Identification of protease-activated receptor-4 (PAR-4) in puromycin-purified brain capillary endothelial cells cultured on Matrigel

An improved method is described for culturing primary rat brain capillary endothelial cells (RBCEC) on glass, covered by Matrigel. The procedure using Matrigel yields spindle-shaped endothelial cells exhibiting close cell-cell appositions seen on electron microscopic sections. These cells permanently express tight junction proteins ZO-1, claudin-5 and the adherent junction protein beta-catenin, as revealed by immunofluorescence. Furthermore, glass coverslips covered with Matrigel provide a stable and low-background fluorescent base for microfluorimetric calcium measurements. By this method, hereby we show that the PAR-4 agonist peptide induces transient [Ca2+]i changes with different kinetics compared to that due to activation of the PAR-1 receptor. This indicates that RBCE cells grown on Matrigel express PAR-4 receptors.     

Genetic Variation and Differentiation of Peat-Bog and Dry-Meadow Populations of the Dwarf Mountain Pine Pinus mugo Turra in the Highlands of the Ukrainian Carpathians

Based on electrophoretic analysis of 21 isozyme loci controlling 10 enzyme systems, the intra- and interpopulation variation was studied in two peat-bog and three dry-meadow populations of the dwarf mountain pine Pinus mugoTurra from the highlands of the Ukrainian Carpathians. In the studied samples (a total of 164 trees), on average 62% of the studied genes were polymorphic; the mean heterozygosity was 21.3%. The dry-meadow populations differed from the peat-bog populations by allele and genotype diversity and by heterozygosity although the indices characterizing population heterogeneity (F _st and G _st) were small (0.027 and 0.032, respectively). Nei's genetic distances between the populations ranged of 0.011 to 0.032 with the mean of 0.018.     

Investigation toward multi-epitope vaccine candidates using native chemical ligation

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.     

Biochemical assay-based selectivity profiling of clinically relevant kinase inhibitors on mutant forms of EGF receptor

Gefitinib and erlotinib are potent EGFR tyrosine kinase inhibitors (potentially) useful for the treatment of non-small-cell lung cancer (NSCLC). Clinical responses, however, in NSCLC patients have been linked to the presence of certain activating mutations of EGFR. We used an ELISA-based biochemical assay to confirm the selective inhibitory efficacy of gefitinib and erlotinib on the activated mutant receptor. Our results are in line with the clinical observations providing evidence for the predictive power of the kinase assay. Four additional compounds were also investigated: CI-1033 and EKB-569 had dramatic inhibitory effects on all EGFR forms, whereas PD153035 and AG1478 were active on wild-type and activating mutant protein. In docking simulations with wild-type EGFR, our inhibitory data are in good agreement with the binding scores. These data confirm that anilinoquinazolines are good starting structures for the next generation of selective drugs against mutant EGFR, whereas CI-1033 and EKB-569 may represent advances for patients with both wild-type and anilinoquinazoline-resistant mutant tumors.     

Root-Zone-Specific Oxygen Tolerance of Azospirillum spp. and Diazotrophic Rods Closely Associated with Kallar Grass

The effect of oxygen on N₂-dependent growth of two Azospirillum strains and two diazotrophic rods closely associated with roots of Kallar grass (Leptochloa fusca) was studied. To enable precise comparison, bacteria were grown in dissolved-oxygen-controlled batch and continuous cultures. Steady states were obtained from about 1 to 30 μM O₂, some of them being carbon limited. All strains needed a minimum amount of oxygen for N₂-dependent growth. Nitrogen contents between 10 and 13% of cell dry weight were observed. The response of steady-state cultures to increasing O₂ concentrations suggested that carbon limitation shifted to internal nitrogen limitation when N₂ fixation became so low that the bacteria could no longer meet their requirements for fixed nitrogen. For Azospirillum lipoferum Rp5, increase of the dilution rate resulted in decreased N₂ fixation in steady-state cultures with internal nitrogen limitation. Oxygen tolerance was found to be strain specific in A. lipoferum with strain Sp59b as a reference organism. Oxygen tolerance of strains from Kallar grass was found to be root zone specific. A. halopraeferens Au 4 and A. lipoferum Rp5, predominating on the rhizoplane of Kallar grass, and strains H6a2 and BH72, predominating in the endorhizosphere, differed in their oxygen tolerance profiles. Strains H6a2 and BH72 still grew and fixed nitrogen in steady-state cultures at O₂ concentrations exceeding those which absolutely inhibited nitrogen fixation of both Azospirillum strains. It is proposed that root-zone-specific oxygen tolerance reflects an adaptation of the isolates to the microenvironments provided by the host plant.     

Identification of allelic state of leaf rust-resistance Lr34 gene in the cultivars of soft winter wheat of Ukrainian breeding

The distribution of alleles at the Lr34 locus associated with leaf rust resistance among winter common wheat (Triticum aestivum L.) cultivars of Ukrainian breeding was studied. Co-dominant molecular-genetic marker cssfr5 was used for detection of the allelic condition of the Lr34 locus. The cultivars with detected allele Lr34(+) were identified as potentially "resistant" and the cultivars in which detected allele Lr34(-) were identified as potentially "susceptible". The collection of the cultivars (81 ones), created in the main plant breeding centers of Ukraine was analyzed. The allele Lr34(+) was identified in 44% cultivars. Results were compared with the data about the distribution of the Lr34(+) in cultivars created in different countries.     

Protective effects of resveratrol on calcium-induced oxidative stress in rat heart mitochondria

Trans-resveratrol is a nutraceutical with known antioxidant, anti-inflammatory, cardioprotective, and anti-apoptotic properties. The aim of this study was to evaluate the effects of resveratrol on heart mitochondria. Resveratrol significantly decreased Fe²⁺ + ascorbate oxidant system-induced lipid peroxide levels, preserved physiological levels of glutathione, and increased nitric oxide (NO) levels in mitochondria. Under calcium-mediated stress, there was a 2.7-fold increase in the NO levels, and a mild decoupling in the mitochondrial respiratory chain. These results provide a mechanism for and support the beneficial effects of resveratrol under pathological conditions induced by oxidative stress and calcium overload. In addition, these findings underscore the usefulness of resveratrol in the prevention of cardiovascular diseases.     

Common Buzzards wintering strategies as an effect of weather conditions and geographic barriers

AimMigration is a constantly changing adaptation due to the climate condition evolution. The struggle for surviving during harsh winter season is different across Europe, being more complex toward the inner parts of the continent. The current approach explores the Common Buzzard number variation during the cold season and the climatic predictors of birds of prey wintering movements in relation to the possible influences of the Carpathian Mountains, which may act as a geographical barrier providing shelter from cold air outbreak from north and northeast of the continent.LocationRomania (45°N25°E).TaxonBirds of Prey.MethodsWe applied a GLMM to investigate the relation between continental and local climatic factors with the number of Common Buzzard observations in two regions. The first region is located inside the Carpathian Arch and the other one outside, east of this large mountains chain.ResultsThe Common Buzzard numbers wintering Eastern from the Carpathian Mountains are highly influenced by AO (Z = 2.87, p < .05%), while those wintering western are influenced by NAO (Z = 2.17, p < .05%). This is the first proof of separating influences for biodiversity of AO and NAO at continental scale, outlining the influence limit placed over the Eastern Carpathian Mountains.Main conclusionsThe Carpathian Mountains act like a geographic barrier, separating the wintering Common Buzzard populations from both sides of the mountain range. While the high number of individuals in Moldova is related to their eastern and northeastern Europe origins, in Transylvania the large number of individuals observed is related to the more sheltered characteristics of the region attracting individuals from central Europe. Also, since Transylvania region is well sheltered during cold air outbreak, it represents a more favorable region for wintering. From this point of view, we can consider that the Carpathian Mountains are a geographic barrier for wintering birds of prey.     

cDNA library construction from meristematic tissue of finger millet panicle

A protocol to obtain full-length cDNA using a SuperScript® Full-Length cDNA Library Construction Kit II (Invitrogen, United States) was developed, and a high quality cDNA library of meristem tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was constructed. The titer of the constructed cDNA library was 3.01 × 10⁵ CFU/mL, the average length of the insert was approximately 1070 base pairs, and the average efficiency of insertion of cDNA fragments was 99.5%. The sequencing of randomly selected clones created cDNA library was carried out. The cDNA sequences of clones were identified by BLAST search. The cDNA library analysis and selective sequencing indicate good functionality and full size of cDNA inserts of the clones. The constructed cDNA library from meristematic tissue of finger millet panicle is a good and reliable source for isolation and identification of key genes of metabolism and development of meristem as well for creation of new genetic markers for genetic research and molecular selection.