Modeling of molecular interaction between apoptin, BCR-Abl and CrkL--an alternative approach to conventional rational drug design

In this study we have calculated a 3D structure of apoptin and through modeling and docking approaches, we show its interaction with Bcr-Abl oncoprotein and its downstream signaling components, following which we confirm some of the newly-found interactions by biochemical methods. Bcr-Abl oncoprotein is aberrantly expressed in chronic myelogenous leukaemia (CML). It has several distinct functional domains in addition to the Abl kinase domain. The SH3 and SH2 domains cooperatively play important roles in autoinhibiting its kinase activity. Adapter molecules such as Grb2 and CrkL interact with proline-rich region and activate multiple Bcr-Abl downstream signaling pathways that contribute to growth and survival. Therefore, the oncogenic effect of Bcr-Abl could be inhibited by the interaction of small molecules with these domains. Apoptin is a viral protein with well-documented cancer-selective cytotoxicity. Apoptin attributes such as SH2-like sequence similarity with CrkL SH2 domain, unique SH3 domain binding sequence, presence of proline-rich segments, and its nuclear affinity render the molecule capable of interaction with Bcr-Abl. Despite almost two decades of research, the mode of apoptin's action remains elusive because 3D structure of apoptin is unavailable. We performed in silico three-dimensional modeling of apoptin, molecular docking experiments between apoptin model and the known structure of Bcr-Abl, and the 3D structures of SH2 domains of CrkL and Bcr-Abl. We also biochemically validated some of the interactions that were first predicted in silico. This structure-property relationship of apoptin may help in unlocking its cancer-selective toxic properties. Moreover, such models will guide us in developing of a new class of potent apoptin-like molecules with greater selectivity and potency.     

Population genetics of the hazel hen <Emphasis Type=Italic>Bonasa bonasia</Emphasis> in Poland assessed with non-invasive samples

Despite a severe decrease in the number of hazel hens during the 20^th century, nowadays this grouse species is rather common in the forests of Northeastern and Southern Poland. We used mitochondrial control region and microsatellite markers to examine the genetic variability of Polish populations of hazel hens. We used non-invasively collected faeces to estimate genetic variability within populations, genetic differentiation among populations as well as genetic differentiation between two regions inhabited by two different subspecies of hazel hens. Our results confirm the usefulness of DNA from faeces to obtain reliable information on the population genetics of hazel hens. We found a rather high level of genetic variability in the Polish population. Genetic variability was higher in birds from continuous forests in the South of the country than in birds from fragmented habitats in the Northeast. Genetic differentiation was higher among subpopulations from Northeastern Poland. Additionally, both classes of molecular markers suggested the presence of two distinct genetic groups of birds, corresponding to previously described subspecies. We conclude that the genetic variability of the Polish hazel hen population has been influenced by habitat fragmentation and the history of the population during its post-glacial colonization of Poland from different glacial refugia.     

Vertical flux of particulate matter in an Arctic fjord: the case of lack of the sea-ice cover in Adventfjorden 2006–2007

Seasonal dynamics of suspended minerals, organic matter, particulate, and dissolved organic carbon (DOC), chlorophyll, and their vertical fluxes were studied in a small Arctic fjord (Adventfjorden, Spitsbergen) from November 2006 to October 2007. The fjord was usually covered with fast ice in winter, but remained open throughout the year since 2005. The open-water winter period caused increased wave action and resuspension of organic and mineral particles. The lack of sea-ice in spring accelerated the onset of the productive season. The earlier light signal also caused an earlier appearance of mesozooplanktonic organisms, accompanied by a significant increase of the DOC pool in the water. In the cold period (winter and spring) 47% of the annual organic matter settled on the sampling site, a lot coming from the spring diatom bloom in April. Summer melt (July, August) resulted in turbid and brackish fjord surface water with stratification and increase of both suspended particles and sedimentation, causing 60% of the annual minerals and 53% of the annual organic matter to settle at the sampling site. Increased dissolved organic carbon (DOC) through sloppy feeding on the mixo- and heterotrophic protista by the abundant mesozooplankton indicated intensive secondary production, resulting in a maximum fecal pellets flux of >5 mg C m⁻² day⁻¹. A warmer climate with reduced sea-ice cover in fjords will advance the onset of the spring bloom and will also result in a larger input of turbid melt water in summer, restricting the light availability and enhancing flocculation and thereby sedimentation.     

Expression and genome organization of resistance gene analogs in soybean.

Sequence analysis of cloned plant disease-resistance genes reveals a number of conserved domains. Researchers have used these domains to amplify analogous sequences, resistance gene analogs (RGAs), from soybean and other crops. Many of these RGAs map in close proximity to known resistance genes. While this technique is useful in identifying potential disease resistance loci, identifying the functional resistance gene from a cluster of homologs requires sequence information from outside of these conserved domains. To study RGA expression and to determine the extent of their similarity to other plant resistance genes, two soybean cDNA libraries (root and epicotyl) were screened by hybridization with RGA class-specific probes. cDNAs hybridizing to RGA probes were detected in each library. Two types of cDNAs were identified. One type was full-length and contained several disease-resistance gene (R-gene) signatures. The other type contained several deletions within these signatures. Sequence analyses of the cDNA clones placed them in the Toll-Interleukin-1 receptor, nucleotide binding domain, and leucine-rich repeat family of disease-resistance genes. Using clone-specific primers from within the 3' end of the LRRs, we were able to map two cDNA clones (LM6 and MG13) to a BAC contig that is known to span a cluster of disease-resistance genes.     

Richness of ancient forest plant species indicates suitable habitats for macrofungi

Macrofungal species richness generally increases with forest continuity as does the richness of so-called ancient forest plant species (AFS). Based on this assumption, we examined the ability of AFS to indicate macrofungal diversity in six study areas covering a range of elevations and environments in the Czech Republic. In total, we used data from 106 sampling plots (2,500 m² each) distributed over six types of forest stands reflecting different intensities and temporal stages of forest management. Species composition of vascular plants and macrofungi was recorded by a single inventory and regular 2-year monitoring, respectively. In total, we found 71 AFS and 1,413 macrofungal species, of which 150 were red-listed macrofungal species. We documented that AFS show potential for being used in the prediction of macrofungi species richness, including endangered species, at the local scale (α-diversity). Additionally, we found significant differences in macrofungal species richness depending on study area and type of forest management, which did not, however, derogate the effect of AFS. Spatial congruence between species composition of AFS and macrofungi communities (β-diversity) increased with forest age and decreased with intensity of forest management. If we consider the simplicity of monitoring AFS in comparison to regular monitoring of macrofungi, we found a widely usable tool for estimating macrofungal diversity in all dominant types of managed forest in central Europe. However, we should be aware of the limited ability of AFS to capture macrofungal diversity across a broader spatial context (γ-diversity), especially in areas with a low diversity of AFS.     

Dealing with Discordant Genetic Signal Caused by Hybridisation,Incomplete Lineage Sorting and Paucity of Primary Nucleotide Homologies: A Case Study of Closely Related Members of the Genus Picris Subsection Hieracioides (Compositae)

We investigated genetic variation and evolutionary history of closely related taxa of Picris subsect. Hieracioides with major focus on the widely distributed P. hieracioides and its closely related congeners, P. hispidissima, P. japonica, P. olympica, and P. nuristanica. Accessions from 140 sample sites of the investigated Picris taxa were analyzed on the infra- and the inter-specific level using nuclear (ITS1-5.8S-ITS2 region) and chloroplast (rpl32-trnL ^(UAG) region) DNA sequences. Genetic patterns of P. hieracioides, P. hispidissima, and P. olympica were shown to be incongruent and, in several cases, both plastid and nuclear alleles transcended borders of the taxa and genetic lineages. The widespread P. hieracioides was genetically highly variable and non-monophyletic across both markers, with allele groups having particular geographic distributions. Generally, all gene trees and networks displayed only a limited and statistically rather unsupported resolution among ingroup taxa causing their phylogenetic relationships to remain rather unresolved. More light on these intricate evolutionary relationships was cast by the Bayesian coalescent-based analysis, although some relationships were still left unresolved. A combination of suite of phylogenetic analyses revealed the ingroup taxa to represent a complex of genetically closely related and morphologically similar entities that have undergone a highly dynamic and recent evolution. This has been especially affected by the extensive and recurrent gene flow among and within the studied taxa and/or by the maintenance of ancestral variation. Paucity of phylogenetically informative signal further hampers the reconstruction of relationships on the infra- as well as on the inter-specific level. In the present study, we have demonstrated that a combination of various phylogenetic analyses of datasets with extremely complex and incongruent phylogenetic signal may shed more light on the interrelationships and evolutionary history of analysed species groups.     

Photosynthetic responses of sun- and shade-grown barley leaves to high light: is the lower PSII connectivity in shade leaves associated with protection against excess of light?

In this study, we have compared photosynthetic performance of barley leaves (Hordeum vulgare L.) grown under sun and shade light regimes during their entire growth period, under field conditions. Analyses were based on measurements of both slow and fast chlorophyll (Chl) a fluorescence kinetics, gas exchange, pigment composition; and of light incident on leaves during their growth. Both the shade and the sun barley leaves had similar Chl a/b and Chl/carotenoid ratios. The fluorescence induction analyses uncovered major functional differences between the sun and the shade leaves: lower connectivity among Photosystem II (PSII), decreased number of electron carriers, and limitations in electron transport between PSII and PSI in the shade leaves; but only low differences in the size of PSII antenna. We discuss the possible protective role of low connectivity between PSII units in shade leaves in keeping the excitation pressure at a lower, physiologically more acceptable level under high light conditions.     

Effects of zebra mussels on cladoceran communities under eutrophic conditions

The purpose of this study was to determine how zebra mussels affected cladoceran community structure under eutrophic conditions. We conducted a mesocosm study where we manipulated the presence of zebra mussels and the presence of large-bodied Daphnia (Daphnia magna and Daphnia pulicaria). We also conducted a complimentary life-table experiment to determine how water from the zebra mussel treatment affected the life history characteristics of the cladoceran species. We anticipated that small- and large-bodied cladoceran species would respond differently to changes in algal quality and quantity under the effects of zebra mussels. Large-bodied Daphnia successfully established in the zebra mussel treatment but failed to grow in the control. We did not observe positive relationships between food concentrations and cladoceran abundances. However, the phosphorus content in the seston indicated that food quality was below the threshold level for large-bodied cladocerans at the beginning of the experiment. We believe that zebra mussels quickly enhanced the phosphorus content in the seston due to the excretion of inorganic phosphorus, thus facilitating the development of large-bodied Daphnia. In conclusion, our results suggest that zebra mussels can alter the phosphorus content of seston in lakes and this can affect the dynamics of crustacean zooplankton.     

Benthic macroinvertebrates along the Czech part of the Labe and lower section of the Vltava rivers from 1996–2005, with a particular focus on rare and alien species

In the Czech part of the Labe River and the lower part of the Vltava River, we examined if the benthic macroinvertebrate composition changed from 1996 to 2005 due to expected improvements in water quality resulting from socioeconomic changes in the Czech Republic since the 1990s. Special attention was given to rare and alien species. The four biological metrics used (Number of taxa, BMWP, Number of sensitive taxa, and Number of EPT taxa) demonstrated that there was indeed an improvement in water quality as well as a slight improvement of the Labe microhabitats during the investigated period. An increasing Number of taxa over time was observed at most sites. Two main concurrent ecological processes are recently in progress in the Labe: a recovery of native species and an expansion of alien species, some of which are considered invasive. The caddisfly Setodes punctatus and the beetle Pomatinus substriatus, considered as regionally extinct in the Czech Republic until 2005, were rediscovered during our investigations. Findings of the crustacean Hemimysis anomala (invasive) and the chironomids Stenochironomus sp. and Lipiniella sp. were the first records of these taxa in the Czech Republic.     

An accurate,sensitive, and scalable method to identify functional sites in protein structures

Functional sites determine the activity and interactions of proteins and as such constitute the targets of most drugs. However, the exponential growth of sequence and structure data far exceeds the ability of experimental techniques to identify their locations and key amino acids. To fill this gap we developed a computational Evolutionary Trace method that ranks the evolutionary importance of amino acids in protein sequences. Studies show that the best-ranked residues form fewer and larger structural clusters than expected by chance and overlap with functional sites, but until now the significance of this overlap has remained qualitative. Here, we use 86 diverse protein structures, including 20 determined by the structural genomics initiative, to show that this overlap is a recurrent and statistically significant feature. An automated ET correctly identifies seven of ten functional sites by the least favorable statistical measure, and nine of ten by the most favorable one. These results quantitatively demonstrate that a large fraction of functional sites in the proteome may be accurately identified from sequence and structure. This should help focus structure-function studies, rational drug design, protein engineering, and functional annotation to the relevant regions of a protein.