Epinephrine, through its action on beta-adrenoceptors, may induce endothelium-dependent vascular dilation, and this action is partly mediated by a cytosolic Ca(2+) ([Ca(2+)](i)) change in endothelial cells. In the present study, we explored the molecular identity of the channels that mediate epinephrine-induced endothelial Ca(2+) influx and subsequent vascular relaxation. Patch clamp recorded an epinephrine- and cAMP-activated cation current in the primary cultured bovine aortic endothelial cells (BAECs) and H5V endothelial cells. L-cis-diltiazem and LY-83583, two selective inhibitors for cyclic nucleotide-gated channels, diminished this cation current. Furthermore, this cation current was greatly reduced by a CNGA2-specific siRNA in H5V cells. With the use of fluorescent Ca(2+) dye, it was found that epinephrine and isoprenaline, a beta-adrenoceptor agonist, induced endothelial Ca(2+) influx in the presence of bradykinin. This Ca(2+) influx was inhibited by L-cis-diltiazem and LY-83583, and by a beta(2)-adrenoceptor antagonist ICI-118551. CNGA2-specific siRNA also diminished this Ca(2+) influx in H5V cells. Furthermore, L-cis-diltiazem and LY-83583 inhibited the endothelial Ca(2+) influx in isolated mouse aortic strips. L-cis-diltiazem also markedly reduced the endothelium-dependent vascular dilation to isoprenaline in isolated mouse aortic segments. In summary, CNG channels, CNGA2 in particular, mediate beta-adrenoceptor agonist-induced endothelial Ca(2+) influx and subsequent vascular dilation.

Heart formation requires a highly balanced network of transcriptional activation of genes. The homeodomain transcription factor, Shox2, is essential for the formation of the sinoatrial valves and for the development of the pacemaking system. The elucidation of molecular mechanisms underlying the development of pacemaker tissue has gained clinical interest as defects in its patterning can be related to atrial arrhythmias. We have analyzed putative targets of Shox2 and identified the Bmp4 gene as a direct target. Shox2 interacts directly with the Bmp4 promoter in chromatin immunoprecipitation assays and activates transcription in luciferase-reporter assays. In addition, ectopic expression of Shox2 in Xenopus embryos stimulates transcription of the Bmp4 gene, and silencing of Shox2 in cardiomyocytes leads to a reduction in the expression of Bmp4. In Tbx5(del/+) mice, a model for Holt-Oram syndrome, and Shox2(-/-) mice, we show that the T-box transcription factor Tbx5 is a regulator of Shox2 expression in the inflow tract and that Bmp4 is regulated by Shox2 in this compartment of the embryonic heart. In addition, we could show that Tbx5 acts cooperatively with Nkx2.5 to regulate the expression of Shox2 and Bmp4. This work establishes a link between Tbx5, Shox2 and Bmp4 in the pacemaker region of the developing heart and thus contributes to the unraveling of the intricate interplay between the heart-specific transcriptional machinery and developmental signaling pathways.

Interest in grape seed proanthocyanidin (GSP) as a cholesterol-lowering nutraceutical is growing. This study was to investigate the effect of GSP on blood cholesterol level and gene expression of cholesterol-regulating enzymes in Golden Syrian hamsters maintained on a 0.1% cholesterol diet. Results affirmed supplementation of 0.5% or 1.0% GSP could decrease plasma total cholesterol and triacylglycerol level. Western blot and real-time polymerase chain reaction analyses demonstrated GSP did not affect sterol regulatory element binding protein-2 and low-density lipoprotein receptor; however, it increased mRNA 3-hydroxy-3-methylglutaryl coenzyme A reductase. GSP had no effect on the protein mass of liver X receptor alpha (LXRα) but it decreased mRNA LXRα. Most importantly, GSP increased not only the protein level of cholesterol-7α-hydroxylase (CYP7A1) but also mRNA CYP7A1. It was concluded that the hypocholesterolemic activity of GSP was most likely mediated by enhancement of bile acid excretion and up-regulation of CYP7A1.

Previous results showed that over-expression of the WTH3 gene in MDR cells reduced MDR1 gene expression and converted their resistance to sensitivity to various anticancer drugs. In addition, the WTH3 gene promoter was hypermethylated in the MCF7/AdrR cell line and primary drug resistant breast cancer epithelial cells. WTH3 was also found to be directly targeted and up regulated by the p53 gene. Furthermore, over expression of the WTH3 gene promoted the apoptotic phenotype in various host cells.

Hypertensive patients have been found to be associated with elevated levels of homocysteine, known as hyperhomocysteinemia. Homocysteine (Hcy) can induce endoplasmic reticulum (ER) stress in endothelial cells. This study aims to investigate whether black tea (BT) protects against hypertension-associated endothelial dysfunction through alleviation of ER stress. Rat aortae and cultured rat aortic endothelial cells were treated with Hcy, BT extract, and theaflavin-3,3'-digallate (TF3). Male Sprague Dawley rats were infused with angiotensin II (Ang II) to induce hypertension and orally administrated with BT extract at 15 mg/kg/day for 2 weeks. Hcy impaired endothelium-dependent relaxations of rat aortae and led to ER stress in endothelial cells, which were ameliorated by co-incubation of BT extract and TF3. The blood pressure of Ang II-infused rats and plasma Hcy level were normalized by BT consumption. Impaired endothelium-dependent relaxations in renal arteries, carotid arteries and aortae, and flow-mediated dilatations in third-order mesenteric resistance arteries were improved. Elevations of ER stress markers and ROS level, plus down-regulation of Hcy metabolic enzymes in aortae from Ang II-infused rats were prevented by BT treatment. Our data reveal the novel cardiovascular benefits of BT in ameliorating vascular dysfunctions, providing insight into developing BT into beneficial dietary supplements in hypertensive patients.

RPB1, the largest subunit of RNA polymerase II, contains a highly modifiable C-terminal domain (CTD) that consists of variations of a consensus heptad repeat sequence (Y1S2P3T4S5P6S7). The consensus CTD repeat motif and tandem organization represent the ancestral state of eukaryotic RPB1, but across eukaryotes CTDs show considerable diversity in repeat organization and sequence content. These differences may reflect lineage-specific CTD functions mediated by protein interactions. Mammalian CTDs contain eight non-consensus repeats with a lysine in the seventh position (K7). Posttranslational acetylation of these sites was recently shown to be required for proper polymerase pausing and regulation of two growth factor-regulated genes.

Seizures were induced by flurothyl inhalation. Rats were intramuscularly treated with progesterone after each seizure. Results demonstrated that glutamate transporter 2 and γ-aminobutyric acid transporter 1 expression levels were significantly increased in the cerebral cortex and hippocampus of the developing rat brain following recurrent seizures. After progesterone treatment, glutamate transporter 2 protein expression was upregulated, but γ-aminobutyric acid transporter 1 levels decreased. These results suggest that glutamate transporter 2 and γ-aminobutyric acid transporter 1 are involved in the pathological processes of epilepsy. Progesterone can help maintain a balance between excitatory and inhibitory systems by modulating the amino acid transporter system, and protect the developing brain after recurrent seizures.

Fibro-adipogenic progenitors (FAPs) are important components of the skeletal muscle regenerative environment. Whether FAPs support muscle regeneration or promote fibro-adipogenic degeneration is emerging as a key determinant in the pathogenesis of muscular diseases, including Duchenne muscular dystrophy (DMD). However, the molecular mechanism that controls FAP lineage commitment and activity is currently unknown. We show here that an HDAC-myomiR-BAF60 variant network regulates the fate of FAPs in dystrophic muscles of mdx mice. Combinatorial analysis of gene expression microarray, genome-wide chromatin remodeling by nuclease accessibility (NA) combined with next-generation sequencing (NA-seq), small RNA sequencing (RNA-seq), and microRNA (miR) high-throughput screening (HTS) against SWI/SNF BAF60 variants revealed that HDAC inhibitors (HDACis) derepress a "latent" myogenic program in FAPs from dystrophic muscles at early stages of disease. Specifically, HDAC inhibition induces two core components of the myogenic transcriptional machinery, MYOD and BAF60C, and up-regulates the myogenic miRs (myomiRs) (miR-1.2, miR-133, and miR-206), which target the alternative BAF60 variants BAF60A and BAF60B, ultimately directing promyogenic differentiation while suppressing the fibro-adipogenic phenotype. In contrast, FAPs from late stage dystrophic muscles are resistant to HDACi-induced chromatin remodeling at myogenic loci and fail to activate the promyogenic phenotype. These results reveal a previously unappreciated disease stage-specific bipotency of mesenchimal cells within the regenerative environment of dystrophic muscles. Resolution of such bipotency by epigenetic intervention with HDACis provides a molecular rationale for the in situ reprogramming of target cells to promote therapeutic regeneration of dystrophic muscles.

The accurate and comprehensive identification of functional regulatory sequences in mammalian genomes remains a major challenge. Here we describe site-specific integration fluorescence-activated cell sorting followed by sequencing (SIF-seq), an unbiased, medium-throughput functional assay for the discovery of distant-acting enhancers. Targeted single-copy genomic integration into pluripotent cells, reporter assays and flow cytometry are coupled with high-throughput DNA sequencing to enable parallel screening of large numbers of DNA sequences. By functionally interrogating >500 kilobases (kb) of mouse and human sequence in mouse embryonic stem cells for enhancer activity we identified enhancers at pluripotency loci including NANOG. In in vitro-differentiated cardiomyocytes and neural progenitor cells, we identified cardiac enhancers and neuronal enhancers, respectively. SIF-seq is a powerful and flexible method for de novo functional identification of mammalian enhancers in a potentially wide variety of cell types.

KV 7.1 voltage-gated potassium channels are expressed in vascular smooth muscle cells (VSMC) of diverse arteries, including mesenteric arteries. Based on pharmacological evidence using R-L3 (KV 7.1 channel opener), HMR1556, chromanol 293B (KV 7.1 channel blockers), stimulation of these channels has been suggested to evoke profound relaxation in various vascular beds of rats. However, the specificity of these drugs in vivo is uncertain.

Hydrogen sulfide (H(2)S) is a potent vasodilator. However, the complex mechanisms of vasoregulation by H(2)S are not fully understood. We tested the hypotheses that (1) H(2)S exerts vasodilatory effects by opening KCNQ-type voltage-dependent (K(v)) K(+) channels and (2) that H(2)S-producing cystathionine-γ-lyase (CSE) in perivascular adipose tissue plays a major role in this pathway.

Hyperhomocysteinemia is associated with an increased risk for cardiovascular diseases through increased oxidative stress. Salidroside is an active ingredient of the root of Rhodiola rosea with documented antioxidative, antihypoxia and neuroprotective properties. However, the vascular benefits of salidroside against endothelial dysfunction have yet to be explored. The present study, therefore, aimed to investigate the protective effect of salidroside on homocysteine-induced endothelial dysfunction. Functional studies on the rat aortas were performed to delineate the vascular effect of salidroside. DHE imaging was used to evaluate the reactive oxygen species (ROS) level in aortic wall and endothelial cells. Western blotting was performed to assess the protein expression associated with oxidative stress and nitric oxide (NO) bioavailability. Exposure to homocysteine attenuated endothelium-dependent relaxations in rat aortas while salidroside pretreatment rescued it. Salidroside inhibited homocystein-induced elevation in the NOX2 expression and ROS overproduction in both aortas and cultured endothelial cells and increased phosphorylation of eNOS which was diminished by homocysteine. The present study shows that salidroside is effective in preserving the NO bioavailability and thus protects against homocysteine-induced impairment of endothelium-dependent relaxations, largely through inhibiting the NOX2 expression and ROS production. Our results indicate a therapeutic potential of salidroside in the management of oxidative-stress-associated cardiovascular dysfunction.

Surface-enhanced Raman scattering (SERS) effect is quite preferred to detect trace pollutants, and reusable SERS substrate is of important practical value. In this research, a kind of effective SiO2 nanorods (NRs)@ Au nanoparticles (NPs) substrate was fabricated completely with physical methods, and it was quite sensitive so that 1 × 10(-6) M monochlorobiphenyl (CB) could be detected. Furthermore, congeners of CB could be detected by reusing this kind of SERS substrate, and the cleaning treatment between every two detections was very simple. The excellent performance of the reusable SERS substrate indicated its great application potential.

Cytosolic Ca2+ ([Ca2+]i) is an important signal that regulates cardiomyocyte differentiation during cardiogenesis. TRPV1 is a Ca2+-permeable channel that is expressed in cardiomyocytes. In the present study, we utilized mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) as a model to investigate the functional role of TRPV1 in cardiomyocyte differentiation. Induction of embryonic stem cells into cardiomyocytes was achieved using embryoid body (EB)-based differentiation method. Quantitative PCRs showed an increased TRPV1 expression during the differentiation process. In [Ca2+]i measurement study, application of TRPV1 agonists, capsaicin and camphor, elicited a [Ca2+]i rise in mESC-CMs, the effect of which was abolished by TRPV1-shRNA. In functional study, treatment of EBs with TRPV1 antagonists (capsazepine and SB366791) and TRPV1-shRNA reduced the size of the EBs and decreased the percentage of spontaneously beating EBs. TRPV1 antagonists and TRPV1-shRNA also suppressed the expression of cardiomyocyte marker genes, including cardiac actin, c-TnT, c-TnI, and α-MHC. Taken together, this study demonstrated an important functional role of TRPV1 channels in the differentiation of mESCs into cardiomyocytes.

Blood pressure is maintained within a normal physiological range by a sophisticated regulatory mechanism. Baroreceptors serve as a frontline sensor to detect the change in blood pressure. Nerve signals are then sent to the cardiovascular control centre in the brain in order to stimulate baroreflex responses. Here, we identify TRPC5 channels as a mechanical sensor in aortic baroreceptors. In Trpc5 knockout mice, the pressure-induced action potential firings in the afferent nerve and the baroreflex-mediated heart rate reduction are attenuated. Telemetric measurements of blood pressure demonstrate that Trpc5 knockout mice display severe daily blood pressure fluctuation. Our results suggest that TRPC5 channels represent a key pressure transducer in the baroreceptors and play an important role in maintaining blood pressure stability. Because baroreceptor dysfunction contributes to a variety of cardiovascular diseases including hypertension, heart failure and myocardial infarction, our findings may have important future clinical implications.

Phospholipase C, β1 (PLCB1) plays critical roles in intracellular transduction and regulating signal activation which are important to tumorigenesis. However, the mechanism of PLCB1 in hepatocellular carcinoma (HCC) is still unknown. This study aims to investigate whether its expression is associated with the clinicopathological parameters and prognosis of the patients with HCC.

E-selectin is a surface marker of endothelial cell (EC) inflammation, one of the hallmarks of atherogenesis. Thus, we tested the hypothesis that delivery of microRNA (miR)-146a and miR-181b with an E-selectin-targeting multistage vector (ESTA-MSV) to inflamed endothelium covering atherosclerotic plaques inhibits atherosclerosis. Cy5-conjugated miR-146a and miR-181b were packaged in polyethylene glycol-polyethyleneimine (PEG/PEI) nanoparticles and loaded into ESTA-MSV microparticles. Both miRs were downregulated in tumor necrosis factor (TNF)-α-treated ECs. Transfection of TNF-α-treated mouse aortas and cultured ECs with miRs was more efficient with ESTA-MSV than with the PEG/PEI. Likewise, miR-146a/-181b packaged in ESTA-MSV efficiently suppressed the chemokines, CCL2, CCL5, CCL8, and CXCL9, and monocyte adhesion to ECs. Complementary in vivo tests were conducted in male apolipoprotein E-deficient mice fed a Western diet and injected intravenously with the particles prepared as above biweekly for 12 weeks. Treatment with miRs packaged in ESTA-MSV but not in PEG/PEI reduced atherosclerotic plaque size. Concurrently, vascular inflammation markers, including macrophages in aortic root lesions and chemokine expression in aortic tissues were reduced while the vascular smooth muscle cells and collagen increased in plaques from ESTA-MSV/miRs-treated vs. vehicle-treated mice. Our data supported our hypothesis that ESTA-MSV microparticle-mediated delivery of miR-146a/-181b ameliorates endothelial inflammation and atherosclerosis.

Haematococcus pluvialis is a freshwater species of Chlorophyta, family Haematococcaceae. It is well known for its capacity to synthesize high amounts of astaxanthin, which is a strong antioxidant that has been utilized in aquaculture and cosmetics. To improve astaxanthin yield and to establish genetic resources for H. pluvialis, we performed whole-genome sequencing, assembly, and annotation of this green microalga. A total of 83.1 Gb of raw reads were sequenced. After filtering the raw reads, we subsequently generated a draft assembly with a genome size of 669.0 Mb, a scaffold N50 of 288.6 kb, and predicted 18,545 genes. We also established a robust phylogenetic tree from 14 representative algae species. With additional transcriptome data, we revealed some novel potential genes that are involved in the synthesis, accumulation, and regulation of astaxanthin production. In addition, we generated an isoform-level reference transcriptome set of 18,483 transcripts with high confidence. Alternative splicing analysis demonstrated that intron retention is the most frequent mode. In summary, we report the first draft genome of H. pluvialis. These genomic resources along with transcriptomic data provide a solid foundation for the discovery of the genetic basis for theoretical and commercial astaxanthin enrichment.

DNA shape adds specificity to sequence motifs but has not been explored systematically outside this context. We hypothesized that DNA-binding proteins (DBPs) preferentially occupy DNA with specific structures ("shape motifs") regardless of whether or not these correspond to high information content sequence motifs. We present ShapeMF, a Gibbs sampling algorithm that identifies de novo shape motifs. Using binding data from hundreds of in vivo and in vitro experiments, we show that most DBPs have shape motifs and can occupy these in the absence of sequence motifs. This "shape-only binding" is common for many DBPs and in regions co-bound by multiple DBPs. When shape and sequence motifs co-occur, they can be overlapping, flanking, or separated by consistent spacing. Finally, DBPs within the same protein family have different shape motifs, explaining their distinct genome-wide occupancy despite having similar sequence motifs. These results suggest that shape motifs not only complement sequence motifs but also facilitate recognition of DNA beyond conventionally defined sequence motifs.

Chromatin remodeling complexes instruct cellular differentiation and lineage specific transcription. The BRG1/BRM-associated factor (BAF) complexes are important for several aspects of differentiation. We show that the catalytic subunit gene Brg1 has a specific role in cardiac precursors (CPs) to initiate cardiac gene expression programs and repress non-cardiac expression. Using immunopurification with mass spectrometry, we have determined the dynamic composition of BAF complexes during mammalian cardiac differentiation, identifying several cell-type specific subunits. We focused on the CP- and cardiomyocyte (CM)-enriched subunits BAF60c (SMARCD3) and BAF170 (SMARCC2). Baf60c and Baf170 co-regulate gene expression with Brg1 in CPs, and in CMs their loss results in broadly deregulated cardiac gene expression. BRG1, BAF60c and BAF170 modulate chromatin accessibility, to promote accessibility at activated genes while closing chromatin at repressed genes. BAF60c and BAF170 are required for proper BAF complex composition, and BAF170 loss leads to retention of BRG1 at CP-specific sites. Thus, dynamic interdependent BAF complex subunit assembly modulates chromatin states and thereby participates in directing temporal gene expression programs in cardiogenesis.