Distinct Distribution of Ectopically Expressed Histone Variants H2A.Bbd and MacroH2A in Open and Closed Chromatin Domains

Background

It becomes increasingly evident that nuclesomes are far from being identical to each other. This nucleosome diversity is due partially to the existence of histone variants encoded by separate genes. Among the known histone variants the less characterized are H2A.Bbd and different forms of macroH2A. This is especially true in the case of H2A.Bbd as there are still no commercially available antibodies specific to H2A.Bbd that can be used for chromatin immunoprecipitation (ChIP).

Methods

We have generated HeLa S3 cell lines stably expressing epitope-tagged versions of macroH2A1.1, H2A.Bbd or canonical H2A and analyzed genomic distribution of the tagged histones using ChIP-on-chip technique.

Results

The presence of histone H2A variants macroH2A1.1 and H2A.Bbd has been analyzed in the chromatin of several segments of human chromosomes 11, 16 and X that have been chosen for their different gene densities and chromatin status. Chromatin immunoprecipitation (ChIP) followed by hybridization with custom NimbleGene genomic microarrays demonstrated that in open chromatin domains containing tissue-specific along with housekeeping genes, the H2A.Bbd variant was preferentially associated with the body of a subset of transcribed genes. The macroH2A1.1 variant was virtually absent from some genes and underrepresented in others. In contrast, in closed chromatin domains which contain only tissue-specific genes inactive in HeLa S3 cells, both macroH2A1.1 and H2A.Bbd histone variants were present and often colocalized.

Conclusions

Genomic distribution of macro H2A and H2A.Bbd does not follow any simple rule and is drastically different in open and closed genomic domains.     

Induction of N–malonyl–D–tryptophan by drought stress. Is D–tryptophan the only D–amino acid appeared in wilted leaves?

D-amino acid were searched in wilted tomato leaves. D-Isomers of free amino acids were not revealed by the treatment with L- and D-amino acid oxidases. The noncationic fraction of the extract contained N-malonyl-D-tryptophan and no other N-acylated amino acids. A special search for endogenous N-malonyl-D-phenylalanine gave negative results. Exogenous¹⁴C-malonate was only incorporated in one Chromatographic zone corresponding to N-malonyl-D-tryptophan. It is concluded that drought stress does not induce the appearance of D-amino acids except for D-tryptophan which is accumulated in the malonylated form.     

Spontaneous aneuploidy level in blood cells of fertile females

In this study, we presented results of molecular cytogenetic assay of blood cells of fertile women of reproductive age. Cultivated and uncultivated cells and two sets of FISH probes with direct and indirect labeling were used. The middle level of aneuploidy for four chromosomes and statistical limits for aneuploidy detection were estimated. Aneuploidy determined with direct multicolor FISH in cultivated lymphocytes varied from 0.1 to 1.3%. The middle level of aneuploidy for all four chromosomes (13, 18, 21, and X) was 1.39%. The limit of mutation detection was 3.4%. We found differences in results with direct and indirect labeling. It was shown that the cultivation process influenced the level of aneuploidy. The absolute error in FISH technique was 0.13% and the relative error was 4.08%.     

Lipid Metabolism of Caddisfly Larvae at Low pH

The influence of low pH (5.0 and 4.0) on lipid metabolism of caddisfly larvae Hydropsyche contubernalis L. (Trichoptera) was studied in 48 h toxicity experiments. The results were correlated with lipid composition of caddisfly larvae directly isolated from natural water. Phospholipids, cholesterol, mono-, di-, triacylglycerols, and fatty acids were detected by thin-layer and liquid chromatography. Minimal environmental changes were shown to initiate the biochemical adaptation mechanisms strengthening the cellular membranes through their condensation due to additional phospholipid and cholesterol synthesis. In the natural medium the adaptation processes are more active than in the artificial medium. More serious changes, such as pH decrease to 4.0, suppress the adaptation processes in the first medium and terminate them in the second one.     

Variation in seed protein and isoenzyme patterns in Cucurbita cultivars

The genetic variability in the seed proteins and the enzyme alcohol dehydrogenase (ADH) in representative species of the genus Cucurbita was studied. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gel. A specific protein components and ADH isoenzymes were established in the polymorphic banding patterns which can be applied individually or in combination as potential biochemical markers for breeding purposes.     

Inhibitory and enhancing effects of piroxicam on whole blood chemiluminescence.

The effects of piroxicam on the production of reactive oxygen species by stimulated phagocytes was studied in whole blood by a chemiluminescence (CL) technique in relation to maximum activity, localization and kinetics of radical generation. We found that piroxicam dose-dependently inhibited total (intra- and extracellular) zymosan-stimulated luminol CL (LCL) at a high stimulant concentration (p = 0.0001). Piroxicam additionally decreased cytochalasin B-reduced LCL, which shows that the effect of the drug should be sought in the extracellular component of the response. Piroxicam inhibited the first phase of extracellular LCL in a dose-dependent manner (p = 0.0001) and revealed itself as an enhancing agent of CL in later time intervals after the start of respiratory burst, in a model system containing horseradish peroxidase (HRP) and sodium azide. It enhanced LCL of a cell-free system, i.e. influenced the CL due to HRP-catalysed decomposition of hydrogen peroxide. It also dose-dependently inhibited the early extracellular superoxide production, evaluated by lucigenin CL (p = 0.022). Piroxicam inhibited the total fMLP-stimulated LCL by 70% approximately and, only by about 30%, the first phase of fMLP-stimulated extracellular LCL, which presupposes an effect on myeloperoxidase-catalysed formation of hypochloric acid. Piroxicam slightly increased the intracellular LCL by phagocytes (p = 0.02), an effect that is probably connected with its ability to induce the release of secondary messengers in signal transduction. In conclusion, the anti-inflammatory effect of piroxicam is probably related to the inhibition of the extracellular generation of superoxide and hypochloric acid in the early stages of phagocyte activation.     

Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.     

Polyfluorinated salicylic acid derivatives as analogs of known drugs: Synthesis,molecular docking and biological evaluation

We have developed the convenient methods for synthesis of polyfluorosalicylic acids and their derivatives. For the first time the biological properties of polyfluorosalicylates were investigated in vitro (permeability through the biological membranes, COX-1 inhibitory action) and in vivo (anti-inflammatory, analgesic activities, acute toxicity). Molecular docking of polyfluorinated salicylates confirmed in vitro and in vivo experiments.