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Sample records for plasmids e2 induced

  1. Prostaglandin E2(PGE2) induces headache in healthy subjects

    DEFF Research Database (Denmark)

    Wienecke, T; Olesen, Jes; Oturai, P S;

    2009-01-01

    The role of prostanoids in nociception is well established. The headache-eliciting effects of prostaglandin E(2) (PGE(2)) and its possible mechanisms have previously not been systematically studied in man. We hypothesized that infusion of PGE(2) might induce headache and vasodilation of cranial v...

  2. Humoral Immune Response Elicited by Plasmid DNA Containing HGV E2 Gene Fragment%含庚型肝炎病毒E2基因片段质粒DNA能诱发相当强烈的体液免疫应答

    Institute of Scientific and Technical Information of China (English)

    Fethia Ben Yebdri; Abderrahmane AAZAZ; 叶凯; 马辉文; 童立恒

    2004-01-01

    研究了庚型肝炎病毒E2(HGV E2)基因片段作为DNA疫苗的可行性.将来自于质粒pThioHis-E2编码HGV E2的基因片段(559bp)亚克隆到质粒pCMV-S中,使之和HBsAg基因位于同一阅读框,形成重组质粒pCMV-S-E2.用纯化的质pCMV-S-E2 DNA注射到昆明小鼠后腿四头肌中来免疫小鼠,同进用pCMV-S作为对照.间隔14天再加强一次免疫.在加强免疫后8天眼眶取血.用E2-GST融合蛋白作为固定化抗原,通过ELISA检测受试小鼠的体液免疫应答.结果表明,用质粒pCMV-S-E DNA免疫的小鼠可以产生很强的体液免疫应答.%In order to study the feasibility of E2 gene fragment of hepatitis virus G(HGV)as a component of DNA vaccine against the hepatitis virus G infection,a 559bp DNA fragment encoding HGV E2 was cloned into plasmid pCMV-S from pThioHis-E2 in the same reading frame with HBsAg gene to form a recombinant plasmid named pCMV-S-E2.BALB/c mice of Kunming strain were immunized with purified plasmid DNA of pCMV-S-E2 by intra-muscularly inoculation.The immunizations were boosted twice at an interval of 14 days.The whole blood was collected from mice orbit on the day-8 after the last boost.Mice sera were screened by ELISA to determine the humoral immune response using E2-GST fusion protein as the immobilized antigen and the sera from mice immunized with pCMVS as control.The result indicated that the immunization with plasmid DNA of pCMV-S-E2 could induce quite strong humoral immune response.

  3. The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

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    Thulasi Raveendrannair Resmi

    2015-05-01

    Full Text Available Cassava mosaic disease is a major constraint to cassava cultivation worldwide. In India, the disease is caused by Indian cassava mosaic virus (ICMV and Sri Lankan cassava mosaic virus (SLCMV. The Agrobacterium Ti plasmid virulence gene virE2, encoding a nuclear-localized, single-stranded DNA binding protein, was introduced into Nicotiana benthamiana to develop tolerance against SLCMV. Leaf discs of transgenic N. benthamiana plants, harboring the virE2 gene, complemented a virE2 mutation in A. tumefaciens and produced tumours. Three tested virE2 transgenic plants displayed reduction in disease symptoms upon agroinoculation with SLCMV DNA A and DNA B partial dimers. A pronounced reduction in viral DNA accumulation was observed in all three virE2 transgenic plants. Thus, virE2 is an effective candidate gene to develop tolerance against the cassava mosaic disease and possibly other DNA virus diseases.

  4. Prostaglandin E2-induced inflammation: Relevance of prostaglandin E receptors.

    Science.gov (United States)

    Kawahara, Kohichi; Hohjoh, Hirofumi; Inazumi, Tomoaki; Tsuchiya, Soken; Sugimoto, Yukihiko

    2015-04-01

    Prostaglandin E2 (PGE2) is one of the most typical lipid mediators produced from arachidonic acid (AA) by cyclooxygenase (COX) as the rate-limiting enzyme, and acts on four kinds of receptor subtypes (EP1-EP4) to elicit its diverse actions including pyrexia, pain sensation, and inflammation. Recently, the molecular mechanisms underlying the PGE2 actions mediated by each EP subtype have been elucidated by studies using mice deficient in each EP subtype as well as several compounds highly selective to each EP subtype, and their findings now enable us to discuss how PGE2 initiates and exacerbates inflammation at the molecular level. Here, we review the recent advances in PGE2 receptor research by focusing on the activation of mast cells via the EP3 receptor and the control of helper T cells via the EP2/4 receptor, which are the molecular mechanisms involved in PGE2-induced inflammation that had been unknown for many years. We also discuss the roles of PGE2 in acute inflammation and inflammatory disorders, and the usefulness of anti-inflammatory therapies that target EP receptors. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance". Copyright © 2014 Elsevier B.V. All rights reserved.

  5. The Role of the E2F Transcription Factor Family in UV-Induced Apoptosis

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    Orla Gannon

    2011-12-01

    Full Text Available The E2F transcription factor family is traditionally associated with cell cycle control. However, recent data has shown that activating E2Fs (E2F1-3a are potent activators of apoptosis. In contrast, the recently cloned inhibitory E2Fs (E2F7 and 8 appear to antagonize E2F-induced cell death. In this review we will discuss (i the potential role of E2Fs in UV-induced cell death and (ii the implications of this to the development of UV-induced cutaneous malignancies.

  6. A Semliki Forest Virus RNA Replicon-based DNA Plasmid Encoding Classical Swine Fever Virus E2 Gene Induces Immune Response in Mice%基于Semliki Forest病毒RNA复制子的猪瘟RNA疫苗的初步研究

    Institute of Scientific and Technical Information of China (English)

    李娜; 仇华吉; 李国新; 朱庆虎; 罗玉子; 贾洪林; 童光志

    2005-01-01

    将猪瘟病毒E2基因克隆于我们此前构建的衍生于Semliki Forest病毒(semliki forest virus,SFV)RNA复制子的新型真核表达载体pSFV1CS中,获得重组质粒pSFV1CS-E2.用纯化的pSFV1CS-E2分别转染BHK-21细胞和293T细胞,经间接免疫荧光试验检测显示,CSFV E2基因在转染细胞中得到表达.小鼠接种试验结果表明,10μg或100μg pSFV1CS-E2可诱导小鼠产生猪瘟特异性抗体.

  7. CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase

    DEFF Research Database (Denmark)

    Vigo, E; Müller, H; Prosperini, E

    1999-01-01

    in the absence of protein synthesis. Furthermore, CDC25A is defined as a novel E2F target whose expression can be directly regulated by E2F-1. Data showing that CDC25A is an essential target for E2F-1, since its activity is required for efficient induction of S phase by E2F-1, are provided. Finally, our results...... show that expression of two E2F target genes, namely CDC25A and cyclin E, is sufficient to induce entry into S phase in quiescent fibroblasts. Taken together, our results provide an important step in defining how E2F activity leads to deregulated proliferation....

  8. Absence of pRb facilitates E2F1-induced apoptosis in breast cancer cells.

    Science.gov (United States)

    Sun, Baohua; Wingate, Hannah; Swisher, Stephen G; Keyomarsi, Khandan; Hunt, Kelly K

    2010-03-15

    The transcription factor E2F1 is known for its interaction with pRb, controlling cell proliferation; however, E2F1 also has a pivotal role in regulating apoptosis.  The relationship between pRb and E2F1 balances cell proliferation and apoptosis giving pRb tumor suppressive properties. The intricacies of the pRb/E2F1 relationship and thus the regulation of cell fate is cell context dependent. To explore the role of pRb in the E2F1-induced apoptosis of human breast cancer cells, we examined cell growth and apoptosis induction in isogenic cell systems of immortalized breast epithelial cells lacking either pRb (76NE7) or p53 (76NE6). We found that E2F1 caused accumulation of cells in G2 and S phases of the cell cycle along with apoptosis in 76NE7 but not 76NE6 cells.  Variants of 76NE6 cells with functional p53 did not rescue the apoptotic response in these cells, whereas knocking down pRb resulted in significant E2F1-induced apoptosis. We also determined that the effect of E2F1 overexpression in two breast cancer cell lines, MDA-MB-436 and MDA-MB-468, which lack pRb and functional p53, was accumulation of cells in G2/S phase and apoptosis. However, E2F did not cause apotosis  in MCF-7 cells which harbor a functional pRb. Therefore, we conclude that in the absence of Rb, E2F1 overexpression results in apoptosis, not proliferation, and that this effect is independent of p53.

  9. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages

    OpenAIRE

    Woolard, Matthew D.; Barrigan, Lydia M.; Fuller, James R.; Buntzman, Adam S.; Bryan, Joshua; Manoil, Colin; Kawula, Thomas H.; Frelinger, Jeffrey A.

    2013-01-01

    Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cell...

  10. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

    OpenAIRE

    Matthew Dale Woolard; Barrigan, Lydia M.; Fuller, James R.; Buntzman, Adam S.; Joshua eBryan; Colin eManoil; Tom eKawula; Frelinger, Jeffrey A.

    2013-01-01

    Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS) induces macrophages to synthesize prostaglandin E2 (PGE2). Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the ce...

  11. Regression of human papillomavirus intraepithelial lesions is induced by MVA E2 therapeutic vaccine.

    Science.gov (United States)

    Rosales, Ricardo; López-Contreras, Mario; Rosales, Carlos; Magallanes-Molina, Jose-Roberto; Gonzalez-Vergara, Roberto; Arroyo-Cazarez, Jose Martin; Ricardez-Arenas, Antonio; Del Follo-Valencia, Armando; Padilla-Arriaga, Santiago; Guerrero, Miriam Veronica; Pirez, Miguel Angel; Arellano-Fiore, Claudia; Villarreal, Freddy

    2014-12-01

    Human papilloma viruses can induce warts, condylomas, and other intraepithelial cervical lesions that can progress to cancer. Cervical cancer is a serious problem in developing countries because early detection is difficult, and thus proper early treatment is many times missing. In this phase III clinical trial, we evaluated the potential use of MVA E2 recombinant vaccinia virus to treat intraepithelial lesions associated with papillomavirus infection. A total of 1176 female and 180 male patients with intraepithelial lesions were studied. They were injected with 10(7) MVA E2 virus particles directly into their uterus, urethra, vulva, or anus. Patients were monitored by colposcopy and cytology. Immune response was determined by measuring the antibody titer against MVA E2 virus and by analyzing the cytotoxic activity against cancer cells bearing papillomavirus DNA. Papillomavirus was determined by the Hybrid Capture method or by polymerase chain reaction analysis. By histology, 1051 (89.3%) female patients showed complete elimination of lesions after treatment with MVA E2. In 28 (2.4%) female patients, the lesion was reduced to CIN 1. Another 97 (8.3%) female patients presented isolated koilocytes after treatment. In men, all lesions were completely eliminated. All MVA E2-treated patients developed antibodies against the MVA E2 vaccine and generated a specific cytotoxic response against papilloma-transformed cells. Papillomavirus DNA was not detected after treatment in 83% of total patients treated. MVA E2 did not generate any apparent side effects. These data suggest that therapeutic vaccination with MVA E2 vaccine is an excellent candidate to stimulate the immune system and generate regression in intraepithelial lesions when applied locally.

  12. Regression of Human Papillomavirus Intraepithelial Lesions Is Induced by MVA E2 Therapeutic Vaccine

    Science.gov (United States)

    López-Contreras, Mario; Rosales, Carlos; Magallanes-Molina, Jose-Roberto; Gonzalez-Vergara, Roberto; Arroyo-Cazarez, Jose Martin; Ricardez-Arenas, Antonio; del Follo-Valencia, Armando; Padilla-Arriaga, Santiago; Guerrero, Miriam Veronica; Pirez, Miguel Angel; Arellano-Fiore, Claudia; Villarreal, Freddy

    2014-01-01

    Abstract Human papilloma viruses can induce warts, condylomas, and other intraepithelial cervical lesions that can progress to cancer. Cervical cancer is a serious problem in developing countries because early detection is difficult, and thus proper early treatment is many times missing. In this phase III clinical trial, we evaluated the potential use of MVA E2 recombinant vaccinia virus to treat intraepithelial lesions associated with papillomavirus infection. A total of 1176 female and 180 male patients with intraepithelial lesions were studied. They were injected with 107 MVA E2 virus particles directly into their uterus, urethra, vulva, or anus. Patients were monitored by colposcopy and cytology. Immune response was determined by measuring the antibody titer against MVA E2 virus and by analyzing the cytotoxic activity against cancer cells bearing papillomavirus DNA. Papillomavirus was determined by the Hybrid Capture method or by polymerase chain reaction analysis. By histology, 1051 (89.3%) female patients showed complete elimination of lesions after treatment with MVA E2. In 28 (2.4%) female patients, the lesion was reduced to CIN 1. Another 97 (8.3%) female patients presented isolated koilocytes after treatment. In men, all lesions were completely eliminated. All MVA E2–treated patients developed antibodies against the MVA E2 vaccine and generated a specific cytotoxic response against papilloma-transformed cells. Papillomavirus DNA was not detected after treatment in 83% of total patients treated. MVA E2 did not generate any apparent side effects. These data suggest that therapeutic vaccination with MVA E2 vaccine is an excellent candidate to stimulate the immune system and generate regression in intraepithelial lesions when applied locally. PMID:25275724

  13. Molecular mechanism of immune response induced by foreign plasmid DNA after oral administration in mice

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    AIM: To study immune response induced by foreign plasmid DNA after oral administration in mice.METHODS: Mice were orally administered with 200 μg of plasmid pcDNA3 once and spleen was isolated 4 h and 18 h after administration. Total RNA was extracted from spleen and gene expression profile of BALB/c mice spleen was analyzed by using Affymetrix oligonucleotide GeneChip. Functional cluster analysis was conducted by GenMAPP software.RESULTS: At 4 h and 18 h after oral plasmid pcDNA3 administration, a number of immune-related genes,including cytokine and cytokine receptors, chemokines and chemokine receptor, complement molecule,proteasome, histocompatibility molecule, lymphocyte antigen complex and apoptotic genes, were up-regulated. Moreover, MAPPFinder results also showed that numerous immune response processes were up-regulated. In contrast, the immunoglobulin genes were down-regulated.CONCLUSION: Foreign plasmid DNA can modulate the genes expression related to immune system via the gastrointestinal tract, and further analysis of the related immune process may help understand the molecular mechanisms of immune response induced by foreign plasmid via the gastrointestinal tract.

  14. Analysis of heavy-ion-induced DNA strand breaks in plasmid pUC18

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Plasmid DNA was irradiated or implanted by mixed particle field(CR) or lithium-ion-beam to detect strand breaks.The primary results showed that mixed particle field could induce single and double strand breaks with positive linear-dose-effects;most of sequence changes induced by CR were point mutant.Lithium-ion-beam could induce strand breaks also,but it was only at dose of 20Gy.

  15. The Retinoblastoma Tumor Suppressor Protein (pRb)/E2 Promoter Binding Factor 1 (E2F1) Pathway as a Novel Mediator of TGFβ-induced Autophagy.

    Science.gov (United States)

    Korah, Juliana; Canaff, Lucie; Lebrun, Jean-Jacques

    2016-01-29

    TGFβ is a multifunctional cytokine that regulates cell proliferation, cell immortalization, and cell death, acting as a key homeostatic mediator in various cell types and tissues. Autophagy is a programmed mechanism that plays a pivotal role in controlling cell fate and, consequently, many physiological and pathological processes, including carcinogenesis. Although autophagy is often considered a pro-survival mechanism that renders cells viable in stressful conditions and thus might promote tumor growth, emerging evidence suggests that autophagy is also a tumor suppressor pathway. The relationship between TGFβ signaling and autophagy is context-dependent and remains unclear. TGFβ-mediated activation of autophagy has recently been suggested to contribute to the growth inhibitory effect of TGFβ in hepatocarcinoma cells. In the present study, we define a novel process of TGFβ-mediated autophagy in cancer cell lines of various origins. We found that autophagosome initiation and maturation by TGFβ is dependent on the retinoblastoma tumor suppressor protein/E2 promoter binding factor (pRb/E2F1) pathway, which we have previously established as a critical signaling axis leading to various TGFβ tumor suppressive effects. We further determined that TGFβ induces pRb/E2F1-dependent transcriptional activation of several autophagy-related genes. Together, our findings reveal that TGFβ induces autophagy through the pRb/E2F1 pathway and transcriptional activation of autophagy-related genes and further highlight the central relevance of the pRb/E2F1 pathway downstream of TGFβ signaling in tumor suppression.

  16. Major role of adipocyte prostaglandin E2 in lipolysis-induced macrophage recruitment[S

    Science.gov (United States)

    Hu, Xiaoqian; Cifarelli, Vincenza; Sun, Shishuo; Kuda, Ondrej; Abumrad, Nada A.; Su, Xiong

    2016-01-01

    Obesity induces accumulation of adipose tissue macrophages (ATMs), which contribute to both local and systemic inflammation and modulate insulin sensitivity. Adipocyte lipolysis during fasting and weight loss also leads to ATM accumulation, but without proinflammatory activation suggesting distinct mechanisms of ATM recruitment. We examined the possibility that specific lipid mediators with anti-inflammatory properties are released from adipocytes undergoing lipolysis to induce macrophage migration. In the present study, we showed that conditioned medium (CM) from adipocytes treated with forskolin to stimulate lipolysis can induce migration of RAW 264.7 macrophages. In addition to FFAs, lipolytic stimulation increased release of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2), reflecting cytosolic phospholipase A2 α activation and enhanced cyclooxygenase (COX) 2 expression. Reconstituted medium with the anti-inflammatory PGE2 potently induced macrophage migration while different FFAs and PGD2 had modest effects. The ability of CM to induce macrophage migration was abolished by treating adipocytes with the COX2 inhibitor sc236 or by treating macrophages with the prostaglandin E receptor 4 antagonist AH23848. In fasted mice, macrophage accumulation in adipose tissue coincided with increases of PGE2 levels and COX1 expression. Collectively, our data show that adipocyte-originated PGE2 with inflammation suppressive properties plays a significant role in mediating ATM accumulation during lipolysis. PMID:26912395

  17. [Induced abortion using prostaglandin E2 and F2alpha gel].

    Science.gov (United States)

    Lippert, T H; Modly, T

    1974-01-01

    In this study of 20 patients in the 13th-17th week of pregnancy abortion was induced with intrauterine, extraamniotic application of prostaglandins (PG) E2 or F2 in gel form. The gel composition was as follows: 4% tylose MH 300, 2% glycerine, 1% chlorhexidine digluconate, 83% sterile distilled water and 10% PG stock solution. Both PGE2 and PGF2 gels were used. Final concentration was 2.5 mg E2 or 2.5 mg F2 per g of gel. Gel was applied via transcervical, extraamniotic polyethylene catheter every 2-3 hours. Results: PGE2-gel was used in 14 cases. After 3-4 applications both fetus and placenta were expelled. Average dose used was 4.6 mg E2/patient. First contractions started in 30 minutes; induction to expulsion time was 11 hours 35 minutes. F2-gel given to 6 patients resulted in expulsion of the fetus in all cases but placenta needed removal by curettage in 4 patients. Average dose per patient was 17.7 mg of F2; first contractions in 30 minutes, average expulsion time 17 hours 38 minutes. With both PGs there were painful contractions which were controlled with a combination of pentazocine and Valium. PGE2 caused vomiting in 5 patients. No increased bleeding or postabortion infection occurred. Follow-up curettage was done in all patients to ensure removal of all tissues. Overall evaluation of the PG-gels was considered good. PG stability in gel form is good; during 8 months of preservation in sterile aluminum tubes at -25 degrees Celsius no decline in clinical effectiveness was noted. The gel application is less expensive than the slow-injection pump method.

  18. Deregulated E2F activity induces hyperplasia and senescence-like features in the mouse pituitary gland

    DEFF Research Database (Denmark)

    Lazzerini Denchi, Eros; Attwooll, Claire; Pasini, Diego

    2005-01-01

    The retinoblastoma gene, RB1, is one of the most frequently mutated genes in human cancer. Rb heterozygous mice develop pituitary tumors with 100% incidence, and the E2F transcription factors are required for this. To assess whether deregulated E2F activity is sufficient to induce pituitary tumor...

  19. Cyclic Mechanical Stretching Induces Autophagic Cell Death in Tenofibroblasts Through Activation of Prostaglandin E2 Production

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    Hua Chen

    2015-04-01

    Full Text Available Background/Aims: Autophagic cell death has recently been implicated in the pathophysiology of tendinopathy. Prostaglandin E2 (PGE2, a known inflammatory mediator of tendinitis, inhibits tenofibroblast proliferation in vitro; however, the underlying mechanism is unclear. The present study investigated the relationship between PGE2 production and autophagic cell death in mechanically loaded human patellar tendon fibroblasts (HPTFs in vitro. Methods: Cultured HPTFs were subjected to exogenous PGE2 treatment or repetitive cyclic mechanical stretching. Cell death was determined by flow cytometry with acridine orange/ethidium bromide staining. Induction of autophagy was assessed by autophagy markers including the formation of autophagosomes and autolysosomes (by electron microscopy, AO staining, and formation of GPF-LC3-labeled vacuoles and the expression of LC3-II and BECN1 (by western blot. Stretching-induced PGE2 release was determined by ELISA. Results: Exogenous PGE2 significantly induced cell death and autophagy in HPTFs in a dose-dependent manner. Blocking autophagy using inhibitors 3-methyladenine and chloroquine, or small interfering RNAs against autophagy genes Becn-1 and Atg-5 prevented PGE2-induced cell death. Cyclic mechanical stretching at 8% and 12% magnitudes for 24 h significantly stimulated PGE2 release by HPTFs in a magnitude-dependent manner. In addition, mechanical stretching induced autophagy and cell death. Blocking PGE2 production using COX inhibitors indomethacin and celecoxib significantly reduced stretching-induced autophagy and cell death. Conclusion: Taken together, cyclic mechanical stretching induces autophagic cell death in tenofibroblasts through activation of PGE2 production.

  20. Prostaglandin E2 Promotes UV Radiation-Induced Immune Suppression through DNA Hypermethylation

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    Ram Prasad

    2013-07-01

    Full Text Available Exposure of mice to UV radiation results in suppression of the contact hypersensitivity (CHS response. Here, we report that the UV-induced suppression of CHS is associated with increases in the levels of cyclooxygenase-2 (COX-2, prostaglandin E2 (PGE2, and PGE2 receptors in the exposed skin. UV radiation.induced suppression of CHS was inhibited by topical treatment of the skin with celecoxib or indomethacin (inhibitors of COX-2 or AH6809 (an EP2 antagonist. Moreover, mice deficient in COX-2 were found to be resistant to UV-induced suppression of CHS. The exposure of wild-typemice to UVB radiation resulted in DNA hypermethylation, increased DNA methyltransferase (Dnmt activity, and elevated levels of Dnmt1, Dnmt3a, and Dnmt3b proteins in the skin, and these responses were downregulated on topical treatment of the site of exposure after irradiation with indomethacin or EP2 antagonist. Topical treatment of UVB-exposed COX-2.deficient mice with PGE2 enhanced the UVB-induced suppression of CHS as well as global DNA methylation and elevated the levels of Dnmt activity and Dnmt proteins in the skin. Intraperitoneal injection of 5-aza-2′-deoxycytidine (5-Aza-dc, a DNA demethylating agent, restored the CHS response to 2,4-dinitrofluorobenzene in UVB-exposed skin and this was associated with the reduction in global DNA methylation and Dnmt activity and reduced levels of Dnmt proteins. Furthermore, treatment with 5-Aza-dc reversed the effect of PGE2 on UV-induced suppression of CHS in COX-2.deficient mice. These findings reveal a previously unrecognized role for PGE2 in the promotion of UVB-induced immunosuppression and indicate that it is mediated through PGE2 regulation of DNA methylation.

  1. Ketoprofen impairs immunosuppression induced by severe sepsis and reveals an important role for prostaglandin E2.

    Science.gov (United States)

    Brogliato, Ariane Rennó; Antunes, Carlos A; Carvalho, Renato S; Monteiro, Ana Paula T; Tinoco, Rodrigo F; Bozza, Marcelo T; Canetti, Claudio; Peters-Golden, Marc; Kunkel, Steven L; Vianna-Jorge, Rosane; Benjamim, Claudia Farias

    2012-12-01

    The mechanism of immunosuppression induced by severe sepsis is not fully understood. The production of prostaglandin E2 (PGE2) during sepsis is well known, but its role in long-term consequences of sepsis has not been explored. The current study evaluates the role of PGE2 in the development of immunosuppression secondary to sepsis and its potential as therapeutic target. Cecal ligation and puncture was used as an experimental model for sepsis induction in Balb/c and C57BL/6 mice. Immunosuppression was evaluated by the response to secondary infection with Aspergillus fumigatus in sepsis survivors. The role of prostanoids was evaluated in vivo and in vitro by treatment with the cyclooxygenase inhibitor ketoprofen. Balb/c mice were more susceptible than C57BL/6 to severe sepsis and to secondary infection, with a greater mortality rate. Prostaglandin E2 concentrations found in bronchoalveolar lavage in sham and cecal ligation and puncture group after fungal challenge were much higher in Balb/c than in C57BL/6 mice. Ketoprofen treatment improved survival of septic Balb/c mice subjected to secondary infection, while also enhancing macrophage phagocytosis and neutrophil recruitment to the lungs. We identified a pivotal role for PGE2 acting on EP4 receptors in modulating cytokine production differentially by sham and septic macrophages. Furthermore, sepsis also altered key enzymes in PGE2 synthesis and degradation. Our results indicate the involvement of PGE2 in severe sepsis-induced immunosuppression. Inhibition of PGE2 production represents an attractive target to improve innate immune response against secondary infection in the immunocompromised host.

  2. Identification of Francisella novicida mutants that fail to induce prostaglandin E2 synthesis by infected macrophages.

    Directory of Open Access Journals (Sweden)

    Matthew Dale Woolard

    2013-02-01

    Full Text Available Francisella tularensis is the causative agent of tularemia. We have previously shown that infection with F. tularensis Live Vaccine Strain (LVS induces macrophages to synthesize prostaglandin E2 (PGE2. Synthesis of PGE2 by F. tularensis infected macrophages results in decreased T cell proliferation in vitro and increased bacterial survival in vivo. Although we understand some of the biological consequences of F. tularensis induced PGE2 synthesis by macrophages, we do not understand the cellular pathways (neither host nor bacterial that result in up-regulation of the PGE2 biosynthetic pathway in F. tularensis infected macrophages. We took a genetic approach to begin to understand the molecular mechanisms of bacterial induction of PGE2 synthesis from infected macrophages. To identify F. tularensis genes necessary for the induction of PGE2 in primary macrophages, we infected cells with individual mutants from the closely related strain Francisella tularensis subspecies novicida U112 (U112 two allele mutant library. Twenty genes were identified that when disrupted resulted in U112 mutant strains unable to induce the synthesis of PGE2 by infected macrophages. Fourteen of the genes identified are located within the Francisella pathogenicity island (FPI. Genes in the FPI are required for F. tularensis to escape from the phagosome and replicate in the cytosol, which might account for the failure of U112 with transposon insertions within the FPI to induce PGE2. This implies that U112 mutant strains that do not grow intracellularly would also not induce PGE2. We found that U112 clpB::Tn grows within macrophages yet fails to induce PGE2, while U112 pdpA::Tn does not grow yet does induce PGE2. We also found that U112 iglC::Tn neither grows nor induces PGE2. These findings indicate that there is dissociation between intracellular growth and the ability of F. tularensis to induce PGE2 synthesis. These mutants provide a critical entrée into the pathways used

  3. E2F and p53 induce apoptosis independently during Drosophila development but intersect in the context of DNA damage.

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    Nam-Sung Moon

    2008-08-01

    Full Text Available In mammalian cells, RB/E2F and p53 are intimately connected, and crosstalk between these pathways is critical for the induction of cell cycle arrest or cell death in response to cellular stresses. Here we have investigated the genetic interactions between RBF/E2F and p53 pathways during Drosophila development. Unexpectedly, we find that the pro-apoptotic activities of E2F and p53 are independent of one another when examined in the context of Drosophila development: apoptosis induced by the deregulation of dE2F1, or by the overexpression of dE2F1, is unaffected by the elimination of dp53; conversely, dp53-induced phenotypes are unaffected by the elimination of dE2F activity. However, dE2F and dp53 converge in the context of a DNA damage response. Both dE2F1/dDP and dp53 are required for DNA damage-induced cell death, and the analysis of rbf1 mutant eye discs indicates that dE2F1/dDP and dp53 cooperatively promote cell death in irradiated discs. In this context, the further deregulation in the expression of pro-apoptotic genes generates an additional sensitivity to apoptosis that requires both dE2F/dDP and dp53 activity. This sensitivity differs from DNA damage-induced apoptosis in wild-type discs (and from dE2F/dDP-induced apoptosis in un-irradiated rbf1 mutant eye discs by being dependent on both hid and reaper. These results show that pro-apoptotic activities of dE2F1 and dp53 are surprisingly separable: dp53 is required for dE2F-dependent apoptosis in the response to DNA damage, but it is not required for dE2F-dependent apoptosis caused simply by the inactivation of rbf1.

  4. Prostaglandin E2 potentiates interferon-γ-induced nitric oxide production in cultured rat microglia.

    Science.gov (United States)

    Nagano, Takayuki; Nishiyama, Ryo; Sanada, Ayaka; Mutaguchi, Yukiko; Ioku, Anna; Umeki, Hirohisa; Kishimoto, Satoshi; Yamanaka, Daisuke; Kimura, Shinya H; Takemura, Motohiko

    2017-02-01

    Prostaglandin E2 (PGE2 ) plays crucial roles in managing microglial activation through the prostanoid EP2 receptor, a PGE2 receptor subtype. In this study, we report that PGE2 enhances interferon-γ (IFN-γ)-induced nitric oxide production in microglia. IFN-γ increased the release of nitrite, a metabolite of nitric oxide, which was augmented by PGE2 , although PGE2 by itself slightly affects nitrite release. The potentiating effect of PGE2 was positively associated with increased expression of inducible nitric oxide synthase. In contrast to nitrite release induced by IFN-γ, lipopolysaccharide-induced nitrite release was not affected by PGE2 . An EP2 agonist, ONO-AE1-259-01 also augmented IFN-γ-induced nitrite release, while an EP1 agonist, ONO-DI-004, an EP3 agonist, ONO-AE-248, or an EP4 agonist, ONO-AE1-329, did not. In addition, the potentiating effect of PGE2 was inhibited by an EP2 antagonist, PF-04418948, but not by an EP1 antagonist, ONO-8713, an EP3 antagonist, ONO-AE3-240, or an EP4 antagonist, ONO-AE3-208, at 10(-6)  M. Among the EP agonists, ONO-AE1-259-01 alone was able to accumulate cyclic adenosine monophosphate (AMP), and among the EP antagonists, PF-04418948 was the only one able to inhibit PGE2 -increased intracellular cyclic AMP accumulation. On the other hand, IFN-γ promoted phosphorylation of signal transducer and activator of transcription 1, which was not affected by PGE2 . Furthermore, other prostanoid receptor agonists, PGD2 , PGF2α , iloprost, and U-46119, slightly affected IFN-γ-induced nitrite release. These results indicate that PGE2 potentiates IFN-γ-induced nitric oxide production in microglia through the EP2 receptor, which may shed light on one of the pro-inflammatory aspects of PGE2 . © 2016 International Society for Neurochemistry.

  5. Study on Plasmid and Damage Induced by Low-energy Neon Ion Irradiation

    Institute of Scientific and Technical Information of China (English)

    MaQiufeng; WangXiao; JinGenming; LiWenjian; DangBingrong; XieHongmei; ZhouLibin; MaoShuhong

    2003-01-01

    DNA is considered to be the most important and sensitive target in biological systems. Beside base damage, DNA strand breaks are the major lesion in the genome due to exposure to ionizing radiation. Mutation can be introduced to DNA as a result of enzymatic processing of DNA lesions or post-irradiation replication. However, the mechanisms of radiation-induced mutations are not well clarified at the molecular level. A good way to approach the mechanism is to irradiate the plasmid DNA of heavy ion, then transfect the DNA to host cells to determine the mutation spectra. So to study the effect of heavy ions on the simple plasmid DNA is even predominant or more feasible.

  6. E2/ER β inhibit ISO-induced cardiac cellular hypertrophy by suppressing Ca2+-calcineurin signaling.

    Science.gov (United States)

    Tsai, Cheng-Yen; Kuo, Wei-Wen; Shibu, Marthandam Asokan; Lin, Yueh-Min; Liu, Chien-Nam; Chen, Yi-Hui; Day, Cecilia-Hsuan; Shen, Chia-Yao; Viswanadha, Vijaya Padma; Huang, Chih-Yang

    2017-01-01

    Cardiovascular incidences are markedly higher in men than in pre-menstrual women. However, this advantage in women declines with aging and therefore can be correlated with the sex hormone 17β-Estradiol (E2) which is reported to protect heart cells by acting though estrogen receptors (ERs). In this study we have determined the effect of E2/ERβ against ISO induced cellular hypertrophy in H9c2 cardiomyoblast cells. The results confirm that ISO induced cardiac-hypertrophy by elevating the levels of hypertrophy associated proteins, ANP and BNP and further by upregulating p-CaMKII, calcineurin, p-GATA4 and NFATc3 which was correlated with a significant enlargement of the H9c2 cardiomyoblast. However, overexpression of ERβ and/or administration of E2 inhibited ISO-induced hypertrophy in H9c2 cells. In addition, E2/ERβ also inhibited ISO-induced NFATc3 translocation, and reduced the protein level of downstream marker, BNP. Furthermore, by testing with the calcineurin inhibitor (CsA), it was confirmed that calcineurin acted as a key mediator for the anti-hypertrophic effect of E2/ERβ. In cells treated with calcium blocker (BATPA), the inhibitory effect of E2/ERβ on ISO-induced Ca2+ influx and hypertrophic effects were totally blocked suggesting that E2/ERβ inhibited calcineurin activity to activate I-1 protein and suppress PP1, then induce PLB protein phosphorylation and activation, resulting in Ca2+ reuptake into sarcoplasmic reticulum through SR Ca2+ cycling modification. In conclusion, E2/ERβ suppresses the Ca2+ influx and calcineurin activity induced by ISO to enhance the PLB protein activity and SR Ca2+ cycling.

  7. E2F1 activation is responsible for pituitary adenomas induced by HMGA2 gene overexpression

    Directory of Open Access Journals (Sweden)

    Fusco Alfredo

    2006-08-01

    Full Text Available Abstract The High Mobility Group protein HMGA2 is a nuclear architectural factor that plays a critical role in a wide range of biological processes including regulation of gene expression, embryogenesis and neoplastic transformation. Several studies are trying to identify the mechanisms by which HMGA2 protein is involved in each of these activities, and only recently some new significant insights are emerging from the study of transgenic and knock-out mice. Overexpression of HMGA2 gene leads to the onset of prolactin and GH-hormone induced pituitary adenomas in mice, suggesting a critical role of this protein in pituitary tumorigenesis. This was also confirmed in the human pathology by the finding that HMGA2 amplification and/or overexpression is present in human prolactinomas. This review focuses on recent data that explain the mechanism by which HMGA2 induces the development of pituitary adenomas in mice. This mechanism entails the activation of the E2F1 protein by the HMGA2-mediated displacement of HDAC1 from pRB protein.

  8. Prostaglandin E2 induces expression of MAPK phosphatase 1 (MKP-1) in airway smooth muscle cells.

    Science.gov (United States)

    Rumzhum, Nowshin N; Ammit, Alaina J

    2016-07-05

    Prostaglandin E2 (PGE2) is a prostanoid with diverse actions in health and disease. In chronic respiratory diseases driven by inflammation, PGE2 has both positive and negative effects. An enhanced understanding of the receptor-mediated cellular signalling pathways induced by PGE2 may help us separate the beneficial properties from unwanted actions of this important prostaglandin. PGE2 is known to exert anti-inflammatory and bronchoprotective actions in human airways. To date however, whether PGE2 increases production of the anti-inflammatory protein MAPK phosphatase 1 (MKP-1) was unknown. We address this herein and use primary cultures of human airway smooth muscle (ASM) cells to show that PGE2 increases MKP-1 mRNA and protein upregulation in a concentration-dependent manner. We explore the signalling pathways responsible and show that PGE2-induces CREB phosphorylation, not p38 MAPK activation, in ASM cells. Moreover, we utilize selective antagonists of EP2 (PF-04418948) and EP4 receptors (GW 627368X) to begin to identify EP-mediated functional outcomes in ASM cells in vitro. Taken together with earlier studies, our data suggest that PGE2 increases production of the anti-inflammatory protein MKP-1 via cAMP/CREB-mediated cellular signalling in ASM cells and demonstrates that EP2 may, in part, be involved. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. Plasmid-based generation of induced neural stem cells from adult human fibroblasts

    Directory of Open Access Journals (Sweden)

    Philipp Capetian

    2016-10-01

    Full Text Available Direct reprogramming from somatic to neural cell types has become an alternative to induced pluripotent stem cells. Most protocols employ viral expression systems, posing the risk of random genomic integration. Recent developments led to plasmid-based protocols, lowering this risk. However, these protocols either relied on continuous presence of a variety of small molecules or were only able to reprogram murine cells. We therefore established a reprogramming protocol based on vectors containing the Epstein-Barr virus (EBV-derived oriP/EBNA1 as well as the defined expression factors Oct3/4, Sox2, Klf4, L-myc, Lin28, and a small hairpin directed against p53. We employed a defined neural medium in combination with the neurotrophins bFGF, EGF and FGF4 for cultivation without the addition of small molecules. After reprogramming, cells demonstrated a temporary increase in the expression of endogenous Oct3/4. We obtained induced neural stem cells (iNSC 30 days after transfection. In contrast to previous results, plasmid vectors as well as a residual expression of reprogramming factors remained detectable in all cell lines. Cells showed a robust differentiation into neuronal (72% and glial cells (9% astrocytes, 6% oligodendrocytes. Despite the temporary increase of pluripotency-associated Oct3/4 expression during reprogramming, we did not detect pluripotent stem cells or non-neural cells in culture (except occasional residual fibroblasts. Neurons showed electrical activity and functional glutamatergic synapses. Our results demonstrate that reprogramming adult human fibroblasts to iNSC by plasmid vectors and basic neural medium without small molecules is possible and feasible. However, a full set of pluripotency-associated transcription factors may indeed result in the acquisition of a transient (at least partial pluripotent intermediate during reprogramming. In contrast to previous reports, the EBV-based plasmid system remained present and active inside

  10. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties.

    Science.gov (United States)

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.

  11. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

    Science.gov (United States)

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. PMID:26966906

  12. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties.

    Directory of Open Access Journals (Sweden)

    Elodie Beaumont

    Full Text Available Various strategies involving the use of hepatitis C virus (HCV E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system.

  13. The Candida Th17 response is dependent on mannan- and beta-glucan-induced prostaglandin E2.

    NARCIS (Netherlands)

    Smeekens, S.P.; Veerdonk, F.L. van de; Meer, J.W.M. van der; Kullberg, B.J.; Joosten, L.A.B.; Netea, M.G.

    2010-01-01

    The fungus Candida albicans is a potent inducer of the T(h)17 response. Prostaglandin E2 (PGE2) is a strong pro-inflammatory mediator, which has proven to be essential for the T(h)17 response. In this study, we have investigated the role of PGE2 in the T(h)17 response induced by C. albicans in human

  14. E2F-1-Induced p53-independent apoptosis in transgenic mice

    DEFF Research Database (Denmark)

    Holmberg, Christian Henrik; Helin, K.; Sehested, M.;

    1998-01-01

    involving increased apoptosis in the germinal epithelium. This effect was potentiated by simultaneous overexpression of DP-1. Testicular atrophy as a result of overexpression of E2F-1 and DP-1 is independent of functional p53, since p53-nullizygous transgenic mice overexpressing E2F-1 and DP-1 also suffered...

  15. Comprehensive set of integrative plasmid vectors for copper-inducible gene expression in Myxococcus xanthus.

    Science.gov (United States)

    Gómez-Santos, Nuria; Treuner-Lange, Anke; Moraleda-Muñoz, Aurelio; García-Bravo, Elena; García-Hernández, Raquel; Martínez-Cayuela, Marina; Pérez, Juana; Søgaard-Andersen, Lotte; Muñoz-Dorado, José

    2012-04-01

    Myxococcus xanthus is widely used as a model system for studying gliding motility, multicellular development, and cellular differentiation. Moreover, M. xanthus is a rich source of novel secondary metabolites. The analysis of these processes has been hampered by the limited set of tools for inducible gene expression. Here we report the construction of a set of plasmid vectors to allow copper-inducible gene expression in M. xanthus. Analysis of the effect of copper on strain DK1622 revealed that copper concentrations of up to 500 μM during growth and 60 μM during development do not affect physiological processes such as cell viability, motility, or aggregation into fruiting bodies. Of the copper-responsive promoters in M. xanthus reported so far, the multicopper oxidase cuoA promoter was used to construct expression vectors, because no basal expression is observed in the absence of copper and induction linearly depends on the copper concentration in the culture medium. Four different plasmid vectors have been constructed, with different marker selection genes and sites of integration in the M. xanthus chromosome. The vectors have been tested and gene expression quantified using the lacZ gene. Moreover, we demonstrate the functional complementation of the motility defect caused by lack of PilB by the copper-induced expression of the pilB gene. These versatile vectors are likely to deepen our understanding of the biology of M. xanthus and may also have biotechnological applications.

  16. Selective Activation of the Prostaglandin E2 Circuit in Chronic Injury-Induced Pathologic Angiogenesis

    Science.gov (United States)

    Liclican, Elvira L.; Nguyen, Van; Sullivan, Aaron B.

    2010-01-01

    Purpose. Cyclooxygenase (COX)-derived prostaglandin E2 (PGE2) is a prevalent and established mediator of inflammation and pain in numerous tissues and diseases. Distribution and expression of the four PGE2 receptors (EP1-EP4) can dictate whether PGE2 exerts an anti-inflammatory or a proinflammatory and/or a proangiogenic effect. The role and mechanism of endogenous PGE2 in the cornea, and the regulation of EP expression during a dynamic and complex inflammatory/reparative response remain to be clearly defined. Methods. Chronic or acute self-resolving inflammation was induced in mice by corneal suture or epithelial abrasion, respectively. Reepithelialization was monitored by fluorescein staining and neovascularization quantified by CD31/PECAM-1 immunofluorescence. PGE2 formation was analyzed by lipidomics and polymorphonuclear leukocyte (PMN) infiltration quantified by myeloperoxidase activity. Expression of EPs and inflammatory/angiogenic mediators was assessed by real-time PCR and immunohistochemistry. Mice eyes were treated with PGE2 (100 ng topically, three times a day) for up to 7 days. Results. COX-2, EP-2, and EP-4 expression was upregulated with chronic inflammation that correlated with increased corneal PGE2 formation and marked neovascularization. In contrast, acute abrasion injury did not alter PGE2 or EP levels. PGE2 treatment amplified PMN infiltration and the angiogenic response to chronic inflammation but did not affect wound healing or PMN infiltration after epithelial abrasion. Exacerbated inflammatory neovascularization with PGE2 treatment was independent of the VEGF circuit but was associated with a significant induction of the eotaxin-CCR3 axis. Conclusions. These findings place the corneal PGE2 circuit as an endogenous mediator of inflammatory neovascularization rather than general inflammation and demonstrate that chronic inflammation selectively regulates this circuit at the level of biosynthetic enzyme and receptor expression. PMID:20610836

  17. Prostaglandin E2 Prevents Ovariectomy-Induced Cancellous Bone Loss in Rats

    Science.gov (United States)

    Ke, Hua Zhu; Li, Mei; Jee, Webster S. S.

    1992-01-01

    The object of this study was to determine whether prostaglandin E2, (PGE2) can prevent ovariectomy induced cancellous bone loss. Thirty-five 3-month-old female Sprague-Dawley rats were divided into two groups. The rats in the first group were ovariectomized (OVX) while the others received sham operation (sham-OVX). The OVX group was further divided into three treatment groups. The daily doses for the three groups were 0,1 and 6 mg PGE2/kg for 90 days. Bone histomorphometric analyses were performed on double-fluorescent-labeled undecalcified proximal tibial metaphysis (PTM). We confirmed that OVX induces massive cancellous bone loss (-80%) and a higher bone turnover (+143%). The new findings from the present study demonstrate that bone loss due to ovarian hormone deficiency can be prevented by a low-dose (1 mg) daily administration of PGE2. Furthermore, a higher-dose (6 mg) daily administration of PGE2 not only prevents bone loss but also adds extra bone to the proximal tibial metaphyses. PGE, at the 1-mg dose level significantly increased trabecular bone area, trabecular width, trabecular node density, density of node to node, ratio of node to free end, and thus significantly decreased trabecular separation from OVX controls. At this dose level, these same parameters did not differ significantly from sham-OVX controls. However, at the 6-mg dose level PGE2, there were significant increases in trabecular bone area, trabecular width, trabecular node density, density of node to node, and ratio of node to free end, while there was significant decrease in trabecular separation from both OVX and sham-operated controls. The changes in indices of trabecular bone microanatomical structure indicated that PGE2 prevented bone loss as well as the disconnection of existing trabeculae. In summary, PGE2, administration to OVX rats decreased bone turnover and increased bone formation parameters resulting in a positive bone balance that prevented bone loss (in both lower and higher

  18. Mutations induced in a shuttle vector plasmid exposed to monofunctionally activated mitomycin C.

    Science.gov (United States)

    Maccubbin, A E; Mudipalli, A; Nadadur, S S; Ersing, N; Gurtoo, H L

    1997-01-01

    Reductive activation of mitomycin C leads to its covalent binding to DNA, forming monoadducts and cross-links. The cytotoxicity of mitomycin C has been attributed to cross-link formation, whereas monoadducts are assumed to cause mutagenicity. We have developed a 32P-postlabeling technique to measure mitomycin C DNA adducts. Using this technique, we have measured monoadduct formation in the shuttle vector plasmid pSP189 and have determined mutations induced by monoadduct formation. The shuttle vector plasmid was incubated with mitomycin C under conditions favoring monofunctional activation of mitomycin C. The plasmid was then replicated in human Ad293 cells, rescued in bacteria, and analyzed for mutations in the supF tRNA gene sequence of pSP189. One major mitomycin C/DNA adduct was observed by 32P-postlabeling and was characterized as a monoadduct of guanine. When pSP189 was exposed to monofunctionally activated mitomycin C, increases in adduct levels and mutation frequency were found to be related to mitomycin C concentration. The majority of the mutations involved single bases, with base substitutions making up 59.1% of the total mutations observed. Of the base substitutions, 67.2% were transversions and 32.8% were transitions, with nearly 80% of all base substitutions involving G:C base pairs. Deletions, either as single bases or large deletions, also involved G:C base pairs the majority of the time. The observed bias of mutations at G:C and the formation of a mitomycin C/DNA monoadduct involving guanine suggests that monoadduct formation may be responsible for the mutations.

  19. HMGA2 induces pituitary tumorigenesis by enhancing E2F1 activity

    DEFF Research Database (Denmark)

    Fedele, Monica; Visone, Rosa; De Martino, Ivana

    2006-01-01

    HMGA2 gene amplification and overexpression in human prolactinomas and the development of pituitary adenomas in HMGA2 transgenic mice showed that HMGA2 plays a crucial role in pituitary tumorigenesis. We have explored the pRB/E2F1 pathway to investigate the mechanism by which HMGA2 acts. Here we......2 mice. Thus, HMGA2-mediated E2F1 activation is a crucial event in the onset of these tumors in transgenic mice and probably also in human prolactinomas....

  20. Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

    Science.gov (United States)

    Ruiz-Masó, José A.; Bordanaba-Ruiseco, Lorena; Sanz, Marta; Menéndez, Margarita; del Solar, Gloria

    2016-01-01

    Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site

  1. Bone formation induced in an infant by systemic prostaglandin-E2 administration

    DEFF Research Database (Denmark)

    Jørgensen, H R; Svanholm, H; Høst, A

    1988-01-01

    We report a case of long-term systemic administration of prostaglandin E2 (PGE2) to a newborn infant with ductus-dependent congenital heart disease. After 46 days of treatment, radiography showed cortical hyperostosis of the long bones. The child died 62 days after discontinuation of prostaglandin...

  2. Prostaglandin (E2) induces immediate migraine-like attack in migraine patients without aura

    DEFF Research Database (Denmark)

    Antonova, Maria; Wienecke, Troels; Olesen, Jes

    2012-01-01

    without aura were randomly allocated to receive 0.4 µg/kg/min PGE(2) (Prostin®E2, dinoprostone) or placebo over 25 minutes in a two-way, crossover study. Headache intensity was recorded on a verbal rating scale, middle cerebral artery blood flow velocity (V(MCA)) was measured by transcranial Doppler (TCD...

  3. Aqueous extract of Pinus caribaea inhibits the damage induced by ultraviolet radiations, in plasmid DNA

    Directory of Open Access Journals (Sweden)

    Marioly Vernhes Tamayo

    2017-08-01

    Full Text Available Context: The incidence of solar ultraviolet radiation (UV on Earth has increased due to diminish of the ozone layer. This enviromental agent is highly genotoxic causing numerous damage in DNA molecule. Nowadays there is a growing interest in the search of compounds capable to minimize these effects. In particular, phytocompounds have been tested as excelent candidates for their antigenotoxic properties. Aims: To evaluate the protective effect of the aqueous extract of Pinus caribaea (EPC against the damage induced by the UVB and UVC radiation. Methods: The cell-free plasmid DNA assay was employed. The forms of plasmid were separated electrophoretically in agarose gel. For genotoxic and photoprotective evaluation of P. caribaea, different concentrations of the extract (0.1 – 2.0 mg/mL and exposure times were evaluated. The CPD lesions were detected enzymatically. Additionally, the transmittance of the aqueous extract against 254 nm and 312 nm was measured. Results: None of the concentrations were genotoxic in 30 min of treatment, for superior times a clastogenic effect was observed. The EPC despite inhibiting the activity of the enzyme T4 endo V, impedes photolesions formation in DNA at concentrations ≥ 0.1 mg/mL. Conclusions: The EPC has photoprotective properties, this effect could be related with its antioxidants and absorptives capacities.

  4. Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.

    Science.gov (United States)

    Meraviglia, Viviana; Zanon, Alessandra; Lavdas, Alexandros A; Schwienbacher, Christine; Silipigni, Rosamaria; Di Segni, Marina; Chen, Huei-Sheng Vincent; Pramstaller, Peter P; Hicks, Andrew A; Rossini, Alessandra

    2015-06-05

    Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

  5. Cyclin E-induced S phase without activation of the pRb/E2F pathway

    DEFF Research Database (Denmark)

    Lukas, J; Herzinger, T; Hansen, Klaus

    1997-01-01

    In cells of higher eukaryotes, cyclin D-dependent kinases Cdk4 and Cdk6 and, possibly, cyclin E-dependent Cdk2 positively regulate the G1- to S-phase transition, by phosphorylating the retinoblastoma protein (pRb), thereby releasing E2F transcription factors that control S-phase genes. Here we...... performed microinjection and transfection experiments using rat R12 fibroblasts, their derivatives conditionally overexpressing cyclins D1 or E, and human U-2-OS cells, to explore the action of G1 cyclins and the relationship of E2F and cyclin E in S-phase induction. We demonstrate that ectopic expression...... that the cyclin E-induced S phase and completion of the cell division cycle can occur in the absence of E2F-mediated transactivation. Together with the ability of cyclin E to overcome a G1 block induced by expression of dominant-negative mutant DP-1, a heterodimeric partner of E2Fs, these results provide evidence...

  6. Prostaglandin E2 Prevents Hyperosmolar-Induced Human Mast Cell Activation through Prostanoid Receptors EP2 and EP4

    OpenAIRE

    Ivonne Torres-Atencio; Erola Ainsua-Enrich; Fernando de Mora; César Picado; Margarita Martín

    2014-01-01

    BACKGROUND: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB) in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2) are probably also released and could explain the r...

  7. Prostaglandin E2-induced colonic secretion in patients with and without colorectal neoplasia

    Directory of Open Access Journals (Sweden)

    Poulsen Steen S

    2010-01-01

    Full Text Available Abstract Background The pathogenesis for colorectal cancer remains unresolved. A growing body of evidence suggests a direct correlation between cyclooxygenase enzyme expression, prostaglandin E2 metabolism and neoplastic development. Thus further understanding of the regulation of epithelial functions by prostaglandin E2 is needed. We hypothesized that patients with colonic neoplasia have altered colonic epithelial ion transport and express functionally different prostanoid receptor levels in this respect. Methods Patients referred for colonoscopy were included and grouped into patients with and without colorectal neoplasia. Patients without endoscopic findings of neoplasia served as controls. Biopsy specimens were obtained from normally appearing mucosa in the sigmoid part of colon. Biopsies were mounted in miniaturized modified Ussing air-suction chambers. Indomethacin (10 μM, various stimulators and inhibitors of prostanoid receptors and ion transport were subsequently added to the chamber solutions. Electrogenic ion transport parameters (short circuit current and slope conductance were recorded. Tissue pathology and tissue damage before and after experiments was assessed by histology. Results Baseline short circuit current and slope conductance did not differ between the two groups. Patients with neoplasia were significantly more sensitive to indomethacin with a decrease in short circuit current of 15.1 ± 2.6 μA·cm-2 compared to controls, who showed a decrease of 10.5 ± 2.1 μA·cm-2 (p = 0.027. Stimulation or inhibition with theophylline, ouabain, bumetanide, forskolin or the EP receptor agonists prostaglandin E2, butaprost, sulprostone and prostaglandin E1 (OH did not differ significantly between the two groups. Histology was with normal findings in both groups. Conclusions Epithelial electrogenic transport is more sensitive to indomethacin in normal colonic mucosa from patients with previous or present colorectal neoplasia compared

  8. Prostaglandin E2-induced colonic secretion in patients with and without colorectal neoplasia

    DEFF Research Database (Denmark)

    Kaltoft, Nicolai; Tilotta, Maria C; Witte, Anne-Barbara;

    2010-01-01

    BACKGROUND: The pathogenesis for colorectal cancer remains unresolved. A growing body of evidence suggests a direct correlation between cyclooxygenase enzyme expression, prostaglandin E2 metabolism and neoplastic development. Thus further understanding of the regulation of epithelial functions...... colorectal neoplasia. Patients without endoscopic findings of neoplasia served as controls. Biopsy specimens were obtained from normally appearing mucosa in the sigmoid part of colon. Biopsies were mounted in miniaturized modified Ussing air-suction chambers. Indomethacin (10 microM), various stimulators...... and inhibitors of prostanoid receptors and ion transport were subsequently added to the chamber solutions. Electrogenic ion transport parameters (short circuit current and slope conductance) were recorded. Tissue pathology and tissue damage before and after experiments was assessed by histology. RESULTS...

  9. Transfer of the pheromone-inducible plasmid pCF10 among Enterococcus faecalis microorganisms colonizing the intestine of mini-pigs

    DEFF Research Database (Denmark)

    Licht, Tine Rask; Laugesen, D.; Jensen, Lars Bogø;

    2002-01-01

    A new animal model, the streptomycin-treated mini-pig, was developed in order to allow colonization of defined strains of Enterococcus faecalis in numbers sufficient to study plasmid transfer. Transfer of the pheromone-inducible pCF10 plasmid between streptomycin-resistant strains of E. faecalis OG...... until the end of the experiment. These observations showed that even in the absence of selective tetracycline pressure, plasmid pCF10 was transferred from ingested E. faecalis cells to other E. faecalis organisms already present in the intestinal environment and that the plasmid subsequently persisted...

  10. Prostaglandin E2 induces spontaneous rhythmic activity in mouse urinary bladder independently of efferent nerves

    Science.gov (United States)

    Kobayter, S; Young, JS; Brain, KL

    2012-01-01

    BACKGROUND AND PURPOSE The acute effects of PGE2 on bladder smooth muscle and nerves were examined to determine the origin of PGE2-induced spontaneous rhythmic contractions. EXPERIMENTAL APPROACH Contraction studies, confocal Ca2+ imaging and electrophysiological recordings in strips of mouse urinary bladder were used to differentiate the effects of PGE2 on bladder smooth muscle and efferent nerves. KEY RESULTS PGE2 (50 µM) increased the tone and caused phasic contractions of detrusor smooth muscle strips. Confocal Ca2+ imaging showed that PGE2 increased the frequency of whole-cell Ca2+ transients (WCTs) (72 ± 5%) and intracellular recordings showed it increased the frequency of spontaneous depolarizations, from 0.31·s−1 to 0.90·s−1. Non-selective inhibition of EP receptors using SC-51322 and AH-6809 (10 µM), or the L-type Ca2+ channel blocker nifedipine (1 µM), prevented these phasic contractions and WCTs, and reduced the tone (by 45 ± 7% and 59 ± 6%, respectively). Blocking P2X1 receptors with NF449 (10 µM) caused a small but significant reduction in the frequency of PGE2-induced phasic contractions (24 ± 9%) and WCTs (28 ± 17%) but had no significant effect on spontaneous depolarizations or tone. Inhibiting muscarinic receptors with cyclopentolate (1 µM) had no significant effect on these measures. Spontaneous WCTs became synchronous in PGE2, implying enhanced functional coupling between neighbouring cells. However, the electrical input resistance was unchanged. CONCLUSIONS AND IMPLICATIONS It was concluded that depolarization alone is sufficient to explain a functional increase in intercellular coupling and the ability of PGE2 to increase detrusor spontaneous rhythmic activity does not require parasympathetic nerves. PMID:21671904

  11. Possible involvement of brain prostaglandin E2 and prostanoid EP3 receptors in prostaglandin E2 glycerol ester-induced activation of central sympathetic outflow in the rat.

    Science.gov (United States)

    Shimizu, Takahiro; Tanaka, Kenjiro; Nakamura, Kumiko; Taniuchi, Keisuke; Yawata, Toshio; Higashi, Youichirou; Ueba, Tetsuya; Dimitriadis, Fotios; Shimizu, Shogo; Yokotani, Kunihiko; Saito, Motoaki

    2014-07-01

    We recently reported that intracerebroventricularly administered 2-arachidonoylglycerol elevated plasma noradrenaline and adrenaline by brain monoacylglycerol lipase- (MGL) and cyclooxygenase-mediated mechanisms in the rat. These results suggest that 2-arachidonoylglycerol is hydrolyzed by MGL to free arachidonic acid, which is further metabolized to prostaglandins (PGs) by cyclooxygenase in the brain, thereby elevating plasma noradrenaline and adrenaline. On the other hand, 2-arachidonoylglycerol can be also metabolized by cyclooxygenase to PG glycerol esters (PG-Gs), which seems to be hydrolyzed by MGL to free PGs. Here, we examined the involvement of brain PG-Gs in the elevation of plasma noradrenaline and adrenaline regarding PGE2-G and prostanoid EP receptors using anesthetized male Wistar rats. Intracerebroventricularly administered PGE2-G (1.5 and 3 nmol/animal) dose-dependently elevated plasma noradrenaline but not adrenaline. PGE2-G also elevated systolic, mean and diastolic blood pressure and heart rate. The PGE2-G-induced elevation of plasma noradrenaline was attenuated by JZL184 (MGL inhibitor). Intracerebroventricularly administered PGE2 (0.3 and 1.5 nmol/animal) and sulprostone (0.1 and 0.3 nmol/animal) (EP1/EP3 agonist) also elevated plasma noradrenaline but not adrenaline in a dose-dependent manner. The sulprostone-induced elevation was attenuated by L-798,106 (EP3 antagonist), but not by SC-51322 (EP1 antagonist). L-798,106 also attenuated the PGE2-G- and PGE2-induced elevation of plasma noradrenaline, while PF-04418948 (EP2 antagonist) and L-161,982 (EP4 antagonist) had no effect on the PGE2-G-induced response. These results suggest a possibility that brain PGE2-G produced from 2-arachidonoylglycerol can be hydrolyzed to free PGE2, thereby activating central sympathetic outflow by brain prostanoid EP3 receptor-mediated mechanisms in the rat. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. EFFECTS OF HBV preS AS A HUMORAL ENHANCER ON THE ABILITIES OF HCV E2 PROTEIN TO INDUCE IMMUNE RESPONSES IN THE DNA-IMMUNIZED MICE

    Institute of Scientific and Technical Information of China (English)

    谢尧; 陶其敏; 高建恩

    2003-01-01

    Objective.To study whether the abilities of hepatitis C virus(HCV)E2 gene immunization to induce humoral and cellular immune responses to E2 protein were affected by hepatitis B virus(HBV)preS gene when they were fused in DNA-immunized mice.Methods.Mice were immunized with E2,preS-E2(preS gene was upstream of E2 gene),and E2-preS(preS gene was downstream of E2 gene)gene by their eukaryotic expression vectors,respectively.The anti-E2 or anti-preS antibodies were detected using the E2 and preS antigens.The cellular immune response to E2 pro-tein in immunized mice was presented by its survival time after injecting SP2/O myeloma cells expressing HCV E2 protein into the abdominal cavity.Results. Chimeric E2 and preS gene immunization can induce mice to develop anti-preS and anti-E2 antibodies.The number of the mice developing anti-E2 antibody and the antibody titers in preS-E2 gene-injected group were higher than those in E2-preS gene-immunized group.However,the mice injected with E2 gene did not develop the detectable anti-E2 antibodies until 12 weeks after DNA immunization.After the mice was injected with target cells,the average survival time of the mice in the group immunized with E2 gene alone was longer than that of the group injected with E2 gene fused with HBV preS and was significantly longer than that of the control(P< 0.05).Conclusion.HBV preS might be a humoral enhancer that can affect the abilities of HCV E2 protein to in-duce immune responses in DNA-immunized mice.

  13. Sulforaphane induces cell cycle arrest by protecting RB-E2F-1 complex in epithelial ovarian cancer cells

    Directory of Open Access Journals (Sweden)

    Morris Robert

    2010-03-01

    Full Text Available Abstract Background Sulforaphane (SFN, an isothiocyanate phytochemical present predominantly in cruciferous vegetables such as brussels sprout and broccoli, is considered a promising chemo-preventive agent against cancer. In-vitro exposure to SFN appears to result in the induction of apoptosis and cell-cycle arrest in a variety of tumor types. However, the molecular mechanisms leading to the inhibition of cell cycle progression by SFN are poorly understood in epithelial ovarian cancer cells (EOC. The aim of this study is to understand the signaling mechanisms through which SFN influences the cell growth and proliferation in EOC. Results SFN at concentrations of 5 - 20 μM induced a dose-dependent suppression of growth in cell lines MDAH 2774 and SkOV-3 with an IC50 of ~8 μM after a 3 day exposure. Combination treatment with chemotherapeutic agent, paclitaxel, resulted in additive growth suppression. SFN at ~8 μM decreased growth by 40% and 20% on day 1 in MDAH 2774 and SkOV-3, respectively. Cells treated with cytotoxic concentrations of SFN have reduced cell migration and increased apoptotic cell death via an increase in Bak/Bcl-2 ratio and cleavage of procaspase-9 and poly (ADP-ribose-polymerase (PARP. Gene expression profile analysis of cell cycle regulated proteins demonstrated increased levels of tumor suppressor retinoblastoma protein (RB and decreased levels of E2F-1 transcription factor. SFN treatment resulted in G1 cell cycle arrest through down modulation of RB phosphorylation and by protecting the RB-E2F-1 complex. Conclusions SFN induces growth arrest and apoptosis in EOC cells. Inhibition of retinoblastoma (RB phosphorylation and reduction in levels of free E2F-1 appear to play an important role in EOC growth arrest.

  14. Deregulated expression of E2F family members induces S-phase entry and overcomes p16INK4A-mediated growth suppression

    DEFF Research Database (Denmark)

    Lukas, J; Petersen, B O; Holm, K

    1996-01-01

    the coexpression of the heterodimeric partner DP-1 to promote S-phase entry and accelerate G1 progression. Furthermore, the pRB-associated E2Fs were all able to overcome a G1 arrest mediated by the p16INK4 tumor suppressor protein, and E2F-1 was shown to override a G1 block mediated by a neutralizing antibody...... to cyclin D1. The p16INK4-induced G1 arrest was not affected by expression of E2F-4, E2F-5, or DP-1 alone, but simulataneous expression of E2F-4 and DP-1 could overcome this block. Our results demonstrate that the generation of E2F activity is rate limiting for G1 progession, is sufficient to induce S...

  15. E2F1 impairs all-trans retinoic acid-induced osteogenic differentiation of osteosarcoma via promoting ubiquitination-mediated degradation of RARα

    Science.gov (United States)

    Zhang, Lei; Zhou, Qian; Zhang, Ning; Li, Weixu; Ying, Meidan; Ding, Wanjing; Yang, Bo; He, Qiaojun

    2014-01-01

    All-trans retinoic acid (ATRA) is a widely used differentiation drug that can effectively induce osteogenic differentiation of osteosarcoma cells, but the underlying mechanism remains elusive, which limits the clinical application for ATRA in osteosarcoma patients. In this study, we identified E2F1 as a novel regulator involved in ATRA-induced osteogenic differentiation of osteosarcoma cells. We observed that osteosarcoma cells are coupled with individual differences in the expression levels of E2F1 in patients, and E2F1 impairs ATRA-induced differentiation of osteosarcoma cells. Moreover, remarkable anti-proliferative and differentiation-inducing effects of ATRA treatment are only observed in E2F1 low to negative expressed primary osteosarcoma cultures. These results strongly suggested that E2F1 may serve as a potent indicator for the effectiveness of ATRA treatment in osteosarcoma. Interestingly, E2F1 is found to downregulate retinoic acid receptor α (RARα), a key factor determines the effectiveness of ATRA. E2F1 specifically binds to RARα and promotes its ubiquitination-mediated degradation; as a consequence, RARα-mediated differentiation is inhibited in osteosarcoma. Therefore, our studies present E2F1 as a potent biomarker, as well as a therapeutic target for ATRA-based differentiation therapeutics, and raise the hope of using differentiation-based approaches for osteosarcoma patients. PMID:24608861

  16. Single extra-amniotic injection of prostaglandin E2 in Tylose gel to induce first trimester abortion in young nullipara.

    Science.gov (United States)

    Djahanbakhch, O; Bassan, T S; Marshall, D E; Gardner, N H

    1982-01-01

    A single extra-amniotic injection of 2 or 4 mg of prostaglandin E2 in Tylose gel was administered to 30 primigravidae between the ages of 14 and 20 years to induce first-trimester abortion. Twenty-three patients (76.7%) aborted, though incompletely, within 9 hours, with a mean induction-abortion interval of 6.3 hours. In the remaining 7 (23.3%) the cervical os was found to be open and no mechanical dilatation was required at the time of vacuum aspiration. The only side effect was vomiting, which occurred in 2 patients. The method was shown to be safe and effective and may be employed in young primigravidae, thus eliminating the cervical complications attending vacuum aspiration.

  17. Role of prostaglandin E2 and leukotriene B4 in skin reaction induced by transdermal application of propranolol.

    Science.gov (United States)

    Kobayashi, I; Hosaka, K; Maruo, H; Saeki, Y; Kamiyama, M; Konno, C; Gemba, M

    2000-02-01

    Dermal application of propranolol (PRL) induced formation of erythema and edema, and pseudoeosinophil infiltration in epidermis and dermis at the application site in guinea pigs. We investigated the production of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) at the application site of PRL and the role of these inflammatory chemical mediators in the occurrence of the skin reactions. PGE2 was found to be produced at the application site slightly after the accumulation of PRL released from the adhesive bandage in the patch test, and the amount of PGE2 increased continuously, with a peak value obtained at 24 h after application. The time-course changes resembled those of delta a* value, the index of erythema formation determined by colorimetric measurement, and edema formation. The production of PGE2 by dermal application of PRL was suppressed by local pretreatment with dexamethasone or indomethacin. However, no notable production of LTB4 was observed at the application site of PRL.

  18. On the mechanism of noradrenaline-induced prostaglandin E2-Synthesis in primary cell cultures from rabbit splenic pulpa.

    Science.gov (United States)

    Brückner-Schmidt, R; Jackisch, R; Hertting, G

    1981-10-01

    The role of Ca2+ and phospholipase A2 in alpha-adrenoceptor mediated stimulation of prostaglandin (PG)E2-release was investigated in primary cell cultures of rabbit splenic pulpa. Noradrenaline enhanced PGE2-release only in the presence of extracellular Ca2+. In contrast, PGE2-release induced by arachidonic acid was unchanged when Ca2+ was omitted. In the presence of Ca2+, the ionophore A 23187 increased PGE2-release concentration-dependently. During incubation in Ca2+-free medium, the ionophore was ineffective. Inhibitors of phospholipase A2 (mepacrine, p-bromophenacyl bromide) abolished the noradrenaline-induced PGE2-release and reduced the effect of A 23187; the stimulation of PGE2-release by arachidonic acid was not affected. Addition of exogenous phospholipase A2 enhances release of PGE2. From these results we suggest that noradrenaline-induced PGE2-release in rabbit splenic fibroblasts via alpha-adrenoceptors involves the following steps: influx of Ca2+, activation of a Ca2+-dependent phospholipase and liberation of arachidonic acid which is transformed into PGs.

  19. Prostaglandin E2 activates the histaminergic system via the EP4 receptor to induce wakefulness in rats.

    Science.gov (United States)

    Huang, Zhi-Li; Sato, Yo; Mochizuki, Takatoshi; Okada, Tetsuya; Qu, Wei-Min; Yamatodani, Atsushi; Urade, Yoshihiro; Hayaishi, Osamu

    2003-07-01

    Prostaglandin (PG)E2 promotes the wakeful state when administered into the posterior hypothalamus, in which the histaminergic tuberomammillary nucleus (TMN) is located. To explore the neurotransmitter mechanisms responsible for PGE2-induced wakefulness in rats, we examined the effect of PGE2 on the activity of the histaminergic system and the involvement of PGE2 receptor subtypes in the response. PGE2 perfusion in the TMN at doses of 100, 200, and 400 pmol/min for 2 hr significantly increased histamine release from the medial preoptic area and frontal cortex in a dose-dependent manner, as measured by in vivo microdialysis. Among the agonists of the four distinct subtypes of PGE2 receptors (EP1-4) tested, only the EP4 receptor agonist (ONO-AE1-329) mimicked the excitatory effect of PGE2 on histamine release from both the medial preoptic area and frontal cortex. Perfusion of either PGE2 or the EP4 agonist into the TMN at a dose of 200 pmol/min for 1 hr increased histidine decarboxylase activity, histidine decarboxylase mRNA level, and histamine content in the hypothalamus. In situ hybridization revealed that EP4 receptor mRNA was expressed in histidine decarboxylase-immunoreactive neurons of the TMN region. Furthermore, EP4 agonist perfusion into the TMN induced wakefulness. These findings indicate that PGE2 induces wakefulness through activation of the histaminergic system via EP4 receptors.

  20. Papillomavirus-Associated Tumor Formation Critically Depends on c-Fos Expression Induced by Viral Protein E2 and Bromodomain Protein Brd4.

    Directory of Open Access Journals (Sweden)

    Maria Delcuratolo

    2016-01-01

    Full Text Available We investigated the mechanism of how the papillomavirus E2 transcription factor can activate promoters through activator protein (AP1 binding sites. Using an unbiased approach with an inducible cell line expressing the viral transcription factor E2 and transcriptome analysis, we found that E2 induces the expression of the two AP1 components c-Fos and FosB in a Brd4-dependent manner. In vitro RNA interference confirmed that c-Fos is one of the AP1 members driving the expression of viral oncogenes E6/E7. Mutation analysis and in vivo RNA interference identified an essential role for c-Fos/AP1 and also for the bromodomain protein Brd4 for papillomavirus-induced tumorigenesis. Lastly, chromatin immunoprecipitation analysis demonstrated that E2 binds together with Brd4 to a canonical E2 binding site (E2BS in the promoter of c-Fos, thus activating c-Fos expression. Thus, we identified a novel way how E2 activates the viral oncogene promoter and show that E2 may act as a viral oncogene by direct activation of c-Fos involved in skin tumorigenesis.

  1. STUDY REGARDING EFFICIENCY OF INDUCED GENETIC TRANSFORMATION IN BACILLUS LICHENIFORMIS WITH PLASMID DNA

    Directory of Open Access Journals (Sweden)

    VINTILĂ T.

    2007-01-01

    Full Text Available A strain of Bacillus licheniformis was subject to genetic transformation with plasmidvectors (pLC1 and pNC61, using electroporation technique, protoplasttransformation and bivalent cations (CaCl2 mediated transformation. In the case oftransformation by electroporation of Bacillus licheniformis B40, the highest numberof transformed colonies (3 were obtained only after a 1,79 KV electric shock, for 2,2milliseconds. Using this transformation technique we have obtained six kanamycinresistant transformants. The frequency of Bacillus licheniformis B40 protoplaststransformation using pLC1 and pNC61 plasmid vectors is approximately 10% (TF =10%. As a result of pLC1 plasmid integration in Bacillus licheniformis protoplasts,six kanamycin resistant transformants were obtained. The pNC61 plasmid, whichconfers trimethoprim resistance, does not integrate in receiver cells by protoplasttransformation. The direct genetic transformation in the presence of bivalent cations(CaCl2, mediated by pLC1 and pNC61 plasmid vectors, produce a lowtransformation frequency. Using this technique, we have obtained three trimethoprimresistant colonies and four kanamycin resistant colonies. The chemical way oftransformation is the only technique, which realizes the integration of pNC61 in B.licheniformis B40 cells.

  2. Disruption of mutually negative regulatory feedback loop between interferon-inducible p202 protein and the E2F family of transcription factors in lupus-prone mice

    Science.gov (United States)

    Panchanathan, Ravichandran; Xin, Hong; Choubey, Divaker

    2010-01-01

    Summary Studies have identified interferon-inducible Ifi202 gene as a lupus susceptibility gene (encoding p202 protein) in mouse models of lupus disease. However, signaling pathways that regulate the Ifi202 expression in cells remain to be elucidated. We found that steady-state levels of Ifi202 mRNA and protein were high in mouse embryonic fibroblasts (MEFs) from E2F1-knockout (E2F1-/-) and E2F1 and E2F2 double knockout (E2F1-/- E2F2-/-) mice than isogenic wild type MEFs. Moreover, overexpression of E2F1 in mouse fibroblasts decreased expression of p202. Furthermore, expression of E2F1, but not E2F4, transcription factor in mouse fibroblasts repressed the activity of 202-luc-reporter in promoter-reporter assays. Interestingly, the E2F1-mediated transcriptional repression of the 202-luc-reporter was independent of p53 and pRb expression. However, the repression was dependent on the ability of E2F1 to bind DNA. We have identified a potential E2F DNA-binding site in the 5′-regulatory region of the Ifi202 gene and mutations in this E2F DNA-binding site reduced the E2F1-mediated transcriptional repression of 202-luc-reporter. Because p202 inhibits the E2F1-mediated transcriptional activation of genes, we compared the expression of E2F1 and its target genes in splenic cells from lupus-prone B6.Nba2 congenic mice, which express increased levels of p202, with age-matched C57BL/6 mice. We found that increased expression of Ifi202 in the congenic mice was associated with inhibition of E2F1-mediated transcription and decreased expression of E2F1 and its target genes that encode pro-apoptotic proteins. Our observations support for the idea that increased Ifi202 expression in certain strain of mice contributes to lupus susceptibility in part by inhibiting E2F1-mediated functions. PMID:18424712

  3. Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

    Directory of Open Access Journals (Sweden)

    Lough John W

    2010-08-01

    Full Text Available Abstract Background The use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry. Results Here we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture. Conclusions Simple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

  4. Hepatitis C Virus E2 Protein Induces Upregulation of IL-8 Pathways and Production of Heat Shock Proteins in Human Thyroid Cells.

    Science.gov (United States)

    Hammerstad, Sara Salehi; Stefan, Mihaela; Blackard, Jason; Owen, Randall P; Lee, Hanna J; Concepcion, Erlinda; Yi, Zhengzi; Zhang, Weijia; Tomer, Yaron

    2017-02-01

    Thyroiditis is one of the most common extrahepatic manifestations of hepatitis C virus (HCV) infection. By binding to surface cell receptor CD81, HCV envelope glycoprotein E2 mediates entry of HCV into cells. Studies have shown that different viral proteins may individually induce host responses to infection. We hypothesized that HCV E2 protein binding to CD81 expressed on thyroid cells activates a cascade of inflammatory responses that can trigger autoimmune thyroiditis in susceptible individuals. Human thyroid cell lines ML-1 and human thyrocytes in primary cell culture were treated with HCV recombinant E2 protein. The expression of major proinflammatory cytokines was measured at the messenger RNA and protein levels. Next-generation transcriptome analysis was used to identify early changes in gene expression in thyroid cells induced by E2. HCV envelope protein E2 induced strong inflammatory responses in human thyrocytes, resulting in production of interleukin (IL)-8, IL-6, and tumor necrosis factor-α. Furthermore, the E2 protein induced production of several heat shock proteins including HSP60, HSP70p12A, and HSP10, in human primary thyrocytes. In thyroid cell line ML-1, RNA sequencing identified upregulation of molecules involved in innate immune pathways with high levels of proinflammatory cytokines and chemokines and increased expression of costimulatory molecules, specifically CD40, known to be a major thyroid autoimmunity gene. Our data support a key role for HCV envelope protein E2 in triggering thyroid autoimmunity through activation of cytokine pathways by bystander mechanisms.

  5. Lipopolysaccharide Enhances the Production of Nicotine-Induced Prostaglandin E2 by an Increase in Cyclooxygenase-2 Expression in Osteoblasts

    Institute of Scientific and Technical Information of China (English)

    Maiko SHOJI; Natsuko TANABE; Narihiro MITSUI; Naoto SUZUKI; Osamu TAKEICHI; Tomoko KATONO; Akira MOROZUMI; Masao MAENO

    2007-01-01

    Previous studies have indicated that lipopolysaccharide (LPS) from Gram-negative bacteria in plaque induces the release of prostaglandin E2 (PGE2),which promotes alveolar bone resorption in periodontitis,and that tobacco smoking might be an important risk factor for the development and severity of periodontitis.We determined the effect of nicotine and LPS on alkaline phosphatase (ALPase)activity,PGE2 production,and the expression of cyclooxygenase (COX-1,COX-2),PGE2 receptors Ep1-4,and macrophage colony stimulating factor(M-CSF)in human osteoblastic Saos-2 cells.The cells were cultured with 10-3 M nicotine in the presence of 0,1,or 10 μg/ml LPS,or with LPS alone.ALPase activity decreased in cells cultured with nicotine or LPS alone,and decreased further in those cultured with both nicotine and LPS,whereas PGE2 production significantly increased in the former and increased further in the latter.By itself,nicotine did not affect expression of COX-1,COX-2,any of the PGE2 receptors,or M-CSF,but when both nicotine and LPS were present,expression of COX-2,Ep3,Ep4,and M-CSF increased significantly.Simultaneous addition of 10-4 M indomethacin eliminated the effects of nicotine and LPS on ALPase activity,PGE2 production,and MCSF expression.Phosphorylation of protein kinase A was high in cells cultured with nicotine and LPS.These results suggest that LPS enhances the production of nicotine-induced PGE2 by an increase in COX-2 expression in osteoblasts,that nicotine-LPS-induced PGE2 interacts with the osteoblast Ep4 receptor primarily in autocrine or paracrine mode,and that the nicotine-LPS-induced PGE2 then decreases ALPase activity and increases M-CSF expression.

  6. Prostaglandin E2 prevents hyperosmolar-induced human mast cell activation through prostanoid receptors EP2 and EP4.

    Directory of Open Access Journals (Sweden)

    Ivonne Torres-Atencio

    Full Text Available BACKGROUND: Mast cells play a critical role in allergic and inflammatory diseases, including exercise-induced bronchoconstriction (EIB in asthma. The mechanism underlying EIB is probably related to increased airway fluid osmolarity that activates mast cells to the release inflammatory mediators. These mediators then act on bronchial smooth muscle to cause bronchoconstriction. In parallel, protective substances such as prostaglandin E2 (PGE2 are probably also released and could explain the refractory period observed in patients with EIB. OBJECTIVE: This study aimed to evaluate the protective effect of PGE2 on osmotically activated mast cells, as a model of exercise-induced bronchoconstriction. METHODS: We used LAD2, HMC-1, CD34-positive, and human lung mast cell lines. Cells underwent a mannitol challenge, and the effects of PGE2 and prostanoid receptor (EP antagonists for EP(1-4 were assayed on the activated mast cells. Beta-hexosaminidase release, protein phosphorylation, and calcium mobilization were assessed. RESULTS: Mannitol both induced mast cell degranulation and activated phosphatidyl inositide 3-kinase and mitogen-activated protein kinase (MAPK pathways, thereby causing de novo eicosanoid and cytokine synthesis. The addition of PGE2 significantly reduced mannitol-induced degranulation through EP(2 and EP(4 receptors, as measured by beta-hexosaminidase release, and consequently calcium influx. Extracellular-signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38 phosphorylation were diminished when compared with mannitol activation alone. CONCLUSIONS: Our data show a protective role for the PGE2 receptors EP(2 and EP(4 following osmotic changes, through the reduction of human mast cell activity caused by calcium influx impairment and MAP kinase inhibition.

  7. Episomal plasmid-based generation of induced pluripotent stem cells from fetal femur-derived human mesenchymal stromal cells.

    Science.gov (United States)

    Megges, Matthias; Oreffo, Richard O C; Adjaye, James

    2016-01-01

    Human bone mesenchymal stromal cells derived from fetal femur 55 days post-conception were reprogrammed to induced pluripotent stem cells using episomal plasmid-based expression of OCT4, SOX2, NANOG, LIN28, SV40LT, KLF4 and c-MYC and supplemented with the following pathway inhibitors - TGFβ receptor inhibitor (A-83-01), MEK inhibitor (PD325901), GSK3β inhibitor (CHIR99021) and ROCK inhibitor (HA-100). Successful induction of pluripotency in two iPS-cell lines was demonstrated in vitro and by the Pluritest.

  8. Increased expression of bHLH transcription factor E2A (TCF3) in prostate cancer promotes proliferation and confers resistance to doxorubicin induced apoptosis

    Energy Technology Data Exchange (ETDEWEB)

    Patel, Divya [Center for Cancer Research and Therapeutics Development, Clark Atlanta University, 223 James P. Brawley Dr. SW, Atlanta, GA 30314 (United States); Chaudhary, Jaideep, E-mail: jchaudhary@cau.edu [Center for Cancer Research and Therapeutics Development, Clark Atlanta University, 223 James P. Brawley Dr. SW, Atlanta, GA 30314 (United States); Dept. of Biological Sciences, Clark Atlanta University, 223 James P. Brawley Dr. SW, Atlanta, GA 30314 (United States)

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer E2A, considered as a tumor suppressor is highly expressed in prostate cancer. Black-Right-Pointing-Pointer Silencing of E2A attenuates cell proliferation and promotes apoptosis. Black-Right-Pointing-Pointer E2A regulates c-myc, Id1, Id3 and CDKN1A expression. Black-Right-Pointing-Pointer Loss of E2A promotes doxorubicin dependent apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Results suggest that E2A acts as a tumor promoter at least in prostate cancer. -- Abstract: E2A (TCF3) is a multifunctional basic helix loop helix (bHLH), transcription factor. E2A regulates transcription of target genes by homo- or heterodimerization with cell specific bHLH proteins. In general, E2A promotes cell differentiation, acts as a negative regulator of cell proliferation in normal cells and cancer cell lines and is required for normal B-cell development. Given the diverse biological pathways regulated/influenced by E2A little is known about its expression in cancer. In this study we investigated the expression of E2A in prostate cancer. Unexpectedly, E2A immuno-histochemistry demonstrated increased E2A expression in prostate cancer as compared to normal prostate. Silencing of E2A in prostate cancer cells DU145 and PC3 led to a significant reduction in proliferation due to G1 arrest that was in part mediated by increased CDKN1A(p21) and decreased Id1, Id3 and c-myc. E2A silencing in prostate cancer cell lines also resulted in increased apoptosis due to increased mitochondrial permeability and caspase 3/7 activation. Moreover, silencing of E2A increased sensitivity to doxorubicin induced apoptosis. Based on our results, we propose that E2A could be an upstream regulator of Id1 and c-Myc which are highly expressed in prostate cancer. These results for the first time demonstrate that E2A could in fact acts as a tumor promoter at least in prostate cancer.

  9. Plasmid Biopharmaceuticals.

    Science.gov (United States)

    Prazeres, Duarte Miguel F; Monteiro, Gabriel A

    2014-12-01

    Plasmids are currently an indispensable molecular tool in life science research and a central asset for the modern biotechnology industry, supporting its mission to produce pharmaceutical proteins, antibodies, vaccines, industrial enzymes, and molecular diagnostics, to name a few key products. Furthermore, plasmids have gradually stepped up in the past 20 years as useful biopharmaceuticals in the context of gene therapy and DNA vaccination interventions. This review provides a concise coverage of the scientific progress that has been made since the emergence of what are called today plasmid biopharmaceuticals. The most relevant topics are discussed to provide researchers with an updated overview of the field. A brief outline of the initial breakthroughs and innovations is followed by a discussion of the motivation behind the medical uses of plasmids in the context of therapeutic and prophylactic interventions. The molecular characteristics and rationale underlying the design of plasmid vectors as gene transfer agents are described and a description of the most important methods used to deliver plasmid biopharmaceuticals in vivo (gene gun, electroporation, cationic lipids and polymers, and micro- and nanoparticles) is provided. The major safety issues (integration and autoimmunity) surrounding the use of plasmid biopharmaceuticals is discussed next. Aspects related to the large-scale manufacturing are also covered, and reference is made to the plasmid products that have received marketing authorization as of today.

  10. Adenosine and Prostaglandin E2 Production by Human Inducible Regulatory T cells (iTreg in Health and Disease

    Directory of Open Access Journals (Sweden)

    Theresa L Whiteside

    2013-07-01

    Full Text Available Regulatory T cells (Treg play a key role in maintaining the balance of immune responses in human health and in disease. Treg come in many flavors and can utilize a variety of mechanisms to modulate immune responses. In cancer, inducible (i or adaptive Treg expand, accumulate in tissues and peripheral blood of patients and represent a functionally-prominent component of CD4+ T lymphocytes. Phenotypically and functionally, iTreg are distinct from natural (n Treg. A subset of iTreg expressing ectonucleotidases CD39 and CD73 is able to hydrolyze ATP to 5’-AMP and adenosine (ADO and thus mediate suppression of those immune cells which express ADO receptors. iTeg can also produce prostaglandin E2 (PGE2. The mechanisms responsible for iTeg-mediated suppression involve binding of ADO and PGE2 produced by iTreg to their respective receptors expressed on Teff, leading to the up-regulation of adenylate cyclase and cAMP activities in Teff and to their functional inhibition. The potential for regulating these mechanisms by the use of pharmacologic inhibitors to relieve iTreg-mediated suppression in cancer suggests the development of therapeutic strategies targeting the ADO and PGE2 pathways.

  11. Prostaglandin E2 reduces radiation-induced epithelial apoptosis through a mechanism involving AKT activation and bax translocation.

    Science.gov (United States)

    Tessner, Teresa G; Muhale, Filipe; Riehl, Terrence E; Anant, Shrikant; Stenson, William F

    2004-12-01

    Prostaglandin E2 (PGE2) synthesis modulates the response to radiation injury in the mouse intestinal epithelium through effects on crypt survival and apoptosis; however, the downstream signaling events have not been elucidated. WT mice receiving 16,16-dimethyl PGE2 (dmPGE2) had fewer apoptotic cells per crypt than untreated mice. Apoptosis in Bax(-/-) mice receiving 12 Gy was approximately 50% less than in WT mice, and the ability of dmPGE2 to attenuate apoptosis was lost in Bax(-/-) mice. Positional analysis revealed that apoptosis in the Bax(-/-) mice was diminished only in the bax-expressing cells of the lower crypts and that in WT mice, dmPGE2 decreased apoptosis only in the bax-expressing cells. The HCT-116 intestinal cell line and Bax(-/-) HCT-116 recapitulated the apoptotic response of the mouse small intestine with regard to irradiation and dmPGE2. Irradiation of HCT-116 cells resulted in phosphorylation of AKT that was enhanced by dmPGE2 through transactivation of the EGFR. Inhibition of AKT phosphorylation prevented the reduction of apoptosis by dmPGE2 following radiation. Transfection of HCT-116 cells with a constitutively active AKT reduced apoptosis in irradiated cells to the same extent as in nontransfected cells treated with dmPGE2. Treatment with dmPGE2 did not alter bax or bcl-x expression but suppressed bax translocation to the mitochondrial membrane. Our in vivo studies indicate that there are bax-dependent and bax-independent radiation-induced apoptosis in the intestine but that only the bax-dependent apoptosis is reduced by dmPGE2. The in vitro studies indicate that dmPGE2, most likely by signaling through the E prostaglandin receptor EP2, reduces radiation-induced apoptosis through transactivation of the EGFR and enhanced activation of AKT and that this results in reduced bax translocation to the mitochondria.

  12. (E)-2-benzylidene-4-phenyl-1,3-diselenole ameliorates signals of renal injury induced by cisplatin in rats.

    Science.gov (United States)

    Bortolatto, Cristiani F; Wilhelm, Ethel A; Roman, Silvane S; Nogueira, Cristina W

    2014-01-01

    The present study investigated the protective role of antioxidant (E)-2-benzylidene-4-phenyl-1,3-diselenole (BPD), an organoselenium compound, against the renal injury induced by cisplatin in rats. Canola oil or BPD (50 mg kg(-1)) was administered orally by gavage once a day for 6 days to rats. The first dose of BPD was given 24 h before a single intraperitoneal injection of saline or cisplatin (7 mg kg(-1)). At day 7, animals were killed and parameters related to renal injury were determined. The histological analysis showed that cisplatin caused renal injury in rats, which was accompanied by an increase in urea and creatinine levels in plasma. The increase of plasma creatinine levels negatively correlated with renal antioxidant defenses including ascorbic acid (AA) and reduced glutathione (GSH) content as well as glutathione S-transferase (GST), glutathione peroxidase (GPx) and catalase (CAT) activities. As revealed by histological analysis, BPD ameliorated tubular injury in rat kidney and reduced plasma markers altered by cisplatin. The administration of BPD to rats attenuated the reduction of renal AA and GSH content in animals exposed to cisplatin. The decrease of GST activity, but not GPx and CAT activities, in rats exposed to cisplatin was totally reversed by BPD administration. BPD was also effective in attenuating the inhibition of a sulfhydryl enzyme sensitive to oxidative stress, δ-aminolevulinic dehydratase, in kidneys of rats exposed to cisplatin. The present study demonstrated that BPD reduced renal injury induced by cisplatin in rats and this effect seems to be related to antioxidant mechanisms.

  13. Structural and electronic transitions in G e2S b2T e5 induced by ion irradiation damage

    Science.gov (United States)

    Privitera, S. M. S.; Mio, A. M.; Smecca, E.; Alberti, A.; Zhang, W.; Mazzarello, R.; Benke, J.; Persch, C.; La Via, F.; Rimini, E.

    2016-09-01

    G e2S b2T e5 polycrystalline films either in the trigonal stable phase or in the metastable rock-salt structure have been irradiated with 150 keV Ar+ ions. The effects of disorder are studied by electrical, optical, and structural measurements and density functional theory (DFT) simulations. In the metastable structure, the main effect of ion irradiation is a progressive amorphization, with an optical threshold at a fluence of 3 ×1013c m-2 . For the trigonal structure, a metal-insulator transition and a crystalline transition to rock-salt structure occur prior to amorphization, which requires a fluence of 8 ×1013c m-2 . The bonds of Te atoms close to the van der Waals gaps, present in the trigonal phase and identified by Raman spectroscopy, change as a function of the disorder induced by the irradiation. Comparison with DFT simulations shows that ion irradiation leads to the gradual filling of the van der Waals gaps with displaced Ge and Sb lattice atoms, giving rise first to a metal-insulator transition (9 % of displaced atoms) correlated to the modification of the Te bonds and then induces a structural transition to the metastable rock-salt phase (15 % of displaced atoms). The data presented here not only show the possibility to tune the degree of order, and therefore the electrical properties and the structure of phase change materials by ion irradiation, but also underline the importance of the van der Waals gaps in determining the transport mechanisms and the stability of the crystalline structure.

  14. Role of the prostaglandin E2 receptor agonists in TGF-β1-induced mesangial cell damage

    Science.gov (United States)

    Xi, Pei-pei; Xu, Yu-yin; Chen, Xiao-lan; Fan, Ya-ping; Wu, Jian-hua

    2016-01-01

    PGE2 exerts its biological effect through binding to various EP receptors that result inactivation of various signal transduction pathways. It also plays an important role in mice glomerular mesangial cells (MCs) damage induced by transforming growth factor-β1 (TGF-β1); however, the molecular mechanisms remain unknown. In the present study, we tested the efficacy of four selective agonists of PGE2 receptor, EP1A (17-phenyl trinor prostaglandin E2 ethyl amid), EP2A (butaprost), EP3A (sulprostone) and EP4A (cay10580), on mice MCs. Compared with the cAMP produced by TGF-β1, additional pretreatment of EP3A decreased the cAMP level. MCs treated with EP1A and EP3A augmented PGE2, cyclooxygenase-2 (COX-2), membrane-bound PGE synthase 1 (mPGES1), laminin (LN), connective tissue growth factor (CTGF) and cyclin D1 expression stimulated by TGFβ1. EP1A and EP3A increased the number of cells in S+G2/M phase and reduced cells in G0/G1 phase. EP1 and EP3 agonists also strengthened TGFβ1-induced mitogen-activated protein kinase (p38MAPK) and extracellular-signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Whereas MCs treated with EP2A and EP4A weakened PGE2, COX-2, mPGES1, LN, CTGF and cyclin D1 expression stimulated by TGFβ1. EP2A and EP4A decreased the number of cells in S+G2/M phase and increased cells in G0/G1 phase. EP2 and EP4 agonists weakened TGFβ1-induced p38MAPK and ERK1/2 phosphorylation. These findings suggest that PGE2 has an important role in the progression of kidney disease via the EP1/EP3 receptor, whereas EP2 and EP4 receptors are equally important in preserving the progression of chronic kidney failure. Thus, agonists of EP2 and EP4 receptors may provide a basis for treating kidney damage induced by TGF-β1. PMID:27512093

  15. Recombinant E2 glycoprotein of bovine viral diarrhea virus induces a solid humoral neutralizing immune response but fails to confer total protection in cattle

    Directory of Open Access Journals (Sweden)

    S. Chimeno Zoth

    2007-06-01

    Full Text Available Two recombinant baculoviruses were produced in order to obtain a bovine viral diarrhea virus (BVDV immunogen: AcNPV/E2 expressing E2 glycoprotein, and AcNPV/E0E1E2 expressing the polyprotein region coding for the three structural proteins of BVDV (E0, E1, and E2. Mice were immunized with Sf9 cells infected with the recombinant baculoviruses in a water in oil formulation and the production of neutralizing antibodies was evaluated. Since E2 elicited higher neutralizing antibody titers than E0-E1-E2 polyprotein, it was selected to immunize cattle. Calves received two doses of recombinant E2 vaccine and were challenged with homologous BVDV 37 days later. The recombinant immunogen induced neutralizing titers which showed a mean value of 1.5 ± 0.27 on the day of challenge and reached a top value of 3.36 ± 0.36, 47 days later (84 days post-vaccination. On the other hand, sera from animals which received mock-infected Sf9 cells did not show neutralizing activity until 25 days post-challenge (62 days post-vaccination, suggesting that these antibodies were produced as a consequence of BVDV challenge. Even when no total protection was observed in cattle, in vitro viral neutralization assays revealed that the recombinant immunogen was able to induce neutralizing antibody synthesis against the homologous strain as well as against heterologous strains in a very efficient way.

  16. HPV-18 E2circumflexE4 chimera: 2 new spliced transcripts and proteins induced by keratinocyte differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Chye Ling [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Gunaratne, Jayantha [Mass Spectrometry and Systems Biology Laboratory, Institute of Molecular and Cell Biology, A-STAR, Biopolis, 61 Biopolis Drive, Proteos, Singapore 138673 (Singapore); Lai, Deborah [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Carthagena, Laetitia [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Wang, Qian [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Xue, Yue Zhen; Quek, Ling Shih [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Doorbar, John [MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London N10 3UE (United Kingdom); Bachelerie, Francoise [UMR-S996, Universite Paris-Sud 11, 32 rue des Carnets, 92140 Clamart (France); Thierry, Francoise, E-mail: francoise.thierry@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore); Bellanger, Sophie, E-mail: sophie.bellanger@imb.a-star.edu.sg [Papillomavirus Regulation and Cancer, Institute of Medical Biology, Agency for Science, Technology and Research (A-STAR), Biopolis, 8A Biomedical Grove, Immunos, Singapore 138648 (Singapore)

    2012-07-20

    The Human Papillomavirus (HPV) E4 is known to be synthesized as an E1circumflexE4 fusion resulting from splice donor and acceptor sites conserved across HPV types. Here we demonstrate the existence of 2 HPV-18 E2circumflexE4 transcripts resulting from 2 splice donor sites in the 5 Prime part of E2, while the splice acceptor site is the one used for E1circumflexE4. Both E2circumflexE4 transcripts are up-regulated by keratinocyte differentiation in vitro and can be detected in clinical samples containing low-grade HPV-18-positive cells from Pap smears. They give rise to two fusion proteins in vitro, E2circumflexE4-S and E2circumflexE4-L. Whereas we could not differentiate E2circumflexE4-S from E1circumflexE4 in vivo, E2circumflexE4-L could be formally identified as a 23 kDa protein in raft cultures in which the corresponding transcript was also found, and in a biopsy from a patient with cervical intraepithelial neoplasia stage I-II (CINI-II) associated with HPV-18, demonstrating the physiological relevance of E2circumflexE4 products.

  17. Suppression of the p53- or pRB-mediated G1 checkpoint is required for E2F-induced S-phase entry

    DEFF Research Database (Denmark)

    Lomazzi, Marina; Moroni, M Cristina; Jensen, Michael R;

    2002-01-01

    fibroblasts, increased E2F1 activity can result in S-phase entry in diploid fibroblasts in which the p53-mediated G1 checkpoint is suppressed. In addition, we show that E2F1 can induce S phase in primary mouse fibroblasts lacking pRB. These results indicate that, in addition to acting as an E2F......Deregulation of the retinoblastoma protein (pRB) pathway is a hallmark of cancer. In the absence of other genetic alterations, this deregulation results in lack of differentiation, hyperproliferation and apoptosis. The pRB protein acts as a transcriptional repressor by targeting the E2F...... transcription factors, whose functions are required for entry into S phase. Increased E2F activity can induce S phase in quiescent cells--this is a central element of most models for the development of cancer. We show that although E2F1 alone is not sufficient to induce S phase in diploid mouse and human...

  18. Effect of intramuscular injection of hepatocyte growth factor plasmid DNA with electroporation on bleomycin-induced lung fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    LONG Xiang; XIONG Sheng-dao; XIONG Wei-ning; XU Yong-jian

    2007-01-01

    Background So far, there is no efficient treatment for pulmonary fibrosis. The objective of this study was to determine whether intramuscular injection of the hepatocyte growth factor (HGF) plasmid DNA by in vivo electroporation could prevent bleomycin-induced pulmonary fibrosis in rats, and to investigate the possible mechanisms.Methods Twenty male Wistar rats were randomly divided into four groups: control group(group C), model group (group M), early intervention group (group Ⅰ ) and late intervention group (groupⅡ). Groups M, Ⅰ and Ⅱ were intratracheally infused with bleomycin, then injected the plasmid pcDNA3.1-hHGF to group Ⅰ on day 7, 14 and 21. Group Ⅱ received the same treatment like Group Ⅰ on day 14 and 21. All the rats were killed on day 28 after bleomycin injection. We detected Homo HGF expression in the rats with ELISA method and estimated the pathological fibrosis score of lung tissue using hematoxylin eosin (HE) and Massion staining. The mRNA expression of transforming growth factor-β1 (TGF-β1),cycloxygenase-2 (COX-2), and rat HGF in rat pulmonary parenchyma were evaluated by RT-PCR.Immunohistochemistry and Western blotting were performed to determine the protein expression of transforming TGF-β1 and COX-2 in lung parenchyma.Results The plasmid pcDNA3.1-hHGF could express hHGF in NIH3T3 cells and the hHGF protein is secreted into the culture medium. The expression of hHGF protein could be monitored in quadriceps muscle, plasma and lung in Groups Ⅰ and Ⅱ. Pulmonary fibrosis levels of Groups Ⅰ and Ⅱ were obviously lower than that of group M (P<0.05).Expression of TGF-β1 protein and mRNA in lung tissue was markedly decreased in Groups Ⅰ and Ⅱ compared with Group M (P<0.05). The level of expression of HGF and COX-2 mRNA was higher in Groups Ⅰ and Ⅱ than in Group M (P<0.05).Conclusions Injection of the plasmid pcDNA3.1-hHGF into skeletal muscle with electroporation has a potential role in the treatment of bleomycin-induced

  19. Metabolite profiling uncovers plasmid-induced cobalt limitation under methylotrophic growth conditions.

    Directory of Open Access Journals (Sweden)

    Patrick Kiefer

    Full Text Available BACKGROUND: The introduction and maintenance of plasmids in cells is often associated with a reduction of growth rate. The reason for this growth reduction is unclear in many cases. METHODOLOGY/PRINCIPAL FINDINGS: We observed a surprisingly large reduction in growth rate of about 50% of Methylobacterium extorquens AM1 during methylotrophic growth in the presence of a plasmid, pCM80 expressing the tetA gene, relative to the wild-type. A less pronounced growth delay during growth under non-methylotrophic growth conditions was observed; this suggested an inhibition of one-carbon metabolism rather than a general growth inhibition or metabolic burden. Metabolome analyses revealed an increase in pool sizes of ethylmalonyl-CoA and methylmalonyl-CoA of more than 6- and 35-fold, respectively, relative to wild type, suggesting a strongly reduced conversion of these central intermediates, which are essential for glyoxylate regeneration in this model methylotroph. Similar results were found for M. extorquens AM1 pCM160 which confers kanamycin resistance. These intermediates of the ethylmalonyl-CoA pathway have in common their conversion by coenzyme B(12-dependent mutases, which have cobalt as a central ligand. The one-carbon metabolism-related growth delay was restored by providing higher cobalt concentrations, by heterologous expression of isocitrate lyase as an alternative path for glyoxylate regeneration, or by identification and overproduction of proteins involved in cobalt import. CONCLUSIONS/SIGNIFICANCE: This study demonstrates that the introduction of the plasmids leads to an apparent inhibition of the cobalt-dependent enzymes of the ethylmalonyl-CoA pathway. Possible explanations are presented and point to a limited cobalt concentration in the cell as a consequence of the antibiotic stress.

  20. The Study on The ImmuneResponse Induced by Expressing Recombinant Plasmid of Dengue Virus Type 2 NS3 Protein

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    The PSV · NS3, an expressing recombinant plasmid of dengue virus type 2 NS3 protein, was in Jected directly into the quadriceps of Balb/C mice to explore whether it could inducing immune response. The splenic T cell subsets of two groups was analysed by flow cytometry. It was found that the percentage of CD4+ and CD8+ T cells of experimental group were significantly higher than those of the control group. The titer of IgG antibody was as high as 1:S 120 in experimental group, but it couldn't be detected in control group by ELISA. The western blot further proved that the IgG antibody was specific for NS3 protein. Those results Suggested that inoculation Balb/C mice with PSV · NS3 could inducing immune response, and the NS3 protein might be used as the candidate protein of DNA vaccine of dengue virus.

  1. Prostaglandin E2 EP2 Receptor Deletion Attenuates Intracerebral Hemorrhage-Induced Brain Injury and Improves Functional Recovery

    Directory of Open Access Journals (Sweden)

    Jenna L. Leclerc

    2015-04-01

    Full Text Available Intracerebral hemorrhage (ICH is a devastating type of stroke characterized by bleeding into the brain parenchyma and secondary brain injury resulting from strong neuroinflammatory responses to blood components. Production of prostaglandin E2 (PGE2 is significantly upregulated following ICH and contributes to this inflammatory response in part through its E prostanoid receptor subtype 2 (EP2. Signaling through the EP2 receptor has been shown to affect outcomes of many acute and chronic neurological disorders; although, not yet explored in the context of ICH. Wildtype (WT and EP2 receptor knockout (EP2−/− mice were subjected to ICH, and various anatomical and functional outcomes were assessed by histology and neurobehavioral testing, respectively. When compared with age-matched WT controls, EP2−/− mice had 41.9 ± 4.7% smaller ICH-induced brain lesions and displayed significantly less ipsilateral hemispheric enlargement and incidence of intraventricular hemorrhage. Anatomical outcomes correlated with improved functional recovery as identified by neurological deficit scoring. Histological staining was performed to begin investigating the mechanisms involved in EP2-mediated neurotoxicity after ICH. EP2−/− mice exhibited 45.5 ± 5.8% and 41.4 ± 8.1% less blood and ferric iron accumulation, respectively. Furthermore, significantly less striatal and cortical microgliosis, striatal and cortical astrogliosis, blood–brain barrier breakdown, and peripheral neutrophil infiltration were seen in EP2−/− mice. This study is the first to suggest a deleterious role for the PGE2-EP2 signaling axis in modulating brain injury, inflammation, and functional recovery following ICH. Targeting the EP2 G protein-coupled receptor may represent a new therapeutic avenue for the treatment of hemorrhagic stroke.

  2. In vitro analysis of the factors contributing to the antiviral state induced by a plasmid encoding the viral haemorrhagic septicaemia virus glycoprotein G in transfected trout cells.

    Science.gov (United States)

    Ortega-Villaizan, M; Chico, V; Martinez-Lopez, A; Falco, A; Perez, L; Coll, J M; Estepa, A

    2011-01-17

    We have found out that transfection of the RTG-2 cell line with the viral haemorrhagic septicaemia virus (VHSV) glycoprotein G (G(VHSV))-coding plasmid induces an anti-VHSV state, similar to that induced by poly I:C. Taking the advantage of the constitutive expression of toll-like receptor 9 gene (tlr9) in RTG-2 cells, we have investigated whether this antiviral state was induced by the cytosine-phosphodiester-guanine (CpG) motifs present in the plasmid DNA, by the endogenous expression of G(VHSV) protein or by both elements. For that, we have analysed the expression profile of the rainbow trout tlr9 and several genes related to TLR9-mediated immune response in the absence or presence of a lysosomotropic drug that specifically blocks TLR9-CpG DNA interaction. The results suggested that the high levels of cell protection conferred by a plasmid encoding G(VHSV) gene are due to G(VHSV) rather than to the CpG motifs within plasmid DNA. Therefore, plasmid DNA might not play a key role in the immune response elicited by DNA vaccines or perhaps other receptors instead TLR9 could be implicated in CpG motifs recognition and signalling. In addition, since RTG-2 cells express tlr9 gene, this cell line could be a good tool for screening TLR9 agonists, such as the immunomodulatory oligonucleotides (IMOs), as fish DNA vaccine adjuvants.

  3. E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

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    Kuan-Ho Lin

    2017-04-01

    Full Text Available Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2, for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs. In addition, protein phosphatase such as protein phosphatase 1 (PP1 and calcineurin (PP2B are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

  4. Multiple linear B-cell epitopes of classical swine fever virus glycoprotein E2 expressed in E.coli as multiple epitope vaccine induces a protective immune response

    Directory of Open Access Journals (Sweden)

    Wei Jian-Chao

    2011-07-01

    Full Text Available Abstract Classical swine fever is a highly contagious disease of swine caused by classical swine fever virus, an OIE list A pathogen. Epitope-based vaccines is one of the current focuses in the development of new vaccines against classical swine fever virus (CSFV. Two B-cell linear epitopes rE2-ba from the E2 glycoprotein of CSFV, rE2-a (CFRREKPFPHRMDCVTTTVENED, aa844-865 and rE2-b (CKEDYRYAISSTNEIGLLGAGGLT, aa693-716, were constructed and heterologously expressed in Escherichia coli as multiple epitope vaccine. Fifteen 6-week-old specified-pathogen-free (SPF piglets were intramuscularly immunized with epitopes twice at 2-week intervals. All epitope-vaccinated pigs could mount an anamnestic response after booster vaccination with neutralizing antibody titers ranging from 1:16 to 1:256. At this time, the pigs were subjected to challenge infection with a dose of 1 × 106 TCID50 virulent CSFV strain. After challenge infection, all of the rE2-ba-immunized pigs were alive and without symptoms or signs of CSF. In contrast, the control pigs continuously exhibited signs of CSF and had to be euthanized because of severe clinical symptoms at 5 days post challenge infection. The data from in vivo experiments shown that the multiple epitope rE2-ba shown a greater protection (similar to that of HCLV vaccine than that of mono-epitope peptide(rE2-a or rE2-b. Therefore, The results demonstrated that this multiple epitope peptide expressed in a prokaryotic system can be used as a potential DIVA (differentiating infected from vaccinated animals vaccine. The E.coli-expressed E2 multiple B-cell linear epitopes retains correct immunogenicity and is able to induce a protective immune response against CSFV infection.

  5. Hepatitis C virus E2 protein induce reactive oxygen species (ROS)-related fibrogenesis in the HSC-T6 hepatic stellate cell line.

    Science.gov (United States)

    Ming-Ju, Hsieh; Yih-Shou, Hsieh; Tzy-Yen, Chen; Hui-Ling, Chiou

    2011-01-01

    Chronic infection of hepatitis C virus (HCV) leads to hepatic fibrosis and subsequently cirrhosis, although the underlying mechanisms have not been established. Previous studies have indicated that the binding of HCV E2 protein and CD81 on the surface of hepatic stellate cells (HSCs) lead to the increased protein level and activity of matrix metallopeptidase (MMP) 2, indicating that E2 may involve in the HCV-induced fibrosis. This study was designed to investigate the involvement of HCV E2 protein in the hepatic fibrogenesis. Results showed that E2 protein may promote the expression levels of α-smooth muscle actin (α-SMA) and collagen α(I). Furthermore, several pro-fibrosis or pro-inflammatory cytokines, including transforming growth factor (TGF)-β1, connective tissue growth factor (CTGF), interleukin (IL)-6 and IL-1β, were significantly increased in E2 transfected-HSC cell lines, while the expression of MMP-2 are also considerably increased. Moreover, the significant increases of CTGF and TGF-β1 in a stable E2-expressing Huh7 cell line were also observed the same results. Further molecular studies indicated that the impact of E2 protein on collagen production related to higher production of ROS and activated Janus kinase (JAK)1, JAK2 and also enhance the activation of ERK1/2 and p38, while catalase and inhibitors specific for JAK, ERK1/2, and p38 abolish E2-enhanced expression of collagen α(I). Taken together, this study indicated that E2 protein involve in the pathogenesis of HCV-mediated fibrosis via an up-regulation of collagen α(I) and oxidative stress, which is JAK pathway related.

  6. Chromosomal and plasmid-encoded factors of Shigella flexneri induce secretogenic activity ex vivo.

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    Christina S Faherty

    Full Text Available Shigella flexneri is a Gram-negative, facultative intracellular pathogen that causes millions of cases of watery or bloody diarrhea annually, resulting in significant global mortality. Watery diarrhea is thought to arise in the jejunum, and subsequent bloody diarrhea occurs as a result of invasion of the colonic epithelium. Previous literature has demonstrated that Shigella encodes enterotoxins, both chromosomally and on the 220 kilobase virulence plasmid. The ShigellaEnterotoxins 1 and 2 (ShET1 and ShET2 have been shown to increase water accumulation in the rabbit ileal loop model. In addition, these toxins increase the short circuit current in rabbit tissue mounted in Ussing chambers, which is a model for the ion exchange that occurs during watery diarrhea. In this study, we sought to validate the use of mouse jejunum in Ussing chamber as an alternative, more versatile model to study bacterial pathogenesis. In the process, we also identified enterotoxins in addition to ShET1 and ShET2 encoded by S. flexneri. Through analysis of proteins secreted from wildtype bacteria and various deletion mutants, we have identified four factors responsible for enterotoxin activity: ShET1 and Pic, which are encoded on the chromosome; ShET2 (encoded by sen or ospD3, which requires the type-III secretion system for secretion; and SepA, an additional factor encoded on the virulence plasmid. The use of mouse jejunum serves as a reliable and reproducible model to identify the enterotoxins elaborated by enteric bacteria. Moreover, the identification of all Shigella proteins responsible for enterotoxin activity is vital to our understanding of Shigella pathogenicity and to our success in developing safe and effective vaccine candidates.

  7. Hepatitis C virus E2 protein encapsulation into poly d, l-lactic-co-glycolide microspheres could induce mice cytotoxic T-cell response.

    Science.gov (United States)

    Roopngam, Piyachat; Liu, Kewei; Mei, Lin; Zheng, Yi; Zhu, Xianbing; Tsai, Hsiang-I; Huang, Laiqiang

    Hepatitis C virus (HCV) is known to cause hepatitis and hepatocellular carcinoma. E2 envelope glycoprotein of HCV type (HCV-E2) has been reported to bind human host cells and is a major target for developing anti-HCV vaccines. However, the therapeutic vaccine for infected patients still needs further development. The vaccine aims to provide cytotoxic T-cells to eliminate infected cells and hepatocellular carcinoma. Currently, there is no effective HCV therapeutic vaccine because most chronically infected patients rarely generate cytotoxic T-cells, even though they have high levels of neutralizing antibodies. Therefore, the adjuvant must be applied to enhance the efficacy of the therapeutic vaccine. In this study, we constructed HCV1b-E2 recombinant protein, a truncated form of peptide, to combine with an effective vaccine adjuvant and delivery system by using poly d,l-lactic-co-glycolide (PLGA) microspheres. HCV1b-E2 protein was effectively encapsulated into PLGA microspheres (HCV1b-E2-PLGA) as a strategy to deliver an insoluble form of HCV1b-E2 protein. The size and shape of PLGA microspheres were generated properly to carry an insoluble form of viral peptide in vivo. The encapsulated viral protein was slowly and continuously released from PLGA microspheres, which indicated the property of the adjuvant. HCV1b-E2-PLGA can trigger a cell-mediated immune response by inducing an expression of mice CD8(+) T-cells. Our results demonstrated that HCV1b-E2-PLGA-immunized mice have a significantly increased CD8(+) T-cell number, whereas HCV1b-E2-immunized mice have a lower number of CD8(+) T-cells. Moreover, HCV1b-E2-PLGA could induce a specific antibody to viral protein, and the immune cells could secrete IFN-γ, which is a significant cytokine for viral response. Thus, HCV1b-E2-PLGA is shown to have adjuvant property and efficacy in the murine model, which is a good strategy to develop HCV prophylactic and therapeutic vaccines.

  8. E2F is involved in radioresistance of carbon ion induced apoptosis via Bax/caspase 3 signal pathway in human hepatoma cell.

    Science.gov (United States)

    Xie, Yi; Si, Jing; Wang, Yu-Pei; Li, Hong-Yan; Di, Cui-Xia; Yan, Jun-Fang; Ye, Yan-Cheng; Zhang, Yan-Shan; Zhang, Hong

    2017-05-13

    Deletion of p53, most common genetic alteration, is observed in human tumors and reported to lead to improve in cell radioresistance. Heavy-ion irradiation (IR) could induce p53(-/-) cancer cells apoptosis. However, little is known regarding the molecular mechanism in this type of cell apoptosis. The present studies have focused on mechanisms state of signaling pathways as an activator of the cell fate decisions induced by heavy ion IR without p53. Carbon ion IR could induce up-regulation of E2F1 expression in cancer cells. This phenomenon was not observed in X-ray IR group. Up-regulation of E2F1 could cause a higher reduction in clonogenic survival, low level of cellular activity, G2 /M phase arrest, promotion of apoptosis rate, up-regulation of phosphor-Rb, Bax, and cleaved-caspase 3 proteins expressions without p53. Changes of E2F1 expressions could partly alter radioresistance in cancer cells. The results were suggested that heavy ion IR could induce p53(-/-) cancer cells apoptosis via E2F1 signal pathway. Our study provides a scientific rationale for the clinical use of heavy ion as radiotherapy in patients with p53-deficient tumors, which are often resistant to radiotherapy. © 2017 Wiley Periodicals, Inc.

  9. Prostaglandin E2 induces hypoxia-inducible factor-1alpha stabilization and nuclear localization in a human prostate cancer cell line.

    Science.gov (United States)

    Liu, Xin Hua; Kirschenbaum, Alexander; Lu, Min; Yao, Shen; Dosoretz, Amy; Holland, James F; Levine, Alice C

    2002-12-20

    Hypoxia-induced up-regulation of vascular endothelial growth factor (VEGF) expression is a critical event leading to tumor neovascularization. Hypoxia stimulates hypoxia-inducible factor-1alpha (HIF-1alpha), a transcriptional activator of VEGF. Cyclooxygenase (COX)-2, an inducible enzyme that catalyzes the formation of prostaglandins (PGs) from arachidonic acid, is also induced by hypoxia. We reported previously that COX-2 inhibition prevents hypoxic up-regulation of VEGF in human prostate cancer cells and that prostaglandin E(2) (PGE(2)) restores hypoxic effects on VEGF. We hypothesized that PGE(2) mediates hypoxic effects on VEGF by modulating HIF-1alpha expression. Addition of PGE(2) to PC-3ML human prostate cancer cells had no effect on HIF-1alpha mRNA levels. However, PGE(2) significantly increased HIF-1alpha protein levels, particularly in the nucleus. This effect of PGE(2) largely results from the promotion of HIF-1alpha translocation from the cytosol to the nucleus. PGE(2) addition to PC-3 ML cells transfected with a GFP-HIF-1alpha vector induced a time-dependent nuclear accumulation of the HIF-1alpha protein. Two selective COX-2 inhibitors, meloxicam and NS398, decreased HIF-1alpha levels and nuclear localization, under both normoxic and hypoxic conditions. Of several prostaglandins tested, only PGE(2) reversed the effects of a COX-2 inhibitor in hypoxic cells. Finally, PGE(2) effects on HIF-1alpha were specifically inhibited by PD98059 (a MAPK inhibitor). These data demonstrate that PGE(2) production via COX-2-catalyzed pathway plays a critical role in HIF-1alpha regulation by hypoxia and imply that COX-2 inhibitors can prevent hypoxic induction of HIF-mediated gene transcription in cancer cells.

  10. A Histone-Like Protein Induces Plasmid DNA to Form Liquid Crystals in Vitro and Gene Compaction in Vivo

    Directory of Open Access Journals (Sweden)

    Shiyong Sun

    2013-12-01

    Full Text Available The liquid crystalline state is a universal phenomenon involving the formation of an ordered structure via a self-assembly process that has attracted attention from numerous scientists. In this study, the dinoflagellate histone-like protein HCcp3 is shown to induce super-coiled pUC18 plasmid DNA to enter a liquid crystalline state in vitro, and the role of HCcp3 in gene condensation in vivo is also presented. The plasmid DNA (pDNA-HCcp3 complex formed birefringent spherical particles with a semi-crystalline selected area electronic diffraction (SAED pattern. Circular dichroism (CD titrations of pDNA and HCcp3 were performed. Without HCcp3, pUC18 showed the characteristic B conformation. As the HCcp3 concentration increased, the 273 nm band sharply shifted to 282 nm. When the HCcp3 concentration became high, the base pair (bp/dimer ratio fell below 42/1, and the CD spectra of the pDNA-HCcp3 complexes became similar to that of dehydrated A-form DNA. Microscopy results showed that HCcp3 compacted the super-coiled gene into a condensed state and that inclusion bodies were formed. Our results indicated that HCcp3 has significant roles in gene condensation both in vitro and in histone-less eukaryotes in vivo. The present study indicates that HCcp3 has great potential for applications in non-viral gene delivery systems, where HCcp3 may compact genetic material to form liquid crystals.

  11. The bovine viral diarrhea virus E2 protein formulated with a novel adjuvant induces strong, balanced immune responses and provides protection from viral challenge in cattle.

    Science.gov (United States)

    Snider, Marlene; Garg, Ravendra; Brownlie, Robert; van den Hurk, Jan V; van Drunen Littel-van den Hurk, Sylvia

    2014-11-28

    Bovine viral diarrhea virus (BVDV) is still one of the most serious pathogens in cattle, meriting the development of improved vaccines. Recently, we developed a new adjuvant consisting of poly[di(sodium carboxylatoethylphenoxy)]-phosphazene (PCEP), either CpG ODN or poly(I:C), and an immune defense regulator (IDR) peptide. As this adjuvant has been shown to mediate the induction of robust, balanced immune responses, it was evaluated in an E2 subunit vaccine against BVDV in lambs and calves. The BVDV type 2 E2 protein was produced at high levels in a mammalian expression system and purified. When formulated with either CpG ODN or poly(I:C), together with IDR and PCEP, the E2 protein elicited high antibody titers and production of IFN-γ secreting cells in lambs. As the immune responses were stronger when poly(I:C) was used, the E2 protein with poly(I:C), IDR and PCEP was subsequently tested in cattle. Robust virus neutralizing antibodies as well as cell-mediated immune responses, including CD8(+) cytotoxic T cell (CTL) responses, were induced. The fact that CTL responses were demonstrated in calves vaccinated with an E2 protein subunit vaccine indicates that this adjuvant formulation promotes cross-presentation. Furthermore, upon challenge with a high dose of virulent BVDV-2, the vaccinated calves showed almost no temperature response, weight loss, leukopenia or virus replication, in contrast to the control animals, which had severe clinical disease. These data suggest that this E2 subunit formulation induces significant protection from BVDV-2 challenge, and thus is a promising BVDV vaccine candidate; in addition, the adjuvant platform has applications in bovine vaccines in general.

  12. Conditional Expression of E2A-HLF Induces B-Cell Precursor Death and Myeloproliferative-Like Disease in Knock-In Mice.

    Directory of Open Access Journals (Sweden)

    Jesús Duque-Afonso

    Full Text Available Chromosomal translocations are driver mutations of human cancers, particularly leukemias. They define disease subtypes and are used as prognostic markers, for minimal residual disease monitoring and therapeutic targets. Due to their low incidence, several translocations and their biological consequences remain poorly characterized. To address this, we engineered mouse strains that conditionally express E2A-HLF, a fusion oncogene from the translocation t(17;19 associated with 1% of pediatric B-cell precursor ALL. Conditional oncogene activation and expression were directed to the B-cell compartment by the Cre driver promoters CD19 or Mb1 (Igα, CD79a, or to the hematopoietic stem cell compartment by the Mx1 promoter. E2A-HLF expression in B-cell progenitors induced hyposplenia and lymphopenia, whereas expression in hematopoietic stem/progenitor cells was embryonic lethal. Increased cell death was detected in E2A-HLF expressing cells, suggesting the need for cooperating genetic events that suppress cell death for B-cell oncogenic transformation. E2A-HLF/Mb1.Cre aged mice developed a fatal myeloproliferative-like disorder with low frequency characterized by leukocytosis, anemia, hepatosplenomegaly and organ-infiltration by mature myelocytes. In conclusion, we have developed conditional E2A-HLF knock-in mice, which provide an experimental platform to study cooperating genetic events and further elucidate translational biology in cross-species comparative studies.

  13. Adenovirus E4 open reading frame 4-induced dephosphorylation inhibits E1A activation of the E2 promoter and E2F-1-mediated transactivation independently of the retinoblastoma tumor suppressor protein

    DEFF Research Database (Denmark)

    Mannervik, M; Fan, S; Ström, A C

    1999-01-01

    Previous studies have shown that the cell cycle-regulated E2F transcription factor is subjected to both positive and negative control by phosphorylation. Here we show that in transient transfection experiments, adenovirus E1A activation of the viral E2 promoter is abrogated by coexpression...... of the viral E4 open reading frame 4 (E4-ORF4) protein. This effect does not to require the retinoblastoma protein that previously has been shown to regulate E2F activity. The inhibitory activity of E4-ORF4 appears to be specific because E4-ORF4 had little effect on, for example, E4-ORF6/7 transactivation...... of the E2 promoter. We further show that the repressive effect of E4-ORF4 on E2 transcription works mainly through the E2F DNA-binding sites in the E2 promoter. In agreement with this, we find that E4-ORF4 inhibits E2F-1/DP-1-mediated transactivation. We also show that E4-ORF4 inhibits E2 mRNA expression...

  14. Lichen metabolites prevent UV light and nitric oxide-mediated plasmid DNA damage and induce apoptosis in human melanoma cells.

    Science.gov (United States)

    Russo, A; Piovano, M; Lombardo, L; Garbarino, J; Cardile, V

    2008-09-26

    In humans both UV-A and UV-B can cause gene mutations and suppress immunity, which leads to skin cancer, including melanoma. Inhibition of reactive oxygen species (ROS) and reactive nitrogen species (RNS) appears particularly promising as ROS and RNS production by both UV-A and UV-B contributes to inflammation, immunosuppression, gene mutation and carcinogenesis. We evaluated the effect of two lichen compounds, sphaerophorin (depside) and pannarin (depsidone) on pBR322 DNA cleavage induced by hydroxyl radicals (()OH), and by nitric oxide (NO), and their superoxide anion (O(2)(-)) scavenging capacity. In addition, we investigated the growth inhibitory activity of these compounds against human melanoma cells (M14 cell line). Sphaerophorin and pannarin showed a protective effect on plasmid DNA and exhibited a superoxide dismutase like effect. The data obtained in cell culture show that these lichen metabolites inhibit the growth of melanoma cells, inducing an apoptotic cell death, demonstrated by the fragmentation of genomic DNA (COMET and TUNEL Assays) and by a significant increase of caspase-3 activity, and correlated, at least in part, to the increase of ROS generation, These results confirm the promising biological properties of sphaerophorin and pannarin and encourage further investigations on their molecular mechanisms.

  15. Inhibition of lipopolysaccharide-induced nitric oxide and prostaglandin E2 production by chloroform fraction of Cudrania tricuspidata in RAW 264.7 macrophages

    OpenAIRE

    Yang Gabsik; Lee Kyungjin; Lee Mihwa; Ham Inhye; Choi Ho-Young

    2012-01-01

    Abstract Background Cudrania tricuspidata extract is an important traditional herbal remedy for tumors, inflammation, gastritis, and liver damage and is predominantly used in Korea, China, and Japan. However, the anti-inflammatory effects of the extract have not yet been conclusively proved. Methods In this study, we investigated the effects of the CHCl3 fraction (CTC) of a methanol extract of C. tricuspidata on the lipopolysaccharide (LPS)-induced nitric oxide (NO) and prostaglandin E2 (PGE2...

  16. Plasmid DNA Vaccine Co-Immunisation Modulates Cellular and Humoral Immune Responses Induced by Intranasal Inoculation in Mice.

    Directory of Open Access Journals (Sweden)

    Deborah F L King

    Full Text Available An effective HIV vaccine will likely require induction of both mucosal and systemic cellular and humoral immune responses. We investigated whether intramuscular (IM delivery of electroporated plasmid DNA vaccine and simultaneous protein vaccinations by intranasal (IN and IM routes could be combined to induce mucosal and systemic cellular and humoral immune responses to a model HIV-1 CN54 gp140 antigen in mice.Co-immunisation of DNA with intranasal protein successfully elicited both serum and vaginal IgG and IgA responses, whereas DNA and IM protein co-delivery did not induce systemic or mucosal IgA responses. Cellular IFNγ responses were preserved in co-immunisation protocols compared to protein-only vaccination groups. The addition of DNA to IN protein vaccination reduced the strong Th2 bias observed with IN protein vaccination alone. Luminex analysis also revealed that co-immunisation with DNA and IN protein induced expression of cytokines that promote B-cell function, generation of TFH cells and CCR5 ligands that can reduce HIV infectivity.These data suggest that while IN inoculation alone elicits both cellular and humoral responses, co-administration with homologous DNA vaccination can tailor these towards a more balanced Th1/Th2 phenotype modulating the cellular cytokine profile while eliciting high-levels of antigen-specific antibody. This work provides insights on how to generate differential immune responses within the same vaccination visit, and supports co-immunisation with DNA and protein by a mucosal route as a potential delivery strategy for HIV vaccines.

  17. Generation of integration-free human induced pluripotent stem cells from postnatal blood mononuclear cells by plasmid vector expression.

    Science.gov (United States)

    Dowey, Sarah N; Huang, Xiaosong; Chou, Bin-Kuan; Ye, Zhaohui; Cheng, Linzhao

    2012-11-01

    Several human postnatal somatic cell types have been successfully reprogrammed to induced pluripotent stem cells (iPSCs). Blood mononuclear cells (MNCs) offer several advantages compared with other cell types. They are easily isolated from umbilical cord blood (CB) or adult peripheral blood (PB), and can be used fresh or after freezing. A short culture allows for more efficient reprogramming, with iPSC colonies forming from blood MNCs in 14 d, compared with 28 d for age-matched fibroblastic cells. The advantages of briefly cultured blood MNCs may be due to favorable epigenetic profiles and gene expression patterns. Blood cells from adults, especially nonlymphoid cells that are replenished frequently from intermittently activated blood stem cells, are short-lived in vivo and may contain less somatic mutations than skin fibroblasts, which are more exposed to environmental mutagens over time. We describe here a detailed, validated protocol for effective generation of integration-free human iPSCs from blood MNCs by plasmid vectors.

  18. A small antigenic determinant of the Chikungunya virus E2 protein is sufficient to induce neutralizing antibodies which are partially protective in mice.

    Directory of Open Access Journals (Sweden)

    Christopher Weber

    2015-04-01

    Full Text Available The mosquito-borne Chikungunya virus (CHIKV causes high fever and severe joint pain in humans. It is expected to spread in the future to Europe and has recently reached the USA due to globalization, climate change and vector switch. Despite this, little is known about the virus life cycle and, so far, there is no specific treatment or vaccination against Chikungunya infections. We aimed here to identify small antigenic determinants of the CHIKV E2 protein able to induce neutralizing immune responses.E2 enables attachment of the virus to target cells and a humoral immune response against E2 should protect from CHIKV infections. Seven recombinant proteins derived from E2 and consisting of linear and/or structural antigens were created, and were expressed in and purified from E. coli. BALB/c mice were vaccinated with these recombinant proteins and the mouse sera were screened for neutralizing antibodies. Whereas a linear N-terminally exposed peptide (L and surface-exposed parts of the E2 domain A (sA alone did not induce neutralizing antibodies, a construct containing domain B and a part of the β-ribbon (called B+ was sufficient to induce neutralizing antibodies. Furthermore, domain sA fused to B+ (sAB+ induced the highest amount of neutralizing antibodies. Therefore, the construct sAB+ was used to generate a recombinant modified vaccinia virus Ankara (MVA, MVA-CHIKV-sAB+. Mice were vaccinated with MVA-CHIKV-sAB+ and/or the recombinant protein sAB+ and were subsequently challenged with wild-type CHIKV. Whereas four vaccinations with MVA-CHIKV-sAB+ were not sufficient to protect mice from a CHIKV infection, protein vaccination with sAB+ markedly reduced the viral titers of vaccinated mice.The recombinant protein sAB+ contains important structural antigens for a neutralizing antibody response in mice and its formulation with appropriate adjuvants might lead to a future CHIKV vaccine.

  19. Androgen receptor or estrogen receptor-beta blockade alters DHEA-, DHT-, and E(2)-induced proliferation and PSA production in human prostate cancer cells.

    Science.gov (United States)

    Arnold, Julia T; Liu, Xunxian; Allen, Jeffrey D; Le, Hanh; McFann, Kimberly K; Blackman, Marc R

    2007-08-01

    Dehydroepiandrosterone (DHEA) is an endogenous steroid that is metabolized to androgens and/or estrogens in the human prostate. DHEA levels decline with age, and use of DHEA supplements to retard the aging process is of unproved effectiveness and safety. LNCaP and LAPC-4 prostate cancer cells were used to determine whether DHEA-modulated proliferation and prostate specific antigen (PSA) production were mediated via the androgen receptor (AR) and/or ERbeta. Cells were treated with DHEA, DHT, or E(2) and antagonists to AR (Casodex-bicalutamide) or ER (ICI 182,780) or siRNA to the respective receptors. Proliferation was assessed by MTT assay and PSA mRNA and protein secretion were measured by quantitative real-time PCR and ELISA. Associations of AR and ERbeta were analyzed by co-immunoprecipitation studies and fluorescent confocal microscopy. DHEA-, T-, and E(2)-induced proliferation of LNCaP cells was blunted by Casodex but not by ICI treatment. In LNCaP cells, Casodex and ICI suppressed hormone-induced PSA production. In LAPC-4 cells, DHT-stimulated PSA mRNA was inhibited by Casodex and ICI, and the minimal stimulation by DHEA was inhibited by ICI. Use of siRNAs confirmed involvement of AR and ERbeta in hormone-induced PSA production while AR-ERbeta co-association was suggested by immunoprecipitation and nuclear co-localization. These findings support involvement of both AR and ERbeta in mediating DHEA-, DHT-, and E(2)-induced PSA expression in prostate cancer cells. (c) 2007 Wiley-Liss, Inc.

  20. Prostaglandin E2 is critical for the development of niacin-deficiency-induced photosensitivity via ROS production

    Science.gov (United States)

    Sugita, Kazunari; Ikenouchi-Sugita, Atsuko; Nakayama, Yasuko; Yoshioka, Haruna; Nomura, Takashi; Sakabe, Jun-Ichi; Nakahigashi, Kyoko; Kuroda, Etsushi; Uematsu, Satoshi; Nakamura, Jun; Akira, Shizuo; Nakamura, Motonobu; Narumiya, Shuh; Miyachi, Yoshiki; Tokura, Yoshiki; Kabashima, Kenji

    2013-10-01

    Pellagra is a photosensitivity syndrome characterized by three ``D's'': diarrhea, dermatitis, and dementia as a result of niacin deficiency. However, the molecular mechanisms of photosensitivity dermatitis, the hallmark abnormality of this syndrome, remain unclear. We prepared niacin deficient mice in order to develop a murine model of pellagra. Niacin deficiency induced photosensitivity and severe diarrhea with weight loss. In addition, niacin deficient mice exhibited elevated expressions of COX-2 and PGE syntheses (Ptges) mRNA. Consistently, photosensitivity was alleviated by a COX inhibitor, deficiency of Ptges, or blockade of EP4 receptor signaling. Moreover, enhanced PGE2 production in niacin deficiency was mediated via ROS production in keratinocytes. In line with the above murine findings, human skin lesions of pellagra patients confirmed the enhanced expression of Ptges. Niacin deficiency-induced photosensitivity was mediated through EP4 signaling in response to increased PGE2 production via induction of ROS formation.

  1. The E(2) Particle

    CERN Document Server

    Ghosh, Subir

    2009-01-01

    Recently it has been advocated [1] that for describing nature within the minimal symmetry requirement, certain subgroups of Lorentz group (along with translation invariance) may play a fundamental role, (instead of the full Poincare group). One such group is E(2) which induces a Lie algebraic Non-Commutative spacetime [4]. In this new kind of Non-Commutative phase space, (which however is not translation invariant in all directions), we construct a point particle action. Interestingly, contrary to some of the other Lie algebraic Non-Commutative spacetimes, no change in the Einstein dispersion relation $p^2=m^2$ is needed. The model is constructed by exploiting a dual canonical phase space following the scheme developed by us earlier [8].

  2. Role of EP2 and EP4 receptors in airway microvascular leak induced by prostaglandin E2.

    Science.gov (United States)

    Jones, Victoria C; Birrell, Mark A; Maher, Sarah A; Griffiths, Mark; Grace, Megan; O'Donnell, Valerie B; Clark, Stephen R; Belvisi, Maria G

    2016-03-01

    Airway microvascular leak (MVL) involves the extravasation of proteins from post-capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGE2, a product of COX-mediated metabolism of arachidonic acid, binds to four receptors, termed EP1–4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Evans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor-deficient mice to define the receptor subtype involved. PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2-induced MVL was demonstrated in Ptger2−/− and Ptger4−/− mice and in wild-type mice pretreated simultaneously with EP2 (PF-04418948) and EP4 (ER-819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2−/− and Ptger4−/− mice. PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2.

  3. Tissue differences in vascular permeability induced by leukotriene B4 and prostaglandin E2 in the rat

    Energy Technology Data Exchange (ETDEWEB)

    Stenson, W.F.; Chang, K.; Williamson, J.R.

    1986-07-01

    The activity of synthetic LTB4 and PGE2, in increasing vascular permeability was tested simultaneously in seventeen different organs in the rat. Rats were injected in the aortic arch through a cannula in the carotid artery with /sup 125/I-albumin, /sup 51/Cr-erythrocytes, and /sup 57/Co-EDTA. The rats were then injected through the carotid artery cannula with LTB4, PGE2 or a combination of LTB4 and PGE2. Eight minutes later the rats were killed and the activity of /sup 125/I, /sup 51/Cr, and /sup 57/Co measured in different organs. Changes in vascular permeability were inferred from changes in the ratios of the isotope activities. LTB4 (15 micrograms/kg) induced enhanced permeability in caecum, small bowel, skin, fat pad, stomach, pancreas, and aorta, but not in the heart, brain, colon, testes, diaphragm, forelimb, cremaster muscle, lung, kidney or eye. A lower dose of LTB4, 3 micrograms/kg, enhanced vascular permeability in caecum, small bowel, skin, stomach, and aorta. PGE2 (1 microgram/kg) enhanced vascular permeability only in the caecum. A combination of LTB4 (3 micrograms/kg) and PGE2 (1 microgram/kg) was more potent than either alone. Rats depleted of neutrophils with anti-neutrophil serum were less sensitive to LTB4 than intact rats. These findings suggest that the vasculatures of different tissues in the rat vary markedly in their susceptibility to LTB4 induced increases in permeability.

  4. Rabbits immunised with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L2E7E2 genes induces regression of established papillomas.

    Science.gov (United States)

    Govan, V A; Williamson, A-L

    2007-07-01

    We previously demonstrated in a cottontail rabbit papillomavirus (CRPV) challenge model that recombinant Bacille Calmette-Guerin (rBCG) could potentially be used as a prophylactic vaccine vehicle to deliver papillomavirus proteins. In this study we investigated whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with rBCG expressing CRPVL2, CRPVE2, CRPVE7 or CRPVL2E7E2 proteins. Rabbits immunised with rBCG/CRPVL2E7E2 had papillomas that were largely suppressed and were significantly smaller compared to the rBCG negative control group (Prabbits immunised with rBCG/CRPVL2E7E2 had papillomas that completely regressed 1.5 weeks post third immunisation. Rabbits immunised with rBCG/CRPVL2, rBCG/CRPVE7, or rBCG/CRPVE2 had papillomas that were significantly smaller than the negative control rabbits (P

  5. Plasmid required for virulence of Agrobacterium tumefaciens

    Energy Technology Data Exchange (ETDEWEB)

    Watson, B.; Currier, T.C.; Gordon, M.P.; Chilton, M.D.; Nester, E.W.

    1975-07-01

    The irreversible loss of crown gall-inducing ability of Agrobacterium tumefaciens strain C-58 during growth at 37/sup 0/C is shown to be due to loss of a large plasmid (1.2 x 10/sup 8/ daltons). The gene responsible for this high rate of plasmid loss at elevated temperatures seems to be located on the plasmid. In addition, another spontaneous avirulent variant, A. tumefaciens strain IIBNV6, is shown to lack the virulence plasmid which its virulent sibling strain, IIBV7, possesses. Deoxyribonucleic acid reassociation measurements prove that the plasmid is eliminated, not integrated into the chromosome, in both of the avirulent derivatives. Transfer of virulence from donor strain C-58 to avirulent recipient strain A136 results from the transfer of a plasmid, which appears identical to the donor plasmid by deoxyribonucleic acid reassociation measurements. The transfer of virulence in another cross, K27 x A136, was also shown to result from the transfer of a large plasmid. These findings establish unequivocally that the large plasmid determines virulence. Two additional genetic determinants have been located on the virulence plasmid of A. tumefaciens strain C-58: the ability to utilize nopaline and sensitivity to a bacteriocin produced by strain 84. The latter trait can be exploited for selection of avirulent plasmid-free derivatives of strain C-58. The trait of nopaline utilization appears to be on the virulence plasmid also in strains IIBV7 and K27.

  6. The syndecan-4/protein kinase Cα pathway mediates prostaglandin E2-induced extracellular regulated kinase (ERK) activation in endothelial cells and angiogenesis in vivo.

    Science.gov (United States)

    Corti, Federico; Finetti, Federica; Ziche, Marina; Simons, Michael

    2013-05-03

    Prostaglandin E2 (PGE2) is regarded as the main mediator of inflammatory symptoms. In addition, it also plays an important role in tumor growth and angiogenesis. In this study, we examined the mechanism of PGE2-induced angiogenic response. We show that in the absence of proteoglycan syndecan-4 (Sdc4), PGE2-induced ERK activation is decreased significantly, as is endothelial cell migration and cord formation in a two-dimensional Matrigel assay. In vivo, PGE2-induced angiogenesis is reduced dramatically in Sdc4(-/-) mice. The mechanism was traced to Sdc4-dependent activation of protein kinase Cα (PKCα). Transduction of an Sdc4 S183E mutant (a cytoplasmic domain mutation that blocks Sdc4-dependent PKCα activation) into Sdc4(-/-) endothelial cells was not able to rescue the loss of PGE2-induced ERK activation, whereas a transduction with full-length Sdc4 resulted in full rescue. Furthermore, PGE2-induced angiogenesis was also reduced in PKCα(-/-) mice. Taken together, these results demonstrate that PGE2-induced activation of angiogenesis is mediated via syndecan-4-dependent activation of PKCα.

  7. Inducible colistin resistance via a disrupted plasmid-borne mcr-1 gene in a 2008 Vietnamese Shigella sonnei isolate

    Science.gov (United States)

    Pham Thanh, Duy; Thanh Tuyen, Ha; Nguyen Thi Nguyen, To; Chung The, Hao; Wick, Ryan R.; Thwaites, Guy E.; Baker, Stephen; Holt, Kathryn E.

    2016-01-01

    Objectives The objective of this study was to assess the presence of mcr-1 in Shigella sonnei isolated in Vietnam. Methods WGS data were analysed for the presence of the mcr-1 gene sequence. The association of mcr-1 with a plasmid was assessed by PCR and by conjugation. Results Through genome sequencing we identified a plasmid-associated inactive form of mcr-1 in a 2008 Vietnamese isolate of Shigella sonnei. The plasmid was conjugated into Escherichia coli and mcr-1 was activated upon exposure to colistin, resulting in highly colistin-resistant transconjugants. Conclusions This is the first description of the mcr-1 gene in Shigella, which is atypical given that colistin is not ordinarily used to treat diarrhoea. Our data suggest the mcr-1 gene has been circulating in human-restricted pathogens for some time but likely carries a selective fitness cost. PMID:27246235

  8. Effect of intense, ultrashort laser pulses on DNA plasmids in their native state: strand breakages induced by {\\it in-situ} electrons and radicals

    CERN Document Server

    D'Souza, J S; Dharmadhikari, A K; Rao, B J; Mathur, D

    2011-01-01

    Single strand breaks are induced in DNA plasmids, pBR322 and pUC19, in aqueous media exposed to strong fields generated using ultrashort laser pulses (820 nm wavelength, 45 fs pulse duration, 1 kHz repetition rate) at intensities of 1-12 TW cm$^{-2}$. The strong fields generate, {\\it in situ}, electrons and radicals that induce transformation of supercoiled DNA into relaxed DNA, the extent of which is quantified. Introduction of electron and radical scavengers inhibits DNA damage; results indicate that OH radicals are the primary (but not sole) cause of DNA damage.

  9. Coimmunization with IL-15 plasmid enhances the longevity of CD8 T cells induced by DNA encoding hepatitis B virus core antigen

    Institute of Scientific and Technical Information of China (English)

    Wei Zhang; Sheng-Fu Dong; Shu-Hui Sun; Yuan Wang; Guang-Di Li; Di Qu

    2006-01-01

    AIM: To test the feasibility of delivering a plasmid encoding IL-15 as a DNA vaccine adjuvant for improving the immune responses induced by hepatitis B virus core gene DNA vaccine.METHODS: We used RT-PCR based strategies to develop IL-15 expression constructs. We first confirmed that the gene could be expressed in Escherichia coli due to the poor expression of IL-15. Then the bioactivity of IL-15 plasmid expression product was identified by CTLL-2 proliferation assay. One hundred micrograms of DNA from each of the IL-15 eukaryotic expressed plasmid and the recombinant plasmid harboring DNA encoding the 144 amino acids of the N-terminus of HBV core gene (abbreviated pHBc144) was used to co-immunize C57 BL/6 mice. The titer of anti-HBcIgG was detected by ELISA and the antigen-specific CD8+T cells (CD8+IFN-γ+ T cells) were detected by intracellular cytokine staining at different time points.RESULTS: After co-immunization by pIL-15 and pHBc144 DNA vaccine the antigen-specific CD8+ cells of mice increased gradually, the first peak of immune response appeared 14 d later, then the number of antigen-specific CD8+ Ts cells decreased gradually and maintained at a steady level in 3 mo. After boosting, the number of antigen-specific CD8+ T cells reached the second peak 10 d later with a double of the 1st peak, then the number of antigen-specific CD8+T cells decreased slowly. IL-15 as a gene adjuvant had no significant effect on humoral immune responses induced by hepatitis B virus core gene DNA vaccine, but increased the memory antigen-specific CD8+ T cells induced by hepatitis B virus core gene DNA vaccine.CONCLUSION: DNA vaccine constructed by HBc Ag 1-144 amino acid induces effective cell immunity, and cytokine plasmid-delivered IL-15 enhances the longevity of CD8+ T cells.

  10. Transfer of bcl-xs plasmid is effective in preventing and inhibiting rat hepatocellular carcinoma induced by N-nitrosomorpholine.

    Science.gov (United States)

    Baba, M; Iishi, H; Tatsuta, M

    2001-08-01

    To examine the effect of the bcl-xs gene on the sequence from hepatic precancerous lesions, foci and neoplastic nodules, to hepatocellular carcinomas, Sprague-Dawley rats were given water containing 175 mg/l N-nitrosomorpholine (NNM) for 8 weeks. At weeks 1, 4 and 7, the left lobe of the rat liver was exposed and injected with the bcl-xs plasmid (pCR3.1-rat bcl-xs cDNA) or pCR3.1 encapsulated in cationic empty liposomes each at a dose of 80 microg plasmid/kg body weight. One minute later, low-field-strength, long-duration electric pulses were applied to the left lobe using a pincette electrode with circular poles 1 cm in diameter. The in vivo electroporation procedure significantly increased the transfer of the reporter gene chloramphenicol acetyl transferase (CAT) plasmid via empty liposomes. Thus, CAT mRNA was expressed not only at the sites of electrode contact but at sites 0.5-1.0 cm away from the electrode, and expression also increased with increasing doses of plasmid, meaning that in vivo electroporation enabled the expression of plasmid DNA throughout an extensive area of the rat liver. By week 11, the neoplastic nodules were significantly fewer and smaller in the bcl-xs group than in the pCR3.1 group at the two sites, one with and the other without electrode contact. No hepatocellular carcinomas were found in the rats that had received the bcl-xs plasmid, whereas these tumors were observed in 30% of the rats given pCR3.1. Moreover, overexpression of the bcl-xs protein was detected, and apoptotic activity was significantly increased in the neoplastic nodules, foci and hepatocytes adjacent to the hepatic lesions. These results indicate that the bcl-xs plasmid inhibits the occurrence and growth of rat hepatocellular carcinoma and may thus be effective for the prevention and treatment of human liver tumors.

  11. [Induced sputum supernatant prostaglandin E2 during oral aspirin challenge of asthmatic patients with and without aspirin hypersensitivity and healthy controls--pilot study].

    Science.gov (United States)

    Ignacak, Maria; Celejewska-Wójcik, Natalia; Wójcik, Krzysztof; Sałapa, Kinga; Konduracka, Ewa; Sanak, Marek; Tyrak, Katarzyna; Sładek, Krzysztof; Musiał, Jacek; Mastalerz, Lucyna

    2016-01-01

    The aim of this pilot study was to evaluate changes in the concentration of prostaglandin E2 (PGE2) in induced sputum supernatant in 3 groups: sub- jects with NSAID-exacerbated respira- tory disease (NERD), aspirin tolerant asthma (ATA) and healthy controls (HC), before and after oral aspirin chal- lenge test. The study was conducted in the years 2014-2015 at the Clinical Department of the Pulmonology Clinic at the University Hospital in Cracow. 43 patients were enrolled in the study (NERD - n = 15, ATA - n = 15 and HC - n = 13). All of them underwent a placebo-controlled oral aspirin challenge. Sputum was induced 24 hours before the challenge and immediately after the test. Induced sputum was processed in order to obtain cystospin slides to depict inflammatory cell patterns and supernatants, in which PGE2 was measured. The concentration of PGE2 was determined using mass spectrometry coupled with gas chromatography (gas chromatography/mass spectrometry - GC/MS). After aspirin challenge, the concentration of PGE2 in induced sputum supernatant decreased in both asthmatics hypersensitive to aspirin (p = 0.01) and those who tolerated aspirin well (p = 0.17). The change in the healthy control group was not statistically significant. These results support the cyclooxygenase theory of PGE2 inhibition by aspirin. However, the mechanism of bronchoconstriction after aspirin administration alone in patients with NSAID-exacerbated respiratory disease remains unclear.

  12. Attenuation of β-Amyloid-Induced Oxidative Cell Death by Sulforaphane via Activation of NF-E2-Related Factor 2

    Directory of Open Access Journals (Sweden)

    Chan Lee

    2013-01-01

    Full Text Available β-amyloid peptide (Aβ, a major component of senile plaques, plays important roles in neuropathology of Alzheimer's disease (AD. An array of in vitro and in vivo data indicates that Aβ-induced neuronal death is mediated by oxidative stress. In this study, we aimed to investigate effects of sulforaphane (SUL, an isothiocyanate in cruciferous vegetables, on Aβ-induced oxidative cell death in SH-SY5Y cells. Cells treated with Aβ25–35 exhibited decreased cell viability and underwent apoptosis as determined by MTT assay and TUNEL, respectively. Aβ25–35-induced cytotoxicity and apoptotic characteristics such as activation of c-JNK, dissipation of mitochondrial membrane potential, altered expression of Bcl-2 family proteins, and DNA fragmentation were effectively attenuated by SUL pretreatment. The antiapoptotic activity of SUL seemed to be mediated by inhibition of intracellular accumulation of reactive oxygen species and oxidative damages. SUL exerted antioxidant potential by upregulating expression of antioxidant enzymes including γ-glutamylcysteine ligase, NAD(PH:quinone oxidoreductase-1, and heme oxygenase-1 via activation of NF-E2-related factor 2(Nrf2. The protective effect of SUL against Aβ25–35-induced apoptotic cell death was abolished by siRNA of Nrf2. Taken together, the results suggest that pharmacologic activation of Nrf2 signaling pathway by SUL might be a practical prevention and/or protective treatment for the management of AD.

  13. DNA-damage response control of E2F7 and E2F8

    OpenAIRE

    Panagiotis Zalmas, L.; Zhao, Xiujie; Graham, Anne L; FISHER Rebecca; Reilly, Carmel; Coutts, Amanda S; La Thangue, Nicholas B

    2008-01-01

    Here, we report that the two recently identified E2F subunits, E2F7 and E2F8, are induced in cells treated with DNA-damaging agents where they have an important role in dictating the outcome of the DNA-damage response. The DNA-damage-dependent induction coincides with the binding of E2F7 and E2F8 to the promoters of certain E2F-responsive genes, most notably that of the E2F1 gene, in which E2F7 and E2F8 coexist in a DNA-binding complex. As a consequence, E2F7 and E2F8 repress E2F target genes...

  14. Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

    DEFF Research Database (Denmark)

    Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla

    2005-01-01

    . Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show that E2F transcription factors bind the E2F8 promoter in vivo and that expression of E2F8 is being induced...... at the G1/S transition. Purified recombinant E2F8 binds specifically to a consensus E2F-DNA-binding site indicating that E2F8, like E2F7, binds DNA without the requirement of co-factors such as DP1. E2F8 inhibits E2F-driven promoters suggesting that E2F8 is transcriptional repressor like E2F7. Ectopic...

  15. Single -and double-strand breaks induced in plasmid DNA irradiated by ultra -soft X-ray; Cassures simple- et double-brin induites par X ultra-mous dans l`ADN plasmidique

    Energy Technology Data Exchange (ETDEWEB)

    Fayard, B.; Touati, A.; Abel, F.; Champion, C.; Chetoui, A. [Paris-6 Univ., 75 (France)]|[Paris-7 Univ., 75 (France); Sage, E. [Institut Curie, UMR 218 CNRS, 75 - Paris (France). Section de Recherche

    1999-01-01

    In order to investigate the molecular consequences of a carbon K photo-ionization located on DNA, dry pBS plasmid samples were irradiated with ultra-soft X-rays at energies below and above the carbon K-threshold (E{sub k}=278 eV). Single- and double-strand breaks (ssb and dsb) were quantified after resolution of the three plasmid forms (supercoiled, relaxed circular, linear) by gel electrophoresis. A factor of 1.2 was found between the doses required at 250 eV and 380 eV to induce the same number of dsb per plasmid. (authors) 6 refs.

  16. Elucidating the role of highly homologous Nicotiana benthamiana ubiquitin E2 gene family members in plant immunity through an improved virus-induced gene silencing approach.

    Science.gov (United States)

    Zhou, Bangjun; Zeng, Lirong

    2017-01-01

    Virus-induced gene silencing (VIGS) has been used in many plant species as an attractive post transcriptional gene silencing (PTGS) method for studying gene function either individually or at large-scale in a high-throughput manner. However, the specificity and efficiency for knocking down members of a highly homologous gene family have remained to date a significant challenge in VIGS due to silencing of off-targets. Here we present an improved method for the selection and evaluation of gene fragments used for VIGS to specifically and efficiently knock down members of a highly homologous gene family. Using this method, we knocked down twelve and four members, respectively of group III of the gene family encoding ubiquitin-conjugating enzymes (E2) in Nicotiana benthamiana. Assays using these VIGS-treated plants revealed that the group III E2s are essential for plant development, plant immunity-associated reactive oxygen species (ROS) production, expression of the gene NbRbohB that is required for ROS production, and suppression of immunity-associated programmed cell death (PCD) by AvrPtoB, an effector protein of the bacterial pathogen Pseudomons syringae. Moreover, functional redundancy for plant development and ROS production was found to exist among members of group III E2s. We have found that employment of a gene fragment as short as approximately 70 base pairs (bp) that contains at least three mismatched nucleotides to other genes within any 21-bp sequences prevents silencing of off-target(s) in VIGS. This improved approach in the selection and evaluation of gene fragments allows for specific and efficient knocking down of highly homologous members of a gene family. Using this approach, we implicated N. benthamiana group III E2s in plant development, immunity-associated ROS production, and suppression of multiple immunity-associated PCD by AvrPtoB. We also unraveled functional redundancy among group III members in their requirement for plant development and

  17. Luteinizing Hormone-Induced Expression of Ptger4b, a Prostaglandin E2 Receptor Indispensable for Ovulation of the Medaka Oryzias latipes, Is Regulated by a Genomic Mechanism Involving Nuclear Progestin Receptor1

    National Research Council Canada - National Science Library

    Akane Hagiwara; Katsueki Ogiwara; Yoshinao Katsu; Takayuki Takahashi

    2014-01-01

    ABSTRACT We previously reported that the prostaglandin E2 receptor subtype Ptger4b plays a role in ovulation in a teleost species, medaka and that ptger4b mRNA is drastically induced in preovulatory...

  18. Inhibition of the prostaglandin E2 receptor EP2 prevents status epilepticus-induced deficits in the novel object recognition task in rats.

    Science.gov (United States)

    Rojas, Asheebo; Ganesh, Thota; Manji, Zahra; O'neill, Theon; Dingledine, Raymond

    2016-11-01

    Survivors of exposure to an organophosphorus nerve agent may develop a number of complications including long-term cognitive deficits (Miyaki et al., 2005; Nishiwaki et al., 2001). We recently demonstrated that inhibition of the prostaglandin E2 receptor, EP2, attenuates neuroinflammation and neurodegeneration caused by status epilepticus (SE) induced by the soman analog, diisopropylfluorophosphate (DFP), which manifest within hours to days of the initial insult. Here, we tested the hypothesis that DFP exposure leads to a loss of cognitive function in rats that is blocked by early, transient EP2 inhibition. Adult male Sprague-Dawley rats were administered vehicle or the competitive EP2 antagonist, TG6-10-1, (ip) at various times relative to DFP-induced SE. DFP administration resulted in prolonged seizure activity as demonstrated by cortical electroencephalography (EEG). A single intraperitoneal injection of TG6-10-1 or vehicle 1 h prior to DFP did not alter the development of seizures, the latency to SE or the duration of SE. Rats administered six injections of TG6-10-1 starting 90 min after the onset of DFP-induced SE could discriminate between a novel and familiar object 6-12 weeks after SE, unlike vehicle treated rats which showed no preference for the novel object. By contrast, behavioral changes in the light-dark box and open field assays were not affected by TG6-10-1. Delayed mortality after DFP was also unaffected by TG6-10-1. Thus, selective inhibition of the EP2 receptor may prevent SE-induced memory impairment in rats caused by exposure to a high dose of DFP.

  19. Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

    Science.gov (United States)

    Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

    2014-01-01

    Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

  20. Advanced glycation end-products induce apoptosis in pancreatic islet endothelial cells via NF-κB-activated cyclooxygenase-2/prostaglandin E2 up-regulation.

    Directory of Open Access Journals (Sweden)

    Kuo-Cheng Lan

    Full Text Available Microvascular complications eventually affect nearly all patients with diabetes. Advanced glycation end-products (AGEs resulting from hyperglycemia are a complex and heterogeneous group of compounds that accumulate in the plasma and tissues in diabetic patients. They are responsible for both endothelial dysfunction and diabetic vasculopathy. The aim of this study was to investigate the cytotoxicity of AGEs on pancreatic islet microvascular endothelial cells. The mechanism underlying the apoptotic effect of AGEs in pancreatic islet endothelial cell line MS1 was explored. The results showed that AGEs significantly decreased MS1 cell viability and induced MS1 cell apoptosis in a dose-dependent manner. AGEs dose-dependently increased the expressions of cleaved caspase-3, and cleaved poly (ADP-ribose polymerase in MS1 cells. Treatment of MS1 cells with AGEs also resulted in increased nuclear factor (NF-κB-p65 phosphorylation and cyclooxygenase (COX-2 expression. However, AGEs did not affect the expressions of endoplasmic reticulum (ER stress-related molecules in MS1 cells. Pretreatment with NS398 (a COX-2 inhibitor to inhibit prostaglandin E2 (PGE2 production reversed the induction of cleaved caspase-3, cleaved PARP, and MS1 cell viability. Moreover, AGEs significantly increased the receptor for AGEs (RAGE protein expression in MS1 cells, which could be reversed by RAGE neutralizing antibody. RAGE Neutralizing antibody could also reverse the induction of cleaved caspase-3 and cleaved PARP and decreased cell viability induced by AGEs. These results implicate the involvement of NF-κB-activated COX-2/PGE2 up-regulation in AGEs/RAGE-induced islet endothelial cell apoptosis and cytotoxicity. These findings may provide insight into the pathological processes within the pancreatic islet microvasculature induced by AGEs accumulation.

  1. Bauer Ketones 23 and 24 from Echinacea paradoxa var. paradoxa Inhibit Lipopolysaccharide-induced Nitric Oxide, Prostaglandin E2 and Cytokines in RAW 264.7 Mouse Macrophages

    Science.gov (United States)

    Zhang, Xiaozhu; Rizshsky, Ludmila; Hauck, Catherine; Qu, Luping; Widrlechner, Mark P.; Nikolau, Basil J.; Murphy, Patricia A.; Birt, Diane F.

    2011-01-01

    Among the nine Echinacea species, E. purpurea, E. angustifolia and E. pallida, have been widely used to treat the common cold, flu and other infections. In our study, ethanol extracts of these three Echinacea species and E. paradoxa, including its typical variety, E. paradoxa var. paradoxa, were screened in lipopolysaccharide (LPS)-stimulated macrophage cells to assess potential anti-inflammatory activity. Echinacea paradoxa var. paradoxa, rich in polyenes/polyacetylenes, was an especially efficient inhibitor of LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), interleukin-1 beta (IL-1β) and interleukin-6 (IL-6) by 46%, 32%, 53% and 26%, respectively, when tested at 20 μg/ml in comparison to DMSO control. By bioactivity-guided fractionation, pentadeca-8Z-ene-11, 13-diyn-2-one (Bauer ketones 23, compound 1) and pentadeca-8Z, 13Z-dien-11-yn-2-one (Bauer ketone 24, compound 2) from E. paradoxa var. paradoxa were found primarily responsible for inhibitory effects on NO and PGE2 production. Moreover, Bauer ketone 24 (compound 2) was the major contributor to inhibition of inflammatory cytokine production in LPS-induced mouse macrophage cells. These results provide a rationale for exploring the medicinal effects of the Bauer ketone-rich taxon, E. paradoxa var. paradoxa, and confirm the anti-inflammatory properties of Bauer ketones 23 and 24. PMID:22133644

  2. Induced pluripotent stem cells generated from human adipose-derived stem cells using a non-viral polycistronic plasmid in feeder-free conditions.

    Directory of Open Access Journals (Sweden)

    Xinjian Qu

    Full Text Available Induced pluripotent stem cells (iPSCs can be generated from somatic cells by ectopic expression of defined transcription factors (TFs. However, the optimal cell type and the easy reprogramming approaches that minimize genetic aberrations of parent cells must be considered before generating the iPSCs. This paper reports a method to generate iPSCs from adult human adipose-derived stem cells (hADSCs without the use of a feeder layer, by ectopic expression of the defined transcription factors OCT4, SOX2, KLF4 and C-MYC using a polycistronic plasmid. The results, based on the expression of pluripotent marker, demonstrated that the iPSCs have the characteristics similar to those of embryonic stem cells (ESCs. The iPSCs differentiated into three embryonic germ layers both in vitro by embryoid body generation and in vivo by teratoma formation after being injected into immunodeficient mice. More importantly, the plasmid DNA does not integrate into the genome of human iPSCs as revealed by Southern blotting experiments. Karyotypic analysis also demonstrated that the reprogramming of hADSCs by the defined factors did not induce chromosomal abnormalities. Therefore, this technology provides a platform for studying the biology of iPSCs without viral vectors, and can hopefully overcome immune rejection and ethical concerns, which are the two important barriers of ESC applications.

  3. Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice.

    Science.gov (United States)

    Gühring, H; Görig, M; Ates, M; Coste, O; Zeilhofer, H U; Pahl, A; Rehse, K; Brune, K

    2000-09-01

    It is widely accepted that peripheral injury increases spinal inducible cyclooxygenase (COX-2) expression and prostaglandin E(2) (PGE(2)) formation as key mediators of nociceptive sensitization. Here, we used inducible nitric oxide synthase (iNOS) gene-deficient (iNOS-/-) mice to determine the contribution of iNOS-derived nitric oxide (NO) to this process. iNOS-/- mice exhibited reduced thermal hyperalgesia after zymosan injection. Spinal NO and PGE(2) formation both remained at baseline levels, in contrast to wild-type (wt) mice. In wt mice reduced hyperalgesia similar to that seen in iNOS-/- mice was induced by local spinal, but not by systemic treatment with the iNOS inhibitor l-NIL, suggesting that the reduced heat sensitization in iNOS-/- mice was attributable to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that the antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOS-/- mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOS-/- mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia.

  4. Acanthopanax koreanum Fruit Waste Inhibits Lipopolysaccharide-Induced Production of Nitric Oxide and Prostaglandin E2 in RAW 264.7 Macrophages

    Directory of Open Access Journals (Sweden)

    Eun-Jin Yang

    2010-01-01

    Full Text Available The Acanthopanax koreanum fruit is a popular fruit in Jeju Island, but the byproducts of the alcoholic beverage prepared using this fruit are major agricultural wastes. The fermentability of this waste causes many economic and environmental problems. Therefore, we investigated the suitability of using A. koreanum fruit waste (AFW as a source of antiinflammatory agents. AFWs were extracted with 80% EtOH. The ethanolic extract was then successively partitioned with hexane, CH2Cl2, EtOAc, BuOH, and water. The results indicate that the CH2Cl2 fraction (100 g/mL of AFW inhibited the LPS-induced nitric oxide (NO and prostaglandin E2 (PGE2 production in RAW 264.7 cells by 79.6% and 39.7%, respectively. These inhibitory effects of the CH2Cl2 fraction of AFWs were accompanied by decreases in the expression of inducible nitric oxide synthase (iNOS and cyclooxygenase-2 (COX-2 proteins and iNOS and COX-2 mRNA in a dose-dependent pattern. The CH2Cl2 fraction of AFWs also prevented degradation of IB- in a dose-dependent manner. Ursolic acid was identified as major compound present in AFW, and CH2Cl2 extracts by high performance liquid chromatography (HPLC. Furthermore using pure ursolic acid as standard and by HPLC, AFW and CH2Cl2 extracts was found to contain 1.58 mg/g and 1.75 mg/g, respectively. Moreover, we tested the potential application of AFW extracts as a cosmetic material by performing human skin primary irritation tests. In these tests, AFW extracts did not induce any adverse reactions. Based on these results, we suggest that AFW extracts be considered possible anti-inflammatory candidates for topical application.

  5. Counteracting effect of TRPC1-associated Ca2+ influx on TNF-α-induced COX-2-dependent prostaglandin E2 production in human colonic myofibroblasts.

    Science.gov (United States)

    Hai, Lin; Kawarabayashi, Yasuhiro; Imai, Yuko; Honda, Akira; Inoue, Ryuji

    2011-08-01

    TNF-α-NF-κB signaling plays a central role in inflammation, apoptosis, and neoplasia. One major consequence of this signaling in the gut is increased production of prostaglandin E(2) (PGE(2)) via cyclooxygenase-2 (COX-2) induction in myofibroblasts, which has been reported to be dependent on Ca(2+). In this study, we explored a potential role of canonical transient receptor potential (TRPC) proteins in this Ca(2+)-mediated signaling using a human colonic myofibroblast cell line CCD-18Co. In CCD-18Co cell, treatment with TNF-α greatly enhanced Ca(2+) influx induced by store depletion along with increased cell-surface expression of TRPC1 protein (but not of the other TRPC isoforms) and induction of a Gd(3+)-sensitive nonselective cationic conductance. Selective inhibition of TRPC1 expression by small interfering RNA (siRNA) or functionally effective TRPC1 antibody targeting the near-pore region of TRPC1 (T1E3) antagonized the enhancement of store-dependent Ca(2+) influx by TNF-α, whereas potentiated TNF-α induced PGE(2) production. Overexpression of TRPC1 in CCD-18Co produced opposite consequences. Inhibitors of NF-κB (curcumin, SN-50) attenuated TNF-α-induced enhancement of TRPC1 expression, store-dependent Ca(2+) influx, and COX-2-dependent PGE(2) production. In contrast, inhibition of calcineurin-nuclear factor of activated T-cell proteins (NFAT) signaling by FK506 or NFAT Activation Inhibitor III enhanced the PGE(2) production without affecting TRPC1 expression and the Ca(2+) influx. Finally, the suppression of store-dependent Ca(2+) influx by T1E3 antibody or siRNA knockdown significantly facilitated TNF-α-induced NF-κB nuclear translocation. In aggregate, these results strongly suggest that, in colonic myofibroblasts, NF-κB and NFAT serve as important positive and negative transcriptional regulators of TNF-α-induced COX-2-dependent PGE(2) production, respectively, at the downstream of TRPC1-associated Ca(2+) influx.

  6. Deficiency in the nuclear factor E2-related factor 2 renders pancreatic β-cells vulnerable to arsenic-induced cell damage

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Bei [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Department of Histology and Embryology, College of Basic Medical Sciences, China Medical University, Shenyang 110001 (China); Fu, Jingqi; Zheng, Hongzhi; Xue, Peng; Yarborough, Kathy; Woods, Courtney G.; Hou, Yongyong; Zhang, Qiang; Andersen, Melvin E. [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States); Pi, Jingbo, E-mail: jpi@thehamner.org [Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, 6 Davis Drive, Research Triangle Park, NC 27709 (United States)

    2012-11-01

    Chronic human exposure to inorganic arsenic (iAs), a potent environmental oxidative stressor, is associated with increased prevalence of type 2 diabetes, where impairment of pancreatic β-cell function is a key pathogenic factor. Nuclear factor E2-related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. However, persistent activation of Nrf2 in response to chronic oxidative stress, including inorganic arsenite (iAs{sup 3+}) exposure, blunts glucose-triggered reactive oxygen species (ROS) signaling and impairs glucose-stimulated insulin secretion (GSIS). In the current study, we found that MIN6 pancreatic β-cells with stable knockdown of Nrf2 (Nrf2-KD) by lentiviral shRNA and pancreatic islets isolated from Nrf2-knockout (Nrf2−/−) mice exhibited reduced expression of several antioxidant and detoxification enzymes in response to acute iAs{sup 3+} exposure. As a result, Nrf2-KD MIN6 cells and Nrf2−/− islets were more susceptible to iAs{sup 3+} and monomethylarsonous acid (MMA{sup 3+})-induced cell damage, as measured by decreased cell viability, augmented apoptosis and morphological change. Pretreatment of MIN6 cells with Nrf2 activator tert-butylhydroquinone protected the cells from iAs{sup 3+}-induced cell damage in an Nrf2-dependent fashion. In contrast, antioxidant N‐acetyl cysteine protected Nrf2-KD MIN6 cells against acute cytotoxicity of iAs{sup 3+}. The present study demonstrates that Nrf2-mediated antioxidant response is critical in the pancreatic β-cell defense mechanism against acute cytotoxicity by arsenic. The findings here, combined with our previous results on the inhibitory effect of antioxidants on ROS signaling and GSIS, suggest that Nrf2 plays paradoxical roles in pancreatic β-cell dysfunction induced by environmental arsenic exposure. -- Highlights: ► Lack of Nrf2 reduced expression of antioxidant genes induced by iAs{sup 3+} in β-cells. ► Deficiency of Nrf2 in

  7. Non-DSB clustered DNA lesions induced by ionizing radiation are largely responsible for the loss of plasmid DNA functionality in the presence of cisplatin.

    Science.gov (United States)

    Kouass Sahbani, S; Rezaee, M; Cloutier, P; Sanche, L; Hunting, D J

    2014-06-25

    The combination of cisplatin and ionizing radiation (IR) increases cell toxicity by both enhancing DNA damage and inhibiting repair mechanisms. Although the formation of cluster DNA lesions, particularly double-strand breaks (DSB) at the site of cisplatin-DNA-adducts has been reported to induce cell death, the contribution of DSB and non-DSB cluster lesions to the cellular toxicity is still unknown. Although both lesions are toxic, it is not always possible to measure their frequency and cell survival in the same model system. To overcome this problem, here, we investigate the effect of cisplatin-adducts on the induction of DSB and non-DSB cluster DNA lesions by IR and determine the impact of such lesions on plasmid functionality. Cluster lesions are two or more lesions on opposite DNA strands with a short distance such that error free repair is difficult or impossible. At a ratio of two cisplatin per plasmid, irradiation of platinated DNA in solution with (137)Cs γ-rays shows enhancements in the formation of DNA DSB and non-DSB cluster lesions by factors of 2.6 and 2.1, respectively, compared to unmodified DNA. However, in absolute terms, the yield for non-DSB cluster lesions is far larger than that for DSB, by a factor of 26. Unmodified and cisplatin-modified DNA were irradiated and subsequently transformed into Escherichia coli to give survival curves representing the functionality of the plasmid DNA as a function of radiation dose. Our results demonstrate that non-DSB cluster lesions are the only toxic lesions present at a sufficient frequency to account for the loss of DNA functionality. Our data also show that Frank-DSB lesions are simply too infrequent to account for the loss of DNA functionality. In conclusion, non-DSB cluster DNA damage is known to be difficult to repair and is probably the lesion responsible for the loss of functionality of DNA modified by cisplatin.

  8. Chemotherapy of Bacterial Plasmids

    Science.gov (United States)

    1979-01-29

    render them non-susceptible to K: z plasmid-encoded enzymes. (3) Development of drugs which are selective inhibitor! 1 4, of plasmid DNA replication. (4... Development of drugs which inhibit phenotypic as expression of plasmid genes, and (5) Development of drugs which are inhibitors o, drug-inactivating...Barnes [2] them non-susceptible to plasmid-encoded enzymes, tabulated data on the incidence of Gram-negative 3) development of drugs which are

  9. Activation of prostaglandin E2-receptor EP2 and EP4 pathways induces growth inhibition in human gastric carcinoma cell lines.

    Science.gov (United States)

    Okuyama, T; Ishihara, S; Sato, H; Rumi, M A K; Kawashima, K; Miyaoka, Y; Suetsugu, H; Kazumori, H; Cava, C F Ortega; Kadowaki, Y; Fukuda, R; Kinoshita, Y

    2002-08-01

    The effect of prostaglandin E2 (PGE2) on the proliferation of gastric cancer cells is still unclear. PGE2 receptors are divided into four subtypes - EP1, EP2, EP3, and EP4 - which are coupled to three different intracellular signal-transduction systems. Stimulation of EP2 and EP4 is linked with cyclic adenosine 3', 5'-monophosphate (cAMP)-dependent protein kinase A (PKA). In some human gastric cancer cells, PGE2 has been suggested to have an antiproliferative effect by way of increased cAMP production. Expression of EP2 and EP4 in human gastric carcinoma cells, however, has not been examined. We examined the expression of EP2 and EP4 and the antiproliferative effects of specific EP2 and EP4 agonists on four different human gastric cancer cell lines. Our data clarified that all the cell lines investigated in this study expressed EP2 and EP4 and that the specific agonists of these receptors induced growth inhibition with an accompanying increase in cAMP production. In summary, gastric cancer cells have EP2 and EP4 receptors, and their selective activation is linked with the decreased cell proliferation.

  10. Prostaglandin E2 induces chloride secretion through crosstalk between cAMP and calcium signaling in mouse inner medullary collecting duct cells

    Science.gov (United States)

    Rajagopal, Madhumitha; Thomas, Sheela V.; Kathpalia, Paru P.; Chen, Yu

    2013-01-01

    Under conditions of high dietary salt intake, prostaglandin E2 (PGE2) production is increased in the collecting duct and promotes urinary sodium chloride (NaCl) excretion; however, the molecular mechanisms by which PGE2 increases NaCl excretion in this context have not been clearly defined. We used the mouse inner medullary collecting duct (mIMCD)-K2 cell line to characterize mechanisms underlying PGE2-regulated NaCl transport. When epithelial Na+ channels were inhibited, PGE2 exclusively stimulated basolateral EP4 receptors to increase short-circuit current (IscPGE2). We found that IscPGE2 was sensitive to inhibition by H-89 and CFTR-172, indicating that EP4 receptors signal through protein kinase A to induce Cl− secretion via cystic fibrosis transmembrane conductance regulator (CFTR). Unexpectedly, we also found that IscPGE2 was sensitive to inhibition by BAPTA-AM (Ca2+ chelator), 2-aminoethoxydiphenyl borate (2-APB) (inositol triphosphate receptor blocker), and flufenamic acid (FFA) [Ca2+-activated Cl− channel (CACC) inhibitor], suggesting that EP4 receptors also signal through Ca2+ to induce Cl− secretion via CACC. Additionally, we observed that PGE2 stimulated an increase in Isc through crosstalk between cAMP and Ca2+ signaling; BAPTA-AM or 2-APB inhibited a component of IscPGE2 that was sensitive to CFTR-172 inhibition; H-89 inhibited a component of IscPGE2 that was sensitive to FFA inhibition. Together, our findings indicate that PGE2 activates basolateral EP4 receptors and signals through both cAMP and Ca2+ to stimulate Cl− secretion in IMCD-K2 cells. We propose that these signaling pathways, and the crosstalk between them, may provide a concerted mechanism for enhancing urinary NaCl excretion under conditions of high dietary NaCl intake. PMID:24284792

  11. Adjuvant effect of caffeine on acetylsalicylic acid anti-nociception: prostaglandin E2 synthesis determination in carrageenan-induced peripheral inflammation in rat.

    Science.gov (United States)

    Fernández-Dueñas, Víctor; Sánchez, Sílvia; Planas, Eulàlia; Poveda, Raquel

    2008-02-01

    In the present study, we report a synergistic interaction between acetylsalicylic acid (ASA) and caffeine (CAF) on the inhibition of nociception in a model of peripheral inflammation in rat; on the contrary no interaction have been found on anti-inflammatory effects and peripheral prostaglandin E2 (PGE-2) synthesis inhibition. Acute inflammation was induced by the subplantar injection of carrageenan into the right hind paw, and the effects of the drugs were evaluated from 0 to 5h. Nociception was assessed using the Randall & Selitto test, and the inflammatory response by plethismometry. Oral administration of ASA (10-400mg/kg) induced dose-related anti-nociceptive and anti-inflammatory effects. On the other hand, oral CAF administration (5-50mg/kg) did not show a dose-related inhibitory effect, neither on the inhibition of nociception nor on the inflammatory response. To analyze a possible interaction between both drugs a dose-response curve to ASA plus a fixed dose of CAF (5mg/kg) was obtained 3h after the injection of carrageenan, when the inflammatory pain peaked. A fixed dose of CAF was able to improve the anti-nociceptive, but not the anti-inflammatory, effects of ASA. We also assessed, by enzyme immunoassay, the effects of the combination on peripheral PGE-2 levels: CAF did not alter the inhibitory effect of ASA on PGE-2 synthesis. Our results corroborate the well-known clinical effects of combining ASA and CAF; on the other hand, we rule out that prostaglandin synthesis inhibition at peripheral sites would be the mechanism responsible of the adjuvant anti-nociceptive effect of CAF.

  12. Plasmid segregation mechanisms

    DEFF Research Database (Denmark)

    Ebersbach, Gitte; Gerdes, Kenn; Charbon, Gitte Ebersbach

    2005-01-01

    Bacterial plasmids encode partitioning (par) loci that ensure ordered plasmid segregation prior to cell division. par loci come in two types: those that encode actin-like ATPases and those that encode deviant Walker-type ATPases. ParM, the actin-like ATPase of plasmid R1, forms dynamic filaments ...

  13. Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

    Science.gov (United States)

    Hadjicharalambous, Chrystalleni; Kozlova, Diana; Sokolova, Viktoriya; Epple, Matthias; Chatzinikolaidou, Maria

    2015-12-01

    Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

  14. Deacylation of the transmembrane domains of Sindbis virus envelope glycoproteins E1 and E2 does not affect low-pH-induced viral membrane fusion activity

    NARCIS (Netherlands)

    Smit, JM; Bittman, R; Wilschut, J

    2001-01-01

    The envelope glycoproteins E1 and E2 of Sindbis virus are palmitoylated at cysteine residues within their transmembrane domains (E1 at position 430, and E2 at positions 388 and 390), Here, we investigated the in vitro membrane fusion activity of Sindbis virus variants (derived from the Tote 1101 inf

  15. Immunogenicity and Protective Response Induced by Recombinant Plasmids Based on the BAB1_0267 and BAB1_0270 Open Reading Frames of Brucella abortus 2308 in BALB/c Mice

    Science.gov (United States)

    Gómez, Leonardo A.; Alvarez, Francisco I.; Fernández, Pablo A.; Flores, Manuel R.; Molina, Raúl E.; Coloma, Roberto F.; Oñate, Angel A.

    2016-01-01

    Immunogenicity induced by recombinant plasmids based on the BAB1_0267 and BAB1_0270 open reading frames (ORFs) of Brucella abortus 2308 was evaluated. Bioinformatics analyses indicate that the BAB1_0267 and BAB1_0270 ORFs encode a protein with a SH3 domain and a Zn-dependent metalloproteinase, respectively. Both ORFs have important effects on intracellular survival and replication of B. abortus 2308, mediated via professional and non-professional phagocytic cells. Our results show that immunization with the recombinant plasmid based on the BAB1_0267 ORF significantly increases the production of IgG1, levels of IFN-γ and the lymphoproliferative response of splenocytes. However, BAB1_0267 did not provide significant levels of protection. The plasmid based on the BAB1_0270 significantly increased IgG2a production, levels of IFN-γ and TNF-α, and the lymphoproliferative response of splenocytes. These results demonstrate that immunization with the BAB1_0270 derived recombinant plasmid induce a Th1-type immune response, correlated with a heightened resistance to B. abortus 2308 infection in mice. It is concluded that the Th1-type immune response against bacterial Zn-dependent metalloproteinase induces a protective response in mice, and that pV270 recombinant plasmid is an effective candidate microbicide against brucellosis. PMID:27747197

  16. Correlation between plasmid DNA damage induced by PM10 and trace metals in inhalable particulate matters in Beijing air

    Institute of Scientific and Technical Information of China (English)

    Lü; Senlin

    2006-01-01

    [1]Wei F S,Chapman R S.Study on Effect of Air Pollution on Respiratory Health Effect (in Chinese).Beijing:China Environmental Science Press,2001.1-16[2]Chapman R S,Watkinson W P,Dreher K L,et al.Ambient particulate matter and respiratory and cardiovascular illness in adults:particle-borne transition metals and the heart-lung axis.Environ Toxicol and Pharmacol,1997,4:331-338[3]Donaldson K,Beswick P H,Gilmour P S.Free radical activity associated with the surface of particles:a unifying factor in determining biological activity? Toxicol Lett,1996,293-298[4]Donaldson K,Brown D M,Mitchell C,et al.Free radical activity of PM10:iron-mediated generation of hydroxyl radicals.Environ Health Perspect,1997,105(S5):1285-1289[5]Donaldson K,Li X,MacNee W.Ultrafine (Nanometer) particle mediated lung injury.J Aero Sci,1998,29:553-560[6]Li X,Gilmour P S,Donaldson K,et al.In vivo and in vitro proinflammatory effects of particulate air pollution (PM10).Environ Health Perspect,1997,105(S5):1279-1283[7]Greenwell L L,Moreno T,Jones T P,et al.Particle-induced oxidative damage is ameliorated by pulmonary antioxidants.Free Radical Biol Medi,2002,32,898-905[8]Greenwell L L,Moreno T,Richard R J.Pulmonary antioxidants exert differential protective effects against urban and industrial particulate matter.J Biosci,2003,28(1):101-107[9]Poli G,Parola M.Oxidative damage and fibrogenesis.Free Radical Biol Medi,1996,22:287-305[10]Costa D L,Dreher K L.Bioavailable transition metals in particulate matter mediate cardiopulmonary injury in healthy and compromised animal models.Environ Health Perspect,1997,105(Supp.5):1053-1060[11]Richards R J,Atkins J,Marrs T C,et al.The biochemical and pathological changes produced by the intraacheal instillation of certain components of zinc hexachloroethane smoke.Toxicol Lett,1989,54:79-88[12]Adamson I Y R,Prieditis H,Hedgecock C,et al.Zinc is the toxic factor in the lung response to an atmospheric particulate sample.Toxicol Appl Pharmacol,2000,166:111-119[13]Wang Y Q

  17. Prostaglandin E2-induced intercellular adhesion molecule-1 expression is mediated by cAMP/Epac signalling modules in bEnd.3 brain endothelial cells

    Science.gov (United States)

    Park, Tae Yeop; Baik, Eun Joo; Lee, Soo Hwan

    2013-01-01

    Background and Purpose Prostaglandin E2 (PGE2) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. Experimental Approach bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE2 with or without agonists/antagonists of PGE2 receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. Key Results PGE2 significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE2 effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N6-Bnz-cAMP). PGE2-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11–7082 and MG-132) attenuated the induction of ICAM-1 by PGE2. PGE2, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti. Conclusion and Implications Our findings suggest that PGE2 induces ICAM-1 expression via EP4 receptor and Epac/Akt/NF-κB signalling pathway in bEnd.3 brain endothelial cells, supporting its pathophysiological role in brain inflammation. PMID:23317035

  18. Soluble iron modulates iron oxide particle-induced inflammatory responses via prostaglandin E2 synthesis: In vitro and in vivo studies

    Directory of Open Access Journals (Sweden)

    Mayer Paula

    2009-12-01

    Full Text Available Abstract Background Ambient particulate matter (PM-associated metals have been shown to play an important role in cardiopulmonary health outcomes. To study the modulation of PM-induced inflammation by leached off metals, we investigated intracellular solubility of radio-labeled iron oxide (59Fe2O3 particles of 0.5 and 1.5 μm geometric mean diameter. Fe2O3 particles were examined for the induction of the release of interleukin 6 (IL-6 as pro-inflammatory and prostaglandin E2 (PGE2 as anti-inflammatory markers in cultured alveolar macrophages (AM from Wistar Kyoto (WKY rats. In addition, we exposed male WKY rats to monodispersed Fe2O3 particles by intratracheal instillation (1.3 or 4.0 mg/kg body weight to examine in vivo inflammation. Results Particles of both sizes are insoluble extracellularly in the media but moderately soluble in AM with an intracellular dissolution rate of 0.0037 ± 0.0014 d-1 for 0.5 μm and 0.0016 ± 0.0012 d-1 for 1.5 μm 59Fe2O3 particles. AM exposed in vitro to 1.5 μm particles (10 μg/mL for 24 h increased IL-6 release (1.8-fold; p 2 synthesis (1.9-fold; p 2 synthesis (2.5-fold, p 2 synthesis by indomethacin caused a pro-inflammatory phenotype as noted by increased IL-6 release from AM exposed to 0.5 μm particles (up to 3-fold; p Conclusions Fe2O3 particle-induced neutrophilic inflammatory response in vivo and pro-inflammatory cytokine release in vitro might be modulated by intracellular soluble iron via PGE2 synthesis. The suppressive effect of intracellular released soluble iron on particle-induced inflammation has implications on how ambient PM-associated but soluble metals influence pulmonary toxicity of ambient PM.

  19. Protective Role of Nuclear Factor E2-Related Factor 2 against Acute Oxidative Stress-Induced Pancreatic β-Cell Damage

    Directory of Open Access Journals (Sweden)

    Jingqi Fu

    2015-01-01

    Full Text Available Oxidative stress is implicated in the pathogenesis of pancreatic β-cell dysfunction that occurs in both type 1 and type 2 diabetes. Nuclear factor E2-related factor 2 (NRF2 is a master regulator in the cellular adaptive response to oxidative stress. The present study found that MIN6 β-cells with stable knockdown of Nrf2 (Nrf2-KD and islets isolated from Nrf2-knockout mice expressed substantially reduced levels of antioxidant enzymes in response to a variety of stressors. In scramble MIN6 cells or wild-type islets, acute exposure to oxidative stressors, including hydrogen peroxide (H2O2 and S-nitroso-N-acetylpenicillamine, resulted in cell damage as determined by decrease in cell viability, reduced ATP content, morphology changes of islets, and/or alterations of apoptotic biomarkers in a concentration- and/or time-dependent manner. In contrast, silencing of Nrf2 sensitized MIN6 cells or islets to the damage. In addition, pretreatment of MIN6 β-cells with NRF2 activators, including CDDO-Im, dimethyl fumarate (DMF, and tert-butylhydroquinone (tBHQ, protected the cells from high levels of H2O2-induced cell damage. Given that reactive oxygen species (ROS are involved in regulating glucose-stimulated insulin secretion (GSIS and persistent activation of NRF2 blunts glucose-triggered ROS signaling and GSIS, the present study highlights the distinct roles that NRF2 may play in pancreatic β-cell dysfunction that occurs in different stages of diabetes.

  20. Effect of c-fos antisense probe on prostaglandin E2-induced upregulation of vascular endothelial growth factor mRNA in human liver cancer cells

    Institute of Scientific and Technical Information of China (English)

    Yong-Qi Li; Kai-Shan Tao; Ning Ren; Yi-Hu Wang

    2005-01-01

    AIM: To examine the effect of prostaglandin E2 (PGE2) on the expression of vascular endothelial growth factor (VEGF) mRNA in the human hepatocellular carcinoma (HCC) HepG2 cells and the possible involvement of c-fos protein in this process.METHODS: Human HCC HepG2 cells were divided into three groups treated respectively with PGE2, a combination of PGE2 and c-fos antisense oligodeoxynucleotide (ASO),and PGE2 plus c-fos sense oligodeoxynudeotide (SO). The expression of VEGF mRNA in HepG2 cells after different treatments was detected by reverse transcriptase-polymerase chain reaction (RT-PCR). The relative expression level of VEGF mRNA in HepG2 cells in each group was measured.RESULTS: Administration of PGE2 resulted in an increased expression of c-fosand VEGF mRNA in HepG2 cells. The relative expression level of c-fos mRNA reached the peak at 3 h (68.4±4.7%) after PGE2 treatment, which was significantly higher than that at 0 h (20.6±1.7%, P<0.01).Whereas, the highest expression level of VEGF mRNA was observed at 6 h (100.5±6.1%) after PGE2 treatment, which was significantly higher than that at 0 h (33.2±2.4%,P<0.01). C-fos ASO significantly reduced PGE2-induced VEGF mRNA expression in HepG2 cells.CONCLUSION: PGE2 increases the expression and secretion of VEGF in HCC cells by activating the transcription factor c-fos, promotes the angiogenesis of HCC and plays an important role in the pathogenesis of liver cancer.

  1. Firocoxib on aqueous humor prostaglandin E 2 levels for controlling experimentally-induced breakdown of blood-aqueous barrier in healthy and Toxoplasma gondii -seropositive cats

    Directory of Open Access Journals (Sweden)

    Deise Cristine Schroder

    2016-06-01

    Full Text Available ABSTRACT: This study aimed to evaluate the effects of firocoxib for controlling experimentally-induced breakdown of the blood-aqueous barrier in healthy and Toxoplasma gondii -seropositive cats. Thirty two cats with no ocular abnormalities were used. Groups (n=8/each were formed with healthy cats that received 5mg g-1 of oral firocoxib (FH or no treatment (CH on day 0; seropositive cats for anti -T. gondii specific immunoglobulin G (IgG were grouped (n=8/each and treated in a similar fashion (FT and CT. On day 1, cats of all groups received the same treatment protocol, and 1h later, aqueocentesis was performed under general anesthesia (M0. Following 1h, the same procedure was repeated (M1. Quantitation of aqueous humor total protein and prostaglandin E2 (PGE2 were determined. Aqueous samples of seropositive cats were tested for anti- T. gondii specific IgG. In M0, aqueous samples of CT showed a significantly higher concentration of PGE2 in comparison with other groups (P<0.05. In all groups, PGE2 concentration increased significantly from M0 to M1 (P=0.001. PGE2 values did not change significantly between groups in M1 (P=0.17. Anti- T. gondii specific IgG were reported only in samples of M1, and aqueous titers did not change significantly between FT and CT (P=0.11. Although we have observed that aqueous humor PGE2 levels were significantly higher in cats of CT group during M0, such increase was not able to break the blood-aqueous barrier and cause anterior uveitis. Firocoxib did not prevent intraocular inflammation after aqueocentesis, in healthy and toxoplasmosis-seropositive cats.

  2. Identification of DHA-23, a Novel Plasmid-mediated and Inducible AmpC beta-Lactamase from Enterobacteriaceae in Northern Taiwan

    Directory of Open Access Journals (Sweden)

    Wen-Shyang eHsieh

    2015-05-01

    Full Text Available Objectives: AmpC β-lactamases are classified as Amber Class C and Bush Group 1. AmpC β-lactamases can hydrolyze broad and extended-spectrum cephalosporins, and are not inhibited by β-lactamase inhibitors such as clavulanic acid. This study was conducted to identify DHA-23, a novel plasmid-mediated and inducible AmpC β-lactamase obtained from Enterobacteriaceae. Methods: A total of 210 carbapenem-resistant Enterobacteriaceae isolates were collected from a medical center (comprising 2 branches in Northern Taiwan during 2009–2012. AmpC β-lactamase genes were analyzed through a polymerase chain reaction using plasmid DNA templates and gene sequencing. The genetic relationships of the isolates were typed using pulsed-field gel electrophoresis following the digestion of intact genomic DNA by using XbaI. Results: Three enterobacterial isolates (one Escherichia coli and 2 Klebsiella pneumoniae were obtained from 3 hospitalized patients. All 3 isolates were resistant or intermediately susceptible to all β-lactams, and exhibited reduced susceptibility to carbapenems. These 3 isolates expressed a novel AmpC β-lactamase, designated DHA-23, approved by the curators of the Lahey website. DHA-23 differs from DHA-1 and DHA-6 by one amino acid substitution (Ser245Ala, exhibiting 2 amino acid changes compared with DHA-7 and DHA-Morganella morganii; 3 amino acid changes compared with DHA-3; 4 amino acid changes compared with DHA-5; and 8 amino acid changes compared with DHA-2 (> 97% identity. This AmpC β-lactamase is inducible using a system involving ampR. Conclusion: This is the first report to address DHA-23, a novel AmpC β-lactamase. DHA-type β-lactamases are continuous threat in Taiwan.

  3. Activation of the Prostaglandin E2 receptor EP2 prevents house dust mite-induced airway hyperresponsiveness and inflammation by restraining mast cells' activity.

    Science.gov (United States)

    Serra-Pages, M; Torres, R; Plaza, J; Herrerias, A; Costa-Farré, C; Marco, A; Jiménez, M; Maurer, M; Picado, C; de Mora, F

    2015-10-01

    Prostaglandin E2 (PGE2 ) has been proposed to exert antiasthmatic effects in patients, to prevent antigen-induced airway pathology in murine models, and to inhibit mast cells (MC) activity in vitro. To assess in a murine model whether the protective effect of PGE2 may be a consequence of its ability to activate the E-prostanoid (EP)2 receptor on airway MC. Either BALB/c or C57BL/6 mice were exposed intranasally (i.n.) to house dust mite (HDM) aeroallergens. Both strains were given PGE2 locally (0.3 mg/kg), but only BALB/c mice were administered butaprost (EP2 agonist: 0.3 mg/kg), or AH6809 (EP2 antagonist; 2.5 mg/kg) combined with the MC stabilizer sodium cromoglycate (SCG: 25 mg/kg). Airway hyperresponsiveness (AHR) and inflammation, along with lung MC activity, were evaluated. In addition, butaprost's effect was assessed in MC-mediated passive cutaneous anaphylaxis (PCA) in mice challenged with 2,4-dinitrophenol (DNP). Selective EP2 agonism attenuated aeroallergen-caused AHR and inflammation in HDM-exposed BALB/c mice, and this correlated with a reduced lung MC activity. Accordingly, the blockade of endogenous PGE2 by means of AH6809 worsened airway responsiveness in sensitive BALB/c mice, and such worsening was reversed by SCG. The relevance of MC to PGE2 -EP2 driven protection was further highlighted in MC-dependent PCA, where butaprost fully prevented MC-induced ear swelling. Unlike in BALB/c mice, PGE2 did not protect the airways of HDM-sensitized C57BL/6 animals, a strain in which we showed MC to be irrelevant to aeroallergen-driven AHR and inflammation. The beneficial effect of both exogenous and endogenous PGE2 in aeroallergen-sensitized mice may be attributable to the activation of the EP2 receptor, which in turn acts as a restrainer of airway MC activity. This opens a path towards the identification of therapeutic targets against asthma along the 'EP2 -MC-airway' axis. © 2015 John Wiley & Sons Ltd.

  4. Genome-based genetic tool development for Bacillus methanolicus: theta- and rolling circle-replicating plasmids for inducible gene expression and application to methanol-based cadaverine production

    Directory of Open Access Journals (Sweden)

    Marta Irla

    2016-09-01

    Full Text Available Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 g/L to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

  5. CCY-1a-E2 induces G2/M phase arrest and apoptotic cell death in HL-60 leukemia cells through cyclin-dependent kinase 1 signaling and the mitochondria-dependent caspase pathway.

    Science.gov (United States)

    Lin, Chin-Fen; Yang, Jai-Sing; Lin, Chingju; Tsai, Fuu-Jen; Lu, Chi-Cheng; Lee, Miau-Rong

    2016-09-01

    Our previous study demonstrated that 2-[(3-methoxybenzyl)oxy]benzaldehyde (CCY-1a-E2) is a potent compound that acts against multiple human leukemia cell lines. CCY-1a-E2 was also shown to have efficacious anti‑leukemic activity in vivo. However, the molecular mechanism of action of CCY‑1a‑E2 attributed to its anticancer effect remains poorly understood. In the present study, CCY‑1a‑E2 suppressed cell viability in multiple leukemia cell lines (HL‑60, K562, KG‑1 and KG‑1a) via inhibition of cell proliferation, cell cycle arrest and induction of apoptosis. CCY‑1a‑E2 exhibited a marked toxic effect on HL‑60 cells and displayed low cytotoxicity in normal human peripheral blood mononuclear cells (PBMCs). Results from flow cytometric analysis indicated that CCY‑1a‑E2 promoted G2/M phase arrest and promoted apoptosis in the HL‑60 cells. CCY‑1a‑E2 treatment upregulated cyclin B, cyclin‑dependent kinase 1 (CDK1), cell division cycle 25C (cdc25C) and p21 protein expression. CCY‑1a‑E2 caused apoptotic cell death and DNA fragmentation as determined by 4',6‑diamidino‑2‑phenylindole (DAPI) staining and DNA gel electrophoresis. Elevated activities of caspase‑8, ‑9 and ‑3 were observed during CCY‑1a‑E2induced cell apoptosis; their specific inhibitors were found to block CCY‑1a‑E2induced apoptosis, respectively. Moreover, CCY‑1a‑E2 time‑dependently disrupted the mitochondrial membrane potential (ΔΨm), and it enhanced the protein levels of Fas/CD95, cytochrome c, Bax, cleaved PARP, as well as attenuated Bcl‑2 expression in the HL‑60 cells. Our results provide direct evidence that supports the future potential therapeutic application of CCY-1a-E2 in leukemia.

  6. Curing of plasmid pXO1 from Bacillus anthracis using plasmid incompatibility.

    Directory of Open Access Journals (Sweden)

    Xiankai Liu

    Full Text Available The large plasmid pXO1 encoding the anthrax toxin is important for the virulence of Bacillus anthracis. It is essential to cure pXO1 from B. anthracis to evaluate its role in the pathogenesis of anthrax infection. Because conventional methods for curing plasmids (e.g., curing agents or growth at elevated temperatures can induce mutations in the host chromosomal DNA, we developed a specific and reliable method to eliminate pXO1 from B. anthracis using plasmid incompatibility. Three putative replication origins of pXO1 were inserted into a temperature-sensitive plasmid to generate three incompatible plasmids. One of the three plasmids successfully eliminated the large plasmid pXO1 from B. anthracis vaccine strain A16R and wild type strain A16. These findings provided additional information about the replication/partitioning of pXO1 and demonstrated that introducing a small incompatible plasmid can generate plasmid-cured strains of B. anthracis without inducing spontaneous mutations in the host chromosome.

  7. Main: E2FAT [PLACE

    Lifescience Database Archive (English)

    Full Text Available tial E2F target genes; most potential E2F targets identified in silico show a cell cycle-regulated expression; Y=T/C; see S000366; E2F; cell cycle; Arabidopsis thaliana TYTCCCGCC ...

  8. E2F activates late-G1 events but cannot replace E1A in inducing S phase in terminally differentiated skeletal muscle cells

    DEFF Research Database (Denmark)

    Pajalunga, D; Tognozzi, D; Tiainen, M

    1999-01-01

    We have previously shown that the adenovirus E1A oncogene can reactivate the cell cycle in terminally differentiated cells. Current models imply that much or all of this E1A activity is mediated by the release of the E2F transcription factors from pocket-protein control. In contrast, we show here...

  9. E2F1 is crucial for E2F-dependent apoptosis

    DEFF Research Database (Denmark)

    Lazzerini Denchi, Eros; Helin, Kristian

    2005-01-01

    Loss of the retinoblastoma protein, pRB, leads to apoptosis, and several results have suggested that this is dependent on the E2F transcription factors. However, so far, the ability of the different E2F family members to contribute to apoptosis is controversial. Here, we show that ectopic...... expression of E2F3 results in apoptosis in both primary mouse fibroblasts and transgenic mice. Apoptosis induced by E2F3 is associated with the accumulation of E2F1 and, strikingly, we found that E2F3-induced apoptosis is dependent on E2F1. On the basis of these results, we propose that the accumulation...... of crucial levels of E2F1 activity, and not total E2F activity, is essential for the induction of apoptosis in response to a deregulated pRB pathway. These results are consistent with previous findings that E2F1, but not other E2Fs, can have tumour-suppressing activities....

  10. Gene synthesis,cloning,prokaryotic expression and analysis of chikungunya virus E2 glycoprotein%基孔肯雅病毒包膜蛋白E2的全基因合成及原核表达

    Institute of Scientific and Technical Information of China (English)

    章萍萍; 邓松华; 潘卫; 曹洁; 陈秋莉; 王锦红; 张华群; 葛宜兵; 祁培培; 刘超

    2012-01-01

    目的 通过对全长基孔肯雅病毒包膜蛋白E2(CHIKV-E2,1~404 aa)及其跨膜疏水区(351~378 aa)缺失突变体E2(1~350 aa)进行原核表达,分析跨膜疏水区对E2蛋白在大肠杆菌中表达的影响.方法 利用ExPasy预测软件对E2蛋白跨膜疏水区进行预测分析,根据GenBank数据库中CHIKV-E2氨基酸序列获得其对应的基因序列.结合重叠延伸PCR(OE-PCR) 原理设计用于全基因合成的核酸引物对CHIKV-E2全长基因(404 aa)进行体外合成,构建全长E2蛋白及其缺失突变体原核表达质粒,将序列正确的两种重组质粒分别转至E.coli BL21(DE3),经IPTG诱导表达,SDS-PAGE检测重组质粒的表达情况.结果 OE-PCR法成功合成了大小为1 212 bp的编码CHIKV-E2(1~404 aa)蛋白的全长基因,构建了全长E2蛋白及其缺失突变体重组表达质粒 pET21b-E2(1~404)和pET21b-E2(1~350),经IPTG诱导表达后,SDS-PAGE结果显示缺失突变体pET21b-E2(1~350)融合蛋白表达量较pET21b-E2(1~404)有明显提高.结论 E2蛋白跨膜疏水区(351~378 aa)对该蛋白的原核表达具有重要影响,缺失该疏水区的突变体在大肠杆菌中表达量比全长E2蛋白表达量明显提高.%Objective To analyze the impact of hydrophobic transmembrane domain of chikimgunya vims E2 glyco-proteins on E2 expression in E. Coli by comparing E2 expression with the hydrophobic transmembrane domain-deleted mutant. Methods On-line software ExPasy was used to predict transmembrane domain of E2 protein and optimized gene sequence encoding E2 protein was obtained according to amino acids of the protein from GenBank; Primers were designed to synthesize E2 gene according to the principle of OE-PCR and prokaryotic expression plasmids of full-length E2 protein and its mutant were constructed. After being transfromed into E. Coli BL21( DE3 ), two plasmids in E. Coli were induced to express by IPTG and protein expression was identified by SDS-PAGE. Results In the study, the gene encoding CHIKV-E

  11. Dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells upon drug-induced DNA damage under hypoxia.

    Science.gov (United States)

    Wu, Minghui; Wang, Xue; McGregor, Natalie; Pienta, Kenneth J; Zhang, Jingsong

    2014-06-01

    Intratumoral hypoxia has been proposed to create a "mutator" phenotype through downregulation of DNA repair, leading to increased genomic instability and drug resistance. Such downregulation of DNA repair has been proposed to sensitize hypoxic cancer cells to DNA-damaging agents and inhibitors of DNA repair. Here, we showed that prostate cancer cells with mutant p53 were resistant to the poly(ADP-ribose) polymerase inhibitor, veliparib (2-[(2R)-2-methylpyrrolidin-2-yl]-1H-benzimidazole-4-carboxamide, dihydrochloride; Abbott Laboratories, Abbott Park, IL), and the DNA-damaging topoisomerase I inhibitor camptothecin-11 (CPT-11) or SN38 (7-ethyl-10-hydroxycamptothecin) under hypoxia. Upregulation of Rad51 by E2F1 upon DNA damage under hypoxia contributed to such resistance, which was reversed by either inhibiting RAD51 transcription with small interfering RNA or by expressing wild-type p53 in the p53 null prostate cancer line. Accumulation of endogenous p53 but not E2F1 and suppressed RAD51 transcription was observed in prostate cancer line with wild-type p53 after DNA damage under hypoxia. Combining veliparib with CPT-11 significantly enhanced DNA damage and apoptosis under both hypoxic and normoxic culture conditions. Such enhanced DNA damage and antitumor activities were seen in the presence of Rad51 upregulation and confirmed in vivo with PC3 mouse xenografts. These data illustrate a dynamic regulation of Rad51 by E2F1 and p53 in prostate cancer cells' response to hypoxia and DNA damage. The veliparib and CPT-11 combination can be further explored as a treatment of metastatic castration-resistant prostate cancers that have frequent p53 mutations and enriched genomic instability.

  12. HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network

    Science.gov (United States)

    Ramos-Montoya, Antonio; Lamb, Alastair D; Russell, Roslin; Carroll, Thomas; Jurmeister, Sarah; Galeano-Dalmau, Nuria; Massie, Charlie E; Boren, Joan; Bon, Helene; Theodorou, Vasiliki; Vias, Maria; Shaw, Greg L; Sharma, Naomi L; Ross-Adams, Helen; Scott, Helen E; Vowler, Sarah L; Howat, William J; Warren, Anne Y; Wooster, Richard F; Mills, Ian G; Neal, David E

    2014-01-01

    Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies. PMID:24737870

  13. HES6 drives a critical AR transcriptional programme to induce castration-resistant prostate cancer through activation of an E2F1-mediated cell cycle network.

    Science.gov (United States)

    Ramos-Montoya, Antonio; Lamb, Alastair D; Russell, Roslin; Carroll, Thomas; Jurmeister, Sarah; Galeano-Dalmau, Nuria; Massie, Charlie E; Boren, Joan; Bon, Helene; Theodorou, Vasiliki; Vias, Maria; Shaw, Greg L; Sharma, Naomi L; Ross-Adams, Helen; Scott, Helen E; Vowler, Sarah L; Howat, William J; Warren, Anne Y; Wooster, Richard F; Mills, Ian G; Neal, David E

    2014-05-01

    Castrate-resistant prostate cancer (CRPC) is poorly characterized and heterogeneous and while the androgen receptor (AR) is of singular importance, other factors such as c-Myc and the E2F family also play a role in later stage disease. HES6 is a transcription co-factor associated with stem cell characteristics in neural tissue. Here we show that HES6 is up-regulated in aggressive human prostate cancer and drives castration-resistant tumour growth in the absence of ligand binding by enhancing the transcriptional activity of the AR, which is preferentially directed to a regulatory network enriched for transcription factors such as E2F1. In the clinical setting, we have uncovered a HES6-associated signature that predicts poor outcome in prostate cancer, which can be pharmacologically targeted by inhibition of PLK1 with restoration of sensitivity to castration. We have therefore shown for the first time the critical role of HES6 in the development of CRPC and identified its potential in patient-specific therapeutic strategies.

  14. Vaccination of Plasmid DNA Encoding Somatostatin Gene Fused with GP5 Gene of Porcine Reproductive and Respiratory Syndrome Virus Induces Anti-GP5 Antibodies and Promotes Growth Performance in Immunized Pigs

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Somatostatin (SS) is a hormone that inhibits the secretion of growth hormone. Immunization against SS can promote the growth of animals. This paper described the effects of DNA immunization on the growth and antibody response in mice and pigs immunized with a plasmid DNA encoding SS fused with GP5 of porcine reproductive and respiratory syndrome virus (PRRSV). A fragment of 180 bp encoding partial SS gene was amplified by PCR from the genomic DNA of peripheral blood mononuclear cells of pigs, and cloned as a fusion gene with PRRSV GP5 in plasmid pISGRTK3. Three times of immunization with the resulting plasmid pISG-SS/GP5 induced anti-GP5 antibodies in BALB/c mice and pigs, as demonstrated by GP5-specific ELISA and immunoblotting. Compared with pigs immunized with empty vector pISGRTK3, the growth performance of pigs immunized with pISG-SS/GP5 was increased by 11.1% on the 13th week after the last vaccination. The results indicated the plasmid DNA encoding SS and PRRSV GP5 fusion gene elicited anti-GP5 antibodies and improved the growth performance of immunized pigs.

  15. Immunization with chlamydial plasmid protein pORF5 DNA vaccine induces protective immunity against genital chlamydial infection in mice

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    To validate the immune protective efficacy of pORF5 DNA vaccine and to analyze potential mechanisms related to this protection. In this study, pORF5 DNA vaccine was constructed and evaluated for its protective immunity in a mouse model of genital chlamydial infection. Groups of BALB/c mice were immunized intranasally with pORF5 DNA vaccine. Humoral and cell mediated immune responses were evaluated. The clearance ability of chlamydial challenge from the genital tract and the chlamy- dia-induced upper genital tract gross pathology and histopathological characterization were also de- tected. The results showed that the total and the IgG2a anti-pORF5 antibody levels in serum were sig- nificantly elevated after pcDNA3.1-pORF5 vaccination, as were the total antibody and IgA levels in vaginal fluids. pcDNA3.1-pORF5 induced a significantly high level of Th1 response as measured by robust gamma interferon (IFN-γ). Minimal IL-4 was produced by immune T cells in response to the re-stimulation with pORF5 protein or the inactive elementary body in vitro. pcDNA3.1-pORF5-vacci- nated mice displayed significantly reduced bacterial shedding upon a chlamydial challenge and an accelerated resolution of infection. 100% of pcDNA3.1-pORF5 vaccinated mice successfully resolved the infection by day 24. pcDNA3.1-pORF5-immunized mice also exhibited protection against patho- logical consequences of chlamydial infection. The stimulated index was significantly higher than that of mice immunized with pcDNA3.1 and PBS (P<0.05). Together, these results demonstrated that immu- nization with pORF5 DNA vaccine is a promising approach for eliciting a protective immunity against a genital chlamydial challenge.

  16. Chlamydial plasmids and bacteriophages.

    Science.gov (United States)

    Pawlikowska-Warych, Małgorzata; Śliwa-Dominiak, Joanna; Deptuła, Wiesław

    2015-01-01

    Chlamydia are absolute pathogens of humans and animals; despite being rather well recognised, they are still open for discovery. One such discovery is the occurrence of extrachromosomal carriers of genetic information. In prokaryotes, such carriers include plasmids and bacteriophages, which are present only among some Chlamydia species. Plasmids were found exclusively in Chlamydia (C.) trachomatis, C. psittaci, C. pneumoniae, C. suis, C. felis, C. muridarum and C. caviae. In prokaryotic organisms, plasmids usually code for genes that facilitate survival of the bacteria in the environment (although they are not essential). In chlamydia, their role has not been definitely recognised, apart from the fact that they participate in the synthesis of glycogen and encode proteins responsible for their virulence. Furthermore, in C. suis it was evidenced that the plasmid is integrated in a genomic island and contains the tetracycline-resistance gene. Bacteriophages specific for chlamydia (chlamydiaphages) were detected only in six species: C. psittaci, C. abortus, C. felis, C. caviae C. pecorum and C. pneumoniae. These chlamydiaphages cause inhibition of the developmental cycle, and delay transformation of reticulate bodies (RBs) into elementary bodies (EBs), thus reducing the possibility of infecting other cells in time. Plasmids and bacteriophages can be used in the diagnostics of chlamydioses; although especially in the case of plasmids, they are already used for detection of chlamydial infections. In addition, bacteriophages could be used as therapeutic agents to replace antibiotics, potentially addressing the problem of increasing antibiotic-resistance among chlamydia.

  17. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2.

    Science.gov (United States)

    Erdmann, Susanne; Shah, Shiraz A; Garrett, Roger A

    2013-12-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2 that was co-infected with the virus SMV1 (Sulfolobus monocaudavirus 1). Although we reaffirm that protospacer selection is essentially a random process with respect to the pMGB1 genome, we identified single spacer sequences specific for each of CRISPR loci C, D and E that, exceptionally, occurred in many sequenced clones. Moreover, the same sequence was reproducibly acquired for a given locus in independent experiments, consistent with it being the first protospacer to be selected. There was also a small protospacer bias (1.6:1) to the antisense strand of protein genes. In addition, new experiments demonstrated that spacer acquisition in the previously inactive CRISPR locus A could be induced on freeze-thawing of the infected cells, suggesting that environmental stress can facilitate activation. Coincidentally with spacer acquisition, a mobile OrfB element was deleted from pMGB1, suggesting that interplay can occur between spacer acquisition and transposition.

  18. Prostaglandin E2 potentiates mesenchymal stem cell-induced IL-10+IFN-γ+CD4+ regulatory T cells to control transplant arteriosclerosis.

    Science.gov (United States)

    Hsu, Wan-Tseng; Lin, Cheng-Hsin; Chiang, Bor-Luen; Jui, Hsiang-Yiang; Wu, Kenneth Kun-Yu; Lee, Chii-Ming

    2013-03-01

    Mesenchymal stem cells (MSCs) are known for their immunomodulatory functions. We previously demonstrated that bone marrow-derived MSCs effectively control transplant arteriosclerosis (TA) by enhancing IL-10(+) and IFN-γ(+) cells. The objective of this study is to elucidate the mechanism by which MSCs induce IL-10(+)IFN-γ(+)CD4(+) regulatory T type 1 (T(R)1)-like cells. In an MLR system using porcine PBMCs, MSC-induced IL-10(+)IFN-γ(+)CD4(+) cells, which confer resistance to allogeneic proliferation in an IL-10-dependent manner, resemble T(R)1-like cells. Both cyclooxygenase-derived PGE(2) and IDO help to induce T(R)1-like cells by MSCs. MSCs constitutively secrete PGE(2), which is augmented in allogeneic reactions. However, T(R)1-like cells were deficient in PGE(2) and 4-fold less potent than were MSCs in suppressing MLR. PGE(2) mimetic supplements can enhance the immunosuppressive potency of T(R)1-like cells. In a porcine model of allogeneic femoral arterial transplantation, MSC-induced T(R)1-like cells combined with PGE(2), but not either alone, significantly reduced TA at the end of 6 wk (percentage of luminal area stenosis: T(R)1-like cells + PGE(2): 11 ± 10%; PGE(2) alone: 93 ± 8.7%; T(R)1-like cells alone: 88 ± 2.4% versus untreated 94 ± 0.9%, p < 0.001). These findings indicate that PGE(2) helps MSC-induced IL-10(+)IFN-γ(+)CD4(+) T(R)1-like cells inhibit TA. PGE(2) combined with MSC-induced T(R)1-like cells represents a new approach for achieving immune tolerance.

  19. The flexibility of UV-inducible mutation in Deinococcus ficus as evidenced by the existence of the imuB-dnaE2 gene cassette and generation of superior feather degrading bacteria.

    Science.gov (United States)

    Zeng, You-Hong; Shen, Fo-Ting; Tan, Chen-Chung; Huang, Chieh-Chen; Young, Chiu-Chung

    2011-12-20

    The lexA-imuB-dnaE2 gene cassette contributing to the TLS (translesion synthesis) polymerase activity and can easily cause mutation after DNA damage in many bacteria. But it was previously thought that TLS polymerase activity was unlikely to exist in the radio-resistant genus Deinococcus. In our preliminary studies, the lexA-imuB-dnaE2 gene cassette was found in a newly isolated feather-degrading Deinococcus ficus. Here we have attempted to determine the imuB gene sequence from another Deinococcus species namely D. grandis, by using the newly designed primers. The destroying of either imuB or dnaE2 gene in D. ficus leads to the increase in UV sensitivity and decrease in UV-induced mutations, which demonstrated the existence of TLS polymerase activity in D. ficus. In the presence of lexA-imuB-dnaE2, it is possible to obtain mutants with various keratinolytic activities after UV exposure. The keratinolytic activity of mutant strain CC-ZG207 increased by approximately twofold during growth in liquid feather medium. In contrast, the mutant strain CC-ZG227 showed only half of the keratinolytic activity compared with the wild type strain. By utilizing SDS-PAGE and zymogram profile analysis, the change in the protease activity was observed. We have proposed that the superior mutants of D. ficus can be created under UV stress, which is mediated by the lexA-imuB-dnaE2 gene cassette.

  20. Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth, Angiogenesis, and Metastasis*

    Science.gov (United States)

    Inada, Masaki; Takita, Morichika; Yokoyama, Satoshi; Watanabe, Kenta; Tominari, Tsukasa; Matsumoto, Chiho; Hirata, Michiko; Maru, Yoshiro; Maruyama, Takayuki; Sugimoto, Yukihiko; Narumiya, Shuh; Uematsu, Satoshi; Akira, Shizuo; Murphy, Gillian; Nagase, Hideaki; Miyaura, Chisato

    2015-01-01

    The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1−/−) mice, we show that prostaglandin E2 (PGE2) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE2 production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE2 further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE2 production by the osteoblasts. PGE2, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE2 acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE2 effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE2 signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors. PMID:26475855

  1. Anti-tumor Effects of pNEgr-mIL-12 Recombinant Plasmid Induced by X-irradiation and Its Mechanisms

    Institute of Scientific and Technical Information of China (English)

    YING YANG; SHU-ZHENG LIU; SHI-BO FU

    2004-01-01

    Objective To study the effect of gene radiotherapy combining injection of recombinant plasmid pNEgr-mIL-12 with local X-irradiation on cancer growth and to elucidate the mechanisms of tumor inhibition. Methods Alkaline lysis was used to extract the plasmid and polyethylene glycol 8000 (PEG 8000) was applied for further purification of plasmids. Enzyme-linked immunosorbent assay (ELISA) was used to detect the expression of IL-12 protein. C57BL/6J mice were subcutaneously inoculated with B16 melanoma cells and the plasmid was injected directly into the tumor. Gene-radiotherapy combining pNEgr-mIL-12 recombinant plasmid with X-irradiation was given three times to C57BL/6J mice bearing B16 melanoma. Changes in immunologic parameters of tumor-bearing mice were detected with relevant immunologic assays. Results Results showed a significant decrease in tumor growth rate (P<0.05-0.001) after 3 times of gene-radiotherapy with IL-12 and X-irradiation. Immunologic studies showed a significant increase in CTL and NK cytolytic activity (P<0.05-0.001) and an up-regulated secretion of IFN-γ and TNF-α (P<0.01-0.001). Moreover, the expression of mIL-12 in B16 melanoma cells of the treated tumor-bearing mice was found to be higher than that of control. Conclusion pNEgr-mIL-12 plasmid combined with X-irradiation can increase tumor control and the mechanism of increased tumor inhibition is related to the enhancement of anticancer immunity in tumor-bearing mice.

  2. Reference: E2FBNTRNR [PLACE

    Lifescience Database Archive (English)

    Full Text Available E2FBNTRNR Chaboute ME, Clement B, Sekine M, Philipps G, Chaubet-Gigot N Cell cycle re...gulation of the tobacco ribonucleotide reductase small subunit gene is mediated by E2F-like elements Plant Cell 12: 1987-2000 (2000) PubMed: 11041892; ...

  3. Effect of a Korean traditional formulation, Hwaotang, on superoxide generation in human neutrophils, platelet aggregation in human blood, and nitric oxide, prostaglandin E2 production and paw oedema induced by carrageenan in mice.

    Science.gov (United States)

    Park, Won-Hwan; Park, Soo-Young; Kim, Hyung-Min; Kim, Cheorl-Ho

    2004-02-01

    Hwaotang, a traditional Korean medicinal formulation, is a dried decoctum of a mixture of 7 herbal medicines, consisting of Angelica gigantis Radix, Rehmanniae radix, Paeoniae radix, Ciniamomi cortex, Cnidii rhizoma, Persicae semen and Carthami flos. We have investigated that Hwaotang water extract (HOT) has various effects on stimulus-induced superoxide generation in human neutrophils. The effects of HOT on superoxide generation in human neutrophils were investigated. HOT significantly inhibited N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced superoxide generation in a concentration-dependent manner, but not that induced by arachidonic acid (AA). On the other hand, HOT enhanced superoxide generation induced by phorbol 12-myristate 13-acetate (PMA) in a concentration-dependent manner. The superoxide generation induced by PMA with HOT was suppressed by staurosporine, an inhibitor of protein kinase C, but was not suppressed by genistein, an inhibitor of protein tyrosine kinase. Tyrosyl phosphorylation of a 58 kDa protein, which was increased by fMLP, was inhibited by HOT. HOT also inhibited the generation of a 47 kDa protein and platelet aggregation in human blood. The results suggest that protein tyrosine kinase participates in fMLP-mediated superoxide generation by HOT-treated human neutrophils. HOT inhibited neutrophil functions, including degranulation, superoxide generation, and leukotriene B4 production, without any effect on 5-lipoxygenase activity. HOT reduced nitric oxide (NO) and prostaglandin E2 production in mouse peritoneal macrophages stimulated with lipopolysaccharide, whereas no influence on the activity of iNOS, COX-2 or COX-1 was observed. HOT significantly reduced mouse paw oedema induced by carrageenan. Western blot analysis showed that HOT reduced the expression of iNOS and COX-2. The results indicate that HOT exerts anti-inflammatory effects related to the inhibition of neutrophil functions and of NO and prostaglandin E2 production, which

  4. Inhibition by prostaglandin E(2) of anaphylatoxin C5a- but not zymosan-induced prostanoid release from rat Kupffer cells.

    Science.gov (United States)

    Pestel, Sabine; Jungermann, Kurt; Götze, Otto; Schieferdecker, Henrike L

    2002-04-01

    The proinflammatory anaphylatoxin C5a induces the release of prostanoids, ie, prostaglandins (PG) and thromboxane (TX), from the resident liver macrophages (Kupffer cells [KC]). Because KC themselves express prostanoid receptors, prostanoids--besides having paracrine functions--might regulate their own release in an autocrine loop. So far, such a possible feedback regulation has not been investigated systematically, probably because of methodological difficulties to measure newly synthesized prostanoids in the presence of added prostanoids. Here, after prelabeling of phospholipids with [(14)C]arachidonate, cellularly formed [(14)C]prostanoids were determined in the presence of added unlabelled prostanoids by thin layer chromatography. In cultured KC, recombinant rat C5a (rrC5a) rapidly increased PGD(2), PGE(2), and TXA(2) release, which was strongly reduced by PGE(2), but neither by PGD(2) nor by the TXA(2) analog U46619. The inhibitory effect of PGE(2) was mimicked by cAMP, indicating that the G(s)-coupled PGE(2) receptors type 2 or 4 were involved. Zymosan also enhanced prostanoid release from KC, but with slightly slower kinetics; this action was neither inhibited by PGE(2) nor by cAMP. Also in perfused rat livers, rrC5a enhanced prostanoid release from KC as shown by prostanoid overflow and thereby indirectly increased glucose output from hepatocytes. Again, PGE(2), but not PGD(2), inhibited rrC5a-elicited prostanoid overflow. This resulted in a complete inhibition of rrC5a-induced, prostanoid-mediated glucose output. Thus, PGE(2) can inhibit specifically the C5a-induced prostanoid release from KC via a feedback mechanism and thereby limit prostanoid-mediated hepatocellular defense reactions, eg, glucose release.

  5. Dominant role of prostaglandin E2 EP4 receptor in furosemide-induced salt-losing tubulopathy: a model for hyperprostaglandin E syndrome/antenatal Bartter syndrome

    DEFF Research Database (Denmark)

    Nüsing, Rolf M; Treude, Antje; Weissenberger, Christian

    2005-01-01

    -/-, EP2-/-, EP3-/-, and EP4-/-), the effect of chronic furosemide administration (7 d) on urine output, sodium and potassium excretion, and renin secretion was determined. Furthermore, furosemide-induced diuresis and renin activity were analyzed in mice with defective PGI2 receptors (IP-/-). In all...... was significantly decreased in EP4-/- mice and to a lesser degree also in IP-/- mice. Pharmacologic inhibition of EP4 receptors in furosemide-treated WT mice with the specific antagonist ONO-AE3-208 mimicked the changes in renin mRNA expression, plasma renin concentration, diuresis, and sodium excretion seen in EP4...

  6. Inhibitory effects of β-chamigrenal, isolated from the fruits of Schisandra chinensis, on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages [corrected].

    Science.gov (United States)

    Shin, Ji-Sun; Ryu, Suran; Cho, Young-Wuk; Kim, Hyun Ji; Jang, Dae Sik; Lee, Kyung-Tae

    2014-06-01

    Much is known about the bioactive properties of lignans from the fruits of Schisandra chinensis. However, very little work has been done to determine the properties of sesquiterpenes in the fruits of S. chinensis. The aim of the present study was to investigate the anti-inflammatory potential of new sesquiterpenes (β-chamigrenal, β-chamigrenic acid, α-ylangenol, and α-ylangenyl acetate) isolated from the fruits of S. chinensis and to explore their effect on macrophages stimulated with lipopolysaccharide. Of these four sesquiterpenes, β-chamigrenal most significantly suppressed lipopolysaccharide-induced nitric oxide and prostaglandin E2 production in RAW 264.7 macrophages (47.21 ± 4.54 % and 51.61 ± 3.95 % at 50 µM, respectively). Molecularly, the inhibitory activity of β-chamigrenal on nitric oxide production was mediated by suppressing inducible nitric oxide synthase activity but not its expression. In the prostaglandin E2 synthesis pathway, β-chamigrenal prevented the upregulation of inducible microsomal prostaglandin E synthase-1 expression after stimulation with lipopolysaccharide. Conversely, β-chamigrenal had no effect on the expression and enzyme activity of cyclooxygenase-2. In addition, the expression of early growth response factor-1, a key transcription factor of microsomal prostaglandin E synthase-1 expression, was inhibited by β-chamigrenal. These results may suggest a possible anti-inflammatory activity of β-chamigrenal which has to be proven in in vivo experiments.

  7. 前列腺素E2致兔盆腔炎模型中子宫主韧带Ⅰ、Ⅲ胶原含量的变化%Changes of contents of type Ⅰ collagen and type Ⅲ collagen in cardinal ligament of uterus of prostaglandin E2 -induced rabbit pelvic inflammation models

    Institute of Scientific and Technical Information of China (English)

    姜永杰; 白杨; 任琛琛; 任瑞芳

    2011-01-01

    目的:探讨因前列腺素E2 (prostaglandin E2,PGE2)干预而形成的盆腔炎动物模型中,PGE2作用时间长短对兔子宫韧带中Ⅰ、Ⅲ胶原含量的变化.方法:注射同等剂量和浓度的PGE2,制备符合筛查标准的盆腔炎动物模型.在模型形成2周、4周、8周时,每组随机选取6只实验动物,解剖获取子宫韧带.染色、蜡块包埋,运用偏光显微镜和电镜观察Ⅰ、Ⅲ胶原的含量和变化,结果:①偏振光显微镜能有效观察Ⅰ、Ⅲ胶原胶原的表现差异.②正常盆底韧带和炎症侵袭韧带中Ⅰ型和Ⅲ型胶原含量差异有统计学意义(P<0.01).③炎症模型中证实PGE2在第4周促进胶原的分泌.④电镜下观察结果与偏光显微镜下的结果无明显差异,表现为随着PGE2干预时间的增加盆腔炎症状加重,子宫韧带中各时段Ⅰ、Ⅲ胶原的合成先是增加后又急速下降,形成单峰状改变.结论:炎症因子PGE2通过调节Ⅰ、Ⅲ胶原的代谢及比例,改变韧带成纤维细胞的分化和活性,说明盆腔炎可能参与盆底功能障碍性疾病的发生.%Objective: To explore the effects of different administration times of prostaglandin E2 on the changes of contents of type I collagen and type III collagen in cardinal ligament of uterus of prostaglandin E2 - induced rabbit pelvic inflammation models. Methods: The pelvic inflammation models were established by injecting prostaglandin E2 (the same dose and the same concentration) into rabbits. At 2, 4 and 8 weeks after establishment of pelvic inflammation models, 6 rabbits were selected from each group randomly, then their cardinal ligaments of uterus were obtained. Staining and paraffin imbedding were used in the study; polarization microscope and electron microscope were used to observe the contents and changes of type I collagen and type III collagen. Results: Polarization microscope could observe the difference between type I collagen and type ID collagen effectively

  8. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  9. Mechanisms of plasmid segregation: have multicopy plasmids been overlooked?

    Science.gov (United States)

    Million-Weaver, Samuel; Camps, Manel

    2014-09-01

    Plasmids are self-replicating pieces of DNA typically bearing non-essential genes. Given that plasmids represent a metabolic burden to the host, mechanisms ensuring plasmid transmission to daughter cells are critical for their stable maintenance in the population. Here we review these mechanisms, focusing on two active partition strategies common to low-copy plasmids: par systems type I and type II. Both involve three components: an adaptor protein, a motor protein, and a centromere, which is a sequence area in the plasmid that is recognized by the adaptor protein. The centromere-bound adaptor nucleates polymerization of the motor, leading to filament formation, which can pull plasmids apart (par I) or push them towards opposite poles of the cell (par II). No such active partition mechanisms are known to occur in high copy number plasmids. In this case, vertical transmission is generally considered stochastic, due to the random distribution of plasmids in the cytoplasm. We discuss conceptual and experimental lines of evidence questioning the random distribution model and posit the existence of a mechanism for segregation in high copy number plasmids that moves plasmids to cell poles to facilitate transmission to daughter cells. This mechanism would involve chromosomally-encoded proteins and the plasmid origin of replication. Modulation of this proposed mechanism of segregation could provide new ways to enhance plasmid stability in the context of recombinant gene expression, which is limiting for large-scale protein production and for bioremediation.

  10. Plasmid-to-plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1986-01-01

    No recombination between plasmids was observed after conjugal transfer of a plasmid into a cell carrying another plasmid. Two types of such recombination took place after transformation, one type being Rec/sup +/ dependent and suggesting a preferred site of recombination. The other much rarer type was at least partially Rec/sup +/ independent.

  11. Plasmid interference for curing antibiotic resistance plasmids in vivo.

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M; Partridge, Sally R; Iredell, Jonathan R

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing ('addiction') systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative 'interference plasmids' were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored.

  12. Fine-tuning synthesis of Yersinia pestis LcrV from runaway-like replication balanced-lethal plasmid in a Salmonella enterica serovar typhimurium vaccine induces protection against a lethal Y. pestis challenge in mice.

    Science.gov (United States)

    Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Gunn, Bronwyn M; Branger, Christine G; Tinge, Steven A; Curtiss, Roy

    2010-06-01

    A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like high-copy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal DeltaasdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain chi9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/promoter of the P22 cro gene (O/P(cro)) (P(R)), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P(BAD) c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of beta-lactamase, and cloned into pYA4534 under the control of the P(trc) promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain chi9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

  13. Plasmid interference for curing antibiotic resistance plasmids in vivo

    Science.gov (United States)

    Kamruzzaman, Muhammad; Shoma, Shereen; Thomas, Christopher M.; Partridge, Sally R.

    2017-01-01

    Antibiotic resistance increases the likelihood of death from infection by common pathogens such as Escherichia coli and Klebsiella pneumoniae in developed and developing countries alike. Most important modern antibiotic resistance genes spread between such species on self-transmissible (conjugative) plasmids. These plasmids are traditionally grouped on the basis of replicon incompatibility (Inc), which prevents coexistence of related plasmids in the same cell. These plasmids also use post-segregational killing (‘addiction’) systems, which poison any bacterial cells that lose the addictive plasmid, to guarantee their own survival. This study demonstrates that plasmid incompatibilities and addiction systems can be exploited to achieve the safe and complete eradication of antibiotic resistance from bacteria in vitro and in the mouse gut. Conjugative ‘interference plasmids’ were constructed by specifically deleting toxin and antibiotic resistance genes from target plasmids. These interference plasmids efficiently cured the corresponding antibiotic resistant target plasmid from different Enterobacteriaceae in vitro and restored antibiotic susceptibility in vivo to all bacterial populations into which plasmid-mediated resistance had spread. This approach might allow eradication of emergent or established populations of resistance plasmids in individuals at risk of severe sepsis, enabling subsequent use of less toxic and/or more effective antibiotics than would otherwise be possible, if sepsis develops. The generalisability of this approach and its potential applications in bioremediation of animal and environmental microbiomes should now be systematically explored. PMID:28245276

  14. Construction of targeted plasmid vector pcDNA3.1-Egr.1p-p16 and its expression in pancreatic cancer JF305 cells induced by radiation in vitro

    Institute of Scientific and Technical Information of China (English)

    Hong-Bing Ma; Ming-Hua Bai; Xi-Jing Wang; Zheng-Li Di; Hui Xia; Zheng Li; Jie Liu; Jie Ma; Hua-Fen Kang; Cong-Mei Wu

    2007-01-01

    AIM: To construct pcDNA3.1-Egr.1p-p16 recombinant plasmid and investigate the expression of p16 in pancreatic cancer JF305 cells induced by radiation and the feasibility of gene radiotherapy for pancreatic carcinoma.METHODS: Human p16 cDNA was ligated to th edownstream of Egr-1 promotor to construct pcDNA3.1-Egr.1p-p16 plasmid by restriction enzyme digested. The recombined plasmids were transfected into pancreatic cancer JF305 cells with lipofectamine. p16 mRNA level was detected by RT-PCR. The expression of p16 after different doses of X-ray radiation was detected by Western blot technique. Cell survival was assessed by clonogenic assays and cell viability was analysed by trypen blue exclusion. Flow cytometry was performed to study the apoptosis of JF305 cells.RESULTS: Restriction enzyme digestion showed the correctly constructed pcDNA3.1-Egr.1p-p16. The p16expression in cells transfected with pcDNA3.1-Egr.1p-p16induced by different doses of radiation was higher than that in the control group (P < 0.05). Eight hours after 2 Gy X-ray radiation, the expression reached its peak(87.00 ng/L), and was significantly higher than that in the control group (P < 0.0.5). Clonogenic analysis and trypan blue extraction test showed that the pcDNA3.1-Egr.1p-p16 transfer enhanced radiation-induced cell killing in p16-null JF305 cell lines. The induction of apoptosis was lower in combined transfection and irradiation group than that in irradiation alone.CONCLUSION: X-ray can induce the recombinant plasmid pcDNA3.1-Egr.1p-p16 expression in JF305 cells.The detection of dose and time provides an experimental basis for in vivo study in future.

  15. Effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Pembroke, J.T.; Stevens, E. (University Coll., Galway (Ireland))

    1984-07-01

    The presence of the IncJ plasmids R391, R997, R705, R706, R748, and R749 was shown to sensitize Escherichia coli AB1157 and both its uvr A and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA/sup +/ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

  16. Cutting edge: retrobulbar inflammation, adipogenesis, and acute orbital congestion in a preclinical female mouse model of Graves' orbitopathy induced by thyrotropin receptor plasmid-in vivo electroporation.

    Science.gov (United States)

    Moshkelgosha, Sajad; So, Po-Wah; Deasy, Neil; Diaz-Cano, Salvador; Banga, J Paul

    2013-09-01

    Graves' orbitopathy (GO) is a complication in Graves' disease (GD) but mechanistic insights into pathogenesis remain unresolved, hampered by lack of animal model. The TSH receptor (TSHR) and perhaps IGF-1 receptor (IGF-1R) are considered relevant antigens. We show that genetic immunization of human TSHR (hTSHR) A-subunit plasmid leads to extensive remodeling of orbital tissue, recapitulating GO. Female BALB/c mice immunized with hTSHR A-subunit or control plasmids by in vivo muscle electroporation were evaluated for orbital remodeling by histopathology and magnetic resonance imaging (MRI). Antibodies to TSHR and IGF-1R were present in animals challenged with hTSHR A-subunit plasmid, with predominantly TSH blocking antibodies and were profoundly hypothyroid. Orbital pathology was characterized by interstitial inflammation of extraocular muscles with CD3+ T cells, F4/80+ macrophages, and mast cells, accompanied by glycosaminoglycan deposition with resultant separation of individual muscle fibers. Some animals showed heterogeneity in orbital pathology with 1) large infiltrate surrounding the optic nerve or 2) extensive adipogenesis with expansion of retrobulbar adipose tissue. A striking finding that underpins the new model were the in vivo MRI scans of mouse orbital region that provided clear and quantifiable evidence of orbital muscle hypertrophy with protrusion (proptosis) of the eye. Additionally, eyelid manifestations of chemosis, including dilated and congested orbital blood vessels, were visually apparent. Immunization with control plasmids failed to show any orbital pathology. Overall, these findings support TSHR as the pathogenic antigen in GO. Development of a new preclinical model will facilitate molecular investigations on GO and evaluation of new therapeutic interventions.

  17. Presence of Glycopeptide-Encoding Plasmids in Enterococcal Isolates from Food and Humans in Denmark

    DEFF Research Database (Denmark)

    Migura, Lourdes Garcia; Valenzuela, Antonio Jesus Sanchez; Jensen, Lars Bogø

    2011-01-01

    developed techniques for classification of plasmids. Replicons associated with sex pheromone-inducible plasmids were detected in all GR E. faecalis, whereas GR Enterococcus faecium contained plasmids known to be widely distributed among enterococci. vanA resistance is common in E. faecium isolates from meat...... and animals in Europe and is rarely found in E. faecalis. This article describes the first characterization of MGE from vanA mediated E. faecalis, thus linking this resistance genotype to pheromone responding plasmids....

  18. E2F-6: a novel member of the E2F family is an inhibitor of E2F-dependent transcription

    DEFF Research Database (Denmark)

    Cartwright, P; Müller, H; Wagener, C;

    1998-01-01

    promoter (TTTCGCGC). In contrast to the other members of the E2F family, ectopic expression of E2F-6 inhibits transcription from promoters possessing E2F recognition sites rather than activating transcription. In addition, overexpression of E2F-6 suppresses the transactivational effects of coexpression......The E2F family of transcription factors are essential for the regulation of genes required for appropriate progression through the cell cycle. Five members of the E2F family have been previously reported, namely E2F1-5. All five are key elements in transcriptional regulation of essential genes......, and they can be divided into two functional groups, those that induce S-phase progression when overexpressed in quiescent cells (E2Fs 1-3), and those that do not (E2Fs 4-5). Here, we describe the identification of a novel member of this family, which we refer to as E2F-6. E2F-6 shares significant homology...

  19. DcE2F, a functional plant E2F-like transcriptional activator from Daucus carota

    DEFF Research Database (Denmark)

    Albani, D; Mariconti, L; Ricagno, S;

    2000-01-01

    In animal cells the progression of the cell cycle through G(1)/S transition and S phase is under the control of the pRB/E2F regulatory pathway. The E2F transcription factors are key activators of genes coding for several regulatory proteins and for enzymes involved in nucleotide and DNA synthesis....... In this report we have detected the presence of E2F-like DNA binding activities in carrot nuclear extracts, and we have isolated a carrot cDNA (DcE2F) encoding a plant E2F homologue. The DcE2F gene is expressed in proliferating cells and is induced during the G(1)/S transition of the cell cycle. Supershift...... assays have revealed that DcE2F is a functional transcription factor that can transactivate, together with a DP partner, an E2F-responsive reporter gene in both plant and mammalian cells....

  20. TNF-alpha/IFN-gamma-induced iNOS expression increased by prostaglandin E2 in rat primary astrocytes via EP2-evoked cAMP/PKA and intracellular calcium signaling.

    Science.gov (United States)

    Hsiao, Han-Yun; Mak, Oi-Tong; Yang, Chung-Shi; Liu, Yu-Peng; Fang, Kuan-Ming; Tzeng, Shun-Fen

    2007-01-15

    Astrocytes, the most abundant glia in the central nervous system (CNS), produce a large amount of prostaglandin E(2) (PGE(2)) in response to proinflammatory mediators after CNS injury. However, it is unclear whether PGE(2) has a regulatory role in astrocytic activity under the inflamed condition. In the present work, we showed that PGE(2) increased inducible nitric oxide synthase (iNOS) production by tumor necrosis factor-alpha and interferon-gamma (T/I) in astrocytes. Pharmacological and RNA interference approaches further indicated the involvement of the receptor EP2 in PGE(2)-induced iNOS upregulation in T/I-treated astrocytes. Quantitative real-time polymerase chain reaction and gel mobility shift assays also demonstrated that PGE(2) increased iNOS transcription through EP2-induced cAMP/protein kinase A (PKA)-dependent pathway. Consistently, the effect of EP2 was significantly attenuated by the PKA inhibitor KT-5720 and partially suppressed by the inhibitor (SB203580) of p38 mitogen-activated protein kinase (p38MAPK), which serves as one of the downstream components of the PKA-dependent pathway. Interestingly, EP2-mediated PKA signaling appeared to increase intracellular Ca(2+) release through inositol triphosphate (IP3) receptor activation, which might in turn stimulate protein kinase C (PKC) activation to promote iNOS production in T/I-primed astrocytes. By analyzing the expression of astrocytic glial fibrillary acidic protein (GFAP), we found that PGE(2) alone only triggered the EP2-induced cAMP/PKA/p38MAPK signaling pathway in astrocytes. Collectively, PGE(2) may enhance T/I-induced astrocytic activation by augmenting iNOS/NO production through EP2-mediated cross-talk between cAMP/PKA and IP3/Ca(2+) signaling pathways.

  1. Persistence Mechanisms of Conjugative Plasmids

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Hansen, Lars H.; Sørensen, Søren Johannes

    2009-01-01

    Are plasmids selfish parasitic DNA molecules or an integrated part of the bacterial genome? This chapter reviews the current understanding of the persistence mechanisms of conjugative plasmids harbored by bacterial cells and populations. The diversity and intricacy of mechanisms affecting...... the successful propagation and long-term continued existence of these extra-chromosomal elements is extensive. Apart from the accessory genetic elements that may provide plasmid-harboring cells a selective advantage, special focus is placed on the mechanisms conjugative plasmids employ to ensure their stable...... maintenance in the host cell. These importantly include the ability to self-mobilize in a process termed conjugative transfer, which may occur across species barriers. Other plasmid stabilizing mechanisms include the multimer resolution system, active partitioning, and post-segregational-killing of plasmid...

  2. Hydrogen gas protects against serum and glucose deprivation‑induced myocardial injury in H9c2 cells through activation of the NF‑E2‑related factor 2/heme oxygenase 1 signaling pathway.

    Science.gov (United States)

    Xie, Qiang; Li, Xue-Xiang; Zhang, Peng; Li, Jin-Cao; Cheng, Ying; Feng, Yan-Ling; Huang, Bing-Sheng; Zhuo, Yu-Feng; Xu, Guo-Hua

    2014-08-01

    Ischemia or hypoxia‑induced myocardial injury is closely associated with oxidative stress. Scavenging free radicals and/or enhancing endogenous antioxidative defense systems may be beneficial for the impediment of myocardial ischemic injury. Hydrogen (H2) gas, as a water‑ and lipid‑soluble small molecule, is not only able to selectively eliminate hydroxyl (·OH) free radicals, but also to enhance endogenous antioxidative defense systems in rat lungs and arabidopsis plants. However, thus far, it has remained elusive whether H2 gas protects cardiomyocytes through enhancement of endogenous antioxidative defense systems. In the present study, the cardioprotective effect of H2 gas against ischemic or hypoxic injury was investigated, along with the underlying molecular mechanisms. H9c2 cardiomyoblasts (H9c2 cells) were treated in vitro with a chemical hypoxia inducer, cobalt chloride (CoCl2), to imitate hypoxia, or by serum and glucose deprivation (SGD) to imitate ischemia. Cell viability and intracellular ·OH free radicals were assessed. The role of an endogenous antioxidative defense system, the NF‑E2‑related factor 2 (Nrf2)/heme oxygenase 1 (HO‑1) signaling pathway, was evaluated. The findings revealed that treatment with CoCl2 or SGD markedly reduced cell viability in H9c2 cells. H2 gas‑rich medium protected against cell injury induced by SGD, but not that induced by CoCl2. When the cells were exposed to SGD, levels of intracellular ·OH free radicals were markedly increased; this was mitigated by H2 gas‑rich medium. Exposure of the cells to SGD also resulted in significant increases in HO‑1 expression and nuclear Nrf2 levels, and the HO‑1 inhibitor ZnPP IX and the Nrf2 inhibitor brusatol aggravated SGD‑induced cellular injury. H2 gas‑rich medium enhanced SGD‑induced upregulation of HO‑1 and Nrf2, and the HO‑1 or Nrf2 inhibition partially suppressed H2 gas‑induced cardioprotection. Furthermore, following genetic silencing of Nrf

  3. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  4. Differential Salt-Induced Dissociation of the p53 Protein Complexes with Circular and Linear Plasmid DNA Substrates Suggest Involvement of a Sliding Mechanism

    Directory of Open Access Journals (Sweden)

    Peter Šebest

    2015-01-01

    Full Text Available A study of the effects of salt conditions on the association and dissociation of wild type p53 with different ~3 kbp long plasmid DNA substrates (supercoiled, relaxed circular and linear, containing or lacking a specific p53 binding site, p53CON using immunoprecipitation at magnetic beads is presented. Salt concentrations above 200 mM strongly affected association of the p53 protein to any plasmid DNA substrate. Strikingly different behavior was observed when dissociation of pre-formed p53-DNA complexes in increased salt concentrations was studied. While contribution from the p53CON to the stability of the p53-DNA complexes was detected between 100 and 170 mM KCl, p53 complexes with circular DNAs (but not linear exhibited considerable resistance towards salt treatment for KCl concentrations as high as 2 M provided that the p53 basic C-terminal DNA binding site (CTDBS was available for DNA binding. On the contrary, when the CTDBS was blocked by antibody used for immunoprecipitation, all p53-DNA complexes were completely dissociated from the p53 protein in KCl concentrations ≥200 mM under the same conditions. These observations suggest: (a different ways for association and dissociation of the p53-DNA complexes in the presence of the CTDBS; and (b a critical role for a sliding mechanism, mediated by the C-terminal domain, in the dissociation process.

  5. A Recombinant DNA Plasmid Encoding the sIL-4R-NAP Fusion Protein Suppress Airway Inflammation in an OVA-Induced Mouse Model of Asthma.

    Science.gov (United States)

    Liu, Xin; Fu, Guo; Ji, Zhenyu; Huang, Xiabing; Ding, Cong; Jiang, Hui; Wang, Xiaolong; Du, Mingxuan; Wang, Ting; Kang, Qiaozhen

    2016-08-01

    Asthma is a chronic inflammatory airway disease. It was prevalently perceived that Th2 cells played the crucial role in asthma pathogenesis, which has been identified as the important target for anti-asthma therapy. The soluble IL-4 receptor (sIL-4R), which is the decoy receptor for Th2 cytokine IL-4, has been reported to be effective in treating asthma in phase I/II clinical trail. To develop more efficacious anti-asthma agent, we attempt to test whether the Helicobacter pylori neutrophil-activating protein (HP-NAP), a novel TLR2 agonist, would enhance the efficacy of sIL-4R in anti-asthma therapy. In our work, we constructed a pcDNA3.1-sIL-4R-NAP plasmid, named PSN, encoding fusion protein of murine sIL-4R and HP-NAP. PSN significantly inhibited airway inflammation, decreased the serum OVA-specific IgE levels and remodeled the Th1/Th2 balance. Notably, PSN is more effective on anti-asthma therapy comparing with plasmid only expressing sIL-4R.

  6. Conjugative plasmids of Neisseria gonorrhoeae.

    Directory of Open Access Journals (Sweden)

    Emilia Pachulec

    Full Text Available Many clinical isolates of the human pathogen Neisseria gonorrhoeae contain conjugative plasmids. The host range of these plasmids is limited to Neisseria species, but presence of a tetracycline (tetM determinant inserted in several of these plasmids is an important cause of the rapid spread of tetracycline resistance. Previously plasmids with different backbones (Dutch and American type backbones and with and without different tetM determinants (Dutch and American type tetM determinants have been identified. Within the isolates tested, all plasmids with American or Dutch type tetM determinants contained a Dutch type plasmid backbone. This demonstrated that tetM determinants should not be used to differentiate between conjugal plasmid backbones. The nucleotide sequences of conjugative plasmids with Dutch type plasmid backbones either not containing the tetM determinant (pEP5233 or containing Dutch (pEP5289 or American (pEP5050 type tetM determinants were determined. Analysis of the backbone sequences showed that they belong to a novel IncP1 subfamily divergent from the IncP1alpha, beta, gamma, delta and epsilon subfamilies. The tetM determinants were inserted in a genetic load region found in all these plasmids. Insertion was accompanied by the insertion of a gene with an unknown function, and rearrangement of a toxin/antitoxin gene cluster. The genetic load region contains two toxin/antitoxins of the Zeta/Epsilon toxin/antitoxin family previously only found in Gram positive organisms and the virulence associated protein D of the VapD/VapX toxin/antitoxin family. Remarkably, presence of VapX of pJD1, a small cryptic neisserial plasmid, in the acceptor strain strongly increased the conjugation efficiency, suggesting that it functions as an antitoxin for the conjugative plasmid. The presence of the toxin and antitoxin on different plasmids might explain why the host range of this IncP1 plasmid is limited to Neisseria species. The isolated plasmids

  7. Comparison of the effects of IV administration of meloxicam, carprofen, and flunixin meglumine on prostaglandin E(2) concentration in aqueous humor of dogs with aqueocentesis-induced anterior uveitis.

    Science.gov (United States)

    Gilmour, Margi A; Payton, Mark E

    2012-05-01

    To compare the effects of meloxicam, carprofen, and flunixin meglumine administered IV on the concentration of prostaglandin E(2) (PGE(2)) in the aqueous humor of dogs with aqueocentesis-induced anterior uveitis. 15 adult dogs with ophthalmically normal eyes. Each dog was assigned to 1 of 4 treatment groups. Treatment groups were saline (0.9% NaCl) solution (1 mL, IV), meloxicam (0.2 mg/kg, IV), carprofen (4.4 mg/kg, IV), and flunixin meglumine (0.5 mg/kg, IV). Each dog was anesthetized, treatment was administered, and aqueocentesis was performed on each eye at 30 and 60 minutes after treatment. Aqueous humor samples were frozen at -80°C until assayed for PGE(2) concentration with an enzyme immunoassay kit. For all 4 treatment groups, PGE(2) concentration was significantly higher in samples obtained 60 minutes after treatment, compared with that in samples obtained 30 minutes after treatment, which indicated aqueocentesis-induced PGE(2) synthesis. For aqueous humor samples obtained 60 minutes after treatment, PGE(2) concentration did not differ significantly among groups treated with saline solution, meloxicam, and carprofen; however, the PGE(2) concentration for the group treated with flunixin meglumine was significantly lower than that for each of the other 3 treatment groups. Flunixin meglumine was more effective than meloxicam or carprofen for minimizing the PGE(2) concentration in the aqueous humor of dogs with experimentally induced uveitis. Flunixin meglumine may be an appropriate pre-medication for use prior to intraocular surgery in dogs.

  8. SMV1 virus-induced CRISPR spacer acquisition from the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2

    DEFF Research Database (Denmark)

    Erdmann, Susanne; Shah, Shiraz Ali; Garrett, Roger Antony

    2013-01-01

    Organisms of the crenarchaeal order Sulfolobales carry complex CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune systems. These systems are modular and show extensive structural and functional diversity, especially in their interference complexes. The primary...... targets are an exceptional range of diverse viruses, many of which propagate stably within cells and follow lytic life cycles without producing cell lysis. These properties are consistent with the difficulty of activating CRISPR spacer uptake in the laboratory, but appear to conflict with the high...... complexity and diversity of the CRISPR immune systems that are found among the Sulfolobales. In the present article, we re-examine the first successful induction of archaeal spacer acquisition in our laboratory that occurred exclusively for the conjugative plasmid pMGB1 in Sulfolobus solfataricus P2...

  9. Phenotypic plasticity in bacterial plasmids.

    Science.gov (United States)

    Turner, Paul E

    2004-01-01

    Plasmid pB15 was previously shown to evolve increased horizontal (infectious) transfer at the expense of reduced vertical (intergenerational) transfer and vice versa, a key trade-off assumed in theories of parasite virulence. Whereas the models predict that susceptible host abundance should determine which mode of transfer is selectively favored, host density failed to mediate the trade-off in pB15. One possibility is that the plasmid's transfer deviates from the assumption that horizontal spread (conjugation) occurs in direct proportion to cell density. I tested this hypothesis using Escherichia coli/pB15 associations in laboratory serial culture. Contrary to most models of plasmid transfer kinetics, my data show that pB15 invades static (nonshaking) bacterial cultures only at intermediate densities. The results can be explained by phenotypic plasticity in traits governing plasmid transfer. As cells become more numerous, the plasmid's conjugative transfer unexpectedly declines, while the trade-off between transmission routes causes vertical transfer to increase. Thus, at intermediate densities the plasmid's horizontal transfer can offset selection against plasmid-bearing cells, but at high densities pB15 conjugates so poorly that it cannot invade. I discuss adaptive vs. nonadaptive causes for the phenotypic plasticity, as well as potential mechanisms that may lead to complex transfer dynamics of plasmids in liquid environments. PMID:15166133

  10. Plasmid Rolling-Circle Replication.

    Science.gov (United States)

    Ruiz-Masó, J A; MachóN, C; Bordanaba-Ruiseco, L; Espinosa, M; Coll, M; Del Solar, G

    2015-02-01

    Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

  11. Modulation of in utero total body irradiation induced newborn mouse growth retardation by maternal manganese superoxide dismutase-plasmid liposome (MnSOD-PL) gene therapy.

    Science.gov (United States)

    Epperly, M W; Smith, T; Zhang, X; Goff, J P; Franicola, D; Greenberger, B; Komanduri, P; Wang, H; Greenberger, J S

    2011-06-01

    To determine the effects of manganese superoxide dismutase (MnSOD) plasmid liposome (PL) maternal radioprotection on fetal mice, timed pregnant female mice (E14 gestation) were irradiated to 3.0 Gy total body irradiation (TBI) dose, and the number, weight and growth and development over 6 months after birth of newborn mice was quantitated compared with irradiated controls. Maternal MnSOD-PL treatment at E13 improved pup survival at birth (5.4±0.9 per litter) compared with non-irradiated 3.0 Gy controls 4.9±1.1. There was no statistically significant difference in newborn abnormalities, male to female ratio in newborn litters, or other evidence of teratogenesis in surviving newborn mice from MnSOD-PL treated compared with irradiated controls. However, E14 3 Gy irradiated pups from gene therapy-treated mothers showed a significant increase in both growth and overall survival over 6 months after birth (P=0.0022). To determine if transgene product crossed the placenta pregnant E13 mice were injected intravenously with hemagglutinin-epitope-tagged MnSOD (100 μg plasmid in 100 μl liposomes), then after 24 h, fetal mice, placentas and maternal tissues were removed and tested by both immunohistochemistry and reverse transcriptase-PCR for transgene and product. There was no evidence of transgene or product in placenta or any fetal tissue while maternal liver was positive by both assays. The data provide evidence for fetal radioprotection by maternal MnSOD-PL gene therapy before irradiation, which is mediated by an indirect bystander effect and is associated with a significant improvement in both survival at birth and growth and development of newborn mice.

  12. Conjugation efficiency depends on intra and intercellular interactions between distinct plasmids: Plasmids promote the immigration of other plasmids but repress co-colonizing plasmids.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode the genes responsible for the synthesis of conjugative pili and plasmid transfer. Expression of the conjugative machinery (including conjugative pili) may be costly to bacteria, not only due to the energetic/metabolic cost associated with their expression but also because they serve as receptors for certain viruses. Consequently, the presence of two plasmids in the same cell may be disadvantageous to each plasmid, because they may impose a higher fitness cost on the host. Therefore, plasmids may encode mechanisms to cope with co-resident plasmids. Moreover, it is possible that the transfer rate of a plasmid is affected by the presence of a distinct plasmid in the recipient cell. In this work, we measured transfer rates of twelve natural plasmids belonging to seven incompatibility groups in three situations, namely when: (i) donor cells contain a plasmid and recipient cells are plasmid-free; (ii) donor cells contain two unrelated plasmids and recipient cells are plasmid-free; and (iii) half of the cells contain a given plasmid and the other half contain another, unrelated, plasmid. In the third situation, recipient cells of a plasmid are the donor cells of the other plasmid. We show that there are more negative interactions (reduction of a plasmid's conjugative efficiency) between plasmids if they reside in the same cell than if they reside in different cells. However, if plasmids interacted intercellularly, the transfer rate of one of the plasmids was often higher (when the unrelated conjugative plasmid was present in the recipient cell) than if the recipient cell was plasmid-free - a positive effect. Experimental data retrieved from the study of mutant plasmids not expressing conjugative pili on the cell surface suggest that positive effects result from a higher efficiency of mating pair formation. Overall, our results suggest that negative interactions are significantly more frequent when plasmids occupy the same cell. Such

  13. Plasmid recombination in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.

    1982-01-01

    DNA recombination in exponential phase and competent Haemophilus influenzae was measured by an electron microscopic assay that relies on the conversion of plasmid RSF0885 monomers into multimeric forms. Dimer circles were present at a frequency of 2% in plasmid preparations from competent Rd (wild-type) cells; multimers were present at a frequency of 0.2% in preparations from exponential phase cells. Thus, plasmid recombination was stimulated in competent cells. Multimer formation occurred efficiently in cells of the transformation defective mutant rec2, implying that the rec2 gene product is not required for plasmid recombination. However, the absence of multimer plasmids in preparations from competent cells of the transformation defective mutant rec1 suggests that the rec1 gene product is required. Digestion of purified plasmids with restriction endonuclease PvuII, which makes a single cut in the monomer, revealed the presence of recombination intermediates composed of two linear plasmids joined to form two pairs of arms resembling the Greek letter chi. Length measurements of these arms taken from a population of recombination intermediates gave evidence that the plasmids were joined at sites of homology. The distributions of individual DNA strands, at the intersections of the four arms, could be resolved in some recombination intermediates and were of two types. The first type of junction appeared as a single-stranded arm appended to each corner. The second type of junction consisted of a single strand of DNA linking the two linear plasmids at a site of homology. The single-stranded linker was frequently situated at the edge of a short gap on one of the plasmids in the pair. The fine structures of the recombinational joints have been interpreted in terms of previously proposed models of recombination.

  14. Microneedle-mediated transcutaneous immunization with plasmid DNA coated on cationic PLGA nanoparticles

    Science.gov (United States)

    Kumar, Amit; Wonganan, Piyanuch; Sandoval, Michael A.; Li, Xinran; Zhu, Saijie; Cui, Zhengrong

    2012-01-01

    Previously, it was shown that microneedle-mediated transcutaneous immunization with plasmid DNA can potentially induce a stronger immune response than intramuscular injection of the same plasmid DNA. In the present study, we showed that the immune responses induced by transcutaneous immunization by applying plasmid DNA onto a skin area pretreated with solid microneedles were significantly enhanced by coating the plasmid DNA on the surface of cationic nanoparticles. In addition, the net surface charge of the DNA-coated nanoparticles significantly affected their in vitro skin permeation and their ability to induce immune responses in vivo. Transcutaneous immunization with plasmid DNA-coated net positively charged anoparticles elicited a stronger immune response than with plasmid DNA-coated net negatively charged nanoparticles or by intramuscular immunization with plasmid DNA alone. Transcutaneous immunization with plasmid DNA-coated net positively charged nanoparticles induced comparable immune responses as intramuscular injection of them, but transcutaneous immunization was able to induce specific mucosal immunity and a more balanced T helper type 1 and type 2 response. The ability of the net positively charged DNA-coated nanoparticles to induce a strong immune response through microneedle-mediated transcutaneous immunization may be attributed to their ability to increase the expression of the antigen gene encoded by the plasmid and to more effectively stimulate the maturation of antigen-presenting cells. PMID:22921518

  15. Pressure-induced structural evaluation and insulator-metal transition in the mixed spinel ferrite Z n0.2M g0.8F e2O4

    Science.gov (United States)

    Rahman, S.; Samanta, Sudeshna; Errandonea, D.; Yan, Shuai; Yang, Ke; Lu, Junling; Wang, Lin

    2017-01-01

    The effect of pressure on the electronic properties and crystal structure in a mixed spinel ferrite Z n0.2M g0.8F e2O4 was studied for the first time up to 48 GPa at room temperature using x-ray diffraction, Raman spectroscopy, and electrical transport measurements. The sample was cubic (spinel-type F d 3 ¯m ) at ambient pressure and underwent a pressure-induced structural transition to an orthorhombic phase (CaT i2O4-type B b m m ) at 21 GPa. This structural transformation corresponded to a first-order phase transition that involved 7.5% molar volume shrinkage. The onset of the Mott insulator-metal transition (IMT) around 20 GPa was due to a spin crossover mechanism that led to the F e3 + magnetic moment collapse. All the Raman modes disappeared at high pressures, which supported metallization. Analysis of structural and electrical transport measurements showed a simultaneous volume collapse and sharp IMT within a narrow pressure range. The orthorhombic high-pressure phase was found to have a higher conductivity than the cubic phase. The pressure dependence of the conductivity supported the metallic behavior of the high-pressure phase.

  16. Antiasthmatic activity of luteolin-7-O-glucoside from Ailanthus altissima through the downregulation of T helper 2 cytokine expression and inhibition of prostaglandin E2 production in an ovalbumin-induced asthma model.

    Science.gov (United States)

    Jin, Meihua; Yang, Ju Hye; Lee, Eunkyung; Lu, Yue; Kwon, Soonyoul; Son, Kun Ho; Son, Jong Keun; Chang, Hyeun Wook

    2009-09-01

    Previously, we reported that an ethanol extract of Ailanthus altissima has antiinflammatory activity in an ovalbumin (OVA)-sensitized murine asthmatic model. To determine the biological compounds from this plant, luteolin-7-O-glucoside (L7G) was isolated and its antiasthmatic activity was evaluated in an in vivo murine asthmatic model. L7G (10 to 100 mg/kg, per os (p.o.)) reduced the amount of eosinophil infiltration in bronchoalveolar lavage (BAL) fluid in a dose-dependent manner. In comparison, dexamethasone (5 mg/kg, p.o.), which was used as a positive control, also strongly inhibited the number of infiltrating eosinophils. L7G inhibited both the prostaglandin E(2) (PGE(2)) and serum immunoglobulin E level in BAL fluid in a dose-dependent manner. In addition, L7G inhibited the transcript profiles of interleukin (IL)-4, IL-5, and IL-13 mRNA expression levels in the murine asthma model, as determined using reverse transcription-polymerase chain reaction (RT-PCR). These results suggest that the antiasthmatic activity of L7G in OVA-induced lung inflammation may occur in part via the downregulation of T helper 2 cytokine transcripts as well as the inhibition of PGE(2) production.

  17. In Silico Detection and Typing of Plasmids using PlasmidFinder and Plasmid Multilocus Sequence Typing

    DEFF Research Database (Denmark)

    Carattoli, Alessandra; Zankari, Ea; García-Fernández, Aurora

    2014-01-01

    In the work presented here, we designed and developed two easy-to-use Web tools for in silico detection and characterization of whole-genome sequence (WGS) and whole-plasmid sequence data from members of the family Enterobacteriaceae. These tools will facilitate bacterial typing based on draft...... genomes of multidrug-resistant Enterobacteriaceae species by the rapid detection of known plasmid types. Replicon sequences from 559 fully sequenced plasmids associated with the family Enterobacteriaceae in the NCBI nucleotide database were collected to build a consensus database for integration...... sequences identified in the 559 fully sequenced plasmids. For plasmid multilocus sequence typing (pMLST) analysis, a database that is updated weekly was generated from www.pubmlst.org and integrated into a Web tool called pMLST. Both databases were evaluated using draft genomes from a collection...

  18. Cytosolic and Calcium-Independent Phospholipases A2 Activation and Prostaglandins E2 Are Associated with Escherichia coli-Induced Reduction of Insulin Secretion in INS-1E Cells.

    Science.gov (United States)

    Caporarello, Nunzia; Salmeri, Mario; Scalia, Marina; Motta, Carla; Parrino, Cristina; Frittitta, Lucia; Olivieri, Melania; Cristaldi, Martina; Avola, Roberto; Bramanti, Vincenzo; Toscano, Maria Antonietta; Anfuso, Carmelina Daniela; Lupo, Gabriella

    2016-01-01

    It is suspected that microbial infections take part in the pathogenesis of diabetes mellitus type 1 (T1DM). Glucose-induced insulin secretion is accompanied by the release of free arachidonic acid (AA) mainly by cytosolic- and calcium independent phospholipases A2 (cPLA2 and iPLA2). Insulinoma cell line (INS-1E) was infected with E. coli isolated from the blood culture of a patient with sepsis. Invasion assay, Scanning Electron Microscopy and Transmission Electron Microscopy demonstrated the capacity of E. coli to enter cells, which was reduced by PLA2 inhibitors. Glucose-induced insulin secretion was significantly increased after acute infection (8h) but significantly decreased after chronic infection (72h). PLA2 activities, cPLA2, iPLA2, phospho-cPLA2, and COX-2 expressions were increased after acute and, even more, after chronic E. coli infection. The silencing of the two isoforms of PLA2s, with specific cPLA2- or iPLA2-siRNAs, reduced insulin secretion after acute infection and determined a rise in insulin release after chronic infection. Prostaglandins E2 (PGE2) production was significantly elevated in INS-1E after long-term E. coli infection and the restored insulin secretion in presence of L798106, a specific EP3 antagonist, and NS-398, a COX-2 inhibitor, and the reduction of insulin secretion in presence of sulprostone, a specific EP3 agonist, revealed their involvement in the effects triggered by bacterial infection. The results obtained demonstrated that cPLA2 and iPLA2 play a key role in insulin secretion process after E. coli infection. The high concentration of AA released is transformed into PGE2, which could be responsible for the reduced insulin secretion.

  19. JNK suppression is essential for 17β-Estradiol inhibits prostaglandin E2-Induced uPA and MMP-9 expressions and cell migration in human LoVo colon cancer cells

    Directory of Open Access Journals (Sweden)

    Chen Wei-Kung

    2011-08-01

    Full Text Available Abstract Background Epidemiological studies demonstrate that the incidence and mortality rates of colorectal cancer in women are lower than in men. However, it is unknown if 17β-estradiol treatment is sufficient to inhibit prostaglandin E2 (PGE2-induced cellular motility in human colon cancer cells. Methods We analyzed the protein expression of urokinase plasminogen activator (uPA, tissue plasminogen activator (tPA, matrix metallopeptidases (MMPs, plasminogen activator inhibitor-1 (PAI-1 and tissue inhibitor of metalloproteinases (TIMPs, and the cellular motility in PGE2-stimulated human LoVo cells. 17β-Estradiol and the inhibitors including LY294002 (Akt activation inhibitor, U0126 (ERK1/2 inhibitor, SB203580 (p38 MAPK inhibitor, SP600125 (JNK1/2 inhibitor, QNZ (NFκB inhibitor and ICI 182 780 were further used to explore the inhibitory effects of 17β-estradiol on PGE2-induced LoVo cell motility. Student's t-test was used to analyze the difference between the two groups. Results Upregulation of urokinase plasminogen activator (uPA, tissue plasminogen activator (tPA and matrix metallopeptidases (MMPs is reported to associate with the development of cancer cell mobility, metastasis, and subsequent malignant tumor. After administration of inhibitors including LY294002, U0126, SB203580, SP600125 or QNZ, we found that PGE2 treatment up-regulated uPA and MMP-9 expression via JNK1/2 signaling pathway, thus promoting cellular motility in human LoVo cancer cells. However, PGE2 treatment showed no effects on regulating expression of tPA, MMP-2, plasminogen activator inhibitor-1 (PAI-1, tissue inhibitor of metalloproteinase-1, -2, -3 and -4 (TIMP-1, -2, -3 and -4. We further observed that 17β-estradiol treatment inhibited PGE2-induced uPA, MMP-9 and cellular motility by suppressing activation of JNK1/2 in human LoVo cancer cells. Conclusions Collectively, these results suggest that 17β-estradiol treatment significantly inhibits PGE2-induced motility

  20. Effect of 16.16 dimethyl prostaglandin E2, N-acetyl-cysteine and the proton pump inhibitor BY 831-78 on hydrogen peroxide-induced mucosal damage in the rat stomach.

    Science.gov (United States)

    Schürer-Maly, C C; Haussner, V; Halter, F

    1990-01-01

    Reactive oxygen species are noxious to gastrointestinal mucosa and contribute to a variety of gastrointestinal diseases. We examined whether 16.16 dimethyl prostaglandin E2 (PG) is protective against the oxidizing action of 6% H2O2 causing gross hemorrhagic lesions in rat gastric mucosa. Male Wistar rats were treated with PG, 0.005-5 micrograms/kg, either intragastrically (i.g.) or subcutaneously, 30 min prior to i.g. administration of 6% H2O2, 0.5 ml/100 g. Further animals received 25 mg of the mucus dissolvent N-acetyl-cystein (NAC) following oral PG treatment or 30 mumol/kg of the H+K(+)-ATPase inhibitor BY 831-78 (BY), 4 h before onset of the experiments. Volume, pH and beta-N-acetyl-glucosaminidase and lactate dehydrogenase as parameters of cell damage were determined in the gastric juice. i.g. PG treatment achieved 60 and 55% reduction of the mucosal lesions in doses between 5 and 0.05 micrograms/kg, respectively. i.p. PG administration was effective in all doses tested. Gastric juice volume was only slightly and enzymes were not significantly affected by PG treatment. NAC did not diminish PG efficacy or aggravate mucosal lesions. Gastric acid suppression did not increase PG-induced protection but was strongly protective by itself, reducing damage by 75%. Low-dose PG treatment achieves an effective protection against oxidative damage in gastric mucosa, which is not the result of dilution or enhanced mucus production.

  1. Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions.

    Science.gov (United States)

    Slamecka, Jaroslav; Salimova, Lilia; McClellan, Steven; van Kelle, Mathieu; Kehl, Debora; Laurini, Javier; Cinelli, Paolo; Owen, Laurie; Hoerstrup, Simon P; Weber, Benedikt

    2016-01-01

    Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or β-thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

  2. Plasmid and chromosome partitioning: surprises from phylogeny

    DEFF Research Database (Denmark)

    Gerdes, Kenn; Møller-Jensen, Jakob; Bugge Jensen, Rasmus

    2000-01-01

    Plasmids encode partitioning genes (par) that are required for faithful plasmid segregation at cell division. Initially, par loci were identified on plasmids, but more recently they were also found on bacterial chromosomes. We present here a phylogenetic analysis of par loci from plasmids and chr...

  3. Competing ParA structures space bacterial plasmids equally over the nucleoid.

    Directory of Open Access Journals (Sweden)

    Robert Ietswaart

    2014-12-01

    Full Text Available Low copy number plasmids in bacteria require segregation for stable inheritance through cell division. This is often achieved by a parABC locus, comprising an ATPase ParA, DNA-binding protein ParB and a parC region, encoding ParB-binding sites. These minimal components space plasmids equally over the nucleoid, yet the underlying mechanism is not understood. Here we investigate a model where ParA-ATP can dynamically associate to the nucleoid and is hydrolyzed by plasmid-associated ParB, thereby creating nucleoid-bound, self-organizing ParA concentration gradients. We show mathematically that differences between competing ParA concentrations on either side of a plasmid can specify regular plasmid positioning. Such positioning can be achieved regardless of the exact mechanism of plasmid movement, including plasmid diffusion with ParA-mediated immobilization or directed plasmid motion induced by ParB/parC-stimulated ParA structure disassembly. However, we find experimentally that parABC from Escherichia coli plasmid pB171 increases plasmid mobility, inconsistent with diffusion/immobilization. Instead our observations favor directed plasmid motion. Our model predicts less oscillatory ParA dynamics than previously believed, a prediction we verify experimentally. We also show that ParA localization and plasmid positioning depend on the underlying nucleoid morphology, indicating that the chromosomal architecture constrains ParA structure formation. Our directed motion model unifies previously contradictory models for plasmid segregation and provides a robust mechanistic basis for self-organized plasmid spacing that may be widely applicable.

  4. Direct and Auger Electron-Induced, Single- and Double-Strand Breaks on Plasmid DNA Caused by 99mTc-Labeled Pyrene Derivatives and the Effect of Bonding Distance

    Science.gov (United States)

    Reissig, Falco; Mamat, Constantin; Steinbach, Joerg; Pietzsch, Hans-Juergen; Freudenberg, Robert; Navarro-Retamal, Carlos; Caballero, Julio; Kotzerke, Joerg; Wunderlich, Gerd

    2016-01-01

    It is evident that 99mTc causes radical-mediated DNA damage due to Auger electrons, which were emitted simultaneously with the known γ-emission of 99mTc. We have synthesized a series of new 99mTc-labeled pyrene derivatives with varied distances between the pyrene moiety and the radionuclide. The pyrene motif is a common DNA intercalator and allowed us to test the influence of the radionuclide distance on damages of the DNA helix. In general, pUC 19 plasmid DNA enables the investigation of the unprotected interactions between the radiotracers and DNA that results in single-strand breaks (SSB) or double-strand breaks (DSB). The resulting DNA fragments were separated by gel electrophoresis and quantified by fluorescent staining. Direct DNA damage and radical-induced indirect DNA damage by radiolysis products of water were evaluated in the presence or absence of the radical scavenger DMSO. We demonstrated that Auger electrons directly induced both SSB and DSB in high efficiency when 99mTc was tightly bound to the plasmid DNA and this damage could not be completely prevented by DMSO, a free radical scavenger. For the first time, we were able to minimize this effect by increasing the carbon chain lengths between the pyrene moiety and the 99mTc nuclide. However, a critical distance between the 99mTc atom and the DNA helix could not be determined due to the significantly lowered DSB generation resulting from the interaction which is dependent on the type of the 99mTc binding motif. The effect of variable DNA damage caused by the different chain length between the pyrene residue and the Tc-core as well as the possible conformations of the applied Tc-complexes was supplemented with molecular dynamics (MD) calculations. The effectiveness of the DNA-binding 99mTc-labeled pyrene derivatives was demonstrated by comparison to non-DNA-binding 99mTcO4–, since nearly all DNA damage caused by 99mTcO4– was prevented by incubating with DMSO. PMID:27583677

  5. Reference: E2F1OSPCNA [PLACE

    Lifescience Database Archive (English)

    Full Text Available E2F1OSPCNA Kosugi S, Ohashi Y E2F sites that can interact with E2F proteins cloned from rice are require...d for meristematic tissue-specific expression of rice and tobacco proliferating cell nuclear antigen promoters Plant J 29: 45-59 (2002) PubMed: 12060226; ...

  6. Transcription-replication collision increases recombination efficiency between plasmids.

    Science.gov (United States)

    Jialiang, Li; Feng, Chen; Zhen, Xu; Jibing, Chen; Xiang, Lv; Lingling, Zhang; Depei, Liu

    2013-11-01

    It has been proposed that the stalling of the replication forks can induce homologous recombination in several organisms, and that arrested replication forks may offer nuclease targets, thereby providing a substrate for proteins involved in double-strand repair. In this article, we constructed a plasmid with the potential for transcription-replication collision (TRC), in which DNA replication and RNA transcription occur on the same DNA template simultaneously. Theoretically, transcription will impede DNA replication and increase homologous recombination. To validate this hypothesis, another plasmid was constructed that contained a homologous sequence with the exception of some mutated sites. Co-transfection of these two plasmids into 293T cells resulted in increased recombination frequency. The ratio of these two plasmids also affected the recombination frequency. Moreover, we found high expression levels of RAD51, which indicated that the increase in the recombination rate was probably via the homologous recombination pathway. These results indicate that mutant genes in plasmids can be repaired by TRC-induced recombination.

  7. Effect of excessive cadmium chloride on the plasmids of E. coli HB 101 in vivo

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    After Escherichia coli HB101 with plasmid pWH58, pWH98, or pTBa5 were cultered respectively in amp LB broth which contained 50 mg/L CdCl2 constantly for 24 h, these plasmids were isolated from E. coli, and the effect of excessive CdCl2 on the E. coli HB101 and plasmid DNA was studied by surveying the growth of E. coli HB101 and plasmid, argarose gel electrophoresis and analysis of restriction fragment length polymorphism (RFLP) of plasmids, and plasmid transformation. The results showed that 50 mg/L CdCl2 treatment lagged the growth of E. coli HB101 for at least 4h, but after grown for 24h there were not significant differences in the growths of E. coli HB101s and the productions of plasmids between the treatment and control. These results implified that E. coli HB101 have induced adaptability to cadmium stress and excessive CdCl2 did not inhibit the replication and amp+ gene's expression of plasmid DNA in vivo of E. coli significantly. 50 mg/L CdCl2 treatment for 24 hours might cause the sequence's change of plasmid DNA, but could not lead to the random breakage of plasmid DNA strands. Moreover, after 50 mg/L of CdCl2 treatment in vivo the transformation activities of plasmid did not altered, implied excessive CdCl2 could not affect the superhelical structure of plasmid and also not break the loop of plasmid DNA evidently.

  8. Pathogenicity of Vibrio anguillarum serogroup O1 strains compared to plasmids, outer membrane protein profiles and siderophore production

    DEFF Research Database (Denmark)

    Pedersen, K.; Gram, Lone; Austin, D.A.;

    1997-01-01

    The virulence of 18 strains of Vibrio anguillarum serogroup 01 was compared to plasmid content, expression of siderophores and outer membrane proteins. All strains, irrespective of plasmid content, produced siderophores and inducible outer membrane proteins under iron-limited conditions. Only...... strains that carried the 67 kbp virulence plasmid or derivatives of it produced the outer membrane protein, OM2. All virulent strains harboured the 67 kbp plasmid or derivatives of it, indicating its importance for virulence. However, some strains carrying the virulence plasmid or a derivative of it...

  9. Co-resident plasmids travel together.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    Conjugative plasmids encode genes that enable them to transfer, by conjugation, from a given host cell to another cell. Conjugative transfer, despite being an important feature of conjugative plasmids, is not constitutive for most plasmids, the reason being that genes involved in horizontal transfer are mostly repressed. Only upon their transient de-repression are plasmids able to transfer horizontally. If host cells harbour multiple plasmids, their simultaneous transfer depends on simultaneous transient de-repression of all plasmids. If de-repression of different plasmids was random and independent events, simultaneous de-repression should be a rare event because the probability of simultaneous de-repression would be the product of the probabilities of de-repression of each plasmid. Some previous observations support this hypothesis, while others show that co-transfer of plasmids is more frequent than this reasoning indicates. Here, we show that co-transfer of multiple plasmids mainly results from non-independent events: the probability that all plasmids within a cell become de-repressed is much higher than if de-repression of plasmids genes were independent. We found a simple model for the probability of co-transfer: the plasmid having the lowest conjugation rates is the one who limits co-transfer. In this sense, cells receiving the plasmid with the lower transfer rate also receive the other plasmid. If de-repression happens simultaneously on co-resident plasmids, common cues may stimulate de-repression of distinct plasmids. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. The Chlamydophila felis plasmid is highly conserved.

    Science.gov (United States)

    Harley, Ross; Day, Sarinder; Di Rocco, Camillo; Helps, Chris

    2010-11-20

    The presence of a plasmid in the Chlamydiaceae is both species and strain specific. Knowledge of the prevalence of the plasmid in different Chlamydia species is important for future studies aiming to investigate the role of the plasmid in chlamydial biology and disease. Although strains of Chlamydophila felis with or without the plasmid have been identified, only a small number of laboratory-adapted strains have been analysed and the prevalence of the plasmid in field isolates has not been determined. This study aimed to determine the prevalence of the plasmid in C. felis-positive conjunctival and oropharyngeal clinical samples submitted for routine diagnosis of C. felis by real-time (Q)PCR. DNA extracts from four laboratory-adapted strains were also analysed. QPCR assays targeting regions of C. felis plasmid genes pCF01, pCF02 and pCF03 were developed for the detection of plasmid DNA. QPCR analysis of DNA extracts from C. felis-positive clinical samples found evidence of plasmid DNA in 591 of 595 samples representing 561 of 564 (99.5%) clinical cases. Plasmid DNA was also detected by QPCR in laboratory-adapted strains 1497V, K2487 and K2490, but not strain 905. We conclude that the plasmid is highly conserved in C. felis, and plasmid-deficient strains represent a rare but important population for future studies of chlamydial plasmid function.

  11. Expression and Characterization of HGV E2 cDNA in Pichia pastoris

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    A 559 base pair fragment of cDNA locating at the putative E2 region of GBV-C/HGV was in-serted into Pichia pastoris expression vector pPICgK in the reading frame of α-factor secreting signal pep-tide. The recombinant expression plasmid pPIC9K-E2 was introduced into P. pastoris GSll5 with electro-poration and recombined with the host genome by homological recombination. The His+Mut+ recombinantyeasts were selected and cultivated in the BMMY medium. After 3 days induction with 0. 5% methanol,the target protein(E2) accumulated up to 30% of total proteins in the supernatant. The expressed E2 pro-tein was proved possessing antigenicity and high specificity with Western blot and ELISA probed with serafrom the immunized rabbits and the patients infected by GBV-C/HGV.

  12. 前列腺素E2在环氧合酶-2促胰腺癌新生血管生成中的介导作用%Cyclooxygenase-2 inducing angiogenesis in pancreatic carcinoma is mediated by prostaglandin E2

    Institute of Scientific and Technical Information of China (English)

    王兴鹏; 谢传高; 董育玮; 张汝玲; 吴丽颖; 吴凯

    2003-01-01

    目的探讨环氧合酶-2(COX-2)促进胰腺癌新生血管生成过程中前列腺素E2(PGE2)的介导作用,进一步揭示COX-2促进胰腺癌生长的机制.方法体外培养胰腺癌细胞株PC-3,分别应用酶联免疫吸附(ELISA)和放射免疫(RIA)等方法,检测选择性COX-2抑制剂Celebrex对胰腺癌PC-3细胞血管内皮生长因子(VEGF)和PGE2表达的调节作用,并观察外源性PGE2对Celebrex调节VEGF表达的干预.建立裸鼠PC-3细胞移植瘤,Western印迹检测Celebrex对胰腺癌组织VEGF表达的影响,RIA测定Celebrex对胰腺癌组织PGE2变化的调节.结果随着Celebrex作用浓度的提高以及作用时间的延长,PC-3细胞分泌的VEGF和PGE2受到抑制,呈时间和剂量依赖性.外源性PGE2显著上调Cele-brex作用后PC-3细胞VEGF蛋白表达,呈剂量依赖性,体内实验表明,Celebrex可显著抑制移植瘤组织VEGF和PGE2的表达.结论COX-2参与了PC-3细胞VEGF分泌的调节,进而促进胰腺癌新生血管形成,而PGE2则在该过程中起着重要的介导作用.

  13. PLASMIDS FROM ANAEROCELLUM THERMOPHILUM AND USES THEREOF

    DEFF Research Database (Denmark)

    2003-01-01

    The present invention concerns the isolation of plasmids from extremely thermophilic anaerobic microorganisms and their use in genetic transformation of thermophilic and mesophilic microorganisms. More particular the invention concerns the use of thermostable plasmid vectors as tools for creating...

  14. Evidence for plasmid-encoded virulence factors in the phytopathogenic bacterium Clavibacter michiganensis subsp. michiganensis NCPPB382.

    Science.gov (United States)

    Meletzus, D; Bermphol, A; Dreier, J; Eichenlaub, R

    1993-01-01

    The tomato pathogen Clavibacter michiganensis subsp. michiganensis NCPPB382, which causes bacterial wilt, harbors two plasmids pCM1 (27.5 kb) and pCM2 (72 kb). After curing of the plasmids, bacterial derivatives were still proficient in the ability to colonize the host plant and in the production of exopolysaccharides but exhibited a reduced virulence. When one of the two plasmids is lost, there is a significant delay in the development of wilting symptoms after infection and a plasmid-free derivative is not able to induce disease symptoms. By cloning of restriction fragments of both plasmids in the plasmid-free strain CMM100, two DNA fragments which restored the virulent phenotype were identified. Further analysis suggested that a fragment of plasmid pCM1 encodes an endocellulase which is involved in the expression of the pathogenic phenotype. Images PMID:8458855

  15. Frequency and diversity of small cryptic plasmids in the genus Rahnella

    Directory of Open Access Journals (Sweden)

    Summers David K

    2010-02-01

    Full Text Available Abstract Background Rahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids. Results In total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified. Conclusions For the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the

  16. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum.

    Science.gov (United States)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian; Tang, I-Ching

    2012-01-01

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at ~6.0.

  17. Effects of different replicons in conjugative plasmids on transformation efficiency, plasmid stability, gene expression and n-butanol biosynthesis in Clostridium tyrobutyricum

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Mingrui; Du, Yinming; Jiang, Wenyan; Chang, Wei-Lun; Yang, Shang-Tian [Ohio State Univ., Columbus, OH (United States). William G. Lowrie Dept. of Chemical and Biomolecular Engineering; Tang, I-Ching [Bioprocessing Innovative Company, Dublin, OH (United States)

    2012-01-15

    Clostridium tyrobutyricum ATCC 25755 can produce butyric acid, acetic acid, and hydrogen as the main products from various carbon sources. In this study, C. tyrobutyricum was used as a host to produce n-butanol by expressing adhE2 gene under the control of a native thiolase promoter using four different conjugative plasmids (pMTL82151, 83151, 84151, and 85151) each with a different replicon (pBP1 from C. botulinum NCTC2916, pCB102 from C. butyricum, pCD6 from Clostridium difficile, and pIM13 from Bacillus subtilis). The effects of different replicons on transformation efficiency, plasmid stability, adhE2 expression and aldehyde/alcohol dehydrogenase activities, and butanol production by different mutants of C. tyrobutyricum were investigated. Among the four plasmids and replicons studied, pMTL82151 with pBP1 gave the highest transformation efficiency, plasmid stability, gene expression, and butanol biosynthesis. Butanol production from various substrates, including glucose, xylose, mannose, and mannitol were then investigated with the best mutant strain harboring adhE2 in pMTL82151. A high butanol titer of 20.5 g/L with 0.33 g/g yield and 0.32 g/L h productivity was obtained with mannitol as the substrate in batch fermentation with pH controlled at {proportional_to}6.0. (orig.)

  18. Loop 7 of E2 enzymes

    DEFF Research Database (Denmark)

    Papaleo, Elena; Casiraghi, Nicola; Arrigoni, Alberto;

    2012-01-01

    The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3...

  19. Loop 7 of E2 enzymes

    DEFF Research Database (Denmark)

    Papaleo, Elena; Casiraghi, Nicola; Arrigoni, Alberto

    2012-01-01

    The ubiquitin (Ub) system controls almost every aspect of eukaryotic cell biology. Protein ubiquitination depends on the sequential action of three classes of enzymes (E1, E2 and E3). E2 Ub-conjugating enzymes have a central role in the ubiquitination pathway, interacting with both E1 and E3...... hydrophobic and acidic residues in a finely orchestrate mechanism....

  20. Main: E2F1OSPCNA [PLACE

    Lifescience Database Archive (English)

    Full Text Available E2F1OSPCNA S000396 21-May-2002 (last modified) uchi re2f-1 found in the promoter of rice PCN...ividing cells and tissue; E2F; PCNA; meristematic tissue; cell cycle; rice (Oryza sativa); tobacco (Nicotiana tabacum) GCGGGAAA ...

  1. Origin and Evolution of Rickettsial Plasmids.

    Science.gov (United States)

    El Karkouri, Khalid; Pontarotti, Pierre; Raoult, Didier; Fournier, Pierre-Edouard

    2016-01-01

    Rickettsia species are strictly intracellular bacteria that have undergone a reductive genomic evolution. Despite their allopatric lifestyle, almost half of the 26 currently validated Rickettsia species have plasmids. In order to study the origin, evolutionary history and putative roles of rickettsial plasmids, we investigated the evolutionary processes that have shaped 20 plasmids belonging to 11 species, using comparative genomics and phylogenetic analysis between rickettsial, microbial and non-microbial genomes. Plasmids were differentially present among Rickettsia species. The 11 species had 1 to 4 plasmid (s) with a size ranging from 12 kb to 83 kb. We reconstructed pRICO, the last common ancestor of the current rickettsial plasmids. pRICO was vertically inherited mainly from Rickettsia/Orientia chromosomes and diverged vertically into a single or multiple plasmid(s) in each species. These plasmids also underwent a reductive evolution by progressive gene loss, similar to that observed in rickettsial chromosomes, possibly leading to cryptic plasmids or complete plasmid loss. Moreover, rickettsial plasmids exhibited ORFans, recent gene duplications and evidence of horizontal gene transfer events with rickettsial and non-rickettsial genomes mainly from the α/γ-proteobacteria lineages. Genes related to maintenance and plasticity of plasmids, and to adaptation and resistance to stress mostly evolved under vertical and/or horizontal processes. Those involved in nucleotide/carbohydrate transport and metabolism were under the influence of vertical evolution only, whereas genes involved in cell wall/membrane/envelope biogenesis, cycle control, amino acid/lipid/coenzyme and secondary metabolites biosynthesis, transport and metabolism underwent mainly horizontal transfer events. Rickettsial plasmids had a complex evolution, starting with a vertical inheritance followed by a reductive evolution associated with increased complexity via horizontal gene transfer as well as

  2. Chlamydophila felis: plasmid detection in Italian isolates.

    Science.gov (United States)

    Di Francesco, Antonietta; Donati, Manuela; Salvatore, Daniela; Cevenini, Roberto; Di Paolo, Maria; Baldelli, Raffaella

    2010-04-01

    Plasmids have been detected in the majority of strains in the genus Chlamydia and in many Chlamydophila species. Previous studies showed that FP Pring and FP Cello Chlamydophila felis strains have an extrachromosomial plasmid, whereas the FP Baker strain does not. Azuma et al. recently sequenced the entire genomic DNA sequence of the Japanese Cp. felis strain Fe/C-56 and described a 7,552 base pair circular plasmid. In the present study a highly conserved plasmid gene was detected in 11 Italian Cp. felis isolates, showing 100% nucleotide identity with the plasmid gene of Fe/C-56 Cp. felis strain.

  3. Feedback regulation between atypical E2Fs and APC/CCdh1 coordinates cell cycle progression.

    Science.gov (United States)

    Boekhout, Michiel; Yuan, Ruixue; Wondergem, Annelotte P; Segeren, Hendrika A; van Liere, Elsbeth A; Awol, Nesibu; Jansen, Imke; Wolthuis, Rob M F; de Bruin, Alain; Westendorp, Bart

    2016-03-01

    E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

  4. MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer

    DEFF Research Database (Denmark)

    Christensen, Lise Lotte; Tobiasen, Heidi; Holm, Anja;

    2013-01-01

    -3p in a second independent cohort of 43 CRC patients, using single TaqMan® microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified...... as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel...

  5. An Asp49 Phospholipase A2 from Snake Venom Induces Cyclooxygenase-2 Expression and Prostaglandin E2 Production via Activation of NF-κB, p38MAPK, and PKC in Macrophages

    Directory of Open Access Journals (Sweden)

    Vanessa Moreira

    2014-01-01

    Full Text Available Phospholipases A2 (PLA2 are key enzymes for production of lipid mediators. We previously demonstrated that a snake venom sPLA2 named MT-III leads to prostaglandin (PGE2 biosynthesis in macrophages by inducing the expression of cyclooxygenase-2 (COX-2. Herein, we explored the molecular mechanisms and signaling pathways leading to these MT-III-induced effects. Results demonstrated that MT-III induced activation of the transcription factor NF-κB in isolated macrophages. By using NF-κB selective inhibitors, the involvement of this factor in MT-III-induced COX-2 expression and PGE2 production was demonstrated. Moreover, MT-III-induced COX-2 protein expression and PGE2 release were attenuated by pretreatment of macrophages with SB202190, and Ly294002, and H-7-dihydro compounds, indicating the involvement of p38MAPK, PI3K, and PKC pathways, respectively. Consistent with this, MT-III triggered early phosphorylation of p38MAPK, PI3K, and PKC. Furthermore, SB202190, H-7-dihydro, but not Ly294002 treatment, abrogated activation of NF-κB induced by MT-III. Altogether, these results show for the first time that the induction of COX-2 protein expression and PGE2 release, which occur via NF-κB activation induced by the sPLA2-MT-III in macrophages, are modulated by p38MAPK and PKC, but not by PI3K signaling proteins.

  6. Moist convection scheme in Model E2

    CERN Document Server

    Kim, Daehyun; Yao, Mao-Sung

    2013-01-01

    This documentation describes the version of the Del Genio - Yao cumulus parameterization used in the NASA Goddard Institute for Space Studies Model E2 GCM. This version was used for the official GISS submissions to the CMIP5 archive.

  7. Gyrase-dependent stabilization of pSC101 plasmid inheritance by transcriptionally active promoters.

    Science.gov (United States)

    Beaucage, S L; Miller, C A; Cohen, S N

    1991-09-01

    The pSC101 plasmid encodes a cis-acting genetic locus termed par that ensures the stable inheritance of plasmids in a population of dividing cells. In the absence of selection, par-defective plasmids are lost rapidly from the bacterial population. We report here that the stability of par-deleted pSC101 derivatives is restored by introducing certain adventitious bacterial promoters onto the plasmid. Stabilization requires active transcription from the inserted promoter and is affected by the site and orientation of the insertion, the length of the nascent transcript and DNA gyrase activity. While a promotor-associated overall increase in negative superhelicity of plasmid DNA was observed, stabilized inheritance appeared to be dependent on localized rather than generalized supercoiling. Our demonstration that promoter-induced DNA supercoiling can mimic the effects of the pSC101 par locus provides evidence that the previously reported superhelicity-generating effects of par are intrinsic to its function.

  8. Bifunctional effect of E2 on macrophage

    Institute of Scientific and Technical Information of China (English)

    MinHONG; QuanZHU

    2004-01-01

    AIM: Our previous study showed that the effect of 1713-estradiol(E2) on macrophage does not strengthen when concentrationincreased. So the effect of E2 on cytokines, intracellular free Ca2+([Ca2+]i) and morphological change of macrophages at differentconcentrations were studied. METHODS: TNF-α was measured by MTT via L929 cell. Nitrate and nitrite level(NO) wasmeasured by the method of Griess. [Ca2+]i was examined by laser scanning confocal microscopy(LSCM). Fluorescent microscopy

  9. Theory of (e, 2e) reactions

    Science.gov (United States)

    Byron, F. W.; Joachain, C. J.

    1989-08-01

    A comprehensive survey is given of the theory of (e, 2e) reactions. We begin by discussing the kinematics of these reactions, with special attention devoted to the coplanar asymmetric (Ehrhardt-type) geometry and the fully symmetric geometry in which most of the recent (e, 2e) coincidence measurements have been performed. We then review the foundations of the theory of the ionization of atoms by electron impact, first for one-electron atoms and then for target atoms with N electrons. Next, we discussed the Wannier theory of threshold ionization and its excitations. We then turn to the analysis of (e, 2e) reactions at intermediate and high energies. The theory of fast coplanar asymmetric (e, 2e) reactions is analyzed and it is shown that the eikonal-Born series method successfully accounts for all the dynamical features of these reactions. In particular, it is shown that second order effects are essential in explaining all the features exhibited by the measured by the measured triple differential cross sections at intermediate energies. Finally, we review the theory of fast symmetric (e, 2e) reactions. We consider first the (e, 2e) spectroscopy regime in which the momentum transfer Δ is large, but the recoil momentum Q of the ion is small or moderate. We then turn to the regime of large Δ and large Q, for which second order effects are of paramount importance, so that the coplanar symmetric triple differential cross section exhibits a striking behaviour in the large angle region.

  10. The muc+ gene of plasmid pKM101 prevents respiration shutoff in far ultraviolet-irradiated Salmonella typhimurium.

    Science.gov (United States)

    Swenson, P A

    1981-01-01

    The plasmid PKM101 is known to protect Escherichia coli and Salmonella typhimurium against killing by far UV irradiation and to enhance UV-induced mutagenesis. The muc+ gene of the plasmid is responsible for both of these effects. This paper shows that respiration of S. typhimurium shuts off about an hour after UV irradiation and that pKM101 prevents the shutoff. Plasmids which contained Tn5 translocatable elements, either in (and having produced a muc mutation) or flanking the muc+ gene, have been introduced into S. typhimurium. The muc mutant plasmid, which does not protect its host against UV killing and does not enhance UV induced mutagenesis, also does not protect against UV induced respiration shutoff. Likewise, plasmids in which the Tn5 translocatable elements flank the muc+ gene protect against shutoff of respiration. Thus the muc+ gene of pKM101 is responsible for protection against UV induced shutoff of respiration in S. typhimurium.

  11. E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes

    OpenAIRE

    Di Stefano, Luisa; Jensen, Michael Rugaard; Helin, Kristian

    2003-01-01

    The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7...

  12. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Science.gov (United States)

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

  13. Large scale genotype comparison of human papillomavirus E2-host interaction networks provides new insights for e2 molecular functions.

    Directory of Open Access Journals (Sweden)

    Mandy Muller

    Full Text Available Human Papillomaviruses (HPV cause widespread infections in humans, resulting in latent infections or diseases ranging from benign hyperplasia to cancers. HPV-induced pathologies result from complex interplays between viral proteins and the host proteome. Given the major public health concern due to HPV-associated cancers, most studies have focused on the early proteins expressed by HPV genotypes with high oncogenic potential (designated high-risk HPV or HR-HPV. To advance the global understanding of HPV pathogenesis, we mapped the virus/host interaction networks of the E2 regulatory protein from 12 genotypes representative of the range of HPV pathogenicity. Large-scale identification of E2-interaction partners was performed by yeast two-hybrid screenings of a HaCaT cDNA library. Based on a high-confidence scoring scheme, a subset of these partners was then validated for pair-wise interaction in mammalian cells with the whole range of the 12 E2 proteins, allowing a comparative interaction analysis. Hierarchical clustering of E2-host interaction profiles mostly recapitulated HPV phylogeny and provides clues to the involvement of E2 in HPV infection. A set of cellular proteins could thus be identified discriminating, among the mucosal HPV, E2 proteins of HR-HPV 16 or 18 from the non-oncogenic genital HPV. The study of the interaction networks revealed a preferential hijacking of highly connected cellular proteins and the targeting of several functional families. These include transcription regulation, regulation of apoptosis, RNA processing, ubiquitination and intracellular trafficking. The present work provides an overview of E2 biological functions across multiple HPV genotypes.

  14. MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

    Science.gov (United States)

    Christensen, Lise Lotte; Tobiasen, Heidi; Holm, Anja; Schepeler, Troels; Ostenfeld, Marie S; Thorsen, Kasper; Rasmussen, Mads H; Birkenkamp-Demtroeder, Karin; Sieber, Oliver M; Gibbs, Peter; Lubinski, Jan; Lamy, Philippe; Laurberg, Søren; Oster, Bodil; Hansen, Kristian Q; Hagemann-Madsen, Rikke; Byskov, Kristina; Ørntoft, Torben F; Andersen, Claus L

    2013-07-01

    Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.

  15. Persistence of Antibiotic Resistance Plasmids in Biofilms

    Science.gov (United States)

    2014-10-01

    plasmids* in*populations*of* Gram > negative *bacteria*grown*in*biofilms*and*well>mixed*liquid*cultures.** * Task2:*Characterize*the*evolution*of*plasmid...R.! Edwards.! 2005.! Overview! of! nosocomial! infections! caused! by! gramP negative ! bacilli .!Clin.!Infect.!Dis.!41:848P854.! LoftiePEaton,!W.,!A... negative ! interaction!between!one!of! its!chromosomal!segments!and!the!plasmid! by!simply!deleting!the!appropriate!chromosomal!segment.!! 7. None

  16. Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

    Directory of Open Access Journals (Sweden)

    H.G.P. van Gennip

    2008-01-01

    Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vfl c36BVDVII 684–796 seemed to be reduced compared to vfl c9 (with the complete antigenic region of BVDVII.

  17. Mechanism of prostaglandin (PG)E2-induced prolactin expression in human T cells: cooperation of two PGE2 receptor subtypes, E-prostanoid (EP) 3 and EP4, via calcium- and cyclic adenosine 5'-monophosphate-mediated signaling pathways.

    Science.gov (United States)

    Gerlo, Sarah; Verdood, Peggy; Gellersen, Birgit; Hooghe-Peters, Elisabeth L; Kooijman, Ron

    2004-11-15

    We previously reported that prolactin gene expression in the T-leukemic cell line Jurkat is stimulated by PGE(2) and that cAMP acts synergistically with Ca(2+) or protein kinase C on the activation of the upstream prolactin promoter. Using the transcription inhibitor actinomycin D, we now show that PGE(2)-induced prolactin expression requires de novo prolactin mRNA synthesis and that PGE(2) does not influence prolactin mRNA stability. Furthermore, PGE(2)-induced prolactin expression was inhibited by protein kinase inhibitor fragment 14-22 and BAPTA-AM, which respectively, inhibit protein kinase A- and Ca(2+)-mediated signaling cascades. Using specific PGE(2) receptor agonists and antagonists, we show that PGE(2) induces prolactin expression through engagement of E-prostanoid (EP) 3 and EP4 receptors. We also found that PGE(2) induces an increase in intracellular cAMP concentration as well as intracellular calcium concentration via EP4 and EP3 receptors, respectively. In transient transfections, 3000 bp flanking the leukocyte prolactin promoter conferred a weak induction of the luciferase reporter gene by PGE(2) and cAMP, whereas cAMP in synergy with ionomycin strongly activated the promoter. Mutation of a C/EBP responsive element at -214 partially abolished the response of the leukocyte prolactin promoter to PGE(2), cAMP, and ionomycin plus cAMP.

  18. Plasmid profiles of Moraxella bovis isolates.

    Science.gov (United States)

    McDonald, T J; Pugh, G W

    1986-04-01

    Two-hundred isolates of Moraxella bovis were selected at random and examined for the presence of plasmid DNA by a rapid alkaline-detergent lysis method. All isolates contained from 1 to 6 plasmids, with varying agarose-gel electrophoretic migration patterns. Most (80%) isolates carried 2 to 4 plasmids, which ranged in molecular weight from 2.6 to 80 megadaltons. Seemingly, plasmid profiles can be used as a simple, reliable epizootiologic tool to establish a strain identification scheme for M bovis.

  19. Plasmid transfer systems in the rhizobia.

    Science.gov (United States)

    Ding, Hao; Hynes, Michael F

    2009-08-01

    Rhizobia are agriculturally important bacteria that can form nitrogen-fixing nodules on the roots of leguminous plants. Agricultural application of rhizobial inoculants can play an important role in increasing leguminous crop yields. In temperate rhizobia, genes involved in nodulation and nitrogen fixation are usually located on one or more large plasmids (pSyms) or on symbiotic islands. In addition, other large plasmids of rhizobia carry genes that are beneficial for survival and competition of rhizobia in the rhizosphere. Conjugative transfer of these large plasmids thus plays an important role in the evolution of rhizobia. Therefore, understanding the mechanism of conjugative transfer of large rhizobial plasmids provides foundations for maintaining, monitoring, and predicting the behaviour of these plasmids during field release events. In this minireview, we summarize two types of known rhizobial conjugative plasmids, including quorum sensing regulated plasmids and RctA-repressed plasmids. We provide evidence for the existence of a third type of conjugative plasmid, including pRleVF39c in Rhizobium leguminosarum bv. viciae strain VF39SM, and we provide a comparison of the different types of conjugation genes found in members of the rhizobia that have had their genomes sequenced so far.

  20. Effect of chromosome homology an plasmid transformation and plasmid conjugal transfer in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Balganesh, M.; Setlow, J.K.

    1984-05-14

    The pairing between plasmid and the homologous part of the chromosome associated with plasmid establishment may differ from the pairing which results from integration of a homologous region of the plasmid into the chromosome. Thus the rate of novobiocin transformation decreases with duplication of the chromosomal portion in pMB2, but the rate of establishment of the plasmid increases with this duplication. A model to explain these data is given. 17 references, 5 figures, 4 tables.

  1. The novel triterpenoid RTA 408 protects human retinal pigment epithelial cells against H2O2-induced cell injury via NF-E2-related factor 2 (Nrf2 activation

    Directory of Open Access Journals (Sweden)

    Xiaobin Liu

    2016-08-01

    Full Text Available Oxidative stress-induced retinal pigment epithelial (RPE cell damage is an important factor in the pathogenesis of age-related macular degeneration (AMD. Previous studies have shown that RTA 408, a synthetic triterpenoid compound, potently activates Nrf2. This study aimed to investigate the protective effects of RTA 408 in cultured RPE cells during oxidative stress and to determine the effects of RTA 408 on Nrf2 and its downstream target genes. Primary human RPE cells were pretreated with RTA 408 and then incubated in 200 μM H2O2 for 6 h. Cell viability was measured with the WST-8 assay. Apoptosis was quantitatively measured by annexin V/propidium iodide (PI double staining and Hoechst 33342 fluorescent staining. Reduced (GSH and oxidized glutathione (GSSG were measured using colorimetric assays. Nrf2 activation and its downstream effects on phase II enzymes were examined by Western blot. Treatment of RPE cells with nanomolar ranges (10 and 100 nM of RTA 408 markedly attenuated H2O2-induced viability loss and apoptosis. RTA 408 pretreatment significantly protected cells from oxidative stress-induced GSH loss, GSSG formation and decreased ROS production. RTA 408 activated Nrf2 and increased the expression of its downstream genes, such as HO-1, NQO1, SOD2, catalase, Grx1, and Trx1. Consequently, the enzyme activities of NQO1, Grx1, and Trx1 were fully protected by RTA 408 pretreatment under oxidative stress. Moreover, knockdown of Nrf2 by siRNA significantly reduced the cytoprotective effects of RTA 408. In conclusion, our data suggest that RTA 408 protect primary human RPE cells from oxidative stress-induced damage by activating Nrf2 and its downstream genes.

  2. The expression of a plasmid-specified exported protein causes structural plasmid instability in Bacillus subtilis

    NARCIS (Netherlands)

    Cordes, C.; Meima, R; Twiest, B; Kazemier, B; Venema, G; vanDijl, JM; Bron, S

    The rolling-circle plasmid pGP1 was used to study the effects of the expression of a plasmid-specified exported protein on structural plasmid stability in Bacillus subtilis. pGP1 contains a fusion between the Bacillus licheniformis penP gene, encoding a C-terminally truncated penicillinase, and the

  3. Stress responses in pathogenic Yersinia enterocolitica with reference to the stability of the virulence plasmid in food

    Science.gov (United States)

    Yersinia enterocolitica has been associated with food-borne illness, most often due the ingestion of pork products. The pathogenic effects induced by a Y. enterocolitica infection are caused by the interplay of chromosomal genes and a virulence plasmid, pYV. Generally, the plasmid is lost during g...

  4. Effects of E2 and E1-E2 Interference on Coulomb Dissociation of 19C

    Institute of Scientific and Technical Information of China (English)

    Rajesh Kharab; Pardeep Singh; Ravinder Kumar

    2007-01-01

    We investigate the effects of higher order multipole transitions, in particular electric quadrupole (E2) and E1-E2interference, on the Coulomb dissociation of 19 C within the framework of the first order eikonal approximation.The sensitivity of the total Coulomb breakup cross section and the longitudinal momentum distribution of the core fragment to these effects are checked. The breakup occurs predominately through the dipole transition and the contribution of E2 transition to the total cross section is found to be within the range from 1 to 3% of that of E1. It is further observed that the E1-E2 interference term contributes nothing to the integrated cross section.On the other hand, the longitudinal momentum distribution is observed to be insensitive to the E2 transition while the E1-E2 interference introduces a small asymmetry in its shape.

  5. The Salmonella enterica Serotype Typhimurium Effector Proteins SipA, SopA, SopB, SopD, and SopE2 Act in Concert To Induce Diarrhea in Calves

    OpenAIRE

    2002-01-01

    Salmonella enterica serotype Typhimurium requires a functional type III secretion system encoded by Salmonella pathogenicity island 1 (SPI1) to cause diarrhea. We investigated the role of genes encoding secreted target proteins of the SPI1-associated type III secretion system for enteropathogenicity in calves. Salmonella serotype Typhimurium strains having mutations in sptP, avrA, sspH1, or slrP induced fluid secretion in the bovine ligated ileal loop model at levels similar to that of the wi...

  6. Media of rat macrophage NR8383 cells with prostaglandins E2-induced VEGF over-expression promotes migration and tube formation of human umbilical vein endothelial cells%前列腺素E2对大鼠巨噬细胞株NR8383合成血管内皮生长因子促进人脐静脉血管内皮细胞成管、迁移的影响

    Institute of Scientific and Technical Information of China (English)

    刘勉; 龚艺; 韦锦燕; 谢多; 王京; 余艳红; 全松

    2016-01-01

    Objective To investigate the effect of prostaglandins E2 (PGE2) in enhancing vascular endothelial growth factor (VEGF) expression in a rat macrophage cell line and the effect of the media from PGE2-inuced rat macrophages on angiogenetic ability of human umbilical vein endothelial cells (HUVECs) in vitro. Methods Western blotting and qPCR were employed to investigate the expressions of VEGF protein and mRNAs in rat macrophage cell line NR8383 stimulated by PGE2 in the presence or absence of EP2 receptor inhibitor (AH6809) and EP4 receptor inhibitor (AH23848). Conditioned supernatants were obtained from different NR8383 subsets to stimulate HUVECs, and the tube formation ability and migration of the HUVECs were assessed with Transwell assay. Results PGE2 stimulation significantly enhanced the expression of VEGF protein and mRNAs in NR8383 cells in a dose-dependent manner. The supernatants from NR8383 cells stimulated by PGE2 significantly enhanced tube formation ability of HUVECs (P<0.05) and promoted the cell migration. Such effects of PGE2 were blocked by the application of AH6809 and AH23848. Conclusion PGE2 can dose-dependently increase VEGF expression in NR8383 cells, and the supernatants derived from PGE2-stimulated NR8383 cells can induce HUVEC migration and accelerate the growth of tube like structures. PGE2 are essential to corpus luteum formation by stimulating macrophages to induce angiogenesis through EP2/EP4.%目的:探究前列腺素E2(PGE2)对大鼠巨噬细胞株NR8383细胞合成血管内皮生长因子(VEGF)的调控作用以及对人脐静脉内皮细胞(HUVEC)趋化成管的影响。方法分别采用0.1 nmol/L PGE2、1 nmol/L PGE2、1 nmol/L PGE2+10 nmol/L EP2受体抑制剂AH6809+10 nmol/L EP4受体抑制剂AH23848处理的NR8383细胞作为各实验组,选择未经PGE2以及其特异性受体抑制剂处理的NR8383细胞作为对照组,采用Western blot和qPCR方法检测各组NR8383细胞内VEGF蛋白以及mRNA的表达水

  7. Inhibition of E2F-mediated transcription by p202.

    Science.gov (United States)

    Choubey, D; Li, S J; Datta, B; Gutterman, J U; Lengyel, P

    1996-01-01

    Many of the antimicrobial, immunomodulatory and cell growth inhibitory activities of the interferons are mediated by interferon-inducible proteins. Earlier we characterized an interferon-inducible murine protein, p202, whose expression in transfected cells inhibits cell proliferation and which can form a complex with retinoblastoma protein (pRb). Here we report that in transfected cells expression of p202 inhibits E2F-stimulated transcription of a reporter gene and of endogenous genes. Inhibition of the transcriptional activity of E2F by p202 does not depend on fully functional pRb and is correlated with inhibition of the sequence-specific DNA binding of E2F. p202 interacts with the transcription factor E2F (E2F-1/DP-1) in vitro and in vivo. Inhibition of E2F activity by p202 may contribute to growth inhibition by the interferons. Images PMID:8896460

  8. Characterization of E2F8, a novel E2F-like cell-cycle regulated repressor of E2F-activated transcription

    OpenAIRE

    Christensen, Jesper; Cloos, Paul; Toftegaard, Ulla; Klinkenberg, David; Bracken, Adrian P.; Trinh, Emmanuelle; Heeran, Mel; Di Stefano, Luisa; Helin, Kristian

    2005-01-01

    The E2F family of transcription factors are downstream effectors of the retinoblastoma protein, pRB, pathway and are essential for the timely regulation of genes necessary for cell-cycle progression. Here we describe the characterization of human and murine E2F8, a new member of the E2F family. Sequence analysis of E2F8 predicts the presence of two distinct E2F-related DNA binding domains suggesting that E2F8 and, the recently, identified E2F7 form a subgroup within the E2F family. We show th...

  9. E2F-4 and E2F-5, two members of the E2F family, are expressed in the early phases of the cell cycle.

    OpenAIRE

    Sardet, C; Vidal, M.; Cobrinik, D; Y. Geng; Onufryk, C; Chen, A; Weinberg, R A

    1995-01-01

    The E2F transcription factors play a role in regulating the expression of genes required for cell proliferation. Their activity appears to be regulated by association with the retinoblastoma protein (pRb) and the pRb-related proteins p107 and p130. In vivo, pRb is found in complex with a subset of E2F components--namely, E2F-1, E2F-2, and E2F-3. Here we describe the characterization of cDNAs encoding two unusual E2Fs, E2F-4 and E2F-5, each identified by the ability of their gene product to in...

  10. E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes.

    Science.gov (United States)

    Di Stefano, Luisa; Jensen, Michael Rugaard; Helin, Kristian

    2003-12-01

    The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7 results in suppression of E2F target genes and accumulation of cells in G1. Furthermore, E2F7 associates with E2F-regulated promoters in vivo, and this association increases in S phase. Interestingly, however, E2F7 binds only a subset of E2F-dependent promoters in vivo, and in agreement with this, inhibition of E2F7 expression results in specific derepression of these promoters. Taken together, these data demonstrate that E2F7 is a unique repressor of a subset of E2F target genes whose products are required for cell cycle progression.

  11. RepA and RepB exert plasmid incompatibility repressing the transcription of the repABC operon.

    Science.gov (United States)

    Pérez-Oseguera, Angeles; Cevallos, Miguel A

    2013-11-01

    Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.

  12. 猪瘟病毒E2基因自杀性DNA疫苗的构建及动物免疫试验%Construction of suicidal DNA vaccine of CSFV E2 gene and immunity induction in animals

    Institute of Scientific and Technical Information of China (English)

    郭抗抗; 温肖会; 汤智慧; 侯勃; 张彦明; 谢林红; 王晶钰

    2011-01-01

    从含有猪瘟病毒(CSFV)Shimen株E2全长基因的重组质粒pMD18-T-E2上扩增得到带有BamHⅠ酶切位点的E2基因片段,构建重组克隆质粒pMD19-T-E2.用BamH Ⅰ酶切pMD19-T-E2和"自杀性"DNA疫苗表达载体pSCA1,构建"自杀性"DNA疫苗重组质粒pSCA1-E2.对目的基因E2的大小、插入位置、方向和读码框经PCR、酶切和序列测定进行鉴定.用纯化的阳性质粒pSCA1-E2转染PK-15细胞,转染后48 h荧光抗体法检测E2蛋白的表达;纯化的pSCA1-E2质粒分别免疫小鼠(10 μg/只)和试验猪(600μg/头),二免后分别检测小鼠脾淋巴细胞刺激指数和猪血清抗猪瘟特异性抗体水平.结果表明,构建的重组质粒中E2基因的大小、插入位置、方向和读码框均正确,获得了重组表达质粒pSCA1-E2;转染后48 h可检测到转染的PK-15细胞中E2蛋白的表达.免疫小鼠产生较高水平的淋巴细胞刺激指数,免疫猪可检测到抗CSFV特异性血清抗体;表明构建的pSCA1-E2重组质粒能激发实验动物的免疫反应.%The complete E2 gene of classical swine fever virus (CSFV) with the BamH I restriction site was cloned from the recombinant plasmid pMD18-T-E2 and inserted into pMD19-T plasmid to construct the recombinant plas-mid pMD19-T-E2. E2 gene was obtained from pMD19-T-E2 digested with BamH I ,and was inserted into the suicidal DNA vaccine vector pSCAl which was treated with the same enzyme. The recombinant plasmid pSCAl-E2 was obtained and identified the length. Insert sites, orientation and reading frame of E2 gene in pSCAl-E2 by polymerase chain reaction (PCR),enzyme digesting and sequencing. E2 protein was determined in PK15 cells transfected with purified recombinant plasmid pSCAl-E2 at 48 h post-transfection. The stimulation index (SI) of T lymphoid cell in experimental mice immunized with pSCAl-E2 (10 μg/each) were analyzed at 10 d,20 d,30 d after the second vaccination. Experimental pigs were immunized with pSCAl-E2 (600 μg/each) and

  13. E2F-5, a new E2F family member that interacts with p130 in vivo

    NARCIS (Netherlands)

    Hijmans, E.M.; Voorhoeve, P.M.; Beijersbergen, R.L.; Veer, L.J. van 't; Bernards, R.A.

    1995-01-01

    E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2

  14. Accurate determination of plasmid copy number of flow-sorted cells using droplet digital PCR.

    Science.gov (United States)

    Jahn, Michael; Vorpahl, Carsten; Türkowsky, Dominique; Lindmeyer, Martin; Bühler, Bruno; Harms, Hauke; Müller, Susann

    2014-06-17

    Many biotechnological processes rely on the expression of a plasmid-based target gene. A constant and sufficient number of plasmids per cell is desired for efficient protein production. To date, only a few methods for the determination of plasmid copy number (PCN) are available, and most of them average the PCN of total populations disregarding heterogeneous distributions. Here, we utilize the highly precise quantification of DNA molecules by droplet digital PCR (ddPCR) and combine it with cell sorting using flow cytometry. A duplex PCR assay was set up requiring only 1000 sorted cells for precise determination of PCN. The robustness of this method was proven by thorough optimization of cell sorting, cell disruption, and PCR conditions. When non plasmid-harboring cells of Pseudomonas putida KT2440 were spiked with different dilutions of the expression plasmid pA-EGFP_B, a PCN from 1 to 64 could be accurately detected. As a proof of principle, induced cultures of P. putida KT2440 producing an EGFP-fused model protein by means of the plasmid pA-EGFP_B were investigated by flow cytometry and showed two distinct subpopulations, fluorescent and nonfluorescent cells. These two subpopulations were sorted for PCN determination with ddPCR. A remarkably diverging plasmid distribution was found within the population, with nonfluorescent cells showing a much lower PCN (≤1) than fluorescent cells (PCN of up to 5) under standard conditions.

  15. E2-25K SUMOylation inhibits proteasome for cell death during cerebral ischemia/reperfusion

    Science.gov (United States)

    Jeong, Eun Il; Chung, Hae Won; Lee, Won Jea; Kim, Seo-Hyun; Kim, Hyunjoo; Choi, Seon-Guk; Jung, Yong-Keun

    2016-01-01

    Cerebral ischemia/reperfusion (I/R) causes brain damage accompanied by ubiquitin accumulation and impairment of proteasome activity. In this study, we report that E2-25K, an E2-conjugating enzyme, is SUMOylated during oxidative stress and regulates cerebral I/R-induced damage. Knockdown of E2-25K expression protects against oxygen/glucose deprivation and reoxygenation (OGD/R)-induced neuronal cell death, whereas ectopic expression of E2-25K stimulates it. Compared with the control mice, cerebral infarction lesions and behavioral/neurological disorders are ameliorated in E2-25K knockout mice during middle cerebral artery occlusion and reperfusion. In particular, E2-25K is SUMOylated at Lys14 under oxidative stress, OGD/R and I/R to prompt cell death. Further, E2-25K downregulates the proteasome subunit S5a to impair proteasome complex and thus restrain proteasome activity under oxidative stress. This proteasome inhibitory activity of E2-25K is dependent on its SUMOylation. These results suggest that E2-25K has a crucial role in oxidative stress and cerebral I/R-induced damage through inhibiting proteasome via its SUMOylation. PMID:28032866

  16. Zyflamend对口腔黏膜癌变过程中前列腺素E2和白三烯B4水平影响的动物实验%Detection of prostaglandin E2 and leukotriene B4 in 7,12-dimethyibenz (a) anthracene-induced oral pre-cancerous lesions in hamsters treated with Zyflamend by liquid chromatographic tandem mass spectrometry

    Institute of Scientific and Technical Information of China (English)

    李芳; 孙正

    2011-01-01

    目的 通过测定抗炎天然植物混合剂Zyflamend对地鼠口腔黏膜癌变过程中花生四烯酸代谢产物前列腺素( prostaglandin,PG) E2和白三烯B4水平的影响,探讨其可能的作用机制.方法 将27只金黄地鼠分为5组,阴性对照组3只,不涂药;其余24只用0.5%的二甲基苯并蒽( dimethylbenzanthracene,DMBA)涂于左侧颊囊3次/周,3周后将其按随机数字表随机分为4组(每组6只),阳性对照组不涂药,其余3组金黄地鼠分别于左侧颊囊分别涂低、中、高浓度Zyflamend(稀释比例分别为1∶3、1∶1和原液).每周涂药3次,第4周末处死动物取左侧颊囊速冻于液氮中行液相色谱-串联质谱法(liquid chromatographic tandem mass spectrometry,LC-MS/MS)分析.结果 阳性对照组PGE2为(0.377±0.290) μg/g,低、中、高浓度Zyflamend组的PGE2水平分别为(0.523±0.286)、(0.488±0.147)和(0.774±0.314) μg/g,低、中浓度组与阳性对照组相比差异无统计学意义(P>0.05),高浓度组与阳性对照组相比差异有统计学意义(P<0.05).与阳性对照组(0.135±0.046) μg/g相比,低、中、高浓度组白三烯B4水平均显著降低(P<0.05),分别为(0.094±0.066)、(0.096±0.077)和(0.067±0.012) μg/g.结论 癌前病变的早期炎症阶段,Zyflamend能够抑制5-脂氧合酶途径中代谢产物白三烯B4的水平.%Objective To determine the level of prostaglandin E2 (PGE2 ) and leukotriene B4 (LTB4 ) after treatment with a unique anti-inflammatory herbal preparation (Zyflamend) in 7,12-dimethylbenz(a) anthracene (DMBA)-induced oral precancerous lesions in golden Syrian hamsters.Methods The hamsters were divided into five groups.The Syrian hamsters were applied with 0.5% DMBA solution topically to the left cheek pouch three times per week for three consecutive weeks.The negative control group was not treated.After the last treatment by DMBA,the 0.5% DMBA treated hamsters were divided into four groups at random.The positive group was

  17. The Human p73 Promoter: Characterization and Identification of Functional E2F Binding Sites

    Directory of Open Access Journals (Sweden)

    Ratnam S. Seelan

    2002-01-01

    Full Text Available p73, a member of the p53 family, is overexpressed in many cancers. To understand the mechanism(s underlying this overexpression, we have undertaken a detailed characterization of the human p73 promoter. The promoter is strongly activated in cells expressing exogenous E2F1 and suppressed by exogenous Rb. At least three functional E2F binding sites, located immediately upstream of exon 1 (at-284,-155 and-132 mediate this induction. 5' serially deleted promoter constructs and constructs harboring mutated E2F sites were analyzed for their response to exogenously expressed E2F1 or Rb to establish functionality of these sites. Authenticity of E2F sites was further confirmed by electrophoretic mobility shift assay (EMSA using E2F1 /DP1 heterodimers synthesized in vitro, followed by competition assays with unlabeled wild-type or mutant oligonucleotides and supershift analysis using anti-E2F1 antibodies. In vivo binding of E2F1 to the p73 promoter was demonstrated using nuclear extracts prepared from E2F1-inducible Saos2 cells. The region conferring the highest promoter activity was found to reside between-113 to-217 of the p73 gene. Two of the three functional E2F sites (at-155 and-132 reside within this region. Our results suggest that regulation of p73 expression is primarily mediated through binding of E2 F1 to target sites at-155 and-132.

  18. Regulation of human genome expression and RNA splicing by human papillomavirus 16 E2 protein.

    Science.gov (United States)

    Gauson, Elaine J; Windle, Brad; Donaldson, Mary M; Caffarel, Maria M; Dornan, Edward S; Coleman, Nicholas; Herzyk, Pawel; Henderson, Scott C; Wang, Xu; Morgan, Iain M

    2014-11-01

    Human papillomavirus 16 (HPV16) is causative in human cancer. The E2 protein regulates transcription from and replication of the viral genome; the role of E2 in regulating the host genome has been less well studied. We have expressed HPV16 E2 (E2) stably in U2OS cells; these cells tolerate E2 expression well and gene expression analysis identified 74 genes showing differential expression specific to E2. Analysis of published gene expression data sets during cervical cancer progression identified 20 of the genes as being altered in a similar direction as the E2 specific genes. In addition, E2 altered the splicing of many genes implicated in cancer and cell motility. The E2 expressing cells showed no alteration in cell growth but were altered in cell motility, consistent with the E2 induced altered splicing predicted to affect this cellular function. The results present a model system for investigating E2 regulation of the host genome.

  19. Study on the construction of recombinant plasmid coexpressing newcastle disease virus F protein and chicken IL-2

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    This study investigated the protection against the ND in chickens by a recombinant DNA vaccine. A plasmid vector encoding NDV F protein, which is reqired for virus cell fusion and is important for vaccine induced immunity, was used as a model to study how DNA vaccines may be modulated by the simulaneous expression of chicken IL-2. The NDV D26 strain F gene with CMV promotor and BGH polyA signal sequence was amplified by PCR from eukaryotic plasmid pcDNA-F, which contains the full-length NDV F gene, and clond into reconstructed eukaryotic plasmid pcDNA-IL2, which contains chicken IL-2 gene. Restriction endonuclease cleavage and PCR amplification showed that a bicistronic plasmid encoding NDV F gene and chicken IL-2 separately was successfully constructed. Two-week-old SPF chickens were intramuscularly innoculated the recombinant plasmid. Antibody and lymphocyte proliferative assay showed that the humoral and cellular immunity of chickens vaccinated the recombinant plasmid greatly increased compared with those innoculated only plasmid expressing NDV F protein. Challenged with the lethal dose of NDV F48E9 strain, 72% chickens vaccinated recombinant plasmid were survived, and 30% chickens vaccinated plasmid expressing F protein were survived. These results proved the adjuvant effect of chicken IL-2, and further showed that the efficacy of a DNA vaccine can be greatly improved by simultaneous expression of IL-2.

  20. Expression and Purification of E2 Glycoprotein from Insect Cells (Sf9) for Use in Serology.

    Science.gov (United States)

    Chua, Chong Long; Sam, I-Ching; Chan, Yoke Fun

    2016-01-01

    Chikungunya virus (CHIKV) is a mosquito-borne arbovirus which poses a major threat to global public health. Definitive CHIKV diagnosis is crucial, especially in distinguishing the disease from dengue virus, which co-circulates in endemic areas and shares the same mosquito vectors. Laboratory diagnosis is mainly based on serological or molecular approaches. The E2 glycoprotein is a good candidate for serological diagnosis since it is the immunodominant antigen during the course of infection, and reacts with seropositive CHIKV sera. In this chapter, we describe the generation of stable clone Sf9 (Spodoptera frugiperda) cells expressing secreted, soluble, and native recombinant CHIKV E2 glycoprotein. We use direct plasmid expression in insect cells, rather than the traditional technique of generating recombinant baculovirus. This recombinant protein is useful for serological diagnosis of CHIKV infection.

  1. Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker.

    Science.gov (United States)

    Bingle, W H

    1988-05-01

    The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.

  2. Colonization of the respiratory tract by a virulent strain of avian Escherichia coli requires carriage of a conjugative plasmid.

    Science.gov (United States)

    Ginns, C A; Benham, M L; Adams, L M; Whithear, K G; Bettelheim, K A; Crabb, B S; Browning, G F

    2000-03-01

    The E3 strain of E. coli was isolated in an outbreak of respiratory disease in broiler chickens, and experimental aerosol exposure of chickens to this strain induced disease similar to that seen in the field. In order to establish whether the virulent phenotype of this strain was associated with carriage of particular plasmids, four plasmid-cured derivatives, each lacking two or more of the plasmids carried by the wild-type strain, were assessed for virulence. Virulence was found to be associated with one large plasmid, pVM01. Plasmid pVM01 was marked by introduction of the transposon TnphoA, carrying kanamycin resistance, and was then cloned by transformation of E. coli strain DH5alpha. The cloned plasmid was then reintroduced by conjugation into an avirulent plasmid-cured derivative of strain E3 which lacked pVM01. The conjugant was shown to be as virulent as the wild-type strain E3, establishing that this plasmid is required for virulence following aerosol exposure. This virulence plasmid conferred expression of a hydroxamate siderophore, but not colicins, on both strain E3 and strain DH5alpha. Carriage of this plasmid was required for strain E3 to colonize the respiratory tracts of chickens but was not necessary for colonization of the gastrointestinal tract. However, the virulence plasmid did not confer virulence, or the capacity to colonize the respiratory tract, on strain DH5alpha. Thus, these studies have established that infection of chickens with E. coli strain E3 by the respiratory route is dependent on carriage of a conjugative virulence plasmid, which confers the capacity to colonize specifically the respiratory tract and which also carries genes for expression of a hydroxymate siderophore. These findings will facilitate identification of the specific genes required for virulence in these pathogens.

  3. Novel functions for atypical E2Fs, E2F7 and E2F8, in polyploidization and liver cancer

    NARCIS (Netherlands)

    Pandit, Shusil Kumar

    2014-01-01

    Atypical E2F transcription factors, E2F7 and E2F8, function as transcriptional repressors of E2F target genes and are crucial for controlling the cell proliferation. In this thesis, we reveal that these two factors are crucial for liver cell polyploidization, embryonic development and prevention of

  4. E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes

    DEFF Research Database (Denmark)

    Di Stefano, Luisa; Jensen, Michael Rugaard; Helin, Kristian

    2003-01-01

    The E2F family of transcription factors play an essential role in the regulation of cell cycle progression. In a screen for E2F-regulated genes we identified a novel E2F family member, E2F7. Like the recently identified E2F-like proteins of Arabidopsis, E2F7 has two DNA binding domains and binds...... to the E2F DNA binding consensus site independently of DP co-factors. Consistent with being an E2F target gene, we found that the expression of E2F7 is cell cycle regulated. Ectopic expression of E2F7 results in suppression of E2F target genes and accumulation of cells in G1. Furthermore, E2F7 associates...... with E2F-regulated promoters in vivo, and this association increases in S phase. Interestingly, however, E2F7 binds only a subset of E2F-dependent promoters in vivo, and in agreement with this, inhibition of E2F7 expression results in specific derepression of these promoters. Taken together, these data...

  5. Modulation of the E2F1-driven cancer cell fate by the DNA damage response machinery and potential novel E2F1 targets in osteosarcomas

    DEFF Research Database (Denmark)

    Liontos, Michalis; Niforou, Katerina; Velimezi, Georgia

    2009-01-01

    Osteosarcoma is the most common primary bone cancer. Mutations of the RB gene represent the most frequent molecular defect in this malignancy. A major consequence of this alteration is that the activity of the key cell cycle regulator E2F1 is unleashed from the inhibitory effects of pRb. Studies...... in animal models and in human cancers have shown that deregulated E2F1 overexpression possesses either "oncogenic" or "oncosuppressor" properties, depending on the cellular context. To address this issue in osteosarcomas, we examined the status of E2F1 relative to cell proliferation and apoptosis...... in a clinical setting of human primary osteosarcomas and in E2F1-inducible osteosarcoma cell line models that are wild-type and deficient for p53. Collectively, our data demonstrated that high E2F1 levels exerted a growth-suppressing effect that relied on the integrity of the DNA damage response network...

  6. E2 multimeric scaffold for vaccine formulation: immune response by intranasal delivery and transcriptome profile of E2-pulsed dendritic cells.

    Science.gov (United States)

    Trovato, Maria; Maurano, Francesco; D'Apice, Luciana; Costa, Valerio; Sartorius, Rossella; Cuccaro, Fausta; McBurney, Sean P; Krebs, Shelly J; Prisco, Antonella; Ciccodicola, Alfredo; Rossi, Mauro; Haigwood, Nancy L; De Berardinis, Piergiuseppe

    2016-07-16

    The E2 multimeric scaffold represents a powerful delivery system able to elicit robust humoral and cellular immune responses upon systemic administrations. Here recombinant E2 scaffold displaying the third variable loop of HIV-1 Envelope gp120 glycoprotein was administered via mucosa, and the mucosal and systemic immune responses were analysed. To gain further insights into the molecular mechanisms that orchestrate the immune response upon E2 vaccination, we analysed the transcriptome profile of dendritic cells (DCs) exposed to the E2 scaffold with the aim to define a specific gene expression signature for E2-primed immune responses. The in vivo immunogenicity and the potential of E2 scaffold as a mucosal vaccine candidate were investigated in BALB/c mice vaccinated via the intranasal route. Fecal and systemic antigen-specific IgA antibodies, cytokine-producing CD4(+) and CD8(+) cells were induced assessing the immunogenicity of E2 particles via intranasal administration. The cytokine analysis identified a mixed T-helper cell response, while the systemic antibody response showed a prevalence of IgG1 isotype indicative of a polarized Th2-type immune response. RNA-Sequencing analysis revealed that E2 scaffold up-regulates in DCs transcriptional regulators of the Th2-polarizing cell response, defining a type 2 DC transcriptomic signature. The current study provides experimental evidence to the possible application of E2 scaffold as antigen delivery system for mucosal immunization and taking advantages of genome-wide approach dissects the type of response induced by E2 particles.

  7. Influenza Plasmid DNA Vaccines: Progress and Prospects.

    Science.gov (United States)

    Bicho, Diana; Queiroz, João António; Tomaz, Cândida Teixeira

    2015-01-01

    Current influenza vaccines have long been used to fight flu infectious; however, recent advances highlight the importance of produce new alternatives. Even though traditional influenza vaccines are safe and usually effective, they need to be uploaded every year to anticipate circulating flu viruses. This limitation together with the use of embryonated chicken eggs as the substrate for vaccine production, is time-consuming and could involve potential biohazards in growth of new virus strains. Plasmid DNA produced by prokaryote microorganisms and encoding foreign proteins had emerged as a promising therapeutic tool. This technology allows the expression of a gene of interest by eukaryotic cells in order to induce protective immune responses against the pathogen of interest. In this review, we discuss the strategies to choose the best DNA vaccine to be applied in the treatment and prevention of influenza. Specifically, we give an update of influenza DNA vaccines developments, all involved techniques, their main characteristics, applicability and technical features to obtain the best option against influenza infections.

  8. Plasmid typing of Shigella sonnei epidemic strains and molecular relationship of their R-plasmids.

    Science.gov (United States)

    Mendoza, M C; Gonzalez, A J; Mendez, F J; Hardisson, C

    1988-06-01

    We conducted a surveillance program on epidemic and/or endemic Shigella strains in Asturias (Spain), their frequency and dispersion in our community, and their R-plasmids. We analyzed initial isolates of Shigella sonnei from two epidemic outbreaks using antibiotic resistance patterns and plasmid profile analysis as epidemiological markers. We found that the 2 outbreaks were caused by different S. sonnei strains, which respectively carried one and two R-plasmids together with other plasmids. The molecular relationship among these and three other R-plasmids from two S. sonnei strains isolated during a previous outbreak, were studied by restriction enzyme analysis and DNA-DNA hybridizations. We were able to establish different levels of relationship among the six R-plasmids.

  9. Prevalence and molecular characterization of plasmid- mediated ...

    African Journals Online (AJOL)

    lactamase genes among nosocomial Staphylococcus aureus drug resistance isolates in Taiwan. .... Table 2: Plasmid profiles of the clinical antibiotic-resistant pathogens. Strain. Profile .... Madec J. Characterization of clinical canine methicillin-.

  10. antimicrobial susceptibility and plasmids from escherichia coli ...

    African Journals Online (AJOL)

    2001-10-10

    Oct 10, 2001 ... transmission to humans of E. coli containing antibiotic resistance plasmids ... resistant micro-organisms, which may in turn transfer resistance to .... cells were washed with sterile normal saline to remove leached. Я-lactamase ...

  11. Protein diversity confers specificity in plasmid segregation.

    Science.gov (United States)

    Fothergill, Timothy J G; Barillà, Daniela; Hayes, Finbarr

    2005-04-01

    The ParG segregation protein (8.6 kDa) of multidrug resistance plasmid TP228 is a homodimeric DNA-binding factor. The ParG dimer consists of intertwined C-terminal domains that adopt a ribbon-helix-helix architecture and a pair of flexible, unstructured N-terminal tails. A variety of plasmids possess partition loci with similar organizations to that of TP228, but instead of ParG homologs, these plasmids specify a diversity of unrelated, but similarly sized, partition proteins. These include the proteobacterial pTAR, pVT745, and pB171 plasmids. The ParG analogs of these plasmids were characterized in parallel with the ParG homolog encoded by the pseudomonal plasmid pVS1. Like ParG, the four proteins are dimeric. No heterodimerization was detectable in vivo among the proteins nor with the prototypical ParG protein, suggesting that monomer-monomer interactions are specific among the five proteins. Nevertheless, as with ParG, the ParG analogs all possess significant amounts of unordered amino acid residues, potentially highlighting a common structural link among the proteins. Furthermore, the ParG analogs bind specifically to the DNA regions located upstream of their homologous parF-like genes. These nucleoprotein interactions are largely restricted to cognate protein-DNA pairs. The results reveal that the partition complexes of these and related plasmids have recruited disparate DNA-binding factors that provide a layer of specificity to the macromolecular interactions that mediate plasmid segregation.

  12. Plasmid and chromosome segregation in prokaryotes

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Bugge Jensen, Rasmus; Gerdes, Kenn

    2000-01-01

    Recent major advances in the understanding of prokaryotic DNA segregation have been achieved by using fluorescence microscopy to visualize the localization of cellular components. Plasmids and bacterial chromosomes are partitioned in a highly dynamic fashion, suggesting the presence of a mitotic......-like apparatus in prokaryotes. The identification of chromosomal homologues of the well-characterized plasmid partitioning genes indicates that there could be a general mechanism of bacterial DNA partitioning. Udgivelsesdato: July 1...

  13. Multiple plasmid interference - Pledging allegiance to my enemy's enemy.

    Science.gov (United States)

    Gama, João Alves; Zilhão, Rita; Dionisio, Francisco

    2017-08-24

    As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Membrane-initiated actions of estradiol (E2 in the regulation of LH secretion in ovariectomized (OVX ewes

    Directory of Open Access Journals (Sweden)

    Oakley Amy E

    2010-05-01

    Full Text Available Abstract Background We demonstrated that E2 conjugated to BSA (E2BSA induces a rapid membrane-initiated inhibition of LH secretion followed hours later by a slight increase in LH secretion. Whether these actions of E2BSA are restricted to the pituitary gland and whether the membrane-initiated pathway of E2BSA contributes to the up-regulation of the number of GnRH receptors during the positive feedback effect of E2 were evaluated here. We have shown that the suppression of LH secretion induced by E2 and E2BSA is the result of a decreased responsiveness of the pituitary gland to GnRH. In this study we further tested the ability of E2BSA to decrease the responsiveness of the pituitary gland to GnRH under the paradigm of the preovulatory surge of LH induced by E2. Methods For the first experiment GnRH and LH secretions were determined in samples of pituitary portal and jugular blood, respectively, in ewes treated with 12 mg E2BSA. In the second experiment, the number of GnRH receptors was quantified in ewes 12 h after administration of 25 micrograms E2 (the expected time for the increase in the number of GnRH receptors and the positive feedback effect of E2 in LH secretion or 12 mg E2BSA. In the third experiment, the preovulatory-like surge of LH was characterized in ewes injected with 25 micrograms E2 alone or followed 8 h later (before the beginning of the LH surge with 60 mg E2BSA. Results a the decrease in LH secretion induced by E2BSA was not accompanied by changes in the pulsatile pattern of GnRH, b E2BSA increased the number of GnRH receptors, and c the presence of E2BSA in E2-treated ewes delayed the onset, reduced the length, and decreased the amount of LH released during the preovulatory surge of LH. Conclusions a the rapid suppression of LH secretion induced by E2BSA is mediated only via a direct action on the pituitary gland, b E2 acting via a membrane-initiated pathway contributes to increase the number of GnRH receptors and, c

  15. Clostridium perfringens type A–E toxin plasmids

    Science.gov (United States)

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  16. Historical Events That Spawned the Field of Plasmid Biology.

    Science.gov (United States)

    Kado, Clarence I

    2014-10-01

    This chapter revisits the historical development and outcome of studies focused on the transmissible, extrachromosomal genetic elements called plasmids. Early work on plasmids involved structural and genetic mapping of these molecules, followed by the development of an understanding of how plasmids replicate and segregate during cell division. The intriguing property of plasmid transmission between bacteria and between bacteria and higher cells has received considerable attention. The utilitarian aspects of plasmids are described, including examples of various plasmid vector systems. This chapter also discusses the functional attributes of plasmids needed for their persistence and survival in nature and in man-made environments. The term plasmid biology was first conceived at the Fallen Leaf Lake Conference on Promiscuous Plasmids, 1990, Lake Tahoe, California. The International Society for Plasmid Biology was established in 2004 (www.ISPB.org).

  17. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Directory of Open Access Journals (Sweden)

    Mette Burmølle

    Full Text Available Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids into a laboratory strain (Escherichia coli Genehogs® for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54 and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33 were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  18. Sequencing of IncX-plasmids suggests ubiquity of mobile forms of a biofilm-promoting gene cassette recruited from Klebsiella pneumoniae.

    Science.gov (United States)

    Burmølle, Mette; Norman, Anders; Sørensen, Søren J; Hansen, Lars Hestbjerg

    2012-01-01

    Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.

  19. Preparation and characterization of antisera against recombinant E2 protein of bovine viral diarrhea virus%牛病毒性腹泻病毒E2蛋白的多克隆抗体制备及鉴定

    Institute of Scientific and Technical Information of China (English)

    高欲燃; 朱远茂; 康健; 史鸿飞; 李娇; 任宪刚; 冯军科; 于作; 薛飞

    2011-01-01

    To prepare the polyclonal antibody against recombinant E2 protein of bovine viral diarrhea virus (BVDV) in rabbits, E. coli BL21 (DE3) was transformed with the recombinant plasmid pET30a-E2. The recombinant E2 protein was expressed in E. coli after cultivation and induction. The purified recombinant E2 protein could be recognized by specific BVDV antisera in western blot. Then the purified recombinant E2 protein was used as antigen for immunizing rabbit to prepare polyclonal antibody against the recombinant E2 protein. The result of virus nentralization test showed that the titer of the polyclonal antibody to neutralize BVDV was 1:2,048. The polyclonal antibody against the recombinant E2 protein of BVDV also had highly reactivity and specialty in immunofluorescence analysis and western blot. The polyclonal antibody against recombinant E2 protein of BVDV developed in rabbits could be used in detection of BVDV in China and provided a good basis for establishing an ELISA for detecting of E2 protein of BVDV.%为制备牛病毒性腹泻病毒(BVDV)重组E2蛋白的兔源多克隆抗体,本研究利用表达BVDV E2蛋白的重组质粒pET30a-E2转化E.coli BL21(DE3),经诱导表达获得重组E2蛋白.Western blot检测显示纯化蛋白能够与BVDV参考阳性血清反应.以纯化的重组E2蛋白免疫新西兰白兔制备多克隆抗体,病毒中和试验测定其中和效价为1:2 048,间接免疫荧光和western blot试验表明其具有良好的反应性和特异性.本研究制备的BVDV重组E2蛋白兔源多克隆抗体可应用于BVDV的检测,同时为进一步建立检测BVDV E2蛋白的ELISA方法奠定基础.

  20. Construction of pTM series plasmids for gene expression in Brucella species.

    Science.gov (United States)

    Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

    2016-04-01

    Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp.

  1. DNA repair in bacterial cultures and plasmid DNA exposed to infrared laser for treatment of pain

    Science.gov (United States)

    Canuto, K. S.; Sergio, L. P. S.; Marciano, R. S.; Guimarães, O. R.; Polignano, G. A. C.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-06-01

    Biostimulation of tissues by low intensity lasers has been described on a photobiological basis and clinical protocols are recommended for treatment of various diseases, but their effects on DNA are controversial. The objective of this work was to evaluate effects of low intensity infrared laser exposure on survival and bacterial filamentation in Escherichia coli cultures, and induction of DNA lesions in bacterial plasmids. In E. coli cultures and plasmids exposed to an infrared laser at fluences used to treat pain, bacterial survival and filamentation and DNA lesions in plasmids were evaluated by electrophoretic profile. Data indicate that the infrared laser (i) increases survival of E. coli wild type in 24 h of stationary growth phase, (ii) induces bacterial filamentation, (iii) does not alter topological forms of plasmids and (iv) does not alter the electrophoretic profile of plasmids incubated with exonuclease III or formamidopyrimidine DNA glycosylase. A low intensity infrared laser at the therapeutic fluences used to treat pain can alter survival of E. coli wild type, induce filamentation in bacterial cells, depending on physiologic conditions and DNA repair, and induce DNA lesions other than single or double DNA strand breaks or alkali-labile sites, which are not targeted by exonuclease III or formamidopyrimidine DNA glycosylase.

  2. Position effect on expression of dsd genes cloned onto multicopy plasmids.

    Science.gov (United States)

    Carothers, A M; Heincz, M C; McFall, E

    1980-04-01

    In the D-serine deaminase system of Escherichia coli, which is regulated by positive control, we have fouand a complete lack of trans activation in vivo with multicopy dsd hybrid plasmids. A PLASmid carrying the regulatory gene, dsdC+, did not promote expression of chromosomal dsdCO+A+ loci, nor did a chromosomal dsdC+ gene promote expression of plasmid-borne dsdC delta O+A+ (dsd regulatory gene negative) restriction fragments. However, hybrid plasmids that comprise the entire dsd system (dsdC+O+A+) are highly inducible for the enzyme. These dsd hybrid plasmid deoxyribonucleic acids functioned well as templates in the in vitro coupled transcription-translation system. In vitro-synthesized dsdC+ protein promoted expression of the dsdA+ operation efficiently. Exogenously purified dsdC+ protein also activated expression of several dsdC delta O+A+ plasmid deoxyribonucleic acid templates in vitro. An explanation that reconciles these results with previous dominance studies is presented.

  3. Loss of plasmids containing cloned inserts coding for novobiocin resistance or novobiocin sensitivity in Haemophilus influenzae

    Energy Technology Data Exchange (ETDEWEB)

    Setlow, J.K.; Spikes, D.; Ledbetter, M.

    1984-06-01

    Plasmids pNov1 and pNov1s, coding for resistance and sensitivity to novobiocin, respectively, were readily lost from wild-type Haemophilus influenzae but retained in a strain lacking an inducible defective prophage. The plasmid loss could be partly or wholly eliminated by a low-copy-number mutation in the plasmid or by the presence of certain antibiotic resistance markers in the host chromosome. Release of both phage HP1c1, measured by plaque assay, and defective phage, measured by electron microscopy, was increased when the plasmids were present. The frequency of recombination between pNov1 and the chromosome, causing the plasmid to be converted to pNov1s, could under some circumstances be decreased from the normal 60 to 70% to below 10% by the presence of a kanamycin resistance marker in the chromosome. This suggested that a gene product coded for by the plasmid, the expression of which was affected by the kanamycin resistance marker, was responsible for the high recombination frequency. Evidence was obtained from in vitro experiments that the gene product was a gyrase.

  4. Induction of DNA synthesis and apoptosis are separable functions of E2F-1

    DEFF Research Database (Denmark)

    Phillips, A C; Bates, S; Ryan, K M;

    1997-01-01

    The family of E2F transcription factors have an essential role in mediating cell cycle progression, and recently, one of the E2F protein family, E2F-1, has been shown to participate in the induction of apoptosis. Cooperation between E2F and the p53 tumor suppressor protein in this apoptotic...... response had led to the suggestion that cell cycle progression induced by E2F-1 expression provides an apoptotic signal when placed in conflict with an arrest to cell cycle progression, such as provided by p53. We show here that although apoptosis is clearly enhanced by p53, E2F-1 can induce significant...... apoptosis in the absence of p53. Furthermore, this apoptotic function of E2F-1 is separable from the ability to accelerate entry into DNA synthesis. Analysis of E2F-1 mutants indicates that although DNA-binding is required, transcriptional transactivation is not necessary for the induction of apoptosis by E...

  5. Polymorphic genetic characterization of E2 gene of bovine viral diarrhea virus in China.

    Science.gov (United States)

    Lang, Yifei; Gao, Shandian; Du, Junzheng; Shao, Junjun; Cong, Guozheng; Lin, Tong; Zhao, Furong; Liu, Lihong; Chang, Huiyun

    2014-12-05

    Bovine viral diarrhea virus (BVDV) is one of the wide distributed pathogenic viruses of livestock and wild animals worldwide. E2 glycoprotein is a major structural component of the BVDV virion and plays a key role in viral attachment to host cells and inducing immune responses against viral infection. In order to gain detailed information of the E2 coding region of BVDV circulating in China, 46 positive samples were tested by RT-PCR for the E2 coding region. The 1122 nt nucleotide sequences of full-length E2 were harvested and analyzed. The results suggested that full-length E2 was an ideal target for BVDV genotyping and divided the domestic BVDV isolates into 9 subgenotypes, namely BVDV-1a, -1b1, -1c, -1d, -1o, -1m, -1p, -1q and BVDV-2a, showing great diversity. The difference of nonsynonymous and synonymous substitution rates (dN-dS) inferred both positive and purifying selection of the E2. However, combination of positive and purifying selection at different points indicated purifying selection within the complete E2. Protein properties analysis based on glycosylation sites and epitope prediction demonstrated that the biological character of E2 among individual BVDV subgenotype was similar, but may alter due to amino acid changes. For the first time, the comprehensive collection of E2 sequences of Chinese BVDV isolates was elucidated, which would provide information for future vaccine design and BVD control in China.

  6. E2F1 transcription factor and its impact on growth factor and cytokine signaling.

    Science.gov (United States)

    Ertosun, Mustafa Gokhan; Hapil, Fatma Zehra; Osman Nidai, Ozes

    2016-10-01

    E2F1 is a transcription factor involved in cell cycle regulation and apoptosis. The transactivation capacity of E2F1 is regulated by pRb. In its hypophosphorylated form, pRb binds and inactivates DNA binding and transactivating functions of E2F1. The growth factor stimulation of cells leads to activation of CDKs (cyclin dependent kinases), which in turn phosphorylate Rb and hyperphosphorylated Rb is released from E2F1 or E2F1/DP complex, and free E2F1 can induce transcription of several genes involved in cell cycle entry, induction or inhibition of apoptosis. Thus, growth factors and cytokines generally utilize E2F1 to direct cells to either fate. Furthermore, E2F1 regulates expressions of various cytokines and growth factor receptors, establishing positive or negative feedback mechanisms. This review focuses on the relationship between E2F1 transcription factor and cytokines (IL-1, IL-2, IL-3, IL-6, TGF-beta, G-CSF, LIF), growth factors (EGF, KGF, VEGF, IGF, FGF, PDGF, HGF, NGF), and interferons (IFN-α, IFN-β and IFN-γ).

  7. Using Plasmids as DNA Vaccines for Infectious Diseases.

    Science.gov (United States)

    Tregoning, John S; Kinnear, Ekaterina

    2014-12-01

    DNA plasmids can be used to induce a protective (or therapeutic) immune response by delivering genes encoding vaccine antigens. That naked DNA (without the refinement of coat proteins or host evasion systems) can cross from outside the cell into the nucleus and be expressed is particularly remarkable given the sophistication of the immune system in preventing infection by pathogens. As a result of the ease, low cost, and speed of custom gene synthesis, DNA vaccines dangle a tantalizing prospect of the next wave of vaccine technology, promising individual designer vaccines for cancer or mass vaccines with a rapid response time to emerging pandemics. There is considerable enthusiasm for the use of DNA vaccination as an approach, but this enthusiasm should be tempered by the successive failures in clinical trials to induce a potent immune response. The technology is evolving with the development of improved delivery systems that increase expression levels, particularly electroporation and the incorporation of genetically encoded adjuvants. This review will introduce some key concepts in the use of DNA plasmids as vaccines, including how the DNA enters the cell and is expressed, how it induces an immune response, and a summary of clinical trials with DNA vaccines. The review also explores the advances being made in vector design, delivery, formulation, and adjuvants to try to realize the promise of this technology for new vaccines. If the immunogenicity and expression barriers can be cracked, then DNA vaccines may offer a step change in mass vaccination.

  8. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids

    OpenAIRE

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Jaana K.H. Bamford; Buckling, Angus

    2011-01-01

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple ant...

  9. E2F1-Mediated Induction of NFYB Attenuates Apoptosis via Joint Regulation of a Pro-Survival Transcriptional Program.

    Directory of Open Access Journals (Sweden)

    Xiaolei Jiang

    Full Text Available The E2F1 transcription factor regulates cell proliferation and apoptosis through the control of a considerable variety of target genes. Previous work has detailed the role of other transcription factors in mediating the specificity of E2F function. Here we identify the NF-YB transcription factor as a novel direct E2F1 target. Genome-wide expression analysis of the effects of NFYB knockdown on E2F1-mediated transcription identified a large group of genes that are co-regulated by E2F1 and NFYB. We also provide evidence that knockdown of NFYB enhances E2F1-induced apoptosis, suggesting a pro-survival function of the NFYB/E2F1 joint transcriptional program. Bioinformatic analysis suggests that deregulation of these NFY-dependent E2F1 target genes might play a role in sarcomagenesis as well as drug resistance.

  10. E2F-5, a new E2F family member that interacts with p130 in vivo

    OpenAIRE

    Hijmans, E.M.; Voorhoeve, P.M.; Beijersbergen, R.L.; Veer, L J Van't; Bernards, R.A.

    1995-01-01

    E2F DNA binding sites are found in a number of genes whose expression is tightly regulated during the cell cycle. The activity of E2F transcription factors is regulated by association with specific repressor molecules that can bind and inhibit the E2F transactivation domain. For E2F-1, E2F-2, and E2F-3, the repressor is the product of the retinoblastoma gene, pRb. E2f-4 interacts with pRb-related p107 and not with pRb itself. Recently, a cDNA encoding a third member of the retinoblastoma gene...

  11. The effects of a low-intensity red laser on bacterial growth, filamentation and plasmid DNA

    Science.gov (United States)

    Roos, C.; Santos, J. N.; Guimarães, O. R.; Geller, M.; Paoli, F.; Fonseca, A. S.

    2013-07-01

    Exposure of nonphotosynthesizing microorganisms to light could increase cell division in cultures, a phenomenon denominated as biostimulation. However, data concerning the importance of the genetic characteristics of cells on this effect are as yet scarce. The aim of this work was to evaluate the effects of a low-intensity red laser on the growth, filamentation and plasmids in Escherichia coli cells proficient and deficient in DNA repair. E. coli cultures were exposed to a laser (658 nm, 10 mW, 1 and 8 J cm-2) to study bacterial growth and filamentation. Also, bacterial cultures hosting pBSK plasmids were exposed to the laser to study DNA topological forms from the electrophoretic profile in agarose gels. Data indicate the low-intensity red laser: (i) had no effect on the growth of E. coli wild type and exonuclease III deficient cells; (ii) induced bacterial filamentation, (iii) led to no alteration in the electrophoretic profile of plasmids from exonuclease III deficient cells, but plasmids from wild type cells were altered. A low-intensity red laser at the low fluences used in phototherapy has no effect on growth, but induces filamentation and alters the topological forms of plasmid DNA in E. coli cultures depending on the DNA repair mechanisms.

  12. Distribution of small native plasmids in Streptococcus pyogenes in India.

    Science.gov (United States)

    Bergmann, René; Nerlich, Andreas; Chhatwal, Gursharan S; Nitsche-Schmitz, D Patric

    2014-05-01

    Complete characterization of a Streptococcus pyogenes population from a defined geographic region comprises information on the plasmids that circulate in these bacteria. Therefore, we determined the distribution of small plasmids (pyogenes isolates from India, where diversity of strains and incidence rates of S. pyogenes infections are high. The collection comprised 77 emm-types. For plasmid detection and discrimination, we developed PCRs for different plasmid replication initiation protein genes, the putative repressor gene copG and bacteriocin genes dysA and scnM57. Plasmid distribution was limited to 13 emm-types. Co-detection analysis using aforementioned PCRs revealed four distinct plasmid sub-types, two of which were previously unknown. Representative plasmids pA852 and pA996 of the two uncharacterized plasmid sub-types were sequenced. These two plasmids could be assigned to the pMV158 and the pC194/pUB110 family of rolling-circle plasmids, respectively. The majority of small plasmids found in India belonged to the two newly characterized sub-types, with pA852- and pA996-like plasmids amounting to 42% and 22% of all detected plasmids, respectively. None of the detected plasmids coded for a known antibiotic resistance gene. Instead, all of the four plasmid sub-types carried known or potential bacteriocin genes. These genes may have influence on the evolutionary success of certain S. pyogenes genotypes. Notably, pA852-like plasmids were found in all isolates of the most prevalent emm-type 11.0. Together, a priori fitness of this genotype and increased fitness due to the acquired plasmids may have rendered type emm11.0 successful and caused the prevalence of pA852-like plasmids in India.

  13. 口服sST2质粒DNA在炎症性肠病小鼠中的免疫水平%Effect of orally delivered plasmid DNA expressing sST2 on inflammatory Th2 cells in intestine of mice with DSS-induced colitis

    Institute of Scientific and Technical Information of China (English)

    朱俊丰; 徐莹; 桑力轩; 杨芳莉; 李岩; 尉冰; 高云峰; 孙逊; 吕昌龙

    2016-01-01

    目的:制备口服可溶性ST2(Soluble ST2,sST2)质粒,观察其对于小鼠炎症性结肠黏膜免疫应答水平。方法提取小鼠脾脏总RNA,扩增sST2基因,并克隆至pcDNA3.1/myc-HisA载体,构建pcDNA3.1-sST2-myc-HisA质粒,借此转染COS-7细胞,使之表达 sST2-myc-HisA融合蛋白。利用脂质体LipofectamineTM 2000包裹,制备口服sST2质粒,经灌胃给予用葡聚糖硫酸钠( Dextran sulfate sodium, DSS)诱生溃疡性结肠炎的C57BL/6实验小鼠,并用组织病理学方法观察结肠黏膜组织形态变化。用Real-timePCR检测sST2基因表达,用免疫印迹检测sST2质粒融合蛋白的表达;用ELISA检测小鼠肠固有层淋巴细胞培养上清中细胞因子( IL-4、IL-5和IL-13)的分泌水平。结果 Real-time PCR检测到sST2基因的正确表达,经双酶切及测序鉴定,证明质粒pcDNA3.1-sST2-myc-HisA构建成功,免疫印迹试验在转染的COS-7细胞检测到sST2-myc-HisA融合蛋白的表达,其相对分子质量与预期的相符;观察到结肠黏膜组织形态发生的变化;Real-time PCR结果显示,sST2质粒组在结肠黏膜组织内sST2的表达水平明显高于pcDNA3.1组和生理盐水组(P<0.01);ELISA结果显示sST2质粒组结肠固有层淋巴细胞培养上清中IL-4、IL-5和IL-13的分泌水平高于pcDNA3.1组和生理盐水组(P<0.05)。结论实验成功制备口服sST2质粒;该质粒可抑制小鼠炎症性结肠黏膜组织内Th2型免疫应答。%Objective To prepare an oral sST2 DNA plasmid and observe its effect on the immuno-regulation of intestinal mucosa of mice with DSS induced colitis. Methods The gene coding sST2 was amplified from total RNA of mice spleen and then inserted into the vector pcDNA3. 1/myc-HisA to construct a recombinant expression plasmid pcDNA3. 1-sST2-myc-HisA, which was transfected to COS-7 cells and made up of a capsule wrapped by LipofectamineTM2000. The capsule was orally dilivered to C57BL/6 mouse with DSS induced colitis, and

  14. p53 represses human papillomavirus type 16 DNA replication via the viral E2 protein

    Directory of Open Access Journals (Sweden)

    Morgan Iain M

    2008-01-01

    Full Text Available Abstract Background Human papillomavirus (HPV DNA replication can be inhibited by the cellular tumour suppressor protein p53. However, the mechanism through which p53 inhibits viral replication and the role that this might play in the HPV life cycle are not known. The papillomavirus E2 protein is required for efficient HPV DNA replication and also regulates viral gene expression. E2 represses transcription of the HPV E6 and E7 oncogenes and can thereby modulate indirectly host cell proliferation and survival. In addition, the E2 protein from HPV 16 has been shown to bind p53 and to be capable of inducing apoptosis independently of E6 and E7. Results Here we use a panel of E2 mutants to confirm that mutations which block the induction of apoptosis via this E6/E7-independent pathway, have little or no effect on the induction of apoptosis by the E6/E7-dependent pathway. Although these mutations in E2 do not affect the ability of the protein to mediate HPV DNA replication, they do abrogate the repressive effects of p53 on the transcriptional activity of E2 and prevent the inhibition of E2-dependent HPV DNA replication by p53. Conclusion These data suggest that p53 down-regulates HPV 16 DNA replication via the E2 protein.

  15. NRIP enhances HPV gene expression via interaction with either GR or E2

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Szu-Wei; Lu, Pei-Yu; Guo, Jih-Huong [Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China); Tsai, Tzung-Chieh [Department of Microbiology and Immunology, National Chiayi University, Chiayi 600-04, Taiwan (China); Tsao, Yeou-Ping [Department of Ophthalmology, Mackay Memorial Hospital, Taipei 104, Taiwan (China); Chen, Show-Li, E-mail: showlic@ha.mc.ntu.edu.tw [Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (China)

    2012-02-05

    We previously identified a gene, nuclear receptor-interaction protein (NRIP), which functions as a transcription cofactor in glucocorticoid receptor (GR) and human papillomavirus E2 (HPV E2)-driven gene expression. Here, we comprehensively evaluated the role of NRIP in HPV-16 gene expression. NRIP acts as a transcription cofactor to enhance GR-regulated HPV-16 gene expression in the presence of hormone. NRIP also can form complex with E2 that caused NRIP-induced HPV gene expression via E2-binding sites in a hormone-independent manner. Furthermore, NRIP can associate with GR and E2 to form tri-protein complex to activate HPV gene expression via GRE, not the E2-binding site, in a hormone-dependent manner. These results indicate that NRIP and GR are viral E2-binding proteins and that NRIP regulates HPV gene expression via GRE and/or E2 binding site in the HPV promoter in a hormone-dependent or independent manner, respectively.

  16. Diversity and homogeneity among small plasmids of Aeromonas salmonicida subsp. salmonicida linked with geographical origin

    Directory of Open Access Journals (Sweden)

    Sabrina A Attéré

    2015-11-01

    Full Text Available Furunculosis, which is caused by Aeromonas salmonicida subsp. salmonicida, is a major salmonid disease in fish farms worldwide. Several plasmids found in this bacterium confer phenotypes such drug resistance and virulence. Small plasmids (pAsa1, pAsa2, pAsa3, and pAsal1 related to ColE1- and ColE2-type replicons are usually present in its normal plasmidome. In the present study, with the objective to investigate if these plasmids display particularities related to the origin of the isolates bearing them, a total of 153 isolates, including 78 new and 75 previously described, were analyzed for the presence of small plasmids by PCR and DNA restriction fragment profiling. A geographical dichotomy between Canadian and European isolates for their propensity to do not have pAsa3 or pAsal1 was found. In addition, the genotyping analysis led to the identification of two European isolates harboring an unusual pAsal1. An investigation by next-generation sequencing (NGS of these two isolates shed light on two pAsal1 variants (pAsal1C and pAsal1D. As with pAsal1B, another pAsal1 variant previously described, these two new variants bore a second insertion sequence (ISAS5 in addition to the usual ISAS11. The characterization of these variants suggested that they could predominate over the wild-type pAsal1 in stressful conditions such as growth at temperatures of 25°C and above. To obtain a comprehensive portrait of the mutational pressure on small plasmids, 26 isolates whose DNA had been sequenced by NGS were investigated. pAsa3 and pAsal1 were more prone to mutations than pAsa1 and pAsa2, especially in the mobA gene, which encodes a relaxase and a primase. Lastly, the average copy number of each plasmid per cell was assessed using raw sequencing data. A clear trend with respect to the relative proportion per cell of each plasmid was identified. Our large-scale study revealed a geographical dichotomy in small plasmid repertoire in addition to a clear trend

  17. Stress responses and replication of plasmids in bacterial cells

    Directory of Open Access Journals (Sweden)

    Wegrzyn Alicja

    2002-05-01

    Full Text Available Abstract Plasmids, DNA (or rarely RNA molecules which replicate in cells autonomously (independently of chromosomes as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

  18. The immunogenicity of viral haemorragic septicaemia rhabdovirus (VHSV) DNA vaccines can depend on plasmid regulatory sequences.

    Science.gov (United States)

    Chico, V; Ortega-Villaizan, M; Falco, A; Tafalla, C; Perez, L; Coll, J M; Estepa, A

    2009-03-18

    A plasmid DNA encoding the viral hemorrhagic septicaemia virus (VHSV)-G glycoprotein under the control of 5' sequences (enhancer/promoter sequence plus both non-coding 1st exon and 1st intron sequences) from carp beta-actin gene (pAE6-G(VHSV)) was compared to the vaccine plasmid usually described the gene expression is regulated by the human cytomegalovirus (CMV) immediate-early promoter (pMCV1.4-G(VHSV)). We observed that these two plasmids produced a markedly different profile in the level and time of expression of the encoded-antigen, and this may have a direct effect upon the intensity and suitability of the in vivo immune response. Thus, fish genetic immunisation assays were carried out to study the immune response of both plasmids. A significantly enhanced specific-antibody response against the viral glycoprotein was found in the fish immunised with pAE6-G(VHSV). However, the protective efficacy against VHSV challenge conferred by both plasmids was similar. Later analysis of the transcription profile of a set of representative immune-related genes in the DNA immunized fish suggested that depending on the plasmid-related regulatory sequences controlling its expression, the plasmid might activate distinct patterns of the immune system. All together, the results from this study mainly point out that the selection of a determinate encoded-antigen/vector combination for genetic immunisation is of extraordinary importance in designing optimised DNA vaccines that, when required for inducing protective immune response, could elicit responses biased to antigen-specific antibodies or cytotoxic T cells generation.

  19. Construction of disarmed Ti plasmids transferable between Escherichia coli and Agrobacterium species.

    Science.gov (United States)

    Kiyokawa, Kazuya; Yamamoto, Shinji; Sakuma, Kei; Tanaka, Katsuyuki; Moriguchi, Kazuki; Suzuki, Katsunori

    2009-04-01

    Agrobacterium-mediated plant transformation has been used widely, but there are plants that are recalcitrant to this type of transformation. This transformation method uses bacterial strains harboring a modified tumor-inducing (Ti) plasmid that lacks the transfer DNA (T-DNA) region (disarmed Ti plasmid). It is desirable to develop strains that can broaden the host range. A large number of Agrobacterium strains have not been tested yet to determine whether they can be used in transformation. In order to improve the disarming method and to obtain strains disarmed and ready for the plant transformation test, we developed a simple scheme to make certain Ti plasmids disarmed and simultaneously maintainable in Escherichia coli and mobilizable between E. coli and Agrobacterium. To establish the scheme in nopaline-type Ti plasmids, a neighboring segment to the left of the left border sequence, a neighboring segment to the right of the right border sequence of pTi-SAKURA, a cassette harboring the pSC101 replication gene between these two segments, the broad-host-range IncP-type oriT, and the gentamicin resistance gene were inserted into a suicide-type sacB-containing vector. Replacement of T-DNA with the cassette in pTiC58 and pTi-SAKURA occurred at a high frequency and with high accuracy when the tool plasmid was used. We confirmed that there was stable maintenance of the modified Ti plasmids in E. coli strain S17-1lambdapir and conjugal transfer from E. coli to Ti-less Agrobacterium strains and that the reconstituted Agrobacterium strains were competent to transfer DNA into plant cells. As the modified plasmid delivery system was simple and efficient, conversion of strains to the disarmed type was easy and should be applicable in studies to screen for useful strains.

  20. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and

  1. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  2. Plasmid mediated quinolone resistance in Enterobacteriaceae

    NARCIS (Netherlands)

    Veldman, K.T.; LS Klinisch Onderzoek Wagenaar

    2014-01-01

    This thesis describes the occurrence of Plasmid Mediated Quinolone Resistance (PMQR) in Salmonella and E. coli from The Netherlands and other European countries. Furthermore, the genetic background of these genes was characterized. Fluoroquinolones are widely used antibiotics in both human and veter

  3. Plasmids spread very fast in heterogeneous bacterial communities.

    Science.gov (United States)

    Dionisio, Francisco; Matic, Ivan; Radman, Miroslav; Rodrigues, Olivia R; Taddei, François

    2002-01-01

    Conjugative plasmids can mediate gene transfer between bacterial taxa in diverse environments. The ability to donate the F-type conjugative plasmid R1 greatly varies among enteric bacteria due to the interaction of the system that represses sex-pili formations (products of finOP) of plasmids already harbored by a bacterial strain with those of the R1 plasmid. The presence of efficient donors in heterogeneous bacterial populations can accelerate plasmid transfer and can spread by several orders of magnitude. Such donors allow millions of other bacteria to acquire the plasmid in a matter of days whereas, in the absence of such strains, plasmid dissemination would take years. This "amplification effect" could have an impact on the evolution of bacterial pathogens that exist in heterogeneous bacterial communities because conjugative plasmids can carry virulence or antibiotic-resistance genes. PMID:12524329

  4. A Type III protein-RNA toxin-antitoxin system from Bacillus thuringiensis promotes plasmid retention during spore development.

    Science.gov (United States)

    Short, Francesca L; Monson, Rita E; Salmond, George P C

    2015-01-01

    Members of the Bacillus cereus sensu lato group of bacteria often contain multiple large plasmids, including those encoding virulence factors in B. anthracis. Bacillus species can develop into spores in response to stress. During sporulation the genomic content of the cell is heavily compressed, which could result in counterselection of extrachromosomal genomic elements, unless they have robust stabilization and segregation systems. Toxin-antitoxin (TA) systems are near-ubiquitous in prokaryotes and have multiple biological roles, including plasmid stabilization during vegetative growth. Here, we have shown that a Type III TA system, based on an RNA antitoxin and endoribonuclease toxin, from plasmid pAW63 in Bacillus thuringiensis serovar kurstaki HD-73 can dramatically promote plasmid retention in populations undergoing sporulation and germination, and we provide evidence that this occurs through the post-segregational killing of plasmid-free forespores. Our findings show how an extremely common genetic module can be used to ensure plasmid maintenance during stress-induced developmental transitions, with implications for plasmid dynamics in B. cereus s.l. bacteria.

  5. The E2F family: specific functions and overlapping interests

    DEFF Research Database (Denmark)

    Attwooll, Claire; Lazzerini Denchi, Eros; Helin, Kristian

    2004-01-01

    The E2F transcription factors are key regulators of cell cycle progression and the E2F field has made rapid advances since its advent in 1986. Yet, while our understanding of the roles and functions of the E2F family has made enormous progress, with each discovery new questions arise. In this rev...

  6. Endogenous mutagenesis in recombinant sulfolobus plasmids.

    Science.gov (United States)

    Sakofsky, Cynthia J; Grogan, Dennis W

    2013-06-01

    Low rates of replication errors in chromosomal genes of Sulfolobus spp. demonstrate that these extreme thermoacidophiles can maintain genome integrity in environments with high temperature and low pH. In contrast to this genetic stability, we observed unusually frequent mutation of the β-D-glycosidase gene (lacS) of a shuttle plasmid (pJlacS) propagated in Sulfolobus acidocaldarius. The resulting Lac(-) mutants also grew faster than the Lac(+) parent, thereby amplifying the impact of the frequent lacS mutations on the population. We developed a mutant accumulation assay and corrections for the effects of copy number and differential growth for this system; the resulting measurements and calculations yielded a corrected rate of 5.1 × 10(-4) mutational events at the lacS gene per plasmid replication. Analysis of independent lacS mutants revealed three types of mutations: (i) G · C-to-A · T transitions, (ii) slipped-strand events, and (iii) deletions. These mutations were frequent in plasmid-borne lacS expressed at a high level but not in single-copy lacS in the chromosome or at lower levels of expression in a plasmid. Substitution mutations arose at only two of 12 potential priming sites of the DNA primase of the pRN1 replicon, but nearly all these mutations created nonsense (chain termination) codons. The spontaneous mutation rate of plasmid-borne lacS was 175-fold higher under high-expression than under low-expression conditions. The results suggest that important DNA repair or replication fidelity functions are impaired or overwhelmed in pJlacS, with results analogous to those of the "transcription-associated mutagenesis" seen in bacteria and eukaryotes.

  7. CDK4, pRB and E2F1: connected to insulin

    Directory of Open Access Journals (Sweden)

    Blanchet Emilie

    2010-02-01

    Full Text Available Abstract Pancreatic β-cells are metabolic sensors involved in the control of glucose homeostasis. This particular cell type controls insulin secretion through a fine-tuned process, which dregulation have important pathological consequences, such as observed during type 2 diabetes. We recently implicated E2F1 in the control of glucose homeostasis. First we showed that E2f1-/- mice have decreased pancreatic size, as the result of impaired postnatal pancreatic growth. We observed in this study that E2F1 was highly expressed in non-proliferating pancreatic β-cells, suggesting that E2F1, besides the control of β-cell number could have a role in pancreatic β-cell function. We demonstrate in our recent study, both in vitro and in vivo that E2F1 directly regulates the expression of Kir6.2, a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion in pancreatic β-cells. Expression of Kir6.2 is lost in pancreas of E2f1-/- mice, resulting in insulin secretion defects in these mice. Furthermore, we demonstrated by in tissue chromatin immunoprecipitation analysis that regulation of Kir6.2 expression by E2F1 follows the same regulatory pathway that the classical E2F1 target genes, implicating the participation of CDK4 and retinoblastoma protein. Moreover, in this context, E2F1 transcriptional activity is regulated by glucose and insulin through the CDK4-dependent inactivation of the pRB protein. In summary we provide evidence that the CDK4-pRB-E2F1 regulatory pathway is involved in glucose homeostasis. In our recent study we decipher a new function for these factors in the control of insulin secretion and open up new avenues for the treatment of metabolic diseases, in particular type 2 diabetes.

  8. Plasmid-mediated tetracycline resistance in Haemophilus ducreyi.

    OpenAIRE

    Albritton, W L; Maclean, I W; Slaney, L A; Ronald, A. R.; Deneer, H G

    1984-01-01

    Clinical isolates of Haemophilus ducreyi were shown to be resistant to tetracycline. Resistance was associated in some strains with a 30-megadalton plasmid capable of transferring resistance in conjugative matings with other strains of H. ducreyi and other species of Haemophilus. Restriction endonuclease digestion patterns suggest a relationship between H. ducreyi plasmids and other tetracycline resistance plasmids in Haemophilus. The presence of plasmid-mediated resistance to the tetracyclin...

  9. Plasmid DNA entry into postmitotic nuclei of primary rat myotubes.

    OpenAIRE

    Dowty, M E; Williams, P.; G. Zhang; Hagstrom, J E; Wolff, J A

    1995-01-01

    These studies were initiated to elucidate the mechanism of DNA nuclear transport in mammalian cells. Biotin- or gold-labeled plasmid and plasmid DNA expression vectors for Escherichia coli beta-galactosidase or firefly luciferase were microinjected into the cytoplasm of primary rat myotubes in culture. Plasmid DNA was expressed in up to 70% of the injected myotubes, which indicates that it entered intact, postmitotic nuclei. The nuclear transport of plasmid DNA occurred through the nuclear po...

  10. Replication of plasmids in gram-negative bacteria.

    OpenAIRE

    1989-01-01

    Replication of plasmid deoxyribonucleic acid (DNA) is dependent on three stages: initiation, elongation, and termination. The first stage, initiation, depends on plasmid-encoded properties such as the replication origin and, in most cases, the replication initiation protein (Rep protein). In recent years the understanding of initiation and regulation of plasmid replication in Escherichia coli has increased considerably, but it is only for the ColE1-type plasmids that significant biochemical d...

  11. Temperature Depended Role of Shigella flexneri Invasion Plasmid on the Interaction with Acanthamoeba castellanii

    Directory of Open Access Journals (Sweden)

    Amir Saeed

    2012-01-01

    Full Text Available Shigella flexneri is a Gram-negative bacterium causing the diarrhoeal disease shigellosis in humans. The virulence genes required for invasion are clustered on a large 220 kb plasmid encoding type three secretion system (TTSS apparatus and virulence factors such as adhesions and invasion plasmid antigens (Ipa. The bacterium is transmitted by contaminated food, water, or from person to person. Acanthamoebae are free-living amoebae (FLA which are found in diverse environments and isolated from various water sources. Different bacteria interact differently with FLA since Francisella tularensis, Vibrio cholerae, Shigella sonnei, and S. dysenteriae are able to grow inside A. castellanii. In contrast, Pseudomonas aeruginosa induces both necrosis and apoptosis to kill A. castellanii. The aim of this study is to examine the role of invasion plasmid of S. flexneri on the interaction with A. castellanii at two different temperatures. A. castellanii in the absence or presence of wild type, IpaB mutant, or plasmid-cured strain S. flexneri was cultured at 30∘C and 37∘C and the interaction was analysed by viable count of both bacteria and amoebae, electron microscopy, flow cytometry, and statistical analysis. The outcome of the interaction was depended on the temperature since the growth of A. castellanii was inhibited at 30∘C, and A. castellanii was killed by invasion plasmid mediated necrosis at 37∘C.

  12. E2F1蛋白的表达及与Sedlin蛋白共定位研究%The expression of human E2F1 and the co-localization with Sedlin

    Institute of Scientific and Technical Information of China (English)

    汪燕燕; 潘林鑫; 王梦媛; 刘晓颖; 范礼斌

    2015-01-01

    目的 研究人E2F1 蛋白在真核细胞内的表达及其与 Sedlin 蛋白的共定位. 方法 构建 pcDNA3. 1-E2F1-FLAG真核表达质粒,然后瞬时转染至 HEK 293T 细胞内, Western blot法检测E2F1蛋白的表达;瞬时单转E2F1-FLAG和共转E2F1-FLAG+Sedlin-绿色荧光蛋白( GFP)至非洲绿猴肾细胞( COS7 )内,免疫荧光制片,荧光显微镜下观察E2F1-FLAG在细胞内的定位及其和Sedlin的共定位. 结果真核表达质粒 pcDNA3. 1-E2F1-FLAG 构建成功,且在HEK 293T细胞中能够成功表达,在COS7 细胞中主要定位在细胞核,且与Sedlin共定位于细胞核. 结论 人E2F1 在HEK 293T、COS7细胞中都能够正常表达,且与 Sedlin蛋白存在共定位现象,为进一步了解人E2F1的功能及其蛋白质相互作用提供了一定的细胞学基础.%Objective To investigate the expression and localization of E2F1 in eukaryotic cells and the co-locali-zation with Sedlin. Methods To construct eukaryotic expression plasmid pcDNA3. 1-E2F1-FLAG. The plsamid was transfected into HEK 293T cells and the expression was checked by Western blot. Its expression in COS7 cells was detected by fluorescence microscopy. Results The eukaryotic expression plasmid of pcDNA3. 1-E2F1-FLAG was constructed successfully, which could be effectively expressed in HEK 293T and COS7 cells. E2F1 was pre-dominantly distributed in the nucleus, and it co-localized in the nucleus with Sedlin. Conclusion E2F1 can over-express efficiently in HEK 293T and COS7 cells, and it can colocalize with Seldin, which provides important bases for further study on E2F1 including its functions and associated proteins.

  13. Plasmid Segregation: Spatial Awareness at the Molecular Level

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Gerdes, Kenn

    2007-01-01

    In bacteria, low-copy number plasmids ensure their stable inheritance by partition loci (par), which actively distribute plasmid replicates to each side of the cell division plane. Using time-lapse fluorescence microscopic tracking of segregating plasmid molecules, a new study provides novel insi...

  14. Cloning of Two Bacteriocin Genes from a Lactococcal Bacteriocin Plasmid

    NARCIS (Netherlands)

    Belkum, Marco J. van; Hayema, Bert Jan; Geis, Arnold; Kok, Jan; Venema, Gerard

    1989-01-01

    Lactococcus lactis subsp. cremoris 9B4 plasmid p9B4-6 (60 kilobases [kb]), which specifies bacteriocin production and immunity, was analyzed with restriction endonucleases, and fragments of this plasmid were cloned into shuttle vectors based on the broad-host-range plasmid pWVO1. Two regions on p9B4

  15. Multilocus sequence typing of IncN plasmids

    DEFF Research Database (Denmark)

    García-Fernández, Aurora; Villa, Laura; Moodley, Arshnee

    2011-01-01

    categorization of IncN plasmids. METHODS: Twelve fully sequenced IncN plasmids available at GenBank were analysed in silico for selecting the loci for the IncN-specific pMLST. A total of 58 plasmids originating from different reservoirs (human, pig, poultry, cattle and horses) and geographic regions (Italy...

  16. 4-Hydroxybenzyl modification of the highly teratogenic retinoid, 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB), yields a compound that induces apoptosis in breast cancer cells and shows reduced teratogenicity.

    Science.gov (United States)

    Anding, Allyson L; Nieves, Nirca J; Abzianidze, Victoria V; Collins, Michael D; Curley, Robert W; Clagett-Dame, Margaret

    2011-11-21

    Retinoids are a class of compounds with structural similarity to vitamin A. These compounds inhibit the proliferation of many cancer cell lines but have had limited medical application as they are often toxic at therapeutic levels. Efforts to synthesize retinoids with a greater therapeutic index have met with limited success. 4-[(1E)-2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthalenyl)-1-propen-1-yl]benzoic acid (TTNPB) is one of the most biologically active all-trans-retinoic acid (atRA) analogues and is highly teratogenic. In this study, we show that modification of the TTNPB carboxyl group with an N-(4-hydroxyphenyl)amido (4HPTTNPB) or a 4-hydroxybenzyl (4HBTTNPB) group changes the activity of the compound in cell culture and in vivo. Unlike TTNPB, both compounds induce apoptosis in cancer cells and bind poorly to the retinoic acid receptors (RARs). Like the similarly modified all-trans-retinoic acid (atRA) analogues N-(4-hydroxyphenyl)retinamide (4-HPR/fenretinide) and 4-hydroxybenzylretinone (4-HBR), 4HBTTNPB is a potent activator of components of the ER stress pathway. The amide-linked analogue, 4HPTTNPB, is less toxic to developing embryos than the parent TTNPB, and most significantly, the 4-hydroxybenzyl-modified compound (4HBTTNPB) that cannot be hydrolyzed in vivo to the parent TTNPB compound is nearly devoid of teratogenic liability.

  17. Analysis list: E2f4 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E2f4 Blood,Embryonic fibroblast,Liver,Muscle,Pluripotent stem cell + mm9 http://dba...rchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4....5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f4.10.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/mm9/colo/E2f4.Blood.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f4....Embryonic_fibroblast.tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f4.Liver.tsv,htt

  18. An E2-Substituted Chimeric Pestivirus With DIVA Vaccine Properties

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Nielsen, Jens

    An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...... engineered specifically for this purpose. The E2-substituted Riems26_E2gif was derived by homologues recombination of the complete E2 protein encoding genome region from Border disease strain Gifhorn into a bacterial artificial chromosome (BAC) harbouring the genome of the CSFV vaccine strain C......-Riems. The virulence, immunogenicity and vaccine properties of Riems26_E2gif were tested in a vaccine-challenge experiment in pigs. Riems26_E2gif vaccinated pigs could be differentiated from infected pigs using a CSFV-E2 specific ELISA. Following challenge infection with highly virulent CSFV strain Koslov, all...

  19. Bacteriophages limit the existence conditions for conjugative plasmids.

    Science.gov (United States)

    Harrison, Ellie; Wood, A Jamie; Dytham, Calvin; Pitchford, Jonathan W; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A

    2015-06-02

    Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. Conjugative plasmids are infectious loops of DNA capable of transmitting DNA between bacterial cells and between species. Because plasmids often carry extra genes that allow bacteria to live in otherwise-inhospitable environments, their dynamics are central to understanding bacterial adaptive evolution. The plasmid-bacterium interaction has typically been studied in isolation, but in natural bacterial communities, bacteriophages, viruses that infect bacteria, are ubiquitous. Using experiments, mathematical models, and computer simulations we show that bacteriophages drive plasmid dynamics through their ecological and evolutionary effects on bacteria and ultimately

  20. Amplification of the E2F1 transcription factor gene in the HEL erythroleukemia cell line

    DEFF Research Database (Denmark)

    Saito, M; Helin, K; Valentine, M B;

    1995-01-01

    and overexpressed in HEL erythroleukemia cells and translocated to other chromosomes in several established human leukemia cell lines. This study provides the first evidence of gene amplification involving a member of the E2F family of transcription factors. We propose that E2F1 overexpression in erythroid......The E2F transcription factor plays an important regulatory role in cell proliferation, mediating the expression of genes whose products are essential for inducing resting cells to enter the cell cycle and synthesize DNA. To investigate the possible involvement of E2F in hematopoietic malignancies...... progenitors may stimulate abnormal cell proliferation by overriding negative regulatory signals mediated by tumor suppressor proteins such as pRb....

  1. Bacteriophage selection against a plasmid-encoded sex apparatus leads to the loss of antibiotic-resistance plasmids.

    Science.gov (United States)

    Jalasvuori, Matti; Friman, Ville-Petri; Nieminen, Anne; Bamford, Jaana K H; Buckling, Angus

    2011-12-23

    Antibiotic-resistance genes are often carried by conjugative plasmids, which spread within and between bacterial species. It has long been recognized that some viruses of bacteria (bacteriophage; phage) have evolved to infect and kill plasmid-harbouring cells. This raises a question: can phages cause the loss of plasmid-associated antibiotic resistance by selecting for plasmid-free bacteria, or can bacteria or plasmids evolve resistance to phages in other ways? Here, we show that multiple antibiotic-resistance genes containing plasmids are stably maintained in both Escherichia coli and Salmonella enterica in the absence of phages, while plasmid-dependent phage PRD1 causes a dramatic reduction in the frequency of antibiotic-resistant bacteria. The loss of antibiotic resistance in cells initially harbouring RP4 plasmid was shown to result from evolution of phage resistance where bacterial cells expelled their plasmid (and hence the suitable receptor for phages). Phages also selected for a low frequency of plasmid-containing, phage-resistant bacteria, presumably as a result of modification of the plasmid-encoded receptor. However, these double-resistant mutants had a growth cost compared with phage-resistant but antibiotic-susceptible mutants and were unable to conjugate. These results suggest that bacteriophages could play a significant role in restricting the spread of plasmid-encoded antibiotic resistance.

  2. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    Science.gov (United States)

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins.

  3. The transcription factors CREB and c-Fos play key roles in NCAM-mediated neuritogenesis in PC12-E2 cells

    DEFF Research Database (Denmark)

    Jessen, U; Novitskaya, V; Pedersen, N;

    2001-01-01

    roles in this process by estimating NCAM-dependent neurite outgrowth from PC12-E2 cells grown in co-culture with NCAM-negative or NCAM-positive fibroblasts. PC12-E2 cells were transiently transfected with expression plasmids encoding wild-type or dominant negative forms of CREB and c-Fos or an activated...... thus present evidence that NCAM-mediated neurite outgrowth involves a series of signal transduction pathways, including the cAMP/PKA pathway, targeting c-Fos and CREB....

  4. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET, a new method for plasmid reconstruction from whole genome sequences.

    Directory of Open Access Journals (Sweden)

    Val F Lanza

    2014-12-01

    Full Text Available Bacterial whole genome sequence (WGS methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage, comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC, comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  5. Plasmid flux in Escherichia coli ST131 sublineages, analyzed by plasmid constellation network (PLACNET), a new method for plasmid reconstruction from whole genome sequences.

    Science.gov (United States)

    Lanza, Val F; de Toro, María; Garcillán-Barcia, M Pilar; Mora, Azucena; Blanco, Jorge; Coque, Teresa M; de la Cruz, Fernando

    2014-12-01

    Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

  6. INSCRIPTION OF UYUK-ARJAN (E 2 E 2 - UYUK-ARJAN (Tuva YAZITI

    Directory of Open Access Journals (Sweden)

    Osman Fikri SERTKAYA

    2011-01-01

    Full Text Available Texts of the tombstone texts, known as Yenisei (Enisei inscriptions, which were found near the Yenisei river banks, are important in terms of Turkic philology and dialectology. The inscription Uyuk-Arjan (E-2, which was found in 1888 by J. R. Aspelin near the Uyuk riverbed, was published by Radloff, Orkun, Malov, Batmanov, Amonjolov and Kormushin previously. This paper is an attempt for the re-reading and re-interpretation of this tombstone text. To this paper resourches in the field are given a resource. Yenisey nehri kıyıları boyunca bulunan Yenisey yazıtlarının metinleri, genel olarak Eski Türk filolojisi, özel olarak da Eski Türk diyalektolojisi bakımından önemlidir. Bu yazıya konu olan mezar yazıtı Uyuk-Arjan (E 2, J. R. Aspelin tarafından 1888’de Uyuk nehri yatağı yakınlarında bulunmuş ve daha önce Radloff, Orkun, Malov, Batmanov, Amonjolov ve Kormuşin tarafından yayımlanmıştır. Bu makale, yazıt metninin yeniden okuma ve anlamlandırma denemesidir. Daha önceki okuma ve anlamlandırmalar da değerlendirilerek, alan araştırmacılarının dikkatlerine sunulmuştur.

  7. Role of Plasmid in Production of Acetobacter Xylinum Biofilms

    Directory of Open Access Journals (Sweden)

    Abbas Rezaee

    2005-01-01

    Full Text Available Acetobacter xylinum has the ability to produce cellulotic biofilms. Bacterial cellulose is expected to be used in many industrial or biomedical materials for its unique characteristics. A. xylinum contains a complex system of plasmid DNA molecules. A 44 kilobases (kb plasmid was isolated in wild type of A. xylinum. To improve the cellulose producing ability of A. xylinum, role of the plasmid in production of cellulose was studied. The comparisons between wild type and cured cells of A. xylinum showed that there is considerably difference in cellulose production. In order to study the relationship between plasmid and the rate of cellulose production, bacteria were screened for plasmid profile by a modified method for preparation of plasmid. This method yields high levels of pure plasmid DNA that can be used for common molecular techniques, such as digestion and transformation, with high efficiency.

  8. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    at high frequencies from diverse donors, I showed plasmid or donor dependence of plasmid transfer to other species. Additionally, environmental factors like stress also impact the permissiveness of phylogenetic groups towards plasmids. The developed method and results increase our ability to predict......Horizontal transfer of mobile genetic elements facilitates adaptive and evolutionary processes in bacteria. Among the known mobile genetic elements, plasmids can confer their hosts with accessory adaptive traits, such as antibiotic or heavy metal resistances, or additional metabolic pathways...... and the extent of bacterial phyla permissive towards plasmid receipt are largely unknown. Historically, methods exploring the underlying genetic and environmental factors of plasmid transfer have been heavily reliant on cultivation and expression of plasmid encoded phenotypes. This has provided an incomplete...

  9. Modeling sRNA-Regulated Plasmid Maintenance

    Science.gov (United States)

    Klumpp, Stefan

    2017-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin’s mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, a short half-life of the protein toxin is also beneficial to the function of the toxin-antitoxin system. In addition, we study a therapeutic scenario in which a competitor mRNA is introduced to sequester the sRNA antitoxin, causing the toxic protein to be expressed. PMID:28085919

  10. Modeling sRNA-regulated Plasmid Maintenance

    CERN Document Server

    Gong, Chen Chris

    2016-01-01

    We study a theoretical model for the toxin-antitoxin (hok/sok) mechanism for plasmid maintenance in bacteria. Toxin-antitoxin systems enforce the maintenance of a plasmid through post-segregational killing of cells that have lost the plasmid. Key to their function is the tight regulation of expression of a protein toxin by an sRNA antitoxin. Here, we focus on the nonlinear nature of the regulatory circuit dynamics of the toxin-antitoxin mechanism. The mechanism relies on a transient increase in protein concentration rather than on the steady state of the genetic circuit. Through a systematic analysis of the parameter dependence of this transient increase, we confirm some known design features of this system and identify new ones: for an efficient toxin-antitoxin mechanism, the synthesis rate of the toxin's mRNA template should be lower that of the sRNA antitoxin, the mRNA template should be more stable than the sRNA antitoxin, and the mRNA-sRNA complex should be more stable than the sRNA antitoxin. Moreover, ...

  11. Analysis list: E2f1 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available E2f1 Blood,Liver + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.1....tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/E2f1.5.tsv http://dbarchive.biosciencedbc.jp/kyus...hu-u/mm9/target/E2f1.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/E2f1.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/E2f1.Liver.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Liver.gml ...

  12. Analysis list: Gtf2e2 [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Gtf2e2 Blood,Liver + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gtf2e2.1.tsv http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Gtf2e2.5.tsv http://dbarchive.biosciencedbc.j...p/kyushu-u/mm9/target/Gtf2e2.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gtf2e2.Blood.tsv,http:...//dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Gtf2e2.Liver.tsv http://dbarchiv...e.biosciencedbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Liver.gml ...

  13. ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines.

    Directory of Open Access Journals (Sweden)

    Lilia Espada

    Full Text Available In this study we demonstrate that accumulation of reactive oxygen species (ROS is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell

  14. PGE2受体EP2和EP4调节CIA小鼠脾B细胞表面分子和细胞因子表达%Prostaglandin E2 receptors,EP2 and EP4,regulate expression of surface molecules and cytokines in B-cells of collagen-induced arthritic mice

    Institute of Scientific and Technical Information of China (English)

    张敬各; 陈海英; 秦瑾; 丛斌; 李巧霞; 贾娴娴; 马春玲; 于峰

    2011-01-01

    目的:探讨前列腺素E2(PGE2)受体EP2和EP4在胶原诱导性关节炎(CIA)小鼠脾B细胞免疫调节中的作用.方法:建立CIA小鼠模型,用CD19+ 免疫磁珠分选脾B细胞,流式细胞术检测MHCⅡ、CD80和CD86的表达,实时荧光定量PCR技术检测EPs 和细胞因子IFN-γ、TNF-α、IL-6、IL-4、IL-10和TGF-β的表达.结果:小鼠脾B细胞表达EP的4个亚型,CIA模型小鼠EP2和EP4表达增加;EP2阻断剂可以降低MHCⅡ、CD80和CD86的表达,而EP4阻断剂对CD80没有明显影响;EP2和EP4阻断剂均可以降低IFN-γ、TNF-α 和IL-6的表达(P<0.05或P<0.01),促进IL-10的表达(P<0.01或P<0.05),并可以分别促进IL-4和TGF-β的表达(P<0.01).结论:PGE2可通过EP2/EP4调节B细胞表面分子和细胞因子参与CIA发病,EP2/EP4有可能成为类风湿关节炎治疗的新靶点.%AIM : To observe the immunoregulatory effects of prostaglandin E2 receptor ( EP ) subtypes EP2/EP4 on the B - cells of collagen - induced arthritic( CIA )mice.METHODS : DBA/1 mice were immunized with chicken type Ⅱ collagen emulsified in Freund's complete adjuvant to induce arthritis.B - cells were isolated from the splenocyte suspension by positive selection using anti - CD19 monoclonal antihody immunomagnetic heads.The expression of MHC Ⅱ,CD 80 and CD86 was examined by flow cytometry.The mRNA levels of EPs, interferon γ ( IFN -γ ), tumor necrosis factor α ( TNF - α ), IL -6, IL -4, IL - 10 and transforming growth factor - β ( TGF - β ) were detected by real - time RT PCR.RESULTS : The rank of the mRNA levels of EPs was EP2 > EP1 > EP3 > EP4 in B - cells and EP2/EP4 mRNA expression was obviously increased in CIA mice.EP2 antagonists inhihited the expression of MHC Ⅱ.CD80 and CD86.EP4 antagonist had little effect on CD80.EP2/EP4 antagonists inhibited the mRNA expression of IFN - γ , TNF - α , and IL 6 ( P < 0.05 or P <0.01 ) and increased the expression of IL - 10 ( P <0.01 or P <0.05 ).Furthermore, the

  15. E2F-1 as an anticancer drug target

    Directory of Open Access Journals (Sweden)

    Joseph R. Bertino

    2011-12-01

    Full Text Available Mounting evidence indicates that the E2F transcription factors play an essential role in all aspects of cellular functions. Many human malignancies have been shown to overexpress one or more of the ‘‘activating’’ E2Fs. In some circumstances, down regulation as well as overexpression of E2F-1, leads to inhibition of cell growth. The emphasis in this review is placed on new data implicating microRNAs in the regulation of E2F activity and the efforts thus far to target this activity in order to cause tumor regression.

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

    Science.gov (United States)

    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.

  17. pRB-E2F1 complexes are resistant to adenovirus E1A-mediated disruption.

    Science.gov (United States)

    Seifried, L A; Talluri, S; Cecchini, M; Julian, L M; Mymryk, J S; Dick, F A

    2008-05-01

    Disruption of pRB-E2F interactions by E1A is a key event in the adenoviral life cycle that drives expression of early viral transcription and induces cell cycle progression. This function of E1A is complicated by E2F1, an E2F family member that controls multiple processes besides proliferation, including apoptosis and DNA repair. Recently, a second interaction site in pRB that only contacts E2F1 has been discovered, allowing pRB to control proliferation separately from other E2F1-dependent activities. Based on this new insight into pRB-E2F1 regulation, we investigated how E1A affects control of E2F1 by pRB. Our data reveal that pRB-E2F1 interactions are resistant to E1A-mediated disruption. Using mutant forms of pRB that selectively force E2F1 to bind through only one of the two binding sites on pRB, we determined that E1A is unable to disrupt E2F1's unique interaction with pRB. Furthermore, analysis of pRB-E2F complexes during adenoviral infection reveals the selective maintenance of pRB-E2F1 interactions despite the presence of E1A. Our experiments also demonstrate that E2F1 functions to maintain cell viability in response to E1A expression. This suggests that adenovirus E1A's seemingly complex mechanism of disrupting pRB-E2F interactions provides selectivity in promoting viral transcription and cell cycle advancement, while maintaining cell viability.

  18. E2F1-mediated transcriptional inhibition of the plasminogen activator inhibitor type 1 gene

    DEFF Research Database (Denmark)

    Koziczak, M; Müller, H; Helin, K;

    2001-01-01

    -sensitive retinoblastoma protein (pRB), a shift to a permissive temperature induced PAI-1 mRNA expression. In U2OS cells stably expressing an E2F1-estrogen receptor chimeric protein that could be activated by tamoxifen, PAI-1 gene transcription was markedly reduced by tamoxifen even in the presence of cycloheximide...

  19. CARTOGRAPHIE DU PLASMIDE pSU100, PLASMIDE CRYPTIQUE DE LACTOBACILLUS CASEI

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    F BENSALAH

    2003-06-01

    Ce plasmide appelé pSU100 a été cloné dans le vecteur de transformation pUC18 au site EcoRI chez E. coli JM103. Les profils électrophorétiques de restriction obtenus par des digestions simples, doubles et triples sous l’action de 33 endonucléases, ont contribué à l’élaboration d’une carte de restriction de ce plasmide. Cinq sites uniques ont été identifiés, ainsi que d’autres sites doubles et multiples. Une étude préliminaire du rôle physiologique de ce plasmide a permis de déceler une résistance à la kanamycine.

  20. Plasmid-cured Chlamydia caviae activates TLR2-dependent signaling and retains virulence in the guinea pig model of genital tract infection.

    Directory of Open Access Journals (Sweden)

    Lauren C Frazer

    Full Text Available Loss of the conserved "cryptic" plasmid from C. trachomatis and C. muridarum is pleiotropic, resulting in reduced innate inflammatory activation via TLR2, glycogen accumulation and infectivity. The more genetically distant C. caviae GPIC is a natural pathogen of guinea pigs and induces upper genital tract pathology when inoculated intravaginally, modeling human disease. To examine the contribution of pCpGP1 to C. caviae pathogenesis, a cured derivative of GPIC, strain CC13, was derived and evaluated in vitro and in vivo. Transcriptional profiling of CC13 revealed only partial conservation of previously identified plasmid-responsive chromosomal loci (PRCL in C. caviae. However, 2-deoxyglucose (2DG treatment of GPIC and CC13 resulted in reduced transcription of all identified PRCL, including glgA, indicating the presence of a plasmid-independent glucose response in this species. In contrast to plasmid-cured C. muridarum and C. trachomatis, plasmid-cured C. caviae strain CC13 signaled via TLR2 in vitro and elicited cytokine production in vivo similar to wild-type C. caviae. Furthermore, inflammatory pathology induced by infection of guinea pigs with CC13 was similar to that induced by GPIC, although we observed more rapid resolution of CC13 infection in estrogen-treated guinea pigs. These data indicate that either the plasmid is not involved in expression or regulation of virulence in C. caviae or that redundant effectors prevent these phenotypic changes from being observed in C. caviae plasmid-cured strains.

  1. Plasmid transfer between bacteria in soil microcosms and the field

    Directory of Open Access Journals (Sweden)

    Eric Smit

    1997-01-01

    Full Text Available In ibis review factors influencing conjugal plasmid transfer between bacteria and the possible role of naturally occurring selftransmissible plasmide for the dissemination of recombinant DNA in soil will be discussed. In microcosm studies, plasmid transfer between various species of introduced bacteria has been detected. Moreover, plamid transfer to indigenous soil micoorganisms was observed. Soil is an oligotrophic environment and plasmid transfer occurred mainly under conditions which were nutritionally favourable for bacteria, such as in the plant rhizosphere and in the presence of clay minerais or added nutrients. Mobilizable plasmids, lacking the ability to transfer themselves, have been reported to be transferred in the presence of selftransmissible plasmids. A study comparing conjugal transfer in microcosme with those in the field revealed that the transfer rates found in microcosme and in the field were similar. Transfer of chromosomal DNA by plasmid RP4 could only be shown on filters and was not observed in soil. Transfer of plasmids carrying biodegradative genes appeared to be favoured in the presence of the compound that can be degraded. Evidence was found for the presence of naturally-occurring selftransmissible plasmids in bacteria in the rhizosphere which could mobilize recombinant plasmids.

  2. Isolation and screening of plasmids from the epilithon which mobilize recombinant plasmid pD10.

    OpenAIRE

    Hill, K E; A. J. Weightman; Fry, J C

    1992-01-01

    This study examined the potential of bacteria from river epilithon to mobilize a recombinant catabolic plasmid, pD10, encoding 3-chlorobenzoate degradation and kanamycin resistance. Fifty-four mobilizing plasmids were exogenously isolated by triparental matings between strains of Pseudomonas putida and epilithic bacteria from the River Taff (South Wales, United Kingdom). Frequencies for mobilization ranged from 1.7 x 10(-8) to 4.5 x 10(-3) per recipient at 20 degrees C. The sizes of the mobil...

  3. Effect of cumin (Cuminum cyminum) seed essential oil on biofilm formation and plasmid Integrity of Klebsiella pneumoniae.

    Science.gov (United States)

    Derakhshan, Safoura; Sattari, Morteza; Bigdeli, Mohsen

    2010-01-01

    Seeds of the cumin plant (Cuminum cyminum L.) have been used since many years in Iranian traditional medicine. We assessed the effect of cumin seed essential oil on the biofilm-forming ability of Klebsiella pneumoniae strains and on the integrity of a native resistance plasmid DNA from K. pneumoniae isolates, treated with essential oil. Antibacterial coaction between the essential oil and selected antibiotic disks were determined for inhibiting K. pneumoniae. The essential oil of the cumin seeds was obtained by hydrodistillation in a Clavenger system. A simple method for the formation of biofilms on semiglass lamellas was established. The biofilms formed were observed by scanning electron microscopy (SEM). The effect of essential oil on plasmid integrity was studied through the induction of R-plasmid DNA degradation. The plasmid was incubated with essential oil, and agarose gel electrophoresis was performed. Disk diffusion assay was employed to determine the coaction. The essential oil decreased biofilm formation and enhanced the activity of the ciprofloxacin disk. The incubation of the R-plasmid DNA with essential oil could not induce plasmid DNA degradation. The results of this study suggest the potential use of cumin seed essential oil against K. pneumoniae in vitro, may contribute to the in vivo efficacy of this essential oil.

  4. Effect of cumin (Cuminum cyminum seed essential oil on biofilm formation and plasmid Integrity of Klebsiella pneumoniae

    Directory of Open Access Journals (Sweden)

    Safoura Derakhshan

    2010-01-01

    Full Text Available Seeds of the cumin plant (Cuminum cyminum L. have been used since many years in Iranian traditional medicine. We assessed the effect of cumin seed essential oil on the biofilm-forming ability of Klebsiella pneumoniae strains and on the integrity of a native resistance plasmid DNA from K. pneumoniae isolates, treated with essential oil. Antibacterial coaction between the essential oil and selected antibiotic disks were determined for inhibiting K. pneumoniae. The essential oil of the cumin seeds was obtained by hydrodistillation in a Clavenger system. A simple method for the formation of biofilms on semiglass lamellas was established. The biofilms formed were observed by scanning electron microscopy (SEM. The effect of essential oil on plasmid integrity was studied through the induction of R-plasmid DNA degradation. The plasmid was incubated with essential oil, and agarose gel electrophoresis was performed. Disk diffusion assay was employed to determine the coaction. The essential oil decreased biofilm formation and enhanced the activity of the ciprofloxacin disk. The incubation of the R-plasmid DNA with essential oil could not induce plasmid DNA degradation. The results of this study suggest the potential use of cumin seed essential oil against K. pneumoniae in vitro, may contribute to the in vivo efficacy of this essential oil.

  5. Expression of Structural Protein E2 of Hepatitis C Virus

    Institute of Scientific and Technical Information of China (English)

    GUO Tai-lin; YE Lin-bo; MAO Can-quan

    2005-01-01

    The E2 glycoprotein is one of the structural components of the hepatitis C virus (HCV) virion. It elicits production of neutralising antibodies against the virus, and is involved in viral morphogenesis. The protein is considered as a major candidate for anti- HCV vaccine. Despitethis, little is known about this protein. Previous studies have focused on the and functional analysis of the glycosylated forms. This report describes expression of the E2 (recE2) in different forms and in different expression systems in Escherichia coli cells and in mammalian cells in order to obtain enough protein efficiently in vitro, in addition we also analysed the usage of rare codons in the genes of E2 and CORE. All results have shown that great efforts should be made to improve the expression efficiency of E2 in bacteria or mammalian cells.

  6. E2F-dependent induction of p14ARF during cell cycle re-entry in human T cells

    DEFF Research Database (Denmark)

    del Arroyo, Ana Gutierrez; El Messaoudi, Selma; Clark, Paula A

    2007-01-01

    The ARF protein, encoded by alternate exon usage within the CDKN2A locus, provides a link between the retinoblastoma (pRb) and p53 tumor suppressor pathways. Agents that disable pRb or otherwise impinge on the E2F family of transcription factors induce expression of ARF, resulting in stabilization...... of p53 and activation of p53-regulated genes. However, in some cell types ARF is not induced upon cell cycle re-entry, as expected of a conventional E2F target gene, leading to the suggestion that the ARF promoter only responds to supra-physiological or aberrant levels of E2F. These properties have...

  7. The E2A-HLF Oncoprotein Activates Groucho-Related Genes and Suppresses Runx1

    Science.gov (United States)

    Dang, Jinjun; Inukai, Takeshi; Kurosawa, Hidemitsu; Goi, Kumiko; Inaba, Toshiya; Lenny, Noel T.; Downing, James R.; Stifani, Stefano; Look, A. Thomas

    2001-01-01

    The E2A-HLF fusion gene, formed by the t(17;19)(q22;p13) chromosomal translocation in leukemic pro-B cells, encodes a chimeric transcription factor consisting of the transactivation domain of E2A linked to the bZIP DNA-binding and protein dimerization domain of hepatic leukemia factor (HLF). This oncoprotein blocks apoptosis induced by growth factor deprivation or irradiation, but the mechanism for this effect remains unclear. We therefore performed representational difference analysis (RDA) to identify downstream genetic targets of E2A-HLF, using a murine FL5.12 pro-B cell line that had been stably transfected with E2A-HLF cDNA under the control of a zinc-regulated metallothionein promoter. Two RDA clones, designated RDA1 and RDA3, were differentially upregulated in E2A-HLF-positive cells after zinc induction. The corresponding cDNAs encoded two WD40 repeat-containing proteins, Grg2 and Grg6. Both are related to the Drosophila protein Groucho, a transcriptional corepressor that lacks DNA-binding activity on its own but can act in concert with other proteins to regulate embryologic development of the fly. Expression of both Grg2 and Grg6 was upregulated 10- to 50-fold by E2A-HLF. Immunoblot analysis detected increased amounts of two additional Groucho-related proteins, Grg1 and Grg4, in cells expressing E2A-HLF. A mutant E2A-HLF protein with a disabled DNA-binding region also mediated pro-B cell survival and activated Groucho-related genes. Among the transcription factors known to interact with Groucho-related protein, only RUNX1 was appreciably downregulated by E2A-HLF. Our results identify a highly conserved family of transcriptional corepressors that are activated by E2A-HLF, and they suggest that downregulation of RUNX1 may contribute to E2A-HLF-mediated leukemogenesis. PMID:11486032

  8. Isolation of clinical strains of Pseudomonas aeruginosa harboring different plasmids.

    Science.gov (United States)

    Ranjbar, R; Owlia, P; Saderi, H; Bameri, Z; Izadi, M; Jonaidi, N; Morovvati, S

    2007-09-01

    Aim of this study was to investigate the presence of plasmids among the strains of P. aeruginosa isolated from clinically diagnosed cases in Tehran in 2006. A total of 38 strains of P. aeruginosa were isolated. With the exception of one isolate, all P. aeruginosa strains harbored at least one plasmid band. The electrophoretic analysis of plasmid DNAs showed different number of plasmid bands among the strains tested. The DNA band of 1.4 kbp was evident in 84.2% of the strains. Approximately 71 and 21% of the isolates harbored concomitantly two and three plasmids, respectively. Isolation of strains with diverse types of plasmids suggests the different cluster of P. aeruginosa might be disseminated during the current study period.

  9. Transformation of Haemophilus influenzae by plasmid RSF0885

    Energy Technology Data Exchange (ETDEWEB)

    Notani, N.K.; Setlow, J.K.; McCarthy, D.; Clayton, N.L.

    1981-12-01

    Plasmid RSF0885, which conferred ampicillin resistance, transformed competent Haemophilus influenzae cells with low efficiency (maximun, less than 0.01%). As judged by competition experiments and uptake of radioactivity, plasmid RSF0885 deoxyribonucleic acid was taken up into competent H. influenzae cells several orders of magnitude less efficiently than H. influenzae chromosomal deoxyribonucleic acid. Plasmid RSF0885 transformed cells with even lower efficiency than could be accounted for by the low uptake. Transformation was not affected by rec-1 and rec-2 mutations in the recipient, and strains cured of the plasmid did not show increased transformation. Plasmid molecules cut once with a restriction enzyme that made blunt ends did not transform. Transformation was favored by the closed circular form of the plasmid.

  10. Comparison of prostaglandin E2 gel, prostaglandin E2 pessary and extra-amniotic saline infusion with oxytocin for induction of labour.

    Science.gov (United States)

    Qamar, Saima; Bashir, Adeela; Ibrar, Faiza

    2012-01-01

    Induction of labour is the intentional initiation of cervical ripening and uterine contraction for the purpose of accomplishing delivery, prior to onset of spontaneous parturition. This study was conducted to compare maternal and neonatal outcome in women induced with Prostaglandin E2 gel, Prostaglandin E2 pessary and extra-amniotic saline infusion with oxytocin at Bishops score prostaglandin gel, prostaglandinE2 pessary and extra-amniotic saline infusion with oxytocin) were collected. Systematic sampling was done. First woman admitted was induced with prostaglandin gel, the second one with prostaglandin pessary and the third was induced with extra amniotic saline infusion and oxytocin. The most common indication for induction was post dates followed by PIH. The induction labour interval was less in EASI with oxytocin group (5.18 +/- 3.4) hours, as compared to prostaglandin pessary (8.81 +/- 5.60) hours and prostaglandin gel (8.32 +/- 5.18) hours. Induction delivery interval in EASI with oxytocin was (10 +/- 5.6) hours as compared to prostaglandin pessary (14 +/- 6.3) hours and prostaglandin gel (13 +/- 7.1) hours. This difference was statistically significant. The primigravidas had longer duration of labour than multigravidas. Induction labour interval in primigravidas was (8.2 +/- 5.1) hours while in multigravidas it was (6.7 +/- 5.02) hours. Induction delivery interval was also more in primigravidas (13.6 +/- 6.80) hours as compared to multigravidas (11.4 +/- 6.20) hours. Vaginal delivery rate was 89.2% while the caesarean section rate was 10.4%. The most common indication for caesarean section was foetal distress. There was no significant difference in perinatal morbidity and mortality in the three groups. EASI with oxytocin is a better method of induction than prostaglandin E2 gel and pessary. Moreover it is more economical in our country.

  11. Lesion of the tuberomammillary nucleus E2-region attenuates postictal seizure protection in rats.

    Science.gov (United States)

    Jin, Chun-Lei; Zhuge, Zheng-Bing; Wu, Deng-Chang; Zhu, Yuan-Yuan; Wang, Shuang; Luo, Jian-Hong; Chen, Zhong

    2007-03-01

    Postictal seizure protection (PSP) is an endogenous anticonvulsant phenomenon that follows an epileptic seizure and inhibits the induction of further seizures. The tuberomammillary nucleus (TM), located in the posterior hypothalamus, consists of five subregions and is the sole source of histaminergic neurons in the brain. To determine whether the TM is involved in PSP in rats, we tested the effects of bilateral electrolytic lesions of the TM E2-region on seizures induced by intermittent maximal electroshock (MES). The TM E2-region lesions significantly attenuated PSP during the intermittent MES procedure. Furthermore, intracerebroventricular injection of alpha-fluoromethylhistidine (100 microg), a selective and irreversible histidine decarboxylase inhibitor, mimicked the attenuation of PSP induced by the lesion of TM E2-region. In addition, neurochemical experiments revealed that the TM E2-region lesions markedly decreased basal histamine levels in the cortex, hippocampus, brainstem and hypothalamus, but had no significant effect on basal glutamate and GABA levels. Moreover, intermittent MES induced a persistent decrease of brain histamine levels in both sham-operated and lesioned rats. These results indicate that through its intrinsic histaminergic system, the TM may exert powerful inhibitory function during the intermittent MES procedure and actively participate in the mechanisms of PSP.

  12. Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum.

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    Kristin M Marshall

    Full Text Available BACKGROUND: Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs. The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative. METHODOLOGY/PRINCIPAL FINDINGS: C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3, pCLJ (strain 657Ba and pCLL (strain Eklund 17B were tagged with the erythromycin resistance marker (Erm using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor:recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism. CONCLUSIONS/SIGNIFICANCE: This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.

  13. E2F1 promote the aggressiveness of human colorectal cancer by activating the ribonucleotide reductase small subunit M2

    Energy Technology Data Exchange (ETDEWEB)

    Fang, Zejun [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Gong, Chaoju [Department of Pathology and Pathophysiology, Zhejiang University School of Medicine, Hangzhou, Zhejiang, 310058 (China); Liu, Hong [Zhejiang Normal University – Jinhua People' s Hospital Joint Center for Biomedical Research, Jinhua, Zhejiang, 321004 (China); Zhang, Xiaomin; Mei, Lingming [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Song, Mintao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (CAMS), School of Basic Medicine, Peking Union Medical College (PUMC), Beijing, 100005 (China); Qiu, Lanlan; Luo, Shuchai; Zhu, Zhihua; Zhang, Ronghui; Gu, Hongqian [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China); Chen, Xiang, E-mail: sychenxiang@126.com [Sanmen People' s Hospital of Zhejiang, Sanmen, Zhejiang, 317100 (China)

    2015-08-21

    As the ribonucleotide reductase small subunit, the high expression of ribonucleotide reductase small subunit M2 (RRM2) induces cancer and contributes to tumor growth and invasion. In several colorectal cancer (CRC) cell lines, we found that the expression levels of RRM2 were closely related to the transcription factor E2F1. Mechanistic studies were conducted to determine the molecular basis. Ectopic overexpression of E2F1 promoted RRM2 transactivation while knockdown of E2F1 reduced the levels of RRM2 mRNA and protein. To further investigate the roles of RRM2 which was activated by E2F1 in CRC, CCK-8 assay and EdU incorporation assay were performed. Overexpression of E2F1 promoted cell proliferation in CRC cells, which was blocked by RRM2 knockdown attenuation. In the migration and invasion tests, overexpression of E2F1 enhanced the migration and invasion of CRC cells which was abrogated by silencing RRM2. Besides, overexpression of RRM2 reversed the effects of E2F1 knockdown partially in CRC cells. Examination of clinical CRC specimens demonstrated that both RRM2 and E2F1 were elevated in most cancer tissues compared to the paired normal tissues. Further analysis showed that the protein expression levels of E2F1 and RRM2 were parallel with each other and positively correlated with lymph node metastasis (LNM), TNM stage and distant metastasis. Consistently, the patients with low E2F1 and RRM2 levels have a better prognosis than those with high levels. Therefore, we suggest that E2F1 can promote CRC proliferation, migration, invasion and metastasis by regulating RRM2 transactivation. Understanding the role of E2F1 in activating RRM2 transcription will help to explain the relationship between E2F1 and RRM2 in CRC and provide a novel predictive marker for diagnosis and prognosis of the disease. - Highlights: • E2F1 promotes RRM2 transactivation in CRC cells. • E2F1 promotes the proliferation of CRC cells by activating RRM2. • E2F1 promotes the migration and

  14. Photonic plasmid stability of transformed Salmonella Typhimurium: A comparison of three unique plasmids

    Directory of Open Access Journals (Sweden)

    Lay Donald

    2009-07-01

    Full Text Available Abstract Background Acquiring a highly stable photonic plasmid in transformed Salmonella Typhimurium for use in biophotonic studies of bacterial tracking in vivo is critical to experimental paradigm development. The objective of this study was to determine stability of transformed Salmonella Typhimurium (S. typh-lux using three different plasmids and characterize their respective photonic properties. Results In presence of ampicillin (AMP, S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 plasmids exhibited 100% photon-emitting colonies over a 10-d study period. Photon emitters of S. typh-lux with pCGLS-1, pAK1-lux and pXEN-1 without AMP selection decreased over time (P 7 to 1 × 109 CFU, P 0.05; although photonic emissions across a range of bacterial concentrations were not different (1 × 104 to 1 × 106 CFU, P > 0.05. For very low density bacterial concentrations imaged in 96 well plates photonic emissions were positively correlated with bacterial concentration (P 3 to 1 × 105 CFU low to high were different in the 96-well plate format (P Conclusion These data characterize photon stability properties for S. typh-lux transformed with three different photon generating plasmids that may facilitate real-time Salmonella tracking using in vivo or in situ biophotonic paradigms.

  15. Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

    Science.gov (United States)

    Aps, Luana R M M; Tavares, Milene B; Rozenfeld, Julio H K; Lamy, M Teresa; Ferreira, Luís C S; Diniz, Mariana O

    2016-06-20

    Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

  16. Bacteriophages Limit the Existence Conditions for Conjugative Plasmids

    Science.gov (United States)

    Wood, A. Jamie; Dytham, Calvin; Pitchford, Jonathan W.; Truman, Julie; Spiers, Andrew; Paterson, Steve; Brockhurst, Michael A.

    2015-01-01

    ABSTRACT Bacteriophages are a major cause of bacterial mortality and impose strong selection on natural bacterial populations, yet their effects on the dynamics of conjugative plasmids have rarely been tested. We combined experimental evolution, mathematical modeling, and individual-based simulations to explain how the ecological and population genetics effects of bacteriophages upon bacteria interact to determine the dynamics of conjugative plasmids and their persistence. The ecological effects of bacteriophages on bacteria are predicted to limit the existence conditions for conjugative plasmids, preventing persistence under weak selection for plasmid accessory traits. Experiments showed that phages drove faster extinction of plasmids in environments where the plasmid conferred no benefit, but they also revealed more complex effects of phages on plasmid dynamics under these conditions, specifically, the temporary maintenance of plasmids at fixation followed by rapid loss. We hypothesized that the population genetic effects of bacteriophages, specifically, selection for phage resistance mutations, may have caused this. Further mathematical modeling and individual-based simulations supported our hypothesis, showing that conjugative plasmids may hitchhike with phage resistance mutations in the bacterial chromosome. PMID:26037122

  17. Plasmid genes required for microcin B17 production.

    Science.gov (United States)

    San Millán, J L; Kolter, R; Moreno, F

    1985-09-01

    The production of the antibiotic substance microcin B17 (Mcc) is determined by a 3.5-kilobase DNA fragment from plasmid pMccB17. Several Mcc- mutations on plasmid pMccB17 were obtained by both transposon insertion and nitrosoguanidine mutagenesis. Plasmids carrying these mutations were tested for their ability to complement Mcc- insertion or deletion mutations on pMM102 (pMM102 is a pBR322 derivative carrying the region encoding microcin B17). Results from these experiments indicate that at least four plasmid genes are required for microcin production.

  18. Plasmid P1 replication: negative control by repeated DNA sequences.

    OpenAIRE

    Chattoraj, D; Cordes, K.; Abeles, A

    1984-01-01

    The incompatibility locus, incA, of the unit-copy plasmid P1 is contained within a fragment that is essentially a set of nine 19-base-pair repeats. One or more copies of the fragment destabilizes the plasmid when present in trans. Here we show that extra copies of incA interfere with plasmid DNA replication and that a deletion of most of incA increases plasmid copy number. Thus, incA is not essential for replication but is required for its control. When cloned in a high-copy-number vector, pi...

  19. [Isolation of the R'his plasmids of Vibrio cholerae].

    Science.gov (United States)

    Rusina, O Iu; Tiganova, I G; Aleshkin, G I; Andreeva, I V; Skavronskaia, A G

    1987-06-01

    V. cholerae strain VT5104 capable of donor activity in conjugation has been constructed by the genetic technique based on plasmid RP4::Mucts62 integration into V. cholerae chromosome due to plasmid homology with Mucts62 inserted into the chromosome. The gene for histidine synthesis has been mobilized and transferred into the recipient cells from VT5104 donor. The conjugants obtained are able to efficiently transfer his+ gene included into the plasmid structure in conjugation with eltor recipient. Thus, the constructed strain VT5104 generates R' plasmids carrying V. cholerae chromosomal genes.

  20. Degradative Plasmid and Heavy Metal Resistance Plasmid Naturally Coexist in Phenol and Cyanide Assimilating Bacteria

    Directory of Open Access Journals (Sweden)

    Bahig E.  Deeb

    2009-01-01

    Full Text Available Problem statement: Heavy metals are known to be powerful inhibitors of xenobiotics biodegradation activities. Alleviation the inhibitory effect of these metals on the phenol biodegradation activities in presence of heavy metals resistant plasmid was investigated. Approach: Combination of genetic systems of degradation of xenobiotic compound and heavy metal resistance was one of the approaches to the creation of polyfunctional strains for bioremediation of soil after co-contamination with organic pollutants and heavy metals. Results: A bacterial strain Pseudomonas putida PhCN (pPhCN1, pPhCN2 had been obtained. This bacterium contained two plasmids, a 120 Kb catabolic plasmid that encode for breakdown of phenol (pPhCN1 and pPhCN2 plasmid (100 Kb that code for cadmium and copper resistant. Cyanide assimilation by this bacterium was encoded by chromosomal genes. The inhibitory effect of cadmium (Cd2+ or copper (Cu2+ on the degradation of phenol and cyanide by P. putida strains PhCN and PhCN1 (contained pPhCN1 were investigated. The resistant strain PhCN showed high ability to degrade phenol and cyanide in presence of Cd2+ or Cu2+ comparing with the sensitive strain PhCN1. In addition, Cd2+ or Cu2+ was also found to exert a strong inhibitory effect on the C23O dioxygenase enzyme activity in the presence of cyanide as a nitrogen source. Conclusion: The presence of heavy metal resistance plasmid alleviated the inhibitory effect of metals on the phenol and cyanide assimilation by resistant strain.

  1. Gamma-ray spectrometer onboard Chang'E-2

    Science.gov (United States)

    Ma, T.; Chang, J.; Zhang, N.; Jian, W.; Cai, M. S.; Gong, Y. Z.; Tang, H. S.; Zhang, R. J.; Wang, N. S.; Yu, M.; Mao, J. P.; Hu, Y. M.; Xu, A. A.; Zhu, M. H.

    2013-10-01

    Chang'E-2 gamma-ray spectrometer (GRS) is included in the payload of Chinese second lunar mission Chang'E-2 that has been launched in October 2010. Specific objectives of the GRS are to map abundance of O, Si, Fe, Ti, U, Th, K, and, perhaps, Mg, Al, and Ca, to depth of about 20 cm. The energy resolution and detection efficiency were improved compared with Chang'E-1 GRS. We will describe the design of GRS, which used LaBr3 for its main detector, and present its performance in this paper. Moreover, the initial result of Chang'E-2 GRS is reported.

  2. A Broad View of the Chang'e 2 Mission

    Institute of Scientific and Technical Information of China (English)

    Pang Dan

    2010-01-01

    @@ China's second lunar exploration satellite Chang'e 2 was launched on October 1 2010 from the Xichang Satellite Launch Center.The satellite was sent directly into an Earth-moon transfer orbit on a LM-3C launch vehicle.Five days later, the satellite reached a preliminary orbit 100km above the moon.All the payloads onboard Chang'e 2 have been operational since October 15,signifying a good start to Chang'e 2's six-month observation mission.

  3. E2F6 Impairs Glycolysis and Activates BDH1 Expression Prior to Dilated Cardiomyopathy

    Science.gov (United States)

    Major, Jennifer L.; Dewan, Aaraf; Salih, Maysoon; Leddy, John J.; Tuana, Balwant S.

    2017-01-01

    Rationale The E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme β-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker. Objective To define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1. Methods and Results We previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the Bdh1 transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone β-hydroxybutyrate (β-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to β-OHB. Conclusions The data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium

  4. [A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance].

    Science.gov (United States)

    Sahin, Fikret; Karasartova, Djursun; Gerçeker, Devran; Aysev, A Derya; Erdem, Birsel

    2008-07-01

    In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

  5. WRKY40 and WRKY6 act downstream of the green leaf volatile E-2-hexenal in Arabidopsis.

    Science.gov (United States)

    Mirabella, Rossana; Rauwerda, Han; Allmann, Silke; Scala, Alessandra; Spyropoulou, Eleni A; de Vries, Michel; Boersma, Maaike R; Breit, Timo M; Haring, Michel A; Schuurink, Robert C

    2015-09-01

    Plants are known to be responsive to volatiles, but knowledge about the molecular players involved in transducing their perception remains scarce. We study the response of Arabidopsis thaliana to E-2-hexenal, one of the green leaf volatiles (GLV) that is produced upon wounding, herbivory or infection with pathogens. We have taken a transcriptomics approach to identify genes that are induced by E-2-hexenal, but not by defence hormones or other GLVs. Furthermore, by studying the promoters of early E-2-hexenal-induced genes we determined that the only statistically enriched cis-element was the W-box motif. Since members of the plant-specific family of WRKY transcription factors act in trans on this cis-element, we focused on WRKY6, 40 and 53 that were most strongly induced by E-2-hexenal. Root elongation of Arabidopsis seedlings of the wrky40 wrky6 double mutant was much less inhibited than in wt plants, similar to the E-2-hexenal-responsive mutant her1, which is perturbed in γ-amino butyric acid (GABA) metabolism. The induction of several of the E-2-hexenal-specific genes was much higher in the wrky40, wrky6 or wrky40 wrky6 mutants, including GAD4, a glutamate decarboxylase that catalyzes the formation of GABA from glutamate. In conclusion, WRKY6 and 40 seem to act as important players transducing E-2-hexenal perception.

  6. Resveratrol enhances the radiosensitivity of nasopharyngeal carcinoma cells by downregulating E2F1.

    Science.gov (United States)

    Tan, Yuhui; Wei, Xianli; Zhang, Wenyin; Wang, Xiaolan; Wang, Kun; Du, Biaoyan; Xiao, Jianyong

    2017-03-01

    Identification of safe, effective radiosensitizing agents is urgently needed to improve the outcome of radiotherapy in nasopharyngeal cancer (NPC). In this study, we assessed the ability of the polyphenol resveratrol to act as a radiosensitizer in vitro and in vivo. CNE-1 cells were treated with 50 µM resveratrol for 24 h, then irradiated. E2F transcription factor 1 (E2F1) was stably knocked down and overexpressed using lentiviruses. A xenograft model of NPC was established in nude mice using CNE-1 cells. Compared to control DMSO‑treated CNE-1 cells, resveratrol inhibited colony-forming ability and induced G1 phase cell cycle arrest. Radiation survival curves confirmed resveratrol significantly sensitized CNE-1 cells, and resveratrol in combination with 2 Gy irradiation synergistically increased apoptosis. Immunoblotting showed resveratrol dose- and time-dependently downregulated E2F1 and phospho-AKT (p-AKT). Knockdown of E2F1 significantly increased radiosensitivity and downregulated p-AKT; overexpression of E2F1 reversed resveratrol-induced radiosensitivity and upregulated p-AKT. In vivo, 50 mg/kg/day resveratrol and 4 Gy irradiation led to significantly lower tumor volume and tumor weight compared to resveratrol or irradiation alone. Our findings show that resveratrol increases the radiosensitivity of NPC cells by downregulating E2F1 and inhibiting p-AKT, and therefore has potential as a radiosensitizer for NPC.

  7. Energy Efficiency Management Platform (E2MP) Project

    Data.gov (United States)

    National Aeronautics and Space Administration — The Energy Efficiency Management Platform (E2MP) is a power-aware, "Green" computing technology for managing energy consumption on High End Computing (HEC) systems...

  8. Sustaining protein synthesis in the absence of rapid cell division: an investigation of plasmid-encoded protein expression in Escherichia coli during very slow growth.

    Science.gov (United States)

    Flickinger, M C; Rouse, M P

    1993-01-01

    The minimum growth rate capable of supporting plasmid-encoded gene expression is determined using continuous cultures of Escherichia coli MZ9387 at dilution rates (D) as low as 5% of the maximum specific growth rate. Expression from a low copy number plasmid, pMPR166, encoding cyanase under the control of P(lac) is investigated in order to study plasmid-encoded gene expression under conditions approaching starvation. Plasmid copy number was stabilized by selection in the presence of 500 micrograms/mL chloramphenicol by constitutive expression of chloramphenicol acetyl transferase (CAT). Plasmid retention was determined by dot-blot hybridization and chloramphenicol resistance. The contribution of plasmid maintenance and cyanase expression to the maximum cell yield (Y'x/s) and the maintenance coefficient (ms) was determined for MZ9387 and MZ9387:pMPR166 under uninduced and IPTG-induced conditions. The values of Y'x/s and ms for non-plasmid-bearing cultures were 0.56 g of cell dry mass (DCM)/g of glucose and 0.26 g of glucose/g of DCM.h, respectively. The cell yield for plasmid-bearing cultures under uninduced conditions (Y 0'x/s) was 0.28 g of DCM/g of glucose, with m0s = 0.08 g of glucose/g of DCM.h. These values decreased following induction of cyanase expression. Glucose consumption in the presence of IPTG was linearly related to the growth rate at D cyanase expression alters metabolism and glucose consumption. The fraction of plasmid-free cells decreased with decreasing Damköhler number (Da). These data confirm the usefulness of Da for predicting the relationship between plasmid-free and plasmid-bearing cells where plasmids are stabilized by concentrations of antibiotic greater than the minimum plasmid-free host cell growth inhibitory concentration. Specific cyanase expression increased as the dilution rate decreased to D = 0.15 h-1. Between D = 0.15 h-1 and D = 0.14 h-1, expression decreased 7-fold. At very low dilution rates (D < or = 0.06 h-1), nonseptated

  9. Nematicidal activity of (E,E)-2,4-decadienal and (E)-2-decenal from Ailanthus altissima against Meloidogyne javanica.

    Science.gov (United States)

    Caboni, Pierluigi; Ntalli, Nikoletta G; Aissani, Nadhem; Cavoski, Ivana; Angioni, Alberto

    2012-02-01

    Methanol extracts of various plant parts of Ailanthus altissima were tested against the root knot nematode Meloidogyne javanica . Extracts of bark (ABE), wood (AWE), roots (ARE), and leaves (ALE) from A. altissima were investigated against freshly hatched second-stage juveniles (J(2)). AWE was the most active extract, with EC(50/3d) of 58.9 mg/L, while ALE, ARE, and ABE did not show nematicidal activity. The chemical composition of the extracts of A. altissima was determined by gas chromatography-mass spectrometry, and (E,E)-2,4-decadienal, (E)-2-undecenal, (E)-2-decenal, hexanal, nonanal, and furfural were the most prominent constituents. (E,E)-2,4-Decadienal, (E)-2-decenal, and furfural showed the highest nematicidal activity against M. javanica , with EC(50/1d) = 11.7, 20.43, and 21.79 mg/L, respectively, while the other compounds were inactive at the concentrations tested. The results obtained showed that AWE and its constituents (E,E)-2,4-decadienal and (E)-2-decenal could be considered as potent botanical nematicidal agents.

  10. E2F1 in renal cancer: Mr Hyde disguised as Dr Jekyll?

    Science.gov (United States)

    Tian, Weihua; Cui, Fenggong; Esteban, Miguel A

    2013-10-01

    The transcription factor E2F1 has both oncogenic and tumour suppressor properties, depending on the context. Clarifying the function of E2F1 in different types of cancer is relevant because in those situations in which it acts as an oncogene there may be a route for therapeutic interference. Renal cell carcinoma is the most frequent form of kidney cancer in adults and inactivation of the von Hippel-Lindau (VHL) gene underlies most cases. This malignancy represents a challenge for standard therapies due to drug- and radio-resistance, effects that fit well within the scope of functions of E2F1. A new report by Mans et al postulates that up-regulation of E2F1 in VHL-defective renal cell carcinoma induces cell senescence and can thus be considered a good prognostic factor. Here we discuss these findings in a wider context and propose that E2F1 may actually not play a uniform role in renal cell carcinoma but rather an ambiguous one whose deeper understanding could have practical implications. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  11. Synergistic cooperation of MDM2 and E2F1 contributes to TAp73 transcriptional activity

    Energy Technology Data Exchange (ETDEWEB)

    Kasim, Vivi, E-mail: vivikasim78@gmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Huang, Can; Zhang, Jing; Jia, Huizhen; Wang, Yunxia [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Yang, Li [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China); Miyagishi, Makoto [Molecular Composite Medicine Research Group, Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba 305-8566 (Japan); Wu, Shourong, E-mail: shourongwu@hotmail.com [The Key Laboratory of Biorheological Science and Technology, Ministry of Education, College of Bioengineering, Chongqing University, Chongqing 400044 (China); The 111 Project Laboratory of Biomechanics and Tissue Repair, College of Bioengineering, Chongqing University, Chongqing 400044 (China)

    2014-07-04

    Highlights: • MDM2 is a novel positive regulator of TAp73 transcriptional activity. • MDM2 colocalizes together and physically interacts with E2F1. • Synergistic cooperation of MDM2 and E2F1 is crucial for TAp73 transcription. • MDM2 regulates TAp73 transcriptional activity in a p53-independent manner. - Abstract: TAp73, a structural homologue of p53, plays an important role in tumorigenesis. E2F1 had been reported as a transcriptional regulator of TAp73, however, the detailed mechanism remains to be elucidated. Here we reported that MDM2-silencing reduced the activities of the TAp73 promoters and the endogenous TAp73 expression level significantly; while MDM2 overexpression upregulated them. We further revealed that the regulation of TAp73 transcriptional activity occurs as a synergistic effect of MDM2 and E2F1, most probably through their physical interaction in the nuclei. Furthermore, we also suggested that MDM2 might be involved in DNA damage-induced TAp73 transcriptional activity. Finally, we elucidated that MDM2-silencing reduced the proliferation rate of colon carcinoma cells regardless of the p53 status. Our data show a synergistic effect of MDM2 and E2F1 on TAp73 transcriptional activity, suggesting a novel regulation pathway of TAp73.

  12. Chang'e 2's Journey to the Moon

    Institute of Scientific and Technical Information of China (English)

    Zong He

    2010-01-01

    @@ OCTOBER 1:INITIAL STAGE OF THE FLIGHT TO THE MOON At 18:59, October 1, 2010, a LM-3C launch vehicle blasted off from the Xichang Satellite Launch Center (XSLC) and precisely placed China's second lunar probe Chang'e 2 into the Earth-moon transfer orbit.Chang'e 2 began its 112-hour journey to the moon and the second phase of China Lunar Exploration Program was formally started.

  13. The vallue of AMH,FSH,E2 in POF%AMH、FSH、E2在卵巢早衰诊断中的价值

    Institute of Scientific and Technical Information of China (English)

    徐琳; 任莉; 韩雪松; 刘攀

    2011-01-01

    目的 研究顺铂所致化疗损伤性卵巢早衰大鼠血清AMH、FSH、E2水平变化,探讨血清AMH、FSH、E2能否作为评估成年雌性大鼠卵巢储备的早期预测指标.方法 通过给成熟雌性SD大鼠腹腔注射生理盐水、4.5mg/kg和6.0mg/kg顺铂,建立大鼠化疗损伤性卵巢早衰模型.检测血清AMH、FSH、E2水平.结果 腹腔注射后,大鼠血清AMH水平均明显降低;血清E2水平与对照组相比,低剂量组明显升高,高剂量组无统计学意义,高剂量组与低剂量组相比明显降低;血清FSH水平:低剂量组与时照组相比无统计学意义,高剂量组与对照组、低剂量组相比均升高.腹腔注射前后,各顺铂组血清AMH水平注射后均较注射前明显降低;血清E2水平于注射低剂量顺铂后升高,而注射高剂量顺铂后差异无统计学意义.各顺铂组血清FSH水平注射后均较注射前明显升高.结论 顺铂所致的卵巢损害早期血清E2代偿性升高,AMH明显降低,此两种激素的变化早于FSH的变化.血清E2的升高和AMH的降低可以作为暴露于化疗后的卵巢损害的早期预测指标,两者结合起来将有助于早期诊断卵巢早衰.%Obejective: To study the changes of serum AMH, FSH, E2 in rats of cisplatin - induced premature ovarian failure in order to investigate serum AMH, FSH, E2 which can be assessed to the early prediction indicators of ovarian reserve in adult female rats. Methods: Adult female SD rats were treated with saline, 4. 5mg/kg and 6. 0mg/kg cisplatin by intraperitoneal injection to establish successfully in rat model of chemotherapy induced premature ovarian failure. The serum AMH, FSH, E2 were measured. Results: The serum AMH levels were significantly lower in both cisplatin groups after intraperitoneal injection; The serum E2 levels were significantly higher in low - dose group but there were not significantly in high - dose group compared with the control group. The serum E2 levels were significantly

  14. The E2 Domains of APP and APLP1 Share a Conserved Mode of Dimerization

    Energy Technology Data Exchange (ETDEWEB)

    S Lee; Y Xue; J Hulbert; Y Wang; X Liu; B Demeler; Y Ha

    2011-12-31

    Amyloid precursor protein (APP) is genetically linked to Alzheimer's disease. APP is a type I membrane protein, and its oligomeric structure is potentially important because this property may play a role in its function or affect the processing of the precursor by the secretases to generate amyloid {beta}-peptide. Several independent studies have shown that APP can form dimers in the cell, but how it dimerizes remains controversial. At least three regions of the precursor, including a centrally located and conserved domain called E2, have been proposed to contribute to dimerization. Here we report two new crystal structures of E2, one from APP and the other from APLP1, a mammalian APP homologue. Comparison with an earlier APP structure, which was determined in a different space group, shows that the E2 domains share a conserved and antiparallel mode of dimerization. Biophysical measurements in solution show that heparin binding induces E2 dimerization. The 2.1 {angstrom} resolution electron density map also reveals phosphate ions that are bound to the protein surface. Mutational analysis shows that protein residues interacting with the phosphate ions are also involved in heparin binding. The locations of two of these residues, Arg-369 and His-433, at the dimeric interface suggest a mechanism for heparin-induced protein dimerization.

  15. Development of new plasmid DNA vaccine vectors with R1-based replicons

    Directory of Open Access Journals (Sweden)

    Bower Diana M

    2012-08-01

    Full Text Available Abstract Background There has been renewed interest in biopharmaceuticals based on plasmid DNA (pDNA in recent years due to the approval of several veterinary DNA vaccines, on-going clinical trials of human pDNA-based therapies, and significant advances in adjuvants and delivery vehicles that have helped overcome earlier efficacy deficits. With this interest comes the need for high-yield, cost-effective manufacturing processes. To this end, vector engineering is one promising strategy to improve plasmid production. Results In this work, we have constructed a new DNA vaccine vector, pDMB02-GFP, containing the runaway R1 origin of replication. The runaway replication phenotype should result in plasmid copy number amplification after a temperature shift from 30°C to 42°C. However, using Escherichia coli DH5α as a host, we observed that the highest yields of pDMB02-GFP were achieved during constant-temperature culture at 30°C, with a maximum yield of approximately 19 mg pDNA/g DCW being observed. By measuring mRNA and protein levels of the R1 replication initiator protein, RepA, we determined that RepA may be limiting pDMB02-GFP yield at 42°C. A mutant plasmid, pDMB-ATG, was constructed by changing the repA start codon from the sub-optimal GTG to ATG. In cultures of DH5α[pDMB-ATG], temperature-induced plasmid amplification was more dramatic than that observed with pDMB02-GFP, and RepA protein was detectable for several hours longer than in cultures of pDMB02-GFP at 42°C. Conclusions Overall, we have demonstrated that R1-based plasmids can produce high yields of high-quality pDNA without the need for a temperature shift, and have laid the groundwork for further investigation of this class of vectors in the context of plasmid DNA production.

  16. Induction of S-phase entry by E2F transcription factors depends on their nuclear localization

    DEFF Research Database (Denmark)

    Müller, H; Moroni, M C; Vigo, E

    1997-01-01

    , and they are in a pathway that is frequently found altered in human cancers. There are five members of the E2F family, and they can be divided into two functional subgroups. Whereas, upon overexpression, E2F-1, -2, and -3 induce S phase in quiescent fibroblasts and override G1 arrests mediated by the p16INK4A tumor...... cytoplasmic after the pRB family members have become phosphorylated. We propose a novel mechanism for the regulation of E2F-dependent transcription in which E2F-4 regulates transcription only from G0 until mid- to late G1 phase whereas E2F-1 is active in late G1 and S phases, until it is inactivated by cyclin...

  17. Chromosomal targeting of replicating plasmids in the yeast Hansenula polymorpha

    NARCIS (Netherlands)

    Faber, Klaas Nico; Swaving, Gert Jan; Faber, Folkert; Ab, Geert; Harder, Willem; Veenhuis, Marten; Haima, Pieter

    1992-01-01

    Using an optimized transformation protocol we have studied the possible interactions between transforming plasmid DNA and the Hansenula polymorpha genome. Plasmids consisting only of a pBR322 replicon, an antibiotic resistance marker for Escherichia coli and the Saccharomyces cerevisiae LEU2 gene we

  18. Genomic comparison of archaeal conjugative plasmids from Sulfolobus

    DEFF Research Database (Denmark)

    Greve, Bo Bjørn

    2004-01-01

    All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence...

  19. Linear plasmid in the genome of Clavibacter michiganensis subsp. sepedonicus.

    Science.gov (United States)

    Brown, Susan E; Knudson, Dennis L; Ishimaru, Carol A

    2002-05-01

    Contour-clamped homogeneous electric field gel analysis of genomic DNA of the plant pathogen Clavibacter michiganensis subsp. sepedonicus revealed the presence of a previously unreported extrachromosomal element. This new element was demonstrated to be a linear plasmid. Of 11 strains evaluated, all contained either a 90-kb (pCSL1) or a 140-kb (pCSL2) linear plasmid.

  20. Homology of plasmids in strains of unicellular cyanobacteria

    NARCIS (Netherlands)

    Hondel, C.A.M.J.J. van den; Keegstra, W.; Borrias, W.E.; Arkel, G.A. van

    1979-01-01

    Six strains of unicellular cyanobacteria were examined for the presence of plasmids. Analysis of lysates of these strains by CsCl-ethidium bromide density centrifugation yielded a major chromosomal DNA band and a minor band containing covalently closed circular plasmid DNA, as shown by electron micr

  1. Examination of uropathogenic Escherichia coli strains conferring large plasmids

    Directory of Open Access Journals (Sweden)

    SUHARTONO

    2010-04-01

    Full Text Available Suhartono (2010 Examination of uropathogenic Escherichia coli strains conferring large plasmids. Biodiversitas 11: 59-64. Of major uropathogens, Escherichia coli has been widely known as a main pathogen of UTIs globally and has considerable medical and financial consequences. A strain of UPEC, namely E. coli ST131, confers a large plasmid encoding cephalosporinases (class C β-lactamase or AmpC that may be disseminated through horizontal transfer among bacterial populations. Therefore, it is worth examining such large plasmids by isolating, purifying, and digesting the plasmid with restriction enzymes. The examination of the large plasmids was conducted by isolating plasmid DNA visualized by agarose gel electrophoresis as well as by PFGE. The relationship of plasmids among isolates was carried out by HpaI restriction enzyme digestion. Of 36 isolates of E. coli ST 131, eight isolates possessed large plasmids, namely isolates 3, 9, 10, 12, 17, 18, 26 and 30 with the largest molecular size confirmed by agarose gel electrophoresis and PFGE was ~42kb and ~118kb respectively. Restriction enzyme analysis revealed that isolates 9, 10, 12, 17 and 18 have the common restriction patterns and those isolates might be closely related.

  2. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  3. Production and pharmaceutical formulation of plasmid DNA vaccines

    NARCIS (Netherlands)

    van der Heijden, I.

    2013-01-01

    Research leading to the thesis ‘Production and pharmaceutical formulation of plasmid DNA vaccines‘ can be divided into two parts. The first part describes the development of a Good Manufacturing Practice (GMP) compliant plasmid DNA production process of pDNA vaccines for the treatment of Human papil

  4. Plasmid cloning vehicle for Haemophilus influenzae and Escherichia coli

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, D.; Clayton, N.L.; Setlow, J.K.

    1982-09-01

    A new plasmid cloning vehicle (pDM2) was used to introduce a library of Haemophilus influenzae chromosomal fragments into H. influenzae. Transformants of the higly recombination-defective rec-1 mutant were more likely to contain exclusively recombinant plasmids after exposure to ligated DNA mixtures than was the wild type. pDM2 could replicate in Escherichia coli K-12.

  5. Functional analysis of three plasmids from Lactobacillus plantarum

    NARCIS (Netherlands)

    Kranenburg, R. van; Golic, N.; Bongers, R.; Leer, R.J.; Vos, W.M. de; Siezen, R.J.; Kleerebezem, M.

    2005-01-01

    Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. Th

  6. Identification of IncA/C Plasmid Replication and Maintenance Genes and Development of a Plasmid Multilocus Sequence Typing Scheme.

    Science.gov (United States)

    Hancock, Steven J; Phan, Minh-Duy; Peters, Kate M; Forde, Brian M; Chong, Teik Min; Yin, Wai-Fong; Chan, Kok-Gan; Paterson, David L; Walsh, Timothy R; Beatson, Scott A; Schembri, Mark A

    2017-02-01

    Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies. Copyright © 2017 American Society for Microbiology.

  7. Deciphering conjugative plasmid permissiveness in wastewater microbiomes

    DEFF Research Database (Denmark)

    Jacquiod, Samuel Jehan Auguste; Brejnrod, Asker Daniel; Milani, Stefan Morberg

    2017-01-01

    Wastewater treatment plants (WWTPs) are designed to robustly treat polluted water. They are characterized by ceaseless flows of organic, chemical and microbial matter, followed by treatment steps before environmental release. WWTPs are hotspots of horizontal gene transfer between bacteria via...... still remains largely uncharted. Furthermore, current in vitro methods used to assess conjugation in complex microbiomes do not include in situ behaviours of recipient cells, resulting in partial understanding of transfers. We investigated the in vitro conjugation capacities of WWTP microbiomes from...... diversity of recipient bacterial phyla for the plasmid was observed, especially in WWTP outlets. We also identified permissive bacteria potentially able to cross WWTPs and engage in conjugation before and after water treatment. Bacterial activity and lifestyle seem to influence conjugation extent...

  8. Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

    DEFF Research Database (Denmark)

    Johnson, Timothy J.; Bielak, Eliza Maria; Fortini, Daniela;

    2012-01-01

    and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid...

  9. The retinoblastoma protein binds to a family of E2F transcription factors

    DEFF Research Database (Denmark)

    Lees, J A; Saito, M; Vidal, M;

    1993-01-01

    for E2F-2 and E2F-3 were mapped to 1p36 and 6q22, respectfully, confirming their independence from E2F-1. However, the E2F-2 and E2F-3 proteins are closely related to E2F-1. Both E2F-2 and E2F-3 bound to wild-type but not mutant E2F recognition sites, and they bound specifically to the retinoblastoma...

  10. [Influence of spv plasmid genes group in Salmonella Enteritidis virulence for chickens. I. Occurrence of spv plasmid genes group in Salmonella Enteritidis large virulence plasmid].

    Science.gov (United States)

    Madajczak, Grzegorz; Binek, Marian

    2005-01-01

    Many Salmonella Enteritidis virulence factors are encoded by genes localized on plasmids, especially large virulence plasmid, in highly conserved fragment, they create spv plasmid gene group. The aims of realized researches were spv genes occurrence evaluation and composition analysis among Salmonella Enteritidis strains caused infection in chickens. Researches were realized on 107 isolates, where in every cases large virulence plasmid 59 kbp size were detected. Specific nucleotides sequences of spv genes (spvRABCD) were detected in 47.7% of isolates. In the rest of examined bacteria spv genes occurred variably. Most often extreme genes of spv group, like spvR and spvD were absent, what could indicate that factors encoded by them are not most important for Salmonella Enteritidis live and their expressed virulence.

  11. Sample displacement chromatography of plasmid DNA isoforms.

    Science.gov (United States)

    Černigoj, Urh; Martinuč, Urška; Cardoso, Sara; Sekirnik, Rok; Krajnc, Nika Lendero; Štrancar, Aleš

    2015-10-02

    Sample displacement chromatography (SDC) is a chromatographic technique that utilises different relative binding affinities of components in a sample mixture and has been widely studied in the context of peptide and protein purification. Here, we report a use of SDC to separate plasmid DNA (pDNA) isoforms under overloading conditions, where supercoiled (sc) isoform acts as a displacer of open circular (oc) or linear isoform. Since displacement is more efficient when mass transfer between stationary and mobile chromatographic phases is not limited by diffusion, we investigated convective interaction media (CIM) monoliths as stationary phases for pDNA isoform separation. CIM monoliths with different hydrophobicities and thus different binding affinities for pDNA (CIM C4 HLD, CIM-histamine and CIM-pyridine) were tested under hydrophobic interaction chromatography (HIC) conditions. SD efficiency for pDNA isoform separation was shown to be dependent on column selectivity for individual isoform, column efficiency and on ammonium sulfate (AS) concentration in loading buffer (binding strength). SD and negative mode elution often operate in parallel, therefore negative mode elution additionally influences the efficiency of the overall purification process. Optimisation of chromatographic conditions achieved 98% sc pDNA homogeneity and a dynamic binding capacity of over 1mg/mL at a relatively low concentration of AS. SDC was successfully implemented for the enrichment of sc pDNA for plasmid vectors of different sizes, and for separation of linear and and sc isoforms, independently of oc:sc isoform ratio, and flow-rate used. This study therefore identifies SDC as a promising new approach to large-scale pDNA purification, which is compatible with continuous, multicolumn chromatography systems, and could therefore be used to increase productivity of pDNA production in the future. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. A MUB E2 structure reveals E1 selectivity between cognate ubiquitin E2s in eukaryotes

    Science.gov (United States)

    Lu, Xiaolong; Malley, Konstantin R.; Brenner, Caitlin C.; Koroleva, Olga; Korolev, Sergey; Downes, Brian P.

    2016-08-01

    Ubiquitin (Ub) is a protein modifier that controls processes ranging from protein degradation to endocytosis, but early-acting regulators of the three-enzyme ubiquitylation cascade are unknown. Here we report that the prenylated membrane-anchored ubiquitin-fold protein (MUB) is an early-acting regulator of subfamily-specific E2 activation. An AtMUB3:AtUBC8 co-crystal structure defines how MUBs inhibit E2~Ub formation using a combination of E2 backside binding and a MUB-unique lap-bar loop to block E1 access. Since MUBs tether Arabidopsis group VI E2 enzymes (related to HsUbe2D and ScUbc4/5) to the plasma membrane, and inhibit E2 activation at physiological concentrations, they should function as potent plasma membrane localized regulators of Ub chain synthesis in eukaryotes. Our findings define a biochemical function for MUB, a family of highly conserved Ub-fold proteins, and provide an example of selective activation between cognate Ub E2s, previously thought to be constitutively activated by E1s.

  13. Permissiveness of soil microbial communities towards broad host range plasmids

    DEFF Research Database (Denmark)

    Klümper, Uli

    larger than previously assumed. I was able to show abundant plasmid transfer from the Gram negative donor strains to a wide diversity of Gram positive soil bacteria, formerly thought to constitute distinct clusters of gene transfer. Moreover, among the observed transconjugants, I identified a core super...... environmental factors that modulate plasmid transfer in soil microbial communities. In order to attain these goals, I developed a high-throughput method that enabled me to evaluate the permissiveness of bacterial communities towards introduced plasmids. This new approach is based on the introduction...... fraction of soil the bacteria (up to 1 in 10,000) were able to take up any of these broad host range conjugal plasmids. The transconjugal pools comprised 11 bacterial phyla. This finding indicates that the realized transfer range of broad host range plasmids in environmental microbial communities is much...

  14. Induction of immune responses in mice and pigs by oral administration of classical swine fever virus E2 protein expressed in rice calli.

    Science.gov (United States)

    Jung, Myunghwan; Shin, Yun Ji; Kim, Ju; Cha, Seung-Bin; Lee, Won-Jung; Shin, Min-Kyoung; Shin, Seung Won; Yang, Moon-Sik; Jang, Yong-Suk; Kwon, Tae-Ho; Yoo, Han Sang

    2014-12-01

    Classical swine fever (CSF), caused by the CSF virus (CSFV), is a highly contagious disease in pigs. In Korea, vaccination using a live-attenuated strain (LOM strain) has been used to control the disease. However, parenteral vaccination using a live-attenuated strain still faces a number of problems related to storage, cost, injection stress, and differentiation of CSFV infected and vaccinated pigs. Therefore, two kinds of new candidates for oral vaccination have been developed based on the translation of the E2 gene of the SW03 strain, which was isolated from an outbreak of CSF in 2002 in Korea, in transgenic rice calli (TRCs) from Oriza sativa L. cv. Dongjin to express a recombinant E2 protein (rE2-TRCs). The expression of the recombinant E2 protein (rE2) in rE2-TRCs was confirmed using Northern blot, SDS-PAGE, and Western blot analysis. Immune responses to the rE2-TRC in mice and pigs were investigated after oral administration. The administration of rE2-TRCs increased E2-specific antibodies titers and antibody-secreting cells when compared to animals receiving the vector alone (p Pigs receiving rE2-TRCs also showed an increase in IL-8, CCL2, and the CD8+ subpopulation in response to stimulation with prE2. These results suggest that oral administration of rE2-TRCs can induce E2-specific immune responses.

  15. Both viral E2 protein and the cellular factor PEBP2 regulate transcription via E2 consensus sites within the bovine papillomavirus type 4 long control region.

    OpenAIRE

    Jackson, M E; Campo, M S

    1995-01-01

    The bovine papillomavirus type 4 (BPV4) long control region (LCR) contains three consensus binding sites, E2(1), E2(2), and E2(3) (ACCN6GGT), for the viral E2 transcription factor and a fourth degenerate site, dE2 (ATCN6GGT), which lies 3 bp upstream of E2(3). The E2(2) site was found to bind the cellular transcription factor PEBP2, and mutations at this site reduced basal promoter activity by as much as 60%, indicating an important role for PEBP2 in LCR function. Mutation of the E2(3) or dE2...

  16. Implication of the E. coli K12 uvrA and recA genes in the repair of 8-methoxypsoralen-induced mono adducts and crosslinks on plasmid DNA; Implicacion de los genes uvrA de E. coli K12 en la reparacion de monoaductos y entrecruzamien tos inducidos en DNA plasmidico por 8-metoxipso raleno mas luz ultravioleta A

    Energy Technology Data Exchange (ETDEWEB)

    Paramio, J.M.; Bauluz, C.; Vidania, R. de

    1986-07-01

    Genotoxicity of psoralen damages on plasmid DNA has been studied. pBR322 DNA was randomly modified with several concentrations of 8-methoxypsoralen plus 365 nm-UV light. After transformation into E. coli strains (wild-type, uvrA and recA) plasmid survival and mutagenesis were analyzed. To study the influence of the SOS response on plasmid recovery, preirradiation of the cells was performed. In absence of cell preirradiation, crosslinks were not repaired in any strain. Mono adducts were also lethal but in part removed by the excision-repair pathway. Preirradiation of the cells significantly. increased plasmid recovery in recA+ celia. In uvrA- only the mutagenic pathway seemed to be involved in the repair of the damaged DNA. Wild type strain showed the highest increase in plasmid survival, involving the repair of mono adducts and some fraction of crosslinks mainly through an error-free repair pathway. This suggests an enhancement of the excision repair promoted by the induction of SOS functions. (Author) 32 refs.

  17. A role of NF-E2 in chronic inflammation and clonal evolution in essential thrombocythemia, polycythemia vera and myelofibrosis?

    Science.gov (United States)

    Hasselbalch, Hans C

    2014-02-01

    A novel murine model for myeloproliferative neoplasms (MPNs) generated by overexpression of the transcription factor NF-E2 has recently been described. Sustained overexpression of NF-E2 in this model induced myeloid expansion with anemia, leukocytosis and thrombocytosis. Herein, it is debated if NF-E2 overexpression also might have induced a sustained state of in vivo leukocyte and platelet activation with chronic and self-perpetuating production of inflammatory products from activated leukocytes and platelets. If so, this novel murine model also may excellently describe the deleterious impact of sustained chronic NF-E2 overexpression during uncontrolled chronic inflammation upon the hematopoietic system--the development of clonal myeloproliferation. Accordingly, this novel murine model may also have delivered the proof of concept of chronic inflammation as a trigger and driver of clonal evolution in MPNs.

  18. E2F-Rb complexes assemble and inhibit cdc25A transcription in cervical carcinoma cells following repression of human papillomavirus oncogene expression

    DEFF Research Database (Denmark)

    Wu, L; Goodwin, E C; Naeger, L K

    2000-01-01

    . To explore the mechanism of repression of cell cycle genes in cervical carcinoma cells following E6/E7 repression, we analyzed regulation of the cdc25A promoter, which contains two consensus E2F binding sites and a consensus E2 binding site. The wild-type E2 protein inhibited expression of a luciferase gene...... E2F4-Rb DNA binding complexes. Importantly, these experiments revealed that HPV-induced alterations in E2F transcription complexes that occur during cervical carcinogenesis are reversed by repression of HPV E6/E7 expression....

  19. Complex nature of enterococcal pheromone-responsive plasmids.

    Science.gov (United States)

    Wardal, Ewa; Sadowy, Ewa; Hryniewicz, Waleria

    2010-01-01

    Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.

  20. Antibiotic resistance of vibrio cholerae: special considerations of R-plasmids.

    Science.gov (United States)

    Kuwahara, S

    1978-09-01

    Studies on the transmission of R plasmid by conjugation between enterobacteria and vibrio or related bacteria were reviewed. The majority of the reports confirmed successful transmission from enterobacteria to Vibrio cholerae and related species, although the transmission frequencies were extremely low and the transmitted R plasmid was very unstable except for thermosensitive kanamycin plasmid and usual R plasmid coexisting with P plasmid. Strains of V. cholerae and Aeromonas liquefaciens as well as A. salmonicida bearing R plasmid were detected in nature. R plasmid was relatively unstable in V. cholerae strains with which transmission of R plasmid to enterobacteria was confirmed. At present, only 3 R plasmids have been obtained from naturally occurring strains of V. cholerae. Although the 2 European plasmids belong to the C incompatibility group with 98 megadalton closed covalent circular DNA molecule, one plasmid belongs to the J group with more than 25 megadalton molecular weight, and no CCC of satelite DNA was detected in bacteria harboring this plasmid.

  1. Bacterial Mitosis: ParM of Plasmid R1 Moves Plasmid DNA by an Actin-like Insertional Polymerization Mechanism

    DEFF Research Database (Denmark)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette

    2003-01-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system...

  2. The challenges of classical swine fever control: modified live and E2 subunit vaccines.

    Science.gov (United States)

    Huang, Yu-Liang; Deng, Ming-Chung; Wang, Fun-In; Huang, Chin-Cheng; Chang, Chia-Yi

    2014-01-22

    Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E(rns) allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA.

  3. Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

    2014-08-26

    Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

  4. Efficient expression of human factor Ⅸ cDNA in livermediated by hydrodynamics-based plasmid administration

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    Hydrodynamics-based administration via tail vein was used to deliver naked plasmid with human factor Ⅸ (hFⅨ) cDNA in 2.2 mL Ringer's solution into mice within 7 s. The peak level of expression of hFⅨ was 2921 ng/mL in mouse plasma. The hFⅨ cDNA expression increased with increasing the amount of plasmid DNA injected. The peak level of gene expression declined after repeated injection of plasmid (1459 ng/mL). The hFⅨ cDNA was detected in various organs, but the highest level of gene expression appeared in liver. Transaminase levels and liver histological results showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage, which was rapidly repaired within 3-10 d. These results suggested that high-level expression of hFⅨ cDNA can be achieved by hydrodynamics-based plasmid transfer and this method is now further used for gene therapy and gene function study in our lab.

  5. Toward the identification of a type I toxin-antitoxin system in the plasmid DNA of dairy Lactobacillus rhamnosus.

    Science.gov (United States)

    Folli, Claudia; Levante, Alessia; Percudani, Riccardo; Amidani, Davide; Bottazzi, Stefania; Ferrari, Alberto; Rivetti, Claudio; Neviani, Erasmo; Lazzi, Camilla

    2017-09-21

    Plasmids carry genes that give bacteria beneficial traits and allow them to survive in competitive environments. In many cases, they also harbor toxin-antitoxin (TA) systems necessary for plasmid maintenance. TA systems are generally characterized by a stable "toxin", a protein or peptide capable of killing the cell upon plasmid loss and by an unstable "antitoxin", a protein or a non-coding RNA that inhibits toxin activity. Here we report data toward the identification of a RNA-regulated TA system in the plasmid DNA of L. rhamnosus isolated from cheese. The proposed TA system comprises two convergently transcribed RNAs: a toxin RNA encoding a 29 amino acid peptide named Lpt and an antitoxin non-coding RNA. Both toxin and antitoxin RNAs resulted upregulated under conditions mimicking cheese ripening. The toxicity of the Lpt peptide was demonstrated in E. coli by cloning the Lpt ORF under the control of an inducible promoter. Bioinformatics screening of the bacterial nucleotide database, shows that regions homologous to the Lpt TA locus are widely distributed in the Lactobacillus genus, particularly within the L. casei group, suggesting a relevant role of TA systems in plasmid maintenance of cheese microbiota.

  6. Plasmid DNA is internalized from the apical plasma membrane of the salivary gland epithelium in live animals.

    Science.gov (United States)

    Sramkova, Monika; Masedunskas, Andrius; Weigert, Roberto

    2012-08-01

    Non-viral-mediated gene delivery represents an alternative way to express the gene of interest without inducing immune responses or other adverse effects. Understanding the mechanisms by which plasmid DNAs are delivered to the proper target in vivo is a fundamental issue that needs to be addressed in order to design more effective strategies for gene therapy. As a model system, we have used the submandibular salivary glands in live rats and we have recently shown that reporter transgenes can be expressed in different cell populations of the glandular epithelium, depending on the modality of administration of plasmid DNA. Here, by using a combination of immunofluorescence and intravital microscopy, we have explored the relationship between the pattern of transgenes expression and the internalization of plasmid DNA. We found that plasmid DNA is internalized: (1) by all the cells in the salivary gland epithelium, when administered alone, (2) by large ducts, when mixed with empty adenoviral particles, and (3) by acinar cells upon stimulation of compensatory endocytosis. Moreover, we showed that plasmid DNA utilizes different routes of internalization, and evades both the lysosomal degradative pathway and the retrograde pathway towards the Golgi apparatus. This study clearly shows that in vivo approaches have the potential to address fundamental questions on the cellular mechanisms regulating gene delivery.

  7. Impact of carbondiimide crosslinker used for magnetic carbon nanotube mediated GFP plasmid delivery

    Energy Technology Data Exchange (ETDEWEB)

    Hao Yuzhi; He Chuan; Yang Xiaoyan; Huang Min; Chen Jie [Department of Electrical and Computer Engineering, University of Alberta, Edmonton, AB (Canada); Xu Peng [National Institute of Nanotechnology, Edmonton, AB (Canada); Xing, James [IntelligentNano Incorporated, Edmonton, AB (Canada)

    2011-07-15

    1-ethyl-3-(3-dimethylaminopropyl) carbondiimide hydrochloride (EDC) is commonly used as a crosslinker to help bind biomolecules, such as DNA plasmids, with nanostructures. However, EDC often remains, after a crosslink reaction, in the micro-aperture of the nanostructure, e.g., carbon nanotube. The remaining EDC shows positive green fluorescent signals and makes a nanostructure with a strong cytotoxicity which induces cell death. The toxicity of EDC was confirmed on a breast cancer cell line (MCF-7) and two leukemic cell lines (THP-1 and KG-1). The MCF-7 cells mainly underwent necrosis after treatment with EDC, which was verified by fluorescein isothiocyanate (FITC) annexin V staining, video microscopy and scanning electronic microscopy (SEM). If the EDC was not removed completely, the nanostructures with remaining EDC produced a green fluorescent background that could interfere with flow cytometry (FACS) measurement and result in false information about GFP plasmid delivery. Effective methods to remove residual EDC on macromolecules were also developed.

  8. [Effect of He-Ne-laser irradiation on plasmid transformation of Escherichia coli bacteria].

    Science.gov (United States)

    Tiflova, O A; Leonov, P G; Karbysheva, E A; Shakhnabatian, L G

    1997-01-01

    The influence of the of radiation a He-Ne laser (632.8 nm, 30 W/m2, 5-20 J/m2) on the transformation of Escherichia coli cells with plasmid DNA was studied. The irradiation of a mixture of bacterial cells and plasmid DNA increased the transformation efficiency 2.5-3 times, thus offering an alternative to the heat treatment commonly used. In contrast to the standard techniques, the laser-induced increase in the transformation efficiency was accompanied by a 1.7- to 2-fold increase in cell survival. The effect of the 632.8-nm light, know to be absorbed by membrane porphyrin components, is supposed to be mediated via a modification in the replication and transformation DNA-membrane complexes in E. coli cells.

  9. Extra-amniotic prostaglandin E2 and the unfavourable cervix.

    Science.gov (United States)

    Shepherd, J; Sims, C; Craft, I

    1976-10-02

    A small dose of prostaglandin E2 suspended in a viscous medium was instilled as a single application into the extra-amniotic space of patients with unfavourable induction features the day before planned induction in an attempt to improve the condition of the cervix. Two groups of 15 patients were studied, one receiving prostaglandin E2 250 mug suspended in methyl ethyl cellulose ('Tylose') 6% solution, and the other tylose alone. Cervical status did not change in those receiving tylose alone, whereas a significant improvement occurred in 14 out of 15 patients receiving the prostaglandin. Labour began before formal induction in 1 patient receiving tylose and in 8 receiving prostaglandin.

  10. Elicitation of strong immune responses by a DNA vaccine expressing a secreted form of hepatitis C virus envelope protein E2 in murine and porcine animal models

    Institute of Scientific and Technical Information of China (English)

    Yi-Ping Li; Hye Na Kang; Lorne A Babiuk; Qiang Liu

    2006-01-01

    AIM: To characterize the immunogenicity of a hepatitis C virus (HCV) E2 DNA vaccine alone or with a protein vaccine boost in murine and porcine animal models.METHODS: A DNA vaccine expressing a secreted form of HCV E2 protein was constructed and used to vaccinate mice and piglets with or without boosting with a recombinant E2 protein vaccine formulated with CpG ODN and 10% Emulsigen. The immunogenicity of HCV E2 vaccines was analyzed by ELISA for antibody responses, MTT assay for lymphocyte proliferation,ELISPOT for the number of interferon-γ secreting cells,and cytotoxic T lymphocyte assays.RESULTS: Intradermal injection of E2 DNA vaccine induced strong Th1-like immune responses in mice. In piglets, E2 DNA vaccine elicited moderate and more balanced immune responses. A DNA vaccine prime and protein boost vaccination strategy induced significantly higher E2-specific antibody levels and shifted the immune response towards Th2-like ones in piglets.CONCLUSION: A DNA vaccine expressing a secreted form of HCV E2 protein elicited E2-specific immune responses in mice and piglets. Recombinant E2 protein vaccination following DNA immunization significantly increased the antibody response in piglets. These HCV E2 vaccines may represent promising hepatitis C vaccine candidates for further investigations.

  11. Translating DNA damage into cancer cell death-A roadmap for E2F1 apoptotic signalling and opportunities for new drug combinations to overcome chemoresistance.

    Science.gov (United States)

    Engelmann, David; Pützer, Brigitte M

    2010-01-01

    The cellular transcription factor E2F1 has been identified as a tumor suppressor regulating the activities of p53 and its homologue TAp73, and promoting apoptosis by the activation of a plethora of death pathways. More than 15 years of experimentation recognized E2F1 as the key player in apoptosis induced by DNA damage in all types of human cancer. This occurs by several mechanisms that affect RB-E2F1 interaction, E2F1 stability and its binding to promoters of E2F1-regulated genes. Recent progress has been made in revealing new proapoptotic genes regulated by E2F1 and it seems that many still remain to be discovered. However, whereas in the past one focused mainly on identifying E2F1 target genes translating cellular stress signals into cell death, today the DNA damage-induced regulatory network governing E2F1's ability to induce apoptosis is rapidly gaining attention as well. Notably, the lately uncovered role of pRB and E2F3 in triggering E2F1-dependent apoptosis through chemotherapy gains our understanding of the DNA damage response in normal and tumor cells. In this context a large body of evidence indicates that nuclear cofactors targeting E2F1 seem to have a major impact on its tumor suppressor function. These new findings are discussed in the context of preclinical studies applying E2F1 overexpression in combination with genotoxic anticancer agents - called chemogene therapy, thereby providing new mechanistic links between the E2F1-induced apoptotic programming and advanced cancer phenotype. Copyright © 2010 Elsevier Ltd. All rights reserved.

  12. PGE2对LPS诱导小鼠骨髓源性树突细胞成熟及EP4受体表达影响%The effect of prostaglandin E2 on maturation and EP4 expression on dendritic cell induced by LPS

    Institute of Scientific and Technical Information of China (English)

    盛康亮; 李影; 付静静; 陈镜宇; 张玲玲; 魏伟

    2015-01-01

    Objective To investigate the effects of PGE2 in the different concentrations on the maturation of bone marrow-derived dendritic cells ( DCs) of mouse stimulated by LPS and EP4 expression. Methods The bone mar-row-derived DCs were induced in the presence of recombinant granulocyte macrophage colony stimulating factor (rmGM-CSF) and rmIL-4. Maturated DCs were induced by LPS (100 ng/ml), and then were treated with PGE2 in different concentrations (0. 625, 1. 25, 2. 5, 5, 10, 20 nmol/L) for 24 h. The ability of antigen uptake and the expressions of CD40 , CD83 and MHC-Ⅱon DCs surface were analyzed by flow cytometry;EP4 R expression was al-so measured by flow cytometry. T cell proliferation in a mixed lymphocyte reaction was analyzed by MTT assay. Re-sults PGE2 (2. 5, 5, 10 nmol/L) significantly enhanced the expression of CD40, CD83, MHC class II molec- ules. However,PGE2 had no effect on CD80 expression in all of concentrations. PGE2(1. 25,2. 5,5,10,20 nmol/L) significantly inhibited the ability of antigen uptake of DCs. DCs stimulated by PGE2 (2. 5,5,10 nmol/L) could promote T cell proliferation. PGE2 (2. 5,5,10 nmol/L) increased EP4 expression on DCs surface. Conclusion PGE2 could regulate DCs function,which might be related to EP4 receptor expression affected by PGE2.%目的:观察不同浓度前列腺素E2(PGE2)刺激对脂多糖( LPS)诱导小鼠骨髓源性树突细胞( DCs)成熟及EP4受体表达的影响。方法采用重组小鼠粒细胞-巨噬细胞集落刺激因子( rmGM-CSF)、重组小鼠白细胞介素4( rmIL-4)刺激小鼠骨髓源细胞,诱导生成DCs;经LPS(100 ng/ml)诱导成熟后,用不同浓度 PGE2(0.625、1.25、2.5、5、10、20 nmol/L)刺激 DCs 24 h,流式细胞术检测 DCs 表型 CD40、CD83、MHC-Ⅱ的表达和抗原摄取功能,荧光间标法检测小鼠骨髓源DCs细胞膜上EP4的表达,以平均荧光强度表示表达的高低;MTT法检测经PGE2及LPS诱导成熟的DCs对T细胞增殖的作

  13. Optimazation of antibiotic therapy in patients with chronic pyelonephritis combined with pancreatic diabetes of the 2 type by evaluation of the expression of plasmid resistance genes

    Directory of Open Access Journals (Sweden)

    Ольга Игоревна Чуб

    2015-07-01

    Full Text Available The resistance caused by the transfer of resistance genes among the uropathogenes with a help of plasmids in the treatment of chronic pyelonephritis (CP combined with pancreatic diabetes (PD of the 2 type increased last years. The aim of research was an optimization of methods of differentiated prescription of antibiotic therapy (ABT for patients with CP and contaminant PD of the 2 type depending on expression of plasmid-induced genic mechanisms of antibiotic resistance.Methods. There were examined 105 patients with chronic pyelonephritis, 73 of them – against the background of PD of the 2 type. The sensitivity of isolated cultures to antibiotics was carried out by the disco-diffuse method of Bauer-Kirbi on the medium of Hinton-Muller using commercial discs. The study of plasmid-induced mechanisms of resistance was carried out by the method of polymerase chain reaction (PCR.Results. Detectability of plasmid-induced mechanisms in patients with CP and PD of the 2 type is 31,5 %. Β-lactamase of extended spectrum (βLES – were the most diffuse detected genes. The highest inhibitory activity against the strains with plasmid genes was shown by meropenem, fosfomicyn, nitroxolinum, 4th generation of cephalosporins and the 3th generation of fluoroquinolones. According to received results it was elaborated an algorithm of differentiated prescription of ABT for patients with CP and PD of the 2 type depending on expression of plasmid-induced resistance genes.Conclusions. There was studied the prevalence of plasmid-induced resistance mechanisms and elaborated the methods for increase an efficiency of empirical ABT for patients with CP and contaminant PD of the 2 type. 

  14. Plasmid DNA Manufacturing for Indirect and Direct Clinical Applications.

    Science.gov (United States)

    Schmeer, Marco; Buchholz, Tatjana; Schleef, Martin

    2017-10-01

    Plasmid DNA is currently gaining increasing importance for clinical research applications in gene therapy and genetic vaccination. For direct gene transfer into humans, good manufacturing practice (GMP)-grade plasmid DNA is mandatory. The same holds true if the drug substance contains a genetically modified cell, for example chimeric antigen receptor (CAR) T cells, where these cells as well as the contained plasmids are used. According to the responsible regulatory agencies, they have to be produced under full GMP. On the other hand, for GMP production of, for example, mRNA or viral vectors (lentiviral vectors, adeno-associated virus vectors, etc.), in many cases, High Quality Grade plasmid DNA is accepted as a starting material. The manufacturing process passes through different production steps. To ensure the right conditions are used for the plasmid, a pilot run must be conducted at the beginning. In this step, a followed upscaling with respect to reproducibility and influences on product quality is performed. Subsequently, a cell bank of the transformed productions strain is established and characterized. This cell bank is used for the cultivation process. After cell harvesting and lysis, several chromatography steps are conducted to receive a pure plasmid product. Depending on the respective required quality grade, the plasmid product is subject to several quality controls. The last step consists of formulation and filling of the product.

  15. Plasmid-associated sensitivity of Bacillus thuringiensis to UV light

    Energy Technology Data Exchange (ETDEWEB)

    Benoit, T.G.; Wilson, G.R.; Bull, D.L.; Aronson, A.I. (Department of Agriculture, College Station, TX (USA))

    1990-08-01

    Spores and vegetative cells of Bacillus thuringiensis were more sensitive to UV light than were spores or cells of plasmid-cured B. thuringiensis strains or of the closely related Bacillus cereus. Introduction of B. thuringiensis plasmids into B. cereus by cell mating increased the UV sensitivity of the cells and spores. Protoxins encoded by one or more B. thuringiensis plasmids were not involved in spore sensitivity, since a B. thuringiensis strain conditional for protoxin accumulation was equally sensitive at the permissive and nonpermissive temperatures. In addition, introduction of either a cloned protoxin gene, the cloning vector, or another plasmid not containing a protoxin gene into a plasmid-cured strain of B. thuringiensis all increased the UV sensitivity of the spores. Although the variety of small, acid-soluble proteins was the same in the spores of all strains examined, the quantity of dipicolinic acid was about twice as high in the plasmid-containing strains, and this may account for the differences in UV sensitivity of the spores. The cells of some strains harboring only B. thuringiensis plasmids were much more sensitive than cells of any of the other strains, and the differences were much greater than observed with spores.

  16. In vitro replication of cyanobacterial plasmids from Synechocystis PCC 6803.

    Science.gov (United States)

    Yang, X; Daniell, H; McFadden, B

    1994-09-01

    Little knowledge of DNA replication in cyanobacteria is available. In this study, we report the development and characterization of an in vitro system for studies of replication of the endogenous plasmids from the unicellular cyanobacterium Synechocystis 6803. This system (fraction III) was isolated at high salt concentrations and partially purified on a heparin-agarose column. DNA polymerases in Synechocystis 6803 appeared to be associated with membranes and could be released by the addition of ammonium sulfate to 20% saturation. DNA synthesis in fraction III was dependent on the addition of cyanobacterial plasmids isolated from the same strain. The in vitro replication products consist mostly of the supercoiled form of the plasmids. Unlike replication of many Escherichia coli plasmids, replication of cyanobacterial plasmids did not require added ATP, was not inhibited by omission of the ribonucleotides, and was insensitive to the RNA polymerase inhibitor rifampicin and the gyrase inhibitor novobiocin, but was inhibited by ethidium bromide. These data suggest that RNA may not be involved in the initiation of replication of cyanobacterial plasmids from Synechocystis 6803. In addition, intermediates of replication have been detected by two-dimensional gel electrophoresis. Density labeling experiments also indicate that cyanobacterial plasmid synthesis in vitro occurs by a semiconservative replication.

  17. Routine induction of labour with extra-amniotic prostaglandin E2 in a viscous gel.

    Science.gov (United States)

    Sims, C D; Mellows, H J; Spencer, P J; Craft, I L

    1979-07-01

    Prostaglandin E2, 350 microgram dispersed in a viscous gel, tylose, was introduced into the extra-amniotic space as a single dose in 285 patients to induce labour. With a favourable cervix, 82 per cent of multiparae and 50 per cent of primiparae were successfully induced. With unfavourable induction features, the success rates were 48 per cent and 24 per cent respectively. In the remaining patients, all but four were successfully delivered when intravenous oxytocin was also used. The method was safe, simple and inexpensive and had many advantages for patients and nursing staff.

  18. Influence of HPV16 E2 and its localisation on the expression of matrix metalloproteinase-9.

    Science.gov (United States)

    Mühlen, Sabrina; Behren, Andreas; Iftner, Thomas; Simon, Christian

    2010-08-01

    Infection with the high-risk HPV types 16 and 18 is the major cause of cervical cancer and plays a role in the development of certain head and neck and skin cancers. We have previously demonstrated that the Early Protein 2 of the Cottontail Rabbit papillomavirus (CRPV), required for skin carcinogenesis in a rabbit model, is able to induce the expression of a matrix metalloproteinase (MMP-9); a protease known to play a key role in invasion and metastasis. However, as of now we do not understand the underlying mechanism of activation nor relevance for the human system. Here, we report that high-risk human papillomavirus HPV16 E2 similar to our previously reported results on CRPV E2 activates the human MMP-9 promoter predominantly via the MEK1-ERK1/2-AP-1-signaling pathway. In addition this activation is associated with a nuclear sub-localisation of HPV16-E2 suggesting a nuclear protein-protein or protein-DNA interaction of E2 as the underlying mechanism of activation.

  19. Antibegomoviral activity of the agrobacterial virulence protein VirE2.

    Science.gov (United States)

    Sunitha, Sukumaran; Marian, Dolly; Hohn, Barbara; Veluthambi, Karuppannan

    2011-12-01

    Mungbean yellow mosaic geminivirus (MYMV) causes severe yellow mosaic disease in blackgram, mungbean, Frenchbean, pigeonpea, soybean and mothbean. We attempted to induce resistance against this virus using the transcriptional activator protein gene deleted in the C-terminal activation domain (TrAP-∆AD) and Agrobacterium tumefaciens virE2. MYMV is known to replicate in agroinoculated tobacco leaf discs. Three transgenic tobacco plants which harboured a truncated MYMV transcriptional activator protein gene and two tobacco plants transformed with the octopine type A. tumefaciens virE2 gene were agroinoculated with an A. tumefaciens strain which harboured the partial dimers of both DNA A and DNA B of MYMV. The level of viral DNA accumulation in leaf discs of transgenic plants correlated inversely to the level of the MYMV TrAP-∆AD transcript. Two VirE2-transgenic plants, which complemented tumorigenesis of a virE2 mutant A. tumefaciens strain, effectively reduced MYMV DNA accumulation in the leaf disc agroinoculation assay.

  20. Aberrant E2F activation by polyglutamine expansion of androgen receptor in SBMA neurotoxicity.

    Science.gov (United States)

    Suzuki, Eriko; Zhao, Yue; Ito, Saya; Sawatsubashi, Shun; Murata, Takuya; Furutani, Takashi; Shirode, Yuko; Yamagata, Kaoru; Tanabe, Masahiko; Kimura, Shuhei; Ueda, Takashi; Fujiyama, Sally; Lim, Jinseon; Matsukawa, Hiroyuki; Kouzmenko, Alexander P; Aigaki, Toshiro; Tabata, Tetsuya; Takeyama, Ken-ichi; Kato, Shigeaki

    2009-03-10

    Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by a polyglutamine repeat (polyQ) expansion within the human androgen receptor (AR). Unlike other neurodegenerative diseases caused by abnormal polyQ expansion, the onset of SBMA depends on androgen binding to mutant human polyQ-AR proteins. This is also observed in Drosophila eyes ectopically expressing the polyQ-AR mutants. We have genetically screened mediators of androgen-induced neurodegeneration caused by polyQ-AR mutants in Drosophila eyes. We identified Rbf (Retinoblastoma-family protein), the Drosophila homologue of human Rb (Retinoblastoma protein), as a neuroprotective factor. Androgen-dependent association of Rbf or Rb with AR was remarkably potentiated by aberrant polyQ expansion. Such potentiated Rb association appeared to attenuate recruitment of histone deacetyltransferase 1 (HDAC1), a corepressor of E2F function. Either overexpression of Rbf or E2F deficiency in fly eyes reduced the neurotoxicity of the polyQ-AR mutants. Induction of E2F function by polyQ-AR-bound androgen was suppressed by Rb in human neuroblastoma cells. We conclude that abnormal expansion of polyQ may potentiate innate androgen-dependent association of AR with Rb. This appears to lead to androgen-dependent onset of SBMA through aberrant E2F transactivation caused by suppressed histone deacetylation.

  1. E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding.

    Science.gov (United States)

    Rogers, Jason H; Owens, Kristin S; Kurkewich, Jeffrey; Klopfenstein, Nathan; Iyer, Sangeeta R; Simon, M Celeste; Dahl, Richard

    2016-01-01

    Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1's ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

  2. E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

    Directory of Open Access Journals (Sweden)

    Jason H. Rogers

    2016-01-01

    Full Text Available Antagonistic interactions between transcription factors contribute to cell fate decisions made by multipotent hematopoietic progenitor cells. Concentration of the transcription factor PU.1 affects myeloid/lymphoid development with high levels of PU.1 directing myeloid cell fate acquisition at the expense of B cell differentiation. High levels of PU.1 may be required for myelopoiesis in order to overcome inhibition of its activity by transcription factors that promote B cell development. The B cell transcription factors, E2A and EBF, are necessary for commitment of multipotential progenitors and lymphoid primed multipotential progenitors to lymphocytes. In this report we hypothesized that factors required for early B cell commitment would bind to PU.1 and antagonize its ability to induce myeloid differentiation. We investigated whether E2A and/or EBF associate with PU.1. We observed that the E2A component, E47, but not EBF, directly binds to PU.1. Additionally E47 represses PU.1-dependent transactivation of the MCSFR promoter through antagonizing PU.1’s ability to bind to DNA. Exogenous E47 expression in hematopoietic cells inhibits myeloid differentiation. Our data suggest that E2A antagonism of PU.1 activity contributes to its ability to commit multipotential hematopoietic progenitors to the lymphoid lineages.

  3. Api5 contributes to E2F1 control of the G1/S cell cycle phase transition.

    Directory of Open Access Journals (Sweden)

    Marina Garcia-Jove Navarro

    Full Text Available BACKGROUND: The E2f transcription factor family has a pivotal role in controlling the cell fate in general, and in particular cancer development, by regulating the expression of several genes required for S phase entry and progression through the cell cycle. It has become clear that the transcriptional activation of at least one member of the family, E2F1, can also induce apoptosis. An appropriate balance of positive and negative regulators appears to be necessary to modulate E2F1 transcriptional activity, and thus cell fate. METHODOLOGY/PRINCIPAL FINDINGS: In this report, we show that Api5, already known as a regulator of E2F1 induced-apoptosis, is required for the E2F1 transcriptional activation of G1/S transition genes, and consequently, for cell cycle progression and cell proliferation. Api5 appears to be a cell cycle regulated protein. Removal of Api5 reduces cyclin E, cyclin A, cyclin D1 and Cdk2 levels, causing G1 cell cycle arrest and cell cycle delay. Luciferase assays established that Api5 directly regulates the expression of several G1/S genes under E2F1 control. Using protein/protein and protein/DNA immunoprecipitation studies, we demonstrate that Api5, even if not physically interacting with E2F1, contributes positively to E2F1 transcriptional activity by increasing E2F1 binding to its target promoters, through an indirect mechanism. CONCLUSION/SIGNIFICANCE: The results described here support the pivotal role of cell cycle related proteins, that like E2F1, may act as tumor suppressors or as proto-oncogenes during cancer development, depending on the behavior of their positive and negative regulators. According to our findings, Api5 contributes to E2F1 transcriptional activation of cell cycle-associated genes by facilitating E2F1 recruitment onto its target promoters and thus E2F1 target gene transcription.

  4. E2F target genes: unraveling the biology

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Ciro, Marco; Cocito, Andrea

    2004-01-01

    The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target...

  5. E2F target genes: unraveling the biology

    DEFF Research Database (Denmark)

    Bracken, Adrian P; Ciro, Marco; Cocito, Andrea

    2004-01-01

    The E2F transcription factors are downstream effectors of the retinoblastoma protein (pRB) pathway and are required for the timely regulation of numerous genes essential for DNA replication and cell cycle progression. Several laboratories have used genome-wide approaches to discover novel target ...... transcription and cellular homeostasis....

  6. Coexistence and B(E2)'s in 98Sr

    Science.gov (United States)

    Fortune, H. T.

    2017-01-01

    I have used a simple two-state model to fit E2 strengths connecting the first two 0+ states to the first two 2+ states in 98Sr. Results for mixing parameters are in excellent agreement with those from a recent analysis. Perhaps surprisingly, they are also in remarkable agreement with results from 1980, despite the wide variation reported in the intervening years.

  7. E-2C Loads Calibration in DFRC Flight Loads Lab

    Science.gov (United States)

    Schuster, Lawrence S.

    2008-01-01

    Objectives: a) Safely and efficiently perform structural load tests on NAVAIR E-2C aircraft to calibrate strain gage instrumentation installed by NAVAIR; b) Collect load test data and derive loads equations for use in NAVAIR flight tests; and c) Assist flight test team with use of loads equations measurements at PAX River.

  8. Plasmid-determined resistance to fosfomycin in Serratia marcescens.

    Science.gov (United States)

    Mendoza, C; Garcia, J M; Llaneza, J; Mendez, F J; Hardisson, C; Ortiz, J M

    1980-08-01

    Multiple-antibiotic-resistant strains of Serratia marcescens isolated from hospitalized patients were examined for their ability to transfer antibiotic resistance to Escherichia coli by conjugation. Two different patterns of linked transferable resistance were found among the transconjugants. The first comprised resistance to carbenicillin, streptomycin, and fosfomycin; the second, and more common, pattern included resistance to carbenicillin, streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, sulfonamide, and fosfomycin. The two types of transconjugant strains carried a single plasmid of either 57 or 97 megadaltons in size. Both of these plasmids are present in parental S. marcescens strains resistant to fosfomycin. The 57-megadalton plasmid was transformed into E. coli.

  9. [Epidemiologic study of 2 S. typhimurium outbreaks using plasmid fingerprints].

    Science.gov (United States)

    Baumgartner, A; Breer, C; Schopfer, K

    1989-04-05

    An outbreak of salmonellosis in an old people's home is reported. The infectious agent, S. typhi-murium, was isolated not only from several inmates but also from sick cows of the farm belonging to the home, in animal feed, from employees of the local butcher's shop, and finally in sludge from the local sewage plant. Plasmid analysis provided evidence of a common origin for the isolated S. typhi-murium strains. The incriminated strains harboured, together with two low-molecular-weight plasmids, a plasmid of approximately 50 Mdal, which was also demonstrated in some other S. typhi-murium strains isolated from clinical cases in the area around St. Gallen.

  10. Effect of Plasmid Incompatibility on DNA Transfer to Streptococcus cremoris

    OpenAIRE

    Van Der Lelie, Daniel; Vossen, Jos M.B.M. van der; Venema, Gerard

    1988-01-01

    Several Streptococcus cremoris strains were used in protoplast transformation and interspecific protoplast fusion experiments with Streptococcus lactis and Bacillus subtilis, with pGKV110, pGKV21, and ΔpAMβ1 as the marker plasmids. ΔpAMβ1 is a 15.9-kilobase nonconjugative, deletion derivative of pAMβ1, which is considerably larger than the pGKV plasmids (approximately 4.5 kilobases). In general, ΔpAMβ1 was transferred more efficiently than the pGKV plasmids. Using electroporation, we were abl...

  11. Separation of plasmid DNA topoisomers by multimodal chromatography.

    Science.gov (United States)

    Silva-Santos, A Rita; Alves, Cláudia P A; Prazeres, Duarte Miguel F; Azevedo, Ana M

    2016-06-15

    The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Atypical Enteropathogenic Escherichia coli Secretes Plasmid Encoded Toxin

    Directory of Open Access Journals (Sweden)

    Rita C. Ruiz

    2014-01-01

    Full Text Available Plasmid encoded toxin (Pet is a serine protease originally described in enteroaggregative Escherichia coli (EAEC prototype strain 042 whose entire characterization was essentially obtained from studies performed with the purified toxin. Here we show that Pet is not exclusive to EAEC. Atypical enteropathogenic Escherichia coli (aEPEC strains, isolated from diarrhea cases, express Pet and its detection in supernatants of infected HEp-2 cells coincides with the appearance of cell damage, which, in turn, were similar to those described with purified Pet. Pet secretion and the cytotoxic effects are time and culture medium dependent. In presence of DMEM supplemented with tryptone cell rounding and detachment were observed after just 5 h of incubation with the bacteria. In the absence of tryptone, the cytotoxic effects were detected only after 24 h of infection. We also show that, in addition to the prototype EAEC, other pet+ EAEC strains, also isolated from diarrhea cases, induce cellular damage in the same degree as the aEPEC. The cytotoxic effects of EAEC and aEPEC strains were significantly reduced in the presence of a serine protease inhibitor or anti-Pet IgG serum. Our results show a common aspect between the aEPEC and EAEC and provide the first evidence pointing to a role of Pet in aEPEC pathogenesis.

  13. A naturally occurring substitution in the E2 protein of Salmonid alphavirus subtype 3 changes viral fitness.

    Science.gov (United States)

    Karlsen, Marius; Andersen, Linda; Blindheim, Steffen H; Rimstad, Espen; Nylund, Are

    2015-01-22

    Phylogenetic analyses of the Salmonid alphavirus subtype 3 (SAV3) epizootic have suggested that a substitution from proline to serine in the receptor binding protein E2 position 206 has occurred after the introduction of virus from a wild reservoir to farmed salmonid fish in Norway. We modelled the 3D structure of P62, the uncleaved E3-E2 precursor, of SAVH20/03 based on its sequence homology to the Chikungunya virus (CHIKV), and studied in vitro and in vivo effects of the mutation using reverse genetics. E2(206) is located on the surface of the B-domain of E2, which is associated with receptor attachment in alphaviruses. Recombinant virus expressing the E2(206S) codon replicated slower and produced significantly less genomic copies than virus expressing the ancestral E2(206P) codon in vitro in Bluegill Fry (BF2) cells. The E2(206S) mutant was out-competed by the E2(206P) mutant after 5 passages in an in vitro competition assay, confirming that the substitution negatively affects the efficacy of virus multiplication in cell culture. Both mutants were highly infectious to Atlantic salmon (Salmo salar), produced similar viral RNA loads in gills, heart, kidney and brain, and induced similar histopathologic changes in these organs. The E2(206S) mutant produced a less persistent infection in salmon and was shed more rapidly to water than the E2(206P) mutant. Reduced generation time through more rapid shedding could therefore explain why a serine in this position became dominant in the viral population after SAV3 was introduced to farmed salmon from the wild reservoir.

  14. Prostaglandin I2 and Prostaglandin E2 Modulate Human Intrarenal Artery Contractility Through Prostaglandin E2-EP4, Prostacyclin-IP, and Thromboxane A2-TP Receptors

    DEFF Research Database (Denmark)

    Eskildsen, Morten P; Hansen, Pernille B L; Stubbe, Jane

    2014-01-01

    receptors. Intrarenal arteries were microdissected from human nephrectomy samples (n=53, median diameter ≈362 μm, 88% viable, 76% relaxed in response to acetylcholine). Rings were suspended in myographs to record force development. In vessels with K(+)-induced tension (EC70: -log [mol/L]=1.36±0.03), PGE2....../L elicited increased tension. This was abolished by thromboxane receptor (TP) antagonist (S18886, 10(-6) mol/L). A TP agonist (U46619, n=6) evoked tension (EC50=8.1±0.2) that was inhibited by S18886. Polymerase chain reaction and immunoblotting showed EP4, IP, and TP receptors in intrarenal arteries......Cyclooxygenase inhibitors decrease renal blood flow in settings with decreased effective circulating volume. The present study examined the hypothesis that prostaglandins, prostaglandin E2 (PGE2) and prostacyclin (PGI2), induce relaxation of human intrarenal arteries through PGE2-EP and PGI2-IP...

  15. Bacterial mitosis: ParM of plasmid R1 moves plasmid DNA by an actin-like insertional polymerization mechanism.

    Science.gov (United States)

    Møller-Jensen, Jakob; Borch, Jonas; Dam, Mette; Jensen, Rasmus B; Roepstorff, Peter; Gerdes, Kenn

    2003-12-01

    Bacterial DNA segregation takes place in an active and ordered fashion. In the case of Escherichia coli plasmid R1, the partitioning system (par) separates paired plasmid copies and moves them to opposite cell poles. Here we address the mechanism by which the three components of the R1 par system act together to generate the force required for plasmid movement during segregation. ParR protein binds cooperatively to the centromeric parC DNA region, thereby forming a complex that interacts with the filament-forming actin-like ParM protein in an ATP-dependent manner, suggesting that plasmid movement is powered by insertional polymerization of ParM. Consistently, we find that segregating plasmids are positioned at the ends of extending ParM filaments. Thus, the process of R1 plasmid segregation in E. coli appears to be mechanistically analogous to the actin-based motility operating in eukaryotic cells. In addition, we find evidence suggesting that plasmid pairing is required for ParM polymerization.

  16. Heterodimerization of the transcription factors E2F-1 and DP-1 is required for binding to the adenovirus E4 (ORF6/7) protein

    DEFF Research Database (Denmark)

    Helin, K; Harlow, E

    1994-01-01

    Adenovirus infection leads to E1A-dependent activation of the transcription factor E2F. E2F has recently been identified in complexes with cellular proteins such as the retinoblastoma protein (pRB) and the two pRB family members p107 and p130. E1A dissociates E2F from these cellular proteins......, and another viral protein, E4 (ORF6/7), can bind to E2F. The binding of E4 to E2F induces the formation of a stable DNA-binding complex containing the two proteins, and stimulation of the adenovirus E2 early promoter can occur. Recent studies have shown that E2F is the combined activity of several proteins...

  17. E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes.

    Directory of Open Access Journals (Sweden)

    Lisbeth Hansen

    Full Text Available Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs to NOD islets around 8-9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse.

  18. E2-2 Dependent Plasmacytoid Dendritic Cells Control Autoimmune Diabetes

    Science.gov (United States)

    Hansen, Lisbeth; Schmidt-Christensen, Anja; Gupta, Shashank; Fransén-Pettersson, Nina; Hannibal, Tine D.; Reizis, Boris; Santamaria, Pere; Holmberg, Dan

    2015-01-01

    Autoimmune diabetes is a consequence of immune-cell infiltration and destruction of pancreatic β-cells in the islets of Langerhans. We analyzed the cellular composition of the insulitic lesions in the autoimmune-prone non-obese diabetic (NOD) mouse and observed a peak in recruitment of plasmacytoid dendritic cells (pDCs) to NOD islets around 8–9 weeks of age. This peak coincides with increased spontaneous expression of type-1-IFN response genes and CpG1585 induced production of IFN-α from NOD islets. The transcription factor E2-2 is specifically required for the maturation of pDCs, and we show that knocking out E2-2 conditionally in CD11c+ cells leads to a reduced recruitment of pDCs to pancreatic islets and reduced CpG1585 induced production of IFN-α during insulitis. As a consequence, insulitis has a less aggressive expression profile of the Th1 cytokine IFN-γ and a markedly reduced diabetes incidence. Collectively, these observations demonstrate a disease-promoting role of E2-2 dependent pDCs in the pancreas during autoimmune diabetes in the NOD mouse. PMID:26624013

  19. E2 regulates epigenetic signature on Neuroglobin enhancer-promoter in neuronal cells

    Directory of Open Access Journals (Sweden)

    Michela eGuglielmotto

    2016-06-01

    Full Text Available Estrogens are neuroprotective factors in several neurological diseases. Neuroglobin (NGB is one of the estrogen target gene involved in neuroprotection, but little is known about its transcriptional regulation. Estrogen genomic pathway in gene expression regulation is mediated by estrogen receptors (ERα and ERβ that bind to specific regulatory genomic regions. We focused our attention on E2-induced NGB expression in human differentiated neuronal cell lines (SK-N-BE and NT-2. Previously, using bioinformatics analysis we identified a putative enhancer in the first intron of NGB locus. Therefore, we observed that E2 increased the enrichment of the H3K4me3 epigenetic marks at the promoter and of the H3K4me1 and H3K27Ac at the intron enhancer. In these NGB regulatory regions, we found estrogen receptor alpha (ER binding suggesting that ER may mediate chromatin remodeling to induce NGB expression upon E2 treatment. Altogether our data show that NGB expression is regulated by ERa binding on genomic regulatory regions supporting hormone therapy applications for the neuroprotection against neurodegenerative disease.

  20. Genome-wide analysis reveals NRP1 as a direct HIF1α-E2F7 target in the regulation of motorneuron guidance in vivo

    NARCIS (Netherlands)

    de Bruin, Alain; Cornelissen, Peter W A; Kirchmaier, Bettina C; Mokry, Michal; Iich, Elhadi; Nirmala, Ella; Liang, Kuo-Hsuan; Végh, Anna M D; Scholman, Koen T; Groot Koerkamp, Marian J; Holstege, Frank C; Cuppen, Edwin; Schulte-Merker, Stefan; Bakker, Walbert J

    2016-01-01

    In this study, we explored the existence of a transcriptional network co-regulated by E2F7 and HIF1α, as we show that expression of E2F7, like HIF1α, is induced in hypoxia, and because of the previously reported ability of E2F7 to interact with HIF1α. Our genome-wide analysis uncovers a transcriptio

  1. Mechanisms of Evolution in High-Consequence Drug Resistance Plasmids

    Directory of Open Access Journals (Sweden)

    Susu He

    2016-12-01

    Full Text Available The dissemination of resistance among bacteria has been facilitated by the fact that resistance genes are usually located on a diverse and evolving set of transmissible plasmids. However, the mechanisms generating diversity and enabling adaptation within highly successful resistance plasmids have remained obscure, despite their profound clinical significance. To understand these mechanisms, we have performed a detailed analysis of the mobilome (the entire mobile genetic element content of a set of previously sequenced carbapenemase-producing Enterobacteriaceae (CPE from the National Institutes of Health Clinical Center. This analysis revealed that plasmid reorganizations occurring in the natural context of colonization of human hosts were overwhelmingly driven by genetic rearrangements carried out by replicative transposons working in concert with the process of homologous recombination. A more complete understanding of the molecular mechanisms and evolutionary forces driving rearrangements in resistance plasmids may lead to fundamentally new strategies to address the problem of antibiotic resistance.

  2. Plasmid Conjugation in E. coli and Drug Resistance

    African Journals Online (AJOL)

    Prof. Ogunji

    respiratory infections etc) or prescribing the 'newest' antibiotics in the market when older “brands” may ..... influence an increase in mortality rate; high economic burden and longer hospital ... Conjugating plasmids into bacteria; Tri Parental.

  3. Construction and Identification of Plasmid pTA-TUB2

    Institute of Scientific and Technical Information of China (English)

    2003-01-01

    An about 1.40 Kb target gene fragment was yielded by PCR amplification with the plasmid pRB 129,which was identified by restriction enzyme digestion that the PCR product was TU B2 gene.The gene was digested by the restriction enzyme and was linked with pTA plasmid to construct pTA-TU B2 plasmid.The plasmid was transformed into Chaetomium spp.by PEG method and the transformation rate was 27/(2×105) and it is nine times higher than that of pRB 129.The transformants can grow on the PDA containing 1 000 μg*mL-1 carbendazim,which is 1 000 times higher than the original Chaetomium spp.The resistance was stable after 10 times transfer on non-selective medium.

  4. Fed-batch microbioreactor platform for scale down and analysis of a plasmid DNA production process.

    Science.gov (United States)

    Bower, Diana M; Lee, Kevin S; Ram, Rajeev J; Prather, Kristala L J

    2012-08-01

    The rising costs of bioprocess research and development emphasize the need for high-throughput, low-cost alternatives to bench-scale bioreactors for process development. In particular, there is a need for platforms that can go beyond simple batch growth of the organism of interest to include more advanced monitoring, control, and operation schemes such as fed-batch or continuous. We have developed a 1-mL microbioreactor capable of monitoring and control of dissolved oxygen, pH, and temperature. Optical density can also be measured online for continuous monitoring of cell growth. To test our microbioreactor platform, we used production of a plasmid DNA vaccine vector (pVAX1-GFP) in Escherichia coli via a fed-batch temperature-inducible process as a model system. We demonstrated that our platform can accurately predict growth, glycerol and acetate concentrations, as well as plasmid copy number and quality obtained in a bench-scale bioreactor. The predictive abilities of the micro-scale system were robust over a range of feed rates as long as key process parameters, such as dissolved oxygen, were kept constant across scales. We have highlighted plasmid DNA production as a potential application for our microbioreactor, but the device has broad utility for microbial process development in other industries as well. Copyright © 2012 Wiley Periodicals, Inc.

  5. Construtcion of Neisseria Gonorrhoeae Porin B Plasmid Recombinant and Its Expression in E.coli

    Institute of Scientific and Technical Information of China (English)

    SONG Qifa; LIAO Fang; YE Siying; CUI Bing; XIONG Ping

    2005-01-01

    Summary: A prokaryotic expression recombinant plasmid pET-PIB to express porin B (PIB) of Neisseria gonorrhoeae in E.coli DE3 was constructed in order to provide a basis of research in detection, prophylactic and therapeutic vaccine against the pathogen infection. The gene encoding PIB was amplified by PCR from Neisseria gonorrhoeae and cloned into prokaryotic expression plasmid pET-28a(+) to construct a pET-PIB recombinant, which was verified by restriction endonuclease and DNA sequencing. Protein PIB was expressed in E.coli DE3 induced with IPTG. The antigenicity of the expressed protein was evaluated by indirect ELISA. Rabbits were immunized with the protein and serum was collected after immunization. To assess the immunogenicity of the protein, the titer of serum to protein PIB was determined by ELISA. DNA sequence analysis showed that the nucleic acid sequence of PIB gene was 99.28 % of homology compared with that (NGPIB18) published in GenBank. A 41 kD fused protein was detected by SDS-PAGE and was proven to have reactivity with anti-PIB polyclonal antibody from mouse. A polyclonal antibody to PIB of 1:4000 titer determined by indirect ELISA was obtained from rabbit immunized with the purified product. Recombinant plasmid encoding PIB of Neisseria gonorrhoeae was constructed. Protein PIB with antigenicity and immunogenicity was successfully expressed.

  6. PPARβ Regulates Liver Regeneration by Modulating Akt and E2f Signaling.

    Directory of Open Access Journals (Sweden)

    Hui-Xin Liu

    Full Text Available The current study tests the hypothesis that peroxisome proliferator-activated receptor β (PPARβ has a role in liver regeneration due to its effect in regulating energy homeostasis and cell proliferation. The role of PPARβ in liver regeneration was studied using two-third partial hepatectomy (PH in Wild-type (WT and PPARβ-null (KO mice. In KO mice, liver regeneration was delayed and the number of Ki-67 positive cells reached the peak at 60 hr rather than at 36-48 hr after PH shown in WT mice. RNA-sequencing uncovered 1344 transcriptomes that were differentially expressed in regenerating WT and KO livers. About 70% of those differentially expressed genes involved in glycolysis and fatty acid synthesis pathways failed to induce during liver regeneration due to PPARβ deficiency. The delayed liver regeneration in KO mice was accompanied by lack of activation of phosphoinositide-dependent kinase 1 (PDK1/Akt. In addition, cell proliferation-associated increase of genes encoding E2f transcription factor (E2f 1-2 and E2f7-8 as well as their downstream target genes were not noted in KO livers 36-48 hr after PH. E2fs have dual roles in regulating metabolism and proliferation. Moreover, transient steatosis was only found in WT, but not in KO mice 36 hr after PH. These data suggested that PPARβ-regulated PDK1/Akt and E2f signaling that controls metabolism and proliferation is involved in the normal progression of liver regeneration.

  7. Fasciation induction by the phytopathogen Rhodococcus fascians depends upon a linear plasmid encoding a cytokinin synthase gene.

    Science.gov (United States)

    Crespi, M; Messens, E; Caplan, A B; van Montagu, M; Desomer, J

    1992-01-01

    Rhodococcus fascians is a nocardiform bacteria that induces leafy galls (fasciation) on dicotyledonous and several monocotyledonous plants. The wild-type strain D188 contained a conjugative, 200 kb linear extrachromosomal element, pFiD188. Linear plasmid-cured strains were avirulent and reintroduction of this linear element restored virulence. Pulsed field electrophoresis indicated that the chromosome might also be a linear molecule of 4 megabases. Three loci involved in phytopathogenicity have been identified by insertion mutagenesis of this Fi plasmid. Inactivation of the fas locus resulted in avirulent strains, whereas insertions in the two other loci affected the degree of virulence, yielding attenuated (att) and hypervirulent (hyp) bacteria. One of the genes within the fas locus encoded an isopentenyltranferase (IPT) with low homology to analogous proteins from Gram-negative phytopathogenic bacteria. IPT activity was detected after expression of this protein in Escherichia coli cells. In R.fascians, ipt expression could only be detected in bacteria induced with extracts from fasciated tissue. R.fascians strains without the linear plasmid but containing this fas locus alone could not provoke any phenotype on plants, indicating additional genes from the linear plasmid were also essential for virulence. These studies, the first genetic analysis of the interaction of a Gram-positive bacterium with plants, suggest that a novel mechanism for plant tumour induction has evolved in R.fascians independently from the other branches of the eubacteria. Images PMID:1547783

  8. Silica Vesicle Nanovaccine Formulations Stimulate Long-Term Immune Responses to the Bovine Viral Diarrhoea Virus E2 Protein.

    Directory of Open Access Journals (Sweden)

    Karishma T Mody

    Full Text Available Bovine Viral Diarrhoea Virus (BVDV is one of the most serious pathogen, which causes tremendous economic loss to the cattle industry worldwide, meriting the development of improved subunit vaccines. Structural glycoprotein E2 is reported to be a major immunogenic determinant of BVDV virion. We have developed a novel hollow silica vesicles (SV based platform to administer BVDV-1 Escherichia coli-expressed optimised E2 (oE2 antigen as a nanovaccine formulation. The SV-140 vesicles (diameter 50 nm, wall thickness 6 nm, perforated by pores of entrance size 16 nm and total pore volume of 0.934 cm3 g(-1 have proven to be ideal candidates to load oE2 antigen and generate immune response. The current study for the first time demonstrates the ability of freeze-dried (FD as well as non-FD oE2/SV140 nanovaccine formulation to induce long-term balanced antibody and cell mediated memory responses for at least 6 months with a shortened dosing regimen of two doses in small animal model. The in vivo ability of oE2 (100 μg/SV-140 (500 μg and FD oE2 (100 μg/SV-140 (500 μg to induce long-term immunity was compared to immunisation with oE2 (100 μg together with the conventional adjuvant Quil-A from the Quillaja saponira (10 μg in mice. The oE2/SV-140 as well as the FD oE2/SV-140 nanovaccine generated oE2-specific antibody and cell mediated responses for up to six months post the final second immunisation. Significantly, the cell-mediated responses were consistently high in mice immunised with oE2/SV-140 (1,500 SFU/million cells at the six-month time point. Histopathology studies showed no morphological changes at the site of injection or in the different organs harvested from the mice immunised with 500 μg SV-140 nanovaccine compared to the unimmunised control. The platform has the potential for developing single dose vaccines without the requirement of cold chain storage for veterinary and human applications.

  9. Transfer of conjugative plasmids among bacteria under environmentally relevant conditions

    DEFF Research Database (Denmark)

    Musovic, Sanin

    at spredningskapacitet af en konjugerbare plasmid, der koder for kviksølv resistens via merA genet, finder sted under substrat begrænsede forhold til syntetisk bakterielt samfund. Plasmid overførsel var meget forhøjet ved kontinuert udsættelse af mikrokosms for en høj koncentration af kviksølv. De forskellige vækstrater...

  10. The Native Plasmid pML21 Plays a Role in Stress Tolerance in Enterococcus faecalis ML21, as Analyzed by Plasmid Curing Using Plasmid Incompatibility.

    Science.gov (United States)

    Zuo, Fang-Lei; Chen, Li-Li; Zeng, Zhu; Feng, Xiu-Juan; Yu, Rui; Lu, Xiao-Ming; Ma, Hui-Qin; Chen, Shang-Wu

    2016-02-01

    To investigate the role of the native plasmid pML21 in Enterococcus faecalis ML21's response to abiotic stresses, the plasmid pML21 was cured based on the principle of plasmid incompatibility and segregational instability, generating E. faecalis mutant strain ML0. The mutant and the wild strains were exposed to abiotic stresses: bile salts, low pH, H2O2, ethanol, heat, and NaCl, and their survival rate was measured. We found that curing of pML21 lead to reduced tolerance to stress in E. faecalis ML0, especially oxidative and osmotic stress. Complementation analysis suggested that the genes from pML21 played different role in stress tolerance. The result indicated that pML21 plays a role in E. faecalis ML21's response to abiotic stresses.

  11. Chang'e-2 spacecraft observations of asteroid 4179 Toutatis

    Science.gov (United States)

    Ji, Jianghui; Jiang, Yun; Zhao, Yuhui; Wang, Su; Yu, Liangliang

    2016-01-01

    On 13 December 2012, Chang'e-2 completed a successful flyby of the near-Earth asteroid 4179 Toutatis at a closest distance of 770 meters from the asteroid's surface. The observations show that Toutatis has an irregular surface and its shape resembles a ginger-root of a smaller lobe (head) and a larger lobe (body). Such bilobate shape is indicative of a contact binary origin for Toutatis. In addition, the high-resolution images better than 3 meters provide a number of new discoveries about this asteroid, such as an 800-meter depression at the end of the large lobe, a sharply perpendicular silhouette near the neck region, boulders, indicating that Toutatis is probably a rubble-pile asteroid. Chang'e-2 observations have significantly revealed new insights into the geological features and the formation and evolution of this asteroid. In final, we brief the future Chinese asteroid mission concept.

  12. Chang'e-2 spacecraft observations of asteroid 4179 Toutatis

    CERN Document Server

    Ji, Jianghui; Zhao, Yuhui; Wang, Su; Yu, Liangliang

    2015-01-01

    On 13 December 2012, Chang'e-2 completed a successful flyby of the near-Earth asteroid 4179 Toutatis at a closest distance of 770 meters from the asteroid's surface. The observations show that Toutatis has an irregular surface and its shape resembles a ginger-root of a smaller lobe (head) and a larger lobe (body). Such bilobate shape is indicative of a contact binary origin for Toutatis. In addition, the high-resolution images better than 3 meters provide a number of new discoveries about this asteroid, such as an 800-meter depression at the end of the large lobe, a sharply perpendicular silhouette near the neck region, boulders, indicating that Toutatis is probably a rubble-pile asteroid. Chang'e-2 observations have significantly revealed new insights into the geological features and the formation and evolution of this asteroid. In final, we brief the future Chinese asteroid mission concept.

  13. The E2/M1 ratio in {Delta} photoproduction

    Energy Technology Data Exchange (ETDEWEB)

    Hoblit, S. [Brookhaven National Lab., Upton, NY (United States). Physics Dept.]|[Univ. of Virginia, Charlottesville, VA (United States). Dept. of Physics; Blanpied, G. [Univ. of South Carolina, Columbia, SC (United States). Dept. of Physics; Blecher, M. [Virginia Polytechnic Inst. and State Univ., Blacksburg, VA (United States). Physics Dept.] [and others; LEGS Collaboration

    1997-10-01

    New high-precision measurements of p({rvec {gamma}}, {pi}) and p({rvec {gamma}}, {gamma}) cross sections and beam asymmetries have been combined with other polarization ratios in a simultaneous analysis of both reactions. The E2/M1 mixing ratio for the n {r_arrow} {Delta} transition extracted from this analysis is EMR = {minus}3.0% {+-} 0.3 (stat+sys) {+-} 0.2 (model).

  14. Generalized seniority and E 2 transitions in the tin isotopes

    Science.gov (United States)

    Morales, Irving O.; Van Isacker, P.; Talmi, I.

    2011-09-01

    Recently, a shallow minimum was discovered in B(E2) values in even Sn isotopes around the middle of the neutron major shell. A peak in that region was expected according to calculations using generalized seniority. In a model calculation we show that the observed shape is consistent with generalized seniority. It seems to be due to the order of filling of j-orbits.

  15. A New Shuttle Plasmid That Stably Replicates in Clostridium acetobutylicum.

    Science.gov (United States)

    Lee, Sang-Hyun; Kwon, Min-A; Choi, Sunwha; Kim, Sooah; Kim, Jungyeon; Shin, Yong-An; Kim, Kyoung Heon

    2015-10-01

    We have developed a new shuttle plasmid, designated as pLK1-MCS that can replicate in both Clostridium acetobutylicum and Escherichia coli, by combining the pUB110 and pUC19 plasmids. Plasmid pLK1-MCS replicated more stably than previously reported plasmids containing either the pIM13 or the pAMβ1 replicon in the absence of antibiotic selective pressure. The transfer frequency of pLK1-MCS into C. acetobutylicum was similar to the transfer frequency of other shuttle plasmids. We complemented C. acetobutylicum ML1 (that does not produce solvents such as acetone, butanol, and ethanol owing to loss of the megaplasmid pSOL1 harboring the adhE1-ctfAB-adc operon) by introducing pLK1-MCS carrying the adhE1-ctfAB-adc operon into C. acetobutylicum ML1. The transformed cells were able to resume anaerobic solvent production, indicating that the new shuttle plasmid has the potential for practical use in microbial biotechnology.

  16. Plasmid copy number noise in monoclonal populations of bacteria

    Science.gov (United States)

    Wong Ng, Jérôme; Chatenay, Didier; Robert, Jérôme; Poirier, Michael Guy

    2010-01-01

    Plasmids are extra chromosomal DNA that can confer to their hosts’ supplementary characteristics such as antibiotic resistance. Plasmids code for their copy number through their own replication frequency. Even though the biochemical networks underlying the plasmid copy number (PCN) regulation processes have been studied and modeled, no measurement of the heterogeneity in PCN within a whole population has been done. We have developed a fluorescent-based measurement system, which enables determination of the mean and noise in PCN within a monoclonal population of bacteria. Two different fluorescent protein reporters were inserted: one on the chromosome and the other on the plasmid. The fluorescence of these bacteria was measured with a microfluidic flow cytometry device. We show that our measurements are consistent with known plasmid characteristics. We find that the partitioning system lowers the PCN mean and standard deviation. Finally, bacterial populations were allowed to grow without selective pressure. In this case, we were able to determine the plasmid loss rate and growth inhibition effect.

  17. Construction of Recombinant Plasmid Harboring APP717 Mutation and Preliminary Study of APP Proteolysis

    Institute of Scientific and Technical Information of China (English)

    LI Xiaoqing; XUE Zheng; ZHANG Suming

    2007-01-01

    In order to investigate the pathogenesis of Alzheimer disease (AD) and study the enzy-matic progress of amyloid precursor protein (APP), the fluorescent eukaryotic expression plasmid of C99 was constructed containing APP717 mutation. The fragment encoding the last 99-aa of APP(which was named C99 containing APP717 mutation), together with the fragment encoding yellow fluorescence protein (which was named YFP) were amplified by PCR. The two fragments (YFP andC99) were inserted into the vector pcDNA3.0. The recombinant plasmid pcDNA3.0-YFP-C99 was accomplished and its authenticity was confirmed by enzyme digestion and sequencing. Then SH-SY5Y cells were transiently transfected with the recombinant plasmid pcDNA3.0-YFP-C99. The expression of the fusion gene was detected by laser confocalmicroscopy. Amyloid-β (Aβ) was de- tected by both microscopy and immunochemistry. The authenticity of the construct was confirmed by the endonuclease digestion and DNA sequencing. The YFP fluorescence could be seen and proved the expression of fusion gene. Aβ labeled by YFP was detected by confocalmicroscopy and confirmed by immunocytochemistry. It was found that Aβ accumulated and deposited in the intracytoplasm, mem-brane and outside of the cell. Furthermore, Aβ accumulated mainly within the cell ahead of the depo-sition in the cell space and the cell shape was rough. It was suggested that Aβ could be generatedwithin the cells. Aβ accumulated in the cell at the early stage before the deposition outside of the cells.Intracellular Aβ accumulation induced the secondary damage to the cells and caused the cell shape rough. Taken together, the recombinant plasmid, pcDNA3.0-YFP-C99 could be a useful tool to fur-ther study the cleavage mechanism of APP and to explore the pathogenesis of AD.

  18. Construction and Use of Flow Cytometry Optimized Plasmid-Sensor Strains

    DEFF Research Database (Denmark)

    Bahl, Martin Iain; Oregaard, Gunnar; Sørensen, Søren Johannes;

    2009-01-01

    stability of the plasmid is high. The method presented here relies on a phenotypic (green fluorescence protein) marker, which is switched on if the host bacteria loses the residing plasmid. The incorporation of flow cytometry for single-cell detection and discrimination between plasmid-free and plasmid...

  19. Allelopathy of plasmid-bearing and plasmid-free organisms competing for two complementary resources in a chemostat.

    Science.gov (United States)

    Bhattacharyya, Joydeb; Smith, Hal L; Pal, Samares

    2012-01-01

    We consider a model of competition between plasmid-bearing and plasmid-free organisms for two complementary nutrients in a chemostat. We assume that the plasmid-bearing organism produces an allelopathic agent at the cost of its reproductive abilities which is lethal to plasmid-free organism. Our analysis leads to different thresholds in terms of the model parameters acting as conditions under which the organisms associated with the system cannot thrive even in the absence of competition. Local stability of the system is obtained in the absence of one or both the organisms. Also, global stability of the system is obtained in the presence of both the organisms. Computer simulations have been carried out to illustrate various analytical results.

  20. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Science.gov (United States)

    Kotzerke, Joerg; Punzet, Robert; Runge, Roswitha; Ferl, Sandra; Oehme, Liane; Wunderlich, Gerd; Freudenberg, Robert

    2014-01-01

    (99m)Tc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99m)Tc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs) or double-strand breaks (DSBs); the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99m)Tc-labeled HYNIC-DAPI compound with that of (99m)Tc pertechnetate ((99m)TcO4(-)). pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03) was twice that caused by (99m)TcO4(-) (0.51), and the number of DSBs increased fivefold in the (99m)Tc-HYNIC-DAPI-treated sample compared with the (99m)TcO4(-) treated sample (0.02 to 0.10). In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99m)TcO4(-) treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99m)Tc-HYNIC-DAPI-treated samples. These results indicated that (99m)Tc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99m)Tc-labeled compound with DNA. In contrast to these results, (99m)TcO4(-) induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99m)Tc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by

  1. 99mTc-labeled HYNIC-DAPI causes plasmid DNA damage with high efficiency.

    Directory of Open Access Journals (Sweden)

    Joerg Kotzerke

    Full Text Available (99mTc is the standard radionuclide used for nuclear medicine imaging. In addition to gamma irradiation, (99mTc emits low-energy Auger and conversion electrons that deposit their energy within nanometers of the decay site. To study the potential for DNA damage, direct DNA binding is required. Plasmid DNA enables the investigation of the unprotected interactions between molecules and DNA that result in single-strand breaks (SSBs or double-strand breaks (DSBs; the resulting DNA fragments can be separated by gel electrophoresis and quantified by fluorescent staining. This study aimed to compare the plasmid DNA damage potential of a (99mTc-labeled HYNIC-DAPI compound with that of (99mTc pertechnetate ((99mTcO4(-. pUC19 plasmid DNA was irradiated for 2 or 24 hours. Direct and radical-induced DNA damage were evaluated in the presence or absence of the radical scavenger DMSO. For both compounds, an increase in applied activity enhanced plasmid DNA damage, which was evidenced by an increase in the open circular and linear DNA fractions and a reduction in the supercoiled DNA fraction. The number of SSBs elicited by 99mTc-HYNIC-DAPI (1.03 was twice that caused by (99mTcO4(- (0.51, and the number of DSBs increased fivefold in the (99mTc-HYNIC-DAPI-treated sample compared with the (99mTcO4(- treated sample (0.02 to 0.10. In the presence of DMSO, the numbers of SSBs and DSBs decreased to 0.03 and 0.00, respectively, in the (99mTcO4(- treated samples, whereas the numbers of SSBs and DSBs were slightly reduced to 0.95 and 0.06, respectively, in the (99mTc-HYNIC-DAPI-treated samples. These results indicated that (99mTc-HYNIC-DAPI induced SSBs and DSBs via a direct interaction of the (99mTc-labeled compound with DNA. In contrast to these results, (99mTcO4(- induced SSBs via radical formation, and DSBs were formed by two nearby SSBs. The biological effectiveness of (99mTc-HYNIC-DAPI increased by approximately 4-fold in terms of inducing SSBs and by approximately

  2. Poster 14: Explorer of Enceladus and Titan (E2T)

    Science.gov (United States)

    Mitri, Giuseppe; Tobie, Gabriel; Postberg, Frank; Soderblom, Jason M.; Wurz, Peter; Barnes, Jason W.; Berga, Marco; Coustenis, Athena; D'Ottavio, Andrea Hayes, Alexander G.; Hayne, Paul O.; Lebreton, Jean-Pierre; Lorenz, Ralph D.; Martelli, Andrea; Petropoulos, Anastassios E.; Yen, Chen-wan L.; Reh, Kim R.; Sotin, Christophe; Srama, Ralf; Tortora, Paolo

    2016-06-01

    The NASA-ESA Cassini-Huygens mission has revealed Titan and Enceladus to be two of the most enigmatic worlds in the Solar System. Titan, with its organically rich and dynamic atmosphere and geology, and Enceladus, with its active plume, both harboring subsurface oceans, are prime environments in which to investigate the conditions for the emergence of life and the habitability of Ocean Worlds. Explorer of Enceladus and Titan (E2T) is dedicated to investigating the evolution and habitability of these Saturnian satellites and will be proposed as a medium-class mission led by ESA in collaboration with NASA in response to ESA's M5 Call. E2T has a focused payload that will provide in-situ sampling and high-resolution imaging during multiple flybys of Enceladus and Titan using a solar-electric powered spacecraft in orbit around Saturn. The E2T mission will provide high-resolution mass spectroscopy of the plume emanating from Enceladus' south po