We systematically investigate the applicability of a molecular dynamics-based setup for the calculations of standard binding free energies of biologically relevant protein-DNA complexes. The free energies are extracted from a potential of mean force calculated using umbrella sampling simulations. Two protein-DNA systems derived from a homeodomain transcription factor complex are studied in order to investigate the binding of both disordered and globular proteins. Free energies and trajectories obtained using two modern molecular mechanical force fields are compared to each other and to experimental data. The temperature dependence of the calculated standard binding free energies is investigated by performing all simulations over a range of temperatures. We show that the values of standard binding free energies obtained from these simulations are overestimated compared to experimental results. Significant differences are observed between the two protein-DNA systems and between the two force fields, which are explained by different propensities to form inter- and intramolecular contacts. The number of protein-DNA contacts increases with increasing temperature, in agreement with the experimentally known temperature dependence of enthalpies of binding. However, conclusions about the temperature dependence of the standard binding free energies cannot be made with confidence, as the differences among the values are on the order of statistical uncertainty.

• MeSH
• DNA chemie   metabolismus   MeSH
• entropie MeSH
• homeoboxový protein Nkx-2.5 chemie   metabolismus   MeSH
• homeodoménové proteiny chemie   metabolismus   MeSH
• lidé MeSH
• simulace molekulární dynamiky MeSH
• simulace molekulového dockingu MeSH
• teplota MeSH
• termodynamika * MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• homeoboxový protein Nkx-2.5 MeSH
• homeodoménové proteiny MeSH
• NKX2-5 protein, human MeSH Prohlížeč

Water molecules can interact with aromatic moieties using either their O-H bonds or their lone-pairs of electrons. In proteins, water-π interactions have been reported to occur with tryptophan and histidine residues, and dynamic exchange between O-Hπ hydrogen bonding and lone-pairπ interactions was suggested to take place, based on ab initio calculations. Here we used classical and QM/MM molecular dynamics simulations, complemented with an NMR study, to examine a specific water-indole interaction observed in the engrailed homeodomain and in its mutants. Our simulations indicate that the binding mode between water and indole can adapt to the potential created by the surrounding amino acids (and by the residues at the DNA surface in protein-DNA complexes), and support the model of dynamic switching between the O-Hπ hydrogen bonding and lone-pairπ binding modes.

• MeSH
• Drosophila MeSH
• homeodoménové proteiny chemie   genetika   metabolismus   MeSH
• kvantová teorie MeSH
• magnetická rezonanční spektroskopie MeSH
• molekulární struktura MeSH
• mutace MeSH
• proteinové domény MeSH
• proteiny Drosophily MeSH
• simulace molekulární dynamiky MeSH
• transkripční faktory chemie   genetika   metabolismus   MeSH
• tryptofan chemie   metabolismus   MeSH
• vazba proteinů MeSH
• voda chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• En protein, Drosophila MeSH Prohlížeč
• homeodoménové proteiny MeSH
• proteiny Drosophily MeSH
• transkripční faktory MeSH
• tryptofan MeSH
• voda MeSH

Formation of all metazoan bodies is controlled by a group of selector genes including homeobox genes, highly conserved across the entire animal kingdom. The homeobox genes from Pou and Six classes are key members of the regulation cascades determining development of sensory organs, nervous system, gonads and muscles. Besides using common bilaterian models, more attention has recently been targeted at the identification and characterization of these genes within the basal metazoan phyla. Cnidaria as a diploblastic sister group to bilateria with simple and yet specialized organs are suitable models for studies on the sensory organ origin and the associated role of homeobox genes. In this work, Pou and Six homeobox genes, together with a broad range of other sensory-specific transcription factors, were identified in the transcriptome of hydrozoan jellyfish Craspedacusta sowerbyi. Phylogenetic analyses of Pou and Six proteins revealed cnidarian-specific sequence motifs and contributed to the classification of individual factors. The majority of the Craspedacusta sowerbyi Pou and Six homeobox genes are predominantly expressed in statocysts, manubrium and nerve ring, the tissues with sensory and nervous activities. The described diversity and expression patterns of Pou and Six factors in hydrozoan jellyfish highlight their evolutionarily conserved functions. This study extends the knowledge of the cnidarian genome complexity and shows that the transcriptome of hydrozoan jellyfish is generally rich in homeodomain transcription factors employed in the regulation of sensory and nervous functions.

• MeSH
• faktory domény POU chemie   genetika   metabolismus   MeSH
• fylogeneze * MeSH
• genetická variace * MeSH
• Hydrozoa genetika   MeSH
• molekulární evoluce MeSH
• orgánová specificita MeSH
• terciární struktura proteinů MeSH
• transkriptom * MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory domény POU MeSH

Estimation of binding free energies is one of the central aims of simulations of biomolecular complexes. We explore the accuracy and efficiency of setups based on nonequilibrium pulling simulations applied to the estimation of binding affinities of DNA-binding proteins. Absolute binding free energies are calculated over a range of temperatures and compared to results obtained previously using an equilibrium method. We show that realistic binding affinities can be obtained with the presented nonequilibrium approach, which also entails lower computational requirements. Errors of the binding free energy estimates are investigated and are shown to be comparable to those observed previously. Bounds are provided on the convergence of the errors with respect to the number of pulling simulations performed and with respect to the applied pull rate.

• MeSH
• DNA vazebné proteiny chemie   MeSH
• DNA chemie   MeSH
• entropie MeSH
• počítačová simulace MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA vazebné proteiny MeSH
• DNA MeSH

Pax transcription factors are evolutionarily conserved regulators of eye development and can be distinguished on the basis of three functional domains: two DNA-binding domains (the paired domain and the paired-type homeodomain), and the octapeptide motif. PaxB of the eyed cubozoan jellyfish, Tripedalia cystophora, is characterized by a Pax2-like paired domain and octapeptide, and a Pax6-like homeodomain. In mice, functionally distinct Pax6 and Pax2 proteins have unique as well as redundant roles in eye morphogenesis. Here, we show that expression of the jellyfish PaxB gene in mouse embryonic eye tissues impairs normal development of lens and retina. Our data show that PaxB misexpression leads to a downregulation of endogenous Pax6 protein in the prospective lens and in subsets of cells within the inner nuclear layer of transgenic retina. In addition to Pax6 downregulation, the expression of PaxB leads to an almost complete loss of amacrine cells in the adult transgenic retina, a phenotype that differs from a loss-of-function of the Pax6 gene. The present data suggest that PaxB, due to its Pax2-like paired domain and Pax-6 like homeodomain, disturbs the transcriptional network regulated by Pax6 in the developing lens and retina. Taken together, our data suggest that molecular properties of individual mouse Pax2 and Pax6 proteins are essential determinants of mouse eye development and cannot be substituted for by jellyfish PaxB which possesses elements of vertebrate Pax2 and Pax6.

• MeSH
• down regulace MeSH
• embryo savčí embryologie   metabolismus   MeSH
• fenotyp MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• myši inbrední C57BL MeSH
• myši transgenní MeSH
• myši MeSH
• oči embryologie   metabolismus   MeSH
• oční proteiny genetika   metabolismus   MeSH
• represorové proteiny genetika   metabolismus   MeSH
• Scyphozoa genetika   metabolismus   MeSH
• transkripční faktor PAX2 genetika   metabolismus   MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory Otx genetika   metabolismus   MeSH
• transkripční faktory paired box genetika   metabolismus   MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• homeodoménové proteiny MeSH
• oční proteiny MeSH
• Pax6 protein, mouse MeSH Prohlížeč
• represorové proteiny MeSH
• transkripční faktor PAX2 MeSH
• transkripční faktor PAX6 MeSH
• transkripční faktory Otx MeSH
• transkripční faktory paired box MeSH

The programming of cell fate by transcription factors requires precise regulation of their time and level of expression. The LIM-homeodomain transcription factor Islet1 (Isl1) is involved in cell-fate specification of motor neurons, and it may play a similar role in the inner ear. In order to study its role in the regulation of vestibulo-motor development, we investigated a transgenic mouse expressing Isl1 under the Pax2 promoter control (Tg +/- ). The transgenic mice show altered level, time, and place of expression of Isl1 but are viable. However, Tg +/- mice exhibit hyperactivity, including circling behavior, and progressive age-related decline in hearing, which has been reported previously. Here, we describe the molecular and morphological changes in the cerebellum and vestibular system that may cause the hyperactivity of Tg +/- mice. The transgene altered the formation of folia in the cerebellum, the distribution of calretinin labeled unipolar brush cells, and reduced the size of the cerebellum, inferior colliculus, and saccule. Age-related progressive reduction of calbindin expression was detected in Purkinje cells in the transgenic cerebella. The hyperactivity of Tg +/- mice is reduced upon the administration of picrotoxin, a non-competitive channel blocker for the γ-aminobutyric acid (GABA) receptor chloride channels. This suggests that the overexpression of Isl1 significantly affects the functions of GABAergic neurons. We demonstrate that the overexpression of Isl1 affects the development and function of the cerebello-vestibular system, resulting in hyperactivity.

• Klíčová slova
• Age-related deterioration of Purkinje cells, Attention deficit hyperactivity disorder, Calcium homeostasis, Cerebellum, Foliation defects, GABA signaling, Hyperactivity, Islet1 transcription factor, Purkinje cells, Transgenic mouse, Vestibular system,
• MeSH
• hyperkineze metabolismus   patologie   MeSH
• mozeček metabolismus   patologie   MeSH
• myši transgenní MeSH
• myši MeSH
• proteiny s homeodoménou LIM biosyntéza   MeSH
• transkripční faktor PAX2 biosyntéza   MeSH
• transkripční faktory biosyntéza   MeSH
• vestibulární aparát metabolismus   patologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• insulin gene enhancer binding protein Isl-1 MeSH Prohlížeč
• Pax2 protein, mouse MeSH Prohlížeč
• proteiny s homeodoménou LIM MeSH
• transkripční faktor PAX2 MeSH
• transkripční faktory MeSH

Mutual interactions of the phytohormones, cytokinins and auxin determine root or shoot identity during postembryonic de novo organogenesis in plants. However, our understanding of the role of hormonal metabolism and perception during early stages of cell fate reprogramming is still elusive. Here we show that auxin activates root formation, whereas cytokinins mediate early loss of the root identity, primordia disorganisation and initiation of shoot development. Exogenous and endogenous cytokinins influence the initiation of newly formed organs, as well as the pace of organ development. The process of de novo shoot apical meristem establishment is accompanied by accumulation of endogenous cytokinins, differential regulation of genes for individual cytokinin receptors, strong activation of AHK4-mediated signalling and induction of the shoot-specific homeodomain regulator WUSCHEL. The last is associated with upregulation of isopentenyladenine-type cytokinins, revealing higher shoot-forming potential when compared with trans-zeatin. Moreover, AHK4-controlled cytokinin signalling negatively regulates the root stem cell organiser WUSCHEL RELATED HOMEOBOX 5 in the root quiescent centre. We propose an important role for endogenous cytokinin biosynthesis and AHK4-mediated cytokinin signalling in the control of de novo-induced organ identity.

• Klíčová slova
• Arabidopsis, Cytokinin metabolism, Cytokinin signalling, De novo organogenesis, Root, Shoot,
• MeSH
• Arabidopsis cytologie   embryologie   genetika   MeSH
• cytokininy genetika   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• meristém cytologie   embryologie   MeSH
• organogeneze rostlin fyziologie   MeSH
• proteinkinasy genetika   metabolismus   MeSH
• proteiny huseníčku genetika   metabolismus   MeSH
• receptory buněčného povrchu genetika   metabolismus   MeSH
• signální transdukce fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• homeodoménové proteiny MeSH
• proteinkinasy MeSH
• proteiny huseníčku MeSH
• receptory buněčného povrchu MeSH
• WOL protein, Arabidopsis MeSH Prohlížeč
• WUSCHEL protein, Arabidopsis MeSH Prohlížeč

OBJECTIVE: To present the results of molecular genetics analysis in men with reproductive disorders focusing on the DNA segments and genes which affect spermatogenesis. DESIGN: Original article. SETTING: Institute of Biology and Medical Genetics of the First Faculty of Medicine and General Teaching Hospital, Prague. METHODS: One hundred and twenty-three patients identified with a fertility disorder were screened for mutations of the CFTR gene. In all patients were performed cytogenic analysis and assessment of Y-chromosome microdeletions. In 107 patients where the fertility was not detected by routine examination we performed an analysis for X-chromosome microdeletions (CNV64, CNV67, CNV69) and in certain genes necessary for normal spermatogenesis (AGFG1, CAPZA3, CNTROB, HOOK1, GOPC, SPATA16). RESULTS: Our results did not reveal any negative efffects of X-chromosome microdeletion on spermatogenesis. Analysis of six genes showed in two patients in gene SPATA16 a homozygotic haplotype [1526C>T + 1577T>C] which can be most probably responsible for the fertility in two examined patients. CONCLUSION: According to our results we do not recommend introduction of X-chromosome microdeletions assays in areas CNV64 , CNV67 and CNV69 into routine diagnostic. Regarding the selected genes affecting spermatogenesis, our results showed that homozygotic haplotype [ 1526C>T + 1577T>C] in SPATA16 gene is very likely responsible for infertility in two of our patients. The above mentioned haplotype deserves attention in the investigation of male infertility.

• Klíčová slova
• AGFG1 gene, CAPZA3 gene, CNTROB gene, GOPC gene SPATA16 gene., HOOK1 gene, X-chromosome microdeletions, male infertility,
• MeSH
• haplotypy MeSH
• homeodoménové proteiny genetika   MeSH
• lidé MeSH
• mužská infertilita etiologie   MeSH
• spermatogeneze genetika   MeSH
• vezikulární transportní proteiny MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• homeodoménové proteiny MeSH
• SPATA16 protein, human MeSH Prohlížeč
• vezikulární transportní proteiny MeSH

Detailed knowledge of human B-cell development is crucial for the proper interpretation of inborn errors of immunity and malignant diseases. It is of interest to understand the kinetics of protein expression changes during development, but also to properly interpret the major and possibly alternative developmental trajectories. We have investigated human samples from healthy individuals with the aim of describing all B-cell developmental trajectories. We validated a 30-parameter mass cytometry panel and demonstrated the utility of "vaevictis" visualization of B-cell developmental stages. We used the trajectory inference tool "tviblindi" to exhaustively describe all trajectories leading to all developmental ends discovered in the data. Focusing on Natural Effector B cells, we demonstrated the dynamics of expression of nuclear factors (PAX-5, TdT, Ki-67, Bcl-2), cytokine and chemokine receptors (CD127, CXCR4, CXCR5) in relation to the canonical B-cell developmental stage markers. We observed branching of the memory development, where follicular memory formation was marked by CD73 expression. Lastly, we performed an analysis of two example cases of abnormal B-cell development caused by mutations in RAG-1 and Wiskott-Aldrich syndrome gene in patients with primary immunodeficiency. In conclusion, we developed, validated, and presented a comprehensive set of tools for the investigation of B-cell development in the bone marrow compartment.

• Klíčová slova
• B‐cell development, CD73, Mass cytometry, RAG‐1, Trajectory inference, WAS,
• MeSH
• algoritmy * MeSH
• B-lymfocyty * imunologie   MeSH
• buněčná diferenciace * imunologie   genetika   MeSH
• homeodoménové proteiny * genetika   metabolismus   MeSH
• lidé MeSH
• mutace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• homeodoménové proteiny * MeSH
• RAG-1 protein MeSH Prohlížeč

The diverse forms of today's dominant vascular plant flora are generated by the sustained proliferative activity of sporophyte meristems at plants' shoot and root tips, a trait known as indeterminacy [1]. Bryophyte sister lineages to the vascular plants lack such indeterminate meristems and have an overall sporophyte form comprising a single small axis that ceases growth in the formation of a reproductive sporangium [1]. Genetic mechanisms regulating indeterminacy are well characterized in flowering plants, involving a feedback loop between class I KNOX genes and cytokinin [2, 3], and class I KNOX expression is a conserved feature of vascular plant meristems [4]. The transition from determinate growth to indeterminacy during evolution was a pre-requisite to vascular plant diversification, but mechanisms enabling the innovation of indeterminacy are unknown [5]. Here, we show that class I KNOX gene activity is necessary and sufficient for axis extension from an intercalary region of determinate moss shoots. As in Arabidopsis, class I KNOX activity can promote cytokinin biosynthesis by an ISOPENTENYL TRANSFERASE gene, PpIPT3. PpIPT3 promotes axis extension, and PpIPT3 and exogenously applied cytokinin can partially compensate for loss of class I KNOX function. By outgroup comparison, the results suggest that a pre-existing KNOX-cytokinin regulatory module was recruited into vascular plant shoot meristems during evolution to promote indeterminacy, thereby enabling the radiation of vascular plant shoot forms.

• Klíčová slova
• ISOPENTENYL TRANSFERASE, KNOX-cytokinin, evo-devo, indeterminacy, plant evolution, vascular plant origins,
• MeSH
• biologická evoluce MeSH
• cytokininy genetika   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• mechy genetika   metabolismus   MeSH
• molekulární evoluce * MeSH
• rostlinné proteiny genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cytokininy MeSH
• homeodoménové proteiny MeSH
• rostlinné proteiny MeSH