Southern montane populations did not contribute to the recolonization of West Siberian Plain by Siberian larch (Larix sibirica): a range‐wide analysis of cytoplasmic markers

While many species were confined to southern latitudes during the last glaciations, there has lately been mounting evidence that some of the most cold‐tolerant species were actually able to survive close to the ice sheets. The contribution of these higher latitude outposts to the main recolonization thrust remains, however, untested. In the present study, we use the first range‐wide survey of genetic diversity at cytoplasmic markers in Siberian larch (Larix sibirica; four mitochondrial (mt) DNA loci and five chloroplast (cp) DNA SSR loci) to (i) assess the relative contributions of southern and central areas to the current L. sibirica distribution range; and (ii) date the last major population expansion in both L. sibirica and adjacent Larix species. The geographic distribution of cpDNA variation was uninformative, but that of mitotypes clearly indicates that the southernmost populations, located in Mongolia and the Tien‐Shan and Sayan Mountain ranges, had a very limited contribution to the current populations of the central and northern parts of the range. It also suggests that the contribution of the high latitude cryptic refugia was geographically limited and that most of the current West Siberian Plain larch populations likely originated in the foothills of the Sayan Mountains. Interestingly, the main population expansion detected through Approximate Bayesian Computation (ABC) in all four larch species investigated here pre‐dates the LGM, with a mode in a range of 220 000–1 340 000 years BP. Hence, L. sibirica, like other major conifer species of the boreal forest, was strongly affected by climatic events pre‐dating the Last Glacial Maximum.     

Dimethylaminopyridine derivatives of lupane triterpenoids cause mitochondrial disruption and induce the permeability transition

Triterpenoids are a large class of naturally occurring compounds, and some potentially interesting as anticancer agents have been found to target mitochondria. The objective of the present work was to investigate the mechanisms of mitochondrial toxicity induced by novel dimethylaminopyridine (DMAP) derivatives of pentacyclic triterpenes, which were previously shown to inhibit the growth of melanoma cells in vitro. MCF-7, Hs 578T and BJ cell lines, as well as isolated hepatic mitochondria, were used to investigate direct mitochondrial effects. On isolated mitochondrial hepatic fractions, respiratory parameters, mitochondrial transmembrane electric potential, induction of the mitochondrial permeability transition (MPT) pore and ion transport-dependent osmotic swelling were measured. Our results indicate that the DMAP triterpenoid derivatives lead to fragmentation and depolarization of the mitochondrial network in situ, and to inhibition of uncoupled respiration, induction of the permeability transition pore and depolarization of isolated hepatic mitochondria. The results show that mitochondrial toxicity is an important component of the biological interaction of DMAP derivatives, which can explain the effects observed in cancer cells.     

Synthesis,biological evaluation,X-ray molecular structure and molecular docking studies of RGD mimetics containing 6-amino-2,3-dihydroisoindolin-1-one fragment as ligands of integrin αIIbβ3

A series of novel RGD mimetics containing phthalimidine fragment was designed and synthesized. Their antiaggregative activity determined by Born’s method was shown to be due to inhibition of fibrinogen binding to α_IIbβ₃. Molecular docking of RGD mimetics to α_IIbβ₃ receptor showed the key interactions in this complex, and also some correlations have been observed between values of biological activity and docking scores. The single crystal X-ray data were obtained for five mimetics.     

Optimization of norbornyl‐based carbocyclic nucleoside analogs as cyclin‐dependent kinase 2 inhibitors

We report on the discovery of norbornyl moiety as a novel structural motif for cyclin‐dependent kinase 2 (CDK2) inhibitors which was identified by screening a carbocyclic nucleoside analogue library. Three micromolar hits were expanded by the use of medicinal chemistry methods into a series of 16 novel compounds. They had prevailingly micromolar activities against CDK2 and the best compound of the series attained IC₅₀ of 190 nM. The binding modes were explored in molecular details by modeling and docking. Quantum mechanics‐based scoring was used to rationalize the affinities. In conclusion, the discovered 9‐hydroxymethylnorbornyl moiety was shown by joint experimental‐theoretical efforts to be able to serve as a novel substituent for CDK2 inhibitors. This finding opens door to the exploration of chemical space towards more effective derivatives targeting this important class of protein kinases.     

Cryo‐EM map interpretation and protein model‐building using iterative map segmentation

A procedure for building protein chains into maps produced by single‐particle electron cryo‐microscopy (cryo‐EM) is described. The procedure is similar to the way an experienced structural biologist might analyze a map, focusing first on secondary structure elements such as helices and sheets, then varying the contour level to identify connections between these elements. Since the high density in a map typically follows the main‐chain of the protein, the main‐chain connection between secondary structure elements can often be identified as the unbranched path between them with the highest minimum value along the path. This chain‐tracing procedure is then combined with finding side‐chain positions based on the presence of density extending away from the main path of the chain, allowing generation of a C_α model. The C_α model is converted to an all‐atom model and is refined against the map. We show that this procedure is as effective as other existing methods for interpretation of cryo‐EM maps and that it is considerably faster and produces models with fewer chain breaks than our previous methods that were based on approaches developed for crystallographic maps.     

Competition among native and invasive Phragmites australis populations: An experimental test of the effects of invasion status,genome size,and ploidy level

Among the traits whose relevance for plant invasions has recently been suggested are genome size (the amount of nuclear DNA) and ploidy level. So far, research on the role of genome size in invasiveness has been mostly based on indirect evidence by comparing species with different genome sizes, but how karyological traits influence competition at the intraspecific level remains unknown. We addressed these questions in a common‐garden experiment evaluating the outcome of direct intraspecific competition among 20 populations of Phragmites australis, represented by clones collected in North America and Europe, and differing in their status (native and invasive), genome size (small and large), and ploidy levels (tetraploid, hexaploid, or octoploid). Each clone was planted in competition with one of the others in all possible combinations with three replicates in 45‐L pots. Upon harvest, the identity of 21 shoots sampled per pot was revealed by flow cytometry and DNA analysis. Differences in performance were examined using relative proportions of shoots of each clone, ratios of their aboveground biomass, and relative yield total (RYT). The performance of the clones in competition primarily depended on the clone status (native vs. invasive). Measured in terms of shoot number or aboveground biomass, the strongest signal observed was that North American native clones always lost in competition to the other two groups. In addition, North American native clones were suppressed by European natives to a similar degree as by North American invasives. North American invasive clones had the largest average shoot biomass, but only by a limited, nonsignificant difference due to genome size. There was no effect of ploidy on competition. Since the North American invaders of European origin are able to outcompete the native North American clones, we suggest that their high competitiveness acts as an important driver in the early stages of their invasion.     

Involvement of the recoverin C-terminal segment in recognition of the target enzyme rhodopsin kinase

NCS (neuronal Ca2+ sensor) proteins belong to a family of calmodulin-related EF-hand Ca2+-binding proteins which, in spite of a high degree of structural similarity, are able to selectively recognize and regulate individual effector enzymes in a Ca2+-dependent manner. NCS proteins vary at their C-termini, which could therefore serve as structural control elements providing specific functions such as target recognition or Ca2+ sensitivity. Recoverin, an NCS protein operating in vision, regulates the activity of rhodopsin kinase, GRK1, in a Ca2+-dependent manner. In the present study, we investigated a series of recoverin forms that were mutated at the C-terminus. Using pull-down assays, surface plasmon resonance spectroscopy and rhodopsin phosphorylation assays, we demonstrated that truncation of recoverin at the C-terminus significantly reduced the affinity of recoverin for rhodopsin kinase. Site-directed mutagenesis of single amino acids in combination with structural analysis and computational modelling of the recoverin-kinase complex provided insight into the protein-protein interface between the kinase and the C-terminus of recoverin. Based on these results we suggest that Phe3 from the N-terminal helix of rhodopsin kinase and Lys192 from the C-terminal segment of recoverin form a cation-π interaction pair which is essential for target recognition by recoverin. Taken together, the results of the present study reveal a novel rhodopsin-kinase-binding site within the C-terminal region of recoverin, and highlights its significance for target recognition and regulation.