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InformatIon ...2 IntroductIon ...3 dIrectors ...4 InnovatIon ...6 InstItute of BIophysIcs ...7 Pál ormos ...8 andrás dÉr ...10 Géza Groma ...13 Lóránd KeLemen ...15 Ilona LacZKÓ ...17 Zsolt toKaJI ...18 György vÁrÓ ...20 László ZImÁnYI ...21 tibor PÁLI...23 alajos BÉrcZI ...25 Balázs sZaLontaI ...26 csaba BaGYInKa ...28 László sIKLÓs ...30 Árpád PÁrducZ ...32 mária deLI ...34 István KrIZBaI ...36 Kornél L. KovÁcs...38

Kornél L. KovÁcs and Gábor rÁKHeLY ...39

Gábor rÁKHeLY and Balázs LeItGeB ...41

selected publications ...42 InstItute of BIochemIstry ...47 antal KIss ...48 György PÓsfaI ...50 csaba PÁL ...52 Balázs PaPP ...54

sándor BenYHe and anna BorsodI ...56

mária sZŰcs ...58

csaba tÖmBÖLY and Géza tÓtH...60

miklós sÁntHa ...62 Imre m. Boros ...64 Péter deÁK ...65 László PoLGÁr ...93 István sImon...95 Gergely sZaKÁcs ...97 Péter tomPa ...99 mária vas ...100 andrás vÁradI ...102 Beáta G. vÉrtessY ...105 Péter ZÁvodsZKY ...107

Péter ZÁvodsZKY and Péter GÁL ...109

selected publications ... 111 InstItute of genetIcs ... 117 Henrik GYurKovIcs ... 118 László sIPos ...120 József mIHÁLY ...122 Géza ÁdÁm ...124 miklós erdÉLYI ...126 István KIss ...129 István andÓ ...130 Ilona dusHa ...132 Gabriella endre ...134 Lajos HaracsKa ...136 Ildikó unK ...138 Éva monostorI ...140 István rasKÓ ...142 Gyula HadLacZKY ...144 Zsolt PÉnZes ...146 selected publications ...148

InstItute of plant BIology ... 153

ferenc naGY ...154 Imre vass ...157 Győző GaraB ...159 Zoltán GomBos ...162 Gábor v. HorvÁtH ...164 Zoltán maGYar ...167 attila feHÉr ...169

Table of ConTenTs

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BIOLOGICAL RESEARCH CENTER, HUNGARIAN ACADEMY OF SCIENCES

CENTER OF EXCELLENCE OF THE EUROPEAN UNION H-6726 szeged, temesvári krt. 62.

H-6701 szeged, P.o. Box 521, Hungary Phone: +36-62-599-600

fax: +36-62-432-576 http://www.szbk.u-szeged.hu

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In 1962, the Hungarian academy of sciences decided to concentrate a substantial part of its means on supporting research in the field of modern biology. Professor Bruno straub f. initiated the foundation of the Biological research center. szeged was chosen as the site for a number of reasons. It is an university town with numerous excellent traditions. Work began in the Brc in 1971, but the official opening ceremony was held in 1973, when the construction of the whole building was finished.

Based on the concept of interdisciplinarity, scien-tific work in the Brc is carried out in 5 institutes:

Biophysics, Biochemistry, Enzymology, Genetics and

Plant Biology. In 2009, the Brc employs 479 persons in the four institutes located in szeged (and 90 persons in the Institute of enzymology located in Budapest), 257 (68) of whom belong to the scientific staff in addition to 66 (16) students in various Phd programs and 9 gradu-ate students of the International training course.

In 1998, the European Molecular Biology Organization (EMBO) and in 2001, the International scientific advisory Board performed a detailed evaluation of research groups in the Brc institutes. Both committees concluded that in certain fields such as developmental genetics, enzymology and plant biology, the research performance meets the highest international standards. In 2000, the Brc was selected as a Center of Excellence of the European Union. scientists from Brc have published 250–300 papers with 1000–1100 impact factors yearly. The Brc institutes in szeged have been supported by the Hungarian academy of sciences with eur 5,75 million every year as basic funding, and a similar amount has been generated from governmental and european grants. The research priorities as listed in this book are changing continuously according to the international

important components of the educational activities of the Biological research center. Phd students are active contributors to the research projects and, after receiving their Phd degrees from the university, they continue their training in laboratories abroad. several successful postdoctoral scientists return to the center and play key roles in the initiation of new competitive projects. The intellectual and infrastructural capacity of the Brc provides a unique situation to serve as a regional center both in Hungary and in the central european region.

a few years ago the Brc initiated the organization of the Biopolisz Szeged—Life Science Consortium

that integrates activities in the field of biology, medicine, biotechnology and plant breeding in the town. functional genomics and bioinformatics serve as a common platform to improve teaching and research performance. furthermore, it is essential to organize an efficient technology transfer procedure presently carried out by the Biopolisz Szeged Innovation Services Ltd. (www.biopolisz.hu). during the last years 14 spin-off companies have been founded by scientists from Brc. as a response to the grant possibilities reflecting the governmental r&d policy, applied research activities have been increased in the center. This stimulated the organization of various consortia, primarily between partners from the szeged university (Knowledge Center of Neurobiology) and from the cereal research non-Profit company (Wheat Consortia). There is a consensus that the industrial development in szeged city can be primarily based on bioindustry and biotechnology. to reach this goal, the Brc proposed the establishment of a bioincubator (Genomic Innovation Center and Szeged Bioinnovation Park). The collaborations of Brc scientists with pharmaceutical companies

bIoloGICal ReseaRCH CenTeR,

Hungarian academy of sciences

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Péter ZávODSZKY

Director (2007– ), Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences

H-1113 Budapest, Karolina út 29.

H-1518 Budapest, P.o.Box 7, H-1518, Hungary Phone: 36-1-209-3535

fax: 36-1-466-5465 e-mail: zxp@enzim.hu secretary: Ágnes szikra Phone: 36-1-279-3113

financial officer: mária Wagner Phone: 36-1-209-3536

DIReCToRs

György PóSFAI

Director (2004– ), Institute of Biochemistry, Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-653

fax: 36-62-433-506 e-mail: posfai@brc.hu

secretary: olga miklós and mónika Bali Phone: 36-62-599-654

financial officer: mónika Kordás Phone: 36-62-599-656

Pál ORMOS

Director General (2010– ), Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-613

fax: 36-62-433-133 e-mail: pormos@brc.hu foigtitk@brc.hu

Director (1994– ), Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences

secretary: andrea Gémes Phone: 36-62-599-614

Dénes DUDITS

Director General (1997–2009), Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-768

fax: 36-62-433-188 e-mail: dudits@brc.hu secretary: Zsuzsa Keczán Phone: 36-62-599-769 office manager: Judit szabad

Phone: 36-62-599-761; e-mail: szabadne@brc.hu

financial officer: anikó Hrk Phone: 36-62-599-609

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Miklós ERDÉLYI

Director (2010– ), Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-686

fax: 36-62-433-503 e-mail: erdelyi@brc.hu

secretaries: csilla soltész and marinetta Herczeg Phone: 36-62-599-657

financial officer: tünde abonyi Phone: 36-62-599-656

István RASKó

Director (1994–2009), Institute of Genetics, Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-681

fax: 36-62-433-503 e-mail: rasko@brc.hu

secretary: csilla soltész and marinetta Herczeg Phone: 36-62-599-657

financial officer: tünde abonyi Phone: 36-62-599-656

Imre vASS

Director (2000– ), Institute of Plant Biology, Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone: 36-62-599-700

fax: 36-62-433-434 e-mail: imre@brc.hu secretary: mariann Károlyi Phone: 36-62-599-714

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László vÍGH

Deputy Director General for Innovation

Biological Research Center, Hungarian Academy of Sciences

H-6726 szeged, temesvári krt. 62. H-6701 szeged, P.o. Box 521, Hungary Phone/fax: 36-62-432-048

e-mail: vigh@brc.hu secretary: mónika Bali Phone: 36-62-599-654

Spin-off CompanIes lInkeD To bRC

Company Contact persons

acheuron Hungary Ltd. tamás Letoha

avIcor Ltd. László Puskás

avIdIn Ltd. László Puskás

Biofotonika Ltd. Győző Garab

curamach Ltd. Gyula Hadlaczky

creative Labor Ltd. vilmos tubak

delta Bio 2000 Ltd. Lajos Haracska

JsW Hungary Ltd. miklós sántha

Lipidart Ltd. Zsolt török, László vígh

Pharmacoidea Ltd. tamás Letoha

Planta cosmetix Ltd. Gábor v. Horváth, dénes dudits

Therbiogen Ltd. vilmos tubak, róbert Katona

transmentIX Ltd. László Puskás

t-sejt Ltd. vilmos tubak, Éva monostori

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Institute of Biophysics

H-6726 Szeged, Temesvári krt. 62.

H-6701 Szeged, P.O. Box 521, Hungary

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Light carries momentum, i.e. it exerts force upon objects. In everyday life this force is negligible. In the microworld, however, it is different: if a micrometer-sized particle is illuminated by light of moderate in-tensity (10 mW), the light pressure may be significant. If a particle with an index of refraction higher than that of its surroundings is placed in a focussed light beam, it will be trapped in the focus. micrometer-sized particles can be trapped this way. typical forces fall in the pn range: this is just the range to manipu-late biological objects (cells, cell organelles) in water and also of forces exterted by biological machines. consequently, this is a procedure with immense po-tential in biology.

In the basic case the position of spherical objects is controlled by optical tweezers. It would offer an addi-tional degree of control if the orientation could also be controlled, thereby extending the manipulation pos-sibilities. In our laboratory we investigate the interac-tion of optical tweezers with objects of special shape.

We can produce micrometer-sized particles of arbi-trary shape by two-photon excited photopolymeriza-tion of light-hardening photoresits, and we use such objects to study new phenomena of trapping. In addi-tion, based on the trapping of such objects we develop novel tools of micromanipulation. We present two typical methods.

Particles of helical, propeller shape will rotate in optical tweezers. objects to which such rotors are at-tached can be rotated, and micromechanical machines can be driven by them. They can be used for the gen-eration of complex machines for use in biology.

If the optical tweezers is generated by linearly po-larized light, flat objects will be trapped such that they will orient along the plane of polarization. With this method objects can be oriented. If we attach a mole-cule to such a particle, we can exert or measure torque on this molecule. In this way we can twist molecules, and their torsional properties can be determined. This is very important in biology, since there are numerous processes involving rotation. for example, to access the information stored in dna it has to be untwisted, consequently information about the torsional prop-erties of dna is fundamental for understanding the encoded information. With our method we can de-termine the torsional elasticity of dna, an important parameter for the function. numerous dna/protein interactions and protein motions involving rotation can be studied by this method.

Figure 1. Twisting a macromolecule by the optical tweezers. By rotat-ing the plane of polarisation, we rotate the flat object at the end of the DNA

opTICal mICRomanIpUlaTIon

Pál ORMOS /

research Professor, Group Leader

László OROSZI / staff scientist Sándor vALKAI / staff scientist András BÚZáS / Phd student

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Optical control in microfluidics

In modern biochemical and medical diagnostical research there is a need for devices that handle small amounts of material but are able to process large numbers of samples in a short time. These requirements can be met by decreasing size, and microfluidics (lab-on-a-chip) is the development seeking solution in this direction. In the size range of these devices the physics of processes is quite different from that of the macroscopic world, consequently the solution is not merely a scaling down of known cases: development is progressing in different directions.

We believe that optical control can be very useful in microfluidics and we perform research in this area studying the processes on which future devices can be based.

using the photopolymerisation technique, we build microfluidics systems where channels and optical waveguides are integrated into a single system. Here light is used to investigate material (cells, molecules) in the channel; in addition, light is also used to manipu-late objects. for example, the fluorescence of cells can be measured using the integrated optical waveguides and selected ones are separated by the pressure of light also carried in integrated waveguides. disposable all-optical microfluidic cell sorters were built this way.

to extend optical control, we developed the con-cept of light-controlled electroosmosis. In a liquid-filled channel the surface charge of the wall is neutral-ized by opposite charges collecting at the wall that can be moved by an electric field parallel to the wall. In microchannels the total volume can be agitated this

nel wall with photosensitive material, and in this way electroosmosis can be modulated by light.

We have developed different flow control schemes: in a single channel we can turn the flow on and off. We have built a light-controlled flow switch: in a bifurcating channel, the direction of flow is selected by light. In microfluidics flow is always laminar, therefore mixing, a key process in biochemical reactions is always a problem. Light-controlled electroosmosis again offers a solution: by illuminating the light-sensitive channel wall with light of appropriate pattern, the flow pattern can be controlled within a single microchannel.

The elaborated methods are useful for studying the flow properties of microchannels, at the same time they have great potential offering methods for controlling mi-crofluidics systems by light. our aim is the development of completely light-controlled microfluidics systems.

Figure 2. Simulation of a light-controlled flow switch. The distribution of electric field and flow pattern are determined by the finite element method

Contact: pormos@brc.hu

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Bioelectronics has a double meaning in scientific lit-erature. on the one hand, as a branch of basic biophysi-cal sciences, it deals with electric phenomena appear-ing on any organization level of livappear-ing systems (a). on the other hand, as a recently developed discipline of in-formation technological science, it explores the poten-tial of biological materials for application in molecular electronics (B). These two areas of research are in close interaction not only with each other, but also with oth-er disciplines of basic and applied sciences. our main goal is to develop novel methods on integrated micro- and nanotechnological platforms for the investigation of light-induced processes in biological membranes, and utilize them in both branches of bioelectronic sci-ence. Besides its impact on basic biophysical science, this research is expected to have applications in various branches of molecular electronics.

A) Electric signals associated with

membrane transport processes

electric phenomena, ubiquitous in living systems, carry a lot of information about basic physiological processes (see, e.g., ecG or eeG) that are inaccessible to other techniques. They can all be traced back to the cellular level, namely to membrane-coupled signal- and energy transduction processes. The importance of methodological developments aiming at the detection of the associated electric signals is underpinned, e.g., by the nobel Prize given for the patch clamp technique (neher and sackmann, 1991).

However, application of the most commonly used microelectrode methods to the investigation of trans-membrane ionic currents often fails due to technical limitations, whereas alternative optical techniques still suffer from fundamental sensitivity and time resolution problems. active pump currents, therefore, are still measured on suspensions of cell organelles or cells by macroelectrode methods, in whose elabora-tion our institute in szeged played a determining role. The generalization of one of our techniques allowed the detection of intramolecular electric signals in all the three spatial dimensions.

bIoeleCTRonICs of Ion-TRanspoRTInG

membRane pRoTeIns

András DÉR /

Principal Investigator

Lajos KESZTHELYI / Professor emeritus Rudolf TóTH-BOCONáDI / staff scientist László FáBIáN / Phd student

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from the application of the technology and its com-bination with molecular dynamics simulations, we

B) Protein-based integrated optical switching

since the start of integrated electronics, the expan-sion of development has been described by “moore’s law”: the density (performance) of integrated elec-tronic circuits doubles about every 1.8 years. While this “law” has remained valid for a remarkable peri-od of 30 years, there is a general perception that the evolutionary development has reached a limit. It is agreed that future development needs revolutionary new principles. Presently, all possible candidates are explored in the search for new routes. molecular elec-tronics combined with optical data processing is re-garded as being among the most promising emerging alternative technologies.

coupling of optical data-processing devices with microelectronics, as well as sensory functions, is one of the biggest challenges in molecular electronics. suit-able nonlinear optical (nLo) materials with high sta-bility and sensitivity are being intensively researched. In addition to organic and inorganic crystals, biologi-cal molecules have also been considered for use in op-toelectronics, among which br has generated the most interest.

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goal is to elaborate br-based films supporting applica-tions of optical and optoelectronical components and devices.

Hofmeister effects

Water is the third most abundant molecule in the universe (after H and co), and the most abundant on earth. The major part of living organisms is made up of water (on each level of organization). If water is ex-tracted, proteins do not function. “Water is a matrix providing stability and flexibility of proteins at the same time.” (Philip Ball)

some unique physical-chemical properties: high electric dipole moment, network of H-bonds, fast proton exchange. according to molecular dynamics modelling, such cluster-formations are more frequent at lower than at higher temperatures:

The reason is the change of H-bond strength versus temperature. What consequences could this have on proteins? temperature change is the most straightfor-ward tool to change the strength of H-bonds; however, this has an impact on the Brownian motion of protein molecules as well. addition of salts which do not af-fect pH, and do not specifically interact with proteins might help this problem. The non-specific effects of neutral salts on protein aggregation and conformation have been known for a long time, and are called Hof-meister effects after their first investigator. according

to the investigations, the effects are dominated by ani-ons rather than catiani-ons. In 1888, Hofmeister ordered the anions according to their ability of precipitating globular proteins from water:

SO4-- > F- > CH3COO- > Cl- > Br- > I- > ClO4-, SCN- (1)

cl- has the least effect (in the middle of the row), while those on the left-hand side of (1) are called ko-smotrops (increase in aggregation: “salting out”), and the right-hand-side ones are called chaotrops (increase in solubility: “salting in”). Interestingly, the same series was found for protein conformation and activity, too: normally, kosmotrops stabilize conformation and in-crease activity, whereas chaotrops destabilize confor-mation and decrease activity. disturbingly, however, the tendency is just the opposite in some cases. such exceptions make the elaboration of a coherent theory of Hofmeister effects rather difficult. The main goal of our research is to develop and apply a comprehen-sive theory of Hofmeister effects. The starting point is that both in aggregation and conformational changes, there is a change in water-exposed protein surface area. according to our hypothesis already supported by a growing line of experimental evidence, the salt-dependence of protein-water interfacial tension holds the explanation of Hofmeister effects.

We are going to provide a microscopic interpre-tation of the effects by the investigation of protein conformational fluctuations. another goal is to use Hofmeister effects as an experimental tool to pinpoint large conformational changes during protein func-tion.

Contact: derandra@brc.hu

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Géza GROMA /

Principal Investigator

Zsuzsanna HEINER / staff scientist Ferenc SARLóS / staff scientist András MAKAI / Programmer

femtobiology is a novel branch of science, focusing on ultrafast processes in biological systems, occurring on a femtosecond (10-15 s) timescale. although com-mon biological reactions are much slower, elemen-tary molecular events, such as chemical bonding and unbonding, as well as vibrational and rotational mo-tions take place in this time range. In this context all chemistry is femtochemistry and all biology is femto-biology. By the methods of classical spectroscopy the above processes could be investigated only indirectly in the frequency domain. The advent of ultrafast lasers made possible studying these events directly in time domain, yielding detailed, previously unobtainable information. (The spectrum can be calculated from the time-evolution, but not vice versa.)

cated that these ultrafast charge motions – as sources of the familiar Hertz dipole radiation – could emit electromagnetic radiation at an experimentally detect-able level. Based on this finding, we started spectro-scopic experiments with femtosecond time-resolution to study the components of this radiation falling to the far infrared and terahertz regime. The main results of this study are the following:

Terahertz radiation of bacteriorhodopsin and its modelling by electron and proton translocation.

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others about the isomerisation of retinal, a functionally important process in the energy conversion.

also for the first time we detected coherent terahertz radiation from a biological sample by a measuring apparatus built directly for this purpose and ensuring the investigation of somewhat slower (200 fs – 5 ps) charge motions. This system made feasible to follow the complete process of the excited-state charge redistribution in retinal. In addition, in the emission signal we observed an additional component of several ps life-time, attributable to the appearance of the primary functional proton motions.

for the analysis of the above and further experi-ments, we also studied the ultrafast absorption ki-netics of native and chemically modified br samples. furthermore, we experimentally determined the opti-cal refractive index of br in a wide spectral range, and

calculated the corresponding sellmeier coefficients, describing the dispersion properties of the protein.

Further research plans, possible utilization of

the results

In our laboratory the construction of an ultrafast optical pump-probe unit is in progress, with the capa-bility of measuring absorption kinetics as well as fluo-rescence up-conversion in the time range of 100 fs – 1 ns. our aim is to operate this laboratory as a national service for solving appropriate problems in the area of both research and development.

Contact: groma@brc.hu

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Lóránd KELEMEN /

Principal Investigator

Badri PRASHAD / Phd student

recent technological developments enable the re-placement of large, complicated and expensive instru-mentation with cheaper and smaller ones. among these are the widely studied lab-on-a-chip systems ap-plying microfluidic methods. In these systems chan-nels, reactors and reservoirs are built on a microscope slide within a few mm2 area in order to carry, mix, react and detect minute volumes of sample. These microscopic labs require the application of mechani-cal devices of a few micrometer dimension.

In our lab we are developing micromachinery for microfluidic applications. The structures with a typi-cal size of less than ten micrometer and sub-micro-meter resolution are made of polymer that hardens in a pre-defined 3d shape upon illumination with fo-cused laser light. The beam consisting of femtosecond pulses initiates two-photon absorption in the polymer exclusively in the vicinity of the focal spot.

Polymerization of light-driven

microstructures

The first group of polymerized structures is actu-ated by radiation pressure when the impulse of light is transferred to the objects via reflection, thereby ini-tiating their movement. a typical example for these structures is a surface-attached micro-motor of 10µm diameter. The motor rotates on an axis when illumi-nated by a beam emerging from a light guide, also polymerized to the surface by a focused laser beam. similar wheels are intended to serve as power sources for complex structures performing various microflu-idic tasks. We are developing gear shift systems for this kind of machinery, similar to those of the mac-roscopic world. Besides the successful polymerization of various gears, we have polymerized other mechani-cal parts, such as spiral springs and tested their

per-DevelopInG aRTIfICIal mICRoTools

foR bIoloGICal applICaTIons

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Lóránd KELEMEN

DEVELOPING ARTIFICIAL MICROTOOLS FOR BIOLOGICAL APPLICATIONS

chemically functionalized microbeads. The beads en-able only translational manipulative motion. With the tools we polymerize, the optical trap will be ca-pable of manipulation with more complicated forms of motion. due to the variability of the tools’ shape, non-trivial movements such as the twisting of the sample will become possible. We tested the manipula-tion capabilities of the microtool-optical trap system in an experiment where a complex 3d superstructure was assembled, an operation which was only possi-ble with high-precision translational and rotational movements.

Electron microscopic images of the basic units polymerized for the 3D assembly experiment, and optical microscopic image of the process of the assembly

Polymerization with

modified laser beams

The most important reason for the investigation of the application of modified laser beams is the accelera-tion and simplificaaccelera-tion of the polymerizaaccelera-tion process. Here the initial single laser beam is altered by a spatial Light modulator (sLm) such that several identical la-ser beams or extended illuminated patterns are pro-jected into the sample and perform polymerization. When we create several distinct but identical focal spots, the result of this parallel-type polymerization is the same number of identical objects (a couple of tens of them). When a complex and continuous light pat-tern is focused into the sample, the polymer hardens in the same shape, without the need of scanning of the beam.

Surface activation of the

polymerized microstructures

The su8 polymer used for polymerization is an epoxy-based resin which is chemically quite inert, therefore it practically does not interact with biologi-cal objects without further treatment. In many cases this is a requirement, but for the micromanipulators designed for optical trapping applications, the inter-action is a must. for this the surface of the polymer has to be activated through one of the processes de-scribed in the literature. We are adopting the surface treatment protocols to the microstructures, equipping them with small functional groups, macromolecules, proteins, as well as metal colloids. our initial results show the coating of microrods’ and manipulators’ sur-face with the protein streptavidin in high density.

Electron and fluorescent optical microscopic image of polymerized heli-cal rods. The optiheli-cal image shows the fluorescence of the labeled protein streptavidin applied on the rods’ surface

Future plans, applications

The polymerized microstructures can be used in microchannel environment to pump or mix liquids of picoliter volumes. The manipulators made for optical trapping systems, depending on their artificially cre-ated surface quality, will enable researchers for various localized measurements on cell surfaces.

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Ilona LACZKó /

Principal Investigator

The main field of chiroptical spectroscopy is the investigation of the steric structure of biopolymers such as proteins and nucleic acids. circular dichroism (cd) is a phenomenon that results when chromo-phores in an asymmetrical environment interact with polarized light. In proteins the major optically active groups are the amide bonds of the peptide backbone. The far-uv cd is generally reflective of the backbone conformation and different secondary structures in polypeptides and proteins give characteristic far-uv cd spectra. cd is a simple and quick method, its time-scale is below the femtosecond region. due to the low time-scale, any cd spectrum can be resolved into the component spectra of the main conformer types (α-helix, β-sheet, random, etc.). This method can also be successfully combined with other vibra-tional spectroscopic methods of similar time-scale [fourier transform infrared (ftIr), raman, vibra-tional circular dichroism].

recently several studies have been performed in national and international cooperations, the most

im-– Complex-formation between antisense oligonu-cleotides (AON) and cell-penetrating peptides (CPP).

The application of aons modulating gene expression is a promising approach in medicinal therapeutics. The method is based on the delivery of aon into the cells by peptides capable of translocation through the cell membrane. The major limitation of the technique is the low efficiency of membrane translocation and targeting. Hence, the main purpose of our work in this field is screening for cPPs which are highly effective in membrane translocation and targeting. complex for-mation between cPP and aon can be followed by cd, ftIr spectroscopies and atomic force microscopy and the cPP/aon molar ratio optimal for cell penetration also can be estimated.

– Secondary and tertiary structures of different hy-drophilic enzymes. There is growing interest in the use of enzymes in aqueous organic solutions. as compared with other catalysts, enzymes are stereoselective. non-aqueous enzymology is of particular relevance when the desired reactants are poorly soluble in aqueous

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movement is a fundamental feature of living or-ganisms and systems, and the same is true for man, the most developed living system. The importance of the information content of these movements (fre-quently representing deeper, e.g. molecular or even in-tramolecular processes as well) have long been known intuitively. (Think about e.g. the effects of ethanol con-tained in alcoholic drinks). However, methods that are really suitable for the quantitative study of these mo-tions have either been developed only recently, or are now in the process of being developed.

Presently the two main (methodological) focuses of our research activities are to study human body move-ments also affected voluntarily by actigraphy, and an internal movement that is practically uncontrollable voluntarily, the continuous changes in pupil size by video-pupillography.

The actigraph usually is a wrist-wearable watch-sized device that is capable of recording movement activi-ties for a relatively long period of time (for several days or weeks), and the collected time-activity data can be transferred into a computer for the purpose of analysis. actograms carry a lot of information about the biologi-cal clock, daily routine, jet lag and various psychiatric disorders. some of their characteristics are already rou-tinely used in medical therapy, but the complex struc-ture of actograms is still mostly unexplored. The reason for this paradox is that while fourier analysis, the main tool used for the evaluation of actograms, is effective in revealing periodic components, it fails to analyse sto-chastic events—resulting in fluctuations—that seem to be prevalent on the ultradian scale (fig. 1a ).

Figure 1: A) Activity recording of a typical day. B) Integral function of the curve in A)

It is a key question in life sciences whether the stochastic dynamics of physiological rhythms are an essential feature for their function, or they are either simple consequences of environmental fluctuations or associated with certain malfunctions. our aim is to find and characterize some new, general features in human physical activity patterns that are expected to shed more light on the nature of complex physiologi-cal processes underlying activity signals.

In the case of video-pupillography, the movements of the subject’s pupil(s) are recorded for a relatively short or long period of time (usually for 1–15 minutes) by a video-camera system, and analyzed by computer programs.

HUman bIopHYsICs anD movemenT analYTICs

Zsolt TOKAJI /

Principal Investigator

András DÉR / Principal Investigator Krisztina SZABó / Phd student

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Figure 2: Probabilities of the appearance of different pupil diameters as a function of time during an 11-minute pupillographic sleepiness test in the case of an alert and a sleepy subject.

Both of these main methods were able to find and show differences between healthy state and the one accompanying affective disorder (depression). Both of these methods are extremely suitable for the urement of alertness, which is presently hardly meas-urable by other objective methods. In these alertness measurements, the strength of video-pupillography is the ability to measure the actual/short term level of alertness as well, whereas actigraphy is excellent for measuring average alertness and its periodicity.

using actigraphy, we have proved and characterized the appearance of a higher order structure of human daily activity. figure 3 shows the result of a statistical analysis of the spikes in fig. 1a.

average day (fig.1b). It is apparent that there are two characteristic slopes of this function, corresponding to the two peaks in the Pdf. during the active periods average activities are similar, and centered to a higher value (right peak in fig. 2a, or the bigger slope in fig. 1b), while it is easy to distinguish resting periods with activities close to zero (left peak in fig. 2a, or plateaus in fig. 1b). In other words, on this time scale the hu-man physical activity is quasi-binary: it is distributed such that well-defined bursts are followed by passive periods. This novel finding, generally characteristic of all cases studied, is expected to inspire new math-ematical models of human physical activity.

although video-pupillography is a relatively rap-idly performable experimental method, we have used it successfully in darkness for the detection of changes of alertness due to natural reasons (e.g. in the case of children with attention-deficit hyperactivity disorder) or drug effects (e.g. nicotine). under room light con-ditions video-pupillography can provide information about changes in the equilibrium of the sympathetic/ parasympathetic nervous activities originating either in natural causes, or resulting from drug use (e.g. methyl-phenidate) or a treatment (e.g. bright light exposure).

In our laboratories, unconventional methods (e.g., distribution function or wavelet analysis) for the evalu-ation of actigraphic and video-pupillographic data are also applied. several new methods are under develop-ment or are being currently introduced into practice.

Further research plans, possible utilization of

the results

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In the last years wonderful results have been pub-lished in the field of single molecule visualization and manipulation techniques. The atomic force microscope is increasingly used in biological research. The instru-ment was developed in the 20th century. a small tip at the end of a µm-size cantilever scans the studied sur-face. during the scan the deflection of the cantilever is proportional to the force acting between the tip and the surface. The spatial resolution of the instrument is de-termined by the sharpness of the tip. on the highest res-olution images even atoms can be distinguished. com-pared with the electron microscope a great advantage of the instrument is that biological samples can be studied in fluids, in their natural environment. This makes pos-sible to observe proteins or cells during function.

With the aid of atomic force microscope results impossible to attain by other techniques have been achieved in biological systems.

Here we present several results from our atomic force microscopy research:

– By studying oligonucleotides we observed that short sequences of nucleic acids self-assemble on mica

surface in a long chain-like formation. This self-assem-bly could have a role in the origin of life, by forming dna or rna, the information carrying molecules.

– on protein level we studied the interaction of the photosynthetic reaction center with carbon nano-tubes. The complex formed is a promising material for biotechnological applications.

– studying the proton pumping protein bacteri-orhodopsin, we measured the size change during its function, which was direct detection of the conforma-tional change of the protein.

– during the study of endothelial cells we observed that mannitol treatment influences their volume and elasticity. upon prolonged calcium treatment a change in cell shape could be detected.

– We demonstrated a difference in shape and elas-ticity between wild-type and mutant bacteria.

Further developments

The accumulated knowledge helps develop new nanotechnological materials and contributes to un-derstanding the action mechanism of drugs during medical treatments.

Contact: varo@brc.hu

bIoloGICal applICaTIon of

THe aTomIC foRCe mICRosCope

György váRó /

Principal Investigator

Zsolt SZEGLETES / staff scientist Attila Gergely vÉGH / Phd student Krisztina NAGY / Phd student

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László ZIMáNYI /

Principal Investigator

Katalin TENGER / staff scientist Petro KHOROSHYY / staff scientist

cytochromes are heme-containing proteins, which carry out diverse physiological functions, such as electron transfer, an important process in the energy metabolism of living cells. c type cyto-chromes are distinguished from other cytocyto-chromes by the covalent attachment of the heme group to the protein. The advantages of this covalent binding are so far unclear. The maturation of mitochondrial cy-tochrome c, i.e. the covalent binding of the heme co-factor is catalysed by the enzyme cytochrome c heme lyase. despite the importance and the widespread oc-currence of these proteins from yeast to human, we know little about the catalytic process or about the interaction of the two proteins and the heme. recent studies have also shown that the mitochondrial cyto-chrome c has another fundamental role besides elec-tron transfer in triggering programmed cell death (apoptosis).

c heme lyase) at a much lower efficiency than in the presence of heme lyase. However, the spontaneously matured cytochrome c has slightly different physico-chemical parameters as compared to the native pro-tein. This indicates that the function of heme lyase is not only the catalysis (acceleration) of covalent heme attachment, but also to facilitate the formation of the final, native conformation (structure) of cytochrome c. Without heme lyase the first process can take place (albeit slowly, with low efficiency), but the second one cannot.

maTURaTIon, sTRUCTURe, anD eleCTRon TRansfeR

pRopeRTIes of CYToCHRome C

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László ZIMáNYI

MATURATION, STRUCTURE, AND ELECTRON TRANSFER

PROPERTIES OF CYTOCHROME C

pole. There is, however, a basic difference between the mechanism of electronic conductance in a metal wire and in a protein. our goal is to better understand the mechanism of electronic conductance in proteins, and to study whether or not nature has optimized (and how) the conductance in certain important proteins. our experiments are mainly performed on cytochrome c. We label the surface of the cyto-chrome with a molecule which becomes an electron source after its irradiation with a short laser pulse, and which donates its electron to the heme group within the protein, and recovers the electron after-wards. We can measure the rate of electron transfer and thereby compare various regions on the protein surface as well as various prospective routes (direc-tions) within the protein in terms of electronic con-ductance. We attempt to explain the link between the efficiency of electronic conductance and the structure of the protein with model calculations. In the figure we coloured the surface of cytochrome c according to the calculated efficiency of electron transfer from the heme (in the middle) to the various regions of the surface as green, red, and blue for average, good, and poor conductance, respectively.

Further research plans, possible utilization

of the results

We plan to investigate the electron transfer routes, the efficiency of electron (and charge) transfer and its energetic utilization in the more complex but simi-larly important physiological oxidizing partner of cytochrome c, the cytochrome c oxidase protein. cy-tochrome c itself is a promising candidate as a com-ponent of biomolecule-based sensors, bioelectronic designs, therefore the understanding of its electric conductance is a high priority. Within the framework of an international collaboration we also plan to incor-porate cytochrome c into hybrid biophotonic archi-tectures – photonic crystals based on porous silicon. Thereby we expect to tune the optical properties of the semiconductor-based photonic crystals using the col-oured cytochrome protein. conversely, we intend to study the effects of the interesting nonlinear optical phenomena characteristic of the photonic crystals on the optical properties of the protein.

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Tibor PáLI /

Principal Investigator, Group Leader

Balázs SZALONTAI / senior scientist Zoltán KóTA / staff scientist Csilla FERENCZ / Phd student Erika KóNYA / technician

Proton pumping by a membranous molecular

motor, the vacuolar proton-ATPase (V-ATPase)

The v-atPase is a membrane-bound molecular ro-tary engine, which converts the chemical energy from atP hydrolysis to the rotation of the rotor domain via a torque between specific subunits. This leads to trans-membrane proton pumping in the interface between the stator and rotor domains. The v-atPase plays an important role in diseases like osteoporosis and in the metastasis of tumours. Therefore, specific inhi-bition of certain sub-classes of the v-atPase family has direct medical and pharmaceutical relevance. to date there is no atomic resolution structure of the v-atPase known and its mechanism of function is not known either.

and found that highly specific v-atPase inhibitors perturb the lipid-protein interface of the Vo domain. We localized a unique cysteine and the glutamic acid residue, essential for proton transport, which were found to face lipids.

The primary long-term objective is the better un-derstanding of structure-function relationship and the identification of functionally relevant structural changes in the engine. This project is aimed at the study of the proton pumping and atP hydrolyzing functions of the Vo and V1domains, respectively, their connection and interaction; the arrangement and interactions, also with lipids, of the Vo subunits; the rotation of the rotor domain; structural stability and the effect of structural and functional agents, e.g. spe-cific inhibitors, on all these features in intact vacuoles, vacuolar vesicles and in reconstituted v-atPase-lipid vesicles. We aim to develop the structural models of the intra-membranous a and c subunits based on our structural data, whereas the function will be

inter-speCTRosCopY-baseD sTRUCTURal bIoloGY of

bIomembRanes anD membRane pRoTeIns

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Tibor PáLI

SPECTROSCOPY-BASED STRUCTURAL BIOLOGY OF

BIOMEMBRANES AND MEMBRANE PROTEINS

for biological membranes the protein-lipid interface may be either polar in the case of surface-bound or absorbed peripheral proteins, or apolar in the case of integral trans-membrane proteins. focusing on the protein-lipid interface requires studies on the struc-ture, the dynamics and the function of both mem-brane proteins and lipids. We are also interested in this problem, because the work on membrane proteins requires they be inserted and assembled properly in the bilayer to achieve functional reconstitution. our objective is to obtain experimental data on factors guiding insertion, folding and assembly of proteins and polypeptides in membranes and on membrane surfaces. These data are used to guide and constrain molecular and physical models (as shown in the fig-ures taken from our publications). We pay increas-ing attention to theoretical approaches to membrane protein folding too. at present, we focus on three groups of proteins: trans-membrane helix (v-atPase subunits and polypeptides), beta-barrel (E. coli outer membrane proteins) and soluble proteins interact-ing with bio- and model membranes (lysozyme).

Structure prediction for a 4 trans-membrane helix electron transport membrane protein unit based on homology and structural constraints.

Bovine rhodopsin (PDB ID: 1L9H) surrounded by a single (partly hidden) shell of energy-minimised bilayer lipids.

Approaches and techniques

The working strategy for the above membrane-pro-tein systems is that structural, dynamic and thermo-dynamic data on native and reconstituted membranes are measured, during permanent control of the bio-logical function, wherever possible, using a range of biophysical techniques, which are then consistently interpreted in detailed molecular models and related to the biological function. data are obtained with a variety of techniques and their combinations. These include fourier-transform infrared (ftIr), site-spe-cific spin-labelling and spin-trapping electron para-magnetic resonance (ePr), polarized attenuated total internal reflection ftIr, uv, visible and fluorescence spectroscopy; high-sensitivity differential scanning calorimetry (dsc); theory and computation (physical models, spectrum simulations and molecular model-ling). This approach can be termed as function-con-trolled spectroscopy-based structural biology.

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Alajos BÉRCZI /

Principal Investigator

In 1971 scandinavian researchers discovered a

b-type cytochrome in bovine chromaffin granule membranes that (1) participated in trans-membrane electron transport but was not a member of the mito-chondrial electron transport chain, (2) was essential for the synthesis of neurotransmitters, and (3) needed the presence of ascorbate (vitamin c) for its biological activity. The newly identified protein was named cyto-chrome b561 (cyt-b561) protein.

It has recently become obvious that proteins simi-lar to the bovine chromaffin granule cyt-b561 protein

protein family. The electron acceptor is ascorbate free radical (aox≡asc*) for the chromaffin granule

cyt-b561; however, the electron acceptors of the other

cyt-b561 proteins are not known. at present 5 members of the cyt-b561 protein family are known and charac-terized in some detail. one major aim of the present research is to identify the electron acceptors for and the physical-chemical properties of the new members of cyt-b561 protein family, since this information is essential and needed for understanding the biological function of the newly discovered cyt-b561 proteins.

It is already evident that members of the cyt-b561 protein family take part in vitamin c metabolism. Preliminary laboratory experiments suggest that (1) some cyt-b561 proteins also play a role in fe-metab-olism, and (2) one member of the cyt-b561 protein family is expressed with other tumour suppressor proteins in cancerous cells and thus participates in their elimination.

Future possibilities

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Without membranes, there are no living organ-isms. membranes separate the cells from the outside world, but at the same time membranes embed those proteins, which sense the signals of the outside world and perform material transport through them. In the interiors of the cells, the generation of energy takes place in membranes, including the primary energy source for all forms of life on earth, photosynthesis. after sequencing the complete genome of several or-ganisms, it seems that about 30% of their sequences encode membrane proteins. In contrast, from the sev-eral tens of thousands of proteins whose 3d structure is known in atomic resolution, only a few dozens are membrane proteins. The reason for this discrepancy between abundance and knowledge is that membranes are very complex assemblies, where proteins and lip-ids together form functional units. The liplip-ids supply the insulation capacity of the membranes, and their hydrophobic double layer provides the working con-ditions for membrane proteins. Isolated membrane proteins are therefore very difficult, and frequently practically impossible to study, and there is always the intriguing question: to what extent the obtained struc-tures, features correlate with those existing under in-membrane conditions.

for the resolution of this problem, two interrelated lines of research can be pursued. on the one hand, whole, possibly intact biological membranes can be studied as they are, by applying methods selectively sensitive for different membrane properties. on the other hand, model systems can be developed which, by mimicking natural conditions as close as possible, permit the study of individual components or particu-lar processes. Then, by compiling the data obtained by

the two approaches, one may hope to obtain a com-prehensive picture of the lipid-protein interactions in biological membranes.

according to this strategy, we apply and further de-velop the perfectly non-invasive fourier transform in-frared (ftIr) spectroscopy in biological membranes. This method has the advantage of having the lipid- and protein-related spectral regions well separated. Thus, both lipids and proteins can be studied individ-ually and, furthermore, via the correlations between changes in the lipid- and protein-related regions the lipid-protein interaction in the membranes can also be addressed.

Figure 1: Schematic view of the attenuated total reflection (ATR) mode of the Fourier transform infrared (FTIR) measurement. PEI – a positively charged polyelectrolyte, which adheres very well to surfac-es. PGA – Poly(glutamic acid) – a negatively charged poly amino acid; PLL – Poly(lysine) – a positively charged poly amino acid.

In the other direction, developing better model sys-tems, we use a nano-technological approach. We build protein-like surfaces layer-by-layer from oppositely charged polyelectrolytes. Thus, we can obtain surfac-es, which mimic the cytoskeleton of the cells. Then, biological and model membranes can be adsorbed

lIpID-pRoTeIn InTeRaCTIons In bIoloGICal

anD moDel sYsTems

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onto these surfaces. In this way, transport processes and lipid-protein interactions can be studied on a stable support on both fully natural membranes and “hybrid” systems built from natural and synthetic components. Infrared spectroscopy also plays an im-portant role in these studies, since ftIr spectroscopy when used in attenuated total reflection mode (atr) (which means that we measure the adsorbed sample on the surface of an internal total reflection element) can follow the structural changes associated step-by-step with the buildup of the polyelectrolyte film and the adsorption of any further component.

concerning the complexity of biological membranes and the need for developing new techniques for their study, at the beginning our research is purely basic re-search, and we are in this phase now. We keep in mind, however, that detailed knowledge and effective experi-mental techniques can have important bio-medical applications. Therefore, when selecting the systems for study, we consider biologically, medically important ob-jects (selected lipid species, ion-channel proteins, etc.).

Contact: balazs@brc.hu

Balázs SZALONTAI

LIPID-PROTEIN INTERACTIONS IN BIOLOGICAL

AND MODEL SYSTEMS

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1. Structure and function of redox proteins

redox proteins play an important role in the bio-energetic processes of living organisms. energy pro-ducing, utilizing, transforming processes are present in all living systems; the research of redox proteins can hardly be overestimated. most of the redox proteins contain some kind of metal (iron, copper, etc.).

our group isolates proteins from the purple, sul-phur, photosynthetic organism Thiocapsa roseoper-sicina. several metal-containing and redox active proteins have already been purified. The long-time favourite and most widely investigated protein is Hyn

hydrogenase (see next section). We have also identified and characterized a number of other metal-containing proteins, including a flavocytochrome, a cytochrome c4 (yet unknown from photosynthetic organism) and

a blue copper protein. We have determined their char-acteristics (redox potential, molecular mass, etc.).

despite the fact that the organism does not survive temperatures above 30 °c, the cytochrome c4 is stable and functional at high temperatures (up to 60 °c). The protein undergoes conformational changes upon in-creases in temperature. These conformational changes are reversible under anaerobic conditions and irre-versible if oxygen is present.

Development and exploitation of the results

The investigation of these proteins helps to under-stand the temperature tolerance of the proteins. on the other hand the techniques developed in the course of research are also important and can be used for other research. The most important technique is a new

pro-tein-sequencing methodology, which was developed in cooperation with an american ms group in texas. The protein sequence is determined with the help of a mass spectrometer. usually the protein sequence is determined by determining the nucleic acid sequence and translating it to protein sequence. It is a quite time-consuming, complicated and not always success-ful method for genetically unmapped organisms (and most organisms belong to this group). furthermore the sequence does not necessarily fit the sequence of the working protein. our new method is competitive with sequence determination from the gene, and we hope that with some improvement it will be possible to decrease the required amount of protein to the level of two-dimensional gel spots. The drawback of this method is that it needs expensive mass spectrometers.

2. Investigation of autocatalytic

and oscillating enzyme reactions

autocatalytic reactions are quite common in living systems; they are very easy to observe macroscopi-cally. Proliferation and reproduction are typical auto-catalytic processes. In order to produce posteriors, in addition to food one or two parents are necessary.

autocatalytic processes are easily recognized by their characteristic spatial patterns. In the absence of other disturbing effects the reaction fronts are spher-ical (or, in the case of flat reaction arrangements, circular). The radii of the objects are continuously increasing in time (see the figure). The spread of hu-mankind on earth has a very similar pattern.

although autocatalytic reactions are common in living systems, they are quite rare in elementary

re-sTRUCTURe anD fUnCTIon of ReDox meTallopRoTeIns

Csaba BAGYINKA /

Principal Investigator

Gabriella PANKOTAI-BODó / staff scientist Rui Miguel MAMEDE BRANCA / staff scientist Judit ŐSZ / staff scientist

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actions. We investigate such elementary reactions on bio-molecules and enzymes.

The main object of our study is the hydrogenase enzyme. It can be found mostly in prokaryotes and ar-chae, but some eukaryotes might contain it as well.

Hydrogenase catalyzes a very simple reaction: it splits hydrogen gas into protons and electrons and, as all catalysts, it works in the reverse direction as well, i.e. it produces hydrogen gas from electrons and protons. The enzyme contains metal cofactors, iron, and some hydrogenases also contain nickel. We have found that during the enzyme reaction there is at least one autocatalytic step involved.

The autocatalytic reaction of hydrogenase in thin layer.

Development and exploitation of the results

autocatalytic reactions are also present in other biological processes. the development of “prion”

diseases (creutzfeldt-Jakob disease, scrapie, bovine spongiform encephalopathy, etc.) is also described as an autocatalytic process. In the case of prion proteins, however, the investigation of the kinet-ics of the autocatalytic reaction (how the reaction proceeds, what happens during the reaction) is very difficult. since the reaction of hydrogenase is easy to follow and the concentration of different com-ponents can easily be changed, there is hope that the information and the techniques developed in research on this reaction can be applied to the case of prion reactions.

the autocatalytic reaction of hydrogenase has biotechnological importance. We do not think that with the help of hydrogenase bio-hydrogen would be produced in industrial quantity by splitting the water. But we do think that hydrogenase can be uti-lized as a key constituent of fuel cells. It might sub-stitute the expensive platinum, because it is cheaper, and it doesn’t have to be mined, because it can be produced utilizing solar energy. for its utilization, however, it is essential to precisely know the reac-tion kinetics.

Contact: csaba@brc.hu

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one paradoxical consequence of the increase of the life expectancy of mankind is the increase of the incidence of those intractable diseases whose risk fac-tor increases with age. The degenerative alterations of the nervous system, associated with such diseases, are at the top of this list. a disease of the motor system, amyotrophic lateral sclerosis (aLs), which currently does not have a cure, deserves special attention com-pared to the other neurodegenerative disorders. first, since it is relatively easy to design predictive clinical trials in this case, this disease is preferred for drug testing by the pharmaceutical companies compared to the other diseases in which the putative drugs are ex-pected to be distributed in a larger market (e.g. among alzheimer’s disease patients).

a reason for this policy is based on the document-ed similarity in the majority of destructive processes observed in the degenerative diseases of the nervous system, thus the results can be generalized. secondly, since the disease also affects the motor axon terminals

located in the skeletal muscle, these parts of the nerv-ous system can be sampled without any complications in the patients, and the results of these tests are easily comparable to those obtained from the animal experi-ments. on the other hand, since neither the cause, nor the exact pathomechanism of the disease is known, explorative basic research is still necessary.

The research of the mechanisms of aLs is built around the anatomy of the voluntary motor system: the pyramidal cells project to the lower motor neurons, which are located in distinct anatomical regions of the brainstem and the ventral horn of the spinal cord. The innervating projections of the lower motor neurons, called peripheral nerves are terminating at the skeletal muscles of the body. during the disease, the progres-sive destruction of the motor neurons causes the loss of innervation of the muscles and leads to the death of the patients if vital muscles are affected. although the complete machinery leading to the destruction of the motor neurons is still unknown, some of the details are already disclosed. such mechanisms are:

exhaus-neURonal DeGeneRaTIon

László SIKLóS /

Principal Investigator, Group Leader

Ngo Thi Kue DUNG / staff scientist Melinda PAIZS / staff scientist

Erika MARáCZINÉ RáCZ / technician Szabolcs áBRAHáM / technician

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tion of the motor neurons due to over-excitation, dam-age of protein and membranous constituents of motor neurons due to the insufficient antioxidant defense, development of deformed cellular structure due to in-adequate assembly of the cytoskeleton, immunologi-cal and auto-immune reactions, and self- amplifying toxic processes due to the impairment of the calcium homeostasis. according to most recent research data, we are aware that during all these pathological proc-esses not only the motor neurons are involved, but the neighbouring astrocytic and microglial cells also take an active role.

our recent studies are aimed at unravelling the role of the stability of calcium homeostasis of the motor neurons during degeneration, and to examine if the neighbouring astrocytic and microglial cells could

modify their resistance. methodically, our experi-ments are based on in vivo, i.e. animal experiexperi-ments: either production of suitable transgenic animals, or pharmacological treatment or surgery of wild-type and/or transgenic animals would help to answer our specific questions. detection will be mainly by elec-tron microscopic techniques. This technique, in ad-dition to ultra-structural resolution, could provide information on the composition of the sample under investigation on the micro-analytical scale.

In our light microscopic studies we intend to in-vestigate—in a similar experimental paradigm—the structural and molecular changes in the neighbour-ing cells. as an example: after characterizneighbour-ing the mor-phology and distribution of the resting microglial cells in the spinal cord (left panel), motor neurons are experimentally injured and start to express a distress signal (red colour, middle panel), then the change in the microglial population could be determined (mid-dle panel green signal; right panel).

Contact: siklos@brc.hu

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our understanding of the role of gonadal steroids is actually undergoing a major reassessment. experi-mental data indicate that apart from being neuroen-docrine regulators, these hormones exert a variety of morphogenetic and organizing effects in the nervous system. They influence the development of certain brain regions and also affect the differentiation of spe-cific neuronal and glial populations and the establish-ment of synaptic connectivity. moreover, it has been discovered that these hormones can be produced in the central nervous system as well and they act locally, resulting in well-defined structural and functional changes. These data have led to the birth of two cat-egories: neurosteroids, i. e. brain-derived steroids and neuroactive steroids, i.e. steroids acting on the brain but produced in the gonads.

The research interest of the Group is focused on the role of gonadal steroids in neuro-glial plasticity, more specifically on the mechanism of neurodegeneration and neuroprotection. our aim is to perform basic research to study the cellular and molecular mecha-nisms responsible for the neuroprotective effects of certain neurosteroids, namely estrogen and one of its precursors, dehydroepiandrosterone (dHea). at this phase of research the aim is not the develop-ment of new drugs, but on the long run we focus on the possible therapeutic use of neuroactive steroids and neurosteroids. according to our opinion the de-tailed knowledge of hormonal action may provide new pieces of evidence for the understanding of the regen-erative capacity of the central nervous system. on the basis of our results we may work out a model system

to study the neuroprotective effect of new synthetic steroids. any knowledge in this field will be especially useful for the elder generation, because at the time of approaching senescence there is a significant decrease in the plasma concentrations of dHea, IGf-I and es-tradiol (and its precursor testosterone) both in women and men, consequently the neuroprotective effect of these molecules is also diminished.

classical studies supported the idea of extensive neuronal loss even in normal ageing, and neuronal degeneration in the hippocampus and cerebral cor-tex was thought to contribute directly to age-related cognitive deficits. It is now recognised that biological

neURonal plasTICITY anD neURopRoTeCTIon:

Role of HoRmones anD neURosTeRoIDs

árpád PáRDUCZ /

Principal Investigator

Zsófia HOYK / staff scientist Tibor HAJSZáN / staff scientist Eszter CSáKváRI / staff scientist Andrea GYENES / staff scientist

Figure

Figure 2:  Probabilities of the appearance of different pupil diameters as  a function of time during an 11-minute pupillographic sleepiness test in  the case of an alert and a sleepy subject.
Figure  1:  Schematic  view  of  the  attenuated  total  reflection  (ATR)  mode  of  the  Fourier  transform  infrared  (FTIR)  measurement
Figure  1:  Co-culture  model  of  the  BBB  (Nakagawa,  Deli  et  al.,  2007;
Figure 3:  A2058 melanoma cells (green) in contact with brain endothe- endothe-lial cells (red: ZO-1 staining).
+7

References

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