Improved procedure for detection of superoxide dismutase isoforms in potato,Solanum tuberosum L.

Superoxide dismutase (SOD) in-gel activity assay with selective inhibitors (KCN and H₂O₂) is one of the most commonly used methods for identification of SOD isoform types, i.e., FeSOD, MnSOD or Cu/ZnSOD, and evaluation of oxidative stress response in plants. However, there are potential pitfalls that surround this assay, such as problem to detect isoforms with low activity, comigration of SOD isoforms or application of inappropriate inhibitor concentration. We propose an improved method based on the combination of in-gel analysis of SOD activity and native-PAGE immunoblotting for identification of isoforms and determination of SOD isoenzyme activity pattern in potato. Depending on cultivar and growing conditions, one MnSOD, 3 FeSOD and 5–6 Cu/ZnSOD isoforms were identified in potato leaves. The most important qualitative difference between ex vitro- and in vitro-grown plants was the presence of additional FeSOD and Cu/ZnSOD isoforms in plantlets grown in vitro. Compared with results of in-gel activity assay with selective inhibitors, new method allowed accurate identification of comigrating FeSOD and Cu/ZnSOD isoforms and two protein bands of ambiguous identities. Potato SODs were also characterized by SDS-PAGE immunoblotting and single MnSOD (23.6 kDa), three Cu/ZnSOD polypeptides (17.9, 17 and 16.3 kDa) and single FeSOD (25.1 kDa) polypeptide were detected in leaves of four examined cultivars. The difference in the number of FeSOD and Cu/ZnSOD isoforms/polypeptides between native-PAGE and SDS-PAGE immunoblots suggests that SOD proteins may have undergone post-translational modifications affecting protein mobility or existence of isoforms that differ from each other in total protein charge, but not in molecular weight.     

Global warming effects on benthic macroinvertebrates: a model case study from a small geothermal stream

The aim of this study was to predict global warming effects on benthic macroinvertebrate community structure by using a small temperate geothermal stream as a model system. We collected benthic macroinvertebrates, measured physical and chemical water properties at eight localities up the Kudo?ki stream steep water temperature gradient, and used 11 metrics and indexes to characterize community structure. Species richness and evenness decreased, but total abundance increased with the increase of average annual water temperature (t _av), with species richness being most and total abundance least sensitive to this parameter. The increase of Gastropoda relative abundance and the decrease of Ephemeroptera, Plecoptera and Trichoptera richness, respectively, were the earliest responses of taxonomic groups to t _av increase. Relative abundance of Orthocladiinae decreased and that of Chironomini increased with the increase of t _av. This indicates that Chironomidae are not reliable predictors of global warming effects in running waters, and that lower taxonomic levels, subfamily or tribe, are more suitable for that purpose. Changes in community structure did not linearly follow t _av increase, since a great community shift was observed at t _av ≈ 20°C indicating that present trends of community responses to changes in climatic conditions should not be linearly extrapolated to future warming.     

Acute and subchronic co-administrations to cadmium,diazinon and selenium induce apparent osteoporotic symptoms in adult male rats

Cadmium (Cd) and diazinon (DZN) are known to be environmental risk factors for various bone diseases including osteoporosis. Selenium (Se), an essential constituent of many antioxidant enzymes, has in higher concentrations negative effects on the bone. The present study was aimed to investigate possible changes in femoral bone of adult male rats after their acute and subchronic exposures to Cd, DZN and Se. A total of 30 male Wistar rats were randomized into three experimental groups. The rats in the group A (4-months-old) were injected intraperitoneally with a mixture of 2 mg CdCl₂ kg^?1, 20 mg DZN kg^?1 and 2 mg Na₂SeO₃ kg^?1 body weight and killed 36 h after xenobiotics had been injected. In the group B, young males (1-month-old) were administered with a combination of 30 mg CdCl₂ L^?1, 40 mg DZN L^?1 and 5 mg Na₂SeO₃ L^?1 in their drinking water, for 90 days. Ten 4-months-old males without toxicant supplementation served as a control group (C). After treatment period, detailed histological analysis of femoral bone was performed in each group. Our results revealed apparent osteoporotic symptoms (resorption lacunae, osteoporotic fractures) in rats from groups A and B. Moreover, histomorphometrical evaluation showed reduced bone vascularization (constricted primary osteons’ vascular canals and Haversian canals) and weakness mechanical properties of bones (smaller size of the secondary osteons) in these rats in comparison with those of the control group. Our study demonstrates for the first time that acute and subchronic co-administrations to Cd, DZN and Se induce evident manifestation characteristics of osteoporosis in male rats.     

Transmembrane Adaptor Protein PAG/CBP Is Involved in both Positive and Negative Regulation of Mast Cell Signaling

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.     

High genetic diversity in isolated populations of Thesium ebracteatum at the edge of its distribution range

The aim of this study was to estimate the degree and distribution of genetic diversity within Central-European populations of Thesium ebracteatum—one of the most endangered plant species in Europe. By analyzing allozymes from 17 populations, we estimated the distribution of genetic diversity and suggest the most valuable populations for conservation. Analysis of molecular variance results showed the highest variance existed between populations (54 %), whereas the mean variance within populations was 46 %. A surprisingly low degree of variance (less than 1 %) was found between the six studied regions. We also observed no correlation between geographical and genetic distance, which supports the idea that individual populations are strongly isolated. T. ebracteatum undergoes extensive clonal growth and may survive for very long periods of time without generative reproduction. Consistent with this, we found a strong and significant relationship between genetic diversity and population size. All populations occupying an area greater than 300 m² showed high genetic diversity, whereas small populations contained less genetic diversity. Therefore, conservation priorities could generally be decided based on population size. Because this species is a weak competitor, existing localities should also be managed to prevent species loss from habitat degradation, by mowing or from time to time otherwise disturbing population areas to create open areas for growth.     

Population densities and habitat use of the golden jackal (Canis aureus) in farmlands across the Balkan Peninsula

The main objective of this study was to analyze the habitat use and population densities of the golden jackal in four countries across lowland regions of the Balkan Peninsula, known as the core area of the species' distribution in Europe. Using indirect (acoustic) method for detecting territorial golden jackals, we surveyed jackal presence and densities on 331 monitoring sites in four countries, covering area an of 4,296 km² in total during April and May 2007–2012. We used GIS to assess landscape and environmental characteristics in a 2-km circular buffer (12.6 km²) around calling stations. Average population density of golden jackals in the study areas ranged between 0.6 and 1.1 territorial groups/10 km² (mean ± SE, 0.6?±?0.06 groups/10 km²), with several high-density areas with up to 4.8 territorial groups/10 km². Analysis of habitat use showed that for both jackal occurrence and number of jackal groups, the only significant parameter was the interaction between country and intensity of agriculture, indicating that jackals adapt their habitat selection patterns in relation to the habitat availability. We observed that selection of the more suitable habitats (shrub–herbaceous vegetation/heterogeneous agricultural vegetation) increased with lower proportion of these habitat types in the study area. Our study confirms high habitat plasticity of the golden jackal and offers explanation for its recent range expansion, which might be connected with the land use changes during the last decades in the Balkan Peninsula.     

In vitro antimicrobial activity of propolis and synergism between propolis and antimicrobial drugs

The aim of this study was to investigate antimicrobial properties of ethanolic extract of 13 propolis (EEP) samples from different regions of Serbia against 39 microorganisms (14 resistant or multiresistant to antibiotics), and to determine synergistic activity between antimicrobials and propolis. Antimicrobial activity of propolis samples was evaluated by agar diffusion and agar dilution method. The synergistic action of propolis with antimicrobial drugs was assayed by the disc diffusion method on agar containing subinhibitory concentrations of propolis. Obtained results indicate that EEP, irrespectively of microbial resistance to antibiotics, showed significant antimicrobial activities against Gram-positive bacteria (MIC 0.078%–1.25% of EEP) and yeasts (0.16%–1.25%), while Gram-negative bacteria were less susceptible (1.25&%ndash;>5%). Enterococcus faecalis was the most resistant Gram-positive bacterium, Salmonella spp. the most resistant Gram-negative bacteria, and Candida albicans the most resistant yeast. EEP showed synergism with selected antibiotics, and displayed ability to enhance the activities of antifungals. The shown antimicrobial potential of propolis alone or in combination with certain antibiotics and antifungals is of potential medical interest.     

GFAP-positive astrocytes are rare or absent in primary adult human brain tissue cultures

Traditionally, astrocytes are divided into fibrous and protoplasmic types based on their morphologic appearance. Here the cultures were prepared separately from the adult human cortical gray and white matter of brain biopsies. Both cultures differed only in the number of glial fibrillary acidic protein (GFAP)-positive cells. In the gray matter these were absent or rare, whereas in confluent cultures from the white matter they reached 0.1% of all cells. Three main morphologic types of GFAP-positive cells were found in this study: stellate, bipolar and large flat cells. GFAP-positive cells with two or three long processes mimic a neuron-like morphology. We did not find process-bearing cells expressing neuronal markers (MAP-2, NF, and N-CAM). The conflicting reports concerning GFAP immunostaining and the study dealing with the presence of putative neurons in adult human brain cultures are discussed with respect to these findings. The latter classification of astrocytes into type 1 and type 2 is based on immunostaining to A2B5 antigen: type 1 (GFAP+/A2B5−) and type 2 (GFAP+/A2B5+) astrocytes are proposed to be analogous to protoplasmic and fibrous astrocytes, respectively. In adult human brain cultures we found only small amount of A2B5-positive cells. Double immunofluorescence revealed that astroglial cells of similar fibrous or bipolar shape grown on one coverslip were either GFAP+/A2B5+ or GFAP+/A2B5−. On the other hand, the A2B5+/GFAP− immunophenotype was not observed. These results indicate that in general the cell phenotype from adult human brain tissue is not well established when they are in culture.