Short repeats in the heme oxygenase 1 gene promoter is associated with increased levels of inflammation, ferritin and higher risk of type-2 diabetes mellitus.

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Andrews, Mónica; Leiva, Elba; Arredondo-Olguín, Miguel

2016-09-01

We evaluated the relationship between the HO1 genotype, ferritin levels and the risk of type-2 diabetes and inflammation. Eight hundred thirty-five individuals were evaluated and classified according to their nutritional status and the presence of type-2 diabetes: 153 overweight (OW); 62 obese (OB); 55 type-2 diabetes mellitus (DM); 202 OWDM; 239 OBDM and 124 controls (C). We studied biochemical (glycemia, insulin, lipid profile, liver enzyme, creatinine, hsCRP), hematological (hemoglobin, free erythrocyte protoporphyrin, transferrin receptor and serum Fe and ferritin) and oxidative stress (SOD, GHS and TBARS) parameters. We determined heme oxygenase activity and the (GT)n polymorphism in its gene promoter. Individuals with diabetes, independent of nutritional status, showed high levels of ferritin and HO activity compared to control subjects. Allelic frequency was not different between the groups (Chi(2), NS) however, genotypes were different (Chi(2), P1). The SS (short-short) genotype was higher in all DM individuals compared to controls and MM was higher in controls. SM (short-medium) genotype was an independent risk factor for DM in logistic regression analysis. We observed high risk for type-2 diabetes mellitus in the presence of SM genotype and high levels of ferritin (OR adjusted: 2.7; 1.9-3.6; p1; compared to control group). It was also significantly related to inflammation. The SM genotype in HO1 gene promoter and ferritin levels were associated with higher risk for type-2 diabetes and for having a higher marker of inflammation, which is the main risk factor for the development of chronic diseases. Copyright © 2016 Elsevier GmbH. All rights reserved.

Paracrine action of HO-1-modified mesenchymal stem cells mediates cardiac protection and functional improvement.

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Zeng, Bin; Ren, Xiaofeng; Lin, Guosheng; Zhu, Chengang; Chen, Honglei; Yin, Jiechao; Jiang, Hong; Yang, Bo; Ding, Danhua

2008-10-01

The aim has been to determine whether the supernatants of mesenchymal stem cells (MSCs) transfected with adenovirus carrying human heme oxygenase-1 (hHO-1) gene protect cardiomyocytes from ischemic injury. We have found that hHO-1 infected MSCs (hHO-1-MSCs) increased expression of hHO-1 protein. Apoptosis of cultured hHO-1-MSCs exposed to hypoxia was suppressed. Several cytokines, including HGF, bFGF, TGF-beta, VEGF and IL-1beta, were produced by hHO-1-MSCs, some being significantly enhanced under hypoxia stimulation. Meanwhile, those cytokines reduced caspase-3 level and activity in cultured adult rat ventricular cardiomyocytes (ARVCs) exposed to hypoxia. Supernatants obtained from hHO-1-MSCs improved left ventricular function, limited myocardial infarct size, increased microvessel density, and inhibited apoptosis of cardiomyocytes in rat myocardial infarction. It can be concluded hHO-1-modified MSCs prevent myocardial cell injury via secretion of paracrine-acting mediators.

Regulation of hemeoxygenase-1 gene expression by Nrf2 and c-Jun in tertiary butylhydroquinone-stimulated rat primary astrocytes

International Nuclear Information System (INIS)

Park, Jin-Sun; Kim, Hee-Sun

2014-01-01

Highlights: • tBHQ increased HO-1 mRNA and protein levels in rat primary astrocytes. • tBHQ enhanced HO-1 gene transcription in an ARE-dependent manner. • tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. • Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. • Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction. - Abstract: Hemeoxygenase-1 (HO-1) is a phase II antioxidant enzyme that is primarily involved in detoxification and cytoprotection in a variety of tissues. However, the mechanism underlying HO-1 gene expression remains unclear. In the present study, we investigated the regulation of HO-1 expression in primary cultured astrocytes by using the natural antioxidant compound tertiary butylhydroquinone (tBHQ). We found that tBHQ increased HO-1 mRNA and protein levels. Promoter analysis revealed that tBHQ enhanced HO-1 gene transcription in an antioxidant response element (ARE)-dependent manner. In addition, tBHQ increased the nuclear translocation and DNA binding of Nrf2 and c-Jun to ARE. Small interfering RNA (siRNA) experiments demonstrated that Nrf2 and c-Jun are involved in the differential modulation of HO-1 expression. Thus, Nrf2 knockdown reduced the basal level of HO-1 expression but did not affect the fold induction by tBHQ. On the other hand, knockdown of c-Jun diminished tBHQ-mediated induction of HO-1 without affecting basal expression. The data suggest that Nrf2 generally modulates the basal expression of HO-1, while c-Jun mediates HO-1 induction in response to tBHQ. The results of co-immunoprecipitation assays demonstrated a physical interaction between Nrf2 and c-Jun in tBHQ-treated astrocytes. The results suggest that Nrf2 and c-Jun regulate HO-1 expression via their coordinated interaction in tBHQ-treated rat primary astrocytes

3D nuclear organization of telomeres in the Hodgkin cell lines U-HO1 and U-HO1-PTPN1: PTPN1 expression prevents the formation of very short telomeres including "t-stumps"

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Lemieux Bruno

2010-12-01

Full Text Available Abstract Background In cancer cells the three-dimensional (3D telomere organization of interphase nuclei into a telomeric disk is heavily distorted and aggregates are found. In Hodgkin's lymphoma quantitative FISH (3D Q-FISH reveals a major impact of nuclear telomere dynamics during the transition form mononuclear Hodgkin (H to diagnostic multinuclear Reed-Sternberg (RS cells. In vitro and in vivo formation of RS-cells is associated with the increase of very short telomeres including "t-stumps", telomere loss, telomeric aggregate formation and the generation of "ghost nuclei". Results Here we analyze the 3D telomere dynamics by Q-FISH in the novel Hodgkin cell line U-HO1 and its non-receptor protein-tyrosine phosphatase N1 (PTPN1 stable transfectant U-HO1-PTPN1, derived from a primary refractory Hodgkin's lymphoma. Both cell lines show equally high telomerase activity but U-HO1-PTPN differs from U-HO1 by a three times longer doubling time, low STAT5A expression, accumulation of RS-cells (p As expected, multinuclear U-HO1-RS-cells and multinuclear U-HO1-PTPN1-RS-cells differ from their mononuclear H-precursors by their nuclear volume (p Conclusion Abundant RS-cells without additional very short telomeres including "t-stumps", high rate of apoptosis, but low STAT5A expression, are hallmarks of the U-HO1-PTPN1 cell line. These characteristics are independent of telomerase activity. Thus, PTPN1 induced dephosphorylation of STAT5 with consecutive lack of Akt/PKB activation and cellular arrest in G2, promoting induction of apoptosis, appears as a possible pathogenetic mechanism deserving further experimental investigation.

Adenovirus-mediated heme oxygenase-1 gene transfer into rabbit ocular tissues.

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Abraham, N G; da Silva, J L; Lavrovsky, Y; Stoltz, R A; Kappas, A; Dunn, M W; Schwartzman, M L

1995-10-01

Heme oxygenase-1 (HO-1) is a stress protein induced up to 100-fold within a few hours after exposure to oxidative stress, and it has been shown to counteract oxidative injury induced by ultraviolet light or free radicals. The current study was undertaken to determine whether the HO-1 gene can be introduced into adult rabbit ocular tissues by microinjection of a recombinant replication-deficient adenovirus human HO-1 cDNA (Adv-HHO). Human HO-1 gene was used for transfection studies to differentiate endogenous from transfected HO. The purified Adv-HHO construct (10(8) pfu/ml) was mixed with lipofectamine and microinjected into the anterior chamber, vitreous cavity, and subretinal space of New Zealand rabbit eyes. After 2 weeks, total RNA was extracted from different ocular tissues, reverse transcription-polymerase chain reaction was performed using specific human HO-1 primers, and amplification products were subjected to Southern hybridization. Transfection with the Adv-HHO construct into rabbit corneal epithelial cells in culture resulted in a functional expression of the human HO-1 gene; the human HO-1 mRNA was detected, and enzyme activity increased threefold. Human HO-1 mRNA was detected in the retina after microinjection of the Adv-HHO construct into the subretinal space. Microinjection into the vitreous resulted in HO-1 mRNA expression in the corneal endothelium, iris, lens, and retina; after intracameral injection of the Adv-HHO construct, human HO-1 mRNA was detected in corneal epithelium and endothelium, ciliary body, lens, and iris. Regardless of the injection site, transfected human HO-1 mRNA was undetectable in tissues outside the eye, that is, brain, liver, and kidney. These results demonstrated a tissue-selective functional transfer of the human HO-1 gene into rabbit ocular tissues in vivo. This technique may be a promising means for delivering HO-1 gene in vivo as a protective mechanism against oxidative stress that contributes to the pathogenesis of

GEC-targeted HO-1 expression reduces proteinuria in glomerular immune injury.

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Duann, Pu; Lianos, Elias A

2009-09-01

Induction of heme oxygenase (HO)-1 is a key defense mechanism against oxidative stress. Compared with tubules, glomeruli are refractory to HO-1 upregulation in response to injury. This can be a disadvantage as it may be associated with insufficient production of cytoprotective heme-degradation metabolites. We, therefore, explored whether 1) targeted HO-1 expression can be achieved in glomeruli without altering their physiological integrity and 2) this expression reduces proteinuria in immune injury induced by an anti-glomerular basement membrane (GBM) antibody (Ab). We employed a 4.125-kb fragment of a mouse nephrin promoter downstream to which a FLAG-tagged hHO-1 cDNA sequence was inserted and subsequently generated transgenic mice from the FVB/N parental strain. There was a 16-fold higher transgene expression in the kidney than nonspecific background (liver) while the transprotein immunolocalized in glomerular epithelial cells (GEC). There was no change in urinary protein excretion, indicating that GEC-targeted HO-1 expression had no effect on glomerular protein permeability. Urinary protein excretion in transgenic mice with anti-GBM Ab injury (days 3 and 6) was significantly lower compared with wild-type controls. There was no significant change in renal expression levels of profibrotic (TGF-beta1) or anti-inflammatory (IL-10) cytokines in transgenic mice with anti-GBM Ab injury. These observations indicate that GEC-targeted HO-1 expression does not alter glomerular physiological integrity and reduces proteinuria in glomerular immune injury.

Heme oxygenase-1 (HO-1 expression in prostate cancer cells modulates the oxidative response in bone cells.

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Mercedes Ferrando

Full Text Available Prostate cancer (PCa is a leading cause of death among males. It is currently estimated that inflammatory responses are linked to 15-20% of all deaths from cancer worldwide. PCa is dominated by complications arising from metastasis to the bone where the tumor cells interact with the bone microenvironment impairing the balance between bone formation and degradation. However, the molecular nature of this interaction is not completely understood. Heme oxygenase-1 (HO-1 counteracts oxidative damage and inflammation. Previous studies from our laboratory showed that HO-1 is implicated in PCa, demonstrating that endogenous HO-1 inhibits bone derived-prostate cancer cells proliferation, invasion and migration and decreases tumor growth and angiogenesis in vivo. The aim of this work was to analyze the impact of HO-1 modulated PCa cells on osteoblasts proliferation in vitro and on bone remodeling in vivo. Using a co-culture system of PC3 cells with primary mice osteoblasts (PMOs, we demonstrated that HO-1 pharmacological induction (hemin treatment abrogated the diminution of PMOs proliferation induced by PCa cells and decreased the expression of osteoclast-modulating factors in osteoblasts. No changes were detected in the expression of genes involved in osteoblasts differentiation. However, co-culture of hemin pre-treated PC3 cells (PC3 Hem with PMOs provoked an oxidative status and activated FoxO signaling in osteoblasts. The percentage of active osteoblasts positive for HO-1 increased in calvarias explants co-cultured with PC3 Hem cells. Nuclear HO-1 expression was detected in tumors generated by in vivo bone injection of HO-1 stable transfected PC3 (PC3HO-1 cells in the femur of SCID mice. These results suggest that HO-1 has the potential to modify the bone microenvironment impacting on PCa bone metastasis.

HO-1 Upregulation Attenuates Adipocyte Dysfunction, Obesity, and Isoprostane Levels in Mice Fed High Fructose Diets

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Zeid Khitan

2014-01-01

Full Text Available Background. Fructose metabolism is an unregulated metabolic pathway and excessive fructose consumption is known to activate ROS. HO-1 is a potent antioxidant gene that plays a key role in decreasing ROS and isoprostanes. We examined whether the fructose-mediated increase in adipocyte dysfunction involves an increase in isoprostanes and that pharmacological induction of HO-1 would decrease both isoprostane levels and adipogenesis. Methods and Results. We examined the effect of fructose, on adipogenesis in human MSCs in the presence and absence of CoPP, an inducer of HO-1. Fructose increased adipogenesis and the number of large lipid droplets while decreasing the number of small lipid droplets (P<0.05. Levels of heme and isoprostane in fructose treated MSC-derived adipocytes were increased. CoPP reversed these effects and markedly increased HO-1 and the Wnt signaling pathway. The high fructose diet increased heme levels in adipose tissue and increased circulating isoprostane levels (P<0.05 versus control. Fructose diets decreased HO-1 and adiponectin levels in adipose tissue. Induction of HO-1 by CoPP decreased isoprostane synthesis (P<0.05 versus fructose. Conclusion. Fructose treatment resulted in increased isoprostane production and adipocyte dysfunction, which was reversed by the increased expression of HO-1.

Molecular mechanism and functional consequences of lansoprazole-mediated heme oxygenase-1 induction

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Schulz-Geske, Stephanie; Erdmann, Kati; Wong, Ronald J; Stevenson, David K; Schröder, Henning; Grosser, Nina

2009-01-01

AIM: To investigate the molecular mechanism and functional consequences of heme oxygenase-1 (HO-1) activation by lansoprazole in endothelial cells and macrophages. METHODS: Expression of HO-1 mRNA was analyzed by Northern blotting. Western blotting was used to determine the HO-1 and ferritin protein levels. NADPH-dependent reactive oxygen species (ROS) formation was measured with lucigenin-enhanced chemiluminescence. HO-1 promoter activity in mouse fibroblasts, stably transfected with a 15-kb HO-1 gene that drives expression of the reporter gene luciferase, was assessed using in vivo bioluminescence imaging. RESULTS: Lansoprazole increased HO-1 mRNA levels in endothelial cells and HO-1 protein levels in macrophages. In addition, lansoprazole-induced ferritin protein levels in both cell systems. Moreover, induction of the antioxidant proteins HO-1 and ferritin by lansoprazole was followed by a decrease in NADPH-mediated ROS formation. The radical scavenging properties of lansoprazole were diminished in the presence of the HO inhibitor, chromium mesoporphyrin IX. Induction of HO-1 gene expression by lansoprazole was not related to oxidative stress or to the activation of the mitogen-activated protein kinase pathway. However, the phosphatidylinositol 3-kinase inhibitor LY294002 showed a concentration-dependent inhibition of HO-1 mRNA and promoter activity. CONCLUSION: Activation of HO-1 and ferritin may account for the gastric protection of lansoprazole and is dependent on a pathway blocked by LY294002. PMID:19764090

Adenoviral transfer of the heme oxygenase-1 gene protects striatal astrocytes from heme-mediated oxidative injury.

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Teng, Zhi-Ping; Chen, Jing; Chau, Lee-Young; Galunic, Nicholas; Regan, Raymond F

2004-11-01

Heme oxygenase-1 (HO-1) is induced in the CNS after hemorrhage, and may have an effect on injury to surrounding tissue. Hemin, the preferred substrate of HO, is a neurotoxin that is present in intracranial hematomas. In a prior study, we observed that HO inhibitors increased the vulnerability of cultured cortical astrocytes to heme-mediated oxidative injury. To investigate the effect of HO more specifically, we used an adenoviral vector encoding the human HO-1 gene to specifically increase HO-1 expression. Incubation with 100 MOI of the HO-1 adenovirus (Adv-HHO-1) for 24 h increased both HO-1 protein and HO activity; a control adenovirus lacking the HO-1 gene had no effect. Using a DNA probe that was specific for human HO-1, 80.5 +/- 7.2% of astrocytes were observed to be infected by in situ hybridization. The cell death produced by 30-60 microM hemin was significantly reduced by pretreatment with 100 MOI Adv-HHO-1, as assessed by LDH release, propidium iodide exclusion, and MTT reduction assay. The threefold increase in cell protein oxidation produced by hemin was also attenuated in cultures pretreated with Adv-HHO-1. These results support the hypothesis that HO-1 protects astrocytes from heme-mediated oxidative injury. Specifically increasing astrocytic HO-1 by gene transfer may have a beneficial effect on hemorrhagic CNS injury.

Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet.

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Komal Sodhi

Full Text Available Oxidative stress underlies the etiopathogenesis of nonalcoholic fatty liver disease (NAFLD, obesity and cardiovascular disease (CVD. Heme Oxygenase-1 (HO-1 is a potent endogenous antioxidant gene that plays a key role in decreasing oxidative stress. Sirtuin1 (SIRT1 belongs to the family of NAD-dependent de-acyetylases and is modulated by cellular redox.We hypothesize that fructose-induced obesity creates an inflammatory and oxidative environment conducive to the development of NAFLD and metabolic syndrome. The aim of this study is to determine whether HO-1 acts through SIRT1 to form a functional module within hepatocytes to attenuate steatohepatitis, hepatic fibrosis and cardiovascular dysfunction.We examined the effect of fructose, on hepatocyte lipid accumulation and fibrosis in murine hepatocytes and in mice fed a high fructose diet in the presence and absence of CoPP, an inducer of HO-1, and SnMP, an inhibitor of HO activity. Fructose increased oxidative stress markers and decreased HO-1 and SIRT1 levels in hepatocytes (p<0.05. Further fructose supplementation increased FAS, PPARα, pAMPK and triglycerides levels; CoPP negated this increase. Concurrent treatment with CoPP and SIRT1 siRNA in hepatocytes increased FAS, PPARα, pAMPK and triglycerides levels suggesting that HO-1 is upstream of SIRT1 and suppression of SIRT1 attenuates the beneficial effects of HO-1. A high fructose diet increased insulin resistance, blood pressure, markers of oxidative stress and lipogenesis along with fibrotic markers in mice (p<0.05. Increased levels of HO-1 increased SIRT1 levels and ameliorated fructose-mediated lipid accumulation and fibrosis in liver along with decreasing vascular dysfunction (p<0.05 vs. fructose. These beneficial effects of CoPP were reversed by SnMP.Taken together, our study demonstrates, for the first time, that HO-1 induction attenuates fructose-induced hepatic lipid deposition, prevents the development of hepatic fibrosis and abates

The progress of tumor gene-radiotherapy induced by Egr-1 promoter

International Nuclear Information System (INIS)

Guo Rui; Li Biao

2010-01-01

The promoter of early growth response gene-1 (Egr-1) is a cis-acting element of Egr-1, and its activity is regulated by inducers such as ionizing radiation, free radical. In designated gene-radiotherapy system, radiation combined with therapeutic gene (such as tumor necrosis factor-α gene, suicide gene) can spatially and temporally regulate therapeutic gene expression in the irradiated field, produced a marked effect, while little systemic toxicities were observed. The combination of radiotherapy and gene therapy is promising in tumor therapy. (authors)

Characterization and functional analysis of the Paralichthys olivaceus prdm1 gene promoter.

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Li, Peizhen; Wang, Bo; Cao, Dandan; Liu, Yuezhong; Zhang, Quanqi; Wang, Xubo

2017-10-01

PR domain containing protein 1 (Prdm1) is a transcriptional repressor identified in various species and plays multiple important roles in immune response and embryonic development. However, little is known about the transcriptional regulation of the prdm1 gene. This study aims to characterize the promoter of Paralichthys olivaceus prdm1 (Po-prdm1) gene and determine the regulatory mechanism of Po-prdm1 expression. A 2000bp-long 5'-flanking region (translation initiation site designated as +1) of the Po-prdm1 gene was isolated and characterized. The regulatory elements in this fragment were then investigated and many putative transcription factor (TF) binding sites involved in immunity and multiple tissue development were identified. A 5'-deletion analysis was then conducted, and the ability of the deletion mutants to promote luciferase and green fluorescent protein (GFP) expression in a flounder gill cell line was examined. The results revealed that the minimal promoter is located in the region between -446 and -13bp, and the region between -1415 and -13bp enhanced the promoter activity. Site-directed mutation analysis was subsequently performed on the putative regulatory elements sites, and the results indicated that FOXP1, MSX and BCL6 binding sites play negative functional roles in the regulation of the Po-prdm1 expression in FG cells. In vivo analysis demonstrated that a GFP reporter gene containing 1.4kb-long promoter fragment (-1415/-13) was expressed in the head and trunk muscle fibres of transient transgenic zebrafish embryos. Our study provided the basic information for the exploration of Po-prdm1 regulation and expression. Copyright © 2017 Elsevier Inc. All rights reserved.

SHB1/HY1 Alleviates Excess Boron Stress by Increasing BOR4 Expression Level and Maintaining Boron Homeostasis in Arabidopsis Roots

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Qiang Lv

2017-05-01

Full Text Available Boron is an essential mineral nutrient for higher plant growth and development. However, excessive amounts of boron can be toxic. Here, we report on the characterization of an Arabidopsis mutant, shb1 (sensitive to high-level of boron 1, which exhibits hypersensitivity to excessive boron in roots. Positional cloning demonstrated that the shb1 mutant bears a point mutation in a gene encoding a heme oxygenase 1 (HO1 corresponding to the HY1 gene involved in photomorphogenesis. The transcription level of the SHB1/HY1 gene in roots is up-regulated under excessive boron stimulation. Either overexpressing SHB1/HY1 or applying the HO1 inducer hematin reduces boron accumulation in roots and confers high boron tolerance. Furthermore, carbon monoxide and bilirubin, catalytic products of HO1, partially rescue the boron toxicity-induced inhibition of primary root growth in shb1. Additionally, the mRNA level of BOR4, a boron efflux transporter, is reduced in shb1 roots with high levels of boron supplementation, and hematin cannot relieve the boron toxicity-induced root inhibition in bor4 mutants. Taken together, our study reveals that HO1 acts via its catalytic by-products to promote tolerance of excessive boron by up-regulating the transcription of the BOR4 gene and therefore promoting the exclusion of excessive boron in root cells.

Protection from ischemic heart injury by a vigilant heme oxygenase-1 plasmid system.

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Tang, Yao Liang; Tang, Yi; Zhang, Y Clare; Qian, Keping; Shen, Leping; Phillips, M Ian

2004-04-01

Although human heme oxygenase-1 (hHO-1) could provide a useful approach for cellular protection in the ischemic heart, constitutive overexpression of hHO-1 may lead to unwanted side effects. To avoid this, we designed a hypoxia-regulated hHO-1 gene therapy system that can be switched on and off. This vigilant plasmid system is composed of myosin light chain-2v promoter and a gene switch that is based on an oxygen-dependent degradation domain from the hypoxia inducible factor-1-alpha. The vector can sense ischemia and switch on the hHO-1 gene system, specifically in the heart. In an in vivo experiment, the vigilant hHO-1 plasmid or saline was injected intramyocardially into myocardial infarction mice or sham operation mice. After gene transfer, expression of hHO-1 was only detected in the ischemic heart treated with vigilant hHO-1 plasmids. Masson trichrome staining showed significantly fewer fibrotic areas in vigilant hHO-1 plasmids-treated mice compared with saline control (43.0%+/-4.8% versus 62.5%+/-3.3%, PhHO-1 expression in peri-infarct border areas, concomitant with higher Bcl-2 levels and lower Bax, Bak, and caspase 3 levels in the ischemic myocardium compared with saline control. By use of a cardiac catheter, heart from vigilant hHO-1 plasmids-treated mice showed improved recovery of contractile and diastolic performance after myocardial infarction compared with saline control. This study documents the beneficial regulation and therapeutic potential of vigilant plasmid-mediated hHO-1 gene transfer. This novel gene transfer strategy can provide cardiac-specific protection from future repeated bouts of ischemic injury.

Type 1 plaminogen activator inhibitor gene: Functional analysis and glucocorticoid regulation of its promoter

International Nuclear Information System (INIS)

Van Zonneveld, A.J.; Curriden, S.A.; Loskutoff, D.J.

1988-01-01

Plasminogen activator inhibitor type 1 is an important component of the fibrinolytic system and its biosynthesis is subject to complex regulation. To study this regulation at the level of transcription, the authors have identified and sequenced the promoter of the human plasminogen activator inhibitor type 1 gene. Nuclease protection experiments were performed by using endothelial cell mRNA and the transcription initiation (cap) site was established. Sequence analysis of the 5' flanking region of the gene revealed a perfect TATA box at position -28 to position -23, the conserved distance from the cap site. Comparative functional studies with the firefly luciferase gene as a reporter gene showed that fragments derived from this 5' flanking region exhibited high promoter activity when transfected into bovine aortic endothelial cells and mouse Ltk - fibroblasts but were inactive when introduced into HeLa cells. These studies indicate that the fragments contain the plasminogen activator inhibitor type 1 promoter and that it is expressed in a tissue-specific manner. Although the fragments were also silent in rat FTO2B hepatoma cells, their promoter activity could be induced up to 40-fold with the synthetic glucocorticoid dexamethasone. Promoter deletion mapping experiments and studies involving the fusion of promoter fragments to a heterologous gene indicated that dexamethasone induction is mediated by a glucocorticoid responsive element with enhancer-like properties located within the region between nucleotides -305 and +75 of the plasminogen activator inhibitor type 1 gene

HO-1 inhibits IL-13-induced goblet cell hyperplasia associated with CLCA1 suppression in normal human bronchial epithelial cells.

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Mishina, Kei; Shinkai, Masaharu; Shimokawaji, Tadasuke; Nagashima, Akimichi; Hashimoto, Yusuke; Inoue, Yoriko; Inayama, Yoshiaki; Rubin, Bruce K; Ishigatsubo, Yoshiaki; Kaneko, Takeshi

2015-12-01

Mucus hypersecretion and goblet cell hyperplasia are common features that characterize asthma. IL-13 increases mucin (MUC) 5AC, the major component of airway mucus, in airway epithelial cells. According to the literature, IL-13 receptor activation leads to STAT6 activation and consequent induction of chloride channel accessory 1 (CLCA1) gene expression, associated with the induction of MUC5AC. Heme oxygenase-1 (HO-1) is an enzyme that catalyzes oxidation of heme to biliverdin, and has anti-inflammatory and anti-oxidant properties. We examined the effects of HO-1 on mucin production and goblet cell hyperplasia induced by IL-13. Moreover, we assessed the cell signaling intermediates that appear to be responsible for mucin production. Normal human bronchial epithelial (NHBE) cells were grown at air liquid interface (ALI) in the presence or absence of IL-13 and hemin, a HO-1 inducer, for 14 days. Protein concentration was analyzed using ELISA, and mRNA expression was examined by real-time PCR. Histochemical analysis was performed using HE staining, andWestern blotting was performed to evaluate signaling transduction pathway. Hemin (4 μM) significantly increased HO-1 protein expression (p b 0.01) and HO-1 mRNA expression (p b 0.001). IL-13 significantly increased goblet cells, MUC5AC protein secretion (p b 0.01) and MUC5AC mRNA (p b 0.001), and these were decreased by hemin by way of HO-1. Tin protoporphyrin (SnPP)-IX, a HO-1 inhibitor, blocked the effect of hemin restoring MUC5AC protein secretion (p b 0.05) and goblet cell hyperplasia. Hemin decreased the expression of CLCA1 mRNA (p b 0.05) and it was reversed by SnPP-IX, but could not suppress IL-13-induced phosphorylation of STAT6 or SAM pointed domain-containing ETS transcription factor (SPDEF) and Forkhead box A2 (FOXA2) mRNA expression. In summary, HO-1 overexpression suppressed IL-13-induced goblet cell hyperplasia and MUC5AC production, and involvement of CLCA1 in the mechanism was suggested.

Production and characterization of soluble human TNFRI-Fc and human HO-1(HMOX1) transgenic pigs by using the F2A peptide.

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Park, Sol Ji; Cho, Bumrae; Koo, Ok Jae; Kim, Hwajung; Kang, Jung Taek; Hurh, Sunghoon; Kim, Su Jin; Yeom, Hye Jung; Moon, Joonho; Lee, Eun Mi; Choi, Ji Yei; Hong, Ju Ho; Jang, Goo; Hwang, Joing-Ik; Yang, Jaeseok; Lee, Byeong Chun; Ahn, Curie

2014-06-01

Generation of transgenic pigs for xenotransplantation is one of the most promising technologies for resolving organ shortages. Human heme oxygenase-1 (hHO-1/HMOX1) can protect transplanted organs by its strong anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Soluble human TNFRI-Fc (shTNFRI-Fc) can inhibit the binding of human TNF-α (hTNF-α) to TNF receptors on porcine cells, and thereby, prevent hTNF-α-mediated inflammation and apoptosis. Herein, we successfully generated shTNFRI-Fc-F2A-HA-hHO-1 transgenic (TG) pigs expressing both shTNFRI-Fc and hemagglutinin-tagged-human heme oxygenase-1 (HA-hHO-1) by using an F2A self-cleaving peptide. shTNFRI-Fc and HA-hHO-1 transgenes containing the F2A peptide were constructed under the control of the CAG promoter. Transgene insertion and copy number in the genome of transgenic pigs was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. Expressions of shTNFRI-Fc and HA-hHO-1 in TG pigs were confirmed using PCR, RT-PCR, western blot, ELISA, and immunohistochemistry. shTNFRI-Fc and HA-hHO-1 were expressed in various organs, including the heart, lung, and spleen. ELISA assays detected shTNFRI-Fc in the sera of TG pigs. For functional analysis, fibroblasts isolated from a shTNFRI-Fc-F2A-HA-hHO-1 TG pig (i.e., #14; 1 × 10(5) cells) were cultured with hTNF-α (20 ng/mL) and cycloheximide (10 μg/mL). The viability of shTNFRI-Fc-F2A-HA-hHO-1 TG pig fibroblasts was significantly higher than that of the wild type (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 24 h, 31.6 ± 3.2 vs. 60.4 ± 8.3 %, respectively; p hHO-1 TG pig fibroblasts was lower than that of the wild type pig fibroblasts (wild type vs. shTNFRI-Fc-F2A-HA-hHO-1 TG at 12 h, 812,452 ± 113,078 RLU vs. 88,240 ± 10,438 RLU, respectively; p hHO-1 TG pigs generated by the F2A self-cleaving peptide express both shTNFRI-Fc and HA-hHO-1 molecules, which provides protection against oxidative and inflammatory injury

The study on Egr-1 promoter which is radioactive promoter

International Nuclear Information System (INIS)

Zhang Chunzhi; Guo Yang; Lv Zhonghong

2006-01-01

Radiogenetic therapy is a heated reaseach on oncotherapy. Early growth response gene-1 (Egr-1) gene promoter is a probably means in radiogenetic therapy. The article review studying on Egr-1 gene promoter and constructing regulating gene expressing system by radiation-inducible Egr-1 gene promoter. (authors)

Isolating Barley ( Hordeum vulgare L.) B1 Hordein Gene Promoter ...

African Journals Online (AJOL)

Promoters play the most important role in determining the temporal and spatial expression pattern and transcript level of a gene. Some strong constitutive promoters, such as cauliflower mosaic virus 35s promoter, are widely used in plant genetic engineering research. However, the expression levels of the foreign genes in ...

Medicago truncatula SOC1 Genes Are Up-regulated by Environmental Cues That Promote Flowering

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Jared B. Fudge

2018-04-01

Full Text Available Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC and CONSTANS (CO, being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1, plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a–MtSOC1c. All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Protectin DX suppresses hepatic gluconeogenesis through AMPK-HO-1-mediated inhibition of ER stress.

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Jung, Tae Woo; Kim, Hyung-Chun; Abd El-Aty, A M; Jeong, Ji Hoon

2017-06-01

Several studies have shown that protectins, which are ω-3 fatty acid-derived proresolution mediators, may improve insulin resistance. Recently, protectin DX (PDX) was documented to attenuate insulin resistance by stimulating IL-6 expression in skeletal muscle, thereby regulating hepatic gluconeogenesis. These findings made us investigate the direct effects of PDX on hepatic glucose metabolism in the context of diabetes. In the current study, we show that PDX regulates hepatic gluconeogenesis in a manner distinct from its indirect glucoregulatory activity via IL-6. We found that PDX stimulated AMP-activated protein kinase (AMPK) phosphorylation, thereby inducing heme oxygenase 1 (HO-1) expression. This induction blocked hepatic gluconeogenesis by suppressing endoplasmic reticulum (ER) stress in hepatocytes under hyperlipidemic conditions. These effects were significantly dampened by silencing AMPK or HO-1 expression with small interfering RNA (siRNA). We also demonstrated that administration of PDX to high fat diet (HFD)-fed mice resulted in increased hepatic AMPK phosphorylation and HO-1 expression, whereas hepatic ER stress was substantially attenuated. Furthermore, PDX treatment suppressed the expression of gluconeogenic genes, thereby decreasing blood glucose levels in HFD-fed mice. In conclusion, our findings suggest that PDX inhibits hepatic gluconeogenesis via AMPK-HO-1-dependent suppression of ER stress. Thus, PDX may be an effective therapeutic target for the treatment of insulin resistance and type 2 diabetes through the regulation of hepatic gluconeogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Estimation of human absorbed dose for (166)Ho-PAM: comparison with (166)Ho-DOTMP and (166)Ho-TTHMP.

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Vaez-Tehrani, Mahdokht; Zolghadri, Samaneh; Yousefnia, Hassan; Afarideh, Hossein

2016-10-01

In this study, the human absorbed dose of holmium-166 ((166)Ho)-pamidronate (PAM) as a potential agent for the management of multiple myeloma was estimated. (166)Ho-PAM complex was prepared at optimized conditions and injected into the rats. The equivalent and effective absorbed doses to human organs after injection of the complex were estimated by radiation-absorbed dose assessment resource and methods proposed by Sparks et al based on rat data. The red marrow to other organ absorbed dose ratios were compared with these data for (166)Ho-DOTMP, as the only clinically used (166)Ho bone marrow ablative agent, and (166)Ho-TTHMP. The highest absorbed dose amounts are observed in the bone surface and bone marrow with 1.11 and 0.903 mGy MBq(-1), respectively. Most other organs would receive approximately insignificant absorbed dose. While (166)Ho-PAM demonstrated a higher red marrow to total body absorbed dose ratio than (166)Ho-1,4,7,10-tetraazacyclo dodecane-1,4,7,10 tetra ethylene phosphonic acid (DOTMP) and (166)Ho-triethylene tetramine hexa (methylene phosphonic acid) (TTHMP), the red marrow to most organ absorbed dose ratios for (166)Ho-TTHMP and (166)Ho-PAM are much higher than the ratios for (166)Ho-DOTMP. The result showed that (166)Ho-PAM has significant characteristics than (166)Ho-DOTMP and therefore, this complex can be considered as a good agent for bone marrow ablative therapy. In this work, two separate points have been investigated: (1) human absorbed dose of (166)Ho-PAM, as a potential bone marrow ablative agent, has been estimated; and (2) the complex has been compared with (166)Ho-DOTMP, as the only clinically used bone marrow ablative radiopharmaceutical, showing significant characteristics.

Two distinct promoters drive transcription of the human D1A dopamine receptor gene.

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Lee, S H; Minowa, M T; Mouradian, M M

1996-10-11

The human D1A dopamine receptor gene has a GC-rich, TATA-less promoter located upstream of a small, noncoding exon 1, which is separated from the coding exon 2 by a 116-base pair (bp)-long intron. Serial 3'-deletions of the 5'-noncoding region of this gene, including the intron and 5'-end of exon 2, resulted in 80 and 40% decrease in transcriptional activity of the upstream promoter in two D1A-expressing neuroblastoma cell lines, SK-N-MC and NS20Y, respectively. To investigate the function of this region, the intron and 245 bp at the 5'-end of exon 2 were investigated. Transient expression analyses using various chloramphenicol acetyltransferase constructs showed that the transcriptional activity of the intron is higher than that of the upstream promoter by 12-fold in SK-N-MC cells and by 5.5-fold in NS20Y cells in an orientation-dependent manner, indicating that the D1A intron is a strong promoter. Primer extension and ribonuclease protection assays revealed that transcription driven by the intron promoter is initiated at the junction of intron and exon 2 and at a cluster of nucleotides located 50 bp downstream from this junction. The same transcription start sites are utilized by the chloramphenicol acetyltransferase constructs employed in transfections as well as by the D1A gene expressed within the human caudate. The relative abundance of D1A transcripts originating from the upstream promoter compared with those transcribed from the intron promoter is 1.5-2.9 times in SK-N-MC cells and 2 times in the human caudate. Transcript stability studies in SK-N-MC cells revealed that longer D1A mRNA molecules containing exon 1 are degraded 1.8 times faster than shorter transcripts lacking exon 1. Although gel mobility shift assay could not detect DNA-protein interaction at the D1A intron, competitive co-transfection using the intron as competitor confirmed the presence of trans-acting factors at the intron. These data taken together indicate that the human D1A gene has

Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

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Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

2009-01-01

The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 laccase gene contains a putative site for xenobiotics (XRE). (Author)

Cloning and expression of Icc1 Laccase gene promoter in Aspergillus niger

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Marqueda-Galvez, A. P.; Loera Carrol, O.; Xaconostle cazares, B.; Tellez-Jurado, A.; Arana-Cuenca, A.

2009-07-01

The white rot fungus Trametes sp. I-62 is a strain with laccase activity and a great potential for biotechnological applications given its ability to detoxify distillery effluents. The Icc1, Icc2 and Icc3 laccase genes of this basidiomycetes have been cloned and sequenced. The promoter region of Icc1 kaccase gene contains a putative site for xenobiotics (XRE). (Author)

HO-1 gene overexpression enhances the beneficial effects of superparamagnetic iron oxide labeled bone marrow stromal cells transplantation in swine hearts underwent ischemia/reperfusion: an MRI study.

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Jiang, Yibo; Chen, Lijuan; Tang, Yaoliang; Ma, Genshan; Shen, Chengxing; Qi, Chunmei; Zhu, Qi; Yao, Yuyu; Liu, Naifeng

2010-05-01

To determine the effect of intracoronary transfer of superparamagnetic iron oxide (SPIO) labeled heme oxygenase-1 (HO-1) overexpressed bone marrow stromal cells (BMSCs) in a porcine myocardial ischemia/reperfusion model. Cell apoptosis was assayed and supernatant cytokine concentrations were measured in BMSCs that underwent hypoxia/reoxygen in vitro. Female mini-swines that underwent 1 h LAD occlusion followed by 1 h reperfusion were randomly allocated to receive intracoronary saline (control), 1 x 10(7) SPIO-labeled BMSCs transfected with pcDNA3.1-Lacz plasmid (Lacz-BMSCs), pcDNA3.1-human HO-1 (HO-1-BMSCs), pcDNA3.1-hHO-1 pretreated with a HO inhibitor, tin protoporphyrin (SnPP, n = 10 each). MRI and postmortem histological analysis were made at 1 week or 3 months thereafter. Post hypoxia/reoxygen in vitro, apoptosis was significantly reduced, supernatant VEGF significantly increased while TNF-alpha and IL-6 significantly reduced in HO-1-BMSCs group compared with Lacz-BMSCs group (all p < 0.05). Myocardial expression of VEGF was significantly higher in HO-1-BMSCs than in Lacz-BMSCs group at 1 week post transplantation (all p < 0.05). Signal voids induced by the SPIO were detected in the peri-infarction region in all BMSC groups at 1 week but not at 3 months post transplantation and the extent of the hypointense signal was the highest in HO-1-BMSCs group, and histological analysis showed that signal voids represented cardiac macrophages that engulfed the SPIO-labeled BMSCs. Pretreatment with SnPP significantly attenuated the beneficial effects of HO-1-BMSCs. Transplantation of HO-1-overexpressed BMSCs significantly enhanced the beneficial effects of BMSCs on improving cardiac function in this model.

Prognostic Significance of Promoter DNA Hypermethylation of cysteine dioxygenase 1 (CDO1 Gene in Primary Breast Cancer.

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Naoko Minatani

Full Text Available Using pharmacological unmasking microarray, we identified promoter DNA methylation of cysteine dioxygenase 1 (CDO1 gene in human cancer. In this study, we assessed the clinicopathological significance of CDO1 methylation in primary breast cancer (BC with no prior chemotherapy. The CDO1 DNA methylation was quantified by TaqMan methylation specific PCR (Q-MSP in 7 BC cell lines and 172 primary BC patients with no prior chemotherapy. Promoter DNA of the CDO1 gene was hypermethylated in 6 BC cell lines except SK-BR3, and CDO1 gene expression was all silenced at mRNA level in the 7 BC cell lines. Quantification of CDO1 methylation was developed using Q-MSP, and assessed in primary BC. Among the clinicopathologic factors, CDO1 methylation level was not statistically significantly associated with any prognostic factors. The log-rank plot analysis elucidated that the higher methylation the tumors harbored, the poorer prognosis the patients exhibited. Using the median value of 58.0 as a cut-off one, disease specific survival in BC patients with CDO1 hypermethylation showed significantly poorer prognosis than those with hypomethylation (p = 0.004. Multivariate Cox proportional hazards model identified that CDO1 hypermethylation was prognostic factor as well as Ki-67 and hormone receptor status. The most intriguingly, CDO1 hypermethylation was of robust prognostic relevance in triple negative BC (p = 0.007. Promoter DNA methylation of CDO1 gene was robust prognostic indicator in primary BC patients with no prior chemotherapy. Prognostic relevance of the CDO1 promoter DNA methylation is worthy of being paid attention in triple negative BC cancer.

Heme oxygenase-1 and abscisic acid effects MAPK´s gene expression in soybean seeds

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Giacometti, R.; Santa Cruz, D.; Noriega, G.; Balestrasse, K.

2012-01-01

In soybean previous studies enabled the identification of MAPK3 and 6 whose activity is enhanced within the signaling pathway leading to defense reactions. In this study the effects of different compounds related to hemeoxygenase (HO-1) biosynthesis on mitogen-activated protein kinase (MAPK’s) genes expression in soybean seeds were tested. To this end, 20μM hemine, 22μM ZnPPIX, 0.5mM furidine or 100μM 8-bromoguanosine 3',5'-cyclic monophosphate (8Br) were added to pre-hydrated seeds for 5 days. MAPK’s genes expression was enhanced in seeds treated with hemine. This result indicates that heme catabolism could be involved in the signaling mediated by this cascade pathway. To confirm this hypothesis experiments were carried out in the precsence of ZnPPIX, a potent irreversible HO-1 inhibitor. In this case, no gene induction was observed. On the other hand, 8Br, a cGMP analog, induced HO-1 gene expression but did not modulate MAPK’s, indicating that this effect could not be mediated by cGMP. When the action of furidine, an abscisic acid inhibitor, was tested a diminution of HO-1 gene expression was observed. In this regard, MAPK’s showed a different response, being MAPK6 the only transcript that showed a diminished respect to controls, while MAPK3 mRNA as well as MAPKK1 was enhanced. These results were confirmed by western blotting and activity determinations. (authors)

Diode-Pumped Thulium (Tm)/Holmium (Ho) Composite Fiber 2.1-Micrometers Laser

Science.gov (United States)

2015-09-01

Schematic of the 800-nm diode pumped Tm/Ho composite fiber laser 8 Under quasi-continuous wave (Q- CW ) pumping conditions of 1-ms duration and a...Fig. 9 (Top) Schematic of the 800-nm diode -pumped Tm/Ho composite fiber laser with outcoupler. (Left) Q- CW laser performance of the Tm/Ho composite...ARL-TR-7452 ● SEP 2015 US Army Research Laboratory Diode -Pumped Thulium (Tm)/Holmium (Ho) Composite Fiber 2.1-μm Laser by G

A Heme Oxygenase-1 Transducer Model of Degenerative and Developmental Brain Disorders

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Hyman M. Schipper

2015-03-01

Full Text Available Heme oxygenase-1 (HO-1 is a 32 kDa protein which catalyzes the breakdown of heme to free iron, carbon monoxide and biliverdin. The Hmox1 promoter contains numerous consensus sequences that render the gene exquisitely sensitive to induction by diverse pro-oxidant and inflammatory stimuli. In “stressed” astroglia, HO-1 hyperactivity promotes mitochondrial iron sequestration and macroautophagy and may thereby contribute to the pathological iron deposition and bioenergetic failure documented in Alzheimer disease, Parkinson disease and certain neurodevelopmental conditions. Glial HO-1 expression may also impact neuroplasticity and cell survival by modulating brain sterol metabolism and the proteasomal degradation of neurotoxic proteins. The glial HO-1 response may represent a pivotal transducer of noxious environmental and endogenous stressors into patterns of neural damage and repair characteristic of many human degenerative and developmental CNS disorders.

AN ELISA ASSAY FOR HEME OXYGENASE (HO-1)

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An ELISA assay for heme oxygenase (HO-l ) Abstract A double antibody capture ELISA for the HO-l protein has been developed to separately quantitate HO-I protein. The use of 2.5% NP40 detergent greatly assists in freeing HO-l protein from membranes and/or other cel...

Upregulation of human heme oxygenase gene expression by Ets-family proteins.

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Deramaudt, B M; Remy, P; Abraham, N G

1999-03-01

Overexpression of human heme oxygenase-1 has been shown to have the potential to promote EC proliferation and angiogenesis. Since Ets-family proteins have been shown to play an important role in angiogenesis, we investigated the presence of ETS binding sites (EBS), GGAA/T, and ETS protein contributing to human HO-1 gene expression. Several chloramphenicol acetyltransferase constructs were examined in order to analyze the effect of ETS family proteins on the transduction of HO-1 in Xenopus oocytes and in microvessel endothelial cells. Heme oxygenase promoter activity was up-regulated by FLI-1ERGETS-1 protein(s). Chloramphenicol acetyltransferase (CAT) assays demonstrated that the promoter region (-1500 to +19) contains positive and negative control elements and that all three members of the ETS protein family were responsible for the up-regulation of HHO-1. Electrophoretic mobility shift assays (EMSA), performed with nuclear extracts from endothelial cells overexpressing HHO-1 gene, and specific HHO-1 oligonucleotides probes containing putative EBS resulted in a specific and marked bandshift. Synergistic binding was observed in EMSA between AP-1 on the one hand, FLI-1, ERG, and ETS-1 protein on the other. Moreover, 5'-deletion analysis demonstrated the existence of a negative control element of HHO-1 expression located between positions -1500 and -120 on the HHO-1 promoter. The presence of regulatory sequences for transcription factors such as ETS-1, FLI-1, or ERG, whose activity is associated with cell proliferation, endothelial cell differentiation, and matrix metalloproteinase transduction, may be an indication of the important role that HO-1 may play in coronary collateral circulation, tumor growth, angiogenesis, and hemoglobin-induced endothelial cell injuries.

[hHO-1 structure prediction and its mutant construct, expression, purification and activity analysis].

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Xia, Zhen Wei; Cui, Wen Jun; Zhou, Wen Pu; Zhang, Xue Hong; Shen, Qing Xiang; Li, Yun Zhu; Yu, Shan Chang

2004-10-01

Human Heme Oxygenase-1 (hHO-1) is the rate-limiting enzyme in the catabolism reaction of heme, which directly regulates the concentration of bilirubin in human body. The mutant structure was simulated by Swiss-pdbviewer procedure, which showed that the structure of active pocket was changed distinctly after Ala25 substituted for His25 in active domain, but the mutated enzyme still binded with heme. On the basis of the results, the expression vectors, pBHO-1 and pBHO-1(M), were constructed, induced by IPTG and expressed in E. coli DH5alpha strain. The expression products were purified with 30%-60% saturation (NH4)2SO4 and Q-Sepharose Fast Flow column chromatography. The concentration of hHO-1 in 30%-60% saturation (NH4)2SO4 components and in fractions through twice column chromatography was 3.6-fold and 30-fold higher than that in initial product, respectively. The activity of wild hHO-1 (whHO-1) and mutant hHO-1 (deltahHO-1) showed that the activity of deltahHO-1 was reduced 91.21% compared with that of whHO-1. The study shows that His25 is of importance for the mechanism of hHO-1, and provides the possibility for effectively regulating the activity to exert biological function.

Over-expression of heme oxygenase-1 promotes oxidative mitochondrial damage in rat astroglia.

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Song, Wei; Su, Haixiang; Song, Sisi; Paudel, Hemant K; Schipper, Hyman M

2006-03-01

Glial heme oxygenase-1 is over-expressed in the CNS of subjects with Alzheimer disease (AD), Parkinson disease (PD) and multiple sclerosis (MS). Up-regulation of HO-1 in rat astroglia has been shown to facilitate iron sequestration by the mitochondrial compartment. To determine whether HO-1 induction promotes mitochondrial oxidative stress, assays for 8-epiPGF(2alpha) (ELISA), protein carbonyls (ELISA) and 8-OHdG (HPLC-EC) were used to quantify oxidative damage to lipids, proteins, and nucleic acids, respectively, in mitochondrial fractions and whole-cell compartments derived from cultured rat astroglia engineered to over-express human (h) HO-1 by transient transfection. Cell viability was assessed by trypan blue exclusion and the MTT assay, and cell proliferation was determined by [3H] thymidine incorporation and total cell counts. In rat astrocytes, hHO-1 over-expression (x 3 days) resulted in significant oxidative damage to mitochondrial lipids, proteins, and nucleic acids, partial growth arrest, and increased cell death. These effects were attenuated by incubation with 1 microM tin mesoporphyrin, a competitive HO inhibitor, or the iron chelator, deferoxamine. Up-regulation of HO-1 engenders oxidative mitochondrial injury in cultured rat astroglia. Heme-derived ferrous iron and carbon monoxide (CO) may mediate the oxidative modification of mitochondrial lipids, proteins and nucleic acids in these cells. Glial HO-1 hyperactivity may contribute to cellular oxidative stress, pathological iron deposition, and bioenergetic failure characteristic of degenerating and inflamed neural tissues and may constitute a rational target for therapeutic intervention in these conditions. Copyright 2005 Wiley-Liss, Inc.

Epigenetics in type 1 diabetes: TNFa gene promoter methylation status in Chilean patients with type 1 diabetes mellitus.

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Arroyo-Jousse, Viviana; Garcia-Diaz, Diego F; Codner, Ethel; Pérez-Bravo, Francisco

2016-12-01

TNF-α is a pro-inflammatory cytokine that is involved in type 1 diabetes (T1D) pathogenesis. The TNFa gene is subject of epigenetic regulation in which folate and homocysteine are important molecules because they participate in the methionine cycle where the most important methyl group donor (S-adenosylmethionine) is formed. We investigated whether TNFa gene promoter methylation status in T1D patients was related to blood folate, homocysteine and TNF-α in a transversal case-control study. We studied T1D patients (n 25, mean=13·7 years) and healthy control subjects (n 25, mean=31·1 years), without T1D and/or other autoimmune diseases or direct family history of these diseases. A blood sample was obtained for determination of serum folate, plasma homocysteine and TNF-α concentrations. Whole blood was used for the extraction of DNA to determine the percentage of methylation by real-time PCR and melting-curve analysis. Results are expressed as means and standard deviations for parametric variables and as median (interquartile range) for non-parametric variables. T1D patients showed a higher TNFa gene promoter methylation (39·2 (sd 19·5) %) when compared with control subjects (25·4 (sd 13·7) %) (P=0·008). TNFa gene promoter methylation was positively associated only with homocysteine levels in T1D patients (r 0·55, P=0·007), but not in control subjects (r -0·122, P=0·872). To our knowledge, this is the first work that reports the methylation status of the TNFa gene promoter and its relationship with homocysteine metabolism in Chilean T1D patients without disease complications.

A vigilant, hypoxia-regulated heme oxygenase-1 gene vector in the heart limits cardiac injury after ischemia-reperfusion in vivo.

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Tang, Yao Liang; Qian, Keping; Zhang, Y Clare; Shen, Leping; Phillips, M Ian

2005-12-01

The effect of a cardiac specific, hypoxia-regulated, human heme oxygenase-1 (hHO-1) vector to provide cardioprotection from ischemia-reperfusion injury was assessed. When myocardial ischemia and reperfusion is asymptomatic, the damaging effects are cumulative and patients miss timely treatment. A gene therapy approach that expresses therapeutic genes only when ischemia is experienced is a desirable strategy. We have developed a cardiac-specific, hypoxia-regulated gene therapy "vigilant vector'' system that amplifies cardioprotective gene expression. Vigilant hHO-1 plasmids, LacZ plasmids, or saline (n = 40 per group) were injected into mouse heart 2 days in advance of ischemia-reperfusion injury. Animals were exposed to 60 minutes of ischemia followed by 24 hours of reperfusion. For that term (24 hours) effects, the protein levels of HO-1, inflammatory responses, apoptosis, and infarct size were determined. For long-term (3 week) effects, the left ventricular remodeling and recovery of cardiac function were assessed. Ischemia-reperfusion resulted in a timely overexpression of HO-1 protein. Infarct size at 24 hours after ischemia-reperfusion was significantly reduced in the HO-1-treated animals compared with the LacZ-treated group or saline-treated group (P < .001). The reduction of infarct size was accompanied by a decrease in lipid peroxidant activity, inflammatory cell infiltration, and proapoptotic protein level in ischemia-reperfusion-injured myocardium. The long-term study demonstrated that timely, hypoxia-induced HO-1 overexpression is beneficial in conserving cardiac function and attenuating left ventricle remodelling. The vigilant HO-1 vector provides a protective therapy in the heart for reducing cellular damage during ischemia-reperfusion injury and preserving heart function.

c-Myc activates BRCA1 gene expression through distal promoter elements in breast cancer cells

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Chen, Yinghua; Xu, Jinhua; Borowicz, Stanley; Collins, Cindy; Huo, Dezheng; Olopade, Olufunmilayo I

2011-01-01

The BRCA1 gene plays an important role in the maintenance of genomic stability. BRCA1 inactivation contributes to breast cancer tumorigenesis. An increasing number of transcription factors have been shown to regulate BRCA1 expression. c-Myc can act as a transcriptional activator, regulating up to 15% of all genes in the human genome and results from a high throughput screen suggest that BRCA1 is one of its targets. In this report, we used cultured breast cancer cells to examine the mechanisms of transcriptional activation of BRCA1 by c-Myc. c-Myc was depleted using c-Myc-specific siRNAs in cultured breast cancer cells. BRCA1 mRNA expression and BRCA1 protein expression were determined by quantitative RT-PCR and western blot, respectively and BRCA1 promoter activities were examined under these conditions. DNA sequence analysis was conducted to search for high similarity to E boxes in the BRCA1 promoter region. The association of c-Myc with the BRCA1 promoter in vivo was tested by a chromatin immunoprecipitation assay. We investigated the function of the c-Myc binding site in the BRCA1 promoter region by a promoter assay with nucleotide substitutions in the putative E boxes. BRCA1-dependent DNA repair activities were measured by a GFP-reporter assay. Depletion of c-Myc was found to be correlated with reduced expression levels of BRCA1 mRNA and BRCA1 protein. Depletion of c-Myc decreased BRCA1 promoter activity, while ectopically expressed c-Myc increased BRCA1 promoter activity. In the distal BRCA1 promoter, DNA sequence analysis revealed two tandem clusters with high similarity, and each cluster contained a possible c-Myc binding site. c-Myc bound to these regions in vivo. Nucleotide substitutions in the c-Myc binding sites in these regions abrogated c-Myc-dependent promoter activation. Furthermore, breast cancer cells with reduced BRCA1 expression due to depletion of c-Myc exhibited impaired DNA repair activity. The distal BRCA1 promoter region is associated with c

Identification and functional characterisation of the promoter of the calcium sensor gene CBL1 from the xerophyte Ammopiptanthus mongolicus

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Yin Weilun

2010-01-01

Full Text Available Abstract Background CBL1 is a calcium sensor that regulates drought, cold and salt signals in Arabidopsis. Overexpression of CBL1 gene in Arabidopsis and in Ammopiptanthus mongolicus showed different tolerant activities. We are interested in understanding the molecular mechanism of the upstream region of the CBL1 gene of A. mongolicus (AmCBL1. We investigated and characterized the promoter of the AmCBL1 gene, for promoters play a very important role in regulating gene expression in eukaryotes. Results A 1683-bp 5' flanking region was isolated from A. mongolicus. The sequence was identified as AmCBL1 promoter. Analysis of the promoter sequence indicated a 690-bp intron and some basic cis-acting elements were related to various environmental stresses and plant hormones. To identify the functional region of the AmCBL1 promoter, five plant expression vectors fused with the GUS (β-glucuronidase gene, driven by series deleted fragments of AmCBL1 promoter at different lengths from -1659, -1414, -1048, -296 to -167 bp relative to the transcriptional start site were constructed and transformed into Nicotiana tabacum L. cv. 89. Functional properties of each promoter segment were examined by GUS staining and fluorescence quantitative analyses using at least three single-copy PCR-positive plants of transgenic tobacco, treated with various environmental stresses and plant hormones for different times. We demonstrated that the AmCBL1 promoter was a vascular-specific and multiple-stress-inducible promoter. Our results further imply that the promoter fragment B1S3 possessed sufficient essential cis-acting elements, accounting for vascular-specific and stress-induced expression patterns. It may also indicate that for response to some stresses certain cis-elements are required in tissues outside the region of the B1S3 construct. Conclusions To help resolve uncertainties about the upstream regulatory mechanism of the CBL1 gene in desert plants, we suggest that

Heme-Oxygenase-1 Expression Contributes to the Immunoregulation Induced by Fasciola hepatica and Promotes Infection

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Paula Carasi

2017-07-01

Full Text Available Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1, the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand, cells expressing intermediate levels of HO-1 expressed high levels of F4/80, CD68, Ly6C, and FIZZ-1, indicating that they might correspond to alternatively activated macrophages. Furthermore, the pharmacological induction of HO-1 with the synthetic metalloporphyrin CoPP promoted F. hepatica infection increasing the clinical signs associated with the disease. In contrast, treatment with the HO-1 inhibitor SnPP protected mice from parasite infection, indicating that HO-1 plays an essential role during F. hepatica infection. Finally, HO-1 expression during F. hepatica infection was associated with TGFβ and IL-10 levels in liver and peritoneum, suggesting that HO-1 controls the expression of these immunoregulatory cytokines during infection favoring parasite survival in the host. These results contribute to the elucidation of the immunoregulatory mechanisms induced by F. hepatica in the host and provide alternative checkpoints to control fasciolosis.

NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

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Pecorelli, Alessandra; Bocci, Velio; Acquaviva, Alessandra; Belmonte, Giuseppe; Gardi, Concetta; Virgili, Fabio; Ciccoli, Lucia; Valacchi, Giuseppe

2013-01-01

During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O 3 per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H 2 O 2 ) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H 2 O 2 are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment

NRF2 activation is involved in ozonated human serum upregulation of HO-1 in endothelial cells

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Pecorelli, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Child Neuropsychiatry Unit, University Hospital, AOUS, Siena (Italy); Bocci, Velio [Department of Physiology, University of Siena (Italy); Acquaviva, Alessandra [Department of Molecular and Developmental Medicine, University of Siena (Italy); Belmonte, Giuseppe [Department of Biomedical Sciences, University of Siena (Italy); Gardi, Concetta [Department of Molecular and Developmental Medicine, University of Siena (Italy); Virgili, Fabio [INRAN, Rome (Italy); Ciccoli, Lucia [Department of Molecular and Developmental Medicine, University of Siena (Italy); Valacchi, Giuseppe, E-mail: giuseppe.valacchi@unife.it [Department of Life Sciences and Biotechnology, University of Ferrara (Italy); Department of Food and Nutrition, Kyung Hee University, Seoul (Korea, Republic of)

2013-02-15

During the last decade, it has been shown that the activation of NRF2 and the binding to electrophile-responsive element (EpREs), stimulates the expression of a great number of genes responsible for the synthesis of phase I and phase II proteins, including antioxidants enzymes and heme oxygenase-1 (HO-1). This critical cell response occurs in cardiovascular, degenerative and chronic infective diseases aggravated by a chronic oxidative stress. In our previous reports we have shown that ozonated plasma is able to up-regulate HO-1 expression in endothelial cells. In the present work we investigated a candidate mechanism involved in this process. After treatment with increasing doses of ozonated serum (20, 40 and 80 μg/mL O{sub 3} per mL of serum), a clear dose dependent activation of NRF2 and the subsequent induction of HO-1 and NAD(P)H quinone oxidoreductase 1(NQO1) was observed. This effect was also present when cells were treated with serum and hydrogen peroxide (H{sub 2}O{sub 2}) or serum and 4-hydroxynonenal (4HNE). Moreover, the treatment with ozonated serum was associated with a dose-dependent activation of extracellular-signal-regulated kinases (ERK1/2) and p38 MAP kinases (p38), not directly involved in NRF2 activation. These data, provide a new insight on the mechanism responsible for the induction of HO-1 expression by ozonated serum in the endothelium, and have a practical importance as an expedient approach to the treatment of patients with both effective orthodox drugs and ozonated autohemotherapy, targeted to the restoration of redox homeostasis. - Highlights: ► Endothelial HO1 is upregulated by ozonated plasma ► This activation is induced by NRF2 and it is ERK independent. ► 4HNE and H{sub 2}O{sub 2} are the main molecules involved in this process. ► Ozonated plasma induced a hormetic effect ► Combination of orthodox medicine and ozonated plasma can be a useful treatment.

Involvement of heme oxygenase-1 in β-cyclodextrin-hemin complex-induced cucumber adventitious rooting process.

Science.gov (United States)

Lin, Yuting; Li, Meiyue; Huang, Liqin; Shen, Wenbiao; Ren, Yong

2012-09-01

Our previous results showed that β-cyclodextrin-hemin complex (CDH) exhibited a vital protective role against cadmium-induced oxidative damage and toxicity in alfalfa seedling roots by the regulation of heme oxygenase-1 (HO-1) gene expression. In this report, we further test whether CDH exhibited the hormonal-like response. The application of CDH and an inducer of HO-1, hemin, were able to induce the up-regulation of cucumber HO-1 gene (CsHO1) expression and thereafter the promotion of adventitious rooting in cucumber explants. The effect is specific for HO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blocked the above responses triggered by CDH, and the inhibitory effects were reversed further when 30% saturation of CO aqueous solution was added together. Further, molecular evidence showed that CDH triggered the increases of the HO-1-mediated target genes responsible for adventitious rooting, including one DnaJ-like gene (CsDNAJ-1) and two calcium-dependent protein kinase (CDPK) genes (CsCDPK1 and CsCDPK5), and were inhibited by ZnPP and reversed by CO. The calcium (Ca2+) chelator ethylene glycol-bis (2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and the Ca2+ channel blocker lanthanum chloride (LaCl3) not only compromised the induction of adventitious rooting induced by CDH but also decreased the transcripts of above three target genes. However, the application of ascorbic acid (AsA), a well-known antioxidant in plants, failed to exhibit similar inducible effect on adventitious root formation. In short, above results illustrated that the response of CDH in the induction of cucumber adventitious rooting might be through HO-1-dependent mechanism and calcium signaling. Physiological, pharmacological and molecular evidence showed that β-cyclodextrin-hemin complex (CDH) was able to induce cucumber adventitious rooting through heme oxygenase-1 (HO-1)-dependent mechanism and calcium signaling.

[Gene transfer-induced human heme oxygenase-1 over-expression protects kidney from ischemia-reperfusion injury in rats].

Science.gov (United States)

Lü, Jin-xing; Yan, Chun-yin; Pu, Jin-xian; Hou, Jian-quan; Yuan, He-xing; Ping, Ji-gen

2010-12-14

To study the protection of gene transfer-induced human heme oxygenase-1 over-expression against renal ischemia reperfusion injury in rats. The model of kidney ischemia-reperfusion injury was established with Sprague-Dawley rats. In the therapy group (n=18), the left kidney was perfused and preserved with Ad-hHO-1 at 2.5×10(9) pfu/1.0 ml after flushed with 0-4°C HC-A organ storage solution via donor renal aorta. The rats in control groups were perfused with 0.9% saline solution (n=12) or the vector carrying no interest gene Ad-EGFP 2.5×10(9) pfu/1.0 ml (n=18) instead of Ad-hHO-1. BUN and Cr in serum were measured by slide chemical methods. The kidney samples of rats were harvested for assay of histology, immunohistochemistry and quantification of HO enzymatic activity. Apoptosis cells in the kidney were measured by TUNEL. Ad-hHO-1 via donor renal aorta could transfect renal cells of rats effectively, enzymatic activity of HO in treated group [(1.62±0.07) nmol×mg(-1)×min(-1)] is higher than in control groups treated with saline solution team [(1.27±0.07) nmol×mg(-1)×min(-1)] and vector EGFP team [(1.22±0.06) nmol×mg(-1)×min(-1)] (PhHO-1 expressed hHO-1 in kidneys at a high level. Corresponding to this, the level of BUN and Cr, as well as the number of apoptosis cells, were decreased, and the damage in histology by HE staining was ameliorated. Over-expression of human HO-1 can protect the kidney from ischemia/reperfusion injury in rats.

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 mediates environmental stress responses in plants.

Science.gov (United States)

Hong, Jeum Kyu; Hwang, Byung Kook

2009-01-01

The promoter of the pepper pathogen-induced membrane protein gene CaPIMP1 was analyzed by an Agrobacterium-mediated transient expression assay in tobacco leaves. Several stress-related cis-acting elements (GT-1, W-box and ABRE) are located within the CaPIMP1 promoter. In tobacco leaf tissues transiently transformed with a CaPIMP1 promoter-beta-glucuronidase (GUS) gene fusion, serially 5'-deleted CaPIMP1 promoters were differentially activated by Pseudomonas syringae pv. tabaci, ethylene, methyl jasmonate, abscisic acid, and nitric oxide. The -1,193 bp region of the CaPIMP1 gene promoter sequence exhibited full promoter activity. The -417- and -593 bp promoter regions were sufficient for GUS gene activation by ethylene and methyl jasmonate treatments, respectively. However, CaPIMP1 promoter sequences longer than -793 bp were required for promoter activation by abscisic acid and sodium nitroprusside treatments. CaPIMP1 expression was activated in pepper leaves by treatment with ethylene, methyl jasmonate, abscisic acid, beta-amino-n-butyric acid, NaCl, mechanical wounding, and low temperature, but not with salicylic acid. Overexpression of CaPIMP1 in Arabidopsis conferred hypersensitivity to mannitol, NaCl, and ABA during seed germination but not during seedling development. In contrast, transgenic plants overexpressing CaPIMP1 exhibited enhanced tolerance to oxidative stress induced by methyl viologen during germination and early seedling stages. These results suggest that CaPIMP1 expression may alter responsiveness to environmental stress, as well as to pathogen infection.

Analysis of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene and promoter in Hodgkin's disease isolates

DEFF Research Database (Denmark)

Sandvej, K; Andresen, B S; Zhou, X G

2000-01-01

AIMS: To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. METHODS: Sequence...... analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). RESULTS: Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1...... promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell...

DNMT 1 maintains hypermethylation of CAG promoter specific region and prevents expression of exogenous gene in fat-1 transgenic sheep.

Science.gov (United States)

Yang, Chunrong; Shang, Xueying; Cheng, Lei; Yang, Lei; Liu, Xuefei; Bai, Chunling; Wei, Zhuying; Hua, Jinlian; Li, Guangpeng

2017-01-01

Methylation is an important issue in gene expression regulation and also in the fields of genetics and reproduction. In this study, we created fat-1 transgenic sheep, investigated the fine-mapping and the modulatory mechanisms of promoter methylation. Sheep fetal fibroblasts were transfected by pCAG-fat1-IRES-EGFP. Monoclonal cell line was screened as nuclear donor and carried out nuclear transfer (441 transgenic cloned embryos, 52 synchronism recipient sheep). Six offsprings were obtained. Expressions of exogenous genes fat-1 and EGFP were detectable in 10 examined tissues and upregulated omega-3 fatty acid content. Interestingly, more or less EGFP negative cells were detectable in the positive transgenic fetal skin cells. EGFP negative and positive cells were sorted by flow cytometry, and their methylation status in the whole promoter region (1701 nt) were investigated by bisulphate sequencing. The fine-mapping of methylation in CAG promoter were proposed. The results suggested that exogenous gene expression was determined by the methylation status from 721-1346 nt and modulated by methylation levels at 101, 108 and 115 nt sites in CAG promoter. To clarify the regulatory mechanism of methylation, examination of four DNA methyltransferases (DNMTs) demonstrated that hypermethylation of CAG promoter is mainly maintained by DNMT 1 in EGFP negative cells. Furthermore, investigation of the cell surface antigen CD34, CD45 and CD166 indicated that EGFP positive and negative cells belong to different types. The present study systematically clarified methylation status of CAG promoter in transgenic sheep and regulatory mechanism, which will provide research strategies for gene expression regulation in transgenic animals.

Analysis of tissue-specific region in sericin 1 gene promoter of Bombyx mori

Energy Technology Data Exchange (ETDEWEB)

Yan, Liu [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Lian, Yu [College of Biomedical Engineering and Instrument Science, Zhejiang University, Hangzhou 310027 (China); Zhejiang Province Key Laboratory of Preventive Veterinary Medicine, Institute of Preventive Veterinary Medicine, Zhejiang University, Hangzhou 310029 (China); Xiuyang, Guo [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Tingqing, Guo [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shengpeng, Wang [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Changde, Lu [Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

2006-03-31

The gene encoding sericin 1 (Ser1) of silkworm (Bombyx mori) is specifically expressed in the middle silk gland cells. To identify element involved in this transcription-dependent spatial restriction, truncation of the 5' terminal from the sericin 1 (Ser1) promoter is studied in vivo. A 209 bp DNA sequence upstream of the transcriptional start site (-586 to -378) is found to be responsible for promoting tissue-specific transcription. Analysis of this 209 bp region by overlapping deletion studies showed that a 25 bp region (-500 to -476) suppresses the ectopic expression of the Ser1 promoter. An unknown factor abundant in fat body nuclear extracts is shown to bind to this 25 bp fragment. These results suggest that this 25 bp region and the unknown factor are necessary for determining the tissue-specificity of the Ser1 promoter.

Characterization of promoter of EgPAL1, a novel PAL gene from the oil palm Elaeis guineensis Jacq.

Science.gov (United States)

Yusuf, Chong Yu Lok; Abdullah, Janna Ong; Shaharuddin, Noor Azmi; Abu Seman, Idris; Abdullah, Mohd Puad

2018-02-01

The oil palm EgPAL1 gene promoter and its regulatory region were functional as a promoter in the heterologous system of Arabidopsis according to the cis-acting elements present in that region. The promoter was developmentally regulated, vascular tissue specific and responsive to water stress agents. Phenylalanine ammonia lyase (PAL, EC 4.3.1.24) is the key enzyme of the phenylpropanoid pathway which plays important roles in plant development and adaptation. To date, there is no report on the study of PAL from oil palm (Elaeis guineensis), an economically important oil crop. In this study, the 5' regulatory sequence of a highly divergent oil palm PAL gene (EgPAL1) was isolated and fused with GUS in Arabidopsis to create two transgenic plants carrying the minimal promoter with (2302 bp) and without its regulatory elements (139 bp). The regulatory sequence contained cis-acting elements known to be important for plant development and stress response including the AC-II element for lignin biosynthesis and several stress responsive elements. The promoter and its regulatory region were fully functional in Arabidopsis. Its activities were characterised by two common fundamental features of PAL which are responsive to plant internal developmental programme and external factors. The promoter was developmentally regulated in certain organs; highly active in young organs but less active or inactive in mature organs. The presence of the AC elements and global activity of the EgPAL1 promoter in all organs resembled the property of lignin-related genes. The existence of the MBS element and enhancement of the promoter activity by PEG reflected the behaviour of drought-responsive genes. Our findings provide a platform for evaluating oil palm gene promoters in the heterologous system of Arabidopsis and give insights into the activities of EgPAL1 promoter in oil palm.

Analysis of Msx1 and Msx2 transactivation function in the context of the heat shock 70 (Hspa1b) gene promoter.

Science.gov (United States)

Zhuang, Fengfeng; Nguyen, Manuel P; Shuler, Charles; Liu, Yi-Hsin

2009-04-03

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent transactivation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and heat shock factors may play a contributing role.

Glutaredoxin 1 (GRX1) inhibits oxidative stress and apoptosis of chondrocytes by regulating CREB/HO-1 in osteoarthritis.

Science.gov (United States)

Sun, Jie; Wei, Xuelei; Lu, Yandong; Cui, Meng; Li, Fangguo; Lu, Jie; Liu, Yunjiao; Zhang, Xi

2017-10-01

GRX1 (glutaredoxin1), a sulfhydryl disulfide oxidoreductase, is involved in many cellular processes, including anti-oxidation, anti-apoptosis, and regulation of cell differentiation. However, the role of GRX1 in the oxidative stress and apoptosis of osteoarthritis chondrocytes remains unclear, prompting the current study. Protein and mRNA expressions were measured by Western blot and RT-qPCR. Oxidative stress was detected by the measurement of MDA and SOD contents. Cells apoptosis were detected by Annexin V-FITC/PI and caspase-3 activity assays. We found that the mRNA and protein expressions of GRX1 were significantly down-regulated in osteoarthritis tissues and cells. GRX1 overexpression increased the mRNA and protein expression of CREB and HO-1. Meanwhile, GRX1 overexpression inhibited oxidative stress and apoptosis in osteoarthritis chondrocytes. Furthermore, we found that GRX1 overexpression regulated HO-1 by increasing CREB, and that HO-1 regulated oxidative stress and apoptosis in osteoarthritis chondrocytes. Thus, GRX1 overexpression constrains oxidative stress and apoptosis in osteoarthritis chondrocytes by regulating CREB/HO-1, providing a novel insight into the molecular mechanism and potential treatment of osteoarthritis. Copyright © 2017. Published by Elsevier Ltd.

Evaluation of promoter methylation status of MLH1 gene in Iranian patients with colorectal tumors and adenoma polyps.

Science.gov (United States)

Zarandi, Ashkan; Irani, Shiva; Savabkar, Sanaz; Chaleshi, Vahid; Ghavideldarestani, Maryam; Mirfakhraie, Reza; Khodadoostan, Mahsa; Nazemalhosseini-Mojarad, Ehsan; Asadzadeh Aghdaei, Hamid

2017-01-01

The aim of this study was to evaluate the methylation status of the promoter region of MLH1 gene in colorectal cancer (CRC) and its precursor lesions as well as elucidate its association with various clinicopathological characteristics among Iranian population. Epigenetic silencing of mismatch repair genes, such as MLH1 , by methylation of CpG islands of their promoter region has been proved to be an important mechanism in colorectal carcinogenesis. Fifty colorectal cancer and polyp tissue samples including 13 Primary colorectal tumor and 37 Adenoma polyp samples were enrolled in this study. Methylation-specific polymerase chain reaction (MSP) was performed to find the frequency of MLH1 Promoter Methylation. Promoter methylation of MLH1 gene was detected in 5 out of 13 tumor tissues and 4 out of 37 adenoma polyp. The frequency of MLH1 methylation in tumor samples was significantly higher compared to that in polyp tissues (P= 0.026). No significant association was observed between MLH1 promoter methylation and clinicopathological characteristics of the patients. The frequency of  MLH1  promoter methylation in CRC and colon polyp was 18%. Our findings indicated that methylation of MLH1 promoter region alone cannot be considered as a biomarker for early detection of CRC.

Mediator subunit MED1 is a T3-dependent and T3-independent coactivator on the thyrotropin β gene promoter

Energy Technology Data Exchange (ETDEWEB)

Matsui, Keiji; Oda, Kasumi; Mizuta, Shumpei; Ishino, Ruri; Urahama, Norinaga; Hasegawa, Natsumi [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Roeder, Robert G. [Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Ito, Mitsuhiro, E-mail: itomi@med.kobe-u.ac.jp [Laboratory of Hematology, Division of Medical Biophysics, Kobe University Graduate School of Health Sciences, 7-10-2 Tomogaoka, Suma-ku, Kobe 654-0142 (Japan); Laboratory of Biochemistry and Molecular Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065 (United States); Department of Family and Community Medicine, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 654-0142 (Japan); Consolidated Research Institute for Advanced Science and Medical Care, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo 159-8555 (Japan)

2013-10-11

Highlights: •MED1 is a bona fide T3-dependent coactivator on TSHB promoter. •Mice with LxxLL-mutant MED1 have attenuated TSHβ mRNA and thyroid hormone levels. •MED1 activates TSHB promoter T3-dependently in cultured cells. •T3-dependent MED1 action is enhanced when SRC1/SRC2 or HDAC2 is downregulated. •MED1 is also a T3-independent GATA2/Pit1 coactivator on TSHB promoter. -- Abstract: The MED1 subunit of the Mediator transcriptional coregulator complex is a nuclear receptor-specific coactivator. A negative feedback mechanism of thyroid-stimulating hormone (TSH, or thyrotropin) expression in the thyrotroph in the presence of triiodothyronine (T3) is employed by liganded thyroid hormone receptor β (TRβ) on the TSHβ gene promoter, where conventional histone-modifying coactivators act as corepressors. We now provide evidence that MED1 is a ligand-dependent positive cofactor on this promoter. TSHβ gene transcription was attenuated in MED1 mutant mice in which the nuclear receptor-binding ability of MED1 was specifically disrupted. MED1 stimulated GATA2- and Pit1-mediated TSHβ gene promoter activity in a ligand-independent manner in cultured cells. MED1 also stimulated transcription from the TSHβ gene promoter in a T3-dependent manner. The transcription was further enhanced when the T3-dependent corepressors SRC1, SRC2, and HDAC2 were downregulated. Hence, MED1 is a T3-dependent and -independent coactivator on the TSHβ gene promoter.

Study of the Role of siRNA Mediated Promoter Methylation in DNMT3B Knockdown and Alteration of Promoter Methylation of CDH1, GSTP1 Genes in MDA-MB -453 Cell Line.

Science.gov (United States)

Naghitorabi, Mojgan; Mir Mohammad Sadeghi, Hamid; Mohammadi Asl, Javad; Rabbani, Mohammad; Jafarian-Dehkordi, Abbas

2017-01-01

Promoter methylation is one of the main epigenetic mechanisms that leads to the inactivation of tumor suppressor genes during carcinogenesis. Due to the reversible nature of DNA methylation, many studies have been performed to correct theses epigenetic defects by inhibiting DNA methyltransferases (DNMTs). In this case novel therapeutics especially siRNA oligonucleotides have been used to specifically knock down the DNMTs at mRNA level. Also many studies have focused on transcriptional gene silencing in mammalian cells via siRNA mediated promoter methylation. The present study was designed to assess the role of siRNA mediated promoter methylation in DNMT3B knockdown and alteration of promoter methylation of Cadherin-1 (CDH1), Glutathione S-Transferase Pi 1(GSTP1), and DNMT3B genes in MDA-MB-453 cell line. MDA-MB-453 cells were transfected with siDNMT targeting DNMT3B promoter and harvested at 24 and 48 h post transfection to monitor gene silencing and promoter methylation respectively. DNMT3B expression was monitored by quantitative RT-PCR method. Promoter methylation was quantitatively evaluated using differential high resolution melting analysis. A non-significant 20% reduction in DNMT3B mRNA level was shown only after first transfection with siDNMT, which was not reproducible. Promoter methylation levels of DNMT3B, CDH1, and GSTP1 were detected at about 15%, 70% and 10% respectively, in the MDA-MB-453 cell line, with no significant change after transfection. Our results indicated that siDNMT sequence were not able to affect promoter methylation and silencing of DNMT3B in MDA-MB-453 cells. However, quantitation of methylation confirmed a hypermethylated phenotype at CDH1 and GSTP1 promoters as well as a differential methylation pattern at DNMT3B promoter in breast cancer.

Inhibition of microRNA-153 protects neurons against ischemia/reperfusion injury in an oxygen-glucose deprivation and reoxygenation cellular model by regulating Nrf2/HO-1 signaling.

Science.gov (United States)

Ji, Qiong; Gao, Jianbo; Zheng, Yan; Liu, Xueli; Zhou, Qiangqiang; Shi, Canxia; Yao, Meng; Chen, Xia

2017-07-01

MicroRNAs are emerging as critical regulators in cerebral ischemia/reperfusion injury; however, their exact roles remain poorly understood. miR-153 is reported to be a neuron-related miRNA involved in neuroprotection. In this study, we aimed to investigate the precise role of miR-153 in regulating neuron survival during cerebral ischemia/reperfusion injury using an oxygen-glucose deprivation and reoxygenation (OGD/R) cellular model. We found that miR-153 was significantly upregulated in neurons subjected to OGD/R treatment. Inhibition of miR-153 significantly attenuated OGD/R-induced injury and oxidative stress in neurons. Nuclear factor erythroid 2-related factor 2 (Nrf2) was identified as a target gene of miR-153. Inhibition of miR-153 significantly promoted the expression of Nrf2 and heme oxygenase-1 (HO-1). However, silencing of Nrf2 significantly blocked the protective effects of miR-153 inhibition. Our study indicates that the inhibition of miR-153 protects neurons against OGD/R-induced injury by regulating Nrf2/HO-1 signaling and suggests a potential therapeutic target for cerebral ischemia/reperfusion injury. © 2017 Wiley Periodicals, Inc.

Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

Science.gov (United States)

Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

2009-01-01

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in probing Msx-dependent mechanisms of gene activation. We showed that Msx protein activates Hspa1b promoter via its C-terminal domain. The activation absolutely depends on the HSEs and physical interactions between Msx proteins and Heat shock factors may play a contributing role. PMID:19338779

Genomic organization and promoter cloning of the human X11α gene APBA1.

LENUS (Irish Health Repository)

Chai, Ka-Ho

2012-05-01

X11α is a brain specific multi-modular protein that interacts with the Alzheimer\\'s disease amyloid precursor protein (APP). Aggregation of amyloid-β peptide (Aβ), an APP cleavage product, is believed to be central to the pathogenesis of Alzheimer\\'s disease. Recently, overexpression of X11α has been shown to reduce Aβ generation and to ameliorate memory deficit in a transgenic mouse model of Alzheimer\\'s disease. Therefore, manipulating the expression level of X11α may provide a novel route for the treatment of Alzheimer\\'s disease. Human X11α is encoded by the gene APBA1. As evidence suggests that X11α expression can be regulated at transcription level, we have determined the gene structure and cloned the promoter of APBA1. APBA1 spans over 244 kb on chromosome 9 and is composed of 13 exons and has multiple transcription start sites. A putative APBA1 promoter has been identified upstream of exon 1 and functional analysis revealed that this is highly active in neurons. By deletion analysis, the minimal promoter was found to be located between -224 and +14, a GC-rich region that contains a functional Sp3 binding site. In neurons, overexpression of Sp3 stimulates the APBA1 promoter while an Sp3 inhibitor suppresses the promoter activity. Moreover, inhibition of Sp3 reduces endogenous X11α expression and promotes the generation of Aβ. Our findings reveal that Sp3 play an essential role in APBA1 transcription.

Deletion of P2 promoter of GJB1 gene a cause of Charcot-Marie-Tooth disease.

Science.gov (United States)

Kulshrestha, R; Burton-Jones, S; Antoniadi, T; Rogers, M; Jaunmuktane, Z; Brandner, S; Kiely, N; Manuel, R; Willis, T

2017-08-01

X-linked Charcot-Marie-Tooth disease (CMT) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene. This gene has nerve specific P2 promoter that work synergistically with SOX10 and EGR2 genes to initiate transcription. Mutation in this region is known to cause Schwann cell dysfunction. A single large family of X linked peripheral neuropathy was identified in our practice. Next generation sequencing for targeted panel assay identified an upstream exon-splicing deletion identified extending from nucleotide c.-5413 to approximately - c.-49. This matches the sequence of 32 nucleotides at positions c.*218-*249 in the 3'UTR downstream of the GJB1 gene. The deleted fragment included the entire P2 promoter region. The deletion segregated with the disease. To our knowledge a deletion of the P2 promoter alone as a cause of CMT has not been reported previously. Copyright © 2017 Elsevier B.V. All rights reserved.

Association of a Human FABP1 Gene Promoter Region Polymorphism with Altered Serum Triglyceride Levels.

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Xian-E Peng

Full Text Available Liver fatty acid-binding protein (L-FABP, also known as fatty acid-binding protein 1 (FABP1, is a key regulator of hepatic lipid metabolism. Elevated FABP1 levels are associated with an increased risk of cardiovascular disease (CVD and metabolic syndromes. In this study, we examine the association of FABP1 gene promoter variants with serum FABP1 and lipid levels in a Chinese population. Four promoter single-nucleotide polymorphisms (SNPs of FABP1 gene were genotyped in a cross-sectional survey of healthy volunteers (n = 1,182 from Fuzhou city of China. Results showed that only the rs2919872 G>A variant was significantly associated with serum TG concentration(P = 0.032.Compared with the rs2919872 G allele, rs2919872 A allele contributed significantly to reduced serum TG concentration, and this allele dramatically decreased the FABP1 promoter activity(P < 0.05. The rs2919872 A allele carriers had considerably lower serum FABP1 levels than G allele carriers (P < 0.01. In the multivariable linear regression analysis, the rs2919872 A allele was negatively associated with serum FABP1 levels (β = -0.320, P = 0.003, while serum TG levels were positively associated with serum FABP1 levels (β = 0.487, P = 0.014. Our data suggest that compared with the rs2919872 G allele, the rs2919872 A allele reduces the transcriptional activity of FABP1 promoter, and thereby may link FABP1 gene variation to TG level in humans.

Andrographolide inhibits TNFα-induced ICAM-1 expression via suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression through the PI3K/Akt/Nrf2 and PI3K/Akt/AP-1 pathways in human endothelial cells.

Science.gov (United States)

Lu, Chia-Yang; Yang, Ya-Chen; Li, Chien-Chun; Liu, Kai-Li; Lii, Chong-Kuei; Chen, Haw-Wen

2014-09-01

Andrographolide, the major bioactive component of Andrographis paniculata, has been demonstrated to have various biological properties including anti-inflammation, antioxidation, and anti-hepatotoxicity. Oxidative stress is considered a major risk factor in aging, inflammation, cancer, atherosclerosis, and diabetes mellitus. NADPH oxidase is a major source of endogenous reactive oxygen species (ROS). In this study, we used EA.hy926 endothelial-like cells to explore the anti-inflammatory activity of andrographolide. Andrographolide attenuated TNFα-induced ROS generation, Src phosphorylation, membrane translocation of the NADPH oxidase subunits p47(phox) and p67(phox), and ICAM-1 gene expression. In the small hairpin RNA interference assay, shp47(phox) abolished TNFα-induced p65 nuclear translocation, ICAM-1 gene expression, and adhesion of HL-60 cells. Andrographolide induced the gene expression of heme oxygenase 1 (HO-1) and glutamate cysteine ligase modifier subunit (GCLM) in a time-dependent manner. Cellular glutathione (GSH) content was increased by andrographolide. shGCLM attenuated the andrographolide-induced increase in GSH content and reversed the andrographolide inhibition of HL-60 adhesion. shHO-1 showed a similar effect on andrographolide inhibition of HL-60 adhesion to shGCLM. The mechanism underlying the up-regulation of HO-1 and GCLM by andrographolide was dependent on the PI3K/Akt pathway, and both the Nrf2 and AP-1 transcriptional factors were involved. Our results suggest that andrographolide attenuates TNFα-induced ICAM-1 expression at least partially through suppression of NADPH oxidase activation and induction of HO-1 and GCLM expression, which is PI3K/Akt pathway-dependent. Copyright © 2014. Published by Elsevier Inc.

Prunella vulgaris Suppresses HG-Induced Vascular Inflammation via Nrf2/HO-1/eNOS Activation

Directory of Open Access Journals (Sweden)

Ho Sub Lee

2012-01-01

Full Text Available Vascular inflammation is an important factor which can promote diabetic complications. In this study, the inhibitory effects of aqueous extract from Prunella vulgaris (APV on high glucose (HG-induced expression of cell adhesion molecules in human umbilical vein endothelial cells (HUVEC are reported. APV decreased HG-induced expression of intercellular adhesion molecule-1 (ICAM-1, vascular cell adhesion molecule-1 (VCAM-1, and E-selectin. APV also dose-dependently inhibited HG-induced adhesion of HL-60 monocytic cells. APV suppressed p65 NF-κB activation in HG-treated cells. APV significantly inhibited the formation of intracellular reactive oxygen species (ROS. HG-stimulated HUVEC secreted gelatinases, however, APV inhibited it. APV induced Akt phosphorylation as well as activation of heme oxygenase-1 (HO-1, eNOS, and nuclear factor E2-related factor 2 (Nrf2, which may protect vascular inflammation caused by HG. In conclusion, APV exerts anti-inflammatory effect via inhibition of ROS/NF-κB pathway by inducing HO-1 and eNOS expression mediated by Nrf2, thereby suggesting that Prunella vulgaris may be a possible therapeutic approach to the inhibition of diabetic vascular diseases.

Fetal Microsatellite in the Heme Oxygenase 1 Promoter Is Associated With Severe and Early-Onset Preeclampsia.

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Kaartokallio, Tea; Utge, Siddheshwar; Klemetti, Miira M; Paananen, Jussi; Pulkki, Kari; Romppanen, Jarkko; Tikkanen, Ilkka; Heinonen, Seppo; Kajantie, Eero; Kere, Juha; Kivinen, Katja; Pouta, Anneli; Lakkisto, Päivi; Laivuori, Hannele

2018-01-01

Preeclampsia is a vascular pregnancy disorder that often involves impaired placental development. HO-1 (heme oxygenase 1, encoded by HMOX1 ) is a stress response enzyme crucial for endothelial and placental function. Long version of the guanine-thymine (GT n ) microsatellite in the HMOX1 promoter decreases HO-1 expression, and the long maternal repeat is associated with late-onset preeclampsia. Our aim was to study whether the length of fetal repeat is associated with mother's preeclampsia, whether the length of fetal and maternal repeats affect HO-1 levels in placenta and maternal serum, and whether HO-1 levels are altered in preeclampsia. We genotyped the repeat in the cord blood of 609 preeclamptic and 745 nonpreeclamptic neonates. HO-1 levels were measured in 36 placental samples, and in the first (222 cases/243 controls) and third (176 cases/53 controls) pregnancy trimester serum samples using enzyme-linked immunosorbent assay. The long fetal GT n repeat was associated with preeclampsia and its severe and early-onset subtypes. Interaction analysis suggested the maternal and fetal effects to be independent. Placental or serum HO-1 levels were not altered in preeclamptics, possibly reflecting heterogeneity of preeclampsia. Carriers of the long fetal and maternal repeats had lower placental and serum HO-1 levels, respectively, providing functional evidence for the association. We conclude that the long fetal GT n repeat may increase mother's risk for especially severe and early-onset preeclampsia. The fetal and maternal risk alleles likely predispose to different disease subtypes. © 2017 American Heart Association, Inc.

Cordycepin alleviates lipopolysaccharide-induced acute lung injury via Nrf2/HO-1 pathway.

Science.gov (United States)

Qing, Rui; Huang, Zezhi; Tang, Yufei; Xiang, Qingke; Yang, Fan

2018-04-24

The present study is to investigate the protective effect of cordycepin on inflammatory reactions in rats with acute lung injury (ALI) induced by lipopolysaccharide (LPS), as well as the underlying mechanism. Wistar rat model of ALI was induced by intravenous injection of LPS (30 mg/kg body weight). One hour later, intravenous injection of cordycepin (1, 10 or 30 mg/kg body weight) was administered. The wet-to-dry weight ratio of lung tissues and myeloperoxidase activity in the lung tissues were measured. The contents of nitrite and nitrate were measured by reduction method, while chemiluminescence was used to determine the content of superoxide. Quantitative real-time polymerase chain reaction and Western blotting were used to determine the expression of mRNA and protein, respectively. Colorimetry was performed to determine the enzymatic activity of heme oxygenase-1 (HO-1). Nuclear translocation of Nrf2 was identified by Western blotting. The plasma contents of cytokines were measured by enzyme-linked immunosorbent assay. Cordycepin enhanced the expression and enzymatic activity of HO-1 in ALI rats, and activated Nrf2 by inducing the translocation of Nrf2 from cytoplasm to nucleus. In addition, cordycepin regulated the secretion of TNF-α, IL-6 and IL-10 via HO-1, and suppressed inflammation in lung tissues of ALI rats by inducing the expression of HO-1. HO-1 played important roles in the down-regulation of superoxide levels in lung tissues by cordycepin, and HO-1 expression induced by cordycepin affected nitrite and nitrate concentrations in plasma and iNOS protein expression in lung tissues. Cordycepin showed protective effect on injuries in lung tissues. The present study demonstrates that cordycepin alleviates inflammation induced by LPS via the activation of Nrf2 and up-regulation of HO-1 expression. Copyright © 2018. Published by Elsevier B.V.

High Glucose Promotes Tumor Invasion and Increases Metastasis-Associated Protein Expression in Human Lung Epithelial Cells by Upregulating Heme Oxygenase-1 via Reactive Oxygen Species or the TGF-β1/PI3K/Akt Signaling Pathway

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Xiaowen Kang

2015-02-01

Full Text Available Background: Growing evidence indicates that heme oxygenase-1 (HO-1 is up-regulated in malignancies and subsequently alters tumor aggressiveness and various cancer-related factors, such as high glucose (HG levels. HO-1 expression can be induced when glucose concentrations are above 25 mM; however, the role of HO-1 in lung cancer patients with diabetes remains unknown. Therefore, in this study we investigated the promotion of tumor cell invasion and the expression of metastasis-associated proteins by inducing the up-regulation of HO-1 expression by HG treatment in A549 human lung epithelial cells. Methods: The expression of HO-1and metastasis-associated protein expression was explored by western blot analysis. HO-1 enzymatic activity, reactive oxygen species (ROS production and TGF-β1 production were examined by ELISA. Invasiveness was analyzed using a Transwell chamber. Results: HG treatment of A549 cells induced an increase in HO-1 expression, which was mediated by the HG-induced generation of reactive oxygen species (ROS and transforming growth factor-β1 (TGF-β1 in a concentration- and time-dependent manner. Following the increase in HO-1 expression, the enzymatic activity of HO-1 also increased in HG-treated cells. Pretreatment with N-acetyl-L-cysteine (NAC or with phosphatidylinositol 3-kinase (PI3K/Akt inhibitors attenuated the HG-induced increase in HO-1 expression. HG treatment of A549 cells enhanced the invasion potential of these cells, as shown with a Transwell assay, and increased metastasis-associated protein expression. However, HO-1 siRNA transfection significantly decreased these capabilities. Conclusion: this study is the first to demonstrate that HG treatment of A549 human lung epithelial cells promotes tumor cell invasion and increases metastasis-associated protein expression by up-regulating HO-1 expression via ROS or the TGF-β1/PI3K/Akt signaling pathway.

Heme Oxygenase-1 Gene Therapy Provides Cardioprotection Via Control of Post-Ischemic Inflammation: An Experimental Study in a Pre-Clinical Pig Model.

Science.gov (United States)

Hinkel, Rabea; Lange, Philipp; Petersen, Björn; Gottlieb, Elena; Ng, Judy King Man; Finger, Stefanie; Horstkotte, Jan; Lee, Seungmin; Thormann, Michael; Knorr, Maike; El-Aouni, Chiraz; Boekstegers, Peter; Reichart, Bruno; Wenzel, Philip; Niemann, Heiner; Kupatt, Christian

2015-07-14

Heme oxygenase-1 (HO-1) is an inducible stress-responsive enzyme converting heme to bilirubin, carbon monoxide, and free iron, which exerts anti-inflammatory and antiapoptotic effects. Although efficient cardioprotection after HO-1 overexpression has been reported in rodents, its role in attenuating post-ischemic inflammation is unclear. This study assessed the efficacy of recombinant adenoassociated virus (rAAV)-encoding human heme oxygenase-1 (hHO-1) in attenuating post-ischemic inflammation in a murine and a porcine ischemia/reperfusion model. Murine ischemia was induced by 45 min of left anterior descending occlusion, followed by 24 h of reperfusion and functional as well as fluorescent-activated cell sorting analysis. Porcine hearts were subjected to 60 min of ischemia and 24h of reperfusion before hemodynamic and histologic analyses were performed. Human microvascular endothelial cells transfected with hHO-1 displayed an attenuated interleukin-6 and intercellular adhesion molecule 1 expression, resulting in reduced monocytic THP-1 cell recruitment in vitro. In murine left anterior descending occlusion and reperfusion, the post-ischemic influx of CD45(+) leukocytes, Ly-6G(+) neutrophils, and Ly-6C(high) monocytes was further exacerbated in HO-1-deficient hearts and reversed by rAAV.hHO-1 treatment. Conversely, in our porcine model of ischemia, the post-ischemic influx of myeloperoxidase-positive neutrophils and CD14(+) monocytes was reduced by 49% and 87% after rAAV.hHO-1 transduction, similar to hHO-1 transgenic pigs. Functionally, rAAV.hHO-1 and hHO-1 transgenic left ventricles displayed a smaller loss of ejection fraction than control animals. Whereas HO-1 deficiency exacerbates post-ischemic cardiac inflammation in mice, hHO-1 gene therapy attenuates inflammation after ischemia and reperfusion in murine and porcine hearts. Regional hHO-1 gene therapy provides cardioprotection in a pre-clinical porcine ischemia/reperfusion model. Copyright © 2015 American

Edaravone Improves Septic Cardiac Function by Inducing an HIF-1α/HO-1 Pathway

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Chao He

2018-01-01

Full Text Available Septic myocardial dysfunction remains prevalent and raises mortality rate in patients with sepsis. During sepsis, tissues undergo tremendous oxidative stress which contributes critically to organ dysfunction. Edaravone, a potent radical scavenger, has been proved beneficial in ischemic injuries involving hypoxia-inducible factor- (HIF- 1, a key regulator of a prominent antioxidative protein heme oxygenase- (HO- 1. However, its effect in septic myocardial dysfunction remains unclarified. We hypothesized that edaravone may prevent septic myocardial dysfunction by inducing the HIF-1/HO-1 pathway. Rats were subjected to cecal ligation and puncture (CLP with or without edaravone infusion at three doses (50, 100, or 200 mg/kg, resp. before CLP and intraperitoneal injection of the HIF-1α antagonist, ME (15 mg/kg, after CLP. After CLP, rats had cardiac dysfunction, which was associated with deformed myocardium, augmented lipid peroxidation, and increased myocardial apoptosis and inflammation, along with decreased activities of catalase, HIF-1α, and HO-1 in the myocardium. Edaravone pretreatment dose-dependently reversed the changes, of which high dose most effectively improved cardiac function and survival rate of septic rats. However, inhibition of HIF-1α by ME demolished the beneficial effects of edaravone at high dose, reducing the survival rate of the septic rats without treatments. Taken together, edaravone, by inducing the HIF-1α/HO-1 pathway, suppressed oxidative stress and protected the heart against septic myocardial injury and dysfunction.

Identification of an ovine atadenovirus gene whose product activates the viral E2 promoter: possible involvement of E2F-1

International Nuclear Information System (INIS)

Kuemin, Daniel; Hofmann, Christian; Uckert, Wolfgang; Both, Gerald W.; Loeser, Peter

2004-01-01

Activation of the adenoviral E2 promoter is an early step in adenovirus gene expression. For members of the mast- and aviadenoviruses, this requires induction of the cellular transcription factor E2F by virally encoded gene products such as E1A, E4orf6/7 and orf22/GAM-1. The newly recognized genus atadenovirus, of which the ovine isolate OAdV is the prototype, lacks any sequence homology to those genes. To find a possible link between E2 promoter activation and OAdV gene expression, we utilized a screening method to search for genes within the OAdV genome that were capable of stimulating the viral E2 promoter. One such gene, E43, was identified within the proposed E4 region toward the right-hand end of the OAdV genome. The E43 gene product was also found to be capable of stimulating E2F-1-dependent gene expression. A closer inspection of the E2 promoter revealed the presence of a non-palindromic E2F binding site within the OAdV E2 promoter. Mutation of this site markedly reduced both E2F-1- and E43-dependent promoter activation. Moreover, a direct protein-protein interaction of the E43 gene product with E2F, but not with the retinoblastoma protein pRb, suggested a possible cooperation between these two proteins in activating the E2 promoter. The importance of the E43 gene product for virus replication is also underlined by the finding that an OAdV recombinant with a functionally inactivated E43 gene showed severely inhibited virus growth

Methylation in the promoter regions of WT1, NKX6-1 and DBC1 genes in cervical cancer tissues of Uygur women in Xinjiang

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Dan Wu

Full Text Available Abstract This study aimed to explore: 1 DNA methylation in the promoter regions of Wilms tumor gene 1 (WT1, NK6 transcription factor related locus 1 gene (NKX6-1 and Deleted in bladder cancer 1 (DBC1 gene in cervical cancer tissues of Uygur women in Xinjiang, and 2 the correlation of gene methylation with the infection of HPV16/18 viruses. We detected HPV16/18 infection in 43 normal cervical tissues, 30 cervical intraepithelial neoplasia lesions (CIN and 48 cervical cancer tissues with polymerase chain reaction (PCR method. Methylation in the promoter regions of the WT1, NKX6-1 and DBC1 genes in the above-mentioned tissues was measured by methylation-specific PCR (MSP and cloning sequencing. The expression level of these three genes was measured by real-time PCR (qPCR in 10 methylation-positive cervical cancer tissues and 10 methylation-negative normal cervical tissues. We found that the infection of HPV16 in normal cervical tissues, CIN and cervical cancer tissues was 14.0, 36.7 and 66.7%, respectively. The infection of HPV18 was 0, 6.7 and 10.4%, respectively. The methylation rates of WT1, NKX6-1 and DBC1 genes were 7.0, 11.6 and 23.3% in normal cervical tissues, 36.7, 46.7 and 30.0% in CIN tissues, and 89.6, 77.1 and 85.4% in cervical cancer tissues. Furthermore, WT1, NKX6-1 and DBC1 genes were hypermethylated in the high-grade squamous intraepithelial lesion (CIN2, CIN3 and in the cervical cancer tissues with infection of HPV16/18 (both P< 0.05. The expression of WT1, NKX6-1 and DBC1 was significantly lower in the methylation-positive cervical cancer tissues than in methylation-negative normal cervical tissues. Our findings indicated that methylation in the promoter regions of WT1, NKX6-1 and DBC1 is correlated with cervical cancer tumorigenesis in Uygur women. The infection of HPV16/18 might be correlated with methylation in these genes. Gene inactivation caused by methylation might be related to the incidence and development of cervical

Neurospora crassa glucose - repressible gene -1(Grg-1) promoter controls the expression of neurospora tyrosinase gene in a clock-controlled manner

International Nuclear Information System (INIS)

Tarawneh, A. K

1997-01-01

In this study sphareroplastes of white Neurospora crassa mutant auxotroph for aromatic am no acids a rom 9 q a-2 inv, was transformed by the pKF-Tyr7-wt DNA construct. This construct contains the promoter of neurospora crassa glucose-repressible gene-1 (G rg-1) usp stream of Neurospora tyrosinase gene. The co transformation of this mutant with pKF-Tyr-7-wt cincture's and the pKAL-1, a plasmid which contains the Neurospora q a-2+ gene transform it to photophor. The transform ant contains the tyrosinase gene which catalyzes the unique step in the synthesis of the black pigment melanin. The activity of the tyrosinase in this transform ant was followed by measuring the absorbance of the dark coloured pigment at 332 nm. The maximum of the tyrosinase activity was shown at 16.36 and 56 hours after the shift of the transformed mycelia from constant light (L L) to constant dark (Dd). The rate of the enzyme activity was changed according to ci radian cycle of 20 hours. This G rg 1/tyrosinase construct provides a good system to study to study the temporal control of gene expression and the interaction between the different environmental c uses that affects gene expression. (author). 20 refs., 4 figs

Heme Oxygenase-1 Induction by Carbon Monoxide Releasing Molecule-3 Suppresses Interleukin-1β-Mediated Neuroinflammation

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Chih-Chung Lin

2017-11-01

Full Text Available Neurodegenerative disorders and brain damage are initiated by excessive production of reactive oxygen species (ROS, which leads to tissue injury, cellular death and inflammation. In cellular anti-oxidant systems, heme oxygenase-1 (HO-1 is an oxidative-sensor protein induced by ROS generation or carbon monoxide (CO release. CO releasing molecules (CORMs, including CORM-3, exert anti-oxidant and anti-inflammatory effects. However, the molecular mechanisms of CORM-3-induced HO-1 expression and protection against interleukin (IL-1β-induced inflammatory responses have not been fully elucidated in rat brain astrocytes (RBA-1. To study the regulation of CORM-3-induced HO-1 expression, signaling pathways, promoter activity, mRNA and protein expression were assessed following treatment with pharmacological inhibitors and gene-specific siRNA knockdown. We found that CORM-3 mediated HO-1 induction via transcritional and translational processes. Furthermore, CORM-3-induced HO-1 expression was mediated by phosphorylation of several protein kinases, such as c-Src, Pyk2, protein kinase Cα (PKCα and p42/p44 mitogen-activated protein kinase (MAPK, which were inhibited by respective pharmacological inhibitors or by gene-specific knockdown with siRNA transfections. Next, we found that CORM-3 sequentially activated the c-Src/Pyk2/PKCα/p42/p44 MAPK pathway, thereby up-regulating mRNA for the activator protein (AP-1 components c-Jun and c-Fos; these effects were attenuated by an AP-1 inhibitor (Tanshinone IIA; TSIIA and other relevant inhibitors. Moreover, CORM-3-induced upregulation of HO-1 attenuated the IL-1β-induced cell migration and matrix metallopeptidase-9 mRNA expression in RBA-1 cells. These effects were reversed by an matrix metalloproteinase (MMP2/9 inhibitor or by transfection with HO-1 siRNA.

The human luteinizing hormone receptor gene promoter: activation by Sp1 and Sp3 and inhibitory regulation.

Science.gov (United States)

Geng, Y; Tsai-Morris, C H; Zhang, Y; Dufau, M L

1999-09-24

To understand the transcriptional mechanism(s) of human LH receptor (LHR) gene expression, we have identified the dominant functional cis-elements that regulate the activity of the promoter domain (-1 to -176 bp from ATG). Mutagenesis demonstrated that the promoter activity was dependent on two Sp1 domains (-79 bp, -120 bp) in a transformed normal placental cell (PLC) and the choriocarcinoma JAR cell. Both elements interacted with endogenous Sp1 and Sp3 factors but not with Sp2 or Sp4. In Drosophila SL2 cells, the promoter was activated by either Sp1 or Sp3. An ERE half-site (EREhs) at -174 bp was inhibitory (by 100%), but was unresponsive to estradiol and did not bind the estrogen receptor or orphan receptors ERR1 and SF-1. The 5' upstream sequence (-177 to -2056 bp) inhibited promoter activity in PLC by 60%, but only minimally in JAR cells. Activation of the human LHR promoter through Sp1/3 factors is negatively regulated through EREhs and upstream sequences to exert control of gene expression. Copyright 1999 Academic Press.

Anyalysis of Msx1 and Msx2 Transactivation Function in the Context of the Heat Shock 70 (Hspa1b) Gene Promoter

OpenAIRE

Zhuang, Fengfeng; Nguyen, Manuel P.; Shuler, Charles; Liu, Yi-Hsin

2009-01-01

Previous studies have shown that Msx proteins control gene transcription predominantly through repression mechanisms. However, gene expression studies using either the gain-of-function or the loss-of-function mutants revealed many gene targets whose expression require functional Msx proteins. To date, investigations into the mechanisms of Msx-dependent trans-activation have been hindered by the lack of a responsive promoter. Here, we demonstrated the usefulness of the mouse Hspa1b promoter in...

AMPK activation represses the human gene promoter of the cardiac isoform of acetyl-CoA carboxylase: Role of nuclear respiratory factor-1

Energy Technology Data Exchange (ETDEWEB)

Adam, Tasneem; Opie, Lionel H. [Hatter Cardiovascular Research Institute, Faculty of Health Sciences, University of Cape Town, Observatory 7925 (South Africa); Essop, M. Faadiel, E-mail: mfessop@sun.ac.za [Cardio-Metabolic Research Group (CMRG), Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600 (South Africa)

2010-07-30

Research highlights: {yields} AMPK inhibits acetyl-CoA carboxylase beta gene promoter activity. {yields} Nuclear respiratory factor-1 inhibits acetyl-CoA carboxylase beta promoter activity. {yields} AMPK regulates acetyl-CoA carboxylase beta at transcriptional level. -- Abstract: The cardiac-enriched isoform of acetyl-CoA carboxylase (ACC{beta}) produces malonyl-CoA, a potent inhibitor of carnitine palmitoyltransferase-1. AMPK inhibits ACC{beta} activity, lowering malonyl-CoA levels and promoting mitochondrial fatty acid {beta}-oxidation. Previously, AMPK increased promoter binding of nuclear respiratory factor-1 (NRF-1), a pivotal transcriptional modulator controlling gene expression of mitochondrial proteins. We therefore hypothesized that NRF-1 inhibits myocardial ACC{beta} promoter activity via AMPK activation. A human ACC{beta} promoter-luciferase construct was transiently transfected into neonatal cardiomyocytes {+-} a NRF-1 expression construct. NRF-1 overexpression decreased ACC{beta} gene promoter activity by 71 {+-} 4.6% (p < 0.001 vs. control). Transfections with 5'-end serial promoter deletions revealed that NRF-1-mediated repression of ACC{beta} was abolished with a pPII{beta}-18/+65-Luc deletion construct. AMPK activation dose-dependently reduced ACC{beta} promoter activity, while NRF-1 addition did not further decrease it. We also investigated NRF-1 inhibition in the presence of upstream stimulatory factor 1 (USF1), a known transactivator of the human ACC{beta} gene promoter. Here NRF-1 blunted USF1-dependent induction of ACC{beta} promoter activity by 58 {+-} 7.5% (p < 0.001 vs. control), reversed with a dominant negative NRF-1 construct. NRF-1 also suppressed endogenous USF1 transcriptional activity by 55 {+-} 6.2% (p < 0.001 vs. control). This study demonstrates that NRF-1 is a novel transcriptional inhibitor of the human ACC{beta} gene promoter in the mammalian heart. Our data extends AMPK regulation of ACC{beta} to the transcriptional level.

Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

Energy Technology Data Exchange (ETDEWEB)

Palsamy, Periyasamy [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Ayaki, Masahiko [Shizuoka National Hospital, Saitama (Japan); Elanchezhian, Rajan [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States); Shinohara, Toshimichi, E-mail: tshinohara@unmc.edu [Department of Ophthalmology and Visual Sciences, University of Nebraska Medical Center, Omaha, NE (United States)

2012-07-06

Highlights: Black-Right-Pointing-Pointer We found significant Keap1 promoter demethylation in diabetic cataractous lenses. Black-Right-Pointing-Pointer Demethylation of Keap1 gene upregulated the expression of Keap1 mRNA and protein. Black-Right-Pointing-Pointer Elevated levels of Keap1 are known to decrease the levels of Nrf2. Black-Right-Pointing-Pointer Thereby, the levels of antioxidant enzymes are suppressed by decreased Nrf2 level. -- Abstract: Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2 Prime deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which

Promoter demethylation of Keap1 gene in human diabetic cataractous lenses

International Nuclear Information System (INIS)

Palsamy, Periyasamy; Ayaki, Masahiko; Elanchezhian, Rajan; Shinohara, Toshimichi

2012-01-01

Highlights: ► We found significant Keap1 promoter demethylation in diabetic cataractous lenses. ► Demethylation of Keap1 gene upregulated the expression of Keap1 mRNA and protein. ► Elevated levels of Keap1 are known to decrease the levels of Nrf2. ► Thereby, the levels of antioxidant enzymes are suppressed by decreased Nrf2 level. -- Abstract: Age-related cataracts (ARCs) are the major cause of visual impairments worldwide, and diabetic adults tend to have an earlier onset of ARCs. Although age is the strongest risk factor for cataracts, little is known how age plays a role in the development of ARCs. It is known that oxidative stress in the lens increases with age and more so in the lenses of diabetics. One of the central adaptive responses against the oxidative stresses is the activation of the nuclear transcriptional factor, NF-E2-related factor 2 (Nrf2), which then activates more than 20 different antioxidative enzymes. Kelch-like ECH associated protein 1 (Keap1) targets and binds to Nrf2 for proteosomal degradation. We hypothesized that hyperglycemia will lead to a dysfunction of the Nrf2-dependent antioxidative protection in the lens of diabetics. We studied the methylation status of the CpG islands in 15 clear and 21 diabetic cataractous lenses. Our results showed significant levels of demethylated DNA in the Keap1 promoter in the cataractous lenses from diabetic patients. In contrast, highly methylated DNA was found in the clear lens and tumorized human lens epithelial cell (HLEC) lines (SRA01/04). HLECs treated with a demethylation agent, 5-aza-2′deoxycytidine (5-Aza), had a 10-fold higher levels of Keap1 mRNA, 3-fold increased levels of Keap1 protein, produced higher levels of ROS, and increased cell death. Our results indicated that demethylation of the CpG islands in the Keap1 promoter will activate the expression of Keap1 protein, which then increases the targeting of Nrf2 for proteosomal degradation. Decreased Nrf2 activity represses the

Inactivation of promoter 1B of APC causes partial gene silencing: evidence for a significant role of the promoter in regulation and causative of familial adenomatous polyposis

DEFF Research Database (Denmark)

Rohlin, A; Engwall, Y; Fritzell, K

2011-01-01

inactivation of promoter 1B is disease causing in FAP; (ii) expression of transcripts from promoter 1B is generated at considerable higher levels compared with 1A, demonstrating a hitherto unknown importance of 1B; (iii) adenoma formation in FAP, caused by impaired function of promoter 1B, does not require......Familial adenomatous polyposis (FAP) is caused by germline mutations in the adenomatous polyposis coli (APC) gene. Two promoters, 1A and 1B, have been recognized in APC, and 1B is thought to have a minor role in the regulation of the gene. We have identified a novel deletion encompassing half...... of this promoter in the largest family (Family 1) of the Swedish Polyposis Registry. The mutation leads to an imbalance in allele-specific expression of APC, and transcription from promoter 1B was highly impaired in both normal colorectal mucosa and blood from mutation carriers. To establish the significance...

Tissue specific promoters improve the localization of radiation-inducible gene expression

International Nuclear Information System (INIS)

Hallahan, Dennis; Kataoka, Yasushi; Kuchibhotla, Jaya; Virudachalam, Subbu; Weichselbaum, Ralph

1996-01-01

Purpose: Site-specific activation of gene expression can be achieved by the use of a promoter that is induced by physical agents such as x-rays. The purpose of the present study was to determine whether site-specific activation of gene therapy can also be achieved within the vascular endothelium by use of radiation-inducible promoters. We studied induction of promoter-reporter gene constructs using previously identified radiation-promoters from c-jun, c-fos, Egr-1, ICAM-1, ELAM-1 after transfection into in the vascular endothelium. Methods: The following radiation-inducible genetic constructs were created: The ELAM-1 promoter fragment was cloned into pOGH to obtain the pE-sel(-587 +35)GH reporter construct. The ICAM-1 promoter fragment (-1162/+1) was cloned upstream of the CAT coding region of the pCAT-plasmid (Promega) after removal of the SV40 promoter by Bgl2/Stu1 digestion to create the pBS-CAT plasmid. The 132 to +170 bp segment of the 5' untranslated region of the c-jun promoter was cloned to the CAT reporter gene to create the -132/+170 cjun-CAT. The Egr-1 promoter fragment (-425/+75) was cloned upstream of the CAT coding region to create the pE425-CAT plasmid. Tandem repeats of the AP-1 binding site were cloned upstream of the CAT coding region (3 xTRE-CAT). Tandem repeats of the Egr binding site (EBS) were cloned upstream of the CAT coding region (EBS-CAT). Human vascular endothelial cells from both large vessel and small vessel origin (HUVEC and HMEC), as well as human tumor cell lines were transfected with plasmids -132/+170 cjun-CAT, pE425-CAT, 3 xTRE-CAT, EBS-CAT, pE-sel-GH and pBS-CAT by use of liposomes. Humor tumor cell lines included SQ20B (squamous), RIT3 (sarcoma), and HL525 (leukemia). Each plasmid was cotransfected with a plasmid containing a CMV promoter linked to the LacZ gene (1 μg). Transfected cells were treated with mock irradiation or x-rays. Cell extracts were assayed for reporter gene expression. Results: Radiation-induced gene

Heme oxygenase-1 delays gibberellin-induced programmed cell death of rice aleurone layers subjected to drought stress by interacting with nitric oxide

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Huangming eWu

2016-01-01

Full Text Available Cereal aleurone layers undergo a gibberellin (GA-regulated process of programmed cell death (PCD following germination. Heme oxygenase-1 (HO-1 is known as a rate-liming enzyme in the degradation of heme to biliverdin IXα (BV, carbon monoxide (CO, and free iron ions (Fe2+. It is a critical component in plant development and adaptation to environment stresses. Our previous studies confirmed that HO-1 inducer hematin (Ht promotes the germination of rice seeds in drought (20% polyethylene glycol-6000, PEG conditions, but the corresponding effects of HO-1 on the alleviation of germination-triggered PCD in GA-treated rice aleurone layers remain unknown. The present study has determined that GA co-treated with PEG results in lower HO-1 transcript levels and HO activity, which in turn results in the development of vacuoles in aleurone cells, followed by PCD. The pharmacology approach illustrated that up- or down-regulated HO-1 gene expression and HO activity delayed or accelerated GA-induced PCD. Furthermore, the application of the HO-1 inducer hematin and nitric oxide (NO donor sodium nitroprusside (SNP not only activated HO-1 gene expression, HO activity, and endogenous NO content, but also blocked GA-induced rapid vacuolation and accelerated aleurone layers PCD under drought stress. However, both HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX and NO scavenger 2-(4-carboxyphenyl0-4, 4, 5, 5-tetramethylimidazoline-l-oxyl-3-oxide potassium salt (cPTIO reserved the effects of hematin and SNP on rice aleurone layer PCD under drought stress by down-regulating endogenous HO-1 and NO, respectively. The inducible effects of hematin and SNP on HO-1 gene expression, HO activity, and NO content were blocked by cPTIO. Together, these results clearly suggest that HO-1 is involved in the alleviation of GA-induced PCD of drought-triggered rice aleurone layers by associating with NO.

Spectroscopy and microchip laser operation of Tm, Ho:KYW crystals with different Ho concentrations

Science.gov (United States)

Gusakova, N. V.; Kurilchik, S. V.; Yasukevich, A. S.; Kisel, V. E.; Dashkevich, V. I.; Orlovich, V. A.; Pavlyuk, A. A.; Vatnik, S. M.; Bagaev, S. N.; Kuleshov, N. V.

2018-02-01

The spectroscopic properties of Tm, Ho:KYW crystals with different Ho concentrations were investigated. The diode-pumped microchip laser operation of Tm (5 at.%), Ho (0.5 at.%):KYW and Tm (5 at.%), Ho (1 at.%):KYW was demonstrated. The highest, to our knowledge, output power of 480 mW with slope efficiency of 31% for CW Tm (5 at.%), Ho (0.5 at.%):KYW microchip laser was obtained.

Embryotoxicity Caused by DON-Induced Oxidative Stress Mediated by Nrf2/HO-1 Pathway

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Miao Yu

2017-06-01

Full Text Available Deoxynivalenol (DON belongs to the type B group of trichothecenes family, which is composed of sesquiterpenoid metabolites produced by Fusarium and other fungi in grain. DON may cause various toxicities, such as cytotoxicity, immunotoxicity, genotoxicity as well as teratogenicity and carcinogenicity. In the present study, we focus on a hypothesis that DON alters the expressions of Nrf2/HO-1 pathway by inducing embryotoxicity in C57BL/6 mouse (5.0, 2.5, 1.0, and 0 mg/kg/day and BeWo cell lines (0 and 50 nM; 3 h, 12 h and 24 h. Our results indicate that DON treatment in mice during pregnancy leads to ROS accumulation in the placenta, which results in embryotoxicity. At the same time Nrf2/HO-1 pathway is up-regulated by ROS to protect placenta cells from oxidative damage. In DON-treated BeWo cells, the level of ROS has time–effect and dose–effect relationships with HO-1 expression. Moderate increase in HO-1 protects the cell from oxidative damage, while excessive increase in HO-1 aggravates the oxidative damage, which is called in some studies the “threshold effect”. Therefore, oxidative stress may be the critical molecular mechanism for DON-induced embryotoxicity. Besides, Nrf2/HO-1 pathway accompanied by the “threshold effect” also plays an important role against DON-induced oxidative damage in this process.

Arctigenin Increases Hemeoxygenase-1 Gene Expression by Modulating PI3K/AKT Signaling Pathway in Rat Primary Astrocytes.

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Jeong, Yeon-Hui; Park, Jin-Sun; Kim, Dong-Hyun; Kim, Hee-Sun

2014-11-01

In the present study, we found that the natural compound arctigenin inhibited hydrogen peroxide-induced reactive oxygen species (ROS) production in rat primary astrocytes. Since hemeoxygenase-1 (HO-1) plays a critical role as an antioxidant defense factor in the brain, we examined the effect of arctigenin on HO-1 expression in rat primary astrocytes. We found that arctigenin increased HO-1 mRNA and protein levels. Arctigenin also increases the nuclear translocation and DNA binding of Nrf2/c-Jun to the antioxidant response element (ARE) on HO-1 promoter. In addition, arctigenin increased ARE-mediated transcriptional activities in rat primary astrocytes. Further mechanistic studies revealed that arctigenin increased the phosphorylation of AKT, a downstream substrate of phosphatidylinositol 3-kinase (PI3K). Treatment of cells with a PI3K-specific inhibitor, LY294002, suppressed the HO-1 expression, Nrf2 DNA binding and ARE-mediated transcriptional activities in arctigenin-treated astrocyte cells. The results collectively suggest that PI3K/AKT signaling pathway is at least partly involved in HO-1 expression by arctigenin via modulation of Nrf2/ARE axis in rat primary astrocytes.

Loss of the NKX3.1 tumorsuppressor promotes the TMPRSS2-ERG fusion gene expression in prostate cancer

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Thangapazham, Rajesh; Saenz, Francisco; Katta, Shilpa; Mohamed, Ahmed A; Tan, Shyh-Han; Petrovics, Gyorgy; Srivastava, Shiv; Dobi, Albert

2014-01-01

In normal prostate epithelium the TMPRSS2 gene encoding a type II serine protease is directly regulated by male hormones through the androgen receptor. In prostate cancer ERG protooncogene frequently gains hormonal control by seizing gene regulatory elements of TMPRSS2 through genomic fusion events. Although, the androgenic activation of TMPRSS2 gene has been established, little is known about other elements that may interact with TMPRSS2 promoter sequences to modulate ERG expression in TMPRSS2-ERG gene fusion context. Comparative genomic analyses of the TMPRSS2 promoter upstream sequences and pathway analyses were performed by the Genomatix Software. NKX3.1 and ERG genes expressions were evaluated by immunoblot or by quantitative Real-Time PCR (qRT-PCR) assays in response to siRNA knockdown or heterologous expression. QRT-PCR assay was used for monitoring the gene expression levels of NKX3.1-regulated genes. Transcriptional regulatory function of NKX3.1 was assessed by luciferase assay. Recruitment of NKX3.1 to its cognate elements was monitored by Chromatin Immunoprecipitation assay. Comparative analysis of the TMPRSS2 promoter upstream sequences among different species revealed the conservation of binding sites for the androgen inducible NKX3.1 tumor suppressor. Defects of NKX3.1, such as, allelic loss, haploinsufficiency, attenuated expression or decreased protein stability represent established pathways in prostate tumorigenesis. We found that NKX3.1 directly binds to TMPRSS2 upstream sequences and negatively regulates the expression of the ERG protooncogene through the TMPRSS2-ERG gene fusion. These observations imply that the frequently noted loss-of-function of NKX3.1 cooperates with the activation of TMPRSS2-ERG fusions in prostate tumorigenesis

LuFLA1PRO and LuBGAL1PRO promote gene expression in the phloem fibres of flax (Linum usitatissimum).

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Hobson, Neil; Deyholos, Michael K

2013-04-01

Cell type-specific promoters were identified that drive gene expression in an industrially important product. To identify flax (Linum usitatissimum) gene promoters, we analyzed the genomic regions upstream of a fasciclin-like arabinogalactan protein (LuFLA1) and a beta-galactosidase (LuBGAL1). Both of these genes encode transcripts that have been found to be highly enriched in tissues bearing phloem fibres. Using a beta-glucuronidase (GUS) reporter construct, we found that a 908-bp genomic sequence upstream of LuFLA1 (LuFLA1PRO) directed GUS expression with high specificity to phloem fibres undergoing secondary cell wall development. The DNA sequence upstream of LuBGAL1 (LuBGAL1PRO) likewise produced GUS staining in phloem fibres with developing secondary walls, as well as in tissues of developing flowers and seed bolls. These data provide further evidence of a specific role for LuFLA1 in phloem fibre development, and demonstrate the utility of LuFLA1PRO and LuBGAL1PRO as tools for biotechnology and further investigations of phloem fibre development.

Structural and functional analysis of mouse Msx1 gene promoter: sequence conservation with human MSX1 promoter points at potential regulatory elements.

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Gonzalez, S M; Ferland, L H; Robert, B; Abdelhay, E

1998-06-01

Vertebrate Msx genes are related to one of the most divergent homeobox genes of Drosophila, the muscle segment homeobox (msh) gene, and are expressed in a well-defined pattern at sites of tissue interactions. This pattern of expression is conserved in vertebrates as diverse as quail, zebrafish, and mouse in a range of sites including neural crest, appendages, and craniofacial structures. In the present work, we performed structural and functional analyses in order to identify potential cis-acting elements that may be regulating Msx1 gene expression. To this end, a 4.9-kb segment of the 5'-flanking region was sequenced and analyzed for transcription-factor binding sites. Four regions showing a high concentration of these sites were identified. Transfection assays with fragments of regulatory sequences driving the expression of the bacterial lacZ reporter gene showed that a region of 4 kb upstream of the transcription start site contains positive and negative elements responsible for controlling gene expression. Interestingly, a fragment of 130 bp seems to contain the minimal elements necessary for gene expression, as its removal completely abolishes gene expression in cultured cells. These results are reinforced by comparison of this region with the human Msx1 gene promoter, which shows extensive conservation, including many consensus binding sites, suggesting a regulatory role for them.

Anti-Inflammatory Effect of Recreational Exercise in TNBS-Induced Colitis in Rats: Role of NOS/HO/MPO System

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Zita Szalai

2014-01-01

Full Text Available There are opposite views in the available literature: Whether physical exercise has a protective effect or not on the onset of inflammatory bowel disease (IBD. Therefore, we investigated the effects of recreational physical exercise before the induction of colitis. After 6 weeks of voluntary physical activity (running wheel, male Wistar rats were treated with TNBS (10 mg. 72 hrs after trinitrobenzene sulphonic acid (TNBS challenge we measured colonic gene (TNF-α, IL-1β, CXCL1 and IL-10 and protein (TNF-α expressions of various inflammatory mediators and enzyme activities of heme oxygenase (HO, nitric oxide synthase (NOS, and myeloperoxidase (MPO enzymes. Wheel running significantly increased the activities of HO, constitutive NOS (cNOS isoform. Furthermore, 6 weeks of running significantly decreased TNBS-induced inflammatory markers, including extent of lesions, severity of mucosal damage, and gene expression of IL-1β, CXCL1, and MPO activity, while IL-10 gene expression and cNOS activity were increased. iNOS activity decreased and the activity of HO enzyme increased, but not significantly, compared to the sedentary TNBS-treated group. In conclusion, recreational physical exercise can play an anti-inflammatory role by downregulating the gene expression of proinflammatory mediators, inducing anti-inflammatory mediators, and modulating the activities of HO and NOS enzymes in a rat model of colitis.

A preliminary study of the relationship between promoter methylation of the ABCG1, GALNT2 and HMGCR genes and coronary heart disease.

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Ping Peng

Full Text Available To investigate the association of ABCG1, GALNT2 and HMGCR genes promoter DNA methylation with coronary heart disease (CHD and explore the interaction between their methylation status and the CHD patients' clinical characteristics in Han Chinese population.Methylation-specific polymerase chain reaction (MSP technology was used to examine the role of the aberrant gene promoter methylation in CHD in Han Chinese population. A total of 85 CHD patients and 54 participants without CHD confirmed by angiography were recruited. 82.8% of the participants with ABCG1 gene promoter hypermethylation have CHD, while only 17.4% of the participants without hypermethylation have it. The average age of the participants with GALNT2 gene promoter hypermethylation is 62.10 ± 8.21, while that of the participants without hypermethylation is 57.28 ± 9.87; in the former group, 75.4% of the participants have CHD, compared to only 50% in the latter group. As for the HMGCR gene, the average age of the participants with promoter hypermethylation is 63.24 ± 8.10 and that of the participants without hypermethylation is 57.79 ± 9.55; its promoter hypermethylation is likely to be related to smoking. Our results indicated a significant statistical association of promoter methylation of the ABCG1 gene with increased risk of CHD (OR = 19.966; 95% CI, 7.319-54.468; P*<0.001; P*: adjusted for age, gender, smoking, lipid level, hypertension, and diabetes. Similar results were obtained for that of the GALNT2 gene (OR = 2.978; 95% CI, 1.335-6.646; P* = 0.008, but not of HMGCR gene (OR = 1.388; 95% CI, 0.572-3.371; P*  = 0.469.The present work provides evidence to support the association of promoter DNA methylation status with the risk profile of CHD. Our data indicates that promoter DNA hypermethylation of the ABCG1 and GALNT2 genes, but not the HMGCR gene, is associated with an increased risk of CHD. CHD, smoking and aging are likely to be the important factors influencing DNA

Transcriptional coactivator NT-PGC-1α promotes gluconeogenic gene expression and enhances hepatic gluconeogenesis.

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Chang, Ji Suk; Jun, Hee-Jin; Park, Minsung

2016-10-01

The transcriptional coactivator PGC-1α plays a central role in hepatic gluconeogenesis. We previously reported that alternative splicing of the PGC-1α gene produces an additional transcript encoding the truncated protein NT-PGC-1α NT-PGC-1α is co-expressed with PGC-1α and highly induced by fasting in the liver. NT-PGC-1α regulates tissue-specific metabolism, but its role in the liver has not been investigated. Thus, the objective of this study was to determine the role of hepatic NT-PGC-1α in the regulation of gluconeogenesis. Adenovirus-mediated expression of NT-PGC-1α in primary hepatocytes strongly stimulated the expression of key gluconeogenic enzyme genes (PEPCK and G6Pase), leading to increased glucose production. To further understand NT-PGC-1α function in hepatic gluconeogenesis in vivo, we took advantage of a previously reported FL-PGC-1α -/- mouse line that lacks full-length PGC-1α (FL-PGC-1α) but retains a slightly shorter and functionally equivalent form of NT-PGC-1α (NT-PGC-1α 254 ). In FL-PGC-1α -/- mice, NT-PGC-1α 254 was induced by fasting in the liver and recruited to the promoters of PEPCK and G6Pase genes. The enrichment of NT-PGC-1α 254 at the promoters was closely associated with fasting-induced increase in PEPCK and G6Pase gene expression and efficient production of glucose from pyruvate during a pyruvate tolerance test in FL-PGC-1α -/- mice. Moreover, FL-PGC-1α -/- primary hepatocytes showed a significant increase in gluconeogenic gene expression and glucose production after treatment with dexamethasone and forskolin, suggesting that NT-PGC-1α 254 is sufficient to stimulate the gluconeogenic program in the absence of FL-PGC-1α Collectively, our findings highlight the role of hepatic NT-PGC-1α in stimulating gluconeogenic gene expression and glucose production. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

Promoter sequence of 3-phosphoglycerate kinase gene 1 of lactic acid-producing fungus rhizopus oryzae and a method of expressing a gene of interest in fungal species

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Gao, Johnway [Richland, WA; Skeen, Rodney S [Pendleton, OR

2002-10-15

The present invention provides the promoter clone discovery of phosphoglycerate kinase gene 1 of a lactic acid-producing filamentous fungal strain, Rhizopus oryzae. The isolated promoter can constitutively regulate gene expression under various carbohydrate conditions. In addition, the present invention also provides a design of an integration vector for the transformation of a foreign gene in Rhizopus oryzae.

Polymorphisms in the Haem Oxygenase-1 promoter are not associated with severity of Plasmodium falciparum malaria in Ghanaian children.

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Hansson, Helle H; Maretty, Lasse; Balle, Christina; Goka, Bamenla Q; Luzon, Elisa; Nkrumah, Francis N; Schousboe, Mette L; Rodrigues, Onike P; Bygbjerg, Ib Christian; Kurtzhals, Jørgen A L; Alifrangis, Michael; Hempel, Casper

2015-04-11

Haem oxygenase-1 (HO-1) catabolizes haem and has both cytotoxic and cytoprotective effects. Polymorphisms in the promoter of the Haem oxygenase-1 (HMOX1) gene encoding HO-1 have been associated with several diseases including severe malaria. The objective of this study was to determine the allele and genotype frequencies of two single nucleotide polymorphisms; A(-413)T and G(-1135)A, and a (GT)n repeat length polymorphism in the HMOX1 promoter in paediatric malaria patients and controls to determine possible associations with malaria disease severity. Study participants were Ghanaian children (n=296) admitted to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Accra, Ghana during the malaria season from June to August in 1995, 1996 and 1997, classified as having uncomplicated malaria (n=101) or severe malaria (n=195; defined as severe anaemia (n=63) or cerebral malaria (n=132)). Furthermore, 287 individuals without a detectable Plasmodium infection or asymptomatic carriers of the parasite were enrolled as controls. Blood samples from participants were extracted for DNA and allele and genotype frequencies were determined with allele-specific PCR, restriction fragment length analysis and microsatellite analysis. The number of (GT)n repeats in the study participants varied between 21 and 46 with the majority of alleles having lengths of 26 (8.1%), 29/30 (13.2/17.9%) and 39/40 (8.0/13.8%) repeats, and was categorized into short, medium and long repeats. The (-413)T allele was very common (69.8%), while the (-1135)A allele was present in only 17.4% of the Ghanaian population. The G(-1135)A locus was excluded from further analysis after failing the Hardy-Weinberg equilibrium test. No significant differences in allele or genotype distribution of the A(-413)T and (GT)n repeat polymorphisms were found between the controls and the malaria patients, or between the disease groups, for any of the analysed polymorphisms and no associations with

Viral promoters can initiate expression of toxin genes introduced into Escherichia coli

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Jacob Daniela

2005-06-01

Full Text Available Abstract Background The expression of recombinant proteins in eukaryotic cells requires the fusion of the coding region to a promoter functional in the eukaryotic cell line. Viral promoters are very often used for this purpose. The preceding cloning procedures are usually performed in Escherichia coli and it is therefore of interest if the foreign promoter results in an expression of the gene in bacteria. In the case molecules toxic for humans are to be expressed, this knowledge is indispensable for the specification of safety measures. Results We selected five frequently used viral promoters and quantified their activity in E. coli with a reporter system. Only the promoter from the thymidine kinase gene from HSV1 showed no activity, while the polyhedrin promoter from baculovirus, the early immediate CMV promoter, the early SV40 promoter and the 5' LTR promoter from HIV-1 directed gene expression in E. coli. The determination of transcription start sites in the immediate early CMV promoter and the polyhedrin promoter confirmed the existence of bacterial -10 and -35 consensus sequences. The importance of this heterologous gene expression for safety considerations was further supported by analysing fusions between the aforementioned promoters and a promoter-less cytotoxin gene. Conclusion According to our results a high percentage of viral promoters have the ability of initiating gene expression in E. coli. The degree of such heterologous gene expression can be sufficient for the expression of toxin genes and must therefore be considered when defining safety measures for the handling of corresponding genetically modified organisms.

Murine homeobox-containing gene, Msx-1: analysis of genomic organization, promoter structure, and potential autoregulatory cis-acting elements.

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Kuzuoka, M; Takahashi, T; Guron, C; Raghow, R

1994-05-01

Detailed molecular organization of the coding and upstream regulatory regions of the murine homeodomain-containing gene, Msx-1, is reported. The protein-encoding portion of the gene is contained in two exons, 590 and 1214 bp in length, separated by a 2107-bp intron; the homeodomain is located in the second exon. The two-exon organization of the murine Msx-1 gene resembles a number of other homeodomain-containing genes. The 5'-(GTAAGT) and 3'-(CCCTAG) splicing junctions and the mRNA polyadenylation signal (UAUAA) of the murine Msx-1 gene are also characteristic of other vertebrate genes. By nuclease protection and primer extension assays, the start of transcription of the Msx-1 gene was located 256 bp upstream of the first AUG. Computer analysis of the promoter proximal 1280-bp sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements for Ap-1, Ap-2, Ap-3, Sp-1, a possible binding site for RAR:RXR, and a number of TCF-1 consensus motifs. Importantly, a perfect reverse complement of (C/G)TTAATTG, which was recently shown to be an optimal binding sequence for the homeodomain of Msx-1 protein (K.M. Catron, N. Iler, and C. Abate (1993) Mol. Cell. Biol. 13:2354-2365), was also located in the murine Msx-1 promoter. Binding of bacterially expressed Msx-1 homeodomain polypeptide to Msx-1-specific oligonucleotide was experimentally demonstrated, raising a distinct possibility of autoregulation of this developmentally regulated gene.

ERalpha and AP-1 interact in vivo with a specific sequence of the F promoter of the human ERalpha gene in osteoblasts.

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Lambertini, Elisabetta; Tavanti, Elisa; Torreggiani, Elena; Penolazzi, Letizia; Gambari, Roberto; Piva, Roberta

2008-07-01

Estrogen-responsive genes often have an estrogen response element (ERE) positioned next to activator protein-1 (AP-1) binding sites. Considering that the interaction between ERE and AP-1 elements has been described for the modulation of bone-specific genes, we investigated the 17-beta-estradiol responsiveness and the role of these cis-elements present in the F promoter of the human estrogen receptor alpha (ERalpha) gene. The F promoter, containing the sequence analyzed here, is one of the multiple promoters of the human ERalpha gene and is the only active promoter in bone tissue. Through electrophoretic mobility shift (EMSA), chromatin immunoprecipitation (ChIP), and re-ChIP assays, we investigated the binding of ERalpha and four members of the AP-1 family (c-Jun, c-fos, Fra-2, and ATF2) to a region located approximately 800 bp upstream of the transcriptional start site of exon F of the human ERalpha gene in SaOS-2 osteoblast-like cells. Reporter gene assay experiments in combination with DNA binding assays demonstrated that F promoter activity is under the control of upstream cis-acting elements which are recognized by specific combinations of ERalpha, c-Jun, c-fos, and ATF2 homo- and heterodimers. Moreover, ChIP and re-ChIP experiments showed that these nuclear factors bind the F promoter in vivo with a simultaneous occupancy stimulated by 17-beta-estradiol. Taken together, our findings support a model in which ERalpha/AP-1 complexes modulate F promoter activity under conditions of 17-beta-estradiol stimulation. (c) 2008 Wiley-Liss, Inc.

Hepatic expression of heme oxygenase-1 and antioxidant response element-mediated genes following administration of ethinyl estradiol to rats

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Morio, Lisa A.; Leone, Angelique; Sawant, Sharmilee P.; Nie, Alex Y.; Brandon Parker, J.; Taggart, Peter; Barron, Alfred M.; McMillian, Michael K.; Lord, Peter

2006-01-01

Heme oxygenase-1 (HO-1) is one of several enzymes induced by hepatotoxicants, and is thought to have an important protective role against cellular stress during liver inflammation and injury. The objective of the present study was to evaluate the role of HO-1 in estradiol-induced liver injury. A single dose of ethinyl estradiol (500 mg/kg, po) resulted in mild liver injury. Repeated administration of ethinyl estradiol (500 mg/kg/day for 4 days, po) resulted in no detectable liver injury or dysfunction. Using RT-PCR analysis, we demonstrate that HO-1 gene expression in whole liver tissue is elevated (> 20-fold) after the single dose of ethinyl estradiol. The number and intensity of HO-1 immunoreactive macrophages were increased after the single dose of ethinyl estradiol. HO-1 expression was undetectable in hepatic parenchymal cells from rats receiving Methocel control or a single dose of ethinyl estradiol, however cytosolic HO-1 immunoreactivity in these cells after repeated dosing of ethinyl estradiol was pronounced. The increases in HO-1 mRNA and HO-1 immunoreactivity following administration of a single dose of ethinyl estradiol suggested that this enzyme might be responsible for the observed protection of the liver during repeated dosing. To investigate the effect of HO-1 expression on ethinyl estradiol-induced hepatotoxicity, rats were pretreated with hemin (50 μmol/kg, ip, a substrate and inducer of HO-1), with tin protoporphyrin IX (60 μmol/kg, ip, an HO-1 inhibitor), or with gadolinium chloride (10 mg/kg, iv, an inhibitor/toxin of Kupffer cells) 24 h before ethinyl estradiol treatment. Pretreatment with modulators of HO-1 expression and activity had generally minimal effects on ethinyl estradiol-induced liver injury. These data suggest that HO-1 plays a limited role in antioxidant defense against ethinyl estradiol-induced oxidative stress and hepatotoxicity, and suggests that other coordinately induced enzymes are responsible for protection observed with

A Baculovirus immediate-early gene, ie1, promoter drives efficient expression of a transgene in both Drosophila melanogaster and Bombyx mori.

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Mika Masumoto

Full Text Available Many promoters have been used to drive expression of heterologous transgenes in insects. One major obstacle in the study of non-model insects is the dearth of useful promoters for analysis of gene function. Here, we investigated whether the promoter of the immediate-early gene, ie1, from the Bombyx mori nucleopolyhedrovirus (BmNPV could be used to drive efficient transgene expression in a wide variety of insects. We used a piggyBac-based vector with a 3xP3-DsRed transformation marker to generate a reporter construct; this construct was used to determine the expression patterns driven by the BmNPV ie1 promoter; we performed a detailed investigation of the promoter in transgene expression pattern in Drosophila melanogaster and in B. mori. Drosophila and Bombyx belong to different insect orders (Diptera and Lepidoptera, respectively; however, and to our surprise, ie1 promoter-driven expression was evident in several tissues (e.g., prothoracic gland, midgut, and tracheole in both insects. Furthermore, in both species, the ie1 promoter drove expression of the reporter gene from a relatively early embryonic stage, and strong ubiquitous ie1 promoter-driven expression continued throughout the larval, pupal, and adult stages by surface observation. Therefore, we suggest that the ie1 promoter can be used as an efficient expression driver in a diverse range of insect species.

LiHo(PO34

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Mokhtar Férid

2009-02-01

Full Text Available Lithium holmium(III polyphosphate(V, LiHo(PO34, belongs to the type I of polyphosphates with general formula ALn(PO34, where A is a monovalent cation and Ln is a trivalent rare earth cation. In the crystal structure, the polyphosphate chains spread along the b-axis direction, with a repeat period of four tetrahedra and 21 internal symmetry. The Li and Ho atoms are both located on twofold rotation axes and are surrounded by four and eight O atoms, leading to a distorted tetrahedral and dodecahedral coordination, respectively. The HoO8 polyhedra are isolated from each other, the closest Ho...Ho distance being 5.570 (1 Å.

Synergistic regulation of the mouse orphan nuclear receptor SHP gene promoter by CLOCK-BMAL1 and LRH-1

International Nuclear Information System (INIS)

Oiwa, Ako; Kakizawa, Tomoko; Miyamoto, Takahide; Yamashita, Koh; Jiang, Wei; Takeda, Teiji; Suzuki, Satoru; Hashizume, Kiyoshi

2007-01-01

Small heterodimer partner (SHP; NR0B2) is an orphan nuclear receptor and acts as a repressor for wide variety of nuclear hormone receptors. We demonstrated here that mouse SHP mRNA showed a circadian expression pattern in the liver. Transient transfection of the mSHP promoter demonstrated that CLOCK-BMAL1, core circadian clock components, bound to E-box (CACGTG), and stimulated the promoter activity by 4-fold. Liver receptor homologue-1 (LRH-1; NR5A2) stimulated the mSHP promoter, and CLOCK-BMAL1 synergistically enhanced the LRH-1-mediated transactivation. Interestingly, SHP did not affect the CLOCK-BMAL1-mediated promoter activity, but strongly repressed the synergistic activation of CLOCK-BMAL1 and LRH-1. Furthermore, in vitro pull-down assays revealed the existence of direct protein-protein interaction between LRH-1 and CLOCK. In summary, this study shows that CLOCK-BMAL1, LRH-1 and SHP coordinately regulate the mSHP gene to generate the circadian oscillation. The cyclic expression of mSHP may affect daily activity of other nuclear receptors and contribute to circadian liver functions

THE ABERRANT PROMOTER HYPERMETHYLATION PATTERN OF THE ANTI - ANGIOGENIC TSP1 GENE IN EPITHELIAL OVARIAN CARCINOMA: AN INDIAN STUDY

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Ramesh

2015-06-01

Full Text Available PURPOSE: The promoter hypermethylation patterns of Thrombospodin - 1 gene in 50 EOC patients were studied and the methylation pattern was correlated with various clinic pathological parameters. METHODS: The promoter hypermethylation pattern of the TSP - 1 gene was assessed using nested PCR and Methylation specific PCR. STATISTICAL ANALYSIS: All the available data was statistically analyzed using the Chi square test or Fisher Exact Test on the SPSS software version 22.0 and a value <0.0 5 was considered statistically significant. RESULTS: Forty of the fifty ovarian carcinoma samples reported positive for methylation corresponding to a methylation frequency of 80%. A methylation frequency of 89.2%, 83.3% and 42.8% was observed in malignant , Low malignant potential (borderline and benign sample cohorts. CONCLUSION: From the results drawn from this study, it clearly shows that the anti angiogenic protein TSP - 1 is extensively hypermethylated in ovarian carcinoma and that it accumulates over t he progression of the disease from benign to malignant. As previous reports suggest that there is no evidence of mutation of this gene, promoter hypermethylation may be a crucial factor for the down regulation of the gene. Further by clubbing together the promoter hypermethylation pattern of TSP - 1 gene with hypermethylation patterns of other TSG may provide a better insight into the application of using methylation profiles of TSG as a biomarker in the detection of ovarian carcinoma.

Spatially and Temporally Regulated NRF2 Gene Therapy Using Mcp-1 Promoter in Retinal Ganglion Cell Injury

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Kosuke Fujita

2017-06-01

Full Text Available Retinal ganglion cell degeneration triggered by axonal injury is believed to underlie many ocular diseases, including glaucoma and optic neuritis. In these diseases, retinal ganglion cells are affected unevenly, both spatially and temporally, such that healthy and unhealthy cells coexist in different patterns at different time points. Herein, we describe a temporally and spatially regulated adeno-associated virus gene therapy aiming to reduce undesired off-target effects on healthy retinal neurons. The Mcp-1 promoter previously shown to be activated in stressed retinal ganglion cells following murine optic nerve injury was combined with the neuroprotective intracellular transcription factor Nrf2. In this model, Mcp-1 promoter-driven NRF2 expression targeting only stressed retinal ganglion cells showed efficacy equivalent to non-selective cytomegalovirus promoter-driven therapy for preventing cell death. However, cytomegalovirus promoter-mediated NRF2 transcription induced cellular stress responses and death of Brn3A-positive uninjured retinal ganglion cells. Such undesired effects were reduced substantially by adopting the Mcp-1 promoter. Combining a stress-responsive promoter and intracellular therapeutic gene is a versatile approach for specifically targeting cells at risk of degeneration. This strategy may be applicable to numerous chronic ocular and non-ocular conditions.

Upland cotton gene GhFPF1 confers promotion of flowering time and shade-avoidance responses in Arabidopsis thaliana.

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Xiaoyan Wang

Full Text Available Extensive studies on floral transition in model species have revealed a network of regulatory interactions between proteins that transduce and integrate developmental and environmental signals to promote or inhibit the transition to flowering. Previous studies indicated FLOWERING PROMOTING FACTOR 1 (FPF1 gene was involved in the promotion of flowering, but the molecular mechanism was still unclear. Here, FPF1 homologous sequences were screened from diploid Gossypium raimondii L. (D-genome, n = 13 and Gossypium arboreum L. genome (A-genome, n = 13 databases. Orthologous genes from the two species were compared, suggesting that distinctions at nucleic acid and amino acid levels were not equivalent because of codon degeneracy. Six FPF1 homologous genes were identified from the cultivated allotetraploid Gossypium hirsutum L. (AD-genome, n = 26. Analysis of relative transcripts of the six genes in different tissues revealed that this gene family displayed strong tissue-specific expression. GhFPF1, encoding a 12.0-kDa protein (Accession No: KC832319 exerted more transcripts in floral apices of short-season cotton, hinting that it could be involved in floral regulation. Significantly activated APETALA 1 and suppressed FLOWERING LOCUS C expression were induced by over-expression of GhFPF1 in the Arabidopsis Columbia-0 ecotype. In addition, transgenic Arabidopsis displayed a constitutive shade-avoiding phenotype that is characterized by long hypocotyls and petioles, reduced chlorophyll content, and early flowering. We propose that GhFPF1 may be involved in flowering time control and shade-avoidance responses.

Clinical Utility of promoter methylation of the tumor suppressor genes DKK3, and RASSF1A in breast cancer patients

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Marwa H. Saied

2018-04-01

Full Text Available Background: DNA methylation is the commonest known epigenetic change that results in silencing of tumor suppressor genes. Promoter methylation of tumor suppressor genes has the potential for early detection of breast cancer. Aim: Aim is to examine the potential usefulness of blood based methylation specific polymerase chain reaction (MSP of methylated DKK3 and RASSF1A genes in early detection of breast cancer. Method: Methylation status of DKK3 and RASSF1 was investigated in forty breast cancer patients, twenty fibroadenoma patients and twenty healthy ladies as control group using MSP. Results: Methylation of DKK3 promoter was found in 22.5% of breast cancer patients, while DKK3 methylation was absent in both fibroadenoma patients and control group. Similarly, methylation of RASSF1 promoter was found in 17.5% of breast cancer patients and in none of fibroadenoma and control group. Conclusion: Promoter methylation of DKK3 and RASSF1 was found in breast cancer patients while absent in control group suggesting that tumorspecific methylation of the two genes (DKK3 and RASSF1A might be a valuable biomarker for the early detection of breast cancer. Keywords: DNA methylation, Breast cancer, DKK3, RASSF1

Regulatory polymorphisms in the bovine Ankyrin 1 gene promoter are associated with tenderness and intramuscular fat content

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Sweeney Torres

2010-12-01

Full Text Available Abstract Background Recent QTL and gene expression studies have highlighted ankyrins as positional and functional candidate genes for meat quality. Our objective was to characterise the promoter region of the bovine ankyrin 1 gene and to test polymorphisms for association with sensory and technological meat quality measures. Results Seven novel promoter SNPs were identified in a 1.11 kb region of the ankyrin 1 promoter in Angus, Charolais and Limousin bulls (n = 15 per breed as well as 141 crossbred beef animals for which meat quality data was available. Eighteen haplotypes were inferred with significant breed variation in haplotype frequencies. The five most frequent SNPs and the four most frequent haplotypes were subsequently tested for association with sensory and technological measures of meat quality in the crossbred population. SNP1, SNP3 and SNP4 (which were subsequently designated regulatory SNPs and SNP5 were associated with traits that contribute to sensorial and technological measurements of tenderness and texture; Haplotype 1 and haplotype 4 were oppositely correlated with traits contributing to tenderness (P Conclusion The conclusion from this study is that alleles defining haplotypes 2 and 4 could usefully contribute to marker SNP panels used to select individuals with improved IMF/juiciness or tenderness in a genome-assisted selection framework.

Cloning and characterization of a heme oxygenase-2 gene from alfalfa (Medicago sativa L.).

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Fu, Guang-Qing; Jin, Qi-Jiang; Lin, Yu-Ting; Feng, Jian-Fei; Nie, Li; Shen, Wen-Biao; Zheng, Tian-Qing

2011-11-01

Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H(2)O(2) (especially) treatment, respectively.

Iron depletion in HCT116 cells diminishes the upregulatory effect of phenethyl isothiocyanate on heme oxygenase-1

International Nuclear Information System (INIS)

Bolloskis, Michael P.; Carvalho, Fabiana P.; Loo, George

2016-01-01

Some of the health-promoting properties of cruciferous vegetables are thought to be partly attributed to isothiocyanates. These phytochemicals can upregulate the expression of certain cytoprotective stress genes, but it is unknown if a particular nutrient is involved. Herein, the objective was to ascertain if adequate iron is needed for enabling HCT116 cells to optimally express heme oxygenase-1 (HO-1) when induced by phenethyl isothiocyanate (PEITC). PEITC increased HO-1 expression and also nuclear translocation of Nrf2, which is a transcription factor known to activate the HO-1 gene. However, in HCT116 cells that were made iron-deficient by depleting intracellular iron with deferoxamine (DFO), PEITC was less able to increase HO-1 expression and nuclear translocation of Nrf2. These suppressive effects of DFO were overcome by replenishing the iron-deficient cells with the missing iron. To elucidate these findings, it was found that PEITC-induced HO-1 upregulation can be inhibited with thiol antioxidants (glutathione and N-acetylcysteine). Furthermore, NADPH oxidase inhibitors (diphenyleneiodonium and apocynin) and a superoxide scavenger (Tiron) each inhibited PEITC-induced HO-1 upregulation. In doing so, diphenyleneiodonium was the most potent and also inhibited nuclear translocation of redox-sensitive Nrf2. Collectively, the results imply that the HO-1 upregulation by PEITC involves an iron-dependent, oxidant signaling pathway. Therefore, it is concluded that ample iron is required to enable PEITC to fully upregulate HO-1 expression in HCT116 cells. As such, it is conceivable that iron-deficient individuals may not reap the full health benefits of eating PEITC-containing cruciferous vegetables that via HO-1 may help protect against multiple chronic diseases. - Highlights: • PEITC increased HO-1 expression in HCT116 cells. • PEITC-induced HO-1 upregulation was impaired in iron-depleted HCT116 cells. • Impairment of PEITC-induced HO-1 upregulation was

Neutron diffraction studies of Ho1-xYxNi2B2C compounds

DEFF Research Database (Denmark)

Chang, L.J.; Tomy, C.V.; Paul, D.M.K.

1996-01-01

Neutron diffraction measurements have been carried out to investigate the nature of magnetic ordering in Ho(1-x)Y(x)Ni(2)B(2)C (x = 0, 0.1 and 0.2) compounds. HoNi(2)B(2)C shows a complex type of magnetic ordering below the superconducting transition, with a commensurate antiferromagnetic ordering...

Genome-wide analysis of regions similar to promoters of histone genes

KAUST Repository

Chowdhary, Rajesh

2010-05-28

Background: The purpose of this study is to: i) develop a computational model of promoters of human histone-encoding genes (shortly histone genes), an important class of genes that participate in various critical cellular processes, ii) use the model so developed to identify regions across the human genome that have similar structure as promoters of histone genes; such regions could represent potential genomic regulatory regions, e.g. promoters, of genes that may be coregulated with histone genes, and iii/ identify in this way genes that have high likelihood of being coregulated with the histone genes.Results: We successfully developed a histone promoter model using a comprehensive collection of histone genes. Based on leave-one-out cross-validation test, the model produced good prediction accuracy (94.1% sensitivity, 92.6% specificity, and 92.8% positive predictive value). We used this model to predict across the genome a number of genes that shared similar promoter structures with the histone gene promoters. We thus hypothesize that these predicted genes could be coregulated with histone genes. This hypothesis matches well with the available gene expression, gene ontology, and pathways data. Jointly with promoters of the above-mentioned genes, we found a large number of intergenic regions with similar structure as histone promoters.Conclusions: This study represents one of the most comprehensive computational analyses conducted thus far on a genome-wide scale of promoters of human histone genes. Our analysis suggests a number of other human genes that share a high similarity of promoter structure with the histone genes and thus are highly likely to be coregulated, and consequently coexpressed, with the histone genes. We also found that there are a large number of intergenic regions across the genome with their structures similar to promoters of histone genes. These regions may be promoters of yet unidentified genes, or may represent remote control regions that

Control of phenylalanine ammonia-lyase gene promoters from pea by UV radiation

International Nuclear Information System (INIS)

Pluskota, W.E.; Michalczyk, D.J.; Gorecki, R.J.

2005-01-01

The gene fusion system was used to study UV light-control of PS PAL1 and PS PAL2 genes encoding phenylalanine ammonia-lyase of pea. The induction of pea PAL promoters was analysed in transgenic tobacco plants. Binary plasmids (derivatives of pBI101.2 vector) containing 5' regulatory fragments of PS PAL1 and PS PAL2 linked to reporter genes (GUS, LUC) were constructed. The analyses were performed with the use of single constructs (containing one variant of PS PAL promoter and one reporter gene) and dual constructs (containing both PS PAL1 and PS PAL2 promoters connected with different reporter genes). The use of dual constructs enabled the evaluation of both PS PAL promoters activity in the same plant. The analyses of in vitro grown plants have shown that both PAL promoters are strongly induced in leaves subjected to UV radiation. In some cases, the UV-stimulated expression exceeded the exposed areas. This phenomenon was observed more often in the leaves of plants containing the PS PAL1::GUS than PS PAL2::GUS construct. Removal of boxes 2, 4, 5 from PS PAL1 promoter and deletion of its 5' end region (-339 to -1394) decreases the level of gene expression but does not eliminate its responsiveness to UV

Role and molecular mechanism of HO-1-mediated NF-κB modulation in fibrosis progression of nonalcoholic steatohepatitis

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NAN Yuemin

2013-05-01

Full Text Available ObjectiveTo investigate the potential effects and molecular mechanisms of heme oxygenase-1 (HO-1-mediated modulation of nuclear factor-kappa B (NF-κB, and its downstream activation of intercellular adhesion molecule 1 (ICAM-1 and platelet derived growth factor (PDGF, in fibrosis progression of non-alcoholic steatohepatitis (NASH using the methionine and choline deficient (MCD mouse model of NASH. Methods Forty C57BL/6J male mice (18-20 g were randomly divided into four groups (n=10 each: NASH model group, administered the MCD diet; HO-1 agonist group, administered the MCD diet with intraperitoneal (ip injections of hemin (30 μmol/kg every other day; HO-1 inhibitor group, administered the MCD diet with ip injections of zinc protoporphyrin (ZnPP-IX; 20 μmol/kg every other day; and control group, administered a methionine and choline sufficient (MCS diet, without agonist nor inhibitor injections. After eight weeks, the mice were sacrificed and resected liver tissues used to assess successful model establishment by histological analysis (hematoxylin-eosin and Masson staining and the differential mRNA expression of HO-1, NF-κB, ICAM-1, and PDGF by real-time quantitative PCR (GAPDH normalized and protein expression of HO-1 and PDGF by western blotting ( β-actin normalized. Significance of an intergroup difference was assessed by single-factor analysis of variance test, and the Student-Newman-Keuls test was used for pairwise comparisons. ResultsThe NASH model group showed the appropriate histologic features of hepatic steatosis, necroinflammation and fibrogenesis, while the control group showed normal lobular architecture. In addition, the NASH model group showed significantly higher expression of HO-1, NF-κB, ICAM-1 and PDGF mRNA (all P＜0.05, and concomitant increases in HO-1 and PDGF protein. The group treated with HO-1 agonist showed significant down-regulation of the NASH-induced NF-κB, ICAM-1 and PDGF expressions, while the opposite

Insulators form gene loops by interacting with promoters in Drosophila.

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Erokhin, Maksim; Davydova, Anna; Kyrchanova, Olga; Parshikov, Alexander; Georgiev, Pavel; Chetverina, Darya

2011-09-01

Chromatin insulators are regulatory elements involved in the modulation of enhancer-promoter communication. The 1A2 and Wari insulators are located immediately downstream of the Drosophila yellow and white genes, respectively. Using an assay based on the yeast GAL4 activator, we have found that both insulators are able to interact with their target promoters in transgenic lines, forming gene loops. The existence of an insulator-promoter loop is confirmed by the fact that insulator proteins could be detected on the promoter only in the presence of an insulator in the transgene. The upstream promoter regions, which are required for long-distance stimulation by enhancers, are not essential for promoter-insulator interactions. Both insulators support basal activity of the yellow and white promoters in eyes. Thus, the ability of insulators to interact with promoters might play an important role in the regulation of basal gene transcription.

Promoter polymorphisms in genes involved in porcine myogenesis influence their transcriptional activity.

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Bongiorni, Silvia; Tilesi, Francesca; Bicorgna, Silvia; Iacoponi, Francesca; Willems, Daniela; Gargani, Maria; D'Andrea, MariaSilvia; Pilla, Fabio; Valentini, Alessio

2014-11-07

Success of meat production and selection for improvement of meat quality is among the primary aims in animal production. Meat quality traits are economically important in swine; however, the underlying genetic nature is very complex. Therefore, an improved pork production strongly depends on identifying and studying how genetic variations contribute to modulate gene expression. Promoters are key regions in gene modulation as they harbour several binding motifs to transcription regulatory factors. Therefore, polymorphisms in these regions are likely to deeply affect RNA levels and consequently protein synthesis. In this study, we report the identification of single nucleotide polymorphisms (SNPs) in promoter regions of candidate genes involved in development, cellular differentiation and muscle growth in Sus scrofa. We identified SNPs in the promoter regions of genes belonging to the Myogenic Regulatory Factors (MRF) gene family (the Myogenic Differentiation gene, MYOD1) and to Growth and Differentiation Factors (GDF) gene family (Myostatin gene, MSTN, GDF8), in Casertana and Large White breeds. The purpose of this study was to investigate if polymorphisms in the promoters could affect the transcriptional activity of these genes. With this aim, we evaluated in vitro the functional activity of the luciferase reporter gene luc2 activity, driven by two constructs carrying different promoter haplotypes. We tested the effects of the G302A (U12574) transition on the promoter efficiency in MYOD1 gene. We ascertained a difference in transcription efficiency for the two variants. A stronger activity of the A-carrying construct is more evident in C2C12. The luciferase expression driven by the MYOD1-A allelic variant displayed a 3.8-fold increased transcriptional activity. We investigated the activity of two haplotype variants (AY527152) in the promoter of GDF8 gene. The haploptype-1 (A435-A447-A879) up-regulated the expression of the reporter gene by a two-fold increase, and

Andrographolide Activates Keap1/Nrf2/ARE/HO-1 Pathway in HT22 Cells and Suppresses Microglial Activation by Aβ42 through Nrf2-Related Inflammatory Response.

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Seo, Ji Yeon; Pyo, Euisun; An, Jin-Pyo; Kim, Jinwoong; Sung, Sang Hyun; Oh, Won Keun

2017-01-01

Therapeutic approach of Alzheimer's disease (AD) has been gradually diversified. We examined the therapeutic and preventive potential of andrographolide, which is a lactone diterpenoid from Andrographis paniculata , and focused on the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated heme oxygenase (HO)-1-inducing effects and the inhibitory activity of amyloid beta (A β ) 42 -induced microglial activation related to Nrf2 and nuclear factor κ B (NF- κ B)-mediated inflammatory responses. Andrographolide induced the expression and translocation of Nrf2 from the cytoplasm to the nucleus, thereby activating antioxidant response element (ARE) gene transcription and HO-1 expression in murine hippocampal HT22 cells. Andrographolide eliminated intracellular A β 42 in BV-2 cells and decreased the production of interleukin (IL)-6, IL-1 β , prostaglandin (PG)E 2 , and nitric oxide (NO) because of artificial phagocytic A β 42 . It decreased pNF- κ B accumulation in the nucleus and the expression of inducible nitric oxide synthase (i-NOS) and cyclooxygenase II (COX-II) in the microglial BV-2 cell line. In summary, andrographolide activates Nrf2-mediated HO-1 expression and inhibits A β 42 -overexpressed microglial BV-2 cell activation. These results suggested that andrographolide might have the potential for further examination of the therapeutics of AD.

Andrographolide Activates Keap1/Nrf2/ARE/HO-1 Pathway in HT22 Cells and Suppresses Microglial Activation by Aβ42 through Nrf2-Related Inflammatory Response

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Ji Yeon Seo

2017-01-01

Full Text Available Therapeutic approach of Alzheimer’s disease (AD has been gradually diversified. We examined the therapeutic and preventive potential of andrographolide, which is a lactone diterpenoid from Andrographis paniculata, and focused on the Kelch-like ECH-associated protein 1 (Keap1/nuclear factor (erythroid-derived 2-like 2 (Nrf2-mediated heme oxygenase (HO-1-inducing effects and the inhibitory activity of amyloid beta (Aβ42-induced microglial activation related to Nrf2 and nuclear factor κB (NF-κB-mediated inflammatory responses. Andrographolide induced the expression and translocation of Nrf2 from the cytoplasm to the nucleus, thereby activating antioxidant response element (ARE gene transcription and HO-1 expression in murine hippocampal HT22 cells. Andrographolide eliminated intracellular Aβ42 in BV-2 cells and decreased the production of interleukin (IL-6, IL-1β, prostaglandin (PGE2, and nitric oxide (NO because of artificial phagocytic Aβ42. It decreased pNF-κB accumulation in the nucleus and the expression of inducible nitric oxide synthase (i-NOS and cyclooxygenase II (COX-II in the microglial BV-2 cell line. In summary, andrographolide activates Nrf2-mediated HO-1 expression and inhibits Aβ42-overexpressed microglial BV-2 cell activation. These results suggested that andrographolide might have the potential for further examination of the therapeutics of AD.

Transactivation of the proenkephalin gene promoter by the Tax sub 1 protein of human T-cell lymphotropic virus type I

Energy Technology Data Exchange (ETDEWEB)

Joshi, J.B. (National Heart, Lung, and Blood Inst., Bethesda, MD (United States)); Dave, H.P.G. (National Inst. of Health, Bethesda, MD (United States))

1992-02-01

Human T-cell lymphotropic virus type I (HTLV-I), an etiologic agent for adult T-cell leukemia, is strongly associated with certain neurological diseases. The HTLV-I genome encodes a protein, Tax{sub 1}, that transactivates viral gene transcription. CD4-positive T helper lymphocytes express the proenkephalin gene, and enkephalins have been implicated as neuroimmunomodulators. The authors have investigated the effect of Tax{sub 1} on the proenkephalin gene promoter in C6 rat glioma cells and demonstrated its transactivation. Analysis using 5{prime} deletion mutants of the promoter region showed that sequences upstream of base pair - 190 are necessary for maximal transactivation. Forskolin, a cAMP modulator, synergistically increased Tax{sub 1}-mediated transactivation of the proenkephalin promoter. Neither Tax{sub 1} transactivation alone nor Tax{sub 1}/cAMP synergism exclusively involved cAMP-responsive elements. Endogenous proenkephalin gene expression increased in Tax{sub 1}-expressing C6 cells. Since HTLV-I infects lymphocytes, which express proenkephalin mRNA, Tax{sub 1} transregulation of proenkephalin expression may provide bidirectional communication between the nervous and immune systems in HTLV-I-related diseases.

PMK-1 p38 MAPK promotes cadmium stress resistance, the expression of SKN-1/Nrf and DAF-16 target genes, and protein biosynthesis in Caenorhabditis elegans.

Science.gov (United States)

Keshet, Alex; Mertenskötter, Ansgar; Winter, Sarah A; Brinkmann, Vanessa; Dölling, Ramona; Paul, Rüdiger J

2017-12-01

The mechanisms of cadmium (Cd) resistance are complex and not sufficiently understood. The present study, therefore, aimed at assessing the roles of important components of stress-signaling pathways and of ABC transporters under severe Cd stress in Caenorhabditis elegans. Survival assays on mutant and control animals revealed a significant promotion of Cd resistance by the PMK-1 p38 MAP kinase, the transcription factor DAF-16/FoxO, and the ABC transporter MRP-1. Transcriptome profiling by RNA-Seq on wild type and a pmk-1 mutant under control and Cd stress conditions revealed, inter alia, a PMK-1-dependent promotion of gene expression for the translational machinery. PMK-1 also promoted the expression of target genes of the transcription factors SKN-1/Nrf and DAF-16 in Cd-stressed animals, which included genes for molecular chaperones or immune proteins. Gene expression studies by qRT-PCR confirmed the positive effects of PMK-1 on DAF-16 activity under Cd stress and revealed negative effects of DAF-16 on the expression of genes for MRP-1 and DAF-15/raptor. Additional studies on pmk-1 RNAi-treated wild type and mutant strains provided further information on the effects of PMK-1 on SKN-1 and DAF-16, which resulted in a model of these relationships. The results of this study demonstrate a central role of PMK-1 for the processing of cellular responses to abiotic and biotic stressors, with the promoting effects of PMK-1 on Cd resistance mostly mediated by the transcription factors SKN-1 and DAF-16.

Cytoprotective effect exerted by geraniin in HepG2 cells is through microRNA mediated regulation of BACH-1 and HO-1.

Science.gov (United States)

Aayadi, Hoda; Mittal, Smriti P K; Deshpande, Anjali; Gore, Makarand; Ghaskadbi, Saroj S

2017-11-01

Geraniin, a hydrolysable tannin, used in traditional medicine in Southeast Asia, is known to exhibit various biological activities. As an antioxidant it is known to up-regulate phase II enzyme Heme oxygenase-1 (HO-1). However its mechanism is not clearly understood. Nuclear factor erythroid-derived 2 related factor 2 (Nrf-2) is transcriptionally up-regulated by Extracellular signal-regulated kinase (ERK) 1/2 and retained in nucleus due to inactivated Glycogen synthase kinase 3 beta (GSK-3β). Geraniin additionally down-regulates expression of microRNA 217 and 377 (miR-217 and miR-377) which target HO-1 mRNA. Expression of BTB and CNC homolog 1 (BACH-1), another regulator of HO-1, is also down-regulated by up-regulating microRNA 98 (miR-98), a negative regulator of BACH-1. Thus, geraniin up-regulates HO-1 expression both through activating its positive regulator Nrf-2 and by down-regulating its negative regulator BACH-1. Up-regulation of HO-1 also confers protection to HepG2 cells from tertiary butyl hydroperoxide (TBH) induced cytotoxicity. [BMB Reports 2017; 50(11): 560-565].

Activation of the Nrf2/HO-1 Antioxidant Pathway Contributes to the Protective Effects of Lycium Barbarum Polysaccharides in the Rodent Retina after Ischemia-Reperfusion-Induced Damage

Science.gov (United States)

Chang, Raymond Chuen-Chung; So, Kwok-Fai; Brecha, Nicholas C.; Pu, Mingliang

2014-01-01

Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are protective to retina after ischemia-reperfusion (I/R). The antioxidant response element (ARE)–mediated antioxidant pathway plays an important role in maintaining the redox status of the retina. Heme oxygenase-1 (HO-1), combined with potent AREs in its promoter, is a highly effective therapeutic target for the protection against neurodegenerative diseases, including I/R-induced retinal damage. The aim of our present study was to investigate whether the protective effect of LBP after I/R damage was mediated via activation of the Nrf2/HO-1-antioxidant pathway in the retina. Retinal I/R was induced by an increase in intraocular pressure to 130 mm Hg for 60 minutes. Prior to the induction of ischemia, rats were orally treated with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. For specific experiments, zinc protoporphyrin (ZnPP, 20 mg/kg), an HO-1 inhibitor, was intraperitoneally administered at 24 h prior to ischemia. The protective effects of LBP were evaluated by quantifying ganglion cell and amacrine cell survival, and by measuring cell apoptosis in the retinal layers. In addition, HO-1 expression was examined using Western blotting and immunofluorescence analyses. Cytosolic and nuclear Nrf2 was measured using immunofluorescent staining. LBP treatment significantly increased Nrf2 nuclear accumulation and HO-1 expression in the retina after I/R injury. Increased apoptosis and a decrease in the number of viable cells were observed in the ganglion cell layer (GCL) and inner nuclear layer (INL) in the I/R retina, which were reversed by LBP treatment. The HO-1 inhibitor, ZnPP, diminished the LBP treatment-induced protective effects in the retina after I/R. Taken together, these results suggested that LBP partially exerted its beneficial neuroprotective effects via the activation of Nrf2 and an increase in HO-1 protein expression. PMID:24400114

A novel BDNF gene promoter directs expression to skeletal muscle

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Heinrich Gerhard

2003-06-01

Full Text Available Abstract Background Cell-specific expression of the gene that encodes brain-derived neurotrophic factor (BDNF is required for the normal development of peripheral sensory neurons and efficient synaptic transmission in the mature central and peripheral nervous system. The control of BDNF gene expression involves multiple tissue and cell-specific promoters that are differentially regulated. The molecular mechanisms that are responsible for tissue and cell-specific expression of these promoters are still incompletely understood. Results The cloning and analysis of three additional zebrafish (Danio rerio BDNF gene exons and two associated promoters, is reported. Among them are two exons that generate a novel tripartite mature transcript. The exons were located on the transcription unit, whose overall organization was determined by cloning, Southern blot hybridization and sequence analysis, and compared with the pufferfish (Fugu rubripes and mammalian BDNF loci, revealing a conserved but more compact organization. Structural and functional analysis of the exons, their adjacent promoters and 5' flanks, showed that they are expressed cell-specifically. The promoter associated with the 5' exon of the tripartite transcript is GC-rich, TATA-less and the 5' flank adjacent to it contains multiple Sp1, Mef2, and AP1 elements. A fusion gene containing the promoter and 1.5 KB of 5' flank is directed exclusively to skeletal muscle of transiently transfected embryos. The second promoter, whose associated 5' exon contains a 25-nucleotide segment of identity with a mammalian BDNF gene exon, was transiently expressed in yolk of the early embryo. RT-PCR analysis of total RNA from whole juvenile fish and adult female skeletal muscle revealed tissue-specific expression of the 5' exons but the novel exon could not be detected even after two rounds of nested PCR. Conclusion The zebrafish BDNF gene is as complex as the mammalian gene yet much more compact. Its exons are

Hořava Gravity is Asymptotically Free in 2+1 Dimensions.

Science.gov (United States)

Barvinsky, Andrei O; Blas, Diego; Herrero-Valea, Mario; Sibiryakov, Sergey M; Steinwachs, Christian F

2017-11-24

We compute the β functions of marginal couplings in projectable Hořava gravity in 2+1 spacetime dimensions. We show that the renormalization group flow has an asymptotically free fixed point in the ultraviolet (UV), establishing the theory as a UV-complete model with dynamical gravitational degrees of freedom. Therefore, this theory may serve as a toy model to study fundamental aspects of quantum gravity. Our results represent a step forward towards understanding the UV properties of realistic versions of Hořava gravity.

New PAH gene promoter KLF1 and 3'-region C/EBPalpha motifs influence transcription in vitro.

Science.gov (United States)

Klaassen, Kristel; Stankovic, Biljana; Kotur, Nikola; Djordjevic, Maja; Zukic, Branka; Nikcevic, Gordana; Ugrin, Milena; Spasovski, Vesna; Srzentic, Sanja; Pavlovic, Sonja; Stojiljkovic, Maja

2017-02-01

Phenylketonuria (PKU) is a metabolic disease caused by mutations in the phenylalanine hydroxylase (PAH) gene. Although the PAH genotype remains the main determinant of PKU phenotype severity, genotype-phenotype inconsistencies have been reported. In this study, we focused on unanalysed sequences in non-coding PAH gene regions to assess their possible influence on the PKU phenotype. We transiently transfected HepG2 cells with various chloramphenicol acetyl transferase (CAT) reporter constructs which included PAH gene non-coding regions. Selected non-coding regions were indicated by in silico prediction to contain transcription factor binding sites. Furthermore, electrophoretic mobility shift assay (EMSA) and supershift assays were performed to identify which transcriptional factors were engaged in the interaction. We found novel KLF1 motif in the PAH promoter, which decreases CAT activity by 50 % in comparison to basal transcription in vitro. The cytosine at the c.-170 promoter position creates an additional binding site for the protein complex involving KLF1 transcription factor. Moreover, we assessed for the first time the role of a multivariant variable number tandem repeat (VNTR) region located in the 3'-region of the PAH gene. We found that the VNTR3, VNTR7 and VNTR8 constructs had approximately 60 % of CAT activity. The regulation is mediated by the C/EBPalpha transcription factor, present in protein complex binding to VNTR3. Our study highlighted two novel promoter KLF1 and 3'-region C/EBPalpha motifs in the PAH gene which decrease transcription in vitro and, thus, could be considered as PAH expression modifiers. New transcription motifs in non-coding regions will contribute to better understanding of the PKU phenotype complexity and may become important for the optimisation of PKU treatment.

Prolonged triglyceride storage in macrophages: pHo trumps pO2 and TLR4.

Science.gov (United States)

Lu, Mingfang; Kho, Terry; Munford, Robert S

2014-08-01

Lipid-laden macrophages contribute to pathologies as diverse as atherosclerosis and tuberculosis. Three common stimuli are known to promote macrophage lipid storage: low tissue oxygen tension (pO2), low extracellular pH (pHo), and exposure to agonists such as bacterial LPS. Noting that cells responding to low pO2 or agonistic bacterial molecules often decrease pHo by secreting lactic and other carboxylic acids, we studied how pHo influences the stimulation of triacylglycerol (TAG) storage by low pO2 and LPS. We found that TAG retention after incubation for 48-72 h was inversely related to pHo when primary macrophages were cultured in 21% oxygen, 4% oxygen, or with LPS at either oxygen concentration. Maintaining pHo at ~7.4 was sufficient to prevent the increase in prolonged TAG storage induced by either low pO2 or LPS. The strong influence of pHo on TAG retention may explain why lipid-laden macrophages are found in some tissue environments and not in others. It is also possible that other long-term cellular changes currently attributed to low pO2 or bacterial agonists may be promoted, at least in part, by the decrease in pHo that these stimuli induce.

Promoter DNA hypermethylation and gene repression in undifferentiated Arabidopsis cells.

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María Berdasco

Full Text Available Maintaining and acquiring the pluripotent cell state in plants is critical to tissue regeneration and vegetative multiplication. Histone-based epigenetic mechanisms are important for regulating this undifferentiated state. Here we report the use of genetic and pharmacological experimental approaches to show that Arabidopsis cell suspensions and calluses specifically repress some genes as a result of promoter DNA hypermethylation. We found that promoters of the MAPK12, GSTU10 and BXL1 genes become hypermethylated in callus cells and that hypermethylation also affects the TTG1, GSTF5, SUVH8, fimbrin and CCD7 genes in cell suspensions. Promoter hypermethylation in undifferentiated cells was associated with histone hypoacetylation and primarily occurred at CpG sites. Accordingly, we found that the process specifically depends on MET1 and DRM2 methyltransferases, as demonstrated with DNA methyltransferase mutants. Our results suggest that promoter DNA methylation may be another important epigenetic mechanism for the establishment and/or maintenance of the undifferentiated state in plant cells.

Baicalein inhibition of oxidative-stress-induced apoptosis via modulation of ERKs activation and induction of HO-1 gene expression in rat glioma cells C6

International Nuclear Information System (INIS)

Chen, Y.-C.; Chow, J.-M.; Lin, C.-W.; Wu, C.-Y.; Shen, S.-C.

2006-01-01

In the present study, we examined the protective mechanism of baicalein (BE) and its glycoside, baicalin (BI), on hydrogen-peroxide (H 2 O 2 )-induced cell death in rat glioma C6 cells. Results of the MTT assay, LDH release assay, and morphological observation showed that H 2 O 2 addition reduced the viability of C6 cells, and this was prevented by the addition of BE but not BI. Incubation of C6 cells with BE significantly decreased the intracellular peroxide level induced by H 2 O 2 according to flow cytometric analysis using DCHF-DA as a fluorescent substrate. Suppression of H 2 O 2 -induced apoptotic events including DNA ladders, hypodiploid cells, and activation of caspases 3, 8, and, 9 by BE but not BI was identified in C6 cells. The cytotoxicity and phosphorylation of ERK proteins induced by H 2 O 2 were blocked by the ERK inhibitor PD98059. Catalase addition prevented H 2 O 2 -induced ROS production, ERKs protein phosphorylation, and cell death, and BE dose-dependently inhibited H 2 O 2 -induced ERK protein phosphorylation in C6 cells. These data suggest that ROS-scavenging activity is involved in BE prevention of H 2 O 2 -induced cell death via blocking ERKs activation. Additionally, BE but not BI induced heat shock protein 32 (HSP32; HO-1) protein expression in both time- and dose-dependent manners, but not heme oxygenase 2 (HO-2), heat shock protein 70 (HSP70), or heat shock protein 90 (HSP90) protein expression. In the absence of H 2 O 2 , BE induces ERKs protein phosphorylation, and HO-1 protein expression induced by BE was blocked by the addition of cycloheximide, actinomycin D, and the ERK inhibitor PD98059. The addition of the HO inhibitor ZnPP inhibited the protective effect of BE against H 2 O 2 -induced cytotoxicity in C6 cells according to the MTT assay and apoptotic morphology under microscopic observation, accompanied by blocking the ROS-scavenging activity of BE in C6 cells. However, BE treatment was unable to protect C6 cells from C2-ceramide

Gap junctional communication modulates gene transcription by altering the recruitment of Sp1 and Sp3 to connexin-response elements in osteoblast promoters

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Stains, Joseph P.; Lecanda, Fernando; Screen, Joanne; Towler, Dwight A.; Civitelli, Roberto

2003-01-01

Loss-of-function mutations of gap junction proteins, connexins, represent a mechanism of disease in a variety of tissues. We have shown that recessive (gene deletion) or dominant (connexin45 overexpression) disruption of connexin43 function results in osteoblast dysfunction and abnormal expression of osteoblast genes, including down-regulation of osteocalcin transcription. To elucidate the molecular mechanisms of gap junction-sensitive transcriptional regulation, we systematically analyzed the rat osteocalcin promoter for sensitivity to gap junctional intercellular communication. We identified an Sp1/Sp3 containing complex that assembles on a minimal element in the -70 to -57 region of the osteocalcin promoter in a gap junction-dependent manner. This CT-rich connexin-response element is necessary and sufficient to confer gap junction sensitivity to the osteocalcin proximal promoter. Repression of osteocalcin transcription occurs as a result of displacement of the stimulatory Sp1 by the inhibitory Sp3 on the promoter when gap junctional communication is perturbed. Modulation of Sp1/Sp3 recruitment also occurs on the collagen Ialpha1 promoter and translates into gap junction-sensitive transcriptional control of collagen Ialpha1 gene expression. Thus, regulation of Sp1/Sp3 recruitment to the promoter may represent a potential general mechanism for transcriptional control of target genes by signals passing through gap junctions.

Effect of TNFα on activities of different promoters of human apolipoprotein A-I gene

International Nuclear Information System (INIS)

Orlov, Sergey V.; Mogilenko, Denis A.; Shavva, Vladimir S.; Dizhe, Ella B.; Ignatovich, Irina A.; Perevozchikov, Andrej P.

2010-01-01

Research highlights: → TNFα stimulates the distal alternative promoter of human apoA-I gene. → TNFα acts by weakening of promoter competition within apoA-I gene (promoter switching). → MEK1/2 and nuclear receptors PPARα and LXRs take part in apoA-I promoter switching. -- Abstract: Human apolipoprotein A-I (ApoA-I) is a major structural and functional protein component of high-density lipoproteins. The expression of the apolipoprotein A-I gene (apoA-I) in hepatocytes is repressed by pro-inflammatory cytokines such as IL-1β and TNFα. Recently, two novel additional (alternative) promoters for human apoA-I gene have been identified. Nothing is known about the role of alternative promoters in TNFα-mediated downregulation of apoA-I gene. In this article we report for the first time about the different effects of TNFα on two alternative promoters of human apoA-I gene. Stimulation of HepG2 cells by TNFα leads to activation of the distal alternative apoA-I promoter and downregulation of the proximal alternative and the canonical apoA-I promoters. This effect is mediated by weakening of the promoter competition within human apoA-I 5'-regulatory region (apoA-I promoter switching) in the cells treated by TNFα. The MEK1/2-ERK1/2 cascade and nuclear receptors PPARα and LXRs are important for TNFα-mediated apoA-I promoter switching.

Epigallocatechin-3-gallate (EGCG) protects skin cells from ionizing radiation via heme oxygenase-1 (HO-1) overexpression

International Nuclear Information System (INIS)

Zhu Wei; Xu Jing; Ge Yangyang

2014-01-01

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent of green tea, is a potent antioxidant and free radical scavenger that may have therapeutic applications for the treatment of many disorders. Radiation therapy is widely used for the treatment of various types of cancers; however, radiation-induced skin injury remains a serious concern. EGCG has not yet been reported as protecting skin cells against ionizing radiation. In the present study, we investigated whether EGCG confers cytoprotection against ionizing radiation. We found that, compared with the control, pretreatment with EGCG significantly enhanced the viability of human skin cells that were irradiated with X-rays, and decreased apoptosis induced by X-ray irradiation. Mito-Tracker assay showed that EGCG suppressed the damage to mitochondria induced by ionizing radiation via upregulation of SOD2. Reactive oxygen species (ROS) in HaCaT cells were significantly reduced when pretreated with EGCG before irradiation. Radiation-induced γH2AX foci, which are representative of DNA double-strand breaks, were decreased by pretreatment with EGCG. Furthermore, EGCG induced the expression of the cytoprotective molecule heme oxygenase-1 (HO-1) in a dose-dependent manner via transcriptional activation. HO-1 knockdown or treatment with the HO-1 inhibitor tin protoporphyrin (SnPPIX) reversed the protective role of EGCG, indicating an important role for HO-1. These results suggest that EGCG offers a new strategy for protecting skin against ionizing radiation. (author)

Effects of heme oxygenase-1 gene modulated mesenchymal stem cells on vasculogenesis in ischemic swine hearts.

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Jiang, Yi-Bo; Zhang, Xiao-Li; Tang, Yao-Liang; Ma, Gen-Shan; Shen, Cheng-Xing; Wei, Qin; Zhu, Qi; Yao, Yu-Yu; Liu, Nai-Feng

2011-02-01

Mesenchymal stem cells (MSCs) transplantation may partially restore heart function in the treatment of acute myocardial infarction (AMI). The aim of this study was to explore the beneficial effects of MSCs modified with heme xygenase-1 (HO-1) on post-infarct swine hearts to determine whether the induction of therapeutic angiogenesis is modified by the angiogenic cytokines released from the implanted cells. In vitro, MSCs were divided into four groups: (1) non-transfected MSCs (MSCs group), (2) MSCs transfected with the pcDNA3.1-Lacz plasmid (Lacz-MSCs group), (3) MSCs transfected with pcDNA3.1-hHO-1 (HO-1-MSCs group), and (4) MSCs transfected with pcDNA3.1-hHO-1 and pretreatment with an HO inhibitor, tin protoporphyrin (SnPP) (HO-1-MSCs + SnPP group). Cells were cultured in an airtight incubation bottle for 24 hours, in which the oxygen concentration was maintained at < 1%, followed by 12 hours of reoxygenation. After hypoxia/reoxygen treatment, ELISA was used to measure transforming growth factor (TGF-β) and fibroblast growth factor (FGF-2) in the supernatant. In vivo, 28 Chinese mini-pigs were randomly allocated to the following treatment groups: (1) control group (saline), (2) Lacz-MSCs group, (3) HO-1-MSCs group, and (4) HO-1-MSCs + SnPP group. About 1 × 10(7) of autologous stem cells or an identical volume of saline was injected intracoronary into porcine hearts 1 hour after MI. Magnetic resonance imaging (MRI) assay and postmortem analysis were assessed four weeks after stem cell transplantation. Post hypoxia/reoxygenation in vitro, TGF-β in the supernatant was significantly increased in the HO-1-MSCs ((874.88 ± 68.23) pg/ml) compared with Lacz-MSCs ((687.81 ± 57.64) pg/ml, P < 0.001). FGF-2 was also significantly increased in the HO-1-MSCs ((1106.48 ± 107.06) pg/ml) compared with the Lacz-MSCs ((853.85 ± 74.44) pg/ml, P < 0.001). In vivo, at four weeks after transplantation, HO-1 gene transfer increased the capillary density in the peri-infarct area

CypA, a gene downstream of HIF-1α, promotes the development of PDAC.

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Huan Zhang

Full Text Available Hypoxia-inducible factor-1α (HIF-1α is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC. In response to tumor hypoxia, cyclophilin A (CypA is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.

Generation of an efficient artificial promoter of bovine skeletal muscle α-actin gene (ACTA1) through addition of cis-acting element.

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Hu, Qian; Tong, Huili; Zhao, Dandan; Cao, Yunkao; Zhang, Weiwei; Chang, Shuwei; Yang, Yu; Yan, Yunqin

2015-03-01

The promoter of skeletal muscle α-actin gene (ACTA1) is highly muscle specific. The core of the bovine ACTA1 promoter extends from +29 to -233, about 262 base pairs (bp), which is sufficient to activate transcription in bovine muscle satellite cells. In this study, analysis by PCR site-specific mutagenesis showed that the cis-acting element SRE (serum response element binding factor) was processed as a transcriptional activator. In order to enhance the bovine ACTA1 promoter's activity, we used a strategy to modify it. We cloned a fragment containing three SREs from the promoter of ACTA1, and then one or two clones were linked upstream of the core promoter (262 bp) of ACTA1. One and two clones increased the activity of the ACTA1 promoter 3-fold and 10-fold, respectively, and maintained muscle tissue specificity. The modified promoter with two clones could increase the level of ACTA1 mRNA and protein 4-fold and 1.1-fold, respectively. Immunofluorescence results showed that green fluorescence of ACTA1 increased. Additionally, the number of total muscle microfilaments increased. These genetically engineered promoters might be useful for regulating gene expression in muscle cells and improving muscle mass in livestock.

Energy levels of Ho3+ in HoCl63-

International Nuclear Information System (INIS)

Tanner, P.A.

1987-01-01

The 5 S 2 , 5 F 4 , 5 F 2 , 3 K 8 , 5 G 6 , 5 F 1 , 5 G 5 5 I 8 absorption spectra of Cs 2 NaHoCl 6 have been recorded at temperatures down to that of liquid helium and new assignments are made for the 5 S 2 , 5 F 4 , 3 K 8 and 5 G 5 terms of HoCl 6 3- . The 5 F 3 → 5 I 8 , 5 F 4 , 5 S 2 → 5 I 7 , 5 I 8 and 5 F 5 → 5 I 7 luminescence transitions of HoCl 6 3- in various elpasolite lattices are reported and assigned, together with luminescence decay measurements. Including the previously reported data, these results enable 57 of the first 60 crystal-field levels of HoCl 6 3- , comprising 14 Lsub(j) terms, to be firmly assigned and tentative assignments are made for the remaining three levels. The agreement between the calculated and observed crystal-field splittings is reasonable. (author)

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

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Meier, Daniel; Schindler, Detlev

2011-01-01

The Fanconi anemia (FA) gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M) that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS). In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs), and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

Fanconi anemia core complex gene promoters harbor conserved transcription regulatory elements.

Directory of Open Access Journals (Sweden)

Daniel Meier

Full Text Available The Fanconi anemia (FA gene family is a recent addition to the complex network of proteins that respond to and repair certain types of DNA damage in the human genome. Since little is known about the regulation of this novel group of genes at the DNA level, we characterized the promoters of the eight genes (FANCA, B, C, E, F, G, L and M that compose the FA core complex. The promoters of these genes show the characteristic attributes of housekeeping genes, such as a high GC content and CpG islands, a lack of TATA boxes and a low conservation. The promoters functioned in a monodirectional way and were, in their most active regions, comparable in strength to the SV40 promoter in our reporter plasmids. They were also marked by a distinctive transcriptional start site (TSS. In the 5' region of each promoter, we identified a region that was able to negatively regulate the promoter activity in HeLa and HEK 293 cells in isolation. The central and 3' regions of the promoter sequences harbor binding sites for several common and rare transcription factors, including STAT, SMAD, E2F, AP1 and YY1, which indicates that there may be cross-connections to several established regulatory pathways. Electrophoretic mobility shift assays and siRNA experiments confirmed the shared regulatory responses between the prominent members of the TGF-β and JAK/STAT pathways and members of the FA core complex. Although the promoters are not well conserved, they share region and sequence specific regulatory motifs and transcription factor binding sites (TBFs, and we identified a bi-partite nature to these promoters. These results support a hypothesis based on the co-evolution of the FA core complex genes that was expanded to include their promoters.

HoYbBIG epitaxial thick films used for Faraday rotator in the 1.55μm band

International Nuclear Information System (INIS)

Zhong, Z.W.; Xu, X.W.; Chong, T.C.; Yuan, S.N.; Li, M.H.; Zhang, G.Y.; Freeman, B.

2005-01-01

Ho 3-x-y Yb y Bi x Fe 5 O 12 (HoYbBIG) garnet thick films with Bi content of x=0.9-1.5 were prepared by the liquid phase epitaxy (LPE) method. Optical properties and magneto-optical properties were characterized. The LPE-grown HoYbBIG thick films exhibited large Faraday rotation coefficients up to 1540 o /cm at 1.55μm, and good wavelength and temperature stability

The HoMBReS and HoMBReS Por un Cambio Interventions to Reduce HIV Disparities Among Immigrant Hispanic/Latino Men.

Science.gov (United States)

Rhodes, Scott D; Leichliter, Jami S; Sun, Christina J; Bloom, Fred R

2016-02-12

Hispanics/Latinos in the United States are affected disproportionately by human immunodeficiency virus (HIV) infection, acquired immunodeficiency syndrome (AIDS), and other sexually transmitted diseases (STDs); however, few effective evidence-based prevention interventions for this population exist. This report describes the Hombres Manteniendo Bienestar y Relaciones Saludables (Men Maintaining Wellbeing and Healthy Relationships) (HoMBReS) intervention, which was developed by a community-based, participatory research partnership in North Carolina and initially implemented during 2005-2009. HoMBReS is an example of an effective intervention that uses lay health advisors (known as Navegantes [navigators]) in the context of existing social networks (i.e., recreational soccer teams) to promote consistent condom use and HIV and STD testing among Hispanic/Latino men. In 2012, HoMBReS was classified as a best-evidence community-level HIV prevention intervention (CDC. Compendium of evidence-based behavioral interventions and best practices for HIV prevention. Atlanta, GA: US Department of Health and Human Services, CDC; 2015). The intervention has been implemented elsewhere, enhanced, and further evaluated in longitudinal intervention and implementation studies. HoMBReS has been adapted for other populations, including men who have sex with men and transgender persons. Additional evaluation has found that Navegantes continue in their roles as health advisors, opinion leaders, and community advocates after study support ends. Hispanic/Latino men's social networks can be leveraged to promote sexual health within the community by decreasing HIV risk behaviors among Hispanics/Latinos in the United States.

Novel strong tissue specific promoter for gene expression in human germ cells

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Kuzmin Denis

2010-08-01

Full Text Available Abstract Background Tissue specific promoters may be utilized for a variety of applications, including programmed gene expression in cell types, tissues and organs of interest, for developing different cell culture models or for use in gene therapy. We report a novel, tissue-specific promoter that was identified and engineered from the native upstream regulatory region of the human gene NDUFV1 containing an endogenous retroviral sequence. Results Among seven established human cell lines and five primary cultures, this modified NDUFV1 upstream sequence (mNUS was active only in human undifferentiated germ-derived cells (lines Tera-1 and EP2102, where it demonstrated high promoter activity (~twice greater than that of the SV40 early promoter, and comparable to the routinely used cytomegaloviral promoter. To investigate the potential applicability of the mNUS promoter for biotechnological needs, a construct carrying a recombinant cytosine deaminase (RCD suicide gene under the control of mNUS was tested in cell lines of different tissue origin. High cytotoxic effect of RCD with a cell-death rate ~60% was observed only in germ-derived cells (Tera-1, whereas no effect was seen in a somatic, kidney-derived control cell line (HEK293. In further experiments, we tested mNUS-driven expression of a hyperactive Sleeping Beauty transposase (SB100X. The mNUS-SB100X construct mediated stable transgene insertions exclusively in germ-derived cells, thereby providing further evidence of tissue-specificity of the mNUS promoter. Conclusions We conclude that mNUS may be used as an efficient promoter for tissue-specific gene expression in human germ-derived cells in many applications. Our data also suggest that the 91 bp-long sequence located exactly upstream NDUFV1 transcriptional start site plays a crucial role in the activity of this gene promoter in vitro in the majority of tested cell types (10/12, and an important role - in the rest two cell lines.

[Divergence of paralogous growth-hormone-encoding genes and their promoters in Salmonidae].

Science.gov (United States)

Kamenskaya, D N; Pankova, M V; Atopkin, D M; Brykov, V A

2017-01-01

In many fish species, including salmonids, the growth-hormone is encoded by two duplicated paralogous genes, gh1 and gh2. Both genes were already in place at the time of divergence of species in this group. A comparison of the entire sequence of these genes of salmonids has shown that their conserved regions are associated with exons, while their most variable regions correspond to introns. Introns C and D include putative regulatory elements (sites Pit-1, CRE, and ERE), that are also conserved. In chars, the degree of polymorphism of gh2 gene is 2-3 times as large as that in gh1 gene. However, a comparison across all Salmonidae species would not extent this observation to other species. In both these chars' genes, the promoters are conserved mainly because they correspond to putative regulatory sequences (TATA box, binding sites for the pituitary transcription factor Pit-1 (F1-F4), CRE, GRE and RAR/RXR elements). The promoter of gh2 gene has a greater degree of polymorphism compared with gh1 gene promoter in all investigated species of salmonids. The observed differences in the rates of accumulation of changes in growth hormone encoding paralogs could be explained by differences in the intensity of selection.

Reaction rate constant of HO2+O3 measured by detecting HO2 from photofragment fluorescence

Science.gov (United States)

Manzanares, E. R.; Suto, Masako; Lee, Long C.; Coffey, Dewitt, Jr.

1986-01-01

A room-temperature discharge-flow system investigation of the rate constant for the reaction 'HO2 + O3 yields OH + 2O2' has detected HO2 through the OH(A-X) fluorescence produced by photodissociative excitation of HO2 at 147 nm. A reaction rate constant of 1.9 + or - 0.3 x 10 to the -15th cu cm/molecule per sec is obtained from first-order decay of HO2 in excess O3; this agrees well with published data.

Functional Analysis of Promoter Region from Eel Cytochrome P450 1A1 Gene in Transgenic Medaka.

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Ogino; Itakura; Kato; Aoki; Sato

1999-07-01

: Transcription of the CYP1A1 genes in mammals and fish is stimulated by polyaromatic hydrocarbons. DNA sequencing analysis revealed that CYP1A1 gene in eel (Anguilla japonica) contains two kinds of putative cis-acting regulatory elements, XRE (xenobiotic-responsive element) and ERE (estrogen-responsive element). XRE is known as the enhancer that is responsible for the inducibility of the genes of CYP1A1 and some other drug-metabolizing enzymes. In the eel CYP1A1 gene, XRE motifs are distributed as follows: five times in the region from -2136 to -1125 bp, XRE(-6) to (-2); once in the proximal basal promoter region, XRE(-1); and once in the first intron, XRE(+1). The region between XRE(-2) and XRE(-1) contains three ERE motifs. To investigate the function of the cis-acting regulatory elements in the eel CYP1A1 gene, recombinant plasmids prepared with its 5' upstream sequence and the structural gene for luciferase were microinjected into fertilized eggs of medaka at the one-cell stage. Hatched fry were treated with 3-methylcholanthrene, and the transcription efficiency was assayed using competitive polymerase chain reaction analysis. Deletion of the region containing the five XREs, XRE(-6) to XRE(-2), and the point mutation of XRE(-1) reduced the inducible expressions by 75% and 56%, respectively, showing apparent dependency of the drug induction on the XREs. Constitutive expression, however, was not significantly affected by deletion or disruption of the XREs. When the region between XRE(-2) and XRE(-1) containing no XREs but three ERE motifs was internally deleted, the inducible expression and the constitutive expression were reduced by 88% and 75%, respectively. Replacement of this region with a partial fragment of eel CYP1A1 complementary DNA, with slight alteration of the distance between the five XREs and XRE(-1), reduced the inducible expression and the constitutive expression by 91% and 60%, respectively. These results strongly suggest that not only XRE but

[The Role of 5-Aza-CdR on Methylation of Promoter in RASSF1A Gene in Endometrial Carcinoma].

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Huang, Li-ping; Chen, Chen; Wang, Xue-ping; Liu, Hui

2015-05-01

To explore the effect of demethylating drug 5-Aza-2'-deoxycytidine (5-Aza-CdR) on methtylation status of the Ras-association domain familylA gene (RASSF1A) in human endometrial carcinoma. Randomly'assign the human endometrial carcinoma cell line HEC-1-B into groups and use demethylating drug 5-Aza-CdR of different concentration to treat them. Then Methylation-specific polymerase chain reaction (MSP), real-time PCR, Western blot, TUNEL technology were used to analyze methylation status of RASSF1A promoter CpG islands, RASSF1A mRNA expression, RASSF1A protein expression and apoptosis of HEC-1-B cell. High DNA methylation in RASSF1A gene promoter region, low RASSF1A mRNA level and protein expression and out of control of human endometrial carcinoma cell HEC-1-B apoptosis were observed. 5-Aza-CdR of different concentration could reverse RASSF1A gene's methylation status, recover the expression of mRNA and protein, and control the growth of HEC-1-B by inducing apoptosis. Aberrant methylation of RASSF1A in endometrial cancer as a therapeutic target, demethylating agent 5-Aza-CdR could be an effective way of gene therapy.

Mechanism of estrogen-mediated attenuation of hepatic injury following trauma-hemorrhage: Akt-dependent HO-1 up-regulation.

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Hsu, Jun-Te; Kan, Wen-Hong; Hsieh, Chi-Hsun; Choudhry, Mashkoor A; Schwacha, Martin G; Bland, Kirby I; Chaudry, Irshad H

2007-10-01

Protein kinase B (Akt) is known to be involved in proinflammatory and chemotactic events in response to injury. Akt activation also leads to the induction of heme oxygenase (HO)-1. Up-regulation of HO-1 mediates potent, anti-inflammatory effects and attenuates organ injury. Although studies have shown that 17beta-estradiol (E2) prevents organ damage following trauma-hemorrhage, it remains unknown whether Akt/HO-1 plays any role in E2-mediated attenuation of hepatic injury following trauma-hemorrhage. To study this, male rats underwent trauma-hemorrhage (mean blood pressure, approximately 40 mmHg for 90 min), followed by fluid resuscitation. At the onset of resuscitation, rats were treated with vehicle, E2 (1 mg/kg body weight), E2 plus the PI-3K inhibitor (Wortmannin), or the estrogen receptor (ER) antagonist (ICI 182,780). At 2 h after sham operation or trauma-hemorrhage, plasma alpha-GST and hepatic tissue myeloperoxidase (MPO) activity, IL-6, TNF-alpha, ICAM-1, cytokine-induced neutrophil chemoattractant-1, and MIP-2 levels were measured. Hepatic Akt and HO-1 protein levels were also determined. Trauma-hemorrhage increased hepatic injury markers (alpha-GST and MPO activity), cytokines, ICAM-1, and chemokine levels. These parameters were markedly improved in the E2-treated rats following trauma-hemorrhage. E2 treatment also increased hepatic Akt activation and HO-1 expression compared with vehicle-treated, trauma-hemorrhage rats, which were abolished by coadministration of Wortmannin or ICI 182,780. These results suggest that the salutary effects of E2 on hepatic injury following trauma-hemorrhage are in part mediated via an ER-related, Akt-dependent up-regulation of HO-1.

Magnetic excitations in Ho2Co17 and Ho2Fe17

International Nuclear Information System (INIS)

Clausen, K.N.

1981-01-01

The low energy part ( 2 Co 17 and Ho 2 Fe 17 have been measured along the three high symmetry directions at a temperature of 4.2 K, using the inelastic neutron scattering technique. The resulting magnon dispersion relations have been interpreted using linear spin wave theory with a Hamiltonian including single ion crystal field anisotropy and isotropic exchange between spatially well localized spins. The R 2 T 17 structure contains two different Ho sites, with the same point symmetry, and from the spin wave results it was concluded that the crystal field anisotropy of the two Ho sites in both Ho 2 Co 17 and Ho 2 Fe 17 were identical. The deduced crystal field parameters for Ho 2 Fe 17 were slightly larger than for Ho 2 Co 17 , and the parameters were of the same order of magnitude as for pure Ho. For Ho 2 Fe 17 the Fe-Fe exchange was found to be anisotropic, and for both compounds the magnetic ordering temperatures of 1178 K for Ho 2 Co 17 and 335 K for Ho 2 Fe 17 were determined by the strong positive 3d-3d exchange. (Auth.)

Promoter for the late gene encoding Vp5 of herpes simplex virus type 1 is recognized by cell extracts derived from uninfected cells

International Nuclear Information System (INIS)

Chisholm, G.E.; Summers, W.C.

1986-01-01

The ability of whole-cell extracts from unidentified HeLa cells to recognize the promoter for the herpes simplex virus type 1 late gene encoding the major capsid protein Vp5 was investigated by using both in vitro transcriptional and S1 nuclease protection analysis. This gene promoter was recognized by the cell extracts and produced abundant amounts of transcript in the absence of any other virus-encoded factors. This transcript was shown to arise, in vitro, from specific initiation at or very near the physiological mRNA start site. Thus, it appears that cell extracts from uninfected HeLa cells can efficiently recognize both early- and late-gene promoters

Gene therapy strategy for long-term myocardial protection using adeno-associated virus-mediated delivery of heme oxygenase gene.

Science.gov (United States)

Melo, Luis G; Agrawal, Reitu; Zhang, Lunan; Rezvani, Mojgan; Mangi, Abeel A; Ehsan, Afshin; Griese, Daniel P; Dell'Acqua, Giorgio; Mann, Michael J; Oyama, Junichi; Yet, Shaw-Fang; Layne, Matthew D; Perrella, Mark A; Dzau, Victor J

2002-02-05

Ischemia and oxidative stress are the leading mechanisms for tissue injury. An ideal strategy for preventive/protective therapy would be to develop an approach that could confer long-term transgene expression and, consequently, tissue protection from repeated ischemia/reperfusion injury with a single administration of a therapeutic gene. In the present study, we used recombinant adeno-associated virus (rAAV) as a vector for direct delivery of the cytoprotective gene heme oxygenase-1 (HO-1) into the rat myocardium, with the purpose of evaluating this strategy as a therapeutic approach for long-term protection from ischemia-induced myocardial injury. Human HO-1 gene (hHO-1) was delivered to normal rat hearts by intramyocardial injection. AAV-mediated transfer of the hHO-1 gene 8 weeks before acute coronary artery ligation and release led to a dramatic reduction (>75%) in left ventricular myocardial infarction. The reduction in infarct size was accompanied by decreases in myocardial lipid peroxidation and in proapoptotic Bax and proinflammatory interleukin-1beta protein abundance, concomitant with an increase in antiapoptotic Bcl-2 protein level. This suggested that the transgene exerts its cardioprotective effects in part by reducing oxidative stress and associated inflammation and apoptotic cell death. This study documents the beneficial therapeutic effect of rAAV-mediated transfer, before myocardial injury, of a cytoprotective gene that confers long-term myocardial protection from ischemia/reperfusion injury. Our data suggest that this novel "pre-event" gene transfer approach may provide sustained tissue protection from future repeated episodes of injury and may be beneficial as preventive therapy for patients with or at risk of developing coronary ischemic events.

Promoter polymorphisms of ST3GAL4 and ST6GAL1 genes and associations with risk of premalignant and malignant lesions of the cervix.

Science.gov (United States)

Rivera-Juarez, Maria de Los Angeles; Rosas-Murrieta, Nora Hilda; Mendieta-Carmona, Victoriano; Hernandez-Pacheco, Raquel Esneidy; Zamora-Ginez, Irma; Rodea-Avila, Carlos; Apresa-Garcia, Teresa; Garay-Villar, Onix; Aguilar-Lemarroy, Adriana; Jave-Suarez, Luis Felipe; Diaz-Orea, Maria Alicia; Milflores-Flores, Lorena; Reyes-Salinas, Juan Salvador; Ceja-Utrera, Francisco Javier; Vazquez-Zamora, Victor Javier; Vargas-Maldonado, Tomas; Reyes-Carmona, Sandra; Sosa-Jurado, Francisca; Santos-Lopez, Gerardo; Reyes-Leyva, Julio; Vallejo-Ruiz, Veronica

2014-01-01

Sialyltransferase gene expression is altered in several cancers, including examples in the cervix. Transcriptional regulation of the responsible genes depends on different promoters. We aimed to determine the association of single-nucleotide polymorphisms in the B3 promoter of the ST3GAL4 gene and the P1 promoter of the ST6GAL1 gene with cervical premalignant lesions or cervical cancer. A blood sample and/or cervical scrapes were obtained from 104 women with normal cytology, 154 with premalignant lesions and 100 with cervical cancer. We also included 119 blood samples of random donors. The polymorphisms were identified by sequencing from PCR products. For the B3 promoter, a fragment of 506 bp (from nucleotide -408 to +98) was analyzed, and for the P1 promoter a 490 bp (-326 to +164) fragment. The polymorphism analysis showed that at SNP rs10893506, genotypes CC and CT of the ST3GAL4 B3 promoter were associated with the presence of premalignant lesions (OR=2.89; 95%CI 1.72-4.85) and cervical cancer (OR=2.23; 95%CI 1.27-3.91). We detected only one allele of each polymorphism in the ST6GAL1 P1 promoter. We did not detect any genetic variability in the P1 promoter region in our study population. Our results suggest that the rs10893506 polymorphism -22C/T may increase susceptibility to premalignant and malignant lesions of the cervix.

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

Science.gov (United States)

Hall, Jessica A.; Tabata, Mitsuhisa; Rodgers, Joseph T.

2014-01-01

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes. PMID:24694308

Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

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Farré Domènec

2007-12-01

Full Text Available Abstract Background The arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes. Results We observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters. Conclusion The study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

International Nuclear Information System (INIS)

Lawal, Akeem O.; Zhang, Min; Dittmar, Michael; Lulla, Aaron; Araujo, Jesus A.

2015-01-01

Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4 h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNA or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25 μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM. - Highlights: • We examined the role of HO-1 expression on diesel exhaust particle (DEP) in endothelial cells. • DEPs exert cytotoxic and inflammatory effects on human microvascular endothelial cells (HMECs). • DEPs induce HO-1 expression in HMECs. • HO-1 protects against the oxidative stress induced by DEps. • HO-1 attenuates the proinflammatory effects

Heme oxygenase-1 protects endothelial cells from the toxicity of air pollutant chemicals

Energy Technology Data Exchange (ETDEWEB)

Lawal, Akeem O.; Zhang, Min; Dittmar, Michael [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Lulla, Aaron [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Molecular Toxicology Interdepartmental Program, University of California, Los Angeles (United States); Araujo, Jesus A., E-mail: JAraujo@mednet.ucla.edu [Division of Cardiology, David Geffen School of Medicine, University of California, Los Angeles, 10833 Le Conte Avenue, CHS 43-264, Los Angeles, CA 90095 (United States); Molecular Toxicology Interdepartmental Program, University of California, Los Angeles (United States); Molecular Biology Institute, University of California, Los Angeles (United States)

2015-05-01

Diesel exhaust particles (DEPs) are a major component of diesel emissions, responsible for a large portion of their toxicity. In this study, we examined the toxic effects of DEPs on endothelial cells and the role of DEP-induced heme oxygenase-1 (HO-1) expression. Human microvascular endothelial cells (HMECs) were treated with an organic extract of DEPs from an automobile engine (A-DEP) or a forklift engine (F-DEP) for 1 and 4 h. ROS generation, cell viability, lactate dehydrogenase leakage, expression of HO-1, inflammatory genes, cell adhesion molecules and unfolded protein respone (UPR) gene were assessed. HO-1 expression and/or activity were inhibited by siRNA or tin protoporphyrin (Sn PPIX) and enhanced by an expression plasmid or cobalt protoporphyrin (CoPPIX). Exposure to 25 μg/ml of A-DEP and F-DEP significantly induced ROS production, cellular toxicity and greater levels of inflammatory and cellular adhesion molecules but to a different degree. Inhibition of HO-1 enzymatic activity with SnPPIX and silencing of the HO-1 gene by siRNA enhanced DEP-induced ROS production, further decreased cell viability and increased expression of inflammatory and cell adhesion molecules. On the other hand, overexpression of the HO-1 gene by a pcDNA 3.1D/V5-HO-1 plasmid significantly mitigated ROS production, increased cell survival and decreased the expression of inflammatory genes. HO-1 expression protected HMECs from DEP-induced prooxidative and proinflammatory effects. Modulation of HO-1 expression could potentially serve as a therapeutic target in an attempt to inhibit the cardiovascular effects of ambient PM. - Highlights: • We examined the role of HO-1 expression on diesel exhaust particle (DEP) in endothelial cells. • DEPs exert cytotoxic and inflammatory effects on human microvascular endothelial cells (HMECs). • DEPs induce HO-1 expression in HMECs. • HO-1 protects against the oxidative stress induced by DEps. • HO-1 attenuates the proinflammatory effects

Tumor Restrictive Suicide Gene Therapy for Glioma Controlled by the FOS Promoter.

Directory of Open Access Journals (Sweden)

Jianqing Pan

Full Text Available Effective suicide gene delivery and expression are crucial to achieving successful effects in gene therapy. An ideal tumor-specific promoter expresses therapeutic genes in tumor cells with minimal normal tissue expression. We compared the activity of the FOS (FBJ murine osteosarcoma viral oncogene homolog promoter with five alternative tumor-specific promoters in glioma cells and non-malignant astrocytes. The FOS promoter caused significantly higher transcriptional activity in glioma cell lines than all alternative promoters with the exception of CMV. The FOS promoter showed 13.9%, 32.4%, and 70.8% of the transcriptional activity of CMV in three glioma cell lines (U87, U251, and U373. Importantly, however, the FOS promoter showed only 1.6% of the transcriptional activity of CMV in normal astrocytes. We also tested the biologic activity of recombinant adenovirus containing the suicide gene herpes simplex virus thymidine kinase (HSV-tk driven by the FOS promoter, including selective killing efficacy in vitro and tumor inhibition rate in vivo. Adenoviral-mediated delivery of the HSV-tk gene controlled by the FOS promoter conferred a cytotoxic effect on human glioma cells in vitro and in vivo. This study suggests that use of the FOS-tk adenovirus system is a promising strategy for glioma-specific gene therapy but still much left for improvement.

Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in mice

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Fu Na

2010-10-01

Full Text Available Abstract Objective Heme oxygenase-1 (HO-1, the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant properties. However, the role of HO-1 on hepatocyte apoptosis remains unclear. We aim to elucidate the effects of HO-1 on oxidative stress related hepatocellular apoptosis in nutritional steatohepatitis in mice. Methods C57BL/6J mice were fed with methionine-choline deficient (MCD diet for four weeks to induce hepatic steatohepatitis. HO-1 chemical inducer (hemin, HO-1 chemical inhibitor zinc protoporphyrin IX (ZnPP-IX and/or adenovirus carrying HO-1 gene (Ad-HO-1 were administered to mice, respectively. Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL assay, the mRNA and protein expression of apoptosis related genes were assayed by quantitative real-time PCR and Western blot. Results Hepatocyte signs of oxidative related apoptotic injury were presented in mice fed with MCD diet for 4 weeks. Induction of HO-1 by hemin or Ad-HO-1 significantly attenuated the severity of liver histology, which was associated with decreased hepatic lipid peroxidation content, reduced number of apoptotic cells by TUNEL staining, down-regulated expression of pro-apoptosis related genes including Fas/FasL, Bax, caspase-3 and caspase-9, reduced expression of cytochrome p4502E1 (CYP2E1, inhibited cytochrome c (Cyt-c release, and up-regulated expression of anti-apoptosis gene Bcl-2. Whereas, inhibition of HO-1 by ZnPP-IX caused oxidative stress related hepatic injury, which concomitant with increased number of TUNEL positive cells and up-regulated expression of pro-apoptosis related genes. Conclusions The present study provided evidences for the protective role of HO-1 in preventing nutritional steatohepatitis through suppressing hepatocyte apoptosis in mice.

Regulatory polymorphisms in the bovine Ankyrin 1 gene promoter are associated with tenderness and intra-muscular fat content

LENUS (Irish Health Repository)

Aslan, Ozlem

2010-12-15

Abstract Background Recent QTL and gene expression studies have highlighted ankyrins as positional and functional candidate genes for meat quality. Our objective was to characterise the promoter region of the bovine ankyrin 1 gene and to test polymorphisms for association with sensory and technological meat quality measures. Results Seven novel promoter SNPs were identified in a 1.11 kb region of the ankyrin 1 promoter in Angus, Charolais and Limousin bulls (n = 15 per breed) as well as 141 crossbred beef animals for which meat quality data was available. Eighteen haplotypes were inferred with significant breed variation in haplotype frequencies. The five most frequent SNPs and the four most frequent haplotypes were subsequently tested for association with sensory and technological measures of meat quality in the crossbred population. SNP1, SNP3 and SNP4 (which were subsequently designated regulatory SNPs) and SNP5 were associated with traits that contribute to sensorial and technological measurements of tenderness and texture; Haplotype 1 and haplotype 4 were oppositely correlated with traits contributing to tenderness (P < 0.05). While no single SNP was associated with intramuscular fat (IMF), a clear association with increased IMF and juiciness was observed for haplotype 2. Conclusion The conclusion from this study is that alleles defining haplotypes 2 and 4 could usefully contribute to marker SNP panels used to select individuals with improved IMF\\/juiciness or tenderness in a genome-assisted selection framework.

Relationship of interleukin-1B gene promoter region polymorphism with Helicobacter pylori infection and gastritis.

Science.gov (United States)

Ramis, Ivy Bastos; Vianna, Júlia Silveira; Halicki, Priscila Cristina Bartolomeu; Lara, Caroline; Tadiotto, Thássia Fernanda; da Silva Maciel, João Batista; Gonçalves, Carla Vitola; von Groll, Andrea; Dellagostin, Odir Antônio; da Silva, Pedro Eduardo Almeida

2015-09-29

Helicobacter pylori infection is associated with gastritis, peptic ulcer disease and gastric carcinoma. The severity of damage is determined by the interplay between environmental/behavioral factors, bacterial pathogenicity genes and host genetic polymorphisms that can influence the secretion levels of inflammatory cytokines. Accordingly, this study aimed to identify polymorphisms in the IL-1B and IL-1RN genes and their associations with H. pylori infection, cagA gene of H. pylori, and gastroduodenal diseases. Gastric biopsy samples from 151 patients infected with H. pylori and 76 uninfected individuals were analyzed. H. pylori infection was diagnosed by histology and PCR. Polymorphisms at positions -511, -31 and +3954 of the IL-1B gene were detected by PCR-RFLP, and an analysis of the VNTR polymorphism of the IL-1RN gene was performed by PCR. It was observed that the presence of the T/T genotype at position -511 and the C/C genotype at position -31 were associated with H. pylori infection and with an increased risk of gastritis in H. pylori-positive patients. Additionally, strains from patients H. pylori-positive carrying the cagA gene was significantly related with the T/T genotype at position -511 of IL-1B.  No association of polymorphisms at position +3954 of IL-1B and in the IL-1RN with H. pylori infection and with risk of severe gastric diseases was found. We demonstrated that polymorphisms in the promoter region of the IL-1B gene (at positions -511 and -31) are associated with an enhanced risk of H. pylori infection as well as gastritis in H. pylori-positive patients.

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

International Nuclear Information System (INIS)

Kurayoshi, Kenta; Ozono, Eiko; Iwanaga, Ritsuko; Bradford, Andrew P.; Komori, Hideyuki; Ohtani, Kiyoshi

2014-01-01

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

Cancer cell specific cytotoxic gene expression mediated by ARF tumor suppressor promoter constructs

Energy Technology Data Exchange (ETDEWEB)

Kurayoshi, Kenta [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan); Ozono, Eiko [Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary, University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ (United Kingdom); Iwanaga, Ritsuko; Bradford, Andrew P. [Department of Obstetrics and Gynecology, University of Colorado School of Medicine, Anschutz Medical Campus, 12800 East 19th Avenue, Aurora, CO 80045 (United States); Komori, Hideyuki [Center for Stem Cell Biology, Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109 (United States); Ohtani, Kiyoshi, E-mail: btm88939@kwansei.ac.jp [Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337 (Japan)

2014-07-18

Highlights: • ARF promoter showed higher responsiveness to deregulated E2F activity than the E2F1 promoter. • ARF promoter showed higher cancer cell-specificity than E2F1 promoter to drive gene expression. • HSV-TK driven by ARF promoter showed higher cancer cell-specific cytotoxicity than that driven by E2F1 promoter. - Abstract: In current cancer treatment protocols, such as radiation and chemotherapy, side effects on normal cells are major obstacles to radical therapy. To avoid these side effects, a cancer cell-specific approach is needed. One way to specifically target cancer cells is to utilize a cancer specific promoter to express a cytotoxic gene (suicide gene therapy) or a viral gene required for viral replication (oncolytic virotherapy). For this purpose, the selected promoter should have minimal activity in normal cells to avoid side effects, and high activity in a wide variety of cancers to obtain optimal therapeutic efficacy. In contrast to the AFP, CEA and PSA promoters, which have high activity only in a limited spectrum of tumors, the E2F1 promoter exhibits high activity in wide variety of cancers. This is based on the mechanism of carcinogenesis. Defects in the RB pathway and activation of the transcription factor E2F, the main target of the RB pathway, are observed in almost all cancers. Consequently, the E2F1 promoter, which is mainly regulated by E2F, has high activity in wide variety of cancers. However, E2F is also activated by growth stimulation in normal growing cells, suggesting that the E2F1 promoter may also be highly active in normal growing cells. In contrast, we found that the tumor suppressor ARF promoter is activated by deregulated E2F activity, induced by forced inactivation of pRB, but does not respond to physiological E2F activity induced by growth stimulation. We also found that the deregulated E2F activity, which activates the ARF promoter, is detected only in cancer cell lines. These observations suggest that ARF promoter

Reduced caveolin-1 promotes hyper-inflammation due to abnormal heme oxygenase-1 localizationin LPS challenged macrophages with dysfunctional CFTR

Science.gov (United States)

Zhang, Ping-Xia; Murray, Thomas S.; Villella, Valeria Rachela; Ferrari, Eleonora; Esposito, Speranza; D'Souza, Anthony; Raia, Valeria; Maiuri, Luigi; Krause, Diane S.; Egan, Marie E.; Bruscia, Emanuela M.

2013-01-01

We have previously reported that TLR4 signaling is increased in lipopolysaccharide (LPS) -stimulated Cystic Fibrosis (CF) macrophages (MΦs), contributing to the robust production of pro-inflammatory cytokines. The heme oxygenase (HO-1)/carbon monoxide (CO) pathway modulates cellular redox status, inflammatory responses, and cell survival. The HO-1 enzyme, together with the scaffold protein caveolin 1 (CAV-1), also acts as a negative regulator of TLR4 signaling in MΦs. Here, we demonstrate that in LPS-challenged CF MΦs, HO-1 does not compartmentalize normally to the cell surface and instead accumulates intracellularly. The abnormal HO-1 localization in CF MΦs in response to LPS is due to decreased CAV-1 expression, which is controlled by the cellular oxidative state, and is required for HO-1 delivery to the cell surface. Overexpression of HO-1 or stimulating the pathway with CO-releasing molecules (CORM2)enhancesCAV-1 expression in CF MΦs, suggesting a positive-feed forward loop between HO-1/CO induction and CAV-1 expression. These manipulations reestablished HO-1 and CAV-1 cell surface localization in CF MΦ's. Consistent with restoration of HO-1/CAV-1 negative regulation of TLR4 signaling, genetic or pharmacological (CORM2)-induced enhancement of this pathway decreased the inflammatory response of CF MΦs and CF mice treated with LPS. In conclusion, our results demonstrate that the counter-regulatory HO-1/CO pathway, which is critical in balancing and limiting the inflammatory response, is defective in CF MΦs through a CAV-1-dependent mechanism, exacerbating the CF MΦ's response to LPS. This pathway could be a potential target for therapeutic intervention for CF lung disease. PMID:23606537

Duodenal ulcer promoting gene of Helicobacter pylori.

Science.gov (United States)

Lu, Hong; Hsu, Ping-I; Graham, David Y; Yamaoka, Yoshio

2005-04-01

Identification of a disease-specific H pylori virulence factors predictive of the outcome of infection remains unachieved. We used the polymerase chain reaction and Southern blot to compare the presence of 14 vir homologue genes with clinical presentation of H pylori infection, mucosal histology, and mucosal interleukin (IL)-8 levels. We examined 500 H pylori strains from East Asia and South America, including 120 with gastritis, 140 with duodenal ulcer (DU), 110 with gastric ulcer (GU), and 130 with gastric cancer. Only 1 gene that encompassed both jhp0917 and jhp0918 called dupA (duodenal ulcer promoting gene) was associated with a specific clinical outcome. dupA was present in 42% of DU vs. 21% of gastritis (adjusted odds ratio [OR] = 3.1, 95% confidence interval [CI]: 1.7-5.7). Its presence was also associated with more intense antral neutrophil infiltration and IL-8 levels and was a marker for protection against gastric atrophy, intestinal metaplasia, and gastric cancer (OR for gastric cancer = 0.42, 95% CI: 0.2-0.9 compared with gastritis). In vitro studies in gastric epithelial cells using dupA -deleted and -complemented mutants showed that the dupA plays roles in IL-8 production, in activation of transcription factors responsible for IL-8 promoter activity, and in increased survivability at low pH. dupA is a novel marker associated with an increased risk for DU and reduced risk for gastric atrophy and cancer. Its association with DU-promoting and -protective effects against atrophy/cancer was evident in both Asian and Western countries.

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells.

Science.gov (United States)

Foresti, Roberta; Bains, Sandip K; Pitchumony, Tamil Selvi; de Castro Brás, Lisandra E; Drago, Filippo; Dubois-Randé, Jean-Luc; Bucolo, Claudio; Motterlini, Roberto

2013-10-01

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5-20 μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration. Copyright © 2013 Elsevier Ltd. All rights reserved.

Heterologous gene expression driven by carbonic anhydrase gene promoter in Dunaliella salina

Science.gov (United States)

Yurong, Chai; Yumin, Lu; Tianyun, Wang; Weihong, Hou; Lexun, Xue

2006-12-01

Dunaliella salina, a halotolerant unicellular green alga without a rigid cell wall, can live in salinities ranging from 0.05 to 5 mol/L NaCl. These features of D. salina make it an ideal host for the production of antibodies, oral vaccine, and commercially valuable polypeptides. To produce high level of heterologous proteins from D. salina, highly efficient promoters are required to drive expression of target genes under controlled condition. In the present study, we cloned a 5' franking region of 1.4 kb from the carbonic anhydrase ( CAH) gene of D. salina by genomic walking and PCR. The fragment was ligated to the pMD18-T vector and characterized. Sequence analysis indicated that this region contained conserved motifs, including a TATA- like box and CAAT-box. Tandem (GT)n repeats that had a potential role of transcriptional control, were also found in this region. The transcription start site (TSS) of the CAH gene was determined by 5' RACE and nested PCR method. Transformation assays showed that the 1.4 kb fragment was able to drive expression of the selectable bar (bialaphos resistance) gene when the fusion was transformed into D. salina by biolistics. Northern blotting hybridizations showed that the bar transcript was most abundant in cells grown in 2 mol/L NaCl, and less abundant in 0.5 mol/L NaCl, indicating that expression of the bar gene was induced at high salinity. These results suggest the potential use of the CAH gene promoter to induce the expression of heterologous genes in D. salina under varied salt condition.

Fatty acid elongase 1 (FAE1) promoter as a candidate for genetic ...

African Journals Online (AJOL)

As an important cis-regulatory element, a promoter plays a key role in plant gene expression and regulation, and has been widely used in plant genetic engineering. The fatty acid elongase 1 (FAE1) promoter was isolated from Arabidopsis thaliana. Sequence analysis showed that the FAE1 promoter contains two Skn-1 ...

Magnetic properties and magnetocaloric effect in the HoNi1−xCuxIn (x=0, 0.1, 0.3, 0.4) intermetallic compounds

International Nuclear Information System (INIS)

Mo, Zhao-Jun; Shen, Jun; Yan, Li-Qin; Tang, Cheng-Chun; He, Xiao-Nan; Zheng, Xinqi; Wu, Jian-Feng; Sun, Ji-Rong; Shen, Bao-Gen

2014-01-01

The magnetic properties and magnetocaloric effect (MCE) in HoNi 1−x Cu x In (x=0, 0.1, 0.3, 0.4) compounds have been investigated. With the substitution of Cu for Ni, the Ho magnetic moment will cant from the c-axis, and form a complicated magnetic structure. These compounds exhibit two successive magnetic transitions with the increase in temperature. The large reversible magnetocaloric effects have been observed in HoNi 1−x Cu x In compounds around T ord , with no thermal and magnetic hysteresis loss. The large reversible isothermal magnetic entropy change (−ΔS M ) is 20.2 J/kg K and the refrigeration capacity (RC) reaches 356.7 J/kg for field changes of 5 T for HoNi 0.7 Cu 0.3 In. Especially, the value of −ΔS M (12.5 J/kg K) and the large RC (132 J/kg) are observed for field changes of 2 T for HoNi 0.9 Cu 0.1 In. Additionally, the values of RC are improved to 149 J/K for the field changes of 2 T due to a wide temperature span for the mix of HoNi 0.9 Cu 0.1 In and HoNi 0.7 Cu 0.3 In compounds with the mass ratio of 1:1. These compounds with excellent MCE are expected to have effective applications in magnetic refrigeration around 20 K. - Highlights: • For magnetic-field changes of 2 T, the values of RC are improved to 149 J/K. • MCEs of these compounds show no thermal and magnetic hysteresis. • Compounds show two successive magnetic transitions with the increase in temperature. • With the substitution of Cu for Ni, compounds form a complicated magnetic structure

Epigenetic Alteration by DNA Methylation of ESR1, MYOD1 and hTERT Gene Promoters is Useful for Prediction of Response in Patients of Locally Advanced Invasive Cervical Carcinoma Treated by Chemoradiation.

Science.gov (United States)

Sood, S; Patel, F D; Ghosh, S; Arora, A; Dhaliwal, L K; Srinivasan, R

2015-12-01

Locally advanced invasive cervical cancer [International Federation of Gynecology and Obstetrics (FIGO) IIB/III] is treated by chemoradiation. The response to treatment is variable within a given FIGO stage. Therefore, the aim of the present study was to evaluate the gene promoter methylation profile and corresponding transcript expression of a panel of six genes to identify genes which could predict the response of patients treated by chemoradiation. In total, 100 patients with invasive cervical cancer in FIGO stage IIB/III who underwent chemoradiation treatment were evaluated. Ten patients developed systemic metastases during therapy and were excluded. On the basis of patient follow-up, 69 patients were chemoradiation-sensitive, whereas 21 were chemoradiation-resistant. Gene promoter methylation and gene expression was determined by TaqMan assay and quantitative real-time PCR, respectively, in tissue samples. The methylation frequency of ESR1, BRCA1, RASSF1A, MLH1, MYOD1 and hTERT genes ranged from 40 to 70%. Univariate and hierarchical cluster analysis revealed that gene promoter methylation of MYOD1, ESR1 and hTERT could predict for chemoradiation response. A pattern of unmethylated MYOD1, unmethylated ESR1 and methylated hTERT promoter as well as lower ESR1 transcript levels predicted for chemoradiation resistance. Methylation profiling of a panel of three genes that includes MYOD1, ESR1 and hTERT may be useful to predict the response of invasive cervical carcinoma patients treated with standard chemoradiation therapy. Copyright © 2015 The Royal College of Radiologists. Published by Elsevier Ltd. All rights reserved.

Genes that cooperate with tumor promoters in transformation

International Nuclear Information System (INIS)

Colburn, N.H.; Smith, B.M.

1988-01-01

Tumor-promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro-1 confers lifespan extension of these cells. Recently, we have found tat a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JPB P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced syntheses of two proteins of 15 and 16 kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation

Quercetin Protects Primary Human Osteoblasts Exposed to Cigarette Smoke through Activation of the Antioxidative Enzymes HO-1 and SOD-1

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Karl F. Braun

2011-01-01

Full Text Available Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS. The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO- 1 and superoxide-dismutase- (SOD- 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.

Crystal structure and magnetic state of pseudo-binary intermetallic compounds Ho(Cosub(1-x)Nisub(x))sub(5)

International Nuclear Information System (INIS)

Chuev, V.V.; Kelarev, V.V.; Pirogov, A.N.; Sidorov, S.K.; Koryakova, V.S.

1983-01-01

In the range of 1.8-1000 K intermetallic compounds Ho(Cosub(1-x)Nisub(x))sub(5) have been investigated neutronographically and roentgenographically. Crystal structure of two series of samples: HoCosub(5.5-5.5x)Nisub(5x) and HoCosub(5-5x)Nisub(5x) is studied. It is shown that Ni atoms mainly occupy positions 2c, Co atoms - positions 3g; coordinates of atoms and position occupation of TbCu 7 type structure are specified. Analysis of magnetic structure is made, angles of magnetic momenta orientation as to crystallographic axes are determined. Magnetic phase diagram is built. Concentrational dependences of sublattice magnetization: Msub(Ho)(x), Mdsub(2c)(x), Mdsub(3g)(x) are determined

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

International Nuclear Information System (INIS)

Kusunoki, Chisato; Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi; Nishio, Yoshihiko; Kashiwagi, Atsunori; Maegawa, Hiroshi

2013-01-01

Highlights: ► Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. ► EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. ► Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. ► Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid (ω3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of ω3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with ω3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). ω3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with ω3-PUFA prevented H 2 O 2 -induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

Catalase overexpression prevents nuclear factor erythroid 2-related factor 2 stimulation of renal angiotensinogen gene expression, hypertension, and kidney injury in diabetic mice.

Science.gov (United States)

Abdo, Shaaban; Shi, Yixuan; Otoukesh, Abouzar; Ghosh, Anindya; Lo, Chao-Sheng; Chenier, Isabelle; Filep, Janos G; Ingelfinger, Julie R; Zhang, Shao Ling; Chan, John S D

2014-10-01

This study investigated the impact of catalase (Cat) overexpression in renal proximal tubule cells (RPTCs) on nuclear factor erythroid 2-related factor 2 (Nrf2) stimulation of angiotensinogen (Agt) gene expression and the development of hypertension and renal injury in diabetic Akita transgenic mice. Additionally, adult male mice were treated with the Nrf2 activator oltipraz with or without the inhibitor trigonelline. Rat RPTCs, stably transfected with plasmid containing either rat Agt or Nrf2 gene promoter, were also studied. Cat overexpression normalized systolic BP, attenuated renal injury, and inhibited RPTC Nrf2, Agt, and heme oxygenase-1 (HO-1) gene expression in Akita Cat transgenic mice compared with Akita mice. In vitro, high glucose level, hydrogen peroxide, and oltipraz stimulated Nrf2 and Agt gene expression; these changes were blocked by trigonelline, small interfering RNAs of Nrf2, antioxidants, or pharmacological inhibitors of nuclear factor-κB and p38 mitogen-activated protein kinase. The deletion of Nrf2-responsive elements in the rat Agt gene promoter abolished the stimulatory effect of oltipraz. Oltipraz administration also augmented Agt, HO-1, and Nrf2 gene expression in mouse RPTCs and was reversed by trigonelline. These data identify a novel mechanism, Nrf2-mediated stimulation of intrarenal Agt gene expression and activation of the renin-angiotensin system, by which hyperglycemia induces hypertension and renal injury in diabetic mice. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

N-myc downstream-regulated gene 1 promotes oxaliplatin-triggered apoptosis in colorectal cancer cells via enhancing the ubiquitination of Bcl-2.

Science.gov (United States)

Yang, Xiao; Zhu, Fan; Yu, Chaoran; Lu, Jiaoyang; Zhang, Luyang; Lv, Yanfeng; Sun, Jing; Zheng, Minhua

2017-07-18

N-myc downstream-regulated gene1 (NDRG1) has been identified as a potent tumor suppressor gene. The molecular mechanisms of anti-tumor activity of NDRG1 involve its suppressive effects on a variety of tumorigenic signaling pathways. The purpose of this study was to investigate the role of NDRG1 in the apoptosis of colorectal cancer (CRC) cells. We first collected the clinical data of locally advanced rectal cancer (LARC) patients receiving oxaliplatin-based neoadjuvant chemotherapy in our medical center. Correlation analysis revealed that NDRG1 positively associated with the downstaging rates and prognosis of patients. Then, the effects of over-expression and depletion of NDRG1 gene on apoptosis of colorectal cancer were tested in vitro and in vivo. NDRG1 over-expression promoted apoptosis in colorectal cancer cells whereas depletion of NDRG1 resulted in resistance to oxaliplatin treatment. Furthermore, we observed that Bcl-2, a major anti-apoptotic protein, was regulated by NDRG1 at post-transcriptional level. By binding Protein kinase Cα (PKCα), a classical regulating factor of Bcl-2, NDRG1 enhanced the ubiquitination and degradation of Bcl-2, thus promoting apoptosis in CRC cells. In addition, NDRG1 inhibited tumor growth and promoted apoptosis in mouse xenograft model. In conclusion,NDRG1 promotes oxaliplatin-triggered apoptosis in colorectal cancer. Therefore, colorectal cancer patients can be stratified by the expression level of NDRG1. NDRG1-positive patients may benefit from oxaliplatin-containing chemotherapy regimens whereas those with negative NDRG1 expression should avoid the usage of this cytotoxic drug.

Andrographolide protects liver cells from H2O2 induced cell death by upregulation of Nrf-2/HO-1 mediated via adenosine A2a receptor signalling.

Science.gov (United States)

Mittal, Smriti P K; Khole, Swati; Jagadish, Nidhi; Ghosh, Debjani; Gadgil, Vijay; Sinkar, Vilas; Ghaskadbi, Saroj S

2016-11-01

Andrographolide, principle constituent of Andrographis paniculata Nees is used in traditional medicine in Southeast Asia and is known to exhibit various biological activities. Its antioxidant activity is due to its ability to activate one of the antioxidant enzymes, heme oxygenase-1 (HO-1) which is regulated transcriptionally through Nrf-2. However, molecular mechanism underlying activation of Nrf-2/HO-1 has not yet been clearly understood. Protective effect of andrographolide against H2O2 induced cell death, reactive oxygen species and lipid peroxidation was observed in HepG2 cells. Ability of andrographolide to modulate G-protein coupled receptor (GPCR) mediated signalling was determined using in silico docking and gene expression was analyzed by qRT-PCR, confocal microscopy and western blot analysis. We clearly show that andrographolide via adenosine A2A receptor signalling leads to activation of p38 MAP kinase, resulting in upregulation of Nrf-2, its translocation to nucleus and activation of HO-1. Additionally, it activates adenylate cyclase resulting in cAMP formation which in turn activates protein kinase A leading to inhibition of GSK-3β by phosphorylation. Inactivated GSK-3β leads to retention of Nrf-2 in the nucleus leading to sustained expression of HO-1 by binding to its antioxidant response element (ARE). Thus, andrographolide probably by binding to adenosine A2a receptor activates Nrf-2 transcription and also inhibits its exclusion from the nucleus by inactivating GSK-3β, together resulting in activation of HO-1. We speculate that andrographolide can be used as a therapeutic drug to combat oxidative stress implicated in pathogenesis of various diseases such as diabetes, osteoporosis, neurodegenerative diseases etc. Copyright © 2016 Elsevier B.V. All rights reserved.

The role of germline promoters and I exons in cytokine-induced gene-specific class switch recombination.

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Dunnick, Wesley A; Shi, Jian; Holden, Victoria; Fontaine, Clinton; Collins, John T

2011-01-01

Germline transcription precedes class switch recombination (CSR). The promoter regions and I exons of these germline transcripts include binding sites for activation- and cytokine-induced transcription factors, and the promoter regions/I exons are essential for CSR. Therefore, it is a strong hypothesis that the promoter/I exons regions are responsible for much of cytokine-regulated, gene-specific CSR. We tested this hypothesis by swapping the germline promoter and I exons for the murine γ1 and γ2a H chain genes in a transgene of the entire H chain C-region locus. We found that the promoter/I exon for γ1 germline transcripts can direct robust IL-4-induced recombination to the γ2a gene. In contrast, the promoter/I exon for the γ2a germline transcripts works poorly in the context of the γ1 H chain gene, resulting in expression of γ1 H chains that is level. Nevertheless, the small amount of recombination to the chimeric γ1 gene is induced by IFN-γ. These results suggest that cytokine regulation of CSR, but not the magnitude of CSR, is regulated by the promoter/I exons.

The effects of lead exposure on the expression of HMGB1 and HO-1 in rats and PC12 cells.

Science.gov (United States)

Yang, Meiyuan; Li, Yaobin; Wang, Ying; Cheng, Nuo; Zhang, Yi; Pang, Shimin; Shen, Qiwei; Zhao, Lijuan; Li, Guilin; Zhu, Gaochun

2018-05-15

Lead (Pb) is an environmental neurotoxic metal. Chronic exposure to Pb causes deficits of learning and memory in children and spatial learning deficits in developing rats. In this study we investigated the effects of Pb exposure on the expression of HMGB1 and HO-1 in rats and PC12 cells. The animals were randomly divided to three groups: control group; low lead exposure group; high lead exposure group; PC12 cells were divided into 3 groups: 0 μM (control group), 1 μM and 100 μM Pb acetate. The results showed that Pb levels in blood and brain of Pb exposed groups were significantly higher than that of the control group (p < 0.05). The expression of HMGB1 and HO-1 were increased in Pb exposed groups than that of the control group (p < 0.05). Moreover, we found that the up-regulation of HO-1 in Pb exposure environment inhibited the expression of HMGB1. Copyright © 2018 Elsevier B.V. All rights reserved.

The electron capture in 163Ho experiment - ECHo

Science.gov (United States)

Gastaldo, L.; Blaum, K.; Chrysalidis, K.; Day Goodacre, T.; Domula, A.; Door, M.; Dorrer, H.; Düllmann, Ch. E.; Eberhardt, K.; Eliseev, S.; Enss, C.; Faessler, A.; Filianin, P.; Fleischmann, A.; Fonnesu, D.; Gamer, L.; Haas, R.; Hassel, C.; Hengstler, D.; Jochum, J.; Johnston, K.; Kebschull, U.; Kempf, S.; Kieck, T.; Köster, U.; Lahiri, S.; Maiti, M.; Mantegazzini, F.; Marsh, B.; Neroutsos, P.; Novikov, Yu. N.; Ranitzsch, P. C. O.; Rothe, S.; Rischka, A.; Saenz, A.; Sander, O.; Schneider, F.; Scholl, S.; Schüssler, R. X.; Schweiger, Ch.; Simkovic, F.; Stora, T.; Szücs, Z.; Türler, A.; Veinhard, M.; Weber, M.; Wegner, M.; Wendt, K.; Zuber, K.

2017-06-01

Neutrinos, and in particular their tiny but non-vanishing masses, can be considered one of the doors towards physics beyond the Standard Model. Precision measurements of the kinematics of weak interactions, in particular of the 3H β-decay and the 163Ho electron capture (EC), represent the only model independent approach to determine the absolute scale of neutrino masses. The electron capture in 163Ho experiment, ECHo, is designed to reach sub-eV sensitivity on the electron neutrino mass by means of the analysis of the calorimetrically measured electron capture spectrum of the nuclide 163Ho. The maximum energy available for this decay, about 2.8 keV, constrains the type of detectors that can be used. Arrays of low temperature metallic magnetic calorimeters (MMCs) are being developed to measure the 163Ho EC spectrum with energy resolution below 3 eV FWHM and with a time resolution below 1 μs. To achieve the sub-eV sensitivity on the electron neutrino mass, together with the detector optimization, the availability of large ultra-pure 163Ho samples, the identification and suppression of background sources as well as the precise parametrization of the 163Ho EC spectrum are of utmost importance. The high-energy resolution 163Ho spectra measured with the first MMC prototypes with ion-implanted 163Ho set the basis for the ECHo experiment. We describe the conceptual design of ECHo and motivate the strategies we have adopted to carry on the present medium scale experiment, ECHo-1K. In this experiment, the use of 1 kBq 163Ho will allow to reach a neutrino mass sensitivity below 10 eV/ c 2. We then discuss how the results being achieved in ECHo-1k will guide the design of the next stage of the ECHo experiment, ECHo-1M, where a source of the order of 1 MBq 163Ho embedded in large MMCs arrays will allow to reach sub-eV sensitivity on the electron neutrino mass.

Regulation of the Osem gene by abscisic acid and the transcriptional activator VP1: analysis of cis-acting promoter elements required for regulation by abscisic acid and VP1.

Science.gov (United States)

Hattori, T; Terada, T; Hamasuna, S

1995-06-01

Osem, a rice gene homologous to the wheat Em gene, which encodes one of the late-embryogenesis abundant proteins was isolated. The gene was characterized with respect to control of transcription by abscisic acid (ABA) and the transcriptional activator VP1, which is involved in the ABA-regulated gene expression during late embryo-genesis. A fusion gene (Osem-GUS) consisting of the Osem promoter and the bacterial beta-glucuronidase (GUS) gene was constructed and tested in a transient expression system, using protoplasts derived from a suspension-cultured line of rice cells, for activation by ABA and by co-transfection with an expression vector (35S-Osvp1) for the rice VP1 (OSVP1) cDNA. The expression of Osem-GUS was strongly (40- to 150-fold) activated by externally applied ABA and by over-expression of (OS)VP1. The Osem promoter has three ACGTG-containing sequences, motif A, motif B and motif A', which resemble the abscisic acid-responsive element (ABRE) that was previously identified in the wheat Em and the rice Rab16. There is also a CATGCATG sequence, which is known as the Sph box and is shown to be essential for the regulation by VP1 of the maize anthocyanin regulatory gene C1. Focusing on these sequence elements, various mutant derivatives of the Osem promoter in the transient expression system were assayed. The analysis revealed that motif A functions not only as an ABRE but also as a sequence element required for the regulation by (OS)VP1.

Synthesis and electrical characterization of BaZr0.9Ho0.1O3-δ electrolyte ceramic for IT - SOFCs

Science.gov (United States)

Saini, Deepash S.; Singh, Lalit K.; Bhattacharya, D.

2018-04-01

A cost-effective modified combustion method using citric acid and glycine has recently been developed to synthesize high quality, and nanosized BaZr0.9Ho0.1O3 ceramic powder. BaZr0.9Ho0.1O3-δ ceramic powder was characterized by X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM) and field emission scanning electron microscopy (FESEM). XRD pattern of BaZr0.9Ho0.1O3-δ ceramic sintered at 1600 °C has shown that pure phase of BaZr0.9Ho0.1O3-δ with cubic Pm3¯m space group symmetry. The transmission electron microscopic investigation has shown that the particle size of the powder calcined at 1100 °C was in the range 30-80 nm. The FESEM image of sintered pellet at 1600 °C for 4 h reveals porous nature of BaZr0.9Ho0.1O3-δ with 83.7 relative density. Impedance analysis reveal three type relaxations in the temperature range 250 °C to 500 °C as studied at different frequencies over 100 Hz to 1 MHz in air. The grain boundary conductivity of BaZr0.9Ho0.1O3-δ ceramic is found lower then grain (bulk) conductivity due to core-space charge layer behavior in grain boundary.

Omega-3 polyunsaturated fatty acid has an anti-oxidant effect via the Nrf-2/HO-1 pathway in 3T3-L1 adipocytes

Energy Technology Data Exchange (ETDEWEB)

Kusunoki, Chisato, E-mail: yosizaki@belle.shiga-med.ac.jp [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Yang, Liu; Yoshizaki, Takeshi; Nakagawa, Fumiyuki; Ishikado, Atsushi; Kondo, Motoyuki; Morino, Katsutaro; Sekine, Osamu; Ugi, Satoshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan); Nishio, Yoshihiko [Division of Diabetes, Metabolism and Endocrinology, Department of Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544 (Japan); Kashiwagi, Atsunori; Maegawa, Hiroshi [Department of Medicine, Shiga University of Medical Science, Seta Tsukinowa-Cho, Otsu, Shiga 520-2192 (Japan)

2013-01-04

Highlights: Black-Right-Pointing-Pointer Omega-3 PUFA has a direct anti-oxidant effect in adipocytes. Black-Right-Pointing-Pointer EPA and DHA induce HO-1 expression in 3T3-L1 adipocytes. Black-Right-Pointing-Pointer Omega-3 PUFA and its end-product, 4-HHE, activates the Nrf-2/HO-1 pathway. Black-Right-Pointing-Pointer Omega-3 PUFA protects against oxidative stress-induced cytotoxicity. -- Abstract: Oxidative stress is produced in adipose tissue of obese subjects and has been associated with obesity-related disorders. Recent studies have shown that omega-3 polyunsaturated fatty acid ({omega}3-PUFA) has beneficial effects in preventing atherosclerotic diseases and insulin resistance in adipose tissue. However, the role of {omega}3-PUFA on adipocytes has not been elucidated. In this study, 3T3-L1 adipocytes were treated with {omega}3-PUFA and its metabolites, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or 4-hydroxy hexenal (4-HHE). {omega}3-PUFA and its metabolites dose-dependently increased mRNA and protein levels of the anti-oxidative enzyme, heme oxygenase-1 (HO-1); whereas no changes in the well-known anti-oxidant molecules, superoxide dismutase, catalase, and glutathione peroxidase, were observed. Knockdown of nuclear factor erythroid 2-related factor 2 (Nrf-2) significantly reduced EPA, DHA or 4-HHE-induced HO-1 mRNA and protein expression. Also, pretreatment with {omega}3-PUFA prevented H{sub 2}O{sub 2}-induced cytotoxicity in a HO-1 dependent manner. In conclusion, treatment with EPA and DHA induced HO-1 through the activation of Nrf-2 and prevented oxidative stress in 3T3-L1 adipocytes. This anti-oxidant defense may be of high therapeutic value for clinical conditions associated with systemic oxidative stress.

Maresin 1 Ameliorates Lung Ischemia/Reperfusion Injury by Suppressing Oxidative Stress via Activation of the Nrf-2-Mediated HO-1 Signaling Pathway

Directory of Open Access Journals (Sweden)

Quanchao Sun

2017-01-01

Full Text Available Lung ischemia/reperfusion (I/R injury occurs in various clinical conditions and heavily damaged lung function. Oxidative stress reaction and antioxidant enzymes play a pivotal role in the etiopathogenesis of lung I/R injury. In the current study, we investigated the impact of Maresin 1 on lung I/R injury and explored the possible mechanism involved in this process. MaR 1 ameliorated I/R-induced lung injury score, wet/dry weight ratio, myeloperoxidase, tumor necrosis factor, bronchoalveolar lavage fluid (BALF leukocyte count, BALF neutrophil ratio, and pulmonary permeability index levels in lung tissue. MaR 1 significantly reduced ROS, methane dicarboxylic aldehyde, and 15-F2t-isoprostane generation and restored antioxidative enzyme (superoxide dismutase, glutathione peroxidase, and catalase activities. Administration of MaR 1 improved the expression of nuclear Nrf-2 and cytosolic HO-1 in I/R-treated lung tissue. Furthermore, we also found that the protective effects of MaR 1 on lung tissue injury and oxidative stress were reversed by HO-1 activity inhibitor, Znpp-IX. Nrf-2 transcription factor inhibitor, brusatol, significantly decreased MaR 1-induced nuclear Nrf-2 and cytosolic HO-1 expression. In conclusion, these results indicate that MaR 1 protects against lung I/R injury through suppressing oxidative stress. The mechanism is partially explained by activation of the Nrf-2-mediated HO-1 signaling pathway.

Four inducible promoters for controlled gene expression in the oleaginous yeast Rhodotorula toruloides

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Alexander Michael Bedford Johns

2016-10-01

Full Text Available Rhodotorula (Rhodosporidium toruloides is an oleaginous yeast with great biotechnological potential, capable of accumulating lipid up to 70 % of its dry biomass, and of carotenoid biosynthesis. However, few molecular genetic tools are available for manipulation of this basidiomycete yeast and its high genomic GC content can make routine cloning difficult. We have developed plasmid vectors for transformation of R. toruloides which include elements for Saccharomyces cerevisiae in-yeast assembly; this method is robust to the assembly of GC-rich DNA and of large plasmids. Using such vectors we screened for controllable promoters, and identified inducible promoters from the genes NAR1, ICL1, CTR3 and MET16. These four promoters have independent induction/repression conditions and exhibit different levels and rates of induction in R. toruloides, making them appropriate for controllable transgene expression in different experimental situations. Nested deletions were used to identify regulatory regions in the four promoters, and to delimit the minimal inducible promoters, which are as small as 200 bp for the NAR1 promoter. The NAR1 promoter shows very tight regulation under repressed conditions as determined both by an EGFP reporter gene and by conditional rescue of a leu2 mutant. These new tools facilitate molecular genetic manipulation and controllable gene expression in R. toruloides.

Induction of HO-1 in tissue macrophages and monocytes in fatal falciparum malaria and sepsis

Directory of Open Access Journals (Sweden)

Liomba N

2003-11-01

Full Text Available Abstract Background As well as being inducible by haem, haemoxygenase -1 (HO-1 is also induced by interleukin-10 and an anti-inflammatory prostaglandin, 15d PGJ2, the carbon monoxide thus produced mediating the anti-inflammatory effects of these molecules. The cellular distribution of HO-1, by immunohistochemistry, in brain, lung and liver in fatal falciparum malaria, and in sepsis, is reported. Methods Wax sections were stained, at a 1:1000 dilution of primary antibody, for HO-1 in tissues collected during paediatric autopsies in Blantyre, Malawi. These comprised 37 acutely ill comatose patients, 32 of whom were diagnosed clinically as cerebral malaria and the other 5 as bacterial diseases with coma. Another 3 died unexpectedly from an alert state. Other control tissues were from Australian adults. Results Apart from its presence in splenic red pulp macrophages and microhaemorrhages, staining for HO-1 was confined to intravascular monocytes and certain tissue macrophages. Of the 32 clinically diagnosed cerebral malaria cases, 11 (category A cases had negligible histological change in the brain and absence of or scanty intravascular sequestration of parasitized erythrocytes. Of these 11 cases, eight proved at autopsy to have other pathological changes as well, and none of these eight showed HO-1 staining within the brain apart from isolated moderate staining in one case. Two of the three without another pathological diagnosis showed moderate staining of scattered monocytes in brain vessels. Six of these 11 (category A cases exhibited strong lung staining, and the Kupffer cells of nine of them were intensely stained. Of the seven (category B cases with no histological changes in the brain, but appreciable sequestered parasitised erythrocytes present, one was without staining, and the other six showed strongly staining, rare or scattered monocytes in cerebral vessels. All six lung sections not obscured by neutrophils showed strong staining of

Inhibitory effect of leptin on rosiglitazone-induced differentiation of primary adipocytes prepared from TallyHO/Jng mice

Energy Technology Data Exchange (ETDEWEB)

Kim, Ki Young; Kim, Joo Young; Sung, Yoon-Young; Jung, Won Hoon; Kim, Hee-Youn; Park, Ji Seon; Cheon, Hyae Gyeong [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of); Rhee, Sang Dal, E-mail: sdrhee@krict.re.kr [Medicinal Science Division, Korea Research Institute of Chemical Technology, 100 Jang-dong, Yuseong, 305-600 Daejon (Korea, Republic of)

2011-03-25

Research highlights: {yields} In this study, we investigated the effects of leptin on adipocyte differentiation prepared from subcutaneous fat of TallyHo mice. {yields} Leptin inhibited the adipocytes differentiation at physiological concentration via inhibition of PPAR{gamma} expression. {yields} Inhibitors of ERK and STAT1 restored the leptin's inhibitory activity both in vitro and in vivo. -- Abstract: The effects of leptin on rosiglitazone-induced adipocyte differentiation were investigated in the primary adipocytes prepared from subcutaneous fat of TallyHO/Jng (TallyHO) mouse, a recently developed model animal for type 2 diabetes mellitus (T2DM). The treatment of leptin inhibited the rosiglitazone-induced adipocyte differentiation with a decreased expression of peroxisome proliferator-activated receptor {gamma} (PPAR{gamma}) a key adipogenic transcription factor, both in mRNA and protein levels. Leptin (10 nM) was sufficient to inhibit the adipocyte differentiation, which seemed to come from increased expression of leptin receptor genes in the fat of TallyHO mice. The inhibition of adipogenesis by leptin was restored by the treatment of inhibitors for extracellular-signal-regulated kinase (ERK) (PD98059) and signal transducer and activator of transcription-1 (STAT1) (fludarabine). Furthermore, in vivo intraperitoneal administration of PD98059 and fludarabine increased the PPAR{gamma} expression in the subcutaneous fat of TallyHO mice. These data suggest that leptin could inhibit the PPAR{gamma} expression and adipocyte differentiation in its physiological concentration in TallyHO mice.

Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

Energy Technology Data Exchange (ETDEWEB)

Park, Sun Young; Kim, Ji-Hee [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of); Lee, Sang Joon [Department of Microbiology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of); Kim, YoungHee, E-mail: yheekim@pusan.ac.kr [Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Busan 609-735 (Korea, Republic of)

2013-04-01

Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E{sub 2} (PGE{sub 2}), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates

Involvement of PKA and HO-1 signaling in anti-inflammatory effects of surfactin in BV-2 microglial cells

International Nuclear Information System (INIS)

Park, Sun Young; Kim, Ji-Hee; Lee, Sang Joon; Kim, YoungHee

2013-01-01

Surfactin, one of the most powerful biosurfactants, is a bacterial cyclic lipopeptide. Here, we investigated the anti-neuroinflammatory properties of surfactin in lipoteichoic acid (LTA)-stimulated BV-2 microglial cells. Surfactin significantly inhibited excessive production of the pro-inflammatory mediators TNF-α, IL-1β, IL-6, monocyte chemoattractant protein-1 (MCP-1), prostaglandin E 2 (PGE 2 ), nitric oxide (NO) and reactive oxygen species (ROS), and suppressed the expression of matrix metalloproteinase-9 (MMP-9), inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Subsequent mechanistic studies revealed that surfactin inhibited LTA-induced nuclear factor-kappaB (NF-κB) and signal transducer and activator of transcription-1 (STAT-1) activation. However, surfactin increases the phosphorylation of the STAT-3, a component of the homeostatic mechanism causing anti-inflammatory events. We also demonstrated that surfactin induces heme oxygenase-1 (HO-1) expression and nuclear factor-regulated factor-2 (Nrf-2) activation, and that the anti-inflammatory effects of surfactin are abrogated by small interfering RNA-mediated knock-down of HO-1 or Nrf-2. Interestingly, we found that surfactin increased the level of cAMP and induced phosphorylation of cAMP responsive element binding protein (CREB) in microglial cells. Furthermore, treatment with the protein kinase A (PKA) inhibitor, H-89, blocked HO-1 induction by surfactin and abolished surfactin's suppressive effects on ROS and NO production. These results indicate that HO-1 and its upstream effector, PKA, play a pivotal role in the anti-neuroinflammatory response of surfactin in LTA-stimulated microglia. Therefore, surfactin might have therapeutic potential for neuroprotective agents to treat inflammatory and neurodegenerative diseases. - Highlights: ► Surfactin inhibits proinflammatory mediator synthesis in LTA-activated BV-2 cells. ► Surfactin suppresses NF-κB and STAT-1, but potentiates phosphorylation

The Mouse Solitary Odorant Receptor Gene Promoters as Models for the Study of Odorant Receptor Gene Choice.

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Andrea Degl'Innocenti

Full Text Available In vertebrates, several anatomical regions located within the nasal cavity mediate olfaction. Among these, the main olfactory epithelium detects most conventional odorants. Olfactory sensory neurons, provided with cilia exposed to the air, detect volatile chemicals via an extremely large family of seven-transmembrane chemoreceptors named odorant receptors. Their genes are expressed in a monogenic and monoallelic fashion: a single allele of a single odorant receptor gene is transcribed in a given mature neuron, through a still uncharacterized molecular mechanism known as odorant receptor gene choice.Odorant receptor genes are typically arranged in genomic clusters, but a few are isolated (we call them solitary from the others within a region broader than 1 Mb upstream and downstream with respect to their transcript's coordinates. The study of clustered genes is problematic, because of redundancy and ambiguities in their regulatory elements: we propose to use the solitary genes as simplified models to understand odorant receptor gene choice.Here we define number and identity of the solitary genes in the mouse genome (C57BL/6J, and assess the conservation of the solitary status in some mammalian orthologs. Furthermore, we locate their putative promoters, predict their homeodomain binding sites (commonly present in the promoters of odorant receptor genes and compare candidate promoter sequences with those of wild-caught mice. We also provide expression data from histological sections.In the mouse genome there are eight intact solitary genes: Olfr19 (M12, Olfr49, Olfr266, Olfr267, Olfr370, Olfr371, Olfr466, Olfr1402; five are conserved as solitary in rat. These genes are all expressed in the main olfactory epithelium of three-day-old mice. The C57BL/6J candidate promoter of Olfr370 has considerably varied compared to its wild-type counterpart. Within the putative promoter for Olfr266 a homeodomain binding site is predicted. As a whole, our findings

Transfection of the Human Heme Oxygenase Gene Into Rabbit Coronary Microvessel Endothelial Cells: Protective Effect Against Heme and Hemoglobin Toxicity

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Abraham, N. G.; Lavrovsky, Y.; Schwartzman, M. L.; Stoltz, R. A.; Levere, R. D.; Gerritsen, M. E.

1995-07-01

Heme oxygenase (HO) is a stress protein and has been suggested to participate in defense mechanisms against agents that may induce oxidative injury such as metals, endotoxin, heme/hemoglobin, and various cytokines. Overexpression of HO in cells might therefore protect against oxidative stress produced by certain of these agents, specifically heme and hemoglobin, by catalyzing their degradation to bilirubin, which itself has antioxidant properties. We report here the successful in vitro transfection of rabbit coronary microvessel endothelial cells with a functioning gene encoding the human HO enzyme. A plasmid containing the cytomegalovirus promoter and the human HO cDNA complexed to cationic liposomes (Lipofectin) was used to transfect rabbit endothelial cells. Cells transfected with human HO exhibited an ≈3.0-fold increase in enzyme activity and expressed a severalfold induction of human HO mRNA as compared with endogenous rabbit HO mRNA. Transfected and nontransfected cells expressed factor VIII antigen and exhibited similar acetylated low-density lipoprotein uptake (two important features that characterize endothelial cells) with >85% of cells staining positive for each marker. Moreover, cells transfected with the human HO gene acquired substantial resistance to toxicity produced by exposure to recombinant hemoglobin and heme as compared with nontransfected cells. The protective effect of HO overexpression against heme/hemoglobin toxicity in endothelial cells shown in these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Archaeal promoter architecture and mechanism of gene activation

DEFF Research Database (Denmark)

Peng, Nan; Ao, Xiang; Liang, Yun Xiang

2011-01-01

element named ara box directing arabinose-inducible expression and the basal promoter element TATA, serving as the binding site for the TATA-binding protein. Strikingly, these promoters possess a modular structure that allows an essentially inactive basal promoter to be strongly activated. The invoked...... mechanisms include TFB (transcription factor B) recruitment by the ara-box-binding factor to activate gene expression and modulation of TFB recruitment efficiency to yield differential gene expression....

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data

DEFF Research Database (Denmark)

Kannan, Soumya; Sams, Thomas; Maury, Jérôme

2018-01-01

activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system......Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds......, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter...

Heme oxygenase-1 gene expression modulates angiotensin II-induced increase in blood pressure.

Science.gov (United States)

Yang, Liming; Quan, Shuo; Nasjletti, Alberto; Laniado-Schwartzman, Michal; Abraham, Nader G

2004-06-01

The heme-heme oxygenase (HO) system has been implicated in the regulation of vascular reactivity and blood pressure. This study examines the notion that overexpression of HO decreases pressor responsiveness to angiotensin II (Ang II). Five-day-old Sprague-Dawley rats received an intraleft ventricular injection of approximately 5x10(9) cfu/mL of retroviruses containing human HO-1 sense (LSN-HHO-1), rat HO-1 antisense (LSN-RHO-1-AS), or control retrovirus (LXSN). Three months later, rats were instrumented with femoral arterial and venous catheters for mean arterial pressure (MAP) determination and Ang II administration, respectively. Rats injected with LSN-HHO-1, but not with LXSN, expressed human HO-1 mRNA and protein in several tissues. BP increased with administration of Ang II in rats expressing and not expressing human HO-1. However, the Ang II-induced pressor response (mm Hg) in LSN-HHO-1 rats (16+/-3, 27+/-3, and 38+/-3 at 0.5, 2, and 10 ng) was surpassed (PHHO-1 rats with the HO inhibitor tin mesoporphyrin (SnMP) enhanced (P<0.05) the Ang II-induced pressor response to a level not different from that observed in LXSN rats. Rats injected with LSN-RHO-1-AS showed a decrease in renal HO-1 protein expression and HO activity relative to control LXSN rats. Administration of Ang II (0.1 to 2 ng) caused small (4 to 5 mm Hg) but significant increases in MAP in rats injected with LSN-RHO-1-AS (P<0.05) compared with rats injected with LXSN. These data demonstrate that overexpression of HO-1 brings about a reduction in pressor responsiveness to Ang II, which is most likely due to increased generation of an HO-1 product, presumably CO, with the ability to inhibit vascular reactivity to constrictor stimuli.

Precise regulation of gene expression dynamics favors complex promoter architectures.

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Dirk Müller

2009-01-01

Full Text Available Promoters process signals through recruitment of transcription factors and RNA polymerase, and dynamic changes in promoter activity constitute a major noise source in gene expression. However, it is barely understood how complex promoter architectures determine key features of promoter dynamics. Here, we employ prototypical promoters of yeast ribosomal protein genes as well as simplified versions thereof to analyze the relations among promoter design, complexity, and function. These promoters combine the action of a general regulatory factor with that of specific transcription factors, a common motif of many eukaryotic promoters. By comprehensively analyzing stationary and dynamic promoter properties, this model-based approach enables us to pinpoint the structural characteristics underlying the observed behavior. Functional tradeoffs impose constraints on the promoter architecture of ribosomal protein genes. We find that a stable scaffold in the natural design results in low transcriptional noise and strong co-regulation of target genes in the presence of gene silencing. This configuration also exhibits superior shut-off properties, and it can serve as a tunable switch in living cells. Model validation with independent experimental data suggests that the models are sufficiently realistic. When combined, our results offer a mechanistic explanation for why specific factors are associated with low protein noise in vivo. Many of these findings hold for a broad range of model parameters and likely apply to other eukaryotic promoters of similar structure.

Genipin inhibits TNF-α-induced vascular smooth muscle cell proliferation and migration via induction of HO-1.

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Fengrong Jiang

Full Text Available Vascular smooth muscle cell (VSMC proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1, a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.

A mutation in a functional Sp1 binding site of the telomerase RNA gene (hTERC promoter in a patient with Paroxysmal Nocturnal Haemoglobinuria

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Mason Philip J

2004-06-01

Full Text Available Abstract Background Mutations in the gene coding for the RNA component of telomerase, hTERC, have been found in autosomal dominant dyskeratosis congenita (DC and aplastic anemia. Paroxysmal nocturnal hemoglobinuria (PNH is a clonal blood disorder associated with aplastic anemia and characterized by the presence of one or more clones of blood cells lacking glycosylphosphatidylinositol (GPI anchored proteins due to a somatic mutation in the PIGA gene. Methods We searched for mutations in DNA extracted from PNH patients by amplification of the hTERC gene and denaturing high performance liquid chromatography (dHPLC. After a mutation was found in a potential transcription factor binding site in one patient electrophoretic mobility shift assays were used to detect binding of transcription factors to that site. The effect of the mutation on the function of the promoter was tested by transient transfection constructs in which the promoter is used to drive a reporter gene. Results Here we report the finding of a novel promoter mutation (-99C->G in the hTERC gene in a patient with PNH. The mutation disrupts an Sp1 binding site and destroys its ability to bind Sp1. Transient transfection assays show that mutations in this hTERC site including C-99G cause either up- or down-regulation of promoter activity and suggest that the site regulates core promoter activity in a context dependent manner in cancer cells. Conclusions These data are the first report of an hTERC promoter mutation from a patient sample which can modulate core promoter activity in vitro, raising the possibility that the mutation may affect the transcription of the gene in hematopoietic stem cells in vivo, and that dysregulation of telomerase may play a role in the development of bone marrow failure and the evolution of PNH clones.

Sequence analysis of the Epstein-Barr virus (EBV) latent membrane protein-1 gene and promoter region

DEFF Research Database (Denmark)

Sandvej, Kristian; Gratama, J W; Munch, M

1997-01-01

Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which...... include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European...... wild-type virus isolates, we sequenced the LMP-1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D...

The complexes of Ho with methylenediphosphonate and 1 hydroxyethylidenephosphonate

International Nuclear Information System (INIS)

Vanura, P.; Jedinakova-Krizova, V.; Hakenova, L.; Munesawa, Y.

1999-01-01

The composition and stability of holmium methylenediphosphonate (MDP) and 1-hydroxyethylidenephosphonate (HEDP) complexes in the aqueous solution of 0.1 M NaCl at the temperature of 25 grad C were studied by potentiometric titration methods. The complexes of the composition HoH n L have been found in the aqueous solution if the concentration of the ligand is higher than the concentration of holmium. The protonation constants of both acids and stability constants of all complexes were determined and the comparison with literature data of analogical complexes of other lanthanides was performed. (authors)

Totarol prevents neuronal injury in vitro and ameliorates brain ischemic stroke: Potential roles of Akt activation and HO-1 induction

Energy Technology Data Exchange (ETDEWEB)

Gao, Yuanxue; Xu, Xiaojun; Chang, Sai; Wang, Yunjie; Xu, Yazhou; Ran, Siqi [Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Huang, Zhangjian [Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China Pharmaceutical University, Nanjing 210009 (China); Li, Ping [Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Li, Jia [National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guo Shoujing Road, Shanghai 201203 (China); Zhang, Luyong [Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Saavedra, Juan M. [Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC 20057 (United States); Liao, Hong, E-mail: liaohong56@hotmail.com [Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Pang, Tao, E-mail: tpang@cpu.edu.cn [Jiangsu Key Laboratory of Drug Screening, State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009 (China); Department of Pharmacology and Physiology, Georgetown University Medical Center, Washington, DC 20057 (United States)

2015-12-01

The natural product totarol, a phenolic diterpenoid and a major constituent isolated from the sap of Podocarpus totara, has been reported to have a potent antimicrobial activity. In this study, we determined whether totarol possessed an additional neuroprotective activity in vitro and in vivo. We found that totarol prevented glutamate- and oxygen and glucose deprivation-induced neuronal death in primary rat cerebellar granule neuronal cells and cerebral cortical neurons. Totarol increased Akt and GSK-3β phosphorylation, Nrf2 and heme oxygenase-1 (HO-1) protein expressions and suppressed oxidative stress by increasing GSH and SOD activities. The PI3K/Akt inhibitor LY294002 prevented totarol neuroprotective effect by suppressing the totarol-induced changes in HO-1 expression and the activities of GSH and SOD. The HO-1 inhibitor ZnPPIX also prevented totarol-increased GSH and SOD activities. In a model of acute cerebral ischemic injury in Sprague–Dawley rats, produced by occlusion of the middle cerebral artery for 2 h followed by 22 h or 46 h of reperfusion, totarol significantly reduced infarct volume and improved the neurological deficit. In this model, totarol increased HO-1 expression and the activities of GSH and SOD. These observations suggest that totarol may be a novel activator of the Akt/HO-1 pathway protecting against ischemic stroke through reduction of oxidative stress. - Graphical abstract: It is unknown whether the natural product totarol has neuroprotective effects in vitro and in vivo. This study underscores that totarol prevents neuronal injury in vitro, not only by activating PI3K/Akt pathway, but also via induction of Nrf2, HO-1, GSH and SOD expressions. Totarol also ameliorated acute cerebral ischemic injury in a rat ischemic stroke model. The findings highlight that totarol may be exploited for protecting against ischemic stroke through Akt/HO-1 pathway. - Highlights: • Totarol protects glutamate- and OGD-induced neuronal injury in vitro. �

Totarol prevents neuronal injury in vitro and ameliorates brain ischemic stroke: Potential roles of Akt activation and HO-1 induction

International Nuclear Information System (INIS)

Gao, Yuanxue; Xu, Xiaojun; Chang, Sai; Wang, Yunjie; Xu, Yazhou; Ran, Siqi; Huang, Zhangjian; Li, Ping; Li, Jia; Zhang, Luyong; Saavedra, Juan M.; Liao, Hong; Pang, Tao

2015-01-01

The natural product totarol, a phenolic diterpenoid and a major constituent isolated from the sap of Podocarpus totara, has been reported to have a potent antimicrobial activity. In this study, we determined whether totarol possessed an additional neuroprotective activity in vitro and in vivo. We found that totarol prevented glutamate- and oxygen and glucose deprivation-induced neuronal death in primary rat cerebellar granule neuronal cells and cerebral cortical neurons. Totarol increased Akt and GSK-3β phosphorylation, Nrf2 and heme oxygenase-1 (HO-1) protein expressions and suppressed oxidative stress by increasing GSH and SOD activities. The PI3K/Akt inhibitor LY294002 prevented totarol neuroprotective effect by suppressing the totarol-induced changes in HO-1 expression and the activities of GSH and SOD. The HO-1 inhibitor ZnPPIX also prevented totarol-increased GSH and SOD activities. In a model of acute cerebral ischemic injury in Sprague–Dawley rats, produced by occlusion of the middle cerebral artery for 2 h followed by 22 h or 46 h of reperfusion, totarol significantly reduced infarct volume and improved the neurological deficit. In this model, totarol increased HO-1 expression and the activities of GSH and SOD. These observations suggest that totarol may be a novel activator of the Akt/HO-1 pathway protecting against ischemic stroke through reduction of oxidative stress. - Graphical abstract: It is unknown whether the natural product totarol has neuroprotective effects in vitro and in vivo. This study underscores that totarol prevents neuronal injury in vitro, not only by activating PI3K/Akt pathway, but also via induction of Nrf2, HO-1, GSH and SOD expressions. Totarol also ameliorated acute cerebral ischemic injury in a rat ischemic stroke model. The findings highlight that totarol may be exploited for protecting against ischemic stroke through Akt/HO-1 pathway. - Highlights: • Totarol protects glutamate- and OGD-induced neuronal injury in vitro. �

Porcine synapsin 1: SYN1 gene analysis and functional characterization of the promoter

DEFF Research Database (Denmark)

Hedegaard, Claus; Kjaer-Sorensen, Kasper; Madsen, Lone Bruhn

2013-01-01

of elements responsible for neuron-specific expression. Expression analysis of SYN1 demonstrated presence of transcript during embryonic development. Analysis of GFP expression in transgenic zebrafish embryos suggests that the pig SYN1 promoter directs expression in neuronal cells. Thus, the SYN1 promoter...

Exogenous Hydrogen Peroxide Contributes to Heme Oxygenase-1 Delaying Programmed Cell Death in Isolated Aleurone Layers of Rice Subjected to Drought Stress in a cGMP-Dependent Manner.

Science.gov (United States)

Wang, Guanghui; Xiao, Yu; Deng, Xiaojiang; Zhang, Heting; Li, Tingge; Chen, Huiping

2018-01-01

Hydrogen peroxide (H 2 O 2 ) is a reactive oxygen species (ROS) that plays a dual role in plant cells. Here, we discovered that drought (20% polyethylene glycol-6000, PEG)-triggered decreases of HO-1 transcript expression and HO activity. However, exogenous H 2 O 2 contributed toward the increase in HO-1 gene expression and activity of the enzyme under drought stress. Meanwhile, the HO-1 inducer hematin could mimic the effects of the H 2 O 2 scavengers ascorbic acid (AsA) and dimethylthiourea (DMTU) and the H 2 O 2 synthesis inhibitor diphenyleneiodonium (DPI) for scavenging or diminishing drought-induced endogenous H 2 O 2 . Conversely, the zinc protoporphyrin IX (ZnPPIX), an HO-1-specific inhibitor, reversed the effects of hematin. We further analyzed the endogenous H 2 O 2 levels and HO-1 transcript expression levels of aleurone layers treated with AsA, DMTU, and DPI in the presence of exogenous H 2 O 2 under drought stress, respectively. The results showed that in aleurone layers subjected to drought stress, when the endogenous H 2 O 2 level was inhibited, the effect of exogenous H 2 O 2 on the induction of HO-1 was enhanced. Furthermore, exogenous H 2 O 2 -activated HO-1 effectively enhanced amylase activity. Application of 8-bromoguanosine 3',5'-cyclic guanosine monophosphate (8-Br-cGMP) (the membrane permeable cGMP analog) promoted the effect of exogenous H 2 O 2 -delayed PCD of aleurone layers in response to drought stress. More importantly, HO-1 delayed the programmed cell death (PCD) of aleurone layers by cooperating with nitric oxide (NO), and the delayed effect of NO on PCD was achieved via mediation by cGMP under drought stress. In short, in rice aleurone layers, exogenous H 2 O 2 (as a signaling molecule) triggered HO-1 and delayed PCD via cGMP which possibly induced amylase activity under drought stress. In contrast, as a toxic by-product of cellular metabolism, the drought-generated H 2 O 2 promoted cell death.

PROMOTER POLYMORPHISM OF IL-1β GENE IN PATIENTS WITH A HISTORY OF ACUTE MYOCARDIAL INFARCTION

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A. V. Shevchenko

2010-01-01

Full Text Available We have performed analysis of associations between IL-1β gene promoter polymorphism (-511C/T and -31 T/C variants, and conventional cardiovascular risk factors in the patients living in the West Siberia who had previously a history of myocardial infarction (MI. We are shown a strong linkage disequilibrium between IL-1β -31C/T (rs1143627, and IL-1β-511T/C (rs16944. Significant differences in frequency distributions of some compound genotypes were observed between healthy and patients with a history of MI. E.g., frequency of IL-1β-31CC/-511CT genotype was detected in 5.5 % of healthy population, while being absent among MI patients. A frequency of IL-1β (-31/-511 CC/CT genotype showed significant differences between MI patients under 55 years, as compared to healthy persons. Hence, the analyzed IL-1β promoter polymorphisms may be considered as an additional constitutional factor predisposing for vascular alterations.

Senataxin Mutation Reveals How R-Loops Promote Transcription by Blocking DNA Methylation at Gene Promoters.

Science.gov (United States)

Grunseich, Christopher; Wang, Isabel X; Watts, Jason A; Burdick, Joshua T; Guber, Robert D; Zhu, Zhengwei; Bruzel, Alan; Lanman, Tyler; Chen, Kelian; Schindler, Alice B; Edwards, Nancy; Ray-Chaudhury, Abhik; Yao, Jianhua; Lehky, Tanya; Piszczek, Grzegorz; Crain, Barbara; Fischbeck, Kenneth H; Cheung, Vivian G

2018-02-01

R-loops are three-stranded nucleic acid structures found abundantly and yet often viewed as by-products of transcription. Studying cells from patients with a motor neuron disease (amyotrophic lateral sclerosis 4 [ALS4]) caused by a mutation in senataxin, we uncovered how R-loops promote transcription. In ALS4 patients, the senataxin mutation depletes R-loops with a consequent effect on gene expression. With fewer R-loops in ALS4 cells, the expression of BAMBI, a negative regulator of transforming growth factor β (TGF-β), is reduced; that then leads to the activation of the TGF-β pathway. We uncovered that genome-wide R-loops influence promoter methylation of over 1,200 human genes. DNA methyl-transferase 1 favors binding to double-stranded DNA over R-loops. Thus, in forming R-loops, nascent RNA blocks DNA methylation and promotes further transcription. Hence, our results show that nucleic acid structures, in addition to sequences, influence the binding and activity of regulatory proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Magnetic and hyperfine interactions in HoFe{sub 1−x}Cr{sub x}O{sub 3} (0≤x≤1) compounds

Energy Technology Data Exchange (ETDEWEB)

Kotnana, Ganesh [Magnetic Materials and Device Physics Laboratory, Department of Physics, Indian Institute of Technology Hyderabad, Hyderabad 502285 (India); Reddy, V. Raghavendra [UGC-DAE Consortium for Scientific Research, Khandwa Road, Indore 452001 (India); Jammalamadaka, S. Narayana, E-mail: surya@iith.ac.in [Magnetic Materials and Device Physics Laboratory, Department of Physics, Indian Institute of Technology Hyderabad, Hyderabad 502285 (India)

2017-05-01

We report on the magnetic and Mössbauer properties of polycrystalline HoFe{sub 1-x}Cr{sub x}O{sub 3} (0≤x≤1) compounds. Magnetization data reveals the continuous tailoring of magnetic transition due to weakening of Ho{sup 3+}-Fe{sup 3+} and Fe{sup 3+}-Fe{sup 3+} interactions in the entire temperature range by replacing the Fe{sup 3+} ions with Cr{sup 3+} ions. The observed decrease in Néel temperature (T{sub N}) and increase in spin re-orientation transition temperature (T{sub SR}) with the replacement of Fe{sup 3+} with Cr{sup 3+} is ascribed to the weakening of Fe(Cr)-O-Fe(Cr) antiferromagnetic exchange interaction. In addition, we also attribute such a change in T{sub N} to the enhancement of ferromagnetic interaction of adjacent Cr{sup 3+} moments through t-e hybridization as a result of the structural distortion. The decrease in isomer shift (IS) suggests enhancement of the interaction between nuclear charge with the 3s electrons as a result of decrease in radial part of 3d wave function with Cr addition. In this paper we also discuss about the variation of quadrupole splitting (QS) and hyperfine fields (H{sub hf}) with Cr addition in HoFe{sub 1-x}Cr{sub x}O{sub 3} (0≤x≤1) compounds. - Highlights: • Magnetic and Mössbauer properties of HoFe{sub 1-x}Cr{sub x}O{sub 3} (0≤x≤1) compounds. • T{sub N} changes due to weakening of Ho{sup 3+}-Fe{sup 3+} and Fe{sup 3+}-Fe{sup 3+} interactions with Cr. • The decrease in isomer shift (IS) is due to decrease in radial part of 3d wavefunction. • Octahedral distortion leads to increase in quadrupole splitting.

High Power Narrow Linewidth 1.26 Micron Ho-Doped Fiber Amplifier, Phase II

Data.gov (United States)

National Aeronautics and Space Administration — This proposal is for the development of an innovative, high power, and extremely reliable 1.26-micron Ho-doped fluoride fiber amplifier. The proposed fiber amplifier...

High Power Narrow Linewidth 1.26 Micron Ho-Doped Fiber Amplifier, Phase I

Data.gov (United States)

National Aeronautics and Space Administration — This proposal is for the development of an innovative, high power, and extremely reliable 1.26-micron Ho-doped fluoride fiber amplifier. The proposed fiber amplifier...

Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

Science.gov (United States)

Fauteux, François; Strömvik, Martina V

2009-01-01

Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP) gene promoters from three plant families, namely Brassicaceae (mustards), Fabaceae (legumes) and Poaceae (grasses) using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L.) Heynh.), soybean (Glycine max (L.) Merr.) and rice (Oryza sativa L.) respectively. We have identified three conserved motifs (two RY-like and one ACGT-like) in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination of conserved motifs

Seed storage protein gene promoters contain conserved DNA motifs in Brassicaceae, Fabaceae and Poaceae

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Fauteux François

2009-10-01

Full Text Available Abstract Background Accurate computational identification of cis-regulatory motifs is difficult, particularly in eukaryotic promoters, which typically contain multiple short and degenerate DNA sequences bound by several interacting factors. Enrichment in combinations of rare motifs in the promoter sequence of functionally or evolutionarily related genes among several species is an indicator of conserved transcriptional regulatory mechanisms. This provides a basis for the computational identification of cis-regulatory motifs. Results We have used a discriminative seeding DNA motif discovery algorithm for an in-depth analysis of 54 seed storage protein (SSP gene promoters from three plant families, namely Brassicaceae (mustards, Fabaceae (legumes and Poaceae (grasses using backgrounds based on complete sets of promoters from a representative species in each family, namely Arabidopsis (Arabidopsis thaliana (L. Heynh., soybean (Glycine max (L. Merr. and rice (Oryza sativa L. respectively. We have identified three conserved motifs (two RY-like and one ACGT-like in Brassicaceae and Fabaceae SSP gene promoters that are similar to experimentally characterized seed-specific cis-regulatory elements. Fabaceae SSP gene promoter sequences are also enriched in a novel, seed-specific E2Fb-like motif. Conserved motifs identified in Poaceae SSP gene promoters include a GCN4-like motif, two prolamin-box-like motifs and an Skn-1-like motif. Evidence of the presence of a variant of the TATA-box is found in the SSP gene promoters from the three plant families. Motifs discovered in SSP gene promoters were used to score whole-genome sets of promoters from Arabidopsis, soybean and rice. The highest-scoring promoters are associated with genes coding for different subunits or precursors of seed storage proteins. Conclusion Seed storage protein gene promoter motifs are conserved in diverse species, and different plant families are characterized by a distinct combination

Inverse correlation between promoter strength and excision activity in class 1 integrons.

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Thomas Jové

2010-01-01

Full Text Available Class 1 integrons are widespread genetic elements that allow bacteria to capture and express gene cassettes that are usually promoterless. These integrons play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. They typically consist of a gene (intI encoding an integrase (that catalyzes the gene cassette movement by site-specific recombination, a recombination site (attI1, and a promoter (Pc responsible for the expression of inserted gene cassettes. The Pc promoter can occasionally be combined with a second promoter designated P2, and several Pc variants with different strengths have been described, although their relative distribution is not known. The Pc promoter in class 1 integrons is located within the intI1 coding sequence. The Pc polymorphism affects the amino acid sequence of IntI1 and the effect of this feature on the integrase recombination activity has not previously been investigated. We therefore conducted an extensive in silico study of class 1 integron sequences in order to assess the distribution of Pc variants. We also measured these promoters' strength by means of transcriptional reporter gene fusion experiments and estimated the excision and integration activities of the different IntI1 variants. We found that there are currently 13 Pc variants, leading to 10 IntI1 variants, that have a highly uneven distribution. There are five main Pc-P2 combinations, corresponding to five promoter strengths, and three main integrases displaying similar integration activity but very different excision efficiency. Promoter strength correlates with integrase excision activity: the weaker the promoter, the stronger the integrase. The tight relationship between the aptitude of class 1 integrons to recombine cassettes and express gene cassettes may be a key to understanding the short-term evolution of integrons. Dissemination of integron-driven drug resistance is therefore more complex than previously thought.

A novel binary T-vector with the GFP reporter gene for promoter characterization.

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Shu-Ye Jiang

Full Text Available Several strategies have been developed to clone PCR fragments into desired vectors. However, most of commercially available T-vectors are not binary vectors and cannot be directly used for Agrobacterium-mediated plant genetic transformation. In this study, a novel binary T-vector was constructed by integrating two AhdI restriction sites into the backbone vector pCAMBIA 1300. The T-vector also contains a GFP reporter gene and thus, can be used to analyze promoter activity by monitoring the reporter gene. On the other hand, identification and characterization of various promoters not only benefit the functional annotation of their genes but also provide alternative candidates to be used to drive interesting genes for plant genetic improvement by transgenesis. More than 1,000 putative pollen-specific rice genes have been identified in a genome-wide level. Among them, 67 highly expressed genes were further characterized. One of the pollen-specific genes LOC_Os10g35930 was further surveyed in its expression patterns with more details by quantitative real-time reverse-transcription PCR (qRT-PCR analysis. Finally, its promoter activity was further investigated by analyzing transgenic rice plants carrying the promoter::GFP cassette, which was constructed from the newly developed T-vector. The reporter GFP gene expression in these transgenic plants showed that the promoter was active only in mature but not in germinated pollens.

Magnetic interactions in HoCr{sub 1-x}Fe{sub x}O{sub 3} (x = 0, 0.2) investigated by neutron powder diffraction

Energy Technology Data Exchange (ETDEWEB)

Liu, Xinzhi, E-mail: liuxinzhi1984.cn@163.com [Department of Nuclear Physics, China Institute of Atomic Energy, Beijing 102413 (China); Hao, Lijie; Ma, Xiaobai [Department of Nuclear Physics, China Institute of Atomic Energy, Beijing 102413 (China); Wang, Chin-Wei [Neutron Group, National Synchrotron Radiation Research Center, Hsinchu 30077, Taiwan (China); Klose, Frank [Australian Nuclear Science and Technology Organization, Lucas Heights, New South Wales 2234 (Australia); Department of Physics and Materials Science, The City University of Hong Kong, Hong Kong Special Administrative Region (Hong Kong); Liu, Yuntao, E-mail: ytliu@ciae.ac.cn [Department of Nuclear Physics, China Institute of Atomic Energy, Beijing 102413 (China); Sun, Kai; Li, Yuqing [Department of Nuclear Physics, China Institute of Atomic Energy, Beijing 102413 (China); Chen, Dongfeng, E-mail: dongfeng@ciae.ac.cn [Department of Nuclear Physics, China Institute of Atomic Energy, Beijing 102413 (China)

2017-07-01

Highlights: • The temperature dependent magnetism of HoCr{sub 1-x}Fe{sub x}O{sub 3} (x = 0, 0.2) were investigated by neutron diffraction. • Cr{sup 3+} moment follows a mean field theory while Ho{sup 3+} follows a spin 1/2 model. • An magneto-elastic strain was observed accompanying with the ordering of Cr{sup 3+}. - Abstract: The temperature dependent magnetism of Fe-doped rare earth orthochromite HoCr{sub 1-x}Fe{sub x}O{sub 3}(x = 0, 0.2) was investigated by neutron powder diffraction. It is found that the magnetism of Cr(Fe){sup 3+} can be well understood within mean field theory, while the ordering of Ho{sup 3+} was induced by the Cr(Fe){sup 3+} sublattice and can be satisfyingly described by an effective S = 1/2 model. The absences of both the most common G{sub x}F{sub z} configuration of Cr{sup 3+} and the ordering of Ho{sup 3+} caused by Ho-Ho interaction evidence a strong Ho{sup 3+}-Cr{sup 3+} interaction which dominates this system. On the other hand, a remarkable magnetoelastic strain was observed accompanying the Cr(Fe){sup 3+} ordering. An analysis based on the equation of state with a Grüneisen approximation was performed and revealed magnetic origin of this strain.

Heme oxygenase-1 accelerates tumor angiogenesis of human pancreatic cancer.

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Sunamura, Makoto; Duda, Dan G; Ghattas, Maivel H; Lozonschi, Lucian; Motoi, Fuyuhiko; Yamauchi, Jun-Ichiro; Matsuno, Seiki; Shibahara, Shigeki; Abraham, Nader G

2003-01-01

Angiogenesis is necessary for the continued growth of solid tumors, invasion and metastasis. Several studies clearly showed that heme oxygenase-1 (HO-1) plays an important role in angiogenesis. In this study, we used the vital microscope system, transparent skinfold model, lung colonization model and transduced pancreatic cancer cell line (Panc-1)/human heme oxygenase-1 (hHO-1) cells, to precisely analyze, for the first time, the effect of hHO-1 gene on tumor growth, angiogenesis and metastasis. Our results revealed that HO-1 stimulates angiogenesis of pancreatic carcinoma in severe combined immune deficient mice. Overexpression of human hHO-1 after its retroviral transfer into Panc-1 cells did not interfere with tumor growth in vitro. While in vivo the development of tumors was accelerated upon transfection with hHO-1. On the other hand, inhibition of heme oxygenase (HO) activity by stannous mesoporphyrin was able transiently to delay tumor growth in a dose dependent manner. Tumor angiogenesis was markedly increased in Panc-1/hHO-1 compared to mock transfected and wild type. Lectin staining and Ki-67 proliferation index confirmed these results. In addition hHO-1 stimulated in vitro tumor angiogenesis and increased endothelial cell survival. In a lung colonization model, overexpression of hHO-1 increased the occurrence of metastasis, while inhibition of HO activity by stannous mesoporphyrin completely inhibited the occurrence of metastasis. In conclusion, overexpression of HO-1 genes potentiates pancreatic cancer aggressiveness, by increasing tumor growth, angiogenesis and metastasis and that the inhibition of the HO system may be of useful benefit for the future treatment of the disease.

Cardiac-Specific Gene Expression Facilitated by an Enhanced Myosin Light Chain Promoter

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Wolfgang Boecker

2004-04-01

Full Text Available Background: Adenoviral gene transfer has been shown to be effective in cardiac myocytes in vitro and in vivo. A major limitation of myocardial gene therapy is the extracardiac transgene expression. Methods: To minimize extracardiac gene expression, we have constructed a tissue-specific promoter for cardiac gene transfer, namely, the 250-bp fragment of the myosin light chain-2v (MLC-2v gene, which is known to be expressed in a tissue-specific manner in ventricular myocardium followed by a luciferase (luc reporter gene (Ad.4 × MLC250.Luc. Rat cardiomyocytes, liver and kidney cells were infected with Ad.4 × MLC.Luc or control vectors. For in vivo testing, Ad.4 × MLC250.Luc was injected into the myocardium or in the liver of rats. Kinetics of promoter activity were monitored over 8 days using a cooled CCD camera. Results: In vitro: By infecting hepatic versus cardiomyocyte cells, we found that the promoter specificity ratio (luc activity in cardiomyocytes per liver cells was 20.4 versus 0.9 (Ad.4 × MLC250.Luc vs. Ad.CMV. In vivo: Ad.4 × MLC250.Luc significantly reduced luc activity in liver (38.4-fold, lung (16.1-fold, and kidney (21.8-fold versus Ad.CMV (p = .01; whereas activity in the heart was only 3.8-fold decreased. The gene expression rate of cardiomyocytes versus hepatocytes was 7:1 (Ad.4 × MLC.Luc versus 1:1.4 (Ad.CMV.Luc. Discussion: This new vector may be useful to validate therapeutic approaches in animal disease models and offers the perspective for selective expression of therapeutic genes in the diseased heart.

Analysis of an osmotically regulated pathogenesis-related osmotin gene promoter.

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Raghothama, K G; Liu, D; Nelson, D E; Hasegawa, P M; Bressan, R A

1993-12-01

Osmotin is a small (24 kDa), basic, pathogenesis-related protein, that accumulates during adaptation of tobacco (Nicotiana tabacum) cells to osmotic stress. There are more than 10 inducers that activate the osmotin gene in various plant tissues. The osmotin promoter contains several sequences bearing a high degree of similarity to ABRE, as-1 and E-8 cis element sequences. Gel retardation studies indicated the presence of at least two regions in the osmotin promoter that show specific interactions with nuclear factors isolated from cultured cells or leaves. The abundance of these binding factors increased in response to salt, ABA and ethylene. Nuclear factors protected a 35 bp sequence of the promoter from DNase I digestion. Different 5' deletions of the osmotin promoter cloned into a promoter-less GUSNOS plasmid (pBI 201) were used in transient expression studies with a Biolistic gun. The transient expression studies revealed the presence of three distinct regions in the osmotin promoter. The promoter sequence from -108 to -248 bp is absolutely required for reporter gene activity, followed by a long stretch (up to -1052) of enhancer-like sequence and then a sequence upstream of -1052, which appears to contain negative elements. The responses to ABA, ethylene, salt, desiccation and wounding appear to be associated with the -248 bp sequence of the promoter. This region also contains a putative ABRE (CACTGTG) core element. Activation of the osmotin gene by various inducers is discussed in view of antifungal activity of the osmotin protein.

Aberrant gene promoter methylation associated with sporadic multiple colorectal cancer.

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Victoria Gonzalo

Full Text Available BACKGROUND: Colorectal cancer (CRC multiplicity has been mainly related to polyposis and non-polyposis hereditary syndromes. In sporadic CRC, aberrant gene promoter methylation has been shown to play a key role in carcinogenesis, although little is known about its involvement in multiplicity. To assess the effect of methylation in tumor multiplicity in sporadic CRC, hypermethylation of key tumor suppressor genes was evaluated in patients with both multiple and solitary tumors, as a proof-of-concept of an underlying epigenetic defect. METHODOLOGY/PRINCIPAL FINDINGS: We examined a total of 47 synchronous/metachronous primary CRC from 41 patients, and 41 gender, age (5-year intervals and tumor location-paired patients with solitary tumors. Exclusion criteria were polyposis syndromes, Lynch syndrome and inflammatory bowel disease. DNA methylation at the promoter region of the MGMT, CDKN2A, SFRP1, TMEFF2, HS3ST2 (3OST2, RASSF1A and GATA4 genes was evaluated by quantitative methylation specific PCR in both tumor and corresponding normal appearing colorectal mucosa samples. Overall, patients with multiple lesions exhibited a higher degree of methylation in tumor samples than those with solitary tumors regarding all evaluated genes. After adjusting for age and gender, binomial logistic regression analysis identified methylation of MGMT2 (OR, 1.48; 95% CI, 1.10 to 1.97; p = 0.008 and RASSF1A (OR, 2.04; 95% CI, 1.01 to 4.13; p = 0.047 as variables independently associated with tumor multiplicity, being the risk related to methylation of any of these two genes 4.57 (95% CI, 1.53 to 13.61; p = 0.006. Moreover, in six patients in whom both tumors were available, we found a correlation in the methylation levels of MGMT2 (r = 0.64, p = 0.17, SFRP1 (r = 0.83, 0.06, HPP1 (r = 0.64, p = 0.17, 3OST2 (r = 0.83, p = 0.06 and GATA4 (r = 0.6, p = 0.24. Methylation in normal appearing colorectal mucosa from patients with multiple and solitary CRC showed no relevant

A variant on promoter of the cannabinoid receptor 1 gene (CNR1) moderates the effect of valence on working memory.

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Fairfield, Beth; Mammarella, Nicola; Franzago, Marica; Di Domenico, Alberto; Stuppia, Liborio; Gatta, Valentina

2018-02-01

Cannabinoid receptor 1 gene (CNR1) variants have been related to affective information processing and, in particular, to stress release. Here, we aimed to examine whether the endocannabinoid system via CNR1 signaling modulates affective working memory, the memory system that transiently maintains and manipulates emotionally charged material. We focused on rs2180619 (A > G) polymorphism and examined genotype data collected from 231 healthy females. Analyses showed how a general positivity bias in working memory (i.e., better memory for positive words) emerged as task strings lengthened only in carriers of the major allele (AA/AG). Differently, GG carriers showed better memory for affective items in general (i.e., positive and negative words). These findings are some of the first to directly highlight the role of variant on promoter of the CNR1 gene in affective working memory and to evidence a differentiation among CNR1 genotypes in terms of larger difficulties in disengaging from negative stimuli in GG carriers.

Reconstructing Dynamic Promoter Activity Profiles from Reporter Gene Data.

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Kannan, Soumya; Sams, Thomas; Maury, Jérôme; Workman, Christopher T

2018-03-16

Accurate characterization of promoter activity is important when designing expression systems for systems biology and metabolic engineering applications. Promoters that respond to changes in the environment enable the dynamic control of gene expression without the necessity of inducer compounds, for example. However, the dynamic nature of these processes poses challenges for estimating promoter activity. Most experimental approaches utilize reporter gene expression to estimate promoter activity. Typically the reporter gene encodes a fluorescent protein that is used to infer a constant promoter activity despite the fact that the observed output may be dynamic and is a number of steps away from the transcription process. In fact, some promoters that are often thought of as constitutive can show changes in activity when growth conditions change. For these reasons, we have developed a system of ordinary differential equations for estimating dynamic promoter activity for promoters that change their activity in response to the environment that is robust to noise and changes in growth rate. Our approach, inference of dynamic promoter activity (PromAct), improves on existing methods by more accurately inferring known promoter activity profiles. This method is also capable of estimating the correct scale of promoter activity and can be applied to quantitative data sets to estimate quantitative rates.

Eukaryotic genomes may exhibit up to 10 generic classes of gene promoters

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Gagniuc Paul

2012-09-01

Full Text Available Abstract Background The main function of gene promoters appears to be the integration of different gene products in their biological pathways in order to maintain homeostasis. Generally, promoters have been classified in two major classes, namely TATA and CpG. Nevertheless, many genes using the same combinatorial formation of transcription factors have different gene expression patterns. Accordingly, we tried to ask ourselves some fundamental questions: Why certain genes have an overall predisposition for higher gene expression levels than others? What causes such a predisposition? Is there a structural relationship of these sequences in different tissues? Is there a strong phylogenetic relationship between promoters of closely related species? Results In order to gain valuable insights into different promoter regions, we obtained a series of image-based patterns which allowed us to identify 10 generic classes of promoters. A comprehensive analysis was undertaken for promoter sequences from Arabidopsis thaliana, Drosophila melanogaster, Homo sapiens and Oryza sativa, and a more extensive analysis of tissue-specific promoters in humans. We observed a clear preference for these species to use certain classes of promoters for specific biological processes. Moreover, in humans, we found that different tissues use distinct classes of promoters, reflecting an emerging promoter network. Depending on the tissue type, comparisons made between these classes of promoters reveal a complementarity between their patterns whereas some other classes of promoters have been observed to occur in competition. Furthermore, we also noticed the existence of some transitional states between these classes of promoters that may explain certain evolutionary mechanisms, which suggest a possible predisposition for specific levels of gene expression and perhaps for a different number of factors responsible for triggering gene expression. Our conclusions are based on

Kidney injury and heme oxygenase-1

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Hai-xing MAI

2012-02-01

Full Text Available     Heme oxygenase-1 (HO-1 is one of the main pathways to degrade heme in mammals, and the main degradation products are free iron (Fe2+, carbon monoxide (CO, and bilirubin. Heme plays an important role in promoting cell survival, circulation of intracellular substrates, and immune regulation. Previous studies suggest that HO-1 pathway is an important internal factor in determining the susceptibility and severity of acute kidney injury (AKI. The induction of HO-1 expression can attenuate the severity of renal ischemia-reperfusion injury (IRI, and the inhibition of HO-1 expression will aggravate IRI. The present article summarizes the latest advances in research abroad and at home on protective mechanism by which HO-1 prevents AKI to further deepen our understanding of the role of HO-1 in the treatment of AKI.   

Mechanosensitive promoter region in the human HB-GAM gene

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Liedert, Astrid; Kassem, Moustapha; Claes, Lutz

2009-01-01

Mechanical loading is essential for maintaining bone mass in the adult skeleton. However, the underlying process of the transfer of the physical stimulus into a biochemical response, which is termed mechanotransduction is poorly understood. Mechanotransduction results in the modulation of gene...... cells. Analysis of the human HB-GAM gene upstream regulatory region with luciferase reporter gene assays revealed that the upregulation of HB-GAM expression occurred at the transcriptional level and was mainly dependent on the HB-GAM promoter region most upstream containing three potential AP-1 binding...

HMGB1-mediated DNA bending: Distinct roles in increasing p53 binding to DNA and the transactivation of p53-responsive gene promoters.

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Štros, Michal; Kučírek, Martin; Sani, Soodabeh Abbasi; Polanská, Eva

2018-03-01

HMGB1 is a chromatin-associated protein that has been implicated in many important biological processes such as transcription, recombination, DNA repair, and genome stability. These functions include the enhancement of binding of a number of transcription factors, including the tumor suppressor protein p53, to their specific DNA-binding sites. HMGB1 is composed of two highly conserved HMG boxes, linked to an intrinsically disordered acidic C-terminal tail. Previous reports have suggested that the ability of HMGB1 to bend DNA may explain the in vitro HMGB1-mediated increase in sequence-specific DNA binding by p53. The aim of this study was to reinvestigate the importance of HMGB1-induced DNA bending in relationship to the ability of the protein to promote the specific binding of p53 to short DNA duplexes in vitro, and to transactivate two major p53-regulated human genes: Mdm2 and p21/WAF1. Using a number of HMGB1 mutants, we report that the HMGB1-mediated increase in sequence-specific p53 binding to DNA duplexes in vitro depends very little on HMGB1-mediated DNA bending. The presence of the acidic C-terminal tail of HMGB1 and/or the oxidation of the protein can reduce the HMGB1-mediated p53 binding. Interestingly, the induction of transactivation of p53-responsive gene promoters by HMGB1 requires both the ability of the protein to bend DNA and the acidic C-terminal tail, and is promoter-specific. We propose that the efficient transactivation of p53-responsive gene promoters by HMGB1 depends on complex events, rather than solely on the promotion of p53 binding to its DNA cognate sites. Copyright © 2018 Elsevier B.V. All rights reserved.

Development of chimeric gene promoters responsive to hypoxia and ionizing radiation

International Nuclear Information System (INIS)

Zheng Aiqing; Yu Jinming

2004-01-01

The authors describe two systems that make use of gene-directed enzyme prodrug therapy, regulated by radiation or hypoxic-responsive promoters. The use of treatment-, condition- or tumor-specific promoters to control gene-directed enzyme prodrug therapy is one such method for targeting gene expression to the tumor. The development of such strategies that achieve tumor targeted expression of genes via selective promoters will enable improved specificity and targeting thereby addressing one of the major limitations of cancer gene therapy

hTERT promoter mediating gene therapy in laryngeal squamous carcinomas cells in vitro

International Nuclear Information System (INIS)

Liao Zhengkai; Zhou Yunfeng; Zhou Fuxiang; Luo Zhiguo; Xiong Jie; Bao Jie; Xie Conghua; Liu Shiquan

2007-01-01

Objective: To investigate the relationship among hTERT promoter activity, hTERT mRNA expression, and telomerase activity (TA) in laryngeal squamous carcinomas cell lines, and to evaluate the usefulness of hTERT promoter mediated gene therapy. Methods: After plasmids pGL3-hTERTp were transfected, hTEBT promoter activity, hTERT mRNA expression and TA were determined by luciferase assay, RT-PCR and TRAP-PCR-ELISA, respectively. Plasmid phTERTp-HRP was constructed and transfected, HRP expression was determined by RT-PCR and competent peroxidase activity was confirmed by enzyme activity assay. The cytotoxicity and radiosensitivity of phTERTp-HRP/IAA were determined by clonogenic assay. Results: The relative levels of hTERT promoter activity, hTERT mRNA expression and TA in Hep2R cells were 1.37-fold, 1.43-fold and 1.81-fold compared with Hep2R cells, hTERT promoter activity was closely associated with hTERT mRNA expression and TA levels (P SF 2 ) was 1.24 (Hep2R cells) and 1.20 (Hep 2cells), the parameter a of with or without IAA incubation were 0.090, 0.020 (Hep2R)and 0.099, 0.042 (Hep2). Conclusions: hTERT promoter is applicable in mediating gene therapy in different radiosensitive laryngeal squamous carcinomas cells. hTERTp-HRP/IAA gene therapy may be a promising supplementary method for radiotherapy of laryngeal squamous-cell carcinomas. (authors)

Effect of heme oxygenase-1 on radiation-induced skin injury

International Nuclear Information System (INIS)

Song Chuanjun; Meng Xingjun; Xie Ling; Chen Qing; Zhou Jundong; Zhang Shuyu; Wu Jinchang

2012-01-01

Objective: To investigate the effect of heme oxygenase-1 (HO-1) on the acute radiation-induced skin injury by gene transfer. Methods: Thirty-three male SD rats were randomly divided into three groups as PBS-injected group, Ad-EGFP-injected group and Ad-HO-1-injected group (n=11). In each group, three rats were used for determining the expression of target gene and the other rats were irradiated on the buttock skin with 40 Gy electron beam generated by a linear accelerator. Immediately after irradiation, rats were administered with a subcutaneous injection of PBS, Ad-EGFP or Ad-HO-1, respectively. Subsequently, the skin reactions were measured twice a week using the semi-quantitative skin injury scale. Results: The strong positive expression of HO-1 was observed in subcutaneous dermal tissue after injection of Ad-HO-1. Compared to the PBS-injected group or the Ad-EGFP-injected group, a significant mitigation of skin injury was observed in Ad-HO-1-injected mice 14 d after irradiation (q=0.000-0.030, P<0.05). Conclusions: HO-1 could significantly mitigate radiation-induced acute skin injury and Ad-HO-1 could be used to treat radiation-induced skin injury. (authors)

Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study.

Science.gov (United States)

McCormick, Thaís M; Canedo, Nathalie H S; Furtado, Yara L; Silveira, Filomena A; de Lima, Roberto J; Rosman, Andréa D F; Almeida Filho, Gutemberg L; Carvalho, Maria da Glória da C

2015-06-02

Human papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions. Sixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP. HPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%). The methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was

Promotion of Metastasis-associated Gene Expression in Survived PANC-1 Cells Following Trichostatin A Treatment.

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Chen, Zongjing; Yang, Yunxiu; Liu, Biao; Wang, Benquan; Sun, Meng; Zhang, Ling; Chen, Bicheng; You, Heyi; Zhou, Mengtao

2015-01-01

Histone deacetylase inhibitors represent a promising class of potential anticancer agents for the treatment of human malignancies. In this study, the effects of trichostatin A (TSA) on apoptosis, metastasis-associated gene expression, and activation of the Notch pathway in human pancreatic cancer cell lines were investigated. After treatment with TSA, cell viability and apoptosis were evaluated using the MTT [3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide] assay, Hoechst 33258 staining, and flow cytometry. Moreover, RT-PCR and western blot analyses were performed to measure the expression levels of apoptosis-associated genes (Bcl-2, Bax, and caspase-3), metastasis-associated genes (E-cadherin, vimentin, and matrix metalloproteinases), and Notch pathway activation (Notch intracellular domain, NICD). The levels of matrix metalloproteinase 2 and NICD were also semi-quantified by immunoassay. Following treatment with TSA for 24 h, PANC-1, SW1990, and MIATACA-2 cells exhibited cell death. The MTT assay revealed that TSA significantly decreased cell viability in a dose-dependent manner in PANC-1 cells. The Hoechst 33258 staining and flow cytometry results evidenced a significant increase in PANC-1 cell apoptosis following TSA treatment. The expression levels of Bax and caspase-3 were increased significantly, whereas Bcl-2 was down-regulated after TSA treatment. In the PANC-1 cells that survived after TSA treatment, the expression levels of vimentin, E-cadherin, and MMP genes were altered by the promotion of potential metastasis and increased expression of NICD. TSA can induce apoptosis of pancreatic cancer cells. In addition, the up-regulation of metastasis-related genes and the activation of the Notch pathway in the survived PANC-1 cells may be associated with a too-low level of TSA or resistance to TSA.

Identification of distal silencing elements in the murine interferon-A11 gene promoter.

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Roffet, P; Lopez, S; Navarro, S; Bandu, M T; Coulombel, C; Vignal, M; Doly, J; Vodjdani, G

1996-08-01

The murine interferon-A11 (Mu IFN-A11) gene is a member of the IFN-A multigenic family. In mouse L929 cells, the weak response of the gene's promoter to viral induction is due to a combination of both a point mutation in the virus responsive element (VRE) and the presence of negatively regulating sequences surrounding the VRE. In the distal part of the promoter, the negatively acting E1E2 sequence was delimited. This sequence displays an inhibitory effect in either orientation or position on the inducibility of a virus-responsive heterologous promoter. It selectively represses VRE-dependent transcription but is not able to reduce the transcriptional activity of a VRE-lacking promoter. In a transient transfection assay, an E1E2-containing DNA competitor was able to derepress the native Mu IFN-A11 promoter. Specific nuclear factors bind to this sequence; thus the binding of trans-regulators participates in the repression of the Mu IFN-A11 gene. The E1E2 sequence contains an IFN regulatory factor (IRF)-binding site. Recombinant IRF2 binds this sequence and anti-IRF2 antibodies supershift a major complex formed with nuclear extracts. The protein composing the complex is 50 kDa in size, indicating the presence of IRF2 or antigenically related proteins in the complex. The Mu IFN-A11 gene is the first example within the murine IFN-A family, in which a distal promoter element has been identified that can negatively modulate the transcriptional response to viral induction.

Radioprotective effect of hematopoietic growth factor gene therapy regulated by Egr-1 promoter on radiation injury of SCID mice

International Nuclear Information System (INIS)

Du Nan; Pei Xuetao; Luo Chengji; Su Yongping; Cheng Tianmin

2002-01-01

Objective: To explore the radioprotective effect of the expression of hematopoietic growth factors regulated by radio-inducible promoter on radiation injury. Methods: The human FL cDNA and EGFP cDNA were linked together with an internal ribosome entry site (IRES) and then inserted into the eukaryotic expression vector pCI-neo with the Egr-1 promoter (Egr-EF), and further transduced into bone marrow stromal cell lines HFCL (HFCL/EF). The HFCL/EF and CD34 + cells from human umbilical cord blood were transplanted i.v. one after the other into sublethally irradiated severe combined immunodeficient (SCID) mice. The number of peripheral blood WBC and human cells engrafted in recipient mice were detected by flow cytometry and CFU-GM assay. Results: In contrast to two control groups (HFCL and HFCL/F), HFCL/EF (the Egr-1 regulatory element-driven expression of FL gene therapy) resulted in a proportionally obvious increase in the number of the WBC at early stage after irradiation. Significant differences were found for CD45 + , CD34 + , CFU-GM, and nucleated cells in the bone marrow. Conclusion: Hematopoietic growth factor gene therapy regulated by radio-inducible promoter has radioprotective effect on radiation hematopoietic injury

Heme oxygenase up-regulation under ultraviolet-B radiation is not epigenetically restricted and involves specific stress-related transcriptions factors

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Diego Santa-Cruz

2017-08-01

Full Text Available Heme oxygenase-1 (HO-1 plays a protective role against oxidative stress in plants. The mechanisms regulating its expression, however, remain unclear. Here we studied the methylation state of a GC rich HO-1 promoter region and the expression of several stress-related transcription factors (TFs in soybean plants subjected to ultraviolet-B (UV-B radiation. Genomic DNA and total RNA were isolated from leaves of plants irradiated with 7.5 and 15 kJ m-2 UV-B. A 304 bp HO-1 promoter region was amplified by PCR from sodium bisulfite-treated DNA, cloned into pGEMT plasmid vector and evaluated by DNA sequencing. Bisulfite sequencing analysis showed similar HO-1 promoter methylation levels in control and UV-B-treated plants (C: 3.4±1.3%; 7.5: 2.6±0.5%; 15: 3.1±1.1%. Interestingly, HO-1 promoter was strongly unmethylated in control plants. Quantitative RT-PCR analysis of TFs showed that GmMYB177, GmMYBJ6, GmWRKY21, GmNAC11, GmNAC20 and GmGT2A but not GmWRK13 and GmDREB were induced by UV-B radiation. The expression of several TFs was also enhanced by hemin, a potent and specific HO inducer, inferring that they may mediate HO-1 up-regulation. These results suggest that soybean HO-1 gene expression is not epigenetically regulated. Moreover, the low level of HO-1 promoter methylation suggests that this antioxidant enzyme can rapidly respond to environmental stress. Finally, this study has identified some stress-related TFs involved in HO-1 up-regulation under UV-B radiation. Keywords: Heme oxygenase, DNA methylation, Transcription factors, Ultraviolet-B radiation, Glycine max

The Cytoprotective Effects of E-α-(4-Methoxyphenyl-2',3,4,4'-Tetramethoxychalcone (E-α-p-OMe-C6H4-TMC--A Novel and Non-Cytotoxic HO-1 Inducer.

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Kai B Kaufmann

Full Text Available Cell protection against different noxious stimuli like oxidative stress or chemical toxins plays a central role in the treatment of many diseases. The inducible heme oxygenase isoform, heme oxygenase-1 (HO-1, is known to protect cells against a variety of harmful conditions including apoptosis. Because a number of medium strong electrophiles from a series of α-X-substituted 2',3,4,4'-tetramethoxychalcones (α-X-TMCs, X = H, F, Cl, Br, I, CN, Me, p-NO2-C6H4, Ph, p-OMe-C6H4, NO2, CF3, COOEt, COOH had proven to activate Nrf2 resulting in HO-1 induction and inhibit NF-κB downstream target genes, their protective effect against staurosporine induced apoptosis and reactive oxygen species (ROS production was investigated. RAW264.7 macrophages treated with 19 different chalcones (15 α-X-TMCs, chalcone, 2'-hydroxychalcone, calythropsin and 2'-hydroxy-3,4,4'-trimethoxychalcone prior to staurosporine treatment were analyzed for apoptosis and ROS production, as well as HO-1 protein expression and enzyme activity. Additionally, Nrf2 and NF-κB activity was assessed. We found that amongst all tested chalcones only E-α-(4-methoxyphenyl-2',3,4,4'-tetramethoxychalcone (E-α-p-OMe-C6H4-TMC demonstrated a distinct, statistically significant antiapoptotic effect in a dose dependent manner, showing no toxic effects, while its double bond isomer Z-α-p-OMe-C6H4-TMC displayed no significant activity. Also, E-α-p-OMe-C6H4-TMC induced HO-1 protein expression and increased HO-1 activity, whilst inhibition of HO-1 by SnPP-IX abolished its antiapoptotic effect. The only weakly electrophilic chalcone E-α-p-OMe-C6H4-TMC reduced the staurosporine triggered formation of ROS, while inducing the translocation of Nrf2 into the nucleus. Furthermore, staurosporine induced NF-κB activity was attenuated following E-α-p-OMe-C6H4-TMC treatment. Overall, E-α-p-OMe-C6H4-TMC demonstrated its effective cytoprotective potential via a non-toxic induction of HO-1 in RAW264

Promoter Methylation Analysis of IDH Genes in Human Gliomas

International Nuclear Information System (INIS)

Flanagan, Simon; Lee, Maggie; Li, Cheryl C. Y.; Suter, Catherine M.; Buckland, Michael E.

2012-01-01

Mutations in isocitrate dehydrogenase (IDH)-1 or -2 are found in the majority of WHO grade II and III astrocytomas and oligodendrogliomas, and secondary glioblastomas. Almost all described mutations are heterozygous missense mutations affecting a conserved arginine residue in the substrate binding site of IDH1 (R132) or IDH2 (R172). But the exact mechanism of IDH mutations in neoplasia is not understood. It has been proposed that IDH mutations impart a “toxic gain-of-function” to the mutant protein, however a dominant-negative effect of mutant IDH has also been described, implying that IDH may function as a tumor suppressor gene. As most, if not all, tumor suppressor genes are inactivated by epigenetic silencing, in a wide variety of tumors, we asked if IDH1 or IDH2 carry the epigenetic signature of a tumor suppressor by assessing cytosine methylation at their promoters. Methylation was quantified in 68 human brain tumors, including both IDH-mutant and IDH wildtype, by bisulfite pyrosequencing. In all tumors examined, CpG methylation levels were less than 8%. Our data demonstrate that inactivation of IDH function through promoter hypermethylation is not common in human gliomas and other brain tumors. These findings do not support a tumor suppressor role for IDH genes in human gliomas.

Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9.

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Matsunaga, Taichi; Yamashita, Jun K

2014-02-07

Specific gene knockout and rescue experiments are powerful tools in developmental and stem cell biology. Nevertheless, the experiments require multiple steps of molecular manipulation for gene knockout and subsequent rescue procedures. Here we report an efficient and single step strategy to generate gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 genome editing technology. We inserted a tetracycline-regulated inducible gene promoter (tet-OFF/TRE-CMV) upstream of the endogenous promoter region of vascular endothelial growth factor receptor 2 (VEGFR2/Flk1) gene, an essential gene for endothelial cell (EC) differentiation, in mouse embryonic stem cells (ESCs) with homologous recombination. Both homo- and hetero-inserted clones were efficiently obtained through a simple selection with a drug-resistant gene. The insertion of TRE-CMV promoter disrupted endogenous Flk1 expression, resulting in null mutation in homo-inserted clones. When the inserted TRE-CMV promoter was activated with doxycycline (Dox) depletion, Flk1 expression was sufficiently recovered from the downstream genomic Flk1 gene. Whereas EC differentiation was almost completely perturbed in homo-inserted clones, Flk1 rescue with TRE-CMV promoter activation restored EC appearance, indicating that phenotypic changes in EC differentiation can be successfully reproduced with this knockout-rescue system. Thus, this promoter insertion strategy with CRISPR/Cas9 would be a novel attractive method for knockout-rescue experiments. Copyright © 2014 Elsevier Inc. All rights reserved.

The Relationship between FHIT Gene Promoter Methylation and Lung Cancer Risk: 
a Meta-analysis

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Yichang SUN

2014-03-01

Full Text Available Background and objective Tumor-suppressor gene promoter DNA methylation in tumor cells is associated with its reduced expression. FHIT (fragile histindine triad was one of the important tumor suppressor genes which was found hypermethylated in the promoter region in most of tumors. The aim of this study is to evaluate the relationship between FIHT gene promother methylation and lung cancer risk by meta-analysis. Methods By searching Pubmed, CNKI and Wanfang, the open published articles related to FHIT gene promoter methylation and lung carcinoma risk were collected. The odds ratio (OR and range of FHIT gene of cancer tissue of lung cancer patients compared with normal lung tissue, plasma and the bronchial lavage fluid were pooled by statistical software Stata 11.0. Results Eleven studies were finally included in this meta-analysis. The median methylation rate were Pmedian=40.0% (0-68.3%, Pmedian=8.7% (0-35.0%, Pmedian=33.3% (17.1%-38.3% and Pmedian=35.9% (31.1%-50.8% in cancer tissue, NLT, BALF and plasm respectively. The pooled results showed the methylation rate in tumor tissue was much higer than that of NLT OR=5.82 (95%CI: 3.74-9.06, P0.05 and plasma OR=1.41 (95%CI: 0.90-2.20, P>0.05. Conclusion Hypermethylation of FHIT gene promoter region was found more frequent in cancer tissue than that of NLT which may demonstrated association between lung cancer risk and FHIT gene promoter methylation.

Regulation of the intronic promoter of rat estrogen receptor alpha gene, responsible for truncated estrogen receptor product-1 expression.

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Schausi, Diane; Tiffoche, Christophe; Thieulant, Marie-Lise

2003-07-01

We have characterized the intronic promoter of the rat estrogen receptor (ER) alpha gene, responsible for the lactotrope-specific truncated ER product (TERP)-1 isoform expression. Transcriptional regulation was investigated by transient transfections using 5'-deletion constructs. TERP promoter constructs were highly active in MMQ cells, a pure lactotrope cell line, whereas a low basal activity was detected in alphaT3-1 gonadotrope cells or in COS-7 monkey kidney cells. Serial deletion analysis revealed that 1) a minimal -693-bp region encompassing the TATA box is sufficient to allow lactotrope-specific expression; 2) the promoter contains strong positive cis-acting elements both in the distal and proximal regions, and 3) the region spanning the -1698/-1194 region includes repressor elements. Transient transfection studies, EMSAs, and gel shifts demonstrated that estrogen activates the TERP promoter via an estrogen-responsive element (ERE1) located within the proximal region. Mutation of ERE1 site completely abolishes the estradiol-dependent transcription, indicating that ERE1 site is sufficient to confer estrogen responsiveness to TERP promoter. In addition, ERalpha action was synergized by transfection of the pituitary-specific factor Pit-1. EMSAs showed that a single Pit-1 DNA binding element in the vicinity of the TATA box is sufficient to confer response by the TERP promoter. In conclusion, we demonstrated, for the first time, that TERP promoter regulation involves ERE and Pit-1 cis-elements and corresponding trans-acting factors, which could play a role in the physiological changes that occur in TERP-1 transcription in lactotrope cells.

Synthesis and characterization of composites HoMn_1_-_x(Ni,Co)_xO_3

International Nuclear Information System (INIS)

Santos, Cassio Morilla dos

2011-01-01

In this work was accomplished the synthesis process and structural and magnetic characterization of HoMn_1_-_X(Ni,Co)_XO_3 compounds of perovskite structure. The samples synthesis were performed through modified polymeric precursor method. After synthesis and solvent removal, the polymer resin formed was treated at 350 deg C/4h for organic constituents removal, followed by heating treatment at 500 deg C/4h and 900 deg C/20h to obtain the crystalline phase. For structural characterization, it was used D10B-XPD beam line of Laboratorio Nacional de Luz Sincrotron (LNLS), where X-rays wavelengths below cobalt, manganese and nickel absorption edge, were used. The formation of HoNi_0_._5_0Mn_0_._5_0O_3, HoCo_0_._5_0Mn_0_._5_0O_3 and HoNi_0_._2_5Co_0_._2_5Mn_0_._5_0O_3 phases were observed by X-ray diffraction technique. By Rietveld refinement method for sample HoNi_0_._2_5Co_0_._2_5Mn_0_._5_0O_3, it was determined that cobalt and nickel had similar occupations at the top and bottom of unit cell, while the manganese preferentially occupied plan 002. The magnetic response of samples was studied through magnetization curves according to the temperature function and the applied magnetic field. The ZFC curves showed a paramagnetic response associated to holmium magnetic moment, and ferromagnetism, antiferromagnetism and ferrimagnetism coexistence, due to sublattices formed by transition metals. The FC curves evidenced the spin reversal phenomenon, associated to the interaction between the sublattice formed by transition metals with sublattices formed by rare-earth, considering a mechanism of antiferromagnetic exchange interaction. (author)

Identification of cis-acting regulatory elements in the human oxytocin gene promoter.

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Richard, S; Zingg, H H

1991-12-01

The expression of hormone-inducible genes is determined by the interaction of trans-acting factors with hormone-inducible elements and elements mediating basal and cell-specific expression. We have shown earlier that the gene encoding the hypothalamic nonapeptide oxytocin (OT) is under the control of an estrogen response element (ERE). The present study was aimed at identifying cis-acting elements mediating basal expression of the OT gene. A construct containing sequences -381 to +36 of the human OT gene was linked to a reporter gene and transiently transfected into a series of neuronal and nonneuronal cell lines. Expression of this construct was cell specific: it was highest in the neuroblastoma-derived cell line, Neuro-2a, and lowest in NIH 3T3 and JEG-3 cells. By 5' deletion analysis, we determined that a segment from -49 to +36 was capable of mediating cells-pecific promoter activity. Within this segment, we identified three proximal promoter elements (PPE-1, PPE-2, and PPE-3) that are each required for promoter activity. Most notably, mutation of a conserved purine-rich element (GAGAGA) contained within PPE-2 leads to a 10-fold decrease in promoter strength. Gel mobility shift analysis with three different double-stranded oligonucleotides demonstrated that each proximal promoter element binds distinct nuclear factors. In each case, only the homologous oligonucleotide, but neither of the oligonucleotides corresponding to adjacent elements, was able to act as a competitor. Thus, a different set of factors appears to bind independently to each element. By reinserting the homologous ERE or a heterologous glucocorticoid response element upstream of intact or altered proximal promoter segments we determined that removal or mutation of proximal promoter elements decreases basal expression, but does not abrogate the hormone responsiveness of the promoter. In conclusion, these results indicate that an important component of the transcriptional activity of the OT

Gene Expression in Class 2 Integrons Is SOS-Independent and Involves Two Pc Promoters.

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Jové, Thomas; Da Re, Sandra; Tabesse, Aurore; Gassama-Sow, Amy; Ploy, Marie-Cécile

2017-01-01

Integrons are powerful bacterial genetic elements that permit the expression and dissemination of antibiotic-resistance gene cassettes. They contain a promoter Pc that allows the expression of gene cassettes captured through site-specific recombination catalyzed by IntI, the integron-encoded integrase. Class 1 and 2 integrons are found in both clinical and environmental settings. The regulation of intI and of Pc promoters has been extensively studied in class 1 integrons and the regulatory role of the SOS response on intI expression has been shown. Here we investigated class 2 integrons. We characterized the P intI2 promoter and showed that intI2 expression is not regulated via the SOS response. We also showed that, unlike class 1 integrons, class 2 integrons possess not one but two active Pc promoters that are located within the attI2 region that seem to contribute equally to gene cassette expression. Class 2 integrons mostly encode an inactive truncated integrase, but the rare class 2 integrons that encode an active integrase are associated with less efficient Pc2 promoter variants. We propose an evolutionary model for class 2 integrons in which the absence of repression of the integrase gene expression led to mutations resulting in either inactive integrase or Pc variants of weaker activity, thereby reducing the potential fitness cost of these integrons.

Promoter hypermethylation of mismatch repair gene hMLH1 predicts the clinical response of malignant astrocytomas to nitrosourea.

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Fukushima, Takao; Katayama, Yoichi; Watanabe, Takao; Yoshino, Atsuo; Ogino, Akiyoshi; Ohta, Takashi; Komine, Chiaki

2005-02-15

In certain types of human cancers, transcriptional inactivation of hMLH1 by promoter hypermethylation plays a causal role in the loss of mismatch repair functions that modulate cytotoxic pathways in response to DNA-damaging agents. The aim of the present study was to investigate the role of promoter methylation of the hMLH1 gene in malignant astrocytomas. We examined the hMLH1 promoter methylation in a homogeneous cohort of patients with 41 malignant astrocytomas treated by 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-2(2-chloroethyl)-3-nitrosourea chemotherapy in combination with radiation and interferon therapy, and assessed the correlation of such methylation with clinical outcome. hMLH1 promoter methylation was found in 6 (15%) of the 41 newly diagnosed malignant astrocytomas. Hypermethylation of the hMLH1 promoter corresponded closely with a loss of immunohistochemical staining for hMLH1 protein (P = 0.0013). Patients with hMLH1-methylated tumors displayed a greater chance of responding to adjuvant therapy as compared with those with hMLH1-unmethylated tumors (P = 0.0150). The presence of hMLH1 hypermethylation was significantly associated with a longer progression-free survival on both univariate analysis (P = 0.0340) and multivariate analysis (P = 0.0161). The present study identified hMLH1 methylation status as a predictor of the clinical response of malignant astrocytomas to chloroethylnitrosourea-based adjuvant therapy. The findings obtained suggest that determination of the methylation status of hMLH1 could provide a potential basis for designing rational chemotherapeutic strategies, as well as for predicting prognosis.

Constitutive expression of the K-domain of a Vaccinium corymbosum SOC1-like (VcSOC1-K) MADS-box gene is sufficient to promote flowering in tobacco.

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Song, Guo-qing; Walworth, Aaron; Zhao, Dongyan; Hildebrandt, Britton; Leasia, Michael

2013-11-01

The K-domain of a blueberry-derived SOC1 -like gene promotes flowering in tobacco without negatively impacting yield, demonstrating potential for manipulation of flowering time in horticultural crops. The SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and SOC1-likes, belonging to the MIKC(c) (type II) MADS-box gene subfamily, are major floral activators and integrators of plant flowering. Both MADS-domains and K (Keratin)-domains are highly conserved in MIKC(c)-type MADS proteins. While there are many reports on overexpression of intact MIKC(c)-type MADS-box genes, few studies have been conducted to investigate the effects of the K-domains. In this report, a 474-bp K-domain of Vaccinium SOC1-like (VcSOC1-K) was cloned from the cDNA library of the northern highbush blueberry (Vaccinium corymbosum L.). Functional analysis of the VcSOC1-K was conducted by ectopically expressing of 35S:VcSOC1-K in tobacco. Reverse transcription PCR confirmed expression of the VcSOC1-K in T0 plants. Phenotypically, T1 transgenic plants (10 T1 plants/event) flowered sooner after seeding, and were shorter with fewer leaves at the time of flowering, than nontransgenic plants; but seed pod production of transgenic plants was not significantly affected. These results demonstrate that overexpression of the K-domain of a MIKC(c)-type MADS-box gene alone is sufficient to promote early flowering and more importantly without affecting seed production.

Expression of the neutral protease gene from a thermophilic Bacillus sp BT1 strain in Bacillus subtilis and its natural host : Identification of a functional promoter

NARCIS (Netherlands)

Vecerek, B; Venema, G

The expression of the neutral protease gene (npr) from the thermophilic Bacillus sp. BT1 strain was studied in its natural host and in mesophilic Bacillus subtilis. In the thermophilic BT1 strain, the transcription of the protease gene is initiated from its own promoter, just 5' to the gene. In

Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein

International Nuclear Information System (INIS)

Wu, C.C.; Hsieh, C.W.; Lai, P.H.; Lin, J.B.; Liu, Y.C.; Wung, B.S.

2006-01-01

Acrolein is a highly electrophilic α,β-unsaturated aldehyde that is present in cigarette smoke. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various such electrophilic compounds. In this study, the regulatory effects of acrolein upon the expression of HO-1 were investigated in endothelial cells (ECs). We demonstrate that acrolein induces the elevation of HO-1 protein levels, and subsequent enzyme activity, at non-cytotoxic concentrations. An additional α,β-unsaturated aldehyde, cinnamaldehyde, was also found to increase HO-1 expression and have less cytotoxicity than acrolein. Moreover, acrolein-mediated HO-1 induction is abrogated in the presence of actinomycin D and cycloheximide. Nrf2 is a transcription factor involved in the induction of HO-1 through an antioxidant response element (ARE) in the promoter region of the HO-1 gene. We show that acrolein induces Nrf2 translocation and ARE-luciferase reporter activity. Acrolein was also found to induce the production of both superoxide and H 2 O 2 at levels greater than 100 μM. However, with the exception of NAC, no antioxidant generated any effect upon acrolein-dependent HO-1 expression in ECs. Our present findings suggest that reactive oxygen species (ROS) may not be a major modulator for HO-1 induction. Using buthionine sulfoximine to deplete the intracellular GSH levels further enhanced the effects of acrolein. We also found that cellular GSH level was rapidly reduced after both 10 and 100 μM acrolein treatment. However, after 6 h of exposure to ECs, only 10 μM acrolein treatment increases GSH level. In addition, only the JNK inhibitor SP600125 and tyrosine kinase inhibitor genistein had any significant inhibitory impact upon the upregulation of HO-1 by acrolein. Pretreatment with a range of other PI3 kinase inhibitors, including wortmannin and LY294002, showed no effects. Hence, we show in our current experiments that a sublethal concentration of acrolein is in fact a novel HO-1 inducer

Upregulation of endothelial heme oxygenase-1 expression through the activation of the JNK pathway by sublethal concentrations of acrolein.

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Wu, C C; Hsieh, C W; Lai, P H; Lin, J B; Liu, Y C; Wung, B S

2006-08-01

Acrolein is a highly electrophilic alpha,beta-unsaturated aldehyde that is present in cigarette smoke. Heme oxygenase-1 (HO-1) is a cytoprotective enzyme activated by various such electrophilic compounds. In this study, the regulatory effects of acrolein upon the expression of HO-1 were investigated in endothelial cells (ECs). We demonstrate that acrolein induces the elevation of HO-1 protein levels, and subsequent enzyme activity, at non-cytotoxic concentrations. An additional alpha,beta-unsaturated aldehyde, cinnamaldehyde, was also found to increase HO-1 expression and have less cytotoxicity than acrolein. Moreover, acrolein-mediated HO-1 induction is abrogated in the presence of actinomycin D and cycloheximide. Nrf2 is a transcription factor involved in the induction of HO-1 through an antioxidant response element (ARE) in the promoter region of the HO-1 gene. We show that acrolein induces Nrf2 translocation and ARE-luciferase reporter activity. Acrolein was also found to induce the production of both superoxide and H2O2 at levels greater than 100 microM. However, with the exception of NAC, no antioxidant generated any effect upon acrolein-dependent HO-1 expression in ECs. Our present findings suggest that reactive oxygen species (ROS) may not be a major modulator for HO-1 induction. Using buthionine sulfoximine to deplete the intracellular GSH levels further enhanced the effects of acrolein. We also found that cellular GSH level was rapidly reduced after both 10 and 100 microM acrolein treatment. However, after 6 h of exposure to ECs, only 10 microM acrolein treatment increases GSH level. In addition, only the JNK inhibitor SP600125 and tyrosine kinase inhibitor genistein had any significant inhibitory impact upon the upregulation of HO-1 by acrolein. Pretreatment with a range of other PI3 kinase inhibitors, including wortmannin and LY294002, showed no effects. Hence, we show in our current experiments that a sublethal concentration of acrolein is in fact a

Spatiotemporal expression of heme oxygenase-1 detected by in vivo bioluminescence after hepatic ischemia in HO-1/luc mice

NARCIS (Netherlands)

Su, Huawei; van Dam, Gooitzen M.; Buis, Carlijn I.; Visser, Dorien S.; Hesselink, Jan Willem; Schuurs, Theo A.; Leuvenink, Henri G. D.; Contag, Christopher H.; Porte, Robert J.

Upregulation of heme oxygenase-1 (HO-1) has been proposed as a critical mechanism protecting against cellular stress during liver transplantation, providing a potential target for new therapeutic interventions. We investigated the feasibility of in vivo bioluminescence imaging (BLI) to noninvasively

[Inactivation of PMS2 gene by promoter methylation in nasopharyngeal carcinoma].

Science.gov (United States)

Ni, H F; Jiang, B; Zhou, Z; Li, Y; Yuan, X Y; Cao, X L; Huang, G W

2016-11-23

Objective: To investigate the inactivation of PMS2 gene mediated by promoter methylation and its regulatory mechanism in nasopharyngeal carcinoma (NPC). Methods: Fifty-four NPC tissues, 16 normal nasopharyngeal epithelia (NNE), 5 NPC cell lines (CNE1, CNE2, TWO3, HNE1 and HONE1) and 1 normal nasopharyngeal epithelial cell line (NP69) were collected.Methylation-specific PCR (MSP) was used to detect the PMS2 promoter methylation, semi-quantitative reverse transcription PCR (qRT-PCR) was applied to determine its mRNA expression, and immunohistochemistry (IHC) was used to detect the protein expression of PMS2. The expressions of PMS2 mRNA in CNE1 and CNE2 cells before and after treated with methyltransferase inhibitor 5-aza-2-deoxycytidine were analyzed by qRT-PCR. The impact of methylation and demethylation on the mRNA expression of PMS2, and the association of mRNA and protein expression of PMS2 with clinicopathological features of nasopharyngeal cancer were analyzed. Results: Methylation of PMS2 gene was detected in all of the five NPC cell lines, but not in normal nasopharyngeal epithelial NP69 cells. The methylation rate of PMS2 gene in NPC tissues was 63% (34/54), significantly higher than that of the normal nasopharyngeal epithelia (0/16, P PMS2 mRNA and protein were significantly down-regulated in the 54 NPC tissues when compared with those in the 16 NNE tissues ( P PMS2 mRNA was restored in the CNE1 and CNE2 cells.However, the expressions of PMS2 mRNA and protein were not significantly correlated with patients' age, gender, TNM stage, histopathologic type or lymph node metastasis ( P >0.05 for all). Conclusions: Promoter methylation-mediated inactivation of PMS2 gene participates in carcinogenesis and development of NPC. PMS2 may be a candidate tumor suppressor in the treatment for patients with inactivation of PMS2 promoter methylation.

Human Leukemic Cells performing Oxidative Phosphorylation (OXPHOS Generate an Antioxidant Response Independently of Reactive Oxygen species (ROS Production

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Abrar Ul Haq Khan

2016-01-01

Full Text Available Tumor cell metabolism is altered during leukemogenesis. Cells performing oxidative phosphorylation (OXPHOS generate reactive oxygen species (ROS through mitochondrial activity. To limit the deleterious effects of excess ROS, certain gene promoters contain antioxidant response elements (ARE, e.g. the genes NQO-1 and HO-1. ROS induces conformational changes in KEAP1 and releases NRF2, which activates AREs. We show in vitro and in vivo that OXPHOS induces, both in primary leukemic cells and cell lines, de novo expression of NQO-1 and HO-1 and also the MAPK ERK5 and decreases KEAP1 mRNA. ERK5 activates the transcription factor MEF2, which binds to the promoter of the miR-23a–27a–24-2 cluster. Newly generated miR-23a destabilizes KEAP1 mRNA by binding to its 3′UTR. Lower KEAP1 levels increase the basal expression of the NRF2-dependent genes NQO-1 and HO-1. Hence, leukemic cells performing OXPHOS, independently of de novo ROS production, generate an antioxidant response to protect themselves from ROS.

Magnetic, transport and magnetocaloric properties in the Laves phase intermetallic Ho (Co1−xAlx)2 compounds

International Nuclear Information System (INIS)

Ivanova, T.I.; Nikitin, S.A.; Tskhadadze, G.A.; Koshkid’ko, Yu.S.; Suski, W.; Iwasieczko, W.; Badurski, D.

2014-01-01

Highlights: • The Al influence on magnetic properties of the Ho (Co 1-x Al x ) 2 compounds is analyzed. • The first-order magnetic transition appears in sample with Al concentrations x ≤ 0.06. • The MCE and Curie temperature TC demonstrate complex Al concentration dependences. • The magnetoresistance for sample with Al concentration x = 0.06 (58%) is maximum. • High magnetic fields changes the Curie temperature T c of the Ho (Co 1−x Al x ) 2 compounds. - Abstract: The magnetization, magnetoresistivity and magnetocaloric effect (MCE) of the Ho (Co 1−x Al x ) 2 Laves phase intermetallic compounds for x ⩽ 0.2 have been investigated. Complex measurements have been carried out in order to determine the influence of substitution in the Co sublattice by Al on the Co moment, type of the magnetic transition and related properties of these compounds. A comparative analysis of the magnetic, transport and magnetocaloric properties of Ho (Co 1−x Al x ) 2 alloys under various Al concentration is represented. Substitutions at the Co site by Al are found to result in the appearance of itinerant electron metamagnetism (IEM) at the small Al concentrations and in positive magnetovolume effect, leading to an initial increase in the ordering temperature; on the other hand the magnetic phase transition temperature as well as ΔT (MCE) do not depend in direct way on the Al concentration. The 16% increase of magnetocaloric effect for the alloy with x = 0.02 is detected in relation to maternal HoCo 2 . A giant value of magnetoresistivity (58%) is observed for the alloy with the same Al concentration

Selenolate complexes of CYP101 and the heme-bound hHO-1/H25A proximal cavity mutant.

Science.gov (United States)

Jiang, Yongying; Ortiz de Montellano, Paul R

2008-05-05

Thiolate and selenolate complexes of CYP101 (P450cam) and the H25A proximal cavity mutant of heme-bound human heme oxygenase-1 (hHO-1) have been examined by UV-vis spectroscopy. Both thiolate and selenolate ligands bound to the heme distal side in CYP101 and gave rise to characteristic hyperporphyrin spectra. Thiolate ligands also bound to the proximal side of the heme in the cavity created by the H25A mutation in hHO-1, giving a Soret absorption similar to that of the H25C hHO-1 mutant. Selenolate ligands also bound to this cavity mutant under anaerobic conditions but reduced the heme iron to the ferrous state, as shown by the formation of a ferrous CO complex. Under aerobic conditions, the selenolate ligand but not the thiolate ligand was rapidly oxidized. These results indicate that selenocysteine-coordinated heme proteins will not be stable species in the absence of a redox potential stabilizing effect.

Quantitative promoter methylation analysis of multiple cancer-related genes in renal cell tumors

International Nuclear Information System (INIS)

Costa, Vera L; Henrique, Rui; Ribeiro, Franclim R; Pinto, Mafalda; Oliveira, Jorge; Lobo, Francisco; Teixeira, Manuel R; Jerónimo, Carmen

2007-01-01

Aberrant promoter hypermethylation of cancer-associated genes occurs frequently during carcinogenesis and may serve as a cancer biomarker. In this study we aimed at defining a quantitative gene promoter methylation panel that might identify the most prevalent types of renal cell tumors. A panel of 18 gene promoters was assessed by quantitative methylation-specific PCR (QMSP) in 85 primarily resected renal tumors representing the four major histologic subtypes (52 clear cell (ccRCC), 13 papillary (pRCC), 10 chromophobe (chRCC), and 10 oncocytomas) and 62 paired normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters. Significant differences in methylation levels among the four subtypes of renal tumors were found for CDH1 (p = 0.0007), PTGS2 (p = 0.002), and RASSF1A (p = 0.0001). CDH1 hypermethylation levels were significantly higher in ccRCC compared to chRCC and oncocytoma (p = 0.00016 and p = 0.0034, respectively), whereas PTGS2 methylation levels were significantly higher in ccRCC compared to pRCC (p = 0.004). RASSF1A methylation levels were significantly higher in pRCC than in normal tissue (p = 0.035). In pRCC, CDH1 and RASSF1A methylation levels were inversely correlated with tumor stage (p = 0.031) and nuclear grade (p = 0.022), respectively. The major subtypes of renal epithelial neoplasms display differential aberrant CDH1, PTGS2, and RASSF1A promoter methylation levels. This gene panel might contribute to a more accurate discrimination among common renal tumors, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses

HOX Gene Promoter Prediction and Inter-genomic Comparison: An Evo-Devo Study

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Marla A. Endriga

2010-10-01

Full Text Available Homeobox genes direct the anterior-posterior axis of the body plan in eukaryotic organisms. Promoter regions upstream of the Hox genes jumpstart the transcription process. CpG islands found within the promoter regions can cause silencing of these promoters. The locations of the promoter regions and the CpG islands of Homeo sapiens sapiens (human, Pan troglodytes (chimpanzee, Mus musculus (mouse, and Rattus norvegicus (brown rat are compared and related to the possible influence on the specification of the mammalian body plan. The sequence of each gene in Hox clusters A-D of the mammals considered were retrieved from Ensembl and locations of promoter regions and CpG islands predicted using Exon Finder. The predicted promoter sequences were confirmed via BLAST and verified against the Eukaryotic Promoter Database. The significance of the locations was determined using the Kruskal-Wallis test. Among the four clusters, only promoter locations in cluster B showed significant difference. HOX B genes have been linked with the control of genes that direct the development of axial morphology, particularly of the vertebral column bones. The magnitude of variation among the body plans of closely-related species can thus be partially attributed to the promoter kind, location and number, and gene inactivation via CpG methylation.

Genome-wide screening identifies Plasmodium chabaudi-induced modifications of DNA methylation status of Tlr1 and Tlr6 gene promoters in liver, but not spleen, of female C57BL/6 mice.

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Al-Quraishy, Saleh; Dkhil, Mohamed A; Abdel-Baki, Abdel Azeem S; Delic, Denis; Santourlidis, Simeon; Wunderlich, Frank

2013-11-01

Epigenetic reprogramming of host genes via DNA methylation is increasingly recognized as critical for the outcome of diverse infectious diseases, but information for malaria is not yet available. Here, we investigate the effect of blood-stage malaria of Plasmodium chabaudi on the DNA methylation status of host gene promoters on a genome-wide scale using methylated DNA immunoprecipitation and Nimblegen microarrays containing 2,000 bp oligonucleotide features that were split into -1,500 to -500 bp Ups promoters and -500 to +500 bp Cor promoters, relative to the transcription site, for evaluation of differential DNA methylation. Gene expression was analyzed by Agilent and Affymetrix microarray technology. Challenging of female C57BL/6 mice with 10(6) P. chabaudi-infected erythrocytes resulted in a self-healing outcome of infections with peak parasitemia on day 8 p.i. These infections induced organ-specific modifications of DNA methylation of gene promoters. Among the 17,354 features on Nimblegen arrays, only seven gene promoters were identified to be hypermethylated in the spleen, whereas the liver exhibited 109 hyper- and 67 hypomethylated promoters at peak parasitemia in comparison with non-infected mice. Among the identified genes with differentially methylated Cor-promoters, only the 7 genes Pigr, Ncf1, Klkb1, Emr1, Ndufb11, and Tlr6 in the liver and Apol6 in the spleen were detected to have significantly changed their expression. Remarkably, the Cor promoter of the toll-like receptor Tlr6 became hypomethylated and Tlr6 expression increased by 3.4-fold during infection. Concomitantly, the Ups promoter of the Tlr1 was hypermethylated, but Tlr1 expression also increased by 11.3-fold. TLR6 and TLR1 are known as auxillary receptors to form heterodimers with TLR2 in plasma membranes of macrophages, which recognize different pathogen-associated molecular patterns (PAMPs), as, e.g., intact 3-acyl and sn-2-lyso-acyl glycosylphosphatidylinositols of P. falciparum

Embedding of $^{163}$Ho and $^{166m}$ Ho in the energy absorbers of low temperature metallic magnetic calorimeters

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The calorimetric measurement of the $^{163}$Ho electron capture spectrum is a promising tool to investigate the electron neutrino mass. A suitable method to embed the source in the detectors is the ion-implantation. This process has already been used to embed $^{163}$Ho ions in micro-fabricated low temperature metallic magnetic prototypes. The $^{163}$Ho electron capture spectrum obtained with these first prototypes is presently the most precise with an energy resolution of $\\Delta$$\\textit{E}$$_{FWHM}$ = 7.6 eV. In order to test the performance of the new generation of low temperature metallic magnetic calorimeters, we propose to perform a $^{163}$Ho ion-implantation on the new chip having two arrays consisting of 32 pixels each. An activity of about 1 Bq per pixel is required. With this new detector array we will be able to achieve a better energy resolution and to acquire a higher statistics which allows for studying the $^{163}$Ho spectral shape. We propose also to perform an ion-implantation of $^{166m}$...

DNA-binding site of major regulatory protein alpha 4 specifically associated with promoter-regulatory domains of alpha genes of herpes simplex virus type 1.

OpenAIRE

Kristie, T M; Roizman, B

1986-01-01

Herpes simplex virus type 1 genes form at least five groups (alpha, beta 1, beta 2, gamma 1, and gamma 2) whose expression is coordinately regulated and sequentially ordered in a cascade fashion. Previous studies have shown that functional alpha 4 gene product is essential for the transition from alpha to beta protein synthesis and have suggested that alpha 4 gene expression is autoregulatory. We have previously reported that labeled DNA fragments containing promoter-regulatory domains of thr...

Exogenous Hydrogen Peroxide Contributes to Heme Oxygenase-1 Delaying Programmed Cell Death in Isolated Aleurone Layers of Rice Subjected to Drought Stress in a cGMP-Dependent Manner

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Wang, Guanghui; Xiao, Yu; Deng, Xiaojiang; Zhang, Heting; Li, Tingge; Chen, Huiping

2018-01-01

Hydrogen peroxide (H2O2) is a reactive oxygen species (ROS) that plays a dual role in plant cells. Here, we discovered that drought (20% polyethylene glycol-6000, PEG)-triggered decreases of HO-1 transcript expression and HO activity. However, exogenous H2O2 contributed toward the increase in HO-1 gene expression and activity of the enzyme under drought stress. Meanwhile, the HO-1 inducer hematin could mimic the effects of the H2O2 scavengers ascorbic acid (AsA) and dimethylthiourea (DMTU) and the H2O2 synthesis inhibitor diphenyleneiodonium (DPI) for scavenging or diminishing drought-induced endogenous H2O2. Conversely, the zinc protoporphyrin IX (ZnPPIX), an HO-1-specific inhibitor, reversed the effects of hematin. We further analyzed the endogenous H2O2 levels and HO-1 transcript expression levels of aleurone layers treated with AsA, DMTU, and DPI in the presence of exogenous H2O2 under drought stress, respectively. The results showed that in aleurone layers subjected to drought stress, when the endogenous H2O2 level was inhibited, the effect of exogenous H2O2 on the induction of HO-1 was enhanced. Furthermore, exogenous H2O2-activated HO-1 effectively enhanced amylase activity. Application of 8-bromoguanosine 3′,5′-cyclic guanosine monophosphate (8-Br-cGMP) (the membrane permeable cGMP analog) promoted the effect of exogenous H2O2-delayed PCD of aleurone layers in response to drought stress. More importantly, HO-1 delayed the programmed cell death (PCD) of aleurone layers by cooperating with nitric oxide (NO), and the delayed effect of NO on PCD was achieved via mediation by cGMP under drought stress. In short, in rice aleurone layers, exogenous H2O2 (as a signaling molecule) triggered HO-1 and delayed PCD via cGMP which possibly induced amylase activity under drought stress. In contrast, as a toxic by-product of cellular metabolism, the drought-generated H2O2 promoted cell death. PMID:29449858

A primary study of high performance transgenic rice through maize UBI-1 promoter fusing selective maker gene

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