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.     

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.     

Estimating intrinsic structural preferences of de novo emerging random-sequence proteins: Is aggregation the main bottleneck?

Present-day proteins are believed to have evolved features to reduce the risk of aggregation. However, proteins can emerge de novo by translation of non-coding DNA segments. In this study we assess the aggregation, disorder and transmembrane propensity of protein sequences generated by translating random nucleotide sequences of varying GC-content. Potential de novo random-sequence proteins translated from regions with GC content between 40% and 60% do not show stronger aggregation propensity than existing ones and exhibit similar tendency to be disordered. We suggest that de novo emerging proteins do not mean an unavoidable aggregation threat to evolving organisms.     

The antimotility action of a trifluoromethyl ketone on some gram-negative bacteria

The inhibition of bacterial motility was studied by a trifluoro methyl ketone derivative on two Escherichia coli strains (wild strain having a proton pump system and the proton pump-deficient mutant strain) and two Helicobacter pylori strains (clarithromycin susceptible and clarithromycin resistant). Evidence is presented of the inhibitory action of 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone (TF18) on the proton motive forces of the two bacterial strains by affecting the action of biological motor and proton efflux in the membranes. The swimming, the forward motion was more sensitive than the vibration or tumbling to the inhibition. We suppose that the inhibiton of bacterial motility is related to the virulence of bacteria: consequently the pathogenicity can be reduced in the presence of TF18.     

Structure, enzymatic stability and antitumor activity of sea lamprey GnRH-III and its dimer derivatives

Direct antitumor activity of sea lamprey (Petromyzon marinus) gonadotropin-releasing hormone III (Glp-His-Trp-Ser-His-Asp-Trp-Lys-Pro-Gly-NH(2); lGnRH-III) was described on several tumor cells. To improve the selectivity of antitumor effects without increasing the hormone releasing activity and to enhance the enzymatic stability, lGnRH-III dimers were prepared via disulfide bond formation. Our results demonstrate that the lGnRH-III dimer derivatives exhibited higher antiproliferative effect and enzymatic stability in comparison with the native lGnRH-III, while lower LH-releasing potency was determined. In order to find a correlation between the biological and structural features of these compounds, the conformation of lGnRH-III and its dimer derivatives was determined by ECD, VCD, FT-IR and (1)H NMR.     

Complete mitochondrial genome of freshwater pearl mussel Lamellidens marginalis (Lamarck, 1819) and its phylogenetic relation within unionidae family

Molecular Biology Reports - Freshwater mussels play a key role in ecology and are often considered as ecological indicators. Conversely, these molluscs are one of the most threatened groups due to...     

Improved extraction procedure for the isolation of β-D-galactosidase from Escherichia coli

Summary A fast 4-step isolation procedure for -D-galactosidase from E. coli has been developed: cell disruption, two-stage aqueous two-phase extraction and ultrafiltration. A 60-fold purification of the enzyme with a total yield of 75% was achieved.