The List of the Most Important Scientific Results of the Institution obtained in the Period 2005–2009

Department 1     Molecular Biophysics and Pharmacology (DMBP)

[1] DNA-protein cross-links by antitumor cisplatin

(a) K. Chvalova, V. Brabec, and J. Kasparkova:  Mechanism of the formation of DNA-protein cross-links by antitumor cisplatin. Nucleic Acids Research, 35,1812-1821, 2007, IBP: all authors. IF 6.9; 15 citations on WOS

(b)100% contribution by Brno laboratory.

(c) DNA–protein cross-links are formed by various DNA-damaging agents including antitumor platinum drugs. The origin of these DPCLs and their cellular processing on molecular level was investigated. It has been shown that in cell-free media cisplatin forms DPCLs more effectively than ineffective transplatin and that the DPCLs are formed by the transformation of DNA monofunctional and intrastrand cross-links of cisplatin. The DPCLs formed by cisplatin inhibit DNA polymerization or removal of these ternary lesions from DNA by nucleotide excision repair system more effectively than plain DNA intrastrand or monofunctional adducts. Thus, the bulky DNA–protein cross-links formed by cisplatin represent a more distinct and persisting structural motif recognized by the components of downstream cellular systems processing DNA damage considerably differently than the plain DNA adducts of this metallodrug. The results of this work also demonstrate that cross-linking DNA and proteins by platinum complexes can be also applied to studies of specific protein–DNA interactions both in vitro and in vivo.

[2] Activation of the trans geometry in platinum antitumor complexes

(a) V. Marini, P. Christofis, O. Novakova, J. Kasparkova, N. Farrell, and V. Brabec: Conformation, protein recognition and repair of DNA interstrand and intrastrand cross-links of antitumor trans-[PtCl₂(NH₃)(thiazole)]. Nucleic Acids Research, 33,5819-5828, 2005,  IBP: corresponding author, first author, 3 co-authors of 4 others. IF 6.9; 15 citations on WOS

F.J. Ramos-Lima, O. Vrana, A.G. Quiroga, C. Navarro-Ranninger, A. Halamikova, H. Rybnickova, L. Hejmalova, and V. Brabec: Structural characterization, DNA interactions, and cytotoxicity of new transplatin analogues containing one aliphatic and one planar heterocyclic amine ligand.  Journal of  Medicinal Chemistry, 49, 2640-2651, 2006, IBP: corresponding author, first author, 4 co-authors of 6 others. IF 4.9; 15 citations on WOS

T. Suchankova, M. Vojtiskova, J. Reedijk, V. Brabec, and J. Kasparkova: DNA and glutathione interactions in cell-free media of asymmetric platinum(II) complexes cis- and trans-[PtCl₂(isopropylamine)(1-methylimidazole)]: relations to their different antitumor effects. Journal of Biological Inorganic Chemistry 14, 75-87, 2009,IBP: corresponding author, first author, 2 co-authors of 3 others. IF 3.6; 1 citation on WOS

A. Halámiková, P. Heringová, J. Kašpárková, F.P. Intini, G. Natile, A. Nemirovski, D. Gibson, and V. Brabec: Cytotoxicity, mutagenicity, cellular uptake, DNA and glutathione interactions of lipophilic trans-platinum complexes tethered to 1-adamantylamine. Journal of Inorganic Biochemistry, 102,1077–1089, 2008, IBP: corresponding author, first author, 2 co-authors of 6 others. IF 3.1; 15 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratories supplied some platinum compounds for this study. These cooperating laboratories were included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) The cytotoxicity of transplatinum complexes of structural formula trans-PtCl₂(L)(L')] (L or L' are sterically hindered heterocyclic planar, nonplanar ligands or aliphatic ligands is greatly enhanced, in comparison to ineffective parent transplatin (L = L' = NH₃) such that cytotoxicity equivalent to that ofthe clinically used cisplatin is obtained. Moreover, an intriguing property of these transplatinum complexes is that they exhibit antitumor activity in various cisplatin resistant cells. Mechanistic studies carried out to delineate the reasons for both thedramatically enhanced cytotoxicity and the lack of cross-resistance withthe clinically used agents revealed that these transplatinum complexes modify DNA, which is the major pharmacological target of platinum drugs, in a unique way. In addition, lesions induced in DNA by these Pt^II compounds are recognized and repaired differently from lesions induced by conventional cisplatin or transplatin. Thus, these results are consistent with the thesis systematically tested by us that platinum drugs that bind to DNA in a manner, which is distinctly different from that of cisplatin, may exhibit altered biological properties, including a different spectrum and intensity of antitumor activity.

[3] DNA modifications by novel Ru^II arene complexes, recognition and repair of these modifications. Relationship to cytotoxicity

(a) O. Novakova, J. Kasparkova, V. Bursova, C. Hofr, M. Vojtiskova, H. Chen, P. J. Sadler, and V. Brabec: Conformation of DNA modified by monofunctional Ru(II) arene complexes: Recognition by DNA-binding proteins and repair. Relationship to cytotoxicity. Chemistry & Biology, 12,121-129, 2005, IBP: corresponding author, first author, 4 co-authors of 6 others. IF 5.6; 33 citations on WOS

T. Bugarcic, O. Novakova, A. Halamikova, L. Zerzankova, O. Vrana, J. Kasparkova, A. Habtemariam, S. Parsons, P. J. Sadler, and V. Brabec: Cytotoxicity, cellular uptake, and DNA interactions of new monodentate ruthenium(II) complexes containing terphenyl arenes. Journal of Medicinal Chemistry, 51, 5310-5319, 2008, IBP: corresponding author, joint first author, 4 co-authors of 8 others. IF 4.9; 8 citations on WOS

O. Novakova, J. Malina, T. Suchankova, J. Kasparkova, T. Bugarcic, P. J. Sadler, and V. Brabec: Energetics, conformation, and recognition of DNA duplexes modified by monodentate Ru^II complexes containing terphenyl arenes. Chemistry-A European Journal, 16,5744-5754, 2010, IBP: corresponding author, first author, 3 co-authors of 5 others. IF 5.4; 0 citations on WOS

O. Nováková, A.A. Nazarov, C. G. Hartinger, B. K. Keppler, and V. Brabec: DNA interactions of dinuclear Ru^II arene antitumor complexes in cell-free media. Biochemical Pharmacology, 77, 364-374, 2009,IBP: corresponding author, first author. IF 4.8;

9 citations on WOS

(b)Contribution to this project by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratories supplied ruthenium compounds for this study. These cooperating laboratories were included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) The results demonstrate that new organoruthenium complexes of the type [(η⁶-arene)Ru^II(en)Cl]⁺ exhibits promising cytotoxic effects in several human tumor cell

lines including those resistant to conventional cisplatin and concomitantly their DNA binding mode involves combined intercalative and monofunctional (coordination) binding modes. These results represent a further improvement in the structure-pharmacological relationship needed for the design of new antitumor ruthenium drugs and chemotherapeutic strategies.

[4] Insights into the resistance of tumors to platinum anticancer drugs

(a) V. Brabec and J. Kasparkova: Modifications of DNA by platinum complexes:  Relation to resistance of tumors to platinum antitumor drugs. Drug Resistance Updates, 8, 131-146, 2005,IBP: all authors. IF 9.4; 72 citations on WOS

(b) Contribution to the writing of the review by authors from Brnois 100%.

(c) Key review article summarizing knowledge on the molecular mechanisms of resistance of tumor cells to platinum anticancer drugs, based primarily on the work of the authors.

[5] A new generation of antitumor ruthenium drugs

 (a) V. Brabec and O. Novakova. DNA binding mode of ruthenium complexes and relationship to tumor cell toxicity. Drug Resistance Updates,  9,111-122, 2006,IBP: all authors. IF 9.4; 63 citations on WOS

(b) Contribution to the writing of the review by authors from Brnois 100%.

(c) Key review article summarizing knowledge on the molecular mechanisms of antitumor activity of a novel generation of ruthenium anticancer drugs, based primarily on the work of the authors.

[6] Unique properties of DNA adducts of oxaliplatin, the second generation antitumor platinum drug

(a) J. Kasparkova, M. Vojtiskova, G. Natile, and V. Brabec: Unique properties of DNA interstrand cross-links of antitumor oxaliplatin and the effect of chirality of the carrier ligand. Chemistry-A European Journal, 14,1330-1341, 2008. IBP: corresponding author, first author, 1 co-author of 2 others. IF 5.5; 10 citations on WOS

J. Malina, O. Novakova, M. Vojtiskova, G. Natile, and V. Brabec: Conformation of DNA GG intrastrand cross-link of antitumor oxaliplatin and its enantiomeric analog. Biophysical Journal, 93, 3950-3962, 2007,IBP: corresponding author, first author, 2 co-authors of 3 others. IF 4.7; 12 citations on WOS

O. Delalande, J. Malina, V. Brabec, and J. Kozelka: Chiral differentiation of DNA adducts formed by enantiomeric analogues of antitumor cisplatin is sequence-dependent. Biophysical Journal,  88:4159-4169, 2005, IBP: joint corresponding author, first author, 1 coauthor of  2 others. IF 4.7; 10 citations on WOS

(b)Contribution to this study by the IBP author’s team is 90% and includes design of the research, all experiments and writing. The foreign laboratories supplied some platinum compounds for this study and partially participated in molecular modelling. These cooperating laboratories were includedas because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This work revealed, for thefirst time, thenewstructural and biochemicalcharacteristics of the cross-links formed in DNA byoxaliplatin. Thisinformationisuseful for a better understandingof how the stereochemistry of the carrieramine ligands of cisplatin analogues

can modulate their anticancer and mutagenicproperties.The unique properties of thecross-links of oxaliplatin are responsible for this drug'sunique antitumor effects.

[7] Thermodynamic analysis and energetics of DNA modified by Pt^II complexes

(a) V. Bursova, J. Kasparkova, C. Hofr, and V. Brabec: Effects of monofunctional adducts of platinum(II) complexes on thermodynamic stability and energetics of DNA duplexes. Biophysical Journal, 88, 1207-1214, 2005,IBP: all authors. IF 4.7; 4citations on WOS

O. Nováková, J. Malina, J. Kašpárková, A. Halámiková, V. Bernard, F. Intini, G. Natile, and V. Brabec:  Energetics, conformation, and recognition of DNA duplexes modified by methylated analogues of [PtCl(dien)]⁺. Chemistry-A European Journal, 15, 6211-6221, 2009, IBP: corresponding author, first author, 4 co-authors of 6 others. IF 5.5; 0 citations on WOS

C. Hofr, and V. Brabec: Thermal stability and energetics of 15-mer DNA duplex interstrand cross-linked by trans-diamminedichloroplatinum(II). Biopolymers 77,222-229, 2005. IBP: all authors. IF 2.4; 10 citations on WOS

J. Kasparkova, V. Marini, V. Bursova, and V. Brabec:  Biophysical studies on the stability of DNA intrastrand cross-links of transplatin. Biophysical  Journal, 95, 4361-4371, 2008, IBP: all authors. IF 4.7; 1 citation on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratory supplied some platinum compounds for this study. This cooperating laboratory was includedas because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) It is shown that the stability of various DNA adducts of platinum complexes depends on the sequence context and correlates with the extent of conformational distortion and thermodynamic destabilization induced in double-helical DNA by these lesions. In addition, it is also demonstrated that rather thermodynamic than thermal characterization of DNA adducts of platinum compounds is a property implicated in the modulation of downstream effects such as protein recognition and repair.

[8] Molecular mechanism underlying antitumor effects of a new tetravalent platinum drug

(a) J. Kasparkova, O. Novakova, O. Vrana, F. Intini, G. Natile, and V. Brabec: Molecular aspects of antitumor effects of a new platinum(IV) drug. Molecular Pharmacology, 70, 1708-1719, 2006,IBP: corresponding author, first author, 2 co-authors of 4 others. IF 4.7; 15 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and inludes design of the research, all experiments and writing. The external laboratories supplied platinum compounds for this study. The cooperating laboratories were included because of the lack of laboratory or unit at the Instituteof Biophysicshaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) It was of great interest to understand details of mechanisms underlying biological efficacy of a new Pt^IV complex, cis,trans,cis-[PtCl₂(CH₃COO)₂(NH₃)(C₁₀H₁₇N)] which seems promising for the perspective application in therapy of corresponding tumors. It was demonstrated that the effects of pharmacological factors associated with antitumor effects of platinum complexes, such as enhanced accumulation of the drug in cells, strong inhibition of DNA polymerization by these adducts, enhanced persistence of the adducts due to their less efficient repair and DNA-protein cross-linking are different from the effects of these factors in the mechanism underlying activity of conventional cisplatin. Hence, the differences between the effects of the new Pt^IV complex and cisplatin observed in this work may help understand the unique activity of this new antitumor metallodrug.

[9] Mechanistic insights into early phases of the mechanism by which cisplatin analogues exert their anticancer activity

(a) J. Kasparkova, T. Suchankova, A. Halamikova, L. Zerzankova, O. Vrana, N. Margiotta, G. Natile, and V. Brabec: Cytotoxicity, cellular uptake, glutathione and DNA interactions of an antitumor large-ring Pt^II chelate complex incorporating the cis-1,4-diaminocyclohexane carrier ligand. Biochemical Pharmacology, 79, 552-564, 2010, IBP: corresponding author, first author, 4 co-authors of 6 others. IF 4.8; 0 citations on WOS

J. Malina, M. Vojtiskova, V. Brabec, C. I. Diakos, and T. W. Hambley: DNA adducts of the enantiomers of the Pt(II) complexes of the ahaz ligand (ahaz = 3-aminohexahydroazepine) and recognition of these adducts by HMG domain proteins. Biochemical and Biophysical Research Communications, 332,1034-1041, 2005, IBP: joint corresponding author, first author, 1 co-author of 2 others. IF 2.7; 5 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratories supplied some platinum compounds for this study. These cooperating laboratories were included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This study was aimed at a deeper understanding of four early steps in the mechanism of biological activity of selected new antitumor analogues of conventional cisplatin (large-ring Pt^II chelate complex incorporating the cis-1,4-diaminocyclohexane carrier ligand or Pt^II complex containing the ahaz ligand (ahaz = 3-aminohexahydroazepine)tested as potential new generation platinum antitumor drugs. The results obtained when the effects of the two Pt^II complexes were tested demonstrated that cell entry, reactions with sulfur-containing compounds, platinum–DNA binding along with processing platinated DNA by proteins (enzymes) and DNA repair were different from the effects of cisplatin. These differences have been suggested to be associated with more favorable biological (pharmacological) effects of the cisplatin analogues.

[10] Metallosupramolecular cylinders - specific DNA major groove binders

(a) J. Malina, M. J. Hannon, and V. Brabec: Recognition of DNA three-way junctions by metallosupramolecular cylinders: Gel electrophoresis studies. Chemistry-A European Journal, 13, 3871-3877, 2007, IBP: corresponding author, first author. IF 5.5; 9 citations on WOS

J. Malina, M. J. Hannon, and V. Brabec: DNA binding of dinuclear iron(II) metallosupramolecular cylinders. DNA unwinding and sequence preference. Nucleic Acids Research, 36, 3630-3638, 2008, IBP: corresponding author, first author. IF 6.9; 4 citations on WOS

J. Malina, M. J. Hannon, and V. Brabec: Interaction of dinuclear ruthenium(II) supramolecular cylinders with DNA: Sequence-specific binding, unwinding, and photocleavage. Chemistry-A European Journal, 14, 10408-10414, 2008, IBP: corresponding author, first author. IF 5.5; 1 citation on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratory supplied supramolecular cylinders for this study. This cooperating laboratory was included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This work describes DNA binding mode synthetic tetracationic supramolecular cylinders (with a triple helical architecture) that target the major groove of DNA and can bind to DNA Y-shaped junctions. In addition, the observation of sequence-preference in the binding of these supramolecular cylinders suggests that a concept based on the use of metallosupramolecular cylinders might result in molecular designs that recognize the genetic code in a sequence-dependent manner with a potential ability to affect the processing of the genetic code. Moreover, RuII metallosupramolecular cylinder binds and cleaves DNA in a sequence-dependent manner, which may provide a useful tool for basic and applied biology, such as for controlled manipulation of the genome.

[11] Photoactivatable Pt^IV compounds. Potential for a new localised, less toxic therapy for cancer

(a) F. S. Mackay, J. A. Woods, P. Heringova, J. Kasparkova, A. M. Pizarro, S. A. Moggach, S. Parsons, V. Brabec, and P. J. Sadler: A potent cytotoxic photoactivated platinum complex. Proceedings of National Academy of Sciences, USA, 104,20743-20748, 2007,IBP: joint corresponding author, 2 co-authors of 7 others. IF 9.4; 42 citations on WOS

P. Heringova, J. Woods, F. S. Mackay, J. Kasparkova, P. J. Sadler, and V. Brabec: Transplatin is cytotoxic when photoactivated: Enhanced formation of DNA cross-links. Journal of Medicinal Chemistry, 49, 7792-7798, 2006,IBP: corresponding author, first author, 1 co-authors of 4 others. IF 4.9; 14 citations on WOS

(b) The contribution of the authors from IBP Brno was ~50% and includes all work on DNA level in cell-free media. The design was done jointly with P.J. Sadler and J.A. Woods, writing was done jointly with P.J. Sadler.

(c) The light-activated platinum complexes tested in this work are effective in killing human cancer cells, including cisplatin-resistant cells, but only upon irradiation, thus combining the potent cytotoxic ability of platinum compounds with the selectivity of a lightactivated drug. The results also demonstrated an unusual mechanism of action of these photoactivatable comppounds which appear candidates for use in photoactivated cancer chemotherapy.

[12] Novel approaches to antitumor polynuclear platinum drugs

(a) V. Brabec, P. Christofis, M. Slamova, H. Kostrhunova, O. Novakova, Y. Najajreh, D. Gibson, and J. Kasparkova: DNA interactions of new cytotoxic tetrafunctional dinuclear platinum complex trans,trans-[{PtCl₂(NH₃)}₂(piperazine)]. Biochemical Pharmacology, 73,1887-1900, 2007, IBP: corresponding author, first author, 4 co-authors of 6 others. IF 4.8; 1 citation on WOS

L. Zerzankova, T. Suchankova, O. Vrana, N. P. Farrell, V. Brabec, and J. Kasparkova: Conformation and recognition of DNA modified by a new antitumor dinuclear Pt^II complex resistant to decomposition by sulfur nucleophiles. Biochemical Pharmacology, 79, 112-121, 2010, IBP: corresponding author, first author, 3 co-authors of 4 others. IF 4.8; 0 citations on WOS

B. Moriarity, O. Novakova, N. Farrell, V. Brabec, and J. Kasparkova: 1,2-GG intrastrand cross-link of antitumor dinuclear bifunctional platinum compound with spermidine linker inhibits DNA polymerization more effectively than the cross-link of conventional cisplatin. Archives of Biochemistry and Biophysics, 459, 264-272, 2007, IBP: corresponding author, first author, 2 co-authors of 3 others. IF 2.6; 9 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing were executed. The foreign laboratories supplied some platinum compounds for this study. These cooperating laboratories were included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This study demonstrates some new features of DNA binding mode of antitumor dinuclear Pt^II complexes and their downstream cellular processing. The results confirm that the DNA conformational distortions induced by polynuclear agents are thus structurally distinct from those of cis-[PtX2(amine)2], and are not necessarily susceptible to the same cellular processing as cisplatin and othercis-[PtX2(amine)2] chemotypes.

[13] Osmium containing metallodrugs

(a) H. Kostrhunova, J. Florian, O. Novakova, A. F. A. Peacock, P. J. Sadler, and V. Brabec: DNA interactions of monofunctional organometallic osmium(II) antitumor complexes in cell-free media. J. Med. Chem. 51, 3635-3643, 2008, IBP: corresponding author, first author, 2 co-authors of 4 others. IF 4.9; 9 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing were executed. The foreign laboratory provided osmium-arene compounds for this study. This cooperating laboratory was included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. The results also indicate both a mechanism of action and a detoxification mechanism for Os^II-arene compounds different from those of conventional cisplatin.

[14] Anew type of platinum complex that has a trans configuration, is asymmetric, and has a singly charged nonplanar semifluxional bicyclic ligand

(a) Y. Najajreh, D. Prilutski, Y. Ardeli-Tzaraf, J.-M. Perez, E. Khazanov, Y. Barenholz, J. Kasparkova, V. Brabec, and D. Gibson: Structure and unique interactions with DNA of a cationic trans-platinum complex with the nonplanar bicyclic piperidinopiperidine ligand. Angewangte Chemie-International Edition, 44, 2-4, 2005, IBP: 2 coauthors of 7. IF 10.9, 12 citations on WOS

(b) Contribution to this study by the IBP author’s team is ~30%. This work reports on X-ray crystal structure, DNA-binding and pharmacological properties of a new Pt^II complex - all data on DNA binding and some pharmacological properties were obtained by the authors from IBP Brno.

(c) An intriguing and unprecedented feature of this new complex is that it binds extremely rapidly to DNA by direct substitution (not requiring initial aquation as does cisplatin), distorts DNA in a unique manner and circumvents all of the known cisplatin resistance mechanism in human ovarian cancer cell lines.

[15] Activity of topoisomerase I is uniquelly affected by DNA adducts of antitumor cisplatin.

(a)J. Malina, O. Vrana, and V. Brabec: Mechanistic studies of the modulation of cleavage activity of topoisomerase I by DNA adducts of mono- and bi-functional Pt^II complexes. Nucleic Acids Research 37, 5432-5442, 2009, IBP: all authors. IF 6.9; 0 citations on WOS

(b) 100% contribution by Brnolaboratory.

(c) This work demonstrates for the first time that mechanisms underlying antitumor effects of cisplatin and perhaps of other platinum drugs may also involve effects of these metallodrugs

on topoisomerase I catalytic activity.

[16] The bifunctional mononuclear Pt^II complex containing the leaving ligands in the trans configuration inhibits binding of the tumor suppressor protein p53 to its consensus DNA sequence

(a) K. Stehlíková, J. Kašpárková, O. Nováková, A. Martínez, V. Moreno, and V. Brabec: Recognition of DNA modified by trans-[PtCl₂NH₃(4-hydroxymethylpyridine)] by tumor suppressor protein p53 and character of DNA adducts of this cytotoxic complex. FEBS Journal, 273, 301-314, 2006, IBP: corresponding author, first author, 3 co-authors of 5 others. IF 3.1; 14 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing were executed. The foreign laboratory supplied the platinum compound for this study. This cooperating laboratory was included because of the lack of a laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c) This new Pt^II complex distort DNA conformation in a unique way. In addition, this work also demonstrates for the first time the efficiency of the bifunctional mononuclear platinum(II) complex containing the leaving ligands in the trans configuration to inhibit binding of the tumor suppressor protein p53 to its consensus DNA sequence. Thus, in this respect the new analogue of clinically ineffective transplatin resembles to antitumor cisplatin, although the reasons for this resemblance may be different.

[17] Effects of cisplatin on the Raman signatures of DNA

(a) O. Vrana, V. Masek, V. Drazan, and V. Brabec: Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin. Journal of Structural Biology 159,1-8, 2007, IBP: all authors. IF 4.0; 3 citations on WOS

(b)100% contribution by Brno laboratory.

(c) Raman spectroscopy was employed to characterize the perturbations to DNA conformation induced in DNA by adducts of antitumor cisplatin. The results demonstrated significant perturbations to the B-form DNA backbone due to the major intrastrand lesions and that several nucleotides changed their conformation from C2'-endo to C3'-endo. Evidence for a partial transition from B- to A-form was found in several regions of the Raman spectra as well.The results of this study demonstrate that Raman spectroscopy represents a suitable tool to provide insights into structural factors involved in the mechanisms underlying antitumor effects of platinum drugs.

[18] The effects of antitumor cisplatin on telomers

(a) P. Heringova, J. Kasparkova, and V. Brabec: DNA adducts of antitumor cisplatin preclude telomeric sequences from forming G-quadruplexes. Journal of  Biological Inorganic Chemistry, 14, 959-968, 2009, IBP: all authors. IF 3.6; 0 citations on WOS

(b)100% contribution by Brno laboratory.

(c) The results indicate that cisplatin is more efficient than transplatin in disturbing the quadruplex structure, thereby precluding telomeric sequences from forming quadruplexes. On the other hand, the results of this work also demonstrate that in absence of free platinum complex, DNA adducts of antitumor cisplatin inhibit telomerase catalysis, so the mechanism

underlying this inhibition does not involve formation of the G quadruplexes which are not elongated by telomerase.

[19] Molecular mechanism of a combined antitumor effect of cisplatin and mild hyperthermia

(a) A. Halamikova, O. Vrana, J. Kasparkova, and V. Brabec: Biochemical studies of the thermal effects on DNA modifications by the antitumor cisplatin and their repair. ChemBioChem 8, 2008-2015, 2007, IBP: all authors. IF 3.3; 1 citation on WOS

(b)100% contribution by Brno laboratory.

(c) This study reveals enhanced DNA-cross-linking efficiency of cisplatin and the lower

efficiency of native DNA repair mechanisms at higher temperature which play an important role in the potentiation of the antitumor effects of cisplatin under conditions of mild hyperthermia.

[20] Deoxyribonuclease I footprinting of DNA binding modes of bifunctional platinum complexes

(a) K. Chválová, J. Kašpárková, N. Farrell, and V. Brabec: Deoxyribonuclease I footprinting reveals different DNA binding modes of bifunctional platinum complexes. FEBS Journal 273,3467-3478, 2006,IBP: corresponding author, first author, 1 co-authors of 2 others. IF 3.1; 3 citations on WOS

(b)Contribution to this study by the IBP author’s team is 95% and includes design of the research, all experiments and writing. The foreign laboratory supplied some platinum compounds for this study. This cooperating laboratory was included because of the lack of laboratory or unit in Brnohaving adequate equipment and experience in synthetic and bioinorganic chemistry.

(c)It was shown in this work that various conformational perturbations induced by platinum binding in the major groove translated into the minor groove, allowing their detection by

DNase I probing. The results also demonstrate the very high sensitivity of DNase I to DNA conformational alterations induced by antitumor platinum complexes so that the platinum adducts which induce specific local conformational alterations in DNA are differently recognized by DNase I.

Department 2     Biophysical Chemistry and Molecular Oncology (DBCMO)

[1] Novel strategies of labeled nucleic acids construction and their application in electrochemical DNA sensing

P. Brazdilova, M. Vrabel, R. Pohl, H. Pivonkova, L. Havran, M. Hocek, and M. Fojta, Ferrocenylethynyl derivatives of nucleoside triphosphates: Synthesis, incorporation, electrochemistry, and bioanalytical applications. Chemistry-a European Journal 13,  9527-9533, 2007, IBP: joint corresponding author, first author; 29 citations on WOS, IF 5.454

H. Cahova, L. Havran, P. Brazdilova, H. Pivonkova, R. Pohl, M. Fojta, and M. Hocek, Aminophenyl- and nitrophenyl-labeled nucleoside triphosphates: Synthesis, enzymatic incorporation, and electrochemical detection. Angewandte Chemie-International Edition 47, 2059-2062, 2008, IBP: joint corresponding author; 27 citations on WOS, IF 10.879

M. Vrabel, P. Horakova, H. Pivonkova, L. Kalachova, H. Cernocka, H. Cahova, R. Pohl, P. Sebest, L. Havran, M. Hocek, and M. Fojta, Base-Modified DNA Labeled by [Ru(bpy)(3)](2+) and [Os(bpy)(3)](2+) Complexes: Construction by Polymerase Incorporation of Modified Nucleoside Triphosphates, Electrochemical and Luminescent Properties, and Applications. Chemistry-a European Journal 15, 1144-1154, 2009, IBP: joint corresponding author; 14 citations on WOS, IF 5.454

About 50 % of the work was done at the IBP (main contributions from L. Havran, H. Pivoňková and P. Horáková under leadership of M. Fojta); crucial cooperation with the group of M. Hocek, IOCB ASCR Prague (pursuing the organic synthesis of modified nucleotides).

A fruitful collaboration with the group of M. Hocek at the IOCB ASCR in Pragueresulted in development of novel facile techniques of enzymatic construction of modified, functionalized and labeled nucleic acids using modified deoxynucleotide triphosphates (the synthesis of which has been developed in the Hocek lab). Particularly in the area of electrochemical DNA sensing, including DNA minisequencing/SNP typing based on “multicolor” electrochemical DNA coding, promising results were obtained and published in prestigious journals. These papers have gained considerable number of citations within relatively short time.

[2] Changes in interfacial properties of a-synuclein preceding its aggregation

E. Palecek, V. Ostatna, M. Masarik, K. Bertoncini, and T.M. Jovin, Changes in interfacial properties of a-synuclein preceding its aggregation. Analyst 133, 76-84, 2008, IBP: corresponding author, first author; 15 citations on WOS, IF 3.761

About 70 % of the work was done at the IBP by V. Ostatná and M. Masařík under leadership of E. Paleček, who also contributed by the major part of writing. The cooperating group (that possesses additional experimental techniques unavailable at the IBP) was involved on the basis on a joint project.

An important application of label-free electrochemical analysis of proteins based on their electrocatalytic activity giving rise to so called “peak H”, which has been established as a useful signal sensitively responding to various changes in the protein structure. In this work, the peak H is used to monitor pre-aggregation and aggregation processes of a-synuclein, a protein the aggregation of which is connected with Parkinson’s disease. Electrochemistry has proved superior to other analytical techniques regarding its sensitivity to changes taking place in early stages of the aggregation process, and appeared useful tool for e.g., seeking substances (potential drugs) affecting the aggregation trigger.

[3] Unfolding/denaturation of proteins viewed by electrochemical methods

V. Ostatna, F. Kuralay, L. Trnkova, and E. Palecek, Constant current chronopotentiometry and voltammetry of native and denatured serum albumin at mercury and carbon electrodes. Electroanalysis 20,  1406-1413, 2008, IBP: corresponding author, first author; 10 citations on WOS, IF 2.901

V. Ostatna, and E. Palecek, Native, denatured and reduced BSA - Enhancement of chronopotentiometric peak H by guanidinium chloride. Electrochimica Acta 53, 4014-4021, 2008, IBP: corresponding author, first author; 14 citations on WOS, IF 3.078

E. Palecek, and V. Ostatna, Potential-dependent surface denaturation of BSA in acid media. Analyst 134, 2076-2080, 2009, IBP: corresponding author, first author; 3 citations on WOS, IF 3.761

E. Palecek, and V. Ostatna, Ionic strength-dependent structural transition of proteins at electrode surfaces. Chemical Communications 13, 1685-1687, 2009, IBP: corresponding author, first author; 4 citations on WOS, IF 5.34

Over 90 % of the work was pursued at the IBP, mostly by V. Ostatná under leadership of E. Paleček. The cooperating group from the MasarykUniversityin Brno(L. Trnková) has contributed by application of a specific electrochemical approach (elimination voltammetry).

Another application of the protein catalytic signal, peak H. This series of papers represents pioneering work in application of simple but highly sensitive electrochemical approaches to study folding/unfolding of non-conjugated proteins. In contrast to traditional view considering proteins adsorbed at mercury electrode surface inherently denatured, it has been shown that adsorbed proteins retain (at least partly) their folded structure. Sensitivity of the peak H to protein unfolding (denaturation) can be utilized to monitor unfolding transitions in solution. Moreover, it has been shown that protein denaturation at the electrode surface can be modulated by ionic strength and electrode potential. Further preliminary results suggest applicability of the electrochemical techniques in studies of folding/unfolding differences in mutant proteins (such as the tumor suppressor p53).

[4] Osmium tetroxide, 2,2'-bipyridine (Os,bipy): Electroactive marker for probing accessibility of tryptophan residues in proteins.

M. Fojta, S. Billova, L. Havran, H. Pivonkova, H. Cernocka, P. Horakova, and E. Palecek, Osmium tetroxide, 2,2 '-bipyridine: Electroactive marker for probing accessibility of tryptophan residues in proteins. Analytical Chemistry 80, 4598-4605, 2008, IBP: corresponding author, first author; 5 citations on WOS, IF 5.712

100 % of the work was done at the IBP under leadership of M. Fojta and partly of E. Paleček.

Osmium tetroxide complexes (Os,L) belong to traditional tools developed and applied by DMCMO in studies of DNA structure and in DNA labeling, based on facile covalent modification of unpaired thymine residues by the Os,L reagents. Besides thymine and other pyrimidines, covalent adducts with Os,L are formed under physiological conditions also with tryptophan residues, making it applicable in probing proteins structure. As a proof of principle, Os,bipy was used here to study reactivity of tryptophan residues in avidin a streptavidin and their complexes with biotin. This work attracted attention of editors of prestigious Analytical Chemistry journal, resulting in publishing the work as “accelerated article” and in publishing of “research profile” related to the article (Anal. Chem., 2008, 80 (13), p 4785).

[5] Electrochemical labeling of DNA and PNA by osmium tetroxide complexes

M. Fojta, P. Kostecka, M. Trefulka, L. Havran, and E. Palecek, "Multicolor" electrochemical labeling of DNA hybridization probes with osmium tetroxide complexes. Analytical Chemistry 79, 1022-1029, 2007, IBP: corresponding author, first author; 29 citations on WOS, IF 5.712

E. Palecek, M. Trefulka, and M. Fojta, End-labeling of peptide nucleic acid with osmium complex. Voltammetry at carbon and mercury electrodes. Electrochemistry Communications 11, 359-362, 2009, IBP: corresponding author, first author; 7 citations on WOS, IF 4.194

100 % of the work was done at the IBP under leadership of M. Fojta and E. Paleček(about equal contribution of both).

Osmium tetroxide complexes (Os,L) are excellent tools for electrochemical DNA labeling due to facile modification of unpaired thymine residues under physiological conditions and to useful electrochemical properties of the Os,L-nucleic acid adducts. The labeling reaction can be performed in any biochemical or biological laboratory without any special requirements for equipment or expertise, making it widely accessible and versatile. In this work we introduced a technique of electrochemical “multicolor” DNA labeling with the Os,L, based on choice of ligands L influencing the label redox potential. Os,L labeling of PNA, which was performed for the first time, provided particularly high sensitivity of electrochemical detection, and together with specific recognition features of the PNA probes appears promising for applications in practical sequence-specific DNA sensing.

[6] Six valent osmium complexes as tools for specific labeling of sugar residues in biopolymers

M. Trefulka, V. Ostatna, L. Havran, M. Fojta, and E. Palecek, Covalent labeling of nucleosides with VIII- and VI-valent osmium complexes. Electroanalysis 19, 1281-1287, 2007, IBP: corresponding author, first author; 17 citations on WOS, IF 2.901

M. Trefulka, and E. Palecek, Voltammetry of Os(VI)-Modified Polysaccharides at Carbon Electrodes. Electroanalysis 21, 1763-1766, 2009, IBP: corresponding author, first author; 2 citations on WOS, IF 2.901

100 % of the work was done at the IBP (the major part of experimental work by M. Trefulka) under leadership of E. Paleček.

Six valent osmium complexes, Os(VI),L, react with vicinal (usually cis-) diols, forming osmate glycolates which exhibit similar electrochemical properties as modification products of nucleobases or tryptophan residues with complexes of osmium tetroxide. The Os(VI) reagents thus can be utilized for labeling of sugar moieties in biopolymers. It has been shown here that 3’-terminal ribose in ribonucleotides can specifically (without simultaneous modification of nucleobases) be labeled with Os(VI),L, making it promising for specific end-labeling of RNA. Pioneering work was done in the area of electrochemical labeling of natural polysaccharides, opening new possibilities in analysis of e.g., glycoproteins or other biomolecules possessing sugar moieties.

[7] Sensitive and specific analysis of purine nucleobases, metabolites and drugs with mechanically roughened carbon electrodes

S. Hason, S. Stepankova, A. Kourilova, V. Vetterl, J. Lata, M. Fojta, and F. Jelen, Simultaneous Electrochemical Monitoring of Metabolites Related to the Xanthine Oxidase Pathway Using a Grinded Carbon Electrode. Analytical Chemistry 81, 4302-4307, 2009, IBP: corresponding author, first author; 1 citation on WOS, IF 5.712

S. Hason, V. Vetterl, F. Jelen, and M. Fojta, Improved sensitivity and selectivity of uric acid voltammetric sensing with mechanically grinded carbon/graphite electrodes. Electrochimica Acta 54, 1864-1873, 2009, IBP: corresponding authors, first author; 5 citations on WOS, IF 3.078

S. Hason, L. Fojt, P. Sebest, and M. Fojta, Improved Electrochemical Detection of Purine Nucleobases at Mechanically Roughened Edge-Plane Pyrolytic Graphite Electrode. Electroanalysis 21, 666-670, 2009, IBP: corresponding authors, first author; 1 citation on WOS, IF 2.901

Over 90 % of the work was done at the IBP, mostly by S. Hasoň who developed the key technique of electrode roughening and constructed other devices allowing electrochemical analysis in small volumes. M. Fojta contributed by design of biochemical experiments and by final writing. The cooperating group (UniversityHospitaland Faculty of Medicine, MU Brno) provided clinical samples and pursued some parallel assays.

This work resulted in a considerable improvement of voltammetric determination of purine derivatives, metabolites and analogues used as drugs by their direct electrochemical oxidation. At mechanically roughened (“grinded”) carbon electrodes (in contrast to other types of electrodes), excellent separation of oxidation peaks of uric acid, xanthine, hypoxanthine, adenine, guanine, allopurinol and oxypurinol from each other and from signals of interferents naturally occurring in biological matrices (such as urine), was attained. This allowed highly selective detection of these species in mixtures, monitoring of their consecutive enzymatic conversions as well as determination in clinical samples. These proposed electrochemical approaches are clearly superior to other, in general rather time consuming and more expensive, analytical techniques.

[8] Detecting DNA damage and monitoring of enzymatic DNA processing by electrochemical methods

K. Cahova-Kucharikova, M. Fojta, T. Mozga, and E. Palecek, Use of DNA repair enzymes in electrochemical detection of damage to DNA bases in vitro and in cells. Analytical Chemistry 77, 2920-2927, 2005, IBP: corresponding author, first author; 16 citations on WOS, IF 5.712

J. Vacek, K. Cahova, E. Palecek, D.R. Bullard, M. Lavesa-Curto, R.P. Bowater, and M. Fojta, Label-free electrochemical monitoring of DNA ligase activity. Analytical Chemistry 80, 7609-7613, 2008, IBP: joint corresponding author, first author; 2 citations on WOS, IF 5.712

85 % of the work was done at the IBP, mostly by K. Cahová and J. Vacek under leadership of M. Fojta. The cooperating group (at Universityof East Anglia, Norwich, UK, leader R.P. Bowater who partly contributed to writing) prepared recombinant DNA ligases used in the latter work.

Electrochemical studies of DNA damage belong to traditionally successful topics of the IBP bioelectrochemistry school. Recently the electrochemical approaches were combined with enzymatic DNA cleavage to propose techniques of sensitive detection of specific damage to nucleobases, and to elaborate techniques for rapid screening of the activities of nucleic acids processing enzymes such as nucleases or DNA ligases. Proof-of-principle results were published in prestigious Analytical Chemistry journal and promising collaboration with a UKpartner was established to focus on further application of electrochemistry in studies of enzymatic activities of recombinant protein constructs.

[9] Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences

M. Brazdova, T. Quante, L. Togel, K. Walter, C. Loscher, V. Tichy, L. Cincarova, W. Deppert, and G.V. Tolstonog, Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences. Nucleic Acids Research 37, 1486-1500, 2009, IBP: first author;5citations on WOS; , IF 6.878

The majority of the work was pursued by M. Brázdová and her students (about 50 % at the IBP and 50 % during her postdoctoral stay at the HPI in Hamburgunder supervision of W. Deppert).

Wild type p53 tumor suppressor protein plays crucial roles in defense against malignant transformation, while some of the p53 mutants may contribute to tumorigenesis through gain of oncogenic functions. Mutant p53 binding to intronic and intergenic DNA sequences was correlated with modulation of the expression of several cancer-related genes in human glioblastoma cells. This work thus represents a contribution to the understanding of molecular mechanisms behind the mutant p53 functions.

[10] Self-assembled monolayers of thiol-end-labeled DNA at mercury electrodes

V. Ostatna, and E. Palecek, Self-Assembled Monolayers of Thiol-End-Labeled DNA at Mercury Electrodes. Langmuir 22, 6481-6484, 2006, IBP: corresponding author, first author; 16 citations on WOS, IF 4.194

100 % of the work was done at the IBP by V. Ostatná under leadership of E. Paleček.

Chemisorption of end-thiolated oligonucleotides at gold substrates is one of most prominent techniques of construction of DNA biosensors. In this work it was demonstrated that preparation of self-assembled monolayers (SAM) of the thiol-terminated DNA at the mercury electrode is faster and more facile than at gold. Further preliminary results showed that similar SAMs can be prepared at solid amalgam electrodes. With respect to a high hydrogen overvoltage at the mercury-based electrodes, results of this work are promising regarding the possibility of construction of novel electrochemical biosensors with wide negative working windows.

[11] Electrochemical monitoring of phytochelatin accumulation in plant cells exposed to heavy metals

M. Fojta, M. Fojtova, L. Havran, H. Pivonkova, V. Dorcak, and I. Sestakova, Electrochemical monitoring of phytochelatin accumulation in Nicotiana tabacum cells exposed to sub-cytotoxic and cytotoxic levels of cadmium. Analytica Chimica Acta 558, 171-178, 2006, IBP: corresponding author, first author; 18 citations on WOS, IF 3.146

Over 90 % of the work was done at the IBP (M. Fojtová prepared biological material, L. Havran and H. Pivoňková performed the analyses under leadership of M. Fojta). V. Dorčák and I.Šestáková (JH IPC ASCR, Prague) contributed by expertise in peptide electrochemistry.

Phytochelatins (PC) are plant peptides involved in detoxification of heavy metals, the exposure to which induces their synthesis. The PC are thus considered as biomarkers of heavy metal contamination. In this work, a simple electrochemical technique for the detection of PC in plant extracts was presented. The paper gained reasonable citation record and attracted attention of several potential users (including those from commercial sphere) involved in plant molecular biology/agrobiology or ecotoxicology. 

[12] Two-dimensional condensation of pyrimidine oligonucleotides during their self-assemblies at mercury based surfaces

S. Hason, V. Vetterl, and M. Fojta, Two-dimensional condensation of pyrimidine oligonucleotides during their self-assemblies at mercury based surfaces. Electrochimica Acta 53, 2818-2824, 2008, IBP: corresponding authors, first author; 3 citations on WOS, IF 3.078

100 % of the work was done at the IBP by S. Hasoň under leadership of V. Vetterl and M. Fojta who contributed by expertise in interfacial and electrochemical properties of nucleic acid components and by final writing, respectively.

Electrical potential-induced 2D condensation of monomer nucleic acids components (bases or nucleosides) is known for decades owing to mainly the pioneering work of V. Vetterl at the IBP. On the other hand, oligo- and polynucleotides were believed not to be capable of the 2D condensation. This work provided the first evidence of formation of 2D-condensed layers of pyrimidine oligonucleotides at negatively charged electrode surfaces of mercury or amalgam electrodes. This observation may have fundamental importance for understanding how various DNA sequence elements may be arranged at electrically charged surface, with expectable applications in the design and construction of DNA-functionalized surfaces and biosensor development.

[13] Electrochemical determination of redox states of peptides and proteins

V. Dorcak, and E. Palecek, Chronopotentiometric Determination of Redox States of Peptides. Electroanalysis 19, 2405–2412, 2007, IBP: corresponding author, first author; 11 citations on WOS, IF 2.901

V. Dorcak, and E. Palecek, Electrochemical Determination of Thioredoxin Redox States. Analytical Chemistry 81, 1543-1548, 2009, IBP: corresponding author, first author; 7 citations on WOS, IF 5.712

100 % of the work was done at the IBP by V. Dorčák under leadership of E. Paleček.

An efficient electrochemical technique for the determination of redox states of peptides or proteins containing cysteine/cystine (in the sense of thiol/disulfide interconversion) was developed. The technique relies in differential interactions of reduced or oxidized peptides/proteins with positively charged surface of the mercury electrode and sensitivity of the catalytic peak H to arrangement of the adsorbed peptide/protein at the surface. This technique is more facile than other approaches used for the distinction between the thiol/disulfide forms, being promising for e.g., the determination of glutathione redox pool or studies of specific proteins behavior upon changes of the redox conditions.

[14] Mechanisms of DNA binding by tumor suppressor protein p53

V. Brazda, E.B. Jagelska, M. Fojta, and E. Palecek, Searching for target sequences by p53 protein is influenced by DNA length. Biochemical and Biophysical Research Communications 341, 470-477, 2006, IBP: corresponding author, first author; 6 citations on WOS, IF 2.648

E.B. Jagelska, V. Brazda, P. Pecinka, E. Palecek, and M. Fojta, DNA topology influences p53 sequence-specific DNA binding through structural transitions within the target sites. Biochemical Journal 412, 57-63, 2008, IBP: corresponding author, first author; 6 citations on WOS, IF 4.371

100 % of the work was done at the IBP, main contribution was from E. Jagelská and V. Brázda. M. Fojta was partially involved in design of the experiments and contributed to final writing.

DNA binding by the tumor suppressor protein p53 is crucial for its biological functions, and the recognition of specific DNA elements by p53 is given not only by nucleotide sequence of the binding site, but also by local and global DNA conformation and by DNA stretches flanking the binding site. The DCMBO has contributed considerably to these findings through complex studies of p53 interactions with large topologically distinct DNA substrates which mimic some features of DNA in vivo better than isolated short target sequences (used in most of other studies).

[15] Recognition of cisplatin-damaged DNA by p53-family proteins

H. Pivonkova, M. Brazdova, J. Kasparkova, V. Brabec, and M. Fojta, Recognition of cisplatin-damaged DNA by p53 protein: Critical role of the p53 C-terminal domain. Biochemical and Biophysical Research Communications 339, 477-484, 2006, IBP: corresponding author, first author; 7 citations on WOS, IF 2.648

H. Pivonkova, P. Pecinka, P. Ceskova, and M. Fojta, DNA modification with cisplatin affects sequence-specific DNA binding of p53 and p73 proteins in a target site-dependent manner.Febs Journal 273, 4693-4706,2006, IBP: corresponding author, first author; 5 citations on WOS, IF 3.139

Over 90 % of the work was done at the DBCMO IBP by H. Pivoňková under leadership of M. Fojta. Cooperating groups, DMBP IBP and Masaryk Memorial Cancer Institute, contributed by expertise in cisplatin-DNA modification and provided material for the experiments, respectively.

DNA damage is important factor influencing protein-DNA interactions. It has been shown that DNA modification with antitumor platinum complexes affects p53-DNA interactions, and that cisplatin-modified DNA is selectively bound by wild type p53 protein primarily through its C-terminal regulatory domain. Since such binding may potentially interfere with p53 activation mechanisms, it may be of importance in cisplatin-treated cancer cells. Results of this work, together with further observations within the ongoing studies with p73 protein isoforms and p53/p73-DNA binding at surfaces, extend also the knowledge about mechanisms of the protein-DNA interactions in more general sense. 

[16] Application of electrochemical analysis in monitoring of gene expression

P. Horakova-Brazdilova, M. Fojtova, K. Vytras, and M. Fojta, Enzyme-linked electrochemical detection of PCR-amplified nucleotide sequences using disposable screen-printed sensors. Applications in gene expression monitoring. Sensors 8, 193-210, 2008, IBP: corresponding author, first author; 6 citations on WOS, IF 1.87

P. Horakova, E. Simkova, Z. Vychodilova, M. Brazdova, and M. Fojta, Detection of Single Nucleotide Polymorphisms in p53 Mutation Hotspots and Expression of Mutant p53 in Human Cell Lines Using an Enzyme-Linked Electrochemical Assay. Electroanalysis 21, 1723-1729, 2009, IBP: corresponding author, first author; IF 2.901

Over 90 % of the work was done at the IBP by P. Horáková and students under leadership of M. Fojta. Cooperating group at the Universityof Pardubicecontributed by expertise in application of screen printed carbon electrodes.

Electrochemical DNA sensing belongs to hot topics of current bioanalytical chemistry. Despite the considerable progress in this field, the vast majority of detection systems have been demonstrated using model analytes. In this work we presented fully functional, electrochemical assays of real gene expression in plants and cultured human cells using RT PCR amplification and primer extension incorporation of labeled nucleotides, as well as SNP typing of expressed p53 gene.

[17] DNA microanalysis through stripping voltammetry of purine complexes with copper ions

S. Hason, and V. Vetterl, Amplified oligonucleotide sensing in microliter volumes containing copper ions by solution streaming. Analytical Chemistry 78, 5179-5183, 2006, IBP: corresponding author, first author; 9 citations on WOS, IF 5.712

S. Hason, and V. Vetterl, Microanalysis of oligodeoxynucleotides by cathodic stripping voltammetry at amalgam-alloy surfaces in the presence of copper ions. Talanta 69, 572-580, 2006, IBP: corresponding author, first author; 15 citations on WOS, IF 3.206

S. Hason, H. Pivonkova, V. Vetterl, and M. Fojta, Label-free sequence-specific DNA sensing using copper-enhanced anodic stripping of purine bases at boron-doped diamond electrodes. Analytical Chemistry 80, 2391-2399, 2008, IBP: corresponding authors, first author; 5 citations on WOS, IF 5.712

100 % of the work was done at the IBP; the main contribution was from S. Hasoň, some biochemical experiments were designed and pursued by M. Fojta and H. Pivoňková.

Stripping voltammetric determination of purine nucleobases in the presence of copper ions proved useful in analysis of synthetic oligonucleotides as well as natural DNAs (after their acid hydrolysis) as a highly sensitive approach. In connection with carbon-based electrodes it in principle enables determination of relative contents of A and G in the DNA, and is compatible with DNA sequence-specific bioassays using magnetic beads, representing a useful, widely applicable low-cost analytical technique.

[18] Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics

E. Palecek, F. Scheller, and J. Wang, (Eds.), Electrochemistry of nucleic acids and proteins. Towards electrochemical sensors for genomics and proteomics., Elsevier, Amsterdam, 2005, IBP: joint editor of the book

The editorial work was done by at least 70 % by E. Paleček. Other members of DBCMO contributed by chapters (M. Fojta, F. Jelen, V. Vetterl, S. Hasoň).

Representative book summarizing most important recent achievements in the area of nucleic acids and protein electrochemistry and biosensor development. 

[19] Magnetic beads as versatile tools for electrochemical DNA and protein biosensing.

E. Palecek, and M. Fojta, Magnetic beads as versatile tools for electrochemical DNA and protein biosensing. Talanta 74,  276-290, 2007, IBP: corresponding author, first author; 47 citations on WOS, IF 3.206

100 % of the work was done at the IBP, about equal contributions from E. Paleček and M. Fojta

Key review of recent progress in the application of magnetic beads-based techniques in nucleic acids and protein biosensing (to which authors of the article contributed considerably). 

 [20] Electroactivity of non-conjugated proteins and interactions of biomacromolecules with surfaces viewed by electrochemical methods

E. Palecek, and V. Ostatna, Electroactivity of nonconjugated proteins and peptides. Towards electroanalysis of all proteins. Electroanalysis 19  2383-2403, 2007, IBP: corresponding author, first author; 25 citations on WOS, IF 2.901

V. Dorcak, M. Bartosik, V. Ostatna, E. Palecek, and M. Heyrovsky, Interaction of Biomacromolecules with Surfaces Viewed by Electrochemical Methods. Electroanalysis 21  662-665, 2009, IBP: corresponding author, first author; 2 citations on WOS, IF 2.901

90 % of the work was done at the IBP, with the main contribution by E. Paleček. M. Heyrovský (JH IPC ASCR, Prague) was involved as an expert in cytalytic hydrogen evolution at mercury electrodes.

Review/tutorial review of recent progress in the area of electrochemistry of non-conjugated protein, achieved primarily at the IBP.

Department 3     Molecular Epigenetics (DME)

[1] The genetics and epigenetics of invasive allopolyploid species

(a) Kovarik, A., Pires, J.C., Leitch, A.R.,  Lim, K.Y., Sherwood, A., Matyasek, R., Rocca, J., Soltis, D.E., Soltis, P.S. (2005) Rapid concerted evolution of nuclear ribosomal DNA in two allopolyploids of recent and recurrent origin. Genetics 169: 931-944. IF 4.2, 63 citations on WOS,  IBP: corresponding author, first author.

Matyasek R, Tate JA, Lim YK, Srubarova H, Koh J, Leitch AR, Soltis DE, Soltis PS, Kovarik A. (2007)Concerted Evolution of rDNA in Recently Formed Tragopogon Allotetraploids Is Typically Associated With an Inverse Correlation Between Gene Copy Number and Expression. Genetics 176(4):2509-2519. IF 4.2, 16 citations on WOS,  IBP: corresponding author, first author.

(b) The contribution of the IBP is estimated to be 80%: the unit structure and gene expression analysis at the population levels (>100 individuals)

(c) Using molecular methods we identified parental rRNA gene alleles in Tragopogon allotetraploids thought to be 80 years old (1). However the homeolog gene ratio varied between the populations and the alelle of T. dubius origin is often depleted. The result is significant for our better understanding of the speciation, in general. We found that expression of parental rRNA genes differ between the populations (2). In most populations we observed an inverse correlation between the gene copy number and transcription activity.

[2]  Preparation of synthetic hybrids and allopolyploids of Nicotiana, their genetic and epigenetic characterization

(a) Skalicka K, Lim KY, Matyasek R, Matzke MA, Leitch AR, Kovarik A. (2005)  Preferential elimination of repeated DNA sequences from the paternal, N. tomentosiformis genome donor of a synthetic, allotetraploid tobacco. New Phytologist 166, 291- 303. IF 5.2, 48 citations on WOS,  IBP: corresponding author, first author.

Lim KY, Skalicka K, Sarasan V, Clarkson J, Chase MW, Kovarik A, Leitch AR (2006) A genetic apprasial of a new synthetic Nicotiana tabacum and of Kostoff (1938) artificial tobacco. American Journal of Botany 93, 875-883. IF 5.2, 20 citations on WOS,  IBP: coauthors

(b) The IBP has contributed ~80%: cloning and sequencing, genomic characterization of satellite repeats, crossing experiments.

(c) In these works we have studied repetitive sequences in the synthetic tobacco genome. While the most repeats were faithfully inherited from the parents few repeats underwent reduction of copy numbers and even physical elimination (1). The sequences that suffered from eliminations were mostly satellites, endogenous retroviruses and pararetroviruses. The common feature of these labile sequences was their N. tomentosiformis origin. The work showed that genetic changes occur relatively rapidly after the formation of allopolyploid nucleus (probably after the first meiosis) and may be targeted to the one subgenomic compartment (2).

[3] Nicotiana allopolyploid genomes after the 5 million years of evolution

(a) Clarkson, J.J., Lim, K.Y., Kovarik, A., Chase, M.W., Knapp, S., Leitch, A.R. (2005). Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae). New Phytologist 168, 241-252. IF 5.2, 29 citations on WOS,  IBP: coauthors

Lim KY, Kovarik A, Matyasek R, Chase MW, Clarkson JJ, Grandbastien MA, Leitch AR.  (2007) Sequence of events leading to near-complete genome turnover in allopolyploid Nicotiana within five million years. New Phytologist 175, 756-763. IF 5.2, 33 citations on WOS,  IBP: coauthors

(b)  This work was 20% done in Brno: DNA extraction, Southern hybridization, cloning

(c)   In this project we have studied genomes of several ancient Nicotiana allotetraploids. Using genomic in situ hybridization and satellite repeat analysis we found that most genomic sequences of progenitor genomes have been replaced/overwritten by novel one (1). Our results show that progenitor genomes have their limited lifespan in allopolyploids. Comparison with younger allopolyploids showed that “genome replacement” occurs between 1-5 myrs (2)

[4]  Epigenetic factors influencing between homogenization of rRNA genes

(a) Dadejová, M., Yoong, Y.K., Kamila Součková-Skalická, K., Matyášek, R., Grandbastien, M.-A., Leitch, A.R. and Kovařík, A (2007). Transcription activity of rRNA genes correlates with their tendency towards intergenomic homogenisation in Nicotianaallotetraploids. New Phytologist 174, 658-668. IF 5.2, 17 citations on WOS,  IBP: corresponding author, first author.

Kovarik, A., Lim, K.Y., Dadejová M., Matyasek, R., Chase, M., Knapp, S., Clarkson, J., Leitch, A.R. (2008)Evolution of rDNA inNicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics. Annals of Botany 101:815-23. IF 2.7, 17 citations on WOS,  IBP: first author.

(b) This work was 70% done in Brno: cell culture, methylation and expression analysis, gen copy number estimation

(c) Based on several recent experimental data (1) we have postulated hypothesis that epigenetic modification of ribosomal rRNA genes (rDNA)can significantly influence stability of these loci in allopolyploids after the merger of two diverged genomes (2).

[5]  Retroelement activity in natural and synthetic Nicotiana allopolyploids

(a) Petit, M, Lim, K.Y., Julio, E., Poncet, C., Dorlhac de Borne, F., Kovarik, A., Leitch, A.R., Grandbastien, M.-A.,and Mhiri, C. (2007)Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (N. tabacum)Molecular Genomics and Genetics 278, 1-15. IF 2.7, 20 citations on WOS,  IBP: coauthor.

PetitM, Guidat C, Daniel J, Denis E, Montoriol E, LimKY, Kovařík A, Leitch AR, M.-A.  Grandbastien M-A, Mhiri C (2010)Mobilization of retrotransposons in synthetic allotetraploid tobacco. New Phytologist 186, 135-147, IF 5.2, 2 citations on WOS

(b) This work was 10% done in Brno: crossing experiments, plant cultivation, DNA extraction

(c )  It is reported that some retroelement families became activated in synthetic lines of Nicotiana tabacum. Newly transposed copies originate from maternal genome donor -Nicotiana sylvestris.

[6] Genomic organization, expression and chromatin of 5S rRNA genes

(a) Garcia S, Lim KY, Chester M, Garnatje T, Pellicer J, Vallès J, Leitch AR, Kovařík A.(2009) Linkage of 35S and 5S rRNA genes in Artemisia (family Asteraceae): first evidence from angiosperms. Chromosoma 118 :85-97. IF 5.1, 3 citations on WOS,  IBP: corresponding author.

Fulnecek J, Kovarik A.(2007)Low abundant spacer 5S rRNA transcripts are frequently polyadenylated in Nicotiana. Molecular Genomics and Genetics 278: 565-573. IF 2.7, 4 citations on WOS,  IBP: corresponding author, first author.

Fulnecek, J., Matyasek, R., Kovarik, A. (2006) Plant 5S rDNA has multiple alternative nucleosome positions. Genome 49, 840-850. IF 1.9, 3 citations on WOS, IBP: corresponding author, first author.

(b) This work was 80% done in Brno: cloning, sequencing, nucleosome mapping, computer predictions, expression analysis,Southern blot hybridization, in situ hybridisation

(c) We have identified linked 35S and 5S rDNA units in several plant genomes (1). This is a very unusual situation since both genes were found separated in all angiosperm species analyse so far. Concerted evolution homogenizing rDNA to a linked genotype across the chromosomes. In Nicotiana (2),we showed that transcription of 5S genes by polymerse III may proceed to the intergenic spacers. Longer spacer transcripts are specifically polyadenylated. Nucleosome positioning does not seem to correlate with the repeat lengths of 5S tandems (3)

[7] Epigenetic memory of plant cells

(a) Lunerová-Bedrichova J., Bleys, A., Fojtová, M., Khaitová L., Depicker A., Kovařík, A. (2008)Trans-generation inheritance of methylation patterns in a tobacco transgene following a posttranscriptional silencing event. The Plant Journal 54: 1049-1062. IF 6.8, 2 citations on WOS,  IBP: corresponding author, first author.

Fojtová, M., Bleys, A., Bedrichova,B., Van Houdt, H., Krizova, K., Depicker, A., Kovařík, A. (2006) The trans‑silencing capacity of invertedly repeated transgenes depends on their epigenetic state in tobacco. Nucleic Acids Research 34, 2280-2293. IF 5.2, 10 citations on WOS,  IBP: corresponding author, first author.

(b) The contribution of Brno laboratory is estimated to be ~80%: DNA methylation and expression analysis, crossing experiments

(c) In these works we have carried out experiments to investigate mechanisms of epigenetic memory in plants. We studied trans-generation inheritance of DNA methylation that appears to be one of the most important epigenetic tools in higher eukaryotes. Using a transgenic system we have demonstrated, for the first time, meiotic inheritance of DNA methylation induced by RNA signals during a posttranscriptional silencing process. Detailed analysis of DNA methylation by bisulfite sequencing showed that the methylation in CG but not in non-CG motifs may be inherited for at least three generations following segregation of the inductor locus.  We conclude that epialleles generated by RNA-directed processes might exhibit long-term epigenetic memory in plant genomes.

[8]  Epigenetic reprogramming of transgenes and endogenes during cell culture process

(a) Krizova K, Fojtova M, Depicker A, Kovařík A (2009)Cell culture-induced gradual and frequent epigenetic reprogramming of invertedly repeated tobacco transgene epialleles. Plant Physiology 149: 1493-1504. IF 6.8, 2 citations on WOS,  IBP: corresponding author, first author.

Koukalova B, Fojtova M, Lim KY, Fulnecek J, Leitch AR, Kovarik A (2005) Dedifferentiation of tobacco cells is associated with rRNA genes hypomethylation, their increased transcription and chromatin alterations. Plant Physiology 139, 275-286. IF 6.2, 17 citations on WOS,  IBP: corresponding author, first author.

(b) This work was 90% done in Brno: cell culture, methylation and expression analysis, small RNA detection

(c) The ability of mature plant cells to regenerate a whole organism is probably the most remarkable growth attribute of plant cells that distinguishes them from mammalian cells. The results demonstrated sensitivity of transgenes containing inverted structures towards epigenetic changes imposed by cell culture (1). We propose that many examples of tissue culture-induced phenotypic variability might originate from epigenetic alterations at repeated loci influencing their transcription status. Indeed, we demonstrated epigenetic reprogramming and expression and chromatin changes of tandemly arranged ribosomal RNA genes in tobacco cell culture (2).

[9] Dynamics of satellite repeats in plant genomes

(a) Lim KY, Matyasek R, Fulnecek J, Kovarik A, Leitch AR (2005). No evidence of intergenomic translocations or male-genome instability in the allopolyploid Nicotiana rustica. Cytogenetic and Genome Research 109:298-309. IF 1.7, 11 citations on WOS,  IBP: coauthors

KoukalovaB, Moraes AP, Renny-Byfield S, MatyasekR, Leitch AR, Kovařík A (2010)Fall and rise of satellite repeats in allopolyploids of Nicotiana over c. 5 million years. New Phytologist 186, 148-160. IF 5.2,  IBP: corresponding author, first author.

(b) The contribution of IBP is estimated to ~70%: cloning and sequencing, genomic characterization of satellite repeats

(c) We isolated subtelomeric, tandemly repeated satellite DNA from Nicotiana diploid and allopolyploid species and analysed patterns of inheritance and divergence by sequence analysis. We observed that parental satellite sequence redistribute around the genome in allopolyploids of Nicotiana section Polydicliae, formed c. 1 million years ago (mya), and that new satellite evolved and amplified in section Repandae, formed c. 5 mya. In some cases that process involved the complete replacement of parental satellite sequences. In contrast, a younger allopolyploid, N., rustica, showed contraction of satellites only.

[10] Plant comparative genomics and chromosome divergence

(a) Lim KY, Soltis DE, Soltis PS, Tate J, Matyasek R, Srubarova H, Kovarik A, Pires JC, Xiong Z, Leitch AR. Rapid chromosome evolution in recently formed polyploids in tragopogon (asteraceae). PLoS ONE. 2008;3(10):e3353. 15 citations on WOS,  IBP: coauthors.

Leitch IJ, Hanson L, Lim KY, Kovarik, A, Chase MW, Clarkson J, Leitch AR(2008)The ups and downs of genome size evolution in polyploid Nicotiana (Solanaceae). Annals of Botany 101:805-14. ) IF 2.7, 11 citations on WOS,  IBP: coauthor.

Lim, K.Y., Kovarik, A., Matyasek, R., Chase, M., Knapp, S., Clarkson, J., Leitch, A.R. (2006) Comparative genomics and repetitive sequence divergence in the diploid Nicotiana section Alatae. The Plant Journal 48, 907-919. IF 6.8; 18 citations on WOS,  IBP: coauthors.

(b) These works were 30% done in IBP: cloning ,sequencing, genomic analysis of repeats

(c) Chromosomes of recently formed Tragopogon allotetraploids show frequent translocation and uniparental monosomies/trisomies (1). We further showed that three ancient Nicotiana allotetraploids have larger genomes than the sum of diploid progenitor genomes (2). This result violates general paradigma on genome downsizing in allopolyploids. We characterized species from section Alatae by comparative molecular cytogenetics.

[11] Genome characterization of polyploid species with a non-symmetrical meiosis

(a) Kovarik A, WerlemarkG, Leitch AR, Souckova-Skalicka K, Lim YK, Koukalova B Nybom H. (2008). Asymmetric meiosis in pentaploid dogroses (Rosa sect. Caninae)is associated with a skewed distribution of rRNA gene families in the gametes. Heredity 101: 359-367. IF 3.8, 1 citation on WOS,  IBP: corresponding author, first author.  

Khaitová L, Werlemark G, Nybom H, Kovařík A (2010) Frequent silencing of rDNA loci on the univalent-forming genomes contrasts with their stable expression on the bivalent-forming genomes in polyploid dogroses (Rosa sect. Caninae).Heredity,104:113-120. IBP: corresponding author, first author.

(b) The contribution of Brno laboratory is estimated to be ~80%: Southern blot hybridization, RT-PCR, cloning, sequencing, Bioinformatic analysis

(c) It is reported that pollen cells (male gametes)contain different spectrum of rRNA gene families than somatic tissues (leaf). The data support the hypothesis that only a subset of chromosomes (bivalent-forming) are transmitted to gametes in the course of male meiosis (1). The RT-PCR data show that the families on bivalent-forming chromosomes dominate rDNA expression in all dogrose species (2). We hypothesize that genes on bivalent genomes are stably expressed, whereas those on univalent genomes undergo variable levels of epigenetic silencing. Nonetheless, mosaic expression of univalent genomes could contribute to phenotypic variation between the species.

[12] Gene expression studies in tobacco genome

a) Šámalová, M., Brzobohatý, B., and Moore, I. (2005) pOp6/LhGR: a stringently regulated and highly responsive dexamethasone-inducible gene expression system for tobacco. Plant Journal 41: 919-935, IF 6.8, 20 citations on WOS,  IBP: first author and one co-author

Fulneček J, Matyášek R, Kovařík A. (2009)Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes. Molecular Genetics and Genomics 281:407-420. IF 2.7, IBP: corresponding author, first author.

(b) The contribution of Brno laboratory is estimated to 70%: cloning, sequencing, expression analysis, T-DNA transformation

(c) We developed a stringently regulated and highly responsive dexamethasone-inducible gene expression system for plants (1) that have been successfully used by several laboratories. We further cloned and sequenced three major DNA methyltransferase families (METI, CMT3 and DRM)from tobacco and the diploid progenitor species (2). Homeologous expression was found in all tissues examined (leaf, root, flower)suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing or rearrangement for over thousands of generations of allotetraploid evolution.

[13] Structure and function of satellite repeats in plant genomes - review

Hemleben, V., Kovarik, A., Torres-Ruiz, R.A., Volkov, R.A., Beridze, T. (2007) Plant highly repeated satellite DNA: molecular evolution, distribution, and use for identification of hybrids. Systematics and Biodiversity  5, 277–289. IF 2.7, 7 citations on WOS,  IBP: coauthor

The contribution of IBP is estimated to ~20%, mainly writing

In this paper we have reviewed literature on plant satellite sequences. We discussed the structure and origin of satellite repeats, their position on chromosomes. Several hypotheses on the stability and dynamics in interspecific hybrids and allopolyploidy have been proposed.

[14] New epigenetic drugs acting on DNA methylation

(a) Fojtova, M., Piskala, A., Votruba, I., Otmar, M., Bartova, E., Kovarik A (2007)Efficacy of DNA hypomethylation capacities of alpha and beta anomers of 5-aza-2‘-deoxycytidine. Pharmacological Research 55, 16-22. IF 2.2, 6 citations on WOS , IBP: corresponding author, first author.

Fojtova, M., Boudny, V., Kovarik, A. Lauerova, L, Adamkova, L., Souckova, K., Jarkovsky, J., Kovarik, J. (2007) Development of IFN gamma resistance is associated with attenuation of SOCS genes induction and constitutive expression of SOCS 3 in melanoma cell. British Journal of Cancer 16, 231-237. IF 4.4, 12 citations on WOS, IBP: first author, coauthor.

Patent (WO 2008101448 20080828).

(b) This work was 60% done at IBP: DNA methylation and expression analysis

(c) We have investigated hypomethylation capacities of alpha and beta anomers of 5-aza-2´-deoxycytidine (1). Surprisingly, alpha anomer induced DNA hypomethylation of the extent comparable with that of beta anomer. However, alpha anomer appeared to be approx. 5 x less toxic than the beta anomer in leukemic cells in vitro. Thus alpha anomer seems to be a promising agent for epigenetic therapy. Epigenetic properties of interferons were studied in melanoma cells (2).

[15] Molecular mechanisms of cytokinin action in plants

(a) Lochmanová, G., Zdráhal, Z., Konečná, H., Koukalová, Š., Malbeck, J., Souček, P., Válková, M., Kiran, N.S., Brzobohatý, B. (2008) Cytokinin-induced photomorphogenesis in dark-grown Arabidopsis: a proteomic analysis. Journal of Experimental Botany 59: 3705-3719, ) IF₂₀₀₈ = 4.001, 2 citations on WOS, IBP: corresponding author, first author and two co-authors

Souček, P., Klíma, P., Reková, A., and Brzobohatý, B. (2007) Involvement of hormones and KNOXI genes in early Arabidopsisseedling development. Journal of Experimental Botany 58: 3797-3810, IF₂₀₀₇ = 3.917, 4 citations on WOS, IBP: corresponding author, first author and two co-authors

Kuderová, A., Urbánková, I., Válková, M., Malbeck, J., Brzobohatý, B., Némethová, D., and Hejátko, J.: Effects of conditional IPT-dependent cytokinin overproduction on root architecture of Arabidopsis seedlings (2008) Plant and Cell Physiology 49: 570-582, IF₂₀₀₈ = 3.542, 11 citations on WOS, IBP: one co-author

Pernisová, M., Klíma, P., Horák, J., Válková, M., Malbeck, J., Souček, P., Reichman, P., Hoyerová, K., Dubová, J., Friml, J., Zažímalová, E., Hejátko, J. (2009) Cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux. Proceedings of the National Academy of Sciences of the United States of America 106: 3609-3614, IF₂₀₀₈ = 9.380, 8 citations on WOS, IBP: one co-author

Hejátko, J., Ryu, H., Kim, G.-T., Dobešová, R., Choi, S., Choi S.M., Souček, P., Horák, J., Pekárová, B., Palme, K., Brzobohatý, B., Hwang, I. (2009) The histidine kinases CYTOKININ-INDEPENDENT1 and ARABIDOPSIS HISTIDINE KINASE2 and 3 regulate vascular tissue development in Arabidopsis shoot. Plant Cell 21: 2008-2021, IF₂₀₀₈ = 9.296, 2 citations on WOS,  IBP: two co-authors

(b) The contribution of IBP is estimated to 50% (a1), 70% (a1), 70% (a2), 10% (a3), 10% (a4) and 20% (a5).

(c) We found that even modest increases in endogenous levels of cytokinins can result in many typical features of light-induced de-etiolation, and identified a number of proteins involved in this developmental switch. We obtained novel information on interactions of plant hormones and KNOXI homeo genes in seedling development. We identified a distinct developmental interval during which primary root elongation is susceptible to regulation by cytokinins. We showed that cytokinins modulate auxin-induced organogenesis in plants via regulation of the auxin efflux. Finally, we found that cytokinin perception via AHK2 and AHK3 cytokinin receptors, and cytokinin-independent activity of the histidine kinase CYTOKININ-INDEPENDENT 1 play a key role in vascular bundle formation in Arabidopsis.

[16] Subcellular compartmentation of cytokinin metabolism in plants

(a) Kiran, N.S., Polanská, L., Fohlerová, R., Mazura, P., Válková, M., Šmeral, M., Zouhar, J., Malbeck, J., Dobrev, P., Macháčková, I., and Brzobohatý, B. (2006) Ectopic over-expression of the maize beta glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco. Journal of Experimental Botany 57: 985-996, IF₂₀₀₆ = 3.630, 10 citations on WOS, IBP: corresponding author, first author and 4 co-authors

Polanská, L., Vičánková, A., Nováková, M., Malbeck, J., Dobrev, P.I., Brzobohatý, B., Vaňková, R., and Macháčková, I. (2007) Altered cytokinin metabolism affects cytokinin, auxin and abscisic acid contents in leaves and chloroplasts and chloroplast ultrastructure in transgenic tobacco. Journal of Experimental Botany 58: 637-649, IF₂₀₀₇ = 3.917, 5 citations on WOS, IBP: one co-author

(b) The contribution of IBP is estimated to 70% (a1) and 10% (a2).

(c) We showed that plant hormone compartmentation plays an important role in hormone homeostasis and that chloroplasts are rather independent organelles with respect to regulation of cytokinin metabolism.

[17] Structure-function relationships in plant beta-glucosidases

(a) Dopitová, R., Mazura, P., Janda, L., Chaloupková, R., Jeřábek, P., Damborský, J., Filipi, T., Kiran, N.S., Brzobohatý, B. (2008) Functional analysis of the aglycone-binding site of the maize beta-glucosidase Zm-p60.1. FEBS Journal 275: 6123-6135, IF₂₀₀₈ = 3.139, 2 citations on WOS, IBP: corresponding author, first author and two co-authors

Mazura, P., Fohlerová, R., Brzobohatý, B., Kiran, N.S., and Janda, L. (2006) A new, sensitive method for enzyme kinetic studies of scarce glucosides. Journal of Biochemical and Biophysical Methods 68: 55-63, IF₂₀₀₆ = 1.403, 3 citations on WOS, IBP: first author and three co-authors

(b) The contribution of IBP is estimated to 70% (a1) and 50% (a2).

(c) We obtained a detailed knowledge on the dual role of F193-aglycone-W373 interactions in the catalytic action of the Zm-p60.1 -glucosidase – contributing both to the enzymes affinity for substrates with aromatic aglycones and co-determination of the catalytic rate by fixing the glucosidic bond in a favorable orientation for attack by catalytic pair. We developed a new, sensitive method for enzyme kinetic studies of scarce glucosides.

Department 4     Molecular Cytology and Cytometry (DMCC)

[1] Effect of HDAC inhibitors on chromatin structure and epigenetics.

(a) Bártová E.,Pacherník J., Harničarová A., Kovařík A., Kovaříková M., Hofmanová J., Skalníková M., Kozubek M., Kozubek S. Nuclear levels and patterns of histone H3 modifications and HP1 proteins after inhibition of histone deacetylases. J.  Cell Sci. 118 (21), 5035-5046,2005.  IF= 6.91; group contribution 90%; DMCC: first author and corresponding author, citations: 38 on WOS.

Bártová E., Pacherník J., Kozubík A., Kozubek S. Differentiation-specific association of HP1alpha and HP1beta with chromocentres is correlated with clustering of TIF1beta at these sites. Histochem. Cell Biol. 127(4):375-88, 2007. IF= 3.22; DMCC contribution 90%, first author and corresponding author form DMCC, citations: 13 on WOS.

(b) 80% of the work was done by researchers from DMCC. E.B. designed the experiments and wrote the paper, made immunofluorescence and confocal microscopy; measured the cell cycle profiles by FACS; A.H. cultivated the cells and performed the immuno-detections. J.P. provided the results of western blots; S.K. was responsible for data analysis and interpretation. Other co-authors provided the cells, cell lysates, and protocols related to NaBt-induced cell differentiation. A.K. detected the level of DNA methylation by the use of DNA methylation-sensitive enzymes.

(c) Inhibition of histone deacetylases by TSA or sodium butyrate caused dynamic reorganization of chromatin in parallel with changes in its epigenetic modifications. After TSA and NaBt treatment, the HP1 proteins were repositioned more internally in the nucleus, being closely associated with interchromatin compartments, while centromeric heterochromatin was relocated closer to the nuclear periphery. These findings strongly suggest dissociation of HP1 proteins from peripherally located centromeres in a hyperacetylated and H3(K4) dimethylated environment.Our findings also showed that the nuclear arrangement of HP1 subtypes and TIF1beta is differentiation specific, and seems to be more important than changes in the levels of these proteins, which were relatively stable during all the induced differentiation processes. The results were discussed from the potential point of view of HDAC inhibitors as anti-tumour drugs.

[2] Relationship between chromatin structure and gene expression.

 (a) Bártová E., Kozubek S. Nuclear architecture in the light of gene expression and cell differentiation studies. Biol. Cell., 98(6):323-36, 2006.  Review. IF= 4.3; DMCC contribution 100%, S.K. corresponding author, citations: 25 on WOS.

(b) 100% of the work was done by researchers from DMCC.

(c) It is evident that primary DNA sequences, that define genomes, are responsible for genome functions. However, the functional properties of chromatin are additionally regulated by heritable modifications known as epigenetic factors and, therefore, genomes should be also considered with respect to their ‘epigenomes’. Nucleosome remodelling, DNA methylation and histone modifications are the most prominent epigenetic changes that play fundamental roles in the chromatin-mediated control of gene expression. Another important nuclear feature with functional relevance is the organization of mammalian chromatin into distinct chromosome territories which are surrounded by the interchromatin compartment that is necessary for transport of regulatory molecules to the targeted DNA. The inner structure of the chromosome territories, as well as the arrangement of the chromosomes within the interphase nuclei, has been found to be non-randomly organized. Therefore, a specific nuclear arrangement can be observed in many cellular processes, such as differentiation and tumour cell transformation.

[3] Nuclear radial distribution of the c-myc transcription sites.

(a) Harničarová A., Kozubek S., Pacherník J., Krejčí J., Bártová E. Distinct nuclear arrangement of active and inactive c-myc genes in control and differentiated colon carcinoma cells. Exp. Cell Res., 312(20):4019-35, 2006. IF=3.77; DMCC contribution: 95%, A.H. first author, S.K. corresponding author,citations: 21 on WOS.

Bártová E., Harničarová A., Krejčí J., Strašák L. and Kozubek S. (2008) Single-cell c-myc gene expression in relationship to nuclear domains, accepted for publication in Chromosome Research, 16(2):325-43, 2008. IF= 3.0; E.B. first and corresponding author;DMCC contribution: 100%, citations: 5 on WOS.

(b) 95% of the work was performed by researchers from DMCC.

(c) We addressed nuclear distribution of the c-myc gene transcripts in human colon cancer cells characterized by normal and derivative chromosome 8, which is resided by the c‑myc protooncogene. Our experiments demonstrate strikingly specific nuclear and territorial arrangements of active genes as compared with inactive ones: on the periphery of their territories facing to the very central region of the cell nucleus. Nuclear arrangement of c-myc genes and transcripts was conserved during cell differentiation and, therefore, independent of the level of differentiation-specific c-myc gene expression. However, after the induction of differentiation, a more internal territorial location was found for the single copy c-myc gene of normal chromosome 8, while amplicons conserved their territorial topography. Majority of transcripts associated with periphery of nucleoli, which seems to be very important nuclear compartment playing an important role in structural regulation of transcription.

[4] Chromatin structure and epigenetics of human embryonic stem cells.

(a) Bártová E., Krejčí J., Harničarová A., Kozubek S. Differentiation of human embryonic stem cells induces condensation of chromosome territories and formation of heterochromatin protein 1 foci. Differentiation, 76(1):24-32, 2008. IF= 2.9; group contribution 100%, S.K. Corresponding author, citations: 11 on WOS.

Krejčí J, Uhlířová R, Galiová G, Kozubek S, Šmigová J, Bártová E. Genome-wide reduction in H3K9 acetylation during human embryonic stem cell differentiation. J. Cell Physiol.;219(3):677-87, 2009. IF= 4.3; DMCC contribution 95%, E.B. corresponding author

(b) 95% of the work was done by researchers from DMCC.

(c) Substantial decondensation of higher-order chromatin could be considered as a mark of stem cell pluripotency, due to the accessibility of the ‘‘open’’ chromatin configuration to regulatory molecules. In our experiments, the Oct4 gene and all the variants of heterochromatin protein 1 (HP1) proteins were affected by chromatin decondensation in undifferentiated hES cells. The heterochromatin protein 1 (HP1) was dispersed hogeneously within interphase nuclei of pluripotent stem cells. Differentiation caused focal rearrangement of HP1 subtypes. This finding was accompanied by the decreased level of H3K9 acetylation. Taken together, differentiation of hESCs is accompanied by a global rearrangement of the cell nucleus, which is of fundamental significance for the cell function.

[5] Epigenetics and transcriptional regulation of rDNA genes

 (a) Harničarová Horáková A., Bártová E., Galiová G., Uhlířová R., Matula P., Kozubek S. SUV39h-independent association of HP1β with fibrilarin-positive nucleolar region. Accepted to Chromosoma 2009, IF= 5.1,

(b) Department contribution: 95%, A.H. first author, E.B. corresponding author. E.B. designed the experiments and wrote the paper. A.H. cultivated cells, made immunofluorescence, image acquisition and PCR. G.G. is responsible for ChIP-PCR results. S.K. is responsible for data analysis and interpretation. External co-operation with P.M. was related to the design of software for data analysis and calculation of some parameters characterizing the nuclear radial distribution of selected genomic elements.

(c) Deficiency of histone methyltransferase SUV39h, and/or inhibition of histone deacetylases (HDACi) decreased HP1band H3K9 trimethylation at chromocenters, but not in fibrillarin-positive regions that co-localized with RNA Pol I. Similarly, SUV39h- and HDACi-dependent nucleolar rearrangement and inhibition of rDNA transcription did not affect the stability of the HP1b-fibrillarin interaction. We conclude that epigenetic processes in nucleoli are different from the rest of the cell nucleus.

[6] Chromatin structure of healthy neutrophil granulocytes and its changes in myeloid  leukemia

(a) E. Lukášová, Z. Kořistek, M. Falk, S. Kozubek, S. Grigoryev, M. Kozubek, V. Ondřej,I. Kroupová: Methylation of histones in myeloid leukemias as a potential marker of granulocyte abnormalities. Journal of Leukocyte Biology 77, 100-111, 2005, DMCC: corresponding author, first author, co-authors. IF 4.63; 13 citations on WOS.

E.Y. Popova, D.E. Clacston, E. Lukasova, P.I. Bird, S.A. Grigoryev: Epigenetic chromatin markers distinguish terminally differentiated leukocytes from incompletely differentiated leukemia cells in human blood. Experimental Hemalology 34, 2006, 453-462. DMCC: co-author. IF 4.15; 10 citations on WOS

(b) The design of the project was done by 90 % by researcher from IBP, where 100% of experiments published in J.L.B. were executed and the manuscript completely written. The cooperating partners were included for participation on design, blood delivery of  healthy donors and leukemia patients and software for results processing. The participation of IBP on the results published in E.H. was on the project design and represents ~ 10%.

(c) This study showed for the first time that the structure of heterochromatin of terminally differentiated neutrophils is substantially different from the structure in other differentiated cells. The basic heterochromatin proteins HP1 are absent and replaced by a serpin MNEI during the cell differentiation. Very high chromatin condensation prevents immunodetection of dimethylH3K9. At advanced phases of CML and in many types of AML, the differentiation of neutrophils is incomplete which is manifested by the expression of small amount of HP1 proteins and partial chromatin decondensation. The changes in the chromatin structure of these leukemia cells are reflected in the loss their function consisting in capture of bacterial infection into relaxed chromatin fibers. The presence of HP1 protein prevents chromatin unwinding and thus cell function.

[7] Higher order chromatin structure in DSB induction, repair and misrepair

(a) M. Falk, E. Lukášová, B. Gabrielová, V. Ondřej, S. Kozubek: Chromatin dynamics during  DSB repair. Biochimica et Biophysica Acta - Molecular Cell Research 773, 1534- 1545, 2007. IF 6.9; 9 citations on WOS

M. Falk, E. Lukášová, S. Kozubek: Chromatin structure influences the sensitivity of DNA to γ-radiation. Biochimica et Biophysica Acta - Molecular Cell Research 1783, 2398-2414, 2008. IBP complete contribution. IF 4.89; 4 citations on WOS

M. Falk, E. Lukášová, S. Kozubek: Higher-order chromatin structure in DSB induction, repair and misrepair. Mutation Research/Reviews in Mutation Research 704, 88-100, 2010. IF 5.91. This review was requested by the editor in chef of the Mut Res Rev on the base of our contribution on the ESF-EMBO Symposium on Spatio-Temporal Radiation Biology held in Spainon 16-21st May 2009. IF 6.9

(b) 100% contribution from IBP.

(c) DNA double-strand breaks (DSBs) lead to chromosomal fragmentation and genomic rearrangements if not accurately repaired. The direct evidence of DSB induction in functionally different chromatin domains of human cells obtained by our experiments shows that genetically inactive condensed chromatin is much less susceptible to the induction of this damage by γ-rays than active decondensed chromatin. Thus, the most active regions of human genome are subjected to the highest risk of radiation damage. We show that higher order chromatin structure has a very important role in DSB repair. Slight movement of sporadic DSB loci for short distances was noticed in living cells associated with chromatin decondensation. A model explaining the formation of chromosome translocations is proposed.

[8] Homeostatic role of adenosine A₁ and A₃ receptor agonists in regulation of hematopoiesis

(a) Pospíšil M., Hofer M., Vacek A., Holá J., Pipalová I., Znojil V.: N⁶-Cyclopentyladenosine inhibits proliferation of murine haematopoietic progenitor cells in vivo. Eur. J. Pharmacol. 507, 1-6, 2005.

Hofer M., Pospíšil M., Vacek A., Holá J., Znojil V., Weiterová L., Štreitová D.: Effects of adenosine A₃ receptor agonist on bone marrow granulocytic system in 5-fluorouracil-treated mice. Eur. J. Pharmacol. 538, 163-167, 2006.

Hofer M., Pospíšil M., Znojil V., Holá J., Štreitová D., Vacek A.: Homeostatic action of adenosine A₃ and A₁ receptor agonists on proliferation of hematopoietic precursor cells. Exp. Biol. Med. 233, 897-900, 2008.

(b) 100% of the work was done in our department (with the exception of statistical evaluations /V. Znojil/).

(c) This series of articles represents a fundamental for investigations of the role of  adenosine A₁ and A₃ receptor signaling in hematopoiesis in vivo.  Whereas adenosine A₁ receptor activation  causes inhibition of proliferation of hematopoietic progenitor and precursor cells,  stimulation od adenosine A₃ receptors intensifies cell multiplication in these important cellular systems. Opposite effects of adenosine A₁ and A₃ receptor agonists can act in accord to maintain homeostasis in production of key hematopoietic cells under the states of suppressed and regenerating hematopoiesis.

[9] Pharmacologically induced inhibition of cyclooxygenase-2 supports hematopoietic recovery following ionizing radiation-induced myelosuppression

(a) Hofer M., Pospíšil M., Znojil V., Holá J., Vacek A., Weiterová L., Štreitová D., Kozubík A.: Meloxicam, a cyclooxygenase 2 inhibitor, supports hematopoietic recovery in gamma-irradiated mice. Radiat. Res. 166, 556-560, 2006.

Hofer M., Pospíšil M., Holá J., Vacek A., Štreitová D., Znojil V.: Inhibition of cyclooxygenase 2 in mice increases production of G-CSF and induces radioprotection. Radiat. Res. 170, 566-571, 2008.

(b) 100% of the work was done in our department (with the exception of statistical evaluations /V. Znojil/ and some consultations in the first publication /A. Kozubík/ )

(c) This set of two papers summarizes the first results on the possibilities of utilizing cyclooxygenase-2 inhibition for supporting regeneration of hematopoiesis in a mammalian organism exposed to sublethal or lethal doses of ionizing radiation. A complex analysis of hematopoiesis in experimental mice revealed a bi-lineage hematopoietic regeneration following administration of a cyclooxygenase-2 inhibitor shortly before or shortly after irradiation. The stimulatory efficacy of cyclooxygenase-2 inhibition was found  to be reflected in enhacement of survival of the animals after a lethal radiation exposure. These findings can find their practical use in the treatment of myelosuppresion in victims of radiation accidents or in oncological patients administered cytotoxic antitumor drugs.

[10] Minisatellite telomeres occur in the family Alliaceae but are lost in Allium.

(a) Sykorova, E., Fajkus, J., Meznikova, M., Lim, K.Y., Neplechova, K., Blattner, F.R., Chase, M.W. and Leitch, A.R. (2006a) Minisatellite telomeres occur in the family Alliaceae but are lost in Allium. American Journal of Botany, 93, 814-823. IF 2.642, 11 citations

Fajkus J, Sykorova E, Leitch A.R.,(2005).Telomeres in evolution and evolution of telomeres. Chromosome Res 13, 469-479.IF 3.405, 38 citations

(b) The authors ES and MM contributed to this result as full employees of the Instituteof Biophysics, JF as part-time employee. Design of the study was done by ES, JF and ARL. The other participating institutions were Masaryk University, Brno, CZ, Leibniz Institute IPK Gatersleben, Germany, and Queen Mary University of London, UK. In total, contribution of IBP is 50%.

(c) The result concludes a set of consecutive studies, pioneered by the group of Ingo Schubert, Gatersleben, in 1995, and then continued in collaborative projects at the Institute of Biophysics and Queen Mary University of London (since 2000). The results obtained during these studies explain the course of evolution of telomeres within a large taxonomic group of plants and exceeds similar studies in its extent and precision.

[11] Asparagales telomerases which synthesize the human type of telomeres

(a) Sykorova, E., Leitch, A.R. and Fajkus, J. (2006b) Asparagales telomerases which synthesize the human type of telomeres. Plant Molecular Biology, 60, 633-646. IF: 3.541, 9 citations

Sykorova, E. and Fajkus, J. (2009) Structure-function relationships in telomerase genes. Biology of the Cell, 101, 375-392.IF: 3.438, 0 citations

(b) The first author (ES) did most of experimental work reported in this study as the full employee of the Institute, all the authors contributed to the design of the study and MS preparation. All experiments were performed at the Instituteof Biophysics. The other participating institutions were MasarykUniversityand Queen Mary University of London.

The overall contribution of the IBP is 70%.

(c) This study resulted in complete characterisation of telomerase genes from number of species possessing “typical” (Arabidopsis-type) and alternative (human-type) telomeres and identified mutations associated with the change in telomere sequence synthesised by these telomerases.

[12] Characterisation of plant-specific telomere-binding proteins from SMH (Single-Myb-Histone) family – AtTRB proteins

(a) Dvorackova, M., Rossignol, P., Shaw, P.J., Koroleva, O.A., Doonan, J.H. and Fajkus, J. (2010) AtTRB1, a telomeric DNA-binding protein from Arabidopsis, is concentrated in the nucleolus and shows highly dynamic association with chromatin. Plant Journal, 61, 637-649. IF: 6.493, 0 citations

Hofr, C., Sultesova, P., Zimmermann, M., Mozgova, I., Schrumpfova, P.P., Wimmerova, M. and Fajkus, J. (2009) Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and kinetics. Biochemical Journal, 419, 221-228. IF: 4.371, 1 citation

Rotkova, G., Sykorova, E. and Fajkus, J. (2009) Protect and regulate: Recent findings on plant POT1-like proteins. Biologia Plantarum, 53, 1-4.IF: 1.426, 1 citation

Mozgova, I., Schrumpfova, P.P., Hofr, C. and Fajkus, J. (2008) Functional characterization of domains in AtTRB1, a putative telomere-binding protein in Arabidopsis thaliana. Phytochemistry, 69, 1814-1819. IF:2.946, 4 citations

Schrumpfova, P.P., Kuchar, M., Palecek, J. and Fajkus, J. (2008) Mapping of interaction domains of putative telomere-binding proteins AtTRB1 and AtPOT1b from Arabidopsis thaliana. Febs Letters, 582, 1400-1406. IF: 3.264, 5 citations

Rotkova, G., Sykorova, E. and Fajkus, J. (2007) Characterization of nucleoprotein complexes in plants with human-type telomere motifs. Plant Physiology and Biochemistry, 45, 716-721.IF: 1.905, 3 citations

(b) Contribution of the Institute – GR and MD performed the experiments as employees of the Inst. Biophys and students of MU. JF has a part-time contract at the IBP, as well as his students, MD and GR. Most of the results were obtained at the Dept. Functional genomics and proteomics at the Masaryk University headed by JF, by his PhD students (MK, MD, IM, GR) and postdocs (CH, JP, PPS). The other participating institution was John Innes Centre, Norwich. The overall contribution of the IBP was 30%.

(c) The SMH proteins (AtTRB proteins) are the only known plant-specific telomere proteins.

Therefore, understanding their structure and functions is of primary importance in searching for biologically active compounds involved in telomere regulation which could be prospectively used to treat immortality of cancer cells.

[13] Improvement of methods for telomere and telomerase analysis and their application in oncology diagnostics

(a) Fajkus, J., M. Dvořáčková, and E. Sýkorová. (2008). Analysis of Telomeres and Telomerase. In The Nucleus: Nuclei and Subnuclear Components (ed. R. Hancock), pp. 267-296. Humana Press, Totowa, NJ, USA.

Fajkus, J. (2006) Detection of telomerase activity by the.TRAP assay and its variants and alternatives. Clinica Chimica Acta, 371, 25-31.IF:2.960, 10 citations

Kunicka, Z., Mucha, I. and Fajkus, J. (2008) Telomerase Activity in Head and Neck Cancer. Anticancer Research, 28, 3125-3129.IF 1.390, 1 citation

Neplechova, K., Sykorova, E. and Fajkus, J. (2005) Comparison of different kinds of probes used for analysis of variant telomeric sequences. Biophysical Chemistry, 117, 225-231.IF 2.362, 2 citations

(b) Studies were designed and evaluated by JF and ES, most experiments were performed by ZK, MDand ES. The overall contribution of the IBP was 40%, the other participating institutions were MU and UniversityHospitalat St. Anne

(c) Quantitative evaluation of telomerase activity, described by Fajkus et al., 2003, was introduced into molecular oncology diagnostics. Standard protocols of telomere and telomerase analysis were described in detail.

[14] HMGB proteins regulate cellular activity of human topoisomerase II alpha

(a) M. Štros, A. Bačíková, E. Polanská, J. Štokrová and F. Strauss: HMGB1 interacts with human topoisomerase II alpha and stimulates its catalytic activity. Nucleic Acids Research, 35, 5001-5013, 2007, IBP: corresponding author, first author. IF 7,55; 4 citations on WOS

M. Štros, E. Polanská, S. Štruncová, S. Pospíšilová: HMGB1 and HMGB2 proteins up regulate cellular expression of human topoisomerase II alpha. Nucleic Acids Research, 37, 2070-2086, 2009, IBP: corresponding author, first author. IF 6.88; 2 citations on WOS

 (b) The first contribution was done by ~75 % by researchers from DMCC. M. Štros proposed the project and together with A. Bačíková executed most experiments. M. Štros has also completely written the manuscript. J. Štokrová (IMB AS CR, Prague) executed EM experiments.The second contribution was done by ~90 % by DMCC. M. Štros proposed the project and together with E. Polanskáexecuted most experiments. M. Štros has also completely written the manuscript.

(c) The first study established for the first time the modulatory effect of HMGB1/2 on cellular activity of topo IIalpha. The latter effect of HMGB1/2 was inhibited in cells with functional, retinoblastoma proteins.In the second study HMGB1 binding to topoisomerase IIalpha was detected as well as enhancement of its activity by HMGB1. A mechanism of HMGB1-mediated stimulation of topo IIalpha activity was presented.

[15] The HMG-box: a versatile protein domain

M. Štros, D. Launholt, K.D. Grasser: The HMG-box: a versatile protein domain occurring in a wide variety of DNA-binding proteins. Cellular and Molecular Life Sciences, 64, 2590-2606, 2007, IBP: corresponding author, first author. IF 5.24, 13 citations on WOS

(b) M. Štros contributed ~65% to this work.

(c) This study presents a review on HMG-box containing proteins in human and plant genomes.

Department 5     Cytokinetics (DC)

[1]  Novel platinum-based drugs – mechanisms of their action in solid cancer models

a)  Kozubík A, Horváth V, Švihálková-Šindlerová L, Souček K, Hofmanová J, Sova P, Kroutil A, Žák F, Mistr A, Turánek J. High effectiveness of platinum(IV) complex with adamantylamine in overcoming resistance to cisplatin and suppressing proliferation of ovarian cancer cells in vitro. Biochem Pharmacol. 69(3): 373-83, 2005. IF 4.84, number of citations (WOS): 25; IBP: corresponding author, first author.

Horváth V, Blanářová O, Švihálková-Šindlerová L, Souček K, Hofmanová J, Sova P, Kroutil A, Fedoročko P, Kozubík A. Platinum(IV) complex with adamantylamine overcomes intrinsic resistance to cisplatin in ovarian cancer cells. Gynecol Oncol. 102(1): 32-40, 2006. IF 2.92, number of citations (WOS): 6;  IBP: corresponding author, first author.

Horváth V, Souček K, Švihálková-Šindlerová L, Vondráček J, Blanářová O, Hofmanová J, Sova P, Kozubík A. Different cell cycle modulation following treatment of human ovarian carcinoma cells with a new platinum(IV) complex vs cisplatin. Invest New Drugs.  25(5): 435-43, 2007. IF 3.40, number of citations (WOS): 3; IBP: corresponding author, first author.

(b) This work was done 90% by the researchers from Department of Cytokinetics.

(c) LA-12 is a novel platinum(IV) compound with adamantylamine, which is able to overcome intrinsic and acquired resistance to widely used platinum compound cisplatin in ovarian cancer cell lines. We intensively examined the mechanisms of LA-12 effects and compared its effects with  cisplatin and oxaliplatin (III. generation of platinum drug) repeatedly confirming high effectiveness of LA-12. Our original papers describe mechanisms of cell cycle regulation (involving cyclins, Cdks etc.) and the effects on cell death. Moreover,  expression of various proteins involved in DNA-damage-responsive signalling were assesed. These results have wide implications for understanding of mechanisms of action of this novel class of platinum cytostatics.

[2] Description of the role of the estrogen receptor in deregulation of cell proliferation induced by polycyclic aromatic hydrocarbons.

a) Plíšková M, Vondráček J, Vojtěšek B, Kozubík A, Machala M: Deregulation of cell proliferation by polycyclic aromatic hydrocarbons in human breast carcinoma MCF-7 cells reflects both genotoxic and nongenotoxic events. Toxicological Sciences 83(2): 246-256, 2005. IF 4.44, number of citations (WOS): 29; IBP: corresponding author, joint first author.

Kummer V, Mašková V, Zralý Z, Neča J, Šimečková P, Vondráček J, Machala M: Estrogenic activity of environmental polycyclic aromatic hydrocarbons in uterus of immature Wistar rats. Toxicology Letters 180(3): 212-221, 2008. IF 3.25, number of citations (WOS): 5; IBP: co-author.

b) This work was performed in collaboration with our long-term partner at the Veterinary Research Institute, and Masaryk Memorial Cancer Institute. For the first part, J. Vondráček provided experimental design and carried experiments (60% of data), and wrote the manuscript. The second study was conceived as an in vivo extension of the original work and has been carried out at the animal facility of VRI. J. Vondráček participated in experimental design and wrote the manuscript.

c) This work described, for the first time, that polycyclic aromatic hydrocarbons (PAHs) may exert positive effects on proliferation of estrogen-sensitive cells and tissues, and that this effect is primarily determined by their relative ability to activate estrogen receptor-alpha and to exert genotoxic effects leading to activation of p53 pathway. These data are of primary importance to our understanding of the pro-carcinogenic effects of PAHs.

[3] Aryl hydrocarbon receptor as regulator of contact inhibition.

a) Andrysík Z, Vondráček J, Machala M, Krčmář P, Švihálková-Šindlerová L, Kranz A, Weiss C, Faust D, Kozubík A, Dietrich C: The aryl hydrocarbon receptor-dependent deregulation of cell cycle control induced by polycyclic aromatic hydrocarbons in rat liver epithelial cells. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis 615(1-2): 87-97, 2007. IF 3.20, number of citations (WOS): 22;  IBP: corresponding author, first author.

Weiss C, Faust D, Schreck I, Ruff A, Farwerck T, Melenberg A, Schneider S, Oesch-Bartlomowicz B, Zatloukalová J, Vondráček J, Oesch F, Dietrich C: TCDD deregulates contact-inhibition in rat liver oval cells via Ah receptor, JunD and Cyclin A. Oncogene 27(15): 2198-2207, 2008. IF 7.22, number of citations (WOS): 15;  IBP: co-author.

b) The first study had been carried out by 80 % at IBP, where most of the experiments were performed and the major part of writing was done. For the second study, we developed ARNT-dominant negative cell line and carried out work at additional cell models to provide further support for our general hypothesis. The overall idea of the project was formulated jointly by collaborating labs in Brnoand JGU Mainz, Germany.

c) This set of studies defined effects of different types of the aryl hydrocarbon receptor (AhR) ligands on cell cycle control in contact – inhibited cells. Activation of AhR has been found to activate a novel pathway leading to increased cyclin A/cdk2 complex activity, which is independent on the AhR transcriptional partner, ARNT. The importance of our results lies in the fact that we described a novel type of effect supporting the current hypothesis that AhR plays a much wider role both in carcinogenesis and in embryonic development, including regulation of cell proliferation, differentiation, apoptosis and senescence.

[4] Interactions of pro-inflammatory cytokines and aryl hydrocarbon receptor in control of xenobiotic metabolism and  proliferation in epithelial cells.

a) Umannová L, Zatloukalová J, Machala M, Krčmář P, Májková Z, Hennig B, Kozubík A, Vondráček J. Tumor necrosis factor-alpha modulates effects of aryl hydrocarbon receptor ligands on cell proliferation and expression of cytochrome P450 enzymes in rat liver ‘stem-like’ cells. Toxicological Sciences 99(1): 79-89, 2007. IF 4.44, number of citations (WOS): 12; IBP: corresponding author, first author.

Umannová L, Machala M, Topinka J, Nováková Z, Milcová A, Kozubík A, Vondráček J. Tumor necrosis factor-alpha potentiates genotoxic effects of benzo[a]pyrene in rat liver epithelial cells through upregulation of cytochrome P450 1B1 expression. Mutation Research – Fundamental and Molecular Mechanisms of Mutagenesis 640(1-2): 162-169, 2008. IF 3.20, number of citations (WOS): 8; IBP: corresponding author, first author.

b) The two studies have been primarily (90%) carried out at the IBP, including majority of experiments and writing the manuscript. The collaborating labs provided especially unavailable techniques.

c) The importance of this project lies in the fact that we documented, for the first time that pro-inflammatory cytokines may disrupt the balance of expression of individual enzymes responsible for metabolic activation of promutagens, such as PAHs, thus leading to enhanced DNA damage and possibly increased mutagenicity of these compounds, primarily through increased cytochrome P450 1B1 expression and activity. Thus, inflammation may enhance mutagenicity alter non-genotoxic pro-carcinogenic effects of AhR ligands, both findings having important consequences for our understanding of the mechanisms of carcinogenicity of environmental pollutants.

[5] Disruption of intercellular communication by non-dioxin-like polychlorinated biphenyls

a) Šimečková P, Vondráček J, Andrysík Z, Zatloukalová J, Krčmář P, Kozubík A, Machala M. The 2,2‘,4,4‘,5,5‘-hexachlorobiphenyl (PCB 153)-enhanced degradation of connexin 43 involves both proteasomal and lysosomal activities. Toxicological Sciences 107: 9-18, 2009. IF 4.443, number of citations (WOS): 3; IBP: co-authors.

Šimečková P, Vondráček J, Procházková J, Kozubík A, Krčmář A, Machala M. 2,2‘,4,4‘,5,5‘-Hexachlorobiphenyl (PCB 153) induces degradation of adherens junction proteins and inhibits β-catenin-dependent transcription in liver epithelial cells. Toxicology 260: 104-111, 2009. IF 2.836; IBP: co-authors.

b) Contribution of IBP was approximately 30 and 40% to the above studies, respectively. We carried out selected measurements and wrote significant parts of both manuscripts.

c) The present recent studies documented, for the first time, that NDL-PCBs may degrade proteins involved in cell-to-cell communication and modulate related intracellular signaling in liver cells, and described contribution of protein degrading machineries to observed toxic events. These results may help to explain tumor promoting effects of NDL-PCBs observed in in vivo models.

[6] Molecular mechanisms responsible for regulation of TRAIL-induced apoptosis in colon cancer cells.

a) Vaculová A, Hofmanová J, Zatloukalová J, Kozubík A. Differences in TRAIL-induced changes of Mcl-1 expression among distinct human colon epithelial cell lines. Exp Cell Res. 315(19): 3259-66, 2009. IF 3.95, IBP: corresponding author, first author.

Vaculová A, Hofmanová J, Souček K, Kozubík A. Different modulation of TRAIL-induced apoptosis by inhibition of pro-survival pathways in TRAIL-sensitive and TRAIL-resistant colon cancer cells. FEBS Lett. 580(28-29): 6565-9, 2006. IF 3.26, number of citations (WOS): 14; IBP: corresponding author, first author.

b) This work was done 100% by the researchers from Department of Cytokinetics.

c) Investigation of the mechanisms responsible for cancer cell resistance to TRAIL is an essential prerequisite for its successful clinical application. We demonstrated the crucial role of prosurvival pathways (MAPK/ERK and PI3K/Akt) and antiapoptotic Mcl-1 protein in protection of colon cancer cells from TRAIL-induced apoptosis. Our results suggest the intracellular targets important for colon cancer cell sensitization to the killing effects of this therapeutically attractive cytokine, and describe the mechanisms of their regulation.

[7] Molecular mechanisms involved in crosstalk of signaling pathways induced by TNF family cytokines and polyunsaturated fatty acids

a) Vaculová A, Hofmanová J, Anděra L, Kozubík A. TRAIL and docosahexaenoic acid cooperate to induce HT-29 colon cancer cell death. Cancer Lett. 229(1): 43-8, 2005. IF 3.5, number of citations (WOS): 8; IBP: corresponding author, first author.

Hofmanová J, Vaculová A, Kozubík A. Polyunsaturated fatty acids sensitize human colon adenocarcinoma HT-29 cells to death receptor-mediated apoptosis. Cancer Lett. 218(1): 33-41, 2005. IF 3.5, number of citations (WOS): 14; IBP: corresponding author, first author.

b) This work was done by 95 % by the researchers from Department of Cytokinetics.

c) The resistance of some cancer cells to apoptosis induced by TRAIL and other TNF family cytokines is a serious obstacle in their clinical application. We demonstrated a significant potentiation of cytokine-induced cell death of human colon cancer cells by polyunsaturated fatty acids (PUFAs), associated with enhancement of caspase activation and involvement of mitochondria. Our studies highlight the efficient modulatory action of PUFAs in regulation of cancer cell sensitivity to killing effects of TNF family cytokines.

[8] Beneficial effects of short chain fatty acid butyrate in modulation of colon cancer cell differentiation and apoptosis 

a) Hofmanová J, Vaculová A, Lojek A, Kozubík A. Interaction of polyunsaturated fatty acids and sodium butyrate during apoptosis in HT-29 human colon adenocarcinoma cells. Eur J Nutr.  44(1):  40-51, 2005; IF 1.9, number of citations (WOS): 12; IBP: corres-ponding author, first author.

HýžďalováM. , Hofmanová J., Pacherník J. , Vaculová A., Kozubík A. The interaction of butyrate with TNF-α during differentiation and apoptosis of colon epithelial cells: Role of NF-κB activation. Cytokine 44(1): 33-43, 2008. IF 2.21, number of citations (WOS): 3;IBP: corresponding author, first author.

b) This work was done by 90% by the researchers from Department of Cytokinetics.

c) These studies illustrate mutual interactions of sodium butyrate (NaBt) with PUFAs (arachidonic acid, AA or docosahexaenoic acid, DHA) showing modulation of cell cycle and shift of the balance between differentiation and apoptosis depending on the cell transformation level (adenoma-carcinoma sequence). In addition to nutrient-nutrient interactions, we evidenced interaction of NaBt with TNF family cytokines (namely TNF alpha) potentiating apoptosis, but suppressing differentiation in various types of colon epithelial cells. We confirmed participation of activation of NF-кB transcription factor in these effects, which was different in cells of non-cancer and cancer origin, suggesting that the NF-кB pathway may be more effectively involved in these processes in cancer cells.

[9] Enhancement of differentiation and apoptosis of leukemic cells by drug interaction

a) Štika J., Vondráček J., Hofmanová J., Šimek V., Kozubík A. MK-886 enhances tumour necrosis factor-a-induced differentiation and apoptosis. Cancer Letters 237(2): 263-271, 2006. IF 3.50, number of citations (WOS): 5; IBP: corresponding author, first author.

Procházková, J., Stixová, L., Souček, K., Hofmanová, J., and Kozubík, A. Monocytic differentiation of leukemic HL-60 cells induced by co-treatment with TNF-alpha and MK886 requires activation of pro-apoptotic machinery. Eur J Haematol. 83: 35-47, 2009. IF 2.24; IBP: corresponding author, first author.

b) First study was performed in the Dept. of Cytokinetics (90%) in collaboration with Faculty of Sciences, MasarykUniversity, Brno, during PhD. study of the first author at IBP. The second study was carried out at IBP by 100%.

c) This set of studies evidenced new possibilities of potentiation of differentiation and/or apoptosis of human leukemia cells by combined treatment with inhibitors of arachidonic acid metabolism and physiological regulators such as TNF alpha. We clarified participation of specific signaling pathways and cell cycle and apoptosis regulatory proteins in these effects.

[10]  TGF-b1 in regulation cancer cell phenotype and apoptosis

a) Souček K, Pacherník J, Kubala L, Vondráček J, Hofmanová J, Kozubík A. Transforming growth factor-beta1 inhibits all-trans retinoic acid-induced apoptosis. Leuk Res 30(5): 607-23, 2006. IF 2.39, number of citations (WOS): 4; IBP: corresponding author, first author

Staršíchová A, Kubala L, Lincová E, Pernicová Z, Kozubík A, Souček K. Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-beta1. Biol Proced Online 2009; IF=2.27; IBP: corresponding author, first author.

b) 95% contribution by Department of Cytokinetics, IBP.

c) In the first study, we demostrated interactions between retinoids, such as  all-trans retinoic acid (ATRA) and transforming growth factor-beta1 (TGF-b1) in regulation of proliferation, differentiation and apoptosis leading to the enhancement of ATRA-induced suppression of cell proliferation, which is accompanied by inhibition of ATRA-induced apoptosis in human leukemia cells. TGF-b family is also a potent inducer of epithelial-mesenchymal transition. We developed application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. This novel application of such approach demonstrates a great potential of for noninvasive and real-time monitoring of cellular fate.

[11] The role of GDF-15 in regulations of osteoclasts

a) Vaňhara P, Lincová E, Kozubík A, Jurdič P, Souček K, Šmarda J. Growth/differentiation factor-15 inhibits differentiation into osteoclasts - a novel factor involved in control of osteoclast differentiation. Differentiation 78(4): 213-22, 2009. IF 3.18, number of citations (WOS): 1; IBP: joint first author; joint  corresponding author.

b) 50% contribution by Department of Cytokinetics, IBP.

c) GDF-15 is a divergent member of TGF-bfamily. We unveiled unprecedented role of GDF-15 in modulation of osteoclast differentiation and possibly in therapy of bone metastases.

[12] The role of PKB/Akt in sensitivity to NSAIDs

a) Lincová, E., Hampl, A., Pernicová, Z., Staršíchová, A., Krčmář, P., Machala, M., Kozubík, A., and Souček, K. Multiple defects in negative regulation of the PKB/Akt pathway sensitise human cancer cells to the antiproliferative effect of non-steroidal anti-inflammatory drugs. Biochem Pharmacol 78(6):561-572, 2009. IF 4.84; IBP: corresponding author, first author.

b) 80% contribution by Department of Cytokinetics, IBP.

c) Role of GDF-15 in proces of chemoprevention and cancer development remains unclear. Here we observed that anti-proliferative effects of non-steroidal anti-inflammatory drugs is not dependent on GDF-15 expressiom. However, our data suggest novel mechanisms of NSAIDs antiproliferative action in cancer epithelial cells, which depends on the status of negative regulation of the PKB/Akt pathway and the isoform-specific action of Akt2. Thus, unexpectedly, multiple defects in negative regulation of the PKB/Akt pathway may contribute to increased sensitivity to chemopreventive effects of these widely used drugs.

[13] The role of dishevelled in the Wnt signaling:

a) Andersson T, Södersten E, Duckworth JK, Cascante A, Fritz N, Sacchetti P, Cervenka I, Bryja V, Hermanson O. CXXC5 is a novel BMP4-regulated modulator of Wnt-signaling in neural stem cells. J Biol Chem. 284(6): 3672-81, 2009. IF 5.52, number of citations (WOS): 11; IBP: joint author.

b) Contribution of V. Bryja (15%) – selected experiments. The projects has been carried in collaboration with the lab of Ernest Arenas, Ola Hermansson (both Karolinska Institute, Stockholm).

 c) The study describes novel interaction partners of Dishevelled, a key mediator of the Wnt signaling pathway. We uncover several previously unknown regulators of the Wnt signaling – CXXC5, which acts as the negative player in the pathway.

[14] The role of Wnt signaling in neural development

a) Čajánek L, Ribeiro D, Liste I, Parish CL, Bryja V, Arenas E. Wnt/beta-catenin signaling blockade promotes neuronal induction and dopaminergic differentiation in embryonic stem cells.Stem Cells. 27(12): 2917-2927, 2009. IF 7.74; IBP: joint author.

b) V. Bryja and his group had significant (35%) contribution to the study. 

c) In the presented work we looked for the role of Wnt signaling in the neural development and in the differentiation of neural stem cells. Special attention has been paid to the role of Wnts in the development of dopaminergic midbrain neurons. We show that Wnt signaling can control several aspects of dopaminergic neuron biology – it controls primary differentiation events, cell proliferation, general morphogenesis of the tissue, and cell-cell contacts.

[15] The function of Wnt receptors in non-canonical Wnt signaling

a) Bryja V, Andersson ER, Schambony A, Esner M, Bryjová L, Biris KK, Hall AC, Kraft B, Čajánek L, Yamaguchi TP, Buckingham M, Arenas E. The Extracellular Domain of Lrp5/6 Inhibits Non-Canonical Wnt Signaling in vivo. Mol Biol Cell. 20(3): 924-936, 2009. Cover article. IF 5.56, number of citations (WOS): 12; IBP: first author, corresponding author.

Schulte G, Bryja V. The Frizzled family of unconventional GPCRs. Trends Pharmacol. Sci. 28(10): 518-525, 2007. IF 9.34, number of citations (WOS): 25; 50% contribution of V. Bryja.

b) V. Bryja is the first and corresponding author of the study published in Molecular Biology of the Cell. In the review article, V. Bryja contributed by 50%.

c) In the study by Bryja et al. we discovered that Lrp6, a single transmembrane protein associated with the Wnt/beta-catenin signaling, can act also in non-canonical Wnt signaling pathway. Lrp6 can bind and sequester non-canonical Wnt ligands, as such it protects non-canonical Wnts from binding to its cognate receptors. We demonstrate this function of Lrp6 in several systems including mammalian cells in culture, Xenopus embryos and mouse. This work was preceded by detailed review focused on the Wnt receptors.

[16] Role of STAT transcription factors in pathological FGFR3 signaling in disease.

a) Krejčí P, Kashiwada TA, Goodridge HS, Salazar L, Schibler MJ, Jelínková P, Thompson LM, Wilcox WR. STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes. J Cell Sci 121: 272-281, 2008. IF 6.247, number of citations (WOS): 5; IBP: first and corresponding author.    

Krejčí P, Procházková J, Bryja V, Jelínková P, Pejchalová K, Kozubík A, Thompson LM, Wilcox WR.Fibroblast growth factor inhibits interferon gamma-STAT1 and interleukin 6-STAT3 signaling in chondrocytes. Cellular Signalling 21(1):151-60, 2009. IF 4.31, number of citations (WOS): 2;  IBP: first and corresponding author.

b) Both articles were mostly executed by P. Krejci, who designed and carried-on most of the experiments, wrote manuscripts and handled the submission and publication processes. The total contribution of IBP team equaled ~80% of the work.

c) We excluded the STAT1 and STAT3 transcription factors from direct participation in FGFR3 signaling pathways in cartilage (Krejci et al., 2008). Prior to this work, the activation of STAT1 was believed to be the major mechanism of FGFR3-mediated inhibition of chondrocyte proliferation. In these two articles we presented evidence that FGFR3-mediated chondrocyte inhibition occurs independently of STAT1 or STAT3, and that the capacity to activate STAT1 is restricted to only a limited number of FGFR3 mutants associated with skeletal dysplasias. During the above mentioned work on STAT, we discovered potent inhibition of canonical STAT-cytokine signaling pathways by FGFR3. Since STAT-cytokine signaling represents an important positive regulator of cartilage, its inhibition may contribute to the growth inhibitory effect of FGFR3 on cartilage (Krejci et al., 2009).

[17] Novel approach to therapeutic FGFR3 inhibition.

a)  Krejčí P, Pejchalová K, Wilcox WR. Simple, mammalian cell-based assay for identification of inhibitors of the Erk MAP kinase pathway. Invest New Drugs 25(4): 391-395, 2007. IF 3.396, number of citations  (WOS): 1.  IBP: First and corresponding author.

Krejčí P, MurakamiS, Procházková J, Smutný J, Chlebová K, Aklian A, Trantirek L, Bryja V,Kozubík A, Wilcox WR. NF449 is a novel inhibitor of fibroblast growth factor receptor 3 (FGFR3) signaling active in chondrocytes and myltiple myeloma cells. J Biol Chem, In press. IBP: First and corresponding author. IF 5.52.

b) P. Krejci contributed serving both main experimentator and principal investigator roles. Total contribution of IBP team equaled ~80% of this work.

c) We developed a chondrocyte-based high-throughput screening assay for identification of novel inhibitors of FGFR3 signalling in a chondrocyte environment. Using this assay, we identified and patented a molecule named NF449 as a novel, potentially theraputic inhibitor of FGFR3.

[18] Characterization of novel tissue fenotype of pathological FGFR3 signaling in cartilage.

a) Matsushita T, Wilcox WR, Chan YY, Kawanami A, Bükülmez H, Balmes G, Krejčí P, Mekikian PB, Otani K, Yamaura I, Warman ML,Givol D, Murakami S. FGFR3 promotes synchondrosis closure and fusion of ossification centers through the MAPK pathway. HumMol Genet 18, 227-40, 2009. IF 7.249, 3 citations on WOS; IBP: co-author. 

b) P. Krejčí contributed ~10% to this article, mostly preparation of the human material included in the study.

c) We described a novel phenotype of FGFR3 signaling in TD in vivo, i.e. premature synchondrosis closure in the spine and cranial base, which may adversely affect potential treatment outcomes in FGFR3-related skeletal dysplasias.

Department 6     Free Radical Pathophysiology (DFRP)

[1]Fabrication of modified porphyrinic microsensorfor electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7

(a) Hrbac J, Gregor C, Machova M, Kralova J, Bystron T, Ciz M, Lojek A. (2007): Nitric oxide sensor based on carbon fiber covered with nickel porphyrin layer deposited using optimized electropolymerization procedure. Bioelectrochemistry. 71(1):46-53.IF 3,0; 11 citations on WOS.

Pekarova M., Kralova, J., Kubala L., Ciz, M., Lojek A., Gregor C., Hrbac J.(2009): Continuous electrochemical monitoring of nitric oxide production in murine macrophage cell line RAW 264.7. Anal. Bioanal. Chem., 394(5):1497-1504.IF 3,3; 1 citation on WOS.

(b) The authors from the department contributed ~30% and 80% to the part of the study related to the preparation of microsensors and the measurement of nitric oxide production by macrophages, resp.

(c) We prepared the sensor composed of electrochemically oxidized carbon fiber, covered solely with nickel porphyrin derivative layer electropolymerized using our improved method. The sensor is characterized by high selectivity towards nitrite, ascorbate and dopamine.Using this modified porphyrinic sensor we realized for the first time the continual and long-term electrochemical detection of NO production by stimulated macrophages. The capability of our microsensor to instantaneously monitor the changes in the NO production by stimulated RAW 264.7 cells was demonstrated by the immediate decrease in the NO signal as a response to the addition of iNOS inhibitor into the cell culture medium.

[2] Various food constituents exert strong antioxidative properties with the potential to decrease oxidative stress in animal cells and tissues

(a) Komrsková D., Lojek A., Hrbáč J., Číž M. (2006): A comparison of chemical systems for luminometric determination of antioxidant activity towards individual reactive oxygen species. Luminescence 21, 239-244. IF 1,2; 5 citations according to WOS

Lopez D, Pavelková M, Gallová L, Simonetti P, Gardana C, Lojek A, Loaiza R, Mitjavila MT. (2007): Dealcoholized red and white wines decrease oxidative stress associated with inflammation in rats. Br J Nutr 98 (3), 611-619.IF 2,8; 3 citations according to WOS

Číž M, Pavelková M, Gallová L, Králová J, Kubala L, Lojek A. (2008): The influence of wine polyphenols on reactive oxygen and nitrogen species production by rat macrophages RAW 264.7. Physiol Res 57, 393-402. IF 1,7; 12 citations according to WOS

(b) The authors from the department contributed 90% to the part of the study related to the evaluation of luminometric determination of antioxidant activity towards individual reactive oxygen species, and 65% to the part of the study related to the determination of antioxidative properties of wine constituents.

(c) In the first part of the study the panel of methods for the generation of reactive oxygen and nitrogen species and the determination of antioxidative activity was optimized and set up. Then antioxidant properties of wine constituents (e.g. polyphenols) were evaluated and compared. Effects of wine constituents on the production of reactive oxygen and nitrogen species by isolated macrophages as well as on the oxidative stress associated with inflammation in vivowere also investigated. The results indicate that the phenolic compounds not only improve antioxidant status in an inflammatory situation, but also limit cell infiltration, possibly through a decrease in superoxide anion and an increase in nitric oxide production.

[3] Myeloperoxidase modulates the balance of pro- and anti-inflammatory lipid mediators during acute inflammation

(a) Kubala L, Schmelzer KR, Klinke A, Kolarova H, Baldus S, Hammock BD, Eiserich JP. Modulation of arachidonic and linoleic acid metabolites in myeloperoxidase-deficient mice during acute inflammation. Free Radic Biol Med. 2010 May 15;48(10):1311-20.IF 5,4.

(b) The authors from the department contributed ~70% and 30% to the part of the study related to animal experiments, in vitro experiments and analysis of samples.

(c) We investigated the role of myeloperoxidase (MPO) in modulating biologically active arachidonic acid (AA) and linoleic acid (LA) metabolites during acute inflammation. Wild-type and MPO-knockout (KO) mice were exposed to intraperitoneally injected endotoxin for 24 h. Compared to wild-type mice, MPO-KO mice had significantly lower plasma levels of LA epoxides and corresponding LA- and AA-derived fatty acid diols and hydroxy intermediates. Conversely, MPO-deficient mice had significantly higher plasma levels of cysteinyl-leukotrienes with well-known proinflammatory properties. In vitro experiments revealed significantly lower amounts of AA and LA epoxides, LA- and AA-derived fatty acid diols, and AA and LA hydroxy intermediates in stimulated polymorphonuclear neutrophils isolated from MPO-KO mice. Our results demonstrate that MPO modulates the balance of pro- and anti-inflammatory lipid mediators during acute inflammation and, in this way, may control acute inflammatory diseases.

[4] Immunomodulating effects of carvedilol.

(a) Nosal R., Jancinova V., Ciz M., Drabikova K., Lojek A., Fabryova V.(2005): Inhibition of chemiluminescence by carvedilol in the cell-free system, whole human blood and blood cells. Scand J Clin Lab Invest 65, 55-64.IF 0,9; 9 citations on WOS.

Pečivová J., Mačičková T., Lojek A., Gallova L., Číž M., Nosáľ R., Holomáňová D.(2007):In vitro effect of carvedilol on professional phagocytes. Pharmacology,  Dec 7;79(2):86-92.IF 1,2; 3 citations on WOS.

Mačičková T., Pečivová J., Nosál R., Lojek A., Pekarová M., Cupaníková D.: Inhibition of superoxide generation and myeloperoxidase release by carvedilol after receptor and nonreceptor stimulation of human neutrophils. Neuroendocrinol. Lett. 2008 Oct 11;29(5):790-793.IF 1,4; 2 citations on WOS.

Pekarova M., Kralova, J., Kubala, L., Ciz, M., Papezikova, I., Macicková T., Pecivova J., Nosal R., Lojek A. (2009): Carvedilol and adrenergic agonists suppress the lipopolysaccharide-induced NO production in RAW 264.7 macrophages via the adrenergic receptor activation. J. Physiol. Pharmacol.,60 (1),143-150.IF 2,6;  1 citation on WOS.

Ben Mosbah I, Roselló-Catafau J, Alfany-Fernandez I, Rimola A, Puig Parellada P, Mitjavila MT, Lojek A, Ben Abdennebi H, Boillot O, Rodes J, Peralta C (2010): Addition of carvedilol to University Wisconsin solution improves steatotic and non-steatotic liver preservation. Liver Transplant.,16:  s. 163-171.IF 4,1.

(b) The average contribution of the department is ~ 50%, in the case of the last article ~ 10%

(c) The antioxidative and immunomodulating properties of carvedilol were studied and described. Our results imply that carvedilol, a unique vasodilating beta-adrenergic antagonist, suppresses metabolic activity of phagocytes through the activation and modulation of signaling pathways connected with adrenergic receptors. Furthermore, carvedilol decreased liver injury and oxidative stress and had significant favourable effect on liver function in the model used. It proves the potential use of carvedilol as the constituent of preservation solutions.

[5] Role of selected signaling pathways and influence of intracellular redox status on embryonal stem cells and embryonal carcinoma stem cells differentiation

(a) Pacherník J, Horváth V, Kubala L, Dvorák P, Kozubík A, Hampl A.Neural differentiation potentiated by the leukaemia inhibitory factor through STAT3 signalling in mouse embryonal carcinoma cells. Folia Biol (Praha). 2007;53(5):157-63. IF 0,6.3 citations according to WOS

Pacherník J, Bryja V, Esner M, Kubala L, Dvorák P, Hampl A.Neural differentiation of pluripotent mouse embryonal carcinoma cells by retinoic acid: inhibitory effect of serum. Physiol Res. 2005;54(1):115-22. IF 1,8.28 citations according to WOS

Konopka R., Lojek A., Kubala L., Pachernik J.: Retinoic acid induced neuronal differentiation of P19 embrynonal carcinoma cells is modulated by intracellular redox state. Neuro Endocrinol Lett. 2008 Oct 11;29(5):770-774. IF 1,4.3 citations according to WOS

Dvorakova J, Hruba A, Velebny V, Kubala L.Isolation and characterization of mesenchymal stem cell population entrapped in bone marrow collection sets. Cell Biol Int. 2008 Sep;32(9):1116-25. IF 1,6.3 citations according to WOS

(b) The authors from the department contributed ~20%, ~20%, 90% and 40% to the individual studies (the analysis of cell signaling pathways and determination of redox status).

(c) We have shown that leukaemia inhibitory factor (LIF) potentiated retinoic acid-induced neural differentiation of pluripotent mouse embryonal carcinoma P19 cells. We also demonstrated that pro-neural effects of LIF and partially also of retinoic acid are abolished by inhibition of the JAK2→STAT3 signalling pathway. In contrast, inhibition of the MEK1→ERK signalling pathway does not exhibit any effect. These results suggest that in neurogenic regions, cooperative action of LIF and other neuro-differentiation-inducing factors, such as retinoic acid, may be mediated by the STAT3 signalling pathway. Furthermore, our data outline a role of reactive oxygen species as important molecules in the transduction of an intracellular signal during the neuronal differentiation of embryonal stem cells. And finally, we showed that washing out bone marrow collection sets may constitute a highly ethical source of mesenchymal stem cells for research purposes and may be utilized also in clinical applications.

[6] Serotonin modulates the oxidative burst of human phagocytes, but not isolated neutrophils, via various mechanisms

(a) Číž M., Komrsková D., Prachařová L., Okénková K., Čížová H., Moravcová A., Jančinová V., Petríková M., Lojek A., Nosáľ R. (2007): Serotonin modulates the oxidative burst of human phagocytes via various mechanisms. Platelets 18(8), 583–590. IF 2,271; 3 citations according to WOS

Okénková K., Lojek A.,Kubala, L., Číž M. (2007): Modulation of rat blood phagocyte activity by serotonin. Chem Listy 101, 245-246. IF 0,593.

Prachařová L, Okénková K, Lojek A, Číž M. (2010):Serotonin and its 5-HT(2) receptor agonist DOI hydrochloride inhibit the oxidative burst in total leukocytes but not in isolated neutrophils.Life Sci 86 (13-14), 518-523. IF 2,583.

(b) The authors from the department contributed 90% to the study related to the evaluation of the effects of serotonin on the oxidative burst of professional phagocytes.

(c) The aim of the study was to clarify the mechanisms of serotonin action on human phagocytes.Serotonin inhibited the production of reactive oxygen species and  myeloperoxidase activity in a dose dependent manner. The hypothesis that the inhibitory activity of serotonin might be also receptor mediated was evaluated using various serotonin receptor agonists and antagonists. Results confirmed that the responsibility for serotonin inhibitory effects lies with both the decrease in the generation of reactive oxygen species (due to the inhibition of myeloperoxidase activity) and with direct scavenging of reactive oxygen species. The effect of serotonin on phagocytes might also be partially mediated by 5-HTR₂ receptor.

[7] Immunomodulating effects of hyaluronan and possible applications in the biology and medicine.

(a) Frankova J., Kubala L., Velebny V., Ciz M., Lojek A. (2006): The effect of Hyiodine (hyaluronan combined with KI₃ complex) on keratinocytes and immune cells.  J Mater Sci Mater Med. 17(10):891-898.IF 1,6.4 citations according to WOS

Krejcova D, Pekarova M, Safrankova B, Kubala L. The effect of different molecular weight hyaluronan on macrophage physiology. Neuro Endocrinol Lett. 2009;30 Suppl 1:106-11.IF 1,4.

Dvorakova J, Velebny V, Kubala L. Hyaluronan influence on the onset of chondrogenic differentiation of mesenchymal stem cells. Neuro Endocrinol Lett. 2008 Oct;29(5):685-90. IF 1,4.2 citations according to WOS

(b) The authors from the department contributed ~90% and 100% to the studies except the preparation of hyaluronan-iodide complex. In the last study the contribution was ~50 % including cell cultures work, protein analysis and gene expression analysis..

(c) We investigated the immunomodulating effects of highly purified pharmacological grade hyaluronan of defined molecular weights 11, 52, 87, 250 and 970 kilodaltons on mouse macrophage cell lines RAW 264.7 and MHS. Hyaluronan of any molecular weight neither exerted stimulatory nor inhibitory effects on macrophages pre-treated by lipopolysaccharide. However, we observed that hyaluronan content of Hyiodine (high molecular weight hyaluronan combined with KI3 complex) reduces the toxic effect of KI3 complex on cells and speeds up the wound healing process by increasing the production of inflammatory cytokines. Our data from further experiments prove that hyaluronan of molecular weights 100, 600 and 1 500 kDa does not significantly modulate early chondrogenesis of mesenchymal stem cells.  

[8] Myeloperoxidase is an important diagnostic marker in cardiovascular diseases

(a) Kubala L, Lu G, Baldus S, Berglund L, Eiserich JP.Plasma levels of myeloperoxidase are not elevated in patients with stable coronary artery disease. Clin Chim Acta. 2008 Aug;394(1-2):59-62. IF 2,96, 4 citations according to WOS

Rudolph V, Rudolph TK, Kubala L, Clauberg N, Maas R, Pekarova M, Klinke A, Lau D, Szöcs K, Meinertz T, Böger RH, Baldus S.A myeloperoxidase promoter polymorphism is independently associated with mortality in patients with impaired left ventricular function. Free Radic Biol Med. 2009 Dec 1;47(11):1584-90. IF 5,4.

(b) The authors from the department contributed ~10% and 20% to the part of the study related to analysis of myeloperoxidase enzymatic activity and catabolism of nitric oxide.

(c) We assessed the prognostic information derived from MPO polymorphism and MPO levels in patients with impaired left ventricular function or acute coronary syndromes. Plasma and serum levels of MPO did not differ significantly between patients with or without coronary artery disease, suggesting that systemic release of MPO was not a characteristic feature of coronary artery disease. On the other hand, we observed thatMPO polymorphism was linked to adverse clinical outcome of patients with impaired left ventricular function.

[9] Immunomodulatory effects of polysaccharides

(a) Ruszova E, Pavek S, Hajkova V, Jandova S, Velebny V, Papezikova I, Kubala L.Photoprotective effects of glucomannan isolated from Candida utilis. Carbohydr Res. 2008 Feb 25;343(3):501-11.IF 2,0.3 citations according to WOS

Hájková V, Svobodová A, Krejčová D, Cíž M, Velebný V, Lojek A, El-Benna J, Kubala L.Soluble glucomannan isolated from Candida utilis primes blood phagocytes. Carbohydr Res. 2009 Oct 12;344(15):2036-41. IF 2,0.

(b) The authors from the department contributed ~80% and 80% to the part of the studies related to the in vitro cell culture experiments, protein analysis, flow cytometric analysis and gene expression analysis.

(c) Highly branched glucomannan (GM) was tested for its photoprotective effects in an in vitro model of UVB-irradiated human keratinocytes and an in vivo model of UV-induced erythema formation in human volunteers. GM suppressed the UVB-induced decrease in keratinocyte viability, which was connected with the suppression of UVB-induced keratinocyte apoptosis. On the other hand, the soluble GM induces priming of phagocytes connected with their degranulation, the increase of surface receptor expression, and potentiation of oxidative burst response to opsonized particles through the activation of NADPH oxidase.

[10] Nitro-fatty acids as important regulators of inflammary processes

(a) Rudolph TK, Rudolph V, Edreira MM, Cole MP, Bonacci G, Schopfer FJ, Woodcock SR, Franek A, Pekarova M, Khoo NK, Hasty AH, Baldus S, Freeman BA.Nitro-fatty acids reduce atherosclerosis in apolipoprotein E-deficient mice. Arterioscler Thromb Vasc Biol. 2010 May;30(5):938-45. Epub 2010 Feb 18.IF 6,9.

(b) The authors from the department contributed ~10% to the part of the study related the measurement of nitric oxide production by macrophages..

(c) In this study, we demonstrated that subcutaneously administered 9- and 10-nitro-octadecenoic acid (nitro-oleic acid) potently reduced atherosclerotic lesion formation in apolipoprotein E-deficient mice. Electrophilic nitro-fatty acids exhibit antiinflammatory signaling actions in inflammatory and vascular cell model systems. Immunostaining and gene expression analyses revealed that nitro-oleic acid attenuated lesion formation by suppressing tissue oxidant generation, inhibiting adhesion molecule expression, and decreasing vessel wall infiltration of inflammatory cells. In addition, nitro-oleic acid reduced foam cell formation by attenuating oxidized low-density lipoprotein-induced phosphorylation of signal transducer and activator of transcription-1, a transcription factor linked to foam cell formation in atherosclerotic plaques.

[11]The effects of H1-antihistamines on phagocytes

(a) Kralova J., Ciz M., Nosal R., Drabikova K., Lojek A. (2006):The Effect of H₁-Antihistamines on Oxidative Burst of Phagocytes. Inflamm. Res., 55 (Suppl. 1), S15-S16.IF 1,5; 3 citations on WOS.

Králová, J., Račková L., Pekarová M., Kubala, L., Nosál R., Jančinová V., Číž M., Lojek A. (2009): The effects of H₁-antihistamines on the nitric oxide production by RAW 264.7 cells with respect to their lipophilicity. Int. Immunopharmacol.  9(7-8):990-5.IF 2,2.

(b) The average contribution of the department is ~ 75%.

(c) Based on our results, H1-antihistamines differentially modulate reactive oxygen and nitrogen species production by phagocytes. The modulation of nitric oxide production is caused by the downregulation of iNOS protein expression and/or the iNOS protein activity. Interestingly, the inhibition of reactive oxygen species production is caused at least partly via non H1-receptor pathway. We suggest that antihistamines interfering with oxidative burst should be usedpreferentially under pathological conditions accompanied by the overproduction of reactive oxygen species.On the other hand, antihistamines which did not interfere with microbicidal mechanisms of leukocytes could be applied preferentiallyin situations where the organism has to cope with an infection.

[12] Oxidatively modified collagen lost its activating properties towards platelets and phagocytes

(a) Číž M, Čížová H, Pejchalová K, Jančinová V, Goshev I, Mihaylova B, Nosáľ R, Lojek A. (2009): Interactions of oxidatively modified calf skin collagen with platelets and phagocytes. Neuro Endocrinol Lett 30 (Suppl 1), 128-132. IF 1,4.

(b) The authors from the department contributed 70% to the study related to the oxidative modification of collagen, its characterisation and its effects on professional phagocyte activity.

(c) The effects of non-modified and oxidatively modified calf skin collagen type I on platelet aggregation in the framework of a general hypothesis that collagen, platelets and professional phagocytes cooperate to modulate the oxidative burst of phagocytes and the extent of oxidative stress were examined. Oxidative modification of collagen samples was characterized by a decrease in denaturation transition temperature, evidencing a significant destruction of the collagen. All oxidatively modified collagen samples, independent of the oxidation treatment applied, lost their platelet-aggregating and phagocyte oxidative burst-inducing activity. The results suggest that reactive oxygen species were able to modify collagen, a major constituent of the extracellular matrix. On the other hand, oxidatively modified collagen lost its activating properties towards platelets and phagocytes.

[13] The small molecular antioxidants contribute to the plasma total peroxyl radical-trapping antioxidant capacity during intestinal ischemia/reperfusion, but insufficiently to prevent the progression of lipid peroxidation and oxidative stress

(a) Čížová H., Papežíková I., Kubala L., Lojek A., Číž M. (2006): Increased antioxidant capacity of serum did not prevent lipid peroxidation in the intermittent ischemia/reperfusion of rat small intestine. Dig Dis Sci 51(4), 657– 661. IF 1,583; 3 citations according to WOS

Papežíková I., Lojek A., Čížová H., Číž M. (2006): Alterations in plasma antioxidants during reperfusion of the ischemic small intestine in rats. Res Vet Sci 81(1), 140-147. IF 1,384; 3 citations according to WOS

Nosáľ R., Jančinová V., Nosáľová V., Perečko T., Číž M., Lojek A. (2009): Pheniramines and oxidative burst of blood phagocytes during ischaemia/reperfusion. Inflamm Res 58(Suppl 1), 66-67. IF 1,457.

(b) The authors from the department contributed 100% to the part of the study related to the changes in plasma antioxidative capacity during ischemia/reperfusion, and 20% to the part of the study related to the evaluation of the effects of pheniramines on the oxidative burst of phagocytes during ischemia/reperfusion.

(c) Changes in small molecular antioxidants were monitored in a model of small intestinal ischemia/reperfusion in Wistar rats with the aim to evaluate their possible role in ischemic preconditioning. An increase in total antioxidant capacity observed after 60-minute ischemia was prevented in preconditioned animals. There was an overall increase in ascorbic acid and uric acid concentrations observed in comparison to intact control, but this increase was not sufficient to prevent lipid peroxidation in serum and intestinal mucosa. We can conclude that even such a large increase in antioxidant capacity is not sufficient to prevent the progression of lipid peroxidation and oxidative stress. We also observed that pheniramines may improve rat mesenteric ischemia/reperfusion by depressing H₁ receptor-mediated effects of histamine on the microcirculation and/or by decreasing oxidative burst of blood phagocytes; the latter may represent a receptor-independent action of pheniramines.

[14] Polyunsaturated fatty acids influence enzymes responsible for the reactive species production by phagocytes

(a) Ambrozova G., Pekarova M., Lojek A. (2010): The effect of polyunsaturated fatty acids on the generation of reactive oxygen and nitrogen species by RAW 264.7 cells.Eur. J. Nutr., 49(3), 133 – 139.IF 1,9;  1 citation on WOS.

(b) All authors are from the department.

(c) We showed that higher concentrations of polyunsaturated fatty acids of both ω-3 and ω-6 groups can inhibit reactive oxygen and nitrogen formation by stimulated macrophages. The expression of inducible nitric oxide can also be inhibited. This effect together with the absence of antioxidant activity and cytotoxic properties indicates that polyunsaturated fatty acids influence enzymes responsible for the reactive species production.

[15] Endothelial dysfunction - mechanisms and their modulation

(a) Papežíková I., Pekarová M., Chatzopoulou M., Nicolaou I., Demopoulos V., Lojek A.: Aldose reductase inhibition abolishes glucose-induced endothelial dysfunction. Neuro Endocrinol Lett. 2008 Oct 11;29(5):775-778.IF 1,4; 2 citations on WOS.

Papezikova I, Pekarova M, Lojek A, Kubala L. (2009): The effect of uric acid on homocysteine-induced endothelial dysfunction in bovine aortic endothelial cells. Neuro Endocrinol Lett. 2009 Dec 21;30(Suppl):112-115.IF 1,4.

(b) Department contributed ~ 85% (JMC2004 was prepared and provided by the partner).

(c) The effect of a novel aldose reductase inhibitor JMC2004 on hyperglycemia-induced endothelial dysfunction was studied. Bovine aortic endothelial cells (BAEC) were treated with glucose, JMC2004 or glucose + JMC2004 for 24 h. Then the cells were stimulated with calcium ionophore A23187 and NO production was measured.  The incubation of the cells with glucose strongly diminished calcium ionophore-induced response. Aldose reductase inhibition with JMC2004 was able to abolish hyperglycemia-induced endothelial dysfunction in bovine aortic endothelial cells. We also proved, that uric acid decreased nitric oxide bioavailability and enhanced superoxide generation in A23187-stimulated BAEC.

[16] Oxidative stress in patients with severe diseases.

(a) Pavelková M., Kubala L., Číž M., Pavlík P., Wagner R., Slavík J., Ondrášek J., Černý J., Lojek A. (2006): Blood phagocyte activation during open heart surgery with cardiopulmonary bypass. Physiol. Res., 55, 165-173.IF 2,1; 3 citations on WOS.

Soska V, Ciz M, Kubala L, Sobotova D, Lojek A. Phagocyte-derived oxidants and plasma antioxidants in haemodialysed patients. Scand J Clin Lab Invest. 2007; 67(3): 343-51.IF 1,2; 1 citation on WOS.

Podborska M, Sevcikova A, Trna J, Dite P, Lojek A, Kubala L. (2009): Increased markers of oxidative stress in plasma of patients with chronic pancreatitis. Neuro Endocrinol Lett. 2009 Dec 21;30:116-120.IF 1,4.

(b) The authors from the department contributed ~60% (sample collection was performed in the hospital, the majority of experimental work at the department).

(c) In collaboration with FacultyHospitalin Brnowe showed that open heart surgery with a cardiopulmonary bypass is associated with a rapid and pronounced activation of blood phagocytes and complement activation. Similarly, the higher generation of phagocyte-derived oxidants and the decline in plasma antioxidative properties after haemodialysis was observed in patients with chronic renal failure. It confirms insufficient antioxidant defence. The significantly increased markers of oxidative stress were also present in patients with chronic pancreatitis. It may play a role in initiation and maintenance of inflammation within the pancreatic tissue.

Department 7     Structure and Dynamics of Nucleic Acids (DSDNA)

[1]  Quantum-chemical analysis of the nature and magnitude of B-DNA base stacking.

(a) J. Šponer, P. Jurečka, I. Marchan, F. J. Luque, M. Orozco, P. Hobza: Nature of base stacking. Reference quantum chemical stacking energies in ten unique B-DNA base pair steps. Chemistry - A European Journal 12, 2854-2865, 2006, IBP: corresponding author, first author, IF 5.5; 77 citations on WOS.

(b) The work was done by 80 % by J. Šponer, who did the primary design, executed most calculations and has completely written the ms. Inclusion of the cooperating laboratories was partially necessitated simply because of the lack of adequate equipment in Brno, as the cooperating laboratories provided all the computers.

(c) This study presents currently the most accurate quantum-chemical calculations on energetics of base stacking in B-DNA. The paper has been widely accepted as standard values and utilized for parametrization of other methods in many laboratories. The data is also of primary importance to understand the basic physical chemistry of base stacking and helps to interpret thermodynamics experiments.

[2] Molecular dynamics simulations of RNA Kink-turn motifs.

(a) F. Rázga, J. Koča, J. Šponer and N. B. Leontis: Hinge-Like Motions in RNA Kink-Turns: The Role of the Second A-Minor Motif and Nominally Unpaired Bases Biophysical Journal  88, 2005, 3466-3485, IBP: joint corresponding author, first author, IF 4.7; 50 citations on WOS.

K. Réblová, F. Rázga, W. Li, H. Gao, J. Frank, J. Šponer: Dynamics of the base of ribosomal A-site finger revealed by molecular dynamics simulations and Cryo-EM. Nucleic Acids Research 38, 2010, 1325-1340, IBP: joint corresponding author, first author, IF 6.9

(b) The project was done by 75 % by researchers from IBP, where all computations were executed and major part of writing was done. The idea of the project was formulated jointly by J. Šponer, N.B. Leontis and J. Frank.

(c) RNA based molecular machines like ribosome work in the regime of thermal fluctuations. The thermal fluctuations provide energy for the directional movements which are rectified by chemical energy. Molecular simulations discovered that recurrent RNA building blocks Kink-turns can act as flexible molecular elbows mediating large-scale movements of the ribosome during protein synthesis. In direct cooperation with leading RNA structural bioinformatics and cryo-electron microscopy laboratories, this suggestion has been documented for Kink-turns occurring at the base of ribosomal GTPase associated center and A-site finger, which are known dynamical ribosomal elements. Molecular simulations represent a useful complement to experiments, because the currently available structural experiments reveal only averaged static structures of the molecular machines.

[3] Creation of database of accurate molecular structures and energies for molecular clusters.

P. Jurečka, J. Šponer, J. Černý and P. Hobza: Benchmark database of accurate (MP2 and CCSD(T) complete basis set limit) interaction energies of small model complexes, DNA base pairs, and amino acid paris. PhysicalChemistry Chemical Physics 8, 1985-1993, 2006, IBP: co-author, IF 4.1, 261 citations on WOS.

(b) J. Šponer contributed ~10% to this work. Larger involvement was hampered by lack of adequate equipment in Brno.

(c) This study presents an unprecedented set of accurate structure-energy data for a wide range of molecular complexes, with the aim to provide reference data of experimental-quality accuracy for parametrization of all other classes of computational methods. The study has major impact in the field and became immediately an established standard which has been used as reference dataset for parametrizaton and verification of other methods in dozens of laboratories around the globe. Note that, in contrast to experiments, modern quantum chemical methods can simultaneously reveal structures and energies of molecular clusters, providing thus the much needed direct structure-energy relationship.

[4] Reparametrization of the  main empirical force field for molecular simulations of DNA.

(a) A. Pérez, I.Marchán, D. Svozil, J. Šponer, T. E. Cheatham, III, C.A.Laughton, M. Orozco: Refinement of the AMBER Force Field for Nucleic Acids: Improving the Description of /Conformers. Biophysical Journal 2007 92: 3817-3829, IBP: co-author, IF 4.7, 128 citation on WOS.

(b) J. Šponer contributed ~10% to this work, mainly by helping to design the project. Larger involvement was hampered by lack of adequate equipment in Brnowhich was necessary for testing of the new force field. Thus although J. Šponer substantially contributed to the design he could not execute the project.  

(c) Relevance of molecular dynamics simulations critically depends on the quality of the molecular mechanical force field describing the atomistic structure of the biopolymers. This work is a seminal methodological contribution in the field of molecular simulations of nucleic acids, as it presents major re-parametrization of the leading force field for nucleic acids. The new force field corrects a detrimental imbalance of the original force field. The new force field became immediately the new standard for DNA simulations and for the first time guarantees fully stable simulations of B-DNA. 

[5] Molecular dynamics characterization of Hepatitis Delta Virus ribozyme.

(a) M. V. Krasovska, J. Sefcikova, K. Réblová, B. Schneider, N. G. Walter, J. Šponer: Cations and hydration in catalytic RNA: Molecular dynamics of the hepatitis delta virus ribozyme. Biophysical Journal 91, 2006, 626-638. IBP: joint corresponding author, first author, IF 4.7, 45 citations on WOS.

M.A. Ditzler, M. Otyepka, J. Šponer, N.G. Walter: Molecular dynamics and quantum mechanics of RNA: conformational and chemical change we can believe in. Accounts of Chemical Research, 43, 2010, 40-47.  IBP: joint corresponding author, IF 12.2.

(b) The contribution of Brnolaboratory is ~50%. The design was done jointly with N.G. Walter, writing was done jointly with N.G. Walter, most calculations were done in Brno.

(c) Hepatitis Delta Virus ribozyme (HDVr) is one of the best studied small catalytic RNA molecules. HDVr catalyzes its own self-cleavage. Despite two decades of intense research, its exact catalytic mechanism is still debated. This work for the first time applied advanced molecular simulations to HDVr. The simulations provided unique insights into structural dynamics of this small catalytic RNA, investigated structural features that could lead to general base catalytic mechanism of this ribozyme and discovered binding of monovalent ions in the catalytic pocket of the ribozyme, which was a year later confirmed by X-ray crystallography. The study is important for basic understanding of the principles of RNA catalysis.

[6] Quantum-chemical evaluation of RNA base pairs.

(a) J.E. Šponer, N.Špačková, P. Kulhánek, J. Leszczynski, J. Šponer: Non-Watson-Crick base pairing in RNA. Quantum chemical analysis of the cis Watson-Crick/sugar edge base pair family. Journal of Physical Chemistry B 109, 2005,  2292-2301, IBP: corresponding author, first author, co-author, IF 4.2, 29 citations on WOS.

J.E. Šponer, N.Špačková, J. Leszczynski, J. Šponer:Principles of RNA base pairing: Structures and energies of the trans Watson-Crick/sugar edge base Paris. Journal of Physical Chemistry B 109, 11399-11410, 2005, IBP: corresponding author, first author, co-author, IF 4.2, 26 citations on WOS.

(b) This work was done 95% in Brno, where design of the project, majority of calculations and all writing were executed.

(c) In contrast to DNA, RNA molecules are capable to form wide range of complex molecular interactions using the 2’OH group of ribose that is absent in DNA. Such extended base pairs are of primary importance for building up of the complex RNA molecular architectures. This work provides the very first quantum-chemical characterization of RNA base pairing patterns with direct involvement of ribose. The calculations provide basic physical chemistry characterization of such interactions. 

[7] Application of modern computational tools to nucleic acids.

(a) J. Šponer, F. Lankaš, Eds., Computational studies of RNA and DNA. Dordrecht: Springer, 2006, 630 pages. IBP: Editor.

(b) The editorial work was done by 75% by J. Šponer

(c) Representative book summarizing the current status in quantum chemical, simulation and mesoscopic modeling studie of nucleic acids, with contribution of basically all leading groups in the respective fields.

[8] Quantum chemical studies of aromatic base stacking.

(a) J. Šponer, K. E. Riley, P. Hobza: Nature and magnitude of aromatic stacking of nucleic

acid bases. Physical Chemistry Chemical Physics 10, 2008, 2595-2610. IBP: first author, IF 4.1, 40 citations on WOS.

(b) Contribution to the writing of the review by J. Šponer is ~50%.

(c) Key review article summarizing current status of quantum chemical studies of base stacking, based primarily on the work of the authors.

[9] Molecular simulations of RNA molecules.

(a) S.E. McDowell, N. Špačková, J. Šponer, N.G. Walter: Molecular dynamics simulations of RNA: An in silico single molecule approach.Biopolymers, 2006, 85, 169-184, IBP: joint corresponding author, co-author, IF 2.8, 38 citations on WOS.

(b) Contribution to the writing of the review by J. Šponer and N. Špačková is ~45%.

(c) Key review article summarizing current status of molecular simulations of RNA molecules, where the authors represent the leading groups.

[10] Molecular simulations of quadruplex DNA.

(a) J. Šponer, N. Špačková: Molecular dynamics simulations and their application to four-stranded DNA. Methods, 43, 2007, 278-290, IBP: complete contribution, IF 3.3, 18 citations on WOS.

(b) 100% contribution by IBP laboratory.

(c) Key review article summarizing current status of molecular simulations of four-stranded nucleic acids (guanine quadruplex and i-DNA) where the IPB group is established international leader.

[11] Molecular dynamics simulations of Hairpin ribozyme.

(a) M.M Rhodes, K. Réblová, J. Šponer, N.G. Walter: Trapped water molecules are essential to structural dynamics and function of a ribozyme. Proceedings of the National Academy of Sciences of the USA103, 2006, 13380-13385, IBP: joint corresponding author, co-author, IF 9.4, 35 citations on WOS.

(b) The overall contribution by J. Šponer and K. Réblová is ~30%, and includes contribution to the project design, smaller part of the actual calculations, providing know-how for the analysis, and some contribution to the writing. 

(c) Hairpin ribozyme (HrRz) is one of the best studied small catalytic RNA molecules that catalyze their own self-cleavage. Despite two decades of intense research, its exact catalytic mechanism is still debated. This work for the first time applied advanced molecular simulations to HrRz. The study discovered (simultaneously with X-ray crystallography experiments) string of water molecules lining up in the closed catalytic pocket of HrRz. The study suggests that the water molecules may be directly involved in catalysis.

[12] Classification of base – phosphate interactions in ribosome.

(a) C.L. Zirbel, J.E. Šponer, J. Šponer, J. Stombaugh, N.B. Leontis: Classification and energetics of the base-phosphate interactions in RNA. Nucleic Acids Research, 37, 2009, 4898-4918, IBP: joint first author, co-author, IF 6.9.

(b) The contribution by J. Šponer and J.E. Šponer is altogether ~40%, and consisted of designing and execution all theoretical calculations, refinement of the final classification, and some other contribution to the interpretation of the data and writing.

(c) Extension of RNA base pairing classification to include base-phosphate interactions, the first joint study applying tools of quantum chemistry and structural bioinformatics to a major class of RNA molecular interactions. The study reveals that i) ~12% of nucleotides in ribosome participate in base-phosphate hydrogen bonding. ii) These hydrogen bonds are sequence specific and represent a major constraint on evolution of RNA sequences. iii) The predicted binding energies of different types of base-phosphate interactions correlate with the frequency of their occurrence in the evolution.

[13] Quantum chemical studies of chemical reactions and molecular interactions in the prebiotioc period. 

(a) J.E. Šponer, B. G. Sumpter, J. Leszczynski, J. Šponer, M. Fuentes-Cabrera: Theoretical Study on the Factors Controlling the Stability of the Borate Complexes of Ribose, Arabinose, Lyxose, and Xylose. Chemistry - A European Journal 14, 2008, 9990-9998. IBP: joint corresponding author, first author, co-author, IF 5.5.

J.E. Šponer, Á. Vázquez-Mayagoitia, B.G. Sumpter, J. Leszczynski, J. Šponer, M. Otyepka, P. Banáš, M. Fuentes-Cabrera: Theoretical study on the intermolecular interactions of potentially primordial base pair analogues, Chemistry - A European Journal 16, 2010, 3057-3065,  IBP: joint corresponding author, first author, co-author, IF 5.5.  

(b) The contribution by J.E. Šponer is altogether ~70%, and consisted of designing of the project together with the cooperating USlab, execution of 90% of computations, interpretation of the data and major part of writing, together with J. Šponer for the second study. 

(c) Application of quantum-chemical methods to study compounds relevant to the prebiotic synthesis of the components of nucleic acids. The first study discusses the prebiotic stabilization of aldopentoses via complexation with borates and shows that, in agreement with experimental findings, ribose forms the most stable sugar-borate complex among the four aldopentoses. This is due to a fortuitous interplay of inter- and intramolecular  hydrogen bonds as well as steric and electrostatic interactions present in the ribose-borate complexes.  The second paper analyzes the intermolecular interactions stabilizing the duplexes of various primordial nucleobase analogues. We show that if the acid/base properties of the bases have ever had a role at the prebiotic selection of nucleobases, it is not because of their impact on the intrinsic stacking and hydrogen bonding properties.

[14] Molecular dynamics simulation of elasticity of RNA motifs.

(a) N. Špačková, J. Šponer: Molecular Dynamics simulations of Sarcin Ricin rRNA motif.  Nucleic Acids Research 34, 697-708, 2006 IBP all authors, IF 6.9, 34 citations on WOS.

K. Réblová, E. Fadrné, J. Sarzynska, T. Kulinski, P. Kulhánek, E. Ennifar, J. Koča, J. Šponer: Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by Molecular Dynamics. Biophysical Journal 93, 2007, 3932-3949, IBP: corresponding author, first author, co-author, IF 4.7, 13 citations on WOS.

(b) Contribution to this project by the IBP author’s team is ~95% and includes the design, vast majority of computations, major interpretation of the data and major part of writing.

(c) The study demonstrates that distinct RNA building blocks are associated with specific molecular flexibilities which can be captured by the simulation technique. In many cases the elasticity is important for function and is not apparent from structural experiments. It is part of the effort to catalogue elasticity of RNA modular blocks using molecular simulation technique.

Department 8     CD Spectroscopy of Nucleic Acids (DSNA)

[1]Arrangement of long human telomere DNA molecules

(a) Vorlickova, M., Chladkova, J., Kejnovska, I., Fialova, M. and Kypr, J. (2005) Guanine tetraplex topology of human telomere DNA is governed by the number of (TTAGGG) repeats.Nucleic Acids Research,33, 5851-5860. IF= 6.968, 52 citations in WOS

Vorlickova, M., Chladkova, J., Kejnovska, I.and Kypr, J. (2007) Quadruplexes of     human telomere DNA. Journal of Biomolecular Structure & Dynamics, 24, 710.

(b) All the authors are from IBP, 100%.

(c) Only short DNA fragments can be studied by X-ray analysis or by NMR. Therefore, atomic structures of quadruplexes formed by only 22 – 25 nucleotides have been published while, however, the telomere DNA is thousands of nucleotides long. We have shown that the way of telomere DNA folding into quadruplex depends on the number of the basic motif repeats. Surprisingly, the quadruplex stability decreases with the motif length. On the basis of a stepwise decrease of enthalpy values in dependence on the repeat number we have suggested that long telomere DNA molecules have, similarly to nucleosomes, a beads-on-a-string–like arrangement. This finding is important as it enables an explanation for the gradual shortening of telomere DNA in the course of ageing.

[2]  CD spectroscopy – a useful tool for studies of nucleic acids

(a) Kypr, J., Kejnovska, I., Renciuk, D. and Vorlickova, M. (2009) Circular dichroism     spectroscopy and conformational properties of DNA. Nucleic Acids Research, 37, 1713-1725. IF= 6.968, by now 18 citations on WOS, all authors from IBP.

Vorlickova, M., Kypr, J. and Sklenar, V. (2005) Nucleic Acids: (c)Spectroscopic Methods,Encyclopedia of Analytical Science. Elsevier, Oxford, vol. 6, 2nd ed., 391-399. (corresponding author, first author, and co-author)

Doneux, T., Bouffier, L., Mello, L.V., Rigden, D.J., Kejnovska, I., Fernig, D.G., Higgins, S.J. and Nichols, R.J. (2009) Molecular dynamics and electrochemical investigations of a pH-responsive peptide monolayer.J. Phys. Chem.,113, 6792-6799. (co-author)

Necas, M., Dostal, J., Kejnovska, I., Vorlickova, M. and Slavik, J. (2005) Molecular and crystal structures of (+)-homochelidonine, (+)-chelamine and (-)-norchelidonine. Journal of Molecular Structure, 734,1-6. (two co-authors)

(b) The contribution of the laboratory is 100 %, 80 %, 10 %, and 20 %, respectively.

(c) The paper reviews studies that provided important information about conformational properties of DNA using circular dichroic spectroscopy. The conformational properties include the B-family of structures, A-form, Z-form, guanine quadruplexes, cytosine i-tetraplexes, triplexes, and other structures. The paper is especially valuable for beginning researchers and students.

The paper published in the Encyclopedia describes the principles of various spectroscopic methods and summarizes their advantages and pitfalls. It may be useful for researchers from distinct fields of science.

[3] Structure of the human telomere DNA sequence

(a) Renciuk, D., Kejnovska, I., Skolakova, P., Bednarova, K., Motlova, J. and Vorlickova, M. (2009) Arrangements of human telomere DNA quadruplex in physiologically relevant K⁺ solutions.Nucleic Acids Research, 37, 6625-6634. IF= 6.968, by now 4 citations in WOS(all authors from IBP)

(b) The contribution of the laboratory is 100%.

(c) DNA at the very ends of the chromosomes (i.e. in the telomeric region) was shown to adopt quadruplex structure, whose formation is related to ageing and carcinogenesis. Quadruplex stabilizing agents suppress proliferation of tumour cells and are thus important for cancer treatment. To enhance the efficacy of these compounds the structure of their target must be known. The arrangement of the human telomeric quadruplex has not yet been unambiguously determined. Our paper has shown that the structure of the quadruplex is polymorphous, which has explained distinctions in structures observed in various laboratories. This paper is important from the medical point of view.

[4]Effect of natural lesions on the structure of the human telomere DNA quadruplex

(a) Tomasko, M., Vorlickova, M. and Sagi, J. (2009) Substitution of adenine for guanine in the quadruplex-forming human telomere sequence G3(T2AG3)3. Biochimie, 91, 171-179. IF= 3.168 (first author and co-author from IBP)  

(b) The initial project of the study of the human telomere DNA structure comes from the IBP laboratory. The idea of the study of the influence of spontaneously occurring DNA lesions on quadruplex formation is a contribution of the collaborating laboratory: The experiments were undertaken in the IBP laboratory and the publications were compiled by both sides. The project was done in ~70 % by researchers from IBP.

(c) The paper is the initial part (a sequel paper was published in 2010 and two following ones were sent for publication) of a project that will study the influence of various lesions on the human telomere DNA structure. Apart from G for A mutations, the project includes a substitution of G by 8-oxoG, which mutation is generated as a consequence of oxidative stress, and, further on, abasic places instead of particular guanines in tetrads, or abasic sites instead of particular adenines in quadruplex loops. Primary structure mutations were intensively studied in DNA duplexes, whereas this project belongs to the pioneering ones that study the influence of lesions on DNA quadruplexes.

[5] Regularities of quadruplex formation

(a)Vorlickova, M., Bednarova, K., Kejnovska, I.and Kypr, J. (2007) Intramolecular and intermolecular guanine quadruplexes of DNA in aqueous salt and ethanol solutions. Biopolymers, 86, 1-10. IF = 2.631

Vorlickova, M., Bednarova, K. and Kypr, J. (2006) Ethanol is a better inducer of DNA guanine tetraplexes than potassium cations. Biopolymers, 82, 253-260(in total 25 citations on WOS).

(b) All the authors are from IBP, 100%.

(c) Sequence analyses, including those of ours, have shown that the human genome contains tens of hundreds of regions formed by sequences capable of forming quadruplexes. They particularly occur in gene promoters and, in a number of cases, were shown to control gene expression. For the possibility of searching for quadruplex along genomes, it is necessary to know the rules of their formation. The two papers contribute to uncovering these rules, which concerns conditions of quadruplex formation as well as suitable primary structure of DNA.

[6] Possibilities of quadruplex structure modifications: RNA-DNA nucleotide substitutions

(a) Vondrusková, J., Kypr, J., Kejnovska, I., Fialova, M. and Vorlickova, M. (2008) Guanine   quadruplex formation by RNA/DNA hybrid analogs of Oxytricha telomere G₄T₄G₄ fragment. Biopolymers, 89,797-806.

Vondruskova, J., Kypr, J., Kejnovska, I., Fialova, M. and Vorlickova, M. (2008) Role of loops in the guanine quadruplex formation by DNA/RNA hybrid analogs of G₄T₄G₄. Int. J. Biol. Macromol., 43,463-467.

(b) All the authors are from IBP, 100%.

(c) The exchange of even a single deoxyribonucleotide for ribonucleotide and vice versa, both in the quadruplex core and in quadruplex loops changes distinctly the type of quadruplex folding. The ability to control quadruplex structure will allow further studies of biophysical and biological properties of the various folding topologies. These findings are useful for engineering particular quadruplex structures in different quadruplex-forming sequences. IF= 2.631, and 2.275 (the number of citations lower than 10 is not indicated).

[7] Quadruplex of the thrombin aptamer

(a) Fialova, M., Kypr, J. and Vorlickova, M. (2006) The thrombin binding aptamer   GGTTGGTGTGGTTGG forms a bimolecular guanine tetraplex.Biochem. Biophys. Res. Commun., 344, 50-54. IF = 2.823, in total 19 citations in WOS

Kejnovská, I., Kypr, J. and Vorlíčková, M. (2007) Oligo(dT) is not a correct native PAGE marker for single-stranded DNA. Biochem. Biophys. Res. Commun., 353, 776-779.

(b) All the authors are from IBP, 100%.

(c) The aptamer binds thrombin and as such represents a useful tool for therapeutic purposes. In the literature, the aptamer is generally taken as a prototype of an intramolecular guanine quadruplex of DNA. We, however, show that the thrombin aptamer quadruplex is formed by two molecules. Only an extension of the aptamer by a sequence containing further guanines, or an elongation of loop regions, cause that quadruplex folding is intramolecular. This change probably also changes the functional properties of the aptamer. The previous conclusions on thrombin structure followed from erroneously selected electrophoretic markers. This is described in the second publication.

[8] Anomalous structures of microsatellite DNA repeats

(a) Kejnovska, I., Kypr, J., Vondruskova, J. and Vorlickova, M. (2007) Towards a better understanding of the unusual conformations of the alternating guanine-adenine repeat strands of DNA. Biopolymers, 85, 19-27.

Kypr, J., Kejnovska, I.and Vorlickova, M. (2007) Conformations of DNA strands containing GAGT, GACA, or GAGC tetranucleotide repeats. Biopolymers, 87, 218-224.

Zemanek, M., Kypr, J. and Vorlickova, M. (2005) Conformational properties of DNA containing (CCA)_nand (TGG)_ntrinucleotide repeats. Int. J. Biol. Macromol. 36, 23-32.

(b) All the authors are from IBP, 100%.

(c) A prevailing part of the non-coding genome regions is formed by repetitive sequences. Just these regions are characteristic of the ability to form anomalous DNA structures, distinct from classical W-C models, which enables their recognition by proteins. The (GA)n sequences form several anomalous structures and the first paper contributes to revealing the role of the two purines, G and A, in the particular structures. The second paper investigates the influence of pyrimidine neighbours on the formation of the structures. The (CCA)n trinucleotide and (TGG)n in the complementary strand belong to the most frequent microsatellites of the human genome. The C-rich strand is able to adopt i-quadruplexes, and the G-rich strand, guanine quadruplexes. IF= 2.631, 2.631 and 2.275, respectively.

[9] Conformational properties of (CGG) repeats associated with the Fragile X Syndrome

(a) Renciuk, D., Zemanek, M., Kejnovska, I., and Vorlickova, M. (2009) Quadruplex-forming properties of FRAXA (CGG) repeats interrupted by (AGG) triplets. Biochimie, 91, 416-422.

Vorlickova, M., Renciuk, D., Fojtik, P., Zemanek, M. and Kejnovska, I.(2007) Conformational properties of trinucleotide repeats associated with human neurodegenerative diseases. J. Biomol. Struct. Dynam., 24, 745.

(b) All the authors are from IBP, 100%.

(c) Trinucleotide motifs expand in genomes while this expansion is associated with serious neurological diseases. We studied (CAG)n, (CTG)n (NAR 1999), (CCG) (NAR 2001), and (CGG)n (NAR 2004). This work is a continuation of our effort to find the structure responsible for the expansion. We have demonstrated that this structure is not a quadruplex as was suggested by other authors. On the contrary, quadruplex formation is stabilized by (AGG) triplets, which interrupt the CGG runs in genomes of healthy individuals and hinder their expansion. These studies contribute to discovering the mechanism of the pathological trinucleotide motif expansions.IF= 3.168 and 1.332, resp.    

[10] Detection of the B-A transition along genomic DNA

(a)Nejedly, K., Chladkova, J., Vorlickova, M., Hrabcova, I.and Kypr, J. (2005) Mapping the B-A conformational transition along plasmid DNA. Nucleic Acids Research, 33, e5.

Nejedlý, K., Chládková, J. and Kypr, J. (2007) Photochemical probing of the B-A conformational transition in a linearized pUC19 DNA and its polylinker region. Biophysical Chemistry, 125, 237-246.

(b) All the authors are from IBP, 100%.

(c) The A-form is adopted by RNA and the B-A transition is a basic manifestation of the conformational polymorphism of DNA. We have developed a new photochemical method to study the transition, which is applicable in genomic DNA studies. IF = 6.968 and 2.133. Number of citations: 13.

[11] The longest blocks of (G+C) and (A+T) in the human genome

(a) Hrabcova, I.and Kypr, J. (2007) The longest (A+T) and (G+C) blocks in the human and other genomes. Journal of Biomolecular Structure and Dynamics 25, 337-345.

(b) This result has in 100% been achieved in the IBP.

(c) Bioinformatic analysis has revealed that in genomes there are long blocks exclusively composed of guanine and cytosine, or of adenine and thymine. We found that such blocks are very frequent and extremely long. Their length even exceeds one kilobase. Randomized human genome sequences do not contain such blocks at all. It is interesting that the human and chimp genomes much differ as far as the blocks are concerned. IF = 1.332.

[12] DNA fragment expansion during PCR

(a) Vondruskova J., Parizkova, N. and Kypr, J. (2007) Factors influencing DNA expansion in the course of polymerase chain reaction. Nucleosides, Nucleotides and Nucleic Acids 26, 65-82.

(b) This result has in 100% been achieved in the IBP.

(c) Some DNA regions expand in the human genome. In some cases the expansion has serious pathological consequences. We have simulated the expansion by PCR of DNA fragments having 6-40 nucleotides in length. It was found that some fragments expanded into kilobase lengths during the PCR. This phenomenon depended on the base content of the fragment, terminal nucleotides, and other factors.

[13] Computer modelling of the tertiary structures of the human chromosome DNA

(a) Hanzalek, P. and Kypr, J. (2005) DNA in phosphorus atom representation: The heteronomous double helices of poly(dA).poly(dT) and poly(dG).poly(dC) and simulation of the yeast genome and of a human chromosome DNA. Journal of Theoretical Biology, 232, 83-91.IF = 2.490

(b) This result has been obtained exclusively at the IBP.

(c) The database of DNA fragment crystal structures is already so rich that it contains sufficient data to extrapolate information on the level of human chromosome DNA molecules containing millions of nucleotides. We created a computer program that enabled us to simulate tertiary structures of the smallest human chromosome 21 and 22 DNA. The simulations showed that the tertiary structures are very compact and have a characteristic domain structure.

Department 9     Plant Developmental Genetics (DPDG)

[1] Evolution of the plant sex chromosomes

(a) Nicolas M., Marais G., Hykelová V., Janoušek B., Laporte V., Vyskot B., Mouchiroud D., Negrutiu I., Charlesworth D., Moneger F.: A gradual and ongoing process of recombination restriction in the evolutionary history of the sex chromosomes in dioecious plants. - Public Library of Science, Biology 3, 2005, 47-56 [IF 14.662]

Marais, G.A.B., Nicolas, M., Bergero, R., Chambrier, P., Kejnovský, E., Moneger, F., Hobza, R., Widmer, A., Charlesworth, D.:Evidence for degeneration of the Y chromosome in the dioecious plant Silene latifolia. – Current Biology, 18, 2008, 545-549 [IF 11.142]

(b) These two papers surely belong to most important papers recently published in the field of plant sex determination and evolution. They come from our long-term collaboration with a number of foreign labs working in this field – especially LyonUniversity, Universityof Edinburgh, and Universityof Zurich. Since 2006 they have been cited at least 71times.

(c) We have studied four X-Y gene pairs in plants in a group of closely related dioecious species of Silene. Except for a recent rearrangement in S. dioica, the gene order is the same in the X chromosomes of all three species. Silent site divergence between the DNA sequences of the X and Y copies of the different genes increases with the genes’ distances from the PAR. Analysis of amino acid replacements vs. synonymous changes showed that the Y-linked copies appear to be functional in all three species. We have also tested whether the S. latifolia Y chromosome is undergoing genetic degeneration by analyzing seven sex-linked genes. S. latifolia Y-linked genes tend to evolve faster at the protein levelthan their X-linked homologs, and they have lower expressionlevels. Wedetect signs of degeneration in most of the Y-linked genesequences analyzed, similar to those of animal Y-linked genes.

[2]Genetics and origins of dioecy

(a) Mráčková, M., Nicolas, M., Hobza, R., Negrutiu, I., Monéger, F., Widmer, A., Vyskot, B., Janoušek, B.: Independent origin of sex chromosomes in two species of the genus Silene. – Genetics, 179, 2008, 1129-1133 [IF 4.389]

Žlůvová J., Janoušek B., Negrutiu I., Vyskot B.: Comparison of the X- and Y chromosome organisation in Silene latifolia. – Genetics, 170, 2005, 1431-1434 [IF 4.389]

(b) These works come nearly exclusively from our lab and deal with genetics, structure, and organization of the sex chromosomes. These papers present very original data and have been cited at least 27times since 2006.

(c) We show that S. colpophylla is, similarly to S. latifolia, a male heterogametic species, but its sex chromosomes have evolved from a different pair of autosomes than in S. latifolia. The results of our phylogenetic study and mapping of homologs of S. latifoliaX-linked genes indicate that the sex determination system in S. colpophylla evolved independently from that in S. latifolia. We have also compared gene orders on the Silene latifolia sex chromosomes. On the basis of  the deletion mapping we conclude that a part of the Y chromosome has been inverted. The gradient in silent-site divergence suggests that this inversion took place after the recombination arrest in this region. Because recombination arrest events followed by Y chromosome rearrangements also have been found in the human Y chromosome, this process seems to be a general evolutionary pathway.

[3] Laser microdissection of plant chromosomes

(a) Hobza, R., Vyskot, B.: Laser microdissection-based analysis of plant sex chromosomes. – Methods in Cell Biology (Academic Press) 82, 2007, 433-453 (ISSN: 0091-679X)

Hobza, R., Kejnovský, E., Vyskot, B., Widmer, A.: The role of chromosomal rearrangements in the evolution of Silene latifolia sex chromosomes. - Molecular Genetics and Genomics, 278, 2007, 633-638 [IF 2.733]

Hobza, R., Hrušáková, P., Šafář, J., Bartoš, J., Janoušek, B., Žlůvová, J., Michu, E., Doležel, J., Vyskot, B.: MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia. - Theoretical and Applied Genetics, 113, 2006, 280-287 [IF 3.949]

(b) These papers first describe an improved technology forlaser dissection of plant mitotic chromosomes. It was exclusively done in our lab. This technique enables to isolate individual chromosomes and cell compartments. These papers have been cited at least 17times since 2007.

(c) Laser technology enables to handle with specific subcellular structures and individual chromosomes. We present a comprehensive protocol to isolate DNA sequences derived from the sex chromosomes of campion. The protocol includes a versatile technique to prepare metaphase chromosomes from germinating seeds or cultured roots. Such slides can be used for laser chromosome microdissection, FISH mapping, and immunostaining. We also demonstrate some applications of the laser-dissected chromosome template, especially a modified FAST-FISH technique to paint individual chromosomes, and to screen chromosome-specific DNA libraries. We have also combined results from physical mapping using PCR on microdissected arms of the X chromosome, and FISH analysis of a new cytogenetic marker. We suggest that the evolution of the Y chromosome has been accompanied by at least one paracentric and one pericentric inversion.

[4]Global analytical comparison of eukaryotic genomes

(a) Kejnovský, E., Leitch, I.J., Leitch, A.R.: Contrasting evolutionary dynamics between angiosperm and mammalian genomes. Trends in Ecology and Evolution, 24, 2009, 572-582 [IF 17.188]

(b) This is a deep analytical comparison of plant and mammalian genomes substantially done by a fellow from our lab E. Kejnovský. It is a comprehensive review in an excellent journal summarizing our new data in genome research.

(c) Continuing advances in genomics are revealing substantial differences between genomes of major eukaryotic lineages. Because most data are available for angiosperms and mammals, we explore differences between these groups and show that angiosperms have less highly compartmentalized and more diverse genomes than mammals. In considering the causes of these differences, four mechanisms are highlighted: polyploidy, recombination, retrotransposition and genome silencing, which have different modes and time scales of activity. Angiosperm genomes are evolutionarily more dynamic and labile, whereas mammalian genomes are more stable at both the sequence and chromosome level. We suggest that fundamentally different life strategies and development feedback on the genome exist, influencing dynamics and evolutionary trajectories at all levels from the gene to the genome.

[5] Roles of repetitive DNA elements in sex chromosomes

(a) Kejnovský, R., Hobza, R., Čermák, T., Kubát, Z., Vyskot, B.: The role of repetitive DNA in structure and evolution of sex chromosomes in plants. Heredity, 102, 2009, 533-541 [IF 3.616]

Mariotti, B., Manzano, S., Kejnovsky, E., Vyskot, B., Jamilena, M.: Accumulation of Y-specific satellite DNAs during the evolution of Rumex acetosa sex chromosomes. – Molecular Genetics and Genomics, 281, 2009, 249-259 [IF 2.733]

(b) A comprehensive study of the roles of repetitive elements in the evolution of plant sex chromosomes is presented. The first paper comes uniqely from our lab, while the other work is based on collaboration with our Spanish partner (Universityof Almería). Since 2010 the two papers have been cited at least 5times.

(c) Eukaryotic genomes contain a large proportion of repetitive DNA sequences, mostly transposable elements and tandem repeats. These repetitive sequences often colonize specific chromosomal (sex chromosomes, B chromosomes) or subchromosomal (telomeres, centromeres) niches. Sex chromosomes, especially non recombining regions of the Y chromosome, are subject to different evolutionary forces compared with autosomes. In nonrecombining regions of the Y chromosome repetitive DNA sequences are accumulated, representing a dominant and early process forming the Y chromosome, probably before genes start to degenerate. Here we analysed the occurrence and role of repetitive DNA in the Y chromosome evolution in various species with a focus on dioecious plants. We also discuss the potential link between recombination and transposition in shaping genomes.

[6] Accumulation of microsatellites and transposable elements on the sex chromosomes

(a) Kubát, Z., Hobza, R., Vyskot, B., Kejnovský, Z.: Microsatellite accumulation on the Y chromosome of Silene latifolia.  - Genome, 51, 2008, 350-356 [IF 2.205]

Čermák, T., Kubát,Z., Hobza,R., Koblížková,A., Widmer,A., Macas,J., VyskotB.,Kejnovský, E.:Survey of repetitive sequences in Silene latifolia and their distribution in connection with evolution. - Chromosome Research, 16, 2008, 961-976 [IF 3.151] 

(b) These results bring many pieces of evidence that the Y chromosome accumulates microsatellites and other repetitive DNA sequences: this process can represent the first indication for genetic degeneration. This research is mainly done in our lab with minor collaborations of two other labs (Instituteof Plant Molecular Biology, České Budějovice, and Universityof Zurich). These two papers have been cited at least 7times since 2009.

(c) We carried out a global survey of all major types of transposable elements in Silene latifolia, a model species with sex chromosomes that are in the early stages of their evolution. We found that the most common types of elements were the subtelomeric tandem repeat X-43.1 and Gypsy retrotransposons, followed by Copia retrotransposons and LINE non-LTR elements. SINE elements and DNA transposons were less abundant. The localization of elements by FISH revealed that most of the Copia elements were accumulated on the Y chromosome. Surprisingly, one type of Gypsy element, which was similar to Ogre elements known from legumes, was almost absent on the Y chromosome but otherwise uniformly distributed on all chromosomes. Altogether, our data provide an overview of the repetitive sequences in Silene latifolia and revealed that genomic distribution and evolutionary dynamics differ among various repetitive elements.

[7] Chromosomal restructuralizations of sex chromosomes

(a) Hobza, R., Lengerová, M., Svoboda, J., Kubeková, H., Kejnovský, E., Vyskot, B.: An accumulation of tandem DNA repeats on the Y chromosome in Silene latifolia during early stages of sex chromosome evolution. - Chromosoma, 115, 2006, 376-382 [IF 4.202]

(b) These results show how chromosome restructuralizations play a role in the evolution of the Y chromosomes in Silene latifolia. This work was exclusively realized in our lab. This paper have been cited at least 17times since 2007.

(c) The sex chromosomes in the dioecious plant Silene latifolia represent an early stage of evolution in which functional X–Y gene pairs are still frequent. In this study, we characterize a novel tandem repeat called TRAYC, which has accumulated on the Y chromosome in S. latifolia. Its presence demonstrates that processes of satellite accumulation are at work even in this early stage of sex chromosome evolution. The presence of TRAYC in other species of the Elisanthe section suggests that this repeat had spread after the sex chromosomes evolved but before speciation within this section. TRAYC possesses a palindromic character and a strong potential to form secondary structures, which could play a role in satellite evolution. TRAYC accumulation is most prominent near the centromere of the Y chromosome. We propose a role for the centromere as a starting point for the cessation of recombination between the X and Y

[8] Retroelements and gene conversion

(a) Kejnovský, E., Hobza, R., Kubát, Z., Widmer, A., Marais, G.A.B., Vyskot, B.: High intrachromosomal similarity of retrotransposon long terminal repeats: evidence for homogenization by gene conversion on plant sex chromosomes? – Gene, 390, 2007, 92-97 [IF 2.699]

Kejnovský, E., Kubát, Z., Macas, J., Hobza, R., Mráček, J., Vyskot, B.: Retand: a novel family of gypsy-like retrotransposons harboring an amplified tandem repeat. - Molecular Genetics and Genomics, 276, 2006, 254-263 [IF 2.733]

(b) These papers reveal peculiar activities of some retroelements in plant genomes. This work was largely realized in our lab with a minor contribution from foreign and domestic colleagues. Since 2007 these papers were cited at least 28times.

(c) Retrotransposons are ubiquitous in the plant genomes and are responsible for their plasticity. Recently, we described a novel family of gypsy-like retrotransposons, named Retand, in the dioecious plant Silene latifolia possessing evolutionary young sex chromosomes of the mammalian type. Here we have analyzed long terminal repeats (LTRs) of Retand that were amplified from laser microdissected X and Y sex chromosomes and autosomes of S. latifolia. A majority of X and Y-derived LTRs formed a few separate clades in phylogenetic analysis reflecting their high intrachromosomal similarity. Moreover, the LTRs localized on the Y chromosome were less divergent than the X chromosome-derived or autosomal LTRs. These data can be explained by a homogenization process, such as gene conversion, working more intensively on the Y chromosome.

[9] Mapping of the sex chromosomes

(a) Yu, Q., Hou, S., Hobza, R., Feltus, F.A., Wang, X., Jin, W., Skelton, R.L., Blas, A., Lemke, C., Saw, J.H., Moore, P.H., Alam, M., Jiang, J., Paterson, A.H., Vyskot, B., Ming, R.: Chromosomal location and gene paucity of the male specific region on papaya Y chromosome. – Molecular Genetics and Genomics, 278, 2007, 177-185 [IF 2.733]
Matsunaga, S., Sabine Lebel-Hardenack, S., Kejnovský, E., Vyskot, B., Grant, S.R., Kawano, S.: An anther- and petal-specific gene SlMF1 is located on plant sex chromosomes. - Genes and Genetic Systems 80, 2005, 395-401 [IF 1.265]

(b) These papers intend to physically map the sex chromosomes of papaya and white campion. In the former case the work is based on collaboration with the University of Honolulu (USA), but cytogenetic analyses were done in our lab. The second paper was realized in collaboration with the Universityof Tokyo. These two papers have been cited at least 18times since 2007.

(c) We investigated the chromosomal location of papaya’s small male specific region of the hermaphrodite Y (Yh) chromosome (MSY) and its genomic features. We conducted chromosome fluorescence in situ hybridization mapping of Yh-specific bacterial artificial chromosomes (BACs) and placed the MSY near the centromere of the papaya Y chromosome. Then we sequenced five MSY BACs to examine the genomic features of this specialized region, which resulted in the largest collection of contiguous genomic DNA sequences of a Y chromosome in flowering plants. Extreme gene paucity was observed in the

papaya MSY with no functional gene identified in 715 kb MSY sequences. A high density of retroelements and local sequence duplications were detected in the MSY that is suppressed for recombination. Location of the papaya MSY near the centromere might have provided recombination suppression and fostered paucity of genes in the male specific region of the Y chromosome.

[10] Accumulation of plastid DNA sequences on the Y chromosome

(a) Kejnovský, E., Kubát, Z., Hobza, R., Lengerová, M.,Sato, S., Tabata, S., Fukui, K., Matsunaga, S., Vyskot, B.: Accumulation of chloroplast DNA sequences on the Y chromosome of Silene latifolia. - Genetica, 128, 2006, 167-175 [IF 2.034]

(b) This paper demonstrates accumulation of plastid DNA sequences as an example of horizontal gene transfer. Both papers come from our long-term collaboration with the Universityof Tokyo. The work was largely done in our lab, only the BAC sequncing was realised in Japan. Since 2007 this paper has been cited at least 15times.

(c) Silene latifolia is a model dioecious plant with heteromorphic sex chromosomes. Theoretical models propose an accumulation of repetitive DNA sequences in nonrecombining parts of the Y chromosome. In this study, we isolated a BAC7H5 clone preferentially hybridizing to the Y chromosome of S. latifolia. Sequence analysis revealed that this BAC7H5 contains part of the chloroplast genome, indicating that these chloroplast sequences have accumulated on the Y chromosome and also may contribute to its large size. We constructed Y chromosome- and X chromosome-specific libraries and screened them to find Y- and/or X-linked copies of chloroplast sequences. Sequence analysis revealed higher divergence of a non-genic region of the chloroplast sequences located on the Y chromosome while genic regions tested showed only very low (max 0.9%) divergence from their chloroplast homologues.

[11] Survey of plant sex determination

(a) Hobza, R., Vyskot, B.: Sex chromosomes in plants. In: FLORICULTURE, ORNAMENTAL AND PLANT BIOTECHNOLOGY. Advances and Topical Issues, Volume I, da Silva, J.A.T. (Ed.), Global Science Books, Ltd., London 2006, 224-235 (ISSN: 1749-0294)

(b) This is an invited book chapter focused on sex determination mechanisms in dioecious plants. It was exclusively done by people from our lab.

(c) A majority of plant species are cosexuals, but about 5% of species are strictly dioecious. A high number of plant species represent intermediate stages, i.e., different forms of flowers are present on one individual or sexually different individuals occur in populations of plants. Similarly as in animals, there are two basic mechanisms of sex determination in plants: genetic and environmental. Among the dioecious species, only a few of them have evolved heteromorphic sex chromosomes, especially white campion and common sorrel. In these two classical species, different sex chromosome-based mechanisms have been described: white campion has the male dominant chromosome Y, while in sorrel the sexuality is controlled by a ratio between the number of X chromosomes and the number of autosomal sets. Molecular analyses show that the plant sex chromosomes are evolutionarily much younger compared with the sex chromosomes in animal species. This fact makes them the optimum models to study early stages of sex chromosome evolution.

[12] Genomic in situ hybridization and phylogenetics

(a) Marková, M., Vyskot, B.: New horizons of genomic in situ hybridization (GISH). Cytogenetic and Genome Research, 126: 368-375, 2009 [IF 2.279]
Marková, M., Michu, E., Vyskot, B., Janoušek, B., Žlůvová, J.: An interspecific hybrid as a tool to study phylogenetics relationship in plants. Chromosome Research, 15, 2007, 1051-1059 [IF 3.151]
Marková, M., Lengerová, M., Žlůvová, J., Janoušek, B., Vyskot, B.: Karyological analysis of an interspecific hybrid between the dioecious Silene latifolia and the hermaphroditic Silene viscosa. - Genome, 49, 2006, 373-379 [IF 2.205]

(b) This group of papers describes a new cytogenetic technique to support phylogenetic tools. All these papers were exclusively designed and realized in our lab. The newly developed technology is protected by a Czech patent. Since 2007 these papers were cited at least 6times.

(c) We established a new auxiliary phylogenetic approach based on genomic in situ hybridization technique (GISH). We used an interspecific hybrid Silene latifolia x Silene viscosa to compare two different genomes simultaneously on one slide. By using GISH with genomic DNA from another closely related species as a probe, we directly compared the level of relatedness between the genomes of the studied species and parental species. This experimental design enabled us to approximately estimate evolutionary relationships between the genome of tested plant species and genomes of both parental species of the hybrid by using the ratio of intensities of fluorescence signals. We tested this technique in various Silene species and the results were in accordance with the topology of the phylogenetic tree we constructed based on rDNA sequences. This approach could help to improve tree topology and serve as a useful complementary tool in molecular phylogenetic studies in related species.

[13] Evolution of the sex chromosomes

(a) Žlůvová, J., Georgiev, S., Janoušek, B., Charlesworth, D., Vyskot, B., Negrutiu, I.: Early events in the evolution of the Silene latifolia Y chromosome: male specialization and recombination arrest. – Genetics, 177, 2007, 375-386 [IF 4.389]

(b) The paper describes evolution of the sex chromosomes with a special attention to the arrest of recombination with the X chromosome. It is a result of a long-term collaboration with the Universityof Lyon, Universityof Edinburgh. Since 2008 this paper has been cited at least 9times.

(c) Understanding the origin and evolution of sex chromosomes requires studying recently evolved X–Y chromosome systems such as those in some flowering plants. Here we describe Y chromosome deletion mutants of Silene latifolia. The combination of results from new and previously described deletions with histological descriptions of their stamen development defects indicates the presence of two distinct Y regions containing loci with indispensable roles in male reproduction. We determined their positions relative to the two main sex determination functions. A region proximal to the centromere on the Y p arm containing the putative stamen promoting sex determination locus includes additional early stamen developmental factors. Analysis of meiotic X–Y pairing in one of the male-sterile mutants indicates that the Y carries sequences specifically affecting sex chromosome pairing.

[14] Functional analysis of the Y chromosome

(a) Žlůvová J., Lengerová M., Marková M., Hobza R., Nicolas M., Vyskot B., Charlesworth D., Negrutiu I.,Janoušek B.: The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution. – Evolution & Development, 7, 2005, 327-336 [IF 3.679]

(b) This work demonstrates how an autosome from a bisexual species can substitute the Y chromosome from a dioecious plant. This work was invented by dr. Janoušek and dr. Žlůvová and largely realized in Brno. This important result has been cited at least 10times since 2006.

(c) We used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females, so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation.  The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females.

[15] Surveys on the model dioecious plant Silene

(a) Žlůvová, J., Janoušek, B., Hobza, R., Mráček, J., Widmer, A., Vyskot, B.: Genus Silene (Caryophyllaceae) – evolutionary diversification and sex chromosome formation. In:PLANT GENOME – BIODIVERSITY AND EVOLUTION, Vol. 1E – Phanerogam –Angiosperm, Ed. Sharma, A.K., Sharma A., Science Publishers, New Hampshire,USA, 2008, pp. 173-226, ISBN 978-1-57808-507-1

Bernasconi, G.,Antonovics, J., Biere, A., Charlesworth, D., Delph, L.F., Filatov, D., Giraud, T., Hood, M.E., Marais, G.A.B., McCauley, D., Pannell, J.R., Shykoff, J.A., Vyskot, B., Wolfe, L., Widmer, A.: Silene as a model system in ecology and evolution. Heredity, 103, 2009, 5-14 [IF 3.616]

(b) These papers are invited reviews to a book on genome diversity and a consorcium overview representing a world society of people working with the model Silene, respectively. The first paper comes from Brno, the second represents a world spectrum of lab representatives (largely from USA, France, Great Britain, and Switzerland).

(c) The genus Silene, studied by Darwin, Mendel and other early scientists, is re-emerging as a system for studying interrelated questions in ecology, evolution and developmental biology. These questions include sex chromosome evolution, epigenetic control of sex expression, genomic conflict and speciation. Its well-studied interactions with the pathogen Microbotryum has made Silenea model for the evolution and dynamics of disease in natural systems, and its interactions with herbivores have increased our understanding of multi-trophic ecological processes and the evolution of invasiveness. Molecular tools are now providing new approaches to many of these classical yet unresolved problems, and new progress is being made through combining phylogenetic, genomic and molecular evolutionary studies with ecological and phenotypic data.

[16] Control of meiotic division as studied by cytogenetic tools

(a) Riehs, N., Akimcheva, S., Puizina, J., Bulánková, P., Odol, R.A., Široký, J., Sleiffer, A., Schweizer, D., Shippen, D.E., Říha, K.:Arabidopsis SMG7 protein is required for exit from meiosis. – Journal of Cell Science, 121, 2008, 2208-2216 [IF 6.847]

Široký, J.: Chromosome landmarks as a tool to study the genome of Arabidopsis thaliana.– Cytogenetic and Genome Research, 120, 2008, 202-209 [IF 2.279]

(b) This paper describes some molecular aspects of plant meiosis. It was basically done in the lab of our former student dr. K. Říha at the Gregor Mendel Institute in Vienna. Our lab performed some cytogenetic analyses. These papers have been cited at least 4times since 2009.

(c) Meiosis consists of two nuclear divisions that are separated by a short interkinesis. Here we show that the SMG7 protein is essential for the progression from anaphase to telophase in the second meiotic division in Arabidopsis. Arabidopsis SMG7 is an essential gene, the disruption of which causes embryonic lethality. Plants carrying a hypomorphic smg7 mutation exhibit an elevated level of transcripts containing premature stop codons. This suggests that the role of SMG7 is conserved in plants. Furthermore, hypomorphic smg7 alleles render mutant plants sterile by causing an unusual cell-cycle arrest in anaphase II that is characterized by delayed chromosome decondensation and aberrant rearrangement of the meiotic spindle. Together, these data indicate that SMG7 counteracts cyclin-dependent kinase activity at the end of meiosis, and reveal a novel link between SMG7 and regulation of the meiotic cell cycle.

[17] Regulation of telomere length

(a) Vespa, L., Warrington, R.T., Široký, J., Mokroš, P., Shippen, D.E.: ATM regulates the length of individual telomere tracts inArabidopsis. Proc Natl Acad Sci USA104, 2007, 18145–18150 [IF 10.228]

Mokroš, P., Vrbský, J., Široký, J.: Identification of chromosomal fusion sites in Arabidopsis mutants using sequential bicolour BAC-FISH. - Genome, 49, 2006, 1036-1042 [IF 2.205]

(b) These papers are focused on structure and function of plant telomeres. The first paper was basically done by the Texas A&M University, our lab performed especially cytogenetic analyses. These papers have been cited at least 9times since 2007.

(c) Telomeres have the ability of protecting linear chromosome ends from DNA damage sensors by using these same proteins as essential components. We have previously shown that the absence of ataxia telangiectasia mutated (ATM) accelerates the onset of genome instability in telomerase-deficient Arabidopsis. Unexpectedly, we found that the onset of profound developmental defects and abundant end-to-end chromosome fusions in atm tert mutants required the presence of only one critically shortened telomere. Notably alternative lengthening of telomeres was suppressed in the absence of ATM. Finally, we show that size differences between telomeres on homologous chromosome ends are greater for atm tert than tert plants. These findings suggest a dual role for ATM in regulating telomere size by promoting elongation of short telomeres and by preventing the accumulation of cells that harbor large telomere deletions.

[18] Cell survival after telomerase blockade

(a) Watson J.M., Bulánková P., Říha K., Shippen D.E., Vyskot B.: Telomerase-independent cell survival in Arabidopsis thaliana. – Plant Journal 43, 2005, 662-674 [IF 6.951]

Široký, J.: Cytogenetics for the study of telomere function in plants. – Cytogenetic and Genome Research, 122, 2008, 374-379 [IF 2.279]

(b) This work represents how plant cells can overcome the telomerase arrest. The first paper was realized in Brno, but the original material with the knock-down telomerase gene comes from Texas. The telomerase action represents a hot topic in molecular biology: The Plant Journal paper has been cited 10times since 2006.

(c) Inactivation of telomerase causes telomere shortening and results in the loss of the telomere’s protective function. Experiments performed on Arabidopsis mutants lacking telomerase activity revealed their unusually high tolerance for genome instability. We present molecular and cytogenetic analysis of two cell lines derived from late-generation telomerase-deficient mutant. These cultures have survived for 3 years, but neither culture has adapted mechanisms to maintain terminal telomeric repeats. One culture suffers from severe growth irregularities and a high degree of mortality. Karyological analysis revealed dramatic genomic rearrangements, a large variation in ploidy, and an extremely high percentage of anaphase bridges. The second cell line survived an apparent crisis and phenotypically appears wild-type with respect to growth and morphology. We conclude that the restructured chromosome termini provide the cell lines with partial protection from end-joining repair activities.