Methodology to label mixed carbohydrate components by APTS

Labelling of carbohydrates with fluorescent dyes is of importance in the analysis of complex oligo- or polysaccharides. The complexity of the hydrolization mixtures of the oligosaccharides can be studied with separation methods after labelling. However, the procedures might provide unexpected phenomena. The experimental circumstances for labelling carbohydrates with 8-aminopyrene-1,3,6-trisulfonic acid were studied and the labelling efficiency of mono and oligosaccharides present in endotoxins was followed by capillary electrophoresis using LIF detection. Significant differences were observed in the labelling of the different sugar molecules, and the lowest reactivity with the dye was observed in the cases of amino-sugars. The results provide a basis for studies in determining the structures of the core parts and the O-chains of bacterial endotoxins.     

Protein kinase C modulates negatively the hepatocyte growth factor-induced migration, integrin expression and phosphatidylinositol 3-kinase activation

Previously, we reported that, in hepatocyte growth factor (HGF)-induced HepG2 cells, protein kinase C (PKC) decreased the duration of intensive Erk1/Erk2 MAP kinase activation. This study shows that the inhibition of PKC enhanced significantly the HGF-induced integrin expression. Beside the prolonged activation of Erk1/Erk2, the activity of phosphatidylinositol 3-kinase (PI 3K) was required for growth factor-induced integrin expression. PI 3-kinase was activated to a higher extent in response to HGF than to epidermal growth factor (EGF), though the activation was transient in both cases. In EGF-induced cells, PI 3K activation was terminated by the loss of phosphotyrosine docking sites for PI 3K. To the contrary, the decrease of PI 3K activation, which followed the HGF-induced increase was not accompanied by the loss of phosphotyrosine docking sites and was prevented by the inhibition of PKC. The negative modulator effects of PKC on integrin expression and PI 3-kinase activation correlated with its ability to limit the HGF-induced motogen response.     

Bacterial models for tumor development. Mini-review

The tumor-inducing effects of Agrobacterium, Bartonella and Helicobacter bacterial species are compared step by step. An analogy for the existence of these individual steps is considered in connection with the development of cancer. The transformations of eukaryotic cells occur in particular in the type IV secretion system, i.e. involving the simultaneous transmission of DNA and protein from bacterial cells to eukaryotic cells. Thus, transfected cells facilitate the indefinite growth of tissue cells and additionally produce growth factors, triggering further bacterial multiplication. The higher numbers of bacteria then produce more transfection and the cycle repeats as long as the host lives. The main limiting factor is the frequency of bacterial infection, while the secondary rate-limiting factors are the levels of transforming growth factors and factors triggering bacteria growth. CONCLUSIONS: Analogous processes are probably responsible for the tumor induction by the three different bacterial species; however, the critical points for eradication are different. The early eradication or limitation of B. henselae or H. pylori can prevent hemangiomas, stomach cancer and malignant cell proliferation. The crown gall formation by A. tumefaciens can only be avoided by prevention of the transforming activity of a single bacterial infection. Questions arise as to what is common in the three processes, and the nature of the rate-limiting step in the three different models. The frequency of transformation is the rate-limiting step, but the co-transmission of the DNA-protein complex is common in the three systems.     

Nitrogen deficiency hinders etioplast development in stems of dark‐grown pea (Pisum sativum) shoot cultures

The effects of nitrogen (N) deprivation were studied in etiolated pea plants (Pisum sativum cv. Zsuzsi) grown in shoot cultures. The average shoot lengths decreased and the stems significantly altered considering their pigment contents, 77 K fluorescence spectra and ultrastructural properties. The protochlorophyllide (Pchlide) content and the relative contribution of the 654–655 nm emitting flash‐photoactive Pchlide form significantly decreased. The etioplast inner membrane structure characteristically changed: N deprivation correlated with a decrease in the size and number of prolamellar bodies (PLBs). These results show that N deficiency directly hinders the pigment production, as well as the synthesis of other etioplast inner membrane components in etiolated pea stems.     

Prescribed burning as an alternative management in grasslands of temperate Europe: The impact on seeds

Fire is not an integral part of terrestrial ecosystems in temperate Europe, nevertheless prescribed burning is proposed to be an alternative to traditional management applied to grasslands. Thus, anthropogenic fire represents a serious challenge to plant species, and there is no information on how the recruitment of species responds to fire. The aim of our study was to assess the effect of fire on seed germination of 16 herbaceous grassland and ruderal species belonging to four common families. We first assessed the fuel load in open habitats in early spring and measured soil temperatures during experimental fires. After that we performed a controlled pot experiment involving heat treatments and experimental fire applied to seeds and followed their germination. Our measurements showed that maximum temperatures are between 73 °C and 264 °C on the ground surface and fire passage is fast, with short residence times of high temperatures. In deeper soil layers (1 cm and 5 cm), temperature increase is negligible. Seed germination of half of the species was decreased by the passage of fire, and a heat shock of 100 °C for 5 min had an even stronger adverse effect. Seeds of three Fabaceae species were stimulated by heat or fire, while negative effects prevailed among species belonging to other families. Anthropogenic fire in grasslands of temperate Europe might reduce recruitment by seed, particularly in species of the Asteraceae and Poaceae, two very important families with a large representation in temperate grasslands. Our results indicate that prescribed burning should be carefully applied in order not to endanger the local persistence of grassland species whose seedling emergence is negatively affected by fire.     

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH)

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant–fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period.     

Etioplasts with protochlorophyll and protochlorophyllide forms in the under‐soil epicotyl segments of pea (Pisum sativum) seedlings grown under natural light conditions

To study if etiolation symptoms exist in plants grown under natural illumination conditions, under‐soil epicotyl segments of light‐grown pea (Pisum sativum) plants were examined and compared to those of hydroponically dark‐grown plants. Light‐, fluorescence‐ and electron microscopy, 77 K fluorescence spectroscopy, pigment extraction and pigment content determination methods were used. Etioplasts with prolamellar bodies and/or prothylakoids, protochlorophyll (Pchl) and protochlorophyllide (Pchlide) forms (including the flash‐photoactive 655 nm emitting form) were found in the (pro)chlorenchyma of epicotyl segments under 3 cm soil depth; their spectral properties were similar to those of hydroponically grown seedlings. However, differences were found in etioplast sizes and Pchlide:Pchl molar ratios, which indicate differences in the developmental rates of the under‐soil and of hydroponically developed cells. Tissue regions closer to the soil surface showed gradual accumulation of chlorophyll, and in parallel, decrease of Pchl and Pchlide. These results proved that etioplasts and Pchlide exist in soil‐covered parts of seedlings even if they have a 3–4‐cm long photosynthetically active shoot above the soil surface. This underlines that etiolation symptoms do develop under natural growing conditions, so they are not merely artificial, laboratory phenomena. Consequently, dark‐grown laboratory plants are good models to study the early stages of etioplast differentiation and the Pchlide–chlorophyllide phototransformation.     

Inducible phosphorylation of cortactin is not necessary for cortactin-mediated actin polymerisation

Cortactin is an SH3 domain-containing protein that contributes to the formation of dynamic cortical actin-associated structures, such as lamellipodia and membrane ruffles. It was originally identified as a substrate for the protein kinase Src; however, the role of tyrosine phosphorylation in the translocation of cortactin to the cell periphery and in the subsequent actin polymerisation is still unclear. Recently, two serine/threonine kinases, Pak1 and Erk, have been implicated in the regulation of cortactin. Therefore, we systematically investigated whether phosphorylation on either tyrosine or serine/threonine residues is necessary for cortactin function. In COS7 cells over-expressing Vav2 or treated with EGF, we could not detect tyrosine phosphorylation, although cortactin was translocated to cell periphery and induced membrane ruffle formation. In addition, the selective MEK inhibitor, PD98059, did not influence in vivo the ability of cortactin to bind to and induce membrane ruffles upon Vav2 over-expression or short-term EGF treatment. Finally, using a constitutively active Pak1 mutant, Pak1 T423E, we showed that Pak1 is not capable of phosphorylating cortactin either in vitro or in COS7 cells. These results suggest that cortactin-mediated actin polymerisation at cell periphery requires only Rac activation but neither tyrosine nor serine/threonine phosphorylation.