipid molecules, a feature that may
modulate the molecular mobility and via that also the sensitivity and reactivity of membrane-
bound transporters, receptors and enzyme systems. It is much evidence available that diverse
impulses targeting the membranes may increase or decrease MF. Chemical compounds,
including pharmaceuticals, may immerse into the membrane, depending on hydrophobicity of
their molecules and may modulate MF. Moreover, spontaneous or evoked changes in the
chemical composition of membranes may also alter the MF. Specificity in composition of
single membranes, high sensitivity of MF to various influences as well as the differences in
time course of the development of changes induced by the respective impulse represent the
reason why the physiological range of changes in MF was not yet demarcated sufficiently.
Aims of the study: In present study we are aimed to demonstrate the intimate coherence
between changes in MF occurring in cardiac sarcolemma and mitochondria in reponse to
physiological impulses and diverset pathologies [1, 2].
Methodical aspects: Membrane dynamics covers virtually all modes of molecular motion in
the membrane such as CH2-bond vibratios, lipid rotation along the long axis, lateral
movements of lipids and proteins and rotation of proteins, etc. Study of these parameters
requests carefully selected methods particularly from the point of view of proper ratio
between the speed of molecular movements and the speed of their detection. Taking all this in
account in our sudy of MF we applied the method off the steady-state DPH fluorescence
anisotropy [3-4].
Results and Discussion. In last 20 years the role of MF became investigated more intensively
also in subcellular membranes of the myocardium. Results confirmed that an increase in MF
is almost unconditionally accompanied with considerable increase in activities of membrane-
bound enzymes, particularly of the ATPases of cardiac sarcolemma and mitochondria [1, 5].
This corollary is also valid vice versa; decrease in MF yields in drop off of membrane
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activities. This was approved to act not only whithin the range of physiological impulses [1],
but also in hypoxia, ischemia, hypertension [2] and irradiation as well as in streptozotocin-
diabetes [2-5]. Investigation of the relationship between MF and the activities of membrane-
embedded systems also revealed several new mechanisms of endogenous protection of the
heart against hypoxia, ischemia, Ca2+-overload and diabetes [2-5]. Special importance seem to
have the control over entry of excess calcium into the cardiomyocytes in diabetic and post-
ichemic reperfused hearts provided by decreased MF of the sarcolemma [6] and the increase
in mitochondrial MF associated with enhanced formation of mitochondrial energy transition
pores that occurs in heart cells in consequence to strong Ca2+-signaling to mitochondria [7].
Acknowledgement. Supported by Grants VEGA 2/0101/12; 2/0054/11;2/0207/11 and APVV
0102-11; 0241-11
References
1. Mujkošová, J., Ferko, M., Humeník, P., Waczulíková, I., Ziegelhöffer, A. (2008)
Physiol. Res. 57, (Suppl.2), S75-S82.
2. Ziegelhöffer, A., Waczulíková, I., Ferko, M., Šikurová, L., Mujkošová, J.,
Ravingerová, T. (2012) Physiol. Res. 61, (Suppl 2), S11-S21.
3. Waczulíková, I., Habodászová, D., Cagalínec, M., Ferko, M., Uličná, O., Mateašík,
A., Šikurová, L., Ziegelhöffer, A. (2007) Can. J. Physiol. Pharmacol. 85, 372-381.
4. Waczulíková I., Cagalinec, M., Uličná, O., Slezák, P., Ziegelhöffer, A. (2010)
Physiol. Res. 59, (Suppl. 1), S9-S17.
5. Ferko, M., Gvozdjaková, A., Mujkošová, J., Kucharská, J., Waczulíková, I., Styk, J.,
Ravingerová, T., Ziegelhöffer-Mihalovičová B., Ziegelhöffer, A. (2006) Gen.
Physiol. Biophys. 25, 397-413.
6. Ziegelhöffer, A., Ravingerová, T., Styk, J., Šeboková, J., Waczulíková, I., Breier, A.,
Džurba, A., Volkovová, K., Čársky, J., Turecký, L. (1997) Mol. Cell. Biochem.176,
191-198.
7. Ziegelhöffer, A., Ravingerová, T., Waczulíková, I., Čársky, J., Neckář, J.,
Ziegelhöffer-Mihalovičová, B., Styk, J. (2002) Ann. N Y Acad. Sci., 967, 463-468.
51
The Study of CE16 Acetyl Esterase from Fungus Hypocrea jecorina
aL. URBANIKOVA, aB. VIDOVA, aA. GODANY, bP.BIELY
aInstitute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta 21, 845 51 Bratislava, Slovak Republic bInstitute of Chemistry, Slovak Academy of Sciences, Dubravska
cesta 9, 945 38 Bratislava, Slovak Republic
Fungus Hypocrea jecorina (anamorph: Trichoderma reesei) secretes a cocktail of
cellulolytic enzymes during its growth on cellulose. Acetylesterase Aes1 showing the unique
structure and properties is one of the cocktail’s components. Based on its primary structure
new family of carbohydrate esterases CE16 has been organised in 2008 [1]. At present, the
family comprises 40 proteins, however, acetylesterase Aes1 is still the only characterised
member.
The enzyme is composed of 348 amino acid residues from which the first 19 form
a secretion signal peptide. The amino acid sequence contains four cysteines which probably
form two disulphide bridges. The molecular mass of the purified enzyme, 45 kDa, is larger
than the mass 37 kDa calculated from the mature polypeptide, due to enzyme glycosylation.
Six potential glycosylated sites can be found in its amino acid sequence. In contrast to
acetylesterases from the CE families 1, 4 and 5, which deacetylate xylopyranosides in
positions 2 and 3, CE16 acetylesterase prefers position 3 and 4 acetyl groups [2].
Highly purified recombinant enzyme Aes1 produced by H. jecorina Rut-C30 has been
crystallized and in spite of the protein glycosylation, the crystals were obtained. One complete
set of diffraction data was collected to 3.98 Å. After crystal annealing another data set was
collected to 2.98 Å resolution using synchrotron radiation source at EMBL, DESY, Hamburg.
Unfortunately, structure solution by molecular replacement method failed as the
acetylesterase primary structure is quite unique and there is no suitable model structure in
PDB. Isomorphous crystals prepared by soaking in the solution of NaBr did not diffract et al.
To make the future structure-function study of the enzyme easier, also synthetic gene
coding acetylesterase was prepared and the recombinant enzyme containing C-terminal His6
tag was produced in E. coli cells. The enzyme has been isolated using metallochelate
chromatography and its crystallization is under work.
In the interim, the enzyme has been studied bioinformatically. It has been found that
the studied acetylesterase belongs to group of serine hydrolase and is homologous to GDSY
(newly SNDH) lipases. Amino acids of the catalytic triad has been detected as well as other
amino acid residues which might be important in substrate binding and hydrolysis.
Acknowledgement. This work was supported by the research grants from the Slovak Grant
Agency VEGA 2/0189/11.
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References
1. Li, X-L., Skory, C., Cotta, M.A., Puchart, V., Biely, P. (2008) Appl. Environ.
Microbiol. 74, 7482-7489.
2. Uhliariková, I., Vršanská, M., McCleary, B.V., Biely, P. (2013) Biochim. Biophys.
Acta 1830, 3365-72.
53
Computer-aided drug design: basic concepts and applications to influenza virus
and Alzheimer’s disease
M. S. LI
Institute of Physics, Polish Academy of Sciences, Al. Lotnikow 32/46, 02-668 Warsaw, Poland
The basic concepts and methods used in the computer-aided drug design such as
docking, steered molecular dynamics (SMD) will be discussed. The main idea of using SMD
to screen out potential leads is that instead of binding free energy, the rupture force defined as
the maximum on the force-time/displacement profile, is employed as a score function. The
particular attention will be drawn to recent results obtained by SMD for top-leads for
influenza viruses.
According to the amyloid cascade hypothesis, the aggregation of amyloid beta peptides is
believed to be associated with the Alzheimer's disease. Therefore, one of possible therapeutic
approaches is to prevent fibril formation of these peptides. Applying the virtual screening to
data base of about 36000 compounds, derived from Eastern plants, we have found several
very promising leads for treatment of the Alzheimer's disease. The possibility of using the
derivatives of vitamin K3 as potential candidates for treating this disease will be explored.
Our in silico results are in line with the experimental ones. General structural properties of
ligands that may control their binding affinity will be discussed.
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Glassy behavior of proteins
C.-K. HU
Institute of Physics of Academia Sinica, Taipei 11529, Taiwan
It is pointed out that that the slow relaxation of a spin glass model [1] at low
temperatures can be slower than the critical slowing down of the Ising model [2] at the critical
temperature [3].
Molecular dynamics simulations of simple models for polymer chains [4] and experimental
studies of the native collagen fibril and the hemoglobin crystal [5] indicate that such systems
can show glassy behavior. It is pointed that glassy behaviors of polymer chains and proteins
are crucial for understanding why a biological system can maintain in a non-equilibrium state.
References
1. Dasgupta, C., Ma, S.-K., Hu, C.-K. (1979) Phys. Rev. B 20, 3837-3849.
2. Wang, F. G., Hu, C.-K. Phys. Rev. E
3. Hu, C.-K. (2013) AIP Conference Proceedings 1518, 541.
4. Ma, W.-J., Hu, C.-K. (2010) J. Phys. Soc. Jpn. 79, 024005 and 024006.
5. Gevorkian, S. G., Allahverdyan, A. E., Gevorgyan, D. S., Hu, C.-K. (2011) EPL 95,
23001 and unpublished data.
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SHORT COMMUNICATIONS
abstracts
57
Activation of Vertebrate Transglutaminases; What We Can Learn from Molecular Dynamics Simulations?
a,bI. KOMÁROMI, bA. FEKETE, bD. MUCS and a,bL. MUSZBEK
aHaemostasis, Thrombosis and Vascular Biology Research Group of the Hungarian Academy of Sciences bClinical Research Center, University of Debrecen, Medical and Health Science
Center, Debrecen, Hungary
Transglutaminases (protein-glutamine γ-glutamyltransferases, EC 2.3.2.13) are Ca2+
dependent enzymes[1]. Their main physiological role is to form inter- or intramolecular ε(γ-
glutamyl)-lysyl crosslinks. All but the human tissue transglutaminase (TG2) the vertebrate
transglutaminase structures have been resolved so far related to the inactive (“closed”)
conformation of the protein (Fig. 1A). The atomic resolution 3D structure for the “open”
conformation, fixed with substrate analog inhibitor [2], is available only for human TG2 (Fig.
1B). The difference between these two types of X-ray structures is surprisingly large.
Nevertheless, only the relative orientations of the constituting domains were changed while
the domains kept their substructures (Fig. 1.).
Because of the structural and functional similarity existing between the inactive
(“closed” conformation) vertebrate transglutaminases, significant similarity between their
“open” conformations can be assumed as well. It was the basis of the theoretical model for the
“open” conformation of FXIII-A2* we proposed [3].
While the whole conformational transition can be considered as „rare events” on the
time scale can be reached even on nowadays high-end supercomputers, these simulations can
provide us valuable data on the early events of conformational changes. Our main aim was to
extract information from molecular dynamics trajectory on how these large conformational
changes begin. Our aim was also to gain information on the binding mode of Ca2+ and to
reveal their role in triggering the large scale conformational transition.
SC1
Figure 1.: Structural alignment oftransglutaminases with known„closed” three-dimensional structures (A) and the „open”structure of human transglutaminase 2 (B). The constituting domains are marked.
58
1μs NPT molecular dynamics simulations using explicit solvent molecules and
periodic boundary condition have been carried out on the „closed” form of human TG2 in the
presence and in the absence of Ca2+ ions. The long range electrostatic forces were calculated
by means of the particle mesh Ewald protocol implemented in the GROMACS molecular
dynamics package. OPLS-AA/L force field was applied for the protein while for the solvent
molecules the TIP3P water model was used. The low frequency modes were extracted from
the covariance matrices which were constructed from the trajectories of dynamic simulations.
They represented motions along which the conformational changes are assumed to occur. The
most probable Ca2+ binding sites were proposed from simulation and they are in good
accordance with those ones which were obtained from systematic mutational experiments [4].
In addition to the primary Ca2+ binding sites were found experimentally and which were
found characteristic ones even from simulations, other, less characteristic Ca2+ binding sites
were obtained as well.
Acknowledgments: This work was partially supported by the Hungarian Scientific Research
Fund (OTKA K-106294), the Hungarian National Infrastructure Development Program
(grant: NIIF-1057) as well as TÁMOP-4.2.2.A-11/1/KONV-2012-0045 and TAMOP-4.2.2.C-
11/1/KONV-2012-0010 grants.
References
1. Iismaa, S.E., Mearns, B.M., Lorand, L., Graham, R.M. (2009) Physiol. Rev. 89, 991-
1023.
2. Pinkas, D.M., Strop, P., Brunger, A.T., Khosla, C. (2007) PLoS Biol. 5, e327.
3. Komaromi, I., Bagoly, Z., Muszbek, L. J. (2011) Thromb. Haemost. 9, 9-20.
4. Kiraly, R., Csosz, E., Kurtan, T., Antus, S., Szigeti, K., Simon-Vecsei, Z., Korponay-
Szabo, I.R., Keresztessy, Z., Fesus ,L. (2009) FEBS J. 276, 7083-96.
59
Factors of virulence of C. burnetii, the etiological agent of Q fever
a,b,cL. SKULTETY, aR. TOMAN, aG. FLOREZ-RAMIREZ
aInstitute of Virology, Slovak Academy of Sciences, Bratislava, Slovakia, bCentre of
Molecular Medicine, Slovak Academy of Sciences, Bratislava, Slovakia cInstitute of
Microbiology, Academy of Science of Czech Republic, Prague, Czech Republic
Coxiella burnetii, the etiological agent of Q fever, is a broadly occurring intracellular
parasite that can infect a wide range of hosts. Many reservoirs have been reported, including
mammals, birds, and arthropods (mainly ticks), but infectious aerosols produced by farm
animals and pets, including those from parturition products where the bacterium can be found
in high numbers as feces, milk, hides and wool, are the most frequent sources of human
infection [1]. The bacterium is responsible for an acute and potentially severe symptoms
characterized by pneumonitis, hepatitis, and a significant incidence of neurological
complications [2]. Because several clinical symptoms of Q fever in humans are similar to
those of commonly occurring infections, an unambiguous clinical diagnosis of the disease is
quite difficult. Various, mainly serological methods are currently used for a rapid and
sensitive diagnosis of the disease, but in numerous cases have yielded ambiguous results [3].
The bacterium is listed as a category B biological warfare agent. It is extremely resistant to
harsh environmental conditions such as heat, dryness, UV rays, and disinfectants. It
replicates within the phagolysosomal vacuoles of animal cells, primarily macrophages,
although recently the successful propagation on axenic medium has been reported [4].
During the last years, noticeable progress has been achieved in gaining a better understanding
of the role of two major outer membrane components – lipopolysaccharide (LPS) and proteins
in virulence of the bacterium. Detailed glycomic studies of the virulent phase I and avirulent
phase II variants of the Nine Mile RSA 493 and 439 strains of C. burnetii have brought
indispensable structural and functional information on their LPS. In addition, our
investigations have focused also on the identification of differentially expressed C. burnetii
proteins. The virulent phase I and avirulent phase II variants of C. burnetii were propagated in
embryonated hen eggs and then purified by centrifugation through Renografin gradients.
Total protein fractions were isolated from each phase and subjected to analysis by gel
electrophoresis plus tandem mass spectrometry. A total of 235 and 215 non-redundant
proteins were unambiguously identified from the phase I and II cells [5], respectively. Several
of the identified proteins are involved in the biosynthesis and metabolism of components of
the extracellular matrix. We have found forty-four proteins which were annotated as having
distinct roles in the pathogenesis or survival of C. burnetii within the harsh phagolysosomal
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environment. Finally, we propose that nine enzymes specifically involved with
lipopolysaccharide biosynthesis and metabolism, and that are distinctively present in phase I
cells, are virulence-associated proteins. Some of these proteins we suggest to be associated
with biosynthesis of the unique C. burnetii virulence factor, virenose.
Acknowledgement. Part of the cost of this study was supported by the following grants:
2/0156/11 of the Scientific Grant Agency of the Ministry of Education of the SR and SAS, the
26240120030, 26240220045 and 26240220062 supported by the Research & Development
Operational Programme funded by the ERDF as well as the IMIC institutional research
concept RVO61388971.
References
1. Angelakis, E., Raoult, D. (2010) Veterinary Microbiology 140, 297-309.
2. Brouqui, P., Marrie, T., Raoult, D. (2007) Coxiella.
3. Slaba, K., Skultety, L., Toman, R. (2005) Acta Virologica 49, 123-127.
4. Omsland, A., Cockrell, D. C., Howe, D., Fischer, E. R., et al. (2009) Proc. Natl. Acad.
Sci. USA 106, 4430-4434.
5. Skultety, L., Hajduch, M., Flores-Ramirez, G., Miernyk, J. A., et al. (2011) Journal of
Proteomics 74, 1974-1984.
6. Flores-Ramirez, G., Janecek, S., Miernyk, J. A., Skultety, L. (2012) Proteome Sci. 10.
61
Multi-view 3D reconstruction of microscopic objects
aR. GARGALIK, bZ. TOMORI
aInstitute of Computer Science, Faculty of Natural Sciences UPJŠ, Košice bInstitute of Experimental Physics SAS, Košice
3D reconstruction from multiple images (also known as structure from motion) is a
widely used technique to reconstruct different objects, such as statues, archaeological
excavations or even some parts of cities. Objects used for reconstruction have in general
bigger dimensions. Sometimes we need to reconstruct smaller objects, or even microscopic
objects. Besides the structure from motion, there are different approaches how to achieve this
goal, such as 3D reconstruction from focus [3], where depth of the scene is estimated from the
stack of images having different focus. Our goal was to evaluate structure from motion
approach on microscopic objects datasets to determine availability and quality for such
datasets. Our application consists from different modules but all are written in C++ using two
third party libraries: OpenCV [4] and Point Cloud library [5]. The whole process consists of
several steps: acquisition, camera calibration, pose estimation, point cloud generation and
mesh generation. In our experiments we use the following microscopic objects: crystal sugar,
sea salt, eyelash, piece of pepper and a piece of bread. The first three were acquired with
constant focal length and the last two with variable focal length.
Acquisition: We used digital microscope DinoXLite camera from AnMo Electronics
Corporation to acquire images at resolution 1280 × 1024 pixels.
Camera calibration: In case the focal length is constant, then the default approach using the
calibration pattern can be used. We printed a small chessboard pattern (approx. 4 × 4 mm) and
used OpenCV [4] to estimate intrinsic camera matrix . In case the focal length was variable,
we used the Bundler software [2] to obtain intrinsic camera matrices.
Pose estimation: First the correspondences between images must be found. We used SIFT
algorithm [7] to detect key points and MSER algorithm [8] to detect regions and we took the
center of gravity of such region to represent the key point. Next, we matched those features
against images to obtain correspondences. For each consecutive image pair we robustly
estimated fundamental matrix from all available correspondences using RANSAC
algorithm (see [1] for more details) and then an essential matrix . Following approach
described in [1], decomposition of essential matrix is done using singular value
decomposition and finally the estimation of rotation matrix and translation vector is
performed. Calculation of projection matrix from , and is straightforward.
Point cloud generation: We implemented iterative linear triangulation described in [1] to
obtain 3D location of each point from each image pair. The final 3D coordinates of a point is
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calculated as a centroid from its 3D locations. The color of a point is calculated as an average
from all projections of the point in images.
Mesh generation: To generate the final mesh from the point cloud, we utilize the algorithm
described in [6], which is already implemented in the Point Cloud library [5]. The result mesh
is then exported so it can be viewed in an external application.
Results: Results can be seen at http://ics.upjs.sk/~gargalik/ssb2013/. The first three datasets
result in good quality. The structure is reconstructed quite precisely and the visual impression
is also quite good. The last two datasets do not result in good quality. We attach it to variable
focal length during the acquisition phase and consecutive improper calibration. The structure
is therefore reconstructed purely.
Acknowledgement: This work was supported by APVV 0526-11 project and VVGS-PF-2012-
60 grant.
References
1. Hartley, R., Zisserman, A. (2003) Multiple View Geometry in Computer Vision.Cambridge, 2nd edition.
2. Snavely, N., Seitz, S.M., Szeliski, R. (2008) Photo Tourism: Exploring image collections in 3D. ACM Transactions on Graphics (Proceedings of SIGGRAPH 2006).
3. Cacciari, I., Mencaglia, A. A., Siano, S. (2013) Micromorphology of gold jewels: a novel algorithm for 3D reconstruction and its quality assessment. Proc. SPIE 8790, Optics for Arts, Architecture, and Archaeology IV, 87900B.
4. Bradski, G. R., Kaehler, A. (2008) Learning OpenCV, O'Reilly, Sebastopol, CA. 5. Rusu, R. B., Cousins, S.,(2011) 3D is here: Point Cloud Library (PCL). IEEE
International Conference on Robotics and Automation (ICRA), Shanghai, China. 6. Marton, Z. C., Rusu, R. B., Beetz, M. (2009) On Fast Surface Reconstruction Methods
for Large and Noisy Datasets. In Proc. of the IEEE International Conference on Robotics and Automation (ICRA), Kobe, Japan.
7. Lowe, D. G. (1999) Object recognition from local scale-invariant features. In Proceedings of the International Conference on Computer Vision 2. pp. 1150–1157.
8. Matas, J., Chum, O., Urba, M., Pajdla, T. (2002) Robust wide baseline stereo from maximally stable extremal regions. In Proc. of British Machine Vision Conference, pp. 384-396.
63
The influence of quercetin on lipid membranes with cholesterol
D. IONESCU
Department of Biophysics, "Carol Davila" University of Medicine and Pharmacy,
Bucharest, 050474, Romania
Among the wide class of flavonoids, widely spread in plants’ kingdom, Quercetin
(Que; 3,5,7,3’,4’-pentahydroxyflavone) is one of the most important antioxidants frequently
found in daily human diet; Que is known due to its remarkable chemo-preventive,
antimutagenic, anti-cancer, cardioprotective, anti-inflammatory, anti-allergenic and anti-
microbial properties [1, 4]. Although the effects of this polyphenol on living cells were quite a
lot investigated, the direct interaction of Que with lipid fraction of the cell membrane is not
fully understood. Therefore, our study pursuit concerns the effects of this flavonoid on the
lipid fraction of cell membranes.
For this purpose a spectrofluorimetric approach was applied, whilst the main parameters
employed in data handling were the generalized polarization [5] and the decomposition in two
Gaussian functions of the emission spectra of Laurdan [3] loaded into phospholipids
liposomes containing different amounts of cholesterol. As a fluorescent probe, Laurdan is able
to detect changes in membrane phase properties. When incorporated in lipid bilayers, Laurdan
emits from two different excited states, a non-relaxed one when the bilayer packing is tight
and a relaxed state when the bilayer packing is loose. The calculation of the generalized
polarization values (GP) leaded us to the conclusion that Que is decreasing phase transition
temperature in pure 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, with 4
degrees in a range of 0-60 M Que concentration .
In quantifying Que effects on phospholipids membranes containing cholesterol, the
decomposition of Laurdan emission spectra in sums of two Gaussian functions on energy
proved to be an excellent tool that allowed a good analysis of the balance between the two
emitting states of Laurdan (the maximum of Laurdan fluorescence emission moved from
440 nm at low temperatures, below the main phase transition of the lipids, to 490 nm at high
temperature). Our results showed that both Laurdan emission states are present, with different
shares, in a wide temperature range for DMPC liposomes with cholesterol. The experiments
disclosed the fact that Que quenches Laurdan fluorescence, depending on temperature and on
the amount of cholesterol at the level of the membrane. Inasmuch, quenching of fluorescence
requires a close approach of fluorophore and quencher, from quenching parameters one being
able to obtain information regarding the relative positioning of the two types of molecules
involved in the quenching process. In our case, both Laurdan and QCT have a hydrophobic
character and therefore are supposed to be found inside the membrane core. At low
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64
temperatures, when lipid packing is tight, water molecules from membrane regions where
Laurdan molecules reside are not that numerous and QCT quenches Laurdan fluorescence
with a higher efficiency. At high temperatures, where lipid membrane is in the liquid-crystal
phase and lipid packing is loose, the amount of water in Laurdan molecules vicinity is large
and probably QCT molecules are repelled, therefore the quenching becomes less efficient.
Stern-Volmer constants were calculated for different lipid membrane composition and the
conclusion was that the nonradiative transitions are favored from the relaxed state of the
fluorophore. The classical Stern-Volmer plot was fitted with an exponential function, with
two free parameters representing the dynamic quenching rate and the radius of the quenching
circle of action. The geometry of quenching, given by the second parameter, is characterized
by an average value of (15 3) Å for all types of studied liposomes that did not depend either
on liposomes composition, or on temperature [2].
Acknowledgement. This work was supported by the projects: The grant from the Romanian
National Authority for Scientific Research CNCS–UEFISCDI, project number PNII-IDEI-
PCE-2011-3-0800 and by Sectorial Operational Programme Human Resources Development,
financed from the European Social Fund and by the Romanian Government under the
contract number POSDRU/89/1.5/S/64109.
References
1. Baran, I., Ganea, C., Ursu, I., Baran, V., Calinescu, O., Iftime, A., Ungureanu, R.,
Tofolean, I.T. (2011) Rom. J. Phys. 56, 388–398.
2. Ionescu, D., Ganea, C. (2012) Eur. Biophys. J. 41, 307–318.
3. Lucio, A.D., De Vequi-Suplicy, C.C. (2010) J. Fluoresc. 20, 473-482.
4. Murakami, A., Ashida, H., Terao, J. (2008) Cancer Letters 269, 315-325.
5. Parasassi,T., De Stasio,G., Dubaldo, A., Gratton, E. (1990) Biophys. J. 57, 1179-1186.
65
How hydrogen peroxide is broken down by oxidized cytochrome c oxidase
aD. JANCURA, bJ. STANICOVA, cG. PALMER and c,dM. FABIAN
a Department of Biophysics, University of P. J. Safarik, Kosice b Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine, Kosice c Department of
Biochemistry and Cell Biology, Rice University, Houston dCenter of Interdisciplinary Biosciences, University of P. J. Safarik, Kosice
The physiological function of cytochrome oxidases is the reduction of dioxygen to
water; this process is associated with the building of a transmembrane proton gradient. The
overall reaction, reduction of O2 by four electrons, proceeds through a well-defined sequence
of intermediates at the catalytic binuclear heme a3-CuB center. This sequence can be
mimicked by the reaction of oxidized cytochrome oxidase (CcO) with excess hydrogen
peroxide. This unphysiological reaction results in a continued and complete H2O2 degradation
yielding back the oxidized enzyme. To explain the ability of CcO to break down peroxide by
turnover and the formation of particular intermediates it was suggested that the enzyme
possesses a catalase-like and /or a peroxidase function [1-3]. These two possible activities
were associated with turnover at the catalytic site of CcO. We show here that the there are two
sites of peroxide decompositions by CcO and that the catalytic site does not exhibit a simple
catalase or catalase-like function. Our kinetic evidence indicates that during a single turnover
two molecules of H2O2 are disintegrated at the catalytic center. This reaction is associated
with the irreversible oxidation of the enzyme leading to radical formation in the vicinity of the
heme a3-CuB site. The migration of these radicals to the surface of the enzyme [3] produces
the second center where H2O2 is decomposed.
Acknowledgement. This work was supported by the National Institutes of Health (GM
084348) and FP7 EU project CELIM 316310.
References
1. Bolshakov, I. A., Vygodina, T. V., Gennis, R., Karyakin, A. A., and Konstantinov, A.
A. (2010) Biochemistry (Mosc) 75, 1352-1360.
2. von der Hocht, I., van Wonderen, J. H., Hilbers, F., Angerer, H., MacMillan, F., and
Michel, H. (2011) Proc. Natl. Acad. Sci. U.S.A. 108, 3964-3969.
3. Musatov, A., Hebert, E., Carroll, C. H., Weintraub, S. T., Robinson. N. C. (2004)
Biochemistry 43, 1003–1009.
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66
Figure 1. Stereo image of the aromatic amino acid networks within Complex IV that connect the binuclear center to W334(I), W19(VIIc) and W48(IV) [1].
Hypothesis: Mitochondrial Electron-Transfer Proteins are a Part of Defense
Mechanism Against Oxidative Damage
a,bE. SEDLÁK, a,bR. VARHAČ and a,cA. MUSATOV
aDepartment of Biochemistry, The University of Texas Health Science Center at San Antonio,
Texas, USA bDepartment of Biochemistry, University of P. J. Safarik, Kosice, Slovakia cDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Science,
Kosice, Slovakia
Approximately 1-2 % of all oxygen consumed by the mitochondria is converted to
reactive oxygen species (ROS). Cellular defenses fight a continuing battle to protect the
mitochondria against this onslaught of ROS. We have demonstrated the existence of the
aromatic amino acid rich network within subunit I of Complex IV (cytochrome c oxidase,
Figure 1) that may be a mitochondrial defense mechanism to scavenge damaging radicals
generated at the binuclear center [1, 2]. A new experimental data suggest that all major
mitochondrial electron-transfer proteins are involved in mitochondrial defense mechanism.
The protection mechanism involves peroxidase/catalase-like activity of Complex I, Complex
III, Complex IV [3-5], and ferricytochrome c [3], which catalyze the decomposition of H2O2,
with the apparent bimolecular rate constants of 55.7 ± 2.7, 249.0 ± 9.0, 63.2 ± 2.5, and 5.1 ±
1.0 M-1s-1, respectively. We hypothesize that although these values obtained for isolated
detergent-solubilized complexes are
lower than the rate of a specialized
peroxidase/catalase, decomposition of
hydrogen peroxide in situ by
mitochondrial electron-transfer
complexes may have an important
role in mitochondrial defense
mechanisms against oxidative
damage. At the very least the ability
to consume H2O2 protects
mitochondrial electron-transport
complexes themselves against severe oxidative damage.
Acknowledgement. The research was supported by GM024795 grant from the National
Institutes of Health and VEGA 0181 grant.
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References
1. Lemma-Gray, P., Weintraub, S. T., Carroll, C. A., Musatov, A., and Robinson, N.
C. (2007) FEBS Lett. 581, 437-442.
2. Musatov, A., Hebert, E., Carroll, C. A., Weintraub, S. T., and Robinson, N.C.
(2004) Biochemistry 43, 1003-1009.
3. Sedlák, E., Fabian, M, Robinson, N. C., Musatov, A. (2010) Free Radic Biol Med.
49, 1574-1581.
4. Bolshakov, I. A., Vygodina, T. V., Gennis, R., Karyakin, A. A., and Konstantinov,
A. A. (2010) Biochemistry (Moscow), 75, 1352-1360.
5. Borisov, V. B., Forte, E., Davletshin, A., Mastronicola, D., Sarti, P., Giuffrè, A.
(2013) FEBS Lett. 587, 2214-2218.
68
Structure of tau protein in the complexes with monoclonal antibodies studied by X-ray crystallography
a,bR. SKRABANA, aO. CEHLAR, cR. DVORSKY, a,bB. KOVACECH, a,bA. KOVAC,
dJ. SEVCIK, a,bM. NOVAK
aInstitute of Neuroimmunology SAS, Bratislava, Slovakia bAxon Neuroscience SE, Bratislava, Slovakia cMax Planck Institute for Molecular Physiology, Dortmund, Germany dInstitute of
Molecular Biology SAS, Bratislava, Slovakia
Neuronal microtubule-associated tau protein is the constitutive component of
neurofibrillary pathology in the Alzheimer’s disease and other tauopathies [1]. Under
physiological conditions, tau protein is in a globally disordered state, which is populated by
transient and/or local motifs of defined secondary and tertiary structure [2]. The proportion of
individual structures in the conformational ensemble of tau protein determines its interaction
propensities and cellular function. Consequently, an enrichment of pathological structural
signatures likely conduces to the creation of toxic species which could precipitate
neurodegenerative process. The knowledge of conformational features of tau protein at the
atomic level is of capital importance for understanding its physiological function and its
pathologic transformation in disease.
Recently we succeeded in structure determination of several complexes between short tau
protein peptides and Fab fragments of specific anti-tau monoclonal antibodies, which
conferred structural snapshots of important regions of tau protein in the free state and/or under
pathological conditions (Fig. 1). Particularly, we have determined the structure of the C-
terminus of the core of paired helical filaments (PHF), the structural subunits of
neurofibrillary pathology [3, 4]. This work conferred the first atomic detail information about
the structure of the genuine tau protein pathological assembly. Furthermore, we have
determined the structure of highly conserved motifs occurring in each of the four repeats of
tau protein and the structure of 14-aminoacid long stretch in the proline-rich region. All
studied motifs are important for the interaction of tau protein with microtubules under
physiological conditions; however, they are also involved in the assembly of tau protein in the
neurodegeneration. Our results are supplemented with the recently published structure of tau
phosphopeptide in the complex with recombinant chicken antibody Fab fragment [5].
Obtained structures are complementary to the NMR-determined structural propensities of tau
protein [6]. Presented results underline the important role of monoclonal antibodies as the tool
for crystallographic investigation of the structure of tau protein.
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Figure 1. Regions of tau protein (red bars) studied by X-ray crystallography of tau-Fab
antibody fragments complexes. The position of motifs is shown on the schematic
representation of the longest human tau protein isoform, tau40 (tau1-441; I1, I2 – amino-
terminal inserts; R1, R2, R3, R4 - microtubule-binding repeats; R’ - region of lower repeat
homology). The extent of the PHF core is delimited by vertical dashed lines.
Acknowledgement. This work was supported by the Slovak Research and Development
Agency under the contracts Nos. APVV-0399-10, LPP-0038-09 and by the Slovak Grant
Agency VEGA grants Nos. 2/0130/12, 2/163/13.
References
1. Wischik, C.M., Novak, M., Thogersen, H.C., Edwards, P.C., Runswick, M.J., Jakes,
R., Walker, J.E,, Milstein, C., Roth, M. & Klug, A. (1988) Proc. Natl. Acad. Sci. USA
85, 4506-4510.
2. Skrabana, R., Sevcik, J., Novak, M. (2006) Cell. Mol. Neurobio.l 26, 1085-1097.
3. Sevcik, J., Skrabana, R., Dvorsky, R., Csokova, N., Iqbal, K., Novak, M (2007) FEBS
Lett 581, 5872-5878.
4. Skrabana, R., Dvorsky, R., Sevcik, J., Novak, M. (2010) J. Struct. Bio.l 171, 74-81.
5. Shih, H.H,, Tu, C., Cao, W., Klein, A., Ramsey, R., Fennell, B.J., Lamber,t M., Ni
Shuilleabhain, D., Autin, B., Kouranova, E., et al. (2012) J. Biol. Chem. 287, 44425-
44434.
6. Mukrasch, M.D., Bibow, S., Korukottu, J., Jeganathan, S., Biernat, J., Griesinger, C.,
Mandelkow, E., & Zweckstetter, M. (2009) PLoS Biol. 7, e34
70
Non-trivial decisioning in Bcl-2 family-mediated regulation of Apoptosis
T. TOKAR, J. ULICNY
Department of Biophysics, University of P. J. Safarik, Kosice, Slovakia
One of the most important signaling checkpoints of apoptosis is the Mitochondrial
Outer Membrane Permeabilization (MOMP), which is controlled by Bcl-2 family of proteins.
Bcl-2 proteins are regulated by variety of incoming pro- & antiapoptotic signals and as a
response to these, Bcl-2 proteins may initiate MOMP and thus allow apoptosis commitment.
Using computational modeling & simulations we found that interplay between the
Bcl-2 family proteins form a regulatory network which can integrate a multitude of
continuous inputs into single binary output. Particular Bcl-2 proteins may serve as a "toggles",
up-/downregulation of whose can "switch on" the MOMP. We have discovered that Bcl-2
family performs pattern recognition - nontrivial behavior, often associated with neural
networks and artificial intelligence.
We conclude that Bcl-2 proteins constitute molecular device, controlling apoptosis
commitment in much more sophisticated manner than previously thought.
Acknowledgement. This work was supported by: Agency of the Ministry of Education of
Slovak Republic for the Structural funds of the EU, Operational program Research and
Development (SEPO II, CEVA), Slovak Research and Development Agency (APVV-0242-11)
and Scientific Grant Agency of the Ministry of Education of Slovak Republic (VEGA-
1/1246/12)
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Galectins-1 and -3 differently modulate wound microenvironment with different impact
on incisional and excisional skin wound healing in rats
aP. GÁL, bV. PERŽEĽOVÁ, bT. VASILENKO, bJ. JAKUBČO, cH.-J. GABIUS, dB. DVOŘÁNKOVÁ, eF. SABOL AND dK. SMETANA JR.
aInstitute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovak Republic bFaculty of Medicine, Pavol Jozef Šafárik University, Košice, Slovak Republic
cFaculty of Veterinary Medicine, Ludwig-Maximilian University, Munich, Germany d1st Faculty of Medicine, Charles University, Prague, Czech Republic
eEast-Slovak Institute of Cardiovascular Diseases, Košice, Slovak Republic
Introduction: Galectins belong to a family of endogenous lectins specifically recognizing
sugar motives and play an important role in cell proliferation, differentiation, migration, and
extracellular matrix (ECM) production. Among many galectins it has been shown that
galectin-1 is up-regulated in tumor stroma and granulation tissue of healing wounds [1,2].
This lectin plays an important role in angiogenesis and tumor metastasis. On the other hand,
galectin-3 is a well known inductor of tissue fibrosis [3]. Therefore, the aim of present
investigation was to evaluate the effects of galectins-1 and -3 on wound healing in vitro and in
vivo.
Methods: In general anesthesia one incisional sutured and one excisional wound were made
on the back of each rat. Wounds were daily treated with human recombinant galectin-1 or
galectin-3 (20 ng/ml) [4]. Control group was not treated and the negative control group was
daily treated with a biological inactive galectin-1-E71Q. Rats were killed at day 7 and 21
post-wounding. Excisions were submitted for histological examination and wound contraction
measurement, while the incisions were proceed for tensile strength measurement. In the in
vitro experiment the interaction between ECM produced by dermal fibroblasts and human
keratinocytes was studied.
Results: We showed that galectins-1 and -3 differently modulate wound healing. Galectin-1
created a suitable microenvironment for fibroblast to myofibroblast transition and biologically
active matrix production that induced poor differentiation phenotype of keratinocytes. These
processes led to significantly increased wound contraction in vivo. In contrast, galectin-3
increased wound tensile strength and collage organization in healing skin wounds.
Discussion and Conclusion: Since eighties it has been suggested that there exist certain
parallels between wound healing and tumor growth [5]. Therapeutic approach of both
pathological states includes a complex treatment that should include wound/tumor
microenvironment modulation. Crucial role in the tumor stroma formation play fibroblasts
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[2]. They also have capability to differentiate into myofibroblasts, that have important role in
wound contraction and biological properties of tumors. We showed that galectins-1 and -3
significantly modulate wound/tumor microenvironment and, thus may be considered as new
therapeutic targets in the process of new drug development.
Acknowledgement. Study supported by grants no. APVV-0526-11 and VEGA-1/1095/11.
References
1. Gál, P., Vasilenko, T., Kostelníková, M., Jakubco, J., Kovác, I., Sabol, F., André, S.,
Kaltner, H., Gabius, H.J., Smetana, K. Jr. (2011) Acta Histochem. Cytochem. 44, 191-
199.
2. Valach, J., Fík, Z., Strnad, H., Chovanec, M., Plzák, J., Cada, Z., Szabo, P., Sáchová,
J., Hroudová, M., Urbanová, M., Steffl, M., Pačes, J., Mazánek, J., Vlček, C., Betka,
J., Kaltner, H., André, S., Gabius, H.J., Kodet, R., Smetana, K. Jr., Gál, P., Kolář, M.
(2012) Int. J. Cancer 131, 2499-2508.
3. Henderson, N.C., Mackinnon, A.C., Farnworth, S.L., Kipari, T., Haslett, C., Iredale,
J.P., Liu, F.T., Hughes, J., Sethi, T. (2008) Am. J. Pathol. 172, 288-298.
4. Cao, Z., Said, N., Amin, S., Wu, H.K., Bruce, A., Garate, M., Hsu, D.K., Kuwabara,
I., Liu, F.T., Panjwani, N. (2002) J. Biol. Chem. 277, 42299-42305
5. Dvorak, H.F. (1986) N. Engl. J. Med. 315, 1650-1659.
73
Using protein engineering to understand the molecular mechanism underlying enzyme
deficiency in clinical mutants of G6PD
M. A. HAMZA, A. A.-T. IDRIS, A. MUSTAFA, M. AL-MAHAREB
Faculty of Medicine, King Fahad Medical City, King Saud Bin Abdul Aziz University for Health Sciences, Riyadh, KSA.
Glucose 6 phosphate dehydrogenase (G6PD) (EC: 1.1.1.49) is a rate-controlling,
ubiquitous enzyme which catalyses the first reaction in the pentose phosphate pathway (ppp)
[1]. The main physiological role of this enzyme is production of NADPH necessary for many
biosynthetic reactions and protection of the cells against oxidative stress [1].
G6PD is a housing-keeping, X-linked gene in the arm of chromosome 13. Human G6PD
deficiency is inherited as an X-linked recessive trait and highly polymorphic with more than
300 know-variants so far resulting from single point mutations [2]. This represents the most
common human enzymopathy with more than 400 million people throughout the world.
Currently, more than 140 variants have significantly reduced activity and result in a condition
known as G6PD deficiency in different populations and the list is still rapidly growing [1].
Most of the polymorphic G6PD deficient patients present clinical manifestations ranging from
mild and asymptomatic to blood transfusion dependent with chronic nonspherocytic
haemolytic anaemia [2]. It has been known that G6PD deficiency offers protection against
malaria. The frequency of G6PD deficiency is correlated with past or present history of
malaria (WHO report, 1989).
In Saudi Arabia several research groups have conducted thorough investigations about the
frequency of G6PD deficiency (Prof. Mohsen El-Hazmi & Dr. Souad Khalil). It was reported
that the frequency of the G6PD deficiency is very high in some parts of the Kingdom [3].
G6PD Mediterranean was found to be the most frequent among the Saudi population causing
severe G6PD deficiency [3]. Despite, extensive efforts to study this enzyme, these have been
directed to the phenotype and less has been done to understand the molecular basis of this
disease.
The main intention of this study was to use protein engineering to introduce clinical mutations
causing haemolytic anaemia into a recombinant DNA of normal human G6PD gene. To this
end, site-directed mutagenesis has been employed to introduce the mutation found in the
pateint's g6pd gene. This allows us of getting abundant amount of protein enabled us of better
scrutinizing the mutated enzyme. Moreover, the engineered enzyme was of identical age
which can not be extracted from patients' blood and might give false result.
This presentation will shed light on protein engineering as a powerful tool of understanding
enzymes characteristics and paving the way through to think on effective therapeutics.
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References
1. Beutler, E., Vulliamy, T., and Luzzato, L. (1996) Blood Cells Mol. Dis. 22, 49-56.
2. Mason, P. J., Bautista, J. M., and Gilsanz, F. (2007) Blood Rev. 21, 267-283.
3. Warsy.Arjumand, A., and El-Hazmi, Mohesn, F. A. (2001) Annals of Saudi Medicine.
21, 174-177.
75
Aliskiren loaded PLA nanospheres: preparation, physicochemical characterization,
in vitro release and in vivo effect
aI. ANTAL, aM. KUBOVCIKOVA, bO. PECHANOVA, bA. BARTA, bM. CEBOVÁ, bM.
KOVÁCSOVÁ, aM. KONERACKA, aV. ZAVISOVA, aA. JURIKOVA, aP. KOPCANSKY
aInstitute of Experimental Physics Slovak Academy of Sciences, 040 01 Kosice, Slovakia bInstitute of Normal and Pathological Physiology Slovak Academy of Sciences, Bratislava
In our study, we prepared and characterized polymer nanospheres with loaded drug
Aliskiren (ALIS). ALIS is a renin inhibitor used to treat high blood pressure (hypertension).
It works by blocking an enzyme in the body that is necessary to produce a substance that
causes blood vessels to tighten. Disadvantage of common oral use of ALIS is its low stability
in pH of gastric medium. We suggest encapsulating ALIS into the polymer nanospheres to
prevent drug degradation and to increase bioavailability of the ALIS.
Polymer nanospheres were prepared by modified nanoprecipitation method. The
principle of this method is based on the adding of the organic phase consisted of mixture of
poly(lactic acid) (PLA, Mw 75 000 – 120 000 g/mol) and the drug ALIS in acetone to the
aqueous phase drop by drop under magnetic stirring until the organic solvent is evaporated
under atmospheric pressure and room temperature.
0 200 400 600 800 1000
0.0
0.2
0.4
0.6
0.8
1.0ALIS/PLANPs
DDCS= 297 nm DDLS= 227 nm
Nor
mal
ized
Inte
nsity
Diameter (nm) Fig. 1: SEM image of ALIS loaded PLANPs Fig. 2: Size distribution obtained by DLS and DCS
Size and morphology of prepared ALIS loaded PLA nanospheres (ALIS/PLANPs) was
studied by several techniques. By scanning electron microscopy (SEM) approximately
spherical shape of nanospheres with typical diameter ca. 250 nm was confirmed (fig. 1).
Dynamic light scattering (DLS) based on the light scattered by colloidal particles which are
undergoing thermal motion and differential centrifugal sedimentation (DCS) based on
measuring the time required for the colloidal particles to settle in a density gradient were
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76
used to determine hydrodynamic particle size and particle size distributions of ALIS loaded
PLANPs. DLS yielded a monomodal distribution with DDLS = 227 nm and the size
determined by DCS was 297 nm (fig.2). The physical state of ALIS encapsulated in the
polymeric nanoparticles was investigated by the differential scanning calorimetry (DSC).
The DSC traces indicated that ALIS was amorphously or molecularly distributed in the
polymeric matrices. The Fourier Transform Infrared Spectrophotometry (FTIR) was taken
in order to qualitatively analyze spectrum of drug ALIS, ALIS loaded and unloaded PLA
nanospheres and physical mixture of ALIS with PLANPs. The FTIR spectrum of
ALIS/PLANPs in comparison with the physical mixture spectrum give us a combination of
PLA and ALIS peaks with partial shift or shape changing. These results confirm that ALIS
have been successfully loaded in the PLANPs. In vitro ALIS release from ALIS/PLANPs to
the surrounding medium at pH 2.0, 4.0 and 7.4 was studied during one week. A biphasic
release of ALIS from PLANPs was observed. During the first phase in 24 hours about 70%
(w/w) of loaded drug was released. This phase was followed by the slower release profile.
Cumulative release amount of ALIS in five days was around 93%. For the study of effect of
the encapsulated ALIS on systolic blood pressure male spontaneously hypertensive rats aged
12 weeks were assigned to untreated group and groups treated with non-encapsulated and
encapsulated ALIS (25 mg/kg per day) for 3 weeks by gavage. Blood pressure was
measured by the plethysmographic method on the tail artery. At the end of experiment,
systolic blood pressure in non-encapsulated ALIS group was significantly lower (178.7 ±
1.8 mm Hg) than in the controls (203.4 ± 4 mm Hg). Encapsulated ALIS, however,
decreased blood pressure even more significantly from the first week of the treatment (160 ±
5 mm Hg vs. 203.4 ± 4 mm Hg in the control rats).
Prepared PLA/ALIS nanospheres are spherical in shape with mean diameter ca. 250 nm with
good colloidal stability and are from this point of view suitable for effective in vitro cellular
uptake of NPs. Encapsulated ALIS decreased blood pressure of the studied male
spontaneously hypertensive rats even more significantly then common administered drug.
Acknowledgement. This work was supported within the projects Nos. 26110230061,
26220120033 in frame of Structural Funds of European Union, VEGA 0041, 0045, APVV-
99-026505, APVV-0742-10 and SK-SRB-0038-11.
77
Magnetosomes - a new type of magnetic nanoparticles. The methods of preparation,
characterization, and their applications
aM. MOLCAN, aA. HASHIM, aJ. KOVAC, aP. KOPCANSKY, bH. GOJZEWSKI, cA.
SKUMIEL, aM. TIMKO
aInstitute of Experimental Physics, Slovak Academy of Sciences, Watsonova 47, 040 01
Košice,Slovakia bInstitute of Physics, Poznan University of Technology, Nieszawska 13A, 60-
965 Poznań, Poland cInstitute of Acoustics, Adam Mickiewicz University, Umultowska 85,
61-614 Poznań, Poland
Magnetic nanoparticles are key components for development of many novel bio- and
nanotechnological applications. One of them is “new” biological magnetic particles, the so-
called magnetosomes, which were found as a product of the biomineralization process from
magnetotactic bacteria. At present they are under investigation especially in biomedical
applications. This ability depends on the presence of intracellular magnetosomes which are
composed of two parts: first, nanometer-sized magnetite (Fe3O4) or greigite (Fe3S4) crystals
(magnetosome crystal), depending on the bacterial species; and second, the bilayer membrane
surrounding the crystal (magnetosome membrane). Interesting is that particles are arranged by
a membrane in the form of chains, which gives unique characteristics.
The methods of bacteria cultivation and magnetic separation of magnetosomes are
successfully managed. Our particles are characterized by morphological, magnetic and
hyperthermic measurements. The magnetic properties and hyperthermia effect were studied in
solution of magnetosomes obtained by changing conditions during biomineralization of
magnetotactic bacteria Magnetospirillum sp. AMB-1. It was shown that adding a higher
amount of Wolfe’s vitamin solution (WVS) or ferric quinate (FQ) cause increase of the mean
diameter from 47 nm (normal condition - NP) up to 52 nm and 58 nm respectively. As a
consequence of these changes of the preparation conditions some properties have been
modified. The magnetization measurements have shown that no hysteresis can be observed at
room temperature on suspension of NP magnetosomes, and this suspension behaves also
superparamagnetically. Small increase in hysteresis was observed for sample WVS (Hc = 6.5
Oe) and for sample FQ (Hc = 20 Oe) what means that these samples show partly
ferromagnetic behaviour too. By using of sonication and ultracentrifugation methods we are
trying to break the chains and get the magnetosome as individual particles. Individual
particles open space for the study of changes in their physical properties, there is also the
possibility for new applications.
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Acknowledgement. This work was supported by the Ministry of Education Agency for the
Structural Funds No. 26220120021, 26220120033, 26110230061 and by the grant VEGA
2/0045/12.
79
POSTERS abstracts
81
Application of surface-enhanced Raman spectroscopy for the detection of trace amounts
of pesticides aldrin, α-endosulfan and lindane
aJ. KUBACKOVA, aP. MISKOVSKY, aG. FABRICIOVA, bS. SANCHEZ-CORTES, aD. JANCURA
a Department of Biophysics, Faculty of Science, P.J. Safarik University, Kosice, Slovakia b Instituto de Estructura de la Materia, CSIC, Madrid, Spain
Surface-enhanced Raman scattering (SERS) can be used as an analytical technique
because of its high sensitivity and selectivity in molecular identification [1]. SERS provides
an enhancement of up - to about 1010 in relation to normal Raman scattering [1] and in certain
cases this techniques leads to an enhancement of up - to about 1014-1015, what corresponds
with single molecular detection [2]. SERS enhancement is mainly based on giant
electromagnetic (EM) field induced by nanostructured noble metal surfaces and associated
with the localized surface plasmon resonance [3]. Most currently employed SERS substrates
are metal (Ag, Au, Cu) nanoparticles (NPs) in suspension (colloids). The EM intensification
occurs mostly in localized regions of the metallic surface called hot spots (HS), such as
interparticle junctions or gaps between NPs [1]. An ideal situation for building interparticle
HS is the use of bifunctional molecules which act as NPs linkers [4]. The linkers adsorbed on
metal surfaces are not only able to induce the formation of the HS but can also act as
molecular hosts of specific analytes.
Aldrin, α-endosulfan and lindane are pesticides belonging to organochlorine family. These
pesticides break down slowly in the environment and can accumulate in the fatty tissues of
animals. Organochlorine pesticides may cause long- or short-term damage to living organisms
by changing the growth rate of some plants and animal species. Moreover, they also represent
certain risks for human health (carcinogenicity). With respect to these facts, the detection and
identification of these chemicals (even at very low concentration) is very important.
By using various types of metal colloids and aliphatic α,ω-dithiols as bifunctional linkers, we
have determined the fingerprint region (300-400 cm-1) for the SERS detection of the above
mentioned pesticides and the limit of detection for the individual molecules (aldrine (5.1x10-8
M), α-endosulfan (28.5x10-8 M), lindane (11.6x10-8 M)). The most suitable substrate for
SERS detection of the studied pesticides appears to be citrate silver colloid particles covered
by 1,8-octanedithiol.
In conclusion, our recent results confirm the high sensitivity of SERS for the detection of low
quantities (~10-8M) of organochlorine pesticides like aldrin, α-endosulfan and lindane. This
fact provides solid basis for the construction of suitable nano-sensors for the detection and
identification of this type of chemicals.
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Acknowledgement. This work was supported by the (i) Agency of the Ministry of Education of
Slovak Republic for the Structural funds of the European Union, Operational program
Education (Doctorand, ITMS code: 26110230013) and Operational program Research and
Development (NanoBioSens, ITMS code: 26220220107 and CEVA II, ITMS code:
26220120040, (ii) Slovak Research and Development Agency under the contract APVV-0242-
11, and (iii) by the project CELIM funded by7FP EU (REGPOT).
References
1. Lopez-Tocon, I., Otero, J.C., Arenas, J.F., Garcia-Ramos, J.V., and Sanchez-Cortes, S.
(2011) Anal. Chem. 83, 2518-2525.
2. Nie, S., and Emory, S.R. (1997) Science 257, 1102-1006.
3. Giannini, V., Fernandez-Dominguez, A.I., Heck, S.C.,and Maier, S.A. (2011) Chem.
Rev. 111, 3888-3912.
4. Guerrini, L., Izquierdo-Lorenzo, I., Garcia-Ramos, J.V., Domingo, C., and Sanchez-
Cortes, S. (2009) Phys. Chem. Chem. Phys. 11, 7363-7371.
83
Adsorption and detection of anthraquinone drug parietin on Ag nanoparticles by
surface-enhanced Raman scattering
aG. FABRICIOVÁ, bE. LOPEZ-TOBAR, bM. V. CAÑAMARES, cM. BAČKOR,
bS. SANCHEZ-CORTES
aDepartment of Biophysics, Faculty of Science P. J. Šafárik University, Košice bInstituto de
Estructura de la Materia, CSIC, Madrid, Spain cDepartment of Botany, Faculty of Science P.
J. Šafárik University, Košice, Slovakia
Many lichens produce secondary metabolites by the mycobionts and these metabolites
absorb radiation in the ultraviolet region. One of the metabolites is an orange coloured
anthraquinone pigment named parietin (Fig. 1). Parietin protects lichen photobiont cells
against excessive photosynthetic active light [1] and also possesses some important biological
activities, including antibacterial and antifungal effects [2]. It has been shown that parietin can
induce apoptosis and inhibit cell proliferation in various human cancer cells [3]. The structure
of parietin is very similar to the one of emodin (Fig. 1). This drug has been employed as
anticancer, antiagregant, antiulcer, anti-inflammatory, myorelaxant and antiseptic agent. The
difference between these two molecules is in position C3 where parietin has a methoxy group
instead of a hydroxyl group.
Fig. 1: Molecular structure of parietin (a) and emodin (b).
In this work, we have successfully applied SERS spectroscopy to the study of the vibrational
properties of the highly fluorescent anthraquinone molecules parietin and emodin on silver
nanoparticles. The comparison between the SERS spectra of parietin and emodin allowed the
identification of the markers (Fig. 2). These bands allow the distinction between these two
similar anthraquinone molecules. SERS spectra of parietin are sensitive to the concentration
of the molecule in a medium. A decrease of parietin concentration leads to the reorientation of
the molecule on the metal surface from perpendicular to parallel. The high value of adsorption
binding constant (Kad = 3.4 × 104 L mol−1) indicates a high affinity of parietin to the metal
surface. The low limit of detection (LOD = 1.25 × 10−8 M) leads to the possibility of using
SERS for the detection of trace quantities of this molecule in aqueous solutions.
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84
A B
A-Fig.2: Comparison of the SERS spectra ofemodin (a) and (c) and parietin (b) and (d) at
different laser excitations: 785 nm (a) and (b), 514.5 nm (c) and (d). Spectra were recorded at
pH 5.5.
B-Fig 3: Langmuir plot of parietin (top). SERS spectra of parietin at pH 8: 5 × 10−5 M (a), 8 ×
10−6 M (b), 2 × 10−6 M (c), 5 × 10−7 M (d) and 2 × 10−7 M (e). Laser excitation at 442 nm.
Acknowledgement. This work was supported (i) by the Agency of the Ministry of Education of
Slovak Republic for the Structural funds of the European Union, Operational program
Research and Development (Contracts: NanoBioSens ITMS code: 26220220107, SEPO II
ITMS code: 26220120039 and CEVA ITMS code: 26220120040), and (ii) by the project
CELIM funded by 7FP EU (REGPOT).
References
1. Solhaug, K.A., Gauslaa, Y. (1996) Oecologia 108, 412–418.
2. Agarwal,S.K., Singh,S.S., Verma,S., Kumar, S. (2000) J. Ethnopharmacol. 72, 43–46.
3. Bačkorová, M., Bačkor, M., Mikes, J., Jendzelovsky, R., Fedoročko, P. (2011)
Toxicol. In Vitro 25, 37–44.
85
Preliminary results of in vitro cytotoxicity testing of bacterial magnetic nanoparticles
aZ. VARCHULOVÁ NOVÁKOVÁ, bM. TIMKO, bA. HASHIM, bM. MOLČAN, aM. KUNIAKOVÁ, a L. ORAVCOVÁ, cM. CSÖBÖNYEIOVÁ, aD. BÖHMER,
aĽ. DANIŠOVIČ
aInstitute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, SK-811 08 Bratislava, Slovakia; bInstitute of Experimental Physics,
SAS, Watsonova 47, 040 04 Košice, Slovakia, cInstitute of Histology and Embryology, Sasinkova 4, , 811 08 Bratislava, Slovakia
Permanent effort in the application of drugs, particularly drugs in the treatment of
cancers is to maximize their therapeutic effect and minimize adverse effects. New approaches
focus on the targeted effect to malignant cells without damaging normal tissue and the
development of resistance [1]. There is emerging new treatment using magnetic particles
(magnetosomes), which are characterized by superparamagnetism due to external magnetic
field. Magnetosomes arise in the process of biomineralization inside the magnetotactic
bacteria. They can offer attractive possibilities of biomedical application, including targeted
transport of biologically active substances, hyperthermia, MRI, etc. The main goal of present
study was to test cytotoxicity of magnetosomes to verify functionality (effect on cancer cells)
and to assess biocompatibility (harmless to healthy cells). Bacterial magnetosomes were
synthesized by biomineralization process of magnetotactic bacteria Magnetospirillum strain
AMB-1 at Institute of Experimental Physics, SAV in Košice [2]. The cell lines HCT-116
(colon cancer cells) and A549 (human lung adenocarcinoma epithelial cell line) were used as
biological model in cytotoxicity assays performed by MTT test. Moreover, we analyzed
apoptosis by staining the nuclei by propidium iodide as well as the kinetics of proliferation by
plotting the growth curves. Preliminary results proved that analyzed magnetosomes did not
affect the proliferation of selected cells and did not induce apoptosis in comparison with
control. According to obtained results it should be emphasized that after further investigations
magnetosomes should be safely used in biomedicine.
References
1. Gupta, P.K., Hung. C.T. (1993) in Magnetically controlled targeted chemotherapy, Microspheres and regional cancer therapy. CRC Press Inc. Boca Raton, FL, 1-59.
2. Hashim, A., Molčan, M., Kováč, J., Varchulová, Z., Gojzewski, H., Makowski, M., Kopčansky, P., Tomori, Z., Timko, M. (2012) Acta Physica Polonica A. 121, 1250-1252.
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Activated Charcoals Supporting Nanomagnets with Antimicrobial Character
aE. VALUŠOVÁ, bP. PRISTAŠ, bP. JAVORSKÝ, a, cM. ANTALÍK, aM. TIMKO
a Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Watsonova 47, Košice, 040 01, Slovakia bInstitute of Animal Physiology Slovak Academy of
Sciences, Šoltésovej 4-6, 040 01 Košice, Slovakia cDepartment of Biochemistry, Faculty of
Science, P. J. Šafárik University, Moyzesova 11, Košice, 040 01, Slovakia
Materials that contain nanomagnets have shown potential for use in advanced applications
because of their particularly interesting properties [1]. Of special interest are those materials
that exhibit superparamagnetic behavior at room temperature. In groundwater treatment
applications, the development of superparamagnetic composites has gained considerable
attention.
Activated carbon supporting magnetic carrier may be additionally combined with silver to
develop composites. Magnetic separation would be a rapid and effective method of removing
these magnetic composites supporting silver as antimicrobial agents from the treated water.
Using a magnet, the thus-removed magnetic composites supporting silver could therefore be
reused for water disinfection avoiding any adverse environmental effects which would be
involved in their disposal [2].
This work presents a technique for the preparation of bifunctional silver-magnetite
activated carbon composites (labelled MCAG) with both antimicrobial and magnetic
properties. The magnetic property is provided by magnetite (Fe3O4), which has been
immobilized onto porous carbon material (charcoal) using an approach which has been
described previously [3]. A permanent magnet is used in the preparation of final silver co-
adsorbed active magnetic carbon particles, a process which negates the need for centrifugation
or filtration procedures. The targeted antimicrobial properties of MCAG were evidenced
against all river water bacteria or Pseudomonas koreensis and Bacillus mycoides cultures
isolated from river water. The bacterial counts in river water samples were reduced by five
orders of magnitude following 30 min of treatment using 1 g l-1 of MCAG at room
temperature. The MCAG thus act as a magnetic filter uptaking bacteria. This idea confirmed
the PCR analysis in which the bacterial DNA was detectable in the sediment after treatment
processing.
The magnetic behavior of MCAG composites has been validated by the hysteresis loop
measured using SQUID magnetometer at 282 K. The magnetic properties of MCAG
composites allow them to be removed easily from water after the bactericidal effect has
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occurred. These initial results indicate that the combination of magnetic character and biocidal
efficiency introduces MCAG as an excellent candidate for the simple ambulatory disinfection
of surface water.
Acknowledgement. The authors wish to thank the Slovak Grant Agency for support through
VEGA grants No. 2/0025/12, 2/0016/12, APVV grants No. APVV-0171-10,APVV-0526-11and
the European Union Structural Funds under ITMS project code 26220120033, 26220120021
and project code 26220220061.
References
1. Fuertes, A.B., Tartaj, P. (2006) Chem. Mater. 18, 1675-1679.
2. Marambio-Jones, C., Hoek, E.M.V. (2010) J. Nanopart. Res. 12, 1531-1551.
3. Valušová, E., Vandžurová, A., Pristaš, P., Antalík, M., Javorský, P. (2012) Water
Sci. Technol. 66 (12), 2772-2778.
88
Use of the modified glassy carbon CuO/ graphene electrode for the glucose determination
aP. KEŠA, bI. HRMO, a,bM. ANTALÍK
aDepartment of Biochemistry, Faculty of Natural Science UPJŠ, Košice, Slovakia
bInstitute of Experimental Physics SAS, Košice, Slovakia
Nanostructures CuO is promising in the development of non-enzymatic glucose sensor
because of its highly specific surface area, good electrochemical activity and the possibility of
promoting electron transfer reactions at a lower over potential [1, 2]. The mechanism of nano-
copper oxide film formation and factors affecting its electrochemical activity may be studied
by cyclic voltammetry, electrochemical impedance spectroscopy and scanning electron
microscopy [3]. CuO/graphene composite may be used for the construct of sensor for the non-
enzymatic detection of glucose. Detection of glucose is important in the fields of biological,
environmental, and clinical analysis [4]. Raman spectroscopy is a widely used tool for the
characterization of carbon materials, especially considering the fact that conjugated and
double carbon-carbon bonds lead to high Raman intensities. It is used to determine the
number and orientation of layers of graphene, the quality and types of edge, and the effects of
perturbations, such as doping, disorder and functional groups [5]. Copper (II) oxide –
graphene composite materials were prepared on the electrode surface at the solution of CuCl2.
The resulting composite material was characterized by cyclic voltammetry on surface of the
glassy carbon electrode. Prepared copper (II) oxide – graphene nanocomposite was scraped
from the electrode surface and was measured Raman spectra by optical tweezers combined
with micro Raman spectroscopy. This composite nanomaterial was used on determination of
glucose and can be used to construct bio batteries too.
Acknowledgement. This work was supported by the research grants from the Slovak Grant
Agency VEGA 2/0025/12 and VEGA 2/191/11, VVGS-PF-2013-114 and VVGS-PF-2013-127
grants and APVV project 0526/11.
References
1. Batchelor-McAuley, C., Du, Y., Wildgoose, G. G., Compton, R. G. (2008) Sens.
Actuators B: Chem. 135, 230-235.
2. Zhuang, Z. J., Su, X. D., Yuan, H. Y., Sun, Q., Xiao, D., Choi, M. M. F. (2008)
Analyst. 133, 126-132.
3. Wen-Zhi, L., You-Qin, L. (2009) Sensors and Actuators B 141, 147-153.
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4. Yu-Wei, H., Ting-Kang, H., Chia-Liang, S., Yung-Tang, N., Nen-Wen, P., Ming-Der,
G. (2012) Electrochimica Acta 82, 152-157.
5. Basko, D. M. (2013) Nature Nanotechnology 8, 235-246.
90
Change of fluorescence characteristics Ca2+-independent discharged photoprotein obelin
under expose to 40°C
aR. ALIEVA, a,bN. BELOGUROVA, aA. PETROVA, a,bN. KUDRYASHEVA
aSiberian Federal University, Krasnoyarsk, Russia bInstitute of Biophysics SB RAS, Krasnoyarsk, Russia
Photoprotein obelin, an enzyme-substrate complex of polypeptide and 2-
hydroperoxycoelenterazine, is responsible for bioluminescence of the marine hydroid Obelia
longissima. Addition of Ca2+ to the obelin triggers a bioluminescent reaction. Obelin is not
fluorescent, but the product of the bioluminescent reaction, enzyme-bound coelenteramide, is
a fluorescent protein called “Ca2+-discharged” obelin. Discharged obelin is stable and
nontoxic and its spectra are variable, it is a perspective fluorescence marker for biological and
medical research. Hence, spectral characteristics of discharged obelin are of high interest.
Here we examined light-induced fluorescence of Ca2+-independent discharged obelin
(obtained without addition of Ca2+). Ca2+-independent discharged obelin was produced by
lyophilization procedure, than it was exposed to 40°C, time of exposure varied from 0 to 12.5
hours. Study of fluorescence peculiarities of Ca2+-independent discharged obelin has not been
carried out yet.
Emission and excitation spectra of Ca2+-independent discharged obelin were analyzed under
variation of wavelengths of excitation (260-390 nm) and emission (400-700 nm). Emission
spectra depended on excitation wavelength, but excitation spectra did not depended on
emission wavelength. Emission spectra at 310-380 nm excitation (coelenteramide absorption
region) included one peak ( max= 500 nm) with a short wavelength shoulder. Emission spectra
at 260÷300 nm excitation (tryptophan absorption region) included three peaks with 355, 500
and 660 nm maxima, corresponding to fluorescence of tryptophan, coelenteramide, and
hypothetical indole-coelenteramide exciplex, respectively. The red peak (λmax=660 nm) has
been recently found in the photoprotein and reported in [1]. The addition of red color to the
battery of known (violet-yellow) colors increases a potential of obelin application as a
fluorescent biomarker.
It was found that exposure to 40°C decreased fluorescent intensity of coelenteramide and
increased that of tryptophan and exciplex. This is probably due to cessation of energy transfer
from tryptophan to coelenteramide as a result of active center destruction under higher
temperature.
Thus it was found that two factors – high-temperature exposure and excitation wavelength –
can change the emission spectra of discharged obelin in the visible region, and hence the color
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of its fluorescence. All results form a basis for development of new medical techniques
involving obelin as a colored biomarker.
Acknowledgement. This work was supported by: the Program ‘Molecular and Cellular
Biology’ of the RAS; the theme №B-14 от 01.03.2013; Federal Target Program ‘Research
and scientific-pedagogical personnel of innovation in Russia’ for 2009-2013, contract
N 02.740.11.0766 and the Grant of Ministry of Education and Science RF 11.G34.31.0058.
References
1. Alieva, R., Belogurova, N., Petrova, A., Kudryasheva, N. (2013) Anal. & Bioanal.
Chem. 405, 10, 3351-3358.
92
Fluorescence properties of Ca2+-discharged obelin in the presence of ethanol and
glycerine
aА.S. PETROVA, bN.V. BELOGUROVA, aR.R. ALIEVA
aSiberian Federal University, Krasnoyarsk bInstitute of Biophysics SB RAS, Krasnoyarsk
Photoprotein obelin, the enzyme-substrate complex of polypeptide with 2-
hydroperoxycoelenterazine, is responsible for bioluminescence of marine hydroid Obelia
longissima. Addition of Ca2+ to the photoprotein triggers the bioluminescent reaction with
light emission. The product of the bioluminescent reaction – enzyme-bound coelenteramide –
is a fluorescent protein called ‘discharged’ obelin. Because discharged obelin is stable and
nontoxic it can be used as fluorescent marker and indicator [1] of calcium ions in biological
and medical investigations, for example in cytology, histology, and cryology. Therefore, it is
very important to know how accompanying compounds influence the fluorescent
characteristics of the discharged obelin. We chose glycerine and ethanol as accompanying
compounds extensively used in cytology, histology, and cryology.
Fluorescent spectra of discharged obelin were studied under addition of glycerin (С = 0.06 –
0.36 М) and ethanol (С = 0.01 – 1.18 М). Addition of the compounds reduced of fluorescence
intensity, and increased shortwave shoulder (λmax = 420 nm) in emission spectra of discharged
obelin. Excitation spectra did not show the dependency on addition of compounds. Emission
spectra were deconvolved into components using Gauss-based distribution and method of
second derivative. It was found that the emission spectra (λexc = 340 nm) of discharged obelin
were a superposition of three components. Spectral components were attributed to different
fluorescent forms of coelenteramide: protonated, partly protonated and deprotonated forms
with maxima at 420, 503 and 565 nm respectively. It should be noted that increase of
glycerine concentration gradually shifted maximum of protonated form from 417 to 407 nm.
Contributions of the components to experimental spectra were calculated. Addition of
glycerine and ethanol increased contribution of the protonated form, and reduced contribution
of the partly protonated form, contribution of the deprotonated form of coelenteramide did not
change. We suggest that reduction of efficiency of proton transfer in the active centre is due to
destruction of the photoprotein in the presence of glycerine and ethanol. Permissible ranges of
the compound concentrations were defined.
Emission spectra under 280 nm excitation consisted of additional red peak (λmax=660 nm) [2].
It was hypothetically attributed to indole-coelenteramide. Addition of glycerine and ethanol
increased intensity of the red peak. Thus, discharged obelin is stable enough in wide
concentration range of glycerine and ethanol. At the same time, its spectra depend on the
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compound concentrations; this fact should be taken into account using discharged obelin as
fluorescent marker in medical and biological investigations.
References
1. Belogurova, N., Kudryasheva, N. (2010) J.Photochem. Photobiol.B. 101, 103–108.
2. Alieva, R.R., Belogurova, N.V., Petrova, А.S., Kudryasheva, N.S. (2013) Analytical
and Bioanalytical Chemistry 405, 3351-3358.
94
Temperature effects on optical absorption, fluorescence and circular dichroism of heparin-safranin T complex
aJ. KUDLÁČOVÁ, a,bM. ANTALÍK
aDepartment of Biochemistry, Faculty of Natural Sciences UPJŠ, Košice
bInstitute of Experimental Physics SAS, Košice
Heparin is a non-cytotoxic, biodegradable and water soluble natural polysaccharide
that is found exclusively in the granules of subsets of mast cells. It is the most complex
member of the glycosaminoglycan family that assumes a helical conformation and is
-(1→4)
linkages. It has a high negative charge density, the result of sulphate and carboxylate residues
that are present in its structure [1]. An extended linear polysaccharide chain of heparin has
been used extensively as an anticoagulant for decades and is also being investigated as a
possible agent to regulate complement activity and inflammation. Furthermore, heparin can
interfere with the activity of growth factors such as bFGF and VEGF, resulting in the
inhibition of angiogenesis and tumor development.
Heparin has recently received an increasing amount of attention as a drug carrier in
macromolecule–anticancer drug conjugate models. After conjugation to drug is anticoagulant
activity of heparin decreased what reduces the risk of hemorrhagic complications in clinical
applications [2]. A great effort has been put into study physico-chemical properties of heparin
because of its wide use in the treatment different types of diseases. It was experimentally and
clinically found out that heparin has anti-inflammatory effect in a number diseases such as
asthma, cystic fibrosis, ulcerative colitis and Alzheimer diseases but its standardisation for use
in these non-anticoagulant applications is not yet well developed [3].
In this work, the interactions of unfractionated heparin with a fluorescent dye safranin
T, a biological stain used in histology and cytology, were investigated using
spectrophotometric methods, such as UV-vis absorption spectroscopy, fluorescence
spectroscopy and circular dichroism.
Acknowledgement. This work was supported by the Slovak Research and Development
Agency APVV 0526-11, APVV-0171-10, by VEGA 2/0025/12, VVGS-2013-127 and VVGS PF-
2013-114.
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References
1. Ampofo, S.A., Wang, H.M. and Linhardt, R.J. (1991) Anal. Biochem. 199, 249-255.
2. Goodarzi, N., Varshochian, R., Kamalinia, G., Atyabi, F. and Dinarvand, R. (2013)
Carbohydr. Polym. 92, 1280-1293.
3. Mulloy, B., Hogwood, J. and Gray, E. (2010) Biologicals. 38, 459-466.
96
Interaction between carbosilane dendrimers and α-synuclein
aK. MILOWSKA, aE. BARTCZAK, aM. BRYSZEWSKA, bR. GOMEZ, bJ. DE LA MATA, cJ.P. MAJORAL, aT. GABRYELAK
aDepartment of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland bLaboratoire de Chimie de Coordination CNRS, Toulouse,
France cDepartamento de Quimica Inorganica, Universidas de Alcala, Alcala, Spain
α-Synuclein (ASN) is a small cytosol protein found predominantly in the central
nervous system, particularly at the ends of presynaptic neurons of the cerebral cortex,
hippocampus and dopaminergic neurons in the striatum. ASN properties depend on its
conformation and degree of aggregation. In physiological conditions, ASN is involved in the
regulation of dopaminergic system and several reports suggest its potential role in regulation
of synaptic function and neuronal plasticity. Physiological functions of this protein are
disturbed by its aggregation. In the body, oxidative stress, posttranslational modification,
catabolism defects and genetic factors (mutation) can promote ASN aggregation. This process
is accompanied by a conformational transition from random coil or α-helical structure to β-
sheet structure [1-3].
Proteins aggregation reduces their bioavailability, which interferes with its
physiological function, and aggregates may have toxic effects on neurons. Disorders of the
ASN structure play a key role in the pathogenesis of Parkinson’s disease, Alzheimer’s disease
with Lewy bodies, dementia with Lewy bodies, multiple system atrophy and other
neurodegenerative diseases collectively called synucleinopathies [4].
Currently, it is necessary to search for factors contributing to the inhibition of ASN
aggregation, which could have therapeutic significance in neurodegenerative diseases. We
decided to focus on dendrimers, because in the previous studies PAMAM, phosphorus and
viologen-phosphorous dendrimers showed the properties preventing the ASN fibrillation [5-
7].
In this study the interaction between two types of dendrimers (carbosilane and
viologen-carbosilane-phosphorus) and α-synuclein was examined.
We investigated whether used dendrimers can interact with α-synuclein, affect its
conformation and can form protein-dendrimer complexes. The interactions between
dendrimers and ASN were examined by zeta potential measurements and circular dichroism
(CD) techniques.
Based on the obtained results the molar ratios of protein-dendrimer complex were
calculated. Furthermore, the effect of dendrimers on protein secondary structure was studied
using circular dichroism spectroscopy. It was shown, that secondary structure of protein was
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changed as a result of its interaction with dendrimers. This fact reflects binding of dendrimers
to the ASN molecules.
The obtained results contribute to the understanding of the mechanisms of
dendrimers/ASN interactions.
Acknowledgement. This work was supported by the research grant UMO-
2012/04/M/NZ1/00059
References
1. Murphy, D.D., Reuter, S.M., Trojanowski, J.Q., Lee, V.M. (2000) J. Neurosci. 20,
3214-3220.
2. Hong, D.P., Xiong, W., Chang, J.Y, Jiang, Ch. (2011) FEBS Let. 585, 561-566
3. Solecka, J., Adamczyk, A., Strosznajder, J.B. (2005), Postepy Biol. Komorki 2, 343-
357.
4. Beyer, K. (2006) Acta Neuropathol. 112, 237-251.
5. Milowska, K., Malachowska, M., Gabryelak, T. (2011) Int. J. Biol. Macromol. 48,
742–746.
6. Milowska, K., Gabryelak, T., Bryszewska, M., Caminade, A.M., Majoral, J.P. (2012)
Int. J. Biol. Macromol. 50, 1138– 1143.
7. Milowska, K., Grochowina, J., Katir, N., El Kadib, A., Majoral, J.P., Bryszewska, M.,
Gabryelak, T. (2013) Mol. Pharmaceutics, 10, 1131-1137.
98
Influence of PAMAM dendrimers on human insulin aggregation processes.
O. NOWACKA, M. BRYSZEWSKA
Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz, Lodz, Poland
In this study the mechanisms of interaction between PAMAM dendrimers and human
insulin have been investigated by circular dichroism and spectrophotometric methods. The
insulin - blood-glucose-regulating peptide-hormone - consists of 51 amino acids. It is known
that insulin undergoes aggeregation and fibrillation and this is the reason of numerous serious
problems for pharmaceutical production and clinical formulation. The aggeregation and
fibrillation processes lead to reduction of insulin biological activity. The best way to oppose
deposition processes is to prevent them and improve insulin variants. It is known that
dendrimers can prevent the aggregation and fibrillation processes of various peptides and
proteins.
The PAMAM dendrimers are based on ethylenediamine core and branched units which are
built from methyl acrylate and ethylenediamine. We have used PAMAM dendrimers with the
same surface groups (–NH2) of three different generations: 2nd, 3rd and 4th.
The aggregation of insulin molecule was induced by dithiotreitol (DTT) which causes
reduction of disulfide bridges and inactivation of the insulin B-chain. The aggregation was
studied spectrophotometrically at 360nm and by circular dichroism (CD) spectroscopy
between 200-250 nm.
The spectrophotometric results show that in the presence of 2nd and 3rd generations of
PAMAM dendrimers the time of insulin aggregation process is similar to control but in the
presence of 4th generation dendrimer the process is significantly slower.
Experiments have shown that the effect is concentration-dependent: application of
dendrimers in 0.1μM concentration enhances the aggregation process as compared to the
samples containing other concentrations of dendrimers, whereas in samples containing
dendrimers of generation 2nd and 3rd in concentration of 0.01μM the aggregation process was
weakened.
Analysis of the circular dichroism results shows the smallest changes in insulin α and β
structures content for PAMAM dendrimers of generation 2nd in the presence of DTT.
The most visible changes in CD spectrum were observed for dendrimers of 3rd generation.
That can be due to the difference in size of dendrimers. Control experiments show that
PAMAM dendrimers do not trigger aggregation processes in human insulin.
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Formation of cytochrome c fibrils as a result of heme destruction
K. GARAJOVÁ, R. VARHAČ
Department of Biochemistry, Faculty of Science, Pavol Jozef Šafárik University in Košice
Formation of fibrillar structures under specific conditions is typical for many proteins.
The vigorous interest for their detailed study is mainly due to their impact on protein-involved
diseases [1]. Within the proteins with the tendency to form in vitro fibrils are the c-type
cytochromes [2, 3]. They are
electron carriers that contain
a heme group covalently attached
to a polypeptide chain. The
attachment of this group is
necessary for correct holoprotein
fold, function and stability.
It has been shown that
solution conditions play an
important role in fibrillation
process [3, 4]. Our data
demonstrate that cytochrome c
forms structures commonly
associated with protein deposition
diseases in the presence of
hydrogen peroxide under mild
conditions (pH 6, 22 °C). When
hydrogen peroxide is in excess, it
has a destructive effect on heme
group evidenced by the loss of
the Soret peak in the visible
absorption spectrum. As a result,
the polypeptide chain is
susceptible to irreversible
conformational changes leading
to almost complete loss of α-helical content as indicated by a significant decrease in the CD
signal strength of the 222 nm minimum. The investigation of thus modified cytochrome c
using AFM method has revealed the formation of the fibril-like structures (Figure 1B). These
structures are distinct from the typical amyloid fibrils in that they miss both the characteristic
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A B
C
Figure 1 AFM images of (A) a control (native cytochrome c)
and (B) cytochrome c after 1-hour incubation with 60 mM
hydrogen peroxide in 5 mM cacodylate buffer (pH 6) at 22 °C.
(C) Detailed structure of the cytochrome c fibrils along with
their line profile. Concentration of the protein was 27 μM. After
incubation, 5 μl of the protein solution was deposited on a
freshly cleaved mica surface and left dry on air. Images were
taken by a Scanning Probe Microscope (Veeco di Innova, Bruker
AXS Inc., Madison) in a tapping mode under ambient
conditions, using silicon cantilevers (Bruker AFM Probes,
Camarillo) with a nominal frequency of 320 kHz and a spring
f 42 N/
100
β-sheet architecture of the monomer units as well as the helical twist of the entire fibrils.
Instead, unstructured polypeptide chain regions are predominant as observed by the far UV
CD spectrum. Our observations support the idea of a ribbon-like alternative fibrillar structure
that the peptides might form [5]. The structures are long, smooth and branched with a width
of 80-150 nm and height of 3-4 nm (Figure 1C).
The data demonstrate that the destruction of the heme in cytochrome c by hydrogen
peroxide results in the loss of the protein native structure. This might lead to at least partial
unfolding of the protein, giving rise to an increased exposure of the polypeptide backbone and
an enhanced propensity to form atypical fibrillar structures.
Acknowledgements. This work was supported by the research grants VEGA 1/0521/12 and
APVV-0280-11. AFM data were acquired in the Department of Biophysics, Institute of
Experimental Physics SAS in Košice.
References
1. Uversky, V. N., and Fink, A. L. (2004) Biochim. Biophys. Acta 1698, 131-153.
2. Pertinhez, T. A., Bouchard, M., Tomlinson, E. J., Wain, R., Ferguson, S. J., Dobson,
C. M., and Smith, L. J. (2001) FEBS Lett. 495, 184-186.
3. Ramakrishna, D., Prasad, M. D., and Bhuyan, A. K. (2012) Arch. Biochem. Biophys.
528, 67-71.
4. Alakoskela, J.-M., Jutila, A., Simonsen, A. C., Pirneskoski, J., Pyhäjoki, S., Turunen,
R., Marttila, S., Mouritsen, O. G., Goormaghtigh, E., Kinnunen, P. K. J. (2006)
Biochemistry 45, 13447-13453.
5. Zhang, S., Andreasen, M., Nielsen, J. T., Liu, L., Nielsen, E. H., Song, J., Ji, G., Sun,
F., Skrydstrup, T., Besenbacher, F., Nielsen, N. C., Otzen, D. E., Dong, M. (2013)
Proc. Natl. Acad. Sci. U.S.A. 110, 2798-2803.
101
Effect of Nanospheres on Insulin Fibril Formation
a, b Z. BEDNARIKOVA, a M. KONERACKA, a V. ZAVISOVA, a K. SIPOSOVA, a M.
KUBOVCIKOVA, a P. KOPCANSKY, c V. GIRMAN, a Z. GAZOVA
aInstitute of Experimental Physics SAS, Košice, Slovakia bDepartment of Biochemistry,
Faculty of Science, Safarik University, Košice, Slovakia cDepartment of Condensed Matter
Physics, Faculty of Science, Safarik University, Košice, Slovakia
Amyloid fibrils are highly ordered protein aggregates appearing in a variety of more than
20 human amyloid-related diseases including Alzheimer´s and Parkinson´s diseases, diabetes
or systemic amyloidosis. The insoluble amyloid aggregates are formed mainly from one type
of poly/peptide depending on the particular disease [1]. Despite the variation in the amino
acid sequence and native structure of these proteins, amyloid fibrils share a several common
structural and spectroscopic properties [2]. Insulin amyloid aggregates have been observed in
patients long-term treated with injected insulin and cause problems also in the storage of this
drug and in application of insulin pumps.
Finding of new substances able to inhibit fibril formation or destroy amyloid fibrils have
great diagnostic and therapeutic impact. It was found that nanoparticles interfere with protein
aggregation significantly. Their potential to affect the protein fibrillization is a function of the
physico-chemical properties of nanoparticles, such as the surface charge, size or
surface/volume ratio [3].
We investigated the effect of a two types of polylactic acid (PLA) nanospheres: i) PLA -
spherical nanospheres formed from PLA with size of ~123 nm, ii) BSA_NS - PLA
nanospheres dosed by Fe3O4 nanoparticles and modified with different amount of bovine
serum albumin (BSA) (BSA/ Fe3O4 ratio = 0.1, 1.0 or 10.0) with size of ~ 134, 127 and 150
nm, respectively on the fibrillization of insulin. Nanospheres were prepared by modified
nanoprecipitation method where Fe3O4 nanoparticles were loaded into biopolymer PLA
nanospheres. Insulin fibrils were achieved by incubation of soluble protein at neutral pH, high
temperature and constant stirring. The insulin fibril formation and effect of nanospheres were
verified through Thioflavin T (ThT) fluorescence assay and transmission electron
microscopy.
It was observed that PLA nanospheres are able to inhibit insulin amyloid aggregation. The
extent of anti-amyloidogenic activity was affected by structure and physico-chemical features
of studied nanospheres. PLA nanospheres without magnetite core and modification showed
low inhibition activity. Nanospheres dosed by Fe3O4 nanoparticles and modified with
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different amount of BSA showed a diverse inhibitory activities. An interesting finding is that
the inhibitory activity of nanospheres is inversely correlated with their size. For the largest
particles with BSA/ Fe3O4 ratio = 10 the lowest inhibitory activity was proved. The most
intensive inhibitory activity was detected for the smallest nanospheres with BSA/ Fe3O4 ratio
= 1.
We concluded that PLA nanospheres are able to inhibit insulin fibrillization and the
extent of this anti-amyloidogenic activity is affected by their size and presence of Fe3O4
nanoparticles. The most effective inhibitors were PLA nanospheres dosed by Fe3O4
nanoparticles and modified BSA with BSA/ Fe3O4 ratio = 1 probably due to their lowest size.
Acknowledgement. This work was supported by VEGA 0181, 0041, APVV0171-10,
VVGS98/13-14 and ESF 26110230061, 26220220005 and 2622012033.
References
1. Dobson, C.M., Karplus, M., (1999) Curr. Opin. Struct. Biol. 9, 92-101.
2. Sunde, M., Blake, C.C.F. (1997) Adv. Prot. Chem. 50, 123–159.
3. Siposova, K., Kubovcikova, M., Bednarikova, Z., Koneracka, M., Zavisova, V.,
Antosova, A., Kopcansky, P., Daxnerova, Z., Gazova, Z. (2012) Nanotechnology 23, 1-
10.
103
Experimental conditions of in vitro lysozyme amyloid fibrillization determine their
properties
aA. ANTOSOVA,a,bZ. BEDNARIKOVA, aK. SIPOSOVA, aE. DEMJEN, aJ. MAREKaZ. GAZOVA
aDepartment of Biophysics, Institute of Experimental Physics SAS, Kosice, Slovakia bDepartment of Biochemistry, Faculty of Science, P. J. Safarik University, Kosice, Slovakia
The knowledge of the propensity of protein molecules to self-assemble into highly
ordered fibrillar aggregates is important for understanding of the origin of many disorders
ranging from Alzheimer’s disease to systemic lysozyme amyloidosis. Lysozyme systemic
amyloidosis is a fatal hereditary disease associated with the deposition of amyloid fibrils in
the spleen, liver and kidney [1]. Mature amyloid fibrils of lysozyme prepared in vitro are
relatively straight and often contain several protofilaments in a periodic twist. The number of
protofilaments and the period of twisting are determined by experimental conditions of fibril
self-assembly [2].
Using a combination of spectroscopic (ThT fluorescence assay, circular dichroism)
and microscopic (atomic force microscopy) techniques we studied in vitro fibrillization and
morphology of hen egg white lysozyme amyloid fibrils at two different experimental
conditions. Lysozyme fibrils were formed at i) pH 2.7, 65 °C in the presence of a saline
solution (80 mM NaCl) after 2 h intensive agitation (LF-NaCl) and at ii) pH 2.5, 65 °C in
ultrapure water without addition of salts after 27 h vigorous stirring (LF-UPW). The kinetics
of the lysozyme fibrillization was monitored by ThT assay to examine the process of protein
polymerization at both experimental conditions. The shape of both growth curves retains the
typical sigmoidal profile of amyloid fibrillization with lag phase of about 1 h (for LF-NaCl
fibrils) and 10 h for LF-UPW fibrils. The differences were observed also for time at which the
values of fluorescence intensities achieved the steady-state values; namely, 2 h for LF-NaCl
fibrils and 25 h for LF-UPW fibrils.
AFM and image analysis using a modified live-wire algorithm [3, 4] were used to
characterize the morphology of obtained lysozyme fibrils. As it is apparent from Figure 1., the
morphology of the LF-NaCl and LF-UPW fibrils is significantly different. Fibrils prepared in
presence of salt (LF-NaCl) are shorter and have higher tendency to associate into the bundles
(Figure 1A) compare to fibrils prepared in ultrapure water (LF-UPW) (Figure 1B). For both
types of fibrils the twisting typical for amyloid fibrillar structures is observed.
To obtain more data about formed lysozyme fibrils the CD spectroscopy was used to
examine the secondary structure in more details. The CD spectra detected for LF-NaCl and
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LF-UPW were substantially different than that detected for native lysozyme. In case of both
types of fibrils the content of β–sheet increased to 33% (LF-NaCl) or 27% (LF-UPW)
compare to native lysozyme (16%).
The obtained data suggest the influence of experimental conditions on the kinetics
lysozyme amyloid polymerization and morphology of mature fibrils. Lysozyme protein
aggregation was facilitated in presence of salt (LF-NaCl), the fibrils are shorter compare to
those ones prepared in ultrapure water (LF-UPW), which are longer and more separated. The
obtained results may contribute to a better understanding of the processes of amyloid self-
assembly of globular proteins.
Acknowledgement: This work was supported by the research grant from the Slovak
Grant Agency VEGA 0181 and the project ESF 26110230061, 26220220005 and
26110230097 and APVV 0171-10, APVV 0526-11.
References
1. Buell, A. K. (2011) J. Am. Chem. Soc. 133, 7737–7743.
2. Kodali, R. and Wetzel, R. Current Opinion in Structural Biology 17, 2007,48–57.
3. Marek, J., Demjenova, E., Tomori, Z., Janacek, J., Zolotova, I., Valle, F., Favre, M.,
and Dietler, G. (2005) Cytometry Part A 63, 87-93.
4. Matas, J., Shao, Z., Kittler, J. Lecture Notes in Computer Science 974, 1995, 83-88.
Figure 1. AFM images of mature lysozyme fibrils LF-NaCl (A) and LF-UPW (B).
1 μm
A B
105
time (min)0 50 100 150 200 250 300 350 400Th
T flu
ores
cenc
e in
tens
ity (a
. u.)
0
2e+4
4e+4
6e+4
8e+4
1e+5EMIM BF4 EMIM acultrapure H2O
Fig.1: Time-dependence of amyloid fibrillization of 10 μM lysozyme (black triangels) at pH 2,5 and in the presence of both ILs 1% v/v detected by ThT assay.
Utilization of imidazolium-based ionic liquids as trigger for lysozyme amyloid
fibrillization
aD. FEDUNOVÁ, aA ANTOŠOVÁ, aJ. MAREK, aE. DEMJÉN, a,bZ. BEDNÁRIKOVÁ, aK. ŠIPOŠOVÁ, aJ. BÁGEĽOVÁ, aZ. GAŽOVÁ
aDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Kosice, Slovakia bDepartment of Biochemistry, Faculty of Science, P. J. Šafárik University,
Košice, Slovakia
Formation of protein amyloid fibrils has been shown to be a generic property of
polypeptide chain. The specific conditions have already been established for direct growth of
amyloid fibrils in vitro for wide range of proteins. Solvent properties play an important role
in controlling of fibrillization process. Recently, ionic liquids (ILs) – compounds consisting
only from ions and possessing melting point below 100oC – have been identified as useful
solvents and additives for diverse processes due to their unique properties [1]. Several studies
have shown that ILs can effectively promote or alter process of protein amyloid self-assembly
[2]. One of the main factors affecting amyloid fibrillization is destabilization of protein native
state. Therefore, it was assumed that direct selection of stabilizing or destabilizing ions (based
on Hoffmeister-type characteristics) could lead to predictable effects on inhibition or
acceleration of amyloid fibrillization. However, various contradictory examples, well-
correlated as well as with no correlation with Hofmeister ranking, were reported for effects of
ILs on amyloid fibrillization [2].
In this work we have studied the effect of
1-ethyl-3-methylimidazolium ionic liquids with
two different anions – tetrafluoroborate (as
chaotropic) [EMIMBF4] and acetate (as
kosmotropic) [EMIMac] on kinetics of lysozyme
amyloid fibrillization and morphology of formed
fibrils. Thioflavin T fluorescence assay has been
used to monitor the kinetics of fibrillization
process. We have found that lysozyme at 2
mg/ml concentration dissolved in water acidified
to pH 2.5 and incubated at 65 oC with intense stirring doesn’t transform into fibrils during
more than 8 days. However, addition of either of ILs [1% (v/v)] into the solution effectively
triggers amyloid fibrillization. At the presence of EMIMBF4, the growth curve has typical
sigmoidal profile with lag phase about 50 minutes. The steady-state fluorescence intensity
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value is reached at 1.5 hours. Even though shorter lag phase is observed at the presence of
EMIMac, the kinetics pattern is broader with lesser steepness and longer time needed for
achievement of steady-state intensity (250 min). Studied ILs have also slightly different effect
on lysozyme secondary structure. CD measurements suggest that with increasing incubation
time the increase of fraction of β-sheet structures is observed, with slightly higher content for
EMIMac (37%) than for EMIMBF4 (31%) for mature fibrils.
AFM method and image analysis using modified live-wire algorithm [3] were used to
visualize and characterize the morphology of obtained fibrils. For both types of fibrils the
twisting typical for amyloid fibrilar structures is observed. The morphology of both type of
fibrils differ slightly in apparent width and high. From image analysis follows that at presence
of EMIMac the two most abundant populations of fibrils are formed with apparent widths
about 70-80 nm and 120-130 nm and the height of 3 and 6 nm. For EMIMBF4, the apparent
width distribution is characterized by curve with main peak at 130 nm and height about 6 nm.
In this work we have found that both ILs are able to trigger lysozyme fibrillization at
low protein concentration as well as low concentration of ILs itself (3-4 mM). Different
properties of used anions have certain effect on lysozyme fibrillization manifested by
different kinetics profile, higher abundance of β-sheet secondary structure and variation of
fibril apparent width and high. Comparing to tetrafluoroborate anion, presence of acetate
anion leads to slowing down the fibrillization process and the formation of fibrils with
variable width and height. The acceleration of fibrillization by ILs is of importance not only
for screening of potential therapeutic compounds against amyloid-related diseases, but also
for preparation of novel biomaterials.
Acknowledgement. This work was supported by research grants from the Slovak Grant
Agency VEGA, 0155, 0181, ESF 26110230097 and APVV 0171-10 and 0526-11.
References
1. Ionic Liquids: From Knowledge to Applications, ed. R. D. Rogers, N. V. Plechkova and K. R. Seddon, (2009) ACS Symposium Series, vol. 1030.
2. Kalhor,H. R., Kamizi,M., Akbari,J., Heydari, A. (2009) Biomacromolecules 10, 2468–2475.
3. Marek, J., Demjenova, E., Tomori, Z., Janacek, J., Zolotova, I., Valle, F., Favre, M., and Dietler, G. (2005) Cytometry Part A 63, 87-93.
107
Effect of small molecule spirobrassinin and magnetite nanoparticle on ThT fluorescence
in cerebrospinal fluid from patients with Alzheimer disease and multiple sclerosis
aZ. KRISTOFIKOVA, bK. SIPOSOVA, a,cA. BARTOS, cJ. KOTOUCOVA, aD. RIPOVA,b,dZ. GAZOVA
aPrague Psychiatric Centre, Alzheimer Disease Centre, Ustavni 91, 181 03 Prague 8, Czech
Republic bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of
Sciences, Kosice, Slovakia cCharles Universtiy in Prague, Third Faculty of Medicine,
University Hospital Kralovske Vinohrady, Department of Neurology, Srobarova 50, 100 34
Prague 10, Czech Republic dDepartment of Medical and Clinical Biochemistry and
LABMED, Faculty of Medicine, Safarik University, Kosice, Slovakia
Since cerebrospinal fluid (CSF) is in direct contact with the environment of the central
nervous system, it is suggested that changes in biochemical composition of brain parenchyma
are reflected here. Therefore, diagnosis of neurological disorders (including of multiple
sclerosis) as well as of neurodegenerative diseases (including of Alzheimer disease) is based
among others on investigations of CSF samples. In addition, CSF samples can be
advantageously used to an evaluation either of new specific biomarkers of various diseases or
of actions of various drugs in experiments in vitro. Previously we have evaluted in vitro
effects of anti-aggregation operated magnetite nanoparticles on the samples from patients
with Alzheimer disease and multiple sclerosis by means of thioflavin T (ThT) based
fluorescence. We have observed attenuated actions of nanoparticles on misfolded
peptides/proteins preaggregated under conditions in vivo in patients with Alzheimer diseases
when compared to old controls but no differences between patients with multiple sclerosis
and young controls [1].
In this study, we have evaluated in vitro effects of ferrofluid suspension or of phytoalexin
spirobrassinin in cerebrospinal fluid of patients with multiple sclerosis, Alzheimer disease and
brain infarction. We have found significant differences in native fluorescence (λ excitation =
440 nm, λ emission = 485 nm) among particular groups (young controls < multiple sclerosis <
Alzheimer disease = brain infarction = old controls). Differences among groups were
observed also in thioflavin T based fluorescence (young controls = brain infarction = multiple
sclerosis < Alzheimer disease < old controls), however, the most marked change from native
to thioflavin T based fluorescence was found in young controls. Both ferrofluid and
spirobrassinin evoked drops in thioflavin T based fluorescence, nevertheless, ferrofluid was
more efficient in old and spirobrassinin in multiple sclerosis group, both compared to young
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controls. The results are discussed in relation to aggregated peptides/proteins and to
liposoluble fluorescent products of lipid peroxidation.
Acknowledgement. This work was supported by the projects: in Slovakia - VEGA 0181, APVV
SK-RO-0016-12, APVV-0171-10 SF 26220220005, 26110230061 and 26110230097; in the
Czech Republic - IGA NT 11225 and IGA NT 13183 grants and by MH CZ - DRO (PCP,
00023752) project. The authors thank M. Koneracka and V. Zavisova for preparing of the
ferrofluid and P. Kutschy for synthesis and providing of the spirobrassinin.
References
1. Gazova, Z., Antosova, A., Kristofikova, Z., Bartos, A., Ricny, J., Cechova, L.,
Klaschka, J. and Ripova, D. (2010) Mol. BioSyst. 6, 2200-2205.
109
Inhibition of Aβ peptide fibrillization by tripeptides – importance of Proline
aM. H. VIET, bK. SIPOSOVA, aM. SUAN LI, b,cZ. BEDNARIKOVA, dT. TRANG
NGUYEN, b,eZ. GAZOVA
aInstitute of Physics, Polish Academy of Sciences Al. Lotnikow 32/46, 02-668 Warsaw, Poland bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Kosice, Slovakia cDepartment of Biochemistry, Faculty of Science, Safarik University,
Kosice, Slovakia dInstitute for Computational Science and Technology, 6 Quarter, Linh
Trung Ward, Thu Duc District, Ho Chi Minh City, Vietnam eDepartment of Medical and
Clinical Biochemistry and LABMED, Faculty of Medicine, Safarik University, Kosice,
Slovakia
Aβ peptide, produced through endo-proteolytic cleavage of amyloid precursor protein, is
thought to be involved in the death of neural cells in Alzheimer's disease (AD), which is an
age-related cognitive impairment. Presently, there is no cure or treatment for AD, and
significant effort has been made to find efficient drugs to cope with it. Recent studies indicate
that amyloid aggregates of Aβ peptide are the main toxic agents involved in pathology of this
disease. Therefore, one of the most straightforward therapeutical approaches is the targeting
of Aβ amyloid fibrillization.
In our study we perform comprehensive study of the effect of all possible three-amino
acid peptides (8000 tripeptides in total) on Aβ peptide amyloid aggregation using the
molecular modeling and experimental methods. By in silico screening we investigated the
binding affinity of tripeptides to Aβ. Combining the docking and molecular mechanics-
Poisson-Boltzman surface area methods we predict that WWW, PWW, WPW and WWP have
significant binding affinity and ability to inhibit fibrillization with inhibition constants in the
rage of μM - nM.
By in vitro experiments the abilities of selected the most effective tripeptides to inhibit
Aβ peptide amyloid fibrillization were investigated by ThT fluorescence assay. Binding of
ThT to assembled β-structures of amyloid fibrils leads to significantly increased fluorescence
intensity of ThT that is not detected for the native Aβ peptide. The inhibitory activities of
tripeptides were examined at three concentrations (1 μM, 60 μM and 200 μM) at constant Aβ
concentration (10μM). The highest fluorescence intensity corresponding to the lowest
inhibitory activity was obtained for WWW tripeptide. Presence of Proline in tripeptide caused
significant decline of fluorescence indicating the increase of inhibition of amyloid
fibrillization. Our results suggest that all examined tripeptides significantly inhibit Aβ
fibrillization as the ThT fluorescence decreases to intensities lower than 50 % of the control
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sample (Aβ alone). The highest inhibitory effect was detected for tripeptid WWP (~ 85 %)
with Proline at the last position of amino acid chain. The similar inhibitory efficiency was
observed for WPW. AFM microscopy was used to visualize the inhibitory activities. The
obtained images confirmed that presence of tripeptides caused extensive reduction of fibrillar
aggregates and changes in typical amyloid morphology of fibrils compared to fibrils produced
by Aβ alone. For the most effective tripeptides the IC50values were determined.
Using in silico and in vitro methods we have showed that tripeptides can affect Aβ
amyloid fibrillization. The molecular binding approach suggests that tripeptides are preferably
located near hydrophobic residues of Aβ. The IC50values determined from dose-response
curves were found in micromolar range, the lowest values were found for tripeptides
containing Proline. Experimental results fully confirmed theoretically predicted significance
of Proline in tripeptide sequence.
Acknowledgement: This work was supported by grants VEGA 0181, No
2011/01/B/NZ1/01622, ESF 26110230061and 26110230097, APVV 0171-10, APVV SK-RO-
0016-12, and VVGS 38/12-13, 98/13-14.
111
Morphological diversity of lysozyme amyloid fibrils induce different cytotoxicity
aM.-M. MOCANU, aC. GANEA, b,cK. SIPOSOVA, aA. FILIPPI, dE. RADU, bE. DEMJEN, bJ. MAREK, b,cZ. BEDNARIKOVA, bA. ANTOSOVA, b,e *Z. GAZOVA
aDepartment of Biophysics "Carol Davila" University of Medicine and Pharmacy, Bucharest,
Romania bDepartment of Biophysics, Institute of Experimental Physics, Slovak Academy of
Sciences, Kosice, Slovakia cDepartment of Biochemistry, Faculty of Science, Safarik
University, Kosice, Slovakia dDepartment of Cellular and Molecular Medicine, "Carol
Davila" University of Medicine and Pharmacy, Bucharest eDepartment of Medical and
Clinical Biochemistry and LABMED, Faculty of Medicine, Safarik University, Kosice,
Slovakia
The ability to form amyloid structures was considered to be an unusual property of the
polypeptide chains found in the deposits associated with the various amyloid diseases [1].
Although the basic structural arrangement of the cross-β structure is conserved for different
fibrils, there are different possibilities how they can pack into the three-dimensional fibril
structure. Morphological variability of amyloid structure probably depends on the number and
disposition of protofilaments [2].
In our study, we characterized polymorphism of lysozyme amyloid fibrils and its
implications in the cytotoxicity. The cytotoxic effect of both types of fibrils on the LLC-PK1
renal cells was evaluated by growth curves and apoptotic/necrotic assays. Lysozyme amyloid
fibrillization was performed at two different pH values, namely at pH 2.7 (strongly
destabilizing conditions - LAF2) and at pH 6.0 (moderate, more physiological conditions -
LAF6). Presence of fibrils was confirmed by increased ThT fluorescence intensity upon
binding of ThT to amyloid fibrillar aggregates.
The shape of both curves of lysozyme fibrillization indicates a cooperative, nucleation-
dependent growth of amyloid fibrils with a typical lag phase (formation of nuclei) and steep
increase of fluorescence intensity corresponding to fibril polymerization. Different shape of
curves suggests differences in morphology of amyloid fibrils, created under different pH
conditions during fibrillization, namely the formation of different amyloid intermediates.
AFM microscopy confirmed different morphology of fibrils. LAF2 and LAF6 formed the
linear non-branched fibrils with features typical for amyloid species. LAF2, prepared at acid
pH, consist of the long fibrils whereas LAF6 fibrils, formed at pH 6, were thicker and shorter
with strong tendency to associate laterally.
LLC-PK1 epithelial renal cells were used to evaluate the cytotoxicity of lysozyme amyloid
fibrils with different morphology. We observed that mature lysozyme fibrils are harmful to
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the renal cells and their morphology significantly changed in the presence of both types of
fibrils. Study of the effect of lysozyme fibrils on cell proliferation indicates a dose-dependent
inhibition of LLC-PK1 cell growth by both types of fibrils (Tab. 1). To clarify the mechanism
of the cell death we have been interested if LAF promoted the cell death by apoptotic or
necrotic processes.
time
hours
presence of LAF6 presence of LAF2
control 0.1μg/ml 10μg/ml 100μg/ml control 0.1μg/ml 10μg/ml 100μg/ml
0 5000 5000 5000 5000 5000 5000 5000 5000
24 7100 4750 3550 1900 21900 13650 10650 12550
72 41900 27500 3350 1650 46650 48000 20500 21050
120 59550 66750 3600 350 62900 48700 25150 14100
Our studies revealed that mature lysozyme amyloid fibrils are actively involved in the
cytotoxic processes, the impairment of viability of cells exposed to the fibrillar aggregates
depends on the morphology of fibrils. The inhibition of cell growth and accumulation of the
cells in late apoptotic/ necrotic stages were significantly depended on the structural properties
of studied fibrils.
Acknowledgement. This work was supported by the projects: VEGA 0181, APVV SK-RO-
0016-12, APVV-0171-10 and 0526-11, ESF 26110230061 and the grant from the Romanian
National Authority for Scientific Research CNCS – UEFISCDI, project number PN-II-RU-
TE-2011-3-0204.
References
1. Dobson, C.M. (2003) Nature 426, 884-890.2. Fändrich, M., Meinhardt, J., Grigorieff, N. (2009) Prion 3, 89-93.
Table 1. Number of LLC-PK1 cells incubated with increasing concentrations of LAF2 and LAF6; control - untreated cells.
113
Preparation of recombinant fragments of human cardiac ryanodine receptor
J. NOVAKOVA, V. BAUEROVA-HLINKOVA, E. SCHILLEROVA, J. GASPERIK, E. HOSTINOVA, L. BORKO, A. ZAHRADNIKOVA, E. KUTEJOVA, J. SEVCIK
Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, 845 51 Bratislava, Slovak Republic
Human ryanodine receptor2 (hRyR2) is a transmembrane protein belonging to the
family of ryanodine receptors (RyRs), which play an important role in transport of Ca2+ ions,
required for proper muscular contraction. RyRs form Ca2+ channels which are located in the
membrane of the sarcoplasmatic/endopasmatic reticulum and enable the flow of calcium ions
from the sarcoplasmatic reticulum into the cytoplasm. They are the largest known ion
channels (>2MDa) and exist as three mammalian isoforms (RyR 1–3), all of which are
homotetrameric proteins that are regulated by phosphorylation, redox modifications, and a
variety of small proteins and ions. A hypothesis has been postulated that the N-terminal and
central part of ryanodine receptor interact together leading to proper opening/closing of the
RyR channel. Moreover, N-terminal, central and C-terminal regions contain mutation clusters
causing cardiac diseases-arrhythmogenic right ventricular dysplasia or catecholaminergic
polymorphic ventricular tachycardia (in RyR2 isoform) or malignant hyperthermia (in RyR1
isoform) which are the most common causes of human death.
The structure of the whole ryanodine receptor has been solved only by electron microscopy.
In our study we have focused on the central part of cardiac hRyR2. We have cloned,
expressed and purified several fragments from the region of 2000 and 2400 aa residues for
further structural studies.
Acknowledgement. This work was supported by Slovak Research and Development Agency
APVV-0628-10.
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The proteolytic reaction of papain; ONIOM-type hybrid QM/MM study
aA. FEKETE, a,bL. MUSZBEK and a,bI. KOMÁROMI
aClinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary and bVascular Biology, Haemosthasis and Thrombosis Research Group
of The Hungarian Academy of Sciences and University of Debrecen, Debrecen, Hungary
The papain is a widely studied cysteine protease with sequential, structural and
functional similarity to e.g. human cathepsins and even to transglutaminases. Owing to the
experimental and theoretical works1,2 have been carried out on this enzyme during the last two
decades, a semi-quantitative model for the cysteine protease reactions could be derived.
Nevertheless, some details, especially the exact sequence of bond breakage and formation and
the relative energies of the stationary points (Michaelis complex (Figure 1.), intermedieres,
transition states and products) on the potential energy surfaces have to be revealed.
Figure 1. The structure of papain (ribbons), N-Methylacetamide and the catalytic triad
(licorice). Visualized with Chimera (http://www.cgl.ucsf.edu/chimera/), the ESP surface was
calculated with DelPhi (http://compbio.clemson.edu/delphi.php).
Our aim was to determine a feasible reaction path for the protease reaction of papain
which connects the Michaelis complex to the acyl-enzyme complex as well as the acyl-
enzyme complex to the products. Instead of the hybrid QM/MM molecular dynamics
simulation, in which only semi-empirical or relatively low level ab initio methods can be used
even on modern supercomputers, a static model allowing higher levels of theories and larger
basis sets was used.
ONIOM-type hybrid QM/MM calculations with electronic embedding
approximation3,4 were used to map the potential energy surfaces and to find the local minima
and first order saddle points (transition states) on its. The existences of these specific points
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were proven by frequency analysis. Modern density functional theories, taking into account
the dispersion effect as well, were applied. Standard valence double zeta and valence triple
zeta quality basis sets with and without diffuse functions were used throughout the study.
Only the vicinity of catalytic site and the catalytic site itself were allowed to move during
optimization. Poisson-Boltzmann (PB) model was used to calculate the electrostatic part of
solvent effect on energy differences.
Besides the “exact” (at the levels of theories we used) stationary point geometries,
energies and relative energies the most important results we obtained the instability of the
“classical” tetrahedral intermediate state. Instead, we found that the proton from the
protonated histidine spontaneously goes to the N atom of the scissile peptide bond resulting in
a zwitterionic tetrahedral intermediate. The finding that the PB reaction field theory without
any specific hydrogen bond is sufficient to favor the zwitterionic catalytic center over the
neutral one can be emphasized as well.
Acknowledgement. This work was partially supported by the Hungarian Scientific Research Fund (OTKA K-106294), the Hungarian National Infrastructure Development Program (grant: NIIF-1057) as well as TÁMOP-4.2.2.A-11/1/KONV-2012-0045 and the European Union and the European Social Fund through project Supercomputer, the national virtual lab (grant no.: TAMOP-4.2.2.C-11/1/KONV-2012-0010).
References
1. Beynon, R., and Bond, J.S. (1994) Proteolytic enzymes: a practical approach, Oxford University Press
2. Mladenovic, M., Fink, R.F., Thiel, W., Schirmeister, T., and Engels, B. (2008) J. Am. Chem. Soc. 130, 8696-8705.
3. Dapprich, S., Komaromi, I., Byun, K.S., Morokuma, K. and Frisch, M.J. (1999) THEO. CHEM. 461-462, 1-21.
4. Vreven, T., Byun, K.S., Komaromi, I., Dapprich, S., Montgomery Jr. J.A., Morokuma, K. and Frisch, M.J. (2006) J. Chem. Theory Comput. 3, 815-26.
116
Mechanism of the irreversible inhibition of human cyclooxygenase-1 by Aspirin as predicted by QM/MM calculations
aL. TÓTH, a,b L. MUSZBEK, b I. KOMÁROMI
a, Clinical Research Center, Medical and Health Science Center, University of Debrecen, Debrecen, Hungary, b, Vascular Biology, Thrombosis and Haemostasis Research Group of
the Hungarian Academy of Sciences at the University of Debrecen, Debrecen, Hungary
Acetylsalicylic acid (aspirin) suppresses the generation of prostaglandin H2, which is
the precursor of thromboxane A2. Aspirin acts as an acetylating agent in which its acetyl
group is covalently attached to a serine residue (S530) in the active site of the
cyclooxygenase-1 enzyme. The exact reaction mechanism has not been revealed by
experimental methods [1-2]. In this study [3] the putative structure of human cyclooxygenase-
1 was constructed from ovine cyclooxygenase-1 by homology modeling and the
acetylsalicylic acid was docked into the arachidonic acid binding cavity of the enzyme. To
characterize the shape of the potential energy surface of the acetylating reaction and to
determine the relative energies of the stationary points on the surface, a series of ONIOM-
type quantummechanical/molecular-mechanical (QM/MM) calculations were carried out at
different QM levels of theories applying electronic embedding approximations. The
acetylsalicylic acid and the surrounding amino acids were included in these calculations.
Frequency analyses were performed to prove the existence of first order saddle points
(representing transition states) and local minima on the potential energy surface [4-6]. It was
found that all levels of theories predicted similar transition state geometries. The activation
energy values, however, demonstrated significant dependence on the methods that were
applied. All the applied “dependable” ab initio and DFT methods predicted that the breakage
of the S530 Oγ-Hγ and formation of the Oγ-C(acetylsalicylic acid carbonyl) bonds occur in a
single elementary step.
Acknowledgments: Support was provided by the National Office of Research and Technology
(JedlikÁnyos grant, NKFP-07-A1-2008-0127), the Hungarian National Infrastructure
Development Program, by the TÁMOP-4.2.2/B-10/1-2010-0024 project and the Hungarian
Academy of Sciences (MTA 11003).This research was realized also in the frames of TÁMOP
4.2.4. A/2-11-1-2012-0001 „National Excellence Program – Elaborating and operating an
inland student and researcher personal support system convergence program”. The project
was subsidized by the European Union and co-financed by the European Social Fund.
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References
1. Bingham, S., Beswick, P.J., Blum, D.E., et al. (2007) Semin. Cell Dev. Biol. 17, 544–
554.
2. Colman, R.W., Marder, V.J., Clowes, A.W., George, J.N. (2005) in Hemostasis and
Thrombosis:Basic Principles and Clinical Practice Fifth edition. Philadelphia:
Lippincott Williams &Wilkins
3. Tóth. L.,Muszbek, L., Komáromi, I. (2013) J. Mol. Graph. Model. 40, 99-109.
4. Vreven, T., Morokuma, K., Farkas, Ö., Schlegel, H.B., Frisch, M.J. (2003) J. Comput.
Chem. 24, 760–769.
5. Dapprich, S., Komáromi, I., Byun, K.S., Morokuma, K., Frisch, M.J. (1999) THEO.
CHEM. 461–462, 1–21.
6. Vreven, T., Byun, K.S., Komáromi, I., Dapprich, S., Montgomery, J.A. Jr, Morokuma,
K., Frisch, M.J. (2006) J. Chem. Theory Comput. 2, 815–826.
118
GLYCOGRID Initiative for Computational Studies of Glycan-Protein Interactions
T. KOZAR
Department of Biophysics, Institute of Experimental Physics, Slovak Academy of Sciences,
Watsonova 47, 040 01 Kosice, Slovakia
Small carbohydrates, oligosaccharides, glycans and glycoconjugates are “friendly”
molecules with a keen interest to “socialize” i.e. interact. Complex glycans are either linear or
branched molecules. They can exist in the cell independently or attached to other
biomolecules, such as glycoproteins, proteoglycans, glycolipids, etc. Carbohydrate oligomers
have an enormous potential for serving as informational macromolecules. Their informational
content is far higher than the informational content of any other biomolecule. The
complexities of glycans and their conjugates, together with the insufficient amount of
structural data in the PDB, complicate our understanding of carbohydrate-protein interactions.
Glycomics [1] is an emerging field, which deals with the structure, function and
interactions of saccharides. Comparable to proteomics, glycomics deals with the structure,
function and interactions of glycans [1]. The increasing importance of glycomics to medicine
was outlined in a recent
review which described the
current view that glycomics is
a novel method that will aid
in the development of new
and improved glyco-
therapeutics for cancer and
inflammation [2]. Detailed
knowledge of the variable
class of glycans and their
interaction patterns (mainly
with proteins and enzymes)
will improve our
understanding of molecular
forces driving many biological and physiological processes.
Proteins interacting with carbohydrates can be grouped into two groups, namely
carbohydrate-binding proteins (CBP) and carbohydrate-modifying (processing) proteins
(CMP). The differences in the specificity, complexity and in the behavior of the members of
these two classes will be used as internal controls in our selection of the appropriate tools,
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Figure 1. Scheme of computational task related to theGLYCOGRIDinitiative.
119
suitable to model their interaction patterns. No changes in the chemical structure of the
carbohydrate occurs in the case of carbohydrate-binding proteins such as lectins for example,
whereas carbohydrate-modifying proteins (enzymes) are modifying the carbohydrate
structure, either adding or detaching a carbohydrate from the glycan structure.
Glycosyltransferases and glycosidases are typical examples of the second class, exhibiting a
remarkably higher level of specificity towards their ligands in comparison to the
carbohydrate-binding proteins.
Computational modeling of complex biological macromolecules on atomic level is a
complex task that requires HPC resources. This is due to number of atoms, environment
(solvent molecules), simulation time and temperature, as all of these variables need to be
accounted for realistic outcomes.
Our recent interest in the glycomics field is concentrated on the use of microarray
technologies, on both, experimental and computational level [3]. For this we started to build
the GLYCOGRID initiative in order to faciliate such studies. Figure 1 shows the major tasks
involved in this initiative. In adition to “GLYCANS” we intended to find glycan analogues by
screening of large libraries of chemical compunds. Results on lectin-ligand interaction
profiles will be presented, illustrating differences in ligand binding to gallectins.
Acknowledgement. This work was supported by Slovak APVV (projects APVV-0171-10,
APVV-0282-11) and VEGA (project 2/0073/10) grant agencies. Supports from EU projects
26220120021, 2622012033, 2611230061 and 26210120002 are gratefully acknowledged as
well
References
1. Feizi, T., Mulloy, B. (2003) Curr. Opin. Struct. Biol. 13, 602-604.
2. Shriver, Z., Raguram, S., Sasisekharan, R. (2004) Nat. Rev. Drug Discov. 3, 863-873.
3. Tkac, J., Katrlik,J., Svitel, J., Gemeiner, P., Kozar, T. (2010) Medicinal Research
Reviews 30, 394-418.
120
Primary processes in photosynthetic bacterial reaction centers:initial terms
M. PUDLÁK, R.PINČÁK
Institute of Experimental Physics SAS, Košice, Slovakia
Photosynthesis is a reaction in which light energy is converted into chemical energy.
The primary process of photosynthesis is carried out by a pigment-protein complex embedded
in the membrane. This complex is named the reaction center (RC). The photosynthetic RC
serves as a photochemical trap. The precise details of the charge separations reactions and
subsequent dark electron transport (ET) form the central question of the conversion of solar
energy into the usable chemical energy. The first RC structurally resolved was of the purple
bacterial RC from Rhodopseudomonas viridis [1]. A remarkable aspect of the RC structures is
the occurrence of two almost identical electron acceptor (labeled A and B) pathways arranged
along the 2C axis relative to the primary charge-separating bacteriochlorophyl dimer (Fig.1).
Fig.1. The RC of purple bacteria. 1 represents bacteriochlorophyl dimer, 2 and 3 represent
bacteriochlorophyl molecules on B and A sides, 4 and 5 represent bacteriopheophitin
molecules on B and A sides, 6 represents quinone molecule. Cytochrome C serve as source of
electron for reaction center.
The initial electron transfer in the bacterial photosynthetic RC occurs almost exclusively
along the cofactors on the A side. The function of the B-side cofactors in RC is unknown.
Here we focus on the initial term which can change the models of primary electron transfer in
photosynthetic bacterial reaction centers. To elucidate the ET in the reaction center we take
into account the initial term which describe the unrelaxed phonon modes. We used the model
presented in [2]. Time evolution of the occupation probabilities is shown in the Fig.2.
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Fig.2, The probabilities to find electron at site 1,2,3.
Acknowledgment. This study was supported by grants from VEGA Grant No. 2/0037/13 and
Ministry of Education Agency for Structural Funds of EU in frame of project 26220120021,
26220120033 and 26110230061.
References
1. Deisenhofer, J., at al. (1985) Nature. 318, 618.
2. Pudlak, M., Pichugin, K.N., Nazmitdinov, R.G., Pincak, R. (2011) Phys.Rev. E 84,
051912.
122
Different effects of low-level laser therapy at 635 and 670 nm on the healing of excisional
and incisional skin wounds in rats
aP. GÁL, bM. MOKRÝ, bB. VIDINSKÝ, bT. VASILENKO, bK. LACJAKOVÁ,bM. SLEZÁK, bM. POLÁKOVÁ, I. bKOVÁČ, aZ. TOMORI
aInstitute of Experimental Physics, Slovak Academy of Sciences, Košice, Slovak RepublicbFaculty of Medicine, Pavol Jozef Šafárik University, Košice, Slovak Republic
Introduction: Although, the exact mechanism of action of LLLT in wound healing is still not
fully clarified, it has been documented that red lasers at a dose of either 4 or 5 J/cm2
accelerate wound closure, increase collagen deposition and reduce inflammation during
wound healing, and thus positively modulate repair processes [1]. In contrast, in in vitro
studies [2,3] and an in vivo study [4] reductions in the rate of healing were observed after
increasing the dose to 10 and 20 J/cm2, respectively. Hence, our study was aimed to compare
effects of different power densities of LLLT at 635 and 670 nm (daily dose = 5 J/cm2) on
excisional and incisional wound healing.
Methods: Four, round, full-thickness, skin wounds were made on the backs of 48 rats that
were divided into two groups (635 and 670 nm laser-treated). Wounds were daily stimulated
with different power densities. Two, six, and 14 days after surgery, eight animals from each
group were killed and samples removed for histological evaluation. Moreover, one full-
thickness skin incision was performed on the back of another 40 rats and immediately
sutured. Wounds were daily stimulated either with 635 or 670 nm laser light. Seven days after
surgery each wound was removed for wound tensile strength measurement.
Results: Whereas in the excisional wound healing model both the 635 and 670 nm lasers
significantly improved wound healing by using higher tested power densities (shortened
process of inflammation, accelerated process of re-epithelization, granulation tissue formation
and collagen deposition), in the incisional healing model the wound tensile strength was
significantly increased differently, i.e. the 635 nm laser improved healing by using higher
tested power density the 670 nm laser improved healing using lower power density.
Discussion and Conclusion: By comparing the effects of different wavelengths (670 and 685
nm) and intensities (2, 15 and 25 mW), achieving a total dose of 10 J/cm2, LLLT was found
to be more effective with the higher intensity combined with the shorter wavelength and with
the lower intensity combined with the longer wavelength [5]. In addition, evaluating the effect
of LLLT at 635 nm during the proliferation phase, re-epithelization and collagen synthesis
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were significantly accelerated in a power density-dependent manner. Moreover, we have also
found that red lasers are able to increase wound tensile strength. In contrast, to open wound
healing; however, the 635 nm laser had a better effect on wound tensile strength at the higher
tested power density and the 670 nm laser at the lower power density. In conclusion, LLLT,
by the method we used, improved wound healing in rats. Nevertheless, different LLLT
parameters were suggested by the investigation for excisional and incisional wounds which
might have a significant clinical impact.
Acknowledgement. Study supported by grants no. APVV-0526-11 and VEGA-2/0191/11.
References
1. Reis, S.R., Medrado, A.P., Marchionni, A.M., Figueira, C., Fracassi, L.D., Knop, L.A.
(2008) Photomed. Laser Surg. 26:307-313
2. Hawkins, D.H., Abrahamse, H. (2006) Lasers Surg. Med. 38, 74–83.
3. Houreld, N.N., Abrahamse, H. (2007) Lasers Med. Sci. 23, 11–18.
4. Kana, J.S., Hutschenreiter, G., Haina, D., Waidelich, W. (1981) Arch. Surg. 116, 293–
296
5. do Nascimento, P.M., Pinheiro, A.L., Salgado, M.A., Ramalho, L.M. (2004)
Photomed. Laser Surg. 22, 513–518
124
DNA-DOPE-Gemini Surfactants Complexes: Structure and Transfection Activity
aL. HUBČÍK, a,bP. PULLMANNOVÁ, cĽ. LACINOVÁ, cZ. SULOVÁ, dS.S. FUNARI, aF. DEVÍNSKY, aD. UHRÍKOVÁ
aFaculty of Pharmacy, Comenius University, Bratislava, bDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Hradec Králové, cInstitute of Molecular
Physiology and Genetics SAV, Bratislava, dHASYLAB at DESY, Hamburg
Cationic liposomes formed by cationic lipid or surfactant and neutral (helper) lipid are
intensively studied as vectors for gene delivery. We have studied the structure and
transfection activity of complexes formed due to DNA interaction with cationic liposomes
prepared from cationic gemini surfactants alkane-α,ω-diyl-bis(dodecyl-dimethylammonium)
bromides (CnGS12) and neutral phospholipid dioleylphosphatidylethanolamine (DOPE) at
molar ratio CnGS12:DOPE=0.3. CnGS12 are gemini surfactants with two quaternary
ammonium groups connected by a polymethylene spacer (n is the number of carbons in the
spacer) and two dodecyl chains. Transfection activity in vitro and in vivo have been reported
for complexes DNA-DOPE-CnGS12 with short spacer (n=2,3) [1].
Synchrotron small angle X-ray diffraction (SAXD) was employed to examine the structure of
DNA-DOPE-CnGS12 complexes. DNA-DOPE-CnGS12 (n=2, 3, 4, 5, 6, 8, 10 and 12)
complexes were prepared using herring testes DNA keeping the isoelectric composition
(DNA:CnGS12 = 2 mol/mol) in solution of sodium chloride (c =150 mmol/l). SAXD has
shown a condensed lamellar phase Lαc with DNA strands regularly packed between lipid
bilayers at 20°C. We determined the repeat distance d of lamellar phase and DNA-DNA
distance dDNA. With increasing n, the lamellar periodicity d linearly decreases from 6.73 nm at
n=2 to 6.31 nm at n=12. The dDNA=f(n) shows non-linear dependence with a minimum at n=3,
and increases from 4.01 nm to 4.44 nm with increasing n.
Transfection experiments using CnGS12-DOPE liposomes were conducted on HEK-293T
cell line. CnGS12-DOPE liposomes were prepared at molar ratio CnGS12: DOPE=0.3 using
CnGS12 with the spacer length n=3, 6 and 12. The CnGS12-DOPE mixture was hydrated by
Opti-MEM® transfection medium and extruded through 100 nm filter. Extruded liposomes
were mixed with green fluorescent protein encoding plasmid (pEGFP) and incubated for 20
minutes before transfection. “Naked” plasmid, as well as plasmid DNA mixed with
commercial transfection mixture Lipofectamine™ 2000 was used as controls. The GFP
expression efficiency was evaluated after 48 h incubation using flow cytometry. C12GS12-
DOPE liposomes have shown the best transfection efficiency for DNA plasmid that was
comparable to transfection efficiency of Lipofectamine™ 2000, while transfection
efficiencies of complexes prepared using either C3GS12 or C6GS12 were insignificant.
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Acknowledgement. The research leading to these results has received funding from the
European Community's Seventh Framework Programme (FP7/2007-2013) under grant
agreement n° 226716 (HASYLAB project II-20100372 EC), by the APVV 02-12-10 and VEGA
grant 1/1224/12 to DU and grant UK/642/2013 to LH.
References
1. Wettig, D., Verrall, R. E. and Foldvari, M. (2008) Current Gene Therapy 8, 9-23.
126
Singlet oxygen luminescence and mitochondrial autofluorescence after illumination of
Hyp/mitochondria complex
aZ. NADOVA, aD. PETROVAJOVA, aD. JANCURA, bD. CHORVAT, bA. CHORVATOVA, cX. RAGAS, cM. GARCIA-DIAZ, cS. NONELL, aP. MISKOVSKY
aDepartment of Biophysics, UPJS Kosice, Slovakia bInternational Laser Centre, Bratislava,
Slovakia cInstitut Quimic de Sarria, Universitat Ramon LLull, Spain
A study of hypericin (Hyp) interaction with mitochondria (mito) as well as the time-
resolved measurement of singlet oxygen (1O2) formation and annihilation after illumination of
Hyp/mito complex is presented. High concentration of Hyp leads to its aggregation inside the
mitochondria and the relative population of the monomeric form of Hyp decreases
concomitantly. Production of 1O2 in mitochondria after illumination of the complex is
characterized by a rise lifetime of ~8μs and shows saturation behavior with respect to Hyp
concentration. Our results confirm that only the monomeric form of Hyp is able to produce its
excited triplet state, which consequently leads to 1O2 production. An influence of photo-
activated Hyp on mitochondrial respiration chain was evaluated by monitoring of time-
resolved NAD(P)H fluorescence.
Acknowledgements: (i) FP7 EU CELIM 316312 (ii) Structural funds of EU, Op. program Res.
And Dev. (NanoBioSens 26220220107 (40%), SEPO II 26220120039 (60%)), APVV-0242-11,
VEGA 1/1246/12
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New bis-tacrine derivatives as topoisomerase I inhibitors
aJ. JANOČKOVÁ, bS. HAMUĽÁKOVÁ, cJ. KORÁBEČNÝ, dK. KUČA, aM. KOŽURKOVÁ
a Department of Biochemistry, b Department of Organic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic, c Department of Toxicology, d Centre of Advanced Studies, Faculty of Military Health Sciences, University of Defense, Hradec
Králové, Czech Republic
DNA is the pharmacological target of many cytotoxic drugs that are currently in
clinical use or in advanced clinical trials. Small ligands bind to DNA and can change and/or
inhibit role of DNA. These ligands can act as drugs when changing or inhibit DNA function;
are required for treatment or control of disease. The study of DNA interaction with ligands is
interesting not only for understanding the mechanism of interaction but also for the design of
new drugs. Drugs can interact with DNA in the several ways – electrostatic interactions,
binding to minor/ major groove of DNA, intercalation, covalent modification, blocking or
poisoning of DNA topoisomerase, decreasing the precursor concentration, blocking mitotic
spindle, inhibition of DNA/ RNA polymerases, blocking or poisoning DNA topoisomerases
[1].
Intercalation involves the insertion of a planar molecule between DNA pairs. The
ligands usually bind to the minor groove of DNA and are stabilized by intermolecular
interaction. Intercalation process is often associated with topoisomerase inhibition. DNA
topoisomerases are essential cellular enzymes that act as very good targets for
chemotherapeutic drugs due to their nature and mechanisms of actions in essential processes
that control the topological state of DNA in cells. There are two classes of topoisomerases:
type I which acts by transiently nicking one of two DNA strands and type II which nicks both
DNA strands and is dependent on ATP. These enzymes are involved in many vital cellular
processes that influence DNA replication, transcription, recombination, integration and
chromosomal segregation [2].
In this research work we focused on the study of DNA binding properties with
selected new synthesized bis-tacrine derivatives–1-(x-(7-methoxy-1,2,3,4-tetrahydroacridine-
9-yl-amino)alkyl)-3-(1,2,3,4-tetrahydroacridine-9-yl)-urea (x = etyl-, propyl-, butyl-, pentyl-,
hexyl-) (Fig.1). Tacrine is characterized by significant physicochemical properties, as strong
intercalation to DNA or intensive fluorescence. It was first drug for treatment of Alzheimer´s
disease [3]. Recently are published some research works where tacrine and their derivatives
act as topoisomerase inhibitors. Bis-tacrine derivatives consist of two planar chromophores
tethered by flexible linker groups which are capable of simultaneously inserting into the
DNA-base stack. In comparison with monomer, the two planar structures connecting
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appropriate linker length are considered to be two major factors for increasing cancer cell
cytotoxicity. The type of DNA interactions with derivatives was studied by spectroscopic
techniques (UV-Vis, fluorescence spectroscopy, circular dichroism). From UV-Vis
spectroscopic titrations were determinate binding constants for complexes of studied bis-
tacrine derivatives with DNA. The ability of bis-tacrine derivatives to influence
topoisomerase I activity was studied by electrophoretic techniques.
Fig.1 Structure of new synthesized bis-tacrine derivatives 1-(x-(7-methoxy-1,2,3,4-
tetrahydroacridine-9-yl-amino)alkyl)-3-(1,2,3,4-tetrahydroacridine-9-yl)-urea, (x = etyl-,
propyl-, butyl-, pentyl-, hexyl-).
Acknowledgement. This study was supported by VEGA 1/0001/13, APVV-0280-11, Internal
Grant Program of the P. J. Šafárik University in Kosice (VVGS 40/12–13, VVGS-PF-2012–
16, VVGS-PF-2013-78), University Hospital in Hradec Kralove (long term development plan)
and University Hradec Kralove (long term development plan).
References
1.Sirajuddin, M., Ali,S., Badshah, A. (2013) J. Photochem. Photobiol. B. 124, 1-19.
2.Palchaudhuri, R., Hergenrother, P. J. (2007) Curr. Opin. Biotechnol. 18, 497 – 503.
3.Kozurkova, M., Hamulakova, S., Gazova, Z., Paulikova, H., Kristian, P. (2011)
Pharmaceuticals 4, 382-418.
129
Investigation of DNA binding activities with new oxime-type ligands
aJ. JANOČKOVÁ, b-c K. MUSILEK, cK. KUČA, aM. KOŽURKOVÁ
a Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic, b Department of Toxicology, c Centre of Advanced Studies, Faculty of Military
Health Sciences, University of Defense, Hradec Králové, Czech Republic
The cholinesterase modulators possess considerable biological activities; their
prominent effects are for example antidepressants, analgetics, anti-inflammatory, fungicidal,
herbicides, hepatitis, antiulcer, anti-androgens activity [1]. Pyridinium oximes are used
mainly as antidotes; other oximes can also act as agents with positive effect on the
cardiovascular system or for their anti-inflammatory, anti-viral, antibacterial and antifungal
activity. The rational for their DNA interaction screening lie in their mice toxicity that they
previously presented in vivo [2]. Thus, the interaction of presented compounds 1-4 with DNA
was supposed due to their structural factors (e.g. condensed or closely connected aromatic
rings).
In this presentation, the biochemical and biological activities of selected cholinesterase
modulators are described. Their capacity to bind to DNA and interfere with human
topoisomerase I was examined.
Acknowledgement. This study was supported by VEGA 1/0001/13, APVV-0280-11, Internal
Grant Program of the P. J. Šafárik University in Kosice (VVGS 40/12–13, VVGS-PF-2012–
16, VVGS-PF-2013-78), University Hospital in Hradec Kralove (long term development plan)
and University Hradec Kralove (long term development plan).
References
1. Hart, W., Sharp,B. W. (1991) Chemical Abstracts 115, 92075.
2. Komloova, M., Musilek, K., Horova, A., Holas, O., Dohnal ,V., Gunn-More, F., Kuca,
K. Bioorg Med. Chem. Lett. (2011) 21, 2505–2509.
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Interaction of bis-3,6-alkylamido-acridines with calf thymus DNA
aD. SABOLOVÁ, aJ. KUDLÁČOVÁ, bL. JANOVEC, bJ. IMRICH
aDepartment of Biochemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 04167 Košice, Slovak Republic bDepartment of Organic
Chemistry, Institute of Chemistry, Faculty of Science, P.J. Šafárik University, Moyzesova 11, 04167 Košice, Slovak Republic
Extensive chemical and biochemical studies over the past thirty years have
characterized many small molecules which are able to interact with nucleic acids and cause
changes in DNA structure. The binding of peptides, synthetic organic molecules and small
fluorescent dyes to DNA can interfere with the numerous processes in which DNA
participates, such as transcription, replication and the DNA repair process. Acridine and
their derivatives are well-known probes for nucleic acids as well as being relevant in the
field of drug development to establish new chemotherapeutic agents [1-3]. Here we report
the interaction of a classic intercalator proflavine (1) and new bis-3,6-alkylamidoacridines
(R: methyl-(2), ethyl-(3), propyl-(4), butyl-(5) with calf thymus DNA (ctDNA) based on
various biophysical studies. The inhibition effect to topoisomerase was also investigated.
UV-Vis and fluorescence spectroscopic measurements indicated that all investigated
compounds bind to ctDNA. The results of CD measurements showed that compounds (1),
(2) and (3) directly interact with ctDNA through intercalation between base-pairs and (4)
and (5) bind with ctDNA not only by intercalation mode, but also by groove binding mode.
The binding constants of proflavine derivatives determined by UV-Vis spectroscopy were
decreased with the length of the 3,6-di-substitued linear chains (from 2.10×105 to 0.68×105
M-1). The DNA melting experiments revealed that the transition temperature, Tm, of the
ctDNA in presence of (1) - (5) increased what indicates that the molecules have bound to
ctDNA and therefore stabilized DNA duplex. Separation of plasmid pUC19 by the
substances and inhibition effect of tested derivatives to topoisomerase I was studied by
agarose gel electrophoresis. An electrophoretic separation proved that ligands (1) - (5)
inhibited topoisomerase I in
Acknowledgement. This work was supported by the Slovak Research and Development
Agency under the contract No. APVV-0280-11, by VEGA 1/0001/13, 1/0672/11 and VVGS
PF-2013-114.
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References
1. Haq, I. (2002) Arch. Biochem. Biophys. 403, 1-15.
2. Pons, O.R., Gregorio, D.M., Mateo, J.V.G., Calatayud, J.M. (2001) Anal. Chim. Acta
438, 149-156.
3. Gosh, R., Bhowmik, S., Bagchi, A., Das, D., Gosh, S. (2010) Eur. Biophys. J. 39,
1243-1249.
132
The effect of new acridine derivatives – alkyl (2E)-3-(acridin-9-yl)-prop-2-enoates - on
DNA
aO. SALEM, bM. VILKOVÁ, bM. PROKAIOVÁ, bJ. IMRICH, aM. KOŽURKOVÁ
aDepartment of Biochemistry, bDepartment of Organic Chemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovak Republic
Acridine compounds belong among the best-studied DNA-binding small heterocyclic
skeletons [1], which can interact with DNA via intercalation. Depending on the nature of a
particular compound and its substituents, the results and cellular consequences of acridine–
DNA interaction may vary significantly. There are several reasons for a wide usefulness of
acridines as broadly active chemotherapeutic agents, which include their ready synthesis,
biological stability, and ability to bind efficiently to DNA and disrupt cellular DNA function
[2]. As the result, acridines have been intensively studied as potential anticancer drugs.
Though they are well-known for their high cytotoxic activity, their clinical application is
limited or even excluded because of unwanted side effects. In addition, acridines have
received intense interest and been also used as anti-inflammatory and antibacterial agents.
In this work we describe the synthesis and biochemical activities of new propyl or butyl
(2E)-3-(acridin-9-yl)-prop-2-enoates as potential anticancer agents. We have focused
especially on a capacity of the derivatives to bind to DNA and interfere with human
topoisomerase I.
Acknowledgement. This work was supported by the research grants from the Slovak Grant
Agency VEGA No. 1/0001/13 and 1/0672/11, and the grant APVV-0280-11.
References
1. Gurova, K. (2009) Future Oncol. 5, 1-28.
2. Plšíková, J., Janovec, L., Kovaľ, J., Ungvarský, J., Mikeš, J., Jenželovský, R.,
Fedoročko, P., Imrich, J., Kristian, P., Kašpárková, J., Brabec, V., Kožurková, M.
(2012) Eur. J. Med. Chem. 57, 283-295.
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Determination of interaction mode and binding constant of emodin incorporated into
DNA
Z. VOJNOVÁ, V. VEREBOVÁ, J. STANIČOVÁ
Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Košice, Slovakia
Emodin, a plant derived anthraquinone, was found to be a photosensitizer which
possess anti-tumor, anti-bacterial, anti-viral, and anti-fungal activity [1]. However, the
detailed mechanism underlying the biological effects of emodin remains unknown. The aim
of this study was to examine the molecular interactions of emodin with calf thymus (ct) DNA
in aqueous solution at physiological pH using spectrophotometric methods.
We used fluorescence spectroscopy for measurement of Langmuir isotherm to determine
association constant which expresses strength of interaction [2]. Fluorescence quenching of
emodin as a result of adding ctDNA and the value of association constant (Ka = 8,0925 x 104
M-1) can lead us to assumption that emodin interacts with DNA by non-intercalative but
groove binding mode of interaction. Absorption analysis has confirmed our results obtained
by fluorescence measurements.
In addition, we proved our results by denaturation of pure DNA and emodin – DNA
complexes, respectively by absorption spectroscopy connected with Peltier module.
Fig. 1 Normalized melting curves of ctDNA and complexes emodin/DNA in TE buffer and
concentration ratios: 1/1, 2/1, 3/1
Melting curves that can be seen in Fig. 1 show a destabilizing effect of the drug on calf
thymus DNA. They were fitted using Van´t Hoff equation to obtain main thermodynamic
characteristics (Tm, ΔT, ΔH).
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Melting temperature Tm is shifted from 67.5°C (pure DNA) to 57°C for complex
emodin/DNA in 2/1 concentration ratio. Also Van´t Hoff enthalpy ΔH is decreasing from 388
kJ (pure DNA) to 126 kJ (emodin/DNA = 2/1) which means that lower energy is needed for
denaturation 50% base pairs of DNA in complex with emodin.The changes in all
thermodynamic parameters lead us to claim that emodin destabilizes ctDNA molecule and
this destabilization is associated with groove binding interaction mode [3]. Our findings can
be support by the fact that an intercalative mode of interaction manifests in thermal
stabilization of DNA molecule due to drug binding [3].
It is generally supposed that small molecules like emodin bind preferentially into minor
groove of DNA, especially to regions which are rich in AT base pairs [4]. In our experiments
the melting curve of 3/1 complex (Fig. 1) shows two phase character with an expressive
destabilization of AT regions in DNA.
It can be concluded that emodin interacts with ctDNA molecule by groove binding mode and
obtained association constant corresponds to this mode of interaction. Emodin incorporation
takes place probably into DNA minor groove by hydrophobic or hydrogen interactions.
Acknowledgement. Authors are pleased to thank Dr. R. Varhač for the possibility to use
experimental equipment in his laboratory and Dr. D. Jancura for inspiring discussions.
References
1. Radovic, J., et al. (2012) Food and chemical toxicology 50, 9.
2. Copeland, R. A. (2000) in Enzymes - a practical introduction to structure, mechanism
and data analysis. NY: John Wiley & Sons, INC.
3. Bi, S., et al. (2008) Spectrochimica Acta part A. 69, 1.
4. Lavery, R., Pullman, B. (1981) International Journal of Quantum Chemistry 20, 1.
135
Selected antioxidant properties of known or commonly used plant extracts
A. FEJERČÁKOVÁ, K. KREMPASKÁ, J. VAŠKOVÁ, L. VAŠKO
Department of Medical and Clinical Biochemistry and LABMED ojsc., Faculty of Medicine, UPJŠ, Košice
Plant products are widely used in testing because of their low toxicity and great
medicinal value. They play important role in alleviating many diseases. Much research has
focused on their different abilities to induce antioxidant effects. Since polyphenolic
compounds have high antioxidant potential, the ability of Siberian ginseng, Stevia rebaudiana
and Red grapes extract to act as scavengers of hydroxyl radical was investigated in
comparison to quercetine. Hydroxyl radicals are highly reactive biological molecules and the
major active oxygen species produced via the Fenton reaction in the presence of transition
metals in living systems. They react with lipids, polypeptides, saccharides, nucleotides, and
organic acids, especially thiamine and guanosine, thereby causing cell damage [4]. Their
scavenging property may provide an important therapeutic approach against oxidative stress
induced ailments.
The present study aims to spectrophotometric quantification of hydroxyl radical scavenging
activity at different concentrations (5, 25, 50, 75, 100 μg.ml-1) of plant extracts
(Eleutherococcus senticosus known as siberian ginseng, Stevia rebaudiana, Vitis vinifera L. -
red grape pomace) in comparison to in general known antioxidant, quercetine. Hydroxyl
radical was generated by the Fe3+-EDTA/ascorbate Fenton system, and assayed by evaluating
deoxyribose degradation using the thiobarbituric acid method [3].
The % inhibition increased with increasing concentration of tested extracts (Table 1).
Quercetine exhibited the strongest scavenging capacity (87%) towards hydroxyl radicals at
the concentration 100 μg.ml-1. Quercetine, as the most common dietary flavonol, is a potent
antioxidant due to the right structural features for free radical scavenging activity. Besides its
antioxidant potential, exerts also anti-inflammatory, antiproliferative and gene expression
regulating effects [5]. Other tested plant extracts (stevia and siberian ginseng) reached
approximately 2.5 lower antioxidant capacity at same concentration (33%). Recent studies
suggest that stevia and ginseng, besides offering many functional benefits, may also have
antioxidant properties [2]. It�s well known that red grapes have antioxidant capacity and this
capacity is likely due to their high contents of phenolics and flavonoids. Phenolic compounds
presented in tested extracts may work by providing hydrogen atoms from their phenolic
hydroxyl groups to scavenge hydroxyl radical generated from hydrogen peroxide [1]. In our
study red grape extract showed a relatively high antioxidant activity towards hydroxyl
radicals (78 %) in concentration dependent manner.
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136
This study suggests that tested extracts might be useful for the development of novel, but first
more potent natural antioxidants. However, further investigations on the isolation and
identification of individual compounds, with the following monitoring their in vivo
antioxidant activities as well as different antioxidant mechanisms is needed to ascertain its
potency.
Table 1: Scavenging of hydroxyl radicals as percentage (%) by tested extracts.
Concentration
μg/mL
Quercetine Vitis
vinifera
Stevia
rebaudiana
Eleutherococcus
senticosus
5 10.55 4.75 10.25 2.60
25 36.49 30.00 12.80 13.30
50 65.14 26.95 6.90 18.45
75 78.67 51.80 18.09 30.25
100 86.69 78.20 36.65 31.80
Acknowledgement. Quercetine was kindly provided by prof. P. Perjési (Pécs, Hungary). This
work was supported by the research grant from the Slovak Grant Agency VEGA No.
1/1236/12.
References
1. Croft, K. D. (1998) Ann. N. Y. Acad. Sci. 854, 435-442.
2. Ghanta, S., et al. (2007) J. Agric. Food Chem. 55, 10962-10967.
3. Halliwell, B., Guttridge, J. M. C., Aruoma, O. I. (1987) Anal. Bioch. 165, 215-219.
4. Jiao, Z., Liu, J., Wang, S. (2005) Food Technol. Biotechnol. 43, 97-102.
5. Wu, L. C., Lu, I. W., Chung, C. E. (2011) Food Funct., 2, 204-212.
137
Comparison of the effects of some natural and synthetic hydroxyl substituted chalcones
against hydroxyl radical
aK. KREMPASKÁ, aJ. VAŠKOVÁ, aA. FEJERČÁKOVÁ, aL. VAŠKO, bP. PERJÉSI
aDepartment of Medical and Clinical Biochemistry and LABMED ojsc., Faculty of Medicine, UPJŠ, Košice, Slovak Republic bInstitute of Pharmaceutical Chemistry, Faculty of Medicine,
University of Pécs, Pécs, Hungary
All aerobic organisms produce reactive oxygen species physiologically. The five most
productive pathways are involved in regulating the production of ROS/RNS and the resulting
effects on signaling cascades. The five mechanisms produce ROS in a non-regulated mode.
Massive production of radicals initiates in biological systems a vicious circle of oxidation and
cyclic production of radicals of various substrates [3]. Clinically significant radicals include
superoxide anion, nitric oxide or a hydroxyl radical. Amounts of natural substances are
characterized by antioxidant properties, result in a variety of biological effects, with the
spectrum of cytoprotective and cytotoxic effects. What is determined by the spatial
arrangement of molecules, structural and lipophilic properties. The term “flavonoid” is
generally used to describe a broad collection of natural products that include a C6-C3-C6
carbon framework, or more specifically a phenylbenzopyran functionality. Depending on the
position of the linkage of the aromatic ring to the benzopyrano moiety, this group of natural
products may be divided into three classes. These groups usually share a common chalcone
precursor, and therefore are biogenetically and structurally related [2].
In this work, we compared the ability to scavenge the hydroxyl radical by the
frequently occurring dietary flavonoids (quercetin, naringin, phlorizine dihydrate) with
synthetically prepared chalcone hydroxyl derivatives (4'-hydroxychalcone, 4'-
hydroxyindanone, 4'-hydroxytetralone, 4'-hydroxybenzosuberone). We used the method
originally described by Halliwell [1] with slight modifications. Solutions 2.8mm 2-deoxy-2-
ribose, 200μM FeCl3, 1.04mM EDTA (1:1 v/v), 1 mM H2O2 1.0mM L-ascorbic acid were
prepared directly before use. Test substances were added at concentrations of 5, 25, 50, 70
and 100 μg.ml-1.
Our measured results show that the highest inhibitory activity against hydroxyl radical
(60%) among all the compounds tested showed 4'-hydroxybenzosuberone, while its
antioxidant effect against pursued radical increase with increasing concentration. Other
synthetic derivatives showed lower ability to inhibit the hydroxyl radical, with a maximum
(approximately 30% namely 4'-hydroxyindanone) made up using the concentration of 100
mg.ml-1. Natural compounds were less effective antioxidants against hydroxyl radical,
whereas the maximum inhibitory effect was approximately 20%. Their activity against
pursued radical was not significantly affected by the concentration used.
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138
These results show that synthetic chalcones demonstrated improved antioxidant
properties to the hydroxyl radical compared to natural chalcones in vitro. Significantly highest
activity was found in 4'-hydroxybenzosuberone, also compared with other natural substances
as were qurcetine, naringin and phlorizin dihydrate. The activity has been rising by increasing
concentration, while the highest was at the highest concentration of 100 μg.ml-1 used.
Acknowledgement. Study was supported by the research grant from the Slovak Grant Agency
VEGA No. 1/1236/12.
References
1. Halliwell, B., Guttridge, J. M. C., Aruoma, O. I. (1987) Anal. Bioch. 165, 215-219.
2. Marais, J.P.J., Deavours, B., Dixon, R.A., Ferreira D. (2006) in The Science of
Flavonoids (Grotewold, E., Ed.). Springer, 1-46.
3. Vašková, J., Vaško, L., Kron I. (2012) in Antioxidant Enzyme (Amr El-Missiry, M.,
Ed.), InTech, DOI: 10.5772/50995.
Available from: http://www.intechopen.com/books/antioxidant-enzyme/oxidative-
processes-and-antioxidative-metaloenzymes
139
The effect of model carcinogen on the antioxidant enzyme status in rats
aĽ. LOHAJOVÁ, bA. SOBEKOVÁ
a Institute of Biophysics, b Institute of Medical Chemistry, Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice
The relations among reactive oxygen species, oxidative stress and carcinogenesis were
observed in this study. Reactive oxygen species (ROS) are unstable molecules which can
oxidize other molecules in order to become stable [3]. They play an important role in the body
but can also lead to oxidative stress. Oxidative stress is a random process of cell damage
resulting from aerobic metabolism. At high concentration, free radicals and non-radical
reactive species are hazardous for living organisms and damage all major cellular constituents
[2]. At moderate concentration, reactive oxygen species play an important role as regulatory
mediators in signaling processes. Many of the ROS-mediated responses actually protect the
cells against oxidative stress and reestablish “redox homeostasis” [2].
In aerobic life, oxidative stress arises from both endogenous and exogenous sources. Cell
damage from oxygen free radicals is ubiquitous. Oxygen free radicals may be considered as
an important class of carcinogens [1]. Superoxide, peroxide, hydroxyl radicals react with
the cell components, including lipids, proteins and DNA. Free radical attack results in a loss
of cell integrity, enzyme features, and genomic stability [4].
An excessive or continuous increasing of ROS production can participate in
the pathogenesis of malignant diseases, atherosclerosis, chronic inflammation, diabetes
mellitus, human immunodeficiency virus (HIV) infection, ischemia-reperfusion injury, and
sleep apnea. N-nitroso-N-methylurea (NMU) induces estrogen-dependent mammary tumors
in female Lewis rats. The aim of the study was to monitor changes in activities of antioxidant
enzymes in the heart and muscles of female rats after induction of mammary carcinoma.
18 rats were divided into three groups in the study. The first group of the animals were treated
with NMU, the second one obtained NMU and estrogen. The third group was a control. The
specific activities of antioxidant enzymes and level of thiobarbituric acid reactive species
(TBARS) – products of lipid peroxidation were determined in heart and muscle tissues of
experimental animals. The high differences in activities of antioxidant enzymes were
observed. Superoxide dismutase (SOD) and catalase (CAT) activities were significantly
increased in the heart and muscle of rats treated with NMU or/and NMU and estrogen when
compare to the control group. Activity of glutathione peroxidases (GPx) did not change in any
experimental groups. The glutathione-S-transferases (GSTs) are family of cytosolic enzymes
involved in the detoxification of a range of xenobiotic compounds by conjugation to
glutathione which is essential in the maintenance of normal physiological processes [3].
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140
Specific activity of GST in heart was significantly decreased. Glutathione reductase (GR)
plays an important role in cellular antioxidant protection because of catalysis the GSH
regeneration. Activity of GR in heart was significantly increased when compare with the
control group. Significantly increased levels of TBARS in the organs indicate oxidative
damage of heart and muscle. The alterations in the activities of antioxidative enzymes and
increased TBARS values showed that both organs were exposed to the action of free radicals.
The protective effect of estrogens is not shown in any investigated tissues. The effect of NMU
is clearly associated with the formation of reactive oxygen species. Formation of tumor in
mammary gland was accompanied with oxidative damage of non target organs, organs with
low effectiveness of antioxidant defense system.
Acknowledgement. The study was supported by the project KEGA 020UVLF-4/2012 and
KEGA 014UVLF-4/2013.
References
1. Dreher, D., Junod, A.F. (1996) Eur.J.Cancer. 32A, 30-8.
2. Dröge, W. (2002) Physiol. Rev. 82, 47-95.
3. Hayes, J. D., Pulford, D. J. (1995) Crit.Rev.Biochem.Mol.Biol. 30, 445-600.
4. Hensley, K., Robinson, K.A., Gabbita, S.P., Salsman, S., Floyd, R.A. (2000) Free
Radical Biol. and Med. 28, 1456–1462.
141
PARTICIPANT ADRESSES
143
1. Alieva Roza Siberian Federal University, Krasnoyarsk, Russia2. Antal Irina Institute of Experimental Physics Slovak Academy of
Sciences, Košice, Slovakia3. Antalík Marián Department of Biochemistry, Faculty of Science, P. J.
Šafárik University in Košice, SlovakiaInstitute of Experimental Physics, Slovak Academy of Sciences. Kosice, Slovakia
4. Antošová Andrea Institute of Experimental Physics Slovak Academy of Sciences, Košice, Slovakia
5. Bágeľová Jaroslava Institute of Experimental Physics Slovak Academy of Sciences, Košice, Slovakia
6. Bednáriková Zuzana Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, SlovakiaInstitute of Experimental Physics, Slovak Academy of Sciences. Kosice, Slovakia
7. Berka Vladimír Division of Hematology, University of Texas Medical School, Houston, USA
8. Berkova Zuzana Department of Lymphoma and Myeloma, The University of Texas - MD Anderson Cancer Center, Houston, USA
9. Biscarini Fabio Dip. Scienze della Vita, Università di Modena e Reggio Emilia, Via Campi 183, 41125 Modena, Italy
10. Csöbönyeiova Mária Institute of Histology and Embryology, Sasinkova 4, 811 08 Bratislava, Slovakia
11. Danišovič Ľuboš Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, SK-811 08 Bratislava, Slovakia
12. Demjén Erna Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
13. Fabian Marián Department of Biochemistry and Cell Biology, Rice University, Houston, USACenter of Interdisciplinary Biosciences, University of P. J. Safarik, Kosice, Slovakia
14. Fabriciová Gabriela Department of Biophysics, Faculty of Sciences, P. J. Šafárik University in Košice, Slovakia
15. Fedunová Diana Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
16. Fejerčáková Andrea Department of Medical and Clinical Biochemistry and LABMED ojsc., P. J. Šafárik University in Košice, Slovakia
17. Fekete Attila Clinical Research Center, Medical and Health ScienceCenter, University of Debrecen, Debrecen, Hungary
18. Gál Peter Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
19. Gargalík Radoslav Institute of Computer Science, Faculty of Science, P. J. Šafárik University in Košice, Slovakia
20. Gažová Zuzana Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
21. Hamza Muaawia A. Faculty of Medicine, King Fahad Medical City, King Saud Bin Abdul Aziz University for Health Sciences, Riyadh, KSA
22. Hritz Jozef Department of Biophysics, “Carol Davila“ University of Medicine and Pharmacy, Bucharest, Romania
23. Hu Chin-Kun Institute of Physics, Academia Sinica, Nankang, Taipei,
144
TaiwanCenter for Nonlinear and Complex System and Department of Physics, Chung Yuan Christian University, Chungli, Taiwan
24. Hubčík Lukáš Faculty of Pharmacy, Comenius University, Bratislava25. Ionescu Diana Department of Biophysics, "Carol Davila" University of
Medicine and Pharmacy,Bucharest, 050474, Romania26. Jancura Daniel Department of Biophysics, Faculty of Sciences, P. J.
Šafárik University in Košice, Slovakia27. Janočková Jana Department of Biochemistry, Faculty of Science, P. J.
Šafárik University in Košice, Slovak Republic28. Keša Peter Department of Biochemistry, P. J. Šafárik University in
Košice, Slovakia29. Komáromi István Haemostasis, Thrombosis and vascular Biology Research
Group, Hungarian Academy of Sciences, University of Debrecen, Debrecen, Hungary
30. Kožár Tibor Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
31. Kožurková Mária Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovakia
32. Krempaská Klára Department of Medical and Clinical Biochemistry and LABMED ojsc., Faculty of Medicine, P. J. Šafárik University in Košice, Slovakia
33. Kudláčová Júlia Department of Biochemistry, Faculty of Science, P. J. Šafárik University in Košice, Slovakia
34. Kutejová Eva Department of Biochemistry and Structural Biology, Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague , Czech Republic
35. Li Mai Suan Institute of Physics, Polish Academy of Science, Warsaw, Poland
36. Lohajová Ľuboslava Institute of Biophysics, Department of Chemistry, Biochemistry and Biophysics, University of Veterinary Medicine and Pharmacy in Košice, Slovakia
37. Lütteke Thomas Justus-Liebig-University Giessen, Germany38. Marek Jozef Institute of Experimental Physics, Slovak Academy of
Sciences. Košice, Slovakia39. Milowska Katarzyna Department of General Biophysics, Faculty of Biology
and Environmental Protection, University of Lodz, Lodz, Poland
40. Miškovský Pavol Department of Biophysics and Center for Interdisciplinary Biosciences, P. J. Šafárik University in Košice, Slovakia
41. Mocanu Maria-Magdalena Department of Biophysics, “Carol Davila“ University of Medicine and Pharmacy, Bucharest, Romania
42. Molčan Matúš Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
43. Musatov Andrey Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
44. Naďová Zuzana Department of Biophysics, Faculty of Sciences, P. J. Šafárik University in Košice, Slovakia
45. Nováková Jana Institute of Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia
46. Nowacka Olga Department of General Biophysics, Faculty of Biology and Environmental Protection, University of Lodz,
145
Poland47. Permyakov Eugene Institute for Biological Instrumentation of the Russian
Academy of Sciences, Pushchino, Moscow region 142290, Russia
48. Permyakov Sergei Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
49. Petrova Alena Siberian Federal University, Krasnoyarsk, Russia50. Pudlák Michal Institute of Experimental Physics, Slovak Academy of
Sciences. Košice, Slovakia51. Roosen-Runge Felix Institute for Applied Physics, University of Tübingen,
Tübingen, Germany52. Sabolová Danica Department of Biochemistry, Faculty of Science, P. J.
Šafárik University in Košice, Slovakia53. Salem Othman Department of Biochemistry, Faculty of Science, P. J.
Šafárik University in Košice, Slovakia54. Sedlák Erik Department of Biochemistry, P. J. Šafárik University in
Košice, SlovakiaInstitute of interdisciplinary biosciences, P. J. Šafárik University in Košice, Slovakia
55. Schreiber Frank Institute for Applied Physics, University of Tübingen, Tübingen, Germany
56. Siebert Hans - Christian Research Institute for Bioinformatics and Nanotechnology (RI-B-NT) Kiel, Germany
57. Škrabana Rostislav Institute of Neuroimunology, Slovak Academy of Sciences, Bratislava, Slovakia
58. Škultéty Ľudovít Institute of Virology, Slovak Academy of Sciences,Bratislava, Slovakia
59. Šipošová Katarína Department of Biochemistry, Faculty of Sciences, P. J. Šafárik University in Košice, SlovakiaInstitute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
60. Tokár Tomáš Department of Biophysics, P. J. Šafárik University in Košice, Slovakia
61. Tóth László Clinical Research Center, Medical and Health Science Center, University of Debrecen,Debrecen, Hungary
62. Tomori Zoltán Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
63. Urbániková Ľubica Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
64. Valušová Eva Institute of Experimental Physics, Slovak Academy of Sciences. Košice, Slovakia
65. Varhač Rastislav Department of Biochemistry, Faculty of Sciences, P. J. Šafárik University in Košice, Slovakia
66. Varchulová Nováková Zuzana
Institute of Medical Biology, Genetics and Clinical Genetics, Faculty of Medicine, Comenius University, Sasinkova 4, SK-811 08 Bratislava, Slovakia
67. Verebová Valéria Institute of Biophysics, University of Veterinary Medicine and Pharmacy, Košice, Slovakia
68. Ziegelhöffer Attila Institute of Heart Research, Slovak Academy of Sciences, Bratislava, Slovakia
69. Žoldák Gabriel Physik Department E22, Technische Universität München, 85748 Garching, Germany
147
AUTHORS INDEX
149
ALBONETTI C. PL8 ALIEVA R. PO6, PO7 ANTAL I. SC11 ANTALÍK M. PO4, PO5, PO8 ANTOŠOVÁ A. PO13, PO14, PO17 AMBRO Ľ. PL9 BAČKOR M. PO2 BAKUNTS A. G. PL1 BARAN I. PL5 BARTA A. SC11 BARTCZAK E. PO9 BARTOŚ A. PO15 BAUER J. PL9 BAUEROVÁ- HLINKOVÁ PO18 BÁGEĽOVÁ J. PO14 BEDNÁRIKOVÁ Z. PO12, PO13,
PO14, PO16, PO17 BELOGUROVA N. PO6, PO7 BERKOVA Z. PL6 BIELY P. PL16 BISCARINI F. PL8 BORKO L. PO18 BÖHMER D. PO3 BRYSZEWSKA M. PO9, PO10 CAÑAMARES M. V. PO2 CEBOVÁ M. SC11 CEHLÁR O. SC7 CIESIELSKI A. PL8 CSÖBÖNYEIOVÁ M. PO3 CHORVÁT D. PO25 CHORVÁTOVÁ A. PO25 DANILUK U. PL6 DANIŠOVIĆ PO3 DE FEYTER S. PL8 DE LA MATA J. PO9 DEMJEN E. PO13, PO14, PO17 DEVÍNSKY F. PO24 DOMINIK A. PL4 DVORSKÝ R. SC7 DVORANKOVA B. SC9 FABIAN M. SC5 FABRICIOVÁ G. PO1, PO2 FEDUNOVÁ D. PO14 FEJERČÁKOVÁ A. PO31, PO32 FEKETE A. SC1, PO19 FERKO M. PL15 FILIPPI A. PO17 FLOREZ-RAMIREZ G. SC2 FUNARI S. S. PO24 GABIUS H. J. SC9 GABRYELAK T. PO9 GANEA C. PL5, PO17 GARAJOVÁ K. PO11
GARCIA-DIAZ M. PO25 GARGALÍK R. SC3 GAŠPERÍK J. PO18 GAŽOVÁ Z. PO12, PO13, PO14 PO15, PO16, PO17 GÁL P. SC9, PO23 GEORGESCU L. PL5 GIRMAN V. PO12 GODÁNY PL16 GOJZEWSKI H. SC12 GOMEZ R. PO9 HAMUĽÁKOVÁ S. PO26 HAMZA M. A. SC10 HASHIM A. SC12, PO3 HOSTINOVÁ E. PO18 HRITZ J. PL7 HRMO I. PO5 HU C. K. PL18 HUBČÍK L. PO24 IDRIS A. A. T SC10 IMRICH J. PO28, PO29 IONESCU D. SC4 JAKUBČO J. SC9 JANCURA D. SC5, PO1, PO25 JANOČKOVÁ J. PO26, PO27 JANOVEC L. PO28 JAVORSKÝ P. PO4 JOOSTEN R. P. PL4 JURÍKOVÁ A. SC11 KATONA E. PL5 KERROS C. PL6 KEŠA P. PO5 KOMÁROMI I. SC1, PO19, PO20 KONERACKÁ M. SC11, PO12 KOPČANSKÝ P. SC11, SC12,PO12 KORÁBEČNÝ J. PO26 KOTOUČOVÁ J. PO15 KOVACECH B. SC7 KOVÁCSOVÁ M. SC11 KOVÁČ A. SC7 KOVÁČ I. PO23 KOVÁČ J. SC12 KOŽÁR T. PO21 KOŽURKOVÁ M. PO26, PO27, PO29 KREMPASKÁ K. PO31, PO32 KRIŠTOFÍKOVÁ Z. PO15 KUBAČKOVÁ J. PO1 KUBOVČÍKOVÁ M. SC10, PO12 KUČA K. PO26, PO27 KUDLÁČOVÁ J. PO8, PO28 KUDRYASHEVA N. PO6 KUNIAKOVÁ M. PO3
150
KUTEJOVÁ E. PL9, PO18 LACINOVÁ Ľ. PO24 LACJAKOVÁ K. PO23 LI M. S. PL17, PO16 LISCIO F. PL8 LOHAJOVÁ Ľ. PO33 LONGOBARDI M PL8 LOPEZ-TOBAR E. PO2 LÜTTEKE T. PL4 MAHAREB M. Al. SC10 MAJORAL J. P. PO9 MALI K.S. PL8 MAREK J. PO13, PO14, PO17 MILOWSKA K. PO9 MISKOVSKY P. PL14, PO1, PO25 MOCANU M. M. PL5, PO17 MOKROS D. PL4 MOKRÝ M. PO23 MOLČAN M. SC12, PO3 MUCS D. SC1 MUJKOŠOVÁ J. PL15 MURÁRIKOVÁ M. PL15 MUSATOV A. SC6 MUSILEK K. PO27 MUSTAFA A. SC10 MUSZBEK L. SC1, PO19, PO20 NAĎOVÁ Z. PO25 NAGY P. PL5 NEMASHKALOVA E. L. PL2 NGUYEN T. T. PO16 NONELL S. PO25 NOVÁK M. SC7 NOVÁKOVÁ J. PO18 NOWACKA O. PO10 ONDROVIČOVÁ G. PL9 ONG Q. K. PL8 ORAVCOVÁ L. PO3 PALMER G. SC5 PECHANOVÁ O. SC11 PELZ B. PL11 PERJÉŠI PO32 PERMYAKOV E. A. PL1, PL2 PERMYAKOV S. E. PL1, PL2 PERŽEĽOVÁ V. SC9 PETROVA A. PO6, PO7 PETROVAJOVÁ D. PO25 PEVALA V. PL9 PINČÁK R. PO22 POLÁKOVÁ M. PO23 PRISTAŠ P. PO4 PROKAIOVÁ M. PO29 PUDLÁK M. PO22 PULLMANOVÁ P. PO24
RADU E. PO17 RAGAS X. PO25 RAVINGEROVÁ T. PL15 REGUERA J. PL8 RENNER C. PL8 RIEF M. PL11 RIPOVÁ D. PO15 ROOSEN-RUNGE F. PL12, PL13 SABOL F. SC9 SABOLOVÁ D. PO28 SALEM O. PO29 SAMANIEGO F. PL6 SAMORI P. PL8 SANCHEZ-CORTES S. PO1, PO2 SAUTER A. PL12, PL13 SCHILLEROVA E. PO18 SCHREIBER F. PL12, PL13 SEDLÁK E. PL10, SC6 SIEBERT H. C. PL3 SHRESTHA D. PL5 SKUMIEL A. SC12 SLEZÁK J. PL15 SLEZÁK M. PO23 SMETANA K. Jr. SC9 STANIČOVÁ J. SC5, PO30 SOBEKOVÁ A. PO33 STELLACCI F. PL8 SULOVÁ Z. PO24 SZÖLLOSI J. PL5 ŠEVČÍK J. SC7, PO18 ŠIPOŠOVÁ K. PO12, PO13, PO14,
PO15, PO16, PO17 ŠKRABANA R. SC7 ŠKULTÉTY Ľ. SC2 TIMKO M. SC12, PO3, PO4 TOKÁR T. SC8 TOMAN R. SC2 TOMORI Z. SC3, PO23 TÓTH L. PO20 UHRÍKOVÁ D. PO24 ULIČNÝ J. SC8 URBÁNIKOVÁ Ľ. PL16 VALUŠOVÁ E. PO4 VARADI T. PL5 VARCHUĽOVÁ-NOVÁKOVÁ PO3 VARHAČ R. SC6, PO11 VASILENKO T. SC9, PO23 VAŠKO PO31, PO32 VAŠKOVÁ J. PO31, PO32 VEREBOVÁ V. PO30 VIDINSKÝ B. PO23 VIDOVÁ B. PL16 VIET M. H. PO16
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VILKOVÁ M. PO29 VOJNOVÁ Z. PO20 VOLOGZHANNIKOVA A. A. PL1 VRIEND G. PL4 WACZULÍKOVÁ I. PL15 WANG S. PL6 WISE J. F. PL6 WOLF M. PL12 ZÁHRADNÍKOVÁ . PO18 ZÁVIŠOVÁ V. SC11, PO12 ZIEGELHÖFFER A. PL15 ZIEGELHÖFFER B. PL15 ZHANG F. PL12, PL13 ŽOLDÁK G. PL11
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Od roku 1993 Vám
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Môžete sa na nás spoľahnúť.
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Laboratórne prístroje a zariadeniaDrieňová 34, 821 02 Bratislava 2
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Využite kalibra�ný servis pipiet všetkých zna�iek bežne dostupných na slovenskom trhu v našomj akreditovanom kalibra�nom laboratóriu vybavenom špi�kovou technológiou METTLER TOLEDO. Akreditovaná kalibrácia je vykonávaná pod�a ISO 8655 a 17025.
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Book of Contribution, 8th International Conference Structure and Stability of Biomacromolecules SSB2013, September 10-13, 2013, Košice, Slovakia Editors: Ing. Jaroslava Bágeľová, CSc., RNDr. Diana Fedunová, PhD., RNDr. Zuzana Gažová, CSc. Reviewers: Doc. MUDr. Marek Dudáš, PhD., Doc. RNDr. Erik Sedlák, PhD. © Institute of Experimental Physics, Slovak Academy of Sciences ISBN: 978-80-89656-01-1 EAN: 9788089656011
ISBN: 978 - 80 - 89656 - 01-1
EAN: 978809656011
SSB 2013