The genus Polyarthra in Lake Peipsi

In Lake Peipsi (Estonia) Polyarthra species often dominate the rotifer community, and are represented by 6 species: Polyarthra dolichoptera Idelson, P. longiremis Carlin, P. luminosa Kutikova, P. major Burckhardt, P. remata Skorikov, P. vulgaris Carlin. The highest diversity occurs in August–September, with P. remata and P. luminosa most abundant. Morphometric data including measurements of body, fins and various types of eggs are given. The seasonal development of the different species and their life cycles are considered.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Transfection of Infectious RNA and DNA/RNA Layered Vectors of Semliki Forest Virus by the Cell-Penetrating Peptide Based Reagent PepFect6

Viral vectors have a wide variety of applications ranging from fundamental studies of viruses to therapeutics. Recombinant viral vectors are usually constructed using methods of reverse genetics to obtain the genetic material of the viral vector. The physicochemical properties of DNA and RNA make them unable to access cells by themselves, and they require assistance to achieve intracellular delivery. Non-viral delivery vectors can be used for this purpose if they enable efficient intracellular delivery without interfering with the viral life cycle. In this report, we utilize Semliki Forest virus (genus alphavirus) based RNA and DNA vectors to study the transfection efficiency of the non-viral cell-penetrating peptide-based delivery vector PepFect6 in comparison with that of the cationic liposome-based Lipofectamine 2000, and assess their impact on viral replication. The optimal conditions for transfection were determined for both reagents. These results demonstrate, for the first time, the ability of PepFect6 to transport large (13-19 kbp) constructs across the cell membrane. Curiously, DNA molecules delivered using the PepFect6 reagent were found to be transported to the cell nucleus approximately 1.5 hours later than DNA molecules delivered using the Lipofectamine 2000 reagent. Finally, although both PepFect6 and Lipofectamine 2000 reagents can be used for alphavirus research, PepFect6 is preferred because it does not induce changes in the normal cellular phenotype and it does not affect the normal replication-infection cycle of viruses in previously transfected cells.     

Soluble CD44 interacts with intermediate filament protein vimentin on endothelial cell surface

CD44 is a cell surface glycoprotein that functions as hyaluronan receptor. Mouse and human serum contain substantial amounts of soluble CD44, generated either by shedding or alternative splicing. During inflammation and in cancer patients serum levels of soluble CD44 are significantly increased. Experimentally, soluble CD44 overexpression blocks cancer cell adhesion to HA. We have previously found that recombinant CD44 hyaluronan binding domain (CD44HABD) and its non-HA-binding mutant inhibited tumor xenograft growth, angiogenesis, and endothelial cell proliferation. These data suggested an additional target other than HA for CD44HABD. By using non-HA-binding CD44HABD Arg41Ala, Arg78Ser, and Tyr79Ser-triple mutant (CD443MUT) we have identified intermediate filament protein vimentin as a novel interaction partner of CD44. We found that vimentin is expressed on the cell surface of human umbilical vein endothelial cells (HUVEC). Endogenous CD44 and vimentin coprecipitate from HUVECs, and when overexpressed in vimentin-negative MCF-7 cells. By using deletion mutants, we found that CD44HABD and CD443MUT bind vimentin N-terminal head domain. CD443MUT binds vimentin in solution with a Kd in range of 12-37 nM, and immobilised vimentin with Kd of 74 nM. CD443MUT binds to HUVEC and recombinant vimentin displaces CD443MUT from its binding sites. CD44HABD and CD443MUT were internalized by wild-type endothelial cells, but not by lung endothelial cells isolated from vimentin knock-out mice. Together, these data suggest that vimentin provides a specific binding site for soluble CD44 on endothelial cells.     

The role of endocytosis on the uptake kinetics of luciferin-conjugated cell-penetrating peptides

Cell-penetrating peptides (CPPs) are short cationic/amphipathic peptides that can be used to deliver a variety of cargos into cells. However, it is still debated which routes CPPs employ to gain access to intracellular compartments. To assess this, most previously conducted studies have relied on information which is gained by using fluorescently labeled CPPs. More relevant information whether the internalized conjugates are biologically available has been gathered using end-point assays with biological readouts. Uptake kinetic studies have shed even more light on the matter because the arbitrary choice of end-point might have profound effect how the results could be interpreted. To elucidate uptake mechanisms of CPPs, here we have used a bioluminescence based assay to measure cytosolic delivery kinetics of luciferin-CPP conjugates in the presence of endocytosis inhibitors. The results suggest that these conjugates are delivered into cytosol mainly via macropinocytosis; clathrin-mediated endocytosis and caveolae/lipid raft dependent endocytosis are involved in a smaller extent. Furthermore, we demonstrate how the involved endocytic routes and internalization kinetic profiles can depend on conjugate concentration in case of certain peptides, but not in case of others. The employed internalization route, however, likely dictates the intracellular fate and subsequent trafficking of internalized ligands, therefore emphasizing the importance of our novel findings for delivery vector development.     

Characterization of heterotrimeric nucleotide-depleted Gα(i)-proteins by Bodipy-FL-GTPγS fluorescence anisotropy

Recombinant heterotrimeric G-protein α_i1, α_i2 and α_i3 subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β₁γ₂ subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α_i were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg²⁺, Mn²⁺ inhibited nucleotide binding to all α_i-heterotrimers studied and accelerated nucleotide release. Mn²⁺ had stabilizing effect on the nucleotide free state of the α_i1 subunit, whereas both Mn²⁺ as well as G-protein activation by mastoparan destabilized the α_i2 subunit.     

Both spatiotemporal connectivity and habitat quality limit the immigration of forest plants into wooded corridors

Extensive afforestation of agricultural areas has increased the importance of green corridors as a dispersal network. We tested the effect of spatiotemporal connectivity, edge effect and habitat structural quality of wooded corridors on the long-term immigration success of forest specialist plants relative to the performance of forest generalists. In agricultural landscapes of central and southern Estonia, we sampled 28 historically connected and 52 isolated tree lines and alleys with a minimum age of 50 years, and 93 edges of ancient forests. The regional pool of common forest plants was compiled using species’ frequency data in 91 ancient forests. Both landscape connectivity and habitat quality affected the richness of response groups, but specialists and generalists responded to different drivers. Forest specialists required long-term neighbourhoods of ancient forest and benefited from a direct connection between forest and corridor. Habitat generalists reacted positively to the recently modified structure of the landscape. When a corridor was connected to forest, the dual edge in the corridor did not result in an increased negative edge effect on forest specialist arrival. Even if both specialists and generalists required wide corridors with optimum shade, forest specialists also benefited from mature overstorey and outward overhanging branches, whereas forest generalists used disturbance-created microhabitats. We conclude that only wooded corridors with long-term connectivity to seed source forests and widely branched tree canopies will function as a green infrastructure supporting forest-specific biodiversity.     

Long‐term colonization ecology of forest‐dwelling species in a fragmented rural landscape – dispersal versus establishment

Species colonization in a new habitat patch is an efficiency indicator of biodiversity conservation. Colonization is a two‐step process of dispersal and establishment, characterized by the compatibility of plant traits with landscape structure and habitat conditions. Therefore, ecological trait profiling of specialist species is initially required to estimate the relative importance of colonization filters. Old planted parks best satisfy the criteria of a newly created and structurally matured habitat for forest‐dwelling plant species. We sampled species in 230 ancient deciduous forests (source habitat), 74 closed‐canopy manor parks (target habitats), 151 linear wooded habitats (landscape corridors), and 97 open habitats (isolating matrix) in Estonia. We defined two species groups of interest: forest (107 species) and corridor specialists (53 species). An extra group of open habitat specialists was extracted for trait scaling. Differing from expectations, forest specialists have high plasticity in reproduction mechanisms: smaller seeds, larger dispersules, complementary selfing ability, and diversity of dispersal vectors. Forest specialists are shorter, less nutrient‐demanding and mycorrhizal‐dependent, stress‐tolerant disturbance‐sensitive competitors, while corridor specialists are large‐seeded disturbance‐tolerant competitors. About 40% of species from local species pools have immigrated into parks. The historic forest area, establishment‐related traits, and stand quality enhance the colonization of forest specialists. The openness of landscape and mowing in the park facilitate corridor specialists. Species traits in parks vary between a forest and corridor specialist, except for earlier flowering and larger propagules. Forest species are not dispersal limited, but they continue to be limited by habitat properties even in the long term. Therefore, the shady parts of historic parks should be appreciated as important forest biodiversity‐enhancing landscape structures. The habitat quality of secondary stands can be improved by nurturing a heterogeneous shrub and tree layer, and modest herb layer management.     

General description of Lake Peipsi-Pihkva

Neural networks and multiple linear regression models of the abundance of brown trout (Salmo trutta L.) on the mesohabitat scale were developed from combinations of physical habitat variables in 220 channel morphodynamic units (pools, riffles, runs, etc.) of 11 different streams in the central Pyrenean mountains. For all the 220 morphodynamic units, the determination coefficients obtained between the estimated and observed values of density or biomass were significantly higher for the neural network (r ² adjusted= 0.93 and r ² adjusted=0.92 (p<0.01) for biomass and density respectively with the neural network, against r ² adjusted=0.69 (p<0.01) and r ² adjusted = 0.54 (p<0.01) with multiple linear regression). Validation of the multivariate models and learning of the neural network developed from 165 randomly chosen channel morphodynamic units, was tested on the 55 other channel morphodynamic units. This showed that the biomass and density estimated by both methods were significantly related to the observed biomass and density. Determination coefficients were significantly higher for the neural network (r ² adjusted =0.72 (p<0.01) and 0.81 (p<0.01) for biomass and density respectively) than for the multiple regression model (r ² adjusted=0.59 and r ² adjusted=0.37 for biomass and density respectively). The present study shows the advantages of the backpropagation procedure with neural networks over multiple linear regression analysis, at least in the field of stochastic salmonid ecology.