P2Y1 and P2Y12 receptor cross-talk in calcium signalling: Evidence from nonstarved and long-term serum-deprived glioma C6 cells

The current work presents results of experiments on the calcium response evoked by the stimulation by extracellular nucleotides occurring in control, nonstarved glioma C6 cells and in cells after long-term (96 h) serum starvation. Three nucleotide receptors were studied: P2Y₁, P2Y₂ and P2Y₁₂. Two of them, P2Y₁ and P2Y₂, directly stimulate calcium response. The protein level of the P2Y₂ receptor did not change during the serum starvation, while P2Y₁ protein level fell dramatically. Observed changes in the calcium response generated by P2Y₁ are directly correlated with the receptor protein level as well as with the amount of calcium present in the intracellular calcium stores, partially depleted during starvation process. The third receptor, P2Y₁₂, did not directly evoke calcium response, however it is activated by the same ligand as P2Y₁. The experiments with AR-C69941MX, the P2Y₁₂-specific antagonist, indicated that in control and serum-starved cells, calcium response evoked by P2Y₁ receptor is potentiated by the activity of P2Y₁₂-dependent signaling pathways. This potentiation may be mediated by P2Y₁₂ inhibitory effect on the plasma membrane calcium pump. The calcium influx enhanced by the cooperation of P2Y₁ and P2Y₁₂ receptor activity directly depends on the capacitative calcium entrance mechanism.     

Disentangling effects of disturbance severity and frequency: Does bioindication really work?

Ecological disturbances are recognized as a crucial factor influencing the attributes of ecological communities. Depending on the specific adaptation or life cycle, plant species show different responses to disturbances of different magnitudes. Herben et al. (Journal of Vegetation Science, 27, 628–636) proposed six disturbance indicator values (DIVs) that describe the niches of Central‐European plant species along gradients of disturbance frequency and severity. Here, we ask if the DIVs can be used in community ecology for bioindication of disturbance regime?We used a dataset of riparian forests sampled within mountain catchments (the Sudetes, SW Poland). As the regime of disturbance is driven by changes in floods from the spring toward the mouth, we calculated the position of every plot along longitudinal (upstream–downstream) gradient and used it as a proxy for the disturbance severity and frequency. We then calculated the community‐weighted means (CWMs) for each of the six indices for each plot and analyzed whether these indices reflected the position of the plots along the rivers. We expected an increase in the severity indices and a decrease in the frequency indices downstream along the rivers. Moreover, we analyzed relationships between disturbance indices and species optima along longitudinal gradient.Surprisingly, means for all analyzed indices increased along the rivers. Severity indices showed the strongest association with the longitudinal gradient. The disturbance severity index for herbs was the only index that differed significantly among species with different responses along longitudinal gradient. On these results, we identified a strong correlation between the severity and frequency indices as the main problem.We conclude that the DIVs have considerable applicative potential; however, the determination of ecological niches separately for disturbance severity and frequency is difficult because different components interact to shape the realized niche of each species. All analyzed indices encompass different attributes of the disturbance regime including both severity and frequency.     

Polymorphism of nuclear DNA in selected species of Taraxacum sect. Palustria

This paper presents the results of research on nuclear DNA polymorphism in six apomictic species of marsh dandelions (Taraxacum sect. Palustria): Taraxacum bavaricum, T. belorussicum, T. brandenburgicum, T. paucilobum, T. subdolum and T. vindobonense. The studies demonstrated the existence of clear genetic differences between species and the existence of nuclear DNA polymorphism within each of the studied species.     

The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

The complexity of chromatin architecture presents a significant barrier to the ability of the DNA repair machinery to access and repair DNA double-strand breaks (DSBs). Consequently, remodeling of the chromatin landscape adjacent to DSBs is vital for efficient DNA repair. Here, we demonstrate that DNA damage destabilizes nucleosomes within chromatin regions that correspond to the γ-H2AX domains surrounding DSBs. This nucleosome destabilization is an active process requiring the ATPase activity of the p400 SWI/SNF ATPase and histone acetylation by the Tip60 acetyltransferase. p400 is recruited to DSBs by a mechanism that is independent of ATM but requires mdc1. Further, the destabilization of nucleosomes by p400 is required for the RNF8-dependent ubiquitination of chromatin, and for the subsequent recruitment of brca1 and 53BP1 to DSBs. These results identify p400 as a novel DNA damage response protein and demonstrate that p400-mediated alterations in nucleosome and chromatin structure promote both chromatin ubiquitination and the accumulation of brca1 and 53BP1 at sites of DNA damage.     

Structural and Biochemical Insights into the Peptidoglycan Hydrolase Domain of FlgJ from Salmonella typhimurium

FlgJ is a glycoside hydrolase (GH) enzyme belonging to the Carbohydrate Active enZyme (CAZy) family GH73. It facilitates passage of the bacterial flagellum through the peptidoglycan (PG) layer by cleaving the β-1,4 glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid sugars that comprise the glycan strands of PG. Here we describe the crystal structure of the GH domain of FlgJ from bacterial pathogen Salmonella typhimurium (StFlgJ). Interestingly, the active site of StFlgJ was blocked by the C-terminal α-helix of a neighbouring symmetry mate and a β-hairpin containing the putative catalytic glutamic acid residue Glu223 was poorly resolved and could not be completely modeled into the electron density, suggesting it is flexible. Previous reports have shown that the GH73 enzyme Auto from Listeria monocytogenes is inhibited by an N-terminal α-helix that may occlude the active site in similar fashion. To investigate if the C-terminus of StFlgJ inhibits GH activity, the glycolytic activity of StFlgJ was assessed with and without the C-terminal α-helix. The GH activity of StFlgJ was unaffected by the presence or absence of the α-helix, suggesting it is not involved in regulating activity. Removal of the C-terminal α-helix did, however, allow a crystal structure of the domain to be obtained where the flexible β-hairpin containing residue Glu223 was entirely resolved. The β-hairpin was positioned such that the active site groove was fully solvent-exposed, placing Glu223 nearly 21.6 Å away from the putative general acid/base residue Glu184, which is too far apart for these two residues to coordinate glycosidic bond hydrolysis. The mobile nature of the StFlgJ β-hairpin is consistent with structural studies of related GH73 enzymes, suggesting that a dynamic active site may be common to many GH73 enzymes, in which the active site opens to capture substrate and then closes to correctly orient active site residues for catalysis.