Recognition of different nucleotidyl-derivatives as substrates of reactions catalyzed by various HIT-proteins

Article abstract of DOI:10.1039/b9nj00660e

Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily. They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their hydrolysis and/or phosphorolysis liberating either the corresponding 5'-NMP or 5'-NDP, respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine 5'-phosphosulfate (1), adenosine 5'-phosphoramidate (2), adenosine 5'-phosphorothioate (3), adenosine 5'-phosphorofluoride (4), diadenosine 5',5'-P¹,P³-triphosphate (5), di(7-methylguanosine) 5',5'-P¹,P³-triphosphate (6) and adenosine 5'-hypophosphate (7). Preferences for the recognition of these compounds as substrates by individual proteins differed. All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and Trypanosoma brucei HIT-45, cleaved (1), (2), (4), (5) and (6). Caenorhabditis elegans decapping protein DcpS degraded (1), (5), (6) and poorly (4). A. thaliana aprataxin-like protein and Hint4 hydrolyzed only (1), (2) and (4), in that order of efficiency. Velocities of those reactions and some K _m values were determined. Applicability of this study to the metabolism of certain nucleotidyl-derivatives is discussed.

Full text of DOI:10.1039/b9nj00660e

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PAPER
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Recognition of different nucleotidyl-derivatives as substrates of reactions
catalyzed by various HIT-proteinswz
Andrzej Guranowski,*a Anna Maria Wojdy$a,a Jaros$aw Zimny,a
Anna Wypijewska,b Joanna Kowalska,b Maciej Łukaszewicz,b
Jacek Jemielity,b Edward Darz˙ynkiewicz,b Agata Jagie$$oc and
Pawe$ Bieganowskid
Received (in Montpellier, France) 12th November 2009, Accepted 8th January 2010
First published as an Advance Article on the web 12th February 2010
DOI: 10.1039/b9nj00660e
Proteins that have a histidine triad in their active sites belong to the HIT-protein superfamily.
They are ubiquitous, are involved in the metabolism of different nucleotides and catalyze their
hydrolysis and/or phosphorolysis liberating either the corresponding 50-NMP or 50-NDP,
respectively. We studied substrate specificity of nine recombinant HIT-proteins with adenosine
50-phosphosulfate (1), adenosine 50-phosphoramidate (2), adenosine 50-phosphorothioate (3),
adenosine 50-phosphorofluoride (4), diadenosine 50,50 0 0-P1,P3-triphosphate (5),
di(7-methylguanosine) 50,50 0 0-P1,P3-triphosphate (6) and adenosine 50-hypophosphate (7).
Preferences for the recognition of these compounds as substrates by individual proteins differed.
All the proteins hydrolyzed (1) but the Arabidopsis thaliana Hint1 did it very slowly. None of the
proteins cleaved (7). Only A. thaliana Hint1 and Escherichia coli HinT hydrolyzed (3). Three
proteins known as dinucleoside triphosphatases, human and A. thaliana Fhit-proteins and
Trypanosoma brucei HIT-45, cleaved (1), (2), (4), (5) and (6). Caenorhabditis elegans decapping
protein DcpS degraded (1), (5), (6) and poorly (4). A. thaliana aprataxin-like protein and Hint4
hydrolyzed only (1), (2) and (4), in that order of efficiency. Velocities of those reactions and
some Km values were determined. Applicability of this study to the metabolism of certain
nucleotidyl-derivatives is discussed.
(EC 2.6.1.29)2,3 and nucleoside phosphoramidases,2,4 or by
Introduction
phosphorylated sugar, in the case of galactose-1-phosphate
uridylyltransferase (EC 2.7.7.10).2 Also dinucleoside tetra-
Proteins that have a histidine triad in their active sites belong to
the HIT-protein superfamily. Sequence alignment of amino acid
phosphate phosphorylase (EC 2.7.7.53) probably functions
fragments that contain a histidine-triad-motif of the proteins
according the same mechanism. One of its histidine residues
used in this study and phylogenic relations of these proteins are
occurs in the region that resembles the active sites of the enzymes
depicted in Fig. 1A and B, respectively.1 HIT-proteins are
mentioned above. Formation of the enzyme-adenylate has been
proved experimentally5,6 and splitting of that intermediate could
ubiquitous and interact with different mono- and dinucleotides.
occur in the presence of phosphate (yielding ADP, ppA)7 or
The two-step mechanism according to which HIT-proteins
ATP (yielding Ap4A, AppppA).8
(HIT-enzymes) act involves first the formation of an enzyme-
nucleotidyl and then splitting of this intermediate. In the first
This study stemmed from the investigation of substrate
specificity of Fhit-proteins (from ꢀfragile histidine ꢀtriad)9 that
step, one (central) of the triad histidines participates in a
ꢀꢀ
covalent nucleotidyl phosphohistydyl intermediate P–N bond
form one of at least four branches of the HIT-protein super-
formation and in the second, the bond is cleaved either by
family. Fhit-proteins, were known primarily as typical dinucleo-
side triphosphatases.10,11 However, our recent study showed
water, in the case of Fhit-proteins/dinucleoside-triphosphatases
that these proteins possess also activities which had been
a Department of Biochemistry and Biotechnology,
assigned to nucleoside phosphoramidases, adenylylsulfatases
´
The University of Life Sciences, 35 Wo!ynska Street,
and phosphodiesterases. The investigated human and Arabidopsis
thaliana Fhit-proteins effectively catalyzed liberation of 50-AMP
(pA) from such naturally occurring nucleotides as adenosine
50-phosphoramidate (NH2-pA) and adenosine 50-phosphosulfate
(SO4-pA, commonly known as APS), as well as from unnatural
nucleotidyl-derivative, adenosine 50-phosphofluoride (F-pA).9
Earlier, similar enzymatic activities have been demonstrated
for some other HIT-proteins. Hint-proteins exhibited adenosine
phosphoramidase activity2,4,12,13 and rabbit Hint1 was
shown to catalyze liberation of 50-NMP (pN) from nucleoside
´
60-637 Poznan, Poland
b Division of Biophysics, Institute of Experimental Physics,
Faculty of Physics, The University of Warsaw, 02-089 Warsaw,
Poland
c Department of Bioorganic Chemistry, Center of Molecular and
Macromolecular Studies, Polish Academy of Sciences,
´
´
90-363 Łodz, Poland
d Department of Pharmacology, Mossakowski Medical Research
´
Centre, 5 Pawinskiego Street, 02-106 Warsaw, Poland
w This article is part of a themed issue on Biophosphates.
z This paper is dedicated to Professor Wojciech J. Stec on the occasion
of his 70th birthday.
ꢁc
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888 | New J. Chem., 2010, 34, 888–893

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nucleotides there is at least one that could be recognized as a
substrate by all tested HIT-proteins, and if among these
proteins there is/are enzyme(s) that exhibit rather narrow
substrate specificity. We have collected for this study nine
recombinant HIT-proteins representing four branches of the
HIT superfamily: three Fhit-proteins (human HsFhit,9 Arabidopsis
thaliana AtFhit,9 and Trypanosoma brucei TbHIT-4518), four
Hint-proteins (AtHint1, AtHint3 and AtHint4 from A. thaliana,
and EcHinT from Escherichia coli), one decapping protein
from Caenorhabditis elegans, CeDcpS,19 and aprataxin-like
protein from A. thaliana, AtAtpx. Their hydrolytic activity
was tested using the following seven nucleotides: adenosine
50-phosphosulfate (1), adenosine 50-phosphoramidate (2),
adenosine 50-phosphrothioate (3), adenosine 50-phosphoro-
fluoride (4), diadenosine 50,50 0 0-P1,P3-triphosphate (5), di(7-
methylguanosine) 50,50 00-P1,P3-triphosphate (6) and adenosine
50-hypophosphate (7). Their structures are shown in Fig. 2. The
results of these studies are presented and the potential role of
the HIT-proteins in the metabolism of nucleotide pro-drugs is
discussed.
Results and discussion
At the beginning of this study, we performed pilot experiments
aimed to identify, amidst the nucleotidyl-derivatives, 1–7,
potential substrates for the reactions catalyzed by nine
HIT-proteins. Each protein was incubated with 1 mM nucleo-
tide in 50 mM potassium phosphate (pH 6.8) containing 5 mM
MgCl2. For the qualitative assays the protein concentration
was sufficient to obtain an enzymatic activity that allowed the
progress of each reaction to be monitored over a reasonable
range of time by analyzing reaction mixtures on thin-layer
chromatograms. Such an approach allowed us to determine if
a given nucleotide was recognized as a substrate by a parti-
cular protein and to select protein/enzyme dilution for quanti-
tative measurements (see below). As shown in Fig. 3A–D, all
four HIT-proteins that recognize dinucleoside triphosphates
(human and Arabidopsis Fhit-proteins, T. brucei HIT-45 and
C. elegans DcpS) easily split ApppA (5) to AMP (pA) and
ADP (ppA). They also hydrolyzed another dinucleoside tri-
phosphate tested, m7Gpppm7G (6), which is an analog of the
eukaryotic mRNA 50 end (cap structure). Whereas HsFhit and
TbHIT-45 hydrolyzed the latter compound 3–5-fold more
slowly than ApppA, the AtFhit cleaved m7Gpppm7G about
4-fold faster than ApppA (Table 1). In this respect AtFhit
resembled the yellow lupin nucleoside triphosphate hydrolase
whose substrate specificity had been described in 1996. Generally,
the capped, i.e. the m7G-containing dinucleoside triphosphates
were much better substrates of the lupin enzyme than ApppA
or GpppG and of all 11 dinucleotides tested m7Gpppm7G
was hydrolyzed with the highest velocity; 4.5-fold faster
than ApppA.20 In contrast to the aforementioned Fhit-
proteins and CeDcpS, neither the four investigated Hint-
proteins (Fig. 3E–H) nor aprataxin-like protein (Fig. 3I)
hydrolyzed ApppA or m7Gpppm7G. However, the AtHint4
catalyzed slow phosphorolysis rather than hydrolysis of
ApppA and this yielded ADP as the reaction product
(Fig. 3G). The Hint4 exhibited most clearly its dual catalytic
activity when incubated with adenosine 50-phosphosulfate (1).
Fig. 1 (A) Sequence alignment of amino acid fragments that contain
histidine triad motif of the proteins used in this study. Abbreviations
are as follows: At: Arabidopsis thaliana, Ec: Escherichia coli, Ce:
Caenorhabditis elegans, Hs: Homo sapiens, Tb: Trypanosoma brucei.
F represents a hydrophobic amino acid. (B) Phylogenetic tree of the
HIT-proteins used in this study. It was plotted using Clusta1X and
Treeview software according to Thompson et al.1 The ‘‘tree’’ shows
also positions of human congeners of some HIT-proteins which were
not investigated but are closely related to the studied ones.
50-O-phosphorothioates (S-pNs) with concomitant release of
H2S.14,15 In addition to Hint- and Fhit-proteins, two other
enzymes that belong to the HIT family were recently described.
One of them is aprataxin—an enzyme that removes AMP from
DNA breaks. Such an adenylylated DNA results from aborted
DNA ligation.16 The other enzyme, DcpS/DCS1, functions as a
‘‘decapping’’ enzyme that hydrolyzes the cap structure during
mRNA degradation.17 We wondered if among the different
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Fig. 2 Structures of the nucleotidyl-derivatives used in this study.
Under the assay conditions, i.e. in the presence of 0.1 M
orthophosphate, AtHint4 catalyzed both the hydrolysis of
(1) (forming AMP) and the phosphorolysis (forming ADP).
The catalytic duality of AtHint4 was less visible with (4)
and (2) (Fig. 3G). CeDcpS also exhibited catalytic duality
but to a lesser extent than the AtHint4 (see enzyme incubated
with (1), Fig. 3D). Very recently, more details on the dual
catalytic activities of these two HIT-proteins have been
described elsewhere.21 All nine HIT-family proteins hydro-
lyzed adenosine 50-phosphosulfate (1) and all except CeDcpS
cleaved adenosine 50-phosphoramidate (2). The latter property
distinguishes CeDcpS from other Npp-pN-cleaving enzymes
which eagerly hydrolyzed (2). Compound (1) appeared to
be a rather good substrate for all tested proteins, except
AtHint1 which hydrolyzed (1) very slowly (Table 1)
(Fig. 3E). Only AtHint1 and EcHinT catalyzed desulfuration
of adenosine 50-phosphrothioate (3) (Fig. 3E and H1). How-
ever, in case of AtHint1 the hydrolysis of that compound
proceeded at least 25-fold more slowly than hydrolysis of
adenosine 50-phosphoramidate (2) and in case of EcHinT
100-times more slowly (Table 1). To show that slow reaction,
two EcHinT concentrations, differing 50-fold, were used;
compare Fig. 3H1 and H2.
Initial velocities of the catalyzed reaction have been esti-
mated for each nucleotide-substrate by the use of HPLC
(see Experimental). Table 1 summarizes results of these measure-
ments. For each HIT-protein, the percent hydrolysis of a given
substrate was expressed relative to the hydrolysis of the
compound that was degraded with the highest velocity. These
were: NH2-pA (2) for HsFhit, AtHint1, AtHint3 and EcHinT,
m7Gpppm7G (6) for AtFhit, and SO4-pA (1) for TbHIT-45,
CeDcpS, AtHint4 and AtAptx. Using radio-labeled adenosine-
8-3H 50-phosphoramidate, we have estimated the Km values
for this nucleotide which, as mentioned above, appeared to be
a good substrate for all the HIT-proteins except CeDcpS. As
shown in Table 2, most of the Kms for NH2-pA fall in the
micromolar range. The same concerns App-pA; the Kms
estimated earlier for that compound were 1.2 mM for HsFhit10
Table 1 Initial velocities of hydrolysis of different nucleotides catalyzed by different HIT-proteins
HIT-proteins and relative initial velocities of degradation (%)a
HsFhit
AtFhit
TbHIT-45
CeDcpS
AtHint1
AtHint3
AtHint4
EcHinT
AtAptx
Nucleotide (no.)
SO4-pA (1)
NH2-pA (2)
S-pA (3)
65
100
0
48
64
0
100
96
0
100
0
0
0.1
100
4
33
100
0
100
33
0
11
100
1
100
13
0
F-pA (4)
App-pA (5)
m7Gpp-pm7G (6)
25
58
8
19
27
100
97
72
1
2
8
10
70
0
0
2
0
0
12
0
0
9
0
0
4
0
0
a
The initial velocities of the hydrolysis were measured by the HPLC analysis of reaction mixture withdrawn at various time intervals. Incubation
was performed at 30 1C and the mixture (0.075 ml final volume) contained 50 mM potassium phosphate (pH 6.8), 5 mM MgCl2, 1 mM substrate
and appropriately diluted HIT-protein. To stop the reaction, 10 ml aliquots were transferred to the test-tubes preheated to 95 1C and kept in that
temperature for 3 min. Then the samples were brought to 0.1 ml water and 25 ml of the resulted solution subjected to HPLC under the conditions
described in Experimental. For each HIT-protein, the values have been related to the value obtained for substrate that was hydrolyzed with the
highest velocity. The corresponding numbers were as follows: 22 nmol NH2-pA/min mgꢀ1 HsFhit; 17.6 nmol m7Gpppm7G/min mgꢀ1 AtFhit; 0.8 nmol
SO4-pA/min mgꢀ1 TbHIT-45; 11.5 nmol SO4-pA/min mgꢀ1 CeDcpS; 8.35 nmol NH2-pA/min mgꢀ1 AtHint1; 4.4 nmol NH2-pA/min mgꢀ1 AtHint3;
3.3 nmol SO4-pA/min mgꢀ1 AtHint4, 56 nmol NH2-pA/min mgꢀ1 EcHinT, and 0.85 nmol SO4-pA/min mgꢀ1 AtAptx.
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Fig. 3 Susceptibility of certain (di)nucleotides to cleavage catalyzed by different HIT-proteins. The reaction mixture (25 ml) contained 0.1 M
potassium phosphate (pH 6.8), 5 mM MgCl2, 1 mM (di)nucleotide indicated and the following amounts of the analyzed HIT-protein: (A) 0.085 mg
of human Fhit (HsFit); (B) 0.11 mg of Arabidopsis thaliana Fhit (AtFhit); (C) 10 mg of Trypanosoma brucei HIT-45 (TbHIT-45); (D) 0.2 mg of
Caenorhabditis elegans DcpS (CeDcpS); (E) 0.24 mg of A. thaliana Hint1 (AtHint1); (F) 0.5 mg of A. thaliana Hint3 (AtHint3); (G) 0.3 mg of
A. thaliana Hint4 (AtHint4); (H1) 1 mg of Escherichia coli HinT (EcHinT); (H2) 0.02 mg of E. coli HinT (EcHinT); (I) 1.6 mg of A. thaliana
aprataxin-like protein (AtAptx). Incubation was carried out at 30 1C. At time intervals (1, 10, 30 and 90 min) 3 ml aliquots were spotted on the
origin and chromatogram developed in dioxane : 25% ammonia : water (6 : 1 : 4, by volume) for 40 min. Spots of the substrates effectively separated
from corresponding products were visualized on the chromatograms under short-wave UV light and photographed. In each case the substrate
(not pointed at by an arrow) migrated faster than the reaction product(s).
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Table 2 The Km values estimated for adenosine 50-phosphoramidate
with some HIT-proteins
molecule. In addition to nitric oxide (NO) and carbon
monoxide (CO), hydrogen sulfide (H2S) has emerged recently
as a potentially important mediator of cardiovascular homeo-
stasis, cytoprotection and signal transduction in the central
nervous system.22–24
HIT-protein
Km for NH2-pA (mM)
AtHint1
AtHint3
AtHint4
AtAptx
EcHinT
TbHIT-45
CeDcpS
HsFhita
AtFhita
1
12
135
28
0.4
15
N/A
3
Experimental
Chemicals
AMP (pA), ADP (ppA), ApppA (5), NH2-pA (2), S-pA (3)
(commonly abbreviated as AMPS) and most of the bulky
reagents were from Sigma, St. Louis, MO, USA. F-pA (4) was
synthesized as described.25 Radio-labeled adenosine-8-3H
50-phosphoramidate was purchased from Moravek Biochemicals,
Brea, CA, USA. Syntheses of adenosine 50-phosphosulfate
(1)21 and of di(7-methylguanosine) 50,500 0-P1,P3-triphosphate26
(6) were described previously. Adenosine 50-hypophosphate
(7) was synthesized according to Setondji et al.:27 Tri-n-
butylammonium hypophosphate (tetrahydrate) (0.41 g, 1 mmol)
and 20,30-isopropylideneadenosine (76 mg, 0.25 mmol) were
dissolved in 5 ml of dimethylformamide (DMF). The mixture
was cooled down to 4 1C and a solution of dicyclohexyl-
carbodiimide (DCC) (0.29 g, 1.37 mmol) in DMF (0.4 ml) was
added. The reaction mixture was kept at 4 1C for 1 h and the
stirring continued for 12 h at room temperature. Then, 50 ml
of water was added and the mixture filtered through a Schott
funnel. 1 M HCl (5 ml) was added to the filtrate and the
reaction to remove the isopropylidene group was continued
for 2 h. The product was isolated on a Sephadex A-25 column
eluted with a gradient of 0.05–0.5 M triethylammonium
bicarbonate. The appropriate fractions were collected and
concentrated to dryness. To remove the residual buffer,
methanol (5 ml) was added and the sample concentrated to
yield ca. 95 mg of the adenosine 50-hypophosphate (triethyl-
ammonium salt, M.W. 725; 0.13 mmol, 52% yield). Then, using
Dowex cation-exchanger, the product was quantitatively con-
verted into the trisodium salt, concentrated and lyophilized
yielding 80 mg; (M.W. 477, d 31P NMR (D2O) 17.09 ppm (d),
4.93 ppm (d), 1JP-P = 660 Hz; MALDI TOF MS m/z 410.0 Mꢀ).
3
a
N/A: Not applicable, given nucleotide is not a substrate. The Km
values for HsFhit and AtFhit were published earlier.9
and 6.5 mM for TbHIT-45.18 This suggests that different
HIT-proteins exhibit high affinity at least for those two
naturally occurring nucleotides.
Regarding newly synthesized adenosine 50-hypophosphate
(7), none of the HIT-proteins investigated cleaved its P-P bond
and liberated pA. Compounds with such a bond do not occur
in living systems and proteins probably do not recognize this
structure. Earlier, we found that (7) was also refractory to
other hydrolases (bacterial alkaline phosphatase, lupin apyrase
and snake venom phosphodiesterase) and that (7) did not inhibit
these enzymes. Only snake venom 50-nucleotidase slowly hydro-
lyzed this compound, releasing adenosine (A. Guranowski,
unpublished observations).
Except for AtHint1, the HIT-proteins also did not release pA
from such a common metabolite as ADP (ppA). AtHint1 was
the only protein that catalyzed noticeable release of pA from
ppA but velocity of that reaction was more than 250-fold lower
than that of the hydrolysis of adenosine 50-phosphoramidate (2).
Conclusions
This study extends our earlier observations concerning Fhit
proteins,9 AtHint4 and CeDcpS,21 and confirms that different
HIT-proteins may be involved in the metabolism of SO4-pA
and hence affect the sulfur metabolism in different organisms.
Also, the results presented in this communication should be
useful for those who study fates of nucleotide prodrugs
delivered to target cells. Exchange of the oxygen atom in the
nucleotide phosphate moiety to such residues as -F, -S, -NH2,
-SO4 or -ppA apparently lowers the nucleotide’s polarity.
(Compare, for example, markedly lower chromatographic
mobility of adenine nucleotides with the mobility of the
adenylyl-derivatives; Fig. 3.) Therefore such synthetically
modified nucleotidyl-derivatives may serve as model com-
pounds for prodrugs which may undergo hydrolysis in cellular
environment, e.g. due to activity of different HIT-proteins,
and liberate the corresponding 50-NMPs which easily enter
common metabolic pathways.
Recombinant proteins
Origin of the recombinant HIT-proteins and procedures used
for their purification were described previously.9,18,19,21
Chromatographic systems
Thin-layer chromatography was performed on aluminium
plates precoated with silica gel containing fluorescent indicator
(Merck). The chromatograms were developed in dioxane: 25%
ammonia : water (6 : 1 : 5, by volume) for 30–40 min. Two
HPLC systems were used. Most of the reaction mixtures were
chromatographed on a reverse phase column (Teknokroma C18,
150 mm ꢂ 4.6 mm; 5 mm). The column was eluted with linear
gradient of 50 mM triethylamine buffer (pH 7.4 adjusted
with CO2) : acetonitrile (97 : 3, v/v) (solvent A) and solvent
A : acetonitrile (60 : 40, v/v, solvent B); 0–15 min, 20% solvent
B, at a flow-rate of 1 ml minꢀ1. In that system the retention
times (min) were as follows: AMP (pA) 3.5, NH2-pA 4.0, ADP
(ppA) 4.5, S-pA 4.6, SO4-pA 5.5, Ap3A (App-pA) 9.8 and
Special attention should be given to nucleoside 50-phosphoro-
thioates, e.g. compound (3). Their administration to animals
or humans can appear beneficial. Desulfuration of those thio-
compounds catalyzed by certain HIT-proteins (rabbit Hint1
described earlier15 and AtHint1 and EcHinT reported in this
paper) releases hydrogen sulfide; a new gaseous signaling
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F-pA 12. Reaction mixtures containing m7Gpppm7G were
chromatographed on an anion exchange column (Hamilton
PRP-X100). Two solutions A and B were prepared. Solution
A was 25 mM citric acid pH 3.5 and solution B was A :
acetonitrile (17 : 3). The column was eluted for 1 min with A
and then 0–5 min with linear gradient 0–50% B. The retention
times (min) were 1.1 for m7pG, 2.1 for m7ppG, and 4.7 for
m7Gpppm7G.
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Enzyme assays
The reaction mixture (usually 50 ml) contained 0.1 M potassium
phosphate (pH 6.8), 5 mM MgCl2, 1 mM di(nucleotide)
indicated and appropriately diluted protein solution. Incu-
bations were carried out at 30 1C and at time intervals 3 or 10 ml
aliquots were analyzed; either by TLC or HPLC, respectively.
Incubation time and protein concentrations were appropriately
adjusted to obtain reliable values of the initial velocities. For
more details see the legend to Fig. 3 and Table 1.
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Acknowledgements
This work was supported by grant PBZ-MNiSW-07/1/2007
from the Polish Ministry of Sciences and Higher Education.
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