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Sample records for tnf receptor cdna

  1. Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

    Science.gov (United States)

    Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

    1990-10-01

    The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

  2. Soluble forms of tumor necrosis factor receptors (TNF-Rs). The cDNA for the type I TNF-R, cloned using amino acid sequence data of its soluble form, encodes both the cell surface and a soluble form of the receptor

    DEFF Research Database (Denmark)

    Nophar, Y; Kemper, O; Brakebusch, C

    1990-01-01

    the extracellular domain of the type I TNF-R matches the COOH-terminal sequence of TBPI. Amino acid sequences in the extracellular domain also fully match other sequences found in TBPI. On the other hand, amino acid sequences in the soluble form of the type II TNF-R (TBPII), while indicating a marked homology...... found to have effects characteristic of TNF, including stimulating phosphorylation of specific cellular proteins. Oligonucleotide probes designed on the basis of the NH2-terminal amino acid sequence of TBPI were used to clone the cDNA for the structurally related cell surface type 1 TNF-R. It is notable...... that although this receptor can signal the phosphorylation of cellular proteins, it appears from its amino acid sequence to be devoid of intrinsic protein kinase activity. The extracellular domain of the receptor is composed of four internal cysteine-rich repeats, homologous to structures repeated four times...

  3. Origin and evolution of TNF and TNF receptor superfamilies

    Science.gov (United States)

    The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in g...

  4. Regulation of tumour necrosis factor (TNF) induced apoptosis by soluble TNF receptors in Helicobacter pylori infection

    OpenAIRE

    Shibata, J; Goto, H; Arisawa, T; Niwa, Y; Hayakawa, T; Nakayama, A; Mori, N

    1999-01-01

    BACKGROUND—Tumour necrosis factor (TNF) is a predominant cytokine produced in the gastric mucosa of patients with Helicobacter pylori infection. TNF induces apoptosis in a variety of cells. The soluble TNF receptors (sTNF-Rs) can be divided into sTNF-RI and sTNF-RII, both of which inhibit TNF activity. However, their precise mechanisms remain unclear.
AIM—To investigate the role of sTNF-Rs in H pylori infection.
METHODS—In 40 patients, production of TNF and sTNF-Rs in gastric mucosa was measu...

  5. Neurodegenerative and neuroprotective effects of tumor necrosis factor (TNF) in retinal ischemia : Opposite roles of TNF receptor 1 and TNF receptor 2

    NARCIS (Netherlands)

    Fontaine, Sharon; Mohand-Said, S; Hanoteau, N; Fuchs, L; Pfizenmaier, K; Eisel, U

    2002-01-01

    Tumor necrosis factor (TNF) is an important factor in various acute and chronic neurodegenerative disorders. In retinal ischemia, we show early, transient upregulation of TNF, TNF receptor 1 (TNF-R1), and TNF-R2 6 hr after reperfusion preceding neuronal cell loss. To assess the specific role of TNF

  6. Increased concentrations of tumour necrosis factor (TNF) and soluble TNF receptors in biliary obstruction in mice; soluble TNF receptors as prognostic factors for mortality

    NARCIS (Netherlands)

    Bemelmans, M. H.; Greve, J. W.; Gouma, D. J.; Buurman, W. A.

    1996-01-01

    Systemic tumour necrosis factor (TNF) is present in jaundiced mice. Two soluble TNF receptors, sTNFr-P55 and sTNFr-P75 are reported to play a part in the natural defence against TNF. This study investigated the properties of circulating TNF and sTNFr in jaundiced mice. The data show that TNF in

  7. Cloning of the human androgen receptor cDNA

    International Nuclear Information System (INIS)

    Govindan, M.V.; Burelle, M.; Cantin, C.; Kabrie, C.; Labrie, F.; Lachance, Y.; Leblanc, G.; Lefebvre, C.; Patel, P.; Simard, J.

    1988-01-01

    The authors discuss how in order to define the functional domains of the human androgen receptor, complementary DNA (cDNA) clones encoding the human androgen receptor (hAR) have been isolated from a human testis λgtll cDNA library using synthetic oligonnucleotide probes, homologous to segments of the human glucocorticoid, estradiol and progesterone receptors. The cDNA clones corresponding to the human glucocorticoid, estradiol and progesterone receptors were eliminated after cross-hybridization with their respective cDNA probes and/or after restriction mapping of the cDNA clones. The remaining cDNA clones were classified into different groups after analysis by restriction digestion and cross-hybridization. Two of the largest cDNA clones from each group were inserted into an expression vector in both orientations. The linearized plasmids were used as templates in in vitro transcription with T7 RNA polymerase. Subsequent in vitro translation of the purified transcripts in rabbit reticulocyte lysate followed by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) permitted the characterization of the encoded polyeptides. The expressed proteins larger than 30,000 Da were analyzed for their ability to bind tritium-labelled dihydrotestosterone ([ 3 H] DHT) with high affinity and specificity

  8. Targeting TNF and TNF Receptor Pathway in HIV-1 Infection: from Immune Activation to Viral Reservoirs.

    Science.gov (United States)

    Pasquereau, Sébastien; Kumar, Amit; Herbein, Georges

    2017-03-30

    Several cellular functions such as apoptosis, cellular proliferation, inflammation, and immune regulation involve the tumor necrosis factor-α (TNF)/TNF receptor (TNFR) pathway. Human immunodeficiency virus 1 (HIV-1) interacts with the TNF/TNFR pathway. The activation of the TNF/TNFR pathway impacts HIV-1 replication, and the TNF/TNFR pathway is the target of HIV-1 proteins. A hallmark of HIV-1 infection is immune activation and inflammation with increased levels of TNF in the plasma and the tissues. Therefore, the control of the TNF/TNFR pathway by new therapeutic approaches could participate in the control of immune activation and impact both viral replication and viral persistence. In this review, we will describe the intricate interplay between HIV-1 proteins and TNF/TNFR signaling and how TNF/TNFR activation modulates HIV-1 replication and discuss new therapeutic approaches, especially anti-TNF therapy, that could control this pathway and ultimately favor the clearance of infected cells to cure HIV-infected patients.

  9. TNF and TNF Receptor Superfamily Members in HIV infection: New Cellular Targets for Therapy?

    Directory of Open Access Journals (Sweden)

    Amit Kumar

    2013-01-01

    Full Text Available Tumor necrosis factor (TNF and TNF receptors (TNFR superfamily members are engaged in diverse cellular phenomena such as cellular proliferation, morphogenesis, apoptosis, inflammation, and immune regulation. Their role in regulating viral infections has been well documented. Viruses have evolved with numerous strategies to interfere with TNF-mediated signaling indicating the importance of TNF and TNFR superfamily in viral pathogenesis. Recent research reports suggest that TNF and TNFRs play an important role in the pathogenesis of HIV. TNFR signaling modulates HIV replication and HIV proteins interfere with TNF/TNFR pathways. Since immune activation and inflammation are the hallmark of HIV infection, the use of TNF inhibitors can have significant impact on HIV disease progression. In this review, we will describe how HIV infection is modulated by signaling mediated through members of TNF and TNFR superfamily and in turn how these latter could be targeted by HIV proteins. Finally, we will discuss the emerging therapeutics options based on modulation of TNF activity that could ultimately lead to the cure of HIV-infected patients.

  10. Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity

    DEFF Research Database (Denmark)

    Bigda, J; Beletsky, I; Brakebusch, C

    1994-01-01

    Whereas there is ample evidence for involvement of the p55 tumor necrosis factor (TNF) receptor (p55-R) in the cytocidal effect of TNF, the role of the p75 TNF receptor (p75-R) in this effect is a matter of debate. In this study, we probed the function of p75-R in cells sensitive...... to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross...... against p55-R. Moreover, it seemed to reverse induced desensitization to the cytocidal effect of anti p55-R Abs, suggesting that it involves mechanisms different from those of the signaling by the p55 TNF-R. In addition, the Abs affected the response to TNF in a way that does not involve the signaling...

  11. Dual role of the p75 tumor necrosis factor (TNF) receptor in TNF cytotoxicity

    DEFF Research Database (Denmark)

    Bigda, J; Beletsky, I; Brakebusch, C

    1994-01-01

    to the cytotoxicity of TNF using a wide panel of antibodies (Abs) against the receptor's extracellular domain. Two distinct Ab effects were observed. The Abs triggered signaling for cytotoxicity. This effect: (a) was correlated with the extent of p75-R expression by the cells; (b) was dependent on receptor cross...

  12. Tumour necrosis factor-alpha (TNF), lymphotoxin and TNF receptor levels in serum from patients with Wegener's granulomatosis

    DEFF Research Database (Denmark)

    Jónasdóttir, O; Petersen, J; Bendtzen, K

    2001-01-01

    Wegener's granulomatosis (WG) is a systemic inflammatory disease with vasculitis as the key feature. Abnormal expression of tumour necrosis factor alpha (TNFalpha) is considered of prime pathogenic importance in several inflammatory diseases. The effects of TNFa are mediated by TNF receptors (TNF......-R), and these receptors are often found in soluble forms (sTNF-R), which can modulate TNFalpha actions. To evaluate the clinical importance of the TNF family of cytokines, the serum levels of TNFalpha, TNFbeta, now termed lymphotoxin (LTalpha), and sTNF-R1 and sTNF-R2 were measured by ELISA in 8 patients with WG during...... of the relative amounts of TNFalpha and sTNF-R indicated that TNFalpha was mostly bound to its soluble receptors. In WG, the serum levels of sTNF-R1 and sTNF-R2 were dramatically increased (p...

  13. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    Immunological cross-reactivity between tumor necrosis factor (TNF) binding proteins which are present in human urine (designated TBPI and TBPII) and two molecular species of the cell surface receptors for TNF is demonstrated. The two TNF receptors are shown to be immunologically distinct, to differ...... in molecular weight (58,000 and 73,000), and to be expressed differentially in different cells. It is further shown that polyclonal antibodies against one of the TNF binding proteins (TBPI) display, by virtue of their ability to bind the TNF receptor, activities which are very similar to those of TNF....... These antibodies are cytotoxic to cells which are sensitive to TNF toxicity, induce resistance to TNF toxicity, enhance the incorporation of thymidine into normal fibroblasts, inhibit the growth of chlamydiae, and induce the synthesis of prostaglandin E2. Monovalent F(ab) fragments of the polyclonal antibodies...

  14. Antibodies to a soluble form of a tumor necrosis factor (TNF) receptor have TNF-like activity

    DEFF Research Database (Denmark)

    Engelmann, H; Holtmann, H; Brakebusch, C

    1990-01-01

    lack TNF-like activities, but acquire them upon cross-linking with anti-F(ab)2 antibodies, suggesting that the ability of the anti-TBPI antibodies to mimic TNF correlates with their ability to cross-link the TNF receptors. This notion was further supported by data obtained in a comparative study...

  15. TNF RECEPTOR 2 AND DISEASE: Autoimmunity and Regenerative Medicine

    Directory of Open Access Journals (Sweden)

    Denise Louise Faustman

    2013-12-01

    Full Text Available The regulatory cytokine tumor necrosis factor (TNF exerts its effects through two receptors: TNFR1 and TNFR2. Defects in TNFR2 signaling are evident in a variety of autoimmune diseases. One new treatment strategy for autoimmune disease is selective destruction of autoreactive T cells by administration of TNF, TNF inducers, or TNFR2 agonism. A related strategy is to rely on TNFR2 agonism to induce T regulatory cells (Tregs that suppress cytotoxic T cells. Targeting TNFR2 as a treatment strategy is likely superior to TNFR1 because of its more limited cellular distribution on T cells, subsets of neurons, and a few other cell types, whereas TNFR1 is expressed throughout the body. This review focuses on TNFR2 expression, structure, and signaling; TNFR2 signaling in autoimmune disease and treatment strategies targeting TNFR2 in autoimmunity and the potential for TNFR2 to facilitate end organ regeneration.

  16. DMPD: Is HIV infection a TNF receptor signalling-driven disease? [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18178131 Is HIV infection a TNF receptor signalling-driven disease? Herbein G, Khan... KA. Trends Immunol. 2008 Feb;29(2):61-7. (.png) (.svg) (.html) (.csml) Show Is HIV infection a TNF receptor signalling-driven dise...ase? PubmedID 18178131 Title Is HIV infection a TNF receptor signalling-driven diseas

  17. Tumour necrosis factor-alpha (TNF), lymphotoxin and TNF receptor levels in serum from patients with Wegener's granulomatosis

    DEFF Research Database (Denmark)

    Jónasdóttir, O; Petersen, J; Bendtzen, K

    2001-01-01

    Wegener's granulomatosis (WG) is a systemic inflammatory disease with vasculitis as the key feature. Abnormal expression of tumour necrosis factor alpha (TNFalpha) is considered of prime pathogenic importance in several inflammatory diseases. The effects of TNFa are mediated by TNF receptors (TNF......-R), and these receptors are often found in soluble forms (sTNF-R), which can modulate TNFalpha actions. To evaluate the clinical importance of the TNF family of cytokines, the serum levels of TNFalpha, TNFbeta, now termed lymphotoxin (LTalpha), and sTNF-R1 and sTNF-R2 were measured by ELISA in 8 patients with WG during...... active disease and during immunosuppressive treatment, and in 11 healthy controls in parallel. Serum concentrations of TNFalpha were undetectable in all except two controls (18%) and three patients with WG (37%). After 7 days of therapy, six of the WG patients had measurable TNFalpha levels. Examination...

  18. Tumour necrosis factor-alpha (TNF), lymphotoxin and TNF receptor levels in serum from patients with Wegener's granulomatosis

    DEFF Research Database (Denmark)

    Jónasdóttir, O; Petersen, J; Bendtzen, K

    2001-01-01

    Wegener's granulomatosis (WG) is a systemic inflammatory disease with vasculitis as the key feature. Abnormal expression of tumour necrosis factor alpha (TNFalpha) is considered of prime pathogenic importance in several inflammatory diseases. The effects of TNFa are mediated by TNF receptors (TNF...

  19. TNF

    Science.gov (United States)

    Jamil, Kaiser; Jayaraman, Archana; Ahmad, Javeed; Joshi, Sindhu; Yerra, Shiva Kumar

    2017-09-01

    Several reports document the role of tumor necrosis factor alpha ( TNF-α ) and lipid metabolism in the context of acute inflammation as a causative factor in obesity-associated insulin resistance and as one of the causative parameter of type 2 diabetes mellitus (T2DM). Our aim was to investigate the association between -308G/A and -238G/A polymorphisms located in the promoter region of the TNF-α gene in T2DM in the Indian population with bioinformatics analysis of TNF-α protein networking with an aim to find new target sites for the treatment of T2DM. Demographics of 100 diabetes patients and 100 healthy volunteers were collected in a structured proforma and 3 ml blood samples were obtained from the study group, after approval of Institutional Ethics Committee of the hospital (IEC). The information on clinical parameters was obtained from medical records. Genomic DNA was extracted; PCR-RFLP was performed using TNF-α primers specific to detect the presence of SNPs. Various bioinformatics tools such as STRING software were used to determine its network with other associated genes. The PCR-RFLP studies showed that among the -238G/A types the GG genotype was 87%, GA genotype was 12% and AA genotype was 1%. Almost a similar pattern of results was obtained with TNF-α -308G/A polymorphism. The results obtained were evaluated statistically to determine the significance. By constructing TNF-α protein interaction network we could analyze ontology and hubness of the network to identify the networking of this gene which may influence the functioning of other genes in promoting T2DM. We could identify new targets in T2DM which may function in association with TNF-α . Through hub analysis of TNF-α protein network we have identified three novel proteins RIPK1, BIRC2 and BIRC3 which may contribute to TNF- mediated T2DM pathogenesis. In conclusion, our study indicated that some of the genotypes of TNF-α -308G/A, -238G/A were not significantly associated to type 2 diabetes

  20. SPATA2 links CYLD to the TNFreceptor signaling complex and modulates the receptor signaling outcomes

    DEFF Research Database (Denmark)

    Wagner, Sebastian A; Satpathy, Shankha; Beli, Petra

    2016-01-01

    TNF-α is a key regulator of innate immune and proinflammatory responses. However, the composition of the TNFreceptor-associated signaling complexes (TNF-RSC) and the architecture of the downstream signaling networks are incompletely understood. We employed quantitative mass spectrometry...... to demonstrate that TNF-α stimulation induces widespread protein phosphorylation and that the scope of phosphorylation expands in a temporal manner. TNF-α stimulation also induces rapid ubiquitylation of components of the TNF-RSC Temporal analysis of the TNF-RSC composition identified SPATA2 as a novel component...

  1. Palmitoylation of TNF alpha is involved in the regulation of TNF receptor 1 signalling.

    Science.gov (United States)

    Poggi, Marjorie; Kara, Imène; Brunel, Jean-Michel; Landrier, Jean-François; Govers, Roland; Bonardo, Bernadette; Fluhrer, Regina; Haass, Christian; Alessi, Marie-Christine; Peiretti, Franck

    2013-03-01

    The pleiotropic pro-inflammatory cytokine tumour necrosis factor alpha (TNF) is synthesised as a transmembrane protein that is subject to palmitoylation. In this study, the roles of this acylation on TNF-mediated biological effects were investigated. We found that the lipid raft partitioning of TNF is regulated by its palmitoylation. Furthermore, we demonstrated that this palmitoylation process interferes with the cleavage/degradation of TNF intracellular fragments but is not involved in the regulation of its ectodomain shedding. Moreover, we found that the palmitoylation of TNF hinders the binding of soluble TNF to TNFR1 and regulates the integration/retention of TNFR1 into lipid rafts. Finally, we demonstrate that the transmembrane forms of wild-type and palmitoylation-defective TNF interact differently with TNFR1 and regulate NFκB activity, Erk1/2 phosphorylation and interleukin-6 synthesis differently, strongly suggesting that palmitoylation of TNF is involved in the regulation of TNFR1 signalling. An evidence for the physiological intervention of this regulation is provided by the fact that, in macrophages, the binding of endogenous soluble TNF to TNFR1 is enhanced by inhibition of palmitoylation. Therefore, our data introduce the new concept that palmitoylation of TNF is one of the means by which TNF-producing cells regulate their sensitivity to soluble TNF. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Circulating tumour necrosis factor alpha & soluble TNF receptors in patients with Guillain-Barre syndrome.

    Science.gov (United States)

    Radhakrishnan, V V; Sumi, M G; Reuben, S; Mathai, A; Nair, M D

    2003-05-01

    Tumour necrosis factor-alpha (TNF-alpha) is regarded as one of the immune factors that can induce demyelination of peripheral nerves in patients with Guillian-Barre syndrome (GBS). This present study was undertaken to find out the role of TNF-alpha and soluble TNF receptors in the pathogenesis of GBS; and to study the effect of intravenous immunoglobulin (ivIg) therapy on the serum TNF-alpha and soluble TNF receptors in patients with GBS. Thirty six patients with GBS in progressive stages of motor weakness were included in this study. The serum TNF-alpha and soluble TNF receptors (TNF-RI, TNF-RII) were measured in the serum samples of these patients before and after ivIg therapy by a sandwich ELISA. Of the 36 patients with GBS, 26 (72.2%) showed elevated serum TNF-alpha levels prior to ivIg therapy. Following a complete course of ivIg therapy there was a progressive decrease in the serum TNF-alpha concentrations in these 26 patients. On the other hand, the soluble TNF receptors, particularly TNF-RII showed an increase in the serum of GBS patients following ivIg therapy. The results indicate that ivIg reduces the serum TNF-alpha concentrations in the GBS patients having elevated levels prior to ivIg therapy. Elevated serum levels of soluble TNF receptors following ivIg therapy may play a protective role by inhibiting the demyelinating effect of TNF-alpha in the peripheral nerves of patients with GBS.

  3. Down-regulation of surface receptors for TNF and IL-1 on circulating monocytes and granulocytes during human endotoxemia: effect of neutralization of endotoxin-induced TNF activity by infusion of a recombinant dimeric TNF receptor

    NARCIS (Netherlands)

    van der Poll, T.; Coyle, S. M.; Kumar, A.; Barbosa, K.; Agosti, J. M.; Lowry, S. F.

    1997-01-01

    Leukocytes rapidly lose their surface receptors for TNF and IL-1 upon exposure to various stimuli in vitro. We sought to determine by FACS analysis changes in the expression of TNF receptors (TNFR) and type II IL-1R on circulating monocytes and granulocytes during endotoxemia in vivo, and the role

  4. Cloning and expression of full-length cDNA encoding human vitamin D receptor

    Energy Technology Data Exchange (ETDEWEB)

    Baker, A.R.; McDonnell, D.P.; Hughes, M.; Crisp, T.M.; Mangelsdorf, D.J.; Haussler, M.R.; Pike, J.W.; Shine, J.; O' Malley, B.W. (California Biotechnology Inc., Mountain View (USA))

    1988-05-01

    Complementary DNA clones encoding the human vitamin D receptor have been isolated from human intestine and T47D cell cDNA libraries. The nucleotide sequence of the 4605-base pair (bp) cDNA includes a noncoding leader sequence of 115 bp, a 1281-bp open reading frame, and 3209 bp of 3{prime} noncoding sequence. Two polyadenylylation signals, AATAAA, are present 25 and 70 bp upstream of the poly(A) tail, respectively. RNA blot hybridization indicates a single mRNA species of {approx} 4600 bp. Transfection of the cloned sequences into COS-1 cells results in the production of a single receptor species indistinguishable from the native receptor. Sequence comparisons demonstrate that the vitamin D receptor belongs to the steroid-receptor gene family and is closest in size and sequence to another member of this family, the thyroid hormone receptor.

  5. The TNF-alpha system in heart failure and after heart transplantation : plasma protein levels, mRNA expression, soluble receptors and plasma buffer capacity

    NARCIS (Netherlands)

    van Riemsdijk-van Overbeeke, I C; Baan, C C; Niesters, H G; Hesse, C J; Loonen, E H; Balk, A H; Maat, A P; Weimar, W

    BACKGROUND: The two soluble tumour necrosis factor (TNF) receptors (sTNF-R1, sTNF-R2) can bind TNF-alpha, which is a cytokine with cardiodepressant properties. In heart failure and after heart transplantation, the TNF-alpha system is unbalanced, due to elevated levels of sTNF receptors. AIM: To

  6. Forward and Reverse Signaling Mediated by Transmembrane Tumor Necrosis Factor-Alpha and TNF Receptor 2: Potential Roles in an Immunosuppressive Tumor Microenvironment

    OpenAIRE

    Qu, Yang; Zhao, Gang; Li, Hui

    2017-01-01

    Tumor necrosis factor-alpha (TNF-α) is a pleiotropic inflammatory cytokine produced mainly by activated macrophages, lymphocytes and other cell types. Two distinct forms of TNF-α have been identified: soluble TNF-α (sTNF-α) and transmembrane TNF-α (mTNF-α). mTNF-α, which is the precursor of sTNF-α, can be cleaved by the TNF-α converting enzyme (TACE) and is released as sTNF-α. sTNF-α binds primarily to TNF receptor 1 (TNFR1) and plays an important role in the inflammatory immune response, whe...

  7. ST6Gal-I sialyltransferase promotes tumor necrosis factor (TNF)-mediated cancer cell survival via sialylation of the TNF receptor 1 (TNFR1) death receptor.

    Science.gov (United States)

    Holdbrooks, Andrew T; Britain, Colleen M; Bellis, Susan L

    2018-02-02

    Activation of the tumor necrosis factor receptor 1 (TNFR1) death receptor by TNF induces either cell survival or cell death. However, the mechanisms mediating these distinct outcomes remain poorly understood. In this study, we report that the ST6Gal-I sialyltransferase, an enzyme up-regulated in numerous cancers, sialylates TNFR1 and thereby protects tumor cells from TNF-induced apoptosis. Using pancreatic and ovarian cancer cells with ST6Gal-I knockdown or overexpression, we determined that α2-6 sialylation of TNFR1 had no effect on early TNF-induced signaling events, including the rapid activation of NF-κB, c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt (occurring within 15 min). However, upon extended TNF treatment (6-24 h), cells with high ST6Gal-I levels exhibited resistance to TNF-induced apoptosis, as indicated by morphological evidence of cell death and decreased activation of caspases 8 and 3. Correspondingly, at these later time points, high ST6Gal-I expressers displayed sustained activation of the survival molecules Akt and NF-κB. Additionally, extended TNF treatment resulted in the selective enrichment of clonal variants with high ST6Gal-I expression, further substantiating a role for ST6Gal-I in cell survival. Given that TNFR1 internalization is known to be essential for apoptosis induction, whereas survival signaling is initiated by TNFR1 at the plasma membrane, we examined TNFR1 localization. The α2-6 sialylation of TNFR1 was found to inhibit TNF-induced TNFR1 internalization. Thus, by restraining TNFR1 at the cell surface via sialylation, ST6Gal-I acts as a functional switch to divert signaling toward survival. These collective findings point to a novel glycosylation-dependent mechanism that regulates the cellular response to TNF and may promote cancer cell survival within TNF-rich tumor microenvironments. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Bilateral muscle fiber and nerve influences by TNF-alpha in response to unilateral muscle overuse - studies on TNF receptor expressions.

    Science.gov (United States)

    Renström, Lina; Stål, Per; Song, Yafeng; Forsgren, Sture

    2017-11-28

    TNF-alpha is suggested to be involved in muscle damage and muscle inflammation (myositis). In order to evaluate whether TNF-alpha is involved in the myositis that occurs in response to muscle overuse, the aim was to examine the expression patterns of TNF receptors in this condition. A rabbit muscle overuse model leading to myositis in the soleus muscle was used. The expression patterns of the two TNF receptors Tumor Necrosis Factor Receptor type 1 (TNFR1) and Tumor Necrosis Factor Receptor type 2 (TNFR2) were investigated. In situ hybridization and immunofluorescence were utilized. Immunostainings for desmin, NK-1R and CD31 were made in parallel. Immunoreactions (IR) for TNF receptors were clearly observed in white blood cells, fibroblasts and vessel walls, and most interestingly also in muscle fibers and nerve fascicles in the myositis muscles. There were very restricted reactions for these in the muscles of controls. The upregulation of TNF receptors was for all types of structures seen for both the experimental side and the contralateral nonexperimental side. TNF receptor expressing muscle fibers were present in myositis muscles. They can be related to attempts for reparation/regeneration, as evidenced from results of parallel stainings. Necrotic muscle fibers displayed TNFR1 mRNA and TNFR2 immunoreaction (IR) in the invading white blood cells. In myositis muscles, TNFR1 IR was observed in both axons and Schwann cells while TNFR2 IR was observed in Schwann cells. Such observations were very rarely made for control animals. The findings suggest that there is a pronounced involvement of TNF-alpha in the developing myositis process. Attempts for reparation of the muscle tissue seem to occur via both TNFR1 and TNFR2. As the myositis process also occurs in the nonexperimental side and as TNF receptors are confined to nerve fascicles bilaterally it can be asked whether TNF-alpha is involved in the spreading of the myositis process to the contralateral side via the

  9. Serotonin 5-HT2A receptor activation blocks TNF-α mediated inflammation in vivo.

    Directory of Open Access Journals (Sweden)

    Felix Nau

    Full Text Available Tumor necrosis factor alpha (TNF-α plays a key role in inflammation, and its production and signaling contribute to many inflammatory related diseases. Recently, we discovered that selective activation of serotonin 5-HT2A receptors with the agonist (R-DOI produces a super-potent blockade of proinflammatory markers in primary rat aortic smooth muscle cells. Here, we demonstrate that systemic administration of (R-DOI can block the systemic effects of TNF-α in whole animal, with potent anti-inflammatory effects in the aortic arch and small intestine. This includes blockade of TNF-α-induced expression of pro-inflammatory cell adhesion (Icam-1, Vcam-1, cytokine (Il-6, IL-1b, and chemokine (Mcp-1, Cx3cl1 genes, and expression of VCAM-1 protein in the intestine. Further, systemic (R-DOI also prevents the TNF-α-induced increase of circulating IL-6. Importantly, utilizing receptor selective antagonists, we have demonstrated that the mechanism underlying the systemic anti-inflammatory effects of (R-DOI is activation of serotonin 5-HT2A receptors. Our results highlight a powerful new role for the serotonin 5-HT2A receptor in inflammatory processes, and indicate that agonism of serotonin receptors may represent an effective and novel approach to develop powerful small molecule therapeutics for inflammatory diseases and conditions such as atherosclerosis and inflammatory bowel disease.

  10. Serotonin 5-HT2A receptor activation blocks TNF-α mediated inflammation in vivo.

    Science.gov (United States)

    Nau, Felix; Yu, Bangning; Martin, David; Nichols, Charles D

    2013-01-01

    Tumor necrosis factor alpha (TNF-α) plays a key role in inflammation, and its production and signaling contribute to many inflammatory related diseases. Recently, we discovered that selective activation of serotonin 5-HT2A receptors with the agonist (R)-DOI produces a super-potent blockade of proinflammatory markers in primary rat aortic smooth muscle cells. Here, we demonstrate that systemic administration of (R)-DOI can block the systemic effects of TNF-α in whole animal, with potent anti-inflammatory effects in the aortic arch and small intestine. This includes blockade of TNF-α-induced expression of pro-inflammatory cell adhesion (Icam-1, Vcam-1), cytokine (Il-6, IL-1b), and chemokine (Mcp-1, Cx3cl1) genes, and expression of VCAM-1 protein in the intestine. Further, systemic (R)-DOI also prevents the TNF-α-induced increase of circulating IL-6. Importantly, utilizing receptor selective antagonists, we have demonstrated that the mechanism underlying the systemic anti-inflammatory effects of (R)-DOI is activation of serotonin 5-HT2A receptors. Our results highlight a powerful new role for the serotonin 5-HT2A receptor in inflammatory processes, and indicate that agonism of serotonin receptors may represent an effective and novel approach to develop powerful small molecule therapeutics for inflammatory diseases and conditions such as atherosclerosis and inflammatory bowel disease.

  11. RANKL cytokine enhances TNF-induced osteoclastogenesis independently of TNF receptor associated factor (TRAF) 6 by degrading TRAF3 in osteoclast precursors.

    Science.gov (United States)

    Yao, Zhenqiang; Lei, Wei; Duan, Rong; Li, Yanyun; Luo, Lu; Boyce, Brendan F

    2017-06-16

    Cytokines, including receptor activator of nuclear factor κB ligand (RANKL) and TNF, induce increased osteoclast (OC) formation and bone loss in postmenopausal osteoporosis and inflammatory arthritides. RANKL and TNF can independently induce OC formation in vitro from WT OC precursors via TNF receptor-associated factor (TRAF) adaptor proteins, which bind to their receptors. Of these, only TRAF6 is required for RANKL-induced osteoclastogenesis in vitro However, the molecular mechanisms involved remain incompletely understood. Here we report that RANKL induced the formation of bone-resorbing OCs from TRAF6 -/- OC precursors when cultured on bone slices but not on plastic. The mechanisms involved increased TNF production by TRAF6 -/- OC precursors resulting from their interaction with bone matrix and release of active TGFβ from the resorbed bone, coupled with RANKL-induced autophagolysosomal degradation of TRAF3, a known inhibitor of OC formation. Consistent with these findings, RANKL enhanced TNF-induced OC formation from TRAF6 -/- OC precursors. Moreover, TNF induced significantly more OCs from mice with TRAF3 conditionally deleted in myeloid lineage cells, and it did not inhibit RANKL-induced OC formation from these cells. TRAF6 -/- OC precursors that overexpressed TRAF3 or were treated with the autophagolysosome inhibitor chloroquine formed significantly fewer OCs in response to TNF alone or in combination with RANKL. We conclude that RANKL can enhance TNF-induced OC formation independently of TRAF6 by degrading TRAF3. These findings suggest that preventing TRAF3 degradation with drugs like chloroquine could reduce excessive OC formation in diseases in which bone resorption is increased in response to elevated production of these cytokines. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. CDNA CLONING OF FATHEAD MINNOW (PIMEPHALES PROMELAS) ESTROGEN AND ANDROGEN RECEPTORS FOR USE IN STEROID RECEPTOR EXTRAPOLATION STUDIES FOR ENDOCRINE DISRUPTING CHEMICALS

    Science.gov (United States)

    cDNA Cloning of Fathead minnow (Pimephales promelas) Estrogen and Androgen Receptors for Use in Steroid Receptor Extrapolation Studies for Endocrine Disrupting Chemicals. Wilson, V.S.1,, Korte, J.2, Hartig P. 1, Ankley, G.T.2, Gray, L.E., Jr 1, , and Welch, J.E.1. 1U.S...

  13. Cloning and functional characterization of a testicular TSH receptor cDNA from the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Vischer, H F; Bogerd, J.

    A cDNA encoding a putative thyroid-stimulating hormone receptor (cfTSH-R) was cloned from the testis of the African catfish (Clarias gariepinus). The cfTSH-R showed the highest amino acid sequence identity with the TSH-Rs of other fish species. In addition, an insertion of approximately 50 amino

  14. Astrocyte reactivity to unconjugated bilirubin requires TNF-α and IL-1β receptor signaling pathways.

    Science.gov (United States)

    Fernandes, Adelaide; Barateiro, Andreia; Falcão, Ana Sofia; Silva, Sandra Leit-Ao; Vaz, Ana Rita; Brito, Maria Alexandra; Silva, Rui Fernando Marques; Brites, Dora

    2011-01-01

    Jaundice and sepsis are common neonatal conditions that can lead to neurodevelopment sequelae, namely if present at the same time. We have reported that tumor necrosis factor (TNF)-α and interleukin (IL)-1β are produced by cultured neurons and mainly by glial cells exposed to unconjugated bilirubin (UCB). The effects of these cytokines are mediated by cell surface receptors through a nuclear factor (NF)-κB-dependent pathway that we have showed to be activated by UCB. The present study was designed to evaluate the role of TNF-α and IL-1β signaling on astrocyte reactivity to UCB in rat cortical astrocytes. Exposure of astrocytes to UCB increased the expression of both TNFreceptor (TNFR)1 and IL-1β receptor (IL-1R)1, but not TNFR2, as well as their activation, observed by augmented binding of receptors' molecular adaptors, TRAF2 and TRAF6, respectively. Silencing of TNFR1, using siRNA technology, or blockade of IL-1β cascade, using its endogenous antagonist, IL-1 receptor antagonist (IL-1ra), prevented UCB-induced cytokine release and NF-κB activation. Interestingly, lack of TNF-α signal transduction reduced UCB-induced cell death for short periods of incubation, although an increase was observed after extended exposure; in contrast, inhibition of IL-1β cascade produced a sustained blockade of astrocyte injury by UCB. Together, our data show that inflammatory pathways are activated during in vitro exposure of rat cortical astrocytes to UCB and that this activation is prolonged in time. This supports the concept that inflammatory pathways play a role in brain damage by UCB, and that they may represent important pharmacological targets. Copyright © 2010 Wiley-Liss, Inc.

  15. Effect of gene-targeted mutation in TNF receptor (p55) on contact hypersensitivity and ultraviolet B-induced immunosuppression

    Energy Technology Data Exchange (ETDEWEB)

    Kondo, Seiji; Wang, Binghe; Fujisawa, Hiroshi [Univ. of Toronto, Ontario (Canada)] [and others

    1995-10-15

    Tumor necrosis factor {alpha} (TNF-{alpha}) is a pleiotropic proinflammatory cytokine. TNF-{alpha} has been implicated in the pathogenesis of delayed-type hypersensitivity reactions such as allergic contact hypersensitivity and has been suggested as a mediator of ultraviolet B (UVB)-induced immunosuppression. Conflicting reports, however, exist concerning the effects of TNF-{alpha} on contact hypersensitivity (CHS). To determine the role of TNF-{alpha} in the generation and regulation of CHS, gene-targeted mutant mice lacking TNF-receptor (p55) gene (TNF-R1(-) mice) were treated with dinitrofluorobenzene (DNFB) to induce CHS. TNF-R1(-) mice showed significant hyperresponsiveness in CHS (152.8 {+-} 20.9%, p < 0.025) compared with normal syngeneic mice (C57BL/6) assessed by ear swelling. To determine whether UVB can induce suppression in TNF-R1(-) mice, mice were irradiated on the shaved abdomen with 96 ml/cm{sup 2} UVB and 3 days later they were painted with 0.5% DNFB (sensitization dose), followed 5 days later with 0.2% DNFB to the left ear (challenge dose). Significant suppression of CHS was observed both locally (sensitization on irradiated site) and systemically (sensitization on unirradiated site) in UVB-irradiated TNF-R1(-) mice as well as in normal mice. To rule out possible signaling through p75 TNF-R, the mice were treated with anti-TNF-{alpha} Ab (V1q), which can neutralize any TNF effects through either receptor. V1q had no effect on these phenomena observed in TNF-R1(-) mice. These results suggest that TNF-{alpha} plays a regulatory role in CHS but is not required to induce UVB-mediated immunosuppression. 45 refs., 5 figs.

  16. Soluble tumour necrosis factor (TNF)-receptor levels in serum as markers of anti-viral host reactivity

    DEFF Research Database (Denmark)

    Bartholdy, C; Nansen, A; Marker, O

    1999-01-01

    -gamma. A simple correlation between release of sTNF-Rs in vivo and macrophage activation in vitro was not present. These findings indicate that sTNF-R75 is indeed a sensitive marker of both innate and specific cell-mediated host reactivity during viral infection, but it is not correlated to a single immunological......The role of soluble receptors for TNF-alpha (sTNF-Rs) as markers of virus-induced host responses was studied by the use of murine model infections. A marked elevation in serum levels of sTNF-R75, but not sTNF-R55, was found 1 day after infection with vesicular stomatitis virus (VSV). In mice...

  17. Nerve growth factor (NGF) induces neuronal differentiation in neuroblastoma cells transfected with the NGF receptor cDNA

    International Nuclear Information System (INIS)

    Matsushima, H.; Bogenmann, E.

    1990-01-01

    Human nerve growth factor (NGF) receptor (NGFR) cDNA was transfected into a neuroblastoma cell line (HTLA 230) which does not express a functional NGF-NGFR signal transduction cascade. Short-term treatment of stably transfected cells (98-3) expressing membrane-bound NGF receptor molecules resulted in a cell cycle-dependent, transient expression of the c-fos gene upon treatment with NGF, suggesting the presence of functional high-affinity NGFR. Extensive outgrowth of neurites and cessation of DNA synthesis occurred in transfectants grown on an extracellular matrix after long-term treatment with NGF, suggesting terminal differentiation. Our data support the idea that introduction of a constitutively expressed NGFR cDNA into cells with neuronal background results in the assembly of a functional NGF-NGFR signal cascade in a permissive extracellular environment

  18. TNF receptor 1, IL-1 receptor, and iNOS genetic knockout mice are not protected from anthrax infection.

    Science.gov (United States)

    Kalns, John; Scruggs, Julie; Millenbaugh, Nancy; Vivekananda, Jeeva; Shealy, David; Eggers, Jeffrey; Kiel, Johnathan

    2002-03-22

    Anthrax produces at least two toxins that cause an intense systemic inflammatory response, edema, shock, and eventually death. The relative contributions of various elements of the immune response to mortality and course of disease progression are poorly understood. We hypothesized that knockout mice missing components of the immune system will have an altered response to infection. Parent strain mice and knockouts were challenged with LD95 of anthrax spores (5 x 10(6)) administered subcutaneously. Our results show that all genetic knockouts succumbed to anthrax infection at the same frequency as the parent. TNF antibody delayed death but TNF receptor 1 knockout had no effect. IL-1 receptor or iNOS knockouts died sooner. Anthrax was more abundant in the injection site of TNF-alpha and iNOS knockouts compared to parent suggesting that attenuated cellular response increases rate of disease progression. With the exception of edema and necrosis at the injection site pathological changes in internal organs were not observed. (C)2002 Elsevier Science (USA).

  19. The role of tumor necrosis factor-α and TNFreceptors in cerebral arteries following cerebral ischemia in rat

    DEFF Research Database (Denmark)

    Maddahi, Aida; Kruse, Lars S; Chen, Qing-Wen

    2011-01-01

    Tumour necrosis factor-α (TNF-α) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expre...... expression of TNF-α and TNFreceptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway....

  20. The role of tumor necrosis factor-α and TNFreceptors in cerebral arteries following cerebral ischemia in rat

    DEFF Research Database (Denmark)

    Maddahi, Aida; Kruse, Lars S; Chen, Qing-Wen

    2011-01-01

    Tumour necrosis factor-a (TNF-a) is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-a acts by binding to its receptors, TNF-R1 (p55) and TNF-R2 (p75), on the cell surface. The aim of this study was first to investigate if there is altered expre...... expression of TNF-a and TNF-a receptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway....

  1. Common gene variants in the tumor necrosis factor (TNF and TNF receptor superfamilies and NF-kB transcription factors and non-Hodgkin lymphoma risk.

    Directory of Open Access Journals (Sweden)

    Sophia S Wang

    Full Text Available A promoter polymorphism in the pro-inflammatory cytokine tumor necrosis factor (TNF (TNF G-308A is associated with increased non-Hodgkin lymphoma (NHL risk. The protein product, TNF-alpha, activates the nuclear factor kappa beta (NF-kappaB transcription factor, and is critical for inflammatory and apoptotic responses in cancer progression. We hypothesized that the TNF and NF-kappaB pathways are important for NHL and that gene variations across the pathways may alter NHL risk.We genotyped 500 tag single nucleotide polymorphisms (SNPs from 48 candidate gene regions (defined as 20 kb 5', 10 kb 3' in the TNF and TNF receptor superfamilies and the NF-kappaB and related transcription factors, in 1946 NHL cases and 1808 controls pooled from three independent population-based case-control studies. We obtained a gene region-level summary of association by computing the minimum p-value ("minP test". We used logistic regression to compute odds ratios and 95% confidence intervals for NHL and four major NHL subtypes in relation to SNP genotypes and haplotypes. For NHL, the tail strength statistic supported an overall relationship between the TNF/NF-kappaB pathway and NHL (p = 0.02. We confirmed the association between TNF/LTA on chromosome 6p21.3 with NHL and found the LTA rs2844484 SNP most significantly and specifically associated with the major subtype, diffuse large B-cell lymphoma (DLBCL (p-trend = 0.001. We also implicated for the first time, variants in NFKBIL1 on chromosome 6p21.3, associated with NHL. Other gene regions identified as statistically significantly associated with NHL included FAS, IRF4, TNFSF13B, TANK, TNFSF7 and TNFRSF13C. Accordingly, the single most significant SNPs associated with NHL were FAS rs4934436 (p-trend = 0.0024, IRF4 rs12211228 (p-trend = 0.0026, TNFSF13B rs2582869 (p-trend = 0.0055, TANK rs1921310 (p-trend = 0.0025, TNFSF7 rs16994592 (p-trend = 0.0024, and TNFRSF13C rs6002551 (p-trend = 0.0074. All associations were

  2. Inhibition of Epithelial TNFReceptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis

    OpenAIRE

    Zhou, Zijuan; Wang, Liang; Feng, Panpan; Yin, Lianhong; Wang, Chen; Zhi, Shengxu; Dong, Jianyi; Wang, Jingyu; Lin, Yuan; Chen, Dapeng; Xiong, Yongjian; Peng, Jinyong

    2017-01-01

    Activation of the TNFreceptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracol...

  3. Local delivery of soluble TNF-alpha receptor 1 gene reduces infarct size following ischemia/reperfusion injury in rats.

    Science.gov (United States)

    Sugano, Masahiro; Hata, Tomoji; Tsuchida, Keiko; Suematsu, Nobuhiro; Oyama, Jun-Ichi; Satoh, Shinji; Makino, Naoki

    2004-11-01

    Apoptosis in the myocardium is linked to ischemia/reperfusion injury, and TNF-alpha induces apoptosis in cardiomyocytes. A significant amount of TNF-alpha is detected after ischemia and reperfusion. Soluble TNF-alpha receptor 1 (sTNFR1) is an extracellular domain of TNF-alpha receptor 1 and is an antagonist to TNF-alpha. In the present study, we examined the effects of sTNFR1 on infarct size in acute myocardial infarction (AMI) following ischemia/reperfusion. Male Wistar rats were subjected to left coronary artery (LCA) ligation. After 30 min of LCA occlusion, the temporary ligature on the LCA was released and blood flow was restored. Immediately after reperfusion, a total of 200 microg of sTNFR1 or LacZ plasmid was injected into three different sites of the left ventricular wall. At 6 h, 1 and 2 days after reperfusion, the TNF-alpha bioactivity in the myocardium was significantly higher in rats receiving LacZ plasmid than in sham-operated rats, whereas sTNFR1 plasmid significantly suppressed the increase in the TNF-alpha bioactivity. The sTNFR1 plasmid significantly reduced DNA fragmentation and caspase activity compared to the LacZ plasmid. Finally, the sTNFR1 expression-plasmid treatment significantly reduced the area of myocardial infarction at 2 days after ischemia/reperfusion compared to LacZ plasmid. In conclusion, the TNF-alpha bioactivity in the heart increased from the early stage of ischemia/reperfusion, and this increase was thought to contribute in part to the increased area of myocardial infarction. Suppression of TNF-alpha bioactivity with the sTNFR1 plasmid reduced the infarct size in AMI following ischemia and reperfusion.

  4. TNF-alpha promoter, Nod2 and toll-like receptor-4 polymorphisms and the in vivo and ex vivo response to endotoxin

    NARCIS (Netherlands)

    Schippers, Emile F.; van 't Veer, Cornelis; van Voorden, Sjaak; Martina, Cerithsa A. E.; le Cessie, Saskia; van Dissel, Jaap T.

    2004-01-01

    Humans exhibit substantial inter-individual differences in TNF-alpha production upon endotoxin stimulation. To determine to what extent the lipopolysaccharide-induced TNF-alpha production capacity in vivo and ex vivo is determined by polymorphisms in toll-like receptor-4 (TLR4), the TNF-alpha

  5. HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein–Coupled Receptor

    OpenAIRE

    Feng, Yu; Broder, Christopher C.; Kennedy, Paul E.; Berger, Edward A.

    2011-01-01

    A cofactor for HIV-1 (human immunodeficiency virus-type 1) fusion and entry was identified with the use of a novel functional complementary DNA (cDNA) cloning strategy. This protein, designated “fusin,” is a putative G protein–coupled receptor with seven transmembrane segments. Recombinant fusin enabled CD4-expressing nonhuman cell types to support HIV-1 Env-mediated cell fusion and HIV-1 infection. Antibodies to fusin blocked cell fusion and infection with normal CD4-positive human target ce...

  6. Ethanol increases tumor necrosis factor-alpha receptor-1 (TNF-R1) levels in hepatic, intestinal, and cardiac cells.

    Science.gov (United States)

    Rodriguez, Diego A; Moncada, Claudio; Núñez, Marco T; Lavandero, Sergio; Ponnappa, Biddanda C; Israel, Yedy

    2004-05-01

    Chronic ethanol consumption leads to cell injury in virtually every tissue. Tumor necrosis factor-alpha (TNF-alpha) constitutes a major factor in the development of alcohol-induced liver injury. In alcohol-dependent subjects, elevated levels of plasma TNF-alpha are strongly predictive of mortality. Binding of TNF-alpha to TNF-alpha receptor-1 (TNF-R1) activates death domain pathways, leading to necrosis and apoptosis in most tissues, and it also increases the expression of intercellular adhesion molecules (i.e., ICAM-1), which promote inflammation. We determined whether ethanol exposure leads to increases in cellular TNF-R1. We incubated HepG2 human hepatoma cells and H4-II-E-C3 rat hepatoma cells with 25, 50, and 100 mM ethanol for various intervals of time up to 48 h. Human colonic adenocarcinoma cells (Caco-2 cells) and neonatal rat primary cardiomyocytes were also incubated with different concentrations of ethanol. Levels of TNF-R1 were measured either by a sandwich enzyme-linked immunosorbent assay (ELISA) method or by determining the extracellular transmembrane domain of TNF-R1 by an intact-cell ELISA method. Ethanol exposure for 48 h increased TNF-R1 levels in human hepatoma cells in a dose-dependent manner. Levels increased significantly by 164% at 50 mM and by 240% at 100 mM ethanol. Effects were time dependent and did not reach a plateau at 48 h. Similar increases in TNF-R1 were also observed in rat hepatoma cells (90% at 50 mM and 230% at 100 mM ethanol). Under similar conditions, Caco-2 cells showed a significant 80% increase in TNF-R1 levels at 200 mM ethanol, a concentration found in intestine. Neonatal rat primary cardiomyocytes showed TNF-R1 increases of 36% at 50 mM and 44% at 100 mM ethanol. These results indicate that exposure of different cell types to pharmacologic concentrations of ethanol increases TNF-R1 levels and may augment TNF-alpha-mediated cell injury in different tissues.

  7. Total and partial sleep deprivation: Effects on plasma TNF-αRI, TNF-αRII, and IL-6, and reversal by caffeine operating through adenosine A2 receptor

    Science.gov (United States)

    Shearer, William T.; Reuben, James M.; Lee, Bang-Ning; Mullington, Janet; Price, Nicholas; Dinges, David F.

    2000-01-01

    Plasma levels of IL-6 and TNF-α are elevated in individuals who are deprived of sleep. TNF-α regulates expression of its soluble receptors, sTNF-αRI and sTNF-αRII. Sleep deprivation (SD) also increases extracellular adenosine that induces sedation and sleep. An antagonist of adenosine, caffeine, raises exogenous adenosine levels, stimulates the expression of IL-6 and inhibits the release of TNF-α. Our objective was to determine the effect of total SD (TSD) or partial SD (PSD) on the levels of these sleep regulatory molecules in volunteers who experienced SD with or without the consumption of caffeine. Plasma levels of IL-6, sTNF-αRI and sTNF-αRII were assayed by ELISA in samples collected at 90-min intervals from each subject over an 88-hour period. The results were analyzed by the repeated measures ANOVA. Whereas only TSD significantly increased sTNF-αRI over time, caffeine suppressed both sTNFreceptors in TSD and PSD subjects. The selective increase in the expression of sTNF-αRI and not sTNF-αRII in subjects experiencing TSD with caffeine compared with others experiencing PSD with caffeine has not been previously reported. Moreover, caffeine significantly increased IL-6 in TSD subjects compared with those who did not receive caffeine. However, subjects who were permitted intermittent naps (PSD) ablated the effects of caffeine and reduced their level of IL-6 to that of the TSD group. These data further lend support to the hypothesis that the sTNF-αRI and not the sTNF-αRII plays a significant role in sleep regulation by TNF-α. .

  8. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    Energy Technology Data Exchange (ETDEWEB)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-08-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10/sup 6/ receptors per cell. The cell line with the highest /sup 125/I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10/sup 6/ receptors with a K/sub d/ of 10/sup -9/ M. This level was not dependent on exposure to metals but could be increased further to 2 x 10/sup 7/ receptors per cell by addition of sodium butyrate to the culture medium. The ..cap alpha.. and ..beta.. subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the ..cap alpha.. and ..beta.. subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  9. High-level expression of human insulin receptor cDNA in mouse NIH 3T3 cells

    International Nuclear Information System (INIS)

    Whittaker, J.; Okamoto, A.K.; Thys, R.; Bell, G.I.; Steiner, D.F.; Hofmann, C.A.

    1987-01-01

    In order to develop a simple, efficient system for the high-level expression of human insulin receptors in eukaryotic cells, a full-length human kidney insulin receptor cDNA was inserted into a bovine papilloma virus vector under the control of the mouse metallothionein promoter. After transfection of mouse NIH 3T3 cells with this construct, seven cell lines expressing insulin receptors were isolated; two cell lines had more than 10 6 receptors per cell. The cell line with the highest 125 I-insulin binding (NIH 3T3 HIR3.5) had 6 x 10 6 receptors with a K/sub d/ of 10 -9 M. This level was not dependent on exposure to metals but could be increased further to 2 x 10 7 receptors per cell by addition of sodium butyrate to the culture medium. The α and β subunits had apparent molecular weights of 147,000 and 105,000, respectively (compared to 135,000 and 95,000 in IM-9 human lymphocytes), values identical to those of the α and β subunits of the insulin receptors of nontransformed NIH 3T3 cells. This size difference was due to altered carbohydrate composition, as N-glycanase digestion reduced the apparent receptor subunit size of the transfected cells and IM-9 lymphocytes to identical values. The alteration in N-linked oligosaccharide composition could not be ascribed to differences in the kinetics of posttranslational processing of the insulin receptors, which was comparable to that of other cells studied. The basal rate of glycogen synthesis in the cells overexpressing insulin receptors was increased 4- to 5-fold compared with controls. Low levels of added insulin (0.1 nM) caused a 50% increase in the rate of glycogen synthesis

  10. The effect of a metalloproteinase inhibitor (GI5402) on tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha receptors during human endotoxemia

    NARCIS (Netherlands)

    Dekkers, P. E.; Lauw, F. N.; ten Hove, T.; te Velde, A. A.; Lumley, P.; Becherer, D.; van Deventer, S. J.; van der Poll, T.

    1999-01-01

    Tumor necrosis factor-alpha (TNF-alpha) is released from the cell surface by cleavage of pro-TNF-alpha by metalloproteinases (MPs). In cell cultures, inhibition of MPs has been found not only to reduce the release of TNF-alpha, but also to enhance the surface expression of TNF-alpha and TNF-alpha

  11. The role of tumor necrosis factor-α and TNFreceptors in cerebral arteries following cerebral ischemia in rat

    Directory of Open Access Journals (Sweden)

    Chen Qing-Wen

    2011-08-01

    Full Text Available Abstract Background Tumour necrosis factor-α (TNF-α is a pleiotropic pro-inflammatory cytokine, which is rapidly upregulated in the brain after injury. TNF-α acts by binding to its receptors, TNF-R1 (p55 and TNF-R2 (p75, on the cell surface. The aim of this study was first to investigate if there is altered expression of TNF-α and TNFreceptors in cerebral artery walls following global or focal ischemia, and after organ culture. Secondly, we asked if the expression was regulated via activation of the MEK-ERK1/2 pathway. Methods The hypothesis was tested in vivo after subarachnoid hemorrhage (SAH and middle cerebral artery occlusion (MCAO, and in vitro by organ culture of isolated cerebral arteries. The localization and amount of TNF-α, TNFreceptor 1 and 2 proteins were analysed by immunohistochemistry and western blot after 24 and 48 h of organ culture and at 48 h following SAH or MCAO. In addition, cerebral arteries were incubated for 24 or 48 h in the absence or presence of a B-Raf inhibitor (SB386023-b, a MEK- inhibitor (U0126 or an NF-κB inhibitor (IMD-0354, and protein expression evaluated. Results Immunohistochemistry revealed enhanced expression of TNF-α, TNF-R1 and TNF-R2 in the walls of cerebral arteries at 48 h after MCAO and SAH compared with control. Co-localization studies showed that TNF-α, TNF-R1 and TNF-R2 were primarily localized to the cell membrane and the cytoplasm of the smooth muscle cells (SMC. There was, in addition, some expression of TNF-R2 in the endothelial cells. Immunohistochemistry and western blot analysis showed that these proteins were upregulated after 24 and 48 h in culture, and this upregulation reached an apparent maximum at 48 h of organ culture. Treatment with U0126 significantly reduced the enhanced SMC expression of TNF-α, TNF-R1 and TNF-R2 immunoreactivities after 24 and 48 h of organ culture. The Raf and NF-κB inhibitors significantly reduced organ culture induced TNF-α expression

  12. Dynamic Arginine Methylation of Tumor Necrosis Factor (TNF) Receptor-associated Factor 6 Regulates Toll-like Receptor Signaling.

    Science.gov (United States)

    Tikhanovich, Irina; Kuravi, Sudhakiranmayi; Artigues, Antonio; Villar, Maria T; Dorko, Kenneth; Nawabi, Atta; Roberts, Benjamin; Weinman, Steven A

    2015-09-04

    Arginine methylation is a common post-translational modification, but its role in regulating protein function is poorly understood. This study demonstrates that, TNF receptor-associated factor 6 (TRAF6), an E3 ubiquitin ligase involved in innate immune signaling, is regulated by reversible arginine methylation in a range of primary and cultured cells. Under basal conditions, TRAF6 is methylated by the methyltransferase PRMT1, and this inhibits its ubiquitin ligase activity, reducing activation of toll-like receptor signaling. In response to toll-like receptor ligands, TRAF6 is demethylated by the Jumonji domain protein JMJD6. Demethylation is required for maximal activation of NF-κB. Loss of JMJD6 leads to reduced response, and loss of PRMT1 leads to basal pathway activation with subsequent desensitization to ligands. In human primary cells, variations in the PRMT1/JMJD6 ratio significantly correlate with TRAF6 methylation, basal activation of NF-κB, and magnitude of response to LPS. Reversible arginine methylation of TRAF6 by the opposing effects of PRMT1 and JMJD6 is, therefore, a novel mechanism for regulation of innate immune pathways. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Strong and selective glomerular localization of CD134 ligand and TNF receptor-1 in proliferative lupus nephritis

    NARCIS (Netherlands)

    Aten, J.; Roos, A.; Claessen, N.; Schilder-Tol, E. J.; ten Berge, I. J.; Weening, J. J.

    2000-01-01

    CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor (TNFR) family that can be expressed on activated T lymphocytes. Interaction between CD134 and its ligand (CD134L) is involved in costimulation of T and B lymphocyte activation, and in T cell adhesion to endothelium. To examine the

  14. Cloning of a cDNA encoding the human cation-dependent mannose 6-phosphate-specific receptor

    International Nuclear Information System (INIS)

    Pohlmann, R.; Nagel, G.; Schmidt, B.

    1987-01-01

    Complementary DNA clones for the human cation-dependent mannose 6-phosphate-specific receptor have been isolated from a human placenta library in λgt11. The nucleotide sequence of the 2463-base-pair cDNA insert includes a 145-base-pair 5' untranslated region, an open reading frame of 831 base pairs corresponding to 277 amino acids, and a 1487-base-pair 3' untranslated region. The deduced amino acid sequence is colinear with that determined by amino acid sequencing of the N-terminus peptide (41 residues) and nine tryptic peptides (93 additional residues). The receptor is synthesized as a precursor with a signal peptide of 20 amino acids. The hydrophobicity profile of the receptor indicates a single membrane-spanning domain, which separates an N-terminal region containing five potential N-glycosylation sites from a C-terminal region lacking N-glycosylation sites. Thus the N-terminal (M/sub r/ = 18,299) and C-terminal (M/sub r/ ≤ 7648) segments of the mature receptor are assumed to be exposed to the extracytosolic and cytosolic sides of the membrane, respectively. Analysis of a panel of somatic cell (mouse-human) hybrids shows that the gene for the receptor is located on human chromosome 12

  15. Receptor-Mediated and Lectin-Like Activities of Carp (Cyprinus carpio) TNF-¿

    NARCIS (Netherlands)

    Forlenza, M.; Magez, S.; Scharsack, J.P.; Westphal, A.H.; Savelkoul, H.F.J.; Wiegertjes, G.F.

    2009-01-01

    Functional characterization of TNF- in species other than mammalian vertebrates is limited, and TNF- has been studied in a limited number of fish species, primarily in vitro using recombinant proteins. Studies on TNF- from different fish species so far pointed to several inconsistencies, in

  16. Apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ.

    Directory of Open Access Journals (Sweden)

    Jiaojiao Chu

    Full Text Available Apelin, a novel adipokine, is the specific endogenous ligand of G protein-coupled receptor APJ. Consistent with its putative role as an adipokine, apelin has been linked to states of insulin resistance. However, the function of apelin in hepatic insulin resistance, a vital part of insulin resistance, and its underlying mechanisms still remains unclear. Here we define the impacts of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes. Our studies indicate that apelin reversed TNF-α-induced reduction of glycogen synthesis in HepG2 cells, mouse primary hepatocytes and liver tissues of C57BL/6J mice by improving JNK-IRS1-AKT-GSK pathway. Moreover, Western blot revealed that APJ, but not apelin, expressed in the hepatocytes and liver tissues of mice. We found that F13A, a competitive antagonist for G protein-coupled receptor APJ, suppressed the effects of apelin on TNF-α-induced reduction of glycogen synthesis in the hepatocytes, suggesting APJ is involved in the function of apelin. In conclusion, we show novel evidence suggesting that apelin ameliorates TNF-α-induced reduction of glycogen synthesis in the hepatocytes through G protein-coupled receptor APJ. Apelin appears as a beneficial adipokine with anti-insulin resistance properties, and thus as a promising therapeutic target in metabolic disorders.

  17. Analysis of TNF-related apoptosis-inducing ligand and receptors and implications in thymus biology and myasthenia gravis.

    Science.gov (United States)

    Kanatli, Irem; Akkaya, Bahar; Uysal, Hilmi; Kahraman, Sevim; Sanlioglu, Ahter Dilsad

    2017-02-01

    Myasthenia Gravis is an autoantibody-mediated, neuromuscular junction disease, and is usually associated with thymic abnormalities presented as thymic tumors (~10%) or hyperplastic thymus (~65%). The exact role of thymus in Myasthenia Gravis development is not clear, yet many patients benefit from thymectomy. The apoptotic ligand TNF-Related Apoptosis-Inducing Ligand is thought to be involved in the regulation of thymocyte counts, although conflicting results are reported. We investigated differential expression profiles of TNF-Related Apoptosis-Inducing Ligand and its transmembrane receptors, Nuclear Factor-kB activation status, and apoptotic cell counts in healthy thymic tissue and pathological thymus from Myasthenia Gravis patients. All tissues expressed TNF-Related Apoptosis-Inducing Ligand and its receptors, with hyperplastic tissue having the highest expression levels of death receptors DR4 and DR5. No detectable Nuclear Factor-kB activation, at least via the canonical Protein Kinase A-mediated p65 Ser276 phosphorylation, was evident in any of the tissues studied. Apoptotic cell counts were higher in MG-associated tissue compared to the normal thymus. Possible use of the TNF-Related Apoptosis-Inducing Ligand within the concept of an apoptotic ligand-mediated medical thymectomy in thymoma- or thymic hyperplasia-associated Myasthenia Gravis is also discussed. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Expression and function of TNF and IL-1 receptors on human regulatory T cells.

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    Frances Mercer

    2010-01-01

    Full Text Available Regulatory T cells (Tregs suppress immune activation and are critical in preventing autoimmune diseases. While the ability of Tregs to inhibit proliferation of other T cells is well established, it is not yet clear whether Tregs also modulate inflammatory cytokines during an immune response. Here, we show that the expression of inflammatory cytokine receptors IL-1R1 and TNFR2 were higher on resting mature Tregs compared to naïve or memory T cells. While upon activation through the T cell receptor (TCR, expression of IL-1R1 and TNFR2 were upregulated on all T cell subsets, IL-1R1 maintained significantly higher expression on activated Tregs as compared to other T cell subsets. The decoy receptor for IL-1 (IL-1R2 was not expressed by any of the resting T cells but was rapidly upregulated and preferentially expressed upon TCR-stimulation on Tregs. In addition, we found that Tregs also expressed high levels of mRNA for IL-1 antagonist, IL-1RA. TCR-stimulation of naïve T cells in the presence of TGFbeta, which induces FOXP3 expression, however did not result in upregulation of IL-1R1 or IL-1R2. In addition, ectopic expression of FOXP3 in non-Tregs, while causing significant upregulation of IL-1R1 and IL-1R2, did not achieve the levels seen in bona fide Tregs. We also determined that resting human Tregs expressing IL-1R1 did not have higher suppressive capacity compared to IL-1R1- Tregs, suggesting that IL-1R1 does not discriminate suppressive resting Tregs in healthy individuals. Functionally, activated human Tregs displayed a capacity to neutralize IL-1beta, which suggests a physiological significance for the expression of IL-1 decoy receptor on Tregs. In conclusion, our findings that human Tregs preferentially express receptors for TNF and IL-1 suggest a potential function in sensing and dampening local inflammation.

  19. Full-length cDNA cloning of Toll-like receptor 4 in dogs and cats.

    Science.gov (United States)

    Asahina, Yuka; Yoshioka, Noriyuki; Kano, Rui; Moritomo, Tadaaki; Hasegawa, Atsuhiko

    2003-12-15

    In the present study, full length of canine and feline Toll-like receptor 4 (TLR4) cDNAs were sequenced, and the expression of canine and feline TLR4 mRNAs in dog and cat tissues were investigated. The full-length cDNA of TLR4 of dog and cat was 2709 bp encoding 637 amino acids and 3113 bp encoding 833 amino acids, respectively. The similarity of canine and feline TLR4 were 83.6% at the nucleotide sequence level and 77.6% at the amino acid sequence level. At the amino acid sequence level, canine and feline TLR4 showed sequence similarities of approximately 62-78% with those of Homo sapiens, Mus musculus, Bos taurus and Equus caballus, respectively. Southern hybridization analyses with TLR4 cDNA probes gave one distinct band in BamHI, EcoRI and HindIII digests of genomic DNA from dogs and cats, respectively, indicating the likely presence of a single TLR4 gene in each species. By RT-PCR analysis, mRNA of canine TLR4 was expressed highly in peripheral blood leukocytes (PBL), moderately in spleen, stomach and small intestine, at low levels in liver, with no expression in kidney, large intestine and skin. On the other hand, mRNA of feline TLR4 was expressed highly in lung, bladder and PBL, moderately in kidney, liver, spleen and large intestine and at low levels in pancreas and small intestine.

  20. Durable Expression of Minicircle DNA-Liposome-Delivered Androgen Receptor cDNA in Mice with Hepatocellular Carcinoma

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    Tian-You Chang

    2014-01-01

    Full Text Available Background. The most common gene-based cancer therapies involve the suppression of oncogenic molecules and enhancement of the expression of tumor-suppressor genes. Studies in noncancer disease animal models have shown that minicircle (MC DNA vectors are easy to deliver and that the proteins from said MC-carrying DNA vectors are expressed over a long period of time. However, delivery of therapeutic genes via a liposome-mediated, MC DNA complex has never been tested in vascular-rich hepatocellular carcinoma (HCC. Liposome-mediated DNA delivery exhibits high in vivo transfection efficiency and minimal systemic immune response, thereby allowing for repetitive interventions. In this study, we evaluated the efficacy of delivering an MC-liposome vector containing a 3.2 kb androgen receptor (AR; HCC metastasis suppressor cDNA into Hepatitis B Virus- (HBV- induced HCC mouse livers. Results. Protein expression and promoter luciferase assays revealed that liposome-encapsulated MC-AR resulted in abundant functional expression of AR protein (100 kD for up to two weeks. The AR cDNA was also successfully delivered into normal livers and diseased livers, where it was persistently expressed. In both normal livers and livers with tumors, the expression of AR was detectable for up to 60 days. Conclusion. Our results show that an MC/liposome delivery system might improve the efficacy of gene therapy in patients with HCC.

  1. Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease.

    Science.gov (United States)

    Mulay, Shrikant R; Eberhard, Jonathan N; Desai, Jyaysi; Marschner, Julian A; Kumar, Santhosh V R; Weidenbusch, Marc; Grigorescu, Melissa; Lech, Maciej; Eltrich, Nuru; Müller, Lisa; Hans, Wolfgang; Hrabě de Angelis, Martin; Vielhauer, Volker; Hoppe, Bernd; Asplin, John; Burzlaff, Nicolai; Herrmann, Martin; Evan, Andrew; Anders, Hans-Joachim

    2017-03-01

    Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2- , and Tnfr1/2- deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2- , and Tnfr1/2 -deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo , and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria. Copyright © 2017 by the American Society of Nephrology.

  2. Experimental Disc Herniation in the Rat Causes Downregulation of Serotonin Receptor 2c in a TNF-dependent Manner.

    Science.gov (United States)

    Jonsson, Daniel; Finskas, Oscar; Fujioka, Yuki; Ståhlberg, Anders; Olmarker, Kjell

    2015-06-01

    During recent decades, the knowledge of the pathophysiology of disc herniation and sciatica has drastically improved. What previously was considered a strict biomechanical process is now considered a more complex interaction between leaked nucleus pulposus and the tissue in the spinal canal. An inflammatory reaction, with tumor necrosis factor (TNF) playing an essential role, has been demonstrated. However, the exact mechanisms of the pathophysiology of disc herniation remain unknown. In this study we use an animal model to investigate (1) if and/or how experimental disc herniation affects gene expression in the early phase (24 hours postsurgery) in the dorsal root ganglion; and (2) if TNF inhibition can reduce any observed changes. A rat model of disc herniation was used. Twenty rats were evenly divided into four groups: naïve, sham, disc herniation, and disc herniation with TNF inhibition. The dorsal root ganglion of the affected nerve root was harvested 24 hours after surgery and analyzed with a TaqMan Low Density Array(®) quantitative polymerase chain reaction assay. Gene expression levels in sham were compared with disc herniation to assess question 1 and disc herniation to disc herniation with TNF inhibition to assess question 2. Experimental disc herniation caused a decrease in the expression of the serotonin receptor 2c gene (p = 0.022). TNF inhibition was found to reduce the observed decrease in expression of serotonin receptor 2c (p = 0.037). Our results suggest that a decrease in the expression of the serotonin receptor 2c gene may contribute to the pathophysiology of disc herniation. Further research on its involvement is warranted. This pilot study gives a brief insight into cellular changes that may contribute to the pathophysiology of disc herniation. This knowledge may contribute to the development of more and better treatment options for patients with disc herniation and sciatica.

  3. Both functional LTbeta receptor and TNF receptor 2 are required for the development of experimental cerebral malaria.

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    Dieudonnée Togbe

    Full Text Available BACKGROUND: TNF-related lymphotoxin alpha (LTalpha is essential for the development of Plasmodium berghei ANKA (PbA-induced experimental cerebral malaria (ECM. The pathway involved has been attributed to TNFR2. Here we show a second arm of LTalpha-signaling essential for ECM development through LTbeta-R, receptor of LTalpha1beta2 heterotrimer. METHODOLOGY/PRINCIPAL FINDINGS: LTbetaR deficient mice did not develop the neurological signs seen in PbA induced ECM but died at three weeks with high parasitaemia and severe anemia like LTalphabeta deficient mice. Resistance of LTalphabeta or LTbetaR deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and angiography, associated with lack of microvascular obstruction, while wild-type mice developed distinct microvascular pathology. Recruitment and activation of perforin(+ CD8(+ T cells, and their ICAM-1 expression were clearly attenuated in the brain of resistant mice. An essential contribution of LIGHT, another LTbetaR ligand, could be excluded, as LIGHT deficient mice rapidly succumbed to ECM. CONCLUSIONS/SIGNIFICANCE: LTbetaR expressed on radioresistant resident stromal, probably endothelial cells, rather than hematopoietic cells, are essential for the development of ECM, as assessed by hematopoietic reconstitution experiment. Therefore, the data suggest that both functional LTbetaR and TNFR2 signaling are required and non-redundant for the development of microvascular pathology resulting in fatal ECM.

  4. Soluble TNF-alpha receptor 1 and IL-6 plasma levels in humans subjected to the sleep deprivation model of spaceflight

    Science.gov (United States)

    Shearer, W. T.; Reuben, J. M.; Mullington, J. M.; Price, N. J.; Lee, B. N.; Smith, E. O.; Szuba, M. P.; Van Dongen, H. P.; Dinges, D. F.

    2001-01-01

    BACKGROUND: The extent to which sleep loss may predispose astronauts to a state of altered immunity during extended space travel prompts evaluation with ground-based models. OBJECTIVE: We sought to measure plasma levels of selected cytokines and their receptors, including the putative sleep-regulation proteins soluble TNF-alpha receptor (sTNF-alpha R) I and IL-6, in human subjects undergoing 2 types of sleep deprivation during environmental confinement with performance demands. METHODS: Healthy adult men (n = 42) were randomized to schedules that varied in severity of sleep loss: 4 days (88 hours) of partial sleep deprivation (PSD) involving two 2-hour naps per day or 4 days of total sleep deprivation (TSD). Plasma samples were obtained every 6 hours across 5 days and analyzed by using enzyme-linked immunoassays for sTNF-alpha RI, sTNF-alpha RII, IL-6, soluble IL-2 receptor, IL-10, and TNF-alpha. RESULTS: Interactions between the effects of time and sleep deprivation level were detected for sTNF-alpha RI and IL-6 but not for sTNF-alpha RII, soluble IL-2 receptor, IL-10, and TNF-alpha. Relative to the PSD condition, subjects in the TSD condition had elevated plasma levels of sTNF-alpha RI on day 2 (P =.04), day 3 (P =.01), and across days 2 to 4 of sleep loss (P =.01) and elevated levels of IL-6 on day 4 (P =.04). CONCLUSIONS: Total sleep loss produced significant increases in plasma levels of sTNF-alpha RI and IL-6, messengers that connect the nervous, endocrine, and immune systems. These changes appeared to reflect elevations of the homeostatic drive for sleep because they occurred in TSD but not PSD, suggesting that naps may serve as the basis for a countermeasures approach to prolonged spaceflight.

  5. Role of B61, the Ligand for the Eck Receptor Tyrosine Kinase, in TNF- α-Induced Angiogenesis

    Science.gov (United States)

    Pandey, Akhilesh; Shao, Haining; Marks, Rory M.; Polverini, Peter J.; Dixit, Vishva M.

    1995-04-01

    B61, a cytokine-inducible endothelial gene product, is the ligand for the Eck receptor protein tyrosine kinase (RPTK). Expression of a B61-immunoglobulin chimera showed that B61 could act as an angiogenic factor in vivo and a chemoattractant for endothelial cells in vitro. The Eck RPTK was activated by tumor necrosis factor-α (TNF-α) through induction of B61, and an antibody to B61 attenuated angiogenesis induced by TNF-α but not by basic fibroblast growth factor. This finding suggests the existence of an autocrine or paracrine loop involving activation of the Eck RPTK by its inducible ligand B61 after an inflammatory stimulus, the net effect of which would be to promote angiogenesis, a hallmark of chronic inflammation.

  6. Molecular cloning and expression of a cDNA encoding a hybrid histidine kinase receptor in tropical periwinkle Catharanthus roseus.

    Science.gov (United States)

    Papon, N; Bremer, J; Vansiri, A; Glévarec, G; Rideau, M; Creche, J

    2006-09-01

    Signalling pathways involving histidine kinase receptors (HKRs) are widely used by prokaryotes and fungi to regulate a large palette of biological processes. In plants, HKRs are known to be implicated in cytokinin, ethylene, and osmosensing transduction pathways. In this work, a full length cDNA named CRCIK was isolated from the tropical species CATHARANTHUS ROSEUS (L.) G. Don. It encodes a 1205 amino acid protein that belongs to the hybrid HKR family. The deduced amino acid sequence shows the highest homology with AtHK1, an osmosensing HKR in ARABIDOPSIS THALIANA. In return, CrCIK protein shares very low identity with the other 10 ARABIDOPSIS HKRs. Southern blot analysis indicates that the CRCIK corresponding gene is either present in multiple copies or has very close homologues in the genome of the tropical periwinkle. The gene is widely expressed in the plant. In C. ROSEUS C20D cell suspension, it is slightly induced after exposure to low temperature, pointing to a putative role in cold-shock signal transduction.

  7. Direct bone formation during distraction osteogenesis does not require TNF alpha receptors and elevated serum TNF alpha fails to inhibit bone formation in TNFR1 deficient mice

    Science.gov (United States)

    Distraction osteogenesis (DO) is a process which induces direct new bone formation as a result of mechanical distraction. Tumor necrosis factor-alpha (TNF) is a cytokine that can modulate osteoblastogenesis. The direct effects of TNF on direct bone formation in rodents are hypothetically mediated th...

  8. Early and exudative age-related macular degeneration is associated with increased plasma levels of soluble TNF receptor II

    DEFF Research Database (Denmark)

    Faber, Carsten; Jehs, Tina; Juel, Helene Baek

    2015-01-01

    techniques. Plasma was isolated from freshly drawn peripheral venous blood samples and analysed for interleukin (IL)15, IL18, interferon (IFN)γ, soluble tumour necrosis factor (TNF) receptor II (sTNFRII) and complement factor H (CFH) Y402H genotype. RESULTS: We included 136 individuals with early or late...... at 3.0 in the middle and 3.6 in the highest tertiles. Levels of IL15, IL18 and IFNγ were low and not associated with AMD. CONCLUSIONS: Increased plasma level of sTNFRII is found to be associated with AMD. The data supports the observations of low-grade, systemic inflammatory alterations in patients...

  9. Transmembrane Tumor Necrosis Factor Controls Myeloid-Derived Suppressor Cell Activity via TNF Receptor 2 and Protects from Excessive Inflammation during BCG-Induced Pleurisy

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    Leslie Chavez-Galan

    2017-08-01

    Full Text Available Pleural tuberculosis (TB is a form of extra-pulmonary TB observed in patients infected with Mycobacterium tuberculosis. Accumulation of myeloid-derived suppressor cells (MDSC has been observed in animal models of TB and in human patients but their role remains to be fully elucidated. In this study, we analyzed the role of transmembrane TNF (tmTNF in the accumulation and function of MDSC in the pleural cavity during an acute mycobacterial infection. Mycobacterium bovis BCG-induced pleurisy was resolved in mice expressing tmTNF, but lethal in the absence of tumor necrosis factor. Pleural infection induced MDSC accumulation in the pleural cavity and functional MDSC required tmTNF to suppress T cells as did pleural wild-type MDSC. Interaction of MDSC expressing tmTNF with CD4 T cells bearing TNF receptor 2 (TNFR2, but not TNFR1, was required for MDSC suppressive activity on CD4 T cells. Expression of tmTNF attenuated Th1 cell-mediated inflammatory responses generated by the acute pleural mycobacterial infection in association with effective MDSC expressing tmTNF and interacting with CD4 T cells expressing TNFR2. In conclusion, this study provides new insights into the crucial role played by the tmTNF/TNFR2 pathway in MDSC suppressive activity required during acute pleural infection to attenuate excessive inflammation generated by the infection.

  10. Role of tumour necrosis factor receptor-1 and nuclear factor-κB in production of TNF-α-induced pro-inflammatory microparticles in endothelial cells.

    Science.gov (United States)

    Lee, S K; Yang, S-H; Kwon, I; Lee, O-H; Heo, J H

    2014-09-02

    Tumour necrosis factor-α (TNF-α) is upregulated in many inflammatory diseases and is also a potent agent for microparticle (MP) generation. Here, we describe an essential role of TNF-α in the production of endothelial cell-derived microparticles (EMPs) in vivo and the function of TNF-α-induced EMPs in endothelial cells. We found that TNF-α rapidly increased blood levels of EMPs in mice. Treatment of human umbilical vein endothelial cells (HUVECs) with TNF-α also induced EMP formation in a time-dependent manner. Silencing of TNF receptor (TNFR)-1 or inhibition of the nuclear factor-κB (NF-κB) in HUVECs impaired the production of TNF-α-induced EMP. Incubation of HUVECs with PKH-67-stained EMPs showed that endothelial cells readily engulfed EMPs, and the engulfed TNF-α-induced EMPs promoted the expression of pro-apoptotic molecules and upregulated intercellular adhesion molecule-1 level on the cell surface, which led to monocyte adhesion. Collectively, our findings indicate that the generation of TNF-α-induced EMPs was mediated by TNFR1 or NF-κB and that EMPs can contribute to apoptosis and inflammation of endothelial cells.

  11. Histamine H4 Receptor mediates interleukin-8 and TNF-α release in human mast cells via multiple signaling pathways.

    Science.gov (United States)

    Chen, X-F; Zhang, Z; Dou, X; Li, J-J; Zhang, W; Yu, Y-Y; Yu, B; Yu, B

    2016-01-27

    Histamine, mainly produced by mast cells, is an important inflammatory mediator in immune response. Recently Histamine H4 Receptor (H4R) was also identified in mast cells, from which pro-inflammatory cytokines and chemokines are released. However, the mechanism of how H4R mediates these cytokines and chemokines release in mast cells was still unclear. To further explore the role of H4R in the immune inflammatory response in mast cells, we tested the release of inflammatory cytokine tumor necrosis factor-α (TNF-α), chemokine interleukin-8 (IL-8) and the relevant signaling pathways activated by H4R on LAD2 cells (a human mast cell line). We found that the release of IL-8 and TNF-α were blocked by inhibitors of PI3K, ERK and Ca2+-Calcineurin-NFAT signaling pathways, while the release of these cytokines and chemokines were enhanced by the inhibitor of P38 signaling pathway. However, inhibitors of the JNK and NF-κB signaling pathways had little effect on the expression of the pro-inflammatory mediators. Moreover, activation of the H4R could induce phosphorylation of ERK, p38 and AKT in mast cells. In conclusion, we found that H4R mediates the release of inflammatory cytokine TNF-α and chemokine IL-8 in human mast cells via PI3K, Ca2+-Calcineurin-NFAT and MAPKs signaling pathways.

  12. Tumor Necrosis Factor (TNF) Receptor Superfamily Member 1b on CD8+ T Cells and TNF Receptor Superfamily Member 1a on Non-CD8+ T Cells Contribute Significantly to Upper Genital Tract Pathology Following Chlamydial Infection.

    Science.gov (United States)

    Manam, Srikanth; Thomas, Joshua D; Li, Weidang; Maladore, Allison; Schripsema, Justin H; Ramsey, Kyle H; Murthy, Ashlesh K

    2015-06-15

    We demonstrated previously that tumor necrosis factor α (TNF-α)-producing Chlamydia-specific CD8(+) T cells cause oviduct pathological sequelae. In the current study, we used wild-type C57BL/6J (WT) mice with a deficiency in genes encoding TNF receptor superfamily member 1a (TNFR1; TNFR1 knockout [KO] mice), TNF receptor superfamily member 1b (TNFR2; TNFR2 KO mice), and both TNFR1 and TNFR2 (TNFR1/2 double KO [DKO] mice) and mix-match adoptive transfers of CD8(+) T cells to study chlamydial pathogenesis. TNFR1 KO, TNFR2 KO, and TNFR1/2 DKO mice displayed comparable clearance of primary or secondary genital Chlamydia muridarum infection but significantly reduced oviduct pathology, compared with WT animals. The Chlamydia-specific total cellular cytokine response in splenic and draining lymph nodes and the antibody response in serum were comparable between the WT and KO animals. However, CD8(+) T cells from TNFR2 KO mice displayed significantly reduced activation (CD11a expression and cytokine production), compared with TNFR1 KO or WT animals. Repletion of TNFR2 KO mice with WT CD8(+) T cells but not with TNFR2 KO CD8(+) T cells and repletion of TNFR1 KO mice with either WT or TNFR1 KO CD8(+) T cells restored oviduct pathology to WT levels in both KO groups. Collectively, these results demonstrate that TNFR2-bearing CD8(+) T cells and TNFR1-bearing non-CD8(+) T cells contribute significantly to oviduct pathology following genital chlamydial infection. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  13. Beta-adrenergic receptor 1 selective antagonism inhibits norepinephrine-mediated TNF-alpha downregulation in experimental liver cirrhosis.

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    Pedro Zapater

    Full Text Available BACKGROUND: Bacterial translocation is a frequent event in cirrhosis leading to an increased inflammatory response. Splanchnic adrenergic system hyperactivation has been related with increased bacterial translocation. We aim at evaluating the interacting mechanism between hepatic norepinephrine and inflammation during liver damage in the presence of bacterial-DNA. ANIMALS AND METHODS: Forty-six mice were included in a 16-week protocol of CCl(4-induced cirrhosis. Laparotomies were performed at weeks 6, 10, 13 and 16. A second set of forty mice injected with a single intraperitoneal dose of CCl(4 was treated with saline, 6-hydroxidopamine, Nebivolol or Butoxamine. After 5 days, mice received E. coli-DNA intraperitoneally. Laparotomies were performed 24 hours later. Liver bacterial-DNA, norepinephrine, TNF-alpha, IL-6 and beta-adrenergic receptor levels were measured. RESULTS: Bacterial-DNA translocation was more frequent in CCl(4-treated animals compared with controls, and increased as fibrosis progressed. Liver norepinephrine and pro-inflammatory cytokines were significantly higher in mice with vs without bacterial-DNA (319.7 ± 120.6 vs 120.7 ± 68.6 pg/g for norepinephrine, 38.4 ± 6.1 vs 29.7 ± 4.2 pg/g for TNF-alpha, 41.8 ± 7.4 vs 28.7 ± 4.3 pg/g for IL-6. Only beta-adrenergic receptor-1 was significantly increased in treated vs control animals (34.6 ± 7.3 vs 12.5 ± 5.3, p=0.01 and correlated with TNF-alpha, IL-6 and norepinephrine hepatic levels in animals with bacterial-DNA. In the second set of mice, cytokine levels were increased in 6-hydroxidopamine and Nebivolol (beta-adrenergic receptor-1 antagonist treated mice compared with saline. Butoxamine (beta-adrenergic receptor-2 antagonist didn't inhibit liver norepinephrine modulation of pro-inflammatory cytokines. CONCLUSIONS: Beta-adrenergic receptor-1 mediates liver norepinephrine modulation of the pro-inflammatory response in CCl(4-treated mice with bacterial-DNA.

  14. Tumor necrosis factor (TNF) receptor shedding controls thresholds of innate immune activation that balance opposing TNF functions in infectious and inflammatory diseases

    DEFF Research Database (Denmark)

    Xanthoulea, Sofia; Pasparakis, Manolis; Kousteni, Stavroula

    2004-01-01

    Tumor necrosis factor (TNF) is a potent cytokine exerting critical functions in the activation and regulation of immune and inflammatory responses. Due to its pleiotropic activities, the amplitude and duration of TNF function must be tightly regulated. One of the mechanisms that may have evolved ...... shedding as a mechanism setting up thresholds of cytokine function to balance resistance and susceptibility to disease. Assessment of p55TNFR shedding may thus be of prognostic value in infectious, inflammatory, and autoimmune diseases....

  15. Different expression of PGE synthase, PGF receptor, TNF, Fas and oxytocin in the bovine corpus luteum of the estrous cycle and pregnancy.

    Science.gov (United States)

    Sakumoto, R; Hayashi, K-G; Takahashi, T

    2014-04-01

    Functional differences between the corpus luteum (CL) of pregnancy and CL of the cycle in cows were examined. Messenger RNA and protein levels of prostaglandin (PG) E synthase (PGES), PGF2α receptor (PGFR), tumor necrosis factor-α (TNF) and Fas were found to be higher in the CL of pregnancy than in CL of the cycle. Oxytocin (OT) mRNA and protein levels were lower in the CL of pregnancy. Messenger RNA levels of progesterone receptor (PR), luteinizing hormone receptor (LHR), PGE2 receptor (PGER), PGF synthase (PGFS), TNF receptor type I (TNFRI) and TNF receptor type II (TNFRII) did not differ between the cycle and pregnancy. PGE2 and PGF2α production by cultured bovine endometrial tissues was decreased by a supernatant derived from the homogenized CL of pregnancy but not by that of the CL of the cycle, suggesting that specific substances in the CL of pregnancy affect endometrial PG production in cows. Collectively, PGES, PGFR, TNF, Fas or OT may contribute to differences between the CL of pregnancy and CL of the estrous cycle in cows. Copyright © 2014 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  16. Construction and characterization of a cDNA library from head kidney of Japanese sea bass (Lateolabrax japonicus).

    Science.gov (United States)

    Shao, Zhan-tao; Cong, Xiao; Yuan, Jin-duo; Yang, Gui-wen; Chen, Ying; Pan, Jie; An, Li-guo

    2009-09-01

    In this paper, a cDNA expression library from head kidney of Japanese sea bass (Lateolabrax japonicus) was constructed for the first time. The first-strand cDNA was synthesized with Moloney Murine Leukaemia virus reverse transcriptase and the double-stranded cDNA was digested by Xho I enzyme. Size fractionation was performed on CHROMA SPIN-400 columns. cDNA fragments longer than 500 bps were ligated into the lambdaZAPExpress vector. The recombinant DNA was packaged in vitro with Gigapack III gold packaging extract. The titers of the primary and amplified library were 1.0 x 10(5) and 5.0 x 10(9) pfu/ml, respectively. To characterize the constructed cDNA library, 15 phage plaques were selected randomly to test the inserted fragments. The results showed that the inserts were mostly longer than 500 bps. To test the utility, the library was screened with primers designed for three immune-related genes of, Myxovirus resistant (Mx), tumor necrosis factor-alpha (TNF-alpha) and Toll-like receptor (TLR). Results of Blastn and alignment showed that they are members of Mx, TNF-alpha and TLR gene families, respectively, which meets our anticipates for this cDNA library as an immune-related one. These results confirmed that the cDNA library constructed will provide a useful tool for gene cloning and expression analysis in immune system of Japanese sea bass.

  17. Retinopathy and nephropathy in type 1 diabetic patients--association with polymorphysms of vitamin D-receptor, TNF, Neuro-D and IL-1 receptor 1 genes.

    Science.gov (United States)

    Bućan, Kajo; Ivanisević, Milan; Zemunik, Tatijana; Boraska, Vesna; Skrabić, Veselin; Vatavuk, Zoran; Galetović, Davor; Znaor, Ljubo

    2009-12-01

    Retinopathy and nephropathy are common late type 1 diabetes mellitus (T1D) complications. In this study we investigated whether individual differences in 4 candidate genes significantly contribute to development and progression of late complications in T1D patients. We examined 121 patients for the presence of diabetic retinopathy and nephropathy. We genotyped variants in vitamin D receptor (VDR) and tumor necrosis factor (TNF) genes in 47 patients and in NeuroD1 and interleukin-1 receptor 1 (IL1R1) genes in 35 patients. Diabetic retinopathy had 66 (55%) patients after a median of 13.0 years after diagnosis. Diabetic nephropathy had 14 (11.66%) patients, all of whom had already developed retinopathy. A significant correlation between the degree of diabetic retinopathy and mean microalbuminuria (MA) value has been found (chi2 = 54.18, p diabetic retinopathy, while no investigated genetic polymorphysms could reliably predict diabetic nephropathy.

  18. Genetic Variations in Pattern Recognition Receptor Loci Are Associated with Anti-TNF Response in Patients with Rheumatoid Arthritis.

    Directory of Open Access Journals (Sweden)

    Jacob Sode

    Full Text Available To determine whether genetic variation within genes related to the Toll-like receptor, inflammasome and interferon-γ pathways contributes to the differences in treatment response to tumour necrosis factor inhibitors (anti-TNF in patients with rheumatoid arthritis (RA.In a retrospective case-case study, we assessed 23 functional single nucleotide polymorphisms (SNPs in 15 genes. We included 538 anti-TNF naïve Danish RA patients from the nationwide DANBIO database. Multivariable logistic regression analyses were performed to detect associations (p-value<0.05 between genotypes and European League Against Rheumatism (EULAR treatment responses. False Discovery Rate corrections for multiple testing (q-value and stratified analyses were performed to investigate association with individual therapies and IgM-rheumatoid factor (RF status.Six of twenty successfully genotyped polymorphisms were nominally associated with EULAR treatment response. Three of these were in weak to moderate linkage disequilibrium with polymorphisms previously reported associated with anti-TNF treatment response. TLR5(rs5744174 variant allele carriers (odds ratio(OR = 1.7(1.1-2.5,p = 0.010,q = 0.46 and TLR1(rs4833095 homozygous variant carriers (OR = 2.8(1.1-7.4,p = 0.037,q = 0.46 had higher odds for a positive treatment response. NLRP3(rs10754558 variant allele carriers (odds ratio(OR = 0.6(0.4-1.0,p = 0.045,q = 0.46 were more likely to have a negative treatment response. The association in TLR5(rs5744174 remained significant after correction for multiple comparisons among patients negative for RF (OR = 6.2(2.4-16.3,p = 0.0002,q = 0.024. No other association withstood correction for multiple testing. Post hoc analyses showed that change in Patient Global score on a visual analogue scale (VAS and change in pain VAS were the main factors responsible for the association.We reproduced previously reported associations between genetic variation in the TLR10/1/6 gene cluster, TLR5

  19. Liganded thyroid hormone receptor inhibits phorbol 12-O-tetradecanoate-13-acetate-induced enhancer activity via firefly luciferase cDNA.

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    Hiroko Misawa

    Full Text Available Thyroid hormone receptor (TR belongs to the nuclear hormone receptor (NHR superfamily and regulates the transcription of its target genes in a thyroid hormone (T3-dependent manner. While the detail of transcriptional activation by T3 (positive regulation has been clarified, the mechanism of T3-dependent repression (negative regulation remains to be determined. In addition to naturally occurring negative regulations typically found for the thyrotropin β gene, T3-bound TR (T3/TR is known to cause artificial negative regulation in reporter assays with cultured cells. For example, T3/TR inhibits the transcriptional activity of the reporter plasmids harboring AP-1 site derived from pUC/pBR322-related plasmid (pUC/AP-1. Artificial negative regulation has also been suggested in the reporter assay with firefly luciferase (FFL gene. However, identification of the DNA sequence of the FFL gene using deletion analysis was not performed because negative regulation was evaluated by measuring the enzymatic activity of FFL protein. Thus, there remains the possibility that the inhibition by T3 is mediated via a DNA sequence other than FFL cDNA, for instance, pUC/AP-1 site in plasmid backbone. To investigate the function of FFL cDNA as a transcriptional regulatory sequence, we generated pBL-FFL-CAT5 by ligating FFL cDNA in the 5' upstream region to heterologous thymidine kinase promoter in pBL-CAT5, a chloramphenicol acetyl transferase (CAT-based reporter gene, which lacks pUC/AP-1 site. In kidney-derived CV1 and choriocarcinoma-derived JEG3 cells, pBL-FFL-CAT5, but not pBL-CAT5, was strongly activated by a protein kinase C activator, phorbol 12-O-tetradecanoate-13-acetate (TPA. TPA-induced activity of pBL-FFL-CAT5 was negatively regulated by T3/TR. Mutation of nt. 626/640 in FFL cDNA attenuated the TPA-induced activation and concomitantly abolished the T3-dependent repression. Our data demonstrate that FFL cDNA sequence mediates the TPA-induced transcriptional

  20. The Glucagon-Like Peptide-1 Receptor Agonist Exendin-4 Inhibits Lipopolysaccharide-Induced Osteoclast Formation and Bone Resorption via Inhibition of TNF-α Expression in Macrophages

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    Wei-Ren Shen

    2018-01-01

    Full Text Available Glucagon-like peptide-1 (GLP-1 receptor agonists are an effective treatment approach for type 2 diabetes. Recently, anti-inflammatory effects of GLP-1 receptor agonists have also been reported. Lipopolysaccharide (LPS induces inflammation and osteoclast formation. In this study, we investigated the effect of exendin-4, a widely used GLP-1 receptor agonist, in LPS-induced osteoclast formation and bone resorption. LPS with or without exendin-4 was administered on mouse calvariae by daily subcutaneous injection. The number of osteoclasts, the ratio of bone resorption pits, and the level of C-terminal cross-linked telopeptide of type I collagen (CTX were significantly lower in LPS- and exendin-4-coadministered mice than in mice administered with LPS alone. RANKL and TNF-α mRNA expression levels were lower in the exendin-4- and LPS-coadministered group than in the LPS-administered group. Our in vitro results showed no direct effects of exendin-4 on RANKL-induced osteoclast formation, TNF-α-induced osteoclast formation, or LPS-induced RANKL expression in stromal cells. Conversely, TNF-α mRNA expression was inhibited in the exendin-4- and LPS-cotreated macrophages compared with cells treated with LPS alone. These results indicate that the GLP-1 receptor agonist exendin-4 may inhibit LPS-induced osteoclast formation and bone resorption by inhibiting LPS-induced TNF-α production in macrophages.

  1. TNFreceptor 1 knockdown in the subfornical organ ameliorates sympathetic excitation and cardiac hemodynamics in heart failure rats.

    Science.gov (United States)

    Yu, Yang; Wei, Shun-Guang; Weiss, Robert M; Felder, Robert B

    2017-10-01

    In systolic heart failure (HF), circulating proinflammatory cytokines upregulate inflammation and renin-angiotensin system (RAS) activity in cardiovascular regions of the brain, contributing to sympathetic excitation and cardiac dysfunction. Important among these is the subfornical organ (SFO), a forebrain circumventricular organ that lacks an effective blood-brain barrier and senses circulating humors. We hypothesized that the tumor necrosis factor-α (TNF-α) receptor 1 (TNFR1) in the SFO contributes to sympathetic excitation and cardiac dysfunction in HF rats. Rats received SFO microinjections of a TNFR1 shRNA or a scrambled shRNA lentiviral vector carrying green fluorescent protein, or vehicle. One week later, some rats were euthanized to confirm the accuracy of the SFO microinjections and the transfection potential of the lentiviral vector. Other rats underwent coronary artery ligation (CL) to induce HF or a sham operation. Four weeks after CL, vehicle- and scrambled shRNA-treated HF rats had significant increases in TNFR1 mRNA and protein, NF-κB activity, and mRNA for inflammatory mediators, RAS components and c-Fos protein in the SFO and downstream in the hypothalamic paraventricular nucleus, along with increased plasma norepinephrine levels and impaired cardiac function, compared with vehicle-treated sham-operated rats. In HF rats treated with TNFR1 shRNA, TNFR1 was reduced in the SFO but not paraventricular nucleus, and the central and peripheral manifestations of HF were ameliorated. In sham-operated rats treated with TNFR1 shRNA, TNFR1 expression was also reduced in the SFO but there were no other effects. These results suggest a key role for TNFR1 in the SFO in the pathophysiology of systolic HF. NEW & NOTEWORTHY Activation of TNFreceptor 1 in the subfornical organ (SFO) contributes to sympathetic excitation in heart failure rats by increasing inflammation and renin-angiotensin system activity in the SFO and downstream in the hypothalamic

  2. Inhibition of Epithelial TNFReceptors by Purified Fruit Bromelain Ameliorates Intestinal Inflammation and Barrier Dysfunction in Colitis.

    Science.gov (United States)

    Zhou, Zijuan; Wang, Liang; Feng, Panpan; Yin, Lianhong; Wang, Chen; Zhi, Shengxu; Dong, Jianyi; Wang, Jingyu; Lin, Yuan; Chen, Dapeng; Xiong, Yongjian; Peng, Jinyong

    2017-01-01

    Activation of the TNFreceptor (TNFR) leads to an inflammatory response, and anti-TNF therapy has been administered to reduce inflammation symptoms and heal mucosal ulcers in inflammatory bowel disease (IBD). Bromelain, a complex natural mixture of proteolytic enzymes, has been shown to exert anti-inflammatory effects. This study aimed to investigate the effect of purified fruit bromelain (PFB)-induced inhibition of epithelial TNFR in a rat colitis model. Colitis was established by intracolonic administration of 2, 4, 6-trinitrobenzene sulfonic acid. Expression of TNFR1 and TNFR2 was measured by quantitative RT-PCR and western blotting. The effect of PFB on colitis was evaluated by examining the inflammatory response and intestinal epithelial barrier function. Our results showed that both TNFR1 and TNFR2 expression were significantly increased in a colitis model, and the increase was significantly reversed by PFB. Colitis symptoms, including infiltration of inflammatory cells, cytokine profiles, epithelial cell apoptosis, and epithelial tight junction barrier dysfunction were significantly ameliorated by PFB. Compared with fruit bromelain and stem bromelain complex, the inhibition of TNFR2 induced by PFB was stronger than that exhibited on TNFR1. These results indicate that PFB showed a stronger selective inhibitory effect on TNFR2 than TNFR1. In other words, purification of fruit bromelain increases its selectivity on TNFR2 inhibition. High expression of epithelial TNFRs in colitis was significantly counteracted by PFB, and PFB-induced TNFR inhibition ameliorated colitis symptoms. These results supply novel insights into potential IBD treatment by PFB.

  3. Gene Expression of Tumor Necrosis Factor α and TNFReceptors (p55 and p75) in Nonalcoholic Steatohepatitis Patients

    International Nuclear Information System (INIS)

    Soliman, S.ET.; Abo-Madyan, A.A.

    2010-01-01

    The main objective of this study was to analyze the pathogenic role of the tumor necrosis factor (TNF-) system in the development of nonalcoholic steatohepatitis (NASH). Fifty obese patients were studied. We investigated: 1) the expression of mRNA of TNF- and their p55 and p75 receptors by quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) in hepatic and adipose tissues; and 2) the relationship between TNF-, p55, and p75 and the severity of NASH. Obese patients without NASH were the control group. A remarkable increase in the expression of mRNA of TNF- was found in patients with NASH in hepatic tissue (0.65 ± 0.54) and in peripheral fat (0.43 ± 0.45); in the control samples, the mRNA expression was 0.30 ± 0.32, P < .006, and 0.28 ± 0.22, P < .016, respectively. Furthermore, we found significant increase in the mRNA levels of p55 receptor (2.94 ± 1.71 vs. 1.46 ± 1.27; P<.04); however, the mRNA expression of the p75 receptor was similar in both patients. Those patients with NASH with significant fibrosis presented an increase in the expression of mRNA TNF- in comparison with those with slight or nonexistent fibrosis. The levels of mRNA-p55 are increased in the liver tissue of NASH patients. This over expression is more elevated in patients with more advanced NASH. These findings suggest that the TNF- system may be involved in the pathogenesis of NASH.

  4. Transmembrane TNF-dependent uptake of anti-TNF antibodies.

    Science.gov (United States)

    Deora, Arun; Hegde, Subramanya; Lee, Jacqueline; Choi, Chee-Ho; Chang, Qing; Lee, Cheryl; Eaton, Lucia; Tang, Hua; Wang, Dongdong; Lee, David; Michalak, Mark; Tomlinson, Medha; Tao, Qingfeng; Gaur, Nidhi; Harvey, Bohdan; McLoughlin, Shaun; Labkovsky, Boris; Ghayur, Tariq

    TNF-α (TNF), a pro-inflammatory cytokine is synthesized as a 26 kDa protein, anchors in the plasma membrane as transmembrane TNF (TmTNF), and is subjected to proteolysis by the TNF-α converting enzyme (TACE) to release the 15 kDa form of soluble TNF (sTNF). TmTNF and sTNF interact with 2 distinct receptors, TNF-R1 (p55) and TNF-R2 (p75), to mediate the multiple biologic effects of TNF described to date. Several anti-TNF biologics that bind to both forms of TNF and block their interactions with the TNF receptors are now approved for the treatment of a variety of immune-mediated diseases. Several reports suggest that binding of anti-TNFs to TmTNF delivers an outside-to-inside 'reverse' signal that may also contribute to the efficacy of anti-TNFs. Some patients, however, develop anti-TNF drug antibody responses (ADA or immunogenicity). Here, we demonstrate biochemically that TmTNF is transiently expressed on the surface of lipopolysaccharide-stimulated primary human monocytes, macrophages, and monocyte-derived dendritic cells (DCs) and expression of TmTNF on the cell surface is enhanced following treatment of cells with TAPI-2, a TACE inhibitor. Importantly, binding of anti-TNFs to TmTNF on DCs results in rapid internalization of the anti-TNF/TmTNF complex first into early endosomes and then lysosomes. The internalized anti-TNF is processed and anti-TNF peptides can be eluted from the surface of DCs. Finally, tetanus toxin peptides fused to anti-TNFs are presented by DCs to initiate T cell recall proliferation response. Collectively, these observations may provide new insights into understanding the biology of TmTNF, mode of action of anti-TNFs, biology of ADA response to anti-TNFs, and may help with the design of the next generation of anti-TNFs.

  5. TNF-α triggers rapid membrane insertion of Ca(2+) permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury.

    Science.gov (United States)

    Yin, Hong Z; Hsu, Cheng-I; Yu, Stephen; Rao, Shyam D; Sorkin, Linda S; Weiss, John H

    2012-12-01

    Excitotoxicity (caused by over-activation of glutamate receptors) and inflammation both contribute to motor neuron (MN) damage in amyotrophic lateral sclerosis (ALS) and other diseases of the spinal cord. Microglial and astrocytic activation in these conditions results in release of inflammatory mediators, including the cytokine, tumor necrosis factor-alpha (TNF-α). TNF-α has complex effects on neurons, one of which is to trigger rapid membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, and in some cases, specific insertion of GluA2 lacking, Ca(2+) permeable AMPA receptors (Ca-perm AMPAr). In the present study, we use a histochemical stain based upon kainate stimulated uptake of cobalt ions ("Co(2+) labeling") to provide the first direct demonstration of the presence of substantial numbers of Ca-perm AMPAr in ventral horn MNs of adult rats under basal conditions. We further find that TNF-α exposure causes a rapid increase in the numbers of these receptors, via a phosphatidylinositol 3 kinase (PI3K) and protein kinase A (PKA) dependent mechanism. Finally, to assess the relevance of TNF-α to slow excitotoxic MN injury, we made use of organotypic spinal cord slice cultures. Co(2+) labeling revealed that MNs in these cultures possess Ca-perm AMPAr. Addition of either a low level of TNF-α, or of the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to the cultures for 48 h resulted in little MN injury. However, when combined, TNF-α+PDC caused considerable MN degeneration, which was blocked by the AMPA/kainate receptor blocker, 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), or the Ca-perm AMPAr selective blocker, 1-naphthyl acetylspermine (NASPM). Thus, these data support the idea that prolonged TNF-α elevation, as may be induced by glial activation, acts in part by increasing the numbers of Ca-perm AMPAr on MNs to enhance injurious excitotoxic effects of deficient

  6. TNF-α triggers rapid membrane insertion of Ca2+ permeable AMPA receptors into adult motor neurons and enhances their susceptibility to slow excitotoxic injury

    Science.gov (United States)

    Yin, Hong Z.; Hsu, Cheng-I; Yu, Stephen; Rao, Shyam D.; Sorkin, Linda S.; Weiss, John H.

    2012-01-01

    Excitotoxicity (caused by over-activation of glutamate receptors) and inflammation both contribute to motor neuron (MN) damage in amyotrophic lateral sclerosis (ALS) and other diseases of the spinal cord. Microglial and astrocytic activation in these conditions results in release of inflammatory mediators, including the cytokine, tumor necrosis factor–alpha (TNF-α). TNF-α has complex effects on neurons, one of which is to trigger rapid membrane insertion of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, and in some cases, specific insertion of GluA2 lacking, Ca2+ permeable AMPA receptors (Ca-perm AMPAr). In the present study, we use a histochemical stain based upon kainate stimulated uptake of cobalt ions (“Co2+ labeling”) to provide the first direct demonstration of the presence of substantial numbers of Ca-perm AMPAr in ventral horn MNs of adult rats under basal conditions. We further find that TNF-α exposure causes a rapid increase in the numbers of these receptors, via a phosphatidylinositol 3 kinase (PI3K) and protein kinase A (PKA) dependent mechanism. Finally, to assess the relevance of TNF-α to slow excitotoxic MN injury, we made use of organotypic spinal cord slice cultures. Co2+ labeling revealed that MNs in these cultures possess Ca-perm AMPAr. Addition of either a low level of TNF-α, or of the glutamate uptake blocker, trans-pyrrolidine-2,4-dicarboxylic acid (PDC) to the cultures for 48 h resulted in little MN injury. However, when combined, TNF-α+PDC caused considerable MN degeneration, which was blocked by the AMPA/kainate receptor blocker, 2,3-Dihydroxy-6-nitro-7-sulfamoylbenzo (F) quinoxaline (NBQX), or the Ca-perm AMPAr selective blocker, 1-naphthyl acetylspermine (NASPM). Thus, these data support the idea that prolonged TNF-α elevation, as may be induced by glial activation, acts in part by increasing the numbers of Ca-perm AMPAr on MNs to enhance injurious excitotoxic effects of deficient

  7. Peptides identify multiple hotspots within the ligand binding domain of the TNF receptor 2

    Directory of Open Access Journals (Sweden)

    Lennick Michael

    2003-01-01

    Full Text Available Abstract Background Hotspots are defined as the minimal functional domains involved in protein:protein interactions and sufficient to induce a biological response. Results Here we describe the use of complex and high diversity phage display libraries to isolate peptides (called Hotspot Ligands or HSPLs which sub-divide the ligand binding domain of the tumor necrosis factor receptor 2 (TNFR2; p75 into multiple hotspots. We have shown that these libraries could generate HSPLs which not only subdivide hotspots on protein and non-protein targets but act as agonists or antagonists. Using this approach, we generated peptides which were specific for human TNFR2, could be competed by the natural ligands, TNFα and TNFβ and induced an unexpected biological response in a TNFR2-specific manner. Conclusions To our knowledge, this is the first report describing the dissection of the TNFR2 into biologically active hotspots with the concomitant identification of a novel and unexpected biological activity.

  8. Circulating TNF receptors are significant prognostic biomarkers for idiopathic membranous nephropathy.

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    Su Mi Lee

    Full Text Available Idiopathic membranous nephropathy (iMN is a common cause of nephrotic syndrome in adults. A biomarker to accurately indicate the severity of iMN and predict long-term prognosis is insufficient. Here, we evaluated the clinical significance of circulating tumor necrosis factor receptors (cTNFRs as prognostic biomarkers of iMN with nephrotic syndrome. A total of 113 patients with biopsy-proven iMN and 43 healthy volunteers were enrolled in this study. Ninety patients with iMN had nephrotic range proteinuria. Levels of cTNFRs were measured by using serum samples collected at the time of initial diagnosis. Levels of cTNFRs were higher in the patients with nephrotic syndrome than in those with subnephrotic range proteinuria or in the healthy volunteers (P for trend <0.001. Estimated glomerular filtration rate and proteinuria tended to worsen as the cTNFRs levels increased. Having a cTNFR1 level within the highest tertile was a significant risk factor for renal progression after adjustment, in comparison with the other tertiles (hazard ratio [HR], 3.39; 95% confidence interval [95% CI], 1.48-7.78; P = 0.004. The cTNFR2 level within the highest tertile also significantly increased the risk of renal progression (HR, 3.29; 95% CI, 1.43-7.54; P = 0.005. Renal tubular TNFRs expression was associated with cTNFRs level. However, the cTNFRs levels were not associated with autoantibody against phospholipase A2 receptor reactivity/levels or treatment response. This study demonstrated that cTNFRs levels at the time of initial diagnosis could predict renal progression in patients with iMN.

  9. The α7 nicotinic acetylcholine receptor ligands methyllycaconitine, NS6740 and GTS-21 reduce lipopolysaccharide-induced TNF-α release from microglia

    DEFF Research Database (Denmark)

    Thomsen, Morten Skøtt; Mikkelsen, Jens D

    2012-01-01

    The anti-inflammatory properties of, particularly the α7, nicotinic acetylcholine receptors (nAChRs) in the peripheral immune system are well documented. There are also reports of anti-inflammatory actions of nicotine in the CNS, but it is unclear, whether this is due to activation or inhibition...... of nAChRs. Here we investigate the mechanisms behind α7 nAChR-mediated modulation of TNF-α release. We show that α7 nAChR agonists or positive allosteric modulators do not affect LPS-induced release of the pro-inflammatory cytokine TNF-α from cultured microglia. This suggests that classical activation...... of, i.e. ion-flux through, the α7 nAChR does not reduce TNF-α release from activated microglia. Contrarily, the α7 nAChR antagonist methyllycaconitine and the weak (...

  10. Activation of AMPA receptor promotes TNF-α release via the ROS-cSrc-NFκB signaling cascade in RAW264.7 macrophages

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiu-Li [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Ding, Fan [Office of Scientific R& D, Tsinghua University, Beijing (China); Li, Hui; Tan, Xiao-Qiu [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Liu, Xiao [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Cao, Ji-Min, E-mail: caojimin@126.com [Department of Physiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China); Gao, Xue, E-mail: longlongnose@163.com [Department of Pathophysiology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, School of Basic Medicine, Peking Union Medical College, Beijing (China)

    2015-05-29

    The relationship between glutamate signaling and inflammation has not been well defined. This study aimed to investigate the role of AMPA receptor (AMPAR) in the expression and release of tumor necrosis factor-alpha (TNF-α) from macrophages and the underlying mechanisms. A series of approaches, including confocal microscopy, immunofluorescency, flow cytometry, ELISA and Western blotting, were used to estimate the expression of AMPAR and downstream signaling molecules, TNF-α release and reactive oxygen species (ROS) generation in the macrophage-like RAW264.7 cells. The results demonstrated that AMPAR was expressed in RAW264.7 cells. AMPA significantly enhanced TNF-α release from RAW264.7 cells, and this effect was abolished by CNQX (AMPAR antagonist). AMPA also induced elevation of ROS production, phosphorylation of c-Src and activation of nuclear factor (NF)-κB in RAW264.7 cells. Blocking c-Src by PP2, scavenging ROS by glutathione (GSH) or inhibiting NF-κB activation by pyrrolidine dithiocarbamate (PDTC) decreased TNF-α production from RAW264.7 cells. We concluded that AMPA promotes TNF-α release in RAW264.7 macrophages likely through the following signaling cascade: AMPAR activation → ROS generation → c-Src phosphorylation → NF-κB activation → TNF-α elevation. The study suggests that AMPAR may participate in macrophage activation and inflammation. - Highlights: • AMPAR is expressed in RAW264.7 macrophages and is upregulated by AMPA stimulation. • Activation of AMPAR stimulates TNF-α release in macrophages through the ROS-cSrc-NFκB signaling cascade. • Macrophage AMPAR signaling may play an important role in inflammation.

  11. Lansoprazole Upregulates Polyubiquitination of the TNF Receptor-Associated Factor 6 and Facilitates Runx2-mediated Osteoblastogenesis

    Directory of Open Access Journals (Sweden)

    Kenichi Mishima

    2015-12-01

    Full Text Available The transcription factor, runt-related transcription factor 2 (Runx2, plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP-transforming growth factor-beta (TGF-β activated kinase-1 (TAK1–p38 mitogen-activated protein kinase (MAPK pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6 and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD. Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

  12. Lansoprazole Upregulates Polyubiquitination of the TNF Receptor-Associated Factor 6 and Facilitates Runx2-mediated Osteoblastogenesis.

    Science.gov (United States)

    Mishima, Kenichi; Kitoh, Hiroshi; Ohkawara, Bisei; Okuno, Tatsuya; Ito, Mikako; Masuda, Akio; Ishiguro, Naoki; Ohno, Kinji

    2015-12-01

    The transcription factor, runt-related transcription factor 2 (Runx2), plays a pivotal role in the differentiation of the mesenchymal stem cells to the osteochondroblast lineages. We found by the drug repositioning strategy that a proton pump inhibitor, lansoprazole, enhances nuclear accumulation of Runx2 and induces osteoblastogenesis of human mesenchymal stromal cells. Systemic administration of lansoprazole to a rat femoral fracture model increased osteoblastogenesis. Dissection of signaling pathways revealed that lansoprazole activates a noncanonical bone morphogenic protein (BMP)-transforming growth factor-beta (TGF-β) activated kinase-1 (TAK1)-p38 mitogen-activated protein kinase (MAPK) pathway. We found by in cellulo ubiquitination studies that lansoprazole enhances polyubiquitination of the TNF receptor-associated factor 6 (TRAF6) and by in vitro ubiquitination studies that the enhanced polyubiquitination of TRAF6 is attributed to the blocking of a deubiquitination enzyme, cylindromatosis (CYLD). Structural modeling and site-directed mutagenesis of CYLD demonstrated that lansoprazole tightly fits in a pocket of CYLD where the C-terminal tail of ubiquitin lies. Lansoprazole is a potential therapeutic agent for enhancing osteoblastic differentiation.

  13. Anti-Drug Antibodies, Drug Levels, Interleukin-6 and Soluble TNF Receptors in Rheumatoid Arthritis Patients during the First 6 Months of Treatment with Adalimumab or Infliximab

    DEFF Research Database (Denmark)

    Eng, Grith Petersen; Bouchelouche, Pierre; Bartels, Else Marie

    2016-01-01

    -TNFi Abs). METHODS: Blood samples from 26 patients with established RA were taken at baseline and following 6 months of treatment with adalimumab or infliximab. Samples were analyzed for levels of TNFi, interleukin (IL)-6, and soluble TNF-receptors 1 and -2 (sTNF-R1 and -2) and for presence of anti......-TNFi Abs. Clinical and demographic data were recorded as well. RESULTS: During the initial 6 months treatment, DAS28(CRP) (Disease activity score in 28 joints using C-reactive protein) and levels of IL-6 and sTNF-R2 decreased significantly in patients without anti-TNFi Abs and in patients retaining...... detectable drug levels. The levels of other tested cytokines (TNF-α, TNF-β, IL-1ra, IL-1b, IL-8, IL-10, IL-12(p70), IL-13, IL-17A, IL-17F, and IL-33) were generally below detection limits. Higher baseline levels of IL-6 associated with undetectable levels of TNFi at follow-up. Anti-TNFi Abs were associated...

  14. Isolation of the human anionic glutathione S-transferase cDNA and the relation of its gene expression to estrogen-receptor content in primary breast cancer

    International Nuclear Information System (INIS)

    Moscow, J.A.; Townsend, A.J.; Goldsmith, M.E.; Whang-Peng, J.; Vickers, P.J.; Poisson, R.; Legault-Poisson, S.; Myers, C.E.; Cowan, K.H.

    1988-01-01

    The development of multidrug resistance in MCF7 human breast cancer cells is associated with overexpression of P-glycoprotein, changes in activities of several detoxication enzymes, and loss of hormone sensitivity and estrogen receptors (ERs). The authors have cloned the cDNA for one of the drug-detoxifying enzymes overexpressed in multidrug-resistant MCF7 cells (Adr R MCF7), the anionic isozyme of glutathione S-transferase (GSTπ). Hybridization with this GSTπ cDNA, GSTπ-1, demonstrated that increased GSTπ activity in Adr R MCF7 cells is associated with overexpression but not with amplification of the gene. They mapped the GSTπ gene to human chromosome 11q13 by in situ hybridization. Since multidrug resistance and GSTπ overexpression are associated with the loss of ERs in Adr R MCF7 cells, they examined several other breast cancer cell lines that were not selected for drug resistance. In each of these cell lines they found an inverse association between GSTπ expression and ER content. They also examined RNA from 21 primary breast cancers and found a similar association between GSTπ expression and ER content in vivo. The finding of similar patterns of expression of a drug-detoxifying enzyme and of ERs in vitro as well as in vivo suggests that ER-negative breast cancer cells may have greater protection against antineoplastic agents conferred by GSTπ than ER-positive tumors

  15. Directed evolution of a three-finger neurotoxin by using cDNA display yields antagonists as well as agonists of interleukin-6 receptor signaling

    Directory of Open Access Journals (Sweden)

    Naimuddin Mohammed

    2011-01-01

    Full Text Available Abstract Background Directed evolution of biomolecules such as DNA, RNA and proteins containing high diversity has emerged as an effective method to obtain molecules for various purposes. In the recent past, proteins from non-immunoglobulins have attracted attention as they mimic antibodies with respect to binding potential and provide further potential advantages. In this regard, we have attempted to explore a three-finger neurotoxin protein (3F. 3F proteins are small (~7 kDa, structurally well defined, thermally stable and resistant to proteolysis that presents them as promising candidates for directed evolution. Results We have engineered a snake α-neurotoxin that belongs to the 3F family by randomizing the residues in the loops involved in binding with acetylcholine receptors and employing cDNA display to obtain modulators of interleukin-6 receptor (IL-6R. Selected candidates were highly specific for IL-6R with dissociation constants and IC50s in the nanomolar range. Antagonists as well as agonists were identified in an IL-6 dependent cell proliferation assay. Size minimization yielded peptides of about one-third the molecular mass of the original proteins, without significant loss of activities and, additionally, lead to the identification of the loops responsible for function. Conclusions This study shows 3F protein is amenable to introduce amino acid changes in the loops that enable preparation of a high diversity library that can be utilized to obtain ligands against macromolecules. We believe this is the first report of protein engineering to convert a neurotoxin to receptor ligands other than the parent receptor, the identification of an agonist from non-immunoglobulin proteins, the construction of peptide mimic of IL-6, and the successful size reduction of a single-chain protein.

  16. Establishment of the first WHO International Standard for etanercept, a TNF receptor II Fc fusion protein: Report of an international collaborative study.

    Science.gov (United States)

    Wadhwa, Meenu; Bird, Chris; Dilger, Paula; Rigsby, Peter; Jia, Haiyan; Gross, Marie Emmanuelle Behr

    2017-08-01

    Etanercept, a recombinant human tumor necrosis factor (TNF) receptor Fc fusion protein is an effective treatment option in adults with rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or plaque psoriasis and paediatrics with juvenile idiotypic arthritis and plaque psoriasis. Patent expiration in Europe and intense development of various etanercept products worldwide triggered a need for an international reference standard to facilitate determination of biological activity. Therefore, three candidate preparations of etanercept were lyophilized and evaluated in a multi-centre collaborative study comprising twenty eight laboratories from 15 countries for their suitability to serve as an international standard for the bioactivity of TNF receptor II Fc fusion proteins (international nonproprietary name, Etanercept). The preparations were tested for neutralization activity against the third TNF-α international standard (IS) in different in vitro cell-based assays, e.g., cytotoxicity, apoptosis and reporter gene methods. Regardless of the assay and the amount of TNF-α IS used, potency estimates for the different preparations were very similar. An indication of the inhibitory activity of etanercept in terms of the biological activity of the TNF-α IS based on ED50 data derived from a limited number of laboratories using a cytotoxicity assay was also derived. Results indicated that the candidate preparation coded 13/204 was stable and suitable to serve as an international standard for the biological activity of etanercept. Therefore, the preparation coded 13/204 was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2015 as the WHO first International Standard for TNF receptor II Fc fusion protein (INN, etanercept) with an assigned in vitro bioactivity of 10,000IU per ampoule. It should be noted that this first-in-class international standard for a Fc fusion protein, available from the National Institute for Biological

  17. Recombinant human growth-regulated oncogene-alpha induces T lymphocyte chemotaxis. A process regulated via IL-8 receptors by IFN-gamma, TNF-alpha, IL-4, IL-10, and IL-13

    DEFF Research Database (Denmark)

    Jinquan, T; Frydenberg, Jane; Mukaida, N

    1995-01-01

    receptors on the cells. This process can be augmented by IFN-gamma and TNF-alpha, and inhibited by IL-4, IL-10, and IL-13. In addition, we also document that on T lymphocytes there exist IL-8 receptors that can be up-regulated by IFN-gamma, TNF-alpha, and IL-2. Our results demonstrate that rhGRO-alpha gene...

  18. Identification and expression analysis of peroxisome proliferator-activated receptors cDNA in a reptile, the leopard gecko (Eublepharis macularius).

    Science.gov (United States)

    Kato, Keisuke; Oka, Yoshitaka; Park, Min Kyun

    2008-05-01

    Despite the physiological and evolutionary significance of lipid metabolism in amniotes, the molecular mechanisms involved have been unclear in reptiles. To elucidate this, we investigated peroxisome proliferators-activated receptors (PPARs) in the leopard gecko (Eublepharis macularius). PPARs belong to a nuclear hormone-receptor family mainly involved in lipid metabolism. Although PPARs have been widely studied in mammals, little information about them is yet available from reptiles. We identified in the leopard gecko partial cDNA sequences of PPARalpha and beta, and full sequences of two isoforms of PPARgamma. This is the first report of reptilian PPARgamma mRNA isoforms. We also evaluated the organ distribution of expression of these genes by using RT-PCR and competitive PCR. The expression level of PPARalpha mRNA was highest in the large intestine, and moderate in the liver and kidney. The expression level of PPARbeta mRNA was highest in the kidney and large intestine, and moderate in the liver. Similarly to the expression of human PPARgamma isoforms, PPARgammaa was expressed ubiquitously, whereas the expression of PPARgammab was restricted. The highest levels of their expression, however, were observed in the large intestine, rather than in the adipose tissue as in mammals. Taken together, these results showed that the profile of PPARbeta mRNA expression in the leopard gecko is similar to that in mammals, and that those of PPAR alpha and gamma are species specific. This may reflect adaptation to annual changes in lipid storage due to seasonal food availability.

  19. Serial analysis of clinical and imaging indices reveals prolonged efficacy of TNF-α and IL-6 receptor targeted therapies in refractory Takayasu arteritis.

    Science.gov (United States)

    Youngstein, Taryn; Peters, James E; Hamdulay, Shahir S; Mewar, Devesh; Price-Forbes, Alec; Lloyd, Mark; Jeffery, Rachel; Kinderlerer, Anne R; Mason, Justin C

    2014-01-01

    We analysed a large cohort of patients with Takayasu arteritis, seeking robust clinical evidence for prolonged responses to tumour necrosis factor-α (TNF-α) and interleukin-6 receptor (IL-6R) antagonists in severe refractory disease. Case notes from ninety-eight patients with Takayasu arteritis were retrospectively reviewed. Drug treatment, laboratory and serial non-invasive imaging data were analysed, and the Indian Takayasu arteritis activity (ITAS) and damage scores (TADs) calculated. Nine patients were treated with biologic therapies. All had previously received high dose prednisolone and ≥1 conventional immunosuppressant. Five patients had failed cyclophosphamide. The patients prescribed biologics had more extensive arterial injury than the remainder of the cohort and persistent active disease (ITAS range 2-9, CRP 12-206 mg/L, TADs 3--1). Eight patients were prescribed anti-TNF-α therapy, three IL-6R blockade. The mean duration of anti-TNF-α treatment was 42 months (maximum 8 years). One patient developed new arterial stenoses while receiving anti-TNF-α and subsequently achieved disease remission with tocilizumab. Two patients have now demonstrated sustained responses to IL-6R inhibition at 19 and 20 months. Following introduction of biologic therapy, serial non-invasive imaging has revealed no significant progression in arterial injury. A significant fall in CRP (p<0.01), prednisolone dose (p<0.01) and ITAS (p<0.01) was observed, with no increase in TADs. We report for the first time sustained responses to both anti-TNF-α and IL6R antagonists in refractory Takayasu arteritis. As 5/9 patients were cyclophosphamide non-responders, we propose that biologics should now be considered ahead of cyclophosphamide in these young patients.

  20. Suberoylanilide hydroxamic acid increases anti-cancer effect of tumor necrosis factor-α through up-regulation of TNF receptor 1 in lung cancer cells.

    Science.gov (United States)

    You, Bo Ra; Han, Bo Ram; Park, Woo Hyun

    2017-03-14

    Suberoylanilide hydroxamic acid (SAHA) as a histone deacetylase (HDAC) inhibitor has anti-cancer effect. Here, we evaluated the effect of SAHA on HDAC activity and cell growth in many normal lung and cancer cells. We observed that the HDAC activities of lung cancer cells were higher than that of normal lung cells. SAHA inhibited the growth of lung cancer cells regardless of the inhibitory effect on HDAC. This agent induced a G2/M phase arrest and apoptosis, which was accompanied by mitochondrial membrane potential (MMP: ΔΨm) loss in lung cancer cells. However, SAHA did not induce cell death in normal lung cells. All tested caspase inhibitors prevented apoptotic cell death in SAHA-treated A549 and Calu-6 lung cancer cells. Treatment with tumor necrosis factor-alpha (TNF-α) enhanced apoptosis in SAHA-treated lung cancer cells through caspase-8 and caspase-9 activations. Especially, SAHA increased the expression level of TNFreceptor 1 (TNFR1), especially acetylation of the region of TNFR1 promoter -223/-29 in lung cancer cells. The down-regulation of TNFR1 suppressed apoptosis in TNF-α and SAHA-treated lung cancer cells. In conclusion, SAHA inhibited the growth of lung cancer cells via a G2/M phase arrest and caspase-dependent apoptosis. SAHA also enhanced apoptotic effect of TNF-α in human lung cancer cells through up-regulation of TNFR1. TNF-α may be a key to improve anti-cancer effect of HDAC inhibitors.

  1. Structural and functional characterization of a novel molluskan ortholog of TRAF and TNF receptor-associated protein from disk abalone (Haliotis discus discus).

    Science.gov (United States)

    Lee, Youngdeuk; Elvitigala, Don Anushka Sandaruwan; Whang, Ilson; Lee, Sukkyoung; Kim, Hyowon; Zoysa, Mahanama De; Oh, Chulhong; Kang, Do-Hyung; Lee, Jehee

    2014-09-01

    Immune signaling cascades have an indispensable role in the host defense of almost all the organisms. Tumor necrosis factor (TNF) signaling is considered as a prominent signaling pathway in vertebrate as well as invertebrate species. Within the signaling cascade, TNF receptor-associated factor (TRAF) and TNF receptor-associated protein (TTRAP) has been shown to have a crucial role in the modulation of immune signaling in animals. Here, we attempted to characterize a novel molluskan ortholog of TTRAP (AbTTRAP) from disk abalone (Haliotis discus discus) and analyzed its expression levels under pathogenic stress. The complete coding sequence of AbTTRAP consisted of 1071 nucleotides, coding for a 357 amino acid peptide, with a predicted molecular mass of 40 kDa. According to our in-silico analysis, AbTTRAP resembled the typical TTRAP domain architecture, including a 5'-tyrosyl DNA phosphodiesterase domain. Moreover, phylogenetic analysis revealed its common ancestral invertebrate origin, where AbTTRAP was clustered with molluskan counterparts. Quantitative real time PCR showed universally distributed expression of AbTTRAP in selected tissues of abalone, from which more prominent expression was detected in hemocytes. Upon stimulation with two pathogen-derived mitogens, lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly I:C), transcript levels of AbTTRAP in hemocytes and gill tissues were differentially modulated with time. In addition, the recombinant protein of AbTTRAP exhibited prominent endonuclease activity against abalone genomic DNA, which was enhanced by the presence of Mg(2+) in the medium. Collectively, these results reinforce the existence of the TNF signaling cascade in mollusks like disk abalone, further implicating the putative regulatory behavior of TTRAP in invertebrate host pathology. Copyright © 2014 Elsevier Ltd. All rights reserved.

  2. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells.

    Science.gov (United States)

    Dyari, Herryawan Ryadi Eziwar; Rawling, Tristan; Chen, Yongjuan; Sudarmana, William; Bourget, Kirsi; Dwyer, Julie M; Allison, Sarah E; Murray, Michael

    2017-12-01

    A saturated analog of the cytochrome P450-mediated ω-3-17,18-epoxide of ω-3-eicosapentaenoic acid (C20E) activated apoptosis in human triple-negative MDA-MB-231 breast cancer cells. This study evaluated the apoptotic mechanism of C20E. Increased cytosolic cytochrome c expression and altered expression of pro- and antiapoptotic B-cell lymphoma-2 proteins indicated activation of the mitochondrial pathway. Caspase-3 activation by C20E was prevented by pharmacological inhibition and silencing of the JNK and p38 MAP kinases (MAPK), upstream MAPK kinases MKK4 and MKK7, and the upstream MAPK kinase kinase apoptosis signal-regulating kinase 1 (ASK1). Silencing of the death receptor TNF receptor 1 (TNFR1), but not Fas, DR4, or DR5, and the adapters TRADD and TNF receptor-associated factor 2, but not Fas-associated death domain, prevented C20E-mediated apoptosis. B-cell lymphoma-2 homology 3-interacting domain death agonist (Bid) cleavage by JNK/p38 MAPK linked the extrinsic and mitochondrial pathways of apoptosis. In further studies, an antibody against the extracellular domain of TNFR1 prevented apoptosis by TNF-α but not C20E. These findings suggest that C20E acts intracellularly at TNFR1 to activate ASK1-MKK4/7-JNK/p38 MAPK signaling and to promote Bid-dependent mitochondrial disruption and apoptosis. In in vivo studies, tumors isolated from C20E-treated nu/nu mice carrying MDA-MB-231 xenografts showed increased TUNEL staining and decreased Ki67 staining, reflecting increased apoptosis and decreased proliferation, respectively. ω-3-Epoxy fatty acids like C20E could be incorporated into treatments for triple-negative breast cancers.-Dyari, H. R. E., Rawling, T., Chen, Y., Sudarmana, W., Bourget, K., Dwyer, J. M., Allison, S. E., Murray, M. A novel synthetic analogue of ω-3 17,18-epoxyeicosatetraenoic acid activates TNF receptor-1/ASK1/JNK signaling to promote apoptosis in human breast cancer cells. © FASEB.

  3. OSCAR Is a Receptor for Surfactant Protein D That Activates TNF-α Release from Human CCR2+ Inflammatory Monocytes

    DEFF Research Database (Denmark)

    Barrow, Alexander D; Palarasah, Yaseelan; Bugatti, Mattia

    2015-01-01

    of recombinant SP-D and captured native SP-D from human bronchoalveolar lavage. OSCAR localized in an intracellular compartment of alveolar macrophages together with SP-D. Moreover, we found OSCAR on the surface of interstitial lung and blood CCR2(+) inflammatory monocytes, which secreted TNF-α when exposed...

  4. Circulating cytokines and cytokine receptors in infliximab treatment failure due to TNF-α independent Crohn disease

    DEFF Research Database (Denmark)

    Steenholdt, Casper; Coskun, Mehmet; Buhl, Sine

    2016-01-01

    The inflammatory response at infliximab (IFX) treatment failure due to tumor necrosis factor (TNF)-α-independent Crohn disease activity is unknown.This is an exploratory, hypothesis-generating study based on samples collected in a clinical trial among patients failing conventional IFX dosages...

  5. Low pH inactivation for xenotropic gamma retrovirus in recombinant human TNFreceptor immunoglobulin G and mechanism of inactivation.

    Science.gov (United States)

    Ma, Rong; Cui, Xiaolan

    2014-01-01

    CHO-derived recombinant proteins for human therapeutic are used commonly. There are noninfectious endogenous retroviruses in CHO cells. Validation study for inactivation process is required. Murine xenotropic gamma retrovirus (X-MulV) is a model virus in validation study. In our previous study, optimum conditions for X-MulV inactivation were sifted. In this study, we performed a further research on low pH inactivation for evaluation of X-MulV clearance in manufacturing of recombinant human TNFreceptor immunoglobulin G fusion proteins (rhTNF-α) for injection. Cell-based infectivity assay was used for the evaluation of X-MulV clearance. RhTNF-α were spiked with X-MulV and were inactivated at pH 3.60 ∼ 3.90, 25 ± 2 °C, and 0 ∼ 240 min, respectively. Samples incubated at the conditions for 15 ∼ 180 min were not inactivated effectively. For 4 h incubation, log10 reductions were achieved 5.0 log10. Biological activity of rhTNF-α incubated at pH 3.60, 25 °C for 4 h, which was assayed on murine L929 fibroblasts cells, was not affected by low pH. Env gene of X-MulV, which was detected by conventional PCR method for the first time, was not detected after incubation at pH 3.60, and it may be the mechanism of low pH inactivation. Copyright © 2013. Published by Elsevier Ltd.

  6. Comparative Expression Analysis of HSP70, HSP90, IL-4, TNF, KITLG and KIT-receptor Gene between Varicocele-Induced and Non-Varicocele Testes of Dog

    Directory of Open Access Journals (Sweden)

    Hossein Hassanpour

    2017-09-01

    Full Text Available Background This study was designed to create an experimental varicocele model by a simple surgical procedure in dog with minimum invasion and to investigate the effect of varicocele-induced infertility on the expression of six related genes (HSP90, HSP70, IL-4, TNF, KITLG and KIT receptor. Materials and Methods In this experimental study, the proximal part of the pampini-form plexus of dog testes was partially occluded without abdominal incision which was confirmed by venographic examination. To evaluate varicocele in its acute form, dogs were castrated after 15 days and testes were dissected. Histopathologic evaluation was undertaken and the relative expression of the six genes was assessed by quantitative realtime polymerase chain reaction (PCR. Results Microscopic changes showed tubule degeneration. The Johnson score was significantly decreased in the varicocele testes when compared with non-varicocele testes. Expressions of HSP90, TNF, KITLG and the KIT-receptor gene were significantly downregulated (P=0.029, 0.047, 0.004 and 0.035 respectively in varicocele-induced testes while HSP70 was upregulated (P=0.018. IL-4 did not show differential expression (P=0.377. Conclusion We conclude that partial occlusion of the proximal part of the pampiniform plexus induces varicocele in the testis of dog. Differential expression of the mentioned genes may be responsible for the pathophysiology of varicocele and related subfertility.

  7. Serum levels of soluble TNFreceptors but not BDNF are associated with apathy symptoms in mild Alzheimer's disease and amnestic mild cognitive impairment

    Directory of Open Access Journals (Sweden)

    Henrique Cerqueira Guimarães

    Full Text Available ABSTRACT Apathy is intimately associated with dementia. Unfortunately, its pathophysiology remains poorly understood. The motivational impairment that characterizes this disorder might share the same inflammatory mechanisms, as suggested by the sickness behavior theory. Objective: The primary aim of this study was to investigate the association between apathy symptoms and serum levels of tumor necrosis factor alpha (TNF-α and its soluble receptors. Brain-derived neurotrophic factor (BDNF levels were also analyzed since these have been associated with depression, a condition which shares abulic features with apathy. Methods: The sample consisted of 27 subjects with mild Alzheimer's disease or amnestic mild cognitive impairment, who were submitted to specific apathy evaluation using the Apathy Scale (AS and provided blood samples for biomarker analysis. Participants were categorized into two groups according to median AS scores (17 points. Results: Subjects with higher apathy symptoms (n=13 displayed higher levels of TNF-α soluble receptors (type 1: p=0.03; type 2: p=0.04. No other difference was found between groups. Conclusion: These findings point to the involvement of inflammatory mediators in the genesis of apathy symptoms, as suggested by the sickness behavior theory.

  8. Diverging mechanisms for TNF-alpha receptors in normal mouse brains and in functional recovery after injury: From gene to behavior.

    Science.gov (United States)

    Quintana, Albert; Molinero, Amalia; Florit, Sergi; Manso, Yasmina; Comes, Gemma; Carrasco, Javier; Giralt, Mercedes; Borup, Rehannah; Nielsen, Finn Cilius; Campbell, Iain L; Penkowa, Milena; Hidalgo, Juan

    2007-09-01

    Cytokines, such as tumour necrosis factor (TNF)-alpha and lymphotoxin-alpha, have been described widely to play important roles in the brain in physiologic conditions and after traumatic injury. However, the exact mechanisms involved in their function have not been fully elucidated. We give some insight on their role by using animals lacking either Type 1 receptor (TNFR1KO) or Type 2 (TNFR2KO) and their controls (C57Bl/6). Both TNFR1KO and to a greater extent TNFR2KO mice showed increased exploration/activity neurobehavioral traits in the hole board test, such as rearings, head dippings, and ambulations, compared with wild-type mice, suggesting an inhibitory role of TNFR1/TNFR2 signaling. In contrast, no significant differences were observed in the elevated plus maze test, ruling out a major role of these receptors in the control of anxiety. We next evaluated the response to a freeze injury to the somatosensorial cortex. The effect of the cryolesion on motor function was evaluated with the horizontal ladder beam test, and the results showed that both TNFR1KO and TNFR2KO mice made fewer errors, suggesting a detrimental role for TNFR1/TNFR2 signaling for coping with brain damage. Expression of approximately 22600 genes was analyzed using an Affymetrix chip (MOE430A) at 0 (unlesioned), 1, or 4 days post-lesion in the three strains. The results show a unique and major role of both TNF receptors on the pattern of gene expression elicited by the injury but also in normal conditions, and suggest that blocking of TNFR1/TNFR2 receptors may be beneficial after a traumatic brain injury. (c) 2007 Wiley-Liss, Inc.

  9. TNF Receptor-2 Facilitates an Immunosuppressive Microenvironment in the Liver to Promote the Colonization and Growth of Hepatic Metastases

    DEFF Research Database (Denmark)

    Ham, Boram; Wang, Ni; D'Costa, Zarina

    2015-01-01

    Successful colonization by a cancer cell of a distant metastatic site requires immune escape in the new microenvironment. TNF signaling has been implicated broadly in the suppression of immune surveillance that prevents colonization at the metastatic site and therefore must be blocked...... chemotherapy-naïve colon cancer patients confirmed the presence of CD33(+)HLA-DR(-)TNFR2(+) myeloid cells in the periphery of hepatic metastases. Overall, our findings implicate TNFR2 in supporting MDSC-mediated immune suppression and metastasis in the liver, suggesting the use of TNFR2 inhibitors...... as a strategy to prevent metastatic progression to liver in colon, lung, and various other types of cancer....

  10. In vivo T cell activation by anti-CD3 monoclonal antibody induces soluble TNF receptor release in mice. Effects of pentoxifylline, methylprednisolone, anti-TNF, and anti-IFN-gamma antibodies

    NARCIS (Netherlands)

    Bemelmans, M. H.; Abramowicz, D.; Gouma, D. J.; Goldman, M.; Buurman, W. A.

    1994-01-01

    Injection of anti-CD3 is accompanied by an increase in systemic TNF, a mediator of the physiologic changes induced by anti-CD3 injection. Various mechanisms have been reported to be responsible for TNF inactivation and clearance. Recently, it has become evident that circulating soluble TNFRs (P55

  11. In vivo T cell activation by anti-CD3 monoclonal antibody induces soluble TNF receptor release in mice. Effects of pentoxifylline, methylprednisolone, anti-TNF, and anti-IFN-gamma antibodies.

    NARCIS (Netherlands)

    Bemelmans, M.H.; Abramowicz, D.; Gouma, D.J.; Goldman, M.; Buurman, W.A.

    1994-01-01

    Injection of anti-CD3 is accompanied by an increase in systemic TNF, a mediator of the physiologic changes induced by anti-CD3 injection. Various mechanisms have been reported to be responsible for TNF inactivation and clearance. Recently, it has become evident that circulating soluble TNFRs (P55

  12. A Model of Post-Infection Fatigue Is Associated with Increased TNF and 5-HT2A Receptor Expression in Mice.

    Science.gov (United States)

    Couch, Yvonne; Xie, Qin; Lundberg, Louise; Sharp, Trevor; Anthony, Daniel C

    2015-01-01

    It is well documented that serotonin (5-HT) plays an important role in psychiatric illness. For example, myalgic encephalomyelitis (ME/CFS), which is often provoked by infection, is a disabling illness with an unknown aetiology and diagnosis is based on symptom-specific criteria. However, 5-HT2A receptor expression and peripheral cytokines are known to be upregulated in ME. We sought to examine the relationship between the 5-HT system and cytokine expression following systemic bacterial endotoxin challenge (LPS, 0.5 mg/kg i.p.), at a time when the acute sickness behaviours have largely resolved. At 24 hours post-injection mice exhibit no overt changes in locomotor behaviour, but do show increased immobility in a forced swim test, as well as decreased sucrose preference and reduced marble burying activity, indicating a depressive-like state. While peripheral IDO activity was increased after LPS challenge, central activity levels remained stable and there was no change in total brain 5-HT levels or 5-HIAA/5-HT. However, within the brain, levels of TNF and 5-HT2A receptor mRNA within various regions increased significantly. This increase in receptor expression is reflected by an increase in the functional response of the 5-HT2A receptor to agonist, DOI. These data suggest that regulation of fatigue and depressive-like moods after episodes of systemic inflammation may be regulated by changes in 5-HT receptor expression, rather than by levels of enzyme activity or cytokine expression in the CNS.

  13. A Model of Post-Infection Fatigue Is Associated with Increased TNF and 5-HT2A Receptor Expression in Mice.

    Directory of Open Access Journals (Sweden)

    Yvonne Couch

    Full Text Available It is well documented that serotonin (5-HT plays an important role in psychiatric illness. For example, myalgic encephalomyelitis (ME/CFS, which is often provoked by infection, is a disabling illness with an unknown aetiology and diagnosis is based on symptom-specific criteria. However, 5-HT2A receptor expression and peripheral cytokines are known to be upregulated in ME. We sought to examine the relationship between the 5-HT system and cytokine expression following systemic bacterial endotoxin challenge (LPS, 0.5 mg/kg i.p., at a time when the acute sickness behaviours have largely resolved. At 24 hours post-injection mice exhibit no overt changes in locomotor behaviour, but do show increased immobility in a forced swim test, as well as decreased sucrose preference and reduced marble burying activity, indicating a depressive-like state. While peripheral IDO activity was increased after LPS challenge, central activity levels remained stable and there was no change in total brain 5-HT levels or 5-HIAA/5-HT. However, within the brain, levels of TNF and 5-HT2A receptor mRNA within various regions increased significantly. This increase in receptor expression is reflected by an increase in the functional response of the 5-HT2A receptor to agonist, DOI. These data suggest that regulation of fatigue and depressive-like moods after episodes of systemic inflammation may be regulated by changes in 5-HT receptor expression, rather than by levels of enzyme activity or cytokine expression in the CNS.

  14. Stimulation of nAchRα7 Receptor Inhibits TNF Synthesis and Secretion in Response to LPS Treatment of Mast Cells by Targeting ERK1/2 and TACE Activation.

    Science.gov (United States)

    Guzmán-Mejía, F; López-Rubalcava, C; González-Espinosa, C

    2018-03-01

    The cholinergic anti-inflammatory pathway is recognized as one of the main mechanisms of neuromodulation of the immune system. Activation of the α7 nicotinic acetylcholine receptor (nAchRα7) suppresses cytokine synthesis in distinct immune cells but the molecular mechanisms behind this effect remain to be fully described. Mast cells (MCs) are essential players of allergic reactions and innate immunity responses related to chronic inflammation. Activation of TLR4 receptor in MCs leads to the rapid secretion of pre-synthesized TNF from intracellular pools and to the activation of NFκB, necessary for de novo synthesis of TNF and other cytokines. Here we report that the nAchRα7 receptor specific agonist GTS-21 inhibits TLR4-induced secretion of preformed TNF from MCs in vivo and in vitro. Utilizing bone marrow-derived mast cells (BMMCs) it was found that GTS-21 also diminished secretion of de novo synthesized TNF, TNF mRNA accumulation and IKK-dependent p65-NFκB phosphorylation in response to LPS. nAchRα7 triggering prevented TLR4-induced ERK1/2 phosphorylation, which resulted an essential step for TNF secretion due to the phosphorylation of the metallopeptidase responsible for TNF maturation (TACE). Main inhibitory actions of GTS-21 were prevented by AG490, an inhibitor of JAK-2 kinase. Our results show for the first time, that besides the prevention of NFκB-dependent transcription, inhibitory actions of nAchRα7 triggering include the blockade of pathways leading to exocytosis of granule-stored cytokines in MCs.

  15. Plasma TNF binding capacity in patients with juvenile idiopathic arthritis

    DEFF Research Database (Denmark)

    Bjørnhart, Birgitte; Svenningsen, Pernille; Gudbrandsdottir, Sif

    2005-01-01

    Tumour necrosis factor (TNF)-alpha and TNF-beta, also called lymphotoxin (LT), are bound by soluble truncated TNF receptors (sTNFRI and II) that are released from cell surfaces and act as natural inhibitors of TNF-induced inflammation. We investigated the plasma levels of sTNFRI and II in parallel...

  16. Cocaine Induces Nuclear Export and Degradation of Neuronal Retinoid X Receptor-γ via a TNF-α/JNK-Mediated Mechanism

    Science.gov (United States)

    Kovalevich, Jane; Yen, William; Ozdemir, Ahmet; Langford, Dianne

    2015-01-01

    Cocaine abuse represents an immense societal health and economic burden for which no effective treatment currently exists. Among the numerous intracellular signaling cascades impacted by exposure to cocaine, increased and aberrant production of pro-inflammatory cytokines in the CNS has been observed. Additionally, we have previously reported a decrease in retinoid-X-receptor-gamma (RXR-γ) in brains of mice chronically exposed to cocaine. Through obligate heterodimerization with a number of nuclear receptors, RXRs serve as master regulatory transcription factors, which can potentiate or suppress expression of a wide spectrum of genes. Little is known about the regulation of RXR levels, but previous studies indicate cellular stressors such as cytokines negatively regulate levels of RXRs in vitro. To evaluate the mechanism underlying the cocaine-induced decreases in RXR-γ levels observed in vivo, we exposed neurons to cocaine in vitro and examined pathways which may contribute to disruption in RXR signaling, including activation of stress pathways by cytokine induction. In these studies, we provide the first evidence that cocaine exposure disrupts neuronal RXR-γ signaling in vitro by promoting its nuclear export and degradation. Furthermore, we demonstrate this effect may be mediated, at least in part, by cocaine-induced production of TNF-α and its downstream effector c-Jun-NH-terminal kinase (JNK). Findings from this study are therefore applicable to both cocaine abuse and to pathological conditions characterized by neuroinflammatory factors, such as neurodegenerative disease. PMID:25586717

  17. Lenalidomide-based maintenance therapy reduces TNF receptor 2 on CD4 T cells and enhances immune effector function in acute myeloid leukemia patients.

    Science.gov (United States)

    Govindaraj, Chindu; Madondo, Mutsa; Kong, Ying Ying; Tan, Peter; Wei, Andrew; Plebanski, Magdalena

    2014-08-01

    A major limitation to improved outcomes in acute myelogenous leukemia (AML) is relapse resulting from leukemic cells that persist at clinical remission. Regulatory T cells (Tregs), which are increased in AML patients, can contribute to immune evasion by residual leukemic cells. Tumor necrosis factor (TNF), a pro-inflammatory cytokine present at high levels within patients, can induce TNF receptor-2 (TNFR2) expression on Tregs. We hypothesized that since TNFR2 is required for Treg stabilization and TNFR2+ Tregs are potent suppressors, targeting TNFR2+ Tregs may restore the effectiveness of immune-surveillance mechanisms. In this pilot study, we report AML patients in clinical remission have substantially increased levels of TNFR2+ T cells, including TNFR2+ Tregs and impaired effector CD4 T cell function with reduced IL-2 and IFNγ production. The immunomodulatory drug, lenalidomide, and the demethylating agent, azacitidine have been moderately successful in treating AML patients, but their combined effects on TNFR2+ T cells, including Tregs are currently unknown. Our data indicates that although treatment with lenalidomide and azacitidine increased cytokine production by effector T cells in all patients, durable clinical remissions may be observed in patients with a concomitant reduction in TNFR2+ T cells and TNFR2+ Tregs. In vitro studies further demonstrated that lenalidomide can reduce TNFR2 expression and can augment effector cytokine production by T cells, which can be further enhanced by azacitidine. These results indicate that reduction of TNFR2+ T cells in AML postremission phase may result from combined azacitidine/lenalidomide therapy and may contribute to an improved clinical outcome. © 2014 Wiley Periodicals, Inc.

  18. Reducing TNF receptor 2+ regulatory T cells via the combined action of azacitidine and the HDAC inhibitor, panobinostat for clinical benefit in acute myeloid leukemia patients.

    Science.gov (United States)

    Govindaraj, Chindu; Tan, Peter; Walker, Patricia; Wei, Andrew; Spencer, Andrew; Plebanski, Magdalena

    2014-02-01

    Acute myeloid leukemia (AML) provides an environment that enables immune suppression, resulting in functionally defective effector T cells; regulatory T cells (Treg) are significant contributors to the impaired antitumor immune response. As TNF is present at high levels in AML and TNF receptor-2 (TNFR2)-expressing Tregs identify highly functional Tregs, we examine the hypothesis that TNFR2(+) Tregs are a relevant Treg subset in this cancer. We also determine the effect of the novel combinatorial therapy of the demethylating agent, azacitidine with the histone deacetylase inhibitor, panobinostat on Tregs, particularly TNFR2(+) Tregs. Thirty healthy donors and 14 patients with AML were enrolled in this study. Patients were treated with azacitidine and panobinostat for 28-day cycles. The frequency and functional relevance of TNFR2(+) Tregs were analyzed subsequently. We report that TNFR2(+) Tregs are increased in AML and have a high migration potential toward the bone marrow. Furthermore, we demonstrate that the level of TNFR2(+) Tregs in the peripheral blood and the bone marrow of patients are decreased in vivo after exposure to panobinostat and azacitidine. Reductions in TNFR2(+) Tregs were associated with increases in Interferon (IFN)-γ and interleukin (IL)-2 production by effector T cells within the bone marrow and beneficial clinical responses. In vitro mechanistic studies indicated panobinostat as the primary driver for the reduction of Tregs. Our study provides for the first time, in vivo validation of the ability of panobinostat in combination with azacitidine to suppress prevalent TNFR2(+) Tregs, resulting in clinical benefits within patients with AML. ©2013 AACR.

  19. Clonorchis sinensis excretory/secretory products promote the secretion of TNF-alpha in the mouse intrahepatic biliary epithelial cells via Toll-like receptor 4.

    Science.gov (United States)

    Yan, Chao; Wang, Yan-Hong; Yu, Qian; Cheng, Xiao-Dan; Zhang, Bei-Bei; Li, Bo; Zhang, Bo; Tang, Ren-Xian; Zheng, Kui-Yang

    2015-10-24

    Toll-like receptor 4 (TLR4), as one of the most important pathogen pattern recognitions (PPRs) plays a central role in elicitation of innate immunity and mediation of adaptive responses against foreign antigens. However, little is known of the roles of TLR4 in the immune responses of biliary epithelial cells (BECs) induced by Clonorchis sinensis, a parasite of significance in human health. In the present study, the primary mouse intrahepatic biliary epithelial cells (MIBECs) were pre-treated with TLR4 inhibitor peptide or control peptide and then stimulated by excretory/secretory products (ESP) of C. sinensis, respectively. The expressions of TLR4 and relative cytokines were determined using western blot and a bead-based analytic detection system, respectively. The results showed that ESP of C. sinensis significantly increased the expression of TLR4 which promoted the expression of MyD88 and NF-κB in BECs; the levels of TNF-α but not IL-6 from MIBECs stimulated by ESP alone were also considerably increased, compared with the group of the medium stimulated. However, the concentration of TNF-α was significantly decreased when MIBECs were pre-treated with TLR4 inhibitor. In addition, ESP could depress the level of IL-6 in MIBECs which was elevated by LPS. Our data for the first time demonstrate that ESP of C. sinensis can potently induce secretion of pro-inflammatory cytokines via TLR4 in MIBECs, which suggests that TLR4 plays an important role in host defenses against C. sinensis and the pathogenesis of clonorchiasis.

  20. Up-regulation of p55 TNF alpha-receptor in dorsal root ganglia neurons following lumbar facet joint injury in rats.

    Science.gov (United States)

    Sakuma, Yoshihiro; Ohtori, Seiji; Miyagi, Masayuki; Ishikawa, Tetsu; Inoue, Gen; Doya, Hideo; Koshi, Takana; Ito, Toshinori; Yamashita, Masaomi; Yamauchi, Kazuyo; Suzuki, Munetaka; Moriya, Hideshige; Takahashi, Kazuhisa

    2007-08-01

    The rat L5/6 facet joint is multisegmentally innervated from the L1 to L6 dorsal root ganglia (DRG). Tumor necrosis factor (TNF) is a known mediator of inflammation. It has been reported that satellite cells are activated, produce TNF and surround DRG neurons innervating L5/6 facet joints after facet injury. In the current study, changes in TNF receptor (p55) expression in DRG neurons innervating the L5/6 facet joint following facet joint injury were investigated in rats using a retrograde neurotransport method followed by immunohistochemistry. Twenty rats were used for this study. Two crystals of Fluorogold (FG; neurotracer) were applied into the L5/6 facet joint. Seven days after surgery, the dorsal portion of the capsule was cut in the injured group (injured group n = 10). No injury was performed in the non-injured group (n = 10). Fourteen days after the first application of FG, bilateral DRGs from T13 to L6 levels were resected and sectioned. They were subsequently processed for p55 immunohistochemistry. The number of FG labeled neurons and number of FG labeled p55-immunoreactive (IR) neurons were counted. FG labeled DRG neurons innervating the L5/6 facet joint were distributed from ipsilateral L1 to L6 levels. Of FG labeled neurons, the ratio of DRG neurons immunoreactive for p55 in the injured group (50%) was significantly higher than that in the non-injured group (13%). The ratio of p55-IR neurons of FG labeled DRG neurons was significantly higher in total L1 and L2 DRGs than that in total L3, 4, 5 and 6 DRGs in the injured group (L1 and 2 DRG, 67%; L3, 4, 5 and 6 DRG, 37%, percentages of the total number of p55-IR neurons at L1 and L2 level or L3-6 level/the total number of FG-labeled neurons at L1 and L2 level or L3-6 level). These data suggest that up-regulation of p55 in DRG neurons may be involved in the sensory transmission from facet joint injury. Regulation of p55 in DRG neurons innervating the facet joint was different between upper DRG innervated

  1. 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine inhibit TNF-α and CXCL10 production from activated primary murine microglia via A2A receptors.

    Science.gov (United States)

    Newell, Elizabeth A; Exo, Jennifer L; Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Kochanek, Patrick M; Jackson, Edwin K

    2015-01-12

    Some cells, tissues and organs release 2',3'-cAMP (a positional isomer of 3',5'-cAMP) and convert extracellular 2',3'-cAMP to 2'-AMP plus 3'-AMP and convert these AMPs to adenosine (called the extracellular 2',3'-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2',3'-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2',3'-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 μM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N(6)-cyclopentyladenosine (CCPA) (10 μM; selective A1 agonist), 5'-N-ethylcarboxamide adenosine (NECA) (10 μM; agonist for all adenosine receptor subtypes) and CGS21680 (10 μM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. (1) 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; (2) DPSPX nearly eliminated the inhibitory effects of 2',3'-cAMP, 3'-AMP, 2'-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; (3) CCPA did not affect LPS-induced TNF-α and CXCL10; (4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. 2',3'-cAMP and its metabolites (3'-AMP, 2'-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. 2’,3’-cAMP, 3’-AMP, 2’-AMP and Adenosine Inhibit TNF-α and CXCL10 Production From Activated Primary Murine Microglia via A2A Receptors

    Science.gov (United States)

    Newell, Elizabeth A.; Exo, Jennifer L.; Verrier, Jonathan D.; Jackson, Travis C.; Gillespie, Delbert G.; Janesko-Feldman, Keri; Kochanek, Patrick M.

    2014-01-01

    Background Some cells, tissues and organs release 2’,3’-cAMP (a positional isomer of 3’,5’-cAMP) and convert extracellular 2’,3’-cAMP to 2’-AMP plus 3’-AMP and convert these AMPs to adenosine (called the extracellular 2’,3’-cAMP-adenosine pathway). Recent studies show that microglia have an extracellular 2’,3’-cAMP-adenosine pathway. The goal of the present study was to investigate whether the extracellular 2’,3’-cAMP-adenosine pathway could have functional consequences on the production of cytokines/chemokines by activated microglia. Methods Experiments were conducted in cultures of primary murine microglia. In the first experiment, the effect of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production was determined. In the next experiment, the first protocol was replicated but with the addition of 1,3-dipropyl-8-p-sulfophenylxanthine (DPSPX) (0.1 µM; antagonist of adenosine receptors). The last experiment compared the ability of 2-chloro-N6-cyclopentyladenosine (CCPA) (10 µM; selective A1 agonist), 5’-N-ethylcarboxamide adenosine (NECA) (10 µM; agonist for all adenosine receptor subtypes) and CGS21680 (10 µM; selective A2A agonist) to inhibit LPS-induced TNF-α and CXCL10 production. Results 1) 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine similarly inhibited LPS-induced TNF-α and CXCL10 production; 2) DPSPX nearly eliminated the inhibitory effects of 2’,3’-cAMP, 3’-AMP, 2’-AMP and adenosine on LPS-induced TNF-α and CXCL10 production; 3) CCPA did not affect LPS-induced TNF-α and CXCL10; 4) NECA and CGS21680 similarly inhibited LPS-induced TNF-α and CXCL10 production. Conclusions 2’,3’-cAMP and its metabolites (3’-AMP, 2’-AMP and adenosine) inhibit LPS-induced TNF-α and CXCL10 production via A2A-receptor activation. Adenosine and its precursors, via A2A receptors, likely suppress TNF-α and CXCL10 production by activated microglia in brain diseases. PMID:25451117

  3. Genistein enhances the effect of trichostatin A on inhibition of A549 cell growth by increasing expression of TNF receptor-1

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Tzu-Chin [Chest Clinic, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC (China); Yang, Ying-Chihi; Huang, Pei-Ru [Department of Nutritional Science, Chung Shan Medical University, Taichung, Taiwan, ROC (China); Wen, Yu-Der [Department of Biology, National Changhua University of Education, Changhua, Taiwan, ROC (China); Yeh, Shu-Lan, E-mail: suzyyeh@csmu.edu.tw [Department of Nutritional Science, Chung Shan Medical University, Taichung, Taiwan, ROC (China); Department of Nutrition, Chung Shan Medical University Hospital, Taichung, Taiwan, ROC (China)

    2012-08-01

    Our previous study has shown that genistein enhances apoptosis in A549 lung cancer cells induced by trichostatin A (TSA). The precise molecular mechanism underlying the effect of genistein, however, remains unclear. In the present study, we investigated whether genistein enhances the anti-cancer effect of TSA through up-regulation of TNF receptor-1 (TNFR-1) death receptor signaling. We incubated A549 cells with TSA (50 ng/mL) alone or in combination with genistein and then determined the mRNA and protein expression of TNFR-1 as well as the activation of downstream caspases. Genistein at 5 and 10 μM significantly enhanced the TSA-induced decrease in cell number and apoptosis in a dose-dependent manner. The combined treatment significantly increased mRNA and protein expression of TNFR-1 at 6 and 12 h, respectively, compared with that of the control group; while TSA alone had no effect. TSA in combination with 10 μM of genistein increased TNFR-1 mRNA and protein expression by about 70% and 40%, respectively. The underlying mechanism for this effect of genistein may be partly associated with the estrogen receptor pathway. The combined treatment also increased the activation of caspase-3 and ‐10 as well as p53 protein expression in A549 cells. The enhancing effects of genistein on the TSA-induced decrease in cell number and on the expression of caspase-3 in A549 cells were suppressed by silencing TNFR-1 expression. These data demonstrated that the upregulation of TNFR-1 death receptor signaling plays an important role, at least in part, in the enhancing effect of genistein on TSA-induced apoptosis in A549 cells. -- Highlights: ► TSA combined with genistein rather than TSA alone increases the expression of TNFR-1. ► Genistein may exert such an effect partly through estrogen receptor pathway. ► The combined treatment increases the activation of caspase-10 and caspase-3. ► The combined treatment also increases the expression of p53 protein. ► TNFR-1 si

  4. Early hCG addition to rFSH for ovarian stimulation in IVF provides better results and the cDNA copies of the hCG receptor may be an indicator of successful stimulation

    Directory of Open Access Journals (Sweden)

    Paraskevis Dimitris

    2009-10-01

    Full Text Available Abstract A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58 were administered hCG and Group B rLH (n = 56 in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.

  5. Liver Tumor Promotion by 2,3,7,8-Tetrachlorodibenzo-p-dioxin Is Dependent on the Aryl Hydrocarbon Receptor and TNF/IL-1 Receptors

    Science.gov (United States)

    Kennedy, Gregory D.; Nukaya, Manabu; Moran, Susan M.; Glover, Edward; Weinberg, Samuel; Balbo, Silvia; Hecht, Stephen S.; Pitot, Henry C.; Drinkwater, Norman R.; Bradfield, Christopher A.

    2014-01-01

    We set out to better understand the signal transduction pathways that mediate liver tumor promotion by 2,3,7,8-tetrachlorodibenzo-p-dioxn (“dioxin”). To this end, we first employed congenic mice homozygous for either the Ahrb1 or Ahrd alleles (encoding an aryl hydrocarbon receptor (AHR) with high or low binding affinity for dioxin, respectively) and demonstrated that hepatocellular tumor promotion in response to dioxin segregated with the Ahr locus. Once we had genetic evidence for the importance of AHR signaling, we then asked if tumor promotion by dioxin was influenced by “interleukin-1 (IL-1)-like” inflammatory cytokines. The importance of this question arose from our earlier observation that aspects of the acute hepatocellular toxicity of dioxin are dependent upon IL1-like cytokine signaling. To address this issue, we employed a triple knock-out (TKO) mouse model with null alleles at the loci encoding the three relevant receptors for tumor necrosis factors α and β and IL-1α and IL-1β (i.e., null alleles at the Tnfrsf1a, Tnfrsf1b, and Il-1r1 loci). The observation that TKO mice were resistant to the tumor promoting effects of dioxin in liver suggests that inflammatory cytokines play an important step in dioxin mediated liver tumor promotion in the mouse. Collectively, these data support the idea that the mechanism of dioxin acute hepatotoxicity and its activity as a promoter in a mouse two stage liver cancer model may be similar, i.e., tumor promotion by dioxin, like acute hepatotoxicity, are mediated by the linked action of two receptor systems, the AHR and the receptors for the “IL-1-like” cytokines. PMID:24718703

  6. α/sub i/-3 cDNA encodes the α subunit of G/sub k/, the stimulatory G protein of receptor-regulated K+ channels

    International Nuclear Information System (INIS)

    Codina, J.; Olate, J.; Abramowitz, J.; Mattera, R.; Cook, R.G.; Birnbaumer, L.

    1988-01-01

    cDNA cloning has identified the presence in the human genome of three genes encoding α subunits of pertussis toxin substrates, generically called G/sub i/. They are named α/sub i/-1, α/sub i/-2 and α/sub i/-3. However, none of these genes has been functionally identified with any of the α subunits of several possible G proteins, including pertussis toxin-sensitive G/sub p/'s, stimulatory to phospholipase C or A 2 , G/sub i/, inhibitory to adenylyl cyclase, or G/sub k/, stimulatory to a type of K + channels. The authors now report the nucleotide sequence and the complete predicted amino acid sequence of human liver α/sub i/-3 and the partial amino acid sequence of proteolytic fragments of the α subunit of human erythrocyte G/sub k/. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of α/sub i/-3, thus identifying it as α/sub k/. The probable identity of α/sub i/-1 with α/sub p/ and possible roles for α/sub i/-2, as well as additional roles for α/sub i/-1 and α/sub i/-3 (α/sub k/) are discussed

  7. Assessment of Anti-TNF-α Activities in Keratinocytes Expressing Inducible TNF- α: A Novel Tool for Anti-TNF-α Drug Screening

    Science.gov (United States)

    Udommethaporn, Sutthirat; Tencomnao, Tewin; McGowan, Eileen M.

    2016-01-01

    Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine important in normal and pathological biological processes. Newly synthesized pro-TNF-α is expressed on the plasma membrane and cleaved to release soluble TNF-α protein: both are biologically active. Secreted TNF-α signals through TNF receptors and the membrane-bound TNF-α acts by cell contact-dependent signaling. Anti-TNF-α antibodies have been used effectively for treatment of chronic inflammation, however with adverse side effects. Thus, there is a need for new anti-TNF-α small molecule compounds. Anti-TNF-α activity assays involve treatment of keratinocytes with exogenous TNF-α before or after anti-TNF-α incubation. However, this model fails to address the dual signaling of TNF-α. Here we describe a Doxycycline (Dox)-inducible TNF-α (HaCaT-TNF-α) expression system in keratinocytes. Using this in-vitro model, we show cell inhibition and induced expression of pro-inflammatory cytokines and markers, including IL-1β, IL-6, IL-8, NF-κB1, and KRT-16, similar to cells treated with exogenous TNF-α. Sufficient secreted TNF-α produced also activated IL-1β and IL-8 expression in wt HaCaT cells. Importantly, stimulated expression of IL-1β and IL-8 in HaCaT-TNF-α were blocked by Quercetin, a flavanol shown to possess anti-TNF-α activities. This novel in vitro cell model provides an efficient tool to investigate the dual signaling of TNF-α. Importantly, this model provides an effective, fast, and simple screening for compounds with anti-TNF-α activities for chronic inflammatory disease therapies. PMID:27415000

  8. A role for the non-receptor tyrosine kinase ACK1 in TNF-alpha-mediated apoptosis and proliferation in human intestinal epithelial caco-2 cells.

    Science.gov (United States)

    Zhao, Xinmei; Lv, Chaolan; Chen, Shengbo; Zhi, Fachao

    2017-09-16

    The roles of tumor necrosis factor alpha (TNF-alpha) and its mediators in cellular processes related to intestinal diseases remain elusive. In this study, we aimed to determine the biological role of activated Cdc42-associated kinase 1 (ACK1) in TNF-alpha-mediated apoptosis and proliferation in Caco-2 cells. ACK1 expression was knocked down using ACK1-specific siRNAs, and ACK1 activity was disrupted using a small molecule ACK1 inhibitor. The Terminal deoxynucleotidyl transferase biotin-dUTP Nick End Labeling (TUNEL) and the BrdU incorporation assays were used to measure apoptosis and cell proliferation, respectively. ACK1-specific siRNA and the pharmacological ACK1 inhibitor significantly abrogated the TNF-alpha-mediated anti-apoptotic effects and proliferation of Caco-2 cells. Interestingly, TNF-alpha activated ACK1 at tyrosine 284 (Tyr284), and the ErbB family of proteins was implicated in ACK1 activation in Caco-2 cells. ACK1-Tyr284 was required for protein kinase B (AKT) activation, and ACK1 signaling was mediated through recruiting and phosphorylating the down-stream adaptor protein AKT, which likely promoted cell proliferation in response to TNF-alpha. Moreover, ACK1 activated AKT and Src enhanced nuclear factor-кB (NF-кB) activity, suggesting a correlation between NF-кB signaling and TNF-alpha-mediated apoptosis in Caco-2 cells. Our results demonstrate that ACK1 plays an important role in modulating TNF-alpha-induced aberrant cell proliferation and apoptosis, mediated in part by ACK1 activation. ACK1 and its down-stream effectors may hold promise as therapeutic targets in the prevention and treatment of gastrointestinal cancers, in particular, those induced by chronic intestinal inflammation. © 2017 The Authors. Cell Biology International Published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.

  9. [Construction of chicken embryo fibroblasts cDNA expression library].

    Science.gov (United States)

    Liu, Wei; Gao, Yu-long; Gao, Hong-lei; Wang, Xiao-mei; Xu, Xiu-hong

    2007-06-01

    Chicken embryo fibroblast (CEF) is a primary cellular material to research the infectious bursal disease virus (IBDV). Constructing the cDNA expression library of CEF is the foundation to research cell tropism and find cell receptors of IBDV from CEF. In order to achieve that purpose, a high-quality cDNA expression library of CEF was constructed by Gateway technology, which could avoid using the restriction enzyme for cloning to solve technical limitation of roution method. The mRNA was extracted from chicken embryonic fibroblast. Moreover, single-strand cDNA and double-strand cDNA were synthesized by using biotin-conjugated Oligo (dT) primer in turn. The double-strand cDNA was ligated Adapter and then purified by the cDNA Size Fractionation Columns. After BP recombination reaction, a cDNA entry library was constructed with a titer of 1 x 10(6) cfu/mL, total clones of 1.2 x 10(7) cfu and an average insertion size of about 2243 bp. After LR recombination reaction, the cDNA entry library was transformed into expression library which took on a titer of 5 x 10(5) cfu/mL, total clones of 5.5 x 10(6) cfu and an average insertion size of about 2411bp. The results indicate that the constructed cDNA expression library performs a remarkable high value in both recombination rate and library coverage. As a result, the cDNA expression library, with its good quality, may facilitate to identify the receptors associated with the resistance against IBDV in chicken embryonic fibroblast and to cast new light on the mechanism of cellular tropism. Moreover, it may also provide data of chicken embryonic fibroblast in transcription level and may be helpful to study its biological functions.

  10. TNF and LT binding capacities in the plasma of arthritis patients: effect of etanercept treatment in juvenile idiopathic arthritis

    DEFF Research Database (Denmark)

    Gudbrandsdottir, S; Larsen, R; Sørensen, L K

    2004-01-01

    necrosis factor (TNF, previously termed TNF alpha) and lymphotoxin (LT, previously termed TNF beta) may be mediated by etanercept itself as well as by naturally occurring soluble TNF receptors. In light of this, it was of interest to study the total TNF neutralizing capacity in plasma before and during...

  11. Normalizing cDNA libraries.

    Science.gov (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A

    2010-04-01

    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  12. cDNA library preparation.

    Science.gov (United States)

    Kooiker, Maarten; Xue, Gang-Ping

    2014-01-01

    The construction of full-length cDNA libraries allows researchers to study gene expression and protein interactions and undertake gene discovery. Recent improvements allow the construction of high-quality cDNA libraries, with small amounts of mRNA. In parallel, these improvements allow for the incorporation of adapters into the cDNA, both at the 5' and 3' end of the cDNA. The 3' adapter is attached to the oligo-dT primer that is used by the reverse transcriptase, whereas the 5' adapter is incorporated by the template switching properties of the MMLV reverse transcriptase. This allows directional cloning and eliminates inefficient steps like adapter ligation, phosphorylation, and methylation. Another important step in the construction of high-quality cDNA libraries is the normalization. The difference in the levels of expression between genes might be several orders of magnitude. Therefore, it is essential that the cDNA library is normalized. With a recently discovered enzyme, duplex-specific nuclease, it is possible to normalize the cDNA library, based on the fact that more abundant molecules are more likely to reanneal after denaturation compared to rare molecules.

  13. Normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1997-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to moderate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library.

  14. Dichotomal role of TNF in experimental pulmonary edema reabsorption

    NARCIS (Netherlands)

    Braun, C; Hamacher, J; Morel, DR; Wendel, A; Lucas, R

    2005-01-01

    Distinct from its receptor binding sites, TNF carries a lectin-like domain, situated at the tip of the molecule, which specifically binds oligosaccharides, such as NN'-diacetylchitobiose. In view of the apparently conflicting data concerning TNF actions in pulmonary edema, we investigated the

  15. Molecular probing of TNF: From identification of therapeutic target to guidance of therapy in inflammatory diseases.

    Science.gov (United States)

    Chu, Cong-Qiu

    2018-01-01

    Therapy by blocking tumor necrosis factor (TNF) activity is highly efficacious and profoundly changed the paradigm of several inflammatory diseases. However, a significant proportion of patients with inflammatory diseases do not respond to TNF inhibitors (TNFi). Prediction of therapeutic response is required for TNFi therapy. Isotope labeled anti-TNF antibodies or TNF receptor have been investigated to localize TNF production at inflammatory tissue in animal models and in patients with inflammatory diseases. The in vivo detection of TNF has been associated with treatment response. Recently, fluorophore labeled anti-TNF antibody in combination with confocal laser endomicroscopy in patients with Crohn's disease yielded more accurate and quantitative in vivo detection of TNF in the diseased mucosa. More importantly, this method demonstrated high therapeutic predication value. Fluorophore labeled TNF binding aptamers in combination with modern imaging technology offers additional tools for in vivo TNF probing. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Functional cloning using pFB retroviral cDNA expression libraries.

    Science.gov (United States)

    Felts, Katherine A; Chen, Keith; Zaharee, Kim; Sundar, Latha; Limjoco, Jamie; Miller, Anna; Vaillancourt, Peter

    2002-09-01

    Retroviral cDNA expression libraries allow the efficient introduction of complex cDNA libraries into virtually any mitotic cell type for screening based on gene function. The cDNA copy number per cell can be easily controlled by adjusting the multiplicity of infection, thus cell populations may be generated in which >90% of infected cells contain one to three cDNAs. We describe the isolation of two known oncogenes and one cell-surface receptor from a human Burkitt's lymphoma (Daudi) cDNA library inserted into the high-titer retroviral vector pFB.

  17. Diverging mechanisms for TNF-alpha receptors in normal mouse brains and in functional recovery after injury: From gene to behavior

    DEFF Research Database (Denmark)

    Quintana, Albert; Molinero, Amalia; Florit, Sergi

    2007-01-01

    , and ambulations, compared with wild-type mice, suggesting an inhibitory role of TNFR1/TNFR2 signaling. In contrast, no significant differences were observed in the elevated plus maze test, ruling out a major role of these receptors in the control of anxiety. We next evaluated the response to a freeze injury...

  18. Molecular mechanisms of action of anti-TNF-α agents - Comparison among therapeutic TNF-α antagonists.

    Science.gov (United States)

    Mitoma, Hiroki; Horiuchi, Takahiko; Tsukamoto, Hiroshi; Ueda, Naoyasu

    2018-01-01

    Tumor necrosis factor (TNF)-α is a potent pro-inflammatory and pathological cytokines in inflammatory diseases such as rheumatoid arthritis and inflammatory bowel diseases. Anti-TNF-α therapy has been established as an efficacious therapeutic strategy in these diseases. In clinical settings, three monoclonal anti-TNF-α full IgG1 antibodies infliximab, adalimumab, and golimumab, PEGylated Fab' fragment of anti-TNF-α antibody certolizumab pegol, extracellular domain of TNF receptor 2/IgG1-Fc fusion protein etanercept, are almost equally effective for rheumatoid arthritis. Although monoclonal full IgG1 antibodies are able to induce clinical and endoscopic remission in inflammatory bowel diseases, certolizumab pegol without Fc portion has been shown to be less effective for inflammatory bowel diseases compared to full IgG1 antibodies. In addition, there are no evidences that etanercept leads clinical remission in inflammatory bowel diseases. Besides the common effect of anti-TNF-α agents on neutralization of soluble TNF-α, each anti-TNF-α agent has its own distinctive pharmacological properties which cause the difference in clinical efficacies. Here we focus on the distinctions of action of anti-TNF-α agents especially in following points; (1) blocking ability against ligands, transmembrane TNF-α and lymphotoxin, (2) effects toward transmembrane TNF-α-expressing cells, (3) effects toward Fcγ receptor-expressing cells, (4) degradation and distribution in inflamed tissue. Accumulating evidence will give us the idea how to modify anti-TNF-α agents to enhance the clinical efficacy in inflammatory diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors

    OpenAIRE

    1992-01-01

    The receptors for tumor necrosis factor (TNF) exist in cell-associated as well as soluble forms, both binding specifically to TNF. Since the soluble forms of TNF receptors (sTNF-Rs) can compete with the cell- associated TNF receptors for TNF, it was suggested that they function as inhibitors of TNF activity; at high concentrations, the sTNF-Rs indeed inhibit TNF effects. However, we report here that in the presence of low concentrations of the sTNF-Rs, effects of TNF whose induction depend on...

  20. Protective effect of chlorpromazine on TNF-mediated hapten-induced irritant reaction.

    Science.gov (United States)

    Erroi, A; Fantuzzi, G; Demitri, M T; Echtenacher, B; Gnocchi, P; Isetta, A; Ghezzi, P

    1995-01-01

    Picryl chloride-induced irritant reaction (IR) was shown to be mediated by tumor necrosis factor (TNF). Anti-TNF monoclonal antibodies, but not interleukin 1 receptor antagonist (IL-1 Ra), had a protective effect. Chlorpromazine (CPZ), an inhibitor of TNF synthesis, protected against IR and inhibited the IR-associated TNF induction in ear homogenates. Investigation of the role of polymorphonuclear leukocyte (PMN) in neutropenic mice showed that neutropenia did not prevent the development of the IR.

  1. TNF biology, pathogenic mechanisms and emerging therapeutic strategies

    Science.gov (United States)

    Kalliolias, George D.; Ivashkiv, Lionel B.

    2016-01-01

    TNF is a pleiotropic cytokine with important functions in homeostasis and disease pathogenesis. Recent discoveries have provided insights into TNF biology that introduce new concepts for the development of therapeutics for TNF-mediated diseases. The model of TNF receptor signalling has been extended to include linear ubiquitination and the formation of distinct signalling complexes that are linked with different functional outcomes, such as inflammation, apoptosis and necroptosis. Our understanding of TNF-induced gene expression has been enriched by the discovery of epigenetic mechanisms and concepts related to cellular priming, tolerization and induction of ‘short-term transcriptional memory’. Identification of distinct homeostatic or pathogenic TNF-induced signalling pathways has introduced the concept of selectively inhibiting the deleterious effects of TNF while preserving its homeostatic bioactivities for therapeutic purposes. In this Review, we present molecular mechanisms underlying the roles of TNF in homeostasis and inflammatory disease pathogenesis, and discuss novel strategies to advance therapeutic paradigms for the treatment of TNF-mediated diseases. PMID:26656660

  2. Skin cancer associated with commonly prescribed drugs: tumor necrosis factor alpha inhibitors (TNF-αIs), angiotensin-receptor blockers (ARBs), phosphodiesterase type 5 inhibitors (PDE5Is) and statins -weighing the evidence.

    Science.gov (United States)

    Nardone, Beatrice; Orrell, Kelsey A; Vakharia, Paras P; West, Dennis P

    2018-02-01

    Skin cancers, including both malignant melanoma (MM) and nonmelanoma skin cancer (NMSC), are the most commonly diagnosed cancers in the US. The incidence of both MM and NMSC continues to rise. Areas covered: Current evidence for an association between four of the most commonly prescribed classes of drugs in the U.S. and risk for MM and NMSC is reported. Medline was searched (January 2000 to May 2017) for each drug in the classes and for 'basal cell carcinoma', 'squamous cell carcinoma', 'non-melanoma skin cancer', 'skin cancer' and 'melanoma'. Skin cancer risk information was reported for: tumor necrosis factor alpha inhibitors (TNF-αIs), angiotensin-receptor blockers (ARBs), phosphodiesterase type 5 inhibitors (PDE5Is) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA)-reductase inhibitors (statins). Expert opinion: Since skin cancer risk is associated with all four classes of these commonly prescribed drugs that represent nearly 20% of the Top 100 drugs in the U.S., these important findings warrant enhanced education, especially for prescribers and those patients at high risk for skin cancer.

  3. Evaluation of preventive and therapeutic activity of novel non-steroidal anti-inflammatory drug, CG100649, in colon cancer: Increased expression of TNF-related apoptosis-inducing ligand receptors enhance the apoptotic response to combination treatment with TRAIL.

    Science.gov (United States)

    Woo, Jong Kyu; Kang, Ju-Hee; Jang, Yeong-Su; Ro, Seonggu; Cho, Joong Myung; Kim, Hwan-Mook; Lee, Sang-Jin; Oh, Seung Hyun

    2015-04-01

    Non-steroidal anti-inflammatory drugs (NSAIDs) have been suggested as the potential new class of preventive or therapeutic antitumor agents. The aim of the present study was to evaluate the antitumor activity of the novel NSAID, CG100649. CG100649 is a novel NSAID dual inhibitor for COX-2 and carbonic anhydrase (CA)-I/-II. In the present study, we investigated the alternative mechanism by which CG100649 mediated suppression of the colon cancer growth and development. The anchorage‑dependent and -independent clonogenic assay showed that CG100649 inhibited the clonogenicity of human colon cancer cells. The flow cytometric analysis showed that CG100649 induced the G2/M cell cycle arrest in colon cancer cells. Animal studies showed that CG100649 inhibited the tumor growth in colon cancer xenograft in nude mice. Furthermore, quantitative PCR and FACS analysis demonstrated that CG100649 upregulated the expression of TNF-related apoptosis-inducing ligand (TRAIL) receptors (DR4 and DR5) but decreased the expression of decoy receptors (DcR1 and DcR2) in colon cancer cells. The results showed that CG100649 treatment sensitized TRAIL‑mediated growth suppression and apoptotic cell death. The combination treatment resulted in significant repression of the intestinal polyp formation in APCmin/+ mice. Our data clearly demonstrated that CG100649 contains preventive and therapeutic activity for colon cancer. The present study may be useful for identification of the potential benefit of the NSAID CG100649, for the achievement of a better treatment response in colon cancer.

  4. HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2.

    Directory of Open Access Journals (Sweden)

    Giorgio Mangino

    Full Text Available BACKGROUND: HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs. We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3. METHODOLOGY/PRINCIPAL FINDINGS: Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2. CONCLUSIONS: Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.

  5. NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1989-01-01

    The restriction fragment length polymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5...

  6. NcoI restriction fragment length polymorphism (RFLP) of the tumour necrosis factor (TNF alpha) region in primary biliary cirrhosis and in healthy Danes

    DEFF Research Database (Denmark)

    Fugger, L; Morling, N; Ryder, L P

    1989-01-01

    The restriction fragment length polymorphism of the human tumour necrosis factor (TNF alpha) region was investigated by means of 20 different restriction enzymes and a human TNF alpha cDNA probe. Only one of the enzymes, NcoI, revealed a polymorphic pattern consisting of fragments of 10.5 and 5.5...

  7. Efficacy Testing of H56 cDNA Tattoo Immunization against Tuberculosis in a Mouse Model.

    Science.gov (United States)

    Platteel, Anouk C M; Nieuwenhuizen, Natalie E; Domaszewska, Teresa; Schürer, Stefanie; Zedler, Ulrike; Brinkmann, Volker; Sijts, Alice J A M; Kaufmann, Stefan H E

    2017-01-01

    -derived epitopes were predominantly IFN-γ + TNF-α + and TNF-α + IL-2 + , respectively. In conclusion, despite inducing appreciable immune responses to Ag85B and ESAT-6, intradermal H56 cDNA tattoo immunization did not substantially enhance the protective effect of BCG under the conditions tested.

  8. Astrocyte galectin-9 potentiates microglial TNF secretion.

    Science.gov (United States)

    Steelman, Andrew J; Li, Jianrong

    2014-08-27

    Aberrant neuroinflammation is suspected to contribute to the pathogenesis of myriad neurological diseases. As such, determining the pathways that promote or inhibit glial activation is of interest. Activation of the surface glycoprotein T-cell immunoglobulin and mucin-domain containing protein 3 (Tim-3) by the lectin galectin-9 has been implicated in promoting innate immune cell activation by potentiating or synergizing toll-like receptor (TLR) signaling. In the present study we examined the role of the Tim-3/galectin-9 pathway in glial activation in vitro. Primary monocultures of microglia or astrocytes, co-cultures containing microglia and astrocytes, and mixed glial cultures consisting of microglia, astrocytes and oligodendrocytes were stimulated with poly(I:C) or LPS, and galectin-9 up-regulation was determined. The effect of endogenous galectin-9 production on microglial activation was examined using cultures from wild-type and Lgals9 null mice. The ability for recombinant galectin-9 to promote microglia activation was also assessed. Tim-3 expression on microglia and BV2 cells was examined by qPCR and flow cytometry and its necessity in transducing the galectin-9 signal was determined using a Tim-3 specific neutralizing antibody or recombinant soluble Tim-3. Astrocytes potentiated TNF production from microglia following TLR stimulation. Poly(I:C) stimulation increased galectin-9 expression in microglia and microglial-derived factors promoted galectin-9 up-regulation in astrocytes. Astrocyte-derived galectin-9 in turn enhanced microglial TNF production. Similarly, recombinant galectin-9 enhanced poly(I:C)-induced microglial TNF and IL-6 production. Inhibition of Tim-3 did not alter TNF production in mixed glial cultures stimulated with poly(I:C). Galectin-9 functions as an astrocyte-microglia communication signal and promotes cytokine production from microglia in a Tim-3 independent manner. Activation of CNS galectin-9 likely modulates neuroinflammatory

  9. Multiple signal transduction pathways regulate TNF-induced actin reorganization in macrophages: inhibition of Cdc42-mediated filopodium formation by TNF

    NARCIS (Netherlands)

    Peppelenbosch, M.; Boone, E.; Jones, G. E.; van Deventer, S. J.; Haegeman, G.; Fiers, W.; Grooten, J.; Ridley, A. J.

    1999-01-01

    TNF is known to regulate macrophage (Mphi) migration, but the signaling pathways mediating this response have not been established. Here we report that stimulation of the 55-kDa TNF receptor (TNFR-1) induced an overall decrease in filamentous actin (F-actin), inhibited CSF-1- and Cdc42-dependent

  10. TNF signaling inhibition in the CNS: implications for normal brain function and neurodegenerative disease

    Directory of Open Access Journals (Sweden)

    Tansey Malú G

    2008-10-01

    Full Text Available Abstract The role of tumor necrosis factor (TNF as an immune mediator has long been appreciated but its function in the brain is still unclear. TNF receptor 1 (TNFR1 is expressed in most cell types, and can be activated by binding of either soluble TNF (solTNF or transmembrane TNF (tmTNF, with a preference for solTNF; whereas TNFR2 is expressed primarily by microglia and endothelial cells and is preferentially activated by tmTNF. Elevation of solTNF is a hallmark of acute and chronic neuroinflammation as well as a number of neurodegenerative conditions including ischemic stroke, Alzheimer's (AD, Parkinson's (PD, amyotrophic lateral sclerosis (ALS, and multiple sclerosis (MS. The presence of this potent inflammatory factor at sites of injury implicates it as a mediator of neuronal damage and disease pathogenesis, making TNF an attractive target for therapeutic development to treat acute and chronic neurodegenerative conditions. However, new and old observations from animal models and clinical trials reviewed here suggest solTNF and tmTNF exert different functions under normal and pathological conditions in the CNS. A potential role for TNF in synaptic scaling and hippocampal neurogenesis demonstrated by recent studies suggest additional in-depth mechanistic studies are warranted to delineate the distinct functions of the two TNF ligands in different parts of the brain prior to large-scale development of anti-TNF therapies in the CNS. If inactivation of TNF-dependent inflammation in the brain is warranted by additional pre-clinical studies, selective targeting of TNFR1-mediated signaling while sparing TNFR2 activation may lessen adverse effects of anti-TNF therapies in the CNS.

  11. Increasing TNF levels solely in the rat hippocampus produces persistent pain-like symptoms.

    Science.gov (United States)

    Martuscello, Regina T; Spengler, Robert N; Bonoiu, Adela C; Davidson, Bruce A; Helinski, Jadwiga; Ding, Hong; Mahajan, Supriya; Kumar, Rajiv; Bergey, Earl J; Knight, Paul R; Prasad, Paras N; Ignatowski, Tracey A

    2012-09-01

    The manifestation of chronic, neuropathic pain includes elevated levels of the cytokine tumor necrosis factor-alpha (TNF). Previously, we have shown that the hippocampus, an area of the brain most notable for its role in learning and memory formation, plays a fundamental role in pain sensation. Using an animal model of peripheral neuropathic pain, we have demonstrated that intracerebroventricular infusion of a TNF antibody adjacent to the hippocampus completely alleviated pain. Furthermore, intracerebroventricular infusion of rTNF adjacent to the hippocampus induced pain behavior in naïve animals similar to that expressed during a model of neuropathic pain. These data support our premise that enhanced production of hippocampal-TNF is integral in pain sensation. In the present study, TNF gene expression was induced exclusively in the hippocampus, eliciting increased local bioactive TNF levels, and animals were assessed for pain behaviors. Male Sprague-Dawley rats received stereotaxic injection of gold nanorod (GNR)-complexed cDNA (control or TNF) plasmids (nanoplasmidexes), and pain responses (i.e., thermal hyperalgesia and mechanical allodynia) were measured. Animals receiving hippocampal microinjection of TNF nanoplasmidexes developed thermal hyperalgesia bilaterally. Sensitivity to mechanical stimulation also developed bilaterally in the rat hind paws. In support of these behavioral findings, immunoreactive staining for TNF, bioactive levels of TNF, and levels of TNF mRNA per polymerase chain reaction analysis were assessed in several brain regions and found to be increased only in the hippocampus. These findings indicate that the specific elevation of TNF in the hippocampus is not a consequence of pain, but in fact induces these behaviors/symptoms. Copyright © 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  12. TNF Counterbalances the Emergence of M2 Tumor Macrophages

    Directory of Open Access Journals (Sweden)

    Franz Kratochvill

    2015-09-01

    Full Text Available Cancer can involve non-resolving, persistent inflammation where varying numbers of tumor-associated macrophages (TAMs infiltrate and adopt different activation states between anti-tumor M1 and pro-tumor M2 phenotypes. Here, we resolve a cascade causing differential macrophage phenotypes in the tumor microenvironment. Reduction in TNF mRNA production or loss of type I TNF receptor signaling resulted in a striking pattern of enhanced M2 mRNA expression. M2 gene expression was driven in part by IL-13 from eosinophils co-recruited with inflammatory monocytes, a pathway that was suppressed by TNF. Our data define regulatory nodes within the tumor microenvironment that balance M1 and M2 populations. Our results show macrophage polarization in cancer is dynamic and dependent on the balance between TNF and IL-13, thus providing a strategy for manipulating TAMs.

  13. Transglutaminase-catalyzed covalent multimerization of Camelidae anti-human TNF single domain antibodies improves neutralizing activity.

    Science.gov (United States)

    Plagmann, Ingo; Chalaris, Athena; Kruglov, Andrei A; Nedospasov, Sergei; Rosenstiel, Philip; Rose-John, Stefan; Scheller, Jürgen

    2009-06-15

    Tumor necrosis factor (TNF) plays an important role in chronic inflammatory disorders, such as Rheumatoid Arthritis and Crohn's disease. Recently, monoclonal Camelidae variable heavy-chain domain-only antibodies (V(H)H) were developed to antagonize the action of human TNF (hTNF). Here, we show that hTNF-V(H)H does not interfere with hTNF trimerization, but competes with hTNF for hTNF-receptor binding. Moreover, we describe posttranslational dimerization and multimerization of hTNF-V(H)H molecules in vitro catalyzed by microbial transglutaminases (MTG). The ribonuclease S-tag-peptide was shown to act as a peptidyl substrate in covalent protein cross-linking reactions catalyzed by MTG from Streptomyces mobaraensis. The S-tag sequence was C-terminally fused to the hTNF-V(H)H and the fusion protein was expressed and purified from Escherichia coli culture supernatants. hTNF-V(H)H-S-tag fusion proteins were efficiently dimerized and multimerized by MTG whereas hTNF-V(H)H was not susceptible to protein cross-linking. Cell cytotoxicity assays, using hTNF as apoptosis inducing cytokine, revealed that dimerized and multimerized hTNF-V(H)H proteins were much more active than the monomeric hTNF-V(H)H. We hypothesize that improved inhibition by dimeric and multimeric single chain hTNF-V(H)H proteins is caused by avidity effects.

  14. Vibration induced hearing loss in guinea pig cochlea: expression of TNF-alpha and VEGF.

    Science.gov (United States)

    Zou, Jing; Pyykkö, Ilmari; Sutinen, Päivi; Toppila, Esko

    2005-04-01

    Transcranial vibration was applied for seven animals at a frequency of 250 Hz for 15 min, and five animals were used as normal controls to investigate cellular and molecular mechanism linked to vibration-induced hearing loss in animal model. Compound action potential (CAP) thresholds were measured by round window niche electrode. The expression of tumour necrosis factor alpha (TNF-alpha) and its receptors (TNF R1, TNF R2), vascular endothelium growth factor (VEGF) and its receptors (VEGF R1, VEGF R2) were analysed by immunohistochemistry. Transcranial vibration caused expression of TNF-alpha, TNF R1 and TNF R2 in the cochlea and the expression of TNF R2 was stronger than that of TNF R1. Vibration also induced VEGF and VEGF R2 expression in the cochlea. The average immediate hearing loss was 62 dB and after three days still 48 dB. It is concluded that transcranial vibration as during temporal bone drilling produces cochlear shear stress that is connected with up-regulation of TNF-alpha and its receptors. Also VEGF and VEGF R2 are up-regulated. These responses may be linked to both the damage and repair process of the cochlea.

  15. TNF activity and T cells.

    Science.gov (United States)

    Mehta, Amit K; Gracias, Donald T; Croft, Michael

    2018-01-01

    TNF (tumor necrosis factor) is both a pro-inflammatory and anti-inflammatory cytokine that is central to the development of autoimmune disease, cancer, and protection against infectious pathogens. As well as a myriad other activities, TNF can be a product of T cells and can act on T cells. Here we review old and new data on the importance of TNF produced by T cells and how TNF signaling via TNFR2 may directly impact alternate aspects of T cell biology. TNF can promote the activation and proliferation of naïve and effector T cells, but also can induce apoptosis of highly activated effector T cells, further determining the size of the pathogenic or protective conventional T cell pool. Moreover, TNF can have divergent effects on regulatory T cells. It can both downregulate their suppressive capacity, but also contribute in other instances to their development or accumulation. Biologics that block TNF or stimulate TNFR2 therefore have the potential to strongly modulate the balance between effector T cells and Treg cells which could impact disease in both positive and negative manners. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. TNF signaling in non-alcoholic fatty liver disease

    NARCIS (Netherlands)

    Vergara, Marcela Aparicio

    2013-01-01

    Tumornecrosefactor-alpha (TNF-α, een eiwit dat verantwoordelijk is voor het proces van celdood en cel-ontsteking), speelt via receptor TNFR1 een centrale rol in het ontstaan van niet-alcoholische leververvetting (NAFLD). Maar anders dan tot nu toe gedacht, bestaat er geen verband tussen

  17. Disease causing mutations in the TNF and TNFR superfamilies: Focus on molecular mechanisms driving disease

    NARCIS (Netherlands)

    Lobito, Adrian A.; Gabriel, Tanit L.; Medema, Jan Paul; Kimberley, Fiona C.

    2011-01-01

    The tumor necrosis factor (TNF) and TNF receptor (TNFR) superfamilies comprise multidomain proteins with diverse roles in cell activation, proliferation and cell death. These proteins play pivotal roles in the initiation, maintenance and termination of immune responses and have vital roles outside

  18. TNF-α Deficiency Prevents Renal Inflammation and Oxidative Stress in Obese Mice

    Directory of Open Access Journals (Sweden)

    Huaiguo Wang

    2017-07-01

    Full Text Available Background/Aims: Obese patients and experimental animals exhibit high levels of inflammatory cytokines, such as tumor necrosis factor (TNF-α. However, the role of TNF-α in the pathophysiologic process in obesity induced kidney damage is still unknown. Methods: We used TNF-α deficient mice and wild-type (WT C57/BJ6 mice controls to study the effect of TNF-α on inflammation and oxidative stress in kidney by the model of high-fat diet (HFD and primary isolated mouse renal proximal tubule cells treated with a mixture of free fatty acids (FFA. Results: Compared with the chow diet group, HFD-fed WT mice had higher urinary albumin and increased levels of renal fibrosis, glomerulosclerosis, inflammation, oxidative stress and apoptosis in the kidney. These changes were co-related with increased expression of TNF-α in the kidney and were attenuated by TNF-α deficiency. In vitro, accumulation of intracellular lipids induced TNF-α expression and oxidative stress in FFA treated primary proximal tubule cells. However, TNF-α inhibition with siRNA or TNF-α deficiency decreased the lipid induced oxidative stress in these cells. Conclusion: These findings suggest that TNF-α plays an important role in the HFD induced kidney damage, and targeting TNF-α and/or its receptors could be a promising therapeutic regimen for progressive nephropathy.

  19. MK2-TNF-Signaling Comes Full Circle.

    Science.gov (United States)

    Menon, Manoj B; Gaestel, Matthias

    2017-12-21

    MK2 (p38 MAPK -activated protein kinase 2) is essential for tumor necrosis factor (TNF) biosynthesis, mainly operating by post-transcriptional regulation. Deletion of the gene encoding MK2 strongly reduced serum TNF and protected against endotoxic shock, demonstrating the positive role of p38 MAPK /MK2 in TNF signaling at the level of ligand expression. Recent evidence indicates that MK2 directly phosphorylates the TNF receptor interactor RIPK1 and suppresses its activity, thereby limiting TNF-mediated apoptosis and necroptosis - pointing to a more complex, double-edged role of MK2 in TNF signaling. In addition, novel MK2 substrates have emerged in the DNA damage response, autophagy, and obesity, making MK2 a multifunctional kinase at the crossroads of stress response and cell death. We therefore propose a more general role of p38 MAPK /MK2 signaling in the timely coordinated onset and resolution of inflammation and beyond. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. TNF Superfamily: A Growing Saga of Kidney Injury Modulators

    Directory of Open Access Journals (Sweden)

    Maria D. Sanchez-Niño

    2010-01-01

    Full Text Available Members of the TNF superfamily participate in kidney disease. Tumor necrosis factor (TNF and Fas ligand regulate renal cell survival and inflammation, and therapeutic targeting improves the outcome of experimental renal injury. TNF-related apoptosis-inducing ligand (TRAIL and its potential decoy receptor osteoprotegerin are the two most upregulated death-related genes in human diabetic nephropathy. TRAIL activates NF-kappaB in tubular cells and promotes apoptosis in tubular cells and podocytes, especially in a high-glucose environment. By contrast, osteoprotegerin plays a protective role against TRAIL-induced apoptosis. Another family member, TNF-like weak inducer of apoptosis (TWEAK induces inflammation and tubular cell death or proliferation, depending on the microenvironment. While TNF only activates canonical NF-kappaB signaling, TWEAK promotes both canonical and noncanonical NF-kappaB activation in tubular cells, regulating different inflammatory responses. TWEAK promotes the secretion of MCP-1 and RANTES through NF-kappaB RelA-containing complexes and upregulates CCl21 and CCL19 expression through NF-kappaB inducing kinase (NIK- dependent RelB/NF-kappaB2 complexes. In vivo TWEAK promotes postnephrectomy compensatory renal cell proliferation in a noninflammatory milieu. However, in the inflammatory milieu of acute kidney injury, TWEAK promotes tubular cell death and inflammation. Therapeutic targeting of TNF superfamily cytokines, including multipronged approaches targeting several cytokines should be further explored.

  1. Efficacy of the combined therapy with etanercept and methotrexat at the female patient with generalized pustular psoriasis and arthritis psoriasis after the therapeutic failure of two the TNFreceptor inhibitors. Case study

    Directory of Open Access Journals (Sweden)

    V. V. Vustina

    2016-01-01

    Full Text Available The article presents the clinical observation effectiveness of TNF-a inhibitor etanercept in patients with generalized pustular psoriasis and PsA, resistant to treatment by other drugs of this class. It presents the current recommendations for the management of patients with pustular psoriasis.

  2. Anti-TNF-α biotherapies

    DEFF Research Database (Denmark)

    Bendtzen, Klaus

    2012-01-01

    This article discusses the rationale behind recommending immunopharmacological guidance of long-term therapies with genetically engineered anti-TNF-α immunoglobulin constructs. Arguments why therapeutic decision-making should not rely on clinical outcome alone are presented. Central...... to this is that the use of theranostics (i.e., monitoring circulating levels of functional anti-TNF-α drugs and antidrug antibodies) would markedly improve treatment because therapies can be tailored to individual patients and provide more effective and economical long-term therapies with minimal risk of side effects....... Large-scale immunopharmacological knowledge of how patients 'handle' TNF-α biopharmaceuticals would also help industry develop more effective and safer TNF-α inhibitors....

  3. Inhibiting TNF-α signaling does not attenuate induction of endotoxin tolerance

    Directory of Open Access Journals (Sweden)

    Loosbroock C

    2014-12-01

    Full Text Available Christopher Loosbroock, Kenneth W Hunter Department of Microbiology and Immunology, University of Nevada School of Medicine, Reno, NV, USA Abstract: Tumor necrosis factor-alpha (TNF-α is a central mediator of inflammatory responses elicited by Toll-like receptor agonists, such as the Gram-negative bacterial outer membrane antigen lipopolysaccharide (LPS. TNF-α is responsible for altering vascular permeability and activating infiltrating inflammatory cells, such as monocytes and neutrophils. Interestingly, TNF-α has also demonstrated the ability to induce tolerance to subsequent challenges with TNF-α or LPS in monocyte and macrophage cell populations. Tolerance is characterized by the inability to mount a typical inflammatory response during subsequent challenges following the initial exposure to an inflammatory mediator such as LPS. The ability of TNF-α to induce a tolerant-like state with regard to LPS is most likely a regulatory mechanism to prevent excessive inflammation. We hypothesized that the induction of tolerance or the degree of tolerance is dependent upon the production of TNF-α during the primary response to LPS. To investigate TNF-α-dependent tolerance, human monocytic THP-1 cells were treated with TNF-α-neutralizing antibodies or antagonistic TNFreceptor antibodies before primary LPS stimulation and then monitored for the production of TNF-α during the primary and challenge stimulation. During the primary stimulation, anti-TNF-α treatment effectively attenuated the production of TNF-α and interleukin-1β; however, this reduced production did not impact the induction of endotoxin tolerance. These results demonstrate that interfering with TNF-α signaling attenuates production of inflammatory cytokines without affecting the induction of tolerance. Keywords: endotoxin tolerance, lipopolysaccharide, tumor necrosis factor-alpha, anti-tumor necrosis factor-alpha, THP-1 cells

  4. Anti-TNF drives regulatory T cell expansion by paradoxically promoting membrane TNF–TNF-RII binding in rheumatoid arthritis

    Science.gov (United States)

    2016-01-01

    The interplay between inflammatory and regulatory pathways orchestrates an effective immune response that provides protection from pathogens while limiting injury to host tissue. Tumor necrosis factor (TNF) is a pivotal inflammatory cytokine, but there is conflicting evidence as to whether it boosts or inhibits regulatory T cells (T reg cells). In this study, we show that the therapeutic anti-TNF antibody adalimumab, but not the soluble TNF receptor etanercept, paradoxically promoted the interaction between monocytes and T reg cells isolated from patients with rheumatoid arthritis (RA). Adalimumab bound to monocyte membrane TNF from RA patients and unexpectedly enhanced its expression and its binding to TNF-RII expressed on T reg cells. As a consequence, adalimumab expanded functional Foxp3+ T reg cells equipped to suppress Th17 cells through an IL-2/STAT5-dependent mechanism. Our data not only highlight the beneficial effect of membrane TNF on T reg cell numbers during chronic inflammation, but in addition reveal how a therapeutic antibody that is thought to act by simply blocking its target can enhance the regulatory properties of this proinflammatory cytokine. PMID:27270893

  5. Androgen receptor abnormalities

    NARCIS (Netherlands)

    A.O. Brinkmann (Albert); G.G.J.M. Kuiper (George); C. Ris-Stalpers (Carolyn); H.C.J. van Rooij (Henri); G. Romalo (G.); G. Trifiro (Gianluca); E. Mulder (Eppo); L. Pinsky (L.); H.U. Schweikert (H.); J. Trapman (Jan)

    1991-01-01

    markdownabstract__Abstract__ The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of

  6. Androgen receptor abnormalities

    NARCIS (Netherlands)

    Brinkmann, A. O.; Kuiper, G. G.; Ris-Stalpers, C.; van Rooij, H. C.; Romalo, G.; Trifiro, M.; Mulder, E.; Pinsky, L.; Schweikert, H. U.; Trapman, J.

    1991-01-01

    The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the

  7. cAMP prevents TNF-induced apoptosis through inhibiting DISC complex formation in rat hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharjee, Rajesh [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Xiang, Wenpei [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States); Family Planning Research Institute, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, People' s Republic of China (China); Wang, Yinna [Vascular Medicine Institute, University of Pittsburgh School of Medicine, 10051-5A BST 3, 3501 Fifth Avenue, Pittsburgh, PA 15261 (United States); Zhang, Xiaoying [Department of Medicine/Endocrinology Division, University of Pittsburgh Medical Center, 200 Lothrop St., Pittsburgh, PA 15213 (United States); Billiar, Timothy R., E-mail: billiartr@upmc.edu [Department of Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA 15213 (United States)

    2012-06-22

    Highlights: Black-Right-Pointing-Pointer cAMP blocks cell death induced by TNF and actinomycin D in cultured hepatocytes. Black-Right-Pointing-Pointer cAMP blocks NF-{kappa}B activation induced by TNF and actinomycin D. Black-Right-Pointing-Pointer cAMP blocks DISC formation following TNF and actinomycin D exposure. Black-Right-Pointing-Pointer cAMP blocks TNF signaling at a proximal step. -- Abstract: Tumor necrosis factor {alpha} (TNF) is a pleiotropic proinflammatory cytokine that plays a role in immunity and the control of cell proliferation, cell differentiation, and apoptosis. The pleiotropic nature of TNF is due to the formation of different signaling complexes upon the binding of TNF to its receptor, TNF receptor type 1 (TNFR1). TNF induces apoptosis in various mammalian cells when the cells are co-treated with a transcription inhibitor like actinomycin D (ActD). When TNFR1 is activated, it recruits an adaptor protein, TNF receptor-associated protein with death domain (TRADD), through its cytoplasmic death effector domain (DED). TRADD, in turn, recruits other signaling proteins, including TNF receptor-associated protein 2 (TRAF2) and receptor-associated protein kinase (RIPK) 1, to form a complex. Subsequently, this complex combines with FADD and procaspase-8, converts into a death-inducing signaling complex (DISC) to induce apoptosis. Cyclic AMP (cAMP) is a second messenger that regulates various cellular processes such as cell proliferation, gene expression, and apoptosis. cAMP analogues are reported to act as anti-apoptotic agents in various cell types, including hepatocytes. We found that a cAMP analogue, dibutyryl cAMP (db-cAMP), inhibits TNF + ActD-induced apoptosis in rat hepatocytes. The protein kinase A (PKA) inhibitor KT-5720 reverses this inhibitory effect of cAMP on apoptosis. Cytoprotection by cAMP involves down-regulation of various apoptotic signal regulators like TRADD and FADD and inhibition of caspase-8 and caspase-3 cleavage. We also found

  8. Antibodies against amino acids 1-15 of tumor necrosis factor block its binding to cell-surface receptor.

    OpenAIRE

    Socher, S H; Riemen, M W; Martinez, D; Friedman, A; Tai, J; Quintero, J C; Garsky, V; Oliff, A

    1987-01-01

    Human tumor necrosis factor (hTNF) mediates a variety of biologic activities, which are dependent on the attachment of hTNF to cell-surface receptors. To identify regions of the hTNF protein involved in binding hTNF to its receptor, we prepared five synthetic peptides [hTNF-(1-15), hTNF-(1-31), hTNF-(65-79), hTNF-(98-111), and hTNF-(124-141)] and two hydroxylamine cleavage fragments [hTNF-(1-39) and hTNF-(40-157)] of hTNF. The hTNF-synthetic peptides and hTNF fragments were tested in hTNF rec...

  9. Chronic ethanol exposure inhibits distraction osteogenesis in a mouse model: Role of the TNF signaling axis

    International Nuclear Information System (INIS)

    Wahl, Elizabeth C.; Aronson, James; Liu, Lichu; Liu, Zhendong; Perrien, Daniel S.; Skinner, Robert A.; Badger, Thomas M.; Ronis, Martin J.J.; Lumpkin, Charles K.

    2007-01-01

    Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that modulates osteoblastogenesis. In addition, the demonstrated inhibitory effects of chronic ethanol exposure on direct bone formation in rats are hypothetically mediated by TNF-α signaling. The effects in mice are unreported. Therefore, we hypothesized that in mice (1) administration of a soluble TNF receptor 1 derivative (sTNF-R1) would protect direct bone formation during chronic ethanol exposure, and (2) administration of recombinant mouse TNF-α (rmTNF-α) to ethanol naive mice would inhibit direct bone formation. We utilized a unique model of limb lengthening (distraction osteogenesis, DO) combined with liquid diets to measure chronic ethanol's effects on direct bone formation. Chronic ethanol exposure resulted in increased marrow TNF, IL-1, and CYP 2E1 RNA levels in ethanol-treated vs. control mice, while no significant weight differences were noted. Systemic administration of sTNF-R1 during DO (8.0 mg/kg/2 days) to chronic ethanol-exposed mice resulted in enhanced direct bone formation as measured radiologically and histologically. Systemic rmTNF-α (10 μg/kg/day) administration decreased direct bone formation measures, while no significant weight differences were noted. We conclude that chronic ethanol-associated inhibition of direct bone formation is mediated to a significant extent by the TNF signaling axis in a mouse model

  10. Local TNF causes NFATc1-dependent cholesterol-mediated podocyte injury

    Science.gov (United States)

    Pedigo, Christopher E.; Ducasa, Gloria Michelle; Leclercq, Farah; Sloan, Alexis; Hashmi, Tahreem; Molina-David, Judith; Ge, Mengyuan; Lassenius, Mariann I.; Groop, Per-Henrik; Kretzler, Matthias; Martini, Sebastian; Reich, Heather; Wahl, Patricia; Ghiggeri, GianMarco; Burke, George W.; Kretz, Oliver; Huber, Tobias B.; Mendez, Armando J.; Merscher, Sandra

    2016-01-01

    High levels of circulating TNF and its receptors, TNFR1 and TNFR2, predict the progression of diabetic kidney disease (DKD), but their contribution to organ damage in DKD remains largely unknown. Here, we investigated the function of local and systemic TNF in podocyte injury. We cultured human podocytes with sera collected from DKD patients, who displayed elevated TNF levels, and focal segmental glomerulosclerosis (FSGS) patients, whose TNF levels resembled those of healthy patients. Exogenous TNF administration or local TNF expression was equally sufficient to cause free cholesterol–dependent apoptosis in podocytes by acting through a dual mechanism that required a reduction in ATP-binding cassette transporter A1–mediated (ABCA1-mediated) cholesterol efflux and reduced cholesterol esterification by sterol-O-acyltransferase 1 (SOAT1). TNF-induced albuminuria was aggravated in mice with podocyte-specific ABCA1 deficiency and was partially prevented by cholesterol depletion with cyclodextrin. TNF-stimulated free cholesterol–dependent apoptosis in podocytes was mediated by nuclear factor of activated T cells 1 (NFATc1). ABCA1 overexpression or cholesterol depletion was sufficient to reduce albuminuria in mice with podocyte-specific NFATc1 activation. Our data implicate an NFATc1/ABCA1-dependent mechanism in which local TNF is sufficient to cause free cholesterol–dependent podocyte injury irrespective of TNF, TNFR1, or TNFR2 serum levels. PMID:27482889

  11. Construction and characteristics of 3-end enriched cDNA library from individual embryos of cattle.

    Science.gov (United States)

    Long, Jian-Er; He, Li-Qiang; Cai, Xia; Ren, Zhao-Rui; Huang, Shu-Zhen; Zeng, Yi-Tao

    2006-11-01

    To analyze stage-specific gene expression profiles of pre-implantation embryos and evaluate potential viability, techniques were adapted to generate 3-end enriched cDNA libraries from individual embryos of cattle based on RT-PCR methodology. The reproducibility of constructing a cDNA library was tested by five independent PCR experiments with specific primers for the presence of several rare genes such as DNMT1 (DNA methylation transferase 1), DNMT2, DNMT3A, Oct-4/3 (octmer-binding transcription factor), IFN-iota, IGF-2r (insulin like growth factor 2 receptor), and the housekeeping genes, H2A and beta-actin. Results indicated repeatability and that a proportion of expressed genes in the cDNA library from an individual embryo was not affected by limited PCR amplification. From the cDNA library, 134 clones were randomly selected for sequencing and showed that structure related elements accounted for 33.5% of transcripts and the energy- and metabolism-related genes were also an important component being 11.9% in the cDNA library. Approximately 14% of genes in the library were functionally unknown including greater than 5% of genes that were likely novel because there was no identity in Genbank. The frequency of structure-related genes such as beta-actin and ribosomal proteins in the cDNA library corresponded to other reports and suggested that the cDNA library constructed by RT-PCR might be proportional to the mRNA populations. The cDNA libraries constructed from different stage embryos will provide a powerful tool to explore novel genes relevant to embryogenesis, determine the profiling of stage-specific gene expression, and evaluate the potential viability of embryos.

  12. TNF-alpha, leptin, and lymphocyte function in human aging

    DEFF Research Database (Denmark)

    Bruunsgaard, H.; Pedersen, Agnes Nadelmann; Schroll, M.

    2000-01-01

    Aging is associated with increased inflammatory activity and concomitant decreased T cell mediated immune responses. Leptin may provide a link between inflammation and T cell function in aging. The aim of the study was to investigate if plasma levels of tumor necrosis factor (TNF)-alpha were...... associated with leptin, circulating interleukin-2 receptors (sIL-2R), and phytohaemagglutinin (PHA) induced IL-2 production in whole blood in elderly humans. Circulating levels of TNF-alpha and sIL-2R were higher in elderly humans (N=42) compared to a young control group (N=37) whereas...... there was no difference with regard to IL-2 production. Furthermore, there were no age-related differences in serum levels of leptin, However, women had higher levels than men. In the elderly people, serum levels of leptin were correlated with TNF-alpha in univariate regression analysis and in a multiple linear...

  13. Differences in reactivation of tuberculosis induced from anti-TNF treatments are based on bioavailability in granulomatous tissue.

    Directory of Open Access Journals (Sweden)

    Simeone Marino

    2007-10-01

    Full Text Available The immune response to Mycobacterium tuberculosis (Mtb infection is complex. Experimental evidence has revealed that tumor necrosis factor (TNF plays a major role in host defense against Mtb in both active and latent phases of infection. TNF-neutralizing drugs used to treat inflammatory disorders have been reported to increase the risk of tuberculosis (TB, in accordance with animal studies. The present study takes a computational approach toward characterizing the role of TNF in protection against the tubercle bacillus in both active and latent infection. We extend our previous mathematical models to investigate the roles and production of soluble (sTNF and transmembrane TNF (tmTNF. We analyze effects of anti-TNF therapy in virtual clinical trials (VCTs by simulating two of the most commonly used therapies, anti-TNF antibody and TNF receptor fusion, predicting mechanisms that explain observed differences in TB reactivation rates. The major findings from this study are that bioavailability of TNF following anti-TNF therapy is the primary factor for causing reactivation of latent infection and that sTNF--even at very low levels--is essential for control of infection. Using a mathematical model, it is possible to distinguish mechanisms of action of the anti-TNF treatments and gain insights into the role of TNF in TB control and pathology. Our study suggests that a TNF-modulating agent could be developed that could balance the requirement for reduction of inflammation with the necessity to maintain resistance to infection and microbial diseases. Alternatively, the dose and timing of anti-TNF therapy could be modified. Anti-TNF therapy will likely lead to numerous incidents of primary TB if used in areas where exposure is likely.

  14. PCR-based cDNA library construction: general cDNA libraries at the level of a few cells.

    OpenAIRE

    Belyavsky, A; Vinogradova, T; Rajewsky, K

    1989-01-01

    A procedure for the construction of general cDNA libraries is described which is based on the amplification of total cDNA in vitro. The first cDNA strand is synthesized from total RNA using an oligo(dT)-containing primer. After oligo(dG) tailing the total cDNA is amplified by PCR using two primers complementary to oligo(dA) and oligo(dG) ends of the cDNA. For insertion of the cDNA into a vector a controlled trimming of the 3' ends of the cDNA by Klenow enzyme was used. Starting from 10 J558L ...

  15. Increased levels of circulating (TNF-α) is associated with (-308G/A) promoter polymorphism of TNF-α gene in Diabetic Nephropathy.

    Science.gov (United States)

    Umapathy, Dhamodharan; Krishnamoorthy, Ezhilarasi; Mariappanadar, Vairamani; Viswanathan, Vijay; Ramkumar, Kunka Mohanram

    2018-02-01

    The crucial role of Tumor Necrosis Factor-α (TNF-α) on renal function in patients with Diabetic Nephropathy (DN) has been well documented. The present study was designed to investigate the association of TNF-α [-308G/A, (rs1800629)] single nucleotide polymorphism (SNP) on the susceptibility to DN subjects and to correlate it with the plasma levels of TNF-α along with circulatory TNFreceptor super family cytokines (sTNFR-1 and sTNFR-2). A total of 756 subjects, were recruited and divided into groups [Group-I, Control (n=218), Group-II, Normoalbuminuria (n=196), Group-IIIa, Microalbuminuria (n=178), Group-IIIb, Macroalbuminuria (n=164)] and were genotyped by PCR-restriction fragment length polymorphism (RFLP). Circulatory levels of TNF-α and sTNFR-1 & sTNFR-2 were measured using multiplex bead based assay. The 'A' allele of TNF-α (-308 G/A) SNP was associated with a significant risk for macroalbuminuria subjects (OR: 2.1; 95% CI: 0.8-3.7; PTNF-α, sTNF-R1 and sTNF-R2 from normo to macroalbuminuria subjects. In DN subjects, the TNF-α level was higher in individuals who had mutant AA, than the wild GG genotype of TNF-α gene. Our results conclude that rs1800629 polymorphism in TNF-α gene is associated with renal complications in T2DM subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. The controversial role of TNF in melanoma

    DEFF Research Database (Denmark)

    Donia, Marco; Kjeldsen, Julie Westerlin; Svane, Inge Marie

    2016-01-01

    ABSTRACT: TNF has been associated with both inhibition and promotion of tumor growth. We recently described a mechanism by which tumor cells attract TNF producing cells via expression of MHC class II molecules.......ABSTRACT: TNF has been associated with both inhibition and promotion of tumor growth. We recently described a mechanism by which tumor cells attract TNF producing cells via expression of MHC class II molecules....

  17. Evidence that TNF-β (lymphotoxin α) can activate the inflammatory environment in human chondrocytes

    Science.gov (United States)

    2013-01-01

    Introduction Inflammatory cytokines play a key role in the pathogenesis of joint diseases such as rheumatoid arthritis (RA). Current therapies target mainly tumor necrosis factor α (TNF-α) as this has proven benefits. However, a large number of patients do not respond to or become resistant to anti-TNF-α therapy. While the role of TNF-α in RA is quite evident, the role of TNF-β, also called lymphotoxin-α (LT-α), is unclear. In this study we investigated whether TNF-β and its receptor play a role in chondrocytes in the inflammatory environment. Methods An in vitro model of primary human chondrocytes was used to study TNF-β-mediated inflammatory signaling. Results Cytokine-induced inflammation enhances TNF-β and TNF-β-receptor expression in primary human chondrocytes accompanied by the up-regulation of inflammatory (cyclooxygenase-2), matrix degrading (matrix metalloproteinase-9 and -13) and apoptotic (p53, cleaved caspase-3) signaling pathways, all known to be regulated by NF-κB. In contrast, anti-TNF-β, similar to the natural NF-κB inhibitor (curcumin, diferuloylmethane) or the knockdown of NF-κB by using antisense oligonucleotides (ASO), suppressed IL-1β-induced NF-κB activation and its translocation to the nucleus, and abolished the pro-inflammatory and apoptotic effects of IL-1β. This highlights, at least in part, the crucial role of NF-κB in TNF-β-induced-inflammation in cartilage, similar to that expected for TNF-α. Finally, the adhesiveness between TNF-β-expressing T-lymphocytes and the responding chondrocytes was significantly enhanced through a TNF-β-induced inflammatory microenvironment. Conclusions These results suggest for the first time that TNF-β is involved in microenvironment inflammation in chondrocytes during RA parallel to TNF-α, resulting in the up-regulation of NF-κB signaling and activation of pro-inflammatory activity. PMID:24283517

  18. Genetic profile of Egyptian hepatocellular-carcinoma associated with hepatitis C virus Genotype 4 by 15 K cDNA microarray: Preliminary study

    Directory of Open Access Journals (Sweden)

    Mansour Tarek

    2008-10-01

    Full Text Available Abstract Background Hepatocellular carcinoma (HCC is a preventable disease rather than a curable one, since there is no well-documented effective treatment modality until now, making the molecular study of this disease mandatory. Findings We studied gene expression profile of 17 Egyptian HCC patients associated with HCV genotype-4 infection by c-DNA microarray. Out of the 15,660 studied genes, 446 were differentially expressed; 180 of them were up regulated and 134 were down regulated. Seventeen genes out of the 180 up-regulated genes are involved in 28 different pathways. Protein phosphatase 3 (PPP3R1 is involved in 10 different pathways followed by fibroblast growth factor receptor 1 (FGFR1, Cas-Br-M ecotropic retroviral transforming sequence b (CBLB, spleen tyrosine kinase (SYK involved in three pathways; bone morphogenetic protein 8a (BMP8A, laminin alpha 3 (LAMA3, cell division cycle 23 (CDC23 involved in 2 pathways and NOTCH4 which regulate Notch signaling pathway. On the other hand, 25 out of the 134 down-regulated genes are involved in 20 different pathways. Integrin alpha V alpha polypeptide antigen CD51 (ITGVA is involved in 4 pathways followed by lymphotoxin alpha (TNF superfamily, member 1 (LTA involved in 3 pathways and alpha-2-macroglobulin (A2M, phosphorylase kinase alpha 2-liver (PHKA2 and MAGI1 membrane associated guanylate kinase 1 (MAGI1 involved in 2 pathways. In addition, 22 genes showed significantly differential expression between HCC cases with cirrhosis and without cirrhosis. Confirmation analysis was performed on subsets of these genes by RT-PCR, including some up-regulated genes such as CDK4, Bax, NOTCH4 and some down-regulated genes such as ISGF3G, TNF, and VISA. Conclusion This is the first preliminary study on gene expression profile in Egyptian HCC patients associated with HCV-Genotype-4 using the cDNA microarray. The identified genes could provide a new gate for prognostic and diagnostic markers for HCC associated

  19. Identification and expression analysis of two pro-inflammatory cytokines, TNF-α and IL-8, in cobia (Rachycentron canadum L.) in response to Streptococcus dysgalactiae infection.

    Science.gov (United States)

    Nguyen, Thuy Thi Thu; Nguyen, Hai Trong; Wang, Pei-Chyi; Chen, Shih-Chu

    2017-08-01

    Tumor necrosis factor-alpha (TNF-α) and interleukin-8 (IL-8/CXCL8) play pivotal roles in mediating inflammatory responses to invading pathogens. In this study, we identified and analyzed expressions of cobia TNF-α and IL-8 during Streptococcus dysgalactiae infection. The cloned cDNA transcript of cobia TNF-α comprised of 1281 base pairs (bp), with a 774 bp open reading frame (ORF) encoding 257 amino acids. The deduced amino acid sequence of cobia TNF-α showed a close relationship (84% similarity) with TNF-α of yellowtail amberjack. The cloned IL-8 cDNA sequence was 828 bp long, including a 300-bp ORF encoding 99 amino acids. The deduced amino acid sequence of cobia IL-8 shared 90% identity with IL-8 of striped trumpeter. Cobia challenged with a virulent S. dysgalactiae strain displayed an early significant up-regulation of TNF-α and IL-8 in head kidney, liver, and spleen. Notably, IL-8 expression level increased dramatically in the liver at the severe stage of infection (72 h). In conclusion, a better understanding of TNF-α and IL-8 allows more detailed investigation of immune responses in cobia and furthers study on controlling the infectious disease caused by S. dysgalactiae. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Treatment of experimental colitis in mice with LMP-420, an inhibitor of TNF transcription

    Directory of Open Access Journals (Sweden)

    Cianciolo George

    2008-03-01

    Full Text Available Abstract Background LMP-420 is a boronic acid-containing purine nucleoside analogue that transcriptionally inhibits TNF production but is non-cytotoxic to TNF-producing cells. Methods This study investigated the efficacy of LMP-420 as an anti-inflammatory agent in acute and chronic colitis induced by oral administration of dextran sulfate sodium (DSS to mice and in chronic colitis following piroxicam administration to IL-10-deficient mice. The severity of colon inflammation was assessed histologically. TNF levels were measured by enzyme immunoassay. Results Administration of DSS for 7 days resulted in severe acute colitis that was associated with a marked increase in stool and colon tissue TNF levels. Initiation of therapy with intraperitoneal (i.p. LMP-420 on day 4 of DSS exposure decreased colonic TNF to near normal levels on day 7. However, neither i.p. nor oral treatment with LMP-420 affected the development or severity of acute DSS colitis. Initiation of LMP-420 therapy after 3 cycles of DSS administration to establish chronic colitis also had no effect on the severity of chronic colitis. Analysis of colonic TNF combined with longitudinal analysis of TNF and TNF receptor (TNF-RII levels in stool during the development of chronic DSS colitis demonstrated that the initially elevated colonic TNF levels returned to normal despite intense on-going inflammation in mice with chronic colitis. RAG-2-/- mice deficient in T and B cells also developed severe ongoing colitis in response to 3 cycles of DSS, but showed marked differences vs. wild type mice in stool TNF and TNF-RII in response to DSS exposure. Systemic and oral LMP-420 treatment for 16 days decreased colonic TNF levels in IL-10-deficient mice with chronic colitis, with a trend to decreased histologic inflammation for oral LMP-420. Conclusion These studies demonstrate that short-term treatment with a transcriptional inhibitor of TNF production can decrease systemic and local colonic levels

  1. Plasma TNF binding capacity profiles during treatment with etanercept in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Gudbrandsdottir, S; Bliddal, H; Petri, A

    2004-01-01

    occurring soluble TNF receptors. However, the clinical response to treatment with etanercept may vary. Previously, pharmacokinetic studies have focused on the molar concentrations of etanercept, but very little is known about the kinetics of bioactive etanercept in patients treated with etanercept....... The purpose of this study was to evaluate kinetics, including inter- and intraindividual variations of the total TNF binding capacity, in RA patients who were on a standard treatment schedule with etanercept....

  2. Impact of TNF-R1 and CD95 internalization on apoptotic and antiapoptotic signaling.

    Science.gov (United States)

    Schütze, Stefan; Schneider-Brachert, Wulf

    2009-01-01

    Internalization of cell surface receptors has long been regarded as a pure means to terminate signaling via receptor degradation. A growing body of information points to the fact that many internalized receptors are still in their active state and that signaling continues along the endocytic pathway. Thus endocytosis orchestrates cell signaling by coupling and integrating different cascades on the surface of endocytic vesicles to control the quality, duration, intensity, and distribution of signaling events. The death receptors tumor necrosis factor-receptor 1 (TNF-R1) and CD95 (Fas, APO-1) are known not only to signal for cell death via apoptosis but are also capable of inducing antiapoptotic signals via transcription factor NF-kappaB induction or activation of the proliferative mitogen-activated protein kinase (MAPK)/ERK (extracellular signal-regulated kinase) protein kinase cascades, resulting in cell protection and tissue regeneration. A clue to the understanding of these contradictory biological phenomena may arise from recent findings which reveal a regulatory role of receptor internalization and intracellular receptor trafficking in selectively transmitting signals, which lead either to apoptosis or to the survival of the cell. In this chapter, we discuss the dichotomy of pro- and antiapoptotic signaling of the death receptors TNF-R1 and CD95. First, we will address the role of lipid rafts and post-translational modifications of death receptors in regulating the formation of receptor complexes. Then, we will discuss the role of internalization in determining the fate of the receptors and subsequently the specificity of signaling events. We propose that fusion of internalized TNF-receptosomes with trans-Golgi vesicles should be recognized as a novel mechanism to transduce death signals along the endocytic route. Finally, the lessons learnt from the strategy of adenovirus to escape apoptosis by targeting death receptor internalization demonstrate the

  3. Lectin cDNA and transgenic plants derived therefrom

    Science.gov (United States)

    Raikhel, Natasha V.

    2000-10-03

    Transgenic plants containing cDNA encoding Gramineae lectin are described. The plants preferably contain cDNA coding for barley lectin and store the lectin in the leaves. The transgenic plants, particularly the leaves exhibit insecticidal and fungicidal properties.

  4. Drugs for Autoimmune Inflammatory Diseases: From Small Molecule Compounds to Anti-TNF Biologics

    Directory of Open Access Journals (Sweden)

    Ping Li

    2017-07-01

    Full Text Available Although initially described as an anti-tumor mediator, tumor necrosis factor-alpha (TNF is generally considered as the master pro-inflammatory cytokine. It plays a crucial role in the pathogenesis of inflammatory diseases, such as rheumatoid arthritis (RA, inflammatory bowel disease, ankylosing spondylitis (AS, and psoriasis. Consequently, anti-TNF therapy has become mainstay treatment for autoimmune diseases. Historically, anti-inflammatory agents were developed before the identification of TNF. Salicylates, the active components of Willow spp., were identified in the mid-19th century for the alleviation of pain, fever, and inflammatory responses. Study of this naturally occurring compound led to the discovery of aspirin, which was followed by the development of non-steroidal anti-inflammatory drugs (NSAIDs due to the chemical advances in the 19th–20th centuries. Initially, the most of NSAIDs were organic acid, but the non-acidic compounds were also identified as NSAIDs. Although effective in the treatment of inflammatory diseases, NSAIDs have some undesirable and adverse effect, such as ulcers, kidney injury, and bleeding in the gastrointestinal tract. In the past two decades, anti-TNF biologics were developed. Drugs belong to this class include soluble TNF receptor 2 fusion protein and anti-TNF antibodies. The introduction of anti-TNF therapeutics has revolutionized the management of autoimmune diseases, such as RA, psoriatic arthritis (PsA, plaque psoriasis (PP, AS, CD and ulcerative colitis (UC. Nevertheless, up to 40% of patients have no response to anti-TNF treatment. Furthermore, this treatment is associated with some adverse effects such as increased risk of infection, and even triggered the de novo development of autoimmune diseases. Such harmful effect of anti-TNF treatment is likely caused by the global inhibition of TNF biological functions. Therefore, specific inhibition of TNF receptor (TNFR1 or TNFR2 may represent a safer and

  5. Molecular networks discriminating mouse bladder responses to intravesical bacillus Calmette-Guerin (BCG, LPS, and TNF

    Directory of Open Access Journals (Sweden)

    Dozmorov Igor

    2008-02-01

    Full Text Available Abstract Background Despite being a mainstay for treating superficial bladder carcinoma and a promising agent for interstitial cystitis, the precise mechanism of Bacillus Calmette-Guerin (BCG remains poorly understood. It is particularly unclear whether BCG is capable of altering gene expression in the bladder target organ beyond its well-recognized pro-inflammatory effects and how this relates to its therapeutic efficacy. The objective of this study was to determine differentially expressed genes in the mouse bladder following chronic intravesical BCG therapy and to compare the results to non-specific pro inflammatory stimuli (LPS and TNF-α. For this purpose, C57BL/6 female mice received four weekly instillations of BCG, LPS, or TNF-α. Seven days after the last instillation, the urothelium along with the submucosa was removed from detrusor muscle and the RNA was extracted from both layers for cDNA array experiments. Microarray results were normalized by a robust regression analysis and only genes with an expression above a conditional threshold of 0.001 (3SD above background were selected for analysis. Next, genes presenting a 3-fold ratio in regard to the control group were entered in Ingenuity Pathway Analysis (IPA for a comparative analysis in order to determine genes specifically regulated by BCG, TNF-α, and LPS. In addition, the transcriptome was precipitated with an antibody against RNA polymerase II and real-time polymerase chain reaction assay (Q-PCR was used to confirm some of the BCG-specific transcripts. Results Molecular networks of treatment-specific genes generated several hypotheses regarding the mode of action of BCG. BCG-specific genes involved small GTPases and BCG-specific networks overlapped with the following canonical signaling pathways: axonal guidance, B cell receptor, aryl hydrocarbon receptor, IL-6, PPAR, Wnt/β-catenin, and cAMP. In addition, a specific detrusor network expressed a high degree of overlap with the

  6. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length ...

  7. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    317. 2.4 cDNA sequencing and analysis. The nucleotide sequence of the cloned H. fossilis GH. cDNA was determined by Sanger's dideoxy chain termi- nation method, using Perkin Elmer bigdye terminator kit in an ABI Prism 377 automated DNA sequencer. All other computational analysis of the GH cDNA was done using.

  8. Priming astrocytes with TNF enhances their susceptibility to Trypanosoma cruzi infection and creates a self-sustaining inflammatory milieu.

    Science.gov (United States)

    Silva, Andrea Alice; Silva, Rafael Rodrigues; Gibaldi, Daniel; Mariante, Rafael Meyer; Dos Santos, Jessica Brandão; Pereira, Isabela Resende; Moreira, Otacílio Cruz; Lannes-Vieira, Joseli

    2017-09-06

    In conditions of immunosuppression, the central nervous sty 5ystem (CNS) is the main target tissue for the reactivation of infection by Trypanosoma cruzi, the causative agent of Chagas disease. In experimental T. cruzi infection, interferon gamma (IFNγ) + microglial cells surround astrocytes harboring amastigote parasites. In vitro, IFNγ fuels astrocyte infection by T. cruzi, and IFNγ-stimulated infected astrocytes are implicated as potential sources of tumor necrosis factor (TNF). Pro-inflammatory cytokines trigger behavioral alterations. In T. cruzi-infected mice, administration of anti-TNF antibody hampers depressive-like behavior. Herein, we investigated the effects of TNF on astrocyte susceptibility to T. cruzi infection and the regulation of cytokine production. Primary astrocyte cultures of neonatal C57BL/6 and C3H/He mice and the human U-87 MG astrocyte lineage were infected with the Colombian T. cruzi strain. Cytokine production, particularly TNF, and TNF receptor 1 (TNFR1/p55) expression were analyzed. Recombinant cytokines (rIFNγ and rTNF), the anti-TNF antibody infliximab, and the TNFR1 modulator pentoxifylline were used to assess the in vitro effects of TNF on astrocyte susceptibility to T. cruzi infection. To investigate the role of TNF on CNS colonization by T. cruzi, infected mice were submitted to anti-TNF therapy. rTNF priming of mouse and human astrocytes enhanced parasite/astrocyte interaction (i.e., the percentage of astrocytes invaded by trypomastigote parasites and the number of intracellular parasite forms/astrocyte). Furthermore, T. cruzi infection drove astrocytes to a pro-inflammatory profile with TNF and interleukin-6 production, which was amplified by rTNF treatment. Adding rTNF prior to infection fueled parasite growth and trypomastigote egression, in parallel with increased TNFR1 expression. Importantly, pentoxifylline inhibited the TNF-induced increase in astrocyte susceptibility to T. cruzi invasion. In T. cruzi-infected mice

  9. cDNA sequence quality data - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project cDNA sequence quality data Data detail Data name cDNA sequence quality... data DOI 10.18908/lsdba.nbdc00838-003 Description of data contents Phred's quality score. P...tion Download License Update History of This Database Site Policy | Contact Us cDNA sequence quality

  10. Macrocyclic θ-defensins suppress tumor necrosis factor-α (TNF-α) shedding by inhibition of TNF-α-converting enzyme.

    Science.gov (United States)

    Schaal, Justin B; Maretzky, Thorsten; Tran, Dat Q; Tran, Patti A; Tongaonkar, Prasad; Blobel, Carl P; Ouellette, André J; Selsted, Michael E

    2018-02-23

    Theta-defensins (θ-defensins) are macrocyclic peptides expressed exclusively in granulocytes and selected epithelia of Old World monkeys. They contribute to anti-pathogen host defense responses by directly killing a diverse range of microbes. Of note, θ-defensins also modulate microbe-induced inflammation by affecting the production of soluble tumor necrosis factor (sTNF) and other proinflammatory cytokines. Here, we report that natural rhesus macaque θ-defensin (RTD) isoforms regulate sTNF cellular release by inhibiting TNF-α-converting enzyme (TACE; also known as a d isintegrin a nd m etalloprotease 17; ADAM17), the primary pro-TNF sheddase. Dose-dependent inhibition of cellular TACE activity by RTDs occurred when leukocytes were stimulated with live Escherichia coli cells as well as numerous Toll-like receptor agonists. Moreover, the relative inhibitory potencies of the RTD isoforms strongly correlated with their suppression of TNF release by stimulated blood leukocytes and THP-1 monocytes. RTD isoforms also inhibited ADAM10, a sheddase closely related to TACE. TACE inhibition was abrogated by introducing a single opening in the RTD-1 backbone, demonstrating that the intact macrocycle is required for enzyme inhibition. Enzymologic analyses showed that RTD-1 is a fast binding, reversible, non-competitive inhibitor of TACE. We conclude that θ-defensin-mediated inhibition of pro-TNF proteolysis by TACE represents a rapid mechanism for the regulation of sTNF and TNF-dependent inflammatory pathways. Molecules with structural and functional features mimicking those of θ-defensins may have clinical utility as TACE inhibitors for managing TNF-driven diseases. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. SDF-1 induces TNF-mediated apoptosis in cardiac myocytes.

    Science.gov (United States)

    Jarrah, Andrew A; Schwarskopf, Martina; Wang, Edward R; LaRocca, Thomas; Dhume, Ashwini; Zhang, Shihong; Hadri, Lahouria; Hajjar, Roger J; Schecter, Alison D; Tarzami, Sima T

    2018-01-01

    Chemokines are small secreted proteins with chemoattractant properties that play a key role in inflammation. One such chemokine, Stromal cell-derived factor-1 (SDF-1) also known as CXCL12, and its receptor, CXCR4, are expressed and functional in cardiac myocytes. SDF-1 both stimulates and enhances the cellular signal which attracts potentially beneficial stem cells for tissue repair within the ischemic heart. Paradoxically however, this chemokine is known to act in concert with the inflammatory cytokines of the innate immune response which contributes to cellular injury through the recruitment of inflammatory cells during ischemia. In the present study, we have demonstrated that SDF-1 has dose dependent effects on freshly isolated cardiomyocytes. Using Tunnel and caspase 3-activation assays, we have demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations (pathological concentrations) induced apoptosis. Furthermore, ELISA data demonstrated that the treatment of isolated adult rat cardiac myocyte with SDF-1 at higher concentrations upregulated TNF-α protein expression which directly correlated with subsequent apoptosis. There was a significant reduction in SDF-1 mediated apoptosis when TNF-α expression was neutralized which suggests that SDF-1 mediated apoptosis is TNF-α-dependent. The fact that certain stimuli are capable of driving cardiomyocytes into apoptosis indicates that these cells are susceptible to clinically relevant apoptotic triggers. Our findings suggest that the elevated SDF-1 levels seen in a variety of clinical conditions, including ischemic myocardial infarction, may either directly or indirectly contribute to cardiac cell death via a TNF-α mediated pathway. This highlights the importance of this receptor/ligand in regulating the cardiomyocyte response to stress conditions.

  12. TNF and ROS Crosstalk in Inflammation

    DEFF Research Database (Denmark)

    Blaser, Heiko; Dostert, Catherine; Mak, Tak W

    2016-01-01

    Tumor necrosis factor (TNF) is tremendously important for mammalian immunity and cellular homeostasis. The role of TNF as a master regulator in balancing cell survival, apoptosis and necroptosis has been extensively studied in various cell types and tissues. Although these findings have revealed...... the role of TNF in the control of ROS/RNS signaling that influences innate immune cells under both physiological and inflammatory conditions. © 2015 Elsevier Ltd....

  13. Biological anti-TNF drugs

    DEFF Research Database (Denmark)

    Prado, Mônica Simon; Bendtzen, Klaus; Andrade, Luis Eduardo Coelho

    2017-01-01

    is frequently caused by antibodies against immune-biologicals, known as anti-drug antibodies (ADA). ADA that neutralize circulating immune-biologicals and/or promote their clearance can reduce treatment efficacy. Furthermore, ADA can induce adverse events by diverse immunological mechanisms. This review...... provides a comprehensive overview of ADA in rheumatoid arthritis patients treated with anti-TNF immune-biologicals, and explores the concept of therapeutic drug monitoring (TDM) as an effective strategy to improve therapeutic management. Expert opinion: Monitoring circulating ADA and therapeutic immune......-biological drugs is helpful when evaluating patients with secondary failure. However, immunological tests for ADA vary considerably regarding their ability to detect different types of ADA. Several assays are not designed to determine ADA-induced drug neutralizing capacity, and they may report clinically non...

  14. TNFR1 and TNFR2 differentially mediate TNF-α-induced inflammatory responses in rheumatoid arthritis fibroblast-like synoviocytes.

    Science.gov (United States)

    Zhang, Hongfeng; Xiao, Weiguo

    2017-04-01

    TNF-α has long been implicated in the progression of rheumatoid arthritis (RA). However, how the receptors of TNF-α, namely TNFR1 and TNFR2, mediate TNF-α-induced inflammatory responses in fibroblast-like synoviocytes (FLS) in RA has not been elucidated. In the present study, primary FLS cells were isolated from RA patients and treated with TNF-α in vitro. The exogenous TNF-α induced the expression and release of endogenous TNF-α in FLS. In addition, TNF-α led to gradual downregulation of TNFR1 following 1 h treatment. By contrast, the expression of TNFR2 was markedly upregulated after 12 h treatment with TNF-α. Moreover, following TNF-α treatment, the expression of interleukin (IL)-2, IL-6, and IL-8 was gradually increased with time, but their mRNA levels dropped significantly at 48 h. We further investigated the differential functions of TNFR1 and TNFR2 in FLS by conducting siRNA-mediated knockdown. The TNF-α autocrine was inhibited to a greater extent in TNFR1-silenced FLS compared with TNFR2-silenced FLS. Silencing of TNFR1, not TNFR2, activated intrinsic apoptosis and inhibited TNF-α-induced cytokine production in FLS. These results suggest that TNFR1 is the major pro-inflammatory mediator of TNF-α in FLS, whereas TNFR2, which is upregulated in response to prolonged TNF-α stimulation, may act as an immunosuppressor in FLS for the prevention of overwhelming inflammatory reactions. © 2017 International Federation for Cell Biology.

  15. Postnatal administration of memantine rescues TNF-α-induced decreased hippocampal precursor proliferation.

    Science.gov (United States)

    Wang, Zhongke; He, Xie; Fan, Xiaotang

    2018-01-01

    Pro-inflammatory cytokine exposure in early postnatal life triggers clear neurotoxic effects on the developing hippocampus. Tumor necrosis factor alpha (TNF-α) is one of the inflammatory mediators and is a potent inhibitor of neurogenesis. Memantine (MEM) is an uncompetitive N-methyl-d-aspartate (NMDA) receptor antagonist that has been demonstrated to increase the proliferation of hippocampal progenitor cells. However, the effects of MEM on TNF-α-mediated impairment of hippocampal precursor proliferation remain unclear. In this study, mice were exposed to TNF-α and later treated with MEM to evaluate its protective effects on TNF-α-mediated toxicity during hippocampal development. The results indicated that brief exposure to TNF-α on postnatal days 3 and 5 resulted in a significant impairment of hippocampal precursor proliferation and a depletion of hippocampal neural precursor cells (NPCs). This effect was attenuated by MEM treatment. We further confirmed that MEM treatment reversed the TNF-α-induced microglia activation and up-regulation of hippocampal NF-κB, MCP-1 and IL-6 mRNA levels, which may be related to the proliferation and maintenance of NPCs. Overall, our results suggest that MEM treatment protects against TNF-α-induced repression of hippocampal precursor proliferation in postnatal mice by partially attenuating neuroinflammatory responses. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression.

    Science.gov (United States)

    Tortarolo, Massimo; Vallarola, Antonio; Lidonnici, Dario; Battaglia, Elisa; Gensano, Francesco; Spaltro, Gabriella; Fiordaliso, Fabio; Corbelli, Alessandro; Garetto, Stefano; Martini, Elisa; Pasetto, Laura; Kallikourdis, Marinos; Bonetto, Valentina; Bendotti, Caterina

    2015-10-01

    Changes in the homeostasis of tumor necrosis factor α (TNFα) have been demonstrated in patients and experimental models of amyotrophic lateral sclerosis (ALS). However, the contribution of TNFα to the development of ALS is still debated. TNFα is expressed by glia and neurons and acts through the membrane receptors TNFR1 and TNFR2, which may have opposite effects in neurodegeneration. We investigated the role of TNFα and its receptors in the selective motor neuron death in ALS in vitro and in vivo. TNFR2 expressed by astrocytes and neurons, but not TNFR1, was implicated in motor neuron loss in primary SOD1-G93A co-cultures. Deleting TNFR2 from SOD1-G93A mice, there was partial but significant protection of spinal motor neurons, sciatic nerves, and tibialis muscles. However, no improvement of motor impairment or survival was observed. Since the sciatic nerves of SOD1-G93A/TNFR2-/- mice showed high phospho-TAR DNA-binding protein 43 (TDP-43) accumulation and low levels of acetyl-tubulin, two indices of axonal dysfunction, the lack of symptom improvement in these mice might be due to impaired function of rescued motor neurons. These results indicate the interaction between TNFR2 and membrane-bound TNFα as an innovative pathway involved in motor neuron death. Nevertheless, its inhibition is not sufficient to stop disease progression in ALS mice, underlining the complexity of this pathology. We show evidence of the involvement of neuronal and astroglial TNFR2 in the motor neuron degeneration in ALS. Both concur to cause motor neuron death in primary astrocyte/spinal neuron co-cultures. TNFR2 deletion partially protects motor neurons and sciatic nerves in SOD1-G93A mice but does not improve their symptoms and survival. However, TNFR2 could be a new target for multi-intervention therapies. © 2015 International Society for Neurochemistry.

  17. TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

    Science.gov (United States)

    Ito, Shiori; Iwaki, Soichiro; Kondo, Rie; Satoh, Masashi; Iwabuchi, Kazuya; Ohkawa, Ryunosuke; Mishima, Yuko; Yatomi, Yutaka; Furumoto, Tomoo; Tsutsui, Hiroyuki; Fujii, Satoshi

    2014-06-01

    Natural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas and mouse NKT cells. NKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and α-galactosylceramide (α-GalCer), the major ligand to produce cytokines in NKT cells. TNF-α mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. Hybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and α-GalCer increased TNF-α mRNA expression and protein production. S1P enhanced TNF-α induction by α-GalCer. S1P receptor antagonists decreased the TNF-α mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-α mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-α mRNA expression in mouse NKT cells. Plasma TNF-α levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (NKT cells and enhances TNF-α production. TNF-α overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

  18. receptores

    Directory of Open Access Journals (Sweden)

    Salete Regina Daronco Benetti

    2006-01-01

    Full Text Available Se trata de un estudio etnográfico, que tuvo lo objetivo de interpretar el sistema de conocimiento y del significado atribuidos a la sangre referente a la transfusión sanguínea por los donadores y receptores de un banco de sangre. Para la colecta de las informaciones se observaron los participantes y la entrevista etnográfica se realizó el análisis de dominio, taxonómicos y temáticos. Los dominios culturales fueron: la sangre es vida: fuente de vida y alimento valioso; creencias religiosas: fuentes simbólicas de apoyos; donación sanguínea: un gesto colaborador que exige cuidarse, gratifica y trae felicidad; donación sanguínea: fuente simbólica de inseguridad; estar enfermo es una condición para realizar transfusión sanguínea; transfusión sanguínea: esperanza de vida; Creencias populares: transfusión sanguínea como riesgo para la salud; donadores de sangre: personas benditas; donar y recibir sangre: como significado de felicidad. Temática: “líquido precioso que origina, sostiene, modifica la vida, provoca miedo e inseguridad”.

  19. Histamine Derived from Probiotic Lactobacillus reuteri Suppresses TNF via Modulation of PKA and ERK Signaling

    Science.gov (United States)

    Thomas, Carissa M.; Hong, Teresa; van Pijkeren, Jan Peter; Hemarajata, Peera; Trinh, Dan V.; Hu, Weidong; Britton, Robert A.; Kalkum, Markus; Versalovic, James

    2012-01-01

    Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s) produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC) separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA), histidine/histamine antiporter (hdcP), and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2)-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA) and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases. PMID:22384111

  20. Histamine derived from probiotic Lactobacillus reuteri suppresses TNF via modulation of PKA and ERK signaling.

    Directory of Open Access Journals (Sweden)

    Carissa M Thomas

    Full Text Available Beneficial microbes and probiotic species, such as Lactobacillus reuteri, produce biologically active compounds that can modulate host mucosal immunity. Previously, immunomodulatory factors secreted by L. reuteri ATCC PTA 6475 were unknown. A combined metabolomics and bacterial genetics strategy was utilized to identify small compound(s produced by L. reuteri that were TNF-inhibitory. Hydrophilic interaction liquid chromatography-high performance liquid chromatography (HILIC-HPLC separation isolated TNF-inhibitory compounds, and HILIC-HPLC fraction composition was determined by NMR and mass spectrometry analyses. Histamine was identified and quantified in TNF-inhibitory HILIC-HPLC fractions. Histamine is produced from L-histidine via histidine decarboxylase by some fermentative bacteria including lactobacilli. Targeted mutagenesis of each gene present in the histidine decarboxylase gene cluster in L. reuteri 6475 demonstrated the involvement of histidine decarboxylase pyruvoyl type A (hdcA, histidine/histamine antiporter (hdcP, and hdcB in production of the TNF-inhibitory factor. The mechanism of TNF inhibition by L. reuteri-derived histamine was investigated using Toll-like receptor 2 (TLR2-activated human monocytoid cells. Bacterial histamine suppressed TNF production via activation of the H(2 receptor. Histamine from L. reuteri 6475 stimulated increased levels of cAMP, which inhibited downstream MEK/ERK MAPK signaling via protein kinase A (PKA and resulted in suppression of TNF production by transcriptional regulation. In summary, a component of the gut microbiome, L. reuteri, is able to convert a dietary component, L-histidine, into an immunoregulatory signal, histamine, which suppresses pro-inflammatory TNF production. The identification of bacterial bioactive metabolites and their corresponding mechanisms of action with respect to immunomodulation may lead to improved anti-inflammatory strategies for chronic immune-mediated diseases.

  1. TNF-α-induced protein 3 (TNFAIP3)/A20 acts as a master switch in TNF-α blockade-driven IL-17A expression.

    Science.gov (United States)

    Urbano, Paulo C M; Aguirre-Gamboa, Raúl; Ashikov, Angel; van Heeswijk, Bennie; Krippner-Heidenreich, Anja; Tijssen, Henk; Li, Yang; Azevedo, Valderilio F; Smits, Lisa J T; Hoentjen, Frank; Joosten, Irma; Koenen, Hans J P M

    2017-12-14

    Anti-TNF inhibitors successfully improve the quality of life of patients with inflammatory disease. Unfortunately, not all patients respond to anti-TNF therapy, and some patients show paradoxical immune side effects, which are poorly understood. Surprisingly, anti-TNF agents were shown to promote IL-17A production with as yet unknown clinical implications. We sought to investigate the molecular mechanism underlying anti-TNF-driven IL-17A expression and the clinical implications of this phenomenon. Fluorescence-activated cell sorting, RNA sequencing, quantitative real-time PCR, Western blotting, small interfering RNA interference, and kinase inhibitors were used to study the molecular mechanisms in isolated human CD4 + T cells from healthy donors. The clinical implication was studied in blood samples of patients with inflammatory bowel disease (IBD) receiving anti-TNF therapy. Here we show that anti-TNF treatment results in inhibition of the anti-inflammatory molecule TNF-α-induced protein 3 (TNFAIP3)/A20 in memory CD4 + T cells. We found an inverse relationship between TNFAIP3/A20 expression levels and IL-17A production. Inhibition of TNFAIP3/A20 promotes kinase activity of p38 mitogen-activated protein kinase and protein kinase C, which drives IL-17A expression. Regulation of TNFAIP3/A20 expression and cognate IL-17A production in T cells are specifically mediated through TNF receptor 2 signaling. Ex vivo, in patients with IBD treated with anti-TNF, we found further evidence for an inverse relationship between TNFAIP3/A20 expression levels and IL-17A-producing T cells. Anti-TNF treatment interferes in the TNFAIP3/A20-mediated anti-inflammatory feedback loop in CD4 + T cells and promotes kinase activity. This puts TNFAIP3/A20, combined with IL-17A expression, on the map as a potential tool for predicting therapy responsiveness or side effects of anti-TNF therapy. Moreover, it provides novel targets related to TNFAIP3/A20 activity for superior therapeutic regimens

  2. Cloning the interleukin 1 receptor from human T cells

    International Nuclear Information System (INIS)

    Sims, J.E.; Acres, R.B.; Grubin, C.E.; McMahan, C.J.; Wignall, J.M.; March, C.J.; Dower, S.K.

    1989-01-01

    cDNA clones of the interleukin 1 (IL-1) receptor expressed in a human T-cell clone have been isolated by using a murine IL-1 receptor cDNA as a probe. The human and mouse receptors show a high degree of sequence conservation. Both are integral membrane proteins possessing a single membrane-spanning segment. Similar to the mouse receptor, the human IL-1 receptor contains a large cytoplasmic region and an extracellular, IL-1 binding portion composed of three immunoglobulin-like domains. When transfected into COS cells, the human IL-1 receptor cDNA clone leads to expression of two different affinity classes of receptors, with K a values indistinguishable from those determined for IL-1 receptors in the original T-cell clone. An IL-1 receptor expressed in human dermal fibroblasts has also been cloned and sequenced and found to be identical to the IL-1 receptor expressed in T cells

  3. Collagen I-induced dendritic cells activation is regulated by TNF-α ...

    Indian Academy of Sciences (India)

    2015-02-04

    Feb 4, 2015 ... Boks MA, Kager-Groenland JR, Mousset CM, van Ham SM and. Ten Brinke A 2014 Inhibition of TNF receptor signaling by anti-. TNFα biologicals primes naïve CD4(+) T cells towards IL-10(+). T cells with a regulatory phenotype and function. Clin. Immunol. 151 136–145. Brand U, Bellinghausen I, Enk AH, ...

  4. Analysis of cognition, motor performance and anxiety in young and aged tumor necrosis factor alpha receptor 1 and 2 deficient mice

    NARCIS (Netherlands)

    Naude, Petrus J W; Dobos, Nikoletta; van der Meer, Dennis; Mulder, Cornelis; Pawironadi, Kim G D; den Boer, Johan A; van der Zee, Eddy A; Luiten, Paulus; Eisel, Ulrich L M

    2014-01-01

    TNF-α plays important functional roles in the central nervous system during normal physiological circumstances via intricate signaling mechanisms between its receptors, TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). Although the roles of TNFR1 and TNFR2 in the diseased brain have received

  5. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis

    DEFF Research Database (Denmark)

    Gregory, Adam P; Dendrou, Calliope A; Attfield, Kathrine E

    2012-01-01

    Although there has been much success in identifying genetic variants associated with common diseases using genome-wide association studies (GWAS), it has been difficult to demonstrate which variants are causal and what role they have in disease. Moreover, the modest contribution that these varian...

  6. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin cDNA cloned from the liver was ...

  7. cDNA structure, genomic organization and expression patterns of ...

    African Journals Online (AJOL)

    use

    2011-11-23

    Nov 23, 2011 ... Visfatin was a newly identified adipocytokine, which was involved in various physiologic and pathologic processes of organisms. The cDNA structure, genomic organization and expression patterns of silver Prussian carp visfatin were described in this report. The silver Prussian carp visfatin. cDNA cloned ...

  8. Immunogenicity of Anti-TNF-α Biotherapies

    DEFF Research Database (Denmark)

    Bendtzen, Klaus

    2015-01-01

    Specific inhibition of the cytokine, tumor necrosis factor-α (TNF), has revolutionized the treatment of patients with several autoimmune diseases, and genetically engineered anti-TNF antibody constructs now constitute a heavy medicinal expenditure in many countries. Unfortunately, up to 30...... of protein drugs. The use of therapeutic strategies based on anti-TNF drug levels and ADA rather than dose-escalation has also proven to be cost-effective, as this allows individualized patient-tailored strategies rather than the current universal approach to loss of response. The objective of the present...... article - and the accompanying article - is to discuss the reasons for recommending assessments of circulating drug and ADA levels in patients treated with anti-TNF biopharmaceuticals and to detail some of the methodological issues that obscure cost-effective and safer therapies....

  9. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... specific for nodules were selected by differential colony hybridization using 32P-labeled cDNA synthesized either from nodule poly(A)+ RNA or from poly(A)+ RNA of uninfected root as probes. Among the recombinant plasmids, the cDNA gene for leghemoglobin was identified. The protein structure derived from...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  10. [The construction of rapid amplification of cDNA ends cDNA libraries from human fetal bone and joint].

    Science.gov (United States)

    Liang, X; Gong, Y; Liu, Q; Li, J; Chen, B; Guo, C

    2001-02-01

    To construct rapid amplification cDNA ends(RACE) cDNA libraries from human fetal bone and joint and provide resources for isolation of bone- and joint-specific development-related genes. Total RNA of bone and joint were extracted with the modified single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. The double-stranded end-blunted cDNA were synthesized using TaKaRa's cDNA synthesis kit and ligated to cassette adaptors. All of the cDNA molecules were amplified by a pair of common primers. A protocol for RACE cDNA library construction from bone and joint was established and two RACE cDNA libraries from human fetal bone and joint were successfully constructed. The protocol of RACE cDNA library construction from limited materials proved to be simple and efficient and the library was suitable for RACE to isolate tissue-specific genes.

  11. Serum progranulin levels in Hispanic rheumatoid arthritis patients treated with TNF antagonists: a prospective, observational study.

    Science.gov (United States)

    Johnson, Jennifer; Yeter, Karen; Rajbhandary, Rosy; Neal, Rebekah; Tian, Qingyun; Jian, Jinlong; Fadle, Natalie; Thurner, Lorenz; Liu, Chuanju; Stohl, William

    2017-03-01

    Since progranulin (PGRN) is a natural ligand of TNF receptors, we assessed whether serum PGRN levels predict and/or reflect responsiveness of RA patients to TNF-antagonist therapy. TNF-antagonist-naïve RA patients (N = 35) were started on TNF-antagonist therapy. At baseline and at follow-up visits, DAS28-ESR, DAS28-CRP, and CDAI were calculated, and venous blood was collected for serum PGRN determination. Disease activity and clinical response were based on EULAR criteria. Baseline serum PGRN levels varied considerably and correlated with ESR and CRP. DAS28-ESR, DAS28-CRP, and CDAI were greater in "PGRN-high" than in "PGRN-low". Baseline serum PGRN levels did not predict clinical responsiveness to TNF-antagonist therapy. Nevertheless, changes in serum PGRN levels at 274+ days following initiation of TNF-antagonist therapy correlated with changes in ESR, CRP, DAS28-ESR, DAS28-CRP, and CDAI. At this time, DAS28-ESR, DAS28-CRP, and CDAI in PGRN-high and PGRN-low equalized, but serum PGRN levels remained greater in PGRN-high than in PGRN-low. To our knowledge, the present report is the first prospective study to longitudinally assess changes in serum PGRN levels following initiation of TNF-antagonist therapy. Although pre-treatment serum PGRN levels may not predict clinical responsiveness to TNF-antagonist therapy, changes in serum PGRN levels correlate with changes in disease metrics over time. By inference, administration of PGRN may represent an effective therapeutic option for development in RA patients.

  12. In vitro and in vivo conditional sensitization of hepatocellular carcinoma cells to TNF-induced apoptosis by Taxol

    Science.gov (United States)

    Minero, VG; De Stefanis, D; Costelli, P; Baccino, FM; Bonelli, G

    2015-01-01

    High mortality among hepatocellular carcinoma (HCC) patients reflects both late diagnosis and low curability, due to pharmacoresistance. Taxol (TAX) is toxic for many human HCC-derived cell lines, yet its clinical efficacy on HCCs is poor. Combining TAX with other drugs appears a promising possibility to overcome such refractoriness. We analyzed whether combining tumor necrosis factor (TNF) with TAX would improve their toxicity. Human HCC-derived cell lines were treated with TAX or TNF, alone or combined. Apoptosis was assessed by morphology and flow-cytometry. Several pro- and anti-apoptotic molecules were evaluated by western blotting and/or enzymatic assay. After a 24 hour treatment, TNF was ineffective and TAX modestly cytotoxic, whereas HCC cells were conditionally sensitized to TNF by TAX. Indeed some relevant parameters were shifted to a prodeath setting: TNF-receptor 1 was increased, SOCS3, c-FLIP and pSTAT3 were markedly downregulated. These observations provide a significant clue to critically improve the drug susceptibility of HCC cells by combining 2 agents, TAX and TNF. The sequential application of TAX at a low dosage followed by TNF for only a short time triggered a strong apoptotic response. Of interest, prior TAX administration could also sensitize to TNF-induced apoptosis in the Yoshida AH-130 hepatoma transplanted in mice. Therefore, scrutinizing the possibility to develop similar combination drug regimens in suitable preclinical models seems highly advisable. PMID:25564714

  13. Role of TNF-α in lung tight junction alteration in mouse model of acute lung inflammation

    Directory of Open Access Journals (Sweden)

    Cuzzocrea Salvatore

    2007-10-01

    Full Text Available Abstract In the present study, we used tumor necrosis factor-R1 knock out mice (TNF-αR1KO to understand the roles of TNF-α on epithelial function in models of carrageenan-induced acute lung inflammation. In order to elucidate whether the observed anti-inflammatory status is related to the inhibition of TNF-α, we also investigated the effect of etanercept, a TNF-α soluble receptor construct, on lung TJ function. Pharmacological and genetic TNF-α inhibition significantly reduced the degree of (1 TNF-α production in pleural exudates and in the lung tissues, (2 the inflammatory cell infiltration in the pleural cavity as well as in the lung tissues (evaluated by MPO activity, (3 the alteration of ZO-1, Claudin-2, Claudin-4, Claudin-5 and β-catenin (immunohistochemistry and (4 apoptosis (TUNEL staining, Bax, Bcl-2 expression. Taken together, our results demonstrate that inhibition of TNF-α reduces the tight junction permeability in the lung tissues associated with acute lung inflammation, suggesting a possible role of TNF-α on lung barrier dysfunction.

  14. Activated platelets enhance IL-10 secretion and reduce TNF-α secretion by monocytes

    DEFF Research Database (Denmark)

    Gudbrandsdottir, Sif; Hasselbalch, Hans C; Nielsen, Claus H

    2013-01-01

    ), Escherichia coli LPS, or intact Porphyromonas gingivalis. Addition of platelets activated by thrombin-receptor-activating peptide enhanced IL-10 production induced by LPS (p ....05), and P. gingivalis (p IL-10 and TNF-α production were observed on addition of platelet supernatant to mononuclear cells, whereas addition of recombinant soluble CD40L mimicked the effects on IL-10...... production. Moreover, Ab-mediated blockade of CD40L counteracted the effect of platelets and platelet supernatants on TNF-α production. Monocytes separated into two populations with respect to IL-10 production induced by TG; the high-secreting fraction increased from 0.8 to 2.1% (p

  15. Radiation targeted TNF gene therapy in TNF-resistant prostate cancer

    International Nuclear Information System (INIS)

    Chung, Theodore D.K.; Mauceri, Helena J.; Hallahan, Dennis E.; Yu, Jenny; Yajnik, Santosh; Weichselbaum, Ralph R.

    1997-01-01

    Purpose/Objectives: Control of gene transcription by ionizing radiation represents a novel method of spatial and temporal regulation of genetic-based medical treatment. We previously reported improved tumor control using this approach in the radioresistant, TNF-sensitive squamous cell carcinoma, SQ-20B. Histopathologic evaluation of treated tumor specimens demonstrated tumor necrosis and vascular obstruction suggesting that the combined TNF and radiation treatment inhibits tumor growth through both direct and indirect mechanisms. The purpose of the present study was to determine the therapeutic potential of this approach in a TNF-resistant prostate cancer xenograft. Materials and Methods: The hormone-refractory human prostate cancer cell line PC-3 was transfected with Ad.Egr-TNF (adenovirus type 5 vector expressing TNFα under the transcriptional regulation of the radiation-inducible Egr-1 promoter) and TNF production was quantified by ELISA. TNF-induced cytotoxicity and radiosensitization were studied using rhTNF and PC-3 in an MTT assay and standard clonogenic survival assays. PC-3 xenografts were established approximately 2 weeks after injecting 2 x 10 7 cells S.C. in the right hind limb of female nude mice. Animals were treated with intratumoral injections of either Ad.Egr-TNF or buffer. Xenografts were irradiated with 5 Gy/fx 4 times per week to a total dose of 40 Gy using a Maxitron 250 x-ray generator (1.88 Gy/min). Results: TNF production in vitro. PC-3 cells infected with Ad.Egr-TNF released >1000 pg/ml TNF. Uninfected cells did not produce detectable levels of TNF. TNF mediated cytotoxicity in vitro. PC-3 cells were highly resistant to rhTNF with 78% cell survival observed at the highest concentration tested (10,000 U/ml). TNF mediated radiosensitization in vitro. rhTNF (1000 U/ml) had no significant effect on clonogenic survival of PC-3 cells following irradiation (D 0 =1.5). Combined Ad.Egr-TNF and 40 Gy in vivo. Combined treatment of PC-3 xenografts

  16. Molecular cloning of feline tumour necrosis factor receptor type I (TNFR I) and expression of TNFR I and TNFR II in lymphoid cells in cats.

    Science.gov (United States)

    Mizuno, T; Goto, Y; Baba, K; Masuda, K; Ohno, K; Tsujimoto, H

    2003-04-01

    Tumour necrosis factor (TNF)-alpha is a pro-inflammatory cytokine produced by many types of cells. It has been shown that two distinct TNF receptors (TNFRs), TNFR type I (TNFR I) and TNFR type II (TNFR II), have different functions in signal transduction, which is possibly associated with the development of a variety of diseases. In this study, we isolated a feline TNFR I cDNA clone and analysed the expression of TNFR I and TNFR II mRNA in feline lymphoid cells. The deduced amino acid sequence of feline TNFRI cDNA showed 75.8, 62.5 60.9 and 72.1% similarity with those of its human, mouse, rat, and pig counterparts, respectively. The feline TNFR I cDNA was shown to encode extracellular, transmembrane and intracellular domains fundamentally conserved in the homologues of other species. Expression of TNFR I and TNFR II mRNAs was shown to be up-regulated in feline peripheral blood mononuclear cells (PBMC) by stimulation with concanavalin A. Five of six feline lymphoma cell lines were shown to express both TNFR I and TNFR II mRNAs. The expression of TNFR I in PBMC was up-regulated in cats infected with feline immunodeficiency virus (FIV), whereas the expression of TNFR II in PBMC was not different between FIV-infected cats and uninfected cats. The present study indicate that expression of TNFR I and TNFR II may be associated with disease progression, especially in retrovirus infections in cats.

  17. Molecular cloning of lupin leghemoglobin cDNA

    DEFF Research Database (Denmark)

    Konieczny, A; Jensen, E O; Marcker, K A

    1987-01-01

    Poly(A)+ RNA isolated from root nodules of yellow lupin (Lupinus luteus, var. Ventus) has been used as a template for the construction of a cDNA library. The ds cDNA was synthesized and inserted into the Hind III site of plasmid pBR 322 using synthetic Hind III linkers. Clones containing sequences...... its nucleotide sequence was consistent with known amino acid sequence of lupin Lb II. The cloned lupin Lb cDNA hybridized to poly(A)+ RNA from nodules only, which is in accordance with the general concept, that leghemoglobin is expressed exclusively in nodules. Udgivelsesdato: 1987-null...

  18. Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

    Science.gov (United States)

    van Schie, Karin A.; Ooijevaar-de Heer, Pleuni; Dijk, Lisanne; Kruithof, Simone; Wolbink, Gertjan; Rispens, Theo

    2016-01-01

    Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab. PMID:27605058

  19. Molecular cloning and characterization of a cDNA encoding ...

    African Journals Online (AJOL)

    enoh

    2012-03-29

    Nanjing) co., Ltd. The nucleotide sequences of these primers are as follows: ..... Ebizuka Y (2000). Molecular cloning and characterization of a cDNA for Glycyrrhiza glabra cycloartenol synthase. Biol. Pharm. Bull. 23(2):231-234.

  20. Method for construction of normalized cDNA libraries

    Science.gov (United States)

    Soares, Marcelo B.; Efstratiadis, Argiris

    1998-01-01

    This invention provides a method to normalize a directional cDNA library constructed in a vector that allows propagation in single-stranded circle form comprising: (a) propagating the directional cDNA library in single-stranded circles; (b) generating fragments complementary to the 3' noncoding sequence of the single-stranded circles in the library to produce partial duplexes; (c) purifying the partial duplexes; (d) melting and reassociating the purified partial duplexes to appropriate Cot; and (e) purifying the unassociated single-stranded circles, thereby generating a normalized cDNA library. This invention also provides normalized cDNA libraries generated by the above-described method and uses of the generated libraries.

  1. Screening of cDNA libraries on glass slide microarrays.

    Science.gov (United States)

    Berger, Dave K; Crampton, Bridget G; Hein, Ingo; Vos, Wiesner

    2007-01-01

    A quantitative screening method was developed to evaluate the quality of cDNA libraries constructed by suppression subtraction hybridization (SSH) or other enrichment techniques. The SSH technique was adapted to facilitate screening of the resultant library on a small number of glass slide microarrays. A simple data analysis pipeline named SSHscreen using "linear models for microarray data" (limma) functions in the R computing environment was developed to identify clones in the cDNA libraries that are significantly differentially expressed, and to determine if they were rare or abundant in the original treated sample. This approach facilitates the choice of clones from the cDNA library for further analysis, such as DNA sequencing, Northern blotting, RT-PCR, or detailed expression profiling using a custom cDNA microarray. Furthermore, this strategy is particularly useful for studies of nonmodel organisms for which there is little genome sequence information.

  2. Constructing and detecting a cDNA library for mites.

    Science.gov (United States)

    Hu, Li; Zhao, YaE; Cheng, Juan; Yang, YuanJun; Li, Chen; Lu, ZhaoHui

    2015-10-01

    RNA extraction and construction of complementary DNA (cDNA) library for mites have been quite challenging due to difficulties in acquiring tiny living mites and breaking their hard chitin. The present study is to explore a better method to construct cDNA library for mites that will lay the foundation on transcriptome and molecular pathogenesis research. We selected Psoroptes cuniculi as an experimental subject and took the following steps to construct and verify cDNA library. First, we combined liquid nitrogen grinding with TRIzol for total RNA extraction. Then, switching mechanism at 5' end of the RNA transcript (SMART) technique was used to construct full-length cDNA library. To evaluate the quality of cDNA library, the library titer and recombination rate were calculated. The reliability of cDNA library was detected by sequencing and analyzing positive clones and genes amplified by specific primers. The results showed that the RNA concentration was 836 ng/μl and the absorbance ratio at 260/280 nm was 1.82. The library titer was 5.31 × 10(5) plaque-forming unit (PFU)/ml and the recombination rate was 98.21%, indicating that the library was of good quality. In the 33 expressed sequence tags (ESTs) of P. cuniculi, two clones of 1656 and 1658 bp were almost identical with only three variable sites detected, which had an identity of 99.63% with that of Psoroptes ovis, indicating that the cDNA library was reliable. Further detection by specific primers demonstrated that the 553-bp Pso c II gene sequences of P. cuniculi had an identity of 98.56% with those of P. ovis, confirming that the cDNA library was not only reliable but also feasible.

  3. Characterization in vitro of a human tumor necrosis factor-binding protein. A soluble form of a tumor necrosis factor receptor.

    OpenAIRE

    Lantz, M; Gullberg, U; Nilsson, E; Olsson, I

    1990-01-01

    Tumor necrosis factor (TNF) is a pleiotropic mediator of inflammatory responses. A cysteine-rich, highly glycosylated 30-kD TNF-binding protein (TNF-BP) purified from urine may have a role in regulation because it protects in vitro against the biological effects of TNF. The cytotoxic effect of TNF on the fibrosarcoma cell line WEHI 164 was inhibited by 50% at a 10-fold excess of TNF-BP. The binding of TNF to the receptor was partially reversed after the addition of TNF-BP. Results from biosyn...

  4. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    International Nuclear Information System (INIS)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon

    2003-01-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology

  5. Radioactive cDNA microarray (II): Gene expression profiling of antidepressant treatment by human cDNA microarray

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Ji Hye; Kang, Rhee Hun; Ham, Byung Joo; Lee, Min Su; Shin, Kyung Ho; Choe, Jae Gol; Kim, Meyoung Kon [College of Medicine, Univ. of Korea, Seoul (Korea, Republic of)

    2003-07-01

    Major depressive disorder is a prevalent psychiatric disorder in primary care, associated with impaired patient functioning and well-being. Fluoxetine is a selective serotonin-reuptake inhibitors (SSRIs) and is a commonly prescribed antidepressant compound. Its action is primarily attributed to selective inhibition of the reuptake of serotonin (5-hydroxytryptamine) in the central nervous system. Objectives ; the aims of this study were two-fold: (1) to determine the usefulness for investigation of the transcription profiles in depression patients, and (2) to assess the differences in gene expression profiles between positive response group and negative response groups by fluoxetine treatment. This study included 53 patients with major depression (26 in positive response group with antidepressant treatment, 27 in negative response group with antidepressant treatment), and 53 healthy controls. To examine the difference of gene expression profile in depression patients, radioactive complementary DNA microarrays were used to evaluate changes in the expression of 1,152 genes in total. Using 33p-labeled probes, this method provided highly sensitive gene expression profiles including brain receptors, drug metabolism, and cellular signaling. Gene transcription profiles were classified into several categories in accordance with the antidepressant gene-regulation. The gene profiles were significantly up-(22 genes) and down-(16 genes) regulated in the positive response group when compared to the control group. Also, in the negative response group, 35 genes were up-regulated and 8 genes were down-regulated when compared to the control group. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  6. High-Throughput Plasmid cDNA Library Screening

    Energy Technology Data Exchange (ETDEWEB)

    Wan, Kenneth H.; Yu, Charles; George, Reed A.; Carlson, JosephW.; Hoskins, Roger A.; Svirskas, Robert; Stapleton, Mark; Celniker, SusanE.

    2006-05-24

    Libraries of cDNA clones are valuable resources foranalysing the expression, structure, and regulation of genes, as well asfor studying protein functions and interactions. Full-length cDNA clonesprovide information about intron and exon structures, splice junctionsand 5'- and 3'-untranslated regions (UTRs). Open reading frames (ORFs)derived from cDNA clones can be used to generate constructs allowingexpression of native proteins and N- or C-terminally tagged proteins.Thus, obtaining full-length cDNA clones and sequences for most or allgenes in an organism is critical for understanding genome functions.Expressed sequence tag (EST) sequencing samples cDNA libraries at random,which is most useful at the beginning of large-scale screening projects.However, as projects progress towards completion, the probability ofidentifying unique cDNAs via EST sequencing diminishes, resulting in poorrecovery of rare transcripts. We describe an adapted, high-throughputprotocol intended for recovery of specific, full-length clones fromplasmid cDNA libraries in five days.

  7. Cloning of the cDNA for human 12-lipoxygenase

    International Nuclear Information System (INIS)

    Izumi, T.; Hoshiko, S.; Radmark, O.; Samuelsson, B.

    1990-01-01

    A full-length cDNA clone encoding 12-lipoxygenase was isolated from a human platelet cDNA library by using a cDNA for human reticulocyte 15-lipoxygenase as probe for the initial screening. The cDNA had an open reading frame encoding 662 amino acid residues with a calculated molecular weight of 75,590. Three independent clones revealed minor heterogeneities in their DNA sequences. Thus, in three positions of the deduced amino acid sequence, there is a choice between two different amino acids. The deduced sequence from the clone plT3 showed 65% identity with human reticulocyte 15-lipoxygenase and 42% identity with human leukocyte 5-lipoxygenase. The 12-lipoxygenase cDNA recognized a 3.0-kilobase mRNA species in platelets and human erythroleukemia cells (HEL cells). Phorbol 12-tetradecanoyl 13-acetate induced megakaryocytic differentiation of HEL cells and 12-lipoxygenase activity and increased mRNA for 12-lipoxygenase. The identity of the cloned 12-lipoxygenase was assured by expression in a mammalian cell line (COS cells). Human platelet 12-lipoxygenase has been difficult to purify to homogeneity. The cloning of this cDNA will increase the possibilities to elucidate the structure and function of this enzyme

  8. Leptin potentiates Prevotella intermedia lipopolysaccharide-induced production of TNF-alpha in monocyte-derived macrophages.

    Science.gov (United States)

    Kim, Sung-Jo

    2010-06-01

    In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-alpha production in differentiated THP-1 cells, a human monocytic cell line. LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-alpha and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-alpha and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Leptin enhanced P. intermedia LPS-induced TNF-alpha production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-alpha expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-alpha production was not mediated by the leptin receptor. The ability of leptin to enhance P. intermedia LPS-induced TNF-alpha production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

  9. TNF-α potentiates uric acid-induced interleukin-1β (IL-1β) secretion in human neutrophils.

    Science.gov (United States)

    Yokose, Kohei; Sato, Shuzo; Asano, Tomoyuki; Yashiro, Makiko; Kobayashi, Hiroko; Watanabe, Hiroshi; Suzuki, Eiji; Sato, Chikako; Kozuru, Hideko; Yatsuhashi, Hiroshi; Migita, Kiyoshi

    2017-09-14

    Monosodium urate (MSU) has been shown to promote interleukin-1β (IL-1β) secretion in human monocytes, but the priming signals for NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome pathway remains elusive. In this study, we investigated the role of Tumor necrosis factor-alpha (TNF-α) on MSU-mediated IL-1β induction in human neutrophils. Human neutrophils were stimulated with MSU, in the presence or absence of TNF-α priming. The cellular supernatants were analyzed for IL-1β, IL-18, and caspase-1 by enzyme-linked immunosorbent assay (ELISA) methods. Pro-IL-1β mRNA expressions in human neutrophils were analyzed by real-time PCR method. TNF-α stimulation induced pro-IL-1β mRNA expression; however, MSU stimulation did not induce pro-IL-1β mRNA expression in human neutrophils. TNF-α alone or MSU stimulation did not result in efficient IL-1β secretion in human neutrophils, whereas in TNF-α-primed neutrophils, MSU stimulation resulted in a marked IL-1β and IL-18 secretion. TNF-α-primed neutrophils secreted cleaved caspase-1 (p20), in response to MSU stimulation. Our data demonstrate that priming of human neutrophils with TNF-α promotes uric acid-mediated IL-1β secretion in the absence of microbial stimulation. These findings provide insights into the neutrophils-mediated inflammatory processes in gouty arthritis.

  10. TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis

    Science.gov (United States)

    Noti, Mario; Corazza, Nadia; Mueller, Christoph; Berger, Barbara

    2010-01-01

    Although tumor necrosis factor (α) (TNF) exerts proinflammatory activities in a variety of diseases, including inflammatory bowel disease, there is increasing evidence for antiinflammatory actions of TNF. In contrast, glucocorticoids (GCs) are steroid hormones that suppress inflammation, at least in part by regulating the expression and action of TNF. We report that TNF induces extraadrenal production of immunoregulatory GCs in the intestinal mucosa during acute intestinal inflammation. The absence of TNF results in a lack of colonic GC synthesis and exacerbation of dextran sodium sulfate–induced colitis. TNF seems to promote local steroidogenesis by directly inducing steroidogenic enzymes in intestinal epithelial cells. Therapeutic administration of TNF induces GC synthesis in oxazolone-induced colitis and ameliorates intestinal inflammation, whereas inhibition of intestinal GC synthesis abrogates the therapeutic effect of TNF. These data show that TNF suppresses the pathogenesis of acute intestinal inflammation by promoting local steroidogenesis. PMID:20439544

  11. Intracellular regulation of TNF activity in health and disease.

    Science.gov (United States)

    Varfolomeev, Eugene; Vucic, Domagoj

    2018-01-01

    Tumor Necrosis Factor alpha (TNFα, TNF) is a key mediator and regulator of mammalian immune responses in healthy organisms and in diseased conditions. TNF governs development of the immune system, cell survival signaling pathways, proliferation and regulates metabolic processes. Whereas TNF-induced NF-κB and MAP pro-survival kinase activities constitute its major biochemical functions, TNF can also stimulate cell death in certain pathological situations. TNF-induced signal transduction pathways are tightly regulated through ubiquitination and phosphorylation of molecules partaking in all TNF-dependent membrane-associated and intracellular protein signaling complexes. Deregulated TNF signaling in individuals carrying naturally occurring genetic mutations in proteins that mediate TNF signaling, or in corresponding genetically modified animal models, results in severe pathologies. In this review we will describe the current knowledge of TNF signaling and its relevance for human health. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. β2-adrenergic agonists modulate TNF-α induced astrocytic inflammatory gene expression and brain inflammatory cell populations

    Science.gov (United States)

    2014-01-01

    Background The NF-κB signaling pathway orchestrates many of the intricate aspects of neuroinflammation. Astrocytic β2-adrenergic receptors have emerged as potential regulators in central nervous system inflammation and are potential targets for pharmacological modulation. The aim of this study was to elucidate the crosstalk between astrocytic β2-adrenergic receptors and the TNF-α induced inflammatory gene program. Methods Proinflammatory conditions were generated by the administration of TNF-α. Genes that are susceptible to astrocytic crosstalk between β2-adrenergic receptors (stimulated by clenbuterol) and TNF-α were identified by qPCR-macroarray-based gene expression analysis in a human 1321 N1 astrocytoma cell line. Transcriptional patterns of the identified genes in vitro were validated by RT-PCR on the 1321 N1 cell line as well as on primary rat astrocytes. In vivo expression patterns were examined by intracerebroventricular administration of clenbuterol and/or TNF-α in rats. To examine the impact on the inflammatory cell content of the brain we performed extensive FACS analysis of rat brain immune cells after intracerebroventricular clenbuterol and/or TNF-α administration. Results Parallel transcriptional patterns in vivo and in vitro confirmed the relevance of astrocytic β2-adrenergic receptors as modulators of brain inflammatory responses. Importantly, we observed pronounced effects of β2-adrenergic receptor agonists and TNF-α on IL-6, CXCL2, CXCL3, VCAM1, and ICAM1 expression, suggesting a role in inflammatory brain cell homeostasis. Extensive FACS-analysis of inflammatory cell content in the brain demonstrated that clenbuterol/TNF-α co-administration skewed the T cell population towards a double negative phenotype and induced a shift in the myeloid brain cell population towards a neutrophilic predominance. Conclusions Our results show that astrocytic β2-adrenergic receptors are potent regulators of astrocytic TNF-α-activated genes in

  13. TNF-α/TNFR1 Signaling is Required for the Full Expression of Acute and Chronic Itch in Mice via Peripheral and Central Mechanisms.

    Science.gov (United States)

    Miao, Xiuhua; Huang, Ya; Liu, Teng-Teng; Guo, Ran; Wang, Bing; Wang, Xue-Long; Chen, Li-Hua; Zhou, Yan; Ji, Ru-Rong; Liu, Tong

    2018-02-01

    Increasing evidence suggests that cytokines and chemokines play crucial roles in chronic itch. In the present study, we evaluated the roles of tumor necrosis factor-alpha (TNF-α) and its receptors TNF receptor subtype-1 (TNFR1) and TNFR2 in acute and chronic itch in mice. Compared to wild-type (WT) mice, TNFR1-knockout (TNFR1-KO) and TNFR1/R2 double-KO (DKO), but not TNFR2-KO mice, exhibited reduced acute itch induced by compound 48/80 and chloroquine (CQ). Application of the TNF-synthesis inhibitor thalidomide and the TNF-α antagonist etanercept dose-dependently suppressed acute itch. Intradermal injection of TNF-α was not sufficient to evoke scratching, but potentiated itch induced by compound 48/80, but not CQ. In addition, compound 48/80 induced TNF-α mRNA expression in the skin, while CQ induced its expression in the dorsal root ganglia (DRG) and spinal cord. Furthermore, chronic itch induced by dry skin was reduced by administration of thalidomide and etanercept and in TNFR1/R2 DKO mice. Dry skin induced TNF-α expression in the skin, DRG, and spinal cord and TNFR1 expression only in the spinal cord. Thus, our findings suggest that TNF-α/TNFR1 signaling is required for the full expression of acute and chronic itch via peripheral and central mechanisms, and targeting TNFR1 may be beneficial for chronic itch treatment.

  14. TNF-α Mediates the Intrinsic and Extrinsic Pathway in Propofol-Induced Neuronal Apoptosis Via PI3K/Akt Signaling Pathway in Rat Prefrontal Cortical Neurons.

    Science.gov (United States)

    Deng, Xiaoyuan; Chen, Bo; Wang, Bin; Zhang, Junfang; Liu, Hongliang

    2017-10-01

    Propofol can cause developing neuronal apoptosis in both in vivo and in vitro studies, and the mechanism is unclear till now. Our previous study has demonstrated that propofol can increase the TNF-α expression in the prefrontal cortex in rat developing brain, the TNF-α antagonist, etanercept, can inhibit propofol-induced neuronal apoptosis, but little is known about how TNF-α mediates that process. This study reveals that propofol at clinically relevant concentrations increases the TNF-α synthesis and release in neurons, and induces neuronal apoptosis; etanercept significantly reduces neuronal apoptosis, the elevation of cleaved caspase-8 and cleaved caspase-9, or the Akt phosphorylation induced by propofol, while the selective PI3K antagonist blocks the neuroprotection of etanercept. Propofol does not change the expression of P2X7 receptor in neurons, and the P2X7 receptor antagonist cannot affect the TNF-α synthesis or release after propofol treatment. These results suggest that propofol can increase the synthesis and release of TNF-α in the primary cultured prefrontal cortical neurons, TNF-α contributes to the intrinsic and extrinsic pathway in propofol-induced neuronal apoptosis via PI3K/Akt signaling pathway, and P2X7R is not involved in the synthesis and release of TNF-α induced by propofol.

  15. Effect of TNF{alpha} on activities of different promoters of human apolipoprotein A-I gene

    Energy Technology Data Exchange (ETDEWEB)

    Orlov, Sergey V., E-mail: serge@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Mogilenko, Denis A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Shavva, Vladimir S. [Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation); Dizhe, Ella B.; Ignatovich, Irina A. [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Perevozchikov, Andrej P., E-mail: app@iem.sp.ru [Department of Biochemistry, Institute of Experimental Medicine, Russian Academy of Medical Sciences, 197376 St. Petersburg (Russian Federation); Department of Embryology, St. Petersburg State University, 199034 St. Petersburg (Russian Federation)

    2010-07-23

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

  16. Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells

    Energy Technology Data Exchange (ETDEWEB)

    Vy Tran, An Hue; Hahm, Soo-Hyun; Han, Se Hee [Department of Advanced Technology Fusion, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of); Chung, Ji Hyung [Department of Applied Bioscience, College of Life Science, CHA University, Gyeonggi-do 463-836 (Korea, Republic of); Park, Geon Tae [Cornell University, Ithaca, NY 14850 (United States); Han, Ye Sun, E-mail: yshan@konkuk.ac.kr [College of Global Integrated Studies, Division of Interdisciplinary Studies, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul 143-701 (Korea, Republic of)

    2015-07-15

    Highlights: • We determine the interaction between hMYH and hTRADD. • We examine changes in the level of hMYH–hTRADD interaction under TNF-α treatment. • hTRADD–hMYH association is involved in the nuclear translocation of NFκB. • hTRADD–hMYH complex influences the TNFR1–TRADD association. - Abstract: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD–hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1–TRADD complex. Thus, this study identified a novel mechanism through which the hMYH–hTRADD interaction may affect the TNF-α signaling pathway. Implications: In HeLa cells, the hTRADD–hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

  17. PGBR extract ameliorates TNF-α induced insulin resistance in hepatocytes.

    Science.gov (United States)

    Chen, Fu-Chih; Shen, Kuo-Ping; Chen, Jin-Bor; Lin, Hui-Li; Hao, Chi-Long; Yen, Hsueh-Wei; Shaw, Shyh-Yu

    2018-01-01

    Pre-germinated brown rice (PGBR) could ameliorate metabolic syndrome, however, not much research estimates the effect of PGBR extract on insulin resistance. The aim of this study is to examine the effects of PGBR extract in TNF-α induced insulin resistance. HepG2 cells, hepatocytes, were cultured in DMEM medium and added with 5 μM insulin or with insulin and 30 ng/ml TNF-α or with insulin, TNF-α and PGBR extract (50, 100, 300 μg/ml). The glucose levels of the medium were decreased by insulin, demonstrating insulin promoted glucose uptake into cell. However, TNF-α inhibited glucose uptake into cells treated with insulin. Moreover, insulin increased the protein expressions of AMP-activated protein kinase (AMPK), insulin receptor substrate-1 (IRS-1), phosphatidylinositol-3-kinase-α (PI3K-α), serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB), glucose transporter-2 (GLUT-2), glucokinase (GCK), peroxisome proliferator activated receptor-α (PPAR-α) and PPAR-γ. TNF-α activated p65 and MAPKs (JNK1/2 and ERK1/2) which worsened the expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, glycogen synthase kinase-3 (GSK-3), PPAR-α and PPAR-γ. Once this relationship was established, we added PGBR extract to cell with insulin and TNF-α. We found glucose levels of medium were lowered and that the protein expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, GSK-3, PPAR-α, PPAR-γ and p65, JNK1/2 were also recovered. In conclusion, this study found that TNF-α inhibited insulin stimulated glucose uptake and aggravated related proteins expressions, suggesting that it might cause insulin resistance. PGBR extract was found to ameliorate this TNF-α induced insulin resistance, suggesting that it might be used in the future to help control insulin resistance. Copyright © 2017. Published by Elsevier Taiwan.

  18. PGBR extract ameliorates TNF-α induced insulin resistance in hepatocytes

    Directory of Open Access Journals (Sweden)

    Fu-Chih Chen

    2018-01-01

    Full Text Available Pre-germinated brown rice (PGBR could ameliorate metabolic syndrome, however, not much research estimates the effect of PGBR extract on insulin resistance. The aim of this study is to examine the effects of PGBR extract in TNF-α induced insulin resistance. HepG2 cells, hepatocytes, were cultured in DMEM medium and added with 5 μM insulin or with insulin and 30 ng/ml TNF-α or with insulin, TNF-α and PGBR extract (50, 100, 300 μg/ml. The glucose levels of the medium were decreased by insulin, demonstrating insulin promoted glucose uptake into cell. However, TNF-α inhibited glucose uptake into cells treated with insulin. Moreover, insulin increased the protein expressions of AMP-activated protein kinase (AMPK, insulin receptor substrate-1 (IRS-1, phosphatidylinositol-3-kinase-α (PI3K-α, serine/threonine kinase PI3K-linked protein kinase B (Akt/PKB, glucose transporter-2 (GLUT-2, glucokinase (GCK, peroxisome proliferator activated receptor-α (PPAR-α and PPAR-γ. TNF-α activated p65 and MAPKs (JNK1/2 and ERK1/2 which worsened the expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, glycogen synthase kinase-3 (GSK-3, PPAR-α and PPAR-γ. Once this relationship was established, we added PGBR extract to cell with insulin and TNF-α. We found glucose levels of medium were lowered and that the protein expressions of AMPK, IRS-1, PI3K-α, Akt/PKB, GLUT-2, GCK, GSK-3, PPAR-α, PPAR-γ and p65, JNK1/2 were also recovered. In conclusion, this study found that TNF-α inhibited insulin stimulated glucose uptake and aggravated related proteins expressions, suggesting that it might cause insulin resistance. PGBR extract was found to ameliorate this TNF-α induced insulin resistance, suggesting that it might be used in the future to help control insulin resistance.

  19. Smoking, white blood cell counts, and TNF system activity in Japanese male subjects with normal glucose tolerance

    Directory of Open Access Journals (Sweden)

    Watanabe Naoya

    2011-11-01

    Full Text Available Abstract Background Cigarette smokers have increased white blood cell (WBC counts and the activation of tumor necrosis factor (TNF. The effect of smoking on WBC counts and TNF system activity, however, has not been separately investigated yet. Subjects and Methods One hundred and forty-two Japanese male subjects with normal glucose tolerance were recruited. They were stratified into two groups based on the questionnaire for smoking: one with current smokers (n = 48 and the other with current non-smokers (n = 94. Whereas no significant differences were observed in age, BMI, high molecular weight (HMW adiponectin, and TNF-α between the two groups, current smokers had significantly higher soluble TNF receptor 1 (sTNF-R1 (1203 ± 30 vs. 1116 ± 21 pg/ml, p = 0.010 and increased WBC counts (7165 ± 242 vs. 5590 ± 163/μl, p p = 0.031 as compared to current non-smokers. Next, we classified 48 current smokers into two subpopulations: one with heavy smoking (Brinkman index ≥ 600 and the other with light smoking (Brinkman index Results Whereas no significant difference was observed in age, BMI, HMW adiponectin, WBC counts and TNF-α, sTNF-R1 and sTNF-R2 were significantly higher in heavy smoking group (1307 ± 44 vs. 1099 ± 30 pg/ml, p p = 0.005 than in light smoking group, whose sTNF-R1 and sTNF-R2 were similar to non-smokers (sTNF-R1: 1116 ± 15 pg/ml, p = 0.718, sTNF-R2; 1901 ± 32 pg/ml, p = 0.437. In contrast, WBC counts were significantly increased in heavy (7500 ± 324/μl, p p = 0.001 smoking group as compared to non-smokers (5590 ± 178/μl. There was no significant difference in WBC counts between heavy and light smoking group (p = 0.158. Conclusion We can hypothesize that light smoking is associated with an increase in WBC counts, while heavy smoking is responsible for TNF activation in Japanese male subjects with normal glucose tolerance.

  20. cDNA library Table - KAIKOcDNA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data List Contact us KAIKOcDNA... cDNA library Table Data detail Data name cDNA library Table DOI 10.18908/lsdba.nbd...c00951-005 Description of data contents List of Bombyx mori cDNA libraries. Data file File name: kaiko_cdna_...iption Registered library name Registered name of the partial cDNA library Library synonym Another name for cDNA... Download License Update History of This Database Site Policy | Contact Us cDNA library Table - KAIKOcDNA | LSDB Archive ...

  1. Study on construction of cDNA library of the treated changliver cell and quality analysis

    OpenAIRE

    Juntang, Lin; Pramanik, Jogenananda; Congrui, Wang; Huiyong, Zhang; Huigen, Feng; Baosheng, Yang; Yuchang, Li; Cunshuan, Xu

    2004-01-01

    The study aims to construct cDNA library of Changliver cell by SMART (switching mechanism at 5′ end of RNA transcript) technique and analyze its quality. cDNA of Changliver cell was made with RT-PCR and LD-PCR (long-distance PCR), the cDNA library was constructed with SMART cDNA library construction kit. Through testing, the high quality cDNA library containing whole long cDNA of Changliver cell had been constructed. The titer of the amplified cDNA library was 4.5 × 1010 pfu/ml and the averag...

  2. TNF-alpha stimulation increases dental pulp stem cell migration in vitro through integrin alpha-6 subunit upregulation.

    Science.gov (United States)

    Shi, Lei; Fu, Shanqi; Fahim, Sidra; Pan, Shuang; Lina, He; Mu, Xiaodan; Niu, Yumei

    2017-03-01

    The dissemination of stem cells into tissues requiring inflammatory and reparative response is fundamentally dependent upon their chemotactic migration. Expression of TNF-α is up regulated in inflamed pulps. Dental pulp cells are also known to express integrin α6 subunit. Expression of integrin subunit α6 has been linked to the acquisition of migratory potential in a wide variety of cell types in both pathological and physiological capacities. Therefore, in this study we examined the effects of a pleiotropic cytokine TNF-α on the migration of hDPSCs and investigated its relationship with expression of integrin α6 in hDPSCs during chemotactic migration. hDPSC cultures were established. Protein expression profile of α6 integrin subunit was determined. Effect of exogenous TNF-α (50ng/mL) on hDPSCs' migration potential was evaluated by transwell inserts and in vitro scratch assay. Upregulation/downregulation of TNF-α mediated migration was assayed in presence/absence of integrin α6 respectively. To suppress integrin α6 expression, cells were transfected with integrin α6 siRNA and then cell migration and cytoskeletal changes were evaluated. Our results showed significant increase of hDPSCs' migration after stimulation with TNF-α. By knockdown of integrin α6, which is upregulated by TNF-α, we observed a decrease in the TNF-α directed chemotaxis of hDPSCs. In this study, we show that activation of integrin α6 brought about by TNF-α led to an increase in migratory activity in DPSCs in vitro thus describing a novel association between a cytokine TNF-α and α6 chain of an adhesion receptor integrin in regulating migration of hDPSCs. Copyright © 2016. Published by Elsevier Ltd.

  3. cDNA library construction of two human Demodexspecies.

    Science.gov (United States)

    Niu, DongLing; Wang, RuiLing; Zhao, YaE; Yang, Rui; Hu, Li; Lei, YuYang; Dan, WeiChao

    2017-06-01

    The research of Demodex, a type of pathogen causing various dermatoses in animals and human beings, is lacking at RNA level. This study aims at extracting RNA and constructing cDNA library for Demodex. First, P. cuniculiand D. farinaewere mixed to establish homogenization method for RNA extraction. Second, D. folliculorumand D. breviswere collected and preserved in Trizol, which were mixed with D. farinaerespectively to extract RNA. Finally, cDNA library was constructed and its quality was assessed. The results indicated that for D. folliculorum& D. farinae, the recombination rate of cDNA library was 90.67% and the library titer was 7.50 × 104 pfu/ml. 17 of the 59 positive clones were predicted to be of D. folliculorum; For D. brevis& D. farinae, the recombination rate was 90.96% and the library titer was 7.85 x104 pfu/ml. 40 of the 59 positive clones were predicted to be of D. brevis. Further detection by specific primers demonstrated that mtDNA cox1, cox3and ATP6 detected from cDNA libraries had 96.52%-99.73% identities with the corresponding sequences in GenBank. In conclusion, the cDNA libraries constructed for Demodexmixed with D. farinaewere successful and could satisfy the requirements for functional genes detection.

  4. Second-strand cDNA synthesis: classical method

    International Nuclear Information System (INIS)

    Gubler, U.

    1987-01-01

    The classical scheme for the synthesis of double-stranded cDNA as it was reported in 1976 is described. Reverse transcription of mRNA with oligo(dT) as the primer generates first strands with a small loop at the 3' end of the cDNA (the end that corresponds to the 5' end of the mRNA). Subsequent removal of the mRNA by alkaline hydrolysis leaves single-stranded cDNA molecules again with a small 3' loop. This loop can be used by either reverse transcriptase or Klenow fragment of DNA polymerase I as a primer for second-strand synthesis. The resulting products are double-stranded cDNA molecules that are covalently closed at the end corresponding to the 5' end of the original mRNA. Subsequent cleavage of the short piece of single-stranded cDNA within the loop with the single-strand-specific S 1 nuclease generate open double-stranded molecules that can be used for molecular cloning in plasmids or in phage. Useful variations of this scheme have been described

  5. Complications of TNF-α antagonists and iron homeostasis

    Science.gov (United States)

    TNF-α is a central regulator of inflammation and its blockade downregulates other proinflammatory cytokines, chemokines, and growth factors. Subsequently, TNF-α antagonists are currently used in treatment regimens directed toward several inflammatory diseases. Despite a beneficia...

  6. Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

    Science.gov (United States)

    Murata, H; Hattori, T; Maeda, H; Takashiba, S; Takigawa, M; Kido, J; Nagata, T

    2015-08-01

    Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel

  7. Confirmation of an IRAK3 polymorphism as a genetic marker predicting response to anti-TNF treatment in rheumatoid arthritis

    DEFF Research Database (Denmark)

    Sode, Jacob; Vogel, U; Bank, Steffen

    2018-01-01

    Several genetic variants in Toll-like receptor (TLR) and nuclear factor (NF)-κB signalling pathways have been reported associated with responsiveness to tumour necrosis factor inhibitor (anti-TNF) treatment in rheumatoid arthritis (RA). The present study was undertaken to replicate these findings...... online publication, 4 October 2016; doi:10.1038/tpj.2016.66....

  8. cDNA libraries for virus-induced gene silencing.

    Science.gov (United States)

    Todd, Andrea T; Liu, Enwu; Page, Jonathan E

    2010-01-01

    Virus-induced gene silencing (VIGS) exploits endogenous plant antiviral defense mechanisms to posttranscriptionally silence the expression of targeted plant genes. VIGS is quick and relatively easy to perform and therefore serves as a powerful tool for high-throughput functional genomics in plants. Combined with the use of subtractive cDNA libraries for generating a collection of VIGS-ready cDNA inserts, VIGS can be utilized to screen a large number of genes to determine phenotypes resulting from the knockdown/knockout of gene function. Taking into account the optimal insert design for VIGS, we describe a methodology for producing VIGS-ready cDNA libraries enriched for inserts relevant to the biological process of interest.

  9. Procedure for normalization of cDNA libraries

    Science.gov (United States)

    Bonaldo, Maria DeFatima; Soares, Marcelo Bento

    1997-01-01

    This invention provides a method to normalize a cDNA library constructed in a vector capable of being converted to single-stranded circles and capable of producing complementary nucleic acid molecules to the single-stranded circles comprising: (a) converting the cDNA library in single-stranded circles; (b) generating complementary nucleic acid molecules to the single-stranded circles; (c) hybridizing the single-stranded circles converted in step (a) with complementary nucleic acid molecules of step (b) to produce partial duplexes to an appropriate Cot; (e) separating the unhybridized single-stranded circles from the hybridized single-stranded circles, thereby generating a normalized cDNA library.

  10. Curcumin: an orally bioavailable blocker of TNF and other pro-inflammatory biomarkers

    Science.gov (United States)

    Aggarwal, Bharat B; Gupta, Subash C; Sung, Bokyung

    2013-01-01

    TNFs are major mediators of inflammation and inflammation-related diseases, hence, the United States Food and Drug Administration (FDA) has approved the use of blockers of the cytokine, TNF-α, for the treatment of osteoarthritis, inflammatory bowel disease, psoriasis and ankylosis. These drugs include the chimeric TNF antibody (infliximab), humanized TNF-α antibody (Humira) and soluble TNF receptor-II (Enbrel) and are associated with a total cumulative market value of more than $20 billion a year. As well as being expensive ($15 000–20 000 per person per year), these drugs have to be injected and have enough adverse effects to be given a black label warning by the FDA. In the current report, we describe an alternative, curcumin (diferuloylmethane), a component of turmeric (Curcuma longa) that is very inexpensive, orally bioavailable and highly safe in humans, yet can block TNF-α action and production in in vitro models, in animal models and in humans. In addition, we provide evidence for curcumin's activities against all of the diseases for which TNF blockers are currently being used. Mechanisms by which curcumin inhibits the production and the cell signalling pathways activated by this cytokine are also discussed. With health-care costs and safety being major issues today, this golden spice may help provide the solution. Linked Articles This article is part of a themed section on Emerging Therapeutic Aspects in Oncology. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-8 PMID:23425071

  11. Restriction landmark cDNA scanning (RLCS): a novel cDNA display system using two-dimensional gel electrophoresis.

    Science.gov (United States)

    Suzuki, H; Yaoi, T; Kawai, J; Hara, A; Kuwajima, G; Wantanabe, S

    1996-01-01

    We have developed a new method, designated restriction landmark cDNA scanning (RLCS), which displays many cDNA species quantitatively and simultaneously as two-dimensional gel spots. In this method cDNA species of uniform length were prepared for each mRNA species using restriction enzymes. After the restriction enzyme sites were radiolabeled as landmarks, the labeled fragments were subjected to high resolution two-dimensional gel electrophoresis. In analyses of cDNA samples from adult mouse liver and brain (cerebral cortex, cerebellum and brain stem) we detected approximately 500 and >1000 discrete gel spots respectively of various intensities at a time. The spot patterns of the three brain regions were very similar, although not identical, but were quite different from the pattern for the liver. RNA blot hybridization analysis using several cloned spot DNAs as probes showed that differences in intensity of the spots among RLCS profiles correlated well with expression levels of the corresponding mRNA species in the brain regions. Because the spots and their intensities reflect distinct mRNA species and their expression level respectively, the RLCS is a novel cDNA display system which provides a great deal of information and should be useful for systematic documentation of differentially expressed genes. PMID:8628652

  12. Taming TNF : strategies to restrain this proinflammatory cytokine

    NARCIS (Netherlands)

    Eigler, A; Sinha, B; Hartmann, G; Endres, S

    1997-01-01

    Recent studies have demonstrated the essential role of tumor necrosis factor alpha (TNF-alpha) in rheumatoid arthritis and Crohn's disease. This article discusses agents known to suppress the formation or activity of TNF-alpha, and summarizes clinical studies using anti-TNF-alpha antibodies.

  13. Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

    Directory of Open Access Journals (Sweden)

    Colo Anna

    2008-06-01

    Full Text Available Abstract Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45 is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7, SOD2 (superoxide dismutase 2, 100P (S100 calcium binding protein P, PI3 (protease inhibitor 3, skin-derived, CSTA (cystatin A, RARRES1 (retinoic acid receptor responder 1, and LXN (latexin. The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in

  14. Mycobacterium tuberculosis lipomannan blocks TNF biosynthesis by regulating macrophage MAPK-activated protein kinase 2 (MK2) and microRNA miR-125b.

    Science.gov (United States)

    Rajaram, Murugesan V S; Ni, Bin; Morris, Jessica D; Brooks, Michelle N; Carlson, Tracy K; Bakthavachalu, Baskar; Schoenberg, Daniel R; Torrelles, Jordi B; Schlesinger, Larry S

    2011-10-18

    Contact of Mycobacterium tuberculosis (M.tb) with the immune system requires interactions between microbial surface molecules and host pattern recognition receptors. Major M.tb-exposed cell envelope molecules, such as lipomannan (LM), contain subtle structural variations that affect the nature of the immune response. Here we show that LM from virulent M.tb (TB-LM), but not from avirulent Myocobacterium smegmatis (SmegLM), is a potent inhibitor of TNF biosynthesis in human macrophages. This difference in response is not because of variation in Toll-like receptor 2-dependent activation of the signaling kinase MAPK p38. Rather, TB-LM stimulation leads to destabilization of TNF mRNA transcripts and subsequent failure to produce TNF protein. In contrast, SmegLM enhances MAPK-activated protein kinase 2 phosphorylation, which is critical for maintaining TNF mRNA stability in part by contributing microRNAs (miRNAs). In this context, human miRNA miR-125b binds to the 3' UTR region of TNF mRNA and destabilizes the transcript, whereas miR-155 enhances TNF production by increasing TNF mRNA half-life and limiting expression of SHIP1, a negative regulator of the PI3K/Akt pathway. We show that macrophages incubated with TB-LM and live M.tb induce high miR-125b expression and low miR-155 expression with correspondingly low TNF production. In contrast, SmegLM and live M. smegmatis induce high miR-155 expression and low miR-125b expression with high TNF production. Thus, we identify a unique cellular mechanism underlying the ability of a major M.tb cell wall component, TB-LM, to block TNF biosynthesis in human macrophages, thereby allowing M.tb to subvert host immunity and potentially increase its virulence.

  15. Association of the TNF-alpha G-308A polymorphism with TNF-inhibitor response in sarcoidosis

    NARCIS (Netherlands)

    Wijnen, P.A.; Cremers, J.P.; Nelemans, P.J.; Erckens, R.J.; Hoitsma, E.; Jansen, T.L.; Bekers, O.; Drent, M.

    2014-01-01

    Responsiveness to tumour necrosis factor (TNF) inhibitors has been associated with the TNF-alpha G-308A polymorphism in rheumatoid arthritis. The aim of this study was to examine the association between the presence of this polymorphism and the response to TNF inhibitors in patients with refractory

  16. Relation of serum TNF-α and TNF-α genotype with delayed cerebral ischemia and outcome in subarachnoid hemorrhage

    NARCIS (Netherlands)

    Beeftink, Martine M. A.; Ruigrok, Ynte M.; Rinkel, Gabriel J. E.; van den Bergh, Walter M.

    2011-01-01

    The pathogenesis of delayed cerebral ischemia (DCI) after subarachnoid hemorrhage (SAH) remains obscure. The authors assessed the relationship of tumor necrosis factor alpha (TNF-α) and TNF-α gene polymorphisms with occurrence of DCI and poor outcome at 3 months. Serum levels of TNF-α were measured

  17. ASSOCIATION OF MYCOBACTERIUM TUBERCULOSIS LINEAGES WITH IFN-Γ AND TNF-Α GENE POLYMORPHISMS AMONG PULMONARY TUBERCULOSIS PATIENT

    Directory of Open Access Journals (Sweden)

    Mohammad Varahram

    2014-02-01

    Full Text Available The six major lineages of Mycobacterium tuberculosis [MTB] are found to be strongly associated with specific geographical outbreaks. But whether these bacterial lineages influence the host genetic polymorphism is uncertain. The present study was designed to evaluate the relevance of strain diversity and host genetic polymorphisms in susceptibility to pulmonary tuberculosis [PTB]. For this reason, single –nucleotide polymorphisms [SNPs] in interferon- γ [IFN-γ] receptor-1[G-611A], IFNG [G+ 2109A] and tumor necrosis factors [TNF-α] genes [at -238, 308,-857position] in patients [n=151] were analyzed and compared with controls [n=83]. The genetic diversity of M. tuberculosis isolates were performed using spacer oligonucleotide typing. Thereafter, the profile of IFN-γ and TNF-α allele frequency were investigated in each subtype of M .tuberculosis. The results showed C allele of TNF 857 and A allele of TNF 238 were more frequent in PTB cases [[TNF 857 C allele OR [CI95%] 0.6[0.4-0.9], p= 0.02] for TNF 238 A allele OR [CI95%] 5.5[3.4-9.0], p= 0.00

  18. cDNA sequences of two inducible T-cell genes

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, B.S. (Indiana Univ. School of Medicine, Indianapolis (USA) Guthrie Research Institute, Sayre, PA (USA)); Weissman, S.M. (Yale Univ., New Haven, CT (USA))

    1989-03-01

    The authors have previously described a set of human T-lymphocyte-specific cDNA clones isolated by a modified differential screening procedure. Apparent full-length cDNAs containing the sequences of 14 of the 16 initial isolates were sequenced and were found to represent five different species of mRNA; three of the five species were identical to previously reported cDNA sequences of preproenkephalin, T-cell-replacing factor, and a serine esterase, respectively. The other two species, 4-1BB and L2G25B, were inducible sequences found in mRNA from both a cytolytic T-lymphocyte and a helper T-lymphocyte clone and were not previously described in T-cell mRNA; these mRNA sequences encode peptides of 256 and 92 amino acids, respectively. Both peptides contain putative leader sequences. The protein encoded by 4-1BB also has a potential membrane anchor segment and other features also seen in known receptor proteins.

  19. Radioactive cDNA microarrys for gene expression profiles in antidepressant therapy

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M. S.; Han, B. J.; Cha, J. H.; Ryu, Y. M.; Shin, E. K.; Park, J. H.; Park, Y. H.; Kim, M. K. [Korea University Medical College, Seoul (Korea, Republic of)

    2002-07-01

    Using radioactive cDNA microarray, we investigated a pattern of gene regulation under treatment of antidepressant on patients of depressive disoder. Basic microarray technology was performed as previously described in our research. The bioinformatic selection of human cDNAs, which is specifically designed for psychiatry, neurology, and signal transduction, were arrayed on nylon membranes. Using with 33P-labeled probes, this method provided highly sensitive gene expression profiles of our interest including brain receptors, drug metabolism, and cellular signalings. Gene expression profiles were also classified into several categories in accordance with the gene-regulation of antidepressant. The gene profiles of our interest were significantly up- (16 genes, >2.0 of Z-ratio) or down- (24 genes, <-2.0 of Z ratio) regulated when compared the good responsed group with the bad-responsed one. Consequently, we demonstrated that radioactive human cDNA microarray is highly likely to be an efficient technology for evaluating the gene regulation of antidepressants, such as selective serotonin-reuptake inhibitors (SSRIs), by using high-throughput biotechnology.

  20. Genomic cloning of the mouse LDL receptor related protein/_2-macroglobulin receptor gene

    NARCIS (Netherlands)

    Zee, A. van der; Stas, L.; Hilleker, C.; Leuven, F. van; Dijk, K.W. van; Havekes, L; Frants, R.A.; Hofker, M.H.

    1994-01-01

    The LDL receptor-related protein (LRP) or alpha 2-macroglobulin receptor (A2mr) is encoded by a 15-kb mRNA in mouse and human. Probes encompassing different regions of the mouse cDNA were used to isolate clones from a cosmid library of mouse strain 129. Four overlapping cosmids were used for

  1. Upregulating Nonneuronal Cholinergic Activity Decreases TNF Release from Lipopolysaccharide-Stimulated RAW264.7 Cells

    Directory of Open Access Journals (Sweden)

    Yi Lv

    2014-01-01

    Full Text Available Nonneuronal cholinergic system plays a primary role in maintaining homeostasis. It has been proved that endogenous neuronal acetylcholine (ACh could play an anti-inflammatory role, and exogenous cholinergic agonists could weaken macrophages inflammatory response to lipopolysaccharide (LPS stimulation through activation of α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR. We assumed that nonneuronal cholinergic system existing in macrophages could modulate inflammation through autocrine ACh and expressed α7nAChR on the cells. Therefore, we explored whether LPS continuous stimulation could upregulate the nonneuronal cholinergic activity in macrophages and whether increasing autocrine ACh could decrease TNF release from the macrophages. The results showed that, in RAW264.7 cells incubated with LPS for 20 hours, the secretion of ACh was significantly decreased at 4 h and then gradually increased, accompanied with the enhancement of α7nAChR expression level. The release of TNF was greatly increased from RAW264.7 cells at 4 h and 8 h exposure to LPS; however, it was suppressed at 20 h. Upregulating choline acetyltransferase (ChAT expression through ChAT gene transfection could enhance ACh secretion and reduce TNF release from the infected RAW264. 7cells. The results indicated that LPS stimulation could modulate the activity of nonneuronal cholinergic system of RAW264.7 cells. Enhancing autocrine ACh production could attenuate TNF release from RAW264.7 cells.

  2. TNF-α Genetic Predisposition and Higher Expression of Inflammatory Pathway Components in Keratoconus.

    Science.gov (United States)

    Arbab, Muneeza; Tahir, Saira; Niazi, Muhammad Khizar; Ishaq, Mazhar; Hussain, Alamdar; Siddique, Pir Muhammad; Saeed, Sadia; Khan, Wajid Ali; Qamar, Raheel; Butt, Azeem Mehmood; Azam, Maleeha

    2017-07-01

    To date keratoconus (KC) pathogenesis is undefined; however, the involvement of inflammatory pathways in disease development is becoming apparent. In the present study, we investigated the role of a promoter region polymorphism rs1800629 (-308G>A) in the inflammatory pathway component TNF-α and its effects on the expression of TNF-α and downstream molecules tumor necrosis factor receptor 1 and 2 (TNFR1 and TNFR2), v-rel avian reticuloendotheliosis viral oncogene homolog A (RELA), and interleukin 6 (IL-6) in KC development. TNF-α promoter polymorphism rs1800629 (-308G>A), was genotyped in 257 sporadic KC patients and 253 healthy controls. Enzyme-linked immunosorbent assay (ELISA) was performed to assess for the -308G>A genotypes. Quantitative polymerase chain reaction (qPCR) was carried out to compare the mRNA expression of TNF-α, TNFR1, TNFR2, RELA, and IL6 in the corneal tissues of 20 KC patients and 20 donor controls. The -308G>A genotype GA was found to be significantly associated with KC development (dominant model [odds ratio (OR) = 6.67 (95% confidence interval [CI] = 4.28-10.42), P A polymorphism is a significant genetic risk factor for the pathogenesis of KC. Moreover, this single nucleotide polymorphism (SNP) was observed to be associated with deregulated expression of downstream molecules, thus further reinforcing the role of the inflammatory pathway components in the development of KC.

  3. TNF-α and Microalbuminuria in Patients with Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    I.-Th. Lampropoulou

    2014-01-01

    Full Text Available Aim. Recent evidence suggests that chronic subclinical inflammation plays a key role in the pathogenesis and progression of diabetic nephropathy. Aim of the present study was to investigate possible correlation between the presence and degree of microalbuminuria and markers of inflammation in patients with type 2 diabetes mellitus (DM. Patients-Methods. Eighty patients were enrolled and clinical and laboratory data were recorded. Albumin-creatinine ratio (ACR was calculated in first-morning urine samples. Serum and urinary tumor necrosis factor-α (TNF-α levels were determined by ELISA. Results. Forty-five patients had normoalbuminuria, 33 microalbuminuria, and 2 macroalbuminuria. Patients with microalbuminuria were older, with higher glycosylated hemoglobin levels (HbA1c and they more frequently had diabetic retinopathy, neuropathy, and cardiovascular disease and were on treatment with angiotensin converting enzyme inhibitors (ACEi and/or angiotensin receptor blockers (ARBs. ACR was significantly correlated with the presence of cardiovascular disease, hypertension, and HbA1c levels and the administration of clopidogrel and ACEi or ARBs. ACR was not correlated with C-reactive protein, fibrinogen, or serum TNF-α levels but had a strong correlation with urinary TNF-α levels. Conclusions. In patients with type 2 DM, urinary, but not serum, TNF-α levels are associated with the presence and severity of microalbuminuria.

  4. Sex Differences of Human Cardiac Progenitor Cells in the Biological Response to TNF-α Treatment

    Directory of Open Access Journals (Sweden)

    Elisabetta Straface

    2017-01-01

    Full Text Available Adult cardiac progenitor cells (CPCs, isolated as cardiosphere-derived cells (CDCs, represent promising candidates for cardiac regenerative therapy. CDCs can be expanded in vitro manyfolds without losing their differentiation potential, reaching numbers that are appropriate for clinical applications. Since mechanisms of successful CDC survival and engraftment in the damaged myocardium are still critical and unresolved issues, we aimed at deciphering possible key factors capable of bolstering CDC function. In particular, the response and the phenotype of CDCs exposed to low concentrations of the multifunctional cytokine tumor necrosis factor α (TNF-α, known to be capable of activating cell survival pathways, have been investigated. Furthermore, differential biological responses of CDCs from male and female donors, in terms of cell cycle progression and cell spreading, have also been assessed. The results obtained indicate that (i the intracellular signaling activated in our experimental conditions is most likely due to the prosurvival and proliferative signaling of TNFreceptor 2 and that (ii cells from female patients appear more responsive to TNF-α treatment in terms of cell cycle progression and migration ability. In conclusion, the present report highlights the hypothesis that TNF-stimulated CDCs isolated from females may represent a promising candidate for cardiac regenerative therapy applications.

  5. Cloning, sequencing and expression of cDNA encoding growth ...

    Indian Academy of Sciences (India)

    Unknown

    cell embryo and the expression was monitored continuously. The expression shown here is in developing embryo and freshly hatched fish. The intensity of green colour indicate the strong expression of EGFP in all the tissues of the embryo/fry. The expression of EGPF indicates the co-expression of catfish GH cDNA and the ...

  6. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    Home; Journals; Journal of Genetics; Volume 96; Issue 2. cDNA cloning, structural analysis, SNP detection and tissue ... Abstract. Insulin-like growth factor 1 (IGF1) plays an important role in growth, reproduction, foetal development and cell proliferation. The present study was conducted to clone and sequence the ...

  7. Cloning and characterization of cDNA encoding xyloglucan ...

    African Journals Online (AJOL)

    Jane

    2011-08-22

    Aug 22, 2011 ... construction and restructuring of xyloglucan cross-links, thereby controlling the mechanical properties of cell wall. We cloned complete cDNA of an ..... are marked by horizontal lines. The conserved cysteine residues (amino acids 220, 229, 274 and 288 in P. glaucum) are marked by vertical blue arrows.

  8. cDNA, genomic sequence cloning and overexpression of ribosomal ...

    African Journals Online (AJOL)

    RPS16 of eukaryote is a component of the 40S small ribosomal subunit encoded by RPS16 gene and is also a homolog of prokaryotic RPS9. The cDNA and genomic sequence of RPS16 was cloned successfully for the first time from the Giant Panda (Ailuropoda melanoleuca) using reverse transcription-polymerase chain ...

  9. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    [Naicy T., Venkatachalapathy T., Aravindakshan T., Raghavan K. C., Mini M. and Shyama K. 2017 cDNA cloning, structural analysis, SNP detection and tissue expression profile of the IGF1 gene in Malabari and Attappady Black goats of India. J. Genet. 96, xx–xx]. Introduction. Insulin-like growth factor 1 (IGF1), an important ...

  10. Construction of yeast surface-displayed cDNA libraries.

    Science.gov (United States)

    Bidlingmaier, Scott; Liu, Bin

    2011-01-01

    Using yeast display, heterologous protein fragments can be efficiently displayed at high copy levels on the Saccharomyces cerevisiae cell wall. Yeast display can be used to screen large expressed protein libraries for proteins or protein fragments with specific binding properties. Recently, yeast surface-displayed cDNA libraries have been constructed and used to identify proteins that bind to various target molecules such as peptides, small molecules, and antibodies. Because yeast protein expression pathways are similar to those found in mammalian cells, human protein fragments displayed on the yeast cell wall are likely to be properly folded and functional. Coupled with fluorescence-activated cell sorting, yeast surface-displayed cDNA libraries potentially allow the selection of protein fragments or domains with affinity for any soluble molecule that can be fluorescently detected. In this report, we describe protocols for the construction and validation of yeast surface-displayed cDNA libraries using preexisting yeast two-hybrid cDNA libraries as a starting point.

  11. A BIOINFORMATIC STRATEGY TO RAPIDLY CHARACTERIZE CDNA LIBRARIES

    Science.gov (United States)

    A Bioinformatic Strategy to Rapidly Characterize cDNA LibrariesG. Charles Ostermeier1, David J. Dix2 and Stephen A. Krawetz1.1Departments of Obstetrics and Gynecology, Center for Molecular Medicine and Genetics, & Institute for Scientific Computing, Wayne State Univer...

  12. Transmembrane TNF-α is sufficient for articular inflammation and hypernociception in a mouse model of gout.

    Science.gov (United States)

    Amaral, Flávio A; Bastos, Leandro F S; Oliveira, Thiago H C; Dias, Ana C F; Oliveira, Vívian L S; Tavares, Lívia D; Costa, Vivian V; Galvão, Izabela; Soriani, Frederico M; Szymkowski, David E; Ryffel, Bernhard; Souza, Danielle G; Teixeira, Mauro M

    2016-01-01

    Gout manifests as recurrent episodes of acute joint inflammation and pain due to the deposition of monosodium urate (MSU) crystals within the affected tissue in a process dependent on NLRP3 inflammasome activation. The synthesis, activation, and release of IL-1β are crucial for MSU-induced inflammation. The current study evaluated the mechanism by which TNF-α contributed to MSU-induced inflammation. Male C57BL/6J or transgenic mice were used in this study and inflammation was induced by the injection of MSU crystals into the joint. TNF-α was markedly increased in the joint after the injection of MSU. There was inhibition in the infiltration of neutrophils, production of CXCL1 and IL-1β, and decreased hypernociception in mice deficient for TNF-α or its receptors. Pharmacological blockade of TNF-α with Etanercept or pentoxyfylline produced similar results. Mechanistically, TNF-α blockade resulted in lower amounts of IL-1β protein and pro-IL-1β mRNA transcripts in joints. Gene-modified mice that express only transmembrane TNF-α had an inflammatory response similar to that of WT mice and blockade of soluble TNF-α (XPro™1595) did not decrease MSU-induced inflammation. In conclusion, TNF-α drives expression of pro-IL-1β mRNA and IL-1β protein in experimental gout and that its transmembrane form is sufficient to trigger MSU-induced inflammation in mice. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Altered levels of laminin receptor mRNA in various human carcinoma cells that have different abilities to bind laminin

    DEFF Research Database (Denmark)

    Wewer, U M; Liotta, L A; Jaye, M

    1986-01-01

    The human laminin receptor was purified and molecularly cloned to investigate its biosynthetic regulation. Laminin receptor from normal and neoplastic tissue was preparatively affinity purified to homogeneity based on the high affinity of the receptor for laminin. The apparent molecular weight...... of the receptor from different carcinoma sources and from normal placental tissue is in the range of 68-72 kDa. Isoelectric focusing and two-dimensional gel electrophoresis indicated that the receptor protein consists of one major polypeptide chain with a pI value of 6.4 +/- 0.2. Laminin receptor cDNA clones were...... isolated after screening a human endothelial lambda gt11 cDNA library with a monoclonal antibody directed against a domain of the laminin receptor involved in ligand binding. Definitive identification of the cDNA clones was based on comparison of cDNA sequence with the amino acid sequence of a cyanogen...

  14. Gene expression profile analysis in human hepatocellular carcinoma by cDNA microarray.

    Science.gov (United States)

    Chung, Eun Jung; Sung, Young Kwan; Farooq, Mohammad; Kim, Younghee; Im, Sanguk; Tak, Won Young; Hwang, Yoon Jin; Kim, Yang Il; Han, Hyung Soo; Kim, Jung-Chul; Kim, Moon Kyu

    2002-12-31

    We performed gene expression profiling of normal and hepatocellular carcinoma (HCC) liver tissues using a high-density microarray that contained 3,063 human cDNA. The results of a microarray hybridization experiment from eight different HCC tissues were analyzed and classified by the Cluster program. Among these differentially-expressed genes, the galectin-3, serine/threonine kinase SGK, translation factor eIF-4A, -4B, -3, fibroblast growth factor receptor, and ribosomal protein L35A were up-regulated; the mRNAs of Nip3, decorin, and the insulin-like growth factor binding protein-3 were down-regulated in HCC. The differential expression of these genes was further confirmed by an RT-PCR analysis. In addition, our data suggest that the gene expression profile of HCC varies according to the histological types.

  15. Dataset of differential lipid raft and global proteomes of SILAC-labeled cystic fibrosis cells upon TNF -α stimulation

    Directory of Open Access Journals (Sweden)

    C. Chhuon

    2016-12-01

    We used the Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC approach to quantify and identify 332 proteins in LRM upon TNF-a stimulation in CF cells and 1381 for the global proteome. We report two detailed tables containing lists of proteins obtained by mass spectrometry and the immunofluorescence validation results for one of these proteins, the G-protein coupled receptor 5A. These results are associated with the article “Changes in lipid raft proteome upon TNF-α stimulation of cystic fibrosis cells” (Chhuon et al., in press [1].

  16. B cells produce less IL-10, IL-6 and TNF-α in myasthenia gravis.

    Science.gov (United States)

    Yilmaz, Vuslat; Oflazer, Piraye; Aysal, Fikret; Parman, Yeşim G; Direskeneli, Haner; Deymeer, Feza; Saruhan-Direskeneli, Güher

    2015-06-01

    B cells from myasthenia gravis (MG) patients with autoantibodies (Aab) against acetylcholine receptor (AChR), muscle-specific kinase (MuSK) or with no detectable Aab were investigated as cytokine producing cells in this study. B cells were evaluated for memory phenotypes and expressions of IL-10, IL-6 and IL-12A. Induced productions of IL-10, IL-6, IL-12p40, TNF-α and LT from isolated B cells in vitro were measured by immunoassays. MG patients receiving immunosuppressive treatment had higher proportions of memory B cells compared with healthy controls and untreated patients. With CD40 stimulation MG patients produced significantly lower levels of IL-10, IL-6. With CD40 and B cell receptor stimulation of B cells, TNF-α production also decreased in addition to these cytokines. The lower levels of these cytokine productions were not related to treatment. Our results confirm a disturbance of B cell subpopulations in MG subgroups on immunosuppressive treatment. B cell derived IL-10, IL-6 and TNF-α are down-regulated in MG, irrespective of different antibody productions. Ineffective cytokine production by B cells may be a susceptibility factor in dysregulation of autoimmune Aab production.

  17. Genetic variation in the TNF gene is associated with susceptibility to severe sepsis, but not with mortality.

    Directory of Open Access Journals (Sweden)

    Zhenju Song

    Full Text Available BACKGROUND: Tumor necrosis factor (TNF and TNF receptor superfamily (TNFR-mediated immune response play an essential role in the pathogenesis of severe sepsis. Studies examining associations of TNF and lymphotoxin-α (LTA single nucleotide polymorphisms (SNPs with severe sepsis have produced conflicting results. The objective of this study was to investigate whether genetic variation in TNF, LTA, TNFRSF1A and TNFRSF1B was associated with susceptibility to or death from severe sepsis in Chinese Han population. METHODOLOGY/PRINCIPAL FINDINGS: Ten SNPs in TNF, LTA, TNFRSF1A and TNFRSF1B were genotyped in samples of patients with severe sepsis (n = 432, sepsis (n = 384 and healthy controls (n = 624. Our results showed that rs1800629, a SNP in the promoter region of TNF, was significantly associated with risk for severe sepsis. The minor allele frequency of rs1800629 was significantly higher in severe sepsis patients than that in both healthy controls (P(adj = 0.00046, odds ratio (OR(adj = 1.92 and sepsis patients (P(adj = 0.002, OR(adj = 1.56. Further, we investigated the correlation between rs1800629 genotypes and TNF-α concentrations in peripheral blood mononuclear cells (PBMCs of healthy volunteers exposed to lipopolysaccharides (LPS ex vivo, and the association between rs1800629 and TNF-α serum levels in severe sepsis patients. After exposure to LPS, the TNF-α concentration in culture supernatants of PBMCs was significantly higher in the subjects with AA+AG genotypes than that with GG genotype (P = 0.007. Moreover, in patients with severe sepsis, individuals with AA+AG genotypes had significantly higher TNF-α serum concentrations than those with GG genotype (P(adj = 0.02. However, there were no significant associations between SNPs in the four candidate genes and 30 day mortality for patients with severe sepsis. CONCLUSIONS/SIGNIFICANCE: Our findings suggested that the functional TNF gene SNP rs1800629 was strongly associated with

  18. Necroptosis Mediates TNF-Induced Toxicity of Hippocampal Neurons

    Directory of Open Access Journals (Sweden)

    Shan Liu

    2014-01-01

    Full Text Available Tumor necrosis factor-α (TNF-α is a critical proinflammatory cytokine regulating neuroinflammation. Elevated levels of TNF-α have been associated with various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. However, the signaling events that lead to TNF-α-initiated neurotoxicity are still unclear. Here, we report that RIP3-mediated necroptosis, a form of regulated necrosis, is activated in the mouse hippocampus after intracerebroventricular injection of TNF-α. RIP3 deficiency attenuates TNF-α-initiated loss of hippocampal neurons. Furthermore, we characterized the molecular mechanism of TNF-α-induced neurotoxicity in HT-22 hippocampal neuronal cells. HT-22 cells are sensitive to TNF-α only upon caspase blockage and subsequently undergo necrosis. The cell death is suppressed by knockdown of CYLD or RIP1 or RIP3 or MLKL, suggesting that this necrosis is necroptosis and mediated by CYLD-RIP1-RIP3-MLKL signaling pathway. TNF-α-induced necroptosis of HT-22 cells is largely independent of both ROS accumulation and calcium influx although these events have been shown to be critical for necroptosis in certain cell lines. Taken together, these data not only provide the first in vivo evidence for a role of RIP3 in TNF-α-induced toxicity of hippocampal neurons, but also demonstrate that TNF-α promotes CYLD-RIP1-RIP3-MLKL-mediated necroptosis of hippocampal neurons largely bypassing ROS accumulation and calcium influx.

  19. Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange

    NARCIS (Netherlands)

    van Schie, Karin A.; Ooijevaar-de Heer, Pleuni; Dijk, Lisanne; Kruithof, Simone; Wolbink, Gertjan; Rispens, Theo

    2016-01-01

    Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Forster resonance energy transfer (FRET) assay that

  20. XIAP Restricts TNF- and RIP3-Dependent Cell Death and Inflammasome Activation

    DEFF Research Database (Denmark)

    Yabal, Monica; Müller, Nicole; Adler, Heiko

    2014-01-01

    of XIAP or deletion of its RING domain lead to excessive cell death and IL-1β secretion from dendritic cells triggered by diverse Toll-like receptor stimuli. Aberrant IL-1β secretion is TNF dependent and requires RIP3 but is independent of cIAP1/cIAP2. The observed cell death also requires TNF and RIP3...... but proceeds independently of caspase-1/caspase-11 or caspase-8 function. Loss of XIAP results in aberrantly elevated ubiquitylation of RIP1 outside of TNFR complex I. Virally infected Xiap(-/-) mice present with symptoms reminiscent of XLP-2. Our data show that XIAP controls RIP3-dependent cell death and IL-1...

  1. TNF-TNFR2/p75 Signaling Inhibits Early and Increases Delayed Nontargeted Effects in Bone Marrow-derived Endothelial Progenitor Cells*

    Science.gov (United States)

    Sasi, Sharath P.; Song, Jin; Park, Daniel; Enderling, Heiko; McDonald, J. Tyson; Gee, Hannah; Garrity, Brittany; Shtifman, Alexander; Yan, Xinhua; Walsh, Kenneth; Natarajan, Mohan; Kishore, Raj; Goukassian, David A.

    2014-01-01

    TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1–5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3–5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general. PMID:24711449

  2. Selective inhibition of TNFR1 reduces osteoclast numbers and is differentiated from anti-TNF in a LPS-driven model of inflammatory bone loss.

    Science.gov (United States)

    Espirito Santo, A I; Ersek, A; Freidin, A; Feldmann, M; Stoop, A A; Horwood, N J

    2015-09-04

    The treatment of autoimmune disorders has been revolutionised by the introduction of biologics such as anti-tumour necrosis factor (anti-TNF). Although in rheumatoid arthritis patients a bone sparing effect of anti-TNF has been shown, the mechanism is not fully understood. Anti-TNF molecules block tumour necrosis factor (TNF) and prevent signalling via both TNF receptor 1 (TNFR1; p55) and TNF receptor 2 (TNFR2; p75). However, signalling via TNFR2 is reported to have protective effects in a number of cell and organ systems. Hence we set out to investigate if pharmacological inhibition of TNFR1 had differential effects compared to pan-TNF inhibition in both an in vitro cell-based model of human osteoclast activity and an in vivo mouse model of lipopolysaccharide (LPS)-induced osteolysis. For the in vitro experiments the anti-human TNFR1 domain antibody (dAb) DMS5541 was used, whereas for the in vivo mouse experiments the anti-mouse TNFR1 dAb DMS5540 was used. We show that selective blocking of TNFR1 signalling reduced osteoclast formation in the presence of TNF. Subcutaneous LPS injection over the calvaria leads to the development of osteolytic lesions within days due to inflammation driven osteoclast formation. In this model, murine TNFR2 genetically fused with mouse IgG1 Fc domain (mTNFR2.Fc), an anti-TNF, did not protect from bone loss in contrast to anti-TNFR1, which significantly reduced lesion development, inflammatory infiltrate, and osteoclast number and size. These results support further exploring the use of TNFR1-selective inhibition in inflammatory bone loss disorders such as osteomyelitis and peri-prosthetic aseptic loosening. Copyright © 2015 Elsevier Inc. All rights reserved.

  3. TRADD, TRAF2, RIP1 and TAK1 are required for TNF-α-induced pro-labour mediators in human primary myometrial cells.

    Science.gov (United States)

    Lim, Ratana; Barker, Gillian; Lappas, Martha

    2017-07-01

    TNF-α plays a central role in the processes of human labour and delivery. This study sought to determine the role of the adaptor proteins TNFR1-associated death domain protein (TRADD), TNF receptor-associated factor 2 (TRAF2), receptor interacting protein 1 (RIP1) and transforming growth factor beta-activated kinase 1 (TAK1) in TNF-α-induced formation of pro-labour mediators. Human primary myometrial cells were transfected with siRNA against TRADD (siTRADD), TRAF2 (siTRAF2), RIP1 (siRIP1) or TAK1 (siTAK1), treated with TNF-α, and assayed for pro-inflammatory mediators expression. siTRADD, siTRAF2, siRIP1 and siTAK1 significantly decreased TNF-α-induced IL-1α, IL-1β, IL-6, IL-8, MCP-1 mRNA expression and release of IL-6, IL-8 and MCP-1; and cyclooxygenase (COX)-2 expression and release of prostaglandin PGF 2α . There was a significant attenuation of TNF-α-induced expression of adhesion molecules ICAM-1 and VCAM-1 mRNA with siTRADD, siTRAF2 or siRIP1. siTRADD and siRIP1 significantly attenuated TNF-α-induced MMP-9 mRNA expression and release and nuclear factor κB (NF-κB) transcriptional activity. There was a significant increase in TNF-α-induced sVCAM-1 release, MMP-9 mRNA expression and NF-κB activity with siTAK1. TRADD, TRAF2, RIP1 and TAK1 are involved in TNF-α signalling in human myometrium. Further studies are required to determine whether inhibition of these proteins can prevent preterm birth. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Diallyl sulfide as a potential dietary agent to reduce TNF-α- and histamine-induced proinflammatory responses in A7r5 cells.

    Science.gov (United States)

    Ho, Cheng-Ying; Weng, Chia-Jui; Jhang, Jhih-Jia; Cheng, Yu-Ting; Huang, Shang-Ming; Yen, Gow-Chin

    2014-05-01

    Oxidative stress-aggravated chronic inflammatory diseases of the airway are well documented; hence, treatment with antioxidants to ameliorate oxidative stress might be an effective strategy to reduce airway complications. The aim of this study was to investigate the effect and molecular mechanism of diallyl sulfide (DAS), which is a natural organosulfuric compound found in garlic, on the inhibition of tumor necrosis factor-alpha (TNF-α)- or histamine-induced inflammation in rat aortic smooth muscle A7r5 cells. A7r5 cells were coincubated with DAS before exposure to TNF-α or histamine. DAS significantly blocked the accumulation of the nuclear p65 protein in TNF-α-induced A7r5 cells by attenuating the TNFreceptor complex through the dissociation of the TNF receptor-associated death domain and TNF receptor-associated factor 2. Moreover, DAS inhibited histamine-induced inflammation by decreasing reactive oxygen species (ROS) levels by enhancing the nuclear factor-erythroid 2-related factor 2-related antioxidative enzyme. DAS also inhibited inflammation by suppressing interleukin-1β and TNF-α through the inhibition of ROS-induced PI3K/Akt and the downstream NF-κB and activator protein-1. Our results demonstrate that DAS is a potential phytochemical to inhibit TNF-α- and histamine-induced inflammation, suggesting that DAS might be an effective dietary agent for the prevention of oxidative stress-induced inflammation of the airway. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. TNF-α Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism

    Science.gov (United States)

    Lee, Jiyoung; Tian, Yongjun; Chan, Stephanie Tze; Kim, Ja Yeon; Cho, Cecilia; Ou, Jing-hsiung James

    2015-01-01

    Invasion by infectious pathogens can elicit a range of cytokine responses from host cells. These cytokines provide the initial host defense mechanism. In this report, we demonstrate that TNF-α, a pro-inflammatory cytokine, can be induced by hepatitis C virus (HCV) in its host cells in a biphasic manner. The initial induction of TNF-α by HCV was prompt and could be blocked by the antibody directed against the HCV E2 envelope protein and by chemicals that inhibit endocytosis, indicating the specificity of endocytic uptake of HCV in this induction. Further studies indicated that the induction of TNF-α was dependent on toll-like receptors 7 and 8 (TLR7/8) but not on other intracellular pattern recognition receptors. Consistently, siRNA-mediated gene silencing of the downstream effectors in the TLR7/8 signaling pathway including MyD88, IRAK1, TRAF6, TAK1 and p65 NF-κB suppressed the expression of TNF-α. The role of p65 NF-κB in the induction of TNF-α via transcriptional up-regulation was further confirmed by the chromatin immunoprecipitation assay. TNF-α induced by HCV could activate its own receptor TNFR1 on hepatocytes to suppress HCV replication. This suppressive effect of TNF-α on HCV was due to its role in supporting interferon signaling, as the suppression of its expression led to the loss of IFNAR2 and impaired interferon signaling and the induction of interferon-stimulated genes. In conclusion, our results indicate that hepatocytes can sense HCV infection via TLR7/8 to induce the expression of TNF-α, which inhibits HCV replication via an autocrine mechanism to support interferon signaling. PMID:26023919

  6. Molecular cloning of growth hormone encoding cDNA of Indian ...

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  7. cDNA cloning and characterization of a mannose-binding lectin from ...

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  8. Isolation and characterization of a Coffea canephora ERF-like c-DNA

    African Journals Online (AJOL)

    A cDNA corresponding to an ERF gene has been isolated from a Coffea canephora fruit cDNA library. The cDNA was 1,317 nucleotides long and has an open reading frame of 987 bp. The predicted polypeptide showed a great similitude with equivalent proteins from others plant species. The binding domain shows 98.3% ...

  9. Molecular cloning of growth hormone encoding cDNA of Indian

    Indian Academy of Sciences (India)

    A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in ...

  10. cDNA cloning and characterization of a mannose-binding lectin

    Indian Academy of Sciences (India)

    Using RNA extracted from Zingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA of Z. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of zoa was 746 bp and contained a ...

  11. cDNA library information - Dicty_cDB | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available (C) 5) sexually fusion-competent KAX3 cells (Gamete phase) (F) cDNA library construction method How to const...dir) 2) Full-length cDNA libraries (oligocapped method)(fl) 3) Gamete-specific subtraction library (sub) cDNA library construction

  12. [CDNA cloning of human leptin and its expression].

    Science.gov (United States)

    Jia, Zhen-Yu; Fu, Xiao-Min; Jin, Ai-Hua; Cao, Jiang

    2003-07-01

    To clone cDNA of human leptin gene and obtain leptin protein for future study on leptin binding proteins. The cDNA of human leptin with 6 x his-tag was cloned by over-hang extension PCR protocol using human genomic DNA as template, and subcloned into in vitro expression vector pIVEX2.3MCS, and the fusion protein was expressed in vitro by Rapid Translation System (RTS) (RTS500 cycle primer Kit and RTS500 ProteoMaster of Roche company). The apparent molecular weight(19.46 kD) and the immuno-specificity of the fusion protein were confirmed by SDS-PAGE and Western blot, and the expressed fusion protein stayed mainly in the supernatant of the reaction mixture in soluble form. This work provides us solid basis for further study on new leptin-associated proteins.

  13. Construction of cDNA library of Pyrocystis lunula (Pyrophyta)

    Science.gov (United States)

    Sui, Zhenghong; Kowallik, Klaus V.

    2004-10-01

    Complementary DNA library of a dinoflagellate Pyrocystis lunula was constructed for the purpose of expression sequence tags analysis. The RNA isolated from this alga was about 20µgg-1 net cells, and the band intensity ratio of 28S/18 S in electrophoresis pattern was nearly 1 to 1. Different cDNA/vector molar ratios were exploited in the ligating reaction to be optimized. The clones produced by cDNA/vector molar ratio of 3.75 to 1 were desirable, most of whose inserts were longer than 300 bp. The recombinants insert length of the unfractionation cDNA library was largely shorter than 500 bp. However, in the fractionation library made from high molecule weight cDNA parts, over seventy percent of the recombinants contained inserts longer than 1 kb, some of which were even longer than 3 kb. Operating concerns were discussed at the end.

  14. Construction of cDNA libraries in vaccinia virus.

    Science.gov (United States)

    Smith, Ernest S; Shi, Shuying; Zauderer, Maurice

    2004-01-01

    Poxvirus expression vectors have gained widespread use for expression of foreign proteins and as delivery vehicles for vaccine antigens. We have developed a novel method using the poxvirus as a library vector for functional selection of specific cDNA. Poxviruses have several unique and useful properties as a library vector. Most importantly, because poxviruses are packaged into fully infectious particles in the cell cytoplasm, specific recombinants can be readily recovered even from a very small number of selected cells. Moreover, in contrast to libraries constructed in retrovirus or plasmid-based vectors, recombinant vaccinia virus can be efficiently recovered even from cells that have been induced to undergo apoptosis or cessation of cell growth. In the past, the major obstacle in this application to poxviruses has been the low frequency with which recombinants can be generated. The most commonly used method to construct recombinant poxvirus is homologous recombination. The frequency of recombinants derived in this manner is of the order of 0.1%, sufficient to recover a recombinant of a purified DNA clone in a transfer plasmid, but far too low to permit construction of a representative cDNA library. We have developed a method that generates nearly 100% recombinant vaccinia viruses at good titer. We have termed this method trimolecular recombination. cDNA libraries of as many as 107 or more independent viral recombinants can be constructed by trimolecular recombination. For the first time, large, diverse, and representative cDNA libraries can be screened in a vaccinia virus-based expression vector.

  15. cDNA cloning, structural analysis, SNP detection and tissue ...

    Indian Academy of Sciences (India)

    THOMAS NAICY

    E-mail: naicy@kvasu.ac.in. and conception rate ... transformed into DH5α strain of Escherichia coli and the clones harbouring ... Primer pairs for caprine IGF1 and GAPDH were designed using Primer3 software (table 1). RTq-PCR was conducted in a 25 μL reaction volume containing 50 ng of cDNA and 2× Max- ima SYBR ...

  16. TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis.

    Science.gov (United States)

    Wen, Shun-Hang; Lin, Luo-Na; Wu, Hu-Jun; Yu, Lu; Lin, Li; Zhu, Li-Li; Li, Hai-Yan; Zhang, Hai-Lin; Li, Chang-Chong

    2018-02-15

    To explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. The A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. S. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. TNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. PENCEGAHAN KOMPLIKASI TUBERCULOSIS AKIBAT PEMBERIAN TNF-α ANTAGONIS

    Directory of Open Access Journals (Sweden)

    Huriatul Masdar

    2009-05-01

    Full Text Available AbstrakTNF-α antagonis telah digunakan secara luas dan menunjukan respon yang baik pada terapi penyakit-penyakit autoimun seperti Rheumatoid Artritis, psoriasis dan Inflammatory Bowel Diseases. Namun, beberapa penelitian melaporkan adanya komplikasi tuberculosis yang terjadi akibat penggunaan obat ini dalam jangka lama. Untuk meminimalisir komplikasi tersebut, skrining adanya TB dengan TST, IGRA dan pemeriksaan radiologi wajib dilakukan sebelum pemberian terapi dengan TNF-α antagonis. Pemeriksaan berkala adanya TB juga harus dilakukan selama pemberian terapi. Selain itu, pemilihan obat yang tepat dengan komplikasi minimal juga harus dilakukan untuk menekan aktivasi TB tersebut.Kata Kunci: TNF-α, TNF-α antagonis, penyakit autoimun, tuberculosis.AbstractTNF-α antagonist has been widely used and showed well responses in autoimmune diseases therapy such as Rheumatoid Arthritis, psoriasis and Inflammatory Bowel Diseases. However, many studies showed that the long time used of those biological agents activate tuberculosis infection. To minimize the complication, first, TB screening by TST, IGRA and radiology examination must be performed to exclude latent TB before starting the therapy using TNF-α antagonist. Second, regular TB check must also be done during TNF-α antagonist treatment. At last, choosing right agents for better treatment with less complication must be considered.Keywords: TNF-α, TNF-α antagonist, autoimmune diseases, tuberculosis.

  18. Weight Gain and Hair Loss during Anti-TNF Therapy

    Directory of Open Access Journals (Sweden)

    Abdo Lutf

    2012-01-01

    Full Text Available Objectives. To investigate the incidence of weight gain and hair loss as adverse effects of anti-TNF therapy in rheumatic diseases. Methods. Patients using anti-TNF therapy, who are followed in rheumatology clinic, were interviewed using a questionnaire to investigate the side effects of anti-TNF therapy. Patients who complained of hair loss and weight gain were asked additional questions concerning the relationship of these adverse effects to anti-TNF use, whether therapy was stopped because of these adverse effects and if the adverse effects reversed after stopping therapy. The files were reviewed to follow the weight change before, during, and after discontinuation of anti-TNF. Results. One hundred fifty consecutive patients (82 RA, 34 ankylosing spondylitis, 32 psoriatic arthritis, and 4 for other indications were interviewed .Weight gain was observed in 20 patients (13.3% with average gain of 5.5 Kg. Anti-TNF was stopped in five patients because of this adverse effect. Hair loss during anti-TNf therapy was reported in five females (3.3% and anti-TNF therapy was stopped in all of them. Conclusion. Weight gain and hair loss appear to be associated with anti-TNF therapy and may be one reason for discontinuing the therapy.

  19. CDNA encoding a polypeptide including a hevein sequence

    Science.gov (United States)

    Raikhel, Natasha V.; Broekaert, Willem F.; Chua, Nam-Hai; Kush, Anil

    1995-03-21

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74-79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  20. cDNA library generation from ribonucleoprotein particles.

    Science.gov (United States)

    Rederstorff, Mathieu; Hüttenhofer, Alexander

    2011-02-01

    Most, if not all, known noncoding RNAs (ncRNAs) are associated with RNA binding proteins, thus forming ribonucleoprotein particles or RNPs. Here we describe a protocol for the generation of a specialized cDNA library from RNPs, thereby increasing the proportion of functional ncRNA species in the library. To that end, cellular extracts are fractionated on 10-30% glycerol gradients. Subsequently, RNP-derived ncRNAs are isolated and 3'-tailed by cytidine triphosphate and poly(A) polymerase; this is followed by 5' adapter ligation by T4 RNA ligase. Reverse transcription of ncRNAs into cDNAs is carried out with an oligo-d(G) anchor primer. The generated cDNA libraries are subsequently submitted to high-throughput sequencing. This RNP selection procedure increases the probability of the presence of biologically relevant ncRNA species in the library compared with libraries generation methods that use size-selected, protein-devoid ncRNAs. The protocol enables the generation of deep-sequencing-compatible cDNA libraries that code for functional ncRNAs within 1 week.

  1. cDNA encoding a polypeptide including a hevein sequence

    Energy Technology Data Exchange (ETDEWEB)

    Raikhel, N.V.; Broekaert, W.F.; Chua, N.H.; Kush, A.

    2000-07-04

    A cDNA clone (HEV1) encoding hevein was isolated via polymerase chain reaction (PCR) using mixed oligonucleotides corresponding to two regions of hevein as primers and a Hevea brasiliensis latex cDNA library as a template. HEV1 is 1018 nucleotides long and includes an open reading frame of 204 amino acids. The deduced amino acid sequence contains a putative signal sequence of 17 amino acid residues followed by a 187 amino acid polypeptide. The amino-terminal region (43 amino acids) is identical to hevein and shows homology to several chitin-binding proteins and to the amino-termini of wound-induced genes in potato and poplar. The carboxyl-terminal portion of the polypeptide (144 amino acids) is 74--79% homologous to the carboxyl-terminal region of wound-inducible genes of potato. Wounding, as well as application of the plant hormones abscisic acid and ethylene, resulted in accumulation of hevein transcripts in leaves, stems and latex, but not in roots, as shown by using the cDNA as a probe. A fusion protein was produced in E. coli from the protein of the present invention and maltose binding protein produced by the E. coli.

  2. TNF-α-induced depressive-like phenotype and p38(MAPK) activation are abolished by ascorbic acid treatment.

    Science.gov (United States)

    Moretti, Morgana; Budni, Josiane; Freitas, Andiara Espíndola; Neis, Vivian Binder; Ribeiro, Camille Mertins; de Oliveira Balen, Grasiela; Rieger, Débora Kurrle; Leal, Rodrigo Bainy; Rodrigues, Ana Lúcia S

    2015-06-01

    We investigated the effects of ascorbic acid on depressive-like behavior induced by tumor necrosis factor (TNF-α) in mice. Additionally, we examined the effects of combined administration of ascorbic acid and antidepressants, MK-801 and 7-nitroindazole in mice exposed or not to TNF-α and the capacity of TNF-α and ascorbic acid to modulate hippocampal and cerebrocortical phosphorylation of extracellular signal-regulated kinase (ERK), p38(MAPK) and c-Jun N-terminal kinase (JNK). In control animals, ascorbic acid reduced the immobility time in the tail suspension test (TST). Unilateral intracerebroventricular administration of TNF-α produced a depressive-like behavior in the TST, and the treatment with ascorbic acid prevented this effect. Sub-effective dose of ascorbic acid combined with sub-effective doses of fluoxetine, imipramine, bupropion, MK-801 or 7-nitroindazole produced a synergistic antidepressant-like effect in mice exposed or not to TNF-α. No treatment caused significant alterations in the locomotor activity of mice. Administration of TNF-α increased the phosphorylation of p38(MAPK) in hippocampus and cerebral cortex, and the treatment with ascorbic acid prevented this effect. Ascorbic acid increased phosphorylation of ERK1 in the hippocampus of saline- and TNF-α-treated animals, however it did not produce alterations in the cerebral cortex. No effects on phosphorylation of ERK2 or JNK were found. The observed effect of ascorbic acid seems to be associated, at least partially, with a reduced p38(MAPK) phosphorylation, activation of the monoaminergic systems as well as inhibition of N-methyl-D-aspartate (NMDA) receptors and nitric oxide (NO) synthesis. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

  3. Dynamic effects of TNF-α on synaptic transmission in mice over time following sciatic nerve chronic constriction injury.

    Science.gov (United States)

    Zhang, Hongmei; Zhang, Haijun; Dougherty, Patrick M

    2013-10-01

    Nerve injury-induced central sensitization can manifest as an increase in excitatory synaptic transmission and/or as a decrease in inhibitory synaptic transmission in spinal dorsal horn neurons. Cytokines such as tumor necrosis factor-α (TNF-α) are induced in the spinal cord under various injury conditions and contribute to neuropathic pain. In this study we examined the effect of TNF-α in modulating excitatory and inhibitory synaptic input to spinal substantia gelatinosa (SG) neurons over time in mice following chronic constriction injury (CCI) of the sciatic nerve. Whole cell patch-clamp studies from SG neurons showed that TNF-α enhanced overall excitability of the spinal cord early in time following nerve injury 3 days after CCI compared with that in sham control mice. In contrast, the effects of TNF were blunted 14 days after CCI in nerve-injured mice compared with sham surgery mice. Immunohistochemical staining showed that the expression of TNFreceptor 1 (TNFR1) was increased at 3 days but decreased at 14 days following CCI in the ipsilateral vs. the contralateral spinal cord dorsal horn. These results suggest that TNF-α acting at TNFR1 is important in the development of neuropathic pain by facilitating excitatory synaptic signaling in the acute phases after nerve injury but has a reduced effect on spinal neuron signaling in the later phases of nerve injury-induced pain. Failure of the facilatory effects of TNF-α on excitatory synaptic signaling in the dorsal horn to resolve following nerve injury may be an important component in the transition between acute and chronic pain conditions.

  4. TNF-α upregulates HIF-1α expression in pterygium fibroblasts and enhances their susceptibility to VEGF independent of hypoxia.

    Science.gov (United States)

    Kim, Kyoung Woo; Lee, Soo Jin; Kim, Jae Chan

    2017-11-01

    The clinical manifestations of pterygium are characterized by rapid growth and postoperative recurrences. We had previously proposed that hypoxia-inducible factor (HIF)-1α recruits progenitor cells during the development and progression of pterygia. Recently, it was reported that various stimuli, including inflammation, could activate HIF-1α even under normoxic conditions. The ocular surface directly faces external environments, and is thus frequently exposed to inflammatory insults. First, we examined the gene expression of HIF-1α, its downstream molecule, vascular endothelial growth factor (VEGF)-A, and VEGF receptor (VEGFR)-2 in corneal and conjunctival cells compared with cultured human umbilical vein endothelial cells. Corneal fibroblasts had high expression of VEGFR-2 in the presence of TNF-α, and HIF-1α was activated by TNF-α in diverse ocular surface cells. The HIF-1α/VEGF/VEGFR signaling pathway in response to TNF-α was evaluated in cultured human pterygium fibroblasts (HPFs) at the gene and protein levels and was compared to treatment with cobalt chloride (CoCl 2 ), a hypoxic mimetic, to exclude the effect of hypoxia. Although VEGF-A expression was not changed by TNF-α, expression of HIF-1α and VEGFR-2 was enhanced in HPFs treated with TNF-α, independent of hypoxia conditioning. In addition, VEGF-C gene expression was activated solely by TNF-α in HPF, but VEGF-B levels were not significantly affected. These results may provide mechanistic explanations for the uniquely vigorous proliferation of pterygium fibrovascular tissue during TNF-α-induced ocular surface inflammation. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Hypothalamic CRF and Norepinephrine Mediate Sympathetic and Cardiovascular Responses to Acute Intracarotid Injection of TNF-α in the Rat

    Science.gov (United States)

    Zhang, Zhi-Hua; Felder, Robert B.

    2009-01-01

    Systemic administration of tumour necrosis factor - alpha (TNF-α) induces the release of norepinephrine (NE) in the paraventricular nucleus (PVN) of hypothalamus and an increase in expression of corticotrophin-releasing-factor (CRF) and CRF type 1 receptors. We explored the hypothesis that CRF and NE in PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α. In anaesthetised rats, the increases in arterial pressure and heart rate induced by intracarotid artery injection of TNF-α were attenuated by intracerebroventricular (ICV) injection of either the α1-adrenergic antagonist prazosin or the CRF antagonist α-helical CRF. Prazosin blocked the TNF-α-induced increase in renal sympathetic nerve activity (RSNA), while α-helical CRF substantially reduced the RSNA response. Conversely, CRF and the α1-adrenergic agonist phenylephrine (PE), administered ICV, both elicited increases in PVN neuronal activity, RSNA, arterial pressure and heart rate. Microinjection of CRF and PE directly into PVN evoked smaller responses. These results are consistent with the hypothesis that NE and CRF in the PVN mediate the cardiovascular and sympathetic responses to acute systemic administration of TNF-α. PMID:18777604

  6. Direct relationship between levels of TNF-α expression and endothelial dysfunction in reperfusion injury

    OpenAIRE

    Zhang, Cuihua; Wu, Junxi; Xu, Xiangbin; Potter, Barry J.; Gao, Xue

    2010-01-01

    We previously found that myocardial ischemia/reperfusion (I/R) initiates expression of tumor necrosis factor-α (TNF) leading to coronary endothelial dysfunction. However, it is not clear whether there is a direct relationship between levels of TNF expression and endothelial dysfunction in reperfusion injury. We studied levels of TNF expression by using different transgenic animals expressing varying amounts of TNF in I/R. We crossed TNF overexpression (TNF++/++) with TNF knockout (TNF−/−) mic...

  7. Transcriptome analysis of ankylosing spondylitis patients before and after TNF-α inhibitor therapy reveals the pathways affected.

    Science.gov (United States)

    Wang, X B; Ellis, J J; Pennisi, D J; Song, X; Batra, J; Hollis, K; Bradbury, L A; Li, Z; Kenna, T J; Brown, M A

    2017-09-01

    Tumor necrosis factor-α (TNF-α) inhibitors are highly effective in suppressing inflammation in ankylosing spondylitis (AS) patients, and operate by suppression of TFN-α and downstream immunological pathways. To determine the mechanisms of action of TNF-α inhibitors in AS patients, we used transcriptomic and bioinformatic approaches on peripheral blood mononuclear cells from AS patients pre and post treatment. We found 656 differentially expressed genes, including the genome-wide significant AS-associated genes, IL6R, NOTCH1, IL10, CXCR2 and TNFRSF1A. A distinctive gene expression profile was found between male and female patients, mainly because of sex chromosome-linked genes and interleukin 17 receptor C, potentially accounting for the differences in clinical manifestation and treatment response between the genders. In addition to immune and inflammation regulatory pathways, like intestinal immune network for IgA production, cytokine-cytokine receptor interaction, Ras signaling pathway, allograft rejection and hematopoietic cell lineage, KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analyses revealed that infection-associated pathways (influenza A and toxoplasmosis) and metabolism-associated pathways were involved in response to TNF-α inhibitor treatment, providing insight into the mechanism of TNF-α inhibitors.

  8. [cDNA library construction from panicle meristem of finger millet].

    Science.gov (United States)

    Radchuk, V; Pirko, Ia V; Isaenkov, S V; Emets, A I; Blium, Ia B

    2014-01-01

    The protocol for production of full-size cDNA using SuperScript Full-Length cDNA Library Construction Kit II (Invitrogen) was tested and high quality cDNA library from meristematic tissue of finger millet panicle (Eleusine coracana (L.) Gaertn) was created. The titer of obtained cDNA library comprised 3.01 x 10(5) CFU/ml in avarage. In average the length of cDNA insertion consisted about 1070 base pairs, the effectivity of cDNA fragment insertions--99.5%. The selective sequencing of cDNA clones from created library was performed. The sequences of cDNA clones were identified with usage of BLAST-search. The results of cDNA library analysis and selective sequencing represents prove good functionality and full length character of inserted cDNA clones. Obtained cDNA library from meristematic tissue of finger millet panicle represents good and valuable source for isolation and identification of key genes regulating metabolism and meristematic development and for mining of new molecular markers to conduct out high quality genetic investigations and molecular breeding as well.

  9. The role of scavenger receptor B1 in infection with Mycobacterium tuberculosis in a murine model.

    Directory of Open Access Journals (Sweden)

    Georgia Schäfer

    2009-12-01

    Full Text Available The interaction between Mycobacterium tuberculosis (Mtb and host cells is complex and far from being understood. The role of the different receptor(s implicated in the recognition of Mtb in particular remains poorly defined, and those that have been found to have activity in vitro were subsequently shown to be redundant in vivo.To identify novel receptors involved in the recognition of Mtb, we screened a macrophage cDNA library and identified scavenger receptor B class 1 (SR-B1 as a receptor for mycobacteria. SR-B1 has been well-described as a lipoprotein receptor which mediates both the selective uptake of cholesteryl esters and the efflux of cholesterol, and has also recently been implicated in the recognition of other pathogens. We show here that mycobacteria can bind directly to SR-B1 on transfected cells, and that this interaction could be inhibited in the presence of a specific antibody to SR-B1, serum or LDL. We define a variety of macrophage populations, including alveolar macrophages, that express this receptor, however, no differences in the recognition and response to mycobacteria were observed in macrophages isolated from SR-B1(-/- or wild type mice in vitro. Moreover, when wild type and SR-B1(-/- animals were infected with a low dose of Mtb (100 CFU/mouse there were no alterations in survival, bacterial burdens, granuloma formation or cytokine production in the lung. However, significant reduction in the production of TNF, IFNgamma, and IL10 were observed in SR-B1(-/- mice following infection with a high dose of Mtb (1000 CFU/mouse, which marginally affected the size of inflammatory foci but did not influence bacterial burdens. Deficiency of SR-B1 also had no effect on resistance to disease under conditions of varying dietary cholesterol. We did observe, however, that the presence of high levels of cholesterol in the diet significantly enhanced the bacterial burdens in the lung, but this was independent of SR-B1.SR-B1 is involved in

  10. Tumour necrosis factor alpha (TNF-α) genetic polymorphisms and ...

    Indian Academy of Sciences (India)

    2Department of Occupational Health and Environmental Health, School of Public Health at Guangxi Medical University,. Nanning, Guangxi 530021, People's Republic of China. Abstract. Epidemiological studies have evaluated the association between tumour necrosis factor alpha (TNF-α)-308G/A and (TNF-α)-. 238G/A ...

  11. Kadar TNF-α dalam Zalir Peritoneal Penderita Endometriosis

    Directory of Open Access Journals (Sweden)

    TEDJA DANUDJA OEPOMO

    2005-11-01

    Full Text Available The aim of this research was to expose the role of tumor necrotic factor alpha (TNF-α in the pathogenetic endometriosis. This research had been done in dr. Muwardi Hospital Surakarta. Twenty patients undergoing laparoscopic operation because of endometriosis indication (Group I, 20 women (aged 23 to 40 who undergo interval sterilization by means of laparoscopic technique (Group II. During laparoscopic operation, peritoneal fluid is taken to examine TNF-α by ELISA technique. The results indicated that by independent t-test, a significant difference of concentration of TNF-α in the peritoneal fluid is found between endometriosis patients and normal women (who are sterilized (P=0.00. By chi-square test, the Ratio Odds value 171 shows that the high concentration of TNF-α will increase the possibility of endometriosis 171 times rather than the low TNF-α. It could be concluded the high concentration of TNF-α is the risk factor of endometriosis in comparison with the low TNF-α. It shows that quite possibly TNF-α has a role in the pathogenic endometriosis.

  12. [Construction and identification of the expression library of album pollen allergens cDNA].

    Science.gov (United States)

    Zhang, Jie; Sun, Xiu-zhen; Yan, Hong; Zhang, Ni; Feng, Xiang-li

    2011-05-01

    To construct and identify the express library of album pollen allergens cDNA. Total RNA were extracted from the album pollen with TRIzol reagent and the mRNA was isolate for the amplify followed. A double stranded cDNA (ds cDNA) was synthesized using primers containing Xho I and Poly(dT) sequence by ZAP Express®cDNA synthesis kit. The ds cDNA was modified and purified by gel chromatography, and then the cDNA fragment with the length of more than 400 bp containing sticky ends was obtained. The cDNA fragment was ligated with Uni-ZAP XR vector and subsequently treated with in vitro packaging using phage by ZAP-cDNA express GigapackIII Gold cloning kit. The express library of album pollen cDNA was constructed by in vitro packaging. The recombination rate and the lengths of fragments inserted of the cDNA library were detected by polymerase chain reaction. The titer and the recombination rate of cDNA expression library constructed were 9.7×10(5) and 100%, respectively. The capacity of the library was 4.85 Pfu. The average length of cDNA fragments inserted was about 1.0 kb. Based on the capacity of cDNA expression library constructed and the length of cDNA insertion fragments, the cDNA expression library constructed is qualified to screening target cDNA clone, laying the foundation for preparation of gene recombinant allergen pollen vaccine.

  13. CD40L and TNF both activate the classical NF-κB pathway, which is not required for the CD40L induced alternative pathway in endothelial cells.

    Science.gov (United States)

    Seigner, J; Basilio, J; Resch, U; de Martin, R

    2018-01-01

    CD40L and TNF signal through engagement of their respective receptors, which are both members of the TNF receptor family. They use partially common signaling molecules leading, among others, to activation of the NF-κB pathway. However, whereas TNF activates the classical, CD40L has been reported to activate the alternative NF-κB pathway, leading to the anticipation that differences in the pattern of inflammatory gene expression would occur. Here, we have compared the gene expression repertoire of CD40L (CD154) and TNF stimulated HUVEC and report that unexpectedly, apart from a stronger response to TNF, no major qualitative differences could be observed. This applies for the period of up to 6 h, a time where the alternative pathway has already been activated. Analysis of the early events after receptor engagement revealed that both TNF and CD40L activate the classical NF-κB pathway, and confirm activation of the alternative by the latter. Furthermore, using genetic and pharmacological inhibition of the classical pathway we show that activation of the alternative occurs independently of the former. This reveals novel insights into NF-κB signaling by CD40L and TNF in endothelial cells. Copyright © 2017. Published by Elsevier Inc.

  14. Prolactin receptor and signal transduction to milk protein genes

    Energy Technology Data Exchange (ETDEWEB)

    Djiane, J.; Daniel, N.; Bignon, C. [Unite d`Endocrinologie Moleculaire, Jouy en Josas (France)] [and others

    1994-06-01

    After cloning of the mammary gland prolactin (PRL) receptor cDNA, a functional assay was established using co-transfection of PRL receptor cDNA together with a milk protein promoter/chloramphenicol acetyl transferase (CAT) construct in Chinese hamster ovary (CHO) cells. Different mutants of the PRL receptor were tested in this CAT assay to delimit the domains in the receptor necessary for signal transduction to milk protein genes. In CHO cells stably transfected with PRL receptor cDNA, high numbers of PRL receptor are expressed. By metabolic labeling and immunoprecipitation, expressed PRL receptor was identified as a single species of 100 kDa. Using these cells, we analyzed the effects of PRL on intracellular free Ca{sup ++} concentration. PRL stimulates Ca{sup ++} entry and induces secondary Ca{sup ++} mobilization. The entry of Ca{sup ++} is a result of an increase in K{sup +} conductance that hyperpolarizes the membranes. We have also analyzed tyrosine phosphorylation induced by PRL. In CHO cells stably transfected with PRL receptor cDNA, PRL induced a very rapid and transient tyrosine phosphorylation of a 100-kDa protein which is most probably the PRL receptor. The same finding was obtained in mammary membranes after PRL injection to lactating rabbits. Whereas tyrosine kinase inhibitors genistein and lavendustin were without effect, PRL stimulation of milk protein gene promoters was partially inhibited by 2 {mu}M herbimycin in CHO cells co-transfected with PRL receptor cDNA and the {Beta} lactoglobulin CAT construct. Taken together these observations indicate that the cytoplasmic domain of the PRL receptor interacts with one or several tyrosine kinases, which may represent early postreceptor events necessary for PRL signal transduction to milk protein genes. 14 refs., 4 figs.

  15. Dynamics of RASSF1A/MOAP-1 association with death receptors.

    Science.gov (United States)

    Foley, Caitlin J; Freedman, Holly; Choo, Sheryl L; Onyskiw, Christina; Fu, Nai Yang; Yu, Victor C; Tuszynski, Jack; Pratt, Joanne C; Baksh, Shairaz

    2008-07-01

    RASSF1A is a tumor suppressor protein involved in death receptor-dependent apoptosis utilizing the Bax-interacting protein MOAP-1 (previously referred to as MAP-1). However, the dynamics of death receptor recruitment of RASSF1A and MOAP-1 are still not understood. We have now detailed recruitment to death receptors (tumor necrosis factor receptor 1 [TNF-R1] and TRAIL-R1/DR4) and identified domains of RASSF1A and MOAP-1 that are required for death receptor interaction. Upon TNF-alpha stimulation, the C-terminal region of MOAP-1 associated with the death domain of TNF-R1; subsequently, RASSF1A was recruited to MOAP-1/TNF-R1 complexes. Prior to recruitment to TNF-R1/MOAP-1 complexes, RASSF1A homodimerization was lost. RASSF1A associated with the TNF-R1/MOAP-1 or TRAIL-R1/MOAP-1 complex via its N-terminal cysteine-rich (C1) domain containing a potential zinc finger binding motif. Importantly, TNF-R1 association domains on both MOAP-1 and RASSF1A were essential for death receptor-dependent apoptosis. The association of RASSF1A and MOAP-1 with death receptors involves an ordered recruitment to receptor complexes to promote cell death and inhibit tumor formation.

  16. Tumor necrosis factor and its receptors in the neuroretina and retinal vasculature after ischemia-reperfusion injury in the pig retina

    DEFF Research Database (Denmark)

    Gesslein, Bodil; Håkansson, Gisela; Gustafsson, Lotta

    2010-01-01

    Numerous studies have been performed aimed at limiting the extent of retinal injury after ischemia, but there is still no effective pharmacological treatment available. The aim of the present study was to examine the role of tumor necrosis factor (TNF)α and its receptors (TNF-R1 and TNF-R2), espe...

  17. Increased levels of soluble tumour necrosis factor receptor-I (P55) and decreased IgG1 reactivities in HIV-1 patients with cytomegalovirus disease

    DEFF Research Database (Denmark)

    Jakobsen, Palle Høy; Dodt, K K; Meyer, C N

    1998-01-01

    The purpose of the study was to investigate potential associations between tumour necrosis factor (TNF), soluble TNF receptors (sTNF-Rs), immunoglobulin (Ig)G subclasses and development of cytomegalovirus (CMV) disease amongst human immunodeficiency virus (HIV)-1 patients. We enrolled HIV-1...

  18. Pre-Transplantation Blockade of TNF-α-Mediated Oxygen Species Accumulation Protects Hematopoietic Stem Cells.

    Science.gov (United States)

    Ishida, Takashi; Suzuki, Sachie; Lai, Chen-Yi; Yamazaki, Satoshi; Kakuta, Shigeru; Iwakura, Yoichiro; Nojima, Masanori; Takeuchi, Yasuo; Higashihara, Masaaki; Nakauchi, Hiromitsu; Otsu, Makoto

    2017-04-01

    Hematopoietic stem cell (HSC) transplantation (HSCT) for malignancy requires toxic pre-conditioning to maximize anti-tumor effects and donor-HSC engraftment. While this induces bone marrow (BM)-localized inflammation, how this BM environmental change affects transplanted HSCs in vivo remains largely unknown. We here report that, depending on interval between irradiation and HSCT, residence within lethally irradiated recipient BM compromises donor-HSC reconstitution ability. Both in vivo and in vitro we demonstrate that, among inflammatory cytokines, TNF-α plays a role in HSC damage: TNF-α stimulation leads to accumulation of reactive oxygen species (ROS) in highly purified hematopoietic stem/progenitor cells (HSCs/HSPCs). Transplantation of flow-cytometry-sorted murine HSCs reveals damaging effects of accumulated ROS on HSCs. Short-term incubation either with an specific inhibitor of tumor necrosis factor receptor 1 signaling or an antioxidant N-acetyl-L-cysteine (NAC) prevents TNF-α-mediated ROS accumulation in HSCs. Importantly, pre-transplantation exposure to NAC successfully demonstrats protective effects in inflammatory BM on graft-HSCs, exhibiting better reconstitution capability than that of nonprotected control grafts. We thus suggest that in vivo protection of graft-HSCs from BM inflammation is a feasible and attractive approach, which may lead to improved hematopoietic reconstitution kinetics in transplantation with myeloablative conditioning that inevitably causes inflammation in recipient BM. Stem Cells 2017;35:989-1002. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.

  19. Molecular Cloning and Functional Characterization of Mannose Receptor in Zebra Fish (Danio rerio during Infection with Aeromonas sobria

    Directory of Open Access Journals (Sweden)

    Feifei Zheng

    2015-05-01

    Full Text Available Mannose receptor (MR is a member of pattern-recognition receptors (PRRs, which plays a significant role in immunity responses. Much work on MR has been done in mammals and birds while little in fish. In this report, a MR gene (designated as zfMR was cloned from zebra fish (Danio rerio, which is an attractive model for the studies of animal diseases. The full-length cDNA of zfMR contains 6248 bp encoding a putative protein of 1428 amino acids. The predicted amino acid sequences showed that zfMR contained a cysteine-rich domain, a single fibronectin type II (FN II domain, eight C-type lectin-like domains (CTLDs, a transmembrane domain and a short C-terminal cytoplasmic domain, sharing highly conserved structures with MRs from the other species. The MR mRNA could be detected in all examined tissues with highest level in kidney. The temporal expression patterns of MR, IL-1β and TNF-α mRNAs were analyzed in the liver, spleen, kidney and intestine post of infection with Aeromonas sobria. By immunohistochemistry assay, slight enhancement of MR protein was also observed in the spleen and intestine of the infected zebra fish. The established zebra fish-A. sobria infection model will be valuable for elucidating the role of MR in fish immune responses to infection.

  20. Increased mRNA expression of a laminin-binding protein in human colon carcinoma: Complete sequence of a full-length cDNA encoding the protein

    International Nuclear Information System (INIS)

    Yow, Hsiukang; Wong, Jau Min; Chen, Hai Shiene; Lee, C.; Steele, G.D. Jr.; Chen, Lanbo

    1988-01-01

    Reliable markers to distinguish human colon carcinoma from normal colonic epithelium are needed particularly for poorly differentiated tumors where no useful marker is currently available. To search for markers the authors constructed cDNA libraries from human colon carcinoma cell lines and screened for clones that hybridize to a greater degree with mRNAs of colon carcinomas than with their normal counterparts. Here they report one such cDNA clone that hybridizes with a 1.2-kilobase (kb) mRNA, the level of which is ∼9-fold greater in colon carcinoma than in adjacent normal colonic epithelium. Blot hybridization of total RNA from a variety of human colon carcinoma cell lines shows that the level of this 1.2-kb mRNA in poorly differentiated colon carcinomas is as high as or higher than that in well-differentiated carcinomas. Molecular cloning and complete sequencing of cDNA corresponding to the full-length open reading frame of this 1.2-kb mRNA unexpectedly show it to contain all the partial cDNA sequence encoding 135 amino acid residues previously reported for a human laminin receptor. The deduced amino acid sequence suggests that this putative laminin-binding protein from human colon carcinomas consists of 295 amino acid residues with interesting features. There is an unusual C-terminal 70-amino acid segment, which is trypsin-resistant and highly negatively charged

  1. Preparation of a differentially expressed, full-length cDNA expression library by RecA-mediated triple-strand formation with subtractively enriched cDNA fragments

    NARCIS (Netherlands)

    Hakvoort, T. B.; Spijkers, J. A.; Vermeulen, J. L.; Lamers, W. H.

    1996-01-01

    We have developed a fast and general method to obtain an enriched, full-length cDNA expression library with subtractively enriched cDNA fragments. The procedure relies on RecA-mediated triple-helix formation of single-stranded cDNA fragments with a double-stranded cDNA plasmid library. The complexes

  2. Toward a cDNA map of the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Korenberg, J.R.; Chen, X.N. [Cedars-Sinai Research Institute, Los Angeles, CA (United States); Adams, M.D.; Venter, J.C. [Institute for Genomic Research, Gaithersburg, MD (United States)

    1995-09-20

    Advances in the Human Genome Project are shaping the strategies for identifying the 50,000-100,000 human genes. High-resolution genetic maps of the human genome combined with sequencing herald an era of rapid regional definition of disease genes. However, only once their chromosomes band location is known will the systematic partial sequencing of thousands of random cDNA clones provide the reagents for the rapid assessment of the genes responsible for the inherited disorders. We now present an approach to the rapid determination of map position and therefore to the creation of a transcribed map of the human genome. Sensitive fluorescence in situ hybridization has been combined with high-resolution chromosome banding and random cDNA sequencing to 41 cDNAs with an average insert size of < 2 kb to single human chromosome bands. The results provide 15 new genes, with database and functional information, as candidates for human disease. These include the large extracellular single-related kinase (HUMERK), the ERK activator kinase (PRKMK1), a new member of the RAS oncogene family, protein phosphotase 2 regulatory subunit B alpha isoform (PPP2R2A), and a novel human gene with very high homology to a plant membrane transport family. Further, an analysis of expressed genes associated with pseudogenes showed that by using these techniques, it is possible to detect accurately the transcribed locus within a multigene or processed pseudogene family in most cases. These findings suggest that direct cDNA mapping using fluorescence in situ hybridization provides an accurate and rapid approach to the definition of a transcribed map of the human genome. This low-cost, high-resolution (205 Mb) mapping greatly enhances the speed with which these genes can be subsequently assigned to contigs. This assignment provides a necessary first step in understanding the relationship of the genes to both acquired and inherited human diseases. 16 refs., 1 fig., 3 tabs.

  3. The antibody response against human and chimeric anti-TNF therapeutic antibodies primarily targets the TNF binding region

    NARCIS (Netherlands)

    van Schie, K. A.; Hart, M. H.; de Groot, E. R.; Kruithof, S.; Aarden, L. A.; Wolbink, G. J.; Rispens, T.

    2015-01-01

    In a subset of patients, anti tumour necrosis factor (TNF) therapeutic antibodies are immunogenic, resulting in the formation of antidrug antibodies (ADAs). Neutralising ADAs compete with TNF for its binding site and reduces the effective serum concentration, causing clinical non-response. It is

  4. Construction and characterization of a normalized cDNA library.

    OpenAIRE

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-01-01

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each...

  5. [Construction and Identification of the cDNA Expression Library for Human Esophageal Cancer Cells].

    Science.gov (United States)

    Zhang, Zhe; Wu, Xiang-Yu; Feng, Lu; Huang, Shang-Ke; Luo, Min-Na; Shao, Shan; Zhao, Xin-Han

    2016-09-01

    To construct a cDNA phage expression library for human esophageal cancer cells. After the total RNA were obtained from esophageal cancer cells, the mRNA were separated with magnetic beads adsorption method, and the single-strand and double-strand cDNA were synthesized through reverse transcription. With the undesirable cDNA fragments removed, the remaining cDNA (linked with Eco R1 aptamer and phosphorylated its 5'end) combined with the carrier of T7 Select10-3b. The recombinant phage were packaged in vitro for preliminary cDNA library. PCR was used to identify the size of inserted cDNA. The constructed original cDNA phage expression library for human esophageal cancer cells was consisted of 2.01×10⁶ pfu/mL bacteriophages with a recombination rate of 100%. The length of the inserted cDNA fragments were range from 300 bp to 1 500 bp. The cDNA phage expression library of human esophageal cell is successfully constructed to meet the currently recognized standards, and can be well used to screen cDNA-cloned genes of human esophageal cancer antigens by serological analysis of recombinantly expressed cDNA clone (SEREX).

  6. Gallic acid induces necroptosis via TNF-α signaling pathway in activated hepatic stellate cells.

    Directory of Open Access Journals (Sweden)

    Ya Ju Chang

    Full Text Available Gallic acid (3, 4, 5-trihydroxybenzoic acid, GA, a natural phenolic acid widely found in gallnuts, tea leaves and various fruits, possesses several bioactivities against inflammation, oxidation, and carcinogenicity. The beneficial effect of GA on the reduction of animal hepatofibrosis has been indicated due to its antioxidative property. However, the cytotoxicity of GA autoxidation causing cell death has also been reported. Herein, we postulated that GA might target activated hepatic stellate cells (aHSCs, the cell type responsible for hepatofibrosis, to mitigate the process of fibrosis. The molecular cytotoxic mechanisms that GA exerted on aHSCs were then analyzed. The results indicated that GA elicited aHSC programmed cell death through TNF-α-mediated necroptosis. GA induced significant oxidative stress through the suppression of catalase activity and the depletion of glutathione (GSH. Elevated oxidative stress triggered the production of TNF-α facilitating the undergoing of necroptosis through the up-regulation of key necroptotic regulatory proteins TRADD and receptor-interacting protein 3 (RIP3, and the inactivation of caspase-8. Calmodulin and calpain-1 activation were engaged, which promoted subsequent lysosomal membrane permeabilization (LMP. The TNF-α antagonist (SPD-304 and the RIP1 inhibitor (necrostatin-1, Nec-1 confirmed GA-induced TNFR1-mediated necroptosis. The inhibition of RIP1 by Nec-1 diverted the cell death from necroptosis to apoptosis, as the activation of caspase 3 and the increase of cytochrome c. Collectively, this is the first report indicating that GA induces TNF signaling-triggered necroptosis in aHSCs, which may offer an alternative strategy for the amelioration of liver fibrosis.

  7. An oral administration of a recombinant anti-TNF fusion protein is biologically active in the gut promoting regulatory T cells: Results of a phase I clinical trial using a novel oral anti-TNF alpha-based therapy.

    Science.gov (United States)

    Almon, Einat; Khoury, Tawfik; Drori, Ariel; Gingis-Velitski, Svetlana; Alon, Sari; Chertkoff, Raul; Mushkat, Mordechai; Shaaltiel, Yoseph; Ilan, Yaron

    2017-07-01

    An orally administered BY-2 plant cell-expressed recombinant anti-TNF fusion protein (PRX-106) consists of the soluble form of the human TNF receptor (TNFR) fused to the Fc component of a human IgG1 domain. Aim This study aim at determining the safety and the immune modulatory effect of an oral administration of PRX-106 in humans. Three different doses (2, 8 or 16mg/day) of PRX-106 were orally administered for five consecutive days in 14 healthy volunteered participants. Subjects were followed for safety parameters and for an effect on T lymphocytes subsets and cytokine levels. An oral administration of PRX-106 was safe and well tolerated. The PK study showed that PRX106 is not absorbed. No effect on white blood cells and lymphocytes counts were noted. A dose dependent effect was noted on systemic lymphocytes. The oral administration of all three dosages was associated with an increase in CD4+CD25+ and CD8+CD25+ subset of suppressor lymphocytes. A marked increase in CD4+CD25+FoxP3 regulatory T cells was noted in the 8mg treated group. In addition, NKT regulatory cells, CD3+CD69+ and CD4+CD62 lymphocyte subsets increased with treatment. No changes in serum TNF alpha were observed. An oral administration of the non-absorbable recombinant anti-TNF fusion protein, PRX-106, is safe, not associated with immune suppression, while inducing a favorable anti-inflammatory immune modulation. The PRX-106 may provide a safe orally administered effective anti-TNF alpha-based immune therapy for inflammatory bowel diseases and non-alcoholic steatohepatitis, as well as other autoimmune, TNF-mediated diseases. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular cloning and functional expression of a Drosophila receptor for the neuropeptides capa-1 and -2

    DEFF Research Database (Denmark)

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael

    2002-01-01

    The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end of the ann......The Drosophila Genome Project website contains an annotated gene (CG14575) for a G protein-coupled receptor. We cloned this receptor and found that the cloned cDNA did not correspond to the annotated gene; it partly contained different exons and additional exons located at the 5(')-end...... of the annotated gene. We expressed the coding part of the cloned cDNA in Chinese hamster ovary cells and found that the receptor was activated by two neuropeptides, capa-1 and -2, encoded by the Drosophila capability gene. Database searches led to the identification of a similar receptor in the genome from...

  9. Characterization of a cDNA encoding cottonseed catalase.

    Science.gov (United States)

    Ni, W; Turley, R B; Trelease, R N

    1990-06-21

    A 1.7 kb cDNA clone was isolated from our lambda gt11 library constructed from poly(A) RNA of 24-h-old cotyledons. The cDNA encodes a full-length catalase peptide (492 amino acid residues). The calculated molecular mass is 56,800, similar to that determined for purified enzyme (57,000 SDS-PAGE). Among higher plant catalases, this cotton catalase shows the highest amino acid sequence identity (85%) to the subunit of homotetrameric maize CAT 1, a developmental counterpart to the homotetrameric CAT A isoform of cotton seeds. Comparison of sequences from cotton, sweet potato, maize CAT 1, and yeast with bovine catalase revealed that the amino acid residues and regions that are involved in catalytic activity and/or required to maintain basic catalase structure, are highly conserved. The C-terminus region, which has the lowest nucleotide sequence identity between plant and mammalian catalases, does not terminate with a tripeptide, S-K/R/H-L, a putative targeting signal for peroxisomal proteins.

  10. Infectious Maize rayado fino virus from Cloned cDNA.

    Science.gov (United States)

    Edwards, Michael C; Weiland, John J; Todd, Jane; Stewart, Lucy R

    2015-06-01

    A full-length cDNA clone was produced from a U.S. isolate of Maize rayado fino virus (MRFV), the type member of the genus Marafivirus within the family Tymoviridae. Infectivity of transcripts derived from cDNA clones was demonstrated by infection of maize plants and protoplasts, as well as by transmission via the known leafhopper vectors Dalbulus maidis and Graminella nigrifrons that transmit the virus in a persistent-propagative manner. Infection of maize plants through vascular puncture inoculation of seed with transcript RNA resulted in the induction of fine stipple stripe symptoms typical of those produced by wild-type MRFV and a frequency of infection comparable with that of the wild type. Northern and Western blotting confirmed the production of MRFV-specific RNAs and proteins in infected plants and protoplasts. An unanticipated increase in subgenomic RNA synthesis over levels in infected plants was observed in protoplasts infected with either wild-type or cloned virus. A conserved cleavage site motif previously demonstrated to function in both Oat blue dwarf virus capsid protein and tymoviral nonstructural protein processing was identified near the amino terminus of the MRFV replicase polyprotein, suggesting that cleavage at this site also may occur.

  11. Effect of TNF-Alpha on Caveolin-1 Expression and Insulin Signaling During Adipocyte Differentiation and in Mature Adipocytes

    Directory of Open Access Journals (Sweden)

    Sara Palacios-Ortega

    2015-07-01

    Full Text Available Background/Aims: Tumor necrosis factor-α (TNF-α-mediated chronic low-grade inflammation of adipose tissue is associated with obesity and insulin resistance. Caveolin-1 (Cav-1 is the central component of adipocyte caveolae and has an essential role in the regulation of insulin signaling. The effects of TNF-α on Cav-1 expression and insulin signaling during adipocyte differentiation and in mature adipocytes were studied. Methods: 3T3-L1 cells were differentiated (21 days in the presence TNF-α (10 ng/mL and mature adipocytes were also treated with TNF-α for 48 hours. Cav-1 and insulin receptor (IR gene methylation were determined as well as Cav-1, IR, PKB/AKT-2 and Glut-4 expression and activation by real time RT-PCR and western blot. Baseline and insulin-induced glucose uptake was measured by the 2-[C14]-deoxyglucose uptake assay. Results: TNF-α slowed down the differentiation program, hindering the expression of some insulin signaling intermediates without fully eliminating insulin-mediated glucose uptake. In mature adipocytes, TNF-α did not compromise lipid-storage capacity, but downregulated the expression of the insulin signaling intermediates, totally blocking insulin-mediated glucose uptake. Insulin sensitivity correlated with the level of activated phospho-Cav-1 in both situations, strongly suggesting the direct contribution of Cav-1 to the maintenance of this physiological response. Conclusion: Cav-1 activation by phosphorylation seems to be essential for the maintenance of an active and insulin-sensitive glucose uptake.

  12. Different roles of TNF inhibitors in acute anterior uveitis associated with ankylosing spondylitis: state of the art.

    Science.gov (United States)

    Fabiani, Claudia; Vitale, Antonio; Lopalco, Giuseppe; Iannone, Florenzo; Frediani, Bruno; Cantarini, Luca

    2016-11-01

    The purpose of the present review was to provide a comprehensive picture of the efficacy of the different tumor necrosis factor (TNF)-α inhibiting agents in the treatment of acute anterior uveitis (AAU), the most common extra-articular manifestation of ankylosing spondylitis (AS). AS related, AAU may lead to severe visual impairment, due to frequent flare recurrences, anterior, and posterior segment complications and traditional treatment side effects. Considerably higher levels of tumor necrosis factor (TNF) have been assessed in the aqueous humor and inflamed joints of patients with AS. Anti-TNF drugs have shown efficacy in preventing relapses of rheumatological manifestations of spondyloarthropathies. Several studies have underlined the sustained efficacy of the monoclonal anti-TNF antibodies also in reducing the recurrence of anterior chamber flares in patients with AS-related AAU. On the other hand, retrospective studies and observational reports have indicated lower effectiveness and some paradoxical occurrence of uveitis following treatment with the soluble receptor agent etanercept. Growing evidence suggests that a prophylactic strategy could be advocated in subjects with frequent and recalcitrant attacks of AS-AAU. In this regard, the administration of monoclonal anti-TNF antibodies such as adalimumab (ADA) has been shown to significantly reduce the rate of AAU recurrences. Indeed, during ADA treatment about 90 % of patients have shown to remain completely free of attacks for the entire follow-up period, in most studies. Further studies are needed to confirm the long-term efficacy of TNF inhibitors in AS related AAU and also their role in preventing ocular complications and visual impairment.

  13. Molecular cloning and chromosomal localization of one of the human glutamate receptor genes

    OpenAIRE

    Puckett, Carmie; Gomez, Christopher M.; Korenberg, Julie R.; Tung, Howard; Meier, Timothy J.; Chen, Xiao Ning; Hood, Leroy

    1991-01-01

    Glutamate receptors are the predominant excitatory neurotransmitter receptors in the mammalian brain and are classified on the basis of their activation by different agonists. The agonists kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxasolepropionic acid define a class of glutamate receptors termed kainate receptors. We have isolated and sequenced a human glutamate receptor (GluHI) cDNA and determined the chromosomal localization of its gene. The DNA sequence of GluHI would encode a 907-am...

  14. Preparation of full-length cDNA libraries: focus on metazoans.

    Science.gov (United States)

    Harada, Masako; Hayashizaki, Yoshihide

    2009-01-01

    Critical steps in a cDNA library preparation include efficient cDNA synthesis, selection of full-length cDNAs, normalizing their abundance, and the subtraction of redundant transcripts. The use of trehalose and sorbiol stabilizes the activity of the reverse transcriptase leading to efficient cDNA synthesis and the cap-trapping method is used for efficient full-length cDNA selection. Through the incorporation of additional normalization and subtraction steps that eliminate the size bias and expressed gene frequency, it is possible to attain cDNA libraries that include larger or rarely expressed genes. This chapter describes an efficient method to construct a full-length cDNA library, with a focus on metazoan samples.

  15. Tumour necrosis factor (TNF and interleukin-1 (IL-1 induce muscle proteolysis through different mechanisms

    Directory of Open Access Journals (Sweden)

    Oded Zamir

    1992-01-01

    Full Text Available The purpose of this study was to test the hypothesis that muscle proteolysis induced by TNF or IL-1 is mediated by glucocorticoids. Rats were treated with 300 μg kg−1 of recombinant human preparations of IL-1α (rIL-1α or TNFα (rTNFα divided into three equal intraperitoneal doses given over 16 h. Two hours before each cytokine injection, rats were given 5 mg kg−1 of the glucocorticoid receptor blocker mifepristone RU 38486, by gavage or were gavaged with the vehicle. Eighteen hours after the first cytokine injection, total and myofibrillar protein breakdown rates were determined in incubated extensor digitorum longus muscles as release of tyrosine and 3-methylhistidine, respectively. Total and myofibrillar proteolytic rates were increased following injection of rIL-1α or rTNFα. Proteolysis induced by rIL-1α was not altered by treatment with RU 38486. In contrast, the glucocorticoid receptor blocker inhibited the proteolytic effect of rTNFα. The results suggest that the proteolytic effect of TNF is mediated by glucocorticoids and that IL-1 induces muscle proteolysis through a glucocorticoid independent pathway.

  16. Solid-phase cDNA library construction, a versatile approach.

    OpenAIRE

    Roeder, T

    1998-01-01

    A rapid and versatile method for cDNA library construction was developed. It is based on conventional cDNA library synthesis including all enzymatic steps usually required, but is performed on a solid support. The cDNA is immobilised via a biotin residue to streptavidin coupled magnetic beads, which allows rapid and easy to perform changes of buffers and enzymes. Therefore, it combines speed (library construction within a single day) with high quality libraries, making it ideally suited for m...

  17. [Software development in data analysis and mining for cDNA microarray].

    Science.gov (United States)

    Wu, Bin; Wang, Jianguo; Wang, Miqu

    2007-12-01

    Data analysis and mining is a key issue to microarray technology and is usually implemented through software development. This paper summarizes the state-of-art software development in cDNA microarray data analysis and mining. The updated software developments are discussed in three stages: data inquisition from cDNA microarray tests, statistical treatment of cDNA data and data mining from gene network.

  18. Molecular cloning and mammalian expression of human beta 2-glycoprotein I cDNA

    DEFF Research Database (Denmark)

    Kristensen, Torsten; Schousboe, Inger; Boel, Espen

    1991-01-01

    Human β2-glycoprotein (β2gpI) cDNA was isolated from a liver cDNA library and sequenced. The cDNA encoded a 19-residue hydrophobic signal peptide followed by the mature β2gpI of 326 amino acid residues. In liver and in the hepatoma cell line HepG2 there are two mRNA species of about 1.4 and 4.3 kb...

  19. The role of TNF-α and TNF-β gene polymorphism in the pathogenesis of Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Hannan Al- Rayes

    2013-01-01

    Full Text Available Rheumatoid arthritis (RA is a chronic systemic inflammatory disorder of unknown etiology that affects the synovial membrane of multiple joints. The clinical presentation of RA may vary from mild to severe with excessive erosions of periarticular bone leading to the loss of functional capacity. Both genetic and environmental factors are important in the development of this disorder. The genetic contribution to susceptibility for RA is underlined by a three-to four-fold higher concordance percentage for clinically expressed disease in monozygotic twins compared to dizygotic twins. The severity and long term outcome of RA have also been related to various genetic factors. Tumor necrosis factor (TNF, a pro-inflammatory cytokine, is involved in the pathogenesis of a variety of autoimmune disorders, including RA. A large number of studies have been undertaken to determine the role of TNF-α promoter polymorphisms in the pathogenesis of RA. On the other hand few attempts have been made to identify the association between TNF-α (lymphotoxin-alfa polymorphism and RA. In this narrative review of published literature, an attempt has been made to determine the association between TNF-α promoter polymorphisms at positions –308, –238, –489, –857, –863 and TNF-β at +252 with respect to susceptibility to and severity of RA, as well as response to drug therapy. In spite of intra-and inter-ethnic variations, analysis of data suggests a significant role of TNF-α/TNF-β polymorphisms in determining the susceptibility/severity of RA and responsiveness to anti-TNF drug therapy. The TNF gene polymorphisms may be an interesting target for novel strategies to prevent RA and/or in its early treatment. Further studies using larger samples are needed to pinpoint the regulatory polymorphisms or haplotypes and their effects on the development of certain manifestations in RA.

  20. Functional expression of the 5-HT1c receptor in neuronal and nonneuronal cells

    International Nuclear Information System (INIS)

    Julius, D.; MacDermott, A.B.; Jessel, T.M.; Huang, K.; Molineaux, S.; Schieren, I.; Axel, R.

    1988-01-01

    The isolation of the genes encoding the multiple serotonin receptor subtypes and the ability to express these receptors in new cellular environments will help to elucidate the molecular mechanisms of action of serotonin in the mammalian brain. The cloning of most neurotransmitter receptors has required the purification of receptor, the determination of partial protein sequence, and the synthesis of oligonucleotide probes with which to obtain cDNA or genomic clones. However, the serotonin receptors have not been purified and antibodies have not been generated. The authors therefore designed a cDNA expression system that permits the identification of functional cDNA clones encoding serotonin receptors in the absence of protein sequence information. They have combined cloning in RNA expression vectors with an electrophysiological assay in oocytes to isolate a functional cDNA clone encoding the entire 5-HT 1c receptor. The sequence of this clone reveals that the 5-HT 1c receptor belongs to a family of G-protein-coupled receptors that are thought to traverse the membrane seven times. Mouse fibroblasts transformed with this clone bind serotonergic ligands and respond to serotonin with an elevation in intracellular calcium. Moreover, in situ hybridization and Northern blot analysis indicate that the 5-HT 1c receptor mRNA is expressed in a wide variety of neurons in the rat central nervous system, suggesting that this receptor plays a prominent role in neuronal function

  1. NF-kappaB is involved in SHetA2 circumvention of TNF-alpha resistance, but not induction of intrinsic apoptosis.

    Science.gov (United States)

    Chengedza, Shylet; Benbrook, Doris Mangiaracina

    2010-03-01

    Treatment of cancer with tumor necrosis factor-alpha (TNF-alpha) is hindered by resistance and toxicity. The flexible heteroarotinoid, SHetA2, sensitizes resistant ovarian cancer cells to TNF-alpha-induced extrinsic apoptosis, and also induces intrinsic apoptosis as a single agent. This study tested the hypothesis that nuclear factor-kappaB (NF-kappaB) is involved in SHetA2-regulated intrinsic and extrinsic apoptosis. SHetA2 inhibited basal and TNF-alpha-induced or hydrogen peroxide-induced NF-kappaB activity through counter-regulation of upstream kinase (IkappaB kinase) activity, inhibitor protein (IkappaB-alpha) phosphorylation, and p-65 NF-kappaB subunit nuclear translocation, but independently of reactive oxygen species generation. Ectopic over-expression of p-65, or treatment with TNF-alpha receptor 1 (TNFR1) small interfering RNA or a caspase-8 inhibitor, each attenuated synergistic apoptosis by SHetA2 and TNF-alpha, but did not affect intrinsic apoptosis caused by SHetA2. In conclusion, NF-kappaB repression is involved in SHetA2 circumvention of resistance to TNF-alpha-induced extrinsic apoptosis, but not in SHetA2 induction of intrinsic apoptosis.

  2. TNF-alpha, leptin, and lymphocyte function in human aging

    DEFF Research Database (Denmark)

    Bruunsgaard, H.; Pedersen, Agnes Nadelmann; Schroll, M.

    2000-01-01

    Aging is associated with increased inflammatory activity and concomitant decreased T cell mediated immune responses. Leptin may provide a link between inflammation and T cell function in aging. The aim of the study was to investigate if plasma levels of tumor necrosis factor (TNF)-alpha were...... there was no difference with regard to IL-2 production. Furthermore, there were no age-related differences in serum levels of leptin, However, women had higher levels than men. In the elderly people, serum levels of leptin were correlated with TNF-alpha in univariate regression analysis and in a multiple linear...... regression analysis adjusting for the effect of gender and body mass index. Furthermore, TNF-alpha, but not leptin, was positively correlated to sIL-2R and negatively correlated to IL-2 production. In conclusion, increased plasma levels of TNF-alpha in aging is associated with poor IL-2 production ex vivo...

  3. Pro-Resolving Effects of Resolvin D2 in LTD4 and TNF-α Pre-Treated Human Bronchi.

    Directory of Open Access Journals (Sweden)

    Rayan Khaddaj-Mallat

    Full Text Available Inflammation is a major burden in respiratory diseases, resulting in airway hyperresponsiveness. Our hypothesis is that resolution of inflammation may represent a long-term solution in preventing human bronchial dysfunctions. The aim of the present study was to assess the anti-inflammatory effects of RvD2, a member of the D-series resolving family, with concomitant effects on ASM mechanical reactivity. The role and mode of action of RvD2 were assessed in an in vitro model of human bronchi under pro-inflammatory conditions, induced either by 1 μM LTD4 or 10 ng/ml TNF-α pre-treatment for 48h. TNF-α and LTD4 both induced hyperreactivity in response to pharmacological stimuli. Enhanced 5-Lipoxygenase (5-LOX and cysteinyl leukotriene receptor 1 (CysLTR1 detection was documented in LTD4 or TNF-α pre-treated human bronchi when compared to control (untreated human bronchi. In contrast, RvD2 treatments reversed 5-LOX/β-actin and CysLTR1/β-actin ratios and decreased the phosphorylation levels of AP-1 subunits (c-Fos, c-Jun and p38-MAP kinase, while increasing the detection of the ALX/FPR2 receptor. Moreover, various pharmacological agents revealed the blunting effects of RvD2 on LTD4 or TNF-α induced hyper-responsiveness. Combined treatment with 300 nM RvD2 and 1 μM WRW4 (an ALX/FPR2 receptor inhibitor blunted the pro-resolving and broncho-modulatory effects of RvD2. The present data provide new evidence regarding the role of RvD2 in a human model of airway inflammation and hyperrresponsiveness.

  4. Characterization of ovine hepatic gene expression profiles in response to Escherichia coli lipopolysaccharide using a bovine cDNA microarray

    Directory of Open Access Journals (Sweden)

    Boermans Herman J

    2006-11-01

    Full Text Available Abstract Background During systemic gram-negative bacterial infections, lipopolysaccharide (LPS ligation to the hepatic Toll-like receptor-4 complex induces the production of hepatic acute phase proteins that are involved in the host response to infection and limit the associated inflammatory process. Identifying the genes that regulate this hepatic response to LPS in ruminants may provide insight into the pathogenesis of bacterial diseases and eventually facilitate breeding of more disease resistant animals. The objective of this research was to profile the expression of ovine hepatic genes in response to Escherichia coli LPS challenge (0, 200, 400 ng/kg using a bovine cDNA microarray and quantitative real-time PCR (qRT-PCR. Results Twelve yearling ewes were challenged iv with E. coli LPS (0, 200, 400 ng/kg and liver biopsies were collected 4–5 hours post-challenge to assess hepatic gene expression profiles by bovine cDNA microarray and qRT-PCR analyses. The expression of CD14, C3, IL12R, NRAMP1, SOD and IGFBP3 genes was down regulated, whereas the expression of ACTHR, IFNαR, CD1, MCP-1 and GH was increased during LPS challenge. With the exception of C3, qRT-PCR analysis of 7 of these genes confirmed the microarray results and demonstrated that GAPDH is not a suitable housekeeping gene in LPS challenged sheep. Conclusion We have identified several potentially important genes by bovine cDNA microarray and qRT-PCR analyses that are differentially expressed during the ovine hepatic response to systemic LPS challenge. Their potential role in regulating the inflammatory response to LPS warrants further investigation.

  5. Activation of α7nAChR Promotes Diabetic Wound Healing by Suppressing AGE-Induced TNF-α Production.

    Science.gov (United States)

    Dong, Miao-Wu; Li, Ming; Chen, Jie; Fu, Tong-Tong; Lin, Ke-Zhi; Ye, Guang-Hua; Han, Jun-Ge; Feng, Xiang-Ping; Li, Xing-Biao; Yu, Lin-Sheng; Fan, Yan-Yan

    2016-04-01

    Diabetes frequently presents accumulation of advanced glycation end products (AGEs), which might induce excessive TNF-α production from macrophages to cause impaired wound healing. Recent studies have shown that activation of α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages efficiently suppressed TNF-α synthesis. The aim of this study was to investigate the accumulation of AGEs in the wounds and determine whether PNU282987, an α7nAChR agonist, can improve wound repair by inhibiting AGE-mediated TNF-α production in a streptozotocin (STZ)-induced diabetic mouse model. Animals were assigned into four groups: wounded control group, wounded diabetic group, wounded diabetic group treated intraperitoneally with PNU282987, or wounded diabetic group treated intraperitoneally with vehicle. Compared with the non-diabetic control mice, the diabetic mice exhibited delayed wound healing that was characterized by elevated accumulation of AGEs, increased TNF-α level and macrophage infiltration, and decreased fibroblast number and collagen deposition at the late stage of repair. Besides, macrophages of diabetic wounds showed expression of α7nAChR. During late repair, PNU282987 treatment of diabetic mice significantly reduced the level of TNF-α, accelerated wound healing, and elevated fibroblast number and collagen deposition. To investigate the cellular mechanism of these observations, RAW 264.7 cells, a macrophage cell line, were incubated with AGEs in the presence or absence of PNU282987. TNF-α production from AGE-stimulated macrophages was significantly decreased by PNU282987 in a dose-dependent manner. Furthermore, PNU282987 significantly inhibited AGE-induced nuclear factor-κB (NF-κB) activation and receptor for AGE (RAGE) expression. These results strongly suggest that activating α7nAChR can promote diabetic wound healing by suppressing AGE-induced TNF-α production, which may be closely associated with the blockage of NF-κB activation in macrophages.

  6. Safety of TNF-α inhibitors during IBD pregnancy

    DEFF Research Database (Denmark)

    Nielsen, Ole Haagen; Loftus, Edward V; Jess, Tine

    2013-01-01

    Tumor necrosis factor (TNF)-alpha inhibitors are increasingly being used in inflammatory bowel disease (IBD). Because this chronic intestinal disorder often affects women of fertile age, it is essential to assess the effect of biologics on pregnancy outcome.......Tumor necrosis factor (TNF)-alpha inhibitors are increasingly being used in inflammatory bowel disease (IBD). Because this chronic intestinal disorder often affects women of fertile age, it is essential to assess the effect of biologics on pregnancy outcome....

  7. A TNF-p100 pathway subverts noncanonical NF-κB signaling in inflamed secondary lymphoid organs.

    Science.gov (United States)

    Mukherjee, Tapas; Chatterjee, Budhaditya; Dhar, Atika; Bais, Sachendra S; Chawla, Meenakshi; Roy, Payel; George, Anna; Bal, Vineeta; Rath, Satyajit; Basak, Soumen

    2017-12-01

    Lymphotoxin-beta receptor (LTβR) present on stromal cells engages the noncanonical NF-κB pathway to mediate RelB-dependent expressions of homeostatic chemokines, which direct steady-state ingress of naïve lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF-κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection-inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non-infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naïve lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTβR-stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF-mediated inhibitions in inflamed SLOs of immunized Nfkb2 -/- mice. In sum, we reveal that an inhibitory TNF-p100 pathway modulates the adaptive compartment during immune responses. © 2017 The Authors.

  8. Discrepancy between molecular structure and ligand selectivity of a testicular follicle-stimulating hormone receptor of the African catfish (Clarias gariepinus)

    NARCIS (Netherlands)

    Bogerd, J.; Blomenröhr, M.; Andersson, E.; van der Putten, H.; Tensen, C.P.; Vischer, H F; Granneman, Joke C M; Janssen-Dommerholt, C; Goos, H.J.; Schulz, Rüdiger W

    A putative FSH receptor (FSH-R) cDNA was cloned from African catfish testis. Alignment of the deduced amino acid sequence with other (putative) glycoprotein hormone receptors and analysis of the African catfish gene indicated that the cloned receptor belonged to the FSH receptor subfamily. Catfish

  9. Expression analysis of a ''Cucurbita'' cDNA encoding endonuclease

    International Nuclear Information System (INIS)

    Szopa, J.

    1995-01-01

    The nuclear matrices of plant cell nuclei display intrinsic nuclease activity which consists in nicking supercoiled DNA. A cDNA encoding a 32 kDa endonuclease has been cloned and sequenced. The nucleotide and deduced amino-acid sequences show high homology to known 14-3-3-protein sequences from other sources. The amino-acid sequence shows agreement with consensus sequences for potential phosphorylation by protein kinase A and C and for calcium, lipid and membrane-binding sites. The nucleotide-binding site is also present within the conserved part of the sequence. By Northern blot analysis, the differential expression of the corresponding mRNA was detected; it was the strongest in sink tissues. The endonuclease activity found on DNA-polyacrylamide gel electrophoresis coincided with mRNA content and was the highest in tuber. (author). 22 refs, 6 figs

  10. The TNF-alpha transgenic mouse model of inflammatory arthritis.

    Science.gov (United States)

    Li, Ping; Schwarz, Edward M

    2003-08-01

    Rheumatoid arthritis is a chronic inflammatory disorder that affects multiple peripheral joints. It is the most common form of inflammatory arthritis and is characterized by synovial hyperplasia, immune cell infiltration, cartilage destruction, and bone erosion. To gain insight into the etiology of the disease, a variety of animal models have been established. Twelve years ago George Kollias' laboratory generated a transgenic (Tg) mouse that over-expresses human TNF-alpha, and develops an erosive polyarthritis with many characteristics observed in rheumatoid arthritis patients. The phenotype of this mouse model validated the theory that TNF-alpha is at the apex of the pro-inflammatory cascade in rheumatoid arthritis, and foreshadowed the remarkable success of anti-TNF-alpha therapy that has transformed the effective management of this disease. As such, the TNF-Tg mice are very useful tools for dissecting the molecular mechanisms of the pathogenic process and evaluating the efficacy of novel therapeutic strategies for rheumatoid arthritis. In this review we (1) provide a brief summary of TNF-alpha biology and the role of this dominant cytokine in rheumatoid arthritis, (2) describe the various TNF-Tg models and their phenotypes, and (3) give examples of how this model has been used experimentally.

  11. Inflammatory factor TNF-α promotes the growth of breast cancer via the positive feedback loop of TNFR1/NF-κB (and/or p38)/p-STAT3/HBXIP/TNFR1.

    Science.gov (United States)

    Cai, Xiaoli; Cao, Can; Li, Jiong; Chen, Fuquan; Zhang, Shuqin; Liu, Bowen; Zhang, Weiying; Zhang, Xiaodong; Ye, Lihong

    2017-08-29

    In the connection between inflammation and cancer development, tumor necrosis factor-alpha (TNF-α) contributes to the tumorigenesis. However, the underlying mechanism remains poorly understood. In this study, we report that TNF-α enhances the growth of breast cancer through up-regulation of oncoprotein hepatitis B X-interacting protein (HBXIP). Our data showed that the levels of TNF-α were positively related to those of HBXIP in clinical breast cancer tissues. Moreover, TNF-α could up-regulate HBXIP in breast cancer cells. Interestingly, silencing of TNFreceptor 1 (TNFR1) blocked the effect of TNF-α on HBXIP. Mechanistically, we revealed that TNF-α could increase the activities of HBXIP promoter through activating transcriptional factor signal transducer and activator of transcription 3 (STAT3). In addition, nuclear factor kappa B (NF-κB) and/or p38 signaling increased the levels of p-STAT3 in the cells. Strikingly, HBXIP could also up-regulate TNFR1, forming a positive feedback loop of TNFR1/NF-κB (and/or p38)/p-STAT3/HBXIP/TNFR1. Notably, TNF-α was able to up-regulate TNFR1 through driving the loop. In function, we demonstrated that the knockdown of HBXIP remarkably abolished the growth of breast cancer mediated by TNF-α in vitro and in vivo . Thus, we conclude that TNF-α promotes the growth of breast cancer through the positive feedback loop of TNFR1/NF-κB (and/or p38)/p-STAT3/HBXIP/TNFR1.Our finding provides new insights into the mechanism by which TNF-α drives oncoprotein HBXIP in the development of breast cancer.

  12. Probing the mechanical properties of TNF-α stimulated endothelial cell with atomic force microscopy

    Directory of Open Access Journals (Sweden)

    Sei-Young Lee

    2011-01-01

    Full Text Available Sei-Young Lee1,2, Ana-Maria Zaske3, Tommaso Novellino1,4*, Delia Danila3, Mauro Ferrari1,5*, Jodie Conyers3, Paolo Decuzzi1,6*1Department of Nanomedicine and Biomedical Engineering, The University of Texas Medical School at Houston, Houston, TX, USA; 2Department of Mechanical Engineering, The University of Texas at Austin, Austin, TX, USA; 3CeTIR – Center for Translational Injury Research, The University of Texas Health Science Center at Houston, Houston, TX, USA; 4Department of Biomedical Engineering, Biomedical Campus University of Rome, Italy; 5MD Anderson Cancer Center, Houston, TX, USA; 6BioNEM – Center of Bio-Nanotechnology and Engineering for Medicine, University of Magna Graecia, Catanzaro, Italy; *Currently at Department of Nanomedicine and Biomedical Engineering, The Methodist Hospital Research Institute, Houston, TX, USAAbstract: TNF-α (tumor necrosis factor-α is a potent pro-inflammatory cytokine that regulates the permeability of blood and lymphatic vessels. The plasma concentration of TNF-α is elevated (> 1 pg/mL in several pathologies, including rheumatoid arthritis, atherosclerosis, cancer, pre-eclampsia; in obese individuals; and in trauma patients. To test whether circulating TNF-α could induce similar alterations in different districts along the vascular system, three endothelial cell lines, namely HUVEC, HPMEC, and HCAEC, were characterized in terms of 1 mechanical properties, employing atomic force microscopy; 2 cytoskeletal organization, through fluorescence microscopy; and 3 membrane overexpression of adhesion molecules, employing ELISA and immunostaining. Upon stimulation with TNF-α (10 ng/mL for 20 h, for all three endothelial cells, the mechanical stiffness increased by about 50% with a mean apparent elastic modulus of E ~5 ± 0.5 kPa (~3.3 ± 0.35 kPa for the control cells; the density of F-actin filaments increased in the apical and median planes; and the ICAM-1 receptors were overexpressed compared with

  13. Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA cloning

    International Nuclear Information System (INIS)

    Han, J.H.; Stratowa, C.; Rutter, W.J.

    1987-01-01

    The authors have cloned a full-length putative rat pancreatic lysophospholipase cDNA by an improved mRNA isolation method and cDNA cloning strategy using [ 32 P]-labelled nucleotides. These new methods allow the construction of a cDNA library from the adult rat pancreas in which the majority of recombinant clones contained complete sequences for the corresponding mRNAs. A previously recognized but unidentified long and relatively rare cDNA clone containing the entire sequence from the cap site at the 5' end to the poly(A) tail at the 3' end of the mRNA was isolated by single-step screening of the library. The size, amino acid composition, and the activity of the protein expressed in heterologous cells strongly suggest this mRNA codes for lysophospholipase

  14. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  15. Effect of PPAR-α and -γ agonist on the expression of visfatin, adiponectin, and TNF-α in visceral fat of OLETF rats

    International Nuclear Information System (INIS)

    Choi, K.C.; Ryu, O.H.; Lee, K.W.; Kim, H.Y.; Seo, J.A.; Kim, S.G.; Kim, N.H.; Choi, D.S.; Baik, S.H.; Choi, K.M.

    2005-01-01

    A variety of adipocytokines and peptides secreted from adipocytes have been considered to play a crucial role in obesity, insulin resistance, and type 2 diabetes. Recently, visfatin, a new adipocytokine, known as a pre-B cell colony-enhancing factor, has been isolated from visceral fat deposits. It has been shown to activate insulin receptors in a manner different from insulin. To understand the role of adipocytokines in improving insulin sensitivity via activation of the nuclear receptor peroxisome proliferator-activated receptor-α (PPAR-α) and -γ (PPAR-γ), we examined the expression of visfatin, adiponectin, and TNF-α in visceral fat depots of Otsuka Long-Evans Tokushima fatty (OLETF) rats from early to advanced diabetic stage (from 28 to 40 weeks of age). Serum glucose and insulin concentrations significantly (P < 0.05) decreased in rosiglitazone or fenofibrate-treated OLETF rats compared to untreated OLETF rats. Rosiglitazone significantly increased serum adiponectin concentration from 20 to 40 weeks of age (P < 0.05), whereas fenofibrate reduced TNF-α concentration. The expression of visfatin and adiponectin mRNA in visceral fat deposits was elevated by rosiglitazone or fenofibrate treatments when compared to untreated OLETF rats (P < 0.05), whereas, TNF-α mRNA was down-regulated by these drugs (P < 0.05). These results suggest that rosiglitazone and fenofibrate may prevent type 2 diabetes by regulating adipocytokines including visfatin, adiponectin, and TNF

  16. Construction and analysis of a cDNA library from yellow-fruit ginseng

    African Journals Online (AJOL)

    The total RNA was isolated from yellow-fruit ginseng (Panax ginseng C.A. Meyer) leaf tissue. A cDNA library of panax ginseng leaves was constructed by using pDNR-LIB vector according to the SMART cDNA library construction kit protocol. We obtained 378 high quality sequences (GenBank accession number: ...

  17. cDNA table - RPD | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available switchLanguage; BLAST Search Image Search Home About Archive Update History Data ...ile URL: ftp://ftp.biosciencedbc.jp/archive/rpd/LATEST/rpd_cdna.zip File size: 15 KB Simple search URL http:...age About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA table - RPD | LSDB Archive ...

  18. [Construction of cDNA library of Magnaporthe grisea with magnetic bead].

    Science.gov (United States)

    Feng, Xu; Xiaoli, Wu; Dewen, Qiu

    2008-06-01

    We constructed cDNA library of Magnaporthe grisea. The good quality cDNA library could facilitate finding proteinaceous elicitors of M. grisea, and elucidating the mechanisms of the M. grisea--rice interaction. The Oligo(dT) combined with the magnetic bead was used to extract mRNA from total RNA of Magnaporthe grisea and as primers to synthesize the first-strand cDNA. Terminal transferase introduced PolyA into 3'terminal of the first cDNA strand, then the PolyA was used for amplifying the second-strand cDNA. Restriction enzyme and adapter were avoided in this research, which could solve technical limitation of the traditional method. Because all reactions were done in one centrifuge tube, this process could reduce the risk of cDNA loss and cross-contamination. The primers designed in this research could clone the amplified cDNAs into expression vector in a desirable orientation. The cDNA library constructed had a high titer of 8.9 x 10(6) cfu/mL, and contained a total clones of 8.9 x 10(7) cfu, with an average inserts size of about 1380 bp. Constructing cDNA library with magnetic bead was a highly efficient method using only small amount of experimental materials within a short period.

  19. Development of a simple and powerful method, cDNA AFLP-SSPAG ...

    African Journals Online (AJOL)

    Differential cDNAs were easily obtained from silver stained cDNA-AFLP separated on polyacylamide gels. The cDNA was then reamplified, cloned and fragments were sequenced. Sequenced clones were used as probes in northern dot blot analyses and library screening. Full-length cDNA was cloned from a library ...

  20. Genomic and cDNA cloning of a novel mouse lipoxygenase gene

    NARCIS (Netherlands)

    Willems van Dijk, K.; Steketee, K.; Havekes, L.; Frants, R.; Hofker, M.

    1995-01-01

    A novel 12- and 15-lipoxygenase related gene was isolated from a mouse strain 129 genomic phage library in a screen with a human 15-lipoxygenase cDNA probe. The complete genomic sequence revealed 14 exons and 13 introns covering 7.3 kb of DNA. The splice junctions were verified from the cDNA

  1. Construction of full-length cDNA library of white flower Salvia ...

    African Journals Online (AJOL)

    In order to screen and isolate secondary metabolite biosynthesis related gene, we construct a cDNA library of white flower Salvia miltiorrhiza bge. f.alba. High quality of total RNA was successfully isolated from roots of white flower S. miltiorrhiza using modified CTAB method. Double strand cDNA was cloned into pDNR-LIB ...

  2. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    International Nuclear Information System (INIS)

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M.

    1989-01-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K m , low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus

  3. Cloning and sequence analysis of H. contortus HC58cDNA gene ...

    African Journals Online (AJOL)

    Phylogenetic analysis revealed close evolutionary proximity of the protein sequence to counterpart sequences in the cathepsin B like proteases, suggesting that HC58cDNA was a member of the papain family. Keywords:Haemonchus contortus, HC58cDNA, cathepsin B like protease, papain family. Kenya Veterinarian Vol.

  4. [A novel vector for construction of a cDNA library].

    Science.gov (United States)

    Fedchenko, V I; Kaloshin, A A; Medvedev, A E

    2010-01-01

    A new original vector pEM-(dT)40(f+) has been prepared. It can be used for cDNA library construction from polyadenylated mRNA, isolated from various sources. The pGEM-(dT)40f(+) is initially transformed into single stranded and then into a linear form and its (dT)40 tail at 3'-end is used as the vector-primer for synthesis of the first strand cDNA. The use of a synthetic oligonucleotide complementary to the vector and recombinant DNA results in vector cyclization and synthesis of the second strand cDNA. This approach significantly simplifies cDNA library construction, it does not require PCR reaction (which can induce artifact mutations in cDNA sequences) and restrictase treatment.

  5. Immune complexes induce TNF-α and BAFF production from U937 cells by HMGB1 and RAGE.

    Science.gov (United States)

    Gao, X-J; Qu, Y-Y; Liu, X-W; Zhu, M; Ma, C-Y; Jiao, Y-L; Cui, B; Chen, Z-J; Zhao, Y-R

    2017-04-01

    This study investigated the effects of immune complexes (ICs) on tumor necrosis factor α (TNF-α) and B cell-activating factor (BAFF) production from U937 cells and further explored the mechanism. U937 cells were incubated with necrosis supernatant or systemic lupus erythematosus (SLE) sera alone, or their combination. The expression of TNF-α and BAFF was determined by Real-time polymerase chain reaction and enzyme-linked immunosorbent assay. High mobility group box protein 1(HMGB1) A-box was produced by gene recombination. HMGB1 A-box and anti-receptor for advanced glycation end products (RAGE) antibody were adopted in the blocking experiments. The importance of DNA for cytokine induction was investigated by DNase treatment. The combination of necrosis supernatant and SLE sera induced the expression of TNF-α and BAFF significantly increased compared to necrosis supernatant or SLE sera alone. Recombinant HMGB1 A-box protein was purified, and TNF-α and BAFF production, which were induced by this combination, was blocked via HMGB1 A-box and anti-RAGE antibody. Moreover, we found that DNA component is important for the immunostimulatory activity of this combination. ICs containing DNA can promote TNF-α and BAFF production in U937 cells, and this process can be mediated by HMGB1 and RAGE. One possible mechanism of increasing BAFF production in SLE is proposed in this study whereby B cell activation, antibody production and ICs stimulated monocytes may create a vicious cycle that leads to B cell hyperactivity, which can be of importance for SLE etiopathogenesis.

  6. Blood-Brain Barrier Penetrating Biologic TNF-α Inhibitor for Alzheimer's Disease.

    Science.gov (United States)

    Chang, Rudy; Knox, Jillian; Chang, Jae; Derbedrossian, Aram; Vasilevko, Vitaly; Cribbs, David; Boado, Ruben J; Pardridge, William M; Sumbria, Rachita K

    2017-07-03

    Tumor necrosis factor alpha (TNF-α) driven processes are involved at multiple stages of Alzheimer's disease (AD) pathophysiology and disease progression. Biologic TNF-α inhibitors (TNFIs) are the most potent class of TNFIs but cannot be developed for AD since these macromolecules do not cross the blood-brain barrier (BBB). A BBB-penetrating TNFI was engineered by the fusion of the extracellular domain of the type II human TNF receptor (TNFR) to a chimeric monoclonal antibody (mAb) against the mouse transferrin receptor (TfR), designated as the cTfRMAb-TNFR fusion protein. The cTfRMAb domain functions as a molecular Trojan horse, binding to the mouse TfR and ferrying the biologic TNFI across the BBB via receptor-mediated transcytosis. The aim of the study was to examine the effect of this BBB-penetrating biologic TNFI in a mouse model of AD. Six-month-old APPswe, PSEN 1dE9 (APP/PS1) transgenic mice were treated with saline (n = 13), the cTfRMAb-TNFR fusion protein (n = 12), or etanercept (non-BBB-penetrating biologic TNFI; n = 11) 3 days per week intraperitoneally. After 12 weeks of treatment, recognition memory was assessed using the novel object recognition task, mice were sacrificed, and brains were assessed for amyloid beta (Aβ) load, neuroinflammation, BBB damage, and cerebral microhemorrhages. The cTfRMAb-TNFR fusion protein caused a significant reduction in brain Aβ burden (both Aβ peptide and plaque), neuroinflammatory marker ICAM-1, and a BBB disruption marker, parenchymal IgG, and improved recognition memory in the APP/PS1 mice. Fusion protein treatment resulted in low antidrug-antibody formation with no signs of either immune reaction or cerebral microhemorrhage development with chronic 12-week treatment. Chronic treatment with the cTfRMAb-TNFR fusion protein, a BBB-penetrating biologic TNFI, offers therapeutic benefits by targeting Aβ pathology, neuroinflammation, and BBB-disruption, overall improving recognition memory in a transgenic mouse model

  7. Role of xanthine oxidase and reactive oxygen intermediates in LPS- and TNF-induced pulmonary edema.

    Science.gov (United States)

    Faggioni, R; Gatti, S; Demitri, M T; Delgado, R; Echtenacher, B; Gnocchi, P; Heremans, H; Ghezzi, P

    1994-03-01

    We studied the role of reactive oxygen intermediates (ROI) in lipopolysaccharide (LPS)-induced pulmonary edema. LPS treatment (600 micrograms/mouse, IP) was associated with a marked induction of the superoxide-generating enzyme xanthine oxidase (XO) in serum and lung. Pretreatment with the antioxidant N-acetylcysteine (NAC)--1 gm/kg orally, 45 minutes before LPS--or with the XO inhibitor allopurinol (AP)--50 mg/kg orally at -1 hour and +3 hours--was protective. On the other hand nonsteroidal antiinflammatory drugs (ibuprofen, indomethacin, and nordihydroguaiaretic acid) were ineffective. These data suggested that XO might be involved in the induction of pulmonary damage by LPS. However, treatment with the interferon inducer polyriboinosylic-polyribocytidylic acid, although inducing XO to the same extent as LPS, did not cause any pulmonary edema, indicating that XO is not sufficient for this toxicity of LPS. To define the possible role of cytokines, we studied the effect of direct administration of LPS (600 micrograms/mouse, IP), tumor necrosis factor (TNF, 2.5 or 50 micrograms/mouse, IV), interleukin-1 (IL-1 beta, 2.5 micrograms/mouse, IV), interferon-gamma (IFN-gamma, 2.5 micrograms/mouse, IV), or their combination at 2.5 micrograms each. In addition to LPS, only TNF at the highest dose induced pulmonary edema 24 hours later. LPS-induced pulmonary edema was partially inhibited by anti-IFN-gamma antibodies but not by anti-TNF antibodies, anti-IL-1 beta antibodies, or IL-1 receptor antagonist (IL-1Ra).

  8. Ghrelin Receptor in Two Species of Anuran Amphibian, Bullfrog (Rana catesbeiana), and Japanese Tree Frog (Hyla japonica)

    OpenAIRE

    Kaiya, Hiroyuki; Koizumi, Yasushi; Konno, Norifumi; Yamamoto, Kazutoshi; Uchiyama, Minoru; Kangawa, Kenji; Miyazato, Mikiya

    2011-01-01

    We identified cDNA encoding a functional growth hormone secretagogue-receptor 1a (GHS-R1a, ghrelin receptor) in two species of anuran amphibian, bullfrog (Rana catesbeiana) and Japanese tree frog (Hyla japonica). Deduced receptor protein for bullfrog and Japanese tree frog (tree frog) was comprised of 374- and 371-amino acids, respectively. The two receptors showed 86% identity with each other, and phylogenetic analysis revealed that the two receptors belong to the same category with tetrapod...

  9. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K

    1996-01-01

    the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine...... density lipoprotein receptor gene family receptors, and 3) six tandemly arranged fibronectin type III repeats also found in certain neural adhesion proteins. sorLA-1 may therefore be classified as a hybrid receptor. Northern blotting revealed specific mRNA transcripts in brain, spinal cord, and testis...

  10. The NRAMPI, VDR and TNF-α gene polymorphisms in Iranian tuberculosis patients: the study on host susceptibility

    Directory of Open Access Journals (Sweden)

    Muayad Merza

    Full Text Available The natural resistance-associated macrophage protein (NRAMP1, Vitamin-D receptor (VDR and Tumor necrosis factor (TNF-α have been associated in susceptibility to tuberculosis, but the results have been inconsistent. This study aimed to determine the association of NRAMP1, VDR, and TNF-á variant with development of pulmonary tuberculosis (PTB among Iranian patients. The single nucleotide polymorphisms (SNPs at INT4, D543, 3'UTR of NRAMP1 gene, SNPs in restriction sites of BsmI, and FokI of the VDR gene and SNPs of TNF-α at -238,-308, -244,857,-863 positions were analyzed by PCR-RFLP among two groups of individual; patients with PTB (n=117 and healthy controls (n=60. Thereafter, the frequencies of extended haplotypes and diplotypes were estimated. No statistically significant differences were observed in allele frequencies of INT4, D543, 3'UTR of NRAMPI, FokI of VDR and TNF-α at -238, -244,-863 and -857 position. Although, the frequency of b allele of BsmI [ORs: 0.24 CI95% (0.07-0.67 (p=0.001] and -308 A variant in TNF-α promoter region [ORs:0.26 CI95%( 0.07-0.77 (p=0.006] were significantly more in PTB patients than healthy controls. The frequency of extended diplotypes of NRAMP [GG TGTG++GA; 0.02(0.001-0.0035], VDR [FFBB; 0.2(0.6-0.6] and TNF-α [CCCCGGGGGG; 0.49(0.25-0.97] were statistically different in patients and control subjects (p<0.05. This study confirmed the association of SNPs in BsmI (B/b + b/b of VDR and SNPs in -308A (G/A +G/G of TNF-α genes with susceptibility to tuberculosis in Iranian PTB patients. Furthermore, the extended haplotypes and diplotypes analysis can be considered as an alternative way to determine the host susceptibility to TB.

  11. TNF-TNFR2/p75 signaling inhibits early and increases delayed nontargeted effects in bone marrow-derived endothelial progenitor cells.

    Science.gov (United States)

    Sasi, Sharath P; Song, Jin; Park, Daniel; Enderling, Heiko; McDonald, J Tyson; Gee, Hannah; Garrity, Brittany; Shtifman, Alexander; Yan, Xinhua; Walsh, Kenneth; Natarajan, Mohan; Kishore, Raj; Goukassian, David A

    2014-05-16

    TNF-α, a pro-inflammatory cytokine, is highly expressed after being irradiated (IR) and is implicated in mediating radiobiological bystander responses (RBRs). Little is known about specific TNF receptors in regulating TNF-induced RBR in bone marrow-derived endothelial progenitor cells (BM-EPCs). Full body γ-IR WT BM-EPCs showed a biphasic response: slow decay of p-H2AX foci during the initial 24 h and increase between 24 h and 7 days post-IR, indicating a significant RBR in BM-EPCs in vivo. Individual TNF receptor (TNFR) signaling in RBR was evaluated in BM-EPCs from WT, TNFR1/p55KO, and TNFR2/p75KO mice, in vitro. Compared with WT, early RBR (1-5 h) were inhibited in p55KO and p75KO EPCs, whereas delayed RBR (3-5 days) were amplified in p55KO EPCs, suggesting a possible role for TNFR2/p75 signaling in delayed RBR. Neutralizing TNF in γ-IR conditioned media (CM) of WT and p55KO BM-EPCs largely abolished RBR in both cell types. ELISA protein profiling of WT and p55KO EPC γ-IR-CM over 5 days showed significant increases in several pro-inflammatory cytokines, including TNF-α, IL-1α (Interleukin-1 alpha), RANTES (regulated on activation, normal T cell expressed and secreted), and MCP-1. In vitro treatments with murine recombinant (rm) TNF-α and rmIL-1α, but not rmMCP-1 or rmRANTES, increased the formation of p-H2AX foci in nonirradiated p55KO EPCs. We conclude that TNF-TNFR2 signaling may induce RBR in naïve BM-EPCs and that blocking TNF-TNFR2 signaling may prevent delayed RBR in BM-EPCs, conceivably, in bone marrow milieu in general. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. Construction and characterization of a normalized cDNA library.

    Science.gov (United States)

    Soares, M B; Bonaldo, M F; Jelene, P; Su, L; Lawton, L; Efstratiadis, A

    1994-09-27

    We have developed a simple procedure based on reassociation kinetics that can reduce effectively the high variation in abundance among the clones of a cDNA library that represent individual mRNA species. For this normalization, we used as a model system a library of human infant brain cDNAs that were cloned directionally into a phagemid vector and, thus, could be easily converted into single-stranded circles. After controlled primer extension to synthesize a short complementary strand on each circular template, melting and reannealing of the partial duplexes at relatively low C0t, and hydroxyapatite column chromatography, unreassociated circles were recovered from the flow through fraction and electroporated into bacteria, to propagate a normalized library without a requirement for subcloning steps. An evaluation of the extent of normalization has indicated that, from an extreme range of abundance of 4 orders of magnitude in the original library, the frequency of occurrence of any clone examined in the normalized library was brought within the narrow range of only 1 order of magnitude.

  13. Targeting cellular adhesion molecules, chemokines and chemokine receptors in rheumatoid arthritis

    NARCIS (Netherlands)

    Haringman, Jasper J.; Oostendorp, Roos L.; Tak, Paul P.

    2005-01-01

    The development of specific targeted therapies, such as anti-TNF-alpha treatment, for chronic inflammatory disorders such as rheumatoid arthritis, has significantly improved treatment, although not all patients respond. Targeting cellular adhesion molecules and chemokines/chemokine receptors as

  14. Sensitization of vascular smooth muscle cell to TNF-α-mediated death in the presence of palmitate

    International Nuclear Information System (INIS)

    Rho, Mun-Chual; Ah Lee, Kyeong; Mi Kim, Sun; Sik Lee, Chang; Jeong Jang, Min; Kook Kim, Young; Sun Lee, Hyun; Hyun Choi, Yung; Yong Rhim, Byung; Kim, Koanhoi

    2007-01-01

    Saturated free fatty acids (FFAs), including palmitate, can activate the intrinsic death pathway in cells. However, the relationship between FFAs and receptor-mediated death pathway is still unknown. In this study, we have investigated whether FFAs are able to trigger receptor-mediated death. In addition, to clarify the mechanisms responsible for the activation, we examined the biochemical changes in dying vascular smooth muscle cell (VSMC) and the effects of various molecules to the receptor-mediated VSMC death. Tumor necrosis factor (TNF)-α-mediated VSMC death occurred in the presence of sub-cytotoxic concentration of palmitate as determined by assessing viability and DNA degradation, while the cytokine did not influence VSMC viability in the presence of oleate. The VSMC death was inhibited by the gene transfer of a dominant-negative Fas-associated death domain-containing protein and the baculovirus p35, but not by the bcl-xL or the c-Jun N-terminal kinase (JNK) binding domain of JNK-interacting protein-1, in tests utilizing recombinant adenoviruses. The VSMC death was also inhibited by a neutralizing anti-TNF receptor 1 antibody, the caspase inhibitor z-VAD, and the cathepsin B inhibitor CA074, a finding indicative of the role of both caspases and cathepsin B in this process. Consistent with this finding, caspase-3 activation and an increase in cytosolic cathepsin B activity were detected in the dying VSMC. Palmitate inhibited an increase of TNF-α-mediated nuclear factor kappa B (NF-κB) activity, the survival pathway activated by the cytokine, by hindering the translocation of the NF-κB subunit of p65 from the cytosol into the nucleus. The gene transfer of inhibitor of NF-κB predisposed VSMC to palmitate-induced cell death. To the best of our knowledge, this study is the first report to demonstrate the activation of TNF-α-mediated cell death in the presence of palmitate. The current study proposes that FFAs would take part in deleterious vascular

  15. Construction and analysis of SSH cDNA library of human vascular endothelial cells related to gastrocarcinoma

    OpenAIRE

    Liu, Yong-Bo; Wei, Zhao-Xia; Li, Li; Li, Hang-Sheng; Chen, Hui; Li, Xiao-Wen

    2003-01-01

    AIM: To construct subtracted cDNA libraries of human vascular endothelial cells (VECs) related to gastrocarcinoma using suppression substractive hybridization (SSH) and to analyze cDNA libraries of gastrocarcinoma and VECs in Cancer Gene Anatomy Project (CGAP) database.

  16. Effect of deficiency of tumor necrosis factor alpha or both of its receptors on Streptococcus pneumoniae central nervous system infection and peritonitis.

    Science.gov (United States)

    Wellmer, A; Gerber, J; Ragheb, J; Zysk, G; Kunst, T; Smirnov, A; Brück, W; Nau, R

    2001-11-01

    Tumor necrosis factor alpha (TNF-alpha) and TNF-beta are key mediators in bacterial inflammation. We therefore examined the role of TNF-alpha and its two receptors in murine pneumococcal central nervous system infection. TNF-alpha knockout mice and age- and sex-matched controls and TNF receptor (p55 and p75)-deficient mice and heterozygous littermates were infected intracerebrally with a Streptococcus pneumoniae type 3 strain. Mice were monitored until death or were killed 36 h after infection. Bacterial titers in blood, spleen, and brain homogenates were determined. Leukocyte infiltration and neuronal damage were assessed by histological scores. TNF-alpha-deficient mice died earlier than the controls after intracerebral infection although overall survival was similar. TNF-alpha deficiency did not inhibit leukocyte recruitment into the subarachnoid space and did not lead to an increased density of bacteria in brain homogenates. However, it caused a substantial rise of the concentration of S. pneumoniae cells in blood and spleen. Spleen bacterial titers were also increased in p55- and p75-deficient mice. TNF receptor-deficient mice showed decreased meningeal inflammation. Neuronal damage was not affected by either TNF-alpha or TNF receptor deficiency. In a murine model of pneumococcal peritonitis, 10(2) CFU of S. pneumoniae produced fatal peritonitis in TNF-alpha-deficient, but not wild-type, mice. Early leukocyte influx into the peritoneum was impaired in TNF-alpha-deficient mice. The lack of TNF-alpha or its receptors renders mice more susceptible to S. pneumoniae infections.

  17. Cathepsin-D And Tnf-α in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    T. Salman

    1996-01-01

    Full Text Available In a study of 34 normal healthy controls, 35 patients with urinary tract bilharziasis and 93 bladder cancer patients (62 of them are operable cases and 31 are non-operable ones, serum tumor necrosis factor alpha (TNF-α and cytosolic Cathepsin-D were estimated. Though both potential markers were elevated in bladder cancer patients, neither Cathepsin-D nor TNF-α showed associations of prognostic value since there were no positive correlations with tumor stages, grades or association of tumors with bilharzia ova or lymph node involvement.

  18. cDNA amplification by SMART-PCR and suppression subtractive hybridization (SSH)-PCR.

    Science.gov (United States)

    Hillmann, Andrew; Dunne, Eimear; Kenny, Dermot

    2009-01-01

    The comparison of two RNA populations that differ from the effects of a single-independent variable, such as a drug treatment or a specific genetic defect, can identify differences in the abundance of specific transcripts that vary in a population-dependent manner. There are a variety of methods for identifying differentially expressed genes, including microarray, SAGE, qRT-PCR, and DDGE. This protocol describes a potentially less sensitive yet relatively easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under investigation and is particularly applicable when minimal levels of starting material, RNA, are available. RNA input can often be a limiting factor when analyzing RNA from, for example, rigorously purified blood cells. This protocol describes the use of SMART-PCR to amplify cDNA from sub-microgram levels of RNA. The amplified cDNA populations under comparison are then subjected to suppression subtractive hybridization (SSH-PCR), a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The final products are cDNA populations enriched for significantly over-represented transcripts in either of the two input RNA preparations. These cDNA populations may then be cloned to make subtracted cDNA libraries and/or used as probes to screen subtracted cDNA, global cDNA, or genomic DNA libraries.

  19. [Construction of a cDNA library from liver tissue of rhesus monkey, Macaca mulatta].

    Science.gov (United States)

    Qin, Sheng-fang; Tan, Wei-dong; Chen, You-nan; Ding, Yang; Li, Sheng-fu; Li, Hong-xia; Wang, Li; Yang, Rong; Lu, Yan-rong

    2007-06-01

    To screen the target rhesus genes and give some basic genetic evidences to its value as one of the most important animal model in biomedical study, we constructed a cDNA expression library from liver tissue of a healthy rhesus monkey. With Trizol reagent, the total RNA was extracted from healthy rhesus liver tissue. By mutant Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT), the first-strand cDNA was synthesized from purified mRNA, and subsequently the second-strand cDNA was generated via E. coli DNA polymerase I . Then, the EcoR I adapter was added to the synthesized double-strand cDNA, which was subsequently digested by Xho I restriction enzyme and fractionated with CHROMA APIN-400 column. The fractionated cDNA fragments to be longer than 0. 5 kb were ligated into lambda ZAP express vector to form the phagemid cDNA recombinants, which were further packaged into the lambda ZAP cDNA library according to the standard protocol with phage lambda Gold packaging extract. In order to get more stable clones with larger quantity, the primary library was amplified through infecting the host strain XL1-Blue MRF'. Then, the library titre, recombinant rate and length of inserted cDNA were measured, respectively. The capacity of the primary stand or unamplified library was 1. 2X 10(6) pfu. The titers of the unamplified library or the amplified library was 1.1 X 10(6) mixture, pfu/mL or 7. 7 X 10(9) pfu/mL respectively, the percentages of recombinants were 99. 3% and 98. 2%, and the average lengths of the inserts were 2.0 kb and 2. 3 kb, respectively. An excellent cDNA expression library has been constructed successfully, which would lay solid foundation for transplantation study and pre-clinic evaluation of related drugs.

  20. [cDNA libraries construction and screening in gene expression profiling of disease resistance in wheat].

    Science.gov (United States)

    Luo, Meng; Kong, Xiu-Ying; Liu, Yue; Zhou, Rong-Hua; Jia, Ji-Zeng

    2002-09-01

    A wheat line, Bai Nong 3217/Mardler BC5F4 with resistance to powdery mildew, was used to construct a conventional cDNA library and a suppression subtractive hybridization (SSH) cDNA library from wheat leaves inoculated by Erysiphe graminis DC. Three hundred and eighty-seven non-redundant ESTs from the conventional cDNA library and 760 ESTs from the SSH cDNA library were obtained, and the ESTs similarity analysis using BLASTn and BLASTx were conducted by comparing these ESTs with sequences in GenBank. The results showed that the redundancy of some kinds of genes such as photosynthesis related genes and ribosome related genes was higher in the conventional cDNA library but the varieties and quantities of disease resistance genes were less than those in the SSH cDNA library. The SSH cDNA library was found to have obvious advantages in gene expression profiling of disease resistance such as simple library construction procedure, rich specific DRR (disease-resistance-related) genes and decreased sequencing amount. To acquire genes that were involved in the powdery mildew resistance of wheat, hybridization with high-density dots membranes was used to screen the two libraries. The result showed that the method was relatively simple in operation, and the membranes could be used for many times. But some problems also existed with this screening method. For instance, a large amount of mRNA and radioactive isotope were needed and the hybridization procedure must be repeated several times to obtain stable hybridization results. About 54.1% function-known ESTs in the SSH cDNA library were identified to be DRR genes by screening. There were 247 clones of the SSH cDNA library that had positive signal in the repeated hybridizations with the pathogen uninfected probe. The identified DRR genes distributed in the whole procedure of powdery mildew resistance, but mainly focused on the SAR (systemic of acquired resistance).

  1. Chronicles of a death foretold: dual sequential cell death checkpoints in TNF signaling.

    Science.gov (United States)

    O'Donnell, Marie Anne; Ting, Adrian T

    2010-03-15

    The kinase RIP1 wears a coat of many colors during TNF receptor signaling and can regulate both activation of pro-survival NFkB and programmed cell death pathways. In this review, we outline how coating RIP1 with K63-linked ubiquitin chains forms a protective layer that prevents RIP1 from binding apoptotic regulators and serves as an early guard against cell death. Further on, binding of NFkB signaling components to the ubiquitin coat of RIP1 activates long-term pro-survival signaling and forms a more impenetrable suit of armor against cell death. If RIP1 is not decorated with ubiquitin chains it becomes an unstoppable harbinger of bad news: programmed cell death.

  2. Isolation of cDNA clones coding for human tissue factor: primary structure of the protein and cDNA

    International Nuclear Information System (INIS)

    Spicer, E.K.; Horton, R.; Bloem, L.

    1987-01-01

    Tissue factor is a membrane-bound procoagulant protein that activates the extrinsic pathway of blood coagulation in the presence of factor VII and calcium. λ Phage containing the tissue factor gene were isolated from a human placental cDNA library. The amino acid sequence deduced from the nucleotide sequence of the cDNAs indicates that tissue factor is synthesized as a higher molecular weight precursor with a leader sequence of 32 amino acids, while the mature protein is a single polypeptide chain composed of 263 residues. The derived primary structure of tissue factor has been confirmed by comparison to protein and peptide sequence data. The sequence of the mature protein suggests that there are three distinct domains: extracellular, residues 1-219; hydrophobic, residues 220-242; and cytoplasmic, residues 243-263. Three potential N-linked carbohydrate attachment sites occur in the extracellular domain. The amino acid sequence of tissue factor shows no significant homology with the vitamin K-dependent serine proteases, coagulation cofactors, or any other protein in the National Biomedical Research Foundation sequence data bank (Washington, DC)

  3. Genetic ablation of soluble TNF does not affect lesion size and functional recovery after moderate spinal cord injury in mice

    DEFF Research Database (Denmark)

    Ellman, Ditte Gry; Degn, Matilda; Lund, Minna C.

    2016-01-01

    Traumatic spinal cord injury (SCI) is followed by an instant increase in expression of the microglial-derived proinflammatory cytokine tumor necrosis factor (TNF) within the lesioned cord. TNF exists both as membrane-anchored TNF (mTNF) and as cleaved soluble TNF (solTNF). We previously demonstra......, and MHCII), lesion size, and functional outcome after moderate SCI were comparable between genotypes. Collectively, our data demonstrate that genetic ablation of solTNF does not significantly modulate postlesion outcome after SCI....

  4. Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis.

    Directory of Open Access Journals (Sweden)

    Kate Welsh

    Full Text Available Members of the Inhibitor of APoptosis (IAP protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC, which binds to selected baculovirus IAP repeat (BIR domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α. Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to

  5. Characterization of Potent SMAC Mimetics that Sensitize Cancer Cells to TNF Family-Induced Apoptosis.

    Science.gov (United States)

    Welsh, Kate; Milutinovic, Snezana; Ardecky, Robert J; Gonzalez-Lopez, Marcos; Ganji, Santhi Reddy; Teriete, Peter; Finlay, Darren; Riedl, Stefan; Matsuzawa, Shu-Ichi; Pinilla, Clemencia; Houghten, Richard; Vuori, Kristiina; Reed, John C; Cosford, Nicholas D P

    2016-01-01

    Members of the Inhibitor of APoptosis (IAP) protein family suppress apoptosis within tumor cells, particularly in the context of immune cell-mediated killing by the tumor necrosis factor (TNF) superfamily cytokines. Most IAPs are opposed endogenously by the second mitochondrial activator of caspases (SMAC), which binds to selected baculovirus IAP repeat (BIR) domains of IAPs to displace interacting proteins. The development of SMAC mimetics as novel anticancer drugs has gained impetus, with several agents now in human clinical trials. To further understand the cellular mechanisms of SMAC mimetics, we focused on IAP family members cIAP1 and cIAP2, which are recruited to TNF receptor complexes where they support cell survival through NF-κB activation while suppressing apoptosis by preventing caspase activation. We established fluorescence polarization (FP) assays for the BIR2 and BIR3 domains of human cIAP1 and cIAP2 using fluorochrome-conjugated SMAC peptides as ligands. A library of SMAC mimetics was profiled using the FP assays to provide a unique structure activity relationship (SAR) analysis compared to previous assessments of binding to XIAP. Potent compounds displayed mean inhibitory binding constants (Ki) of 9 to 27 nM against the BIR3 domains of cIAP1 and cIAP2, respectively. Selected compounds were then characterized using cytotoxicity assays in which a cytokine-resistant human tumor cell line was sensitized to either TNF or lymphotoxin-α (LT-α). Cytotoxicity correlated closely with cIAP1 and cIAP2 BIR3 binding activity with the most potent compounds able to reduce cell viability by 50%. Further testing demonstrated that active compounds also inhibit RIP1 binding to BIR3 of cIAP1 and cIAP2 in vitro and reduce steady-state cIAP1 protein levels in cells. Altogether, these data inform the SAR for our SMAC mimetics with respect to cIAP1 and cIAP2, suggesting that these IAP family members play an important role in tumor cell resistance to cytotoxicity

  6. Cloning of human purine-nucleoside phosphorylase cDNA sequences by complementation in Escherichia coli.

    OpenAIRE

    Goddard, J M; Caput, D; Williams, S R; Martin, D W

    1983-01-01

    We have obtained cDNA clones that contain the entire coding region of the human purine-nucleoside phosphorylase (PNP; EC 2.4.2.1) mRNA. The cDNA sequences were generated by reverse transcription of PNP-enriched mRNA obtained by immunoadsorption of HeLa cell polyribosomes with monospecific antibody to human PNP. cDNA molecules that were close in length to PNP mRNA were separated by agarose gel electrophoresis and inserted into the Pst I site of the plasmid pBR322. Plasmid DNA from the pooled c...

  7. Generation of cDNA expression libraries enriched for in-frame sequences

    OpenAIRE

    Davis, Claytus A.; Benzer, Seymour

    1997-01-01

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore deve...

  8. Generation of full-length cDNA libraries: focus on plants.

    Science.gov (United States)

    Seki, Motoaki; Kamiya, Asako; Carninci, Piero; Hayashizaki, Yoshihide; Shinozaki, Kazuo

    2009-01-01

    Full-length cDNAs are essential for the correct annotation of transcriptional units and gene products from genomic sequence data and for functional analysis of the genes. Full-length cDNA libraries are very important resources for isolation of the full-length cDNAs. The biotinylated cap trapper method using the trehalose-thermostabilized reverse transcriptase has been developed and has become an efficient method for construction of high-content full-length cDNA libraries. We have constructed full-length cDNA libraries from various plants and animals using this method. The protocol of the method is described in this chapter.

  9. Cloning and sequencing of dolphinfish (Coryphaena hippurus, Coryphaenidae) growth hormone-encoding cDNA.

    Science.gov (United States)

    Peduel, A D; Elizur, A; Knibb, W

    1994-01-01

    The cDNA encoding the preprotein growth hormone from the dolphinfish (Coryphaena hippurus) has been cloned and sequenced. The cDNA was derived by reverse transcription of RNA from the pituitary of a young fish using the method known as Rapid Amplification of cDNA Ends (RACE). An oligonucleotide primer corresponding to the 5' region of Pagrus major and the universal RACE primer enabled amplification using the Polymerase Chain Reaction (PCR). The dolphinfish and yellow-tail, Seriola quineqeradiata, are both members of the sub-order Percoidei (Perciforme) and their GH sequences show a high level of homology.

  10. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    International Nuclear Information System (INIS)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H.

    1990-01-01

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the α-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor

  11. Changing the insulin receptor to possess insulin-like growth factor I ligand specificity

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, A.S.; Kjeldsen, T.; Wiberg, F.C.; Christensen, P.M.; Rasmussen, J.S.; Norris, K.; Moeller, K.B.; Moeller, N.P.H. (Biopharmaceuticals Div., Bagsvaerd (Denmark))

    1990-08-14

    To examine the role of the N-terminal part of the insulin-like growth factor I (IGF-I) receptor and insulin receptor in determining ligand specificity, the authors prepared an expression vector encoding a hybrid receptor where exon 1 (encoding the signal peptide and seven amino acids of the {alpha}-subunit), exon 2, and exon 3 of the insulin receptor were replaced with the corresponding IGF-I receptor cDNA (938 nucleotides). To allow direct quantitative comparison of the binding capabilities of this hybrid receptor with those of the human IGF-I receptor and the insulin receptor, all three receptors were expressed in baby hamster kidney (BHK) cells as soluble molecules and partially purified before characterization. The hybrid IGF-I/insulin receptor bound IGF-I with an affinity comparable to that of the wild-type IGF-I receptor. In contrast, the hybrid receptor no longer displayed high-affinity binding of insulin. These results directly demonstrate that it is possible to change the specificity of the insulin receptor to that of the IGF-I receptor and, furthermore, that the binding specificity for IGF-I is encoded within the nucleotide sequence from 135 to 938 of the IGF-I receptor cDNA. Since the hybrid receptor only bound insulin with low affinity, the insulin binding region is likely to be located within exons 2 and 3 of the insulin receptor.

  12. Antiallodynic Activity of Ceftriaxone and Clavulanic Acid in Acute Administration is Associated with Serum TNF-α Modulation and Activation of Dopaminergic and Opioidergic Systems.

    Science.gov (United States)

    Ochoa-Aguilar, A; Sotomayor-Sobrino, M A; Jaimez, R; Rodríguez, R; Plancarte-Sánchez, R; Ventura-Martinez, R

    2017-03-01

    Preclinical Research The aim of this study was to determine the antiallodynic effect of acute administration of the β-lactam antimicrobials, ceftriaxone (CFX) and clavulanic acid (CLAV), for the control of established pain on a model of neuropathic pain (NP). We also investigated the involvement of dopaminergic and opioidergic pathways as well as alterations in serum concentrations of TNF-α in the antiallodynic actions of these drugs. CFX, CLAV, or gabapentin (GAP), a reference drug, were administered i.p. twelve days after constriction of the sciatic nerve in rats. Mechanic and cold allodynia were evaluated for 3 h and alterations in serum concentration of TNF-α determined. Both CFX and CLAV had antiallodynic effects in response to mechanical and cold stimulation, similar to GAP. The antiallodynic effects of CFX and CLAV were blocked by haloperidol (HAL), a D2 receptor antagonist, and by naloxone (NLX), an opioid receptor antagonist. Additionally, serum TNF-α levels were attenuated following CFX and CLAV administration. These results suggest that acute administration of CFX and CLAV may represent a promising approach for treating the acute allodynia of NP, and that the mechanisms involved in these effects involve activation of dopaminergic and opioidergic pathways as well as modulation of TNF-α production. Drug Dev Res 78 : 105-115, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  13. 5'-end sequences of budding yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available List Contact us Budding yeast cDNA sequencing project 5'-end sequences of budding yeast full-length cDNA clones and quality... scores Data detail Data name 5'-end sequences of budding yeast full-length cDNA clones and quality...or-capping method, the sequence quality score generated by the Phred software, and links to SGD, dbEST and U...es. FASTA format. Quality Phred's quality score About This Database Database Desc...g yeast full-length cDNA clones and quality scores - Budding yeast cDNA sequencing project | LSDB Archive ...

  14. Basal cell carcinoma is associated with high TNF-alpha release but nor with TNF-alpha polymorphism at position--308

    DEFF Research Database (Denmark)

    Skov, Lone; Allen, Michael H; Bang, Bo

    2003-01-01

    secretion of TNF-alpha has been identified in humans. We have therefore investigated the association of the --308 polymorphism with the risk of basal cell carcinoma (BCC) in humans. The frequency of TNF G and TNF A alleles among Caucasian patients with a previous BCC (n=191) and health adults (n-107) were...... compared. For the TNF--308 polymorphism there was significant association between the genotype or allele frequencies and having BCC. To determine whether patients with a previous BCC had an increased capacity to secrete TNF-alpha, mononuclear cells were stimulated with lipopolysaccharide. Mononuclear cells...... from patients with a previous BCC (n=15) demonstrated a significantly increased release of TNF-alpha upon stimulation with lipopolysaccharide (Pcells age-matched control subjects (n=16). Further studies of other polymorphisms of the TNF-alpha gene associated...

  15. Selective TNF-α inhibitor-induced injection site reactions.

    Science.gov (United States)

    Murdaca, Giuseppe; Spanò, Francesca; Puppo, Francesco

    2013-03-01

    During the last decade, many new biological immune modulators entered the market as new therapeutic principles. TNF-α is a pro-inflammatory cytokine known to a have a key role in the pathogenic mechanisms of various immune-mediated or inflammatory diseases. TNF-α blockers have demonstrated efficacy in large, randomized controlled clinical trials either as monotherapy or in combination with other anti-inflammatory or disease-modifying anti-rheumatic drugs. Although generally well tolerated and safe, potential adverse events may be associated with TNF-α inhibitor treatment. The authors will briefly review the potential adverse drug reactions and the immunological mechanisms of injection site reactions (ISRs) in patients treated with etanercept and adalimumab. Patients treated with TNF-α inhibitors can develop ISR around the sites of injections. 'Type IV delayed type reaction' or 'recall ISRs'. Eosinophilic cellulitis or 'Wells syndrome', 'type III' and 'type I' reactions are reported. Long-term studies are necessary to determine the durability of response and the real risk of ISRs with golimumab and certolizumab pegol. Further studies are also necessary to evaluate the immunogenicity of these drugs.

  16. Immunogenicity of anti-TNF biologic therapies for rheumatoid arthritis

    NARCIS (Netherlands)

    van Schouwenburg, Pauline A.; Rispens, Theo; Wolbink, Gerrit Jan

    2013-01-01

    Currently, five anti-TNF biologic agents are approved for the treatment of rheumatoid arthritis (RA): adalimumab, infliximab, etanercept, golimumab and certolizumab pegol. Formation of anti-drug antibodies (ADA) has been associated with all five agents. In the case of adalimumab and infliximab,

  17. Tumour necrosis factor alpha (TNF-α) genetic polymorphisms and ...

    Indian Academy of Sciences (India)

    Sensitivity analysis of the summary odds ratio coefficients on the association between TNF-α-308G/A polymorphism and AILD risk using a random effects model. (A allele vs G allele). Results were computed by omitting each study in turn. Error bars are 95% confidence interval. Journal of Genetics, Vol. 92, No. 3, December ...

  18. Polimorfismo del TNF-alpha en autoinmunidad y tuberculosis.

    Directory of Open Access Journals (Sweden)

    Paula A. Correa

    2004-06-01

    Full Text Available El factor de necrosis tumoral alfa (TNF-a está incriminado tanto en enfermedades autoinmunes como en infecciosas. En el presente estudio se examinó el polimorfismo de la región promotora -308 del gen del TNF-a en enfermedades autoinmunes [lupus eritematoso sistémico (LES, artritis reumatoidea (AR, síndrome de Sjögren primario (SSp] y en tuberculosis. La genotipificación del polimorfismo -308 del TNF-a se realizó en ADN de pacientes con AR (N=165, LES (N=118, SSp (N=67, tuberculosis (N=138 y controles sanos (N=419, mediante reacción en cadena de la polimerasa con polimorfismos en los tamaños de los fragmentos de restricción (PCR-RFLP. El alelo TNF2 se asoció con la AR (OR=1,6; IC95% 1,2-2,3, p=0,008, el LES (OR=2,3; IC95% 1,6-3,3, p

  19. AIP1: a new player in TNF signaling

    OpenAIRE

    Guicciardi, M. Eugenia; Gores, Gregory J.

    2003-01-01

    Apoptosis signal–regulating kinase 1 (ASK1) is an upstream activator of JNK and p38 MAPK signaling cascades. Evidence now shows that the ASK1-interacting protein, AIP1, plays an important role in TNF-α–induced ASK1 activation by facilitating dissociation from its inhibitor.

  20. Concluding remarks: Reflections on the forty year's history of TNF

    International Nuclear Information System (INIS)

    Martin, L.W.

    1994-01-01

    The History of NATO's TNF policy is much wider than it may first appear, for NATO TNF policy has been the heart of the whole nuclear question since shortly after it was first so dramatically posed in 1945. Admittedly the ultimate foundation of deterrence has been the overarching balance of strategic nuclear weapons between the two Superpowers. These have provided the strategic open-quotes monitorclose quotes of ultimately unacceptable damage that inspires cautious behavior at lower levels of conflict. But if the strategic weapons inculcate the caution, it has been with TNF that the opposing blocs have chiefly tried to harness nuclear weapons to particular situations and manipulate them to their advantage. The key characteristic of nuclear weapons that distinguishes them from all others is their virtually unlimited destructive potential. Powers disposing of large strategic nuclear forces cannot readily, perhaps ever, be deprived of an ultimate capacity to do outrageous harm. If they are to be defeated, it must therefore be by outmaneuvering them so that they yield the essential prizes without recourse to their worst possibilities. If nuclear weapons are to be used in this process, whether actually or by latent influence on particular issues, it must be in some limited form. NATO's TNF policy constitutes the best example of this so far in nuclear history

  1. Molecular identification of the first insect ecdysis triggering hormone receptors

    DEFF Research Database (Denmark)

    Iversen, Annette; Cazzamali, Giuseppe; Williamson, Michael

    2002-01-01

    The Drosophila Genome Project website (www.flybase.org) contains an annotated gene sequence (CG5911), coding for a G protein-coupled receptor. We cloned the cDNA corresponding to this sequence and found that the gene has not been correctly predicted. The corrected gene CG5911 has five introns and...

  2. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  3. Muscarinic acetylcholine receptor subtypes: localization and structure/function

    DEFF Research Database (Denmark)

    Brann, M R; Ellis, J; Jørgensen, H

    1993-01-01

    Based on the sequence of the five cloned muscarinic receptor subtypes (m1-m5), subtype selective antibody and cDNA probes have been prepared. Use of these probes has demonstrated that each of the five subtypes has a markedly distinct distribution within the brain and among peripheral tissues. The...

  4. Decreased inducibility of TNF expression in lipid-loaded macrophages

    Directory of Open Access Journals (Sweden)

    Kallin Bengt

    2002-10-01

    Full Text Available Abstract Background Inflammation and immune responses are considered to be very important in the pathogenesis of atherosclerosis. Lipid accumulation in macrophages of the arterial intima is a characteristic feature of atherosclerosis which can influence the inflammatory potential of macrophages. We studied the effects of lipid loading on the regulation of TNF expression in human monocyte-derived macrophages. Results In macrophages incubated with acetylated low density lipoprotein (ac-LDL for 2 days, mRNA expression of TNF in cells stimulated with TNF decreased by 75%. In cell cultures stimulated over night with IL-1β, lipid loading decreased secretion of TNF into culture medium by 48%. These results suggest that lipid accumulation in macrophages makes them less responsive to inflammatory stimuli. Decreased basal activity and inducibility of transcription factor AP-1 was observed in lipid-loaded cells, suggesting a mechanism for the suppression of cytokine expression. NF-κB binding activity and inducibility were only marginally affected by ac-LDL. LDL and ac-LDL did not activate PPARγ. In contrast, oxidized LDL stimulated AP-1 and PPARγ but inhibited NF-κB, indicating that the effects of lipid loading with ac-LDL were not due to oxidation of lipids. Conclusions Accumulation of lipid, mainly cholesterol, results in down-regulation of TNF expression in macrophages. Since monocytes are known to be activated by cell adhesion, these results suggest that foam cells in atherosclerotic plaques may contribute less potently to an inflammatory reaction than newly arrived monocytes/macrophages.

  5. TNF-α -238, -308, -863 polymorphisms, and brucellosis infection.

    Science.gov (United States)

    Eskandari-Nasab, Ebrahim; Moghadampour, Mehdi; Sepanj-Nia, Adel

    2016-01-01

    Brucella abortus is an intracellular bacterium that affects humans and domestic animals. Tumor necrosis factor-alpha (TNF-α) has been shown as a key player in the induction of cell-mediated resistance against Brucella infection. We aimed to evaluate the possible influence of the TNF-α promoter polymorphisms (-308 G/A, -238 G/A, and -863 C/A) on the susceptibility of human brucellosis. A total of 153 patients with active brucellosis and 128 healthy individuals were recruited. All subjects were genotyped for the polymorphisms in the TNF-α gene by Allele-Specific polymerase chain reaction analysis. Our results showed that the TNF-α -308 GG genotype was significantly more frequently present in controls than in brucellosis patients (91% vs. 75%), thus was a protective factor against developing brucellosis (OR=0.313, p=0.001). In contrast, the -308 GA genotype (OR=3.026, p=0.002) and minor allele (A) (OR=3.058, p=0.001) as well as AAG haplotype (OR=4.014, p=0.001) conferred an increased risk of brucellosis. However, the -238 G/A and -863 C/A polymorphisms were not associated with the risk of brucellosis at both allelic and genotypic levels (p>0.05). Our study revealed that the TNF-α -308 A allele or GA heterozygosity or AAG haplotype were associated with an increased risk of brucellosis in our population. Copyright © 2015 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  6. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76...... in human. Although additional minor bands of 1.5 and 3.3 kb were found in Northern blots, RT-PCR (reverse transcription polymerase chain reaction) analysis failed to provide evidence that these minor bands are products of the tetranectin gene. Finally, the genetic map location for this gene, Tna...

  7. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    length τ-crystallin cDNA from crocodilian lens and α-enolase from other tissues. ... human (Acc. No. NM_001428). The sequences were used to construct a phylogenetic tree depicting gene lineage, using the clustering program DNAML.

  8. Construction and characterization of a cDNA expression library from the endangered Hu sheep.

    Science.gov (United States)

    Hu, P-F; Li, X-C; Liu, H-K; Guan, W-J; Ma, Y-H

    2014-10-31

    Hu sheep is one of the most important species in China; it is also listed as one of the 78 nationally protected domestic animals by the Chinese government in 2000. The construction of cDNA expression library of Hu sheep is of great significance for protecting individual genomes, generating transgenic sheep, and conducting clinical research using cDNA from Hu sheep. In this study, the total RNA from the ear tissue of Hu sheep was extracted, and a cDNA expression library was constructed using the SMART(TM) technique. The titer of amplified cDNA library was 1.09 x 10(10) PFU/mL, the rate of recombination was above 91.6%, and the average size of fragments was 1.1 kb. This study has an important significance for the preservation of Hu sheep resources at the genome level.

  9. Mouse tetranectin: cDNA sequence, tissue-specific expression, and chromosomal mapping

    DEFF Research Database (Denmark)

    Ibaraki, K; Kozak, C A; Wewer, U M

    1995-01-01

    regulation, mouse tetranectin cDNA was cloned from a 16-day-old mouse embryo library. Sequence analysis revealed a 992-bp cDNA with an open reading frame of 606 bp, which is identical in length to the human tetranectin cDNA. The deduced amino acid sequence showed high homology to the human cDNA with 76......(s) of tetranectin. The sequence analysis revealed a difference in both sequence and size of the noncoding regions between mouse and human cDNAs. Northern analysis of the various tissues from mouse, rat, and cow showed the major transcript(s) to be approximately 1 kb, which is similar in size to that observed...

  10. cDNA - ASTRA | LSDB Archive [Life Science Database Archive metadata

    Lifescience Database Archive (English)

    Full Text Available ing/transcriptional initiation regionss About This Database Database Description Download License Update History of This Database Site Policy | Contact Us cDNA - ASTRA | LSDB Archive ...

  11. Functionality of intrinsic disorder in tumor necrosis factor-α and its receptors.

    Science.gov (United States)

    Uversky, Vladimir N; El-Baky, Nawal Abd; El-Fakharany, Esmail M; Sabry, Amira; Mattar, Ehab H; Uversky, Alexey V; Redwan, Elrashdy M

    2017-11-01

    Tumor necrosis factor-α (TNF-α) is a pleiotropic inflammatory cytokine that exerts potent cytotoxic effects on solid tumor cells, while not affecting their normal counterparts. It is also known that TNF-α exerts many of its biological functions via interaction with specific receptors. To understand the potential roles of intrinsic disorder in the functioning of this important cytokine, we explored the peculiarities of intrinsic disorder distribution in human TNF-α and its homologs from various species, ranging from zebrafish to chimpanzee. We also studied the peculiarities of intrinsic disorder distribution in human TNFreceptors, TNFR1 and TNFR2. Analysis revealed that cytoplasmic domains of TNF-α and its receptors are expected to be highly disordered. Furthermore, although the sequence identities of analyzed TNF-α homologs range from 99.57% (between human and chimpanzee proteins) to 22.33% (between frog and fish proteins), their intrinsic disorder profiles are characterized by a remarkable similarity. These observations indicate that the peculiarities of distribution of the intrinsic disorder propensity within the amino acid sequences are evolutionary conserved, and therefore could be of functional importance for this family of proteins. We also show that disordered and flexible regions of human TNF-α and its TNFR1 and TNFR2 receptors are crucial for some of their biological activities. © 2017 Federation of European Biochemical Societies.

  12. Construction of primary and subtracted cDNA libraries from early embryos.

    Science.gov (United States)

    Rothstein, J L; Johnson, D; Jessee, J; Skowronski, J; DeLoia, J A; Solter, D; Knowles, B B

    1993-01-01

    By modifying current cDNA cloning and electroporation methods, large and representative murine cDNA libraries were synthesized from 10 to 100 ng mRNA isolated from unfertilized egg and preimplantation mouse embryos. High cloning efficiency is essential for complete representation of genes expressed in egg and preimplantation embryos and for the isolation of stage-specific genes using subtractive hybridization. Because the mouse embryo contains no more than 50 pg of poly(A)+ mRNA at any stage of preimplantation development, approximately 5000-10,000 embryos are required to obtain enough mRNA to synthesize libraries using current methods. To obtain a representative library that also includes rare transcripts, the size of the library should be at least 10(6) clones. The average percent conversion of mRNA to single-stranded cDNA was 20-40%, so that a cloning efficiency of nearly 2 x 10(8) cfu/microgram cDNA is required for such a cDNA library. No previous methods have provided directional cloning of cDNA into plasmids with these high efficiencies. The advent of electroporation methods for the introduction of nucleic acids into bacteria has made possible the use of standard plasmid vectors for high-efficiency cDNA cloning. Plasmid vectors are currently available that can accommodate the directional cloning of cDNA such that T7 and T3 RNA polymerase promoter sequences can be used to generate sense and anti-sense transcripts for subtractive hybridization and riboprobe synthesis. The cDNA libraries we derived using this methodology are a reusable and abundant source of genetic information about the control of preimplantation development. Specialized subtractive cDNA libraries enriched for genes expressed exclusively at a predetermined time in development give access to genes expressed in a stage-specific manner. The ability to construct new cDNA libraries from limited amounts of starting material ensures the provision of new and important resources for the identification

  13. TNF-mediated damage to glomerular endothelium is an important determinant of acute kidney injury in sepsis.

    Science.gov (United States)

    Xu, Chang; Chang, Anthony; Hack, Bradley K; Eadon, Michael T; Alper, Seth L; Cunningham, Patrick N

    2014-01-01

    Severe sepsis is often accompanied by acute kidney injury (AKI) and albuminuria. Here we studied whether the AKI and albuminuria associated with lipopolysaccharide (LPS) treatment in mice reflects impairment of the glomerular endothelium with its associated endothelial surface layer. LPS treatment decreased the abundance of endothelial surface layer heparan sulfate proteoglycans and sialic acid, and led to albuminuria likely reflecting altered glomerular filtration permselectivity. LPS treatment decreased the glomerular filtration rate (GFR), while also causing significant ultrastructural alterations in the glomerular endothelium. The density of glomerular endothelial cell fenestrae was 5-fold lower, whereas the average fenestrae diameter was 3-fold higher in LPS-treated than in control mice. The effects of LPS on the glomerular endothelial surface layer, endothelial cell fenestrae, GFR, and albuminuria were diminished in TNF receptor 1 (TNFR1) knockout mice, suggesting that these LPS effects are mediated by TNF-α activation of TNFR1. Indeed, intravenous administration of TNF decreased GFR and led to loss of glomerular endothelial cell fenestrae, increased fenestrae diameter, and damage to the glomerular endothelial surface layer. LPS treatment decreased kidney expression of vascular endothelial growth factor (VEGF). Thus, our findings confirm the important role of glomerular endothelial injury, possibly by a decreased VEGF level, in the development and progression of AKI and albuminuria in the LPS model of sepsis in the mouse.

  14. NLRX1 Acts as an Epithelial-Intrinsic Tumor Suppressor through the Modulation of TNF-Mediated Proliferation

    Directory of Open Access Journals (Sweden)

    Ivan Tattoli

    2016-03-01

    Full Text Available The mitochondrial Nod-like receptor protein NLRX1 protects against colorectal tumorigenesis through mechanisms that remain unclear. Using mice with an intestinal epithelial cells (IEC-specific deletion of Nlrx1, we find that NLRX1 provides an IEC-intrinsic protection against colitis-associated carcinogenesis in the colon. These Nlrx1 mutant mice have increased expression of Tnf, Egf, and Tgfb1, three factors essential for wound healing, as well as increased epithelial proliferation during the epithelial regeneration phase following injury triggered by dextran sodium sulfate. In primary intestinal organoids lacking Nlrx1, stimulation with TNF resulted in exacerbated proliferation and expression of the intestinal stem cell markers Olfm4 and Myb. This hyper-proliferation response was associated with increased activation of Akt and NF-κB pathways in response to TNF stimulation. Together, these results identify NLRX1 as a suppressor of colonic tumorigenesis that acts by controlling epithelial proliferation in the intestine during the regeneration phase following mucosal injury.

  15. [Primary culture of cat intestinal epithelial cell and construction of its cDNA library].

    Science.gov (United States)

    Ye, L; Gui-Hua, Z; Kun, Y; Hong-Fa, W; Ting, X; Gong-Zhen, L; Wei-Xia, Z; Yong, C

    2017-04-12

    Objective To establish the primary cat intestinal epithelial cells (IECs) culture methods and construct the cDNA library for the following yeast two-hybrid experiment, so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection, by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMART™ technology, and then the double-strand cDNAs were acquired by LD-PCR, which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recombination. Matchmaker™ Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calculation of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of collagenase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1×10 6 independent clones. The titer was 2.8×10 9 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research, and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

  16. cDNA fingerprinting of osteoprogenitor cells to isolate differentiation stage-specific genes.

    OpenAIRE

    Candeliere, G A; Rao, Y; Floh, A; Sandler, S D; Aubin, J E

    1999-01-01

    A cDNA fingerprinting strategy was developed to identify genes based on their differential expression pattern during osteoblast development. Preliminary biological and molecular staging of cDNA pools prepared by global amplification PCR allowed discrim-inating choices to be made in selection of expressed sequence tags (ESTs) to be isolated. Sequencing of selected ESTs confirmed that both known and novel genes can be isolated from any developmental stage of interest, e.g. from primitive progen...

  17. CDNA cloning, characterization and expression of an endosperm-specific barley peroxidase

    DEFF Research Database (Denmark)

    Rasmussen, Søren Kjærsgård; Welinder, K.G.; Hejgaard, J.

    1991-01-01

    A barley peroxidase (BP 1) of pI ca. 8.5 and M(r) 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C...

  18. Construction of an infectious cDNA clone of foot-and-mouth disease ...

    Indian Academy of Sciences (India)

    A stable, full-length cDNA clone of FMDV type O1BFS 1860 preceded by a bacteriophage T7 polymerase promoter was assembled in a plasmid vector pGEMR-7Zf(–). An ∼8.2 kb PCR product was amplified from the cDNA clone and a full-length RNA was generated from it by in vitro transcription. Transfection of BHK-21 ...

  19. cDNA cloning and mRNA expression of heat shock protein 70 gene ...

    African Journals Online (AJOL)

    In this study, the full-length heat shock protein 70 of Tegillarca granosa was cloned from cDNA library by rapid amplification of cDNA end (RACE). The open reading frame (ORF) of heat shock protein 70 was 1968 bp, and it encoded a protein of 655 amino acids with a predicted molecular weight of 71.48 kDa and an ...

  20. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  1. Trehalose as a good candidate for enriching full-length cDNAs in cDNA library construction.

    Science.gov (United States)

    Chen, Lei; Cao, Lixue; Zhou, Longhai; Jing, Yudong; Chen, Zuozhou; Deng, Cheng; Shen, Yu; Chen, Liangbiao

    2007-01-10

    It has been reported that the disaccharide trehalose is capable of increasing the thermostability and thermoactivity of reverse transcriptase, and therefore improving the length of cDNA synthesis. However, no test has been done on how the disaccharide trehalose performs in the context of the entire cDNA synthesis processes, or whether it can seamlessly integrate into the commercially available cDNA synthesis kit. In this report, we optimized a protocol to incorporate trehalose in the Stratagene's cDNA library construction kit in order to demonstrate great improvement in cDNA's length (average length of 1.8 kb in the trehalose group versus 1.0 kb in the control). Sequence analysis of the cDNA clones showed that the addition of trehalose did not increase the error rate of the RT products but greatly increase the quantity of full-length in cDNA library.

  2. Construction of Infectious cDNA Clone of a Chrysanthemum stunt viroid Korean Isolate

    Science.gov (United States)

    Yoon, Ju-Yeon; Cho, In-Sook; Choi, Gug-Seoun; Choi, Seung-Kook

    2014-01-01

    Chrysanthemum stunt viroid (CSVd), a noncoding infectious RNA molecule, causes seriously economic losses of chrysanthemum for 3 or 4 years after its first infection. Monomeric cDNA clones of CSVd isolate SK1 (CSVd-SK1) were constructed in the plasmids pGEM-T easy vector and pUC19 vector. Linear positive-sense transcripts synthesized in vitro from the full-length monomeric cDNA clones of CSVd-SK1 could infect systemically tomato seedlings and chrysanthemum plants, suggesting that the linear CSVd RNA transcribed from the cDNA clones could be replicated as efficiently as circular CSVd in host species. However, direct inoculation of plasmid cDNA clones containing full-length monomeric cDNA of CSVd-SK1 failed to infect tomato and chrysanthemum and linear negative-sense transcripts from the plasmid DNAs were not infectious in the two plant species. The cDNA sequences of progeny viroid in systemically infected tomato and chrysanthemum showed a few substitutions at a specific nucleotide position, but there were no deletions and insertions in the sequences of the CSVd progeny from tomato and chrysanthemum plants. PMID:25288987

  3. Construction of a T7 Human Lung Cancer cDNA Library

    Directory of Open Access Journals (Sweden)

    Wentao YUE

    2008-10-01

    Full Text Available Background and objective Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC diagnosis, new biomarker, such as serum autoantibody may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocarcinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library.Results Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106 pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5×1010 pfu/mL. PCR amplification of random plaque show insert ratio were 100% (24/24 in adenocarcinoma library and 95.8% in human lung squamas carcinoma library (23/24. Insert range from 300 bp to 1 500 bp. Conclusion Two phage display cDNA library from NSCLC were constructed.

  4. [Construction of suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus].

    Science.gov (United States)

    Wu, Jia-hong; Zhao, Tong-yan; Dong, Yan-de

    2006-08-01

    To construct the suppression subtracted cDNA library of deltamethrin-resistant Aedes albopictus. Total RNA was extracted from the deltamethrin-resistant (R-lab) and -sensitive (S-lab) isolates, mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Two subtractions were performed by suppression subtractive hybridization with S-lab as tester and R-lab as driver or S-lab as driver and R-lab as tester. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries contained 580 and 477 positive clones respectively. The PCR results of 150 clones picked randomly from each library showed that the positive ratio of constructed cDNA libraries was 93%, with a length of cDNA fragments ranged from 150bp to 750bp. The suppression subtracted cDNA library of deltamethrin-resistant Ae. albopictus is constructed.

  5. [Construction of the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei].

    Science.gov (United States)

    Bei, Zhu-chun; Wang, Jing-yan

    2004-06-01

    To construct the subtracted cDNA libraries related to artemisinin-resistance of Plasmodium berghei using suppression subtractive hybridization PCR (SSH PCR). Total RNA was extracted from the artemisinin-sensitive (NS) and artemisinin-resistant (AR) strains of Plasmodium berghei K173. The cDNA synthesis followed the protocol of super SMART cDNA synthesis kit. Taking the NS as driver, AR as tester and reverse, two subtractions were performed by SSH PCR. Enriched different expressed cDNA was cloned into pMD18-T vector to construct subtractive libraries. The subtracted cDNA libraries of NS-AR and AR-NS contained 395 and 506 positive clones respectively. The PCR results of 108 clones picked randomly from each library showed 100 and 104 positive inserts contained in the plasmids respectively, and distributing in 250-2000 bp. The successful construction of the subtracted cDNA libraries related to artemisinin-resistance of P. berghei enable us to identify the different expressed genes involved in the resistance mechanism.

  6. [cDNA library constructing and specific antigen expression of Streptomyces thermohydroscopicus].

    Science.gov (United States)

    Xu, Lei; Wang, Ling-ling; Liu, Shuo; Ling, Yuan; Ma, Lie; Wang, Qun; Zhang, Li-jiao; He, Xiao-yu; Zhao, Ming-jing; Wang, Xiao-ge

    2012-03-01

    To construct a cDNA library from Streptomyces thermohydroscopicus and screen genes with virulence, obtain the recombinant fusion virulence proteins by prokaryotic expression system. The Streptomyces thermohydroscopicus cDNA library was constructed by switching mechanism at 5'end of RNA transcript approach. A total of 1020 clones randomly selected from the cDNA library were sequenced and these expressed sequence tags (EST) were further analyzed for the screen of antigen-specific genes. The two candidate genes were subcloned into expression vector pET-28a. The recombinants were transformed into BL2 and proteins were expressed by the induction of isopropyl-β-D-1-thiogalactopyranoside (IPTG). A high-quality cDNA library from Streptomyces thermohydroscopicus was constructed and a set of 978 valid sequences were obtained. Clustering and assembly of these cDNA sequences resulted in 347 unique genes, among which 2 potential antigen-specific genes were highly allied with outer membrane lipoprotein (51%) and transferring-binding protein B (42%) from Actinobacillus pleuropneumoniae serotype (APP). The open reading frame (ORF) of the two candidate genes are 1554 bp and 726 bp, which coded two peptides with 517 and 241 amino acids, respectively. The molecular weights of the recombinant fusion proteins were 63 000 and 30 000. The cDNA library of Streptomyces thermohydroscopicus reached the quality requirement of gene library. EST database in the library would greatly facilitate further screening of virulence genes.

  7. Cell lysis-free quantum dot multicolor cellular imaging-based mechanism study for TNF-α-induced insulin resistance.

    Science.gov (United States)

    Kim, Min Jung; Rangasamy, Sabarinathan; Shim, Yumi; Song, Joon Myong

    2015-01-27

    TNF-α is an inflammatory cytokine that plays an important role in insulin resistance observed in obesity and chronic inflammation. Many cellular components involved in insulin signaling cascade are known to be inhibited by TNF-α. Insulin receptor substrate (IRS)-1 is one of the major targets in TNF-α-induced insulin resistance. The serine phosphorylation of IRS-1 enables the inhibition of insulin signaling. Until now, many studies have been conducted to investigate the mechanism of TNF-α-induced insulin resistance based on Western blot. Intracellular protein kinase crosstalk is commonly encountered in inflammation-associated insulin resistance. The crosstalk among the signaling molecules obscures the precise role of kinases in insulin resistance. We have developed a cell lysis-free quantum dots (QDots) multicolor cellular imaging to identify the biochemical role of multiple kinases (p38, JNK, IKKβ, IRS1ser, IRS1tyr, GSK3β, and FOXO1) in inflammation-associated insulin resistance pathway with a single assay in one run. QDot-antibody conjugates were used as nanoprobes to simultaneously monitor the activation/deactivation of the above seven intracellular kinases in HepG2 cells. The effect of the test compounds on the suppression of TNF-α-induced insulin resistance was validated through kinase monitoring. Aspirin, indomethacin, cinnamic acid, and amygdalin were tested. Through the measurement of the glycogen level in HepG2 cell treated with TNF-α, it was found that aspirin and indomethacin increased glycogen levels by almost two-fold compared to amygdalin and cinnamic acid. The glucose production assay proved that cinnamic acid was much more efficient in suppressing glucose production, compared with MAP kinase inhibitors and non-steroidal anti-inflammatory drugs. QDot multicolor cellular imaging demonstrated that amygdalin and cinnamic acid selectively acted via the JNK1-dependent pathway to suppress the inflammation-induced insulin resistance and improve

  8. Insulin receptors

    International Nuclear Information System (INIS)

    Kahn, C.R.; Harrison, L.C.

    1988-01-01

    This book contains the proceedings on insulin receptors. Part A: Methods for the study of structure and function. Topics covered include: Method for purification and labeling of insulin receptors, the insulin receptor kinase, and insulin receptors on special tissues

  9. cDNA cloning and expression of Bacillus thuringiensis Cry1Aa toxin binding 120 kDa aminopeptidase N from Bombyx mori.

    Science.gov (United States)

    Yaoi, K; Nakanishi, K; Kadotani, T; Imamura, M; Koizumi, N; Iwahana, H; Sato, R

    1999-01-18

    Bacillus thuringiensis Cry1Aa toxin binds to a 120 kDa putative receptor protein in the Bombyx mori midgut. Recently, this protein was purified and identified as glycosyl-phosphatidylinositol (GPI) anchored aminopeptidase N (APN). In this study, a full-length cDNA thought to encode this 120 kDa APN was isolated and sequenced. It has a 2958 bp ORF encoding 986 amino acids. In the deduced amino acid sequence, we identified GPI-anchor and zinc-metallopeptidase signals, which are the same as those of APNs of other insects that are reported to be putative Cry1 toxin receptors. The B. mori APN amino acid sequence also has a high similarity with those of the other APNs. Subsequently, the recombinant APN was expressed by Escherichia coli and its Cry1Aa toxin binding ability was analyzed. Ligand blotting showed that Cry1Aa toxin bound to the recombinant APN.

  10. TNF-α is involved in the abnormal thymocyte migration during experimental Trypanosoma cruzi infection and favors the export of immature cells.

    Directory of Open Access Journals (Sweden)

    Ana Rosa Pérez

    Full Text Available Previous studies revealed a significant production of inflammatory cytokines together with severe thymic atrophy and thymocyte migratory disturbances during experimental Chagas disease. Migratory activity of thymocytes and mature T cells seem to be finely tuned by cytokines, chemokines and extracellular matrix (ECM components. Systemic TNF-α is enhanced during infection and appears to be crucial in the response against the parasite. However, it also seems to be involved in disease pathology, since it is implicated in the arrival of T cells to effector sites, including the myocardium. Herein, we analyzed the role of TNF-α in the migratory activity of thymocytes in Trypanosoma cruzi (T. cruzi acutely-infected mice. We found increased expression and deposition of TNF-α in the thymus of infected animals compared to controls, accompanied by increased co-localization of fibronectin, a cell migration-related ECM molecule, whose contents in the thymus of infected mice is also augmented. In-vivo studies showed an enhanced export of thymocytes in T. cruzi-infected mice, as ascertained by intrathymic injection of FITC alone or in combination with TNF-α. The increase of immature CD4(+CD8(+ T cells in secondary lymphoid organs was even more clear-cut when TNF-α was co-injected with FITC. Ex-vivo transmigration assays also revealed higher number of migrating cells when TNF-α was added onto fibronectin lattices, with higher input of all thymocyte subsets, including immature CD4(+CD8(+. Infected animals also exhibit enhanced levels of expression of both mRNA TNFreceptors in the CD4(+CD8(+ subpopulation. Our findings suggest that in T. cruzi acute infection, when TNF-α is complexed with fibronectin, it favours the altered migration of thymocytes, promoting the release of mature and immature T cells to different compartments of the immune system. Conceptually, this work reinforces the notion that thymocyte migration is a multivectorial biological event

  11. TNF-α inhibitory effect of Euphorbia hirta in rats.

    Science.gov (United States)

    Ahmad, Sheikh Fayaz; Bani, Sarang; Sultan, Phalisteen; Ali, Sheikh Abid; Bakheet, Saleh A; Attia, Sabry M; Abd-Allah, Adel R A

    2013-04-01

    Euphorbia hirta L. (Euphorbiaceae) (E. hirta) is a tree locally used as a traditional medicine in Africa and Australia to treat numerous diseases such as hypertension, respiratory ailments, tumors, and wounds, and it has reported antiallergic, antipyretic, anti-inflammatory activities, etc. This study evaluated the ability of fresh leaves of E. hirta ethanol extract to inhibit the intracellular tumor necrosis factor α (TNF-α) level in the synovial fluid and neutrophils in lipopolysaccharide (LPS)-induced inflamed rat knees. Female Wister albino rats 140-160 g were used. E. hirta ethanol extract was given orally at 25, 50, 100, and 200 mg/kg, 2 h before an intra-articular (i.a.) injection of LPS. Two and three hours later, synovial fluid and neutrophils levels of intracellular TNF-α production were measured. In the time course of the experiment, E. hirta maximum inhibition at 100 and 200 mg/kg (p.o.) dose showed 16.5 ± 1.34 and 14.4 ± 1.30% of synovial fluid, 4.26 ± 0.36 and 3.78 ± 0.29% of neutrophils levels of intracellular TNF-α productions at 2 h after LPS injection. LPS control displayed 22.97 ± 1.61 and 6.78 ± 0.34% of synovial fluid and neutrophils levels of intracellular TNF-α at 2 h after LPS injection. Intracellular TNF-α was also estimated at 3 h after LPS injection. The LPS-injected rat knee model gives a comparative study of acute anti-inflammatory responses. E. hirta inhibition of proinflammatory intracellular cytokine TNF-α production with LPS-induced inflamed rat knee is of great importance in defining the anti-arthritic potential of E. hirta.

  12. Interleukin-1beta and TNF-alpha: reliable targets for protective therapies in Parkinson´s Disease?

    Directory of Open Access Journals (Sweden)

    María Celeste Leal

    2013-04-01

    Full Text Available Neuroinflammation has received increased attention as a target for putative neuroprotective therapies in Parkinson´s Disease (PD. Two prototypic pro-inflammatory cytokines Interleukin-1beta (IL-1 and Tumor necrosis factor-alpha (TNF have been implicated as main effectors of the functional consequences of neuroinflammation on neurodegeneration in PD models. In this review, we describe that the functional interaction between these cytokines in the brain differs from the periphery (e.g. their expression is not induced by each other and present data showing predominantly a toxic effect of these cytokines when expressed at high doses and for a sustained period of time in the substantia nigra pars compacta (SN. In addition, we highlight opposite evidence showing protective effects of these two main cytokines when conditions of duration, amount of expression or state of activation of the target or neighboring cells are changed. Furthermore, we discuss these results in the frame of previous disappointing results from anti-TNF clinical trials against Multiple Sclerosis, another neurodegenerative disease with a clear neuroinflammatory component. In conclusion, we hypothesize that the available evidence suggests that the duration and dose of IL-1 or TNF expression is crucial to predict their functional effect on the SN. Since these parameters are not amenable for measurement in the SN of PD patients, we call for an in-depth analysis to identify downstream mediators that could be common to the toxic (and not the protective effects of these cytokines in the SN. This strategy could spare the possible neuroprotective effect of these cytokines operative in the patient at the time of treatment, increasing the probability of efficacy in a clinical setting. Alternatively, receptor-specific agonists or antagonists could also provide a way to circumvent undesired effects of general anti-inflammatory or specific anti IL-1 or TNF therapies against PD.

  13. Influence of NKG2D Genetic Variants on Response to Anti-TNF Agents in Patients with Rheumatoid Arthritis

    Directory of Open Access Journals (Sweden)

    Milena Iwaszko

    2018-01-01

    Full Text Available A natural killer group 2 member D (NKG2D acts as a powerful activating and co-stimulatory receptor on immune effector cells including NK and T cells. Disruptions within the NKG2D signalling pathway may trigger an exacerbated immune response and promote autoimmune reactions. The objective of the study was to evaluate a plausible role of polymorphisms within the NKG2D gene as a predictor of how effective anti-tumor necrosis factor (TNF therapy is in rheumatoid arthritis (RA patients. A total of 280 RA patients receiving anti-TNF therapy were genotyped for NKG2D rs2255336 (A > G, rs1049174 (C > G, and rs1154831 (C > A. Clinical response was evaluated according to the European League against Rheumatism (EULAR criteria at the 12th and 24th week. Both the NKG2D rs225336 and rs1049174 polymorphisms were significantly associated with efficacy of TNF inhibitors. Inefficient therapy was more frequently observed in patients with rs2255336 GG or rs1049174 CC genotype as compared to other genotypes (p-value = 0.003 and p-value = 0.004, respectively. The presence of the rs2255336 G or the rs1049174 C allele correlated with a worse EULAR response (p-value = 0.002, p-value = 0.031, respectively. Moreover, patients carrying the rs2255336 or rs1049174 heterozygous genotype achieved better EULAR responses than patients with homozygous genotypes (p-value = 0.010 and p-value = 0.002, respectively. Data from the present study provides evidence that NKG2D polymorphisms may affect response to anti-TNF inhibitors in RA patients.

  14. Insights into deregulated TNF and IL-10 production in malaria

    DEFF Research Database (Denmark)

    Boeuf, Philippe S; Loizon, Séverine; Awandare, Gordon A

    2012-01-01

    ABSTRACT: BACKGROUND: Severe malarial anaemia (SMA) is a major life-threatening complication of paediatric malaria. Protracted production of pro-inflammatory cytokines promoting erythrophagocytosis and depressing erythropoiesis is thought to play an important role in SMA, which is characterized...... the activation status of those cells in SMA patients. METHODS: The IL-10 and TNF production capacity and the activation phenotype of monocytes and T cells were compared in samples collected from 332 Ghanaian children with non-overlapping SMA (n = 108), cerebral malaria (CM) (n = 144) or uncomplicated malaria (UM......) (n = 80) syndromes. Activation status of monocytes and T cells was ascertained by measuring HLADR + and/or CD69+ surface expression by flow cytometry. The TNF and IL-10 production was assessed in a whole-blood assay after or not stimulation with lipopolysaccharide (LPS) or phytohaemaglutinin (PHA...

  15. iRhom2 deficiency relieves TNF-α associated hepatic dyslipidemia in long-term PM2.5-exposed mice.

    Science.gov (United States)

    Ge, Chen-Xu; Qin, Yu-Ting; Lou, De-Shuai; Li, Qiang; Li, Yuan-Yuan; Wang, Zhong-Ming; Yang, Wei-Wei; Wang, Ming; Liu, Nan; Wang, Zhen; Zhang, Peng-Xing; Tu, Yan-Yang; Tan, Jun; Xu, Min-Xuan

    2017-12-02

    Accumulating researches reported that particulate matter (PM2.5) is a risk factor for developing various diseases, including metabolic syndrome. Recently, inactive rhomboid protein 2 (iRhom2) was considered as a necessary modulator for shedding of tumor necrosis factor-α (TNF-α) in immune cells. TNF-α, a major pro-inflammatory cytokine, was linked to various pathogenesis of diseases, including dyslipidemia. Here, wild type (WT) and iRhom2-knockout (iRhom2 -/- ) mice were used to investigate the effects of iRhom2 on PM2.5-induced hepatic dyslipidemia. The hepatic histology, inflammatory response, glucose tolerance, serum parameters and gene expressions were analyzed. We found that long-term inhalation of PM2.5 resulted in hepatic steatosis. And a significant up-regulation of iRhom2 in liver tissues was observed, accompanied with elevated TNF-α, TNF-α converting enzyme (TACE), TNFα receptor (TNFR)2 and various inflammatory cytokines expressions. Additionally, PM2.5 treatment caused TG and TC accumulation in serum and liver, probably attributed to changes of genes modulating lipid metabolism. Intriguingly, hepatic injury and dyslipidemia were attenuated by iRhom2 -/- in mice with PM2.5 challenge. In vitro, iRhom2-knockdwon reduced TNF-α expressions and its associated inflammatory cytokines in Kupffer cells, implying that liver-resident macrophages played an important role in regulating hepatic inflammation and lipid metabolism in cells treated with PM2.5. The findings indicated that long-term PM2.5 exposure caused hepatic steatosis and dyslipidemia through triggering inflammation, which was, at least partly, dependent on iRhom2/TNF-α pathway in liver-resident macrophages. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Cinnamon Extract Improves TNF-a Induced Overproduction of Intestinal ApolipoproteinB-48 Lipoproteins

    Science.gov (United States)

    TNF-alpha stimulates the overproduction of intestinal apolipoproteins. We evaluated whether a water extract of cinnamon (Cinnulin PF®) improved the dyslipidemia induced by TNF-alpha in Triton WR-1339 treated hamsters, and whether Cinnulin PF® inhibits the TNF-alpha-induced over the secretion of apoB...

  17. Improved survival of TNF-deficient mice during the zymosan-induced multiple organ dysfunction syndrome.

    NARCIS (Netherlands)

    Volman, T.J.H.; Hendriks, T.; Verhofstad, A.A.J.; Kullberg, B.J.; Goris, R.J.A.

    2002-01-01

    The purpose of the study was to investigate the course of the zymosan-induced multiple organ dysfunction syndrome (MODS) in the absence of tumor necrosis factor (TNF) in a murine model. Tumor Necrosis Factor-alpha-lymphotoxin-a knockout (TNF/LT-/-) mice (n = 36) and wild-type (TNF/LT+/+) mice (n =

  18. Tumor necrosis factor alpha (TNF alpha) in human skin : A comparison of different antibodies for immunohistochemistry

    NARCIS (Netherlands)

    van der Laan, N; de Leij, LFMH; Buurman, W; Timens, W; ten Duis, HJ

    Conflicting results have been reported regarding the localization and presence of TNF alpha in normal human skin, To study TNF alpha expression, we tested a panel of antibodies directed against human TNF alpha, First, antibodies were tested for immunoreactivity on cytospots of isolated

  19. Tailored anti-TNF therapy during pregnancy in patients with IBD : Maternal and fetal safety

    NARCIS (Netherlands)

    de Lima, A.; Zelinkova, Z.; van der Ent, C.; Steegers, E. A P; van der Woude1, C. J.

    Objective Antitumour necrosis factor (TNF) during pregnancy in patients with IBD is related to high fetal anti-TNF levels. We evaluated maternal and child safety on discontinuing anti-TNF in the second trimester of pregnancy. Design Two groups of women with IBD were prospectively followed-up during

  20. Tailored anti-TNF therapy during pregnancy in patients with IBD: Maternal and fetal safety

    NARCIS (Netherlands)

    A. de Lima (Alison); Z. Zelinkova (Zuzana); C. van der Ent (Cokkie); E.A.P. Steegers (Eric); van der Woude1, C.J.

    2016-01-01

    textabstractObjective Antitumour necrosis factor (TNF) during pregnancy in patients with IBD is related to high fetal anti-TNF levels. We evaluated maternal and child safety on discontinuing anti-TNF in the second trimester of pregnancy. Design Two groups of women with IBD were prospectively

  1. cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

    International Nuclear Information System (INIS)

    Szybka, Malgorzata; Kordek, Radzislaw; Zakrzewska, Magdalena; Rieske, Piotr; Pasz-Walczak, Grazyna; Kulczycka-Wojdala, Dominika; Zawlik, Izabela; Stawski, Robert; Jesionek-Kupnicka, Dorota; Liberski, Pawel P

    2009-01-01

    Recently published data showed discrepancies beteween P53 cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers. To this end, we analyzed 23 colorectal cancers for P53 mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry. We found P53 gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of P53 mRNA was present in samples with and without P53 mutations. In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated P53 mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without P53 mutation (normal cells and cells showing K-RAS and/or APC but not P53 mutation) in samples presenting P53 mutation; 3, heterozygous or hemizygous mutations of P53 gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in P53 cDNA and DNA sequencing analysis

  2. [Construction of phage display cDNA library from adult worms of Schistosoma japonicum].

    Science.gov (United States)

    Sun, Yi; Jia, Ren-chu; Liu, Jin-ming; Yuan, Chun-xiu; Shi, Yao-jun; Lu, Ke; Fu, Zhi-qiang; Sun, Huan; Cai, You-min; Lin, Jiao-jiao

    2007-10-01

    To screen protective antigen genes and construct the T7 phage display library from adult worms of Schistosoma japonicum. Total RNA was extracted from adult worms of S. japonicum by Trizol reagent anti mRNA was isolated from the total RNA. The ds cDNA was synthesized by reverse transcription using random primer. Directional EcoR I/ Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was digested with EcoR I anti Hind III, which resulted in ds cDNA with EcoR I and Hind III adhering ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated and ligated into T7 Select 10-3b vector. After packaging in citro, the T7 Select 10-3b vector was transformed into BLT5403 to construct the T7 phage display cDNA library. Plaque assay and PCR were used to evaluate the library. Seven known objective genes of S. japonicum were screened by PCR to detect the representation of the library. Primary library capacity was 4.98 x 10(6) pfu, and the titer of amplified library was 3.85 x 10(11) pfu/mL. The PCR identification result of 96 clones picked at random showed that recombination rate was 93.8%, in which 95.6% inserted cDNA fragments were longer than 300 bp in length. All the seven known objective genes of S. japonicum were amplified from the library. The T7 phage display library from adult worms of Schistosoma japonicum was constructed.

  3. [Construction of cDNA expression library of unfed female Haemaphysalis longicornis and immuno-screening].

    Science.gov (United States)

    Chai, Hui-ping; Liu, Guang-yuan; Zhang, Lin; Gong, Zhen-li; Xie, Jun-ren; Tian, Zhan-cheng; Wang, Lu; Jia, Ning

    2009-02-28

    To construct a cDNA expression library from unfed female tick Haemaphysalis longicornis for screening and cloning potential antigenic genes. Total RNA was isolated from unfed female ticks, mRNA was purified and a library of oligo (dT) -primed cDNA with added directional EcoR I /Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I /Hind III arms of the lambda SCREEN vector. Pure phage stocks were harvested by plaque purification and converted to plasmid subclones by plating phage on host strain BM25.8. Recombinant plasmids that were subcloned to E. coli BM25.8 were isolated and transformed into E. coli JM109. Recombinant plasmids abstracted from JM109 were identified by PCR and sequencing. The recombinant phage DNA was packaged by using phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.8 x 10(6) pfu. Data showed 100% of the library were recombinant and the titer of the amplified library was 2.4 x 10(9) pfu/ml. Forty-two clones of encoding immunodominant antigens were obtained from the cDNA library. Sequence analysis revealed 12 unique cDNA sequences and the encoded putative proteins showed similarities to H. longicornis tropomyosin mRNA, Rhipicephalus annulatus unknown larval protein mRNA, chromosome 2R of Drosophila melanogaster, mitochondrial DNA of H. flava, clones HqL09 unkown mRNA and Hq05 mRNA of H. qinghaiensis, and myosin alkali light chain protein mRNA. The cDNA expression library from unfed female H. longicornis was successfully constructed and screening of protective genes may provide candidate antigens of the tick.

  4. Clinical management of paradoxical psoriasiform reactions during TNF- α therapy.

    Science.gov (United States)

    Navarro, R; Daudén, E

    2014-10-01

    There have been reports of paradoxical induction or worsening of psoriasis during treatment with tumor necrosis factor (TNF) α agents (infliximab, etanercept, adalimumab, and certolizumab). It has been hypothesized that an imbalance between TNF-α and interferon α might have a role in the etiology and pathogenesis of these reactions. Paradoxical psoriasiform reactions can be divided clinically into de novo psoriasis and exacerbation of preexisting psoriasis. The first, which is more common and more extensively described in the literature, occurs in patients without a history of psoriasis who are receiving TNF-α therapy for another inflammatory disorder. The second can occur with or without changes in the morphology of the lesions. In this article, we review the literature on the clinical and histologic features of paradoxical psoriasiform reactions, analyze their clinical course and treatment, and propose a clinical management model for use in routine practice. Copyright © 2012 Elsevier España, S.L.U. and AEDV. All rights reserved.

  5. Transport experience of new ''TNF-XI'' powder package

    International Nuclear Information System (INIS)

    Nomura, I.; Fujiwara, T.; Naigeon, P.

    2004-01-01

    Since the Tokai criticality accident in 1999, there has been no specialized manufacturer conducting uranium re-conversion in Japan. For this reason, Nuclear Fuel Industries, Ltd. (NFI) imports from overseas almost all the uranium oxide powder used for manufacturing pellets for nuclear fuel assemblies. To date, an NT-IX package has been used for transporting the uranium oxide powder. However, due to the adoption of IAEA TS-R-1 into Japanese domestic regulations, we have begun to use a new TNF-XI powder package because the NT-IX package can suffer major deformation under the drop test III condition. The TNF-XI package was jointly developed by COGEMA LOGISTICS of France and NFI from 2000, and started to be used for actual transportation in 2003. This package has improved transport efficiency, handling operability and safety performance in comparison to its predecessor. This paper describes the characteristics of the new TNF-XI package and its actual transportation records and performance

  6. Inhibiting HLA-G restores IFN-γ and TNF-α producing T cell in pleural Tuberculosis.

    Science.gov (United States)

    Saurabh, Abhinav; Chakraborty, Sushmita; Kumar, Prabin; Mohan, Anant; Bhatnagar, Anuj K; Rishi, Narayan; Mitra, Dipendra Kumar

    2018-03-01

    Human Leukocyte Antigen-G (HLA-G), a non-classical, class Ib molecule, has been shown to mediate immunoregulatory functions by inducing apoptosis, inhibits cytotoxicity and differentiation by modulating cytokine secretion. Due to its immune-suppressive function, it facilitates tolerance in feto-maternal interface and transplantation. In contrary, it favours immune evasion of microbes and tumors by inhibiting immune and inflammatory responses. In Tuberculosis (TB), we previously reported differential expression of HLA-G and its receptor Ig-like transcript -2 (ILT-2) in disseminated vs. localized Tuberculosis. The present study explores the impact of HLA-G inhibition on the function of T cells and monocytes, in TB Pleural Effusion (PE), a localized form of TB. Blocking of HLA-G resulted in significant increase in IFN-γ and TNF-α production by CD3 + T cells. Additionally, we observed that HLA-G influences the apoptosis and cytotoxic effect of T cells from TB- PE patients. Next, we checked the impact of interaction between HLA-G and ILT-4 receptor in monocytes derived from TB-PE patients upon blocking and observed significant increase in IFN-γ production. The present study reveals for the first time HLA-G mediated suppression of Th1 cytokines, especially, IFN-γ and TNF-α in TB-PE patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

  7. TNF-related apoptosis-inducing ligand (TRAIL) for bone sarcoma treatment: Pre-clinical and clinical data.

    Science.gov (United States)

    Gamie, Zakareya; Kapriniotis, Konstantinos; Papanikolaou, Dimitra; Haagensen, Emma; Da Conceicao Ribeiro, Ricardo; Dalgarno, Kenneth; Krippner-Heidenreich, Anja; Gerrand, Craig; Tsiridis, Eleftherios; Rankin, Kenneth Samora

    2017-11-28

    Bone sarcomas are rare, highly malignant mesenchymal tumours that affect teenagers and young adults, as well as older patients. Despite intensive, multimodal therapy, patients with bone sarcomas have poor 5-year survival, close to 50%, with lack of improvement over recent decades. TNF-related apoptosis-inducing ligand (TRAIL), a member of the tumour necrosis factor (TNF) ligand superfamily (TNFLSF), has been found to induce apoptosis in cancer cells while sparing nontransformed cells, and may therefore offer a promising new approach to treatment. We cover the existing preclinical and clinical evidence about the use of TRAIL and other death receptor agonists in bone sarcoma treatment. In vitro studies indicate that TRAIL and other death receptor agonists are generally potent against bone sarcoma cell lines. Ewing's sarcoma cell lines present the highest sensitivity, whereas osteosarcoma and chondrosarcoma cell lines are considered less sensitive. In vivo studies also demonstrate satisfactory results, especially in Ewing's sarcoma xenograft models. However, the few clinical trials in the literature show only low or moderate efficacy of TRAIL in treating bone sarcoma. Potential strategies to overcome the in vivo resistance reported include co-administration with other drugs and the potential to deliver TRAIL on the surface of primed mesenchymal or immune cells and the use of targeted single chain antibodies such as scFv-scTRAIL. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Analysis of TNF-α (-308) polymorphism and gingival crevicular fluid TNF-α levels in aggressive and chronic periodontitis: A preliminary report.

    Science.gov (United States)

    Özer Yücel, Özlem; Berker, Ezel; Mesci, Lütfiye; Eratalay, Kenan; Tepe, Eser; Tezcan, İlhan

    2015-04-01

    The purpose of this study was to determine the differences in the distribution of TNF-α (-308) gene polymorphism among aggressive periodontitis, chronic periodontitis and periodontally healthy individuals and also to investigate whether this polymorphism is associated with gingival crevicular fluid TNF-α levels and periodontal disease severity. A total of 93 individuals were enrolled in the study including 38 aggressive periodontitis, 29 chronic periodontitis patients, and 26 healthy controls. Single nucleotide polymorphism at TNF-α (-308) is analyzed by PCR-RFLP method. Gingival crevicular fluid samples were analyzed for TNF-α, using ELISA. The distribution of genotypes and allele frequencies for TNF-α (-308) were similar among the groups. After stratification of patients with respect to attachment level, aggressive periodontitis patients with clinical attachment level ⩾4mm was observed to have a higher frequency of TNF-α (-308) allele 2 compared to the chronic periodontitis patients with clinical attachment level ⩾4mm. No significant differences were found between the TNF-α levels of the different genotypes in spite of an insignificant increase in patient groups carrying TNF-α (-308) allele 2. The results of this study revealed an association between TNF-α (-308) allele 2 frequency and aggressive periodontitis patients with clinical attachment level ⩾4mm in the population studied. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. Secukinumab efficacy in anti-TNF-naive and anti-TNF-experienced subjects with active ankylosing spondylitis: results from the MEASURE 2 Study.

    Science.gov (United States)

    Sieper, Joachim; Deodhar, Atul; Marzo-Ortega, Helena; Aelion, Jacob A; Blanco, Ricardo; Jui-Cheng, Tseng; Andersson, Mats; Porter, Brian; Richards, Hanno B

    2017-03-01

    There is significant unmet need in patients with ankylosing spondylitis (AS) who have inadequate response or intolerance to anti-tumour necrosis factor (TNF) treatment. Secukinumab, an anti-interleukin-17A monoclonal antibody, significantly improved signs and symptoms of AS in the MEASURE 2 study (NCT01649375). Subjects with active AS (N=219) received secukinumab (150 or 75 mg) or placebo at baseline, weeks 1, 2, 3 and 4, and every 4 weeks thereafter. Randomisation was stratified by prior anti-TNF use: anti-TNF-naive or inadequate response/intolerance to one anti-TNF (anti-TNF-IR). The primary endpoint was Assessment of SpondyloArthritis International Society criteria (ASAS) 20 at week 16. At week 16, 68.2% of anti-TNF-naive subjects treated with secukinumab 150 mg achieved ASAS20 compared with 31.1% treated with placebo (pTNF-IR group, 50.0% of subjects treated with secukinumab 150 mg achieved an ASAS20 response compared with 24.1% treated with placebo (pTNF-naive and anti-TNF-IR subjects through 52 weeks of therapy. NCT01649375. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

  10. O papel do Fator de Necrose Tumoral Alfa (TNF-alfa no processo de erosão óssea presente no colesteatoma adquirido da orelha média The role of Tumor Necrosis Factor -Alpha (TNF- alpha in bone resorption present in middle ear cholesteatoma

    Directory of Open Access Journals (Sweden)

    Rodrigo Faller Vitale

    2007-02-01

    Full Text Available O colesteatoma adquirido da orelha média causa erosão óssea, com altas taxas de morbidade e mortalidade. O TNF-alfa (TNF-alfa lambda uma das principais citocinas envolvidas neste processo. OBJETIVO: Avaliar o papel do TNF-alfa na reabsorsão óssea e a ação dele no colesteatoma. MATERIAL E MÉTODOS: Foi realizado um levantamento e uma revisão crítica da literatura. RESULTADOS: Todos os autores estudados concordam com a importância do TNF-alfa no processo de reabsorção óssea presente no colesteatoma e com o grau de destruição observado. Diferentes trabalhos demonstraram que o TNF-alfa é capaz de provocar erosão óssea, através de diferentes vias de ação. Ele pode estimular a diferenciação e a maturação dos osteoclastos ou, ainda, agir na matriz óssea expondo-a à ação dos osteoclastos. Existe a possibilidade de inibir a ação do TNF-alfa, diminuindo seus efeitos e prevenindo a perda óssea em doenças como a artrite reumatóide. Não existe, entretanto, trabalhos específicos em colesteatoma. Não existe consenso sobre a sua localização. Estas diferenças, provavelmente, ocorrem devido à distribuição dos receptores. CONCLUSÃO: O TNF-alfa, presente no colesteatoma promove a reabsorsão óssea, juntamente com outras citocinas (RANKL e IL-1, estando relacionado com a presença de complicações.Cholesteatoma may cause bone erosion, with high morbidity and mortality rates. Tumor Necrosis Factor -Alpha (TNF-a is one of the main cytokines involved in this process. Our goal was to evaluate the role of TNF-a in Bone Resorption and its effect on cholesteatoma. MATERIAL AND METHODS: analysis and critical literature review. RESULTS: Different studies have demonstrated that TNF-a is capable of causing bone erosion. It may stimulate the differentiation and maturation of osteoclasts or it may act on the bone matrix, exposing it to the action of the osteoclasts. It is possible to inhibit TNF-a, reducing its effects and prevent

  11. ¿(Anti-TNF-¿ y tuberculosis pulmonar

    Directory of Open Access Journals (Sweden)

    Carlo Vinicio Caballero Uribe

    2006-01-01

    Full Text Available Presentación de una paciente con artritis reumatoide severa en tratamiento con inhibidores del Factor de Necrosis Tumoral (Anti-TNF, quien presenta además un cuadro de tuberculosis pulmonar. La artritis reumatoide es una enfermedad inflamatoria crónica de las articulaciones, que afecta en un inicio la membrana sinovial, pero que si no es tratada oportunamente lleva a daño estructural irreversible del sistema músculo-esquelético y eventualmente de otros sistemas orgánicos. Dentro de los criterios de la American College of Rheumatology se incluyen la Rigidez Matutina, Artritis de 3 o más articulaciones, Artritis simétrica, Nódulos reumáticos, Factor Reumatoideo y hallazgos radiográficos. Dentro de la patogenia de esta enfermedad, el Factor de Necrosis Tumoral es una citocina que juega un papel importante, una producción elevada de TNF-α se ha encontrado en la sinovial de estos pacientes, y por su capacidad de inducir la producción de otras citocinas, como IL-6, IL-17, GM-CSF, M-CSF, e incluso IL-1 y TNF-α (función autócrina, parecería que el TNF-α ejerce una acción “jerárquica” dentro de la llamada red de citocinas y una inhibición de su acción da como resultado un beneficio terapéutico en los pacientes con AR. Sin embargo, es conocido que la infección concurrente más frecuentemente informada con el uso de agentes biológicos (Anti-TNF es la TB, y la incidencia de ésta se ha incrementado desde el advenimiento de la terapia biológica. Por tanto, la descripción de este caso no corresponde a un hecho médico aislado, sino a una problemática actual y real. Este es el primer caso que se reporta en la Costa Caribe.

  12. Signal transduction by the platelet-derived growth factor receptor

    International Nuclear Information System (INIS)

    Williams, L.T.; Escobedo, J.A.; Keating, M.T.; Coughlin, S.R.

    1988-01-01

    The mitogenic effects of platelet-derived growth factor (PDGF) are mediated by the PDGF receptor. The mouse PDGF receptor was recently purified on the basis of its ability to become tyrosine phosphorylated in response to the A-B human platelet form of PDGF, and the receptor amino acid sequence was determined from a full-length cDNA clone. Both the human and mouse receptor cDNA sequences have been expressed in Chinese hamster ovary fibroblast (CHO) cells that normally lack PDGF receptors. This paper summarizes recent results using this system to study signal transduction by the PDGF receptor. Some of the findings show that the KI domain of the PDGF receptor plays an important role in the stimulation of DNA synthesis by PDGF. Surprisingly, the kinase insert region is not essential for PDGF stimulation of PtdIns turnover, pH change, increase in cellular calcium, and receptor autophosphorylation. In addition, PDGF stimulates a conformational change in the receptor

  13. Isolation and characterization of the murine alpha-L-iduronidase cDNA

    Energy Technology Data Exchange (ETDEWEB)

    Clarke, L.A.; Zhang, H. Nasir, J. [Univ. of British Columbia, Vancouver (Canada)] [and others

    1994-09-01

    Mucopolysaccharidosis I (MPS I) are a group of disorders caused by deficiency of the lysosomal enzyme alpha-L-iduronidase. The characterization of the human gene and the identification of mutations underlying MPS I in humans has led to the delineation of the molecular basis of this disorder. Model systems are now needed for the evaluation and development of therapeutics for this disorder. Both canine and feline models for MPS type I have been described but only the canine gene has been isolated and characterized. We report here the cloning and expression of the murine alpha-L-iduronidase cDNA. The murine cDNA was obtained by screening a mouse liver cDNA library with a probe from the human cDNA. The full length murine cDNA is 3120 base pairs in length and thus is considerably larger than both the human and canine transcripts. The increase in size is due to a 1.2 kb 3{prime} untranslated region in the murine cDNA that contains a CA dinucleotide repeat. Within the coding region the murine cDNA shows sequences. At the protein level the murine protein shows 77% similarity with the human protein and 75% similarity with the canine protein. There are significant differences in both the start and stop sites with the murine protein 9 amino acids shorter at both the N terminal signal peptide region and the C terminus. Expression of the murine cDNA in COS-1 cells resulted in a 20 fold increase in intracellular alpha-L-iduronidase activity as well as the detection of considerable enzyme activity in the culture medium. Comparison of the reported missense mutations underlying MPS I in humans (A75T, H82P, R89Q, L218P, P533R, Q310X, T366P) has shown conservation of these amino acid residues in the murine protein. The isolation of the murine iduronidase cDNA will now allow for the development of a murine model for MPS I.

  14. Digital analysis of cDNA abundance; expression profiling by means of restriction fragment fingerprinting

    Directory of Open Access Journals (Sweden)

    Regenbogen Johannes

    2002-03-01

    Full Text Available Abstract Background Gene expression profiling among different tissues is of paramount interest in various areas of biomedical research. We have developed a novel method (DADA, Digital Analysis of cDNA Abundance, that calculates the relative abundance of genes in cDNA libraries. Results DADA is based upon multiple restriction fragment length analysis of pools of clones from cDNA libraries and the identification of gene-specific restriction fingerprints in the resulting complex fragment mixtures. A specific cDNA cloning vector had to be constructed that governed missing or incomplete cDNA inserts which would generate misleading fingerprints in standard cloning vectors. Double stranded cDNA was synthesized using an anchored oligo dT primer, uni-directionally inserted into the DADA vector and cDNA libraries were constructed in E. coli. The cDNA fingerprints were generated in a PCR-free procedure that allows for parallel plasmid preparation, labeling, restriction digest and fragment separation of pools of 96 colonies each. This multiplexing significantly enhanced the throughput in comparison to sequence-based methods (e.g. EST approach. The data of the fragment mixtures were integrated into a relational database system and queried with fingerprints experimentally produced by analyzing single colonies. Due to limited predictability of the position of DNA fragments on the polyacrylamid gels of a given size, fingerprints derived solely from cDNA sequences were not accurate enough to be used for the analysis. We applied DADA to the analysis of gene expression profiles in a model for impaired wound healing (treatment of mice with dexamethasone. Conclusions The method proved to be capable of identifying pharmacologically relevant target genes that had not been identified by other standard methods routinely used to find differentially expressed genes. Due to the above mentioned limited predictability of the fingerprints, the method was yet tested only with

  15. Generation of cDNA expression libraries enriched for in-frame sequences.

    Science.gov (United States)

    Davis, C A; Benzer, S

    1997-03-18

    Bacterial cDNA expression libraries are made to reproduce protein sequences present in the mRNA source tissue. However, there is no control over which frame of the cDNA is translated, because translation of the cDNA must be initiated on vector sequence. In a library of nondirectionally cloned cDNAs, only some 8% of the protein sequences produced are expected to be correct. Directional cloning can increase this by a factor of two, but it does not solve the frame problem. We have therefore developed and tested a library construction methodology using a novel vector, pKE-1, with which translation in the correct reading frame confers kanamycin resistance on the host. Following kanamycin selection, the cDNA libraries contained 60-80% open, in-frame clones. These, compared with unselected libraries, showed a 10-fold increase in the number of matches between the cDNA-encoded proteins made by the bacteria and database protein sequences. cDNA sequencing programs will benefit from the enrichment for correct coding sequences, and screening methods requiring protein expression will benefit from the enrichment for authentic translation products.

  16. [Construction of a yeast two-hybrid cDNA library from the human testis].

    Science.gov (United States)

    Zheng, Ying; Zhang, Lu-Ping; Jia, Xiao-Qin; Wang, Hai-Yan

    2012-04-01

    To construct a human testis cDNA library for yeast two-hybrid screening. Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library. We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length. The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.

  17. [Construction of cDNA expression library of salivary gland from Boophilus microplus].

    Science.gov (United States)

    Tian, Zhan-Cheng; Liu, Guang-Yuan; Xie, Jun-Ren; Gong, Zhen-Li

    2008-10-30

    Total RNA were isolated from salivary gland dissected from partially engorged Boophilus microplus. The mRNA was purified. A library of oligo (dT)-primed cDNA with added directional EcoR I/Hind III linkers was constructed from the purified mRNA. The constructed cDNA was ligated to the EcoR I/Hind III arms of the lambda SCREEN vector. The recombinant phage DNA was packaged by phage-marker packaging extracts, resulting in a primary cDNA library with a size of 1.38x10(6) PFU. Data showed 100% of the library were recombinant and the titer of the amplified library was 2x10(9) PFU/ml. A partial cDNA encoding cytochrome oxidase C subunit II of B. microplus was screened from the expression library with rabbit serum against B. microplus salivary gland proteins. The results is suggested that the cDNA expression library has been constructed.

  18. Construction of equalized short hairpin RNA library from human brain cDNA.

    Science.gov (United States)

    Xu, Lei; Li, Jingqi; Liu, Li; Lu, Lixia; Gao, Jingxia; Li, Xueli

    2007-02-20

    Short hairpin RNA (shRNA) library is a powerful new tool for high-throughput loss-of-function genetic screens in mammalian cells. An shRNA library can be constructed from synthetic oligonucleotides or enzymatically cleaved natural cDNA. Here, we describe a new method for constructing equalized shRNA libraries from cDNA. First, enzymatically digested cDNA fragments are equalized by a suppression PCR-based method modified from suppression subtractive hybridization. The efficiency of equalization was confirmed by quantitative real-time PCR. The fragments are then converted into an shRNA library by a series of enzymatic treatments. With this new technology, we constructed a library from human brain cDNA. Sequence analysis showed that most of the randomly selected clones had inverted repeat sequences converted from different cDNA. After transfecting HEK 293T cells and detecting gene expression, three out of eight clones were demonstrated to significantly inhibit their target genes.

  19. Molecular cloning of goat 20alpha-hydroxysteroid dehydrogenase cDNA.

    Science.gov (United States)

    Jayasekara, Walimuni Samantha Nilanthi; Yonezawa, Tomohiro; Ishida, Maho; Yamanouchi, Keitaro; Nishihara, Masugi

    2004-06-01

    20Alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which catalyzes the conversion of progesterone to its inactive form 20alpha-dihydroprogesterone, is expressed in murine placenta and has been suggested to play roles in maintaining pregnancy. To understand the role of 20alpha-HSD during pregnancy in the goat, as a first step, cloning and sequencing of 20alpha-HSD cDNA were performed. The full nucleotide sequence of 20alpha-HSD cDNA was determined on samples obtained from the corpus luteum at the luteal phase of the estrous cycle and the placenta in late pregnancy by RT-PCR and 3' and 5' RACE systems. Cloned 20alpha-HSD cDNA consisted of 1124 bp and belonged to the aldo-keto reductase superfamily. From the start codon to stop codon there were 323 amino acids, the same as in other species. To verify whether the protein derived from goat 20alpha-HSD cDNA had 20alpha-HSD activity, the cDNA was expressed by bacteria. Bacterially expressed goat 20alpha-HSD protein showed 20alpha-HSD enzyme activity. A tissue distribution study demonstrated that 20alpha-HSD was expressed in the placenta, but not in the adrenal gland, liver and spleen during pregnancy. The present study suggests that goat 20alpha-HSD is another member of the aldo-keto reductase superfamily and that it plays a role in the placenta during pregnancy.

  20. [Combining SSH and cDNA microarray for identification of lung cancer related genes].

    Science.gov (United States)

    Fan, Baoxing; Zhang, Kaitai; Da, Jiping; Xie, Ling; Wang, Shengqi; Wu, Dechang

    2003-04-20

    To screen and identify differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and some other kinds of tumor tissues using suppression subtractive hybridization (SSH) and cDNA Microarray. One cDNA chip was made by gathering clones of three differentially expressed cDNA libraries which came from BEP2D cell lines during three different malignant transformed phases. Then the clones were hybridizated with cDNA probes which extracted from 15 cases of lung cancer tissues, 5 cases of paracancerous pulmonary tissues and 24 cases of other 8 kinds of tumor tissues respectively. Twenty-six cDNAs were obtained which expressed higher in lung cancer tissues than that in paracancerous pulmonary tissues. Thirty-one cDNAs expressed remarkably higher in paracancerous tissues than those in cancer tissues. Compared with other 8 kinds of tumors, paracancerous tissues had 63 overexpressed cDNAs and lung cancer tissues had 87 overexpressed cDNAs. The combination of SSH and cDNA microarray is rapid and effective for screening and identification of differentially expressed genes in different samples. It may be potentially useful for diagnosis of lung cancer to further study the differentially expressed genes among lung cancer tissues, paracancerous pulmonary tissues and other tumor tissues.

  1. Sequence of a cDNA encoding turtle high mobility group 1 protein.

    Science.gov (United States)

    Zheng, Jifang; Hu, Bi; Wu, Duansheng

    2005-07-01

    In order to understand sequence information about turtle HMG1 gene, a cDNA encoding HMG1 protein of the Chinese soft-shell turtle (Pelodiscus sinensis) was amplified by RT-PCR from kidney total RNA, and was cloned, sequenced and analyzed. The results revealed that the open reading frame (ORF) of turtle HMG1 cDNA is 606 bp long. The ORF codifies 202 amino acid residues, from which two DNA-binding domains and one polyacidic region are derived. The DNA-binding domains share higher amino acid identity with homologues sequences of chicken (96.5%) and mammalian (74%) than homologues sequence of rainbow trout (67%). The polyacidic region shows 84.6% amino acid homology with the equivalent region of chicken HMG1 cDNA. Turtle HMG1 protein contains 3 Cys residues located at completely conserved positions. Conservation in sequence and structure suggests that the functions of turtle HMG1 cDNA may be highly conserved during evolution. To our knowledge, this is the first report of HMG1 cDNA sequence in any reptilian.

  2. Telmisartan, a possible PPAR-δ agonist, reduces TNF-α-stimulated VEGF-C production by inhibiting the p38MAPK/HSP27 pathway in human proximal renal tubular cells

    Energy Technology Data Exchange (ETDEWEB)

    Kimura, Hideki, E-mail: hkimura@u-fukui.ac.jp [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Department of Clinical Laboratories and Nephrology, University of Fukui Hospital, Fukui (Japan); Mikami, Daisuke; Kamiyama, Kazuko [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Sugimoto, Hidehiro [Department of Clinical Laboratories and Nephrology, University of Fukui Hospital, Fukui (Japan); Kasuno, Kenji; Takahashi, Naoki [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Yoshida, Haruyoshi [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan); Division of Nephrology, Obama Municipal Hospital, Obama, Fukui (Japan); Iwano, Masayuki [Division of Nephrology, Department of General Medicine, School of Medicine, Faculty of Medical Sciences, University of Fukui, Fukui (Japan)

    2014-11-14

    Highlights: • TNF-α increased VEGF-C expression by enhancing phosphorylation of p38MAPK and HSP27. • Telmisartan decreased TNF-α-stimulated expression of VEGF-C. • Telmisartan suppressed TNF-α-induced phosphorylation of p38MAPK and HSP27. • Telmisartan activated endogenous PPAR-δ protein. • Telmisartan suppressed p38MAPK phosphorylation in a PPAR-δ-dependent manner. - Abstract: Vascular endothelial growth factor-C (VEGF-C) is a main inducer of inflammation-associated lymphangiogenesis in various inflammatory disorders including chronic progressive kidney diseases, for which angiotensin II receptor type 1 blockers (ARBs) are widely used as the main treatment. Although proximal renal tubular cells may affect the formation of lymphatic vessels in the interstitial area by producing VEGF-C, the molecular mechanisms of VEGF-C production and its manipulation by ARB have not yet been examined in human proximal renal tubular epithelial cells (HPTECs). In the present study, TNF-α dose-dependently induced the production of VEGF-C in HPTECs. The TNF-α-induced production of VEGF-C was mediated by the phosphorylation of p38MAPK and HSP27, but not by that of ERK or NFkB. Telmisartan, an ARB that can activate the peroxisome proliferator-activated receptor (PPAR), served as a PPAR-δ activator and reduced the TNF-α-stimulated production of VEGF-C. This reduction was partially attributed to a PPAR-δ-dependent decrease in p38MAPK phosphorylation. Our results indicate that TNF-α induced the production of VEGF-C in HPTECs by activating p38MAPK/HSP27, and this was partially inhibited by telmisartan in a PPAR-δ dependent manner. These results provide a novel insight into inflammation-associated lymphangiogenesis.

  3. Molecular characterization of an. alpha. sub 2B -adrenergic receptor

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, J.K.; Dewan Zeng; D' Angelo, D.D.; Tucker, A.L.; Zhihong Lu; Barber, C.M.; Lynch, K.R. (Univ. of Virginia Health Sciences Center, Charlottesville (United States))

    1990-02-26

    {alpha}{sub 2}-Adrenergic receptors comprise a heterogeneous population based on pharmacologic and molecular evidence. The authors have isolated a cDNA clone (pRNG{alpha}2) encoding a previously undescribed third subtype of an {alpha}{sub 2}-adrenergic receptor from a rat kidney cDNA library. The library was screened with an oligonucleotide encoding a highly conserved region found in all biogenic amine receptors described to date. The deduced amino acid sequence displays many features of G-protein coupled receptors with exception of the absence of the consensus N-linked glycosylation site at the amino terminus. Membranes prepared from COS-1 cells transfected with pRNG{alpha}2 display high affinity and saturable binding to {sup 3}H-rauwolscine (K{sub d}=2 nM).Competition curve data analysis shows that pRNG{alpha}2 protein binds to a variety of adrenergic drugs with the following rank order of potency: yohimbine {ge} cholorpromazine > prazosin {ge} clonidine > norepinephrine {ge} oxymetazoline. pRNG{alpha}2 RNA accumulates in both adult rat kidney and rat neonatal lung (predominant species is 4.0 kb). They conclude that pRNG{alpha}2 likely represents a cDNA for the {alpha}{sub 2B}-adrenergic receptor.

  4. Molecular characterization of thyroid hormone receptor beta from Schistosoma japonicum and assessment of its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice

    Directory of Open Access Journals (Sweden)

    Qiu Chunhui

    2012-08-01

    Full Text Available Abstract Background Thyroid hormones (TH modulate growth, development and differentiation and metabolic processes by interacting with thyroid hormone receptors (THRs. The purpose of this study was to identify a novel thyroid hormone receptor beta encoding gene of Schistosoma japonicum (SjTHRβ and to investigate its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice. Methods The full-length cDNA sequence of SjTHRβ, its gene organization, and its transcript levels were characterized, and the phylogenetic relationship between THR, RAR and RXR from other organisms were analysis, the ability of this protein binding to a conserved DNA core motif, and its potential as a vaccine candidate antigen against schistosomiasis in BALB/c mice were evaluated. Results The SjTHRβ cDNA was cloned, verified by 5’ and 3’ Rapid Amplification of cDNA Ends and shown to be polyadenylated at the 3’end, suggesting the transcript is full-length. SjTHRβ is homologous to THRs from other species and has a predicted conservative DNA binding domain and ligand binding domain that normally characterizes these receptors. A comparative quantitative PCR analysis showed that SjTHRβ was the highest expressed in 21d worms and the lowest in 7 d and 13 d schistosomula. The cDNA corresponding to DNA binding domain (SjTHRβ-DBD and ligand binding domain (SjTHRβ-LBD were cloned and subsequently expressed in E coli. The expressed proteins were used to immunize mice and generate specific serum against recombinant SjTHRβ (rSjTHRβ. Western blotting revealed that anti-rSjTHRβ-LBD serum recognized two protein bands in extracts from 21 d worm with molecular sizes of approximately 95 kDa and 72 kDa. Electrophoretic mobility shift assay (EMSA analysis showed that rSjTHRβ-DBD could bind to a conserved DNA core motif. Immunization of BALB/c mice with rSjTHRβ-LBD could induce partial protective efficacy(27.52% worm reduction and 29.50% liver eggs

  5. B-cell phenotype and IgD-CD27- memory B cells are affected by TNF-inhibitors and tocilizumab treatment in rheumatoid arthritis.

    Directory of Open Access Journals (Sweden)

    Rita A Moura

    Full Text Available The use of TNF-inhibitors and/or the IL-6 receptor antagonist, tocilizumab, in rheumatoid arthritis (RA have pleiotropic effects that also involve circulating B-cells. The main goal of this study was to assess the effect of TNF-inhibitors and tocilizumab on B-cell phenotype and gene expression in RA.Blood samples were collected from untreated early RA (ERA patients, established RA patients under methotrexate treatment, established RA patients before and after treatment with TNF-inhibitors and tocilizumab, and healthy donors. B-cell subpopulations were characterized by flow cytometry and B-cell gene expression was analyzed by real-time PCR on isolated B-cells. Serum levels of BAFF, CXCL13 and sCD23 were determined by ELISA.The frequency of total CD19+ B cells in circulation was similar between controls and all RA groups, irrespective of treatment, but double negative (DN IgD-CD27- memory B cells were significantly increased in ERA and established RA when compared to controls. Treatment with TNF-inhibitors and tocilizumab restored the frequency of IgD-CD27- B-cells to normal levels, but did not affect other B cell subpopulations. TACI, CD95, CD5, HLA-DR and TLR9 expression on B-cells significantly increased after treatment with either TNF-inhibitors and/ or tocilizumab, but no significant changes were observed in BAFF-R, BCMA, CD69, CD86, CXCR5, CD23, CD38 and IgM expression on B-cells when comparing baseline with post-treatment follow-ups. Alterations in B-cell gene expression of BAFF-R, TACI, TLR9, FcγRIIB, BCL-2, BLIMP-1 and β2M were found in ERA and established RA patients, but no significant differences were observed after TNF-inhibitors and tocilizumab treatment when comparing baseline and follow-ups. Serum levels of CXCL13, sCD23 and BAFF were not significantly affected by treatment with TNF-inhibitors and tocilizumab.In RA patients, the use of TNF-inhibitors and/ or tocilizumab treatment affects B-cell phenotype and IgD-CD27- memory B

  6. TNF-α-Induced NOD2 and RIP2 Contribute to the Up-Regulation of Cytokines Induced by MDP in Monocytic THP-1 Cells.

    Science.gov (United States)

    Chen, Xiaobin; Xiao, Zhilin; Xie, Xiumei; Liu, Xueting; Jiang, Manli; Yuan, Chuang; Yang, Li; Hu, Jinyue

    2017-06-21

    Nucleotide-binding oligomerization domain containing 2 (NOD2)-induced signal transduction and cytokine production is regulated by a number of factors. However, the feedback effect of the pro-inflammatory TNF-α on NOD2-induced inflammation is not fully understood. In this study, we found unexpectedly that TNF-α up-regulated NOD2 ligand MDP-induced production of the CXC chemokines, including CXCL1, 2, and 8, and the pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, in a dose-dependent manner at both mRNA and protein levels in monocytic THP-1 cells. Though TNF-α induced the up-regulation of ubiquitin-editing enzyme A20, an important negative regulator for Toll-like receptor- and NOD2-induced inflammatory responses, the over-expression of A20 by gene transfer did not reversed MDP-induced production of cytokines, suggested that A20 did not regulate the functions of NOD2 in THP-1 cells. Meanwhile, we found that TNF-α up-regulated NOD2 and its down-stream adaptor protein RIP2 at both mRNA and protein levels. MDP induced the activation of ERK, JNK, p38 and NF-κB, and TNF-α pre-treatment augmented this activation. The results from pharmacological inhibition assay showed that cytokine production was dependent on MAPK signaling. In addition, we found that the pre-treatment of THP-1 cells with MDP down-regulated the mRNA levels of cytokine induced by MDP re-treatment. MDP pre-treatment up-regulated NOD2, but down-regulated RIP2, and down-regulated NOD2 signal transduction induced by MDP re-stimulation. Taking together, these results suggested that TNF-α is a positive regulator for NOD2 functions via up-regulation of NOD2 and its signal adaptor RIP2, and TNF-α-induced A20 does not regulate MDP-induced inflammatory responses in THP-1 cells. J. Cell. Biochem. 9999: 1-10, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  7. Expressed sequence tags: normalization and subtraction of cDNA libraries expressed sequence tags\\ normalization and subtraction of cDNA libraries.

    Science.gov (United States)

    Soares, Marcelo Bento; de Fatima Bonaldo, Maria; Hackett, Jeremiah D; Bhattacharya, Debashish

    2009-01-01

    Expressed Sequence Tags (ESTs) provide a rapid and efficient approach for gene discovery and analysis of gene expression in eukaryotes. ESTs have also become particularly important with recent expanded efforts in complete genome sequencing of understudied, nonmodel eukaryotes such as protists and algae. For these projects, ESTs provide an invaluable source of data for gene identification and prediction of exon-intron boundaries. The generation of EST data, although straightforward in concept, requires nonetheless great care to ensure the highest efficiency and return for the investment in time and funds. To this end, key steps in the process include generation of a normalized cDNA library to facilitate a high gene discovery rate followed by serial subtraction of normalized libraries to maintain the discovery rate. Here we describe in detail, protocols for normalization and subtraction of cDNA libraries followed by an example using the toxic dinoflagellate Alexandrium tamarense.

  8. Functional discrepancies between tumor necrosis factor and lymphotoxin alpha explained by trimer stability and distinct receptor interactions

    DEFF Research Database (Denmark)

    Schuchmann, M; Hess, S; Bufler, P

    1995-01-01

    interaction with the human p55TNFR. This was demonstrated in NIH 3T3 cells transfected with the human p55TNFR, where cytotoxicity is mediated exclusively by the transfected receptor. Although the p55ATNFR had virtually identical affinities for TNF and LT alpha, as defined by Scatchard analysis......Tumor necrosis factor (TNF) and lymphotoxin alpha (LT alpha) are closely related cytokines which bind with nearly identical affinities to the same pair of cell surface receptors, p55 and p75TNFR. Therefore it is assumed that TNF and LT alpha are redundant cytokines. This study, however...

  9. A novel member of the interferon receptor family complements functionality of the murine interferon gamma receptor in human cells.

    Science.gov (United States)

    Hemmi, S; Böhni, R; Stark, G; Di Marco, F; Aguet, M

    1994-03-11

    Expression of the human interferon gamma receptor (IFN-gamma R) in mouse cells is not sufficient to confer biological responsiveness to human IFN-gamma and vice versa. An additional species-specific component is required for signal transduction. We identified this cofactor by expression cloning in simian COS cells stably transfected with the nonfunctional murine IFN-gamma R and a IFN-gamma-inducible reporter construct encoding the human Tac antigen (interleukin-2 receptor alpha chain, CD25). A cDNA clone was obtained that, upon stable transfection, rendered human HEp-2 cells expressing the murine IFN-gamma R fully responsive to murine IFN-gamma. This cDNA encodes a novel 332 amino acid type I transmembrane protein that belongs to the IFN receptor family and that we designate IFN-gamma R beta chain.

  10. ESTs, cDNA microarrays, and gene expression profiling: tools for dissecting plant physiology and development.

    Science.gov (United States)

    Alba, Rob; Fei, Zhangjun; Payton, Paxton; Liu, Yang; Moore, Shanna L; Debbie, Paul; Cohn, Jonathan; D'Ascenzo, Mark; Gordon, Jeffrey S; Rose, Jocelyn K C; Martin, Gregory; Tanksley, Steven D; Bouzayen, Mondher; Jahn, Molly M; Giovannoni, Jim

    2004-09-01

    Gene expression profiling holds tremendous promise for dissecting the regulatory mechanisms and transcriptional networks that underlie biological processes. Here we provide details of approaches used by others and ourselves for gene expression profiling in plants with emphasis on cDNA microarrays and discussion of both experimental design and downstream analysis. We focus on methods and techniques emphasizing fabrication of cDNA microarrays, fluorescent labeling, cDNA hybridization, experimental design, and data processing. We include specific examples that demonstrate how this technology can be used to further our understanding of plant physiology and development (specifically fruit development and ripening) and for comparative genomics by comparing transcriptome activity in tomato and pepper fruit.

  11. Construction and analysis of full-length and normalized cDNA libraries from citrus.

    Science.gov (United States)

    Marques, M Carmen; Perez-Amador, Miguel A

    2012-01-01

    We have developed an integrated method to generate a normalized cDNA collection enriched in full-length and rare transcripts from citrus, using different species and multiple tissues and developmental stages. Interpretation of ever-increasing raw sequence information generated by modern genome sequencing technologies faces multiple challenges, such as gene function analysis and genome annotation. In this regard, the availability of full-length cDNA clones facilitates functional analysis of the corresponding genes enabling manipulation of their expression and the generation of a variety of tagged versions of the native protein. The development of full-length cDNA sequences has the power to improve the quality of genome annotation, as well as provide tools for functional characterization of genes.

  12. Design and Screening of M13 Phage Display cDNA Libraries

    Directory of Open Access Journals (Sweden)

    Yuliya Georgieva

    2011-02-01

    Full Text Available The last decade has seen a steady increase in screening of cDNA expression product libraries displayed on the surface of filamentous bacteriophage. At the same time, the range of applications extended from the identification of novel allergens over disease markers to protein-protein interaction studies. However, the generation and selection of cDNA phage display libraries is subjected to intrinsic biological limitations due to their complex nature and heterogeneity, as well as technical difficulties regarding protein presentation on the phage surface. Here, we review the latest developments in this field, discuss a number of strategies and improvements anticipated to overcome these challenges making cDNA and open reading frame (ORF libraries more readily accessible for phage display. Furthermore, future trends combining phage display with next generation sequencing (NGS will be presented.

  13. Primate TNF Promoters Reveal Markers of Phylogeny and Evolution of Innate Immunity

    Science.gov (United States)

    Baena, Andres; Ligeiro, Filipa; Diop, Ousmane M.; Brieva, Claudia; Gagneux, Pascal; O'Brien, Stephen J.; Ryder, Oliver A.; Goldfeld, Anne E.

    2007-01-01

    Background Tumor necrosis factor (TNF) is a critical cytokine in the immune response whose transcriptional activation is controlled by a proximal promoter region that is highly conserved in mammals and, in particular, primates. Specific single nucleotide polymorphisms (SNPs) upstream of the proximal human TNF promoter have been identified, which are markers of human ancestry. Methodology/Principal findings Using a comparative genomics approach we show that certain fixed genetic differences in the TNF promoter serve as markers of primate speciation. We also demonstrate that distinct alleles of most human TNF promoter SNPs are identical to fixed nucleotides in primate TNF promoters. Furthermore, we identify fixed genetic differences within the proximal TNF promoters of Asian apes that do not occur in African ape or human TNF promoters. Strikingly, protein-DNA binding assays and gene reporter assays comparing these Asian ape TNF promoters to African ape and human TNF promoters demonstrate that, unlike the fixed differences that we define that are associated with primate phylogeny, these Asian ape-specific fixed differences impair transcription factor binding at an Sp1 site and decrease TNF transcription induced by bacterial stimulation of macrophages. Conclusions/significance Here, we have presented the broadest interspecies comparison of a regulatory region of an innate immune response gene to date. We have characterized nucleotide positions in Asian ape TNF promoters that underlie functional changes in cell type- and stimulus-specific activation of the TNF gene. We have also identified ancestral TNF promoter nucleotide states in the primate lineage that correspond to human SNP alleles. These findings may reflect evolution of Asian and African apes under a distinct set of infectious disease pressures involving the innate immune response and TNF. PMID:17637837

  14. [Screening of specifically expressed genes in amphioxus neurula by construction of a subtractive cDNA library].

    Science.gov (United States)

    Zhang, Lei; Yang, Yong-Jie; Zhang, Yan-Jun

    2010-12-01

    To screen specifically expressed genes in the development of nerve, muscle, and body axis of amphioxus, Branchiostoma belcheri tsingtauenese. A subtractive cDNA library was constructed from the 12-hour amphioxus neurula cDNA after subtractively hybridized with the 6-hour amphioxus gastrula cDNA. The total RNA was extracted from the 12-hour neurula and 6-hour gastrula, then reverse transcribed into cDNA. The 12-hour neurula cDNA was designated as the experimental group (the tester) and the 6-hour gastrula cDNA as the control group (the driver). The differentially expressed sequences were exponentially amplified using suppression PCR. Background was subtracted and differentially expressed sequences were further enriched. The PCR products were ligated to the T Vector. After transformation of the recombinant plasmid carrying inserted amphioxus cDNA into E.coli host cells, the cDNA library was constructed successfully. Two hundred randomly chosen positive clones were sequenced and some of neurula-specifically expressed genes were obtained. SSH is an effective method for searching differentially expressed genes. The subtractive cDNA library we generated provides a tool for further study of regulatory mechanisms of amphioxus early embryonic development.

  15. Gene therapy for bladder pain with gene gun particle encoding pro-opiomelanocortin cDNA.

    Science.gov (United States)

    Chuang, Yao-Chi; Chou, A-K; Wu, P-C; Chiang, Po-Hui; Yu, T-J; Yang, L-C; Yoshimura, Naoki; Chancellor, Michael B

    2003-11-01

    Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

  16. Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA

    Directory of Open Access Journals (Sweden)

    Karlovsky Petr

    2008-10-01

    Full Text Available Abstract Background cDNA-AFLP is a transcriptomics technique which does not require prior sequence information and can therefore be used as a gene discovery tool. The method is based on selective amplification of cDNA fragments generated by restriction endonucleases, electrophoretic separation of the products and comparison of the band patterns between treated samples and controls. Unequal distribution of restriction sites used to generate cDNA fragments negatively affects the performance of cDNA-AFLP. Some transcripts are represented by more than one fragment while other escape detection, causing redundancy and reducing the coverage of the analysis, respectively. Results With the goal of improving the coverage of cDNA-AFLP without increasing its redundancy, we designed a modified cDNA-AFLP protocol. Immobilized cDNA is sequentially digested with several restriction endonucleases and the released DNA fragments are collected in mutually exclusive pools. To investigate the performance of the protocol, software tool MECS (Multiple Enzyme cDNA-AFLP Simulation was written in Perl. cDNA-AFLP protocols described in the literatur and the new sequential digestion protocol were simulated on sets of cDNA sequences from mouse, human and Arabidopsis thaliana. The redundancy and coverage, the total number of PCR reactions, and the average fragment length were calculated for each protocol and cDNA set. Conclusion Simulation revealed that sequential digestion of immobilized cDNA followed by the partitioning of released fragments into mutually exclusive pools outperformed other cDNA-AFLP protocols in terms of coverage, redundancy, fragment length, and the total number of PCRs. Primers generating 30 to 70 amplicons per PCR provided the highest fraction of electrophoretically distinguishable fragments suitable for normalization. For A. thaliana, human and mice transcriptome, the use of two marking enzymes and three sequentially applied releasing enzymes for each

  17. [Construction and characterization of a cDNA library from human liver tissue of cirrhosis].

    Science.gov (United States)

    Chen, Xiao-hong; Chen, Zhi; Chen, Feng; Zhu, Hai-hong; Zhou, Hong-juan; Yao, Hang-ping

    2005-03-01

    To construct a cDNA library from human liver tissue of cirrhosis. The total RNA from human liver tissue of cirrhosis was extracted using Trizol method, and the mRNA was purified using mRNA purification kit. SMART technique and CDSIII/3' primer were used for first-strand cDNA synthesis. Long distance PCR was then used to synthesize the double-strand cDNA that was then digested by proteinase K and Sfi I, and was fractionated by CHOMA SPIN-400 column. The cDNA fragments longer than 0.4 kb were collected and ligated to lambdaTripl Ex2 vector. Then lambda-phage packaging reaction and library amplification were performed. The qualities of both unamplified and amplified cDNA libraries was strictly checked by conventional titer determination. Eleven plaques were randomly picked and tested using PCR with universal primers derived from the sequence flanking the vector. The titers of unamplifed and amplified libraries were 1.03 x 10(6) pfu/ml and 1.36 x 10(9) pfu/ml respectively. The percentages of recombinants from both libraries were 97.24 % in unamplified library and 99.02 % in amplified library. The lengths of the inserts were 1.02 kb in average (36.36 % 1 approximately equals 2 kb and 63.64 % 0.5 approximately equals 1.0 kb). A high quality cDNA library from human liver tissue of cirrhosis was constructed successfully, which can be used for screening and cloning new special genes associated with the occurrence of cirrhosis.

  18. Display of a maize cDNA library on baculovirus infected insect cells.

    Science.gov (United States)

    Meller Harel, Helene Y; Fontaine, Veronique; Chen, Hongying; Jones, Ian M; Millner, Paul A

    2008-08-12

    Maize is a good model system for cereal crop genetics and development because of its rich genetic heritage and well-characterized morphology. The sequencing of its genome is well advanced, and new technologies for efficient proteomic analysis are needed. Baculovirus expression systems have been used for the last twenty years to express in insect cells a wide variety of eukaryotic proteins that require complex folding or extensive posttranslational modification. More recently, baculovirus display technologies based on the expression of foreign sequences on the surface of Autographa californica (AcMNPV) have been developed. We investigated the potential of a display methodology for a cDNA library of maize young seedlings. We constructed a full-length cDNA library of young maize etiolated seedlings in the transfer vector pAcTMVSVG. The library contained a total of 2.5 x 10(5) independent clones. Expression of two known maize proteins, calreticulin and auxin binding protein (ABP1), was shown by western blot analysis of protein extracts from insect cells infected with the cDNA library. Display of the two proteins in infected insect cells was shown by selective biopanning using magnetic cell sorting and demonstrated proof of concept that the baculovirus maize cDNA display library could be used to identify and isolate proteins. The maize cDNA library constructed in this study relies on the novel technology of baculovirus display and is unique in currently published cDNA libraries. Produced to demonstrate proof of principle, it opens the way for the development of a eukaryotic in vivo display tool which would be ideally suited for rapid screening of the maize proteome for binding partners, such as proteins involved in hormone regulation or defence.

  19. Cloning, sequencing, and expression of cDNA for human β-glucuronidase

    International Nuclear Information System (INIS)

    Oshima, A.; Kyle, J.W.; Miller, R.D.

    1987-01-01

    The authors report here the cDNA sequence for human placental β-glucuronidase (β-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH 2 -terminal amino acid sequence determined for human spleen β-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human β-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human β-glucuronidase, demonstrate the existence of two populations of mRNA for β-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length

  20. Pretreatment with a 55-kDa tumor necrosis factor receptor-immunoglobulin fusion protein attenuates activation of coagulation, but not of fibrinolysis, during lethal bacteremia in baboons

    NARCIS (Netherlands)

    van der Poll, T.; Jansen, P. M.; van Zee, K. J.; Hack, C. E.; Oldenburg, H. A.; Loetscher, H.; Lesslauer, W.; Lowry, S. F.; Moldawer, L. L.

    1997-01-01

    Baboons (Papio anubis) receiving a lethal intravenous infusion with live Escherichia coli were pretreated with either a 55-kDa tumor necrosis factor (TNF) receptor-IgG fusion protein (TNFR55:IgG) (n = 4, 4.6 mg/kg) or placebo (n = 4). Neutralization of TNF activity in TNFR55:IgG-treated animals was

  1. Elevated interferon gamma expression in the central nervous system of tumour necrosis factor receptor 1-deficient mice with experimental autoimmune encephalomyelitis

    DEFF Research Database (Denmark)

    Wheeler, Rachel D; Zehntner, Simone P; Kelly, Lisa M

    2006-01-01

    Inflammation in the central nervous system (CNS) can be studied in experimental autoimmune encephalomyelitis (EAE). The proinflammatory cytokines interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF) are implicated in EAE pathogenesis. Signals through the type 1 TNF receptor (TNFR1...

  2. Optimized cDNA libraries for virus-induced gene silencing (VIGS using tobacco rattle virus

    Directory of Open Access Journals (Sweden)

    Page Jonathan E

    2008-01-01

    Full Text Available Abstract Background Virus-induced gene silencing (VIGS has emerged as a method for performing rapid loss-of-function experiments in plants. Despite its expanding use, the effect of host gene insert length and other properties on silencing efficiency have not been systematically tested. In this study, we probed the optimal properties of cDNA fragments of the phytoene desaturase (PDS gene for efficient VIGS in Nicotiana benthamiana using tobacco rattle virus (TRV. Results NbPDS inserts of between 192 bp and 1304 bp led to efficient silencing as determined by analysis of leaf chlorophyll a levels. The region of the NbPDS cDNA used for silencing had a small effect on silencing efficiency with 5' and 3' located inserts performing more poorly than those from the middle. Silencing efficiency was reduced by the inclusion of a 24 bp poly(A or poly(G homopolymeric region. We developed a method for constructing cDNA libraries for use as a source of VIGS-ready constructs. Library construction involved the synthesis of cDNA on a solid phase support, digestion with RsaI to yield short cDNA fragments lacking poly(A tails and suppression subtractive hybridization to enrich for differentially expressed transcripts. We constructed two cDNA libraries from methyl-jasmonate treated N. benthamiana roots and obtained 2948 ESTs. Thirty percent of the cDNA inserts were 401–500 bp in length and 99.5% lacked poly(A tails. To test the efficiency of constructs derived from the VIGS-cDNA libraries, we silenced the nicotine biosynthetic enzyme, putrescine N-methyltransferase (PMT, with ten different VIGS-NbPMT constructs ranging from 122 bp to 517 bp. Leaf nicotine levels were reduced by more than 90% in all plants infected with the NbPMT constructs. Conclusion Based on the silencing of NbPDS and NbPMT, we suggest the following design guidelines for constructs in TRV vectors: (1 Insert lengths should be in the range of ~200 bp to ~1300 bp, (2 they should be positioned in

  3. Construction of cDNA libraries: focus on protists and fungi.

    Science.gov (United States)

    Rodríguez-Ezpeleta, Naiara; Teijeiro, Shona; Forget, Lise; Burger, Gertraud; Lang, B Franz

    2009-01-01

    Sequencing of cDNA libraries is an efficient and inexpensive approach to analyze the protein-coding portion of a genome. It is frequently used for surveying the genomes of poorly studied eukaryotes, and is particularly useful for species that are not easily amenable to genome sequencing, because they are nonaxenic and/or difficult to cultivate. In this chapter, we describe protocols that have been applied successfully to construct and normalize a variety of cDNA libraries from many different species of free-living protists and fungi, and that require only small quantities of cell material.

  4. Development of an HIV-based cDNA expression cloning system.

    Science.gov (United States)

    van Maanen, Marc; Tidwell, Jennie K; Donehower, Lawrence A; Sutton, Richard E

    2003-07-01

    Expression cloning of cDNAs is a powerful tool with which to identify genes based on their specific functional properties. Here we describe the development of a cDNA library transfer system based on the human immunodeficiency virus type-1 (HIV). This system represents an improvement over current oncoretroviral cDNA expression systems in terms of target cell range and the inclusion of a selectable marker. By use of a simple packaging system, we were able to produce high-titer vector stocks from HIV vector-based cDNA libraries and demonstrate highly efficient cDNA expression cloning in three model experiments. First, HOS TK(-) cells, which are null for thymidine kinase (TK) expression, were transduced with an HIV-based cDNA library derived from primary human foreskin fibroblasts (HFFs) and functionally selected for TK expression. In a second experiment, hypoxanthine guanine phosphoribosyltransferase-1-deficient (HPRT(-)) fibroblasts were transduced with a T cell (PM1) line-derived cDNA library and selected for HPRT expression. Both TK (frequency 1 in 5.0 x 10(4)) and HPRT (frequency 1 in 2.0 x 10(4)) cDNAs were readily isolated from these HIV-based cDNA libraries. As a third example, we demonstrated the ability of this vector system to allow functional cDNA library screens to be performed in primary, mitotically inactive cell types. Using senescent HFFs as a target cell population, we were able to isolate SV40 large T antigen cDNA-containing clones (frequency 1 in 2.5 x 10(4)) based on their ability to overcome the senescence-induced block to cell proliferation. Thus, this system can be used to clone relatively low-abundance cDNAs based upon their expression. Because of the ability of HIV-based vectors to transduce primary and nondividing cells efficiently, this vector system will further broaden the range of cell types in which expression cloning studies can be performed.

  5. Expressions of toll-like receptors 2 and 4, and relative cellular ...

    African Journals Online (AJOL)

    Purpose: To investigate the expressions of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), tumor necrosis factor alpha (TNF-α), IFN-γ (IFN- gamma), interleukin 2 (IL-2), interleukin 6 (IL-6) and interleukin 10 (IL-10) in human immunodeficiency virus (HIV) patients with tuberculosis (TB) infection. Methods: Two groups of ...

  6. Matrix metalloproteinase-9 (MMP9) is involved in the TNF-α-induced fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells.

    Science.gov (United States)

    Weiler, Julian; Mohr, Marieke; Zänker, Kurt S; Dittmar, Thomas

    2018-04-10

    In addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α). The gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody. The data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. The matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline

  7. 15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-α by decreasing levels of acetylated histone H3 and H4 at its promoter

    International Nuclear Information System (INIS)

    Engdahl, Ryan; Monroy, M. Alexandra; Daly, John M.

    2007-01-01

    Prostaglandin metabolite 15-Deoxy-Δ 12,14 -prostaglandin J2 (15d-PGJ2) is known to inhibit a number of pro-inflammatory cytokines as well as being a ligand for nuclear receptor PPARγ. We investigated the ability of 15d-PGJ2 to inhibit TNF-α gene expression through mechanisms that involve histone modification. Pretreatment with 15d-PGJ2 (10 μM) inhibited LPS-stimulated TNF-α mRNA in THP-1 monocytes or PMA-differentiated cells to nearly basal levels. A specific PPARγ ligand, GW1929, failed to inhibit LPS-induced TNF-α mRNA expression nor did a PPARγ antagonist, GW9662, alter the repression of TNF-α mRNA in LPS-stimulated cells pretreated with 15d-PGJ2 suggesting a PPARγ-independent inhibition of TNF-α mRNA in THP-1 cells. Transfection studies with a reporter construct and subsequent treatment with 15d-PGJ2 demonstrated a dose-dependent inhibition of the TNF-α promoter. Additional studies demonstrated that inhibition of histone deacetylases with trichostatin A (TSA) or overexpression of histone acetyltransferase CBP could overcome 15d-PGJ2-mediated repression of the TNF-α promoter, suggesting that an important mechanism whereby 15d-PGJ2 suppresses a cytokine is through factors that regulate histone modifications. To examine the endogenous TNF-α promoter, chromatin immunoprecipitations (ChIP) were performed. ChIP assays demonstrated that LPS stimulation induced an increase in histone H3 and H4 acetylation at the TNF-α promoter, which was reduced in cells pretreated with 15d-PGJ2. These results highlight the ability of acetylation and deacetylation factors to affect the TNF-α promoter and demonstrate that an additional important mechanism whereby 15d-PGJ2 mediates TNF-α transcriptional repression by altering levels of acetylated histone H3 and H4 at its promoter

  8. Detection of melatonin receptor mRNA in human muscle

    International Nuclear Information System (INIS)

    Li Lei

    2004-01-01

    To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

  9. Genetic characterization and evolutionary inference of TNF-α through computational analysis

    Directory of Open Access Journals (Sweden)

    Gauri Awasthi

    Full Text Available TNF-α is an important human cytokine that imparts dualism in malaria pathogenicity. At high dosages, TNF-α is believed to provoke pathogenicity in cerebral malaria; while at lower dosages TNF-α is protective against severe human malaria. In order to understand the human TNF-α gene and to ascertain evolutionary aspects of its dualistic nature for malaria pathogenicity, we characterized this gene in detail in six different mammalian taxa. The avian taxon, Gallus gallus was included in our study, as TNF-α is not present in birds; therefore, a tandemly placed duplicate of TNF-α (LT-α or TNF-β was included. A comparative study was made of nucleotide length variations, intron and exon sizes and number variations, differential compositions of coding to non-coding bases, etc., to look for similarities/dissimilarities in the TNF-α gene across all seven taxa. A phylogenetic analysis revealed the pattern found in other genes, as humans, chimpanzees and rhesus monkeys were placed in a single clade, and rats and mice in another; the chicken was in a clearly separate branch. We further focused on these three taxa and aligned the amino acid sequences; there were small differences between humans and chimpanzees; both were more different from the rhesus monkey. Further, comparison of coding and non-coding nucleotide length variations and coding to non-coding nucleotide ratio between TNF-α and TNF-β among these three mammalian taxa provided a first-hand indication of the role of the TNF-α gene, but not of TNF-β in the dualistic nature of TNF-α in malaria pathogenicity.

  10. Molecular cloning and genomic organization of a second probable allatostatin receptor from Drosophila melanogaster

    DEFF Research Database (Denmark)

    Lenz, C; Williamson, M; Grimmelikhuijzen, C J

    2000-01-01

    We (C. Lenz et al. (2000) Biochem. Biophys. Res. Commun. 269, 91-96) and others (N. Birgül et al. (1999) EMBO J. 18, 5892-5900) have recently cloned a Drosophila receptor that was structurally related to the mammalian galanin receptors, but turned out to be a receptor for a Drosophila peptide...... with a Drosophila genomic database clone that contained a DNA sequence coding for a protein having, again, structural similarities with the rat galanin receptors. Using PCR with primers coding for the presumed exons of this second Drosophila receptor gene, 5'- and 3'-RACE, and Drosophila cDNA as template, we...

  11. Molecular cloning of the feline thymus and activation-regulated chemokine cDNA and its expression in lesional skin of cats with eosinophilic plaque.

    Science.gov (United States)

    Maeda, Sadatoshi; Okayama, Taro; Ohmori, Keitaro; Masuda, Kenichi; Ohno, Koichi; Tsujimoto, Hajime

    2003-02-01

    Thymus and activation-regulated chemokine (TARC) is a member of CC chemokine and plays an essential role in recruitment of CC chemokine receptor 4 positive Th2 cells to allergic lesion. To investigate the association of TARC in allergic inflammation of cats, a TARC cDNA was cloned from feline thymus by RT-PCR with 3' rapid amplification of cDNA ends (RACE) method. The feline TARC clone contained a full length open reading frame encoding 99 amino acids which shared 80.8%, 72.5%, 65.6% and 67.8% homology with dog, human, mouse and rat homologues, respectively. Expression of TARC mRNA was detected not only in thymus but also in spleen, lung, lymph node, kidney, small intestine, colon and skin of the normal cat tissues examined. Furthermore, it was found that TARC mRNA was strongly expressed in lesional skin of cats with eosinophilic plaque. The present results demonstrated that TARC might be involved in the pathogenesis of eosinophilic plaque in cats.

  12. Implication of TNF superfamily receptors and their functional antagonists in neuronal apoptotic cell death

    OpenAIRE

    Gozzelino, Raffaella

    2007-01-01

    L'apoptosi pot ser induïda a través de nombrosos estímuls, entre els quals hi ha elsreceptors de mort. Per induir apoptosi TNFα necessita la participació d'inhibidors de latranscripció d'ARN o de la síntesi proteica, com són ActD i CHX. En aquest estudidemostrem com la citotoxicitat de TNFα en cèl·lules PC12 i en neurones corticalssensibilitzades amb ActD es dóna a través de l'activació de la caspasa iniciadora 8, dela generació de tBid i de la conseqüent activació de les pro-caspas...

  13. TNF Receptor 1/2 Predict Heart Failure Risk in Type 2 Diabetes Mellitus Patients.

    Science.gov (United States)

    Ping, Zhang; Aiqun, Ma; Jiwu, Li; Liang, Shao

    2017-04-06

    Inflammation plays an important role in heart failure and diabetes mellitus. Traditional serum markers have limited predictive value in heart failure and diabetes. TNFR1 and TNFR2 (TNFR1/2) have been proven to be strongly associated with heart failure and diabetes complications. This study aimed to assess the association of sTNFR1 and sTNFR2 levels and incidental HF risk in diabetes patients.We detected the mRNA, protein, and serum expression of TNFR1/2, their downstream signaling pathway protein NF-kB, and JNK expression and some traditional serum inflammatory markers in a heart failure group without diabetes mellitus or abnormal glucose tolerance (n = 84), a diabetes mellitus group without heart failure (n = 86), and a heart failure with diabetes mellitus group (n = 86).TNFR1/2 were significantly higher in patients with heart failure and diabetes mellitus based on mRNA expression to protein expression and serum expression. However, there were no differences in mRNA, protein, and serum levels of TNFR1/2 between the HF group and DM group. Furthermore, there were no differences between the groups in some traditional serum inflammatory markers.This study demonstrated higher expressions of TNFR, NF-kB, and JNK in patients with heart failure and diabetes mellitus. Compared with traditional serum markers, TNFR1 and TNFR2 are associated with heart failure risk in type 2 diabetes mellitus patients.

  14. Molecular characterization of a novel human hybrid-type receptor that binds the alpha2-macroglobulin receptor-associated protein

    DEFF Research Database (Denmark)

    Jacobsen, Linda; Madsen, P; Moestrup, S K

    1996-01-01

    the corresponding cDNA. The gene, designated SORL1, maps to chromosome 11q 23/24 and encodes a 2214-residue type 1 receptor containing a furin cleavage site immediately preceding the N terminus determined in the purified protein. The receptor, designated sorLA-1, has a short cytoplasmic tail containing a tyrosine...... but not in several major organs. Both RAP and an antibody against a synthetic peptide derived from a sequence determined in the mature protein detected sorLA-1 in crude human brain extracts. The domain structure suggests that sorLA-1 is an endocytic receptor possibly implicated in the uptake of lipoproteins...

  15. TNF-α and IL-1β Dependent Induction of CCL3 Expression by Nucleus Pulposus Cells Promotes Macrophage Migration through CCR1

    Science.gov (United States)

    Wang, Jianru; Tian, Ye; Phillips, Kate L.E.; Chiverton, Neil; Haddock, Gail; Bunning, Rowena A.; Cross, Alison K.; Shapiro, Irving M.; LeMaitre, Christine L.; Risbud, Makarand V.

    2012-01-01

    Objective To investigate TNF-α and IL-1β regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. Methods qRT-PCR and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, C/EBP-β and MAPK on cytokine mediated CCL3 promoter activity. Effect of NP-conditioned medium on macrophage migration was measured using a transwell system. Results An increase in CCL3 expression and promoter activity was observed in NP cells after TNF-α or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBP-β on CCL3 promoter was confirmed through gain- and loss-of-function studies. Noteworthy, co-transfection of p50 completely blocked cytokine and p65 dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with Sh-p65 and Sh-Ikkβ significantly decreased TNF-α dependent increase in CCL3 expression. Analysis of degenerate human NP tissues showed that CCL3, but not CCL4 expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNF-α or IL-1β promoted their migration; pretreatment of macrophages with antagonist to CCR1, primary receptor for CCL3 and CCL4, blocked cytokine mediated migration. Conclusions By controlling the activation of MAPK, NF-κB and C/EBPβ signaling, TNF-α and IL-1β modulate the expression of CCL3 in NP cells. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerate, herniated discs. PMID:23233369

  16. cDNA, genomic cloning and sequence analysis of ribosomal protein ...

    African Journals Online (AJOL)

    Ribosomal protein S4X (RPS4X) is one of the 40S ribosomal proteins encoded by the RPS4X gene. The cDNA and the genomic sequence of RPS4X were cloned successfully from giant panda (Ailuropoda melanoleuca) using reverse transcriptase-polymerase chain reaction (RT-PCR) and touchdown-PCR technology ...

  17. Cloning and sequencing of complete τ-crystallin cDNA from ...

    Indian Academy of Sciences (India)

    Unknown

    brain, heart and gonad, suggesting both to be the product of the same gene. The study thus provides the first report on cDNA sequence of τ-crystallin from a reptilian species and also re-confirms it to be an example of the phenomenon of gene sharing as was demonstrated earlier in the case of peking duck. Moreover, the ...

  18. Strategies to enhance immunogenicity of cDNA vaccine encoded antigens by modulation of antigen processing

    NARCIS (Netherlands)

    Platteel, Anouk C M; Marit de Groot, A; Andersen, Peter; Ovaa, Huib; Kloetzel, Peter M; Mishto, Michele; Sijts, Alice J A M

    2016-01-01

    Most vaccines are based on protective humoral responses while for intracellular pathogens CD8(+) T cells are regularly needed to provide protection. However, poor processing efficiency of antigens is often a limiting factor in CD8(+) T cell priming, hampering vaccine efficacy. The multistage cDNA

  19. Avoiding cross hybridization by choosing nonredundant targets on cDNA arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Knudsen, Steen

    2002-01-01

    PROBEWIZ designs PCR primers for amplifying probes for cDNA arrays. The probes are designed to have minimal homology to other expressed sequences from a given organism. The primer selection is based on user-defined penalt