Gotowa bibliografia na temat „Relaxation Stimulated Transcription (RST)”

The effects of in vivo treatment with estrogen and progesterone on isoproterenol-induced uterine relaxation and beta(2)-adrenoceptor (beta(2)AR) mRNA production in non-pregnant rat myometrium were investigated. Whether homologous myometrial desensitization of beta(2)AR function was dependent on or modulated by the two steroids was also examined. Estrogen treatment alone or in combination with progesterone reduced maximal relaxation (E(max)) of isolated uterine strips subsequently challenged with isoproterenol whereas progesterone alone had no effect on this parameter. The reduction was accompanied by an enhanced beta(2)AR mRNA concentration. The concentration of isoproterenol giving half-maximal relaxing response (EC(50)) increased following estrogen treatment and this effect was curbed by progesterone. Isoproterenol had no effect on beta(2)AR transcription irrespective of the steroid regimes employed. E(max) of isolated uterine strips was reduced following prolonged in vivo treatment with isoproterenol but the effect was found only when estrogen alone was administered concomitantly. Finally, in vivo treatment with isoproterenol increased EC(50) of uterine strips subsequently stimulated with isoproterenol in vitro. This effect was independent of steroid treatment. We conclude that homologous desensitization of beta(2)AR function in non-pregnant rat myometrium in terms of sensitivity (EC(50)) is independent of sex steroids but in terms of maximal response (E(max)) occurs only in the presence of estrogen. We speculate whether progesterone withdrawal in connection with the well-known estrogen dominance at rat parturition may strengthen the desensitization induced by beta(2)AR activation and thus contribute to the transformation of the uterus from a quiescent to a highly contractile organ.

Salidroside (SAL) is an active component ofRhodiola roseawith documented antioxidative properties. The purpose of this study is to explore the mechanism of the protective effect of SAL on hydrogen peroxide- (H2O2-) induced endothelial dysfunction. Pretreatment of the human umbilical vein endothelial cells (HUVECs) with SAL significantly reduced the cytotoxicity brought by H2O2. Functional studies on the rat aortas found that SAL rescued the endothelium-dependent relaxation and reduced superoxide anion (O2∙-) production induced by H2O2. Meanwhile, SAL pretreatment inhibited H2O2-induced nitric oxide (NO) production. The underlying mechanisms involve the inhibition of H2O2-induced activation of endothelial nitric oxide synthase (eNOS), adenosine monophosphate-activated protein kinase (AMPK), and Akt, as well as the redox sensitive transcription factor, NF-kappa B (NF-κB). SAL also increased mitochondrial mass and upregulated the mitochondrial biogenesis factors, peroxisome proliferator-activated receptor gamma-coactivator-1alpha (PGC-1α), and mitochondrial transcription factor A (TFAM) in the endothelial cells. H2O2-induced mitochondrial dysfunction, as demonstrated by reduced mitochondrial membrane potential (Δψm) and ATP production, was rescued by SAL pretreatment. Taken together, these findings implicate that SAL could protect endothelium against H2O2-induced injury via promoting mitochondrial biogenesis and function, thus preventing the overactivation of oxidative stress-related downstream signaling pathways.

The purpose of this study was to explore the involvement of adenosine receptor(s) in porcine coronary artery (PCA) relaxation and to define the role of MAPK signaling pathways. Isometric tensions were recorded in denuded PCA rings. 5′-( N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine receptor agonist, induced a concentration-dependent relaxation (EC50 = 16.8 nM) of PGF2α (10 μM)-preconstricted arterial rings. NECA-induced relaxation was completely blocked by 0.1 μM SCH-58261 (A2A antagonist) at lower doses (1–40 nM) but not at higher doses (80–1,000 nM). MRS-1706 (1 μM, A2B antagonist) was able to shift the NECA concentration-response curve to the right. CGS-21680 (selective A2A agonist) induced responses similarly to NECA, whereas N6-cyclopentyladenosine (A1 agonist) and Cl-IB-MECA (A3 agonist) did not. Furthermore, the effect of NECA was attenuated by the addition of SB-203580 (10 μM, p38 MAPK inhibitor) but not by PD-98059 (10 μM, MEK inhibitor). Interestingly, SB-203580 had no effect on CGS-21680-induced relaxation. Western blot analysis demonstrated that PGF2α and adenosine agonists stimulated p38 MAPK at a concentration of 40 nM in PCA smooth muscle cells. MRS-1706 (1 μM) significantly reduced NECA-induced p38 MAPK phosphorylation. Addition of NECA and SB-203580 alone or in combination inhibited PGF2α-induced p38 MAPK. Western blot data were further confirmed by p38 MAPK activity measurement using activating transcription factor-2 assay. Our results suggest that the adenosine receptor subtype involved in causing relaxation of porcine coronary smooth muscle is mainly A2A subtype, although A2B also may play a role, possibly through p38 MAPK pathway.

Normal blood vessel tone is maintained by a balance of vasoconstrictors and vasodilators produced by endothelial cells in the vasculature. Nitric oxide (NO) is a potent vasodilator that causes vascular smooth muscle cell relaxation by elevating intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The physiological mechanisms regulating NO production in the vasculature are not completely understood. We report here that production of this vasodilator by vascular endothelial cells can be significantly suppressed by hypoxia. Exposing human endothelial cells to low PO2 results in 40–60% reduction in the steady-state mRNA levels of endothelial constitutive NO synthase (eNOS), the major enzyme responsible for NO production in these cells. The lower levels of eNOS mRNA result from decreased transcription of the gene as well as reduced message stability. In endothelial-smooth muscle cell co-culture experiments, hypoxic endothelial cells stimulated significantly less cGMP production by smooth muscle cells than the corresponding normoxic controls. This inhibitory effect of hypoxia on NOS production by endothelial cells occurs after 24 h of hypoxia and persists for at least 48 h. These new findings suggest that hypoxia might cause changes in blood vessel tone through compound mechanisms: by increasing the production of endothelium-derived vasoconstrictors and, as shown here, by suppressing the production of vasodilators like NO.

Adiponectin is an adipose tissue-secreted adipokine with beneficial effects on the cardiovascular system. In this study, we evaluated a potential role for adiponectin in the protective effects of anthocyanin on diabetes-related endothelial dysfunction. We treated db/db mice on a normal diet with anthocyanin cyanidin-3- O-β-glucoside (C3G; 2 g/kg diet) for 8 wk. Endothelium-dependent and -independent relaxations of the aorta were then evaluated. Adiponectin expression and secretion were also measured. C3G treatment restores endothelium-dependent relaxation of the aorta in db/db mice, whereas diabetic mice treated with an anti-adiponectin antibody do not respond. C3G treatment induces adiponectin expression and secretion in cultured 3T3 adipocytes through transcription factor forkhead box O1 (Foxo1). Silencing Foxo1 expression prevented C3G-stimulated induction of adiponectin expression. In contrast, overexpression of Foxo1-ADA promoted adiponectin expression in adipocytes. C3G activates Foxo1 by increasing its deacetylation via silent mating type information regulation 2 homolog 1 (Sirt1). Furthermore, purified anthocyanin supplementation significantly improved flow-mediated dilation (FMD) and increased serum adiponectin concentrations in patients with type 2 diabetes. Changes in adiponectin concentrations positively correlated with FMD in the anthocyanin group. Mechanistically, adiponectin activates cAMP-PKA-eNOS signaling pathways in human aortic endothelial cells, increasing endothelial nitric oxide bioavailability. These results demonstrate that adipocyte-derived adiponectin is required for anthocyanin C3G-mediated improvement of endothelial function in diabetes.

Abstract Abstract 2996 Histone deacetylase 6 (HDAC6), a cytoplasmic enzyme, is a member of Class IIb HDAC family and catalyses the deacetylation of histones. Acetylation of histones induces chromatin relaxation leading gene transcription; conversely, deacetylation of histones induces chromatin condensation and silencing of gene transcription. HDAC6 regulates important cellular functions including histone modifications, gene transcription, and viral infection, degradation of misfolded proteins, cell migration, and immune synapse formation. HDAC inhibitors have been developed over the last decade as anti-tumor therapeutic agents in solid tumors and hematologic malignancies including multiple myeloma (MM). Myeloma represents a model system for the development of novel drugs which impact tumor cells as well as accessory cells in the tumor microenvironment. In this study we evaluated the effects of selective HDAC6 inhibition on immune accessory cells using a novel HDAC6 small molecule inhibitor, WT-161 compared with a control HDAC inhibitor trichostatin-A (TsA). Peripheral blood (PBMC) or bone marrow mononuclear cells (BMNC) from healthy donors or MM patients were cultured in the absence or presence of WT-161 (0.25-2.5uM) for 6h to 7 days. We first determined whether WT-161 induces HDAC6 inhibition in normal PBMCs by western blot of acetylated (Lys40)-tubulin, a target for HDAC6 activity. WT-161 induced acetylation of tubulin in PBMCs as early as 6 hours of exposure. Immunophenotypic changes were determined by flow cytometric analysis. Immunophenotypic analysis of PBMCs cultured with WT-161 showed significantly increased expression of PD1 in all immune cell subpopulations including CD4T, CD8T, CD3/CD8/CD56+ NKT and NK cells as well as in CD14+ myeloid cells. Conversely, decreased expression of negative costimulatory molecules CTLA4 and PDL1 was also noted, with no significant change in the expression of positive costimulatory molecules CD28, ICOS and ICOSL. Additionally, culture of PBMCs with WT-161 significantly increased expression of effector/memory T cell marker CXCR3 and decreased expression of CXCR4 in CD4T, CD8T, NKT, NK effector cells as well as CD14+ myeloid cells. WT-161 also induced ICAM1 expression on all immune cell subpopulations. WT-161 effects on the proliferation of immune cells were next assessed by CFSE-flow cytometric analysis. CFSE stained PBMCs were stimulated with anti-CD3 ab and cultured with WT-161 for 7 days. While WT-161 triggered proliferation of CD4T and CD8T cells, proliferation of NK and NKT cells decreased and B cells were unaffected. Interestingly, CD4/CD25/Foxp3 coexpressing regulatory T cells were also significantly diminished in both PBMCs and BMNCs cultured with WT-161 for 4 days. These data suggest that HDAC6 may have an immune regulatory function, and that inhibition of HDAC6 induces changes in the immune effector cells in MM microenvironment. Ongoing analysis of effects of HDAC6 inhibition on immune cells in the tumor microenvironment will further define the role of HDAC6 in disease pathogenesis and suggest novel immune-based epigenetic-targeted therapies. Disclosures: Raje: AMGEN: Consultancy; Celgene: Consultancy; Novartis: Consultancy; astraZeneca: Research Funding. Munshi:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Richardson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Johnson&Johnson: Membership on an entity's Board of Directors or advisory committees. Anderson:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

AbstractHydrogen sulfide (H2S) is an endogenous gaseous transmitter synthesized in various cell types. It is well established that H2S functions in many physiological processes, including the relaxation of vascular smooth muscle, mediation of neurotransmission, regulation of inflammation, and modulation of insulin signaling. In recent years, it has been revealed that polysulfides, substances with a varying number of sulfur atoms (H2Sn), are generated endogenously from H2S in the presence of oxygen. A series of studies describes that sulfane sulfur has the unique ability to bind reversibly to other sulfur atoms to form hydropersulfides and polysulfides, and that polysulfides activate ion channels and promote calcium influx. Furthermore, polysulfides regulate tumor suppressor activity, promote the activation of transcription factors targeting antioxidant genes and regulate blood pressure by vascular smooth muscle relaxation. Insulin secretion from pancreatic β cells plays a critical role in response to increased blood glucose concentration. H2S has emerged as an important regulator of glycemic control and exhibits characteristic regulation of glucose homeostasis. However, the effects of polysulfides on glucose-stimulated insulin secretion (GSIS) are largely unknown. In this study, we demonstrated that pharmacological polysulfide salts including Na2S2, Na2S3, and Na2S4 considerably inhibit GSIS in mouse and rat pancreatic β-cell-derived MIN6 and INS-1 cell lines, and that the effect is dependent on the activation of ATP-sensitive potassium channels. In addition, we demonstrated that a mixture of Na2S and diethylamine NONOate inhibits GSIS in a similar way to the pharmacological administration of polysulfide salts.

Abstract Background Many Heart Failure with preserved Ejection Fraction (HFpEF) patients have metabolic syndrome and develop Exercise Induced Pulmonary Hypertension (EIPH). The pathogenesis of EIPH in HFpEF remains unclear as there is no rodent model. As the SGLT2 inhibitor Empagliflozin improves clinical outcome in patients with type 2 diabetes and cardiovascular risk, we tested its effect on EIPH in a novel rat model of HFpEF. Methods Obese ZSF1 (HFpEF model) with leptin receptor mutation have metabolic syndrome and received the VEGF-inhibitor SU5416 to stimulate PH (Obese + Sugen). Half also received Empagliflozin (0.2 mg/kg/day) in drinking water from 8 to 22 weeks old. Lean ZSF1 lacking the mutation served as controls. During treadmill exercise, right/left ventricle (RV/LV) hemodynamics were evaluated via catheters. Pulmonary artery vascular smooth muscle cells (PAVSMC) prepared from normal or diabetic patients were cultured in standard media, or with Palmitate acid, Glucose and Insulin (PGI) to induce metabolic stress. Flow cytometry was used to evaluate reactive oxygen species (ROS) in mitochondria (Mitosox) or cytoplasm (CellROX). Results Relative to Lean, Obese + Sugen had increased body weight and HgA1C (Fig. 1A). Relative to Lean and at rest, Obese + Sugen showed mildly elevated RVSP and LVEDP. After exercise, LVSP and LVEDP rose similarly in Lean and Obese + Sugen. However, after exercise, Obese + Sugen showed a markedly greater increase in RVSP and exercise intolerance consistent with EIPH (Fig. 1B). In MR imaging of PA, Lean showed dobutamine (5 μg/kg/min)-induced PA dilation, which was not seen in Obese + Sugen (Fig. 1C). Protein levels of sGCβ1 (key regulator of PA relaxation) and its transcription factor (NFYA) both were decreased in PA from Obese + Sugen relative to Lean (Fig. 1D). Obese + Sugen + SGLT2 inhibitor treated rats showed marked improvements metabolic syndrome (body weight, HgA1c), exercise induced increase in RVSP, PA response to dobutamine, and increased NFYA and sGCβ1 expression (Fig. 1A–D). We observed greater ROS-induced DNA damage (8-OHdG staining) (Fig. 1E) and mitochondrial complex I, III, and IV activity in Obese + Sugen PA that was normalized in Obese + Sugen + SGLT2 inhibitor (Fig. 1F), suggesting a role of ROS in EIPH. Control human PAVSMC treated with PGI media showed elevated cytoplasmic and mitochondrial ROS, associated with increased mitochondrial complex I, III, IV and V activity (Fig. 1F, G). PGI media also accelerated the degradation of NFYA RNA and protein level in a manner mimicked by H2O2, and prevented by catalase/SOD (Fig. 1H, I), suggesting PGI-induced ROS enhanced NFYA degradation. Diabetic human PAVSMCs cultured in normal media resembled PGI-treated normal cells with respect to sGCb1 and NFYA expression, and in response to catalase/SOD (Fig. 1H, I). Conclusions In this PH-HFpEF model, metabolic syndrome contributes to PA dysfunction and EIPH through mitochondrial dysfunction and enhanced ROS, which were improved by Empagliflozin treatment. Figure 1 Funding Acknowledgement Type of funding source: None

Genus Mycobacterium comprises a large number of species including many pathogens such as Mycobacterium leprae, Mycobacterium abscessus and Mycobacterium tuberculosis (Mtb), the last one is the causative agent of the fatal disease tuberculosis. The unique features of the deadly organism viz, slow growing, tough cell wall, latency and resistance to various drugs demand a systematic understanding of many essential molecular processes including transcription. Studies have been undertaken to understand several aspects of transcription in mycobacteria which revealed its machinery to be conserved with other eubacteria? However, many facets of transcription in mycobacteria and regulation are different. The transcription regulators and them regulation are the basic counter stones which govern gene expression. The present study is aimed to understand better the mechanistic regulation of transcription of important housekeeping functions, DNA gyrase and also to obtain further insights into the role of transcription elongation factor Gre. Chapter 1 of the thesis provides a general introduction of the bacterial transcription machinery, associated transcription regulators and their regulation. It covers the description of the central player- the RNA polymerase (RNAP) followed by each step of the transcription initiation, elongation and various factors involved in their regulation. Finally, an overview of the emerging information on several aspects of mycobacterial transcription is discussed emphasizing on RNAP, promoter architecture, and its regulation. In Chapter 2, the studies are directed to understand the mechanism for topology-dependent regulation of Mtb Gyrase. The gyrase is encoded by two genes gyrB and gyrA which form a bicistronicity operon in Mtb and harbor multiple promoters. The principal promoter PgyrB1 drives the transcription of the dicistron and the weaker divergent promoter PgyrR is engaged in transcription in the opposite direction. The divergent and overlapping PgyrR show decrease in activity when the PgyrB1 was induced upon relaxation of the genome by a phenomenon termed relaxation stimulated transcription (RST). PgyrR plays a role in the fine tuning of gyr gene expression by reiterative transcription (RT), a regulatory mechanism hitherto not described in Mtb. In vitro transcription assays show that RT at PgyrR is dependent on the negatively supercoiled status of the DNA. The principal promoter PgyrB1 is also regulated by DNA topology but does not exhibit RT. It is elucidated that the RNAP binding is efficient at PgyrB1 when the DNA is relaxed whereas binding to PgyrR is preferred when DNA is supercoiled. Thus, a collaboration between RST and RT govern the regulation of gyr operon; the differential topology sensitivity of the overlapping promoters determines and dictate the efficiency of transcription initiation at gyr promoters. In addition, this study suggests a new mechanism of RST distinct from the one observed for other bacteria, such as E. coli or M. smegmatis. Chapter 3 describes studies that have been carried out to delineate the mechanism underlying the differential function of transcription regulator MtbGreA and its homolog Rv3788 (MtbGfh1). MtbGreA binds to RNAP and induces the intrinsic transcript cleavage activity of RNAP thereby allowing RNAP to resume transcription from paused and arrested sites. In spite of having Gre like domains, MtbGfh1 does not stimulate RNA cleavage. Instead, it inhibits transcription by binding to RNAP. Homology modeling and docking data suggest that Gre and MtbGfh1 bind to RNAP in a different orientation. MtbGreA coordinate with the Mg2+ present in the catalytic center of the RNAP while MtbGfh1 was observed to be facing away from Mg2+ Swapping of a stretch of residues from the N-terminus of MtbGreA into MtbGfh1 acquire GreA like transcript cleavage stimulatory activity and enhance promoter clearance for MtbGfh1. Bioinformatics analysis and biochemical assays demonstrate the significance of a stretch of residues in the N-terminus of MtbGreA and MtbGfh1 for their functions. Also, the orientation of the MtbGreA and MtbGfh1 while binding to RNAP is a crucial determinant in governing their respective function. Being the general inhibitor of transcription, overexpression of MtbGfh1 led to the appearance of tiny colonies and slow growth of cells suggesting its regulatory role to maintain the physiology of Mtb. In Chapter 4, the influence of perturbation of GreA level on Mtb growth and physiology has been studied. Mtb contains a single Gre protein (Rv1080c), unlike many other bacteria where both GreA and GreB are present. Further, the GC-rich genome of Mtb may pose an additional challenge to the transcribing RNAP. Hence the role of GreA could be essential to maintain high fidelity of transcription and RNAP distribution in Mtb genome. To validate this, the conditional knockdown strain of MtbGreA was generated. GreA depleted strain exhibited slow growth and caused phenotypical changes in Mtb cells. Moreover, the occupancy of RNAP on the promoter and gene body of candidate gene tested was found to be disrupted upon MtbGreA depletion, suggesting the regulatory role of GreA in modulating Mtb physiology.