CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

Visualizing stable features in live cell nucleus for evaluation of the cell global motion compensation.

The compensation of cell motion is an important step in single-particle tracking analysis of live cells. This step is required in most of the cases, since the movement of subcellular foci is superimposed by the movement and deformation of the cell, while only the local motion of the foci is important to be analysed. The cell motion and deformation compensation is usually performed by means of image registration. There are a number of approaches with different models and properties presented in the literature that perform cell image registration. However, the evaluation of the registration approach quality on real data is a tricky problem due to the fact that some stable features in the images with a priori no local motion are needed. In this paper we propose a methodology for creating live cell nuclei image sequences with stable features imposed. The features are defined using the regions of fluorescence bleaching invoked by the UV laser. Data with different deformations are acquired and can be used for evaluation of the cell image registration methods. Along with that, we describe an image analysis technique and a metric that can characterize the quality of the method quantitatively. The proposed methodology allows building a ground truth dataset for testing and thoroughly evaluating cell image registration methods.

PRMT1 arginine methyltransferase accumulates in cytoplasmic bodies that respond to selective inhibition and DNA damage.

Protein arginine methyltransferases (PRMTs) are responsible for symmetric and asymmetric methylation of arginine residues of nuclear and cytoplasmic proteins. In the nucleus, PRMTs belong to important chromatin modifying enzymes of immense functional significance that affect gene expression, splicing and DNA repair. By time-lapse microscopy we have studied the sub-cellular localization and kinetics of PRMT1 after inhibition of PRMT1 and after irradiation. Both transiently expressed and endogenous PRMT1 accumulated in cytoplasmic bodies that were located in the proximity of the cell nucleus. The shape and number of these bodies were stable in untreated cells. However, when cell nuclei were microirradiated by UV-A, the mobility of PRMT1 cytoplasmic bodies increased, size was reduced, and disappeared within approximately 20 min. The same response occurred after γ-irradiation of the whole cell population, but with delayed kinetics. Treatment with PRMT1 inhibitors induced disintegration of these PRMT1 cytoplasmic bodies and prevented formation of 53BP1 nuclear bodies (NBs) that play a role during DNA damage repair. The formation of 53BP1 NBs was not influenced by PRMT1 overexpression. Taken together, we show that PRMT1 concentrates in cytoplasmic bodies, which respond to DNA injury in the cell nucleus, and to treatment with various PRMT1 inhibitors.

[Higher incidence of thyropathy in patients with oesophageal achalasia. Genetic, autoimmune, regional or just a random association?]. / Vyssí výskyt tyreopatií u achalázie jícnu. Genetická, autoimunitní, regionální ci jen náhodná souvislost?

INTRODUCTION: The etiology of esophageal achalasia is still largely unknown. Inflammatory response to an initial stimulus on the level of genetic and/or immune predisposition may be the underlying cause of the disease. The final result is progressive disappearance of ganglion cells in the myenteric plexus and motility disorder. Autoimmune thyropathy (AIT) is a typical disease involving genetic background and immune response disorder. PATIENTS AND METHODS: 44 patients (of which 30 women and 14 men) with diagnosed esophageal achalasia and a control group of patients with esophageal reflux of corresponding age and sex were screened for thyroid disease. RESULTS: Thyroid disease was diagnosed in 15 out of 44 patients with achalasia (34%). Thyropathy was detected in 11 women (37%) and 4 men (28%). AIT was detected in 10 patients, in 4 of whom with hypfunction, nontoxic cystic or nodular goitre was detected in 4 patients, 1 patient was after strumectomy for benign node. Positive antithyroid antibody was newly detected in 4 patients with achalasia; subclinical hypothyreosis was found in one of them. There were two cases ofAIT with subclinical hypofunction and 1 case of nontoxic goitre in the control group (7%). The difference was statistically significant (p < 0.01). CONCLUSION: The incidence of thyroid disease proved higher in patients with achalasia than in the controls. The rate of occurrence of thyroid disease exceeded significantly the occurrence in the population. The association of achalasia with prevailingly autoimmune thyropathy may corroborate the importance of autoimmunity in the etiopathogenesis of the disease.

Oral N-acetylcysteine attenuates the rat pulmonary inflammatory response to antigen.

Oxidative stress is involved in the pathophysiology of inflammatory airway diseases including asthma; therefore, antioxidants might be of clinical benefit in asthma treatment. In the present study, the effects of N-acetylcysteine on sensitised brown Norway rats were examined. N-Acetylcysteine (3 mmol kg body weight(-1) administered orally) was given daily for 1 week before challenge and various antigen-induced pulmonary responses were studied. Antigen exposure increased lipid peroxidation in bronchoalveolar lavage fluid (BALF) and oxidised glutathione levels in lung tissue 2 h after challenge. Lung nuclear transcription factor-KB-binding activity was increased 2 h after challenge, and BALF tumour necrosis factor-alpha and inducible nitric oxide synthase expression in lungs peaked 4 h after challenge. Expression of intercellular adhesion molecule-1 and mucin MUC5AC was also increased 4 h after challenge. These changes in oxidant status, transcription factor activation, and inflammatory cytokine and gene expression were reduced by N-acetylcysteine. This thiol did not affect the immediate bronchospasm reaction to antigen in anaesthetised rats but inhibited airways hyperresponsiveness to 5-hydroxytryptamine and the augmented eosinophil numbers in BALF, which appear 24 h after exposure of conscious rats to antigen aerosol, and abolished antigen-induced extravasation of Evans blue into BALF. These results indicate that oral N-acetylcysteine exerts an antioxidant protective effect and attenuates pulmonary inflammation in experimental asthma.

Effects of inhaled glaucine on pulmonary responses to antigen in sensitized guinea pigs.

The alkaloid (S)-(+)-1,2,9,10-tetramethoxyaporphine (glaucine) is a phosphodiesterase 4 inhibitor with bronchodilator and anti-inflammatory activity in vitro. In this study, we examined the in vivo effects of glaucine on an animal model of asthma. In ovalbumin sensitized guinea pigs, inhaled glaucine (10 mg ml(-1), 3 min) inhibited the acute bronchoconstriction produced by aerosol antigen (antigen response was 256+/-42 and 95+/-14 cm H(2)O l(-1) s(-1) in control and glaucine-treated animals, respectively; P<0.05). Pretreatment with glaucine (10 mg ml(-1), 10 min inhalation, 30 min pre- and 3 h post-antigen exposure) markedly reduced airway hyperreactivity to histamine, eosinophil lung accumulation, and increased eosinophil peroxidase activity in bronchoalveolar lavage fluid 24 h after exposure of conscious guinea pigs to aerosol antigen. In addition, inhaled glaucine (5-10 mg ml(-1), 3 min) inhibited the microvascular leakage produced after inhaled antigen at all airway levels. These data support the potential interest of phosphodiesterase 4 inhibitors in asthma treatment.

The effects of subchronical exposure to SO2 on biochemical and hematological parameters in guinea pigs.

The effects of subchronical exposure to SO2 (400ppm, 3 hours daily, 28 days) on biochemical and hematological parameters were investigated in guinea pigs. Mostly no significant changes in the values of biochemical parameters and no significant changes in hematological parameters were found. The levels of investigated ions (K+, Na+, Cl-, Ca++, Mg++ and phosphates), proteins (albumines, globulines, total proteins), enzymes (LD, ALT, AST, CK) and other biochemical parameters (urea, creatinine, bilirubin) were not significantly different between groups, with the exception of a significantly higher ALP concentration in the exposed group as compared with controls (2.17 mukat and 1.85 mukat, respectively. It can be concluded that a subchronical exposure to sulphur dioxide mostly did not induce any definite changes in biochemical and hematological parameters in guinea pigs.

[Oxygen radicals in the mechanism of lung injury caused by air pollutants]. / Kyslíkové radikály v mechanizmoch poskodenia pl'úc vyvolaného polutantmi ovzdusia.

Ozone and nitrogen oxides are environmental pollutants especially present in smog. They are oxidants potent to cause bronchial hyperreactivity, lung fibrosis, and emphysema. Other occupational hazards are mineral dust particles (asbestos, silica) which may cause the development of inflammation and fibrosis in the lungs. Inflammatory cells (neutrophils, eosinophils, monocytes, macrophages) become activated during inflammation and produce superoxide anion radicals. Reactive oxygen species developed e.g. in smog have been recognized--at least partly--to be responsible for the observed pulmonary tissue damage. Several mechanisms leading to toxicity, including disbalance of vegetative nervous control, damage of bronchial epithelium, role of mediators of inflammation and other issues are discussed. (Fig. 1, Ref. 32.)

Separation of biologically active peptides by capillary electrophoresis and high-performance liquid chromatography.

HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta- and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C18 stationary phase and mobile phases of 20-40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4-5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50 mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the D- and L-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM beta-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.