I

SCIENTIFIC PUBLICATIONS OF THE INSTITUTE OF BIOPHYSICS OF THE HUNGARIAN ACADEMY OF SCIENCES

2011

LABORATORY OF MEMBRANE BIOENERGETICS

1. Fábián L, Heiner Z, Mero M, Kiss M, Wolff EK, Ormos P, Osvay K, Protein-based ultrafast photonic switching

Dér A

Optics Express, 19:2. Giotta L, Mastrogiacomo D, Italiano F, Milano F, Agostiano A,

18861-18870(2011)

Nagy KValli L, Trotta M

,

Reversible binding of metal ions onto bacterial layers revealed by protonation-induced ATR-FTIR difference spectroscopy Langmuir, 27:

3. 3762–3773(2011)

Groma GIVibrational motions associated with primary processes in

, Colonna A, Martin J-L , Vos MH

bacteriorhodopsin studied by coherent infrared emission spectroscopy Biophysical Journal, 100:

4. Hajdu K, Szabó T, Magyar M, Bencsik G, Németh Z,

1578–1586(2011)

Nagy K, Magrez A, Forró L, Váró

Photosynthetic reaction center protein in nanostructures G, Hernádi K, Nagy L

Physica Status Solidi B, 248:5.

2700-2703(2011)

Kelemen L, Ormos P, Two-photon polymerization with optimized spatial lightmodulator

Vizsnyiczai G

Journal of the European Optical Society – Rapid Publications, 6:11029(2011)

6. Metzger NK, Mazilu M, Kelemen L, Ormos PObservation and stimulation of an optically driven micromotor

, Dholakia K

Journal of Optics,13:7.

044018(2011)

Tóth-Boconádi R, Dér A, Optical and electric signals from dried oriented purple

Keszthelyi L

membrane of bacteriorhodopsins Bioelectrochemistry, 81:

8. 17–21(2011)

Végh AG, Fazakas C, Nagy K, Wilhelm I, Krizbai IA, Nagyőszi P, Szegletes Z, Spatial and temporal dependence of the cerebral endothelial cells elasticity

Váró G

Journal of Molecular Recognition, 24:9.

422–428(2011) Végh AG, Nagy K, Bálint Z, Kerényi Á, Rákhely G, Váró G,

Effect of Antimicrobial Peptide-Amide: Indolicidin on Biological Membranes Szegletes Z

Journal of Biomedicine and Biotechnology, DOI:10.1155/2011/670589

LABORATORY OF MEMBRANE STRUCTURE AND DYNAMICS

10. Balogh G, Maulucci G, Gombos I, Horváth I, Török Z, Péter M, Fodor E, Páli T

Heat stress causes spatially-distinct membrane re-modelling in K562 leukemia cells

, Benkő S, Parasassi T, De Spirito M, Harwood JL, Vígh L

PLoS ONE, 6:e21182(2011)

II

LABORATORY OF MOLECULAR NEUROBIOLOGY 11. Bauer HC, Traweger A, Zweimueller-Mayer J, Lehner C, Tempfer H, Krizbai I,

Wilhelm INew aspects of the molecular constituents of tissue barriers

, Bauer H

Journal of Neural Transmission, 118:12. Ceruti S, Colombo L, Magni G, Viganňa F, Boccazzi M,

7–21(2011)

Deli MA

Oxygen–glucose deprivation increases the enzymatic activity and the

, Sperlágh B, Abbracchio MP, Kittel Á

microvesicle-mediated release of ectonucleotidases in the cells composing the blood–brain barrier Neurochemistry International, 59:

13. 259–271(2011)

Drug transport and the blood-brain barrier Deli MA

Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, (Eds.:K. Tihanyi and M. Vastag), Bentham Science Publishers Ltd., pp.144-165(2011)

14. Elsworth JD, Morrow BA, Hajszan TPhencyclidine-induced Loss of Asymmetric Spine Synapses in Rodent Prefrontal Cortex is Reversed by Acute and Chronic Treatment with Olanzapine

, Leranth C, Roth RH

Neuropsychopharmacology, 36:15. Elsworth JD,

2054–2061(2011)

Hajszan TLoss of asymmetric spine synapses in dorsolateral prefrontal cortex of cognitively impaired phencyclidine-treated monkeys

, Leranth C, Roth RH

International Journal of Neuropsychopharmacology, 14:16.

1411–1415(2011)

Fazakas C, Wilhelm I, Nagyőszi P, Farkas AE, Haskó J, Molnár J, Bauer H, Bauer H-C, Ayaydin F, Dung NTK, Siklós L, Transmigration of melanoma cells through the blood-brain barrier: Role of endothelial tight junctions and melanoma-released serine proteases

Krizbai IA

PLoS ONE, 6:17. Fenyvesi F, Kiss T, Fenyvesi É, Szente L,

e20758(2011) Veszelka S, Deli MA

Randomly methylated β-cyclodextrin derivatives enhance taxol

, Váradi J, Fehér P,Ujhelyi Z, Tósaki Á, Vecsernyés M, Bácskay I

permeability through human intestinal epithelial Caco-2 cell monolayer Journal of Pharmaceutical Sciences, 100:

18. Gesuete R, Orsini F, Zanier ER, Albani D,

4734-4744(2011)

Deli MAGlial cells drive preconditioning-induced blood-brain barrier protection

, Bazzoni G, De Simoni M-G

Stroke, 42:19. Glavinas H, von Richter O, Vojnits K, Mehn D,

1445-1453(2011)

Wilhelm I, Nagy T, Janossy J, Krizbai I

Calcein assay: a high-throughput method to assess P-gp inhibition

, Couraud P, Krajcsi P

Xenobiotica, 41: 712–719(2011)

III

20. György B, Módos K, Pállinger É, Pálóczi K, Pásztói M, Misják P, Deli MA

Detection and isolation of cell-derived microparticles are compromised by protein

, Sipos Á, Szalai A, Voszka I, Polgár A, Tóth K, Csete M, Nagy G, Gay S, Falus A, Kittel Á, Buzás EI

complexes resulting from shared biophysical parameters Blood, 117:

21. e39-48(2011)

Kürti L, Kukovecz Á, Kozma G, Ambrus R, Deli MAStudy of the parameters influencing the co-grinding process for the production of meloxicam nanoparticles

, Szabó-Révész P

Powder Technology, 212:22. Lachenmaier SM,

210–217(2011)

Deli MAIntracellular transport of Toxoplasma gondii through the blood–brain barrier

, Meissner M, Liesenfeld O

Journal of Neuroimmunology, 232:23. Lehner C,Gehwolf R, Tempfer H,

119–130(2011)

Krizbai IOxidative Stress and Blood–Brain Barrier Dysfunction Under Particular Consideration of Matrix Metalloproteinases

, Hennig B, Bauer H-C, Hannelore B

Antioxidants & Redox Signaling, 15:24.

1305-1323(2011)

Paizs M, Tortarolo M, Bendotti C,Engelhardt J, Talampanel reduces the level of motoneuronal calcium in transgenic

Siklós L

mutant SOD1 mice only if applied presymptomatically Amyotrophic Lateral Sclerosis, 12:

25. 340–344(2011)

Commentary - Two Hits with One Shot - A Possibility of Simultaneous Targeting Motor Neuron Loss and Depression in ALS by Upregulating ADAR2

Siklós L

CNS & Neurological Disorders - Drug Targets, 10:26. Sziráki I, Erdő F, Beéry E, Molnár PM,

863(2011)

Fazakas C, Wilhelm I, Makai I, Kis E, Herédi-Szabó K, Abonyi T, Krizbai IQuinidine as an ABCB1 probe for testing drug interactions at the blood–brain barrier: an in vitro in vivo correlation study

, Tóth GK, Krajcsi P

Journal of Biomolecular Screening, 16:27. Szűts A, Láng P, Ambrus R,

886-894(2011)

Kiss L, Deli MAApplicability of sucrose laurate as surfactant in solid dispersions prepared by melt technology

, Szabó-Révész P

International Journal of Pharmaceutics, 410:28.

107–110(2011)

Tóth A, Veszelka S, Nakagawa S, Niwa M, Patented in vitro blood-brain barrier models in CNS drug discovery

Deli M

Recent Patents on CNS Drug Discovery, 6:29.

107-118(2011)

Veszelka S, Kittel Á, Tools for modelling blood-brain barrier penetrability

Deli MA

Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, (Eds.:K. Tihanyi and M. Vastag) Bentham Science Publishers Ltd.,pp.166-188(2011)

IV

30. Wilhelm I, Fazakas C, In vitro models of the blood-brain barrier

Krizbai IA

Acta Neurobiologiae Experimentalis, 71:31.

113–128(2011)

Wilhelm I, Tudjuk, hogy hisszük, vagy hisszük, hogy tudjuk?

Krizbai IA

A tudomány határai, (Eds.:P. Cseke) Komp-Press, pp. 53-58, 2011.

LABORATORY OF ENERGY CONVERTING REDOX ENZYMES

32. Ács N, Bagi Z, Rákhely G, Kovács E, Wirth R, Improvement of biogas production by biotechnological manipulation of the microbial population

Kovács KL

EXPRES 2011 - 3rd IEEE International Symposium on Exploitation of Renewable Energy Sources, Proceedings 2011, DOI: 10.1109/EXPRES.2011.5741813

33. Bódás S, Kovács KLA biogáz jövője

Magyar Energetika, 18:

34. Bódás S,

10-12(2011)

Kovács KLLehetőségek és tennivalók a biogáz-hasznosításában

, Lengyel A, Petis M, Pongrácz P, Somosné Nagy A, Szárszó T

ZIP Magazin,1:35.

32-35(2011)

Janzsó G, Bogár F, Hudoba L, Penke B, Rákhely G, Exploring and characterizing the folding processes of Lys- and Arg-containing Ala-based peptides: A molecular dynamics study

Leitgeb B

Computational Biology and Chemistry, 35:36.

240–250(2011)

The role of GM microbes in the fermentation technology Kovács KL

In: Plain Facts about GMO (Eds.:E. Balázs, D. Dudits, L. Sági) Tisza Press, pp. 75-80, 2011.

37. Kredics L, Leitgeb BDistribution of mono-and disaccharide-releasing extracellular enzyme production abilities within a Trichoderma population from Hungarian winter wheat rhizosphere

, Hatvani L, Manczinger L, Vágvölgyi C, Szekeres A

BIOXEN Seminar "Novel approaches for environmental protection", (Eds.:B. Skrbic), Novi Sad, pp. 56-62, 2011.

38. Characteristic conformational patterns of the Trp- and Arg-rich antimicrobial peptides

Leitgeb B

4th European Conference on Chemistry for Life Sciences, (Eds.: T. Kiss and A. Perczel), Medimond International Proceedings, pp. 59-62, 2011.

39. Leitgeb B, Janzsó G, Hudoba L, Penke B, Rákhely GHelix and H-bond formations of alanine-based peptides containing basic amino acids

, Bogár F

Structural Chemistry, 22:40. Pusztahelyi T, Klement E, Szajli E, Klem J, Miskei M, Karányi Z, Emri T, Kovács S,

Orosz G,

1287–1295(2011)

Kovács KLComparison of transcriptional and translational changes caused by long-term menadione exposure in Aspergillus nidulans

, Medzihradszky KF, Prade RA, Pócsi I

Fungal Genetics and Biology, 48:92-103(2011)

V

41. Sági L, Gócza E,Procedures for the generation of genetically modified organisms

Kovács KL

In: Plain Facts about GMO (Eds.:E. Balázs, D. Dudits, L. Sági), Tisza Press, pp. 18-34, 2011.

LABORATORY OF METALLOPROTEIN BIOPHYSICS

42. Desmet F, Bérczi A, Zimányi LAxial ligation of the high-potential heme center in an Arabidopsis cytochrome b561

, Asard H, Van Doorslaer S

FEBS Letters, 585:

LABORATORY OF BIOINFORMATICS

545–548(2011)

43. Ordas A, Hegedus Z

Deep sequencing of the innate immune transcriptomic response of zebrafish embryos to Salmonella infection

, Henkel CV, Stockhammer OW, Butler D, Jansen HJ, Racz P, Mink M, Spaink HP, Meijer AH

Fish & Shellfish Immunology, 31: 716-724(2011)

Protein-based ultrafast photonic switching

László Fábián,1 Zsuzsanna Heiner,1 Mark Mero,2 Miklós Kiss,3

Elmar K. Wolff,4 Pál Ormos,1 Károly Osvay,3 and András Dér1,* 1Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, P.O. Box 521, 6701

Szeged, Hungary 2HAS Research Group of Laser Physics, P.O. Box 406, 6701 Szeged, Hungary

3Department of Optics and Quantum Electronics, University of Szeged, P.O. Box 406, 6701 Szeged, Hungary 4Institute for Applied Biotechnology and System Analysis at the University of Witten/Herdecke, Herrhausenstrasse 44,

58455 Witten, Germany

*derandra@brc.hu

Abstract: Several inorganic and organic materials have been suggested for

utilization as nonlinear optical material performing light-controlled active

functions in integrated optical circuits, however, none of them is considered

to be the optimal solution. Here we present the first demonstration of a

subpicosecond photonic switch by an alternative approach, where the active

role is performed by a material of biological origin: the chromoprotein

bacteriorhodopsin, via its ultrafast BR->K and BR->I transitions. The

results may serve as a basis for the future realization of protein-based

integrated optical devices that can eventually lead to a conceptual revolution

in the development of telecommunications technologies.

©2011 Optical Society of America

OCIS Codes: (060.1155) All-optical networks; (130.4815) Optical switching devices;

(130.3120) Integrated optics devices; (320.7120) Ultrafast phenomena; (320.7085) Ultrafast

information processing; (160.1435) Biomaterials.

References and links

1. S. A. Haque and J. Nelson, “Toward organic all-optical switching,” Science 327(5972), 1466–1467 (2010).

2. J. M. Hales, J. Matichak, S. Barlow, S. Ohira, K. Yesudas, J.-L. Bredas, J. W. Perry, and R. R. Marder, “Design

of polymethine dyes with large third-order optical nonlinearities and loss figures of merit,” Science 327(5972),

1485–1488 (2010).

3. X. Hu, P. Jiang, C. Ding, H. Yang, and Q. Gong, “Picosecond and low-power all-optical switching based on an

organic photonic bandgap microcavity,” Nat. Photonics 2(3), 185–189 (2008).

4. W. Stoeckenius, R. H. Lozier, and R. A. Bogomolni, “Bacteriorhodopsin and the purple membrane of

halobacteria,” Biochim. Biophys. Acta 505, 215–278 (1979).

5. N. Vsevolodov, Biomolecular electronics (Birkhauser, Boston, 1998).

6. E. Korchemskaya, N. Burykin, S. Bugaychuk, O. Maksymova, T. Ebrey, and S. P. Balashov, “Dynamic

holography in bacteriorhodopsin/gelatin films: Effects of light-dark adaptation at different humidity,”

Photochem. Photobiol. 83(2), 403–408 (2007).

7. J. A. Stuart, D. L. Marcy, and R. R. Birge, “Photonic and optoelectronic application of bacteriorhodopsin,” in

Bioelectronic Applications of Photochromic Pigments, A. Dér, and L. Keszthelyi, eds. (2001), pp. 15–29.

8. D. Zeisel and N. Hampp, “Spectral relationship of light-induced refractive index and absorption changes in

bacteriorhodopsin films containing wildtype BR and the variant BR-D96N,” J. Phys. Chem. 96(19), 7788–7792

(1992).

9. K. J. Wise, N. B. Gillespie, J. A. Stuart, M. P. Krebs, and R. R. Birge, “Optimization of bacteriorhodopsin for

bioelectronic devices,” Trends Biotechnol. 20(9), 387–394 (2002).

10. S. P. Balashov, “Photoreactions of the photointermediates of bacteriorhodopsin,” Isr. J. Chem. 35, 415–428

(1995).

11. P. Ormos, Z. Dancsházy, and L. Keszthelyi, “Electric response of a back photoreaction in the bacteriorhodopsin

photocycle,” Biophys. J. 31(2), 207–213 (1980).

12. A. Colonna, G. I. Groma, and M. H. Vos, “Retinal isomerization dynamics in dry bacteriorhodopsin films,”

Chem. Phys. Lett. 415(1-3), 69–73 (2005).

13. G. Váró and L. Keszthelyi, “Photoelectric signals from dried oriented purple membranes of Halobacterium

halobium,” Biophys. J. 43(1), 47–51 (1983).

14. L. Fábián, E. K. Wolff, L. Oroszi, P. Ormos, and A. Dér, “Fast integrated optical switching by the protein

bacterorhodopsin,” Appl. Phys. Lett. 97(2), 023305 (2010).

#150654 - $15.00 USD Received 6 Jul 2011; revised 19 Aug 2011; accepted 19 Aug 2011; published 13 Sep 2011(C) 2011 OSA 26 September 2011 / Vol. 19, No. 20 / OPTICS EXPRESS 18861

Published: March 11, 2011

r 2011 American Chemical Society 3762 dx.doi.org/10.1021/la104868m | Langmuir 2011, 27, 3762–3773

ARTICLE

pubs.acs.org/Langmuir

Reversible Binding of Metal Ions onto Bacterial Layers Revealed byProtonation-Induced ATR-FTIR Difference SpectroscopyLivia Giotta,*,† Disma Mastrogiacomo,‡ Francesca Italiano,§ Francesco Milano,§ Angela Agostiano,§

Krisztina Nagy,‡,|| Ludovico Valli,‡ and Massimo Trotta§

†Dipartimento di Scienza dei Materiali and ‡Dipartimento di Ingegneria dell’Innovazione, University of Salento, Lecce, I-73100, Italy§CNR - Istituto per i Processi Chimico-Fisici, Sezione di Bari, Bari, I-70126 Italy

)Institute of Biophysics, Biological Research Center of Hungarian Academy of Sciences, H-1250 Budapest, Hungary

bS Supporting Information

’ INTRODUCTION

The development of biofilms onto surfaces in contact withbacterial suspensions represents a problem in industrial andaquatic environments because this phenomenon promotesfouling3 and corrosion.4 Nevertheless, bacterial cell adhesiononto surfaces can be successfully exploited to investigate thestructure of microorganism cell components at the molecularlevel. If the abiotic surface where adhesion of cells occurs is theinternal reflection element (IRE) of an attenuated total reflection(ATR) accessory mounted into a Fourier transform infrared(FTIR) spectrometer, the biological film can indeed be suitablyprobed by vibrational spectroscopy.

In situ monitoring of biofilm formation and modification byATR-FTIR spectroscopy has been reported by several authorswho employed flow-cells placed onto bare,5-10 or suitablycoated11-13 ATR crystals. Flow-through cells were used notonly for flowing the bacterial suspension onto the IRE surface butalso for exposing the developed biofilm to aqueous solutions ofdifferent physical and chemical properties, such as pH, ionicstrength, metal ion concentration, etc., to study the response ofthe biomass to environmental modifications. Comparison ofinfrared absorption spectra of bacterial layers exposed to differentchemical environments allowed getting information on the

functional groups that account for surface chemical propertiesof various microorganisms.

In situ ATR-FTIR spectroscopy proved to be particularly suitablefor investigating biosorption properties of bacterial biomass. Bacter-ial cell walls contain indeed a range of organic acid functional groupsthat can adsorb metal cations and protons from solution, thusmodifying their chemical state and consequently their infraredabsorption spectrum. The interaction of cell envelope with metalcations andprotons can be therefore investigated looking at bacteriallayer spectral changes induced by exposure to these ionic species.

To our knowledge the direct acquisition of difference spectrato study bacterial biosorption properties has not been reportedyet. Several authors investigated the interaction of microorgan-isms with metal ions comparing absolute infrared absorptionspectra recorded on free and metal-bound bacterial cells.6 Never-theless, as pointed out by Heinrich and co-workers,14 the analysisof difference spectra is very helpful for focusing the attentionsolely on chemical groups involved in the transition under investiga-tion. On the other hand, the direct acquisition of ATR-FTIR

Received: December 7, 2010Revised: February 8, 2011

ABSTRACT: The ability of microorganisms to adhere toabiotic surfaces and the potentialities of attenuated total reflec-tion Fourier transform infrared (ATR-FTIR) spectroscopy havebeen exploited to study protonation and heavy metal bindingevents onto bacterial surfaces. This work represents the firstattempt to apply on bacteria the recently developed methodknown as perfusion-induced ATR-FTIR difference spectroscopy.1,2

Such a technique allows measurement of even slight changes inthe infrared spectrum of the sample, deposited as a thin layer onan ATR crystal, while an aqueous solution is perfused over itssurface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surfaceof Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni2þ with surface protonable groups of thismicroorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons.Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterialspectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected revealthat carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni2þ, establishing withthis ion relatively weak coordinative bonds.

1578 Biophysical Journal Volume 100 March 2011 1578–1586

Vibrational Motions Associated with Primary Processes inBacteriorhodopsin Studied by Coherent Infrared Emission Spectroscopy

Geza I. Groma,†‡* Anne Colonna,† Jean-Louis Martin,† and Marten H. Vos†

†Laboratory for Optical Biosciences, Ecole Polytechnique, Institut National de la Sante et de la Recherche Medicale, Centre National de laRecherche Scientifique, Palaiseau, France; and ‡Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences,Szeged, Hungary

ABSTRACT The primary energetic processes driving the functional proton pump of bacteriorhodopsin take place in the form ofcomplex molecular dynamic events after excitation of the retinal chromophore into the Franck-Condon state. These early eventsinclude a strong electronic polarization, skeletal stretching, and all-trans-to-13-cis isomerization upon formation of the J interme-diate. The effectiveness of the photoreaction is ensured by a conical intersection between the electronic excited and groundstates, providing highly nonadiabatic coupling to nuclear motions. Here, we study real-time vibrational coherences associatedwith these motions by analyzing light-induced infrared emission from oriented purple membranes in the 750–1400 cm�1 region.The experimental technique applied is based on second-order femtosecond difference frequency generation onmacroscopicallyordered samples that also yield information on phase and direction of the underlying motions. Concerted use of several analysismethods resulted in the isolation and characterization of seven different vibrational modes, assigned as C-C stretches, out-of-plane methyl rocks, and hydrogen out-of-plane wags, whereas no in-plane H rock was found. Based on their lifetimes andseveral other criteria, we deduce that the majority of the observed modes take place on the potential energy surface of theexcited electronic state. In particular, the direction sensitivity provides experimental evidence for large intermediate distortionsof the retinal plane during the excited-state isomerization process.

INTRODUCTION

Retinal proteins are of high importance in a wide range ofliving organisms. They are involved both in visual percep-tion of vertebrates and many invertebrates and in light-energy transduction of an increasingly documented classof underwater microorganisms (1,2). The best studiedretinal protein utilizing light energy is bacteriorhodopsin(bR). bR builds up a transmembrane potential by a proton-pumping mechanism during its photocycle initiated by lightabsorption (3,4). The early steps of this photocycle can bebriefly summarized in terms of intermediates distinguishedby visual absorption spectroscopy as (5–8):

bR568/hy

FC!200fs I460!500fs J625!3ps K610:

Here, bR568 denotes the electronic ground state (S0) of theresting form of bR, FC and I460 correspond to the Franck-Condon and the energetically minimum structures of thefirst electronically excited state (S1), respectively, and J625and K610 are the two first ground-state intermediates ofthe photocycle. It is evident from extensive experimentaldata that the configuration of the retinal chromophore isall-trans in the bR568 form and 13-cis in the K610 interme-diate (8–13). However, the detailed timing of the isomeriza-tion and its interplay with other ultrafast processes are stillsubjects of intense debate.

Early time-resolved transient absorption experiments(14,15) resulted in an initially widely accepted model

SubmittedDecember 2, 2010, andaccepted for publicationFebruary 4, 2011.

*Correspondence: groma@brc.hu

Editor: Leonid S Brown.

� 2011 by the Biophysical Society

0006-3495/11/03/1578/9 $2.00

proposing molecular motion on the excited-state potentialenergy surface (PES) along a single degree of freedom,the torsion around the C13¼C14 bond, directly coupled tothe optical transition. A more advanced description of thephotoprocesses of retinal is based on the concept of a multi-dimensional conical intersection (CI). Here, beyond thetorsional motion, at least one additional reaction coordinateis required, and the PESs are described as hypersurfaces thatexperience strong nonadiabatic coupling at true crossingpoints, or even seams, allowing the photoreaction to befunneled at a very high rate, efficiency, and selectivity(16,17). Ab initio quantum chemical simulations indicatedthat the relaxation of the S1 state can be described asa two-mode molecular motion: initial relaxation from theFC region is dominated by skeletal stretching, and the CIis formed by coupling this reaction path to the isomerizationprocess (18–20). Unlike S0, which has a covalent Ag-like(dot-dot) character, S1 has a highly ionic Bu-like (hole-pair) structure (19). The corresponding positive charge trans-fer along the hydrocarbon tail upon excitation to the FCregion is known as sudden polarization, from early literature(21–23) speculating about the functional role of this effect inthe energy-transduction process. The charge-transfer natureof S1 is maintained, and even increased, during the two-mode pathway of relaxation (19,20,24). Altogether, isomer-ization through a CI cannot be handled as a separate event,because it is intertwined with other major processes such asintramolecular charge transfer and skeletal stretching.

Recent hybrid ab initio quantum mechanical/molecularmechanical (QM/MM) calculations made possible a detailed

doi: 10.1016/j.bpj.2011.02.011

Photosynthetic reaction centerprotein in nanostructures

Kata Hajdu1, Tibor Szabo1, Melinda Magyar1, Gabor Bencsik2, Zoltan Nemeth3, Krisztina Nagy4,Arnaud Magrez5, Laszlo Forro5, Gyorgy Varo4, Klara Hernadi3, and Laszlo Nagy*,1

1 Department of Medical Physics and Informatics, University of Szeged, 6720 Szeged, Hungary2 Department of Physical Chemistry and Materials Science, University of Szeged, 6720 Szeged, Hungary3 Department of Applied and Environmental Chemistry, University of Szeged, 6720 Szeged, Hungary4 Institute of Biophysics, BRC, Szeged, 6726 Szeged, Hungary5 Institute of Physics of Complex Matter, Ecole Polytechnique Federale de Lausanne, 1015 Lausanne, Switzerland

Received 19 April 2011, revised 28 May 2011, accepted 5 July 2011

Published online 11 August 2011

Keywords carbon nanotubes, ITO, nanocomposites, reaction centers

* Corresponding author: e-mail lnagy@sol.cc.u-szeged.hu, Phone/Fax: 36-62-544121

Photosynthetic reaction center (RC) is one of the most

important proteins, because it is Nature’s solar battery

converting light energy into chemical potential in the photo-

synthetic membrane assuring conditions for carbon reduction in

cells. Although it is developed in nanometer scale, and is

working in nanoscopic power, this is the protein that assures the

energy input practically for the whole biosphere on Earth. The

extremely large quantum yield of the primary charge separation

(close to 100%) in the RC offers a big challenge to use it in

nanodevices. Results of structural (AFM, EM), optical, and

electro chemical investigations on RC bio-nanocomposite

materials based on different carrier matrices (e.g., CNTs,

ITO) will be presented.

� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

1 Introduction Photosynthetic reaction center (RC,Fig. 1) is one of the most important proteins, because it isNature’s solar battery converting light energy into chemicalpotential in the photosynthetic membrane assuring con-ditions for carbon reduction in cells [1, 2]. Although it isdeveloped in nanometer scale, and is working in nanoscopicpower, this is the protein that assures the energy inputpractically for the whole biosphere on Earth. The extremelylarge quantum yield of the primary charge separation (closeto 100% [3]) in the RC offers a big challenge to use it innanodevices. Details of the pigment–protein complexorganization and kinetic components of charge separationand stabilization are given earlier [1, 4, 5].

It is known that biological materials keep the extremesensitivity only in their original environment. When they areisolated from the living organisms their activity usuallydecreases. However, there are evidences that it is possible toattach them to artificial materials so that they are able toretain their activity. Other unique possibility is to attach themto nanomaterials. These unique systems are called bio-nanocomposites. In these materials there is a chance tocombine the advantageous properties of the two

components. These new types of materials might open somany possible directions for new generations of practicalapplications, e.g., energy conversion and storage, integratedoptoelectronics in analytical, memory, and microimagingdevices, etc. For review see, e.g., Giardi and Pace [6].

For example, several organic materials, as well as lightsensitive biological systems were immobilized on inorganiccarrier surfaces, like semiconducting nanoparticle aggre-gates, silicon nanorods, or transition metal-oxides, metal, orcarbon nanowires showing functional activities (for typicalreferences see, e.g. [7–9]). The key questions of researchesthese days are: new applications, reproducibility of themeasurements, and stability – mainly that of the biologicalcomponent.

It was shown recently [10, 11] that RCs can be attachedto carbon nanotubes (CNTs) and there is an electronicconnection between the two types of materials. In our studieswe aimed to show the physico chemical properties (photo-chemical activity, electric conductivity) of the bio-nano-composite materials prepared by RCs and different carriermatrices. The carrier matrices in our experiments are theisolating borosilicate glass, conducting indium tin oxide

Phys. Status Solidi B, 1–4 (2011) / DOI 10.1002/pssb.201100046 p s sbasic solid state physics

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� 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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R A PID PU B LIC AT IO N S

Journal of the European Optical Society - Rapid Publications 6, 11029 (2011) www.jeos.org

Two-photon polymerization with optimized spatial lightmodulator

Lorand Kelemenlkelemen@brc.hu

Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, Temesvari krt. 62,Szeged, Hungary, H-6726

Pal Ormos Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, Temesvari krt. 62,Szeged, Hungary, H-6726

Gaszton Vizsnyiczai Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, Temesvari krt. 62,Szeged, Hungary, H-6726

The application of diffractive optical elements can enhance the efficiency of the two- photon polymerization (TPP) process by multiplyingthe polymerizing beams. Spatial light modulators (SLMs) can dynamically change the light intensity pattern used for polymerization, makingsingle shot polymerization possible. Most reflective, liquid crystal-based instruments, however, suffer from various surface aberrations. Inorder to enable SLMs to generate suitable polymerizing beams for TPP, these aberrations need to be corrected. Several methods wereintroduced earlier to compensate SLM aberrations in different applications. For the nonlinear process of TPP, we developed and specificallycharacterized a correction procedure. We used a simple interferometric method to determine the surface distortion of the SLM, calculateda correcting hologram and confirmed the correction with the polymerization of test structures. The corrected SLM was capable of parallelpolymerization of 3D structures with a quality achievable with non-SLM beams. [DOI: 10.2971/jeos.2011.11029]

Keywords: Diffractive optics, 3D Microstructure fabrication, Spatial light modulators, Laser materials processing

1 INTRODUCTION

The method of two-photon polymerization (TPP) to producepolymer microtools for a wide range of applications [1]-[8]has been used for more than a decade [9]-[11]. It is capa-ble of producing structures with sub-micrometer features andcan create complex 3D structures in a few straightforwardsteps. The method relies on focusing an ultrashort-pulsedlaser beam into a layer of photoresist, where only multi-photon absorption can occur. The polymerization is confinedinto a well-defined volume normally bearing an ellipsoid-likeshape, called the voxel [12, 13], which can have dimensionslower than the diffraction limit. TPP is capable of producingstructures with feature size below 100 nm [14].

The efficiency of TPP can be improved by the multiplicationof the laser beam using various diffractive optical elementssuch as kinoform, microlens array or spatial light modulators(SLM) [15]-[18]. Multiplication of the original laser beam bySLM has been used routinely in optical trapping experimentswhere it creates several, independently movable focal spots,allowing the parallel manipulation of numerous trapped ob-jects in 3D [19]-[21]. In TPP, the control of the position of sev-eral polymerizing focal spots in 3D enables the parallel poly-merization of small identical objects [16] or extended struc-tures [17, 18]; SLM-assisted polymerization of a complex mi-crostructure was realized even with a single, static illumina-tion with specially tailored optical fields [22]. SLM modifiedbeams generally suffer form a certain level of wavefront dis-tortion due to the curvature of the reflecting surface. This ispresent in almost all LCoS (Liquid Crystal on Silicon) SLMs

with different severity [23]-[26], resulting in an irregular in-tensity distribution in the focus. In optical traps, poor fo-cal spot characteristics lead to smaller trap stiffness and dis-torted force field [27, 28]. Wavefront distortion determinationmethods may apply wavefront sensors [29]-[31], interferome-try [24] or monitor focal spot distribution [26, 28]; the correc-tion may apply Zernike polynomials [26, 28, 31], perform thecorrection in domains of the SLM [32, 33] or apply a Laguerre-Gauss beam to optimize the wavefront [25]. The correction caneven be performed for large area SLMs [34]. Some of thesemethods are intended to correct the aberrations of the entireoptical pathway [32, 33], some only that of the SLM separately[24, 31, 34]. What is common is that the methods use exactlythe phase shifting ability of the SLM to achieve the correction:a calculated or approximated correction hologram generatesthe inverse phase shift of what the surface distortion created.

During our TPP experiments, we observed strong distortionsin the geometry of the resulted structures when SLM was usedto create multiple polymerizing focal spots. We attributed thisto the distorted focal intensity distribution that affects the reg-ular shape of the polymerizing voxel inversely and originatesfrom the distorted surface of the instrument. In order to poly-merize microstructures with sub-micrometer features usingSLM-modified beams the distortion needed to be corrected.Due to the nonlinear nature of TPP, the corrections have tobe implemented and critically characterized specifically forthis procedure. Our goal was to show that when a distorted-surface LCoS SLM is used in TPP its adverse effect on the

Received January 26, 2011; published May 31, 2011 ISSN 1990-2573

Bioelectrochemistry 81 (2011) 17–21

Contents lists available at ScienceDirect

Bioelectrochemistry

j ourna l homepage: www.e lsev ie r.com/ locate /b ioe lechem

Optical and electric signals from dried oriented purplemembrane of bacteriorhodopsins

R. Tóth-Boconádi a, A. Dér a, L. Keszthelyi a,b,⁎a Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, P. O. B. 521, H-6701, Hungaryb KFKI Research Institute for Particle and Nuclear Physics, Hungarian Academy of Sciences, Budapest P.O.B. 49, H-1525, Hungary

⁎ Corresponding author. Institute of Biophysics, Bioloian Academy of Sciences, Szeged, P. O. B. 521, H-6701, Hfax:+36 62433133.

E-mail address: kl@brc.hu (L. Keszthelyi).

1567-5394/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.bioelechem.2010.12.003

a b s t r a c t

a r t i c l e i n f o

Article history:Received 11 June 2010Received in revised form 16 December 2010Accepted 19 December 2010Available online 28 December 2010

Keywords:Bacteriorhodopsin mutantsQuasi-continuous illuminationM intermediateBlue excitationLifetimes

All the intermediates of the bacteriorhodopsin photocycle are excitable with light of suitable wavelength. Thisproperty might regulate the activity in the cells when they are exposed in the nature to high light intensity. Onthe other hand this property is involved in many applications. In this study the ground state and Mintermediate of dried oriented samples of wild-type bacteriorhodopsin and its mutant D96N were excitedwith 406 nm laser flashes. Substantial M populations were generated with quasi-continuous illumination. Thedecay of the absorption of M intermediate had three components: their lifetimes were very different for laserflash and quasi-continuous illuminations in cases of both bacteriorhodopsin species. The optical answer forthe excitation of M intermediate had a lifetime of 2.2 ms. Electric signals for M excitation had large fastnegative components and small positive components in the 100 μs time domain. The results are expected tohave important implications for bioelectronic applications of bacteriorhodopsin.

gical Research Centre, Hungar-ungary. Tel.: +36 30 9194685;

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The possible application of the dried oriented purple membranelayers containing the light sensitive protein, bacteriorhodopsin (BR)[1–3] in different electronic and optical tools became evident in thelast years [4–11]. The photocycle of BR in dried samples is restricted: itreaches the M intermediate for light excitation, stops there and finallyreturns to ground state with a lifetime of seconds. Themost importantcharacteristic of these samples is their stability: they maintain thisrestricted function for years or, with other term, not damaged during106–107 photon induced cycles.

It is well known that the intermediates of the BR photocycle can beexcited by light [12–22]. This process is important in the life of thecells. As pointed out first in [12] the blue illumination of the Mintermediate short-cuts the photocycle, i.e. blocks the protonpumping activity, this way protecting the cells from excessivechemical potential in case of high illumination. The special featuresof intermediate excitation, like wavelength or lifetime, are useful insome applications: for example the Q form arising from excited O viaanother intermediate P may serve as a memory element [23]. The Mintermediate is especially important because its excitation terminatesthe photocycle, i.e. reestablishes the BR ground state in short time asinvestigated in the usual wet samples [12–16,22]. In view of the

urgency of a recent boom in bioelectronic applications using dry BRfilms [5–11], it seemed to be worthwhile also to know the timeneeded for this process for dried oriented purple membrane layers.

Hence, we investigated the optical and electric responses of the Mintermediate of the wild-type bacteriorhodopsin (WTBR) and itsmutant D96N in dried oriented samples. First the ground state wasexcited and investigated. It turned out in absorption measurements ataround 400 nm (the characteristic wavelength of M decay) that thelifetimes and amplitudes of the three decay components depend onthe strength and duration of illumination. Studying the optical answerfor M excitation, absorption changes of ms duration emerged duringthe reestablishment of the ground state. In the electric responses forM excitation two negative components appeared in the μs time rangeand a third positive component with a lifetime of around 100 μs.

2. Materials and methods

Dried oriented samples of purple membrane separated from strainR1M1 ofHalobacterium salinarum and strain L-33 containing themutantD96N(suppliedby J. K. Lanyi)wereprepared asdescribed in [1]. In orderto obtain substantial M populations the samples were illuminated withquasi-continuous (Q-C) light (quasi-continuous means continuousillumination for some s) from a 200W tungsten lamp filtered forwavelengths above 500 nm, or by a 1 mJflash of an excimer laser drivendye laser (LambdaPhysik, EMG101MSC, Göttingen,Germany)withdyeRhodamine 6 G, emitting at 570 nm. The M concentration and theabsorption changes for M excitation were determined using a

Received: 4 May 2010, Revised: 10 September 2010, Accepted: 4 October 2010, Published online in Wiley Online Library: 2011

Spatial and temporal dependence of thecerebral endothelial cells elasticityy

Attila G. Vegha, Csilla Fazakasa, Krisztina Nagya, Imola Wilhelma,Istvan A. Krizbaia, Peter Nagyoszia, Zsolt Szegletesa and Gyorgy Varoa*

The reliable determination of the mechanical properties of a living cell is one of the most important challenges ofthe atomic force microscopic measurements. In the present study the spatial and temporal dependency of theforce measurements on cerebral endothelial cells was investigated. Besides imaging the cells, two differentsequences of force measurements were applied: (1) Acquisition of force curves in short time at several pointsacross the cell surface investigating spatial dependence of the elasticity. (2) Acquisition of force curves for long timeat a previously determined place, over the cell nucleus, which provides the temporal stability/variation ofthe measured forces/values. Three different stages of endothelial cell cultures of the hCMEC/D3 cells were used:sub-confluent living, confluent living, and confluent fixed cells. The Young’s modulus was calculated from the forcecurves using the Hertz model and the results were plotted against time or location correspondingly. The rational ofusing the three stage of culture was to clarify whether the observed effect belongs to the individual cell, tothe ensemble of cells or just to some, not living cell component. In case of sub-confluent cells the results revealed asofter nuclear region compared to the periphery, while an attenuated oscillation like fluctuation in time, with aperiod of about 10–30min, was observed. Confluent living cells showed similar tendencies to the sub-confluentcells, but the changes were larger and the temporal oscillations had longer period. The spatial dependency of theelasticity on confluent cells was confirmed by force–volume measurement too. In case of fixed cells neither spatialnor temporal differences were observed between the nuclear and peripheral region, however the Young’s modulusand the error of themeasurement was larger, compared to the sub-confluent living cells. Copyright� 2011 JohnWiley& Sons, Ltd.

Keywords: atomic force microscopy; blood–brain-barrier; cell imaging; Young’s modulus

INTRODUCTION

After the invention of the atomic force microscope (AFM) in 1986(Binnig et al., 1986) the instrument evolved as a high resolutionnoninvasive microscope. Besides of high resolution imaging inthe native environment of biologic samples the AFM is a powerfultool to determine the mechanical properties of the studiedobject, such as elasticity of living cells (Vinckier and Semenza,1998; Santos and Castanho, 2004). A broad range of different celltypes have already been investigated by AFM includingendothelial cells (Oberleithner et al., 2003; Pesen and Hoh,2005b), epithelial cells (Sharma et al., 2005), and platelets(Radmacher et al., 1996).Since endothelial cells are exposed to continuous mechanical

stimuli, when the blood cells force their way through themicrocapillary vessels, numerous studies have been completed inorder to know their mechanical properties. Majority of the AFMstudies were performed on human umbilical vein endothelialcells. A growing amount of data is available about the effect ofdrugs (Oberleithner et al., 2003, 2004; Hillebrand et al., 2006),single molecule binding (Pfister et al., 2005), cell–cell interaction(Zhang et al., 2003), and mechanical characteristics (Mathur et al.,2001; Sato et al., 2004) as well.Despite of numerous studies on endothelial cells only little

information is available about the cerebral endothelial cells.

These cells form a specialized type of endothelial cellscharacterized by low paracellular and transcellular permeabilityand a continuous line of tight junctions at the contact betweenthem, which forms the basis of the blood–brain-barrier,restricting and controlling the free movement of differentsubstances between blood and neural tissue. Therefore it plays akey role in the maintenance of the homeostasis of the centralnervous system (Farkas et al., 2005; Wilhelm et al., 2007). If theclinical treatment requires the blood–brain-barrier can beopened temporarily by different chemicals, such as the mannitol(Kroll and Neuwelt, 1998; Rappoport et al., 2000; Balint et al.,2007). To fulfill all these requirements the endothelial cells shouldbe elastic and tough.

(wileyonlinelibrary.com) DOI:10.1002/jmr.1107

Research Article

* Correspondence to: G. Varo, Biological Research Center, Temesvari krt. 62,Szeged, Hungary.E-mail: varo@brc.hu

a A. G. Vegh, C. Fazakas, K. Nagy, I. Wilhelm, I. A. Krizbai, P. Nagyoszi, Z. Szegletes,

G. Varo

Institute of Biophysics, Biological Research Center of the Hungarian Academy

of Sciences, 6726 Szeged, Hungary

y This article is published in Journal of Molecular Recognition as a focus on AFMon Life Sciences and Medicine, edited by Jean-Luc Pellequer and Pierre Parot(CEA Marcoule, Life Science Division, Bagnols sur Ceze, France).

J. Mol. Recognit. 2011; 24: 422–428 Copyright � 2011 John Wiley & Sons, Ltd.

422

Hindawi Publishing CorporationJournal of Biomedicine and BiotechnologyVolume 2011, Article ID 670589, 6 pagesdoi:10.1155/2011/670589

Research Article

Effect of Antimicrobial Peptide-Amide: Indolicidin on BiologicalMembranes

Attila Gergely Vegh,1 Krisztina Nagy,1 Zoltan Balint,1 Adam Kerenyi,1 Gabor Rakhely,1, 2

Gyorgy Varo,1 and Zsolt Szegletes1

1 Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, P.O. Box 521, 6701 Szeged, Hungary2 Department of Biotechnology, University of Szeged, Kozep fasor 52, 6726 Szeged, Hungary

Correspondence should be addressed to Zsolt Szegletes, zszeglet@brc.hu

Received 20 January 2011; Revised 24 March 2011; Accepted 29 April 2011

Academic Editor: Miguel Castanho

Copyright © 2011 Attila Gergely Vegh et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Indolicidin, a cationic antimicrobial tridecapeptide amide, is rich in proline and tryptophan residues. Its biological activity isintensively studied, but the details how indolicidin interacts with membranes are not fully understood yet. We report here anin situ atomic force microscopic study describing the effect of indolicidin on an artificial supported planar bilayer membraneof dipalmitoyl phosphatidylcholine (DPPC) and on purple membrane of Halobacterium salinarum. Concentration dependentinteraction of the peptide and membranes was found in case of DPPC resulting the destruction of the membrane. Purplemembrane was much more resistant against indolicidin, probably due to its high protein content. Indolicidin preferred the borderof membrane disks, where the lipids are more accessible. These data suggest that the atomic force microscope is a powerful tool inthe study of indolicidin-membrane interaction.

1. Introduction

Antimicrobial cationic peptides are host defense moleculesproduced by the innate immune system of organisms allacross the evolutionary spectrum. They play a key role in thehost defense system of many higher organisms [1]. Indoli-cidin, encoded by a member of cathelicidin gene family, acationic antimicrobial tridecapeptide amide (H-Ile-Leu-Pro-Trp-Lys-Trp-Pro-Trp-Trp-Pro-Trp-Arg-Arg-NH2), was iso-lated from cytoplasmic granules of bovine neutrophils [2]. Itis one of the shortest known natural-occurring antimicrobialpeptide [3], toxic to both prokaryotes and eukaryotes [3, 4].The high percentage of proline and tryptophan residuesmakes indolicidin a unique antimicrobial. Unlike severalother antimicrobial peptides, the structure of indolicidinupon membrane interaction is not a well-defined helix or aβ-turn and does not display their characteristic amphipathicnature [4–7]. It has been reported that indolicidin expressesits antimicrobial activity by creating pores through cell mem-branes [8]. Other studies showed that indolicidin treatmentresulted total disintegration of membrane structures [9] orthat it did not cause cell lysis even at high concentration [4].Compared to α-helical antibiotic peptides, indolicidin is less

able to dissipate the bacterial inner membrane potential andforms smaller pores, yet it kills bacteria rapidly [10]. It wasreported that an interfacial membrane location was preferredby indolicidin [6, 11]. These results point to a mechanism ofaction that is different from well-defined channel formation.

Lipid bilayer on polyelectrolyte films can be used as auseful experimental approach to study basic problems ofbiological membrane structures [12–14]. Bacteriorhodopsinis a light-driven proton pump in the plasma membrane ofHalobacterium salinarum. This integral membrane protein istightly packed in two-dimensional crystalline from termedpurple membrane with high (75% w/w) bacteriorhodopsincontent, with no other protein. The remaining 25% is lipid[15]. In situ atomic force microscopic (AFM) experimentscould provide detailed information about these systems [16].

Here, we present an AFM study on the interaction of in-dolicidin with an artificial and a natural membrane.

2. Materials and Methods

2.1. Sample Preparation. Freshly cleaved mica (SPI-ChemMica Sheets, Structure Probe, Inc., West Chester, PA, USA)

Heat Stress Causes Spatially-Distinct Membrane Re-Modelling in K562 Leukemia CellsGabor Balogh1, Giuseppe Maulucci5, Imre Gombos1, Ibolya Horvath1, Zsolt Torok1, Maria Peter1, Elfrieda

Fodor1, Tibor Pali2, Sandor Benko3, Tiziana Parasassi4, Marco De Spirito5, John L. Harwood6*, Laszlo

Vıgh1*

1 Institute of Biochemistry, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary, 2 Institute of Biophysics, Biological Research Centre, Hungarian

Academy of Sciences, Szeged, Hungary, 3 First Department of Internal Medicine, Albert Szent-Gyorgyi Clinical Center, University of Szeged, Szeged, Hungary, 4 Institute of

Translational Pharmacology, CNR, Rome, Italy, 5 Istituto di Fisica, Universita Cattolica Sacro Cuore, Rome, Italy, 6 School of Biosciences, Cardiff University, Cardiff, Wales,

United Kingdom

Abstract

Cellular membranes respond rapidly to various environmental perturbations. Previously we showed that modulations inmembrane fluidity achieved by heat stress (HS) resulted in pronounced membrane organization alterations which could beintimately linked to the expression and cellular distribution of heat shock proteins. Here we examine heat-inducedmembrane changes using several visualisation methods. With Laurdan two-photon microscopy we demonstrate that, incontrast to the enhanced formation of ordered domains in surface membranes, the molecular disorder is significantlyelevated within the internal membranes of cells preexposed to mild HS. These results were compared with those obtainedby anisotropy, fluorescence lifetime and electron paramagnetic resonance measurements. All probes detected membranechanges upon HS. However, the structurally different probes revealed substantially distinct alterations in membraneheterogeneity. These data call attention to the careful interpretation of results obtained with only a single label. Subtlechanges in membrane microstructure in the decision-making of thermal cell killing could have potential application incancer therapy.

Citation: Balogh G, Maulucci G, Gombos I, Horvath I, Torok Z, et al. (2011) Heat Stress Causes Spatially-Distinct Membrane Re-Modelling in K562 LeukemiaCells. PLoS ONE 6(6): e21182. doi:10.1371/journal.pone.0021182

Editor: Peter Csermely, Semmelweis University, Hungary

Received May 1, 2011; Accepted May 22, 2011; Published June 16, 2011

Copyright: � 2011 Balogh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was funded by the European Union Project LipidomicNet (HEALTH-F4-2008-202272), Hungarian National Scientific Research Foundation(OTKA NN 76716 and K68804), and the Hungarian National Development Agency TAMOP-4.2.2/08/1-2008-0005, TAMOP-4.2.2/08/1-2008-0002 and TAMOP-4.2.2/08/1-2008-0013. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: vigh@brc.hu (LV); Harwood@cardiff.ac.uk (JLH)

Introduction

There has been a renewed interest in the application of

hyperthermia in antitumor therapy. This is a promising treatment

modality for cancer, especially in combination with radiotherapy,

because various tumors are often more thermally sensitive than

normal tissues [1]. However, the primary target of cellular heat

killing is still unknown. It was suggested 30 years ago that mem-

branes of tumor cells are the major targets for heat treatment and

that the effectiveness of hyperthermic cell killing is influenced

basically by the fluidity of membranes [2]. In support of this

proposal, the use of membrane fluidizing agents (typically local

anaesthetics) was shown to potentiate the therapeutic effect of

hyperthermia [2].

On the other hand, cells exposed to non-lethal elevated tem-

peratures or treated with various substances targeting membranes

develop a stronger resistance to a subsequent severe heat stress

(HS) [3] – a phenomenon called acquired thermotolerance. One

of the major obstacles for many types of anticancer therapy is that

they induce a stress response (also called a heat shock response)

making tumors more resistant to subsequent treatments. Amongst

other effects, the heat shock response restores the normal protein

folding environment by upregulating heat shock proteins (Hsps)

and changing their cellular locations [4–7] thus altering pathways

controlling cell survival, growth and metabolism.

The question as to how membrane structural alterations can be

linked to the Janus-like properties of hyperthermia in cancer

therapy has been addressed in recent reviews [1,8]. Cellular mem-

branes have also been implicated as the primary heat sensors

[9,10] as well as in the decision-making for thermal cell killing

[1,11].

Previously we demonstrated that membrane hyperfluidization

acts as a primary signal to initiate the Hsp response in prokaryotic

organisms [12,13], yeast [14], K562 leukemia [15] and B16

melanoma cells [16]. Similarly to HS, exposing K562 cells to

benzyl alcohol (BA), which is a well-established membrane flui-

dizing agent [17,18], elicited nearly identical increases in cytosolic

Ca2+ concentration, Hsp70 synthesis and mitochondrial hyperpo-

larization [15]. Moreover, the microdomain organization of pla-

sma membranes (PM) has been shown to be a decisive factor in the

perception and transduction of heat- or non-proteotoxic chemical

agent-induced membrane stress into signals which then trigger the

transcriptional activation of heat shock genes in B16 melanoma

cells [16]. As reviewed recently, the temporal and spatial

regulation of the membrane hyperfine structure appears to be a

hallmark of cellular stress sensing and signalling events [6,9].

PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e21182

BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - REVIEW ARTICLE

New aspects of the molecular constituents of tissue barriers

H. C. Bauer • A. Traweger • J. Zweimueller-Mayer •

C. Lehner • H. Tempfer • I. Krizbai •

I. Wilhelm • H. Bauer

Received: 17 August 2010 / Accepted: 30 August 2010 / Published online: 24 September 2010

� Springer-Verlag 2010

Abstract Epithelial and endothelial tissue barriers are

based on tight intercellular contacts (Tight Junctions, TJs)

between neighbouring cells. TJs are multimeric com-

plexes, located at the most apical border of the lateral

membrane. So far, a plethora of proteins locating at tight

intercellular contacts have been discovered, the role of

which has just partly been unraveled. Yet, there is con-

vincing evidence that many TJ proteins exert a dual role:

They act as structural components at the junctional site

and they are involved in signalling pathways leading to

alterations of gene expression and cell behaviour (migra-

tion, proliferation). This review will shortly summarize

the classical functions of TJs and TJ-related proteins and

will introduce a new category, termed the ‘‘non-classical’’

functions of junctional proteins. A particular focus will be

directed towards the nuclear targeting of junctional pro-

teins and the downstream effects elicited by their intra-

nuclear activities.

Keywords Tissue-barriers � Epithelia � Endothelia �Tight junctions

Introduction

Vertebrate epithelial and endothelial cells are capable of

forming particularly tight intercellular connections/con-

tacts, referred to as zonulae occludentes (Tight junctions,

TJs). TJs represent the most restrictive type of intercellular

junctions located at the apical border of the lateral mem-

brane of endothelial and epithelial cells. They act as a

paracellular diffusion barrier and thereby separate internal

and external fluid compartments, an indispensable pre-

requisite for every organ development and function. The

term ‘‘barrier’’ not only covers the junctional occlusion

between neighbouring cells but also the concomitant

establishment of many transport systems enabling the

vectorial transport of macromolecules and ions across

epithelial and endothelial cell sheets.

Freeze fracture replicas of well-developed TJs show a

belt-like area of anastomizing strands, created by rows of

proteinaceous particles within the plasma membrane. We

now have evidence that these particles are created by

transmembrane proteins responsible for the intimate cell–

cell contact (Aijaz et al. 2006; Tsukita et al. 2001). The

complexity of the TJ network and the number of TJ strands

vary markedly among different epithelia and endothelia,

and intensive research has revealed that it is primarily the

composition of distinct integral membrane proteins which

determines the specific ‘‘tightness’’ or ‘‘leakiness’’ of an

H. C. Bauer � J. Zweimueller-Mayer � C. Lehner �H. Bauer (&)

Department of Organismic Biology, University of Salzburg,

Hellbrunner Strasse 34, 5020 Salzburg, Austria

e-mail: Hannelore.Bauer@sbg.ac.at

A. Traweger

Baxter Innovations GmbH, 2304 Orth an der Donau, Austria

H. C. Bauer � H. Tempfer

Paracelsus Private Medical University,

Mullner Hauptstrasse 48, 5020 Salzburg, Austria

C. Lehner

Department of Traumatology and Sports Injuries,

Paracelsus Private Medical University, Mullner Hauptstrasse 48,

5020 Salzburg, Austria

I. Krizbai � I. Wilhelm

Biological Research Center, Institute of Biophysics,

Temesvari krt 62, 6726 Szeged, Hungary

123

J Neural Transm (2011) 118:7–21

DOI 10.1007/s00702-010-0484-6

Author's personal copy

Oxygen–glucose deprivation increases the enzymatic activity and themicrovesicle-mediated release of ectonucleotidases in the cellscomposing the blood–brain barrier

Stefania Ceruti a,⇑, Laura Colombo a, Giulia Magni a, Francesca Viganò a, Marta Boccazzi a, Mária A. Deli b,Beáta Sperlágh c, Maria P. Abbracchio a, Ágnes Kittel c

a Laboratory of Molecular and Cellular Pharmacology of Purinergic Transmission, Department of Pharmacological Sciences, Università degli Studi di Milano, Via Balzaretti, 9,20133 Milan, Italyb Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt 62, H-6726 Szeged, Hungaryc Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony u. 43, Budapest 1083, Hungary

a r t i c l e i n f o

Article history:Received 11 February 2011Received in revised form 19 May 2011Accepted 26 May 2011Available online 6 June 2011

Keywords:Brain endothelial cellsPericytesAstrocytesHypoxiaNTPDases

a b s t r a c t

The blood–brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is com-posed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and theirreceptors (the purinergic system) constitute a widely diffused signaling system involved in many path-ophysiological processes. However, the role of this system in controlling BBB functions is still largelyunknown. By using cultures of these three cell types grown separately and a BBB in vitro model consistingof triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymesnucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellularnucleotides) under control and ischemic (oxygen–glucose deprivation in vitro; OGD) conditions. NTP-Dase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytesand, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell deathwhen OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes andpericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activ-ity following exposure to OGD in all three cell types, either when grown separately or when co-culturedtogether to resemble the composition of the BBB. Moreover, electron microscopy analysis showed thatboth endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane,which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by dam-aged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effectfor brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucle-otides in modulating BBB responses to ischemic events, and to develop new effective purinergic-basedapproaches for brain ischemia.

� 2011 Elsevier B.V. All rights reserved.

1. Introduction

The blood–brain barrier (BBB) is the dynamic interface betweenthe blood flow and the central nervous system (CNS), consisting ofendothelial cells of brain capillaries, pericytes and astrocyte end-feet. The four major roles played by the BBB are (i) the creationof a regulated microenvironment for reliable neuronal signalingwithin the CNS, (ii) the supply of nutrients, (iii) the defense from

toxic substances and (iv) the control of the communication be-tween the CNS and the periphery (Abbott et al., 2006). Overall,the BBB acts as a physical, transport and metabolic barrier forthe nervous system (Zlokovic, 2008).

Brain endothelial cells are a specialized type of epithelial cells,which form the inner layer of cerebral blood vessels (Joó, 1996).They are connected with each other through tight junctions andconstitute a physical barrier to restrict the flux of cells and mole-cules between the blood and the brain (Deli, 2009). The morphol-ogy and the molecular composition of these junctions arecontrolled by the brain microenvironment (Hawkins and Davis,2005), the specific properties of brain endothelial cells being alsoinduced and maintained by a cross-talk with neighboring cells.The crucial role of astrocytes in preserving the integrity of theBBB was discovered first (as reviewed in Deli et al. (2005) and

0197-0186/$ - see front matter � 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.neuint.2011.05.013

⇑ Corresponding author. Tel.: +39 02 50318261; fax: +39 02 50318284.E-mail addresses: stefania.ceruti@unimi.it (S. Ceruti), laura.colombo1@unimi.it

(L. Colombo), giulia.magni@unimi.it (G. Magni), francesca.vigano@unimi.it(F. Viganò), marta.boccazzi@unimi.it (M. Boccazzi), deli@brc.hu (M.A. Deli),sperlagh@koki.hu (B. Sperlágh), mariapia.abbracchio@unimi.it (M.P. Abbracchio),kittel007@gmail.com (Á. Kittel).

Neurochemistry International 59 (2011) 259–271

Contents lists available at ScienceDirect

Neurochemistry International

journal homepage: www.elsevier .com/locate /nci

144 Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, 2011, 144-165

K. Tihanyi and M. Vastag (Eds) All rights reserved - © 2011 Bentham Science Publishers Ltd.

CHAPTER 8

Drug Transport and the Blood-Brain Barrier

Mária A. Deli

Biological Research Center, Hungarian Academy of Sciences; E-mail: deli@brc.hu

Abstract: The blood-brain barrier, a dynamic interface separating the brain from systemic circulation, is the major entry route for therapeutic compounds to the central nervous system. The blood-brain barrier phenotype of the endothelial cells of brain microvessels includes tight interendothelial junctions, the lack of pinocytosis and fenestrae, transendothelial transport pathways, and a metabolic barrier. The primary role of the blood-brain barrier is to create ionic homeostasis for neuronal functions, but it also provides the central nervous system with nutrients and protects it from toxic insults. The formation and maintenance of these organ-specific characteristics are based on cross talk between the cells of the neurovascular unit, such as brain endothelial cells, pericytes, astroglia, microglia and neurons. The problem of drug transport at the blood-brain barrier is two-fold: the great majority of neuropharmaceutical candidates, hydrophilic molecules, biopharmaceuticals and efflux transporter ligands do not penetrate the blood-brain barrier, while unwanted side effects develop if a drug with main peripheral action crosses the blood-brain barrier. Overcoming the major mechanisms restricting drug transport at the level of the blood-brain barrier, tight interendothelial junctions, efflux transporters and the enzymatic barrier can lead to better drug penetration to the brain. In addition, there are several physiological transport pathways – the carrier systems and the adsorptive and receptor-mediated transports – which can be exploited for drug targeting. Strategies for drug delivery and targeting to the brain include modification of the molecules, modification of the blood-brain barrier functions, and circumvention of the blood-brain barrier. Some of the techniques based on these strategies are already in clinical use, while others are promising new possibilities for improving the therapy of central nervous system diseases.

OVERVIEW OF THE STRUCTURE AND FUNCTIONS OF THE BLOOD-BRAIN BARRIER

The blood-brain barrier (BBB), a dynamic interface separating the brain from systemic circulation, is the major entry route for therapeutic compounds to the central nervous system (CNS). The estimated total length of human brain capillaries is 650 km, with a total surface area of about 20 m2 [1, 2]. The complex tight junctions (TJs) between brain endothelial cells constitute the morphological basis of the BBB [3]. The primary role of the BBB is to create ionic homeostasis for neuronal functions [4]. It also provides the CNS with nutrients and protects it from toxic insults by sophisticated transport systems [5]. The low level of paracellular flux and transendothelial vesicular trafficking results in a transport barrier for drugs that are hydrophilic and have a molecular mass larger than 400 Da, while the presence of effective efflux transporters at the luminal membrane of brain endothelial cells limits the brain penetration of lipophilic xenobiotics and drugs. The BBB prevents 98% of potential neuropharmaceuticals, especially new biopharmacons, nucleic acids, peptide or protein drugs, from reaching their targets in the CNS [1]. For these reasons, the treatment of CNS diseases, including strokes, Alzheimer’s disease and brain tumours, remains unsatisfactory, and improving drug delivery to the CNS is considered essential for the future success of therapies for neurological disorders [6].

THE ANATOMICAL BASIS OF THE BLOOD-BRAIN BARRIER

It was 125 years ago that Paul Ehrlich and his co-workers discovered, using dye studies, that the mammalian CNS has a unique compartment within the body. In the 1960s, the interendothelial junctions of cerebral capillaries were identified through electron microscopy as the anatomical basis of the vertebrate BBB [7,8]. Brain endothelial cells are characterized by their thin cytoplasm, their complex TJs, the lack of fenestrae, the small number of vesicles and the large number of mitochondria [9]. The freeze-fracture morphology of BBB TJs is unique: in addition to the correlation between the number of TJ strands and the tightness of the junctions, as measured by electrical resistance, the association of the particles with the inner (P-face) is as high as the association with the outer (E-face) lipidic leaflet of the plasma membrane. This high P-face/E-face ratio also reflects the quality of the barrier [9].

Phencyclidine-induced Loss of Asymmetric Spine Synapsesin Rodent Prefrontal Cortex is Reversed by Acute andChronic Treatment with Olanzapine

John D Elsworth*,1, Bret A Morrow1, Tibor Hajszan2,3, Csaba Leranth2 and Robert H Roth1

1Laboratory of Neuropsychopharmacology, Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA; 2Department of

Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA; 3Department of Biophysics, Biological Research Center,

Hungarian Academy of Sciences, Szeged, Hungary

Enduring cognitive deficits exist in schizophrenic patients, long-term abusers of phencyclidine (PCP), as well as in animal PCP models of

schizophrenia. It has been suggested that cognitive performance and memory processes are coupled with remodeling of pyramidal

dendritic spine synapses in prefrontal cortex (PFC), and that reduced spine density and number of spine synapses in the medial PFC of

PCP-treated rats may potentially underlie, at least partially, the cognitive dysfunction previously observed in this animal model. The

present data show that the decrease in number of asymmetric (excitatory) spine synapses in layer II/III of PFC, previously noted at

1-week post PCP treatment also occurs, to a lesser degree, in layer V. The decrease in the number of spine synapses in layer II/III was

sustained and persisted for at least 4 weeks, paralleling the observed cognitive deficits. Both acute and chronic treatment with the atypical

antipsychotic drug, olanzapine, starting at 1 week after PCP treatment at doses that restore cognitive function, reversed the asymmetric

spine synapse loss in PFC of PCP-treated rats. Olanzapine had no significant effect on spine synapse number in saline-treated controls.

These studies demonstrate that the effect of PCP on asymmetric spine synapse number in PFC lasts at least 4 weeks in this model. This

spine synapse loss in PFC is reversed by acute treatment with olanzapine, and this reversal is maintained by chronic oral treatment,

paralleling the time course of the restoration of the dopamine deficit, and normalization of cognitive function produced by olanzapine.

Neuropsychopharmacology (2011) 36, 2054–2061; doi:10.1038/npp.2011.96; published online 15 June 2011

Keywords: asymmetric spine synapse; olanzapine; phencyclidine; prefrontal cortex; pyramidal cell; schizophrenia

����������������������������������������������

INTRODUCTION

Schizophrenia is a chronic mental disorder that affectsabout 1% of the population and is characterized by so-called ‘positive’ and ‘negative’ symptoms, as well ascognitive deficits (Freedman, 2003). Cognitive dysfunction,including prefrontal cortex (PFC)-dependent functions suchas working memory, appears to be the most enduring andtreatment-resistant feature of schizophrenia, which repre-sent a severe clinical problem (Tamminga et al, 1998).

It has been suggested that cognitive performance andmemory processes are coupled with remodeling of pyrami-dal dendritic spine synapses in both the PFC (Hof andMorrison, 2004; Nimchinsky et al, 2002) and hippocampus(Silva, 2003; Toni et al, 1999). By reducing the number ofavailable neuronal circuits for memory storage, loss of spine

synapses is believed to result in poor memory and cognitiveperformance. In fact, a recent study has demonstrated astrong correlation between the loss of asymmetric spinesynapses in monkey PFC and cognitive impairment duringaging (Peters et al, 2008). Several groups have reporteddystrophic changes in frontal cortical pyramidal celldendrites in schizophrenia, including decreases in dendriticlength, branching, and spine density (Black et al, 2004;Broadbelt et al, 2002; Garey et al, 1998; Glantz and Lewis,2000; Kalus et al, 2000; Kolluri et al, 2005), consistent withthe hypothesis that these changes are involved in thecognitive dysfunction that typifies the disorder. It is notcurrently known though whether the pathological changesin the cortex in schizophrenia are fixed or can be altered bytreatment with antipsychotic drugs.

Long-term abusers of phencyclidine (PCP) develop anenduring cognitive dysfunction (Cosgrove and Newell,1991), and animal models that use a subchronic PCPtreatment paradigm simulate the schizophrenia-like lastingcognitive deficit (Jentsch et al, 1997a, b). Our own studiesdemonstrate a significant reduction in the number ofasymmetric synapses on dendritic spines of pyramidalneurons in layer II/III of PFC in the rodent PCP model at a

Received 14 December 2010; revised 25 April 2011; accepted 2 May2011

*Correspondence: Dr JD Elsworth, Laboratory of Neuropsycho-pharmacology, Department of Psychiatry, Yale University School ofMedicine, 300 George Street, Suite 901, New Haven, CT 06511, USA,Tel: + 1 203 785 4506, Fax: + 1 203 785 7357,E-mail: john.elsworth@yale.edu

Neuropsychopharmacology (2011) 36, 2054–2061

& 2011 American College of Neuropsychopharmacology. All rights reserved 0893-133X/11

www.neuropsychopharmacology.org

Loss of asymmetric spine synapses indorsolateral prefrontal cortex of cognitivelyimpaired phencyclidine-treated monkeys

John D. Elsworth1, Tibor Hajszan2,3, Csaba Leranth2 and Robert H. Roth1

1 Laboratory of Neuropsychopharmacology, Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA2 Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA3 Department of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary

Abstract

Schizophrenia patients, long-term abusers of phencyclidine (PCP), and monkeys treated with PCP all

exhibit enduring cognitive deficits. Evidence indicates that loss of prefrontal cortex spine synapses results

in cognitive dysfunction, suggesting the presence of synaptic pathology in the monkey PCP model ;

however, there is no direct evidence of such changes. In this study we use the monkey PCP model of

schizophrenia to investigate at the ultrastructural level whether remodelling of dorsolateral prefrontal

cortex (DLPFC) asymmetric spine synapses occurs following PCP. Subchronic PCP treatment resulted in a

decrease in the number of asymmetric spine synapses, which was greater in layer II/III than layer V of

DLPFC, compared to vehicle-treated controls. This decrease may contribute to PCP-induced cognitive

dysfunction in the non-human primate model and perhaps in schizophrenia. Thus, the synapse loss in the

PCPmodel provides a novel target for the development of potential treatments of cognitive dysfunction in

this model and in schizophrenia.

Received 11 March 2011 ; Reviewed 2 May 2011 ; Revised 9 May 2011 ; Accepted 16 May 2011 ;

First published online 27 June 2011

Key words : Asymmetric synapse, electron microscopy, phencyclidine, prefrontal cortex, schizophrenia.

Introduction

It has been observed in both rodents and primates

that withdrawal from subchronic phencyclidine (PCP)

treatment leads to lasting deficits in frontal lobe-

associated cognitive functions and to decreased pre-

frontal dopamine (DA) utilization (Jentsch et al. 1997).

At the same time, there are no signs of DA system

lesions or any impairment of motor, motivational or

associative learning processes. Damage to the pre-

frontal cortex in humans and monkeys leads to signs

that resemble the negative symptoms of schizophrenia

(Park &Holzman, 1992 ; Ridley et al. 1993), particularly

cognitive disturbance (Tamminga et al. 1998). Because

cognitive performance and memory processes appear

to be associated with remodelling of pyramidal

dendritic spine synapses in prefrontal cortex (Hof

& Morrison, 2004 ; Nimchinsky et al. 2002), loss of

prefrontal spine synapses may contribute to cognitive

dysfunction. Supporting this view, recent studies have

demonstrated a strong correlation between the loss of

asymmetric spine synapses in monkey prefrontal cor-

tex and the impairment of cognitive functions during

ageing (Dumitriu et al. 2010; Peters et al. 2008). Based

on these data, it is hypothesized that loss of prefrontal

spine synapses underlies cognitive dysfunction and

decreased prefrontal cellular activity both in schizo-

phrenia patients and in the PCP schizophrenia model.

Indeed, dendritic spine density of prefrontal pyrami-

dal neurons is decreased in schizophrenia (Glantz &

Lewis, 2000 ; Kolluri et al. 2005), suggestive of spine

synapse loss. More recently, we have described an

extensive reduction in the number of prefrontal

asymmetric (excitatory) spine synapses in the rat PCP

model (Hajszan et al. 2006), at a time when animals

exhibit cognitive deficits and decreased prefrontal

DA turnover. However, electron microscopic demon-

stration of synaptic alterations in either schizophrenia

patients or primate models of schizophrenia is so far

lacking. Because schizophrenia is a disease of higher

Address for correspondence : Dr R. H. Roth, Yale University School of

Medicine, 300 George Street, Suite 901, New Haven, CT 06511, USA.

Tel : (203) 785-4506 Fax : (203) 785-7357

Email : robert.roth@yale.edu

International Journal of Neuropsychopharmacology (2011), 14, 1411–1415. f CINP 2011doi:10.1017/S1461145711000939

BRIEF REPORT

Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of Endothelial Tight Junctions andMelanoma-Released Serine ProteasesCsilla Fazakas1., Imola Wilhelm1., Peter Nagyoszi1, Attila E. Farkas1, Janos Hasko1, Judit Molnar1,

Hannelore Bauer2, Hans-Christian Bauer2, Ferhan Ayaydin3, Ngo Thi Khue Dung1, Laszlo Siklos1,

Istvan A. Krizbai1*

1 Institute of Biophysics, Biological Research Center, Szeged, Hungary, 2 Department of Organismic Biology, University of Salzburg, Salzburg, Austria, 3 Laboratory of

Cellular Imaging, Biological Research Center, Szeged, Hungary

Abstract

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasizeto the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the onlypossibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the greatclinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletelyunderstood. In order to investigate this question we have used an in vitro experimental setup based on the culture ofcerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able toadhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from theluminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was acceleratedwhen they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through theendothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanomacells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at leastpartially) the paracellular transmigration pathway. During this process melanoma cells produced and released largeamounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitorPefablocH was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest thatrelease of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main stepsin the formation of brain metastases in malignant melanoma.

Citation: Fazakas C, Wilhelm I, Nagyoszi P, Farkas AE, Hasko J, et al. (2011) Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of EndothelialTight Junctions and Melanoma-Released Serine Proteases. PLoS ONE 6(6): e20758. doi:10.1371/journal.pone.0020758

Editor: Gilles J. Guillemin, University of New South Wales, Australia

Received January 26, 2011; Accepted May 8, 2011; Published June 2, 2011

Copyright: � 2011 Fazakas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: This work was partially supported by the project REG-DA-09-1-2009-0004, BBBTEER9 (IAK). The funder had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: krizbai@brc.hu

. These authors contributed equally to this work.

Introduction

Malignant melanoma is the third common cause of brain

metastasis behind lung and breast cancer, having the highest

propensity to metastasize to the brain of all primary neoplasms in

adults. Autopsy data indicate a prevalence of 55–75% of brain

metastasis in melanoma. Brain metastases contribute to death in

nearly 95% of patients with a median survival of less than one year

(for review see: [1,2]).

Since the central nervous system (CNS) lacks a lymphatic

system, tumor cells can only reach the brain parenchyma by

hematogenous metastasis formation. During this process meta-

static cells need to traverse brain endothelial cells which in turn

form the morphological basis of the blood-brain barrier (BBB).

The BBB is a complex system with the main function of regulating

the entry of blood-borne substances into the brain and thus

maintaining the homeostasis of the CNS. Cerebral endothelial

cells (CECs) – coming in contact with pericytes and astrocytes –

form a single cell layer lining the blood vessels, and are sealed with

a continuous belt of tight junctions (TJs) (for review see: [3]). TJs

regulate the paracellular permeability of the endothelial layer and

are composed of transmembrane proteins, including occludin,

claudins and junctional adhesion molecules, and cytoplasmic

plaque proteins which comprise zonula occludens proteins (ZO-1,

ZO-2) and associated molecules (for review see: [4]). Development

and maintenance of tight junctions is supported by adherens

junctions (AJs) which are located basolaterally to TJs and also form

a continuous line along cell-cell boundaries. AJs are also composed

of transmembrane proteins (cadherins) and cytoplasmic proteins

(catenins).

The process of transendothelial migration of tumor cells is

largely uncharacterized, and much of our knowledge comes from

endothelial cells of non-cerebral origin, which do not present the

special BBB phenotype. Different cell surface and adhesion

molecules, proteolytic enzymes and signaling pathways have been

shown to facilitate invasive and migratory capacities of melanoma

PLoS ONE | www.plosone.org 1 June 2011 | Volume 6 | Issue 6 | e20758

RESEARCH ARTICLE

Randomly Methylated β-Cyclodextrin Derivatives Enhance TaxolPermeability Through Human Intestinal Epithelial Caco-2 CellMonolayer

FERENC FENYVESI,1 TIMEA KISS,1 EVA FENYVESI,2 LAJOS SZENTE,2 SZILVIA VESZELKA,3 MARIA A. DELI,3 JUDIT VARADI,1

PALMA FEHER,1 ZOLTAN UJHELYI,1 ARPAD TOSAKI,4 MIKLOS VECSERNYES,1 ILDIKO BACSKAY1

1Department of Pharmaceutical Technology, Faculty of Pharmacy, Medical and Health Science Center, University of Debrecen,H-4010 Debrecen, Hungary

2Cyclolab Cyclodextrin Research and Development Laboratory Ltd., H-1097 Budapest, Hungary

3Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences,H-6726 Szeged, Hungary

4Department of Pharmacology, Faculty of Pharmacy, Medical and Health Science Center, University of Debrecen,H-4012 Debrecen, Hungary

Received 10 January 2011; revised 23 March 2011; accepted 25 May 2011

Published online in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22666

ABSTRACT: Intestinal absorption and bioavailability of taxol are limited by its low solubilityand P-glycoprotein (Pgp) activity. Methylated $-cyclodextrins (CDs) effectively form complexeswith paclitaxel but randomly methylated $-cyclodextrin (RAMEB) is cytotoxic in high concen-trations. Second-generation derivatives containing monoamino (MaRAMEB) and succinylated(SuRAMEB) ionic substituents with similar inclusion capacity but less toxicity could be promis-ing alternatives of RAMEB. Our aim was to examine and compare the efficacy of MaRAMEBand SuRAMEB with the parental RAMEB on taxol bidirectional permeability using the Caco-2model. Taxol permeability was not changed by 30-min pretreatment with CDs. In co-treatmentwith $-cyclodextrins, the apical to basolateral taxol flux was 4 to 6 times greater than in un-treated monolayers and it was also higher than in cells treated with Pgp inhibitor cyclosporinA. No decrease in basolateral to apical taxol flux was observed in pretreatment or co-treatmentwith CDs, suggesting no Pgp inhibition. All three CDs showed similar effects on taxol per-meability but RAMEB altered tight junction protein distribution and significantly decreasedtransepithelial electrical resistance. None of the CDs modified paracellular permeability tomannitol and polyethylene glycol 4000. In conclusion, second-generation derivatives of methyl-$-cyclodextrin, especially MaRAMEB, enhanced taxol permeability across Caco-2 cells withless toxicity and similar effectiveness as RAMEB. © 2011 Wiley-Liss, Inc. and the AmericanPharmacists Association J Pharm SciKeywords: Caco-2 cells; P-glycoprotein; tight junction; epithelial delivery/permeability;cyclodextrins; taxol

INTRODUCTION

Taxanes have a significant importance in the treat-ment of various solid tumors and leukemias.1 Onlyparenterally available preparations can be found on

Correspondence to: Miklos Vecsernyes (E-mail: vecsernyes.miklos@pharm.unideb.hu) Tel.: +36-52-512-900/22456, fax: +36-52-512-900/22630

Ferenc Fenyvesi and Tımea Kiss contributed equally to thisstudy.Journal of Pharmaceutical Sciences© 2011 Wiley-Liss, Inc. and the American Pharmacists Association

the drug market; therefore, there is a need for the de-velopment of alternative formulations. Oral admin-istration is convenient, safe, and provides facilitiesto chronic treatment regimens, but poor solubility ofthe active ingredients and the physiological barriersduring the absorption processes in the small intes-tine often cause low permeability of the orally ad-ministrated drugs.2 This is the case with taxol, too,which has bioavailability of approximately 1%–2%.3,4

On the contrary, taxol is a P-glycoprotein (Pgp) sub-strate and this ABC transporter is also responsible

JOURNAL OF PHARMACEUTICAL SCIENCES 1

Glial Cells Drive Preconditioning-Induced Blood-BrainBarrier Protection

Raffaella Gesuete, PhD; Franca Orsini, PhD; Elisa R. Zanier, MD; Diego Albani, MSc;Maria A. Deli, MD, PhD; Gianfranco Bazzoni, MD; Maria-Grazia De Simoni, PhD

Background and Purpose—The cerebrovascular contribution to ischemic preconditioning (IPC) has been scarcelyexplored. Using in vivo and in vitro approaches, we investigated the involvement of the blood-brain barrier and the roleof its cellular components.

Methods—Seven-minute occlusion of the right middle cerebral artery, used as in vivo IPC stimulus 4 days before permanentocclusion of the right middle cerebral artery, significantly reduced brain infarct size (8.45�0.7 versus 13.61�0.08 mm3

measured 7 days after injury) and preserved blood-brain barrier function (Evans blue leakage, 0.54�0.1 versus 0.89�0.1ng/mg). Assessment of neuronal, endothelial, and glial gene expression revealed that IPC specifically increased glial fibrillaryacidic protein mRNA, thus showing selective astrocyte activation in IPC-protected mice.

Results—The blood-brain barrier was modeled by coculturing murine primary brain microvessel endothelial and astroglialcells. One-hour oxygen-glucose deprivation (OGD), delivered 24 hours before a 5-hour OGD, acted as an IPC stimulus,significantly attenuating the reduction in transendothelial electric resistance (199.17�11.7 versus 97.72�3.4 �cm2) andthe increase in permeability coefficients for sodium fluorescein (0.98�0.11�10�3 versus 1.8�0.36�10�3 cm/min) andalbumin (0.12�0.01�10�3 versus 0.29�0.07�10�3 cm/min) induced by severe OGD. IPC also prevented the 5-hourOGD–induced disorganization of the tight junction proteins ZO-1 and claudin-5. IPC on glial (but not endothelial) cellsalone preserved transendothelial electric resistance, permeability coefficients, and ZO-1 localization after 5 hours ofOGD. Astrocyte metabolic inhibition by fluorocitrate abolished IPC protection, confirming the critical role of astrocytes.IPC significantly increased glial fibrillary acidic protein, interleukin-6, vascular endothelial growth factor-a, and ciliaryneurotrophic factor gene expression after OGD in glial cells, indicating that multiple pathways mediate the glialcontribution to IPC.

Conclusions—Our data show that the blood-brain barrier can be directly preconditioned and that astrocytes are majormediators of IPC protection. (Stroke. 2011;42:1445-1453.)

Key Words: astrocytes � blood-brain barrier � ischemia � oxygen-glucose deprivation � preconditioning

The concept of neuroprotection in ischemic precondition-ing (IPC) is based on the observation that a brief,

noninjurious stimulus protects the brain from a subsequentsevere insult.1,2 Experimental IPC also has a human counter-part, as transient ischemic attacks may be associated with abetter prognosis after a subsequent stroke.3,4 In vitro and invivo IPC models have mostly focused on neurons as cellulartargets of cerebral IPC, but little attention has been paid so farto the cerebrovascular compartment, even though it plays acrucial role in the pathogenesis of ischemic brain injury.5

Endothelial cells of the cerebral microvasculature are themain component of the blood-brain barrier (BBB), and theirclose association with astrocyte endfeet, pericytes, and mi-croglia is essential for the maintenance of the nervous systemmicroenvironment.6 It is known that IPC stimuli may atten-uate BBB disruption and brain edema.7,8 In addition, brain

endothelial cells can be preconditioned, an effect associatedwith stabilization of tight junction (TJ) proteins9 and/oractivation of the phosphatidylinositol-3 kinase/Akt path-way.10,11 However, little is known about the contribution ofglial cells to BBB preconditioning. By in vivo and in vitroapproaches, this study aimed at evaluating the functionaleffects of IPC on the BBB and the role of its cellularcomponents in mediating IPC-induced protection.

Methods

AnimalsC57BL/6 mice (Harlan Laboratories, Italy) were housed in anSpecific Pathogen Free (SPF) vivarium. Procedures involving ani-mals and their care were conducted in conformity with institutionalguidelines that are in compliance with national and international lawsand policies.12

Received September 21, 2010; accepted January 11, 2011.From the Mario Negri Institute (R.G., F.O., E.R.Z., D.A., G.B., M.G.D.S.), Milan, Italy, and Biological Research Center (M.A.D.), Szeged, Hungary.The online-only Data Supplement is available at http://stroke.ahajournals.org/content/full/stroke.110.603266/DC1.Correspondence to Maria Grazia De Simoni, Laboratory of Inflammation and Nervous System Diseases, Mario Negri Institute, via G. La Masa 19,

20156 Milan, Italy. E-mail desimoni@marionegri.it© 2011 American Heart Association, Inc.

Stroke is available at http://stroke.ahajournals.org DOI: 10.1161/STROKEAHA.110.603266

1445 at SZTE ALTALANOS ORVOSTUDOMANYI KAR on April 27, 2011 stroke.ahajournals.orgDownloaded from

712

Introduction

Transporter mediated drug–drug interaction (tDDI) has been shown to cause clinically relevant DDI leading to dose adjustments and/or contraindications. Therefore, the assessment of the tDDI potential early in the drug development process has gained interest in recent years. The first efflux transporter shown to play a role in drug absorption was the ATP-binding cassette sub-family B member 1/P-glycoprotein (ABCB1/P-gp; Meyers et al. 1991; Hsing et al. 1992). It is now known that intestinal ABCB1 limits uptake of orally administered drugs and co-administered drugs (Lee et al. 2010), as well as excipi-ents, (Bogman et al. 2005) may modulate absorption through inhibition of ABCB1. A tDDI that has been attrib-uted to inhibition of intestinal ABCB1 recently led to the contraindication of the direct thrombin inhibitor dabiga-tran with the ABCB1 inhibitor quinidine (Pradaxa prod-uct characteristics 2009) and the direct renin inhibitor

aliskiren with potent ABCB1 inhibitors cyclosporine, quinidine and verapamil (Rasilez product characteristics 2009; Rebello et al. 2011).

The selection of in vitro assay systems to measure ABCB1 inhibition is quite broad. The Caco-2 colon car-cinoma line, the first cellular intestinal absorption model (Hunter 1993) that expresses close to physiological levels of ABCB1 (Englund et al. 2006; Taipalensuu et al. 2001) seems to be an obvious choice. However, Caco-2 cells express a plethora of efflux transporters with overlap-ping substrate specificities (Taipalensuu 2001; Englund et al. 2006) and ABCB1 expression varies between labs (Maubon et al. 2007). Although ABCB1 selective/spe-cific inhibitors as multidrug resistance (MDR) reversal agents have been developed during the past two decades (Dantzig et al. 2003) the availability of these inhibitors is still limited (Rajnai et al. 2010; Szerémy et al. 2010). Nevertheless, a number of studies have been carried out

RESEARCH ARTICLE

Calcein assay: a high-throughput method to assess P-gp inhibition

H. Glavinas1, O. von Richter2, K. Vojnits1, D. Mehn1, I. Wilhelm3, T. Nagy1, J. Janossy1, I. Krizbai3, P. Couraud4,5,6, and P. Krajcsi1

1Solvo Biotechnology, Szeged, Hungary, 2AiCuris GmbH & Co KG, Wuppertal, Germany, 3Institute of Biophysics, Biological Research Center, Szeged, Hungary, 4INSERM Institute Cochin, Paris, France, 5Cnrs, UMR8104, Paris, France, and 6Univ Paris Descartes, Paris, France

Abstract1. Transporter mediated drug–drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant.

Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest.2. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout

and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC50 values that varied 10–100-fold. The differences observed for IC50 values for the same compounds were in the following rank order: IC50, MDCKII-MDR1 >IC50, K562MDR >IC50, hCMEC/D3. Higher IC50 values were obtained in cells with higher ABCB1 expression.

3. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC50 values on different ABCB1 expression levels.

Keywords: Transporter, ABCB1, DDI, high-throughput, MDCKII-MDR1, K562MDR, hCMEC/D3

Address for Correspondence: P. Krajcsi, Solvo Biotechnolgy, Gyar u. 2., Budaors 2040, Hungary. E-mail: krajcsi@solvo.com

(Received 07 February 2011; revised 19 April 2011; accepted 06 May 2011)

Xenobiotica, 2011; 41(8): 712–719© 2011 Informa UK, Ltd.ISSN 0049-8254 print/ISSN 1366-5928 onlineDOI: 10.3109/00498254.2011.587033

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IMMUNOBIOLOGY

e-Blood

Detection and isolation of cell-derived microparticles are compromised by proteincomplexes resulting from shared biophysical parametersBence Gyorgy,1 Karoly Modos,2 Eva Pallinger,1 Krisztina Paloczi,1 Maria Pasztoi,1,3 Petra Misjak,1 Maria A. Deli,4

Aron Sipos,5 Aniko Szalai,5 Istvan Voszka,2 Anna Polgar,6 Kalman Toth,7 Maria Csete,5 Gyorgy Nagy,1,8 Steffen Gay,9

Andras Falus,1,3 *Agnes Kittel,10 and *Edit I. Buzas1

1Department of Genetics, Cell- and Immunobiology, Semmelweis University, Budapest, Hungary; 2Department of Biophysics and Radiation Biology,Semmelweis University, Budapest, Hungary; 3Research Group for Inflammation Biology and Immunogenomics, Hungarian Academy of Sciences, Budapest,Hungary; 4Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary;5Department of Optics and Quantum Electronics, University of Szeged, Szeged, Hungary; 6National Institute of Rheumatology and Physiotherapy, Budapest,Hungary; 7Department of Orthopaedics, University of Szeged, Szeged, Hungary; 8Department of Rheumatology, Semmelweis University, Budapest, Hungary;9Center for Experimental Rheumatology, Zurich Center for Integrative Human Physiology, USZ, Zurich, Switzerland; and 10Institute of Experimental Medicine,Hungarian Academy of Sciences, Budapest, Hungary

Numerous diseases, recently reported toassociate with elevated microvesicle/microparticle (MP) counts, have also longbeen known to be characterized by accel-erated immune complex (IC) formation.The goal of this study was to investigatethe potential overlap between parametersof protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which mightperturb detection and/or isolation of MPs.In this work, after comprehensive charac-

terization of MPs by electron microscopy,atomic force microscopy, dynamic light-scattering analysis, and flow cytometry,for the first time, we drive attention tothe fact that protein complexes, espe-cially insoluble ICs, overlap in biophysi-cal properties (size, light scattering, andsedimentation) with MPs. This, in turn,affects MP quantification by flow cytom-etry and purification by differential cen-trifugation, especially in diseases in

which IC formation is common, includ-ing not only autoimmune diseases, butalso hematologic disorders, infections,and cancer. These data may necessitatereevaluation of certain published dataon patient-derived MPs and contributeto correct the clinical laboratory assess-ment of the presence and biologic func-tions of MPs in health and disease.(Blood. 2011;117(4):e39-e48)

Introduction

Membrane vesicles are small subcellular structures surrounded bya phospholipid bilayer. Their release by various cell types isenhanced during activation and apoptosis.1 They represent hetero-geneous structures and can be classified into several groupsdepending on their size, antigenic features, and mechanism ofcellular release.1 The two best characterized categories includeexosomes and microvesicles/microparticles (MPs). Both popula-tions are characterized by the exposure of phosphatidylserine,which allows annexin-V (AX) to bind to these–lipid surfaces.1

Exosomes are composed of small, 50- to 100-nm–sized structuresreleased on exocytosis of multivesicular bodies.1 The diameter ofMPs, formed by membrane blebbing, is described to be 100 to1000 nm2. However, precise definitions of MPs are still lacking.1,2

MPs are found in various biologic fluids, including blood plasma,3

urine,4 and synovial fluid (SF).5,6 Numerous flow cytometry(FC) studies using blood plasma have shown correlation of MPcounts with human cardiovascular7 and autoimmune diseases,8

hematologic disorders,9 and cancer.10 Of particular interest, autoim-mune diseases were reported to be characterized by elevatedlevels of MPs.11

The assessment of exosomes and MPs is complicated by thepresence of further known categories of membrane bound subcellu-

lar structures, such as apoptotic vesicles, exosome-like vesicles,membrane particles, and ectosomes.1 There is a substantial sizeoverlap among the aforementioned vesicle categories, and the sizedistribution of a given vesicle preparation may also be affected by themethod used for their isolation.3,12,13 Recently, attempts have been madeto standardize isolation and detection protocols for membrane vesicles.3,14

Up until now, no systematic study has been carried out concerning MPsize distributions in blood plasma and SF.

In contrast to membrane vesicles, immune complexes (ICs)have been known for a long time and were characterized exten-sively by immunologists. ICs consist of multivalent antigens andantibodies and are either soluble or insoluble.15 Soluble ICs consistof 2 to 10 macromolecules, whereas insoluble ICs form colloidalprecipitates.15 In the presence of a given antigen/antibody ratio,temperature, ionic strength, and pH, soluble ICs lose their solubil-ity. Insoluble IC particles are characterized by significant lightscattering and visible turbidity.15 ICs are well documented to haveimmunoregulatory and immunopathologic functions.16,17 In ourwork, we chose to investigate rheumatoid arthritis (RA) SFsamples reported to contain high amounts of ICs.17

In this study, we demonstrated overlapping biophysical proper-ties (size, light scattering, sedimentation) of protein complexes

Submitted September 21, 2010; accepted October 19, 2010. Prepublished online asBlood First Edition paper, November 1, 2010; DOI 10.1182/blood-2010-09-307595.

*A.K. and E.I.B. contributed equally to this study.

An Inside Blood analysis of this article appears at the front of this issue.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 USC section 1734.

© 2011 by The American Society of Hematology

e39BLOOD, 27 JANUARY 2011 � VOLUME 117, NUMBER 4

only.For personal use at UNIV KENTUCKY MED CTR on April 4, 2011. bloodjournal.hematologylibrary.orgFrom

Author's personal copy

Study of the parameters influencing the co-grinding process for the production ofmeloxicam nanoparticles

Levente Kürti a,c, Ákos Kukovecz b, Gábor Kozma b, Rita Ambrus a, Mária A. Deli c, Piroska Szabó-Révész a,⁎a Department of Pharmaceutical Technology, University of Szeged, Szeged, Hungaryb Department of Applied & Environmental Chemistry, University of Szeged, Szeged, Hungaryc Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged, Hungary

a b s t r a c ta r t i c l e i n f o

Article history:Received 9 July 2010Received in revised form 31 March 2011Accepted 21 May 2011Available online 1 June 2011

Keywords:NanoparticleMeloxicamCo-grindingScanning electron microscopyFactorial experimental designNasal powder system

Co-grinding is a procedure for the preparation of nanoparticles in which the drug is ground together with oneor more excipients. The grinding of meloxicam, a crystalline solid, together with amorphous polyvinylpyrro-lidone (PVP) or semi-crystalline polyethylene glycol (PEG) as excipients, is expected to lead to a drasticreduction in particle size. We optimized meloxicam grinding using a three level full factorial response surfacedesign. In the case of PVP the optimum co-grinding parameter set in our study proved to be a meloxicam toPVP-C30 ratio of 1:1, and a rotation frequency of 400 rpm. The best size reduction was achieved at ameloxicam to PEG 6000 ratio=1:2 at a rotation frequency of 400 rpm: nanoparticles averagingdSEM=174 nm in diameter and with a very narrow size distribution (standard deviation 35% of mean)were obtained. X-ray powder diffraction analysis indicated that the optimized products contained amorphousmeloxicam nanoparticles in the PVP-C30 composition, although meloxicam nanocrystals could also bedetected in the samples which contained PEG 6000. The dissolution properties were significantly increasedunder nasal conditions (pH 5.1, temperature 30 °C), especially in the case of the amorphous product. Such drypowder systems can offer novel opportunities in systemic nasal drug delivery.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Nanonization offers an excellent possibility to overcome mucosalbarriers [1,2] and has several advantages in drug delivery [3,4]. It isbelieved that nanoparticles can be transported across barriers into thebloodstream without prior dissolution [5]. Nano-sized drugs withcarriers can overcome the resistance offered by the physiologicalbarriers in the body because the efficient delivery of drugs to variousparts of the body is directly affected by the particle size [6].

Several techniques are available for the production of drugnanoparticles [7]. Basically, bottom-up and top-down technologiescan be differentiated. The bottom-up technologies start from themolecules, which are precipitated (crystallized) in a controlled fashionto yield the desired particle size. Nowadays, bottom-up techniques arenot the main choice for drug nanoparticle production due to the use oforganic solvents. The top-down technologies are disintegrationmethods e.g. various types of milling (high-intensity ultrasonication,high-pressure homogenization, grinding or co-grinding) are morefrequently used [8].

The size reduction of pharmaceutical materials is often performedby means of dry milling [9,10], but the size reduction possible by dry

milling is known to be limited to around 3 μm due to aggregation ofthe particles. Experiments focusing on a particle size reduction to thesubmicron region by co-grinding with additives have recently beenattempted [11–14].

Nanonization has become a popular approach to produce particlesin the size range of 200–400 nm, to improve both the dissolution rateand the solubility of the compound [15]. The latter phenomenon isdue to the well-known dependency of solubility on particle size asdescribed by the Ostwald–Freundlich equation. Breakage of micron-sized drug crystals into nanoparticles creates an increased particlesurface area, which is thermodynamically unfavorable. Thus, nano-sized particles tend to agglomerate to reduce their surface area.Particle agglomeration can be prevented by steric stabilization usingpolymeric excipients [16].

Co-grinding is a top-down disintegration procedure for thepreparation of nanoparticles by grinding of the drug together withone or more excipients [17,18]. A main advantage of co-grinding ascompared to other methods is that it is a simple procedure and organicsolvents are not needed for the preparation of nanoparticles; it istherefore an economically and environmentally desirable technology[19]. It is important to control the parameters (e.g. duration of grinding,the grinding rate, the material and the volume of the grinding pot, thematerial and the number of the grinding balls, the grinding excipientsand the drug/excipient ratio) by means of a factorial experimentaldesign [20,21].

Powder Technology 212 (2011) 210–217

⁎ Corresponding author at: H-6720 Eötvös utca 6., Szeged, Hungary. Tel.: +36 62545572; fax: +36 62 545571.

E-mail address: revesz@pharm.u-szeged.hu (P. Szabó-Révész).

0032-5910/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.powtec.2011.05.018

Contents lists available at ScienceDirect

Powder Technology

j ourna l homepage: www.e lsev ie r.com/ locate /powtec

Journal of Neuroimmunology 232 (2011) 119–130

Contents lists available at ScienceDirect

Journal of Neuroimmunology

j ourna l homepage: www.e lsev ie r.com/ locate / jneuro im

Intracellular transport of Toxoplasma gondii through the blood–brain barrier

Sabrina M. Lachenmaier a, Mária A. Deli b, Markus Meissner c, Oliver Liesenfeld a,⁎a Institute of Microbiology and Hygiene, Charité—Medical School Berlin, Campus Benjamin Franklin, Berlin, Germanyb Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungaryc Hygiene Institute, Department of Parasitology, Heidelberg University School of Medicine, Germany

⁎ Corresponding author. Tel.: +49 3084453630.E-mail address: oliver.liesenfeld@charite.de (O. Liese

0165-5728/$ – see front matter © 2010 Elsevier B.V. Aldoi:10.1016/j.jneuroim.2010.10.029

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 May 2010Received in revised form 29 August 2010Accepted 25 October 2010

Keywords:Toxoplasma gondiiBlood–brain barrierNeuroinvasionTransendothelial migration

Toxoplasma gondii establishes latent infection in the central nervous system of immunocompentent hosts.Toxoplasmic encephalitis is a life threatening reactivation of latent infection in the brain of immunocom-promised patients. To further understand the mechanisms of entry into the brain of T. gondii we investigatedhost molecules and cells involved in the passage of the parasite through the blood–brain barrier. First, usingmicroarrays brain endothelial cells were found to upregulate, among others, chemokines and adhesionmolecules following infection with tachyzoites. Using flow cytometry we observed upregulated ICAM-1expression on the surface of brain endothelial cells following infection; ICAM-1 expression was furtherincreased after pre-incubation with IFN-γ. Compared to RH tachyzoites, ME49 tachyzoites induced a strongerupregulation of ICAM-1 and an earlier and stronger IL-6 and MCP-1 secretion by brain endothelial cells. Usingan in vitro coculture model of the BBB (primary glia cells and brain endothelial cells) we found a strongermigration of infected antigen-presenting cells compared to lymphocytes (4.63% vs. 0.6% of all cells) across theBBB. Among all antigen-presenting cells CD11b+/CD11c+ cells showed the highest infection rate, whereas themajority of infected cells that migrated through the blood–brain barrier were CD11b+/CD11c− cells. Infectionof PBMCs with type I or type II Toxoplasma strains resulted in similar patterns of cell migration across the invitro BBB model.In conclusion, these results suggest that T. gondii modulates gene expression of brain endothelial cells topromote its own migration through the blood–brain barrier in a ‘Trojan horse’ manner. Cells expressingCD11b either with or without CD11c are likely candidate cells for the intracellular transport of T. gondii acrossthe BBB. T. gondii type I and type II strains induced similar migration patterns of antigen-presenting cellsacross the in vitro BBB.

nfeld).

l rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

The blood–brain barrier is a selective cellular border at the levelof specialized cerebral microvascular endothelial cells that protectsthe central nervous system (CNS) from blood-borne endangerments.The brain endothelial cells interacting with perivascular structureslike pericytes, microglia and astroglia have characteristic propertiesdefined by high transendothelial electrical resistance, the expressionof tight junctions sealing the paracellular spaces, and a low pino-cytotic activity (Abbott et al., 2006; Ge et al., 2005; Rubin and Staddon,1999; Deli et al., 2005). Although the CNS is routinely surveyed bycells of the immune system, inflammatory processes can lead to anexcessive leukocyte infiltration into the brain and cause pathology(Hickey, 2001; Luster et al., 2005) as in patients with multiple scle-rosis or AIDS dementia (McFarland and Martin, 2007; Nottet, 1999).Under healthy conditions the endothelial cells of the blood–brain

barrier express very low levels of adhesion molecules that could beused by leukocytes for transendothelial migration. Upon inflamma-tory stimulation, microvascular endothelial cells induce the expres-sion of cell adhesion molecules including ICAM-1, VCAM-1, andselectins (Kadl and Leitinger, 2005; Coisne et al., 2006). In concertwith the secretion of various chemokines the vascular responseimplicates a decrease of blood–brain barrier function. Successfulleukocyte trafficking thereby demands the interaction of selectins andtheir ligands as well as the cooperation of cell adhesion moleculeswith integrins or chemokines with their receptors (Ransohoff et al.,2003; Pober and Sessa, 2007). Infections of the CNS are caused by avariety of extracellular (mostly causing meningitis) and intracellularpathogens (Kim, 2008; Marra and Brigham, 2001; Katti, 2004; Kim,2002). Whereas extracellular pathogens are believed to use theblood–cerebrospinal fluid barrier in the choroid plexus to gain accessto the brain, intracellular pathogens including Listeria monocytogenes,Cryptococcus neoformans or the human immunodeficiency virusexploit host cells to transmigrate across host barriers (Drevets et al.,2004; Charlier et al., 2009; Kanmogne et al., 2007). Infection with theprotozoan parasite Toxoplasma gondii results in invasion of the brain

FORUM REVIEW ARTICLE

Oxidative Stress and Blood–Brain Barrier DysfunctionUnder Particular Consideration of Matrix

Metalloproteinases

Christine Lehner,1,2 Renate Gehwolf,1,3 Herbert Tempfer,4 Istvan Krizbai,5

Bernhard Hennig,6 Hans-Christian Bauer,1,4 and Hannelore Bauer1

Abstract

A cell’s ‘‘redox’’ (oxidation and reduction) state is determined by the sum of all redox processes yielding reactiveoxygen species (ROS), reactive nitrogen species (RNS), and other reactive intermediates. Low amounts of ROS/RNS are generated by different mechanisms in every cell and are important regulatory mediators in manysignaling processes (redox signaling). When the physiological balance between the generation and elimination ofROS/RNS is disrupted, oxidative/nitrosative stress with persistent oxidative damage of the organism occurs.Oxidative stress has been suggested to act as initiator and/or mediator of many human diseases. The cerebralvasculature is particularly susceptible to oxidative stress, which is critical since cerebral endothelial cells play amajor role in the creation and maintenance of the blood–brain barrier (BBB). This article will only containa focused introduction on the biochemical background of redox signaling, since this has been reported already ina series of excellent recent reviews. The goal of this work is to increase the understanding of basic mechanismsunderlying ROS/RNS-induced BBB disruption, with a focus on the role of matrix metalloproteinases, which,after all, appear to be a key mediator in the initiation and progression of BBB damage elicited by oxidative stress.Antioxid. Redox Signal. 15, 000–000.

Introduction

Oxidative stress: Cellular and physiologicalbackground

Reactive oxygen species (ROS) and reactive nitrogenspecies (RNS), summarized in this article as ROS, are

present at moderate levels in every living cell where theyfulfill vitally important functions. Provided that the redoxhomeostasis is well balanced, redox signaling enables the cellsto respond properly to endogenous and exogenous stimuli ofvarious kinds. Any imbalance between generation and elim-ination of ROS leads to a physiologic phenomenon termedoxidative stress. Oxidative stress processes are involved di-rectly or indirectly in many human pathologies, includingcancers, neurodegenerative diseases, diabetes, atherosclero-sis, ischemic stroke, and many more (for reviews, see Refs. 34,164, 168).

ROS are produced by a great variety of mechanisms andoriginate from many sources. Exogenous sources include

exposure to di-oxygen, ozone (a powerful oxidizing agent thatcan oxidize biological components directly), ionizing andnonionizing irradiation, UV radiation, air pollutants, cigarettesmoke, industrial contaminants, the invasion of pathogens,and food that may contain various kinds of oxidants, in-cluding peroxides, aldehydes, oxidized fatty acids, and tran-sition metals (89). Endogenously, two ROS-generatingsystems exist: a) the leakage of activated oxygen from mito-chondria during oxidative phosphorylation; and b) oxygen-metabolizing enzymatic reactions such as xanthine oxidase,cytochromes P450, NADPH oxidases, myeloperoxidase, andnitric oxidase synthase (140).

ROS can basically be subdivided into two groups: a) Theradical group containing superoxide ion radical, hydroxylradical, nitric oxide radical, peroxyl and alkoxyl radicals, andone form of singlet oxygen; and b) nonradical compoundscomprising a large variety of substances such as hypochlorousacid, hydrogen peroxide, organic peroxides, aldehydes,ozone, and oxygen (89). The terms reactive oxygen species,

1Department of Organismic Biology, Development Biology Group, 2Clinic for Sports Injuries and Trauma Surgery, and 3Christian DopplerClinic, University Hospital of Salzburg, Salzburg, Austria.

4Paracelsus Private Medical University, Salzburg, Austria.5Institute of Biophysics, Biology Research Center, Szeged, Hungary.6Molecular and Cell Nutrition Laboratory, University of Kentucky, Lexington, Kentucky.

ANTIOXIDANTS & REDOX SIGNALINGVolume 15, Number 5, 2011ª Mary Ann Liebert, Inc.DOI: 10.1089/ars.2011.3923

1

Correspondence: L. Sikl ó s, Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre, Szeged, P.O. Box 521, H-6701 Hungary. Fax: 36 62 433133. E-mail: siklos@brc.hu

(Received 10 December 2010 ; accepted 25 April 2011 )

Amyotrophic Lateral Sclerosis, 2011; 12: 340–344

ISSN 1748-2968 print/ISSN 1471-180X online © 2011 Informa HealthcareDOI: 10.3109/17482968.2011.584627

AMPA receptor antagonists. Indeed, different com-petitive (8) or non-competitive (9,10) AMPA recep-tor inhibitors have been reported to prolong the survival of transgenic (Tg) mice with mutant Cu/Zn superoxide dismutase (SOD1) when administered before the onset of motor impairment. Using the non-competitive AMPA receptor inhibitor, we mod-estly reduced the loss of MNs in the lumbar spinal cord of SOD1-G93A mice, while the morphology of the remaining MNs, and especially that of the den-drites was well preserved by the treatment (9). As AMPA receptor-mediated excitotoxicity on MNs in in vitro paradigms is thought to be mediated by an excessive calcium infl ux particularly at the level of the dendrites, we hypothesized that the benefi cial effect of AMPA receptor inhibitors could be a result of a reduced permeability to calcium. The drug used in our previous study belongs in the class of active

Introduction

Amyotrophic lateral sclerosis (ALS) is a multifac-torial (1), multisystem (2), non-cell autonomous disease (3). Several pathomechanisms of the degen-eration of motor neurons (MNs) have been identi-fi ed, which contribute to the progression of the motor dysfunction during the disease. Excitotoxicity has attracted considerable attention since an abnor-mal glutamate metabolism was documented in pathogenesis-oriented clinical studies (4), and was confi rmed in in vitro experiments and in animal models of ALS (5). Moreover, riluzole, the only drug to date to display a potential to increase patient sur-vival, has an anti-excitotoxic effect (6). Since glutamate-induced excitotoxicity to MNs is mediated through calcium-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (7), a protective effect was expected from treatment with

ORIGINAL ARTICLE

Talampanel reduces the level of motoneuronal calcium in transgenic mutant SOD1 mice only if applied presymptomatically

MELINDA PAIZS 1 , MASSIMO TORTAROLO 2 , CATERINA BENDOTTI 2 , J Ó ZSEF I. ENGELHARDT 3 & L Á SZL Ó SIKL Ó S 1

1 Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre, Szeged, Hungary, 2 Laboratory of Molecular Neurobiology, Department of Neuroscience, ‘ Mario Negri ’ Institute for Pharmacological Research, Milan, Italy, and 3 Department of Neurology, University of Szeged, Hungary

Abstract We tested the effi cacy of treatment with talampanel in a mutant SOD1 mouse model of ALS by measuring intracellular calcium levels and loss of spinal motor neurons. We intended to mimic the clinical study; hence, treatment was started when the clinical symptoms were already present. The data were compared with the results of similar treatment started at a presymptomatic stage. Transgenic and wild-type mice were treated either with talampanel or with vehicle, starting in pre-symptomatic or symptomatic stages. The density of motor neurons was determined by the physical disector, and their intracellular calcium level was assayed electron microscopically. Results showed that motor neurons in the SOD1 mice exhibited an elevated calcium level, which could be reduced, but not restored, with talampanel only when the treatment was started presymptomatically. Treatment in either presymptomatic or symptomatic stages failed to rescue the motor neurons. We conclude that talampanel reduces motoneuronal calcium in a mouse model of ALS, but its effi cacy declines as the disease progresses, suggesting that medication initiation in the earlier stages of the disease might be more effective.

Key words: Talampanel , AMPA receptor , mSOD1 model , intracellular calcium , motor neuron

Commentary CNS & Neurological Disorders - Drug Targets, 2011, Vol. 10, No. 8 863

Commentary

Research Highlights

Hideyama, T.; Yamashita, T.; Suzuki, T.; Tsuji, S.; Higuchi, M.; Seeburg, P.H.; Takahashi, R.; Misawa, H.; Kwak, S. Induced loss of ADAR2 engenders slow death of motor neurons from Q/R site-unedited GluR2.

J. Neurosci., 2010, 30, 11917-11925.

Two Hits with One Shot – A Possibility of Simultaneous Targeting Motor Neuron Loss and Depression in ALS by Upregulating ADAR2

A leading hypothesis for the selective death of motor neurons in sporadic amyotrophic lateral sclerosis (ALS) is excitotoxicity mediated by Ca

2+-permeable -amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. The AMPA receptors are assembled from 4 subunits

and their permeability for Ca2+

is determined by the absence of the glutamate receptor subunit type 2 (GluR2), associated with an increased portion of unedited GluR2 mRNA at the glutamine/arginine (Q/R) site. RNA editing at this location is predominantly catalyzed by adenosine deaminase acting on RNA 2 (ADAR2). Thus, the observation that a substantial portion of Q/R site unedited GluR2 mRNA is present in postmortem spinal cord tissue of sporadic ALS patients suggests inefficiency of ADAR2 to be a disease-specific molecular dysfunction in ALS. To decide whether this malfunction could be a cause of motor neuron death in vivo, Hideyama and colleagues developed a conditional ADAR2 knock-out mouse line (designated as AR2), which carries gene-targeted floxed ADAR2 alleles that become functionally ablated by Cre recombinase expressed from a transgene (VAChT-Cre.Fast) in approximately 50% of motor neurons. They demonstrated that only the ADAR2-lacking spinal motor neurons degenerated in these animals, and this could be prevented by generating Q/R site edited GluR2 in the absence of ADAR2 by a point mutation in the endogenous GluR2 alleles. These results convincingly demonstrate that the inefficient GluR2 Q/R site editing found in the spinal cords of patients with sporadic ALS proposes the decreased level of ADAR2 mRNA to be a direct cause of death of motor neurons in mice, which accentuates the detrimental role of elevated Ca

2+ influx in motor neurons as a consequence of

increased Ca2+

-permeability of AMPA receptors.

From therapeutic point of view, an interesting aspect of the Ca2+

-mediated cell death in ALS is connected with certain emotional reactions of the patients such as depression and anxiety, which has received particular attention in ALS. Although many researchers with experience with ALS challenge the lay opinion of an increased risk of developing major depressive disorder in ALS, studies using standardized methodology assessing nonphysical symptoms reported an occurrence exceeding the prevalence of that in the general population. There is growing evidence that besides the monoaminergic system the glutamatergic system, particularly N-methyl-D-aspartate receptor mediated Ca

2+-entry

plays an important role in the pathobiology of major depressive disorder. It is thus intriguing that conventional tricyclic antidepressants, specific serotonin reuptake inhibitors and serotonin/norepinephrine reuptake inhibitors not only down-regulate N-methyl-D-aspartate receptors but – at least in an in vitro model system – have the potency to enhance GluR2 Q/R site-editing either by up-regulating ADAR2 mRNA expression level, or through other unidentified mechanisms. It will now be important to determine whether these drugs exert a similar protective effect on motor neurons in in vivo models of ALS, and thus constitute a new therapeutic approach for this disease.

Laszlo Siklos (Editorial Board Member)

Institute of Biophysics

Biological Research Center

Szeged

Hungary

E-mail: siklos@brc.hu

© 2011 Society for Laboratory Automation and Screening www.slas.org 1

IntroductIon

Drug efflux transporters of the adenosine triphos-

phate (atp)-binding cassette-containing proteins have a major role in the transport of clinically relevant drugs. abCb1/ p-glycoprotein (p-gp)/Mdr1, one of the most important efflux transporters, is present in the apical membranes of endothelial/epithelial cells of the tissues with barrier function, the blood–brain barrier (bbb) among them. abCb1 substrates are translo-cated from the basolateral to the apical side of the cells (for review, see schinkel and Jonker1). abCb1 prevents brain pen-etration of substrate drugs, and abCb1 inhibition may lead to increased brain exposure and Cns toxicity. therefore, testing

for abCb1-mediated drug interaction is important to assess the toxicity potential of coadministered drugs.

digoxin is the prototypic abCb1 probe substrate recom-mended by the regulatory agencies.2,3 however, a consensus has not been reached on this issue.4 Quinidine is also considered an abCb1-specific probe.4 several studies suggest that the entry of quinidine into the brain is restricted5 and controlled by abCb1 expressed in the bbb.6–8 Yet, even in recent reviews, quinidine is classified as an abCb1 inhibitor.2,9 the aim of the present study was to provide further evidence that quinidine can be used as a probe substrate for abCb1 at the bbb employing a battery of test systems, including membrane assays, cell-based assays, and dual-/triple-probe microdialysis assays in anesthetized and awake rats. We propose using quinidine and psC-833 (valspodar), a clinically relevant abCb1 inhibitor,10,11 as a probe substrate/reference inhib-itor combination to assess investigational drugs for their interac-tions on abCb1 in the in vitro and in vivo bbb models.

MAterIALS And MethodS

Chemicals and supplies for in vitro and in vivo assays

Quinidine, fetal calf serum (fCs), Cpt-caMp, hydrocortisone, collagen type iV, fibronectin, heparin, insulin-transferrin-sodium selenite media supplement, and puromycin were purchased from

1solvo biotechnology, szeged, hungary2department of biochemistry, faculty of Medicine, university of szeged, hungary3institute of biophysics, biological research Center, szeged, hungary4department of Medical Chemistry, faculty of Medicine, university of szeged, szeged, hungary5trans-neuropharma ltd, budapest, hungary

received Jan 26, 2011, and in revised form May 13, 2011. accepted for publi-cation May 25, 2011.

Journal of biomolecular screening xx(x); xxxxdoi: 10.1177/1087057111414896

Quinidine as an ABcB1 Probe for testing drug Interactions at the Blood–Brain Barrier: An In Vitro

In Vivo correlation Study

IStVán SzIrákI,1 FrAncISkA erdo,1 erzSéBet Beéry,1 PetrA MAgdoLnA MoLnár,2 cSILLA FAzAkAS,3 IMoLA WILheLM,3 ILdIkó MAkAI,1 eMeSe kIS,1 krISztInA herédI-SzABó,1 tIBor

ABonyI,1 IStVán krIzBAI,3 gáBor k. tóth,4 and Péter krAjcSI1

this study provides evidence that quinidine can be used as a probe substrate for abCb1 in multiple experimental systems both in vitro and in vivo relevant to the blood–brain barrier (bbb). the combination of quinidine and psC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on abCb1. effects of quinidine and substrate–inhibitor interac-tions were tested in a membrane assay and in monolayer assays. the authors compared quinidine and digoxin as abCb1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in atpase and monolayer efflux assays employing MdCKii-Mdr1 and the rat brain microcapillary endothelial cell system. brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of abCb1 at the bbb, resulting in an increased brain pen-etration of quinidine. in sum, quinidine is a good probe to study abCb1 function at the bbb. Moreover, quinidine/psC-833 is an abCb1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo. (Journal of Biomolecular Screening 2011:000-000)

key words: bbb, abCb1/p-gp, quinidine, psC-833, microdialysis

at EGIS GYOEGYSZERGYAR on August 11, 2011jbx.sagepub.comDownloaded from

Author's personal copy

International Journal of Pharmaceutics 410 (2011) 107–110

Contents lists available at ScienceDirect

International Journal of Pharmaceutics

journa l homepage: www.e lsev ier .com/ locate / i jpharm

Rapid communication

Applicability of sucrose laurate as surfactant in solid dispersions prepared bymelt technology

Angéla Szutsa, Péter Lánga, Rita Ambrusa, Lóránd Kissa,b, Mária A. Delib, Piroska Szabó-Révésza,∗

a Department of Pharmaceutical Technology, University of Szeged, H-6720 Szeged, Eötvös u. 6, Hungaryb Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, H-6726 Szeged, Temesvári krt. 62., Hungary

a r t i c l e i n f o

Article history:Received 16 December 2010Received in revised form 7 March 2011Accepted 13 March 2011Available online 21 March 2011

Keywords:Sucrose laurateSurfactantSolid dispersionMelt technologyCytotoxicityCaco-2 cellsGemfibrozilPEG 6000

a b s t r a c t

This study focused on an investigation of the applicability of sucrose laurate as surfactant in solid dis-persions. Although this surfactant has a US Drug Master File, it has not been used so far in internalpharmaceutical products. High drug-loaded solid dispersion systems consisting of gemfibrozil as a modeldrug and PEG 6000 as a carrier, with or without sucrose laurate (D1216), were prepared by the meltingmethod. Cytotoxicity studies on Caco-2 monolayer cells were also performed, in order to gain informationon the applicability of D1216 in oral formulations. The results showed that the presence of the surface-active agent did not affect the solid-state characteristics of the model drug significantly. A markedlyimproved dissolution of gemfibrozil from the ternary solid dispersion systems was observed as com-pared with the binary solid dispersion systems. The optimum concentration range of the D1216 in theformulations was determined to be 5–10%. The effective final concentrations of D1216 in the dissolutionexperiments proved to be non-toxic towards CaCo-2 cells. The results suggest the potential use of D1216in innovative internal pharmaceutical formulations.

© 2011 Elsevier B.V. All rights reserved.

The poor water solubility of drug substances and their lowrates of dissolution in the aqueous gastrointestinal fluids often leadto insufficient bioavailability, and this remains a problem to thepharmaceutical industry. Solid dispersions of hydrophobic drugsin water-soluble carriers have attracted considerable interest as ameans of improving dissolution behaviour, and hence enhancingbioavailability. Water-soluble carriers such as high-molecular-weight polyethylene glycols (PEGs) and polyvinylpyrrolidones(PVPs) have been most commonly used for solid dispersions(Bikiaris et al., 2005; Craig and Newton, 1991; Leuner andDressman, 2000; Saharan et al., 2009; Serajuddin, 1999). The useof surfactants with solubilizing properties, such as polysorbates,poloxamers, Gelucires (polyethylene glycol glycerides), sodiumlauryl sulfate or vitamin E TPGS have also attracted consider-able interest recently (Dehghan and Jafar, 2006; Jagdale et al.,2010; Liu and Wang, 2007; Mura et al., 1999; Okonogi andPuttipipatkhachorn, 2006; Owusu-Ababio et al., 1998; Sethia andSquillante, 2002; Vasconcelos et al., 2007). As described in thereview by Vasconcelos et al. (2007), the third-generation soliddispersion systems contain a surfactant carrier, or a mixture ofamorphous polymers and surfactants as carriers. These third-generation solid dispersions are intended to achieve the highestdegree of bioavailability for poorly soluble drugs. The inclusion

∗ Corresponding author. Tel.: +36 6254 5572; fax: +36 6254 5571.E-mail address: revesz@pharm.u-szeged.hu (P. Szabó-Révész).

of surfactants in the solid dispersions may help to avoid drugrecrystallization and to stabilize the systems (Vasconcelos et al.,2007).

Sucrose esters (SEs) are widely used in the food and cosmet-ics industries, and there has recently been great interest in theirapplicability in different pharmaceutical fields. They are biodegrad-able, natural, non-ionic surface-active agents consisting of sucroseas hydrophilic moiety and fatty acids as lipophilic groups (Abd-Elbary et al., 2008; Csóka et al., 2007; Ganem Quintanar et al., 1998;Okamoto et al., 2005; Otomo, 2009; Ntawukulilyayo et al., 1993;Shibata et al., 2002).

In an earlier study we investigated, the structure and ther-mal behaviour of SE in order to predict their applicability in hotmelt technology (Szuts et al., 2007). Our results revealed that SEsare semicrystalline carriers, with both amorphous and crystallineregions. During the preparation of solid dispersions, the drugs arebuilt into the amorphous phases of the SEs (Szuts et al., 2008). Inmelt technology, mainly the lipophilic SEs may be suggested as car-riers. They display characteristic melting, whereas SEs with highor moderate HLB values only soften during heating (Szuts et al.,2007). It has also been found that hydrophilic SEs exhibit gellingbehaviour at body temperatures, which can influence the drugrelease (Szuts et al., 2010a,b). In view of these results, the appli-cability of hydrophilic SEs alone as carriers in hot melt technologyis not suggested. Dispersion or dissolution of the drugs in the soft-ened SEs is difficult, and a high amount of swelling SEs can reducethe rate of dissolution of a drug.

0378-5173/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.ijpharm.2011.03.033

Recent Patents on CNS Drug Discovery, 2011, 6, 107-118 107

1574-8898/11 $100.00+.00 © 2011 Bentham Science Publishers

Patented In Vitro Blood-Brain Barrier Models in CNS Drug Discovery

Andrea Tóth1, Szilvia Veszelka1, Shinsuke Nakagawa2,3, Masami Niwa2,3 and Mária A. Deli1,2*

1Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre of the Hungarian Academy

of Sciences, Szeged, Hungary; 2PharmaCo-Cell Co. Ltd., Nagasaki, Japan;

3Department of Pharmacology 1, Nagasaki

University Graduate School of Biomedical Sciences, Nagasaki, Japan

Received: April 12, 2011; Accepted: April 21, 2011; Revised: May 5, 2011

Abstract: The blood-brain barrier (BBB) is a regulatory interface between the circulation and the central nervous system (CNS). Therapy of neurological diseases is limited due to restricted penetration of pharmacons across the BBB. Models for screening the brain penetration of drug candidates are needed early in drug discovery. Culture-based models are useful tools for both basic research on BBB, and testing the permeability of new therapeutical molecules. This review focuses on patented in vitro BBB models and their potential application in CNS drug discovery. Cell culture models using primary and immortalized brain endothelial cells of non-human and human origin, in co-culture or mono-culture setting, in static or dynamic conditions are discussed, as well as methods to induce BBB properties in such in vitro models. The aim of these models is to reproduce as many aspects as possible of the in vivo BBB. All models should show some elements of general endothelial and specific BBB properties, like physiologically realistic cell architecture, restrictive paracellular pathway, and functional expression of transport mechanisms. Though no “ideal in vitro BBB model” has been constructed yet, the currently available models provide valuable information on BBB permeability and are useful tools in CNS drug discovery.

Keywords: Blood-brain barrier, brain endothelial cell, co-culture, in vitro blood-brain barrier model, model for CNS drug testing, prediction model for CNS permeability.

INTRODUCTION

Endothelial cells lining the microvessels of the central nervous system (CNS) differ fundamentally from peripheral vascular endothelium both in their morphological and functional features including the regulation of the exchange of molecules and cells between the blood and the neural parenchyma. The blood-brain barrier (BBB) is constituted by brain capillaries, which possess specialized structural and functional characteristics. In humans, the estimated total length of cerebral capillaries is about 650 km and the total surface area is about 20 m2 [1, 2].

Cerebral endothelial cells form the anatomical basis of the BBB. There is a dynamic interaction between brain endothelial and other neighboring cells, such as astroglia, pericytes, perivascular microglia, and neurons as depicted on Fig. (1). The cross-talk between these cells induce a unique BBB phenotype in endothelial cells including (i) a mor-phological barrier based on interendothelial tight junctions (TJ) that markedly limit paracellular permeability, (ii) a unique pattern of receptors, transporters and drug efflux pumps, and (iii) enzymatic and metabolic barriers [3]. The three major functions of the BBB are the creation and maintenance of ionic homeostasis for neuronal functions, supply of the CNS with nutrients, and protection from toxic insults [4].

*Address correspondence to this author at the Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungary; Tel: +36 62 599602; Fax: +36 62 433133; E-mail: deli@brc.hu

By separating the brain from systemic circulation the BBB is the major entry gate for therapeutic compounds to the CNS. The low level of paracellular flux and transen-dothelial vesicular trafficking results in a transport barrier for drugs which are hydrophilic and have a molecular mass bigger than 400 Da. Efflux pumps at the luminal membrane of brain endothelial cells limit the brain penetration of lipophilic xenobiotics and drugs [5]. These properties of the BBB and other factors contributing to the rate and extent of drug disposition within the brain prevent 98% of potential neuropharmaceuticals, especially new biopharmacons, nucleic acids, peptide or protein drugs, to reach therapeutical levels in the CNS [1, 6]. Despite efforts to increase drug delivery to brain through strategies to overcome the junctional or efflux barriers, to exploit BBB transport mechanisms or to circumvent the BBB [7], still the majority of newly developed neuropharmaceuticals fail due to poor CNS pharmacokinetics [2]. Therefore, the early screening of potential drug candidate molecules for brain penetration is a very important task in drug discovery and development.

BBB models have been widely used in pharmaceutical industry for testing the permeability of cerebral capillaries and investigating brain penetration. Cell culture-based models proved to be one of the most versatile tools in basic BBB research and also in testing CNS drug penetration [8]. In drug development, several sequential and parallel steps for screening BBB permeability are suggested [9]. Computa-tional approaches to predict drug penetrability can be used to screen big compound libraries with the advantages of being quick and high throughput. Non-cell based in vitro models of permeability can further refine and specify penetration

166 Solubility, Delivery and ADME Problems of Drugs and Drug Candidates, 2011, 166-188

K. Tihanyi and M. Vastag (Eds) All rights reserved - © 2011 Bentham Science Publishers Ltd.

CHAPTER 9

Tools for Modelling Blood-Brain Barrier Penetrability

Szilvia Veszelka1, Ágnes Kittel2 and Mária A. Deli1

1Biological Research Center, Hungarian Academy of Sciences and 2Institute of Experimental Medicine; E-mail: deli@brc.hu

Abstract: As the blood-brain barrier (BBB) prevents the majority of potential neurotherapeutics from reaching the central nervous system, early screening for BBB penetrability is very important in drug development. There are an abundance of available methods, from in silico screening through non-cell-based and cell-based in vitro methods to animal studies, with different predictive values, speed and throughput. Computational models and in vitro methods measuring physico-chemical properties, the octanol/water partition coefficient, and penetration through artificial membranes are accurate for predictions of passive permeability methods. Cell cultures including epithelial cell lines and their transporter transfected or drug-treated subclones and brain endothelial cell-based models in mono- and co-cultures with glial cells and/or pericytes are versatile tools for bi-directional active and passive transcellular transport. In vivo techniques to measure brain uptake, influx or efflux transport include brain perfusion, microdialysis, magnetic resonance imaging, positron emission tomography and, more recently, near-infrared time-domain optical imaging. These research asnd screening tools for BBB permeability will be reviewed with a special emphasis on culture-based BBB models. While none of these methods can be used alone to generate a reliable prediction of drug transport through the BBB, a combination of different models can give a useful estimate of the brain penetrability of drug candidates in preclinical screening.

INTRODUCTION TO BLOOD-BRAIN BARRIER PERMEABILITY

The blood-brain barrier (BBB) restricts the free penetration of molecules and cells from systemic circulation to brain tissue. Due to the very limited paracellular flux and the presence of efflux pumps, hydrophilic molecules, including biopharmaceuticals and a large number of lipophilic drugs, cannot reach their target in the central nervous system (CNS), resulting in ineffective treatment for many neurological diseases [1]. Despite efforts to increase drug delivery to the brain through strategies to overcome the junctional or efflux barriers to exploit BBB transport mechanisms or to circumvent the BBB, the majority of newly developed neuropharmaceuticals still fail due to poor CNS pharmacokinetics [2]. Early screening of drug candidates for brain penetrability is therefore a very important task in drug development [3].

Several models have been developed to examine BBB permeability, its physiology, pathology and pharmacology. Cell culture-based BBB models have proved to be one of the most versatile tools in both basic research and permeability testing [4]. In drug development, several sequential and parallel steps for screening BBB permeability have been suggested [3, 5]. Computational approaches for predicting drug penetrability can be used to screen big compound libraries, with the advantage of being quick and high throughput. Non-cell-based in vitro permeability models can further refine and specify penetration properties. Epithelial cell line models offer better estimation of the efflux transport of drug candidates, while brain endothelial cell culture models best mimic the properties and complexity of the BBB. In vivo models are physiologically the most complex, and should be used for validation of other preclinical screens. Combining these methods can give reliable estimates of BBB penetrability and help in selecting the most promising CNS drug candidates [3, 5].

IN SILICO MODELS

In silico prediction of BBB permeability provides a quick, cheap, high throughput filtering method for new compounds in the drug discovery pipeline. In silico permeation modelling is based on the physico-chemical characteristics of the molecule; however, this widely-used method has both strengths and limitations [3, 5-8]. The computational models are based on experimental data from previous in vivo and in vitro studies; the selection of datasets is therefore a critical component of the predictive power of the estimations. The original assumption used for in silico predictions was that passive diffusion is the major route for a compound through the BBB. This simplification, however, does not take the

Review Acta Neurobiol Exp 2011, 71: 113–128

© 2011 by Polish Neuroscience Society - PTBUN, Nencki Institute of Experimental Biology

INTRODUCTION

The blood-brain barrier (BBB) is an active interface between the circulation and the central nervous system (CNS) which restricts the free movement of different substances between the two compartments and plays a crucial role in the maintenance of the homeostasis of the CNS. The BBB has a dual, a barrier and a carrier function. The barrier function means that the BBB restricts the transport of potentially toxic or harmful substances from the blood to the brain which is achieved through a fourfold defense line:

1) The paracellular barrier formed by interendothe-lial junctions restricts the free movement of water soluble compounds between two adjacent cells.

2) The transcellular barrier is made possible by the low level of endocytosis and transcytosis characteristic for brain endothelial cells and inhibits transport of

substances through the cytoplasm.3) The enzymatic barrier is provided by a complex

set of enzymes, including acetylcholinesterase, alka-line phosphatase, gamma-glutamyl transpeptidase, monoamine oxidases, and other drug metabolizing enzymes capable to degrade different chemical com-pounds.

4) In addition to these, the cerebral endothelium expresses a large number of efflux transporters (ABC, ATP-binding cassette transporters) like ABCB1 (P-glycoprotein), ABCC1, ABCC4 and ABCG2 (BCRP).

Besides the barrier function the BBB possesses an important carrier function which is responsible for the transport of nutrients to the brain and removal of metab-olites. Small lipid-soluble molecules and blood gases like oxygen and carbon dioxide diffuse passively the BBB, while essential polar nutrients like glucose and amino acids require specific transport proteins (solute carriers, SLC transporters) in order to reach the brain.

The BBB plays a crucial role in the clinical practice as well. On the one side there is a large number of neu-rological disorders including cerebral ischemia, brain

In vitro models of the blood-brain barrierImola Wilhelm, Csilla Fazakas, and Istvan A. Krizbai*

Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary, *Email: krizbai@brc.hu

The blood-brain barrier (BBB) is an active interface between the circulation and the central nervous system (CNS) with a dual function: the barrier function restricts the transport from the blood to the brain of potentially toxic or harmful substances; the carrier function is responsible for the transport of nutrients to the brain and removal of metabolites. The BBB plays a crucial role in the clinical practice as well. On the one side there is a large number of neurological disorders including cerebral ischemia, brain trauma and tumors, neurodegenerative disorders, in which the permeability of the BBB is increased. On the other hand due to the relative impermeability of the barrier many drugs are unable to reach the CNS in therapeutically relevant concentration, making the BBB one of the major impediments in the treatment of CNS disorders. The significant scientific and industrial interest in the physiology and pathology of the BBB led to the development of several in vitro models of the BBB. These models are mainly based on the culture of cerebral endothelial cells. The best in vitro models which mimic the best way the in vivo anatomical conditions are the co-culture models in which brain endothelial cells are co-cultured with astrocytes and/or pericytes. Our in vitro BBB model is characterized by high transendothelial electrical resistance (TEER regularily above 200 Ohm×cm2), low permeability and expression of several transporters. Our experiments have proven that the model is suitable for basic research and for testing the interaction between the BBB and potential drug candidates (toxicity, permeability, interaction with efflux transporters) as well.

Key words: cerebral endothelial cells, blood-brain barrier, in vitro models, permeability, tight junction

Correspondence should be addressed to: I. A. Krizbai E-mail: krizbai@brc.hu

Received 20 December 2010, accepted 26 January 2011

Improvement of Biogas Production by Biotechnological Manipulation of the Microbial

Population

N. Ács*, Z. Bagi*, G. Rákhely*,**, E. Kovács*, R. Wirth and K. L. Kovács*,**,° * Department of Biotechnology, University of Szeged, Hungary

** Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary acsn@brc.hu, bagiz@brc.hu, rakhely@brc.hu, kovacse@brc.hu, roland.w@freemeil.hu,

°corresponding author: kornel@brc.hu

Abstract—Biogas is a renewable energy carrier and the production of biogas is associated with double benefits: elimination of environmental pollution problems is coupled with the generation of useful energy. Biogas technologies commonly apply natural anaerobic consortia of microbes. This is partly due to the fact that, from a microbiological aspect, this is a very complicated and complex system. Moreover, the population dynamics of the natural ecosystems could not be properly studied before the introduction of molecular biological techniques. Research on the diversity of these microbial communities is needed for the optimization of biogas production technologies as their economic viability is closely related to the efficacy of the concerted microbiological actions. A systematic study, using 5-litre CSTR digesters, disclosed that a key fermentation parameter is the loading rate of organic total solids (OTS) in maintaining an altered population balance.

I. INTRODUCTION Various approaches have been developed for the treatment and elimination of organic waste, often involving biological systems [1, 13]. Technologies converting organic material into biogas or hydrogen in a fermentation process are the only ones bringing about the combined advantages of waste disposal and generation of useful energy at the same time [7, 9, 10, 19]. Biogas is a gaseous renewable energy carrier consisting mainly of methane and carbon dioxide, which is produced as the end product of the anaerobic digestion of organic material [12]. Biogas can be used in several ways. After removal of trace contamination hydrogen sulfide, xyloxanes and water, biogas can be burnt to generate heat or can be used as fuel in gas engines, coupled to a generator to produce electricity and heat. If the carbon dioxide is also withdrawn from biogas, the remaining gas (often called biomethane), has the properties of purified natural gas, and can be utilized as a transportation fuel, a raw material for the chemical industry, or in fuel cells, which convert it to electricity with high efficiency [1].

Biogas technologies commonly apply natural anaerobic consortia of microbes. These communities form an intricate microbiological food chain. Moreover, the population dynamics of the natural ecosystems could not be properly studied before the introduction of molecular biological techniques [2, 6, 11, 17, 18].

Research on the diversity of these microbial communities is needed for the optimization of biogas production technologies as their economic viability is closely related to the efficacy of the concerted microbiological actions [8]. One of the rate-limiting factors in biogas-producing consortia is the actual level of hydrogen in the system [2, 3, 16]. The presence of too much hydrogen inhibits the acetogenic bacteria that generate hydrogen in the system, whereas too little hydrogen has an adverse effect on an important group of methanogens, the hydrogenotrophic methanogens. In natural ecosystems a very low partial pressure of hydrogen is maintained, which may be a limiting factor for the methanogens [2, 5].

We demonstrated earlier that reductant accessibility is indeed a limiting factor in biogas production and presented data supporting the hypothesis that the introduction of hydrogen-producing bacteria into the natural biogas-generating consortium effectively increases biogas production both in batch fermentations and in a scale-up anaerobic digestor [2, 7].

In this study systematic experiments were conducted in 5 litre CSTR (continuous stirred) fermentors, designed for biogas research at laboratory scale. These devices model the real-life, large scale biogas production plants much better than the routinely used batch systems and some of the first results are reported here. Thermophilic conditions were selected because the microbial diversity in the thermophilic natural consortia is lower, which requires a thorough inspection of the microbiological profiles. Introduction of a hydrogen-producing new member into such consortia is therefore somewhat more challenging than altering the composition of a microbial consortium under mesophilic conditions. Caldicellulosiruptor saccharolyticus is a good hydrogen producer and the beneficial effect of adding this strain to the biogas and biohydrogen generating systems has been demonstrated [2, 4, 7, 14]. An important question remained before the implementation of large-scale application: the conditions under which C. saccharolyticus becomes a stable member of the biogas-producing microbial consortium. This can be determined only in systematic experiments with digesters functioning in continuous operation mode. Therefore the survival of C. saccharolyticus in a thermophilic biogas producing consortium was tested by molecular biological methods. The method developed and the results are presented here together with some details of the laboratory

EXPRES 2011 • 3rd IEEE International Symposium on Exploitation of Renewable Energy Sources • March 11-12, 2011, Subotica, Serbia

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Computational Biology and Chemistry 35 (2011) 240–250

Contents lists available at ScienceDirect

Computational Biology and Chemistry

jo ur n al hom ep age : www.elsev ier .com/ locate /compbio lchem

xploring and characterizing the folding processes of Lys- and Arg-containingla-based peptides: A molecular dynamics study

ábor Janzsóa, Ferenc Bogárb,c, Liza Hudobaa, Botond Penkeb,c, Gábor Rákhelya,d, Balázs Leitgeba,∗

Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, HungarySupramolecular and Nanostructured Materials Research Group of the Hungarian Academy of Sciences, University of Szeged, Dóm tér 8, H-6720 Szeged, HungaryDepartment of Medical Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, HungaryDepartment of Biotechnology, University of Szeged, Középfasor 52, H-6726 Szeged, Hungary

r t i c l e i n f o

rticle history:eceived 11 January 2011eceived in revised form 23 May 2011ccepted 24 May 2011

eywords:la-based peptide

a b s t r a c t

In this study, molecular dynamics simulations were carried out on Lys- and Arg-containing Ala-basedpeptides (i.e. Ace-(AAAAK)nA-NH2 and Ace-(AAAAR)nA-NH2, where n = 1–4), in order to explore and char-acterize their folding processes. For the oligopeptides, the evolution of �-helical structure with regard tothe whole conformation, as well as to each residue was investigated, and the helix-forming propensitieswere characterized. On the basis of the helicity curves, representing the alteration of average helicityas a function of time, the typical time values describing the folding processes and subprocesses were

olding processolecular dynamicselical structure

ntramolecular H-bondolding pathway

identified. In the case of each peptide, the evolution and role of helix-stabilizing, non-local and side-chain-to-backbone H-bonds were examined. The appearing i ← i + 4 H-bonds pointed out the role ofthese interactions in the stabilization of �-helical conformations, while the occurring i ← i + 3 H-bondsindicated the presence of �-turn or 310-helical structures. Studying the formation and role of non-localand side-chain-to-backbone H-bonds led to the observation that these types of interactions produced aneffect on the evolution of helical conformations, as well as on the folding processes.

. Introduction

The energetics and kinetics of the folding processes of peptidesnd proteins are in the focus of interest for a long time (Baldwin,007; Daggett and Fersht, 2003; De Mori et al., 2005; Dill et al.,008; Eaton et al., 2000; Ferguson and Fersht, 2003; Morra et al.,008; Osterhout, 2005; Shea and Brooks, 2001). One of the mosttudied fields is the evolution of different secondary structural ele-ents (Eaton et al., 2000; Ferguson and Fersht, 2003; Osterhout,

005), which play an important role in stabilizing the conforma-ion of peptides, as well as they can be responsible for the biologicalctivity of numerous molecules. Since the formation of �-helix ishe fastest among the periodic secondary structures, thus, the struc-ural evolution of peptide sequences consisting of various aminocids into this helical conformation has extensively been investi-ated (Aurora et al., 1997; Makhatadze, 2005; Osterhout, 2005).mong the different amino acids, alanine is a well-known repre-entative possessing a high propensity to form helical (especially

-helical) structure. However, the determination of the conforma-

ional features of polyalanine peptides by experimental methods isery difficult. The reason for this is that these molecules are consid-

∗ Corresponding author. Tel.: +36 62 599 726; fax: +36 62 433 133.E-mail address: leitgeb@brc.hu (B. Leitgeb).

476-9271/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.compbiolchem.2011.05.004

© 2011 Elsevier Ltd. All rights reserved.

erably insoluble in aqueous solution, and this insolubility inducesthe aggregation of these oligopeptides. In order to avoid this hin-drance, other amino acids can be inserted into the sequencesof polyalanine peptides, which possess positively and negativelycharged side-chains, respectively. As the earlier studies pointed out,the substitution of Ala amino acids by Lys and Glu residues in certainpositions of the sequences resulted in the formation of salt-bridgesbetween the charged groups of side-chains, which contributed tothe structural stabilization of the helical conformation (Marquseeand Baldwin, 1987; Marqusee et al., 1989). Another method to solvethe above-mentioned solubility problem is the incorporation ofonly one type of the hydrophilic amino acids (e.g. Lys, Arg or Glu)into the sequences of polyalanine peptides (Marqusee et al., 1989).The inclusion of these residues into the oligopeptides provides thedesired aqueous solubility, and additionally, the presence of thesebasic or acidic amino acids can prevent the formation of aggregates.

Previously, a series of Ala-based peptides was designed byMarqusee et al. (1989), in which Lys residues were located incertain positions of the sequences. These molecules proved to bevery useful to elucidate the formation of the secondary structureof Ala-based peptides by experimental measurements, therefore,

they gained a widespread interest. Accordingly, numerous stud-ies were performed on a variety of Lys-containing peptides, aswell as on their derivatives, in which Arg residues were incorpo-rated instead of the Lys amino acids. In order to investigate the

74 75

market acceptance of GEM, large biotechnology companies are tending to withdraw from the field. Indeed, horizontal gene transfer is a commonplace in bacterial genera containing both human and animal pathogens, and such transfer may also occur between bacteria and fungi. GEMs with new fitness traits may be more resistant against the competitive organisms of their wild-type parents, and therefore their release may cause perturbations in the natural microflora. Furthermore, using gene constructs bearing antibiotic resistance genes may result in unexpected and undesirable gene escape. On the other hand, un-controlled gene transfer can be prevented by genetic engineering (an example was mentioned above for A. radiobacter), and sequences coding for antibiotic resistance can be destroyed in transgenic organisms (like in GE B. thüringiensis). GEMs engineered to have improved anti-microbial activity may exert non-target effects on natural microbial communities, but other measures, like crop monoculture, ploughing or pesticide treatments may certainly cause much stronger adverse effects.

Despite concerns, countries with rapidly growing economies like China, India or the US have less aversion to the introduction of GEM-based biopesticides and biofertilizers. Irrespective of practical applications, research on such organisms is carried on in other parts of the world, as methods, gene constructs, products and technologies developed while working with GEMs are utilized very efficiently in industrial microbiology and molecular plant breeding.

Further reading

Cummings, J. (2003): GM microbes invade North America. Science in •Society, 19: 39.

Garay-Munoz, C. • et al. (2010): Mode of action of Bacillus thüringiensis- genetically modified Cry1AbMod and Cry1AcMod toxins: role of alkaline pH in toxin oligomerization. Southwestern Entomologist, 35: 383–386.

Hirsch, P. E. (2005): Release of transgenic bacterial inoculants – rhizobia as •a case study. Plant and Soil, 266: 1–10.

Hunter, P. (2009): Fight fire with fire. Can biopesticides fill the void left by •banning chemical pesticides and herbicides. EMBO Reports, 10: 433–436.

Thomas, M. B., Read, A. F. (2007): Fungal bioinsecticide with a sting. Nature •Biotechnology, 25: 1367–1368.

12. The role of GM microbes in the fermentation technology

KORNÉL KOVÁCS

Biomass stores the energy captured from sunlight during photosynthesis in the form of chemical bonds holding the molecules together. In the various ways of producing renewable biofuels, this chemical energy is released and converted into a form that is easy to utilize in everyday energy consuming devices, e.g. transportation vehicles, light bulbs etc. The most commonly used fractions of biomass are the ones prepared for energy storage purposes by the photosynthe-tic organisms themselves. These are sugars or their polymers, such as starch or cellulose.

The transformation of starch into sugar is an important branch of the starch industry and one of the most important applications of biotechnology. Countless foods contain ingredients produced by the breakdown of starch. Enzymes are the key to these chemical reactions – enzymes that are predominantly produced with the help of genetically modified microorganisms. Starches are chemically bound clusters of sugar molecules found in plants. Under the right conditions, starch molecules can be broken down into sugar. Sugar is the most preferred carbon and energy source for almost all microbes used in biotechnological processes. This process makes it possible to obtain sugar from the starch of many different plants, rather than just sugar beets or sugar cane. This is now being done by industrial-scale starch saccharification. The most important sources of starch are maize, potatoes, and wheat.

Strong acids were once used to break apart starch molecules and release sugar. Now, enzymes do the job offering many advantages: with enzymes, the process targets the proper chemical bonds much more precisely. Different enzymes can be used to produce syrups with different levels of sweetness and different technical characteristics. The end products are not only used as custom tailored ingredients in countless foods and drinks, they can also be further processed into glucose, artificial sweeteners, or fat substitutes.

For a long time, enzymatic breakdown of starch (saccharification) did not make economic sense. Things changed, however, as soon as the enzymes responsible for this process became available at low cost, high quality, and in practically unlimited quantities. Now, almost all of the enzymes used to break down starch are produced with the help of genetically modified microorganisms.

Plants are used as a starch source. A certain portion of the raw material may be genetically modified. Cultivars of maize and potato have been produced, in which the structural properties of the starch molecules are altered so that saccharification takes place more efficiently. These starch sources are then used in bioenergy production, making subsequent fermentation of the sugar component more efficient and the overall process economically more viable. Next generation renewable energy carriers will utilize lignocellulosic raw material. Lignocellulose

László Hornok, Katalin Posta 12. The role of GM microbes in the fermentation technology

BIOXEN Seminar "Novel approaches for environmental protection", Novi Sad, 8-10 September 2011

DISTRIBUTION OF MONO- AND DISACCHARIDE-RELEASINGEXTRACELLUbAR ENZYME PRODUCTION ABILITlES WITHIN A

TRICHODERMA POPULATION FROM HUNGARIAN WINTER WHEATRHIZOSPHERE

Kredics, L.1, Leitgeb, B.2, Hatvani, L.1, Manczinger, L.1, Vágvölgyi, Cs.1,Szekeres, A.3

1University of Szeged, Faculty of Science and Informatics, Department ofMicrobiology, Hungary

21nstituteof Biophysics, Biological Research Centre of the HungarianAcademy of Sciences, Szeged, Hungary3Fumoprep Ltd., Mórahalom, Hungary

AbstractThe action of fungal hydrolytic enzymes is playing a crucial role in the biocontrolprocess of Trichoderma strains. In the present study, information was collected aboutthe distribution of mono- and disaccharide-releasing extracellular enzyme productionabilities within a Hungarian Trichoderma population from winter wheat rhizosphere.Key Words: a-glucosidase, ~galactosidase, ~glucosidase, 13-1,4-N-acetyl-glucosaminidase, ~xylosidase, cellobiohydrolase, N-acetyl-~glucosaminidase,Trichoderma

Introduction

The efficient control of fungal plant pathogens causing substantial losses inagricultural production is an important issue for ali plant cultivation systems. Speciesbelonging to the genus Trichoderma are imperfect filamentous fungi of multipleimportances. Members of this genus are weil known as cellulase producers ofbiotechnological relevance (Kubicek et al., 1990; Sch moll and Kubicek, 2003).Certain Trichoderma species are on the growing list of potential fungal pathogens inimmunocompromised hosts (Kredics et al., 201Oa), while others are harmful inmushroom cultivation as the causative agents of green mouId epidemics (Hatvani etal., 2008; Kredics et al., 2010b). Furthermore, the genus involves promisingbiocontrol candidates with excellent antagonistic abilities against a number of plantpathogenic fungi. Several modes of action have been proposed to play roles inbiocontrol capabilities, including antibiosis by the production of antifungal metabolites(Szekeres, 2005), competition for space and nutrients (Sivan, 1989), piant growthpromotion, induction of the defence responses in plants (Harman, 2004) andmycoparasitism (Chet, 1987). These processes are supposed to act synergistically(Schirmböck, 1994; Manczinger, 2002). For the study of this complex synergisticsystem it is crucial to clear the relative importance of the individual mechanisms inthe antagonistic process. Both the competition by colonizing the ecological nichefavoured by the pathogen and mycoparasitism by penetration of the host hyphaerequires hydrolytic enzyme systems that are playing important roles in the digestionof the available natural substrates in the soil and the polymers constituting the cell-wall and cytoplasm of the target fungi. Based on the involvement in the biocontrolprocess, the extracellular enzymes secreted by Trichoderma species can be

56

ORIGINAL RESEARCH

Helix and H-bond formations of alanine-based peptides containingbasic amino acids

Balazs Leitgeb • Gabor Janzso • Liza Hudoba •

Botond Penke • Gabor Rakhely • Ferenc Bogar

Received: 25 November 2010 / Accepted: 14 June 2011 / Published online: 26 June 2011

� Springer Science+Business Media, LLC 2011

Abstract We studied comprehensively the helicity and

H-bonding evolutions during the folding processes of Lys-

and Arg-containing alanine-based peptides. The evolution

of a-helical conformation concerning the entire sequence

and each amino acid residue was examined, as well as the

helix-forming propensities were characterized. The for-

mation of various types of the intramolecular H-bonds was

also investigated, pointing out the helix-stabilizing role of

local interactions and the destabilizing role of non-local

interplays. Our study led to the observation that the non-

local H-bonds affected the evolution of helical conforma-

tions, as well as the entire folding processes.

Keywords Alanine-based peptide �Molecular dynamics �Folding � Helical conformation � Intramolecular H-bond

Introduction

Extensive research efforts have been focused on studying

and characterizing the folding processes of peptides and

proteins for a long time [1–9]. Since the various secondary

structural elements play a relevant role in determining and

stabilizing the structures of peptides and proteins, several

attempts have been made to investigate the formation of

different secondary structures as a function of time [1, 4,

6]. Among the periodic conformations, the evolution of

a-helical structures proved to be the fastest, thus, examin-

ing the formation of a variety of peptide sequences into

a-helices became one of the most studied fields of peptide

and protein folding [6, 10, 11]. Since the amino acid ala-

nine proved to be a helix-forming residue, the structural

and conformational features of polyalanine peptides have

been extensively investigated so far. However, to deter-

mine the three-dimensional structure of these homo-

oligopeptides by experimental techniques is a challenging

task, due to their insolubility and aggregation propensity in

aqueous solution. To resolve these problems, basic and/or

acidic amino acids were previously incorporated into the

sequences of polyalanine peptides in the following two

ways. For one of them, the Ala residues located at certain

positions of the sequences were substituted by Lys and Glu

amino acids, resulted in the appearance of helix-stabilizing

salt-bridges formed between the charged side-chains [12,

13]. The other way to avoid the hindrances is the inclusion

of only basic or acidic amino acids (e.g., Lys, Arg, or Glu)

into the sequences of polyalanines [13]. The incorporation

of hydrophilic residues into the homo-oligopeptides pro-

vides the aqueous solubility, as well as their presence in the

sequences can prevent the formation of aggregates. A

series of Lys-containing Ala-based peptides was earlier

designed [13], which gained a widespread interest, due to

B. Leitgeb (&) � G. Janzso � L. Hudoba � G. Rakhely

Institute of Biophysics, Biological Research Center of the

Hungarian Academy of Sciences, Temesvari krt. 62,

6726 Szeged, Hungary

e-mail: leitgeb@brc.hu

B. Penke � F. Bogar

Department of Medical Chemistry, University of Szeged,

Dom ter 8, 6720 Szeged, Hungary

G. Rakhely

Department of Biotechnology, University of Szeged,

Kozepfasor 52, 6726 Szeged, Hungary

B. Penke � F. Bogar

Supramolecular and Nanostructured Materials Research Group

of the Hungarian Academy of Sciences, University of Szeged,

Dom ter 8, 6720 Szeged, Hungary

123

Struct Chem (2011) 22:1287–1295

DOI 10.1007/s11224-011-9824-x

Fungal Genetics and Biology 48 (2011) 92–103

Contents lists available at ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier .com/ locate/yfgbi

Comparison of transcriptional and translational changes caused by long-termmenadione exposure in Aspergillus nidulans

Tünde Pusztahelyi a,⇑, Éva Klement b, Emilia Szajli b, József Klem c,d, Márton Miskei e,f,Zsolt Karányi g, Tamás Emri a, Szilvia Kovács a, Gyula Orosz d, Kornél L. Kovács d, Katalin F. Medzihradszky b,h,Rolf A. Prade i, István Pócsi a,⇑⇑a Department of Microbial Biotechnology and Cell Biology, Faculty of Science and Technology, University of Debrecen, Egyetem tér 1, H-4032 Debrecen, Hungaryb Proteomics Research Group, Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungaryc Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt. 62, H-6726 Szeged, Hungaryd Department of Biotechnology, University of Szeged, Középfasor 52, H-6726 Szeged, Hungarye Department of Horticultural Sciences and Plant Biotechnology, Faculty of Agriculture, Centre of Agricultural Sciences, University of Debrecen,Egyetem tér 1, H-4032 Debrecen, Hungaryf Mycology Group of the Hungarian Academy of Sciences, Institute of Plant Protection, Szent István University, Páter Károly utca. 1, H-2100 Gödöll}o, Hungaryg Department of Medicine, Faculty of Medicine, University of Debrecen, P.O. Box 19, H-4012 Debrecen, Hungaryh Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94158, USAi Department of Biochemistry and Molecular Biology, Oklahoma State University, 348E Noble Research Center, Stillwater, OK 74078, USA

a r t i c l e i n f o a b s t r a c t

Article history:Received 22 February 2010Accepted 19 August 2010Available online 24 August 2010

Keywords:FungiAspergillus (Emericella) nidulansOxidative stressMenadioneProteomicsGenomics

1087-1845/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.fgb.2010.08.006

Abbreviations: GSH, glutathione; GSSG, glutathioxygen species; MSB, menadione sodium bisulfite.⇑ Corresponding author. Address: Department of

Cell Biology, Faculty of Science and Technology, UniveH-4010 Debrecen, Hungary.⇑⇑ Corresponding author. Address: Department ofCell Biology, Faculty of Science and Technology, UniveH-4010 Debrecen, Hungary.

E-mail addresses: pusztahelyi@yahoo.com (T. Pu(I. Pócsi).

Under long-term oxidative stress caused by menadione sodium bisulfite, genome-wide transcriptionaland proteome-wide translational changes were compared in Aspergillus nidulans vegetative cells. Thecomparison of proteomic and DNA microarray expression data demonstrated that global gene expressionchanges recorded with either flip-flop or dendrimer cDNA labeling techniques supported proteomechanges moderately with 40% and 34% coincidence coefficients, respectively. Enzyme levels in the glyco-lytic pathway were alternating, which was a direct consequence of fluctuating gene expression patterns.Surprisingly, enzymes in the vitamin B2 and B6 biosynthetic pathways were repressed concomitantlywith the repression of some protein folding chaperones and nuclear transport elements. Under long-termoxidative stress, the peroxide-detoxifying peroxiredoxins and cytochrome c peroxidase were replaced bythioredoxin reductase, a nitroreductase and a flavohemoprotein, and protein degradation became pre-dominant to eliminate damaged proteins.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction Cano-Dominguez et al., 2008), ROS are also cytotoxic in prokaryotic

In aerobic organisms reactive oxygen species (ROS) are gener-ated continuously as side products of respiration (Li et al., 2009).ROS include hydrogen peroxide (H2O2), superoxide anion (O��2 )and hydroxyl radicals (HO�). In addition to their important signal-ing functions in diverse cellular processes (Lara-Otíz et al., 2003;

ll rights reserved.

one disulfide; ROS, reactive

Microbial Biotechnology andrsity of Debrecen, P.O. Box 63,

Microbial Biotechnology andrsity of Debrecen, P.O. Box 63,

sztahelyi), ipocsi@gmail.com

and eukaryotic organisms. Not surprisingly, significant efforts aremade by the O2-exposed cells to eliminate harmful ROS througha wide array of both enzymatic and non-enzymatic processes(Pócsi et al., 2004; Li et al., 2009). Higher concentrations of ROSthat may originate from exogenous sources or due to intracellularenzyme activities may cause aging and even initiate apoptotic celldeath (Perrone et al., 2008; Scheckhuber et al., 2009). ROS gener-ated at low concentrations can trigger an adaptive stress responsethat makes the cells resistant to lethal concentrations of these toxicoxygen derivatives (Collinson and Dawes, 1992; Jamieson, 1992; Liet al., 2008a).

Gene expression and proteome surveys have identifiednumerous genes and gene products induced or repressed in re-sponse to oxidants in yeasts and filamentous fungi (Godon et al.,1998; Gasch et al., 2000; Chen et al., 2003, 2008; Kim et al.,2006, 2007a). Applications of ROS generating agents, employedat sublethal doses in Aspergillus nidulans (Pócsi et al., 2005)

18 19

László Sági, Elen Gócza, Kornél Kovács

5. Procedures for the generation of geneticallymodified organisms

LÁSZLÓ SÁGI, ELEN GÓCZA, KORNÉL KOVÁCS

Introduction

Life scientists often believe that communicating technical details on their subject – in this case the production of genetically modified (GM) living organisms – is so interesting in itself that it will inevitably convince a reluctant or ignorant public, and as a result, people will be happy to convert to their ‘discoveries’.

That this notion has been a failure and wishful thinking rather than reality can be best demonstrated by the present anti-GM attitude in a number of EU-countries as well as by a strong ‘no’ to cultivation, processing and sale of GM crops. Hungary (and some of the neighbouring countries) is no exception to this type of public reaction, which is partially due to the not-so-neutral presentation of GM issues in national and local media (see also Chapter 17).

While dissemination of scientific information on the GM subject may not have been very successful in the popular media, the provision of comprehensible and unbiased information to people showing an interest in the issue as well as to decision makers is definitely justified. We do not want to be blamed for concealing the “obscure machinations” of scientists from the public.

In this chapter, we will therefore give a concise overview on (i) the major techniques currently employed for the generation of GM organisms (microbes, plants and animals), and (ii) the principles and practice on how to make these techniques ever more efficient and, at the same time, acceptable for the public.

Definition: transgenic or genetically modified living organisms are organisms each of whose cells stably contains the inserted DNA and this transgene is (or these transgenes are) transmitted to the offspring as well. Generation of GM microbes

As scientists analysing basic cellular processes, they often extract enzymes or DNA from whole cells and study their activity and structure outside the cell. When a process is investigated in vitro, it can often be manipulated in ways that would be impossible if it was still taking place inside a cell. Natural processes resulting in the transfer of DNA from one cell to another cell (transformation, transduction, conjugation) have been discovered. A by-product of the quest for knowledge was the accumulation of a versatile tool kit for manipulating and analyzing DNA and proteins. Biological knowledge and biotechnology know-how have increased. Modern biotechnologies have harnessed enzymes and procedures that scientists copied and modified from nature.

The explosion of DNA technologies has in part been the result of the intense research focus on understanding the function of DNA from the beginning of the 1950s. One of the challenges facing the newly born molecular biology was the fact that in nature many genes are found on one DNA molecule. In the 1960s, sci-entists studying how certain bacteria resist infection by bacteriophages observed that the DNA of the infecting virus was cut into pieces at well-defined sequence positions: the corresponding enzymes were named restriction endonucleases or restriction enzymes (Figure 5.1.). By now more than 5,000 restriction enzymes have been identified and isolated from hundreds of bacterial species.

Figure 5.1. Schematic activity of a restriction enzyme from the E. coli bacterium

These enzymes are used like precise DNA scissors to cut DNA molecules into precise pieces, which can then be used either for identification or for making copies of desired genes. Identification of specific DNA sequences is useful in a number of courtroom cases such as the identification of criminal suspects by their DNA pattern, verification of biological fathers when in doubt, or even for clarifying food safety issues. Genes that carry traits, which we decide to exploit, are then multiplied and their gene products can subsequently be generated at will. The polymerase chain reaction (PCR) is now used in many kinds of applications from disease diagnosis to studying ancient DNA. PCR can produce over 1 billion copies of a DNA segment from a single starting molecule in about 1 hour of amplification. It is a molecular copy-machine working at amazing speed.

The term cloning means the production of identical copies of something. For instance, cars rolling off the assembly lines are clones. In biology, cloning specifically refers to the production of genetically identical copies. When applied

5. Procedures for the generation of genetically modified organisms

FEBS Letters 585 (2011) 545–548

journal homepage: www.FEBSLetters .org

Axial ligation of the high-potential heme center in an Arabidopsis cytochrome b561

Filip Desmet a, Alajos Bérczi b, László Zimányi b, Han Asard c, Sabine Van Doorslaer a,⇑a Department of Physics, University of Antwerp, Belgiumb Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungaryc Department of Biology, University of Antwerp, Belgium

a r t i c l e i n f o a b s t r a c t

Article history:Received 29 October 2010Revised 21 December 2010Accepted 4 January 2011Available online 12 January 2011

Edited by Miguel De la Rosa

Keywords:Double mutantEPRCytochrome b561Optical absorption spectroscopyResonance Raman spectroscopy

0014-5793/$36.00 � 2011 Federation of European Biodoi:10.1016/j.febslet.2011.01.006

Abbreviations: ASC, ascorbate; Cyt-b561, cytochtonoplast-Cyt-b561; CGCytb, chromaffin granule cytoparamagnetic resonance; TSCytb, tumor suppressor 1Raman⇑ Corresponding author. Address: Department of Ph

Campus Drie Eiken, Universiteitsplein 1, 2610 Wilrijk,E-mail address: sabine.vandoorslaer@ua.ac.be (S. V

Arabidopsis has four putative, di-heme cytochrome b561 proteins, including one localized in thetonoplast (TCytb). From a comparative electron paramagnetic resonance (EPR), UV–Vis absorptionand resonance Raman study, on wild type, H83A/H156A-TCytb and H83L/H156L-TCytb doublemutants, it follows that the H83 and H156 residues are binding one of the two hemes. These mea-surements show that the high-potential heme site is situated at the cytoplasmic side of the mem-brane and allow the unambiguous differentiation between two models on the heme localizationin cytochrome b561 proteins.� 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction EPR signatures [9–11]. Both centers have EPR spectra typical of

Cytochromes b561 (Cyts-b561) are ascorbate (ASC)-reducible,trans-membrane, di-heme proteins identified in a great variety oforganisms, including invertebrates, vertebrates and plants [1]. AllCyts-b561 are predicted to consist of six trans-membrane helices,with four highly conserved His residues likely involved in the hemecoordination [2]. Arabidopsis and other plants contain several genesencoding putative Cyts-b561; however, little is known about thelocalization and physiological function of the gene products [3].One plant isoform is localized on the tonoplast and is named tono-plast-Cyt-b561 (TCytb or TCB). Cyts-b561 are generally accepted tocatalyze ASC-driven trans-membrane electron transport. Thisactivity has been demonstrated for TCytb by expression in yeastcells and measuring ASC-dependent reduction of extracellular fer-ric-chelates [4].

The chromaffin granule cytochrome b561 (CGCytb) is the moststudied member of this protein family [5–11]. The oxidized CGCytbwas investigated using electron paramagnetic resonance (EPR)spectroscopy, showing that the two heme centers have distinct

chemical Societies. Published by E

rome b561; TCytb or TCB,chrome b561; EPR, electron01F6 protein; RR, resonance

ysics, University of Antwerp,Belgium. Fax: +32 32652470.an Doorslaer).

low-spin ferric heme centers, with characteristic signals atgz = 3.7, and gz = 3.1, for the low and high-potential heme. Combin-ing EPR and site-directed mutagenesis for CGCytb, the hemeligands were identified as His54 and His122 at the matrix (intrave-sicular) side (M-side) for the low-potential heme, while His88 andHis161, located at the cytoplasmic (extravesicular) side (C-side), li-gate the high-potential heme [11]. Furthermore, mutation of theHis92 residue, situated near the C-side heme in CGCytb561, causeda shift in the position of the gz = 3.1 signal, whereas mutation ofHis110, in the vicinity of the M-side heme, induced a change inthe gz = 3.7 signal [12].

In this work, we use a combination of EPR, UV–Vis absorptionand resonance Raman (RR) spectroscopy with site-directed muta-genesis, to investigate the heme ligation of TCytb in Arabidopsis.The results will be compared to those of other Cyt-b561 proteins,such as CGCytb and the recently characterized putative tumor sup-pressor 101F6 protein (TSCytb) [13].

2. Materials and methods

2.1. Materials

Yeast cell growth, microsomal membrane preparation, andprotein purification by His-tag affinity chromatography were per-formed as described [3,4]. H83A/H156A-TCytb and H83L/H156L-TCytb were obtained by site-directed mutagenesis using the

lsevier B.V. All rights reserved.

lable at ScienceDirect

Fish & Shellfish Immunology 31 (2011) 716e724

Contents lists avai

Fish & Shellfish Immunology

journal homepage: www.elsevier .com/locate/ fs i

Deep sequencing of the innate immune transcriptomic responseof zebrafish embryos to Salmonella infection

Anita Ordas a,b, Zoltan Hegedus c,d, Christiaan V. Henkel e, Oliver W. Stockhammer a, Derek Butler f,Hans J. Jansen e, Peter Racz a,b, Matyas Mink b, Herman P. Spaink a, Annemarie H. Meijer a,*a Institute of Biology, Leiden University, Einsteinweg 55, 2333 CC Leiden, The NetherlandsbDepartment of Genetics, Faculty of Science and Informatics, University of Szeged, 52 Kozepfasor, H-6726 Szeged, Hungaryc Zenon Bio Ltd., Maros u 40, H-6721 Szeged, HungarydBioinformatics Laboratory, Biological Research Center, Hungarian Academy of Sciences, Temesvari krt. 62, H-6726 Szeged, Hungarye ZF-Screens B.V., Niels Bohrweg 11, 2333 CA Leiden, The NetherlandsfBaseClear B.V., Einsteinweg 55, 2333 CC Leiden, The Netherlands

a r t i c l e i n f o

Article history:Received 23 June 2010Received in revised form15 August 2010Accepted 24 August 2010Available online 1 September 2010

Keywords:Zebrafish embryoInnate immunitySalmonella typhimuriumTag-SeqRNA-Seq

* Corresponding author. Tel.: þ31 71 5274927; fax:E-mail address: a.h.meijer@biology.leidenuniv.nl (

1050-4648/$ e see front matter � 2010 Elsevier Ltd.doi:10.1016/j.fsi.2010.08.022

a b s t r a c t

Salmonella enterica serovar Typhimurium (S. typhimurium) bacteria cause an inflammatory and lethalinfection in zebrafish embryos. To characterize the embryonic innate host response at the transcriptomelevel, we have extended and validated previous microarray data by Illumina next-generation sequencinganalysis. We obtained 10 million sequence reads from control and Salmonella-infected zebrafish embryosusing a tag-based sequencing method (DGE or Tag-Seq) and 15 million reads using whole transcriptsequencing (RNA-Seq), which respectively mapped to circa 65% and 85% of 28,716 known Ensembl tran-scripts. Both sequencing methods showed a strong correlation of sequence read counts per transcript andan overlap of 241 transcripts differentially expressed in response to infection. A lower overlap of 165transcripts was observed with previous microarray data. Based on the combined sequencing-based andmicroarray-based transcriptome data we compiled an annotated reference set of infection-responsivegenes in zebrafish embryos, encoding transcription factors, signal transduction proteins, cytokines andchemokines, complement factors, proteins involved in apoptosis and proteolysis, proteins with anti-microbial activities, as well as many known or novel proteins not previously linked to the immuneresponse. Furthermore, by comparison of the deep sequencing data of S. typhimurium infection in zebrafishembryos with previous deep sequencing data of Mycobacterium marinum infection in adult zebrafish wederived a common set of infection-responsive genes. This gene set consists of known and putative innatehost defense genes that are expressed both in the absence and presence of a fully developed adaptiveimmune system and that provide a valuable reference for future studies of hostepathogen interactionsusing zebrafish infection models.

� 2010 Elsevier Ltd. All rights reserved.

1. Introduction

In the recent years zebrafish has becomewidely used as a modelfor in vivo studies of hostepathogen interactions. Zebrafish developboth an innate and adaptive immune system with notable similar-ities to that ofmammals [1,2]. Zebrafish embryos can be exploited tostudy innate immunity separately fromadaptive immune functions,since components of the innate immune system are functionalalready at the first day of embryogenesis contrary to the adaptiveimmune system that is not active during the first weeks of zebrafish

þ31 71 5274357.A.H. Meijer).

All rights reserved.

development [3e6]. Furthermore, the externally developing andtransparent zebrafish embryos are highly suited for real-timeanalysis of hostepathogen interactions, which can be combinedwith efficient gene knock-down analysis using antisense morpho-lino oligonucleotides. It has been demonstrated that the compo-nents of the main innate immune signaling pathways are stronglyconserved between zebrafish and mammals [7,8] and severalinfection models for studying innate immune response mecha-nisms in zebrafish embryos have now been developed [9].

Salmonella infections, causing salmonellosis and typhoid fever,are studied in several animalmodels, of which the best studied is themouse model of S. enterica serovar Typhimurium infection (hereafterreferred to as S. typhimurium) [10]. The opportunity of real-timeanalysis led to the development of a S. typhimurium infection model