Tackling the epigenome in the pluripotent stem cells

Embryonic stem cells are unique in their abilities of self-renewal and to differentiate into many, if not all, cellular lineages. Transcrip- tional regulation, epigenetic modifications and chromatin structures are the key modulators in controlling such pluripotency nature of embryonic stem cell genomes, particularly in the developmental decisions and the maintenance of cell fates. Among them, epigenetic regulation of gene expression is mediated partly by covalent modifications of core histone proteins including methylation, phosphoryla- tion and acetylation. Moreover, the chromatins in stem cell genome appear as a highly organized structure containing distinct functional domains. Recent rapid progress of new technologies enables us to take a global, unbiased and comprehensive view of the epigenetic modifications and chromatin structures that contribute to gene expression regulation and cell identity during diverse developmental stages. Here, we summarized the latest advances made by high throughput approaches in profiling epigenetic modifications and chromatin con- formations, with an emphasis on genome-wide analysis of histone modifications and their implications in pluripotency nature of embry- onic stem cells.     

Proteome analysis of hepatocellular carcinoma by two-dimensional difference gel electrophoresis: novel protein markers in hepatocellular carcinoma tissues

Hepatocellular carcinoma (HCC) is a highly malignant tumor, and chronic infection with hepatitis B virus is one of its major risk factors. To identify the proteins involved in HCC carcinogenesis, we used two-dimensional fluorescence DIGE to study the differentially expressed proteins in tumor and adjacent nontumor tissue samples. Samples from 12 hepatitis B virus-associated HCC patients were analyzed. A total of 61 spots were significantly up-regulated (ratio >/= 2, p 相似文献   

Mir-206 Regulates Pulmonary Artery Smooth Muscle Cell Proliferation and Differentiation

Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.     

Meiosis-activating sterol and the maturation of isolated mouse oocytes

This study was carried out to examine the effects of the meiosis-activating C(29) sterol, 4,4-dimethyl-5 alpha-cholesta-8,14, 24-trien-3 beta-ol (FF-MAS), on mouse oocyte maturation in vitro. Cumulus cell-enclosed oocytes (CEO) and denuded oocytes (DO) from hormonally primed, immature mice were cultured 17-18 h in minimum essential medium (MEM) containing 4 mM hypoxanthine plus increasing concentrations of FF-MAS. The sterol induced maturation in DO with an optimal concentration of 3 microg/ml but was without effect in CEO, even at concentrations as high as 10 microg/ml. Some stimulation of maturation in hypoxanthine-arrested CEO was observed when MEM was replaced by MEMalpha. Interestingly, the sterol suppressed the maturation of hypoxanthine-arrested CEO in MEM upon removal of glucose from the medium. FF-MAS also failed to induce maturation in DO when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP). The rate of maturation in FF-MAS-stimulated, hypoxanthine-arrested DO was slow, as more than 6 h of culture elapsed before significant meiotic induction was observed, and this response required the continued presence of the sterol. Although the oocyte took up radiolabeled lanosterol, such accumulation was restricted by the presence of cumulus cells. In addition, lanosterol failed to augment FSH-induced maturation and was even inhibitory at a high concentration. Moreover, the downstream metabolite, cholesterol, augmented the inhibitory action of dbcAMP on maturation in both CEO and DO. Two inhibitors of 14 alpha-demethylase, ketoconazole, and 14 alpha-ethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol that can suppress FF-MAS production from lanosterol failed to block consistently FSH-induced maturation. These results confirm the stimulatory action of FF-MAS on hypoxanthine-arrested DO but do not support a universal meiosis-inducing function for this sterol.     

Solution structure of the first intracellular loop of prostacyclin receptor and implication of its interaction with the C-terminal segment of G alpha s protein

The amino acids (residues 39-51) responsible for the interaction between the first intracellular loop (iLP1) of the human prostacyclin receptor (IP) and G alpha s protein have been identified [Zhang, L., Huang, G., Wu, J., and Ruan, K. H. (2005) Biochemistry 44, 11389-11401]. To further characterize the structural/functional relationship of the iLP1 in coupling with the G alpha s protein, the solution structures of a constrained peptide (IP iLP1) that mimicked the iLP1 of the IP receptor in the absence and presence of a synthetic peptide, corresponding to the C-terminal 11 residues (Q384-L394 in the protein sequence) of the G alpha s protein (G alpha s-Ct), were determined by 2D 1H NMR spectroscopy. The NMR solution structural model of the iLP1 domain showed two turn structures in residues Arg41-Ala44 and Arg45-Phe49 with the conserved Arg45 at the center. The conformational change of the side chain of the Arg45 was observed upon the addition of the G alpha s-Ct peptide. On the other hand, the solution structural models of the G alpha s-Ct peptide in the absence and presence of the IP iLP1 peptide were also determined. The N-terminal domain (Q384-Q390 in the G alpha s protein) of the peptide adopted an alpha-helical conformation. However, the helical structure of the C-terminal domain (Q390-E392 in the G alpha s protein) of the peptide was destabilized upon addition of the IP iLP1 peptide. These structural studies have implied that there are direct or indirect contacts between the IP iLP1 domain and the C-terminal residues of the G alpha s protein in the receptor/G protein coupling. The possible charge and hydrophobic interactions between the two peptides were also discussed. These data prompted intriguing speculations on the IP/G alpha s coupling which mediates vasodilatation and inhibition of platelet aggregation.     

A single amino acid determines the catalytic efficiency of two alkenal double bond reductases produced by the liverwort Plagiochasma appendiculatum

Alkenal double bond reductases (DBRs) catalyze the NADPH-dependent reduction of the α,β-unsaturated double bond of many secondary metabolites. Two alkenal double bond reductase genes PaDBR1 and PaDBR2 were isolated from the liverwort species Plagiochasma appendiculatum. Recombinant PaDBR2 protein had a higher catalytic activity than PaDBR1 with respect to the reduction of the double bond present in hydroxycinnamyl aldehydes. The residue at position 56 appeared to be responsible for this difference in enzyme activity. The functionality of a C56 to Y56 mutation in PaDBR1 was similar to that of PaDBR2. Further site-directed mutagenesis and structural modeling suggested that the phenol ring stacking between this residue and the substrate was an important determinant of catalytic efficiency.     

Risk Factors of Pneumothorax after Endobronchial Ultrasound-Guided Transbronchial Biopsy for Peripheral Lung Lesions

Background

The risk of endobronchial ultrasound-guided transbronchial biopsy-related pneumothorax is a major concern and warrants further studies. The aim of our study was to estimate the risk of pneumothorax after this procedure and identify its risk factors.

Methods

From 2007 to 2011, 399 patients who underwent endobronchial ultrasound-guided transbronchial biopsy for peripheral lung lesions were included in this study. The variables analyzed included patient factors, lesion factors and procedure factors. Multivariate logistic regression analysis was used to identify independent risk factors for pneumothorax.

Results

The incidence of pneumothorax was 3.3% (13/399). Chest tube placement was required for 31% (4/13) of pneumothoraces. Independent risk factors for pneumothorax included pulmonary emphysema (OR, 55.09; 95% CI, 9.37–324.03; p<0.001) and probe position adjacent to the lesion (OR, 17.01; 95% CI, 2.85–101.64; p = 0.002). The number of biopsy specimens, age, sex, history of prior lung surgery and lesion size, location and character did not influence the risk of pneumothorax in our analyses.

Conclusions

The risk of pneumothorax after endobronchial ultrasound-guided transbronchial biopsy is low. To further reduce the risk of pneumothorax, every effort should be made to advance the endobronchial ultrasound probe into the bronchus where it is imaged within the target lesion before embarking on transbronchial biopsy.     

The control of single-celled cotton fiber elongation by developmentally reversible gating of plasmodesmata and coordinated expression of sucrose and K+ transporters and expansin

Each cotton fiber is a single cell that elongates to 2.5 to 3.0 cm from the seed coat epidermis within approximately 16 days after anthesis (DAA). To elucidate the mechanisms controlling this rapid elongation, we studied the gating of fiber plasmodesmata and the expression of the cell wall-loosening gene expansin and plasma membrane transporters for sucrose and K(+), the major osmotic solutes imported into fibers. Confocal imaging of the membrane-impermeant fluorescent solute carboxyfluorescein (CF) revealed that the fiber plasmodesmata were initially permeable to CF (0 to 9 DAA), but closed at approximately 10 DAA and re-opened at 16 DAA. A developmental switch from simple to branched plasmodesmata was also observed in fibers at 10 DAA. Coincident with the transient closure of the plasmodesmata, the sucrose and K(+) transporter genes were expressed maximally in fibers at 10 DAA with sucrose transporter proteins predominately localized at the fiber base. Consequently, fiber osmotic and turgor potentials were elevated, driving the rapid phase of elongation. The level of expansin mRNA, however, was high at the early phase of elongation (6 to 8 DAA) and decreased rapidly afterwards. The fiber turgor was similar to the underlying seed coat cells at 6 to 10 DAA and after 16 DAA. These results suggest that fiber elongation is initially achieved largely by cell wall loosening and finally terminated by increased wall rigidity and loss of higher turgor. To our knowledge, this study provides an unprecedented demonstration that the gating of plasmodesmata in a given cell is developmentally reversible and is coordinated with the expression of solute transporters and the cell wall-loosening gene. This integration of plasmodesmatal gating and gene expression appears to control fiber cell elongation.     

Involvement of phosphatidylinositol 3-kinase-mediated up-regulation of I kappa B alpha in anti-inflammatory effect of gemfibrozil in microglia

The present study underlines the importance of PI3K in mediating the anti-inflammatory effect of gemfibrozil, a prescribed lipid-lowering drug for humans, in mouse microglia. Gemfibrozil inhibited LPS-induced expression of inducible NO synthase (iNOS) and proinflammatory cytokines in mouse BV-2 microglial cells and primary microglia. By overexpressing wild-type and dominant-negative constructs of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) in microglial cells and isolating primary microglia from PPAR-alpha-/- mice, we have demonstrated that gemfibrozil inhibits the activation of microglia independent of PPAR-alpha. Interestingly, gemfibrozil induced the activation of p85alpha-associated PI3K (p110beta but not p110alpha) and inhibition of that PI3K by either chemical inhibitors or dominant-negative mutants abrogated the inhibitory effect of gemfibrozil. Conversely, overexpression of the constitutively active mutant of p110 enhanced the inhibitory effect of gemfibrozil on LPS-induced expression of proinflammatory molecules. Similarly, gemfibrozil also inhibited fibrillar amyloid beta (Abeta)-, prion peptide (PrP)-, dsRNA (poly IC)-, HIV-1 Tat-, and 1-methyl-4-phenylpyridinium (MPP+)-, but not IFN-gamma-, induced microglial expression of iNOS. Inhibition of PI3K also abolished the inhibitory effect of gemfibrozil on Abeta-, PrP-, poly IC-, Tat-, and MPP+-induced microglial expression of iNOS. Involvement of NF-kappaB activation in LPS-, Abeta-, PrP-, poly IC-, Tat-, and MPP+-, but not IFN-gamma-, induced microglial expression of iNOS and stimulation of IkappaBalpha expression and inhibition of NF-kappaB activation by gemfibrozil via the PI3K pathway suggests that gemfibrozil inhibits the activation of NF-kappaB and the expression of proinflammatory molecules in microglia via PI3K-mediated up-regulation of IkappaBalpha.