2012 - BRC

SCIENTIFIC PUBLICATIONS OF THE INSTITUTE OF BIOPHYSICS OF
THE HUNGARIAN ACADEMY OF SCIENCES
2012
DEPARTMENT OF PROTEIN DYNAMICS, BIOLOGICAL ENERGY
CONVERSION AND NANOBIOTECHNOLOGY
1.
Aekbote BL, Jacak J, Schütz GJ, Csányi E, Szegletes Z, Ormos P, Kelemen L
Aminosilane-based functionalization of two-photon polymerized 3D SU-8
microstructures
European Polymer Journal, 48:1745-1754(2012)
2.
Bovino FA, Larciprete MC, Sibilia C, Giardina M, Váró G, Gergely C
Nonlinear ellipsometry by second harmonic generation
Nonlinear Optics
(Eds.: N. Kamanina) Rijeka: InTech, pp. 117-132(2012)
3.
Bovino FA, Larciprete MC, Sibilia C, Váró G, Gergely C
Evidence of multipolar response of Bacteriorhodopsin by noncollinear
second harmonic generation
Optics Express, 20:14621-14631(2012)
4.
Buzás A, Geretovszky Z
Nanosecond laser-induced selective removal of the active layer of
CuInGaSe2 solar cells by stress-assisted ablation
Physical Review B, 85:245304(2012)
5.
Búzás A, Kelemen L, Mathesz A, Oroszi L, Vizsnyiczai G, Vicsek T, Ormos P
Light sailboats: Laser driven autonomous microrobots
Applied Physics Letters, 101:041111(2012)
6.
Chappaz-Gillot C, Marek PL, Blaive BJ, Canard G, Bürck J, Garab G, Hahn H,
Jávorfi T, Kelemen L, Krupke R, Mössinger D, Ormos P, Reddy CM,
Roussel C, Steinbach G, Szabó M, Ulrich AS, Vanthuyne N, Vijayaraghavan A,
Zupcanova A, Balaban TS
Anisotropic organization and microscopic manipulation of self-assembling
synthetic porphyrin microrods that mimic chlorosomes: Bacterial
light-harvesting systems
Journal of the American Chemical Society, 134:944-954(2012)
7.
Di Leonardo R, Búzás A, Kelemen L, Vizsnyiczai G, Oroszi L, Ormos P
Hydrodynamic synchronization of light driven microrotors
Physical Review Letters, 109:034104(2012)
I
8.
Groma GI, Heiner Z, Makai A, Sarlós F
Estimation of kinetic parameters from time-resolved fluorescence data:
A compressed sensing approach
The Royal Society of Chemistry Advances, 2:11481-11490(2012)
9.
Hollósi M, Vass E, Szilvágyi G, Jakas A, Laczkó I
Structure analysis of proteins, peptides and metal complexes by vibrational
circular dichroism
Arkivoc, 291-300(2012)
10.
Keszthelyi L
A physical hypothesis for learning
Journal of Biological Physics and Chemistry, 12:54-55(2012)
11.
Kincses A, Tóth-Boconádi R, Dér A
2D measurement of ion currents associated to the signal transduction of the
phototactic alga Chlamydomonas reinhardtii
Journal of Photochemistry and Photobiology B: Biology, 114:147-152(2012)
12.
Marton A, Vizler C, Kusz E, Temesfoi V, Szathmary Z, Nagy K, Szegletes Z,
Varo G, Siklos L, Katona RL, Tubak V, Howard OMZ, Duda E,
Minarovits J, Nagy K, Buzas K
Melanoma cell-derived exosomes alter macrophage and dendritic cell
functions in vitro
Immunology Letters, 148:34-38(2012)
13.
Palima D, Bañas AR, Vizsnyiczai G, Kelemen L, Ormos P, Glückstad J
Wave-guided optical waveguides
Optics Express, 20:2004-2014(2012)
14.
Simon LM, Laczkó I, Demcsák A, Tóth D, Kotormán M, Fülöp L
The formation of amyloid-like fibrils of alpha-chymotrypsin in
different aqueous organic solvents
Protein and Peptide Letters, 19:544-550(2012)
15.
Váró G, Szegletes Z
Artificial and natural membranes
Atomic Force Microscopy Investigations into Biology – From Cell to Protein
(Eds.: C. L. Frewin), Rijeka:InTech, pp. 219-232(2012)
16.
Végh AG, Fazakas C, Nagy K, Wilhelm I, Molnár J, Krizbai IA,
Szegletes Z, Váró G
Adhesion and stress relaxation forces between melanoma and cerebral
endothelial cells
European Biophysics Journal, 41:139-145(2012)
II
DEPARTMENT OF MEMBRANE STRUCTURE AND DYNAMICS
17.
Nagy K, Váró G, Szalontai B
Kappa-Casein terminates casein micelle build-up by its "soft"
secondary structure
European Biophysics Journal, 41:959-968(2012)
18.
Szalontai B, Domonkos I, Gombos Z
The role of membrane structure in acclimation to low-temperature stress
Photosynthesis: Plastid Biology, Energy Conversion and Carbon Assimilation
(Eds.: J. J. Eaton-Rye, B. C. Tripathy, T. D. Sharkey) Berlin, New York:
SPRINGER, pp. 233-250(2012)
19.
Szűts V, Ötvös F, Dézsi L, Vágvölgyi C, Szalontai B, Dobrzynski H, Boyett M, Zhang
H, Papp JG, Varró A, Benyhe S, Erdélyi L
What have we learned from two-pore potassium channels? Their molecular
configuration and function in the human heart
Acta Biologica Szegediensis, 56:93-107(2012)
DEPARTMENT OF MOLECULAR NEUROBIOLOGY
20.
Adalbert R, Morreale G, Paizs M, Conforti L, Walker SA, Roderick HL,
Bootman MD, Siklós L, Coleman MP
Intra-axonal calcium changes after axotomy in wild-type and slow
wallerian degeneration axons
Neuroscience, 225:44-54(2012)
21.
Cardoso FL, Kittel A, Veszelka S, Palmela I, Tóth A, Brites D, Deli MA, Brito MA
Exposure to lipopolysaccharide and/or unconjugated bilirubin impair
the integrity and function of brain microvascular endothelial cells
PLoS ONE, 7:e35919(2012)
22.
Hellinger E, Veszelka S, Tóth AE, Walter F, Kittel A, Bakk ML, Tihanyi K,
Háda V, Nakagawa S, Thuy DHD, Niwa M, Deli MA, Vastag M
Comparison of brain capillary endothelial cell-based and epithelial
(MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate
blood-brain barrier penetration models
European Journal of Pharmaceutics and Biopharmaceutics,
82:340-351(2012)
23.
Hornok V, Bujdosó T, Toldi J, Nagy K, Demeter I, Fazakas C, Krizbai I,
Vécsei L, Dékány I
Preparation and properties of nanoscale containers for biomedical
application in drug delivery: preliminary studies with kynurenic acid
Journal of Neural Transmission, 119:115-121(2012)
III
24.
Jańczewski D, Song J, Csányi E, Kiss L, Blazsó P, Katona RL, Deli MA,
Gros G, Xu J, Vancso GJ
Organometallic polymeric carriers for redox triggered release
of molecular payloads
Journal of Materials Chemistry, 22:6429-6435(2012)
25.
Kang HJ, Voleti B, Hajszan T, Rajkowska G, Stockmeier CA, Licznerski P,
Lepack A, Majik MS, Jeong LS, Banasr M, Son H, Duman RS
Decreased expression of synapse-related genes and loss of synapses in
major depressive disorder
Nature Medicine, 18:1413-1419(2012)
26.
Krizbai I, Wilhelm I
A sejtek közötti kommunikáció útjai
Korunk, 4:48-54(2012)
27.
Kürti L, Veszelka S, Bocsik A, Ngo TKD, Ózsvári B, Puskás LG, Kittel A,
Szabó-Révész P, Deli MA
The effect of sucrose esters on a culture model of the nasal barrier
Toxicology in Vitro, 26:445-454(2012)
28.
Mallareddy JR, Tóth G, Fazakas C, Molnár J, Nagyőszi P, Lipkowski AW,
Krizbai IA, Wilhelm I
Transport characteristics of endomorphin-2 analogues in brain capillary
endothelial cells
Chemical Biology & Drug Design, 79:507-513(2012)
29.
Miettinen R, Hajszan T, Riedel A, Szigeti-Buck K, Leranth C
Estimation of the total number of hippocampal CA1 pyramidal neurons:
New methodology applied to helpless rats
Journal of Neuroscience Methods, 205:130-138(2012)
30.
Ramirez SH, Haskó J, Skuba A, Fan S, Dykstra H, McCormick R, Reichenbach N,
Krizbai I, Mahadevan A, Zhang M, Tuma R, Son YJ, Persidsky Y
Activation of cannabinoid receptor 2 attenuates leukocyte-endothelial
cell interactions and blood-brain barrier dysfunction under
inflammatory conditions
The Journal of Neuroscience, 32:4004-4016(2012)
31.
Sajben-Nagy E, Maróti G, Kredics L, Horváth B, Párducz A, Vágvölgyi C,
Manczinger L
Isolation of new Pseudomonas tolaasii bacteriophages and genomic
investigation of the lytic phage BF7
FEMS Microbiology Letters, 332:162-169(2012)
IV
32.
Troakes C, Maekawa S, Wijesekera L, Rogelj B, Siklós L, Bell C, Smith B,
Newhouse S, Vance C, Johnson L, Hortobágyi T, Shatunov A, Al-Chalabi A,
Leigh N, Shaw CE, King A, Al-Sarraj S
An MND/ALS phenotype associated with C9orf72 repeat expansion:
Abundant p62-positive, TDP-43-negative inclusions in cerebral cortex,
hippocampus and cerebellum but without associated cognitive decline
Neuropathology, 32:505-514(2012)
33.
Ujhelyi Z, Fenyvesi F, Váradi J, Fehér P, Kiss T, Veszelka S, Deli M,
Vecsernyés M, Bácskay I
Evaluation of cytotoxicity of surfactants used in self-micro emulsifying
drug delivery systems and their effects on paracellular transport in Caco-2
cell monolayer
European Journal of Pharmaceutical Sciences, 47:564-573(2012)
34.
Wilhelm I, Krizbai I
Molekuláris forródrótok
Korunk, 4:43-47(2012)
DEPARTMENT OF BIOPHYSICS OF REDOX PROTEINS
35.
Dömötör D, Becságh P, Rákhely G, Schneider G, Kovács T
Complete Genomic Sequence of Erwinia amylovora Phage PhiEaH2
Journal of Virology, 86:10899(2012)
36.
Fülöp A, Béres R, Tengölics R, Rákhely G, Kovács KL
Relationship between PHA and hydrogen metabolism in the purple sulfur
phototrophic bacterium Thiocapsa roseopersicina BBS
International Journal of Hydrogen Energy, 37:4915-4924(2012)
37.
Hajdu K, Gergely C, Martin M, Cloitre T, Zimányi L, Tenger K,
Khoroshyy P, Palestino G, Agarwal V, Hernádi K, Németh Z, Nagy L
Porous Silicon/Photosynthetic Reaction Center Hybrid Nanostructure
Langmuir, 28:11866-11873(2012)
38.
Hajdu K, Gergely C, Martin M, Zimányi L, Agarwal V, Palestino G,
Hernádi K, Németh Z, Nagy L
Light-harvesting bio-nanomaterial using porous silicon and photosynthetic
reaction center
Nanoscale Research Letters, 7:1-6(2012)
39.
Leitgeb B
Conformational similarities and dissimilarities between the stereoisomeric
forms of endomorphin-2
Chemical Biology & Drug Design, 79:313-325(2012)
V
40.
Leitgeb B
Spatial relationships between the pharmacophores of endomorphin-2:
A comparative study of stereoisomers
Central European Journal of Chemistry, 10:1791-1798(2012)
41.
Leitgeb B, Sokolova V, Schäfer E, Viczián A
Effects of missense mutation on structure and function of photoreceptor
Plant Signaling & Behavior, 7:589-591(2012)
42.
Schkolnik G, Utesch T, Salewski J, Tenger K, Millo D, Kranich A, Zebger I,
Schulz C, Zimányi L, Rákhely G, Mroginski MA, Hildebrandt P
Mapping local electric fields in proteins at biomimetic interfaces
Chemical Communications, 48:70-72(2012)
43.
Szőri-Dorogházi E, Maróti G, Szőri M, Nyilasi A, Rákhely G, Kovács KL
Analyses of the large subunit histidine-rich motif expose an alternative
proton transfer pathway in [nife] hydrogenases
PLoS ONE, 7:e34666(2012)
44.
Wirth R, Kovács E, Maróti G, Bagi Z, Rákhely G, Kovács KL
Characterization of a biogas-producing microbial community by short-read
next generation DNA sequencing
Biotechnology for Biofuels, 5:1-16(2012)
GROUP OF BIOINFORMATICS
45.
Bihary D, Kerényi A, Gelencsér Z, Netotea S, Kertész-Farkas A,
Venturi V, Pongor S
Simulation of communication and cooperation in multispecies bacterial
communities with an agent based model
Scalable Computing: Practice and Experience, 13:21-28(2012)
46.
Gelencsér Z, Choudhary KS, Coutinho BG, Hudaiberdiev S, Galbáts B,
Venturi V, Pongor S
Classifying the topology of AHL-driven quorum sensing circuits in
proteobacterial genomes
Sensors, 12:5432-5444(2012)
47.
Kertész-Farkas A, Reiz B, Myers MP, Pongor S
Database searching in mass spectrometry based proteomics
Current Bioinformatics, 7:221-230(2012)
48.
Reiz B, Kertész-Farkas A, Pongor S, Myers MP
Data preprocessing and filtering in mass spectrometry based proteomics
Current Bioinformatics, 7:212-220(2012)
VI
European Polymer Journal 48 (2012) 1745–1754
Contents lists available at SciVerse ScienceDirect
European Polymer Journal
journal homepage: www.elsevier.com/locate/europolj
Macromolecular Nanotechnology
Aminosilane-based functionalization of two-photon polymerized 3D
SU-8 microstructures
Badri Lakshmanrao Aekbote a, Jaroslaw Jacak b, Gerhard J. Schütz b,1, Erzsébet Csányi c,
Zsolt Szegletes a, Pál Ormos a, Lóránd Kelemen a,⇑
b
c
Biological Research Centre, Hungarian Academy of Sciences, Institute of Biophysics, Temesvári krt. 62, Szeged H-6726, Hungary
Biophysics Institute, Johannes Kepler University Linz, Altenbergerstrasse 69, A-4040 Linz, Austria
University of Szeged, Faculty of Pharmacy, Department of Pharmaceutical Technology, Eötvös u. 6, Szeged H-6721, Hungary
a r t i c l e
i n f o
Article history:
Received 9 January 2012
Received in revised form 4 June 2012
Accepted 18 June 2012
Available online 26 June 2012
Keywords:
Two-photon polymerization
3D microstructures
Optical trap
Surface functionalization
Aminosilane treatment
Gold nanoparticle
a b s t r a c t
There is an increasing interest in functionalized complex 3D microstructures with submicrometer features for micro- and nanotechnology applications in biology. Depending
primarily on the material of the structures various methods exist to create functional layers
of simple chemical groups, biological macromolecules or metal nanoparticles. Here an
effective coating method is demonstrated and evaluated on SU-8 based 3D microstructures
made by two-photon polymerization. Protein streptavidin and gold nanoparticles (NP)
were bound to the microstructures utilizing acid treatment-mediated csilane chemistry.
The protein surface density, quantified with single molecule fluorescence microscopy
revealed that the protein forms a third of a monolayer on the two-photon polymerized
structures. The surface coverage of the gold NPs on the microstructures was simply controlled with a single parameter. The possible degrading effect of the acid treatment on
the sub-micrometer features of the TPP microstructures was analyzed. Our results show
that the silane chemistry-based method, used earlier for the functionalization of large-area
surfaces can effectively be adapted to coat two-photon polymerized SU-8 microstructures
with sub-micrometer features.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Microfabrication using two-photon polymerization
(TPP) can produce a wide range of devices for biological
applications. Polymeric materials of different chemical
characteristics, such as ORMOCER, SU-8, S1813, and PEGda
have been used to create 2D and 3D micro and nanostructures of arbitrary complexity [1–4]. Many applications,
especially in sensory or tissue engineering, require the
functionalization of the microstructure’s surface. Depending on the goal, the coating can consist of small functional
groups, larger biomolecules (protein, DNA) or even metal
⇑ Corresponding author. Tel.: +36 62 599600x419; fax: +36 62 433133.
E-mail address: [email protected] (L. Kelemen).
Present address: Institute of Applied Physics, Vienna University of
Technology, Wiedner Hauptstr. 8, 1040 Vienna, Austria.
1
0014-3057/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.eurpolymj.2012.06.011
nanoparticles. The coating strategy depends primarily on
the material of the photoresist used to make the microstructures: acrylate [5–7], inorganic/organic hybrid polymers (ORMOCER) [8], or the epoxy based SU-8 [9,10]. It
also depends on the material of the coating: different linker molecules are needed for metal nanoparticle coatings
and for specific or even non-specific protein coatings.
SU-8 is a low-cost epoxy-based negative photoresist
that has found widespread use in microfabrication and
MEMS [11–14]. It is so primarily due to its mechanical
rigidity, chemical resistance to a wide range of solvents,
being optically transparent and not cytotoxic [15–17].
The existing coating strategies are numerous, often taking
advantage of the opening of the epoxide groups that remain
unreacted during cross linking [9]. Wang and coauthors
showed UV-illumination mediated covalent linking of various monomers onto the SU-8 without any pre-treatment
MACROMOLECULAR NANOTECHNOLOGY
a
5
Nonlinear Ellipsometry by
Second Harmonic Generation
Fabio Antonio Bovino1, Maria Cristina Larciprete2, Concita Sibilia2
Maurizio Giardina1, G. Váró3 and C. Gergely4
1Quantum Optics Lab Selex-Sistemi Integrati, Genova, Italy
of Basic and Applied Sciences in Engineering, Sapienza University, Rome,
3Institute of Biophysics, Biological Research Center,
Hungarian Academy of Sciences, Szeged,
4Montpellier University, Charles Coulomb Laboratory UMR 5221, Montpellier,
1,2Italy
3Hungary
4France
2Department
1. Introduction
Among the different nonlinear optical processes, second harmonic generation (SHG) is one
of the most investigated. Briefly, polarization in a dielectric material can be expanded in
terms of applied electric field. Second harmonic generation corresponds to an optical
process of coherent radiation from electric-dipoles forming in the nonlinear optical material.
In particular, SHG is related to the second term of the polarization expansion, thus it can be
obtained only in materials which are noncentrosymmetric i.e. posses no centre of inversion
symmetry. From the experimental point of view, the frequency of the incoming –
fundamental- beam, , is doubled by the second order optical susceptibility ijk(2) of the
material. The SHG processes, along with the structure of the nonlinear optical tensor, ijk(2),
are strongly dependent on the crystalline structure of the material, thus by choosing the
appropriate polarization state for the fundamental beam, different amplitude and
polarization state of the nonlinear optical response can be selectively addressed.
As a consequence, several experimental techniques have been developed, for the
determination of the different non-zero components of the third rank tensor ijk(2), with
reference to a well-characterized sample. The Maker fringes technique (Maker et al, 1962),
which is based on the investigation of oscillations of the SH intensity by changing the crystal
thickness, has been without doubt the most employed. Briefly, this technique consists in
measuring the SH signal transmitted trough the nonlinear crystal as a function of the
fundamental beam incidence angle, which is continuously varied by placing the sample
onto a rotation stage. The polarization states of both fundamental and generated beams are
selected by rotating a half-wave plate (polarizer) and a linear polarizer (analyzer),
respectively. On a reference line, a small fraction of the fundamental beam is usually sent
onto a reference crystal, which is hold at a fixed incidence angle, in order to minimize the
influence of laser energy fluctuations. On the measurement line, the second harmonic signal
Evidence of multipolar response of
Bacteriorhodopsin by noncollinear second
harmonic generation
F. A. Bovino,1,* M. C. Larciprete,2 C. Sibilia,2 G. Váró,3 and C. Gergely4,5
1
Quantum Optics Lab., Selex-SI Via Puccini 2 Genova, Italy
Dipartimento SBAI, Università di Roma La sapienza, Via A.Scarpa 16 00161 Roma, Italy
3
Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, H-6701 Szeged,
Hungary
4
Université Montpellier 2, Laboratoire Charles Coulomb UMR 5221, F-34095, Montpellier, France
5
CNRS, Laboratoire Charles Coulomb UMR 5221, F-34095, Montpellier, France
*
[email protected]
2
Abstract: Noncollinear second harmonic generation from a
Bacteriorhodopsin (BR) oriented multilayer film was systematically
investigated by varying the polarization state of both fundamental beams.
Both experimental results and theoretical simulations, show that the
resulting polarization mapping is an useful tool to put in evidence the
optical chirality of the investigated film as well as the corresponding
multipolar contributions to the nonlinear.
©2012 Optical Society of America
OCIS codes: (160.4330) Nonlinear optical materials; (350.4238) Nanophotonics; (190.2620)
Harmonic generation and mixing.
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#166255 - $15.00 USD
(C) 2012 OSA
Received 6 Apr 2012; revised 10 May 2012; accepted 12 May 2012; published 15 Jun 2012
18 June 2012 / Vol. 20, No. 13 / OPTICS EXPRESS 14621
PHYSICAL REVIEW B 85, 245304 (2012)
Nanosecond laser-induced selective removal of the active layer of CuInGaSe2 solar cells
by stress-assisted ablation
András Buzás1,* and Zsolt Geretovszky2
1
Institute of Physics, Biological Research Center, Hungarian Academy of Sciences, H-6701 Szeged, P.O. Box 521, Hungary
2
Department of Optics and Quantum Electronics, University of Szeged, Dóm tér 9, H-6720 Szeged, Hungary
(Received 20 December 2011; published 7 June 2012)
We demonstrate that laser pulses of nanosecond duration (λ = 1064 nm, τ = 25 ns, PRR = 5 kHz) are capable
of the clean removal of the CuInGaSe2 (CIGS) and ZnO:Al layers in the layer structure of chalcogenide-based
solar cells, leaving the underlying Mo layer undamaged and producing excellent crater morphology. Our results
prove that the material removal process is governed by the thermomechanical stress developing in the CIGS layer
due to rapid laser heating. In the mechanical ablation of the active layer, three phenomena play a crucial role,
namely, delamination, buckling, and fracture. Morphological and compositional analysis of the laser-processed
areas is used to identify the experimental parameters where clean mechanical ablation can be achieved. Numerical
calculations, performed in the COMSOL software environment, are also presented to complement the experimental
tendencies and verify the proposed model. Our calculation proves the development of a stress distribution that
drives the delamination of the CIGS and Mo layers. As the delamination front proceeds radially outward, the
separation of the layers ceases in the colder outer regions according to the Griffith’s criterion and defines the
size of the craters produced afterwards. The free-standing chalcogenide layer continues to deform, and buckling
results in a growing tensile stress at the perimeter of the delaminated area, where ultimately fracture will finalize
the removal process and facilitate the clean ablation of the laser-irradiated area.
DOI: 10.1103/PhysRevB.85.245304
PACS number(s): 81.40.Gh, 68.60.Dv, 79.20.Eb, 68.60.Bs
I. INTRODUCTION
Removal of a coating from the surface of a substrate or
an underlying layer stack has many far reaching applications.
Some of these applications, such as paint removal or computerto-press imaging,1 are more forgiving to the quality of the
processed area, while others such as circuit patterning, mask
manufacturing, organic electronics (OLED fabrication),2 or
laser scribing of solar panels are more stringent and can
not be satisfied by the typical, thermally driven ablation
process. Fortunately, ablation is known to occur by a variety
of mechanisms, among which the stress-assisted route has
several appealing features, such as complete removal of a
single or even multiple layers, no surface contamination
by debris redeposition, and preserving the performance of
any underlying functional layers. As a consequence, laser
processing is gradually emerging as a key enabling technology
of photovoltaics in general, and of chalcogenide active layer
patterning in particular.
Thin-film solar cells having CuInGaSe2 (CIGS) as their
active layer are among the most promising due to their
high cell efficiency [approaching 20.5% (Ref. 3)] and low
fabrication cost. The typical substrate-type CIGS solar cell has
the following layer structure: glass/Mo/CIGS/CdS/ZnO:Al,
where the glass is used as a support and the ZnO:Al film
is the so-called window layer. During the fabrication of a
solar module, an array of cells is formed on the substrate.
In order to achieve the desired interconnection schemes,4,5
the layers must be patterned, typically after their deposition
steps. The first scribe is an insulating cut, performed on the
back contact Mo layer (denoted as P1). The next processing
step after the deposition of the absorber CIGS and buffer CdS
layers is to form a conducting scribe (P2). Finally, the front
contact ZnO:Al window layer must also be patterned (P3) to
insulate adjacent cells. The role of the P1 and P3 scribes is
1098-0121/2012/85(24)/245304(14)
to insulate neighboring cells, while that of P2 is to facilitate
the connection of front and back contact layers of neighboring
cells in series. The efficiency of the so-formed module depends
not only on the cell efficiency, but also on the quality of the
scribes. Two major losses originate from the patterned area.
The first is that the patterned area does not take part in energy
conversion, clearly explaining why this area is called the dead
zone. The other source of loss is that scribing may result in a
shunt and resistance increase at the insulation and conducting
scribes, respectively.
It has been proposed that laser processing would be the
optimum choice to realize patterning due to its high accuracy
and high processing speed coupled with a moderate investment
cost.6 Compaan et al. were the first to investigate the pros and
cons of laser scribing of CIGS-based solar cell.6 Nanosecond
laser scribing the P2 line in a flexible solar cell was reported
by Kessler et al.4 where the removal of the CIGS layer was
incomplete, but the remnants of the layer were converted to a
conductor. However, they found that this molten CIGS residue
increases the resistivity at the ZnO:Al/Mo interface and hence
decreases module efficiency.5 An alternative route for realizing
the P2 scribe is the microwelding process where very high laser
fluences (≈50 J cm−2 ) are used to metallize the CIGS layer
and form a conduction channel between the Mo and ZnO:Al
layers.7,8 It was also demonstrated for the P2 and P3 laser
scribes that the use of picosecond and femtosecond lasers9–12
results in good channel characteristics. Murrison and coworkers reported the use of a nanosecond laser for performing
the P2 and P3 scribes and claim that their appealing scribe characteristics are due to the low absorption of the CIGS, which
results in the intensive evaporation at the CIGS/Mo interface.13
During laser ablation, many mechanisms are known that
may lead to material removal in a multilayer structure, such
as evaporation or thermomechanical stress-driven fracture,
245304-1
©2012 American Physical Society
APPLIED PHYSICS LETTERS 101, 041111 (2012)
Light sailboats: Laser driven autonomous microrobots
Anrdás Búzás,1 Lóránd Kelemen,1 Anna Mathesz,1 László Oroszi,1 Gaszton Vizsnyiczai,1
Tamás Vicsek,2 and Pál Ormos1
1
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Temesvári krt 62.
H-6726 Szeged, Hungary
2
Department of Biological Physics, Eötvös University and Statistical and Biological Physics Research Group
of the Hungarian Academy of Sciences, Pázmány P. stny. 1A, H-1117 Budapest, Hungary
(Received 13 May 2012; accepted 3 July 2012; published online 24 July 2012)
We introduce a system of light driven microscopic autonomous moving particles that move on a
flat surface. The design is simple, yet effective: Micrometer sized objects with wedge shape are
produced by photopolymerization, and they are covered with a reflective surface. When the area of
motion is illuminated perpendicularly from above, the light is deflected to the side by the wedge
shaped objects, in the direction determined by the position and orientation of the particles. The
momentum change during reflection provides the driving force for an effectively autonomous
C 2012
motion. The system is an efficient tool to study self propelled microscopic robots. V
American Institute of Physics. [http://dx.doi.org/10.1063/1.4737646]
There is great interest in self propelled microscopic
robots, originating from both technology and fundamental
science. Intensive development is directed towards technological applications primarily in the biomedical field, for
tasks like material transport, local diagnostics, etc. Individual or collective motion of active biological systems (from
bacteria to large animals) is also the target of a wide range
of basic science studies due to remarkable dynamical
phenomena in such non-equilibrium systems (for a recent
review see Ref. 1). The experimental studies require appropriate functional moving models. Different systems have
been developed, depending on the requirements and the
capabilities. The basic examples are, of course, the moving
organisms themselves and they represent the various essential features as well. The mechanism of locomotion is contained in every unit, the energy for propulsion is provided in
the medium, and it is gained by the moving particles upon
demand. Accordingly, in many experiments living bacteria
are used (see, e.g., Refs. 2–5). However, the properties of
the living objects are largely fixed, and it is not possible to
modify them significantly. In the process of testing specific
theories there is also need for objects with different and
controllable properties: size, shape, velocity, interaction
between particles, etc. Consequently, there is need for artificial systems the properties of which can be arbitrarily determined and controlled. Various kinds of self propelled
microscopic swimmers have been introduced with most different propulsion means: chemical,6–8 light induced chemical,9 light induced thermal,10 and electromagnetic.11
However, up till now the microscopic models have produced only limited results.
In this field in many cases size is not a principal requirement, after all the space where the particles are moving may
not be limited. In many experimental approaches, independent, autonomous robots are used, primarily in a two dimensional environment: robots rolling on a solid surface12 or
ships floating on a water surface,13 or even helicopters flying
in 3 dimensions.14,15 They have the obvious advantage that
their properties can be defined in a practically arbitrary way
0003-6951/2012/101(4)/041111/5/$30.00
and even quite complex behavioral patterns (propulsion,
control, interaction) can be realized. These latter systems,
however, are fairly complicated, the experimental area is
also large. There is still need for possibly simple microscopic
swimmers with efficient propulsion and well defined
properties.
Here we introduce a simple yet very effective self propelled swimmer. The energy source for motion is light. The
system is two dimensional, and the particles are moving on a
planar surface.
There are several properties the swimmers should have.
The requirement concerning the propulsion is that it should
result in a motion of the units (i) that is regular to some
extent and (ii) in a direction corresponding to a “forward”
motion of the object (there should exist a forward direction,
fixed to the body of the swimmer), with possibly long persistence length. The energy supply for the motion should be
continuous. The shape of the swimmer should be possible to
vary, but in general an elongated shape is preferred.
Light has proven to be a practical means for the transport of microscopic particles. Optical micromanipulation has
developed into an extremely powerful tool in biology.16
However, in the procedures demonstrated so far, either light
pressure was used to transport particles (in this case objects
are moving in the direction of the light propagation,17 not
determined by their own orientation) or optical traps were
used to grab and move particles in a direction determined by
the motion of the trap itself.18 Such objects cannot be
regarded as autonomous particles.
There is a still not reported possibility to harness the light
energy to drive the microscopic particles in a way that fulfills
the requirement for the motion of self propelled swimmers.
In the approach introduced in this work we use reflective
wedge shaped particles, sliding on a flat horizontal surface
(2D system). The area of motion is illuminated from above
by a collimated homogeneous light beam, from a direction
perpendicular to the surface of motion (see Fig. 1). The light
is reflected on the surface of the wedge in a direction determined by the shape and position of each individual particle.
101, 041111-1
C 2012 American Institute of Physics
V
Downloaded 10 Nov 2012 to 160.114.60.79. Redistribution subject to AIP license or copyright; see http://apl.aip.org/about/rights_and_permissions
ARTICLE
pubs.acs.org/JACS
Anisotropic Organization and Microscopic Manipulation of
Self-Assembling Synthetic Porphyrin Microrods That Mimic
Chlorosomes: Bacterial Light-Harvesting Systems
00 00
Cyril Chappaz-Gillot,†,3 Peter L. Marek,‡,3 Bruno J. Blaive,† Gabriel Canard,† Jochen B€urck,§ Gyoz
o Garab,#
‡,||,^
#
#
‡,^
‡
Horst Hahn,
Tamas Javorfi, Lorand Kelemen, Ralph Krupke, Dennis M€ossinger, Pal Ormos,#
Chilla Malla Reddy,‡,X Christian Roussel,† Gabor Steinbach,# Milan Szabo,# Anne S. Ulrich,§,^
Nicolas Vanthuyne,† Aravind Vijayaraghavan,‡ Anita Zupcanova,#,Δ and Teodor Silviu Balaban*,†
†
)
ISM2-Chirosciences, Faculte des Sciences, Aix-Marseille Univ. UMR 6263, Saint-Jer^ome, Case A62,
Avenue Escadrille Normandie-Niemen, F-13397 Marseille, Cedex 20, France
‡
Institute for Nanotechnology, §Institute for Biological Interfaces, and ^Center for Functional Nanostructures,
Karlsruhe Institute of Technology, Postfach 3640, D-76021 Karlsruhe, Germany
#
Biological Research Center, Hungarian Academy of Sciences, Temesvari k€orut 62, H-6726 Szeged, Hungary
Joint Research Laboratory Nanomaterials, Karlsruhe Institute of Technology and Technische Universit€at Darmstadt,
D-64287 Darmstadt, Germany
Δ
Biological Centre, Academy of Sciences of the Czech Republic, 370 05 Ceske Budejovice, Czech Republic
bS Supporting Information
ABSTRACT: Being able to control in time and space the positioning, orientation, movement, and sense of rotation of nano- to microscale objects is currently an active research area in nanoscience,
having diverse nanotechnological applications. In this paper, we demonstrate unprecedented control and maneuvering of rod-shaped
or tubular nanostructures with high aspect ratios which are formed
by self-assembling synthetic porphyrins. The self-assembly algorithm,
encoded by appended chemical-recognition groups on the periphery
of these porphyrins, is the same as the one operating for chlorosomal
bacteriochlorophylls (BChl's). Chlorosomes, rod-shaped organelles
with relatively long-range molecular order, are the most efficient naturally occurring light-harvesting systems.1,2 They are used by green
photosynthetic bacteria to trap visible and infrared light of minute intensities even at great depths, e.g., 100 m below water surface or in
volcanic vents in the absence of solar radiation. In contrast to most other natural light-harvesting systems, the chlorosomal antennae are
devoid of a protein scaffold to orient the BChl's; thus, they are an attractive goal for mimicry by synthetic chemists, who are able to engineer
more robust chromophores to self-assemble. Functional devices with environmentally friendly chromophores—which should be able to
act as photosensitizers within hybrid solar cells, leading to high photon-to-current conversion efficiencies even under low illumination
conditions—have yet to be fabricated. The orderly manner in which the BChl's and their synthetic counterparts self-assemble imparts
strong diamagnetic and optical anisotropies and flow/shear characteristics to their nanostructured assemblies, allowing them to be
manipulated by electrical, magnetic, or tribomechanical forces.
’ INTRODUCTION
Green photosynthetic bacteria developed some 2.5 billion
years ago in an anoxic atmosphere, and they use for light-harvesting
special organelles called chlorosomes.1,2 This name describes
their “green sac” nature, as these harbor bacteriochlorophyll
(BChl) c, d, or e molecules (Chart 1). In spite of numerous
attempts, it has not been possible to obtain single-crystal diffraction
data from either chlorosomes or various BChl homologues or
derivatives. Solid-state 13C NMR spectroscopy on uniformly
labeled samples,36 as well as elegant mutagenesis studies,7 have
demonstrated that these BChl's self-assemble into highly organized extended nanostructures and that proteins, unlike other
r 2011 American Chemical Society
common photosynthetic antenna systems,1,2 are not involved
in the pigment scaffolding. BChl-containing chlorosomes can
scavenge light of minute intensity, such as at 100 m below the water
surface8 or even in volcanic vents at a depth of over 2300 m,
where a photosynthetic microbe thrives in the absence of solar
radiation by using infrared photons.9 Once a photon is trapped,
one of the BChl molecules becomes excited into the first singlet
excited state. Due to the high degree of ordering of the chromophores,10 the radiant energy is then neatly passed by rapid
Received: May 5, 2011
Published: December 09, 2011
944
dx.doi.org/10.1021/ja203838p | J. Am. Chem. Soc. 2012, 134, 944–954
PRL 109, 034104 (2012)
week ending
20 JULY 2012
PHYSICAL REVIEW LETTERS
Hydrodynamic Synchronization of Light Driven Microrotors
R. Di Leonardo,1,* A. Búzás,2 L. Kelemen,2 G. Vizsnyiczai,2 L. Oroszi,2 and P. Ormos2,*
1
2
IPCF-CNR UOS Roma, Dipartimento di Fisica, Università Sapienza, I-00185 Rome, Italy
Biological Research Center, Hungarian Academy of Sciences, Institute of Biophysics, H-6726 Szeged, Hungary
(Received 17 January 2012; revised manuscript received 29 May 2012; published 17 July 2012)
Hydrodynamic synchronization is a fundamental physical phenomenon by which self-sustained
oscillators communicate through perturbations in the surrounding fluid and converge to a stable
synchronized state. This is an important factor for the emergence of regular and coordinated patterns
in the motions of cilia and flagella. When dealing with biological systems, however, it is always hard to
disentangle internal signaling mechanisms from external purely physical couplings. We have used the
combination of two-photon polymerization and holographic optical trapping to build a mesoscale model
composed of chiral propellers rotated by radiation pressure. The two microrotors can be synchronized by
hydrodynamic interactions alone although the relative torques have to be finely tuned. Dealing with a
micron sized system we treat synchronization as a stochastic phenomenon and show that the phase lag
between the two microrotors is distributed according to a stationary Fokker-Planck equation for an
overdamped particle over a tilted periodic potential. Synchronized states correspond to minima in this
potential whose locations are shown to depend critically on the detailed geometry of the propellers.
DOI: 10.1103/PhysRevLett.109.034104
PACS numbers: 05.45.Xt, 05.10.Gg, 47.63.mf, 87.80.Cc
Synchronization is at the basis of a wide variety of
fascinating and important phenomena in physics, biology,
and engineering. From coupled Josephson junctions [1] to
cardiac pacemaker cells [2], the presence of a weak interaction between two or more self-sustained oscillators often
leads to the emergence of synchronous patterns [3]. At the
micron scale of cells and bacteria, hydrodynamic interactions provide a strong and long-ranged mechanism for
coupling [4]. Since synchronization phenomena are known
to occur even in the presence of extremely weak and subtle
couplings, it is quite natural to expect strong synchronous
behavior in such a strongly coupled regime. The presence
of a strong coupling, however, is not a sufficient condition
for synchronization [5,6], and the role of hydrodynamic
interactions for the emergence of synchronous behaviors in
flagella [7–9] and cilia [10–13] is still the subject of a lively
debate [14]. In the case of waving sheets [5], kinematic
reversibility can destroy synchronization when the sheets
have reflection symmetry. For the same reason, a collection
of rigid rotors, spinning around fixed axes and coupled
through hydrodynamic interactions, will appear as the
same physical system evolving on a time reversed trajectory when we change sign to all applied torques. Such
reversible dynamics cannot give rise to any synchronization behavior that is, by definition, an irreversible process.
This symmetry upon torque reversal can be broken by
using phase dependent torques [6] or, alternatively, by
introducing some degree of mechanical flexibility in the
form of internal degrees of freedom with finite stiffness
[15,16]. In the latter case, when we reverse the sign of
applied torques, internal forces will not change their sign
and the system will not trace back its history. As a consequence, synchronization in uniformly rotating systems is
0031-9007=12=109(3)=034104(5)
driven by small deviations from rigid dynamics and
amounts to a tiny effect, despite the presence of strong
hydrodynamic couplings. In such a situation, synchronization is highly sensitive to a small mismatch in the rotors’
free rotational frequencies [16]. An extremely low
Reynolds number is an important condition in mesoscopic
dynamics, but even more peculiar is the unavoidable presence of noise. However, hydrodynamic synchronization of
rotators has been investigated only by analytical [6,11] and
numerical models [15] or macroscopic experiments [16]
that do not take into account noise. A colloidal model for
rotators, of the kind used [13] for modeling ciliar beating
motions, is still lacking.
In this Letter, we have used two-photon polymerization
to build a microscopic model of hydrodynamically coupled
propellers driven by radiation pressure. We demonstrate
that hydrodynamic interactions alone can synchronize
the two rotors, although the applied torques have to be
finely tuned. The stationary probability distribution for the
phase lag between the two rotors obeys a Fokker-Planck
equation for an overdamped particle over a tilted periodic
potential [17].
Synchronization between two self-sustained oscillators
manifests with the appearance of phase-locked states that
survive even when we detune the two oscillators’ frequencies within some finite range [3]. Those states are even
easily detectable in the presence of noise that would
quickly destroy any accidental phase-locking, driving
phases away in diffusing random walks. On the other
hand, if noise is unbounded, perfect phase-locking never
occurs. However, synchronization still will be clearly visible as an intermittent dynamics where rapid phase slips
interrupt periods of phase-locking whose lifetime increases
034104-1
Ó 2012 American Physical Society
Dynamic Article Links
RSC Advances
Cite this: RSC Advances, 2012, 2, 11481–11490
PAPER
www.rsc.org/advances
Estimation of kinetic parameters from time-resolved fluorescence data: A
compressed sensing approach
Géza I. Groma,* Zsuzsanna Heiner, András Makai and Ferenc Sarlós
Received 10th August 2012, Accepted 24th September 2012
DOI: 10.1039/c2ra21773b
The characterization of fluorescence kinetic measurements by a set of lifetimes and amplitudes is a
well-known, ill-posed problem. The most effective approaches for dealing with this difficulty
generally look for a regularized distribution of amplitudes on a predefined large grid of time
constants. Here we argue that in the absence of any additional a priori knowledge on the underlying
mechanism, the simplest solution of any complex kinetics is the sparsest distribution. We have found
that the basis pursuit denoising procedure is an excellent method for finding very sparse models
describing time-resolved fluorescence data. Our simulation results indicate that for truly sparse
kinetics, this method provides a superior resolution of closely located time constants. Additional
information on a distribution corresponding to a given level of noise can be obtained from the
averaged solution even if the true kinetics are far from sparsity. A case study on a compressed set of
real experimental data taken from the fluorescence of flavin adenine dinucleotide revealed five distinct
time constants, ranging from 500 fs to 3 ns. The obtained time constants were almost independent of
wavelength without any constraint favouring this arrangement.
Aij = exp (2ti /tj)
1 Introduction
(3)
.
Time-resolved fluorescence spectroscopy and its recent extension, fluorescence lifetime imaging (FLIM) are very effective
methods for characterizing the microenvironment of a fluorescent chromophore in a wide range of different systems.1,2
Although in the simplest case, the fluorescence decay kinetics can
be described by a single exponential, in a real heterogenic
environment, the analysis of the kinetics is rather challenging. In
such a case the most general model describing the kinetics can be
expressed in the form of quasi-Laplace transform3
f ðtÞ~
?
ð
gðtÞ expð{t=tÞdt
(1)
0
The difficulty of the analysis lies in the fact that obtaining the
unknown g(t) function requires the inversion of (1), which is a
well-known, ill-posed problem.3,4 In practical computation, one
has to solve the corresponding discrete system of equations:
y = Ax
(2)
, where y (M 6 1) is the vector of data, x (N 6 1) is the vector of
unknown amplitudes, and the matrix A (M 6 N) (known as
experimental matrix) is defined as:
Institute of Biophysics, Biological Research Centre, Hungarian Academy
of Sciences, 6701 Szeged, Hungary. E-mail: [email protected];
Fax: +36 6243 3133; Tel: +36 6259 9620
This journal is ß The Royal Society of Chemistry 2012
Since the experimentally determined y is corrupted by noise,
(2) is generally solved in a least-square manner, by minimizing
either the Euclidean (l2) norm defined as x2 = ||y 2 Ax||22 , or a
properly weighted version of its. The ill-posed nature of the
problem is manifested in the existence of many different
solutions with very similar fitting to the experimental data.
In a traditional approach, one supposes that N % M, and the
data are fitted by a low number of exponentials of unknown
time constants. Generally, however, neither an a priori knowledge exists for choosing the exact number of N, nor the
calculated goodness of fitting helps to determine it.
Alternatively, the fit can be executed on a large grid of
predefined tj values (exponential series).5 The problem with this
advanced approach is that unless the noise level is very low and
the set of equations is kept highly overdetermined (N , M)—
which results in poor resolution—the solution becomes irregular. The stability and reliability of both the discrete exponential
and the exponential series methods can be improved by global
fitting which introduces further independent experimental
variables, such as wavelength.6–8
The most effective way for handling ill-posed problems is to
apply some regularization on the underdetermined (N . M)
least-square solution of (2), by applying a constraint expressed
in a y(x) function. The regularization with y(x) and
regularization parameter l can be expressed as a minimization
problem aiming to find the distribution of the amplitudes in the
form of:
RSC Adv., 2012, 2, 11481–11490 | 11481
Issue in Honor of Prof. Ferenc Fülöp
ARKIVOC 2012 (v) 291-300
Structure analysis of proteins, peptides and metal complexes by vibrational
circular dichroism
Miklós Hollósi,a Elemér Vass,a Gábor Szilvágyi,a Andreja Jakas,b Ilona Laczkóc
a
b
Institute of Chemistry, Eötvös Loránd University, POB 32, H-1518 Budapest, Hungary
Department of Organic Chemistry and Biochemistry, Ruđer Bošković Institute, POB 180, 10002
Zagreb, Croatia
c
Institute of Biophysics, Biological Research Center, POB 521, 6701 Szeged, Hungary
E-mail: [email protected]
Dedicated to Professor Ferenc Fülöp on the occasion of his 60th birthday
Abstract
There are two principal forms of vibrational optical activity (VOA), an IR form referred to as
vibrational circular dichroism (VCD) and Raman form known as Raman optical activity (ROA).
This paper reports examples of the application of VCD spectroscopy for the determination of the
absolute configuration and conformation of chiral molecules, e.g. cyclic -lactams. VCD
spectroscopy can be applied for the characterization of the conformation of proteins and peptides
in solution. VCD based conformational analysis of cyclic peptides is discussed. Examples are the
cyclic hexapeptide cyclo(Pro2-Gly-Pro2-Gly) and cyclic peptides comprising -homoamino acids
(trans-2-aminocyclopentane or trans-2-aminocyclohexane carboxylic acid). Structure analysis by
VCD of opiate peptides, glycopeptides, peptidomimetics and chiral transition metal complexes
are also discussed.
Keywords: Vibrational circular dichroism (VCD); determination of absolute configuration;
characterization of protein conformation; structure analysis of cyclic peptides; VCD of
glycopeptides and chiral transition metal complexes
Introduction
Vibrational optical activity (VOA) is due to the differential response of a molecule to leftcircularly polarized (LCP) versus right-circularly polarized (RCP) radiation during a vibrational
transition. There are two principal forms of VOA, an IR form referred to as vibrational circular
dichroism (VCD) and a Raman form known as Raman optical activity (ROA). Both VCD and
ROA are very sensitive to the stereochemical features of chiral molecules, and they have similar
differences and advantages to their parent spectroscopies, IR absorption and Raman scattering.1
Page 291
©
ARKAT-USA, Inc.
Journal of Photochemistry and Photobiology B: Biology 114 (2012) 147–152
Contents lists available at SciVerse ScienceDirect
Journal of Photochemistry and Photobiology B: Biology
journal homepage: www.elsevier.com/locate/jphotobiol
2D measurement of ion currents associated to the signal transduction
of the phototactic alga Chlamydomonas reinhardtii
András Kincses, Rudolf Tóth-Boconádi, András Dér ⇑
Institute of Biophysics, Biological Research Centre of the 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 26 May 2012
Accepted 5 June 2012
Available online 15 June 2012
Keywords:
Electric measurement
Phototaxis
Chlamydomonas
Channelrhodopsins
Optogenetics
a b s t r a c t
Our objective was to develop a simple procedure for the detection of light-induced ion currents of photomotile cells in two dimensions. The novel technique was based on the light gradient method (LGM), and
the model object was Chlamydomonas reinhardtii, a phototactic unicellular alga, ideal for such experiments. The conventional LGM cuvette was modified such that the electrode pair could be rotated around
the sample and pick up the electric signals from arbitrary directions. The experiments were performed
with and without the application of an auxiliary light beam preorienting the motile cells. The analysis
of the detected traces revealed two main vectorial components of the signal by the help of singular value
decomposition (SVD), in concert with previous experimental findings and theoretical considerations suggesting different origins of the ‘‘fast’’ and ‘‘slow’’ components of the photoelectric response of Chlamydomonas and Haematococcus cells. Using plausible assumptions, our method allowed a quantitative
analysis of the signal, assigning size and direction to the two vectorial components. The method allows
a rapid and accurate way to measure electric signals of photomotive cells in 2D, and particularly, to test
the physiological activity and in vivo-kinetics of site-directed mutants of ChR1 or ChR2, providing novel
photo-electrophysiological methods with important quantitative information.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
One of the most important goals in modern biophysics is to describe membrane-coupled signal and energy transduction processes on different levels of physiological organization. Such
processes govern, e.g., nerve signalization, or the energy conversion in chloroplasts and mitochondria [1,2]. The phototactic unicellular alga, Chlamydomonas reinhardtii is a popular model system to
investigate basic problems concerning both signal and energy
transduction, and electric signals carry a lot of information about
these processes. The light gradient method (LGM) [3,4] is ideally
suited for the in vivo detection of electric signals because it has a
fine time resolution and, unlike microelectrode techniques, it does
not make any demands for the cell size or the consistency of the
cell wall. Cells can be investigated by LGM under their natural living conditions, while the sensitivity and reproducibility of the
measurements is competitive with other methods (patch-clamp,
BLM, SSM, etc.) [5,6]. The results give us direct information about
the kinetics and spatial properties of the underlying transport
processes.
Chlamydomonas cells have a simple light-tracking system [7–9].
During their swim toward the light source, the algae are rotating
counterclockwise around their longitudinal axes because of asym⇑ Corresponding author. Tel.: +36 62 599606; fax: +36 62 433133.
E-mail address: [email protected] (A. Dér).
1011-1344/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jphotobiol.2012.06.001
metric strokes of their flagellae, so sustaining a helical pathway
[7,10]. The two flagellae are radially oriented during the helical
motion (with the dominating flagellum pointing to the outside).
As a consequence of such a motion pattern, the photoreceptors of
the eyespot receive sinusoidally modulated light intensity. This
modulation decreases when the ideal orientation is approached
and disappears when light- and tracking-directions coincide [11].
After reaching the ideal orientation, the algae should protect their
photoreceptors from the absorption of further photons, so as to
avoid disadvantageous direction changes. This is the reason why
the eyespot normal points 45° outside of the beating plane of the
flagellae. It should be noted that, under high intensity illumination,
the algae move not toward the light source, but away from it.
Both types of phototactic motions are mediated by a cascade of
transmembraneous ion currents, starting at the photoreceptor
and ending at the flagella region. The cascade is initiated by two,
rhodopsin-type ion channels called Channelrhodopsin 1 and 2
(ChR1 and ChR2) [5,12–16]. The channelrhodopsins belong to the
G-protein-coupled receptor superfamily, consisting of two parts,
a protein (opsin) and a chromophore (all-trans retinal). The opsin
part of both ChRs is built up by about 700 amino acids, and have
a core region of seven transmembrane alpha helices of the size of
ca. 300 amino acids. In native algae, these ion channels are shown
to be Ca2+-dependent. ChR1 and 2 are light-gated ion channels,
mediating a light-induced depolarization of the plasma membrane.
If the depolarization reaches a threshold, voltage-gated Ca2+
Immunology Letters 148 (2012) 34–38
Contents lists available at SciVerse ScienceDirect
Immunology Letters
journal homepage: www.elsevier.com/locate/immlet
Melanoma cell-derived exosomes alter macrophage and dendritic cell functions
in vitro
Annamaria Marton a , Csaba Vizler a , Erzsebet Kusz a , Viktoria Temesfoi a , Zsuzsa Szathmary b ,
Krisztina Nagy c , Zsolt Szegletes c , Gyorgy Varo c , Laszlo Siklos c , Robert L. Katona d , Vilmos Tubak a ,
O.M. Zack Howard e , Erno Duda a , Janos Minarovits f , Katalin Nagy g , Krisztina Buzas a,g,∗
a
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
Galenbio Kft, Mosonmagyaróvár, Hungary
c
Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
d
Institute of Genetics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, Hungary
e
National Cancer Institute-Frederick, Laboratory of Molecular Immunoregulation, Frederick, MD 21702-1201, United States
f
National Center for Epidemiology, Microbiological Research Group, Budapest, Hungary
g
Faculty of Dentistry, University of Szeged, Hungary
b
a r t i c l e
i n f o
Article history:
Received 23 December 2011
Received in revised form 6 July 2012
Accepted 30 July 2012
Available online xxx
Keywords:
Melanoma
Tumor immunity
Exosomes
Cytokine profile
a b s t r a c t
To clarify controversies in the literature of the field, we have purified and characterized B16F1 melanoma
cell derived exosomes (mcd-exosomes) then we attempted to dissect their immunological activities. We
tested how mcd-exosomes influence CD4+ T cell proliferation induced by bone marrow derived dendritic
cells; we quantified NF-␬B activation in mature macrophages stimulated with mcd-exosomes, and we
compared the cytokine profile of LPS-stimulated, IL-4 induced, and mcd-exosome treated macrophages.
We observed that mcd-exosomes helped the maturation of dendritic cells, enhancing T cell proliferation
induced by the treated dendritic cells. The exosomes also activated macrophages, as measured by NF-␬B
activation. The cytokine and chemokine profile of macrophages treated with tumor cell derived exosomes
showed marked differences from those induced by either LPS or IL-4, and it suggested that exosomes may
play a role in the tumor progression and metastasis formation through supporting tumor immune escape
mechanisms.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
Previous studies showed that cultured human tumor cells
release exosomes, i.e., microvesicles of 20–100 nm diameters. Exosomes bear the molecular markers of the originating tumor cells’
plasma membrane. Among others, exosomes contain structural
proteins, MHC molecules and cell surface molecules typically associated with apoptosis; therefore they are possible inducers of
anti-tumor immune responses [1]. The immunomodulatory activities of tumor exosomes are poorly understood, with reported
activating [2] and inhibitory effects [3]. The wide range of observed
biological effects seems to depend on tumor type and staging.
The size of exosomes may facilitate their in vivo penetration
and interaction with different host cell types, even distant from
the tumor mass. Exosomes have been shown to participate in
∗ Corresponding author at: Biological Research Centre of the Hungarian Academy
of Sciences, Szeged and at the Faculty of Dentistry, University of Szeged, Hungary.
E-mail addresses: [email protected], [email protected] (K. Buzas).
0165-2478/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.imlet.2012.07.006
cell-to-cell communication by various mechanisms. The most obvious mechanism involves membrane proteins, partially identical
with those of the originating tumor cells. Upon endocytosis, exosomes may deliver their active components, proteins and RNA,
directly into the cytoplasm of bystander cells. These transport processes can influence invasion of tumor cells, stimulate antigen
specific T-cell responses, modulate cell polarity and have a role in
the developmental patterning of tissues [4]. Exosomes may also
have an important role in tumor immune evasion by direct suppression of immune cell activation.
By dissecting the immunomodulatory effects of melanoma cell
derived exosomes we wished to clarify some of the controversies in the literature of the field. We tested how mcd-exosomes
influence the CD4+ T cell proliferation induced by bone marrow
derived dendritic cells (DCs). We quantified the NF-␬B activation in
mature macrophages stimulated with mcd-exosomes. We qualified
the production pattern of inflammatory cytokines and chemokines
that have previously been identified as mediators or regulators of
anti-cancer responses. Our findings suggest that, in spite of their
immune activating effects detectable in our model, the concomitant
Wave-guided optical waveguides
D. Palima,1 A. R. Bañas,1 G. Vizsnyiczai,2 L. Kelemen,2 P. Ormos,2 and J. Glückstad1,*
1
DTU Fotonik, Dept. of Photonics Engineering, Technical University of Denmark, DK-2800, Kgs. Lyngby, Denmark
2
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Szeged H-6701, Hungary
*
[email protected]
www.ppo.dk
Abstract: This work primarily aims to fabricate and use two photon
polymerization (2PP) microstructures capable of being optically
manipulated into any arbitrary orientation. We have integrated optical
waveguides into the structures and therefore have freestanding waveguides,
which can be positioned anywhere in the sample at any orientation using
optical traps. One of the key aspects to the work is the change in direction of
the incident plane wave, and the marked increase in the numerical aperture
demonstrated. Hence, the optically steered waveguide can tap from a
relatively broader beam and then generate a more tightly confined light at its
tip. The paper contains both simulation, related to the propagation of light
through the waveguide, and experimental demonstrations using our
BioPhotonics Workstation. In a broader context, this work shows that
optically trapped microfabricated structures can potentially help bridge the
diffraction barrier. This structure-mediated paradigm may be carried
forward to open new possibilities for exploiting beams from far-field optics
down to the subwavelength domain.
©2012 Optical Society of America
OCIS codes: (170.4520) Optical confinement and manipulation; (220.4000) Microstructure
fabrication; (230.7370) Waveguides.
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#158363 - $15.00 USD
(C) 2012 OSA
Received 21 Nov 2011; revised 2 Jan 2012; accepted 4 Jan 2012; published 13 Jan 2012
30 January 2012 / Vol. 20, No. 3 / OPTICS EXPRESS 2004
10
Artificial and Natural Membranes
György Váró and Zsolt Szegletes
Institute of Biophysics, Biological Research Centre, Szeged
Hungary
1. Introduction
Non-cellular forms of life are not known on the earth. Starting with unicellular organisms, such as bacteria - up to multicellular complex organisms, the basic building unit is the cell,
surrounded by the cell membrane. The cell contains all the characteristics of life, having the
genetic program to build up the structure and to keep it functioning.
Membranes are an essential structural component of living objects (Sybesma, 1977;
Volkenstein, 1981). They are formed from different lipids and act as a selective barrier
around the cell and cell organelles. The phospholipid bilayer is the basic structure of all
biological membranes. Besides the phospholipids, some other lipids are generally present in
the membrane, such as glycolipids and cholesterol. Many biological processes require
membranes. Physically and chemically essential functions include metabolism and the
process of the accumulation and usage of energy in the biological system.
An essential function of the membrane is to keep a well-defined chemical composition
inside of the membrane at a limited volume, which is different from the outside. There are
large concentration differences between the two sides. To build up and maintain this
concentration difference, selective passive diffusion and selective active transport
translocate biologically important molecules through the membrane. This is achieved by
different channels and pumps built from a large variety of membrane proteins. The
selectivity is achieved by the composition and structure of the transporter (Sybesma, 1977;
Volkenstein, 1981).
Membrane proteins fall into two categories, depending on how they are bound to the
membrane. One category is that of the peripheral proteins. They are loosely bound to the
membrane through electrostatic interactions and they can be removed in lipid free-form by
relatively mild treatments. In the second category are the integral proteins, embedded inside
the membrane and often spanning it entirely. They are difficult to remove, having large
hydrophobic domains, isolated with bound lipids. The integral membrane proteins occur in
a wide variety of shapes. The most common formations are the α-helix and the β-sheet
structures (Sybesma, 1977).
There are a large variety of structural possibilities and this is reflected in the very different
properties and behaviour of the membrane proteins. Due to the lateral fluidity of the
membrane, proteins are mobile in the surface and they can form aggregates. Large
associations of proteins and lipids form rafts. By interacting with the proteins, new
characteristic properties appear (Engel and Gaub, 2008).
Eur Biophys J (2012) 41:139–145
DOI 10.1007/s00249-011-0765-5
ORIGINAL PAPER
Adhesion and stress relaxation forces between melanoma
and cerebral endothelial cells
Attila G. Végh • Csilla Fazakas • Krisztina Nagy •
Imola Wilhelm • Judit Molnár • István A. Krizbai
Zsolt Szegletes • György Váró
•
Received: 11 August 2011 / Revised: 6 October 2011 / Accepted: 11 October 2011 / Published online: 30 October 2011
Ó European Biophysical Societies’ Association 2011
Abstract Mechanical parameters play a crucial role in
proper cellular functions. This article examines the process
of the appearance and breaking of adhesion forces during
contact between the confluent cerebral endothelial cell
layer and a melanoma cell attached to a tipless cantilever.
This adhesion is the initial phase of melanoma transmigration through the endothelial cell layer. Taking the force
measurement, if the contact was prolonged for several
seconds, a decrease in the load force was observed, which
corresponds to stress relaxation of the cells. The dependence of adhesion force and stress relaxation on dwell time
showed a saturation-like behavior. These stress relaxation
curves could be fitted with the sum of two exponentials,
suggesting that two independent processes take place
simultaneously. The breakup of the adhesion during the
retraction of the cantilever with the attached melanoma cell
is not continuous but shows jumps. Between living endothelial and melanoma cells, a minimum jump size of about
20 pN could be determined. The minimum jump is independent of the dwell time and load force. It seems to be the
elementary binding force between these two cell types. In
case of fixed endothelial cells, the adhesion force was
strongly decreased and the jumps disappeared, whereas the
stress relaxation did not show considerable change upon
fixation.
Keywords Atomic force microscopy Cell elasticity Endothelium Cell–cell interaction Blood-brain barrier
A. G. Végh C. Fazakas K. Nagy I. Wilhelm J. Molnár I. A. Krizbai Z. Szegletes G. Váró (&)
Institute of Biophysics, Biological Research
Centre of the Hungarian Academy of Sciences,
6726 Szeged, Hungary
e-mail: [email protected]
Introduction
Since the invention of the atomic force microscope (AFM) in
1986 (Binnig et al. 1986), the instrument has evolved into a
high-resolution imaging tool capable of determining the micromechanical properties of samples, such as the local elasticity
and viscosity of the studied object (Santos and Castanho 2004;
Vinckier and Semenza 1998; Willemsen et al. 1999). These
properties are increasingly being used in the characterization of
different biological samples (Ando 2003). Proper morphology
and elasticity are essential for multicellular structures. Alterations in cell mechanics induced by various factors can lead to
improper cell or even tissue function (Panorchan et al. 2011;
Moreno-Flores et al. 2010a; Zhang et al. 2009). Understanding
the rules of single cell and intercellular mechanics could open
new insights into cellular processes, including the response to
different stress factors or cell-cell interactions (Moreno-Flores
et al. 2010b; Rabinovich et al. 2005).
Micro-mechanical properties are determined by force
measurement with AFM. The curve resulting from the tip
approaching and pushing the sample surface with a load force
indents the sample, characterized by the local elasticity of the
probed object (Dimitriadis et al. 2002; Fritz et al. 1997; Sen
et al. 2005). The retracting branch characterizes the adhesion
force appearing between the probe and sample (Eastman and
Zhu 1996; Puech et al. 2006; Sen et al. 2005). Adhesion forces
can be measured between the sample and a surface modified
tip also, or by binding a functional group or even a monitoring
cell to the cantilever (Berger et al. 1995; Eastman and Zhu
1996). To study cell–cell interactions, a technique was
developed using a cell bound to the AFM cantilever as the
probe during the measurement (Zhang et al. 2006). It was
shown that the cadherin-mediated cell-cell interaction has a
minimal binding force in the range of 50 pN (Panorchan et al.
2011; Zhang et al. 2009). By acting on the cells, a stress
123
Eur Biophys J (2012) 41:959–968
DOI 10.1007/s00249-012-0854-0
ORIGINAL PAPER
j-Casein terminates casein micelle build-up by its ‘‘soft’’
secondary structure
Krisztina Nagy • György Váró • Balázs Szalontai
Received: 15 February 2012 / Revised: 11 June 2012 / Accepted: 11 June 2012 / Published online: 27 September 2012
Ó European Biophysical Societies’ Association 2012
Abstract In our previous paper (Nagy et al. in J Biol
Chem 285:38811–38817, 2010) by using a multilayered
model system, we showed that, from a-casein, aggregates
(similar to natural casein micelles) can be built up step by
step if Ca-phosphate nanocluster incorporation is ensured
between the protein adsorption steps. It remained, however,
an open question whether the growth of the aggregates can
be terminated, similarly to in nature with casein micelles.
Here, we show that, in the presence of Ca-phosphate
nanoclusters, upon adsorbing onto earlier a-casein surfaces,
the secondary structure of a-casein remains practically
unaffected, but j-casein exhibits considerable changes in
its secondary structure as manifested by a shift toward
having more b-structures. In the absence of Ca-phosphate,
only j-casein can still adsorb onto the underlying casein
surface; this j-casein also expresses considerable shift
toward b-structures. In addition, this j-casein cover terminates casein aggregation; no further adsorption of either
a- or j-casein can be achieved. These results, while
obtained on a model system, may show that the
Ca-insensitive j-casein can, indeed, be the outer layer of
the casein micelles, not only because of its ‘‘hairy’’ extrusion
into the water phase, but because of its ‘‘softer’’ secondary
structure, which can ‘‘occlude’’ the interacting motifs
serving casein aggregation. We think that the revealed nature of the molecular interactions, and the growth mechanism
found here, might be useful to understand the aggregation
process of casein micelles also in vivo.
K. Nagy G. Váró B. Szalontai (&)
Institute of Biophysics, Biological Research Centre,
Hungarian Academy of Sciences, P.O.B. 521,
Szeged 6701, Hungary
e-mail: [email protected]
Keywords a-Casein j-Casein Casein micelle Atomic force microscopy (AFM) Attenuated total
reflection Fourier-transform infrared (ATR-FTIR)
Introduction
Caseins, the most abundant proteins in milk, are responsible for the transport of calcium phosphate (CaP), which is
essential for bone development of mammalian neonates. In
milk, caseins form micelles, which have roughly spherical
shapes and whose sizes may vary between 150 and
300 nm. These casein micelles can be made up from four
different caseins [a(s1), a(s2), b, and j] (Walstra and
Jennes 1984). a-Casein and b-casein are calcium sensitive,
while j-casein is insensitive to calcium. As the principle of
casein micelle organization, two alternative models have
emerged (for reviews see Farrell et al. 2006; Horne 2002,
2006; Walstra 1999). According to the submicelle model,
caseins first form small subunits called submicelles
(*15–20 nm); these units are then connected with each
other, forming the micelles (Walstra 1999). Recently, the
coexistence of submicelles with casein micelles in thin,
spin-coated films has been shown (Müller-Buschbaum
et al. 2007). The other—copolymer—model does not
assume submicelles; it rather considers the CaP nanoclusters as nuclei for micelle growth by making contact with
the phosphoseryl residues of several casein molecules
(Holt et al. 2003).
Previously, we found *20-nm-sized features in
a-casein films, which were built layer by layer from very
dilute (0.1 mg ml-1) solution onto polyelectrolyte films
(Nagy et al. 2010). In all those experiments, first, CaP
nanoclusters had to be formed on the surface of the terminating layer, and then a-casein could adsorb again. If the
123
Chapter 11
The Role of Membrane Structure in Acclimation
to Low-Temperature Stress
Balázs Szalontai
Institute of Biophysics, Biological Research Centre, Hungarian Academy of Sciences,
Szeged H-6701, Hungary
Ildikó Domonkos and Zoltán Gombos*
Institute of Plant Biology, Biological Research Centre, Hungarian Academy of Sciences,
Szeged H-6701, Hungary
Summary ................................................................................................................................................................233
I. Introduction .......................................................................................................................................................234
II. Low-Temperature Stress: Changes in Membrane Composition
in Cyanobacteria and in Higher Plants..............................................................................................................235
A. Cyanobacteria ....................................................................................................................................235
1. Lipids .......................................................................................................................................235
2. Carotenoids ..............................................................................................................................237
B. Higher Plants .....................................................................................................................................238
1. Phosphatidylglycerol, a Key Lipid in Low-Temperature Stress Resistance ..............................238
III. Structural Changes in Thylakoid Membranes ...................................................................................................240
A. Lipid Dynamics ..................................................................................................................................240
B. Protein Structure ................................................................................................................................244
C. Lipid-Protein Interaction .....................................................................................................................245
Acknowledgements.................................................................................................................................................247
References .............................................................................................................................................................247
Summary
All photosynthetic protein complexes are embedded in membranes, and lipids surround these complexes.
These lipids are indispensable not only in maintaining the functional state/conformation of the photosynthetic reaction centers, but are also needed for protection against environmental stress conditions such as
cold and chilling. The redox enzymes, elements of the photosynthetic electron transport chain, which
transform the physical energy of the photons to chemical potential, are also in the photosynthetic complexes. During low-temperature stress conditions, the rate of photosynthetic electron transport decreases.
This decrease can be enhanced further by exposing the photosynthetic apparatus to high light-intensities.
In this chapter, we concentrate on the role of the photosynthetic membrane, especially on the structural
aspects of the lipids involved in the protection against low-temperature stress. Genetic manipulations of
higher plants and cyanobacteria have suggested that unsaturation of lipids plays an important role in preserving photosynthetic functions at low temperatures. Lipids, in particular phosphatidylglycerol, the only
*Author for correspondence, e-mail: [email protected]
J.J. Eaton-Rye, B.C. Tripathy and T.D. Sharkey (eds.), Photosynthesis: Plastid Biology, Energy Conversion
and Carbon Assimilation, Advances in Photosynthesis and Respiration 34, pp. 233–250,
DOI 10.1007/978-94-007-1579-0_11, © Springer Science+Business Media B.V. 2012
233
Volume 56(2):93-107, 2012
Acta Biologica Szegediensis
http://www.sci.u-szeged.hu/ABS
REVIEW ARTICLE
What have we learned from two-pore potassium channels?
Their molecular configuration and function in the human
heart
Viktória Szûts1*, Ferenc Ötvös1, László Dézsi2, Csaba Vágvölgyi3, Balázs Szalontai4,
Halina Dobrzynski5, Mark Boyett5, Henggui Zhang6, Julius G Papp7,8, András Varró7,8,
Sándor Benyhe1, Lajos Erdélyi9
1
Institute of Biochemistry, Biological Research Center, Hungarian Academy of Science, Szeged, Hungary , 2Institute of Human
Physiology and Clinical Experimental Research, University of Semmelweis, Budapest, Hungary 3Department of Microbiology,
Faculty of Science and Informatics, University of Szeged, Szeged, Hungary, 4Institute of Biophysics, Biological Research
Center, Hungarian Academy of Science, Szeged, Hungary, 5Cardiovascular Medicine, Faculty of Medical and Human Sciences,
University of Manchester, Core Technology Facility, Manchester, United Kingdom, 6Physics & Astronomy, The University
of Manchester, Manchester, United Kingdom, 7Division of Cardiovascular Pharmacology, Hungarian Academy of Sciences,
Szeged, Hungary, 8Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, University of Szeged, Hungary,
9
Department of Physiology, Morphology and Neuroscience, Faculty of Science and Informatics, University of Szeged, Szeged,
Hungary
Two-pore domain potassium channels (K2P) control excitability, stabilize the resting
membrane potential below firing threshold, and accelerate repolarisation in different cells. Until
now, fifteen different genes for the six K2P channel subfamily were cloned. The pore-forming
part is translated from two genes and they are built up from a dimer of two two-unit transmembrane domains functioning with a wide spectrum of physiological profiles. K2P ion channels
were discovered in the last two decades and gave novel opportunity to recognize the complex
molecular mechanism of the potassium ion flux, and may lead to the design of individual drug
targeting in the future. In this review, we summarise the structure, function, channelopathies
and pharmacological silhouette of the two-pore potassium channels in the human tissues. In
addition, we present the computer model of the partially reconstructed wild type K2P1/TWIK1
Acta Biol Szeged 56(2):93-107 (2012)
lacking the intracellular C and N terminal loops.
ABSTRACT
The structure of the two-pore domain channels
The inward rectiÞer potassium channels (IKI) are responsible
for the time course of the action potential (AP) (Dhamoon et
al. 2005). IK1 and the background currents (IKb), both called
leak currents, contribute to the resting membrane potential
(Lesage et al. 2000a; Bayliss et al. 2008), and strongly inßuence the Þnal repolarisation in cardiomyocytes, renal and
neuronal cells (PŽrier et al. 1994; Nichols et al. 1997; Karle et
al. 2002; Nerbonne et al. 2005; Millar et al. 2006; Bayliss et
al. 2008). Furthermore, they also regulate diastolic membrane
conductance (Dhamoon et al. 2004; Miake et al. 2003; Zobel
et al. 2003) in the heart. During the time course of the AP,
both IKI and IKb currents are active but their molecular basis
is poorly understood in the heart and kidney, contrary to the
brain. The strong inward rectiÞer current is determined by
Kir2.x ion channels, while the IKb current is conducted by
the two-pore ion channels (K2P) (Lesage et al. 1996a, 2000a;
Accepted May 11, 2012
*Corresponding author. E-mail: [email protected], [email protected]
KEY WORDS
inward rectifier channels
K2P, two-pore domain
potassium ion channels
TWIK-1 prediction model
Goldstein et al. 2001; Gierten et al. 2008). Although, they are
structurally very different (Nerbonne et al. 2001, 2005), both
the Kir2.x and K2P channel subunits are thought to establish
the structural and molecular basis of the IK1 ion channels in
cardiomyocytes. The expression and properties of the above
mentioned K+ channels are altered in cardiac diseases (arrhythmias, Long QT syndromes, hypertrophic cardiomyopathy, heart failure) or other, i.e. renal and neuronal diseases
(Bayliss et al. 2008; Hedley et al. 2009; Gaborit et al. 2009;
Greiser et al. 2009; Es-Salah-Lamoureux et al. 2010).
K2P structure and their nomenclature
In the literature, the conventional name is frequently used
instead of the systematic nomenclature accepted by the
Human Genome Organization (HUGO; Goldstein et al.
2005) for genes (KCNK1-18) and proteins (K2P1-18) of K2P
channels. In this review, we mark both the latest and the old
nomenclature.
Lesage et al. (1996a, b) have reported the structure of the
Þrst member of the two-pore channel family, although it was
93
Neuroscience 225 (2012) 44–54
INTRA-AXONAL CALCIUM CHANGES AFTER AXOTOMY IN
WILD-TYPE AND SLOW WALLERIAN DEGENERATION AXONS
R. ADALBERT, a* G. MORREALE, a à M. PAIZS, b
L. CONFORTI, a§ S. A. WALKER, a H. L. RODERICK, a,c
M. D. BOOTMAN, a L. SIKLÓS b AND M. P. COLEMAN a
We conclude that there is little relationship between calcium
distribution and the early stages of Wallerian degeneration
at the time points studied in vivo or in vitro but that WldS
neurites fail to show a later calcium rise that could be a
cause or consequence of the later stages of Wallerian
degeneration. Ó 2012 IBRO. Published by Elsevier Ltd. All
rights reserved.
a
The Babraham Institute, Babraham Research Campus, Babraham,
Cambridge CB22 3AT, United Kingdom
b
Institute of Biophysics, Biological Research Center,
Szeged H-6701, Hungary
c
Department of Pharmacology, University of Cambridge,
Tennis Court Road, Cambridge CB2 1PD, United Kingdom
Key words: slow Wallerian degeneration, Schmidt–
Lanterman cleft, axotomy, calcium distribution.
Abstract—Calcium accumulation induces the breakdown of
cytoskeleton and axonal fragmentation in the late stages of
Wallerian degeneration. In the early stages there is no
evidence for any long-lasting, extensive increase in intraaxonal calcium but there does appear to be some redistribution. We hypothesized that changes in calcium distribution
could have an early regulatory role in axonal degeneration
in addition to the late executionary role of calcium.
Schmidt–Lanterman clefts (SLCs), which allow exchange
of metabolites and ions between the periaxonal and extracellular space, are likely to have an increased role when
axon segments are separated from the cell body, so we used
the oxalate-pyroantimonate method to study calcium at
SLCs in distal stumps of transected wild-type and slow Wallerian degeneration (WldS) mutant sciatic nerves, in which
Wallerian degeneration is greatly delayed. In wild-type
nerves most SLCs show a step gradient of calcium distribution, which is lost at around 20% of SLCs within 3 mm of the
lesion site by 4–24 h after nerve transection. To investigate
further the association with Wallerian degeneration, we
studied nerves from WldS rats. The step gradient of calcium
distribution in WldS is absent in around 20% of the intact
nerves beneath SLCs but 4–24 h following injury, calcium
distribution in transected axons remained similar to that in
uninjured nerves. We then used calcium indicators to study
influx and buffering of calcium in injured neurites in primary
culture. Calcium penetration and the early calcium increase
in this system were indistinguishable between WldS and
wild-type axons. However, a significant difference was
observed during the following hours, when calcium
increased in wild-type neurites but not in WldS neurites.
INTRODUCTION
Axonal degradation during Wallerian degeneration is a
calcium-mediated event (Schlaepfer, 1974). Calcium
has long been known to activate proteases in the late
stages of Wallerian degeneration (Schlaepfer and
Hasler, 1979) but the intra-axonal calcium distribution is
altered earlier after lesion in myelinated axons, well
before axons fragment at around 36 h after transection
(Beirowski et al., 2004, 2005). Using the oxalatepyroantimonate method to fix and identify calcium at the
ultrastructural level, it was shown that in uninjured, wildtype axons there are discrete gradients of axoplasmic
calcium that produce a characteristic pattern of
precipitate, decreasing in the axoplasm beneath the
Schmidt–Lantermann clefts (SLCs) and in the paranodal
regions at the node of Ranvier (Mata et al., 1987).
Within 4 h of nerve injury, long before any steady
increase in total calcium levels, the uneven distribution
of calcium precipitate below the majority of SLCs is lost
(Mata et al., 1986).
SLCs are funnel-shaped disruptions in the regular
configuration of the myelin sheath of peripheral nerve
fibers, subdividing the nerve into irregular segments,
which were formerly interpreted as shearing defects of
the myelin sheath (Robertson, 1958). Recent findings
that dextran tracers show penetration in intermodal
regions of nerves in a configuration consistent with
movement through SLCs (Mierzwa et al., 2010), support
the view that SLCs are open to slow diffusion of
metabolites (Rosenbluth, 2009) and contribute to
intracellular ionic homeostasis (Kamasawa et al., 2005).
Furthermore, the altered appearance, i.e. increased
prominence of pre-existing incisures during Wallerian
degeneration (Krinke et al., 1986) and the increased
occurrence of SLCs in nerves consisting of new myelin
after chronic nerve compression (Berger and Gupta,
*Corresponding author. Tel: +44-1223-496351; fax: +44-1223496348.
E-mail address: [email protected] (R. Adalbert).
Equal contribution.
à
Present address: Centro di Ricerca per la Viticoltura, CRA-VIT,
Viale XXVIII Aprile 26, 31015 Conegliano (TV), Italy.
§
Present address: School of Biomedical Sciences, University of
Nottingham, Medical School Queen’s Medical Centre, Nottingham NG7
2UH, United Kingdom.
Abbreviations: ANOVA, analysis of variance; DRG, dorsal root ganglia;
EDDs, electron-dense deposits; ESI, electron spectroscopic imaging;
SCG, superior cervical ganglia; SD, Sprague–Dawley; SLCs, Schmidt–
Lanterman clefts; WldS, slow Wallerian degeneration.
0306-4522/12 $36.00 Ó 2012 IBRO. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.neuroscience.2012.08.056
44
Exposure to Lipopolysaccharide and/or Unconjugated
Bilirubin Impair the Integrity and Function of Brain
Microvascular Endothelial Cells
Filipa L. Cardoso1, Ágnes Kittel2, Szilvia Veszelka3, Inês Palmela1, Andrea Tóth3, Dora Brites1,4,
Mária A. Deli3, Maria A. Brito1,4*
1 Research Institute for Medicines and Pharmaceutical Sciences, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal, 2 Institute of Experimental Medicine,
Hungarian Academy of Sciences, Budapest, Hungary, 3 Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre, Hungarian Academy of
Sciences, Szeged, Hungary, 4 Department of Biochemistry and Human Biology, Faculty of Pharmacy, University of Lisbon, Lisbon, Portugal
Abstract
Background: Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though
lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of
unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells
(BMEC).
Methodology/Principal Findings: Monolayers of primary rat BMEC were treated with 1 mg/ml LPS and/or 50 mM UCB, in the
presence of 100 mM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB
model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell
death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in monocultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough
endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were
noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds
triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures.
The functional changes were confirmed by alterations in immunostaining for junctional proteins b-catenin, ZO-1 and
claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure
to LPS and UCB.
Conclusions: LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action.
Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the
brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal
period.
Citation: Cardoso FL, Kittel Á, Veszelka S, Palmela I, Tóth A, et al. (2012) Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and
Function of Brain Microvascular Endothelial Cells. PLoS ONE 7(5): e35919. doi:10.1371/journal.pone.0035919
Editor: Peter Csermely, Semmelweis University, Hungary
Received September 15, 2011; Accepted March 27, 2012; Published May 7, 2012
Copyright: ß 2012 Cardoso et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant PTDC/SAU-FCF/68819/2006 of the Fundaçãopara a Ciência e a Tecnologia (FCT), Lisbon, Portugal. No competing
financial interests exist. The funder (FCT, http://alfa.fct.mctes.pt) 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: [email protected]
cohesive intercellular junctional complexes, composed of tight
junctions (TJs) and adherens junctions (AJs). TJs are formed by
transmembrane proteins like claudins, occludin, tricellulin,
junctional adhesion molecules, and cytoplasmic proteins, like the
zonula occludens (ZO) family [2]. TJs are responsible for high
transendothelial electrical resistance (TEER) and low paracellular
permeability at the BBB [3,4]. AJs are constituted by the
transmembrane proteins vascular endothelial cadherins, nectins,
platelet-endothelial cell adhesion molecule, and by the cytoplasmic catenins, comprising b-catenin [5]. BMEC, pericytes and
astrocytes share a thick basement membrane that is composed of
various extracellular matrix (ECM) classes of molecules [1].
Matrix metalloproteinases (MPPs) are known to digest basement
Introduction
The blood-brain barrier (BBB) is a dynamic interface between
blood and brain compartments that protects nerve tissue from
insults. Brain microvascular endothelial cells (BMEC), possessing
unique properties, are considered the main constituents of the
barrier. They regulate the selective passage of substances through
the expression of specific influx and efflux transport systems [1].
ATP-binding cassette (ABC) transporters, such as the efflux
transporter P-glycoprotein (P-gp), export potentially toxic compounds. A relevant transcellular vesicular transport mechanism at
the BBB occurs through caveolae, which are dynamic pieces of
membrane enriched in cholesterol and sphingolipids, as well as in
the structural protein caveolin-1 [1]. Additionally, BMEC display
PLoS ONE | www.plosone.org
1
May 2012 | Volume 7 | Issue 5 | e35919
Author's personal copy
European Journal of Pharmaceutics and Biopharmaceutics 82 (2012) 340–351
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutics and Biopharmaceutics
journal homepage: www.elsevier.com/locate/ejpb
Research paper
Comparison of brain capillary endothelial cell-based and epithelial
(MDCK-MDR1, Caco-2, and VB-Caco-2) cell-based surrogate blood–brain barrier
penetration models
Éva Hellinger a, Szilvia Veszelka b, Andrea E. Tóth b, Fruzsina Walter b, Ágnes Kittel c, Mónika Laura Bakk a,
Károly Tihanyi a, Viktor Háda d, Shinsuke Nakagawa e,f, Thuy Dinh Ha Duy e,f, Masami Niwa e,f,
Mária A. Deli b, Monika Vastag a,⇑
a
Division of Pharmacology and Drug Safety Research, Gedeon Richter Plc., Budapest, Hungary
Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
c
Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest, Hungary
d
Spectroscopic Research, Gedeon Richter Plc., Budapest, Hungary
e
Department of Pharmacology 1, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan
f
BBB Laboratory, PharmaCo-Cell Co. Ltd., Nagasaki, Japan
b
a r t i c l e
i n f o
Article history:
Received 1 March 2012
Accepted in revised form 31 July 2012
Available online 11 August 2012
Keywords:
Blood–brain barrier
Brain endothelial cell
VB-Caco-2
MDCK-MDR1
Surrogate BBB model
P-glycoprotein
a b s t r a c t
An accurate means of predicting blood–brain barrier (BBB) penetration and blood–brain partitioning of
NCEs (new chemical entities) would fulfill a major need in pharmaceutical research. Currently, an industry-standard BBB drug penetration model is not available. Primary brain capillary endothelial cells,
optionally co-cultured with astrocytes and/or pericytes, are the most valued models of BBB. For routine
use, establishing and maintaining a co-culture system is too costly and labor intensive. Alternatively,
non-cerebral cell lines such as MDCK-MDR1 are used, and most recently, the suitability of native and
modified Caco-2 for predicting brain penetration has also come under investigation. This study provides
comparative data on the morphology and functionality of the high integrity brain capillary endothelial
BBB model (EPA: triple culture of brain capillary endothelial cells with pericytes and astrocytes) and
the epithelial cell-based (native Caco-2, high P-glycoprotein expressing vinblastine-treated VB-Caco-2
and MDCK-MDR1) surrogate BBB models. Using a panel of 10 compounds VB-Caco-2 and MDCK-MDR1
cell lines show restrictive paracellular pathway and BBB-like selective passive permeability that makes
them comparable to the rat brain BBB model, which gave correlation with the highest r2 value with
in vivo permeability data. In bidirectional assay, the VB-Caco-2 and the MDCK-MDR1 models identified
more P-glycoprotein drug substrates than the rat brain BBB model. While the complexity and predictive
value of the BBB model is the highest, for the screening of NCEs to determine whether they are efflux substrates or not, the VB-Caco-2 and the MDCK-MDR1 models may provide a simple and inexpensive tool.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
The insufficient presence of drugs at their brain targets due to
the barrier function of brain capillary endothelial cells is a common
cause of failure of drugs that target the central nervous system
(CNS). An important feature of the brain capillary endothelial cells
that form the blood–brain barrier (BBB) is that they exert a strict
control over molecular movements between the brain and periphery through the expression of a wide range of brain-specific, high
activity uptake, and efflux transporters [1–4].
⇑ Corresponding author. Division of Pharmacology and Drug Safety Research,
} i út. 19-21, Budapest, Hungary. Tel.: +36 1 431
Gedeon Richter Plc., H-1103 Gyömro
4803; fax: +36 1 889 8400.
E-mail address: [email protected] (M. Vastag).
0939-6411/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejpb.2012.07.020
It is clear that BBB transporters such as P-glycoprotein (P-gp,
MDR1), and drug binding to plasma proteins or brain tissue, may
drastically modify the distinct processes of (1) the rate, (2) the extent of drug penetration, and (3) the intra brain drug distribution,
which all affect the success of drug therapy [5,6].
In drug discovery for the prediction of brain penetration, several
types of models are used. Such models are in silico prediction,
PAMPA (parallel artificial membrane permeability assay), cell culture-based approaches, and also animal models (BUI, in situ perfusion, etc.) as reviewed recently [7,8]. PAMPA is a high throughput
and low cost method for prediction of passive brain penetration
in early phase of drug research for screening compounds [9]. Due
to the higher complexity of information derived on both passive
penetration and active transport processes, cell cultures are the
next favored tools for BBB drug penetration modeling [7]. Basically,
J Neural Transm (2012) 119:115–121
DOI 10.1007/s00702-011-0726-2
BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - ORIGINAL ARTICLE
Preparation and properties of nanoscale containers for biomedical
application in drug delivery: preliminary studies with kynurenic
acid
V. Hornok • T. Bujdosó • J. Toldi • K. Nagy
I. Demeter • C. Fazakas • I. Krizbai •
L. Vécsei • I. Dékány
•
Received: 17 June 2011 / Accepted: 13 October 2011 / Published online: 8 November 2011
Ó Springer-Verlag 2011
Abstract The main purpose of this study was to facilitate
the delivery of kynurenic acid (KYNA) across the blood–
brain barrier (BBB) by applying micelles as nanoscale
containers. Non-ionic amphiphilic molecules were used for
preparation of spherical micelles for delivery of kynurenic
acid in aqueous solution in physiological condition. It was
established that Triton X 100 and Lutensol AP 20 nonionic surfactants are able to produce stable nanocontainers
for delivery of kynurenic acid molecules. The incorporation of KYNA molecules was investigated by dynamic
light scattering and the size of micelles were calculated
between 5 and 10 nm in 150 mM NaCl and pH 7.5–7.6
solutions. Encapsulated kynurenic acid showed a significantly higher blood–brain barrier permeability compared
with non-encapsulated kynurenic acid. The in vivo experiments showed that the encapsulated kynurenic acid is able
to display effects within the central nervous system, even
after its peripheral administration.
V. Hornok T. Bujdosó I. Dékány (&)
Supramolecular and Nanostructured Materials Research Group
of the Hungarian Academy of Sciences, Faculty of Medicine,
Institute of Medical Chemistry, and Nanomedicine Program,
University of Szeged, Aradi v.t.1, Szeged 6720, Hungary
e-mail: [email protected]
J. Toldi K. Nagy I. Demeter
Deparment of Physiology, Anatomy and Neuroscience,
University of Szeged, Közép fasor 52, Szeged 6726, Hungary
C. Fazakas I. Krizbai
Institute of Biophysics, Biological Research Centre
of the Hungarian Academy of Sciences,
Temesvári krt. 62, Szeged 6726, Hungary
L. Vécsei
Faculty of Medicine, Institute of Neuroscience,
University of Szeged, Semmelweis u 6, Szeged 6720, Hungary
Keywords Non-ionic surfactants Micelles Kynurenic acid Nanocontainers Blood–brain barrier Encapsulation
Introduction
Molecules with long alkyl chains and polar head groups (so
called amphiphilic surfactant molecules) spontaneously
self-organize into a variety of structures in aqueous solutions (Evans and Wennerström 1994; Lindman and Wennerström 1980; Stauffer et al. 1994; Stilbs et al. 1983;
Szleifer et al. 1987). The simplest and best understood of
these is the spherical micelle (Evans and Wennerström
1994). To characterize the amphiphilic aggregation process, we can begin by considering how adding surfactant to
water leads to the formation of this typical structure (Evans
and Wennerström 1994; Lindman and Wennerström 1980;
Szleifer et al. 1987). It is well known from the surfactant
literature that the dual-character molecule possessing the
well-defined polar head and nonpolar tail needed to produce amphiphilic behaviour is a sodium dodecyl sulfate
(SDS), whose structure is given in Fig. 1a. In aqueous
solutions up to 8 9 10-3 M, most of the properties SDS
displays are similar to those we can observe for a typical
electrolyte such as NaCl. The insolubility of SDS’s
hydrocarbon chain in water causes the molecule to concentrate in the inner part of the micelle producing a
‘‘hydrophobic environment like a nanocontainer for
hydrophobic small molecules‘‘ (Evans and Wennerström
1994; Lindman and Wennerström 1980; Szleifer et al.
1987). If we used higher concentration from surfactant
molecules they can aggregate in bilayer structure and form
liquid crystalline structures as we can see in Fig. 1b or
cylindrical structure like in Fig. 1c. We can write the
123
Journal of
Materials Chemistry
View Online / Journal Homepage / Table of Contents for this issue
C
Dynamic Article Links <
Cite this: J. Mater. Chem., 2012, 22, 6429
PAPER
www.rsc.org/materials
Downloaded by Agency for Science, Technology & Research (A*STAR) on 13 March 2012
Published on 23 February 2012 on http://pubs.rsc.org | doi:10.1039/C2JM15755A
Organometallic polymeric carriers for redox triggered release of molecular
payloads†
Dominik Ja
nczewski,a Jing Song,a Erzsebet Csanyi,b Lorand Kiss,c Peter Blazso,d Robert L. Katona,d
M
aria A. Deli,c Guillaume Gros,a Jianwei Xua and G. Julius Vancso‡*a
Received 9th November 2011, Accepted 12th January 2012
DOI: 10.1039/c2jm15755a
The synthesis and characterization of a novel redox responsive comb-copolymer consisting of
a poly(ferrocenylsilane) backbone and N-dimethylethyl ammonium and N-dimethyldecyl ammonium
substituents are reported. Due to the presence of the side groups featuring cationic amine as well as
decyl hydrocarbon chains the comb copolymer exhibits amphiphilic behaviour and forms micellar
assemblies with typical dimensions of 100 nm. The assemblies display unique, redox induced
morphology change in water, investigated by dynamic light scattering and transmission electron
microscopy. Paclitaxel and Nile Red were encapsulated in the micelles as model guest molecular
payloads. Release of the guests with a high degree of profile control by varying the concentration of
redox agents is presented.
Introduction
Nature provided us with unique, tailored solutions at the nanoscale for molecular delivery and submicrometre compartmentalization. Virus capsids, perfectly crafted for the nucleic acid
delivery, or lysosomes, playing a vital role as intracellular reactors, are just some examples of a wide family of naturally
occurring vehicles,1 with sizes of a few dozen nanometres.2 Drug
and biomolecular delivery, which is a traditional field for application of such devices, is looking for nano-containers with the
ability to release material upon triggered external stimuli.3
Improved delivery control could address fundamental issues of
contemporary medicine like lowering the effective dose, reducing
side effects, as well as providing solutions for an effective way to
deliver biomacromolecules. Applications of amphiphilic block-4
and comb-co-polymers5 have attracted considerable attention,
when molecules for construction of micellar compartments are
requested. Their application may become successful for many
a
Institute of Materials Research and Engineering A*STAR (Agency for
Science, Technology and Research) 3 Research Link, Singapore 117602.
E-mail: [email protected]; Fax: +31 53 4893823; Tel: +31 53 489
2974
b
Department of Pharmaceutical Technology, University of Szeged, E€
otv€
os
u. 6, H-6720 Szeged, Hungary
c
Institute of Biophysics, Biological Research Centre of the Hungarian
Academy of Sciences, Temesv
ari krt. 62, H-6726 Szeged, Hungary
d
Institute of Genetics, Biological Research Centre of the Hungarian
Academy of Sciences, Temesv
ari krt. 62, H-6726 Szeged, Hungary
† Electronic supplementary information (ESI) available: Cyclic
voltammograms and LDH graphs. See DOI: 10.1039/c2jm15755a
‡ Permanent address: Materials Science and Technology of Polymers,
Faculty of Science and Technology, University of Twente and MESA+
Institute for Nanotechnology, P. O. Box 217, AE 7500, Enschede, The
Netherlands.
This journal is ª The Royal Society of Chemistry 2012
types of encapsulation, spanning from inorganic nanoparticles6
to biologically active organic compounds.7
Redox responsive delivery vehicles could provide a suitable
solution for the release directly into intracellular compartments
with local redox gradient.8 They may also serve as materials to
fabricate devices that respond to variations of the electric
potential.9 There are various ways to incorporate the redox
trigger into a molecular delivery vehicle. Obviously, it is necessary for part of the constituting molecules to undergo a reversible
or irreversible reduction–oxidation process.10 Disulfide bridges11
and ferrocene derivatives,12 with oxidation potential versus
standard hydrogen electrode of 0.25 V and 0.6 V respectively,
are two particularly favourable solutions investigated.
Poly(ferrocenylsilane) (PFS),13 an interesting class of redox
responsive polymers, featuring alternating ferrocene and silicon
units within the polymer backbone, was also explored in the
context of redox responsive release vehicles.14 Layer by layer
(LbL) structures composed of PFS polyions15 with alternating
charges and block-copolymeric micelles16 containing the PFS
unit were assembled for this purpose.
In this contribution the synthesis and application of novel
redox active vehicles, constructed with an amphiphilic PFS comb
co-polymer, are reported. The polymer, when suspended in
water, forms composite micelles with hydrophobic pockets
suitable for molecular delivery purpose. Unlike many redox
active systems reported so far, micellar assemblies do not
decompose upon redox stimuli but display reversible
morphology transformation effectively collapsing and expanding
as a molecular sponge. Due to their suitable size, in the 100 nm
range, and their positive charge, the properties of the materials
are investigated in the context of redox triggered release of drugs
and DNA delivery.
J. Mater. Chem., 2012, 22, 6429–6435 | 6429
letters
Decreased expression of synapse-related genes and loss of
synapses in major depressive disorder
npg
© 2012 Nature America, Inc. All rights reserved.
Hyo Jung Kang1, Bhavya Voleti1, Tibor Hajszan2,3, Grazyna Rajkowska4, Craig A Stockmeier4, Pawel Licznerski1,
Ashley Lepack1, Mahesh S Majik5, Lak Shin Jeong5, Mounira Banasr1, Hyeon Son6 & Ronald S Duman1,7
Previous imaging and postmortem studies have reported a
lower brain volume and a smaller size and density of neurons
in the dorsolateral prefrontal cortex (dlPFC) of subjects with
major depressive disorder (MDD)1,2. These findings suggest
that synapse number and function are decreased in the
dlPFC of patients with MDD. However, there has been no
direct evidence reported for synapse loss in MDD, and the
gene expression alterations underlying these effects have not
been identified. Here we use microarray gene profiling and
electron microscopic stereology to reveal lower expression
of synaptic-function–related genes (CALM2, SYN1, RAB3A,
RAB4B and TUBB4) in the dlPFC of subjects with MDD and
a corresponding lower number of synapses. We also identify a
transcriptional repressor, GATA1, expression of which is higher
in MDD and that, when expressed in PFC neurons, is sufficient
to decrease the expression of synapse-related genes, cause
loss of dendritic spines and dendrites, and produce depressive
behavior in rat models of depression.
MDD affects approximately 17 percent of the US population and is
predicted to be the number two cause of illness worldwide by the year
2020 (ref. 3). The symptoms of MDD include cognitive impairment
and memory loss, implicating synaptic dysfunction in the pathophysiology of the disorder4–6. This possibility is supported by studies
using animal models of depression that have shown a reduction in
the number of dendritic spines and in the function of neurons in the
PFC7,8. Postmortem studies also report morphometric changes in the
PFC of subjects with MDD, including a smaller size of the neuronal
bodies1,9. This is also consistent with brain imaging studies reporting a smaller volume of PFC subregions in patients with MDD2,10.
Together, these studies suggest a loss of spine number and dendritic
arbor in MDD, although there is no direct evidence showing this type
of neuronal atrophy in the brains of subjects with MDD.
To gain insight into the mechanisms that underlie the neuronal
atrophy and reduced volume of the PFC, we analyzed the data from a
prior microarray gene expression study that we conducted of the dlPFCs
(Brodmann area 9) of subjects with MDD and matched controls11.
We classified the microarray data with a 5% false discovery rate11 and
found that about 30% of the downregulated genes in the MDD group
could be related to some aspect of synaptic function. A more extended
range (using a 20% false discovery rate cutoff) identified an additional
subset of downregulated genes related to synaptic function in the dlPFC
of the subjects with MDD (Supplementary Table 1). These synaptic functions include the regulation of synaptic vesicle transport and
release (calmodulin 2, synapsin I, synapsin III, Rab3A, amphiphysin and
synaptogyrin 3)12–16, the regulation of synaptic strength (neurogranin)17, dendritic spine formation (Rab4B)18 and axonal outgrowth
and regeneration (tubulins)19. It is notable that other genome-wide
expression studies of MDD have reported gene alterations in similar
functional categories (such as cytoarchitecture, vesicular transport and
synaptic transmission) of some of the same synapse-related genes20–23
that we identified here (for example, genes encoding subtypes of
amphiphysin, synaptogyrin, synapsin and the Ras superfamily)22–24.
A PCR analysis showed significantly lower (P < 0.05) expression
for five of the ten genes we identified and trends for lower expression
for all but one (amphiphysin) of the remaining genes in the dlPFC of
the subjects with MDD as compared to age-matched, nondepressed
healthy controls (Supplementary Table 1). An in situ hybridization
analysis of the five confirmed genes (CALM2, SYN1, RAB3A, RAB4B
and TUBB4) showed lower expression in the gray matter of the dlPFC
of the subjects with MDD, with a laminar distribution in the middle
(synapsin I) or the middle and deep layers (calmodulin 2, Rab3A,
Rab4B and β-tubulin 4) of the dlPFC (Fig. 1a–e). A quantitative ana­
lysis confirmed that the expression of these five genes was significantly
lower in subjects with MDD compared to individuals without MDD
(controls) (Fig. 1a–e). Studies in rats showed that chronic unpredictable
stress (CUS), which is considered one of the most valid rodent models
of depression, decreases the expression of CALM2, SYN1, RAB3A and
RAB4B, but not that of TUBB4, in the PFC (Supplementary Fig. 1),
suggesting that the decreased expression of these synapse-related
genes in MDD results from chronic stress exposure and that these
alterations could contribute to depressive behaviors.
The lower expression of synapse-related genes found here and
a previous report of smaller neuronal cell bodies in the dlPFC of
1Department
of Psychiatry, Yale University, New Haven, Connecticut, USA. 2Department of Obstetrics and Gynecology, Yale University, New Haven, Connecticut,
USA. 3Department of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary. 4Department of Psychiatry and Human Behavior,
University of Mississippi Medical Center, Jackson, Mississippi, USA. 5Department of Bioinspired Science, College of Pharmacy, Ewha Womans University, Seoul,
Korea. 6Department of Biochemistry and Molecular Biology, Hanyang University College of Medicine, Seoul, Korea. 7Department of Neurobiology, Yale University,
New Haven, Connecticut, USA. Correspondence should be addressed to R.S.D. ([email protected]).
Received 7 October 2011; accepted 23 June 2012; published online 12 August 2012; doi:10.1038/nm.2886
nature medicine VOLUME 18 | NUMBER 9 | SEPTEMBER 2012
1413
2012/4
KRIZBAI ISTVÁN – WILHELM IMOLA
A SEJTEK KÖZÖTTI
KOMMUNIKÁCIÓ ÚTJAI
A
48
A sejtek közötti
kommunikáció
különbözõ formái a
szervezetben nincsenek
[...] élesen elválasztva.
Együttesen azonban egy
olyan jól összehangolt
zenekart alkotnak,
amely nélkül
elképzelhetetlen lenne
az élet muzsikája.
földi élet kialakulásának korai fázisában, több milliárd évvel ezelõtt jelentek
meg az elsõ sejtes organizmusok. Ez az
evolúciós lépés igen sikeresnek bizonyult, és az egysejtû élõlények megjelenését hamarosan követte a többsejtû, egyre komplexebb szervezetek kialakulása. A sejt azonban mindenképp az
élõ szervezetek alapkövének tekinthetõ. A többsejtû organizmusok fennmaradásának egyik alapfeltétele azonban, hogy az egyedi sejtek összehangolják
mûködésüket, képesek legyenek kommunikálni
egymással. Írásunkban azon alapelvekre próbálunk
rávilágítani, amelyek meghatározzák a többsejtû
szervezetek sejtjei közötti információcserét.
Az egysejtû és a magasabb rendû többsejtû élõlények között az egyik legnagyobb különbség,
hogy míg az egysejtû élõlényekben az összes életfunkciót egyetlen sejt látja el, a többsejtû organizmusokban egyfajta munkamegosztás jön létre, azaz az egyes sejtek, sejtcsoportok specializált feladatok ellátására „szakosodnak”. Így jöttek létre a
különbözõ szervek és szövetek. Ahhoz azonban,
hogy a munkamegosztás zökkenõmentesen mûködjön, hatékony kommunikációs kapcsolatokra
van szükség – egyrészt olyanokra, amelyek rövid
távon egy sejtcsoport tagjai között mûködnek,
másrészt olyanokra is, amelyek egymástól távol
esõ sejtcsoportok vagy szervek között képesek
üzenet közvetítésére. A sejtek közötti (intercelluláris) kommunikáció nélkül elképzelhetetlenek
olyan élettani folyamatok, mint a fejlõdés, az immunrendszer mûködése vagy szervezetünk alkalmazkodása a külsõ környezet változó feltételeihez. Az evolúció során a sejtek közötti kommuni-
Toxicology in Vitro 26 (2012) 445–454
Contents lists available at SciVerse ScienceDirect
Toxicology in Vitro
journal homepage: www.elsevier.com/locate/toxinvit
The effect of sucrose esters on a culture model of the nasal barrier
Levente Kürti a,b, Szilvia Veszelka a, Alexandra Bocsik a, Ngo Thi Khue Dung a, Béla Ózsvári c,
László G. Puskás c, Ágnes Kittel d, Piroska Szabó-Révész b, Mária A. Deli a,⇑
a
Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Temesvári Krt. 62, H-6726 Szeged, Hungary
Department of Pharmaceutical Technology, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
c
Avidin Ltd., Közép fasor 52, H-6726 Szeged, Hungary
d
Institute of Experimental Medicine, Hungarian Academy of Sciences, Szigony u. 43, H-1083 Budapest, Hungary
b
a r t i c l e
i n f o
Article history:
Received 12 July 2011
Accepted 10 January 2012
Available online 17 January 2012
Keywords:
Cytotoxicity
Sucrose ester
Human nasal epithelial cell
RPMI 2650 cell line
Cell electronic sensing
Epithelial permeability
a b s t r a c t
Sucrose esters are effective solubilizers and there is an interest to use them as pharmaceutical excipients
for nasal drug delivery. We have determined for the first time the non-toxic doses of laurate and
myristate sucrose esters by four independent methods, and their effects on epithelial permeability using
RPMI 2650 human nasal epithelial cell line. Based on real-time cell electronic sensing, MTT dye conversion and lactate dehydrogenase release methods reference surfactant Cremophor RH40 proved to be the
least toxic excipient, and could be used at 5 mg/mL concentration for 1 h in epithelial cells without cellular damage. The non-toxic dose of Tween 80 was 1 mg/mL, while the dose of laurate and myristate
sucrose esters that could be safely used on cells for 1 h was 0.1 mg/mL. Both the reference surfactants
and the sucrose esters significantly enhanced the permeability of epithelial cell layers for the paracellular
marker FITC-labelled 4.4 kDa dextran at 0.1 mg/mL concentration. The effects of sucrose esters on epithelial permeability were dose-dependent. These data indicate that laurate and myristate sucrose esters can
be potentially used as permeability enhancers in nasal formulations to augment drug delivery to the systemic circulation.
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Intranasal administration is an effective way to deliver drugs
into the systemic circulation as an alternative to the oral and
parenteral routes for some therapeutic agents (Chien et al.,
2008). Undoubtedly, the nasal administration of medicines has
been widely used for the treatment of topical nasal conditions such
as nasopharyngitis or allergic rhinitis for many years. The nasal
pathway may circumvent the blood–brain barrier and allow centrally acting pharmacons a direct transport route to the central
nervous system (Illum, 2000; Horvát et al., 2009). Concerning systemic delivery, drugs can be rapidly absorbed through the nasal
mucosa, resulting in a quick onset of action, and avoiding degradation in the gastrointestinal tract and first-pass metabolism in the
liver (Ugwoke et al., 2001; Arora et al., 2002).
Nasal absorption is affected by physicochemical, anatomical
and physiological, as well as formulation factors. The bioavailability of different active agents varies between 1% and 90%. Many
drugs display high bioavailability by the nasal route, particularly
if they have lipophilic characteristics (Davis and Illum, 2003). The
number of nasal formulations which are available on the market
is rapidly growing. The use of nasal administration of drugs is
⇑ Corresponding author. Tel.: +36 62 599602; fax: +36 62 433133.
E-mail address: [email protected] (M.A. Deli).
0887-2333/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tiv.2012.01.015
especially important in the treatment of migraine, severe pain
and hormone replacement (Illum, 2003).
Lipophilic drugs can be expected to demonstrate rapid and efficient absorption when given nasally, but more polar compounds
are poorly absorbed. Bioavailabilities of about 1% are to be expected for biopharmaceuticals such as insulin, calcitonin or leuproline, and even less for higher molecular weight peptides and
proteins such as growth hormone, interferons and growth factors
(Kissel and Werner, 1998). The poor uptake of drugs from the nasal
cavity can be associated with three major factors, (i) poor transport
across the nasal epithelium; (ii) possible enzymatic degradation in
the nasal cavity; (iii) rapid mucociliary clearance from the absorption site (Schmidt et al., 1998).
The critical factor for nasal absorption is the penetration across
the nasal epithelial cell layers. The pathways for absorption across
the nasal respiratory epithelium are similar to those across other
epithelia in the body. Lipophilic drugs are transported transcellularly by passive lipid-mediated free diffusion, whereas hydrophilic
compounds cross the barrier passively via the paracellular pathway,
or use active carrier- or receptor-mediated processes (Deli, 2009).
Since the junctions between epithelial cells constitute the
strongest barrier in the nasal system (Wolburg et al., 2008), considerable effort has been directed towards the development of technologies that can improve the permeation across these cells
(Illum, 2003). Methods to enhance or promote absorption by using
Chem Biol Drug Des 2012; 79: 507–513
ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01306.x
Research Article
Transport Characteristics of Endomorphin-2
Analogues in Brain Capillary Endothelial Cells
Jayapal Reddy Mallareddy1, Géza Tóth1,
Csilla Fazakas2, Judit Molnár2, Péter
} szi2, Andrzej W. Lipkowski3, István
Nagyo
A. Krizbai2 and Imola Wilhelm2,*
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
Mossakowski Medical Research Centre, Polish Academy of
Sciences, Warsaw, Poland
*Corresponding author: Imola Wilhelm, [email protected]
Because of their poor metabolic stability and limited blood–brain barrier permeability, endomorphins have a low analgesic efficacy when
administered systemically. Therefore, it is of great
importance to design analogues with improved
peptidase resistance and better delivery to the
central nervous system. Recently, novel endomorphin-2 analogues have been synthesized, which
proved to bind with high affinity and selectivity to
the l-opioid receptors and showed proteolytic
resistance. In this study, we have analysed the
transport characteristics of endomorphin-2 and
three of its analogues [Dmt-Pro-Phe-Phe-NH2, Tyr(1S,2R)Acpc-Phe-Phe-NH2 and Tyr-(1S,2R)AchcPhe-Phe-NH2] using an in vitro blood–brain barrier
model. The lipophilicity of the analogues, as
assessed by their octanol ⁄ water partition coefficients, was higher than that of endomorphin-2.
The flux of all four peptides from the apical
(blood) side to the basolateral (brain) side was not
saturable in the 10 nM–1 mM concentration range,
suggesting that a passive mechanism plays a
major role in their transport. The permeability
coefficient of the analogues was significantly
higher than that of endomorphin-2, suggesting
increased blood–brain barrier penetration properties. We conclude that because of their good peptidase resistance and improved transport through
brain endothelial cells, these endomorphin-2 analogues will have better analgesic properties
in vivo.
Key words: analogues, blood–brain barrier, cerebral endothelial
cell, endomorphin, influx, opioid peptides, transport
Received 24 August 2011, revised 28 November 2011 and accepted for
publication 3 December 2011
As key elements of the opioid system, l, d and j receptors are the
most extensively studied families among the G-protein-coupled
receptors located in the central nervous system (CNS) and peripheral tissues of various mammalian species. They are involved in a
wide variety of pharmacological and physiological processes including pain perception and modulation (1). Among these receptor classes, l-opioid receptor (MOR) is the main target of analgesics in the
treatment of pain (2). The two potent endogenous opioid peptides
endomorphin-1 (EM-1, H-Tyr-Pro-Trp-Phe-NH2) and endomorphin-2
(EM-2, H-Tyr-Pro-Phe-Phe-NH2) have high affinity and selectivity for
MOR, elicite equivalent analgesia to morphine with fewer side
effects (1). It is widely accepted that the mediation of the analgesic
effects occurs within the CNS; therefore, opioid peptides should be
able to cross the blood–brain barrier (BBB) intact.
The BBB is an active interface between blood and the CNS preventing the free movement of solutes between the two compartments.
The morphological basis of the BBB is formed by cerebral endothelial
cells (CECs). Cerebral endothelial cells form a single cell layer lining
the blood vessels and are sealed with a continuous belt of tight junctions. Transport across the BBB is strictly limited through a fourfold
defence line: paracellular barrier (represented by interendothelial
junctions), transcellular barrier (assured by the low level of endocytosis and transcytosis), enzymatic barrier and efflux transporters (for
review see Ref. 3). This highly differentiated phenotype of CECs is
induced and maintained by the cross-talk with the surrounding cells
such as pericytes, astrocytes and neurons (for review see Ref. 4).
The relative impermeability of the barrier not only protects the brain
from potentially harmful substances, but also prevents different
drugs to reach therapeutically relevant concentrations in the brain.
Exogenous application of most of native opioid peptides in general
has a limited in vivo efficacy, owing to their poor metabolic stability
and limited delivery to the CNS (5). Therefore, it is of great importance to design opioid peptide analogues with improved peptidase
resistance and greater BBB permeation. In case of EM-1, introduction of d-Ala in place of Pro2 (6) and linkage of oligoarginine to the
C-terminal (7) resulted in analogues eliciting antinociceptive effect
when administered peripherally. In case of EM-2, Pro4 substitution
and cationization proved to be promising for the development of
opioid drugs successfully penetrating the brain (8). However, proteolytically stable, pharmacologically active therapeutic drugs entering
the CNS may possibly be achieved only through systematic modification of the peptide sequence.
Recently, we have synthesized EM-2 analogues showing high affinity
and selectivity to MOR and presenting enzymatic stability (9–11). In the
507
Journal of Neuroscience Methods 205 (2012) 130–138
Contents lists available at SciVerse ScienceDirect
Journal of Neuroscience Methods
journal homepage: www.elsevier.com/locate/jneumeth
Basic Neuroscience
Estimation of the total number of hippocampal CA1 pyramidal neurons: New
methodology applied to helpless rats
Riitta Miettinen a,∗ , Tibor Hajszan a,b , Anett Riedel c , Klara Szigeti-Buck a , Csaba Leranth a,d
a
Department of Obstetrics, Gynecology, and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA
Department of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
Department of Zoology and Developmental Neurobiology, Institute of Biology, University of Magdeburg, Germany
d
Department of Neurobiology, Yale University School of Medicine, New Haven, CT, USA
b
c
a r t i c l e
i n f o
Article history:
Received 28 September 2011
Received in revised form 2 December 2011
Accepted 20 December 2011
Keywords:
Depression
Antidepressant
Stereology
Optical disector
Durcupan
a b s t r a c t
We have recently reported that in the learned helplessness model of depression, the less hippocampal
spine synapses rats have, the more helpless they become. It remains unclear, however, whether the
observed synaptic changes are associated with the loss of CA1 pyramidal cells.
Cell bodies in the CA1 pyramidal layer are very densely packed, making cell counting difficult in this
hippocampal subregion. To address this issue, we developed a new approach that (1) yields excellent
preservation of the three-dimensional tissue structure; (2) utilizes osmium tetroxide to unambiguously
label nucleoli; and (3) facilitates and accelerates unbiased, reliable counting of densely packed cell bodies.
Our method provides an improved tool for studies aiming to evaluate hippocampal atrophy and cell
loss, the most characteristic features in many neurodegenerative diseases, such as Alzheimer’s disease,
temporal lobe epilepsy and ischemia, as well as in several psychiatric disorders.
Using this new method, we demonstrated no significant changes in the number of CA1 pyramidal cells
in the rat learned helplessness paradigm. In addition, volumes of the CA1 pyramidal cell layer and the
entire CA1 subfield remained unchanged among treatment groups. We conclude that previously observed
synaptic alterations in helpless rats are not associated with CA1 pyramidal cell loss. This finding suggests
that behavioral outcome in the learned helplessness paradigm is related to plastic events at the synaptic
level, rather than at the level of principal cells.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Major depressive disorder is a devastating illness (Belmaker and
Agam, 2008) with an estimated lifetime prevalence of 17% in the
United States (Kessler et al., 1994). Despite intensive research on
depression neurobiology and antidepressant mechanisms, current
clinical management of the disease remains limited (Trivedi et al.,
2006). Evidence for hippocampal atrophy in depressed patients
(Sheline et al., 1999; Frodl et al., 2002; MacMaster et al., 2008),
as well as derailment of many hippocampus-related functions in
depression (McEwen, 2003; O’Brien et al., 2004; Gallassi et al., 2006)
indicates that the hippocampus is critically involved in the disease
(Campbell and Macqueen, 2004).
Recently, we have reported that the number of hippocampal
spine synapses is inversely correlated with the severity of helpless
behavior in the rat learned helplessness (LH) model of depression,
∗ Corresponding author. Tel.: +358 0 505231458, fax: +1 203 785 7684.
E-mail addresses: riitta.miettinen@tut.fi, [email protected] (R. Miettinen).
0165-0270/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jneumeth.2011.12.017
i.e., the less synapses the animals have, the more helpless they are
(Hajszan et al., 2009, 2010). Considering the fact that neuronal and
glial cell loss (Rajkowska, 2000; Stockmeier et al., 2004) and altered
neurogenesis in the dentate gyrus (Malberg et al., 2000; Santarelli
et al., 2003; Sahay and Hen, 2007) have previously been reported to
be associated with depression and/or antidepressant treatment, it
is important to know whether the observed synapse loss in helpless
animals is associated with the loss of hippocampal pyramidal cells,
or the changes occurred exclusively at the synaptic level. Therefore,
the goal of this study was to investigate whether inescapable stress
and antidepressant treatment cause changes in the number of CA1
pyramidal cells.
This is a methodological challenge, because the somata of CA1
pyramidal cells are relatively small and very densely packed in
vivo. The packing density of these pyramidal cell bodies is further
increased due to the excessive shrinkage and flattening of sections
(especially in the z-dimension) resulting from histological procedures that are required for labeling the cells for counting.
These problems make cell counting a practical challenge and
entail a certain potential for bias even with new and putatively
4004 • The Journal of Neuroscience, March 21, 2012 • 32(12):4004 – 4016
Neurobiology of Disease
Activation of Cannabinoid Receptor 2 Attenuates
Leukocyte–Endothelial Cell Interactions and Blood–Brain
Barrier Dysfunction under Inflammatory Conditions
Servio H. Ramirez,1,3,4 János Haskó,5 Andrew Skuba,4 Shongshan Fan,1 Holly Dykstra,1 Ryan McCormick,1
Nancy Reichenbach,1 Istvan Krizbai,5 Anu Mahadevan,6 Ming Zhang,1 Ronald Tuma,2,3 Young-Jin Son,4
and Yuri Persidsky1,2,3
1Department of Pathology and Laboratory Medicine, 2Department of Physiology, 3Center for Substance Abuse Research, and 4Shriners Hospitals Pediatric
Research Center and Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, 5Institute of
Biophysics, Biological Research Center, Szeged, Hungary 6726, and 6Organix, Woburn, Massachusetts 01801
Previous studies have shown that modulation of the receptor-mediated cannabinoid system during neuroinflammation can produce potent
neuroprotective and anti-inflammatory effects. However, in this context, little is known about how selective activation of the cannabinoid type-2
receptor (CB2R) affects the activated state of the brain endothelium and blood– brain barrier (BBB) function. Using human brain tissues and
primary human brain microvascular endothelial cells (BMVECs), we demonstrate that the CB2R is highly upregulated during inflammatory
insult. We then examined whether the CB2R agonists could attenuate inflammatory responses at the BBB using a mouse model of LPS-induced
encephalitis and highly selective CB2R agonists. Visualization by intravital microscopy revealed that administration of JWH133 [(6aR,10aR)-3(1,1-dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran] or a novel resorcinol-based compound, O-1966 (1-[4-(1,1dimethyl-heptyl)-2,6-dimethoxy-phenyl]-3-methyl-cyclohexanol), greatly attenuated leukocyte adhesion in surface pial vessels and in deep
ascendingcorticalpostcapillaryvenules.BBBpermeabilityassessmentswithsmallandlargefluorescenttracersshowedthatCB2Ragonistswere
effective at preventing barrier leakiness after LPS administration. To determine whether the effects by CB2R agonists on barrier protection are
not only due to the CB2R modulation of immune cell function, we tested the agonists in vitro with barrier-forming primary BMVECs. Remarkably, the addition of CB2R agonist increased transendothelial electrical resistance and increased the amount of tight junction protein present in
membrane fractions. Furthermore, CB2R agonists decreased the induction of intercellular adhesion molecule-1 and vascular cell adhesion
molecule-1 surface expression in BMVECs exposed to various proinflammatory mediators. Together, these results suggest that pharmacological
CB2R ligands offer a new strategy for BBB protection during neuroinflammation.
Introduction
The blood– brain barrier (BBB) shields the brain parenchyma
from immune cells and toxins in the blood, thus allowing the
adequate environment needed for normal neuronal and glial cell
function. During neuroinflammation, the BBB can become impaired by the triggered inflammatory response from all the cells
of the neurovascular unit (Neuwelt et al., 2008), and particularly
from immune– endothelial cell engagement (Persidsky et al.,
Received Sept. 7, 2011; revised Jan. 10, 2012; accepted Jan. 25, 2012.
Author contributions: S.H.R. and Y.P. designed research; S.H.R., J.H., A.S., S.F., R.M., H.D., N.R., and M.Z. performed research; I.K., A.M., R.T., and Y.-J.S. contributed unpublished reagents/analytic tools; S.H.R. analyzed data;
S.H.R. and Y.P. wrote the paper.
This work was supported in part by NIH Grants RO1MH065151 R01DA025566, and R37AA015913 (Y.P.) and a
Temple University development grant (S.H.R.). J.H. was supported by a fellowship from the Hungarian-American
Enterprise Scholarship Fund. We acknowledge the NIH National NeuroAIDS Consortium for brain tissue specimens
used in this study. We thank Dr. Ellen Unterwald (Temple University School of Medicine) for assistance with the CB2R
knock-out animals.
Correspondence should be addressed to Yuri Persidsky, Department of Pathology and Laboratory Medicine,
Temple University School of Medicine, 3401 North Broad Street, Philadelphia, PA 19140. E-mail:
[email protected].
DOI:10.1523/JNEUROSCI.4628-11.2012
Copyright © 2012 the authors 0270-6474/12/324004-13$15.00/0
2006a). Therapeutic strategies that prevent the immune– endothelial interactions and maintain tightness of the barrier are critical in neuroprotection (Greenwood et al., 2002). One promising
therapeutic avenue could be activation of the endogenous cannabinoid system, which has been shown previously to induce both
anti-inflammatory and neuroprotective effects.
There are two well-characterized cannabinoid receptors with distinct physiological properties. The psychoactive effects of cannabinoids are associated with the cannabinoid type-1 receptor (CB1R),
while the CB2R mainly mediates anti-inflammatory actions (Miller
and Stella, 2008). In the brain, the anti-inflammatory and neuroprotective effects of cannabinoids have been confirmed in animal models of multiple sclerosis (MS), Alzheimer’s disease (AD), stroke, and
amyotrophic lateral sclerosis (ALS) (for review, see Cabral and
Griffin-Thomas, 2009; Zhang et al., 2009b). These effects have been
observed with pharmacological ligands that act on either the CB1R,
CB2R, or both. For example, in a viral model of MS, CB1R agonists
have been shown to reduce perivascular CD4 ⫹ T lymphocyte infiltration, inhibit microglial responses, and suppress the upregulation
of adhesion molecules in the brain endothelium (Mestre et al., 2009).
However, because of their psychoactive effects, the uses of CB1R
RESEARCH LETTER
Isolation of new Pseudomonas tolaasii bacteriophages and
genomic investigation of the lytic phage BF7
} Sajben-Nagy1, Gergely Maróti2, László Kredics1, Balázs Horváth2, Árpád Párducz3, Csaba
Eniko
Vágvölgyi1 & László Manczinger1
1
Department of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; 2Bay Zoltán Nonprofit Research Ltd,
Szeged, Hungary; and 3Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Szeged, Hungary
} Sajben-Nagy,
Correspondence: Eniko
Department of Microbiology, Faculty of
Science and Informatics, University of
Szeged, Közép fasor 52, H-6726 Szeged,
Hungary. Tel.: +3662544005; fax:
+3662544823; e-mail: [email protected]
Received 8 August 2011; revised 20 March
2012; accepted 4 May 2012.
Final version published online 28 May 2012.
DOI: 10.1111/j.1574-6968.2012.02592.x
MICROBIOLOGY LETTERS
Editor: Richard Calendar
Keywords
phage; morphological investigation; genomic
analysis; Podoviridae; Autographivirinae;
φKMV-like phages.
Abstract
Sixteen lytic bacteriophages that infect Pseudomonas tolaasii LMG 2342T were
isolated from smashed sporocarps of oyster mushroom (Pleurotus ostreatus)
showing necrotic symptoms. On the basis of the host range investigation of the
phages, they have wide infection abilities against the genus Pseudomonas,
mainly in the case of phages Bf3, Bf7, Bf10, and Bf15. Molecular investigations
have revealed that they all have dsDNA genomes about 40 kbp in size. Identical restriction patterns resulting from restriction enzyme analysis suggest that
the isolates probably belong to the same phage species. However, there was a
difference between these phage isolates in their infecting abilities. Phage isolate
Bf7 was investigated and characterized more deeply. Morphological characterization of Bf7 by transmission electron microscopy (TEM) has shown that it
has a short, noncontractile tail, an icosahedral phage head, and the size is
about 60 nm in diameter, suggesting that it belongs to the Podoviridae family.
Complete genome sequence analysis of the Bf7 phage isolate revealed a
40 058 bp genome, 58.4% G+C content, 46 open reading frames encoding different proteins showing homology to proteins of the bacteriophage Caulobacter
crescentus φCd1 from the Podoviridae family. On the basis of these results and
comparative genomic studies, we classified the Bf7 phage to the subfamily of
Autographivirinae, φKMV-like phages.
Introduction
Pseudomonas tolaasii is a Gram-negative mushroom pathogenic bacterium that is well known as the causative
agent of the yellowing of oyster mushroom (Pleurotus
ostreatus) and the brown blotch disease of champignon,
Agaricus bisporus (Bessette et al., 1985; Lee & Cha, 1998).
The mushroom infecting phenomenon was firstly
described by Tolaas (1915). The bacterium produces a
cellular membrane destructive toxin called tolaasin, which
disrupts the membrane of the mushroom via pore formation (Rainey et al., 1992). Moreover, bacterial blotch diseases can be caused by other fluorescent pseudomonads
such as Pseudomonas agarici, Pseudomonas costantinii,
Pseudomonas gingeri (Geels et al., 1994; Munsch et al.,
2002), and some Pseudomonas fluorescens bv. V strains
(Sajben et al., 2011). The infection processes have different characteristics, but the final result is usually the same:
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
the product becomes unsuitable for sale resulting in serious economic losses. Different studies investigated the
significance of fluorescent pseudomonads in the detrimental processes during cultivation and the discolorations
after harvesting in case of A. bisporus, Pleurotus pulmonarius, and Lentinula edodes (Thorn & Tsuneda, 1996; Wells
et al., 1996). There is an increasing need for appropriate
control as the application of most chemical substances
during cultivation is prohibited. There are numerous
promising investigations for the inactivation of the
browning processes with antagonistic bacteria (Fermor &
Lynch, 1988; Tsukamoto et al., 1998) and toxin neutralizing substances (Soler-Rivas et al., 1999; Tsukamoto et al.,
2002). At the same time, there are some Pseudomonas
species that play an essential role in sporocarp formation
and healthy development of mushrooms (Rainey, 1991),
so the complete exclusion of the genus from cultivation is
not a possible option. According to this, the targeted
FEMS Microbiol Lett 332 (2012) 162–169
bs_bs_banner
Neuropathology 2012; 32, 505–514
O rig i na l Ar t i cl e
neup_1286
doi:10.1111/j.1440-1789.2011.01286.x
505..514
An MND/ALS phenotype associated with C9orf72
repeat expansion: Abundant p62-positive,
TDP-43-negative inclusions in cerebral cortex,
hippocampus and cerebellum but without
associated cognitive decline
Claire Troakes,1 Satomi Maekawa,1 Lokesh Wijesekera,1 Boris Rogelj,1 László Siklós,3
Christopher Bell,1 Bradley Smith,1 Stephen Newhouse,1 Caroline Vance,1 Lauren Johnson,1
Tibor Hortobágyi,1 Aleksey Shatunov,1 Ammar Al-Chalabi,1 Nigel Leigh,1 Christopher E. Shaw,1
Andrew King1,2* and Safa Al-Sarraj1,2*
1
King’s College London, MRC Centre for Neurodegeneration Research, Department of Clinical Neuroscience,
Institute of Psychiatry, De Crespigny Park, and 2Department of Clinical Neuropathology, King’s College Hospital,
Denmark Hill, London, UK, and 3Laboratory of Molecular Neurobiology, Institute of Biophysics, Biological Research
Center, Szeged, Hungary
The transactive response DNA binding protein (TDP-43)
proteinopathies describe a clinico-pathological spectrum
of multi-system neurodegeneration that spans motor
neuron disease/amyotrophic lateral sclerosis (MND/ALS)
and frontotemporal lobar degeneration (FTLD). We have
identified four male patients who presented with the clinical features of a pure MND/ALS phenotype (without
dementia) but who had distinctive cortical and cerebellar
pathology that was different from other TDP-43 proteinopathies. All patients initially presented with weakness of
limbs and respiratory muscles and had a family history of
MND/ALS. None had clinically identified cognitive decline
or dementia during life and they died between 11 and 32
months after symptom onset. Neuropathological investigation revealed lower motor neuron involvement with TDP43-positive inclusions typical of MND/ALS. In contrast, the
cerebral pathology was atypical, with abundant star-shaped
p62-immunoreactive neuronal cytoplasmic inclusions in
the cerebral cortex, basal ganglia and hippocampus, while
Correspondence: Claire Troakes, PhD, Department of Clinical Neuroscience, Institute of Psychiatry, King’s College London, PO65, De
Crespigny Park, London SE5 8AF, UK. Email: claire.troakes@
kcl.ac.uk
*These authors contributed equally to the study.
Received 16 September 2011; revised 21 November 2011 and
accepted 22 November 2011; published online 19 December 2011.
© 2011 Japanese Society of Neuropathology
TDP-43-positive inclusions were sparse. This pattern was
also seen in the cerebellum where p62-positive, TDP-43negative inclusions were frequent in granular cells. Western
blots of cortical lysates, in contrast to those of sporadic
MND/ALS and FTLD-TDP, showed high p62 levels and
low TDP-43 levels with no high molecular weight smearing.
MND/ALS-associated SOD1, FUS and TARDBP gene
mutations were excluded; however, further investigations
revealed that all four of the cases did show a repeat expansion of C9orf72, the recently reported cause of chromosome 9-linked MND/ALS and FTLD. We conclude that
these chromosome 9-linked MND/ALS cases represent a
pathological sub-group with abundant p62 pathology in the
cerebral cortex, hippocampus and cerebellum but with no
significant associated cognitive decline.
Key words: ALS/MND, C9orf72, cerebellum, p62, TDP-43.
INTRODUCTION
The TDP-proteinopathies are a group of neurodegenerative disorders characterised by the mislocalization and
aggregation of the transactive response DNA binding
protein (TDP-43) to form cytoplasmic and nuclear inclusions within neurons and glia.1 TDP-43 regulates RNA
transcription, splicing, trafficking and microRNA biogenesis.2,3 TDP-43 predominantly resides in the nucleus where
Author's personal copy
European Journal of Pharmaceutical Sciences 47 (2012) 564–573
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Evaluation of cytotoxicity of surfactants used in self-micro emulsifying drug
delivery systems and their effects on paracellular transport in Caco-2 cell monolayer
Zoltán Ujhelyi a, Ferenc Fenyvesi a, Judit Váradi a, Pálma Fehér a, Tímea Kiss a, Szilvia Veszelka b,
Mária Deli b, Miklós Vecsernyés a, Ildikó Bácskay a,⇑
a
b
Department of Pharmaceutical Technology, Faculty of Pharmacy, Medical and Health Science Center, University of Debrecen, Hungary
Department of Biophysics, Biological Research Centre, Hungarian Academy of Sciences, Hungary
a r t i c l e
i n f o
Article history:
Received 17 January 2012
Received in revised form 5 July 2012
Accepted 5 July 2012
Available online 27 July 2012
Keywords:
Labrasol
Polysorbate
Caco-2
Cytotoxicity
Permeability
CMC
a b s t r a c t
The objective of this study was to examine the cellular effects of the members of two non-ionic amphiphilic tenside groups and their mixtures on human Caco-2 cell monolayers as dependent upon their
chemical structures and physicochemical properties. The first group of polyethylene glycol esters is
represented by Polysorbates and Labrasol alone and in blends, while the members of the second group.
Capryol 90, Capryol PGMC, Lauroglycol 90 and Lauroglycol FCC were used as propylene glycol esters. They
are increasingly used in SMEDDS as recent tensides or co-tensides to increase hydrophobic bioavailability
of a drug. Critical micelle concentration was measured by determination of surface tension. CMC refers to
the ability of solubilization of surfactants. Cytotoxicity tests were performed on Caco-2 cell monolayers
by MTT and LDH methods. Paracellular permeability as a marker of the integrity of cell monolayers, was
examined with Lucifer yellow assays combined with TransEpithelial Electrical Resistance (TEER) measurements. The effect of these surfactants on tight junctions as evidence for paracellular pathway was
also characterized. The results of cytotoxicity assays were in agreement, and showed significant differences among the cytotoxic properties of surfactants in a concentration-dependent manner. Polysorbates
20, 60, 80 are the most toxic compounds. In the case of Labrasol, the degree of esterification and lack of
sorbit component decreased cytotoxicity. If the hydrophyl head was changed from polyethylene glycol to
propylene glycol the main determined factor of cytotoxicity was the monoester content and the length of
carbon chain. In our CMC experiments, we found that only Labrasol showed expressed cytotoxicity above
the CMC. It refers to good ability of micelle solubilization of Labrasol. In our paracellular transport experiments each of polyethylene glycol surfactants (Polysorbates and Labrasol) altered TEER values, but propylene glycol esters did not modify the monolayer integrity. Polyethylene glycol esters alone and in
blends (0.05% Labrasol – 0.001% Polysorbates 20, 60, 80) were able to increase Lucifer yellow permeability significantly below the IC50 concentration. On the other hand Labrasol and Polysorbates 20 have
expressed effect on tight junctions of Caco-2 monolayer. It could be concluded that polyethylene glycol
ester-type tensides were able to enhance the paracellular permeability by the redistribution of junctional
proteins. Our results might ensure useful data for selection of suitable tensides, co-tensides and tenside
mixtures for SMEDDS formulations.
Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction
Surface-active agents are widely used in new pharmaceutical
dosage form development to improve the bioavailability of drugs
which have low solubility in water. They can influence drug permeability by modifying barriers (Fischer et al., 2011), by micellar
solubilization, membrane fluidization, ion-pair formation, and the
inhibition of efflux transporters such as P-glycoprotein. On the
⇑ Corresponding author. Address: Department of Pharmaceutical Technology,
University of Debrecen, Egyetem Square 1, Debrecen 4010, Hungary. Tel./fax: +36
52512900/22630.
E-mail address: [email protected] (I. Bácskay).
0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.07.005
other hand, surfactants can cause local irritations, membrane damage and cell death and, therefore, during formulation processes
in vitro tests are required for the investigation of cytotoxicity
and irritative effects (Menard et al., 2011).
Tensides are characterized by their critical micelle concentration (CMC) and hydrophilic–lipophilic balance (HLB) values.
Primarily, CMC refers to arrangement of surfactants into micelles
and indirectly to the solubilizing ability of lipophilic molecules
such as membrane lipids (Korhonen et al., 2004). Micellar solubilization on the intestinal membrane permeability above the CMC
can predict some lipophilic drug absorption (Miller et al., 2011).
The other important property of tensides which influences
membrane integrity is the structures of tensides. The relationship
WILHELM IMOLA – KRIZBAI ISTVÁN
MOLEKULÁRIS FORRÓDRÓTOK
Jeltovábbító folyamatok a sejtekben
A
mikor azt mondjuk, kommunikáció az
élõvilágban, általában az egyedek (állatok, emberek) közötti verbális és nonverbális információcserére gondolunk.
Vannak azonban a „kapcsolattartásnak” más szintjei is – olyannyira, hogy az élõ szervezet alapkövei, a sejtek is „társalognak” egymással. Írásunkban azt a kérdést fogjuk körüljárni, hogy milyen
folyamatok zajlanak le egy sejt belsejében, amikor
az „megválaszolja” egy másik sejt „üzenetét”. Az
emberi szervezet ugyanis mintegy 1014 (százszor
egymilliószor egymillió) sejtbõl áll, így alapvetõ
fontosságú, hogy ez a nagyszámú építõelem egymással folyamatosan kommunikáljon – anélkül
azonban, hogy zavarná a többi sejt mûködését.
Hogyan lehetséges ez?
A sejtek az információt „kódolják”, mégpedig
hírszállító molekulák formájában – ezekkel „üzennek” egymásnak. Az üzenetek azonban nemcsak a
célsejtekhez, hanem gyakran az egész szervezetbe
eljutnak (például a vérárammal), így minden sejtnek magának kell kiszûrnie a hírvivõ anyagok áradatából a neki „címzett” információkat. Olyan ez
(csak sokkal komplexebb), mint a telefonálás: egy
adott pillanatban rengeteg hívás indul, de mindegyik csak egy bizonyos kiválasztott telefont csörget meg, bárhol legyen is az. A sejteknek is vannak
„telefonszámai”, a receptorok – ezek „választják
ki” a rengeteg jelzés közül azt, amelyet nekik „címeztek”. Ez úgy történik, hogy a receptor csak azt
a hírmolekulát képes megkötni, amely szerkezeti
szempontból „hozzá illik” – ezt a molekulát nevezzük a receptor ligandjának. A ligand kapcsolódása
után a receptor az információt a sejt belsejébe to-
Napjaink
népbetegségeinek
hátterében – a szív- és
érrendszeri, valamint a
daganatos betegségekre
gondolunk – gyakran
a jelátviteli útvonalak
szabályozásának
megbomlott egyensúlya
áll.
2012/4
GENOME ANNOUNCEMENT
Dóra Dömötör,a Péter Becságh,b Gábor Rákhely,c,d György Schneider,e and Tamás Kovácsa
Department of Biotechnology, Nanophagetherapy Center, Enviroinvest Corporation, Pécs, Hungarya; Roche Magyarország Kft, Budaörs, Hungaryb; Department of
Biotechnology, University of Szeged, Szeged, Hungaryc; Institute of Biophysics, Biological Research Center, Szeged, Hungaryd; and Institute of Medical Microbiology and
Immunology, University of Pécs, Pécs, Hungarye
Erwinia amylovora is the causative agent of fire blight, a serious disease of some Rosaceae plants. The newly isolated bacteriophage PhiEaH2 is able to lyse E. amylovora in the laboratory and has reduced the occurrence of fire blight cases in field experiments. This study presents the sequenced complete genome and analysis of phage PhiEaH2.
E
rwinia amylovora, a member of Enterobacteriaceae, is the causative agent of fire blight, a serious disease of some Rosaceae
plants (8, 9). One alternative treatment to control fire blight could
be the application of bacteriophages (2, 6, 7).
We isolated E. amylovora phage PhiEaH2 from a soil sample in
Hungary. This phage demonstrated strong lytic effect against E.
amylovora in the laboratory and reduced the occurrence of fire
blight cases in a field experiment when no artificial infection was
applied (D. Dömötör, G. Schneider, G. Rákhely, B.G. Polyák, and
T. Kovács, submitted for publication). These observations indicate that this phage might be able to be used as a biocontrol agent
against this plant-pathogenic bacterium.
The genomic DNA of phage PhiEaH2 was extracted and purified by using a Roche High Pure viral nucleic acid kit (Roche
Diagnostics GmbH, Germany) according to the protocol supplied. A shotgun library was created and sequenced using two
platforms (Roche GS Junior, ABI 3500XL genetic analyzer) with
20-fold coverage of the phage genome. Open reading frame (ORF)
prediction was done by using Genemark, Baysys, and Rast. Sequence annotations were performed by Baysys and Rast. The
genomic sequence of PhiEaH2 phage is 243,050 bp in length with
a G⫹C content of 51.28 mol%. No phages against E. amylovora
with such a large genome have been sequenced before (1, 4, 5). The
genome showed 262 ORFs, and 205 ORFs were annotated as encoding hypothetical proteins; most of them had the highest similarity to the Salmonella phage SPN3US (accession no. JN641803)
(3). Additionally, 57 ORFs were annotated as functional genes.
Thirty-five ORFs were predicted to encode proteins involved in
the structure and assembly of virions, and 15 ORFS were found to
encode proteins related to nucleic acid metabolism and modification and DNA replication (thymidylate synthase, thymidylate kinase, DNA adenine methylase, endodeoxyribonuclease, RNase H,
dihydrofolate reductase, a transcriptional regulator, DNA-dependent RNA polymerase beta subunits, helicases, and an SMC domain-containing protein). One ORF encodes a protein containing
an HD domain, two ORFs encode endolysins, and another two are
for acetyltransferases. The product of one ORF is involved in amylovoran biosynthesis, and two proteins contain radical SAM superfamily domains.
In conclusion, we analyzed the complete genomic sequence of
the newly isolated E. amylovora phage PhiEaH2. PhiEaH2 is a
good candidate for use as biocontrol agent against this plantpathogenic bacterium. However, sequencing its genome revealed
the presence of the amsF gene, which codes for a protein that is
essential in amylovoran biosynthesis. This complex polysaccha-
October 2012 Volume 86 Number 19
ride is necessary for E. amylovora to evoke the pathogenic process
in the host plant. The presence of this gene in PhiEaH2 must be
taken into consideration if practical use of this phage strain is
intended.
Nucleotide sequence accession number. The complete genome sequence of E. amylovora phage PhiEaH2 has been submitted to GenBank and assigned accession number JX316028.
ACKNOWLEDGMENTS
This work was funded by the European Union and by the Hungarian
Government; projects GVOP-3.3.3-05/2.-2006-01-0045/3.0, GOP-1.1.107/1-2008-0038, GOP-1.3.2.-09-201-0023. The Hungarian National
Technology Program (projects FAGCNTER and MFCDiagn) also supported this work.
REFERENCES
1. Born Y, et al. 2011. Novel virulent and broad-host-range Erwinia amylovora bacteriophages reveal a high degree of mosaicism and a relationship to
Enterobacteriaceae phages. Appl. Environ. Microbiol. 77:5945–5954.
2. Jones JB, et al. 2007. Bacteriophages for plant disease control. Annu. Rev.
Phytopathol. 45:245–262.
3. Lee JH, Shin H, Kim H, Ryu S. 2011. Complete genome sequence of
Salmonella bacteriophage SPN3US. J. Virol. 85:13470 –13471.
4. Lehman SM, Kropinski AM, Castle AJ, Svircev AM. 2009. Complete
genome of the broad-host-range Erwinia amylovora phage phiEa21-4 and
its relationship to Salmonella phage felix O1. Appl. Environ. Microbiol.
75:2139 –2147.
5. Müller I, Kube M, Reinhardt R, Jelkmann W, Geider K. 2011. Complete
genome sequences of three Erwinia amylovora phages isolated in north
America and a bacteriophage induced from an Erwinia tasmaniensis strain.
J. Bacteriol. 193:795–796.
6. Nagy JK, Király L, Schwarczinger I. 2012. Phage therapy for plant disease
control with a focus on fire blight. Cent. Eur. J. Biol. 7:1–12.
7. Svircev AM, Castle AJ, Lehman SM. 2010. Bacteriophages for control of
phytopathogens in food production systems, 79 –102. In Sabour PM, Griffiths MW (ed), Bacteriophages in the control of food- and waterborne
pathogens. ASM Press, Washington, DC.
8. Van der Zwet T, Beer SV. 1999. Fire blight—its nature, prevention and
control: a practical guide to integrated disease management. Agriculture
Information Bulletin No. 631. U.S. Department of Agriculture, Washington, DC.
9. Vanneste JL. 2000. Fire blight: the disease and its causative agent, Erwinia
amylovora. CABI Publishing, Wallingford, United Kingdom.
Received 18 July 2012 Accepted 18 July 2012
Address correspondence to Tamás Kovács, [email protected].
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.01870-12
Journal of Virology
p. 10899
jvi.asm.org
10899
Downloaded from http://jvi.asm.org/ on November 10, 2012 by MAGYAR TUDOMANYOS AKADEMIA Szegedi Biológiai Központ
Complete Genomic Sequence of Erwinia amylovora Phage PhiEaH2
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 7 ( 2 0 1 2 ) 4 9 1 5 e4 9 2 4
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/he
Relationship between PHA and hydrogen metabolism
in the purple sulfur phototrophic bacterium Thiocapsa
roseopersicina BBS
András Fülöp a, Rita Béres a, Roland Tengölics a, Gábor Rákhely a,b,*, Kornél L. Kovács a,b
a
b
Department of Biotechnology, University of Szeged, Közép Fasor 52, Szeged 6726, Hungary
Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, Temesvári krt 62, Szeged 6726, Hungary
article info
abstract
Article history:
Purple sulfur phototrophic bacteria accumulate various storage materials, such as sulfur
Received 27 September 2011
globules, glycogen or polyhydroxy alkanoates (PHAs) under appropriate conditions. The
Received in revised form
formation of these materials requires reducing power which might be recovered upon their
30 November 2011
breakdown. This work aims at the understanding of the metabolism of PHA and its link to
Accepted 3 December 2011
the nitrogenase mediated in vivo H2 evolution in Thiocapsa roseopersicina BBS. The strain
Available online 29 December 2011
could accumulate 30% of the dry cell weight in the form of PHAs. Analysis of the genome
sequence revealed the loci involved in PHAs synthesis and degradation. Phylogenetic
Keywords:
analysis indicated independent evolution of the anabolic and catabolic proteins. A mutant
Hydrogen
carrying deleted PHA biosynthesis genes has been created in a host containing nitrogenase
Polyhydroxy alkanoates
but none of the hydrogenases. Determination of the H2 evolving capacity of the mutant
Nitrogenase
revealed significantly reduced H2 production in PHA deficient cells. Addition of excess
Thiocapsa roseopersicina
electron sources such as thiosulfate stimulated the H2 production via multiple effects.
Metabolic versatility
Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights
Purple sulfur bacteria
1.
Introduction
Hydrogen is an economically sound energy carrier which can be
produced by various biological pathways [1]. Hydrogenases are
the dedicated enzymes for hydrogen metabolism; they can
produce or oxidize H2. Nitrogenases can also produce substantial amount of H2 as the byproduct of nitrogen fixation and these
are practically unidirectional enzymes. In purple sulfur phototrophic bacteria, both enzyme systems can be involved in biohydrogen evolution while in the case of non-sulfur phototophs
no H2 evolving hydrogenase has been identified so far.
H2 formation involving either enzyme systems requires
excess electrons, which may derive from the central
reserved.
metabolic processes, the quinone pool, NADH or directly from
the oxidation of organic/inorganic compounds, such as
formate or reduced sulfur compounds [1]. Most microorganisms utilize reducing power also for the accumulation of
various storage materials, i.e. glycogen, PHA or globules of
elementary sulfur [2]. Accumulation of such excess materials
constitutes a widespread strategy, which enables the adaptation of microorganisms to the changing environment and
substrate fluctuations. The capability to conserve energy and
carbon source endows the microbes with a selective advantage under nutrient limitations over those lacking this capability. The production of the storage materials competes for
the electrons with the H2 evolving enzymes, but upon their
* Corresponding author. Department of Biotechnology, University of Szeged, Közép fasor 52, Szeged 6726, Hungary. Tel.: þ36 62 546940;
fax: þ36 62 544352.
E-mail address: [email protected] (G. Rákhely).
0360-3199/$ e see front matter Copyright ª 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ijhydene.2011.12.019
Article
pubs.acs.org/Langmuir
Porous Silicon/Photosynthetic Reaction Center Hybrid Nanostructure
Kata Hajdu,† Csilla Gergely,*,§,∥ Marta Martin,§,∥ Thierry Cloitre,§,∥ László Zimányi,⊥ Katalin Tenger,⊥
Petro Khoroshyy,⊥ Gabriela Palestino,# Vivechana Agarwal,▽ Klára Hernádi,‡ Zoltán Németh,‡
and László Nagy†
Departments of †Medical Physics and Informatics and ‡Applied and Environmental Chemistry, University of Szeged, Szeged, Hungary
§
Université Montpellier 2 and ∥CNRS, Laboratoire Charles Coulomb UMR 5221, Montpellier, France
⊥
Institute of Biophysics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
#
Facultad de Ciencias Químicas, Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico
▽
CIICAP - Universidad Autonoma del Estado de Morelos, Cuernavaca, Mexico
ABSTRACT: The purified photosynthetic reaction center protein (RC)
from Rhodobacter sphaeroides R-26 purple bacteria was bound to porous
silicon microcavities (PSiMc) either through silane-glutaraldehyde (GTA)
chemistry or via a noncovalent peptide cross-linker. The characteristic
resonance mode in the microcavity reflectivity spectrum red shifted by several
nanometers upon RC binding, indicating the protein infiltration into the
porous silicon (PSi) photonic structure. Flash photolysis experiments
confirmed the photochemical activity of RC after its binding to the solid
substrate. The kinetic components of the intraprotein charge recombination
were considerably faster (τfast = 14 (±9) ms, τslow = 230 (±28) ms with the
RC bound through the GTA cross-linker and only τfast = 27 (±3) ms through
peptide coating) than in solution (τfast = 120 (±3) ms, τslow = 1387 (±2) ms),
indicating the effect of the PSi surface on the light-induced electron transfer
in the protein. The PSi/RC complex was found to oxidize the externally added electron donor, mammalian cytochrome c, and
the cytochrome oxidation was blocked by the competitive RC inhibitor, terbutryne. This fact indicates that the specific surface
binding sites on the PSi-bound RC are still accessible to external cofactors and an electronic interaction with redox components
in the aqueous environment is possible. This new type of biophotonic material is considered to be an excellent model for new
generation applications at the interface of silicon-based electronics and biological redox systems designed by nature.
■
INTRODUCTION
The application of technical developments in biology (e.g.,
actuators, artificial tissues, and drug delivery systems) and vice
versa, i.e., biological materials in technical developments (e.g.,
environmentally friendly biodegradable items and biosensor
devices) are the focus of current research and industrial
interest.1−3 The use of biological materials developed by nature
to perform extremely efficient and sensitive functions, with
exceptional capacity, would be beneficial in (bio)hybrid
systems. Among these systems, the bionanocomposites are of
special interest because of their combined advantageous
properties and the possibility of the appearance of some new
characteristics. There are many potential applications of
bionanocomposite materials such as biosensors, integrated
(opto)electronic devices (switches or converters), photoelectric
energy conversion, and (single-molecule) imaging.2,4
To fabricate bionanocomposite substances, one of the most
promising materials is silicon because of its abundance and
wide application in semiconductor technology.5 However, lightsensitive biomolecules as the biological components are
promising because of their extremely fast performance and
efficient energy conversion.6,7 In a successful combination of
silicon-based materials and light-sensitive biological molecules
© 2012 American Chemical Society
(e.g., light-energy-converting proteins), a bionanocomposite
can be designed to convert light energy efficiently within a
tunable time (from femtoseconds to seconds) and wavelength
range (from UV to IR).
Different types of silicon and silica-based substances have
been described in the literature for hosting photosynthetic
materials (at any level of organization from whole cells to
molecules) either as a passive environment for assuring proper
conditions for biological structure and function or as an active
component participating in biological functions (charge
separation and stabilization). The encapsulation of whole
plant cells into various silica-based matrices is a promising
target for the development of sustainable technology such as
bioreactors.8 Mesoporous silica materials (MPS) have been
shown to successfully retain the structure and function of the
photosynthetic reaction center protein in FSM (folded-sheet
mesoporous silica material) and SBA-15 (Santa Barbara
Amorphous porous silica material) and those of the lightharvesting pigment protein complex (LHC), which is another
Received: May 8, 2012
Revised: July 16, 2012
Published: July 19, 2012
11866
dx.doi.org/10.1021/la301888p | Langmuir 2012, 28, 11866−11873
Hajdu et al. Nanoscale Research Letters 2012, 7:400
http://www.nanoscalereslett.com/content/7/1/400
NANO EXPRESS
Open Access
Light-harvesting bio-nanomaterial using porous
silicon and photosynthetic reaction center
Kata Hajdu1*, Csilla Gergely2, Marta Martin2, László Zimányi3, Vivechana Agarwal4, Gabriela Palestino5,
Klára Hernádi6, Zoltán Németh6 and László Nagy1
Abstract
Porous silicon microcavity (PSiMc) structures were used to immobilize the photosynthetic reaction center (RC)
purified from the purple bacterium Rhodobacter sphaeroides R-26. Two different binding methods were compared
by specular reflectance measurements. Structural characterization of PSiMc was performed by scanning electron
microscopy and atomic force microscopy. The activity of the immobilized RC was checked by measuring the visible
absorption spectra of the externally added electron donor, mammalian cytochrome c. PSi/RC complex was found to
oxidize the cytochrome c after every saturating Xe flash, indicating the accessibility of specific surface binding sites
on the immobilized RC, for the external electron donor. This new type of bio-nanomaterial is considered as an
excellent model for new generation applications of silicon-based electronics and biological redox systems.
Keywords: Porous silicon functionalization, Peptide, Photosynthetic reaction center, Nanomaterial, Biophotonics
Background
In the last few years, the use of bio-nanocomposites has
been the subject of extensive study. Using a hybrid material, it may be possible to harness the advantages of
two different materials at the same time. Several
attempts to fabricate functional biocomposites by different groups have been reported [1-6]. Photosynthetic reaction center (RC) is one of the proteins of high interest,
because it is nature's solar battery, converting light energy into chemical potential in the photosynthetic membrane, thereby assuring carbon reduction in cells [7,8].
Although RC functions on the nanometer scale, with
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%) [9] in RC presents a
great challenge to use it in artificial light harvesting systems. However, as biological materials are very sensitive
to the external effects and are generally stable only in
their own environment, to keep them functional after
their isolation, a special vehicle is necessary to hold and
protect them from degradation.
* Correspondence: [email protected]
1
Department of Medical Physics and Informatics, University of Szeged,
Szeged, H-6720, Hungary
Full list of author information is available at the end of the article
Numerous investigations have recently focused on
micro- and nanostructured materials due to the drastic
increase in the surface area-to-volume ratio compared
with the bulk materials. One of the promising nanostructured materials is porous silicon (PSi), well known
for photonic applications, sensors, and novel drug delivery methods [10-15]. Various applications of PSi in
bio-nanotechnology are possible due to its advantageous properties namely tunable pore dimensions,
large surface area, multilayered photonic structures,
easy and cheap fabrication method, and biocompatibility. The exceptional electrical and optical properties
and the particular multilayered photonic structures
offer unique application possibilities in integrated
optoelectronic and biosensing (biophotonic) devices as
well [10,13,14]. On the other hand, meso- and macroporous silicon assures good conditions for the penetration of the required biomolecules. The pore size and
optical properties are adjustable during the wet electrochemical etching process, which is used to fabricate
the well-arranged one-dimensional photonic structure
[16].
In this work, RC was immobilized on the surface of
porous silicon microcavities via two different methods:
covalent binding and non-covalent attachment via a specific peptide interface (‘peptide binding’). In both cases,
© 2012 Hajdu et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction
in any medium, provided the original work is properly cited.
Chem Biol Drug Des 2012; 79: 313–325
ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01275.x
Research Article
Conformational Similarities and Dissimilarities
Between the Stereoisomeric Forms of
Endomorphin-2
Balázs Leitgeb*
Institute of Biophysics, Biological Research Center of the Hungarian
Academy of Sciences, Temesvri krt. 62, H-6726 Szeged, Hungary
*Corresponding author: Balzs Leitgeb, [email protected]
In this study, taking into account both the L–D and
cis–trans isomerisms, a comprehensive structural
characterization and a comparative conformational analysis were performed on the 32 stereoisomeric
forms
of
opioid
tetrapeptide,
endomorphin-2. For all stereoisomers, the U-W and
v conformational spaces were explored, in the
course of which the conformational distributions,
as well as the rotamer states of aromatic side
chains were characterized in detail. Furthermore,
the typical b- and c-turn structures, as well as the
characteristic intramolecular interactions (i.e., Hbonds, aromatic–aromatic and proline–aromatic
interplays) were determined. The afore-mentioned
structural and conformational features identified
for each stereoisomeric form were compared with
one another, considering all 32 stereoisomers. The
results obtained from this comparative study indicated that both similarities and dissimilarities
could be observed between the stereoisomeric
forms, with regard to their structural and conformational properties. This theoretical work supplied several valuable observations concerning the
effects of both L–D and cis–trans isomerisms on
the three-dimensional structure of parent peptide
and its stereoisomers. Nevertheless, in the course
of this structural investigation, it was clarified
how the structural and conformational features of
stereoisomeric forms differed from one another.
Key words: aromatic–aromatic interaction, cis–trans isomerism,
conformational analysis, endomorphin, intramolecular H-bond, L–D isomerism, proline–aromatic interaction, turn structure
Received 5 May 2011, revised 1 August 2011 and accepted for publication 17 November 2011
Endomorphins (EMs) are opioid tetrapeptides with high affinity and
selectivity toward the l-opioid receptor (MOR) (1), which possess
important biological effects and modulate different physiological
processes (2–4). The various structural and conformational features
of EMs and their structurally modified analogs have been extensively investigated by a variety of experimental techniques and the-
oretical methods, so far (5). Previously, we performed the detailed
conformational analysis, as well as the comprehensive structural
characterization of both endomorphin-1 (EM1) and endomorphin-2
(EM2) by means of theoretical calculations (6–9). Our computational
studies provided new valuable results with regard to the threedimensional (3D) structure and conformational properties, as well
as to the possible bioactive conformation of these tetrapeptides.
However, taking into consideration all the earlier results, it is
worthwhile to mention that despite the extensive research efforts
focused on studying the 3D structure and bioactivity of EMs and
their derivatives, a definitive model regarding the biologically active
form of EM1 and EM2 is not available yet (5).
It is well-known that the different types of stereoisomerism play a
relevant role in the determination of peptide conformations, and
they are remarkable contributors to the formation of the bioactive
forms of peptides. The cis–trans isomerism of peptide bonds
produces important effects on the conformational properties of
peptides, while the inversion of the chirality of amino acids (i.e.,
the L–D isomerism) contributes significantly to the bioactivity and
selectivity of peptides. From the structural and biological points of
view, the opioid tetrapeptide, EM2 and its stereoisomeric forms
seem to be ideal representatives, to investigate the effects of stereoisomerisms on the structural features and bioactivity of peptides.
Previously, all fifteen stereoisomers of EM2 containing one or more
D-amino acids were synthesized, which exhibited various affinities
toward the MOR (10). Although the 3D structure of this series of
stereoisomeric EM2 analogs was examined by CD spectroscopy, this
study gave only elusive indications about the conformational features of stereoisomers (10). Based on the data derived from the CD
measurements, it was concluded that the inversion of the chirality
of one or more amino acid residues induced conformational
changes, but it was not clarified how the 3D structure of stereoisomeric forms differed from one another. Nevertheless, not only the
afore-mentioned L–D isomerism produces an effect on the structural
properties and bioactivity of EM2, but the cis–trans isomerism
affects also the conformational features and affinity of this tetrapeptide. Namely, as the results obtained from the earlier studies
pointed out, the EM2 and its structurally modified derivatives
existed as an equilibrium mixture of the cis- and trans-isomers
regarding the Tyr1-Pro2 peptide bond (5). Among the EM analogs,
for example, various cis ⁄ trans population ratios were identified in
the case of D-amino acid-containing derivatives, such as [D-Pro2]EM1
(11), [D-Phg3]EM2 (12), [D-Phg4]EM2 (12), [(2R,3R)-bMePhe4]EM2 (13),
[(2R,3S)-bMePhe4]EM2 (13), [D-Ala4-NH-Bn]EM1 (14), [D-Val4-NHBn]EM1 (14), and [D-Val4-NH-Bn]EM2 (14). Although the previous
313
Cent. Eur. J. Chem. • 10(6) • 2012 • 1791-1798
DOI: 10.2478/s11532-012-0105-3
Central European Journal of Chemistry
Spatial relationships between the
pharmacophores of endomorphin-2:
a comparative study of stereoisomers
Short Communication
Balázs Leitgeb*
Institute of Biophysics, Biological Research Centre,
Hungarian Academy of Sciences, H-6726 Szeged, Hungary
Received 19 April 2012; Accepted 31 July 2012
Abstract: The spatial relationships between the pharmacophore elements were investigated in the case of four different stereoisomeric forms
of opioid tetrapeptide, endomorphin-2, taking into account the L-D and cis-trans isomerisms. On the basis of distances and angles
measured between the pharmacophoric points, a comparative analysis of conformational distributions was performed, applying a
variety of distance-angle maps. The results obtained by this theoretical study indicated that the stereoisomers of endomorphin-2 could
be distinguished from one another, based on the comparative analysis of distance-angle maps. Nevertheless, it could be concluded
that this method proved to be suitable to examine the effects of L-D and cis-trans isomerisms on the spatial relationships of the
pharmacophores of tetrapeptide.
Keywords: Endomorphin • Pharmacophore • Conformational analysis • L-D isomerism • Cis-trans isomerism
© Versita Sp. z o.o.
1. Introduction
Endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2) is
an opioid tetrapeptide possessing high activity and
selectivity toward the µ-opioid receptor [1]. The threedimensional (3D) structural features of endomorphins
(EMs) and their analogs have been studied by means of
various experimental and theoretical methods [2]. In our
previous structural investigations, detailed conformational
analyses were carried out on the EMs applying different
computational methods, in order to identify their
characteristic structural and conformational properties
[3-6]. These theoretical studies led to several valuable
observations concerning the 3D structural features of
these tetrapeptides, as well as regarding their possible
biologically active form. In a recent study, a comparative
conformational analysis was performed for all the
stereoisomeric forms of EM2, taking into consideration
both the L-D and cis-trans isomerisms [7]. For the thirtytwo stereoisomers of tetrapeptide, a comprehensive
structural characterization was carried out, and their
typical features were determined, with regard to the Φ-Ψ
conformational distributions and the rotamer states, as
well as to the secondary structural elements and the
intramolecular interactions. Based on the results derived
from these theoretical calculations, it was concluded
that both conformational similarities and dissimilarities
could be observed between the stereoisomeric forms of
EM2, and it was clarified how their 3D structural features
differed from one another.
The data obtained by earlier studies indicated that
four groups could be considered as pharmacophore
elements for the EMs, as follows: (1) the N-terminal
amino group; (2) the aromatic side-chain of the Tyr1
amino acid; (3) the aromatic side-chain of the Trp3/
Phe3 residue; (4) the aromatic side-chain of the Phe4
amino acid [2]. Thus, the relative spatial arrangement of
these putative pharmacophores seems to be important
in the formation of the biologically active form of these
tetrapeptides. Previously, a novel approach (CSP,
Conformationally Sampled Pharmacophore) was
applied for nonpeptidic and peptidic ligands showing
selectivity toward the δ-opioid receptor [8-10]. This
method was developed based on the characterization
of all possible combinations of distances and angles
measured between the pharmacophore elements of
* E-mail: [email protected]
1791
ARTICLE ADDENDUM
Plant Signaling & Behavior 7:5, 1–3; May 2012;
G
2012 Landes Bioscience
Effects of missense mutation on structure and function of photoreceptor
Balázs Leitgeb,1 Vladyslava Sokolova,2 Eberhard Schäfer3 and András Viczián2,*
1
Institute of Biophysics; Biological Research Centre; Hungarian Academy of Sciences; Szeged, Hungary; 2Institute of Plant Biology; Biological Research Centre;
Hungarian Academy of Sciences; Szeged, Hungary; 3Biologie II/Institut für Botanik; University of Freiburg; Freiburg, Germany
P
hytochromes (PHYs) are photoreceptors of the red (R ~660 nm)
and far-red (FR ~730 nm) light, and they
control a wide range of responses affecting crucial aspects of plant life. There are
five genes PHYA-PHYE encoding for
phytochromes of different but overlapping function. One of these, PHYA has
the unique function controlling specific
responses in high irradiance far-red, as
well as in very weak light. Appropriate
PHYA functioning requires not only
the photoreversibility of molecule but
also the proper nuclear localization
and degradation of receptor. Recently,
we identified and described a mutant
PHYA allele (phyA-5) in Arabidopsis
thaliana, which showed reduced binding
affinity to FHY1/FHL, the proteins
regulating its nuclear transport, resulting
in impaired nuclear localization and
altered signaling under certain conditions. We present here a hypothesis to
explain how the identified amino acid
substitution may lead to structural
changes manifested as altered signaling
and phenotype displayed by the phyA-5
mutant.
histidine kinases (HKRD)1 is essential for
the nuclear translocation and dimerization
of molecule.3
PHYs are synthesized in Pr form which
can be converted by R photon absorption
to the Pfr form which can be transformed
back to Pr by FR photon absorption. The
biologically active phyA Pfr conformer
binds directly to nuclear import shuttle
proteins, such as FHY1 (for FAR-RED
ELONGATED HYPOCOTYL1) and
FHL (for FHY1-LIKE)4,5 and it is
transported to the nucleus. PHYA Pfr
is ubiquitinated and undergoes rapid
degradation.6-8 Recent results, supported
with mathematical modeling prove that
not only the unperturbed Pr/Pfr photoconversion but also the nuclear import and
degradation of photoreceptor are required
for the normal PHYA functioning. These
events are tightly linked to each other like
interlacing “cycles”.9 Very recently, we
identified and described a mutant (phyA-5)
expressing PHYA with a missense mutation.10 This mutant line shows a fairly
complex phyA-specific phenotype, including a reduced nuclear import at low
intensities of FR indicating that the point
mutation identified alters the FHY1/FHLphyA Pfr association/dissociation “cycle”.
The fact that PHYA-5 governs wild-typelike signaling when it is fused to a nuclear
localization signal (NLS), strongly supported this conclusion.10
Our knowledge about the interaction of
phyA with the FHY1 and FHL proteins is
rather limited. Yeast two-hybrid assays
showed that the Pfr form of the
phyA406 (1–406 amino acids) specifically
binds the FHY1/FHL transporter molecules.5,11 However, the exact three-dimensional (3D) structure of plant PHYs in
contrast to microbial PHYs is not yet
© 2012 Landes Bioscience.
Do not distribute.
Keywords: Arabidopsis, photoreceptor,
photomorphogenesis, phytochrome A,
nuclear import, FHY1, FHL, 3D structure
Submitted: 02/13/12
Accepted: 02/14/12
http://dx.doi.org/10.4161/psb.19702
*Correspondence to: András Viczián;
Email: [email protected]
Addendum to: Sokolova V, Bindics J, Kircher S,
Ádám E, Schäfer E, Nagy F, et al. Missense
mutation in the amino terminus of phytochrome
A disrupts the nuclear import of the photoreceptor. Plant Physiol 2012; 158:107–18; PMID:
21969386; http://dx.doi.org/10.1104/pp.111.
186288
www.landesbioscience.com
A functional PHY molecule is a dimer of
two ~120 kDa monomers, and each of the
monomers contains the N-terminal photosensory and the C-terminal regulatory
domains (see Fig. 1A). The N-terminal
part is necessary for the photosensory
function, and it contains the NTE, PAS,
GAF and PHY domains. The GAF
domain harbors the linear tetrapyrrol
chromophore attached to a conserved
cysteine residue via thioether bond.1,2
The C-terminal part, containing two PAS
domains and a region similar to the
Plant Signaling & Behavior
1
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Cite this: Chem. Commun., 2012, 48, 70–72
COMMUNICATION
www.rsc.org/chemcomm
Downloaded on 10 November 2012
Published on 11 November 2011 on http://pubs.rsc.org | doi:10.1039/C1CC13186A
Mapping local electric fields in proteins at biomimetic interfacesw
Gal Schkolnik,a Tillmann Utesch,a Johannes Salewski,a Katalin Tenger,b Diego Millo,a
Anja Kranich,a Ingo Zebger,a Claudia Schulz,a László Zimányi,b Gábor Rákhely,c
Maria Andrea Mroginskia and Peter Hildebrandt*a
Received 30th May 2011, Accepted 2nd November 2011
DOI: 10.1039/c1cc13186a
We present a novel approach for determining the strength of the
electric field experienced by proteins immobilised on membrane
models. It is based on the vibrational Stark effect of a nitrile label
introduced at different positions on engineered proteins and
monitored by surface enhanced infrared absorption spectroscopy.
Most biochemical and biophysical processes of proteins take
place at and in membranes and thus under the influence of
electrostatic fields. Particularly strong local electric fields of
the order of 109 V m 1 prevail at the membrane/solution
interface and in the boundary region between the hydrophobic
core and the polar or charged headgroups of the membrane.1
Such high external electric fields may modulate the structure of
integral membrane and membrane-attached proteins as well as
the conformational and reaction dynamics of proteins, such as
in enzymatic processes, ion transport, and electron transfer.1–7
Despite a large body of experimental data, no concept has yet
been established to describe the electric-field dependence of
protein functions on a molecular level. As an essential part of
such a concept, the local electric field strengths at the protein/
membrane interface need to be quantified, which requires
novel methodological strategies.
Electric field strengths may be determined on the basis of the
vibrational Stark effect (VSE), which refers to the electric field
induced frequency shift Dn of a vibrational mode according to
hcDn =
-
D~
mF
(1)
where D~
m is the change in the dipole moment between the
vibrational ground and excited states, F is the electric field
strength, and h and c denote the Planck constant and the speed
of light, respectively. A particularly sensitive VSE reporter
group is the nitrile function. In their pioneering work, Boxer
and co-workers8–10 have introduced a nitrile label at different
a
Technische Universität Berlin, Institut für Chemie, Sekr. PC14,
Straße des 17. Juni 135, D-10623 Berlin, Germany.
E-mail: [email protected]; Fax: +49 30 31421122;
Tel: +49 30 31421419
b
Institute of Biophysics, Biological Research Centre, H-6726 Szeged
Temesvári krt. 62., Hungary
c
Department of Biotechnology, University of Szeged, Köze´p fasor 52,
H-6726 Szeged, Hungary
w Electronic supplementary information (ESI) available: Description
of experimental procedures and the MD simulation as well as further
spectroscopic data on MBN. See DOI: 10.1039/c1cc13186a
70
Chem. Commun., 2012, 48, 70–72
sites on the protein surface and at the active site of the enzyme
human aldose reductase, and probed the respective CRN
stretching modes of the proteins in solution. In the present
work, we have, for the first time, extended this strategy to
proteins immobilised on simple membrane models monitoring
the CRN stretchings using surface enhanced infrared absorption
(SEIRA) spectroscopy.
We have chosen the heme protein cytochrome c (Cyt-c) as a
convenient test protein that can be readily electrostatically
bound to Au electrodes coated with self-assembled monolayers
(SAMs) of carboxyl-terminated alkylthiols. Such devices may
be considered as a simple model for biological membranes,
specifically with respect to the electrostatics at the SAM/
solution (membrane/solution) interface.7 Furthermore, the
structure and reaction dynamics of Cyt-c have been shown
to depend sensitively on the interfacial electric field upon
binding to SAM-coated electrodes and liposomes. It has been
proposed that electric field variations at the mitochondrial
membrane may contribute to the switching of the protein
function from electron transport in the respiratory chain to
lipid peroxidation as one of the first steps in apoptosis.7 Thus,
this work constitutes the starting point for a comprehensive
analysis of the electric field control of Cyt-c’s function.
4-Mercaptobenzonitrile (MBN) has been used as a nitrile
label, covalently attached to a Cys side chain of site-directed
engineered horse heart Cyt-c variants. In this work, we have
introduced a Cys at two different positions, one in the vicinity
of the redox centre (Lys8) and one at a relatively remote
position (Lys39). The two variants, K8C and K39C, were then
treated with MBN such that the aromatic nitrile was selectively
bound to the thiol function of the cysteine (see ESIw for
experimental details).
IR spectra of the labelled Cyt-c variants in solution display
weak but clearly identifiable peaks in an optical window
between 2200 and 2300 cm 1 that is free of any interference
by IR bands of the protein, but which includes a strong
background absorption (Fig. S1, ESIw). To determine the
frequencies of the nitrile stretching modes with higher precision,
we have thus used the second derivatives of the spectra in which
the minima correspond to the peak maxima of the IR absorption
bands (Fig. 1 and 2; for details of the spectra analysis, see
ESIw). For the labelled K39C variant in solution (Fig. 1, top),
the nitrile stretching mode is observed at 2235.1 cm 1.
This journal is
c
The Royal Society of Chemistry 2012
Analyses of the Large Subunit Histidine-Rich Motif
Expose an Alternative Proton Transfer Pathway in
[NiFe] Hydrogenases
Emma Szőri-Dorogházi1, Gergely Maróti2, Milán Szőri3, Andrea Nyilasi4, Gábor Rákhely1,4*,
Kornél L. Kovács1,4
1 Department of Biotechnology, University of Szeged, Szeged, Hungary, 2 BayGen Institute, Bay Zoltán Foundation for Applied Research, Szeged, Hungary, 3 Department
of Chemical Informatics, Juhász Gyula Faculty of Education, University of Szeged, Szeged, Hungary, 4 Institute of Biophysics, Biological Research Centre, Hungarian
Academy of Sciences, Szeged, Hungary
Abstract
A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases.
The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present
in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn
hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue.
Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif
might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the
conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization
was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly
based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our
results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A
novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the
position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton
translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis.
Citation: Szőri-Dorogházi E, Maróti G, Szőri M, Nyilasi A, Rákhely G, et al. (2012) Analyses of the Large Subunit Histidine-Rich Motif Expose an Alternative Proton
Transfer Pathway in [NiFe] Hydrogenases. PLoS ONE 7(4): e34666. doi:10.1371/journal.pone.0034666
Editor: Beata G. Vertessy, Institute of Enzymology of the Hungarian Academy of Science, Hungary
Received September 22, 2011; Accepted March 6, 2012; Published April 12, 2012
Copyright: ß 2012 Szőri-Dorogházi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by EU projects HyVolution FP6-IP-SES6 019825 and FP7 Collaborative Project SOLAR-H2 FP7-Energy-212508, and by domestic
funds (NAP-BIO Teller Ede Program OMFB-00441/2007, GOP-1.1.2.-07/1-2003+8-0007, Asbóth-DAMEC-2007/09, BAROSS-DA07-DA-TECH-07-2008-0012, KoBioCHG
TECH_09_04 and TÁMOP-4.2.1/B-09/1/KONV-2010-0005). 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: [email protected]
9], but they are not expressed in this organism. The Hyn
hydrogenase is a truly bidirectional enzyme with remarkable
stability; it is active even when it is extracted from the
photosynthetic membrane [10].
The crystal structures of periplasmic [NiFe(Se)] hydrogenases
from sulfate-reducing [11–15] bacteria and one of the photosynthetic bacteria [16] have been reported. These structures were
used to model and study several structure-function relationships of
[NiFe] hydrogenases. The structural analysis of the periplasmic
[NiFe] hydrogenase of Desulfovibrio gigas showed that the metal
atoms of the active site are deeply buried inside the protein [11],
and that the Ni and Fe are coordinated by cysteine thiolates of the
L2 and L5 CxxC motifs [17]. The latter consensus sequence is also
involved in the biosynthesis of hydrogenases as the endoproteolytic
cleavage of the carboxy-terminus takes place at the Cx2Cx2H/R
motif. The endopeptidases cleave after the conserved His (or Arg)
amino acid of this motif and this maturation step is essential for the
proper folding and assembly of the large subunit [3]. Following the
removal of an approximately 25–32 amino acid fragment from the
C-terminus of the protein, the matured large and small subunits
form the functional heterodimer.
Introduction
Hydrogenases are the key enzymes of hydrogen metabolism
catalyzing the reversible heterolytic cleavage of molecular
hydrogen according to the reaction: H2«2H++2e2. These
metalloenzymes are widespread in bacteria and archaea and are
present in some eukaryotes. Hydrogenases are classified on the
basis of the metal content of their active site: [NiFe], [FeFe] or [Fe]
hydrogenases [1,2]. The core of a [NiFe] hydrogenase consists of a
small subunit, which is responsible for the electron transfer
between the active center and the surface of the enzyme, and a
large subunit harboring the binuclear active site [3].
Thiocapsa roseopersicina BBS, which belongs to the family of purple
sulfur photosynthetic bacteria [4], has been shown to possess four
functional [NiFe] hydrogenases with differences in their in vivo
function, localization and composition [5,6]. Two of these
enzymes (Hyn and Hup) are membrane-associated, while the
other two are localized in the cytoplasm (Hox1 and Hox2).
Furthermore, the genes of a regulatory hydrogenase (similar to
HupUV in Rhodobacter capsulatus [7] and HoxBC in Ralstonia
eutropha [8]) could also be detected in T. roseopersicina (hupTUV) [7–
PLoS ONE | www.plosone.org
1
April 2012 | Volume 7 | Issue 4 | e34666
Wirth et al. Biotechnology for Biofuels 2012, 5:41
http://www.biotechnologyforbiofuels.com/content/5/1/41
RESEARCH
Open Access
Characterization of a biogas-producing microbial
community by short-read next generation DNA
sequencing
Roland Wirth1, Etelka Kovács1, Gergely Maróti2,3, Zoltán Bagi1, Gábor Rákhely1,4 and Kornél L Kovács1,4*
Abstract
Background: Renewable energy production is currently a major issue worldwide. Biogas is a promising renewable
energy carrier as the technology of its production combines the elimination of organic waste with the formation of
a versatile energy carrier, methane. In consequence of the complexity of the microbial communities and metabolic
pathways involved the biotechnology of the microbiological process leading to biogas production is poorly
understood. Metagenomic approaches are suitable means of addressing related questions. In the present work a
novel high-throughput technique was tested for its benefits in resolving the functional and taxonomical complexity
of such microbial consortia.
Results: It was demonstrated that the extremely parallel SOLiD™ short-read DNA sequencing platform is capable of
providing sufficient useful information to decipher the systematic and functional contexts within a biogasproducing community. Although this technology has not been employed to address such problems previously, the
data obtained compare well with those from similar high-throughput approaches such as 454-pyrosequencing GS
FLX or Titanium. The predominant microbes contributing to the decomposition of organic matter include members
of the Eubacteria, class Clostridia, order Clostridiales, family Clostridiaceae. Bacteria belonging in other systematic
groups contribute to the diversity of the microbial consortium. Archaea comprise a remarkably small minority in
this community, given their crucial role in biogas production. Among the Archaea, the predominant order is the
Methanomicrobiales and the most abundant species is Methanoculleus marisnigri. The Methanomicrobiales are
hydrogenotrophic methanogens. Besides corroborating earlier findings on the significance of the contribution of
the Clostridia to organic substrate decomposition, the results demonstrate the importance of the metabolism of
hydrogen within the biogas producing microbial community.
Conclusions: Both microbiological diversity and the regulatory role of the hydrogen metabolism appear to be the
driving forces optimizing biogas-producing microbial communities. The findings may allow a rational design of
these communities to promote greater efficacy in large-scale practical systems. The composition of an optimal
biogas-producing consortium can be determined through the use of this approach, and this systematic
methodology allows the design of the optimal microbial community structure for any biogas plant. In this way,
metagenomic studies can contribute to significant progress in the efficacy and economic improvement of biogas
production.
Keywords: Biogas, Next-generation sequencing, DNA, Microbial community structure, Bacteria, Methanogens,
SOLiD™, Metagenomics, Hydrogen metabolism
* Correspondence: [email protected]
1
Department of Biotechnology, University of Szeged, Középfasor 52
Szeged H-6726, Hungary
4
Institute of Biophysics, Biological Research Center, Hungarian Academy of
Sciences, Temesvári krt. 62, Szeged H-6726, Hungary
Full list of author information is available at the end of the article
© 2012 Wirth et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Scalable Computing: Practice and Experience
Volume 13, Number 1, pp. 21–28. http://www.scpe.org
ISSN 1895-1767
c 2012 SCPE
1
SIMULATION OF COMMUNICATION AND COOPERATION IN MULTISPECIES
BACTERIAL COMMUNITIES WITH AN AGENT BASED MODEL
DÓRA BIHARY1⋆ ,ÁDÁM KERÉNYI2⋆ , ZSOLT GELENCSÉR1 , SERGIU NETOTEA3 , ATTILA KERTÉSZ-FARKAS4 ,
VITTORIO VENTURI4 AND SÁNDOR PONGOR1,2,4
Abstract.
Members of bacterial communities communicate and cooperate via diffusible chemical materials they emit into the environment,
and at the same time, they also compete for nutrients and space. Agent-based models (ABMs) are useful tools for simulating the
growth of communities containing multiple interacting microbial species. In this work we present numerical indices characterizing
spatial distribution and the fitness of competing bacterial species in an ABM and we present data on how these indices can be used
to visually summarize large scale simulation experiments. Preliminary results show bacterial agents utilizing different nutrients but
sharing communication signals and public goods can form stable mixed communities in which the species grow faster than any of
the single species alone.
Key words: quorum sensing, Pseudomonas aeruginosa, hybrid model, statistics, segregation, fitness
1. Introduction, state-of-the-art. Multispecies microbial communities are now recognized as a major
form of bacterial life. These communities (such as the gastrointestinal flora, the microflora of dental cavities,
the rhizosphere around plant roots or the large microbial mats on the seafloor) contain more than one species.
Computer simulations play an important role in the study of these communities since it is extremely complicated
to collect reliable data on the size and growth dynamics of free-living bacterial communities.
The interaction between individual bacteria in a community is often based on the exchange of diffusible
signals, the best known example of which is a mechanism called quorum sensing (QS) [1, 2]. In this mechanism,
signaling materials secreted by the bacteria are supposed to spread in the environment by diffusion. The
concentration of signals regulates the behavior of bacteria, which results in collective patterns of behavior, such
as coordinated movement (e.g. swarming), secretion of specific materials, resistance to antibiotics, etc. When
the concentration of the secreted signal is greater than a certain threshold, bacteria, such as Pseudomonas
aeruginosa, switch from low to high metabolic activity, they increase the amount of secreted signaling molecules
and they also start to secret other molecules, frequently referred to as ”public goods” or simply ”factors” (e.g.
surfactants, enzymes, siderophores), which facilitates movement and nutrient uptake [2, 3]. As a result, the
colony changes behavior, for instance it starts to grow and expand. In some cases, this is accompanied by a
swarming motion of the cells.
There are various approaches for modeling the growth of bacterial communities. Continuous models represent both the nutrients and the bacterial colony as continuous quantities described by reaction-diffusion
equations [3]. Agent-based models consider bacteria as individuals capable of nutrient uptake, movement and
cell-division [4, 5]. In agent based models, the nutrients are often considered as diffusing materials described by
reaction-diffusion equations - these models are specifically called ”hybrid models” as they combine individualbased agents with diffusing materials. Continuous and agent-based hybrid models were used primarily to show
that colony shapes (especially the well known fractal-like or circular shapes) can be reproduced by simple
models [3, 4, 5].
Recently we adapted the hybrid methodology for describing the behavior of QS bacteria [6, 7]. Briefly,
agents representing individual bacteria move randomly on a 2D plane in this model, and they secrete two kinds
of materials, a signal S and a factor F (public goods material) that both spread via diffusion on the 2D plane.
Agents have different ”physiological” states and they switch between states depending on the local concentration
of S and F . In the ground state (S and F below threshold), nutrient uptake, movement and signal production
is at a low level. In the activated state (S above threshold, F below threshold), the production of public goods
(F ) starts, and signal production is upgraded to a higher level. In the quorum state, signal production is high,
production of public goods is high, and movement and nutrient uptake are also upgraded to a higher level. In
1 Faculty of Information Technology, Pázmány Péter Catholic University, Práter u. 50/a. 1083 Budapest, Hungary, 2 Biological
Research Centre of the Hungarian Academy of Sciences, H-6726, Szeged, Temesvári krt. 62, Hungary, 3 Umeå University, SE901 87 Umeå, Sweden, 4 International Centre for Genetic Engineering and Biotechnology, Padriciano 99, I-34012 Trieste, Italy,
[email protected], ⋆ These authors contributed equally.
21
Sensors 2012, 12, 5432-5444; doi:10.3390/s120505432
OPEN ACCESS
sensors
ISSN 1424-8220
www.mdpi.com/journal/sensors
Review
Classifying the Topology of AHL-Driven Quorum Sensing
Circuits in Proteobacterial Genomes
Zsolt Gelencsér 1, Kumari Sonal Choudhary 2, Bruna Goncalves Coutinho 2,3,
Sanjarbek Hudaiberdiev 2, Borisz Galbáts 1,4, Vittorio Venturi 2,* and Sándor Pongor 2,*
1
2
3
4
Faculty of Information Technology, PázmányPéter Catholic University, Práter u. 50/a,
Budapest 1083, Hungary; E-Mails: [email protected] (Z.G.); [email protected] (B.G.)
International Centre for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99,
Trieste 32149, Italy; E-Mails: [email protected] (K.S.C.); [email protected] (B.G.C.);
[email protected] (S.H.)
The Capes Foundation, Ministry of Education of Brazil, Cx postal 250, Brasilia, DF 70.040-020, Brazil
Biological Research Center, Temesvári krt 62, Szeged 6726, Hungary
* Authors to whom correspondence should be addressed; E-Mails: [email protected] (V.V.);
[email protected] (S.P.); Tel.: +39-040-3751; Fax: +39-040-226-555.
Received: 1 February 2012; in revised form: 24 March 2012 / Accepted: 26 April 2012 /
Published: 27 April 2012
Abstract: Virulence and adaptability of many Gram-negative bacterial species are
associated with an N-acylhomoserine lactone (AHL) gene regulation mechanism called
quorum sensing (QS). The arrangement of quorum sensing genes is variable throughout
bacterial genomes, although there are unifying themes that are common among the various
topological arrangements. A bioinformatics survey of 1,403 complete bacterial genomes
revealed characteristic gene topologies in 152 genomes that could be classified into
16 topological groups. We developed a concise notation for the patterns and show that the
sequences of LuxR regulators and LuxI autoinducer synthase proteins cluster according to
the topological patterns. The annotated topologies are deposited online with links to
sequences and genome annotations at http://bacteria.itk.ppke.hu/QStopologies/.
Keywords: quorum sensing; N-AHL; topology; proteobacteria; bacteria
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1
Current Bioinformatics, 2012, 7, 00-00
Data Preprocessing and Filtering in Mass Spectrometry Based Proteomics
Beáta Reiz1,2,3, Attila Kertész-Farkas1, Sándor Pongor1,2 and Michael P. Myers*,1
1
International Centre for Genetic Engineering and Biotechnology, 34012 Trieste, Italy, 2Szeged Biological Center,
Temesvári krt 67, Szeged, Hungary, 6720 3Institute of Informatics, University of Szeged, Aradi vértanúk tere 1, Szeged,
Hungary, 6720
Abstract: Mass spectrometry based proteomics analysis can produce many thousands of spectra in a single experiment,
and much of this data, frequently greater than 50%, cannot be properly evaluated computationally. Therefore a number of
strategies have been developed to aid the processing of mass spectra and typically focus on the identification and
elimination of noise, which can provide an immediate improvement in the analysis of large data streams. This is mostly
carried out with proprietary software. Here we review the current main principles underlying the preprocessing of mass
spectrometry data give an overview of the publicly available tools.
Keywords: Data filtering, Mass spectrometry, Proteomics.
1. INTRODUCTION
Mass spectrometry coupled with high performance liquid
chromatography has become the de facto experimental
standard for the proteomic analysis of complex biological
materials
such
as
tissue
samples,
biofluids,
immunoprecipitates etc. [1]. Each sample produces several
thousand spectra, and owing to the large amount and
complexity of the data, interpretation of LC-MS/MS relies
almost entirely on computational tools [2]. Despite recent
technological advances, such as the improvement of mass
accuracy and sensitivity, a large part of proteomics data is
uninformative: many of the collected spectra are not easily
interpreted, and it is not unusual to see cases where >50% of
the collected spectra do not result in matches and even good
quality spectra, which result in matches, can carry up to 80
% extraneous peaks [3]. These poor results are the
consequence of the inherent properties of the sample, the
properties of the instrumentation and the drive to extract as
much data as possible from the sample. This results in many
spectra not being derived from true peptides. Removal of
these extraneous data points can improve both the speed of
analysis and the statistical confidence in the final results [3,
4]. Consequently, preprocessing and filtering of the data are
a major challenge. Some of the initial steps of the data
cleaning process are carried out automatically, by the
instrumentation’s proprietary onboard software, so the initial
steps are often partly hidden from the experimenter. In
addition, filtering steps can be included at later stages of the
experimental pipeline, so the limits between filtering and
data interpretation are often blurred.
The methods used for preprocessing MS spectra draw
upon a number of disciplines, not all of which are included
in standard bioinformatics curricula. The methods use
various heuristics taken from diverse fields ranging from
chemical computing to electronic signal processing. Finally,
*Address correspondence to this author at the Protein Networks Group,
International Centre for Genetic Engineering and Biotechnology, 34012
Trieste, Italy; Tel: +39-040 375 7391; Fax: +39-040 226 555;
E-mail: [email protected]
1574-8936/12 $58.00+.00
there are strong ties to pattern classification, in particular to
outlier detection, since data preprocessing can be viewed as
the successive application of models in which part of the
information is discarded at every step. One of the goals of
this review is to place spectrum preprocessing methods into
this general framework. We will concentrate on the most
widely used approach, bottom up proteomics, where proteins
are identified from the mass spectra of their proteolytic
peptides [1]. There are a number of expert reviews on the
general computational approaches of this field [5-7]. The
goal of this article is to provide an introductory overview of
spectrum preprocessing techniques for students and
bioinformaticians who are not experts of LC-MS/MS.
2. A PROTEOMICS EXPERIMENT
The goal of an LC-MS/MS experiment is to identify
proteins in a sample – which can be a single protein, a
relatively simple mixture of proteins, such as from an
immuno-precipitation, or a complex mixture of proteins,
such as from a lysate or biofluid. In a typical experiment, the
protein sample is treated with a protease, typically trypsin, to
create smaller peptides, which are more efficiently analyzed
by the mass spectrometer. It is important to note that mass
spectrometers can only analyze positively or negatively
charged species and that the mass spectrometer does not
directly measure the mass of the ion, rather its mass to
charge ratio (m/z).
Liquid chromatography, or LC, is often used for
introducing the peptides into the mass spectrometer and the
solvents used for LC are largely compatible for this
interface. The LC is also used to simultaneously remove
impurities and concentrate the peptides. Perhaps most
importantly, the chromatographic separation of the peptides
gives the mass spectrometer more time to analyze the
sample. A typical analysis entails an initial measurement of
the m/z of the molecular species, or precursors, that are
eluting from the LC. From this initial measurement, referred
to as the precursor scan, a single precursor ion is selected,
isolated from other precursors, and fragmented. The m/z of
the resulting fragment ions, also called product ions, are
© 2012 Bentham Science Publishers