Sample records for jnk-mediated interleukin-2 mrna

  1. Cytokine mRNA expression in intestinal tissue of interleukin-2 deficient mice with bowel inflammation

    PubMed Central

    Autenrieth, I; Bucheler, N; Bohn, E; Heinze, G; Horak, I

    1997-01-01

    Background—Mice deficient in interleukin-2 (IL-2) develop inflammatory bowel disease resembling ulcerative colitis in humans. Recent studies provided evidence that ?? T cells, particularly CD4 T cells, rather than B cells, are involved in the pathogenesis of bowel inflammation of IL-2 deficient mice. ?Aim—To analyse the pattern of expression of cytokine mRNA in intestinal tissue of normal and IL-2 deficient mice. ?Methods—Expression of ?-actin, IL-1?, IL-1?, IL-6, IL-10, tumour necrosis factor ? (TNF-?), interferon ? (IFN-?) and transforming growth factor ?1 (TGF-?1) mRNA was analysed in colon and small intestinal tissue of both IL-2 deficient (IL-2?/?) mice and normal (wild type) litter mates (IL-2+/+) at different ages by using qualitative, as well as semiquantitative, competitive reverse transcription polymerase chain reaction (RT-PCR). Results were correlated with the phase of progression of the disease, as determined by histology. ?Results—IL-2?/? mice had expressed low levels of IL-1?, IL-1?, IL-6, TNF-?, and IFN-? mRNA in the colon by 1.5 weeks of age. In advance of the development of histologically and clinically detectable bowel inflammation, expression of IL-1?, IL-1?, IL-6, TNF-?, IFN-?, and IL-10, but not TGF-?1, mRNA increased in the colon of IL-2 deficient mice. In contrast, IL-2+/+ mice expressed TGF-?1 mRNA in colon tissue at 13 and 23 weeks of age, but not IL-1?, IL-1?, IL-6, TNF-?, IL-10, or IFN-? mRNA. Levels of expression of cytokine mRNA in tissue from the small intestine were comparable in IL-2?/? and IL-2+/+ mice. ?Conclusions—Bowel inflammation in IL-2 deficient mice is preceded by an increase in IL-1?, IL-1?, TNF-?, and IFN-? mRNA expression in colon tissue. Low levels of TGF-?1, but high levels of IL-1?, IL-1?, IL-6, TNF-?, IFN-?, and IL-10 mRNA expression correlate with the manifestation of severe colitis, and suggest that T cells and macrophages are involved in bowel inflammation of IL-2 deficient mice. ?? Keywords: cytokine; mRNA expression; interleukin-2 deficient mice; bowel inflammation PMID:9462212

  2. Sensitivity of Whole-Blood T Lymphocytes in Individual Patients to Tacrolimus (FK 506): Impact of Interleukin2 mRNA Expression as Surrogate Measure of Immunosuppressive Effect

    Microsoft Academic Search

    Christoph Hartel; Nina Schumacher; Lutz Fricke; Brigitte Ebel; Holger Kirchner; Michael Muller-Steinhardt

    Background: To optimize immunosuppressive treat- ment in individual transplant patients, functional mea- surements of the effects of tacrolimus (FK 506) are of clinical importance. Previous investigations have dem- onstrated the occurrence of tacrolimus-resistant produc- tion of interleukin-2 (IL-2) in vitro, which may explain in part why rejection episodes are still a frequent problem despite attainment of therapeutic blood con- centrations

  3. Targeting of TAK1 by the NF-B protein Relish regulates the JNK-mediated

    E-print Network

    an adaptive immune system, being en- tirely dependent on innate immunity for its resistance to microbialTargeting of TAK1 by the NF- B protein Relish regulates the JNK-mediated immune response, San Diego, California 92121, USA The molecular circuitry underlying innate immunity is constructed

  4. Musca domestica larva lectin induces apoptosis in BEL-7402 cells through a Ca(2+)/JNK-mediated mitochondrial pathway.

    PubMed

    Wang, Chun-Ling; Xia, Yan; Nie, Jian-Zeng; Zhou, Minghui; Zhang, Rong-Ping; Niu, Li-Li; Hou, Li-Hua; Cao, Xiao-Hong

    2013-06-01

    Although Musca domestica larvae lectin (MLL) is able to inhibit cancer cell proliferation and to induce cancer cell apoptosis, the molecular mechanism(s) responsible for these processes remain elusive. In the current study, the signaling network underlying the MLL-induced apoptosis of human hepatoma BEL-7402 cell was investigated. Our data found out that MLL causes a sustained increase of the intracellular Ca(2+) and this process was prevented by the intracellular calcium chelator, BAPTA-AM, suggesting the involvement of intracellular Ca(2+) in MLL-induced cell apoptosis. MLL also causes the production of reactive oxygen species and elevates the phosphorylation status of JNK, processes associated with the increased cytoplasmic Ca(2+). The mitochondrial permeability transition pore (MPTP) opening study showed that MLL treatment of BEL-7402 cells results in the opening of MPTP and a reduction of mitochondrial transmembrane potential. In such condition, cytochrome-c was detected to be released from mitochondria to cytoplasm through the MPTP. This eventually activates caspase-3 and thus results in apoptosis of the tested BEL-7402 cells. According to a comprehensive review of all the evidence, it is concluded that MLL induces apoptosis of BEL-7402 cells through a Ca(2+)/JNK-mediated MPTP pathway. PMID:23247835

  5. A JNK-mediated autophagy pathway that triggers c-IAP degradation and necroptosis for anticancer chemotherapy

    PubMed Central

    He, Weiyang; Wang, Qiong; Srinivasan, Balasubramanian; Xu, Jennings; Padilla, Mabel T.; Li, Zi; Wang, Xia; Liu, Yushi; Gou, Xin; Shen, Han-Ming; Xing, Chengguo; Lin, Yong

    2014-01-01

    Killing cancer cells through the induction of apoptosis is one of the main mechanisms of chemotherapy. However, numerous cancer cells have primary or acquired apoptosis resistance, resulting in chemoresistance. In this study, using a novel chalcone derivative chalcone-24 (Chal-24), we identified a novel anticancer mechanism through autophagy-mediated necroptosis (RIP1- and RIP3-dependent necrosis). Chal-24 potently killed different cancer cells with induction of necrotic cellular morphology while causing no detectable caspase activation. Blocking the necroptosis pathway with either necrostatin-1 or by knockdown of RIP1 and RIP3 effectively blocked the cytotoxicity of Chal-24, suggesting that Chal-24-induced cell death is associated with necroptosis. Chal-24 robustly activated JNK and ERK and blockage of which effectively suppressed Chal-24-induced cytotoxicity. In addition, Chal-24 strongly induced autophagy that is dependent on JNK-mediated phosphorylation of Bcl-2 and Bcl-xL and dissociation of Bcl-2 or Bcl-xL from Beclin1. Importantly, suppression of autophagy, with either pharmacological inhibitors or siRNAs targeting the essential autophagy components ATG7 and Beclin1, effectively attenuated Chal-24-induced cell death. Furthermore, we found that autophagy activation resulted in c-IAP1 and c-IAP2 degradation and formation of the Ripoptosome that contributes to necroptosis. These results thus establish a novel mechanism for killing cancer cells that involves autophagy-mediated necroptosis, which may be employed for overcoming chemoresistance. PMID:23831571

  6. Advances in interleukin 2 receptor targeted treatment

    PubMed Central

    Morris, J.; Waldmann, T.

    2000-01-01

    T cell activation and cellular immune responses are modulated by interleukin 2 (IL2) through binding to its corresponding cell surface receptor. Three forms of the receptor are recognised based on IL2 binding affinity. The high affinity receptor is a heterotrimer composed of ?, ?, and ?c-polypeptide chains. The 55 kDa ?-chain also known as the Tac (T cell activation) antigen or CD-25 is a unique subunit of the high affinity IL2 receptor (IL2R?). Resting T cells express few IL2R?, however, when activated, the expression of ILR2? rapidly increases. The IL2R? is shed from the cell surface and is measurable in the serum as a 45 kDa soluble form (s-Tac or s-IL2R?). Serum concentrations of s-Tac can be used as a surrogate marker for T cell activation and IL2R? expression. IL2R? is over expressed by T cells in a number of autoimmune diseases, allograft rejection and a variety of lymphoid neoplasms. IL2 induced proliferation of T cells can be inhibited by the murine monoclonal antibody (anti-Tac) directed against the ?-chain of the IL2R. Through molecular engineering, murine anti-Tac has been humanised reducing its immunogenicity without changing its specificity. Humanised anti-Tac (HAT) has been shown to reduce the incidence of renal and cardiac allograft rejection as well as decrease the severity of graft versus host disease in patients undergoing HLA matched allogeneic bone marrow transplantation. IL2R? targeted treatment with radioimmunoconjugates of anti-Tac and immunotoxins has shown promise in the treatment of CD25 expressing lymphomas.?? PMID:11053100

  7. Resveratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation.

    PubMed

    Puissant, Alexandre; Robert, Guillaume; Fenouille, Nina; Luciano, Frederic; Cassuto, Jill-Patrice; Raynaud, Sophie; Auberger, Patrick

    2010-02-01

    Autophagy that is induced by starvation or cellular stress can enable cancer cell survival by sustaining energy homeostasis and eliminating damaged organelles and proteins. In response to stress, cancer cells have been reported to accumulate the protein p62/SQSTM1 (p62), but its role in the regulation of autophagy is controversial. Here, we report that the plant phytoalexin resveratrol (RSV) triggers autophagy in imatinib-sensitive and imatinib-resistant chronic myelogenous leukemia (CML) cells via JNK-dependent accumulation of p62. JNK inhibition or p62 knockdown prevented RSV-mediated autophagy and antileukemic effects. RSV also stimulated AMPK, thereby inhibiting the mTOR pathway. AMPK knockdown or mTOR overexpression impaired RSV-induced autophagy but not JNK activation. Lastly, p62 expression and autophagy in CD34+ progenitors from patients with CML was induced by RSV, and disrupting autophagy protected CD34+ CML cells from RSV-mediated cell death. We concluded that RSV triggered autophagic cell death in CML cells via both JNK-mediated p62 overexpression and AMPK activation. Our findings show that the JNK and AMPK pathways can cooperate to eliminate CML cells via autophagy. PMID:20103647

  8. Soluble interleukin-2 receptor as a predictor of neonatal sepsis

    Microsoft Academic Search

    Michael L. Spear; John L. Stefano; Paul Fawcett; Roy Proujansky

    1995-01-01

    We prospectively measured soluble interleukin-2 receptor levels in 56 premature infants with suspected sepsis and demonstrated significant differences between those with positive results on blood, urine, or cerebrospinal fluid cultures, and those with negative results. Soluble interleukin-2 receptor levels can be used to facilitate the diagnosis of sepsis in premature infants with negative blood culture results. (J PEDIATR 1995;126:982-5)

  9. Nicotine exaggerates LPS-induced airway hyperreactivity via JNK-mediated up-regulation of Toll-like receptor 4.

    PubMed

    Xu, Yuan; Zhang, Yaping; Cardell, Lars-Olaf

    2014-09-01

    Tobacco smokers often display increased airway hyperreactivity (AHR) when faced with bacterial infections. The present study uses a murine organ-culture model to dissect the mechanisms involved in this exaggerated smooth muscle response. Nicotine simulates the effects of smoking, and LPS represents bacterial infection. Contractile responses of isolated murine tracheal segments were analyzed in myographs after organ culture with increasing concentrations of LPS and/or nicotine for 4 days with or without specific MAPK inhibitors. Nicotine's effect on the expression of cell surface Toll-like receptors (TLRs), MCP-1, COX-2, and TNF-? were examined by real-time PCR. Increased protein expression was verified by immunohistochemistry. LPS concentration-dependently increased contractile responses to bradykinin and des-Arg(9)-bradykinin. A combination of nicotine and low-dose LPS caused powerful synergistic contractions along with increased kinin receptor expression. Specific kinin B1 and B2 receptor inhibitors blocked this reaction. Nicotine increased mRNA and protein expression of TLR4 and -6 in the epithelium and smooth muscle layer, with MCP-1 and COX-2 mRNA increasing in parallel. Specific inhibition of JNK attenuated nicotine's effects. In conclusion, long-term exposure to nicotine up-regulated the expression of TLR4 and -6 via a JNK-related pathway, causing an exaggeration of the LPS-induced local airway inflammation and increased AHR. This might offer a mechanistic explanation to the increased AHR seen in tobacco smokers confronted with bacterial infections. PMID:24669857

  10. Distribution of interleukin-2, -4, -10, tumour necrosis factor-? and transforming growth factor-? mRNAs in oral lichen planus

    Microsoft Academic Search

    C Simark-Mattsson; G Bergenholtz; M Jontell; C Eklund; G. J Seymour; P. B Sugerman; N. W Savage; U. I Dahlgren

    1999-01-01

    In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-? (TNF-?) and transforming growth factor ?-1 (TGF-?-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with 35S-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-? (IFN-?), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes

  11. c-Jun N-terminal kinase (JNK)-mediated modulation of brain mitochondria function: new target proteins for JNK signalling in mitochondrion-dependent apoptosis.

    PubMed

    Schroeter, Hagen; Boyd, Clinton S; Ahmed, Ruhi; Spencer, Jeremy P E; Duncan, Roger F; Rice-Evans, Catherine; Cadenas, Enrique

    2003-06-01

    The molecular mechanisms underlying the initiation and control of the release of cytochrome c during mitochondrion-dependent apoptosis are thought to involve the phosphorylation of mitochondrial Bcl-2 and Bcl-x(L). Although the c-Jun N-terminal kinase (JNK) has been proposed to mediate the phosphorylation of Bcl-2/Bcl-x(L) the mechanisms linking the modification of these proteins and the release of cytochrome c remain to be elucidated. This study was aimed at establishing interdependency between JNK signalling and mitochondrial apoptosis. Using an experimental model consisting of isolated, bioenergetically competent rat brain mitochondria, these studies show that (i) JNK catalysed the phosphorylation of Bcl-2 and Bcl-x(L) as well as other mitochondrial proteins, as shown by two-dimensional isoelectric focusing/SDS/PAGE; (ii) JNK-induced cytochrome c release, in a process independent of the permeability transition of the inner mitochondrial membrane (imPT) and insensitive to cyclosporin A; (iii) JNK mediated a partial collapse of the mitochondrial inner-membrane potential (Deltapsim) in an imPT- and cyclosporin A-independent manner; and (iv) JNK was unable to induce imPT/swelling and did not act as a co-inducer, but as an inhibitor of Ca-induced imPT. The results are discussed with regard to the functional link between the Deltapsim and factors influencing the permeability transition of the inner and outer mitochondrial membranes. Taken together, JNK-dependent phosphorylation of mitochondrial proteins including, but not limited to, Bcl-2/Bcl-x(L) may represent a potential of the modulation of mitochondrial function during apoptosis. PMID:12614194

  12. Leflunomide or A77 1726 protect from acetaminophen-induced cell injury through inhibition of JNK-mediated mitochondrial permeability transition in immortalized human hepatocytes

    SciTech Connect

    Latchoumycandane, Calivarathan [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Seah, Quee Ming [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Tan, Rachel C.H. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore); Sattabongkot, Jetsumon [Armed Forces Research Institute of Medical Sciences, Bangkok 10400 (Thailand); Beerheide, Walter [Siam Life Science Ltd., Bangkok 10500 (Thailand); Boelsterli, Urs A. [Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117597 (Singapore) and Department of Pharmacy, Faculty of Science, National University of Singapore, Singapore 117597 (Singapore)]. E-mail: phcbua@nus.edu.sg

    2006-11-15

    Leflunomide, a disease-modifying anti-rheumatic drug, protects against T-cell-mediated liver injury by poorly understood mechanisms. The active metabolite of leflunomide, A77 1726 (teriflunomide) has been shown to inhibit stress-activated protein kinases (JNK pathway), which are key regulators of mitochondria-mediated cell death. Therefore, we hypothesized that leflunomide may protect from drugs that induce the mitochondrial permeability transition (mPT) by blocking the JNK signaling pathway. To this end, we exposed cultured immortalized human hepatocytes (HC-04) to the standard protoxicant drug acetaminophen (APAP), which induces CsA-sensitive mPT-mediated cell death. We determined the effects of leflunomide on the extent of APAP-induced hepatocyte injury and the upstream JNK-mediated mitochondrial signaling pathways. We found that leflunomide or A77 1726 concentration-dependently protected hepatocytes from APAP (1 mM)-induced mitochondrial permeabilization and lethal cell injury. This was not due to proximal inhibition of CYP-catalyzed APAP bioactivation to its thiol-reactive metabolite. Instead, we demonstrate that leflunomide (20 {mu}M) inhibited the APAP-induced early (3 h) activation (phosphorylation) of JNK1/2, thus inhibiting phosphorylation of the anti-apoptotic protein Bcl-2 and preventing P-Bcl-2-mediated induction of the mPT. This greatly attenuated mitochondrial cytochrome c release, which we used as a marker for mitochondrial permeabilization. The specific JNK2 inhibitor SP600125 similarly protected from APAP-induced cell death. In conclusion, these findings are consistent with our hypothesis that leflunomide protects from protoxicant-induced hepatocyte injury by inhibiting JNK signaling and preventing mPT induction.

  13. Recent Advances in the Understanding of Interleukin2 Signal Transduction

    Microsoft Academic Search

    Franck Gesbert; Maryvonnick Delespine-Carmagnat; Jacques Bertoglio

    1998-01-01

    Interleukin-2 is one of the critical cytokines that control the proliferation and differentiation of cells of the immune system. The present article briefly reviews the current and recently established knowledge on the intracellular signaling events that convert the initial interaction of IL-2 with its receptor into pathways leading to the various biological functions. A first step in IL-2 signaling is

  14. Ginsenoside Rb3 Protects Cardiomyocytes against Ischemia-Reperfusion Injury via the Inhibition of JNK-Mediated NF-?B Pathway: A Mouse Cardiomyocyte Model

    PubMed Central

    Xie, Zulong; Yang, Shuang; Xu, Wei; Hou, Jingbo; Yu, Bo

    2014-01-01

    Ginsenoside Rb3 is extracted from the plant Panax ginseng and plays important roles in cardiovascular diseases, including myocardial ischemia-reperfusion (I/R) injury. NF-?B is an important transcription factor involved in I/R injury. However, the underlying mechanism of ginsenoside Rb3 in myocardial I/R injury remains poorly understood. In the current study, a model of myocardial I/R injury was induced via oxygen and glucose deprivation (OGD) followed by reperfusion (OGD-Rep) in mouse cardiac myoblast H9c2 cells. Our data demonstrate that ginsenoside Rb3 suppresses OGD-Rep-induced cell apoptosis by the suppression of ROS generation. By detecting the NF-?B signaling pathway, we discover that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is closely related to the inhibition of NF-?B activity. Ginsenoside Rb3 inhibits the upregulation of phospho-I?B-? and nuclear translocation of NF-?B subunit p65 which are induced by ORD-Rep injury. In addition, the extract also inhibits the OGD-Rep-induced increase in the expression of inflammation-related factors, such as IL-6, TNF-?, monocyte chemotactic protein-1 (MCP-1), MMP-2 and MMP-9. However, LPS treatment alleviates the protective roles of ginsenoside Rb3 and activates the NF-?B pathway. Finally, the upstream factors of NF-?B were analyzed, including the Akt/Foxo3a and MAPK signaling pathways. We find that ginsenoside Rb3 pretreatment only decreases the phosphorylation of JNK induced by OGD-Rep injury, an indicator of the MAPK pathway. Importantly, an inhibitor of phospho-JNK, SP600125, protects against OGD-Rep induced apoptosis and inhibited NF-?B signaling pathway, similar to the roles of ginsenoside Rb3. Taken together, our results demonstrate that the protective effect of ginsenoside Rb3 on the OGD-Rep injury is attributed to the inhibition of JNK-mediated NF-?B activation, suggesting that ginsenoside Rb3 has the potential to serve as a novel therapeutic agent for myocardial I/R injury. PMID:25084093

  15. Interleukin-2 and interleukin-2 receptor expression in human corticotrophic adenoma and murine pituitary cell cultures.

    PubMed Central

    Arzt, E; Stelzer, G; Renner, U; Lange, M; Müller, O A; Stalla, G K

    1992-01-01

    The production of IL-1 and IL-6 by pituitary cells has recently been demonstrated. In this study we investigated the expression of IL-2 and its receptor (IL-2R) by pituitary cells of different species. In Northern blots, a single hybridizing band of 1 kb, identical to that in normal stimulated lymphocytes, was obtained with specific IL-2 probes. In the mouse AT-20 pituitary tumor cell line, IL-2 mRNA expression was detected after stimulation with corticotropin-releasing hormone or phorbol myristate acetate. In human corticotrophic adenoma cells, basal IL-2 mRNA expression as well as IL-2 secretion were further stimulated by phorbol myristate acetate. Both adenoma and AtT-20 cells showed detectable amounts of IL-2R mRNA and by immunofluorescence, IL-2R membrane expression. In addition, dual immunofluorescence studies in rat anterior pituitary cells demonstrated colocalization of IL-2R with ACTH-positive cells and other cell types expressing the receptor. In addition to the action of lymphocyte-produced IL-2, this cytokine may have a paracrine or autocrine regulatory role within the pituitary. It remains to be established whether IL-2 production occurs in the normal pituitary or is intrinsic to the process of tumor development of these cells. IL-2 may be involved in the growth control of pituitary cells. Images PMID:1331177

  16. Local interleukin-2 therapy in bovine ocular squamous cell carcinoma

    Microsoft Academic Search

    V. P. M. G. Rutten; W. R. Klein; W. A. C. De Jong; W. Misdorp; W. Den Otter; P. A. Steerenberg; W. H. De Jong; E. J. Ruitenberg

    1989-01-01

    Summary Five cows bearing bovine ocular squamous cell carcinoma (BOSCC) were treated with low doses of recombinant human interleukin-2 (rhIL-2). A dose of 2500 U rhIL-2 was injected intralesionally and another 2500 U were injected into the subparotid regional lymph node once a day during a period of 5 consecutive days. This cycle of 5 days was repeated after an

  17. CDNA clones coding for polypeptides exhibiting murine interleukin-2 activity

    SciTech Connect

    Lee, F.D.; Yokota, T.; Arai, K.

    1989-01-17

    A process is described for producing a polypeptide exhibiting murine interleukin-2 activity, the process comprising the steps of: providing a vector comprising a nucleotide sequence coding for the polypeptide, wherein the nucleotide sequence is capable of being expressed by a host containing the vector and wherein the nucleotide sequence is selected from the group of nucleotide sequences capable of encoding a polypeptide; incorporating the vector into the host; and maintaining the host containing the vector under conditions suitable for expression of the nucleotide sequence into the polypeptide.

  18. Interleukin-2 dependent cytotoxic T-cell clones

    SciTech Connect

    Palladino, M.

    1987-07-28

    A method is described of stimulating production of the lymphokines ..cap alpha..-interferon and ..beta..-interferon by interleukin-2 dependent cytotoxic cultured T-cell lines comprising administering to a T-cell line selected from the group consisting of T-cell lines CTLL-RP (CRL 8201), CTLL-R8 (CRL 8202), CTLL-R9 (CRL 8203), CTLL-R11 (CRL 8204), and CTLL-R12 (CRL 8205). An amount of an antigen selected from the group consists of Newcastle Disease Virus and Sendai Virus sufficient to cause stimulation of production of the lymphokines.

  19. Reversible anergy in circulating lymphocytes of cancer patients during interleukin-2 therapy

    Microsoft Academic Search

    Emilio Clementi; Eraldo Bucci; Giovanni Citterio; Giuseppo Landonio; Giuseppe Consogno; Claudio Fortis

    1994-01-01

    Interleukin-2 plays a crucial role in enhancing the antitumor immune response. Clinical trials, mainly in renal cell carcinoma and melanoma patients, have been carried out with encouraging results. Recent reports demonstrated that interleukin-2 therapy may depress the immune response either in vitro or in vivo. We decided to monitor, in nine renal cancer patients, the proliferative responses and the parallel

  20. Modification of T-Cell Proliferation and Interleukin 2 Production in Mice Infected with Trypanosoma cruzi

    Microsoft Academic Search

    Annick Harel-Bellan; Mireille Joskowicz; Didier Fradelizi; Harvey Eisen

    1983-01-01

    Acute infection of mice with Trypanosoma cruzi results in severe immunodepression and the appearance of autoimmune symptoms. In vitro, concanavalin A-stimulated T cells from spleens of infected animals could neither produce nor respond to interleukin 2. Interleukin 2 production was not restored by addition of exogenous interleukin 1, and proliferative response to concanavalin A was not restored by exogenous interleukin

  1. Ocular inflammatory effects of intravitreally injected interleukin-2.

    PubMed

    Samples, J R; Boney, R S; Rosenbaum, J T

    1993-07-01

    The primary, known physiologic effect of interleukin-2 (IL-2) is to act as a T lymphocyte growth factor. We investigated the potential contribution of IL-2 to intraocular inflammation by studying the inflammation resulting from the intravitreal injection of recombinant, human IL-2 in New Zealand white rabbits. Serial slit lamp observations indicated that 40 microgram of intravitreally injected IL-2 induced an anterior uveitis which was maximal 5 days after the injection. Inflammation was less marked but still significant with amounts of IL-2 as low as 400 ng. Direct examination of aqueous humor confirmed elevations of protein, prostaglandin E2, and mononuclear cells which correlated with the clinical observations. The kinetics of the response to intravitreal IL-2 distinguished it from the responses to other intravitreally injected cytokines such as interleukins 1, 6, or 8 as well as tumor necrosis factor. Intramuscular injection of cyclosporine A significantly reduced the protein extravasation associated with IL-2 injection, but cyclosporine had no effect on inflammation secondary to an intravitreal injection of interleukin-1. These observations implicate IL-2 as a potential contributor to uveitis. In addition, the studies with cyclosporine indicate the heterogeneity of inflammation such that pharmacologic agents which affect one cause of uveitis are not necessarily efficacious in another model. PMID:8222724

  2. Intravenous ascorbic acid as an adjuvant to interleukin-2 immunotherapy

    PubMed Central

    2014-01-01

    Interleukin-2 (IL-2) therapy has been demonstrated to induce responses in 10-20% of advanced melanoma and renal cell carcinoma patients, which translates into durable remissions in up to half of the responsers. Unfortunately the use of IL-2 has been associated with severe toxicity and death. It has been previously observed and reported that IL-2 therapy causes a major drop in circulating levels of ascorbic acid (AA). The IL-2 induced toxicity shares many features with sepsis such as capillary leakage, systemic complement activation, and a relatively non-specific rise in inflammatory mediators such as TNF-alpha, C-reactive protein, and in advanced cases organ failure. Animal models and clinical studies have shown rapid depletion of AA in conditions of sepsis and amelioration associated with administration of AA (JTM 9:1-7, 2011). In contrast to other approaches to dealing with IL-2 toxicity, which may also interfere with therapeutic effects, AA possesses the added advantage of having direct antitumor activity through cytotoxic mechanisms and suppression of angiogenesis. Here we present a scientific rationale to support the assessment of intravenous AA as an adjuvant to decrease IL-2 mediated toxicity and possibly increase treatment efficacy. PMID:24884532

  3. Modeling interleukin-2-based immunotherapy in AIDS pathogenesis.

    PubMed

    Joly, Marcel; Odloak, Darci

    2013-10-21

    In this paper, we sought to identify the CD4(+) T-cell dynamics in the course of HIV infection in response to continuous and intermittent intravenous courses of interleukin-2 (IL-2), the principal cytokine responsible for progression of CD4(+) T-lymphocytes from the G1 to the S phase of the cell cycle. Based on multivariate regression models, previous literature has concluded that the increase in survival of CD4(+) T-cell appears to be the critical mechanism leading to sustained CD4(+) T-cell levels in HIV-infected patients receiving intermittent IL-2 therapy. Underscored by comprehensive mathematical modeling, a major finding of the present work is related to the fact that, rather than due to any increase in survival of CD4(+) T-cells, the expressive, selective and sustained CD4(+) T-cell expansions following IL-2 administration may be related to the role of IL-2 in modulating the dynamics of Fas-dependent apoptotic pathways, such as activation-induced cell death (AICD) or HIV-specific apoptotic routes triggered by viral proteins. PMID:23806696

  4. Dissociation between interleukin-1 and interleukin-2 production in proliferative response to microbial antigens: restorative effect of exogenous interleukin-2.

    PubMed Central

    Vismara, D; Lombardi, G; Piccolella, E; Colizzi, V

    1985-01-01

    The relationship between the production of interleukin-1 (IL-1) and interleukin-2 (IL-2) after stimulation of human mononuclear cells within an antigenic extract from Candida albicans was analyzed in both responder and nonresponder donors. Culture supernatants from responders contained both IL-1 and IL-2 activity, whereas the supernatants from nonresponders contained only IL-1 and no appreciable IL-2. However, the addition of exogenous IL-2 to nonresponder cultures restored the normal proliferative response. Similar observations were made when cells from mice infected intravenously with high doses of Mycobacterium bovis BCG were cultured; these cells showed a marked impairment of the proliferative response to purified protein derivative. Spleen cells from BCG-induced unresponsive mice failed to produce IL-2 despite the fact that normal IL-1 activity was present in the culture. Again, the addition of exogenous IL-2 fully reversed the proliferative unresponsiveness. Thus, the presence of IL-1 does not necessarily induce production of IL-2, and the proliferative unresponsiveness is therefore due to a primary lack of IL-2. PMID:3894232

  5. Increased interleukin-2 levels during standard TRH test in man.

    PubMed

    Komorowski, J; Stepie?, H; Pawlikowski, M

    1994-09-01

    Interleukin-2 (IL-2) is a pluripotential cytokine that, besides its role in the regulation of immunocompetent cells function, also stimulates hormone secretion. On the other hand, several factors, including cytokines (interleukin-1, IL-1; interleukin-6, IL-6) and pituitary hormones (thyrotropin, TSH; prolactin, PRL), exert stimulatory effects on T-cell connected IL-2 production. In order to evaluate the role of both pituitary hormones in the activation of the immune system, the following two standard diagnostic tests were performed: TRH test (0.2 mg) in 8 healthy human subjects (4F/4M) aged 18-50 years, and oral metoclopramide (MCP) test (10 mg) in 8 females with galactorrhea and regular menstruation aged 18-52 years. The mobilization (peak response) of PRL, TSH, triiodothyronine (T3), thyroxin (T4), IL-1 beta, IL-2, IL-6 in TRH test, and PRL, IL-1 beta, IL-2, IL-6 for MCP test were evaluated. The responses of TSH (2.0 +/- 0.3 vs 12.3 +/- 2.2 microlU/ml, p < 0.01), PRL (15.3 +/- 2.3 vs 46.4 +/- 8.8 ng/ml, p < 0.01), T3 (178.0 +/- 16.4 vs 248.7 +/- 21.1 ng/dl, p < 0.001), T4 (7.9 +/- 0.4 vs 9.6 +/- 0.5 micrograms/dl, p < 0.001), and IL-2 (45.6 +/- 7.8 vs 79.9 +/- 16.4 fmol/ml, p < 0.05) in TRH test were noted. The peak response of PRL (16.3 +2- 2.6 vs 107.7 +/- 22.4 ng/ml, p < 0.01) in MCP test was also observed, but without any changes in interleukin concentrations.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7816186

  6. Transcriptional Repression of the Interleukin2 Gene by Vitamin D 3 : Direct Inhibition of NFATp\\/AP1 Complex Formation by a Nuclear Hormone Receptor

    Microsoft Academic Search

    IRIS ALROY; TERRI L. TOWERS; ANDLEONARD P. FREEDMAN

    1995-01-01

    T-lymphocyte proliferation is suppressed by 1,25-dihydroxyvitamin D3(1,25(OH)2D3), the active metabolite of vitamin D3, and is associated with a decrease in interleukin 2 (IL-2), gamma interferon, and granulocyte- macrophage colony-stimulating factor mRNA levels. We report here that 1,25(OH)2D3-mediated repression in Jurkat cells is cycloheximide resistant, suggesting that it is a direct transcriptional repressive effect on IL-2 expression by the vitamin D3

  7. Studies evaluating the antitumor activity and toxicity of interleukin-15, a new T cell growth factor: comparison with interleukin-2.

    PubMed

    Munger, W; DeJoy, S Q; Jeyaseelan, R; Torley, L W; Grabstein, K H; Eisenmann, J; Paxton, R; Cox, T; Wick, M M; Kerwar, S S

    1995-10-15

    Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target cells. It is approximately one-third as potent as interleukin-2 in inducing specific cytolytic cells that lyse allogeneic target cells. Interleukin-15 is approximately half as potent as interleukin-2 in suppressing pulmonary metastasis induced by MCA-205 tumor cells. The dose of interleukin-15 required to induce pulmonary vascular leak in mice is six times higher than that required for interleukin-2. These results support the view that interleukin-15 exhibits a therapeutic index that is superior to interleukin-2. PMID:7553894

  8. Functional interleukin-2 receptors on intestinal epithelial cells.

    PubMed Central

    Ciacci, C; Mahida, Y R; Dignass, A; Koizumi, M; Podolsky, D K

    1993-01-01

    The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa. Images PMID:8326018

  9. In Vitro Interleukin-1 and 2 Production and Interleukin 2 Receptor Expression in the Rhesus Monkey

    NASA Technical Reports Server (NTRS)

    Schmitt, Didier A.; Sonnenfeld, Gerald; Husson, David; Tkaczuk, Jean; Andre, Eric; Schaffar, Laurance

    1996-01-01

    Anti-human monoclonal antibodies were used to detect and quantify interleukins-1 and 2 and interleukin-2 receptor expression in peripheral blood mononuclear cells from a rhesus monkey. Interleukin-1 production could be induced by phorbol esters (PMA) and was potentiated by phytohemagglutinin (PHA). Interleukin-2 secretion could also be induced by the combination of PHA and PMA, but only weakly with PHA alone. Interleukin-2 receptor expression was present in a subpopulation of unstimulated lymphocytes and could be enhanced by PHA or PMA. These data show once again that the rhesus monkey immune system is cross-reactive with the human one and that rhesus macaque could be a good model to study interleukin therapy.

  10. Enhancement by Interleukin 4 of Interleukin 2- or Antibody-induced Proliferation of Lymphocytes from Interleukin 2-treated Cancer Patients1

    Microsoft Academic Search

    Jonathan Treisman; Carl M. Higuchi; John A. Thompson; Steven Gillis; Catherine G. Lindgren; Donald E. Kern; Stanley R. Ridell; Philip D. Greenberg

    Systemic interleukin 2 (IL-2) and IL-2-activated lymphocytes have induced tumor regression in some cancer patients. The IL-2-activated cells have usually been generated by obtaining peripheral blood mono- nuclear cells (PB1V1C) from cancer patients shortly after systemic IL-2 therapy and culturing them with IL-2 in vitro. In an effort to augment the ex vivogeneration of such cells preactivated in vivo, we

  11. DIFFERENTIAL EFFECTS OF HUMAN AND PORCINE INTERLEUKIN 2 ON NATURAL KILLING (NK) ACTIVITY OF NEWBORN PIGLETS

    E-print Network

    Paris-Sud XI, Université de

    DIFFERENTIAL EFFECTS OF HUMAN AND PORCINE INTERLEUKIN 2 ON NATURAL KILLING (NK) ACTIVITY OF NEWBORN killing (NK) activity can be defined as the ability of normal unsensitized lymphoid cells to lyse tumor, Charley et al 1985a), can be stimulated in vitro by natural or recombinant human or por- cine IFNa

  12. Deregulated T Cell Activation and Autoimmunity in Mice Lacking Interleukin 2 Receptor beta

    Microsoft Academic Search

    Haruhiko Suzuki; Thomas M. Kundig; Caren Furlonger; Andrew Wakeham; Emma Timms; Toshifumi Matsuyama; Rudolf Schmits; John J. L. Simard; Pamela S. Ohashi; Henrik Griesser; Tadatsugu Taniguchi; Christopher J. Paige; Tak W. Mak

    1995-01-01

    In mice lacking the interleukin-2 receptor beta chain (IL-2Rbeta), T cells were shown to be spontaneously activated, resulting in exhaustive differentiation of B cells into plasma cells and the appearance of high serum concentrations of immunoglobulins G1 and E as well as autoantibodies that cause hemolytic anemia. Marked infiltrative granulocytopoiesis was also apparent, and the animals died after about 12

  13. Interleukin2 receptor ? chain regulates the size and content of the peripheral lymphoid compartment

    Microsoft Academic Search

    Dennis M. Willerford; Jianzhu Chen; Judith A. Ferry; Laurie Davidson; Averil Ma; Frederick W. Alt

    1995-01-01

    Interleukin-2 receptor ? chain (IL-2R?) expression occurs at specific stages of early T and B lymphocyte development and is induced upon activation of mature lymphocytes. Young mice that lack IL-2R? have phenotypically normal development of T and B cells. However, as adults, these mice develop massive enlargement of peripheral lymphoid organs associated with polyclonal T and B cell expansion, which,

  14. Inhibitors of glycoprotein processing alter T-cell proliferative responses to antigen and to interleukin 2.

    PubMed Central

    Wall, K A; Pierce, J D; Elbein, A D

    1988-01-01

    Most of the cell-surface molecules involved in T-cell immune responses are N-linked glycoproteins. We have investigated the effects of inhibitors of glycoprotein processing on specific T-cell functions, with the dual aims of examining the functional role of carbohydrate and of testing the usefulness of such compounds as immunomodulators. Treatment of a cloned murine helper T-cell line with these inhibitors differentially affects the proliferative response of the cell, depending upon the nature of the stimulus. Treatment with the plant alkaloid swainsonine, which inhibits the processing mannosidase II and causes the accumulation of glycoproteins bearing hybrid-type oligosaccharide structures, enhances the proliferative response of the T-cell clone to antigen and to the mitogen concanavalin A. Treatment with another plant alkaloid, castanospermine, which inhibits glucosidase I and causes the accumulation of glucose-containing high-mannose structures, has the opposite effect and inhibits the proliferative response of the T cell to antigen. Cell-surface oligosaccharide alteration does not affect antigen recognition, as judged by the lack of effect of either drug on interleukin 2 production following antigen stimulation. Cells treated with either alkaloid proliferate poorly to exogenous interleukin 2 and may have defective interleukin 2 receptor function. Swainsonine-treated cells apparently have compensatory alterations that can overcome the reduced responsiveness to interleukin 2. Antibody-binding studies indicate that normal quantities of many cell-surface molecules, including the T-cell receptor for antigen, are expressed by the treated cells. PMID:3135550

  15. Effects of dexamethasone on human natural killer cell cytotoxicity, interferon production, and interleukin-2 receptor expression induced by microbial antigens.

    PubMed Central

    Piccolella, E; Lombardi, G; Vismara, D; Del Gallo, F; Colizzi, V; Dolei, A; Dianzani, F

    1986-01-01

    Dexamethasone inhibits the expression of the interleukin-2 receptor, the synthesis of immune interferon, and the development of natural killer cells when added to peripheral blood mononuclear cells cultured with soluble microbial antigens (purified protein derivative and a polysaccharide extract from Candida albicans [MPPS]) or human recombinant interleukin-2. PMID:2417957

  16. Interleukin-2 receptor monoclonal antibodies in renal transplantation: meta-analysis of randomised trials

    PubMed Central

    Adu, Dwomoa; Cockwell, Paul; Ives, Natalie J; Shaw, Jonathan; Wheatley, Keith

    2003-01-01

    Objective To study the effect of interleukin-2 receptor monoclonal antibodies on acute rejection episodes, graft loss, deaths, and rate of infection and malignancy in patients with renal transplants. Design Meta-analysis of published data. Data sources Medline, Embase, and Cochrane library for years 1996-2003 plus search of medical editors' trial amnesty and contact with manufacturers of the antibodies. Selection of studies Randomised controlled trials comparing interleukin-2 receptor antibodies with placebo or no additional treatment in patients with renal transplants receiving ciclosporin based immunosuppression. Results Eight randomised controlled trials involving 1871 patients met the selection criteria (although only 1858 patients were analysed). Interleukin-2 receptor antibodies significantly reduced the risk of acute rejection (odds ratio 0.51, 95% confidence interval 0.42 to 0.63). There were no significant differences in the rate of graft loss (0.78, 0.58 to 1.04), mortality (0.75, 0.46 to 1.23), overall incidence of infections (0.97, 0.77 to 1.24), incidence of cytomegalovirus infections (0.81, 0.62 to 1.04), or risk of malignancies at one year (0.82, 0.39 to 1.70). The different antibodies had a similar sized effect on acute rejection (test for heterogeneity P=0.7): anti-Tac (0.37, 0.16 to 0.89), BT563 (0.37, 0.1 to 1.38), basiliximab (0.56, 0.44 to 0.72), and daclizumab (0.46, 0.32 to 0.67). The reduction in acute rejections was similar for all ciclosporin based immunosuppression regimens (test for heterogeneity P=1.0). Conclusions Adding interleukin-2 receptor antibodies to ciclosporin based immunosuppression reduces episodes of acute rejection at six months by 49%. There is no evidence of an increased risk of infective complications. Longer follow up studies are needed to confirm whether interleukin-2 receptor antibodies improve long term graft and patient survival. What is already known on this topicEpisodes of acute rejection reduce graft survival in patients with renal transplantsIncreasing immunosuppression to reduce rejection can increase infection and malignancyWhat this study addsAddition of interleukin-2 receptor antibodies to ciclosporin based immunosuppression regimens halves the risk of acute rejectionPatients receiving antibodies did not have an increased risk of infectionThe effects on graft loss and mortality at one year were not significant PMID:12689974

  17. Interleukin 2 production in a family with systemic lupus erythematosus and a C4Q0 heterozygous inheritance.

    PubMed Central

    Gutierrez, C; Cabrero, E; Vicario, J L; Martín Villa, M; Rengel, M A; Gomez Campdera, F J; Yebra, M; Fernández-Cruz, E; Arnaiz Villena, A

    1991-01-01

    Interleukin 2 production was studied in a family with systemic lupus erythematosus (SLE) and a C4Q0 heterozygous inheritance. Autoimmune manifestations seemed to be associated with the HLA haplotype containing the C4Q0 allele, which was shared by all four ill family members. Concentrations of interleukin 2, however, did not associate either with the haplotype or with the clinical or serological manifestations, as diminished concentrations of interleukin 2 were found in only two subjects with SLE. Thus the defect in this family seemed to be acquired rather than genetically conditioned. PMID:1888202

  18. Focal takotsubo cardiomyopathy with high-dose interleukin-2 therapy for malignant melanoma.

    PubMed

    Damodaran, Senthil; Mrozek, Ewa; Liebner, David; Kendra, Kari

    2014-12-01

    High-dose interleukin-2 (IL-2) is an available treatment option for patients with metastatic melanoma or renal cell carcinoma, and is associated with sustained complete and partial responses in a subset of patients. IL-2, however, is not devoid of toxicities, most of which involve the cardiovascular system and manifest as hypotension, arrhythmias, and cardiomyopathy. This report describes an unusual presentation of takotsubo cardiomyopathy in a postmenopausal woman receiving high-dose IL-2 for metastatic melanoma. PMID:25505207

  19. A function for interleukin 2 in Foxp3-expressing regulatory T cells

    Microsoft Academic Search

    Jeffrey P Rasmussen; Marc A Gavin; Jason D Fontenot; Alexander Y Rudensky

    2005-01-01

    Regulatory T cells (Treg cells) expressing the forkhead family transcription factor Foxp3 are critical mediators of dominant immune tolerance to self. Most Treg cells constitutively express the high-affinity interleukin 2 (IL-2) receptor ?-chain (CD25); however, the precise function of IL-2 in Treg cell biology has remained controversial. To directly assess the effect of IL-2 signaling on Treg cell development and

  20. Anti-interleukin 2 receptor antibody attenuates low-dose streptozotocin-induced diabetes in mice

    Microsoft Academic Search

    N. Hatamori; K. Yokono; M. Hayakawa; T. Taki; W. Ogawa; M. Nagata; J. Hari; K. Shii; H. Taniguchi; S. Baba

    1990-01-01

    Summary  Recent evidence indicates that activated T cells and macrophages play an important role in the induction of insulitis and diabetes in certain strains of mice treated with multiple subdiabetogenic doses of streptozotocin. In the present study, we treated C57BL\\/6J mice with five daily doses of 40 mg\\/ml streptozotocin and examined the prophylactic effect of an anti-interleukin 2 receptor monoclonal antibody

  1. Local interleukin-2 and interleukin-12 therapy of bovine ocular squamous cell carcinomas

    Microsoft Academic Search

    Rachel J. E. Stewart; Agnieszka Masztalerz; John. J. L. Jacobs; Willem Den Otter

    2005-01-01

    Interleukin-2 and interleukin-12 have been used independently to successfully treat the induced and the spontaneous tumours in animals. This trial was done to determine if a combination of IL-2 and IL-12 in the treatment of spontaneous bovine ocular squamous cell carcinomas (BOSCC) would be more successful than IL-2 or IL-12 therapy by themselves.For this trial, we selected 25 BOSCC tumours

  2. Novel Immunosuppressant Agents Targeting Activated Lymphocytes by Biocompatible MPC Polymer Conjugated with Interleukin2

    Microsoft Academic Search

    N. Chiba; M. Ueda; T. Shimada; H. Jinno; J. Watanabe; K. Ishihara; M. Kitajima

    2007-01-01

    The immunopharmacological profile of novel biocompatible water-soluble interleukin-2 (IL-2)-conjugated 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer immunosuppressive agents was established. MPC-co-n- butyl methacrylate (BMA)-co-p-nitrophenylcarbonyloxyethyl methacrylate (NPMA) (PMBN) was prepared as a backbone for these novel agents. PMBN contained MPC as a biocompatible unit, BMA as a hydrophobic domain in water, and NPMA as an immobilizable unit with IL-2. This research showed that proliferation

  3. Lymphocyte infiltration of the skin in transgenic mice carrying the human interleukin-2 gene

    Microsoft Academic Search

    M. Akiyama; M. Yokoyama; M. Katsuki; S. Habu; T. Nishikawa

    1993-01-01

    Inflammatory lesions of the skin such as erythema, depigmentation and hair loss were observed in C57\\/BL6(B6) transgenic mice that carried an intact human genomic interleukin-2 gene (gIL-2 transgenic mice). Accumulation of T lymphocytes in the perivascular and periadnexal areas of the dermis was the first change, followed by dermal papillary oedema, which occurred before the development of macroscopic skin lesions.

  4. Adjuvant treatment of breast cancer: A pilot immunochemotherapy study with CMF, interleukin-2 and interferon alpha

    Microsoft Academic Search

    Giuseppe Tonini; Corrado Nunziata; Salvatore P. Prete; Rita Pepponi; Mario Turriziani; Giovanna Masci; Grazia Graziani; Enzo Bonmassar; Liana De Vecchis

    1998-01-01

    Immune responses, including natural immunity (NI), potentiate the antitumor effects of chemotherapy. Since interferons and\\u000a interleukin-2 (IL-2) augment NI, a pilot study was conducted to assess the tolerability and the effects on host immunity of\\u000a adjuvant chemotherapy associated with IL-2?+?interferon alpha (IFN) in breast cancer patients after surgery. Ten patients\\u000a underwent alternating 28-day cycles of chemoimmunotherapy [cyclophosphamide?+?methotrexate?+ 5-fluorouracil (CMF, days

  5. Immobilization and voltammetric detection of human interleukine-2 gene on the pencil graphite electrode

    Microsoft Academic Search

    M. S. Hejazi; E. Alipour; M. H. Pournaghi-Azar

    2007-01-01

    The immobilization and differential pulse anodic voltammetry (DPAV) of a 20-mer oligonucleotide related to the human interleukine-2 (hIL-2) using renewable pencil graphite electrode (PGE) is described. The influences of electrochemical pretreatment of PGE on the ability of the electrode in hIL-2 adsorption, and conditions of hiIL-2 immobilization on PGE including immobilization potential and time, sodium chloride concentration as well as

  6. Effects of Cancer Immunotherapy with Indomethacin and Interleukin2 on Murine Hemopoietic Stem Cells 1

    Microsoft Academic Search

    Mary Nel Saarloos; Nelson K. S. Khoo; Peeyush K. Lala

    1992-01-01

    We examined: (a) whether in vitro-generated lymphocyte-activated killer (LAK) cells from normal mice and splenic killer cells from tumor- bearing mice subjected to interleukin-2 (IL-2) therapy alone or in com- bination with chronic indomethacin therapy have any detrimental ef- fects on the spleen colony-forming units (CFU-S) of the normal bone marrow (BM); and (b) the effects of these immunotherapy protocols

  7. Interleukin 2 Receptors and Detergent-Resistant Membrane Domains Define a Clathrin-Independent Endocytic Pathway

    Microsoft Academic Search

    Christophe Lamaze; Annick Dujeancourt; Takeshi Baba; Charles G. Lo; Alexandre Benmerah; Alice Dautry-Varsat

    2001-01-01

    Clathrin-dependent endocytosis has long been presented as the only efficient mechanism by which transmembrane receptors are internalized. We selectively blocked this process using dominant-negative mutants of Eps15 and showed that clathrin-mediated endocytosis of transferrin was inhibited, while endocytosis of interleukin 2 (IL2) receptors proceeded normally. Ultrastructural and biochemical experiments showed that clathrin-independent endocytosis of IL2 receptors exists constitutively in lymphocytes

  8. Soluble interleukin 2 receptor levels in families of people with schizophrenia

    Microsoft Academic Search

    Fiona Gaughran; Evonne O'Neill; Pak Sham; Robert J. Daly; Fergus Shanahan

    2002-01-01

    Background: Several authors have reported increased soluble interleukin 2 receptor (sIL2R?) concentrations in schizophrenia. The aim of this work was to examine serum sIL2R? in the first degree relatives of patients with schizophrenia.Methods: We sampled 51 first degree relatives of patients with DSM IIIR schizophrenia. These relatives were unaffected by psychosis and included nine fathers, thirteen mothers, seventeen sisters and

  9. Evolution of the levels of soluble interleukin-2 receptors during Plasmodium falciparum and P. vivax malaria.

    PubMed Central

    Deloron, P; Lepers, J P; Coulanges, P

    1989-01-01

    Increased levels of soluble interleukin-2 receptors (IL-2R) in serum were observed in both Plasmodium falciparum- and P. vivax-infected individuals compared with nonparasitemic subjects. Clinical symptoms of P. falciparum malaria were associated with higher levels of soluble IL-2R. Temporal evolution in serum of IL-2R during the course of a malaria attack mimicked the kinetics of soluble IL-2R under experimental conditions. PMID:2671038

  10. Molecular signatures induced by interleukin-2 on peripheral blood mononuclear cells and T cell subsets

    Microsoft Academic Search

    Ping Jin; Ena Wang; Maurizio Provenzano; Sara Deola; Silvia Selleri; Jiaqiang Ren; Sonia Voiculescu; David Stroncek; Monica C Panelli; Francesco M Marincola

    2006-01-01

    : Experimentally, interleukin-2 (IL-2) exerts complex immunological functions promoting the proliferation, survival and activation of T cells on one hand and inducing immune regulatory mechanisms on the other. This complexity results from a cross talk among immune cells which sways the effects of IL-2 according to the experimental or clinical condition tested. Recombinant IL-2 (rIL-2) stimulation of peripheral blood mononuclear

  11. Partial restoration of impaired interleukin-2 production and Tac antigen (putative interleukin-2 receptor) expression in patients with acquired immune deficiency syndrome by isoprinosine treatment in vitro.

    PubMed Central

    Tsang, K Y; Fudenberg, H H; Galbraith, G M; Donnelly, R P; Bishop, L R; Koopmann, W R

    1985-01-01

    The in vitro effects of isoprinosine (ISO) on interleukin-2 (IL-2) production, the expression of Tac antigen (IL-2 receptor) on lymphocytes, and the ability of Leu 3(+) cells to absorb interleukin-1 (IL-1) were investigated in 10 patients with acquired immune deficiency syndrome (AIDS). In 9 of the 10 patients, production of IL-2 from mononuclear cells and Leu 3(+) cells was depressed; expression of Tac antigen on mononuclear cells and Leu 2(+) cells was found to be depressed in 9 of 10 patients. The ability of the Leu 3(+) lymphocytes to absorb IL-1 was depressed in all (four of four) patients studied. After ISO treatment, IL-2 production, Tac antigen expression and IL-1 absorption were restored to normal or near normal levels in most of the patients. These results suggest that ISO has an immunostimulating capacity in AIDS patients and that the potential of ISO in immune response restoration in AIDS patients deserves critical consideration. PMID:2581997

  12. Bovine T lymphocytes. I. Generation and maintenance of an interleukin-2-dependent, cytotoxic T-lymphocyte cell line.

    PubMed Central

    Picha, K S; Baker, P E

    1986-01-01

    Primary and secondary bovine allogeneic mixed leucocyte cultures were examined for the generation of antigen-specific cytotoxic leucocytes. While optimal generation of murine and human cytotoxic T lymphocytes typically requires 4-8 days, alloantigen-specific cytotoxic bovine leucocytes were demonstrated consistently only after prolonged incubation periods, optimally found to be about 15 days. Restimulation of long-term bovine mixed leucocyte cultures with the original stimulator population revealed responder cells demonstrating augmented alloantigen-specific lytic activity. When placed into human recombinant interleukin-2, responder cells expanded and required passaging every 3-4 days. The same was not true of cells placed into interleukin-2-free medium. Cells cultured in interleukin-2-containing medium retained alloantigen specificity after 10 weeks of culture. Moreover, they continued to display total dependence on human, simian or bovine interleukin-2 for growth. PMID:2417937

  13. Inhibition of G-Protein ?? Signaling Enhances T Cell Receptor-Stimulated Interleukin 2 Transcription in CD4+ T Helper Cells

    PubMed Central

    Yost, Evan A.; Hynes, Thomas R.; Hartle, Cassandra M.; Ott, Braden J.; Berlot, Catherine H.

    2015-01-01

    G-protein-coupled receptor (GPCR) signaling modulates the expression of cytokines that are drug targets for immune disorders. However, although GPCRs are common targets for other diseases, there are few GPCR-based pharmaceuticals for inflammation. The purpose of this study was to determine whether targeting G-protein ?? (G??) complexes could provide a useful new approach for modulating interleukin 2 (IL-2) levels in CD4+ T helper cells. Gallein, a small molecule inhibitor of G??, increased levels of T cell receptor (TCR)-stimulated IL-2 mRNA in primary human naïve and memory CD4+ T helper cells and in Jurkat human CD4+ leukemia T cells. G?1 and G?2 mRNA accounted for >99% of G? mRNA, and small interfering RNA (siRNA)-mediated silencing of G?1 but not G?2 enhanced TCR-stimulated IL-2 mRNA increases. Blocking G?? enhanced TCR-stimulated increases in IL-2 transcription without affecting IL-2 mRNA stability. Blocking G?? also enhanced TCR-stimulated increases in nuclear localization of nuclear factor of activated T cells 1 (NFAT1), NFAT transcriptional activity, and levels of intracellular Ca2+. Potentiation of IL-2 transcription required continuous G?? inhibition during at least two days of TCR stimulation, suggesting that induction or repression of additional signaling proteins during T cell activation and differentiation might be involved. The potentiation of TCR-stimulated IL-2 transcription that results from blocking G?? in CD4+ T helper cells could have applications for autoimmune diseases. PMID:25629163

  14. Studies Evaluating the Antitumor Activity and Toxicity of Interleukin15, a New T Cell Growth Factor: Comparison with Interleukin2

    Microsoft Academic Search

    William Munger; Susan Quinn Dejoy; R. Jeyaseelan; Lawrence W. Torley; Kenneth H. Grabstein; June Eisenmann; Ray Paxton; Tom Cox; Michael M. Wick; S. S. Kerwar

    1995-01-01

    Interleukin-15 is a new cytokine that stimulates the proliferation of T cells and other cells of the immune system. Some of the biological properties of interleukin-15 overlap that of interleukin-2. Using murine models, the present studies have shown that interleukin-15, in vivo, is three to four times more potent than interleukin-2 in generating cytolytic effector splenocytes that lyse YAC target

  15. An interleukin-2 enhancer binding factor 2 homolog involved in immune response from Chinese mitten crab Eriocheir sinensis.

    PubMed

    Yang, Jialong; Wang, Lingling; Huang, Mengmeng; Wang, Leilei; Gai, Yunchao; Qiu, Limei; Zhang, Huan; Song, Linsheng

    2011-06-01

    As a transcription factor, Interleukin-2 enhancer binding factor 2 (ILF2) regulates IL-2 gene at level of transcription, splicing and translation in vertebrates and plays significant roles in immune system. In this study, an ILF2 homolog was identified from Chinese mitten crab Eriocheir sinensis (designated as EsILF) by expressed sequence tag (EST) analysis. The full-length cDNA of EsILF was of 2159bp, containing a 5' untranslated region (UTR) of 90bp, a 3' UTR of 866bp with a poly (A) tail, and an open reading frame (ORF) of 1203bp encoding a polypeptide of 400 amino acids with the predicted molecular weight of 44.3kDa, which shared 59.6-64.5% identities with vertebrate ILF2. There were a conserved N-terminal RGG-rich single-stranded RNA-binding domain and a DZF zinc-finger nucleic acid binding domain in the primary structure, strongly suggesting that EsILF was a homolog of vertebrate ILF2. The mRNA of EsILF was constitutively expressed in all tested tissues of untreated crabs, including hepatopancreas, gill, gonad, muscle, heart and hemocytes, with highest expression in muscle and relative lower levels in hemocytes and gonad. The mRNA expression of EsILF in hemocytes was regulated differently after the crabs were stimulated by bacteria Listonella anguillarum and fungi Pichia pastoris GS115. The expression level was significantly (P<0.05) down-regulated to 0.35- and 0.29-fold compared with blank group at 6h and 12h after the stimulation of L. anguillarum, while P. pastoris significantly (P<0.05) up-regulated the expression level to 3.2-fold compared with the blank group at 6h post treatment. The results indicated that EsILF was involved in the immune response of crab toward both L. anguillarum and P. pastoris. PMID:21439385

  16. Pharmacokinetics and stability of the ch14.18–interleukin-2 fusion protein in mice

    Microsoft Academic Search

    Kari Kendra; Jacek Gan; Melody Ricci; Jean Surfus; Anisa Shaker; Michael Super; Jami D. Frost; Alexander Rakhmilevich; Jacquelyn A. Hank; Stephen D. Gillies; Paul M. Sondel

    1999-01-01

    The fusion protein formed from ch14.18 and interleukin-2 (ch14.18–IL-2), shown to exhibit antitumor efficacy in mouse models,\\u000a consists of IL-2 genetically linked to each heavy chain of the ch14.18 chimeric anti-GD2 monoclonal antibody. The purpose\\u000a of this study was to determine the pharmacokinetics of ch14.18–IL-2 in mice and assess its stability in murine serum. Following\\u000a i.v. injection, the fusion protein

  17. Ability of isoprinosine to restore interleukin-2 production and T cell proliferation in autoimmune mice.

    PubMed Central

    Fischbach, M; Talal, N

    1985-01-01

    Autoimmune mice bearing the single autosomal recessive gene 1pr are unable to produce the T cell growth factor, interleukin-2 (IL-2). A physiological consequence of this defect is the inability of T cells from C57B1/6J-lpr/lpr mice to respond to antigen presented by macrophages. In an attempt to reverse these abnormalities, we administered the inosine containing drug isoprinosine. Injection of isoprinosine after antigen immunization restored both antigen presentation and IL-2 production. PMID:2412742

  18. Antibodies to Interleukin-2 Elicit Selective T Cell Subset Potentiation through Distinct Conformational Mechanisms.

    PubMed

    Spangler, Jamie B; Tomala, Jakub; Luca, Vincent C; Jude, Kevin M; Dong, Shen; Ring, Aaron M; Votavova, Petra; Pepper, Marion; Kovar, Marek; Garcia, K Christopher

    2015-05-19

    Interleukin-2 (IL-2) is a pleiotropic cytokine that regulates immune cell homeostasis and has been used to treat a range of disorders including cancer and autoimmune disease. IL-2 signals via interleukin-2 receptor-? (IL-2R?):IL-2R? heterodimers on cells expressing high (regulatory T cells, Treg) or low (effector cells) amounts of IL-2R? (CD25). When complexed with IL-2, certain anti-cytokine antibodies preferentially stimulate expansion of Treg (JES6-1) or effector (S4B6) cells, offering a strategy for targeted disease therapy. We found that JES6-1 sterically blocked the IL-2:IL-2R? and IL-2:IL-2R? interactions, but also allosterically lowered the IL-2:IL-2R? affinity through a "triggered exchange" mechanism favoring IL-2R?(hi) Treg cells, creating a positive feedback loop for IL-2R?(hi) cell activation. Conversely, S4B6 sterically blocked the IL-2:IL-2R? interaction, while also conformationally stabilizing the IL-2:IL-2R? interaction, thus stimulating all IL-2-responsive immune cells, particularly IL-2R?(hi) effector cells. These insights provide a molecular blueprint for engineering selectively potentiating therapeutic antibodies. PMID:25992858

  19. Serum soluble interleukin-2 receptor level as a prognostic indicator in gastric cancer.

    PubMed Central

    Nakata, B.; Chung, K. H.; Kato, Y.; Yamashita, Y.; Inui, A.; Arimoto, Y.; Maeda, K.; Onoda, N.; Sawada, T.; Sowa, M.

    1998-01-01

    T lymphocytes, activated by interleukin 2 during an anti-tumour response, release soluble interleukin 2 receptors (sIL-2R) into the bloodstream. We analysed the prognostic value of the serum sIL-2R level in gastric cancer. Serum concentration of sIL-2R in 96 gastric cancer patients and 100 healthy control subjects' was measured by enzyme-linked immunosorbent assay. All survivors were followed for more than 50 months. Serum sIL-2R level was considered with respect to prognosis, clinicopathological factors, other tumour markers and peripheral blood cell count. Stage III and IV patients had significantly higher sIL-2R levels than lower stage patients and control subjects. Stage III and IV gastric cancer patients were divided into 'high' and 'low' slL-2R groups based upon the control subjects' serum sIL-2R mean value plus one standard deviation. The high group had a significantly worse prognosis than the low group, although clinicopathological features and treatments were similar. Multivariate analysis demonstrated that the serum sIL-2R level is an independent indicator. The sIL-2R level did not correlate with carbohydrate antigen 19-9, however it did correlate with carcinoembryonic antigen (r = 0.22) and with numbers of peripheral blood monocytes (r = 0.54). In conclusion, serum sIL-2R may predict the outcome of gastric cancer patients with stage III or IV disease. PMID:9667652

  20. Pharmacological characterisation of cannabinoid receptors inhibiting interleukin 2 release from human peripheral blood mononuclear cells.

    PubMed

    Ihenetu, Kenneth; Molleman, Areles; Parsons, Mike; Whelan, Clifford

    2003-03-19

    The effects of a range of cannabinoid receptor agonists and antagonists on phytohaemagglutinin-induced secretion of interleukin-2 from human peripheral blood mononuclear cells were investigated. The nonselective cannabinoid receptor agonist WIN55212-2 ((R)-(+)-[2,3-dihydro-5-methyl-3-[4-morpholinylmethyl]pyrrolo[1,2,3-de]1,4-benzoxazin-6-yl](1-naphthyl) methanone mesylate) and the selective cannabinoid CB(2) receptor agonist JWH 015 ((2-methyl-1-propyl-1H-indol-3-yl)-1-napthalenylmethanone) inhibited phytohaemagglutinin (10 microg/ml)-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max), WIN55212-2=8.8 x 10(-7) M, 95% confidence limits (C.L.)=2.2 x 10(-7)-3.5 x 10(-6) M; JWH 015=1.8 x 10(-6) M, 95% C.L.=1.2 x 10(-6)-2.9 x 10(-6) M, n=5). The nonselective cannabinoid receptor agonists CP55,940 ((-)-3-[2-hydroxy-4-(1,1-dimethyl-hepthyl)-phenyl]4-[3-hydroxypropyl]cyclo-hexan-1-ol), Delta(9)-tetrahydrocannabinol and the selective cannabinoid CB(1) receptor agonist ACEA (arachidonoyl-2-chloroethylamide) had no significant (P>0.05) inhibitory effect on phytohaemagglutinin-induced release of interleukin-2. Dexamethasone significantly (P<0.05) inhibited phytohaemagglutinin-induced release of interleukin-2 in a concentration-dependent manner (IC(1/2max)=1.3 x 10(-8) M, 95% C.L.=1.4 x 10(-9)-3.2 x 10(-8) M). The cannabinoid CB(1) receptor antagonist SR141716A (N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride) (10(-6) M) did not antagonise the inhibitory effect of WIN55212-2 whereas the cannabinoid CB(2) receptor antagonist SR144528 (N-(1,S)-endo-1,3,3-trimethyl bicyclo(2,2,1)heptan-2-yl)-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) antagonised the inhibitory effect of WIN55212-2 (pA(2)=6.3+/-0.1, n=5). In addition, CP55,940 (10(-6) M) and Delta(9)-tetrahydrocannabinol (10(-6) M) also antagonised the inhibitory effects of WIN55212-2 (pA(2)=6.1+/-0.1, n=5 and pA(2)=6.9+/-0.2, n=5). In summary, WIN55,212-2 and JWH 015 inhibited interleukin-2 release from human peripheral blood mononuclear cells via the cannabinoid CB(2) receptor. In contrast, CP55,940 and Delta(9)-tetrahydrocannabinol behaved as partial agonists/antagonists in these cells. PMID:12620515

  1. Interleukin-2 stimulates a late increase in phosphatidic acid production in the absence of phospholipase D activation.

    PubMed

    Jones, D R; Flores, I; Díaz, E; Martinez, C; Mérida, I

    1998-08-14

    The signal transduction pathways involving phospholipid metabolism during T-cell proliferation remain partly undefined. Herein we show that interleukin-2 caused a late (> 12 h) rise in the intracellular phosphatidic acid content of CTLL-2 cells which was a consequence of the activation of the enzyme diacylglycerol kinase. No activation of phospholipase D was observed at similar times. Incubation of the cells with a recognized diacylglycerol kinase a isoform inhibitor, R59499, prior to interleukin-2 stimulation was able to block cell cycle entry, diacyglycerol kinase activation and phosphatidic acid accumulation. In contrast, when R59499 was added 3 h after interleukin-2, few or no observable effects on the above three parameters were noticed. These results suggest that the early signaling employed by IL-2 involving the alpha isoform of diacylglycerol kinase is sufficient to control the late increase in phosphatidic acid and that phosphatidic acid is a mitogenic agent in T-cells. PMID:9738925

  2. IGRA-positive patients and interferon-gamma/interleukin-2 signatures: can the Fluorospot assay provide further information?

    PubMed

    Bittel, P; Mayor, D; Iseli, P; Bodmer, T; Suter-Riniker, F

    2014-06-01

    A goal of testing for latent tuberculosis (TB) infection is to identify individuals who are at increased risk for the development of active TB. No laboratory tool is currently available to distinguish between individuals in the process of progressing from latent TB infection towards active disease and those who are not. Determination of the interferon-gamma and interleukin-2 T cell signature might provide an additional and rapid tool to evaluate treatment necessity and clinical management of a patient. Here, we present three cases of interferon-gamma release assay-positive patients with differing interferon-gamma and interleukin-2 signatures when analyzed by the Fluorospot assay. PMID:24477887

  3. Chemical Synthesis of O-Glycosylated Human Interleukin-2 by the Reverse Polarity Protection Strategy.

    PubMed

    Asahina, Yuya; Komiya, Shinobu; Ohagi, Ami; Fujimoto, Rina; Tamagaki, Hiroko; Nakagawa, Katsuhiro; Sato, Takashi; Akira, Shizuo; Takao, Toshifumi; Ishii, Akira; Nakahara, Yoshiaki; Hojo, Hironobu

    2015-07-01

    The chemical synthesis of human interleukin-2 (IL-2)?, having a core 1 sugar, by a ligation method is reported. Although IL-2 is a globular glycoprotein, its C-terminal region, in particular (99-133), is extremely insoluble when synthesized by solid-phase method. To overcome this problem, the side-chain carboxylic acid of the Glu residues was protected by a picolyl ester, thus reversing its polarity from negative to positive. This reverse polarity protection significantly increased the isoelectric point of the peptide segment and made it positive under acidic conditions and facilitated the purification. An efficient method to prepare the prolyl peptide thioester required for the synthesis of the (28-65) segment was also developed. These efforts resulted in the total synthesis of the glycosylated IL-2 having full biological activity. PMID:26012897

  4. Suppressor and cytolytic cell function in multiple sclerosis. Effects of cyclosporine A and interleukin 2.

    PubMed Central

    Bania, M B; Antel, J P; Reder, A T; Nicholas, M K; Arnason, B G

    1986-01-01

    Patients with progressive multiple sclerosis (MS) demonstrated persistent reductions in levels of concanavalin A (Con A)-induced suppressor activity and heightened levels of in vitro pokeweed mitogen (PWM)-induced IgG secretion. The reduced Con A suppressor activity could not be reversed by addition of interleukin 2 (IL-2). Cyclosporine A (CsA) treatment did not alter the defect in Con A-induced suppressor activity, but did markedly inhibit T8+ cell-mediated alloantigen directed cytolytic activity; this latter defect was reversible by in vitro addition of IL-2. CsA-treated patients did not differ from placebo-treated patients with regard to levels of PWM-induced IgG secretion or proliferative responses of their mononuclear cells to Con A. The results indicate that CsA treatment of MS patients reduces cytolytic function from baseline normal values, but does not alter aberrant suppressor cell function. PMID:2942563

  5. The promise of low-dose interleukin-2 therapy for autoimmune and inflammatory diseases.

    PubMed

    Klatzmann, David; Abbas, Abul K

    2015-05-01

    Depletion of regulatory T (TReg) cells in otherwise healthy individuals leads to multi-organ autoimmune disease and inflammation. This indicates that in a normal immune system, there are self-specific effector T cells that are ready to attack normal tissue if they are not restrained by TReg cells. The data imply that there is a balance between effector T cells and TReg cells in health and suggest a therapeutic potential of TReg cells in diseases in which this balance is altered. Proof-of-concept clinical trials, now supported by robust mechanistic studies, have shown that low-dose interleukin-2 specifically expands and activates TReg cell populations and thus can control autoimmune diseases and inflammation. PMID:25882245

  6. Genetically engineered Newcastle disease virus expressing interleukin 2 is a potential drug candidate for cancer immunotherapy.

    PubMed

    Bai, Fuliang; Niu, Zeshan; Tian, Hui; Li, Siming; Lv, Zheng; Zhang, Tianyuan; Ren, Guiping; Li, Deshan

    2014-01-01

    Newcastle disease virus (NDV) is an intrinsically tumor-specific virus, several clinical trials have reported that mesogenic NDV is a safe and effective agent for human cancer therapy. Interleukin 2 (IL2) is a cytokine that stimulates T cell propagation to trigger innate and adaptive immunity. IL2 has been used for cancer therapy and has achieved curative effects. In this study, a recombinant NDV LaSota strain expressing human interleukin 2 (rLaSota/IL2) was generated. The ability of rLaSota/IL2 to express human IL2 was detected in the infected tumor cells. In addition, the activity of IL2 was analyzed. The antitumor potential of rLaSota/IL2 was studied by xenograph mice carrying H22 and B16-F10 cells. Tumor-specific CD4(+) and CD8(+) T cells and MHC II were also analyzed in the two tumor-bearing models. Our study showed that rLaSota/IL2 significantly stimulated tumor-specific cytotoxic T-lymphocyte (CTL) responses and increased regulatory CD4(+) and cytotoxic CD8(+) T cells proliferation. The treatment with rLaSota/IL2 led to tumor regression in tumor-bearing mice and prolonged the survival of tumor-bearing mice. Furthermore, tumor challenging experiments demonstrated that rLaSota/IL2 invoked mice a unique capacity to remember a pathogen through the generation of memory T cells, which protect the host in the event of reinfection and form adaptive immune system. The result indicates that tumor-infiltrating CD4(+) T regulatory cells may denote the effective regression of tumors. Taken together, rLaSota/IL2 has potential for immunotherapy and oncolytic therapy of cancers and may be an ideal candidate for clinical application in future cancer therapy. PMID:24613899

  7. Soluble Interleukin 2 Receptor Levels, Temperament and Character in Formerly Depressed Suicide Attempters Compared with Normal Controls

    ERIC Educational Resources Information Center

    Rothenhausler, Hans-Bernd; Stepan, Alexandra; Kapfhammer, Hans-Peter

    2006-01-01

    An imbalance of the immune system and mixed personality profiles in suicide attempters have been reported. As suicidal behavior is common in patients with psychiatric disorders within the spectrum of depressive features, in this study we measured soluble interleukin-2 receptor concentrations in plasma (sIL-2R) and investigated temperament and…

  8. Further characterization of the binding of human recombinant interleukin 2 to heparin and identification of putative binding sites

    Microsoft Academic Search

    Saloua Najjam; Barbara Mulloy; Jacques Theze; Myrtle Gordon; Roslyn Gibbs; Christopher C. Rider

    1998-01-01

    6To whom correspondence should be addressed We have previously provided compelling evidence that human recombinant interleukin 2 (IL-2) binds to the sulfated polysaccharides heparin, highly sulfated heparan sulfate and fucoidan. Here we show that IL-2 binding is dependent on heparin chain length, but with fragments as small as 15-mers retaining binding activity. The addition of exogenous heparin has no effect

  9. Human cytokine-induced killer cells have enhanced in vitro cytolytic activity via non-viral interleukin-2 gene transfer

    Microsoft Academic Search

    Srinivas Nagaraj; Carsten Ziske; Ingo GH Schmidt-Wolf

    2004-01-01

    Modulation of the immune system by genetically modified immunological effector cells is of potential therapeutic value in the treatment of malignancies. Interleukin-2 (IL-2) is a crucial cytokine which induces potent antitumor response. Cytokine-induced killer cells (CIK) have been described as highly efficient cytotoxic effector cells capable of lysing tumor cell targets and are capable of recognizing these cells in a

  10. Inhibition of Interleukin-2 Gene Expression by Human Herpesvirus 6B U54 Tegument Protein

    PubMed Central

    Iampietro, Mathieu; Morissette, Guillaume; Gravel, Annie

    2014-01-01

    ABSTRACT Human herpesvirus 6B (HHV-6B) is a ubiquitous pathogen causing lifelong infections in approximately 95% of humans worldwide. To persist within its host, HHV-6B has developed several immune evasion mechanisms, such as latency, during which minimal proteins are expressed, and the ability to disturb innate and adaptive immune responses. The primary cellular targets of HHV-6B are CD4+ T cells. Previous studies by Flamand et al. (L. Flamand, J. Gosselin, I. Stefanescu, D. Ablashi, and J. Menezes, Blood 85:1263–1271, 1995) reported on the capacity of HHV-6A as well as UV-irradiated HHV-6A to inhibit interleukin-2 (IL-2) synthesis in CD4+ lymphocytes, suggesting that viral structural components could be responsible for this effect. In the present study, we identified the HHV-6B U54 tegument protein (U54) as being capable of inhibiting IL-2 expression. U54 binds the calcineurin (CaN) phosphatase enzyme, causing improper dephosphorylation and nuclear translocation of NFAT (nuclear factor of activated T cells) proteins, resulting in suboptimal IL-2 gene transcription. The U54 GISIT motif (amino acids 293 to 297), analogous to the NFAT PXIXIT motif, contributed to the inhibition of NFAT activation. IMPORTANCE Human herpesvirus 6A (HHV-6A) and HHV-6B are associated with an increasing number of pathologies. These viruses have developed strategies to avoid the immune response allowing them to persist in the host. Several studies have illustrated mechanisms by which HHV-6A and HHV-6B are able to disrupt host defenses (reviewed in L. Dagna, J. C. Pritchett, and P. Lusso, Future Virol. 8:273–287, 2013, doi:10.2217/fvl.13.7). Previous work informed us that HHV-6A is able to suppress synthesis of interleukin-2 (IL-2), a key immune growth factor essential for adequate T lymphocyte proliferation and expansion. We obtained evidence that HHV-6B also inhibits IL-2 gene expression and identified the mechanisms by which it does so. Our work led us to the identification of U54, a virion-associated tegument protein, as being responsible for suppression of IL-2. Consequently, we have identified HHV-6B U54 protein as playing a role in immune evasion. These results further contribute to our understanding of HHV-6 interactions with its human host and the efforts deployed to ensure its long-term persistence. PMID:25122797

  11. Effects of Interleukin 2 Receptor Blockers on Patient and Graft Survival in Renal-Transplanted Children

    PubMed Central

    Sharifian, Mostafa; Arad, Banafsheh; Simfroosh, Naser; Basiri, Abbas; Otukesh, Hassan; Esfandiar, Nasrin

    2014-01-01

    Background: Monoclonal antibodies block interleukin-2 receptors on alloantigen-reactive T-Lymphocytes and induce selective immunosuppression. It is postulated that induction therapy with these agents in pediatric transplantation may decrease acute rejection and improve graft survival with no significant side effect or increase in the incidence of viral infections. Objectives: The aim of this study was to examine the effects of interleukin 2 receptor blockers on patient and graft survival in renal-transplanted children. Patients and Methods: One hundred and eighty six children aged 7-13 years who received renal transplantation in university-affiliated hospital between 2003 and 2012 were enrolled in the study. All patients received prednisolone, cyclosporine and mycophenolate mofetil or azathioprine as basic immunosuppressive therapy. Patients were divided into two groups according to receiving induction therapy with IL2-receptor blockers. We investigated for acute rejection episodes, Cytomegalovirus (CMV) and BK virus infection and one and three year’s survival of the patients and the grafts Results: From 186 renal-transplanted children included in this study, 36 patients were in treated group (group 1) and 150 patients in control group (group 2). The mean age of the patients was 10.4 ± 2 years and 55.6% were males. In first six months of transplantation, eight patients in group one had one episode of acute rejection and no one had two episodes. Early acute rejection rate was 8.36 (22%). In the control group, 37 patients had one episode and three patients had two episodes of acute rejection (rejection rate 28.6%). Therefore, early acute rejection rates were lower in group one. Late acute rejection rates did not show any difference in group 1 and group 2 (27.7% vs. 27.3% respectively). There was lower prevalence of steroid-resistance rejection in group 1 patients (5.5%) compared with 6.6% in group 2, but it did not reach statistical significance. None of the patients in IL2-R blocker group died at one year follow-up (patient survival 100%). However, in control group, four (2.6%) patients died toward the end of first year (patient survival 97.4%). When patients in group 1 and group 2 were age and sex matched with equal number the difference was significant (P < 0.05). Conclusions: Induction therapy with IL2-R blockers reduced the rate of early acute rejection, but had no effect on late acute rejections. Patient and graft survival were better in treated group, but did not reach statistical significance. A longer period of follow-up may be required to discern a clear advantage for induction therapy with these agents. PMID:25695021

  12. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: Involvement of interleukin-2 system

    SciTech Connect

    Cervia, Davide, E-mail: d.cervia@unitus.it [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy) [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Catalani, Elisabetta; Belardinelli, Maria Cristina [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Perrotta, Cristiana [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy)] [Department of Biomedical and Clinical Sciences, “Luigi Sacco” University Hospital, University of Milan, Milano (Italy); Picchietti, Simona [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Alimenti, Claudio [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)] [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy); Casini, Giovanni; Fausto, Anna Maria [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy)] [Department for Innovation in Biological, Agro-food and Forest systems (DIBAF), University of Tuscia, Viterbo (Italy); Vallesi, Adriana [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)] [Department of Environmental and Natural Sciences, University of Camerino, Camerino (Italy)

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell–cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-?, tumor necrosis factor-?, interleukin (IL)-1?, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ? and ? subunits of IL-2 receptor (IL-2R), while the mRNA levels of the ? subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2R? subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. -- Highlights: ? Euplotes pheromone Er-1 increases the growth of human Jurkat T-cells. ? Er-1 increases the T-cell production of specific cytokines. ? Er-1 activates interleukin-2 receptor and extracellular signal-regulated kinases. ? The immuno-enhancing effect of Er-1 on Jurkat cells translates to an inhibition of human glioma cell growth.

  13. Role of Interleukin-2 in Uremic Pruritus Among Attendants of AL-Zahraa Hospital Dialysis Unit

    PubMed Central

    Azim, Amira Adel Abdel; Farag, Asmaa Saied; El-Maleek Hassan, Doaa Abd; Abdu, Safaa Mahmoud Ismail; Lashin, Somaya Mohamed Abo-Elfetouh; Abdelaziz, Nahla Mohamed

    2015-01-01

    Background: Uremic pruritus (UP) is a very distressing symptom and remains one of the most frustrating and potentially disabling symptoms in patients with end-stage renal disease (ESRD). Its etiopathogenesis remains unclear and complex. The aim of this study was to investigate the possible role of interleukin-2 (IL-2) in UP, and correlate its level with the severity of itching in ESRD patients. Patients and Methods: This study was carried out on 60 patients on maintenance hemodialysis (HD), 30 patients with UP and 30 patients without UP, and 30 apparently healthy age- and sex-matched subjects as controls. Itch intensity was scored as mild, moderate, and severe using five-dimensional itch scale. Some relevant clinical parameters (age, sex, xerosis, presence of neuropathy, duration of dialysis, complete medical history, and history of pruritic skin diseases) and laboratory findings including creatinine, urea, calcium, phosphorus, parathyroid hormone, and serum levels of IL-2 were evaluated. Results: In our study, we found a statistically significant difference in IL-2 level between patients and controls. However, there was no statistically significant difference in IL-2 levels between cases with pruritus and cases without pruritus. Also, there was a statistically significant relation between IL-2 level and duration of the disease. Conclusion: Further studies are needed to understand the contribution of IL-2 and possibly other cytokines in the pathogenesis of this distressing symptom in ESRD. PMID:25814728

  14. Attenuation of interleukin 2-induced pulmonary vascular leak syndrome by low doses of oral methotrexate.

    PubMed

    DeJoy, S Q; Jeyaseelan, R; Torley, L W; Schow, S R; Wick, M M; Kerwar, S S

    1995-11-01

    Pulmonary vascular leak induced in mice by interleukin 2 (IL-2) was attenuated by pretreatment with single or multiple doses of oral methotrexate. Methotrexate also attenuated pulmonary vascular leak when either larger doses of IL-2 or when lymphokine-activated killer (LAK) cells or LAK cells plus IL-2 were administered. Lymphoid infiltrates in the lungs of mice treated with IL-2 and methotrexate were significantly lower. The number of mice surviving treatment with high doses of IL-2 was also significantly increased when these mice were treated with methotrexate. Methotrexate prevented the IL-2-induced increase in the number of splenocytes that were asialo GM1+ but had no effect on Lyt 2+ or L3T4+ cell content. A marginal but significant inhibition in the generation of effector splenocytes that were cytolytic to either YAC or MCA-205 tumor targets was observed in mice treated with methotrexate and IL-2. In vivo studies indicated that methotrexate did not compromise the anti-tumor efficacy of treatment regimens that contained IL-2, LAK cells, or IL-2 and LAK cells. These results demonstrate the potential clinical utility of methotrexate in attenuating pulmonary vascular leak induced by IL-2 without compromising its efficacy. One potential mechanism of action of methotrexate is related to its ability to stimulate the release of adenosine followed by the inhibition of the adhesion of leukocytes to the IL-2-activated endothelium. PMID:7585532

  15. Antibodies to interleukin 2. Effects on immune responses in vitro and in vivo

    PubMed Central

    1984-01-01

    Antibodies to highly purified mouse interleukin 2 (IL-2) were raised in rabbits; a 1:500 dilution of antiserum completely blocked the in vitro mitogenic effect of 10(-9) M IL-2. The antisera functioned effectively to immunoprecipitate biosynthetically labeled IL-2 and the purified immunoglobulins were useful in the construction of affinity columns for the adsorption and one-step immunopurification of IL-2. The antibodies were apparently specific for IL-2 among the lymphokines, they did not block the biological effects of IL-1, IL-3, gamma-IFN, B cell stimulating factor(s), and cytotoxic T cell differentiation factor(s). When anti-IL-2 was added to the in vitro reactions, it blocked mixed leukocyte reactions (MLR) and associated lymphocyte proliferation, the in vitro generation of cytotoxic T cells, and antibody formation as assessed by erythrocyte-specific plaque-forming cells (PFC). When injected into mice, anti-IL-2 antibodies also reduced the formation of cytotoxic lymphocytes in response to allogeneic cells, suggesting that endogenous IL-2 participates in such reactions in vivo. Taken together, the results indicate that these IL-2 antibodies will be useful adjuncts in the analysis of immune response both in vivo and in vitro. PMID:6236276

  16. Integrating Signals from the T-Cell Receptor and the Interleukin-2 Receptor

    PubMed Central

    Beyer, Tilo; Busse, Mandy; Hristov, Kroum; Gurbiel, Slavyana; Smida, Michal; Haus, Utz-Uwe; Ballerstein, Kathrin; Pfeuffer, Frank; Weismantel, Robert; Schraven, Burkhart; Lindquist, Jonathan A.

    2011-01-01

    T cells orchestrate the adaptive immune response, making them targets for immunotherapy. Although immunosuppressive therapies prevent disease progression, they also leave patients susceptible to opportunistic infections. To identify novel drug targets, we established a logical model describing T-cell receptor (TCR) signaling. However, to have a model that is able to predict new therapeutic approaches, the current drug targets must be included. Therefore, as a next step we generated the interleukin-2 receptor (IL-2R) signaling network and developed a tool to merge logical models. For IL-2R signaling, we show that STAT activation is independent of both Src- and PI3-kinases, while ERK activation depends upon both kinases and additionally requires novel PKCs. In addition, our merged model correctly predicted TCR-induced STAT activation. The combined network also allows information transfer from one receptor to add detail to another, thereby predicting that LAT mediates JNK activation in IL-2R signaling. In summary, the merged model not only enables us to unravel potential cross-talk, but it also suggests new experimental designs and provides a critical step towards designing strategies to reprogram T cells. PMID:21829342

  17. Agonistic effects of a monoclonal antibody specific for the interleukin-2 receptor

    SciTech Connect

    Eardley, D.D.; Makrides, V.

    1986-03-05

    Interleukin-2 (IL-2) mediated immune responses can be blocked by monoclonal antibodies to the IL-2 receptor. The monoclonal antibody, M720, is defined as specific for the IL-2 receptor because it blocks /sup 35/S-IL-2 binding to Con A blasts, reacts with lymphoblasts but not resting splenocytes, and inhibits IL-2 induced proliferation to mitogen, antigen, or allogeneic stimuli. Under appropriate culture conditions, the IL-2 receptor-specific antibody can act like IL-2 in that it will induce proliferation in T cells in the absence of additional antigen or mitogen. This agonistic effect is dependent on time, dose of antibody, and requires fetal calf serum (FCS) in the media. Because the FCS is not mitogenic by itself, the authors propose that the FCS components act as incomplete mitogen to induce appearance of IL-2 receptors but lack a factor which would push the majority of the cells into the S phase of the cell cycle. This factor is usually IL-2, but in the authors experiments, the IL-2 receptor-specific antibody can provide the same stimulus. These data indicate that factors like FCS can induce IL-2 receptors, but without additional IL-2 or receptor triggering, the cells will not proceed through the synthetic and proliferative phases of cell growth.

  18. Effect of 1,1-dimethylhydrazine on lymphoproliferation and interleukin 2 immunoregulatory function.

    PubMed

    Bauer, R M; Tarr, M J; Olsen, R G

    1990-01-01

    The studies reported here suggest that the immunomodulatory effects of 1,1-dimethylhydrazine (UDMH) are associated, in part, with interference with interleukin 2 (IL-2) regulatory action. Concanavalin A (Con A)-stimulated DNA synthesis in murine splenocytes was inhibited from 18.6 to 44.1% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml) and IL-2-dependent DNA synthesis in CTLL-20 cells was inhibited from 11.3 to 41.58% at sub-toxic concentrations of UDMH (10 to 50 micrograms/ml). In addition, UDMH suppressed phorbol myristic acetate (PMA)-stimulated IL-2 production in EL-4 cells by up to 30% and slightly suppressed IL-2 production by Con A-stimulated murine splenocytes. In all cases, inhibition was evident at sub-toxic UDMH concentrations and was demonstrated to be independent of inactivation of IL-2 or interference with IL-2 absorption. It is suggested that UDMH has the potential to modify immune function through interference with IL-2 production and especially the lymphoproliferative response to IL-2. PMID:2331149

  19. Role of interleukin-2 in superantigen-induced T-cell anergy

    PubMed Central

    Cornwell, W D; Rogers, T J

    1999-01-01

    T-cell anergy is a state of immunological tolerance characterized by unresponsiveness to antigenic stimulation. Previous studies have shown that anergy is induced in T cells following stimulation in the absence of adequate costimulatory signals. These cells fail to respond to stimulation via the T-cell receptor (TCR), and fail to produce normal levels of interleukin-2 (IL-2). We present results here which show that low concentrations of the superantigen staphylococcal enterotoxin A (SEA) in the absence of antigen-presenting cells induced both proliferation and anergy in the A.E7 T-cell clone. Furthermore, under these conditions, the A.E7 clone remained responsive to exogenous IL-2. Fluorescence-activated cellular cytometry analysis revealed unaltered expression of the TCR/CD3 complex in the anergized clone; however, both CD4 and CD25 expression increased after 24 hr of stimulation by SEA under these conditions. Interestingly, a low level of IL-2 production was measured during the induction of anergy. Most strikingly, stimulation of the A.E7 clone by SEA in combination with exogenous IL-2 resulted in a more pronounced state of anergy. These results suggest that the induction of anergy is a process that is essentially independent of the production of IL-2. PMID:10233695

  20. Development of fetal thymocytes in organ cultures. Effect of interleukin 2

    PubMed Central

    1987-01-01

    Most fetal thymocytes from 14-d mouse embryos are Thy-1+, L3T4-, Ly-2-, and express the receptor for interleukin 2 (IL-2). The development of thymocytes has been followed in fetal thymus organ cultures. When fetal thymus from 14-d embryos were cultured for a 6-d period, thymocytes increased in number 20-40-fold, and 95% became Thy-1+, L3T4+, Ly-2+. The addition of IL-2 to organ cultures of 14-d fetal thymus inhibited, in a dose-dependent manner, cell proliferation and the appearance of Thy-1+, L3T4+, Ly-2+ thymocytes. The addition of IL-2 also resulted in the appearance of a population of cells that were cytotoxic for syngeneic and allogeneic fetal thymocytes and syngeneic tumour targets. While the events that lead to the expression of the IL-2 receptor on 14- d fetal thymocytes are unknown, IL-2 in fetal thymus organ cultures inhibits the normal maturation of fetal thymocytes and raises the question of whether the cytotoxic cells that appear reflect selection through an alternative pathway of development. PMID:3108443

  1. Interleukin-2 activity can be fine tuned with engineered receptor signaling clamps.

    PubMed

    Mitra, Suman; Ring, Aaron M; Amarnath, Shoba; Spangler, Jamie B; Li, Peng; Ju, Wei; Fischer, Suzanne; Oh, Jangsuk; Spolski, Rosanne; Weiskopf, Kipp; Kohrt, Holbrook; Foley, Jason E; Rajagopalan, Sumati; Long, Eric O; Fowler, Daniel H; Waldmann, Thomas A; Garcia, K Christopher; Leonard, Warren J

    2015-05-19

    Interleukin-2 (IL-2) regulates lymphocyte function by signaling through heterodimerization of the IL-2R? and ?c receptor subunits. IL-2 is of considerable therapeutic interest, but harnessing its actions in a controllable manner remains a challenge. Previously, we have engineered an IL-2 "superkine" with enhanced affinity for IL-2R?. Here, we describe next-generation IL-2 variants that function as "receptor signaling clamps." They retained high affinity for IL-2R?, inhibiting binding of endogenous IL-2, but their interaction with ?c was weakened, attenuating IL-2R?-?c heterodimerization. These IL-2 analogs acted as partial agonists and differentially affected lymphocytes poised at distinct activation thresholds. Moreover, one variant, H9-RETR, antagonized IL-2 and IL-15 better than blocking antibodies against IL-2R? or IL-2R?. Furthermore, this mutein prolonged survival in a model of graft-versus-host disease and blocked spontaneous proliferation of smoldering adult T cell leukemia (ATL) T cells. This receptor-clamping approach might be a general mechanism-based strategy for engineering cytokine partial agonists for therapeutic immunomodulation. PMID:25992859

  2. Novel use of hepatobiliary scintigraphy for the diagnosis of interleukin-2 cholangiopathy.

    PubMed

    Marti, Jon K; Banks, Kevin P; Song, Won S

    2010-01-01

    We report a case of interleukin-2 (IL-2) induced cholangiopathy diagnosed with the aid of hepatobiliary scintigraphy. Patient was a 32 years old, male with history of metastatic melanoma. Computed tomography (CT) upon admission demonstrated worsening of patient's metastatic lung disease with a normal appearance of the gallbladder. The patient was started on high dose IL-2 treatment for regression of his disease. Four days after IL-2 treatment was begun, the patient developed severe right upper quadrant pain and elevated liver function tests. A right upper quadrant ultrasound and surgical consultation were requested. Sonographic findings demonstrated diffuse gallbladder wall thickening, mural edema, a positive sonographic Murphy's sign, but no gallstones. The preliminary working diagnosis was acalculous cholecystitis versus IL-2 induced cholangiopathy. To clarify between these two entities, a hepatobiliary scan was obtained that demonstrated filling of the gallbladder with prompt biliary-to-bowel transit and normal liver function. In this case, the clinical presentation and history of recent IL-2 treatment were suggestive of IL-2 cholangiopathy, though the patient's co-morbidities and in-patient status raised concern for acalculous cholecystitis. Given the marked differences in treatment, hepatobiliary imaging was requested and found to be normal, making acalculous cholecystitis very unlikely. In conclusion, we believe this is the first case in which hepatobiliary scintigraphy was used to aid in the diagnosis of IL-2 induced cholangiopathy. PMID:20808991

  3. A Synaptic Basis for Paracrine Interleukin-2 Signaling during homotypic T cell interaction

    PubMed Central

    Sabatos, Catherine A.; Doh, Junsang; Chakravarti, Sumone; Friedman, Rachel S.; Pandurangi, Priya G.; Tooley, Aaron J.; Krummel, Matthew F.

    2015-01-01

    Summary T cells slow their motility, increase adherence and arrest after encounters with antigen-presenting cells (APCs) bearing peptide-MHC complexes. Here, we analyzed the cell-cell communication among activating T cells. In vivo and in vitro, activating T cells associate in large clusters that collectively persist for >30 minutes, but they also engaged in more transient interactions, apparently distal to APCs. Homotypic aggregation was driven by LFA-1 integrin interactions. Ultrastructural analysis revealed that cell-cell contacts between activating T cells were organized as multifocal synapses, and T cells oriented both the microtubule organizing complex and interleukin-2 (IL-2) secretion toward this synapse. T cells engaged in homotypic interactions more effectively captured IL-2 relative to free cells. T cells receiving paracrine synaptic IL-2 polarized their IL-2 signaling subunits into the synaptic region and more efficiently phosphorylated the transcription factor STAT5, likely through a synapse-associated signaling complex. Thus, synapse-mediated cytokine delivery accelerates responses in activating T cells. PMID:18674934

  4. Characterization of interleukin 2 stimulated 65-kilodalton phosphoprotein in human T cells

    SciTech Connect

    Zu, Youli; Kohno, Michiaki; Namba, Yuziro (Kyoto Univ. (Japan)); Kohno, Michiaki (Gifu Pharmaceutical Univ. (Japan)); Kubota, Ichiro (Suntory Bio-Pharam Tech Center (Japan)); Nishida, Eisuke (Univ. of Tokyo (Japan))

    1990-01-30

    The authors have characterized the cellular proteins which are rapidly phosphorylated by interleukin 2 (IL 2) in a human IL 2 dependent cell line. When treated with IL 2, the phosphorylation of five proteins, 65, 50, 37, 24, and 21 kDa, was found in IL 2 dependent cell lines by two-dimensional gel electrophoretic analysis. After cell conversion from an IL 2 dependent state to an IL 2 independent state, one of the five phosphoproteins, the 65-kDa protein, became constitutively phosphorylated even without addition of IL 2. Also, in other IL 2 independent cell lines, such as KUT-2 and HUT-102, constitutive phosphorylation of the 65-kDa protein occurred without IL 2-stimulation. So our researchers were focused on biochemical characterization of the 65-kDa protein. It was found that the 65-kDa protein was one of the major cellular proteins by comparing the results of two-dimensional gel electrophoretic analysis of ({sup 32}P)P{sub i}-labeled and ({sup 3}H)leucine-labeled cellular proteins and peptide mapping analysis. Subcellular fraction studies indicated that the 65-kDa protein is a cytosol protein. The 65-kDa protein was purified from cytosol of a human T cell line, and its amino acid composition and amino acid sequences of its three oligopeptides were determined. It was found that the 65-kDa protein is identical with 1-plastin.

  5. Regulation of Rho signaling pathways in interleukin-2-stimulated human T-lymphocytes.

    PubMed

    Mzali, Rym; Seguin, Laetitia; Liot, Caroline; Auger, Anick; Pacaud, Pierre; Loirand, Gervaise; Thibault, Christelle; Pierre, Josiane; Bertoglio, Jacques

    2005-11-01

    Rho GTPases are key regulators of many cellular functions, including cytoskeleton organization which is important for cell morphology and mobility, gene expression, cell cycle progression, and cytokinesis. In addition, it has recently been recognized that Rho GTPase activity is required for development of the immune system, as well as for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen-specific populations and provides a model to study cell cycle entry and cell cycle progression. We have performed gene expression analysis in a model of human T lymphocytes, which proliferate in response to IL-2. In addition to changes in genes relevant to cell cycling and to the antiapoptotic effects of IL-2, we have analyzed expression and variations of more than 300 genes involved in Rho GTPase signaling pathways. We report here that IL-2 regulates the expression of a number of proteins, which participate in the Rho GTPase pathways, including some of the GTPases themselves, GDP/GTP exchange factors, GTPase activating proteins, as well as GDIs and effectors. Our results suggest that regulation of expression of components of the Rho GTPase pathways may be an important mechanism in assembling specific signal transduction cascades that need to be active at certain times during the cell cycle. Some of our findings may also be relevant to the roles of Rho GTPases in T lymphocyte functions and proliferation. PMID:16148026

  6. Sharing of the Interleukin2 (IL2) Receptor gamma Chain Between Receptors for IL2 and IL4

    Microsoft Academic Search

    Motonari Kondo; Toshikazu Takeshita; Naoto Ishii; Masataka Nakamura; Sumiko Watanabe; Ken-Ichi Arai; Kazuo Sugamura

    1993-01-01

    The gamma chain of the interleukin-2 (IL-2) receptor is an indispensable subunit for IL-2 binding and intracellular signal transduction. A monoclonal antibody to the gamma chain, TUGm2, inhibited IL-2 binding to the functional IL-2 receptors and also inhibited IL-4-induced cell growth and the high-affinity binding of IL-4 to the CTLL-2 mouse T cell line. Another monoclonal antibody, TUGm3, which reacted

  7. Immunotherapy of hepatocellular carcinoma with autologous lymphokine-activated killer cells and\\/or recombinant interleukin-2

    Microsoft Academic Search

    Takashi Ishikawa; Michio Imawari; Takashi Moriyama; Shin Ohnishi; Nobuyuki Matsuhashi; Gen Suzuki; Fumimaro Takaku

    1988-01-01

    Five patients with hepatocellular carcinoma were subjected to immunotherapy: three patients were treated by adoptive immunotherapy with lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2), and two patients by systemic administration of rIL-2 alone. In one patient with diffuse-type hepatocellular carcinoma and portal vein thrombosis who was treated by infusion of LAK cells (a total number of 1.5x1010 cells\\/13 doses)

  8. Serial monitoring of interleukin-1ß, soluble interleukin-2 receptor and lipopolysaccharide binding protein levels after death

    Microsoft Academic Search

    Uta Reichelt; Roman Jung; Axel Nierhaus; Michael Tsokos

    2005-01-01

    We prospectively monitored the postmortem course of interleukin-1? (IL-1?), soluble interleukin-2 receptor (sIL-2R) and lipopolysaccharide binding protein (LBP) in septic and non-septic fatalities to evaluate their potential as biochemical postmortem markers of sepsis. Serum concentrations were determined by chemiluminescent immunometric assays. In both the sepsis group and the control group a postmortem increase of IL-1? levels with the progression of

  9. Identification and Initial Characterization of a Rat Monoclonal Antibody Reactive with the Murine Interleukin 2 Receptor-Ligand Complex

    Microsoft Academic Search

    Thomas R. Malek; Richard J. Robb; Ethan M. Shevach

    1983-01-01

    Xenogeneic monoclonal antibodies were prepared to the murine interleukin 2 (IL-2)-dependent HT2 cell line. One rat IgM monoclonal antibody (7D4) was identified that inhibited proliferation of the HT2 cells and of IL-2-dependent CTLL cells in the presence of crude rat IL-2 as well as of purified human IL-2. The level of inhibition was dependent on both antibody and IL-2 concentration.

  10. Clinical and immunological effects of human recombinant interleukin-2 given by repetitive weekly infusion to normal dogs

    Microsoft Academic Search

    Stuart C. Helfandl; Steve A. Soergell; Peter S. MacWilliams; Jacquelyn A. Hank; Paul M. Sondel

    1994-01-01

    Four normal adult dogs received two consecutive weekly cycles of human recombinant interleukin-2 (IL-2) by continuous infusion for 4 days\\/week. The dose of IL-2 given to each dog was 3×106 units m-2 day-1. Toxicities consisted of mild vomiting, diarrhea, and lethargy to varying degrees in all the dogs. These side-effects were reversed when the treatment was discontinued. Fever, tachypnea, and

  11. Comparative analysis of the expression of c-Fos and interleukin-2 proteins in hypothalamus cells during various treatments

    Microsoft Academic Search

    S. V. Barabanova; Z. E. Artyukhina; K. T. Ovchinnikova; T. V. Abramova; T. B. Kazakova; V. Kh. Khavinson; V. V. Malinin; E. A. Korneva

    2008-01-01

    The aim of the present work was to perform a combined analysis of the degree of activation of the anterior hypothalamus of\\u000a the rat and expression of the interleukin-2 gene during treatments of different types: mild stress (“handling”) and adaption\\u000a to it, as well as intranasal administration of physiological saline and the peptides Vilon (Lys-Glu) and Epitalon (Ala-Glu-Asp-Gly).\\u000a Changes in

  12. Developing an electrochemical deoxyribonucleic acid (DNA) biosensor on the basis of human interleukine-2 gene using an electroactive label

    Microsoft Academic Search

    M. H. Pournaghi-Azar; M. S. Hejazi; E. Alipour

    2006-01-01

    Development of an electrochemical DNA biosensor based on a human interleukine-2 (IL-2) gene probe, using a pencil graphite electrode (PGE) as transducer and methylene blue (MB) as electroactive label is described. The sensor relies on the immobilization of a 20-mer single stranded oligonucleotide probe (hIL-2) related to the IL-2 gene on the electrode. The hybridization between the probe and its

  13. Regression of established pulmonary metastases and subcutaneous tumor mediated by the systemic administration of high-dose recombinant interleukin 2

    Microsoft Academic Search

    S. A. Rosenberg; J. J. Mule; P. J. Spiess; C. M. Reichert; S. L. Schwarz

    1985-01-01

    Incubation of resting lymphoid cells with recombinant interleukin 2 (IL-2) in vitro leads to the generation of lymphokine activated killer (LAK) cells capable of lysing fresh tumor cell suspensions in short-term chromium-release assays. Previous studies have demonstrated that the injection of LAK cells plus low doses of recombinant IL-2 were capable of inhibiting the growth of pulmonary metastases. The authors

  14. Proliferation and Cytolytic Function of Anti-CD3 + Interleukin2 Stimulated Peripheral Blood Mononuclear Cells Following Bone Marrow Transplantation

    Microsoft Academic Search

    Emmanuel Katsanis; Peter M. Anderson; Alexandra H. Filipovich; Diane E. Hasz; Mary L. Rich; Cynthia M. Loeffler; Daniel J. Weisdorf

    1991-01-01

    We evaluated the proliferation, cytolytic function, and pheno- typic characteristics of antLCD3 plus interleukin-2 (IL-2) stimulated peripheral blood mononuclear cells (PBMCs) from 44 patients with leukemia or non-Hodgkin's lymphoma (NHL) treated with multiagent chemotherapy or following bone marrow transplantation (BMT). BMT patients had decreased cell growth with only a 1.35 f 0.25 (autologous BMTfor acute lymphoblastic leukemia (ALL)), 1.24 f

  15. Phase I Evaluation of a Combination of Monoclonal Antibody R24 and Interleukin 2 in Patients with Metastatic Melanoma1

    Microsoft Academic Search

    Dean F. Bajorin; Paul B. Chapman; George Wong; Daniel G. Coit; Jolanta Kunicka; Joseph Dimaggio; Carlos Cordon-Cardo; Carlos Urmacher; Lucy Dantes; Mary Agnes; John Liu Templeton; Herbert F. Oettgen; Alan N. Houghton

    A combination of recombinant human interleukin 2 (rhl I.-2) and mouse monoclonalantibody R24 (recognizing the ganglioside <.,,,) was evaluated in patients with metastatic melanoma in a phase I trial, rhl I -2 was given at a constant daily dose of 1 x 10'units\\/m2i.v. over 6 h on days 1-5 and 8-12. R24 was given on days 8-12 at four dose

  16. The changes of interleukin-2, tumour necrotic factor and gamma-interferon production among patients with Kawasaki disease

    Microsoft Academic Search

    C.-Y. Lin; B. Hwang; B. N. Chiang

    1991-01-01

    Included in this study were 43 cases of mucocutaneous lymph node syndrome (MCLS or Kawasaki disease) treated solely with aspirin. Of these, 19 developed coronary aneurysm. Mononuclear cells (MNC) of these MCLS patients were collected weekly and stimulated either with phytohaemagglutinin (PHA) or PHA plus phorbol myristic acetate. The production of interleukin-2 (IL-2), tumour necrotic factor (TNF) and gamma-interferon (IFN-r)

  17. Les récepteurs solubles de l'interleukine-2 au cours de la radiothérapie des cancers du sein

    Microsoft Academic Search

    H. Galinat; A.-M. Hersant; P. Rambert; J.-L. Floiras; C. Cohen-Solal; B. De La Lande; M.-F. Pichon

    2005-01-01

    We assessed the relationships between soluble interleukin-2 receptor (R-IL2s) circulating concentrations and the clinicobiological characteristics of 80 breast cancer patients treated by radiotherapy. We did not evidenced significant relationships between R-IL2s concentrations before radiotherapy and patient's or tumours characteristics. However, R-IL2s concentrations were found to be positively correlated to lymphocyte levels and cumulated radiations dose during radiotherapy. Radiotherapy-induced lymphocyte lysis

  18. Lipoteichoic Acid Inhibits Interleukin-2 (IL-2) Function by Direct Binding to IL-2

    PubMed Central

    Plitnick, Lisa M.; Jordan, Robert A.; Banas, Jeffrey A.; Jelley-Gibbs, Dawn M.; Walsh, Mary C.; Preissler, Mark T.; Gosselin, Edmund J.

    2001-01-01

    Lipoteichoic acid (LTA) is associated with the cell envelope of most gram-positive bacteria. Although previously thought to act mainly as a virulence factor by virtue of its adhesive nature, evidence is now provided that LTA can also suppress the function of interleukin-2 (IL-2), an autocrine growth factor for T cells. LTA from four separate bacterial strains lowered the levels of detectable IL-2 during a peripheral blood mononuclear cell response to the antigen tetanus toxoid (TT). T-cell proliferation in response to TT was similarly inhibited by LTA. In contrast, levels of detectable gamma interferon increased. In addition, LTA inhibited IL-2 detection by enzyme-linked immunosorbent assay (ELISA) and blocked the proliferative response of an IL-2-dependent T-cell line to soluble IL-2. Further studies using ELISA demonstrated that LTA blocks IL-2 detection and function by binding directly to IL-2. Flow cytometric analysis revealed that IL-2 binding to T cells is inhibited in the presence of purified LTA but not LTA plus anti-LTA monoclonal antibody. In summary, these studies demonstrate a novel effect of LTA on the immune response through direct binding to IL-2 and inhibition of IL-2 function. Importantly, gram-positive organisms from which LTA is obtained not only play an important role in the pathology of diseases such as bacterial endocarditis, septic shock, acute respiratory distress syndrome, and multiple organ failure but also comprise a significant portion of commensal populations within the human host. Inhibition of IL-2 function by LTA may represent yet another mechanism by which gram-positive bacteria dampen the host immune response and facilitate survival. Thus, LTA provides a potential target for therapeutic intervention when gram-positive organisms are involved. PMID:11527813

  19. Modulation of cellular immune responses in mice with disseminated histoplasmosis by recombinant interleukin-2.

    PubMed Central

    Deepe, G S; Taylor, C L; Harris, J E; Bullock, W E

    1986-01-01

    Depression of the cellular immune responses in mice with disseminated histoplasmosis is associated with deficient production of interleukin-2 (IL-2) by splenocytes. Therefore, we examined whether a highly purified preparation of IL-2, recombinant human IL-2 (rIL-2), could modify the cellular immune responses in infected mice and whether this lymphokine could alter the severity of histoplasmosis in animals. Exogenous rIL-2, at concentrations of up to 1,000 U/ml, failed to augment the proliferative responses to concanavalin A by unfractionated splenocytes or splenic T cells from mice infected for 1 week. In addition, rIL-2 did not modulate the plaque-forming cell response to sheep erythrocytes by splenocytes from these same mice. However, at week 3, rIL-2 in concentrations ranging from 10 to 1,000 U/ml considerably augmented the proliferative response to concanavalin A and plaque-forming cell response to sheep erythrocytes by splenocytes from infected mice. Kinetics studies demonstrated that rIL-2 exerted maximal immunoregulatory activity when added on day 0 or 1 to cultures of splenocytes. In vivo administration of rIL-2, 200 to 20,000 U/day, for 10 days to normal and 3-week-infected mice did not alter the proliferative activity of splenocytes to concanavalin A; 200,000 U of rIL-2 per day actually depressed the proliferative responses of splenocytes from normal and infected mice. In vivo, rIL-2 did not modify delayed-type hypersensitivity responses to sheep erythrocytes or to histoplasmin by normal and infected mice. Moreover, treatment with rIL-2 in vivo did not reduce the number of Histoplasma CFU in spleens of mice. Thus, despite the immunoenhancing effect of rIL-2 in vitro, this lymphokine failed to exert similar effects in vivo. PMID:3487507

  20. Interleukin-2 in relation to T cell subpopulations in rheumatic heart disease.

    PubMed Central

    Zedan, M M; el-Shennawy, F A; Abou-Bakr, H M; al-Basousy, A M

    1992-01-01

    Interleukin-2 (IL-2) and T cell subpopulations were evaluated in children with rheumatic heart disease (RHD). Three groups were included: 13 patients with active RHD, 12 with non-active RHD, and 14 control children. Serum IL-2 and T cell subpopulations were measured by radioimmunoassay and monoclonal antibodies respectively. Patients with active RHD showed a significant increase in IL-2 concentrations and helper:suppressor (H:S) ratio compared with controls with a mean (SEM) IL-2 of 3.48 (0.62) v 1.26 (0.16) U/ml and H:S ratio 2.31 (0.14) v 1.66 (0.04). There was a significant decrease in T suppressor (CD8+) and pan T (CD3+) cells compared with controls with a mean (SEM) for CD8+ of 23.75 (1.19) v 32.23 (0.56)% and CD3+ of 79.55 (0.94) v 85.00 (0.11)%. Patients with non-active RHD showed a significant decrease only in the CD3+ cells (78.20 (0.20)%) when compared with controls. A deficiency of CD3+ cells is a constant finding in patients with RHD, whether the disease is active or not. There was a significant increase in IL-2 concentration with a significant decrease in CD8+ cells in patients with active RHD in comparison with the non-active group (mean (SEM) IL-2 of 3.48 (0.62) v 1.85 (0.24) U/ml and CD8+ of 23.75 (1.19) v 28.83 (1.91)%). Thus an increase in IL-2 and a decrease in CD8+ cells may be related to rheumatic activity. T helper (CD4+) cells did not differ significantly between groups. PMID:1471890

  1. The beneficial effects of adjunctive recombinant human interleukin-2 for multidrug resistant tuberculosis

    PubMed Central

    Shen, Hong; Min, Rui; Tan, Qi; Xie, Weiping; Pan, Hongqiu; Zhang, Li; Xu, Hongtao; Zhang, Xia; Dai, Jianzhong

    2015-01-01

    Introduction Multidrug-resistant tuberculosis (MDR-TB) is a hard-to-treat disease with a poor outcome of chemotherapy. In the present study, the efficacy and safety of recombinant human interleukin-2 (rhIL-2) were investigated in patients with MDR-TB. Material and methods Fifty culture-confirmed patients with MDR-TB were included. Twenty-five patients were randomly assigned to the trial group (injection of 500 000 IU of rhIL-2 once every other day at the first, third, fifth and seventh months in addition to standard multidrug therapy) and another 25 patients to the control group with standard multidrug therapy. All patients were monitored clinically, and T-cell subsets were analyzed by flow cytometry. Results The rates of sputum negative conversion and X-ray resolution in the trial group were higher than those of the control, and the improvements were significant by completion of treatment. In addition, CD4+CD25+ T cells in the controls rose gradually during treatment. The levels at the end of the seventh month were significantly higher than before, which were also significantly different when compared with those from the trial group at the same time. However, there were no such changes associated with treatment in the trial group. No significant differences appeared in other T cell subsets. Conclusions Exogenous IL-2 in the present regimen improves immunity status. Adjunctive immunotherapy with a long period of rhIL-2 is a promising treatment modality for MDR-TB.

  2. Bronchoalveolar lavage analysis, gallium-67 lung scanning and soluble interleukin-2 receptor levels in asbestos exposure

    SciTech Connect

    Delclos, G.L.; Flitcraft, D.G.; Brousseau, K.P.; Windsor, N.T.; Nelson, D.L.; Wilson, R.K.; Lawrence, E.C.

    1989-04-01

    This study examined different markers of lung immunologic and inflammatory responses to previous asbestos exposure. We performed bronchoalveolar lavage (BAL) and gallium-67 (/sup 67/Ga) lung scans and measured serum and BAL soluble interleukin-2 receptor (IL-2R) and angiotensin-converting enzyme (SACE) levels in 32 subjects with a history of significant asbestos exposure, 14 without (EXP) and 18 with (ASB) radiographic evidence of asbestosis. BAL analysis revealed increases in neutrophils in both ASB and EXP when compared to controls (P less than 0.01), which persisted after adjustment for smoking category. Although significant abnormalities of macrophage and total lymphocyte profiles were not found in the study population, lymphocyte subpopulation analysis revealed elevation of BAL T4/T8 ratios in the entire study group (ASB + EXP) when compared to controls (P less than 0.05), independent of smoking category. /sup 67/Ga lung scan activity was increased in 56% of ASB and in 36% of EXP: no correlations between positive scans and different radiological and functional parameters could be found. There was no significant elevation of mean SACE, serum, or BAL IL-2R levels in any of the study categories. These data suggest that asbestos exposure may be associated with parenchymal inflammation, even in the absence of clinical criteria for asbestosis. Abnormalities of gallium uptake and of BAL analysis reflect the clinically inapparent inflammation. The increased BAL T4/T8 ratios observed suggest that abnormal local pulmonary immunoregulation may play a role in the pathogenesis of asbestos-related lung diseases.

  3. Administration in vivo of recombinant interleukin 2 protects mice against septic death.

    PubMed Central

    Weyand, C; Goronzy, J; Fathman, C G; O'Hanley, P

    1987-01-01

    Administration in vivo of recombinant interleukin 2 (rIL-2) to mice induces a polyclonal IgM response. When co-administered with a specific antigen, rIL-2 can enhance concentrations of murine IgM antibodies specific for the antigen by fivefold within 7 d of initial treatment. IgM antibodies that are induced after injection of rIL-2 include antibodies specific for J5, a cell wall core lipopolysaccharide (LPS) antigen that is shared by the different members of the Enterobactericeae family. We report here that mice pretreated with rIL-2 or immunized with J5 antigen 7 d before bacterial challenge were protected from septic death that is caused by intraperitoneal challenges with Escherichia coli. Optimal protection was provided by a combined J5 antigen and rIL-2 treatment. Acquisition of the rIL-2 and J5 antigen-induced protection against lethal bacterial infection coincided temporally with maximal serum IgM titers that also contained IgM antibodies specific for the J5 antigen. In passive immunization experiments, the affinity-purified IgM fraction in sera of rIL-2-treated animals was identified as necessary and sufficient for protection. The IgM-depleted serum had no protective effect. The nonspecific augmentation of host-defense mechanisms without the induction of endotoxin manifestations makes rIL-2 a potential candidate to any alternative LPS-containing vaccines for the prevention of bacterial infections by gram-negative organisms since the core LPS antigen is shared among gram-negative bacteria. PMID:3294901

  4. Expression of CD11b and CD18 on polymorphonuclear neutrophils stimulated with interleukin-2

    PubMed Central

    Abdel-Salam, Bahaa K.A.

    2014-01-01

    Background Interleukin-2 (IL-2) is a lymphocyte-activating and growth-promoting factor, and has been widely studied on T-cells and NK-cells. However, the interaction of polymorphonuclear neutrophils (PMNs) with IL-2 is poorly studied and thus, this study aimed at defining IL-2 participation in the expression of CD11b and CD18 on PMNs. Material and methods PMNs were isolated from heparinized whole blood of healthy donors. Purified cells were incubated with IL-2 (10 ng/ml) for 24 hours at 37°C in a humidified incubator with 5% CO2. After 24 hours’ incubation, surface molecules (CD11b and CD18) were measured by flow cytometry. Results Interestingly, the antibodies of IL-2R? chain (CD122-FITC) were found in all observed cells. The induction of CD11b mean fluorescence intensity (MFI) in highly purified PMNs stimulated with IL-2 was clearly increased recording 43% in comparison to the freshly isolated PMNs and the un-stimulated PMNs which were found to be 23% and 28% of CD11b, respectively. Furthermore, flow cytometry analysis demonstrated that the highly purified PMNs exposed to IL-2 showed an increase in CD18 MFI, recording 47% with respect to that of the freshly isolated PMNs and PMNs cultured with the medium alone which showed a small amount of 38% and 27%, respectively. Conclusions Results demonstrated that CD11b and CD18 had been acquired on the surface of the IL-2-in vitro-activated PMNs. These findings indicated that IL-2 may play a crucial role in PMNs migration.

  5. Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

    SciTech Connect

    Atluru, D.; Polam, S.; Atluru, S. (Kansas State Univ., Manhattan (United States)); Woloschak, G.E. (Argonne National Lab., IL (United States))

    1990-01-01

    Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, the authors examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-simulated ({sup 3}H)thymidine uptake, IL-2production, and IL-2R expression in a dose-related manner. Further, they found H-7 inhibited T-cell proliferation, IL-2 production, and IL-2mRNA from PHA plus PMA-stimulated cultures. They also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. The results demonstrate that PKC activation is one major pathway through which T-cells become activated.

  6. Direct and rapid electrochemical biosensing of the human interleukin-2 DNA in unpurified polymerase chain reaction (PCR)-amplified real samples

    Microsoft Academic Search

    Mohammad Hossein Pournaghi-Azar; Esmaeel Alipour; Sepideh Zununi; Haleh Froohandeh; Mohammad Saeid Hejazi

    2008-01-01

    Electrochemical detection of polymerase chain reaction (PCR)-amplified human interleukin-2 (IL-2) coding DNA sample (399bp size) without any purification and pre-treatment is described. To achieve this goal, a sensor was made by immobilization of a 20-mer oligonucleotide (chIL-2) as the probe on the pencil graphite electrode (PGE). This probe is related to the antisense strand of human interleukin-2 gene. The results

  7. Murine interleukin-2 generates glycogen-rich and mucus-secreting NK cells.

    PubMed Central

    Ginsburg, H; Ben-David, E; Kinarty, A; Rofolovitch, M; Amitay, M; Chriqui, E; Kedar, E; Davidson, S

    1983-01-01

    Colonies of cells termed 'giant granular leucocytes' (GGL) displaying natural killer (NK) activity were generated in cell culture. The prominent feature of these cells was the formation of large cytoplasmic pool--the 'theca'--filled up with glycogen. This was demonstrated by the strong positive red staining of the theca with periodic acid Schiff reagent (PAS) which was abolished by prior treatment with amylase. Two different procedures were employed for obtaining colonies of NK-GGL. In the first, mice were injected either with killed Corynebacterium parvum or with killed Bordetella pertusis preparations and their mesenteric lymph-node cells were grown on syngeneic X-irradiated embryonic skin fibroblast monolayers. At the foci of GGL formation the fibroblasts were killed and the cleared areas thus formed were populated by adherent GGL. In the second procedure, supernates from rat or mouse spleen cultures stimulated with concanavalin A (Con A)--Interleukin-2 (IL-2)--were added to cultures of spleen and lymph-node cells prepared from either ordinary or from athymic nude mice. Richest GGL populations developed when rat IL-2 was added to cells of nude mice. Mouse IL-2 was less consistent. With nude mouse cells it stimulated, either mast cells or GGL, or both; rat IL-2 did not stimulate mast-cell differentiation in nude mouse cultures. In contrast, supernates from lymph-node cell cultures prepared from mice infected with Schistosoma mansoni. Mucosal mast cell-stimulating factor (MMSF) stimulated the formation of colonies of mast cells but not GGL. When MMSF was added as late as 23 days, colonies of young mast cells appeared and mast cells progressively increased in number. When rat IL-2 was added to such mature mast-cell cultures on the 30th day, colonies of cytolytic-GGL appeared. These observations indicate that precursors of mast cells and GGL persist in the cultures and preserve their potential to be stimulated by T-cell factors. GGL-NK cells developed on monolayers prepared from whole embryos released substance that displayed morphology and staining characteristic of mucus. Evidence gathered from in-vitro and in-vivo studies links the in-vitro GGL-NK cells to motile cells that inhabit the mucosal epithelium. Based on the observations, a hypothesis on the function of NK cytotoxicity is brought forward. It proposes the replacement of ordinary epithelial cells, which are killed during a proliferative and differentiative response of other cells at the onset of an infection course. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6192079

  8. Interleukin 2 gene therapy for prostate cancer: phase I clinical trial and basic biology.

    PubMed

    Belldegrun, A; Tso, C L; Zisman, A; Naitoh, J; Said, J; Pantuck, A J; Hinkel, A; deKernion, J; Figlin, R

    2001-05-20

    Twenty-four patients with locally advanced prostate cancer (CaP) were enrolled in a phase I clinical trial using gene-based immunotherapy. A functional DNA-lipid complex encoding the interleukin 2 (IL-2) gene (Leuvectin; Vical, San Diego, CA) was administered intraprostatically into the hypoecogenic tumor lesion, using transrectal ultrasound guidance. Two groups of patients having locally advanced tumors were enrolled to receive a treatment regimen composed of two serial intraprostatic injections of the IL-2 gene agent administered 1 week apart. The first groups of patients included radical prostatectomy candidates who subsequently underwent surgery after the completion of the treatment regimen. The second group consisted of patients who had failed a prior therapy. Prostate specimens of the treated areas were attained after treatment and compared with the transrectal biopsies performed at baseline to assess for any responses. IL-2 gene therapy was well tolerated, with no grade 3 or 4 toxic reactions occurring. The most commonly reported symptoms were mild hematuria, transient rectal bleeding, and perineal discomfort that are likely attributable to the injection itself. During the entire course of treatment, there were no significant changes in American Urologic Association (AUA) symptom scores, in hematologic disturbances, electrolyte imbalances, or hepatic functions. Evidence of systemic immune activation was observed after IL-2 gene therapy, based on an increase in the intensity of T cell infiltration seen on immunohistochemical analysis of tissue samples from the injected tumor sites, and based on increased proliferation rates of peripheral blood lymphocytes that were cocultured with patient serum collected after treatment. Furthermore, transient decreases in serum prostate-specific antigen (PSA) (responders) were seen in 16 of 24 patients (67%) on day 1. Fourteen of the patients persisted in this decrease to day 8 (58%). In eight patients the PSA level rose (nonresponders). More patients (9 to 10) in the group that failed prior therapy responded to the IL-2 gene injections (chi-square test, p = 0.04), and 6 of the 9 also had lower than baseline PSA levels at week 10 after treatment. To the best of our knowledge, this is the first clinical study of its kind aimed at exploring the role of IL-2-based gene therapy in CaP patients. This phase I trial demonstrated the safety of intraprostatic Leuvectin injection, with transient PSA-based responses seen after therapy. PMID:11387054

  9. Antitumor effects of polyethylene glycol-modified recombinant human interleukin-2 on mouse uterine cervical carcinoma in vivo

    Microsoft Academic Search

    Lifu Wang; Yuxin Wu; Yongping Zhang; Wei Tang; Xinyuan Liu

    1997-01-01

    Polyethylene glycol (PEG-8000)-modified recombinant human interleukin-2 (PEG-rIL-2) is a cytokine with prolonged circulatory\\u000a half-life. In this paper, the antitumor effects of PEG-rIL-2 against mouse uterine cervical carcinoma (U14) transplanted intraperitoneally or subcutaneously is reported. PEG-rIL-2 at different doses was administered intraperitoneally.\\u000a The results showed that PEG-rIL-2 (4500 IU, i.p., QD×5) prolonged survival time of mice bearing ascites tumor as compared

  10. Induction of hepatitis by JNK-mediated expression of TNF?

    PubMed Central

    Das, Madhumita; Sabio, Guadalupe; Jiang, Feng; Rincón, Mercedes; Flavell, Richard A.; Davis, Roger J.

    2009-01-01

    The cJun NH2-terminal kinase (JNK) signaling pathway has been implicated in the development of tumor necrosis factor (TNF) -dependent hepatitis. Indeed, JNK may play a critical role in hepatocytes during TNF-stimulated cell death in vivo. To test this hypothesis, we examined the phenotype of mice with compound disruption of the Jnk1 and Jnk2 genes. Mice with loss of JNK1/2 expression in hepatocytes exhibited no defects in the development of hepatitis compared with control mice. In contrast, mice with loss of JNK1/2 in the hematopoietic compartment exhibited a profound defect in hepatitis that was associated with markedly reduced expression of TNF?. Together, these data indicate that JNK is required for TNF? expression, but JNK is not required for TNF?-stimulated death of hepatocytes. Indeed, TNF?-induced similar hepatic damage in mice with hepatocyte-specific JNK1/2-deficiency and control mice. These observations confirm a role for JNK in the development of hepatitis, but identify hematopoietic cells as the site of the essential function of JNK. PMID:19167327

  11. [The STAT5 signaling in the expression of alpha-subunit of interleukin-2 receptor in human blood lymphocytes].

    PubMed

    Mitiushova, E V; Shatrova, A N; Zenin, V V; Aksenov, N D; Marakhova, I I

    2013-01-01

    The comparative study of the STAT3 and STAT5 activity (as assessed by tyrosine phosphorylation level) and the expression of a ?-subunit of interleukin-2 receptor (as examined by cytophotometric evaluation of the number of CD25+ cells) during the phytohemagglutinin (PHA)-induced proliferation of human blood lymphocytes (HBL) have been made. It has been revealed that the level of STAT3 phosphorylation is high in both res ting and competent HBL and remains unchanged in the presence of PHA or interleukin-2 (IL-2). In contrast to STAT3, phosphorylation of STAT5 was not seen in both resting and competent HBL. We observed phosphorylation of STAT5 no earlier than 5 h after PHA stimulation and the maximum phosphorylation was detected following 24 h. Exogenous IL-2 induced high level of STAT5 phosphorylation in the competent HBL as early as at 30 min and this level of STAT5 phosphorylation kept in the next 24-48 h. The correlation between alterations in tyrosine phosphorylation level of STAT5 and the expression of CD25 has been established. WHI-P131, an inhibitor of JAK3 kinase, prevents STAT5 activation, cell surface expression of CD25 and lymphocyte proliferation. It has been concluded that JAK3/STAT5 signaling via IL-2 receptor is necessary to maintain the long-term expression of the high-affinity ???(c)-receptor of IL-2 and optimal proliferation of HBL. PMID:25509109

  12. Ability of virus SV40 T-antigen to replace interleukin-2, a specific growth factor of T-lymphocytes

    SciTech Connect

    Kulikov, V.V.; Shlyankevich, M.A.; Drize, O.B.; Shapot, V.S.

    1986-12-10

    The entry of T-lymphocytes into the DNA-synthesizing phase is determined by three successive signals: an antigenic effect, interleukin-2 - a specific growth factor of T-lymphocytes - and by nonspecific serum growth factors, primarily transferrin. This system was used for a study of the effect of the T-antigen of virus SV 40 on the mitotic cycle. Purified T-antigen was injected alternately into T-lymphocytes using vesicles from erythrocyte ghosts instead of one of the control signals. It was established that the T-antigen cannot replace the antigenic effect, but is capable of replacing the specific growth factor interleukin-2. However, both normally proliferating T-lymphocytes and T-lymphocytes induced to divide have an absolute requirement for transferrin and probably for other nonspecific growth factors. It is suggested that the polymorphism of tumors caused by papovaviruses is determined by the ability of their early proteins to imitate the action on cells of growth factors specific for them.

  13. Expression levels and genetic polymorphisms of interleukin-2 and interleukin-10 as biomarkers of Graves’ disease

    PubMed Central

    LIANG, CUIGE; DU, WENHUA; DONG, QINGYU; LIU, XIAOMENG; LI, WENXIA; WANG, YUELI; GAO, GUANQI

    2015-01-01

    The aim of the present study was to determine whether the expression levels of interleukin (IL)-2 and IL-10 may be used as biological markers in Graves’ disease (GD) patients. A total of 256 individuals, including 118 GD patients and 138 healthy individuals, were enrolled into the study. Blood samples were collected from each patient and healthy individual, which were then subjected to enzyme-linked immunosorbent assay (ELISA). Total RNA and total proteins were determined using reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. In addition, restriction fragment length polymorphism (RFLP) analysis was performed to detect the presence of genetic polymorphisms. The ELISA results indicated that the IL-2 and IL-10 serum levels in the GD patients were increased by ~5.2 and ~7-fold when compared with the levels in the healthy controls. The results of RT-qPCR indicated that the mRNA expression levels of IL-2 and IL-10 were upregulated in the GD patients when compared with the healthy controls. Furthermore, the western blot analysis results revealed that the protein expression levels of IL-2 and IL-10 were significantly increased in the GD patients. RFLP analysis indicated that the increased number of GG single nucleotide polymorphisms (SNPs) in the GD group were detected in the ?330 locus of the IL-2 promoter and the ?1082 locus of the IL-10 promoter. In addition, the results indicated that the relatively high rates of homozygous GG SNPs (IL-2 ?330T/G and IL-10 ?1082A/G polymorphisms) on the alleles may be associated with the incidence of GD. The serum, mRNA and protein expression levels of IL-2 and IL-10 were significantly increased in GD patients when compared with the levels in the healthy controls. In conclusion, the expression levels and genetic polymorphisms of IL-2 and IL-10 may be potential biomarkers for the incidence of Graves’ disease in the population studied. PMID:25667655

  14. Diacylglycerol kinase-dependent formation of phosphatidic acid molecular species during interleukin-2 activation in CTLL-2 T-lymphocytes

    PubMed Central

    Mizuno, Satoru; Sakai, Hiromichi; Saito, Masafumi; Kado, Sayaka; Sakane, Fumio

    2012-01-01

    Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species. PMID:23650609

  15. Diacylglycerol kinase-dependent formation of phosphatidic acid molecular species during interleukin-2 activation in CTLL-2 T-lymphocytes.

    PubMed

    Mizuno, Satoru; Sakai, Hiromichi; Saito, Masafumi; Kado, Sayaka; Sakane, Fumio

    2012-01-01

    Although effective liquid chromatography (LC)/mass spectrometry (MS) methods enabling the separation of phospholipid molecular species have been developed, there are still problems with an intracellular signaling molecule, phosphatidic acid (PA). In this study, we optimized LC/MS conditions to improve the quantitative detection of PA molecular species from a cellular lipid mixture. Using the newly developed LC/MS method, we showed that stimulation of CTLL-2 murine T-lymphocytes by interleukin-2 (IL-2) induced a significant increase of 36:1-, 36:2-, 40:5- and 40:6-diacyl-PA. A diacylglycerol kinase (DGK) inhibitor, R59949, attenuated the increase of 36:1-, 40:5-, 40:6-diacyl-PA, suggesting that DGK IL-2-dependently and selectively generated these diacyl-PA species. PMID:23650609

  16. The stimulation of EL-4 cells to produce interleukin-2 and its potential use in immunocytotoxicity testing

    SciTech Connect

    Lasek, W.; Steer, S.; Clothier, R.; Balls, M. (Univ. of Nottingham Medical School, Queen's Medical Centre (England))

    1989-01-01

    The ability of EL-4 thymoma cells to produce interleukin-2 (IL-2) following exposure to phorbol-12-myristate 13-acetate (PMA) and Concanavalin A (Con A) has been studied in vitro using medium containing either 10% or 1% fetal calf serum (FCS). The potent stimulatory effect of PMA on IL-2 production by EL-4 cells has been confirmed by measuring 3H-thymidine incorporation by the IL-2-dependent T cell line, CTLL-2, in the presence of conditioned medium (CM) from stimulated cultures. EL-4 cells produced several times more IL-2 when cultured in medium containing 10% FCS than when only 1% FCS was present. Added together, PMA and Con A acted synergistically in some EL-4 cell cultures. The ability of E:-4 cells to produce IL-2 was maintained after further incubation without stimulants. CM with IL-2 activity from stimulated EL-4 cells could prove useful in immunotoxicity testing.

  17. Intratumoral Injection of an Adenovirus Expressing Interleukin 2 Induces Regression and Immunity in a Murine Breast Cancer Model

    NASA Astrophysics Data System (ADS)

    Addison, Christina L.; Braciak, Todd; Ralston, Robert; Muller, William J.; Gauldie, Jack; Graham, Frank L.

    1995-08-01

    Rodent tumor cells engineered to secrete cytokines such as interleukin 2 (IL-2) or IL-4 are rejected by syngeneic recipients due to an enhanced antitumor host immune response. An adenovirus vector (AdCAIL-2) containing the human IL-2 gene has been constructed and shown to direct secretion of high levels of human IL-2 in infected tumor cells. AdCAIL-2 induces regression of tumors in a transgenic mouse model of mammary adenocarcinoma following intratumoral injection. Elimination of existing tumors in this way results in immunity against a second challenge with tumor cells. These findings suggest that adenovirus vectors expressing cytokines may form the basis for highly effective immunotherapies of human cancers.

  18. NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating function

    SciTech Connect

    Zhao Guohua [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Shi Lingfang [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Qiu Daoming [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Hu Hong [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States); Kao, Peter N. [Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236 (United States)]. E-mail: peterkao@stanford.edu

    2005-05-01

    NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthesy, J. Biol. Chem. 269 (1994) 20691-20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2.

  19. Skin tumor responsiveness to interleukin-2 treatment and CD8 Foxp3+ T cell expansion in an immunocompetent mouse model.

    PubMed

    Foureau, David M; McKillop, Iain H; Jones, Chase P; Amin, Asim; White, Richard L; Salo, Jonathan C

    2011-09-01

    Recombinant human interleukin-2 (rhIL-2) therapy is approved for treating patients with advanced melanoma yet significant responses are observed in only 10-15% of patients. Interleukin-2 induces Foxp3 expression in activated human CD8 T cells in vitro and expands circulating CD8 Foxp3+ T cells in melanoma patients. Employing IL-2 responsive (B16-F1, B16-BL6, JB/MS, MCA-205) and nonresponsive (JB/RH, B16-F10) subcutaneous tumor mouse models, we evaluated CD8 Foxp3+ T cell distribution and changes in response to rhIL-2 (50,000 U, i.p. or s.q., twice daily for 5 days). In tumor-free mice and subcutaneous tumor-bearing mouse models, CD8 Foxp3+ T cells were a rare but naturally occurring cell subset. Primarily located in skin-draining lymph nodes, CD8 Foxp3+ T cells expressed both activated T cell (CD28(+), CD44(+)) and Treg (CTLA4(+), PD1(lo/var), NKG2A(+/var)) markers. Following treatment with rhIL-2, a dramatic increase in CD8 Foxp3+ T cell prevalence was observed in the circulation and tumor-draining lymph nodes (TD.LNs) of animals bearing IL-2 nonresponsive tumors, while no significant changes were observed in the circulation and TD.LNs of animals bearing IL-2 responsive tumors. These findings suggest expansion of CD8 Foxp3+ T cell population in response to rhIL-2 treatment may serve as an early marker for tumor responsiveness to immunotherapy in an immune competent model. Additionally, these data may provide insight to predict response in patients with melanoma undergoing rhIL-2 treatment. PMID:21638127

  20. Effect of vanadium on the subset and proliferation of peripheral blood T cells, and serum interleukin-2 content in broilers.

    PubMed

    Cui, Wei; Cui, Heng-Min; Peng, Xi; Zuo, Zhicai; Liu, Xiaodong; Wu, Bangyuan

    2011-06-01

    The purpose of this 42-day study was to investigate the effects of dietary excess vanadium on immune function by determining changes of the subsets and proliferation function of peripheral blood T cells. Four hundred twenty 1-day-old avian broilers were divided into six groups and fed on a corn-soybean basal diet as control diet or the same diet amended to contain 5, 15, 30, 45, and 60 ppm vanadium supplied as ammonium metavanadate. In comparison with those of the control group, the percentages of CD (3) (+) , CD (3) (+) CD (4) (+) , and CD (3) (+) CD (8) (+) were decreased in 45 and 60 ppm groups from 14 to 42 days of age, and the percentages of CD (3) (+) and CD (3) (+) CD (4) (+) were increased in 5 ppm group at 42 days of age. The CD (4) (+) /CD (8) (+) ratio was increased in 45 and 60 ppm groups at 28 days of age. Meanwhile, the proliferation function of peripheral blood T cell were decreased in 30, 45, and 60 ppm groups from 14 to 42 days of age. Also, the serum interleukin-2 contents were decreased in 45 and 60 ppm groups from 14 to 42 days of age and increased in 5 ppm group at 28 days of age. Histopathologically, hypocellularity appeared in the thymus in 45 and 60 ppm groups. It was concluded that dietary vanadium in excess of 30 ppm reduced the percentages of peripheral blood T-cell subsets and the proliferation function and serum interleukin-2 contents. The cellular immune function was finally impaired in broilers. PMID:20532669

  1. mRNA

    PubMed Central

    Van Lint, Sandra; Heirman, Carlo; Thielemans, Kris; Breckpot, Karine

    2013-01-01

    Two decades ago, mRNA became the focus of research in molecular medicine and was proposed as an active pharmaceutical ingredient for the therapy of cancer. In this regard, mRNA has been mainly used for ex vivo modification of antigen-presenting cells (APCs), such as dendritic cells (DCs). This vaccination strategy has proven to be safe, well tolerated and capable of inducing tumor antigen-specific immune responses. Recently, the direct application of mRNA for in situ modification of APCs, hence immunization was shown to be feasible and at least as effective as DC-based immunization in pre-clinical models. It is believed that application of mRNA as an off-the-shelf vaccine represents an important step in the development of future cancer immunotherapeutic strategies. Here, we will discuss the use of ex vivo mRNA-modified DCs and “naked mRNA” for cancer immunotherapy focusing on parameters such as the employed DC subtype, DC activation stimulus and route of immunization. In addition, we will provide an overview on the clinical trials published so far, trying to link their outcome to the aforementioned parameters. PMID:23291946

  2. Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek's disease virus but fails to increase protection against virulent challenge

    Microsoft Academic Search

    I. Tarpey; P. J. Davis; P. Sondermeijer; C. van Geffen; I. Verstegen; V. E. J. C. Schijns; Jill Kolodsick; R. Sundick

    2007-01-01

    As Marek's disease virus continues to evolve towards greater virulence, more efficacious vaccines will be required in the future. We expressed chicken interleukin-2 (IL-2) from a turkey herpesvirus (HVT) in an attempt to increase the efficacy of HVT as a vaccine against Marek's disease. The recombinant IL-2\\/HVT was safe for in ovo vaccination, although it replicated less in the birds

  3. Increased excretion of soluble interleukin 2 receptors and free light chain immunoglobulins in the urine of patients with active lupus nephritis

    Microsoft Academic Search

    C Y Tsai; T H Wu; K H Sun; W M Lin; C L Yu

    1992-01-01

    Samples of protein from the urine of 23 patients with lupus nephropathy and 15 patients with proteinuria who did not have systemic lupus erythematosus (SLE) were studied for the presence of cytokines, soluble interleukin 2 receptors (sIL-2R), and free light chain immunoglobulins. The patients with lupus nephropathy were divided into two groups with active (nephritis) and inactive inflammation (nephrosis) based

  4. Comparison of the radiosensitivity of interleukin-2 production between species, between tissues, and between young and old individuals

    SciTech Connect

    Peterson, W.J.; Akagawa, T.; Anderson, D.G.; Makinodan, T.

    1985-04-01

    The radiosensitivity of interleukin-2 (IL-2) production was assessed of (a) peripheral blood mononuclear cells (PBMC) of young humans, dogs, and mice (C57BL/6); (b) PBMC and splenic cells of young mice; and (c) PBMC of young and old humans and the splenic cells of young and old mice. The results indicate that (a) large differences in radiosensitivity exist between the PBMC of humans, dogs, and mice (e.g., the radiation doses which resulted in 37% remaining IL-2 activity (D37) of human, dog, and mouse PBMC were 3771, greater than 10,000, and 1398 rads, respectively); (b) only a small difference exists between the PBMC and splenic cells of mice; and (c) no difference exists between the PBMC of young and old humans and between splenic cells of young and old mice. Topological abnormalities, as judged by scanning electron microscopic analysis, could not be detected in dog PBMC after their exposure to 1800 rads, but could be detected in mouse PBMC after their exposure to 400 rads.

  5. Suppressor cells induced by BCG release non-specific factors in vitro which inhibit DNA synthesis and interleukin-2 production.

    PubMed Central

    Colizzi, V; Ferluga, J; Garreau, F; Malkovsky, M; Asherson, G L

    1984-01-01

    Mice injected intravenously with a high dose (5 X 10(7) ) of BCG fail to develop delayed hypersensitivity to BCG and are described as anergic or unresponsive. Spleen cells from these mice release factors on culture which suppress DNA synthesis induced by concanavalin A in vitro. Cell separation experiments showed that both macrophages and T cells produce inhibitory factors. However, the macrophage factor has a molecular weight 10,000-30,000, while the T cell factor has a molecular weight of 50,000-70,000. Further evidence that these two factors are different is provided by the kinetics of their action. The T cell factor only acts when given within 12 hr of stimulation with concanavalin A, while the macrophage factor acts even when given at 48 hr. In the case of the T cell factor, the inhibition of DNA synthesis may be attributed to its ability to block the interleukin-2 production induced by Con A. As similar T cell and macrophage factors are produced in mice responding to simple chemically reactive haptenes (contact sensitizers), it is possible that a similar suppressor circuit is involved in the control of the response to contact sensitizers and in the production of unresponsiveness (anergy) in mice given large doses of BCG. PMID:6228520

  6. Protective efficacy of a Treponema pallidum Gpd DNA vaccine vectored by chitosan nanoparticles and fused with interleukin-2.

    PubMed

    Zhao, Feijun; Wang, Shiping; Zhang, Xiaohong; Gu, Weiming; Yu, Jian; Liu, Shuangquan; Zeng, Tiebing; Zhang, Yuejun; Wu, Yimou

    2012-02-01

    In the present study, immunomodulatory responses of a DNA vaccine constructed by fusing Treponema pallidum (Tp) glycerophosphodiester phosphodiesterase (Gpd) to interleukin-2 (IL-2) and using chitosan (CS) nanoparticles as vectors were investigated. New Zealand white rabbits were immunized by intramuscular inoculation of control DNAs, Tp Gpd DNA vaccine, or Gpd-IL-2 fusion DNA vaccine, which were vectored by CS nanoparticles. Levels of the anti-Gpd antibodies and levels of IL-2 and interferon-? in rabbits were increased upon inoculation of Gpd-IL-2 fusion DNA vaccine, when compared with the inoculation with Gpd DNA vaccine, with CS vectoring increasing the effects. The Gpd-IL-2 fusion DNA vaccine efficiently enhanced the antigen-specific lymphocyte proliferative response. When the rabbits were challenged intradermally with 10(5) Tp (Nichols) spirochetes, the Gpd-IL-2 fusion DNA vaccine conferred better protection than the Gpd DNA vaccine (P < 0.05), as characterized by lower detectable amounts of dark field positive lesions (17.5%), lower ulcerative lesion scores (15%), and faster recovery. Individuals treated with the Tp Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles had the lowest amounts of dark field positive lesions (10%) and ulcerations (5%) observed and the fastest recovery (42 days). These results indicate that the Gpd-IL-2 fusion DNA vaccine vectored by CS nanoparticles can efficiently induce Th1-dominant immune responses, improve protective efficacy against Tp spirochete infection, and effectively attenuate development of syphilitic lesions. PMID:22260167

  7. Transgenic tobacco plants as production platform for biologically active human interleukin 2 and its fusion with proteinase inhibitors.

    PubMed

    Redkiewicz, Patrycja; Wi?syk, Aneta; Góra-Sochacka, Anna; Sirko, Agnieszka

    2012-09-01

    Transgenic plants offer a low-cost approach for the production of pharmaceutically important and commercially valuable recombinant proteins. Our studies were focused on the plant-based production of human interleukin 2 (hIL-2) and its fusion with proteinase inhibitors, either SPI2 from Galleria mellonella or CMTI from Cucurbita maxima. Finally, five plant expression cassettes were obtained. Three of them contained the single cDNA encoding CMTI?I, SPI2 and hIL-2, respectively, while two of them contained the translational fusion, SPI2::hIL-2 and CMTI::hIL-2. In all cases, the transgenes were controlled by the RbcS1 promoter and terminator and the recombinant proteins were targeted to the endoplasmic reticulum. After tobacco transformation, five groups of transgenic plants were obtained and analysed. The level of recombinant proteins was estimated either by Western blot or by ELISA. The biological activity of plant-produced hIL-2 alone or in a fusion with SPI2 or CMTI was confirmed using the mammalian cells proliferation assay. The activities of proteinase inhibitors were confirmed in proteolysis assay using azocoll as a substrate. The usefulness of using proteinase inhibitor CMTI I in a fusion with hIL-2 as a protective agent against trypsin digestion was demonstrated. PMID:22564275

  8. Molecular dissection of the interactions of an antitumor interleukin-2-derived mutein on a phage display-based platform.

    PubMed

    Rojas, Gertrudis; Carmenate, Tania; Leon, Kalet

    2015-04-01

    A mutein with stronger antitumor activity and lower toxicity than wild-type human interleukin-2 (IL-2) has been recently described. The rationale behind its design was to reinforce the immunostimulatory potential through the introduction of four mutations that would selectively disrupt the interaction with the IL-2 receptor alpha chain (thought to be critical for both IL-2-driven expansion of T regulatory cells and IL-2-mediated toxic effects). Despite the successful results of the mutein in several tumor models, characterization of its interactions was still to be performed. The current work, based on phage display of IL-2-derived variants, showed the individual contribution of each mutation to the impairment of alpha chain binding. A more sensitive assay, based on the ability of phage-displayed IL-2 variants to induce proliferation of the IL-2-dependent CTLL-2 cell line, revealed differences between the mutated variants. The results validated the mutein design, highlighting the importance of the combined effects of the four mutations. The developed phage display-based platform is robust and sensitive, allows a fast comparative evaluation of multiple variants, and could be broadly used to engineer IL-2 and related cytokines, accelerating the development of cytokine-derived therapeutics. PMID:25683569

  9. Treatment of Walker ascites tumor cells by combination of photodynamic therapy with cyclophosphamide and interleukin-2 entrapped in liposomes

    NASA Astrophysics Data System (ADS)

    Dima, Vasile F.; Ionescu, Mircea D.; Balotescu, Carmen; Dima, V. S.

    2003-12-01

    The purpose of this study was to investigate the beneficial and adverse local effects of PDT associated with chemoimmunotherapy on rats bearing Walker ascites tumor cells. Experiments were performed on five batches of Wistar inbred rats with ascites tumor cells receiving intraperitoneally PDT (Photofrin II and 18 hrs later HeNe laser irradiation); Cyclophosphamide (CY); interleukin-2 (IL-2) or associated therapy (PDT+CY+IL-2). The control batch consisted of untreated rats (HBSS). The following results were noticed: (a) sole administration of PDT, IL-2 or CY reduced tumor growth, gave survival rates between 28.4 and 56.5% and cure rates ranging from 12.4 to 33.3%; (b) combined therapy (PDT+CY+IL-2) decreased tumor growth, increased survival rates (88.5%) and cure rates were 73.1% forty-two days post-transplantation. Summing up, in this study we noticed that PDT associated with chemoimmunotherapy reduced mortality as well as tumor volumes and increased cure rates in rats with ascites tumor cells. This approach points to the need for further evaluation in patients with peritoneal malignancies.

  10. [Analysis of the molecular characteristics and cloning of full-length coding sequence of interleukin-2 in tree shrews].

    PubMed

    Huang, Xiao-Yan; Li, Ming-Li; Xu, Juan; Gao, Yue-Dong; Wang, Wen-Guang; Yin, An-Guo; Li, Xiao-Fei; Sun, Xiao-Mei; Xia, Xue-Shan; Dai, Jie-Jie

    2013-04-01

    While the tree shrew (Tupaia belangeri chinensis) is an excellent animal model for studying the mechanisms of human diseases, but few studies examine interleukin-2 (IL-2), an important immune factor in disease model evaluation. In this study, a 465 bp of the full-length IL-2 cDNA encoding sequence was cloned from the RNA of tree shrew spleen lymphocytes, which were then cultivated and stimulated with ConA (concanavalin). Clustal W 2.0 was used to compare and analyze the sequence and molecular characteristics, and establish the similarity of the overall structure of IL-2 between tree shrews and other mammals. The homology of the IL-2 nucleotide sequence between tree shrews and humans was 93%, and the amino acid homology was 80%. The phylogenetic tree results, derived through the Neighbour-Joining method using MEGA5.0, indicated a close genetic relationship between tree shrews, Homo sapiens, and Macaca mulatta. The three-dimensional structure analysis showed that the surface charges in most regions of tree shrew IL-2 were similar to between tree shrews and humans; however, the N-glycosylation sites and local structures were different, which may affect antibody binding. These results provide a fundamental basis for the future study of IL-2 monoclonal antibody in tree shrews, thereby improving their utility as a model. PMID:23572362

  11. Effect of acute heat stress on adrenocorticotropic hormone, cortisol, interleukin-2, interleukin-12 and apoptosis gene expression in rats

    PubMed Central

    WANG, LI; LIU, FADONG; LUO, YAN; ZHU, LINGQIN; LI, GUANGHUA

    2015-01-01

    The aim of the present study was to investigate the effect of acute heat stress on the neuroendocrine and immunological function in rats. Male Sprague-Dawley rats were randomly divided into two groups and respectively exposed to heat (32°C) or to room temperature (24°C). After 7 days of heat exposure, the heat-stress rat model was established. The organ coefficients of the pituitary and adrenal glands were determined. The body temperature was measured by telemetry. The average contents of adrenocorticotropic hormone (ACTH), cortisol (Cor), interleukin-2 (IL-2) and IL-12 in serum were detected. The expression of apoptotic genes in the spleen was measured. The results showed that acute heat stress did not evidently affect the body temperature and body weight (P>0.05), but the exposure increased the organ coefficients of the pituitary and adrenal glands (P<0.05). Heat exposure significantly elevated the level of ACTH, Cor, IL-2 and IL-12 (P<0.05). The expression of caspase-3 and Bax were not changed significantly (P>0.05), while Bcl2 was reduced (P<0.05).

  12. Association of interleukin-2 and interferon-? production by blood mononuclear cells in infancy with parental allergy skin tests and with subsequent development of atopy

    Microsoft Academic Search

    Fernando D. Martinez; Debra A. Stern; Anne L. Wright; Catharine J. Holberg; Lynn M. Taussig; Marilyn Halonen

    1995-01-01

    The mechanisms regulating the onset of atopic sensitization in human beings are not yet fully clarified. We assessed the capacity of mitogen-stimulated umbilical and peripheral blood mononuclear cells to produce interferon-? (IFN-?) and interleukin-2 (IL-2) at birth and at 9 months of age in 159 infants. Mononuclear cell production of both IFN-? and IL-2 at 9 months, but not at

  13. Expansion of CD3 + CD56 + lymphocytes correlates with induction of cytotoxicity by interleukin-2 gene transfer in human breast tumor cultures

    Microsoft Academic Search

    David M. Euhus; Lucille Kimura; Bruce Arnold

    1997-01-01

    Background: Mice immunized with murine mammary carcinoma cells genetically engineered to secrete interleukin-2 (IL-2) are rendered resistant\\u000a to subsequent challenge with unmodified tumor cells, and in the case of mice bearing established tumors, the rate of development\\u000a of pulmonary metastases is reduced. Despite these encouraging animal results, little is known about the induction of antitumor\\u000a immunity by IL-2 gene transfer

  14. Defective T-cell response to PHA and mitogenic monoclonal antibodies in male homosexuals with acquired immunodeficiency syndrome and its in vitro correction by interleukin 2

    Microsoft Academic Search

    Niculae Ciobanu; Karl Welte; Gerard Kruger; Salvatore Venuta; Jonathan Gold; Stuart P. Feldman; Chang Yi Wang; Benjamin Koziner; Malcolm A. S. Moore; Bijan Safai; Roland Mertelsmann

    1983-01-01

    We studied the ability of phytohemagglutinin (PHA) and two anti-T-cell monoclonal antibodies OKT3 and Pan T2, to induce proliferation and interleukin 2 (IL2) production in peripheral blood lymphocytes (PBL) from 21 homosexual patients: 12 with Kaposi's sarcoma (KS), 4 with reactive lymphadenopathy, and 5 with opportunistic infections. All patients with KS and opportunistic infections had significantly lower mitogen-stimulated DNA synthesis,

  15. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes

    Microsoft Academic Search

    E. A. Grimm; A. Mazumder; H. Z. Zhang; S. A. Rosenberg

    1982-01-01

    Activation in lectin-free interleukin 2 (IL-2) containing supernatants of peripheral blood mononuclear leukocytes (PBL) from cancer patients or normal individuals resulted in expression of cytotoxicity toward 20 of 21 natural killer (NK)-resistant fresh solid tumor cells tested. Fresh solid tumor cells were resistant to NK-mediated lysis in 10 autologous patients' PBL-tumor interactions, and from 17 normal individuals tested against 13

  16. The effect of interleukin-2 on canine peripheral nerve sheath tumours after marginal surgical excision: a double-blind randomized study

    PubMed Central

    2013-01-01

    Background The objective of this study was to evaluate the effect on outcomes of intraoperative recombinant human interleukin-2 injection after surgical resection of peripheral nerve sheath tumours. In this double-blind trial, 40 patients due to undergo surgical excision (<5 mm margins) of presumed peripheral nerve sheath tumours were randomized to receive intraoperative injection of interleukin-2 or placebo into the wound bed. Results There were no significant differences in any variable investigated or in median survival between the two groups. The median recurrence free interval was 874 days (range 48–2141 days), The recurrence-free interval and overall survival time were significantly longer in dogs that undergone the primary surgery by a specialist-certified surgeon compared to a referring veterinarian regardless of whether additional adjunct therapy was given. Conclusion Overall, marginal excision of peripheral nerve sheath tumours in dogs resulted in a long survival time, but adjuvant treatment with recombinant human interleukin-2 (rhIL-2) did not provide a survival advantage. PMID:23927575

  17. Effects of subcutaneous interleukin-2 therapy on CD4 subsets and in vitro cytokine production in HIV+ subjects.

    PubMed Central

    De Paoli, P; Zanussi, S; Simonelli, C; Bortolin, M T; D'Andrea, M; Crepaldi, C; Talamini, R; Comar, M; Giacca, M; Tirelli, U

    1997-01-01

    HIV infection is characterized by the reduction of the CD4+, CD45RA+, CD26+, and CD28+ lymphocyte subsets and of the in vitro production of IL-2, IL-4, and interferon-gamma; on the contrary, chemokine production is usually increased. These abnormalities are only partially restored by antiretroviral chemotherapy. Therapy with interleukin-2 has been proposed to restore the functions of the immune system, but the mechanisms by which IL-2 exerts its activities are unknown. The aim of this study was to define the effects of rIL-2 administration on CD4+, CD45RA+, CD45R0+, and CD26+ lymphocytes and on the in vitro production of IL-2, IL-4, IL-10, IFN-gamma, RANTES, and sCD30 in HIV+ patients. 10 HIV+ patients with CD4 cell counts between 200 and 500 cells/mm3 were treated with six cycles of subcutaneous recombinant IL-2 administration, in combination with zidovudine and didanosine. This therapeutic regimen resulted in a remarkable increase in the number of CD4+ cells and in the prolonged reduction of the levels of viremia. CD45R01 cells were expanded during the first cycle of therapy, while CD45RA+/CD26+ cells predominated after the third cycle. At this time, the in vitro production of IL-2, IL-4, IFN-gamma, and sCD30 were significantly upregulated. These results demonstrate that rIL-2 in HIV+ patients induces the reconstitution of the CD4/CD45RA lymphocytes subtype. This expanded cell population recovered the ability to produce in vitro IL-2, IL-4, and IFN-gamma. These effects may be beneficial to HIV+ patients by improving their immune response to microorganisms or vaccines. PMID:9389737

  18. Tumour necrosis factor-alpha, interleukin-2 soluble receptor and different inflammatory parameters in patients with rheumatoid arthritis.

    PubMed Central

    Fröde, Tânia Silvia; Tenconi, Patrícia; Debiasi, Marilei Reynaud; Medeiros, Yara Santos

    2002-01-01

    BACKGROUND AND AIMS: Although the participation of cytokines in the pathogenesis of rheumatoid arthritis (RA) seems to be unequivocal, their relationship with current serum markers of this disease is not clear. The present study analyses whether there is any correlation between the levels of tumour necrosis factor-alpha (TNF-alpha), interleukin-2 soluble receptor (sIL-2R) and the concentrations of C-reactive protein (CRP), erythrocyte sedimentation rate (ESR) and beta(2)-microglobulin in a group of 21 patients with RA, all rheumatoid factor positive. METHODS: The levels of TNF-alpha and sIL-2R were analysed in association with other parameters of inflammation (ESR, CRP and beta(2)-microglobulin). RESULTS: In comparison with the control group, RA patients presented high median levels of both cytokines, TNF-alpha (6.4 pg/ml) and sIL-2R (56 pmol/L), as well as of ESR (34 mm/h), CRP (0.9 mg/dl) and beta(2)-microglobulin (1.6 mg/dl) (p < 0.01). However, only ESR levels in the RA group significantly differ from the control group (p < 0.01). No correlation was found between the inflammatory parameters. CONCLUSIONS: These results suggested that TNF-alpha and slL-2R levels are up-regulated in RA patients but did not significantly differ from the control group. Due to the chronic course of this disease, other inflammatory markers must be identified in order to provide early therapeutic strategies to these patients. PMID:12581498

  19. Phase I study of recombinant human interleukin-2 for pediatric malignancies: feasibility of outpatient therapy. A Pediatric Oncology Group Study.

    PubMed

    Pais, R C; Abdel-Mageed, A; Ghim, T T; Ode, D; Melendez, E; Kim, H S; Findley, H; Ragab, A H

    1992-08-01

    Published data indicate that when recombinant interleukin-2 (rIL-2) is administered to children as a 15-min i.v. bolus, doses of 18 x 10(6) IU/m2 are poorly tolerated, requiring intensive care unit (ICU) management of IL-2-induced hypotension. We administered rIL-2 as a 1- or 2-h i.v. infusion to 11 children with malignancies refractory to conventional therapy. IL-2 was given every Monday/Wednesday/Friday for 3 weeks. Four children received 12 x 10(6) IU/m2/dose, four received 18 x 10(6) IU/m2/dose, and three received 24 x 10(6) IU/m2/dose (1 Cetus Unit = 6 IU). Fever, chills, flushing, nausea, vomiting, transient weight gain, and oliguria were observed at all three dose levels (not dose-limiting toxicities). Cardiovascular toxicity was significantly reduced compared to the bolus regimen. Mild hypotension was observed at all three dose levels; however, there was no severe dose-limiting hypotension. Because of reduced cardiovascular toxicity, IL-2 was safely administered on an outpatient basis. This regimen induced marginal transient increases in natural killer cell activity and lymphokine-activated killer cell activity. No measurable clinical tumor response was observed in any of the 11 children. The maximum-tolerated dose has not been reached. This regimen allows for a considerable cost reduction (outpatient care instead of ICU care) and safety, making further clinical trials on the use of IL-2 in children more feasible. PMID:1504055

  20. Interleukin-2 causes an increase in saturated/monounsaturated phosphatidic acid derived from 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol.

    PubMed

    Jones, D R; Pettitt, T R; Sanjuán, M A; Mérida, I; Wakelam, M J

    1999-06-11

    Phosphatidic acid generation through activation of diacylglycerol kinase alpha has been implicated in interleukin-2-dependent T-lymphocyte proliferation. To investigate this lipid signaling in more detail, we characterized the molecular structures of the diradylglycerols and phosphatidic acids in the murine CTLL-2 T-cell line under both basal and stimulated conditions. In resting cells, 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol subtypes represented 44 and 55% of total diradylglycerol, respectively, and both showed a highly saturated profile containing primarily 16:0 and 18:1 fatty acids. 1-O-Alk-1'-enyl-2-acylglycerol represented 1-2% of total diradylglycerol. Interleukin-2 stimulation did not alter the molecular species profiles, however, it did selectively reduce total 1-O-alkyl-2-acylglycerol by over 50% at 15 min while only causing a 10% drop in 1,2-diacylglycerol. When radiolabeled CTLL-2 cells were challenged with interleukin-2, no change in the cellular content of phosphatidylcholine nor phosphatidylethanolamine was observed thereby ruling out phospholipase C activity as the source of diradylglycerol. In addition, interleukin-2 failed to stimulate de novo synthesis of diradylglycerol. Structural analysis revealed approximately equal amounts of 1,2-diacyl phosphatidic acid and 1-O-alkyl-2-acyl phosphatidic acid under resting conditions, both containing only saturated and monounsaturated fatty acids. After acute (2 and 15 min) interleukin-2 stimulation the total phosphatidic acid mass increased, almost entirely through the formation of 1-O-alkyl-2-acyl species. In vitro assays revealed that both 1,2-diacylglycerol and 1-O-alkyl-2-acylglycerol were substrates for 1,2-diacylglycerol kinase alpha, the major isoform in CTLL-2 cells, and that the lipid kinase activity was almost totally inhibited by R59949. In conclusion, this investigation shows that, in CTLL-2 cells, 1,2-diacylglycerol kinase alpha specifically phosphorylates a pre-existing pool of 1-O-alkyl-2-acylglycerol to form the intracellular messenger 1-O-alkyl-2-acyl phosphatidic acid. PMID:10358029

  1. The AP-1 site at -150 bp, but not the NF-kappa B site, is likely to represent the major target of protein kinase C in the interleukin 2 promoter

    PubMed Central

    1992-01-01

    Stimulation of T cells with antigen results in activation of several kinases, including protein kinase C (PKC), that may mediate the later induction of activation-related genes. We have examined the potential role of PKC in induction of the interleukin 2 (IL-2) gene in T cells stimulated through the T cell receptor/CD3 complex. We have previously shown that prolonged treatment of the untransformed T cell clone Ar-5 with phorbol esters results in downmodulation of the alpha and beta isozymes of PKC, and abrogates induction of IL-2 mRNA and protein. Here we show that phorbol ester treatment also abolishes induction of chloramphenicol acetyltransferase activity in Ar-5 cells transfected with a plasmid containing the IL-2 promoter linked to this reporter gene. The IL-2 promoter contains binding sites for nuclear factors including NFAT-1, Oct, NF-kappa B, and AP-1, which are all potentially sensitive to activation of PKC. We show that induction of a trimer of the NFAT and Oct sites is not sensitive to phorbol ester treatment, and that mutations in the NF-kappa B site have no effect on inducibility of the IL-2 promoter. In contrast, mutations in the AP-1 site located at - 150 bp almost completely abrogate induction of the IL-2 promoter, and appearance of an inducible nuclear factor binding to this site is sensitive to PKC depletion. Moreover, cotransfections with c-fos and c- jun expression plasmids markedly enhance induction of the IL-2 promoter in minimally stimulated T cells. Our results indicate that the AP-1 site at -150 bp represents a major, if not the only, site of PKC responsiveness in the IL-2 promoter. PMID:1740667

  2. Interleukin 2 induces the expression of CD45RO and the memory phenotype by CD45RA+ peripheral blood lymphocytes

    PubMed Central

    1994-01-01

    The CD45RA and CD45RO isoforms of the leukocyte common antigen identify functionally distinct "naive" and "memory" T cell subsets. While antigenic and mitogenic stimuli are known to initiate transition from the naive to memory state, little is known about the role of cytokines in this process. This report demonstrates that in vitro exposure of purified CD45RA+/CD45RO- peripheral blood lymphocytes (PBL) to interleukin 2 (IL-2) promotes their conversion to the CD45RA-/CD45RO+ phenotype. Conversion to CD45RO occurs for both the CD3+ and CD3-/CD56+ lymphocyte subsets, but occurs more rapidly, and at lower IL-2 concentrations, in the CD3-/CD56+ population. Expression of CD45RO was observed only in response to IL-2 and was not observed during long-term culture in IL-4, IL-6, or IL-7. We also examined the effect of IL-2 on the expression of adhesion molecules by T cells. The expression of CD2, CD11a, and CDw29 increased, and expression of Leu-8 (LAM-1) decreased, on cultured CD45RA+/CD45RO- cells after they converted to expression of CD45RO. In contrast, lymphocytes that remained CD45RA+/CD45RO- after 10 d in culture exhibited no change from their baseline adhesion molecule profile. Finally, to test the role of endogenous IL-2 during T cell activation we stimulated CD45RA+/CD45RO- PBL with immobilized anti-CD3 in the presence of neutralizing anti-IL-2 antibody and/or cyclosporin A. Both agents significantly reduced the expression of CD45RO and the effect of cyclosporin A was reversed by exogenous IL-2. We conclude that IL-2 promotes CD45RA+ cells to express the memory phenotype and is a mediator of CD45RO expression after stimulation of the T cell receptor/CD3 complex. PMID:8113679

  3. Recombinant Interleukin-2 in Patients Aged Younger Than 60 Years With Acute Myeloid Leukemia in First Complete Remission

    PubMed Central

    Kolitz, Jonathan E.; George, Stephen L.; Benson, Don M.; Maharry, Kati; Marcucci, Guido; Vij, Ravi; Powell, Bayard L.; Allen, Steven L.; DeAngelo, Daniel J.; Shea, Thomas C.; Stock, Wendy; Bakan, Courtney E.; Hars, Vera; Hoke, Eva; Bloomfield, Clara D.; Caligiuri, Michael A.; Larson, Richard A.

    2014-01-01

    BACKGROUND Recombinant interleukin-2 (rIL-2) induces cellular cytotoxicity against leukemia blasts. Patients with acute myeloid leukemia (AML) in first complete remission (CR) may harbor minimal residual disease that is susceptible to rIL-2–activated effector cells. METHODS In the Cancer and Leukemia Group B (CALGB) 19808 study, patients with AML in first CR were randomly assigned after all planned chemotherapy to receive a 90-day course of subcutaneously administered rIL-2 or no further therapy. The primary objective was to compare disease-free survival (DFS) between the 2 treatment arms. A total of 534 patients achieved a CR, 214 of whom were randomized. Six courses of low-dose daily rIL-2 were given for the expansion of cytotoxic effector cells, each followed by 3-day high-dose boluses given to trigger cytotoxicity against minimal residual disease. RESULTS On the protocol-specified intention-to-treat analysis, the hazards ratio for DFS was 0.75 (95% confidence interval, 0.52–1.09; P =.13); the 5-year DFS rate was 42% in the observation arm and 53% in the rIL-2 treatment arm. The hazards ratio for overall survival (OS) was 0.88 (95% confidence interval, 0.54–1.23; P =.34); the 5-year OS rate was 58% for the observation arm and 63% for the rIL-2 treatment arm. Twenty-five of the 107 patients randomized to treatment with rIL-2 either refused or were unable to initiate therapy and 30 patients did not complete their assigned therapy. However, significant toxicities were not commonly observed. The trial design did not anticipate the difficulties patients would encounter with protocol compliance. CONCLUSIONS The efficacy of immunotherapy with rIL-2 administered after intensive postremission treatment was not assessed as planned because of unexpected refusals by patients and/or their physicians to comply with protocol-directed therapy. Neither DFS nor OS was found to be significantly improved. PMID:24382782

  4. CD4+ CD45RA+ T cells modulate allergen-induced interleukin 2 responsiveness in human lymphocytes.

    PubMed

    Kawano, Y; Noma, T; Maeda, K; Yata, J

    1992-03-01

    Peripheral blood lymphocytes from nonallergic individuals acquired responsiveness to interleukin 2 (IL2) after stimulation with ovalbumin (OVA) or Dermatophagoides farinae (Df) antigens when they were pretreated with the CD45RA antibody, which has been shown to define the suppressor inducer subset of CD4+ cells and also to block its suppressor activity. The effect provided by the CD45RA antibody was lost if the lymphocytes had initially been activated with the OVA of Df antigens. The magnitude of the responses was comparable to the allergen-induced responses observed in OVA- or Df-sensitized lymphocytes from allergic patients. The pre-existing IL2 responsiveness in the patients was not increased by the CD45RA antibody pretreatment. However, the CD45RA antibody pretreatment gave rise to Df-induced IL2 responsiveness in the lymphocytes of the patients sensitized with OVA but not with Df; conversely, OVA-induced IL2 responsiveness was enhanced in Df- but not in OVA-sensitized lymphocytes. The CD45RA antibody apparently acts on CD4+ T cells, but not on CD8+ T cells, to induce the IL2 response. A further dissection of normal CD4+ T cells indicated that CD4+45RA- T cells preferentially respond to IL2 after stimulation with OVA or Df antigens. Since normal CD4+45RA+ T cells did not show antigen-induced IL2 responsiveness even after pretreatment with the CD45RA antibody, it is unlikely that the CD45RA antibody stimulates CD4+45RA+ T cells to become responsive to IL2 after antigenic challenge. Alternatively, CD4+45RA+ T cells may modulate the activity of CD4+45RA- T cells, which are potentially responsive to IL2 by antigenic stimulation and thus provide tolerance in nonallergic lymphocytes. Collectively, a defective suppressor activity of CD4+45RA+ T cells may exist in patients with hen-egg allergy and/or bronchial asthma, which may cause lymphocytes to be hyperreactive to OVA or Df antigens. PMID:1531787

  5. Adoptive immunotherapy of human pancreatic cancer with lymphokine-activated killer cells and interleukin-2 in a nude mouse model

    SciTech Connect

    Marincola, F.M.; Da Pozzo, L.F.; Drucker, B.J.; Holder, W.D. Jr. (Stanford Univ. School of Medicine, CA (USA))

    1990-11-01

    A pancreatic cancer cell line was grown in orthotopic and heterotopic positions in young Swiss/NIH nude mice, which were tested with adoptive immunotherapy. Mice were injected with 1 x 10(7) human cancer cells in the subcutaneous tissue and duodenal lobe of the pancreas. The mice were randomly divided into four groups: group IA (LAK + IL-2) (N = 25) received 2 X 10(7) human lymphokine-activated killer (LAK) cells from normal donors by tail vein injection followed by 10,000 units of human recombinant interleukin-2 (IL-2) given intraperitoneally every 12 hours for 28 days; group IB (IL-2) (N = 27) was given the same dose of IL-2 alone; group IC (RPMI-1640) (N = 18) received a placebo consisting of 1 ml of RPMI-1640 intraperitoneally every 12 hours; and group ID (LAK) (N = 14) received 2 X 10(7) LAK cells but no IL-2. Toxicity was significantly higher in group IB, with a mortality rate of 45.5% (10/22 animals) versus a 0% mortality (0/25) in group IA. None of the group IA or IB animals died of pancreatic cancer during the experiment. The animals that did not receive IL-2 died before 28 days in 14.2% of group IC and in 16.7% of group ID. The area under the growth curve of subcutaneous tumors during the course of treatment and the pancreatic tumor weight at the end of treatment were compared in each group. Subcutaneous tumors had a reduced rate of growth in group IA animals compared to all the other treatments. Pancreatic tumor growth was slowed in group IA. The animals treated with IL-2 alone (group IB) showed some slowing of tumor growth that was intermediate between group IA, group IC, and group ID. A similar experiment was done with irradiated (375 rad) mice. Nine nude mice with tumors were treated with LAK + IL-2 (group IIA), eight received IL-2 alone (group IIB), and seven received placebo (group IIC).

  6. Predictors of bacterial pneumonia in the Evaluation of Subcutaneous Interleukin-2 in a Randomized International Trial (ESPRIT)

    PubMed Central

    Pett, SL; Carey, C; Lin, E; Wentworth, D; Lazovski, J; Miró, JM; Gordin, F; Angus, B; Rodriguez-Barradas, M; Rubio, R; Tambussi, G; Cooper, DA; Emery, S

    2010-01-01

    Background and Objectives Bacterial pneumonia still contributes to morbidity/mortality in HIV-infection despite effective combination antiretroviral therapy (cART). ESPRIT, a trial of intermittent recombinant interleukin-2 (rIL-2) with cART vs.cART alone (control arm) in HIV-infected adults with CD4+?300 offered the opportunity to explore associations between bacterial pneumonia and rIL-2, a cytokine which increases some bacterial infections. Methods Baseline and time-updated factors associated with first-episode pneumonia on study were analysed using multivariate proportional hazards regression models. Smoking/pneumococcal vaccination history was not collected. Results IL-2 cycling was most intense in years 1-2. Over ?7 years, 93 IL-2 (rate 0.67/100PY) and 86 control (rate 0.63/100PY) patients experienced a pneumonia-event, (HR=1.06,95%CI=0.79,1.42,p=0.68). Median CD4+ prior to pneumonia was 570 (IL-2 arm) and 463cells/uL (control arm). Baseline risks for bacterial pneumonia included older age, IVDU, detectable HIV viral load (VL), previous recurrent pneumonia; Asian ethnicity was associated with decreased risk. Higher proximal VL (HR for 1 log10 higher VL=1.28,95%CI=1.11,1.47,p=<.001) was associated with increased risk; higher CD4+ prior to the event (HR per 100 cells higher=0.94,95%CI0.89,1.0,p=0.04) decreased risk. Compared to controls, the hazard for a pneumonia-event was higher if rIL-2 was received <180 days prior (HR=1.66,95%CI=1.07,2.60,p=0.02) vs.?180 days (HR=0.98,95%CI=0.70,1.37,p=0.9). Compared to the control group, pneumonia-risk in the IL-2 arm decreased over time with HRs of 1.41, 1.71, 1.16, 0.62 and 0.84 in years 1, 2, 3-4,5-6 and 7, respectively. Conclusions Bacterial pneumonia rates in cART-treated adults with moderate immunodeficiency are high. The mechanism of the association between bacterial pneumonia and recent IL-2 receipt and/or detectable HIV-viraemia deserves further exploration. PMID:20812949

  7. Lectin interactions with the Jurkat leukemic T-cell line: quantitative binding studies and interleukin-2 production

    SciTech Connect

    Dupuis, G.; Bastin, B.

    1988-03-01

    Phytohemagglutinin (PHA), concanavalin A (Con A), pea lectin, and wheat germ agglutinin (WGA) have been used to investigate their binding properties to Jurkat 77 6.8 leukemic human T cells and their ability to induce these cells to produce interleukin-2 (IL-2). Binding studies showed that the Jurkat cells fixed 0.82 +/- 0.11 microgram pea lectin, 2.02 +/- 0.17 micrograms Con A, 1.85 +/- 0.07 micrograms PHA and 8.88 +/- 0.61 micrograms WGA. Scatchard plots were linear, indicating that the binding process was homogeneous with respect to the binding constant. PHA and Con A bound with the highest affinity (Kass (apparent) approximately equal to 9 x 10(9) M-1), followed by pea lectin and WGA (Kass (apparent) approximately equal to 3 x 10(9) M-1). The number of lectin binding sites was in agreement with the results of saturation experiments. We also evaluated the effect of the presence of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the binding process. Results show that there were no gross alterations in the value of (apparent) Kass in the case of PHA and WGA. In contrast, the presence of TPA decreased the affinity of Con A and modified the Scatchard profile for pea lectin, which was curvilinear with a concavity turned upward. In this case, data were (apparent) K1 = 17.7 x 10(9) M-1 (high-affinity sites) and (apparent) K2 = 2.6 x 10(9) M-1 (low-affinity sites). The four lectins shared the ability to stimulate Jurkat 77 6.8 cells to secrete IL-2. Optimal lectin concentrations were 20 micrograms/ml (PHA) and 50 micrograms/ml (WGA and Con A). Pea lectin failed to display a dose-response relationship, and IL-2 production increased proportionally with lectin concentration. Con A was the most efficient stimulator (250 U/ml), followed by WGA (160 U/ml) and PHA (108 U/ml).

  8. An Anti-Interleukin-2 Receptor Drug Attenuates T- Helper 1 Lymphocytes-Mediated Inflammation in an Acute Model of Endotoxin-Induced Uveitis

    PubMed Central

    Navea, Amparo; Almansa, Inmaculada; Muriach, María; Bosch-Morell, Francisco

    2014-01-01

    The aim of the present study was to evaluate the anti-inflammatory efficacy of Daclizumab, an anti-interleukin-2 receptor drug, in an experimental uveitis model upon a subcutaneous injection of lipopolysaccharide into Lewis rats, a valuable model for ocular acute inflammatory processes. The integrity of the blood-aqueous barrier was assessed 24 h after endotoxin-induced uveitis by evaluating two parameters: cell count and protein concentration in aqueous humors. The histopathology of all the ocular structures (cornea, lens, sclera, choroid, retina, uvea, and anterior and posterior chambers) was also considered. Enzyme-linked immunosorbent assays of the aqueous humor samples were performed to quantify the levels of the different chemokine and cytokine proteins. Similarly, a biochemical analysis of oxidative stress-related markers was also assessed. The inflammation observed in the anterior chamber of the eyes when Daclizumab was administered with endotoxin was largely prevented since the aqueous humor protein concentration substantially lowered concomitantly with a significant reduction in the uveal and vitreous histopathological grading. Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon-?, also significantly reduced with related anti-oxidant systems recovery. Daclizumab treatment in endotoxin-induced uveitis reduced Th1 lymphocytes-related cytokines, such as Interleukin-2 and Interferon gamma, by about 60–70% and presented a preventive role in endotoxin-induced oxidative stress. This antioxidant protective effect of Daclizumab may be related to several of the observed Daclizumab effects in our study, including IL-6 cytokine regulatory properties and a substantial concomitant drop in INF?. Concurrently, Daclizumab treatment triggered a significant reduction in both the uveal histopathological grading and protein concentration in aqueous humors, but not in cellular infiltration. PMID:24595020

  9. Expression of chicken interleukin-2 by turkey herpesvirus increases the immune response against Marek's disease virus but fails to increase protection against virulent challenge.

    PubMed

    Tarpey, I; Davis, P J; Sondermeijer, P; van Geffen, C; Verstegen, I; Schijns, V E J C; Kolodsick, Jill; Sundick, R

    2007-02-01

    As Marek's disease virus continues to evolve towards greater virulence, more efficacious vaccines will be required in the future. We expressed chicken interleukin-2 (IL-2) from a turkey herpesvirus (HVT) in an attempt to increase the efficacy of HVT as a vaccine against Marek's disease. The recombinant IL-2/HVT was safe for in ovo vaccination, although it replicated less in the birds compared with the parent HVT strain. Expression of IL-2 increased the neutralizing antibody response against HVT but did not increase the protection against virulent Marek's disease virus challenge. PMID:17364512

  10. Evaluation of murine lymphocyte membrane potential, intracellular free calcium, and interleukin-2 receptor expression upon exposure to 1,1-dimethylhydrazine.

    PubMed

    Frazier, D E; Tarr, M J; Olsen, R G

    1992-06-01

    The effects of 1,1-dimethylhydrazine (UDMH) on several early events associated with lymphocyte activation were examined. The concentration of intracellular calcium ([Ca2+]i) and membrane potential of murine lymphocytes were found to be altered upon exposure to UDMH; [Ca2+]i was increased in murine thymocytes, while splenocytes exhibited membrane hyperpolarization. In addition, interleukin-2 receptor expression induced by in-vitro concanavalin A stimulation of murine splenocytes at 24 and 48 h in the presence of UDMH was not affected. UDMH may interfere with the ability of these two distinct lymphocyte populations to regulate normal ionic fluctuations, thus contributing to altered immune responsiveness. PMID:1609436

  11. Construction, electrochemically biosensing and discrimination of recombinant plasmid (pEThIL-2) on the basis of interleukine-2 DNA insert

    Microsoft Academic Search

    Mohammad Saeid Hejazi; Mohammad Hossein Pournaghi-Azar; Esmaeel Alipour; Farrokh Karimi

    2008-01-01

    Construction, electrochemically biosensing and discrimination of recombinant pEThIL-2 plasmid, with 5839bp size, on the basis of interleukine-2 (IL-2) DNA insert are described. Plasmid pEThIL-2 was constructed by PCR amplification of IL-2 encoding DNA and subcloning into pET21a(+) vector using BamHI and SacI sites. The recombinant pEThIL-2 plasmid was detected with a label-free DNA hybridization biosensor using a non-inosine substituted probe.

  12. The role of cytokines, adhesion molecules, and chemokines in interleukin-2-induced lymphocytic infiltration in C57BL/6 mice.

    PubMed Central

    Anderson, J A; Lentsch, A B; Hadjiminas, D J; Miller, F N; Martin, A W; Nakagawa, K; Edwards, M J

    1996-01-01

    IL-2 mediates the regression of certain malignancies, but clinical use is limited because of associated toxicities, including parenchymal lymphocytic infiltration with multiple organ failure. Secondarily induced cytokines are important mediators of IL-2 toxicity and IL-2-induced lymphocyte-endothelial adherence and trafficking. The recently discovered C-C chemokines, RANTES (regulated on activation, normal T expressed and secreted) and macrophage inflammatory protein-1alpha, have also been implicated in lymphocytic migration. We hypothesized that IL-2 alters cytokine, C-C chemokine, and adhesion molecule expression in association with parenchymal lymphocytic infiltration. C57BL/6 mice were injected with 3x10(5) IU of IL-2 or 0.1 ml of 5% dextrose intraperitoneally every 8 h for 6 d, then killed. IL-2 induced massive lymphocytic infiltration in the liver and lung and moderate infiltration in the kidney in association with organ edema and dysfunction. Immunostaining showed increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression in association with this organ-specific lymphocytic infiltration. Flow cytometry showed increased expression of the corresponding ligands (lymphocyte function-associated antigen-1 and very late antigen-4) on splenocytes. IL-2 increased TNF-alpha mRNA and protein expression in the liver. Organs infiltrated by lymphocytes had increased TNF-alpha mRNA, whereas RANTES mRNA was increased in all organs, regardless of lymphocytic infiltration. IL-2 toxicity involves organ-specific TNF-alpha and RANTES production with increased ICAM-1 and VCAM-1 expression as potential mechanisms facilitating lymphocytic infiltration and organ dysfunction. PMID:8621780

  13. [The surface expression of CD25 at different stages of proliferative response in human lymphocytes. II. The role of interleukin-2].

    PubMed

    Shatrova, A N; Zenin, V V; Aksenov, N D; Mitiushova, E V; Marakhova, I I

    2011-01-01

    The expression of alpha-subunit of interleukin-2 receptor (IL-2Ralpha) was assessed by quantifying activation-induced upregulation of CD25 in human blood lymphocytes (HBL) stimulated by interleukin-2 (IL-2). It was established that exogenous IL-2 induced no surface expression of CD25 neither proliferation at 48 h of IL-2 action. In component HBL, pretreated by sub-mitogenic doses of phytohemagglutinin (PHA), 5-15 % of cell population was revealed to represent the CD2t+ cells, and in the competent cells only, exogenous IL-2 induced the surface expression of CD25 as well as the growth and the proliferative response, which was comparable with those to mitogenic doses of PHA. The JAK3 inhibitor WHI-P131 eliminated IL-2-dependent CD25 expression without influencing the CD25 expression in competent cells. Unlike, PP2 was found to inhibit the IL-2-dependent CD25 expression in a lesser extent than WHI-P131, however this drug was effectively inhibited CD25 expression in PHA-pretreated, competent HBL. These data suggest that Src-dependent signaling participate in the early IL-2Ralpha expression that precedes the IL-2-dependent cell cycle progression of activated HBL. It is concluded that in normal T cells, the IL-2Ralpha expression in firstly induced by antigen (mitogen) and thereafter it is held IL-2 through JAK-dependent signaling pathway. PMID:21961284

  14. Serum levels of endothelin-1 (ET-1), interleukin-2 (IL-2) and amino-terminal propeptide type III procollagen (PIII NP) in patients with acute and chronic filariasis.

    PubMed

    el-Sharkawy, I M; Haseeb, A N; Saleh, W A

    2001-04-01

    Filariasis, a mosquito-borne parasitic disease, is a worldwide health problem. There is still, some controversial concerning the diagnosis of acute and chronic infections. The serum levels of endothelin-1 (ET-1), interleukin-2 (IL-2) and amino-terminal propeptide Type III (PIII NP) was measured in patients with acute and chronic filariasis as compared with controls. The ET-1, IL-2 and PIII NP levels were significantly high in chronic cases than in acute. On the other hand, the serum levels of IL-2 and PIII NP were significantly high in acute cases than in the controls. These three immuno-mediators play role in the pathogenesis of filariasis particularly. The chronic cases. So, these mediators can be used as markers for diagnosis of human cases infected with chronic and acute filariasis. PMID:12557940

  15. Lipopolysaccharide enhances in vivo interleukin-2 production and proliferation by naive antigen-specific CD4 T cells via a Toll-like receptor 4-dependent mechanism

    PubMed Central

    Costalonga, Massimo; Zell, Traci

    2007-01-01

    Microbial adjuvants are essential for the development of T-cell-dependent antibody production, recall T-cell proliferation and interferon-? production following immunization with protein antigens. Using an adoptive transfer approach, we showed that the adjuvant lipopolysaccharide enhanced the frequency of cells producing interleukin-2, enhanced clonal expansion by antigen-specific CD4 T cells and increased CD86 and interleukin-1? production by antigen-presenting cells. All of these effects were dependent on Toll-like receptor-4 (TLR4) expression by cells other than the antigen-specific CD4 T cells. The ability of lipopolysaccharides to increase the number of antigen-specific CD4 T cells that survive after immunization probably explains the previous finding that antigen-specific proliferation by T cells from normal mice depends on previous exposure to antigen and adjuvant. PMID:17484770

  16. Prolonged survival of a patient affected by pancreatic adenocarcinoma with massive lymphocyte and dendritic cell infiltration after interleukin-2 immunotherapy. Report of a case.

    PubMed

    Nobili, Cinzia; Degrate, Luca; Caprotti, Roberto; Franciosi, Claudio; Leone, Biagio Eugenio; Trezzi, Rosangela; Romano, Fabrizio; Uggeri, Fabio; Uggeri, Franco

    2008-01-01

    Several studies have shown that there is a paucity of immune cells within the stroma of pancreatic adenocarcinoma, a very aggressive cancer with a median survival of about 18 months. A 65-year-old man presented with jaundice. Abdominal ultrasound revealed intra- and extrahepatic bile duct dilatation and a 45-mm diameter hypoechoic solid mass within the pancreatic head; a computed tomography scan excluded vascular infiltration and metastatic lesions. The patient received immunotherapy consisting of 6,000,000 IU human recombinant interleukin-2 administered subcutaneously twice a day for 3 consecutive days. Thirty-six hours after the last dose, he underwent a pylorus-preserving pancreatoduodenectomy. Because of the presence of high-grade dysplasia detected by intraoperative histological examination of a distal section, a spleen preserving total pancreatectomy was performed. The postoperative course was uneventful. The patient died 32 months after surgery because of local recurrence. Histopathology showed G3 pancreatic ductal adenocarcinoma infiltrating the anterior and posterior peripancreatic tissue, duodenal wall and intrapancreatic common bile duct, with sarcoma-like foci and a component of intraductal tumor involving the common bile duct. In the distal pancreas, widespread foci of pancreatic intraepithelial neoplasia (PanI2-3) were found. The Ki-67 proliferation index was 16%. TNM staging was pT3 pN1 R1. Sections were immunostained for the T-lymphocyte marker CD3 and for the dendritic cell marker CD1a. Intratumoral infiltration was high for CD1a+ cells and mild for CD3+ cells. Preoperative immunotherapy with interleukin-2 may contribute to massive stromal infiltration of immune cells in pancreatic adenocarcinoma. This may prolong the survival even in the presence of negative prognostic factors (age >65 years, tumor diameter >20 mm, R1, tumor grade G3). PMID:18705415

  17. Elf-1 and Stat5 bind to a critical element in a new enhancer of the human interleukin-2 receptor alpha gene.

    PubMed Central

    Lécine, P; Algarté, M; Rameil, P; Beadling, C; Bucher, P; Nabholz, M; Imbert, J

    1996-01-01

    The interleukin 2 receptor alpha-chain (IL-2R alpha) gene is a key regulator of lymphocyte proliferation. IL-2R alpha is rapidly and potently induced in T cells in response to mitogenic stimuli. Interleukin 2 (IL-2) stimulates IL-2R alpha. transcription, thereby amplifying expression of its own high-affinity receptor. IL-2R alpha transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains NF-kappaB and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific Ets protein Elf-1 and HMG-I(Y) proteins. However, none of these proximal regions account for the induction of IL-2R alpha transcription by IL-2. To find new regulatory regions of the IL-2R alpha gene, 8.5 kb of the 5' end noncoding sequence of the IL-2R alpha gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine IL-2-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers IL-2 responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for IL-2 inducibility of PRRIII/CAT reporter constructs. IL-2 induced the binding of Stat5a and b proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of IL-2R alpha expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in IL-2R alpha regulation and suggest that the T-cell-specific Elf-1 factor can serve as a transcriptional repressor. PMID:8943338

  18. Analysis of human peripheral blood T lymphocytes proliferating in response to sensitizing antigens: effect of interleukin-2 (IL-2)-responsive bystander cells.

    PubMed

    Sia, D Y; Chou, J L

    1988-03-01

    The contribution of interleukin-2 (IL-2)-responsive bystander cells to the proliferative responses of human peripheral blood T lymphocytes to antigens used for sensitization such as Purified Protein Derivative (PPD), Tetanus toxoid (T T) and Influenza virus was investigated. Marked proliferation of the unfractionated peripheral blood mononuclear cells (PBMC) was observed following stimulation with these antigens to which the individuals were known to have been sensitized previously. Depletion of large granular lymphocytes (LGL) from PBMC resulted in substantial reduction in the response of the lymphoid cells in proliferating to the antigens. Proliferation of the T4+T8- (helper)-enriched population, or T4+T8- subset depleted of any IL-2 receptor (IL-2R)-bearing lymphoid cells to these antigens was comparable to that of LGL-depleted PBMC cultures. Cell titration experiments of the blast cells generated from these cultures revealed that PBMC-derived population contained fewer antigen-reactive lymphocytes. These results, therefore, suggested that IL-2-responsive LGL through expansion affected the concentration of antigen-proliferating T cells in the antigen-stimulated PBMC cultures. PMID:3134300

  19. In vivo and in vitro administration of interleukin 2-containing preparation reverses T-cell unresponsiveness in Mycobacterium bovis BCG-infected mice.

    PubMed Central

    Colizzi, V

    1984-01-01

    Mice infected with high doses of Mycobacterium bovis BCG (3 X 10(7)) showed a marked impairment of delayed-type hypersensitivity to PPD in vivo, and their splenic T cells failed to proliferate when cultured in vitro with concanavalin A or PPD. However, this state of unresponsiveness could be reversed both in vitro and in vivo by the administration of an interleukin 2 (IL-2)-containing preparation. IL-2 produced spontaneously by the gibbon lymphosarcoma T-cell line MLA-144 and T-cell-conditioned medium from a mixed lymphocyte reaction were able to increase DNA synthesis of splenic T lymphocytes from BCG-immunosuppressed mice cultured with concanavalin A or PPD. Furthermore, BCG-infected mice treated in vivo with at least 100 U of IL-2 showed a positive skin reaction to PPD, and their spleen cells were fully responsive in vitro. The reversal of BCG-induced immunosuppression was not observed when infected mice were injected with IL-2 preparations previously incubated with blast cells, a procedure known to remove IL-2 activity. These results indicate that the basis of BCG-induced unresponsiveness is a deficiency in the production of IL-2 rather than a lack of reactive T cells. PMID:6376356

  20. Defective interleukin-2 induction of lymphokine-activated killer (LAK) activity in peripheral blood T lymphocytes of patients with monoclonal gammopathies.

    PubMed Central

    Massaia, M; Bianchi, A; Dianzani, U; Camponi, A; Attisano, C; Boccadoro, M; Pileri, A

    1990-01-01

    The recombinant interleukin-2 (rIL-2) generation of lymphokine-activated killer (LAK) cells was investigated in peripheral blood T lymphocytes (PBT) of 16 patients with monoclonal gammopathy of undetermined significance (MGUS) and 32 patients with multiple myeloma (MM). LAK activity was significantly decreased in MM, but not in MGUS patients, and was partially recovered in MM in the remission phase. This finding was unexpected, because CD8+ CD11b+ cells, which contain LAK precursors, are significantly increased in MM. LAK activity was investigated in purified CD8+ CD11b+ lymphocytes to discriminate between an intrinsic defect or a defective regulation by other T cell subsets. These cells were intrinsically unable to generate LAK activity fully following rIL-2 stimulation. MM showed the more pronounced LAK deficiency, while MGUS patients showed intermediate values. Phenotyping revealed significantly increased proportions of Leu7+ and HLA-DR+ cells in MM patients. These data reveal another dysregulation of T cell effector functions in patients with monoclonal gammopathies and offer further evidence of the impairment of their cell-mediated immunity. PMID:2105868

  1. Immunogenicity of oral vaccination with Lactococcus lactis derived vaccine candidate antigen (UreB) of Helicobacter pylori fused with the human interleukin 2 as adjuvant.

    PubMed

    Zhang, Hong-xin; Qiu, Yu-yu; Zhao, Ying-hui; Liu, Xin-ting; Liu, Ming; Yu, Ai-lian

    2014-02-01

    Helicobacter pylori (H. pylori) infection remains a significant global public health problem. Vaccine, especially edible vaccine, is considered to be effective in the management of H. pylori infections. By using recombinant technology, Lactococcus lactis (L. lactis) could serve as an antigen-delivering vehicle for the development of edible vaccine. The aim of this study was to produce edible UreB (urease B) vaccine derived from L. lactis against H. pylori. The UreB subunit is the most effective and common immunogen of all strains of H. pylori. The UreB was produced as a chimeric protein fused with IL-2 (human interleukin 2) as the mucosal adjuvant. Mucosal immunization of mice with recombinant L. lactis NZ9000 containing the UreB-IL-2 protein elicited more anti-UreB antibody that specifically bounded to the purified bacterial UreB protein and more cytokines such as IFN-?, IL-4, and IL-17, and had a lower H. pylori burden and urease activity than control mice. These results suggest that the recombinant L. lactis expressing UreB-IL-2 can be potentially used as an edible vaccine for controlling H. pylori infection. PMID:24036137

  2. T lymphocyte activation signals for interleukin-2 production involve activation of MKK6-p38 and MKK7-SAPK/JNK signaling pathways sensitive to cyclosporin A.

    PubMed

    Matsuda, S; Moriguchi, T; Koyasu, S; Nishida, E

    1998-05-15

    p38/CSBP, a subgroup member of mitogen-activated protein kinase (MAPK) superfamily molecules, is known to be activated by proinflammatory cytokines and environmental stresses. We report here that p38 is specifically activated by signals that lead to interleukin-2 (IL-2) production in T lymphocytes. A p38 activator MKK6 was also markedly activated by the same stimulation. Pretreatment of cells with SB203580, a specific inhibitor of p38, as well as expression of a dominant-negative mutant of MKK6, suppressed the transcriptional activation of the IL-2 promoter. We also demonstrated that MKK7, a recently described MAPK kinase family member, plays a major role in the activation of stress-activated protein kinase (SAPK)/c-Jun NH2-terminal kinase (JNK) in T lymphocytes. Moreover, a dominant-negative mutant of MKK7 abrogated the transcriptional activation of the distal nuclear factor of activated T cells response element in the IL-2 promoter. Cyclosporin A, a potent immunosuppressant, inhibited activation of both p38 and SAPK/JNK pathways but not the MAPK/extracellular signal-regulated kinase (ERK) pathway. Our results indicate that both MKK6 to p38 and MKK7 to SAPK/JNK signaling pathways are activated in a cyclosporin A-sensitive manner and contribute to IL-2 gene expression in T lymphocytes. PMID:9575191

  3. Immunotherapy of established tumors in mice by intratumoral injection of interleukin-2 plasmid DNA: induction of CD8+ T-cell immunity.

    PubMed

    Saffran, D C; Horton, H M; Yankauckas, M A; Anderson, D; Barnhart, K M; Abai, A M; Hobart, P; Manthorpe, M; Norman, J A; Parker, S E

    1998-01-01

    Intratumoral (i.t.) injection of a plasmid DNA vector encoding the murine interleukin-2 (IL-2) gene was used to treat established renal cell carcinoma (Renca) tumors in BALB/c mice. Tumor regression was observed in 60-90% of mice that were injected i.t. for 4 days with IL-2 plasmid DNA complexed with the cationic lipid DMRIE/DOPE ((+/-)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propa naminium bromide/dioleoylphosphatidylethanolamine). The mice remained tumor-free until the conclusion of the study, which was 4 months after tumor challenge. In a rechallenge experiment, mice that were rendered tumor-free for 6 months by IL-2 plasmid DNA treatment rejected a subsequent challenge of Renca cells but could not reject a challenge with the unrelated, syngeneic CT-26 tumor. Spleen cells from cured mice contained Renca-specific cytotoxic T lymphocytes, and adoptive transfer of mixed lymphocyte cultures into naive mice at 2 days after challenge with Renca cells prevented tumor growth. In vivo depletion of T-cell subsets at the time of i.t. injection with IL-2 plasmid DNA demonstrated that CD8+ T cells, but not CD4+ T cells, were the primary effectors of the antitumor response. PMID:9824052

  4. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  5. Transgenic Eimeria mitis expressing chicken interleukin 2 stimulated higher cellular immune response in chickens compared with the wild-type parasites

    PubMed Central

    Li, Zhuoran; Tang, Xinming; Suo, Jingxia; Qin, Mei; Yin, Guangwen; Liu, Xianyong; Suo, Xun

    2015-01-01

    Chicken coccidiosis, caused by Eimeria sp., occurs in almost all poultry farms and causes huge economic losses in the poultry industry. Although this disease could be controlled by vaccination, the reduced feed conservation ratio limits the widespread application of anticoccidial vaccines in broilers because some intermediate and/or low immunogenic Eimeria sp. only elicit partial protection. It is of importance to enhance the immunogenicity of these Eimeria sp. by adjuvants for more effective prevention of coccidiosis. Cytokines have remarkable effects on the immunogenicity of antigens. Interleukin 2 (IL-2), for example, significantly stimulates the activation of CD8+ T cells and other immune cells. In this study, we constructed a transgenic Eimeria mitis line (EmiChIL-2) expressing chicken IL-2 (ChIL-2) to investigate the adjuvant effect of ChIL-2 to enhance the immunogenicity of E. mitis against its infection. Stable transfected EmiChIL-2 population was obtained by pyrimethamine selection and verified by PCR, genome walking, western blotting and indirect immunofluorescence assay. Cellular immune response, E. mitis-specific IFN-? secretion lymphocytes in the peripheral blood mononuclear cells, stimulated by EmiChIL-2 was analyzed by enzyme-linked immunospot assay (ELISPOT). The results showed that EmiChIL-2 stimulated a higher cellular immune response compared with that of the wild-type parasite infection in chickens. Moreover, after the immunization with EmiChIL-2, elevated cellular immune response as well as reduced oocyst output were observed These results indicated that ChIL-2 expressed by Eimeria sp. functions as adjuvant and IL-2 expressing Eimeria parasites are valuable vaccine strains against coccidiosis.

  6. Functional RNAi screen targeting cytokine and growth factor receptors reveals oncorequisite role for interleukin-2 gamma receptor in JAK3-mutation-positive leukemia.

    PubMed

    Agarwal, A; MacKenzie, R J; Eide, C A; Davare, M A; Watanabe-Smith, K; Tognon, C E; Mongoue-Tchokote, S; Park, B; Braziel, R M; Tyner, J W; Druker, B J

    2015-06-01

    To understand the role of cytokine and growth factor receptor-mediated signaling in leukemia pathogenesis, we designed a functional RNA interference (RNAi) screen targeting 188 cytokine and growth factor receptors that we found highly expressed in primary leukemia specimens. Using this screen, we identified interleukin-2 gamma receptor (IL2R?) as a critical growth determinant for a JAK3(A572V) mutation-positive acute myeloid leukemia cell line. We observed that knockdown of IL2R? abrogates phosphorylation of JAK3 and downstream signaling molecules, JAK1, STAT5, MAPK and pS6 ribosomal protein. Overexpression of IL2R? in murine cells increased the transforming potential of activating JAK3 mutations, whereas absence of IL2R? completely abrogated the clonogenic potential of JAK3(A572V), as well as the transforming potential of additional JAK3-activating mutations such as JAK3(M511I). In addition, mutation at the IL2R? interaction site in the FERM domain of JAK3 (Y100C) completely abrogated JAK3-mediated leukemic transformation. Mechanistically, we found IL2R? contributes to constitutive JAK3 mutant signaling by increasing JAK3 expression and phosphorylation. Conversely, we found that mutant, but not wild-type JAK3, increased the expression of IL2R?, indicating IL2R? and JAK3 contribute to constitutive JAK/STAT signaling through their reciprocal regulation. Overall, we demonstrate a novel role for IL2R? in potentiating oncogenesis in the setting of JAK3-mutation-positive leukemia. In addition, our study highlights an RNAi-based functional assay that can be used to facilitate the identification of non-kinase cytokine and growth factor receptor targets for inhibiting leukemic cell growth. PMID:25109334

  7. Pilot trial of interleukin-2 and zoledronic acid to augment ?? T cells as treatment for patients with refractory renal cell carcinoma

    PubMed Central

    Lang, Joshua M.; Kaikobad, Mahazarin R.; Wallace, Marianne; Staab, Mary Jane; Horvath, Dorothea L.; Wilding, George; Liu, Glenn; Eickhoff, Jens C.; Malkovsky, Miroslav

    2011-01-01

    Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human V?9 V?2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these V?9 V?2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of V?9 V?2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in V?9 V?2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of V?9 V?2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of V?9 V?2 T cell in patients with metastatic RCC. PMID:21647691

  8. Pilot trial of interleukin-2 and zoledronic acid to augment ?? T cells as treatment for patients with refractory renal cell carcinoma.

    PubMed

    Lang, Joshua M; Kaikobad, Mahazarin R; Wallace, Marianne; Staab, Mary Jane; Horvath, Dorothea L; Wilding, George; Liu, Glenn; Eickhoff, Jens C; McNeel, Douglas G; Malkovsky, Miroslav

    2011-10-01

    Prior to the advent of VEGF-targeted therapies, renal cell carcinoma (RCC) was among the few solid tumors shown to respond to cytokine-based therapies such as interleukin-2 (IL-2) and interferon alpha. Previous work has shown that aminobisphosphonates, including zoledronic acid (ZA), are capable of activating human V?9 V?2 T cells in vitro, and these cells can be further expanded with IL-2. Moreover, these V?9 V?2 T cells have cytolytic activity in vitro to multiple human tumor cell lines. In the current report, we have conducted a pilot trial in patients with metastatic RCC, evaluating different doses of ZA in combination with low-dose IL-2 to determine whether combining these agents can promote in vivo proliferation of V?9 V?2 T cells and elicit an antitumor response. In 12 patients evaluated, no objective clinical responses were observed by RECIST criteria; however, two patients experienced prolonged stable disease. A modest increase in V?9 V?2 T-cell frequency could be detected by Day 8 of therapy in four of the nine patients who received at least one cycle of therapy, but not to the magnitude anticipated from preclinical models. Repeated administration of IL-2 and ZA resulted in both a diminished in vivo percentage of V?9 V?2 T cells as well as impaired expansion in vitro after the first cycle of therapy. These results suggest that repeated administration of IL-2 and ZA, at the doses and schedules used in this trial, may actually inhibit the proliferative capacity of V?9 V?2 T cell in patients with metastatic RCC. PMID:21647691

  9. A Phase I Study of High-Dose Interleukin-2 With Sorafenib in Patients With Metastatic Renal Cell Carcinoma and Melanoma

    PubMed Central

    Lam, Elaine; Mortazavi, Amir; Kendra, Kari; Lesinski, Gregory B.; Mace, Thomas A.; Geyer, Susan; Carson, William E.; Tahiri, Sanaa; Bhinder, Arvinder; Clinton, Steven K.; Olencki, Thomas

    2014-01-01

    This study was designed to evaluate the safety and feasibility of high-dose interleukin-2 (HD IL-2) followed by sorafenib in patients with metastatic melanoma (MM) and renal cell carcinoma (RCC). Biomarkers relevant to the antitumor effects of IL-2 that may be altered by sorafenib including the percentages of natural T-regulatory cells (Tregs), myeloid-derived suppressor cells (MDSC), and STAT5 phosphorylation (pSTAT5) in T cells were evaluated. We hypothesized that the proposed treatment schedule is feasible and safe and may lead to enhanced tumor response. A phase I dose escalation trial was conducted in patients with either metastatic RCC or MM. HD IL-2 (600,000 IU/kg IV q8h×8–12 doses) was administered on days 1–5 and 15–19, followed by sorafenib on days 29–82. The sorafenib dose was escalated. The percentage of Tregs, MDSC, and pSTAT5 in T cells were evaluated in peripheral blood by flow cytometry. Twelve of the 18 patients were evaluable for dose-limiting toxicity. No dose-limiting toxicity was observed. The treatment-related toxicity was predictable and did not seem to be additive with this schedule of administration. Partial responses were seen in 3 patients. No significant changes in the percentage of circulating Treg and MDSC were observed, whereas sorafenib did not adversely affect the ability of IL-2 to induce pSTAT5 in T cells. HD IL-2 followed by sorafenib was safe and feasible in patients with MM and RCC and did not adversely affect T-cell signaling through STAT5 in response to IL-2. PMID:24598448

  10. The immediate-early gene product Egr-1 regulates the human interleukin-2 receptor beta-chain promoter through noncanonical Egr and Sp1 binding sites.

    PubMed Central

    Lin, J X; Leonard, W J

    1997-01-01

    The interleukin-2 IL-2 receptor beta-chain (IL-2Rbeta) is an essential component of the receptors for IL-2 and IL-15. Although IL-2Rbeta is constitutively expressed by lymphocytes, its expression can be further induced by a number of stimuli, including phorbol 12-myristate 13-acetate (PMA). We have now characterized factors that bind to an enhancer region located between nucleotides -170 and -139 of the human IL-2Rbeta promoter. Both Sp1 and Sp3 bound to the 5' portion of this region, whereas a PMA-inducible factor (PIF) mainly bound to its 3' portion and bound to the Sp binding motifs as well. In Jurkat T cells, induction of PIF DNA binding activity was rapidly induced, required de novo protein synthesis, and was sustained at a high level for at least 23 h. Interestingly, PIF was constitutively activated in human T-cell leukemia virus type 1-transformed MT-2 cells. In this paper, we demonstrate that PIF is Egr-1 based on its recognition by anti-Egr-1 antisera in gel mobility shift assays, even though the IL-2Rbeta DNA binding motif differed substantially from the canonical Egr-1 binding site. In addition, Egr-1 bound to the Sp binding site. In Jurkat cells, both sites were required for maximal IL-2Rbeta promoter activity, and in HeLaS3 cells, transfection of Egr-1 could drive activity of a reporter construct containing both sites. Moreover, Sp1 and Egr-1 could form a complex with kinetics that correlated with the production of Egr-1 in Jurkat cells upon PMA stimulation. Thus, Sp1 and Egr-1 physically and functionally cooperate to mediate maximal IL-2Rbeta promoter activity. PMID:9199305

  11. Structure of the interleukin-2 tyrosine kinase Src homology 2 domain; comparison between X-ray and NMR-derived structures

    PubMed Central

    Joseph, Raji E.; Ginder, Nathaniel D.; Hoy, Julie A.; Nix, Jay C.; Fulton, D. Bruce; Honzatko, Richard B.; Andreotti, Amy H.

    2012-01-01

    The crystal structure of the interleukin-2 tyrosine kinase Src homology domain (Itk SH2) is described and it is found that unlike in studies of this domain using NMR spectroscopy, cis–trans-prolyl isomerization is not readily detected in the crystal structure. Based on similarities between the Itk SH2 crystal form and the cis form of the Itk SH2 NMR structure, it is concluded that it is likely that the prolyl imide bond at least in part adopts the cis conformation in the crystal form. However, the lack of high-resolution data and the dynamic nature of the proline-containing loop mean that the precise imide-bond conformation cannot be determined and prolyl cis–trans isomerization in the crystal cannot be ruled out. Given the preponderance of structures that have been solved by X-ray crystallography in the Protein Data Bank, this result supports the notion that prolyl isomerization in folded proteins has been underestimated among known structures. Interestingly, while the precise status of the proline residue is ambiguous, Itk SH2 crystallizes as a domain-swapped dimer. The domain-swapped structure of Itk SH2 is similar to the domain-swapped SH2 domains of Grb2 and Nck, with domain swapping occurring at the ?-meander region of all three SH2 domains. Thus, for Itk SH2 structural analysis by NMR spectroscopy and X-ray crystallography revealed very different structural features: proline isomerization versus domain-swapped dimerization, respectively. PMID:22297986

  12. Relationship between Serum Level of Interleukin-2 in Patients with Systemic Lupus Erythematosus and Disease Activity in Comparison with Control Group

    PubMed Central

    Aghaei, Mehrdad; Musavi, Sara; Nomali, Mahin

    2014-01-01

    Background: Despite the large number of surveys, there are not any validated biomarkers for SLE disease activity till now. This study aimed to evaluate the relationship between serum level of IL-2 in patients with SLE and disease activity in comparison with control group. Materials and Methods: In this case-control study, 73 patients with lupus and 73 healthy subjects referred to the rheumatology clinic of 5 Azar Hospital in Gorgan (North of Iran).They were studied via convenience sampling during 2011-2012. Blood samples were taken from both groups and serum levels of interleukin -2 measured by Avi Bion Human IL-2 ELISA kit. Serum Level of IL-2 greater than 15 pg/ml defined positive and lesser than this amount defined negative. Disease activity evaluated with SLE disease activity index. Score greater than or equal to three or four defined as active disease. Data analysis conducted by SPSS software (version 16) and by using descriptive statistics and statistical tests. Results: Serum level of IL-2 was positive in 45.2% of sample studied and negative in 54.8% in case group, while in control group, serum level of IL-2 only in 11% of sample studied was positive and in 89% was negative. Statistical analysis indicated a significant relationship between serum level of IL-2 and the SLE disease activity index (p=0.025). Conclusion: This study showed the relationship between serum levels of IL-2 and disease activity, so this biomarker can be used as a clinical indicator for assessing disease activity in patients with SLE. PMID:25177590

  13. Comparisons of CD8+ T Cells Specific for Human Immunodeficiency Virus, Hepatitis C Virus, and Cytomegalovirus Reveal Differences in Frequency, Immunodominance, Phenotype, and Interleukin-2 Responsiveness ? †

    PubMed Central

    Jagannathan, Prasanna; Osborne, Christine M.; Royce, Cassandra; Manion, Maura M.; Tilton, John C.; Li, Li; Fischer, Steven; Hallahan, Claire W.; Metcalf, Julia A.; McLaughlin, Mary; Pipeling, Matthew; McDyer, John F.; Manley, Thomas J.; Meier, Jeffery L.; Altman, John D.; Hertel, Laura; Davey, Richard T.; Connors, Mark; Migueles, Stephen A.

    2009-01-01

    To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8+ T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8+ T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8+ T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8+ T cells were predominantly CD27+45RO+ for HIV and CD27?45RA+ for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8+ T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8+ T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8+ T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained. PMID:19129459

  14. Comparisons of CD8+ T cells specific for human immunodeficiency virus, hepatitis C virus, and cytomegalovirus reveal differences in frequency, immunodominance, phenotype, and interleukin-2 responsiveness.

    PubMed

    Jagannathan, Prasanna; Osborne, Christine M; Royce, Cassandra; Manion, Maura M; Tilton, John C; Li, Li; Fischer, Steven; Hallahan, Claire W; Metcalf, Julia A; McLaughlin, Mary; Pipeling, Matthew; McDyer, John F; Manley, Thomas J; Meier, Jeffery L; Altman, John D; Hertel, Laura; Davey, Richard T; Connors, Mark; Migueles, Stephen A

    2009-03-01

    To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8(+) T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8(+) T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8(+) T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8(+) T cells were predominantly CD27(+)45RO(+) for HIV and CD27(-)45RA(+) for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8(+) T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8(+) T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8(+) T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained. PMID:19129459

  15. Induction of c-fos expression through JNK-mediated TCF/Elk-1 phosphorylation.

    PubMed Central

    Cavigelli, M; Dolfi, F; Claret, F X; Karin, M

    1995-01-01

    Growth factors induce c-fos transcription by stimulating phosphorylation of transcription factor TCF/Elk-1, which binds to the serum response element (SRE). Under such conditions Elk-1 could be phosphorylated by the mitogen-activated protein kinases (MAPKs) ERK1 and ERK2. However, c-fos transcription and SRE activity are also induced by stimuli, such as UV irradiation and activation of the protein kinase MEKK1, that cause only an insignificant increase in ERK1/2 activity. However, both of these stimuli strongly activate two other MAPKs, JNK1 and JNK2, and stimulate Elk-1 transcriptional activity and phosphorylation. We find that the JNKs are the predominant Elk-1 activation domain kinases in extracts of UV-irradiated cells and that immunopurified JNK1/2 phosphorylate Elk-1 on the same major sites recognized by ERK1/2, that potentiate its transcriptional activity. Finally, we show that UV irradiation, but not serum or phorbol esters, stimulate translocation of JNK1 to the nucleus. As Elk-1 is most likely phosphorylated while bound to the c-fos promoter, these results suggest that UV irradiation and MEKK1 activation stimulate TCF/Elk-1 activity through JNK activation, while growth factors induce c-fos through ERK activation. Images PMID:8846788

  16. JNK-mediated phosphorylation of DLK suppresses its ubiquitination to promote neuronal apoptosis

    PubMed Central

    Huntwork-Rodriguez, Sarah; Wang, Bei; Watkins, Trent; Ghosh, Arundhati Sengupta; Pozniak, Christine D.; Bustos, Daisy; Newton, Kim; Kirkpatrick, Donald S.

    2013-01-01

    Neurons are highly polarized cells that often project axons a considerable distance. To respond to axonal damage, neurons must transmit a retrograde signal to the nucleus to enable a transcriptional stress response. Here we describe a mechanism by which this signal is propagated through injury-induced stabilization of dual leucine zipper-bearing kinase (DLK/MAP3K12). After neuronal insult, specific sites throughout the length of DLK underwent phosphorylation by c-Jun N-terminal kinases (JNKs), which have been shown to be downstream targets of DLK pathway activity. These phosphorylation events resulted in increased DLK abundance via reduction of DLK ubiquitination, which was mediated by the E3 ubiquitin ligase PHR1 and the de-ubiquitinating enzyme USP9X. Abundance of DLK in turn controlled the levels of downstream JNK signaling and apoptosis. Through this feedback mechanism, the ubiquitin–proteasome system is able to provide an additional layer of regulation of retrograde stress signaling to generate a global cellular response to localized external insults. PMID:23979718

  17. Cytokine mRNA expression profiles in rats infected with the intestinal nematode Nippostrongylus brasiliensis.

    PubMed Central

    Matsuda, S; Uchikawa, R; Yamada, M; Arizono, N

    1995-01-01

    Although the immune responses to intestinal nematode infection have been well studied and have been shown to be strongly driven by Th2-associated cytokines in mice, such information has been limited with respect to rats. We investigated changes in levels of the mRNAs encoding interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-10, and gamma interferon in the mesenteric lymph nodes of rats infected with Nippostrongylus brasiliensis by reverse transcription-PCR in comparison with immunoglobulin E (IgE)/IgG2a antibody, eosinophil, basophil, and mucosal mast cell responses. In the two rat strains used, Brown Norway and Fischer-344, which show different responses to allergens, serum IgE increased to much higher levels in the former than in the latter 2 weeks after infection. Intestinal mastocytosis was observed much earlier and more intensely in Brown Norway rats than in Fischer-344 rats, but the degrees of peripheral eosinophilia and basophilia did not differ between the two strains. In both strains, IL-3, IL-4, and IL-5 mRNA expression increased and peaked around 7 to 14 days after infection, while expression of IL-2, IL-10, and gamma interferon mRNAs did not change notably throughout the experimental period. The highest IL-4 mRNA expression was observed slightly earlier in Brown Norway than in Fischer-344 rats, but levels of IL-3 and IL-5 mRNAs peaked synchronously in both strains. The amounts of mRNAs encoding these three cytokines were always higher in Brown Norway than in Fischer-344 rats. It is suggested that in rats, Th2 or Th2-like cells are also induced after nematode infection, and IgE elevation is mainly related to increased IL-4 gene expression. PMID:7591119

  18. Effects of modulators of cytosolic Ca2+ on phytohemagglutin-dependent Ca2+ response and interleukin-2 production in Jurkat cells.

    PubMed

    Dupuis, G; Aoudjit, F; Ricard, I; Payet, M D

    1993-01-01

    We have previously reported the presence, in Jurkat T cells, of outward K+ currents and inward currents that have been attributed to Ca2+ channels. Here, we have studied the effects of dimethyl 1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridine-dicarboxylate (nifedipine) and 4-(2,1,3-benzoxadiazol-4-yl)-1,4-dihydro-2,6-dimethyl-5- methoxy-carbonylpyridine-3-carboxylate (PN200-110), two dihydropyridines (DHPs) known to inhibit voltage-dependent Ca2+ channel activity in different types of cells, and two inhibitors of internal Ca2+ release (muscle cells), ryanodine and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), on the Phaseolus vulgaris phytohemagglutinin (PHA)-dependent responses in Jurkat T lymphocytes. Our results show that nifedipine and PN200-110 inhibit the PHA-dependent production of interleukin-2 except when 12-O-tetradecanoyl-13-O-acetyl phorbol is added to the cultures. Ryanodine and TMB-8 are not inhibitors. The PHA-dependent Ca2+ response is significantly reduced when the cells are preincubated in the presence of the DHPs. Under these conditions, ryanodine has only a small inhibitory effect and TMB-8 has no effect. In contrast, only ryanodine (50 microM) decreases the PHA-dependent cytosolic release of Ca2+i when the cells are bathed in a medium containing a low concentration of Ca2+ (60 nM). The inhibitory effects of nifedipine and PN200-110 may result from the binding of these DHPs to specific receptor sites as revealed by studies using [3H]PN200-110 (KD = 8.5 +/- 3.1 nM; 2300 +/- 500 apparent binding sites/cell). Photoaffinity labeling studies using [3H]azidopine as a probe showed specific incorporation of label into three glycoproteins of molecular mass (+/- SD) 170 +/- 13, 110 +/- 25, and 60 +/- 17 kd as analyzed by electrophoresis under reducing conditions. PMID:8426093

  19. Neo-adjuvant chemo-(immuno-)therapy of advanced squamous-cell head and neck carcinoma: a multicenter, phase III, randomized study comparing cisplatin?+?5-fluorouracil (5FU) with cisplatin +?5FU?+?recombinant interleukin 2

    Microsoft Academic Search

    Giovanni Mantovani; Vittorio Gebbia; Mario Airoldi; Cesare Bumma; Paolo Contu; Alessandro Bianchi; Massimo Ghiani; Daniela Dessì; Elena Massa; Luigi Curreli; Biancarosa Lampis; Paola Lai; Carlo Mulas; Antonio Testa; Ernesto Proto; Gabrio Cadeddu; Giorgio Tore

    1998-01-01

    We carried out an open, randomized, phase III, multicenter clinical trial to compare, in neo-adjuvant setting, the clinical\\u000a response and toxicity of the combination chemotherapy cisplatin?+?5-FU with the same combination plus s.c. recombinant interleukin-2\\u000a (rIL-2) in patients with advanced (stage III–IV) head and neck squamous-cell carcinoma (HNSCC). Regimen A was the classical\\u000a Al Sarraf treatment: 100?mg\\/m2 cisplatin i.v. on day

  20. Characterization of visna virus mRNA.

    PubMed Central

    Filippi, P; Brahic, M; Vigne, R; Tamalet, J

    1979-01-01

    Visna virus is a retrovirus responsible for a classical slow infection of the central nervous system of sheep. In the present work we focused our attention on the viral mRNA's. We found that, during the acute infection in vitro, (i) viral mRNA's amount to only 0.1% of the total cytoplasmic RNA, (ii) 20% of the total cytoplasmic viral RNA is found in polyribosomes, and (iii) three viral mRNA's can be identified by sucrose gradient sedimentation or polyacrylamide gel electrophoresis. Their sedimentation coefficients are 36S, 27S, and 21S. PMID:228056

  1. The protein pheromone Er-1 of the ciliate Euplotes raikovi stimulates human T-cell activity: involvement of interleukin-2 system.

    PubMed

    Cervia, Davide; Catalani, Elisabetta; Belardinelli, Maria Cristina; Perrotta, Cristiana; Picchietti, Simona; Alimenti, Claudio; Casini, Giovanni; Fausto, Anna Maria; Vallesi, Adriana

    2013-02-01

    Water-soluble protein signals (pheromones) of the ciliate Euplotes have been supposed to be functional precursors of growth factors and cytokines that regulate cell-cell interaction in multi-cellular eukaryotes. This work provides evidence that native preparations of the Euplotes raikovi pheromone Er-1 (a helical protein of 40 amino acids) specifically increases viability, DNA synthesis, proliferation, and the production of interferon-?, tumor necrosis factor-?, interleukin (IL)-1?, IL-2, and IL-13 in human Jurkat T-cells. Also, Er-1 significantly decreases the mRNA levels of the ? and ? subunits of IL-2 receptor (IL-2R), while the mRNA levels of the ? subunit appeared to be not affected. Jurkat T-cell treatments with Er-1 induced the down-regulation of the IL-2R? subunit by a reversible and time-dependent endocytosis, and increased the levels of phosphorylation of the extracellular signal-regulated kinases (ERK). The cell-type specificity of these effects was supported by the finding that Er-1, although unable to directly influence the growth of human glioma U-373 cells, induced Jurkat cells to synthesize and release factors that, in turn, inhibited the U-373 cell proliferation. Overall, these findings imply that Er-1 coupling to IL-2R and ERK immuno-enhances T-cell activity, and that this effect likely translates to an inhibition of glioma cell growth. PMID:23103669

  2. Th1 cytokine mRNA expression dominates in the skin-draining lymph nodes of C57BL/6 mice following vaccination with irradiated Schistosoma mansoni cercariae, but is down-regulated upon challenge infection.

    PubMed Central

    Betts, C J; Wilson, R A

    1998-01-01

    Vaccination of C57BL/6 mice with irradiated cercariae of Schistosoma mansoni results in the induction of high levels of immunity to subsequent infection. The events occurring in the lymph nodes draining the exposure site have been analysed ex vivo by reverse transcription-polymerase chain reaction (RT-PCR) and the timing of cytokine gene expression following exposure has been established. After vaccination, spatial separation of the T-helper type 1 (Th1) and Th2 responses was evident, with interferon-gamma (IFN-gamma), interleukin-2 (IL-2) and IL-12 mRNA peaking earlier than mRNA for IL-4, IL-5 and IL-10. In contrast to the profiles observed post-vaccination, following challenge the IL-4 mRNA was predominant in the draining lymph nodes, with IFN-gamma message levels barely detectable above the naive level. These observations are confirmed by the analysis of IL-4 and IFN-gamma mRNA using competitive PCR. From these studies it is clear that irradiated cercariae are more able to promote a protective Th1 response, with normal parasites eliciting higher IL-4 and IL-5 expression upon both primary and secondary stimulation. PMID:9536118

  3. Coupling mRNA Synthesis and Decay

    PubMed Central

    Braun, Katherine A.

    2014-01-01

    What has been will be again, what has been done will be done again; there is nothing new under the sun.—Ecclesiastes 1:9 (New International Version) Posttranscriptional regulation of gene expression has an important role in defining the phenotypic characteristics of an organism. Well-defined steps in mRNA metabolism that occur in the nucleus—capping, splicing, and polyadenylation—are mechanistically linked to the process of transcription. Recent evidence suggests another link between RNA polymerase II (Pol II) and a posttranscriptional process that occurs in the cytoplasm—mRNA decay. This conclusion appears to represent a conundrum. How could mRNA synthesis in the nucleus and mRNA decay in the cytoplasm be mechanistically linked? After a brief overview of mRNA processing, we will review the recent evidence for transcription-coupled mRNA decay and the possible involvement of Snf1, the Saccharomyces cerevisiae ortholog of AMP-activated protein kinase, in this process. PMID:25154419

  4. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. PMID:25620012

  5. Biological and biochemical characterization of a factor produced spontaneously by adherent cells of human immunodeficiency virus-infected patients inhibiting interleukin-2 receptor alpha chain (Tac) expression on normal T cells.

    PubMed Central

    Ammar, A; Cibert, C; Bertoli, A M; Tsilivakos, V; Jasmin, C; Georgoulias, V

    1991-01-01

    Adherent cells from human immunodeficiency virus (HIV)-infected subjects but not from normal blood donors, patients with Gram-positive or -negative bacteremia, active tuberculosis, toxoplasmosis, pulmonary aspergillosis, and cytomegalovirus infection produce spontaneously an activity which inhibits alpha chain of interleukin-2 (Tac) expression and interleukin 2 (IL-2) production by normal activated T cells and IL-2 production by these cells. A similar biologic activity was detected in culture supernatants of in vitro HIV-I-infected normal adherent and leukemic U937 cells. Tac-inhibitory activity is not cytotoxic and it could be detected in serum-free conditioned media. Recombinant granulocyte/macrophage colony-stimulating factor and phorbol myristate acetate stimulation of patients' and normal adherent cells did not enhance specifically the production of the Tac inhibitor. Biologically active conditioned media did not contain infectious virus as well as secreted p24, gp120 viral proteins; the biologic activity could not be abolished by anti-p24, anti-gp120, and anti-nef monoclonal antibodies or human purified polyclonal anti-HIV IgG. Gel filtration of conditioned media followed by anion exchange chromatography resulted in a 1,200-fold degree of purification and revealed that the biologically active molecule was cationic. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of this fraction and gel elution of the proteins showed that the biologic activity was associated with a 29-kD protein which was distinct from alpha- or gamma-interferon, tumor necrosis factor-alpha, and prostaglandin E2. The above findings demonstrate the production of inhibitory factor(s) during HIV infection, which might be involved in the pathogenesis of the patients' immune defect. Images PMID:1904071

  6. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  7. mRNA stability in the nucleus*

    PubMed Central

    Liu, Han; Luo, Min; Wen, Ji-kai

    2014-01-01

    Eukaryotic gene expression is controlled by different levels of biological events, such as transcription factors regulating the timing and strength of transcripts production, alteration of transcription rate by RNA processing, and mRNA stability during RNA processing and translation. RNAs, especially mRNAs, are relatively vulnerable molecules in living cells for ribonucleases (RNases). The maintenance of quality and quantity of transcripts is a key issue for many biological processes. Extensive studies draw the conclusion that the stability of RNAs is dedicated-regulated, occurring co- and post-transcriptionally, and translation-coupled as well, either in the nucleus or cytoplasm. Recently, RNA stability in the nucleus has aroused much research interest, especially the stability of newly-made transcripts. In this article, we summarize recent progresses on mRNA stability in the nucleus, especially focusing on quality control of newly-made RNA by RNA polymerase II in eukaryotes. PMID:24793762

  8. Vaccination with Messenger RNA (mRNA)

    Microsoft Academic Search

    Steve Pascolo

    Both DNA and mRNA can be used as vehicles for gene therapy. Because the immune system is naturally activated by foreign nucleic\\u000a acids thanks to the presence of Toll-like Receptors (TLR) in endosomes (TLR3, 7, and 8 detect exogenous RNA, while TLR9 can\\u000a detect exogenous DNA), the delivery of foreign nucleic acids usually induces an immune response directed against the

  9. Staufen-mediated mRNA decay

    PubMed Central

    Park, Eonyoung; Maquat, Lynne E.

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

  10. Selective mRNA translation in erythropoiesis.

    PubMed

    Thiadens, Klaske A M H; von Lindern, Marieke

    2015-06-01

    The daily production of up to 1011 erythrocytes is tightly controlled to maintain the number of erythrocytes in peripheral blood between narrow boundaries. Availability of growth factors and nutrients, particularly iron, control the proliferation and survival of precursor cells partly through control of mRNA translation. General translation initiation mechanisms can selectively control translation of transcripts that carry specific structures in the UTRs. This selective mRNA translation is an important layer of gene expression regulation in erythropoiesis. Ribosome profiling is a recently developed high throughput sequencing technique for global mapping of translation initiation sites across the transcriptome. Here we describe what is known about control of mRNA translation in erythropoiesis and how ribosome profiling will help to further our knowledge. Ribosome footprinting will give insight in transcript-specific translation at codon resolution, which is of great value to understand many cellular processes during erythropoiesis. It will be of particular interest to understand responses to iron availability and reactive oxygen species (ROS), which affects translation initiation of transcripts harbouring upstream ORFs (uORF) and potential alternative downstream ORFs (aORF). PMID:26009174

  11. Gene regulation by mRNA editing

    SciTech Connect

    Ashkenas, J. [Univ. of Washington, Seattle, WA (United States)

    1997-02-01

    The commonly cited figure of 10{sup 5} genes in the human genome represents a tremendous underestimate of our capacity to generate distinct gene products with unique functions. Our cells possess an impressive collection of tools for altering the products of a single gene to create a variety of proteins. The different gene products may have related but distinct functions, allowing cells of different types or at different developmental stages to fine-tune their patterns of gene expression. These tools may act in the cytoplasm, as when proteins undergo post-translational modifications, or in the nucleus, in the processing of pre-mRNA. Two forms of intranuclear fine-tuning are well established and widely studied: alternative splicing of pre-mRNAs and alternative polyadenylation site selection. In recent years it has become clear that cells possess yet another tool to create RNA sequence diversity, mRNA editing. The term {open_quotes}editing{close_quotes} is applied to posttranscriptional modifications of a purine or pyrimidine, which alter an mRNA sequence as it is read, for example, by ribosomes. Covalent changes to the structure of nucleotide bases are well known to occur on tRNA and rRNA molecules, but such changes in mRNA sequence are novel in that they have the capacity to change specific protein sequences. 43 refs., 1 fig.

  12. Understanding regulation of mRNA by RNA binding proteins

    E-print Network

    Robertson, Alexander De Jong

    2014-01-01

    Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA ...

  13. Coupled Evolution of Transcription and mRNA Degradation

    Microsoft Academic Search

    Mally Dori-Bachash; Efrat Shema; Itay Tirosh

    2011-01-01

    mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in

  14. Cytokine mRNA Expression and Proliferative Responses Induced by Pertussis Toxin, Filamentous Hemagglutinin, and Pertactin of Bordetella pertussis in the Peripheral Blood Mononuclear Cells of Infected and Immunized Schoolchildren and Adults

    PubMed Central

    He, Qiushui; Minh, Nhu Nguyen Tran; Edelman, Kati; Viljanen, Matti K.; Arvilommi, Heikki; Mertsola, Jussi

    1998-01-01

    Pertussis infection is increasingly recognized in older children and adults, indicating the need of booster immunizations in these age groups. We investigated the induction of pertussis-specific immunity in schoolchildren and adults after booster immunization and natural infection. The expression of mRNA of gamma interferon (IFN-?), interleukin-2 (IL-2), IL-4, and IL-5 in the peripheral blood mononuclear cells (PBMCs) was assayed by reverse transcription-PCR. The PBMCs of 17 children immunized with one dose of an acellular vaccine containing pertussis toxin (PT), filamentous hemagglutinin (FHA), and pertactin (PRN) significantly proliferated in vitro after stimulation with the vaccine antigens. The PBMCs of seven infected individuals markedly proliferated in the presence of PT and FHA, but the cells of only two of these subjects responded to PRN. At least one of the antigens induced mRNA for IL-4 and/or IL-5 in the cells of 93% of tested vaccinees and patients, and FHA induced IFN-? mRNA in the cells of two-thirds of them. Expression of mRNA for IFN-? correlated with the production of the cytokine protein. Anti-FHA immunoglobulin G antibodies significantly correlated with FHA-induced proliferative responses both before and after immunization. These results show that booster immunization with acellular pertussis vaccine induces both antibody- and cell-mediated immune responses in schoolchildren. Further, booster immunization and natural infection seem to induce the expression of mRNA of T-helper 1 (Th1) and Th2 type cytokines in similar manners. This observation supports the use of acellular pertussis vaccines for booster immunizations of older children, adolescents, and adults. PMID:9673264

  15. Hierarchy of Protein Tyrosine Kinases in Interleukin-2 (IL-2) Signaling: Activation of Syk Depends on Jak3; However, Neither Syk nor Lck Is Required for IL-2-Mediated STAT Activation

    PubMed Central

    Zhou, Yong-Jie; Magnuson, Kelly S.; Cheng, Tammy P.; Gadina, Massimo; Frucht, David M.; Galon, Jerome; Candotti, Fabio; Geahlen, Robert L.; Changelian, Paul S.; O'Shea, John J.

    2000-01-01

    Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by ?c-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines. PMID:10825200

  16. Effect of interleukin-2 on cell proliferation, sister-chromatid exchange induction, and nuclear stress protein phosphorylation in PHA-stimulated Fischer 344 rat spleen lymphocytes: Modulation by 2-mercaptoethanol

    SciTech Connect

    Morris, S.M.; Aidoo, A.; Domon, O.E.; McGarrity, L.J.; Kodell, R.L.; Schol, H.M.; Hinson, W.G.; Pipkin, J.L.; Casciano, D.A. (National Center for Toxicological Research, Jefferson, AR (USA))

    1990-01-01

    The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly. The number of SCE increased significantly from control frequencies, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 {mu}M 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G{sub 1}-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

  17. Intensive chemotherapy with idarubicin, ara-C, etoposide, and m-AMSA followed by immunotherapy with interleukin-2 for myelodysplastic syndromes and high-risk Acute Myeloid Leukemia (AML).

    PubMed

    Ganser, A; Heil, G; Seipelt, G; Hofmann, W; Fischer, J T; Langer, W; Brockhaus, W; Kolbe, K; Ittel, T H; Brack, N; Fuhr, H G; Knuth, P; Höffken, K; Bergmann, L; Hoelzer, D

    2000-01-01

    Intensive chemotherapy followed by treatment with interleukin-2 (IL-2) was evaluated in a prospective, randomized, multicenter trial including 18 patients with refractory anemia with excess of blasts in transformation (RAEB-T), 86 patients with acute myeloid leukemia (AML) evolving from myelodysplastic syndromes, and six patients with secondary AML after previous chemotherapy. Median age was 58 years (range: 18-76 years). Forty-nine patients (45%) achieved a complete remission (CR) after two induction cycles with idarubicin, ara-C, and etoposide, 52% of them aged 60 years (p=0.06). After two consolidation courses, patients were randomized to four cycles of either high- or low-dose IL-2. Patients aged up to 55 years with an HLA-identical sibling donor were eligible for allogeneic bone marrow transplantation. The median relapse-free survival was 12.5 months, with a probability of ongoing CR at 6.5 years of 19%. Overall survival of all patients was 8 months, and 21 months for the CR patients. Median survival was significantly longer among patients aged

  18. Early phosphatidylinositol 3-kinase/Akt pathway activation limits1 poliovirus-induced JNK-mediated cell death2

    E-print Network

    Boyer, Edmond

    . In poliomyelitis, this survival pathway may limit the spread of PV-induced30 damage in the CNS.31 pasteur-00316053,version1-18Sep2008 #12;3 Poliovirus (PV), from the Picornaviridae family, causes paralytic poliomyelitis

  19. Methotrexate Promotes Platelet Apoptosis via JNK-Mediated Mitochondrial Damage: Alleviation by N-Acetylcysteine and N-Acetylcysteine Amide

    PubMed Central

    Paul, Manoj; Hemshekhar, Mahadevappa; Thushara, Ram M.; Sundaram, Mahalingam S.; NaveenKumar, Somanathapura K.; Naveen, Shivanna; Devaraja, Sannaningaiah; Somyajit, Kumar; West, Robert; Basappa; Nayaka, Siddaiah C.; Zakai, Uzma I.; Nagaraju, Ganesh; Rangappa, Kanchugarakoppal S.; Kemparaju, Kempaiah; Girish, Kesturu S.

    2015-01-01

    Thrombocytopenia in methotrexate (MTX)-treated cancer and rheumatoid arthritis (RA) patients connotes the interference of MTX with platelets. Hence, it seemed appealing to appraise the effect of MTX on platelets. Thereby, the mechanism of action of MTX on platelets was dissected. MTX (10 ?M) induced activation of pro-apoptotic proteins Bid, Bax and Bad through JNK phosphorylation leading to ??m dissipation, cytochrome c release and caspase activation, culminating in apoptosis. The use of specific inhibitor for JNK abrogates the MTX-induced activation of pro-apoptotic proteins and downstream events confirming JNK phosphorylation by MTX as a key event. We also demonstrate that platelet mitochondria as prime sources of ROS which plays a central role in MTX-induced apoptosis. Further, MTX induces oxidative stress by altering the levels of ROS and glutathione cycle. In parallel, the clinically approved thiol antioxidant N-acetylcysteine (NAC) and its derivative N-acetylcysteine amide (NACA) proficiently alleviate MTX-induced platelet apoptosis and oxidative damage. These findings underpin the dearth of research on interference of therapeutic drugs with platelets, despite their importance in human health and disease. Therefore, the use of antioxidants as supplementary therapy seems to be a safe bet in pathologies associated with altered platelet functions. PMID:26083398

  20. mRNA quality control goes transcriptional

    PubMed Central

    Kilchert, Cornelia; Vasiljeva, Lidia

    2013-01-01

    Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5? capped, spliced and 3? polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC (quality control) mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs. Recently, with the growing appreciation that mRNA processing occurs concomitantly with polII (RNA polymerase II) transcription, it has become evident that QC acts at the transcriptional level in addition to degrading aberrant RNAs. In the present review, we discuss mechanisms that allow cells to co-transcriptionally initiate the removal of RNAs as well as down-regulate transcription of transcripts where processing repeatedly fails. PMID:24256272

  1. The Majority of Epidermal T Cells in Psoriasis Vulgaris Lesions can Produce Type 1 Cytokines, Interferon-?, Interleukin2, and Tumor Necrosis Factor-?, Defining TC1 (Cytotoxic T Lymphocyte) and TH1 Effector Populations:1 a Type 1 Differentiation Bias is also Measured in Circulating Blood T Cells in Psoriatic Patients

    Microsoft Academic Search

    Lisa M Austin; Maki Ozawa; Toyoko Kikuchi; Ian B Walters; James G Krueger

    1999-01-01

    Psoriasis vulgaris is a skin disease potentially mediated by pro-inflammatory cytokines produced by type 1 lesional T cells. The capability of individual T cells to produce these cytokines in lesional skin is not known. In this study we measured the ability of lesional and peripheral blood T cells to produce intracellular interferon-?, tumor necrosis factor-?, interleukin-2, interleukin-4, and interleukin-10 proteins

  2. Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-gamma.

    PubMed

    Park, Jeong Ho; Sung, Haan Woo; Yoon, Byung Il; Kwon, Hyuk Moo

    2009-06-01

    The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-gamma were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-gamma (ChIFN-gamma) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killedvaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-gamma did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV. PMID:19461208

  3. Increased expression of the interleukin 2 (IL-2) receptor beta chain (p70) on CD56+ natural killer cells after in vivo IL-2 therapy: p70 expression does not alone predict the level of intermediate affinity IL- 2 binding

    PubMed Central

    1990-01-01

    The expression of the 70-kD beta subunit of the interleukin 2 receptor (IL-2R) has been examined on peripheral blood lymphocytes (PBL) obtained from patients receiving systemic infusions of IL-2. Using monoclonal antibodies directed against p70, flow cytometric analyses revealed a greater than threefold increase in expression of the IL-2R beta chain on CD56+ natural killer (NK) cells from post-IL-2 therapy PBL relative to pre-therapy cells. The level of p70 expression on the post-therapy cells was three- to fourfold greater (based on fluorescence intensity) than the level of p70 expression on YT cells, an NK-like cell line that expresses approximately 12,000 intermediate affinity IL-2 binding sites/cell. Despite the high level of p70 expression, in 125I-IL-2 binding assays only 790-1,290 intermediate affinity IL-2 binding sites/cell were detected on post-therapy cells from six patients. These data represent the first report of increased p70 expression after in vivo IL-2 administration and suggest a requirement for at least one additional subunit for the formation of functional intermediate affinity IL-2Rs. Furthermore, the presence on the surface of post-therapy NK cells of excess p70 that does not bind IL-2 with intermediate affinity implies that the formation of intermediate affinity IL-2Rs is not solely determined by the level of p70 expression, and that the response of NK cells to IL-2 might be regulated by altering the expression of p70 or some other IL-2R subunit. PMID:1698909

  4. Anti-CD3 antibody-induced expression of both p55 and p75 chains of the high affinity interleukin-2 receptor on human T lymphocytes is inhibited by cyclosporin A.

    PubMed Central

    Foxwell, B M; Simon, J; Herrero, J J; Taylor, D; Woerly, G; Cantrell, D; Ryffel, B

    1990-01-01

    The inhibitory effect of cyclosporin (CsA) was investigated on human lymphocytes stimulated by anti-T-cell antibodies (anti-CD3 and -CD2) or mitogenic lectins. Whereas inhibition of cell proliferation (50%) occurred at 10 ng/ml CsA after cell activation via CD3 or CD2, higher CsA concentrations (300 ng/ml) were necessary to inhibit lectin-mediated cell activation (PHA, Con A). Exogenous recombinant interleukin-2 (rIL-2) partially reversed the inhibitory effect on antibody-stimulated cells only; however, at higher CsA concentrations (300 ng/ml) proliferation was again inhibited. Thus, CsA affected IL-2R expression and/or function at higher concentrations (300 ng/ml). CsA had no effect on receptor function as measured on IL-2-dependent cell growth of CTLL cells or preactivated lymphocytes. However, CsA inhibited both high and low affinity receptor expression as shown by [125I]IL-2 equilibrium binding studies on anti-CD3-stimulated cells. Cross-linking studies revealed that both p55 (TAC) and p75 chains of the IL-2R were not induced at low CsA concentrations (10 ng/ml). However, addition of rIL-2 reversed CsA inhibition of IL-2R expression. It is concluded that CsA, at least in anti-CD3-stimulated cells, inhibits IL-2R expression and cell proliferation with similar potency. Exogenous rIL-2 reverses CsA inhibition of IL-2R expression. This might be due to binding of rIL-2 to receptors which escape CsA inhibition, thereby up-regulating receptor expression which is drug resistant. Images Figure 4 Figure 5 PMID:2312149

  5. 1?,25-dihydroxyvitamin D3 in combination with transforming growth factor-? increases the frequency of Foxp3? regulatory T cells through preferential expansion and usage of interleukin-2.

    PubMed

    Chambers, Emma S; Suwannasaen, Duangchan; Mann, Elizabeth H; Urry, Zoe; Richards, David F; Lertmemongkolchai, Ganjana; Hawrylowicz, Catherine M

    2014-09-01

    A high prevalence of vitamin D insufficiency and deficiency exists worldwide, which is associated with an increased incidence and severity of a range of immune-mediated diseases. This has resulted in considerable interest in the immunodulatory functions of vitamin D. The active form of vitamin D, 1?,25-dihydroxyvitamin D3 [1,25(OH)2D3], has been shown to increase the frequency of Foxp3(+) CD4(+) T regulatory (Treg) cells when present at high concentrations or under strong T-cell stimulation in culture. Supporting evidence exists in vivo for a positive association between serum 25(OH)D and Foxp3(+) Treg cell numbers in humans. The aim of this work was to identify the cytokine milieu required in vitro to promote Foxp3(+) Treg cells in cultures containing 1,25(OH)2D3 at more moderate concentrations (10(-7) M). Stimulation of human CD4(+) T cells with a combination of 1,25(OH)2D3 and transforming growth factor-? (TGF-?) greatly increased the frequency of Foxp3(+) Treg cells, which is proposed to result from the preferential expansion of Foxp3(+) Treg cells, as compared with the Foxp3(-) effector T cells, in culture. The differential effect on proliferation may result from enhanced availability and usage of interleukin-2 by the Foxp3(+) Treg cells compared with Foxp3(-) effector T cells. In summary, modulation of the cytokine environment to one high in TGF-? in the presence of 1,25(OH)2D3(10(-7) M) significantly increased Foxp3(+) Treg cell frequency. These data provide additional evidence for the important immunomodulatory properties of 1,25(OH)2D3 that exist and may help to control inflammatory diseases. PMID:24673126

  6. Protection of chicken against very virulent IBDV provided by in ovo priming with DNA vaccine and boosting with killed vaccine and the adjuvant effects of plasmid-encoded chicken interleukin-2 and interferon-?

    PubMed Central

    Park, Jeong Ho; Sung, Haan Woo; Yoon, Byung Il

    2009-01-01

    The aim of this study was to examine the efficacy of in ovo prime-boost vaccination against infectious bursal disease virus (IBDV) using a DNA vaccine to prime in ovo followed by a killed-vaccine boost post hatching. In addition, the adjuvant effects of plasmid-encoded chicken interleukin-2 and chicken interferon-? were tested in conjunction with the vaccine. A plasmid DNA vaccine (pcDNA-VP243) encoding the VP2, VP4, and VP3 proteins of the very virulent IBDV (vvIBDV) SH/92 strain was injected into the amniotic sac alone or in combination with a plasmid encoding chicken IL-2 (ChIL-2) or chicken IFN-? (ChIFN-?) at embryonation day 18, followed by an intramuscular injection of a commercial killed IBD vaccine at 1 week of age. The chickens were orally challenged with the vvIBDV SH/92 strain at 3 weeks of age and observed for 10 days. In ovo DNA immunization followed by a killed-vaccine boost provided significantly better immunity than the other options. No mortality was observed in this group after a challenge with the vvIBDV. The prime-boost strategy was moderately effective against bursal damage, which was measured by the bursa weight/body weight ratio, the presence of IBDV RNA, and the bursal lesion score. In ovo DNA vaccination with no boost did not provide sufficient immunity, and the addition of ChIL-2 or ChIFN-? did not enhance protective immunity. In the ConA-induced lymphocyte proliferation assay of peripheral blood lymphocyte collected 10 days post-challenge, there was greater proliferation responses in the DNA vaccine plus boost and DNA vaccine with ChIL-2 plus boost groups compared to the other groups. These findings suggest that priming with DNA vaccine and boosting with killed vaccine is an effective strategy for protecting chickens against vvIBDV. PMID:19461208

  7. Cytoplasmic deadenylation: Regulation of mRNA fate

    PubMed Central

    Wiederhold, Katrin; Passmore, Lori A

    2014-01-01

    The poly(A) tail of mRNA has an important influence on the dynamics of gene expression. On one hand, it promotes enhanced mRNA stability to allow production of the protein, even after inactivation of transcription. On the other hand, shortening of the poly(A) tail (deadenylation) slows down translation of the mRNA, or prevents it entirely, by inducing mRNA decay. Thus deadenylation plays a crucial role in the post-transcriptional regulation of gene expression, deciding the fate of individual mRNAs. It acts both in basal mRNA turnover, as well as temporally- and spatially-regulated translation and decay of specific mRNAs. Here, we discuss mRNA deadenylation in eukaryotes, focusing on the main deadenylase, the Ccr4-Not complex, including its composition, regulation and functional roles. PMID:21118121

  8. Interleukin-2 Engineering for improved therapeutic effectiveness

    E-print Network

    Rao, Balaji Madhav, 1978-

    2004-01-01

    (cont.) (K[d] [approximately] 10pM) for its private alpha receptor subunit, unlike wild-type IL-2 (K[d] [approximately] 10 nM). IL-2 mutants with picomolar affinity for IL-2R? stimulate T cell growth responses quantitatively ...

  9. MECHANISMS OF CYTOPLASMIC mRNA TURNOVER IN EUKARYOTES

    E-print Network

    Bedwell, David M.

    @uab.edu Rm 456, Bevill Bldg 975-6585 1. mRNA stability plays a key role in controlling basal gene expression inefficiently. This increases the quality control of mRNA biogenesis and gene expression. Significance of m 12:1024 Hosoda et al (2003) JBC 278:38287 Deadenylation Is Coupled to Translation Termination -eRF3

  10. Regression of bladder tumors in mice treated with interleukin 2 gene- modified tumor cells [published erratum appears in J Exp Med 1993 Jun 1;177(6):following 1831

    PubMed Central

    1993-01-01

    This study explored the use of interleukin 2 (IL-2) and interferon gamma (IFN-gamma) gene-modified tumor cells as cellular vaccines for the treatment of bladder cancer. The mouse MBT-2 tumor used is an excellent model for human bladder cancer. This carcinogen-induced tumor of bladder origin resembles human bladder cancer in its etiology and histology, and responds to treatment in a manner similar to its human counterpart. Using retroviral vectors, the human IL-2 and mouse IFN- gamma genes were introduced and expressed in MBT-2 cells. The tumor- forming capacity of the cytokine gene-modified MBT-2 cells was significantly impaired, since no tumors formed in mice injected intradermally with either IL-2- or IFN-gamma-secreting cells, using cell doses far exceeding the minimal tumorigenic dose of parental MBT-2 cells. Furthermore, mice that rejected the IL-2- or IFN-gamma-secreting tumor cells became highly resistant to a subsequent challenge with parental MBT-2 cells, but not to 38C13 cells, a B cell lymphoma of the same genetic background. To approximate the conditions as closely as possible to the conditions prevailing in the cancer patient, inactivated cytokine-secreting cells were used to treat animals bearing tumors established by orthotopic implantation of MBT-2 cells into the bladder wall of the animal. Treatment of mice carrying a significant tumor burden with IL-2-secreting MBT-2 cells had a significant inhibitory effect on tumor progression with extended survival. Moreover, in 60% of the mice the tumor regressed completely and the animals remained alive and free of detectable tumor for the duration of the observation period. Treatment of tumor-bearing animals with IL-2- secreting MBT-2 cells was superior to the use of cisplatin, a chemotherapeutic agent used in the treatment of bladder cancer. The therapeutic effect of IFN-gamma-secreting cells was minimal and treatment with unmodified MBT-2 cells had no effect on tumor growth or survival, showing that the parental MBT-2 cells were nonimmunogenic in this experimental setting. Most importantly, mice that exhibited complete tumor regression after treatment with IL-2-secreting MBT-2 cells became resistant to a subsequent challenge with a highly tumorigenic dose of parental MBT-2 cells, indicating that long-term immunological memory was established in the "cured" mice. PMID:8459207

  11. Highly Efficient Expression of Interleukin-2 under the Control of Rabbit ?-Globin Intron II Gene Enhances Protective Immune Responses of Porcine Reproductive and Respiratory Syndrome (PRRS) DNA Vaccine in Pigs

    PubMed Central

    Liu, Jiyu; Hu, Yue; Li, Feng; Wu, Jiaqiang; Guo, Lihui; Liu, Junzhen; Tao, Haiying; Sun, Wenbo; Chen, Lei; Cong, Xiaoyan; Ren, Sufang; Shi, Jianli; Li, Jun; Wang, Jinbao; Huang, Baohua; Wan, Renzhong

    2014-01-01

    Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit ?-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections. PMID:24603502

  12. Ischemic acute tubular necrosis induces an extensive local cytokine response. Evidence for induction of interferon-gamma, transforming growth factor-beta 1, granulocyte-macrophage colony-stimulating factor, interleukin-2, and interleukin-10.

    PubMed

    Goes, N; Urmson, J; Ramassar, V; Halloran, P F

    1995-02-27

    We noted previously that ischemic acute tubular necrosis (ATN) induces local expression of MHC products in renal epithelium. The present investigations were conducted to establish the role of IFN-gamma in the regulation of MHC antigen expression in ATN and to explore the changes in cytokine and growth factor expression induced by ischemic renal injury. We produced unilateral ischemic ATN in mice by clamping the left renal pedicle. MHC class I and II steady state mRNA induction was assessed by northern blot analysis, and MHC product was quantified by the extent of binding of radiolabeled monoclonals to tissue homogenates. The steady state mRNA levels for IFN-gamma, IL-2, IL-10, and granulocyte-macrophage CSF were assessed by reverse transcriptase polymerase chain reaction and the levels for transforming growth factor-beta 1 and prepro-epidermal growth factor (ppEGF) were assessed by Northern blot analysis. In the injured kidneys, steady state mRNA levels for IFN-gamma, IL-2, IL-10, granulocyte-macrophage CSF, and transforming growth factor beta-1 were increased, whereas ppEGF mRNA was markedly decreased. The MHC expression was inhibited by treatment of mice with an anti-IFN-gamma mAb (R4-6A2). Murine EGF, administered in an attempt to accelerate recovery, did not reduce the cytokine and MHC changes. These data indicate that ischemic injury, and possibly other forms of injury, triggers a complex circuit of proinflammatory cytokines. This "injury response" could be relevant to clinical renal transplants, where ATN is associated with poor graft outcome. PMID:7878762

  13. mRNA quantification via second harmonic super resolution microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jing; Cho, Il-Hoon; Kadam, Ulhas; Irudayaraj, Joseph

    2014-03-01

    Cell-specific information on quantity and localization of key mRNA transcripts in single-cell level are critical to the assessment of cancer risk, therapy efficacy, and effective prevention strategies. However, most available technologies for mRNA detection rely on cell extraction that inherently destroys the tissue context and provide only average expression levels from cell populations or whole tissues. In this paper, we proposed a novel super resolution concept, second harmonic generation (SHG) super-resolution microscopy (SHaSM), and apply that to detect single short mRNA transcript, Her2 mRNA, beyond the diffraction limit. Nano-sized SHG crystals, barium titanium oxide BaTiO3 (BTO), were functionalized with two complimentary strands of Her2 mRNA after the chemical surface-modification. Dimer schematic was used to improve the specificity of detection and quantification, where two BTO monomers bind to the Her2 mRNA to form a dimer and being visualized via the SHaSM. SHaSM is able to detect single BTO nanocrystal with ~20 nm spatial resolution, and differentiate BTO dimers (Her2 mRNA) from BTO monomers (non-specific bounded BTO nanocrystal) with high specificity.

  14. Heritable variation of mRNA decay rates in yeast

    PubMed Central

    Andrie, Jennifer M.; Wakefield, Jon

    2014-01-01

    Gene expression levels are determined by the balance between rates of mRNA transcription and decay, and genetic variation in either of these processes can result in heritable differences in transcript abundance. Although the genetics of gene expression has been a subject of intense interest, the contribution of heritable variation in mRNA decay rates to gene expression variation has received far less attention. To this end, we developed a novel statistical framework and measured allele-specific differences in mRNA decay rates in a diploid yeast hybrid created by mating two genetically diverse parental strains. We estimate that 31% of genes exhibit allelic differences in mRNA decay rates, of which 350 can be identified at a false discovery rate of 10%. Genes with significant allele-specific differences in mRNA decay rates have higher levels of polymorphism compared to other genes, with all gene regions contributing to allelic differences in mRNA decay rates. Strikingly, we find widespread evidence for compensatory evolution, such that variants influencing transcriptional initiation and decay have opposite effects, suggesting that steady-state gene expression levels are subject to pervasive stabilizing selection. Our results demonstrate that heritable differences in mRNA decay rates are widespread and are an important target for natural selection to maintain or fine-tune steady-state gene expression levels. PMID:25258386

  15. Role of mRNA Stability during Bacterial Adaptation

    PubMed Central

    Dressaire, Clémentine; Picard, Flora; Redon, Emma; Loubière, Pascal; Queinnec, Isabelle; Girbal, Laurence; Cocaign-Bousquet, Muriel

    2013-01-01

    Bacterial adaptation involves extensive cellular reorganization. In particular, growth rate adjustments are associated with substantial modifications of gene expression and mRNA abundance. In this work we aimed to assess the role of mRNA degradation during such variations. A genome-wide transcriptomic-based method was used to determine mRNA half-lives. The model bacterium Lactococcus lactis was used and different growth rates were studied in continuous cultures under isoleucine-limitation and in batch cultures during the adaptation to the isoleucine starvation. During continuous isoleucine-limited growth, the mRNAs of different genes had different half-lives. The stability of most of the transcripts was not constant, and increased as the growth rate decreased. This half-life diversity was analyzed to investigate determinants of mRNA stability. The concentration, length, codon adaptation index and secondary structures of mRNAs were found to contribute to the determination of mRNA stability in these conditions. However, the growth rate was, by far, the most influential determinant. The respective influences of mRNA degradation and transcription on the regulation of intra-cellular transcript concentration were estimated. The role of degradation on mRNA homeostasis was clearly evidenced: for more than 90% of the mRNAs studied during continuous isoleucine-limited growth of L. lactis, degradation was antagonistic to transcription. Although both transcription and degradation had, opposite effects, the mRNA changes in response to growth rate were driven by transcription. Interestingly, degradation control increased during the dynamic adaptation of bacteria as the growth rate reduced due to progressive isoleucine starvation in batch cultures. This work shows that mRNA decay differs between gene transcripts and according to the growth rate. It demonstrates that mRNA degradation is an important regulatory process involved in bacterial adaptation. However, its impact on the regulation of mRNA levels is smaller than that of transcription in the conditions studied. PMID:23516597

  16. The highways and byways of mRNA decay

    Microsoft Academic Search

    Nicole L. Garneau; Jeffrey Wilusz; Carol J. Wilusz

    2007-01-01

    When considering the control of gene expression, the focus has traditionally been on transcriptional regulation. Recently, however, the large contribution made by mRNA decay has become difficult to ignore. Large-scale analyses indicate that as many as half of all changes in the amounts of mRNA in some responses can be attributed to altered rates of decay. In this article, we

  17. HDAC3 regulates stability of estrogen receptor ? mRNA

    SciTech Connect

    Oie, Shohei; Matsuzaki, Kazuya; Yokoyama, Wataru; Murayama, Akiko; Yanagisawa, Junn, E-mail: junny@agbi.tsukuba.ac.jp

    2013-03-08

    Highlights: ? HDAC inhibitors decrease the stability of ER? mRNA in MCF-7 cells. ? HDAC3 is involved in maintaining ER? mRNA stability in MCF-7 cells. ? ER? mRNA instability by knockdown of HDAC3 reduces the estrogen-dependent proliferation of ER?-positive MCF-7 cells. ? HDAC3 specific inhibitor will be one of new drugs for ER?-positive breast cancers. -- Abstract: Estrogen receptor alpha (ER?) expression is a risk factor for breast cancer. HDAC inhibitors have been demonstrated to down-regulate ER? expression in ER?-positive breast cancer cell lines, but the molecular mechanisms are poorly understood. Here, we showed that HDAC inhibitors decrease the stability of ER? mRNA, and that knockdown of HDAC3 decreases the stability of ER? mRNA and suppresses estrogen-dependent proliferation of ER?-positive MCF-7 breast cancer cells. In the Oncomine database, expression levels of HDAC3 in ER?-positive tumors are higher than those in ER?-negative tumors, thus suggesting that HDAC3 is necessary for ER? mRNA stability, and is involved in the estrogen-dependent proliferation of ER?-positive tumors.

  18. Emerging features of mRNA decay in bacteria.

    PubMed Central

    Steege, D A

    2000-01-01

    The problem of mRNA decay in E. coli has recently seen exciting progress, with the discoveries that key degradation enzymes are associated together in a high molecular weight degradosome and that polyadenylation promotes decay. Recent advances make it clear that mRNA decay in bacteria is far more interesting enzymatically than might have been predicted. In-depth study of specific mRNAs has revealed multiple pathways for degradation. Which pathway a given mRNA follows appears to depend in large part on the location of the initiating endonucleolytic cleavage within the mRNA. During the steps of mRNA decay, stable RNA structures pose formidable barriers to the 3' --> 5' exonucleases. However, polyadenylation is now emerging as a process that plays an important role in maintaining the momentum of exonucleolytic degradation by adding single-stranded extensions to the 3' ends of mRNAs and their decay intermediates, thereby facilitating further exonuclease digestion. PMID:10943888

  19. Endoribonucleases--enzymes gaining spotlight in mRNA metabolism.

    PubMed

    Li, Wai Ming; Barnes, Tavish; Lee, Chow H

    2010-02-01

    The efficient turnover of messenger RNA represents an important mechanism that allows the cell to control gene expression. Until recently, the mechanism of mRNA decay was mainly attributed to exonucleases, comprising enzymes that degrade RNAs from the ends of the molecules. This article summarizes the endoribonucleases, comprising enzymes that cleave RNA molecules internally, which were identified in more recent years in eukaryotic mRNA metabolism. Endoribonucleases have received little attention in the past, based on the difficulty in their identification and a lack of understanding of their physiological significance. This review aims to compare the similarities and differences among this group of enzymes, as well as their known cellular functions. Despite the many differences in protein structure, and thus difficulties in identifying them based on amino acid sequence, most endoribonucleases possess essential cellular functions and have been shown to play an important role in mRNA turnover. PMID:19968858

  20. Apollo 324TM PrepX mRNA Library

    E-print Network

    Apollo 324TM System PrepX mRNA Library Protocol DRAFT ­ P037497, Rev. A ­ 9.14.2012 #12;© Copyright ONLY The Apollo 324 System is for research use only. WARRANTY For warranty and liability information, please see IntegenX terms and conditions of sale. TRADEMARKS Apollo 324, BeadX and PrepX are trademarks

  1. Analysis of mRNA Translation in Cultured Hippocampal Neurons

    Microsoft Academic Search

    Joel D. Richter

    2007-01-01

    Synaptic plasticity, the ability of neuronal synapses to undergo morphological and biochemical changes in response to various stimuli, forms the underlying basis of long?term memory storage. Regulated mRNA translation at synapses is required for this plasticity. However, the mechanism by which translation at synapses is controlled and how the encoded proteins modulate persistent changes in synaptic morphology and functional integration

  2. Analysis of mRNA recognition by human thymidylate synthase.

    PubMed

    Brunn, Nicholas D; Dibrov, Sergey M; Kao, Melody B; Ghassemian, Majid; Hermann, Thomas

    2014-01-01

    Expression of hTS (human thymidylate synthase), a key enzyme in thymidine biosynthesis, is regulated on the translational level through a feedback mechanism that is rarely found in eukaryotes. At low substrate concentrations, the ligand-free enzyme binds to its own mRNA and stabilizes a hairpin structure that sequesters the start codon. When in complex with dUMP (2'-deoxyuridine-5'-monophosphate) and a THF (tetrahydrofolate) cofactor, the enzyme adopts a conformation that is unable to bind and repress expression of mRNA. Here, we have used a combination of X-ray crystallography, RNA mutagenesis and site-specific cross-linking studies to investigate the molecular recognition of TS mRNA by the hTS enzyme. The interacting mRNA region was narrowed to the start codon and immediately flanking sequences. In the hTS enzyme, a helix-loop-helix domain on the protein surface was identified as the putative RNA-binding site. PMID:25423174

  3. Differential protein occupancy profiling of the mRNA transcriptome

    PubMed Central

    2014-01-01

    Background RNA-binding proteins (RBPs) mediate mRNA biogenesis, translation and decay. We recently developed an approach to profile transcriptome-wide RBP contacts on polyadenylated transcripts by next-generation sequencing. A comparison of such profiles from different biological conditions has the power to unravel dynamic changes in protein-contacted cis-regulatory mRNA regions without a priori knowledge of the regulatory protein component. Results We compared protein occupancy profiles of polyadenylated transcripts in MCF7 and HEK293 cells. Briefly, we developed a bioinformatics workflow to identify differential crosslinking sites in cDNA reads of 4-thiouridine crosslinked polyadenylated RNA samples. We identified 30,000 differential crosslinking sites between MCF7 and HEK293 cells at an estimated false discovery rate of 10%. 73% of all reported differential protein-RNA contact sites cannot be explained by local changes in exon usage as indicated by complementary RNA-seq data. The majority of differentially crosslinked positions are located in 3? UTRs, show distinct secondary-structure characteristics and overlap with binding sites of known RBPs, such as ELAVL1. Importantly, mRNA transcripts with the most significant occupancy changes show elongated mRNA half-lives in MCF7 cells. Conclusions We present a global comparison of protein occupancy profiles from different cell types, and provide evidence for altered mRNA metabolism as a result of differential protein-RNA contacts. Additionally, we introduce POPPI, a bioinformatics workflow for the analysis of protein occupancy profiling experiments. Our work demonstrates the value of protein occupancy profiling for assessing cis-regulatory RNA sequence space and its dynamics in growth, development and disease. PMID:24417896

  4. Regulation of CCR2 chemokine receptor mRNA stability.

    PubMed

    Xu, L; Rahimpour, R; Ran, L; Kong, C; Biragyn, A; Andrews, J; Devries, M; Wang, J M; Kelvin, D J

    1997-11-01

    During inflammatory and immunological responses, leukocytes respond to external stimuli by altering the stability of cytokine and cytokine receptor messages. Change in message stability is an effective mechanism for rapidly regulating steady state levels of mRNA. Cytokine messages containing A-U-rich elements located in the 3' untranslated region (ARE) are the best studied examples of this process. AREs have been shown to act as targeting motifs for degradation of cytokine and transcription factor messages. We have recently observed that the interleukin-8 (IL-8) receptor messages, IL-8RA and B (CXCR1 and CXCR2), also undergo changes in stability in response to the inflammatory stimulator lipopolysaccharide (LPS). To determine whether regulation of message stability is a common mechanism for modulation of chemokine receptor mRNA we explored whether the stability of the CC chemokine receptor message for CCR2 (monocyte chemotactic protein-1 receptor) is also regulated by LPS. We found that LPS induces a rapid loss of steady state levels of CCR2 message through message degradation. Furthermore, LPS stimulated the decay of Poly(A) CCR2 mRNA faster than total CCR2 RNA, indicating that deadenylation is the first step in LPS-induced CCR2 RNA degradation. We conclude from these experiments that LPS stimulates the rapid degradation of CCR2 messages through a two-step process, deadenylation followed by degradation of the message body. In contrast to the results obtained for CCR2 mRNA, macrophage inflammatory protein-1alpha messages, which contain an ARE motif, were stabilized by LPS stimulation, indicating that chemokine and chemokine receptor mRNA stability are regulated by different and opposing mechanisms. PMID:9365120

  5. Cytokine mRNA expression in painful radiculopathy

    PubMed Central

    Rothman, Sarah M.; Huang, Zhong; Lee, Kathryn E.; Weisshaar, Christine L.; Winkelstein, Beth A.

    2009-01-01

    Inflammatory cytokines contribute to lumbar radiculopathy. Regulation of cytokines for transient cervical injuries, with or without longer-lasting inflammation, remains to be defined. The C7 root in the rat underwent compression (10gf), chromic gut suture exposure (chr), or their combination (10gf+chr). Ipsilateral C7 spinal cord and dorsal root ganglia (DRG) were harvested at 1 hour after injury for real-time PCR analysis of IL-1?, IL-6 and TNF?. Cytokine mRNA increased following all three injuries. TNF? mRNA in the DRG was significantly increased over sham following 10gf+chr (p=0.026). Spinal IL-1? was significantly increased over sham following 10gf and 10gf+chr (p<0.024); IL-6 was significantly increased after 10gf+chr (p<0.024). In separate studies, the soluble TNF? receptor was administered at injury and again at 6 hours in all injury paradigms. Allodynia was assessed and tissue samples were harvested for cytokine PCR. Allodynia significantly decreased with receptor administration for 10gf and 10gf+chr (p<0.005). Treatment also significantly decreased IL-1? and TNF? mRNA in the DRG for 10gf+chr (p<0.028) at day 1. Results indicate an acute, robust cytokine response in cervical nerve root injury with varying patterns dependent on injury type, and that early increases in TNF? mRNA in the DRG may drive pain-related signaling for transient cervical injuries. Perspective Inflammatory cytokine mRNA in the DRG and spinal cord are defined following painful cervical nerve root injury. Studies describe a role for TNF? in mediating behavioral sensitivity and inflammatory cytokines in transient painful radiculopathy. Results outline an early response of inflammatory cytokine upregulation in cervical pain. PMID:18848809

  6. UIF, a New mRNA export adaptor that works together with REF/ALY, requires FACT for recruitment to mRNA.

    PubMed

    Hautbergue, Guillaume M; Hung, Ming-Lung; Walsh, Matthew J; Snijders, Ambrosius P L; Chang, Chung-Te; Jones, Rachel; Ponting, Chris P; Dickman, Mark J; Wilson, Stuart A

    2009-12-01

    Messenger RNA (mRNA) export adaptors play an important role in the transport of mRNA from the nucleus to the cytoplasm. They couple early mRNA processing events such as 5' capping and 3' end formation with loading of the TAP/NXF1 export receptor onto mRNA. The canonical adaptor REF/ALY/Yra1 is recruited to mRNA via UAP56 and subsequently delivers the mRNA to NXF1 [1]. Knockdown of UAP56 [2, 3] and NXF1 [4-7] in higher eukaryotes efficiently blocks mRNA export, whereas knockdown of REF only causes a modest reduction, suggesting the existence of additional adaptors [8-10]. Here we identify a new UAP56-interacting factor, UIF, which functions as an export adaptor, binding NXF1 and delivering mRNA to the nuclear pore. REF and UIF are simultaneously found on the same mRNA molecules, and both proteins are required for efficient export of mRNA. We show that the histone chaperone FACT specifically binds UIF, but not REF, via the SSRP1 subunit, and this interaction is required for recruitment of UIF to mRNA. Together the results indicate that REF and UIF represent key human adaptors for the export of cellular mRNAs via the UAP56-NXF1 pathway. PMID:19836239

  7. Interplay between Exonic Splicing Enhancers, mRNA Processing, and mRNA Surveillance in the Dystrophic Mdx Mouse

    PubMed Central

    Buvoli, Massimo; Buvoli, Ada; Leinwand, Leslie A.

    2007-01-01

    Background Pre-mRNA splicing, the removal of introns from RNA, takes place within the spliceosome, a macromolecular complex composed of five small nuclear RNAs and a large number of associated proteins. Spliceosome assembly is modulated by the 5? and 3? splice site consensus sequences situated at the ends of each intron, as well as by exonic and intronic splicing enhancers/silencers recognized by SR and hnRNP proteins. Nonsense mutations introducing a premature termination codon (PTC) often result in the activation of cellular quality control systems that reduce mRNA levels or alter the mRNA splicing pattern. The mdx mouse, a commonly used genetic model for Duchenne muscular dystrophy (DMD), lacks dystrophin by virtue of a premature termination codon (PTC) in exon 23 that also severely reduces the level of dystrophin mRNA. However, the effect of the mutation on dystrophin RNA processing has not yet been described. Methodology/Principal Finding Using combinations of different biochemical and cellular assays, we found that the mdx mutation partially disrupts a multisite exonic splicing enhancer (ESE) that is recognized by a 40 kDa SR protein. In spite of the presence of an inefficient intron 22 3? splice site containing the rare GAG triplet, the mdx mutation does not activate nonsense-associated altered splicing (NAS), but induces exclusively nonsense-mediated mRNA decay (NMD). Functional binding sites for SR proteins were also identified in exon 22 and 24, and in vitro experiments show that SR proteins can mediate direct association between exon 22, 23, and 24. Conclusions/Significance Our findings highlight the complex crosstalk between trans-acting factors, cis-elements and the RNA surveillance machinery occurring during dystrophin mRNA processing. Moreover, they suggest that dystrophin exon–exon interactions could play an important role in preventing mdx exon 23 skipping, as well as in facilitating the pairing of committed splice sites. PMID:17487273

  8. Peptide inhibitors of botulinum neurotoxin by mRNA display

    SciTech Connect

    Yiadom, Kwabena P.A.B. [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States); Muhie, Seid [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States); Yang, David C.H. [Department of Chemistry, Georgetown University, Washington, DC 20057 (United States)]. E-mail: yangdc@georgetown.edu

    2005-10-07

    Botulinum neurotoxins (BoNTs) are extremely toxic. The metalloproteases associated with the toxins cleave proteins essential for neurotransmitter secretion. Inhibitors of the metalloprotease are currently sought to control the toxicity of BoNTs. Toward that goal, we produced a synthetic cDNA for the expression and purification of the metalloprotease of BoNT/A in Escherichia coli as a biotin-ubiquitin fusion protein, and constructed a combinatorial peptide library to screen for BoNT/A light chain inhibitors using mRNA display. A protease assay was developed using immobilized intact SNAP-25 as the substrate. The new peptide inhibitors showed a 10-fold increase in affinity to BoNT/A light chain than the parent peptide. Interestingly, the sequences of the new peptide inhibitors showed abundant hydrophobic residues but few hydrophilic residues. The results suggest that mRNA display may provide a general approach in developing peptide inhibitors of BoNTs.

  9. A genomic view of mRNA turnover in yeast.

    PubMed

    Pérez-Ortín, José E; Jordán-Pla, Antonio; Pelechano, Vicent

    2011-01-01

    The steady-state mRNA level is the result of two opposing processes: transcription and degradation; both of which can provide important points to regulate gene expression. In the model organism yeast Saccharomyces cerevisiae, it is now possible to determine, at the genomic level, the transcription and degradation rates, as well as the mRNA amount, using DNA chip or parallel sequencing technologies. In this way, the contribution of both rates to individual and global gene expressions can be analysed. Here we review the techniques used for the genomic evaluation of the transcription and degradation rates developed for this yeast, and we discuss the integration of the data obtained to fully analyse the expression strategies used by yeast and other eukaryotic cells. PMID:21819946

  10. CELL BIOLOGY: TAPping into mRNA Export

    NSDL National Science Digital Library

    Melissa J. Moore (Brandeis University; Department of Biochemistry)

    2001-11-30

    Access to the article is free, however registration and sign-in are required. There seem to be numerous pathways for exporting mRNAs from the nucleus to the cytoplasm. But working out which set of export adaptors and receptors transport individual mRNAs has been very difficult. In a Perspective, Moore and Rosbash discuss a new strategy using cell-penetrating peptide inhibitors for unraveling the routes of mRNA export in living cells (Gallouzi and Steitz).

  11. Vibrational force alters mRNA expression in osteoblasts

    NASA Technical Reports Server (NTRS)

    Tjandrawinata, R. R.; Vincent, V. L.; Hughes-Fulford, M.

    1997-01-01

    Serum-deprived mouse osteoblastic (MC3T3E1) cells were subjected to a vibrational force modeled by NASA to simulate a space shuttle launch (7.83 G rms). The mRNA levels for eight genes were investigated to determine the effect of vibrational force on mRNA expression. The mRNA levels of two growth-related protooncogenes, c-fos and c-myc, were up-regulated significantly within 30 min after vibration, whereas those of osteocalcin as well as transforming growth factor-beta1 were decreased significantly within 3 h after vibration. No changes were detected in the levels of beta-actin, histone H4, or cytoplasmic phospholipase A2 after vibration. No basal levels of cyclooxygenase-2 expression were detected. In addition, the extracellular concentrations of prostaglandin E2 (PGE2), a potent autocrine/paracrine growth factor in bone, were not significantly altered after vibration most likely due to the serum deprivation state of the osteoblasts. In comparison with the gravitational launch profile, vibrational-induced changes in gene expression were greater both in magnitude and number of genes activated. Taken together, these data suggest that the changes in mRNA expression are due to a direct mechanical effect of the vibrational force on the osteoblast cells and not to changes in the local PGE2 concentrations. The finding that launch forces induce gene expression is of utmost importance since many of the biological experiments do not dampen vibrational loads on experimental samples. This lack of dampening of vibrational forces may partially explain why 1-G onboard controls sometimes do not reflect 1-G ground controls. These data may also suggest that scientists use extra ground controls that are exposed to launch forces, have these forces dampened on launched samples, or use facilities such as Biorack that provide an onboard 1-G centrufuge in order to control for space shuttle launch forces.

  12. Oligonucleotide switches and nanomaterials for intracellular mRNA sensing

    NASA Astrophysics Data System (ADS)

    Tombelli, S.; Ballestri, M.; Giambastiani, G.; Giannetti, A.; Guerrini, A.; Sotgiu, G.; Trono, C.; Tuci, G.; Varchi, G.; Baldini, F.

    2013-06-01

    We describe here the conjugation of polymethylmethacrylate nanoparticles to particular oligonucleotide switches, termed molecular beacons (MBs), as potential intracellular nanosensors. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The nanosensors have been characterized in vitro by investigating the analytical performances of the chosen molecular beacon and its functionalities after conjugation to the nanoparticles.

  13. Compilation of E. coli mRNA promoter sequences.

    PubMed Central

    Lisser, S; Margalit, H

    1993-01-01

    An updated compilation of 300 E. coli mRNA promoter sequences is presented. For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally. We comment on the reliability of the sequence databanks and analyze the conservation of known promoter features in the current compilation. This database is available by E-mail. PMID:8479900

  14. The utility of protein and mRNA correlation

    SciTech Connect

    Payne, Samuel H.

    2015-01-01

    Transcriptomic, proteomic and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight.

  15. Nuclear mRNA export: insights from virology

    Microsoft Academic Search

    Bryan R Cullen

    2003-01-01

    To maximize the production of progeny virions, several viruses have evolved mechanisms that promote the selective nuclear export of viral mRNA transcripts while, in some cases, inhibiting the export of cellular mRNAs. To achieve this goal, viruses have evolved regulatory proteins and cis-acting RNA elements that selectively interact with key cellular nuclear export factors. Efforts to identify the cellular targets

  16. The utility of protein and mRNA correlation.

    PubMed

    Payne, Samuel H

    2015-01-01

    Transcriptomic, proteomic, and metabolomic measurements are revolutionizing the way we model and predict cellular behavior, and multi-omic comparisons are being published with increased regularity. Some have expected a trivial and predictable correlation between mRNA and protein; however, the manifest complexity of biological regulation suggests a more nuanced relationship. Indeed, observing this lack of strict correlation provides clues for new research topics, and has the potential for transformative biological insight. PMID:25467744

  17. RNA complementary to ?-amylase mRNA in barley

    Microsoft Academic Search

    John C. Rogers

    1988-01-01

    Two experimental approaches demonstrate that different types of RNA complementary to a-amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B a-amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids.

  18. Sorting signal targeting mRNA into hepatic extracellular vesicles

    PubMed Central

    Szostak, Natalia; Royo, Felix; Rybarczyk, Agnieszka; Szachniuk, Marta; Blazewicz, Jacek; del Sol, Antonio; Falcon-Perez, Juan M

    2014-01-01

    Intercellular communication mediated by extracellular vesicles has proved to play an important role in normal and pathological scenarios. However not too much information about the sorting mechanisms involved in loading the vesicles is available. Recently, our group has characterized the mRNA content of vesicles released by hepatic cellular systems, showing that a set of transcripts was particularly enriched in the vesicles in comparison with their intracellular abundance. In the current work, based on in silico bioinformatics tools, we have mapped a novel sequence of 12 nucleotides C[TA]G[GC][AGT]G[CT]C[AT]GG[GA], which is significantly enriched in the set of mRNAs that accumulate in extracellular vesicles. By including a 3?-UTR containing this sequence in a luciferase mRNA reporter, we have shown that in a hepatic cellular system this reporter mRNA was incorporated into extracellular vesicles. This study identifies a sorting signal in mRNAs that is involved in their enrichment in EVs, within a hepatic non-tumoral cellular model. PMID:24921245

  19. Sequence and expression of ferredoxin mRNA in barley

    SciTech Connect

    Zielinski, R.; Funder, P.M.; Ling, V. (Univ. of Illinois, Urbana (USA))

    1990-05-01

    We have isolated and structurally characterized a full-length cDNA clone encoding ferredoxin from a {lambda}gt10 cDNA library prepared from barley leaf mRNA. The ferredoxin clone (pBFD-1) was fused head-to-head with a partial-length cDNA clone encoding calmodulin, and was fortuitously isolated by screening the library with a calmodulin-specific oligonucleotide probe. The mRNA sequence from which pBFD-1 was derived is expressed exclusively in the leaf tissues of 7-d old barley seedlings. Barley pre-ferredoxin has a predicted size of 15.3 kDal, of which 4.6 kDal are accounted for by the transit peptide. The polypeptide encoded by pBFD-1 is identical to wheat ferredoxin, and shares slightly more amino acid sequence similarity with spinach ferredoxin I than with ferredoxin II. Ferredoxin mRNA levels are rapidly increased 10-fold by white light in etiolated barley leaves.

  20. A Nucleolin-Binding 3' Untranslated Region Element Stabilizes  Globin mRNA In Vivo

    Microsoft Academic Search

    Yong Jiang; Xiang-Sheng Xu; J. Eric Russell

    2006-01-01

    The normal expression of human globin is critically dependent upon the constitutively high stability of its encoding mRNA. Unlike with -globin mRNA, the specific cis-acting determinants and trans-acting factors that participate in stabilizing -globin mRNA are poorly described. The current work uses a linker-scanning strategy to identify a previously unknown determinant of mRNA stability within the -globin 3 untranslated region

  1. Differential regulation of host mRNA translation during obligate pathogen-plant interactions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Virus infection reprograms the plant messenger RNA (mRNA) transcriptome by activating or interfering with a variety of signaling pathways, but the effects on host mRNA translation have not been explored on a genome-wide scale. To address this issue, Arabidopsis thaliana mRNA transcripts were quantif...

  2. RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

    PubMed Central

    Shen, V; Imamoto, F; Schlessinger, D

    1982-01-01

    Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro. Images PMID:6176575

  3. Detection and partial characterization of proenkephalin mRNA.

    PubMed Central

    Gubler, U; Kilpatrick, D L; Seeburg, P H; Gage, L P; Udenfriend, S

    1981-01-01

    We have used an oligodeoxynucleotide of defined sequence to detect and quantitate proenkephalin mRNA in the poly(A)-containing fraction of RNA from bovine adrenal medullas. The decahexamer 5'-d(G-G-T-A-G-T-C-C-A-T-C-C-A-C-C-A)-3' was synthesized to be complementary to the codons specifying the amino acid sequence NH2-Trp-Trp-Met-Asp-Tyr-Gln-COOH. This stretch of amino acids occurs in peptide I, one of the intermediates in the biosynthetic pathway of the enkephalins in bovine adrenal medulla. This pathway starts with a precursor (proenkephalin) of about 45 kilodaltons [Stern, A. S., Jones, B. N., Shively, J. E., Stein, S. & Udenfriend, S. (1981) Proc. Natl. Acad. Sci. USA 78, 1962-1966]. The decahexamer hybridized to adrenal poly(A)+RNA and was extended into cDNA with reverse transcriptase (RNA-dependent DNA nucleotidyltransferase). Five main discrete products ranging in size from 115 to 168 nucleotides were observed. The sequences of these extensions were found to be identical over the approximately 70 nucleotides sequenced from their 5' termini and corresponded exactly to the sequence expected from the amino acid sequence of peptide I. These cDNAs and the decahexamer itself hybridized to an adrenal medullary poly(A)+RNA species of about 1500 nucleotides, sufficient in size to code for the proposed proenkephalin. At saturation, approximately 2 fmol of the decahexamer were bound per microgram of mRNA; thus, the proenkephalin mRNA represents about 0.1% of the total poly(A)+RNA population in the tissue. Images PMID:6946486

  4. Differential regulation of plastid mRNA stability. Progress report

    SciTech Connect

    Stern, D.B.

    1993-09-01

    Our goal is to identify cis-acting sequences and transacting factors that function in plastid mRNA maturation, stabilization, and/or decay through an in vitro and in vivo analysis of mRNA:protein interactions. Our previous results emphasized the study of 3{prime}end inverted repeat sequences (IRs) that serve both as mRNA processing elements and stability determinants, and associate with plastid proteins that potentially play enzymatic, structural and/or regulatory roles. We seek to define, by single base and internal deletion mutagenesis, the sequence and structural requirements for protein binding to the 3{prime} IRs of petD and psbA mRNAs; to purify RNA-binding proteins that demonstrate gene- or sequence-specific binding, or that are implicated in RNA stabilization or decay; and to investigate the native form of mRNA in the plastid, by attempting to purify ribonucleoprotein (RNP) particles from organelles. Our view of mRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNA-binding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with petD 3{prime} IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the petD stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins.

  5. Targeting mRNA for Alzheimer's and Related Dementias

    PubMed Central

    2014-01-01

    Brain deposition of the amyloid beta-protein (A?) and tau are characteristic features in Alzheimer's disease (AD). Mutations in the A? precursor protein (APP) and a protease involved in A? production from APP strongly argue for a pathogenic role of A? in AD, while mutations in tau are associated with related disorders collectively called frontotemporal lobar degeneration (FTLD). Despite intense effort, therapeutic strategies that target A? or tau have not yet yielded medications, suggesting that alternative approaches should be pursued. In recent years, our laboratory has studied the role of mRNA in AD and FTLD, specifically those encoding tau and the A?-producing protease BACE1. As many FTLD-causing tau mutations destabilize a hairpin structure that regulates RNA splicing, we have targeted this structure with small molecules, antisense oligonucleotides, and small molecule-antisense conjugates. We have also discovered that microRNA interaction with the 3?-untranslated region of tau regulates tau expression. Regarding BACE1, we found that alternative splicing leads to inactive splice isoforms and antisense oligonucleotides shift splicing toward these inactive isoforms to decrease A? production. In addition, a G-quadruplex structure in the BACE1 mRNA plays a role in splice regulation. The prospects for targeting tau and BACE1 mRNAs as therapeutic strategies will be discussed. PMID:24876993

  6. Nonstop mRNA Decay Initiates at the Ribosome

    PubMed Central

    Ge, Zhiyun; Mehta, Preeti; Richards, Jamie; Karzai, A. Wali

    2010-01-01

    SUMMARY The translation machinery deciphers genetic information encoded within mRNAs to synthesize proteins needed for various cellular functions. Defective mRNAs that lack in-frame stop codons trigger nonproductive stalling of ribosomes. We investigated how cells deal with such defective mRNAs, and present evidence to demonstrate that RNase R, a processive 3?-to-5? exoribonuclease, is recruited to stalled ribosomes for the specific task of degrading defective mRNAs. The recruitment process is selective for nonstop mRNAs and is dependent on the activities of SmpB protein and tmRNA. Most intriguingly, our analysis reveals that a unique structural feature of RNase R, the C-terminal lysine-rich (K-rich) domain, is required both for productive ribosome engagement and targeted nonstop mRNA decay activities of the enzyme. These findings provide new insights into how a general RNase is recruited to the translation machinery and highlight a novel role for the ribosome as a platform for initiating nonstop mRNA decay. PMID:21091502

  7. Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets

    PubMed Central

    REHWINKEL, JAN; LETUNIC, IVICA; RAES, JEROEN; BORK, PEER; IZAURRALDE, ELISA

    2005-01-01

    Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that degrades mRNAs containing nonsense codons, and regulates the expression of naturally occurring transcripts. While NMD is not essential in yeast or nematodes, UPF1, a key NMD effector, is essential in mice. Here we show that NMD components are required for cell proliferation in Drosophila. This raises the question of whether NMD effectors diverged functionally during evolution. To address this question, we examined expression profiles in Drosophila cells depleted of all known metazoan NMD components. We show that UPF1, UPF2, UPF3, SMG1, SMG5, and SMG6 regulate in concert the expression of a cohort of genes with functions in a wide range of cellular activities, including cell cycle progression. Only a few transcripts were regulated exclusively by individual factors, suggesting that these proteins act mainly in the NMD pathway and their role in mRNA decay has not diverged substantially. Finally, the vast majority of NMD targets in Drosophila are not orthologs of targets previously identified in yeast or human cells. Thus phenotypic differences observed across species following inhibition of NMD can be largely attributed to changes in the repertoire of regulated genes. PMID:16199763

  8. Prolyl carboxypeptidase mRNA expression in the mouse brain.

    PubMed

    Jeong, Jin Kwon; Diano, Sabrina

    2014-01-13

    Prolyl carboxypeptidase (PRCP), a serine protease, is widely expressed in the body including liver, lung, kidney and brain, with a variety of known substrates such as plasma prekallikrein, bradykinin, angiotensins II and III, and ?-MSH, suggesting its role in the processing of tissue-specific substrates. In the brain, PRCP has been shown to inactivate hypothalamic ?-MSH, thus modulating melanocortin signaling in the control of energy metabolism. While its expression pattern has been reported in the hypothalamus, little is known on the distribution of PRCP throughout the mouse brain. This study was undertaken to determine PRCP expression in the mouse brain. Radioactive in situ hybridization was performed to determine endogenous PRCP mRNA expression. In addition, using a gene-trap mouse model for PRCP deletion, X-gal staining was performed to further determine PRCP distribution. Results from both approaches showed that PRCP gene is broadly expressed in the brain. PMID:24161824

  9. An agent-based model for mRNA export through the nuclear pore complex.

    PubMed

    Azimi, Mohammad; Bulat, Evgeny; Weis, Karsten; Mofrad, Mohammad R K

    2014-11-01

    mRNA export from the nucleus is an essential step in the expression of every protein- coding gene in eukaryotes, but many aspects of this process remain poorly understood. The density of export receptors that must bind an mRNA to ensure export, as well as how receptor distribution affects transport dynamics, is not known. It is also unclear whether the rate-limiting step for transport occurs at the nuclear basket, in the central channel, or on the cytoplasmic face of the nuclear pore complex. Using previously published biophysical and biochemical parameters of mRNA export, we implemented a three-dimensional, coarse-grained, agent-based model of mRNA export in the nanosecond regime to gain insight into these issues. On running the model, we observed that mRNA export is sensitive to the number and distribution of transport receptors coating the mRNA and that there is a rate-limiting step in the nuclear basket that is potentially associated with the mRNA reconfiguring itself to thread into the central channel. Of note, our results also suggest that using a single location-monitoring mRNA label may be insufficient to correctly capture the time regime of mRNA threading through the pore and subsequent transport. This has implications for future experimental design to study mRNA transport dynamics. PMID:25253717

  10. Nonsense Mutations in hERG Cause a Decrease in Mutant mRNA Transcripts by Nonsense-Mediated mRNA Decay in Human Long QT Syndrome

    PubMed Central

    Gong, Qiuming; Zhang, Li; Vincent, G. Michael; Horne, Benjamin D.; Zhou, Zhengfeng

    2008-01-01

    Background Long QT syndrome type 2 (LQT2) is caused by mutations in the human ether-a-go-go-related gene (hERG). More than 30% of the LQT2 mutations result in premature termination codons (PTCs). Degradation of PTC-containing mRNA transcripts by nonsense-mediated mRNA decay (NMD) is increasingly recognized as a mechanism for reducing mRNA levels in a variety of human diseases. However, the role of NMD in LQT2 mutations has not been explored. Methods and Results We examined the expression of hERG mRNA in lymphocytes from patients carrying the R1014X mutation using a technique of allele-specific transcript quantification. The R1014X mutation led to a reduced level of mutant mRNA compared to that of the wild-type allele. The decrease in mutant mRNA was also observed in LQT2 nonsense mutations W1001X and R1014X using hERG minigenes expressed in HEK293 cells or neonatal rat ventricular myocytes. Treatment with the protein synthesis inhibitor cycloheximide or RNAi-mediated knockdown of the Upf1 protein resulted in the restoration of mutant mRNA to levels comparable to that of the wild-type minigene, suggesting that hERG nonsense mutations are subject to NMD. Conclusions These results indicate that LQT2 nonsense mutations cause a decrease in mutant mRNA levels by NMD rather than production of truncated proteins. Our findings suggest that the degradation of hERG mutant mRNA by NMD is an important mechanism in LQT2 patients with nonsense or frameshift mutations. PMID:17576861

  11. Imaging and characterizing influenza A virus mRNA transport in living cells

    Microsoft Academic Search

    Wei Wang; Zong-Qiang Cui; Han Han; Zhi-Ping Zhang; Hong-Ping Wei; Ya-Feng Zhou; Ze Chen; Xian-En Zhang

    2008-01-01

    The mechanisms of influenza A virus mRNA intracel- lular transport are still not clearly understood. Here, we visualized the distribution and transport of influ- enza A virus mRNA in living cells using molecular beacon (MB) technology. Confocal-FRAP measure- ments determined that the transport of influenza A virus intronless mRNA, in both nucleus and cyto- plasm, was energy dependent, being similar

  12. GnRH mRNA and protein expression in human preimplantation embryos

    Microsoft Academic Search

    Eva Maria Casan; Francisco Raga; Mary Lake Polan

    1999-01-01

    for human embryos. We postulate that in humans GnRH may play a role in preimplantation embryonic development as well as in the implantation process. To examine this hypothesis, we assessed GnRH and GnRH-receptor mRNA and protein expression in human preimplantation embryos with three pronuclei. GnRH is expressed in peri-implantation human embryos at both the mRNA and protein level. GnRH-receptor mRNA

  13. Differential Responses to Osmotic Stress of Vasopressin-Neurophysin mRNA in Hypothalamic Nuclei

    Microsoft Academic Search

    Peter H. Burbach; Meltsje J. De Hoop; Hartwig Schmale; Dietmar Richter; E. Ronald de Kloet; Jeroen A. Ten Haaf; David De Wied

    1984-01-01

    mRNA encoding the vasopressin-neurophysin precursor was quantitated in individual hypothalamic nuclei of rats by a liquid hybridization assay. Drinking of 2% saline for 14 days, a treatment that increased the plasma vasopressin concentration 9-fold, resulted in a 5- and 2-fold increase in mRNA levels in the supraoptic and paraventricular nucleus, respectively. This osmotic stimulus had no effect on vasopressin-neurophysin mRNA

  14. All things must pass: contrasts and commonalities in eukaryotic and bacterial mRNA decay

    Microsoft Academic Search

    Joel G. Belasco

    2010-01-01

    Despite its universal importance for controlling gene expression, mRNA degradation was initially thought to occur by disparate mechanisms in eukaryotes and bacteria. This conclusion was based on differences in the structures used by these organisms to protect mRNA termini and in the RNases and modifying enzymes originally implicated in mRNA decay. Subsequent discoveries have identified several striking parallels between the

  15. The Mumps Virus Fusion Protein mRNA Sequence and Homology among the Paramyxoviridae Proteins

    Microsoft Academic Search

    NARAYANASAMY ELANGO; TAMAS M. VARSANYI; JAN KOVAMEES; ERLING NORRBY

    1989-01-01

    SUMMARY The complete nucleotide sequence of the fusion protein (F) mRNA of the virulent SBL-1 strain of mumps virus has been determined by sequencing eDNA clones and mRNA and confirmed by partially sequencing the genomic RNA. The mRNA was 1721 nucleotides long excluding the poly(A) sequence and had one long open reading frame which encoded a protein of 538 amino

  16. Urokinase expression by tumor suppressor protein p53: a novel role in mRNA turnover.

    PubMed

    Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Shetty, Rashmi S; Liu, Ming-Cheh; Shetty, Sreerama

    2008-09-01

    Lung carcinoma (H1299) cells deficient in p53 (p53(-/-)) express large amounts of urokinase-type plasminogen activator (uPA) protein and uPA mRNA, and exhibit slower degradation of uPA mRNA than that of p53-expressing nonmalignant Beas2B human airway epithelial cells. Expression of p53 protein in H1299 cells, upon transfection with p53 cDNA, suppressed basal as well as uPA-induced expression of uPA protein in both conditioned media and cell lysates, and decreased the level of steady-state uPA mRNA primarily due to increased uPA mRNA turnover. Inhibition of p53 expression by RNA silencing (SiRNA) in Beas2B cells enhanced basal and uPA-mediated uPA protein and mRNA expression with stabilization of uPA mRNA. Purified p53 binds to the uPA mRNA 3' untranslated region (UTR) in a sequence-specific manner and endogenous uPA mRNA associates with p53 protein isolated from Beas2B cytosolic extracts. p53 binds to a 35-nucleotide uPA 3'UTR sequence and insertion of this sequence into beta-globin mRNA accelerates degradation of otherwise stable beta-globin mRNA. These observations confirm a new role for p53 as a uPA mRNA binding protein that down-regulates uPA mRNA stability and decreases cellular uPA expression. PMID:18390474

  17. Factors that contribute to faecal cyclooxygenase-2 mRNA expression in subjects with colorectal cancer

    Microsoft Academic Search

    Y Hamaya; K Yoshida; T Takai; M Ikuma; A Hishida; S Kanaoka

    2010-01-01

    Background:We previously reported that a faecal cyclooxygenase-2 (COX-2) mRNA assay was useful for identifying colorectal cancer (CRC). This study sought to investigate the factors that contribute to faecal COX-2 mRNA expression in subjects with CRC.Methods:The study cohort comprised 78 patients with CRC and 36 control subjects. The expressions of COX-2, ?-2-microglobulin (B2M), carcinoembryonic antigen (CEA), E-cadherin (E-cad), and CD45 mRNA

  18. Nuclear Imprisonment: Viral Strategies to Arrest Host mRNA Nuclear Export

    PubMed Central

    Kuss, Sharon K.; Mata, Miguel A.; Zhang, Liang; Fontoura, Beatriz M. A.

    2013-01-01

    Viruses possess many strategies to impair host cellular responses to infection. Nuclear export of host messenger RNAs (mRNA) that encode antiviral factors is critical for antiviral protein production and control of viral infections. Several viruses have evolved sophisticated strategies to inhibit nuclear export of host mRNAs, including targeting mRNA export factors and nucleoporins to compromise their roles in nucleo-cytoplasmic trafficking of cellular mRNA. Here, we present a review of research focused on suppression of host mRNA nuclear export by viruses, including influenza A virus and vesicular stomatitis virus, and the impact of this viral suppression on host antiviral responses. PMID:23872491

  19. TOPICAL REVIEW: Mechanisms governing the control of mRNA translation

    NASA Astrophysics Data System (ADS)

    Livingstone, Mark; Atas, Evrim; Meller, Amit; Sonenberg, Nahum

    2010-06-01

    The translation of cellular mRNA to protein is a tightly controlled process often deregulated in diseases such as cancer. Furthering our understanding of mRNA structural elements and the intracellular proteins and signaling pathways that affect protein expression is crucial in the development of new therapies. In this review, we discuss the current state-of-the-art of detecting and determining the role of mRNA sequence elements in regulating the initiation of mRNA translation and the therapeutic strategies that exploit this knowledge to treat disease.

  20. Nitric oxide synthase: mRNA expression of different isoforms in human monocytes/macrophages.

    PubMed

    Reiling, N; Ulmer, A J; Duchrow, M; Ernst, M; Flad, H D; Hauschildt, S

    1994-08-01

    To detect mRNA expression of nitric oxide synthase (NOS) isoforms in human monocytes/macrophages reverse transcription polymerase chain reaction (RT-PCR) was used. mRNA was isolated from stimulated or unstimulated monocytes/macrophages and RT-PCR was performed using oligonucleotide primers derived from mRNA sequences of either human endothelial constitutive (c) or human hepatocyte inducible (i) NOS. RT-PCR of mRNA isolated from resting monocytes and macrophages resulted in the amplification of a cNOS specific mRNA fragment. When the cells were stimulated with lipopolysaccharide (LPS)/interferon-gamma (IFN-gamma) prior to mRNA extraction, RT-PCR yielded an iNOS-specific amplification product. Whereas the activation of both cell types was accompanied by expression of iNOS mRNA, the cNOS signal seemed to be diminished upon immunostimulation. Not only in purified human monocytes but also in the human monocytoid cell lines MonoMac 6, THP-1, and U937 cNOS mRNA was detected. The data clearly demonstrate the presence of iNOS and cNOS mRNA in human monocytes/macrophages and provide the necessary tools to investigate the regulation of NO synthesis in these cell populations. PMID:7520003

  1. Reverse Transcription-Competitive Multiplex PCR Improves Quantification of mRNA in Clinical Samples—Application to the Low Abundance CFTR mRNA

    Microsoft Academic Search

    Stefan M. Loitsch; Stefan Kippenberger; Nurlan Dauletbaev; Thomas O. F. Wagner; Joachim Bargon

    1999-01-01

    Background: To monitor gene therapy, we wished to quantify cystic fibrosis transmembrane conductance regulator (CFTR) mRNA. We developed a PCR-based method to measure CFTR mRNA in clinical samples. Methods: Expression was determined by reverse transcription-competitive multiplex PCR (RCMP) for CFTR and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcripts, and for serial dilutions of two internal cDNA standards consisting of CFTR and GAPDH mutants

  2. Differential regulation of plastid mRNA stability. Progress report

    SciTech Connect

    Not Available

    1992-09-01

    Our view of MRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNAbinding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with penD 3{prime}IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the pend stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins that form complexes of as-yet unknown function.

  3. Differential regulation of plastid mRNA stability

    SciTech Connect

    Not Available

    1992-01-01

    Our view of MRNA decay is that it is regulated by three interactive components: RNA structure, ribonucleases and RNAbinding proteins. We have used mutagenesis to study the role of RNA structure in regulating RNA decay rates, and to identify protein binding and endonuclease recognition sites. We have identified at least three endonuclease activities; one that cleaves psbA RNA; and two whose cleavage patterns with penD 3{prime}IR-RNA has been studied (endoC1 and endoC2). Additionally, we have continued to analyze the properties of the major RNA processing exoribonuclease. We have concentrated our efforts on three RNA-binding proteins. A 100 kd protein with properties suggestive of a mammalian RNP component has been purified. A protein of 55 kd that may also be an endonuclease has been partially purified. We have studied the interaction of a 29 kd protein with the pend stem/loop, and its role in RNA processing. Recently, we have used a novel gel shift/SDS-PAGE technique to identify new RNA-binding proteins that form complexes of as-yet unknown function.

  4. Distinguishing direct from indirect roles for bicoid mRNA localization factors.

    PubMed

    Weil, Timothy T; Xanthakis, Despina; Parton, Richard; Dobbie, Ian; Rabouille, Catherine; Gavis, Elizabeth R; Davis, Ilan

    2010-01-01

    Localization of bicoid mRNA to the anterior of the Drosophila oocyte is essential for patterning the anteroposterior body axis in the early embryo. bicoid mRNA localizes in a complex multistep process involving transacting factors, molecular motors and cytoskeletal components that remodel extensively during the lifetime of the mRNA. Genetic requirements for several localization factors, including Swallow and Staufen, are well established, but the precise roles of these factors and their relationship to bicoid mRNA transport particles remains unresolved. Here we use live cell imaging, super-resolution microscopy in fixed cells and immunoelectron microscopy on ultrathin frozen sections to study the distribution of Swallow, Staufen, actin and dynein relative to bicoid mRNA during late oogenesis. We show that Swallow and bicoid mRNA are transported independently and are not colocalized at their final destination. Furthermore, Swallow is not required for bicoid transport. Instead, Swallow localizes to the oocyte plasma membrane, in close proximity to actin filaments, and we present evidence that Swallow functions during the late phase of bicoid localization by regulating the actin cytoskeleton. In contrast, Staufen, dynein and bicoid mRNA form nonmembranous, electron dense particles at the oocyte anterior. Our results exclude a role for Swallow in linking bicoid mRNA to the dynein motor. Instead we propose a model for bicoid mRNA localization in which Swallow is transported independently by dynein and contributes indirectly to bicoid mRNA localization by organizing the cytoskeleton, whereas Staufen plays a direct role in dynein-dependent bicoid mRNA transport. PMID:20023172

  5. Precision and functional specificity in mRNA decay , Chih Long Liu

    E-print Network

    Herschlag, Dan

    precisely measured the decay of each yeast mRNA, after thermal inactivation of a temperature-sensitive RNA of each mRNA is a fundamental feature of the gene expression program in yeast. Athough initiation of stimuli and cellular signals, including specific hormones (2, 4), iron (5, 6), cell cycle progression (7

  6. Differentiation equations III: mRNA transcription-protein translation model

    NSDL National Science Digital Library

    David Liao

    The first video segment presents a canonical mathematical example from quantitative biology, in which mRNA is transcribed from a gene sequence, and protein is translated from mRNA. The second segment uses eigenvector-eigenvalue analysis to sketch the trajectories of the system in a phase portrait. Finally, the third segment generalizes the linear stability analysis used to study this example.

  7. Enhanced mRNA cap methylation increases Cyclin D1 expression and promotes cell transformation

    PubMed Central

    Cowling, Victoria H.

    2012-01-01

    Cap-dependent mRNA translation requires the methylation of the mRNA guanosine cap by RNA guanine-7-methyltransferase (RNMT). mRNA cap methylation was recently described to be rate-limiting for a subset of mRNAs, and to be enhanced by expression of c-Myc and E2F1, although the biological significance of this finding was not investigated. Here it is reported that increased RNMT expression enhances cellular mRNA cap methyltransferase activity, promotes mammary epithelial cell transformation and co-operates with H-RasV12 or c-Myc to promote fibroblast cell transformation. Cyclin D1 is a prominent oncogene in epithelial tumours. A significant fraction of Cyclin D1 mRNA was found to be unmethylated on the mRNA cap and thus dormant in mammary epithelial cells. Cyclin D1 expression was increased by enhanced mRNA cap methylation. In summary, this report demonstrates that mRNA cap methylation is rate-limiting for expression of an oncogene and cell transformation. PMID:19915615

  8. RELATION OF MRNA REVERSE TRANSCRIPTASE-PCR SIGNAL TO CAMPYLOBACTER SPP. COLONIZATION OF CHICKS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chicken colonization by cells of Campylobacter jejuni having positive mRNA Reverse Transcriptase-PCR (RT-PCR) signal, but which are non-cultivable, would provide a means to relate cell viability with mRNA signal. In addition, the role of viable but non-cultivable (VBNC) forms of Campylobacter spp. f...

  9. mRNA Degradation Machinery in Plants Yukako Chiba & Pamela J. Green

    E-print Network

    Green, Pamela

    Abstract Control of gene expression is exerted by multiple steps such as transcription, mRNA processing, m in the regulation of gene expression. Therefore, mRNA turnover is an impor- tant process not only for setting the basal level of gene expression but also as a regulatory step. Compared to the mechanism of transcription

  10. The use of mRNA display to select high-affinity protein-binding peptides

    E-print Network

    Heller, Eric

    The use of mRNA display to select high-affinity protein-binding peptides David S. Wilson*, Anthony the use of ``mRNA display,'' an in vitro selection technique, to identify peptide aptamers to a protein than is possible with phage display. Starting with a library of 1013 random peptides, 20 different

  11. Regulation of the mRNA half-life in breast cancer

    PubMed Central

    Griseri, Paola; Pagès, Gilles

    2014-01-01

    The control of the half-life of mRNA plays a central role in normal development and in disease progression. Several pathological conditions, such as breast cancer, correlate with deregulation of the half-life of mRNA encoding growth factors, oncogenes, cell cycle regulators and inflammatory cytokines that participate in cancer. Substantial stability means that a mRNA will be available for translation for a longer time, resulting in high levels of protein gene products, which may lead to prolonged responses that subsequently result in over-production of cellular mediators that participate in cancer. The stability of these mRNA is regulated at the 3’UTR level by different mechanisms involving mRNA binding proteins, micro-RNA, long non-coding RNA and alternative polyadenylation. All these events are tightly inter-connected to each other and lead to steady state levels of target mRNAs. Compelling evidence also suggests that both mRNA binding proteins and regulatory RNAs which participate to mRNA half-life regulation may be useful prognostic markers in breast cancers, pointing to a potential therapeutic approach to treatment of patients with these tumors. In this review, we summarize the main mechanisms involved in the regulation of mRNA decay and discuss the possibility of its implication in breast cancer aggressiveness and the efficacy of targeted therapy. PMID:25114848

  12. Expression of IMPDH mRNA after Mycophenolate Administration in Male Volunteers

    PubMed Central

    Min, Won-Ki

    2014-01-01

    Background. Mycophenolic acid (MPA) is the first-line antimetabolic immunosuppressants used in solid organ transplantation. Here, in vivo expressions of the pharmacodynamic marker IMPDH mRNA were analyzed to investigate its usefulness in assessing drug effects. Materials and Methods. Six healthy male volunteers who had the same genotype for genes known to be associated with drug metabolism and effects were selected to remove the confounding effect of these genotypes. Mycophenolate mofetil (MMF, 1?g) was administered once to each subject, and blood samples were collected with certain interval before and after MMF administration to measure lymphocyte expression levels of IMPDH1 and IMPDH2 mRNA. One week later, the experiment was repeated. Results. Whereas IMPDH1 mRNA expression was stable, IMPDH2 mRNA expression showed 2 peaks in the first week. Both IMPDH1 and IMPDH2 mRNA expression in the second week remarkably decreased from the first week. Conclusion. The temporary increase in IMPDH2 mRNA expression in the first week might be due to a reactive reaction against the plasma MPA concentration. In the second week, the intracellular guanosine monophosphate might be depleted, rendering IMPDH2 mRNA synthesis inactive. When MPA is regularly administered to reach a steady state, the IMPDH2 mRNA expression may be kept low and may effectively reflect biological responses regardless of drug intake. PMID:25105143

  13. Multiple Roles of RNase Y in Streptococcus pyogenes mRNA Processing and Degradation

    PubMed Central

    Itzek, Andreas; Malke, Horst; Ferretti, Joseph J.

    2013-01-01

    Control over mRNA stability is an essential part of gene regulation that involves both endo- and exoribonucleases. RNase Y is a recently identified endoribonuclease in Gram-positive bacteria, and an RNase Y ortholog has been identified in Streptococcus pyogenes (group A streptococcus [GAS]). In this study, we used microarray and Northern blot analyses to determine the S. pyogenes mRNA half-life of the transcriptome and to understand the role of RNase Y in global mRNA degradation and processing. We demonstrated that S. pyogenes has an unusually high mRNA turnover rate, with median and mean half-lives of 0.88 min and 1.26 min, respectively. A mutation of the RNase Y-encoding gene (rny) led to a 2-fold increase in overall mRNA stability. RNase Y was also found to play a significant role in the mRNA processing of virulence-associated genes as well as in the rapid degradation of rnpB read-through transcripts. From these results, we conclude that RNase Y is a pleiotropic regulator required for mRNA stability, mRNA processing, and removal of read-through transcripts in S. pyogenes. PMID:23543715

  14. Development of an interleukin 2 receptor targeted gene therapy vehicle 

    E-print Network

    Wattanakaroon, Wanida

    2006-08-16

    antibody to the IL-2R was used to target the oligonucleotide delivery vehicle which consisted of a polyamidoamine dendrimer. Optimization of the delivery vehicle involves understanding the factors that govern its association with oligonucleotide...

  15. Development of an interleukin 2 receptor targeted gene therapy vehicle

    E-print Network

    Wattanakaroon, Wanida

    2006-08-16

    for the malignant phenotype in human cancer. Gene alterations include serial oncogene activation and tumor suppressor gene inactivation (Tripathy, 2000). The leukemias and lymphomas are malignant tumors or cancers of hematopoietic cells of the bone marrow. T... Publishing Group). Allograft rejection Bone marrow Cardiac Liver Renal Autoimmune disease Aplastic anaemia Behcet?s syndrome Crohn?s disease Giant cell arteritis Juvenile rheumatoid arthritis Kawasaki disease Multiple sclerosis Polymalgia...

  16. In the right place at the right time: visualizing and understanding mRNA localization

    PubMed Central

    Buxbaum, Adina R.; Haimovich, Gal

    2015-01-01

    The spatial regulation of protein translation is an efficient way to create functional and structural asymmetries in cells. Recent research has furthered our understanding of how individual cells spatially organize protein synthesis, by applying innovative technology to characterize the relationship between mRNAs and their regulatory proteins, single-mRNA trafficking dynamics, physiological effects of abrogating mRNA localization in vivo and for endogenous mRNA labelling. The implementation of new imaging technologies has yielded valuable information on mRNA localization, for example, by observing single molecules in tissues. The emerging movements and localization patterns of mRNAs in morphologically distinct unicellular organisms and in neurons have illuminated shared and specialized mechanisms of mRNA localization, and this information is complemented by transgenic and biochemical techniques that reveal the biological consequences of mRNA mislocalization. PMID:25549890

  17. Effect of transcription inhibitors on the iron-dependent degradation of transferrin receptor mRNA.

    PubMed

    Seiser, C; Posch, M; Thompson, N; Kühn, L C

    1995-12-01

    Transferrin receptor (TfR) mRNA expression is tightly linked to intracellular iron levels. Upon iron deprivation, the iron regulatory protein (IRP) stabilizes TfR mRNA by binding to stem-loop structures in its 3'-untranslated region, whereas increased iron levels result in inactivation of the mRNA-binding protein and rapid degradation of TfR mRNA. Although IRP and the regulation of its RNA binding activity have been studied intensively, little is known about the mechanism of TfR mRNA degradation. In order to get more information about factors involved in this process we investigated the in vivo IRP-RNA interaction and the effect of transcription inhibitors on the iron-dependent decay of TfR mRNA. Here we demonstrate that part of the active IRP co-localizes with TfR mRNA to the rough endoplasmic reticulum. High intracellular iron levels led to a drastic reduction of this active RNA-bound IRP in vivo, indicating that IRP dissociates prior to TfR mRNA decay. Furthermore, the transcription inhibitor actinomycin D and translation inhibitor cycloheximide suppressed TfR mRNA degradation but did not interfere with the IRP dissociation step. Other inhibitors of RNA polymerase II had no effect on iron-dependent degradation of TfR mRNA. However, high concentrations of alpha-amanitin known to block transcription by RNA polymerase III interfered with mRNA decay suggesting the involvement of polymerase III transcripts in the degradation pathway. PMID:7493976

  18. DNA Replication-Dependent Histone H2A mRNA Expression in Pea Root Tips.

    PubMed Central

    Tanimoto, E. Y.; Rost, T. L.; Comai, L.

    1993-01-01

    Histone H2A mRNA is selectively expressed in scattered subpopulations of cells in the pea (Pisum sativum) root apical meristem. To study whether this specific expression was associated with the cell cycle, a double-labeling technique was used to identify cells replicating DNA during S phase and those expressing H2A mRNA. Cells in S phase were detected by [3H]thymidine incorporation and autoradiography, whereas cells containing H2A mRNA were identified by in situ hybridization using digoxigenin-labeled probes. Approximately 92% of the [3H]thymidine-labeled S-phase cells expressed H2A mRNA and 85% of cells that expressed H2A mRNA were in S phase. In root tissue located basal to the promeristem, synchronous co-located expression was observed in scattered packets of proliferating cells. Furthermore, neither H2A mRNA nor S-phase cells could be detected within the quiescent center or mature root cap. When DNA synthesis was inhibited with hydroxyurea, a commensurate and specific decrease in steady-state levels of H2A mRNA was found. We conclude that cell-specific expression of pea histone H2A mRNA is replication dependent and that H2A mRNA is transiently accumulated during a period of the cell cycle that mostly overlaps the S phase. We propose that the overlap between H2A expression and S phase could occur if H2A mRNA accumulation began in late G1 and abated in late S. PMID:12232021

  19. Experiment K-6-11. Actin mRNA and cytochrome c mRNA concentrations in the tricepts brachia muscle of rats

    NASA Technical Reports Server (NTRS)

    Booth, F. W.; Morrison, P. R.; Thomason, D. B.; Oganov, V. S.

    1990-01-01

    It is well known that some skeletal muscles atrophy as a result of weightlessness (Steffen and Musacchia 1986) and as a result of hindlimb suspension (Tischler et al., 1985, Thomason et al., 1987). Because the content of protein is determined by the rates of protein synthesis and degradation, a decrease in protein synthesis rate, or an increase in the protein degradation, or changes in both could produce the atrophy. Indeed, an increased protein degradation (Tischler et al., 1985) and a decreased protein synthesis (Thomason et al., 1988) have been observed in skeletal muscles of suspended hindlimbs of rats. Any decrease in protein synthesis rate could be caused by decreases in mRNA concentrations. Such decreases in the concentration and content of alpha-actin mRNA and cytochrome c mRNA have been noted in skeletal muscles of hindlimb suspended rats (Babij and Booth, 1988). From these findings researchers hypothesized that alpha-actin mRNA and cytochrome c mRNA would decrease in the triceps brachia muscle of Cosmos 1887 rats.

  20. Single-molecule modeling of mRNA degradation by miRNA: Lessons from data

    PubMed Central

    2015-01-01

    Recent experimental results on the effect of miRNA on the decay of its target mRNA have been analyzed against a previously hypothesized single molecule degradation pathway. According to that hypothesis, the silencing complex (miRISC) first interacts with its target mRNA and then recruits the protein complexes associated with NOT1 and PAN3 to trigger deadenylation (and subsequent degradation) of the target mRNA. Our analysis of the experimental decay patterns allowed us to refine the structure of the degradation pathways at the single molecule level. Surprisingly, we found that if the previously hypothesized network was correct, only about 7% of the target mRNA would be regulated by the miRNA mechanism, which is inconsistent with the available knowledge. Based on systematic data analysis, we propose the alternative hypothesis that NOT1 interacts with miRISC before binding to the target mRNA. Moreover, we show that when miRISC binds alone to the target mRNA, the mRNA is degraded more slowly, probably through a deadenylation-independent pathway. The new biochemical pathway proposed here both fits the data and paves the way for new experimental work to identify new interactions. PMID:26050661

  1. Isolation and In Vitro Translation of ?-Crystallin mRNA from Embryonic Chick Lens Fibers

    PubMed Central

    Zelenka, Peggy; Piatigorsky, Joram

    1974-01-01

    Of the protein synthesized and accumulated during differentiation of embryonic chick lens fibers, 70-80% is the tissue specific protein ?-crystallin. We have isolated and partially characterized the total cytoplasmic mRNA from purified lens fibers of 15-day-old embryos as an initial step toward understanding the regulated expression of ?-crystallin during development. Each lens fiber mass contained an average of 10 ?g of cytoplasmic RNA; approximately 0.1 ?g per fiber mass was recovered in the mRNA fraction by oligo(dT)-cellulose chromatography. The mRNA electrophoresed primarily as a single peak on a polyacrylamide-agarose gel with an apparent molecular weight of about 9 × 105 estimated by comparison with 28S and 18S rRNA markers. Of the protein synthesized in response to the mRNA in cell-free systems derived from Krebs II ascites tumor cells or rabbit reticulocytes, 70-80% comigrated with ?-crystallin on sodium dodecyl sulfatepolyacrylamide-agarose gels. Comparison of the tryptic peptides of ?-crystallin with those of the in vitro products from both heterologous systems established that the lens fiber mRNA contained ?-crystallin mRNA, and that no other functional mRNAs were present in detectable quantities. Thus, the specialization of protein synthesis in embryonic chick lens fibers apparently results from an accumulation of ?-crystallin mRNA in the cytoplasm. Images PMID:4525468

  2. Impact of STAT/SOCS mRNA Expression Levels after Major Injury

    PubMed Central

    Brumann, M.; Matz, M.; Kusmenkov, T.; Stegmaier, J.; Biberthaler, P.; Kanz, K.-G.; Mutschler, W.; Bogner, V.

    2014-01-01

    Background. Fulminant changes in cytokine receptor signalling might provoke severe pathological alterations after multiple trauma. The aim of this study was to evaluate the posttraumatic imbalance of the innate immune system with a special focus on the STAT/SOCS family. Methods. 20 polytraumatized patients were included. Blood samples were drawn 0?h–72?h after trauma; mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3 were quantified by qPCR. Results. IL-10 mRNA expression increased significantly in the early posttraumatic period. STAT 3 mRNA expressions showed a significant maximum at 6?h after trauma. SOCS 1 levels significantly decreased 6?h–72?h after trauma. SOCS 3 levels were significantly higher in nonsurvivors 6?h after trauma. Conclusion. We present a serial, sequential investigation in human neutrophil granulocytes of major trauma patients evaluating mRNA expression profiles of IL-10, STAT 3, SOCS 1, and SOCS 3. Posttraumatically, immune disorder was accompanied by a significant increase of IL-10 and STAT 3 mRNA expression, whereas SOCS 1 mRNA levels decreased after injury. We could demonstrate that death after trauma was associated with higher SOCS 3 mRNA levels already at 6?h after trauma. To support our results, further investigations have to evaluate protein levels of STAT/SOCS family in terms of posttraumatic immune imbalance. PMID:24648661

  3. Context effects on N6-adenosine methylation sites in prolactin mRNA.

    PubMed Central

    Narayan, P; Ludwiczak, R L; Goodwin, E C; Rottman, F M

    1994-01-01

    The methylation of internal adenosine residues in mRNA only occurs within GAC or AAC sequences. Although both of these sequence motifs are utilized, a general preference has been noted for the extended sequence RGACU. Not all RGACU sequences in an mRNA are methylated and the mechanisms that govern the selection of methylation sites in mRNA remain unclear. To address this problem we have examined the methylation of transcripts containing sequences of a natural mRNA, namely, bovine prolactin mRNA. In this mRNA, a specific AGACU sequence in the 3' untranslated region is the predominant site of methylation both in vivo and in vitro. The degree to which N6-adenosine methyltransferase recognizes the sequence context of the consensus methylation site was explored by mutational analysis of the nucleotides adjacent to the core sequence as well as the extended regions in which the core element was found. Our results indicate that efficient methylation depends on the extended five nucleotide consensus sequence but is strongly influenced by the context in which the consensus sequence occurs within the overall mRNA molecule. Furthermore, consensus methylation sites present in an RNA duplex are not recognized by the methyltransferase. Images PMID:8127679

  4. Splicing is required for rapid and efficient mRNA export in metazoans

    PubMed Central

    Luo, Ming-juan; Reed, Robin

    1999-01-01

    Pre-mRNA splicing is among the last known nuclear events before export of mature mRNA to the cytoplasm. At present, it is not known whether splicing and mRNA export are biochemically coupled processes. In this study, we have injected pre-mRNAs containing a single intron or the same mRNAs lacking an intron (?i-mRNAs) into Xenopus oocyte nuclei. We find that the spliced mRNAs are exported much more rapidly and efficiently than the identical ?i-mRNAs. Moreover, competition studies using excess ?i-mRNA indicate that different factor(s) are involved in the inefficient export of ?i-mRNA vs. the efficient export of spliced mRNA. Consistent with this conclusion, spliced mRNA and ?i-mRNA, though identical in sequence, are assembled into different messenger ribonucleoprotein particles (mRNP) in vitro. Strikingly, the mRNA in the spliced mRNP, but not in the ?i-mRNP, is exported rapidly and efficiently. We conclude that splicing generates a specific nucleoprotein complex that targets mRNA for export. Our results, revealing a link between splicing and efficient mRNA export, may explain the reports that an intron is required for efficient expression of many protein-coding genes in metazoans. PMID:10611316

  5. Intranasal mRNA nanoparticle vaccination induces prophylactic and therapeutic anti-tumor immunity

    PubMed Central

    Phua, Kyle K. L.; Staats, Herman F.; Leong, Kam W.; Nair, Smita K.

    2014-01-01

    Direct in vivo administration of messenger RNA (mRNA) delivered in both naked and nanoparticle formats are actively investigated because the use of dendritic cells transfected ex vivo with mRNA for cancer therapy is expensive and needs significant infrastructure. Notably, intravenous and subcutaneous injections are the only routes of administration tested for mRNA nanoparticle tumor vaccination. In this report, we demonstrate that tumor immunity can be achieved via nasal administration of mRNA. Mice nasally immunized with mRNA delivered in nanoparticle format demonstrate delayed tumor progression in both prophylactic and therapeutic immunization models. The observed tumor immunity correlates with splenic antigen-specific CD8+ T cells and is achieved only when mRNA is delivered in nanoparticle but not in naked format. In conclusion, we demonstrate, as a proof-of-concept, a non-invasive approach to mRNA tumor vaccination, increasing its potential as a broadly applicable and off-the-shelf therapy for cancer treatment. PMID:24894817

  6. Definition of global and transcript-specific mRNA export pathways in metazoans

    PubMed Central

    Farny, Natalie G.; Hurt, Jessica A.; Silver, Pamela A.

    2008-01-01

    Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms’ export pathways. Here we report the results of a genome-wide RNAi screen in which we identify 72 factors required for polyadenylated [poly-(A+)] mRNA export from the nucleus in Drosophila cells. Using structural and functional conservation analysis of yeast and Drosophila mRNA export factors, we expose the evolutionary divergence of eukaryotic mRNA export pathways. Additionally, we demonstrate the differential export requirements of two endogenous heat-inducible transcripts—intronless heat-shock protein 70 (HSP70) and intron-containing HSP83—and identify novel export factors that participate in HSP83 mRNA splicing. We characterize several novel factors and demonstrate their participation in interactions with known components of the Drosophila export machinery. One of these factors, Drosophila melanogaster PCI domain-containing protein 2 (dmPCID2), associates with polysomes and may bridge the transition between exported messenger ribonucleoprotein particles (mRNPs) and polysomes. Our results define the global network of factors involved in Drosophila mRNA export, reveal specificity in the export requirements of different transcripts, and expose new avenues for future work in mRNA export. PMID:18086857

  7. Mechanism of decay of the cry1Aa mRNA in Bacillus subtilis.

    PubMed Central

    Vázquez-Cruz, C; Olmedo-Alvarez, G

    1997-01-01

    We undertook the study of the decay process of the cry1Aa mRNA of Bacillus thuringiensis expressed in B. subtilis. The cry1Aa transcript is a 3.7-kb mRNA expressed during sporulation whose transcriptional control has previously been studied in both B. subtilis and B. thuringiensis. We found that the cry1Aa mRNA has a half-life of around 9 min and that its decay occurs through endoribonucleolytic cleavages which result in three groups of high-molecular-weight mRNA intermediates ranging in size from 2.7 to 0.5 kb. A comparative study carried out with Escherichia coli showed a similar pattern of degradation intermediates. Primer extension analysis carried out on RNA from B. subtilis revealed that most cleavages occur within two regions located toward the 5' and 3' ends of the mRNA. The most prominent processing site observed for the cry1Aa mRNA isolated from B. subtilis is only two bases away from that occurring on RNA isolated from E. coli. Most cleavage sites occur at seemingly single-stranded RNA segments rich in A and U nucleotides, suggesting that a common and conserved mechanism may process the cry1Aa mRNA. PMID:9335281

  8. Transcription Control Pathways Decode Patterned Synaptic Inputs into Diverse mRNA Expression Profiles

    PubMed Central

    Jain, Pragati; Bhalla, Upinder S.

    2014-01-01

    Synaptic plasticity requires transcription and translation to establish long-term changes that form the basis for long term memory. Diverse stimuli, such as synaptic activity and growth factors, trigger synthesis of mRNA to regulate changes at the synapse. The palette of possible mRNAs is vast, and a key question is how the cell selects which mRNAs to synthesize. To address this molecular decision-making, we have developed a biochemically detailed model of synaptic-activity triggered mRNA synthesis. We find that there are distinct time-courses and amplitudes of different branches of the mRNA regulatory signaling pathways, which carry out pattern-selective combinatorial decoding of stimulus patterns into distinct mRNA subtypes. Distinct, simultaneously arriving input patterns that impinge on the transcriptional control network interact nonlinearly to generate novel mRNA combinations. Our model combines major regulatory pathways and their interactions connecting synaptic input to mRNA synthesis. We parameterized and validated the model by incorporating data from multiple published experiments. The model replicates outcomes of knockout experiments. We suggest that the pattern-selectivity mechanisms analyzed in this model may act in many cell types to confer the capability to decode temporal patterns into combinatorial mRNA expression. PMID:24787753

  9. Single-molecule modeling of mRNA degradation by miRNA: Lessons from data

    E-print Network

    Celine Sin; Davide Chiarugi; Angelo Valleriani

    2014-10-20

    Recent experimental results on the effect of miRNA on the decay of its target mRNA have been analyzed against a previously hypothesized single molecule degradation pathway. According to that hypothesis, the silencing complex (miRISC) first interacts with its target mRNA and then recruits the protein complexes associated with NOT1 and PAN3 to trigger deadenylation (and subsequent degradation) of the target mRNA. Our analysis of the experimental decay patterns allowed us to refine the structure of the degradation pathways at the single molecule level. Surprisingly, we found that if the previously hypothesized network was correct, only about 7% of the target mRNA would be regulated by the miRNA mechanism, which is inconsistent with the available knowledge. Based on systematic data analysis, we propose the alternative hypothesis that NOT1 interacts with miRISC before binding to the target mRNA. Moreover, we show that when miRISC binds alone to the target mRNA, the mRNA is degraded more slowly, probably through a deadenylation-independent pathway. The new biochemical pathway we propose both fits the data and paves the way for new experimental work to identify new interactions.

  10. T-lymphocyte cytokine mRNA expression in cystic echinococcosis.

    PubMed

    Fauser, S; Kern, P

    1997-04-01

    In the present study we investigated cytokine mRNA expression by peripheral blood mononuclear cells (PBMC) from patients with cystic echinococcosis (CE) after stimulation with different antigens. By using reverse transcriptase polymerase chain reaction (RT-PCR) we could demonstrate that restimulation with crude Echinococcus granulosus antigen (Eg-Ag) induced or enhanced Th2 cytokine mRNA expression, especially IL-5 (by using antigen from sheep cyst fluid) in 23 out of 26 investigated CE patients and IL-10 (by using antigen from camel cyst fluid) in 10 out of 10 investigated CE patients. In contrast, IL-5 mRNA expression was absent in PBMC of healthy controls after Eg-Ag stimulation. To determine the specificity of this reaction we stimulated PBMC from 11 CE patients with crude Echinococcus multilocularis antigen (Em-Ag) and PBMC from 8 CE patients with Toxocara canis antigen (Tc-Ag). We found that the PBMC of patients showed a similar mRNA cytokine pattern on stimulation with Em-Ag when compared with Eg-Ag stimulation. The cytokine mRNA pattern on stimulation with Tc-Ag, however, resembled the cytokine mRNA pattern of unstimulated PBMC. Furthermore, the stimulation of PBMC with crude Mycobacterium tuberculosis antigen (H37Ra) and purified protein derivative (PPD) of M. tuberculosis revealed distinct IL-5 mRNA expression in all investigated CE patients, whereas in healthy controls IL-5 mRNA expression was very weak or totally absent. Thus, our results indicate an induction of Th2 cytokine mRNA expression in CE patients, which is frequently observed in parasite infections. Interestingly, this response persists after stimulation with tuberculosis antigens, which normally induce Th1 response. PMID:9095287

  11. Expression of various insulin-like growth factor-1 mRNA isoforms in colorectal cancer

    PubMed Central

    Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Ma?gorzata; Przybyszewska, Wies?awa; Koczorowska, Maria; Drews, Micha?; Kaczmarek, El?bieta

    2012-01-01

    Aim of the study Several epidemiological studies have attempted to demonstrate a relationship between increased serum level of insulin-like growth factor 1 (IGF-1) and an augmented risk of developing colorectal cancers (CRC). The human IGF-1 gene is composed of 6 exons and demonstrated expression of 6 different splice variants (isoforms) of mRNA (IA, IB, IC, IIA, IIB and IIC). The aim of the study was to evaluate the expression of different isoforms of IGF-1 mRNA in CRC and normal colon tissue. Material and methods 13 paired tissue specimens (colorectal tumor and non-tumor tissues) were analyzed using both quantitative polymerase chain reaction (PCR) and immunocytochemistry methods (IHC). The expression of classes I and II and variants A, B, C of IGF-1 mRNA were measured. Results In CRC higher amounts of IGF-1 class II mRNA than class I mRNA were detected. Among A, B, C isoforms, A variant of IGF-1 mRNA prevailed. The amounts of IGF-1 class I and class II mRNAs and of IGF-1 variant B mRNA were lowered in CRC as compared to the control. In CRC significant correlations were detected between reciprocal expression of class I and class II as well as between I and II isoforms and A, B and C. Conclusions Expression of IGF-1 mRNA isoforms differs between normal and CRC tissues. Even if all isoforms of IGF-1 mRNA manifested correlations with each other in tissues of CRC, expression of all transcripts (except that of isoform A) was significantly decreased as compared to the control. PMID:23788868

  12. Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity

    PubMed Central

    Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

    2014-01-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  13. mRNA on the move: the road to its biological destiny.

    PubMed

    Eliscovich, Carolina; Buxbaum, Adina R; Katz, Zachary B; Singer, Robert H

    2013-07-12

    Cells have evolved to regulate the asymmetric distribution of specific mRNA targets to institute spatial and temporal control over gene expression. Over the last few decades, evidence has mounted as to the importance of localization elements in the mRNA sequence and their respective RNA-binding proteins. Live imaging methodologies have shown mechanistic details of this phenomenon. In this minireview, we focus on the advanced biochemical and cell imaging techniques used to tweeze out the finer aspects of mechanisms of mRNA movement. PMID:23720759

  14. Detection of CK20mRNA in peripheral blood of pancreatic cancer and its clinical significance

    Microsoft Academic Search

    Yun-Li Zhang; Jian-Guo Feng; Jian-Min Gou; Ping Wang; Zhang YL; Feng JG; Gou JM; Zhou LX; Li Xin Zhou

    2005-01-01

    Abstract Abstract Abstract Abstract Abstract AIM:T o detect the expression of CK20mRNA in peripheral blood,of pancreatic ,cancer ,and ,evaluate ,its clinical significance. METHODS:Expression of CK20mRNA in

  15. The Nuclear PolyA-Binding Protein Nab2p Is Essential for mRNA Production.

    PubMed

    Schmid, Manfred; Olszewski, Pawel; Pelechano, Vicent; Gupta, Ishaan; Steinmetz, Lars M; Jensen, Torben Heick

    2015-07-01

    Polyadenylation of mRNA is a key step in eukaryotic gene expression. However, despite the major impact of poly(A) tails on mRNA metabolism, the precise roles of poly(A)-binding proteins (PABPs) in nuclear mRNA biogenesis remain elusive. Here, we demonstrate that rapid nuclear depletion of the S. cerevisiae PABP Nab2p leads to a global loss of cellular mRNA, but not of RNA lacking poly(A) tails. Disappearance of mRNA is a nuclear event, but not due to decreased transcription. Instead, the absence of Nab2p results in robust nuclear mRNA decay by the ribonucleolytic RNA exosome in a polyadenylation-dependent process. We conclude that Nab2p is required to protect early mRNA and therefore constitutes a crucial nuclear mRNA biogenesis factor. PMID:26119729

  16. Expression of facilitative glucose transporter 1 mRNA in colon cancer was not regulated by k-ras

    Microsoft Academic Search

    Yoshikazu Noguchi; Takahide Okamoto; Doulet Marat; Takaki Yoshikawa; Aya Saitoh; Chiharu Doi; Kuniyasu Fukuzawa; Akira Tsuburaya; Shinobu Satoh; Takaaki Ito

    2000-01-01

    The expression of facilitative glucose transporter isoforms in colon adenocarcinoma and the possible role of k-ras in inducing GLUT (glucose transporter) mRNA were studied. RT-PCR demonstrated GLUT2 and GLUT3 expression in 100% of the ten normal colon mucosa samples but detected no GLUT1 mRNA. By contrast, GLUT1 mRNA was detected in all 20 (100%) colon cancer samples examined. GLUT4 mRNA

  17. Visual detection of Akt mRNA in living cell using gold nanoparticle beacon

    NASA Astrophysics Data System (ADS)

    Ma, Yi; Tian, Caiping; Li, Siwen; Wang, Zhaohui; Gu, Yueqing

    2014-09-01

    PI3K-Akt signaling pathway plays the key role in cell apoptosis and survival, and the components of PI3K /Akt signaling pathway are often abnormally expressed in human tumors. Therefore, determination of the Akt (protein kinase B, PKB) messenger ribonucleic acid (mRNA) expression is significantly important in understanding the mechanism of tumor progression. In this study, we designed a special hairpin deoxyribonucleic acid (DNA) functionalized with gold nanoparticles and fluorescein isothiocyanate(FITC) as a beacon for detecting human Akt mRNA. Spectrofluorometer was used to detect the fluorescence quenching and recovery of the beacons, and laser confocal scanning microscopy was adopted to image Akt mRNA in cells. The results showed that this beacon could sensitively and quantitatively measure the Akt mRNA in living cells . This strategy is potentially useful for the cellular imaging of RNA or protein expression in living cells.

  18. Astrocyte cultures derived from human brain tissue express angiotensinogen mRNA

    SciTech Connect

    Milsted, A.; Barna, B.P.; Ransohoff, R.M.; Brosnihan, K.B.; Ferrario, C.M. (Cleveland Clinic Foundation, OH (USA))

    1990-08-01

    The authors have identified human cultured cell lines that are useful for studying angiotensinogen gene expression and its regulation in the central nervous system. A model cell system of human central nervous system origin expressing angiotensinogen has not previously been available. Expression of angiotensinogen mRNA appears to be a basal property of noninduced human astrocytes, since astrocytic cell lines derived from human glioblastomas or nonneoplastic human brain tissue invariably produced angiotensinogen mRNA. In situ hybridization histochemistry revealed that angiotensinogen mRNA production was not limited to a subpopulation of astrocytes because >99% of cells in these cultures contained angiotensinogen mRNA. These cell lines will be useful in studies of the molecular mechanisms controlling angiotensin synthesis and the role of biologically active angiotensin in the human brain by allowing the authors to examine regulation of expression of the renin-angiotensin system in human astrocyte cultures.

  19. Quantitative Imaging of Single mRNA Splice Variants in Living Cells

    PubMed Central

    Lee, Kyuwan; Cui, Yi

    2015-01-01

    Alternative mRNA splicing is a fundamental process of gene regulation via the precise control of the post-transcriptional step that occurs before mRNA translation. Errors in RNA splicing have been known to correlate with different diseases; however, a key limitation is the lack of technologies for live cell monitoring and quantification to understand the process of alternative splicing. Here, we report a spectroscopic strategy for quantitative imaging of mRNA splice variants in living cells, using nanoplasmonic dimer antennas. The spatial and temporal distribution of three selected splice variants of the breast cancer susceptibility gene, BRCA1 were monitored at single copy resolution by measuring the hybridization dynamics of nanoplasmonic antennas targeting complementary mRNA sequences in live cells. Our study provides valuable insights on RNA and its transport in living cells, which has the potential to enhance our understanding of cellular protein complex, pharmacogenomics, genetic diagnosis, and gene therapies. PMID:24747838

  20. Inefficient SRP interaction with a nascent chain triggers a mRNA quality control pathway.

    PubMed

    Karamyshev, Andrey L; Patrick, Anna E; Karamysheva, Zemfira N; Griesemer, Dustin S; Hudson, Henry; Tjon-Kon-Sang, Sandra; Nilsson, IngMarie; Otto, Hendrik; Liu, Qinghua; Rospert, Sabine; von Heijne, Gunnar; Johnson, Arthur E; Thomas, Philip J

    2014-01-16

    Misfolded proteins are often cytotoxic, unless cellular systems prevent their accumulation. Data presented here uncover a mechanism by which defects in secretory proteins lead to a dramatic reduction in their mRNAs and protein expression. When mutant signal sequences fail to bind to the signal recognition particle (SRP) at the ribosome exit site, the nascent chain instead contacts Argonaute2 (Ago2), and the mutant mRNAs are specifically degraded. Severity of signal sequence mutations correlated with increased proximity of Ago2 to nascent chain and mRNA degradation. Ago2 knockdown inhibited degradation of the mutant mRNA, while overexpression of Ago2 or knockdown of SRP54 promoted degradation of secretory protein mRNA. The results reveal a previously unappreciated general mechanism of translational quality control, in which specific mRNA degradation preemptively regulates aberrant protein production (RAPP). PMID:24439374

  1. [Studies on the possibility of mRNA translation for interferon in the heterologic systems].

    PubMed

    Verkhats'ka, A A

    1976-01-01

    It is established that mRNA for interoferon which is isolated from free polysomas bound with endoplasmic reticulum can translate protein in homological and heterological systems. The predominant synthesis of interoferon is observed on the bound polysomas. PMID:1258163

  2. Expression of beta 3-adrenoceptor mRNA in rat tissues.

    PubMed Central

    Evans, B. A.; Papaioannou, M.; Bonazzi, V. R.; Summers, R. J.

    1996-01-01

    1. This study examines the expression of beta 3-adrenoceptor messenger RNA (beta 3-AR mRNA) in rat tissues to allow comparison with atypical beta-adrenoceptors determined by functional and radioligand binding techniques. 2. A reverse transcription/polymerase chain reaction protocol has been developed for determining the relative amounts of beta 3-AR mRNA in rat tissues. 3. Measurement of adipsin and uncoupling protein (UCP) mRNA was used to examine all tissues for the presence of white and brown adipose tissue which may contribute beta 3-AR mRNA. 4. The beta 3-AR mRNA is expressed at high levels in brown and white adipose tissue, stomach fundus, the longitudinal/circular smooth muscle of both colon and ileum, and colon submucosa. There was substantial expression of adipsin in colon submucosa and moderate expression in fundus, suggesting that in these regions at least some of the beta 3-AR signal may be contributed by fat. Pylorus and colon mucosa showed moderate levels of beta 3-AR mRNA with lower levels of adipsin. Ileum mucosa and submucosa showed low but readily detectable levels of beta 3-AR. 5. Expression of adipsin in rat skeletal muscles coupled to very low levels of beta 3-AR mRNA indicates that the observed beta 3-AR may be due to the presence of intrinsic fat. beta 3-AR mRNA was virtually undetectable in heart, lung and liver. These results raise the possibility that the atypical beta-AR demonstrated by functional and/or binding studies in muscle and in heart is not the beta 3-AR. 6. By use of two different sets of primers for amplification of beta 3-AR cDNA, no evidence was found for differential splicing of the mRNA in any of the tissues examined. 7. The detection of beta 3-AR mRNA in the gut mucosa and submucosa suggests that in addition to its established roles in lipolysis, thermogenesis and regulation of gut motility beta 3-AR may subserve other functions in the gastrointestinal tract. The absence of beta 3-AR mRNA in rat heart or its presence with adipsin in skeletal muscle suggests that atypical beta-adrenoceptor responses in heart and skeletal muscle are unlikely to be mediated by beta 3-AR. Images Figure 2 PMID:8825365

  3. Exogenous mRNA delivery and bioavailability in gene transfer mediated by piggyBac transposition

    PubMed Central

    2013-01-01

    Background Up to now, the different uptake pathways and the subsequent intracellular trafficking of plasmid DNA have been largely explored. By contrast, the mode of internalization and the intracellular routing of an exogenous mRNA in transfected cells are poorly investigated and remain to be elucidated. The bioavailability of internalized mRNA depends on its intracellular routing and its potential accumulation in dynamic sorting sites for storage: stress granules and processing bodies. This question is of particular significance when a secure transposon-based system able to integrate a therapeutic transgene into the genome is used. Transposon vectors usually require two components: a plasmid DNA, carrying the gene of interest, and a source of transposase allowing the integration of the transgene. The principal drawback is the lasting presence of the transposase, which could remobilize the transgene once it has been inserted. Our study focused on the pharmacokinetics of the transposition process mediated by the piggyBac transposase mRNA transfection. Exogenous mRNA internalization and trafficking were investigated towards a better apprehension and fine control of the piggyBac transposase bioavailability. Results The mRNA prototype designed in this study provides a very narrow expression window of transposase, which allows high efficiency transposition with no cytotoxicity. Our data reveal that exogenous transposase mRNA enters cells by clathrin and caveolae-mediated endocytosis, before finishing in late endosomes 3 h after transfection. At this point, the mRNA is dissociated from its carrier and localized in stress granules, but not in cytoplasmic processing bodies. Some weaker signals have been observed in stress granules at 18 h and 48 h without causing prolonged production of the transposase. So, we designed an mRNA that is efficiently translated with a peak of transposase production 18 h post-transfection without additional release of the molecule. This confines the integration of the transgene in a very small time window. Conclusion Our results shed light on processes of exogenous mRNA trafficking, which are crucial to estimate the mRNA bioavailability, and increase the biosafety of transgene integration mediated by transposition. This approach provides a new way for limiting the transgene copy in the genome and their remobilization by mRNA engineering and trafficking. PMID:24070093

  4. Novel, Testis-Specific mRNA Transcripts Encoding N-Terminally Truncated Choline Acetyltransferase

    E-print Network

    Ibáñez, Carlos

    Novel, Testis-Specific mRNA Transcripts Encoding N-Terminally Truncated Choline Acetyltransferase of choline acetyltransferase (ChAT) mRNA and protein in the mammalian testis. We have now found that none of the ChAT mRNAs produced in the testis is capable of encoding a full-length ChAT protein. Two ChAT c

  5. Relating mRNA and protein biomarker levels in a Dehalococcoides and Methanospirillum-containing community.

    PubMed

    Rowe, Annette R; Mansfeldt, Cresten B; Heavner, Gretchen L; Richardson, Ruth E

    2015-03-01

    To better understand the quantitative relationships between messenger RNA (mRNA) and protein biomarkers relevant to bioremediation, we quantified and compared respiration-associated gene products in an anaerobic syntrophic community. Respiration biomarkers for Dehalococcoides, an organohalide reducer, and Methanospirillum, a hydrogenotrophic methanogen, were quantified via qRT-PCR for mRNA and multiple reaction monitoring (MRM) of proteotypic peptides for protein. mRNA transcripts of the Dehalococcoides reductive dehalogenases PceA, TceA, and DMC1545, and hydrogenase HupL, as well as the Methanospirillum oxidoreductases MvrD and FrcA were shown to be similarly regulated with respect to their temporal responses to substrate addition. However, MvrD was two orders of magnitude lower in mRNA abundance. Per cell, Dehalococcoides protein biomarkers quantified were more abundant than Methanospirillum proteins. Comparing mRNA with protein abundance, poor correlations were observed between mRNA transcript levels and the net protein produced. For example, Dehalococcoides HupL and TceA transcripts were similarly abundant though TceA was far more abundant at the protein level (167?±?121 vs. 1095?±?337 proteins per cell, respectively). In Methanospirillum, MvrD maintained comparable per-cell protein abundance to FrcA (42?±?14 vs. 60?±?1 proteins per cell, respectively) despite the significantly lower transcript levels. Though no variability in protein decay rates was observed, the mRNA translation rate quantified for TceA was greater than the other Dehalococcoides targets monitored. These data suggest that there is considerable variation in the relationship between mRNA abundance and protein production both across transcripts within an organism and across organisms. This highlights the importance of empirically based studies for interpreting biomarker levels in environmentally relevant organisms. PMID:25467924

  6. Increased atrial natriuretic peptide mRNA expression in the kidney of diabetic rats

    Microsoft Academic Search

    Shyi-Jang Shin; Yau-Jiunn Lee; Mian-Shin Tan; Tusty-Jiuan Hsieh; Juei-Hsiung Tsai

    1997-01-01

    Increased atrial natriuretic peptide mRNA expression in the kidney of diabetic rats. To investigate whether renal synthesis of atrial natriuretic peptide (ANP) is influenced in diabetes, we measured renal ANP mRNA levels, urine volume, urinary ANP and sodium excretion rates in streptozotocin (STZ)-induced diabetic rats. By using reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blot analysis, we found that

  7. Differential expression of BDNF mRNA splice variants in mouse brain and immune cells

    Microsoft Academic Search

    Niels Kruse; Seray Cetin; Andrew Chan; Ralf Gold; Fred Lühder

    2007-01-01

    For the neurotrophin BDNF several splice variants were recently described. We analyzed the expression of mBDNF mRNA splice variants in cells of the immune system in comparison to the central nervous system (CNS). Whereas all splice variants are expressed in the CNS, only mBDNF 3 mRNA could be detected in primary and secondary lymphoid organs as well as in purified

  8. Effects of Ginseng and Echinacea on Cytokine mRNA Expression in Rats

    PubMed Central

    Ulu???k, Deniz; Keskin, Ercan

    2012-01-01

    The aim of the study was to determine the effect of ginseng and echinacea on the mRNA expression of IL-10, TNF-?, and TGF-?1 in healthy rats. Six-week-old male Fischer 344 rats (n = 48) were used. The animals were divided into three equal groups, as follows: control (C); ginseng (G); echinacea (E). While the C group was fed a standard rat diet (Purina) ad libitum for a period of 40 days, the G and E groups animals received the same diet containing 0.5?g/kg of Panax ginseng root powder and 0.75?g/kg of Echinacea purpurea root powder, respectively. Blood samples were obtained from 8 rats in each group after 20 and 40 days of treatment, and the mRNA expression of IL-10, TNF-?, and TGF-?1 was determined. After 20 days of treatment, the expression of IL-10 mRNA in the G group was different from the C group (P < 0.05); however, after 40 days of treatment, there was no difference between the groups. There was no difference after 20 and 40 days of treatment between the groups with respect to the expression of TGF-?1 mRNA. After 20 days of treatment, the expression of TNF-? mRNA in the E group was higher (P < 0.05) than the C group. After 40 days of treatment, the expression of TNF-? mRNA was similar in all of the groups. Based on the current study, the increase in expression of IL-10 mRNA in the G group and the increase in expression of TNF-? mRNA in the E group support the use of these plants for purposes of modulating the immune system. However, a more detailed study regarding the effects of ginseng and echinacea on these cytokines and other cytokines is needed. PMID:22666172

  9. Expression of p53 and mdm2 mRNA and protein in colorectal carcinomas.

    PubMed

    Broll, R; Stark, A; Windhövel, U; Best, R; Strik, M W; Schimmelpenning, H; Schwandner, O; Kujath, P; Bruch, H P; Duchrow, M

    1999-07-01

    The aim of our study was to investigate the expression of p53 and mdm2 mRNA and protein in colorectal adenocarcinoma. For the detection of mRNA, 60 fresh frozen human tumour samples and 12 samples of corresponding normal tissue were examined. After total RNA extraction, reverse transcription (RT) was performed followed by cDNA amplification with specific primers using RT-polymerase chain reaction (PCR). Immunohistochemical detection of protein was examined in 81 formalin-fixed and paraffin-embedded human tumour specimens as well as 15 samples of adjacent normal colorectal tissue. p53 mRNA was detected in 80% (48/60) of the tumours and in 67% (8/12) of normal tissue samples; 87% (52/60) of tumours had mdm2 mRNA in contrast to only 17% (2/12) of normal tissue specimens. Nuclear p53 protein expression was observed in 52% (42/81) of the tumour specimens and in none of the 15 normal specimens, whereas mdm2 protein was found in the nucleus (31%, 25/81) and also in the cytoplasm (86%, 70/81) of tumour samples. In normal tissue, mdm2 protein expression was only observed in the cytoplasm (13%, 2/15) and not in the nucleus. There was a significant correlation between coexpression of p53 and mdm2 protein and the occurrence of lymph node metastases (P = 0.03) as well as between p53 protein expression and the occurrence of distant metastases (P = 0.007). Additionally, significant associations were found between p53 mRNA and p53 protein, p53 mRNA and mdm2 mRNA or protein, and also between mdm2 mRNA and mdm2 protein expression, supporting the existence of a regulatory mechanism involving p53 and mdm2. PMID:10533452

  10. Quality control of mRNA 3'-end processing is linked to the nuclear exosome

    Microsoft Academic Search

    Patricia Hilleren; Terri McCarthy; Michael Rosbash; Roy Parker; Torben Heick Jensen

    2001-01-01

    An emerging theme in messenger RNA metabolism is the coupling of nuclear pre-mRNA processing events, which contributes to mRNA quality control. Most eukaryotic mRNAs acquire a poly(A) tail during 3'-end processing within the nucleus, and this is coupled to efficient export of mRNAs to the cytoplasm. In the yeast Saccharomyces cerevisiae, a common consequence of defective nuclear export of mRNA

  11. mRNA export: an assembly line from genes to nuclear pores

    Microsoft Academic Search

    Patrizia Vinciguerra; Françoise Stutz

    2004-01-01

    mRNAs are transported from the nucleus to the cytoplasm by a machinery conserved from yeast to humans. Previous studies showed that mRNA export factors are loaded on nascent mRNAs during elongation, coupling transcription to export. More recently identified mRNA export factors connect transcription initiation to the export machinery associated with nuclear pores, and potentially tether active genes to the nuclear

  12. Galectin-1 and Galectin-3 mRNA expression in renal cell carcinoma

    PubMed Central

    2014-01-01

    Background Galectins are known to regulate cell differentiation and growth as well as cell adhesion and apoptosis. Galectins have been discussed as possible prognosticators for survival in renal cell cancer (RCC) and other urological tumors. They might also play an emerging role as possible new marker-proteins for RCC. In this study, we analyzed the expression of galectin-1 and galectin-3 mRNA in order to further investigate their clinical significance in RCC. Methods Tissue samples were obtained from 106 patients undergoing surgery for RCC. The expression of galectin-1 and galectin-3 mRNA in normal kidney and corresponding cancer tissue was analyzed using quantitative real time PCR. Differences in expression levels of paired tissue samples were assessed using paired two-sample tests. Associations of relative mRNA expression levels in tumor tissues with clinical findings were analyzed using univariate logistic regression. Results The expression of galectin-1 (p < 0.001) and -3 (p < 0.001) mRNA were significantly higher in RCC when compared to the adjacent normal kidney tissue. For clear cell RCC, an association of male gender with higher galectin-1 and galectin-3 mRNA expression (p = 0.054, p = 0.034) was detected. For all RCCs, galectin-1 mRNA expression failed to show a significant association with advanced disease as well as a higher rate of lymph node metastases (p = 0.058, p = 0.059). Conclusion The mRNA expression of galectin-1 and galectin-3 is significantly increased in RCC cancer tissue. The higher mRNA expression in tumor tissue of male patients raises the question of a functional connection between galectins and the higher prevalence of RCC in men. Associations with advanced disease might lead to new ways of identifying patients at higher risk of recurrent disease and might even facilitate early metastasectomy with curative intent. PMID:24708743

  13. Post-Transcriptional Regulation of 5-Lipoxygenase mRNA Expression via Alternative Splicing and Nonsense-Mediated mRNA Decay

    PubMed Central

    Ochs, Meike J.; Sorg, Bernd L.; Pufahl, Laura; Grez, Manuel; Suess, Beatrix; Steinhilber, Dieter

    2012-01-01

    5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes (LT), a group of inflammatory lipid mediators derived from arachidonic acid. Here, we investigated the regulation of 5-LO mRNA expression by alternative splicing and nonsense-mediated mRNA decay (NMD). In the present study, we report the identification of 2 truncated transcripts and 4 novel 5-LO splice variants containing premature termination codons (PTC). The characterization of one of the splice variants, 5-LO?3, revealed that it is a target for NMD since knockdown of the NMD factors UPF1, UPF2 and UPF3b in the human monocytic cell line Mono Mac 6 (MM6) altered the expression of 5-LO?3 mRNA up to 2-fold in a cell differentiation-dependent manner suggesting that cell differentiation alters the composition or function of the NMD complex. In contrast, the mature 5-LO mRNA transcript was not affected by UPF knockdown. Thus, the data suggest that the coupling of alternative splicing and NMD is involved in the regulation of 5-LO gene expression. PMID:22363630

  14. Sequences of large T1 ribonuclease-resistant oligoribonucleotides from protamine mRNA: the overall architecture of protamine mRNA.

    PubMed Central

    Davies, P L; Dixon, G H; Simoncsits, A; Brownlee, G C

    1979-01-01

    Limited T1 ribonuclease digestion of the family of protamine mRNA's purified from rainbow trout testis yields several large oligoribonucleotide fragments ranging in size from 12--54 nucleotides in length. Several of these fragments purified by two dimensional gel electrophoresis contain several G residues and must represent nuclease-resistant, base-paired regions of the mRNA. Sequence analysis of these oligonucleotides by the method of Simoncsits, A., Brownlee, G.G., Brown, R.S., Rubin, J.R. and Guilley, H. (1977) Nature 269: 833-836, shows that these oligoribonucleotides arise from the 5'- and 3'-non-coding regions of the mRNA. Comparisons of the sequences of the large RNA fragment with DNA sequences obtained after cloning double-stranded protamine cDNA in the plasmids pBr322 and pmB9 show precise correspondence of a 54 nucleotide RNA fragment with positions 49--100 from the 3'-poly(A) tract and extending to within 5 nucleotides of the termination codon. Two other RNA fragments of 21 and 25 nucleotides in length arise from the 5'-non-coding region of the message and possess an AUG-sequence at their 3'-termini which is the initiation codon. The presence of distinct by homologous sequences in several sets of large RNA fragments is consistent with the presence of several closely related protamine mRNA's. Images PMID:118437

  15. microRNA expression in autonomous thyroid adenomas: Correlation with mRNA regulation.

    PubMed

    Floor, Sébastien L; Trésallet, Christophe; Hébrant, Aline; Desbuleux, Alice; Libert, Frédérick; Hoang, Catherine; Capello, Matteo; Andry, Guy; van Staveren, Wilma C G; Maenhaut, Carine

    2015-08-15

    The objective of the study was to identify the deregulated miRNA in autonomous adenoma and to correlate the data with mRNA regulation. Seven autonomous adenoma with adjacent healthy thyroid tissues were investigated. Twelve miRNAs were downregulated and one was upregulated in the tumors. Combining bioinformatic mRNA target prediction and microarray data on mRNA regulations allowed to identify mRNA targets of our deregulated miRNAs. A large enrichment in mRNA encoding proteins involved in extracellular matrix organization and different phosphodiesterases were identified among these putative targets. The direct interaction between miR-101-3p and miR-144-3p and PDE4D mRNA was experimentally validated. The global miRNA profiles were not greatly modified, confirming the definition of these tumors as minimal deviation tumors. These results support a role for miRNA in the regulation of extracellular matrix proteins and tissue remodeling occurring during tumor development, and in the important negative feedback of the cAMP pathway, which limits the consequences of its constitutive activation in these tumors. PMID:25916957

  16. Molecular structure of the human argininosuccinate synthetase gene: Occurrence of alternative mRNA splicing

    SciTech Connect

    Freytag, S.O.; Beaudet, A.L.; Bock, H.G.O.; O'Brien, W.E.

    1984-10-01

    The human genome contains one expressed argininosuccinate synthetase gene and ca. 14 pseudogenes that are dispersed to at least 11 human chromosomes. Eleven clones isolated from a human genomic DNA library were characterized extensively by restriction mapping, Southern blotting, and nucleotide sequencing. These 11 clones represent the entire expressed argininosuccinate synthetase gene that spans 63 kilobases and contains at least 13 exons. The expressed gene codes for two mRNAs that differ in their 5' untranslated sequences and arise by alternative splicing involving the inclusion or deletion of an entire exon. In normal human liver and cultured fibroblasts, the predominant mature argininosuccinate synthetase mRNA lacks sequences encoded by exon 2 in the expressed gene. In contrast, the predominant argininosuccinate synthetase mRNA in baboon liver contains exon 2 sequences. A transformed canavanine-resistant human cell line in which argininosuccinate synthetase activity is 180-fold higher than that in wild-type cells contains abundant amounts of both forms of the argininosuccinate synthetase mRNA. The mRNA lacking exon 2 sequences is the more abundant mRNA species in the canavanine-resistant cells. These observations show that splicing of the argininosuccinate synthetase mRNA is species specific in primates and varies among different human cell types.

  17. Cytokine mRNA expression in intestine from normal and inflammatory bowel disease patients.

    PubMed

    McCabe, R P; Secrist, H; Botney, M; Egan, M; Peters, M G

    1993-01-01

    Cytokines are involved in the regulation of normal immune events and may be important in the development or perpetuation of immune events in inflammatory bowel disease. We have previously shown that normal human mononuclear cells from tonsil, spleen, and peripheral blood exhibit tissue and stimulus-specific patterns of cytokine mRNA expression. The aim of this study was to determine if disease-dependent differences of cytokine mRNA expression could be found in the intestine. Total RNA was isolated from intestinal mucosa and lamina propria mononuclear cells from inflammatory bowel disease patients and controls. cDNA probes specific for interleukins (IL)-1, -4, -5, and -6 and transforming growth factor-beta were used. IL-1 beta mRNA and TGF-beta mRNA steady state expressions were higher in inflammatory bowel disease specimens than in normal intestine. In addition, mononuclear cell specimens had stronger cytokine mRNA expression than mucosal specimens. The steady state mRNA expression of proinflammatory cytokines is higher in inflammatory bowel disease, consistent with the ongoing inflammation seen. PMID:8440073

  18. Ferredoxin-1 mRNA is destabilized by changes in photosynthetic electron transport

    PubMed Central

    Petracek, Marie E.; Dickey, Lynn F.; Nguyen, Tuyen T.; Gatz, Christiane; Sowinski, Dolores A.; Allen, George C.; Thompson, William F.

    1998-01-01

    In transgenic tobacco, pea Ferredoxin-1 (Fed-1) mRNA accumulates rapidly in response to photosynthesis even when the transgene is driven by a constitutive promoter. To investigate the role of photosynthesis on Fed-1 mRNA stability, we used the tetracycline repressible Top10 promoter system to specifically shut off transcription of the Fed-1 transgene. The Fed-1 mRNA has a half-life of approximately 2.4 hr in the light and a half-life of only 1.2 hr in the dark or in the presence of the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). These data indicate that cessation of photosynthesis, either by darkness or DCMU results in a destabilization of the Fed-1 mRNA. Furthermore, the Fed-1 mRNA half-life is reduced immediately upon transfer to darkness, suggesting that Fed-1 mRNA destabilization is a primary response to photosynthesis rather than a secondary response to long-term dark adaptation. Finally, the two different methods for efficient tetracycline delivery reported here generally should be useful for half-life measurements of other mRNAs in whole plants. PMID:9671795

  19. Conserved Non-Coding Sequences are Associated with Rates of mRNA Decay in Arabidopsis

    PubMed Central

    Spangler, Jacob B.; Feltus, Frank Alex

    2013-01-01

    Steady-state mRNA levels are tightly regulated through a combination of transcriptional and post-transcriptional control mechanisms. The discovery of cis-acting DNA elements that encode these control mechanisms is of high importance. We have investigated the influence of conserved non-coding sequences (CNSs), DNA patterns retained after an ancient whole genome duplication event, on the breadth of gene expression and the rates of mRNA decay in Arabidopsis thaliana. The absence of CNSs near ? duplicate genes was associated with a decrease in breadth of gene expression and slower mRNA decay rates while the presence CNSs near ? duplicates was associated with an increase in breadth of gene expression and faster mRNA decay rates. The observed difference in mRNA decay rate was fastest in genes with CNSs in both non-transcribed and transcribed regions, albeit through an unknown mechanism. This study supports the notion that some Arabidopsis CNSs regulate the steady-state mRNA levels through post-transcriptional control mechanisms and that CNSs also play a role in controlling the breadth of gene expression. PMID:23675377

  20. Three-Dimensional Mapping of mRNA Export through the Nuclear Pore Complex

    PubMed Central

    Schnell, Steven J.; Ma, Jiong; Yang, Weidong

    2014-01-01

    The locations of transcription and translation of mRNA in eukaryotic cells are spatially separated by the nuclear envelope (NE). Plenty of nuclear pore complexes (NPCs) embedded in the NE function as the major gateway for the export of transcribed mRNAs from the nucleus to the cytoplasm. Whereas the NPC, perhaps one of the largest protein complexes, provides a relatively large channel for macromolecules to selectively pass through it in inherently three-dimensional (3D) movements, this channel is nonetheless below the diffraction limit of conventional light microscopy. A full understanding of the mRNA export mechanism urgently requires real-time mapping of the 3D dynamics of mRNA in the NPC of live cells with innovative imaging techniques breaking the diffraction limit of conventional light microscopy. Recently, super-resolution fluorescence microscopy and single-particle tracking (SPT) techniques have been applied to the study of nuclear export of mRNA in live cells. In this review, we emphasize the necessity of 3D mapping techniques in the study of mRNA export, briefly summarize the feasibility of current 3D imaging approaches, and highlight the new features of mRNA nuclear export elucidated with a newly developed 3D imaging approach combining SPT-based super-resolution imaging and 2D-to-3D deconvolution algorithms. PMID:25393401

  1. Variations in cytokine mRNA expression during normal human pregnancy

    PubMed Central

    Kruse, N; Greif, M; Moriabadi, N F; Marx, L; Toyka, K V; Rieckmann, P

    2000-01-01

    Epidemiological data provide evidence that disease activity of T cell-mediated, organ-specific autoimmune diseases is reduced during pregnancy. Although there are several experimental animal studies on the effect of pregnancy on the immune system, the situation in humans is less clear. We therefore performed a prospective analysis of cytokine mRNA expression in whole blood by a new on-line reverse transcriptase-polymerase chain reaction technique and of serum hormone levels during pregnancy in healthy women. The control group included age-matched non-pregnant healthy women. Quantitativecytokine mRNA expression revealed significantly reduced IL-18, interferon-gamma (IFN-?), and IL-2 mRNA levels in the first and second trimester in pregnancy compared with non-pregnant women. No difference between groups was detected for tumour necrosis factor-alpha (TNF-?) mRNA. IL-4 and IL-10 mRNA were detected at low levels in only 20% of pregnant women and were reduced to a statistically significant extent in the second and third trimester compared with the control group. Changes in IL-18 mRNA expression correlated inversely with serum values for human choriogonadotropin (HCG) and IL-10 serum levels correlated with increases in serum 17?-oestradiol levels. These data indicate immunomodulatory effects of pregnancy at the cytokine level which may be related to the variations in the clinical course of organ-specific, T cell-mediated autoimmune diseases during pregnancy. PMID:10632669

  2. Creatine kinase and alpha-actin mRNA levels decrease in diabetic rat hearts

    SciTech Connect

    Popovich, B.; Barrieux, A.; Dillmann, W.H.

    1987-05-01

    Diabetic cardiomyopathy is associated with cardiac atrophy and isoenzyme redistribution. To determine if tissue specific changes occur in mRNAs coding for ..cap alpha..-actin and creatine kinase (CK), they performed RNA blot analysis. Total ventricular RNA from control (C) and 4 wk old diabetic (D) rats were hybridized with /sup 32/P cDNA probes for ..cap alpha..-actin and CK. A tissue independent cDNA probe, CHOA was also used. Signal intensity was quantified by photodensitometry. D CK mRNA was 47 +/- 16% lower in D vs C. Insulin increases CK mRNA by 20% at 1.5 hs, and completely reverses the deficit after 4 wks. D ..cap alpha..-actin mRNA is 66 +/- 18% lower in D vs C. Insulin normalized ..cap alpha..-actin mRNA by 5 hs. CHOA mRNA is unchanged in D vs C, but D + insulin CHOA mRNA is 30 +/- 2% lower than C. In rats with diabetic cardiomyopathy, muscle specific CK and ..cap alpha..-actin mRNAs are decreased. Insulin treatment reverses these changes.

  3. Perinuclear Mlp proteins downregulate gene expression in response to a defect in mRNA export.

    PubMed

    Vinciguerra, Patrizia; Iglesias, Nahid; Camblong, Jurgi; Zenklusen, Daniel; Stutz, Françoise

    2005-02-23

    The mRNA export adaptor Yra1p/REF contributes to nascent mRNP assembly and recruitment of the export receptor Mex67p. yra1 mutants exhibit mRNA export defects and a decrease in LacZ reporter and certain endogenous transcripts. The loss of Mlp1p/Mlp2p, two TPR-like proteins attached to nuclear pores, rescues LacZ mRNA levels and increases their appearance in the cytoplasm, without restoring bulk poly(A)+ RNA export. Chromatin immunoprecipitation, FISH and pulse-chase experiments indicate that Mlps downregulate LacZ mRNA synthesis in a yra1 mutant strain. Microarray analyses reveal that Mlp2p also reduces a subset of cellular transcripts in the yra1 mutant. Finally, we show that Yra1p genetically interacts with the shuttling mRNA-binding protein Nab2p and that loss of Mlps rescues the growth defect of yra1 and nab2 but not other mRNA export mutants. We propose that Nab2p and Yra1p are required for proper mRNP docking to the Mlp platform. Defects in Yra1p prevent mRNPs from crossing the Mlp gate and this block negatively feeds back on the transcription of a subset of genes, suggesting that Mlps link mRNA transcription and export. PMID:15692572

  4. Lymphokine mRNA profile and functional analysis of a human CD4 + clone with unique antitumor specificity isolated from renal cell carcinoma ascitic fluid

    Microsoft Academic Search

    Arie Belldegrun; Atan Kasid; Michael Uppenkamp; Steven A. Rosenberg

    1990-01-01

    Summary We here describe the isolation, characterization, profile of lymphokine expression and T-cell-receptor gene rearrangment pattern of 444P.3, a CD3+ CD4+ CD8- 4B4+ interleukin-2 (IL-2)-dependent clone derived from the malignant ascites of a patient with renal cell cancer. The 444P.3 clone exhibited unique antitumor specificity between days 45 and 84 in culture and then lost its lytic, but not its

  5. The iron-sensing aconitase B binds its own mRNA to prevent sRNA-induced mRNA cleavage

    PubMed Central

    Benjamin, Julie-Anna M.; Massé, Eric

    2014-01-01

    Aconitase is an iron–sulfur protein and a major enzyme of the TCA cycle that catalyzes the conversion of citrate to isocitrate under iron-rich conditions. In Escherichia coli, aconitase B (AcnB) is a typical moonlighting protein that can switch to its apo form (apo-AcnB) which favors binding its own mRNA 3?UTR and stabilize it when intracellular iron become scarce. The small regulatory RNA (sRNA) RyhB has previously been shown to promote RNase E-dependent degradation of acnB mRNA when it was expressed from an ectopic arabinose-dependent promoter, independently of intracellular iron levels. In marked contrast, we report here that expression of RyhB under low-iron conditions did not result in acnB mRNA degradation even when RyhB was bound to acnB ribosome binding site (RBS). Genetic and biochemical evidence suggested that, under low-iron conditions, apo-AcnB bound to acnB 3?UTR close to a RNase E cleavage site that is essential for RyhB-induced acnB mRNA degradation. Whereas RyhB can block acnB translation initiation, RNase E-dependent degradation of acnB was prevented by apo-AcnB binding close to the cleavage site. This previously uncharacterized regulation suggests an intricate post-transcriptional mechanism that represses protein expression while insuring mRNA stability. PMID:25092924

  6. Oestradiol reduces Liver Receptor Homolog-1 mRNA transcript stability in breast cancer cell lines

    SciTech Connect

    Lazarus, Kyren A. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia) [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Environmental and Biotechnology Centre, Swinburne University, Hawthorn, Victoria 3122 (Australia); Zhao, Zhe; Knower, Kevin C. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia)] [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); To, Sarah Q. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia) [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia); Chand, Ashwini L. [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia)] [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Clyne, Colin D., E-mail: Colin.clyne@princehenrys.org [Cancer Drug Discovery Laboratory, Prince Henry’s Institute of Medical Research, Clayton, Victoria 3168 (Australia); Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168 (Australia)

    2013-08-30

    Highlights: •LRH-1 is an orphan nuclear receptor that regulates tumor proliferation. •In breast cancer, high mRNA expression is associated with ER+ status. •In ER?ve cells, despite very low mRNA, we found abundant LRH-1 protein. •Our data show distinctly different LRH-1 protein isoforms in ER? and ER+ breast cancer cells. •This is due to differences in LRH-1 mRNA and protein stability rates. -- Abstract: The expression of orphan nuclear receptor Liver Receptor Homolog-1 (LRH-1) is elevated in breast cancer and promotes proliferation, migration and invasion in vitro. LRH-1 expression is regulated by oestrogen (E{sub 2}), with LRH-1 mRNA transcript levels higher in oestrogen receptor ? (ER?) positive (ER+) breast cancer cells compared to ER? cells. However, the presence of LRH-1 protein in ER? cells suggests discordance between mRNA transcript levels and protein expression. To understand this, we investigated the impact of mRNA and protein stability in determining LRH-1 protein levels in breast cancer cells. LRH-1 transcript levels were significantly higher in ER+ versus ER? breast cancer cells lines; however LRH-1 protein was expressed at similar levels. We found LRH-1 mRNA and protein was more stable in ER? compared to ER+ cell lines. The tumor-specific LRH-1 variant isoform, LRH-1v4, which is highly responsive to E{sub 2}, showed increased mRNA stability in ER? versus ER+ cells. In addition, in MCF-7 and T47-D cell lines, LRH-1 total mRNA stability was reduced with E{sub 2} treatment, this effect mediated by ER?. Our data demonstrates that in ER? cells, increased mRNA and protein stability contribute to the abundant protein expression levels. Expression and immunolocalisation of LRH-1 in ER? cells as well as ER? tumors suggests a possible role in the development of ER? tumors. The modulation of LRH-1 bioactivity may therefore be beneficial as a treatment option in both ER? and ER+ breast cancer.

  7. The Ccr4-Not Complex Interacts with the mRNA Export Machinery

    PubMed Central

    Kerr, Shana C.; Azzouz, Nowel; Fuchs, Stephen M.; Collart, Martine A.; Strahl, Brian D.; Corbett, Anita H.; Laribee, R. Nicholas

    2011-01-01

    Background The Ccr4-Not complex is a key eukaryotic regulator of gene transcription and cytoplasmic mRNA degradation. Whether this complex also affects aspects of post-transcriptional gene regulation, such as mRNA export, remains largely unexplored. Human Caf1 (hCaf1), a Ccr4-Not complex member, interacts with and regulates the arginine methyltransferase PRMT1, whose targets include RNA binding proteins involved in mRNA export. However, the functional significance of this regulation is poorly understood. Methodology/Principal Findings Here we demonstrate using co-immunoprecipitation approaches that Ccr4-Not subunits interact with Hmt1, the budding yeast ortholog of PRMT1. Furthermore, using genetic and biochemical approaches, we demonstrate that Ccr4-Not physically and functionally interacts with the heterogenous nuclear ribonucleoproteins (hnRNPs) Nab2 and Hrp1, and that the physical association depends on Hmt1 methyltransferase activity. Using mass spectrometry, co-immunoprecipitation and genetic approaches, we also uncover physical and functional interactions between Ccr4-Not subunits and components of the nuclear pore complex (NPC) and we provide evidence that these interactions impact mRNA export. Conclusions/Significance Taken together, our findings suggest that Ccr4-Not has previously unrealized functional connections to the mRNA processing/export pathway that are likely important for its role in gene expression. These results shed further insight into the biological functions of Ccr4-Not and suggest that this complex is involved in all aspects of mRNA biogenesis, from the regulation of transcription to mRNA export and turnover. PMID:21464899

  8. Real-time monitoring of intracellular mRNA hybridization inside single living cells.

    PubMed

    Perlette, J; Tan, W

    2001-11-15

    A molecular beacon, an oligonucleotide probe with inherent signal transduction mechanisms, is an optimal tool for visualizing real-time mRNA hybridization in single living cells. Each molecular beacon (MB) consists of a single-stranded DNA molecule in a stem-loop conformation with a fluorophore linked to the 5' end and a quencher at the 3' end. In this study, we demonstrate real-time monitoring of mRNA-DNA hybridization inside living cells using molecular beacons. A MB specific for beta-actin mRNA has been designed and synthesized. After microinjection into the cytoplasm of single living kangaroo rat kidney cells (PtK2 cells), the MB hybridizes with beta-actin mRNA as shown by fluorescence measurements over time. Hybridization dynamics have been followed. Strict control experiments have been carried out to confirm that the fluorescence signal increase is indeed due to the hybridization of mRNA inside single living cells. Variation in the MB/mRNA hybridization fluorescent signal has been observed for different PtK2 cells, which indicates the amount of mRNA in different cells is different. We have also monitored the beta-1 andrenergic receptor mRNA inside the PtK2 cells. These studies demonstrate the feasibility of using MBs and the ultrasensitivity achieved in our fluorescence imaging system for real-time detection of mRNA hybridization and for the visualization of oligonucleotide/mRNA interactions inside single living cells. PMID:11816586

  9. mRNA stability as a function of striated muscle oxidative capacity.

    PubMed

    D'souza, Donna; Lai, Ruanne Y J; Shuen, Michael; Hood, David A

    2012-08-15

    A change in mRNA stability alters the abundance of mRNA available for translation and is emerging as a critical pathway influencing gene expression. Variations in the stability of functional and regulatory mitochondrial proteins may contribute to the divergent mitochondrial densities observed in striated muscle. Thus we hypothesized that the stability of mRNAs encoding for regulatory nuclear and mitochondrial transcription factors would be inversely proportional to muscle oxidative capacity and would be facilitated by the activity of RNA binding proteins (RBPs). The stability of mitochondrial transcription factor A (Tfam), peroxisome proliferator-activated receptor gamma coactivator 1? (PGC-1?), and nuclear respiratory factor 2? (NRF-2?) mRNA was assessed in striated muscles with distinct oxidative capacities using in vitro decay assays. All three mitochondrial regulators were rapidly degraded in cardiac and slow-twitch red (STR) muscle, resulting in a ?60-65% lower (P < 0.05) mRNA half-life (t(1/2)) compared with fast-twitch white (FTW) fibers. This accelerated rate of Tfam mRNA decay was matched by a 2.5-fold increase in Tfam transcription in slow- compared with fast-twitch muscle (P = 0.05). Protein expression of four unique RBPs [AU-rich binding factor 1 (AUF1), human antigen R (HuR), KH-homology splicing regulatory protein (KSRP), and CUG binding protein 1 (CUGBP1)] believed to modulate mRNA stability was elevated in cardiac and STR muscles (P < 0.05) and was moderately associated with the decay of Tfam, PGC-1?, and NRF-2? mRNA. Variable rates of transcript degradation were apparent when comparing all transcripts within the same muscle type. Thus the distribution of RBPs appears to follow a fiber-type specific pattern and subsequently functions to alter the stability of specific mitochondrial regulators in a transcript- and tissue-specific fashion. PMID:22718808

  10. Arc mRNA induction in striatal efferent neurons associated with response learning.

    PubMed

    Daberkow, D P; Riedy, M D; Kesner, R P; Keefe, K A

    2007-07-01

    The dorsal striatum is involved in motor-response learning, but the extent to which distinct populations of striatal efferent neurons are differentially involved in such learning is unknown. Activity-regulated, cytoskeleton-associated (Arc) protein is an effector immediate-early gene implicated in synaptic plasticity. We examined arc mRNA expression in striatopallidal vs. striatonigral efferent neurons in dorsomedial and dorsolateral striatum of rats engaged in reversal learning on a T-maze motor-response task. Male Sprague-Dawley rats learned to turn right or left for 3 days. Half of the rats then underwent reversal training. The remaining rats were yoked to rats undergoing reversal training, such that they ran the same number of trials but ran them as continued-acquisition trials. Brains were removed and processed using double-label fluorescent in situ hybridization for arc and preproenkephalin (PPE) mRNA. In the reversal, but not the continued-acquisition, group there was a significant relation between the overall arc mRNA signal in dorsomedial striatum and the number of trials run, with rats reaching criterion in fewer trials having higher levels of arc mRNA expression. A similar relation was seen between the numbers of PPE(+) and PPE(-) neurons in dorsomedial striatum with cytoplasmic arc mRNA expression. Interestingly, in behaviourally activated animals significantly more PPE(-) neurons had cytoplasmic arc mRNA expression. These data suggest that Arc in both striatonigral and striatopallidal efferent neurons is involved in striatal synaptic plasticity mediating motor-response learning in the T-maze and that there is differential processing of arc mRNA in distinct subpopulations of striatal efferent neurons. PMID:17614950

  11. Stability of mRNA influences osteoporotic bone mass via CNOT3

    PubMed Central

    Watanabe, Chiho; Morita, Masahiro; Hayata, Tadayoshi; Nakamoto, Tetsuya; Kikuguchi, Chisato; Li, Xue; Kobayashi, Yasuhiro; Takahashi, Naoyuki; Notomi, Takuya; Moriyama, Keiji; Yamamoto, Tadashi; Ezura, Yoichi; Noda, Masaki

    2014-01-01

    Osteoclastogenesis is under the control of posttranscriptional and transcriptional events. However, posttranscriptional regulation of osteoclastogenesis is incompletely understood. CNOT3 is a component of the CCR4 family that regulates mRNA stability, but its function in bone is not known. Here, we show that Cnot3 deficiency by deletion of a single allele induces osteoporosis. Cnot3 deficiency causes an enhancement in bone resorption in association with an elevation in bone formation, resulting in high-turnover type bone loss. At the cellular level, Cnot3 deficiency enhances receptor activator of NF-?B ligand (RANKL) effects on osteoclastogenesis in a cell-autonomous manner. Conversely, Cnot3 deficiency does not affect osteoblasts directly. Cnot3 deficiency does not alter RANKL expression but enhances receptor activator of NF-?B (RANK) mRNA expression in bone in vivo. Cnot3 deficiency promotes RANK mRNA stability about twofold in bone marrow cells of mice. Cnot3 knockdown also increases RANK mRNA expression in the precursor cell line for osteoclasts. Anti-CNOT3 antibody immunoprecipitates RANK mRNA. Cnot3 deficiency stabilizes luciferase reporter expression linked to the 3?-UTR fragment of RANK mRNA. In contrast, Cnot3 overexpression destabilizes the luciferase reporter linked to RANK 3?-UTR. In aged mice that exhibit severe osteoporosis, Cnot3 expression levels in bone are reduced about threefold in vivo. Surprisingly, Cnot3 deficiency in these aged mice further exacerbates osteoporosis, which also occurs via enhancement of osteoclastic activity. Our results reveal that CNOT3 is a critical regulator of bone mass acting on bone resorption through posttranscriptional down-regulation of RANK mRNA stability, at least in part, even in aging-induced osteoporosis. PMID:24550297

  12. Cyclic nucleotide phosphodiesterase 7B mRNA: an unfavorable characteristic in chronic lymphocytic leukemia.

    PubMed

    Zhang, Lingzhi; Murray, Fiona; Rassenti, Laura Z; Pu, Minya; Kelly, Colleen; Kanter, Joan R; Greaves, Andrew; Messer, Karen; Kipps, Thomas J; Insel, Paul A

    2011-09-01

    A cost- and time-efficient means to define the prognosis of patients with chronic lymphocytic leukemia (CLL) is desirable but does not yet exist. On the basis of the evidence that CLL cells have enhanced expression of the cyclic nucleotide phosphodiesterase isoform 7B (PDE7B), we hypothesized that PDE7B expression might provide such information. We assessed PDE7B mRNA expression using quantitative real-time PCR in peripheral blood mononuclear cells isolated from 85 patients and 30 normal subjects. We compared PDE7B mRNA expression with that of other disease features to determine if its expression correlates with the prognosis of patients with CLL. We found that CLL patients with PDE7B mRNA levels in the top quartile (greater than ninefold elevation relative to normal controls) have a several-year shorter median time-to-treatment (TTT, 36 months) compared to that of patients whose CLL cells express lower levels of PDE7B mRNA (TTT, 77 months, p=0.001). High PDE7B mRNA expression correlates with expression of zeta-chain-associated protein kinase 70 (ZAP-70), unmutated immunoglobulin heavy chain variable (IGHV) region genes and ?2 microglobulin (?2M), but use of a multivariate Cox model revealed that high PDE7B mRNA expression independently predicts a short TTT, even after adjusting for several other disease characteristics (ZAP-70 or CD38 expression, IGHV mutation status and Rai status). High expression of PDE7B is an unfavorable characteristic in CLL. Assessment of PDE7B mRNA expression thus appears to be a clinically useful biomarker to define the prognosis of patients with CLL. PMID:21120911

  13. Circulating resistin protein and mRNA concentrations and clinical severity of coronary artery disease

    PubMed Central

    Sopic, Miron; Spasojevic-Kalimanovska, Vesna; Kalimanovska-Ostric, Dimitra; Andjelkovic, Kristina; Jelic-Ivanovic, Zorana

    2015-01-01

    Introduction Previous studies have implicated a strong link between circulating plasma resistin and coronary artery disease (CAD). The aim of this study was to evaluate the differences in peripheral blood mononuclear cells (PBMC) resistin mRNA and its plasma protein concentrations between the patients with CAD of different clinical severity. Material and methods This study included 33 healthy subjects as the control group (CG) and 77 patients requiring coronary angiography. Of the latter 30 was CAD negative whereas 47 were CAD positive [18 with stable angina pectoris (SAP) and 29 with acute coronary syndrome (ACS)]. Circulating resistin was measured by ELISA; PBMC resistin mRNA was determined by real-time PCR. Results Resistin protein was significantly higher in the ACS group compared to the CG (P = 0.001) and the CAD negative group (P = 0.018). Resistin mRNA expression did not vary across the study groups, despite the positive correlation seen with plasma resistin (? = 0.305, P = 0.008). In patients, plasma resistin and PBMC resistin mRNA negatively correlated with HDL-C (? = -0.404, P < 0.001 and ? = -0.257, P = 0.032, respectively). Furthermore, the highest plasma resistin tertile showed the lowest HDL-C (P = 0.006). Plasma resistin was positively associated with serum creatinine (? = 0.353, P = 0.002). Conclusion Significant increase of plasma resistin in patients with ACS compared to CG and CAD negative patients was observed. Despite no change in PBMC resistin mRNA in different disease conditions a positive association between resistin mRNA and resistin plasma protein was evident. Both plasma resistin and PBMC resistin mRNA were negatively associated with plasma HDL-C, and plasma resistin positively with serum creatinine.

  14. Self-digitization microfluidic chip for absolute quantification of mRNA in single cells.

    PubMed

    Thompson, Alison M; Gansen, Alexander; Paguirigan, Amy L; Kreutz, Jason E; Radich, Jerald P; Chiu, Daniel T

    2014-12-16

    Quantification of mRNA in single cells provides direct insight into how intercellular heterogeneity plays a role in disease progression and outcomes. Quantitative polymerase chain reaction (qPCR), the current gold standard for evaluating gene expression, is insufficient for providing absolute measurement of single-cell mRNA transcript abundance. Challenges include difficulties in handling small sample volumes and the high variability in measurements. Microfluidic digital PCR provides far better sensitivity for minute quantities of genetic material, but the typical format of this assay does not allow for counting of the absolute number of mRNA transcripts samples taken from single cells. Furthermore, a large fraction of the sample is often lost during sample handling in microfluidic digital PCR. Here, we report the absolute quantification of single-cell mRNA transcripts by digital, one-step reverse transcription PCR in a simple microfluidic array device called the self-digitization (SD) chip. By performing the reverse transcription step in digitized volumes, we find that the assay exhibits a linear signal across a wide range of total RNA concentrations and agrees well with standard curve qPCR. The SD chip is found to digitize a high percentage (86.7%) of the sample for single-cell experiments. Moreover, quantification of transferrin receptor mRNA in single cells agrees well with single-molecule fluorescence in situ hybridization experiments. The SD platform for absolute quantification of single-cell mRNA can be optimized for other genes and may be useful as an independent control method for the validation of mRNA quantification techniques. PMID:25390242

  15. GTP cyclohydrolase I mRNA: novel splice variants in the slime mould Physarum polycephalum and in human monocytes (THP-1) indicate conservation of mRNA processing.

    PubMed Central

    Golderer, G; Werner, E R; Heufler, C; Strohmaier, W; Gröbner, P; Werner-Felmayer, G

    2001-01-01

    GTP cyclohydrolase I (EC 3.5.4.16) is the first enzyme in the biosynthesis of tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)-biopterin] in mammals and of folic acid in bacteria. Here we have characterized the GTP cyclohydrolase I gene structure and two mRNA species from Physarum polycephalum, an acellular slime mould that synthesizes H(4)-biopterin and metabolites of the folic acid biosynthetic pathway. Its GTP cyclohydrolase I gene consists of seven exons, and the two GTP cyclohydrolase I cDNA species isolated from Physarum encode for proteins with 228 (25.7 kDa) and 195 (22.1 kDa) amino acids. Furthermore, we identified two previously undescribed mRNA species in interferon-gamma-treated human myelomonocytoma cells (THP-1) in addition to the cDNA coding for the fully functional 250-residue (27.9 kDa) protein, which is identical with that in human phaeochromocytoma cells. One of the new splice variants codes for a 233-residue (25.7 kDa) protein, whereas the other codes for the full-length protein but is alternatively spliced within the 3'-untranslated region. In heterologous expression, the shorter proteins of Physarum as well as of THP-1 cells identified here are degraded by proteolysis. Accordingly, only the 27.9 kDa protein was detectable in Western blots from THP-1 cell extracts. Quantification of GTP cyclohydrolase I mRNA species in different human cell types with and without cytokine treatment showed that in addition to the correct mRNA the two splice variants isolated here, as well as the two splice variants known from human liver, are strongly induced by cytokines in cell types with inducible GTP cyclohydrolase I (THP-1, dermal fibroblasts), but not in cell types with constitutive GTP cyclohydrolase I expression (SK-N-SH, Hep-G2). As in human liver, splicing of the new mRNA variant found in THP-1 cells occurs at the boundary of exons 5 and 6. Strikingly, the 195-residue protein from Physarum is alternatively spliced at a homologous position, i.e. at the boundary of exons 6 and 7. Thus alternative splicing of GTP cyclohydrolase I at this position occurs in two species highly distant from each other in terms of evolution. It remains to be seen whether variant proteins encoded by alternatively spliced GTP cyclohydrolase I mRNA transcripts do occur in vivo and whether they participate in regulation of enzyme activity. PMID:11284739

  16. Differential expression of IGF-1 mRNA isoforms in colorectal carcinoma and normal colon tissue.

    PubMed

    Kasprzak, Aldona; Szaflarski, Witold; Szmeja, Jacek; Andrzejewska, Ma?gorzata; Przybyszewska, Wies?awa; Kaczmarek, El?bieta; Koczorowska, Maria; Ko?ci?ski, Tomasz; Zabel, Maciej; Drews, Micha?

    2013-01-01

    The insulin-like growth factor (IGF)-1 gene consists of 6 exons resulting in the expression of 6 variant forms of mRNA (IA, IB, IC, IIA, IIB and IIC) due to an alternative splicing. The mechanisms of IGF-1 gene splicing and the role of local expression manifested by IGF-1 mRNA variants in colorectal carcinoma (CRC) have not been extensively investigated. Therefore, the aim of our study was to analyse the expression of IGF-1 mRNA isoforms [A, B, C, P1 (class I) and P2 (class II)], as well as the protein expression in CRC and control samples isolated from 28 patients. The expression of Ki-67 was also analysed and clinical data were obtained. For this purpose, we used quantitative real-time PCR (qPCR) and immunocytochemistry. The expression of mRNAs coding for all splicing isoforms of IGF-1 was observed in every tissue sample studied, with a significantly lower expression noted in the CRC as compared to the control samples. The cytoplasmic expression of IGF-1 protein was found in 50% of the CRC and in ~40% of the non-tumor tissues; however, no significant quantitative inter-group differences were observed. The expression of the IGF-1 gene in the 2 groups of tissues was controlled by the P1 and P2 promoters in a similar manner. No significant differences were detected in the expression of the IGF-1 A and B isoforms; however, their expression was significantly higher compared to that of isoform C. No significant differences were observed between the expression of Ki-67 mRNA in the CRC and control tissue even though the expression of the Ki-67 protein was higher in the CRC compared to the control samples. Ki-67 protein expression was associated with the macroscopic and microscopic aspects of CRC. A significant positive correlation was found between the local production of total mRNA and isoform A and the expression of Ki-67 mRNA, although only in the non-tumor tissues. In CRC samples, the local expression of the total IGF-1 mRNA and all splicing isoforms of IGF-1 mRNA decreased as compared to the normal colon tissues, although however, with conservation of both gene promoter activities and with the continued principal splicing IGF-1 mRNA isoforms. PMID:23165777

  17. Expression of human ?-defensin 5 (HD5) mRNA in nasal and bronchial epithelial cells

    PubMed Central

    Frye, M; Bargon, J; Dauletbaev, N; Weber, A; Wagner, T; Gropp, R

    2000-01-01

    Background/Aims—Human defensins are antibiotic peptides expressed in myeloid and epithelial cells. Human ?-defensin 5 (HD5) has been detected in Paneth cell granules in the crypts of Lieberkühn and has recently been identified in the female reproductive tract. The aim of this study was to investigate the presence of HD5 mRNA in nasal and bronchial epithelial cells. Methods/Results—Semiquantitative reverse transcription polymerase chain reaction (RT–PCR) analysis showed that HD5 mRNA was expressed infrequently and to varying degrees in bronchial and nasal epithelial cells. In situ hybridisation resulted in a positive signal in the epithelial layer of nasal polyps. HD5 mRNA was locally restricted to a specific area of epithelial cells and also occurred in submucosal glands. Conclusions—HD5 mRNA expression in nasal and bronchial epithelial cells is rare and seemed to be locally induced. The results indicate that HD5 might play a role in innate defence in nasal and bronchial epithelia. Key Words: antimicrobial peptides • human ?-defensin 5 • mRNA • expression • airways PMID:11064671

  18. Regulation of mRNA transport, localization and translation in the nervous system of mammals (Review)

    PubMed Central

    DI LIEGRO, CARLO MARIA; SCHIERA, GABRIELLA; DI LIEGRO, ITALIA

    2014-01-01

    Post-transcriptional control of mRNA trafficking and metabolism plays a critical role in the actualization and fine tuning of the genetic program of cells, both in development and in differentiated tissues. Cis-acting signals, responsible for post-transcriptional regulation, reside in the RNA message itself, usually in untranslated regions, 5? or 3? to the coding sequence, and are recognized by trans-acting factors: RNA-binding proteins (RBPs) and/or non-coding RNAs (ncRNAs). ncRNAs bind short mRNA sequences usually present in the 3?-untranslated (3?-UTR) region of their target messages. RBPs recognize specific nucleotide sequences and/or secondary/tertiary structures. Most RBPs assemble on mRNA at the moment of transcription and shepherd it to its destination, somehow determining its final fate. Regulation of mRNA localization and metabolism has a particularly important role in the nervous system where local translation of pre-localized mRNAs has been implicated in developing axon and dendrite pathfinding, and in synapse formation. Moreover, activity-dependent mRNA trafficking and local translation may underlie long-lasting changes in synaptic efficacy, responsible for learning and memory. This review focuses on the role of RBPs in neuronal development and plasticity, as well as possible connections between ncRNAs and RBPs. PMID:24452120

  19. Molecular cloning, polymorphism and association analyses of a novel differentially expressed porcine mRNA.

    PubMed

    Liu, G Y; Xiong, Y Z

    2009-11-01

    The mRNA differential display technique was performed to investigate the differences of gene expression in the longissimus muscle tissues from Meishan and Large White pigs. One novel mRNA that was differentially expressed was identified through semi-quantitative RT-PCR and the full-length cDNA sequence was then obtained using the rapid amplification of cDNA ends (RACE) method. The nucleotide sequence of the mRNA is not homologous to any of the known porcine genes. Sequence prediction analysis revealed that this mRNA is no-coding mRNA. Polymorphism analyses revealed that there was a C-T mutation on the position of 505 bp and PCR-HhaI-RFLP analyses revealed that Chinese indigenous pig breeds and exotic pig breeds displayed obvious genotype and allele frequency differences at this locus. Association analyses revealed that this polymorphic locus was significantly associated with the drip loss rate, water holding capacity, dressing percentage, rib numbers, lean meat percentage, estimated lean meat percentage, loin eye width and loin eye area (P < 0.05). PMID:19130294

  20. Abnormal cellular localization of thyroglobulin mRNA associated with hereditary congenital goiter and thyroglobulin deficiency.

    PubMed Central

    Van Voorthuizen, W F; Dinsart, C; Flavell, R A; DeVijlder, J J; Vassart, G

    1978-01-01

    The goiters in a breed of hypothyroid goats contain only minute amounts of thyroblobulin-related antigens (0.01% of normal value). We have analyzed these goiters for the presence of mRNA coding for thyroglobulin. Using DNA complementary to beef 33S thyroglobulin mRNA as a probe, we found that the mRNA sequence is present in the goat goiter but at a concentration 1/10-1/40 that of normal goat thyroid. Hybrids of cDNA with either goiter or normal thyroid RNA exhibited identical sharp melting curves which suggests that the same RNA sequence is responsible for hybridization in both tissues. Normal goat thyroid contains a population of large membrane-bound polysomes engaged in throglobulin synthesis. In contrast, such polysomes are absent in the goiter. In regard to subcellular distribution, the relative amount of the thyroglobulin mRNA sequences from the goiter in nuclear RNA was 42% of normal, in cytoplasmic RNA was 7% of normal, and in the membrane fraction was only 1-2% of normal. Our results suggest that the lack of thyroglobulin in these goiters is due to a defect in thyroglobulin mRNA which leads to aberrant processing and/or transport of it from its site of synthesis to the endoplasmic reticulum. PMID:272675

  1. Multiple forms of mouse antizyme inhibitor 1 mRNA differentially regulated by polyamines.

    PubMed

    Murakami, Yasuko; Ohkido, Makiko; Takizawa, Hiroko; Murai, Noriyuki; Matsufuji, Senya

    2014-03-01

    Antizyme inhibitor 1 (Azin1), a positive regulator of cellular polyamines, is induced by various proliferative stimuli and repressed by polyamines. It has been reported that the translational repression of Azin1 by polyamines involves an upstream open reading frame on the mRNA, but little has been known about polyamine effect on its transcription or splicing. We found multiple forms of Azin1 transcripts formed by alternative splicing and initiation of transcription from putative alternative start sites. One of the novel splice variants, Azin1-X, has a premature termination codon on 5? extension of exon 7, encodes a C-terminal truncated form of protein (Azin1?C), and is subject to nonsense-mediated mRNA decay. 2-Difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis, increased both transcription from the canonical transcription start site and the ratio of the full-length mRNA to Azin1-X mRNA, whereas polyamines show the opposite effect. Thus, polyamines regulate two novel steps of Azin1 expression, namely the transcription and a particular splicing pattern, both of which may affect the level of mRNA encoding the full-length active Azin1 protein. PMID:24077669

  2. Identification of a cis-acting element that localizes mRNA to synapses

    PubMed Central

    Meer, Elliott J.; Wang, Dan Ohtan; Kim, Sangmok; Barr, Ian; Guo, Feng; Martin, Kelsey C.

    2012-01-01

    Messenger RNA (mRNA) localization and regulated translation can spatially restrict gene expression to each of the thousands of synaptic compartments formed by a single neuron. Although cis-acting RNA elements have been shown to direct localization of mRNAs from the soma into neuronal processes, less is known about signals that target transcripts specifically to synapses. In Aplysia sensory-motor neuronal cultures, synapse formation rapidly redistributes the mRNA encoding the peptide neurotransmitter sensorin from neuritic shafts into synapses. We find that the export of sensorin mRNA from soma to neurite and the localization to synapse are controlled by distinct signals. The 3? UTR is sufficient for export into distal neurites, whereas the 5? UTR is required for concentration of reporter mRNA at synapses. We have identified a 66-nt element in the 5? UTR of sensorin that is necessary and sufficient for synaptic mRNA localization. Mutational and chemical probing analyses are consistent with a role for secondary structure in this process. PMID:22383561

  3. Enzymatic synthesis of DNA complementary to mitochondrial mRNA via reverse transcription.

    PubMed Central

    Frolova, L; Arsenyan, S; Avdonina, T; Gaitskhoki, V; Kisselev, O; Neifach, S; Kisselev, L

    1978-01-01

    The poly(A)-containing mitochondrial mRNAs of rat liver were tested for their ability to serve as templates for the DNA synthesis by means of reverse transcription in the presence of the oligo(dT) primer and the RNA-directed DNA-polymerase from avian myeloblastosis virus. The mT-mRNA does not support the DNA synthesis in the standard conditions sufficient for effective reverse transcription of rabbit globin mRNA and of poly(A) in the presence of oligo(dT) primers. After a mild alkaline treatment of the mRNA and subsequent polyadenylation of the 3'-termini of the generated fragments with ATP:RNA adenyltransferase from E.coli the poly(A) (+) polyribonucleotides are able to serve as templates for reverse transcription in the presence of oligo(dT) and the reverse transcriptase. A conclusion is made that a "structural stop" exists in mitochondrial mRNA non-translable regions adjacent to the poly(A) terminal sequence. The "structural stop" is suggested to be caused by post-transcriptional modification of mRNA (methylation, etc.) or by a particularly stable secondary structure in this region of the mRNA molecules. PMID:77007

  4. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

    Kreamer, G.L.; Squibb, K.; Gioeli, D.; Garte, S.J.; Wirgin, I. (New York University Medical Center, Institute of Environmental Medicine, Tuxedo (USA))

    1991-06-01

    The authors sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, they found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of {beta}-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers.

  5. The Streptococcus mutans irvA gene encodes a trans-acting riboregulatory mRNA.

    PubMed

    Liu, Nan; Niu, Guoqing; Xie, Zhoujie; Chen, Zhiyun; Itzek, Andreas; Kreth, Jens; Gillaspy, Allison; Zeng, Lin; Burne, Robert; Qi, Fengxia; Merritt, Justin

    2015-01-01

    In both prokaryotes and eukaryotes, insight into gene function is typically obtained by in silico homology searches and/or phenotypic analyses of strains bearing mutations within open reading frames. However, the studies herein illustrate how mRNA function is not limited to the expression of a cognate protein. We demonstrate that a stress-induced protein-encoding mRNA (irvA) from the dental caries pathogen Streptococcus mutans directly modulates target mRNA (gbpC) stability through seed pairing interactions. The 5' untranslated region of irvA mRNA is a trans riboregulator of gbpC and a critical activator of the DDAG stress response, whereas IrvA functions independently in the regulation of natural competence. The irvA riboregulatory domain controls GbpC production by forming irvA-gbpC hybrid mRNA duplexes that prevent gbpC degradation by an RNase J2-mediated pathway. These studies implicate a potentially ubiquitous role for typical protein-encoding mRNAs as riboregulators, which could alter current concepts in gene regulation. PMID:25574948

  6. Acetaldehyde increases mRNA levels of glucose-6-phosphate dehydrogenase in rat hepatocytes in culture

    SciTech Connect

    Teel, J.F.; Kletzien, R.F.; Ginsberg, L.C.; Stevens, G.J.; Stapleton, S.R. (Western Michigan Univ., Kalamazoo (United States) Upjohn Co., Kalamazoo, MI (United States))

    1991-03-11

    Addition of insulin and glucocorticoids for 48h to rat hepatocytes in culture increased glucose-6-phosphate dehydrogenase (G6PDH) activity and mRNA levels. Ethanol was found to potentiate this increase. The addition of ethanol alone for 48h however, had no effect on G6PDH activity or mRNA level. Since the effect of ethanol on G6PDH appears to be dependent on hormones as well as a long incubation time, the data is suggestive of the effect of ethanol being mediated through a metabolite. These studies were therefore initiated to investigate the effects of a major metabolite of ethanol, acetaldehyde. In rat hepatocytes incubated in a chemically defined medium the addition of acetaldehyde increased G6PDH mRNA levels 2-3 fold over control. This increase was observed in as little as 6h of acetaldehyde incubation. When the cells were incubated in the presence of insulin and hydrocortisone for 6h, G6PDH mRNA levels were doubled. Addition of acetaldehyde under these same conditions caused an additional 2-3 fold accumulation of G6PDH mRNA. These effects of acetaldehyde are selective as the control mRNAs were unaffected. Studies are continuing to investigate the mechanism by which acetaldehyde exerts its effects.

  7. Inhibition of NF-?B, iNOS mRNA, COX2 mRNA, and COX catalytic activity by phenyl- N- tert-butylnitrone (PBN)

    Microsoft Academic Search

    Yashige Kotake; Hong Sang; Takashi Miyajima; Gemma L. Wallis

    1998-01-01

    Previously, the spin trapping agent phenyl-N-tert-butylnitrone (PBN) has been shown to decrease the level of nitric oxide synthase mRNA in vivo. This inhibition is suggested to be an underlying mechanism for PBN’s wide variety of pharmacological actions in animal models. However, the determination of PBN’s cellular pharmacological activities has not been carried out, but is necessary for the understanding of

  8. Regulation of Urokinase Receptor Expression by p53: Novel Role in Stabilization of uPAR mRNA?

    PubMed Central

    Shetty, Sreerama; Velusamy, Thirunavukkarasu; Idell, Steven; Shetty, Praveenkumar; Mazar, Andrew P.; Bhandary, Yashodhar P.; Shetty, Rashmi S.

    2007-01-01

    We found that p53-deficient (p53?/?) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53?/? cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3? untranslated region (3?UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3?UTR sequence, and insertion of the p53 binding sequence into ?-globin mRNA destabilized ?-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric ?-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells. PMID:17548471

  9. Regulation of urokinase receptor expression by p53: novel role in stabilization of uPAR mRNA.

    PubMed

    Shetty, Sreerama; Velusamy, Thirunavukkarasu; Idell, Steven; Shetty, Praveenkumar; Mazar, Andrew P; Bhandary, Yashodhar P; Shetty, Rashmi S

    2007-08-01

    We found that p53-deficient (p53(-/-)) lung carcinoma (H1299) cells express robust levels of cell surface uPAR and uPAR mRNA. Expression of p53 protein in p53(-/-) cells suppressed basal and urokinase (uPA)-induced cell surface uPAR protein and increased uPAR mRNA degradation. Inhibition of p53 by RNA silencing in Beas2B human airway epithelial cells conversely increased basal as well as uPA-mediated uPAR expression and stabilized uPAR mRNA. Purified p53 protein specifically binds to the uPAR mRNA 3' untranslated region (3'UTR), and endogenous uPAR mRNA associates with p53. The p53 binding region involves a 37-nucleotide uPAR 3'UTR sequence, and insertion of the p53 binding sequence into beta-globin mRNA destabilized beta-globin mRNA. Inhibition of p53 expression in these cells reverses decay of chimeric beta-globin-uPAR mRNA. These observations demonstrate a novel regulatory role for p53 as a uPAR mRNA binding protein that down-regulates uPAR expression, destabilizes uPAR mRNA, and thereby contributes to the viability of human airway epithelial or lung carcinoma cells. PMID:17548471

  10. Integrated miRNA and mRNA expression profiling to identify mRNA targets of dysregulated miRNAs in non-obstructive azoospermia

    PubMed Central

    Zhuang, Xuan; Li, Zhiming; Lin, Huinuan; Gu, Long; Lin, Qing; Lu, Zhongxian; Tzeng, Chi-Meng

    2015-01-01

    The aim of this study was to identify mRNA targets of dysregulated miRNAs through the integrated analysis of miRNA and mRNA expression profiling in men with normal versus impaired spermatogenesis. The expression of mRNAs and miRNAs in testicular tissues obtained from males with non-obstructive azoospermia (NOA, n = 4) or obstructive azoospermia (OA, n = 3) with normal spermatogenesis was analyzed using microarray technology. Some of the most interesting results were validated by real time PCR using samples from the same cohort. Ninety-three miRNAs and 4172 mRNAs were differentially expressed in the NOA and normozoospermic OA patients. In addition to confirming that significantly dysregulated genes and miRNAs play pivotal roles in NOA, promising correlation signatures of these miRNA/mRNA pairs were discovered in this study. The functional classification of the miRNA/mRNA pairs revealed that differentially expressed genes were most frequently associated with spermatogenesis, the cell meiosis, the cell cycle, and the development of secondary male sexual characteristics. This is the first systematic profiling of both mRNA and miRNA in testicular tissues of patients with NOA and OA. Our results indicate that the phenotypic status of NOA is characterized by the dysfunction of normal spermatogenesis when compared with OA or normozoospermic males. PMID:25628250

  11. c-jun mRNA expression and profilin mRNA amplification in rat alveolar macrophages exposed to volcanic ash and sulfur dioxide.

    PubMed

    Samukawa, Takuya; Arasidani, Keiichi; Hori, Hajime; Hirano, Hideyasu; Arima, Terukatsu

    2003-10-01

    Local residents exposed to heavy falls of ash discharged by Mt. Sakurajima, an active volcano, have been reported to develop acute and chronic inflammation of the respiratory tract. The present study aimed to determine the primary cause of this inflammation using an experimental model. Wistar rats were exposed for 5 days (4 h/d) to air containing 100 mg/m3 volcanic ash (mass median aerodynamic diameter, 4.3 microm; geometric standard deviation, 1.7) with or without 1.5 ppm sulfur dioxide (SO2). The lungs were then lavaged, and mRNA was extracted from alveolar macrophages and assessed by reverse transcription-polymerase chain reaction (RT-PCR). In the lavage fluid, no change in cellularity or increase in the content of tumor necrosis factor (TNF)-alpha was detected. However, at 1 h following exposure, 80% of macrophages were seen to have phagocytosed the volcanic ash. This percentage was unchanged at 24 h after exposure. Profilin mRNA content of the macrophages was elevated, and c-jun mRNA was expressed. Alveolar macrophages exposed to volcanic ash and SO2, therefore, are likely to have some inflammatory and fibrogenic potential. PMID:14620666

  12. ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue.

    PubMed

    Kurita, Daisuke; Chadani, Yuhei; Muto, Akira; Abo, Tatsuhiko; Himeno, Hyouta

    2014-12-01

    Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3' end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3' end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding. PMID:25355516

  13. ArfA recognizes the lack of mRNA in the mRNA channel after RF2 binding for ribosome rescue

    PubMed Central

    Kurita, Daisuke; Chadani, Yuhei; Muto, Akira; Abo, Tatsuhiko; Himeno, Hyouta

    2014-01-01

    Although trans-translation mediated by tmRNA-SmpB has long been known as the sole system to relieve bacterial stalled ribosomes, ArfA has recently been identified as an alternative factor for ribosome rescue in Escherichia coli. This process requires hydrolysis of nascent peptidyl-tRNA by RF2, which usually acts as a stop codon-specific peptide release factor. It poses a fascinating question of how ArfA and RF2 recognize and rescue the stalled ribosome. Here, we mapped the location of ArfA in the stalled ribosome by directed hydroxyl radical probing. It revealed an ArfA-binding site around the neck region of the 30S subunit in which the N- and C-terminal regions of ArfA are close to the decoding center and the mRNA entry channel, respectively. ArfA and RF2 sequentially enter the ribosome stalled in either the middle or 3? end of mRNA, whereas RF2 induces a productive conformational change of ArfA only when ribosome is stalled at the 3? end of mRNA. On the basis of these results, we propose that ArfA functions as the sensor to recognize the target ribosome after RF2 binding. PMID:25355516

  14. Amicoumacin a inhibits translation by stabilizing mRNA interaction with the ribosome.

    PubMed

    Polikanov, Yury S; Osterman, Ilya A; Szal, Teresa; Tashlitsky, Vadim N; Serebryakova, Marina V; Kusochek, Pavel; Bulkley, David; Malanicheva, Irina A; Efimenko, Tatyana A; Efremenkova, Olga V; Konevega, Andrey L; Shaw, Karen J; Bogdanov, Alexey A; Rodnina, Marina V; Dontsova, Olga A; Mankin, Alexander S; Steitz, Thomas A; Sergiev, Petr V

    2014-11-20

    We demonstrate that the antibiotic amicoumacin A (AMI) is a potent inhibitor of protein synthesis. Resistance mutations in helix 24 of the 16S rRNA mapped the AMI binding site to the small ribosomal subunit. The crystal structure of bacterial ribosome in complex with AMI solved at 2.4 Å resolution revealed that the antibiotic makes contacts with universally conserved nucleotides of 16S rRNA in the E site and the mRNA backbone. Simultaneous interactions of AMI with 16S rRNA and mRNA and the in vivo experimental evidence suggest that it may inhibit the progression of the ribosome along mRNA. Consistent with this proposal, binding of AMI interferes with translocation in vitro. The inhibitory action of AMI can be partly compensated by mutations in the translation elongation factor G. PMID:25306919

  15. Rotation of the head of the 30S ribosomal subunit during mRNA translocation.

    PubMed

    Guo, Zhuojun; Noller, Harry F

    2012-12-11

    Elongation factor-G-catalyzed translocation of mRNA and tRNAs during protein synthesis involves large-scale conformational changes in the ribosome. Formation of hybrid-state intermediates is coupled to counterclockwise (forward) rotation of the body of the 30S subunit. Recent structural studies implicate intrasubunit rotation of the 30S head in translocation. Here, we observe rotation of the head during translocation in real time using ensemble stopped-flow FRET with ribosomes containing fluorescent probes attached to specific positions in the head and body of the 30S subunit. Our results allow ordering of the rates of movement of the 30S subunit body and head during translocation: body forward > head forward > head reverse ? body reverse. The rate of quenching of pyrene-labeled mRNA is consistent with coupling of mRNA translocation to head rotation. PMID:23188795

  16. Promoter-Bound Trinucleotide Repeat mRNA Drives Epigenetic Silencing in Fragile X Syndrome

    PubMed Central

    Colak, Dilek; Zaninovic, Nikica; Cohen, Michael S.; Rosenwaks, Zev; Yang, Wang-Yong; Gerhardt, Jeannine; Disney, Matthew D.; Jaffrey, Samie R.

    2015-01-01

    Epigenetic gene silencing is seen in several repeat-expansion diseases. In fragile X syndrome, the most common genetic form of mental retardation, a CGG trinucleotide–repeat expansion adjacent to the fragile X mental retardation 1 (FMR1) gene promoter results in its epigenetic silencing. Here, we show that FMR1 silencing is mediated by the FMR1 mRNA. The FMR1 mRNA contains the transcribed CGG-repeat tract as part of the 5? untranslated region, which hybridizes to the complementary CGG-repeat portion of the FMR1 gene to form an RNA·DNA duplex. Disrupting the interaction of the mRNA with the CGG-repeat portion of the FMR1 gene prevents promoter silencing. Thus, our data link trinucleotide-repeat expansion to a form of RNA-directed gene silencing mediated by direct interactions of the trinucleotide-repeat RNA and DNA. PMID:24578575

  17. mRNA transport in yeast: time to reinvestigate the functions of the nucleolus.

    PubMed Central

    Schneiter, R; Kadowaki, T; Tartakoff, A M

    1995-01-01

    Nucleocytoplasmic transport of mRNA is vital to gene expression and may prove to be key to its regulation. Genetic approaches in Saccharomyces cerevisiae have led to the identification of conditional mutants defective in mRNA transport. Mutations in approximately two dozen genes result in accumulation of transcripts, trapped at various sites in the nucleus, as detected by in situ hybridization. Phenotypic and molecular analyses of many of these mRNA transport mutants suggest that, in yeast, the function of the nucleus is not limited to the biogenesis of pre-ribosomes but may also be important for transport of poly(A)+ RNA. A similar function of the animal cell nucleolus is suggested by several observations. Images PMID:7626803

  18. Prp19C and TREX: interacting to promote transcription elongation?and mRNA export.

    PubMed

    Chanarat, Sittinan; Burkert-Kautzsch, Cornelia; Meinel, Dominik M; Sträßer, Katja

    2012-01-01

    During transcription of protein coding genes by RNA Polymerase II the mRNA is processed and packaged into an mRNP. Among the proteins binding cotranscriptionally to the mRNP are mRNA export factors. One of the protein complexes thus coupling transcription to mRNA export is the TREX complex. However, despite the fact that TREX was identified and characterized about a decade ago, it had remained enigmatic how TREX is recruited to genes. The conserved Prp19 complex (Prp19C) has long been known for its function in splicing. We recently identified Prp19C to be essential for a second step in gene expression namely TREX occupancy at transcribed genes, answering this long-standing question but also raising new ones. PMID:22456314

  19. Opiate exposure and withdrawal dynamically regulate mRNA expression in the serotonergic dorsal raphe nucleus.

    PubMed

    Lunden, J W; Kirby, L G

    2013-12-19

    Previous results from our lab suggest that hypofunctioning of the serotonergic (5-HT) dorsal raphe nucleus (DRN) is involved in stress-induced opiate reinstatement. To further investigate the effects of morphine dependence and withdrawal on the 5-HT DRN system, we measured gene expression at the level of mRNA in the DRN during a model of morphine dependence, withdrawal and post withdrawal stress exposure in rats. Morphine pellets were implanted for 72h and then either removed or animals were injected with naloxone to produce spontaneous or precipitated withdrawal, respectively. Animals exposed to these conditions exhibited withdrawal symptoms including weight loss, wet dog shakes and jumping behavior. Gene expression for brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB), corticotrophin releasing-factor (CRF)-R1, CRF-R2, alpha 1 subunit of the GABAA receptor (GABAA-?1), ?-opioid receptor (MOR), 5-HT1A receptor, tryptophan hydroxylase2 (TPH2) and the 5-HT transporter was then measured using quantitative real-time polymerase chain reaction at multiple time-points across the model of morphine exposure, withdrawal and post withdrawal stress. Expression levels of BDNF, TrkB and CRF-R1 mRNA were decreased during both morphine exposure and following 7days of withdrawal. CRF-R2 mRNA expression was elevated after 7days of withdrawal. 5-HT1A receptor mRNA expression was decreased following 3h of morphine exposure, while TPH2 mRNA expression was decreased after 7days of withdrawal with swim stress. There were no changes in the expression of GABAA-?1, MOR or 5-HT transporter mRNA. Collectively these results suggest that alterations in neurotrophin support, CRF-dependent stress signaling, 5-HT synthesis and release may underlie 5-HT DRN hypofunction that can potentially lead to stress-induced opiate relapse. PMID:24055683

  20. Mammalian Peptidylglycine ?-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

    PubMed Central

    Brenet, Fabienne; Dussault, Nadège; Borch, Jonas; Ferracci, Géraldine; Delfino, Christine; Roepstorff, Peter; Miquelis, Raymond; Ouafik, L'Houcine

    2005-01-01

    Peptidylglycine ?-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal ?-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3? untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3? UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3? UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3? UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules. PMID:16107699

  1. Evidence for post-initiation regulation of mRNA biogenesis in tuberculosis1

    PubMed Central

    Salamon, Hugh; Qiao, Yaming; Cheng, Jeff C.; Yamaguchi, Ken D.; Soteropoulos, Patricia; Weiden, Michael; Gennaro, Maria Laura; Pine, Richard

    2013-01-01

    Mycobacterium tuberculosis infection alters macrophage gene expression and macrophage response to interferon gamma (IFN?), a critical host defense cytokine. However, regulation of these changes is poorly understood. We report discordance of changes in nascent transcript and total nuclear RNA abundance for the transcription factors STAT1 and IRF1, together with lack of effect on their RNA half-lives, in human THP-1 cells infected with M. tuberculosis and stimulated with IFN?. The results indicate that negative post-initiation regulation of mRNA biogenesis limits the expression of these factors, which mediate host defense against M. tuberculosis through the cellular response to IFN?. Consistent with the results for STAT1 and IRF1, transcriptome analysis reveals down-regulation of post-initiation mRNA biogenesis processes and pathways by infection, with and without IFN? stimulation. Clinical relevance for regulation of post-initiation mRNA biogenesis is demonstrated by studies of donor samples showing that post-initiation mRNA biogenesis pathways are repressed in latent tuberculosis infection compared to cured disease and in active tuberculosis compared to ongoing treatment or to latent tuberculosis. For active disease and latent infection donors from two populations (London, UK, and The Gambia), each analyzed using a different platform, pathway-related gene expression differences were highly correlated, demonstrating substantial specificity in the effect. Collectively, the molecular and bioinformatic analyses point toward down-regulation of post-initiation mRNA biogenesis pathways as a means by which M. tuberculosis infection limits expression of immunologically essential transcription factors. Thus, negative regulation of post-initiation mRNA biogenesis may constrain the macrophage response to infection and overall host defense against tuberculosis. PMID:23378427

  2. An investigation of nutrient-dependent mRNA translation in Drosophila larvae

    PubMed Central

    Nagarajan, Sabarish; Grewal, Savraj S.

    2014-01-01

    ABSTRACT The larval period of the Drosophila life cycle is characterized by immense growth. In nutrient rich conditions, larvae increase in mass approximately two hundred-fold in five days. However, upon nutrient deprivation, growth is arrested. The prevailing view is that dietary amino acids drive this larval growth by activating the conserved insulin/PI3 kinase and Target of rapamycin (TOR) pathways and promoting anabolic metabolism. One key anabolic process is protein synthesis. However, few studies have attempted to measure mRNA translation during larval development or examine the signaling requirements for nutrient-dependent regulation. Our work addresses this issue. Using polysome analyses, we observed that starvation rapidly (within thirty minutes) decreased larval mRNA translation, with a maximal decrease at 6–18?hours. By analyzing individual genes, we observed that nutrient-deprivation led to a general reduction in mRNA translation, regardless of any starvation-mediated changes (increase or decrease) in total transcript levels. Although sugars and amino acids are key regulators of translation in animal cells and are the major macronutrients in the larval diet, we found that they alone were not sufficient to maintain mRNA translation in larvae. The insulin/PI3 kinase and TOR pathways are widely proposed as the main link between nutrients and mRNA translation in animal cells. However, we found that genetic activation of PI3K and TOR signaling, or regulation of two effectors – 4EBP and S6K – could not prevent the starvation-mediated translation inhibition. Similarly, we showed that the nutrient stress-activated eIF2? kinases, GCN2 and PERK, were not required for starvation-induced inhibition of translation in larvae. These findings indicate that nutrient control of mRNA translation in larvae is more complex than simply amino acid activation of insulin and TOR signaling. PMID:25305039

  3. Biomarkers for Monitoring Pre-Analytical Quality Variation of mRNA in Blood Samples

    PubMed Central

    Zhang, Hui; Korenková, Vlasta; Sjöback, Robert; Švec, David; Björkman, Jens; Kruhøffer, Mogens; Verderio, Paolo; Pizzamiglio, Sara; Ciniselli, Chiara Maura; Wyrich, Ralf; Oelmueller, Uwe; Kubista, Mikael; Lindahl, Torbjørn; Lönneborg, Anders; Rian, Edith

    2014-01-01

    There is an increasing need for proper quality control tools in the pre-analytical phase of the molecular diagnostic workflow. The aim of the present study was to identify biomarkers for monitoring pre-analytical mRNA quality variations in two different types of blood collection tubes, K2EDTA (EDTA) tubes and PAXgene Blood RNA Tubes (PAXgene tubes). These tubes are extensively used both in the diagnostic setting as well as for research biobank samples. Blood specimens collected in the two different blood collection tubes were stored for varying times at different temperatures, and microarray analysis was performed on resultant extracted RNA. A large set of potential mRNA quality biomarkers for monitoring post-phlebotomy gene expression changes and mRNA degradation in blood was identified. qPCR assays for the potential biomarkers and a set of relevant reference genes were generated and used to pre-validate a sub-set of the selected biomarkers. The assay precision of the potential qPCR based biomarkers was determined, and a final validation of the selected quality biomarkers using the developed qPCR assays and blood samples from 60 healthy additional subjects was performed. In total, four mRNA quality biomarkers (USP32, LMNA, FOSB, TNRFSF10C) were successfully validated. We suggest here the use of these blood mRNA quality biomarkers for validating an experimental pre-analytical workflow. These biomarkers were further evaluated in the 2nd ring trial of the SPIDIA-RNA Program which demonstrated that these biomarkers can be used as quality control tools for mRNA analyses from blood samples. PMID:25369468

  4. Induction of gadd153 mRNA by nutrient deprivation is overcome by glutamine.

    PubMed

    Huang, Q; Lau, S S; Monks, T J

    1999-07-01

    The growth arrest and DNA damage-inducible (gadd) genes are co-ordinately activated by a variety of genotoxic agents and/or growth-cessation signals. The regulation of gadd153 mRNA was investigated in renal proximal tubular epithelial cells (LLC-PK1) cultured in a nutrient- and serum-deprived medium. The addition of glutamine alone to LLC-PK1 cells cultured in Earl's balanced salt solution (EBSS) is sufficient to suppress gadd153 mRNA expression, and the removal of only glutamine from Dulbecco's modified Eagle's medium (DMEM) is also sufficient to induce gadd153 mRNA expression. Consistent with these findings, the inhibition of glutamine utilization with acivicin and 6-diazo-5-oxo-l-norleucine (DON) in cells grown in a glutamine-containing medium effectively induces gadd153 expression. Glutamine can be used as an energy source in cultured mammalian cells. However, it is unlikely that deficits in cellular energy stores (ATP) are coupled to gadd153 mRNA expression, because concentrations of ATP, UTP and GTP are all elevated in EBSS-exposed cells, and the addition of alpha-oxoglutarate to cells grown in EBSS has no effect on gadd153 mRNA expression. In contrast, concentrations of CTP decline substantially in EBSS and glutamine-deprived DMEM-cultured cells. Glutamine also serves as a precursor for the synthesis of protein and DNA. The addition of glutamine to cells grown in EBSS partly restores CTP concentrations. The addition of pyrimidine ribonucleosides (cytidine and uridine) to LLC-PK1 cells also restores CTP concentrations, in a manner commensurate with their relative abilities to overcome gadd153 expression. Finally, glutamine does not completely suppress DNA damage-induced gadd153 expression, suggesting that multiple signalling pathways lead to the expression of gadd153 mRNA under conditions of nutrient deprivation and DNA damage. PMID:10377266

  5. Intracellular delivery of molecular beacons by PMMA nanoparticles and carbon nanotubes for mRNA sensing

    NASA Astrophysics Data System (ADS)

    Giannetti, A.; Tombelli, S.; Trono, C.; Ballestri, M.; Giambastiani, G.; Guerrini, A.; Sotgiu, G.; Tuci, G.; Varchi, G.; Baldini, F.

    2013-02-01

    We describe here the use of carbon nanotubes (CNTs) and polymeric nanoparticles made of a core of polymethylmethacrylate (PMMA) surrounded by a shell bearing cationic groups, as intracellular delivery tools of molecular beacons (MBs), particular fluorescent DNA probes, for the detection and localization of a specific mRNA. Survivin mRNA targeting MBs have been used with Atto647N and Blackberry 650 as fluorophore/quencher pair. The MB was anchored to the surface of CNTs and PMMA nanoparticles via a commercial sulfhydryl-reactive heterobifunctional crosslinker and the achieved nanomaterials were then characterized in vitro.

  6. A Max-Plus Model of Ribosome Dynamics During mRNA Translation

    E-print Network

    Brackley, Chris A; Romano, M Carmen; Thiel, Marco

    2011-01-01

    We examine the dynamics of the translation stage of cellular protein production, in which ribosomes move uni-directionally along mRNA strands building an amino acid chain as they go. We describe the system using a timed event graph - a class of Petri net useful for studying discrete events which take a finite time. We use max-plus algebra to describe a deterministic version of the model, calculating the protein production rate and density of ribosomes on the mRNA. We find exact agreement between these analytical results and numerical simulations of the deterministic case.

  7. A human promyelocyte mRNA transiently induced by TPA is homologous to yeast IPP isomerase

    SciTech Connect

    Xuan, J.W.; Kowalski, J.; Denhardt, D.T. (Univ. of Western Ontario (Canada)); Chambers, A.F. (London Regional Cancer Centre, Ontario (Canada))

    1994-03-01

    A cDNA clone was isolated from HL60 human promyelocyte cells on the basis of the induction of its cognate mRNA by the phorbol ester TPA (12-O-tetrade-canoyl phorbol-13-acetate) in the presence of cycloheximide. Northern analysis showed a peak of induction of a 1.9-kb mRNA at 3-6 h after TPA addition. Sequence analysis revealed that the cDNA was likely a human homolog of yeast isopentenyl diphosphate:dimethylallyl diphosphate isomerase, an enzyme important for isoprenoid synthesis and the prenylation of proteins such as ras p21. 14 refs., 2 figs.

  8. Accumulation of cell wall hydroxyproline-rich glycoprotein mRNA is an early event in maize embryo cell differentiation.

    PubMed Central

    Ruiz-Avila, L; Burgess, S R; Stiefel, V; Ludevid, M D; Puigdomènech, P

    1992-01-01

    The accumulation of the mRNA coding for a hydroxyproline-rich glycoprotein (HRGP), an abundant component of the wall from the cells of vegetative tissues, has been observed in maize embryo by in situ hybridization. The HRGP mRNA accumulates in the embryo axis and not in the scutellum and preferentially in dividing and provascular cells. The histone H4 mRNA is distributed in similar tissues but is restricted to defined groups of cells, indicating that these two gene products have a different steady-state level of accumulation during the cell cycle. The HRGP mRNA appears to be a useful marker for early formation of the vascular systems. The mRNA accumulation correlates in space and time with cells having a low content of cellulose in their walls, suggesting that the mRNA is produced in the early stages of cell wall formation before complete deposition of cellulose. Images PMID:1549604

  9. Using protein abundance to indicate underlying mRNA expression levels in E. coli: an SEM modelling approach

    PubMed Central

    Harris, Jacqueline J.; Duarte, Christine W.; Mossing, Michael C.

    2012-01-01

    Do steady-state protein levels accurately predict mRNA levels? Based on the central dogma (DNA ? RNA ? protein) current protein levels are representative of mRNA present at an earlier time. However, most cellular mRNA – protein comparative studies try to relate steady-state protein levels to current mRNA levels in cells. Protein steady-states are more correctly related to protein production, protein degradation and other complex cellular conditions. Using Structural Equation Modelling (SEM) we relate linear protein measurements to latent mRNA in E. coli. This method can be used to find the optimal protein measurements that explain underlying mRNA expression, and better understand the proteomic and transcriptomic relationship in E. coli gene expression. PMID:22199038

  10. The tRNA Splicing Endonuclease Complex Cleaves the Mitochondria-localized CBP1 mRNA.

    PubMed

    Tsuboi, Tatsuhisa; Yamazaki, Reina; Nobuta, Risa; Ikeuchi, Ken; Makino, Shiho; Ohtaki, Ayumi; Suzuki, Yutaka; Yoshihisa, Tohru; Trotta, Christopher; Inada, Toshifumi

    2015-06-26

    The tRNA splicing endonuclease (Sen) complex is located on the mitochondrial outer membrane and splices precursor tRNAs in Saccharomyces cerevisiae. Here, we demonstrate that the Sen complex cleaves the mitochondria-localized mRNA encoding Cbp1 (cytochrome b mRNA processing 1). Endonucleolytic cleavage of this mRNA required two cis-elements: the mitochondrial targeting signal and the stem-loop 652-726-nt region. Mitochondrial localization of the Sen complex was required for cleavage of the CBP1 mRNA, and the Sen complex cleaved this mRNA directly in vitro. We propose that the Sen complex cleaves the CBP1 mRNA, which is co-translationally localized to mitochondria via its mitochondrial targeting signal. PMID:25971974

  11. Sensitive nonradioactive detection of mRNA in tissue sections: Novel application of the whole-mount in situ hybridization

    Microsoft Academic Search

    Antoon F. M. Moorman; Arjan C. Houweling; Boer de P. A. J; Vincent M. Christoffels

    2001-01-01

    SUMMARY The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chro- mosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunc- tion with high spatial resolution, we developed a nonradioactive in

  12. Markedly Increased Constitutive CYP1A1 mRNA Levels in the Fertilized Ovum of the Mouse

    Microsoft Academic Search

    Anup Dey; Daniel W. Nebert

    1998-01-01

    Using a highly sensitive RT-PCR technique that measures mRNA (cDNA)-to-DNA ratios, we are able to detect constitutive CYP1A1 mRNA in adult mouse liver as well as in the oocyte. Twelve hours after fertilization of the ovum, there is a more than 100-fold increase in constitutive CYP1A1 mRNA levels; this dramatic increase completely disappears by the 2-cell stage at gestational day

  13. Four-base codon mediated mRNA display to construct peptide libraries that contain multiple nonnatural amino acids

    Microsoft Academic Search

    Norihito Muranaka; Takahiro Hohsaka; Masahiko Sisido

    2006-01-01

    In vitro selection and directed evolution of peptides from mRNA display are powerful strategies to find novel peptide ligands that bind to target bio- molecules. In this study, we expanded the mRNA display method to include multiple nonnatural amino acids by introducing three different four- base codons at a randomly selected single position on the mRNA. Another nonnatural amino acid

  14. Differential Upregulation of Aquaporin4 mRNA Expression in Reactive Astrocytes after Brain Injury: Potential Role in Brain Edema

    Microsoft Academic Search

    M. L. Vizuete; J. L. Venero; C. Vargas; A. A. Ilundáin; M. Echevarr??a; A. Machado; J. Cano

    1999-01-01

    Astrocytes and aquaporin-4 (AQP4) play a significant role in brain ion homeostasis. Consequently the regulation of AQP4 mRNA in the CNS after different neurological insults was of interest. A single intrastriatal injection of ringer or quinolinic acid strongly induced AQP4 mRNA in the striatum, specially at the core of the lesion. Colocalization studies demonstrated that AQP4 mRNA induction was restricted

  15. Quantitation of the mRNA levels of Epo and EpoR in various tissues in the ovine fetus

    Microsoft Academic Search

    R. Bruce David; Gaik Bee Lim; Karen M Moritz; Irene Koukoulas; E. Marelyn Wintour

    2002-01-01

    A partial cDNA of the sheep erythropoietin receptor (EpoR) was obtained and used in real-time PCR to quantitate mRNA levels in placenta, liver and kidney throughout development (term=150 days). This was compared with Epo mRNA levels in the same tissues. Both Epo and EpoR mRNA were present in the placenta throughout gestation at low levels from 66 days onwards and

  16. mRNA Levels in Control Rat Liver Display Strain-Specific, Hereditary, and AHR-Dependent Components

    Microsoft Academic Search

    Paul C. Boutros; Ivy D. Moffat; Allan B. Okey; Raimo Pohjanvirta

    2011-01-01

    Rat is a major model organism in toxicogenomics and pharmacogenomics. Hepatic mRNA profiles after treatment with xenobiotic chemicals are used to predict and understand drug toxicity and mechanisms. Surprisingly, neither inter- and intra-strain variability of mRNA abundances in control rats nor the heritability of rat mRNA abundances yet been established. We address these issues by studying five populations: the popular

  17. Dimeric Structure of a Human Apolipoprotein B mRNA Editing Protein and Cloning and Chromosomal Localization of Its Gene

    Microsoft Academic Search

    Paul P. Lau; Hui-Jia Zhu; Antonio Baldini; Chutaporn Charnsangavej; Lawrence Chan

    1994-01-01

    Apolipoprotein B (apoB) mRNA editing consists of a posttranscriptional C --> U conversion involving the first base of the codon CAA encoding glutamine-2153 to UAA, a stop codon, in apoB mRNA. Using a cloned rat cDNA as a probe, we cloned the cDNA and genomic sequences of the gene for a human apoB mRNA editing protein. Expression of the cDNA

  18. Barentsz is essential for the posterior localization of oskar mRNA and colocalizes with it to the posterior pole

    PubMed Central

    van Eeden, Fredericus J.M.; Palacios, Isabel M.; Petronczki, Mark; Weston, Matthew J.D.; St Johnston, Daniel

    2001-01-01

    The localization of Oskar at the posterior pole of the Drosophila oocyte induces the assembly of the pole plasm and therefore defines where the abdomen and germ cells form in the embryo. This localization is achieved by the targeting of oskar mRNA to the posterior and the localized activation of its translation. oskar mRNA seems likely to be actively transported along microtubules, since its localization requires both an intact microtubule cytoskeleton and the plus end–directed motor kinesin I, but nothing is known about how the RNA is coupled to the motor. Here, we describe barentsz, a novel gene required for the localization of oskar mRNA. In contrast to all other mutations that disrupt this process, barentsz-null mutants completely block the posterior localization of oskar mRNA without affecting bicoid and gurken mRNA localization, the organization of the microtubules, or subsequent steps in pole plasm assembly. Surprisingly, most mutant embryos still form an abdomen, indicating that oskar mRNA localization is partially redundant with the translational control. Barentsz protein colocalizes to the posterior with oskar mRNA, and this localization is oskar mRNA dependent. Thus, Barentsz is essential for the posterior localization of oskar mRNA and behaves as a specific component of the oskar RNA transport complex. PMID:11481346

  19. Identification of sequences in c-myc mRNA that regulate its steady-state levels.

    PubMed Central

    Yeilding, N M; Rehman, M T; Lee, W M

    1996-01-01

    The level of cellular myc proto-oncogene expression is rapidly regulated in response to environmental signals and influences cell proliferation and differentiation. Regulation is dependent on the fast turnover of c-myc mRNA, which enables cells to rapidly alter c-myc mRNA levels. Efforts to identify elements in myc mRNA responsible for its instability have used a variety of approaches, all of which require manipulations that perturb normal cell metabolism. These various approaches have implicated different regions of the mRNA and have led to a lack of consensus over which regions actually dictate rapid turnover and low steady-state levels of c-myc mRNA. To identify these regions by an approach that does not perturb cell metabolism acutely and that directly assesses the effect of a c-myc mRNA region on the steady-state levels of c-myc mRNA, we developed an assay using reverse transcription and PCR to compare the steady-state levels of human myc mRNAs transcribed from two similarly constructed myc genes transiently cotransfected into proliferating C2C12 myoblasts. Deletion mutations were introduced into myc genes, and the levels of their mRNAs were compared with that of a near-normal, reference myc mRNA. Deletion of most of the myc 3' untranslated region (UTR) raised myc mRNA levels, while deletion of sequences in the myc 5' UTR (most of exon 1), exon 2, or the protein-coding region of exon 3 did not, thus demonstrating that the 3' UTR is responsible for keeping myc mRNA levels low. Using a similar reverse transcription-PCR assay for comparing the steady-state levels of two beta-globin-myc fusion mRNAs, we showed that fusion of the myc 3' UTR lowers globin mRNA levels by destabilizing beta-globin mRNA. Surprisingly, fusion of the protein-coding region of myc exon 3 also lowered globin mRNA steady-state levels. Investigating the possibility that exon 3 coding sequences may play some other role in regulating c-myc mRNA turnover, we demonstrated that these sequences, but not myc 3' UTR sequences, are necessary for the normal posttranscriptional downregulation of c-myc mRNA during myoblast differentiation. We conclude that, while two elements within c-myc mRNA can act as instability determinants in a heterologous context, only the instability element in the 3' UTR regulates its steady-state levels in proliferating C2C12 cells. PMID:8668167

  20. Influence of steroids on the hypothalamic corticotropin-releasing factor and preproenkephalin mRNA responses to stress.

    PubMed Central

    Lightman, S L; Young, W S

    1989-01-01

    We have used in situ hybridization histochemistry to investigate the influence of both circulating corticosteroids and the stress paradigm of i.p. hypertonic saline on the levels of mRNAs encoding corticotropin-releasing factor (CRF) and preproenkephalin in parvocellular neurons of the rat hypothalamus. Stress increased both CRF and preproenkephalin mRNAs, whereas adrenalectomy increased only CRF mRNA. After adrenalectomy, even when CRF mRNA had reached peak levels, stress still further increased CRF mRNA and caused an exaggerated rise in preproenkephalin mRNA. Dexamethasone administration in the fast or intermediate-feedback time domains had no effect on CRF or preproenkephalin mRNA responses to stress; however, when administered over a longer period of time in the slow-feedback time domain dexamethasone reduced basal CRF mRNA levels and the stress-stimulated levels of CRF and preproenkephalin mRNA. These results show that different stimuli to the parvocellular paraventricular hypothalamus differentially regulate CRF transcript levels. Furthermore, in spite of the lack of any detectable effect of changes in circulating glucocorticoid levels on basal levels of preproenkephalin mRNA, glucocorticoids markedly alter the preproenkephalin mRNA response to stress. PMID:2786213

  1. Bromocriptine reduces rat thyrotropin beta-subunit mRNA stability.

    PubMed

    Levy, A; Lightman, S L

    1990-01-01

    We have examined the effect of orally administered bromocriptine on TSH beta-subunit messenger (m)RNA in the anterior pituitary glands of Sprague-Dawley rats using in situ and dot-blot hybridization histochemistry. Quantitative in situ hybridization of pituitary sections demonstrated a 60% reduction in TSH beta-subunit mRNA probe binding from rats fed a diet containing bromocriptine 10 mg/kg/day. This was confirmed by dot-blot analysis of nuclear and cytoplasmic pituitary extracts from the same tissue. Hybridization of cytoplasmic extracts of pituitary cells cultured under actinomycin D-induced transcription arrest showed that part of the effect of bromocriptine appeared to be mediated through a change in TSH beta-subunit mRNA stability and implies that the acute influence of dopamine on TSH metabolism may be transduced by control of TSH beta-subunit mRNA catabolism. This suggests a mechanism by which cells with relatively stable tissue specific mRNAs appear to respond rapidly to hormonal effects at the transcriptional level. PMID:2319107

  2. Decreased TIM-3 mRNA expression in peripheral blood mononuclear cells from nephropathy patients.

    PubMed

    Cai, X Z; Liu, N; Qiao, Y; Du, S Y; Chen, Y; Chen, D; Yu, S; Jiang, Y

    2015-01-01

    Increasing evidence shows that TIM-1 and TIM-3 in-fluence chronic autoimmune diseases, and their expression levels in immune cells from nephritic patients are still unknown. Real-time transcription-polymerase chain reaction analysis was used to deter-mine expression levels of TIM-1 and TIM-3 mRNA in peripheral blood mononuclear cells (PBMCs) from 36 patients with minimal change glo-merulopathy (MCG), 65 patients with lupus nephritis (LN), 78 patients with IgA nephropathy (IgAN), 55 patients with membranous nephropa-thy (MN), 22 patients with crescentic glomerulonephritis (CGN), 26 patients with anaphylactoid purpura nephritis (APN), and 63 healthy controls. TIM-3 mRNA expression significantly decreased in PBMCs from nephritic patients (LN, P < 0.0001; MCG, P < 0.0001; MN, P = 0.0031; CGN, P = 0.0464; IgAN, P = 0.0002; APN, P = 0.0392) com-pared with healthy controls. In contrast, there was no significant differ-ence in TIM-1 mRNA expression between the patients and the healthy controls. Our results suggest that insufficient expression of TIM-3 mRNA may be involved in the pathogenesis of nephropathy. PMID:26125859

  3. Analysis of a mod B mutant in Dictyostelium discoideum using mRNA differential display.

    PubMed

    Yoshida, M; Sendai, Y; Sakuragi, N; Hotta, Y

    2000-02-01

    Three differential cDNAs of Dictyostelium, not detected in the mod B mutant defective in O-glycosylation, were isolated by using an mRNA differential display. These cDNAs encode a protein tyrosine kinase, an adenylyl cyclase and a putative protein kinase C inhibitor whose expression is developmentally regulated. PMID:10795321

  4. Roles for transcript leaders in translation and mRNA decay revealed by transcript leader sequencing

    E-print Network

    Arribere, Joshua Alexander

    Transcript leaders (TLs) can have profound effects on mRNA translation and stability. To map TL boundaries genome-wide, we developed TL-sequencing (TL-seq), a technique combining enzymatic capture of m[superscript 7]G-capped ...

  5. Widespread occurrence of N6-methyladenosine in bacterial mRNA.

    PubMed

    Deng, Xin; Chen, Kai; Luo, Guan-Zheng; Weng, Xiaocheng; Ji, Quanjiang; Zhou, Tianhong; He, Chuan

    2015-07-27

    N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m(6)A as well as m(6)A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m(6)A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m(6)A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m(6)A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m(6)A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m(6)A pattern that is distinct from those in eukaryotes. Most m(6)A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m(6)A peaks indicates that the majority of m(6)A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m(6)A in these pathways. PMID:26068471

  6. Amino acid sequence of the mRNA cap-binding protein from human tissues

    SciTech Connect

    Rychlik, W.; Domier, L.L.; Gardner, P.R.; Hellmann, G.M.; Rhoads, R.E.

    1987-02-01

    The 25-kDa mRNA cap-binding protein (CBP) involved in translation was purified by affinity chromatography from human erythrocytes and rabbit reticulocytes. The sequences of eight human and seven rabbit tryptic and V8 proteolytic peptides were determined. Based on the peptide sequence data, oligodeoxynucleotide probes were synthesized and used to screen human fibroblast and lymphocyte lambda cDNA libraries. The DNA sequence obtained from recombinant lambda phage inserts was found to code for all but one peptide. A 23-base oligonucleotide was synthesized based on the DNA sequence and used to prime synthesis of cDNA from human placental mRNA to construct a third library in lambdagt10. Screening with a 22-base oligonucleotide, whose sequence was upstream from the 23-base primer, yielded numerous recombinant phages with approx.250-base inserts. The 1900-base-pair cDNA sequence compiled from all phage inserts appeared to represent the entire primary sequence of CBP. Blot analysis of human placental and HeLa mRNA revealed multiple CBP mRNA species ranging from 1925 to 2250 bases. The amino acid sequence of CBP showed homology to the cap-binding PB2 protein of influenza virus.

  7. ORGANELLES DO NOT COLOCALIZE WITH mRNA GRANULES IN POST-ISCHEMIC NEURONS

    E-print Network

    DeGracia, Donald J.

    of TA in post-ischemic neurons, such as the hippocampal CA1 re- gion, is one of the strongest predictorsRNA. Together, these results shed additional light on the identity of the mRNA granule by ruling out a direct Materials Antisera for -tubulin (T6199) and neurofilament (NF) H/M (N2912) were purchased from Sigma

  8. Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum

    PubMed Central

    Staudacher, Jonas J.; Naarmann-de Vries, Isabel S.; Ujvari, Stefanie J.; Klinger, Bertram; Kasim, Mumtaz; Benko, Edgar; Ostareck-Lederer, Antje; Ostareck, Dirk H.; Bondke Persson, Anja; Lorenzen, Stephan; Meier, Jochen C.; Blüthgen, Nils; Persson, Pontus B.; Henrion-Caude, Alexandra; Mrowka, Ralf; Fähling, Michael

    2015-01-01

    Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5?- and 3?-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5?- as well as 3?-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage. PMID:25753659

  9. Post-transcriptional regulation of satellite cell quiescence by TTP-mediated mRNA decay.

    PubMed

    Hausburg, Melissa A; Doles, Jason D; Clement, Sandra L; Cadwallader, Adam B; Hall, Monica N; Blackshear, Perry J; Lykke-Andersen, Jens; Olwin, Bradley B

    2015-01-01

    Skeletal muscle satellite cells in their niche are quiescent and upon muscle injury, exit quiescence, proliferate to repair muscle tissue, and self-renew to replenish the satellite cell population. To understand the mechanisms involved in maintaining satellite cell quiescence, we identified gene transcripts that were differentially expressed during satellite cell activation following muscle injury. Transcripts encoding RNA binding proteins were among the most significantly changed and included the mRNA decay factor Tristetraprolin. Tristetraprolin promotes the decay of MyoD mRNA, which encodes a transcriptional regulator of myogenic commitment, via binding to the MyoD mRNA 3' untranslated region. Upon satellite cell activation, p38?/? MAPK phosphorylates MAPKAP2 and inactivates Tristetraprolin, stabilizing MyoD mRNA. Satellite cell specific knockdown of Tristetraprolin precociously activates satellite cells in vivo, enabling MyoD accumulation, differentiation and cell fusion into myofibers. Regulation of mRNAs by Tristetraprolin appears to function as one of several critical post-transcriptional regulatory mechanisms controlling satellite cell homeostasis. PMID:25815583

  10. Intermolecular interaction between a branching ribozyme and associated homing endonuclease mRNA.

    PubMed

    Birgisdottir, Ása B; Nielsen, Henrik; Beckert, Bertrand; Masquida, Benoît; Johansen, Steinar D

    2011-04-01

    RNA tertiary interactions involving docking of GNRA (N; any base; R; purine) hairpin loops into helical stem structures on other regions of the same RNA are one of the most common RNA tertiary interactions. In this study, we investigated a tertiary association between a GAAA hairpin tetraloop in a small branching ribozyme (DiGIR1) and a receptor motif (HEG P1 motif) present in a hairpin structure on a separate mRNA molecule. DiGIR1 generates a 2', 5' lariat cap at the 5' end of its downstream homing endonuclease mRNA by catalysing a self-cleavage branching reaction at an internal processing site. Upon release, the 5' end of the mRNA forms a distinct hairpin structure termed HEG P1. Our biochemical data, in concert with molecular 3D modelling, provide experimental support for an intermolecular tetraloop receptor interaction between the L9 GAAA in DiGIR1 and a GNRA tetraloop receptor-like motif (UCUAAG-CAAGA) found within the HEG P1. The biological role of this interaction appears to be linked to the homing endonuclease expression by promoting post-cleavage release of the lariat capped mRNA. These findings add to our understanding of how protein-coding genes embedded in nuclear ribosomal DNA are expressed in eukaryotes and controlled by ribozymes. PMID:21495911

  11. Dynamics of translation by single ribosomes through mRNA secondary structures

    PubMed Central

    Chen, Chunlai; Zhang, Haibo; Broitman, Steven L.; Reiche, Michael; Farrell, Ian; Cooperman, Barry S.; Goldman, Yale E.

    2013-01-01

    During protein synthesis, the ribosome translates nucleotide triplets in single-stranded mRNA into polypeptide sequences. Strong downstream mRNA secondary (2°) structures, which must be unfolded for translation, can slow or even halt protein synthesis. Here we employ single molecule fluorescence resonance energy transfer to determine reaction rates for specific steps within the elongation cycle as the Escherichia coli ribosome encounters stem loop or pseudoknot mRNA 2° structures. Downstream stem-loops containing 100% G-C base pairs decrease the rates of both tRNA translocation within the ribosome and deacylated tRNA dissociation from the ribosomal exit (E) site. Downstream stem-loops or pseudoknots containing both G-C and A-U pairs also decrease the rate of tRNA dissociation, but they have little effect on tRNA translocation rate. Thus, somewhat surprisingly, unfolding of mRNA 2° structures is more closely coupled to E-site tRNA dissociation than to tRNA translocation. PMID:23542154

  12. Decoupled evolution of coding region and mRNA expression patterns after gene

    E-print Network

    Wagner, Andreas

    Decoupled evolution of coding region and mRNA expression patterns after gene duplication perspective on molecular evolution maintains that the vast majority of mutations affecting gene function are neutral or deleterious. After a gene duplication where both genes are retained, it predicts that original

  13. Toward Automatic Annotation of in situ mRNA Expression Patterns of Drosophila Embryos

    Microsoft Academic Search

    Jie Zhou; Hanchuan Peng

    2005-01-01

    The in situ mRNA hybridization gene expression pattern images of Drosophila melanogaster in the course of embryogenesis provide spatial-temporal information of the expression patterns of a target gene. This information is critical for understanding the roles of genes during the development of embryos. Currently, the annotation of these images is often done by manually assigning a subset of image ontology

  14. SMG6 Cleavage Generates Metastable Decay Intermediates from Nonsense-Containing ?-Globin mRNA

    PubMed Central

    Schoenberg, Daniel R.

    2013-01-01

    mRNAs targeted by endonuclease decay generally disappear without detectable decay intermediates. The exception to this is nonsense-containing human ?-globin mRNA, where the destabilization of full-length mRNA is accompanied by the cytoplasmic accumulation of 5?-truncated transcripts in erythroid cells of transgenic mice and in transfected erythroid cell lines. The relationship of the shortened RNAs to the decay process was characterized using an inducible erythroid cell system and an assay for quantifying full-length mRNA and a truncated RNA missing 169 nucleotides from the 5? end. In cells knocked down for Upf1 a reciprocal increase in full-length and decrease in shortened RNA confirmed the role of NMD in this process. Kinetic analysis demonstrated that the 5?-truncated RNAs are metastable intermediates generated during the decay process. SMG6 previously was identified as an endonuclease involved in NMD. Consistent with involvement of SMG6 in the decay process full-length nonsense-containing ?-globin mRNA was increased and the ?169 decay intermediate was decreased in cells knocked down for SMG6. This was reversed by complementation with siRNA-resistant SMG6, but not by SMG6 with inactivating PIN domain mutations. Importantly, none of these altered the phosphorylation state of Upf1. These data provide the first proof for accumulation of stable NMD products by SMG6 endonuclease cleavage. PMID:24086375

  15. Simultaneous in vivo Quantitation of Vascular Endothelial Growth Factor mRNA Splice Variants

    Microsoft Academic Search

    Wilfried Renner; Ernst Pilger

    1999-01-01

    Vascular endothelial growth factor (VEGF) is an important regulator of angiogenesis. In vivo expression of four different VEGF isoforms, consisting of 121, 165, 189 or 206 amino acids, has been found in the human organism, with all isoforms arising from a single gene by alternative mRNA splicing. We developed an assay for simultaneous quantitation of VEGF isoform expression using competitive

  16. Degradation profile of mRNA in a dead rat body: basic semi-quantification study

    Microsoft Academic Search

    Hiroshi Inoue; Akihiko Kimura; Tsutomu Tuji

    2002-01-01

    To profile postmortem degradation of mRNA, total RNA was extracted, at given postmortem intervals, from the brain, lung, heart and liver of rats left at 20°C. In electrophoretic analysis, total RNA was most stable in the brain, moderately stable in the lung and heart, and most unstable in the liver. Northern blot analysis of total RNA extracts from the brain

  17. CYTOKINE MRNA PROFILES FOR ISOCYANATES WITH KNOWN AND UNKNOWN POTENTIAL TO INDUCE RESPIRATORY SENSITIZATION

    EPA Science Inventory

    Cytokine mRNA Profiles for Isocyanates with Known and Unknown Potential to Induce Respiratory Sensitization. Plitnick, L.M., Loveless, S.E., Ladics, G.S., Holsapple, M.P., Smialowicz, R.J., Woolhiser, M.R., Anderson, P.K., Smith, C., Sailstad, D.M. and Selgrade, M.J.K (2002) Tox...

  18. FEEDING FREQUENCY, GROWTH, GRHRELIN, AND NPY MRNA IN NORRIS AND NWAC103 CHANNEL CATFISH (ICTALURUS PUNCTATUS)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fish growth is influenced by feed availability and intake, genetics, environment, and nutrition. Of these factors, feed intake is perhaps the principal factor affecting growth rate of catfish. We examined the effect of feeding frequency on growth performance and abundance of ghrelin and NPY mRNA o...

  19. 2004 NaturePublishing Group Translation of mRNA into protein represents the final

    E-print Network

    Bedwell, David M.

    ©2004 NaturePublishing Group Translation of mRNA into protein represents the final step in the gene without affecting general protein biosynthesis or the translational status of the cellular transcriptome where transcription is silent or when local control over protein accumulation is required. Although many

  20. G-quadruplexes within prion mRNA: the missing link in prion disease?

    PubMed Central

    Olsthoorn, René C.L.

    2014-01-01

    Cellular ribonucleic acid (RNA) plays a crucial role in the initial conversion of cellular prion protein PrPC to infectious PrPSc or scrapie. The nature of this RNA remains elusive. Previously, RNA aptamers against PrPC have been isolated and found to form G-quadruplexes (G4s). PrPC binding to G4 RNAs destabilizes its structure and is thought to trigger its conversion to PrPSc. Here it is shown that PrP messenger RNA (mRNA) itself contains several G4 motifs, located in the octarepeat region. Investigation of the RNA structure in one of these repeats by circular dichroism, nuclear magnetic resonance and ultraviolet melting studies shows evidence of G4 formation. In vitro translation of full-length PrP mRNA, naturally harboring five consecutive G4 motifs, was specifically affected by G4-binding ligands, lending support to G4 formation in PrP mRNA. A possible role of PrP binding to its own mRNA and the role of anti-prion drugs, many of which are G4-binding ligands, in prion disease are discussed. PMID:25030900

  1. Ty1 gag enhances the stability and nuclear export of Ty1 mRNA.

    PubMed

    Checkley, Mary Ann; Mitchell, Jessica A; Eizenstat, Linda D; Lockett, Stephen J; Garfinkel, David J

    2013-01-01

    Retrotransposon and retroviral RNA delivery to particle assembly sites is essential for their replication. mRNA and Gag from the Ty1 retrotransposon colocalize in cytoplasmic foci, which are required for transposition and may be the sites for virus-like particle (VLP) assembly. To determine which Ty1 components are required to form mRNA/Gag foci, localization studies were performed in a Ty1-less strain expressing galactose-inducible Ty1 plasmids (pGTy1) containing mutations in GAG or POL. Ty1 mRNA/Gag foci remained unaltered in mutants defective in Ty1 protease (PR) or deleted for POL. However, Ty1 mRNA containing a frameshift mutation (Ty1fs) that prevents the synthesis of all proteins accumulated in the nucleus. Ty1fs RNA showed a decrease in stability that was mediated by the cytoplasmic exosome, nonsense-mediated decay (NMD) and the processing body. Localization of Ty1fs RNA remained unchanged in an nmd2? mutant. When Gag and Ty1fs mRNA were expressed independently, Gag provided in trans increased Ty1fs RNA level and restored localization of Ty1fs RNA in cytoplasmic foci. Endogenously expressed Gag also localized to the nuclear periphery independent of RNA export. These results suggest that Gag is required for Ty1 mRNA stability, efficient nuclear export and localization into cytoplasmic foci. PMID:22998189

  2. Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem

    E-print Network

    Kimble, Judith

    Genome-wide analysis of mRNA targets for Caenorhabditis elegans FBF, a conserved stem cell Contributed by Judith Kimble, January 14, 2010 (sent for review December 8, 2009) Stem cells are essential in adults. The great therapeutic promise of stem cells makes under- standing their regulation a high

  3. Post-synthetic processing mRNA often undergoes processing after synthesis

    E-print Network

    Park, Sheldon

    ligation · Solid phase synthesis vastly simplified the chemical synthesis of peptides · Chemical synthesis protein fragments in vitro to achieve a longer peptide chain · Peptide ligation and solid phase synthesisPost-synthetic processing mRNA often undergoes processing after synthesis ­ during RNA splicing

  4. Mammalian microRNAs predominantly act to decrease target mRNA levels

    E-print Network

    Bedwell, David M.

    RNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization bolstered the lingering notion that with some exceptions (for example, Drosophila miR-12 regulation of CGRNA-destabilization scenario differences between protein and mRNA changes are mostly attributed to either measurement noise

  5. Genome-Wide Analysis of Host mRNA Translation during Hepatitis C Virus Infection

    PubMed Central

    Colman, Hélène; Le Berre-Scoul, Catherine; Hernandez, Céline; Pierredon, Sandra; Bihouée, Audrey; Houlgatte, Rémi; Vagner, Stephan; Rosenberg, Arielle R.

    2013-01-01

    In the model of Huh-7.5.1 hepatocyte cells infected by the JFH1 hepatitis C virus (HCV) strain, transcriptomic and proteomic studies have revealed modulations of pathways governing mainly apoptosis and cell cycling. Differences between transcriptomic and proteomic studies pointed to regulations occurring at the posttranscriptional level, including the control of mRNA translation. In this study, we investigated at the genome-wide level the translational regulation occurring during HCV infection. Sucrose gradient ultracentrifugation followed by microarray analysis was used to identify translationally regulated mRNAs (mRNAs associated with ribosomes) from JFH1-infected and uninfected Huh-7.5.1 cells. Translationally regulated mRNAs were found to correspond to genes enriched in specific pathways, including vesicular transport and posttranscriptional regulation. Interestingly, the strongest translational regulation was found for mRNAs encoding proteins involved in pre-mRNA splicing, mRNA translation, and protein folding. Strikingly, these pathways were not previously identified, through transcriptomic studies, as being modulated following HCV infection. Importantly, the observed changes in host mRNA translation were directly due to HCV replication rather than to HCV entry, since they were not observed in JFH1-infected Huh-7.5.1 cells treated with a potent HCV NS3 protease inhibitor. Overall, this study highlights the need to consider, beyond transcriptomic or proteomic studies, the modulation of host mRNA translation as an important aspect of HCV infection. PMID:23552407

  6. Reserpine potentiates NMDA-induced c-fos mRNA expression in the mouse brain.

    PubMed

    Ferré, S; Tusell, J M; Barrón, S; Giménez-Llort, L; Martínez, E; Serratosa, J

    1996-07-19

    The systemic administration of a non-convulsant dose of N-methyl-D-aspartate (NMDA; 75 mg/kg i.p.), which was associated with motor activation, induced a regional c-fos mRNA expression in the mouse brain. The NMDA-induced c-fos mRNA expression was predominant in the dentate gyrus and in the medial mammillary nucleus and less pronounced in other hippocampal areas, cortical areas, bed nucleus of the stria terminalis and posterior amygdaloid nuclei. It is suggested that the hippocampus and/or the extended amygdala might be involved in the previously hypothesized dopamine-independent NMDA-mediated motor activation mechanism. No increase in c-fos mRNA expression was observed 21 h after reserpine treatment (5 mg/kg s.c.). However, reserpinization induced a significant potentiation of the NMDA-induced c-fos mRNA expression. These results show the existence of a strong and selective amine-dependent modulation of NMDA neurotransmission in the brain. PMID:8843094

  7. Translational bypassing without peptidyl-tRNA anticodon scanning of coding gap mRNA

    E-print Network

    Bedwell, David M.

    by the smaller eubacterial ribosomal subunit, 30S, is less well known, but in some cases, it may move from Subject Categories: RNA; proteins Keywords: mRNA folding; ORF coupling; ribosomal frame- shifting termination, the 30S subunit scans with the potential to find reinitiation codons (Klovins et al, 1997; Wills

  8. Feedforward regulation of mRNA stability by prolonged extracellular signal-regulated kinase activity

    PubMed Central

    Nagashima, Takeshi; Inoue, Norihiko; Yumoto, Noriko; Saeki, Yuko; Magi, Shigeyuki; Volinsky, Natalia; Sorkin, Alexander; Kholodenko, Boris N; Okada-Hatakeyama, Mariko

    2015-01-01

    Extracellular signal-regulated kinase (ERK) plays a central role in signal transduction networks and cell fate decisions. Sustained ERK activation induces cell differentiation, whereas transient ERK results in the proliferation of several types of cells. Sustained ERK activity stabilizes the proteins of early-response gene products. However, the effect of ERK activity duration on mRNA stability is unknown. We analyzed the quantitative relationship between the duration of four ERK activity kinetics and the mRNA expression profile in growth factor-treated cells. Time-course transcriptome analysis revealed that the cells with prolonged ERK activity generally showed sustained mRNA expression of late response genes but not early or mid genes. Selected late response genes decayed more rapidly in the presence of a specific ERK inhibitor than a general transcription inhibitor and the decay rate was not related to the number of AU-rich elements. Our results suggest that sustained ERK activity plays an important role in the lifespan of the mRNA encoded by late response genes, in addition to the previously demonstrated role in protein stabilization of early-response genes, including transcription factors regulating the transcription of mid and late genes. This double-positive regulation of ligand-induced genes, also termed feedforward regulation, is critical in cell fate decisions. PMID:25491268

  9. RNA UK 2008 497 Recognition of nonsense mRNA: towards a unified

    E-print Network

    Mühlemann, Oliver

    the different cellular surveillance mechanisms that ensure accurate gene expression, nonsense- mediated m that monitor different steps during gene expression and collectively ensure sufficiently high fidelity: alternative splicing, exon junction complex, gene expression, nonsense-mediated mRNA decay, RNA surveillance

  10. Androgen-dependent loss of muscle BDNF mRNA in two mouse models of SBMA.

    PubMed

    Halievski, Katherine; Henley, Casey L; Domino, Laurel; Poort, Jessica E; Fu, Martina; Katsuno, Masahisa; Adachi, Hiroaki; Sobue, Gen; Breedlove, S Marc; Jordan, Cynthia L

    2015-07-01

    Transgenic expression of neurotrophic factors in skeletal muscle has been found to protect mice from neuromuscular disease, including spinal bulbar muscular atrophy (SBMA), triggering renewed interest in neurotrophic factors as therapeutic agents for treating neuromuscular disease. Because SBMA is an androgen-dependent disease, and brain-derived neurotrophic factor (BDNF) mediates effects of androgens on neuromuscular systems, we asked whether BDNF expression is impaired in two different transgenic (Tg) mouse models of SBMA, the so called "97Q" and "myogenic" SBMA models. The 97Q model globally overexpresses a full length human AR with 97 glutamine repeats whereas the myogenic model of SBMA overexpresses a wild-type rat androgen receptor (AR) only in skeletal muscle fibers. Using quantitative PCR, we find that muscle BDNF mRNA declines in an androgen-dependent manner in both models, paralleling changes in motor function, with robust deficits (6-8 fold) in both fast and slow twitch muscles of impaired Tg males. Castration rescues or reverses disease-related deficits in muscle BDNF mRNA in both models, paralleling its effect on motor function. Moreover, when disease is acutely induced in Tg females, both motor function and muscle BDNF mRNA expression plummet, with the deficit in muscle BDNF emerging before overt motor dysfunction. That androgen-dependent motor dysfunction is tightly associated with a robust and early down-regulation of muscle BDNF mRNA suggests that BDNF delivered to skeletal muscle may have therapeutic value for SBMA. PMID:25929689

  11. Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts

    E-print Network

    Vale, Ronald D.

    Widespread cytoplasmic mRNA transport in yeast: Identification of 22 bud-localized transcripts identified two mRNAs that localize to the bud tips of the yeast Saccharomyces cerevisiae. To identify reporter assay, identified 22 mRNAs that are localized to bud tips. These messages encode a wide variety

  12. Long-range Transcriptome Sequencing Reveals Cancer Cell Growth Regulatory Chimeric mRNA12

    PubMed Central

    Plebani, Roberto; Oliver, Gavin R; Trerotola, Marco; Guerra, Emanuela; Cantanelli, Pamela; Apicella, Luana; Emerson, Andrew; Albiero, Alessandro; Harkin, Paul D; Kennedy, Richard D; Alberti, Saverio

    2012-01-01

    mRNA chimeras from chromosomal translocations often play a role as transforming oncogenes. However, cancer transcriptomes also contain mRNA chimeras that may play a role in tumor development, which arise as transcriptional or post-transcriptional events. To identify such chimeras, we developed a deterministic screening strategy for long-range sequence analysis. High-throughput, long-read sequencing was then performed on cDNA libraries from major tumor histotypes and corresponding normal tissues. These analyses led to the identification of 378 chimeras, with an unexpectedly high frequency of expression (?2 x 10-5 of all mRNA). Functional assays in breast and ovarian cancer cell lines showed that a large fraction of mRNA chimeras regulates cell replication. Strikingly, chimeras were shown to include both positive and negative regulators of cell growth, which functioned as such in a cell-type-specific manner. Replication-controlling chimeras were found to be expressed by most cancers from breast, ovary, colon, uterus, kidney, lung, and stomach, suggesting a widespread role in tumor development. PMID:23226102

  13. FOXO1 and c-jun transcription factors mRNA are modulated in endometriosis

    Microsoft Academic Search

    K. Shazand; S. Baban; C. Prive; B. Malette; P. Croteau; M. Lagace; J.-B. Racine; P. Hugo

    2004-01-01

    level. In this perspective, we targeted isolation of differentially expressed genes in the eutopic endometrial tissue. Our assump- tion was that the endometrial cells of patients presented an unusual gene expression profile, allowing their implantation and survival in an ectopic site, leading to endometriotic lesions. Here, we report that mRNA steady-state levels of two key tran- scription factors are modulated

  14. Characterization of the Crithidia fasciculata mRNA Cycling Sequence Binding Proteins

    Microsoft Academic Search

    RIAZ MAHMOOD; BIDYOTTAM MITTRA; JANE C. HINES; DAN S. RAY

    2001-01-01

    The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in

  15. Breaking the seals: efficient mRNA detection from human archival paraffin-embedded tissue.

    PubMed

    Illig, Romana; Fritsch, Helga; Schwarzer, Christoph

    2009-08-01

    During our study on HOXA13, HOXD12, and HOXD13 mRNA expression in human adult and embryonic tissues, we were confronted with the fact that, within our specimen collection, as in other University Departments in Europe, <20% of all samples yielded reliable labeling, while most samples were resistant to hybridization by standard protocols due to over-fixation. Fixation is essential for specimen stability, especially when samples are stored at room temperature and used for histology, and people tend to be more worried about under- than over-fixation. On the other hand fixation inhibits penetration by the probe and may also trap mRNA within ribosomes. Therefore, we developed a nonradioactive in situ hybridization technique, which allows detection of mRNA expressed on low levels from a variety of differentially fixed tissues while maintaining tissue integrity. This was achieved by improving target retrieval and probe detection. In contrast with others, our method allows reliable staining from tissues that are fixed in paraformaldehyde from four hours to over one week, and archived samples that were stored at room temperature for several years (17-19 yr in some cases) and exceeds detection limits of purely fluorescent methods. Our protocol is highly suitable for detecting CDX-2 mRNA in carcinoma specimens, but especially designed to investigate mRNAs in nonpathological adult and embryonic tissues. Due to the use of standardized probes, we do not expect problems in detecting other mRNAs expressed in suitable amounts. PMID:19549718

  16. Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers

    Microsoft Academic Search

    David J. Dix; Brenda R. Eisenberg

    1990-01-01

    Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch- hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher lev- els of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fbers revealed a large

  17. Vanadium-Induced Chemokine mRNA Expression and Pulmonary Inflammation

    Microsoft Academic Search

    Lisa M. Pierce; Francesca Alessandrini; John J. Godleski; Joseph D. Paulauskis

    1996-01-01

    Occupational exposure to vanadium is common in petrochemical, mining, steel, and utilities industries and results in toxic effects largely confined to the respiratory system. Vanadium exposure has been associated with inflammatory changes in the upper and lower respiratory tracts in addition to changes in pulmonary function. We investigated the abilities of several vanadium compounds to increase mRNA levels for selected

  18. Definition of global and transcript-specific mRNA export pathways in metazoans

    Microsoft Academic Search

    Natalie G. Farny; Jessica A. Hurt; Pamela A. Silver

    2008-01-01

    Eukaryotic gene expression requires export of messenger RNAs (mRNAs) from their site of transcription in the nucleus to the cytoplasm where they are translated. While mRNA export has been studied in yeast, the complexity of gene structure and cellular function in metazoan cells has likely led to increased diversification of these organisms' export pathways. Here we report the results of

  19. DEVELOPMENTAL BIOLOGY 146,12-23 (1991) Transcription in Leech: mRNA Synthesis Is Required

    E-print Network

    Weisblat, David A.

    1991-01-01

    DEVELOPMENTAL BIOLOGY 146,12-23 (1991) Transcription in Leech: mRNA Synthesis Is Required for Early was analyzed in embryos of the glossiphoniid leech Helobdella triserialis by autoradiographic detection investigations into the development of glossi- phoniid leeches, we have recently analyzed the onset of zygotic

  20. Burn-induced enhancement of mRNA of lysosomal cathepsins in skeletal muscle

    SciTech Connect

    Odessey, R.

    1987-05-01

    A localized burn injury to a rat hindlimb results in atrophy of soleus muscle (in the absence of cellular damage) which is attributable to an increase in muscle protein breakdown. Previous work has shown that lysosomal enzyme activities (cathepsins B, L, and D) are elevated in muscle from the burned leg by 50% to 100%. Activities are only slightly elevated by 1 day postburn but are maximal by 2 days. Incubation of muscles in the presence of /sup 3/H-mannose show that microsomal glycoprotein synthesis is specifically elevated by 1 day postburn. To determine the cause of the increased protein synthesis, total RNA was extracted from muscles of burned and control legs and electrophoresed in agarose. Northern blots of these samples were hybridized to nick translated cDNA clones which were obtained from several sources. Quantitative analysis of autoradiograms indicated that mRNA of cathepsin B was elevated 5 to 30 fold by one day postburn and remained high during the next two days. Changes in cathepsin D mRNA was less marked. The increased mRNA levels correlate in time with the increased protein synthesis in the microsomal glycoprotein fraction, but precede the increase in cathepsin activity. These results indicate that burns increase the synthesis of lysosomal proteases in skeletal muscle by increasing the level of cathepsin mRNA.

  1. Unification of gene expression data applying standard mRNA quantification references for comparable analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    High throughput quantitative measurements of gene expression data have problems of reproducibility and comparability due to a lack of standard mRNA quantification references. Efforts have been made to safeguard data fidelity, yet generating quality expression data of inherent value remains a challe...

  2. Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia

    PubMed Central

    Axelrod, Felicia B.; Liebes, Leonard; Gold-von Simson, Gabrielle; Mendoza, Sandra; Mull, James; Leyne, Maire; Norcliffe-Kaufmann, Lucy; Kaufmann, Horacio; Slaugenhaupt, Susan A.

    2011-01-01

    Familial dysautonomia (FD) is caused by an intronic splice mutation in the IKBKAP gene that leads to partial skipping of exon 20 and tissue-specific reduction in I-?-B kinase complex associated protein/ elongation protein 1 (IKAP/ELP-1) expression. Kinetin (6-furfurylaminopurine) has been shown to improve splicing and increase wild-type IKBKAP mRNA and IKAP protein expression in FD cell lines and carriers. To determine if oral kinetin treatment could alter mRNA splicing in FD subjects and was tolerable, we administered kinetin to eight FD individuals homozygous for the splice mutation. Subjects received 23.5 mg/Kg/day for 28 days. An increase in wild-type IKBKAP mRNA expression in leukocytes was noted after eight days in six of eight individuals; after 28 days the mean increase as compared to baseline was significant (p=0.002). We have demonstrated that kinetin is tolerable in this medically fragile population. Not only did kinetin produce the desired effect on splicing in FD patients, but also that effect appears to improve with time despite lack of dose change. This is the first report of a drug that produces in vivo mRNA splicing changes in individuals with FD and supports future long-term trials to determine if kinetin will prove therapeutic in FD patients. PMID:21775922

  3. Modularized learning of genetic interaction networks from biological annotations and mRNA expression data

    Microsoft Academic Search

    Phil Hyoun Lee; Doheon Lee

    2005-01-01

    Motivation: Inferring the genetic interaction mechanism using Bayesian networks has recently drawn increasing attention due to its well-established theoretical foundation and statistical robustness. However, the relative insufficiency of experiments with respect to the number of genes leads to many false positive inferences. Results: We propose a novel method to infer genetic networks by alleviating the shortage of available mRNA expression

  4. Nucleotide sequence of 3' untranslated portion of human alpha globin mRNA.

    PubMed Central

    Wilson, J T; deRiel, J K; Forget, B G; Marotta, C A; Weissman, S M

    1977-01-01

    We have determined the nucleotide sequence of 75 nucleotides of the 3'-untranslated portion of normal human alpha globin mRNA which corresponds to the elongated amino acid sequence of the chain termination mutant Hb Constant Spring. This was accomplished by sequence analysis of cDNA fragments obtained by restriction endonuclease or T4 endonuclease IV cleavage of human globin cDNA synthesized from globin mRNA by use of viral reverse transcriptase. Analysis of cRNA synthesized from cDNA by use of RNA polymerase provided additional confirmatory sequence information. Possible polymorphism has been identified at one site of the sequence. Our sequence overlaps with, and extends the sequence of 43 nucleotides determined by Proudfood and coworkers for the very 3'-terminal portion of human alpha globin mRNA. The complete 3'-untranslated sequence of human alpha globin mRNA (112 nucleotides including termination codon) shows little homology to that of the human or rabbit beta globin mRNAs except for the presence of the hexanucleotide sequence AAUAAA which is found in most eukaryotic mRNAs near the 3'-terminal poly (A). Images PMID:909779

  5. Analysis of mRNA Partitioning Between the Cytosol and Endoplasmic Reticulum Compartments

    E-print Network

    Nicchitta, Chris

    14 Analysis of mRNA Partitioning Between the Cytosol and Endoplasmic Reticulum CompartmentsRNAs that are non-canonically partitioned to the ER--although lacking an encoded signal sequence;198 Stephens et al. 1. Introduction In eukaryotic cells, protein synthesis is compartmentalized; mRNAs encoding

  6. MYELIN BASIC PROTEIN-MRNA USED TO MONITOR TRIMETHYLTIN TOXIC NEUROPATHY IN RATS

    EPA Science Inventory

    Trimethyltin (TMT) is an alkyltin that selectively targets neurons of the limbic system. ene probe (i.e., mRNA) for myelin basic protein (MBP) was used to monitor this toxic neuropathy. prague Dawley rats, were dosed (IP) acutely with hydroxide at neuropathic (8.0 mg/kg) or non-n...

  7. Bromouridine triphosphate inhibits transcription termination and mRNA release by vaccinia virions.

    PubMed

    Shuman, S; Moss, B

    1989-12-15

    Termination of transcription in vitro by purified vaccinia virus RNA polymerase occurs downstream of a cis-acting signal UUUUUNU in the nascent RNA strand and requires a trans-acting termination factor, VTF, that is associated with the viral mRNA capping enzyme. Factor-dependent termination can be inhibited specifically by incorporation of BrUMP (from BrUTP) into nascent RNA in place of UMP. The relevance of VTF action to early vaccinia mRNA biogenesis was demonstrated in the present study of the effects of BrUTP on mRNA synthesis and release by permeabilized vaccinia virions. BrUMP incorporation inhibited the release of newly made transcripts from the virus particle, resulting in the accumulation of transcripts within virus cores. This effect was observed also with IUMP, but not with BrCMP or IMP incorporation. Transcripts synthesized in the presence of BrUTP were heterogeneous in size and severalfold larger than transcripts made in the presence of UTP. The progressive increase in the size of the core-associated, BrUMP-containing transcripts indicated that they were still engaged by elongating RNA polymerase. These results are consistent with a predominant pathway of mRNA 3'-end formation by virions that involves VTF-dependent transcription termination. These data do not support an alternative model of 3'-end formation by endonucleolytic cleavage of larger RNA precursors. PMID:2592381

  8. IN VIVO AND IN VITRO CYP1B MRNA EXPRESSION IN CHANNEL CATFISH

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Our goal was to study the induction of CYP1B mRNA expression in channel catfish (Ictalurus punctatus). CYP1B belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to Ah...

  9. Differential expression of human Fas mRNA species upon peripheral blood mononuclear cell activation.

    PubMed Central

    Liu, C; Cheng, J; Mountz, J D

    1995-01-01

    Human Fas/Apo-1 is a cell-surface protein that mediates apoptosis upon ligation with Fas ligand. The gene lies on the long arm of chromosome 10, consists of nine exons, and spans more than 26 kb of DNA. We previously reported the presence of a Fas variant mRNA, designated as Fas delta TM, in human peripheral blood mononuclear cells. Fas delta TM is generated by alternative splicing of the intact exon 6, which encodes the Fas transmembrane domain. In the present study, we describe three novel forms of Fas mRNA that are generated by alternative splicing of exons 3, 4, 6 and 7. These three mRNA variants undergo a frameshift and produce truncated polypeptides because of the appearance of a stop codon in the altered open reading frame. On activation of the peripheral blood mononuclear cells, a decreased expression of alternatively spliced Fas mRNA species correlated with increased cell-surface expression of Fas. These results suggest that differential expression of alternatively spliced Fas mRNAs may play a role in regulation of Fas function via regulation of the production of the membrane-bound and the soluble, secreted Fas protein products. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7575433

  10. Genome-wide analysis of mRNA decay patterns during early Drosophila development

    E-print Network

    Thomsen, Stefan; Anders, Simon; Chandra Janga, Sarath; Huber, Wolfgang; Alonso, Claudio R

    2010-09-21

    of Drosophila embryos and unfertilized eggs with genome-wide microarray technology to determine the degradation patterns of all mRNAs present during early fruit fly development. Our work studies the kinetics of mRNA decay, the contributions of maternally...

  11. A small RNA activates CFA synthase by isoform-specific mRNA stabilization

    PubMed Central

    Fröhlich, Kathrin Sophie; Papenfort, Kai; Fekete, Agnes; Vogel, Jörg

    2013-01-01

    Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5? end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5? untranslated region. Intriguingly, this mechanism is generic such that the activation is equally achieved by seed pairing of unrelated small RNAs, suggesting that this mechanism may be utilized in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. PMID:24141880

  12. Npl3 is an antagonist of mRNA 30 end formation

    E-print Network

    . These results suggest that elongation rate and mRNA packaging can influence polyadenylation and termination polyadenylation/termina- tion sequences that are presumably recognized as RNA. In yeast, these sequences appear upon the Ctk1 kinase in yeast (Lee and Greenleaf, 1991; Cho et al, 2001; Skaar and Greenleaf, 2002; Ahn

  13. A Journal of Integrative Biology Morphology and Estrogen Receptor a mRNA Expression

    E-print Network

    Wade, Juli

    in the Developing Green Anole Forebrain LAUREL AMANDA BECK1Ã AND JULI WADE1­3 1 Neuroscience Program, Michigan State ontogeny of reproductive nuclei in the green anole lizard, including whether steroid hormones influence. Morphology and estrogen receptor a mRNA expression in the developing green anole forebrain. J. Exp. Zool. 311

  14. PHAS\\/4E-BPs as regulators of mRNA translation and cell proliferation

    Microsoft Academic Search

    John C. Lawrence; Robert T. Abraham

    1997-01-01

    Insulin and growth factors elicit rapid increases in protein synthesis by stimulating mRNA translation. PHAS\\/4E-BPs, a recently discovered family of elF4E-binding proteins, appear to play a key role in this process, as well as in the control of cell proliferation.

  15. mdm2 mRNA level is a prognostic factor in soft tissue sarcoma.

    PubMed Central

    Taubert, H.; Koehler, T.; Meye, A.; Bartel, F.; Lautenschläger, C.; Borchert, S.; Bache, M.; Schmidt, H.; Würl, P.

    2000-01-01

    BACKGROUND: The oncogenic properties of murine double minute-2 (mdm2) protein over-expression, which mostly results from the interaction with the tumor suppressor p53, are well described and their negative impacts on the prognosis of affected patients is well characterized. However, clinical relevance of mdm2 mRNA expression is poorly investigated. MATERIALS AND METHODS: In this study, 65 soft tissue sarcoma (STS) samples were analyzed for mdm2 mRNA expression by a quantitative reverse transcription polymerase chain reaction (RT-PCR) approach using available validated ready-to-use assays based on the TaqMan technology (PE Applied Biosystems, Weiterstadt, Germany). Mdm2 data were correlated to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression calculated from the same sample. RESULTS: For patients with a mdm2/GAPDH mRNA ratio below 50 zmol/amol the survival was strikingly reduced in comparison to patients with a ratio of > or =50 (p = 0.0241). Multivariate Cox analysis showed that the difference in prognosis for patients with tumor stage 2 and 3 became even more pronounced between patients with a ratio of <50 zmol/amol and patients with a ratio of > or =50 (p = 0.0041; RR = 5.6). To test if the group with an mdm2 mRNA expression > or =50 is homogenous concerning the prognosis, the group was divided into three subgroups with values of 50 to <100, 100 to <500 and > or =500. The subgroup with values of 100 to <500 showed the best prognosis (p = 0.0164); whereas, the one with values of 50 to <100 showed the worst prognosis in this group and, in between, was the one with values of > or =500. After omitting patients of stage 1 and 4, the subgroup with values of 100 to <500 showed an even more striking best prognosis (p = 0.0015); the other subgroups remained in the same sequence. The risk of tumor-related death over 5 years was most conspicuous in patients with mdm2 mRNA expression <50 than in those with ratios of 100 to <500 displaying a 13.3-fold higher risk. In a comparison between mdm2 mRNA levels and P53 protein expression or p53 mutational status, no relationship was found. CONCLUSIONS: In our study, the mdm2 mRNA level appears to be an independent prognostic factor for STS patients, marking its role in STS genesis and as a potential factor for gene therapeutical approaches. PMID:10803408

  16. mRNA Cap Methylation Influences Pathogenesis of Vesicular Stomatitis Virus In Vivo

    PubMed Central

    Ma, Yuanmei; Wei, Yongwei; Zhang, Xiaodong; Zhang, Yu; Cai, Hui; Zhu, Yang; Shilo, Konstantin; Oglesbee, Michael; Krakowka, Steven; Whelan, Sean P. J.

    2014-01-01

    ABSTRACT One role of mRNA cap guanine-N-7 (G-N-7) methylation is to facilitate the efficient translation of mRNA. The role of mRNA cap ribose 2?-O methylation is enigmatic, although recent work has implicated this as a signature to avoid detection of RNA by the innate immune system (S. Daffis, K. J. Szretter, J. Schriewer, J. Q. Li, S. Youn, J. Errett, T. Y. Lin, S. Schneller, R. Zust, H. P. Dong, V. Thiel, G. C. Sen, V. Fensterl, W. B. Klimstra, T. C. Pierson, R. M. Buller, M. Gale, P. Y. Shi, M. S. Diamond, Nature 468:452-456, 2010, doi:10.1038/nature09489). Working with vesicular stomatitis virus (VSV), we previously showed that a panel of recombinant VSVs carrying mutations at a predicted methyltransferase catalytic site (rVSV-K1651A, -D1762A, and -E1833Q) or S-adenosylmethionine (SAM) binding site (rVSV-G1670A, -G1672A, and -G4A) were defective in cap methylation and were also attenuated for growth in cell culture. Here, we analyzed the virulence of these recombinants in mice. We found that rVSV-K1651A, -D1762A, and -E1833Q, which are defective in both G-N-7 and 2?-O methylation, were highly attenuated in mice. All three viruses elicited a high level of neutralizing antibody and provided full protection against challenge with the virulent VSV. In contrast, mice inoculated with rVSV-G1670A and -G1672A, which are defective only in G-N-7 methylation, were attenuated in vivo yet retained a low level of virulence. rVSV-G4A, which is completely defective in both G-N-7 and 2?-O methylation, also exhibited low virulence in mice despite the fact that productive viral replication was not detected in lung and brain. Taken together, our results suggest that abrogation of viral mRNA cap methylation can serve as an approach to attenuate VSV, and perhaps other nonsegmented negative-strand RNA viruses, for potential application as vaccines and viral vectors. IMPORTANCE Nonsegmented negative-sense (NNS) RNA viruses include a wide range of significant human, animal, and plant pathogens. For many of these viruses, there are no vaccines or antiviral drugs available. mRNA cap methylation is essential for mRNA stability and efficient translation. Our current understanding of mRNA modifications of NNS RNA viruses comes largely from studies of vesicular stomatitis virus (VSV). In this study, we showed that recombinant VSVs (rVSVs) defective in mRNA cap methylation were attenuated in vitro and in vivo. In addition, these methyltransferase (MTase)-defective rVSVs triggered high levels of antibody responses and provided complete protection against VSV infection. Thus, this study will not only contribute to our understanding of the role of mRNA cap MTase in viral pathogenesis but also facilitate the development of new live attenuated vaccines for VSV, and perhaps other NNS RNA viruses, by inhibiting viral mRNA cap methylation. PMID:24371058

  17. Tissue-specific mRNA expression profiling in grape berry tissues

    PubMed Central

    Grimplet, Jerome; Deluc, Laurent G; Tillett, Richard L; Wheatley, Matthew D; Schlauch, Karen A; Cramer, Grant R; Cushman, John C

    2007-01-01

    Background Berries of grape (Vitis vinifera) contain three major tissue types (skin, pulp and seed) all of which contribute to the aroma, color, and flavor characters of wine. The pericarp, which is composed of the exocarp (skin) and mesocarp (pulp), not only functions to protect and feed the developing seed, but also to assist in the dispersal of the mature seed by avian and mammalian vectors. The skin provides volatile and nonvolatile aroma and color compounds, the pulp contributes organic acids and sugars, and the seeds provide condensed tannins, all of which are important to the formation of organoleptic characteristics of wine. In order to understand the transcriptional network responsible for controlling tissue-specific mRNA expression patterns, mRNA expression profiling was conducted on each tissue of mature berries of V. vinifera Cabernet Sauvignon using the Affymetrix GeneChip® Vitis oligonucleotide microarray ver. 1.0. In order to monitor the influence of water-deficit stress on tissue-specific expression patterns, mRNA expression profiles were also compared from mature berries harvested from vines subjected to well-watered or water-deficit conditions. Results Overall, berry tissues were found to express approximately 76% of genes represented on the Vitis microarray. Approximately 60% of these genes exhibited significant differential expression in one or more of the three major tissue types with more than 28% of genes showing pronounced (2-fold or greater) differences in mRNA expression. The largest difference in tissue-specific expression was observed between the seed and pulp/skin. Exocarp tissue, which is involved in pathogen defense and pigment production, showed higher mRNA abundance relative to other berry tissues for genes involved with flavonoid biosynthesis, pathogen resistance, and cell wall modification. Mesocarp tissue, which is considered a nutritive tissue, exhibited a higher mRNA abundance of genes involved in cell wall function and transport processes. Seeds, which supply essential resources for embryo development, showed higher mRNA abundance of genes encoding phenylpropanoid biosynthetic enzymes, seed storage proteins, and late embryogenesis abundant proteins. Water-deficit stress affected the mRNA abundance of 13% of the genes with differential expression patterns occurring mainly in the pulp and skin. In pulp and seed tissues transcript abundance in most functional categories declined in water-deficit stressed vines relative to well-watered vines with transcripts for storage proteins and novel (no-hit) functional assignments being over represented. In the skin of berries from water-deficit stressed vines, however, transcripts from several functional categories including general phenypropanoid and ethylene metabolism, pathogenesis-related responses, energy, and interaction with the environment were significantly over-represented. Conclusion These results revealed novel insights into the tissue-specific expression mRNA expression patterns of an extensive repertoire of genes expressed in berry tissues. This work also establishes an extensive catalogue of gene expression patterns for future investigations aimed at the dissection of the transcriptional regulatory hierarchies that govern tissue-specific expression patterns associated with tissue differentiation within berries. These results also confirmed that water-deficit stress has a profound effect on mRNA expression patterns particularly associated with the biosynthesis of aroma and color metabolites within skin and pulp tissues that ultimately impact wine quality. PMID:17584945

  18. Mononuclear cell metallothionein mRNA levels in human subjects with poor zinc nutrition.

    PubMed

    Kwon, Chong-Suk; Kountouri, Aggeliki M; Mayer, Claus; Gordon, Margaret-Jane; Kwun, In-Sook; Beattie, John H

    2007-02-01

    Human zinc deficiency is thought to be prevalent worldwide, particularly in populations with diets low in zinc and animal protein and high in inhibitors of zinc absorption, such as phytic acid. Confirmation of zinc deficiency is, however, difficult in the absence of a reliable and sensitive marker of zinc status. Under controlled conditions, T-lymphocyte metallothionein-2A (MT-2A) mRNA levels change in relation to zinc status and the objective of the present study was to investigate whether these transcript levels could be related to dietary zinc intake, plasma zinc or other biochemical parameters influenced by, or influencing, zinc metabolism in human subjects likely to be zinc deficient. Rural Koreans (n 110, age 50-80 years) with a range of zinc and phytic acid dietary intake were recruited for the study and blood samples were analysed for plasma zinc, HDL, LDL, alpha-tocopherol and thiobarbituric acid reactive substances, mononuclear cell (MNC) MT-2A mRNA, serum protein and albumin, and blood haematocrit, Hb and glucose. Multiple correlation and principal component analysis showed a significant negative correlation between plasma zinc and MNC MT-2A mRNA levels. Female subjects had higher MT-2A transcript levels than males and MT-2A mRNA levels tended to increase with age. There was no significant association between dietary zinc intake or any index of zinc intake relating to dietary inhibitors of zinc absorption. It is concluded that MNC MT-2A mRNA levels cannot be used to predict poor zinc nutrition. PMID:17298692

  19. Baseline MxA mRNA Expression Predicts Interferon Beta Response in Multiple Sclerosis Patients

    PubMed Central

    Matas, Elisabet; Bau, Laura; Martínez-Iniesta, María; Romero-Pinel, Lucía; Mañé, M. Alba; Cobo-Calvo, Álvaro; Martínez-Yélamos, Sergio

    2014-01-01

    Background Myxovirus resistance protein A (MxA) is a molecule induced after interferon-beta injection, mostly used to evaluate its bioactivity. There is little available data on clinical utility of baseline MxA mRNA status. The objective of the study is to investigate whether baseline MxA mRNA expression can predict relapse and disease progression in multiple sclerosis patients treated with interferon-beta. Methods Baseline blood samples were obtained before the first interferon-beta dose was administered to evaluate MxA mRNA expression using real-time polymerase chain reaction (PCR). Demographic and clinical variables were prospectively recorded to define treatment responder and non responder groups. Results 104 patients were included in the study. Baseline MxA mRNA expression was significantly lower in the group of patients who met the definition of responders (1.07 vs 1.95, Student t test, p<0.0001). A threshold of 1.096 was established using Receiver Operating Characteristic analysis to differentiate between responders and non-responders (sensitivity 73.9%, specificity 69.0%). Survival analysis using this threshold showed that time to next relapse (p<0.0001) and to EDSS progression (p?=?0.01) were significantly higher in patients with lower MxA titers. Conclusion The results suggest that baseline MxA mRNA levels may be useful for predicting whether multiple sclerosis patients will respond or not to interferon-beta treatment. PMID:25396411

  20. Developmental changes of neurotrophin mRNA expression in the layers of rat visual cortex.

    PubMed

    Patz, Silke; Wahle, Petra

    2006-11-01

    Neurotrophins are essential factors for the structural, neurochemical and functional maturation of the brain including developmental and adult plasticity. Northern blots and polymerase chain reaction revealed the expression of neurotrophin 4 (NT4), neurotrophin 3 (NT3), brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cortex. The cellular producers of NT3 and BDNF have been characterized by anatomical methods as being mostly pyramidal, and the tyrosine kinase B (TrkB) receptor is expressed by many cortical neurons. However, these methods have so far failed to identify the cells producing NT4 and NGF mRNA. These factors are much lower in expression than, e.g. BDNF, and apparently remain below detection levels of in situ hybridization. Given their specific actions on cell types and afferent systems, knowledge about the producing cell types is highly desirable. To narrow down on the producing cell types, we quantified by reverse transcriptase-polymerase chain reaction (RT-PCR) the developmental changes of BDNF, NT3, NT4, NGF and TrkB mRNA expression in total visual cortex lysates, and in the cortical layers dissected by tangential cryostat sectioning. We found dramatic changes in laminar expression of NT3 and NGF, mild changes of NT4, and no changes of BDNF and TrkB mRNA. For instance, NT3 is important early on for thalamocortical axons, and we found transient peaks of NT3 mRNA expression first in layer VI, then in layer IV. NT4 mRNA was in layers IV and VI, suggesting NT4 protein production in thalamorecipient layers, but peak expression gradually shifted to upper layers as did NGF expression. The layer-specific developmental expression shifts of neurotrophin mRNAs correlate with morphogenetic processes. PMID:17100834

  1. ABCA1 mRNA expression and cholesterol outflow in U937 cells

    PubMed Central

    Su, Xian-Ming; Wei, Yang; Wang, Ying; Zhang, Wei

    2015-01-01

    Objective: To investigate the oxidized low density lipoprotein (oxLDL) on U937 cell ATP-binding cassette transporter A1 (ABCA1) mRNA expression and cholesterol efflux situation. Methods: Human U937 cells were incubated with gradient concentrations of oxLDL (0, 25, 50, 75, 100, 125 mg/L), and then dyed by oil red O to estimate the content of intracelluar lipid and detect the expressing quantity of ABCA1 mRNA by Real-time Fluorescence quantitative PCR simultaneously. Calculating the cholesterol efflux rates by using the scintillation counter to detect the amount of H3-cholesterol in each well cell culture plate and medium. Results: Real-time Fluorescence quantitative PCR analysis showed that the expression levels of ABCA1 mRNA in monocytes were lower than basal line when not intervened with oxLDL, and increased drastically with oxLDL stimulation, significant difference compared with controls (P < 0.01), and reached the highest level at oxLDL 50 mg/L, nevertheless, continuously increasing the concentration of oxLDL above 50 mg/L, the expression decreased. So is the outflowing rate of intracelluar lipid. Oil red O dyeing results also suggested that celluar lipid content was the highest when intervened with 125 mg/L oxLDL, and increased most obviously at 50 mg/L oxLDL. Cholesterol outflow result also demonstrated that cholesterol outflow rate related with the ABCA1 mRNA expressing quantity. Conclusion: With the increase of intervening concentration of oxLDL on U937cells, the exprssion of ABCAl mRNA represented that rising before 50 mg/L oxLDL, and then decreasing, reaching the top point at 50 mg/L oxLDL. So was the change in the outflowing rate of intracelluar lipid.

  2. Ribosomal position and contacts of mRNA in eukaryotic translation initiation complexes.

    PubMed

    Pisarev, Andrey V; Kolupaeva, Victoria G; Yusupov, Marat M; Hellen, Christopher U T; Pestova, Tatyana V

    2008-06-01

    The position of mRNA on 40S ribosomal subunits in eukaryotic initiation complexes was determined by UV crosslinking using mRNAs containing uniquely positioned 4-thiouridines. Crosslinking of mRNA positions (+)11 to ribosomal protein (rp) rpS2(S5p) and rpS3(S3p), and (+)9-(+)11 and (+)8-(+)9 to h18 and h34 of 18S rRNA, respectively, indicated that mRNA enters the mRNA-binding channel through the same layers of rRNA and proteins as in prokaryotes. Upstream of the P-site, the proximity of positions (-)3/(-)4 to rpS5(S7p) and h23b, (-)6/(-)7 to rpS14(S11p), and (-)8-(-)11 to the 3'-terminus of 18S rRNA (mRNA/rRNA elements forming the bacterial Shine-Dalgarno duplex) also resembles elements of the bacterial mRNA path. In addition to these striking parallels, differences between mRNA paths included the proximity in eukaryotic initiation complexes of positions (+)7/(+)8 to the central region of h28, (+)4/(+)5 to rpS15(S19p), and (-)6 and (-)7/(-)10 to eukaryote-specific rpS26 and rpS28, respectively. Moreover, we previously determined that eukaryotic initiation factor2alpha (eIF2alpha) contacts position (-)3, and now report that eIF3 interacts with positions (-)8-(-)17, forming an extension of the mRNA-binding channel that likely contributes to unique aspects of eukaryotic initiation. PMID:18464793

  3. Constitutive and allergen-induced expression of eotaxin mRNA in the guinea pig lung

    PubMed Central

    1995-01-01

    Eotaxin is a member of the C-C family of chemokines and is related during antigen challenge in a guinea pig model of allergic airway inflammation (asthma). Consistent with its putative role in eosinophilic inflammation, eotaxin induces the selective infiltration of eosinophils when injected into the lung and skin. Using a guinea pig lung cDNA library, we have cloned full-length eotaxin cDNA. The cDNA encodes a protein of 96 amino acids, including a putative 23-amino acid hydrophobic leader sequence, followed by 73 amino acids composing the mature active eotaxin protein. The protein-coding region of this cDNA is 73, 71, 50, and 48% identical in nucleic acid sequence to those of human macrophage chemoattractant protein (MCP) 3, MCP-1, macrophage inflammatory protein (MIP) 1 alpha, and RANTES, respectively. Analysis of genomic DNA suggested that there is a single eotaxin gene in guinea pig which is apparently conserved in mice. High constitutive levels of eotaxin mRNA expression were observed in the lung, while the intestines, stomach, spleen, liver, heart, thymus, testes, and kidney expressed lower levels. To determine if eotaxin mRNA levels are elevated during allergen-induced eosinophilic airway inflammation, ovalbumin (OVA)-sensitized guinea pigs were challenged with aerosolized antigen. Compared with the lungs from saline-challenged animals, eotaxin mRNA levels increased sixfold within 3 h and returned to baseline by 6 h. Thus, eotaxin mRNA levels are increased in response to allergen challenge during the late phase response. The identification of constitutive eotaxin mRNA expression in multiple tissues suggests that in addition to regulating airway eosinophilia, eotaxin is likely to be involved in eosinophil recruitment into other tissues as well as in baseline tissue homing. PMID:7869037

  4. Increased CB2 mRNA and anandamide in human blood after cessation of cannabis abuse.

    PubMed

    Muhl, Daniela; Kathmann, Markus; Hoyer, Carolin; Kranaster, Laura; Hellmich, Martin; Gerth, Christoph W; Faulhaber, Johannes; Schlicker, Eberhard; Leweke, F Markus

    2014-07-01

    In previous studies, long-term cannabis use led to alterations of the endocannabinoid system including an increase in CB1 and/or CB2 receptor messenger RNA (mRNA) in blood cells and an increase in the serum level of the endocannabinoid 2-arachidonoyl glycerol. However, in those studies, cannabis use was stopped only few days before testing or not interrupted at all. Therefore, one cannot decide whether the alterations are due to long-term cannabis abuse or are confounded by acute effects of cannabis. Blood was sampled from donors that had smoked marijuana ?20 times in their lives but had abstained from cannabis for ?6 months (high-frequency users, HFU) and from controls (cannabis use ?5 times lifetime). CB1 and CB2 mRNA was determined in peripheral mononuclear blood cells using the reverse transcriptase polymerase chain reaction. Serum anandamide level was assayed using electrospray tandem mass spectrometry. CB2 mRNA was increased by 45 % in HFU when compared to controls, whereas CB1 mRNA did not differ. The anandamide level in HFU exceeded that in controls by 90 %. Tobacco smoking could be excluded as a confounding factor. In conclusion, marijuana users that had smoked marijuana ?20 times in their lives and stopped cannabis use at least 6 months before the study show an increase in CB2 receptor mRNA in the blood and in serum anandamide level. These alterations resemble those obtained for marijuana smokers that had stopped cannabis use only few days before testing and may be implicated in the pathogenesis of disorders associated with long-term cannabis use. PMID:24788457

  5. Coding elements in exons 2 and 3 target c-myc mRNA downregulation during myogenic differentiation.

    PubMed Central

    Yeilding, N M; Lee, W M

    1997-01-01

    Downregulation in expression of the c-myc proto-oncogene is an early molecular event in differentiation of murine C2C12 myoblasts into multinucleated myotubes. During differentiation, levels of c-myc mRNA decrease 3- to 10-fold despite a lack of change in its transcription rate. To identify cis-acting elements that target c-myc mRNA for downregulation during myogenesis, we stably transfected C2C12 cells with mutant myc genes or chimeric genes in which various myc sequences were fused to the human beta-globin gene or to the bacterial chloramphenicol acetyltransferase (CAT) gene. Deletion of coding sequences from myc exon 2 or exon 3 abolished downregulation of myc mRNA during myogenic differentiation, while deletion of introns or sequences in the 5' or 3' untranslated regions (UTRs) did not, demonstrating that coding elements in both exons 2 and 3 are necessary for myc mRNA downregulation. Fusion of coding sequences from either myc exon 2 or 3 to beta-globin mRNA conferred downregulation onto the chimeric mRNA, while fusion of myc 3' UTR sequences or coding sequences from CAT or ribosomal protein L32 did not, demonstrating that coding elements in myc exons 2 and 3 specifically confer downregulation. These results present the apparent paradox that coding elements in either myc exon 2 or myc exon 3 are sufficient to confer downregulation onto beta-globin mRNA, but neither element alone was sufficient for myc mRNA downregulation, suggesting that some feature of beta-globin mRNA may potentiate the regulatory properties of myc exons 2 and 3. A similar regulatory function is not shared by all mRNAs because fusion of either myc exon 2 or myc exon 3 to CAT mRNA did not confer downregulation onto the chimeric mRNA, but fusion of the two elements together did. We conclude from these results that two myc regulatory elements, one exon 2 and one in exon 3, are required for myc mRNA downregulation. Finally, using a highly sensitive and specific PCR-based assay for comparing mRNA levels, we demonstrated that the downregulation mediated by myc exons 2 and 3 results in a decrease in cytoplasmic mRNA levels, but not nuclear mRNA levels, indicating that regulation is a postnuclear event. PMID:9111340

  6. miR-19b regulates hTERT mRNA expression through targeting PITX1 mRNA in melanoma cells

    PubMed Central

    Ohira, Takahito; Naohiro, Sunamura; Nakayama, Yuji; Osaki, Mitsuhiko; Okada, Futoshi; Oshimura, Mitsuo; Kugoh, Hiroyuki

    2015-01-01

    Human telomerase reverse transcriptase (hTERT) plays a crucial role in cancer development. We previously identified paired-like homeodomain1 (PITX1) as an hTERT suppressor gene. However, the underlying mechanisms that are involved in the regulation of PITX1 remain unknown. Here, we report that the microRNA-19b (miR-19b) regulates hTERT expression and cell proliferation through inhibition of PITX1. Compared with normal melanocyte cells, miR-19b expression was higher in most melanoma cells and was accompanied by downregulation of PITX1. Moreover, overexpression of miR-19b inhibited PITX1 mRNA translation through a miR-19b binding site within the 3?UTR of the PITX1 mRNA. Our combined findings indicate the participation of miR-19b as a novel upstream effector of hTERT transcription via direct targeting of PITX1. PMID:25643913

  7. Icaritin opposes the development of social aversion after defeat stress via increases of GR mRNA and BDNF mRNA in mice.

    PubMed

    Wu, Xiao; Wu, Jinfeng; Xia, Shijin; Li, Bei; Dong, Jingcheng

    2013-11-01

    Icariin is a major constituent of flavonoids isolated from Herba Epimedii. Several previous studies have demonstrated the antidepressant-like effects of icariin. After oral administration of icariin, 19 metabolites of icariin were detected in rat plasma. Icaritin is one such of metabolite of icariin. In this study, a chronic social defeat protocol is used as a mouse model for depression, and the effects of icaritin administration on social avoidance are investigated. The data indicates that icaritin (5mg/kg and 10mg/kg) oral administration opposes the development of social aversion after defeat stress. In vitro corticosterone sensitivity assay demonstrated that icaritin partially restored social defeat-induced impairment of glucocorticoid sensitivity. The expressions of GR mRNA and BDNFmRNA in the hippocampus were increased after icaritin treatment. Meanwhile, the social defeat-induced increases in CRH mRNA in hypothalamus were restored by icaritin administration. Our data also suggests that icaritin administration remarkably attenuated the increases in serum IL-6 and TNF-? level that occur following exposure to social defeat. In conclusion, icaritin is a novel antidepressant. It partially restored social defeat-induced impairment of glucocorticoid sensitivity, HPA axis hyperactivity. These effects are at least partially attributed to normalization of the GR function and increases in BDNF expression. PMID:24064280

  8. Exploring mRNA 3?-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening

    PubMed Central

    Beaudoin, Jean-Denis; Perreault, Jean-Pierre

    2013-01-01

    Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3?-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3?-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo. The 3?-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3?-UTRs of mRNAs are cis-regulatory elements that have a significant impact on gene expression. PMID:23609544

  9. Elimination of cap structures generated by mRNA decay involves the new scavenger mRNA decapping enzyme Aph1/FHIT together with DcpS

    PubMed Central

    Taverniti, Valerio; Séraphin, Bertrand

    2015-01-01

    Eukaryotic 5? mRNA cap structures participate to the post-transcriptional control of gene expression before being released by the two main mRNA decay pathways. In the 3?-5? pathway, the exosome generates free cap dinucleotides (m7GpppN) or capped oligoribonucleotides that are hydrolyzed by the Scavenger Decapping Enzyme (DcpS) forming m7GMP. In the 5?-3? pathway, the decapping enzyme Dcp2 generates m7GDP. We investigated the fate of m7GDP and m7GpppN produced by RNA decay in extracts and cells. This defined a pathway involving DcpS, NTPs and the nucleoside diphosphate kinase for m7GDP elimination. Interestingly, we identified and characterized in vitro and in vivo a new scavenger decapping enzyme involved in m7GpppN degradation. We show that activities mediating cap elimination identified in yeast are essentially conserved in human. Their alteration may contribute to pathologies, possibly through the interference of cap (di)nucleotide with cellular function. PMID:25432955

  10. The Cellular Localization of Increased Atrial Natriuretic Peptide mRNA and Immunoreactivity in Diabetic Rat Kidneys

    Microsoft Academic Search

    Feng J. Lai; Ming C. Hsieh; Shih C. Hsin; Shiu R. Lin; Jinn Y. Guh; Hung C. Chen; Shyi J. Shin

    2002-01-01

    SUMMARY Increased intrarenal atrial natriuretic peptide (ANP) mRNA expression has been reported in several disorders. To further investigate the action of renal ANP, we need to elucidate the exact site of its alteration in diseased kidneys. ANP mRNA and ANP were detected by in situ hybridization and immunohistochemistry in the kidneys from five nor- mal and five diabetic rats. Renal

  11. A bicistronic CYCLIN D1-TROP2 mRNA chimera demonstrates a novel oncogenic mechanism in human cancer.

    PubMed

    Guerra, Emanuela; Trerotola, Marco; Dell' Arciprete, Roberta; Bonasera, Veronica; Palombo, Barbara; El-Sewedy, Tarek; Ciccimarra, Tommaso; Crescenzi, Carlo; Lorenzini, Franco; Rossi, Cosmo; Vacca, Giovanna; Lattanzio, Rossano; Piantelli, Mauro; Alberti, Saverio

    2008-10-01

    A chimeric CYCLIN D1-TROP2 mRNA was isolated from human ovarian and mammary cancer cells. The CYCLIN D1-TROP2 mRNA was shown to be a potent oncogene as it transforms naïve, primary cells in vitro and induces aggressive tumor growth in vivo in cooperation with activated RAS. Silencing of the chimeric mRNA inhibits the growth of breast cancer cells. The CYCLIN D1-TROP2 mRNA was expressed by a large fraction of the human gastrointestinal, ovarian, and endometrial tumors analyzed. It is most frequently detected in intestinal cell aneuploid cancers and it is coexpressed with activated RAS oncogenes, consistent with a cooperative transforming activity in human cancers. The chimeric mRNA is a bicistronic transcript of post transcriptional origin that independently translates the Cyclin D1 and Trop-2 proteins. This is a novel mechanism of CYCLIN D1 activation that achieves the truncation of the CYCLIN D1 mRNA in the absence of chromosomal rearrangements. This leads to a higher CYCLIN D1 mRNA stability, with inappropriate expression during the cell cycle. The stabilized CYCLIN D1 mRNA cooperates with TROP2 in stimulating the growth of the expressing cells. These findings show a novel epigenetic, oncogenic mechanism, which seems to be widespread in human cancers. PMID:18829570

  12. TGF-2 reduces nitric oxide synthase mRNA through a ROCK-dependent pathway in airway epithelial cells

    E-print Network

    George, Steven C.

    TGF- 2 reduces nitric oxide synthase mRNA through a ROCK-dependent pathway in airway epithelial of Chemical Engineering and Material Science, University of California, Irvine, Irvine, California Submitted 3 mRNA through a ROCK-dependent pathway in airway epithelial cells. Am J Physiol Lung Cell Mol Physiol

  13. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system

    PubMed Central

    Koussounadis, Antonis; Langdon, Simon P.; Um, In Hwa; Harrison, David J.; Smith, V. Anne

    2015-01-01

    Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general. PMID:26053859

  14. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system.

    PubMed

    Koussounadis, Antonis; Langdon, Simon P; Um, In Hwa; Harrison, David J; Smith, V Anne

    2015-01-01

    Differential mRNA expression studies implicitly assume that changes in mRNA expression have biological meaning, most likely mediated by corresponding changes in protein levels. Yet studies into mRNA-protein correspondence have shown notoriously poor correlation between mRNA and protein expression levels, creating concern for inferences from only mRNA expression data. However, none of these studies have examined in particular differentially expressed mRNA. Here, we examined this question in an ovarian cancer xenograft model. We measured protein and mRNA expression for twenty-nine genes in four drug-treatment conditions and in untreated controls. We identified mRNAs differentially expressed between drug-treated xenografts and controls, then analysed mRNA-protein expression correlation across a five-point time-course within each of the four experimental conditions. We evaluated correlations between mRNAs and their protein products for mRNAs differentially expressed within an experimental condition compared to those that are not. We found that differentially expressed mRNAs correlate significantly better with their protein product than non-differentially expressed mRNAs. This result increases confidence for the use of differential mRNA expression for biological discovery in this system, as well as providing optimism for the usefulness of inferences from mRNA expression in general. PMID:26053859

  15. A molecular switch from a single mRNA controls skin homeostasis in wound healing and cancer

    E-print Network

    Shyamasundar, R.K.

    A molecular switch from a single mRNA controls skin homeostasis in wound healing and cancer together, the stringent control of miR for skin homeostasis and deregulati to multiple pathologies in skin mRNA controls skin homeostasis in wound healing and cancer Gopinath M Sundaram Institute of Medical

  16. Ubiquitin-Associated domain of Mex67 synchronizes recruitment of the mRNA export machinery with transcription

    E-print Network

    Paris-Sud XI, Université de

    mature and correctly packaged yeast mRNPs are released from the transcription site and transportedRNA binding proteins implicated in the processing and packaging of mRNA into stable and export competent m by promoting correct loading of mRNA binding proteins. In THO mutants, the production of improperly packaged m

  17. Effect of tri-iodothyronine on leptin release and leptin mRNA accumulation in rat adipose tissue.

    PubMed Central

    Fain, J N; Bahouth, S W

    1998-01-01

    Leptin, the product of the obese gene, is produced by white adipocytes. The release of leptin, as well as leptin mRNA content, was enhanced in adipocytes isolated from hypothyroid rats. The administration of tri-iodothyronine (T3) 8 h before death inhibited leptin release by adipocytes incubated for 6 or 24 h. Direct addition of T3 to pieces of adipose tissue enhanced the loss of leptin mRNA seen over 24 h in the presence of dexamethasone plus the beta3-adrenergic agonist Cl 316,243. In contrast, if pieces of adipose tissue were incubated with dexamethasone plus insulin, enhanced the T3 accumulation of leptin mRNA. These results indicate that T3 enhances net adipocyte leptin mRNA accumulation in a condition that approximates the fed state (presence of insulin) but inhibits leptin mRNA accumulation in a condition that approximates the fasted state (absence of insulin). PMID:9601064

  18. Effect of tri-iodothyronine on leptin release and leptin mRNA accumulation in rat adipose tissue.

    PubMed

    Fain, J N; Bahouth, S W

    1998-06-01

    Leptin, the product of the obese gene, is produced by white adipocytes. The release of leptin, as well as leptin mRNA content, was enhanced in adipocytes isolated from hypothyroid rats. The administration of tri-iodothyronine (T3) 8 h before death inhibited leptin release by adipocytes incubated for 6 or 24 h. Direct addition of T3 to pieces of adipose tissue enhanced the loss of leptin mRNA seen over 24 h in the presence of dexamethasone plus the beta3-adrenergic agonist Cl 316,243. In contrast, if pieces of adipose tissue were incubated with dexamethasone plus insulin, enhanced the T3 accumulation of leptin mRNA. These results indicate that T3 enhances net adipocyte leptin mRNA accumulation in a condition that approximates the fed state (presence of insulin) but inhibits leptin mRNA accumulation in a condition that approximates the fasted state (absence of insulin). PMID:9601064

  19. Dis3- and exosome subunit-responsive 3 Prime mRNA instability elements

    SciTech Connect

    Kiss, Daniel L.; Hou, Dezhi [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States)] [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States); Gross, Robert H. [Dartmouth College, Department of Biological Sciences, Life Sciences Center 343, Hanover, NH 03755 (United States)] [Dartmouth College, Department of Biological Sciences, Life Sciences Center 343, Hanover, NH 03755 (United States); Andrulis, Erik D., E-mail: exa32@case.edu [Case Western Reserve University School of Medicine, Department of Molecular Biology and Microbiology, Cleveland, OH 44106 (United States)

    2012-07-06

    Highlights: Black-Right-Pointing-Pointer Successful use of a novel RNA-specific bioinformatic tool, RNA SCOPE. Black-Right-Pointing-Pointer Identified novel 3 Prime UTR cis-acting element that destabilizes a reporter mRNA. Black-Right-Pointing-Pointer Show exosome subunits are required for cis-acting element-mediated mRNA instability. Black-Right-Pointing-Pointer Define precise sequence requirements of novel cis-acting element. Black-Right-Pointing-Pointer Show that microarray-defined exosome subunit-regulated mRNAs have novel element. -- Abstract: Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3 Prime -5 Prime exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3 Prime untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA turnover that involves direct Dis3 and other exosome subunit recruitment to and/or regulation on mRNA substrates.

  20. Protamine mRNA ratio in stallion spermatozoa correlates with mare fecundity.

    PubMed

    Paradowska-Dogan, A; Fernandez, A; Bergmann, M; Kretzer, K; Mallidis, C; Vieweg, M; Waliszewski, P; Zitzmann, M; Weidner, W; Steger, K; Kliesch, S

    2014-07-01

    Highly compacted sperm DNA in protamine toroids and a minor fraction of nucleohistones are prerequisites for the efficient transmission of the paternal genome into the oocyte at fertilization. The objective of this study was to evaluate whether protamines might serve as a prognostic factor for stallion fertility. In situ hybridization detected specific expression of P1 mRNA in the cytoplasm of stage I to VII spermatids, whereas comparable immunohistochemical stainings showed that protein expression was delayed till elongating spermatids in differentiation stages III to VIII. No staining was detectable in cryptorchid testis because of the lack of spermatids in the seminiferous tubules. Using quantitative real-time polymerase chain reaction, we identified mRNA transcripts of P1 and 2 variants of protamine- 2 (P2, P3) in ejaculated spermatozoa from 45 thoroughbred stallions. According to the mare fertility descriptor (i.e. the 'none-return-rate 28 percentage' or NRR28%), stallions were divided into three groups (i.e. high, reduced and low fertility). The P2/P1 mRNA ratio was found to be significantly reduced in the group with lower fertility (p = 0.016) and was slightly correlated with sperm concentration (correlation coefficient r = 0.263). Furthermore, morphologically abnormal sperm count negatively correlated with P2/P1 mRNA ratio, indicating that spermatozoa carrying head defects display a diminished protamine ratio (r = -0.348). Conversely, the P2/P1 ratio was positively correlated with mare fertility or NRR28% (r = 0.274). Interestingly, P3/P1 mRNA ratio remained unaltered in the investigated groups indicating that this variant plays a minor role in equine sperm chromatin compaction. Aberrant protamine transcripts content in equine spermatozoa was not associated with DNA defragmentation rate as measured by flow cytometric acridine orange test. On the basis of these results, we suggest that, similar to human, equine protamine expression constitutes a checkpoint of spermatogenesis and as a corollary the level of protamine mRNA may reflect the quality of spermatogenesis and spermatozoa's fertilizing capacity. PMID:24711287

  1. ?-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis

    PubMed Central

    Fahlgren, A; Hammarström, S; Danielsson, Å; Hammarströ;m, M-L

    2004-01-01

    mRNA expression of two recently described human ?-defensins (hBD-3 and hBD-4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohn's disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD-3 and hBD-4 mRNAs were determined in freshly isolated epithelial cells by real-time quantitative reverse transcription–polymerase chain reaction (QRT-PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD-3 and hBD-4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD-3 and hBD-4 mRNA, with higher expression levels of hBD-3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt- compared to the villus/luminal-compartment. Interferon (IFN)-?, but not tumour necrosis factor (TNF)-? or IL-1?, augmented hBD-3 mRNA expression. None of these agents stimulated hBD-4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD-3 and hBD-4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohn's colitis did not show increased expression of hBD-4 mRNA, while the data are inconclusive for hBD-3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD-3 and hBD-4 mRNA expression in the epithelium, whereas in CD this is less evident. PMID:15270856

  2. Interferon-? regulates cellular metabolism and mRNA translation to potentiate macrophage activation.

    PubMed

    Su, Xiaodi; Yu, Yingpu; Zhong, Yi; Giannopoulou, Eugenia G; Hu, Xiaoyu; Liu, Hui; Cross, Justin R; Rätsch, Gunnar; Rice, Charles M; Ivashkiv, Lionel B

    2015-08-01

    Interferon-? (IFN-?) primes macrophages for enhanced microbial killing and inflammatory activation by Toll-like receptors (TLRs), but little is known about the regulation of cell metabolism or mRNA translation during this priming. We found that IFN-? regulated the metabolism and mRNA translation of human macrophages by targeting the kinases mTORC1 and MNK, both of which converge on the selective regulator of translation initiation eIF4E. Physiological downregulation of mTORC1 by IFN-? was associated with autophagy and translational suppression of repressors of inflammation such as HES1. Genome-wide ribosome profiling in TLR2-stimulated macrophages showed that IFN-? selectively modulated the macrophage translatome to promote inflammation, further reprogram metabolic pathways and modulate protein synthesis. These results show that IFN-?-mediated metabolic reprogramming and translational regulation are key components of classical inflammatory macrophage activation. PMID:26147685

  3. Increased leptin mRNA expression in the blood of dogs naturally infected by Leishmania infantum.

    PubMed

    Di Loria, Antonio; Squillacioti, Caterina; De Luca, Adriana; Veneziano, Vincenzo; Mirabella, Nicola; Guccione, Jacopo; Santoro, Domenico

    2014-12-01

    Canine leishmaniosis (CL) is a severe and potentially fatal zoonosis caused by the protozoan Leishmania infantum. Severe forms of CL are commonly associated with a non-protective, humoral immune-response and high parasitic loads. Leptin, a 16?kD hormone mainly secreted by adipocytes, regulates both the innate and adaptive immunity. The goal of this study was to evaluate leptin mRNA expression levels in blood samples from privately owned dogs with CL (n?=?11) and healthy controls (n?=?10) using quantitative, real-time polymerase chain reaction. Blood samples from dogs with CL expressed significantly higher leptin mRNA levels (two-fold) compared to healthy controls (P?=?0.018). The results suggest a possible involvement of leptin in the pathophysiology of Leishmania infection in dogs and the possible use of leptin as a biomarker for CL. Future studies investigating the immunological role of leptin in dogs with CL are warranted. PMID:25458880

  4. Tentative Mapping of Transcription-Induced Interchromosomal Interaction using Chimeric EST and mRNA Data

    PubMed Central

    Unneberg, Per; Claverie, Jean-Michel

    2007-01-01

    Recent studies on chromosome conformation show that chromosomes colocalize in the nucleus, bringing together active genes in transcription factories. This spatial proximity of actively transcribing genes could provide a means for RNA interaction at the transcript level. We have screened public databases for chimeric EST and mRNA sequences with the intent of mapping transcription-induced interchromosomal interactions. We suggest that chimeric transcripts may be the result of close encounters of active genes, either as functional products or “noise” in the transcription process, and that they could be used as probes for chromosome interactions. We have found a total of 5,614 chimeric ESTs and 587 chimeric mRNAs that meet our selection criteria. Due to their higher quality, the mRNA findings are of particular interest and we hope that they may serve as food for thought for specialists in diverse areas of molecular biology. PMID:17330142

  5. Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

    PubMed

    Geula, Shay; Moshitch-Moshkovitz, Sharon; Dominissini, Dan; Mansour, Abed AlFatah; Kol, Nitzan; Salmon-Divon, Mali; Hershkovitz, Vera; Peer, Eyal; Mor, Nofar; Manor, Yair S; Ben-Haim, Moshe Shay; Eyal, Eran; Yunger, Sharon; Pinto, Yishay; Jaitin, Diego Adhemar; Viukov, Sergey; Rais, Yoach; Krupalnik, Vladislav; Chomsky, Elad; Zerbib, Mirie; Maza, Itay; Rechavi, Yoav; Massarwa, Rada; Hanna, Suhair; Amit, Ido; Levanon, Erez Y; Amariglio, Ninette; Stern-Ginossar, Noam; Novershtern, Noa; Rechavi, Gideon; Hanna, Jacob H

    2015-02-27

    Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable. However, they fail to adequately terminate their naïve state and, subsequently, undergo aberrant and restricted lineage priming at the postimplantation stage, which leads to early embryonic lethality. m(6)A predominantly and directly reduces mRNA stability, including that of key naïve pluripotency-promoting transcripts. This study highlights a critical role for an mRNA epigenetic modification in vivo and identifies regulatory modules that functionally influence naïve and primed pluripotency in an opposing manner. PMID:25569111

  6. Cloning and expression analysis of prohibitin mRNA in canine mammary tumors

    PubMed Central

    MATSUYAMA, Satoshi; NAKANO, Yuko; NAKAMURA, Mieko; YAMAMOTO, Ryohei; SHIMADA, Terumasa; OHASHI, Fumihito; KUBO, Kihei

    2014-01-01

    Prohibitin is an antiproliferative protein that is a product of a putative tumor suppressor gene. However, there is little information on prohibitins in companion animals. In this study, we cloned canine prohibitin mRNA using RT-PCR and 3?-RACE (Rapid Amplification of cDNA Ends). The sequence was well conserved compared with those of other mammals, including human. The deduced amino acid sequence translated from the open reading frame completely corresponded to the human sequence. Canine prohibitin mRNA was expressed in all normal mammary and tumor samples examined. These results suggest that this protein plays a vital role in cell growth mechanisms and may be related to the occurrence of canine mammary tumors. PMID:25312047

  7. A max-plus model of ribosome dynamics during mRNA translation.

    PubMed

    Brackley, Chris A; Broomhead, David S; Romano, M Carmen; Thiel, Marco

    2012-06-21

    We examine the dynamics of the translation stage of cellular protein production, in which ribosomes move uni-directionally along an mRNA strand, building amino acid chains as they go. We describe the system using a timed event graph-a class of Petri net useful for studying discrete events, which have to satisfy constraints. We use max-plus algebra to describe a deterministic version of the model, where the constraints represent steric effects which prevent more than one ribosome reading a given codon at a given time and delays associated with the availability of the different tRNAs. We calculate the protein production rate and density of ribosomes on the mRNA and find exact agreement between these analytical results and numerical simulations of the deterministic model, even in the case of heterogeneous mRNAs. PMID:22441134

  8. Absolute mRNA concentrations from sequence-specific calibration of oligonucleotide arrays

    PubMed Central

    Hekstra, Doeke; Taussig, Alexander R.; Magnasco, Marcelo; Naef, Felix

    2003-01-01

    Oligonucleotide microarrays are based on the hybridization of labeled mRNA molecules to short length oligonucleotide probes on a glass surface. Two effects have been shown to affect the raw data: the sequence dependence of the probe hybridization properties and the chemical saturation resulting from surface adsorption processes. We address both issues simultaneously using a physically motivated hybridization model. Based on publicly available calibration data sets, we show that Langmuir adsorption accurately describes GeneChip hybridization, with model parameters that we predict from the sequence composition of the probes. Because these parameters have physical units, we are able to estimate absolute mRNA concentrations in picomolar. Additionally, by accounting for chemical saturation, we substantially reduce the compressive bias of differential expression estimates that normally occurs toward high concentrations. PMID:12655013

  9. DeltaA mRNA and protein distribution in the zebrafish nervous system

    PubMed Central

    Tallafuss, Alexandra; Trepman, Alissa; Eisen, Judith S.

    2010-01-01

    Physical interaction between the transmembrane proteins Delta and Notch allows only a subset of neural precursors to become neurons, as well as regulating other aspects of neural development. To examine the localization of Delta protein during neural development, we generated an antibody specific to zebrafish DeltaA (Dla). Here we describe for the first time the subcellular localization of Dla protein in distinct puncta at cell cortex and/or membrane, supporting the function of Dla in direct cell-cell communication. In situ RNA hybridization and immunohistochemistry revealed dynamic, coordinated expression patterns of dla mRNA and Dla protein in the developing and adult zebrafish nervous system. Dla expression is mostly excluded from differentiated neurons and is maintained in putative precursor cells at least until larval stages. In the adult brain, dla mRNA and Dla protein are expressed in proliferative zones normally associated with stem cells. PMID:19924821

  10. TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer

    PubMed Central

    Proença, Marcela Alcântara; de Oliveira, Juliana Garcia; Cadamuro, Aline Cristina Targa; Succi, Maysa; Netinho, João Gomes; Goloni-Bertolo, Eny Maria; Pavarino, Érika Cristina; Silva, Ana Elizabete

    2015-01-01

    AIM: To evaluate the effect of promoter region polymorphisms of toll-like receptor (TLR)2-196 to -174del and TLR4-1607T/C (rs10759932) on mRNA and protein expression in tumor tissue and of TLR4+896A/G (rs4986790) on colorectal cancer (CRC) risk. METHODS: The TLR2-196 to -174del polymorphism was investigated using allele-specific polymerase chain reaction (PCR) and the TLR4-1607T/C and TLR4+896A/G by PCR-restriction fragment length polymorphism (RFLP). We genotyped 434 DNA samples from 194 CRC patients and 240 healthy individuals. The mRNA relative quantification (RQ) was performed in 40 tumor tissue samples by quantitative PCR TaqMan assay, using specific probes for TLR2 and TLR4 genes, and ACTB and GAPDH reference genes were used as endogenous controls. Protein expression was analyzed by immunohistochemistry with specific primary antibodies. RESULTS: No association was found for TLR4-1607T/C and TLR4+896A/G by three statistical models (log-additive, dominant and recessive). However, based on dominant and log-additive models, the polymorphic variant TLR2-196 to -174del was associated with increased CRC risk [dominant: odds ratio (OR) = 1.72, 95%CI: 1.03-2.89; P = 0.038 and log-additive: OR =1.59, 95%CI: 1.02-2.48; P = 0.039]. TLR2 mRNA expression was increased in tumor tissue (RQ = 2.36) when compared to adjacent normal tissue (RQ = 1; P < 0.0001), whereas the TLR4 mRNA showed a basal expression (RQ = 0.74 vs RQ = 1, P = 0.452). Immunohistochemistry analysis of TLR2 and TLR4 protein expression was concordant with the findings of mRNA expression. In addition, the TLR2-196 to -174del variant carriers showed mRNA relative expression 2.19 times higher than wild-genotype carriers. The TLR2 protein expression was also higher for the TLR2-196 to -174del variant carriers [117 ± 10 arbitrary unit (a.u.) vs 95 ± 4 a.u., P = 0.03]. However, for the TLR4 -1607T/C polymorphism no significant difference was found for both mRNA (P = 0.56) and protein expression (P = 0.26). CONCLUSION: Our findings suggest that TLR2-196 to -174del polymorphism increases TLR2 mRNA expression and is associated with higher CRC risk, indicating an important role in CRC genetic susceptibility. PMID:26167073

  11. Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

    SciTech Connect

    Jalava, A.M.; Heikkilae, J.E.; Akerman, K.E.O. (Abo Academy (Finland))

    1988-11-01

    The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc RNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC{sub 8}) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC{sub 8} it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.

  12. Induction of aquaporin-4 water channel mRNA after focal cerebral ischemia in rat

    Microsoft Academic Search

    Masaaki Taniguchi; Toshihide Yamashita; Eiji Kumura; Michio Tamatani; Akihiro Kobayashi; Takashi Yokawa; Motohiko Maruno; Amami Kato; Takanori Ohnishi; Eiji Kohmura; Masaya Tohyama; Toshiki Yoshimine

    2000-01-01

    Aquaporin-4 (AQP4) is a member of a water-selective channel aquaporin-family and mainly expressed in the several structures of the brain and in the collecting duct of the kidney. Here we show its functional involvement in the water homeostasis of the ischemic brain. The expression of AQP4–mRNA is increased in the peri-infarcted cortex during the observation period (?7 days) after MCA-occlusion,

  13. Nogo Receptor mRNA Expression in Intact and Regenerating CNS Neurons

    Microsoft Academic Search

    David Hunt; M. R. J. Mason; G. Campbell; R. Coffin; P. N. Anderson

    2002-01-01

    The expression of mRNA for Nogo-66 receptor (NgR) in unoperated adult rats and mice, and rats with nerve grafts placed in the thalamus and cerebellum to stimulate axonal regeneration, was investigated by in situ hybridization. NgR was strongly expressed in neurons of the neocortex, hippocampal formation, and amygdaloid nuclei and dorsal thalamus and moderately expressed in the red nucleus and

  14. Ovarian Steroid Regulation of Tryptophan Hydroxylase mRNA Expression in Rhesus Macaques

    Microsoft Academic Search

    Melanie Pecins-Thompson; Nancy A. Brown; Steven G. Kohama; Cynthia L. Bethea

    Progesterone (P) stimulates prolactin secretion through an un- known neural mechanism in estrogen (E)-primed female mon- keys. Serotonin is a stimulatory neurotransmitter in prolactin regulation, and this laboratory has shown previously that E induces progestin receptors (PR) in serotonin neurons. There- fore, we questioned whether E and\\/or E1P increased serotonin neural function. The expression of mRNA for tryptophan hy- droxylase

  15. Suppression of facilitative glucose transporter 1 mRNA can suppress tumor growth

    Microsoft Academic Search

    Yoshikazu Noguchi; Aya Saito; Yohei Miyagi; Shoji Yamanaka; Doulet Marat; Chiharu Doi; Takaki Yoshikawa; Akira Tsuburaya; Takaaki Ito; Shinobu Satoh

    2000-01-01

    We attempted to suppress glucose transporter 1 (GLUT1) expression by transfecting MKN45 cells with cDNA for antisense GLUT1. Glucose transport was significantly decreased in cells with antisense GLUT1 compared with wild-type cells or cells with vector alone. Suppression of GLUT1 mRNA resulted in a decreased number of cells in the S phase. This was accompanied by overexpression of p21 protein.

  16. Rhythmic expression of Nocturnin mRNA in multiple tissues of the mouse

    PubMed Central

    Wang, Yunxia; Osterbur, David L; Megaw, Pamela L; Tosini, Gianluca; Fukuhara, Chiaki; Green, Carla B; Besharse, Joseph C

    2001-01-01

    Background Nocturnin was originally identified by differential display as a circadian clock regulated gene with high expression at night in photoreceptors of the African clawed frog, Xenopus laevis. Although encoding a novel protein, the nocturnin cDNA had strong sequence similarity with a C-terminal domain of the yeast transcription factor CCR4, and with mouse and human ESTs. Since its original identification others have cloned mouse and human homologues of nocturnin/CCR4, and we have cloned a full-length cDNA from mouse retina, along with partial cDNAs from human, cow and chicken. The goal of this study was to determine the temporal pattern of nocturnin mRNA expression in multiple tissues of the mouse. Results cDNA sequence analysis revealed a high degree of conservation among vertebrate nocturnin/CCR4 homologues along with a possible homologue in Drosophila. Northern analysis of mRNA in C3H/He and C57/Bl6 mice revealed that the mNoc gene is expressed in a broad range of tissues, with greatest abundance in liver, kidney and testis. mNoc is also expressed in multiple brain regions including suprachiasmatic nucleus and pineal gland. Furthermore, mNoc exhibits circadian rhythmicity of mRNA abundance with peak levels at the time of light offset in the retina, spleen, heart, kidney and liver. Conclusion The widespread expression and rhythmicity of mNoc mRNA parallels the widespread expression of other circadian clock genes in mammalian tissues, and suggests that nocturnin plays an important role in clock function or as a circadian clock effector. PMID:11394964

  17. Analysis of myosin heavy chain mRNA expression by RT-PCR

    NASA Technical Reports Server (NTRS)

    Wright, C.; Haddad, F.; Qin, A. X.; Baldwin, K. M.

    1997-01-01

    An assay was developed for rapid and sensitive analysis of myosin heavy chain (MHC) mRNA expression in rodent skeletal muscle. Only 2 microg of total RNA were necessary for the simultaneous analysis of relative mRNA expression of six different MHC genes. We designed synthetic DNA fragments as internal standards, which contained the relevant primer sequences for the adult MHC mRNAs type I, IIa, IIx, IIb as well as the embryonic and neonatal MHC mRNAs. A known amount of the synthetic fragment was added to each polymerase chain reaction (PCR) and yielded a product of different size than the amplified MHC mRNA fragment. The ratio of amplified MHC fragment to synthetic fragment allowed us to calculate percentages of the gene expression of the different MHC genes in a given muscle sample. Comparison with the traditional Northern blot analysis demonstrated that our reverse transcriptase-PCR-based assay was reliable, fast, and quantitative over a wide range of relative MHC mRNA expression in a spectrum of adult and neonatal rat skeletal muscles. Furthermore, the high sensitivity of the assay made it very useful when only small quantities of tissue were available. Statistical analysis of the signals for each MHC isoform across the analyzed samples showed a highly significant correlation between the PCR and the Northern signals as Pearson correlation coefficients ranged between 0.77 and 0.96 (P < 0.005). This assay has potential use in analyzing small muscle samples such as biopsies and samples from pre- and/or neonatal stages of development.

  18. Strain and gender modulate hepatic hepcidin 1 and 2 mRNA expression in mice

    Microsoft Academic Search

    Brice Courselaud; Marie-Berengere Troadec; Severine Fruchon; Gennady Ilyin; Nicolas Borot; Patricia Leroyer; Helene Coppin; Pierre Brissot; Marie-Paule Roth; Olivier Loreala

    2004-01-01

    Hepcidin (HEPC) plays a key role in iron homeostasis and an abnormally low level of hepcidin mRNA has been reported in HFE-1 genetic hemochromatosis. Considering the well-known phenotypic variability of this disease, especially between men and women, it is important to define factors susceptible to modulate hepatic hepcidin expression and, consequently, to influence the development of iron overload in HFE-1

  19. Elevated level of HSPA1L mRNA correlates with graft-versus-host disease.

    PubMed

    Atarod, Sadaf; Turner, Brie; Pearce, Kim Frances; Ahmed, Shaheda S; Norden, Jean; Bogunia-Kubik, Katarzyna; Wang, Xiao-Nong; Collin, Matthew; Dickinson, Anne Mary

    2015-06-01

    Graft-versus-host disease (GVHD) can be a fatal complication of allogeneic stem cell transplantation (allo-HSCT). GVHD can be classified as acute (aGVHD: up to 100days) or chronic (cGVHD: after 100days) based on the time-point of disease occurrence. At present there are a limited number of biomarkers available for use in the clinic. Thus, the aim of this research was to evaluate the biomarker potential of the extensively studied Heat Shock Protein 70 family members (HSPA1A/HSPA1B and HSPA1L) at the messenger RNA (mRNA) level in acute and cGVHD patient cohorts. In the skin biopsies, HSPA1L mRNA expression was lower in patients with severe aGVHD (grades II-III) when compared to those with none or low grade aGVHD (grades 0-I) and normal controls. In whole blood, HSPA1L mRNA expression level was significantly (p=0.008) up-regulated at 28days post-transplant in cGVHD patients with a significant area under the curve (AUC=0.773). In addition, HSPA1B expression in whole blood was significantly higher at 3months post-transplant in both the aGVHD grade II-III (p=0.012) and cGVHD (p=0.027) patients. Our initial results in this small cohort show that quantifying HSPA1L mRNA expression in the whole blood of allo-HSCT patients at day 28 post-allo-HSCT may be a useful predictive biomarker for cGVHD. PMID:25680846

  20. Modulation of mRNA for Microtubule-Associated Proteins during Brain Development

    Microsoft Academic Search

    I. Ginzburg; T. Scherson; D. Giveon; L. Behar; U. Z. Littauer

    1982-01-01

    The heterogeneity of tau microtubule-associated proteins from rat brain is developmentally determined. Newborn rat brain contains two tau polypeptides (tau 0) with somewhat different molecular weights than the five tau components associated with microtubules from 12-day-old brain (tau 12). tau 0 and tau 12 are immuunologically related and crossreact with antibodies against tau 12 proteins. Enrichment of the tau mRNA

  1. Localization of laminin B1 mRNA in retinal ganglion cells by in situ hybridization

    PubMed Central

    1990-01-01

    In the nervous system, neuronal migration and axonal growth are dependent on specific interactions with extracellular matrix proteins. During development of the vertebrate retina, ganglion cell axons extend along the internal limiting (basement) membrane and form the optic nerve. Laminin, a major component of basement membranes, is known to be present in the internal limiting membrane, and might be involved in the growth of ganglion cell axons. The identity of the cells that produce retinal laminin, however, has not been established. In the present study, we have used in situ hybridization to localize the sites of laminin B1 mRNA synthesis in the developing mouse retina. Our results show that there are at least two principal sites of laminin B1 mRNA synthesis: (a) the hyaloid vessels and the lens during the period of major axonal outgrowth, and (b) the retinal ganglion cells at later development stages. Muller (glial) cells, the major class of nonneuronal cells in the retina, do not appear to express laminin B1 mRNA either during development or in the adult retina. In Northern blots, we found a single transcript of approximately 6-kb size that encodes the laminin B1 chain in the retina. Moreover, laminin B1 mRNA level was four- to fivefold higher in the postnatal retina compared to that in the adult. Our results show that in addition to nonneuronal cells, retinal ganglion cells also synthesize laminin. The function of laminin in postnatal retinas, however, remains to be elucidated. Nevertheless, our findings raise the possibility that neurons in other parts of the nervous system might also synthesize extracellular matrix proteins. PMID:2351694

  2. A post-translational regulatory switch on UPF1 controls targeted mRNA degradation

    PubMed Central

    Kurosaki, Tatsuaki; Li, Wencheng; Hoque, Mainul; Popp, Maximilian W.-L.; Ermolenko, Dmitri N.; Tian, Bin

    2014-01-01

    Nonsense-mediated mRNA decay (NMD) controls the quality of eukaryotic gene expression and also degrades physiologic mRNAs. How NMD targets are identified is incompletely understood. A central NMD factor is the ATP-dependent RNA helicase upframeshift 1 (UPF1). Neither the distance in space between the termination codon and the poly(A) tail nor the binding of steady-state, largely hypophosphorylated UPF1 is a discriminating marker of cellular NMD targets, unlike for premature termination codon (PTC)-containing reporter mRNAs when compared with their PTC-free counterparts. Here, we map phosphorylated UPF1 (p-UPF1)-binding sites using transcriptome-wide footprinting or DNA oligonucleotide-directed mRNA cleavage to report that p-UPF1 provides the first reliable cellular NMD target marker. p-UPF1 is enriched on NMD target 3? untranslated regions (UTRs) along with suppressor with morphogenic effect on genitalia 5 (SMG5) and SMG7 but not SMG1 or SMG6. Immunoprecipitations of UPF1 variants deficient in various aspects of the NMD process in parallel with Förster resonance energy transfer (FRET) experiments reveal that ATPase/helicase-deficient UPF1 manifests high levels of RNA binding and disregulated hyperphosphorylation, whereas wild-type UPF1 releases from nonspecific RNA interactions in an ATP hydrolysis-dependent mechanism until an NMD target is identified. 3? UTR-associated UPF1 undergoes regulated phosphorylation on NMD targets, providing a binding platform for mRNA degradative activities. p-UPF1 binding to NMD target 3? UTRs is stabilized by SMG5 and SMG7. Our results help to explain why steady-state UPF1 binding is not a marker for cellular NMD substrates and how this binding is transformed to induce mRNA decay. PMID:25184677

  3. Mammalian tissues defective in nonsense-mediated mRNA decay display highly aberrant splicing patterns

    PubMed Central

    2012-01-01

    Background Nonsense-mediated mRNA decay (NMD) affects the outcome of alternative splicing by degrading mRNA isoforms with premature termination codons. Splicing regulators constitute important NMD targets; however, the extent to which loss of NMD causes extensive deregulation of alternative splicing has not previously been assayed in a global, unbiased manner. Here, we combine mouse genetics and RNA-seq to provide the first in vivo analysis of the global impact of NMD on splicing patterns in two primary mouse tissues ablated for the NMD factor UPF2. Results We developed a bioinformatic pipeline that maps RNA-seq data to a combinatorial exon database, predicts NMD-susceptibility for mRNA isoforms and calculates the distribution of major splice isoform classes. We present a catalog of NMD-regulated alternative splicing events, showing that isoforms of 30% of all expressed genes are upregulated in NMD-deficient cells and that NMD targets all major splicing classes. Importantly, NMD-dependent effects are not restricted to premature termination codon+ isoforms but also involve an abundance of splicing events that do not generate premature termination codons. Supporting their functional importance, the latter events are associated with high intronic conservation. Conclusions Our data demonstrate that NMD regulates alternative splicing outcomes through an intricate web of splicing regulators and that its loss leads to the deregulation of a panoply of splicing events, providing novel insights into its role in core- and tissue-specific regulation of gene expression. Thus, our study extends the importance of NMD from an mRNA quality pathway to a regulator of several layers of gene expression. PMID:22624609

  4. Effects of continuous diazepam administration on GABA A subunit mRNA in rat brain

    Microsoft Academic Search

    C. Heninger; N. Saito; J. F. Tallman; K. M. Garrett; M. P. Vitek; R. S. Duman; D. W. Gallager

    1990-01-01

    Rats treated chronically with diazepam develop tolerance to diazepam effects and show changes in sensitivity of GABAergic\\u000a systems. In order to investigate possible molecular mechanisms associated with these changes, we have evaluated the effects\\u000a of acute and chronic diazepam treatment on levels of mRNA for the ?1 and ?1 subunits of the GABAA receptor. Northern blots were hybridized with32P-labeled GABA

  5. Effects of dietary cholesterol and hypothyroidism on rat apolipoprotein mRNA metabolism

    Microsoft Academic Search

    Jim J. Apostolopoulos; Geoffrey J. Howlett; Noel Fidge

    The effects of dietary cholesterol and hypothyroidism on the mRNA levels of rat apolipoproteins A-I, A-IV, and E were measured in extracts of rat liver and rat intestine by hybridization to specific cDNA. Four groups, each comprised of six rats, were fed diets consisting of normal laboratory rat chow and either i) no supplements (control); ii) 5% lard, 1% cholesterol,

  6. BRCA1 mRNA expression levels as an indicator of chemoresistance in lung cancer

    Microsoft Academic Search

    Miquel Taron; Rafael Rosell; Enriqueta Felip; Pedro Mendez; John Souglakos; Maria Sanchez Ronco; Cristina Queralt; Joaquim Majo; Jose Miguel Sanchez; Jose Javier Sanchez; Jose Maestre

    2004-01-01

    Lung cancer is the most common cancer, with dismal outcome. Treatment approaches, including cisplatin- based chemotherapy and surgery, are currently based on the clinical classification of the tumor, without genetic assessment for predicting differential chemosensitivity. BRCA1 plays a central role in DNA repair, and decreased BRCA1 mRNA expression in the human breast cancer HCC1937 cell line caused cisplatin hypersensitivity, but

  7. Post-transcriptional regulation of placenta growth factor mRNA by hydrogen peroxide

    PubMed Central

    Shaw, Jennifer H.; Lloyd, Pamela G.

    2012-01-01

    In tissues containing pre-existing collateral vessels, occlusion of an upstream supply artery results in diversion of blood flow through these vessels, protecting the distal tissue from ischemia. The sudden rise in blood flow through collateral vessels exerts shear stress upon the vessel wall, thereby providing the initial stimulus for arteriogenesis. Arteriogenesis, the structural expansion of collateral circulation, involves smooth muscle cell (SMC) proliferation which leads to increased vessel diameter and wall thickness. Since shear is sensed at the level of endothelial cells (EC), communication from EC to the underlying SMC must occur as part of this process. We previously reported that endothelial cells (EC) exposed to shear stress release hydrogen peroxide (H2O2), and that H2O2 can signal vascular SMC to increase gene and protein expression of placenta growth factor (PLGF), a known mediator of arteriogenesis. The purpose of the current study was to further elucidate the mechanism whereby PLGF is regulated by H2O2. We found that a single, physiological dose of H2O2 increases PLGF mRNA half-life, but has no effect on PLGF promoter activity, in human coronary artery SMC (CASMC). We further demonstrated that the H2O2–induced increase in PLGF mRNA levels partially relies on p38 MAPK, JNK and ERK1/2 pathways. Finally, we showed that chronic exposure to pathological levels of H2O2 further increases PLGF mRNA levels, but does not result in a corresponding increase in PLGF secreted protein. These data suggest that PLGF regulation has an important translational component. To our knowledge, this is the first study to characterize post-transcriptional regulation of PLGF mRNA by H2O2 in vascular SMC. These findings provide new insights into the regulation of this important growth factor and increase our understanding of PLGF-driven arteriogenesis. PMID:22683469

  8. Analysis of Large-Scale mRNA Expression Data Sets by Genetic Algorithms

    Microsoft Academic Search

    Chia Huey Ooi; Patrick Tan

    DNA microarray experiments typically produce large-scale data sets comprising thousands of mRNA expression values measured\\u000a across multiple biological samples. A common problem in the analysis of this data is the ‘curse of dimensionality’, where\\u000a the number of available samples is often insufficient for reliable analysis due to the large number of individual measurements\\u000a made per sample. Genetic algorithms (GAs) are

  9. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays

    Microsoft Academic Search

    S A Bustin

    2000-01-01

    The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a com- plex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suf- fers from the problems inherent in PCR. The recent introduction

  10. Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain

    Microsoft Academic Search

    Françoise Dandoy-Dron; Louisa Benboudjema; Frédéric Guillo; Alexandre Jaegly; Claude Jasmin; Dominique Dormont; Michael G Tovey; Michel Dron

    2000-01-01

    The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB\\/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine

  11. Analysis of matched mRNA measurements from two different microarray technologies

    Microsoft Academic Search

    Winston Patrick Kuo; Tor-kristian Jenssen; Atul J. Butte; Lucila Ohno-machado; Isaac S. Kohane

    2002-01-01

    Motivation: The existence of several technologies for measuring gene expression makes the question of cross- technology agreement of measurements an important issue. Cross-platform utilization of data from different tech- nologies has the potential to reduce the need to duplicate experiments but requires corresponding measurements to be comparable. Methods: A comparison of mRNA measurements of 2895 sequence-matched genes in 56 cell

  12. Asymmetric Subcellular mRNA Distribution Correlates with Carbonic Anhydrase Activity in Acetabularia acetabulum1

    PubMed Central

    Serikawa, Kyle A.; Porterfield, D. Marshall; Mandoli, Dina F.

    2001-01-01

    The unicellular green macroalga Acetabularia acetabulum L. Silva is an excellent system for studying regional differentiation within a single cell. In late adults, physiologically mediated extracellular alkalinity varies along the long axis of the alga with extracellular pH more alkaline along the apical and middle regions of the stalk than at and near the rhizoid. Respiration also varies with greater respiration at and near the rhizoid than along the stalk. We hypothesized that the apical and middle regions of the stalk require greater carbonic anhydrase (CA) activity to facilitate inorganic carbon uptake for photosynthesis. Treatment of algae with the CA inhibitors acetazolamide and ethoxyzolamide decreased photosynthetic oxygen evolution along the stalk but not at the rhizoid, indicating that CA facilitates inorganic carbon uptake in the apical portions of the alga. To examine the distribution of enzymatic activity within the alga, individuals were dissected into apical, middle, and basal tissue pools and assayed for both total and external CA activity. CA activity was greatest in the apical portions. We cloned two CA genes (AaCA1 and AaCA2). Northern analysis demonstrated that both genes are expressed throughout much of the life cycle of A. acetabulum. AaCA1 mRNA first appears in early adults. AaCA2 mRNA appears in juveniles. The AaCA1 and AaCA2 mRNAs are distributed asymmetrically in late adults with highest levels of each in the apical portion of the alga. mRNA localization and enzyme activity patterns correlate for AaCA1 and AaCA2, indicating that mRNA localization is one mechanism underlying regional differentiation in A. acetabulum. PMID:11161047

  13. tion, the ribosomes will dissociate from the mRNA before they reach the legitimate

    E-print Network

    Lykke-Andersen, Jens

    of the termination complex (eRF1 and eRF3), and trigger degradation of this mRNA. There may be additional functions further light on post-splicing gene regulation. References and Notes 1. M. W. Hentze, A. E. Kulozik, Cell. Acad. Sci. U.S.A. 96, 14937 (1999). 13. H. Le Hir, M. J. Moore, L. E. Maquat, Genes Dev. 14, 1098 (2000

  14. Alternative splicing of parathyroid hormone-related protein mRNA: expression and stability

    Microsoft Academic Search

    R S Sellers; A I Luchin; V Richard; R M Brena; D Lima; T J Rosol

    2004-01-01

    Parathyroid hormone-related protein (PTHrP) is a multifunctional protein that is often dysregulated in cancer. The human PTHrP gene is alternatively spliced into three isoforms, each with a unique 3'-untranslated region (38-UTR), encoding 139, 173 and 141 amino acid proteins. The regulation of PTHrP mRNA isoform expression has not been completely elucidated, but it may be affected by transforming growth factor-1

  15. Long-term dietary restriction influences plasma ghrelin and GOAT mRNA level in rats.

    PubMed

    Reimer, Raylene A; Maurer, Alannah D; Lau, David C W; Auer, Roland N

    2010-04-19

    The objective of this study was to examine the effect of chronic dietary restriction on the physical characteristics of the intestine and gut-derived satiety hormone production. Male Wistar rats (8 weeks) were randomized to ad libitum (AL) or 35% dietary restriction (DR) for 5 months. At the end of the study, physical measurements were made on the intestine and satiety hormone secretion and mRNA expression determined. A comparison group of young, growing AL rats (5 weeks) was also examined. The adult DR rats gained less weight over 5 months and had lower fat mass than adult AL rats (p<0.05). The weight of the small intestine as a percentage of total body weight was greater in adult DR compared to adult AL but lower than young AL rats. Compared to AL, DR down-regulated proglucagon and cholecystokinin mRNA in the duodenum and ghrelin mRNA in the stomach of adult rats but was not different from young AL. Ghrelin-O-acyltransferase (GOAT) mRNA in the stomach was up-regulated 21-fold in adult AL rats compared to young AL and 14-fold compared to adult DR rats. Total and des-acyl ghrelin was approximately 50% higher in adult DR and young AL rats compared to adult AL. Plasma leptin and insulin were lower in adult DR and young AL rats compared to adult AL. Our findings suggest that long-term energy deficits continue to drive up ghrelin levels which may have profound implications for practical implementation of DR as an anti-aging or anti-obesity strategy in humans. PMID:20149910

  16. Comparing Transcription Rate and mRNA Abundance as Parameters for Biochemical Pathway and Network Analysis

    Microsoft Academic Search

    Brewster Hayles; Sailu Yellaboina; Degeng Wang

    2010-01-01

    The cells adapt to extra- and intra-cellular signals by dynamic orchestration of activities of pathways in the biochemical networks. Dynamic control of the gene expression process represents a major mechanism for pathway activity regulation. Gene expression has thus been routinely measured, most frequently at steady-state mRNA abundance level using micro-array technology. The results are widely used in statistical inference of

  17. Analysis of Cytokine mRNA and DNA: Detection and Quantitation by Competitive Polymerase Chain Reaction

    Microsoft Academic Search

    Gary Gilliland; Steven Perrin; Kerry Blanchard; H. Franklin Bunn

    1990-01-01

    The expression of two cytokines, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 3 (IL-3), has been investigated in MLA-144 cells before and after induction with phorbol 12-myristate 13-acetate. We describe an adaptation of the polymerase chain reaction (PCR) for highly accurate quantitation of mRNA or DNA from a small number of cells. Aliquots of the PCR mixture containing cDNA copies of

  18. Induction of Metallothionein mRNA and Protein in Murine Astrocyte Cultures

    Microsoft Academic Search

    Kyle K. Kramer; Jie Liu; Supratim Choudhuri; Curtis D. Klaassen

    1996-01-01

    Astrocytes are known to express metallothionein (MT) and were studied in culture to determine whether MT could be directly induced and which isoforms are induced. Primary astrocyte cultures were established from neonatal CF-1 mice. Both concentration–response and time–course analyses for MT induction at the protein level were determined. At the mRNA level, induction of MT-I, -II, and -III was examined

  19. The mRNA assembly line: transcription and processing machines in the same factory

    Microsoft Academic Search

    David Bentley

    2002-01-01

    Processing of RNA precursors to their mature form often occurs co-transcriptionally. Consequently, the ternary complex of DNA template, RNA polymerase and nascent RNA chain is the physiological substrate for factors that modify the nascent RNA by capping, splicing and cleavage\\/polyadenylation. mRNA production is thought to occur within a ‘factory’ that contains the RNA polymerase II transcription machine and the processing

  20. Dis3- and exosome subunit-responsive 3' mRNA instability elements.

    PubMed

    Kiss, Daniel L; Hou, Dezhi; Gross, Robert H; Andrulis, Erik D

    2012-07-01

    Eukaryotic RNA turnover is regulated in part by the exosome, a nuclear and cytoplasmic complex of ribonucleases (RNases) and RNA-binding proteins. The major RNase of the complex is thought to be Dis3, a multi-functional 3'-5' exoribonuclease and endoribonuclease. Although it is known that Dis3 and core exosome subunits are recruited to transcriptionally active genes and to messenger RNA (mRNA) substrates, this recruitment is thought to occur indirectly. We sought to discover cis-acting elements that recruit Dis3 or other exosome subunits. Using a bioinformatic tool called RNA SCOPE to screen the 3' untranslated regions of up-regulated transcripts from our published Dis3 depletion-derived transcriptomic data set, we identified several motifs as candidate instability elements. Secondary screening using a luciferase reporter system revealed that one cassette-harboring four elements-destabilized the reporter transcript. RNAi-based depletion of Dis3, Rrp6, Rrp4, Rrp40, or Rrp46 diminished the efficacy of cassette-mediated destabilization. Truncation analysis of the cassette showed that two exosome subunit-sensitive elements (ESSEs) destabilized the reporter. Point-directed mutagenesis of ESSE abrogated the destabilization effect. An examination of the transcriptomic data from exosome subunit depletion-based microarrays revealed that mRNAs with ESSEs are found in every up-regulated mRNA data set but are underrepresented or missing from the down-regulated data sets. Taken together, our findings imply a potentially novel mechanism of mRNA turnover that involves direct Dis3 and other exosome subunit recruitment to and/or regulation on mRNA substrates. PMID:22668878